IntroductionCanine diabetes mellitus (CDM) is a relatively common endocrine disease in dogs. Many CDM clinical features resemble human type 1 diabetes mellitus (T1DM), but lack of autoimmune biomarkers makes calling the disease autoimmune controversial. Autoimmune biomarkers linking CDM and T1DM would create an alternative model for drug development impacting both human and canine disease. MethodsWe examined peripheral blood of diagnosed CDM dog patients comparing it to healthy control (HC) dogs. Dogs were recruited to a study at the Colorado State University Veterinary Teaching Hospital and blood samples collected for blood chemistry panels, complete blood counts (CBC), and immunologic analysis. Markers of disease progression such as glycated albumin (fructosamine, the canine equivalent of human HbA1c) and c-peptide were addressed. ResultsSignificant differences in adaptive immune lymphocytes, innate immune macrophages/monocytes and neutrophils and differences in platelets were detected between CDM and HC based on CBC. Significant differences in serum glucose, cholesterol and the liver function enzyme alkaline phosphatase were also detected. A systemic immune inflammation index (SII) and chronic inflammation index (CII) as measures of dynamic changes in adaptive and innate cells between inflammatory and non-inflammatory conditions were created with highly significant differences between CDM and HC. Th40 cells (CD4+CD40+ T cells) that are demonstrably pathogenic in mouse T1DM and able to differentiate diabetic from non-diabetic subjects in human T1DM were significantly expanded in peripheral blood mononuclear cells.ConclusionsBased on each clinical finding, CDM can be categorized as an autoimmune condition. The association of significantly elevated Th40 cells in CDM when compared to HC or to osteoarthritis, a chronic but non-autoimmune disease, suggests peripheral blood Th40 cell numbers as a biomarker that reflects CDM chronic inflammation. The differences in SII and CII further underscore those findings.

Treating MS has been difficult. One successful drug is Ocrelizumab (anti-CD20), used for the chronic relapsing MS (RMS) and the progressive MS (PMS) forms. TH40 cells are pathogenic effector T cells that increase in percentage and numbers during chronic inflammation. Here we show that in the earliest MS course, clinically isolated syndrome (CIS), TH40 cells expand in number. In PMS TH40 cell numbers remain expanded demonstrating sustained chronic inflammation. In RMS TH40 cells were found in CSF and express CD20. Ocrelizumab reduced TH40 cells to healthy control levels in patients. During treatment inflammatory cytokine producing TH40 cells were decreased.

Abstract Canine Diabetes (CD) is identical to human T1DM. Helper T cells expressing the CD40 receptor (TH40) are prominent in human T1DM. TH40 cell expansions occur regardless of HLA haplotype or auto-antibody status and remain elevated throughout disease. TH40 cells from T1DM respond to human islets, TH40 from healthy controls do not. At a Veterinary Hospital 6 female and 2 male dogs that met clinical criteria for T1DM were recruited. Physical exam, blood panels and CBC were done. Dogs were dosed with a OPT501 a drug targeting CD40 mediated inflammation by IV infusion. Doses were 2 and later 4 mg/kg, given on days 1, 4, 7, then weekly for 8 weeks. Diabetic dogs had significantly elevated TH40 numbers compared to non-diabetic dogs (p < 0. 0001). OPT501 reduced TH40 cell numbers to normal, following which insulin requirements reduced on average by 75% and up to 90%. The canine equivalent of HbA1c reduced 40.2% on average; 3 subjects were normal range. Daily blood glucose reduced significantly, 3 subjects maintained normal glucose levels. Time-in-range (TIR) increased to up to 90%, from a previous 10%. Increased c - peptide levels were universally detected after 8 weeks of treatment. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

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.

