U.S. patent application number 11/188577 was filed with the patent office on 2006-01-26 for antibody production methods related to disruption of peripheral tolerance in b lymphocytes.
Invention is credited to Thomas F. Tedder.
Application Number | 20060021069 11/188577 |
Document ID | / |
Family ID | 34752618 |
Filed Date | 2006-01-26 |
United States Patent
Application |
20060021069 |
Kind Code |
A1 |
Tedder; Thomas F. |
January 26, 2006 |
Antibody production methods related to disruption of peripheral
tolerance in B lymphocytes
Abstract
The subject invention relates a method for the production of
monoclonal antibodies. The method utilizes an immunized animal
having antibody-producing cells with disrupted peripheral
tolerance. The invention also provides a method for the use of such
monoclonal antibodies, and polyclonal antibodies derived from an
immunized animal having antibody-producing cells with disrupted
peripheral tolerance, for in vitro and in vivo clinical diagnostics
and therapeutics.
Inventors: |
Tedder; Thomas F.; (Durham,
NC) |
Correspondence
Address: |
JENKINS, WILSON & TAYLOR, P. A.
3100 TOWER BLVD
SUITE 1400
DURHAM
NC
27707
US
|
Family ID: |
34752618 |
Appl. No.: |
11/188577 |
Filed: |
July 25, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09555349 |
Aug 1, 2000 |
6921846 |
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PCT/US98/25253 |
Nov 25, 1998 |
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11188577 |
Jul 25, 2005 |
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60065975 |
Nov 28, 1997 |
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Current U.S.
Class: |
800/4 ;
435/70.21 |
Current CPC
Class: |
C07K 16/44 20130101;
C07K 2317/92 20130101; C07K 16/40 20130101 |
Class at
Publication: |
800/004 ;
435/070.21 |
International
Class: |
C12P 21/04 20060101
C12P021/04; A01K 67/027 20060101 A01K067/027 |
Goverment Interests
GRANT STATEMENT
[0002] This invention was made in part from government support
under Grant Number AI-26872 from the National Institute of Health
(NIH). The U.S. Government has certain rights in the invention.
Claims
1. A method for production of a monoclonal antibody to an antigen
comprising: (a) immunizing an animal, the animal having
antibody-producing cells with a manipulated characteristic that
facilitates the antibody-producing cell's ability to produce
antibodies, with said antigen to permit said antibody-producing
cells to produce antibodies to said antigen; (b) removing at least
a portion of said antibody-producing cells from said animal, (c)
forming a hybridoma by fusing one of said antibody-producing cells
with an immortalizing cell wherein said hybridoma is capable of
producing a monoclonal antibody to said antigen, (d) propagating
said hybridoma, and (e) harvesting the monoclonal antibodies
produced by said hybridoma.
2. The method of claim 1, wherein said manipulated characterisitic
comprises disrupted peripheral tolerance.
3. The method of claim 1, wherein the animal is selected from the
group consisting of a mouse, rat, pig, guinea pig, poultry, a goat,
a sheep, primate and a rabbit.
4. The method of claim 3, wherein said animal is a mouse.
5. The method of claim 4, wherein said mouse is a transgenic mouse
overexpressing CD19.
6. The method of claim 1, wherein said antibody-producing cells
comprise B lymphocytes.
7. The method of claim 1, wherein said monoclonal antibodies
produced comprise antibodies having a high affinity for said
antigen.
8. A method for production of a monoclonal antibody to an antigen
comprising: (a) immunizing an animal, the animal having
antibody-producing cells with disrupted peripheral tolerance, with
said antigen to permit said antibody-producing cells to produce
antibodies to said antigen; (b) removing at least a portion of said
antibody-producing cells from said animal, (c) forming a hybridoma
by fusing one of said antibody-producing cells with an
immortalizing cell wherein said hybridoma is capable of producing a
monoclonal antibody to said antigen, (d) propagating said
hybridoma, and (e) harvesting the monoclonal antibodies produced by
said hybridoma.
9. The method of claim 8, wherein said animal is selected from the
group consisting of a mouse, rat, pig, guinea pig, poultry, a goat,
a sheep, primate and a rabbit.
10. The method of claim 9, wherein said animal is a mouse.
11. The method of claim 10, wherein said mouse is a transgenic
mouse overexpressing CD19.
12. The method of claim 8, wherein said antibody-producing cells
comprise B lymphocytes.
13. The method of claim 8, wherein said monoclonal antibodies
produced comprise antibodies having a high affinity for said
antigen.
14. A method for production of polyclonal antibodies to an antigen
comprising immunizing an animal having antibody-producing cells
with disrupted peripheral tolerance with said antigen to permit
said antibody-producing cells to produce antibodies to said antigen
and separating serum, which contains said polyclonal antibodies,
from said animal.
15. The method of claim 14, wherein said animal is selected from
the group consisting of a mouse, rat, pig, guinea pig, poultry, a
goat, a sheep, primate and a rabbit.
16. The method of claim 15, wherein said animal is a mouse.
17. The method of claim 16, wherein said mouse is a transgenic
mouse overexpressing CD19.
18. The method of claim 14, wherein said antibody-producing cells
comprise B lymphocytes.
19. A diagnostic assay kit for detecting the presence of an antigen
in a biological sample, the kit comprising a first container
containing a first antibody capable of immunoreacting with the
antigen, wherein the first antibody is produced from an animal
having antibody-producing cells with disrupted peripheral tolerance
and the first antibody is present in an amount sufficient to
perform at least one assay.
20. The assay kit of claim 19, further comprising a second
container containing a second antibody that immunoreacts with the
first antibody, wherein second antibody is produced from an animal
having antibody-producing cells with disrupted peripheral
tolerance.
21. The assay kit of claim 20, wherein the first antibody and the
second antibody comprise monoclonal antibodies.
22. The assay kit of claim 21, wherein said first antibody
comprises an antibody having a high affinity for said antigen.
23. The assay kit of claim 20, wherein the first antibody is
affixed to a solid support.
24. The assay kit of claim 20, wherein the first and second
antibodies each further comprise an indicator.
25. An assay kit of claim 24, wherein the indicator is a
radioactive label or an enzyme.
26. A method of producing a non-human animal with an immune system
having cells with a predetermined characteristic, the method
comprising the steps of: (a) obtaining an animal having immune
system cells with a particular characteristic; (b) obtaining
another animal having immune system cells with either a same or a
different characteristic from the animal of step (a); and (c)
breeding the animal of step (a) with the animal of step (b) to
produce an animal with an immune system having cells with a
predetermined characteristic.
27. The method of claim 26, wherein said animals are selected from
the group consisting of a mouse, rat, pig, guinea pig, poultry, a
goat, a sheep, primate and a rabbit.
28. The method of claim 27, wherein said animals are transgenic
animals.
Description
PRIORITY APPLICATION INFORMATION
[0001] This application is a regular U.S. patent application under
37 C.F.R. .sctn. 1.111(a) based on and claiming priority to U.S.
Provisional Application Ser. No. 60/065,975 filed Nov. 28, 1997,
the entire contents of which are herein incorporated by
reference.
TECHNICAL FIELD OF THE INVENTION
[0003] The subject invention relates generally to a method for the
production of monoclonal antibodies. More particularly, the subject
invention utilizes an animal having antibody-forming cells, such as
B lymphocytes, with disrupted peripheral tolerance. Preferably, the
animal comprises a transgenic animal. The invention also provides a
method for the use of such monoclonal antibodies, and polyclonal
antibodies derived from an animal having antibody-forming cells,
such as B lymphocytes, with disrupted peripheral tolerance, for in
vitro and in vivo clinical diagnostics and therapeutics.
[0004] The publications and other materials used herein to
illuminate the background of the invention, and in particular
cases, to provide additional details respecting the practice, are
incorporated herein by reference, and for convenience, are
referenced by author and date in the following text, and
respectively group in the appended list of references.
TABLE-US-00001 Table of Abbreviations Ab antibody AFC
antibody-forming cell Ag antigen ALP alkaline phosphatase BSA
bovine serum albumin Btk Bruton's tyrosine kinase C complement,
usually followed by a number from 1 to 9 when referencing the
factors of the complement system in the immune system CD cluster of
differentiation CD19 cell surface molecule of B lymphocytes CD19KO
CD19-deficient mice CD19TG human CD19-transgenic mice CFA complete
Freund's adjuvant CGG chicken gamma-globulin CJD Creutzfeldt-Jakob
disease 15B3 a monoclonal antibody against bovine, murine and human
prion protein epitope HAT hypoxanthine, aminopterin and thymidine
hCD19 human CD19 HPRT hypoxanthine phosphoribosyl transferase HRP
horseradish peroxidase Ig immuoglobulin Ig.sup.HEL high-affinity
HEL-specific IgM.sup.a and IgD.sup.a-antigen receptors Lyn a
tyrosine kinase MAb monoclonal antibody MHC major
histocompatability complex NP (4-hydroxy-3-nitrophenyl)acetyl PNA
peanut agglutinin PrP prion protein PrPc a normal prion protein
epitope PrPSc a disease related prion protein epitope sHEL soluble
hen egg lysozyme TSE transmissible spongiform encephalopathic
agents TUNEL terminal deoxynucleotidyl transferase (TdT)-mediated
dUTP-biotin nick-end labeling Vav a protooncogene Xid X-linked
immunodeficiency - a mutation in Btk
BACKGROUND OF THE INVENTION
[0005] Kohler and Milstein are generally credited with having
devised the techniques that successfully resulted in the formation
of the first monoclonal antibody-producing hybridomas (G. Kohler
and C. Milstein (1975) Nature 256:495-497; (1976), Eur. J. Immunol.
6:511-519). By fusing antibody-forming cells (spleen B-lymphocytes)
with myeloma cells (malignant cells of bone marrow primary tumors),
they created a hybrid cell line arising from a single fused cell
hybrid (called a hybridoma or clone). The hybridoma had inherited
certain characteristics of both the lymphocytes and the myeloma
cell lines. Like the lymphocytes, the hybridoma secreted a single
type of immunoglobulin; moreover, like the myeloma cells, the
hybridoma had the potential for indefinite cell division. The
combination of these two features offered distinct advantages over
conventional antisera.
[0006] Antisera derived from vaccinated animals are variable
mixtures of polyclonal antibodies which never can be reproduced
identically. Monoclonal antibodies are highly specific
immunoglobulins of a single type. The single type of
immunoglobulins secreted by a hybridoma is specific to one and only
one antigenic determinant, or epitope, on the antigen, a complex
molecule having a multiplicity of antigenic determinants. For
instance, if the antigen is a protein, an antigenic determinant may
be one of the many peptide sequences (generally 6-7 amino acids in
length; Atassi, M. Z. (1980) Molec. Cell. Biochem. 32:21-43) within
the entire protein molecule. Hence, monoclonal antibodies raised
against a single antigen may be distinct from each other depending
on the determinant that induced their formation. For any given
hybridoma, however, all of the antibodies it produces are
identical. Furthermore, the hybridoma cell line is easily
propagated in vitro or in vivo, and yields monoclonal antibodies in
extremely high concentration.
[0007] A monoclonal antibody can be utilized as a probe to detect
its antigen. Thus, monoclonal antibodies have been used in in vitro
diagnostics, for example, radioimmunoassays and enzyme-linked
immunoassays (ELISA), and in in vivo diagnostics, e.g. in vivo
imaging with a radio-labeled monoclonal antibody. Also, a
monoclonal antibody can be utilized as a vehicle for drug delivery
to such antibodies' antigen.
[0008] Before a monoclonal antibody can be utilized for such
purpose, however, it is essential that the monoclonal antibody be
capable of binding to the antigen of interest; i.e., the target
antigen. This procedure is carried out by screening the hybridomas
that are formed to determine which hybridomas, if any, produce a
monoclonal antibody that is capable of binding to the target
antigen. This screening procedure can be very tedious in that
numerous, for example, perhaps several thousand, monoclonal
antibodies may have to be screened before a hybridoma that produces
an antibody that is capable of binding the target antigen is
identified. Accordingly, there is a need for a method for the
production of monoclonal antibodies that increases the likelihood
that the hybridoma will produce an antibody to the target
antigen.
[0009] Additionally, the immune systems of conventional animals
used in the production of monoclonal antibodies cannot recognize
epitopes that are highly conserved among vertebrate, and
particularly mammalian species, as "non-self" because of "self"
tolerance. The term "tolerance" is well known in the art and refers
to the failure of an animal's immune system to respond to its own
tissues. To the animal's immune system, a highly conserved epitope
appears to be "self", and no immune response is generated.
Therefore, conventional animals are ineffective in the production
of antibodies against such highly conserved epitopes.
[0010] There have been attempts in the prior art to address the
problems found in the production of monoclonal antibodies,
particularly with respect to the streamlining of the screening
process for monoclonal antibodies and, to a certain extent, to the
generation of a monoclonal antibody to an epitope that is highly
conservative among animal species, particularly mammalian
species.
[0011] One such attempt is described in U.S. Pat. No. 5,223,410
issued to Gargan et al. on Jun. 29, 1993, assigned to American
Biogenetic Sciences, Inc. This patent describes a method for
producing antibodies using an antigen-free animal. Particularly, it
describes the production of monoclonal antibodies using sterile or
germ-free mice. This patent focuses on the problem of streamlining
of the screening processes for monoclonal antibodies by providing
antigen-free or germ-free animals in which monoclonal antibodies
can be more easily identified.
[0012] Korth et al. (1997) "Prion (PrPSc)-Specific Epitope Defined
by a Monoclonal Antibody" (Letter to Nature) Nature 390:74
describes a monoclonal antibody, 15B3, that can discriminate
between the normal and disease-specific forms of a prion (PrP).
Prions are infectious particles causing transmissible spongiform
encephalopathies. 15B3 specifically precipitates bovine, murine, or
human PrPSc (the disease causing form), but not PrPc (the normal
form), suggesting that it recognizes an epitope common to prions
from different species. The 15B3 epitope was mapped as three
polypeptide segments in PrP using immobilized synthetic peptides.
However, the biological activity of this monoclonal antibody, which
was produced from BALB/c mice, is uncharacterized.
[0013] In light of the above, a need exists for a method for making
monoclonal antibodies against epitopes that are highly conservative
among vertebrate, and particularly, mammalian species.
SUMMARY OF THE INVENTION
[0014] In accordance with the subject invention, a method is
provided for the production of monoclonal antibodies to an antigen
comprising: [0015] (a) immunizing an animal having antibody-forming
cells with disrupted peripheral tolerance with said antigen to
permit said antibody-producing cells to produce antibodies to said
antigen; [0016] (b) removing at least a portion of said
antibody-producing cells from said animal; [0017] (c) forming a
hybridoma by fusing one of said antibody-producing cells with an
immortalizing cell wherein said hybridoma is capable of producing a
monoclonal antibody to said antigen; [0018] (d) propagating said
hybridoma; and [0019] (e) harvesting the monoclonal antibodies
produced by said hybridoma.
[0020] The subject invention also provides methods for utilizing a
monoclonal antibody or a polyclonal antibody derived from an animal
having antibody-forming cells with disrupted peripheral
tolerance.
[0021] Alternatively, the present invention provides a process of
detecting an antigen, wherein the process comprises immunoreacting
the antigen with an antibody prepared according to the process
described above to form an antibody-polypeptide conjugate, and
detecting the conjugate.
[0022] In another aspect, the present invention contemplates a
diagnostic assay kit for detecting the presence of an antigen in a
biological sample, where the kit comprises a first container
containing a first antibody capable of immunoreacting with antigen,
with the first antibody present in an amount sufficient to perform
at least one assay, and wherein the antibody is produced by the
process described above. Preferably, an assay kit of the invention
further comprises a second container containing a second antibody
that immunoreacts with the first antibody, wherein the second
antibody is produced by the processes described above. Thus, more
preferably, the antibodies used in an assay kit of the present
invention are monoclonal antibodies. Even more preferably, the
first antibody is affixed to a solid support. More preferably
still, the first and second antibodies comprise an indicator.
Optionally, the indicator is a radioactive label or an enzyme.
[0023] In another embodiment, the present invention contemplates a
diagnostic assay kit for detecting the presence, in a biological
sample, of an antibody immunoreactive with an antigen, the kit
comprising a first container containing the antigen that
immunoreacts with the antibody, with the antigen present in an
amount sufficient to perform at least one assay, and wherein the
antibody is produced by the processes described above.
[0024] In another embodiment, the present invention contemplates a
method of producing a non-human animal with an immune system having
cells with a predetermined characteristic. The method comprises the
steps of: [0025] (a) obtaining an animal having immune system cells
with a particular characteristic; [0026] (b) obtaining another
animal having immune system cells with either the same or a
different characteristic from the animal of step (a); and [0027]
(c) breeding the animal of step (a) with the animal of step (b) to
produce an animal with an immune system having cells with a
predetermined characteristic.
[0028] Accordingly, it is an object of this invention to provide an
improved method for the production of antibodies, particularly
monoclonal antibodies.
[0029] It is another object of this invention to provide a method
for the production of antibodies, particularly monoclonal
antibodies, using an animal having antibody producing cells with
disrupted peripheral tolerance.
[0030] It is a further object of this invention to provide a method
for the production of antibodies, particularly monoclonal
antibodies, using an animal having antibody producing cells with
disrupted peripheral tolerance.
[0031] It is still a further object of this invention to provide a
method of producing a non-human animal with an immune system having
cells with a predetermined characteristic.
[0032] Some of the aspects and objects of the invention having been
stated hereinabove, other aspects and objects will become evident
as the description proceeds, when taken in connection with the
accompanying drawings as best described hereinbelow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 depicts anti-HEL IgM.sup.a antibody levels in
Ig.sup.HEL and sHEL/Ig.sup.HEL mice that overexpress CD19. Each
value indicates serum levels of HEL-specific IgM.sup.a from
individual 2-month-old (2 mo) or 5- to 10-month old (>5 mo) mice
measured by ELISA. Horizontal bars indicating mean anti-HEL
antibody concentrations for each group are provided for reference.
The dashed horizontal line (arrowhead) delimits the 95% confidence
interval for the log normal distribution of anti-HEL antibody
levels observed in unimmunized 2-month-old sHEL/Ig.sup.HEL mice as
described in Materials and Methods.
[0034] FIG. 2 depicts humoral immune responses of (A)
sHEL/Ig.sup.HEL and (B) Ig.sup.HEL mice that overexpress CD19 in
response to immunization with HEL. Two-month-old mice were injected
i.p. with HEL or PBS mixed with CFA on days 0 and 21 (arrows), and
were bled at the indicated times. Levels of serum anti-HEL
IgM.sup.a antibodies for individual mice (dots or squares) were
determined by ELISA. Mean antibody levels are shown as solid
(hCD19.sup.+/+) or dashed (hCD19.sup.-/-) lines. The dashed
horizontal lines (arrowhead) delimit the 95% confidence interval
for the log normal distribution of anti-HEL antibody levels
observed in unimmunized sHEL/Ig.sup.HEL mice.
[0035] FIG. 3 depicts signal transduction through surface IgM and
CD19 in B cells from (A)sHEL/Ig.sup.HEL/hCD19.sup.+/+ or (B)
Ig.sup.HEL/hCD19.sup.+/+ mice. Relative [Ca.sup.+/+].sub.i levels
were assessed by flow cytometry after gating on the B220.sup.+
population of indo-1 loaded splenocytes. Baseline fluorescence
ratios were collected for 1 min before HEL and/or specific
monoclonal antibodies were added (arrows) at final concentrations
of: HEL, 100 ng/ml; anti-mouse CD19, 40 .mu.g/ml; anti-human CD19,
40 .mu.g/ml. An increase in [Ca.sup.++ ], over time is shown as an
increase in the ratio of indo-1 fluorescence. Values represent the
ratios of fluorescence intensity of cell populations after
treatment relative to the fluorescence intensity of untreated
cells. These results are representative of those obtained from
three littermate pairs of mice.
