AU2011249583A1 - Method of determining kidney transplantation tolerance - Google Patents

Description

METHOD OF DETERMINING KIDNEY TRANSPLANTATION TOLERANCE

The present invention relates to a method of determining an individual's transplantation tolerance by determining the level of a number of biomarkers. The present invention also relates to a kit comprising reagents for detecting the levels of the biomarkersv The present invention also relates to a sensor for detecting the expression levels of a plurality of genes that can be used to determine an individual's transplantation tolerance.

Transplantation tolerance is defined as the stable maintenance of good allograft function in the sustained absence of immunosuppressive therapy. In the clinical arena it.is only visible when patients experience stable allograft function despite having Ceased all immunosuppression for an extended period of time_* This state, defined as operational tolerance, has rarely been reported in renal transplantation (1-S), being more common in liver transplantation (6, 7). Lo g term survival of kidney transplants currently depends on sustained drug-induced immunosuppression. However, this is accompanied by increased morbidity and mortality, mainly due to cardiovascular disease, opportunistic infection and malignancy (S). Currently, we do not have the means to identify priori those patients who are developing tolerance to their transplants and who would therefore benefit from partial or complete cessation of immunosuppression. Hence, there is an increasing need to develop assays and identify biomarkers that would allow clinicians to safely minimise immunosuppression, based on a patient's specific immunological profile.

Previous studies have identified biomarkers of tolerance in liver transplant recipients. In particular, cytokine gene polymorphisms were studied in a cohort of paediatric recipients. All of the immunosuppression-free children and the majority of those on minimal immunosuppression displayed low tumour necrosis factor (TKF)-α and Wgh inteimediate interleukin (IL)-10 profiles in comparison with control patients on maintenance immunosuppression (36), In addition there was a difference in dendritic cell subset ratios between the two groups of patients. In comparison with patients on maintenance immunosuppression, circulating levels of plasmacytoid dendritic cells (pDC2), repotted to selectively induce T-helper (Th) 2 responses, were more prevalent relative to monocytoid dendritic cells (pDCl), which induce Thl-type responses, in the immunosuppression-free or minimally immunosuppressed patients (37). A further attempt at identifying biomarkers of tolerance in adult liver transplant recipients using peripheral Wood gene expression profiling and extensive blood celJ immunophenotyping has been performed. It was demonstrated that operationally tolerant patients could b identified with a signature of genes that encoded γδΤ cell and natural killer (NIC) cell receptors, as well as genes involved in cell proliferation arrest (38). They also found in the tolerant patients greater numbers of circulating potentially regulatory T- cell subsets, CD4-C 2S* T-cells and γδ T cells, in particular the VS1⁺ sub-type that has been implicated in irnmunoregulatory processes in epithelial tissues. Interestingly, previously observed differences in ratios of dendritic cell subsets could not be replicated in this patient cohort. The same group, have studied gene expression profiles in the peripheral blood of liver transplant recipients comparing patients where immunosuppression weaning was successful with those where the weaning process was attempted but led to acute rejection requiring reintroducrion of immunosuppression and with healthy controls (3.9). They identified three distinct gene signatures incorporating a modest number of genes (between 2 and 7) that discriminated tolerant and non-tolerant liver allograft recipients and healthy non-transplanted controls. This genomic footprint of operational tolerance has been validated in an independent cohort of 23 additional liver transplant recipients and is mainly characterized by upregulation of genes encoding for a variety of cell-surface receptors expressed by NK, CD8~, and γδ T cells. The previously observed expansion of putative regulatory T cells (CD4^:i'CD25⁺Foxp3~ ySTCR⁺, and 51TCR* T cells) in peripheral blood was replicated in this new set of tolerant recipients. Taken together it appears that a. combination of transcriptional profiling and flow cytometry in peripheral blood may identify liver transplant recipients, who are able to accept their grafts in the absence of pharmacological immunosuppression.

S ulillou et al analysed the TG repertoire in five operationall tolerant kidney transplant recipients and demonstrated in these patients skewed TCR νβ chain usage, observed mainly in the CD8⁺ subset. These cells were also characterized by a decrease in cytokine transcripts (IL10, IL2, JL1 , IFN-v), suggesting a state of hyporesponsiveness (40). There were in addition significantly fewer circulating C^'D8⁺CD28^~ effector lymphocytes in tolerant patients in comparison with patients with chronic allograft rejection suggesting suppression of cytotoxicity in these patients (Baeten et al, 2005 (45)). Later the same group used expression arrays to identify a set of 33 genes that could correctly distinguish with high specificity operationally tolerant kidney transplant recipients from patients with acute and chronic allograft rejection and healthy age-matched volunteers (41). Expression, of co-stimulatory genes and markers of early and late T cell activation were reduced in tolerant patients compared with controls, and although expression of the anti-inflammatory cytokine transforming growth factor-β (TGFfi) was not upregulated in tolerant patients, many TGFp-regulated genes were.

The same group have analysed blood cell phen types and transcriptional patterns in a group of eight operationally tolerant kidney allograft recipients and demonstrated higher absolute number? of circulating B cells and regulatory T cells (CD25^WCD4") in comparison with a control group of patients with chronic rejection, and a significant decrease in FOXP3 transcript levels in the recipients with, chronic rejection (42). Interestingly in this study the blood cell phenotype of clinically tolerant patients did not differ ftom that of healthy individuals, suggesting that operational tolerance is no due to an increased pool of regulatory T cells but may be due to maintenance of a natural state that is lacking in patients with chronic rejection. By contrast, a different group report a more variable TCR-VP repertoire and a higher percentage of CD4⁺CD25^hi8h in long-term stable kidney transplant recipients, two of whom were immunosuppression free, in comparison with patients with chronic rejection, dialysis patients and healthy controls (43).

In Brouard et tl, 2007 (41) they use a set of 49 genes thai gets a maximum sensitivity of 90% in the training set. A set of mostly different 33 genes is said to classify individuals as being tolerant or chronic rejectors. MS4A1, is a molecule also known as CD20, It is expressed in B lymphocytes on the surface. This molecule is present in both gene sets of the Brouard et t, 2007 (41) paper, i.e., the 33 gene and the 49 gene set, Furthermore, Louyet et ctl, 2005 (44) identify MS4A1 as a marker related to the toleration of grafts in a. rat animal model. It is submitted, that, there is a need for an improved method for effectively determining an individual's tolerance to an organ transplant. The. present invention provides a metliod of determining aft individual's immunological tolerance to a kidney organ transplantation comprising determining the level of expression of at least, two genes seLected from the group consisting of TL 5, PNOC, SH2D1B, CD79B, TCL1A, HS3ST1, MS4A1, FCRL1, SLC8A1 and FCRL2 in a sample obtained from the individual.

Tt has been found that by making the determination set out above it is possible to determine, with high specificity and sensitivity whether an individual is immunologically tolerant to the organ transplantation. Specificity is defined as the proportion of true negatives (individuals that are non-tolerant) identified as non-tolerant in the method. Sensitivity is defined as the proportion of true positives (individuals that are tolerant) identified as tolerant in the method. The method provides a highly accurate test that can be performed relatively easil as only a few biomarkers (i.e., the gene, expression levels) are measured. A simple and. effective test of an individual's tolerance tp an organ transplantation is therefore provided.

The term 'immunological tolerance" is well, known to those skilled in the art and refers to. the stable maintenance of good allograft function in the sustained absence of immunosuppressive therapy, in the clinical arena it is only visible when patients experience stable allograf function despite having ceased all immunosuppression for an extended period of time e.g. at least I year.

The method of the present invention can be used to determine an individual's tolerance to a kidney transplant.

It has been found that in individuals who are tolerant of a transplanted organ the level of expression of SH2D1B, PNOC, CD79B, TCL1A, HS3ST1, MS4A1, FCRLl and FCRL2 are raised and that the level of expression of TLR5 and SLCSAl are reduced. Afty combination of the genes can be used to determine an individual's tolerance to an organ transplant. Although all 10 genes can be used in making such a determination, preferably only 2, 3.. 4 or 5 genes are used, to make such a detennination, more preferably only 3 genes are used to make such a determination. It is particularly preferred that tlie method of the present invention comprises determining the level of expression of genes TLR5, PNOC and SH2D1B in a sample obtained from the indi vidual. A positive prediction of an individual's tolerance to an organ transplantation is given by a high level of expression of SH2D1B and PNOC and a low level of expression of TLR5. As will be appreciated by those skilled in the art, the method of the present invention can additionally include determining the expression level of one or more of the following genes CD79B, TCL1A, HS3ST1, MS4A1, FCRU, SLC8A1 and FCRL2.

The method of the present, invention may additionally comprise determining the level of expression of one or more suitable controls. Suitable controls include HPRT, beta-actin and Beta2Microglobulin. The level of the control should not be significantly different between individuals who are tolerant and individuals who are not tolerant.

In a particularly preferred embodiment of the present invention, the probability of an individual being tolerant (P-Tol) is determined by the following formula; P-Tol = e*/(eft-l) wherein

Z - -4,4347+2.7191 *[SH2DB1] - 4,1.98733*[TLR5] + 3.300620*[PNOC] and a P-Tol score of greater that 0.11 is indicative of an individual being, tolerant.

The formula is designed to be applied to gene expression levels determined using micr array analysis. If gene expression levels are determined using other methods, e.g., RT-PCR, the formula may need to be modified. In particular, in a preferred embodiment of the present invention, when the method is performed using RT-PCR the probability of an individual being tolerant (P-Tol) is determined by the following formula: P-Tol = e^z/(e^z+l) wherein

Z - -14.457-^94.156* [PNOC] + 6.289* [SH2DB1] + 5.054* [TLR5] - 1.523 * [PNOC]* [SH2DB 1 ] - 51.584*[PNOC']n^'l R.5] - 2J39*[Sim>Bl]^TLR5] and a P-Tol score of of greater that 0.0602 is indicative of an indi vidual being tolerant.

