U.S. patent application number 10/045185 was filed with the patent office on 2002-12-05 for use of interleukin - 15.
Invention is credited to Dooms, Hans Peter Raf, Fiers, Walter Charles, Grooten, Johan Adriaan Marc.
Application Number | 20020182178 10/045185 |
Document ID | / |
Family ID | 8228040 |
Filed Date | 2002-12-05 |
United States Patent
Application |
20020182178 |
Kind Code |
A1 |
Grooten, Johan Adriaan Marc ;
et al. |
December 5, 2002 |
Use of interleukin - 15
Abstract
The invention relates to the use of IL-15 or active variants
thereof and/or IL-15 activity enhancing compounds for the
manufacture of a pharmaceutical composition for manipulating memory
cells of the immune system, such as manipulating viability ad/or
responsiveness of said memory cells. The IL-15 activity enhancing
compound is for example lipopolysaccharide (LPS). The invention
further relates to the use of IL-15 inhibiting or eliminating
compounds for the manufacture of a pharmaceutical composition for
manipulating memory cells of the immune system. Such inhibiting or
eliminating compounds are for example anti-IL-15 antibodies,
anti-IL-15R.alpha. antibodies, fragments of these antibodies, e.g.
the Fab or F(ab').sub.2 fragment, soluble IL-15R.alpha., fusion
proteins consisting of soluble IL-15R.alpha., and Fc fragment,
compounds, e.g. peptides, binding-and/or inhibiting functional
IL-15 receptor, IL-15 antisense oligonucleotides.
Inventors: |
Grooten, Johan Adriaan Marc;
(Lovendegem, BE) ; Dooms, Hans Peter Raf; (Ieper,
BE) ; Fiers, Walter Charles; (Destelbergen,
BE) |
Correspondence
Address: |
KNOBBE, MARTENS, OLSON & BEAR, LLP
16 th Floor
620 Newport Center Dr.
Newport Beach
CA
92660
US
|
Family ID: |
8228040 |
Appl. No.: |
10/045185 |
Filed: |
October 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10045185 |
Oct 18, 2001 |
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09380049 |
Aug 23, 1999 |
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6344192 |
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09380049 |
Aug 23, 1999 |
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PCT/EP98/01127 |
Feb 23, 1998 |
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Current U.S.
Class: |
424/85.2 ;
424/85.1; 435/386; 435/69.5; 435/69.52; 514/44A; 530/351; 930/140;
930/141 |
Current CPC
Class: |
Y10S 930/141 20130101;
A61K 38/2086 20130101; A61K 38/1793 20130101; Y10S 930/14
20130101 |
Class at
Publication: |
424/85.2 ;
514/44; 424/85.1; 435/386; 435/69.5; 435/69.52; 530/351; 930/140;
930/141 |
International
Class: |
A61K 031/70; A01N
043/04; C12P 021/02; C12P 021/04; A61K 045/00; C07K 001/00; C07K
014/00; C07K 017/00; C12N 005/00; C12N 005/02 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 21, 1997 |
EP |
9720051.1 |
Claims
1. Use of IL-15 or active variants thereof and/or IL-15 activity
enhancing compounds for the manufacture of a pharmaceutical
composition for manipulating memory cells of the immune system.
2. Use of IL-15 or active variants thereof and/or IL-15 activity
enhancing compounds according to claim 1 for manipulating viability
of memory cells generated during encounter with antigen.
3. Use of IL-15 or active variants thereof and/or IL-15 activity
enhancing compounds according to claim 1 for manipulating
responsiveness of memory cells to a new encounter with antigen.
4. Use of IL-15 or active variants thereof and/or IL-15 activity
enhancing compounds according to claim 1 or 2 to promote an immune
response of said memory cells.
5. Use of IL-15 or active variants thereof and/or IL-15 activity
enhancing compounds for inducing a resting phenotype in ex vivo
propagated T lymphocytes.
6. Use of IL-15 or active variants thereof and/or IL-15 activity
enhancing compounds for protecting ex vivo propagated T lymphocytes
against cells death in absence of growth factor.
7. Use of IL-15 or active variants thereof and/or IL-15 activity
enhancing compounds according to claims 1 to 6 wherein the memory
cells are CD4.sup.+ T lymphocytes.
8. Use according to any one of the claims 1 to 7, wherein the IL-15
activity enhancing compound is lipopolysaccharide (LPS).
9. Use of IL-15 inhibiting or eliminating compounds for the
manufacture of a pharmaceutical composition for manipulating memory
cells of the immune system.
10. Use of IL-15 inhibiting or eliminating compounds according to
claim 9 wherein the memory cells are CD4.sup.+ T lymphocytes.
11. Use of IL-15 inhibiting or eliminating compounds according to
claim 9 or 10 wherein said compound is selected from the group
consisting of an anti-IL-15 antibody, anti-IL-15R.alpha. antibody,
fragments of these antibodies, e.g. the Fab or F(ab').sub.2
fragment, soluble IL-15R.alpha., fusion proteins consisting of
soluble IL-15R.alpha. and Fc fragment, compounds, e.g. peptides,
binding and/or inhibiting functional IL-15 receptor, IL-15
antisense oligonucleotides.
12. Pharmaceutical composition comprising IL-15 or active variants
thereof and/or IL-15 activity enhancing compounds, optionally
together with a suitable excipient.
13. Pharmaceutical composition according to claim 12 for use in
manipulating memory cells of the immune system.
14. Pharmaceutical composition according to claim 12 or 13 for use
in the treatment of auto-immune diseases or in treatment before,
during or after transplantation.
15. Pharmaceutical composition according to claim 12, 13 and 14,
wherein the IL-15 activity enhancing compound is lipopolysaccharide
(LPS).
16. Pharmaceutical composition comprising IL-15 inhibiting or
eliminating compounds, optionally together with a suitable
excipient.
17. Pharmaceutical composition according to claim 16 for use in the
repression of an immune response of memory cells of the immune
system.
18. Pharmaceutical composition according to claim 16 for use in the
treatment of immune deficiency diseases, or in treatment before,
during or after vaccination.
19. Pharmaceutical composition according to claims 16, 17 and 18,
wherein the compound is selected from the group consisting of an
anti-IL-15 antibody, anti-IL-15R.alpha. antibody, fragments of
these antibodies, e.g. the Fab or F(ab').sub.2 fragment, soluble
IL-15R.alpha., fusion proteins consisting of soluble IL-15R.alpha.
and Fc fragment, compounds, e.g. peptides, binding and/or
inhibiting functional IL-15 receptor, IL-15 antisense
oligonucleotides.
20. Pharmaceutical composition comprising ex vivo cultivated T
lymphocytes, treated with IL-15 or active variants thereof and/or
IL-15 activity enhancing compounds.
Description
[0001] The present invention relates to a new use of Interleukin-15
(IL-15). The invention further relates to pharmaceutical
preparations, containing IL-15 itself, IL-15 stimulating compounds
or-IL-15 inhibiting and/or eliminating compounds.
