Gisela M. Vaitaitis

Expression of CD40 identifies a unique pathogenic T cell population in type 1 diabetes

Juvenile diabetes (type 1) is an autoimmune disease in which CD4+ T cells play a major role in pathogenesis characterized by insulitis and β cell destruction leading to clinical hyperglycemia. To date, no marker for autoimmune T cells has been described, although it was previously demonstrated that autoimmune mice have a large population of CD4+ cells that express CD40. We show here that established, diabetogenic T cell clones of either the Th1 or Th2 phenotype are CD40-positive, whereas nondiabetogenic clones are CD40-negative. CD40 functionally signals T cell clones, inducing rapid activation of the transcription factor NFκB. We show that autoimmune diabetes-prone nonobese diabetic mice have high levels of CD40+CD4+ T cells in the thymus, spleen, and importantly, in the pancreas. Finally, as demonstrated by adoptive transfers, CD4+CD40+ cells infiltrate the pancreatic islets causing β-cell degranulation and ultimately diabetes.

Cutting edge: CD40-induced expression of recombination activating gene (RAG) 1 and RAG2: a mechanism for the generation of autoaggressive T cells in the periphery.

It has been speculated that autoimmune diseases are caused by failure of central tolerance. However, this remains controversial. We have suggested that CD40 expression identifies autoaggressive T cells in the periphery of autoimmune prone mice. In this study, we report that CD40 was cloned from autoaggressive T cells and that engagement induces expression and nuclear translocation of the recombinases, recombination activating gene (RAG) 1 and RAG2 in the autoaggressive, but not in the nonautoaggressive, peripheral T cell population. Furthermore, we demonstrate that CD40 engagement induces altered TCR Vα, but not Vβ, expression in these cells. Therefore, CD40-regulated expression of RAG1 and RAG2 in peripheral T cells may constitute a novel pathway for the generation of autoaggressive T cells.

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 CD4loCD40+ 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, CD4loCD40+ 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 Vα3.2+ and Vα8.3+ T cells. Furthermore, peripheral Vα3.2+CD40+ T cells induce diabetes in NOD.scid recipients while Vα8.3+ T cells or Vα3.2+‐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 Vα expansion promotes diabetes.

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.

Galectin-9 controls CD40 signaling through a Tim-3 independent mechanism and redirects the cytokine profile of pathogenic T cells in autoimmunity.

While it has long been understood that CD40 plays a critical role in the etiology of autoimmunity, glycobiology is emerging as an important contributor. CD40 signaling is also gaining further interest in transplantation and cancer therapies. Work on CD40 signaling has focused on signaling outcomes and blocking of its ligand, CD154, while little is known about the actual receptor itself and its control. We demonstrated that CD40 is in fact several receptors occurring as constellations of differentially glycosylated forms of the protein that can sometimes form hybrid receptors with other proteins. An enticing area of autoimmunity is differential glycosylation of immune molecules leading to altered signaling. Galectins interact with carbohydrates on proteins to effect such signaling alterations. Studying autoimmune prone NOD and non-autoimmune BALB/c mice, here we reveal that in-vivo CD40 signals alter the glycosylation status of non-autoimmune derived CD4 T cells to resemble that of autoimmune derived CD4 T cells. Galectin-9 interacts with CD40 and, at higher concentrations, prevents CD40 induced proliferative responses of CD4loCD40+ effector T cells and induces cell death through a Tim-3 independent mechanism. Interestingly, galectin-9, at lower concentrations, alters the surface expression of CD3, CD4, and TCR, regulating access to those molecules and thereby redirects the inflammatory cytokine phenotype and CD3 induced proliferation of autoimmune CD4loCD40+ T cells. Understanding the dynamics of the CD40 receptor(s) and the impact of glycosylation status in immunity will gain insight into how to maintain useful CD40 signals while shutting down detrimental ones.

High distribution of CD40 and TRAF2 in Th40 T cell rafts leads to preferential survival of this auto-aggressive population in autoimmunity.

Background CD40–CD154 interactions have proven critical in autoimmunity, with the identification of CD4loCD40+ T cells (Th40 cells) as harboring an autoaggressive T cell population shedding new insights into those disease processes. Th40 cells are present at contained levels in non-autoimmune individuals but are significantly expanded in autoimmunity. Th40 cells are necessary and sufficient in transferring type 1 diabetes in mouse models. However, little is known about CD40 signaling in T cells and whether there are differences in that signaling and subsequent outcome depending on disease conditions. When CD40 is engaged, CD40 and TNF-receptor associated factors, TRAFs, become associated with lipid raft microdomains. Dysregulation of T cell homeostasis is emerging as a major contributor to autoimmune disease and thwarted apoptosis is key in breaking homeostasis. Methodology/Principal Findings Cells were sorted into CD4hi and CD4lo (Th40 cells) then treated and assayed either as whole or fractionated cell lysates. Protein expression was assayed by western blot and Nf-κB DNA-binding activity by electrophoretic mobility shifts. We demonstrate here that autoimmune NOD Th40 cells have drastically exaggerated expression of CD40 on a per-cell-basis compared to non-autoimmune BALB/c. Immediately ex-vivo, untreated Th40 cells from NOD mice have high levels of CD40 and TRAF2 associated with the raft microdomain while Th40 cells from NOR and BALB/c mice do not. CD40 engagement of Th40 cells induces Nf-κB DNA-binding activity and anti-apoptotic Bcl-XL expression in all three mouse strains. However, only in NOD Th40 cells is anti-apoptotic cFLIPp43 induced which leads to preferential survival and proliferation. Importantly, CD40 engagement rescues NOD Th40 cells from Fas-induced death. Conclusions/Significance CD40 may act as a switch between life and death promoting signals and NOD Th40 cells are poised for survival via this switch. This may explain how they expand in autoimmunity to thwart T cell homeostasis.

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‐1β, IL‐8, and TNFβ. 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 IFNγ 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.

CD40 glycoforms and TNF-receptors 1 and 2 in the formation of CD40 receptor(s) in autoimmunity

The CD40-CD154 dyad is an intensely studied field as is glycosylation status and both impact immunological functions and autoimmune conditions. CD40 has several isoforms, is modified by glycosylation, and trimerizes to form the functional receptor. We described a CD4(+)CD40(+) T cell (Th40) subset which is expanded in autoimmunity and is necessary and sufficient in transferring type 1 diabetes. Glycosylation impacts immunological events and T cells from autoimmune mouse strains express 30-40% less GlcNAc-branched N-glycans than T cells from non-autoimmune strains, a decrease known to activate T cells. Here we demonstrate that several CD40 receptor constellations exist on CD4 T cells. However, rather than containing different isoforms of CD40 they contain different glycoforms of isoform I. The glycoform profile is dependent on availability of CD154 and autoimmune NOD mice express a high level of a less glycosylated form. Interestingly, CD40 stimulation induces some CD40 receptor constellations that contain TNF-receptors 1 and 2 and targeting of those alters CD40 signaling outcomes in NOD Th40 cells. CD40-stimulation in vivo of non-autoimmune BALB/c mice expands the Th40 population and alters the CD40 glycoform profile of those cells to appear more like that of autoimmune prone NOD mice. Further understanding the dynamics and composition of the different CD40 receptor constellations will provide important insights into treatment options in autoimmunity.

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 OVA323–339 peptide as a neo‐self antigen on islet β cells and medullary thymic epithelial cells (mTECs) and a transgenic TCR recognizing the OVA323–339 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.

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.