ECDI‐fixed donor splenocytes prolong skin allograft survival by promoting M2 macrophage polarization and inducing regulatory T cells

Abstract

Rejection is a common complication of allogeneic tissue transplantation. Fixation of splenocytes (SP) with 1‐ethyl‐3‐(3'‐dimethylaminopropyl)‐carbodiimide (ECDI) induces immune tolerance in recipients post‐transplantation; however, the mechanism underlying this effect remains unclear. Here, we determined the mechanisms of ECDI‐fixed donor SP (ECDI‐SP) in inducing tolerance in skin allograft transplantation. C57BL/6‐recipient mice that received Balb/c full‐thickness skin transplants with two infusions of donor‐derived ECDI‐SP, along with rapamycin showed superior skin allograft survival and lower inflammatory cell infiltration than mice that received rapamycin‐only treatment. In ECDI‐SP‐treated mice, the levels of anti‐inflammatory cytokines such as interleukin (IL)‐10 in sera were markedly increased, whereas the expression of inflammatory cytokines was significantly suppressed. Splenic macrophages were significantly polarized to the alternative activated macrophage (M2) phenotype, with expansion of CD4+Foxp3+ regulatory T cells (Tregs) in the spleen and draining lymph nodes. Allostimulatory activity of ECDI‐SP in vitro and donor‐specific ex vivo hyporesponsiveness were observed. C57BL/6 macrophages engulfed allogeneic Balb/c‐derived ECDI‐SP, polarized to the M2 phenotype, with pronounced cAMP response element‐binding (CREB) protein phosphorylation. By facilitating increased IL‐10 expression, ECDI‐SP induced M2 polarization and Treg production, inhibiting effector T‐cell proliferation. Thus, ECDI‐SP modulates macrophage M2 polarization by increasing CREB phosphorylation and promoting Treg production to suppress allogeneic skin graft rejection.

Negative Vaccination Strategies for Promotion of Transplant Tolerance

“…55 In mice, donor apoptotic cells localize to the recipient's splenic marginal zone within hours 56 and are phagocytosed by recipient DCs and macrophages Continued next page through host efferocytic receptor tyrosine kinase pathways. 57,58 Recipient DCs and macrophages that phagocytose apoptotic donor cells broadly cause an increase in anti-inflammatory cytokines, 56,59,60 a reduction in proinflammatory cytokines, 61 and subsequent induction of donor Ag-specific Foxp3 + Treg. 61,62 Reference Model Treatment Outcomes…”

“…Zheng et al 49 Murine models show marked increases in antiinflammatory cytokines such as IL-10, IL-13, and transforming growth factor-β after apoptotic donor leukocyte infusion. 59,60 The effect is rapid, with IL-10 production by splenic macrophages within 10 min of infusion rapidly altering the phenotype of F4/80 + macrophages to favor high PD-L1 expression. 56 In mouse heterotopic heart transplantation, expression of anti-inflammatory cytokines is also increased within allograft tissue.…”

“…61,62 Splenic macrophages are polarized toward the alternatively-activated M2 phenotype, leading to the expansion of Treg in the spleen and allograft-draining lymph nodes. 59 Direct in vitro culture of apoptotic allogeneic APCs with naive CD4 + and CD4 + CD25 – T cells induces a Treg phenotype with upregulation of Foxp3, high expression of CD62L and CD2, and low expression of costimulatory signaling molecules that do not stimulate proliferation or inflammatory cytokine secretion by cocultured CD4 + T cells. 66 Similarly, there appears to be an increase in circulating regulatory B-cell phenotypes, with a simultaneous reduction in allospecific effector B cells.…”

“…In murine skin allograft models, donor ECDI-splenocyte infusions and concurrent rapamycin administration significantly extended graft survival time for both full-thickness and vascularized grafts. 59,61 Long-term mouse cardiac allograft survival (>100 d) has also been achieved after donor ECDI-splenocyte infusion, including full MHC mismatch models and (together with OX40-OX40L blockade) in presensitized recipients. 53,63,68,78 Similarly, donor ECDI-splenocyte infusion alone can induce tolerance of islet allografts, 58,62,64 with coadministration of B-cell blockade using anti-CD20 inducing tolerance in a rat-to-mouse islet xenograft model.…”