Steady-state memory-phenotype conventional CD4+ T cells exacerbating autoimmune neuroinflammation in bystander manner via Bhlhe40/GM-CSF axis

Steady-state memory-phenotype conventional CD4+ T cells exacerbating autoimmune neuroinflammation in bystander manner via Bhlhe40/GM-CSF axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Steady-state memory-phenotype conventional CD4 + T cells exacerbating autoimmune neuroinflammation in bystander manner via Bhlhe40/GM-CSF axis Je-Min Choi, Min-Zi Cho, Hong-Gyun Lee, Jae-Won Yoon, Gil-Ran Kim, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2219047/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 May, 2023 Read the published version in Experimental & Molecular Medicine → Version 1 posted 10 You are reading this latest preprint version Abstract Memory-phenotype (MP) CD4 + T cells are a substantial population of conventional T cells that exist in steady-state mice, and their immunologic functions in autoimmune disease have not yet been studied. In this work, we unveil a unique phenotype of MP CD4 + T cells by analyzing single-cell transcriptomics and T cell receptor (TCR) repertoires. We found that steady-state MP CD4 + T cells exist regardless of germ and food-antigen which are composed of heterogenous effector subpopulations. Distinct subpopulations of MP CD4 + T cells are specifically activated by IL-1 family cytokines and STAT activators, revealing that the cells have TCR-independent bystander effector functions like innate lymphoid cell. Especially, CCR6 high MP CD4 + T cells are major responders to IL-1β and IL-23 without MOG 35 − 55 antigen reactivity, which gives them pathogenic-Th17 characteristics and allows them to contribute to autoimmune encephalomyelitis. We identified Bhlhe40 in CCR6 high MP CD4 + T cells drives the expression of GM-CSF through IL-1β and IL-23 signaling, contributing to CNS pathology in experimental autoimmune encephalomyelitis. Collectively, our findings reveal clearly distinct effector-like heterogeneity of MP CD4 + T cells in steady state and CCR6 high MP CD4 + T cells exacerbate autoimmune neuroinflammation by Bhlhe40/GM-CSF axis in bystander manner synergistically with antigen-specific T cells. Biological sciences/Immunology/Autoimmunity Biological sciences/Immunology/Neuroimmunology Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Lymphocyte activation Memory-phenotype CD4 T cells CCR6 Bystander EAE neuroinflammation Bhlhe40 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The immunological memory of antigen-specific T cells enables faster and more potent responses upon re-exposure to a previously encountered antigen, providing long-lasting immunity 1 . Although a substantial population of memory-phenotype (MP) conventional T cells exists in steady state mice unexposed to foreign antigens, it has been reported that MP T cells are formed before birth in humans and exist in germ-free (GF) and antigen-free (AF) conditioned mice 2 – 6 . After undergoing homeostatic proliferation in a lymphopenic environment, naïve T cells acquire phenotypical, functional, and gene expression–like antigen-specific memory and become MP T cells 7 – 10 . T cell receptor (TCR) and CD28 signaling seem to be required for the conversion of naïve CD4 + T cells into self-derived MP T cells in lympho-sufficient condition. These MP CD4 + T cells express CD5, a marker with high affinity to self-antigen, and resist infection by mediating a Th1-like immune response without antigen stimulation 11 . A previous study confirmed that antigen-non-specific MP CD4 + T cells proliferated more than Lymphocytic choriomeningitis virus (LCMV)-specific T cells in an LCMV infection model. Indeed, treatment with an anti-MHCII antibody did not inhibit the proliferation of MP CD4 + T cells, suggesting that they play a bystander role against infection 12 . Unlike MP CD4 + T cells, MP CD8 + T cells have been well studied for their antigen-specific and bystander functions. MP CD8 + T cells can rapidly produce IFN-γ upon stimulation with IL-12 and IL-18 and without cognate antigen stimulation 13 , 14 . In particular, MP CD8 + T cells are called virtual memory. They can have specific reactions to certain antigens without previous exposure 5,15−18 and increase NKG2D and granzyme B expression upon IL-12, IL-18, and IL-15 stimulation, producing a bystander killing role against infection 16 , 19 , 20 . In addition, MP CD8 + T cells can play an antigen-specific protection role in Listeria monocytogenes , Herpes simplex virus (HSV), and vaccinia infections 5 , 15 , 18 , 21 . Also, MP CD8 + T cells have high affinity to self-antigens, so they can break peripheral tolerance with MP CD4 + T cells and develop autoimmune diabetes 22 , 23 . Overall, previous studies have shown the function of MP CD8 + T cells in disease, but the role of MP CD4 + T cells in autoimmune disease has not yet been studied. Most previous studies have focused on the role of autoantigen-specific T cells in both humans and mice to understand autoimmune diseases and find therapeutic drugs to regulate antigen-specific T cells 24 . Interestingly, antigen-non-specific T cells, including myelin oligodendrocyte glycoprotein (MOG) tetramer–negative CD4 + Th17 cells, also infiltrate the Central nervous system (CNS) in significant proportions and exacerbate experimental autoimmune encephalomyelitis (EAE) pathogenesis 25 – 28 . Bystander-activated T cells and Epstein-Barr virus–specific CD8 + T cells are clonally expanded and correlate with disease pathogenesis in the joints of chronic inflammatory arthritis and Sjogren’s syndrome patients 29 – 31 , raising questions about the role of antigen-nonrelated T cells in autoimmune disease. In this study, we hypothesized that MP conventional CD4 + T cells could be encephalitogenic bystander cells during the development of autoimmune neuroinflammatory disease. First, we examined the heterogeneity of MP CD4 + T cells using single cell RNA-sequencing (scRNA-seq) and TCR sequencing analyses. We found distinct subpopulations of Th1-, Th17-, Treg-, and Tfh-like cells among MP CD4 + T cells. Those cells respond to IL-1 family cytokines and STAT activating cytokines, even without TCR stimulation. We further found that CCR6 high MP CD4 + T cells are the major responders to IL-1β and IL-23, expressing pathogenic signature genes in a bystander manner and thereby contributing to the development of MOG antigen–specific T cell–derived EAE. In this context, we identified Bhlhe40 which regulates the production of GM-CSF in CCR6 high MP CD4 + T cells in bystander manner, exacerbating EAE pathogenesis. Our findings indicate the pathogenic role of antigen independent MP CD4 + T cells along with antigen-specific T cells during autoimmune neuroinflammatory disease. Materials And Methods Mice C57BL/6J mice were purchased from DBL (Chungcheongbuk-do, Korea), and Rag -/- , GM-CSF -/- and 2D2 TCR-transgenic mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). CD45.1 + , Foxp3-GFP mice were provided by Jeehee Youn (Hanyang University). GF and AF mice were purchased from the animal facility of POSTECH Biotech Center (Pohang, Korea). Bhlhe40 -/- and Bhlhe40 GFP mice were provided by Brian T. Edelson (Washington University). Il1r1 -/- mice were provided by Heung-Kyu Lee (KAIST University). The mice were housed and bred in a specific pathogen–free animal facility at Hanyang University under controlled conditions with a constant temperature (21 ± 1°C) and humidity (50 ± 5%) and a 12 h light/dark cycle with regular chow and autoclaved water. All mouse experimental procedures used in this study were approved by the Institutional Animal Care and Use Committee of Hanyang University (2020-0018A,2021-005A, 2021-0158A). MP CD4 + T cell isolation and in vitro activation MP (TCRβ + CD4 + CD1d tetramer - CD25 - CD62L low CD44 high ) CD4 + T cells from the spleens of 8 to 12-week-old mice were isolated using a FACS Aria II and FACS Aria Fusion cell sorter (BD Biosciences, Franklin Lakes, NJ, USA). FACS-sorted MP CD4 + T cells were stimulated with IL-1β (20 ng/mL, R&D Systems, Minneapolis, MN, USA), IL-23 (20 ng/mL, R&D Systems), IL-12 (20 ng/mL, Peprotech, Rocky Hill, NJ, USA), IL-18 (20 ng/mL, R&D Systems), IL-33 (20 ng/mL, R&D Systems), IL-25 (20 ng/mL, R&D Systems), IL-7 (10 ng/mL, Peprotech, Rocky Hill, NJ, USA), or plate-bound anti-CD3/anti-CD28 (2 µg/mL, BD Biosciences) for 5 days at 37°C in an incubator. Active EAE and adoptive transfer EAE In active EAE model, FACS-sorted MP CD4 + T cells (γδTCR - NK1.1 - V β 11 - TCRβ + CD4 + CD1d tetramer - Foxp3 - CD62L low CD44 high , 5 × 10 5 ) from Foxp3-GFP mice were adoptively transferred to 5-week-old female C57BL/6 mice. After transfer, the mice were immunized with 200 µg of MOG 35-55 peptide in complete Freund’s adjuvant (Chondrex, Inc., USA). At 0 and 48 h after immunization, the mice were intraperitoneally treated with 500 ng of pertussis toxin (List Biological Laboratories, Inc., Campbell, CA, USA). The animals were scored daily for clinical disease. In another EAE model by adoptive transfer, naive (CD4 + V β 11 + CD25 - CD62L high CD44 low ) CD45.1 − T cells (1–5 × 10 4 ) from 2D2 TCR-transgenic mice were transferred into Rag -/- mice with or without WT CCR6 high or WT CCR6 low or Bhlhe40 -/- CCR6 high or GM-CSF -/- CCR6 high or GM-CSF -/- CCR6 low or Il1r1 -/- CCR6 high or Il1r1 -/- CCR6 low MP CD4 + T cells (CD45.1 + γδTCR - NK1.1 - V β 11 - TCRβ + CD4 + CD1d tetramer - CD25 - CD62L low CD44 high , 1.0 × 10 5 ). Before transfer, CCR6 high or CCR6 low MP CD4 + T cells were primed in vitro with IL-7 (10 ng/mL) and IL-1β (20 ng/mL) for 5–7 days. After transfer, the mice were immunized with 100 µg of MOG 35 − 55 peptide in complete Freund’s adjuvant (Chondrex, Inc., USA). At 0 and 48 h after immunization, the mice were intraperitoneally treated with 200 ng of pertussis toxin (List Biological Laboratories, Inc., Campbell, CA, USA). The animals were scored daily for clinical disease as follows 32 : partially limp tail, 0.5; completely limp tail, 1; limp tail and waddling gait, 1.5; paralysis of one hind limb, 2; paralysis of one hind limb and partial paralysis of the other hind limb, 2.5; paralysis of both hind limbs, 3; ascending paralysis, 3.5; paralysis of trunk, 4; moribund, 4.5; dead, 5. On day 12 or 13, the mice were sacrificed and perfused with Phosphate buffered saline (PBS). To isolate lymphocytes, the spinal cord and brain were digested with 1 mg/mL of collagenase D (11 088 866 001; Sigma-Aldrich) and DNase I (10 104 159 001; Sigma-Aldrich) and incubated at 80 RPM on a shaker for 35 min. After enzyme digestion, lymphocytes were isolated by Percoll (GE Healthcare, Little Chalfont, UK) density-gradient centrifugation. In vitro MOG reactivity assay (CFSE) For antigen-specific proliferation, 2D2 naïve T cells (CD4 + V β 11 + CD25 - CD62L high CD44 low ) and CCR6 high or CCR6 low MP CD4 + T cells (γδTCR - NK1.1 - V β 11 - TCRβ + CD4 + CD1d tetramer - CD25 - CD62L low CD44 high ) and CD11c + dendritic cells (MHC-II + CD11c + ) (APC) from the spleens of 2D2 TCR-transgenic and C57BL/6 mice were isolated using a FACS Aria Fusion cell sorter. Before co-culture, naïve and MP CD4 + T cells were stained with 1.25 µM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, Carlsbad, CA) for 7 min at room temperature. After incubation, 10% Fetal bovine serum (FBS) was added, and the incubation was continued on ice for 3 min. 2D2 Naïve and CCR6 high or WT CCR6 low MP CD4 + T cells (1 × 10 5 cells/well) were then washed with PBS and co-cultured with CD11c + dendritic cells (5 × 10 4 ) with or without 50 µg/mL of MOG 35-55 peptide for 72 h. Flow cytometry Cell surface staining was performed using the following monoclonal antibodies: anti-CD4 (RM4-5; eBioscience, San Diego, CA, USA, dilution 1:500), anti-CD25 (PC61.5; eBioscience, dilution 1:500), anti-CD44 (IM7; BioLegend, San Diego, CA, USA, dilution 1:500), anti-CD62L (MEL-14; BioLegend, dilution 1:500), anti-CD45 (30-F11, BioLegend, dilution 1:500), anti-CD45.1 (A20; eBioscience, dilution 1:500), anti-TCRβ (H57-597; eBioscience, dilution 1:500), anti-CCR6 (29-2L17; BD, dilution 1:100), anti-CXCR3 (CXCR3-173; BD, dilution 1:100), anti-V α 3.2 (RR3-16; BioLegend, dilution 1:500), anti-V β 11 (RR3-15; BioLegend, dilution 1:500), and PE-conjugated CD1d tetramer (PBS57; NIH, dilution 1:1000). Biotinylated PBS57 loaded and unloaded CD1d monomers were provided by the US National Institutes of Health Tetramer Core facility. For intracellular staining, the cells were stimulated with a cell stimulation cocktail (00-4975-03; eBioscience) for 4 h at 37°C, and then surface markers were stained. After staining of the surface markers, the cells were fixed and permeabilized in Cytofix/Cytoperm (554714; BD Bioscience) or FOXP3/Transcription factor staining buffer set (00-5523-00; eBioscience) for 30 min at 4°C or RT. Intracellular staining was performed using the following monoclonal antibodies: anti-IL-17A (eBio17B7; eBioscience, dilution 1:200), anti-IFN-γ (XMG1.2; eBioscience, dilution 1:400), anti-GM-CSF (MP1-22E9; BD Biosciences, dilution 1:200), anti-Ki67 (SolA15; eBioscience, dilution 1:500), anti-IL-4 (11B11; BioLegend, dilution 1:200), IL-13 (eBio13A; eBioscience, dilution 1:200), anti-TNF-α (MP6-XT22; BioLegend, dilution 1:500), anti-IL-5 (TRFK5; BioLegend, dilution 1:200), anti-IL-1R1 (35F5; BD Biosciences, dilution 1:100), anti-RORγt (Q31-378; BD Biosciences, dilution 1:100), anti-T-bet (4B10; BioLegend, dilution 1:50), anti-GATA3 (L50-823; BD Bioscience, dilution 3 µl per well) and IgG1 (R2-34; BD Biosciences). Stained cells were analyzed by flow cytometry (FACS Canto II, BD Bioscience), and data were analyzed using FlowJo software version 10.8.0 (Tree Star, Ashland, OR, USA). Cytokine (ELISA) Samples were measured using IL-17A ELISA (432501; BioLegend), IL-13 ELISA (88-7137-88; Thermo Fisher), IL-4 ELISA (431104; BioLegend), IL-5 ELISA (431204; BioLegend), IFN-γ ELISA (430801; BioLegend), TNF-α ELISA (430904; BioLegend), and GM-CSF ELISA (432201; BioLegend) kits according to the manufacturers’ instructions. Briefly, microwell plates (Corning Costar; 9018) were coated with capture antibodies overnight at 4°C and blocked with ELISA diluent 1X for 1 h at room temperature. Samples and two-fold serial diluted standards were incubated at room temperature for 2 h, and then detection antibodies and streptavidin-HRP were added. Then, 1X TMB solution and a stop solution (2 N H 2 SO 4 ) was loaded. The optical density was analyzed at 450 nm. Between all procedures, plates were washed at least 3 times with wash buffer (1X PBS, 0.05% Tween-20). Statistical analysis All data were analyzed in non-parametric analyses using the Mann-Whitney test or two-way ANOVA