Antibody immunosuppressive therapy in solid organ transplant

The use of antibodies in transplantation dates back to 1986 when muromonab CD3, a monoclonal antibody (mAb) targeting CD3, was first approved for prevention and treatment of renal allograft rejection. These agents have largely been used in a brief adjunctive manner to provide immunosuppression during the initial period after solid organ transplantation or during an episode of acute rejection. Recent advances in our understanding of transplant immunology have allowed emergence of numerous new mAbs, targeting co-stimulatory signals, cell surface receptors and novel protein constructs. During the next decade, transplant professionals will increasingly require knowledge of the mechanisms and pharmacologic characteristics of these novel therapeutic agents.     

Comparison of long-term immunosuppression for limb transplantation using cyclosporine, tacrolimus, and mycophenolate mofetil: implications for clinical composite tissue transplantation

This study compared the efficacy of long-term intermittent immunosuppression in preventing the rejection of a limb transplant across the strongest histocompatibility barrier in ACI --> Lewis rats using the conventional immunosuppressive agent cyclosporine-A and the newer immunosuppressive agents FK-506 (tacrolimus) and RS-61443 (mycophenolate mofetil). The recipient animals were immunosuppressed daily for 14 days postoperatively, followed by long-term intermittent, twice-weekly immunosuppression using cyclosporine 25 mg/kg, RS-61443 30 mg/kg, or FK-506 2 mg/kg. All three immunosuppressive agents were able to prolong the rejection of the skin component of a limb transplant compared with nonimmunosuppressed controls. Eight of nine animals receiving cyclosporine immunosuppression showed signs of rejection of the skin component of the limb transplant while continuing to receive long-term immunosuppression and had a mean rejection time of 61.6 days. Seven of 10 animals immunosuppressed with RS-61443 also showed signs of rejection while still receiving immunosuppression, with a mean rejection time of 43.6 days. Nine of 10 animals receiving FK-506 immunosuppression showed no signs of skin rejection, but died of bacterial pneumonia between 273 and 334 days after transplantation, with a mean rejection time of 296.1 days. There was no statistically significant difference between intermittent immunosuppression with cyclosporine and RS-61443, but FK-506 was significantly superior to both cyclosporine and RS-61443. The implication of this study is that FK-506, but not cyclosporine or RS-61443, is probably the only single immunosuppressive agent capable of preventing rejection of the skin component of a composite tissue transplant. Combination immunosuppression with FK-506 and RS-61443, therefore, may be required to allow composite tissue transplantation to become a predictable clinical reality in the future.     

Rejection prophylaxis with sequential OKT3 and CSA after kidney transplantation

Sixteen kidney transplant recipients received the IgG2a anti-CD3 monoclonal antibody OKT3 and azathioprine as rejection prophylaxis during the first two postoperative weeks. Concomitant immunosuppression consisted of low dose steroids while cyclosporine A therapy was instituted on day 12. Side effects included fever, bronchospasm, hypotension and diarrhoea. OKT3 caused T cell modulation resulting in CD3 dim +, CD4+ or CD8+, CD5+, WT31– and 11F2–cells. Anti-OKT3 antibodies were found in approximately 50% of the patients. The protocol induced a 100% patient and graft survival and a 81% actuarial freedom of rejection at 18 months. It prevented CsA associated nephrotoxicity in the direct postoperative phase.These beneficial effects outweighed the side effects of OKT3.     

CD8 T cells specific for a donor-derived, self-restricted transplant antigen are nonpathogenic bystanders after vascularized heart transplantation in mice

CD8 T cell cross-priming, an established mechanism of protective antiviral immunity, was originally discovered during studies involving minor transplantation Ags. It is unclear whether or how cross-primed CD8 T cells, reactive to donor-derived, but recipient class I MHC-restricted epitopes, could injure a fully MHC-disparate, vascularized transplant. To address this question we studied host class I MHC-restricted, male transplantation Ag-reactive T cell responses in female recipients of fully MHC-disparate, male heart transplants. Cross-priming to the immune-dominant determinant HYUtyp occurred at low frequency after heart transplantation. CD8 T cell preactivation through immunization with HYUtyp mixed in CFA did not alter the kinetics of acute rejection. Furthermore, neither HYUtyp immunization nor adoptive transfer of HYUtyp-specific TCR-transgenic T cells affected outcome in 1) a model of chronic rejection in the absence of immunosuppression or 2) a model of allograft acceptance induced by costimulatory blockade. The results support the contention that CD8 T cells reactive to host-restricted, but donor-derived, Ags are highly specific and are nonpathogenic bystanders during rejection of MHC-disparate cardiac allografts.     