[0036] FIG. 4 depicts affinity measurements of anti-NP antibodies
from hybridomas of CD19TG and CD19KO mice compared with affinities
of antibodies generated wild-type C57BL/6 mice. Representative
anti-NP antibodies were purified and their affinities for NP (Ka)
measured by fluorescence quenching (bars). For comparison,
affinities of anti-NP antibodies generated by B cells isolated from
foci (filled circles) or germinal centers (filled triangles) are
shown as previously described in the art. The days following NP
immunization that the antibodies were isolated from mice is
indicated. The thick vertical line indicates the lower limit of
detection in the fluorescence quinch assay and the thin vertical
line indicates the average Ka for anti-NP antibodies generated by
germinal center B cells.
[0037] FIG. 5 depicts reactivity of anti-NP antibodies with self
antigens.
[0038] FIG. 5A depicts hybridoma supernatant fluid was assessed for
reactivity with ssDNA by ELISA. Sera from autoimmune
MRL.sup.lpr/lpr mice was used as a positive control. Values
represent mean OD values (.+-.SD) from triplicate wells. Similar
results were obtained in three independent experiments.
[0039] FIG. 5B depicts reactivity of purified TG7-83 antibody with
ssDNA compared with two established anti-ssDNA antibodies of the
IgG1 isotype (452s.69 and 165s.3g). Reactivity was significantly
higher (p<0.05, * p<0.01) than the negative control
antibodies (B1-8 or TG18-161).
DETAILED DESCRIPTION OF THE INVENTION
[0040] In the preferred embodiment of this invention transgenic
mouse models for autoreactive B cells provide a mechanism for
determining the role of CD19 signaling in regulating peripheral
tolerance in autoimmunity. The CD19 cell surface molecule regulates
signal transduction events critical for B lymphocyte development
and humoral immunity. Increasing the density of CD19 expression
renders B lymphocytes hyper-responsive to transmembrane signals,
and transgenic mice that over-express CD19 have increased levels of
autoantibodies. The role of CD19 in tolerance regulation and
auto-antibody generation was therefore examined by crossing mice
that overexpress a human CD19 transgene with transgenic mice
expressing a model autoantigen (soluble hen egg lysozyme, sHEL) and
high-affinity HEL-specific IgM.sup.a and IgD.sup.a (Ig.sup.HEL
antigen receptors).
[0041] In the preferred model of peripheral tolerance, B cells in
sHEL/Ig.sup.HEL double-transgenic mice are functionally anergic and
do not produce autoantibodies. However, it was found that
overexpression of CD19 in sHEL/Ig.sup.HEL double-transgenic mice
resulted in a breakdown of peripheral tolerance and the production
of anti-HEL antibodies at levels similar to those observed in
Ig.sup.HEL mice lacking the sHEL autoantigen. Therefore, altered
signaling thresholds due to CD19 overexpression resulted in the
breakdown of peripheral tolerance. Thus, CD19 overexpression shifts
the balance between tolerance and immunity to autoimmunity by
augmenting antigen receptor signaling.
[0042] This surprising discovery indicates that animals having
antibody producing cells with disrupted peripheral tolerance are
useful in the production of monoclonal antibodies. Transgenic CD19
mice wherein CD19 is over-expressed experience a breakdown in
peripheral tolerance. This renders B lymphocytes in such mice
hyper-responsive to transmembrane signals. Such B lymphocytes are
thus capable of distinguishing highly conserved epitopes in mammals
when such epitopes are introduced to the mouse as an antigen. The
immune system of a normal mouse would perceive such an epitope as
identical to something that occurs naturally or is native to the
mouse ("self"). In contrast, the immune system of the CD19
over-expressing transgenic mouse, and particularly the B
lymphocytes of the mouse's immune system, recognize the highly
conservative mammalian epitope as a particle foreign to the mouse's
system ("non-self"), which would instigate an immune response.
[0043] This immune response is developed to produce monoclonal
antibodies as described more fully herein. The demonstrated ability
to break down peripheral tolerance as described herein and the
breeding experiments described herein provide a method for
manipulating the immune system of an animal such that an animal
having an altered immune system with desired characteristics can be
produced, as more fully described in Example 3.
[0044] While the following terms are believed to have well defined
meanings in the art, the following definitions are set forth to
facilitate explanation of the invention.
[0045] The term "immune system" includes all the cells, tissues,
systems, structures and processes, including non-specific and
specific categories, that provide a defense against "non-self"
molecules, including potential pathogens, in an animal.
[0046] As is well known in the art, the non-specific immune system
includes phagocytositic cells such as neutrophils, monocytes,
tissue macrophages, Kupffer cells, alveolar macrophages and
microglia. The specific immune system refers to the cells and other
structures that impart specific immunity within a host. Included
among these cells are the lymphocytes, particularly the B cell
lymphocytes and the T cell lymphocytes. These cells also include
natural killer (NK) cells. Additionally, antibody-producing cells,
like B lymphocytes, and the antibodies produced by the
antibody-producing cells are also included within the term "immune
system".
[0047] The term "tolerance" is meant to refer to an animal's immune
system's failure to respond to its own tissues or to tissues or
molecules so like its own as to be recognized as its own.
[0048] The term "peripheral" or "peripheral lymphoid tissues" refer
to the lymph node-, spleen-, or gut-associated lymphoid tissues
wherein cells, such as B lymphocytes, of the immune system are
developed.
[0049] Thus, the term "peripheral" in the context of the term
"peripheral tolerance" indicates a tolerance, or failure to
recognize an antigen, by a cell of the immune system, such as a B
lymphocyte, in the peripheral lymphoid tissues wherein such cells
usually react with antigens.
[0050] The term "disrupted peripheral tolerance", as used herein
and in the claims, means any manipulation or alteration of the
peripheral tolerance of the antibody-producing cells of the immune
system. Preferably, the term "disrupted peripheral tolerance" is
meant to refer to the break down of peripheral tolerance, which
facilities monoclonal antibody production in accordance with the
methods of the present invention.
[0051] The term "anergy" means a condition in which the immune
system of an animal fails to respond to the injection of an
antigen. Thus, the term "peripheral anergy" means a condition in
which the peripheral immune system of an animal fails to respond to
the injection of an antigen.
[0052] The term "autoantibody" means an antibody formed against an
epitope native to the animal.
[0053] The term "antibody-producing cell" refers to any
antibody-producing cell within the immune system. Preferably, it is
meant to refer to B lymphocytes.
[0054] The term "complement" is meant to refer to the non-specific
defense system that is activated by the bonding of antibodies to
antigens and by this means is directed against specific invaders
that have been identified by antibodies. Eleven complement proteins
have been characterized in the field and are generally referred to
by those having ordinary skill in the art as C1-C9. The complement
proteins act generally along a cascade wherein they contribute to
(1) recognition (C1); (2) activation (C4, C2, and C3, in that
order); and (3) attack (C5-C9). During the attack phase, complement
proteins attach to the cell membrane and destroy the victim cell in
a process known as complement fixation. The complement system is
well known in the art and is more fully described in Fox, Human
Physiology, William C. Brown Pub., DuBuque, Iowa (1987).
[0055] The term "humoral immunity" is meant to refer to the form of
acquired immunity in which antibody molecules are secreted in
response to antigenic stimulation.
[0056] The term "cell-mediated immunity" is meant to refer to the
immunological defense provided by T cell lymphocytes, which come
into close proximity to their victim cells.
[0057] The terms "B cell lymphocytes" or "B lymphocytes" are meant
to refer to a type of lymphocyte that can be transformed by
antigens into plasma cells that secrete antibodies, and are thus
responsible for humoral immunity.
[0058] The term "T cell lymphocytes" is meant to refer to a type of
lymphocyte that provides cell-mediated immunity, in contrast to B
lymphocytes that provide humoral immunity to the secretion of
antibodies. There are three sub-populations of T cells: cytotoxic,
helper, and suppressor.
[0059] The terms "overexpress", "overexpressing" and
"overexpressed" refer to any level of expression of a gene or
protein, whether the gene be a transgene or a normal gene, that
exceeds normal or expected levels of expression by any amount.
[0060] Following long-standing patent law convention, the terms "a"
and "an" mean "one or more" when used in this application,
including the claims.
[0061] In the following Detailed Description, the use of transgenic
mice which overexpress CD19 is described as a preferred embodiment
of the instant invention. Such transgenic mice have been developed
in the field according to published techniques (see, for example,
Engel et al. (1995) Immunity 3:39-50 and Zhou (1994) Mol. Cell.
Biol. 14:3884-3894). Thus, these mice are conveniently available as
starting materials. However, it also should be noted that there has
been no disclosure of the production of monoclonal antibodies until
the instant disclosure.
[0062] Given advances in transgenic animal techniques, which have
been published in the art, it is believed that any animal can be
utilized in the subject invention, including mouse, pig, rat,
rabbit, guinea pig, goat, sheep, primate, and poultry.
[0063] Moreover, while CD19 overexpressing transgenic mice are
preferred because the break down in peripheral tolerance of
antibody-producing cells found in these mice, other animals having
a manipulated or altered characteristics in the cells of their
immune system are contemplated to be within the scope of this
invention. Moderate levels of disrupted peripheral tolerance have
been described in the art with respect to the manipulation of CD45
and with respect to the manipulation of LPR mice. But, there has
been no disclosure of the production of monoclonal antibodies until
the instant disclosure. Finally, other particular candidate
characteristics for manipulation are provided in Example 3.
Production of Monoclonal Antibodies
[0064] The animal having antibody-forming cells with disrupted
peripheral tolerance is utilized for the production of monoclonal
antibodies. The system can be utilized to produce a monoclonal
antibody to any antigen that the animal not having antibody-forming
cells with disrupted peripheral tolerance could produce. An
exemplary list of antigens appears in U.S. Pat. No. 3,935,074, the
contents of which are herein incorporated by reference.
[0065] However, the animal having antibody-forming cells, such as B
lymphocytes, with disrupted peripheral tolerance provides a much
enhanced immune response to the antigen. Thus, one can increase the
likelihood of locating a B-lymphocyte that produces an antibody
that is capable of binding to a specific epitope of the antigen.
This is a major advantage of the subject invention. In addition,
the system having antibody-forming cells with disrupted peripheral
tolerance system is particularly useful for generating a highly
specific antibody for those antigens with numerous epitopes.
[0066] Preferably, the system having antibody-forming cells with
disrupted peripheral tolerance is used to generate monoclonal
antibodies to epitopes that are highly conserved among vertebrate
and particularly, mammalian species. Animals not having
antibody-forming cells with disrupted peripheral tolerance do not
typically respond to such highly conserved epitopes because of
self-tolerance. Stated differently, the immune systems of
conventional animals cannot recognize highly conserved epitopes as
"non-self" and therefore cannot produce antibodies against such an
epitope. The immune systems of the animals of the instant
invention, can recognize highly conserved epitopes as "non-self"
because of the antibody-forming cells with disrupted peripheral
tolerance.
[0067] The animals of the instant invention can be immunized by
standard techniques. For example, the animal be immunized at least
two times with at least about three weeks between each
immunization, followed by a prefusion booster.
Somatic Cells
[0068] Somatic cells of the animal having the potential for
producing antibody and, in particular B lymphocytes, are suitable
for fusion with a B-cell myeloma line. Those antibody-producing
cells that are in the dividing plasmablast stage fuse
preferentially. Somatic cells can be derived from the lymph nodes,
spleens and peripheral blood of primed animals, and the lymphatic
cells of choice depend to a large extent on their empirical
usefulness in the particular fusion system. However, somatic cells
derived from the spleen are generally preferred. Once primed or
hyperimmunized, animals having antibody-forming cells with
disrupted peripheral tolerance can be used as a source of
antibody-producing lymphocytes. Mouse lymphocytes give a higher
percentage of stable fusions with the mouse myeloma lines described
herein below. Indeed, mice are the preferred animals for use in
making monoclonal antibodies because of the availability of
excellent cell lines to use as fusion partners. However, the use of
antibody-producing cells from other animals is also possible. The
choice of a particular animal depends on the choice of antigen, for
it is important that the animal have a B-lymphocyte in its
repertoire of B-lymphocytes that can produce an antibody to such
antigen.
Immortalizing Cells
[0069] Specialized myeloma cell lines have been developed from
lymphocyte tumors for use in hybridoma-producing fusion procedures
(G. Kohler and C. Milstein (1976) Eur. J. Immunol 6:511-519; M.
Schulman et al. (1978) Nature 276:269-270). The cell lines have
been developed for at least three reasons. The first reason is to
facilitate the selection of fused myeloma cells. Usually, this is
accomplished by using myelomas with enzyme deficiencies that render
them incapable of growing in certain selective media that support
the growth of hybridomas. The second reason arises from the
inherent ability of lymphocyte tumor cells to produce their own
antibodies. The purpose of using monoclonal techniques is to obtain
immortal fused hybrid cell lines that produce the desired single
specific antibody genetically directed by the somatic cell
component of the hybridoma. To eliminate the production of tumor
cell antibodies by the hybridomas, myeloma cell lines incapable of
producing light or heavy immunoglobulin chains or those deficient
in antibody secretion mechanisms are used. A third reason for
selection of these cell lines is their suitability and efficiency
for fusion.
[0070] Several myeloma cell lines can be used for the production of
fused cell hybrids, including NS-1, X63-Ag8, NIS-Ag4/1,
MPC11-45.6TG1.7, X63-Ag8.653, Sp2/O-Agf14, FO, and S194/5XXO.Bu.1.,
all derived from mice, and 210-.RCY3.AgI.+B 2.3+L derived from
rats. (G. J. Hammerling, U. Hammerling and J. F. Kearnly, eds.
(1981), Monoclonal antibodies and hybridomas, J. L. Turk, eds.
Research Monographs in Immunology, Vol. 3, Elsevier/North Holland
Biomedical Press, New York).
Fusion
[0071] Methods for generating hybrids of antibody-producing spleen
or lymph node cells and immortalizing cells generally comprise
mixing somatic cells with immortalizing cells in a proportion which
can vary from about 20:1 to about 1:1 in the presence of an agent
or agents (chemical, viral or electrical) that promote the fusion
of cell membranes. It is often preferred that the same species of
animal serve as the source of the somatic and immortalizing cells
used in the fusion procedure. Fusion methods have been described by
Kohler and Milstein (1975), Nature 256:495-497; (1976), Eur. J.
Immunol. 6:511-519; by Gefter et al. (1977), Somatic Cell Genet.
3:231-236 and by Kozbor et al. (1983), Immunology Today, 4:72. The
fusion-promoting agents used by those investigators were Sendai
virus and polyethylene glycol (PEG), respectively.
[0072] One can also utilize the recently developed
EBV-transformation technique (Cole et al. (1985), Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Isolation of Clones and Antibody Detection
[0073] Fusion procedures usually produce viable hybrids at very low
frequency, about 1.times.10.sup.-5 to 1.times.10.sup.-8. Because of
the low frequency of obtaining viable hybrids, it is essential to
have a means to select fused cell hybrids from the remaining
unfused cells, particularly the unfused myeloma cells. A means of
detecting the desired antibody-producing hybridomas among the other
resulting fused cell hybrids is also necessary.
[0074] Generally, the fused cells are cultured in selective media,
for instance HAT medium, which contains hypoxanthine, aminopterin
and thymidine. HAT medium permits the proliferation of hybrid cells
and prevents growth of unfused myeloma cells which normally would
continue to divide indefinitely. Aminopterin blocks de novo purine
and pyrimidine synthesis by inhibiting the production of
tetrahydrofolate. The addition of thymidine bypasses the block in
pyrimidine synthesis, while hypoxanthine is included in the media
so that inhibited cells can synthesize purine using the nucleotide
salvage pathway. The myeloma cells employed are mutants lacking
hypoxanthine phosphoribosyl transferase (HPRT) and thus cannot
utilize the salvage pathway. In the surviving hybrid, the B
lymphocyte supplies genetic information for production of this
enzyme. Since B lymphocytes themselves have a limited life span in
culture (approximately two weeks), the only cells which can
proliferate in HAT media are hybrids formed from myeloma and spleen
cells.
[0075] To facilitate screening of antibody secreted by the hybrids
and to prevent individual hybrids from overgrowing others, the
mixture of fused myeloma and B-lymphocytes is diluted in HAT medium
and cultured in multiple wells of microtiter plates. In two to
three weeks, when hybrid clones become visible microscopically, the
supernatant fluid of the individual wells containing hybrid clones
is assayed for specific antibody production.
[0076] The assay must be sensitive, simple and rapid. Assay
techniques include radioimmunoassays, enzyme immunoassays,
cytotoxicity assays, and plaque assays.
Cell Propagation and Antibody Production
[0077] Once the desired fused cell hybrids have been selected and
cloned into individual antibody-producing cell lines, each cell
line can be propagated in either of two standard ways. A sample of
the hybridoma can be injected into a histocompatible animal of the
type that was used to provide the somatic and myeloma cells for the
original fusion. The injected animal develops tumors secreting the
specific monoclonal antibody produced by the fused cell hybrid. The
body fluids of the animal, such as serum or ascites fluid, can be
tapped to provide monoclonal antibodies in high concentration.
Alternatively, the individual cell lines can be propagated in vitro
in laboratory culture vessels. The culture medium, containing high
concentrations of a single specific monoclonal antibody, can be
harvested by decantation, filtration or centrifugation.
Use of the Monoclonal Antibody
[0078] The monoclonal antibodies made by the method of the subject
invention can be utilized in any technique known or to be developed
in the future that utilizes a monoclonal antibody.
[0079] A major use of monoclonal antibodies is in an immunoassay,
which is the measurement of the antigen-antibody interaction. Such
assays are generally heterogeneous or homogeneous. In a homogeneous
immunoassay the immunological reaction usually involves the
specific antibody, a labeled analyte, and the sample of interest.
The signal arising from the label is modified, directly or
indirectly, upon the binding of the antibody to the labeled
analyte. Both the immunological reaction and detection of the
extent thereof are carried out in a homogeneous solution.
Immunochemical labels which may be employed include free radicals,
fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
The major advantage of a homogeneous immunoassay is that the
specific antibody need not be separated from the labeled
analyte.
[0080] In a heterogeneous immunoassay, the reagents are usually the
specimen, the specific antibody, and means for producing a
detectable signal. The specimen is generally placed on a support,
such as a plate or a slide, and contacted with the antibody in a
liquid phase. The support is then separated from the liquid phase
and either the support phase or the liquid phase is examined for a
detectable signal employing means for producing such signal. The
signal is related to the presence of the analyte in the specimen.
Means for producing a detectable signal include the use of
radioactive labels, fluorescers, enzymes, and so forth. Exemplary
of heterogeneous immunoassays are the radioimmunossay,
immunofluoroescence methods, enzyme-linked immunoassays, and the
like.
[0081] For a more detailed discussion of the above immunoassay
techniques, see Enzyme-immunoassay, by Edward T. Maggio, CRC Press,
Inc., Boca Raton, Fla., (1980). See also, for example, U.S. Pat.
Nos. 3,690,834; 3,791,932; 3,817,837; 3,850,578; 3,853,987;
3,867,517; 3,901,654; 3,935,074; 3,984,533; 3,996,345; and
4,098,876, the contents of each of which are herein incorporated by
reference, and which listing is not intended to be exhaustive.
[0082] Another major use of monoclonal antibodies are in vivo
imaging and therapeutics. The monoclonal antibodies can be labeled
with radioactive compounds, for instance, radioactive iodine, and
administered to a patient intravenously. The antibody can also be
labeled with a magnetic probe. NMR can then be utilized to pinpoint
the antigen. After localization of the antibodies at the antigen,
the antigen can be detected by emission tomographical and
radionuclear scanning techniques, thereby pinpointing the location
of the antigen.
