Fig 2: CTLA-4, GITR, ICOS and OX40 Are Highly Expressed by Tumor-Infiltrating Treg Cells(A–C) Mice (n = 5) were injected subcutaneously (s.c.) with B16, MCA205, MC38 (C57BL/6 mice) or CT26 (Balb/c mice) cells. Ten days later, cell suspensions of PBMC, draining LNs and tumor-infiltrating lymphocytes (TILs) were stained and analyzed by flow cytometry. (A) Representative histograms of CTLA-4 expression detected by intracellular staining of individual T cell subsets in mice with MCA205 tumors. Dotted lines represent the gates, numbers indicate the percentage of CTLA-4+ cells. (B and C) Percentage (B) and MFI (C) of CTLA-4-expressing cells in murine PBMCs, LNs, and TILs in different tumor models.(D) Representative histograms of CTLA-4 expression detected by intracellular staining of T cell subsets in PBMCs and TILs in a patient with advanced melanoma.(E and F) Percentage (E) and MFI (F) of CTLA-4 expression in T cells in PBMCs and TILs of patients with advanced melanoma (n = 8), early-stage NSCLC (n = 8) and RCC (n = 8).(G) Heatmap demonstrating the percentage of cells expressing co-inhibitory and co-stimulatory molecules within the indicated T cell subsets quantified by flow cytometry. Each row represents an individual murine or human tumor (n = 5).(H) MFI of the indicated co-inhibitory and co-stimulatory molecules in PBMCs and TILs in patients with melanoma. Horizontal bars represent the mean; error bars show ± standard error of the mean (SEM).*p < 0.05; ****p < 0.0001. See also Table S1 and Figure S1.

Fig 3: Prior Nb exposure results in elevated inflammation, eosinophil infiltration, and ILC2 presence in genital tissue, following HSV-2 vaginal infection(A) At day 9 post-Nb infection (Nb only) and day 3 post-HSV-2 infection (HSV-2 only and Nb + HSV-2), numbers of FGT myeloid cells were analyzed by flow cytometry.(B) Vaginal tissue was analyzed by Sirius red staining. Representative images (n = 4) of virus-induced ulcers were taken at ×400 magnification.(C) Black boxes indicate magnified sections of (Ci) vaginal stroma (St) and (Cii) epithelium (Ep), taken at ×1,000 magnification. White arrows indicate eosinophilic cell infiltration and migration in ulcerated vaginal tissue. Black arrowheads indicate eosinophil presence in vaginal epithelial layer.(D) At day 2 post-HSV-2 infection, levels of MBP (25 kDa) and GAPDH (37 kDa) were measured in HSV-2-only and Nb + HSV-2 FGT homogenates. Density of MBP was measured relative to that of GAPDH.(E) MFI of Siglec-F, CD11b, Ly6G, Ly6C, CD62L, and CD49d on FGT eosinophils in Nb 9 dpi and Nb + HSV-2 mice.(F) Levels of IL-5 in vaginal lavages at day 2 post-virus infection, determined by Luminex. Dotted line represents LLOQ.(G) Frequencies (mean ± SEM) and numbers of Lin-IL-7Ra+ICOS+ST2+ cells (ILC2s) in the FGT of Nb 9 dpi, HSV-2-only and Nb + HSV-2 mice.(H and I) Frequency and number of (H) Lin-IL-7Ra+ICOS+ cells and (I) Ly6Chi monocytes that are IL-5+ in the FGT, determined by flow cytometry. Data are representative of two independent experiments with 5–6 mice per group (mean ± SEM).(J) Nb-infected mice were treated with 20 µg a-IL-5 or isotype control, on day -2, 0, and 2 post-HSV-2 infection. Viral progression was determined by daily pathology scoring and HSV-2 ulcerated epithelium was qualified as percentage (%) of ulcerated epithelium. At day 6 post-HSV-2 infection, numbers of eosinophils in the iLN were determined by flow cytometry. Data are representative of two independent experiments with 4 mice per group (mean ± SEM). Statistical significance was calculated by two-way ANOVA with Bonferroni correction for multiple comparisons and Mann-Whitney t test. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.

Fig 4: CXCR5-PD-1+ICOS+ T Cells Are Clonally Related to TFH Cells and Exhibit Epigenetic Similarities(A) Circos plots of TCR sequence overlap among different populations. Each thin slice of the arc represents a unique TCR sequence, ordered by the clone size (darker green for larger clones, inner circle). Outer circle indicate TCR sequences found in naive, CXCR5-PD-1-ICOS-, CXCR5-PD-1+ICOS+, and TFH cells. Each plot represents data from one individual.(B) Bhattacharyya coefficient measurement for the TCR repertoire similarity between CXCR5-PD-1+ICOS+ T cells and other populations within LN. Grey lines connect samples from the same patient (n = 9).(C) Principal-component analysis of ATAC-seq data of LN CD4+ T cell subsets from 6 HIV+ donors. Each symbol represents cells from one donor; cells of the same type are coded by the same color.(D) Representative chromatin accessibility at the CXCR5 upstream region for each CD4+ T cell subset.(E) Example plots showing TCF1 and CD38 staining.(F) Quantification of activated TCF1- T cells among CXCR5-PD-1+ICOS+ T cells and TFH cells in HIV+ LNs (n = 8).(G) Sorted TFH cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and treated with SEB or DMSO for 7 days in the presence of B cells. Bar graph quantifies the fluorescence intensity of CXCR5 staining on TFH cells. Cell division was measured by dilution of CFSE staining (n = 6).For (B) and (G), Friedman test was performed and corrected using Dunn’s multiple comparisons test. For (F), paired t test was used. Data are represented as mean ± SEM.Also see Figures S5A, S5B, S4E, and S4F.

Fig 5: T cell proportions and activation in the draining lymph nodes following combined debulking surgery with IMQ and/or anti-CD40. BALB/c mice bearing AB1-HA tumors underwent debulking surgery and treatment with IMQ (50 µg i.t. q1dx6 on day of surgery) and/or anti-CD40 (100 µg i.p. q1dx1, starting one day after surgery). On day 26, the draining lymph nodes were removed for analysis of T cell subsets by flow cytometry. CD4+ and CD8+ T cells were identified as% of total lymphocytes (based on forward and side scatter), and analysed for ICOS expression (activation status) as% of total CD4+ or CD8+ T cells. *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated; Kruskal-Wallis test with Dunn’s correction for multiple comparisons.