Fabrice Porcheray

B-Cell Immunity in the Context of T-Cell Tolerance after Combined Kidney and Bone Marrow Transplantation in Humans

Five patients with end‐stage kidney disease received combined kidney and bone marrow transplants from HLA haploidentical donors following nonmyeloablative conditioning to induce renal allograft tolerance. Immunosuppressive therapy was successfully discontinued in four patients with subsequent follow‐up of 3 to more than 6 years. This allograft acceptance was accompanied by specific T‐cell unresponsiveness to donor antigens. However, two of these four patients showed evidence of de novo antibodies reactive to donor antigens between 1 and 2 years posttransplant. These humoral responses were characterized by the presence of donor HLA‐specific antibodies in the serum with or without the deposition of the complement molecule C4d in the graft. Immunofluorescence staining, ELISA assays and antibody profiling using protein microarrays demonstrated the co‐development of auto‐ and alloantibodies in these two patients. These responses were preceded by elevated serum BAFF levels and coincided with B‐cell reconstitution as revealed by a high frequency of transitional B cells in the periphery. To date, these B cell responses have not been associated with evidence of humoral rejection and their clinical significance is still unclear. Overall, our findings showed the development of B‐cell allo‐ and autoimmunity in patients with T‐cell tolerance to the donor graft.

Chronic humoral rejection of human kidney allografts associates with broad autoantibody responses.

Background. Chronic humoral rejection (CHR) is a major complication after kidney transplantation. The cause of CHR is currently unknown. Autoantibodies have often been reported in kidney transplant recipients alongside antidonor human leukocyte antigen antibodies. Yet, the lack of comprehensive studies has limited our understanding of this autoimmune component in the pathophysiology of CHR. Methods. By using a series of ELISA and immunocytochemistry assays, we assessed the development of autoantibodies in 25 kidney transplant recipients with CHR and 25 patients with stable graft function. We also compared the reactivity of five CHR and five non-CHR patient sera with 8027 recombinant human proteins using protein microarrays. Results. We observed that a majority of CHR patients, but not non-CHR control patients, had developed antibody responses to one or several autoantigens at the time of rejection. Protein microarray assays revealed a burst of autoimmunity at the time of CHR. Remarkably, microarray analysis showed minimal overlap between profiles, indicating that each CHR patient had developed autoantibodies to a unique set of antigenic targets. Conclusion. The breadth of autoantibody responses, together with the absence of consensual targets, suggests that these antibody responses result from systemic B-cell deregulation.

Combined CD4+ Donor Lymphocyte Infusion and Low-Dose Recombinant IL-2 Expand FOXP3+ Regulatory T Cells following Allogeneic Hematopoietic Stem Cell Transplantation

CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) successfully control graft-versus-host-disease (GVHD) in animal models. In humans, incomplete reconstitution of Treg after allogeneic hematopoietic stem cell transplantation (HSCT) has been associated with chronic GVHD (cGVHD). Recent studies have demonstrated that interleukin (IL)-2 infusions expand Treg in vivo. However, the effectiveness of this therapy depends on the number of cells capable of responding to IL-2. We examined the effect of low-dose IL-2 infusions on Treg populations after HSCT in patients who also received infusions of donor CD4(+) lymphocytes. Utilizing FOXP3 as a Treg marker, we found that patients who received CD4+DLI concomitantly with IL-2 had greater expansion of Treg compared to patients who received IL-2 (P = .03) or CD4(+)DLI alone (P = .001). FOXP3 expression correlated with absolute CD4(+)CD25(+) cell counts. Moreover, expanded CD4(+)CD25(+) T cells displayed normal suppressive function and treatment with CD4(+)DLI and IL-2 was not associated with GVHD. This study suggests that administration of low-dose IL-2 combined with adoptive CD4(+) cellular therapy may provide a mechanism to expand Treg in vivo.

Polyreactive Antibodies Developing Amidst Humoral Rejection of Human Kidney Grafts Bind Apoptotic Cells and Activate Complement

Antibody mediated rejection (AMR) is associated with a variety of graft‐reactive antibodies following kidney transplant. To characterize these antibodies, we immortalized 107 B cell clones from a patient with AMR. In a previous study, we showed that six clones were reacting to multiple self‐antigens as well as to HLA and MICA for two of them, thus displaying a pattern of polyreactivity. We show here that all six polyreactive clones also reacted to apoptotic but not viable cells. More generally we observed a nearly perfect overlap between polyreactivity and reactivity to apoptotic cells. Functionally, polyreactive antibodies can activate complement, resulting in the deposition of C3d and C4d at the surface of target cells. Testing the serum of 88 kidney transplant recipients revealed a significantly higher IgG reactivity to apoptotic cells in AMR patients than in patients with stable graft function. Moreover, total IgG purified from AMR patients had increased complement activating properties compared to IgG from non‐AMR patients. Overall, our studies show the development of polyreactive antibodies cross‐reactive to apoptotic cells during AMR. Further studies are now warranted to determine their contribution to the detection of C4d in graft biopsies as well as their role in the pathophysiology of AMR.

