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.

Kidney allograft rejection is associated with an imbalance of B cells, regulatory T cells and differentiated CD28-CD8+ T cells: analysis of a cohort of 1095 graft biopsies.

Introduction: The human immune system contains cells with either effector/memory or regulatory functions. Besides the well-established CD4+CD25hiCD127lo regulatory T cells (Tregs), we and others have shown that B cells can also have regulatory functions since their frequency and number are increased in kidney graft tolerance and B cell depletion as induction therapy may lead to acute rejection. On the other hand, we have shown that CD28-CD8+ T cells represent a subpopulation with potent effector/memory functions. In the current study, we tested the hypothesis that kidney allograft rejection may be linked to an imbalance of effector/memory and regulatory immune cells. Methods: Based on a large cohort of more than 1000 kidney graft biopsies with concomitant peripheral blood lymphocyte phenotyping, we investigated the association between kidney graft rejection and the percentage and absolute number of circulating B cells, Tregs, as well as the ratio of B cells to CD28-CD8+ T cells and the ratio of CD28-CD8+ T cells to Tregs. Kidney graft biopsies were interpreted according to the Banff classification and divided into 5 biopsies groups: 1) normal/subnormal, 2) interstitial fibrosis and tubular atrophy grade 2/3 (IFTA), 3) antibody-mediated rejection (ABMR), 4) T cell mediated-rejection (TCMR), and 5) borderline rejection. We compared group 1 with the other groups as well as with a combined group 3, 4, and 5 (rejection of all types) using multivariable linear mixed models. Results and discussion: We found that compared to normal/subnormal biopsies, rejection of all types was marginally associated with a decrease in the percentage of circulating B cells (p=0.06) and significantly associated with an increase in the ratio of CD28-CD8+ T cells to Tregs (p=0.01). Moreover, ABMR, TCMR (p=0.007), and rejection of all types (p=0.0003) were significantly associated with a decrease in the ratio of B cells to CD28-CD8+ T cells compared to normal/subnormal biopsies. Taken together, our results show that kidney allograft rejection is associated with an imbalance between immune cells with effector/memory functions and those with regulatory properties.

Antibody-mediated allograft rejection is associated with an increase in peripheral differentiated CD28-CD8+ T cells - Analyses of a cohort of 1032 kidney transplant recipients.

BACKGROUND: CD28-CD8+ T cells represent a differentiated CD8+ T cell subset that is found to be increased in various conditions associated with chronic antigenic stimulation such as aging, chronic viral infections, autoimmune diseases, cancers, and allotransplantation. METHODS: Using multivariate models, we analyzed a large cohort of 1032 kidney transplant patients in whom 1495 kidney graft biopsies were performed concomitant with a peripheral blood leukocyte phenotyping by flow cytometry. We investigated the association between the level of CD28-CD8+ T cells in the blood and the diagnosis of graft rejection according to the recent Banff classification of renal allograft pathology. FINDINGS: We found that antibody-mediated rejection (ABMR) was associated with a significant increase in the percentage as well as the absolute number of CD28-CD8+ T cells in the peripheral blood of kidney transplant patients at the time of biopsy. The confounder-adjusted mean difference of log percentage and log absolute value between the ABMR group and the normal/subnormal histology group were 0.29 (p=0.0004) and 0.38 (p=0.0004), respectively. Moreover, we showed that CD28-CD8+ T cells from the patients diagnosed with ABMR responded more rigorously to TCR and FcγRIIIA (CD16) engagement compared to their CD28+ counterparts as evidenced by an increase in the expression of IFNγ, TNFα, and CD107a. INTERPRETATION: Collectively, our data suggest that differentiated CD28-CD8+ T cells, with increased frequency, number, and function, may participate in the pathobiology of ABMR. Further studies are warranted to clarify the immunological role of this T cell subset in kidney graft rejection. FUNDING: Agence nationale de la recherche (France).

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.