Harold D. Chapman

Th1 to Th2 Cytokine Shifts in Nonobese Diabetic Mice: Sometimes an Outcome, Rather Than the Cause, of Diabetes Resistance Elicited by Immunostimulation

Numerous immunostimulatory protocols inhibit the development of T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse model. Many of these protocols, including treatment with the nonspecific immunostimulatory agents CFA or bacillus Calmette-Guérin (BCG) vaccine, have been reported to mediate protection by skewing the pattern of cytokines produced by pancreatic β-cell autoreactive T cells from a Th1 (IFN-γ) to a Th2 (IL-4 and IL-10) profile. However, most of these studies have documented associations between such cytokine shifts and disease protection rather than a cause/effect relationship. To partially address this issue we produced NOD mice genetically deficient in IFN-γ, IL-4, or IL-10. Elimination of any of these cytokines did not significantly alter the rate of spontaneous IDDM development. Additional experiments using these mice confirmed that CFA- or BCG-elicited diabetes protection is associated with a decreased IFN-γ to IL-4 mRNA ratio within T cell-infiltrated pancreatic islets, but this is a secondary consequence rather than the cause of disease resistance. Unexpectedly, we also found that the ability of BCG and, to a lesser extent, CFA to inhibit IDDM development in standard NOD mice is actually dependent upon the presence of the Th1 cytokine, IFN-γ. Collectively, our studies demonstrate that while Th1 and Th2 cytokine shifts may occur among β-cell autoreactive T cells of NOD mice protected from overt IDDM by various immunomodulatory therapies, it cannot automatically be assumed that this is the cause of their disease resistance.

Identification of a CD8 T Cell That Can Independently Mediate Autoimmune Diabetes Development in the Complete Absence of CD4 T Cell Helper Functions

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic β cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic β cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4αβ Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted β cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.

Activated NKT cells inhibit autoimmune diabetes through tolerogenic recruitment of dendritic cells to pancreatic lymph nodes

NKT cell activation by α-galactosylceramide (α-GalCer) inhibits autoimmune diabetes in NOD mice, in part by inducing recruitment to pancreatic lymph nodes (PLNs) of mature dendritic cells (DCs) with disease-protective effects. However, how activated NKT cells promote DC maturation, and what downstream effect this has on diabetogenic T cells was unknown. Activated NKT cells were found to produce a soluble factor(s) inducing DC maturation. Initially, there was a preferential accumulation of mature DCs in the PLNs of α-GalCer-treated NOD mice, followed by a substantial increase in T cells. Adoptive transfer of a diabetogenic CD8 T cell population (AI4) induced a high rate of disease (75%) in PBS-treated NOD recipients, but not in those pretreated with α-GalCer (8%). Significantly, more AI4 T cells accumulated in PLNs of α-GalCer than PBS-treated recipients, while no differences were found in mesenteric lymph nodes from each group. Compared with those in mesenteric lymph nodes, AI4 T cells entering PLNs underwent greater levels of apoptosis, and the survivors became functionally anergic. NKT cell activation enhanced this process. Hence, activated NKT cells elicit diabetes protection in NOD mice by producing a soluble factor(s) that induces DC maturation and accumulation in PLNs, where they subsequently recruit and tolerize pathogenic T cells.

The preferential ability of B lymphocytes to act as diabetogenic APC in NOD mice depends on expression of self-antigen-specific immunoglobulin receptors

B lymphocytes partially contribute to autoimmune type 1 diabetes (T1D) as a subset of APC with a preferential ability to trigger pathogenic CD4 T cells. We hypothesized that this resulted fromthe unique ability of B lymphocytes to take up pancreatic β cell proteins through Ig mediated capture. T1D was significantly delayed, but not prevented, in a NOD stock in which the B lymphocyteIg repertoire was strongly restricted because of the allelic exclusion induced by transgenic Ig molecules specific for the disease irrelevant hen egg lysozyme (HEL) protein (NOD.IgHEL mice). However, introducing the Igμnull mutation to eliminate the small residual numbers of non‐transgenic B lymphocytes in the NOD.IgHEL stock strongly suppressed T1D to the same low levels that characterize B lymphocyte deficient NOD.Igμnull mice. In contrast to standard NOD mice, both the NOD.IgHEL.Igμnull and NOD.Igμnull stocks were unable to generate T cell responses against the candidate diabetes autoantigen, glutamic acid decarboxylase. These results indicate that Ig‐mediated capture of β cell autoantigens accounts for why B lymphocytes have a greater capacity than other APC subtypes to trigger diabetogenic T cells. Hence, defects in B lymphocyte, as well as T lymphocyte, tolerance induction mechanisms may contribute to T1D in NOD mice.

