Fig 2: Gut V?7+ IELs Respond to Btnl Proteins(A) Left: surface CD25 expression of designated IEL subsets after 12-hr co-culture of total IELs with MODE-K cells transduced with empty vector (EV), Btnl1 (L1), Btnl6 (L6), or Btnl1 plus Btnl6 (L1+6). Right: fold increase in CD25+ cells as percentage of the IEL subset relative to co-culture with MODE-K.EV (n = 21).(B) GFP and CD25 expression by designated IELs from Nur77.gfp mice ex vivo or after 12-hr co-culture of total IELs with designated cells (n = 8).(C) Percentage of V?7+ IELs from Nur77.gfp mice that were CD25+GFP+CD122- directly ex vivo or after 12-hr co-culture of total IELs with designated cells.(D) CD3 expression in V?7+CD25+ or V?7+CD25- IELs after 12-hr co-culture of total IELs with MODE-K.L1+6 (n = 21).(E) Surface CD25 expression as percentage of V?7+ IELs after indicated transwell co-culture conditions (n = 3).(F) Surface CD25 expression on V?7+ IELs from WT and Btnl1-/- mice after indicated culture conditions (n = 7).(G) Surface CD25 expression by designated IEL subsets after incubation with anti-CD3 or control immunoglobulin (Ig) (n = 12).(H) Cytokine concentrations assessed by luminex in supernatants after 48 hr of co-cultures indicated (n = 3).Data are representative of one (H), two (B) or more than three (A, D, and E) independent experiments. Some panels present data pooled from two (C) or more than three (A, F, and G) independent experiments. All error bars represent mean ± SD. See also Figure S6.

Fig 3: Brilliant Blue G (BBG) treatment does not affect the distribution of splenic leukocytes from SOD1G93A mice.Splenic leukocytes from BBG- or saline-treated SOD1G93A mice at end-stage were labelled with fluorochrome-conjugated mAbs. The percentage of (A) B cells (CD19+CD3-), (B) T cells (CD3+CD19-), (C) CD8+ T cells (CD8+CD3+CD19-), (D) CD4+ T cells (CD4+CD3+), (E) regulatory T (Treg) cells (CD25+CD4+CD3+), (F) dendritic cells (DCs; CD11b±CD11c+), (G) macrophages (MOs; CD11b+CD11c-Ly6G-) and (H) polymorphonuclear neutrophils (PMNs; CD11b+CD11c-Ly6G+) of the total CD45+ leukocyte population were determined by flow cytometry. Results shown as means ± SD, n = 6 (male and female). Symbols represent individual animals.

Fig 4: The maturation of DCs in tumors and lymph nodes. (A–C) Cell suspensions from treated Pan02_HVEM tumors were analyzed by flow cytometry after stained with indicated fluorescence-conjugated antibodies. The tumor infiltrating immune cells were presented as the percentages of CD45+ cells in total viable cells (A). DCs (B) and T cells (C) were expressed as the percentages of CD11c+ and CD3+ in CD45+ cells, respectively (n=5). (D–G) Pan02_HVEM cells were mock-infected (black bars), or infected with oHSV (blue bars) or oHSV-CD40L (red bars) at MOI=1. At 12 hpi, bone marrow-derived DCs (BM-DCs) were added and co-cultured for 12 hours. The expression of CD40 (D), CD80 (E), CD86 (F), and MHC-II (G) was analyzed by flow cytometry and presented as the percentages of CD11c+ cells, respectively. (H) The concentration of IL-12 in above co-culture media was quantitated by ELISA and plotted in columns. (I–L) The percentages of CD40+ (I), CD80+ (J), CD86+ (K), and MHC-II+ (L) in CD11c+ cells from treated tumors (on day 20) were plotted in scatter graphs (n=5). (M–P) Cell suspensions from tumor-draining lymph node (tdLN) of treated mice were analyzed by flow cytometry after stained with indicated fluorescence-conjugated antibodies on day 20. The DCs among tdLNs were defined as CD11c+ in CD45+ cells (M), and the percentages of CD40+ (N), CD80+ (O), and CD86+ (P) in CD11c+ cells were plotted in scatter graphs (n=5). (Q) Mice were sacrificed on day 3 or day 6 after completion of treatment. The concentration of IL-12 in the tumors was determined by ELISA (n=3). (R) Mice-bearing Pan02_HVEM tumors received PBS or oHSV-CD40L treatment, and IL-12 was blocked using anti-IL-12 antibody before each virus treatment. The survival of treated mice was plotted using Kaplan-Meier analysis and log-rank test (n=5). Other values were presented as mean±SD and statistics were performed using two-tailed Student’s t-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. DC, dendritic cell; IL, interleukin; MHC, major histocompatibility complex; oHSV, oncolytic herpes simplex virus-1; PBS, phosphate buffered saline.

Fig 5: AN3025 significantly inhibits MC38 tumor growth as a monotherapy in hTNFR2 mouse model. (A–E) Murine colon cancer MC38 cells (5E5) were implanted subcutaneously into homozygous TNFR2 humanized mice (female). Mice were grouped when tumor volume reached approximately 100 mm3. (A, B) MC38 tumor bearing TNFR2 humanized mice were treated with AN3025 at the dosage of 10mg/kg, 3mg/kg or 1mg/kg every 4 days intraperitoneally for 5 doses in total. (A) Tumor volume measurement during the treatment (n=8 each group). (B) Body weight record during the treatment (n=8 each group). (C) MC38 tumor bearing TNFR2 humanized mice were treated with AN3025 at the dosage of 10mg/kg, 3mg/kg every 3 days intraperitoneally for 3 doses in total. Tregs (CD45+CD3+CD4+ Foxp3+) frequency in total CD4+ T cells in the MC38 tumors was quantified by flow cytometry (n=6 each group). (D, E) MC38 tumor bearing TNFR2 humanized mice were treated intraperitoneally with 10mg/kg AN3025 alone or combination of 10mg/kg AN3025, 0.3mg/mouse anti-mouse CD4 depletion antibody and 0.3mg/mouse anti-mouse CD8 depletion antibody every 4 days for 5 doses in total. (D) Tumor volume measurement during the treatment (n=8 each group). (E) Body weight record during the treatment (n=8 each group). Values were expressed as Mean ± SEM. **P < 0.01; ***P < 0.001.