Prevention trials with statins reduce the relative risk of cardiovascular (CV) events by 10 to 40%. This leaves a ‘residual risk’ of 60–90% for which the CANTOS trial (IL1β inhibition) provided proof of concept that targeting inflammation reduces CV event rates; unfortunately, it did not change rates of type 1 diabetes (T1D) and infections in T1D patients were higher. A major molecular driver of auto-inflammation in both diseases is the CD40/CD154 inflammatory dyad with unique T cell subset, CD3+CD4+CD40+ (TH40 cell) known to be significantly expanded in T1D subjects. Normalization of the aberrant contact dependent interaction of this dyad has shown efficacy in animal models using antibody to CD154, reversing T1D and rate of atherosclerosis, but unfortunately has deleterious side effects when given in humans. We measured TH40 cells in T1D subjects with high calcium scores (a measure of atherosclerotic burden) and those with low calcium scores. Additional to the higher percentage of TH40 cells compared to controls, we have found a statistically significant elevation in in INFg production in the high calcium score cohort, indicating a more pathogenic state of these cells. We designed peptides capable of binding directly to the CD40 receptor, which have been shown to reverse T1D in animals. Peptide was administered to ApoE−/− mice, a model of atherosclerosis. Immunohistochemical staining demonstrated significant reduction in plaque as well as smooth muscle and collagen content. In-vitro analysis of TH40 mouse splenic cells showed modulation in IL2, INFγ and IL17, all potent cytokines of atherosclerosis. This new information relays promise for a more directed diagnostic and therapeutic target for T1D related atherosclerosis.

Abstract Multiple Sclerosis (MS) is a neuro-degenerative autoimmune disease, progressive in nature, that lacks biomarkers for diagnosis or progression. We described a population of CD4+ T cells that express the CD40 receptor, Th40 cells, which proved causal in murine models of autoimmunity. Hypothesis Peripheral blood Th40 numbers reflect MS progression and their control correlates with improvement. Peripheral blood and cerebral spinal fluid from MS patients and disease controls were examined for HLA haplotype and PBMC were characterized by flow cytometry. Random sampling of CIS, RRMS, SPMS and PPMS patients demonstrated diverse HLA haplotypes; DR2 / DQ6 were highly represented but surprisingly DR4, DR3 and DQ2 were prominent. In antigen recall, each HLA haplotype preferred unique myelin-based antigens when presenting to Th40 cells. Percentages of Th40 cells in peripheral blood of CIS and RRMS had a wide range while the range in PPMS and SPMS was tight yet significantly elevated. CSF from RRMS patients had Th40 cell percentages mirroring peripheral blood. RRMS subjects had elevated B cells that surprisingly were the major source of IL-17. Rituximab treated subjects demonstrated a significant but temporal reduction in Th40 cell numbers; FoxP3+ cells were elevated while Th40 inflammatory-cytokine production was reduced. Conclusions The wide range of Th40 cell numbers in CIS and RRMS but universally high numbers in SPMS suggest that Th40 cell number expansions in MS reflect progression. Th40 cells in CSF demonstrate access to the CNS and an important correlation between peripheral blood and CNS. Antigen recall to myelin demonstrates Th40 pathogenicity. Rituximab treatment ablates B cells that then affect pathogenic effector Th40 cells.

The incidence of Type 1 Diabetes (T1D) is on the rise. Quests to understand T1D etiology as well as how to predict onset are ongoing and, in many ways, those goals intertwine. CD4+CD40+ T cell (Th40) percentages are expanded in T1D subjects compared to controls. Antibodies to β-cell antigens are considered risk factors for T1D development but risk of seroconversion to antibody-positivity declines after age 10. While genetic components associate with T1D, not all T1D subjects have those components and not all subjects with those components develop T1D. Therefore, more robust methods for prediction of T1D are needed. Can high Th40 cell percentages be a biomarker in addition to other markers? We assessed Th40 cell percentages, along with other parameters, in blood collected from TrialNet preT1D subjects and stratified them according to their percentages. Th40-low versus Th40-high preT1D subjects demonstrate patterns of CD4 T cell levels that parallel those seen between control and T1D. Cytokine patterns are significantly different between the Th40 groups and a CD4+CD8+ population, described previously in Rheumatoid Arthritis, is more common in Th40-high subjects. Subjects experiencing impaired glucose tolerance have significantly higher Th40 cell percentages than controls. PreT1Ds expressing T1D associated HLA DR4/DR4 or DQ8/DQ8 are more likely found among Th40-high subjects. Interestingly, DR4/DR4 subjects were significantly older compared to all other preT1Ds, suggesting that this haplotype together with a high Th40 cell percentage may represent someone who will onset after age 30, which is reported for 42% of T1D cases. It will be important to determine if preT1D subjects with high Th40 cell percentages will develop overt T1D.