The expression of each gene is expressed as 2^-dcr, where dCT is calculated as. the CT difference between each gene and. the control gene. Other formulae can. be used which provide a substantially identical measure of probability. Such alternative formulae will be apparent to those skilled in the art and can be easily calculated. The method of the present invention can additionally include determining the level of B cells and NK cells. By additionally determming the level of the B ceils and the NK cells, the specificity and sensitivity of the method can be further improved. In particular, it has been found that in individuals who are tolerant of a transplanted organ the levels of both the B ceils and the NK cells are raised.

The method of the present invention can additionally include determining the level of CD4.+CD25^im T cells. By additionally determining the level of the CD4+ CD25ⁱⁿ¹ T cells, the specificity and sensitivity of the method can be further improved. In particular, it has been found that in individuals who are tolerant of a transplanted organ that the level of the CD4+ CD25^iw T cells is reduced relative to total CD4+ T cells.

The method of the present invention can additionally include determming the level of donor specific CD4+ T cells. The level of donor specific CD4+ T cells can be determined using an intefer on gamma ELISPot assay as described below. By additionally determining the level of donor specific CD4+ T cells, the specificity and sensitivity of the method can be further improved. By measuring the level of donor specific CD4+ T cells, the response of the individual to the donor organ can be determined. In particular, it has been found that in individuals who are tolerant of a transplanted organ mat the level of such a response (i.e the level of the donor specific CD4+ T cells) is reduced.

The method of the present invention can additionally include deterrniiijiig the ratio of FoxP3 to «-l.,2-mannosidase gene expression level of CD4+ T cells- By additionally determining th& ratio of FoxP3 to a-l^-mannosidase gene expression level of CD4÷ T cells the specificity and sensitivity of the method can be further improved. In particular, it has been found that in. individuals who are tolerant of a transplanted organ that the ratio is increased.

The method of the present invention can additionally include determining the ratio of CD i 9+ tq CD3+ cells. By additionally determining the ratio of CD19+ to CD3+ cells the specificity and sensitivity of the method can be further improved. In particular, it has been found that in individuals who are tolerant of a transplanted organ that the ratio is increased.

The method is performed on a sample obtained from the individual. The sample may be any suitable sample from whii?h it is possible to measure the markers mentioned above, Preferably the sample is blood, Serum Or otter blood fractions, urine or a graft biopsy sample. Most preferably the sample is a peripheral blood sample.

SH2D1B (SH2 domain containing protein IB) is a standard term well known to. those skilled in the art. In particular, the sequences of the polymorphic human forms of SH2D1 B are given in the NCBI protein database under accession number GI:42744572₍ version AAH66595.1; accession number 01:54792745, version NP_444512.2; accession number 01:18490409, version AAH22407.1; and accession number Gl:55960297j version C.AI1578G.1.

TLR5 (ToU-iike receptor 5 protein) is a standard term well known to those, skilled in the art. In particular, a few exemplary sequences of the polymorphic human forms of TLR5 given in the NCBI protein database ate under aceesision number 01:80478954, version AAI09119. accession number 01:80475052, version AAIO9120. accession number 01:13810568, version BAB43955.1; and accession number GT:222875780, version AC 69034.1 ,

PNOC (Nociceptin) is a standard term well known to those skilled in the art. in particular, the sequences of the. polymorphic human forms of PNOC are given in the NCBI protein. database under accession number 01:49456885, version CAG46763.1; and accession number 01:49456835 version CA.G46738.1.

CD79B (B-cell antigen receptor complex-associated protein beta-chain) is a standard term well known to those skilled in the art, In particular, the sequences* of the pol>TOorphic human forms of CB79B are given in the NCBI protein database under accession number. GI: 1087009, version AAC6Q654.1; and accession number GI:20987620, version AAH30210J . TCL1 A (T-cell ieukemia lymphoma 1 A) is a standard term well known to those skilled in the art. In particular, the sequences of the polymorphic, human forms of TCL1A are given in the NCBI protein database under accession number GI:48145709, version CAG33Q77.1 ; accession number GI: 148922879, version NP_001092195.1; accession number 01:11415028, version NP_068801.l; accession number 01:13097750, version AAH03574.1; accession number GI:46255821, version AAH1 024,1; and accession number 01:13543334, version AAH0583 .1.

HS3ST1 (Heparan sulfate (glucosamine) 3-O-sulfotransferase 1) is a standard term well known to those skilled in the art. In particular, the sequences of the polymorphic human forms of HS3ST1 are given in the NCBI protein database under accession number 01:116283706, version AAH25735.1;. and accession number 01:34785943_» version AAH57803.1. MS4A1 (Membrane-spanning 4-domains, subfamily A, member 1; B-lymphocyte antigen CD20) is a standard term well known to those skilled in. the art. In particular, the sequences of the polymorphic human forms of MS4A1 are given in the NCBI protein database under accession number GI:23l 10989, version NP._690605.lj accessio number 01:23110987, version NP_068769.2; and accession number 01:12803921, version AAH028Q7.1.

TC Ll (also referred to as THC2438936 herein) (Near 3' of Fc receptor-like protein ί (FCRL1) gene) is a standard term well known to those skilled in the art. In particular, a few exemplary sequences of the polymorphic human forms of FCRL1 given in the NCBI protein database are under accession number 01:55662454, versio CAH73G53.1; accession number GI:556 1.5.1 , version CAH70234;1; accession number 01^*55661511, versio CAH70232.1; accession number GI:21707303, version AAH33.690.1; and accession number OT:117606520, version ABK.4191^'7.1. SLC8A1 (solute carrier family 8 (sodium/calcium exchanger), member 1) is a standard term well known to those skilled in the art. In reticular, the sequences of the ^· ο!γΐΒθφ^η& human forms of SLC8A1 are given in the NCBI protein database under accession number 01:68087008, version AAH98285.1; and accession number 01:67514242, version AAH98308.1. FC1 L2 (Fc receptor-like protein 2) is a standard term well known to those skilled iri the art. In particular, a tew exemplary sequences of the polymorphic human forms of FCRL2 given in the NCBI protein database are under accession number 01:5.5662464, version CAH73063.1; accession number Gi;55662461, version CAH73060.1; accession number GT:46623042, ersion AAH69185.1; and accession number GI:117606518, version ABK41916.1.

FoxP3 (forkhead box P3) is a standard term well known to those skilled in the art. In particular, the sequences of the polymorphic human forms of FoxP3 are given in the NCBI protein database under accession number GI: 146262391, version number ABQ15210.1; accession number 01:219518921, version AAI43787.1; accession number GI;219S17996, version AAI43786.1; accession number GI: 109731678, version ΑΑΠ3404.Ι; accession number GI: 109730459, version AAI 13402.1; and accession number 01:63028441, version AAY27Q88.1.

1,2-alpha mannosidase is a standard term well known to those skilled in the art. In particular, the term refers to the 1 ,2-alpha mannosidase Al form. Sequence of the human form, of 1,2ralpha mannosidase Al is given in the NCBI protein database under accession number GI:24497519, version number NP_005898.2.

For the avoidance of doubt the. specific sequences of the markers mentioned above are defined with respect to the version present in the database at the priority date of the present application. The specific sequences of the markers are exemplary. Those skilled in the art will appreciate that polymorphic variants exist in the human population.

There are numerous ways of determining the level of expression of the genes, including Northern blotting, mRNA microarrays, RT-PC methods, differential display, RNA interference, reporter gene assays and tag based technologies like serial analysis of gene expression (SAGE). Such methods are well known to those skilled in the art (seje for example Measuring Gene Expression by Matthew Avison, 2007, published by Taylor & Francis Group; ISBN; 978-0-415-37472-9 (paperback) 978-0-203-88987-9 (electronic)), Levels of the encoded protein expressed can also s measured to determine the level of gene expression. Numerous methods of determining the level of protein expression are well know to those skilled in the art.

The levels of the various cell types that can be measured in the present methods as additional biomarkers can be detected using any suitable method. For example, flow cytometry using appropriate antibodies can be used. Such methods ar well known to those, skilled in the art..

The level of donor specific hyporesponsiveness of CD4-'- T cells can be determined using any suitable method. Suitable methods include measuring IFNgamma production by ELISA, Luminex methods or by intracellular cytokine production using flow cytometry. In making such measurements, it is preferred that. the method comprises the following steps; a. Having a set. number of CD4+ T cells from the recipient;

b. Stimulating the CD4- T cells with cells from the donor or ceils from an individual that has the same HLA-class II as the donor (at. serological precision), wherein the cells have been irradiated (preferably the cells are PBMC that, have been depleted of T and NK cells (using CD2 and TCRgd antibodies);

c. Stimulating the CD4~ T cells with cells from a "3^rd party" that has similar HLA-class-II mismatches as those present between donor and recipient (preferably the cells are PBMC that have been depleted of T and NK cells (usin CD2 and

TCRgd antibodies);

d. Stimulating the CD4~ T cells with cells from a "4^th party" that has complete HLA-cJass-II mismatch to both the donor and the recpient (preferably the cells are PBMC that have been depleted Of T and NIC cells (using CD2 and TCRgd antibodies); and

e. determining the relative levels of IFNgamma. production by the CD4+ T cells.

