U.S. patent application number 13/059745 was filed with the patent office on 2011-08-18 for methods and compositions for determining a graft tolerant phenotype in a subject.
Invention is credited to Li Li, Minnie M. Sarwal.
Application Number | 20110201519 13/059745 |
Document ID | / |
Family ID | 41707384 |
Filed Date | 2011-08-18 |
United States Patent
Application |
20110201519 |
Kind Code |
A1 |
Sarwal; Minnie M. ; et
al. |
August 18, 2011 |
Methods and Compositions for Determining a Graft Tolerant Phenotype
in a Subject
Abstract
Methods are provided for determining whether a subject has a
graft tolerant phenotype. In practicing the subject methods, the
expression of at least 5 genes in a sample from the subject, e.g.,
a blood sample, is assayed to obtain a gene expression result for
the at least 5 genes. The obtained gene expression result for the
at least 5 genes is then employed to determine whether the subject
has a graft tolerant phenotype. Also provided are compositions,
systems and kits that find use in practicing the subject methods.
The methods and compositions find use in a variety of applications,
including the determination of an immunosuppressive therapy
regimen.
Inventors: |
Sarwal; Minnie M.; (Portola
Valley, CA) ; Li; Li; (Fremont, CA) |
Family ID: |
41707384 |
Appl. No.: |
13/059745 |
Filed: |
August 17, 2009 |
PCT Filed: |
August 17, 2009 |
PCT NO: |
PCT/US09/04710 |
371 Date: |
May 9, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61089805 |
Aug 18, 2008 |
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Current U.S.
Class: |
506/9 ; 435/6.1;
435/6.12; 435/6.16; 435/6.17; 435/6.18; 435/7.1; 436/501 |
Current CPC
Class: |
C12Q 2600/124 20130101;
C12Q 1/6883 20130101; C12Q 1/6876 20130101; C12Q 2600/158 20130101;
C12Q 2600/118 20130101 |
Class at
Publication: |
506/9 ; 436/501;
435/6.18; 435/6.16; 435/6.17; 435/6.1; 435/6.12; 435/7.1 |
International
Class: |
C40B 30/04 20060101
C40B030/04; G01N 33/566 20060101 G01N033/566; C12Q 1/68 20060101
C12Q001/68; G01N 33/573 20060101 G01N033/573 |
Goverment Interests
GOVERNMENT RIGHTS
[0001] This invention was made with Government support under
contract AI061739 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1. A method of determining whether a subject who has received an
allograft has a graft tolerant phenotype, said method comprising:
(a) evaluating the level of expression of at least 5 genes in a
sample from said subject to obtain a gene expression result,
wherein said at least 5 genes are selected from either Table 2 or
Table 4; and (b) determining whether said subject has a graft
tolerant phenotype based on said gene expression result.
2. The method according to claim 1, wherein said allograft is a
renal allograft.
3. The method according to claim 1, wherein said sample is a blood
sample.
4. The method according to claim 1, wherein said evaluating step
comprises assaying said sample for an expression product of said at
least 5 genes.
5. The method according to claim 4, wherein said expression product
is selected from one both of: a nucleic acid transcript and a
protein.
6. The method of claim 5, wherein said expression product is a
nucleic acid transcript and said evaluating step comprises
performing one or more of the following: a RT-PCR assay, a
microarray assay, and a Northern blot.
7. The method according to claim 1, wherein the expression level of
at least 10 genes from either Table 2 or Table 4 is evaluated.
8. The method according to claim 1, wherein the expression level of
all of the genes from either Table 2 or Table 4 is evaluated.
9. The method according to claim 1, wherein said determining step
comprises comparing said gene expression result to a reference gene
expression profile.
10. The method according to claim 9, wherein said reference gene
expression profile is selected from: a graft tolerant phenotype
gene expression profile and a graft intolerant phenotype gene
expression profile.
11. A method of managing immunosuppressive therapy treatment in a
subject having an allograft, said method comprising: (a)
determining whether said subject has a graft tolerant phenotype
according to claim 1; and (b) managing future immunosuppressive
therapy treatment in said subject based on said determining step
(a).
12. A system for determining whether a subject who has received an
allograft has a graft tolerant phenotype, said system comprising:
(a) a gene expression evaluation element for evaluating the level
of expression of at least 5 genes in a sample from said subject to
obtain a gene expression result, wherein said at least 5 genes are
selected from either Table 2 or Table 4; and (b) a graft tolerance
determination element for employing said gene expression result to
determining whether said subject has a graft tolerant
phenotype.
13. The system according to claim 12, wherein said gene expression
evaluation element comprises at least one reagent for assaying a
sample for an expression product of said at least 5 genes.
14. The system according to claim 12, wherein said expression
product of said at least one gene is selected from: a nucleic acid
transcript and a protein.
15. The system according to claim 12, wherein the expression level
all of the genes in either Table 2 or Table 4 is assessed.
16. A kit for determining whether a subject who has received an
allograft has a graft tolerant phenotype, said kit comprising: (a)
a gene expression evaluation element for evaluating the level of
expression of at least 5 genes in a sample from said subject to
obtain a gene expression result, wherein said at least 5 genes are
selected from either Table 2 or Table 4; and (b) a phenotype
determination element for employing said gene expression result to
determine whether said subject has a graft tolerant phenotype.
17. The kit according to claim 16, wherein said gene expression
evaluation element comprises at least one reagent for evaluating a
sample for an expression product of said gene.
18. The kit according to claim 17, wherein said expression product
of said at least one gene is selected from: a nucleic acid
transcript and a protein.
19. The kit according to claim 17, wherein the expression level all
of the genes in either Table 2 or Table 4 is assessed.
20. A computer program product for determining whether a subject
who has undergone a renal allograft has a graft tolerant phenotype,
wherein said computer program product, when loaded onto a computer,
is configured to employ a gene expression result from a sample
derived from said subject to determining whether a subject who has
undergone a renal allograft has a graft tolerant phenotype, wherein
said gene expression result comprises expression data for at least
one gene from Table 2 or Table 4.
Description
INTRODUCTION
Background of the Invention
[0002] Transplantation of a graft organ or tissue from a donor to a
host patient is a feature of certain medical procedures and
treatment protocols. Despite efforts to avoid graft rejection
through host-donor tissue type matching, in transplantation
procedures where a donor organ is introduced into a host,
immunosuppressive therapy is generally required to the maintain
viability of the donor organ in the host.
[0003] A variety of immunosuppressive agents have been employed in
transplantation procedures, including azathioprine, methotrexate,
cyclophosphamide, FK-506, rapamycin and corticosteroids. Agents
finding increased use in immunosuppressive therapy due to their
preferential effect on T-cell mediated reactions are the
cyclosporins.
[0004] Following transplantation, administration of the
immunosuppressive agent must be continued indefinitely since the
benefits of immunosuppressive therapy are reversible and graft
rejection may occur once administration of the immunosuppressive
agent is discontinued. While use of immunosuppressive agents, such
as Cyclosporin A, has been reported to prolong the survival of
allogeneic transplants involving skin, heart, kidney, pancreas,
bone marrow, small intestine and lung, use of such agents is not
without undesirable side effects. Examples of undesirable side
effects include increased risk of development of neoplastic disease
conditions, e.g., skin cancer, lymphoma, etc.
[0005] While most recipients who discontinue their
immunosuppressive treatment following a graft go on to suffer
rejection, not all subjects suffer graft rejection. In a few cases,
individuals tolerate their graft without immunosuppression,
suggesting that immune non-responsiveness can be achieved in
clinical practice. The mechanisms of this process are not well
understood, but may involve a combination of clonal deletion,
clonal anergy and the generation of active regulatory T cells.
[0006] Because of the undesirable sides effects and risks of long
term immunosuppressive therapy, it would be desirable to be able
identify those individuals who are tolerant to their graft, i.e.,
graft tolerant, so that immunosuppression could be reduced or even
discontinued in those individuals. Of particular interest would be
the development of a way to identify graft tolerant individuals
without first discontinuing immunosuppressive therapy, thereby
avoiding the risk of graft rejection and damage to the graft
associated therewith. The present invention meets this need.
RELEVANT LITERATURE
[0007] Publications of interest include: U.S. Patent Publication
No. 2003/0104371; published PCT application No. WO 2005/070086;
Brouard et al., PNAS 2007, vol. 104, pp. 15448-15453.
SUMMARY OF THE INVENTION
[0008] Methods are provided for determining whether a subject has a
graft tolerant phenotype. In practicing the subject methods, the
expression of at least one gene in a sample from the subject, e.g.,
a blood sample, is assayed to obtain an expression evaluation for
the at least one gene. The obtained expression evaluation is then
employed to determine whether the subject has a graft tolerant
phenotype. Also provided are compositions, systems and kits that
find use in practicing the subject methods. The subject methods and
compositions find use in a variety of applications, including the
determination of an immunosuppressive therapy regimen.
[0009] Aspects of the present invention include methods of
determining whether a subject who has received an allograft has a
graft tolerant phenotype, the method including: (a) evaluating the
level of expression of at least 5 genes in a sample from the
subject to obtain a gene expression result, wherein the at least 5
genes are selected from either Table 2 or Table 4; and (b)
determining whether the subject has a graft tolerant phenotype
based on the gene expression result. In certain embodiments, the
allograft is a renal allograft. In certain embodiments, the sample
is a blood sample, e.g., a peripheral blood sample. In certain
embodiments, the evaluating step comprises assaying the sample for
an expression product of the at least 5 genes. In certain
embodiments, the expression product is selected from one both of: a
nucleic acid transcript and a protein. In certain embodiments, the
expression product is a nucleic acid transcript and the evaluating
step comprises performing one or more of the following: a RT-PCR
assay, a microarray assay, and a Northern blot. In certain of these
embodiments, the microarray is a genomic array. In certain
embodiments, the expression product is a protein and the evaluating
step comprises employing a proteomic array. In certain embodiments,
and the expression level of at least 10 genes from either Table 2
or Table 4 is evaluated. In certain embodiments, the expression
level of all of the genes from either Table 2 or Table 4 is
evaluated. In certain embodiments, the determining step comprises
comparing the gene expression result to a reference gene expression
profile. In certain embodiments, the reference gene expression
profile is selected from: a graft tolerant phenotype gene
expression profile and a graft intolerant phenotype gene expression
profile.
