In vitro recognition and impairment of pig islet cells by baboon immune cells: similarity to human cellular reactions.

BACKGROUND: Grafting pig islets into patients with type 1 diabetes requires control of the strong cellular xenogeneic rejection. This in vitro study compared the cellular reaction of baboons and humans to pig islet cells (PICs) to confirm the validity of using these animals for further in vivo preclinical trials. METHODS: Baboon or human peripheral blood mononuclear cells (PBMCs) or subsets were co-incubated with PICs from specific pathogen-free adult pigs for 7 days to determine the mechanisms and intensity of PBMC proliferation. Interleukin (IL) 10 and interferon (IFN) gamma secretion were assessed by enzyme-linked immunosorbent assay. Because proliferation was not indicative of aggression, a test based on perifusion analysis of the alteration of basal and stimulated insulin releases from PIC incubated with different baboon and human cells was developed. RESULTS: Baboon PBMCs strongly proliferated in response to PICs (stimulation index [SI]=24.8+/-6.9 [n=8] vs. 23.9+/-3.4 [n=34] for human PBMCs), showing considerable variation in intensity among animals (2.3

In vitro co-incubation of pig islet cells with xenogeneic human blood mononuclear cells causes loss of insulin release during perifusion: involvement of non-T-cell- and T-cell-mediated mechanisms.

Because the different steps of the human cellular immune rejection of pig islets are still poorly understood, our previous work concerned the intensity and mechanisms of the proliferation of human peripheral blood mononuclear cells (PBMC) to adult pig islet cells (PIC). As lymphocyte proliferation is not indicative of alteration of PIC, the present in vitro study evaluated cell-mediated immune effectors possibly involved in impairment of adult PIC. A test was thus developed, based on perifusion analysis of the alteration of insulin release from PIC incubated with different human cells. Compared to PIC incubation alone or with autologous pig splenocytes, seven-day co-incubation with whole human peripheral blood mononuclear cells (PBMC) (n = 18) led to almost complete abolition of basal and stimulated insulin releases (p < 0.0001). This effect could not be reversed by extensive sequential washes before perifusion of PIC, and the number of PIC was decreased by 78% after seven-day co-incubation with PBMC. PBMC are a complex mixture of cells involved in different xenogeneic mechanisms, and two components of this PIC impairment were then detected separately. First, the effect of PBMC against PIC was decreased (p < 0.0001) after removal of either MHC class II+ or CD14+ cells from PBMC. On the contrary, decreasing effect (p < 0.001) on insulin secretion was observed when only plastic-adherent or CD14+ cells were co-incubated with PIC. Additionally, alteration of insulin release from PIC cultured with PBMC or plastic-adherent cells was abolished dose-dependently (p < 0.0001 and p < 0.04, respectively) by gadolinium chloride (which inhibits macrophages), but not modified by cyclosporin A or mycophenolate mofetil which did not alter insulin release from PIC but blocked the proliferation of PBMC against PIC. A second mechanism was also detected, since co-incubation of PIC with purified human T cells remixed with antigen-presenting cells led to a decrease (p < 0.0001) of insulin release. This model based on the alteration of dynamic basal and stimulated insulin secretion provides detailed account of in vitro human cell-mediated impairment of PIC. It shows that the xenogeneic effect of whole mononuclear cells was strong and rapid. A crucial role was played by MHC class II+, CD14+, and plastic-adherent cells. Two mechanisms appear to be responsible for the role of these cells: 1) early direct effect, potentially involved in vivo in primary nonfunction of islets aggressed by monocytes/macrophages; and 2) the presentation of PIC xenoantigens leading to impairment by T lymphocytes, which may be involved in in vivo specific cellular rejection.

Intensity and mechanisms of in vitro xenorecognition of adult pig pancreatic islet cells by CD4+ and CD8+ lymphocytes from type I diabetic or healthy subjects.

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 CD4+ T cells were blocked with monoclonal antibodies, whereas the blocking of CD8+ 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 CD4+ cells alone did not proliferate in response to islet cells but recovered their proliferative ability when mixed with antigen-presenting cells, whereas CD8+ 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-gamma 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-gamma/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 CD4 class II-restricted Th2 response, with an indirect recognition pathway, as well as a CD8 T-cell response resulting from direct recognition. This strong reaction constitutes a serious obstacle which may vary in degree among subjects.

Porcine endogenous retroviral mRNAs in pancreas and a panel of tissues from specific pathogen-free pigs.

