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CD40/CD154-interaction is critical in the development of Experimental Autoimmune Encephalomyelitis (EAE; mouse model of Multiple Sclerosis). Culprit CD4+CD40+ T cells drive a more severe form of EAE than conventional CD4 T cells. Blocking CD40/CD154-interaction with CD154-antibody prevents or ameliorates disease but had thrombotic complications in clinical trials. We targeted CD40 using a CD154-sequence based peptide. Peptides in human therapeutics demonstrate good safety. A small peptide, KGYY 6 , ameliorates EAE when given as pretreatment or at first symptoms. KGYY 6 binds Th40 and memory T cells, affecting expression of CD69 and IL-10 in the CD4 T cell compartment, ultimately hampering disease development. Unlabelled Image • A CD154-derived, CD40-targeting, peptide (KGYY 6) ameliorates EAE. • KGYY 6 alters the phenotype of CD4 T cells but does not deplete those cells. • Being a peptide, KGYY 6 avoids problems common to antibodies. • KGYY 6 directly targets CD40, not CD154, avoiding possible thrombotic problems. • KGYY 6 does not include the αIIbβ3 interacting domain of CD154. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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+CD25hiFoxp3+, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

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-kB 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. [ABSTRACT FROM AUTHOR]

Abstract: 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 CD4lo and CD40 expression (TCD40) 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 TCD40 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 TCD40 cells. Non-autoimmune subjects, even DR3+ and DR4+, do not have elevated percentages of TCD40 cells. The majority of TCD40 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 TCD40 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, TCD40 cells produce a Th1, pro-inflammatory cytokine profile. In healthy controls, TCD40 cells have equally Th1 and Th2 profiles. [Copyright &y& Elsevier]