[0010] Aspects of the present invention include methods of managing
immunosuppressive therapy treatment in a subject having an
allograft, the method comprising: (a) determining whether the
subject has a graft tolerant phenotype as described above; and (b)
managing future immunosuppressive therapy treatment in the subject
based on the determining step (a). In certain embodiments, the
method comprises at least reducing immunosuppression in the subject
if said subject is found to have a graft tolerant phenotype. In
certain embodiments, the method comprises discontinuing
immunosuppression in the subject if the subject is found to have a
graft tolerant phenotype.
[0011] Aspects of the present invention include systems for
determining whether a subject who has received an allograft has a
graft tolerant phenotype, the system comprising: (a) a gene
expression evaluation element for evaluating the level of
expression of at least 5 genes in a sample from the subject to
obtain a gene expression result, wherein the at least 5 genes are
selected from either Table 2 or Table 4; and (b) a graft tolerance
determination element for employing the gene expression result to
determining whether the subject has a graft tolerant phenotype. The
output of the subject systems is in a user-readable format. Such
formats include, but are not limited to: a file stored in a memory
that is accessible to a user, displayed to a user on graphical user
interface (GUI), sent to a user via the internet, e.g., on a web
page or as an email, etc. As such, in certain embodiments, systems
of the invention include a communications module having input and
output managers that regulate communication between the system and
a user. In certain embodiments, the gene expression evaluation
element comprises at least one reagent for assaying a sample for an
expression product of the at least 5 genes. In certain embodiments,
the expression product of the at least one gene is selected from: a
nucleic acid transcript and a protein. In certain embodiments, the
expression level all of the genes in either Table 2 or Table 4 is
assessed.
[0012] Aspects of the present invention include kits for
determining whether a subject who has received an allograft has a
graft tolerant phenotype, the kit comprising: (a) a gene expression
evaluation element for evaluating the level of expression of at
least 5 genes in a sample from the subject to obtain a gene
expression result, wherein the at least 5 genes are selected from
either Table 2 or Table 4; and (b) a phenotype determination
element for employing the gene expression result to determine
whether the subject has a graft tolerant phenotype. In certain
embodiments, the kite further includes instructions for using the
gene expression evaluation and phenotype determination elements to
determine whether a subject has a graft tolerant phenotype. In
certain embodiments, the gene expression evaluation element
comprises at least one reagent for evaluating a sample for an
expression product of the gene. In certain embodiments, the
expression product of the at least one gene is selected from: a
nucleic acid transcript and a protein. In certain embodiments the
gene expression evaluation element is selected form: a microarray
and a collection of gene specific primers, where in certain
embodiments the kits include both of these components. In certain
embodiments, the expression level all of the genes in one or both
of Table 2 or Table 4 is assessed. In certain embodiments, the
phenotype determination element comprises a reference expression
value for the at least one gene. In certain embodiments, the
phenotype determination element comprises a reference expression
profile that includes a reference expression value for at least one
additional gene. In certain embodiments, the reference expression
profile is a graft tolerant phenotype expression profile. In
certain embodiments, the reference expression profile is a graft
intolerant phenotype expression profile.
[0013] Aspects of the present invention include computer program
products for determining whether a subject who has undergone a
renal allograft has a graft tolerant phenotype, wherein the
computer program product, when loaded onto a computer, is
configured to employ a gene expression result from a sample derived
from the subject to determining whether a subject who has undergone
a renal allograft has a graft tolerant phenotype, wherein the gene
expression result comprises expression data for at least one gene
from Table 2 or Table 4.
[0014] Also provided are reference expression profiles for a
phenotype that is one of: (a) graft tolerant; or (b) graft
intolerant; wherein the expression profile is recorded on a
computer readable medium that is accessible by a user, e.g., in a
user readable format. In certain embodiments, the expression
profile includes at least 5 genes from either one or both of Tables
2 or 4. In certain embodiments, the expression profile is a profile
for a phenotype that is allograft tolerant. In certain embodiments,
the expression profile is a profile for a phenotype that is graft
intolerant.
[0015] Also provided by the invention is a collection of reagents
for evaluating gene expression, where the collection includes:
reagents specific for at least 5 of the genes in either one or both
of Tables 2 or 4. In certain embodiments, the reagents are gene
specific primers. In certain embodiments, the collection comprises
at least 10 gene specific primers. Also provided are arrays of
probe nucleic acids immobilized on a solid support, where the
arrays include: a plurality of probe nucleic acid compositions,
wherein each probe nucleic acid composition is specific for a gene
whose expression profile is indicative of a graft tolerance, e.g.,
allograft tolerance, wherein at least two of the probe nucleic acid
compositions correspond to genes listed in one or both of Tables 2
and 4.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1A shows LC 49 gene foot prints prediction on a set of
LC samples (see Brouard et al., PNAS 2007, vol. 104, pp.
15448-15453). FIG. 1B shows prediction using 22 gene set of the
present invention to re-classify the same LC samples. All
re-analyzed samples in FIG. 1B were predicted as the same as in
FIG. 1A except for MIS6 and MIS8, which have the trend of the TOL
prediction.
[0017] FIG. 2 provides sample predictions using the top 24 genes
selected from the 99 genes in Table 1. In FIG. 2, the left panel
shows the training set and the right panel shows the test set. All
spontaneous samples are 100% predicted correctly using this 24 gene
set. The LK2 induced patient (TOL_LK2) was predicted as SP TOL,
which has been confirmed by clinical observation. The patient has
been off drug for 2 years.
[0018] FIG. 3 shows the TGF-.beta. signaling network for the top 24
genes from the 99 gene set in Table 1 identified in the analysis
using the Agilent array platform.
[0019] FIG. 4 shows B, T, and NK cell signaling regulation for the
top 24 genes from the 99 gene set in Table 1 identified in the
analysis using the Agilent array platform.
[0020] FIG. 5 shows 3-class prediction of HD vs. TOL vs. CAN
samples based on the 28 gene set of Table 2.
[0021] FIG. 6 shows the gene expression trends between HD vs. SP
vs. IN of the genes in Table 4. Significantly down-regulated genes
are shown in the left panel and up regulated genes are shown in the
right panel.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0022] Methods are provided for determining whether a subject has a
graft tolerant phenotype. In practicing the subject methods, the
expression of at least one gene in a sample from the subject, e.g.,
a blood sample, is assayed to obtain an expression evaluation for
the at least one gene. The obtained expression evaluation is then
employed to determine whether the subject has a graft tolerant
phenotype. Also provided are compositions, systems and kits that
find use in practicing the subject methods. The methods and
compositions find use in a variety of applications, including the
determination of an immunosuppressive therapy regimen.
[0023] Before the present invention is described in greater detail,
it is to be understood that this invention is not limited to
particular embodiments described, as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present invention
will be limited only by the appended claims.
[0024] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0025] Certain ranges are presented herein with numerical values
being preceded by the term "about." The term "about" is used herein
to provide literal support for the exact number that it precedes,
as well as a number that is near to or approximately the number
that the term precedes. In determining whether a number is near to
or approximately a specifically recited number, the near or
approximating unrecited number may be a number which, in the
context in which it is presented, provides the substantial
equivalent of the specifically recited number.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, representative illustrative methods and materials are
now described.
[0026] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed. It is noted that, as used herein and in
the appended claims, the singular forms "a", "an", and "the"
include plural referents unless the context clearly dictates
otherwise. It is further noted that the claims may be drafted to
exclude any optional element. As such, this statement is intended
to serve as antecedent basis for use of such exclusive terminology
as "solely," "only" and the like in connection with the recitation
of claim elements, or use of a "negative" limitation.
[0027] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0028] As summarized above, the subject invention is directed to
methods of determining whether a subject has a graft tolerant
phenotype, as well as reagents and kits for use in practicing the
subject methods. In further describing the invention, the subject
methods are described first, followed by a review of the reagents
and kits for use in practicing the subject methods.
Methods of Determining Whether a Subject has a Graft Tolerant
Phenotype
[0029] The subject invention provides methods of determining
whether a patient or subject has a graft tolerant phenotype. By
graft tolerant phenotype is meant that the subject does not reject
a graft organ, tissue or cell(s) that has been introduced into/onto
the subject. In other words, the subject tolerates or maintains the
organ, tissue or cell(s) that has been transplanted to it. As in
known in the transplantation field, the graft organ, tissue or
cell(s) may be allogeneic or xenogeneic, such that the grafts may
be allografts or xenografts. A feature of the graft tolerant
phenotype detected or identified by the subject methods is that it
is a phenotype which occurs without immunosuppressive therapy,
i.e., it is present in a host that is not undergoing
immunosuppressive therapy such that immunosuppressive agents are
not being administered to the host.