of variance in Prism version 8.0 (GraphPad Software, San Diego, CA). Data are presented as the mean ± S.D. or mean ± S.E.M. For all data, significance was defined as p ≤ 0.05. Sample size and statistical information is provided in each figure legend. Results Single-cell RNA-sequencing identifies clearly distinct effector-like subpopulations in steady-state MP conventional CD4 + T cells. Steady-state, unprimed specific-pathogen-free (SPF)-housed mice have a significant proportion of MP CD4 + T cells (TCRβ + CD1d tetramer − CD8 − CD4 + CD25-CD44 high CD62L low ) in their spleen, thymus, inguinal lymph nodes (iLNs), mesenteric lymph nodes (mLNs), Peyer’s patches (PPs), and lung tissues (Fig. 1 a and Fig. S1a, Supporting information). The proportion and number of CD4 + T cells increased with age in all tissues (Fig. S1, Supporting information). To identify and focus on the characteristics of MP CD4 + T cells, we used fluorescence-activated cell sorting (FACS) to sort MP CD4 + T cells from the spleens of 10-week-old C57BL/6 mice and performed scRNA-seq with paired V(D)J sequencing of the T cell receptor (Fig. S2a-d, Supporting information). Also, we generated a pipeline to filter out PLZF and TCR V α 14-J α 18 (TRAV11-TRAJ18) expressing cells, a well-known key transcription factor for the development of NKT and MAIT cells 33 – 35 . An unbiased clustering analysis revealed clearly distinct effector T cell–like subpopulations (Fig. 1 b) and differentially expressed genes (DEGs) defining each cluster (Fig. 1 c and d). A Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of each cluster indicated that clusters 3 and 5 were Th1- and Th17-like populations, respectively, with significance in “chemokine-mediated signaling pathway” and “cellular response to interleukin-1” (Fig. S3, Supporting information). In addition, lineage-specific transcription factors and chemokine receptors were localized in each cluster (Fig. 1 e and Fig. S4, Supporting information), suggesting that MP CD4 + T cells are composed of Th1-, Th17-, Tfh-, and Treg-like subpopulations. To examine whether the subpopulations of steady-state MP CD4 + T cells depended on germ or food antigens, we analyzed the proportion of CD44 high CD62L low MP CD4 + T cells in the tissues of SPF-, GF-, and AF-housed mice using flow cytometry. The proportions of total MP CD4 + T cells were almost identical in the spleen and other tissues except the mLN and PP (Fig. 1 f, Fig. S5a and S5b, Supporting information), indicating that the generation of splenic MP CD4 + T cells is not affected by the microbiome or food antigen stimulation. Our scRNA-seq analysis further confirmed that the SPF- and GF-housed mice had identical proportions of distinct effector-like subpopulations of splenic MP CD4 + T cells (Fig. 1 g and h), but the proportion of Th17-like MP cells in the mLNs of GF-housed mice was much lower than that in the mLNs of SPF-housed mice (Fig. 1 i and j). In support, the proportion of CCR6 high or RORγt + MP CD4 + T cells in the mLNs of GF-housed mice was lower than that in SPF-housed mice, suggesting that the generation of gut MP CD4 + T cells is specifically dependent on germs (Fig. S6, Supporting information). The TCR clonal diversity of MP CD4 + T cells in GF-housed mice, especially the Th17-like population in the mLNs, was reduced compared with SPF-housed mice, though comparable diversity was observed in the spleen (Fig. 1 k and l). There seemed no significant clonal expansion observed in MP subpopulations. Together, these data indicate that steady-state splenic MP CD4 + T cells contain heterogeneous subpopulations of Th1-, Th17-, Tfh-, and Treg-like cells that express effector molecules and exist independently of the gut microbiome and food antigens. Differential innate-like effector functions of MP CD4 + T cells after exposure to IL-1 family and STAT activating cytokines. To determine the effector functions of MP CD4 + T cells to respond to various cytokines, we performed scRNA-seq of MP CD4 + T cells cultured in conditioned medium with IL-12/IL-18, IL-25/IL-33, and IL-1β/IL-23 cytokines (Fig. 2 a and Fig. S2e, Supporting information). Additionally, IL-7 was added to the culture medium for T-cell survival and maintenance 36 . The merged Uniform Manifold Approximation and Projection (UMAP) plot shows phenotypic characteristics of Th1-, Th2-, Th17-, and Treg-like subpopulations (Fig. 2 b and c), which express lineage specific genes (Fig. 2 d). Each cytokine set induced distinct subpopulations localized exclusively in the plot (Fig. 2 e). We further confirmed the expression of selected gene sets related to the Th1, Th2, and Th17 lineages in each bystander-activated condition, which shows that type 1, 2, and 3 cytokines can upregulate lineage-specific genes in MP CD4 + T cells (Fig. 2 f and g). A single-cell regulatory network inference and clustering (SCENIC) analysis in each cytokine condition revealed a significant level of common or specific transcription factor activity (Fig. 2 h). In addition, an Ingenuity pathway analysis (IPA) showed the predicted transcriptional regulators in each condition and speculated that the target transcription factors included Bhlhe40, which can be a potent regulator of bystander activation in MP CD4 + T cells (Fig. 2 i). To evaluate the effector functions of MP CD4 + T cells, we treated MP CD4 + T cells with those cytokines and/or anti-CD3/anti-CD28 (Fig. 2 j and k). Consistently, the MP CD4 + T cells responded to IL-12/IL-18, IL-25/IL-33, and IL-1β/IL-23 cytokines without TCR stimulation, and T-bet + IFN-γ + , GATA3 + IL-13 + , and RORγt + IL-17 + cells increased compared to TCR-stimulated condition (Fig. 2 j). However, TNF-α was produced only in the presence of TCR stimulation and IFN-γ in the culture supernatant from IL-12/IL-18-conditioned MP CD4 + T cells, and IL-4 was also significantly detected with TCR stimulation but not in the bystander condition with IL-25/IL-33. Similarly, Granulocyte-macrophage colony-stimulating factor (GM-CSF) was secreted more efficiently with IL-1β/IL-23 and TCR stimulation than with the cytokines alone (Fig. 2 k). Collectively, these results indicate that steady state MP CD4 + T cells have functional heterogeneity of innate-like responses to various sets of IL-1 family and STAT activating cytokines even in the absence of T cell receptor stimulation and suggest possible potent transcriptional regulators that control MP CD4 + T cell effector functions. Potential responder MP CD4 + T cells express distinct chemokine receptors upon IL-12/IL-18 and IL-1β/IL-23 stimulation. To determine which MP CD4 + T cells are potential responders to IL-1 family and STAT activating cytokines, we analyzed the subpopulation of expanding or responding clusters and performed trajectory analyses. Control MP CD4 + T cells cultured with IL-7 produced a significantly separate population of CXCR3 high cells. In IL-18/IL-12-conditioned MP CD4 + T cells the CXCR3 high cell population was reduced, and the IFN-γ- or IL-13 high expressing Th1 and proliferating Th1 populations were greatly increased (Fig. 3 a and b). Th1 signature genes, Tbx21, Ifng, Cxcr3, Il2rb1, Il18r1 , and CCR5 were highly expressed by IL-18/IL-12-conditioned MP CD4 + T cells (Fig. 3 c). GO/KEGG analysis indicated that the related gene sets in those cells had increased including “Cellular response to interferon-gamma”, “Cytokine cytokine receptor interaction”, “Alzheimer’s disease”, and “Parkinson’s disease” (Fig. 3 d). The IPA returned terminologies related to cytokines and inflammation such as “JAK/STAT signaling” and “neuroinflammation signaling pathway” and related to cytotoxic response including “Granzyme B signaling” (Fig. 3 e). In a pseudo-time trajectory analysis, the MP CD4 + T cells formed a continuous progression that started in CXCR3 high cells and gradually progressed toward Fate 1, which expressed Ifng, Stat5a, Bhlhe40, Batf3, Irf4 and Irf8 (Fig. 3 f and g). Similarly, a CCR6 high cluster was present in IL-7-conditioned MP CD4 + T cells, and its Th17-like cluster was specifically increased by IL-1β and IL-23 (Fig. 3 h and i). Th17 signature genes, Rorc, Ccr6, Il17a Il1r1, Il23r , and proliferating marker Mki67 were expressed by those cells (Fig. 3 j). GO/KEGG analysis predicted that the related gene sets in IL-1β /IL-23-cultured MP CD4 + T cells had increased “Alzheimer’s disease,” “Parkinson’s disease,” and “Huntington’s disease” which are neurological diseases and increased “cytokine signaling pathway” (Fig. 3 k). The IPA more clearly explained the related pathways, “STAT3 pathway,” “leukocyte extravasation signaling,” “neuroinflammation signaling pathway,” “chemokine signaling,” and “Th17 activation pathway” (Fig. 3 l). In the trajectory analysis, CCR6 high cells seemed to be the starting point, and then the cells gradually differentiated toward Fate 2 (Fig. 3 m), which expresses pathogenic Th17-related genes such as Rorc, Il17a, Csf2, Il22, Bhlhe40, Rora and Cebpb with increased activities (Fig. 3 n) and expression level (Fig. 3 o) of related transcriptomes. These results collectively reveal that Th1-like and Th17-like MP CD4 + T cells expressing different chemokine receptors respond specifically to IL-12/IL-18 and IL-1β/IL-23 cytokines with the effector functions. Steady state CCR6 high memory phenotype CD4 + T cells are bystander-activated by IL-1β and IL-23 to become pathogenic Th17-like cells. Since scRNA-seq predicted that CCR6 high cells were the major cells responding to IL-1β and IL-23, we sorted splenic CCR6 high and CCR6 low MP CD4 + T cells (Fig. 4 a and b) and then determined their proportions in steady-state SPF- and GF-housed mice (Fig. 4 c). We found that splenic CCR6 high MP CD4 + T cells were independent of the gut. CCR6 high MP CD4 + T cells expressed RORγt more highly than CCR6 low cells and had comparable expression of T-bet (Fig. 4 d). Steady-state CCR6 high MP CD4 + T cells, but not CCR6 low , expressed IL-17A (Fig. 4 e), suggesting that CCR6 high MP CD4 + T cells are Th17-like cells. Further stimulation by IL-1β and IL-23 induced IL-17A and GM-CSF expression (Fig. 4 f and g), which confirms that CCR6 high MP CD4 + T cells produce pathogenic cytokines in the bystander manner. The amount of cytokine secreted, as determined by ELISA, consistently showed that CCR6 high MP CD4 + T cells, but not CCR6 low , significantly produced IL-17A, GM-CSF, and IFN-γ and that IL-1β and IL-23 had important synergy for pathogenicity (Fig. 4 h). In addition, IL-1β greatly expanded RORγt expression in CCR6 high MP CD4 + T cells but not CCR6 low cells, whereas IL-23 somewhat inhibited the proportion of Ki67-expressing cells, suggesting that IL-1β is important for the proliferation of CCR6 high MP CD4 + T cells (Fig. 4 i). To further confirm the functions of IL-1β and IL-23 in MP CD4 + T cells, we performed bulk-RNA seq with bystander-activated MP CD4 + T cells exposed to IL-1β and IL-23. In the heatmap DEG analysis, IL-1β increased the expression of proliferation-related gene such as Mki67 and cdk2 , whereas IL-23 alone did not have any significant effects on gene expression (Fig. 4 j). IL-23 and IL-1β together significantly induced the expression of pathogenic genes such as Bhlhe40, Il1r1, Csf2 , Ifng, Il22 and Il17a . In support, Gene set enrichment analysis (GSEA) pathway enrichment plot of IL-1β vs. IL-1β and IL-23 demonstrated that the enrichment score for cell proliferation was higher with IL-1β, and the score of the pathogenic Th17 signature with IL-1β and IL-23 was higher than in the control group (Fig. 4 k). Through these results, we understand that steady-state CCR6 high MP CD4 + T cells, which show Th17-like characteristics, are the major bystander-activated cells responding to IL-1β and IL-23, which potentiate the cells’ pathogenic character. CCR6 high MP CD4 + T cells exacerbate autoimmune neuroinflammation in bystander manner. To reveal the importance of splenic MP CD4 + T cells during an autoimmune disease, we first induced active EAE in 5-week-old mice, who have a lower proportion of MP CD4 + T cells than 10-week-old mice. In this mouse model, we compared the disease severity of control mice with that of those who received an additional adoptive transfer of Treg-deleted (Foxp3 − ) MP CD4 + T cells from 10-week-old Foxp3-GFP mice. EAE was rapidly induced and progressed by transferring additional MP CD4 + T cells from 10-week-old mice, suggesting that MP CD4 + T cells could be an important contributor to MOG 35 − 55 -induced EAE pathogenesis (Fig. S7a, Supporting information). In support, Rag −/− mice that adoptively transferred MOG-TCR transgenic (2D2) naïve CD45.1 − V β 11 + CD4 + T cells with Treg-deleted MP CD4 + T cells (CD45.1 + CD4 + ) showed a more severe phenotype of EAE than the mice that received only 2D2 T cells (Fig. S7b, Supporting information), suggesting that MP CD4 + T cells contribute to the pathogenesis of EAE. Based on our previous results, we hypothesized that CCR6 high MP CD4 + T cells are the major pathogenic subpopulation contributing to EAE disease progression. To test that hypothesis, we transferred CCR6 high and CCR6 low MP CD4 + T cells (CD45.1 + CD4 + , gating strategy and purity is shown in Fig. S8, Supporting information) and 2D2 naïve CD4 + T cells into Rag −/− mice. The additional transfer of CCR6 high MP CD4 + T cells exacerbated EAE development compared with 2D2 transfer alone or the transfer of CCR6 low MP CD4 + T cells (Fig. 5 a). Interestingly, the number of transferred MP CD4 + T cells that appeared in the spinal cord and brain tissue did not differ between conditions (Fig. 5 b). However, CCR6 high MP CD4 + T cells produced significantly more cytokines, particularly IL-17A and GM-CSF, in the spinal cord and brain tissue than CCR6 low MP CD4 + T cells (Fig. 5 c and d). Therefore, CCR6 high MP CD4 + T cells, along with antigen-specific T cells, contribute to the pathogenicity of autoimmune neuroinflammation by expressing pathogenic cytokines such as IL-17A and GM-CSF. To clarify the antigen-independent activation of CCR6 high MP CD4 + T cells in an EAE mouse model, we detected 2D2 TCR (V α 3.2 + and V β 11 + ), which are predominantly expressed in 2D2 naïve CD4 + T cells. Indeed, CCR6 high MP CD4 + T cells barely expressed V α 3.2 + and V β 11 + (Fig. 5 e and f). In support, we confirmed that steady-state CCR6 high and CCR6 low MP CD4 + T cells did not respond to the MOG 35 − 55 antigen (Fig. 5 g). Collectively, these results suggest that CCR6 high MP CD4 + T cells infiltrate in CNS tissue and exacerbate autoimmune neuroinflammation in a bystander manner. Innate-like effector functions of CCR6 high MP CD4 + T cells are conferred by Bhlhe40/GM-CSF axis. Among the candidate genes involved in bystander activation of