Sustained inflammation is a crucial pathologic component in atherosclerosis and type 2 diabetes (T2D). A common molecular player driving auto-inflammation in both diseases is the CD40/CD154 inflammatory dyad. By normalizing the aberrant contact dependent interaction of the CD40/CD154 dyad, and resultant auto-inflammation, multiple studies have shown both prevention and therapeutic efficacy in both diseases. We designed a series of peptides derived from the CD154 protein sequence that are capable of binding directly to the CD40 receptor to interrupt the inflammatory signal pathways. Two peptides, KGYY6 and KGYY15, were highly effective, initially demonstrating efficacy in type one diabetes mouse models. KGYY6 was chosen for additional testing in the T2D model with atherosclerosis. ApoE-/- mice were utilized due to their ability to develop severe vascular disease and acquire the elements of T2D after 16 weeks of a high fat diet (HFD). KGYY6 was administered by IV tail injection at a dose of 1mg/kg and compared to controls given vehicle only. Aortic en-Face analysis with Sudan IV stain demonstrated significant reduction in plaque in KGYY6 treated mice. Decreases in plaque area and changes in both smooth muscle and collagen measurement were additionally noted by sequential 5um aortic cross sections from the aortic valve leaflets into the ascending aorta. In-vitro analysis of CD3+CD4+CD40+ splenic cells demonstrated a reduction in inflammatory cytokine expression in response to KGYY6 treatment, specifically IL2, IFNγ, and IL17, which are potent cytokines in atherosclerosis. Western blot analysis performed on adipose and muscle tissue demonstrated an increased expression of the glucose transport protein GLUT 4, with corresponding glucose tolerance testing demonstrating increased glucose tolerance and improved insulin sensitivity with lowered plasma insulin level. KGYY6 normalizes the aberrant CD40/CD154 interaction, reducing inflammatory cytokines and regulating glucose, all together resulting in abrogating atherosclerosis.

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.

Summary CD 40 plays a critical role in the pathogenesis of type 1 diabetes (T1D). The mechanism of action, however, is undetermined, probably because CD 40 expression has been grossly underestimated. CD 40 is expressed on numerous cell types that now include T cells and pancreatic β cells. CD 40 + CD 4 + cells [T helper type 40 ( TH 40)] prove highly pathogenic in NOD mice and in translational human T1D studies. We generated BDC 2.5. CD 40 −/− and re‐derived NOD . CD 154 −/− mice to better understand the CD 40 mechanism of action. Fully functional CD 40 expression is required not only for T1D development but also for insulitis. In NOD mice, TH 40 cell expansion in pancreatic lymph nodes occurs before insulitis and demonstrates an activated phenotype compared with conventional CD 4 + cells, apparently regardless of antigen specificity. TH 40 T‐cell receptor ( TCR ) usage demonstrates increases in several V α and V β species, particularly V α 3.2 + that arise early and are sustained throughout disease development. TH 40 cells isolated from diabetic pancreas demonstrate a relatively broad TCR repertoire rather than restricted clonal expansions. The expansion of the V α /V β species associated with diabetes depends upon CD 40 signalling; NOD . CD 154 −/− mice do not expand the same TCR species. Finally, CD 40‐mediated signals significantly increase pro‐inflammatory T h1‐ and T h17‐associated cytokines whereas CD 28 co‐stimulus alternatively promotes regulatory cytokines.

Introduction: Type 1 Diabetes (T1D), a classic autoimmune disease, is an independent risk factor for atherosclerosis with CD40 being a major contributor to both diseases. We described a unique CD4+ T cell population initially in T1D that expresses CD40; over time we termed those cells Th40. Th40 cells are significantly expanded in peripheral blood of T1D patients, as well as being necessary and sufficient to cause T1D in mice. In multiple cell types of human coronary atherosclerotic lesions, CD40 or its ligand CD154 are expressed and blocking CD40/CD154 interaction confers a favorable fibrous cap phenotype, abrogates atherosclerosis, and limits neointimal formation and restenosis. Successful human trials at targeting the ligand CD154 in other diseases, were hampered by embolic events due to normal CD154 action in thrombus stabilization. Results: We have found that Th40 cells are similarly expanded in ApoE deficient mice, a mouse model for atherosclerosis. These cells are found within plaque in large numbers. They not only overexpress interferon gamma (INFγ) but, in turn, INFγ feeds back upon the Th40 cell, causing proliferation. When INFγ is blocked, expansion of Th40 cells cease. We developed a novel small therapeutic peptide (KGYY15) that targets the CD40 molecule directly and Th40 cells specifically. Results of this novel method of direct targeting show a reduction of Th40 cells within target organs, reduction of overall plaque formation (p Conclusions: While hyperglycemia is studied world-wide as one factor increasing risk of atherosclerosis in diabetes, we have found an autoimmune mechanism which can, at minimum, augment the risk in T1D, if not be a primary reason for the aggressive atherosclerosis. These findings are ongoing evidence of the importance of inflammatory pathways in atheroma formation and most importantly, demonstrate a novel, effective method of treatment.

