Maria P. Hernandez-Fuentes

Identification of a B cell signature associated with renal transplant tolerance in humans

Establishing long-term allograft acceptance without the requirement for continuous immunosuppression, a condition known as allograft tolerance, is a highly desirable therapeutic goal in solid organ transplantation. Determining which recipients would benefit from withdrawal or minimization of immunosuppression would be greatly facilitated by biomarkers predictive of tolerance. In this study, we identified the largest reported cohort to our knowledge of tolerant renal transplant recipients, as defined by stable graft function and receiving no immunosuppression for more than 1 year, and compared their gene expression profiles and peripheral blood lymphocyte subsets with those of subjects with stable graft function who are receiving immunosuppressive drugs as well as healthy controls. In addition to being associated with clinical and phenotypic parameters, renal allograft tolerance was strongly associated with a B cell signature using several assays. Tolerant subjects showed increased expression of multiple B cell differentiation genes, and a set of just 3 of these genes distinguished tolerant from nontolerant recipients in a unique test set of samples. This B cell signature was associated with upregulation of CD20 mRNA in urine sediment cells and elevated numbers of peripheral blood naive and transitional B cells in tolerant participants compared with those receiving immunosuppression. These results point to a critical role for B cells in regulating alloimmunity and provide a candidate set of genes for wider-scale screening of renal transplant recipients.

Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

Multiparameter immune profiling of operational tolerance in liver transplantation

Immunosuppressive drugs can be completely withdrawn in up to 20% of liver transplant recipients, commonly referred to as ‘operationally’ tolerant. Immune characterization of these patients, however, has not been performed in detail, and we lack tests capable of identifying tolerant patients among recipients receiving maintenance immunosuppression. In the current study we have analyzed a variety of biological traits in peripheral blood of operationally tolerant liver recipients in an attempt to define a multiparameter ‘fingerprint’ of tolerance. Thus, we have performed peripheral blood gene expression profiling and extensive blood cell immunophenotyping on 16 operationally tolerant liver recipients, 16 recipients requiring on‐going immunosuppressive therapy, and 10 healthy individuals. Microarray profiling identified a gene expression signature that could discriminate tolerant recipients from immunosuppression‐dependent patients with high accuracy. This signature included genes encoding for γδ T‐cell and NK receptors, and for proteins involved in cell proliferation arrest. In addition, tolerant recipients exhibited significantly greater numbers of circulating potentially regulatory T‐cell subsets (CD4+CD25+ T‐cells and Vδ1+ T cells) than either non‐tolerant patients or healthy individuals. Our data provide novel mechanistic insight on liver allograft operational tolerance, and constitute a first step in the search for a non‐invasive diagnostic signature capable of predicting tolerance before undergoing drug weaning.

Loss of Direct and Maintenance of Indirect Alloresponses in Renal Allograft Recipients: Implications for the Pathogenesis of Chronic Allograft Nephropathy

Chronic allograft nephropathy (CAN) is the principal cause of late renal allograft failure. This complex process is multifactorial in origin, and there is good evidence for immune-mediated effects. The immune contribution to this process is directed by CD4+ T cells, which can be activated by either direct or indirect pathways of allorecognition. For the first time, these pathways have been simultaneously compared in a cohort of 22 longstanding renal allograft recipients (13 with good function and nine with CAN). CD4+ T cells from all patients reveal donor-specific hyporesponsiveness by the direct pathway according to proliferation or the secretion of the cytokines IL-2, IL-5, and IFN-γ. Donor-specific cytotoxic T cell responses were also attenuated. In contrast, the frequencies of indirectly alloreactive cells were maintained, patients with CAN having significantly higher frequencies of CD4+ T cells indirectly activated by allogeneic peptides when compared with controls with good allograft function. An extensive search for alloantibodies has revealed significant titers in only a minority of patients, both with and without CAN. In summary, this study demonstrates widespread donor-specific hyporesponsiveness in directly activated CD4+ T cells derived from longstanding recipients of renal allografts, whether they have CAN or not. However, patients with CAN have significantly higher frequencies of CD4+ T cells activated by donor Ags in an indirect manner, a phenomenon resembling split tolerance. These findings provide an insight into the pathogenesis of CAN and also have implications for the development of a clinical tolerance assay.

Significant Frequencies of T Cells With Indirect Anti-Donor Specificity in Heart Graft Recipients With Chronic Rejection

BACKGROUND The purpose of this study was to determine whether T cells with indirect allospecificity could be detected in heart transplant recipients with chronic rejection. METHOD AND RESULTS Human T-cell clones were used to determine the most effective way to deliver major histocompatibility complex alloantigens for indirect presentation. Seven allograft recipients with evidence of progressive, chronic rejection were selected. Four heart graft recipients with no evidence of chronic rejection were used as controls. Peripheral blood T cells and antigen-presenting cells from the recipients were cultured with frozen/thawed stored donor cells or major histocompatibility complex class I-derived synthetic peptides in limiting dilution cultures and then compared with controls using tetanus toxoid and frozen/thawed third-party cells with no human leukocyte antigens in common with the donor. In 5 of 7 patients analyzed who had chronic rejection, elevated frequencies of T cells with indirect, anti-donor specificity (iHTLf) were detected. No such elevated iHTLf were detected in recipients without chronic rejection. DISCUSSION iHTLf can be obtained from human transplant recipients, which supports the contention that the indirect pathway is involved in chronic transplant rejection.

