Search

Memory T cells (T mems) represent a major barrier to allograft tolerance in clinical transplantation. Alloreactive T mems form in response to MHC alloantigen exposure from prior transplant, blood transfusion or pregnancy. Here we investigated the ability to achieve cardiac allograft tolerance in multiparous female non-human primates (NHPs) using IL-6 blockade and mixed chimerism. Eight NHPs (Group A (n=6): male recipients; Group B (n=2): multiparous female recipients) underwent heart and donor bone marrow transplantation with non-myeloablative conditioning including total body and thymic irradiation, ATGAM, aCD154 mAb, cyclosporine, and aIL-6R mAb (tocilizumab, 10 mg/kg IV x7) through day 84 post-transplant (pTx). NHPs were then monitored off immunosuppression. In Group A, 4/6 recipients had >400 days allograft survival pTx; 2 failed from rejection (169, 383 days pTx) [Table]. In Group B, 2/2 failed from rejection by 200 days pTx. Group A had substantially lower CD8+ pre-Tx circulating T mems than Group B [Figure], including the 2 Group A rejectors. Group A had similar magnitude but longer duration of lymphocyte chimerism (median 4.9%, 180 days pTx) than Group B (median 5.3%, 90 days pTx). Mixed chimerism failed to achieve cardiac allograft tolerance in multiparous female NHPs using a protocol successful in males, presumably due to higher pre-transplant host T mem burden. This suggests the need for additional therapy for successful clinical application of this tolerance protocol in specific patient populations. [ABSTRACT FROM AUTHOR]

Mixed hematopoietic chimerism can achieve tolerance of kidney but not heart allografts in non-human primates (NHPs). Inhibiting IL-6 signaling has been shown to dampen inflammation and promote regulatory T cells (T regs) and can be achieved by blocking the cytokine (aIL-6 mAb) or its receptor (aIL-6R mAb). We investigated whether adding aIL-6 or aIL-6R mAb therapy to a mixed chimerism protocol could achieve heart allograft tolerance in NHPs. Twelve MHC-mismatched NHPs underwent simultaneous heart and donor bone marrow transplantation with non-myeloablative conditioning including total body and thymic irradiation, ATGAM, aCD154 mAb, and cyclosporine until day 28 post-transplant (pTx). Group A (n=2) received no aIL-6R/ aIL-6 mAb therapy, Group B (n=6) received aIL-6R mAb (tocilizumab, 10 mg/kg IV x7) through day 84 pTx and Group C (n=4) received aIL-6 mAb (clazakizumab, 50 mg/kg IV x1 then 10 mg/kg SC x6) through day 84 pTx. NHPs were then monitored off immunosuppression for graft function and histologic evidence of rejection. Group A recipients (2/2) had allograft failure due to rejection within 130 days pTx [Table]. In Group B, 4/6 recipients experienced allograft survival >400 days pTx; two failed due to rejection (days 169, 383 pTx). Among Group C recipients, 3/4 are ongoing with functioning heart allografts (days 280, 203, and 168 pTx); one was euthanized for CMV infection. Recipients in Group B had greater magnitude and duration of lymphocyte chimerism (median 4.9%, 180 days pTx) than those in Groups A (median 2.5%, 32 days pTx) or C (median 2.6%, 51 days pTx). There was no difference in peripheral T reg expansion among groups (p>0.05). Blocking IL-6 signaling in a mixed chimerism NHP model induced long-term immunosuppression-free cardiac allograft survival. Further investigation will determine the potential benefit of aIL-6 therapy in avoiding rebound alloimmune responses from accumulation of serum IL-6 following cessation of aIL-6R therapy. [ABSTRACT FROM AUTHOR]

In this study, we established a novel isotope-free approach for the detection of cell-mediated lympholysis (CML) in MHC defined peripheral blood mononuclear cells (PBMCs) using multiparameter flow and imaging cytometry. CML is an established in vitro assay to detect the presence of cytotoxic effector T-lymphocytes precursors (CTLp). Current methods employed in the identification of CTLp in the context of transplantation are based upon the quantification of chromium ( 51 Cr) released from target cells. In order to adapt the assay to flow cytometry, primary porcine PBMC targets were labeled with eFluor670 and incubated with major histocompatibility complex (MHC) mismatched effector cytotoxic lymphocytes (CTLs). With this method, we were able to detect target-specific lysis that was comparable to that observed with the 51 Cr-based assay. In addition, the use of quantitative cell imaging demonstrates the presence of accessory cells involved in the cytotoxic pathway. This innovative technique improves upon the standard 51 Cr release assay by eliminating the need for radioisotopes and provides enhanced characterization of the interactions between effector and target cells. This technique has wide applicability to numerous experimental and clinical models involved with effector–cell interactions. [ABSTRACT FROM AUTHOR]