Efficient Expansion of Human Granzyme B-Expressing B Cells with Potent Regulatory Properties.

Granzyme B-expressing B cells have been shown to be an important regulatory B cell subset in humans. However, it is unclear which subpopulations of B cells express GZMB under normal conditions and which protocols effectively induce ex vivo expansion of GZMB+ B cells. We found that in the peripheral blood of normal individuals, plasmablasts were the major B cell subpopulation that expressed GZMB. However, when using an in vitro plasmablast differentiation protocol, we obtained only 2% GZMB+ B cells. Nevertheless, using an expansion mixture containing IL-21, anti-BCR, CpG oligodeoxynucleotide, CD40L, and IL-2, we were able to obtain more than 90% GZMB+ B cells after 3 d culture. GZMB+ B cells obtained through this protocol suppressed the proliferation of autologous and allogenic CD4+CD25- effector T cells. The suppressive effect of GZMB+ B cells was partially GZMB dependent and totally contact dependent but was not associated with an increase in effector T cell apoptosis or uptake of GZMB by effector T cells. Interestingly, we showed that GZMB produced by B cells promoted GZMB+ B cell proliferation in ERK1/2-dependent manner, facilitating GZMB+ B cell expansion. However, GZMB+ B cells tended to undergo apoptosis after prolonged stimulation, which may be considered a negative feedback mechanism to limit their uncontrolled expansion. Finally, we found that expanded GZMB+ B cells exhibited a regulatory phenotype and were enriched in CD307bhi, CD258hiCD72hi, and CD21loPD-1hi B cell subpopulations. Our study, to our knowledge, provides new insight into biology of GZMB+ B cells and an efficient method to expand GZMB+ B cells for future cell therapy applications.

Clinical and immunological follow-up of very long-term kidney transplant recipients treated with calcineurin inhibitors indicates dual phenotypes.

Operationally tolerant kidney transplant recipients harbor an immunological signature, associated with low rejection risk, and focused on B lymphocytes. Here, we investigated whether patients with long-term transplantation and still on immunosuppressive therapy would present such a signature of low immunological rejection risk, compared to more recently transplanted patients. Of 114 kidney transplant recipients enrolled, 38 with more than 25 years of graft survival and stable graft function under calcineurin inhibitors, were matched with two different groups of transplanted patients (10-15 and 5-7 years after transplantation). Three phenotypes associated with low immunological rejection risk (Tfh, B and regulatory T cells), initially found in operationally tolerant kidney transplant recipients, and the composite score of tolerance (combination of six transcriptomic markers, age at transplantation and age at sampling) were analyzed. We found that very long-term patients were characterized by a significantly lower percentage of total B cells, a significantly higher proportion of CD24HiCD38Lo memory B cells, significantly fewer CD24LoCD38Lo naive B cells, and a significantly lower proportion of PD1HiCCR7Lo Tfh lymphocytes than more recently transplanted patients. This phenotype is associated with a positive composite score of tolerance in patients transplanted for more than 25 years. Thus, our study suggests a dual phenotype in very long-term kidney transplanted patients with an immunological profile associated with low rejection risk.

Transcriptional meta-analysis of regulatory B cells.

Regulatory B cells (Bregs) have the ability to regulate inflammation in various pathological situations, making them key players in immune regulation. Several mechanisms have been described and we recently identified a GZMB expressing Breg population in kidney transplanted patients who tolerate a kidney graft. To further investigate their biology and mechanisms, we conducted a transcriptomic analysis by RNAseq of these cells and we performed the first weighted meta-analysis of publicly available transcriptomic data from published Breg studies both in humans and mice. We identified two distinct and unique transcriptional signatures of 126 and 93 genes, respectively, associated with these Bregs. While we highlighted genes coding for proteins with potent involvement in regulatory functions, proliferation, and coding for transcription factors, the comparison between humans and mice did not allow identifying a common pattern. Thus, our results suggest distinct species-restricted Breg transcriptional signatures in humans and mice.

Unique and specific Proteobacteria diversity in urinary microbiota of tolerant kidney transplanted recipients.

