Brigitte Koeberlein

Regulation of pancreatic |[beta]|-cell growth and survival by the serine/threonine protein kinase Akt1/PKB|[alpha]|

The physiological performance of an organ depends on an interplay between changes in cellular function and organ size, determined by cell growth, proliferation and death. Nowhere is this more evident than in the endocrine pancreas, where disturbances in function or mass result in severe disease. Recently, the insulin signal-transduction pathway has been implicated in both the regulation of hormone secretion from β cells in mammals as well as the determination of cell and organ size in Drosophila melanogaster. A prominent mediator of the actions of insulin and insulin-like growth factor 1 (IGF-1) is the 3′-phosphoinositide–dependent protein kinase Akt, also known as protein kinase B (PKB). Here we report that overexpression of active Akt1 in the mouse β cell substantially affects compartment size and function. There was a significant increase in both β-cell size and total islet mass, accompanied by improved glucose tolerance and complete resistance to experimental diabetes.

B lymphocyte–directed immunotherapy promotes long-term islet allograft survival in nonhuman primates

We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Thymoglobulin) and the monoclonal antibody to CD20 rituximab (Rituxan) promoted long-term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy. B lymphocyte reconstitution after rituximab-mediated depletion was characterized by a preponderance of immature and transitional cells, whose persistence was associated with long-term islet allograft survival. Development of donor-specific alloantibodies was abrogated only in the setting of continued rapamycin monotherapy.

B Cell-Mediated Antigen Presentation Is Required for the Pathogenesis of Acute Cardiac Allograft Rejection

Acute allograft rejection requires the activation of alloreactive CD4 T cells. Despite the capacity of B cells to act as potent APCs capable of activating CD4 T cells in vivo, their role in the progression of acute allograft rejection was unclear. To determine the contribution of B cell APC function in alloimmunity, we engineered mice with a targeted deficiency of MHC class II-mediated Ag presentation confined to the B cell compartment. Cardiac allograft survival was markedly prolonged in these mice as compared to control counterparts (median survival time, >70 vs 9.5 days). Mechanistically, deficient B cell-mediated Ag presentation disrupted both alloantibody production and the progression of CD4 T cell activation following heart transplantation. These findings demonstrate that indirect alloantigen presentation by recipients’ B cells plays an important role in the efficient progression of acute vascularized allograft rejection.

Impaired Activation of Islet-Reactive CD4 T Cells in Pancreatic Lymph Nodes of B Cell-Deficient Nonobese Diabetic Mice

Despite the impressive protection of B cell-deficient (μMT−/−) nonobese diabetic (NOD) mice from spontaneous diabetes, existence of mild pancreatic islet inflammation in these mice indicates that initial autoimmune targeting of β cells has occurred. Furthermore, μMT−/− NOD mice are shown to harbor a latent repertoire of diabetogenic T cells, as evidenced by their susceptibility to cyclophosphamide-induced diabetes. The quiescence of this pool of islet-reactive T cells may be a consequence of impaired activation of T lymphocytes in B cell-deficient NOD mice. In this regard, in vitro anti-CD3-mediated stimulation demonstrates impaired activation of lymph node CD4 T cells in μMT−/− NOD mice as compared with that of wild-type counterparts, a deficiency that is correlated with an exaggerated CD4 T cell:APC ratio in lymph nodes of μMT−/− NOD mice. This feature points to an insufficient availability of APC costimulation on a per T cell basis, resulting in impaired CD4 T cell activation in lymph nodes of μMT−/− NOD mice. In accordance with these findings, an islet-reactive CD4 T cell clonotype undergoes suboptimal activation in pancreatic lymph nodes of μMT−/− NOD recipients. Overall, the present study indicates that B cells in the pancreatic lymph node microenvironment are critical in overcoming a checkpoint involving the provision of optimal costimulation to islet-reactive NOD CD4 T cells.

In Vivo BLyS/BAFF Neutralization Ameliorates Islet-Directed Autoimmunity in Nonobese Diabetic Mice

B lymphocytes are required for the pathogenesis of autoimmune diabetes in NOD mice. Previous studies established that a lymphopenic transitional (TR) B cell compartment reduces the competitive constraint on the entry of newly emerging TR B cells into the splenic follicle (FO), thereby disrupting a peripheral negative selection checkpoint in NOD mice. Thus, development of clinically feasible immunotherapeutic approaches for restoration of appropriate negative selection is essential for the prevention of anti-islet autoimmunity. In this study we hypothesized that in vivo neutralization of the B lymphocyte stimulator (BLyS/BAFF) may enhance the stringency of TR→FO selection by increasing TR B cell competition for follicular entry in NOD mice. This study demonstrated that in vivo BLyS neutralization therapy leads to the depletion of follicular and marginal zone B lymphocytes. Long-term in vivo BLyS neutralization caused an increased TR:FO B cell ratio in the periphery indicating a relative resistance to follicular entry. Moreover, in vivo BLyS neutralization: 1) restored negative selection at the TR→FO checkpoint, 2) abrogated serum insulin autoantibodies, 3) reduced the severity of islet inflammation, 4) significantly reduced the incidence of spontaneous diabetes, 5) arrested the terminal stages of islet cell destruction, and 6) disrupted CD4 T cell activation in NOD mice. Overall, this study demonstrates the efficacy of B lymphocyte-directed therapy via in vivo BLyS neutralization for the prevention of autoimmune diabetes.

