Fig 2: Lack of intracranial tumor eradication and Treg depletion with the anti-GITR IgG 2a antibody/SRS combination. C57BL/6 mice were intracranially inoculated with GL261-luc tumor, randomized to groups of =8, and dosed with 200 µl of anti-GITR (2a) (10 mg/kg) on day 10, 13, 16 and/or SRS (10 Gy) on day 10. Mice were followed for survival, a and tumor growth was assessed by bioluminescent imaging b. Mice were sacrificed on day 21, tumor infiltrating Tregs were isolated and analyzed by flow cytometry c. CD11b + CD45+ tumor resident microglia and tumor infiltrating mononuclear cells were isolated on day 21, analyzed by flow cytometry (gated on CD3+ cells), and mean fluorescence intensity (MFI) of Fc?RIII and IV expression was calculated d. Flank tumors were established in C57BL/6 mice by subcutaneous inoculation of 2 × 106 GL261-luc cells in a volume of 100 µl, and intracranial tumors were established as in Fig. 1. Mice were dosed i.p with 200 µl of anti-GITR (2a) (10 mg/kg) on days 10, 13, and 16, sacrificed on day 17, and tumor infiltrating lymphocytes were harvested and analyzed by flow cytometry for FoxP3 expression (gated on CD3+ cells) e-f. Symbol and horizontal bar denote single mouse and average value, respectively. *P < .05, ***P < .001. NS, non-significant

Fig 3: AE promote hematopoietic progenitor generation with T cell potential.(A) Schematic of protocol for investigating the niche effect on hematopoiesis. (B) Representative flow cytometric analysis of the hematopoietic progenitors obtained from H1-derived HE, or HE cocultured with Mes (HE + Mes), VE (HE + VE), or AE (HE + AE) following 7 days of EHT culture (n = 3). (C) The fold change in CD43+CD45+ cell number compared to HE. The cells were generated under the conditions of HE, HE + Mes, HE + VE, or HE + AE. (n = 3; one-way ANOVA). (D) CFC assay of HPCs generated with or without coculture with AE, VE, or Mes. Colony-forming units (CFUs) per 2000 cells plated. (n = 3; two-way ANOVA). (E) Schematic diagram of the protocol for T cell differentiation. MACS, magnetic-activated cell sorting. (F and G) T cell potential of hematopoietic progenitors generated from D5 HE following 7 days of culture in the presence or absence of VE, AE, or Mes. (n = 3; one-way ANOVA). (H) Representative flow cytometric analysis of the expression of CD4 and CD8 by CD3+ cells before and after anti-CD3/CD28 stimulation (n = 3). (I) The proportion of CD107a in hPSC-derived T cells (hPSC-T) and peripheral blood T cells (PB-T) after treatment with phorbol 12-myristate 13-acetate (PMA)–ionomycin. (n = 3; two-way ANOVA). (J) Polyfunctional cytokine production by hPSC-T and PB-T after treatment with PMA-ionomycin. (n = 3; two-way ANOVA). Experiments were performed on H1 hESCs unless otherwise indicated. Data are presented as means ± SEM. *P < 0.05 and ***P < 0.001.

