Bruce M. Hall

INCREASED EXPRESSION OF HLA-DR ANTIGENS ON RENAL TUBULAR CELLS IN RENAL TRANSPLANTS: RELEVANCE TO THE REJECTION RESPONSE

Whether the expression of DR antigens is altered in cadaver renal transplants was examined by the use of monoclonal antibodies to the non-polymorphic region of the DR molecule and an indirect immunoperoxidase stain. Expression of DR antigens increased considerably on renal tubular cells in all 25 biopsy specimens which showed severe cellular rejection, but in only 4 of 14 biopsy specimens with no or minimum evidence of rejection. 2 of these 4 specimens were from patients recently treated for severe rejection and the other 2 subsequently lost their grafts from chronic rejection. DR antigens were also expressed on the cell surface of isolated tubular cells aspirated from transplanted kidneys with acute cellular rejection but not on tubular cells in normal kidneys or aspirates from kidneys without rejection. Biopsy specimens with increased DR expression in tubules usually had an interstitial T cell infiltrate. Expression of DR antigens on tubular cells was not related to HLA-DR incompatibility between donor and host, or to the type of immunosuppressive therapy given. The expression of DR antigens on renal tubular cells may be induced by the infiltrating activated T cells or be a consequence of tubular regeneration following rejection or ischaemic damage. The increased expression of DR antigens on renal tubular cells during rejection makes these cells potential targets for delayed type hypersensitivity responses, which are only effective against DR antigen bearing cells.

Induction of long-term specific tolerance to allografts in rats by therapy with an anti-CD3-like monoclonal antibody

Monoclonal antibodies to CDS have been shown to activate T cells in vivo and in vitro but have also been shown to render T cells anergic in vitro. In this study G4.18, a mouse IgG3 mAb, was produced that appeared to recognize CDS by its binding to all peripheral T cells, including a population not recognized by mAb to TCR-α/β that was presumed to be TCR-γ/δ cells. It precipitated molecules in the 24–26 kd region consistent with the CD3 complex as well as molecules ∼45 and ∼49 kd that corresponded to TCR α and β chains and a 92-kd complex. Incubating T cells for 24 hr with saturating concentrations of G4.18 caused modulation of the TCR complex. In vitro, it activated T cells but only if prebound to plastic. In solution it inhibited MLC and CML, but not PHA or Con A activation. In vivo, G4.18 was not toxic even in high doses, and this was thought to be due to the inability of this mAb to activate T cells in vitro because the rat lacks Fc receptors for mouse IgGS. Therapy with G4.18 resulted in transient modulation of TCR/CD3 on T cells and depletion of these cells from blood. G4.18 had no depleting effects by lymph node or spleen cells but caused marked, transient thymic involution. Therapy with G4.18 also induced indefinite survival (&100 days) of PVG (RTF) heart grafts but not skin grafts in DA (RTFα) hosts. These hosts with long-surviving cardiac transplants, when grafted from PVG skin, accepted these grafts but rejected third-party skin in first-set. Thus G4.18 was shown to induce long-term specific tolerance to an organ allograft.

Elevated Levels of Tumor Necrosis Factor-α in the Nephrotic Syndrome in Humans

To investigate the possible role of cytokines in the mediation of glomerular injury in the nephrotic syndrome, the levels of interleukin (IL)-1β, IL-2, interferon (IFN)-α, IFN-γ, and tumor necrosis factor-α (TNF-α) were measured in patients with primary nephrotic syndrome. These patients had minimal change nephropathy (MCN), focal and segmental glomerulosclerosis (FSGS), or membranous nephropathy (MN) on biopsy. Cytokine levels were assessed by immunoradiometric assays, and specimens consisted of plasma, urine, and the culture supernate of mitogen-stimulated peripheral blood mononuclear cells (PBMC). Only TNF-α was found to be significantly elevated, in the plasma and urine of patients with FSGS and MN, above that found in healthy control subjects and patients with MCN. The elevation of TNF-a could not be shown to correlate with the length or severity of the nephrotic syndrome or with loss of body mass. IL-1β, IL-2, IFN-α, and IFN-γ levels were not elevated. In culture, mitogen-stimulated PBMC from all three groups of nephrotic subjects released an excess of TNF-α compared with controls, a response not consistently observed for the other cytokines measured. The findings of this survey of cytokine levels in nephrotic patients support the possibility that TNF-α may play a pathogenic role in the induction or maintenance of glomerular barrier dysfunction in humans.

Cells mediating allograft rejection.