B Cell Selection Defects Underlie the Development of Diabetogenic APCs in Nonobese Diabetic Mice

One mechanism whereby B cells contribute to type 1 diabetes in nonobese diabetic (NOD) mice is as a subset of APCs that preferentially presents MHC class II-bound pancreatic β cell Ags to autoreactive CD4 T cells. This results from their ability to use cell surface Ig to specifically capture β cell Ags. Hence, we postulated a diabetogenic role for defects in the tolerance mechanisms normally blocking the maturation and/or activation of B cells expressing autoreactive Ig receptors. We compared B cell tolerance mechanisms in NOD mice with nonautoimmune strains by using the IgHEL and Ig3-83 transgenic systems, in which the majority of B cells recognize one defined Ag. NOD- and nonautoimmune-prone mice did not differ in ability to delete or receptor edit B cells recognizing membrane-bound self Ags. However, in contrast to the nonautoimmune-prone background, B cells recognizing soluble self Ags in NOD mice did not undergo partial deletion and were also not efficiently anergized. The defective induction of B cell tolerance to soluble autoantigens is most likely responsible for the generation of diabetogenic APC in NOD mice.

Functional evidence for the mediation of diabetogenic T cell responses by HLA-A2.1 MHC class I molecules through transgenic expression in NOD mice.

Particular major histocompatibility complex (MHC) class II alleles clearly contribute to T cell-mediated autoimmune type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice. However, studies in NOD mice indicate MHC class I-restricted T cell responses are also essential to T1D development. In humans, epidemiological studies have suggested that some common class I alleles, including HLA-A2.1 (A*02011), may confer increased susceptibility to T1D when expressed in conjunction with certain class II alleles. We show here that when HLA-A2.1 molecules are transgenically expressed in NOD mice, A2-restricted T cell responses arise against pancreatic β cells, leading to an earlier onset of T1D. The accelerated onset of T1D in the NOD.HLA-A2.1 transgenic mice is not due to nonspecific effects of expressing a third class I molecule, because a stock of NOD mice transgenically expressing HLA-B27 class I molecules showed no such acceleration of T1D, but rather were significantly protected from disease. These findings provide the first functional evidence that certain human MHC class I molecules can contribute to the development of T1D.

Loss of intra-islet CD20 expression may complicate efficacy of B-cell-directed type 1 diabetes therapies.

OBJECTIVE Consistent with studies in NOD mice, early clinical trials addressing whether depletion of B cells by the Rituximab CD20-specific antibody provides an effective means for type 1 diabetes reversal have produced promising results. However, to improve therapeutic efficacy, additional B-cell–depleting agents, as well as attempts seeking diabetes prevention, are being considered. RESEARCH DESIGN AND METHODS Autoantibodies, including those against insulin (IAAs), are used to identify at-risk subjects for inclusion in diabetes prevention trials. Therefore, we tested the ability of anti-CD20 to prevent diabetes in NOD mice when administered either before or after IAA onset. RESULTS The murine CD20-specific 18B12 antibody that like Rituximab, depletes the follicular (FO) but not marginal zone subset of B cells, efficiently inhibited diabetes development in NOD mice in a likely regulatory T-cell–dependent manner only when treatment was initiated before IAA detection. One implication of these results is that the FO subset of B cells preferentially contributes to early diabetes initiation events. However, most important, the inefficient ability of anti-CD20 treatment to exert late-stage diabetes prevention was found to be attributable to downregulation of CD20 expression upon B cell entry into pancreatic islets. CONCLUSIONS These findings provide important guidance for designing strategies targeting B cells as a potential means of diabetes intervention.