CCR6 high MP CD4 + T cells, we identified that Bhlhe40/GM-CSF axis could potentially give rise to the pathogenic function of CCR6 high MP CD4 + T cells induced by IL-1β and IL-23 without TCR stimulation (Fig. 6 a). By using Bhlhe40 GFP mice, we found that CCR6 high MP CD4 + T cells activated by IL-1β and IL-23 showed significantly increased level of Bhlhe40 (Fig. 6 b). Interestingly, Bhlhe40 GFP positive T cells majorly produced effector cytokines including IL-17A and GM-CSF compared to Bhlhe40 GFP negative T cells (Fig. 6 c). In support, Bhlhe40 −/− CCR6 high MP CD4 + T cells showed markedly reduced IL-17A and GM-CSF production compared to WT (Fig. 6 d and e), suggesting that Bhlhe40 is an important transcriptional regulator for the pathogenic functions of bystander CCR6 high MP CD4 + T cells. To confirm the in vivo relevance, we transferred WT CCR6 high or Bhlhe40 −/− CCR6 high MP CD4 + T cells along with 2D2 naïve CD4 + T cells into Rag −/− mice. Bhlhe40 −/− CCR6 high MP CD4 + T cells showed abrogated functions for exacerbating EAE disease compared by WT CCR6 high MP CD4 + T cells (Fig. 6 f). Interestingly, the number of Bhlhe40 −/− CCR6 high MP CD4 + T cells expressing IL-17A and GM-CSF in the spinal cord and brain tissue was significantly reduced compared to WT CCR6 high MP CD4 + T cells (Fig. 6 g and h). In addition, transfer of GM-CSF-deficient CCR6 high MP CD4 + T cells showed abrogated function of contributing EAE pathogenesis (Fig. 6 i), suggesting that disease aggravation by bystander CCR6 high MP CD4 + T cells is committed by IL-1β and IL-23 via Bhlhe40/GM-CSF axis. Collectively, these results indicate that Bhlhe40 confers innate-like pathogenic functions of CCR6 high MP CD4 + T cells with GM-CSF production. Discussion In this study, we intensively validated the characteristics of steady state MP CD4 + T cells using scRNA-seq to unveil their innate-like effector functions during autoimmune disease. We found clearly distinct effector-like subpopulations in steady-state splenic MP CD4 + T cells that are independent of the microbiome and food antigens. MP CD4 + T cells can be bystander-activated by the different sets of IL-1 family and STAT activating cytokines. Specific chemokine receptor–expressing cells are defined as potential responder cells to each set of cytokines, and we focused on CCR6 high MP CD4 + T cells to further validate their functions in responding to IL-1β/IL-23. We demonstrated that steady state CCR6 high MP CD4 + T cells have innate-like effector functions that exacerbate EAE disease progression in a bystander manner, along with antigen-specific T cells. We suggest Bhlhe40 as a pivotal transcriptional regulator that governs GM-CSF production in bystander-activated CCR6 high MP CD4 + T cells, exacerbating EAE development. Overall, our results reveal the innate lymphoid cell-like immunological functions of steady-state MP CD4 + T cells in autoimmune disease. Innate T cells such as natural killer T (NKT), mucosal-associated invariant T (MAIT), and γδ T cells have limited TCR gene usage compared to conventional T cells, which recognize complexes of non-peptide antigens such as glycolipids, phospho-antigens, and vitamin B metabolites, respectively 37 – 39 . These innate T cells are derived from the thymus, which can evoke robust cytokine production. Previously, innate lymphocytes, such as NKT17, γδT17 cell, and ILC3 subsets have been defined to commonly express IL-17 and RORγt 40 – 42 . Furthermore, a recent study reported a novel subset of αβ-γδ co-expressing T cells which recognize MHC-restricted peptide antigens and produce effector cytokine IL-17A, GM-CSF, and IFN-γ by IL-1β and IL-23 stimulation 43 . Also, another group said that natural Th17 cells and γδ T cells expanded after the candidiasis infection model in oral cavity 44 . In this context, we carefully eliminated the possibility of contamination of innate T cells by sorting conventional MP CD4 + T cells using NKT (CD1d tetramer), γδ T, αβ-γδ T, ILC, and MAIT (TCRβ + CD8 − CD4 + CD25 − CD44 high CD62L low ) exclusion gates. We confirmed that PLZF/CD1d tetramer negative steady-state MP CD4 + T cells still exists as a heterogenous population containing CCR6 high RORγt + IL-17A + cells, which is majorly bystander-activated by IL-1β and IL-23. Therefore, collectively, we provide compelling evidence that conventional CD4 + T cells distinguished from previously known innate T cells exist, which have an innate-like features and contribute to autoimmune neuroinflammation. As our study revealed the heterogeneous characteristics of MP CD4 + T cells by single cell transcriptomic analysis, recent studies revealed the potential heterogeneity of murine MP CD4 + T cells 45 – 47 . CXCR3 + T-bet + Th1-like MP CD4 T cells spontaneously generated from naïve CD4 + T cells in steady state showed innate-like effector functions against T. gondii infection 11 . These cells require DC1-derived tonic IL-12 signal for optimal differentiation of T-bet high MP T cells 46 . Similarly, we confirmed CXCR3 high MP CD4 + T cells are expressing T-bet and they are major responders to IL-12 and IL-18 cytokine stimulation. CXCR3 high MP CD4 + T cells, in high correlation with CCR5 expression, can produce IFN-γ and T-bet in response to IL-12 and IL-18, suggesting a innate-like function of CXCR3 high MP CD4 + T cells. In our previous study, we demonstrated that IL-1β and IL-23 which are derived from innate immune cells 27,48−50 can synergistically potentiate the pathogenicity of memory CD4 + T cells in vitro 26 and that non-myelin-specific CD4 + T cells can infiltrate the CNS with MOG antigen–specific T cells, which significantly contribute to EAE disease progression 25 , 26 , 51 , 52 . In rheumatoid arthritis patients, T cells that infiltrate the synovial fluid mainly express the CD45RO + memory marker and specifically respond to epitopes of Epstein-Barr virus and cytomegalovirus 29 , 30 , 53 , 54 . In type 1 diabetes, infection with rotavirus or coxsackie virus is reported to be involved in accelerated diabetes onset through Toll-like receptor (TLR) signaling without pancreatic infection 55 , 56 , and influenza A virus is linked to diabetes in human patients 57 , 58 . Collectively, those studies suggest that antigen-non-related CD4 + T cells can contribute to disease onset or progression with antigen-specific T cells in various autoimmune diseases. We have identified here that CCR6 high MP CD4 + T cells are the major subpopulation of MP CD4 + T cells that respond to IL-1β and IL-23 by expanding and inducing pathogenic Th17 characteristics. These IL-1β and IL-23 signaling In an adoptive transfer model of EAE, CCR6 high MP CD4 + T cells transferred with MOG-specific T cells induced more severe EAE than CCR6 low cells, with increased production of IL-17 and GM-CSF in the CNS. We further confirmed that MP CD4 + T cells do not respond to MOG 33 − 55 antigen, indicating the innate-like functions of CCR6 high MP CD4 + T cells in autoimmune neuroinflammation. In addition, we confirmed IL-1R1 is required for pathogenic contribution of CCR6 high MP CD4 + T cells in EAE disease (Fig. S9, Supporting information) suggesting IL-1 signal to CCR6 high MP CD4 + T cells could trigger their bystander effector functions in vivo. Further studies should be done to reveal the distinct mechanism between antigen-specific T cells and bystander-activated T cells in autoimmune disease pathogenesis. Analyzing the single cell transcriptomics of IL-1β/IL-23 responding MP CD4 + T cells, we identified that Bhlhe40 could be a potential transcriptional regulator inducing GM-CSF in CCR6 high MP CD4 + T cells. Bhlhe40 has been reported to play pivotal roles in T cells. Bhlhe40-deficient naïve CD4 + T cells show limited response to TCR stimulation 59 . In addition, Bhlhe40 seem to be required for Th1 and Th17 effector cytokine production including IL-17A, GM-CSF and IFN-γ in the context of autoimmune disease, GVHD, and Toxoplasma gondii infection model 27,60−62 . Expression of Bhlhe40 correlate with mouse Csf2 locus, which encodes GM-CSF, 61 , 63 and also positive correlation with GM-CSF expression was reported in human PBMC 64 . We demonstrate that steady-state CCR6 high MP CD4 + T cells are expressing RORγt and IL-1 receptor that they up-regulate Bhlhe40 in response to IL-1β and IL-23. In the absence of TCR engagement, they can produce IL-17 and GM-CSF that are importantly contribute to the pathogenesis of EAE. In support, a previous study reported the majority of Bhlhe40-expressing pathogenic T cells in active EAE are non-MOG-specific 27 , 61 . As Bhlhe40 −/− CCR6 high MP CD4 + T cells showed reduced GM-CSF and GM-CSF −/− CCR6 high MP CD4 + T cells could not exacerbate EAE, Bhlhe40/GM-CSF axis seem to be an important mechanism of bystander-activated MP CD4 + T cells during EAE. Therefore, Bhlhe40 can be a pivotal transcriptional regulator for both antigen-specific and bystander MP CD4 + T cells in the context of CNS inflammation and targeting of Bhlhe40 in CD4 + T cells may serve as a potential novel treatment strategy to control autoimmune diseases. Self-antigen-specific T cells are fundamentally important in triggering autoimmune inflammation; however, antigen-non-related naturally arising steady state MP CD4 + T cells are also importantly contributing to pathogenic inflammation in a bystander manner. Collectively, our studies of the role that MP CD4 + T cells play in neuroinflammatory disease shed light on the bystander function of adaptive immune cells to understand disease pathogenesis and reveal a novel drug development strategy to modulate autoimmune diseases. Declarations Acknowledgements We thank Dr. Dongsoo Kyeong and Dr. Younhee Shin (Insilicogen Inc.) for supporting bioinformatic analysis of the scRNA-seq data. We thank Mr. Yeon-Ho Kim and Ms. In Young Song for technical support in the FACS sorting conducted at Hanyang LINC Analytical Equipment Center (Seoul) and the NIH tetramer core facility (Emory University) for providing mouse CD1d PBS-57 (Biotinylated Monomer). We thank Prof. Jeehee Youn (Hanyang University) for kindly providing CD45.1 + , Foxp3-GFP mice and Prof. Kwang Soon Kim (POSTECH) for helping us purchase germ-free and antigen-free mice. Funding This research was supported by the Basic Science Research Program (NRF-2019R1A2C3006155) of the National Research Foundation funded by the Korean government. A uthor contributions M.-Z.C., H.-G.L., and J.-M.C. conceptualized and designed this study. M.-Z.C., H.-G.L. performed and analyzed most of the experiments including bioinformatic analysis. Y.J.L. supported conceptualization for bioinformatic analyses. J.-W.Y., G.-R.K. and J.-H.K. supported experiments. R.T. and B.T.E. provided Bhlhe40 -/- and Bhlhe40 GFP . 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2219047","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":150714666,"identity":"2c733570-550e-4db8-985e-e506229e0cab","order_by":0,"name":"Je-Min Choi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYBACxgYGhgMgxMbegyx+gBgtPGeI1IJQIZFDpBbm9u7EwwU1d+T4JN8e/FzYZpcn78D88APDmXu4HdZzdsPhGceeGbNJ5yVLz2xLLjY8wGYswXCjGLeWGbkbDvM2HE5sk84xkOZtY07c2MBgxsDwIQG3lvlvoVokzxj/5m2rB2ph/4ZfywxeqBYJHjOgLYcT5zPwAG25gUdLD9BhPMcOG7Px5JhZ85w7nriBmadYIuEMbi2G7Wc3f+apOSwn337G+DZPWXXi/Pb2jR8+HMOjpQHFTjYGBoPDQAZuDQwM8qjcP0CRBqwKR8EoGAWjYAQDAEScWc8We0b5AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9482-710X","institution":"Hanyang University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Je-Min","middleName":"","lastName":"Choi","suffix":""},{"id":150714667,"identity":"389f4c24-55da-4a44-ac71-7446af6fc5f0","order_by":1,"name":"Min-Zi Cho","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min-Zi","middleName":"","lastName":"Cho","suffix":""},{"id":150714668,"identity":"59af9ab4-d640-4754-8dc5-7c316569e890","order_by":2,"name":"Hong-Gyun Lee","email":"","orcid":"https://orcid.org/0000-0001-5960-5504","institution":"Hanyang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hong-Gyun","middleName":"","lastName":"Lee","suffix":""},{"id":150714669,"identity":"f5f37bfe-e142-40c9-9eab-c59df4609399","order_by":3,"name":"Jae-Won Yoon","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jae-Won","middleName":"","lastName":"Yoon","suffix":""},{"id":150714670,"identity":"f2bbeaa2-734e-4551-a57e-c0a914acda15","order_by":4,"name":"Gil-Ran Kim","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gil-Ran","middleName":"","lastName":"Kim","suffix":""},{"id":150714671,"identity":"f919f578-9bc9-46fc-8247-09d944eff7f0","order_by":5,"name":"Ja-Hyun Koo","email":"","orcid":"","institution":"Hanyang University, Department of Life Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ja-Hyun","middleName":"","lastName":"Koo","suffix":""},{"id":150714672,"identity":"d80ed34e-9233-41fa-a449-6bc2afbc6eb0","order_by":6,"name":"Reshma Taneja","email":"","orcid":"https://orcid.org/0000-0001-6214-6177","institution":"University of Singapore","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Reshma","middleName":"","lastName":"Taneja","suffix":""},{"id":150714673,"identity":"9a33860f-d62d-4c32-9bae-d0f7458003df","order_by":7,"name":"Brian Edelson","email":"","orcid":"","institution":"Washington University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Brian","middleName":"","lastName":"Edelson","suffix":""},{"id":150714674,"identity":"6a82f1ef-c270-41bb-a012-8c8a7d59d901","order_by":8,"name":"You Jeong Lee","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"You","middleName":"Jeong","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2022-10-30 16:10:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2219047/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2219047/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-023-00995-1","type":"published","date":"2023-05-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":28973299,"identity":"a884240c-5768-45e8-91e0-dcf6df73d443","added_by":"auto","created_at":"2022-11-11 22:25:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2002772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle cell RNA-sequencing identifies clearly distinct effector-like subpopulations in steady-state MP CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells. \u003c/strong\u003e(a) Representative flow cytometry plots showing the population of memory-phenotype (MP) CD4\u003csup\u003e+\u003c/sup\u003e T cells (TCRβ\u003csup\u003e+\u003c/sup\u003eCD1d tetramer\u003csup\u003e-\u003c/sup\u003eCD8\u003csup\u003e-\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003e) in murine spleens, thymuses, inguinal lymph nodes (iLNs), lungs, mesenteric lymph nodes (mLNs), and Peyer’s patches (PP). (b) tSNE plot of splenic MP CD4\u003csup\u003e+\u003c/sup\u003e T cells isolated from 10-week-old specific-pathogen-free (SPF) mice. (c) Heatmap of differentially expressed transcripts in splenic MP CD4\u003csup\u003e+\u003c/sup\u003e T cells. (d) Expression of selected genes used to define MP CD4\u003csup\u003e+\u003c/sup\u003e T cell clusters. (e) Differential expression of transcription factors and chemokine receptors from splenic MP CD4\u003csup\u003e+ \u003c/sup\u003eT cell clusters. (f) Flow cytometry plots of splenic MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (TCRβ\u003csup\u003e+\u003c/sup\u003eCD1d tetramer\u003csup\u003e-\u003c/sup\u003eCD8\u003csup\u003e-\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003e) from SPF-, GF-, and AF-mice. (g) tSNE plot and (h) proportion of integrated splenic MP CD4\u003csup\u003e+\u003c/sup\u003e T cells isolated from 10-week-old SPF- and GF-mice. (i) tSNE plot and (j) proportion of integrated mLN MP CD4\u003csup\u003e+\u003c/sup\u003e T cells isolated from SPF- and GF-mice. (k) Pie chart representing the clonal size distribution of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells. (l) Diversity of the TCR repertoire in MP CD4\u003csup\u003e+\u003c/sup\u003e T cell subsets from SPF- and GF-mice.