Anti-CD25 Treatment and FOXP3-Positive Regulatory T Cells in Heart Transplantation

G. Vlad

a, E. K. Ho

a, E.R.Vasilescu

a, J. Fan

a, Z. Liu

b, J.W. Cai

a, Z. Jin

c, E. Burke

d,

M. Dengd, M. Cadeiras

d, R. Cortesini

a, S. Itescu

d, C. Marboe

a, D. Mancini

d, and N.

Suciu-Focaa*

The Departments of Pathology

a, Surgery

b, Biostatistics

c, Medicine

d, and of

Columbia University, New York, NY 10032 Running Title: Daclizumab and T Regulatory Cells Word count: 3655 Key Words: Heart Transplantation, T regulatory cells, Rejection, FOXP3, Daclizumab Corresponding Author: Dr. Nicole Suciu-Foca Department of Pathology Columbia University 630 West 168

th Street – P&S 14-401

New York, NY 10032 Tel: (212) 305-6941; Fax: (212) 305-3429 E-mail: ns20@columbia.edu The interleukin-2 receptor alpha chain (IL-2Ra, CD25) plays a major part in shaping the dynamics of T cell populations following immune activation, due to its role in T cell proliferation and survival. Strategies to blunt the effector responses in transplantation have been developed by devising pharmaceutical agents to block the IL-2 pathways. However, such strategies could adversely affect the CD25

+

FOXP3+ Treg populations which also rely on intereukin-2 signaling for survival. The

present study shows that a cohort of heart allograft recipients treated with Daclizumab (a humanized anti-CD25 antibody) display FOXP3 expression patterns consistent with functional T regulatory cell populations. High levels of FOXP3 were observed to correlate with lower incidence of and recovery from acute rejection, as well as lower levels of anti-donor HLA antibody production. Therefore, T reg populations appear fully functional in patients treated with Daclizumab, even when

5 doses were administered. By comparison, patients treated with fewer doses or no Daclizumab had a higher incidence of acute rejection, antibody production and graft failure. Therefore, our data indicates that Daclizumab treatment does not interfere with the generation of regulatory T cells and has a beneficial effect on heart allograft survival. Introduction

The use of calcineurin inhibitors as part of standard immunosuppressive therapy in organ transplantation has resulted in a remarkable increase in the rate of allograft survival. The additional use of various monoclonal (anti-CD3) and polyclonal (antithymocytic/lymphocytic) antibodies (as depletional induction agents) has further contributed to prevention of early episodes of acute rejection (1). However, the risk of infection, malignancy and late rejection remains a looming concern. More recently, a new generation of immunosuppressive agents has been introduced to fine tune T cell responses by specifically targeting activated T cells. The humanized anti-interleukin-2 receptor alpha chain (IL-2R, CD25) monoclonal antibody (Daclizumab, Zenapax®, Hoffmann-La Roche) is such an example. Its efficacy in conjunction with triple immunosuppression has been documented in several studies (1-4).

The underlying hypothesis for the development of this drug has been that blocking of the interleukin-2 receptor may prevent the proliferation and differentiation of effector T cells. However, studies in the field of regulatory T cells have shown that CD25 represents a crucial marker of FOXP3

+ T cells with

suppressor function (5-12). In the light of this data, the obvious question is whether anti-CD25 treatment results in the blockade or depletion of regulatory T cells and whether quiescence can be attained in their absence. To answer this question we have performed serial determinations of markers deemed to be characteristic of regulatory/suppressor T cells (Treg/Ts), such as FOXP3, CTLA-4, and IL-10, in a cohort of 110 heart recipients transplanted between 2003 and 2006 (Table 1). As putative markers for rejection we used IFN-γ and VEGF. In addition, given the fact that development of anti donor HLA antibodies is detrimental to the graft we monitored such antibodies in patients’ sera. We now report on the positive correlation between quiescence and expression of these Treg markers in peripheral blood CD4

+ and CD8

+ T cells, demonstrating that the development of

regulatory T cells is not impaired in patients treated with Daclizumab. Such regulatory T cells were negatively associated with the occurrence of early acute rejection episodes and development of allo-antibodies. (13-15) Material and Methods Patients

110 heart allograft recipients (84 males and 26 females) with a mean age of 49.4 years old (49.4±13.0 SD, range 18-72) were recruited for these studies at the

time of transplantation. The demographic of this study population is shown in Table 1. All patients gave informed consent under the auspices of the appropriate Institutional Review Board. Patients were treated with cyclosporine (CSA), mycophenolate mofetil (MMF), prednisone (MP) and with or without Daclizumab (administered at 1mg/kg body weight). Endomyocardial biopsies (EMB)

Endomyocardial biopsies were performed by the standard transjugular approach weekly for the first month and then at progressively longer intervals to a baseline schedule of every 6 months. The mean number of biopsies per patient was 10 + 2. A minimum of four biopsy fragments were fixed in 4% buffered formalin, paraffin embedded, and multiple hematoxylin and eosin stained sections from the three levels in the block were examined. An additional fragment was frozen for RT-PCR and Lymphocyte Growth Assays (LGA). Histological grades were assigned according to the criteria of the International Society for Heart and Lung Transplantation (ISHLT): no rejection (grade 0), focal (grade 1A) or diffuse interstitial mononuclear infiltrates without myocyte necrosis (grade 1B), multifocal aggressive infiltrates with myocyte damage (grade 3A), diffuse inflammatory process with necrosis (grade 3B) and diffuse aggressive polymorphous/mononuclear infiltrate with edema, hemorrhage, vasculitis and necrosis (grade 4) (16).