Pigs are potential providers of donor tissues for xenotransplantation (e.g. of pancreatic islets) in Type 1 diabetes. In this context, our group has studied the use of islets from specific pathogen-free (SPF) pigs as a means of reducing the risks of "conventional zoonosis". Although this approach does not prevent the transmission of pig endogenous retrovirus (PERV) to humans, we attempted to determine the presence of C-type PERV mRNAs for gag, pol, and env subtypes as a first descriptive step in the retroviral characterisation of SPF pig tissues (especially pancreas). Using semiquantitative reverse-transcriptase polymer chain reaction with 18S rRNA and beta-actin as internal controls, PERV mRNA levels were compared in a large panel of tissues from SPF and conventional pigs. PERV mRNAs for gag, pol, env-A and env-B were present in all tissues studied from the nine SPF pigs tested. Signals for env-C mRNAs were of much lower intensity than those for env-A and B, and most often undetectable in pancreas. The mRNA levels for gag, pol, env-A, env-B and env-C mRNAs were lower in pancreas (p < 0.01) than in all other tissues. Among other porcine tissues likely to be grafted in man, the highest retroviral mRNA levels were detected in kidney (p < 0.01), followed by liver, lung and heart. Amplified PERV mRNA signals were about 17 times less frequent in pig pancreas than in the retroviral-producing porcine cell line G2, while kidney contained about 6 times more PERV mRNAs than pancreas. The levels of gag, pol, env-A, env-B, and env-C mRNAs also varied between tissues of conventional pigs: PERV mRNA levels were lowest in pancreas, and env-C mRNAs were most often undetectable. For all SPF tissues tested, pol, gag, env-A, env-B, and env-C mRNA levels were in the same range or slightly higher than in corresponding tissues of conventional pigs. In summary, this study of C-type PERV mRNAs in a large panel of tissues from SPF pigs, in the context of our strategy of quality assurance and sanitary control, indicated that PERV mRNA levels were in the same range in SPF and corresponding conventional pig tissues, confirming that the use of SPF pigs would not prevent the risk of PERV transmission to human recipients of xenografts. PERV-A and PERV-B may be mainly represented, and PERV-C much less, in these pig tissues (particularly pancreas). The fact that pancreas expressed the lowest PERV mRNA levels and kidney the highest, among porcine tissues likely to be grafted, could be of interest from a clinical point of view. Pig tissues may differ in their loads of PERV sequences, which could be a factor in the risk of PERV transmission during xenotransplantation.

In vitro xenorecognition of adult pig pancreatic islet cells by splenocytes from nonobese diabetic or non-diabetes-prone mice.

BACKGROUND: In vitro studies of the nonobese diabetic (NOD) mouse prone to type 1 autoimmune diabetes were conducted in order to investigate the mechanisms possibly involved in cell-mediated rejection of adult pig islet xenografts. Mouse cellular proliferation in discordant situations was previously investigated only with stimulator lymphocytes and found to be low in intensity and due to an indirect recognition mechanism involving murine antigen-presenting cells (APC). It was also important to characterize murine anti-pig islet response. METHODS AND RESULTS: In the present study, mouse splenocytes responded to pig islet cells since primary proliferations were detected in non-diabetes-prone Balb/c (P<0.04) or NOD (P<0.001) mice. Moreover, NOD mice displayed a higher (P<0.003) splenocyte response to pig islet cells (stimulation index: 5.8+/-0.7) than did Balb/c mice (stimulation index: 2.3+/-0.3), whereas responses to pig stimulator splenocytes were similar in both strains. The proliferation of NOD splenocytes to pig islet cells was lower (P<0.0001) than the allogeneic response to Balb/c islet cells but similar to syngeneic proliferation to NOD islet cells. In both NOD and Balb/c mice, splenocyte proliferation to pig islet cells was abolished (P<0.01) when CD4+ cells were blocked with antibodies, whereas the blocking of CD8+ cells had a nonsignificant effect. The main T-splenocyte subsets involved were restricted to mouse MHC class II molecules as they did not proliferate in the presence of monoclonal antibodies directed at I-A molecules. NOD and Balb/c splenocyte proliferation to pig islet cells was abolished after removal of plastic-adherent APC, which indicates that the major activation pathway was indirect. Purified CD4+ or CD8- cells alone did not proliferate in response to pig islet cells but recovered a proliferative ability when mixed with APC. CD4- cells, alone or in the presence of APC, were not capable of responding to pig islet cells. Both Th1 and Th2 splenocytes were involved in response to pig islet cells since interferon-gamma (IFN-gamma) and interleukin (IL-)-4 production increased significantly (300-fold and 11-fold, respectively, P<0.02 for both), whereas the increase in IL-10 production was much lower (only 1.5-fold). The IFN-gamma/IL-4 and IFN-gamma/IL-10 ratios stimulated by pig islet cells were not different with NOD and Balb/c splenocytes. CONCLUSION: 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.