Co-incubation of pig islet cells with spleen cells from non-obese diabetic mice causes decreased insulin release by non-T-cell- and T-cell-mediated mechanisms

In 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, co-incubation 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-1β 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+ 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+ T lymphocytes)

Feeding NOD mice with pig splenocytes induces transferable mechanisms that modulate cellular and humoral xenogeneic reactions against pig spleen or islet cells

We have reported previously that oral administration of pig cells to NOD mice modified xenogeneic cellular response against pig islet cells (PICs), and hypothesized that it may have induced active suppression. This preliminary report evaluated only the effect of feeding pig cells by ‘primary’ proliferation, i.e. when splenocytes from fed mice are confronted with pig cells in vitro. The present study also considered ‘secondary’ proliferation and cytokine production after feeding and subsequent in vivo graft of pig cells. Additionally, serum IgM and IgG isotypes were quantified by ELISA using pig target cells. Induction of active mechanism by feeding was hypothetical, which led us here to transfer splenocytes from mice fed pig spleen cells (PSC) and evaluate ‘primary’ (after transfer) and ‘secondary’ (after transfer and subsequent graft of pig cells) proliferations and cytokine secretions in recipient mice. We also determined whether the effects of feeding pig cells persisted after depression of suppressor mechanisms by cyclophosphamide. Mice fed with PSC displayed increased ‘primary’ splenocyte proliferation to PSC or PIC (P < 0·0001), while ‘secondary’ responses were decreased (P < 0·03) in those fed PSC and subsequently grafted with PSC. The increased ‘primary’ and decreased ‘secondary’ proliferations were reduced (P < 0·04) by pretreatment with cyclophosphamide. The IL-10/ and IL-4/IFNγ ratios produced in response to PSC increased (P < 0·04) in mice fed and grafted with PSC compared to those grafted only with PSC. IgM and IgG levels against pig cells were, respectively, increased (P < 0·04) and decreased (P < 0·04) in mice fed and grafted with PSC. IgG2a and IgG2b, but not IgG1, levels were lower (P < 0·01). These effects of feeding PSC on ‘secondary’ proliferation, cytokine and antibody productions, were not detected when mice were fed PSC only after graft with PSC. Transfer with splenocytes from mice fed PSC increased ‘primary’ proliferation of splenocytes from recipient mice in response to PSC (P < 0·02) or PIC (P < 0·05). After transfer with splenocytes from PSC-fed mice and graft with PSC, ‘secondary’ proliferation to pig cells were reduced (P < 0·04), and the IL-10/IFNγ ratio produced in response to PSC was increased fourfold. Thus, oral administration of PSC induces active transferable mechanisms, characterized by a biphasic pattern with early increased ‘primary’ xenogeneic cellular reactions to both PSC and PIC, followed by decreased ‘secondary’ responsiveness and a concomitant shift of the Th1/Th2 balance towards greater Th2 influence. Decreased responsiveness may be due to active suppression, even though induction of anergy or deletion cannot be excluded

Achieving permanent survival of islet xenografts by independent manipulation of direct and indirect T-cell responses

Recent success in pancreatic islet allotransplantation has raised expectations but has equally highlighted the acute shortage of donor tissue. The use of xenogeneic tissue would help to address this shortage; however, strong cellular immunity limits the application of this approach. T-cell responses to xenogeneic tissues involve recognition of intact species-mismatched major histocompatibility complex (MHC) molecules, the direct pathway, and xenogeneic proteins presented as peptides by responder-type MHC molecules, the indirect pathway. In this study, we exploited the species difference to selectively and sequentially inhibit direct and indirect xenoresponses after transplantation of porcine islets into mice. Selective inhibition of the direct response was achieved using porcine CTLA4-Ig, which binds preferentially to pig versus mouse B7 molecules. Selective inhibition of the indirect response was achieved using murine CTLA4-Ig, which binds preferentially to mouse B7 molecules. Administration of porcine CTLA4-Ig alone caused modest prolongation of islet survival. Injection of murine CTLA4-Ig alone had a minimal effect. However, the injection of the porcine fusion protein early and the murine homolog late after grafting led to permanent survival of the porcine islets, in the absence of any other immunosuppression. These results suggest that a similar approach could have clinical utility in porcine islet xenotransplantation