Jonathan D. Katz

Following a diabetogenic T cell from genesis through pathogenesis

Nonobese diabetic (NOD) mice spontaneously develop a disease very similar to type 1 diabetes in humans. We have generated a transgenic mouse strain carrying the rearranged T cell receptor genes from a diabetogenic T cell clone derived from a NOD mouse. Self-reactive T cells expressing the transgene-encoded specificity are not tolerized in these animals, resulting in rampant insulitis and eventually diabetes. Features of the disease process emphasize two so-called check-points, recognized previously in the NOD and human diseases but easily misinterpreted. Although NOD mice are protected from insulitis and diabetes by expression of the E molecule encoded in the major histocompatibility complex, the transgenics are not, permitting us to exclude some possible mechanisms of protection.

Bim/Bcl-2 balance is critical for maintaining naive and memory T cell homeostasis

We examined the role of the antiapoptotic molecule Bcl-2 in combating the proapoptotic molecule Bim in control of naive and memory T cell homeostasis using Bcl-2−/− mice that were additionally deficient in one or both alleles of Bim. Naive T cells were significantly decreased in Bim+/−Bcl-2−/− mice, but were largely restored in Bim−/−Bcl-2−/− mice. Similarly, a synthetic Bcl-2 inhibitor killed wild-type, but not Bim−/−, T cells. Further, T cells from Bim+/−Bcl-2−/− mice died rapidly ex vivo and were refractory to cytokine-driven survival in vitro. In vivo, naive CD8+ T cells required Bcl-2 to combat Bim to maintain peripheral survival, whereas naive CD4+ T cells did not. In contrast, Bim+/−Bcl-2−/− mice generated relatively normal numbers of memory T cells after lymphocytic choriomeningitis virus infection. Accumulation of memory T cells in Bim+/−Bcl-2−/− mice was likely caused by their increased proliferative renewal because of the lymphopenic environment of the mice. Collectively, these data demonstrate a critical role for a balance between Bim and Bcl-2 in controlling homeostasis of naive and memory T cells.

Genetic Control of Diabetes Progression

Autoimmune diabetes in both the human and the nonobese diabetic mouse has elaborate genetics; in the latter case, the disease is influenced by at least 15-20 loci. We anticipated that the genetics would be simpler in the BDC2.5 T cell receptor transgenic mouse model of diabetes, wherein many T cells express a particular diabetogenic specificity. Initiation of insulitis in this model was the same on the two genetic backgrounds analyzed, but the kinetics and penetrance of diabetes were strikingly different, permitting us to focus on genetic influences during a defined window of disease progression. The differences correlated with variations in five genomic intervals, certain ones of which have been previously implicated in susceptibility to autoimmune disease. This reductionist approach indeed simplified the analysis of diabetes susceptibility loci.

The countervailing actions of myeloid and plasmacytoid dendritic cells control autoimmune diabetes in the nonobese diabetic mouse.

Islet Ag-specific CD4+ T cells receive antigenic stimulation from MHC class II-expressing APCs. Herein, we delineate the direct in vivo necessity for distinct subsets of macrophages and dendritic cells (DC) in type 1 diabetes mellitus of the NOD mouse by using diphtheria toxin-mediated cell ablation. The ablation of macrophages had no impact on islet Ag presentation or on the induction of insulitis or diabetes in either transfer or spontaneous models. However, the ablation of CD11b+CD11c+ DC led to the loss of T cell activation, insulitis, and diabetes mediated by CD4+ T cells. When the specific myeloid DC subset was “added-back” to mice lacking total DC, insulitis and diabetes were restored. Interestingly, when NOD mice were allowed to progress to the insulitis phase, the ablation of DC led to accelerated insulitis. This accelerated insulitis was mediated by the loss of plasmacytoid DC (pDC). When pDC were returned to depleted mice, the localized regulation of insulitis was restored. The loss of pDC in the pancreas itself was accompanied by the localized loss of IDO and the acceleration of insulitis. Thus, CD11c+CD11b+ DC and pDC have countervailing actions in NOD diabetes, with myeloid DC providing critical antigenic stimulation to naive CD4+ T cells and pDC providing regulatory control of CD4+ T cell function in the target tissue.