A baseline angiogram was performed within one month of transplantation to detect unsuspected vascular disease. Angiograms were repeated annually. The diagnosis of CAV was made when contrast angiography demonstrated diffused small vessel involvement, defined as concentric and symmetrical narrowing of terminal branches.

Molecular HLA typing and testing of anti-HLA antibodies

HLA genotypes of transplant recipient/donor pairs were determined by PCR with sequence specific primers (SSP) using commercially available kits (One Lambda, Los Angeles, CA). Sequential samples of sera obtained from each patient prior to and following transplantation were tested for anti-HLA alloantibodies by lymphocytotoxicity and solid-phase assays to determine the frequency of panel reactive antibodies (PRA). The specificity of the antibodies for the donor’s HLA antigens was assessed by screening and direct cross-matching procedures as previously described (17).

Lymphocyte Growth Assay A biopsy fragment (1mm

3 piece) was placed in medium supplemented with

recombinant IL-2 (5 units/ml) and examined microscopically at 48h. Growth was

scored on a semi quantitative scale from 0 to 3 on the basis of circumferential T cell aggregation. A score of 1 or greater was deemed positive (18). Quantitative Real Time (qRT)-PCR A total of 508 peripheral blood samples from 110 transplant patients were tested by qRT-PCR for expression of various genes at the transcriptional level. CD4 and CD8 cells were isolated from each of these samples by use of commercially available magnetic isolation kits (Miltenyi Biotec, Gladbach, Germany). Additionally, qRT-PCR studies of were performed on 104 EMB samples. Biopsies were stored overnight at 4

oC in 5 volumes of RNAlater tissue collection/RNA

stabilization solution (Ambion, Foster City, California). After removal of the RNA stabilization solution biopsies were stored at -80

oC for subsequent RNA extraction.

Total RNA was isolated from biopsies with the RNAqueous-4PCR kit (Ambion) following the manufacturer’s recommendations. Complementary cDNA was synthesized using the 1

st Strand cDNA Synthesis Kit for RT-PCR (Roche

Diagnostics, Basel, Switzerland). Quantitative real-time PCR was performed using Taqman gene expression assays (Applied Biosystems, Foster City, CA). The gene expression assays used in these studies are identified below, by gene name, manufacturer’s abbreviation and part number: Cytotoxic T Lymphocyte Antigen 4 (CTLA-4, Hs00175480_m1), Forkhead box P3 (FOXP3, Hs00203958_m1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 4326317E), Interleukin-10 (IL-10, Hs00174086_m1), Interferon-gamma (IFN-γ, Hs00174143_m1), and vascular endothelial growth factor (VEGF, Hs00173626_m1) all from Applied Biosystems. Forward and reverse primers are contained in different exons, to prevent amplification of genomic DNA. We used the 7300 RT-PCR instrument (Applied Biosystems), applying the manufacturer’s protocols and recommended amplification conditions, as follows: one cycle at 50°C (2 min), then 95°C (10 min), followed by 50 cycles at 95°C (15 s) and 60°C (1 min). Data were collected and analyzed using the 7300 SDS 1.3.1 software (Applied Biosystems). Each assay plate included “no template” negative controls and a control cDNA. The relative amount of gene expression was calculated according to the formula: 2

−∆Ct, where

∆Ct = [Ct(gene) − Ct(GAPDH)] and Ct is the “crossing threshold” value returned by the PCR instrument for every gene amplification. Statistical Analysis

The generalized estimating equation (GEE) approach with working independence was used to study the association between two variables. Two GEE modeling approaches were used, one with constant intercept and the other with different intercept term for each subject. The identity link was used for continuous variables, while the logit link was used for rejection, which is binary.