Host-microbiota interactions can modulate the immune system both at local and systemic levels, with potential consequences for organ transplantation outcomes. In this study, we hypothesized that differences in the urinary microbiome following kidney transplantation would be associated with posttransplantation status: stable, minimally immunosuppressed, or tolerant. One hundred thirteen urine samples from stable (n = 51), minimally immunosuppressed (n = 19), and spontaneously tolerant (n = 16) patients, paired with age-matched controls (n = 27) were profiled and compared to each other at a taxonomic level with special interest in the immunosuppressive regimen. All comparisons and correlations were adjusted on sex and time posttransplantation. Our results highlighted a unique and specific urinary microbiota associated with spontaneous tolerance characterized by a high diversity and a clear Proteobacteria profile. Finally, we report that this profile is (1) impacted by gender, (2) inversely correlated with immunosuppressive drugs (calcineurin inhibitors and mammalian target of rapamycin inhibitors), and (3) stable in time.

Increased degradation of ATP is driven by memory regulatory T cells in kidney transplantation tolerance.

Regulatory T cells were recently proposed as the central actor in operational tolerance after renal transplantation. Tolerant patients harbor increased FoxP3hi memory Treg frequency and increased demethylation in the Foxp3 Treg-specific demethylated region when compared to stable kidney recipients and exhibit greater memory Treg suppressive capacities and higher expression of the ectonucleotidase CD39. However, in this particular and unique situation the mechanisms of action of Tregs were not identified. Thus, we analyzed the ability of memory Tregs to degrade extracellular ATP in tolerant patients, healthy volunteers, and patients with stable graft function under immunosuppression and determined the role of immunosuppressive drugs on this process. The conserved proportion of memory Tregs leads to the establishment of a pro-tolerogenic balance in operationally tolerant patients. Memory Tregs in tolerant patients display normal capacity to degrade extracellular ATP/ADP. In contrast, memory Tregs from patients with stable graft function do not have this ability. Finally, in vitro, immunosuppressive drugs may favor the lower proportion of memory Tregs in stable patients, but they have no effect on CD39-dependent ATP degradation and do not explain memory Treg lack of extracellular ATP/ADP degradation ability. Thus, intrinsic active regulatory mechanisms may act long after immunosuppressive drug arrest in operationally tolerant patients and may contribute to kidney allograft tolerance via the maintenance of CD39 Treg function.

Biomarkers and possible mechanisms of operational tolerance in kidney transplant patients.

A small number of patients do not reject their graft after weaning from immunosuppressive treatment. Here, we analyze the studies carried out to try to understand the mechanisms involved in this operational transplant tolerance and evaluate the hypotheses proffered on these potential mechanisms.

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.

Regulatory B Cells with a Partial Defect in CD40 Signaling and Overexpressing Granzyme B Transfer Allograft Tolerance in Rodents.

Emerging knowledge regarding B cells in organ transplantation has demonstrated that these cells can no longer be taken as mere generators of deleterious Abs but can also act as beneficial players. We previously demonstrated in a rat model of cardiac allograft tolerance induced by short-term immunosuppression an accumulation in the blood of B cells overexpressing inhibitory molecules, a phenotype also observed in the blood of patients that spontaneously develop graft tolerance. In this study, we demonstrated the presence in the spleen of regulatory B cells enriched in the CD24(int)CD38(+)CD27(+)IgD(-)IgM(+/low) subpopulation, which are able to transfer donor-specific tolerance via IL-10 and TGF-ß1-dependent mechanisms and to suppress in vitro TNF-α secretion. Following anti-CD40 stimulation, IgD(-)IgM(+/low) B cells were blocked in their plasma cell differentiation pathway, maintained high expression of the inhibitory molecules CD23 and Bank1, and upregulated Granzyme B and Irf4, two molecules described as highly expressed by regulatory B cells. Interestingly, these B cells recognized specifically a dominant donor Ag, suggesting restricted specificity that could lead to a particular B cell response. Regulatory B cells were not required for induction of tolerance and appeared following Foxp3(+)CD4(+)CD25(+) regulatory T cells, suggesting cooperation with regulatory T cells for their expansion. Nevertheless, following transfer to new recipients, these B cells migrated to the allograft, kept their regulatory profile, and promoted local accumulation of Foxp3(+)CD4(+)CD25(+) regulatory T cells. Mechanisms of regulatory B cells and their cell therapy potential are important to decipher in experimental models to pave the way for future developments in the clinic.

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.

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.

A common gene signature across multiple studies relate biomarkers and functional regulation in tolerance to renal allograft.