The CD40-CD154 dyad is critically involved in autoimmune diseases including Multiple Sclerosis (MS) and Type 1 Diabetes (T1D). CD4+CD40+ T cells, Th40, are necessary and sufficient to establish T1D in the mouse model. In the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of MS, CD4 T cells are known to play a dominant role in disease development, however, the contribution of Th40 cells has not been determined. Here we reveal that the EAE induction regimen, specifically the Freund’s adjuvant (CFA), causes a significant expansion of Th40 cells, which in turn play a major role in EAE development. While expansion of Th40 cells is not dependent on specific antigen, the precipitation of central nervous system disease symptoms is MOG-antigen dependent, shaping a different TCR repertoire. We show that surface expression of CD40 on T cells is associated with a more rapid and severe disease course. Th40 cells are present in lesions in the brains and spinal cords of EAE mice and their cytokine profile is skewed toward IFNγ. In EAE facilitated by CD4+CD40- T cells, where disease develops with significantly slower kinetics and lesser severity, the cytokine profile is skewed more towards IL-17. Primary Th40 cells can adoptively transfer EAE, without prior in-vitro expansion, requiring only the injection of CFA in the recipient mice. These data demonstrate the importance of T cell CD40 and its relation to differential expression of IFNγ and IL-17 in EAE disease induction.

Multiple Sclerosis is a neurodegenerative disease with autoimmune components. Diagnosis is invasive and time consuming. The etiology is unclear as are clues for disease progression. The autoimmune components have largely been defined in the EAE mouse model. The problem has been defining a reliable biomarker for pathogenic cells. We described a unique T cell subset, Th40 cells that is highly auto-aggressive. We compared Th40 levels in clinically isolated syndrome (CIS), relapsing/remitting (RRMS), secondary progressive (SPMS) and primary progressive (PPMS) patients to non-autoimmune controls. MS subjects have significantly increased percentages of Th40 cells compared to controls. In CIS and RRMS Th40 cell levels cover a broad range while in SPMS and PPMS the range is centralized. Longitudinal studies show that Th40 cell levels remain elevated. Th40 cells produce IFNg, TNFa and IL-17. HLA haplotypes were diverse with only 50% of patients expressing the DR2 (DR15) haplotype or DR3 and DR4. Th40 cells are significantly elevated in MS regardless of HLA haplotype and responded to CNS antigens creating antigen signatures. The ratio of Th40 to or to Tr1 cells is 7 to 1. Th40 cells have a memory phenotype. The chemokine receptors CCR5 and CXCR3 were detected. Samples from patients on disease-modifying-therapy (DMT) demonstrated lymphopenia, but the Th40 percentage was not altered. Th40 cells provide a unique diagnostic tool for MS with significant improvement over current options.

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.

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.

Abstract CD40-CD154 interaction is decisive in directing autoimmune inflammation; therefore, controlling this interaction and hence destructive inflammation is a major goal in a number of autoimmune diseases including Type 1 Diabetes (T1D). Initial attempts to block CD40-CD154 interaction used antibody, random peptides, and small organic molecules, which all were problematic. Therefore an efficacious treatment remains a major goal. Here we reveal a unique targeted peptide, KGYY15, based on the domain of CD154 that binds to the CD40 receptor. Immunoprecipitation utilizing the peptide demonstrates that it binds directly to the CD40 receptor protein which in turn alters the CD40 signaling outcome. KGYY15 prevents T1D in 87% of non-obese diabetic (NOD) mice and histology reveals significant decreases in β-islet infiltration. Interestingly, KGYY15 treatment reverses overt hyperglycemia in 80% of new onset diabetic mice with significant improvement in β-islet insulin granule score. Structure-activity relationship studies demonstrate that the 15-mer peptide is optimal for efficacy and that five specific amino acids are critical in its activity. KGYY15 is well tolerated and functions to control the culprit Th40 effector T cells that are causative in T1D. The peptide sequence is 80% homologous to the human sequence suggesting an important candidate for translational studies.