[0030] In practicing the subject methods, a subject or patient
sample, e.g., cells or collections thereof, e.g., tissues, is
assayed to determine whether the host from which the assayed sample
was obtained is graft tolerant, i.e., has a graft tolerant
phenotype. Accordingly, the first step of the subject methods is to
obtain a suitable sample from the subject or patient of interest,
i.e., a patient on immunosuppressive therapy and having at least
one graft, e.g., allograft. The sample is derived from any initial
suitable source, where sample sources of interest include, but are
not limited to, many different physiological sources, e.g., CSF,
urine, saliva, tears, tissue derived samples, e.g., homogenates,
and blood or derivatives thereof.
[0031] In certain embodiments, a suitable initial source for the
patient sample is blood. As such, the sample employed in the
subject assays of these embodiments is generally a blood-derived
sample. The blood derived sample may be derived from whole blood or
a fraction thereof, e.g., serum, plasma, etc., where in many
embodiments the sample is derived from blood cells harvested from
whole blood. Of particular interest as a sample source are
peripheral blood lymphocytes (PBL). Any convenient protocol for
obtaining such samples may be employed, where suitable protocols
are well known in the art.
[0032] In practicing the subject methods, the sample is assayed to
obtain an expression evaluation, e.g., expression profile, for one
or more genes, where the term expression profile is used broadly to
include a genomic expression profile, e.g., an expression profile
of nucleic acid transcripts, e.g., mRNAs, of the one or more genes
of interest, or a proteomic expression profile, e.g., an expression
profile of one or more different proteins, where the
proteins/polypeptides are expression products of the one or more
genes of interest. As such, in certain embodiments the expression
of only one gene is evaluated. In yet other embodiments, the
expression of two or more, e.g., about 5 or more, about 10 or more,
about 15 or more, about 25 or more, about 50 or more, about 100 or
more, about 200 or more, etc., genes is evaluated. Accordingly, in
the subject methods, the expression of at least one gene in a
sample is evaluated. In certain embodiments, the evaulation that is
made may be viewed as an evaulation of the transcriptosome, as that
term is employed in the art. See e.g., Gomes et al., Blood (2001
Jul. 1) 98(1):93-9.
[0033] In generating the expression profile, in many embodiments a
sample is assayed to generate an expression profile that includes
expression data for at least one gene/protein, usually a plurality
of genes/proteins, where by plurality is meant at least two
different genes/proteins, and often at least about 5, typically at
least about 10 and more usually at least about 20 different
genes/proteins or more, such as 50 or more, 100 or more, etc.
[0034] In the broadest sense, the expression evaluation may be
qualitative or quantitative. As such, where detection is
qualitative, the methods provide a reading or evaluation, e.g.,
assessment, of whether or not the target analyte, e.g., nucleic
acid or expression product, is present in the sample being assayed.
In yet other embodiments, the methods provide a quantitative
detection of whether the target analyte is present in the sample
being assayed, i.e., an evaluation or assessment of the actual
amount or relative abundance of the target analyte, e.g., nucleic
acid in the sample being assayed. In such embodiments, the
quantitative detection may be absolute or, if the method is a
method of detecting two or more different analytes, e.g., target
nucleic acids, in a sample, relative. As such, the term
"quantifying" when used in the context of quantifying a target
analyte, e.g., nucleic acid(s), in a sample can refer to absolute
or to relative quantification. Absolute quantification may be
accomplished by inclusion of known concentration(s) of one or more
control analytes and referencing the detected level of the target
analyte with the known control analytes (e.g., through generation
of a standard curve). Alternatively, relative quantification can be
accomplished by comparison of detected levels or amounts between
two or more different target analytes to provide a relative
quantification of each of the two or more different analytes, e.g.,
relative to each other.
[0035] Genes/proteins of interest are genes/proteins that are
differentially expressed or present at different levels in graft
tolerant versus graft intolerant individuals. Representative
genes/proteins of interest in certain embodiments include, but are
not limited to, the genes/proteins provided in Tables 1, 2, 3 and
4. Note that for all of the gene tables shown herein, detailed
information for each specific gene, including nucleotide sequence
information, can be retrieved through the NCBI Entrez nucleotide
database at located at the website:
http(colon)//www(dot)ncbi.nlm.nih(dot)gov/ by selecting "Gene" as
the database and entering the Entrez Gene ID number listed into the
search window.
[0036] In certain embodiments, at least one of the genes/proteins
in the prepared expression profile is from Tables 1, 2, 3 and/or 4,
where the expression profile may include expression data for 5, 10,
20, 50, 75 or more of, including all of, the genes/proteins listed
in Tables 1, 2, 3 and/or 4. In certain embodiments, at least 5
genes in Table 2 and/or Table 4 are evaluated, including 10 or
more, 20 or more or all of the genes in Table 2 and/or Table 4. The
number of different genes/proteins whose expression and/or quantity
data, i.e., presence or absence of expression, as well as
expression/quantity level, that are included in the expression
profile that is generated may vary, but may be at least 2, and in
many embodiments ranges from 2 to about 100 or more, sometimes from
3 to about 75 or more, including from about 4 to about 70 or
more.
[0037] In certain embodiments, the expression profile obtained is a
genomic or nucleic acid expression profile, where the amount or
level of one or more nucleic acids in the sample is determined,
e.g., the nucleic acid transcript of the gene of interest. In these
embodiments, the sample that is assayed to generate the expression
profile employed in the diagnostic methods is one that is a nucleic
acid sample. The nucleic acid sample includes a plurality or
population of distinct nucleic acids that includes the expression
information of the phenotype determinative genes of interest of the
cell or tissue being diagnosed. The nucleic acid may include RNA or
DNA nucleic acids, e.g., mRNA, cRNA, cDNA etc., so long as the
sample retains the expression information of the host cell or
tissue from which it is obtained. The sample may be prepared in a
number of different ways, as is known in the art, e.g., by mRNA
isolation from a cell, where the isolated mRNA is used as is,
amplified, employed to prepare cDNA, cRNA, etc., as is known in the
differential expression art. The sample is typically prepared from
a cell or tissue harvested from a subject to be diagnosed, e.g.,
via biopsy of tissue, using standard protocols, where cell types or
tissues from which such nucleic acids may be generated include any
tissue in which the expression pattern of the to be determined
phenotype exists, including, but not limited to, peripheral blood
lymphocyte cells, etc., as reviewed above.
[0038] The expression profile may be generated from the initial
nucleic acid sample using any convenient protocol. While a variety
of different manners of generating expression profiles are known,
such as those employed in the field of differential gene expression
analysis, one representative and convenient type of protocol for
generating expression profiles is array-based gene expression
profile generation protocols. Such applications are hybridization
assays in which a nucleic acid that displays "probe" nucleic acids
for each of the genes to be assayed/profiled in the profile to be
generated is employed. In these assays, a sample of target nucleic
acids is first prepared from the initial nucleic acid sample being
assayed, where preparation may include labeling of the target
nucleic acids with a label, e.g., a member of signal producing
system. Following target nucleic acid sample preparation, the
sample is contacted with the array under hybridization conditions,
whereby complexes are formed between target nucleic acids that are
complementary to probe sequences attached to the array surface. The
presence of hybridized complexes is then detected, either
qualitatively or quantitatively. Specific hybridization technology
which may be practiced to generate the expression profiles employed
in the subject methods includes the technology described in U.S.
Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710;
5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732;
5,661,028; 5,800,992; the disclosures of which are herein
incorporated by reference; as well as WO 95/21265; WO 96/31622; WO
97/10365; WO 97/27317; EP 373 203; and EP 785 280. In these
methods, an array of "probe" nucleic acids that includes a probe
for each of the phenotype determinative genes whose expression is
being assayed is contacted with target nucleic acids as described
above. Contact is carried out under hybridization conditions, e.g.,
stringent hybridization conditions, and unbound nucleic acid is
then removed.
[0039] The term "stringent assay conditions" as used herein refers
to conditions that are compatible to produce binding pairs of
nucleic acids, e.g., surface bound and solution phase nucleic
acids, of sufficient complementarity to provide for the desired
level of specificity in the assay while being less compatible to
the formation of binding pairs between binding members of
insufficient complementarity to provide for the desired
specificity. Stringent assay conditions are the summation or
combination (totality) of both hybridization and wash
conditions.
[0040] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization (e.g., as in array, Southern or Northern
hybridizations) are sequence dependent, and are different under
different experimental parameters. Stringent hybridization
conditions that can be used to identify nucleic acids within the
scope of the invention can include, e.g., hybridization in a buffer
comprising 50% formamide, 5.times.SSC, and 1% SDS at 42.degree. C.,
or hybridization in a buffer comprising 5.times.SSC and 1% SDS at
65.degree. C., both with a wash of 0.2.times.SSC and 0.1% SDS at
65.degree. C. Exemplary stringent hybridization conditions can also
include a hybridization in a buffer of 40% formamide, 1 M NaCl, and
1% SDS at 37.degree. C., and a wash in 1.times.SSC at 45.degree. C.
Alternatively, hybridization to filter-bound DNA in 0.5 M
NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C. can be employed. Yet additional stringent hybridization
conditions include hybridization at 60.degree. C. or higher and
3.times.SSC (450 mM sodium chloride/45 mM sodium citrate) or
incubation at 42.degree. C. in a solution containing 30% formamide,
1M NaCl, 0.5% sodium sarcosine, 50 mM MES, pH 6.5. Those of
ordinary skill will readily recognize that alternative but
comparable hybridization and wash conditions can be utilized to
provide conditions of similar stringency.