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2219047/v1/aa3cb5bb50d936d8ec3c2e37.png"},{"id":28973298,"identity":"8e970256-fa40-4c22-a2dc-4daea1994032","added_by":"auto","created_at":"2022-11-11 22:25:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1238771,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential innate-like effector functions of MP CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells after exposure to IL-1 family and STAT activating cytokines. \u003c/strong\u003e(a) Overview of experimental design. (b) UMAP representation of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells stimulated with IL-12 and/or IL-18 and IL-33 and/or IL-25 and IL-1β and/or IL-23 in the presence of IL-7. (c) Heatmap and (d) violin plots of differentially expressed transcripts in cluster. (e) Individual cytokines conditions visualized with UMAP. (f) MA plots of differentially expressed genes comparing IL-7 versus IL-12/18 or IL-33/25 or IL-1β/23. (g) Heatmap representing gene expression of Resting-, Tfh-, Th1-, Th2-, Th17- and Treg-related gene signatures in each cytokine condition. (h) Heatmap representing transcription activity. (i) Venn diagram of transcriptional regulators predicted by the IPA. Numbers indicate the number of gene in each gate. (j) MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (TCRβ\u003csup\u003e+\u003c/sup\u003eCD1d tetramer\u003csup\u003e-\u003c/sup\u003eCD8\u003csup\u003e-\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003e) were cultured for 5 days with IL-12 and/or IL-18 and IL-33 and/or IL-25 and IL-1β and/or IL-23 in the presence of IL-7 or anti-CD3/anti-CD28. Representative flow cytometry plots showing the expression of effector lineage markers in each cytokine condition. (k) IFN-γ, TNF-α, IL-4, IL-5, IL-13, IL-17A, and GM-CSF were measured by ELISA (n=5, 5 independent experiments). Data are presented as the mean ± S.D. P values were calculated using Mann-Whitney U-test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2219047/v1/af05c9efbf38b2547e10a528.png"},{"id":28974029,"identity":"70428b4f-aa87-402f-b7e7-3913669ed8c6","added_by":"auto","created_at":"2022-11-11 22:33:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1615990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePotential responder MP CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells express distinct chemokine receptors upon IL-12/IL-18 and IL-1β /IL-23 stimulation. \u003c/strong\u003e(a) tSNE plots and (b) proportion of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells stimulated with IL-12 and IL-18 in the presence of IL-7 for 5 days. (c) Expression of selected Th1-related genes. (d) Selected KEGG/GO terms in cluster 2,3,4 and 6 and (e) Ingenuity pathway analysis (IPA) in cluster 3,4 and 6 of IL-12- and IL-18-responded MP CD4\u003csup\u003e+\u003c/sup\u003e T cells. (f) Pseudo-time trajectory: each cell is colored by its pseudo-time value and (g) the expression level of the related genes. (h) tSNE plots and (i) proportion of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells stimulated with IL-1β and IL-23 in the presence of IL-7 for 5 days. (j) Expression of selected Th17-related genes. (k) Selected KEGG/GO terms and (l) IPA of IL-1β- and IL-23-stimulated MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (clusters 3, 4, 6). (m) Pseudo-times trajectory: each cell is colored by its pseudo-time value and (n) the transcription factor activity and (o) expression level of the related genes.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2219047/v1/94b2fb19f7955462f99daa10.png"},{"id":28973300,"identity":"f8b7a9e2-fa7e-418e-adbc-f21c023e6e45","added_by":"auto","created_at":"2022-11-11 22:25:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":827874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSteady state CCR6\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ehigh\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e MP CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells are bystander-activated by IL-1β and IL-23 to become pathogenic Th17-like cells. \u003c/strong\u003e(a) Gating strategy of CCR6\u003csup\u003ehigh\u003c/sup\u003e and CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells from SPF mice. (b) The percentage of CCR6 expression in steady-state splenic MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (n=6). (c) The expression of CCR6 in SPF- vs. GF-MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (n=4). (d) Transcription factor expression level of T-bet and RORγt in FACS-sorted CCR6\u003csup\u003ehigh\u003c/sup\u003e and CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells and (e) cytokine expression and the average proportion of CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells vs. CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (n = 6). CCR6\u003csup\u003ehigh\u003c/sup\u003e and CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells were stimulated with IL-1β and/or IL-23 in the presence of IL-7 for 5 days. (f) The representative proportion and (g) the average value showing the cytokine producing cells (n=5). (h) Concentration of IL-17A, GM-CSF, and IFN-γ were analyzed by ELISA (n = 5). (i) The proportion of RORγt\u003csup\u003e+\u003c/sup\u003e and Ki67\u003csup\u003e+ \u003c/sup\u003ecells in CCR6\u003csup\u003ehigh\u003c/sup\u003e and CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells. (j) Heatmap of selected genes (k) Gene set enrichment analysis (GSEA) pathway enrichment plot related to “Cell Proliferation” and “Pathogenic T\u003csub\u003eH\u003c/sub\u003e17 signature” by bulk RNA-seq analysis. \u003cem\u003eq\u003c/em\u003e, false discovery rate; NES, normalized enrichment score. Data are presented as the mean ± S.D. All data of p values were calculated using Mann-Whitney U-test (ND, not detected; NS, not significant; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2219047/v1/86efada5c548ef24bc8eed6f.png"},{"id":28973303,"identity":"07b6c445-bfdc-433a-917c-6cfbae92a875","added_by":"auto","created_at":"2022-11-11 22:25:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":449651,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCR6\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ehigh\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e MP CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells exacerbate autoimmune neuroinflammation in bystander manner. \u003c/strong\u003e(a) Naïve CD4\u003csup\u003e+\u003c/sup\u003e T cells (5 × 10\u003csup\u003e4\u003c/sup\u003e) from 2D2 transgenic mice were adoptively transferred, with or without CCR6\u003csup\u003ehigh\u003c/sup\u003e or CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+ \u003c/sup\u003eT cells (1 × 10\u003csup\u003e5\u003c/sup\u003e,\u003csup\u003e \u003c/sup\u003eCD45.1\u003csup\u003e+\u003c/sup\u003eγδTCR\u003csup\u003e-\u003c/sup\u003eNK1.1\u003csup\u003e-\u003c/sup\u003eV\u003csub\u003eβ\u003c/sub\u003e11\u003csup\u003e-\u003c/sup\u003eTCRβ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD1d tetramer\u003csup\u003e-\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003e), into Rag\u003csup\u003e-/-\u003c/sup\u003e mice who were immunized with MOG\u003csub\u003e35-55\u003c/sub\u003e in CFA. EAE clinical score was monitored daily (n=15). (b) Absolute cell numbers of infiltrated CD45.1\u003csup\u003e+\u003c/sup\u003eMP CD4\u003csup\u003e+\u003c/sup\u003e T cells in the spinal cord and brain (n=8). (c) The representative plots and (d) absolute cell number of IL-17A, GM-CSF, and IFN-γ\u003csup\u003e \u003c/sup\u003eproducing cells were determined in MP CD4\u003csup\u003e+\u003c/sup\u003e T cells from the spinal cords and brains on days 12–13 after immunization (n=8). (e) The representative dot plots and (f) the average value showing the percentage of 2D2 TCR (V\u003csub\u003eα\u003c/sub\u003e3.2\u003csup\u003e+ \u003c/sup\u003eand V\u003csub\u003eβ\u003c/sub\u003e11\u003csup\u003e+\u003c/sup\u003e) in spleens from 2D2 transgenic mice and C57BL/6 wild type mice (n=4). (g) FACS-sorted 2D2 naïve CD4\u003csup\u003e+\u003c/sup\u003e T cells and CCR6\u003csup\u003ehigh\u003c/sup\u003e or CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells were cultured with CD11c\u003csup\u003e+\u003c/sup\u003e dendritic cells (MHC-II\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e) with or without MOG\u003csub\u003e35-55 \u003c/sub\u003epeptide (50 μg/ml) for 3 days. CFSE levels were measured by flow cytometry. Data are presented as the mean ± S.E.M in a and the mean ± S.D in b,d,f. \u003cem\u003eP\u003c/em\u003e values were calculated using two-way ANOVA or Mann-Whitney U-test (NS, not significant; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2219047/v1/8995d462c6b52f77dcd0c499.png"},{"id":28974030,"identity":"08a0decf-bfd2-403d-9732-01706bc40a17","added_by":"auto","created_at":"2022-11-11 22:33:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":867972,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInnate-like effector functions of CCR6\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ehigh\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e MP CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells are conferred by Bhlhe40/GM-CSF axis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Predicted upstream network on IL-1β and IL-23 responded MP CD4\u003csup\u003e+\u003c/sup\u003e T cells by IPA. (b) The representative histogram and average value showing the percentage of Bhlhe40 level in CCR6\u003csup\u003ehigh\u003c/sup\u003e and CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells induced by IL-1β/IL-23 stimulation without TCR engagement (n=3). (c) The representative percentage of IL-17A and GM-CSF expression compared to Bhlhe40\u003csup\u003eGFP\u003c/sup\u003e positive and negative cells in CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells induced by IL-1β /IL-23 without TCR engagement for 5 days (n=3). (d) Representative flow cytometry plots showing the cytokine expression and (e) average proportion of WT CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells vs. Bhlhe40\u003csup\u003e-/-\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (n = 3). (f) Naïve CD4\u003csup\u003e+\u003c/sup\u003e T cells (5 × 10\u003csup\u003e4\u003c/sup\u003e) from 2D2 transgenic mice were adoptively transferred, with or without WT CCR6\u003csup\u003ehigh\u003c/sup\u003e or Bhlhe40\u003csup\u003e-/-\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+ \u003c/sup\u003eT cells (1 × 10\u003csup\u003e5\u003c/sup\u003e,\u003csup\u003e \u003c/sup\u003eCD45.1\u003csup\u003e+\u003c/sup\u003eγδTCR\u003csup\u003e-\u003c/sup\u003eNK1.1\u003csup\u003e-\u003c/sup\u003eV\u003csub\u003eβ\u003c/sub\u003e11\u003csup\u003e-\u003c/sup\u003eTCRβ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD1d tetramer\u003csup\u003e-\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003e), into Rag\u003csup\u003e-/-\u003c/sup\u003e mice who were immunized with MOG\u003csub\u003e35-55\u003c/sub\u003e in CFA. EAE clinical score was monitored daily (n=5). (g) Representative flow cytometry plots and (h) absolute cell numbers of infiltrated 2D2 TCR (V\u003csub\u003eβ\u003c/sub\u003e11\u003csup\u003e+\u003c/sup\u003e)\u003csup\u003e- \u003c/sup\u003eMP CD4\u003csup\u003e+\u003c/sup\u003e T cells (gating from CD45\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e) and IL-17A, GM-CSF, and IFN-γ\u003csup\u003e \u003c/sup\u003eproducing cells from the spinal cords and brain on days 13 after immunization (n=5). (i) Naïve CD4\u003csup\u003e+\u003c/sup\u003e T cells (5 × 10\u003csup\u003e4\u003c/sup\u003e) from 2D2 transgenic mice were adoptively transferred, with or without WT CCR6\u003csup\u003ehigh\u003c/sup\u003e or WT CCR6\u003csup\u003elow\u003c/sup\u003e or GM-CSF\u003csup\u003e-/-\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e or GM-CSF\u003csup\u003e-/-\u003c/sup\u003e CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+ \u003c/sup\u003eT cells (1 × 10\u003csup\u003e5\u003c/sup\u003e,\u003csup\u003e \u003c/sup\u003eCD45.1\u003csup\u003e+\u003c/sup\u003eγδTCR\u003csup\u003e-\u003c/sup\u003eNK1.1\u003csup\u003e-\u003c/sup\u003eV\u003csub\u003eβ\u003c/sub\u003e11\u003csup\u003e-\u003c/sup\u003eTCRβ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD1d tetramer\u003csup\u003e-\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003e), into Rag\u003csup\u003e-/-\u003c/sup\u003e mice who were immunized with MOG\u003csub\u003e35-55\u003c/sub\u003e in CFA. EAE clinical score was monitored daily (n=9). Data are presented as the mean ± S.E.M in F and I and the mean ± S.D in b,c,d,e,g,h values were calculated using two-way ANOVA or Mann-Whitney U-test (NS, not significant; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2219047/v1/753080e6094cd7831d8e53d8.png"},{"id":36487338,"identity":"deac0a45-fbca-488a-ad30-8e0445481ba6","added_by":"auto","created_at":"2023-05-01 07:05:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3322710,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2219047/v1/5f7e5241-5183-431d-84af-76cf7745c0ed.pdf"},{"id":28973304,"identity":"4d5eec5f-7b97-4940-8c27-f2708cfca686","added_by":"auto","created_at":"2022-11-11 22:25:59","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":2685016,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-2219047/v1/fd71d2f7571870a9ef9585de.docx"}],"financialInterests":"(Not answered)","formattedTitle":"\u003cp\u003eSteady-state memory-phenotype conventional CD4\u003csup\u003e+\u003c/sup\u003e T cells exacerbating autoimmune neuroinflammation in bystander