Pierrick Guerif

Unique B Cell Differentiation Profile in Tolerant Kidney Transplant Patients

Operationally tolerant patients (TOL) display a higher number of blood B cells and transcriptional B cell signature. As they rarely develop an allo‐immune response, they could display an abnormal B cell differentiation. We used an in vitro culture system to explore T‐dependent differentiation of B cells into plasma cells. B cell phenotype, apoptosis, proliferation, cytokine, immunoglobulin production and markers of differentiation were followed in blood of these patients. Tolerant recipients show a higher frequency of CD20+CD24hiCD38hi transitional and CD20+CD38loCD24lo naïve B cells compared to patients with stable graft function, correlating with a decreased frequency of CD20−CD38+CD138+ differentiated plasma cells, suggestive of abnormal B cell differentiation. B cells from TOL proliferate normally but produce more IL‐10. In addition, B cells from tolerant recipients exhibit a defective expression of factors of the end step of differentiation into plasma cells and show a higher propensity for cell death apoptosis compared to patients with stable graft function. This in vitro profile is consistent with down‐regulation of B cell differentiation genes and anti‐apoptotic B cell genes in these patients in vivo. These data suggest that a balance between B cells producing IL‐10 and a deficiency in plasma cells may encourage an environment favorable to the tolerance maintenance.

Tolerant Kidney Transplant Patients Produce B Cells with Regulatory Properties

Whereas a B cell-transcriptional profile has been recorded for operationally tolerant kidney graft patients, the role that B cells have in this tolerance has not been reported. In this study, we analyzed the role of B cells from operationally tolerant patients, healthy volunteers, and kidney transplant recipients with stable graft function on T cell suppression. Proliferation, apoptosis, and type I proinflammatory cytokine production by effector CD4(+)CD25(-) T cells were measured after anti-CD3/anti-CD28 stimulation with or without autologous B cells. We report that B cells inhibit CD4(+)CD25(-) effector T cell response in a dose-dependent manner. This effect required B cells to interact with T-cell targets and was achieved through a granzyme B (GzmB)-dependent pathway. Tolerant recipients harbored a higher number of B cells expressing GzmB and displaying a plasma cell phenotype. Finally, GzmB(+) B-cell number was dependent on IL-21 production, and B cells from tolerant recipients but not from other patients positively regulated both the number of IL-21(+) T cells and IL-21 production, suggesting a feedback loop in tolerant recipients that increases excessive B cell activation and allows regulation to take place. These data provide insights into the characterization of B cell-mediated immunoregulation in clinical tolerance and show a potential regulatory effect of B cells on effector T cells in blood from patients with operationally tolerant kidney grafts.

Central Role of CD45RA− Foxp3hi Memory Regulatory T Cells in Clinical Kidney Transplantation Tolerance

The role of Foxp3(+) regulatory T cells (Tregs) in operational tolerance remains elusive, as initial results revealed an increased frequency of this subset in tolerant patients but no functional differences compared with immunosuppressed recipients. In addition, recent studies of regulatory B cells strongly suggest that Tregs may not have a central role in kidney transplantation tolerance. However, recent investigations of the crucial role of Foxp3 demethylation in Treg function and the possibility of identifying distinct Foxp3 T cell subsets prompted us to more thoroughly characterize Tregs in operationally tolerant patients. Thus, we studied the level of demethylation of the Foxp3 Treg-specific demethylated region (TSDR) in circulating CD4(+) T cells and analyzed Treg subset frequency in tolerant patients, healthy volunteers, patients with stable graft function under immunosuppression, and chronically rejecting recipients. We observed a higher proportion of CD4(+) T cells with demethylated Foxp3 and a specific expansion of CD4(+) CD45RA(-) Foxp3(hi) memory Tregs exclusively in tolerant patients. The memory Tregs of tolerant recipients exhibited increased Foxp3 TSDR demethylation, expressed higher levels of CD39 and glucocorticoid-induced TNF-related receptor, and harbored greater suppressive properties than memory Tregs from patients with stable graft function. Taken together, our data demonstrate that operationally tolerant patients mobilize an array of potentially suppressive cells, including not only regulatory B cells but also Tregs. Our results also indicate that tolerant patients have potent CD4(+)CD45RA(-) Foxp3(hi) memory Tregs with a specific Foxp3 TSDR demethylation pattern, which may contribute to the maintenance of graft tolerance.

A composite score associated with spontaneous operational tolerance in kidney transplant recipients

New challenges in renal transplantation include using biological information to devise a useful clinical test for discerning high- and low-risk patients for individual therapy and ascertaining the best combination and appropriate dosages of drugs. Based on a 20-gene signature from a microarray meta-analysis performed on 46 operationally tolerant patients and 266 renal transplant recipients with stable function, we applied the sparse Bolasso methodology to identify a minimal and robust combination of six genes and two demographic parameters associated with operational tolerance. This composite score of operational tolerance discriminated operationally tolerant patients with an area under the curve of 0.97 (95% confidence interval 0.94-1.00). The score was not influenced by immunosuppressive treatment, center of origin, donor type, or post-transplant lymphoproliferative disorder history of the patients. This composite score of operational tolerance was significantly associated with both de novo anti-HLA antibodies and tolerance loss. It was validated by quantitative polymerase chain reaction using independent samples and demonstrated specificity toward a model of tolerance induction. Thus, our score would allow clinicians to improve follow-up of patients, paving the way for individual therapy.