A suitable method for determining the level of donor specific Ct>4+ T cells is described, herein below. In order to determine whether the level of the markers referred to above, is greater than (high) or less than (low) normal, the normal level of a relevant population of non-tolerant individuals is typically determined. The relevant population can be defined based on, for example, organ transplanted, level and type of immunosuppressive medication, ethnic background or any other characteristic that can affect normal levels of the markers. Once the normal levels ar known, the measured levels can be compared and the significance of the difference determined using standard statistical methods. If there is a substantial difference between the measured level and the normal level (i.e. a statistically significant difference), then the individual from, whom the levels have been measured may be considered to be immunologically tolerant.

The technology described herein allows the monitoring of an individual's tolerance to the graft (i.e. transplanted organ) and thereby can identify individuals that can stop taking immunosupression medication or reduce the level of immunosupression medication. The present technology may also assist with the management of immunosupression protocols and the post-transplantation management of transplant organ recipients.

The present invention also provides a sensor for detecting the expression levels of at least 2 genes selected from the group consisting of TL 5, PNOC, SH2D1B, CD79B, TCLIA, HS3ST1, MS4A1* FCRLl, S.LC8A1 and FCRL2. Preferably the sensor is for detecting the expression levels of the '11,115 PNOC and SH2D1B genes. Suitable sensors for monitoring the expression levels of genes in a microarray are well know to those skilled in the art and include mRNA. chips, protein expression sensor, etc. The sensors generally comprises one or more nucleic acid probes specific for the gene being detected adhered to the sensor surface. The nucleic acid probe thereby enables the detection of a gene transcript from the target gene. Preferably the sensor is additionally for detecting the expression of one or more, preferably all, of the following genes CD79B, TCLI , HS3ST1, MS4A1, FCRLl, SLC8A1 and FCRL2. The present invention also provides a kit comprising reagents for detecting the level of expression of at least 2 genes selected from the group consisting of TLR5, PNOC, SH2D1B, CD79B, TCLIA, HS3ST1, MS4A1, FCRLl, SLCSAl and FCRLl Preferably the kit comprises reagents for detecting the level of expression of th TLR5, PNOC and SH2D1B genes. Preferably the kit further comprises reagents for detecting the level of expression of one or more of the following genes CD79B, TCL1A, HS3ST1, MS4A1, FCRL1, SLC8A1 and FGRL2. ^rrhe reagents for detecting the level of expression of the genes are preferably reagents for detecting the level of gene expression of the genes by RT-PCR.

The kit can also include a computer programmed with an algorithm for calculating the individual's probability of being tolerant, instructions and other items useful for performing the method described herein. Particular aspects of this technology are described by way of example, below with reference to the following figures.

Figure 1 shows the flow cytometry analysis of peripheral blood lymphocyte subsets of the Xraining (A-D) and lest set (E-H). Lymphocyte subsets were defined as: B cells as CD19+ lymphocytes (A,E), cells as CD56÷.CD3- lymphocyte (B,F); T cells as CD3+ lymphocytes (C,G). Ratio of CDl9*-;CD3+ is shown (D,H). Median and interquartile range are shown. Two-sided p values for Mann- Whitne U test comparisons between Tol-DF patients and other groups are shown (*** p<0.001, ** p<0.01 or * p<0.05). P values for comparisons petween other study groups are shown in Table 5.

Figure 2 shows the flow cytometry analysis of peripheral blood T cell expression of CD25 of the training (A & B) and test sets (C & D). Median and interquartile range of the percentages of C 4÷ T cells with intermediate (CD4+CD25mt) and high (CD4+CD25w) CD25 expression are shown. Two-sided p values for Mann-Whitney U test comparisons between Tol-DF patients and the rest of the groups are shown (**) p<0_*01. or (*) p<0.05. P values for comparisons between other study groups ate shown in Table 5.

Figure 3 shows (A) Percentage of patients per group with positive detection of serum donor specific (DSA) and.non specific (NDSA) anti-HLA class T (CI) and class II (CII) antibodies in the training s t. (B) Renal function of patients in whom complement-fixing (IgGl, IgG3) or non-complement-fixing (IgG2, IgG4) DSA were present (+ve) or absent (- ve). Median and interquartile range is shown. Two-sided p values for Mann-Whitney U test comparisons between groups are displayed (* p<0.05). Of note, DSA levels were absent in t olerant recipients. Figure 4 shows the IFNy ELI Spots used to detect direct pathway alloresponses in patients of the (A) training and (B) test. set. The number of IFNy producing cells in recipient CD - T cells was calculated (background-deducted) when stimulated with donor cells and third party cells (3rdP), to obtain a frequency of responder cells. Median and interquartile ranges tor the ratio of responder frequencies on donor:3rdP stimulation are shown, Ratio values >L5 indicate hypoiesponsiveness to donor. Two-sided p values for Mann- Whitney U test comparisons between groups are shown (** p 0.01, * p<0,05). individual patient IFNy ELISpot responder frequencies to donor and 3«iP are shown in Figure. 1.0. ,ζ: Wilcoxon test between donor and 3r<»P frequencies p < 0,05.

Figure 5 shows the qRT-PCR gene expression analysis of peripheral blood expression of FoxP3 and a-1,2-mamiosidase, A ratio of the expression values of FoxP3 and -1,2- mannosidase was calculated patients of the training Set (A) and test set (B). Median and interquartile range is shown. Two-sided p values for Mami- Whitney U test comparisons between Tol-DF and other groups are shown (*** p<0.QQl, ** p<0.01). Statistical values for comparisons between other study groups are shown in Table 6. Individual expression values for FoxP3 and a-1,2-mannosidase are also shown in Figures 11 A and 11 B, respectively.

Figure 6 shows the ROC curves of the training (A) and test set (B) generated using up to 10 highest ranked genes (black lines). Significant differential gene expression was detected by microarray analysis of peripheral blood. Using a binary regression model for classification ROC curves (Tol-DF vs non-tolerant groups, excluding HC) were generated using the top 1Q ranked significant genes identified by four-class Kruskal-Wallis analysis of microarra data. Genes were ranked within the training set based on their value with 1% FDR. The same 2-class model was used to assess the diagnostic capabilities of the same genes to detect Tol-DF recipients within the test set. Figure 7 shows the ROG curves of the training set (A) and test se (B) generated using crossplatforin biomarkers and genes identified by microarray analysis_* Two-class RQC curves (Tol-D vs non-tolerant groups, excluding HC) were generated using 4 biomarkers: BAf lymphocyte ratio, % CD4+CD25*nt, ratio of anti-dono.r:anti-3n!P ELISpot frequencies and ratio of FoxP3/a- 1,2-mannosidase expression, combined with sequential addition of 10 most significant genes. Estimated probabilities of patients from each study group of the training set (C) and test set (D) of being Classified as tolerant based on the cross-platform biomarker signature of tolerance (4 biomarkers+lO genes) was calculated using a binary regression procedure.

Figure 8 shows the analysis of peripheral blood B cell subsets in training and test set patietits, (A) B cell subsets were analysed by gating on GDI 9+ lymphocytes and defined as follows: late-memory B cells CD19+CD27+IgD-CD24+CD38-/hu; natve/mature B cells CD19-CD27-CD24intCD38int; T1/T2 transitional B cells 0Ο19+0Ο27.0Ο2 Η·ΟΟ3$Μ. Percentages of na'ive B cells (B), T1 T2 transitional B cells (0) arid memory B cells (D) of total B cells. Ratio of the percentage of ΊΊ/Τ2 transitional: memory B cells (E). Median and interquartile range is shown, When significant, p values for 2-tailed Mann-Whitney U test comparisons between groups are shown (* p<0.05, ** p<0,01). Figure 9 shows the analysts of B cell cytokine production. B cell production of (A) TGFfl, (B) IL-10 and (0) IFNy was assessed by intracellular cytokine staining alter in vitro stimulation of PBMC with phorbol 12-myriState 13-acetate and ionomyein. Results are expressed as the number of cytokine producing B cells (gated on CD 19* lymphocytes) detected per 1x10* B cells analysed by flow cytometry in stimulated (+) and unstimulated (-) cultures, with median and interquartile range shown for each group. Figures (D^'-F) show the number of cytokine producing cells per lxl 0* stimulated B cells expressed as a ratio of each cytokine response. Figure G shows ΤθΡβ and IFNy cell cytokine responses, where each patient is represented by a filled circle and black filled circles represent Tol-DF patients, showing that B cells of Tol-DF patients have a higher capacity to produce TGFp rather than Ε⁷Νγ. P values for Wilcoxon-matched pair test (A-C) and 2-tailed Marm- Whitney ϋ test comparisons between groups (D-F) are shown (*p<0.05, **p<0.01, ***p<0,001). Sample n; HC=8, Tol-DFH 0, s-LP=8, s-C I=16, CR=4.

Figure 10 shows the cellular functional assays detecting direct pathway alldresponses by IFNy ELISpot in the (A) training and (B) test sets. The number of IFNy producing cells in recipient CD4+T cells was calculated. Data shows the frequency (1 responder / n cells) and median group frequency (black bar) of T cell responses to donor APCs (o anti-Donor response) or equally mismatched 3«i party APCs (an inverted triangle indicates an antt-3rdP response) after deducting background. Two-sided p values for Wilcoxon matched paired test (** p<0.01) between donor and 3niP frequencies is shown.