manner via Bhlhe40/GM-CSF axis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe immunological memory of antigen-specific T cells enables faster and more potent responses upon re-exposure to a previously encountered antigen, providing long-lasting immunity \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Although a substantial population of memory-phenotype (MP) conventional T cells exists in steady state mice unexposed to foreign antigens, it has been reported that MP T cells are formed before birth in humans and exist in germ-free (GF) and antigen-free (AF) conditioned mice \u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter undergoing homeostatic proliferation in a lymphopenic environment, na\u0026iuml;ve T cells acquire phenotypical, functional, and gene expression\u0026ndash;like antigen-specific memory and become MP T cells \u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. T cell receptor (TCR) and CD28 signaling seem to be required for the conversion of na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells into self-derived MP T cells in lympho-sufficient condition. These MP CD4\u003csup\u003e+\u003c/sup\u003e T cells express CD5, a marker with high affinity to self-antigen, and resist infection by mediating a Th1-like immune response without antigen stimulation \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. A previous study confirmed that antigen-non-specific MP CD4\u003csup\u003e+\u003c/sup\u003e T cells proliferated more than Lymphocytic choriomeningitis virus (LCMV)-specific T cells in an LCMV infection model. Indeed, treatment with an anti-MHCII antibody did not inhibit the proliferation of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, suggesting that they play a bystander role against infection \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnlike MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, MP CD8\u003csup\u003e+\u003c/sup\u003e T cells have been well studied for their antigen-specific and bystander functions. MP CD8\u003csup\u003e+\u003c/sup\u003e T cells can rapidly produce IFN-γ upon stimulation with IL-12 and IL-18 and without cognate antigen stimulation \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In particular, MP CD8\u003csup\u003e+\u003c/sup\u003e T cells are called virtual memory. They can have specific reactions to certain antigens without previous exposure \u003csup\u003e5,15\u0026minus;18\u003c/sup\u003e and increase NKG2D and granzyme B expression upon IL-12, IL-18, and IL-15 stimulation, producing a bystander killing role against infection \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In addition, MP CD8\u003csup\u003e+\u003c/sup\u003e T cells can play an antigen-specific protection role in \u003cem\u003eListeria monocytogenes\u003c/em\u003e, Herpes simplex virus (HSV), and \u003cem\u003evaccinia\u003c/em\u003e infections \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Also, MP CD8\u003csup\u003e+\u003c/sup\u003e T cells have high affinity to self-antigens, so they can break peripheral tolerance with MP CD4\u003csup\u003e+\u003c/sup\u003e T cells and develop autoimmune diabetes \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Overall, previous studies have shown the function of MP CD8\u003csup\u003e+\u003c/sup\u003e T cells in disease, but the role of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells in autoimmune disease has not yet been studied.\u003c/p\u003e \u003cp\u003eMost previous studies have focused on the role of autoantigen-specific T cells in both humans and mice to understand autoimmune diseases and find therapeutic drugs to regulate antigen-specific T cells \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Interestingly, antigen-non-specific T cells, including myelin oligodendrocyte glycoprotein (MOG) tetramer\u0026ndash;negative CD4\u003csup\u003e+\u003c/sup\u003e Th17 cells, also infiltrate the Central nervous system (CNS) in significant proportions and exacerbate experimental autoimmune encephalomyelitis (EAE) pathogenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Bystander-activated T cells and Epstein-Barr virus\u0026ndash;specific CD8\u003csup\u003e+\u003c/sup\u003e T cells are clonally expanded and correlate with disease pathogenesis in the joints of chronic inflammatory arthritis and Sjogren\u0026rsquo;s syndrome patients \u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, raising questions about the role of antigen-nonrelated T cells in autoimmune disease.\u003c/p\u003e \u003cp\u003eIn this study, we hypothesized that MP conventional CD4\u003csup\u003e+\u003c/sup\u003e T cells could be encephalitogenic bystander cells during the development of autoimmune neuroinflammatory disease. First, we examined the heterogeneity of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells using single cell RNA-sequencing (scRNA-seq) and TCR sequencing analyses. We found distinct subpopulations of Th1-, Th17-, Treg-, and Tfh-like cells among MP CD4\u003csup\u003e+\u003c/sup\u003e T cells. Those cells respond to IL-1 family cytokines and STAT activating cytokines, even without TCR stimulation. We further found that CCR6 \u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells are the major responders to IL-1β and IL-23, expressing pathogenic signature genes in a bystander manner and thereby contributing to the development of MOG antigen\u0026ndash;specific T cell\u0026ndash;derived EAE. In this context, we identified Bhlhe40 which regulates the production of GM-CSF in CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells in bystander manner, exacerbating EAE pathogenesis. Our findings indicate the pathogenic role of antigen independent MP CD4\u003csup\u003e+\u003c/sup\u003e T cells along with antigen-specific T cells during autoimmune neuroinflammatory disease.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eMice\u003c/h2\u003e\n\u003cp\u003eC57BL/6J mice were purchased from DBL (Chungcheongbuk-do, Korea), and Rag\u003csup\u003e-/-\u003c/sup\u003e, GM-CSF\u003csup\u003e-/-\u003c/sup\u003e and 2D2 TCR-transgenic mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). CD45.1\u003csup\u003e+\u003c/sup\u003e, Foxp3-GFP mice were provided by Jeehee Youn (Hanyang University). GF and AF mice were purchased from the animal facility of POSTECH Biotech Center (Pohang, Korea). Bhlhe40\u003csup\u003e-/-\u003c/sup\u003e and Bhlhe40\u003csup\u003eGFP\u003c/sup\u003e mice were provided by Brian T. Edelson (Washington University). Il1r1\u003csup\u003e-/-\u003c/sup\u003e mice were provided by Heung-Kyu Lee (KAIST University). The mice were housed and bred in a specific pathogen\u0026ndash;free animal facility at Hanyang University under controlled conditions with a constant temperature (21\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) and humidity (50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%) and a 12 h light/dark cycle with regular chow and autoclaved water. All mouse experimental procedures used in this study were approved by the Institutional Animal Care and Use Committee of Hanyang University (2020-0018A,2021-005A, 2021-0158A).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eMP CD4\u003csup\u003e+\u003c/sup\u003e T cell isolation and in vitro activation\u003c/h2\u003e\n\u003cp\u003eMP (TCR\u0026beta;\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD1d tetramer\u003csup\u003e-\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003e) CD4\u003csup\u003e+\u003c/sup\u003e T cells from the spleens of 8 to 12-week-old mice were isolated using a FACS Aria II and FACS Aria Fusion cell sorter (BD Biosciences, Franklin Lakes, NJ, USA). FACS-sorted MP CD4\u003csup\u003e+\u003c/sup\u003e T cells were stimulated with IL-1\u0026beta; (20 ng/mL, R\u0026amp;D Systems, Minneapolis, MN, USA), IL-23 (20 ng/mL, R\u0026amp;D Systems), IL-12 (20 ng/mL, Peprotech, Rocky Hill, NJ, USA), IL-18 (20 ng/mL, R\u0026amp;D Systems), IL-33 (20 ng/mL, R\u0026amp;D Systems), IL-25 (20 ng/mL, R\u0026amp;D Systems), IL-7 (10 ng/mL, Peprotech, Rocky Hill, NJ, USA), or plate-bound anti-CD3/anti-CD28 (2 \u0026micro;g/mL, BD Biosciences) for 5 days at 37\u0026deg;C in an incubator.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eActive EAE and adoptive transfer EAE\u003c/h2\u003e\n\u003cp\u003eIn active EAE model, FACS-sorted MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (\u0026gamma;\u0026delta;TCR\u003csup\u003e-\u003c/sup\u003eNK1.1\u003csup\u003e-\u003c/sup\u003eV\u003csub\u003e\u0026beta;\u003c/sub\u003e11\u003csup\u003e-\u003c/sup\u003eTCR\u0026beta;\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD1d tetramer\u003csup\u003e-\u003c/sup\u003eFoxp3\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003e, 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) from Foxp3-GFP mice were adoptively transferred to 5-week-old female C57BL/6 mice. After transfer, the mice were immunized with 200 \u0026micro;g of MOG\u003csub\u003e35-55\u003c/sub\u003e peptide in complete Freund\u0026rsquo;s adjuvant (Chondrex, Inc., USA). At 0 and 48 h after immunization, the mice were intraperitoneally treated with 500 ng of pertussis toxin (List Biological Laboratories, Inc., Campbell, CA, USA). The animals were scored daily for clinical disease.\u003c/p\u003e\n\u003cp\u003eIn another EAE model by adoptive transfer, naive (CD4\u003csup\u003e+\u003c/sup\u003eV\u003csub\u003e\u0026beta;\u003c/sub\u003e11\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003ehigh\u003c/sup\u003eCD44\u003csup\u003elow\u003c/sup\u003e) CD45.1\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells (1\u0026ndash;5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) from 2D2 TCR-transgenic mice were transferred into Rag\u003csup\u003e-/-\u003c/sup\u003e mice with or without WT CCR6\u003csup\u003ehigh\u003c/sup\u003e or WT CCR6\u003csup\u003elow\u003c/sup\u003e or Bhlhe40\u003csup\u003e-/-\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e or GM-CSF\u003csup\u003e-/-\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e or GM-CSF\u003csup\u003e-/-\u003c/sup\u003e CCR6\u003csup\u003elow\u003c/sup\u003e or Il1r1\u003csup\u003e-/-\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e or Il1r1\u003csup\u003e-/-\u003c/sup\u003e CCR6\u003csup\u003elow\u003c/sup\u003eMP CD4\u003csup\u003e+\u003c/sup\u003e T cells (CD45.1\u003csup\u003e+\u003c/sup\u003e\u0026gamma;\u0026delta;TCR\u003csup\u003e-\u003c/sup\u003eNK1.1\u003csup\u003e-\u003c/sup\u003eV\u003csub\u003e\u0026beta;\u003c/sub\u003e11\u003csup\u003e-\u003c/sup\u003eTCR\u0026beta;\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD1d tetramer\u003csup\u003e-\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003e, 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e). Before transfer, CCR6\u003csup\u003ehigh\u003c/sup\u003e or CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells were primed \u003cem\u003ein vitro\u003c/em\u003e with IL-7 (10 ng/mL) and IL-1\u0026beta; (20 ng/mL) for 5\u0026ndash;7 days. After transfer, the mice were immunized with 100 \u0026micro;g of MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e peptide in complete Freund\u0026rsquo;s adjuvant (Chondrex, Inc., USA). At 0 and 48 h after immunization, the mice were intraperitoneally treated with 200 ng of pertussis toxin (List Biological Laboratories, Inc., Campbell, CA, USA). The animals were scored daily for clinical disease as follows \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e: partially limp tail, 0.5; completely limp tail, 1; limp tail and waddling gait, 1.5; paralysis of one hind limb, 2; paralysis of one hind limb and partial paralysis of the other hind limb, 2.5; paralysis of both hind limbs, 3; ascending paralysis, 3.5; paralysis of trunk, 4; moribund, 4.5; dead, 5. On day 12 or 13, the mice were sacrificed and perfused with Phosphate buffered saline (PBS). To isolate lymphocytes, the spinal cord and brain were digested with 1 mg/mL of collagenase D (11 088 866 001; Sigma-Aldrich) and DNase I (10 104 159 001; Sigma-Aldrich) and incubated at 80 RPM on a shaker for 35 min. After enzyme digestion, lymphocytes were isolated by Percoll (GE Healthcare, Little Chalfont, UK) density-gradient centrifugation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eIn vitro MOG reactivity assay (CFSE)\u003c/h2\u003e\n\u003cp\u003eFor antigen-specific proliferation, 2D2 na\u0026iuml;ve T cells (CD4\u003csup\u003e+\u003c/sup\u003eV\u003csub\u003e\u0026beta;\u003c/sub\u003e11\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003ehigh\u003c/sup\u003eCD44\u003csup\u003elow\u003c/sup\u003e) and CCR6\u003csup\u003ehigh\u003c/sup\u003e or CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (\u0026gamma;\u0026delta;TCR\u003csup\u003e-\u003c/sup\u003eNK1.1\u003csup\u003e-\u003c/sup\u003eV\u003csub\u003e\u0026beta;\u003c/sub\u003e11\u003csup\u003e-\u003c/sup\u003eTCR\u0026beta;\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD1d tetramer\u003csup\u003e-\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003e) and CD11c\u003csup\u003e+\u003c/sup\u003e dendritic cells (MHC-II\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e) (APC) from the spleens of 2D2 TCR-transgenic and C57BL/6 mice were isolated using a FACS Aria Fusion cell sorter. Before co-culture, na\u0026iuml;ve and MP CD4\u003csup\u003e+\u003c/sup\u003e T cells were stained with 1.25 \u0026micro;M carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, Carlsbad, CA) for 7 min at room temperature. After incubation, 10% Fetal bovine serum (FBS) was added, and the incubation was continued on ice for 3 min. 2D2 Na\u0026iuml;ve and CCR6\u003csup\u003ehigh\u003c/sup\u003e or WT CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were then washed with PBS and co-cultured with CD11c\u003csup\u003e+\u003c/sup\u003e dendritic cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) with or without 50 \u0026micro;g/mL of MOG\u003csub\u003e35-55\u003c/sub\u003e peptide for 72 h.