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.

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.

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.