Patients tolerant to a kidney graft display a specific blood cell transcriptional pattern but results from five different studies were inconsistent, raising the question of relevance for future clinical application. To resolve this, we sought to identify a common gene signature, specific functional and cellular components, and discriminating biomarkers for tolerance following kidney transplantation. A meta-analysis of studies identified a robust gene signature involving proliferation of B and CD4 T cells, and inhibition of CD14 monocyte related functions among 96 tolerant samples. This signature was further supported through a cross-validation approach, yielding 92.5% accuracy independent of the study of origin. Experimental validation, performed on new tolerant samples and using a selection of the top-20 biomarkers, returned 91.7% of good classification. Beyond the confirmation of B-cell involvement, our data also indicated participation of other cell subsets in tolerance. Thus, the use of the top 20 biomarkers, mostly centered on B cells, may provide a common and standardized tool towards personalized medicine for the monitoring of tolerant or low-risk patients among kidney allotransplant recipients. These data point to a global preservation of genes favoring the maintenance of a homeostatic and 'healthy' environment in tolerant patients and may contribute to a better understanding of tolerance maintenance mechanisms.

Identification of tribbles-1 as a novel binding partner of Foxp3 in regulatory T cells.

In a previous study, we identified TRIB1, a serine-threonine kinase-like molecule, as a biomarker of chronic antibody-mediated rejection of human kidneys when measured in peripheral blood mononuclear cells. Here, we focused our analysis on a specific subset of peripheral blood mononuclear cells that play a dominant role in regulating immune responses in health and disease, so-called CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). We isolated both human and murine Treg and non-Treg counterparts and analyzed TRIB1 and Foxp3 mRNA expression by quantitative PCR on the freshly isolated cells or following 24 h of activation. Physical interaction between the human TRIB1 and Foxp3 proteins was analyzed in live cell lines by protein complementation assay using both flow cytometry and microscopy and confirmed in primary freshly isolated human CD4(+)CD25(hi)CD127(-) Tregs by co-immunoprecipitation. Both TRIB1 and Foxp3 were expressed at significantly higher levels in Tregs than in their CD4(+)CD25(-) counterparts (p < 0.001). Moreover, TRIB1 and Foxp3 mRNA levels correlated tightly in Tregs (Spearman r = 1.0; p < 0.001, n = 7), but not in CD4(+)CD25(-) T cells. The protein complementation assay revealed a direct physical interaction between TRIB1 and Foxp3 in live cells. This interaction was impaired upon deletion of the TRIB1 N-terminal but not the C-terminal domain, suggesting an interaction in the nucleus. This direct interaction within the nucleus was confirmed in primary human Tregs by co-immunoprecipitation. These data show a direct relationship between TRIB1 and Foxp3 in terms of their expression and physical interaction and highlight Tribbles-1 as a novel binding partner of Foxp3 in Tregs.

Unaltered regulatory B-cell frequency and function in patients with multiple sclerosis.

Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) typically characterized by the recruitment of T cells into the CNS. However, certain subsets of B cells have been shown to negatively regulate autoimmune diseases and some data support a prominent role for B cells in MS physiopathology. For B cells in MS patients we analyzed subset frequency, cytokine secretion ability and suppressive properties. No differences in the frequencies of the B-cell subsets or in their ability to secrete cytokines were observed between MS and healthy volunteers (HV). Prestimulated B cells from MS patients also inhibited CD4(+)CD25(-) T cell proliferation with a similar efficiency as B cells from HV. Altogether, our data show that, in our MS patient cohort, regulatory B cells have conserved frequency and function.

Structural insights into the catalytic mechanism of granzyme B upon substrate and inhibitor binding.