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eFlow cytometry\u003c/h2\u003e\n\u003cp\u003eCell surface staining was performed using the following monoclonal antibodies: anti-CD4 (RM4-5; eBioscience, San Diego, CA, USA, dilution 1:500), anti-CD25 (PC61.5; eBioscience, dilution 1:500), anti-CD44 (IM7; BioLegend, San Diego, CA, USA, dilution 1:500), anti-CD62L (MEL-14; BioLegend, dilution 1:500), anti-CD45 (30-F11, BioLegend, dilution 1:500), anti-CD45.1 (A20; eBioscience, dilution 1:500), anti-TCR\u0026beta; (H57-597; eBioscience, dilution 1:500), anti-CCR6 (29-2L17; BD, dilution 1:100), anti-CXCR3 (CXCR3-173; BD, dilution 1:100), anti-V\u003csub\u003e\u0026alpha;\u003c/sub\u003e3.2 (RR3-16; BioLegend, dilution 1:500), anti-V\u003csub\u003e\u0026beta;\u003c/sub\u003e11 (RR3-15; BioLegend, dilution 1:500), and PE-conjugated CD1d tetramer (PBS57; NIH, dilution 1:1000). Biotinylated PBS57 loaded and unloaded CD1d monomers were provided by the US National Institutes of Health Tetramer Core facility. For intracellular staining, the cells were stimulated with a cell stimulation cocktail (00-4975-03; eBioscience) for 4 h at 37\u0026deg;C, and then surface markers were stained. After staining of the surface markers, the cells were fixed and permeabilized in Cytofix/Cytoperm (554714; BD Bioscience) or FOXP3/Transcription factor staining buffer set (00-5523-00; eBioscience) for 30 min at 4\u0026deg;C or RT. Intracellular staining was performed using the following monoclonal antibodies: anti-IL-17A (eBio17B7; eBioscience, dilution 1:200), anti-IFN-\u0026gamma; (XMG1.2; eBioscience, dilution 1:400), anti-GM-CSF (MP1-22E9; BD Biosciences, dilution 1:200), anti-Ki67 (SolA15; eBioscience, dilution 1:500), anti-IL-4 (11B11; BioLegend, dilution 1:200), IL-13 (eBio13A; eBioscience, dilution 1:200), anti-TNF-\u0026alpha; (MP6-XT22; BioLegend, dilution 1:500), anti-IL-5 (TRFK5; BioLegend, dilution 1:200), anti-IL-1R1 (35F5; BD Biosciences, dilution 1:100), anti-ROR\u0026gamma;t (Q31-378; BD Biosciences, dilution 1:100), anti-T-bet (4B10; BioLegend, dilution 1:50), anti-GATA3 (L50-823; BD Bioscience, dilution 3 \u0026micro;l per well) and IgG1 (R2-34; BD Biosciences). Stained cells were analyzed by flow cytometry (FACS Canto II, BD Bioscience), and data were analyzed using FlowJo software version 10.8.0 (Tree Star, Ashland, OR, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eCytokine (ELISA)\u003c/h2\u003e\n\u003cp\u003eSamples were measured using IL-17A ELISA (432501; BioLegend), IL-13 ELISA (88-7137-88; Thermo Fisher), IL-4 ELISA (431104; BioLegend), IL-5 ELISA (431204; BioLegend), IFN-\u0026gamma; ELISA (430801; BioLegend), TNF-\u0026alpha; ELISA (430904; BioLegend), and GM-CSF ELISA (432201; BioLegend) kits according to the manufacturers\u0026rsquo; instructions. Briefly, microwell plates (Corning Costar; 9018) were coated with capture antibodies overnight at 4\u0026deg;C and blocked with ELISA diluent 1X for 1 h at room temperature. Samples and two-fold serial diluted standards were incubated at room temperature for 2 h, and then detection antibodies and streptavidin-HRP were added. Then, 1X TMB solution and a stop solution (2 N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) was loaded. The optical density was analyzed at 450 nm. Between all procedures, plates were washed at least 3 times with wash buffer (1X PBS, 0.05% Tween-20).\u003c/p\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eAll data were analyzed in non-parametric analyses using the Mann-Whitney test or two-way ANOVA of variance in Prism version 8.0 (GraphPad Software, San Diego, CA). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. or mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. For all data, significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05. Sample size and statistical information is provided in each figure legend.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSingle-cell RNA-sequencing identifies clearly distinct effector-like subpopulations in steady-state MP conventional CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e \u003c/sup\u003e \u003cstrong\u003eT cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSteady-state, unprimed specific-pathogen-free (SPF)-housed mice have a significant proportion of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (TCR\u0026beta;\u003csup\u003e+\u003c/sup\u003eCD1d tetramer\u003csup\u003e\u0026minus;\u003c/sup\u003eCD8\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD25-CD44\u003csup\u003ehigh\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003e) in their spleen, thymus, inguinal lymph nodes (iLNs), mesenteric lymph nodes (mLNs), Peyer\u0026rsquo;s patches (PPs), and lung tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea and Fig. S1a, Supporting information). The proportion and number of CD4\u003csup\u003e+\u003c/sup\u003e T cells increased with age in all tissues (Fig. S1, Supporting information). To identify and focus on the characteristics of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, we used fluorescence-activated cell sorting (FACS) to sort MP CD4\u003csup\u003e+\u003c/sup\u003e T cells from the spleens of 10-week-old C57BL/6 mice and performed scRNA-seq with paired V(D)J sequencing of the T cell receptor (Fig. S2a-d, Supporting information). Also, we generated a pipeline to filter out PLZF and TCR V\u003csub\u003e\u0026alpha;\u003c/sub\u003e14-J\u003csub\u003e\u0026alpha;\u003c/sub\u003e18 (TRAV11-TRAJ18) expressing cells, a well-known key transcription factor for the development of NKT and MAIT cells \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. An unbiased clustering analysis revealed clearly distinct effector T cell\u0026ndash;like subpopulations (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb) and differentially expressed genes (DEGs) defining each cluster (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec and d). A Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of each cluster indicated that clusters 3 and 5 were Th1- and Th17-like populations, respectively, with significance in \u0026ldquo;chemokine-mediated signaling pathway\u0026rdquo; and \u0026ldquo;cellular response to interleukin-1\u0026rdquo; (Fig. S3, Supporting information). In addition, lineage-specific transcription factors and chemokine receptors were localized in each cluster (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee and Fig. S4, Supporting information), suggesting that MP CD4\u003csup\u003e+\u003c/sup\u003e T cells are composed of Th1-, Th17-, Tfh-, and Treg-like subpopulations. To examine whether the subpopulations of steady-state MP CD4\u003csup\u003e+\u003c/sup\u003e T cells depended on germ or food antigens, we analyzed the proportion of CD44\u003csup\u003ehigh\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells in the tissues of SPF-, GF-, and AF-housed mice using flow cytometry. The proportions of total MP CD4\u003csup\u003e+\u003c/sup\u003e T cells were almost identical in the spleen and other tissues except the mLN and PP (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef, Fig. S5a and S5b, Supporting information), indicating that the generation of splenic MP CD4\u003csup\u003e+\u003c/sup\u003e T cells is not affected by the microbiome or food antigen stimulation. Our scRNA-seq analysis further confirmed that the SPF- and GF-housed mice had identical proportions of distinct effector-like subpopulations of splenic MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg and h), but the proportion of Th17-like MP cells in the mLNs of GF-housed mice was much lower than that in the mLNs of SPF-housed mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ei and j). In support, the proportion of CCR6\u003csup\u003ehigh\u003c/sup\u003e or ROR\u0026gamma;t\u003csup\u003e+\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells in the mLNs of GF-housed mice was lower than that in SPF-housed mice, suggesting that the generation of gut MP CD4\u003csup\u003e+\u003c/sup\u003e T cells is specifically dependent on germs (Fig. S6, Supporting information). The TCR clonal diversity of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells in GF-housed mice, especially the Th17-like population in the mLNs, was reduced compared with SPF-housed mice, though comparable diversity was observed in the spleen (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ek and l). There seemed no significant clonal expansion observed in MP subpopulations. Together, these data indicate that steady-state splenic MP CD4\u003csup\u003e+\u003c/sup\u003e T cells contain heterogeneous subpopulations of Th1-, Th17-, Tfh-, and Treg-like cells that express effector molecules and exist independently of the gut microbiome and food antigens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential innate-like effector functions of MP CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e \u003c/sup\u003e \u003cstrong\u003eT cells after exposure to IL-1 family and STAT activating cytokines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the effector functions of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells to respond to various cytokines, we performed scRNA-seq of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells cultured in conditioned medium with IL-12/IL-18, IL-25/IL-33, and IL-1\u0026beta;/IL-23 cytokines (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea and Fig. S2e, Supporting information). Additionally, IL-7 was added to the culture medium for T-cell survival and maintenance \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The merged Uniform Manifold Approximation and Projection (UMAP) plot shows phenotypic characteristics of Th1-, Th2-, Th17-, and Treg-like subpopulations (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb and c), which express lineage specific genes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). Each cytokine set induced distinct subpopulations localized exclusively in the plot (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee). We further confirmed the expression of selected gene sets related to the Th1, Th2, and Th17 lineages in each bystander-activated condition, which shows that type 1, 2, and 3 cytokines can upregulate lineage-specific genes in MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef and g). A single-cell regulatory network inference and clustering (SCENIC) analysis in each cytokine condition revealed a significant level of common or specific transcription factor activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eh). In addition, an Ingenuity pathway analysis (IPA) showed the predicted transcriptional regulators in each condition and speculated that the target transcription factors included Bhlhe40, which can be a potent regulator of bystander activation in MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ei). To evaluate the effector functions of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, we treated MP CD4\u003csup\u003e+\u003c/sup\u003e T cells with those cytokines and/or anti-CD3/anti-CD28 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ej and k). Consistently, the MP CD4\u003csup\u003e+\u003c/sup\u003e T cells responded to IL-12/IL-18, IL-25/IL-33, and IL-1\u0026beta;/IL-23 cytokines without TCR stimulation, and T-bet\u003csup\u003e+\u003c/sup\u003eIFN-\u0026gamma;\u003csup\u003e+\u003c/sup\u003e, GATA3\u003csup\u003e+\u003c/sup\u003eIL-13\u003csup\u003e+\u003c/sup\u003e, and ROR\u0026gamma;t\u003csup\u003e+\u003c/sup\u003eIL-17\u003csup\u003e+\u003c/sup\u003e cells increased compared to TCR-stimulated condition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ej). However, TNF-\u0026alpha; was produced only in the presence of TCR stimulation and IFN-\u0026gamma; in the culture supernatant from IL-12/IL-18-conditioned MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, and IL-4 was also significantly detected with TCR stimulation but not in the bystander condition with IL-25/IL-33. Similarly, Granulocyte-macrophage colony-stimulating factor (GM-CSF) was secreted more efficiently with IL-1\u0026beta;/IL-23 and TCR stimulation than with the cytokines alone (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ek). Collectively, these results indicate that steady state MP CD4\u003csup\u003e+\u003c/sup\u003e T cells have functional heterogeneity of innate-like responses to various sets of IL-1 family and STAT activating cytokines even in the absence of T cell receptor stimulation and suggest possible potent transcriptional regulators that control MP CD4\u003csup\u003e+\u003c/sup\u003e T cell effector functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePotential responder MP CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e \u003c/sup\u003e \u003cstrong\u003eT cells express distinct chemokine receptors upon IL-12/IL-18 and IL-1\u0026beta;/IL-23 stimulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine which MP CD4\u003csup\u003e+\u003c/sup\u003e T cells are potential responders to IL-1 family and STAT activating cytokines, we analyzed the subpopulation of expanding or responding clusters and performed trajectory analyses. Control MP CD4\u003csup\u003e+\u003c/sup\u003e T cells cultured with IL-7 produced a significantly separate population of CXCR3\u003csup\u003ehigh\u003c/sup\u003e cells. In IL-18/IL-12-conditioned MP CD4\u003csup\u003e+\u003c/sup\u003e T cells the CXCR3\u003csup\u003ehigh\u003c/sup\u003e cell population was reduced, and the IFN-\u0026gamma;- or IL-13\u003csup\u003ehigh\u003c/sup\u003eexpressing Th1 and proliferating Th1 populations were greatly increased (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea and b). Th1 signature genes, \u003cem\u003eTbx21, Ifng, Cxcr3, Il2rb1, Il18r1\u003c/em\u003e, and \u003cem\u003eCCR5\u003c/em\u003e were highly expressed by IL-18/IL-12-conditioned MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). GO/KEGG analysis indicated that the related gene sets in those cells had increased including \u0026ldquo;Cellular response to interferon-gamma\u0026rdquo;, \u0026ldquo;Cytokine cytokine receptor interaction\u0026rdquo;, \u0026ldquo;Alzheimer\u0026rsquo;s disease\u0026rdquo;, and \u0026ldquo;Parkinson\u0026rsquo;s disease\u0026rdquo; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed). The IPA returned terminologies related to cytokines and inflammation such as \u0026ldquo;JAK/STAT signaling\u0026rdquo; and \u0026ldquo;neuroinflammation signaling pathway\u0026rdquo; and related to cytotoxic response including \u0026ldquo;Granzyme B signaling\u0026rdquo; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee). In a pseudo-time trajectory analysis, the MP CD4\u003csup\u003e+\u003c/sup\u003e T cells formed a continuous progression that started in CXCR3\u003csup\u003ehigh\u003c/sup\u003e cells and gradually progressed toward Fate 1, which expressed \u003cem\u003eIfng, Stat5a, Bhlhe40, Batf3, Irf4\u003c/em\u003e and \u003cem\u003eIrf8\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef and g). Similarly, a CCR6\u003csup\u003ehigh\u003c/sup\u003e cluster was present in IL-7-conditioned MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, and its Th17-like cluster was specifically increased by IL-1\u0026beta; and IL-23 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eh and i). Th17 signature genes, \u003cem\u003eRorc, Ccr6, Il17a Il1r1, Il23r\u003c/em\u003e, and proliferating marker \u003cem\u003eMki67\u003c/em\u003e were expressed by those cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ej). GO/KEGG analysis predicted that the related gene sets in