Figure 11 shows qRT-PCR analysis of (A) FoxP3 and (B) a-l ,2-mannosidase expression in whole peripheral blood samples of the training and test sets. Expression levels of FoxP3 and a-1,2-mannosidase are expressed as units relative to the expression of HPRT. When significant, p values for 2-tailed Mann- Whitney U test comparisons between groups are shown (* p<Q.05, ** ρΟ.ΟΪ, *** pO.OOl). Figure 12 shows the correlation of gene expression by microarray and qRT-PCR for selected genes that displayed significantly differential expression (A - E) for training set. Signal intensity data of each probe on the microarray was calculated for each patient and expression relative to^' HPRT was then calculated for each gene as log2 [gene of interest] - log2 [HPRT]. qRT-PCR data are depicted as units / HPRT . Pearson and Spearman rank correlation coefficients and p- values are shown. RT-PCR gene expression data for the same set of genes is shown in F - J). Median and interquartile range for each group is shown. Statistical comparison between groups was assessed by Mann- Whitney U test and significant p values are shown (*p<0.05, ** pO.01, *** p<0.001). Figure 13 shows the correlation of gene expression by microarray and qRT-PCR for selected genes that displayed significantly differential expression (A - B) for test set. Signal intensity data of each probe on the microarray was calculated for each patient and expression relative to HPRT was then calculated for each gene as log2 [gene of interest] - Iog2 [HPRT]. qRT-PCR data are depicted as unite / HPRT . Pearson and Spearman rank correlation coefficients and p-va ues are shown. RTPCR gene expression data for the same set of genes is shown in F - J). Median and interquartile range for each group is shown. Statistical comparison between groups was assessed by Mann-Whitney U test and significant p values are shown (*p<0.05, ** p<0.01, *** p<0.001). Figure 14 shows the results of a binary regression model used to cross validate selected combinations of biomarkers. MSE == Mean. Squared Error, SD= Standard Deviation.

Figure 15 is a Bdxplot showing expression levels of the 3 genes (SH2DB1, P OC and TLR5) per group. Figure 16 is ROC curve obtained using a Logistic Regression classifier with the main and interaction effects between the three genes TLR5, PNOC and SH2DB1. AUC, Sensrtivity and Specificity are calculated for the optimal cutoff in this sample (Le. 0.0602).

Figure 17 shows Boxplots of Probabilit of Tolerance estimated by logistic regression classifier using the 3 genes (SH2DBU PNOC and TLR5).

Figure 18 shows a classification tree estimated with 3 genes, TLRS, SH2DB.1 and. PNOC. The Figure shows cutoffs at each split and probability of each outcome assigned at each terminal node (0 -above- being non-tolerant, and 1 -below- being tolerant).

Figure 19 shows a series of Boxplots illustrating the application of the tree's cutoffs. Figure 20 shows an ROC curve resulting from the use of the estimated classification tree with 3 genes to predict tolerance.

Figure 21 shows a barplot with frequencies of probability of tolerance assigned by the classification tree by patient group.

EXAMPLES

Materials and Methods

Study Patients:

Training set description:

This oohort of patients recruited by the Indices of Tolerance network (ΪΟΤ) consisted of 71 kidney transplant recipients and 19 age and sex matched healthy controls (HC). Five patient groups were included: eleven functionally stable kidney transplant recipients (serum creatinine (CRT) < 160 μιηοί Ι and < 10% rise in the last 1.2 months) despite having stopped all their immunosuppression for more than one year (Tol-DF), eleven patients with stable renal function (same criteria) maintained on less than 10 mg/day of prednisone as the only immunosuppressive agent (s-LP), ten patients maintained on immunosuppression (Azathioprine and Prednisone) in the absence of a calcineurin inhibitor since transplantation (s-nC I), thirty patients maintained on standard calcineunn- inhibitor therapy (s-CNI), nine patients -with biopsy proven (all reevaluated for this study) and immunologically driven chronic rejection (CR). Patient clinical characteristics are described in Table 1. All samples were processed and analysed in a blinded fashion.

Test set description:

An independent set of kidney transplant recipients were recruited in the USA (the ITN cohort). The ΓΤΝ cohort consisted of (1) "Tol-DF" (n=24) functionally stable kidney transplant recipients (serum creatinine (CRT) within 25% of baseline) despite having stopped all their immunosuppression for more than one ean (2) "Mono" (n=ll) patients with stable renal function that are maintained on monotherapy with steroids; (3) "s-CNT subjects (n - 34), with clinically stable renal function using the same criteria as Tol-DF while on maintenance with a triple drug immunosuppressive regimen (including a. caleineurin or mTOR inhibitor, an. anti-proliferative agent and corticosteroids) and. "CAN'* participants (n - 20), defined as those with chronic allograft nephropathy and impaired renal function (50% increase in their baseline CRT at- time of enrolment relative to their initial post-transplant baseline) due to presumed immune mediated allograft rejection. An additional group of 31 healthy control volunteers (HC) with no known history of renal disease/dysfunction or evidence of acute medical illness was enrolled. Group characteristics are summarised in. Table 3. Whole blood rnRNA and frozen PBMC were received by labs performing the selected validation assays described.

Blood samples:

The training set samples were processed in all cases within 24 hours of venesection. PBMCs were obtained by density gradient centrifugation using Lymphocyte Separating Medium (PAA Laboratories, Somerset UK). Cells were washed and resuspended in 10% DMSO (Sigma, Dorset UK) and human serum (Biowest. France) and frozen immediately at -80°C. After 24 hrs cells were transferred into liquid nitrogen and kept until use. Flow cytometry on PBMC:

Thawed PBMC were washed .and resuspended at 1 x lOVmL. Titrated amounts of fluorochrome-conjugated monoclonal antibodies were used to identify leucocytes, CD45+CD14- for lymphocytes, CD3+ for T ceils, CD19÷ for B cells, CD56+CD3- for NK. cells, CD4_*CD3+for CD4 T cells, CD8+CD3₊for CD8 T cells, B cell subsets ere defined as previously described (3,0), as CD19₊CD27»IgD.CD24fCD38-rtnt for late-meinory B cells, CD19+CD27- CD24intCD38im for naive/mature B cells and CDl9+CD27-CD24-CD38bi for T1/T2 transitional B cells (All from Caltag, Burlingham USA). Cells were fixed with 1% paraformaldehyde/PBS and data acquired on a FACScatibur within 48 hours. CD25 expression was studied on CD4+ T cells as described in (31). B cell production of TGFjJ (from R&D systems), IL-10 and IFNy (both from ©Bioscience UK) was assessed by intracellular cytokine staining on in vitro stimulated PBMC with 500ng/mL phorbol 1 - myristate 1. -acetate and ΙμΜ ionomycin in the presence of 2μΜ monensin and It g/mL brefeldin-A for 5 hours at 37^SC. A minimum of 10,000 CD19 events were acquired for each sample.

Anti-donor antibody detection:

Peripheral blood was collected in clotting activator vacutainers (Becton Dickinson, San Jose USA) and allowed to clot for a minimum of 2 and a maximum of 24 hours. Samples were centrifuged and collected serum stored at -80°C until use.

Screening for IgO anti-HLA antibodies of any specificity by x AP® (Luminex) technology (32). After washing, HLA-coated Luminex screening beads and 12.5μΙ of patient serum or control serum were added on a plate and xed gently for 30 minutes in the dark. Plates were washed three times and PE-conjugated goat anti-human IgG (1:10) added to each test well. Plates were incubated, for 1 hour, wash buffer added and then data collected using theLuminexioo instrument, as recommended by the manufacturers. Screening for IgG subclass and anti-HLA broad specificity:

Positive sera were tested for IgG subclass identification and class 1, and class II broad specificity distinction. Screening was performed using class I and II Luminex identification kits (Quest Biomedical). Secondary antibodies used for detection of bound patient antibodies were as follows: anti-human IgGl conjugated to biotin (clone 8c/6-39, Sigma) anti-human IgG2 conjugated to biotin (clone HP-6014, Sigma), anti-human IgG3 conjugated to biotin (clone HP-6050, Sigma), anti-human IgG4 conjugated to biotin (clone HP-6050, Sigma), and streptavidin-phycoerythrin (Calbiochem).

Cell fractions for functional assays: PBMCs were thawed on the day of the assay. T cell subsets CD4* and CD4+CD25- (CD4+ depleted of CD25+ cells) were separated using standard methods of negative immune- isolation as previously described (33). Purity was verified by flow cytometry. In particular, the inventors have used two specific sets of monoclonal antibodies with a fluorochrome bound to stain isolated peripheral blood mononuclear cells. The following analysis was used on lymphocytes (selected by forward side and on CD45-CD1.4- expression). The first set included: TCR gamma'delta-FITC, CD25-PE, CD4-APC. The level of CD4+CD25^int was obtained selecting the CD4-H T cells and within this subset studying the. intermediate expression of CD25 (defined from GD25negative to CD25 as high as CD4NEG cells showed, CD25 high cells are excluded from this gate). The second set included: CD3- FITC, CD56-PE and CD19-APC. The variable "B.T" was obtained selecting the CD19+ cells and dividing that percentage by the percentage of CD3+ lymphocytes.

Donor, surrogate donor and arty cell s:

Cells from the 31 living kidney donors were used for the 71 donor-specific cellular assays, on the training set, and 28 donors for 64 cell samples on the test set. Where donor blood was unavailable, surrogate donor cells were obtained that had equal HLA class II expression as the original donor. These cells and similarly mismatched 3^r* party cells were used from: healthy volunteers from the Anthony Nolan bone marrow registry, HLA-typed healthy volunteers and splenocytes collected at the time of cadaveric donation at the Hammersmith and Guy's Hospitals in London, Similarly mismatched 3^ri party ceils were selected by the number of HLA mismatches for class II (HLA-DR and HLA-DQ) when compared to the relevant donor and recipient.