[0041] In certain embodiments, the stringency of the wash
conditions that set forth the conditions which determine whether a
nucleic acid is specifically hybridized to a surface bound nucleic
acid. Wash conditions used to identify nucleic acids may include,
e.g.: a salt concentration of about 0.02 molar at pH 7 and a
temperature of at least about 50.degree. C. or about 55.degree. C.
to about 60.degree. C.; or, a salt concentration of about 0.15 M
NaCl at 72.degree. C. for about 15 minutes; or, a salt
concentration of about 0.2.times.SSC at a temperature of at least
about 50.degree. C. or about 55.degree. C. to about 60.degree. C.
for about 15 to about 20 minutes; or, the hybridization complex is
washed twice with a solution with a salt concentration of about
2.times.SSC containing 0.1% SDS at room temperature for 15 minutes
and then washed twice by 0.1.times.SSC containing 0.1% SDS at
68.degree. C. for 15 minutes; or, equivalent conditions. Stringent
conditions for washing can also be, e.g., 0.2.times.SSC/0.1% SDS at
42.degree. C.
[0042] A specific example of stringent assay conditions is rotating
hybridization at 65.degree. C. in a salt based hybridization buffer
with a total monovalent cation concentration of 1.5 M (e.g., as
described in U.S. patent application Ser. No. 09/655,482 filed on
Sep. 5, 2000, the disclosure of which is herein incorporated by
reference) followed by washes of 0.5.times.SSC and 0.1.times.SSC at
room temperature.
[0043] Stringent assay conditions are hybridization conditions that
are at least as stringent as the above representative conditions,
where a given set of conditions are considered to be at least as
stringent if substantially no additional binding complexes that
lack sufficient complementarity to provide for the desired
specificity are produced in the given set of conditions as compared
to the above specific conditions, where by "substantially no more"
is meant less than about 5-fold more, typically less than about
3-fold more. Other stringent hybridization conditions are known in
the art and may also be employed, as appropriate.
[0044] The resultant pattern of hybridized nucleic acid provides
information regarding expression for each of the genes that have
been probed, where the expression information is in terms of
whether or not the gene is expressed and, typically, at what level,
where the expression data, i.e., expression profile (e.g., in the
form of a transcriptosome), may be both qualitative and
quantitative.
[0045] Alternatively, non-array based methods for quantitating the
levels of one or more nucleic acids in a sample may be employed,
including those based on amplification protocols, e.g., Polymerase
Chain Reaction (PCR)-based assays, including quantitative PCR,
reverse-transcription PCR (RT-PCR), real-time PCR, and the
like.
[0046] Where the expression profile is a protein expression
profile, any convenient protein quantitation protocol may be
employed, where the levels of one or more proteins in the assayed
sample are determined. Representative methods include, but are not
limited to: proteomic arrays, flow cytometry, standard immunoassays
(e.g., western blot, ELISA assays), etc.
[0047] Following obtainment of the expression profile from the
sample being assayed, the expression profile is compared with a
reference or control profile to determine the particular graft
tolerant/intolerant phenotype of the cell or tissue, and therefore
host, from which the sample was obtained/derived. The terms
"reference" and "control" as used herein mean a standardized
pattern of gene expression or levels of expression of certain genes
to be used to interpret the expression signature of a given patient
and assign a graft tolerant/intolerant phenotype thereto. The
reference or control profile may be a profile that is obtained from
a cell/tissue known to have the desired phenotype, e.g., tolerant
phenotype, and therefore may be a positive reference or control
profile. In addition, the reference/control profile may be from a
cell/tissue known to not have the desired phenotype, e.g., an
intolerant phenotype, and therefore be a negative reference/control
profile.
[0048] In certain embodiments, the obtained expression profile is
compared to a single reference/control profile to obtain
information regarding the phenotype of the cell/tissue being
assayed. In yet other embodiments, the obtained expression profile
is compared to two or more different reference/control profiles to
obtain more in depth information regarding the phenotype of the
assayed cell/tissue. For example, the obtained expression profile
may be compared to a positive and negative reference profile to
obtain confirmed information regarding whether the cell/tissue has
the phenotype of interest.
[0049] The comparison of the obtained expression profile and the
one or more reference/control profiles may be performed using any
convenient methodology, where a variety of methodologies are known
to those of skill in the array art, e.g., by comparing digital
images of the expression profiles, by comparing databases of
expression data, etc. Patents describing ways of comparing
expression profiles include, but are not limited to, U.S. Pat. Nos.
6,308,170 and 6,228,575, the disclosures of which are herein
incorporated by reference. Methods of comparing expression profiles
are also described above.
[0050] The comparison step results in information regarding how
similar or dissimilar the obtained expression profile is to the
control/reference profile(s), which similarity/dissimilarity
information is employed to determine the phenotype of the
cell/tissue being assayed. For example, similarity with a positive
control indicates that the assayed cell/tissue has a tolerant
phenotype. Likewise, similarity with a negative control indicates
that the assayed cell/tissue has an intolerant phenotype.
[0051] Depending on the type and nature of the reference/control
profile(s) to which the obtained expression profile is compared,
the above comparison step yields a variety of different types of
information regarding the cell/tissue that is assayed. As such, the
above comparison step can yield a positive/negative determination
of a tolerant phenotype of an assayed cell/tissue. In many
embodiments, the above-obtained information about the cell/tissue
being assayed is employed to diagnose a host, subject or patient
with respect to that host's graft tolerance, as described
above.
[0052] The subject methods further find use in pharmacogenomic
applications. In these applications, a subject/host/patient is
first diagnosed for the presence or absence of the graft tolerant
phenotype using a protocol such as the diagnostic protocol
described in the preceding section. The subject is then treated
using a protocol whose suitability is determined using the results
of the diagnosis step. More specifically, where the identified
phenotype is tolerant, a protocol that may include a reduced level
of immunosuppression (i.e., immunosuppression at a level less than
that which is indicated for patients not known to be graft
tolerant), or no immunosuppression, may be employed to manage/treat
the subject. Alternatively, where a patient is identified as having
an intolerant phenotype, full immunosuppressive protocols may be
employed/continued.
[0053] In many embodiments, a host is screened for the presence of
a graft tolerant phenotype following receipt of a graft or
transplant. The host may be screened once or serially following
transplant receipt, e.g., weekly, monthly, bimonthly, half-yearly,
yearly, etc., as long as the host is on immunosuppressive therapy.
In certain embodiments, monitoring of the host expression profile
even after immunosuppressive therapy has been reduced or
discontinued is conducted to determine whether the host has
maintained the tolerogenic expression profile and may continue for
the lifetime of the host.
Databases of Expression Profiles of Phenotype Determinative
Genes
[0054] Also provided are databases of expression profiles of graft
tolerant phenotype determinative genes. Such databases will
typically comprise expression profiles of various cells/tissues
having graft tolerant phenotypes, negative expression profiles,
etc., where such profiles are further described below.
[0055] The expression profiles and databases thereof may be
provided in a variety of media to facilitate their use. "Media"
refers to a manufacture that contains the expression profile
information of the present invention. The databases of the present
invention can be recorded on computer readable media, e.g. any
medium that can be read and accessed directly by a user employing a
computer. Such media include, but are not limited to: magnetic
storage media, such as floppy discs, hard disc storage medium, and
magnetic tape; optical storage media such as CD-ROM; electrical
storage media such as RAM and ROM; and hybrids of these categories
such as magnetic/optical storage media. One of skill in the art can
readily appreciate how any of the presently known computer readable
mediums can be used to create a manufacture comprising a recording
of the present database information. "Recorded" refers to a process
for storing information on computer readable medium, using any such
methods as known in the art. Any convenient data storage structure
may be chosen, based on the means used to access the stored
information. A variety of data processor programs and formats can
be used for storage, e.g. word processing text file, database
format, etc. Thus, the subject expression profile databases are
accessible by a user, i.e., the database files are saved in a
user-readable format (e.g., a computer readable format, where a
user controls the computer).
[0056] As used herein, "a computer-based system" refers to the
hardware means, software means, and data storage means used to
analyze the information of the present invention. The minimum
hardware of the computer-based systems of the present invention
comprises a central processing unit (CPU), input means, output
means, and data storage means. A skilled artisan can readily
appreciate that any one of the currently available computer-based
system are suitable for use in the present invention. The data
storage means may comprise any manufacture comprising a recording
of the present information as described above, or a memory access
means that can access such a manufacture.
[0057] A variety of structural formats for the input and output
means can be used to input and output the information in the
computer-based systems of the present invention, e.g., to and from
a user. One format for an output means ranks expression profiles
possessing varying degrees of similarity to a reference expression
profile. Such presentation provides a skilled artisan with a
ranking of similarities and identifies the degree of similarity
contained in the test expression profile.
Reagents and Kits
[0058] Also provided are reagents and kits thereof for practicing
one or more of the above-described methods. The subject reagents
and kits thereof may vary greatly. Reagents of interest include
reagents specifically designed for use in production of the
above-described expression profiles of phenotype determinative
genes, i.e., a gene expression evaluation element made up of one or
more reagents.
[0059] One type of such reagent is an array of probe nucleic acids
in which the phenotype determinative genes of interest are
represented. A variety of different array formats are known in the
art, with a wide variety of different probe structures, substrate
compositions and attachment technologies (e.g., dot blot arrays,
microarrays, etc.). Representative array structures of interest
include those described in U.S. Pat. Nos. 5,143,854; 5,288,644;
5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270;
5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800,992; the
disclosures of which are herein incorporated by reference; as well
as WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373 203;
and EP 785 280.
