In conclusion, mouse cell-mediated reaction against xenogeneic adult pig islet cells mainly involves class II-restricted CD4+ T lymphocytes of Th1 and Th2 subtypes, with an indirect pathway for the recognition. Although of low intensity, this cell-mediated reaction constitutes an obstacle to pig islet engraftment in the mouse, although one not necessarily more insurmountable than alloreactivity. The peculiarity of NOD mouse splenocytes, in terms of proliferation against pig islets, suggests that the study of islet xenograft rejection should take the immunogenetic context of diabetes into account, in which case the use of non-diabetes-prone mice has its limitations.

Type I diabetes could be treatable by a graft of pig islets [1]. Rejection of this discordant xenograft remains, however, a major problem. Despite advances in the understanding of hyperacute rejection that have made short-term engraftment of porcine tissue a feasible objective, cell-mediated rejection can occur. This cellular rejection could be a serious problem in the case of islets, which are possibly less susceptible to hyperacute rejection [2,3].In vitro human cellular recognition in the discordant human-pig situation has been mainly investigated with stimulator pig lymphocytes or endothelial cells [4±6], i. e. cells which are not intended to be grafted in diabetes. In terms of the disease which islet grafts are intended to combat, it is thus important to characterise human anti-pig islet response since islets have particularities different to lymphocytes, e. g. they do not constitutively express class II molecules Diabetologia (1999) Summary The intensity and mechanisms of cell-mediated rejection of pig islet cells were studied in 49 Type I diabetic and 34 healthy subjects. Human peripheral mononuclear cells proliferated strongly in response to pig islet cells (p < 0.001), though with notable interindividual variations (stimulation index 2 to 215). The variance of stimulation index was higher in diabetic than healthy subjects (p < 0.0001). The response to islet cells was stronger (p < 0.01) than that to pig splenocytes. Proliferation in response to islet cells was strongly decreased (p < 0.01) when CD 4 + T cells were blocked with monoclonal antibodies, whereas the blocking of CD 8 + cells or NK cells gave less pronounced effects. The response to islet cells was decreased (p < 0.01), but not abolished, after antigen-presenting cells were removed. Purified CD 4 + cells alone did not proliferate in response to islet cells but recovered their proliferative ability when mixed with antigen-presenting cells, whereas CD 8 + cells alone proliferated in the presence of interleukin-2 in response to islet cells. Proliferation was blocked (p < 0.01) by anti-DR monoclonal antibodies. During proliferation in response to islet cells, interleukin-10 increased 43-fold (p < 0.01) but interferon-g increased only slightly. No statistical differences were detected between diabetic and control subjects with respect to lymphocyte subsets and the recognition mechanisms or to interferon-g / interleukin-10 production in response to islet cells. These results provide the first detailed information on human cell-mediated xenoreaction to pig islet cells. This situation involves a dominant CD 4 class II-restricted Th2 response, with an indirect recognition pathway, as well as a CD 8 T-cell response resulting from direct recognition. This strong reaction constitutes a serious obstacle which may vary in degree among subjects. [Diabetologia (1999) 42: 330±335]

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SUMMARYIn vitro studies were conducted in the non-obese diabetic (NOD) mouse, prone to Type 1 autoimmune diabetes, to investigate the mechanisms involved in cell-mediated rejection of pig islet xenografts. Our previous work concerning the mechanisms of proliferation of xenogeneic lymphocytes to pig islet cells (PIC) was not indicative of PIC impairment. Consequently, a test was developed based on perifusion analysis of the alteration of basal and stimulated insulin release from adult PIC incubated with mouse splenocytes or subsets. Compared with PIC incubation alone or with syngeneic pig splenocytes, coincubation with mouse whole spleen cells resulted in a decrease of basal and stimulated insulin release (P , 0´001). Two components of this alteration were detected separately: PIC impairment was decreased (P , 0´01) after removal of plastic-adherent cells from spleen cells, but maintained (P , 0´01) when plastic-adherent cells alone were co-incubated with PIC. The increase of murine interleukin-1b when mouse plastic-adherent spleen cells were cultured with PIC (P , 0´04) was indicative of macrophage activation. Soluble factors produced during co-incubation of mouse splenocytes or plastic-adherent cells with PIC were involved in the impairment process, since supernatant fluids collected during previous PIC±mouse cell co-incubations directly altered (P , 0´01) insulin release from PIC. Moreover, impairment of PIC by mouse spleen cells was abolished (P , 0´01) by gadolinium chloride (which inhibits macrophages), but not by cyclosporin A. Another mechanism was apparent, since co-incubation of PIC with purified mouse T cells or CD4 1 T cells, re-mixed with antigen-presenting cells, led to a decrease (P , 0´01) of insulin release. This model, based on the alteration of dynamic basal and stimulated insulin release, is indicative of in vitro cell-mediated alteration of PIC in the NOD mouse. The effect of whole spleen cells was rapid, and a crucial role was played by plastic-adherent cells. Two mechanisms were responsible for the behaviour of these cells: an early direct effect (at least in part via soluble products); and the indirect presentation of PIC xenoantigens (leading to impairment by CD4 1 T lymphocytes).

The BB rat develops a spontaneous type I diabetic syndrome with anti-islet autoimmunity. Sera from diabetic and nondiabetic BB rats (from diabetes-prone litters), nondiabetic BB rats (from low-risk lines), and nondiabetes-prone Sprague-Dawley rats were collected twice a week from age 40 days to 160 days. Sera were tested for: (1) complement-dependent toxicity to 51Cr-labeled islet cells in vitro; (2) immunoglobulin binding to RIN-5 F insulinoma cells; and (3) ability to selectively suppress insulin secretion from normal islets in vitro. All sera from rats that subsequently became diabetic or glucose-intolerant were toxic to islet cells from various rat strains in the presence of complement. They were toxic neither to hepatocytes nor to fibroblasts. The toxic potency was associated with the globulin fraction. It was, in most cases, maximal either before or immediately after the onset of the disease. Sera from the nondiabetes-susceptible BB rats and the rats which, in diabetes-prone litters, died too early to be classified tended toward greater toxicity to islets. Immunoglobulins from diabetic sera bound to RIN-5 F cells more than did the serum globulins from other groups, their maximal binding capacity occurring after the onset of diabetes. Furthermore, BB diabetic sera were capable of selectively inhibiting the insulin secretion from normal rat islets in vitro either in the presence or, in some cases, in the absence of complement. The A- and D-cell functions were not suppressed. The combination of such results suggests the presence of one or more antibodies capable of binding to beta cells, inhibiting their function, and inducing their lysis.(ABSTRACT TRUNCATED AT 250 WORDS)