Interleukin-4 Deficiency Does Not Exacerbate Disease in NOD Mice

To investigate the role of interleukin (IL)-4 in the regulation of autoimmune diabetes, we crossed the IL-4 knock-out mutation onto the NOD genetic background. This experiment was accelerated by typing for microsatellites linked to known diabetes susceptibility (Idd) loci, and included a control backcross of the wildtype 129/SvJ-derived IL-4 gene, the original target locus. We also crossed the mutation into the BDC2.5 transgenic line, a diabetes model that carries the rearranged T-cell receptor genes from a diabetogenic Tcell clone. The IL-4-null mutation did not accelerate or intensify insulitis in regular NOD mice or in the BDC2.5 transgenic model; it also had no effect on the timing or frequency of the transition to overt diabetes. These data indicate that IL-4 plays no required role in controlling the aggressiveness of murine diabetes.

Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system

BackgroundIn this study we have built and mined a gene expression database composed of 65 diverse mouse tissues for genes preferentially expressed in immune tissues and cell types. Using expression pattern criteria, we identified 360 genes with preferential expression in thymus, spleen, peripheral blood mononuclear cells, lymph nodes (unstimulated or stimulated), or in vitro activated T-cells.ResultsGene clusters, formed based on similarity of expression-pattern across either all tissues or the immune tissues only, had highly significant associations both with immunological processes such as chemokine-mediated response, antigen processing, receptor-related signal transduction, and transcriptional regulation, and also with more general processes such as replication and cell cycle control. Within-cluster gene correlations implicated known associations of known genes, as well as immune process-related roles for poorly described genes. To characterize regulatory mechanisms and cis-elements of genes with similar patterns of expression, we used a new version of a comparative genomics-based cis-element analysis tool to identify clusters of cis-elements with compositional similarity among multiple genes. Several clusters contained genes that shared 5–6 cis-elements that included ETS and zinc-finger binding sites. cis-Elements AP2 EGRF ETSF MAZF SP1F ZF5F and AREB ETSF MZF1 PAX5 STAT were shared in a thymus-expressed set; AP4R E2FF EBOX ETSF MAZF SP1F ZF5F and CREB E2FF MAZF PCAT SP1F STAT cis-clusters occurred in activated T-cells; CEBP CREB NFKB SORY and GATA NKXH OCT1 RBIT occurred in stimulated lymph nodes.ConclusionThis study demonstrates a series of analytic approaches that have allowed the implication of genes and regulatory elements that participate in the differentiation, maintenance, and function of the immune system. Polymorphism or mutation of these could adversely impact immune system functions.

BCL-2 allows effector and memory CD8+ T cells to tolerate higher expression of BIM

As acute infections resolve, most effector CD8+ T cells die, whereas some persist and become memory T cells. Recent work showed that subsets of effector CD8+ T cells, identified by reciprocal expression of killer cell lectin-like receptor G1 (KLRG1) and CD127, have different lifespans. Similar to previous reports, we found that effector CD8+ T cells reported to have a longer lifespan (i.e., KLRG1lowCD127high) have increased levels of Bcl-2 compared with their shorter-lived KLRG1highCD127low counterparts. Surprisingly, we found that these effector KLRG1lowCD127high CD8+ T cells also had increased levels of Bim compared with KLRG1highCD127low cells. Similar effects were observed in memory cells, in which CD8+ central memory T cells expressed higher levels of Bim and Bcl-2 than did CD8+ effector memory T cells. Using both pharmacologic and genetic approaches, we found that survival of both subsets of effector and memory CD8+ T cells required Bcl-2 to combat the proapoptotic activity of Bim. Interestingly, inhibition or absence of Bcl-2 led to significantly decreased expression of Bim in surviving effector and memory T cells. In addition, manipulation of Bcl-2 levels by IL-7 or IL-15 also affected expression of Bim in effector CD8+ T cells. Finally, we found that Bim levels were significantly increased in effector CD8+ T cells lacking Bax and Bak. Together, these data indicate that cells having the highest levels of Bim are selected against during contraction of the response and that Bcl-2 determines the level of Bim that effector and memory T cells can tolerate.