Decrease of blood anti-α1,3 Galactose Abs levels in multiple sclerosis (MS) and clinically isolated syndrome (CIS) patients.

The etiology of multiple sclerosis (MS) remains elusive. Among the possible causes, the increase of anti-Neu5Gc antibodies during EBV primo-infection of Infectious mononucleosis (IMN) may damage the integrity of the blood-brain barrier facilitating the transfer of EBV-infected B cells and anti-EBV T cell clones in the brain. We investigated the change in titers of anti-Neu5Gc and anti-α1,3 Galactose antibodies in 49 IMN, in 76 MS, and 73 clinically isolated syndrome (CIS) patients, as well as age/gender-matched healthy individuals. Anti-Gal and anti-Neu5Gc are significantly increased during IMN (p=0.02 and p<1.10-4 respectively), but not in acute CMV primo-infection. We show that, whereas there was no change in anti-Neu5Gc in MS/CIS, the two populations exhibit a significant decrease in anti-Gal (combined p=2.7.10-3), in contrast with patients with non-MS/CIS central nervous system pathologies. Since anti-Gal result from an immunization against α1,3 Gal, lacking in humans but produced in the gut, our data suggest that CIS and MS patients have an altered microbiota or an altered response to this microbiotic epitope.

Is pre-transplant sensitization against angiotensin II type 1 receptor still a risk factor of graft and patient outcome in kidney transplantation in the anti-HLA Luminex era? A retrospective study

We aimed to assess the correlation of anti‐angiotensin II type 1 receptor antibodies (anti‐AT1R‐Abs) before transplantation on a multicentric cohort of kidney transplant recipients (2008–2012), under tacrolimus and mycophenolate mofetil (MMF), screened by Luminex technology for anti‐HLA immunization. Anti‐AT1R antibody levels were measured by ELISA in pretransplantation sera of 940 kidney recipients from three French centers of the DIVAT cohort. Multivariable Cox models estimated the association between pretransplant anti‐angiotensin II type 1 receptor antibodies and time to acute rejection episodes (ARE) or time to graft failure. Within our cohort, 387 patients (41.2%) had pretransplant AT1R‐Abs higher than 10 U/ml and only 8% (72/970) greater than 17 U/ml. The cumulative probability of clinically relevant (cr)‐ARE was 22.5% at 1 year post‐transplantation [95% CI (19.9–25.4%)]. The cumulative probability of graft failure and patient death were 10.6% [95% CI (8.4–13.3%)] and 5.7% [95% CI (4.0–8.1%)] at 3 years post‐transplantation, respectively. Multivariate Cox models indicated that pretransplant anti‐AT1R antibody levels higher than 10 U/ml were not significantly independently associated with higher risks of acute rejection episodes [HR = 1.04, 95% CI (0.80–1.35)] nor with risk of graft failure [HR = 0.86, 95% CI (0.56–1.33)]. Our study did not confirm an association between pretransplant anti‐AT1R antibody levels and kidney transplant outcomes.

HCMV triggers frequent and persistent UL40-specific unconventional HLA-E-restricted CD8 T-cell responses with potential autologous and allogeneic peptide recognition

Immune response against human cytomegalovirus (HCMV) includes a set of persistent cytotoxic NK and CD8 T cells devoted to eliminate infected cells and to prevent reactivation. CD8 T cells against HCMV antigens (pp65, IE1) presented by HLA class-I molecules are well characterized and they associate with efficient virus control. HLA-E-restricted CD8 T cells targeting HCMV UL40 signal peptides (HLA-EUL40) have recently emerged as a non-conventional T-cell response also observed in some hosts. The occurrence, specificity and features of HLA-EUL40 CD8 T-cell responses remain mostly unknown. Here, we detected and quantified these responses in blood samples from healthy blood donors (n = 25) and kidney transplant recipients (n = 121) and we investigated the biological determinants involved in their occurrence. Longitudinal and phenotype ex vivo analyses were performed in comparison to HLA-A*02/pp65-specific CD8 T cells. Using a set of 11 HLA-E/UL40 peptide tetramers we demonstrated the presence of HLA-EUL40 CD8 αβT cells in up to 32% of seropositive HCMV+ hosts that may represent up to 38% of total circulating CD8 T-cells at a time point suggesting a strong expansion post-infection. Host’s HLA-A*02 allele, HLA-E *01:01/*01:03 genotype and sequence of the UL40 peptide from the infecting strain are major factors affecting the incidence of HLA-EUL40 CD8 T cells. These cells are effector memory CD8 (CD45RAhighROlow, CCR7-, CD27-, CD28-) characterized by a low level of PD-1 expression. HLA-EUL40 responses appear early post-infection and display a broad, unbiased, Vβ repertoire. Although induced in HCMV strain-dependent, UL4015-23-specific manner, HLA-EUL40 CD8 T cells are reactive toward a broader set of nonapeptides varying in 1–3 residues including most HLA-I signal peptides. Thus, HCMV induces strong and life-long lasting HLA-EUL40 CD8 T cells with potential allogeneic or/and autologous reactivity that take place selectively in at least a third of infections according to virus strain and host HLA concordance.

