Georg F. Beilhack

Nonobese diabetic mice express aspects of both type 1 and type 2 diabetes

Before the onset of autoimmune destruction, type 1 diabetic patients and an animal model, the nonobese diabetic (NOD) mouse, show morphological and functional abnormalities in target organs, which may act as inciting events for leukocyte infiltration. To better understand these abnormalities, but without the complications associated with lymphocytic infiltrates, we examined genes expressed in autoimmune target tissues of NOD/severe combined immunodeficient (scid) mice and of autoimmune-resistant C57BL/6/scid mice. Our results suggest that the NOD genetic background may predispose them to diabetic complications, including insulin resistance in the absence of high circulating glucose levels and without autoimmune destruction of their β cells. Several of these genes lie within known type 1 and 2 diabetes loci. These data suggest that the NOD mouse may be a good candidate to study an interface between type 1 and type 2 diabetes.

Human Risk Allele HLA-DRB1*0405 Predisposes Class II Transgenic Ab0 NOD Mice to Autoimmune Pancreatitis

BACKGROUND & AIMS Autoimmune pancreatitis (AIP) underlies 5%-11% of cases of chronic pancreatitis. An association between AIP and the human leukocyte antigen (HLA)-DRB1*0405/DQB1*0401 haplotype has been reported, but linkage disequilibrium has precluded the identification of predisposing HLA gene(s). We studied the role of single HLA genes in the development of AIP in transgenic mice. METHODS CD4(+) T-cell-negative I-Abeta chain(-/-) (Ab0) mice develop AIP spontaneously, likely due to dysregulation of CD8(+) T- cell responses. We generated Ab0 nonobese diabetic (NOD) mice transgenic for HLA-DR*0405, leading to rescue of CD4(+) T cells; we compared their susceptibility to AIP with HLA-DQ8 or HLA-DR*0401 (single) transgenic, or HLA-DR*0405/DQ8 (double) transgenic mice. RESULTS CD4(+) T-cell-competent HLA-DR*0405 transgenic Ab0 NOD mice develop AIP with high prevalence after sublethal irradiation and adoptive transfer of CD90(+) T cells, leading to complete pancreatic atrophy. HLA-DR*0405 transgenic mice can also develop unprovoked AIP, whereas HLA-DR*0401, HLA-DQ8, and HLA-DR*0405/DQ8 transgenic Ab0 NOD controls all remained normal, even after irradiation and adoptive transfer of CD90(+) T cells. Pancreas histology in HLA-DR*0405 transgenic mice was characterized by destructive infiltration of the exocrine tissue with CD4(+) and CD8(+) T cells, B cells, and macrophages. Mice with complete pancreatic atrophy lost weight, developed fat stools, and had reduced levels of serum lipase activity. CONCLUSIONS Because HLA-DR*0405 expression fails to protect mice from AIP, the HLA-DRB1*0405 allele appears to be an important risk factor for AIP on the HLA-DRB1*0405/DQB1*0401 haplotype. This humanized mouse model should be useful for studying immunopathogenesis, diagnostic markers, and therapy of human AIP.

A diagnostic window for the treatment of acute graft-versus-host disease prior to visible clinical symptoms in a murine model

BackgroundAcute graft-versus-host disease (aGVHD) poses a major limitation for broader therapeutic application of allogeneic hematopoietic cell transplantation (allo-HCT). Early diagnosis of aGVHD remains difficult and is based on clinical symptoms and histopathological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestation. Therefore, for improved disease prevention it is important to develop predictive assays to identify patients at risk of developing aGVHD. Here we address whether insights into the timing of the aGVHD initiation and effector phases could allow for the detection of migrating alloreactive T cells before clinical aGVHD onset to permit for efficient therapeutic intervention.MethodsMurine major histocompatibility complex (MHC) mismatched and minor histocompatibility antigen (miHAg) mismatched allo-HCT models were employed to assess the spatiotemporal distribution of donor T cells with flow cytometry and in vivo bioluminescence imaging (BLI). Daily flow cytometry analysis of peripheral blood mononuclear cells allowed us to identify migrating alloreactive T cells based on homing receptor expression profiles.ResultsWe identified a time period of 2 weeks of massive alloreactive donor T cell migration in the blood after miHAg mismatch allo-HCT before clinical aGVHD symptoms appeared. Alloreactive T cells upregulated α4β7 integrin and P-selectin ligand during this migration phase. Consequently, targeted preemptive treatment with rapamycin, starting at the earliest detection time of alloreactive donor T cells in the peripheral blood, prevented lethal aGVHD.ConclusionsBased on this data we propose a critical time frame prior to the onset of aGVHD symptoms to identify alloreactive T cells in the peripheral blood for timely and effective therapeutic intervention.