Immunomodulatory effects of BXL-01-0029, a less hypercalcemic vitamin D analogue, in human cardiomyocytes and T cells

Current immunosuppressive protocols have reduced rejection occurrence in heart transplantation; nevertheless, management of heart transplant recipients is accompanied by major adverse effects, due to drug doses close to toxic range. In allograft rejection, characterized by T-helper 1 (Th1) cell-mediated response, the CXCL10-CXCR3 axis plays a pivotal role in triggering a self-promoting inflammatory loop. Indeed, CXCL10 intragraft production, required for initiation and development of graft failure, supports organ infiltration by Th1 cells. Thus, targeting the CXCL10-CXCR3 axis while avoiding generalized immunosuppression, may be of therapeutic significance. Based on preclinical evidence for immunoregulatory properties of vitamin D receptor agonists, we propose that a less hypercalcemic vitamin D analogue, BXL-01-0029, might have the potential to contribute to rejection management. We investigated the effect of BXL-01-0029 on CXCL10 secretion induced by proinflammatory stimuli, both in human isolated cardiomyocytes (Hfcm) and purified CD4+ T cells. Mycophenolic acid (MPA), the active agent of mycophenolate mofetil, was used for comparison. BXL-01-0029 inhibited IFNγ and TNFα-induced CXCL10 secretion by Hfcm more potently than MPA, impairing cytokine synergy and pathways. BXL-01-0029 reduced also CXCL10 protein secretion and gene expression by CD4+ T cells. Furthermore, BXL-01-0029 did not exert any toxic effect onto both cell types, suggesting its possible use as a dose-reducing agent for conventional immunosuppressive drugs in clinical transplantation.     

Anti-CD45RB monoclonal antibody-mediated transplantation tolerance

Currently, lifelong immunosuppression is required for organ transplant recipients. The majority of transplant recipients will eventually develop chronic rejection with resultant graft loss, despite treatment with powerful immunosuppressive agents. These agents are also associated with numerous toxicities including reduced immunity against infection and malignancy. Therefore, the central goal in transplant science is to devise tolerance strategies in an attempt to establish a state of prolonged non-reactivity against the allograft, accompanied with preservation of an intact immune system. Although predictable tolerance induction has been elusive, we found that short course of the novel immunomodulatory agent, anti-CD45RB monoclonal antibody, leads to indefinite acceptance of renal allografts in mice, and has been shown to markedly prolong allograft survival in primates. We review the current state of development of this antibody, and the progress made in defining its mechanism of action.     

Therapeutic implications of NK cell regulation of allogeneic CD8 T cell-mediated immune responses stimulated through the direct pathway of antigen presentation in transplantation

Natural killer (NK) cells are a population of innate type I lymphoid cells essential for early anti-viral responses and are known to modulate the course of humoral and cellular-mediated T cell responses. We assessed the role of NK cells in allogeneic CD8 T cell-mediated responses in an immunocompetent mouse model across an MHC class I histocompatibility barrier to determine its impact in therapeutic clinical interventions with polyclonal or monoclonal antibodies (mAbs) targeting lymphoid cells in transplantation. The administration of an NK cell depleting antibody to either CD8 T cell replete or CD8 T cell-depleted naïve C57BL/6 immunocompetent mice accelerated graft rejection. This accelerated rejection response was associated with an in vivo increased cytotoxic activity of CD8 T cells against bm1 allogeneic hematopoietic cells and bm1 skin allografts. These findings show that NK cells were implicated in the control host anti-donor cytotoxic responses, likely by competing for common cell growth factors in both CD8 T cell replete and CD8 T cell-depleted mice, the latter reconstituting in response to lymphopenia. Our data calls for precaution in solid organ transplantation under tolerogenic protocols involving extensive depletion of lymphocytes. These pharmacological biologics with depleting properties over NK cells may accelerate graft rejection and promote aggressive CD8 T cell cytotoxic alloresponses refractory to current immunosuppression.     

