CD40-mediated signalling influences trafficking, T-cell receptor expression, and T-cell pathogenesis, in the NOD model of type 1 diabetes.

CD40 plays a critical role in the pathogenesis of type 1 diabetes (T1D). The mechanism of action, however, is undetermined, probably because CD40 expression has been grossly underestimated. CD40 is expressed on numerous cell types that now include T cells and pancreatic ß cells. CD40+ CD4+ cells [T helper type 40 (TH40)] prove highly pathogenic in NOD mice and in translational human T1D studies. We generated BDC2.5.CD40-/- and re-derived NOD.CD154-/- mice to better understand the CD40 mechanism of action. Fully functional CD40 expression is required not only for T1D development but also for insulitis. In NOD mice, TH40 cell expansion in pancreatic lymph nodes occurs before insulitis and demonstrates an activated phenotype compared with conventional CD4+ cells, apparently regardless of antigen specificity. TH40 T-cell receptor (TCR) usage demonstrates increases in several Vα and Vß species, particularly Vα3.2+ that arise early and are sustained throughout disease development. TH40 cells isolated from diabetic pancreas demonstrate a relatively broad TCR repertoire rather than restricted clonal expansions. The expansion of the Vα/Vß species associated with diabetes depends upon CD40 signalling; NOD.CD154-/- mice do not expand the same TCR species. Finally, CD40-mediated signals significantly increase pro-inflammatory Th1- and Th17-associated cytokines whereas CD28 co-stimulus alternatively promotes regulatory cytokines.

An alternative role for Foxp3 as an effector T cell regulator controlled through CD40.

The BDC2.5 T cell clone is highly diabetogenic, but the transgenic mouse generated from that clone is surprisingly slow in diabetes development. Although defining pathogenic effector T cells in autoimmunity has been inconsistent, CD4(+) cells expressing the CD40 receptor (Th40 cells) are highly diabetogenic in NOD mice, and NOD.BDC2.5.TCR.Tg mice possess large numbers of these cells. Given the importance of CD40 for pathogenic T cell development, BDC2.5.CD40(-/-) mice were created. Regulatory T cells, CD4(+)CD25(hi)Foxp3(+), develop normally, but pathogenic effector cells are severely reduced in number. Th40 cells from diabetic BDC2.5 mice rapidly induce diabetes in NOD.scid recipients, but Th40 cells from prediabetic mice transfer diabetes very slowly. Demonstrating an important paradigm shift, effector Th40 cells from prediabetic mice are Foxp3(+). As mice age, moving to type 1 diabetes development, Th40 cells lose Foxp3. When Th40 cells that are Foxp3(+) are transferred to NOD.scid recipients, disease is delayed. Th40 cells that are Foxp3(-) rapidly transfer disease. Th40 cells from BDC2.5.CD40(-/-) mice do not transfer disease nor do they lose Foxp3 expression. Mechanistically, Foxp3(+) cells produce IL-17 but do not produce IFN-γ, whereas Foxp3(-) Th40 cells produce IFN-γ and IL-2. This poses a new consideration for the function of Foxp3, as directly impacting effector T cell function.

A CD40-targeted peptide controls and reverses type 1 diabetes in NOD mice.

AIMS/HYPOTHESIS: The CD40-CD154 interaction directs autoimmune inflammation. Therefore, a long-standing goal in the treatment of autoimmune disease has been to control the formation of that interaction and thereby prevent destructive inflammation. Antibodies blocking CD154 are successful in mouse models of autoimmune disease but, while promising when used in humans, unfortunate thrombotic events have occurred, forcing the termination of those studies. METHODS: To address the clinical problem of thrombotic events caused by anti-CD154 antibody treatment, we created a series of small peptides based on the CD154 domain that interacts with CD40 and tested the ability of these peptides to target CD40 and prevent type 1 diabetes in NOD mice. RESULTS: We identified a lead candidate, the 15-mer KGYY15 peptide, which specifically targets CD40-positive cells in a size- and sequence-dependent manner. It is highly efficient in preventing hyperglycaemia in NOD mice that spontaneously develop type 1 diabetes. Importantly, KGYY15 can also reverse new-onset hyperglycaemia. KGYY15 is well tolerated and functions to control the cytokine profile of culprit Th40 effector T cells. The KGYY15 peptide is 87% homologous to the human sequence, suggesting that it is an important candidate for translational studies. CONCLUSIONS/INTERPRETATION: Peptide KGYY15 constitutes a viable therapeutic option to antibody therapy in targeting the CD40-CD154 interaction in type 1 diabetes. Given the involvement of CD40 in autoimmunity in general, it will also be important to evaluate KGYY15 in the treatment of other autoimmune diseases. This alternative therapeutic approach opens new avenues of exploration in targeting receptor-ligand interactions.