MLR cultures for ELISpot :

Human IFNy-ELiSpotPRO (Mabtech, Sweden) kits were used and developed according to manufacturer's instructions. Background deducted positive spots were enumerated using an automatic image analyser for ELISpot plates (AID, Germany). Quantitative assessment of direct pathway donor antigen-specific responder T cell frequencies as made by stimulating recipient CD4+ T cells with T cell- and N cell-depleted PBMCs (APCs) separated from either donor PBMCs or HLA-typed 3^,ld party cells. Allogeneic MLR cultures were performed over 24 hours, Duplicates were set up with three doubling dilutions starting typically at 2x10^s responder cells per well. The. ratio of stimulator to responder cells was kept constant by always using half the number of APCs compared to the number of responder cells used in the top dilution, typically 1x10^s fesporiders per well Donor reactivity was expressed as a ratio of frequency to donor and frequency to 3^rt party. The inverse of the frequency was recorded in the database (i.e. 1 in 54,000 cells Was recorded as 54,000), therefore ratio values >1.5 were defined as indicating a hyporesponse to donor stimulation.

Blood sampling for gene expression analysis:

For the training set cohort peripheral vein blood was drawn directly into PAXgene Blood R A tubes (QIAgeflj Crawley UK). Whole-blood RNA was extracted using the PAXgene Blood RNA Kit including DNAse I treatment (QIAgen). For the test set cohort peripheral vein blood was drawn directly into Terap s™, Blood. RNA tubes (Applied Biosystems Inc.). Whole-blood RNA was extracted according to manufacturer's instructions. Total RNA samples were subjected to gene expression analysis by RT-PCR and microarrays.

Samples for mRNA studies;

95 samples from the training set were used that consisted of: 13 samples from 10 Tol-DF patients, 16 samples from 11 s-LP patients, 8 samples from 8 s-nCNI patients, 40 samples from 28 s-CNI patients, 10 samples from 9 CR, patients and 8 samples from 8 HC. As the test set 142 samples were used that consisted of: 31 samples from 23 Tol-DF patients, 14 samples from 11 Mono patients, 52 samples from 34 s NI patients, 25 samples from 18 CAN patients and 20 samples from 20 HC.

RNA Quality Control :

Quality and integrity of ?AXg,me<§(training set) and Tempus™(te,^y/ set) purified RNA were determined using the Agilent RNA 6000 Nana Kit on t e Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was quantified by measuring a60m on the ND-1000 Spectrophotometer (NanoDrop Technologies). RNA Amplification and Labelling:

Sample labeling was performed as detailed in the "One_^Colour icroarray-Based Gen Expression Analysis" protocol (version 5.5, part number G4140-9004O). Briefly, 0.5 μg of total RNA was used for the amplification and labeling step using the Agilent Low RNA input Linear Amp Kit (Agilent Technologies) in the presence of eyanine 3-CTP. Yields of cRNA and the dye incorporation rate were measured with the ND-1Q00 Spectrophotometer.

Hybridization of RISET 2.0 Agilent custom Microarrays:

All whole blood samples, were hybridized on the RISET 2,0 microarray platform. This is a custom Agilent 8x15K 60mer oligonucleotide microarray comprising 5,069 probes represented in triplicates. Probes selected corresponded to 4607 genes with a valid Entrez Gene ID and an additional 407 probes which could not be assigned to a valid Entrez Gene ID, Probe design was optimised for the detection of multiple transcript variants of a gene, on optimized, hybridization properties of the probes, and avoiding crosshybridization. The hybridization procedure was performed after control of RNA quality and integrity and according to the "One-Colour Microarray-Based Gene Expression Analysis" protocol using the Agilent Gene. Expression Hybridization Kit (Agilent Technologies). Briefly, 0.6 μg C.y3 -labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 hours, 65 °C) to RISET 2.0 microarrays. Following hybridization, the microarrays were washed once with Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated ( TC) Agilent Gene Expression Wash Buffer 2 containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetanitriie for 30 sec,

Scanning and data analysis:

Fluorescence signals of the Agilent Microarrays were detected using Agilent's Microarray Scanner System (Agilent Technologies, Inc.). The Agilent Feature Extraction Software (FES v.9.5.1.1) was used to read out and process the microarray image files. To determine differential gene expression, FES derived output data files were further analyzed using the Rosetta Resolver gene expression data analysis system (v.7.1.0.2., Rosetta Inpharmatics LLC). First, an artificial common reference was computed from all samples included in the IOT dataset. Using this baseline, log2 ratios were calculated for each gene and sample. Additionally, p-values indicating the reliability of an observed difference between a sample and the common reference were calculated for each gene applying the universal error model implemented in the Rosetta Resolver software (34).

Annotation enrichment analysis: Lists of genes found to be discriminatory between different sample groups, and common to both study sets, were analysed for a statistically significant, enrichment of biological pathway annotation terms in comparison to the complete RISET 2,0 microarray configuration. Term, enrichment relative to the expected background distribution was scored using Fisher's exact test. Annotations were derived from different sources, e.g. Gene Ontology (GO, www.geneontology.org), signaling pathway membership, sequence motifs, chromosomal proximity, literature keywords, and cell-specific marker genes.

Quantitative RT-PCR analysis;

200ng of whole blood total R A was reverse transcribed using the qPCR 1st Strand synthesis kit (Stratagene) and synthesised cDNA was subjected to RT-PCR analysis.

Microarray data validation:

A selected set of genes identified by microarray gene expression analysis were validated by quantitative RT-PCR. Quantitative RT-PCR was performed for the following genes using pre-made TaqMan panels from Applied Biosystems: Hs01017452..ml B lymphoid tyrosine kinase (BLK)_S Hs00236881_mlCD79b molecule (CD79b\ Hs01099196 ml heparan sulfate (glucosamine) 3-029 sulfotranaferase 1 (HS3ST1), HsO 1592483_m.l SH2 domain containing IB (SH2D1B), HsQ0172040_ml Tcell leukaemia (TCL1A).

Other assays screened in the training set.'

The inventors also performed indirect pathway IFNyEUSpot, and direct and indirect pathway trans-vivo DTH assays. RT-PCR amplification for cytokine genes was performed, on direct and indirect pathway cultures of donor and recipient cells and TCR-repertoire profiling was achieved by TCR-Landseape analysis (data not shown).

Statistics:

Nan-parametric tests were used to estimate statistical significance as n < 20 in many group comparisons and data did not conform to a normal distribution. Wilcoxon signed rank test was used to compare responses within the same group of patients. Mann- Whitney U tests were used to compare medians between patients groups. To compare associations between clinical variables, usually recorded as categorical data and presence or absence of anti- HLA antibodies, we used the Fisher Exact test. Two sided p values were used to indicate a significant difference when it was < 0.05. Statistical analysis of microarrays and biomarkers;

Significantly altered expression detected by microarray was statistically determined using four-class analysis and the Kruskal-Wallis test with Benjam i-Hochberg adjustment for False Discovery Rate (FDR) at 1%. The inventors chose a non-parametric test for this analysis as the data in some cases appeared to deviate from normality. A similar procedure was used to rank the biomarkers (tested on the log-scale_> with missing values set equal to the sample-wide mean). To evaluate the predictive power of a number of variables to detect tolerant patients the inventors used receiver operating characteristic (ROC) curves. To build these, firstly four class analysis identified differentially expressed probes of Tol- DF within the training set and were ranked using the Kruskal-Wallis test. Then the topmost significantly differentially expressed probes were added in a binary regression model, and used to perform classification within sample. The binary regression procedure was used to compute probabilities p[l],.....,p[n] of being -a Tol-DF patient for each subject. The ROC curve was produced by varying a probability threshold, between zero and one; for each value of the threshold t, a 2x2 classification table of Actual class versus Predicted class for subject i set equal to "Tol-DF" if p[ j > /. Bootstrap resampling of the subjects indicated that the within-sample classification results were robust. For the (est set_f the same probes from the training set analysis were used.

Tolerant renal transplant patient demographics:

The training set comprised of 71 European kidney transplant recipients and 19 age and sex-matched healthy controls (Table 1). The Tol-DF group had a high percentage of cadaveric donors (7 out of 1 1), a high degree of HLA mismatching (median mismatches 4.0), were predominantly male (9 out of 11), had varied causes of end stage renal failure and some evidence of sensitising events, such as blood transfusions and previous transplants (Table 2). These patients had relatively imeventful posttransplant courses with only 1 patient having a documented episode of acute cellular rejection (ACR). The. period of being immunosuppression-free varied from 1 to 21 years. The Tol-DF group of the test set (Table 3) consisted of 24 patients most of whom had received their transplant from a highl HLA-matehed living donor (median mismatches 0.0) and had ceased to take immunosuppression for periods from 1 to 32 years. Tol-DF recipients displayed increased numbers of B and NK lymphocytes. As shown in Figure 1, Tol-DF patients of the training set displayed an increased percentage of peripheral blood B and NK cells, and a corresponding decrease in the percentage of T cells. When expressing the percentages of B cells and T cells as a ratio, Tol-DF patients displayed the highest Tatio compared to all other, study groups including HC. For 6 Tol-DF patients and 10 s-CNI patients it was also possible to calculate the absolute number of cells per lymphocyte subset. This showed that the altered ratio was due to an expansion in B and NK cell numbers and not a reduction in T cell numbers, as none of the Tol-DF group were lymphopenie (Table 2). In line with the findings in the training set, Tol-DF patients of the test set also showed elevated percentages of peripheral bloqd B cells and. a higher tatio of B:T cell percentages (Figures 1 B & H) compared to all other groups except HC. Given the distinct increase in peripheral blood B cells detected in Tol-DF patients, B cell subset analysis (Figure 8) and cytokine production (Figur 9) were further assessed hi selected patients from both study sets. The Tol-DF group displayed a trend in redistribution of B cell subsets* with a decreased late-memory pool and concomitant increase in transitional and naive B cell subsets. When examining the percentages of B cell subsets as a ratio, Tol- DF patients were found to have a significantly lower proportion of memory and higher proportion of transitional B cells compared to CR patients. A significant proportion of B cells from Tol-DF patients were found to produce TGFp upon in vitro stimulation, rather than TL-10 or ΙΚΝγ. However, no significant differences in production of IL-10 were detected for any study group, he capacity of B cells from each patient group to produce either cytokine on stimulation was analysed by calculating a ratio of the number of B cells producing each cytokine. This suggested that B cells of Tol-DF patients had a skewed cytokine response, with a higher propensity for TGFp production than B cells from other study groups.