Enhanced pathogenicity of diabetogenic T cells escaping a non-MHC gene-controlled near death experience

For unknown reasons, the common MHC class I variants encoded by the H2g7 haplotype (Kd, Db) aberrantly elicit autoreactive CD8 T cell responses essential to type 1 diabetes development when expressed in NOD mice, but not other strains. In this study, we show that interactive non-MHC genes allow a NOD-derived diabetogenic CD8 T cell clonotype (AI4) to be negatively selected at far greater efficiency in C57BL/6 mice congenically expressing H2g7 (B6.H2g7). However, the few AI4 T cells escaping negative selection in B6.H2g7 mice are exported from the thymus more efficiently, and are more functionally aggressive than those of NOD origin. This provides mechanistic insight to previous findings that resistant mouse strains carry some genes conferring greater diabetes susceptibility than the corresponding NOD allele. In the B6.H2g7 stock, non-MHC gene-controlled elevations in TCR expression are associated with both enhanced negative selection of diabetogenic CD8 T cells and increased aggressiveness of those escaping this process. An implication of this finding is that the same phenotype, in this case relatively high TCR expression levels, could have double-edged sword effects, contributing to type 1 diabetes resistance at one level of T cell development, but at another actually promoting pathogenesis.

Inhibition of Autoimmune Diabetes in Nonobese Diabetic Mice by Transgenic Restoration of H2-E MHC Class II Expression: Additive, But Unequal, Involvement of Multiple APC Subtypes

Transgenic restoration of normally absent H2-E MHC class II molecules on APC dominantly inhibits T cell-mediated autoimmune diabetes (IDDM) in nonobese diabetic (NOD) mice. We analyzed the minimal requirements for transgenic H2-E expression on APC subtypes (B lymphocytes vs macrophages/dendritic cells (DC)) to inhibit IDDM. This issue was addressed through the use of NOD stocks transgenically expressing high levels of H2-E and/or made genetically deficient in B lymphocytes in a series of genetic intercross and bone marrow/lymphocyte chimera experiments. Standard (H2-Enull) NOD B lymphocytes exert a pathogenic function(s) necessary for IDDM. However, IDDM was inhibited in mixed chimeras where H2-E was solely expressed on all B lymphocytes. Interestingly, this resistance was abrogated when even a minority of standard NOD H2-Enull B lymphocytes were also present. In contrast, in NOD chimeras where H2-E expression was solely limited to approximately half the macrophages/DC, an active immunoregulatory process was induced that inhibited IDDM. Introduction of a disrupted IL-4 gene into the NOD-H2-E transgenic stock demonstrated that induction of this Th2 cytokine does not represent the IDDM protective immunoregulatory process mediated by H2-E expression. In conclusion, high numbers of multiple subtypes of APC must express H2-E MHC class II molecules to additively inhibit IDDM in NOD mice. This raises a high threshold for success in future intervention protocols designed to inhibit IDDM by introduction of putatively protective MHC molecules into hemopoietic precursors of APC.

Genes within the Idd5 and Idd9/11 Diabetes Susceptibility Loci Affect the Pathogenic Activity of B Cells in Nonobese Diabetic Mice

Autoreactive T cells clearly mediate the pancreatic β cell destruction causing type 1 diabetes (T1D). However, studies in NOD mice indicate that B cells also contribute to pathogenesis because their ablation by introduction of an Igμnull mutation elicits T1D resistance. T1D susceptibility is restored in NOD.Igμnull mice that are irradiated and reconstituted with syngeneic bone marrow plus NOD B cells, but not syngeneic bone marrow alone. Thus, we hypothesized some non-MHC T1D susceptibility (Idd) genes contribute to disease by allowing development of pathogenic B cells. Supporting this hypothesis was the finding that unlike those from NOD donors, engraftment with B cells from H2g7 MHC-matched, but T1D-resistant, nonobese-resistant (NOR) mice failed to restore full disease susceptibility in NOD.Igμnull recipients. T1D resistance in NOR mice is mainly encoded within the Idd13, Idd5.2, and Idd9/11 loci. B cells from NOD congenic stocks containing Idd9/11 or Idd5.1/5.2-resistance loci, respectively, derived from the NOR or C57BL/10 strains were characterized by suppressed diabetogenic activity. Immature autoreactive B cells in NOD mice have an impaired ability to be rendered anergic upon Ag engagement. Interestingly, both Idd5.1/5.2 and Idd9/11-resistance loci were found to normalize this B cell tolerogenic process, which may represent a mechanism contributing to the inhibition of T1D.