Everolimus and basiliximab permit suppression by human CD4+CD25+ cells in vitro.

Immunosuppressive drugs are essential for the prevention of acute transplant rejection but some may not promote long‐term tolerance. Tolerance is dependent on the presence and regulatory function of CD4+CD25+ T cells in a number of animal models. The direct effects of immunosuppressive drugs on CD4+CD25+ cells, particularly those that interfere with IL‐2 signaling are uncertain. We studied the effects of the rapamycin derivative everolimus and the anti‐CD25 monoclonal antibody basiliximab on the regulatory capacity of human CD4+CD25+ cells in vitro. Both drugs permitted the suppression of proliferation and IFN‐γ secretion by CD4+CD25− cells responding to allogeneic and other polyclonal stimuli; CTLA‐4 expression was abolished on CD4+CD25+ cells without compromising their suppressive ability. Everolimus reduced IFN‐γ secretion by CD4+CD25− cells before the anti‐proliferative effect: this is a novel finding. Exogenous IL‐2 and IL‐15 could prevent the suppression of proliferation by CD4+CD25+ cells and the drugs could not restore suppression. By contrast, suppression of IFN‐γ secretion was only slightly impeded with the exogenous cytokines. Finally, CD4+CD25+ cells were more resistant than CD4+CD25− cells to the pro‐apoptotic action of the drugs. Together these data suggest that CD4+CD25+ cells may still exert their effects in transplant patients taking immunosuppression that interferes with IL‐2 signaling.

Immunologic monitoring. [Review] [136 refs]

Summary:  The development of reliable in vitro assays that could allow the quantitation and characterization of anti‐donor alloimmune responses has always been a goal in clinical transplantation, both to predict presensitization to the transplanted tissue and to be able to identify rejection without resorting to more invasive tests. With recent development in our understanding of transplantation biology and therapeutics, there is a real expectation that these tests may be used to identify tolerance as much as to predict rejection. The traditional limiting dilution assays still have a contribution to make and are being complemented by an array of tools, such as ELISpot, flow cytometry‐based techniques, and microarray analysis. The assays that have been informative, to date, are discussed in this review. This information will lead, at least, to a better understanding of how and when the rejection process occurs. More interestingly, the objective is to apply this information to evaluate tolerance‐inducing strategies or to identify patients that have become tolerant to their graft and can be weaned of immunosuppression. Of course sensitive, accurate and specific immunologic monitoring has applications well beyond the field of transplantation.

CD4+CD25+ Regulatory T Cells Do Not Significantly Contribute to Direct Pathway Hyporesponsiveness in Stable Renal Transplant Patients

CD4(+)CD25(+) regulatory T cells have been shown to regulate a variety of autoimmune and allogeneic responses in mice and humans. The role of CD4(+)CD25(+) cells in regulating alloresponses in human transplant recipients remains uncertain. Previous research has demonstrated a reduced frequency of direct pathway donor-specific T cells in renal transplant recipients when compared with the frequency of T cells reactive to an HLA-matched third party. A number of mechanisms have been proposed to account for this finding; the purpose of this study was to determine whether CD4(+)CD25(+) cells play a significant role. Twelve stable renal transplant patients were investigated using limiting dilution assay (LDA) and ELISPOT for interferon-gamma to determine the effect of depleting CD4(+)CD25(+) cells on the direct pathway alloresponse. The percentage of CD4(+)CD25(+) cells in the peripheral blood of the study patients was equivalent to that of healthy controls. Furthermore, in no case did depletion of CD4(+)CD25(+) cells significantly increase the frequency of donor-specific T cells detected by LDA. This was also found with ELISPOT in all except one patient, in whom depletion revealed an increased frequency of alloreactive T cell to both donor and third party. Finally, kinetic analysis of the LDA data did not indicate regulation against donor when compared with third party. It is concluded that the action of CD4(+)CD25(+) regulatory cells is not the main mechanism of donor-specific hyporesponsiveness in the direct pathway of allorecognition.

Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated with IL-10-Anergized Donor T Cells.

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.

Cross-validation of IFN-γ Elispot assay for measuring alloreactive memory/effector T cell responses in renal transplant recipients.

Assessment of donor‐specific alloreactive memory/effector T cell responses using an IFN‐γ Elispot assay has been suggested to be a novel immune‐monitoring tool for evaluating the cellular immune risk in renal transplantation. Here, we report the cross‐validation data of the IFN‐γ Elispot assay performed within different European laboratories taking part of the EU RISET consortium. For this purpose, development of a standard operating procedure (SOP), comparisons of lectures of IFN‐γ plates assessing intra‐ and interlaboratory assay variability of allogeneic or peptide stimuli in both healthy and kidney transplant individuals have been the main objectives. We show that the use of a same SOP and count‐settings of the Elispot bioreader allow low coefficient variation between laboratories. Frozen and shipped samples display slightly lower detectable IFN‐γ frequencies than fresh samples. Importantly, a close correlation between different laboratories is obtained when measuring high frequencies of antigen‐specific primed/memory T cell alloresponses. Interestingly, significant high donor‐specific alloreactive T cell responses can be similarly detected among different laboratories in kidney transplant patients displaying histological patterns of acute T cell mediated rejection. In conclusion, assessment of circulating alloreactive memory/effector T cells using an INF‐γ Elispot assay can be accurately achieved using the same SOP, Elispot bioreader and experienced technicians in kidney transplantation.