Human granzyme B (hGzmB), which is present in various immune cells, has attracted much attention due to its role in various pathophysiological conditions. The hGzmB activity is triggered at a catalytic triad (His59, Asp103, Ser198), cleaving its specific substrates. To date, the drug design strategy against hGzmB mainly targets the catalytic triad, which causes the non-specificity problem of inhibitors due to the highly conserved active site in serine proteases. In the present work, microsecond classical molecular dynamics simulations are devoted to exploring the structural dynamics of the hGzmB catalytic cycle in the presence of Ac-IEPD-AMC, a known substrate (active hGzmB), and Ac-IEPD-CHO, a known inhibitor (inactive hGzmB). By comparing active and inactive forms of hGzmB in the six different stages of the hGzmB catalytic cycle, we revealed, for the very first time, an additional network of interactions involving Arg216, a residue located outside the conventional binding site. Upon activation, the His59∙∙∙Asp103 hydrogen bond is broken due to the formation of the Asp103∙∙∙Arg216 salt bridge, expanding the active site to facilitate the substrate-binding. On the contrary, the binding of inhibitor Ac-IEPD-CHO to hGzmB prevents the Arg216-mediated interactions within the catalytic triad, thus preventing hGzmB activity. In silico Arg216Ala mutation confirms the role of Arg216 in enzyme activity, as the substrate Ac-IEPD-AMC failed to bind to the mutated hGzmB. Importantly, as Arg216 is not conserved amongst the various granzymes, the current findings can be a major step to guide the design of hGzmB specific therapeutics.

Urinary metabolomic profiling from spontaneous tolerant kidney transplanted recipients shows enrichment in tryptophan-derived metabolites.

BACKGROUND: Operational tolerance is the holy grail in solid organ transplantation. Previous reports showed that the urinary compartment of operationally tolerant recipients harbor a specific and unique profile. We hypothesized that spontaneous tolerant kidney transplanted recipients (KTR) would have a specific urinary metabolomic profile associated to operational tolerance. METHODS: We performed metabolomic profiling on urine samples from healthy volunteers, stable KTR under standard and minimal immunosuppression and spontaneous tolerant KTR using liquid chromatography in tandem with mass spectrometry. Supervised and unsupervised multivariate computational analyses were used to highlight urinary metabolomic profile and metabolite identification thanks to workflow4metabolomic platform. FINDINGS: The urinary metabolome was composed of approximately 2700 metabolites. Raw unsupervised clustering allowed us to separate healthy volunteers and tolerant KTR from others. We confirmed by two methods a specific urinary metabolomic signature in tolerant KTR mainly driven by kynurenic acid independent of immunosuppressive drugs, serum creatinine and gender. INTERPRETATION: Kynurenic acid and tryptamine enrichment allowed the identification of putative pathways and metabolites associated with operational tolerance like IDO, GRP35 and AhR and indole alkaloids. FUNDING: This study was supported by the ANR, IRSRPL and CHU de Nantes.

New Method for the Expansion of Highly Purified Human Regulatory Granzyme B-Expressing B Cells.

Granzyme B (GZMB)-expressing B cells inhibit CD4+ T-lymphocyte proliferation in a contact- and GZMB-dependent manner, through degradation of TCR zeta or induction of T-cell apoptosis. This regulatory B-cell population is present in human healthy individuals and represents about 1% of circulating B cells. Their small proportion requires the development of expansion methods to enable their study and envision clinical applications. We describe here how to expand GZMB-expressing B cells to obtain more than 90% of highly purified GZMB+ B cells, and the protocol of B/T cells coculture for the evaluation of the suppressive function of the GZMB+ B-cell population.

Connection of BANK1, Tolerance, Regulatory B cells, and Apoptosis: Perspectives of a Reductionist Investigation.

BANK1 transcript is upregulated in whole blood after kidney transplantation in tolerant patients. In comparison to patients with rejection, tolerant patients display higher level of regulatory B cells (Bregs) expressing granzyme B (GZMB+) that have the capability to prevent effector T cells proliferation. However, BANK1 was found to be decreased in these GZMB+ Bregs. In this article, we investigated seven different transcriptomic studies and mined the literature in order to make link between BANK1, tolerance and Bregs. As for GZMB+ Bregs, we found that BANK1 was decreased in other subtypes of Bregs, including IL10+ and CD24hiCD38hi transitional regulatory B cells, along with BANK1 was down-regulated in activated/differentiated B cells, as in CD40-activated B cells, in leukemia and plasma cells. Following a reductionist approach, biological concepts were extracted from BANK1 literature and allowed us to infer association between BANK1 and immune signaling pathways, as STAT1, FcγRIIB, TNFAIP3, TRAF6, and TLR7. Based on B cell signaling literature and expression data, we proposed a role of BANK1 in B cells of tolerant patients that involved BCR, IP3R, and PLCG2, and a link with the apoptosis pathways. We confronted these data with our experiments on apoptosis in total B cells and Bregs, and this suggests different involvement for BANK1 in these two cells. Finally, we put in perspective our own data with other published data to hypothesize two different roles for BANK1 in B cells and in Bregs.