Time to death was used as the end point in overall graft survival analysis,

and patients who were alive were censored at the date of last clinical contact. The Cox proportional hazards regression model was used for multivariate analysis of Daclizumab treatment. Overall graft survival was computed using Kaplan-Meier product-limit estimators and compared by log rank tests. The influence of Daclizumab treatment on the development of anti-HLA antibodies was evaluated by Kaplan-Meier estimators, with P values obtained using log rank statistics. For categorical variables, a 2x2 table was constructed and compared by either chi-square analysis or Fisher’s exact (2-tail) test. A non-parametric Wilcoxon exact test was used for analysis of changes in the level of FOXP3 expression in CD4

+

and CD8+ T cells from patients with resolving acute rejection episodes. P values

less than 0.05 were considered significant. All statistical tests were 2-sided. Statistical analysis of data was performed using the SAS version 9.1 software package (SAS Institute, Cary, NC) (19, 20). Results Expression of FOXP3 in CD4 and CD8 T cells from the peripheral blood of heart allograft recipients

Regulatory T cells are characterized by their capacity to suppress the reactivity of other effector populations of antigen-stimulated T cells. Several distinct populations of regulatory T cells, such as thymus-derived CD4

+CD25

+FOXP3

+

natural T reg (5, 6), antigen-induced CD4+CD25

+ FOXP3

+ peripheral T reg (7-9),

IL-10-producing CD4+CD25

+FOXP3

- Tr1 (10, 11), CD8

+ FOXP3

+ Ts (8, 9, 21-23)

and CD8+ IL-10-producing Ts (12) have been shown to play a role in human

transplantation and experimental models (8). To assess the presence of T reg in human allotransplantation, we analyzed the expression of FOXP3 in peripheral CD4 and CD8 T cell populations as well as in heart allograft biopsies. EMB grade 3A or higher was considered indicative of acute cellular rejection (16). Analysis of 508 peripheral blood samples showed a negative correlation between the level of FOXP3 expression in CD4

+ T cells and EMB (N=1557) grade 3A (p=0.033, Figure

1). Although within the CD8 compartment the level of FOXP3 expression was

5-fold higher during quiescence compared to high-grade rejection, the difference did not reach significance (p>0.296).

Similarly, analysis of FOXP3 expression in biopsy tissue (N=103) showed a negative association with the grade of rejection which did not reach statistical significance (P=0.416). However, the consistent trend for high expression of FOXP3 in peripheral blood and biopsies during quiescence and low expression during episodes of acute rejection suggests that regulatory T cells play a role in allotransplantation.

The dynamic of FOXP3 expression in peripheral blood T cells during resolving rejection

Resolving rejections were defined as lymphocytic infiltrates grade 3A EMB subsiding to grade 1 or 0 within 1 week. We examined the expression of FOXP3 in peripheral blood T cells from 9 such cases. In one of these 9 cases, the CD4

+ T

cells were not testable for technical reasons. Of particular note was the finding that the level of FOXP3 expression went from low levels to higher values during resolving rejection. Thus, in 7/8 rejection episodes, the level of FOXP3 in peripheral blood CD4 T cells increased 7 fold (P=0.036) during the resolving rejection. Similarly, peripheral CD8 T cells showed a mean fold increase of 13 (P=0.028), in 9/9 cases of resolving rejection (Figure 2). An exception seemed to occur in a patient who showed low FOXP3 (in both CD4 and CD8 peripheral cells) in conjunction with EMB grade 3A, followed one week later by a 10-fold decrease in the level of FOXP3 as rejection appeared to resolve to grade 1 EMB. This patient, however, showed a second episode of rejection grade 3, two weeks after the first grade 3 episode (Figure 3) consistent with the low level of FOXP3 seen at the time when EMB showed only grade 1B infiltrates. The finding that during recurrent rejection the level of FOXP3 expression in peripheral blood T cells stays at a low value reinforces the concept that FOXP3 is associated with T regulatory cells that may play a role in protecting the graft. FOXP3 associations with the expression of other markers of T cell function

Cytotoxic lymphocyte associated antigen 4 (CTLA-4) negatively regulates the classical costimulation pathways required for efficient T cell activation. This molecule is highly expressed by both CD4 and CD8 regulatory T cells (6, 17, 18). There is also evidence that interleukin-10 mediates the suppressor function of several types of regulatory T cells (8, 10, 11, 12). Because different populations of regulatory T cells may be simultaneously present in the circulation (24, 25), we analyzed the relationship between the level of expression of FOXP3 and that of these suppressor markers in CD4 and CD8 cells from sequential samples of blood. We found a significant correlation between the level of expression of FOXP3 and CTLA-4 as well as FOXP3 and IL-10, irrespective of rejection status (Table 2).

No association between the level of expression of FOXP3 and that of IFN-γ in CD4 and CD8 T cells from the circulation of transplant patients was found during quiescence or rejection (GEE, P-values >0.05, Table 2).

Studies of EMB tissue showed no significant relationships between the level of expression of FOXP3, IL-10, CTLA-4 or IFN-γ (P values >0.05). This most likely reflects the fact that within the EMB tissue, infiltrating T cells represent only a small fraction of the cell sample. Unexpectedly however, there was a significant correlation between the levels of expression of FOXP3 and VEGF (P=0.007, Table 2). This observation was unexpected because VEGF, a growth factor active in angiogenesis, vasculogenesis and endothelial cell growth, was previously shown to be increased in allograft rejection (26).