A Glucose Sensor Role for Glucokinase in Anterior Pituitary Cells

Enzymatic activity of glucokinase was demonstrated, quantitated, and characterized kinetically in rat and mouse pituitary extracts using a highly specific and sensitive spectrometric assay. A previously proposed hypothesis that the glucokinase gene might be expressed in the pituitary corticotrophic cells was therefore reexamined using mRNA in situ hybridization and immunohistochemical techniques. No evidence was found that corticotrophs are glucokinase positive, and the identity of glucokinase-expressing cells remains to be determined. The findings do, however, suggest a novel hypothesis that a critical subgroup of anterior pituitary cells might function as glucose sensor cells and that direct fuel regulation of such cells may modify the classical indirect neuroendocrine pathways that are known to control hormone secretion from anterior pituitary cells.

Cardiac allograft tolerance induced by intra-arterial infusion of recombinant adenoviral CTLA4Ig.

BACKGROUND Systemic administration of soluble recombinant fusion protein of cytotoxic T lymphocyte antigen 4 (CTLA4Ig) induces blockade of the CD28/B7 costimulatory pathway and promotes survival of allogeneic and xenogeneic grafts. We tested the efficacy of local expression of CTLA4Ig gene in the myocardium, induced by transduction with a recombinant adenovirus encoding the CTLA4Ig gene, on the survival of rat cardiac allografts. METHODS The donor hearts were perfused ex vivo with recombinant adenovirus encoding CTLA4Ig cDNA (AdCTLA4Ig) via intra-aorta coronary artery before transplantation. The distribution and duration of CTLA4Ig transgene expression in the myocardium was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) or in situ RT-PCR after transplantation. RESULTS In situ RT-PCR demonstrated abundant expression of CTLA4Ig transgene in the endo-myocardium of AdCTLA4Ig-perfused cardiac grafts. Lewis and Brown Norway cardiac allografts transduced with AdCTLA4Ig survived indefinitely in nonimmunosuppressed Wistar Furth recipients. However, donor-strain skin grafts were rejected by long-term recipients of cardiac allografts, which also triggered the rejection of the primary heart grafts. CONCLUSIONS A single ex vivo intra-aortic infusion of recombinant adenovirus encoding the CTLA4Ig gene induced efficient transduction of the endo-myocardium and promoted the permanent survival of cardiac allografts in nonimmunosuppressed hosts. Despite the beneficial effect of local immunosuppression on cardiac allograft survival, the strategy failed to promote a state of donor-specific peripheral tolerance.

Acquisition of Humoral Transplantation Tolerance upon De Novo Emergence of B Lymphocytes

A major obstacle to transplantation tolerance is humoral immunity. In this paper, we demonstrate that the intrinsic developmental propensity of the B lymphocyte compartment for acquisition of self-tolerance can be harnessed to induce humoral unresponsiveness to transplanted alloantigens. In the current study, when transitional B cells developed in the presence of donor lymphoid cells, the mature B lymphocyte compartment failed to mount a donor-specific alloantibody response to an organ transplant—despite unrestrained acute T cell-mediated allograft rejection. Specifically, we generated an experimental system wherein a B6 strain B cell compartment developed de novo in the presence of F1 (B6xBALB/c) lymphoid cells and in a T cell-deficient setting. Following establishment of a steady-state B cell compartment, these B6 mice were transplanted with heterotopic cardiac allografts from allogeneic BALB/c donors. The mice were then inoculated with purified syngeneic B6 T cells. As expected, all cardiac allografts were acutely rejected. However, the B lymphocyte compartment of these mice was completely inert in its capacity to form a BALB/c-specific alloantibody response. Using an alloantigen-specific Ig transgenic system, we demonstrated that this profound degree of humoral tolerance was caused by clonal deletion of alloreactive specificities from the primary B cell repertoire. Thus, de novo B cell compartment development at the time of transplantation is of critical importance in recipient repertoire “remodeling” to a humoral tolerant state.

CD4+ T Cells Recognizing a Single Self-Peptide Expressed by APCs Induce Spontaneous Autoimmune Arthritis

We have examined processes leading to the spontaneous development of autoimmune inflammatory arthritis in transgenic mice containing CD4+ T cells targeted to a nominal Ag (hemagglutinin (HA)) and coexpressing HA driven by a MHC class II promoter. Despite being subjected to multiple tolerance mechanisms, autoreactive CD4+ T cells accumulate in the periphery of these mice and promote systemic proinflammatory cytokine production. The majority of mice spontaneously develop inflammatory arthritis, which is accompanied by an enhanced regional immune response in lymph nodes draining major joints. Arthritis development is accompanied by systemic B cell activation; however, neither B cells nor Ab is required for arthritis development, since disease develops in a B cell-deficient background. Moreover, arthritis also develops in a recombinase activating gene-deficient background, indicating that the disease process is driven by CD4+ T cells recognizing the neo-self HA Ag. These findings show that autoreactive CD4+ T cells recognizing a single self-Ag, expressed by systemically distributed APCs, can induce arthritis via a mechanism that is independent of their ability to provide help for autoantibody production.

Genetic mapping of a mouse ocular malformation locus, tcm, to chromosome 4

The Tcm mutation in the mouse is an autosomal dominant ocular malformation manifesting as microphthalmia, iris dysplasia, cataract, and coloboma. As a first step to cloning the Tcm gene, we report the localization of the Tcm mutation with respect to known microsatellite markers. Backcross progeny carrying the Tcm mutation were produced by mating Tcm/+ heterozygous mice to normal C57BL/6 partners. Genomic DNA from each mouse was subjected to PCR analysis to identify simple sequence length polymorphisms. Our results locate Tcm to Chr 4 and suggest candidate genes responsible for the Tcm phenotype. Finally, ocular histopathology was done in 3-week-old animals to define the extent of the malformation.