Behaviour of Non-Donor Specific Antibodies during Rapid Re-Synthesis of Donor Specific HLA Antibodies after Antibody Incompatible Renal Transplantation

Background

HLA directed antibodies play an important role in acute and chronic allograft rejection. During viral infection of a patient with HLA antibodies, the HLA antibody levels may rise even though there is no new immunization with antigen. However it is not known whether the converse occurs, and whether changes on non-donor specific antibodies are associated with any outcomes following HLA antibody incompatible renal transplantation.

Methods

55 patients, 31 women and 24 men, who underwent HLAi renal transplant in our center from September 2005 to September 2010 were included in the studies. We analysed the data using two different approaches, based on; i) DSA levels and ii) rejection episode post transplant. HLA antibody levels were measured during the early post transplant period and corresponding CMV, VZV and Anti-HBs IgG antibody levels and blood group IgG, IgM and IgA antibodies were quantified.

Results

Despite a significant DSA antibody rise no significant non-donor specific HLA antibody, viral or blood group antibody rise was found. In rejection episode analyses, multiple logistic regression modelling showed that change in the DSA was significantly associated with rejection (p = 0.002), even when adjusted for other antibody levels. No other antibody levels were predictive of rejection. Increase in DSA from pre treatment to a post transplant peak of 1000 was equivalent to an increased chance of rejection with an odds ratio of 1.47 (1.08, 2.00).

Conclusion

In spite of increases or decreases in the DSA levels, there were no changes in the viral or the blood group antibodies in these patients. Thus the DSA rise is specific in contrast to the viral, blood group or third party antibodies post transplantation. Increases in the DSA post transplant in comparison to pre-treatment are strongly associated with occurrence of rejection.     

Design,synthesis, and evaluation of 4,6-diaminonicotinamide derivatives as novel and potent immunomodulators targeting JAK3

In organ transplantation, T cell-mediated immune responses play a key role in the rejection of allografts. Janus kinase 3 (JAK3) is specifically expressed in hematopoietic cells and associated with regulation of T cell development via interleukin-2 signaling pathway. Here, we designed novel 4,6-diaminonicotinamide derivatives as immunomodulators targeting JAK3 for prevention of transplant rejection. Our optimization of C4- and C6-substituents and docking calculations to JAK3 protein confirmed that the 4,6-diaminonicotinamide scaffold resulted in potent inhibition of JAK3. We also investigated avoidance of human ether-a-go-go related gene (hERG) inhibitory activity. Selected compound 28 in combination with tacrolimus prevented allograft rejection in a rat heterotopic cardiac transplantation model.     

Structured to reduce the mitogenicity of anti-CD3 antibody based on computer-guided molecular design

The mouse anti-human CD3 monoclonal antibody such as OKT3 is a potent immunosuppressive agent used in clinical transplantation to manipulate T-cell functions and prevent acute allograft rejection. However, the broad use of anti-CD3 antibody in clinical treatment was severely limited by the side effects of human anti-mouse antibody response and cytokine release syndrome. In this study, on the basis of a murine anti-human CD3 antibody yCD3 obtained in our previous work, a novel engineered anti-human CD3 antibody fragment (i.e. V(H)-Linker-V(L)-Hinge-CH(3)) was constructed with computer-guided molecular design method to avoid the clinical side effects. According to the distance geometry and intra-molecular interaction, the hinge region was re-designed and different from the parental hinge region in human IgG1. With the novel hinge region, the cysteine residues in hinge were exposure and prone to form the disulfide bond. Therefore, a novel bivalent antibody fragment named as mini-yCD3 was obtained. Mini-yCD3 displayed similar antigen-binding affinity and specificity to yCD3. Importantly, mini-yCD3 was shown to be much less potent in the induction of T-cell proliferation, cytokine release (interferon-gamma and interleukin-2) and early activation marker expression on the cell surface (CD69 and CD25) than parental yCD3. Furthermore, mini-yCD3 was effective in modulating T-cell receptor/CD3 and inhibiting mixed lymphocyte reaction with similarity as yCD3. In conclusion, the constructed mini-yCD3 was much less mitogenic to T cells but retained potent immunosuppression, suggesting it might be an alternative to yCD3 as an immunosuppressive drug with less immunogenicity and toxicity for clinical application.     