FOXP3 is inversely related to the production of anti-donor HLA antibodies

The generation, differentiation, and maturation of antibody producing B cells are highly dependent on T cell help. Such help can be provided via the production of Th2-type cytokines, or by direct T-B interaction (27). By inhibiting Th (28, 29, and reviewed in ref. 8), T reg can interfere with B cell proliferation and differentiation into Ig secreting plasma cells. To determine whether the presence of T reg may affect the antibody status of the patients, we analyzed the relationship between the level of FOXP3 expression in CD4 and CD8 T cells and the presence of anti-HLA (IgG) antibodies in the patients’ circulation. This analysis revealed a negative correlation between high levels of FOXP3 in the CD4 compartment and the development of anti-HLA IgG antibodies (p=0.014, Figure 4A). Patients developing high PRA following transplantation also showed low expression of FOXP3 in CD8 T cells. However, this correlation was not statistically significant (p= 0.976, Figure 4). These findings are consistent with the hypothesis that regulatory T cells suppress the activation and differentiation of alloantibody producing B cells, either directly or acting on allospecific T helper cells.

Relationship between Daclizumab treatment and immune responses against the graft

Within this study cohort of 110 patients, 7 were not treated with Daclizumab. All 7 patients showed an early, post-transplantation “spike” of anti-HLA Ab. In three recipients this phenomenon was transient, although the patients remained prone to subsequent sporadic, short-term bouts of anti-HLA antibodies as late as one year after transplantation (Figure 5A, B, C). In the remaining four patients not receiving treatment with Daclizumab anti-HLA antibodies persisted in the circulation over the entire period of observation as illustrated in Figure 5D. The anti-donor-HLA specificity of the antibodies was demonstrated by a direct cross match of the patients’ sera with T cells and B cells from the specific donor spleens.

Study of patients treated with Daclizumab showed that only 42% of the patients that have received 4 or more doses developed anti-HLA antibodies within 24 months post transplantation, compared to 69% and 100% of those who received 1-3 doses or no Daclizumab, respectively (P<0.0001) (Figure 5E, F). Thus, it appears that the activation of the humoral arm of the immune response against the graft was inhibited by Daclizumab in a dose related manner. We analyzed the frequency of EMB grade 3A or higher in our cohort of patients, after completion of the Daclizumab treatment course. Patients receiving more than 3 doses had a significantly lower incidence of EMB grade 3A rejection compared to patients receiving 0-3 doses (P<0.04, Figure 6). Acute rejection (EMB grade 3+) was strongly associated with positive LGA results which reflect the growth capacity of graft infiltrating cells and their implicitly aggressive nature (P<0.0001, Figure 7). This data indicates that Daclizumab inhibits both the cellular and humoral immune response against the graft.

Effect of Daclizumab treatment on patient survival Among patients treated with Daclizumab (N= 103), 73.6% received 5

courses of the drug while the remaining 25.4% received lesser amounts. The Cox proportionate hazards model shows that Daclizumab had a protective effect against graft loss, and that this effect was dosage dependent. Thus, receiving three Daclizumab doses or less exposes a patient to 4.3 fold higher risk of graft loss compared to patients receiving four or more treatments (Figure 8A, B). Patients receiving five or more doses of Daclizumab showed the same (about 4-fold) protection from graft loss as those receiving 4 doses, suggesting no further benefit from additional doses. The 12-month actuarial heart allograft survival was 82% among patients receiving 0-3 doses of Daclizumab and 96% among those receiving more (Figure 8C, D). Discussion

Over the last decade, the literature has been dominated by the concept that regulatory T cells which have the CD4

+CD25

+FOXP3

+ phenotype are crucially

important to the maintenance of tolerance to self and non-self antigens (5-8). Studies in rodents have demonstrated unambiguously that this population of regulatory T cells is primarily responsible for protecting the animal from autoimmune diseases (5-8, 30, 31). Evidence that both CD4

+ and CD8

+ FOXP3

+ T

cells mediate allogeneic tolerance has also been provided by numerous studies in rodents (30, 32, 33). In spite of the large body on information about the capacity of T reg to inhibit T cell responses in malignancy (34, 35) and certain autoimmune diseases, the data regarding their mechanism of action and clinical significance is still controversial. The controversy emerges primarily from the finding that in human CD25, FOXP3 and CTLA-4 are markers which are shared by Treg with a stable phenotype and by ‘transient’ regulatory T cells which represent only a stage in the differentiation of activated T cells (36, 37). The promiscuity of FOXP3 expression in activated human T cells may explain our finding that FOXP3 and IFN-γ showed no negative association either in EMB or circulating T cells. Similarly, the positive correlation between the expression of FOXP3 and a putative rejection marker VEGF (26) may be caused by the presence of activated rather than regulatory T cells within the graft. On the other hand, it is possible that, by analogy to the situation encountered in cancer, VEGF and IL-10 create an immunosuppressive environment within the graft, inhibiting T cell reactivity (38). Similarly, the finding of FOXP3 and CTLA-4 expression in peripheral blood CD4 and CD8 T cells even during EMB grade 3 rejection episodes, may also indicate ongoing T cell activation rather than presence of Treg. However, the significant rise in the level of expression of these markers after successful treatment of a rejection episode, together with the negative association between high level of FOXP3 and development of alloantibodies indicates that FOXP3

