Donor CD56~+ Cells Accelerate Human-mouse Xenogeneic Graft-versus-host Disease

Graft-versus-host disease(GVHD) is a major cause of morbidity and mortality during allogeneic bone marrow transplantation(alloBMT). GVHD is mainly mediated by donor T cells expand upon encountering genetically disparate antigens. In concert with pro-inflammatory cytokines, donor alloreactive T cells play critical roles in initiating a dynamic course of tissue destruction. Recent studies showed that allogeneic CD56+cells exhibited protective effect during GVHD. CD56+cells are divided into two subsets according to their phenotype, CD3-CD56+NK cell and CD3+CD56+cells. CD3-CD56+NK cells are a subset of cytotoxic lymphocytes that contribute to approximately 5% to 10% of circulating lymphocytes in healthy subjects, whereas CD3+CD56+cells are predominantly comprised an innate-like NKT cell subsets which possesses NK-like cytolytic activity. Besides being killer cells, both subsets have been revealed to display strong regulatory function through secreting various cytokines. Because of the hardness to get enough grafts from human beings, xenogeneic transplantation has been considered as another way to overcome organ failure. In this study, in order to explore the CD56+cells-related mechanisms involved in xenogeneic GVHD, we intend to discover the function of CD56+cells in xenogeneic GVHD model mice and in vitro xenogeneic co-culture system.1. Depletion of CD56+cells alleviates human PBMC-induced xenogeneic GVHD.In order to explore the role of donor CD56+cells in xenogeneic GVHD, we established an NOD/SCID mice GVHD model by transplanted human PBMC(hPBMC). The average survival time of model mice was 14 days. Infusion of donor CD56+cells sorted from hPBMC did not cause lethal GVHD. However, the NOD/SCID mice transplanted with hPBMC depleted CD56+cells extended average survival time to 30 days. These results suggested that donor CD56+cells accelerated human-mouse xenogeneic GVHD.2. Co-culture with xenogeneic cells promotes the proliferation and activation of sorted CD56+cells.THP-1 cell is a human origin cell line, while J558 L cell is a murine origin cell line. CD56+cell enriched from hPBMC were co-cultured with THP-1 cells or J558 L cells, to set up an allogeneic or xenogeneic co-culture system in vitro, respectively. Flow cytometry was performed to detect the proliferation and activation of CD56+cells. As a result, the enriched CD56+cells co-cultured with xenogeneic cells showed a significant higher proliferation index and increased activation markers, when compared to allogeneic cells.3. Pro-inflammatory cytokines increase significantly after CD56+cells co-cultured with xenogeneic cells.To access the cytokine producing profiles of CD56+cells in xenogeneic stimulation system,supernatant from allogeneic or xenogeneic co-culture system were collected. The level of Th1/Th2/Th17 cytokines were further detected by CBA and/or sandwich ELISA. The results indicated that CD56+cells from xenogeneic group secreted higher level of pro-inflammatory cytokines(IFN-?, TNF, IL-1?) than that from allogeneic group.4. Cytotoxic activity of CD56+cells decreases after co-culture with xenogeneic cells.In order to exam the cytotoxicity of CD56+cells, CD56+cells were cultured with xenogeneic cells for 7 days. The cytotoxic activity of CD56+cells were detected by killing assay.Compared to unstimulated group, the CD56+cells in xenogeneic group showed a decreased cytotoxic activity against the cells derived from both mouse(J558L and Yac-1 cells) and human(T1, THP-1 and K562 cells). The decreased cytotoxic capability of CD56+cells suggested that other mechanisms but not cytotoxicity should be responsible for accelerated human-mouse xenogeneic GVHD in the presence of CD56+cells.5. Upon xenogeneic cell stimulation, CD56+cells facilitate the proliferation and activation of T cells.As T cells are well accepted to be the major mediator for GVHD, we further accessed the effect of CD56+cells on CD4+and CD8+T cells. allogeneic or xenogeneic co-culture system wasset up by co-culture of enriched T cells with THP-1 cells or J558 L cells, respectively. The proliferation index and the level of activation markers of enriched T cells were measured, in the presence or absence of CD56+cells. The results indicated that adding enriched CD56+cells promoted the proliferation of CD8+T cells and the activation of both CD4+and CD8+T cells.In summary, we found that donor CD56+cells did not induce severe xenogeneic GVHD directly, but the presence of CD56+cells accelerated xenogeneic GVHD. Upon xenogeneic stimulation in vitro, CD56+cells rapidly proliferated, activated, and secreted various pro-inflammatory cytokines. Furthermore, CD56+cells facilitated the proliferation of CD8+T cells and the activation of both CD4+and CD8+T cells, which may contribute to accelerate human-mouse xenograft GVHD.