Pretransplant IgG Reactivity to Apoptotic Cells Correlates With Late kidney Allograft Loss

Preexisting serum antibodies have long been associated with graft loss in transplant recipients. While most studies have focused on HLA‐specific antibodies, the contribution of non‐HLA‐reactive antibodies has been largely overlooked. We have recently characterized mAbs secreted by B cell clones derived from kidney allograft recipients with rejection that bind to apoptotic cells. Here, we assessed the presence of such antibodies in pretransplant serum from 300 kidney transplant recipients and examined their contribution to the graft outcomes. Kaplan–Meier survival analysis revealed that patients with high pretransplant IgG reactivity to apoptotic cells had a significantly increased rate of late graft loss. The effect was only apparent after approximately 1 year posttransplant. Moreover, the association between pretransplant IgG reactivity to apoptotic cells and graft loss was still significant after excluding patients with high reactivity to HLA. This reactivity was almost exclusively mediated by IgG1 and IgG3 with complement fixing and activating properties. Overall, our findings support the view that IgG reactive to apoptotic cells contribute to presensitization. Taking these antibodies into consideration alongside anti‐HLA antibodies during candidate evaluation would likely improve the transplant risk assessment.

Expansion of Polyreactive B Cells Cross-Reactive to HLA and Self in the Blood of a Patient With Kidney Graft Rejection

Antibody rejection is often accompanied by nondonor HLA specific antibodies (NDSA) and self‐reactive antibodies that develop alongside donor‐specific antibodies (DSA). To determine the source of these antibodies, we immortalized 107 B‐cell clones from a kidney transplant recipient with humoral rejection. Two of these clones reacted to HLA class I or MICA. Both clones were also reactive to self‐antigens and a lysate of a kidney cell line, hence revealing a pattern of polyreactivity. Monoclonality was verified by the identification of a single rearranged immunoglobulin heavy chain variable region (VH) sequence for each clone. By tracking their unique CDR3 sequence, we found that one such polyreactive clone was highly expanded in the patient blood, representing ∼0.2% of circulating B cells. The VH sequence of this clone showed evidence of somatic mutations that were consistent with its memory phenotype and its expansion. Lastly, the reactivity of the expanded polyreactive B‐cell clone was found in the patient serum at time of rejection. In conclusion, we provide here proof of principle at the clonal level that human antibodies can cross‐react to HLA and self. Our findings strongly suggest that polyreactive antibodies contribute to DSA, NDSA as well as autoantibodies, in transplant recipients.

Combined CD4 T-cell and antibody response to human minor histocompatibility antigen DBY after allogeneic stem-cell transplantation.

Background. Antibody responses to HY antigens in male recipients are frequent after transplantation of stem cells from female donors (Miklos et al., Blood 2005; 105: 2973; Miklos et al., Blood 2004; 103: 353). However, evidence that this B-cell immunity is accompanied by T-cell responses to the cognate antigens is scarce. Here, we examined T- and B-cell responses to DBY antigen in a male patient who received hematopoietic stem cells from a human leukocyte antigen-identical female sibling. Materials and Methods. We used 93 overlapping peptides representing the entire DBY protein to detect and characterize T-cell and antibody responses to DBY by enzyme-linked immunosorbent spot (ELISPOT) and enzyme-linked immunosorbent assay. Results. High frequency CD4+ T cells specific for a unique DBY peptide were detected in the patient blood. We isolated the corresponding T-cell clone and characterized the recognized epitope as an 18-mer peptide restricted by human leukocyte antigen-DRB1*0101. Upon stimulation, this clone produced cytokines with no evidence of Th1 or Th2 polarization. Remarkably, this clone also recognized the DBX homologue peptide and responded to female donor dendritic cells stimulated with poly I/C or lipopolysaccharide, indicating that the peptide was endogenously processed in these cells. High titer DBY-specific antibodies were also found in the patient serum which, in contrast to the T-cell response, did not cross-react with DBX. Conclusion. We show here the development of a coordinated B and T-cell response to DBY in a recipient of sex mismatched allogeneic hematopoietic stem-cell transplantation. Our findings support a role for CD4+ T cells in the development of humoral immunity to minor histocompatibility antigens.