IL-1\u0026beta; /IL-23-cultured MP CD4\u003csup\u003e+\u003c/sup\u003e T cells had increased \u0026ldquo;Alzheimer\u0026rsquo;s disease,\u0026rdquo; \u0026ldquo;Parkinson\u0026rsquo;s disease,\u0026rdquo; and \u0026ldquo;Huntington\u0026rsquo;s disease\u0026rdquo; which are neurological diseases and increased \u0026ldquo;cytokine signaling pathway\u0026rdquo; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ek). The IPA more clearly explained the related pathways, \u0026ldquo;STAT3 pathway,\u0026rdquo; \u0026ldquo;leukocyte extravasation signaling,\u0026rdquo; \u0026ldquo;neuroinflammation signaling pathway,\u0026rdquo; \u0026ldquo;chemokine signaling,\u0026rdquo; and \u0026ldquo;Th17 activation pathway\u0026rdquo; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003el). In the trajectory analysis, CCR6\u003csup\u003ehigh\u003c/sup\u003e cells seemed to be the starting point, and then the cells gradually differentiated toward Fate 2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003em), which expresses pathogenic Th17-related genes such as \u003cem\u003eRorc, Il17a, Csf2, Il22, Bhlhe40, Rora and Cebpb\u003c/em\u003e with increased activities (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003en) and expression level (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eo) of related transcriptomes. These results collectively reveal that Th1-like and Th17-like MP CD4\u003csup\u003e+\u003c/sup\u003e T cells expressing different chemokine receptors respond specifically to IL-12/IL-18 and IL-1\u0026beta;/IL-23 cytokines with the effector functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSteady state CCR6\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ehigh\u003c/strong\u003e \u003c/sup\u003e \u003cstrong\u003ememory phenotype CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003eT cells are bystander-activated by IL-1\u0026beta; and IL-23 to become pathogenic Th17-like cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince scRNA-seq predicted that CCR6\u003csup\u003ehigh\u003c/sup\u003e cells were the major cells responding to IL-1\u0026beta; and IL-23, we sorted splenic CCR6\u003csup\u003ehigh\u003c/sup\u003e and CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea and b) and then determined their proportions in steady-state SPF- and GF-housed mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). We found that splenic CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells were independent of the gut. CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells expressed ROR\u0026gamma;t more highly than CCR6\u003csup\u003elow\u003c/sup\u003e cells and had comparable expression of T-bet (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed). Steady-state CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, but not CCR6\u003csup\u003elow\u003c/sup\u003e, expressed IL-17A (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee), suggesting that CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells are Th17-like cells. Further stimulation by IL-1\u0026beta; and IL-23 induced IL-17A and GM-CSF expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef and g), which confirms that CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells produce pathogenic cytokines in the bystander manner. The amount of cytokine secreted, as determined by ELISA, consistently showed that CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, but not CCR6\u003csup\u003elow\u003c/sup\u003e, significantly produced IL-17A, GM-CSF, and IFN-\u0026gamma; and that IL-1\u0026beta; and IL-23 had important synergy for pathogenicity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eh). In addition, IL-1\u0026beta; greatly expanded ROR\u0026gamma;t expression in CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells but not CCR6\u003csup\u003elow\u003c/sup\u003e cells, whereas IL-23 somewhat inhibited the proportion of Ki67-expressing cells, suggesting that IL-1\u0026beta; is important for the proliferation of CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ei). To further confirm the functions of IL-1\u0026beta; and IL-23 in MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, we performed bulk-RNA seq with bystander-activated MP CD4\u003csup\u003e+\u003c/sup\u003e T cells exposed to IL-1\u0026beta; and IL-23. In the heatmap DEG analysis, IL-1\u0026beta; increased the expression of proliferation-related gene such as \u003cem\u003eMki67\u003c/em\u003e and \u003cem\u003ecdk2\u003c/em\u003e, whereas IL-23 alone did not have any significant effects on gene expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ej). IL-23 and IL-1\u0026beta; together significantly induced the expression of pathogenic genes such as \u003cem\u003eBhlhe40, Il1r1, Csf2\u003c/em\u003e, \u003cem\u003eIfng, Il22\u003c/em\u003e and \u003cem\u003eIl17a\u003c/em\u003e. In support, Gene set enrichment analysis (GSEA) pathway enrichment plot of IL-1\u0026beta; vs. IL-1\u0026beta; and IL-23 demonstrated that the enrichment score for cell proliferation was higher with IL-1\u0026beta;, and the score of the pathogenic Th17 signature with IL-1\u0026beta; and IL-23 was higher than in the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ek). Through these results, we understand that steady-state CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, which show Th17-like characteristics, are the major bystander-activated cells responding to IL-1\u0026beta; and IL-23, which potentiate the cells\u0026rsquo; pathogenic character.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCR6\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ehigh\u003c/strong\u003e \u003c/sup\u003e \u003cstrong\u003eMP CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003eT cells exacerbate autoimmune neuroinflammation in bystander manner.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo reveal the importance of splenic MP CD4\u003csup\u003e+\u003c/sup\u003e T cells during an autoimmune disease, we first induced active EAE in 5-week-old mice, who have a lower proportion of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells than 10-week-old mice. In this mouse model, we compared the disease severity of control mice with that of those who received an additional adoptive transfer of Treg-deleted (Foxp3\u003csup\u003e\u0026minus;\u003c/sup\u003e) MP CD4\u003csup\u003e+\u003c/sup\u003e T cells from 10-week-old Foxp3-GFP mice. EAE was rapidly induced and progressed by transferring additional MP CD4\u003csup\u003e+\u003c/sup\u003e T cells from 10-week-old mice, suggesting that MP CD4\u003csup\u003e+\u003c/sup\u003e T cells could be an important contributor to MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e-induced EAE pathogenesis (Fig. S7a, Supporting information). In support, Rag\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice that adoptively transferred MOG-TCR transgenic (2D2) na\u0026iuml;ve CD45.1\u003csup\u003e\u0026minus;\u003c/sup\u003eV\u003csub\u003e\u0026beta;\u003c/sub\u003e11\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells with Treg-deleted MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (CD45.1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e) showed a more severe phenotype of EAE than the mice that received only 2D2 T cells (Fig. S7b, Supporting information), suggesting that MP CD4\u003csup\u003e+\u003c/sup\u003e T cells contribute to the pathogenesis of EAE. Based on our previous results, we hypothesized that CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells are the major pathogenic subpopulation contributing to EAE disease progression. To test that hypothesis, we transferred CCR6\u003csup\u003ehigh\u003c/sup\u003e and CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (CD45.1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e, gating strategy and purity is shown in Fig. S8, Supporting information) and 2D2 na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells into Rag\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. The additional transfer of CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells exacerbated EAE development compared with 2D2 transfer alone or the transfer of CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Interestingly, the number of transferred MP CD4\u003csup\u003e+\u003c/sup\u003e T cells that appeared in the spinal cord and brain tissue did not differ between conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). However, CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells produced significantly more cytokines, particularly IL-17A and GM-CSF, in the spinal cord and brain tissue than CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec and d). Therefore, CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, along with antigen-specific T cells, contribute to the pathogenicity of autoimmune neuroinflammation by expressing pathogenic cytokines such as IL-17A and GM-CSF. To clarify the antigen-independent activation of CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells in an EAE mouse model, we detected 2D2 TCR (V\u003csub\u003e\u0026alpha;\u003c/sub\u003e3.2\u003csup\u003e+\u003c/sup\u003e and V\u003csub\u003e\u0026beta;\u003c/sub\u003e11\u003csup\u003e+\u003c/sup\u003e), which are predominantly expressed in 2D2 na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells. Indeed, CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells barely expressed V\u003csub\u003e\u0026alpha;\u003c/sub\u003e3.2\u003csup\u003e+\u003c/sup\u003e and V\u003csub\u003e\u0026beta;\u003c/sub\u003e11\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee and f). In support, we confirmed that steady-state CCR6\u003csup\u003ehigh\u003c/sup\u003e and CCR6\u003csup\u003elow\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells did not respond to the MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e antigen (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eg). Collectively, these results suggest that CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells infiltrate in CNS tissue and exacerbate autoimmune neuroinflammation in a bystander manner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInnate-like effector functions of CCR6\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ehigh\u003c/strong\u003e \u003c/sup\u003e \u003cstrong\u003eMP CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003eT cells are conferred by Bhlhe40/GM-CSF axis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the candidate genes involved in bystander activation of CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, we identified that Bhlhe40/GM-CSF axis could potentially give rise to the pathogenic function of CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells induced by IL-1\u0026beta; and IL-23 without TCR stimulation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). By using Bhlhe40\u003csup\u003eGFP\u003c/sup\u003e mice, we found that CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells activated by IL-1\u0026beta; and IL-23 showed significantly increased level of Bhlhe40 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). Interestingly, Bhlhe40\u003csup\u003eGFP\u003c/sup\u003e positive T cells majorly produced effector cytokines including IL-17A and GM-CSF compared to Bhlhe40\u003csup\u003eGFP\u003c/sup\u003e negative T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec). In support, Bhlhe40\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells showed markedly reduced IL-17A and GM-CSF production compared to WT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed and e), suggesting that Bhlhe40 is an important transcriptional regulator for the pathogenic functions of bystander CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells. To confirm the in vivo relevance, we transferred WT CCR6\u003csup\u003ehigh\u003c/sup\u003e or Bhlhe40\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells along with 2D2 na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells into Rag\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Bhlhe40\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells showed abrogated functions for exacerbating EAE disease compared by WT CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef). Interestingly, the number of Bhlhe40\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells expressing IL-17A and GM-CSF in the spinal cord and brain tissue was significantly reduced compared to WT CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg and h). In addition, transfer of GM-CSF-deficient CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells showed abrogated function of contributing EAE pathogenesis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ei), suggesting that disease aggravation by bystander CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells is committed by IL-1\u0026beta; and IL-23 via Bhlhe40/GM-CSF axis. Collectively, these results indicate that Bhlhe40 confers innate-like pathogenic functions of CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells with GM-CSF production.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we intensively validated the characteristics of steady state MP CD4\u003csup\u003e+\u003c/sup\u003e T cells using scRNA-seq to unveil their innate-like effector functions during autoimmune disease. We found clearly distinct effector-like subpopulations in steady-state splenic MP CD4\u003csup\u003e+\u003c/sup\u003e T cells that are independent of the microbiome and food antigens. MP CD4\u003csup\u003e+\u003c/sup\u003e T cells can be bystander-activated by the different sets of IL-1 family and STAT activating cytokines. Specific chemokine receptor\u0026ndash;expressing cells are defined as potential responder cells to each set of cytokines, and we focused on CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells to further validate their functions in responding to IL-1β/IL-23. We demonstrated that steady state CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells have innate-like effector functions that exacerbate EAE disease progression in a bystander manner, along with antigen-specific T cells. We suggest Bhlhe40 as a pivotal transcriptional regulator that governs GM-CSF production in bystander-activated CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, exacerbating EAE development. Overall, our results reveal the innate lymphoid cell-like immunological functions of steady-state MP CD4\u003csup\u003e+\u003c/sup\u003e T cells in autoimmune disease.