[0060] In many embodiments, the arrays include probes for at least
1 of the genes listed in Tables 1, 2, 3 and/or 4. In certain
embodiments, the number of genes that are from Tables 1, 2, 3
and/or 4 that is represented on the array is at least 5, at least
10, at least 25, at least 50, at least 75 or more, including all of
the genes listed in Tables 1, 2, 3 and/or 4. In certain
embodiments, the arrays include at least 5 genes listed in Table 2
and/or Table 4, including 10 or more, 20 or more or all of the
genes in Table 2 and/or Table 4. The subject arrays may include
only those genes that are listed in Tables 1, 2, 3 and/or 4, or
they may include additional genes that are not listed in Tables 1,
2, 3 and/or 4. Where the subject arrays include probes for such
additional genes, in certain embodiments the number % of additional
genes that are represented does not exceed about 50%, usually does
not exceed about 25%. In many embodiments where additional
"non-Table 1" genes are included, a great majority of genes in the
collection are phenotype determinative genes, where by great
majority is meant at least about 75%, usually at least about 80%
and sometimes at least about 85, 90, 95% or higher, including
embodiments where 100% of the genes in the collection are phenotype
determinative genes.
[0061] Another type of reagent that is specifically tailored for
generating expression profiles of phenotype determinative genes is
a collection of gene specific primers that is designed to
selectively amplify such genes. Gene specific primers and methods
for using the same are described in U.S. Pat. No. 5,994,076, the
disclosure of which is herein incorporated by reference. Of
particular interest are collections of gene specific primers that
have primers for at least 1 of the genes listed in one Tables 1, 2,
3 and/or 4, often a plurality of these genes, e.g., at least 2, 5,
10, 15 or more. In certain embodiments, the number of genes that
are from Tables 1, 2, 3 and/or 4 that have primers in the
collection is at least 5, at least 10, at least 25, at least 50, at
least 75 or more, including all of the genes listed in Tables 1, 2,
3 and/or 4. In certain embodiments, the subject gene specific
primers collections include at least 5 genes listed in Table 2
and/or Table 4, including 10 or more, 20 or more or all of the
genes in Table 2 and/or Table 4. The subject gene specific primer
collections may include only those genes that are listed in Tables
1, 2, 3 and/or 4, or they may include primers for additional genes
that are not listed in Tables 1, 2, 3 and/or 4. Where the subject
gene specific primer collections include primers for such
additional genes, in certain embodiments the number % of additional
genes that are represented does not exceed about 50%, usually does
not exceed about 25%. In many embodiments where additional
"non-Table 1", "non-Table 2", "non-Table 3" or "non-Table 4" genes
are included, a great majority of genes in the collection are
phenotype determinative genes, where by great majority is meant at
least about 75%, usually at least about 80% and sometimes at least
about 85, 90, 95% or higher, including embodiments where 100% of
the genes in the collection are phenotype determinative genes.
[0062] The kits of the subject invention may include the
above-described arrays and/or gene specific primer collections. The
kits may further include one or more additional reagents employed
in the various methods, such as primers for generating target
nucleic acids, dNTPs and/or rNTPs, which may be either premixed or
separate, one or more uniquely labeled dNTPs and/or rNTPs, such as
biotinylated or Cy3 or Cy5 tagged dNTPs, gold or silver particles
with different scattering spectra, or other post synthesis labeling
reagent, such as chemically active derivatives of fluorescent dyes,
enzymes, such as reverse transcriptases, DNA polymerases, RNA
polymerases, and the like, various buffer mediums, e.g.
hybridization and washing buffers, prefabricated probe arrays,
labeled probe purification reagents and components, like spin
columns, etc., signal generation and detection reagents, e.g.
streptavidin-alkaline phosphatase conjugate, chemifluorescent or
chemiluminescent substrate, and the like.
[0063] The subject kits may also include a phenotype determination
element, which element is, in many embodiments, a reference or
control expression profile that can be employed, e.g., by a
suitable computing means, to make a phenotype determination based
on an "input" expression profile, e.g., that has been determined
with the above described gene expression evaluation element.
Representative phenotype determination elements include databases
of expression profiles, e.g., reference or control profiles, as
described above.
[0064] In addition to the above components, the subject kits will
further include instructions for practicing the subject methods.
These instructions may be present in the subject kits in a variety
of forms, one or more of which may be present in the kit. One form
in which these instructions may be present is as printed
information on a suitable medium or substrate, e.g., a piece or
pieces of paper on which the information is printed, in the
packaging of the kit, in a package insert, etc. Yet another means
would be a computer readable medium, e.g., diskette, CD, etc., on
which the information has been recorded. Yet another means that may
be present is a website address which may be used via the internet
to access the information at a removed site. Any convenient means
may be present in the kits.
Systems
[0065] Also provided are systems for practicing one or more of the
above-described methods. The subject systems may vary greatly, but
typically include at least a gene expression evaluation element,
e.g., one or more reagents, and a phenotype determination
element.
[0066] Reagents of interest include reagents specifically designed
for use in production of the above-described expression profiles of
phenotype determinative genes, i.e., a gene expression evaluation
element made up of one or more reagents. One type of such reagent
is an array of probe nucleic acids in which the phenotype
determinative genes of interest are represented. A variety of
different array formats are known in the art, with a wide variety
of different probe structures, substrate compositions and
attachment technologies. Representative array structures of
interest include those described in U.S. Pat. Nos. 5,143,854;
5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980;
5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800,992;
the disclosures of which are herein incorporated by reference; as
well as WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373
203; and EP 785 280.
[0067] In many embodiments, the arrays include probes for at least
1 of the genes listed in Tables 1, 2, 3 and/or 4. In certain
embodiments, the number of genes that are from Tables 1, 2, 3
and/or 4 that is represented on the array is at least 5, at least
10, at least 25, at least 50, at least 75 or more, including all of
the genes listed in Tables 1, 2, 3 and/or 4. In certain
embodiments, the arrays include at least 5 genes listed in Table 2
and/or Table 4, including 10 or more, 20 or more or all of the
genes in Table 2 and/or Table 4. The subject arrays may include
only those genes that are listed in Tables 1, 2, 3 and/or 4, or
they may include additional genes that are not listed in Tables 1,
2, 3 and/or 4. Where the subject arrays include probes for such
additional genes, in certain embodiments the number % of additional
genes that are represented does not exceed about 50%, usually does
not exceed about 25%. In many embodiments where additional
"non-Table 1", "non-Table 2", "non-Table 3" or "non-Table 4" genes
are included, a great majority of genes in the collection are
phenotype determinative genes, where by great majority is meant at
least about 75%, usually at least about 80% and sometimes at least
about 85, 90, 95% or higher, including embodiments where 100% of
the genes in the collection are phenotype determinative genes.
[0068] Another type of reagent that is specifically tailored for
generating expression profiles of phenotype determinative genes is
a collection of gene specific primers that is designed to
selectively amplify such genes. Gene specific primers and methods
for using the same are described in U.S. Pat. No. 5,994,076, the
disclosure of which is herein incorporated by reference. Of
particular interest are collections of gene specific primers that
have primers for at least 1 of the genes listed in one Tables 1, 2,
3 and/or 4, often a plurality of these genes, e.g., at least 2, 5,
10, 15 or more. In certain embodiments, the number of genes that
are from Tables 1, 2, 3 and/or 4 that have primers in the
collection is at least 5, at least 10, at least 25, at least 50, at
least 75 or more, including all of the genes listed in Tables 1, 2,
3 and/or 4. In certain embodiments, the subject gene specific
primers collections include at least 5 genes listed in Table 2
and/or Table 4, including 10 or more, 20 or more or all of the
genes in Table 2 and/or Table 4. The subject gene specific primer
collections may include only those genes that are listed in Tables
1, 2, 3 and/or 4, or they may include primers for additional genes
that are not listed in Tables 1, 2, 3 and/or 4. Where the subject
gene specific primer collections include primers for such
additional genes, in certain embodiments the number % of additional
genes that are represented does not exceed about 50%, usually does
not exceed about 25%. In many embodiments where additional
"non-Table 1", "non-Table 2", "non-Table 3" or "non-Table 4" genes
are included, a great majority of genes in the collection are
phenotype determinative genes, where by great majority is meant at
least about 75%, usually at least about 80% and sometimes at least
about 85, 90, 95% or higher, including embodiments where 100% of
the genes in the collection are phenotype determinative genes.
[0069] The systems of the subject invention may include the
above-described arrays and/or gene specific primer collections. The
systems may further include one or more additional reagents
employed in the various methods, such as primers for generating
target nucleic acids, dNTPs and/or rNTPs, which may be either
premixed or separate, one or more uniquely labeled dNTPs and/or
rNTPs, such as biotinylated or Cy3 or Cy5 tagged dNTPs, gold or
silver particles with different scattering spectra, or other post
synthesis labeling reagent, such as chemically active derivatives
of fluorescent dyes, enzymes, such as reverse transcriptases, DNA
polymerases, RNA polymerases, and the like, various buffer mediums,
e.g. hybridization and washing buffers, prefabricated probe arrays,
labeled probe purification reagents and components, like spin
columns, etc., signal generation and detection reagents, e.g.
streptavidin-alkaline phosphatase conjugate, chemifluorescent or
chemiluminescent substrate, and the like.
[0070] The systems may also include a phenotype determination
element, which element is, in many embodiments, a reference or
control expression profile that can be employed, e.g., by a
suitable computing means, to make a phenotype determination based
on an "input" expression profile, e.g., that has been determined
with the above described gene expression evaluation element.
Representative phenotype determination elements include databases
of expression profiles, e.g., reference or control profiles, as
described above.