[0083] By way of illustration, the purified monoclonal antibody is
suspended in an appropriate carrier, e.g., saline, with or without
human albumin, at an appropriate dosage and is administered
intravenously, e.g., by continuous intravenous infusion over
several hours, as in Miller et al., In Hybridomas in Cancer
Diagnosis and Therapy (1982), incorporated herein by reference.
[0084] The monoclonal antibodies of subject invention can be used
therapeutically. Antibodies with the proper biological properties
are useful directly as therapeutic agents. Alternatively, the
antibodies can be bound to a toxin to form an immunotoxin or to a
radioactive material or drug to form a radiopharmaceutical or
pharmaceutical. Methods for producing immunotoxins and
radiopharmaceuticals of antibodies are well-known (see, for
example, Cancer Treatment Reports (1984) 68:317-328).
[0085] It also is believed that polyclonal antibodies derived from
an animal having antibody-producing cells with disrupted peripheral
tolerance also can be utilized in immunoassays and provide an
improved result as compared to polyclonal antibodies derived from a
conventional animal. Polyclonal antibodies derived from an animal
having antibody-producing cells with disrupted peripheral tolerance
can be made by utilizing such an animal, as described hereinabove,
and immunization techniques, as described hereinabove, followed by
separating the polyclonal antibodies from the animal by
conventional techniques, e.g. by separating the serum from the
animal.
[0086] Means for preparing and characterizing antibodies are well
known in the art (See, e.g., Antibodies--A Laboratory Manual, E.
Howell and D. Lane, Cold Spring Harbor Laboratory, 1988).
Monoclonal antibodies can be readily prepared through use of
well-known techniques such as those exemplified in U.S. Pat. No.
4,196,265, herein incorporated by reference.
Pharmaceutical Compositions
[0087] In a preferred embodiment, the present invention provides
pharmaceutical compositions comprising a monoclonal antibody
produced by a process of the present invention and a
physiologically acceptable carrier. Such a composition has a
variety of uses, including, for example but not limited to, use as
a delivery agent for a cytotoxic substance as described herein.
[0088] A composition of the present invention is typically
administered parenterally in dosage unit formulations containing
standard, well-known nontoxic physiologically acceptable carriers,
adjuvants, and vehicles as desired. The term "parenteral" as used
herein includes intravenous, intramuscular, intraarterial
injection, or infusion techniques.
[0089] Injectable preparations, for example sterile injectable
aqueous or oleaginous suspensions, are formulated according to the
known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation can also be a
sterile injectable solution or suspension in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol.
[0090] Among the acceptable vehicles and solvents that may be
employed are water, Ringer's solution, and isotonic sodium chloride
solution. In addition, sterile, fixed oils are conventionally
employed as a solvent or suspending medium. For this purpose any
bland fixed oil can be employed including synthetic mono- or
di-glycerides. In addition, fatty acids such as oleic acid find use
in the preparation of injectables.
[0091] Preferred carriers include neutral saline solutions buffered
with phosphate, lactate, Tris, and the like.
Assay Kits
[0092] In another aspect, the present invention contemplates
diagnostic assay kits for detecting the presence of an antigen in
biological samples, where the kits comprise a first container
containing a first antibody capable of immunoreacting with the
antigen with the first antibody present in an amount sufficient to
perform at least one assay, the antibody obtained from an animal
having antibody-producing cells with disrupted peripheral
tolerance. Preferably, the assay kits of the invention further
comprise a second container containing a second antibody that
immunoreacts with the first antibody, the second antibody obtained
from an animal having antibody-producing cells with disrupted
peripheral tolerance. Thus more preferably, the antibodies used in
the assay kits of the present invention are monoclonal antibodies.
Even more preferably, the first antibody is affixed to a solid
support. More preferably still, the first and second antibodies
comprise an indicator, and, preferably, the indicator is a
radioactive label or an enzyme.
[0093] The following Examples have been included to illustrate
preferred modes of the invention. Certain aspects of the following
Examples are described in terms of techniques and procedures found
or contemplated by the present inventor to work well in the
practice of the invention. These Examples are exemplified through
the use of standard laboratory practices of the inventor. In light
of the present disclosure and the general level of skill in the
art, those of skill will appreciate that the following Examples are
intended to be exemplary only and that numerous changes,
modifications and alterations can be employed without departing
from the spirit and scope of the invention.
EXAMPLE 1
CD19-Regulated Signaling Thresholds Control Peripheral Tolerance
and Autoantibody Production in B Lymphocytes
[0094] B lymphocyte tolerance to self antigens is achieved by the
negative selection and elimination of immature B cells that express
high-affinity IgM receptors for autoantigens. Goodnow, C. C. (1996)
Proc. Natl. Acad. Sci. USA 93:2264-2271; Hartley et al. (1993) Cell
72:325-335; Nemazee et al. (1989) Nature 337:562-566; Goodnow, C.
C. (1992) Annu. Rev. Immunol. 10:489-518. Negative selection is
antigen receptor-dependent but also relies on established
triggering thresholds for intracellular signals (Goodnow, C. C.
(1996) Proc. Natl. Acad. Sci. USA 93:2264-2271; Klinman, N. R.
(1996) Immunity 5:189-195). If antigen receptor ligation generates
inadequate intracellular signals because of a low affinity for
autoantigens, or the valency or concentration of autoantigen is
low, autoreactive B cells mature and leave the bone marrow but are
rendered functionally anergic. Goodnow, C. C. (1996) Proc. Natl.
Acad. Sci. USA 93:2264-2271; Goodnow, C. C. (1992) Annu. Rev.
Immunol. 10:489-518; Klinman, N. R. (1996) Immunity 5:189-195;
Adelstein et al. (1991) Science 251:1223-1225; Nemazee et al.
(1991) Immunol. Rev. 122:117-132. Intracellular signaling
thresholds are likely to also play a major role in the regulation
and maintenance of peripheral tolerance.
[0095] The CD19 cell surface molecule regulates intracellular
signaling thresholds critical for B cell development and humoral
immunity. Tedder et al. (1997) Immunity 6:107-118; Fearon et al.
(1996) Science 272:50-54; Carter et al. (1992) Science 256:105-107;
Dempsey et al. (1996) Science 271:348-350; Engel et al. (1995)
Immunity 3:39-50; Rickert et al. (1995) Nature 376:352-355. B
lymphocytes from mice that overexpress CD19 are hyper-responsive to
antigen receptor crosslinking, which results in serum
immunoglobulin (Ig) levels that are increased by about 40% and
humoral responses that are augmented several fold. Engel et al.
(1995) Immunity 3:39-50; Sato et al. (1995) Proc. Natl. Acad. Sci.
USA 92:11558-11562; Zhou et al. (1994) Mol. Cell. Biol.
14:3884-3894. Based on this, it was expected that CD19
overexpression by autoreactive B cells would either lead to their
augmented negative selection in the bone marrow or result in a more
profound state of peripheral anergy.
[0096] Unexpectedly however, C57BL/6 mice that overexpress CD19
have twofold to fourfold higher levels of anti-DNA autoantibodies
and rheumatoid factor. Tedder et al. (1997) Immunity 6:107-118;
Sato et al. (1996) J. Immunol. 156:4371-4378. Increased
autoantibody production in mice overexpressing CD19 correlates with
dramatic increases in the number of B1 lineage cells. However,
since IgG anti-DNA autoantibodies are preferentially increased in
mice that overexpress CD19, the CD19-induced autoantibodies may
alternatively result from alterations in conventional B-cell
tolerance.
[0097] Transgenic mouse models for autoreactive B cells (Goodnow,
C. C. (1992) Annu. Rev. Immunol 10:489-518; Nemazee et al. (1991)
Immunol. Rev. 122:117-132) provide a mechanism for determining the
role of CD19 signaling in regulating peripheral tolerance and
autoimmunity. B cells from transgenic mice expressing a model
autoantigen (soluble hen egg lysozyme, sHEL) and high-affinity
HEL-specific IgM.sup.a and IgD.sup.a (Ig.sup.HEL) antigen receptors
enter the peripheral pool but are anergic to antigen receptor
ligation and produce little, if any, spontaneous HEL-specific
antibody. Goodnow et al. (1988) Nature 334:676-682. Mice that
express a human CD19 (hCD19) transgene provide a model for
examining augmented CD19 function in vivo. Tedder et al. (1997)
Immunity 6:107-118; Sato et al. (1995) Proc. Natl. Acad. Sci. USA
92:11558-11562; Zhou et al. (1994) Mol. Cell. Biol. 14:3884-3894;
Sato et al. (1996) J. Immunol. 156:4371-4378; Sato et al. (1997) J.
Immunol. 158:4662-4669) Since hCD19 can replace the function of
mouse CD19 in vivo, hemizygous hCD19.sup.+/- transgenic mice
express cell surface CD19 at a twofold higher density while
hCD19.sup.+/+ transgenic mice express threefold higher densities of
CD19. Sato et al. (1996) J. Immunol. 156:4371-4378; Sato et al.
(1997) J. Immunol. 158:4662-4669.
[0098] Therefore, sHEL/Ig.sup.HEL double-transgenic mice were
crossed with hCD19 transgenic mice to determine whether tolerance
would be maintained in sHEL/Ig.sup.HEL/hCD19 transgenic mice or
autoantibodies would be generated. CD19 overexpression in
sHEL/Ig.sup.HEL double-transgenic mice resulted in the production
of anti-HEL antibodies at levels similar to those observed in
Ig.sup.HEL mice lacking this model self antigen. Therefore, lowered
signaling thresholds due to CD19 overexpression resulted in the
breakdown of peripheral tolerance in sHEL/Ig.sup.HEL
double-transgenic mice.
[0099] Mice. hCD19 transgenic mice (h19-1 line, C57BL/6) were
produced as described in Engel et al. (1995) Immunity 3:39-50 and
in Zhou et al. (1994) Mol. Cell. Biol. 14:3884-3894). In the h19-1
line of mice, 9-14 copies of the hCD19 transgene are integrated
into a single (or closely linked) site(s). These h19-1 mice used in
this study were backcrossed onto a wild-type C57BL/6 background for
8 to 10 generations without a diminution of hCD19 expression and
all mice express similar levels of cell-surface hCD19. Mice
expressing sHEL (ML5 line) and Ig.sup.HEL (MD4 line) were as
described (Goodnow et al. (1988) Nature 334:676-682; Hartley et al.
(1991) Nature 353:765-769). sHEL/Ig.sup.HEL/hCD19 triple-transgenic
mice were generated by appropriate backcrosses of sHEL/Ig.sup.HEL
double-transgenic mice with hCD19.sup.+/+ mice. Transgene
expression was assessed as described in (Engel et al. (1995)
Immunity 3:39-50; Zhou et al. (1994) Mol. Cell. Biol. 14:3884-3894;
Goodnow et al. (1988) Nature 334:676-682; Hartley et al. (1991)
Nature 353:765-769.) Mice were housed in a specific pathogen-free
barrier facility. All studies and procedures were approved by the
Duke University Animal Care and Use Committee.
[0100] Immunization of Mice. Two-month-old mice were immunized i.p.
with 100 .mu.g of HEL in complete Freund's adjuvant (CFA, Sigma
Chemical Co.) or PBS in CFA at day 0 and were boosted at day 21.
Animals were bled just before the first immunization and 7, 14, and
28 days later.
[0101] Mouse Ig Isotype-specific ELISAs. Serum levels of
HEL-specific IgM allotype a (IgM.sup.a) antibody were measured by
ELISA on HEL-coated plates as described (Goodnow et al. (1989)
Nature 342:385-391). Absolute antibody concentrations were
determined relative to a standard curve of HEL-specific IgM.sup.a
monoclonal antibody (E1 clone) generated from an Ig.sup.HEL
transgenic mouse immunized with HEL. The ELISA sensitivity limit
was about 20 ng/ml of anti-HEL IgM.sup.a antibody.
[0102] Immunofluorescence Analysis. Antibodies used in this study
included: FITC-conjugated and biotin-coupled goat anti-mouse IgM
isotype-specific antibodies (Southern Biotechnology Associates,
Inc., Birmingham, Ala.); anti-B220 (CD45RA, RA3-6B2, provided by R.
L. Coffman, DNAX Research Inst., Palo Alto, Calif.), anti-I-A
(M5/114.15.2, American Type Culture Collection (ATCC), Bethesda,
Md., clone TIB120), anti-HSA (M1/69, PharMingen, San Diego,
Calif.), anti-CD5 (53-7.313, ATCC, clone TIB104), anti-B7-2 (GL-1,
PharMingen) and anti-mouse IgM.sup.a (DS-1, PharMingen) monoclonal
antibodies. Phycoerythrin-conjugated streptavidin (Fisher
Scientific, Fair Lawn, N.J.) was used to reveal biotin-coupled
monoclonal antibody staining. Phycoerythrin-conjugated goat
anti-rat IgG antibodies (Caltag, Burlingame, Calif.) were used to
visualize anti-CD5 monoclonal antibody staining. Cells reacting
with biotin-coupled HEL were stained with phycoerythrin-conjugated
streptavidin. Isolated lymphocytes were analyzed on a FACScan.RTM.
flow cytometer (Becton-Dickinson, San Jose, Calif.) as described
(Sato et al. (1996) J. Immunol. 156:4371-4378.) Measurement of
Intracellular Calcium. Splenocytes were isolated, loaded with
indo-1 and stained with FITC-labeled anti-B220 antibodies as
described (Sato et al. (1996) Immunity 5:551-562.). Relative
intracellular Ca.sup.++ levels ([Ca.sup.++].sub.i) were assessed by
flow cytometry after gating on the B220.sup.+ population of cells.
Baseline fluorescence ratios were collected for 1 min before HEL
and/or specific monoclonal antibodies were added at final
concentrations of: HEL, 100 ng/ml; anti-mouse CD19, 40 .mu.g/ml
(MB19-1, IgA) (Sato et al. (1996) J. Immunol. 156:4371-4378); and
anti-human CD19, 40 .mu.g/ml (HB12b, IgG1) (Bradbury et al. (1992)
J. Immunol. 149:2841-2850.) An increase in the ratio of indo-1
fluorescence indicates an increase in [Ca.sup.++].sub.i.
[0103] Statistical Analysis. All data are shown as mean
values.+-.SEM. Analysis of variance (ANOVA) was used to analyze the
data, and the Student's t test was used to compare population
sample means. The Mann-Whitney test was also used to compare
population frequency distributions. The 95% confidence interval for
anti-HEL antibody levels observed in sHEL/Ig.sup.HEL mice was
determined using the log normal distribution (mean.+-.2 SD) of
antibody values with undetectable levels (<20 ng/ml) assigned
the value of 10 ng/ml.
[0104] Autoantibodies in sHEL/Ig.sup.HEL/hCD19 transgenic mice.
Serum anti-HEL IgM.sup.a autoantibody levels in Ig.sup.HEL
transgenic, sHEL/Ig.sup.HEL double-transgenic, and
sHEL/Ig.sup.HEL/hCD19 triple-transgenic mice were determined to
assess the status of B cell tolerance in each set of mice. Serum
antibody levels for each individual mouse are shown in FIG. 1 and
mean autoantibody levels for each set of mice are provided to
simplify discussion of the results. Two-month-old sHEL/Ig.sup.HEL
double transgenic mice produced very low or undetectable levels of
anti-HEL IgM.sup.a antibodies (mean levels 31 ng/ml) when compared
with Ig.sup.HEL transgenic mice (mean 16,700 ng/ml, FIG. 1).
However, 45% (14 of 33) of sHEL/Ig.sup.HEL/hCD19.sup.+/- mice had
anti-HEL IgM.sup.a autoantibody levels (mean 2,430 ng/ml) that were
significantly greater than those found in sHEL/Ig.sup.HEL mice
(P.ltoreq.0.001, FIG. 1). Anti-HEL IgM.sup.a antibody levels were
also elevated in 38% (14 of 36) of sHEL/Ig.sup.HEL/hCD19.sup.+/+
mice (mean 10,500 ng/ml, P.ltoreq.0.01, FIG. 1). Autoantibody
levels in some sHEL/Ig.sup.HEL/hCD19 mice were equivalent to those
of Ig.sup.HEL-transgenic mice not expressing sHEL. In fact,
overexpression of CD19 resulted in anti-HEL autoantibody levels in
some mice that were one thousand (1,000)-fold higher than in
sHEL/Ig.sup.HEL mice. By comparison, overexpression of CD19 in
Ig.sup.HEL/CD19.sup.+/+ mice resulted in only a fourfold increase
in anti-HEL antibody levels (mean 77,300 ng/ml, FIG. 1). Thus,
lowered signaling thresholds resulting from the overexpression of
CD19 abrogated peripheral anergy in a significant proportion of
two-month-old sHEL/Ig.sup.HEL mice.
[0105] The breakdown in peripheral tolerance and the development of
autoantibodies in sHEL/Ig.sup.HEL mice that overexpressed CD19
correlated with mouse age. By five to ten months of age, all
sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice produced significantly higher
levels of autoantibodies (mean 144,000 ng/ml) than sHEL/Ig.sup.HEL
mice (300 ng/ml, P<0.01, FIG. 1). The lowest autoantibody level
found in a five-month-old sHEL/Ig.sup.HEL/hCD19.sup.+/+ mouse was
2,300 ng/ml. Therefore, the breakdown of tolerance in
sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice had 100% penetrance by five
months of age.
[0106] Abrogation of peripheral tolerance in sHEL/Ig.sup.HEL/hCD19
mice. Whether B-cell anergy could be surmounted in young mice that
overexpress CD19 was assessed by immunizing two-month-old mice with
HEL in CFA. Mice without detectable levels of spontaneous anti-HEL
antibodies were also injected with CFA alone to mimic a nonspecific
inflammatory stimulus. Immunization of
sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice with HEL generated primary
anti-HEL antibody responses in some mice, and a mean secondary
antibody response that was two hundred-fold higher (P<0.05) than
that of sHEL/Ig.sup.HEL mice (FIG. 2A). A measurable antibody
response was only detected in sHEL/Ig.sup.HEL mice after secondary
immunization (FIG. 2A). A striking result was that the inflammation
induced by CFA alone induced sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice to
produce anti-HEL antibodies in response to endogenous sHEL
autoantigen (FIG. 2A). In this case, the mean secondary antibody
response was 4 thousandfold higher than in sHEL/Ig.sup.HEL mice
(P<0.05). In fact, the anti-HEL antibody levels induced in some
sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice were equivalent to those of
Ig.sup.HEL mice (FIG. 2B). Similar results were obtained with
sHEL/Ig.sup.HEL/hCD19.sup.+/- mice although anti-HEL autoantibody
levels were intermediate. In sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice
that already expressed detectable anti-HEL antibodies, autoantibody
levels were also dramatically augmented following CFA
administration. Therefore, inflammatory responses induced by the
administration of CFA revealed a breakdown in tolerance and
resulted in autoantibody production in anergic mice that
overexpressed CD19.
[0107] Effects of CD19 overexpression on B-cell development. The
effects of CD19 overexpression on B-cell development was assessed
to elucidate the cellular basis for the breakdown of peripheral
tolerance in sHEL/Ig.sup.HEL mice. The breakdown in tolerance did
not result from relaxed negative selection, since the number of
mature IgM.sup.+B220.sup.hi or HSA.sup.lo B220.sup.hi B cells in
the bone marrow, blood, and spleen of Ig.sup.HEL/hCD19.sup.+/+ mice
was significantly reduced in the absence or presence of sHEL (Table
1).
[0108] B cell development in Ig.sup.HEL and sHEL/Ig.sup.HEL mice
that overexpress CD19 was analyzed using flow cytometry. The
results are discussed herein below. Representative two-color
immunofluorescence staining of B cells from A) bone marrow, B)
blood, C) spleens, and D) peritoneum of littermate pairs was
performed. B lymphocytes were revealed by B220 or IgM expression.