\u003c/p\u003e \u003cp\u003eInnate T cells such as natural killer T (NKT), mucosal-associated invariant T (MAIT), and γδ T cells have limited TCR gene usage compared to conventional T cells, which recognize complexes of non-peptide antigens such as glycolipids, phospho-antigens, and vitamin B metabolites, respectively \u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These innate T cells are derived from the thymus, which can evoke robust cytokine production. Previously, innate lymphocytes, such as NKT17, γδT17 cell, and ILC3 subsets have been defined to commonly express IL-17 and RORγt \u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Furthermore, a recent study reported a novel subset of αβ-γδ co-expressing T cells which recognize MHC-restricted peptide antigens and produce effector cytokine IL-17A, GM-CSF, and IFN-γ by IL-1β and IL-23 stimulation \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Also, another group said that natural Th17 cells and γδ T cells expanded after the candidiasis infection model in oral cavity\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In this context, we carefully eliminated the possibility of contamination of innate T cells by sorting conventional MP CD4\u003csup\u003e+\u003c/sup\u003e T cells using NKT (CD1d tetramer), γδ T, αβ-γδ T, ILC, and MAIT (TCRβ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003e) exclusion gates. We confirmed that PLZF/CD1d tetramer negative steady-state MP CD4\u003csup\u003e+\u003c/sup\u003e T cells still exists as a heterogenous population containing CCR6\u003csup\u003ehigh\u003c/sup\u003eRORγt\u003csup\u003e+\u003c/sup\u003eIL-17A\u003csup\u003e+\u003c/sup\u003e cells, which is majorly bystander-activated by IL-1β and IL-23. Therefore, collectively, we provide compelling evidence that conventional CD4\u003csup\u003e+\u003c/sup\u003e T cells distinguished from previously known innate T cells exist, which have an innate-like features and contribute to autoimmune neuroinflammation.\u003c/p\u003e \u003cp\u003eAs our study revealed the heterogeneous characteristics of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells by single cell transcriptomic analysis, recent studies revealed the potential heterogeneity of murine MP CD4\u003csup\u003e+\u003c/sup\u003e T cells \u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. CXCR3\u003csup\u003e+\u003c/sup\u003eT-bet\u003csup\u003e+\u003c/sup\u003e Th1-like MP CD4 T cells spontaneously generated from na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells in steady state showed innate-like effector functions against \u003cem\u003eT. gondii\u003c/em\u003e infection \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These cells require DC1-derived tonic IL-12 signal for optimal differentiation of T-bet\u003csup\u003ehigh\u003c/sup\u003e MP T cells \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Similarly, we confirmed CXCR3\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells are expressing T-bet and they are major responders to IL-12 and IL-18 cytokine stimulation. CXCR3\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, in high correlation with CCR5 expression, can produce IFN-γ and T-bet in response to IL-12 and IL-18, suggesting a innate-like function of CXCR3\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e \u003cp\u003eIn our previous study, we demonstrated that IL-1β and IL-23 which are derived from innate immune cells\u003csup\u003e27,48\u0026minus;50\u003c/sup\u003e can synergistically potentiate the pathogenicity of memory CD4\u003csup\u003e+\u003c/sup\u003e T cells \u003cem\u003ein vitro\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and that non-myelin-specific CD4\u003csup\u003e+\u003c/sup\u003e T cells can infiltrate the CNS with MOG antigen\u0026ndash;specific T cells, which significantly contribute to EAE disease progression \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In rheumatoid arthritis patients, T cells that infiltrate the synovial fluid mainly express the CD45RO\u003csup\u003e+\u003c/sup\u003e memory marker and specifically respond to epitopes of Epstein-Barr virus and cytomegalovirus \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. In type 1 diabetes, infection with rotavirus or coxsackie virus is reported to be involved in accelerated diabetes onset through Toll-like receptor (TLR) signaling without pancreatic infection \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, and influenza A virus is linked to diabetes in human patients \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Collectively, those studies suggest that antigen-non-related CD4\u003csup\u003e+\u003c/sup\u003e T cells can contribute to disease onset or progression with antigen-specific T cells in various autoimmune diseases. We have identified here that CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells are the major subpopulation of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells that respond to IL-1β and IL-23 by expanding and inducing pathogenic Th17 characteristics. These IL-1β and IL-23 signaling In an adoptive transfer model of EAE, CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells transferred with MOG-specific T cells induced more severe EAE than CCR6\u003csup\u003elow\u003c/sup\u003e cells, with increased production of IL-17 and GM-CSF in the CNS. We further confirmed that MP CD4\u003csup\u003e+\u003c/sup\u003e T cells do not respond to MOG\u003csub\u003e33\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e antigen, indicating the innate-like functions of CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells in autoimmune neuroinflammation. In addition, we confirmed IL-1R1 is required for pathogenic contribution of CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells in EAE disease (Fig. S9, Supporting information) suggesting IL-1 signal to CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells could trigger their bystander effector functions in vivo. Further studies should be done to reveal the distinct mechanism between antigen-specific T cells and bystander-activated T cells in autoimmune disease pathogenesis.\u003c/p\u003e \u003cp\u003eAnalyzing the single cell transcriptomics of IL-1β/IL-23 responding MP CD4\u003csup\u003e+\u003c/sup\u003e T cells, we identified that Bhlhe40 could be a potential transcriptional regulator inducing GM-CSF in CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells. Bhlhe40 has been reported to play pivotal roles in T cells. Bhlhe40-deficient na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells show limited response to TCR stimulation \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In addition, Bhlhe40 seem to be required for Th1 and Th17 effector cytokine production including IL-17A, GM-CSF and IFN-γ in the context of autoimmune disease, GVHD, and Toxoplasma gondii infection model \u003csup\u003e27,60\u0026minus;62\u003c/sup\u003e. Expression of Bhlhe40 correlate with mouse \u003cem\u003eCsf2\u003c/em\u003e locus, which encodes GM-CSF, \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e and also positive correlation with GM-CSF expression was reported in human PBMC \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. We demonstrate that steady-state CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells are expressing RORγt and IL-1 receptor that they up-regulate Bhlhe40 in response to IL-1β and IL-23. In the absence of TCR engagement, they can produce IL-17 and GM-CSF that are importantly contribute to the pathogenesis of EAE. In support, a previous study reported the majority of Bhlhe40-expressing pathogenic T cells in active EAE are non-MOG-specific \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. As Bhlhe40\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells showed reduced GM-CSF and GM-CSF\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells could not exacerbate EAE, Bhlhe40/GM-CSF axis seem to be an important mechanism of bystander-activated MP CD4\u003csup\u003e+\u003c/sup\u003e T cells during EAE. Therefore, Bhlhe40 can be a pivotal transcriptional regulator for both antigen-specific and bystander MP CD4\u003csup\u003e+\u003c/sup\u003e T cells in the context of CNS inflammation and targeting of Bhlhe40 in CD4\u003csup\u003e+\u003c/sup\u003e T cells may serve as a potential novel treatment strategy to control autoimmune diseases.\u003c/p\u003e \u003cp\u003eSelf-antigen-specific T cells are fundamentally important in triggering autoimmune inflammation; however, antigen-non-related naturally arising steady state MP CD4\u003csup\u003e+\u003c/sup\u003e T cells are also importantly contributing to pathogenic inflammation in a bystander manner. Collectively, our studies of the role that MP CD4\u003csup\u003e+\u003c/sup\u003e T cells play in neuroinflammatory disease shed light on the bystander function of adaptive immune cells to understand disease pathogenesis and reveal a novel drug development strategy to modulate autoimmune diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Dongsoo Kyeong and Dr. Younhee Shin (Insilicogen Inc.) for supporting bioinformatic analysis of the scRNA-seq data. We thank Mr. Yeon-Ho Kim and Ms. In Young Song for technical support in the FACS sorting conducted at Hanyang LINC Analytical Equipment Center (Seoul) and the NIH tetramer core facility (Emory University) for providing mouse CD1d PBS-57 (Biotinylated Monomer). We thank Prof. Jeehee Youn (Hanyang University) for kindly providing CD45.1\u003csup\u003e+\u003c/sup\u003e, Foxp3-GFP mice and Prof. Kwang Soon Kim (POSTECH) for helping us purchase germ-free and antigen-free mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Basic Science Research Program (NRF-2019R1A2C3006155) of the National Research Foundation funded by the Korean government.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003euthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.-Z.C., H.-G.L., and J.-M.C. conceptualized and designed this study. M.-Z.C., H.-G.L. performed and analyzed most of the experiments including bioinformatic analysis. Y.J.L. supported conceptualization for bioinformatic analyses. J.-W.Y., G.-R.K. and J.-H.K. supported experiments. R.T. and B.T.E. provided Bhlhe40\u003csup\u003e-/-\u003c/sup\u003e and Bhlhe40\u003csup\u003eGFP\u003c/sup\u003e. M.-Z.C., H.-G.L., and J.-M.C. wrote draft manuscript, and all authors reviewed the manuscript. J.-M.C. supervised the analyses and acquired funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request. RNA-seq data have been deposited in the NCBI Gene Expression Omnibus.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKaech, S. M. \u0026amp; Cui, W. Transcriptional control of effector and memory CD8 + T cell differentiation. 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Nat Immunol \u003cb\u003e21\u003c/b\u003e, 388\u0026ndash;399, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41590-020-0622-8\u003c/span\u003e\u003cspan address=\"10.1038/s41590-020-0622-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Memory-phenotype CD4 T cells, CCR6, Bystander, EAE, neuroinflammation, Bhlhe40","lastPublishedDoi":"10.21203/rs.3.rs-2219047/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2219047/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMemory-phenotype (MP) CD4\u003csup\u003e+\u003c/sup\u003e T cells are a substantial population of conventional T cells that exist in steady-state mice, and their immunologic functions in autoimmune disease have not yet been studied. In this work, we unveil a unique phenotype of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells by analyzing single-cell transcriptomics and T cell receptor (TCR) repertoires. We found that steady-state MP CD4\u003csup\u003e+\u003c/sup\u003e T cells exist regardless of germ and food-antigen which are composed of heterogenous effector subpopulations. Distinct subpopulations of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells are specifically activated by IL-1 family cytokines and STAT activators, revealing that the cells have TCR-independent bystander effector functions like innate lymphoid cell. Especially, CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells are major responders to IL-1β and IL-23 without MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e antigen reactivity, which gives them pathogenic-Th17 characteristics and allows them to contribute to autoimmune encephalomyelitis. We identified Bhlhe40 in CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells drives the expression of GM-CSF through IL-1β and IL-23 signaling, contributing to CNS pathology in experimental autoimmune encephalomyelitis. Collectively, our findings reveal clearly distinct effector-like heterogeneity of MP CD4\u003csup\u003e+\u003c/sup\u003e T cells in steady state and CCR6\u003csup\u003ehigh\u003c/sup\u003e MP CD4\u003csup\u003e+\u003c/sup\u003e T cells exacerbate autoimmune neuroinflammation by Bhlhe40/GM-CSF axis in bystander manner synergistically with antigen-specific T cells.\u003c/p\u003e","manuscriptTitle":"Steady-state memory-phenotype conventional CD4+ T cells exacerbating autoimmune neuroinflammation in bystander manner via Bhlhe40/GM-CSF axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-11 22:25:53","doi":"10.21203/rs.3.rs-2219047/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2022-12-20T07:15:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-12-19T10:21:14+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-12-04T21:26:39+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-11-17T10:44:47+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-11-09T22:07:30+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2022-11-09T11:16:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-11-01T00:45:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2022-10-31T07:23:45+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2022-10-31T02:46:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-10-30T16:08:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"105985a2-2a2e-40c9-93e1-c8e869c12896","owner":[],"postedDate":"November 11th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":16902878,"name":"Biological sciences/Immunology/Autoimmunity"},{"id":16902879,"name":"Biological sciences/Immunology/Neuroimmunology"},{"id":16902880,"name":"Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Lymphocyte activation"}],"tags":[],"updatedAt":"2023-05-01T07:05:52+00:00","versionOfRecord":{"articleIdentity":"rs-2219047","link":"https://doi.org/10.1038/s12276-023-00995-1","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2023-05-01 04:00:00","publishedOnDateReadable":"May 1st, 2023"},"versionCreatedAt":"2022-11-11 22:25:53","video":"","vorDoi":"10.1038/s12276-023-00995-1","vorDoiUrl":"https://doi.org/10.1038/s12276-023-00995-1","workflowStages":[]},"version":"v1","identity":"rs-2219047","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2219047","identity":"rs-2219047","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}