Cytokine profiles in early rejection following OKT3 treatment in liver transplant patients

OKT3 , a murine monoclonal antibody specific to the human CD3 complex, induces immunosuppression by depletion of T cells. Administration of OKT3 results in significant release of proinflammatory cytokines, such as TNFalpha and IL1beta. Liver recipients who experience rejection within 3 weeks after transplantation with OKT3 prophylaxis recover their T cells by postoperative day 10 despite complete initial clearance. We sought to analyze the role of proinflammatory and Th-1 cytokines in T cell recovery and rejection after liver transplantation with OKT3 prophylaxis. In plasma samples from 32 patients, we measured TNFalpha, IL1beta and IL6 (before transplant and on postoperative days 1, 2 and 3) and IL2, IFNgamma, sIL2R and slCAM (postoperative days 5, 7 and 10) and examined possible correlations with T-cell recovery and occurrence of rejection within 3 weeks. TNFalpha, IL1beta, and IL6 did not correlate with T-cell recovery. In patients who rejected, IL2 and IFNgamma on postoperative days 5 and 7 correlated with degree of T-cell recovery by day 10; a significant rise in sIL2R over time also correlated with T-cell recovery in this group. Our results emphasize the role of Th-1 cytokines in rejection following OKT3 induction and suggest that markers of T cell activation may predict risk.     

Increased Numbers of Circulating CD8 Effector Memory T Cells before Transplantation Enhance the Risk of Acute Rejection in Lung Transplant Recipients

The effector and regulatory T cell subpopulations involved in the development of acute rejection episodes in lung transplantation remain to be elucidated. Twenty-seven lung transplant candidates were prospectively monitored before transplantation and within the first year post-transplantation. Regulatory, Th17, memory and naïve T cells were measured in peripheral blood of lung transplant recipients by flow cytometry. No association of acute rejection with number of peripheral regulatory T cells and Th17 cells was found. However, effector memory subsets in acute rejection patients were increased during the first two months post-transplant. Interestingly, patients waiting for lung transplant with levels of CD8⁺ effector memory T cells over 185 cells/mm³ had a significant increased risk of rejection [OR: 5.62 (95% CI: 1.08-29.37), p=0.04]. In multivariate analysis adjusted for age and gender the odds ratio for rejection was: OR: 5.89 (95% CI: 1.08-32.24), p=0.04. These data suggest a correlation between acute rejection and effector memory T cells in lung transplant recipients. The measurement of peripheral blood CD8⁺ effector memory T cells prior to lung transplant may define patients at high risk of acute lung rejection.     

ES-cell derived hematopoietic cells induce transplantation tolerance

Background

Bone marrow cells induce stable mixed chimerism under appropriate conditioning of the host, mediating the induction of transplantation tolerance. However, their strong immunogenicity precludes routine use in clinical transplantation due to the need for harsh preconditioning and the requirement for toxic immunosuppression to prevent rejection and graft-versus-host disease. Alternatively, embryonic stem (ES) cells have emerged as a potential source of less immunogenic hematopoietic progenitor cells (HPCs). Up till now, however, it has been difficult to generate stable hematopoietic cells from ES cells.

Methodology/Principal Findings

Here, we derived CD45⁺ HPCs from HOXB4-transduced ES cells and showed that they poorly express MHC antigens. This property allowed their long-term engraftment in sublethally irradiated recipients across MHC barriers without the need for immunosuppressive agents. Although donor cells declined in peripheral blood over 2 months, low level chimerism was maintained in the bone marrow of these mice over 100 days. More importantly, chimeric animals were protected from rejection of donor-type cardiac allografts.

Conclusions

Our data show, for the first time, the efficacy of ES-derived CD45⁺ HPCs to engraft in allogenic recipients without the use of immunosuppressive agents, there by protecting cardiac allografts from rejection.     

Alteration of Innate Immunity by Donor IL-6 Deficiency in a Presensitized Heart Transplant Model

Engraftment of IL-6 deficient donor into wild-type recipient could significantly improve allograft survival through T cell lineage particularly regulatory T cells (Tregs) in non-sensitized transplant host. However, its effect on innate immune responses remains uncertain. Our data revealed that donor IL-6 deficiency significantly increased infiltration of two subsets of MDSCs (CD11b+Gr1+myeloid-derived suppressor cells), CD11b+Gr1^-low and CD11b+Gr1^-int with strong immunosuppression activity in the transplanted graft. It resulted in a dramatic increase of CD11b+Gr1^-low frequency and a significant decrease of the frequency of CD11b+Gr1^-high and CD4-CD8-NK1.1+ cells in the recipient’s spleen. Unexpectedly, donor IL-6 deficiency could not significantly reduce macrophage frequency irrespective of in the host’s spleen or graft. Taken together, suppression of innate immune effector cells and enhanced activity of regulatory MDSCs contributed to tolerance induction by blockade of IL-6 signaling pathway. The unveiled novel mechanism of targeting IL-6 might shed light on clinical therapeutic application in preventing accelerated allograft rejection for those pre-sensitized transplant recipients.     