[0002] The cytokine interleukin-15 (IL-15) was originally
identified in culture supernatants of the simian kidney epithelial
cell line CV-1/EBNA and the T cell leukemia cell line HuT-102
(Grabstein et al., 1994; Burton et al., 1994; Bamford et al.,
1994). The IL-15 cDNA sequence encodes a 162 amino acid (aa)
precursor protein consisting of a 48 aa peptide and a 114 aa mature
protein (Grabstein et al., 1994). Although there is no sequence
homology with IL-2, analysis of the amino acid sequence predicts
that IL-15, like IL-2, is a member of the four .alpha. helix bundle
cytokine family. Furthermore IL-15 and IL-2 exert their biological
activities through binding on the IL-2R.beta. and .gamma.chains,
supplemented by a specific IL-15R.alpha. and IL-2R.alpha.
polypeptide, respectively (Giri et al., 1995). This sharing of
receptor subunits probably accounts for the similar functional
activities of both cytokines observed on T, B and NK cells. IL-15
mRNA is widely distributed in fibroblasts, epithelial cells and
monocytes but not in resting or activated T cells, the predominant
source of IL-2.
[0003] IL-15 and IL2 share various biological functions. IL-15,
like IL-2, has been defined as a T cell growth factor. IL-15 was
originally discovered as a factor that could induce proliferation
of the IL-2 dependent murine cytotoxic T-cell line (CD8.sup.+)
CTLL-2 (Grabstein et al., 1994). Proliferation upon addition with
IL-15 was also observed in phytohaemagglutinin (PHA)-activated
CD4.sup.+ or CD8.sup.+ human peripheral blood T lymphocytes (PBT),
and .gamma..delta. subsets of T cells (Grabstein et al., 1994;
Nishimura et al., 1996). Studies with phenotypically memory
(CD45RO.sup.+) and naive (CD45RO.sup.-) T cells, isolated from
human PBT, revealed that IL-15, like IL-2, induces in memory
CD4.sup.+ and CD8.sup.+ T cells and naive CD8.sup.+ T cells but not
in naive CD4.sup.+ T cells the expression of the CD69 activation
marker and proliferation (Kanegane et al., 1996). IL-15 was as
effective as IL-2 in the in vitro generation of
alloantigen-specific cytotoxic T cells in mixed lymphocyte cultures
and in promoting the induction of lymphokine activated killer (LAK)
cells (Grabstein et al., 1994). Additionally, in vivo studies in a
murine model demonstrated the capacity of IL-15 to augment
CD8.sup.+ T-cell-mediated immunity against Toxoplasma gondii
infection (Khan and Kasper, 1996). Here vaccination of mice with
soluble parasite antigen (Ag) and IL-15 resulted in significant
proliferation of splenocytes expressing the CD8.sup.+ phenotype and
protection against a lethal parasite challenge for at least 1 month
postimmunization.
[0004] Natural Killer (NK) cells are considered an important target
for IL-15 action. Treatment of NK cells with IL-15 results in
proliferation and enhancement of cytotoxic activity and in
production of Interferon .gamma. (IFN.gamma.), tumor necrosis
factor .alpha. (TNF.alpha.) and granulocytmacrophage colony
stimulating factor (GM-CSF) (Carson et al., 1994). Furthermore
IL-15 can substitute for the bone marrow microenvironment during
the maturation of murine NK1.1.sup.+ cells from nonlytic to lytic
effector cells (Puzanov et al., 1996).
[0005] Apart from its activities on T and NK cells, IL-15
costimulates, in a comparable way as IL-2, proliferation of B cells
activated with immobilized anti-IgM or phorbol ester (Armitage
et.al., 1995). Stimulation of B cells with a combination of CD40L
and IL-15 efficiently induces immunoglobulin synthesis. IL-15 has
no stimulatory activity on resting B cells.
[0006] IL-15 was also found to have other biological activities.
Chemoattractant factors are cytokines or chemokines that regulate
the migration of lymphocytes to inflammation regions.
[0007] IL-15 is described as a chemoattractant factor for human
PBT, inducing polarisation, invasion of collagen gels and
redistribution of adhesion receptors (Wilkinson and Liew, 1995;
Nieto et al., 1996). Murine mast cells proliferate in response to
IL-15, but not to IL-2, using a novel receptor/signalling pathway,
not shared with IL-2 (Tagaya et al., 1996). Furthermore, it has
been shown that IL-15 and IL-2 have different effects on
differentiation of bipotential T/NK progenitor cells, with IL-15
predominantly promoting the development of TCR.gamma..delta. T
cells and NK cells (Leclercq et al., 1996). The most striking
difference, however, between IL-15 and IL-2 lies in their
expression patterns. The presence of IL-15 mRNA in a variety of
non-lymfoid tissues indicates that the secretion of the cytokine is
not solely regulated by the immune system and/or that the cytokine
can act outside the immune system itself. Accordingly, addition of
IL-15 to a myoblast cell line affects parameters associated with
skeletal muscle fiber hypertrophy, suggesting IL-15 has anabolic
activities and increases skeletal muscle mass (Quinn et al.,
1995).
[0008] Activated CD4.sup.+ T lymphocytes play a key role in the
development of an effective immune response against pathogens by
providing the growth factors necessary for the expansion of the
activated CD4.sup.+ T lymphocytes (autocrine growth) and for the
expansion of CD8.sup.+ cytolytic cells and the differentiation of B
cells into antibody-secreting plasma cells (paracrine "helper"
activity).
[0009] After clearance of the pathogen, a subfraction of the
generated Ag-specific T cells persist as memory cells, either in
the lymphoid tissue or in the circulation. Throughout this
application, "memory cells" are defined as antigen-experienced
cells. These memory lymphocytes are small, resting cells which are
optimally primed for the generation of a quantitatively and
qualitatively superior, secondary response upon a re-encounter with
the priming Ag. In order to accomplish the transition from
activated CD4.sup.+ effector cell to resting CD4.sup.+ memory cell
and to acquire long-term survival, these effectors need to acquire
the following characteristics:
[0010] (i) being resistant towards, or escaping from,
activation-induced cell death (AICD); AICD is responsible for
attenuation of the immune reaction;
[0011] (ii) being independent from autocrine growth factors,
produced during the immune response. Normally, the disappearance of
these growth factors--a consequence of the ending of immune
activity--results in growth factor depletion-induced cell death by
apoptosis;
[0012] (iii) having the capacity, in case of a renewed contact with
the antigen, to expand maximally by production of the necessary
autocrine- and paracrine-acting helper cytokines such as IL-2.
[0013] The research that resulted in the present invention,
indicated that IL-15 promotes the generation and persistence of
CD4.sup.+ memory cells, by promoting antigen activated CD4.sup.+
T-lymphocytes to acquire the characteristics, mentioned above:
resistance towards AICD, insensitivity towards apoptosis following
growth factor withdrawal at the end of the antigen stimulus and
high responsiveness towards renewed antigen challenge. Resistance
towards AICD and insensitivity towards apoptosis determine the
survival of the CD4.sup.+ T lymphocytes. Responsiveness is
characterised by cell division, expansion of the cell number and
production of helper cytokines.