Tolerant recipients had fewer activated CD4+T cells in peripheral blood. Expression of CD25 by CD4+ T cells was analysed as described above. Tol-DF patients in the training set were found to. have significantly lower percentages of circulating CD4+CD2Sint T cells, broadly thought of as activated T cells (9, 10) (Figure 2A compared to HC, s-LP₃ s-nCNI and CR groups. Interestingly, significant differences in the percentages of CD4+CD25hi regulatory T cells were only detected between patients on full immunosuppression with s- C I and other groups (Figure 2B). Similar results were also found in the test set, with Tol- DF patients having significantly lower percentages of CD4÷CD25im T cells compared to s- CNI and CAN groups, but again no differences in the percentages of CD4+CD25 i Tregs were detected between Tol-DF and any other study group (Figures 2C and 2D). Statistical comparisons between other groups are shown in Table 5. When we tested the ability of enriched CD4-CD.25h> T cells to suppress autologous T cell proliferation Induced by polyclonal stimulation, no significant differences were found between any of the patient groups or healthy controls (data not shown). Furthermore, Tol-DF patients did not display higher percentages of other regulatory T ceil subsets such as CD3-CD8+CD28- or CD3+CD -CD8- T cells (data not shown). The majority of tolerant recipients did not have detectable anti-donor HLA specific antibodies. Serum non-donor specific antibodies (NDSA) were, detectable in some patients from all study groups of the training set (Figure 3A) by Luminex xM AP analysis. Witl jn this cohort, no Tol-DF patients had detectable donor-specific antibodies (DSA),, whereas all other groups had some patients with detectable DSA, with almost half of the CR patients having detectable both donor, and non-donor specific anti-HL A class I and class 11 antibodies. Similar to the training s¾r, only .1 of 22 Tol-DF patients within the test set had detectable DSA (data not shown). Interestingly, in general DSA-positive patients had worse graft function than DSA-negative patients, with an estimated glomerular filtration rate of 31 (range 17-87) in DSA-positive patients compared to 60 (range 13-94) in DSA- negative patients. The possible pathogenicity of detected anti-donor antibodies was tested in the training set (Figure 3B). In 7 of .20 patients with anti -class I antibodies and 4 of 13 patients with anti-class II antibodies, the inventors found complement-fixing isotypes (IgGl and IgG3); the remaining positive cases were exclusively of non-cpmplement-fixing isotypes. Detection of NDSA anti-class I and anti-class H antibodies was significantly associated with having recei ved a previous transplant and having detectable panel reactive antibodies before, transplant (Fisher Exact test p<0.05). but not with previous pregnancies, blood transfusions, graft dysfunction, or episodes of ACR. In contrast DSA anti-class II antibodies were associated with previous epi$odes of ACR and the number of HLA mismatches between donor and recipient (Fisher Exact test <0,05).

Tolerant patients have lower frequencies of direct pathway antirdonor IFNyCD + T cell responses. Comparison of direct pathway CD4+T cell anti-donor and anti-3^rt party (equally mismatched to donor) responses was assessed by IFNy ELISpot. Tol-DF patients had significantly higher ratios of fesponder anti-donor:anti 3^ril-party frequencies indicating donor-specific hyporesponsiveness, compared to all other stable patient groups within the (mining set (Figure 4A; individual responder frequencies against donor and 3^rd party are shown in Figure 10). Donor-specific hyporesponslveness was not mediated by Tregs, as depletion of CD25+ cells from responder T cells did not result in an increase in responder frequencies (data not shown). As the Tol-DF group of the test set was frequently completely HLA matched with their donor, anti-donor and anti-3^rf party IFNyresponses were generally very low (responder frequencies >1 200,0Q0). Despite Ibis, th trend in anti-donor responses in this Tol-DF group was generally reproduced although a significant difference compared to other groups was not detected (Figure 4B).

Tolerant recipients displayed a higher ratio of expression of FoxP3 and a-l ,2-mann0sidase genes in peripheral blood Whole blood gene expression levels of FoxP3 and. a-1,2- mannosidase, both of which have been shown to correlate with anti-donor immune reactivity after transplantation (11) were analysed by q T-PCR (Figure 1 1). When calculating the ratio of FoxP3 and a-1,2-mannosidase expression, a significant difference was detected between Tol-DF and the CR and HC groups of the training set (Figure 5 A). The patient groups displaying the highest ratio were HC, s-LP and Tol-DF whereas the ratio was dramatically lower in C (Mann-Whitney U test p values for comparisons between groups other than Tol-DF are shown in Table. 6). This ratio significantly correlated with eGFR and inversely correlated with serum creatinine (Pearson Coefficient: 0.372 p=0..OQ2 and -.0.299 p=0,014 respectively, data not shown). When the same analysis was performed on the test set, the ratio in Tol-DF patients was significantly higher than in all other patient groups except HC (Figure 51¾), Combining the training and test set observations shows that tolerance is associated with a high ratio of peripheral blood FoxP3 and a- 1 ,2-mannosidase expression.

Tolerant patients exhibited a distinct gene expression profile. The RISET 2.0 custom microarray, designed with a focus on transplantation research, was assembled by the inclusion of 5,069 probes and used to analyse the expression of 4607 genes (valid Entreir Gene ID) in peripheral blood samples. A four-class analysis of microarray data was performed on the training set (Figure 6). Significantly altered gene expression detected between Tol-DF patients and other comparator groups, stable recipients (s-CNI, s-nCNI and s-LP), CR and HC. was statistically determined using, the ruskal-Wallts non- parametric test with adjustment for False Discovery Rate (FDR) at 1% (1 ). The HC group was included in this analysis in order to address the lack of immune-suppression in Toi-D patients compared to the other study groups. Two hundred and sixty probes* corresponding to 255 genes, were identified as being significantly differentially expressed between the study groups. When a similar analysis was performed on the test set, 1 ,378 probes, corresponding to 1352 genes, with significantly altered expression were identified, with 174 probes (170 genes) found to be common between both the training, and test sets (Table 7).

Microarray expression was validated by qRT-PCR analysis of several probes that were highly ranked within the list, and including probes detected to be either down- or up- regulated. Expression of all the genes was highly correlated using both assays (Figure 12 A-E) and qRT-PCR quantitated expression of the selected genes was significantly different to at least one of the other patient groups, depending on the gene studied (Figure 12 F-J), Interestingly, the median expression levels in Tol-DF patient samples for all selected genes was very similar between the training and test set, although due to the higher sample number in the test set thtir correlation coefficients were generally higher (Figure 13 A-E). Furthermore, gene expression in Tol-DF patients Was significantly different to most of the other groups for 4 of 5 genes tested (Figures 12 & 13 F-J). Median probe expression values for top ranked probes are shown in Table 8.

Gene expression diagnostic capabilities for a more precise quantitative approach to gene expression analysis, wim the utility to identify tolerant from non-tolerant individuals, were investigated b the inventors using the top ranked genes identified by microarray analysis,, excluding any overlapping probes for any single gene (e.g. TCL1A ranked 2 and 4, excluding probe ranked 4), in an additive binary regression model to build ROC curves. These probes were used to build a gene expression signature to specifically identify Tol- DF patients by firstly producing predicted classes (within-sample) and hence a classification for each individual. For this analysis, two-class ROC curves (tolerant vs non- tolerant) were built by both including and excluding the HC group from, the non-tolerant comparator groups. This was done because, whilst the comparison of healthy controls to tolerant individuals is of interest in identifying tolerance-specific gene expression, in the context of developing a clinical diagnostic test for tolerance in renal transplant patients, this comparison is not useful. The corresponding R.OC curve built excluding HC (Figure 6A) and based on the expressio of the top 10 ranked genes (Table 4) delivered a peak specificity and. sensitivity of 1, with a threshold of 0.0 a corresponding positive predictive value (PPV) and negative predicative values (NPV) of 100% within t e. training set (ROC including HC; threshold 0.2, specificity 0.853, sensitivity 0.923). Although 3 genes and 6 genes were sufficient to deliver good discrimination of tolerant patients within the training set, the top 10 ranked genes were selected for use, as they improved the specificity and sensitivity of subsequent ROC analysis of the test <«?r (Figure 6B). -Within sample analysis of the test set, delivered a specificity of 0.890 and sensitivity of 0.806, with a threshold of 0,35, a PPV and NPV of 71% and 93%., respectively (ROC including HC; threshold 0.23, specificity 0,801, sensitivity 0.806).

The inventors performed annotation enrichment analyses on the set of 174 overlapping probes identified between the training and test sets. The majorit of genes found to have any significant association with annotated pathways were enriched within B cell related pathways (Table ). In line with these data, of the top 1.1 ranked probes, corresponding to 1Q genes, 6 genes, afe described to be expressed by B cells or related to B cell function (Table 4). In addition to the B cell related pathways enriched within this probe list, other pathways were also significant, including protein-tyrosine kinases, generation of secondary signaling messenger molecules and other T cell activation related pathways (Table 9). Cross-platform biomarfcer diagnostic capabilities.