Forty years ago Medawar (1944) showed that the graft rejection was systemically propogated, adaptive and antigen-specific, and established its immunological nature. Billingham et al. (1954, 1963) identified the response as cell-mediated and akin to delayed-type hypersensitivity (DTH), by showing that accelerated rejection could be transferred from animals which had rejected a graft to naive recipients with lymphoid cells but not serum. Over the intervening years, there has been a massive accumulation of experimental data pertaining to alloimmune responses. In spite of this, the mechanism by which cells of the immune system cause the destruction of foreign tissue is still unknown. It has been established that contact between alloantigen and cells of the immune system triggers an integrated series of interactions between a number of different cell populations (Snell 1978, Bach et al. 1976, Cantor & Boyse 1975a). There is still considerable controversy regarding the precise nature of the interactions and the identity of the cells which are relevant to the effector arm of the response (Mason 1983, McKenzie 1983). A great variety of in vivo and in vitro models have been developed to study alloimmune responses. These have revealed the existence of a number of effector mechanisms with the potential to cause graft rejection. These include cell or tissue destruction mediated by delayed type hypersensitivity reactions, cytotoxic T-cells, antibody augmented by complement, natural killer cells and macrophages (Carpenter et al. 1976, Charpentier 1983, McKenzie 1983). In the immunocompetent host, many or all of these potential effector mechnisms may be induced by allografts. The relative importance of each in the mediation of graft destruction is difficult to establish. In addition, the immune system has inbuilt feedback control mechanisms which are activated to suppress effector responses (Batchelor 1983, Dorsch & Roser 1982a). The cells which mediate these inhibitory responses will be present in host lymphoid tissue and in the graft at the time of rejection and may be difficult to distinguish from the cells mediating rejection.

Microvascular destruction in renal transplant rejection

In normal kidneys, peritubular and glomerular capillaries can be readily identified by their intense expression of HLA class I and class II compared to other cells within the graft. This high density of expression of MHC, plus their exposure to activated circulating lymphocytes, makes these cells the likely early and primary target of rejection responses. The fate of these capillaries during renal allograft rejection was examined using an indirect immunoperoxidase staining technique and monoclonal antibodies to class I and class II MHC antigens as well as other antigens on capillary endothelium including ICAM-1, LFA-3, and a novel antigen identified by E1.5. Expression of HLA-DR by peritubular capillaries was decreased during rejection, and this disappearance of peritubular capillaries with severe rejection was confirmed by loss of other markers of microvascular endothelium. These studies suggest peritubular capillaries may be the major target of the acute rejection response, and the techniques described allow assessment of degree of damage to these structures in renal allograft biopsies.

Induction of specific tolerance to allografts in rats by therapy with non-mitogenic, non-depleting anti-CD3 monoclonal antibody: association with TH2 cytokines not anergy.

BACKGROUND Anti-CD3 monoclonal antibodies (mAb) are potent immunosuppressives in transplantation but most do not induce tolerance. They induce anergy in Th1 cells but, if they bind to Fc receptors on antigen presenting cells, they activate T cells to release cytokines. METHODS This study examined the mechanisms of transplant tolerance induction to PVG fully allogeneic grafts in dark agouti rats by G4.18, a mouse immunoglobulinG3 anti-rat CD3 mAb that does not bind rat Fc receptors. Evidence of T cell activation was assayed by flow cytometry, reverse transcription (RT)-polymerase chain reaction (PCR) for cytokine mRNA, and responsiveness in mixed lymphocyte culture. RESULTS G4.18 treatment modulated T cell receptor/CD3 and CD2 and depleted T cells by <20% but did not induce activation surface markers. mRNA for interleukin (IL)-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and IL-4 in the lymph node, spleen, and thymus was not increased, and IFN-gamma mRNA was reduced. G4.18-treated and naive rat cells had similar proliferation and expression of IL-2, IFN-gamma, and IL-4 in vitro. G4.18-treated allograft recipients had no induction of mRNA for IL-2, IFN-gamma, TNF-alpha, TNF-beta, IL-4, IL-5, IL-10, perforin, and granzyme A & B in the spleen or grafts, with levels similar to those in isografts. The IL-4 and IL-5 mRNA levels in the spleen but not the graft of G4.18-treated recipients were higher than in rejecting and naive animals. Cells from G4.18-treated graft recipients proliferated more rapidly to the donor than to the third party and had increased IL-4 expression. CONCLUSIONS G4.18 induced transplant tolerance by a combination of modulation and blocking of the TCR/CD3, associated with increased Th2 cytokines, without depletion, induction of anergy, or nonspecific activation of T cells.

A Randomized Controlled Trial Of Cyclosporine Withdrawal In Renal-Transplant Recipients: 15-Year Results.

Background. In renal transplantation, the immunosuppressive efficacy of cyclosporine is counterbalanced by its nephrotoxicity. Although cyclosporine improves short-term graft survival, its long-term effects are unclear. Methods. Recipients of first cadaver renal transplants were randomized into three groups between 1983 and 1986: azathioprine and prednisolone alone (AP, n=158), long term cyclosporine alone (Cy, n=166), and short-term cyclosporine followed by azathioprine and prednisolone (CyAP, n=165). All groups received methylprednisolone induction. Results. There were no significant differences in patient survival at 15 years (48 vs. 56 vs. 51%, P=0.14), and 15-year graft survival (censored for death) in those patients in the CyAP group (47 vs. 44 vs. 59%, P=0.06) was not significantly different statistically. When deaths or graft losses before 12 months were censored, the differences in 15-year graft survival between the groups were significant (58%, 51%, 70%, P=0.01). The CyAP group also had lower mean serum creatinine at all time points beyond 3 months posttransplant out to 10 years (143 vs. 169 vs. 131 μmoles/L, P=0.04). Per protocol analysis, after censoring patients at change in therapy, increased the observed differences in 15-year graft survival between the groups (54 vs. 38 vs. 65%, P=0.01). Conclusion. Survival and function of first cadaveric kidney transplants is improved by use of short-term cyclosporine followed by azathioprine and prednisolone. Long-term cyclosporine use reduces long-term graft survival.