+ CD4 and CD8 T cell subsets

have a protective role against rejection in humans, similar to animal models (9, 22, 23, 39, 40). Therefore, our data is consistent with a recently proposed model (36, 37), which suggests that in humans, transient expression of FOXP3 is characteristic of all activated T cells. However, stable expression of FOXP3 differentiates T regulatory cells from activated T cells committed to other effector functions (28, 41). The clinical consequences of this observation cannot be underestimated as they warn against the use of these markers for identifying T suppressors for either diagnostic or prognostic purposes. Although it is difficult to discriminate between the possibility that the cells expressing CD25, FOXP3 and CTLA-4 are precursors of Th or Treg, the quantitative changes which we observed during resolving rejection argue in favor of continuous monitoring of these markers to predict the outcome of a rejection episode. This view is further supported by the finding that declining levels of FOXP3 occurring following treatment of rejection are indicative of recurrence rather than remission of acute rejection. The fact that CD25 is expressed by all T cells upon activation explains the apparent inconsistency between the clinical efficacy of Daclizumab as an immunosuppressive agent and its potential to block or deplete CD25

+ T reg. Our

data contribute evidence that Daclizumab, particularly after repeated administration, inhibits the production of allo-antibodies and improves patient survival, consistent with other authors finding that it reduces the rate of acute rejection. (1-4, 42) Furthermore, the data indicate that CD4

+ and CD8

+ T regulatory

cells develop in patients treated with Daclizumab together with standard calcineurin – inhibitory therapy arguing against the view that this type of immunosuppression prevents the differentiation of regulatory T cells. Acknowledgements This work was supported by RO1 AI55234-04 and the Interuniversitary Organ Transplantation Consortium, Rome, Italy. Figures Figure 1. The level of FOXP3 expression (relative to GAPDH) in peripheral CD4 T cells (A), CD8 T cells (B) and EMB (C) is inversely related to acute rejection grade 3A. Figure 2. The dynamic of FOXP3 expression in peripheral T cells during resolving rejection. Figure 3. The dynamic of FOXP3 expression in CD8+ T cells from a patient with multiple rejection episodes. Figure 4. FOXP3 expression in peripheral T cells is inversely related to the development of anti-HLA antibodies in heart transplant recipients.

Figure 5. Anti-HLA antibody production. Daclizumab non-treated patients recurrent (A, B, C) or prolonged (D) anti-HLA antibody production. Log rank statistics (E) and cumulative incidence of anti-donor antibody production (F) in patients receiving various Daclizumab treatments. Figure 6. Incidence of acute rejection after completion of Daclizumab treatment(s). Figure 7. Relationship between development of acute rejection (EMB grade ≥3A) and LGA results. Figure 8. Protective effect of Daclizumab on heart graft survival. Daclizumab treatment(s) and hazard ratios of graft loss (A, B) and actuarial survival (C, D). References 1. Kirk AD. Induction immunosuppression. Transplantation 2006;82(5):593-602. 2. Sandrini S. Use of IL-2 receptor antagonists to reduce delayed graft function following renal transplantation: a review. Clinical transplantation 2005;19(6):705-710. 3. Figueras J, Prieto M, Bernardos A, Rimola A, Suarez F, de Urbina JO et al. Daclizumab induction and maintenance steroid-free immunosuppression with mycophenolate mofetil and tacrolimus to prevent acute rejection of hepatic allografts. Transpl Int 2006;19(8):641-648. 4. Vo AA, Toyoda M, Peng A, Bunnapradist S, Lukovsky M, Jordan SC. Effect of induction therapy protocols on transplant outcomes in crossmatch positive renal allograft recipients desensitized with IVIG. Am J Transplant 2006;6(10):2384-2390. 5. Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annual review of immunology 2004;22:531-562. 6. Shevach EM. CD4+ CD25+ suppressor T cells: more questions than answers. Nature reviews 2002;2(6):389-400. 7. Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells. Nature reviews 2003;3(3):253-257. 8. Vlad G, Cortesini R, Suciu-Foca N. License to heal: bidirectional interaction of antigen-specific regulatory T cells and tolerogenic APC. J Immunol 2005;174(10):5907-5914. 9. Manavalan JS, Rossi PC, Vlad G, Piazza F, Yarilina A, Cortesini R et al. High expression of ILT3 and ILT4 is a general feature of tolerogenic dendritic cells. Transplant immunology 2003;11(3-4):245-258. 10. Roncarolo MG, Gregori S, Levings M. Type 1 T regulatory cells and their relationship with CD4+CD25+ T regulatory cells. Novartis Foundation symposium 2003;252:115-127; discussion 127-131, 203-110. 11. Levings MK, Gregori S, Tresoldi E, Cazzaniga S, Bonini C, Roncarolo MG. Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25+CD4+ Tr cells. Blood 2005;105(3):1162-1169. 12. Endharti AT, Rifa IMs, Shi Z, Fukuoka Y, Nakahara Y, Kawamoto Y et al. Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells. J Immunol 2005;175(11):7093-7097.