CD40 engagement of CD4+ CD40+ T cells in a neo-self antigen disease model ablates CTLA-4 expression and indirectly impacts tolerance.

Biomarkers defining pathogenic effector T (Teff) cells slowly have been forthcoming and towards this we identified CD4(+) T cells that express CD40 (CD4(+) CD40(+) ) as pathogenic in the NOD type 1 diabetes (T1D) model. CD4(+) CD40(+) T cells rapidly and efficiently transfer T1D to NOD.scid recipients. To study the origin of CD4(+) CD40(+) T cells and disease pathogenesis, we employed a dual transgenic model expressing OVA(323-339) peptide as a neo-self antigen on islet ß cells and medullary thymic epithelial cells (mTECs) and a transgenic TCR recognizing the OVA(323-339) peptide. CD4(+) CD40(+) T cells and Treg cells each recognizing the cognate neo-antigen, rather than being deleted through central tolerance, drastically expanded in the thymus. In pancreatic lymph nodes of DO11.RIPmOVA mice, CD4(+) CD40(+) T cells and Treg cells are expanded in number compared with DO11 mice and importantly, Treg cells remain functional throughout the disease process. When exposed to neo-self antigen, CD4(+) CD40(+) T cells do not express the auto-regulatory CTLA-4 molecule while naïve CD4(+) CD40(+) T cells do. DO11.RIPmOVA mice develop autoimmune-type diabetes. CD40 engagement has been shown to prevent CTLA-4 expression and injecting anti-CD40 in DO11.RIPmOVA mice significantly exacerbates disease. These data suggest a unique means by which CD4(+) CD40(+) T cells thwart tolerance.

Disruption of the homeostatic balance between autoaggressive (CD4+CD40+) and regulatory (CD4+CD25+FoxP3+) T cells promotes diabetes.

Although regulatory T cells (Tregs) are well described, identifying autoaggressive effector T cells has proven more difficult. However, we identified CD4loCD40+ (Th40) cells as being necessary and sufficient for diabetes in the NOD mouse model. Importantly, these cells are present in pancreata of prediabetic and diabetic NOD mice, and Th40 cells but not CD4+CD40(-) T cells transfer progressive insulitis and diabetes to NOD.scid recipients. Nonobese-resistant (NOR) mice have the identical T cell developmental background as NOD mice, yet they are diabetes-resistant. The seminal issue is how NOR mice remain tolerant to diabetogenic self-antigens. We show here that autoaggressive T cells develop in NOR mice and are confined to the Th40 subset. However, NOR mice maintain Treg numbers equivalent to their Th40 numbers. NOD mice have statistically equal numbers of CD4+CD25+forkhead box P3+intrinsic Tregs compared with NOR or nonautoimmune BALB/c mice, and NOD Tregs are equally as suppressive as NOR Tregs. A critical difference is that NOD mice develop expanded numbers of Th40 cells. We suggest that a determinant factor for autoimmunity includes the Th40:Treg ratio. Mechanistically, NOD Th40 cells have low susceptibility to Fas-induced cell death and unlike cells from NOR and BALB/c mice, have predominantly low Fas expression. CD40 engagement of Th40 cells induces Fas expression but further confers resistance to Fas-mediated cell death in NOD mice. A second fundamental difference is that NOD Th40 cells undergo much more rapid homeostatic expansion than Th40 cells from NOR mice.

Biomarker Discovery in Pre-Type 1 Diabetes; Th40 Cells as a Predictive Risk Factor.