Etoposide Selectively Ablates Activated T Cells To Control the Immunoregulatory Disorder Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is an inborn disorder of immune regulation caused by mutations affecting perforin-dependent cytotoxicity. Defects in this pathway impair negative feedback between cytotoxic lymphocytes and APCs, leading to prolonged and pathologic activation of T cells. Etoposide, a widely used chemotherapeutic drug that inhibits topoisomerase II, is the mainstay of treatment for HLH, although its therapeutic mechanism remains unknown. We used a murine model of HLH, involving lymphocytic choriomeningitis virus infection of perforin-deficient mice, to study the activity and mechanism of etoposide for treating HLH and found that it substantially alleviated all symptoms of murine HLH and allowed prolonged survival. This therapeutic effect was relatively unique among chemotherapeutic agents tested, suggesting distinctive effects on the immune response. We found that the therapeutic mechanism of etoposide in this model system involved potent deletion of activated T cells and efficient suppression of inflammatory cytokine production. This effect was remarkably selective; etoposide did not exert a direct anti-inflammatory effect on macrophages or dendritic cells, and it did not cause deletion of quiescent naive or memory T cells. Finally, etoposide’s immunomodulatory effects were similar in wild-type and perforin-deficient animals. Thus, etoposide treats HLH by selectively eliminating pathologic, activated T cells and may have usefulness as a novel immune modulator in a broad array of immunopathologic disorders.

Expression of Diabetes-Associated Genes by Dendritic Cells and CD4 T Cells Drives the Loss of Tolerance in Nonobese Diabetic Mice

In humans and NOD mice, defects in immune tolerance result in the spontaneous development of type-1-diabetes. Recent studies have ascribed a breakdown in tolerance to dysfunction in regulatory T cells that is secondary to reduced IL-2 production by T cells having the NOD diabetes susceptibility region insulin-dependent diabetes 3 (Idd3). In this study, we demonstrate a peripheral tolerance defect in the dendritic cells of NOD mice that is independent of regulatory T cells. NOD CD8 T cells specific for islet Ags fail to undergo deletion in the pancreatic lymph nodes. Deletion was promoted by expression of the protective alleles of both Idd3 (Il2) and Idd5 in dendritic cells. We further identify a second tolerance defect that involves endogenous CD4 T cell expression of the disease-promoting NOD alleles of these genetic regions. Pervasive insulitis can be reduced by expression of the Idd3 and Idd5 protective alleles by either the Ag-presenting cell or lymphocytes.

NKT Cells and IFN-γ Establish the Regulatory Environment for the Control of Diabetogenic T Cells in the Nonobese Diabetic Mouse

In type 1 diabetes mellitus (T1DM), T cell-mediated destruction of insulin-producing pancreatic β cells leads to the acute onset of hyperglycemia. The nonobese diabetic mouse model of human T1DM reveals that T cells capable of inducing diabetes can escape normal central tolerance, and can cause T1DM if left unchecked. However, several regulatory T cell subsets can temper autoaggressive T cells, although it remains undetermined when and how, and by which subset, homeostatic control of diabetogenic T cells is normally achieved in vivo. Using a cotransfer model, we find that NKT cells efficiently dampen the action of diabetogenic CD4+ T cells, and do so in an indirect manner by modifying the host environment. Moreover, the NKT cell-containing population modifies the host via production of IFN-γ that is necessary for driving the inhibition of diabetogenic T cells in vivo.