[0071] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
I. Methods and Materials
[0072] We performed 80 Agilent whole human genome arrays, which
include approximately 43,000 probes, on samples from the following
subjects: 15 Stanford spontaneous tolerances (SP-TOL or SP), 20
Immune Tolerance Network (ITN) spontaneous tolerances, 16 induced
tolerances (IN-TOL or IN), 9 chronic (CAN), 8 healthy donor (HD),
and 12 stable patients. We used 5 Stanford spontaneous tolerance
samples and 5 chronic samples as our training set for 2-class (TOL
vs. CAN) biomarker discovery, all others are as our test set. We
used 14 Stanford spontaneous tolerance samples, 8 healthy donor
samples, and 9 chronic samples as our training set for 3-class (HD
vs. TOL vs. CAN) biomarker discovery, all others are as our test
set for the prediction.
Patient and Materials
[0073] Peripheral whole blood samples were collected from 72 renal
transplant patients (tolerant, stable) and 8 normal controls
enrolled in this study. The protocol was approved by IRB and
required written informed consent. To generate informative
biomarkers by microarray for spontaneous tolerance, samples were
chosen from 4 clinical phenotypes: 1) Immunosuppressive drug-free,
spontaneous tolerant (SP-TOL): patients with long-term stable graft
function, without immunosuppression for at least 2 years. Stable
graft function was defined as stable Schwardz calculated creatinine
clearance >60 mls/min/1.73 m2 with absent or low grade
proteinuria (<1.5 g/day). 2) Induced tolerant (IN-TOL): patients
with stable graft function were induced to accept transplanted
organ through a processing of control weaning immunosuppressive
drug after transplantation. 3) Chronic rejection (CAN): This group
was defined according to clinical and histological criteria. All
CAN patients had a progressive degradation of their renal function
(creatinine clearance <60 mls/min/1.73 m.sup.2 and/or
proteinuria >1.5 g/day) and histological signs of vascular
chronic rejection defined as endarteritis and allograft
glomerulopathy with basement membrane duplication. 4) Healthy
donors (HD) were included as controls. They all had a normal blood
formula and no infectious or other concomitant pathology for at
least 6 months prior to the study.
RNA Extraction
[0074] Whole blood was collected in PAXgene Blood RNA Tubes
(PreAnalytiX, Qiagen), and total RNA was extracted using the
PAXgene Blood RNA Kit (PreAnalytiX, Qiagen). PBMCs were purified
using standard Ficoll-Paque gradient centrifugation according to
the instructions of the manufacturer (GE Healthcare, Sweden). Total
RNA was extracted using RNeasy Plus Mini Kit (Qiagen.) Total RNA
concentration was measured by NanoDrop ND-1000 (NanoDrop
Technologies, Wilmington, Del.), and the integrity of the extracted
total RNA was assessed with the Agilent 2100 Bioanalyzer using RNA
Nano Chips (Agilent Technologies, Santa Clara, Calif.). Total RNA
was stored at -80.degree. C. until sample preparation for the
microarray experiments.
Microarray Hybridization and Data Analysis.
[0075] RNA samples (test and reference) were labeled using the
Agilent low RNA input fluorescent linear amplification Kit (p/n
5188-5340) according to the manufacturer's instructions. To avoid
confounding by extraneous factors, all the experiments were
performed with a single batch and processed by one technician on
the same day for each step. Briefly, 100 ng of total RNA was
reverse transcribed. Amplification and labeling were performed by
T7-polymerase in vitro transcription, to give fluorescent-labeled
cRNA. Test and reference cRNAs were labeled with Cyanine-5 and
Cyanine-3 CTP dyes. The dye incorporation rate was assessed with a
NanoDrop ND-1000 spectrophotometer. Hybridization was carried out
using the Agilent oligonucleotide microarray hybridization kit (p/n
5188-5242), following the manufacturer's instructions. Briefly, 825
ng of test sample cRNA was mixed with 825 ng of reference sample
cRNA in the presence of target controls. This solution was
subjected to fragmentation (30 min at 60.degree. C.) and then
hybridization on 44K Human Whole-Genome 60-mer oligo-chips (G4112F,
Agilent Technologies) in Agilent hybridization. Slides were
disassembled and washed according to the manufacturer's
instructions. Microarrays were scanned with an Agilent microarray
scanner (Agilent dual laser DNA microarray scanner G2565BA, Agilent
technologies, Palo Alto, Calif. USA).
[0076] GeneSpring (Agilent Inc, CA) was used for normalization and
data transformation. Statistical Analysis of Microarray (SAM) and
Prediction Analysis of Microarray (PAM) programs were used to
analyze Agilent microarray data. T test and Jonckheere-Terpstra
trend test were used to determine the specific genes for sub cell
types by the ordinal rank of healthy donor (HD), spontaneous
tolerance (SP), and induced tolerance (IN). Statistical analyses
were done by SAS 9.3 software (Cary, N.C.).
II. Results
[0077] We identified 99 genes using the SAM (see Tusher, et al.
supra) and PAM statistical analysis tool (Predictive Analysis of
Microarray data, Tibshirani et al., Proc. Nat'l Acad. Sci. USA
(2002) 99:6567-72) (FDR<2%) with highest predictive value in
identifying a tolerogenic state in the blood samples. PAM is a
statistical analysis program that identifies a minimum gene set
characteristic of user defined sample groups (the learning set) and
then scores both known and unknown samples based on similarity to
identified expression profile differences.
[0078] The 99 genes identified are listed in Table 1 below. Also
noted in Table I is whether the identified gene is associated with
the TGF-.beta. signaling pathway (p<0.0001 by hypergeometric)
and/or with cell cycle regulation/signaling (p=0.0001 by
hypergeometric); genes in both of these pathways whose expression
correlates with graft tolerance were identified in a previous study
using the Stanford Lymphochip (LC) micro-array (see Brouard et al.,
PNAS 2007, vol. 104, pp. 15448-15453).
[0079] Forty genes in Table 1 were identified in Brouard et al.
using the LC platform, 2 of which were used as part of the 49 gene
`footprint` described therein (P=0.0085 by hypergeometric). FIG. 1A
shows the classification of a set of samples using the LC 49
footprint prediction of the Brouard et al. study. From the
overlapping 40 genes (i.e., between the 99 genes in Table 1 (using
Agilent arrays) and those identified in the LC platform studies of
Brouard et al. (using Stanford Lymphochip arrays)), the top 22
genes using PAM & SAM analyses (FDR <2%) were employed to
re-classify the same LC samples shown in FIG. 1A. As shown in FIG.
1B, the 22 gene set gives similar predictions as the 49 gene set in
FIG. 1A. Indeed, all samples were predicted as the same as 49
footprints, except for MIS6 and MIS8, which have the trend of the
TOL prediction.
[0080] FIG. 2 provides sample predictions using the top 24 genes
selected from the 99 genes in Table 1. In FIG. 2, the left panel
shows the training set and the right panel shows the test set. All
spontaneous samples are 100% predicted correctly using this 24 gene
set. The LK2 induced patient (TOL_LK2) was predicted as SP-TOL,
which has been confirmed by clinical observation. The patient has
been off drug for 2 years.
TABLE-US-00001 TABLE 1 A list of the 99 genes identified that are
differentially expressed in TOL vs. CAN. Genes involved in
TGF-.beta. and cell cycle signaling are indicated. Entrez Cell 24
Symbol GeneID fold FDR (%) TGF-b cycle genes MARCH1 55016 3.0022797
0 ANGPTL1 9068 -2.007214 0 ANKRD15 23189 -2.0878 0 ANTXR1 84168
-2.19655 1.1507317 APOBEC3A 200315 7.3809241 0.5802009 Y APOC3 345
-3.212175 1.1507317 ARL4C 10123 2.4144416 0.5802009 ARRDC2 27106
2.2580325 0.5802009 BARD1 580 -2.139497 0 BMPR1A 657 -2.797208
1.1507317 Y CAGE1 285782 -2.69422 1.1507317 CASP8 841 3.1857514 0 Y
Y CCNY 219771 2.1650647 0.5802009 CD14 929 2.9491052 0 Y Y CD163
9332 3.9154456 0.9863415 Y CD244 51744 2.2280641 0 Y CD36 948
2.8681281 0.5802009 Y CDK5RAP3 80279 2.0183769 0 CNTNAP2 26047
-5.192761 1.1507317 Y CXX1 8933 -2.620834 1.1507317 CYP26A1 1592
-2.769904 1.1507317 CYP4B1 1580 -3.547685 1.1507317 DOCK2 1794
2.3320694 0 Y DYNC2LI1 51626 -2.096258 0 EMP2 2013 -2.701 1.1507317
Y EMR2 30817 3.166663 0.9863415 ENAH 55740 -2.891351 1.1507317 ETS2
2114 2.692491 0.5802009 F13A1 2162 3.1994918 0 Y Y FAM13A1OS 285512
2.6713794 0.5802009 FLJ90086 389389 -2.103278 1.1507317 FOSL2 2355
2.809 0.5802009 Y FOXC1 2296 -6.621625 1.1507317 Y FTMT 94033
2.0330983 0 GGTA1 2681 4.3356754 0.7671545 GNPTAB 79158 2.3641187 0
Y GOLPH2 51280 -2.853767 1.1507317 GPR125 166647 -2.675509
1.1507317 GZMB 3002 2.7880761 0.5802009 GZMK 3003 3.179326 0 Y
HLA-DQA2 3118 3.5716123 0.9863415 HSD17B6 8630 -2.21751 1.1507317
IFNK 56832 -2.913018 0 Y IGHM 3507 2.2203545 0.7671545 Y IPO13 9670
-2.01756 0 ITIH4 3700 1.9524537 0 KRT17 3872 -2.917036 1.1507317 Y
KRT80 144501 -2.621779 0 Y LAIR2 3904 2.7438641 0.5802009 LDHAL6A
160287 -2.371389 1.1507317 LGR6 59352 3.2632785 0.9863415 LOC124245
124245 2.1722114 0.5802009 LOC150371 150371 -3.532812 1.1507317
LOC283278 283278 -2.818204 0 LOC400451 400451 -2.543694 1.1507317
LOC440156 642477 -2.310971 0 LOC541472 541472 -3.541878 0 Y
LOC55908 55908 -1.76498 0 LOXL2 4017 -4.541061 1.1507317 Y LRPAP1
4043 2.2859522 0 LYZ 4069 2.8267647 0 Y MARCO 8685 2.8601166
1.1365251 MLPH 79083 -2.58653 1.1507317 MPEG1 219972 3.2504029 0 Y
MPZL1 9019 -2.331467 1.1507317 MYO3A 53904 -2.950033 1.1507317 NCR1
9437 3.8883374 1.2148487 NEDD4L 23327 -2.623521 1.2148487 NLRP3
114548 3.7234615 0.5802009 PAICS 10606 -4.250718 1.2148487 PBLD
64081 -2.232983 0 PCDHA9 9752 -2.634803 0 Y PHF1 5252 1.6854487 0
PLA2G7 7941 4.9729708 0 Y PLTP 5360 -2.266916 1.1507317 PNPLA7
375775 -3.730196 1.1507317 Y PPBP 5473 2.4655572 0.7671545 PRKCDBP
112464 -4.079189 1.1507317 REL 5966 2.0806223 0 Y RNPEP 6051
2.3243464 0 Y SCHIP1 29970 -2.832874 1.1507317 SGCE 8910 -3.914326
0 Y SLC13A1 6561 -3.412758 1.1507317 SMO 6608 -2.186908 0 SMPX
23676 -2.630361 0 Y SPATS1 221409 -3.279478 1.1507317 STAB1 23166
2.9611082 0 Y STEAP1 26872 -3.380256 1.1507317 TJP1 7082 -3.399506
1.2148487 Y TNFSF13 8741 2.6887683 0.8908891 TPD52L3 89882
-3.766346 1.6627545 TPX2 22974 -1.955087 0 TSPAN32 10077 2.1335027
0 Y TUBA3C 7278 2.4974196 0.5802009 WDR65 149465 -3.843356 0 Y XKRX
402415 -2.744066 1.1507317 XRCC2 7516 -2.979154 1.1507317 Y ZC3H11B
643136 2.4800248 0.8908891 ZFP91 80829 2.2669509 0
[0081] In further analyses, we identified 28 genes using PAM and
SAM analysis tools (FDR<5%) whose expression levels are the best
predictors of 3 distinct subject classes, differentiating HD vs. SP
vs. CAN (see Table 2, below). FIG. 5 shows 3-class prediction of HD
vs. TOL vs. CAN samples based on this 28 gene set.
