Ronald G. Gill

Rosiglitazone promotes development of a novel adipocyte population from bone marrow-derived circulating progenitor cells.

Obesity and weight gain are characterized by increased adipose tissue mass due to an increase in the size of individual adipocytes and the generation of new adipocytes. New adipocytes are believed to arise from resident adipose tissue preadipocytes and mesenchymal progenitor cells. However, it is possible that progenitor cells from other tissues, in particular BM, could also contribute to development of new adipocytes in adipose tissue. We tested this hypothesis by transplanting whole BM cells from GFP-expressing transgenic mice into wild-type C57BL/6 mice and subjecting them to a high-fat diet or treatment with the thiazolidinedione (TZD) rosiglitazone (ROSI) for several weeks. Histological examination of adipose tissue or FACS of adipocytes revealed the presence of GFP(+) multilocular (ML) adipocytes, whose number was significantly increased by ROSI treatment or high-fat feeding. These ML adipocytes expressed adiponectin, perilipin, fatty acid-binding protein (FABP), leptin, C/EBPalpha, and PPARgamma but not uncoupling protein-1 (UCP-1), the CD45 hematopoietic lineage marker, or the CDllb monocyte marker. They also exhibited increased mitochondrial content. Appearance of GFP(+) ML adipocytes was contemporaneous with an increase in circulating levels of mesenchymal and hematopoietic progenitor cells in ROSI-treated animals. We conclude that TZDs and high-fat feeding promote the trafficking of BM-derived circulating progenitor cells to adipose tissue and their differentiation into ML adipocytes.

NK cells promote islet allograft tolerance via a perforin-dependent mechanism

Although major histocompatibility complex (MHC) class II–restricted CD4 T cells are well appreciated for their contribution to peripheral tolerance to tissue allografts, little is known regarding MHC class I–dependent reactivity in this process. Here we show a crucial role for host MHC class I–dependent NK cell reactivity for allograft tolerance in mice induced through either costimulation blockade using CD154-specific antibody therapy or by targeting LFA-1 (also known as CD11a). Tolerance induction absolutely required host expression of MHC class I, but was independent of CD8 T cell–dependent immunity. Rather, tolerance required innate immunity involving NK1.1+ cells, but was independent of CD1d-restricted NKT cells. Therefore, NK cells seem to be generally required for induction of tolerance to islet allografts. Additional studies indicate that CD154-specific antibody–induced allograft tolerance is perforin dependent. Notably, NK cells that are perforin competent are sufficient to restore allograft tolerance in perforin-deficient recipients. Together, these results show an obligatory role for NK cells, through perforin, for induction of tolerance to islet allografts.Note: In the version of this article initially published, the authors inadvertently misquoted a study as evidence that mouse NKT cells can express CD154 (ref. 29). Rather, the cited study concerned CD40-CD40L interactions in human NK cells. By misquoting this study, the authors also omitted an appropriate reference regarding prior evidence of CD40-CD40L interactions by murine NKT cells (Kitamura, H. et al., J. Exp. Med. 189, 1121; 1999). These errors have been corrected in the PDF version.

Evidence for a primary islet autoantigen (preproinsulin 1) for insulitis and diabetes in the nonobese diabetic mouse

It has been reported that an insulin 2 gene knockout, when bred onto nonobese diabetic (NOD) mice, accelerates diabetes. We produced insulin 1 gene knockout congenic NOD mice. In contrast to insulin 2, diabetes and insulitis were markedly reduced in insulin 1 knockout mice, with decreased and delayed diabetes in heterozygous females and no insulitis and diabetes in most homozygous female mice. Lack of insulitis was found for insulin 1 female homozygous knockout mice at 8, 12, and 37 weeks of age. Despite a lack of insulitis, insulin 1 homozygous knockout mice spontaneously expressed insulin autoantibodies. Administration of insulin peptide B:9-23 of both insulin 1 and 2 to NOD mice induced insulin autoantibodies. Insulin 1 is not the only lymphocytic target of NOD mice. Insulin 1 homozygous knockout islets, when transplanted into recently diabetic wild-type NOD mice, became infiltrated with lymphocytes and only transiently reversed diabetes. These observations indicate that loss of either insulin gene can influence progression to diabetes of NOD mice and suggest that the preproinsulin 1 gene is crucial for the spontaneous development of NOD insulitis and diabetes.

