MHC Tetramers Specifically Identify High- and Low-avidity Donor-specific B Cells in Transplantation Tolerance and Rejection.

BACKGROUND: Although donor-specific antibody pre- and posttransplantation is routinely assessed, accurate quantification of memory alloreactive B cells that mediate recall antibody response remains challenging. Major histocompatibility complex (MHC) tetramers have been used to identify alloreactive B cells in mice and humans, but the specificity of this approach has not been rigorously assessed. METHODS: B-cell receptors from MHC tetramer-binding single B cells were expressed as mouse recombinant immunoglobulin G1 (rIgG1) monoclonal antibodies, and the specificity was assessed with a multiplex bead assay. Relative binding avidity of rIgG1 was measured by modified dilution series technique and surface plasmon resonance. Additionally, immunoglobulin heavy chain variable regions of 50 individual B-cell receptors were sequenced to analyze the rate of somatic hypermutation. RESULTS: The multiplex bead assay confirmed that expressed rIgG1 monoclonal antibodies were preferentially bound to bait MHC class II I-E d over control I-A d and I-A b tetramers. Furthermore, the dissociation constant 50 binding avidities of the rIgG1 ranged from 10 mM to 7 nM. The majority of tetramer-binding B cells were low avidity, and ~12.8% to 15.2% from naive and tolerant mice and 30.9% from acute rejecting mice were higher avidity (dissociation constant 50 <1 mM). CONCLUSIONS: Collectively, these studies demonstrate that donor MHC tetramers, under stringent binding conditions with decoy self-MHC tetramers, can specifically identify a broad repertoire of donor-specific B cells under conditions of rejection and tolerance.

CSF-1 in Inflammatory and Arthritic Pain Development.

Pain is one of the most debilitating symptoms in many diseases for which there is inadequate management and understanding. CSF-1, also known as M-CSF, acts via its receptor (CSF-1R, c-Fms) to regulate the development of the monocyte/macrophage lineage and to act locally in tissues to control macrophage numbers and function. It has been implicated in the control of neuropathic pain via a central action on microglia. We report in this study that systemic administration of a neutralizing anti-CSF-1R or CSF-1 mAb inhibits the development of inflammatory pain induced by zymosan, GM-CSF, and TNF in mice. This approach also prevented but did not ameliorate the development of arthritic pain and optimal disease driven by the three stimuli in mice, suggesting that CSF-1 may only be relevant when the driving inflammatory insults in tissues are acute and/or periodic. Systemic CSF-1 administration rapidly induced pain and enhanced the arthritis in an inflamed mouse joint, albeit via a different pathway(s) from that used by systemic GM-CSF and TNF. It is concluded that CSF-1 can function peripherally during the generation of inflammatory pain and hence may be a target for such pain and associated disease, including when the clinically important cytokines, TNF and GM-CSF, are involved. Our findings have ramifications for the selection and design of anti-CSF-1R/CSF-1 trials.

Successful Treatment of T Cell-Mediated Acute Rejection with Delayed CTLA4-Ig in Mice.

Clinical observations that kidney transplant recipients receiving belatacept who experienced T cell-mediated acute rejection can be successfully treated and subsequently maintained on belatacept-based immunosuppression suggest that belatacept is able to control memory T cells. We recently reported that treatment with CTLA4-Ig from day 6 posttransplantation successfully rescues allografts from acute rejection in a BALB/c to C57BL/6 heart transplant model, in part, by abolishing B cell germinal centers and reducing alloantibody titers. Here, we show that CTLA4-Ig is additionally able to inhibit established T cell responses independently of B cells. CTLA4-Ig inhibited the in vivo cytolytic activity of donor-specific CD8+ T cells, and the production of IFNγ by graft-infiltrating T cells. Delayed CTLA4-Ig treatment did not reduce the numbers of graft-infiltrating T cells nor prevented the accumulation of antigen-experienced donor-specific memory T cells in the spleen. Nevertheless, delayed CTLA4-Ig treatment successfully maintained long-term graft acceptance in the majority of recipients that had experienced a rejection crisis, and enabled the acceptance of secondary BALB/c heart grafts transplanted 30 days after the first transplantation. In summary, we conclude that delayed CTLA4-Ig treatment is able to partially halt ongoing T cell-mediated acute rejection. These findings extend the functional efficacy of CTLA4-Ig therapy to effector T cells and provide an explanation for why CTLA4-Ig-based immunosuppression in the clinic successfully maintains long-term graft survival after T cell-mediated rejection.

CTLA4-Ig in combination with FTY720 promotes allograft survival in sensitized recipients.

Despite recent evidence of improved graft outcomes and safety, the high incidence of early acute cellular rejection with belatacept, a high-affinity CTLA4-Ig, has limited its use in clinical transplantation. Here we define how the incomplete control of endogenous donor-reactive memory T cells results in belatacept-resistant rejection in an experimental model of BALB/c.2W-OVA donor heart transplantation into C57BL/6 recipients presensitized to donor splenocytes. These sensitized mice harbored modestly elevated numbers of endogenous donor-specific memory T cells and alloantibodies compared with naive recipients. Continuous CTLA4-Ig treatment was unexpectedly efficacious at inhibiting endogenous graft-reactive T cell expansion but was unable to inhibit late CD4+ and CD8+ T cell infiltration into the allografts, and rejection was observed in 50% of recipients by day 35 after transplantation. When CTLA4-Ig was combined with the sphingosine 1-phosphate receptor-1 (S1PR1) functional antagonist FTY720, alloantibody production was inhibited and donor-specific IFN-γ-producing T cells were reduced to levels approaching nonsensitized tolerant recipients. Late T cell recruitment into the graft was also restrained, and graft survival improved with this combination therapy. These observations suggest that a rational strategy consisting of inhibiting memory T cell expansion and trafficking into the allograft with CTLA4-Ig and FTY720 can promote allograft survival in allosensitized recipients.