Sequential Targeting of CD52 and TNF Allows Early Minimization Therapy in Kidney Transplantation: From a Biomarker to Targeting in a Proof-Of-Concept Trial.

BACKGROUND: There is high medical need for safe long-term immunosuppression monotherapy in kidney transplantation. Selective targeting of post-transplant alloantigen-(re)activated effector-T cells by anti-TNF antibodies after global T cell depletion may allow safe drug minimization, however, it is unsolved what might be the best maintenance monotherapy. METHODS: In this open, prospective observational single-centre trial, 20 primary deceased donor kidney transplant recipients received 2x20 mg Alemtuzumab (d0/d1) followed by 5 mg/kg Infliximab (d2). For 14 days all patients received only tacrolimus, then they were allocated to either receive tacrolimus (TAC, n = 13) or sirolimus (SIR, n = 7) monotherapy, respectively. Protocol biopsies and extensive immune monitoring were performed and patients were followed-up for 60 months. RESULTS: TAC-monotherapy resulted in excellent graft survival (5yr 92%, 95%CI: 56.6-98.9) and function, normal histology, and no proteinuria. Immune monitoring revealed low intragraft inflammation (urinary IP-10) and hints for the development of operational tolerance signature in the TAC- but not SIR-group. Remarkably, the TAC-monotherapy was successful in all five presensitized (ELISPOT+) patients. However, recruitment into SIR-arm was stopped (after n = 7) because of high incidence of proteinuria and acute/chronic rejection in biopsies. No opportunistic infections occurred during follow-up. CONCLUSIONS: In conclusion, our novel fast-track TAC-monotherapy protocol is likely to be safe and preliminary results indicated an excellent 5-year outcome, however, a full-scale study will be needed to confirm our findings. TRIAL REGISTRATION: EudraCT Number: 2006-003110-18.

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-Vß 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.

Expansion of memory-type CD8+ T cells correlates with the failure of early immunosuppression withdrawal after cadaver liver transplantation using high-dose ATG induction and rapamycin.

BACKGROUND: We report on a pilot study investigating the feasibility of early immunosuppression withdrawal after liver transplantation (LT) using antithymocyte globulin (ATG) induction and rapamycin. METHODS: LT recipients received 3.75 mg/kg per day ATG from days 0 to 5 followed by rapamycin-based immunosuppression. In the absence of acute rejection (AR), rapamycin was withdrawn after month 4. Immunomonitoring included analysis of peripheral T-cell phenotypes and clonality, cytokine production in mixed lymphocyte reaction, and characterization of intragraft infiltrating cells. RESULTS: Ten patients were enrolled between October 2009 and July 2010. In the first three patients, complete withdrawal of immunosuppression after month 4 led to AR. No further withdrawals of immunosuppressive were attempted. Two AR occurred in the remaining seven patients. ATG induced profound T-cell depletion followed by CD8(+) T-cell reexpansion exhibiting memory/effector-like phenotype associated with progressive oligoclonal T-cell expansion (Vß/HPRT ratio) and gradually enhanced anti-cytomegalovirus and anti-Epstein-Barr virus T-cell frequencies. Patients developing AR were characterized by decreased TCAIM expression. AR were associated with increased donor-specific production of interferon (IFN)-γ and interleukin (IL)-17, increased intragraft expression of IFN-γ mRNA, and significant CD8(+) T-cell infiltrates colocalizing with IL-17(+) cells. CONCLUSION: High-dose ATG followed by short-term rapamycin treatment failed to promote early operational tolerance to LT. AR correlates with expansion of memory-type CD8(+) T cells and increased levels of IFN-γ and IL-17 in mixed lymphocyte reaction and in the graft. This suggests that resistance and preferential expansion of effector memory T-cell in lymphopenic environment could represent the major barrier for establishment of tolerance to LT in approaches using T-cell-depleting induction.

Cutting Edge: Immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients.

Regulatory macrophages (M regs) were administered to two living-donor renal transplant recipients. Both patients were minimized to low-dose tacrolimus monotherapy within 24 wk of transplantation and subsequently maintained excellent graft function. After central venous administration, most M regs remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, and bone marrow. By 1 y posttransplantation, both patients displayed patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the range consistent with nonrejection. It is concluded that M regs warrant further study as a potential immune-conditioning therapy for use in solid-organ transplantation. The results of this work are being used to inform the design of The ONE Study, a multinational clinical trial of immunomodulatory cell therapy in renal transplantation.

Analysis of the peripheral T-cell repertoire in kidney transplant patients.