CD4 T cell–mediated cardiac allograft rejection requires donor but not host MHC class II

Numerous studies indicate that CD4 T cells are required for acute cardiac allograft rejection. However, the precise role for CD4 T cells in this response has remained ambiguous owing to the multipotential properties of this T-cell subpopulation. In the current study, we demonstrate the capacity of CD4 T cells to serve as direct effector cells of cardiac allograft rejection. We show that CD4 T cells are both necessary and sufficient for acute graft rejection, as indicated by adoptive transfer experiments in immune-deficient SCID and rag1(-/-) recipients. We have analyzed the contribution of direct (donor MHC class II restricted) and indirect (host MHC class II restricted) antigen recognition in CD4-mediated rejection. Acute CD4 T cell-mediated rejection required MHC class II expression by the allograft, indicating the importance of direct graft recognition. In contrast, reciprocal experiments indicate that CD4 T cells can acutely reject allogeneic cardiac allografts established in rag1(-/-) hosts that were also MHC class II deficient. This latter result indicates that indirect presentation of donor antigens by host MHC class II is not required for acute CD4-mediated rejection. Taken together, these results indicate that CD4 T cells can serve as effector cells for primary acute cardiac allograft rejection, predominantly via direct donor antigen recognition and independent of indirect reactivity.

Autoimmune Diabetes in NOD Mouse Is L3T4 T-Lymphocyte Dependent

Cultured BALB/c islets fail to function when transplanted into diabetic nonobesediabetic (NOD) mice; such grafted tissue is rapidly destroyed by disease recurrence. The cellular requirements for this graft damage are unclear. This study was designed to investigate the role of the L3T4+ T-lymphocyte subset in disease recurrence in the NOD mouse. L3T4+ T-lymphocytes were depleted by the in vivo administration of the L3T4-specific monoclonal antibody GK1.5. This treatment reduced the level of L3T4+ T-lymphocytes from an initial 43% of the peripheral blood lymphocytes to 4%. L3T4 levels remained at this low level for ∼2 wk after withdrawal of GK1.5 treatment, after which the L3T4 levels slowly began to increase in the periphery. Grafting of cultured BALB/c islet tissue into GK 1.5-treated diabetic NOD mice resulted in a rapid return to normoglycemia that persisted for 2-4 wk. The gradual return to the hyperglycemic condition roughly correlated with the reappearance of L3T4+ T-lymphocytes in the peripheral circulation. From these findings we conclude that the disease process in the NOD mouse is L3T4 T-lymphocyte dependent.

Evidence Of Recurrent Autoimmunity In Human Allogeneic Islet Transplantation

We transplanted 10,000 isolated, handpicked human pancreatic islets into the subfascial compartment of the forearm muscle of a type I diabetic recipient who had received a successful renal transplant one year prior. The recipient was maintained on his usual immunosuppressive therapy of cyclosporine, azathioprine, and prednisone. A biopsy performed 7 days after transplantation showed normal islets with both insulin- and glucagon-staining cells present and no lymphocytic infiltration. A second biopsy performed 14 days after transplantation showed a dense mononuclear cell infiltrate with a preferential loss of insulin-staining cells relative to glucagon-staining cells in the islets. These data are consistent with recurrent autoimmune diabetes in an isolated islet allograft in an immunosuppressed type I diabetic recipient. In addition, this forearm subfascial site may be a useful means to monitor islet rejection and autoimmune recurrence in therapeutic intraportal islet allografts.

Cellular and humoral autoimmunity directed at bile duct epithelia in murine biliary atresia

Biliary atresia is an inflammatory fibrosclerosing lesion of the bile ducts that leads to biliary cirrhosis and is the most frequent indication for liver transplantation in children. The pathogenesis of biliary atresia is not known; one theory is that of a virus‐induced, subsequent autoimmune‐mediated injury of bile ducts. The aim of this study was to determine whether autoreactive T cells and autoantibodies specific to bile duct epithelia are present in the rotavirus (RRV)‐ induced murine model of biliary atresia and whether the T cells are sufficient to result in bile duct inflammation. In vitro analyses showed significant increases in IFN‐γ–producing T cells from RRV‐diseased mice in response to bile duct epithelial autoantigen. Adoptive transfer of the T cells from RRV‐diseased mice into naïve syngeneic SCID recipients resulted in bile duct–specific inflammation. This induction of bile duct pathology occurred in the absence of detectable virus, indicating a definite response to bile duct autoantigens. Furthermore, periductal immunoglobulin deposits and serum antibodies reactive to bile duct epithelial protein were detected in RRV‐diseased mice. In conclusion, both cellular and humoral components of autoimmunity exist in murine biliary atresia, and the progressive bile duct injury is due in part to a bile duct epithelia–specific T cell–mediated immune response. The role of cellular and humoral autoimmunity in human biliary atresia and possible interventional strategies therefore should be the focus of future research. (HEPATOLOGY 2006;44:1231–1239.)