Quadrants delineated by squares indicated the pre-B cell
(B220.sup.loIgM.sup.-), immature B cell (B220.sup.loIgM.sup.+) and
mature B cell (B220.sup.hiIgM.sup.+) compartments, with numbers
representing the percentage of cells within quadrants. The gates
that defined mature B lymphocytes for sHEL/Ig.sup.HEL mice were
different from the gate used for Ig.sup.HEL mice since surface IgM
levels are downregulated in sHEL/Ig.sup.HEL mice. Spleen cells were
also stained for B220 or IgM and counterstained for sHEL binding or
I-A expression.
[0109] Additional gates were used to determine the frequency of the
CD5.sup.+B220.sup.+ population and CD5-B220.sup.+ population of
cells for Table 1. Populations of cells lacking surface antigen
expression were determined using unreactive monoclonal antibodies
as controls. All samples were stained in parallel and analyzed
sequentially by flow cytometry with identical instrument settings.
Relative fluorescence intensity was shown on a four decade log
scale, with 50% log density contour levels. Horizontal dashed lines
in some histograms were provided for reference. Similar results
were obtained with at least five sets of mice. Equivalent results
were obtained by using anti-IgM.sup.a antibody instead of anti-IgM
antibody.
[0110] A similar decrease in the generation of mature B cells
occurs, presumably as a consequence of increased clonal
elimination, in wild-type mice that overexpress CD19. Zhou et al.
(1994) Mol. Cell. Biol. 14:3884-3894. However, since all B cells
bear the same receptor with the same affinity for antigen, the
partial decrease in generation of mature B cells in the bone marrow
of Ig.sup.HEL/hCD19.sup.+/+ mice and sHEL/Ig.sup.HEL/hCD19.sup.+/+
mice suggests that this developmental bottleneck occurs independent
of antigen receptor ligation. Further, it is difficult to imagine a
deleting antigen that binds to the transgenic receptor better than
HEL, and deletion also occurs in mice lacking sHEL. Therefore,
overexpression of CD19 may alter the generation of mature B cells
through mechanisms in addition to increased negative selection.
Nonetheless, the breakdown in tolerance did not result from relaxed
negative selection.
[0111] Peripheral B cell numbers were significantly reduced in both
Ig.sup.HEL mice and sHEL/Ig.sup.HEL mice overexpressing CD19 (Table
1). Overexpression of CD19 reduced circulating B cell numbers by
87% in Ig.sup.HEL mice and 78% in sHEL/Ig.sup.HEL mice. CD19
overexpression reduced spleen B cell numbers by 42% in Ig.sup.HEL
mice and 48% in sHEL/Ig.sup.HEL mice. Conventional B cells within
the peritoneum were also reduced by >90% in
Ig.sup.HEL/hCD19.sup.+/+ and sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice.
Overexpression of CD19 did not induce the generation of B cells
with the phenotypic characteristics of either B1a or B1b cells and
only small numbers of CD5.sup.+B220.sup.lo B cells were observed in
any of the 2-month-old transgenic mouse lines (Table 1). In
addition, all of the HEL-binding B cells in each line of mice were
conventional B cells since they were CD5.sup.-, CD23.sup.+,
IgD.sup.hi, and B220.sup.hi. Thus, the dramatic increase in the
levels of autoantibodies generated in
sHEL/Ig.sup.HEL/hCD19.sup.+/+-transgenic mice were even more
significant given the >50% reduction in numbers of peripheral B
cells in these mice.
[0112] Chronic stimulation through the B-cell antigen receptor in
sHEL/Ig.sup.HEL mice results in a unique IgM.sup.loIgD.sup.hi
phenotype with increased expression of class II (I-A) antigens
(Goodnow et al. (1988) Nature 334:676-682; Goodnow et al. (1989)
Nature 342:385-391; Mason et al. (1992) Intl. Immunol. 4:163-175).
In comparison, the overexpression of hCD19.sup.+/+ in these mice
resulted in even lower IgM expression and higher I-A expression
(Table 1). Despite the decrease in surface IgM, all B cells from
sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice still bound sHEL in vitro in
proportion to their IgM.sup.a density. B cells from
Ig.sup.HEL/hCD19.sup.+/+ transgenic mice had an intermediate
IgM.sup.loI-A.sup.hi phenotype even in the absence of sHEL (Table
1). B cells from mice that overexpressed CD19 also expressed
significantly elevated levels of cell surface CD86 (B7-2).
Therefore, CD19 overexpression appeared to augment the phenotypic
outcome of signaling through the B cell antigen receptor in the
absence or presence of autoantigen. However, B cells from
sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice still exhibited a phenotype that
is characteristic of anergic B cells.
[0113] [Ca.sup.++].sub.i responses in B cells from
sHEL/Ig.sup.HEL/hCD19 mice. Peripheral tolerance in sHEL/Ig.sup.HEL
mice results in the failure of anergic B cells to mobilize
intracellular Ca.sup.++ in response to HEL-mediated antigen
receptor crosslinking in vitro. Cooke et al. (1994) J. Exp. Med.
179:425-438. B cells from sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice were
equivalent to anergic B cells from sHEL/Ig.sup.HEL mice in their
failure to mobilize Ca.sup.++ in response to HEL (FIG. 3A). B cells
from sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice that generated high levels
of autoantibodies also failed to mobilize Ca.sup.++ in response to
HEL. B cells from Ig.sup.HEL/hCD19.sup.+/+ mice generated normal
Ca.sup.++ responses (FIG. 3B). Therefore, the development of
autoimmunity in sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice does not result
from a CD19-induced recovery of early signaling responses in the
bulk of anergic B cells.
[0114] Although antigen receptor ligation did not induce Ca.sup.++
responses in anergic B cells, crosslinking human and mouse CD19
induced a normal Ca.sup.++ response in anergic B cells from
sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice (FIG. 3A). Crosslinking mouse
CD19 in sHEL/Ig.sup.HEL mice also induced a normal Ca.sup.++
response. The presence of HEL during CD19 crosslinking resulted in
a Ca.sup.++ response that was significantly greater than that
observed with CD19 crosslinking alone in
sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice (P<0.01, FIG. 3A). However,
the magnitude of the CD19/HEL-induced Ca.sup.++ response in
sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice (FIG. 4A) was less than that
observed in Ig.sup.HEL/hCD19.sup.+/+ mice (FIG. 3B). Of interest
was the observation that the magnitude of the CD19-induced
Ca.sup.++ response in sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice was always
significantly higher than the Ca.sup.++ response in
Ig.sup.HEL/hCD19.sup.+/+ mice (n=3, p<0.05). The increased
Ca.sup.++ responses of B cells from sHEL/Ig.sup.HEL/hCD19.sup.+/+
mice presumably results from the endogenous ligation of antigen
receptors by sHEL encountered in vivo. These results indicate that
CD19 ligation can induce a relatively normal Ca.sup.++ response in
anergic B cells. Moreover, CD19 ligation can also augment
transmembrane signals generated through the B-cell antigen receptor
despite clonal anergy.
[0115] Thus, the striking induction of autoantibody production in
sHEL/Ig.sup.HEL mice that are normally functionally anergic
directly implicates CD19 signaling thresholds as a regulator of
peripheral tolerance in B cells. CD19 overexpression by only
twofold to threefold caused a breakdown of peripheral B-cell
tolerance in a clear and dramatic fashion, with autoantibody levels
increased several thousandfold in some sHEL/Ig.sup.HEL/CD19.sup.+/+
mice (FIGS. 1 and 2). These dramatic increases in autoantibody
levels in sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice are even more
significant given the >50% reduction in numbers of peripheral B
cells in mice that overexpress CD19 (Table 1).
[0116] Overexpression of CD19 alone did not induce anergic B cells
to produce autoantibodies, as evidenced by the fact that some
sHEL/Ig.sup.HEL/hCD19 mice were anergic and did not produce
spontaneous autoantibodies until 5 months of age (FIG. 1). However,
significant autoantibody production was induced in 2-month-old
anergic sHEL/Ig.sup.HEL/hCD19 mice by inducing inflammation with
CFA (FIG. 2). Autoantibodies in sHEL/Ig.sup.HEL mice that
overexpressed CD19 are likely to have originated from a breakdown
in tolerance in conventional B cells, since HEL-specific B1a or B1b
cells were not detected in Ig.sup.HEL (Cyster et al. (1995)
Immunity 2:13-24), sHEL/Ig.sup.HEL or sHEL/Ig.sup.HEL/hCD19 mice
(Table 1). These findings suggest that alterations in CD19-related
signaling thresholds breaks peripheral tolerance, which predisposes
B cells to the induction of autoantibodies.
[0117] The levels of autoantibody production observed in
sHEL/Ig.sup.HEL mice that overexpress CD19 (FIGS. 1 and 2) clearly
demonstrates that tolerance was abrogated in a significant portion
of B cells. Since Ig.sup.HEL B cells are constantly exposed to
antigen in transgenic sHEL mice, autoantibody production in
sHEL/Ig.sup.HEL/CD19.sup.+/+ mice is most likely induced through an
antigen receptor-dependent process. Autoantibody production in
sHEL/Ig.sup.HEL/CD19.sup.+/+ mice may relate to the observation
that CD19 ligation can augment transmembrane signals generated
through the antigen receptor despite clonal anergy (FIG. 3).
[0118] Applicant has recently demonstrated that genetic alterations
in CD19 expression have significant effects on the signal
transduction pathways activated following B cell antigen receptor
engagement. Particularly, it was shown that CD19 and CD22
reciprocally regulate Vav tyrosine phosphorylation during B
lymphocyte signaling. Therefore, one pathway to autoantibody
production in sHEL/Ig.sup.HEL mice may be via concomitant CD19
overexpression, chronic antigen receptor ligation, and the
influence of inflammatory mediators triggering the simultaneous
breakdown of tolerance and autoantibody production in anergic
Ig.sup.HEL B cells.
[0119] Alternatively, inflammatory mediators such as those
generated by CFA administration may induce the expansion or
differentiation of antigen-stimulated Ig.sup.HEL B cell clones
subsequent to a CD19-induced breakdown in tolerance. The latter
possibility is supported by the finding that B cells from mice that
overexpressed CD19 maintained a phenotype characteristic of anergic
B cells and failed to generate Ca.sup.++ responses following
antigen receptor ligation (FIG. 3). The spontaneous development of
autoantibodies in sHEL/Ig.sup.HEL/hCD19 mice may also require a
breakdown in T-cell tolerance, since HEL-specific helper T cells
are anergic due to chronic sHEL exposure. Adelstein et al. (1991)
Science 251:1223-1225. Thereby, soluble factors induced by CFA
administration may replace the requirement for T-cell help during
autoantibody production in sHEL/Ig.sup.HEL mice. Thus, the current
results suggest strongly that inappropriate CD19 expression or
function contributes to autoimmunity by disrupting tolerance.
[0120] Variability in the timing and magnitude of autoantibody
production in individual sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice is
similar to what has been observed in many mouse models of
autoimmunity. Theofilopoulos et al., eds. (1992) Murine models of
lupus. Systemic Lupus Erythematosus. Churchill Livingston,
Edinburgh. Overexpression of CD19 in sHEL/Ig.sup.HEL mice resulted
in significant autoantibody production in a large portion of
2-month-old mice, while all triple-transgenic mice produced
significant autoantibodies by 5 months of age (FIG. 1). The
expansion and/or accumulation of B-cell clones that have escaped
tolerance may explain why all sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice
produced high levels of spontaneous autoantibodies by 5 months of
age. This contrasts markedly with old sHEL/Ig.sup.HEL mice which
did not produce significant levels of anti-HEL autoantibodies (FIG.
1).
[0121] Previous studies of sHEL/Ig.sup.HEL mice have demonstrated
that functional inactivation of autoreactive B cells is maintained
throughout life. Rathmell et al. (1994) J. Immunol. 153:2831-2842.
The variability and delayed onset of autoantibody production in
sHEL/Ig.sup.HEL/hCD19.sup.+/+ mice may also result from their
confinement to a specific-pathogen-free barrier facility.
Autoantibodies first appear in some mouse models of lupus (NZB and
MRL strains) around 2 months of age, but are dramatically increased
by 4-5 months of age in all mice. Variability in onset and
magnitude of autoantibody production also occurs in individual mice
of these inbred mouse strains. Therefore, variability between the
triple transgenic mice examined in this study is not surprising
despite their identical genetic background and the fact that the
hCD19 transgene is expressed to the same extent in all animals.
[0122] Consistent with the current studies, an association between
CD19 overexpression and autoimmunity in humans has been suggested.
Oooto et al. (1995) Jpn. J. Clin. Pathol. 43:381-384. The etiology
of autoimmunity in humans has also been historically linked with
the accumulation of inflammatory episodes or infectious agents and
often varies in degree and time of onset. Therefore, many of the
current findings in sHEL/Ig.sup.HEL mice mimic the evolution of
autoreactive B cells and autoantibodies in humans.
[0123] In contrast with CD19 overexpressing B cells, signaling in
response to antigen receptor ligation is diminished in
CD45-deficient Ig.sup.HEL B cells. Cyster et al. (1996) Nature
381:325-328. Diminished signaling in CD45-deficient B cells leads
to reduced negative selection in the bone marrow and prolonged
retention in peripheral lymphoid tissues of mice expressing sHEL.
Since the in vivo functional capacity of peripheral CD45-deficient
Ig.sup.HEL B cells or their production of autoantibodies has not
been examined, it is difficult to assess how diminished signaling
in those studies relates to the results of this Example.
Nonetheless, all of these studies demonstrate that antigen receptor
signaling strength influences positive or negative selection, and
the current studies demonstrate a direct and active role for CD19
in regulating peripheral tolerance and autoantibody generation.
[0124] For several reasons, it is unlikely that the breakdown in
tolerance observed in this study results from the inadvertent
insertion of the CD19 transgene into a locus that controls B cell
tolerance. First, the human CD19 transgenic mice used in these
studies reconstitute normal B cell function when crossed with
CD19-deficient mice. Sato et al. (1997) J. Immunol. 158:4662-4669.
The h19-1 line of mice has also been backcrossed extensively onto a
wild-type C57BL/6 background without a diminution of human CD19
expression. This suggests that only one transgene integration site
exists and that the heterogeneity in autoantibody production
observed between triple transgenic mice does not reflect the
segregated inheritance of transgenes that have integrated into
multiple sites.
[0125] Applicant also has generated and analyzed 7 independent
lines of hCD19 transgenic mice. Zhou et al. (1994) Mol. Cell. Biol.
14:3884-3894. In all cases, B cells from each mouse line
demonstrate identical functional abnormalities. In lines subjected
to analysis, hyper-responsive B cells and enhanced autoantibody
production was observed. The magnitude of these abnormalities
correlates directly and linearly with the level of hCD19
overexpression. Sato et al. (1997) J. Immunol 158:4662-4669. In
addition, the abnormalities observed in hCD19 transgenic mice are
reciprocal of what applicant has observed in CD19-deficient mice
(Engel et al. (1995) Immunity 3:39-50; Sato et al. (1995) Proc.
Natl. Acad. Sci. USA 92:11558-11562; Sato et al. (1996) J. Immunol.
156:4371-4378). Therefore, the effects observed in the current
study most likely relate directly to augmented CD19 function rather
than an interruption of other genes involved in tolerance
regulation.
[0126] CD19 is a signaling component of a multimeric complex that
includes CD21, the receptor for the C3d fragment of complement that
covalently associates with antigens during complement activation.
Bradbury et al. (1992) J. Immunol. 149:2841-2850; Matsumoto et al.
(1991) J. Exp. Med. 173:55-64. C3d binding to CD21 can thereby act
as a ligand for the CD19 complex that links complement activation
with B-cell function. Pepys, M. B. (1976) Transplant Rev.
32:93-120; Melchers et al. (1985) Nature 317:264-267; Fearon et al.
(1995) Annu. Rev. Immunol. 13:127-149.
[0127] Since CD21-deficient mice manifest developmental and
functional defects similar to those of CD19-deficient mice (Engel
et al. (1995) Immunity 3:39-50; Rickert et al. (1995) Nature
376:352-355; Ahearn et al. (1996) Immunity 4:251-262; Molina et al.
(1996) Proc. Natl. Acad. Sci. USA 93:3357-3361) overexpression of
CD19 in vivo may mimic C3d ligation of CD21 by augmented signaling
through the CD19 complex. Tedder et al. (1997) Immunity 6:107-118.
Because the roadblock to B-cell Ca.sup.++ responses in anergic B
cells was transcended in vitro by simultaneous CD19 ligation and
antigen receptor signaling (FIG. 4), C3 cleavage products binding
to the CD19 complex may provide a molecular mechanism for bypassing
peripheral B-cell anergy in vivo. The inappropriate or prolonged
generation of C3d during inflammatory or infectious episodes in
vivo may increase the responsiveness of autoreactive B cells to
weak self antigens through augmented CD19 function, resulting in a
breakdown of tolerance and the clonal amplification of
autoantibody-producing B cells. Because altering CD19 complex
function provides a mechanism for breaking self-tolerance in vivo,
CD19 function may be a molecular mechanism linking inflammation
with the development of autoimmune disease. TABLE-US-00002 TABLE 1
Phenotype and Frequency of B lymphocytes in Lymphoid Tissues sHEL
Ig.sup.HEL sHEL Ig.sup.HEL Ig.sup.HEL hCD19.sup.+/+ Ig.sup.HEL
hCD19.sup.+/+ Frequency (%) and Tissue Phenotype Number (# .times.
10.sup.-6) of B cells* bone % IgM.sup.-B220.sup.lo 14 .+-. 2 14
.+-. 2 13 .+-. 2 13 .+-. 2 marrow % IgM.sup.+B220.sup.lo 32 .+-. 4
49 .+-. 10 46 .+-. 2 53 .+-. 3 % IgM.sup.+B220.sup.hi 16 .+-. 3 8
.+-. 3.sup..sctn. 12 .+-. 2 8 .+-. 1 % HSA.sup.loB220.sup.hi 25
.+-. 3 9 .+-. 3.sup.1 17 .+-. 5 5 .+-. 1.sup..sctn. blood %
B220.sup.+ 43 .+-. 3 7 .+-. 2.sup.1 22 .+-. 3 6 .+-. 2.sup.1
#B220.sup.+ 2.4 .+-. 0.5 0.3 .+-. 0.1.sup.1 0.9 .+-. 0.2 0.2 .+-.
0.1.sup.1 spleen % B220.sup.+ 36 .+-. 6 28 .+-. 5 44 .+-. 4 17 .+-.
3.sup.1 #B220.sup.+ 24 .+-. 5 14 .+-. 3.sup..sctn. 29 .+-. 5 15
.+-. 6.sup..sctn. peritoneum % CD5.sup.+B220.sup.lo 2.2 .+-. 0.3
1.3 .+-. 0.4 4.5 .+-. 1.1 1.9 .+-. 0.7 #CD5.sup.+B220.sup.lo 0.05
.+-. 0.01 0.05 .+-. 0.02 0.10 .+-. 0.02 0.04 .+-. 0.02 %
CD5.sup.-B220.sup.hi 38 .+-. 6 2.7 .+-. 0.7.sup.1 14 .+-. 2 1.2
.+-. 0.4.sup.1 #CD5.sup.-B220.sup.hi 1.0 .+-. 0.3 0.07 .+-.
0.03.sup..sctn. 0.33 .+-. 0.03 0.02 .+-. 0.01.sup.1 Levels Relative
to Expression Source of B cells Ig.sup.HEL Mice (% .+-.
SEM).sup..dagger-dbl. IgM levels bone B220.sup.lo 100 69 .+-.
12.sup.1 41 .+-. 9 22 .+-. 8 marrow: B220.sup.hi 100 63 .+-.
4.sup.1 4.1 .+-. 0.5 4.3 .+-. 0.7 blood: B220.sup.+ 100 68 .+-.
15.sup.1 19 .+-. 3 25 .+-. 4 spleen: B220.sup.+ 100 62 .+-. 4.sup.1
16 .+-. 2 7 .+-. 1.sup.1 I-A levels: blood: IgM.sup.+ 100 126 .+-.