Immunomodulatory effects of mesenchymal stem cells for the treatment of cardiac allograft rejection

Heart transplantation continues to be the gold standard clinical intervention to treat patients with end-stage heart failure. However, there are major complications associated with this surgical procedure that reduce the survival prognosis of heart transplant patients, including allograft rejection, malignancies, infections, and other complications that arise from the use of broad-spectrum immunosuppression drugs. Recent studies have demonstrated the use of mesenchymal stem cells (MSCs) against allotransplantation rejection in both in vitro and in vivo settings due to their immunomodulatory properties. Therefore, utilization of MSCs provides new and exciting strategies to improve heart transplantation and potentially reduce the use of broad-spectrum immunosuppression drugs while alleviating allograft rejection. In this review, we will discuss the current research on the mechanisms of cardiac allograft rejection, the physiological and immunological characteristics of MSCs, the effects of MSCs on the immune system, and immunomodulation of heart transplantation by MSCs.     

Pre-Transplant Donor-Specific T-Cell Alloreactivity Is Strongly Associated with Early Acute Cellular Rejection in Kidney Transplant Recipients Not Receiving T-Cell Depleting Induction Therapy

Preformed T-cell immune-sensitization should most likely impact allograft outcome during the initial period after kidney transplantation, since donor-specific memory T-cells may rapidly recognize alloantigens and activate the effector immune response, which leads to allograft rejection. However, the precise time-frame in which acute rejection is fundamentally triggered by preformed donor-specific memory T cells rather than by de novo activated naïve T cells is still to be established. Here, preformed donor-specific alloreactive T-cell responses were evaluated using the IFN-γ ELISPOT assay in a large consecutive cohort of kidney transplant patients (n = 90), to assess the main clinical variables associated with cellular sensitization and its predominant time-frame impact on allograft outcome, and was further validated in an independent new set of kidney transplant recipients (n = 67). We found that most highly T-cell sensitized patients were elderly patients with particularly poor HLA class-I matching, without any clinically recognizable sensitizing events. While one-year incidence of all types of biopsy-proven acute rejection did not differ between T-cell alloreactive and non-alloreactive patients, Receiver Operating Characteristic curve analysis indicated the first two months after transplantation as the highest risk time period for acute cellular rejection associated with baseline T-cell sensitization. This effect was particularly evident in young and highly alloreactive individuals that did not receive T-cell depletion immunosuppression. Multivariate analysis confirmed preformed T-cell sensitization as an independent predictor of early acute cellular rejection. In summary, monitoring anti-donor T-cell sensitization before transplantation may help to identify patients at increased risk of acute cellular rejection, particularly in the early phases after kidney transplantation, and thus guide decision-making regarding the use of induction therapy.     

Immunological monitoring of renal transplant recipients to predict acute allograft rejection following the discontinuation of tacrolimus

Background

Transplant patients would benefit from reduction of immunosuppression providing that graft rejection is prevented. We have evaluated a number of immunological markers in blood of patients in whom tacrolimus was withdrawn after renal transplantation. The alloreactive precursor frequency of CD4+ and CD8+ T cells, the frequency of T cell subsets and the functional capacity of CD4+CD25+FoxP3+ regulatory T cells (Treg) were analyzed before transplantation and before tacrolimus reduction. In a case-control design, the results were compared between patients with (n = 15) and without (n = 28) acute rejection after tacrolimus withdrawal.

Principal Findings

Prior to tacrolimus reduction, the ratio between memory CD8+ T cells and Treg was higher in rejectors compared to non-rejectors. Rejectors also had a higher ratio between memory CD4+ T cells and Treg, and ratios <20 were only observed in non-rejectors. Between the time of transplantation and the start of tacrolimus withdrawal, an increase in naive T cell frequencies and a reciprocal decrease of effector T cell percentages was observed in rejectors. The proportion of Treg within the CD4+ T cells decreased after transplantation, but anti-donor regulatory capacity of Treg remained unaltered in rejectors and non-rejectors.