LFA-1 (CD11a) as a Therapeutic Target

Leukocyte function associated antigen‐1 (LFA‐1) was one of the earliest of cell‐surface molecules identified by monoclonal antibodies generated against leukocyte immunogens. This integrin heterodimer is perhaps best known as a classic adhesion molecule facilitating the interaction between T cells and antigen‐presenting cells. However, varied studies indicate that LFA‐1 has multi‐faceted roles in the immune response including adhesion, activation and trafficking of leukocyte populations. While there has been long‐standing interest in LFA‐1 as a therapeutic target for regulating immunity, anti‐LFA‐1 therapy is still not a first‐line indication for any clinical condition. Antagonism of LFA‐1 with monoclonal antibodies, either alone or in combination with other agents, can result in regulatory tolerance in vivo. Furthermore, new generation humanized anti‐LFA‐1 monoclonal antibodies (Efalizumab) show at least modest promise for continued application in clinical trials. Thus, anti‐LFA‐1 forms a potential, but still largely unexploited, immunotherapy which may find its greatest application as an agent which augments other therapies.

CD4-Dependent Generation of Dominant Transplantation Tolerance Induced by Simultaneous Perturbation of CD154 and LFA-1 Pathways

CD154 and LFA-1 (CD11a) represent conceptually distinct pathways of receptor/ligand interactions (costimulation and adhesion/homing, respectively) that have been effectively targeted to induce long-term allograft acceptance and tolerance. In the current study, we determined the relative efficacy and nature of tolerance induced by mAbs specific for these pathways. In vitro analysis indicated that simultaneous targeting of CD154 and LFA-1 resulted in profound inhibition of alloreactivity, suggesting that combined anti-CD154/anti-LFA-1 therapy could be highly effective in vivo. Thus, we evaluated combining mAb therapies targeting CD154 and LFA-1 for inducing transplantation tolerance to pancreatic islet allografts. Monotherapy with either anti-CD154 or anti-LFA-1 was partially effective for inducing long-term allograft survival, whereas the combination resulted in uniform allograft acceptance in high-responder C57BL/6 recipients. This combined therapy was not lymphocyte depleting and did not require the long-term deletion of donor-reactive T lymphocytes to maintain allograft survival. Importantly, combined anti-CD154/anti-LFA therapy uniquely resulted in “dominant” transplantation tolerance. Therefore, simultaneous perturbation of CD154 and LFA-1 molecules can result in profound tolerance induction not accomplished through individual monotherapy approaches. Furthermore, results show that such regulatory tolerance can coexist with the presence of robust anti-donor reactivity, suggesting that active tolerance does not require a corresponding deletion of donor-reactive T cells. Interestingly, although the induction of this regulatory state was highly CD4 dependent, the adoptive transfer of tolerance was less CD4 dependent in vivo.

Anti-LFA-1 therapy induces long-term islet allograft acceptance in the absence of IFN-gamma or IL-4.

mAb therapy directed against a variety of cell surface accessory molecules has been effectively utilized to prolong allograft acceptance in various models of tissue and organ transplantation. The purpose of this study was to determine whether transient therapy directed against the adhesion molecule LFA-1 (CD11a) was sufficient to induce donor-specific tolerance to pancreatic islet allografts. Anti-LFA-1 monotherapy was found to be efficacious in inducing long-term islet allograft acceptance in multiple donor-recipient strain combinations. Graft acceptance following anti-LFA-1 therapy was not simply due to clonal ignorance of donor Ags in that the majority of recipients bearing established islet allografts resisted rejection induced by immunization with donor-type APCs. Furthermore, donor-specific tolerance from anti-LFA-1-treated animals could be transferred to secondary immune-deficient animals. Taken together, these results indicated that transient anti-LFA-1 monotherapy resulted in donor-specific tolerance. In vitro, functionally tolerant animals retained normal anti-donor reactivity as assessed by proliferative, cytotoxic, and cytokine release assays that demonstrated that tolerance was not secondary to general clonal deletion or anergy of donor-reactive T cells. Finally, anti-LFA-1 treatment was effective in both IL-4-deficient and IFN-γ-deficient recipients, indicating that neither of these cytokines are universally required for allograft acceptance. These results suggest that anti-adhesion-based therapy can induce a nondeletional form of tolerance that is not overtly dependent on the prototypic Th1 and Th2 cytokines, IFN-γ and IL-4, respectively, in contrast to results in other transplantation models.