Chronic Humoral Rejection of Human Kidney Allografts Is Associated with MMP-2 Accumulation in Podocytes and its Release in the Urine

Chronic humoral rejection (CHR) is an important cause of late graft failures following kidney transplantation. Overall, the pathophysiology of CHR is poorly understood. Matrix metalloproteinase‐2 (MMP‐2), a type IV collagenase, has been implicated in chronic kidney disease and allograft rejection in previous studies. We examined the presence of MMP‐2 in allograft biopsies and in the urine of kidney transplant recipients with CHR. MMP‐2 staining was detected by immunohistochemistry in podocytes for all CHR patients but less frequently in patients with other renal complications. Urinary MMP‐2 levels were also significantly higher in CHR patients (median 4942 pg/mL, N = 27) compared to non‐CHR patients (median 598 pg/mL, N = 65; p < 0.001). Elevated urinary MMP‐2 correlated with higher levels of proteinuria in both CHR and non‐CHR patients. Longitudinal analysis indicated that increase in urine MMP‐2 coincided with initial diagnosis of CHR as documented by the biopsies. Using an enzymatic assay, we demonstrated that MMP‐2 was present in its active form in the urine of patients with CHR. Overall, our findings associate MMP‐2 with glomerular injury as well as interstitial fibrosis and tubular atrophy observed in patients with CHR.

Evidence to Support a Contribution of Polyreactive Antibodies to HLA Serum Reactivity.

Background Assessing the serum reactivity to HLA is essential for the evaluation of transplant candidates and the follow-up of allograft recipients. In this study, we look for evidence at the clonal level that polyreactive antibodies cross-reactive to apoptotic cells and multiple autoantigens can also react to HLA and contribute to the overall serum reactivity. Methods We immortalized B cell clones from the blood of 2 kidney transplant recipients and characterized their reactivity to self-antigens, apoptotic cells as well as native, denatured, and cryptic HLA determinants using enzyme-linked immunosorbent assay (ELISA), immunofluorescence, flow cytometry and Luminex assays. We also assessed the reactivity of 300 pretransplant serum specimens to HLA and apoptotic cells. Results We report here 4 distinct B cell clones cross-reactive to self and HLA class I. All 4 clones reacted to numerous HLA class I alleles but did not appear to target canonical “shared” epitopes. In parallel experiments, we observed a strong correlation between IgG reactivity to HLA and apoptotic cells in pretransplant serum samples collected from 300 kidney transplant recipients. Further analysis revealed that samples with higher reactivity to apoptotic cells displayed significantly higher class I percent panel-reactive antibodies compared to samples with low reactivity to apoptotic cells. Conclusions We provide here (1) proof of principle at the clonal level that human polyreactive antibodies can cross-react to HLA, multiple self-antigens and apoptotic cells and (2) supportive evidence that polyreactive antibodies contribute to overall HLA reactivity in the serum of patients awaiting kidney transplant.

Expansion and somatic hypermutation of B-cell clones in rejected human kidney grafts.

Background B-cell infiltrates are common in rejected kidney allografts, yet their composition is still unclear. The aim of our study was to characterize the clonal composition of B-cell infiltrates of rejected human kidney grafts. Methods We used a molecular approach to characterize the partial B-cell repertoires of 5 failed human kidney grafts with detectable B-cell infiltrates. A comparison between the intragraft and blood repertoire was also conducted for 1 case. Results Redundant sequences were observed in both blood and graft, although the level of clonal amplification was significantly higher for the graft. Somatic hypermutations (SHMs) were also more frequent in sequences found in the graft compared to the blood. The rate of nonsilent mutations was significantly higher in complementarity determining regions (CDRs) compared to framework regions in blood sequences as well as in graft sequences found at low frequency. In contrast, this preferential distribution was lost in sequences found at high frequency in the graft, suggesting a lack of affinity maturation in situ. Lastly, follicular dendritic cells were undetectable in CD20+ infiltrates in all samples examined. Conclusions We provide here evidence that B-cell clones expand and undergo SHMs in situ. However, the even distribution of nonsilent SHM in high-frequency graft sequences together with the absence of follicular dendritic cells do not support the view that infiltrating B cells are part of functional germinal centers.