13. Reed EF, Demetris AJ, Hammond E, Itescu S, Kobashigawa JA, Reinsmoen NL et al. Acute antibody-mediated rejection of cardiac transplants. J Heart Lung Transplant 2006;25(2):153-159. 14. Lietz K, John R, Burke E, Schuster M, Rogers TB, Suciu-Foca N et al. Immunoglobulin M-to-immunoglobulin G anti-human leukocyte antigen class II antibody switching in cardiac transplant recipients is associated with an increased risk of cellular rejection and coronary artery disease. Circulation 2005;112(16):2468-2476. 15. Vasilescu ER, Ho EK, de la Torre L, Itescu S, Marboe C, Cortesini R et al. Anti-HLA antibodies in heart transplantation. Transplant immunology 2004;12(2):177-183. 16. Marboe CC, Billingham M, Eisen H, Deng MC, Baron H, Mehra M et al. Nodular endocardial infiltrates (Quilty lesions) cause significant variability in diagnosis of ISHLT Grade 2 and 3A rejection in cardiac allograft recipients. J Heart Lung Transplant 2005;24(7 Suppl):S219-226. 17. Vasilescu ER, Ho EK, Colovai AI, Vlad G, Foca-Rodi A, Markowitz GS et al. Alloantibodies and the outcome of cadaver kidney allografts. Human immunology 2006;67(8):597-604. 18. Itescu S, Burke E, Lietz K, John R, Mancini D, Michler R et al. Intravenous pulse administration of cyclophosphamide is an effective and safe treatment for sensitized cardiac allograft recipients. Circulation 2002;105(10):1214-1219. 19. Liang KY, Zeger, SL. Longitudinal data analysis using generalized linear models. Bi ometrika 1986 73: 13-22. 20. Kalbfleisch JD, Prentice RL The statistical analysis of failure time data, second edition, Wiley, New York 2002. 21. Liu Z, Tugulea S, Cortesini R, Suciu-Foca N. Specific suppression of T helper alloreactivity by allo-MHC class I-restricted CD8+CD28- T cells. Int Immunol 1998;10(6):775-783. 22. Chang CC, Ciubotariu R, Manavalan JS, Yuan J, Colovai AI, Piazza F et al. Tolerization of dendritic cells by T(S) cells: the crucial role of inhibitory receptors ILT3 and ILT4. Nat Immunol 2002;3(3):237-243. 23. Manavalan JS, Kim-Schulze S, Scotto L, Naiyer AJ, Vlad G, Colombo PC et al. Alloantigen specific CD8+CD28- FOXP3+ T suppressor cells induce ILT3+ ILT4+ tolerogenic endothelial cells, inhibiting alloreactivity. Int Immunol 2004;16(8):1055-1068. 24. Cobbold SP, Graca L, Lin CY, Adams E, Waldmann H. Regulatory T cells in the induction and maintenance of peripheral transplantation tolerance. Transpl Int 2003;16(2):66-75. 25. Scotto L, Naiyer AJ, Galluzzo S, Rossi P, Manavalan JS, Kim-Schulze S et al. Overlap between molecular markers expressed by naturally occurring CD4+CD25+ regulatory T cells and antigen specific CD4+CD25+ and CD8+CD28- T suppressor cells. Human immunology 2004;65(11):1297-1306. 26. Reinders ME, Sho M, Izawa A, Wang P, Mukhopadhyay D, Koss KE et al. Proinflammatory functions of vascular endothelial growth factor in alloimmunity. The Journal of clinical investigation 2003;112(11):1655-1665.