Conclusions

Immunological monitoring revealed an association between acute rejection following the withdrawal of tacrolimus and 1) the ratio of memory T cells and Treg prior to the start of tacrolimus reduction, and 2) changes in the distribution of naive, effector and memory T cells over time. Combination of these two biomarkers allowed highly specific identification of patients in whom immunosuppression could be safely reduced.     

New immunosuppressive drugs in heart transplantation

Only a few randomized clinical trials have been performed so far in heart transplant recipients, mainly because of the relatively small number of heart transplants performed worldwide each year. The main focus of the few controlled trials that have been completed has been the prevention and treatment of heart allograft rejection. In the area of pharmacologic immunosuppression, both biological agents and drugs have been the subject of investigation. Among the biological agents, chimeric monoclonal antibodies directed against the interleukin (IL)-2 receptor, which have been found to be safe and effective in renal transplant recipients, are now undergoing the test of controlled trials in heart transplant recipients. Immunosuppressive drugs that have been studied in controlled trials include calcineurin inhibitors (such as the microemulsion formulation of cyclosporine and tacrolimus) and inhibitors of purine synthesis, such as mycophenolate mofetil. Non-pharmacologic prophylactic immunosuppression with photopheresis has also been tested in a prospective, multicenter, randomized trial. New immunosuppressive regimens, such as mycophenolate mofetil combined with a monoclonal antibody against the IL-2 receptor, are being tested with the aim to reduce or eliminate calcineurin inhibitors or corticosteroids. Although clinical approaches to the induction of tolerance have undergone preliminary clinical evaluation, the ability to induce tolerance to an allograft in humans remains an elusive goal.     

Development of tolerogenic strategies in the clinic

The study of tolerance in the clinic can be divided into three areas: (i) focused evaluation of existing tolerant transplant recipients as to their mechanism of tolerance; (ii) prospective tolerance trials, such as combined bone marrow and kidney transplantation as well as T cell depletion followed by subsequent weaning of immunosuppression; and (iii) immunologic assays to assess the likelihood of rejection or tolerance. Frankly, a very small number of patients have been transplanted with the intention of removing all immunosuppressive therapy, but several clinical trials with this aim are currently in progress, largely sponsored by the Immune Tolerance Network, a joint venture between the National Institutes of Health and the Juvenile Diabetes Research Foundation. Similarly, a reliable assay to assess tolerance has not yet been developed but a variety of approaches towards assessing rejection, and in some cases tolerance, are being developed. It would be accurate to state that many of the experimental and preclinical approaches to the induction of tolerance have resulted in better immunosuppression for human transplantation, but reliable tolerance strategies in humans have not yet been achieved. Combined bone marrow and kidney transplantation may be considered as one exception to this, but such a strategy is not generally applicable to the vast majority of solid organ transplant recipients. This review will summarize efforts to date, particularly focusing on kidney transplantation.     

DR(high+)CD45RA(-)-Tregs potentially affect the suppressive activity of the total Treg pool in renal transplant patients

Recent studies show that regulatory T cells (Tregs) play an essential role in tolerance induction after organ transplantation. In order to examine whether there are differences in the composition of the total CD4(+)CD127(low+/-)FoxP3(+)- Treg cell pool between stable transplant patients and patients with biopsy proven rejection (BPR), we compared the percentages and the functional activity of the different Treg cell subsets (DR(high+)CD45RA(-)-Tregs, DR(low+)CD45RA(-)-Tregs, DR(-)CD45RA(-)-Tregs, DR(-)CD45RA(+)-Tregs). All parameters were determined during the three different periods of time after transplantation (0-30 days, 31-1,000 days, >1,000 days). Among 156 transplant patients, 37 patients suffered from BPR. The most prominent differences between rejecting and non-rejecting patients were observed regarding the DR(high+)CD45RA(-)-Treg cell subset. Our data demonstrate that the suppressive activity of the total Treg pool strongly depends on the presence of these Treg cells. Their percentage within the total Treg pool strongly decreased after transplantation and remained relatively low during the first year after transplantation in all patients. Subsequently, the proportion of this Treg subset increased again in patients who accepted the transplant and reached a value of healthy non-transplanted subjects. By contrast, in patients with acute kidney rejection, the DR(high+)CD45RA(-)-Treg subset disappeared excessively, causing a reduction in the suppressive activity of the total Treg pool. Therefore, both the monitoring of its percentage within the total Treg pool and the monitoring of the HLA-DR MFI of the DR(+)CD45RA(-)-Treg subset may be useful tools for the prediction of graft rejection.