[0014] Thus, treatment of antigen stimulated CD4.sup.+ cells with
IL-15, even at very low concentrations, turns off the program of
cell death running in the absence of growth factor. Unlike with
IL-2, survival of CD4.sup.+ T cells with IL-15 is not accompanied
by DNA synthesis nor proliferation, demonstrating that IL-15
induces a resting phenotype in these cells. Moreover, the
sensitivity towards AICD of CD4.sup.+ T lymphocytes, cultured in
presence of IL-2, is reversed by IL-15. Restimulation of these
IL-15 treated, resting T cells with a suitable antigen (Ag)
presented by Ag presenting cells (APC) results in maximal cell
expansion, driven by a renewed production of helper cytokines. This
cell expansion is not attenuated by a massive cell death as a
consequence of AICD. In contrast to what is observed for cells
cultured in presence of IL-2, the above-mentioned activities of
IL-15 provide a method to achieve survival of immuno competent
CD4.sup.+ T lymphocytes, herewith strongly improving the secondary
restimulation of CD4.sup.+ T lymphocytes. In other words, the
formation of immunological CD4.sup.+ memory cells can be controlled
in a positive sense, by an increased IL-15 activity, or in a
negative sense, by a decreased IL-15 activity.
[0015] A first aspect of the present invention thus relates to the
use of IL-15 in the manufacturing of a pharmaceutical preparation
for the stimulation of the formation of memory cells. Such a
stimulation can be used in a number of applications. It can be
applied before, during or after vaccination to increase the
efficiency of the vaccination against infection or diseases of
which the pathological evolution derives, at least in part, from an
inadequate CD4.sup.+ T cell-dependent immune response. Thus,
diseases, where the existence of sufficient numbers of Ag-specific
CD4.sup.+ memory cells is necessary to control (re-emergent)
pathogens, are suitable indications for IL-15 treatment. Important
but non-limiting examples of such pathogenic conditions are
bacterial, parasitical or viral infections (e.g. HIV) and
cancer.
[0016] Other possible indications of this approach are individuals
showing hyporesponsiveness towards pathogens or vaccins, or
suffering from a chronic infection or from a generally weakened
immune condition. As we assume that the action of IL-15 becomes
even more important towards the end of an acute immune response,
promoting the subsequent quiescent period, therapeutic doses of
IL-15 should preferentially be administered when the immune
response is subsiding, in this way favouring the establishment and
long-term survival of CD4.sup.+ memory cells.
[0017] A second aspect of the present invention relates to those
cases where an unwanted or harmful CD4.sup.+ T cell-dependent
immune response is (co)-responsible for disease. As an example,
several reports demonstrated the involvement of autopathogenic
CD4.sup.+ T cells in autoimmune conditions. As a consequence it is
anticipated that blocking the activity of IL-15 will suppress the
long-term survival of autoreactive CD4.sup.+ effector T cell clones
as well as promote the regression of already formed autoreactive
CD4.sup.+ T cells, thus resulting in beneficial effects for
patients suffering from an auto-immune condition. Therapy aiming at
the inhibition of IL-15 activities can be accomplished by
administration of agents interfering with the binding of IL-15 to
its receptor such as antagonistic anti-IL-15 antibodies or
anti-IL-15R.alpha. antibodies or the Fab or F(ab').sub.2 fragments
of these Ab, soluble IL-15R.alpha., fusion proteins consisting of
soluble IL-15R.alpha. and Fc fragment, or peptides binding with
high affinity on the IL-15R.alpha. without inducing signalling. A
different approach consists of inhibiting IL-15 synthesis by
administration of IL-15 antisense oligonucleotides through direct
vaccination of patients with naked DNA, or by gene therapy
approaches.
[0018] A third embodiment of the invention further relates to a
pharmaceutical preparation promoting the formation of memory cells,
which preparation contains IL-15 or IL15 promoting compounds,
possibly in presence of a suitable excipient. A fourth embodiment
of the invention relates to a pharmaceutical preparation inhibiting
the formation of memory cells, which preparation contains IL-15
inhibiting and/or eliminating compounds, such as IL-15 antibodies
or compounds that interfere with the binding of IL-15 with its
receptor, such as soluble IL15R.alpha., possibly in presence of a
suitable excipient.
[0019] For the use of IL-15 according to the present invention,
IL-15 can be administered by bolus injection, continuous infusion,
sustained release from implants or other suitable technique.
Administration may be by intravenous injection, subcutaneous
injection, or parenteral or intraperitoneal infusion. IL-15
therapeutic agent will be administered in the form of a
pharmaceutical composition comprising purified polypeptide in
conjunction with physiologically acceptable carriers, excipients or
diluents. Such carriers will be nontoxic to patients at the dosages
and concentrations employed. Ordinarily, the preparation of such
compositions entails combining a mammalian IL-15 polypeptide or
derivative thereof with buffers, antioxidants such as ascorbic
acid, low molecular weight (less than about 10 residues)
polypeptides, proteins, amino acids, carbohydrates including
glucose, sucrose or dextrans, chelating agents such as EDTA,
glutathione and other stabilizers and excipients. Neutral buffered
saline or saline mixed with conspecific serum albumin are exemplary
appropriate diluents. Elevated levels of IL-15 can also be obtained
by adoptive transfer of cells ex vivo transfected with constructs
consisting of an IL-15 cDNA sequence driven by a potent promoter,
or by introduction into the target cells of an IL-15 cDNA sequence
after a suitable promoter.
[0020] The meaning of therapeutic in the present application is not
limited to the treatment of an existing disease or condition, but
comprises the use of IL-15 as support during vaccination and other
profylactive treatments, where the formation of immunological
memory cells is essential or helpful.
[0021] The present invention can be executed with isolated IL-15.
Suitable IL-15 sources are the culture medium of constitutively
IL-15 producing human cell lines such as the T-102. Alternatively,
recombinant IL-15 can be applied. WO 95/27722 gives the information
needed to prepare recombinant IL-15. Recombinant IL-15 is
commercial available as well. Alternatively, variants of IL-15 can
be used, as long as they have the activity needed to stimulate the
formation of memory cells. These variants are identified as "active
variants". Active variants further comprise IL-15 fragments
displaying sufficient IL-15 activity to be useful in the invention.
Moreover, the activity of IL-15 can be stimulated in an indirect
way by the addition of IL-15 inducing compounds, such as LPS for
induction of IL-15 production in monocytes, or by IL-15 inducing
methods, such as UV.beta. irradiation for keratinocytes.
[0022] For the second variant of the invention, isolated IL-15
inhibiting or eliminating compounds can be used. If these compounds
are (poly)peptides, they may be produced by recombinant DNA
techniques.
[0023] The present invention will be further elucidated with
reference to the example below, which is only intended by way of
explanation and does not imply any limitation whatever to the scope
of the invention.
LEGENDS TO THE FIGURES
[0024] FIG. 1: IL-15 is mainly a survival factor, while IL-2 is
mainly a proliferation-inducing factor.
[0025] T-HA cells were harvested on day 4 after antigenic
restimulation, and were incubated in 200 .mu.l containing the
indicated concentrations of IL-2 or IL-15 for 72 h.
[0026] (A) 1.times.10.sup.4 T-HA cells were cultured for 72 h with
increasing concentrations of IL-2 or IL-15. Cultures were pulsed
with .sup.3H-thymidine for the last 8 h.
[0027] (B) Viable cell numbers in micro-cultures seeded with
2.10.sup.4 T-HA cells were determined on day 3 of culture by
trypan-blue dye exclusion. Results shown are averages of 2
haemocytometer counts of 2 wells.