5.sup.1 169 .+-. 7 254 .+-. 33.sup.1 spleen: IgM.sup.+ 100 259 .+-.
55.sup..sctn. 184 .+-. 13 283 .+-. 30.sup..sctn. *= Cumulative mean
(.+-. SEM) frequencies of different cell populations from at least
five two-month-old mice of each genotype. Flow cytometry gates were
used to determine the frequency of each cell type within the
lymphocyte population. B-cell numbers for blood indicate the number
of cells/ml. B-cell numbers from spleen and peritoneum were
determined based on the total number of lymphocytes recovered.
.sup..dagger-dbl.= Relative cell surface antigen densities were
determined by comparing the channel numbers of mean linear
fluorescence intensity between Ig.sup.HEL B cells and B cells from
other mice. Values represent the mean expression levels obtained
from at least three sets of mice of each genotype. All samples in
each set of mice were stained in parallel and analyzed sequentially
by flow cytometry with identical instrument settings. .sup..sctn.=
Differences between mice not expressing hCD19 and those expressing
hCD19 were significant, p < 0.05; .sup.1= p < 0.01.
EXAMPLE 2
Preparation of Monoclonal Antibodies Against Prion Epitopes
[0128] As discussed above, monoclonal antibodies (mAbs) have become
powerful tools in research and biotechnology. In these areas, as in
the acquisition of immunity, production of potentially useful
antibodies often depends on two aspects of "self"-"non-self"
discrimination during the antibody response in mice. First,
antibodies are not normally produced against self antigens, and
secondly, antibodies to foreign antigens are normally directed
exclusively at regions of the foreign antigen that differ from
self. The number of monoclonal antibodies generated against
species-specific antigens for use in sensitive immunoassays or
allele-specific antibodies for blood grouping and tissue typing
before transfusion or organ transplantation has been quite
significant. However, the true potential of monoclonal antibodies
has not been realized in many cases because of the difficulty in
generating antibodies directed to self antigens or antigens well
conserved across species barriers.
[0129] This problem has resulted in the development of a number of
alternative approaches to generating monoclonal antibodies such as
phage display and other in vitro approaches. While there is
considerable power to these alternative approaches, the generation
of dramatic antibody diversity and clonal selection of high
affinity antibodies that occur normally during an immune response
in animals are impossible to recapitulate in vitro. Therefore,
effective methods, such as those described in Example 1, have been
developed wherein clonal tolerance is blocked in vivo and wherein
"self"-"non-self" discrimination is negated during the antibody
response in mice. Being able to modulate the molecular basis for
the cellular decision of tolerance is important for controlling the
immunogenicity or tolerogencity of vaccines, tumors, and tissue
transplants, and for understanding the breakdown of self-tolerance
in autoimmune diseases.
[0130] A number of molecules are key to regulating B cell function
and the generation of humoral immune responses. Two examples of
such molecules are CD19 and CD22. Tedder et al. (1997) Immunity
6:107-118; Tedder et al. (1997) Ann. Rev. Immunol. 15:481-504.
Recent studies have demonstrated that CD19 and CD22 are members of
a new class of lymphocyte surface receptors called "response
regulators". This term designates that these molecules regulate the
magnitude and duration of transmembrane signals received by a B
lymphocyte.
[0131] Understanding how these molecules function provides multiple
options for use in regulating humoral immune responses.
Specifically, mice have been generated that are hyporesponsive to
transmembrane signals, while other mice have been generated that
are hyper-responsive to transmembrane signals. This has
considerable ramifications for the abilities of these mice to
recognize and generate humoral antibody responses against a variety
of antigens, particularly self antigens. Moreover, this provides a
powerful new approach to developing a new class of mAbs that react
with molecules which are highly conserved during recent mammalian
evolution.
[0132] Unconventional agents termed prion proteins (PrPsc) are
considered the etiologic agent of Creutzfeldt-Jakob disease (CJD).
The pathologic properties of these proteins lie in their
three-dimensional configuration and their ability to recruit and
influence normal PrPs to undergo similar conformational changes.
Because these proteins are highly conserved across species it has
been difficult to generate effective humoral immune responses
against these agents in animal models.
[0133] The majority of PrPsc antigenic sites are species-directed,
involve non-self sites and are common to both the normal host
precursor (PrPc) and the disease form (PrPsc). Because of this, the
present methods to diagnose CJD-associated diseases are lengthy,
require relatively large quantities of starting material to detect
PrPsc and lack sensitivity (reviewed in Kascsak et al. (1993) Dev.
Biol. Stand. 80:141-151). See also Korth et al. (1997)(Letter to
Nature) Nature 390:74.
[0134] In this Example, development of monoclonal antibodies (mAbs)
reactive with PrPs is described. Development of systems for the
production of mAbs reactive with distinct epitopes or
conformational determinants present on PrPs but not PrPc are also
described.
[0135] Determination of whether tolerance-deficient mice develop
humoral immune responses to PrPc. Initial studies are initiated to
determine whether tolerance-deficient mice are able to generate
humoral immune responses against hamster PrPc or recombinant PrPc
proteins and peptides. Mice are immunized intra-pertioneally (ip)
with the test proteins and both primary and secondary humoral
immune responses are assessed by ELISA. Multiple
tolerance-deficient mice strains are assessed and are compared with
wild-type mice of the same genetic background.
[0136] Next, different immunogens are evaluated for their ability
to generate anti-PrP antibody responses to assess whether any have
specific advantages. One suitable immunogen is described in Korth
et al. (1997) (Letter to Nature) Nature 390:74.
[0137] Different gene-targeted or transgenic mouse models for
tolerance-deficiency are then assessed to determine advantageous
humoral immune responses following immunization. Upon observation
of clear differences between lines of mice, then those mice with
optimal humoral responses are crossed to generate a line of mice
that are optimal for generating humoral responses to PrPs.
[0138] In a second wave of experiments, tolerance-deficient mice
are generated that possess different major histocompatability
complex (MHC) backgrounds. In many experimental models, differences
in MHC genes have a predetermining effect on the ability of mice to
elicit humoral immune responses. MHC differences may account for
some of the difficulties in generating humoral responses to PrP
protein in mice.
[0139] Currently, all tolerance-deficient mice have a C57Bl/6
background. These mice are bred into a BALB/c background for at
least five generations. Humoral immune responses in mice is
assessed following initial crosses to assess whether this has an
advantageous influence on humoral immune responses. Following these
crosses, these mice are used for the generation of mAbs.
[0140] Generate mAbs Reactive with PrPc. The mice generated above
and immunized with optimal antigens are used for the generation of
mAbs using standard approaches, such as those described above.
[0141] A problem has been encountered in the development of
hybridomas producing antibodies reactive with PrP protein in which
the myeloma fusion partner cells express endogenous PrP protein
which leads to the death of the hybridoma. This problem is
circumvented either by genetically engineering the fusion partner
myeloma cell line or introducing the appropriate genetic mutations
in the mice used for fusions. These technologies will be utilized
when it appears that difficulties in generating mAbs results from
the above problem.
[0142] Generate mAbs with PrPO/O tolerance-deficient mice. Mice
rendered deficient in PrPc by homologous recombination gene
targeting are obtained. PrPO/O mice generate sera capable of
specifically precipitating in vitro synthesized human prion
proteins, while wild-type mice do not generate humoral immune
responses. Drasemann et al. (1996) J. Immunol. Methods
199:109-118). This provides considerable advantages for generating
mAbs reactive with this self-protein should responses with
tolerance-deficient mice not be optimal. Furthermore, this may
allow the generation of mAbs reactive with novel epitopes that may
not be optimally identified in tolerance-deficient mice. PrPO/O
tolerance-deficient mice generate vigorous polyclonal immune
responses after immunization with human prion gene sequences. These
mice will be used as spleen donors for mAb production.
[0143] Generate mAbs reactive with "infectious" PrP isoforms. For
this phase, steps are taken to adequately protect laboratory and
animal care personnel against potential infectious materials.
Monoclonal antibodies are then preferably generated by DNA-mediated
immunization of mice, or by other standard protocols known to those
in the art.
[0144] Additionally, high affinity monoclonal antibodies are
prepared by the methods described in this Example. Monoclonal
antibodies having high affinity for an antigen are desirable in
that high affinity provides for biological activity in vivo. Some
monoclonal antibodies produced by conventional methods often react
with antigens in a biological sample, such as blood, but the
affinity of such reactions is low.
[0145] The terms "affinity" and "high affinity" have
well-recognized meanings in the art. Particularly, the term
"affinity" refers to the goodness of the fit of an antigenic
determinant to a single antigen-binding site, and it is independent
of the number of sites. The term "high affinity" refers to a
particular good fit of an antigenic determinant to a single
antigen-binding site.
[0146] Typically, the affinity of an antibody and an antigenic
determinant is judged by the use of an affinity constant K.
Antigens (Ags) and antibodies (Abs) interact according to the
reversible equilibrium equation: Ag+Ab.revreaction.AgAb The
affinity constant K is determined by the equation
K=[AgAb]/[Ag][Ab], wherein the units for K are liters per mole.
[0147] The affinity constant for a particular antigen-antibody
interaction can be determined by measuring the concentration of
free Ag required to fill half the antigen binding sites on the
antibody. When this concentration value is plugged into the
equation presented above, K=1/[Ag]. Thus, the reciprocal of the
antigen concentration that produces half maximal binding is equal
to the affinity constant of the antibody for the antigen. A high
affinity antigen-antibody interaction is therefore typically
described as having an affinity constant K of greater that
1.times.10.sup.5 liters per mole. See Alberts et al. Molecular
Biology of the Cell, Garland Publishing, Inc. (New York and London.
1983), p. 970.
[0148] Such high affinity monoclonal antibodies have application,
for example, in a pharmaceutical setting. Specific binding of an
antigen, such as a cancer antigen, can prevent the antigen from
carrying out its biological activity in the cell with which it is
associated, thereby killing the cell or slowing its growth.
Alternatively, such monoclonal antibodies have application as drug
carriers, for delivery of, for example, a cytotoxic agent to the
cancer cell.
[0149] Production of monoclonal antibodies directed to
cancer-associated antigens are also described in U.S. Pat. Nos.
5,660,834; 5,491,088; 5,665,382; 5,651,991; and 5,242,824; the
entire contents of which is herein incorporated by reference.
EXAMPLE 3
Preparation of a Non-Human Animal Having an Immune System with
Desired Characteristics
[0150] Example 1 describes breeding of transgenic mice, including a
transgenic mouse which over-expresses CD19, wherein progeny of such
mice possess antibody-forming cells having disrupted peripheral
tolerance. Given the disclosure of Example 1, then, a practitioner
having ordinary skill in the art can breed and/or prepare lines
transgenic mice for cross-breeding, wherein each line of mice have
immune systems with certain characteristics, to produce progeny
animals having combinations of the characteristics. Such animals
would be very useful, inter alia, in the preparation of monoclonal
antibodies as described herein or in the preparation of vaccines.
This Example describes the preparation of such an animal.
[0151] An example of a desirable characteristic is
hyper-sensitivity to antigens so that antibodies, and particularly
monoclonal antibodies, can be made against epitopes that are highly
conservative among mammalian or other vertebrate species. Indeed,
an animal having a hyper-sensitive immune system due to a breakdown
in peripheral tolerance is described in Example 1.
[0152] In producing a non-human animal having an immune system with
a predetermined or desired characteristic, it is preferable to
initially prepare a transgenic animal which includes a transgene,
wherein expression of the transgene in the animal imparts a
predetermined or desired characteristic to a cell or a type of
cells within the animal's immune system. As shown in Example 1,
transgenic mice which over-express CD19 are used to provide
disruption of peripheral tolerance in B lymphocytes in mice.
[0153] Preparation of transgenic animals is accomplished through
use of accepted protocols in the art. Such protocols are described,
for example, in Engel et al. (1995) Immunity 3:39-50 and in Zhou
(1994) Mol. Cell. Biol. 14:3884-3894, wherein the production of the
CD19 overexpressing transgenic mice described in Example 1 is
described. Additionally, standard preparation techniques for
transgenic animals, including mice, are also described in U.S. Pat.
No. 5,633,076 (bovine); U.S. Pat. No. 5,573,933 (pigs); U.S. Pat.
No. 5,675,063 (rabbits); U.S. Pat. No. 5,633,425 (mouse); U.S. Pat.
No. 5,661,016 (mice and other animals) and U.S. Pat. No. 4,736,866
(mice and other animals), the entire contents of each of which are
herein incorporated by reference.
[0154] The preferred next step in the method is to prepare a second
line of transgenic animals which include a transgene, wherein
expression of the transgene in the animal imparts a predetermined
or desired characteristic to a cell or a type of cells within the
animal's immune system. The predetermined characteristic of the
cell or cells within the immune system of the second strain of
transgenic animals can be the same or different from the
predetermined characteristic of the cell or cells within the immune
system of the first strain of animals. In Example 1, the second and
subsequent lines of animals included sHEL (ML5 line) and Ig.sup.HEL
(ML5 line) mice, as well as double transgenic sHEL/Ig.sup.HEL
mice.
[0155] Next, the first and second strains of transgenic animals are
bred according to classical breeding techniques. Such techniques
are well known in the art, and are be more fully described in U.S.
Pat. No. 5,633,076 (bovine); U.S. Pat. No. 5,573,933 (pigs); U.S.
Pat. No. 5,675,063 (rabbits); U.S. Pat. No. 5,633,425 (mouse); U.S.
Pat. No. 5,661,016 (mice and other animals) and U.S. Pat. No.
4,736,866 (mice and other animals), the entire contents of each of
which are herein incorporated by reference.
[0156] Additionally, techniques for the care and breeding of a
germ-free colony of mice are described in U.S. Pat. No. 5,223,410,
the entire contents of which is herein incorporated by reference.
Such techniques may optionally be incorporated into the production
of the animal having an immune system with predetermined
characteristics as described in this Example.
[0157] The resulting progeny are then screened to determine that
transgenes are inherited and are subsequently expressed. Screening
protocols include the well known techniques of PCR amplification,
northern blot analysis and southern blot analysis using nucleic
acid probes or segments from the transgene initially used to
prepare the transgenic animal. Additional protocols are described
in Engel et al. (1995) Immunity 3:39-50 and in Zhou (1994) Mol.
Cell. Biol. 14:3884-3894.
[0158] Immune cells within the progeny are then screened to
determine if the predetermined characteristics have been imparted
to the cells. Such screening methods are provided in Example 1 and
include isotype-specific ELISA assays and immunofluorescence
assays. Finally, progeny having immune cell or cells, such as
antibody-producing cells like B lymphocytes, are bred according to
well-known techniques to propagate a line of mice which have within
their immune systems cells which demonstrate the predetermined or
desired characteristics.
[0159] In addition to the lines of mice described in of Example 1,
applicant has prepared a line of CD22 deficient mice and a line of
CD19 deficient mice according to standard "knock-out" methods, such
as those methods described in Sato et al. (1996) Immunity
5:551-562. The CD22 deficient line of mice exhibit a positive
effect on antigen recognition, while the CD19 deficient line of
mice exhibit a negative effect on antigen recognition. The lines of
mice were crossed. The resulting line of mice exhibited normalized
B cell development in contrast to the abnormal B cell development
observed in CD22 deficient and CD19 deficient mice.
[0160] As another example of a predetermined or desired
characteristic to be manipulated, the role of antigen receptor
signaling strength in the development of autoreactive B cells has
recently been examined in sHEL/Ig.sup.HEL mice. Cyster et al.
(1995) Immunity 2:13-24; Cyster et al. (1996) Nature (London)
381:325-328. Mutations in the SHP1 protein tyrosine phosphatase of
motheaten viable (mev) mice abrogates the negative regulatory role
of SHP1 in antigen receptor signaling, resulting in the generation
of autoantibodies in non-transgenic mice. In Ig.sup.HEL mice, the
mev mutation lowers signaling thresholds, which incites the
negative selection of Ig.sup.HEL B cells in the bone marrow of sHEL
mice. Cyster et al. (1995) Immunity 2:13-24. The SHP1 deficiency
thereby prevents autoantibody generation but facilitates the
development of peritoneal B1 cells reactive with HEL. Cyster et al.
(1995) Immunity 2:13-24.
[0161] These characterisitics contrast markedly with the results of
Example 1, in which lowering B-cell signaling thresholds by
increased CD19 expression resulted in a breakdown in tolerance and
autoantibody production rather than the total negative selection of
Ig.sup.HEL B cells in the bone marrow of sHEL mice (FIG. 1). Thus,
SHP1 may play a key role in setting thresholds for negative
selection in the bone marrow, while CD19 regulates peripheral
tolerance. Alternatively, tolerance may be finely tuned, with CD19
and SHP1 altering signaling strengths to differing extents. Thus,
animals can be produced according to the methods of this Example
wherein the animals have cells in their immune systems that display
characteristics associated with altered SHP1 and CD19
expression.
[0162] Other examples of predetermined or desired characteristics
would be apparent to one having ordinary skill in the art. See, for
example, a review article by Tedder et al., entitled "The CD19-CD21
Complex Regulates Signal Transduction Thresholds Governing Humoral
Immunity and Autoimmunity", Immunity 6:107-118 (February 1997),
which discusses the role played by the CD19-CD21 complex in the
immune systems of vertebrates.
[0163] The CD19 molecule forms a complex with CD21 (also known in
the art as complement receptor Type II [CR2]), CD81 and Leu-13.
Tedder et al. (1994) Immunology Today 15:437-442. Additionally, the
structure and in vitro function of the CD19-CD21 complex has been
characterized in the art. Tedder et al. (1994), Immunol. Today
15:437-442. Summarily, CD19 is a member of the immunoglobulin
superfamily with a cytoplasmic region of approximately 240 amino
acids. The amino acid sequences of the cytoplasmic of human CD19
(hCD19), mouse CD19 (mCD19), and guinea pig CD19 are highly
homologous, which is consistent with a critical role for this
region in CD19 function. CD19 physically associates with CD21 on
the surface of human B cells. CD21 contains an extracellular domain
of 15 or 16 repeating structural elements called short consensus
repeats (SCRs), a membrane spanning region, and a 34-amino acid
cytoplasmic domain. Human and mouse forms have been identified. The
human form, hCD21, can physically associate with a structurally
similar complement receptor, CD35 (CR1) and generate a receptor
complex that does not contain CD19. The human CD35 is expressed by
B cells, erythrocytes, neutrophils, monocytes, and some T cells.
Thus, interactions of these molecules can be manipulated to prepare
a non-human animal having an immune system with predetermined or
desired characteristics associated with such a manipulation.
[0164] CD19 also associates directly with CD81, a member of the
tetrospans family of proteins that includes CD9, CD37, CD53, CD63
and CD82. Bradberry et al. (1992), J. Immunol. 149:2841-2850 and
Levy et al. (1991) J. Biol. Chem. 266:14597-14602. CD81 is
over-expressed by most B lineage cells and by a wide variety of
cell types including most lymphocytes, natural killer cells,
thymocytes, eosinophils, neuroblastomas, melanomas, and
fibroblasts. Thus, interactions of CD19 and CD81 within the complex
can be manipulated to prepare a non-human animal having an immune
system with predetermined or desired characteristics associated
with such a manipulation.
[0165] Further, Table 2 presents phenotypic characteristics of mice
with genetically altered response regulators of B lymphocyte signal
transduction. The table presents both negative and positive
effects, each such effect being a potential predetermined
characteristic for an animal prepared according to the methods of
this Example. TABLE-US-00003 TABLE 2 Phenotypic Characteristics of
Mice with Genetically Altered Response Regulators of B Lymphocyte
Signal Transduction Genotype Conventional B Cells CD5+/B-1 Cells
Negative Effects CD19 deficient .dwnarw.50% decrease
.dwnarw..dwnarw.80% decrease CD21 deficient Normal .dwnarw.40%
decrease BTK deficient .dwnarw.40% decrease
.dwnarw..dwnarw..dwnarw.99% decrease Xid .dwnarw..dwnarw.70%
decrease .dwnarw..dwnarw..dwnarw.99% decrease Vav deficient
.dwnarw..dwnarw..about.Normal Undetectable Positive Effects CD19
.dwnarw..dwnarw.70% decrease .uparw..uparw..uparw.210%
Overexpressed increase SHP1 defective .dwnarw..dwnarw..dwnarw.
