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

doi:10.21203/rs.3.rs-2219047/v1

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

https://doi.org/10.21203/rs.3.rs-2219047/v1

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published 01 May, 2023

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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

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.

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^lowCD44^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_β11^-TCRβ⁺CD4⁺CD1d tetramer^-Foxp3^-CD62L^lowCD44^high, 5 × 10⁵) 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_β11⁺CD25^-CD62L^highCD44^low) CD45.1⁻ T cells (1–5 × 10⁴) 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^lowMP CD4⁺ T cells (CD45.1⁺γδTCR^-NK1.1^-V_β11^-TCRβ⁺CD4⁺CD1d tetramer^-CD25^-CD62L^lowCD44^high, 1.0 × 10⁵). 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_β11⁺CD25^-CD62L^highCD44^low) and CCR6^high or CCR6^low MP CD4⁺ T cells (γδTCR^-NK1.1^-V_β11^-TCRβ⁺CD4⁺CD1d tetramer^-CD25^-CD62L^lowCD44^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 × 10⁵ cells/well) were then washed with PBS and co-cultured with CD11c⁺ dendritic cells (5 × 10⁴) 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_α3.2 (RR3-16; BioLegend, dilution 1:500), anti-V_β11 (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 H₂SO₄) 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.

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^highCD62L^low) in their spleen, thymus, inguinal lymph nodes (iLNs), mesenteric lymph nodes (mLNs), Peyer’s patches (PPs), and lung tissues (Fig. 1a 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_α14-J_α18 (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. 1b) and differentially expressed genes (DEGs) defining each cluster (Fig. 1c 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. 1e 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^highCD62L^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. 1f, 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. 1g 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. 1i 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. 1k 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. 2a 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. 2b and c), which express lineage specific genes (Fig. 2d). Each cytokine set induced distinct subpopulations localized exclusively in the plot (Fig. 2e). 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. 2f 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. 2h). 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. 2i). 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. 2j 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. 2j). 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. 2k). 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^highexpressing Th1 and proliferating Th1 populations were greatly increased (Fig. 3a 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. 3c). 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. 3d). 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. 3e). 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. 3f 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. 3h and i). Th17 signature genes, Rorc, Ccr6, Il17a Il1r1, Il23r, and proliferating marker Mki67 were expressed by those cells (Fig. 3j). 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. 3k). The IPA more clearly explained the related pathways, “STAT3 pathway,” “leukocyte extravasation signaling,” “neuroinflammation signaling pathway,” “chemokine signaling,” and “Th17 activation pathway” (Fig. 3l). In the trajectory analysis, CCR6^high cells seemed to be the starting point, and then the cells gradually differentiated toward Fate 2 (Fig. 3m), which expresses pathogenic Th17-related genes such as Rorc, Il17a, Csf2, Il22, Bhlhe40, Rora and Cebpb with increased activities (Fig. 3n) and expression level (Fig. 3o) 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. 4a and b) and then determined their proportions in steady-state SPF- and GF-housed mice (Fig. 4c). 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. 4d). Steady-state CCR6^high MP CD4⁺ T cells, but not CCR6^low, expressed IL-17A (Fig. 4e), 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. 4f 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. 4h). 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. 4i). 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. 4j). 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. 4k). 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_β11⁺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. 5a). Interestingly, the number of transferred MP CD4⁺ T cells that appeared in the spinal cord and brain tissue did not differ between conditions (Fig. 5b). 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. 5c 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_α3.2⁺ and V_β11⁺), which are predominantly expressed in 2D2 naïve CD4⁺ T cells. Indeed, CCR6^high MP CD4⁺ T cells barely expressed V_α3.2⁺ and V_β11⁺ (Fig. 5e 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. 5g). 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. 6a). 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. 6b). Interestingly, Bhlhe40^GFP positive T cells majorly produced effector cytokines including IL-17A and GM-CSF compared to Bhlhe40^GFP negative T cells (Fig. 6c). In support, Bhlhe40^−/− CCR6^high MP CD4⁺ T cells showed markedly reduced IL-17A and GM-CSF production compared to WT (Fig. 6d 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. 6f). 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. 6g and h). In addition, transfer of GM-CSF-deficient CCR6^high MP CD4⁺ T cells showed abrogated function of contributing EAE pathogenesis (Fig. 6i), 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.

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 cavity⁴⁴. 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^highCD62L^low) exclusion gates. We confirmed that PLZF/CD1d tetramer negative steady-state MP CD4⁺ T cells still exists as a heterogenous population containing CCR6^highRORγt⁺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.

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.

Author 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. 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.

Conflict of Interest

The authors declare that they have no competing interests.

Data and materials availability

The 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.

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

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Figures

Introduction

Materials And Methods

Mice

MP CD4⁺ T cell isolation and in vitro activation

Active EAE and adoptive transfer EAE

In vitro MOG reactivity assay (CFSE)

Flow cytometry

Cytokine (ELISA)

Statistical analysis

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

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

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Mice

MP CD4+ T cell isolation and in vitro activation

Active EAE and adoptive transfer EAE

In vitro MOG reactivity assay (CFSE)

Flow cytometry

Cytokine (ELISA)

Statistical analysis

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

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

MP CD4⁺ T cell isolation and in vitro activation