[0028] (C) % apoptotic cells in the source cultures was determined
by flow cytometric quantitation of cells, which had taken up the
exclusion dye PI.
[0029] FIG. 2: IL-15 induces a resting phenotype.
[0030] 2.times.10.sup.4 T-HA cells were cultured with IL-2 (10
ng/ml) or IL-15 (1 ng/ml) for 48-72 h. Cell cycle status, cell size
and expression of activation markers were analysed. (A) PI
fluorescence intensity as a measure of cellular DNA content, and
cell cycle distribution percentages. (B) forward light scatter as a
measure of cell size. (C) (D) CD25 and CD71 expression. Dotted
lines represent labeling with secondary Ab alone. (E) Rhodarmine123
incorporation indicative for mitochondrial membrane potential
values (MFI: mean fluorescence intensity).
[0031] FIG. 3: IL-15 or low-dose IL-2 treated T-HA cells show
increased proliferation in response to Ag/APC stimulation.
[0032] T-HA lymphocytes were harvested on day 12 after antigenic
restimulation and cultured for 48 h with the indicated
concentrations of IL-2 or IL-15 in 24-well plates. Next, pretreated
cells were harvested, cytokine was washed away and 1.times.10.sup.4
cells were stimulated with 2.times.10.sup.5 irradiated spleen cells
as APC and 200 ng/ml purified BHA as antigen. .sup.3H-thymidine was
added for the last 12 h of the 84 h culture period. Data are
expressed as cpm of .sup.3H-thymidine incorporated.
[0033] FIG. 4 : IL-15 or low-dose IL-2 treatment of T-HA cells
results in increased generation of effector cells during an Ag/APC
response.
[0034] T-HA lymphocytes were harvested on day 12 after antigenic
restimulation and were pretreated for 48h with IL-2 (7.7 ng/ml or
0.077 ng/ml) or IL-15 (1 ng/ml) followed by labelling with the
green fluorescent membrane marker PKH2-GL. 12 h later,
1.times.10.sup.4 labeled cells were stimulated with
2.times.10.sup.5 irradiated spleen cells as APC and 200 ng/ml
purified BHA as antigen. Cultures were harvested at the indicated
time points and dead (A) and viable (B) cell numbers of the stained
cell population were determined by flow cytometry and PI uptake,
using the unlabeled spleen cells as an internal standard. Countings
shown are averages of triplicate cultures.
[0035] FIG. 5: Culture of T-HA cells in the presence of IL-15
results both in optimal recovery of cells after primary antigenic
restimulation and in optimal proliferative responsiveness upon
secondary antigenic restimulation.
[0036] 1.times.10.sup.5 T-HA lymphocytes, pretreated for 48 h with
IL-2 (10 or 0.1 ng/ml) or IL-15 (1 ng/ml), were stimulated in 1 ml
with 2.times.10.sup.6 irradiated spleen cells and 200 ng/ml BHA. On
day 4 these cultures were supplemented with the same concentrations
of cytokine as during the pretreatment and incubated under these
conditions for 8 more days.
[0037] (A) Viable cells were counted by trypan blue dye exclusion
on day 12 after starting the antigenic restimulation described
above. Data are represented as a cell recovery index, i.e. the
factor by which the input cell number has multiplied on day 12.
[0038] (B) 1.times.10.sup.4 of the recovered cells were stimulated
a second time with Ag/APC and proliferation was determined by
.sup.3H-thymidine incorporation.
[0039] (C) The total proliferative response expected during the
second antigenic restimulation is represented as a reactivity
index, calculated as follows:
(cell recovery index described in FIG. 5(A)).times.(CPM measured on
day 15 (FIG. 5(B))
[0040] FIG. 6 : Culture of T-HA cells in the presence of IL-15
results in a strongly enhanced generation of immune effector
cells.
[0041] Starting from day-2, T-HA lymphocytes were pretreated for 48
h with the indicated concentrations of IL-2 or IL-15. On day 0,
1.times.10.sup.5 T-HA cells were stimulated in 1 ml with
2.times.10.sup.6 irradiated spleen cells and 200 ng/ml BHA. On day
4 these cultures were supplemented with the same concentrations of
cytokine as during the pretreatment and incubated under these
conditions for 8 more days. On day 12 after the first antigenic
stimulation, 1.times.10.sup.4 cells were restimulated with Ag/APC
and the number of generated effectors was determined on day 15 as
previously described. The total number of effector cells generated
on day 15 per cell stimulated on day 0 is represented as a cell
recovery index calculated as follows
(number of cells harvested from the cultures on day
12).times.(number of cells counted on day 15)
10.sup.5.times.10.sup.4
[0042] FIG. 7 IL-15 protects activated polyclonal CD4.sup.+ T cell
populations against growth factor withdrawal-induced PCD and
TCR-induced death.
[0043] Freshly isolated, unsorted spleen cells from C57Bl/6 mice
were polyclonally activated with soluble anti-CD3 mAb (1 .mu.g/ml).
On day 4, CD4.sup.+ T cells were isolated by magnetic cell sorting
and 7.5.times.10.sup.6 cells were further cultured for 10 days
without exogenously added cytokine or with addition of IL-15 (10
ng/ml) or IL-2 (10 ng/ml). On day 14 after the initial stimulation,
cultures were harvested and survival, sensitivity for TCR-induced
death and TCR-responsiveness were evaluated. (A) Viable CD4.sup.+ T
cell numbers were counted after addition of trypan blue. Survival
is presented as the percentage recovery of the input cell numbers.
3 independent countings were performed, SD<15%. (B)
Susceptibility for TCR-induced death was evaluated by restimulation
of 1.times.10.sup.4 viable IL-15- or IL-2-cultured cells, isolated
by density gradient centrifugation, with plate-bound anti-CD3 mAb
(10 .mu.g/ml) for 24 h and determination of percentages of
apoptotic CD4.sup.+ T cells by PI uptake. Results represent 3
pooled wells. (C) Secondary responsiveness of activated CD4.sup.+ T
cell populations to appropriate TCR-stimulation was measured by
restimulating 1.times.10.sup.4 pretreated T lymphocytes with 1
.mu.g/ml soluble anti-CD3 mAb and 2.times.10.sup.4
IFN-.gamma.-activated macrophages (Mf4/4), either in the absence or
presence of 1 ng/ml IL-15 or IL-2. Naive CD4.sup.+ T cells were
added as a control to assure that these stimulation conditions
could properly induce a proliferative response. Proliferation was
measured by addition of .sup.3H-thymidine for the last 12 h of the
84 h assay period. No proliferation could be detected in cultures
of T cells and Mf4/4 without soluble anti-CD3 Ab (cpm<500)
indicating that the-observed response was strictly dependent on TCR
triggering Results represent means of triplicate cultures.
Experiments on freshly isolated spleen cells were done 2 times with
similar results.
[0044] FIG. 8 In vivo administration of IL-15 during/after a
primary antigenic challenge augments proliferative reponsiveness of
primed LN cells to a secondary antigenic challenge.