27. Ruprecht CR, Lanzavecchia A. Toll-like receptor stimulation as a third signal required for activation of human naive B cells. European journal of immunology 2006;36(4):810-816. 28. Kim-Schulze S, Scotto L, Vlad G, Piazza F, Lin H, Liu Z et al. Recombinant Ig-like transcript 3-Fc modulates T cell responses via induction of Th anergy and differentiation of CD8+ T suppressor cells. J Immunol 2006;176(5):2790-2798. 29. Liu Z, Tugulea S, Cortesini R, Lederman S, Suciu-Foca N. Inhibition of CD40 signaling pathway in antigen presenting cells by T suppressor cells. Human immunology 1999;60(7):568-574. 30. Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance. Nature reviews 2003;3(3):199-210. 31. Zwar TD, van Driel IR, Gleeson PA. Guarding the immune system: suppression of autoimmunity by CD4+CD25+ immunoregulatory T cells. Immunology and cell biology 2006;84(6):487-501. 32. Kapp JA, Honjo K, Kapp LM, Xu X, Cozier A, Bucy RP. TCR transgenic CD8+ T cells activated in the presence of TGFbeta express FoxP3 and mediate linked suppression of primary immune responses and cardiac allograft rejection. Int Immunol 2006;18(11):1549-1562. 33. Liu J, Liu Z, Witkowski P, Vlad G, Manavalan JS, Scotto L et al. Rat CD8+ FOXP3+ T suppressor cells mediate tolerance to allogeneic heart transplants, inducing PIR-B in APC and rendering the graft invulnerable to rejection. Transplant immunology 2004;13(4):239-247. 34. Banham AH, Powrie FM, Suri-Payer E. FOXP3+ regulatory T cells: Current controversies and future perspectives. European journal of immunology 2006;36(11):2832-2836. 35. Beyer M, Schultze JL. Regulatory T cells in cancer. Blood 2006;108(3):804-811. 36. Pillai V, Ortega SB, Wang CK, Karandikar NJ. Transient regulatory T-cells: A state attained by all activated human T-cells. Clin Immunol 2006. 37. Wang J, Ioan-Facsinay A, van der Voort EI, Huizinga TW, Toes RE. Transient expression of FOXP3 in human activated nonregulatory CD4(+) T cells. European journal of immunology 2007;37(1):129-138. 38. Yu H, Kortylewski M, Pardoll D. Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment. Nature reviews 2007;7(1):41-51. 39. Gonzalez-Rey E, Chorny A, Fernandez-Martin A, Ganea D, Delgado M. Vasoactive intestinal peptide generates human tolerogenic dendritic cells that induce CD4 and CD8 regulatory T cells. Blood 2006;107(9):3632-3638. 40. Rezvani K, Mielke S, Ahmadzadeh M, Kilical Y, Savani BN, Zeilah J et al. High donor FOXP3-positive regulatory T-cell (Treg) content is associated with a low risk of GVHD following HLA-matched allogeneic SCT. Blood 2006;108(4):1291-1297. 41. Ziegler SF. FOXP3: of mice and men. Annual review of immunology 2006;24:209-226. 42. Kreijveld E, Koenen HJ, Klasen IS, Hilbrands LB, Joosten I. Following anti-CD25 treatment, a functional CD4+CD25+ regulatory T-cell pool is present in renal transplant recipients. Am J Transplant 2007;7(1):249-255.

Table 1

Study populationtransplanted between 06/21/2003 and 05/12/2006

Total number of patients studied 110

Age (mean +S.D.) 49+13

Age range: 18-72

Gender: Male 84 76%

Female 26 24%Endomyocardial Biopsy GradeTests N (%)

Grade 0 879 57%

Grade 1A 560 35%

Grade 1B 73 5%

Grade 2 9 1%

Grade 3A 36 2%

Total: 1557 100%Anti-HLA antibodies screeningTests N (%)(%)

HLA-class I Positive 76 4%

HLA-class II Positive 159 9%Total 1744 100%Lymphocyte Growth Assay (LGA)Tests N (%)LGA+ 161 11%LGA - 1365 89%Total 1526 100%

Table 1. Demographics and immunopathological findings

Figure 1

A. B.

FoxP

3 ex

pres

sion

in

per

iphe

ral C

D4

cells

01

23

4

Rejection No Rejection

02

46

810

Rejection No rejection

FoxP

3 ex

pres

sion

in

per

iphe

ral C

D8

cells

mean SE

rejection n=51 0.8851 0.8402

no-rejection n=153 6.6945 4.8673

mean SE

rejection n=51 0.0883 0.0200

no-rejection n=155 2.8657 1.3061

C. D.

0.0

0.10

0.20

Rejection No rejection

FoxP

3 ex

pres

sion

in

Hea

rt Bi

opsi

es

Response and Covariate n Estim. 95% CI P-

value*

(A) CD4 / FOXP3

206 -1.323 (-2.476, -0.169) 0.025

(B)CD8 / FOXP3

204 -0.030 (-0.085, 0.026)

0.296 (N.S.)