.uparw..uparw..uparw. CD22 deficient .dwnarw.50% decrease in
.uparw.Increase circulating B cells Lyn deficient .dwnarw.50%
decrease Normal Arrows represent the relative effect of the genetic
alteration on B cell development
[0166] As shown in Table 2, additional examples include
Btk-deficient mice and Xid mice with mutations in Btk both have
diminished numbers of B-1 and conventional B cells. Vav-deficient
mice have similar defects.
[0167] In addition to the examples set forth in Table 2, it is also
optionally desirable to prepare an animal wherein the C3 or C3d
binding component to the CD19-CD21 complex is depleted. It is known
in the art that such a depletion leads to impaired humoral
responses to T-cell dependent and some T-cell independent antigens
in mice. Pepys (1974) J. Exp. Med., 140:126-145.
[0168] In vivo complement depletion suppresses the production of
IgG and other T-cell dependent antibody classes much more
significantly than T-cell independent IgM responses. Transient
depletion of C3 also completely abrogates the development of memory
B cells. Klaus and Humphrey (1977) Immunology 33:31-40.
[0169] Complement deficiencies that effect C3 activation in humans,
guinea pigs, and dogs result in diminished humoral responses to
foreign antigens. See, for example, O'Neil et al., (1988) J.
Immunol. 140:139-145. Additionally, C3- and C4-deficient mice
suffer severe defects in both their primary and secondary antibody
resporises to T-cell dependent antigens, even at high antigen
doses.
[0170] A novel C3 mRNA transcript has been identified that encodes
a truncated C3 protein. Cahen-Kramer et al. (1994), J. Exp. Med.
180:2079-2088. Cell lines transfected with the related cDNA secrete
a co-stimulatory factor that augments the proliferation of B cells
in assays with macrophage-depleted mouse splenic B cells. Such a
cDNA thus provides a candidate for use in the production of a
transgenic animal according to the methods of this
EXAMPLE
[0171] Additionally, CD20 and CD35, or CD21/35 play a role in
immune response, and are thus candidates for manipulation in an
animal according to the method of this Example. It has been
observed that pre-treatment of mice with a mCD21/35 MAb blocks both
T-cell dependent and independent immune responses in the generation
of immunological memory. See, for example, Gustavsson et al. (1995)
J. Immunol. 154:6524-6528. Chimeric mice with normal levels of
CD21/35 on their follicular dendritic cells, but not on their B
cells, have defects in humoral responses to antigens similar to
those of CD21/35 deficient mice. Croix et al. (1996) J. Exp. Med.
183:1857-1864.
[0172] Additional candidates of interest include CD5.sup.- B cells
and CD5.sup.+ B-1 cells, given their documented role in pathogenic
autoantibody responses, such as the pathogenic autoantibody
responses of systemic lupus erythematosus (SLE). SLE is
characterized by production of antibodies to DNA within the body in
association with systemic inflammation. Shirai et al. (1991) Clin.
Immunol. Immunopathol. 59:173-186. See also Murakami and Honjo
(1995), Immunol. Today 16:534-539, discussing that high affinity
pathogenic IgG antibodies are generally produced by CD5.sup.- B
cells.
[0173] In summary, multiple response regulators govern signaling
thresholds in the cells of an animal's immune system, particularly
B cells. Response regulators with positive or negative effects
influence signaling through the B cell antigen-receptor complex.
The resulting signaling thresholds regulate negative selection in
the bone marrow, the magnitude of antibody response in the
periphery, autoimmunity, and peripheral tolerance. Therefore,
preparing an animal having cells in its immune system with
predetermined characteristics derived from manipulation of these
response regulators is highly useful in the characterization of
immune response, development of monoclonal antibodies, and
vaccines, and in the treatment of autoimmune disorders.
[0174] Given that methods for the production of transgenic animals
well-known in the art, it is believed that any animal can be
utilized in the methods of this Example, including mouse, pig, rat,
rabbit, guinea pig, goat, sheep, primate, and poultry, with a mouse
being preferred. Additionally, by the term "transgenic", it is
meant any animal having a genome altered by the hand of man in any
manner. Thus, the term "transgenic" includes the insertion of
desired transgene, any of the well-known "knock-in" approaches, and
any of the well-known "knock-out" approaches.
[0175] Furthermore, the preparation of lines of animals having
immune system cells demonstrating a predetermined or desired
characteristic for use in subsequent breeding is not limited to
transgenic protocols. Any suitable protocol that generates an
alteration in the cells of the animal's is contemplated to be
within the scope of the method of this Example. Such protocols
include, among others, exposure to mutagens, as described in Cyster
et al. (1995) Immunity 2:13-24.
EXAMPLE 4
Alteration of Immune Response to NP Hapten in Mice
[0176] C57BL/6 mice generate T cell-dependent humoral responses to
the (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten that are dominated
by canonical antibodies composed of a single V.sub.H gene, V186.2,
and .lamda.1 light chain. Selection for this receptor is thought to
be driven by its frequency and affinity. However, lowering the
activation threshold of B lymphocytes by overexpression of a single
cell-surface molecule, CD19, resulted in anti-NP antibodies
comprising an unprecedented diverse repertoire of V.sub.H and
V.sub.L rearrangements with no or few mutations. Remarkably, many
exhibited affinities for NP greater than or equal to that of
canonical antibodies. Thus, antigen-receptor selection is regulated
by endogenous B lymphocyte signaling thresholds and not antigen
receptor affinity.
[0177] The unprecedented diverse repertoire of V.sub.H and V.sub.L
rearrangements presented in this Example demonstrates the
flexibility and broad ranging applicability of methods of the
present invention. Indeed, the ability to alter cells of the immune
system in an animal is further exemplified by the detailed
characterization of the unprecedented immune response to NP
presented in this Example. The production of monoclonal antibodies
to highly conserved epitopes and the production of a non-human
animal having cells of the immune system with a predetermined
characteristic in accordance with the methods of the present
invention is thus further illustrated by the data presented in this
Example.
Background
[0178] Despite the enormous diversity of antibodies, inbred strains
of mice often respond to haptens and simple antigens by producing
remarkably homogenous antibodies (Blier and Bothwell, 1988). One of
the best examples is the response of C57BL/6 (Igh.sup.b) mice to
the (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten (Imanishi and
Makela, 1975). C57BL/6 mice immunized with NP coupled to protein
carriers generate serum antibodies which bear the normally rare
.lamda.1 light chain (Cumano and Rajewsky, 1986; Imanishi and
Maakela, 1975; Jacob et al., 1991.; Karjalainen et al., 1980;
Makela and Karjalainen, 1977; Reth et al., 1978; Reth et al., 1979;
Weiss and Rajewsky, 1990). Immunization with carrier protein alone
elicits virtually no .lamda.1 antibody or .lamda.1.sup.+ B cells.
Early in the immune response (days 4-8 post-immunization) a large
proportion of activated .lamda.1.sup.+ B cells express multiple D
gene segments in combination with various members of the large J558
family of V.sub.H genes, including V186.2, C1H4, CH10, V23, 24.8,
V102, and V583.5 (Allen et al., 1988; Bothwell et al., 1981; Jacob
and Kelsoe, 1992; Jacob et al., 1993). By day 10 after
immunization, the majority of .lamda.1.sup.+ B cells express
V186.2-to-DFL16.1 gene rearrangements that encode a tyrosine-rich
CDR3 region with a consensus motif, YYGS (Bothwell et al., 1981;
Cumano and Rajewsky, 1985; Jacob et al., 1993; McHeyzer-Williams et
al., 1993; Weiss and Rajewsky, 1990). The V186.2-to-DFL16.1 heavy
chain rearrangement paired with the .lamda.1 light chain is
referred to as the canonical anti-NP B cell antigen receptor (Reth
et al., 1978; Reth et al., 1979).
[0179] The homogeneity of the anti-NP response in Igh.sup.b mice
(Maizels and Bothwell, 1985) is mirrored in the response of BALB/c
mice to phosphorylcholine (Crews et al., 1981); antibodies produced
against p-azophenylarsonate in strain A mice (Pawlak et al., 1973);
the 2phenyloxazolone response in BALB/c and DBA/2 mice (Makela et
al., 1978); and the response of BALB/c mice to
poly(Glu.sup.60-Ala.sup.30-Tyr.sup.10) (Theze and Somme, 1979). The
cause of low genetic variance in these antibody responses remains
obscure. Linkage of restricted antibody responses to single VDJ
gene segments or Igh alleles suggests an occasional, single best
solution to antigen-complementarity that results in expansion of
that B cell lineage. In this case, strain-specific differences in
the repertoire of germline V.sub.H genes would regulate the
antibody response. Alternatively, several investigators have
suggested that restricted antibody responses are circumscribed by
self-tolerance; others note that clones expressing V(D)J
rearrangements that are robustly tolerant of mutational change
should outgrow mutationally fragile competitors. Nonetheless, the
mechanisms driving repertoire selection has remained a
controversial issue. Together, these observations have suggested
that the great specificity of humoral immune responses is not the
consequence of highly selective clonal activation but of
competitive survival and proliferation of higher affinity B
cells.
[0180] Transmembrane signals generated through the B cell antigen
receptor complex regulate B cell responses to antigen binding and
may thereby also regulate repertoire selection. Other cell surface
molecules can also modifying B cell responses to antigen.
Transmembrane signals generated through the B cell antigen receptor
complex and other surface receptors are critically regulated by
CD19 expression (reviewed in Fearon and Locksley, 1996; Tedder et
al., 1997). CD19 functions as a general regulator of B cell
proliferation, differentiation, clonal expansion in the peripheral
B cell pool, and of peripheral tolerance, as described above.
[0181] Whether differences in endogenous signal transduction
thresholds influence repertoire selection was assessed in this
Example using C57BL/6 mice with single complementary genetic
alterations that result in either the loss or overexpression of the
CD19 cell surface molecule. In general, B lymphocytes from
CD19-deficient (CD19-KO) mice are hypo-responsive to transmembrane
signals while B lymphocytes from transgenic mice that overexpress
CD19 (CD19-TG) are hyper-responsive (Engel et al., 1995; Sato et
al., 1995; Zhou et al., 1994). Indeed, transgenic mice with even
small increases (10-25%) in CD19 receptor density on B cells
exhibit quantitative changes in B cell functional capacity. Thus,
CD19-deficient and CD19-overexpressing mice serve as model systems
where CD19 is a general response regulator of cell-surface receptor
signaling and cellular signal transduction thresholds.
[0182] To assess the contribution of antigen receptor signaling to
repertoire selection, CD19-TG, CD19KO, and wildtype C57BL/6 mice
were immunized with NP coupled to the T-cell-dependent antigen,
chicken gamma globulin (CGG). Mice that overexpressed CD19
generated anti-NP humoral immune responses that were quantitatively
similar to those of wildtype C57BL/6 mice, while CD19KO mice
generated only modest responses. However, the antibody response
generated by mice that overexpressed CD19 were qualitatively
distinct from those of C57BL/6 controls. V.sub.H gene segment and
gene family use were unprecedently diverse in CD19TG mice and none
of the anti-NP antibodies produced carried .lamda.1 light chains.
Significantly, some of the atypical antibodies bound the NP hapten
better than canonical antibodies predominantly generated in
wild-type C57BL/6 mice. In addition, several of the anti-NP
antibodies generated by CD19-TG mice were reactive with a self
antigens. Thus, endogenous signaling thresholds regulate repertoire
diversity and selection during B cell responses to antigen
independently of change in the immunoglobulin loci.
Anti-NP Immune Responses in Mice Overexpressing CD19
[0183] CD19TG and wildtype C57BL/6 mice were immunized with
NP.sub.18-CGG to assess their humoral immune responses to NP. Serum
anti-NP antibody concentrations at the time of immunization (day 0)
and on subsequent days (4, 8, 10, 16, and 58) were determined by
ELISA using NP.sub.25-BSA. Values represented either mean antibody
levels (.+-.SEM) from 4-5 individual mice per time-point following
immunization relative to isotype-matched anti-NP monoclonal
antibodies used to generate standard curves, or represented
antibody concentrations relative to control serum from a C57BL/6
mouse immunized with NP.sub.18-CGG. CD19TG mice generated serum IgM
responses quantitatively similar to those of wildtype mice, despite
an overall (-80%) reduction in peripheral B cell numbers (Engel et
al., 1995). IgG1 responses of CD19TG mice were .about.10-fold lower
than in wildtype mice. IgG2a, IgG2b, and IgG3 responses in CD19TG
mice were also below those of wildtype mice. All antibody responses
were of the Igh.sup.b allotype. Remarkably, .lamda.1 antibody
responses were poor in CD19TG mice, while K.sup.+ antibody
responses were similar in CD19TG and wildtype mice. Therefore, the
primary NP-specific antibody response in CD19TG mice is dominated
by IgM and IgG1, with the vast majority of antibodies bearing light
chains other than .lamda.1.
[0184] The relative affinity/avidity of the antibody response in
CD19TG mice was assessed by comparing antisera binding to highly-
(NP.sub.25-BSA) or sparsely- (NP.sub.5-BSA) substituted NP-bovine
serum albumin (BSA) substrates over a wide range of antibody
concentrations as described previously (Herzenberg et al., 1980).
Values represented mean (.+-.SEM) ratios of anti-NP.sub.5 versus
anti-NP.sub.25 antibody concentrations from 4-5 individual mice per
time-point. In cases where anti-NP.sub.5 antibody levels were not
detectable, values of 0 were used for generating means. Differences
between CD19TG and wildtype mice were significant, p<0.05, at
about 3 days, 10 days and 14 days after immunization for IgM; at
about 11 days, 18 days and 58 days for .lamda.1; and at about 58
days for .kappa..
[0185] Despite similar levels of IgM anti-NP antibodies, the
primary antibody response in CD19TG mice was generally of a lower
affinity compared with antiserum from wildtype mice. However, the
IgG1 antibody responses in CD19TG and wildtype mice exhibited
comparable affinity maturation. Again however, the .lamda.1
antibody response was modest and this response was of lower
affinity than observed in wildtype mice. It therefore appears that
in response to NP, mice overexpressing CD19 generate high affinity
IgG1 antibody that does not bear .lamda.1 light chain.
[0186] The relative frequency of anti-NP antibody-producing B cells
in the spleen and bone marrow of CD19TG mice was assessed using
ELISpot assays. Determined values represented mean AFC numbers
(.+-.SEM) from 4-14 individual mice per time-point following
immunization on day 0. Differences between CD19TG and wildtype mice
were significant, p<0.05, at about days 8 and 11 for IgM in
spleen, and at about day 58 for IgG1 in bone marrow.
[0187] NP-specific IgM antibody producing cells were 2- to 7-fold
higher among CD19TG mouse splenocytes than among wildtype
splenocytes following immunization. The frequency of IgG1 anti-NP
antibody-forming cells among CD19TG splenocytes were not
significantly different from wildtype controls. CD19TG mice also
had consistently higher frequencies of IgM-secreting cells and
lower frequencies of IgG-secreting cells among bone marrow-derived
anti-NP antibody-forming cells when compared with wildtype
controls. Importantly, the overall kinetics of NP-specific
antibody-forming cell responses were similar in CD19TG and wildtype
mice, although the long-lived memory antibody-forming cells
normally found in the marrow of wildtype mice 58 days after
immunization were not present in CD19TG mice. Therefore, the serum
antibody responses observed in CD19TG and wildtype mice generally
mirrored the antibody-forming cell responses.
Anti-NP Immune Responses in CD19KO Mice
[0188] CD19KO mice were immunized with NP.sub.18-CGG to compare
their humoral immune responses with wildtype littermates. Mean
serum anti-NP antibody concentrations (.+-.SEM) from 4-5 individual
mice per time-point following immunization (day 0) were determined
by ELISA using NP.sub.25-BSA as described above. The average
relative affinity of serum anti-NP antibodies was estimated by
determining the mean concentration of NP.sub.5-binding and
NP.sub.25-binding antibodies at each time-point by ELISA as
described above. Differences between CD19KO and wildtype were
significant, p<0.05, at about 3 days and 18 days after
immunization for IgM; at about 18 days and 58 days for IgG1; at
about 58 days for .lamda.1; and at about 18 and 58 days for
.kappa..
[0189] The frequency of spleen and bone marrow cells secreting
anti-NP.sub.25-binding antibody was determined by ELISpot assays as
described above. Values represent mean antibody-forming cell (AFC)
numbers (.+-.SEM) from 4-5 individual mice per time-point following
immunization on day 0. Differences between CD19KO and wildtype mice
were significant, p<0.05, at about days 10 and 18 for IgG1 in
spleen, and at about days 10, 16 and 58 for IgG1 in bone
marrow.
[0190] Serum IgM and IgG1 antibody responses to NP were 10-fold and
>100-fold lower in CD19KO mice than in wildtype mice. .lamda.1-
and .kappa.-bearing antibody responses to NP were also suppressed
in CD19KO mice. Affinity maturation was also delayed and reduced in
CD19KO mice when compared with wildtype mice. The relative
frequency of B cells producing IgG1 anti-NP antibodies in the
spleens and bone marrow of CD19KO mice was markedly lower than in
wildtype mice at each time point following immunization. Therefore,
antibody responses to NP in CD19KO mice were markedly diminished
beyond what would be expected with the overall (40-60%) reduction
in peripheral B cell numbers in these mice.
Germinal Center Responses in CD19TG and CD19KO mice
[0191] Germinal center B cell responses in CD19TG and CD19KO mice
were assessed after immunization with NP.sub.18-CGG. Histologic
sections of spleen were stained with the GL7 monoclonal antibody
and/or peanut agglutinin (PNA) to identify germinal center B cells
(Laszlo et al., 1993; Rose et al., 1980). Antibody specific for
mouse .kappa. light chain was used to visualize the B cell zones
(follicles) within the splenic white pulp. Overall, B cell
follicles were smaller in CD19TG mice than in wildtype mice both
before and after NP immunization. Correspondingly, T cell zones
occupied a larger portion of CD19TG mouse spleens. The overall
frequency of follicles was also significantly (p<0.01) lower in
CD19TG mice than in wildtype mice, reflecting reduced B cell
numbers in these mice.
[0192] The frequency of PNA.sup.+ germinal centers within follicles
of CD19TG mice increased in response to immunization, although the
germinal centers were usually smaller in CD19TG mice than in
wildtype controls. The frequency of germinal centers per follicle
was also significantly reduced in CD19TG mice. Nonetheless, the
percentage of splenic B cells induced to express the GL7 antigen
was similar in CD19TG and wildtype mice as assessed by flow
cytometry, although GL7 expression kinetics were delayed in CD19TG
mice. However, the increase in GL7.sup.+ B cells in CD19TG mice at
day 16 was due to a significant number of GL7.sup.+ B cells found
outside of PNA.sup.+ germinal centers, something not observed in
C57BL/6 mice. In fact, there were relatively few GL7.sup.+ B cells
within the germinal centers of CD19TG mice on day 16 but there were
significant numbers of GL7.sup.+ cells with abundant cytoplasmic
immunoglobulin clustered around the penicilliary arterioles. Thus,
germinal centers form following antigen challenge of CD19TG mice
but they are small in size and dissipate at a faster rate than in
wildtype mice. Moreover, the germinal centers of CD19TG mice
displayed different phenotypic properties than observed in wildtype
mice by the dissociation of the PNA and GL7 markers. In further
contrast with results obtained in normal mice, germinal centers in
CD19TG mice did not contain .lamda.1.sup.+ B cells after
immunization with NP-CGG.