[0045] Mice were immunized with HA and treated for 14 days with
IL-15 or PBS delivered by an ALZET mini-osmotic pump. On day 21
after the initial HA injection, mice were sacrificed and draining
lymph nodes prepared. 2.times.10.sup.5 LN cells were restimulated
in 96-well microtiter plates with 500 ng/ml HA or HEL without
exogenous cytokine. Results show the HA-specific proliferation,
i.e. absolute HA-induced proliferation minus HEL-induced
proliferation, of LN cell cultures from IL-15- (hatched) or
PBS-treated (dotted) mice, measured by addition of
.sup.3H-thymidine after 72 h (A) or 120 h (B) for an additional 12
h. Data shown in (C) also represent HA-specific proliferation at
120 h but here 1 ng/ml exogenous IL-15 was added during the culture
period. Each bar represents proliferation of LN cells derived from
one individual mouse, except for the naive mice (black) where LN of
three individuals were pooled.
[0046] FIG. 9 In vivo administration of IL-15 during/after a
primary antigenic challenge augments Ag-specific Ab titers elicited
by a secondary antigenic challenge.
[0047] Mice were immunized with HA and treated for 10 days with
IL-15 or PBS delivered by daily bolus injections. One day after the
last delivery, IL-15- or PBS-treated mice were rechallenged with
HA. Two weeks later, blood samples were taken, sera prepared and
anti-HA Ab detected with an indirect ELISA. The sera were serially
diluted in Maxisorp 96-well plates (Nunc, Roskilde, Denmark)
previously coated with HA by overnight incubation at 4.degree. C.
with 0,5 .mu.g/ml stock solution of the Ag. Bound Ab was detected
with goat anti-mouse IgG Ab (Sigma) using alkaline
phosphatase-conjugated rabbit anti-goat IgG as detecting Ab
(Sigma). Results are represented as O.D. values as a function of
serum dilution factor. Each curve represents serum of an individual
animal. Sera of naive mice were used as a control on background
signals.
EXAMPLE
1. MATERIALS AND METHODS
1.1. CD4.sup.+ T Cell Clone
[0048] The influenza A/H3 haemagglutinin (HA)-specific and
H-2.sup.b restricted CD4.sup.+ murine T cell clone T-HA was
developed in this laboratory by an initial immunisation of C57Bl/6
mice with 100 .mu.g/ml bromelain cleaved haemagglutinin (BHA) and
0.5 mg/ml adjuvant (Ribi, Immunochem Research Inc., Hamilton,
Mont., USA) and a second immunisation with 32 .mu.g/ml BHA three
weeks later.
[0049] 5 days after this boost immunisation lymph nodes were
isolated and 3.10.sup.7 cells were stimulated in vitro with 0.5
.mu.g/ml BHA in 25 cm.sup.2 culture flasks (Nunclon, Nunc,
Roskilde, Denmark). On day 4 10 U/ml mouse IL-2 (from PMA
stimulated EL4.IL-2 cells) was added to the cultures.
[0050] After 2 additional biweekly restimulations with 0.5 .mu.g/ml
BHA and APC, a pool of optimally HA-reactive T-lymphocytes was
obtained. These T-HA cells were maintained long term in vitro by
biweekly restimulation in 25 cm.sup.2 culture flasks with 10 ng/ml
BHA and 7.times.10.sup.7 syngeneic spleen cells (3000 rad gamma
irradiated). On day 2, 30 IU/ml rhIL-2 was added and T cells were
further cultured and expanded by medium renewal and IL-2 addition
every 4 days. C57Bl/6 mice (Broekman Instituut, Eindhoven,
Netherlands) were used as a source of spleen cells. T-HA cells were
cultured in 12.5, mM Hepes-buffered RPMI 1640 medium (Life
Technologies, Paisley, Scotland) supplemented with 10% FCS (Life
Science International), 2 mM Glutamax-I, penicillin/streptomycin, 1
mM sodium pyruvate (all from Life Technologies, Paisley, Scotland)
and 5.times.10.sup.-5 M 2-ME (BDH, Poole, England).
1.2. Cytokines
[0051] Recombinant human IL-2 (r-IL2) had a specific activity of
1.3.times.10.sup.7 IU/mg as determined in the CTLL-2 assay, hence 1
IU corresponds to 77 pg.
[0052] Recombinant human IL-15 (r-IL15) was purchased from
PeproTech (London, UK) and had a specific activity of
2.times.10.sup.6 U/mg according to the manufacturer.
[0053] Hereafter, `IL-2` and `IL-2`, as well as `r-IL-15` and
`IL-15` are are used interchangeably because it is not essential
for the invention to use a recombinant form.
1.3. IL-2 or IL-15 Pretreatment
[0054] T-HA cells were harvested from cultures by incubation in
non-enzymatic cell dissociation buffer (Sigma) and viable cells
were separated from remaining irradiated-spleen cells and dead
cells by centrifugation on a Histopaque-1077 (Sigma, Irvine, UK)
density gradient for 25 min at 2000 rpm. 2-5.times.10.sup.5 T-HA
cells were cultured for 48 h in 24-well flat-bottom tissue culture
plates (Falcon) in the presence of variable concentrations of IL-2
or IL-15.
1.4. Proliferation Assays
[0055] Induction of proliferation by IL-2, IL-15 or Ag/APC was
measured by incubating 1.times.10.sup.4 T-HA cells with serial
dilutions of IL-2, IL-15 or 200 ng/ml BHA and 2.times.10.sup.5
irradiated C57Bl/6 spleen cells as a source of APC in 96-well,
flat-bottom, microtiter plates (Falcon 3072, Becton Dickinson,
Franklin Lakes, N.J., USA) .sup.3H-thymidine (Amersham) was added
at 0.5 .mu.Ci/well for the last 8-12 h of the indicated incubation
period. Cells were harvested on glass fiber filters and
.sup.3H-thymidine incorporation was measured on a Topcount
betaplate counter (Packard). Results reported are means of
triplicate cultures.
1.5. Cell-labelling with PKH2-GL
[0056] T-HA cells were harvested and washed twice in medium devoid
of serum in polypropylene tubes. 1.times.10.sup.6-1.times.10.sup.7
cells were resuspended in 1 ml diluent A and stained with 2 .mu.M
PKH2-GL (Sigma, St. Louis, Mo., USA) following the instructions of
the manufacturer. Stained cells were washed twice with medium
supplemented with 18% fetal calf serum (FCS) and incubated
overnight in their culture medium to allow dissociation of excess
dye from the membrane.
1.6. Analysis of Viable and Dead Cell Populations
[0057] Viable cell numbers were determined by counting trypan-blue
dye excluding cells in a hemocytometer. Duplicate wells were always
counted twice and results shown are averages of these 4 independent
countings. Apoptosis was analysed by addition of 30 .mu.M propidium
iodide (PI) (ICN) to harvested cells and percentage of cells taking
up PI was measured with an EPICS 753 flow-cytometer (Coulter
Electronics, Luton, UK), equipped with an Argon ion laser emitting
at 488 nm, after gating out cell debris. PI fluorescence was
detected at 610-630 nm. Additionally, percentage of apoptotic cells
was also determined by forward scatter analysis (results not
shown). Data obtained by the latter method correlated well with the
PI dye exclusion data.
[0058] In mixed cultures of PKH2-GL stained T-HA cells and APC
splenocytes, numbers of viable and apoptotic T-HA cells were
obtained by flow-cytometric analysis of PI-negative and -positive
cells respectively that emitted green fluorescence (525 nm) from
the PKH2-GL stain.