IL-15 Harnesses Pro-inflammatory Function of TEMRA CD8 in Kidney-Transplant Recipients

The involvement of TEMRA CD8 is evident in a large array of immunological conditions ranging from auto- to allo-immunity. Nevertheless, the factors leading to their accumulation and activation remain ill-defined and, efficient therapeutics to control their inflammatory response is lacking. Here, we show that IL-15-stimulated TEMRA from kidney-transplant (KT) recipients promote inflammation by inducing the expression of CX3CL1 by endothelial cells in an IFN-γ- and TNF-α-dependent manner. The responsiveness of TEMRA to IL-15 is not restricted to chronic stimulation, as TEMRA from healthy volunteers respond earlier and faster when compared to effector memory (EM). IL-15 induces antiapoptotic signals and promotes proliferation dependent of PI3K/Akt, MAPK, and ERK pathways. Without ex vivo stimulation, TEMRA cells are metabolically more active than naive and EM, as shown by their high ATP reservoir and a high expression of genes involved in glycolysis, glutaminolysis, and the Pentose Phosphate Pathway. Upon stimulation, TEMRA adapt their metabolism by sustaining an increased mitochondrial respiration and glycolysis. Finally, we show that the inhibition of glycolysis is highly effective in preventing endothelial inflammation induced by TEMRA from KT recipients. Together, our findings highlight the metabolic fitness that tightly regulates the immune function of TEMRA in physiological and pathogenic situations.

TEMRA CD8 T Cells from Human Kidney Transplant Recipients Exhibit Potent Anti-Donor Reactivity and Induce GVHD in Humanized Mouse Model

Introduction We have shown that an increase of TEMRA CD8 (CD45RA+CCR7-) evidenced 1 year post-transplantation is associated with a higher risk of kidney graft failure. The functionality of TEMRA CD8 and especially their reactivity upon donor-specific stimulation have never been investigated. In this study, we characterize before and one year after transplantation the functional response of TEMRA CD8 from Kidney Transplantation (KT) recipients after donor-specific stimulation and we setup a model of humanized NSG mice to investigate in vivo their pro-inflammatory response. Methods PBMC from 24 living-donor kidney-transplant recipients have been prospectively collected before and 12 month post-transplantation. Extensive phenotype of CD8 T cell subsets have been performed using multi-parameter flow cytometry. Naïve, TEMRA and EM CD8 from KT recipients have been cultured with irradiated donor-specific CD3-depleted PBMC. Expression of activation marker (CD25, CD69), cytotoxic marker (CD107a) and secretion of pro-inflammatory cytokines (Luminex Assay) have been analyzed after 24h and 48h of stimulation. Proliferation (CPD dilution) has been investigated after 5 days of culture. One to 5.106 TEMRA CD8 purified from KT recipients were co-injected i.v. with 107 T- and NK-depleted autologous PBMC in NSG HLA-A*0201 transgeneic mice. Xenogeneic GVHD was monitored by daily evaluation of the body weight and CD8 T cell infiltration was investigated using fluorescent microscopy in different organs. Results Kidney transplantation results in an increase of TEMRA CD8 (25 vs 39%), characterized by a high expression of GZMb and effector-associated transcription factor T-bet, and a concomitant decrease of NAÏVE CD8 whereas frequency of EM CD8 remains unchanged. Donor-specific stimulation results in the early expression of CD25+CD69+CD107a+ by TEMRA CD8 whereas NAÏVE CD8 failed to upregulate CD25 or CD107a. Expression of cytotoxic molecule CD107a by TEMRA was even enhanced after kidney transplantation. Donor specific stimulation results in the proliferation of TEMRA CD8. Finally, the infusion of TEMRA CD8 from KT recipients induces GVHD in 7 to 10 days with an infiltration of TEMRA in colon. Conclusion We demonstrate that TEMRA CD8 exhibit potent pathogenic function both in vitro when challenged with donor cells and in vivo resulting in an acute GVHD in immunodeficient mice. Our study highlights the need to design TEMRA specific therapy to improve kidney graft outcome. IHU Cesti (ANR-10-IBHU-005). FP7 VISICORT (602470). Fondation Centaure. Labex IGO (ANR-11-LABX-0016-01).

Corrigendum: Broad Impairment of Natural Killer Cells From Operationally Tolerant Kidney Transplanted Patients

[This corrects the article on p. 1721 in vol. 8, PMID: 29312288.].