[0193] The number and size of primary follicles in CD19KO mice were
normal, but generated few germinal centers or GL7.sup.+ B cells
following NP-immunization. Similarly, .lamda.1.sup.+ B cells were
not observed in the few germinal centers that formed in CD19KO mice
after immunization with NP. Therefore, NP-immunization did not
induce a significant germinal center response in CD19KO mice.
Anti-NP Antibody Diversity in CD19TG Mice
[0194] The repertoire of anti-NP antibodies elicited in CD19TG mice
was examined by generating hybridomas from splenocytes of
individual CD19TG mice immunized with NP.sub.18-CGG. The hybridomas
were labeled TG2, TG3, TG7, or TG18 depending on the day of
splenocyte isolation post-immunization (Table 3). Splenocytes from
mice boosted with NP.sub.18-CGG on day 15 were used to generate
TG18 hybridomas. The TG2, TG3, TG7 and TG18 fusions generated 164,
36, 210, and 275 hybridomas total with 3, 1, 31, and 10 monoclonal
hybridoma lines isolated that secreted antibodies reactive with
NP-BSA but not BSA in ELISA assays. Surprisingly, none of these
hybridomas secreted .lamda.1-bearing antibodies (Table 3).
[0195] From the TG2 and TG3 fusions, three of the four hybridomas
(75%) secreted .mu., .kappa. antibody products, while one (1)
produced a .gamma.2a, .kappa. antibody (Table 3). The relative
affinities/avidities of anti-NP antibodies were determined by
calculating the ratio of NP.sub.5-binding antibody concentrations
to NP.sub.25-binding antibody concentrations as previously
described (Herzenberg et al., 1980). The affinity threshold for
IgG1 antibody binding to each NP-BSA conjugate was determined using
monoclonal antibodies with known affinities for NP. The
H33L.gamma.1 antibody (IgG1, .lamda.1; K.sub.a=2.0.times.10.sup.7
M.sup.-1) bound equally well to both NP.sub.5-BSA and NP.sub.25-BSA
conjugates (binding ratio .about.1.0), whereas the B1-8.gamma.1
monoclonal antibody (IgG1, .lamda.1) with a Ka=10.sup.6 M.sup.-1
exhibited 5-fold lower relative binding to NP.sub.5-BSA than to
NP.sub.25-BSA (ratio .about.0.2). The H50G.gamma.1 monoclonal
antibody (IgG1, .lamda.1) with a Ka=1.2.times.10.sup.5 M.sup.-1 did
not bind NP.sub.5-BSA at detectable levels, but bound NP.sub.25-BSA
(ratio<0.01). Based on this analysis, these anti-NP antibodies
generated from CD19TG mice had relatively low affinity/avidity
values for NP.
[0196] From the TG7 fusion, 22 of the 28 hybridomas studied (79%)
secreted .mu. antibodies while the rest secreted .lamda.1
antibodies (Table 3). None of the antibodies bore .lamda.1 light
chains, while 68% bore .lamda.2, 21% bore .lamda.3, and 11% bore
.kappa. light chains. The relative affinities/avidities of the TG7
antibodies were quite heterogeneous, although the IgG1 antibodies
were generally of lower affinity (Table 3). By contrast, all 10
hybridomas from the TG18 fusion secreted G1, .kappa. antibodies
with affinities/avidities equal to that of the
H33L.gamma.1/.lamda.1 control antibody (Table 3).
Sequence Analysis of Anti-NP Antibodies from CD19TG Mice During
Primary Responses
[0197] The heavy chain genes of the TG2, TG3 and TG7 hybridomas
were sequenced by PCR amplification of cDNA made from hybridoma
RNA. Eight of 31 antibodies (26%) were encoded by VDJ
rearrangements containing V.sub.H gene segments common in the
anti-NP B cells of C57BL/6 mice, V186.2, V23, and C.sub.1H.sub.4
(Table 3). Three of these antibodies, TG7-14, -17, and -99, may
have arisen from a common progenitor since each had identical VDJ
sequences. These were the only antibodies to display canonical VDJ
sequences for anti-NP antibodies with the preferred YYGS motif in
CDR3 (Table 3). Two of the three V23 containing antibodies, TG7-26,
and -170, also shared identical VDJ sequences. The V.sub.H regions
used by all eight hybridomas were free of somatic mutations. Thus,
CD19TG mice are capable of generating antibody heavy chains typical
of those obtained in Igh.sup.b mice to NP, although these
represented a minority of the antibodies and none of these heavy
chains paired with .lamda.1.
[0198] The remaining 74% of primary-response antibodies used
V.sub.H genes not normally found in NP-specific B cells from
C57BL/6 mice (Table 3). Sixteen of these antibodies used V.sub.H
segments encoded by known members of the J558 family; 86.22 (1
hybridoma), G4D11 (1 hybridoma), V130 (5 hybridomas, 2 were
related), 671.5 (8 related hybridomas), and C1A4 (1 hybridoma).
Remarkably, the majority of V.sub.H regions did not contain somatic
mutations. One anti-NP antibody, TG7-83, used a previously
unidentified V.sub.H segment similar to the 5D3 gene (Kaartinen et
al., 1988) of the J558 family although six nucleotide differences
at the 5' end were homologous with the 186.2 V.sub.H gene sequence.
This V.sub.H sequence is similar to that of the dC5 antibody from a
C57BL/6 mouse (GenBank accession number AF045488) and the germline
V.sub.HII gene, H30, isolated from BALB/c mice (Schiff et al.,
1985) and is therefore likely to represent a heretofore
unidentified V.sub.H gene segment in C57BL/6 mice.
[0199] TG7 hybridomas utilized V.sub.H gene segments from the 7183,
Q52 and IX gene families (Table 3). The TG7-3 antibody was encoded
by a novel V.sub.H7183 family member most similar to the
V.sub.H61-1P gene of BALB/c mice (Chukwuocha et al., 1994). A
C57BL/6 V.sub.H gene that only differs from TG7-3 at four positions
was identified, although these differences are unlikely to
represent somatic diversification since these residues are found in
other V.sub.H7183 family members of C57BL/6 mice. The TG7-50, -108,
and -110 hybridomas generated unrelated antibody products using
V.sub.H segments almost identical to the OX-2 gene segment, a
V.sub.H Q52 family member of BALB/c mice (Lawler et al., 1987). The
TG7-125 hybridoma utilized a V.sub.H region identical to the BALB/c
germline OX-1 V.sub.H gene, another member of the Q52 family. The
TG7-118 hybridoma utilized a V.sub.H region most homologous with
the VGAM3-8 V.sub.H gene of C57BL/6 mice, an IX gene family member
(Winter et al., 1985). The TG7-188 V.sub.H sequence was also 98%
homologous with V.sub.H regions of two hybridomas, 5G6 and 264,
from C57BL/6 mice (GenBank accession number AF045504, Nottenburg et
al., 1987). The TG7-188 V.sub.H segment may therefore represent a
new member of the IX gene family in C57BL/6 mice. The AGTC changes
at the 5' end may represent somatic diversification since we were
unable to detect similar sequences in the C57BL/6 genome.
Considerable diversity in D.sub.H and J.sub.H use by all of the
hybridomas was apparent (Table 3), but relatively few antibodies
contained the CDR3 YYGS motif typical of anti-NP antibodies in
C57BL/6 mice. All J.sub.H1 sequences were of the b allotype. Thus,
the antibody response in CD19TG mice was quite diverse by day 7
after primary immunization, although the hybridomas primarily used
germline genes with no, or few, somatic mutations (Table 3).
Sequence Analysis of Anti-NP Antibodies from CD19TG Mice During
Secondary Responses
[0200] All ten hybridomas generated from the TG18 fusion produced
antibodies paired with .kappa. light chains (Table 3). One of
these, TG 1843, carried a member of the J558 family, V23, bearing 2
point mutations. The TG18-161 hybridoma V.sub.H gene segment
matched the V23 sequence except for 4 nucleotide differences; 3
were clustered at codons 9, 10 and 11 which were identical to the
V186.2 gene. Thus, the TG18-161 heavy chain gene rearrangement may
be derived from a hybrid of two well characterized V.sub.H gene
segments, V23 and V186.2, or from a previously unknown J558 family
member. Consistent with the second possibility is the full identity
of the TG18-161 V.sub.H gene with that present in the 70.1.4
hybridoma derived from a C57BL/6 mouse (GenBank accession number
AF006576). Of the remaining eight TG18 hybridomas, six were
clonally related (Table 3) with V.sub.H gene segments homologous
(96%) to the germline 22.1 V.sub.H gene of the J606 V.sub.H family
in BALB/c mice (Brodeur and Riblet, 1984; Hartman and Rudikoff,
1984). The two remaining hybridomas, TG18-5 and TG18-259, shared
identical VDJ sequences with V.sub.H segments encoded by OX2-like
genes of the Q52 family. These hybridomas utilized a V.sub.H gene
segment that differed from those utilized by the unrelated TG7-50,
-108 and -110 hybridomas at three positions that are potential
sites of hypermutation. D.sub.H and J.sub.H gene utilization was
also diverse among the TG18 hybridoma set (Table 3), although none
of the antibodies encoded the YYGS motif. Thus, the repertoire of
the anti-NP antibody response of CD19TG mice substantially diverges
from the response of wildtype Igh.sup.b mice with only 20% ( 2/10)
of the TG18 antibodies encoded by members of the J558 V.sub.H gene
family and none carried the .lamda.1 light chain.
.lamda. Light Chain Utilization by Anti-NP Antibodies
[0201] In contrast with the expanded V.sub.H repertoire used to
generate anti-NP antibodies in CD19TG mice, there was a striking
deficiency in .lamda.1 utilization and the .lamda. light chain
repertoire was remarkably compressed (Table 3). .lamda.2 and
.lamda.3 diversification did not occur since only two different
.lamda. light chains were used by sixteen .lamda.-producing
hybridomas. There was no evidence of junctional diversity in any of
the light chains and only one mutation was found in all of the
.lamda.3 light chains. Although heavy chain gene mutations are
commonly greater than ten-fold more frequent than .lamda.mutations
during anti-NP responses (Cumano and Rajewsky, 1986; Ford et al.,
1994), the lack of diversity and mutations in this large panel of
antibodies was unexpected.
V.sub.H Utilization by CD19KO Mice
[0202] The repertoire of anti-NP antibodies elicited in CD19KO mice
was also examined by generating hybridomas from splenocytes of
individual mice. CD19KO mice were immunized with NP.sub.18-CGG on
day 0, boosted on day 7, with hybridomas generated on day 10. The
KO10 fusions generated 615 hybridomas total. Only six clonal
hybridomas were isolated (<1%) that secreted .mu., .kappa.
antibodies with low affinities/avidities for NP.sub.25-BSA, but not
BSA, in ELISA screens (Table 3). Four antibodies were encoded by
non-canonical, germline V.sub.H genes of the J558 family (Table 3).
Of interest, the KO10-613 antibody was encoded by a newly described
member of the J558 family, L350-7 (Kasturi et al., 1994). Two
identical antibodies were encoded by a new member of the IX V.sub.H
gene family. Thus, there was little affinity maturation or
selection for canonical sequences in CD19-deficient mice.
Affinity Analysis of Anti-NP Antibodies
[0203] Antibodies representing most NP-specific hybridomas from
primary and secondary responses were purified and used for NP
affinity determinations by fluorescence quenching (Azuma et al.,
1987; Eisen and McGuigan 1971; Jones et al., 1986). This assay
measures antibody binding to NP-caproate, a monovalent derivative
of the immunizing hapten. Under the conditions utilized the assay
was sensitive to 7.0.times.10.sup.3 M.sup.-1, below which
NP-specific binding was not detected. Of eight TG7 antibodies, the
K.sub.as ranged between a low of 7.2.times.10.sup.3 M-1 for TG7-125
and a high of 1.6.times.10.sup.5 M.sup.-1 for TG7-180, with an
average affinity of 7.2.times.10.sup.4 M.sup.-1 (FIG. 4). The
KO10-613 antibody had a Ka of 1.3.times.10.sup.4 M.sup.-1. By
contrast, all three TG18 antibodies had relatively high affinities,
1.9 to 2.9.times.10.sup.6 M.sup.-1.
[0204] The K.sub.as of the NP-specific hybridomas antibodies were
also compared with K.sub.as of antibodies generated by
transfectomas producing canonical anti-NP antibodies and
representative antibodies utilized by B cells isolated from
NP-specific foci or germinal centers according to art-recognized
techniques. On average, the NP-specific antibodies generated in
CD19TG mice were of lower affinity than canonical anti-NP
antibodies represented by the B1-8.gamma.1 control antibody (FIG.
4). However, the TG18 antibodies were uniformly of higher affinity
than canonical antibodies or antibodies isolated from germinal
centers. Thus, the CD19TG mouse generates noncanonical anti-NP
antibodies of higher affinity than canonical antibodies generated
in wild-type C57BL/6 mice.
B Cell Apoptosis in CD19TG and CD19KO Mice
[0205] Apoptosis regulates the immune response of B lymphocytes and
influences selection for affinity maturation within germinal
centers. Mice that constitutively express Bcl-x.sub.L in B cells
exhibit expanded use of non-canonical anti-NP antigen receptors
following immunization with NP-CGG. To assess whether decreases in
apoptosis caused the dramatic shift in repertoire utilization
observed in mice expressing differing CD19 levels, B cell apoptosis
was assessed in situ and in vitro by two methods, TUNEL analysis
and by determining the frequency of hypodiploid B cells labeled
with propidium iodide. Immunohistological analysis of spleen tissue
sections from CD19TG and CD19KO mice revealed that the overall
frequency of apoptotic cells within follicles of each mouse strain
were not obviously different from wildtype C57BL/6 littermates. The
frequency of apoptotic cells in CD19KO mice was increased above
C57BL/6 controls, but the majority of apoptotic cells were within
the T cell zones of the knockout animals. When B cells were
purified from these mice and assessed for the frequency of
hypodiploid B220.sup.+ B cells labeled with propidium iodide,
0.19.+-.0.03% apoptotic B cells were observed in CD19TG B cells,
0.27% in CD19KO B cells, compared with 0.20.+-.0.03% apoptotic B
cells from wildtype littermates. Culturing the B cells overnight
(16 h) or for 2 days in the presence of varying concentrations of
anti-IgM antibodies did not reveal dramatic differences in the
frequency of apoptotic cells in CD19TG mice and controls. By
contrast, the frequency of apoptotic cells was significantly
reduced in CD19KO B cells. Therefore, the repertoire expansion
observed in CD19TG mice does not appear to result from a
significantly reduced rate of B cell apoptosis.
Reactivity of Anti-NP Antibodies with Autoantigens
[0206] Since tolerance to self antigens may influence the diversity
of the B cell repertoire and CD19TG mice have defects in peripheral
tolerance, the potential for non-canonical anti-NP antibodies to
react with self antigens was assessed. Of interest was that 27 of
the 47 antibodies analyzed in this study had Arg residues located
within their CDR3 regions compared with 12 of 45 NP-specific
antibodies generated in C57BL/6 mice that were chosen randomly from
the GenBank database. The facts that CD19TG mice produce anti-DNA
autoantibodies and that anti-DNA autoantibodies commonly contain
Arg residues within their CDR3 regions (Krishnan et al., 1995;
Shlomchik et al., 1990) prompted an assessment of whether the
anti-NP antibodies produced by CD19TG mice also reacted with ssDNA.
The TG7-83IgG1 antibody reacted strongly with ssDNA, at a level
similar to that of serum from autoimmune MRL.sup.lpr/lpr mice (FIG.
5A). In fact, the relative binding of the TG7-83 antibody for ssDNA
was comparable with the binding of two well-characterized,
isotype-matched anti-ssDNA autoantibodies (Krishnan et al., 1995;
Tillman et al., 1992) over a range of antibody concentrations (FIG.
5B). Two additional antibodies, TG2-354 and TG2-417, also bound
ssDNA at levels significantly higher than non-specific control
antibodies. The KO10-613, TG3471, TG7-75, and TG7-68 antibodies
also reacted with self protein antigens. This finding indicates
that alterations in tolerance regulation in CD19TG mice in part
account for the expanded NP-specific antibody repertoire of CD19TG
mice, in accordance with the methods of the present invention.
Materials and Methods
Mice
[0207] The generation of CD19-deficient mice (CD19KO) and human
CD19 (hCD19) transgenic mice (CD19TG, h19-1 line, C57BL/6) has been
described in the art (Engel et al., 1995; Zhou et al., 1994). B
lymphocytes of the h19-1 line of CD19TG mice express 3-fold higher
levels of total cell surface CD19 (Sato et al., 1996; Sato et al.,
1997) and have 9-14 copies of the hCD19 transgene integrated into a
single (or closely linked) genomic site(s) on chromosome 7. The
h19-1 mice used in this study were backcrossed with C57BL/6 mice
(Jackson laboratory, Bar Harbor, Me.) for 8 to 10 generations
without a diminution of hCD19 expression and all mice expressed
similar levels of cell-surface hCD19. CD19KO mice were backcrossed
with C57BL/6 mice for 8 to 10 generations. Flow cytometric analysis
demonstrated that B lymphocytes from all mice expressed the
IgM.sup.b but not IgM.sup.a allotype, and the mice only produce
antibodies of the b allotype. All mice were 2-3 months of age at
the time of use and were housed under identical conditions in a
specific pathogen free barrier-facility. All studies and procedures
were approved by the Animal Care and Use Committee of Duke
University.
Antigens and Immunizations
[0208] Succinic anhydride esters of (4-hydroxy-3-nitrophenyl)acetyl
(NP; Genosys Biotechnologies, The Woodlands, Tex.) were reacted
with CGG (Sigma Chemical Co., St. Louis, Mo.) or bovine serum
albumin (BSA, Sigma Chemical Co.) as described (Jacob et al.,
1991). The coupling ratio of each hapten/protein conjugate was
determined spectrophotometrically. Eight-week old mice were
immunized with a single intraperitoneal injection of 50 .mu.g
NP.sub.18-CGG conjugate precipitated in alum (Jacob et al., 1991).
Quantification of Serum Anti-NP Antibody Levels Serum IgM, IgG1,
IgG2a, IgG2b, IgG3, IgA, .kappa. light chain, and .lamda.1 light
chain antibodies specific for NP were quantified by ELISA. Wells of
96-well flat bottom plates (Costar, Cambridge, Mass.) were coated
with either 5 .mu.g/ml NP.sub.5BSA or NP.sub.25-BSA in 0.1 M
borate-buffered saline (pH 8.4) at 4.degree. C. overnight before
the wells were blocked with phosphate-buffered saline (pH 7.4)
containing 2% gelatin and 1% BSA. Serially-diluted mouse sera were
then added to each well at room temperature for 1.5 hours. After
washing with Tris-buffered saline (pH 7.5) containing 0.05% Tween
20 (Sigma Chemical Co.), alkaline phosphatase (ALP)-labeled goat
antibody specific for mouse IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, or
.kappa. light chain (Southern Biotechnology Associates, Birmingham,
Ala.) was added and incubated at room temperature for 1.5 hours.
.lamda.1 light chain-bearing antibody binding was assessed using
biotinylated Ls136 (anti-.lamda.1) monoclonal antibody (Reth et
al., 1978) and ALP-conjugated streptavidin (Southern Biotechnology
Associates). ALP activity was visualized using p-nitrophenyl
phosphate substrate (Southern Biotechnology Associates) and optical
densities were determined at 405 nm.