CONTEXT: The incidence of type 1 diabetes (T1D) is increasing worldwide. The quest to understand T1D etiology and how to predict diabetes is ongoing; and, in many ways, those goals intertwine. Although genetic components associate with T1D, not all individuals with T1D have those components, and T1D does not develop in all subjects with those components. OBJECTIVE: More robust methods for prediction of T1D are needed. We investigated if high CD4+CD40+ T-cell (Th40) levels can be used as a biomarker. METHODS: Th40 levels were assessed along with other parameters in blood collected from prediabetic subjects in TrialNet. RESULTS: In prediabetic subjects stratified according to Th40 cell level, patterns paralleled those seen between control subjects and those with T1D. Cytokine patterns were significantly different between those with high Th-40 levels (Th40-high) and those with low levels, and a CD4/CD8 double-positive population was more represented in Th40-high groups. Subjects experiencing impaired glucose tolerance had a significantly higher Th40 level than did control subjects. HLA DR4/DR4 and DQ8/DQ8 were more likely found among Th40-high subjects. Interestingly, HLA DR4/DR4 subjects were significantly older compared with all other subjects, suggesting that this haplotype, together with a high Th40 level, may represent someone in whom T1D will develop after age 30 years, which is reported for 42% of T1D cases. CONCLUSION: Considering the differences found in relation to prediabetic Th40 cell level, it may be possible to devise methods that more accurately predict who will proceed toward diabetes and, possibly, indicate prediabetic stage.

An analytical workflow for investigating cytokine profiles.

Understanding cytokine profiles of disease states has provided researchers with great insight into immunologic signaling associated with disease onset and progression, affording opportunities for advancement in diagnostics and therapeutic intervention. Multiparameter flow cytometric assays support identification of specific cytokine secreting subpopulations. Bead-based assays provide simultaneous measurement for the production of ever-growing numbers of cytokines. These technologies demand appropriate analytical techniques to extract relevant information efficiently. We illustrate the power of an analytical workflow to reveal significant alterations in T-cell cytokine expression patterns in type 1 diabetes (T1D) and breast cancer. This workflow consists of population-level analysis, followed by donor-level analysis, data transformation such as stratification or normalization, and a return to population-level analysis. In the T1D study, T-cell cytokine production was measured with a cytokine bead array. In the breast cancer study, intracellular cytokine staining measured T cell responses to stimulation with a variety of antigens. Summary statistics from each study were loaded into a relational database, together with associated experimental metadata and clinical parameters. Visual and statistical results were generated with custom Java software. In the T1D study, donor-level analysis led to the stratification of donors based on unstimulated cytokine expression. The resulting cohorts showed statistically significant differences in poststimulation production of IL-10, IL-1 beta, IL-8, and TNF beta. In the breast cancer study, the differing magnitude of cytokine responses required data normalization to support statistical comparisons. Once normalized, data showed a statistically significant decrease in the expression of IFN gamma on CD4+ and CD8+ T cells when stimulated with tumor-associated antigens (TAAs) when compared with an infectious disease antigen stimulus, and a statistically significant increase in expression of IL-2 on CD8+ T cells. In conclusion, the analytical workflow described herein yielded statistically supported and biologically relevant findings that were otherwise unapparent.

Th40 cells (CD4+CD40+ Tcells) drive a more severe form of Experimental Autoimmune Encephalomyelitis than conventional CD4 T cells.

CD40-CD154 interaction is critically involved in autoimmune diseases, and CD4 T cells play a dominant role in the Experimental Autoimmune Encephalomyelitis (EAE) model of Multiple Sclerosis (MS). CD4 T cells expressing CD40 (Th40) are pathogenic in type I diabetes but have not been evaluated in EAE. We demonstrate here that Th40 cells drive a rapid, more severe EAE disease course than conventional CD4 T cells. Adoptively transferred Th40 cells are present in lesions in the CNS and are associated with wide spread demyelination. Primary Th40 cells from EAE-induced donors adoptively transfer EAE without further in-vitro expansion and without requiring the administration of the EAE induction regimen to the recipient animals. This has not been accomplished with primary, non-TCR-transgenic donor cells previously. If co-injection of Th40 donor cells with Freund's adjuvant (CFA) in the recipient animals is done, the disease course is more severe. The CFA component of the EAE induction regimen causes generalized inflammation, promoting expansion of Th40 cells and infiltration of the CNS, while MOG-antigen shapes the antigen-specific TCR repertoire. Those events are both necessary to precipitate disease. In MS, viral infections or trauma may induce generalized inflammation in susceptible individuals with subsequent disease onset. It will be important to further understand the events leading up to disease onset and to elucidate the contributions of the Th40 T cell subset. Also, evaluating Th40 levels as predictors of disease onset would be highly useful because if either the generalized inflammation event or the TCR-honing can be interrupted, disease onset may be prevented.