1.7. Ant Dies and Reagents
[0059] For immunofluorescence, rat anti-mouse CD25 (clone PC 61)
and rat anti-mouse CD71 (clone R217 17.1.3, kindly provided by Dr.
G. Leclercq) were used as primary antibodies. Anti-CD25 and
anti-CD71 binding was detected with a FITC-conjugated goat anti-rat
IgG (Sera-Lab, Crawley Down, UK). The mitochondrial membrane
potential was measured by addition of 1 .mu.M Rhodamine123
(Molecular Probes Inc., Eugene, Oreg.) for 30 min to the cells and
subsequent flow-cytometric analysis of the fluorescence
intensity.
1.8. Cell Cycle Analysis
[0060] T-HA cells were harvested, washed once in cold PBS, and
lysed in Krishan's reagent (0.05 mg/ml PI, 0.02 mg/ml ribonuclease
A, 0.3% Nonidet P-40, 0.1% sodium citrate). Cell nuclei were
analysed for DNA content by flow-cytometry. The distribution of
cells along the distinct stages of the cell cycle was calculated
with MDADS Paral software (Coulter Electronics).
1.9. Experiments with Freshly Isolated Spleen Cells
[0061] 8.times.10.sup.8 spleen cells were prepared from spleens of
naive, 8 week old C57Bl/6 mice and were activated in 25 cm.sup.2
tissue culture flasks (Falcon, Becton Dickinson) with 1 .mu.g/ml
soluble anti-CD3 mAb (145-2C11). After 24 h, excess antibody was
removed and cells were further cultured for 72 h without addition
of exogenous cytokine. Following this stimulation period, cultures
were harvested and CD4.sup.+ T cells were isolated by
immunomagnetic cell sorting. A negative selection procedure, using
an Ab cocktail designed for the enrichment of murine CD4.sup.+ T
cells (StemSep, Stem Cell Technologies, Vancouver, Canada), was
followed according to the manufacturer's instructions.
7.5.times.10.sup.6 recovered cells were further cultured for 10
days and supplemented (every fourth day) with their respective
cytokines (no, 10 ng/ml IL-15 or 10 ng/ml IL-2). Viable cell
numbers were determined on day 14 based on trypan blue dye
exclusion. For restimulation 1 .mu.g/ml soluble anti-CD3 mAb and
the immortalized macrophage cell line Mf4/4 (freely available from
De Smedt, Universiteit Gent) were used. Prior to use, Mf4/4 cells
were activated for 24 h with 400 U/ml IFN-.gamma. to enhance
expression of costimulatory molecules. Then, they were treated for
90 min with 30 .mu.g/ml Mitomycin-C (Duchefa, Haarlem, The
Netherlands) in order to block their proliferation, thus avoiding
interference with proliferation-measurements from the restimulated
lymphocytes. Alternatively, for determination of susceptibility to
anti-CD3-induced death, freshly isolated, unsorted spleen cells
were activated for 72 h in 24-well plates with 1 .mu.g/ml soluble
anti-CD3 mAb (145-2C11) without exogenous cytokine and were
supplemented on day 3 with 10 ng/ml IL-15 or IL-2. After an
additional 8 day culture period, the cells were harvested and
restimulated with plate-bound anti-CD3 mAb (10 .mu.g/ml). Apoptotic
cell numbers were determined after 24 h by PI dye uptake. CD4/CD8
ratios were determined by labeling 1.times.10.sup..ident.cells with
0.5 .mu.g PE-conjugated rat anti-mouse CD4 mAb (Pharmingen, San
Diego, Calif.) and 0.5 .mu.g/ml FITC-labeled rat anti-mouse CD8 mAb
(clone 53-6.7, kindly provided by Dr. G. Leclercq, Ghent, Belgium)
and, after gating out dead cells and debris, analysis of stained
populations on a FACScalibur flow cytometer (Becton Dickinson).
Absolute numbers of CD4.sup.+ T cells in the respective cultures
were calculated from the percentages obtained and total viable cell
countings by trypan-blue dye exclusion.
2. RESULTS
2.1. IL-15 is a Survival Factor, Protecting CD4.sup.+ T Cells
Against Cell Death Following Growth Factor Withdrawal without
Inducing Proliferation
[0062] During the acute activation phase by antigen, CD4.sup.+
effector cells transiently produce high levels of IL-2, resulting
in autocrine growth and strong expansion of the lymphocytes.
However, following termination of the effector phase and hence, the
ending of cytokine production, the generated lymphocytes become
dependent on the exogenous supply of growth factors, mainly IL-2,
for their survival. We studied the survival of activated T-HA
cells, harvested four days after stimulation with Ag/APC, in the
presence of increasing concentrations of IL-2 or IL-15. After three
days of treatment with different concentrations of cytokines, the
uptake of .sup.3H-thymidine as a measure for cell division, the
absolute numbers of viable lymphocytes and the percentage apoptotic
cells in the various cultures were determined (FIG. 1).
[0063] In contrast to treatment with IL-2, treatment with IL-15 did
not result in cell division (FIG. 1A). These results are in
contrast with the results obtained by Kanegane & Tosato (1996),
who claimed that CD4.sup.+ cells proliferate upon addition of
IL-15. This discrepancy is probably due to the presence of low
amounts of IL-2 (besides IL-15) in their experiments. Unexpectedly,
notwithstanding the absence of growth factor activity by IL-1S,
IL-15 treated T-HA lymphocytes showed protection towards cell
death, normally occurring in absence of growth factor activity: the
cell numbers remained unchanged at an IL-15 concentration of 0.1
ng/ml or higher (FIG. 1B) and no increase of the percentage of
apoptotic cells was seen (FIG. 1C). This is in clear contrast with
the explicit decrease of the number of viable cells and the
corresponding increase of dead cells in absence of cytokine, a
reflection of the growth factor depletion-induced cell death as
well as with the cell growth-associated survival of IL-2 treated
T-HA lymphocytes (FIG. 1).
[0064] From these data we conclude that IL-15, but not IL-2, is
capable of inducing a survival signal in CD4.sup.30 T lymphocytes
without driving the cells into the proliferative cell cycle. In
this way, IL-15 establishes a resting phenotype in T-HA lymphocytes
that is linked, however, to survival. Moreover, under conditions of
low cytokine concentration, in the range of 0.1 ng/ml, the net
recovery of viable T-HA cells was considerably higher when cells
were cultured with IL-15 instead of IL-2. Based on the preservation
of viability in absence of active cell division upon IL-15
treatment, this example shows that the IL-15 polypeptide gives to
the antigen activated CD4.sup.+ T lymphocytes the property to
survive as a resting cell in absence of cytokine with inherent
growth activity. In this way, IL-15 acts as a factor, enabling the
survival of antigen-primed CD4.sup.+ T lymphocytes, generated by a
preceding antigenic activation, or resting cells.