[0209] The concentrations of IgM, IgG1, .lamda.1, or .kappa.
anti-NP antibodies were estimated by comparisons to standard curves
generated using serially diluted control monoclonal antibodies on
each plate. The standard for IgG1 and .lamda.1 anti-NP antibodies
was H33L.gamma.1, as is known in the art. The standard for IgM was
B1-8, an IgM anti-NP monoclonal antibody (Reth et al., 1978). The
.kappa. antibody standard was TG1843 (Table 3). The standard for
IgG2a, IgG2b, IgG3, IgA anti-NP antibodies was serially diluted
serum from a C57BL/6 mouse obtained 10 days following immunization
with NP.sub.18-CGG.
Enzyme-Linked Immunospot Assays
[0210] The frequency of NP-specific antibody-forming cells (AFC)
from single-cell splenocyte and bone marrow suspensions were
estimated by enzyme-linked immunospot (ELISpot) assays using
NP.sub.5-BSA and NP.sub.25-BSA conjugates as has been described in
the art.
Immunofluorescence Staining and Flow Cytometry Analysis
[0211] Single cell suspensions of mouse splenocytes were incubated
with anti-FcgRI/RII monoclonal antibody (clone 2.4G2, PharMingen,
San Diego, Calif.) for 10 min on ice to block Fc.gamma. receptor
function. To determine the frequency of GL7.sup.+ cells among
B220.sup.+ B cells, splenocytes were subsequently incubated with
FITC-labeled GL7 monoclonal antibody (PharMingen),
phycoerythrin-conjugated anti-B220 monoclonal antibody (RA3-6B2,
Caltag, South San Francisco, Calif.), and 7-aminoactinomycin D
(Molecular Probes Inc., Eugene, Oreg.) for 30 min on ice before
washing. The cells were subsequently analyzed on a FACScan flow
cytometer (Becton Dickinson, San Jose, Calif.). The percentage of
GL7.sup.+, B220.sup.+ cells was calculated from live lymphocytes
selected by forward-side scatter patterns and exclusion of
7-aminoactinomycin D. In additional experiments, splenocytes were
incubated with FITC-labeled anti-B220 antibody (RA3-6B2) and
biotinylated Ls136 antibody, washed, and incubated with
phycoerythrin-conjugated streptavidin. After washing, the frequency
of .lamda.1.sup.+ cells among viable B220.sup.+ lymphocytes was
determined by flow cytometry.
Immunohistochemistry
[0212] Six-.mu.m-thick frozen spleen sections were mounted on
poly-L-lysine (Sigma Chemical Co.)-coated slides, fixed, and stored
at -80.degree. C. as described (Jacob et al., 1991). Frozen
sections were stained with horseradish peroxidase (HRP)-conjugated
peanut agglutinin (PNA; ICN Pharmaceuticals, Costa Mesa, Calif.)
and biotinylated Ls136 antibody, followed by
streptavidin-peroxidase (Southern Biotechnology Associates)
treatment as described (Jacob et al., 1991). Additional serial
frozen sections were also stained with HRP-conjugated PNA and
biotinylated GL7 antibody, followed by streptavidin/alkaline
phosphatase, or stained with HRP-conjugated PNA and ALP-labeled
anti-.kappa. light chain antibodies. HRP and ALP activities were
visualized using 3-aminoethyl carbasole (Sigma Chemical Co.) and
naphthol AS-MX phosphate/fast blue BB (Sigma Chemical Co.),
respectively (Jacob et al., 1991). Terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL;
MEBSTAIN Apoptosis kit; Immunotech, Westbrook, Me.) was used to
identify apoptotic cell nuclei (Gavrieli et al., 1992) and the
sections were counterstained with hematoxylin.
Hybridoma Generation
[0213] Seven to eight-week-old mice were immunized with single
intraperitoneal injections of 100 .mu.g of NP.sub.18-CGG conjugate
precipitated in alum on day 0. Two, three, or seven days after
immunization, splenocytes from one or two immunized mice were fused
with nonsecreting P3X63-Ag8.653 myeloma cells as described (Kearney
et al., 1979) and subdivided into ten 96 well tissue culture
plates. One CD19TG mouse was boosted on day 15 with the same
antigen and its splenocytes were fused with the myeloma cell line
on day 18. Two CD19KO mice were boosted on day 7 with 100 .mu.g of
NP-CGG conjugate and their splenocytes were fused with myeloma
cells on day 10. The hybridomas generated from these fusions were
named based on the splenocyte source and the fusion day following
immunization: for example, KO10 hybridomas were generated from
splenocytes of CD19KO mice 10 days after the first
immunization.
[0214] Monoclonal hybridomas secreting anti-NP antibodies were
identified by ELISA. Culture supernatant fluid from each hybridoma
was added to NP.sub.45-BSA-coated 96-well flat bottom ELISA plates.
After washing, ALP-labeled goat anti-mouse IgM+IgG+IgA antibodies
were added to each well and ALP activity was visualized using
p-nitrophenyl phosphate substrate. Hybridomas generating
BSA-reactive antibodies were identified by ELISA using BSA-coated
plates and were eliminated. The class and isotypes of NP-reactive
antibodies were determined by ELISA with NP.sub.45-BSA coated
plates and by using mouse monoclonal antibody isotyping kits
(Amersham Life Sciences, Arlington Heights, Ill.). Hybridomas
secreting .lamda. light chain antibodies were identified by ELISA
using plates coated with goat anti-mouse whole Ig antibodies and
ALP-labeled goat anti-mouse .lamda. light chain antibodies
(Southern Biotechnology Associates) as the developing reagent.
Hybridomas secreting .lamda. light chain antibodies were identified
by immunohistochemical staining using cytospin preparations of each
hybridoma. Hybridomas were centrifuged onto glass slides, dried for
2 hours, then fixed with acetone at 4.degree. C. for 10 min. The
slides were stained with biotinylated Ls136 antibody, followed by
incubation with HRP-conjugated streptavidin which was visualized as
above.
[0215] Some NP-specific hybridomas were grown in miniPerm
bioreactors (Heraeus, South Plainfield, N.J.). Their culture
supernatant fluid was concentrated and the antibody product was
purified over protein G-Sepharose (Pierce, Rockford, Ill.) or
mannose-binding columns (Pierce, Rockford, Ill.). Antibody protein
concentrations and purity were determined by light absorption and
by antibody isotype-specific sandwich ELISA.
V.sub.H and Light Chain Gene Utilization
[0216] Cytoplasmic RNA was extracted from 0.1-1.times.10.sup.6
hybridoma cells using the RNeasy Mini Kit (Qiagen Chatsworth,
Calif.). First strand cDNA was synthesized from cytoplasmic RNA
using oligo-dT primers (dT.sub.18) and a Superscript Kit (Gibco
BRL, Gaitherburg, Md.). One .mu.l of cDNA solution was used as
template for PCR amplification of V.sub.H genes. PCR reactions were
carried out in a 100-.mu.l volume of a reaction mixture composed of
10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, 200 .mu.M
dNTP (Perkin Elmer, Foster City, Calif.), 50 .mu.mol of each
primer, and 5 U of Taq DNA polymerase (ISC Bioexpress, Kaysville,
Utah). Amplification was for 30 cycles (94.degree. C. for 1 min,
58.degree. C. for 1 min, 72.degree. C. for 1 min; Thermocycler,
Perkin Elmer). V186.2-related V.sub.H genes were amplified using a
sense primer complementary to the 5' region of the V186.2 gene
(primer V186.2; 5' TCTAG AATTC AGGTC CAACT GCAGC AGCC 3'--SEQ ID
NO:1) and antisense primers complementary to the Cp coding region
(primer C.mu.-in; 5' GAGGG GGAAG ACATT TGGGA AGGAC TG 3'--SEQ ID
NO:2) or the Cy region (primer C.gamma.1; 5' GAGTT CCAGG TCACT
GTCAC TGGC 3'--SEQ ID NO:3). V.sub.H genes not amplified using the
V186.2 primer were amplified using a promiscuous 5' V.sub.H primer
(MSVHE; 5' GGGM TTCGA GGTGC AGCTG CAGGA GTCTGG 3'--SEQ ID NO:4) as
previously described (Kantor et al., 1996). .lamda. light chain
cDNA was amplified using a V.lamda. primer (5' MCTG CAGGC TGTTG
TGACT CAGGA ATC--SEQ ID NO:5) and a CA primer (CGGGA TCCGC TCTTC
AGAGG AAGGT GGAAA CA--SEQ ID NO:6). Amplified PCR products were
purified from agarose gels using the QIAquick gel purification kit
(Qiagen) and were sequenced directly in both directions using an
ABI 377 PRISM DNA sequencer after amplification using the Perkin
Elmer Dye Terminator Sequencing system with AmpliTaq DNA polymerase
and the same primers for initial PCR amplification.
[0217] Sequences were compared with known V.sub.H sequences using
the BLAST search program provided by the National Center for
Biotechnology Information. V.sub.H genes belonging to the J558
family were analyzed as described (Bothwell et al., 1981; Gu et
al., 1991).
Anti-NP Antibody Affinity Measurements
[0218] The K.sub.as of purified anti-NP antibodies was determined
by fluorescence quenching as described (Azuma et al., 1987; Eisen
and McGuigan, 1971; Jones et al., 1986). Briefly, K.sub.as for NP
and NIP haptens were measured by fluorescence quenching in a
Shimadzu RF-3501 fluorospectrophotometer (Shimadzu Scientific
Instruments, Columbia, Md.). Excitation and emission wavelengths
were 280 and 340 nm, respectively: temperature (25.degree. C.) and
pH (7.4) were held constant. Titration was conducted by adding NP-
or NIP-caproate (Cambridge Research) over a three-log range
(10.sup.-8-10.sup.-5 M) to a known concentration of antibody (33 mM
in 2.5 ml PBS) in quartz cuvettes. Emission signal loss, or quench,
versus antigen concentration was plotted according to the Scatchard
equation to derive association constants for half-maximal
binding.
Anti-ssDNA ELISA
[0219] Calf thymus DNA (Sigma Chemical Co.) was purified by
repeated phenol/chloroform extraction followed by ethanol
precipitation. The DNA suspension was boiled for 10 min before
immersion in an ice bath to generate ssDNA. ELISA assays were
carried out in 96 well Immulon II microtiter plates (Dynatech
Laboratories, Chantilly, Va.) that were coated overnight at
4.degree. C. with ssDNA (5 mg/ml in 0.1 M Na citrate buffer
containing 0.15 M NaCl, pH 8.0). Plates were washed three times
with PBS (pH 7.3). Supernatant fluid from individual hybridomas was
diluted (generally 1:2) in PBS containing 1% BSA (Sigma) and 0.05%
Tween-20 and added to triplicate wells of the antigen-coated ELISA
plates. Sera from individual MRL autoimmune mice were used as
positive controls which were diluted (1:100) in PBS containing 1%
BSA (Sigma Chemical Co.) and 0.05% Tween-20. Peroxidase-conjugated
goat anti-mouse Ig antibody diluted in PBS containing 1% BSA and
0.05% Tween-20 was added to the wells for 1 h before washing three
times with PBS. Substrate solution containing 0.015% 3,3',
5,5'-tetramethylbenzidine (Sigma Chemical Co.) and 0.01%
H.sub.2O.sub.2 in 0.1 M Na citrate buffer (pH 4.0) was added at
room temperature for 30 min before the OD of the wells were
determined at 380 nm wavelength on a Titertek Plate Reader (Flow
Laboratories, McLean, Va.). OD values within the linear range of
the ELISA were determined using a standard serum obtained from
MRL.sup.lpr/lpr mice with linear regression analysis.
Assessment of Apoptosis
[0220] B cells were purified from single cell splenocyte
suspensions by removing T cells with anti-Thy1.2 antibody-coated
magnetic beads (Dynal, Inc., Lake Success, N.Y.). B cell
suspensions were analyzed by flow cytometry following isolation to
assess purity. B cell preparations from CD19KO and C57BL/6 mice
were >95% B220.sup.+ while preparations from CD19TG mice were
>75% B220.sup.+. B cells were seeded in 24-well flat bottom
plates (Costar) at 1.times.10.sup.6 cells per well with various
concentration of F(ab').sub.2 fragments of goat anti-mouse IgM
antibodies (Cappel, Durham, N.C.) and cultured for 16 hrs or 48 hrs
in a CO.sub.2 incubator. Cultured B cells were washed with PBS
containing 0.2% BSA and TUNEL.sup.+ cells were detected by flow
cytometry analysis using the MEBSTAIN Apoptosis kit (Immunotech).
The frequency of apoptotic cells was calculated: % apoptosis=[(%
TUNEL.sup.+ cells/(% TUNEL.sup.+ cells+% live cells)].times.100.
Cultured cells were also washed with PBS containing 0.2% BSA
followed by PBS containing 1% glucose and fixed with ice-cold 70%
ethanol overnight. The fixed cells were stained with 0.05 mg/ml
propidium iodide (PI; Sigma chemical Co.) solution containing 100
U/ml RNase A (Sigma Chemical Co.). Stained cells were analyzed by
flow cytometry and cells with hypodiploid nuclei were considered
apoptotic.
Data Analysis
[0221] All data are shown as mean values.+-.SEM unless indicated
otherwise. Analysis of variance (ANOVA) was used to analyze the
data, and the Student's t test was used to compare sample means.
The paired Student's t test was used to compare the means of %
apoptotic cells. TABLE-US-00004 TABLE 3 Summary of V.sub.H gene
usage by anti-NP hybridomas. NP.sub.25 V.sub.H Somatic CDR3
Hybridoma Isotype ELISA.sup.a NP.sub.5/NP.sub.25 Family V.sub.H
gene.sup.b Mutation D.sub.H J.sub.H Length TG2-354 M, .kappa. 0.16
<0.01 J558 86.22 0 SP2.3.sup. 1 8 TG2-403 M, .kappa. 0.45
<0.01 J558 186.2 0 SP2.2 2 7 TG2-417 M, .kappa. 2.76 <0.01
J558 G4D11 0 FL16.1 2 12 TG3-471 G2a, .kappa. 0.17 <0.01 J558
C1H4 0 SP2.4/6 2 11 TG7-3 M, .kappa. >3.00 <0.01 7183 (61-1P)
ND.sup.c ND 3 5 TG7-13 M, .lamda.2 2.80 <0.01 J558 V23 0 Q52 2
10 TG7-14 M, .lamda.2 >3.00 0.86 J558 186.2 0 FL16.1 2 10 TG7-17
M, .lamda.2 >3.00 0.29 J558 186.2 0 EL16.1 2 10 TG7-26 G1,
.lamda.2 1.39 <0.01 J558 v23 0 Q52 2 10 TG7-32 M, .lamda.2
>3.00 0.53 J558 130 0 FL16.1 2 10 TG7-50 M, .lamda.2 2.42 0.42
Q52 OX2 ND Q52 4 10 TG7-68 G1, .lamda.2 1.40 <0.01 J558 130 5
FL16.1 1 8 TG7-75 G1, .lamda.2 1.46 <0.01 J558 130 0 FL16.1 1 8
TG7-80 M, .lamda.3 >3.00 1.21 J558 671.5 0 SP2.4/6 2 10 TG7-83
G1, .lamda.2 1.43 0.01 J558 (5D3) ND SP2.6/7 3 5 TG7-88 M, .lamda.2
>3.00 1.31 J558 671.5 0 SP2.4/6 2 10 TG7-93 M, .lamda.3 >3.00
0.98 J558 671.5 0 SP2.4/6 2 10 TG7-99 M, .lamda.2 >3.00 0.92
J558 186.2 0 FL16.1 2 10 TG7-108 M, .lamda.2 >3.00 0.07 Q52 OX2
ND ST4 4 10 TG7-110 M, .lamda.3 >3.00 <0.01 Q52 OX2 ND FL16.1
4 9 TG7-112 M, .lamda.3 >3.00 0.96 J558 671.5 0 SP2.4/6 2 10
TG7-114 M, .lamda.2 >3.00 0.45 J558 130 0 FL16.1 2 10 TG7-118
G1, .kappa. 1.20 0.04 IX (VGAM3-8) ND SP2.5/7 2 8 TG7-125 M,
.lamda.2 >3.00 0.02 Q52 OX-1 ND SP2.9 4 8 TG7-129 M, .lamda.3
>3.00 1.04 J558 671.5 0 SP2.4/6 2 10 TG7-137 G1, .kappa. 0.99
0.04 J558 C1A4 0 FL16.1 4 11 TG7-138 M, .lamda.2 >3.00 0.51 J558
ND ND ND ND ND TG7-159 M, .lamda.2 >3.00 1.06 J558 671.5 0
SP2.4/6 2 10 TG7-167 M, .lamda.3 >3.00 1.38 J558 671.5 0 SP2.4/6
2 10 TG7-170 M, .lamda.2 >3.00 0.05 J558 V23 0 Q52 2 10 TG7-180
M, .lamda.2 >3.00 0.79 J558 130 2 FL16.1 1 12 TG7-186 M,
.lamda.2 >3.00 1.01 J558 671.5 0 SP2.4/6 2 10 TG18-5 G1, .kappa.
1.45 0.97 Q52 OX2 ND SP2 4 7 TG18-41 G1, .kappa. 1.49 1.20 J606
22.1 ND FL16.1 2 10 TG18-43 G1, .kappa. 1.35 0.90 J558 V23 2 FL16.1
2 11 TG18-48 G1, .kappa. 1.58 1.14 J606 22.1 ND FL16.1 2 10 TG18-61
G1, .kappa. 1.64 1.06 J606 22.1 ND FL16.1 2 10 TG18-99 G1, .kappa.
1.64 0.97 J606 22.1 ND FL16.1 2 10 TG18-161 G1, .kappa. 1.54 1.29
J558 (V23) ND Q52 2 9 TG18-223 G1, .kappa. 1.59 1.14 J606 22.1 ND
FL16.1 2 10 TG18-252 G1, .kappa. 1.60 0.82 J606 22.1 ND FL16.1 2 10
TG18-259 G1, .kappa. 1.52 0.73 Q52 OX2 ND SP2 4 7 KO10-613 M,
.kappa. 0.20 <0.01 J558 L350-7 ND SP2.2-5 4 10 KO10-678 M,
.kappa. 0.75 0.16 J558 VGAM3.0 0 FL16.1 3 13 KO10-683 M, .kappa.
0.18 <0.01 IX (VGAM3.8) ND FL16.1 3 10 KO10-863 M, .kappa. 0.55
0.03 J558 vmn2 0 SP2.8 4 12 KO10-1006 M, .kappa. 0.48 <0.01 J558
86.22 1 DST4 3 10 KO10-1121 M, .kappa. 0.15 <0.01 IX (VGAM3.8)
ND FL16.1 3 10 .sup.aValues represent mean ELISA OD results
obtained using hybridoma culture supernatant fluid. A positive
control IgM antibody (B1-8, 10 .mu.g/ml) generated mean OD values
of 1.18 while an IgG1 antibody (H33L.gamma.l, 1.0 .mu.g/ml)
generated OD values of 1.76. All OD values were significantly
greater (p < 0.05) than those obtained with control culture
media (IgM ELISA, 0.072 .+-. 0.001; IgG ELISA 0.077 .+-. 0.001) or
supernatant fluid from isotope-matched # negative control
hybridomas. These results are representative of those obtained in
at least three experiments. .sup.bParenthesis indicate that the
V.sub.H genes used are similar to those cited, but are likely to be
distinct genes. .sup.cND, not determined because the homologous
gene has not been identified in C57BL/6 mice, the size of the D
region was too small, or there were ambiguities in the sequence of
TG7-138.
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Sequence CWU 1
1
6 1 29 DNA Mus sp. 1 tctagaattc aggtccaact gcagcagcc 29 2 27 DNA
Mus sp. 2 gagggggaag acatttggga aggactg 27 3 24 DNA Mus sp. 3
gagttccagg tcactgtcac tggc 24 4 31 DNA Mus sp. 4 gggaattcga
ggtgcagctg caggagtctg g 31 5 28 DNA Mus sp. 5 aactgcaggc tgttgtgact
caggaatc 28 6 32 DNA Mus sp. 6 cgggatccgc tcttcagagg aaggtggaaa ca
32
* * * * *