Defining a new biomarker for the autoimmune component of Multiple Sclerosis: Th40 cells.

Multiple Sclerosis (MS) is a chronic inflammatory, neurodegenerative disease. Diagnosis is very difficult requiring defined symptoms and multiple CNS imaging. A complicating issue is that almost all symptoms are not disease specific for MS. Autoimmunity is evident, yet the only immune-related diagnostic tool is cerebral-spinal fluid examination for oligoclonal bands. This study addresses the impact of Th40 cells, a pathogenic effector subset of helper T cells, in MS. MS patients including relapsing/remitting MS, secondary progressive MS and primary progressive MS were examined for Th40 cell levels in peripheral blood and, similar to our findings in autoimmune type 1 diabetes, the levels were significantly (p<0.0001) elevated compared to controls including healthy non-autoimmune subjects and another non-autoimmune chronic disease. Classically identified Tregs were at levels equivalent to non-autoimmune controls but the Th40/Treg ratio still predicted autoimmunity. The cohort displayed a wide range of HLA haplotypes including the GWAS identified predictive HLA-DRB1*1501 (DR2). However half the subjects did not carry DR2 and regardless of HLA haplotype, Th40 cells were expanded during disease. In RRMS Th40 cells demonstrated a limited TCR clonality. Mechanistically, Th40 cells demonstrated a wide array of response to CNS associated self-antigens that was dependent upon HLA haplotype. Th40 cells were predominantly memory phenotype producing IL-17 and IFNγ with a significant portion producing both inflammatory cytokines simultaneously suggesting an intermediary between Th1 and Th17 phenotypes.

A unique T cell subset described as CD4loCD40+ T cells (TCD40) in human type 1 diabetes.

Human T1D pancreatic lymph nodes contain diabetes-autoantigen responsive T cells but identification of such T cells in the periphery has proven difficult. Here we describe a unique T cell subset defined by CD4(lo) and CD40 expression (T(CD40)) that is significantly expanded in peripheral blood of T1D but not control or T2D subjects. The HLA-DR3 and DR4 alleles are considered high risk factors for T1D and T(CD40) expansion occurs in T1D subjects carrying HLA DR3 or DR4 haplotypes but, T1D subjects who do not carry either DR3 or DR4 haplotypes still have an expanded percentage of T(CD40) cells. Non-autoimmune subjects, even DR3(+) and DR4(+), do not have elevated percentages of T(CD40) cells. The majority of T(CD40) cells in T1D carry a memory phenotype and a portion of those proliferates when exposed to diabetes-associated self-antigens. A greater number of memory T(CD40) cells express CXCR3 when compared to CD40(-) memory cells and that number is significantly expanded in T1D compared to control subjects. If only total CD4(+) T cells are compared no difference in CXCR3 is seen. Furthermore, T(CD40) cells produce a Th1, pro-inflammatory cytokine profile. In healthy controls, T(CD40) cells have equally Th1 and Th2 profiles.

Peripheral CD4loCD40+ auto-aggressive T cell expansion during insulin-dependent diabetes mellitus.

The generation of auto-aggressive T cells involves failure of central or peripheral tolerance. We previously demonstrated that peripheral CD4(lo)CD40(+) T cells give rise to pathogenic T cells in the non-obese diabetic (NOD) model. Here we show that peripheral CD4(+)CD40(+) T cells from diabetic or pre-diabetic NOD mice induce insulin-dependent diabetes mellitus. Consistent with breach of peripheral tolerance, CD4(lo)CD40(+) T cells expand with age in NOD mice but not in MHC-matched non-obese resistant (NOR) or BALB/c controls. Suggestive of a causal role for CD40 in autoimmunity, blocking CD40-CD154 interactions early during NOD development prevents autoaggressive T cell expansion while promoting increases in CD4(+)CD25(+) regulatory T cells. Importantly, CD40 signals promote expansion of V alpha 3.2(+) and V alpha 8.3(+) T cells. Furthermore, peripheral V alpha 3.2(+)CD40(+) T cells induce diabetes in NOD.scid recipients while V alpha 8.3(+) T cells or V alpha 3.2(+)-depleted T cell populations do not. This is the first demonstration that primary T cells transfer disease with the kinetics of auto-aggressive T cell clones and that specific TCR V alpha expansion promotes diabetes.