Fig 1: Pmel-1 x SLAMF6 -/- T cells have a better functional capacity.(A–D) Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml) and then incubated overnight with B16-F10/mhgp100 melanoma cells. (A) The cells were incubated at a 1:1 effector-to-target ratio. IFN-? secretion was measured by ELISA. Each point represents one mouse. (B) The cells were incubated at the indicated effector-to-target ratios. IFN-? secretion was measured by ELISA. (C) The cells were incubated at a 1:1 effector-to-target ratio. GM-CSF secretion was measured by ELISA. Each point represents one mouse. (D) Conditioned medium was collected and analyzed with Quantibody mouse cytokine array. (E, F) Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes were activated for 7 days with gp10025-33 peptide and IL-2 (30 IU/ml) and then incubated for 16 hr with B16-F10/mhgp100 melanoma cells. Granzyme-B expression was detected by flow cytometry. One representative experiment (E) and a summary of triplicates (F) are shown. (G–J) B16-F10/mhgp100 mouse melanoma cells were injected s.c. into the back of C57BL/6 mice. Pmel-1 or Pmel-1 x SLAMF6 -/- mouse splenocytes were expanded with gp10025-33 peptide in the presence of IL-2 (30 IU/ml). On day 7, Pmel-1 cells or Pmel-1 x SLAMF6 -/- cells were adoptively transferred i.v. into the irradiated tumor-bearing mice. N = 8 mice per group. Tumor size was measured three times a week. (G) Scheme of the experimental layout. (H) Spider plots showing tumor volume [calculated as L (length) x W (width)2 x 0.5]. CR, complete response. (I) Normalized tumor volume (Mean ± SEM) until day 45, on which the first mouse had to be sacrificed. Tumor dimensions were normalized to the 1st measurement. (J) Kaplan Meier survival curve. (K) Percent T cells specific for gp10025-33 peptide in the spleen or tumor draining lymph nodes (DLN) of mice sacrificed 7 days post-ACT. Tet, tetramer. Student t test. *, p<0.05, **, p<0.01, ***, p<0.001.
Fig 2: Differential innate-like effector functions of MP CD4+ T cells after exposure to IL-1 family and STAT-activating cytokines.a Overview of the experimental design. b UMAP representation of MP CD4+ T cells stimulated with IL-12 and/or IL-18 and IL-25 and/or IL-33 and IL-23 and/or IL-1ß in the presence of IL-7. c Heatmap and d violin plots of differentially expressed transcripts in a cluster. e Individual cytokine conditions visualized with UMAP. f MA plots of differentially expressed genes comparing IL-7 versus IL-12/18, IL-25/33 or IL-23/1ß. g Heatmap representing gene expression of resting-, Tfh-, Th1-, Th2-, Th17- and Treg-related gene signatures in each cytokine condition. h Heatmap representing transcriptional activity. i Venn diagram of transcriptional regulators predicted by IPA. Numbers indicate the number of genes in each gate. j MP CD4+ T cells (TCRß+CD1d tetramer–CD8–CD4+CD25–CD62LlowCD44high) were cultured for 5 days with IL-12 and/or IL-18 and IL-25 and/or IL-33 and IL-23 and/or IL-1ß 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-?, TNF-a, IL-4, IL-5, IL-13, IL-17A, and GM-CSF concentrations were measured by ELISA (n = 5, 5 independent experiments). Data are presented as the mean ± S.D. p Values were calculated using the Mann?Whitney U-test (*p < 0.05, **p < 0.01).
