Modulating CD40 and integrin signaling in the proinflammatory nexus using a 15-amino-acid peptide, KGYY15.

CD40 signaling has long been a target in autoimmunity. Attempts to block signaling between CD40 and CD154 during clinical trials using monoclonal antibodies suffered severe adverse events. Previously, we developed a peptide, KGYY15, that targets CD40 and, in preclinical trials, prevents type 1 diabetes in >90% of cases and reverses new-onset hyperglycemia in 56% of cases. It did so by establishing normal effector T-cell levels rather than ablating the cells and causing immunosuppression. However, the relationship between KGYY15 and other elements of the complex signaling network of CD40 is not clear. Studying interactions between proteins from autoimmune and nonautoimmune mice, we demonstrate interactions between CD40 and integrin CD11a/CD18, which complicates the understanding of the inflammatory nexus and how to prevent autoinflammation. In addition to interacting with CD40, KGYY15 interacts with the integrins CD11a/CD18 and CD11b/CD18. We argue that modulation of CD40-CD154 signaling may be more advantageous than complete inhibition because it may preserve normal immunity to pathogens.

Importance of CD40/CD40 dyad in the course of infection with Trypanosoma cruzi: Impact of its inhibition.

Chagas heart disease (CHD), caused by the protozoan parasite Trypanosoma cruzi, consists of a progressive myocarditis which may lead to congestive heart failure or sudden death. Previous work from our laboratory has demonstrated that the experimental infection of mice with T. cruzi positively modulates the expression of CD40 by myocardial cells, whose ligation potentiates IFN-γ-induced IL-6 production. Herein, we investigate the role of the CD40/CD40L interaction during T. cruzi infection using a CD40-targeted peptide and evaluating parasitological, histopathological and serological parameters. To reproduce acute and chronic phases of theT. cruzi infection, we used two experimental models: Balb/c mice infected with RA strain of T. cruzi (Balb/c-RA) and C3H/HeN mice infected with Sylvio X-10/4 parasites (C3H/HeN-Sylvio), respectively. Balb/c-RA treated with CD40-tageted peptide since day 0 post infection (pi), were unable to control the acute infection dying within 23-26 days pi with marked tissue damage. In contrast, treatment of C3H/HeN-Sylvio treated with CD40-targeted peptide starting on day 30 pi resulted in amelioration of myocardial and skeletal muscle damage. Altogether, our results indicate a dual role of CD40/CD40L dyad in the control of T.cruzi infection as well as the associated pathology, depending on the timing of treatment initiation.

Awareness and acceptability of HIV pre-exposure prophylaxis (PrEP) among students at two historically Black universities (HBCU): a cross-sectional survey.

BACKGROUND: Despite young African American adults (ages 18-24) being among the highest risk groups for HIV infection, little is known about their awareness of HIV pre-exposure prophylaxis (PrEP) - a once daily pill shown to be > 90% effective in preventing HIV. To explore awareness and acceptability of PrEP among college students in this demographic, we conducted a survey of attendees at two large historically Black universities (HBCU) in North Carolina. METHODS: We administered a 14-item questionnaire to students at two HBCUs in North Carolina between February and April 2018. Questions were formatted in a yes/no or multiple choice format. Questionnaire items specifically addressed PrEP awareness and acceptability. Surveys were administered to students at a campus health fair and while transiting the campus student union via iPad. Response to all questions was optional. We fit a logistic regression model to determine association of key demographic determinants with PrEP acceptability and awareness. Statistical analyses were conducted using SAS 9.4 (SAS, Cary, NC). RESULTS: Overall, 210 students participated in the survey, of which 60 completed all survey items as presented. The survey cohort was 75% female, 89% heterosexual and 39% freshmen. The mean age of respondents was 19.8 years (SD: 1.8). Fifty-two percent of survey respondents reported that they were aware of PrEP prior to the time of survey administration. Only 3% of respondents reported that they were on PrEP. The most common sources of information on PrEP were campus health services (24%) and non-social media advertising (15%). Of respondents who were aware of PrEP, 61% reported that they had heard about in the 6 months prior to survey administration, while only 19% say they were aware of it for more than a year. Regarding acceptability of PrEP, 58% of respondents reported that they would take a once a day pill for HIV if they were at risk. Our logistic regression analysis found no statistically significant associations between key demographic factors and PrEP awareness. However, persons who perceived themselves to be at risk for HIV acquisition were more likely to find once daily oral PrEP (relative risk 2.66 (95% CI 1.31-5.42)) as an acceptable prevention strategy than the rest of the survey cohort. CONCLUSIONS: African American HBCU students are becoming aware of PrEP, and generally perceive the intervention as acceptable and worth consideration.

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.

Ocrevus reduces TH40 cells, a biomarker of systemic inflammation, in relapsing multiple sclerosis (RMS) and in progressive multiple sclerosis (PMS).

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.

Canine diabetes mellitus demonstrates multiple markers of chronic inflammation including Th40 cell increases and elevated systemic-immune inflammation index, consistent with autoimmune dysregulation.

Introduction: Canine 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. Methods: We 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. Results: Significant 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. Conclusions: Based 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.

The pathology of bleomycin-induced fibrosis is associated with loss of resident lung mesenchymal stem cells that regulate effector T-cell proliferation.