Fig 4: hCII707-721–Reactive iNKT Cells Suppress Through Fas-FasL and TNFa-TNFRICFSE+ PBMC cells from HDs were activated with plate-bound anti-CD3 (10 µg/mL) and soluble anti- CD28 (2 µg/mL) for 48 hours as responders (Resp) and cocultured with purified autologous hCII707-721–reactive Va24Ja18+ iNKT cells or control cells (Va24Ja18- cells) for another 24 hours. (A) FACS gating strategy of back-gating and gating for Va24Ja18+ iNKT cells. (B) FACS plot of gating strategy for responder CFSE + cells (hCII707-721–reactive Va24Ja18+ iNKT cells as suppressors). (C) hCII707-721–reactive Va24Ja18+ iNKT cells from PBMCs of a HD were enriched with magnetic beads before coculture with autologous activated responder T lymphocytes for functional suppression assay. Va24Ja18+ iNKT cells were obtained by treatment with hCII707-721 (100 µg/mL) for 48 hours (C–F) or from conventional Va24Ja18+ iNKT (G–K). (D) Representative histograms of CFSE+ gating in Resp CFSE+ responder T cells (Resp) cocultured with reactive hCII707-721 Va24Ja18+ iNKT cells or control cells (Va24Ja18- cells) for another 24 hours. (E) Representative histograms of 7AAD+ gating in Resp cocultured with reactive hCII707- 721 Va24Ja18+ iNKT cells or control cells (Va24Ja18- cells). (F) The percentage of 7AAD+ dead cells in Resp CFSE+ responder T cells (Resp) were then cocultured with reactive hCII707-721 Va24Ja18+ iNKT cells or control cells (Va24Ja18- cells) for another 24 hours. (G) FACS plot of gating strategy for responder CFSE+ cells (conventional Va24Ja18+ iNKT cells as suppressors). (H) Conventional Va24Ja18+ iNKT cells cocultured with autologous activated responder T lymphocytes for functional suppression assay. Representative histograms of CFSE+ gating in Resp CFSE+ responder T cells (Resp) cocultured with conventional Va24Ja18+ iNKT cells or control cells (Va24Ja18- cells) for another 24 hours, with percentages in (I). (J) Representative histograms of 7AAD+ gating in Resp cocultured with conventional Va24Ja18+ iNKT cells or control cells (Va24Ja18- cells). (K) The percentage of 7AAD+ dead cells in Resp CFSE+ responder T cells (Resp) cocultured with conventional Va24Ja18+ iNKT cells or control cells (Va24Ja18- cells) for another 24 hours. One-way ANOVA *p < 0.05. The bars indicate the mean ± SEM, n = 7. (L) TNF pathway (PID, MSidDB) enrichment in hCII707-721–reactive iNKT cells compared with conventional iNKT cells (FDR = 0.038). (M) FAS pathway (PID, MSidDB) enrichment in hCII707-721–reactive iNKT vs conventional iNKT cells (FDR = 0.14). (N) Gating strategy for responder CFSE + cells and proliferating CFSE+ cells. (O) The percentage of proliferating CFSE+ cells. Ratio of Resp-only (without neutralizing antibodies) set to 1; ratios calculated as percentage of proliferating CFSE+ cells in various cocultures/percentage of proliferating CFSE+ cells in Resp-only. The bars indicate the mean ± SEM; n = 6–8. Unpaired student t test was applied; *p < 0.05 between Resp + iNKT (Control) and Resp-only. One-way ANOVA and Dunnett multiple-comparisons test: #p < 0.05, ##p < 0.01 between groups with blocking antibodies and control group. ANOVA = analysis of variance; CFSE = carboxyfluorescein succinimidyl ester; FACS = fluorescence-activated cell sorting; FDR = false discovery rate; HD = healthy donor; MS = multiple sclerosis; NKT = natural killer T; PBMC = peripheral blood mononuclear cell; PID = The Pathway Interaction Database; SEM = standard error of mean; TNF = tumor necrosis factor; TNFRI = TNF receptor I; 7AAD = 7-Aminoactinomycin D.

Fig 5: Synergy between abortive HIV-1 RNA and R848-induced signaling enhances CD8+ T cell IFN? responses. (A and B) DCs were (co)stimulated with R848 and HIV-1 Cap-RNA58 for 48 h and cocultured with PBLs. PBLs were directly treated with anti-CD3 and anti-CD28 as a positive control. After 6 days, cells were restimulated and the percentages of CD3+CD8+ cells with intracellular expression levels of granzyme B, perforin, and IFN? were assessed by flow cytometry. The percentage of cells in each gate is indicated by numbers in the plots (A). Data are representative of 3 donors of different experiments (A) or are collated data of 3 donors with each symbol representing a different donor (B), (mean ± sd). *p < 0.05, **p < 0.01, Student's t-test