Determination of the precursor frequency and the reaction intensity of xenoreactive human T lymphocytes

Department of Surgery, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
Xenotransplantation (Impact Factor: 2.84). 05/2010; 17(3):188-96. DOI: 10.1111/j.1399-3089.2010.00575.x
Source: PubMed


It is acknowledged that the response of human T cells to xenogeneic targets is more potent than that to allogeneic targets. However, it is not clear whether the more vigorous T cell response to xenoantigens than to alloantigens is attributable to a higher frequency or stronger reaction of xenoreactive T cells.
We determined the precursor frequencies (PFs) and stimulation indexes (SIs) of xenoreactive human T cells by performing a mixed lymphocyte reaction (MLR) assay using a carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling technique. Irradiated porcine or human peripheral blood mononuclear cells (PBMCs)used as stimulator cells--were cultured with CFSE-labeled human PBMCs--used as responder cells.
The SIs of the xenoreactive CD4(+) T cells were significantly higher than those of the alloreactive CD4(+) T cells, whereas the PFs of the alloreactive and xenoreactive CD4(+) T cell precursors were almost identical, suggesting a stronger reaction by a single xenoreactive CD4(+) T cell. In contrast, the SIs of the xenoreactive CD8(+) T cells did not differ from those of the alloreactive CD4(+) T cells, and the PFs of the allo- and xenoreactive CD8(+) T cell precursors were also identical. Addition of a soluble human CD47-Fc fusion protein in the porcine-to-human MLR assay caused a statistically significant reduction of the SIs of the xenoreactive CD4(+) T cells. Such an alteration was abrogated by further addition of blocking antibodies (Abs) against either human CD47 or signal regulatory protein-alpha in the porcine-to-human MLR assay. Addition of human CD47-Fc after the depletion of non-T cells from the population of human responder PBMCs in this MLR assay did not influence the SIs of the xenoreactive CD4(+) T cells.
The more vigorous T cell response to xenoantigens than to alloantigens is possibly attributable to a stronger reaction of xenoreactive T cells; the interspecies incompatibility of CD47 may contribute to such xenoreactive CD4(+) T cell responses via an indirect pathway.

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    • "not due to a higher frequency of human xenoreactive CD4 þ T cells than in the allogeneic counterpart , and there was no evidence of any differences in CD8 þ T-cell content between allo-and xeno-related direct reactions (Tahara et al. 2010). These studies also suggest that the CD4 þ helper function is needed for xenoreactive CD8 þ T cells to proliferate (Tahara et al. 2010). Indirect anti-pig T-cell responses appear to be mediated primarily by CD4 þ T cells (Dorling et al. 1996). "
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    • "These observations raise a question as to whether interspecies incompatibility of CD47 affects CD4+ T cell-mediated responses to xenoantigens positively or negatively. In our previous study, recombinant human CD47-Fc fusion protein (which contains the extracellular domain of human CD47 fused to the Fc portion of human immunoglobulin) significantly reduced the indirect response of human CD4+ T cells to porcine antigens, but did not affect the direct response of these cells in in vitro pig-to-human mixed lymphocyte reaction assays [27]. Inhibition of the phagocytic activity of human APCs toward porcine cells by interaction between the human CD47-Fc fusion protein and the corresponding ligand, probably SIRPα, might might attenuate subsequent CD4+ T cell immune responses against porcine antigens. "
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    ABSTRACT: We have previously proven that the interspecies incompatibility of CD47 is responsible for phagocytosis of xenogeneic cells by host macrophages. Utilizing an model in the present study, we investigated whether genetically engineered expression of mouse CD47 in rat insulinoma cells (INS-1E) could inhibit macrophage-mediated xenograft rejection. INS-1E cells transfected with the pRc/CMV-mouse CD47 vector (mCD47-INS-1E) induced SIRPα-tyrosine phosphorylation in mouse macrophages in vitro, whereas cells transfected with the control vector (cont-INS-1E) did not. When these cells were injected into the peritoneal cavity of streptozotocin-induced diabetic Rag2γ chain mice, which lack T, B, and NK cells, the expression of mouse CD47 on the INS-1E cells markedly reduced the susceptibility of these cells to phagocytosis by macrophages. Moreover, these mice became normoglycemic after receiving mCD47-INS-1E, whereas the mice that received cont-INS-1E failed to achieve normoglycemia. Furthermore, injection of an anti-mouse SIRPα blocking monoclonal antibody into the mouse recipients of mCD47-INS-1E cells prevented achievement of normoglycemia. These results demonstrate that interspecies incompatibility of CD47 significantly contributes to rejection of xenogeneic cells by macrophages. Thus, genetic induction of the expression of recipient CD47 on xenogeneic donor cells could provide inhibitory signals to recipient macrophages via SIPRα; this constitutes a novel approach for preventing macrophage-mediated xenograft rejection.
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