Prebiotic Supplementation During Pregnancy Modifies the Gut Microbiota and Increases Metabolites in Amniotic Fluid, Driving a Tolerogenic Environment In Utero.

The gut microbiota is influenced by environmental factors such as food. Maternal diet during pregnancy modifies the gut microbiota composition and function, leading to the production of specific compounds that are transferred to the fetus and enhance the ontogeny and maturation of the immune system. Prebiotics are fermented by gut bacteria, leading to the release of short-chain fatty acids that can specifically interact with the immune system, inducing a switch toward tolerogenic populations and therefore conferring health benefits. In this study, pregnant BALB/cJRj mice were fed either a control diet or a diet enriched in prebiotics (Galacto-oligosaccharides/Inulin). We hypothesized that galacto-oligosaccharides/inulin supplementation during gestation could modify the maternal microbiota, favoring healthy immune imprinting in the fetus. Galacto-oligosaccharides/inulin supplementation during gestation increases the abundance of Bacteroidetes and decreases that of Firmicutes in the gut microbiota, leading to increased production of fecal acetate, which was found for the first time in amniotic fluid. Prebiotic supplementation increased the abundance of regulatory B and T cells in gestational tissues and in the fetus. Interestingly, these regulatory cells remained later in life. In conclusion, prebiotic supplementation during pregnancy leads to the transmission of specific microbial and immune factors from mother to child, allowing the establishment of tolerogenic immune imprinting in the fetus that may be beneficial for infant health outcomes.

Toward a better definition of hematopoietic progenitors suitable for B cell differentiation.

The success of inducing human pluripotent stem cells (hIPSC) offers new opportunities for cell-based therapy. Since B cells exert roles as effector and as regulator of immune responses in different clinical settings, we were interested in generating B cells from hIPSC. We differentiated human embryonic stem cells (hESC) and hIPSC into B cells onto OP9 and MS-5 stromal cells successively. We overcame issues in generating CD34+CD43+ hematopoietic progenitors with appropriate cytokine conditions and emphasized the difficulties to generate proper hematopoietic progenitors. We highlight CD31intCD45int phenotype as a possible marker of hematopoietic progenitors suitable for B cell differentiation. Defining precisely proper lymphoid progenitors will improve the study of their lineage commitment and the signals needed during the in vitro process.

CXCR5+PD1+ICOS+ Circulating T Follicular Helpers Are Associated With de novo Donor-Specific Antibodies After Renal Transplantation.

Donor-specific anti-HLA antibodies (DSAs) are a major risk factor associated with renal allograft outcomes. As a trigger of B cell antibody production, T follicular helper cells (Tfhs) promote DSA appearance. Herein, we evaluated whether circulating Tfhs (cTfhs) are associated with the genesis of antibody-mediated rejection. We measured cTfh levels on the day of transplantation and 1 year after transplantation in blood from a prospective cohort of 237 renal transplantation patients without DSA during the first year post-transplantation. Total cTfhs were characterized as CD4+CD45RA-CXCR5+, and the three following subsets of activated cTfh were analyzed: CXCR5+PD1+, CXCR5+PD1+ICOS+, an CXCR5+PD1+CXCR3-. Immunizing events (previous blood transfusion and/or pregnancy) and the presence of class II anti-HLA antibodies were associated with increased frequencies of activated CXCR5+PD1+, CXCR5+PD1+ICOS+, and CXCR5+PD1+CXCR3- cTfh subsets. In addition, ATG-depleting induction and calcineurin inhibitor treatments were associated with a relative increase of activated cTfh subsets frequencies at 1 year post-transplantation. In multivariate survival analysis, we reported that a decrease in activated CXCR5+PD1+ICOS+ at 1 year after transplantation in the blood of DSA-free patients was significantly associated with the risk of developing de novo DSA after the first year (p = 0.018, HR = 0.39), independently of HLA mismatches (p = 0.003, HR = 3.79). These results highlight the importance of monitoring activated Tfhs in patients early after transplantation and show that current treatments cannot provide early, efficient prevention of Tfh activation and migration. These findings indicate the need to develop innovative treatments to specifically target Tfhs to prevent DSA appearance in renal transplantation.