Fig 3: Innate-like effector functions of CCR6high MP CD4+ T cells are conferred by the Bhlhe40/GM-CSF axis.a Predicted upstream network of IL-23- and IL-1ß-responsive MP CD4+ T cells by IPA. b Representative histogram and average value showing the percentage of Bhlhe40-positive in CCR6high and CCR6low MP CD4+ T cells induced by IL-23/IL-1ß stimulation without TCR engagement (n = 3). c Representative percentages of IL-17A- and GM-CSF-expressing cells in Bhlhe40GFP-positive and Bhlhe40GFP-negative cells in CCR6high MP CD4+ T cells induced by IL-23/IL-1ß without TCR engagement for 5 days (n = 3). d Representative flow cytometry plots showing the cytokine expression and e average proportion of WT CCR6high MP CD4+ T cells vs. Bhlhe40–/– CCR6high MP CD4+ T cells (n = 3). f Naïve CD4+ T cells (5 × 104) from 2D2 transgenic mice were adoptively transferred, with or without WT CCR6high or Bhlhe40–/– CCR6high MP CD4+ T cells (1 × 105, CD45.1+?dTCR–NK1.1–Vß11–TCRß+CD4+CD1d tetramer–CD25–CD62LlowCD44high), into Rag–/– mice that were immunized with MOG35-55 in CFA. The EAE clinical score was monitored daily (n = 5). g Representative flow cytometry plots and h absolute cell numbers of infiltrated 2D2 TCR (Vß11+)– MP CD4+ T cells (gating from CD45+CD4+) and IL-17A-, GM-CSF-, and IFN-?–producing cells in the spinal cord and brain on Day 13 after immunization (n = 5). i Naïve CD4+ T cells (5 × 104) from 2D2 transgenic mice were adoptively transferred, with or without WT CCR6high, WT CCR6low, GM-CSF–/– CCR6high or GM-CSF–/– CCR6low MP CD4+ T cells (1 × 105, CD45.1+?dTCR–NK1.1–Vß11–TCRß+CD4+CD1d tetramer–CD25–CD62LlowCD44high), into Rag–/– mice that were immunized with MOG35-55 in CFA. The 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–e, g, h. Values were calculated using two-way ANOVA or the Mann?Whitney U-test (NS not significant; *p < 0.05, **p < 0.01, ***p < 0.001).
Fig 4: Steady-state CCR6high MP CD4+ T cells undergo bystander activation by IL-23 and IL-1ß to become pathogenic Th17-like cells.a Gating strategy for CCR6high and CCR6low MP CD4+ T cells from SPF mice. b The percentage of CCR6-expressing cells in steady-state splenic MP CD4+ T cells (n = 6). c The expression of CCR6 in SPF mouse- vs. GF mouse-derived MP CD4+ T cells (n = 4). d Expression levels of the transcription factors T-bet and ROR?t in FACS-sorted CCR6high and CCR6low MP CD4+ T cells and e cytokine expression and the average proportion of CCR6high MP CD4+ T cells vs. CCR6low MP CD4+ T cells (n = 6). CCR6high and CCR6low MP CD4+ T cells were stimulated with IL-23 and/or IL-1ß in the presence of IL-7 for 5 days. f The representative proportion and g the average value of cytokine-producing cells (n = 5). h The concentrations of IL-17A, GM-CSF, and IFN-? were analyzed by ELISA (n = 5). i The proportions of ROR?t+ and Ki67+ cells in CCR6high and CCR6low MP CD4+ T cells. j Heatmap of selected genes. k Gene set enrichment analysis (GSEA) pathway enrichment plot related to “Cell Proliferation” and “Pathogenic TH17 signature” by bulk RNA-seq analysis. q false discovery rate, NES normalized enrichment score. Data are presented as the mean ± S.D. All p values were calculated using the Mann?Whitney U-test (ND not detected, NS not significant; *p < 0.05, **p < 0.01, ***p < 0.001).
Fig 5: CCR6 high MP CD4+ T cells exacerbate autoimmune neuroinflammation in a bystander manner.a Naïve CD4+ T cells (5 × 104) from 2D2 transgenic mice were adoptively transferred, with or without CCR6high or CCR6low MP CD4+ T cells (1 × 105, CD45.1+?dTCR–NK1.1–Vß11–TCRß+CD4+CD1d tetramer–CD25–CD62LlowCD44high), into Rag–/– mice that were immunized with MOG35-55 in CFA. The EAE clinical score was monitored daily (n = 15). b Absolute cell numbers of infiltrated CD45.1+MP CD4+ T cells in the spinal cord and brain (n = 8). c Representative plots and d absolute cell numbers of IL-17A-, GM-CSF-, and IFN-?–producing cells in MP CD4+ T cells isolated from the spinal cord and brain on Days 12–13 after immunization (n = 8). e Representative dot plots and f average value showing the percentage of 2D2 TCR (Va3.2+ and Vß11+) in spleens from 2D2 transgenic mice and C57BL/6 wild-type mice (n = 4). g FACS-sorted 2D2 naïve CD4+ T cells and CCR6high or CCR6low MP CD4+ T cells were cultured with CD11c+ dendritic cells (MHC-II+CD11c+) with or without the MOG35-55 peptide (50 µg/ml) for 3 days. CFSE levels were measured by flow cytometry. Data are presented as the mean ± S.E. in a and the mean ± S.D. in b, d, f. p Values were calculated using two-way ANOVA or the Mann?Whitney U-test (NS not significant; *p < 0.05, **p < 0.01, ***p < 0.001).
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