TABLE-US-00002 TABLE 2 List of 28 genes whose expression levels
predict 3 distinct subject classes (HD vs. TOL vs. CAN). HD vs. TOL
vs. Symbol Entrez Gene ID CAN TP73 7161 Y CIRBP 1153 Y VNN1 8876 Y
ATXN1 6310 Y BMP2K 55589 Y KIAA1324 57535 Y PLXNC1 10154 Y CCL3
6348 Y WNK1 65125 Y CCL3L3 414062 Y IL6 3569 Y MYL2 4633 Y KIF15
56992 Y IPO13 9670 Y TMEM117 84216 Y KIAA1609 57707 Y RUNX2 860 Y
CBFA2T2 9139 Y TUBB4 10382 Y PDPN 10630 Y PRPF40B 25766 Y NRTN 4902
Y KRT17 3872 Y TSPAN7 7102 Y CLGN 1047 Y CCL2 6347 Y CENPN 55839 Y
DOPEY2 9980 Y
[0082] We then performed a sub analysis of the data for genes that
are: 1) expressed in distinct T cell subsets, and 2) whose
expression level is significantly different between HD vs. TOL
(SP+IN) phenotypes or has significantly expression rank order by JT
test among HD vs. SP vs. IN. T cell subsets were defined based on
their expression of CD3, CD4, CD8, and CD45 markers (see Table 3).
Table 3 lists 147 genes from JT test and SAM (FDR<5%) that are
significantly differentially regulated for either HD vs. TOL
(SP+IN) or HD vs. SP vs. IN and also shows their specific T cell
subset expression patterns.
TABLE-US-00003 TABLE 3 A gene list (147 genes) of either
significantly differentially expressed between HD vs. TOL (SP + IN)
or has significantly expression rank order by JT test among HD vs.
Spontaneous tolerance (SP) vs. Induced tolerance (IN) with specific
5 different cell types. trends of Entrez FDR_HD-TOL HD vs HD vs SP
Sybmol Gene ID Z_JT P value JT (%) TOL vs IN CD3+ CD4+/CD8+
CD45+/CD45- CNIH 10175 -1.14459 0.25238 3.045626145 Y CD3+ CD4+
CD8B 926 2.55331 0.01067 1.640686369 Y Y CD3+ CD8+ HNRPLL 92906
-1.60242 0.10906 0.215406287 Y CD3+ Naive PVRL3 25945 -3.1168
0.00183 0.121036585 Y Y CD3+ Naive ANK3 288 -2.342 0.01918 50 Y
CD3+ C6orf15 84830 -3.01114 0.0026 0.882047023 Y CD3+ DUSP16 80824
-2.13069 0.03311 50 Y CD3+ IER5L 389792 2.02504 0.04286 50 Y CD3+
INPP4B 8821 -2.02504 0.04286 50 Y CD3+ KIAA391 9692 2.44765 0.01438
50 Y CD3+ NPDC1 56654 2.55331 0.01067 50 Y CD3+ PRKCA 5578 -2.87027
0.0041 1.640686369 Y CD3+ TRPC1 7220 -1.98982 0.04661 50 Y CD3+
ANK3 288 -2.44765 0.01438 4.215733066 Y Y CD3+ C13orf15 28984
-0.33457 0.73795 3.53567017 Y CD3+ C17orf48 56985 -0.93328 0.35068
1.640686369 Y CD3+ C18orf17 125488 -0.86284 0.38822 2.310988327 Y
CD3+ C1orf54 79630 -1.84895 0.06447 0.083225156 Y CD3+ CCDC14
112942 -2.44765 0.01438 0.121036585 Y Y CD3+ CCDC19B 55013 -0.72197
0.47031 4.215733066 Y CD3+ CD28 940 -1.81373 0.06972 1.104381518 Y
CD3+ CD48 962 -0.93328 0.35068 3.045626145 Y CD3+ CD6 923 -1.10937
0.26727 3.53567017 Y CD3+ CDR2 1039 -1.10937 0.26727 2.674788698 Y
CD3+ CITED4 163732 -0.65153 0.5147 1.931533662 Y CD3+ DCTN6 10671
-0.9685 0.3328 0.277544618 Y CD3+ DCTN6 10671 -1.25024 0.21121
0.124017782 Y CD3+ DNAJB1 3337 -0.61632 0.53769 1.104381518 Y CD3+
DYNLT3 6990 -1.32068 0.18661 0.193782756 Y CD3+ EFCAB4A 283229
-1.67286 0.09436 0.193782756 Y CD3+ FAM84B 157638 -2.342 0.01918
0.407822995 Y Y CD3+ FXYD5 53827 -0.36979 0.71154 2.674788698 Y
CD3+ GAS5 60674 -1.5672 0.11707 0.121036585 Y CD3+ GPRASP1 9737
-2.37722 0.01744 0.882047023 Y Y CD3+ HECA 51696 -1.03893 0.29884
0.407822995 Y CD3+ HLF 3131 -2.58853 0.00964 0.121036585 Y Y CD3+
IL23A 51561 -2.06026 0.03937 0.407822995 Y Y CD3+ KIAA48 9729
-2.41244 0.01585 0.596694647 Y Y CD3+ NELL2 4753 -2.23635 0.02533
4.215733066 Y Y CD3+ NGFRAP1L1 340542 -1.98982 0.04661 1.104381518
Y Y CD3+ OCIAD2 132299 -1.9546 0.05063 0.707577565 Y CD3+ PBX4
80714 -0.26414 0.79168 3.045626145 Y CD3+ PVRL3 25945 -1.60242
0.10906 0.407822995 Y CD3+ RGS1 6001 -1.81373 0.06972 0.596694647 Y
CD3+ SFXN1 94081 -0.05283 0.95787 2.310988327 Y CD3+ SGTB 54557
-1.74329 0.08128 1.342763357 Y CD3+ SH3YL1 26751 -1.70807 0.08762
3.53567017 Y CD3+ SLC26A11 284129 -0.9685 0.3328 0.882047023 Y CD3+
SPOCK2 9806 -0.82762 0.40788 2.310988327 Y CD3+ TMEM3B 161291
-1.84895 0.06447 0.596694647 Y CD3+ TMEM66 51669 -1.5672 0.11707
0.707577565 Y CD3+ TNFAIP8 25816 -1.32068 0.18661 2.310988327 Y
CD3+ TULP3 7289 -0.72197 0.47031 0.707577565 Y CD3+ ZC3H6 376940
-2.20113 0.02773 0.407822995 Y Y CD3+ ZNF563 147837 -2.09547
0.03613 1.342763357 Y Y CD3+ TRIB1 10221 -1.03893 0.29884
1.342763357 Y CD4+ Naive ZMIZ1 57178 -1.63764 0.1015 0.407822995 Y
CD4+ Naive ARHGEF1L 55160 2.55331 0.01067 50 Y CD4+ STAB1 23166
2.342 0.01918 50 Y CD4+ TRIM7 81786 2.02504 0.04286 50 Y CD4+
B3GNT5 84002 -1.35589 0.17513 0.707577565 Y CD4+ CD3LF 146722
0.36979 0.71154 4.79008092 Y CD4+ CENTG3 116988 -1.35589 0.17513
1.931533662 Y CD4+ CLEC7A 64581 -0.82762 0.40788 2.310988327 Y CD4+
CSF1R 1436 0.61632 0.53769 4.215733066 Y CD4+ CTSS 1520 2.83505
0.00458 0.124017782 Y Y CD4+ EAF2 55840 -0.51066 0.60959
4.215733066 Y CD4+ ETS2 2114 -1.21502 0.22436 0.882047023 Y CD4+
GNB4 59345 -0.89806 0.36915 1.104381518 Y CD4+ IFNAR2 3455 2.37722
0.01744 0.596694647 Y Y CD4+ IRAK3 11213 -0.65153 0.5147
1.104381518 Y CD4+ KIF13A 63971 -0.15848 0.87408 3.53567017 Y CD4+
MAFB 9935 -1.1798 0.23808 0.707577565 Y CD4+ SMPDL3A 10924 -1.10937
0.26727 0.882047023 Y CD4+ TBC1D8 11138 -0.75719 0.44894
2.674788698 Y CD4+ TRIB1 10221 -1.25024 0.21121 0.215406287 Y CD4+
DKK3 27122 2.06026 0.03937 50 Y CD8+ ARHGAP8 23779 -1.60242 0.10906
1.640686369 Y CD8+ AUTS2 26053 -2.16591 0.03032 2.310988327 Y Y
CD8+ C1orf21 81563 -1.77851 0.07532 0.882047023 Y CD8+ CRTAM 56253
2.02504 0.04286 1.931533662 Y Y CD8+ DKK3 27122 -1.60242 0.10906
1.342763357 Y CD8+ EDG8 53637 2.37722 0.01744 1.931533662 Y Y CD8+
PPP1R16B 26051 -0.29935 0.76467 0.596694647 Y CD8+ RUNX3 864
-0.68675 0.49224 1.342763357 Y CD8+ SYTL2 54843 -1.67286 0.09436