The long-term stability of renal grafts depends on the absence of chronic rejection. As T cells play a key role in rejection processes, analyzing the T-cell repertoire may be useful for understanding graft function outcomes. We have therefore investigated the power of a new statistical tool, used to analyze the peripheral blood TCR repertoire, for determining immunological differences in a group of 229 stable renal transplant patients undergoing immunosuppression. Despite selecting the patients according to stringent criteria, the patients displayed heterogeneous T-cell repertoire usage, ranging from unbiased to highly selected TCR repertoires; a skewed TCR repertoire correlating with an increase in the CD8(+) /CD4(+) T-cell ratio. T-cell repertoire patterns were compared in patients with clinically opposing outcomes i.e. stable drug-free operationally tolerant recipients and patients with the "suspicious" form of humoral chronic rejection and were found significantly different, from polyclonal to highly selected TCR repertoires, respectively. Moreover, a selected TCR repertoire was found to positively correlate with the Banff score grade. Collectively, these data suggest that TCR repertoire categorization might be included in the calculation of a composite score for the follow-up of patients after kidney transplantation.

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.

Failure of glatiramer acetate to modify the peripheral T cell repertoire of relapsing-remitting multiple sclerosis patients.

Glatiramer acetate (GA) is a random copolymer used as an immunomodulatory treatment in relapsing-remitting multiple sclerosis (RR-MS). Its mechanisms of action are poorly understood, and several hypotheses have been put forward, the majority of which rely on in vitro studies. It has been hypothesised that further to processing by APC, GA could provide a large number of different epitopes with a possible sequence similarity to auto-antigens, which are able to stimulate a large proportion of T cells. Given that in a previous study we showed that the circulating T cells of MS patients present more alterations of the Vbeta T cell receptor (TCR) usage than normal individuals, we explored the possible effect of GA on the ex vivo T cell repertoire of MS patients. Here we used quantitative PCR and electrophoresis to longitudinally analyse (and without any ex vivo stimulation), the CDR3 length distribution (LD) and the amount of Vbeta TCR, as well as various cytokines, in the blood T cells of 10 RR-MS patients before and after 3 months and 2 years of GA treatment. In addition, we also determined the status of responder and non-responder patients after 24 months of GA treatment based on clinical and radiological criteria. We found no significant modification of cytokine production, Vbeta TCR mRNA accumulation or CDR3-LD in the patients after short-term and long-term treatment. In addition, we did not observe any difference in CDR3-LD in the GA responder patients (n=6) compared to non-responder patients (n=4). Focusing our study on responder patients, we performed TCR repertoire analysis in the CD4+ and CD8+ compartment. Alterations of CDR3-LD were predominantly found in the CD8+ compartment, without any significant influence of GA treatment. Finally, the T cell repertoire variations in MS patients treated with GA and healthy controls were equivalent. Collectively, our data suggest that GA therapy does not induce significant variations in cytokine production or TCR usage in MS patients.

Statistical analysis of CDR3 length distributions for the assessment of T and B cell repertoire biases.

Complementarity-determining region 3 (CDR3) length distribution analysis explores the diversity of the T cell receptor (TCR) and immunoglobulin (Ig) repertoire at the transcriptome level. Studies of the CDR3, the most hypervariable part of these molecules, have been frequently used to identify recruitment of T and B cell clones involved in immunological responses. CDR3 length distribution analysis gives a clear perception of repertoire variations between individuals and over time. However, the complexity of CDR3 length distribution patterns and the high number of possible repertoire alterations per individual called for the development of robust data analysis methods. The goal of these methods is to identify, quantify and statistically assess differences between repertoires so as to offer a better diagnostic or predictive tool for pathologies involving the immune system. In this review we will explain the benefit of analyzing CDR3 length distribution for the study of immune cell diversity. We will start by describing this technology and its associated data processing, and will subsequently focus on the statistical methods used to compare CDR3 length distribution patterns. Finally, we will address the various methods for assessing CDR3 length distribution gene signatures in pathological states.

Serial blood T cell repertoire alterations in multiple sclerosis patients; correlation with clinical and MRI parameters.

A significant skewing of the peripheral T cell repertoire has been shown in relapsing-remitting multiple sclerosis (MS). Most of the studies already performed in this field are cross-sectional and therefore, little is known of the T cell repertoire evolution over time in MS and the correlation of T cell repertoire variation with clinical and MRI parameters. This study was performed on serially harvested frozen PBMC from nine untreated MS patients (27 samples) and 14 healthy individuals. The blood T cell repertoire of each patient was analysed at the complementarity determining region 3 (CDR3) level and compared with a monthly MRI scan performed over a six month period with assessment of T2 lesion load and gadolinium enhancing lesions. A highly significant blood T cell repertoire skewing was observed in MS patients as compared with healthy controls (p<0.01). In addition, the number of altered Vbeta families correlated significantly with both the T2 lesion volume and the number of gadolinium enhancing lesions as assessed by MRI (Spearman correlation tests, r=0.51 and r=0.44, p<0.01 and p<0.05 respectively). Furthermore, the variation of the number of altered Vbeta families over time also correlated with the appearance of new gadolinium enhancing lesions (r=0.36, p=0.05). These findings which need confirmation on larger serial cohorts, suggest an association between the magnitude of TCRBV CDR3 length distribution alterations in the peripheral blood of MS patients and the disease process.