(C)BX / FOXP3

103 -0.115 (-0.509, 0.280)

0.416(N.S.)

mean SE

rejection n=44 0.0883 0.0499

no-rejection n=57 0.1437 0.0855

* GEE test

Figure 2

CD4 T cells

3A 0 or 1Biopsy Grade

0.001

0.01

0.10

1.00

10.00Fo

xP3

Expr

essi

onCD8 T cells

3A 0 or 1Biopsy Grade

0.001

0.01

0.10

1.00

10.00

100.00

Expression of FoxP3 in CD4+ and CD8+ Peripheral Blood T Cells after Successful Treatment of High Grade (>3A) Rejections

CD4+ FoxP3N= 8

CD8+ FoxP3N= 9

Incidence of increased expression 7/8 9/9

Mean Fold Increase +SD 7.54 + 8.88 13.52 + 30.23

Range 4.41 – 27.88 0.47 – 93.83

P-value * 0.036 0.028

* Wilcoxon Exact Test

Figure 3

CD8 T cells

0.01

0.10

1.00

3A 1B(1wk)

3A(2wks)

Biopsy Grade

FoxP

3 Ex

pres

sion

Table 2

Cells Analyzed Response Covariate n Estimate 95% CI p-value*

CD4 FOXP3

FOXP3

FOXP3

CD8 FOXP3 IL-10 242 2.079 (0.474, 3.684) 0.011

FOXP3

FOXP3

CTLA4 251 0.765 (0.213, 1.317) 0.007

CD8 CTLA4 246 0.594 (0.109, 1.078) 0.016

CD4 IL-10 251 0.351 (0.249, 0.452) <0.0001

CD4 IFN-G 247 2.004 (-0.261,4.270) N.S.

CD8 IFN-G 252 -0.070 (-2.257,2.118) N.S.

Table 2. FoxP3 as a marker of suppression in transplant patients:. Strong associations with the suppressive markers CTLA4 and IL-10 in T cells (irrespective of rejection status)

Cells Analyzed Response Covariate n Estimate 95% CI p-value*

FOXP3

FOXP3

FOXP3

EMB VEGF 102 1.235 (0.853, 1.616) 0.007

EMB IFN-G 100 0.238 (-0.415, 1.892) N.S.

EMB IL-10 99 2.547 (-3.969, 9.064) N.S.

* GEE tests

Figure 4

Association between anti-HLA Ab and FoxP3 expression (GEE test)

Response Covariate n Estimate 95% CI P-value

-0.068 0.014

N.S. (0.976)-0.0003

Anti-HLA IgG CD4 / FOXP3 255 (-0.123, -0.014)

Anti-HLA IgG CD8 / FOXP3 252 (-0.021,-0.020)

FOXP3 expression in peripheral CD4 cells

0.0 0.2 0.4 0.6 0.8 1.0

020

4060

80An

ti H

LA (I

gG) A

ntib

odie

s

FOXP3 expression in peripheral CD8 cells

0.0 0.2 0.4 0.6 0.8 1.00

2040

6080

Figure 5

A.

0

20

40

60

80

100

1 yr post-TxTransplantation

0

20

40

60

80

100

Transplantation 6 mo post-Tx

C.

D.

0

20

40

60

80

100

Transplantation 1 yr post-Tx

Anti-HLA (IgM) abAnti-HLA (IgG) ab

Legend

B.

0

20

40

60

80

100

1 yr post-TxTransplantation

F.E.

Number of Daclizumab treatmentsNone 1-3 Doses >3 Doses

Mo. N=7 N=16 N=87

0 0 0 06 100 38 23

12 100 38 3118 100 69 3924 100 69 42

Log rank statistics: P-value<0.0001

0

20

40

60

80

100

0 6 12 18 24

01-3>3

Number of Daclizumab Treatments

Months (post transplantation)

Cum

ulat

ive

Inci

denc

e (%

) of

anti-

dono

r Ab

prod

uctio

n

Figure 6

Acute Rejection

+ -0-3

> 3

6 16 22

9 78 87

15 94 109

Dac

lizum

abTr

eatm

ent (

dose

s)

Chi square test, P <0.04

Figure 7

LGA

+ -+-

20 14 34

138 1311 1449

158 1325 1483

AcuteRej.

Chi square test, P<0.0001

Figure 8

C.A.

80

85

90

95

100

0 12 24 36

Months post transplantation

Surv

ival

(%)

Group A (0-3 Daclizumab Treatments)

Group B (4+ Daclizumab Treatments)

0

0.1

0.2

0.3

0.4

0.5

0.6

> 0 > 1 > 2 > 3 > 4Number of Daclizumab treatment courses

Gra

ft Lo

ss H

azar

d R

atio

P=0.028

D.B.

Comparison of # of

Daclizumab courses

Hazard Ratio

95% Hazard Ratio Confidence limits

P-value*

( 0 ) vs (>0) 0.420 0.051 3.434 0.419

(≤1) vs (>1) 0.481 0.100 2.321 0.362

(≤2) vs (>2) 0.342 0.085 1.379 0.132

(≤3) vs (>3) 0.234 0.062 0.883 0.032

(≤4) vs (>4) 0.269 0.071 1.016 0.053

Patient Group A Patient Group B(0-3 doses of

Daclizumab) N=23Actuarial Survival

(%)

(4+ doses of Daclizumab) N=87 Actuarial Survival

(%)0 100 1006 86 10012 82 9618 82 9324 82 9330 82 8936 82 89

Mo. Post-Tx

* Cox proportional hazard modelLog rank statistics: P-value =0.028