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.

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.

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.

Neurotrophins modulate monocyte chemotaxis without affecting macrophage function.

Neurotrophins nerve growth factor (NGF), brain-derived growth factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) and their high-affinity tyrosine protein kinase receptor (Trk) family, TrkA, TrkB, TrkC, and low-affinity p75(NTR) receptor, are key molecules implicated in the development of the central nervous system. Increasing evidence suggests that they also have physiological and pathological roles outside the nervous system. In this study we examined the expression of neurotrophins and their receptors in human activated macrophages and to what extent neurotrophins themselves modulate macrophage activation, in a model of primary adult monocyte-derived macrophage. Our data indicate that macrophages express neurotrophin and neurotrophin receptor genes differentially, and respond to cell stimulation by specific inductions. Neurotrophins did not modify the antigen-presenting capacities of macrophages or their production of proinflammatory cytokines, but somehow skewed their activation phenotype. In contrast, NGF clearly increased CXCR-4 expression in macrophage and their chemotactic response to low CXCL-12 concentration. The differential effect of specific macrophage stimuli on neurotrophin expression, in particular NGF and NT-3, and the specific enhancement of CXCR-4 expression suggest that neurotrophins might participate in tissue-healing mechanisms that should be investigated further in vivo.

Macrophage activation switching: an asset for the resolution of inflammation.

Macrophages play a central role in inflammation and host defence against microorganisms, but they also participate actively in the resolution of inflammation after alternative activation. However, it is not known whether the resolution of inflammation requires alternative activation of new resting monocytes/macrophages or if proinflammatory activated macrophages have the capacity to switch their activation towards anti-inflammation. In order to answer this question, we first characterized differential human macrophage activation phenotypes. We found that CD163 and CD206 exhibited mutually exclusive induction patterns after stimulation by a panel of anti-inflammatory molecules, whereas CCL18 showed a third, overlapping, pattern. Hence, alternative activation is not a single process, but provides a variety of different cell populations. The capacity of macrophages to switch from one activation state to another was then assessed by determining the reversibility of CD163 and CD206 expression and of CCL18 and CCL3 production. We found that every activation state was rapidly and fully reversible, suggesting that a given cell may participate sequentially in both the induction and the resolution of inflammation. These findings may provide new insight into the inflammatory process as well as new fields of investigation for immunotherapy in the fields of chronic inflammatory diseases and cancer.

GroEL (Hsp60) of Clostridium difficile is involved in cell adherence.

Previous results have demonstrated that adherence of Clostridium difficile to tissue culture cells is augmented by various stresses; this study focussed on whether the GroEL heat shock protein is implicated in this process. The 1940 bp groESL operon of C. difficile was isolated by PCR. The 1623 bp groEL gene is highly conserved between various C. difficile isolates as determined by RFLP-PCR and DNA sequencing, and the operon is present in one copy on the bacterial chromosome. The 58 kDa GroEL protein was expressed in Escherichia coli in fusion with glutathione S:-transferase and the fusion protein was purified from IPTG-induced bacterial lysates by affinity chromatography on glutathione-Sepharose. A polyclonal, monospecific antiserum was obtained for GroEL which established by immunoelectron microscopy, indirect immunofluorescence and immunoblot analysis that GroEL is released extracellularly after heat shock and can be surface associated. Cell fractionation experiments suggest that GroEL is predominantly cytoplasmic and membrane bound. GroEL-specific antibodies as well as the purified protein partially inhibited C. difficile cell attachment and expression of the protein was induced by cell contact, suggesting a role for GroEL in cell adherence.