TREATMENT WITH INTERLEUKIN-4 PROLONGS ALLOGENEIC NEONATAL HEART GRAFT SURVIVAL BY INDUCING T HELPER 2 RESPONSES

BACKGROUND The T helper (Th) 2 cytokine interleukin (IL)-4 has been implicated as a major regulatory cytokine for the induction of transplant tolerance, but few studies have examined the capacity of IL-4 to induce tolerance. The effect of IL-4 therapy alone or with low doses of anti-CD4 monoclonal antibody (mAb) therapy on survival of fully allogeneic PVG neonatal heart graft in adult DA rats was examined. METHODS Rat recombinant (r) IL-4 was given at 30 microg (10(4) U)/kg daily intraperitoneally for 10 days and MRC OX35 (anti-CD4, nondepleting) or MRC OX81 (anti-IL-4) was given intraperitoneally on days 0, 3, 7, and 10. Semiquantitative reverse transcriptase-polymerase chain reaction was used to assay mRNA for cytokine in the graft, regional node and spleen and fluorescence-activated cell sorting was used to assay alloantibody Ig isotypes. RESULTS Grafts in rIL-4-treated rats survived a median period of 39 days (range, 28-52 days), significantly longer than in both untreated and nontransfected Chinese hamster ovary-K1 supernatant-treated controls (median, 14 days; range, 10-16 days, P=0.009). rIL-4 treatment with a suboptimal dose of anti-CD4 mAb prolonged median survival to 70 days (range, 63-80 days), which was longer than rIL-4 treatment alone or anti-CD4 mAb alone (median, 36 days; range, 30-55 days; P<0.0045). Combining MRC OX81 with MRC OX35 therapy led to earlier rejection at a median period of 26 days (range, 20-28 days); MRC OX81 alone had no effect on graft survival. Alloantibody titers, especially IgG1, were higher in rIL-4-treated animals and lower in anti-CD4 mAb-treated animals than in animals with normal rejection (P<0.05). IL-4 mRNA was increased in regional lymph nodes and spleen of the rIL-4-treated groups compared with all other groups, but there were no differences for IL-2, interferon-gamma, or IL-10. CONCLUSIONS rIL-4 therapy markedly prolonged neonatal cardiac allograft survival, and, with anti-CD4 therapy, it further prolonged survival. It induced IL-4 mRNA in lymphoid tissues and enhanced alloantibody production, especially IgG1, which demonstrated enhanced Th2 responses, but did not affect Th1 cytokines.

Distinct regulatory CD4+T cell subsets; differences between naïve and antigen specific T regulatory cells.

Effector T cells have functional subpopulations with distinct cytokine, cytokine receptor, chemokine receptor and transcription factors. We review how activation of antigen specific Treg induces expression of cytokines, cytokine receptors and chemokine receptors depending upon the effector lineage they are activated by. Activated Treg express receptors that are directly related to the effector T cell lineage. Other classes of Treg are induced in the periphery from effector lineage CD4(+)CD25(-)FOXP3(-)CD127(high)T cells, either by IL-10 or TGF-β or by association with activated CD4(+)CD25(+)FOXP3(+)Treg. Thus Treg are produced and adapt to the specific immune inflammatory environment they are activated within. Activated Treg produce different molecules to mediate suppression, which are tailored to the immune response they are activated by and control.

Suppressor T cells in rats with prolonged cardiac allograft survival after treatment with cyclosporine

DA rats treated with cyclosporine for 2 weeks after being grafted with an RT1-incompatible PVG heart graft did not reject the graft and developed a state of specific unresponsiveness to graft antigens. The cellular mechanisms maintaining this state of unresponsiveness were studied by testing the capacity of lymphocytes from these animals to effect or inhibit graft rejection in irradiated grafted hosts. Whole lymph node and spleen cell populations, and the T cell subpopulation separated from the latter, failed to restore the rejection of PVG hearts in irradiated DA recipients but restored third-party Wistar-Furth (W/F) rejection. Both whole spleen cells and the splenic T cell subpopulation had the capacity to suppress the ability of normal DA lymphocytes to cause graft rejection. Suppression was not dependent upon a state of chimerism in grafted cyclosporine -treated animals, and was not associated with any measurable alterations in the proportion of cytotoxic/suppressor T cells in lymphoid tissues. These studies show that the state of specific unresponsiveness that follows the treatment of heart grafted rats with cyclosporine is dependent, in part, upon active suppression that is induced or mediated by T lymphocytes. Many features of the immune reactivity of cyclosporine -treated grafted rats support the hypothesis that the mechanism of specific suppression in these animals is akin to that of enhancement, rather than to that of transplantation tolerance induced in neonatal rats.