All assays described in the Materials and Methods section were tested in parallel for their diagnostic ability to distinguish Tol-CF patients from all other study groups. Assays performed on the test set were those that were highly predictive of tolerance within the training set and are discussed above. By combining the various biomarkers which indicate the presence of tolerance, the inventors expected that it was possible to significantly improve the diagnostic ability of any such individual test. This was indeed observed for the test set. Indeed when biomarkers and raieroarray data were analysed in combination, using 1) the ratio of B / T lymphocyte subsets, 2) the percentage of CD4- CD25im T cells, 3·) the ratio of anti*donor to anti-3^r<l party ELISpot frequencies, 4) the ratio of FoxP3/a-1,2- mannosidase expression and 5) a signature of the top 10 ranked genes, the specificity and sensitivity for the training set was 1, with a threshold of 0.01. which implied PPV and NPV of 100% (Figure 7A). When analysing the test set a peak specificity of 0.923 and a sensitivity of 0,903 were obtained with a threshold of 0.27, PPV of 80% and NPV of 96% (Figure 7B), which improved the diagnostic capacity compared to that obtained with gene expression alone. Therefore, application of a cross-platform biomarker signature improves the ability to id ntify bona fide tolerance, as in addition to gene expression and phenotype analysis, it can also take into consideration an individual's immunological functional state, which may be more closely related to describing the mechanistic basis of tolerance. In this respect, studying patient T cell, and B cell responses are useful approaches and may also be. used as biomarkers in the present invention. The utility of this Grass-platform biomarker signature lies in its abilit to identity renal transplant patients who may be unknowingly operationally tolerant. As shown in Figures 7 C and D, 5 stable recipients of the lest set could be identified to have the tolerant signature, and therefore may benefit from managed immunosuppression weaning. Interestingly, 2 CAN patients of the test set were also identified as having a high probability of being tolerant. This finding may be explained by difierences in the clinical assessment of chronic rejection, as unlike the CR group of the training set, CAN patients were not proven by biopsy to have immune-mediated rejection, but were defined on thfc basis of poor graft function. It is possible that the cross-platform biomarkers used to test these patients have sufficient sensitivity to detect subtle differences between these two patients groups, a property which may be revealed by serial immune monitoring of patients such as these over time,

Statistical Data

The statistician calculated the following sensitivities and specificities using the training set;

The statistician calculated the following sensitivities and specificities for the listed _:genes using the training set:

The same calculations were then made using the test set

In order to select the best subset of genes and additional biomarkers that would provide the best predictive value, as well as good generalizabil ty, additional analysis was carried out. First, the best subset of each size (1 to 14 biomarkers) was selected based on the Akaike's Information Criterion. Hie biomarkers selected for each subset are shown in the table, below.

A binary regression model was estimated for each of those subsets, and cross validation was used to establish the stability of the solution, in order to avoid overfit in the test set. Figure 14 shows the results of the cross-validation. The results of the cross validation suggest that the_. optimal solution should include a small number of markers (for example 2 to 5 or preferably 2 to 3), since the models seem to start overfUting to the specific characteristics of the test set with the inclusion of additional markers_*

To confirm this, the binary regression models including the best subsets of each size were used to estimate ROC curves, and the corresponding optimal sensitivity and specificity in the training set.

Training set

Subsequently, the probability of tolerance was estimated for the patients in the test set, by using the coefficients obtained in the training set for each subset size. These probabilities where used in combination with the optimal cutoff (also estimated in the training set) to compute the sensitivity and specificity in the test set.

These results confirm those of the cross-validation, and support the use of a model with preferably 2, 3, 4 or 5 biomarkers, more preferably 2 or 3 biomarkers, to best predict the probability of tolerance, in individual patients.

Discussion

The inventors have developed a set of biomarkers that distinguish toleran renal transplant recipients from patients with stable renal function under different degrees of immunosuppression, patients undergoing Chronic rejection and healthy controls. Biomarkers identified in a training set of tolerant patients have been validated in an independent test set. The inventors have found an expansion of B and . cells in peripheral blood of drug-free tolerant recipients, which is similar to the findings of a previous study on a smaller cohort of similar patients (13). Microarray analysis also revealed a clear and strong B cell bias of genes with altered expression between ToI-DF and the other groups. In particular, it has been found that the combination of the SH2JD1B, TL 5 and PNOG genes provides a very effective test for determining an individual's tolerance. The role of T cells in initiating and maintaining allograft rejection (14, 15) and tolerance (16) has been clearly established, whereas the role of B cells and the mechanisms whereby they may contribute to the tolerant state have yet to be elucidated. Interestingly, a murine study of transplantation tolerance, induced by ant GD45RB therapy has shown a mechanistic role for B cells (17). Recent data have also shown the ability of naive B ceils, following antigen-specific cognate interactions, to induce regulatory T cells that inhibit graft rejection in a murine model of heart transplantation (18). Whilst no significant increase in Bf-1 (IL-10 producing B cells) was detected in any patient group within this study, data presented here show altered ratios of B cell transitional and late-memory populations, relative increase of TGFp producing B cells, absence, of serum donor-specific antibodies and donor-specific direct T cell hyporesponsiveness in tolerant, recipients. These observations aUow speculation that renal transplant tolerance may be associated with alterations in both T-cell and B-cell mediated functions, A recent study -by Porcheray ei at, Studying both B cell and T ceil immunity in combined kidney and bone marrow transplant recipients, has however demonstrated the uncoupling of T cell and B cell anti-donor immuni ty in some of their studied toleraftt patients (19). In this respect, the B cell signature observed in tolerant renal patients in this study may indicate an important role for B cells in promoting tolerance. Monitoring of anti-donor responses using functional assays has demonstrated that hyporesponsiveness of direct pathway T cells develops over time after solid organ transplantation (20, 21). I the clinical context, enumerating the frequency of anti-donor T cells has proven useful in steroid withdrawal protocols (22). In the present study, measuring anti-donor direct pathway responses by ELISpot has also proven useful, where determining the ratio of responses against donor and third party T cells reveals donor specific hyporesponsiveness in tolerant patients. This test, however, is more useful when donor and recipient have several HLA-mismatches. Similar studies to this have focused on gene profiling of tolerant liver (23, .24) and also tolerant kidney recipients (25, 26). The set of genes that were differentially expressed in those studies differ to those identified herein and are not as effective in determining whether an individual is immunologically tolerant. This possibly reflects differences in the otgan, the patient groups, the R A source and preparation protocol, of the analysis platfijrm used. Indeed the microarray used in this study was selectively designed based on both published and unpublished data to have a transplantation focus., and therefore included a significant number of immune response related probes.

The two of the most highly ranked genes associated with tolerance found within the training set, TCLIA (rank 2) MS4A1 (CD20) (rank 5), are both B cell related genes. MS4A1 has previously been identified by Braud ei al„ (25) to be associated with tolerant renal transplant patients_*

A possible interpretation of the tolerant, signature described by this study could be that die immunological bioinarJkers detected are merely due to the lack of drug-mediated immune suppression in the Tol-DF group. To address this possibility, the study groups of the training set were specifically selected to include stable renal transplant patients o distinct immunosuppressive regimes and healthy controls as immune suppression-free subjects. Although clear differences between the healthy control and Tol-DF groups were observed in the training s t, these differences were not reproduced in the test set, a finding which may be attributed to the fact the mechanisms of tolerance may be more subtle within the test set,, where tolerant recipients are highl HLA-matched to their donors, in contrast to the training set. As all of these study groups have been taken into consideration, the combination of biomarkers described here appears to be a specific description of transplant- tolerance, rather than simply a consequence of the absence of immunosuppression. It is pertinent to observe that whilst detailed comparison of tolerant patients and healthy controls may reveal more about the mechanistic basis of tolerance, in the clinical context, mis comparison is not relevant.

An interesting comparison is tha of Tol-DF and s-LP patient groups of the training set, which differ in the use of 10 mg/day of prednisone, considered by many clinicians as quasi-physiological. The s-LP group had a higher proportion of female recipients, a higher percentage of cadaveric donors, and poorer kidney function than the Tol-DF group. Rather counter^ntuitively,. in most of the assays described there are clear differences between these two groups in immunophenotype, anti-donor responses, FoxP3/a-I,2-mannosidase ratio and gene expression,. This supports the notion that steroid monotherapy can induce a significant difference in the patient's inirnune status that can be evidenced by biomarkers. One of the Tol-DF patients within the (mining set had received a bone marrow donation 4 years prior to kidney transplantation from the same donor, Immune suppression was initially withdrawn from this patient: as evidence of chimerism was detected. As the mechanism of tolerance induction could be different in this patient, biomarker and ROC curve analysis was performed by inclusion and exclusion of this patient, however this patient did not appear as an outlier within the tolerant group in any of the assays studied, The utility of this tolerant signature depends on its ability to identify transplant recipients that can safel be weaned from, immunosuppression. The inventors have now developed a specific and sensitive set of biomarkers, which when combined, can identify tolerant renal allograft recipients and also several renal transplant, recipients on immunosuppressive drugs. Validation of these biomarkers has been achieved using a completely independent set of patients, and this validation is reinforced by the fact that the test set was derived from, a genetically different population, and that there were also differences in the collection and processing of test set and training set samples. The biomarkers can be implemented as a decisional tool in the clinical setting, which may allow tailored and safe clinical posttransplantation management of renal allograft recipients.

Further Validation using RT-PCR

In order to fUrfher validate the present method of deteixnining tolerance the inventors performed a further study (the "GAMBIT'* study) on a different patient group.