Host-derived CD4+ T cells attenuate stem cell-mediated transfer of autoimmune arthritis in lethally irradiated C57BL/6.g7 mice.

OBJECTIVE In the K/BxN mouse model of inflammatory arthritis, T cells carrying a transgenic T cell receptor initiate disease by helping B cells to produce arthritogenic anti-glucose-6-phosphate isomerase (anti-GPI) autoantibodies. We found that lethally- irradiated lymphocyte-deficient C57BL/6 (B6).g7 (I-A(g7) +) recombinase-activating gene-deficient (Rag(-/-)) mice reconstituted with K/BxN hematopoietic stem and progenitor cells exhibit arthritis by week 4. In contrast, healthy B6.g7 recipients of K/BxN hematopoietic stem and progenitor cells show only mild arthritis, with limited extent and duration. The objective of this study was to investigate the factors responsible for the attenuation of arthritis in B6.g7 recipients. METHODS Antibody responses were measured by enzyme-linked immunosorbent assay. Fluorescence-activated cell sorting analyses were performed for testing chimerism, expression of markers of activation and suppression, tetramer binding, and intracellular cytokines in CD4+ T cells. Suppressive activity of CD4+ T cells was studied by adoptive transfer. RESULTS Titers of anti-GPI antibodies in reconstituted B6.g7 mice were ∼60-fold lower than in reconstituted B6.g7 Rag(-/-) mice. Examination of chimerism in the reconstituted B6.g7 mice showed that B cells and myeloid cells in these mice were donor derived, but CD4+ T cells were primarily host derived and enriched for cells expressing the conventional regulatory markers CD25 and FoxP3. Notably, CD4+CD25-FoxP3- T cells expressed markers of suppressive function (CD73 and folate receptor 4), and delayed disease after adoptive transfer. Activation of donor-derived CD4+ T cells was reduced, and thymic deletion of these cells appeared increased. CONCLUSION Despite myeloablation, host CD4+ T cells having a regulatory phenotype emerge in these mice and attenuate autoimmunity.

B6.g7 mice reconstituted with BDC2·5 non‐obese diabetic (BDC2·5NOD) stem cells do not develop autoimmune diabetes

In BDC2·5 non‐obese diabetic (BDC2·5NOD) mice, a spontaneous model of type 1 diabetes, CD4+ T cells express a transgene‐encoded T cell receptor (TCR) with reactivity against a pancreatic antigen, chromogranin. This leads to massive infiltration and destruction of the pancreatic islets and subsequent diabetes. When we reconstituted lethally irradiated, lymphocyte‐deficient B6.g7 (I‐Ag7+) Rag–/– mice with BDC2·5NOD haematopoietic stem and progenitor cells (HSPC; ckit+Lin–Sca‐1hi), the recipients exhibited hyperglycaemia and succumbed to diabetes. Surprisingly, lymphocyte‐sufficient B6.g7 mice reconstituted with BDC2·5NOD HSPCs were protected from diabetes. In this study, we investigated the factors responsible for attenuation of diabetes in the B6.g7 recipients. Analysis of chimerism in the B6.g7 recipients showed that, although B cells and myeloid cells were 98% donor‐derived, the CD4+ T cell compartment contained ∼50% host‐derived cells. These host‐derived CD4+ T cells were enriched for conventional regulatory T cells (Tregs) (CD25+forkhead box protein 3 (FoxP3)+] and also for host‐ derived CD4+CD25–FoxP3– T cells that express markers of suppressive function, CD73, FR4 and CD39. Although negative selection did not eliminate donor‐derived CD4+ T cells in the B6.g7 recipients, these cells were functionally suppressed. Thus, host‐derived CD4+ T cells that emerge in mice following myeloablation exhibit a regulatory phenoytpe and probably attenuate autoimmune diabetes. These cells may provide new therapeutic strategies to suppress autoimmunity.