2.2. IL-15 Induces a Resting Phenotype
[0065] It is thought that after conclusion of a primary immune
response a fraction of activated effector cells reverts to a
resting state and persists in the animal as a population of small
lymphocytes, ready for a "memory" response in case of re-emergence
of their antigen. It was wondered whether T-HA lymphocytes
surviving with IL-15 without cycling could be phenotyped as small,
resting lymphocytes. Therefore a number of features generally
recognized as parameters for lymphocyte quiescence were studied. It
was determined whether the observed growth-arrest took place in a
specific phase of the cell cycle. Cell cycle analysis by flow
cytometry revealed that IL-15-treated cells accumulated in
G.sub.0/G.sub.1 (FIG. 2), indicative of the induction by IL-15 of
an arrest in cell cycle entry. Thus, cycling cells treated with
IL-is are neither arrested immediately nor randomly, which in fact
would be apoptosis-inducing, but proceed with their cycle until
they reach G.sub.0/G.sub.1 and then exit cell cycle progression in
an orderly manner, i.e. without triggering programmed cell
death.
[0066] Additionally, cell size, expression of activation markers
and mitochondrial transmembrane potential (.DELTA..PSI..sub.m) as
an indicator of the metabolic state of the cells were evaluated.
IL-15-treated T-HA cells exhibited all the hallmarks of resting
cells: the cells were small, expressed low levels of the CD25
(IL-2R.alpha.) and CD71 (transferrin receptor) activation markers,
and had a low .DELTA..PSI..sub.m (FIG. 2). In contrast,
IL-2-cultured cells were large blastoid cells with high CD25 and
CD71 expression levels and a high oxidative metabolism as indicated
by the increased .DELTA..PSI..sub.m. Thus, the IL-15-induced arrest
in G.sub.0/G.sub.1 of T-HA cells is accompanied by acquisition of a
typical quiescent phenotype.
2.3. IL-15 Protects the CD4.sup.+ Helper T Cell Clone T-HA Against
AICD
[0067] AICD of mature T-lymphocytes is generally considered as a
key mechanism, restricting both the strength and duration of an
immune response (Critchfield et al., 1995). It has been shown that
clonal expansion and as a consequence, IL-2 is an important
regulator of susceptibility to AICD as T lymphocytes cultured in
the presence of IL-2 easily undergo apoptosis following
crosslinking of the T-lymphocyte receptor for antigen (TCR)
(Lenardo, 1991). We compared the influence of IL-2 and IL-15
treatment on responsiveness of T-HA cells towards stimulation by
Ag/APC. T-HA cells were treated for 48 h with IL-15 (1 ng/ml) or
IL-2 (100 IU/ml (7.7 ng/ml) or 1 IU/ml (0.077 ng/ml)) prior to
antigenic restimulation.
[0068] As was described above, cells differed functionally at the
moment of restimulation according to the cytokine added: IL-15-kept
the T-HA cells fully viable but non-proliferating, while high-dose
IL-2 (7.7 ng/ml) induced vigorous cell cycling and treatment with
low-dose IL-2 (0.077 ng/ml) resulted in extensive cell death. T-HA
cells pretreated with IL-15 proliferated dramatically stronger in
response to antigenic restimulation as compared to cells cultured
in the presence of 100 IU IL-2 (FIG. 3). The small fraction of T-HA
cells surviving incubation with 1 IU/ml IL-2, exhibited a
reactivity towards Ag/APC similar to cells pretreated with
IL-15.
[0069] In a next step we wanted to answer the question whether this
differential responsiveness was a reflection of a differential
sensitivity towards AICD or rather the consequence of differing
proliferative capacities of IL-2 versus IL-15 pretreated cells. For
this purpose, T-HA cells were labelled with a green fluorescent dye
prior to Ag/APC stimulation, allowing them to be discriminated from
APC present in these cultures during analysis of viable and dead
cell numbers by flow cytometry.
[0070] The results shown in FIG. 4A clearly demonstrate that the
defective immune responsiveness observed with T-HA cells pretreated
with 7.7 ng/ml IL-2 is the consequence of extensive cell death,
apparent 48 h after start of the activation with Ag/APC. This is in
agreement with the generally accepted view that IL-2 sensitizes T
cells for AICD. On the contrary, T-HA cells cultured in the
presence of IL-15 were efficiently protected against cell death
occurring during antigenic activation, resulting in the generation
of high numbers of effector cells (FIG. 4). Comparable results were
obtained with the small population of T-HA cells surviving a
low-dose 0.077 ng/ml IL-2 treatment. This result illustrates the
inhibitory activity of IL-15 on Ag/APC induced cell death (AICD)
and, as a result of this activity, the considerably increased
formation of effector T-lymphocytes after a renewed, secondary
stimulation with antigen of the IL-15 treated, resting CD4.sup.+ T
lymphocytes.
2.4. Protection IL-15 Against AICD and Against Growth Factor
Withdrawal-induced Apoptosis Strongly Potentiates Immune
Responsiveness upon Rechallenge with Antigen
[0071] In this example, the effect of the IL-15 treatment on the
strength of the secondary immune response of CD4.sup.+ T
lymphocytes is illustrated. T-HA lymphocytes were cultured in the
presence of IL-15, or in high- or low-dose IL-2 before and after
restimulation with Ag/APC.
[0072] As shown in FIG. 5A, the presence of IL-15 resulted in the
preservation over a long time of the highest number of
T-lymphocytes, generated in response to a preceeding antigenic
stimulation. Thus, starting from a fixed number of IL-15 treated
T-HA cells, the combination of optimal proliferation in response to
Ag/APC stimulation and optimal persistence of the generated
effector cells by addition of IL-15 after termination of the
response to antigen, resulted in a 31-fold increase of T cells
available for a second Ag/APC response. High-dose IL-2, sensitizing
towards AICD, or low-dose IL-2, insufficiently supporting growth
and viability, raised T cell numbers 27.3 and 6.7 fold respectively
(FIG. 5A).
[0073] T cells that survive with IL-15 remained optimally sensitive
towards subsequent activation by antigen (FIG. 5B). The combination
of a high yield of T lymphocytes after primary stimulation with
antigen, optimal persistence and high reactivity upon renewed
stimulation with antigen of IL-15 treated CD4.sup.+ T lymphocytes
resulted in a maximal reactivity index of the thus treated T-HA
cells (FIG. 5C). Furthermore, the cumulative effect of this
reactivity leads to a 105 fold yield in effector cells as shown in
FIG. 6 T-HA lymphocytes that survive due to high or low doses of
IL-2 generated a considerably weaker reactivity index (FIG. 5C)
and, as a consequence, merely 20 and 8 fold increases in cell
numbers, respectively, 3 days after secondary stimulation (FIG.
6).
[0074] Obviously, IL-15 both enhances the availability as well as
the responsiveness of cells resulting in maximal secondary
responses, features that cannot be achieved by any dose of IL-2.
These results clearly demonstrate that IL-15, but not IL-2,
possesses the properties necessary for generating an efficient
immune memory, i.e. providing a strong survival signal allowing the
persistence of immune effectors in a quiescent state and priming
these memory cells for optimal response in case of renewed Ag
stimulation by inducing insensitivity towards AICD. Taken together
our study defines a unique novel pro-memory function for IL-15,
clearly distinct from the physiological activities exerted by IL-2
or other cytokines with growth factor activity.