New study groups;

Tolerants: new patients that have been, completely off immunosuppression for longer than one year with <10% CRT rise since baseline before weaning. (Corresponds to Index group of the lOT study)

Stable; Adult kidney transplant recipients, with stable function, that have been transplanted for longer than 5 years, that are maintained on any immunosuppression therapy and that have had overall stable kidney function (<15% change in mean eGFR) in the last 5 years. (Corresponds to control groups 1 , 2 and 3 of the IOT study)

Chronic Rejection: Adult and paediatric kidney transplant recipients, more than 1 year posttransplant with increasing dysfunction that have undergone a graft biopsy in the previous 3 months and have been classified as having immunologically-driven chronic allograft nephropathy, (Corresponds to control group 4 of the IOT study).

In this study the inventors collected new samples from the following patient groups:

33 samples from Stable patients

12 samples from, patients on Chronk Rejection, and

5 samples from Tolerant patients

1 sample from a Healthy Control

1 sample from a patient who has lost tolerance

RT-PCR was performed on the 10 genes selected using the following protocol.

RT-PCR protocol

Whole blood was collected directly from the peripheral vein into "Tempus tubes™" (ABI cat number: 4342792), containing a solution that lyses cells and stabilizes mR A.. The tubes were stored at *20C until use.

Whole blood RNA was extracted using the Tempus Spin RNA Isolation Kit (ABI cat mber: 4380204). The quantity and quality of the mRNA was measured using the ND- 1000 Spectrophotometer (NanoDrop Technologies), The RNA was then stored at -50C,

.1 μg of whole blood total RNA Was reverse transcribed using the ABI Taqman Reverse Transcription synthesis kit: (ABI cat number: 4304134) into cDNA for immediate use. cDNA was subjected to RT-PCR analysis using the primers and probes, shown below, in 384-well plates (ABI cat number: 4306737) in 20 μί reaction volumes per well.

[

After the initial RT-PCR step to check the levels of expression of the genes in samples from healthy controls, and to demonstrate that the expression of the same genes is also detected in whole blood samples from patients, RT-PCR was carried out on patient c A for this study.

Data preprocessing steps: - Read in and merge the data from the different plates

- Check for batch effects due to a change in type of plate (between % well plates and 384 well plates). No batch effects found.

- Check wells with non-template controls to detect possible contamination in the plates.

- Code CT values above 35 as undetermined.

- Cheek the coefficient of variation across technical duplicates, considering alarming any above 3%. I have found great quality of duplicates, and no reason for concern.

- Aggregate duplicates using the mean.

- Calculate dCT as the difference in CT values between the gene of interest and HPRT (the control gene).

Rescale dCT to obtain only positive values using 2^{,tl T}.

- Elittiinate data from the gene SLC8A1 due to excessive levels of undetermined expression. This gene is very lowly expressed and badly detected by RT-PCR, It is likely that this gene was a false positive, originally selected, due to outlier values. - Eliminate data from patients with missing values (only stable patients have been eliminated). The data is produced in the form of heatmaps (not shown), wherein dendrograms show the results of unsupervised hierarchies., clustering of patients using either .10 or 3 genes. It is apparent that using 10 genes does not help to group tolerant patients together, whereas using the three genes selected via cross-validation the 5 tolerant patients tend to cluster together on the right side, under the last branch of the dendrogram. Data not shown.

Box plots showing the expression levels of the 3 genes PNOC, SH2DB1 and TLR5 are shown. See Figure .15.

There are several possible ways to combine the three genes to create a classifier to differentiate tolerant from non-tolerant patients, as will b« apparent to those skilled in the art. The inventors present here the results of two classifiers: i) a logistic regression model with main and interaction effects, and 2) a classification tree.

In order to calculate the parameters of these models the inventors used the data from stable, chronic rejectors and tolerant patients, but dichotomize me outcome as tolerant vs non-tolerant.

Results from Logistic Regression fit:

(Dispersion parameter for binomial family taken to be 1) Null deviance: 32.508 on 43 degrees of freedom

Residual deviance! L4.B92 on 4-3 degrees of freedom

AIC: 28.892

The coefficients under "Estimate" column are the ones used to calculate the probability of Tolerance. See Figure 16 for the ROC curve obtained using a logistic regression classifier. Figure 17 shows boxplots of the probability of tolerance estimated using the logistic regression classifier.

Using this method a single chronifc rejector was misclassified as tolerant, and 5 stable patients were classified as tolerant, comprising 15% of the stable population_., falling within the predicted 20% who might be eligible for immunosuppression weaning. Regression Algorithm:

Z=.14.457+94.156*P OC+6.289*SmDBl+5.054*TLR5-l.S23*PNOC*SH2DBl-

51.584*PNOC*TLR5-2.339*SH2DBl*TLR5

P(Tol)= e^z/(e^z+l)

Note: The expression of each gene is expressed asI*⁰⁷, where dCT is calculated as the CT difference between each gene and the control gene (HPRT). A patient is classified as tolerant if P(Tol) is > .0.0602.

Figure 18 shows a. classification tree estimated, with the 3 genes (PNOC, SH2DBI and TLR5), For optimal performance of the classifier, any patient with a probability of tolerance larger than 0 is classified as tolerant. Two of the terminal nodes have associated probability of tolerance larger than 0: 1) Patients with TLR5037 & SH2DB1 >1.02 & TLR5<1.58 (probability of tolerance = 0.600); and .2) Patients with TLR5<&37 & SH2DB1 >1.02 & TLR5>1.58 & PNOC<0.042 (probability of tolerance = 0.333). Figure 1 shows boxplots of the classification tree's cutoffs.

Figure 20 shows the ROC curve resulting from the use of the estimated classification tree.. The sensitivity, specificity and AUG result from classifying as tolerant any patient with a probability of tolerance larger than 0. Figure 21 shows the number of patients of each group assigned to different probabilities. One CR patient was misclassified as tolerant (same misclassified using regression). Equally, 5 stable patients were classified as tolerant.

These results show the. success obtained using these three genes to distinguish between tolerant and nott-tolerant patients. A successful performance can be achieved using different classification methods, two of which are illustrated here.

Table 7 List of 174 probes common to -both training and test sets Identified to have significant" differential expression by study groups following four-class analysis. Ranked on p values by Kruskal-Wallis test, with adjustment for False Discovery Rate. (FDR) at 1 %.

Table 8

Median of log-fold change in expression (relative to median of all samples) of differentially expressed probes detected in patient groups of the training set, ranked on 1% FDR. Plots showing the fold expression for each patient group of the training and test sets, for all genes detected to be significantly differentially expressed available in online material. Stable; combined groups s-CNI, s-nCNI, s-LP.

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Claims (18)

Claims

1. A method of determining an individual^ immunological tolerance to a kidney organ transplantation comprising determining the level of expression of at least 2 genes selected from the group consisting of TLR5, PNOC, SH2D1B, CD79B, TCL1A, HS3ST1, MS4A1, FCRL1, SLC8A1 and FCRL2 in a sample obtained from the individual.

2. The method of claim 1 , wherein the level of expression of SH2D1 B, PNOC CD79B, TCL1.A, HS3ST1, MS4A1, FCRL1 and FCRL2 is higher than a normal level in an individual considered to have immunological tolerance, and the level of expression of TL 5 and SLC8AI is lower than a normal level in an individual considered to have immunological toleranpe.

3. The method of claim 1, wherein the level of expression of genes TLR,5, PNOC and SH2D1B in a sample obtained from the individual is deteririiried.

4. The method of claim 3, wherein a positive prediction of an individual's tolerance to. an organ transplantation is given by a high level of expression of SH2D1B and PNOC and a low level of expression on TLR5.

5. The method claim 3, wherein the expression level of one or more of the following genes CD79B, TCL1.A, HS3ST.1, MS4A1, FCRL.1, SLC8A1 and FCRL2 is additionally determined.

6. The method of any one of the preceding claims, wherein the method comprises detecting the. level of B cells and NK cells, wherein a raised level of such cells is indicative of immunological tolerance.

7. The method of any one of the preceding claims, wherein the method comprises determining the level of CD4+CD25^int T cells, wherein a reduced level of such cells relative to total CD4T T cells is indicati ve of immunological tolerance.

8. The method of any one of the preceding claims, wherein the method comprises detennining the level of donor specific CD4+ T cells, wherein a reduced level of such cells, is indicative of immunological tolerance.

9. The method of any one of the preceding claims, wherein the method comprises determining the ratio of FoxP3 to a-l_>2-mannosidase gene expression level of CD4+ T cells, wherein a high ratio is indicative of immunological tolerance,

10. The method of any one of the preceding claims, wherein the method comprises determining the ratio of CD 19+ to CD3+ cells, wherein a high ratio is indicative of Immunological tolerance,

11. The method of any one of the preceding claims, wherein the expression levels of beta- actin and/or HRPT :are used as controls,

12. A sensor for detecting the expression levels of at least 2 genes selected from the group consisting of TLR5, P QC, SH2D1B, CD79B, TCL1A, HS3ST1, MS.4A1, FCRL1, SLC8A1 and FCRL2.

13. A sensor for detecting the expression levels of the TLR5, PNOC and SH2D1 B, genes.

14. The sensor of claim 13, which is for detecting the expression of one or more of the following genes CD79B, TCL1A, HS3ST1, MS4A1, FCRLl, SLC8AI and FCRL2.

15. A kit comprising reagents for detecting the level of expression of at least 2 genes selected from the grou consisting of TLR5, PNOC, SH2D1B, CD79B, TCL1A, HS3ST1, MS4A1, FCRLl, SLC8A1 and FCRL2.

16. A kit comprising reagents for detecting the level of expression of the LR5, PNOC and SH2D IB genes..

17. The kit of claim 16 that, further comprises reagents for detecting the level of expression of one or more of the following genes CD79B, TGI.1 , HS3ST1, MS4AI , FCRLl, SLC8A1 and FCRL2.

18. The kit of any one of claims 15 to 17, which comprises reagents for detecting the level, of gene expression of the genes by RT-PCR.