Fig 2: Expression of NUP214-ABL1 and TLX1 Is Required to Induce T-ALL in a Transgenic Mouse Model(A) Pie chart representing the percentage of T-ALL (left) or NUP214-ABL1-positive T-ALL (right) with TLX1 or TLX3 expression.(B) Schematic overview of the transgenic mouse models used in this study. Red triangles represent LoxP sites. A conditional loxP-STOP-loxP NUP214-ABL1 knockin mouse model (abbreviated as LSL-NA) was generated. NUP214-ABL1 expression was initiated by crossing LSL-NA mice with CD4-Cre mice. Co-expression of NUP214-ABL1 and TLX1 was achieved by crossing NA mice with Tg(Lck-TLX1) mice, resulting in Tg(CD4 Cre; NUP214-ABL1; Lck TLX1) mice (abbreviated as NA + TLX1).(C) Kaplan-Meier overall survival curve comparing NA + TLX1, TLX1, and NA mice.(D) Representative fluorescence-activated cell sorting (FACS) analysis of GFP expression in NA + TLX1 mice at end-stage disease compared with wild-type (WT) cells for spleen, thymus, peripheral blood (PB), and bone marrow (BM).(E–G) Peripheral white blood cell count (WBC) (E), spleen weight (F), and thymus weight (G) at end-stage disease for NA + TLX1 mice compared with NA and LSL-NA mice (end stage for NA and LSL-NA defined as >360 days). Star indicates NA + TLX1 mouse that presented with an elevated WBC, but did not present with an enlarged spleen or thymus at end stage. Statistical significance was calculated using a Mann-Whitney test. Data are presented as mean ± SD. N.s., not significant.(H) Representative FACS analysis for CD4 and CD8 expression in GFP-positive NA + TLX1 leukemic cells from the peripheral blood compared with NA and LSL-NA peripheral blood cells.(I) H&E and immunohistochemical staining for CD3 and Cre in spleen cells from LSL-NA, NA, and NA + TLX1 mice. Scale bars represent 100 µm.(J) Kaplan-Meier overall survival curve of secondary (using cells from three different primary NA + TLX1 mice) and tertiary transplants.(K) Growth curve of ex vivo primary immature pro T cells expressing EML1-ABL1, TLX1 or both. Data are presented as mean ± SD.(L) Kaplan-Meier overall survival curve of mice transplanted with hematopoietic stem/progenitor cells expressing EML1-ABL1, TLX1 or EML1-ABL1+TLX1.See also Figures S1–S4 and Table S1.

Fig 3: Therapeutic effect of C20 in combination with ICI in two different schedules of vaccination.a Balb/c mice were inoculated s.c. with CT26 cells and treated with C20 and ICI starting from day 2 as depicted in the experimental scheme. Tumor volume and survival curve. b, c Balb/c mice were inoculated s.c. with CT26 cells and treated with ICI and NCV according to the experimental scheme. b Tumor (50–100 mm3) bearing mice were randomized at day 6 and treated with aCTLA-4 and vaccinated with C20 the day after. The treatment was repeated weekly as described in the scheme. b Tumor volume measurements and survival curve. This experiment was conducted only once (C) CT26 tumor growth in CD4 or CD8 depleted mice treated as in panel (b). This experiment was repeated twice with similar results. Six animals per group were utilized with the error bars representing the s.e.m. Significance was determined using Mann–Whitney and Log-rank (Mantel–Cox) test **p < 0.01 ***p < 0.001.

Fig 4: MALT1 plays a profound role in the immune rejection of tumor cells and the response to the anti-PD-1 therapy.a WT Pan T cells were stimulated by plate-bound anti-CD3/28 (5 µg/ml) for the indicated time points. The Malt1 protein level (left) and mRNA level (right) was detected by WB or qPCR, respectively. b Tumor cells were inoculated subcutaneously in Malt1+/- and Malt1-/- mice. Tumor growth was measured using calipers at indicated time points and tumor weights were calculated at the end stage. c The percentage of CD8+ T cells in the tumor-infiltrated lymphocytes (TILs) gated on CD45.2+. d The percentage of IFN-?- and Granzyme B-producing CD8+ T cells gated on CD8+ cells. e The FACS plot of PD-1-expressing CD8+ T cells gated on CD8+ cells, and statistical results of PD-1+CD8+ percentage and mean fluorescence intensity (MFI) of PD-1 expression on CD8+ T cells. f Tumor growth in LysCre/Malt1fl/fl and control mice. g Tumor growth in CD4Cre/Malt1fl/fl and control mice. h Tumor growth in mice treated with anti-PD-1 antibody or IgG control. Statistical analysis was performed by unpaired Student t-test and the error bars represent ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. These results are from one representative experiment of three independent biological replicates.

Fig 5: Repeat Treg depletion. Repeat rounds of DTX medicated Treg depletion in BALB/c FoxP3.dtr mice treated with 25 ng/g/mouse DTX i.p. (A) AB1-HA tumor growth in BALB/c FoxP3.dtr mice treated with two rounds of DTX (black arrows) 1day apart. (B) Transient Treg depletion in blood correlated with increase CD8 T cell activation in DTX-treated mice. (C) Changes in CD4, CD8, and Treg proportions and activation status (ICOS expression, P < 0.01) in peripheral blood over time. T cell activation (white arrows) correlated with transient Treg depletion (grey arrows). All mice remained healthy after receiving second round of the same dose of DTX (R2). Representative data showing mean (n = 5/group) ± SEM from two independent experiments. CD4 (open circle) and CD8 (open square) as proportion of total lymphocytes. Treg (closed diamond) as proportion of total CD4 T cells. Mixed model analysis of variance was used to analyse difference in growth. Mann–Whitney U-test two groups *P < 0.05, **P < 0.01, ***P < 0.001.