2.5. Induction of Quiescence and Protection Against Apoptosis by
IL-15 also Occurs with ex vivo Isolated T Cells
[0075] Fresh, unsorted spleen cells from naive C57Bl/6 mice were
isolated and polyclonally stimulated in vitro. The stimulus
consisted of soluble anti-CD3 mAb (1 .mu.g/ml) which, in the
presence of costimulation by spleen APC, polyclonally activates
naive T cells (Tamura, T., and Nariuchi, H., J. Immunol. 148:2370,
1992). After 24 h, remaining anti-CD3 mAb was removed and the
activated cells were further cultured in the absence of exogenous
cytokine. To confirm that activation occurred, anti-CD3
mAb-activated and unstimulated cells were pulsed with
.sup.3H-thymidine. Soluble anti-CD3 mAb induced a strong
proliferative response: 25,304 cpm as opposed to 2,581 cpm for
unstimulated cells. On day 4, CD4.sup.+ cells were isolated by
immunomagnetic cell sorting and further cultured without cytokine
or in presence of IL-15 (1 ng/ml).
[0076] After 10 days of culture in the absence of exogenous
cytokine, viable cell numbers had dropped to 15% of the cell input,
while IL-15 maintained cell numbers at approximately 60% of cell
input (FIG. 7A). Cells surviving with IL-15 appeared as small
resting lymphocytes and did not reveal DNA synthesis (59 cpm with
10 ng/ml IL-15)-whereas proliferation could be induced with IL-2
(4,184 cpm with 10 ng/ml). Hence, also for freshly isolated and
TCR-activated CD4.sup.+ T cells, IL-15 acts as a survival factor
and induces quiescence.
[0077] Next, the resistance to TCR-induced cell death was
investigated, triggered by immobilized anti-CD3 mAb, in these
polyclonally activated T cell cultures. The CD4.sup.+ T cell
population maintained throughout with IL-15 was largely resistant,
whereas cells cultured with IL-2 showed extensive cell death (FIG.
7B). Finally, CD4.sup.+ T cells residing in an IL-15-induced,
resting state proliferated in response to renewed stimulation with
soluble anti-CD3 and APC, while cells maintained with IL-2 did not
(FIG. 7C). Also here, addition of IL-15 to the IL-15 pretreated
cultures further increased the proliferative response, hence
confirming the growth-promoting activity of IL-15 in the presence
of TCR aggregation. These experiments demonstrate that the
characteristics induced by IL-15 in the clonal CD4.sup.+ T cell
T-HA namely long-term survival as a resting population, resistance
to apoptosis and increased responsiveness to TCR restimulation, are
also acquired by freshly isolated CD4.sup.+ T cells treated with
IL-15.
2.6. In vivo Evaluation of the Capacity of IL-15 to Enhance Memory
Responses
[0078] In vitro, the activities of IL-15 described here show that
this cytokine is a factor promoting the generation and persistence
of memory CD4.sup.+ T cells, thus enhancing secondary/memory immune
responses to an antigen. It was investigated whether administration
of IL-15 in vivo during and/or after a primary immune response
against an antigen could enhance the secondary/memory response
against this antigen.
[0079] Therefore IL-15 was delivered by a slow-release mini-osmotic
pump or by bolus injections to mice immunized with haemaglutinnin
(HA) and responsiveness to a renewed challenge with this antigen
was evaluated. In the first experiment the initial immunisation
consisted of two injections with HA: 5 .mu.g injected
intraperitoneal (IP) on day 0 and another 5 .mu.g injected
subcutaneous (SC) in the right flank on day 3. On day 4, a bolus
injection of 1 .mu.g hIL-15 in a volume of 100 .mu.l PBS was given
(or 100 .mu.l PBS as a control). Two hours after this initial
delivery, an ALZET mini-osmotic pump (model 2002, Alza Corp., Palo
Alto, Calif.), filled with 10 .mu.g hIL-15 in 235 (.+-.5,7) .mu.l
PBS or with PBS without cytokine, was implanted SC on the back of
the mouse. The opening of the pump was oriented towards the place
where the SC injection of HA was given. Filling of the pump was
performed according to the manufacturer's instructions. The pumps
released IL-15 in the animal at a flow rate of 0,52 (.+-.0,03)
.mu.l/hr during 14 days.
[0080] On day 21 after the first HA injection, mice were sacrificed
and the draining lymph nodes (LN) of IL-15 or PBS-treated mice were
isolated as well as LN from naive mice, LN cells were prepared and
restimulated in vitro with 500 ng/ml HA or henn egg lysozyme (HEL)
as an irrelevant antigen. Proliferation induced by these antigens
was measured by .sup.3H-thymidine incorporation. Results shown in
FIG. 8 represent the specific proliferation against HA, i.e. cpm
obtained with HEL is subtracted from the absolute cpm value
obtained with HA, from LN cells of two mice. Proliferation after 72
h (FIG. 8A) as well as 120 h (FIG. 8B) of LN cells from
IL-15-treated mice was markedly increased, as compared to
PBS-treated or naive mice. Also, addition of exogenous IL-15 during
the restimulation period could prolong Ag-specific proliferative
responses (FIG. 8C). Again, LN cells from IL-15-treated mice were
most responsive to this exogenous IL-15.
[0081] The data demonstrate that slow-release delivery of IL-15
during and/or after a primary immune response results in an
augmented proliferative responsiveness of cells from the draining
LN against the immunizing antigen, indicative for the presence of a
higher frequency of memory T cells in the immunized animal.
[0082] In a second experiment, mice were immunized with a single SC
bolus injection of 2,5 .mu.g HA (day 0) at the right flank and were
treated, starting on day 4 after this initial immunization, with
daily bolus injections of IL-15 for 10 consecutive days. The
following doses were administered: day 4, 5 .mu.g IL-15; day 5-7, 4
.mu.g; day 8-10, 3 .mu.g and day 11-13, 2 .mu.g. The cytokine was
injected SC at the same site as the Ag in a volume of 100 .mu.l
PBS. Control mice were injected with PBS without cytokine. On day
14, a new challenge of 2,5 .mu.g HA was given to the IL-15- and
PBS-treated mice, injected SC at the same site as the initial
immunisation. Two weeks later, blood was collected by retro-orbital
bleeding and serum was prepared immediately by incubating the blood
samples at 37.degree. C. for 20 minutes. Titers of antibody (Ab)
directed against HA were determined by indirect ELISA.
[0083] FIG. 9 demonstrates that the anti-HA Ab titer of the two
IL-15-treated mice was enhanced, as compared to the four PBS
treated mice. Titers in naive mice are shown as an additional
control on ELISA background levels. The augmented anti-HA Ab titers
are indicative that an enhanced immune response against the second
HA challenge occurred in mice treated with IL-15 after the primary
response. Taken together, it is shown that administration of IL-15
in vivo during and/or after an ongoing immune response enhances the
secondary/memory response elicited by a renewed contact with the Ag
involved.
1 LIST OF ABBREVIATIONS aa: amino acid Ab: antibody Ag: antigen
AICD: activation-induced cell death APC: antigen-presenting cell
BHA: bromelain cleaved haemagglutinin IL: interleukin LAK:
lymphokine activated killer cell 2-ME: .beta.-mercaptoethanol PBT:
peripheral blood T lymphocytes PHA: phytohaemagglutinin TCR: T cell
receptor Th: T helper lymphocyte cpm: count per minute
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