3.53567017 Y CD8+ VCAM1 7412 -1.74329 0.08128 0.596694647 Y CD8+
ZNF364 27246 -2.02504 0.04286 0 Y Y CD8+ ANXA2 302 2.55331 0.01067
50 Y Naive ANXA2P1 303 2.62374 0.0087 50 Y Naive CD58 965 -2.27156
0.02311 50 Y Naive CD99 4267 2.79983 0.00511 50 Y Naive CYB561 1534
2.44765 0.01438 50 Y Naive CYB561 1534 2.79983 0.00511 50 Y Naive
FAM11C 548322 -2.58853 0.00964 0.882047023 Y Naive FAM38A 9780
2.02504 0.04286 50 Y Naive PDIA6 10130 2.76462 0.0057 50 Y Naive
PLEKHA2 59339 2.09547 0.03613 50 Y Naive TUBB2C 10383 2.27156
0.02311 50 Y Naive AHR 196 -0.54588 0.58515 2.674788698 Y Naive
ARHGDIA 396 -1.77851 0.07532 0.121036585 Y Naive B4GALT1 2683
-0.47544 0.63447 1.640686369 Y Naive CHMP2B 25978 -1.88416 0.05954
0.882047023 Y Naive CREM 1390 -0.79241 0.42812 0.215406287 Y Naive
CREM 1390 -1.53198 0.12553 0 Y Naive DYNLT3 6990 -1.91938 0.05494 0
Y Naive ELL2 22936 -2.09547 0.03613 0.121036585 Y Y Naive EPAS1
2034 -1.14459 0.25238 4.215733066 Y Naive FTH1 2495 -1.32068
0.18661 1.342763357 Y Naive FTH1 2495 -1.10937 0.26727 3.53567017 Y
Naive FTHL17 53940 -1.5672 0.11707 0.124017782 Y Naive GNA13 10672
-0.93328 0.35068 0.277544618 Y Naive GNG2 54331 -1.77851 0.07532
0.882047023 Y Naive HN1 51155 2.23635 0.02533 0.707577565 Y Y Naive
HNRPLL 92906 -0.89806 0.36915 0.277544618 Y Naive IL2 3558 -1.49677
0.13445 0.124017782 Y Naive IL4I1 259307 -1.03893 0.29884
0.882047023 Y Naive IL8 3576 -2.83505 0.00458 0 Y Y Naive ITGB1
3688 -0.36979 0.71154 1.640686369 Y Naive KLF8 11279 2.30678
0.02107 1.342763357 Y Y Naive LATS2 26524 -1.1798 0.23808
2.310988327 Y Naive LRP12 29967 -2.37722 0.01744 0 Y Y Naive MAF
4094 -1.28546 0.19863 4.79008092 Y Naive MAP3K8 1326 -1.25024
0.21121 0.083225156 Y Naive MYO1F 4542 1.5672 0.11707 4.215733066 Y
Naive NAB1 4664 -1.39111 0.16419 0.121036585 Y Naive NRAS 4893
-0.36979 0.71154 2.310988327 Y Naive OGFRL1 79627 -1.5672 0.11707
1.640686369 Y Naive PANX1 24145 -0.9685 0.3328 0.882047023 Y Naive
PBEF1 10135 -2.20113 0.02773 0.407822995 Y Y Naive PIK3R1 5295
-0.75719 0.44894 1.640686369 Y Naive PLEKHA5 54477 -1.9546 0.05063
0.707577565 Y Naive PLXND1 23129 -0.40501 0.68547 2.310988327 Y
Naive PRNP 5621 -1.63764 0.1015 0.193782756 Y Naive SAT1 6303
-1.53198 0.12553 0.121036585 Y Naive SLC39A8 64116 -2.27156 0.02311
0.193782756 Y Y Naive SPTLC1 10558 -2.23635 0.02533 0.124017782 Y Y
Naive SRGN 5552 -2.16591 0.03032 0.707577565 Y Y Naive STAM 8027
-0.89806 0.36915 0.596694647 Y Naive SUB1 10923 -1.49677 0.13445
0.121036585 Y Naive SUB1 10923 -1.25024 0.21121 0.124017782 Y Naive
TGFB1 7040 -1.35589 0.17513 0.121036585 Y Naive TIMP1 7076 -0.29935
0.76467 3.045626145 Y Naive TMED5 50999 -0.86284 0.38822
0.596694647 Y Naive TPM4 7171 -0.5811 0.56117 2.674788698 Y Naive
YWHAZ 7534 -1.39111 0.16419 4.79008092 Y Naive ZNF365 22891 -2.7294
0.00635 1.104381518 Y Y Naive
[0083] We then performed another sub-analysis using PAM and SAM
(FDR <2%) to identify genes whose expression shows significantly
upward or downward trends in HD vs. SP vs. IN (see Table 4, below).
FIG. 6 shows the gene expression trends of these significantly
down-regulated genes (left panel) and up regulated genes (right
panel) between HD vs. SP vs. IN. As shown in Table 4, one gene
(FAM110C) is known to be differentially expressed in Naive T
cells.
TABLE-US-00004 TABLE 4 A gene list (24 genes) of significant
differentially expressed either up or down among 3-class (HD vs. SP
vs. IN) Entrez P value CD4+/ CD45+/ gene Gene ID Z_JT JT
HD->SP->IN CD3+ CD8+ CD45- AGBL3 340351 -3.50419 0.00046 Y
ELAVL1 1994 -3.50419 0.00046 Y NUPL1 9818 -3.39854 0.00068 Y KLF5
688 -3.22245 0.00127 Y MYLIP 29116 -3.22245 0.00127 Y ATXN3 4287
-3.15201 0.00162 Y ZDHHC17 23390 -2.97592 0.00292 Y PGM3 5238
-2.87027 0.0041 Y ZMYM2 7750 -2.76462 0.0057 Y FAM110C 642273
-2.58853 0.00964 Y Naive OR10J3 441911 3.25767 0.00112 Y SSX3 10214
3.25767 0.00112 Y GNG13 51764 3.29289 0.00099 Y KIAA1751 85452
3.3281 0.00087 Y RGMA 56963 3.36332 0.00077 Y IL1F8 27177 3.43376
0.0006 Y GPHA2 170589 3.53941 0.0004 Y MKL2 57496 3.57463 0.00035 Y
TRPV1 7442 3.64507 0.00027 Y KRTAP13-1 140258 3.75072 0.00018 Y
NOTCH4 4855 3.78594 0.00015 Y OR13C4 138804 3.82116 0.00013 Y KCNE1
3753 3.96203 0.00007 Y
[0084] Identification of a large number of differentially expressed
genes reported in this study of spontaneously tolerant adult renal
transplant patients provides several biomarkers for spontaneously
achieved immune tolerance. The gene expression study reported here
shows that expression differences characteristic of spontaneously
achieved tolerance can be detected in whole blood lysates obviating
the need for more invasive methods of sampling.
[0085] It is evident that subject invention provides a convenient
and effective way of determining whether a subject has a graft
tolerant phenotype, without first removing the subject from
immunosuppressive therapy. As such, the subject invention provides
a number of distinct benefits, including the ability to easily
identify subjects undergoing immunosuppressive therapy that have a
graft tolerant phenotype, and therefore may be removed from
immunosuppressive therapy, so that these individuals can avoid the
adverse conditions, as well as costs, associated with such therapy.
As such, the subject invention represents a significant
contribution to the art.
[0086] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
[0087] Accordingly, the preceding merely illustrates the principles
of the invention. It will be appreciated that those skilled in the
art will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
* * * * *