Tissue-resident mesenchymal stem cells (MSCs) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis, and tumor formation. Here, we define a population of resident lung MSCs (luMSCs) that function to regulate the severity of bleomycin injury via modulation of the T-cell response. Bleomycin-induced loss of these endogenous luMSCs and elicited fibrosis (pulmonary fibrosis), inflammation, and pulmonary arterial hypertension (PAH). Replacement of resident stem cells by administration of isolated luMSCs attenuated the bleomycin-associated pathology and mitigated the development of PAH. In addition, luMSC modulated a decrease in numbers of lymphocytes and granulocytes in bronchoalveolar fluid and demonstrated an inhibition of effector T-cell proliferation in vitro. Global gene expression analysis indicated that the luMSCs are a unique stromal population differing from lung fibroblasts in terms of proinflammatory mediators and profibrotic pathways. Our results demonstrate that luMSCs function to protect lung integrity after injury; however, when endogenous MSCs are lost, this function is compromised illustrating the importance of this novel population during lung injury. The definition of this population in vivo in both murine and human pulmonary tissue facilitates the development of a therapeutic strategy directed at the rescue of endogenous cells to facilitate lung repair during injury.

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.

A CD40 targeting peptide prevents severe symptoms in experimental autoimmune encephalomyelitis.

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, KGYY6, ameliorates EAE when given as pretreatment or at first symptoms. KGYY6 binds Th40 and memory T cells, affecting expression of CD69 and IL-10 in the CD4 T cell compartment, ultimately hampering disease development.

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.

Challenges to Reshape the Future of Type 1 Diabetes Research.

Context: Immunotherapy trials to prevent type 1 diabetes have been unsuccessful for >15 years. Understanding pitfalls and knowledge gaps in the immunology of type 1 diabetes should lead us in new directions that will yield better trial outcomes. A proposal is made for precision medicine trial design in future type 1 diabetes studies. Evidence Acquisition: High-quality peer-reviewed basic science and clinical research trials for type 1 diabetes were used in this Perspective article. Type 1 diabetes publications were reviewed from 2000 to 2018 by using Google Scholar and PubMed reference databases. Evidence Synthesis: Personalized medicine for type 1 diabetes should recognize that each individual has phenotypic and genotypic quirks that distinguish them from other study participants. A uniform protocol for antigen-specific immunotherapy has consistently failed to prevent disease. An alternative approach using molecular tools to personalize the preventive treatment strategy might be a road forward for type 1 diabetes research. Assumptions or lack of knowledge about disease stratification (not all type 1 diabetes is the same disease), individualized antigen-specific T cells, regulatory T-cell populations, and T-cell receptor rearrangement are just a few aspects of immunology that require integration with clinical trial design. Conclusions: The type 1 diabetes research community continues to bring forward novel immunotherapy trials to prevent disease, but this approach is unlikely to succeed until several fundamental aspects of clinical immunology are recognized and addressed. Here, we identify several knowledge gaps that could rectify type 1 diabetes trial design and lead to future success.

Are we aiming to miss in translational autoimmunity treatments?

Autoimmunity treatments, fruitfully pioneered in mouse models, can be disappointing or result in immunosuppression and opportunistic infections in translational trials. Many possible reasons exist, but one major, overlooked reason may be the treatment timing in relation to circadian oscillations of the immune system. Mice and humans both have immunological circadian clocks and experience the same circulatory oscillations of immune cells with regards to their sleep/wake phases, but have opposite sleep/wake phases with regard to the daylight cycle. Therefore, researchers mainly study mice and potential autoimmunity treatments during the murine sleep/rest phase, which is when pro-inflammatory mediators and more adaptive immune cells are prevalent in the circulation. In translational trials, however, treatment administration happens primarily during a patient's wake/activity phase, during the daytime, which is when more local and acute immune responses are active in the circulation. Therefore, we believe that the most opportune window for autoimmunity treatment may be missed in translational trials. Shifting the timing, and adjusting dosing to target only immune cells that are active at that time, may result in higher success with minimized immunosuppression or toxicities.

Overlooked Mechanisms in Type 1 Diabetes Etiology: How Unique Costimulatory Molecules Contribute to Diabetogenesis.

Type 1 Diabetes (T1D) develops when immune cells invade the pancreatic islets resulting in loss of insulin production in beta cells. T cells have been proven to be central players in that process. What is surprising, however, is that classic mechanisms of tolerance cannot explain diabetogenesis; alternate mechanisms must now be considered. T cell receptor (TCR) revision is the process whereby T cells in the periphery alter TCR expression, outside the safety-net of thymic selection pressures. This process results in an expanded T cell repertoire, capable of responding to a universe of pathogens, but limitations are that increased risk for autoimmune disease development occurs. Classic T cell costimulators including the CD28 family have long been thought to be the major drivers for full T cell activation. In actuality, CD28 and its family member counterparts, ICOS and CTLA-4, all drive regulatory responses. Inflammation is driven by CD40, not CD28. CD40 as a costimulus has been largely overlooked. When naïve T cells interact with antigen presenting cell CD154, the major ligand for CD40, is induced. This creates a milieu for T cell (CD40)-T cell (CD154) interaction, leading to inflammation. Finally, defined pathogenic effector cells including TH40 (CD4+CD40+) cells can express FOXP3 but are not Tregs. The cells loose FOXP3 to become pathogenic effector cells. Each of these mechanisms creates novel options to better understand diabetogenesis and create new therapeutic targets for T1D.

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.