Fig 2: IRE1a inhibition attenuates EMT6 tumor growth and synergizes with anti–PD-L1 treatment in conjunction with increased DC and CD8+ T cell tumor infiltration and activation. Mice were inoculated orthotopically with EMT6 cells in the mammary fat pad, grouped out 7 d later, and treated with vehicle, G9668 (p.o., 250 mg/kg, BID), anti–PD-L1 antibody (i.v., 10 mg/kg at first dose, i.p., 5 mg/kg BIW thereafter), or the combination. (A) Tumor growth trajectories were measured over 19 d (n = 15). (B and C) Mice were treated with G9668 for 7 d and then sacrificed (n = 9). (B) Abundance of tumor cDC1s and cDC2s, as well as expression of MHC-I and CD59, were measured by flow cytometry. Total DCs were characterized as F4/80low, CD11c+ and MHC-IIhigh, while cDC1s were further characterized as CD103+ XCR1+ CD11b-, and cDC2s were characterized as CD103- XCR1- CD11b+. MFI, mean fluorescence intensity. (C) Abundance and activation marker expression of tumor-infiltrating CD8+ T cells were assayed by flow cytometry. Analysis was performed using one-way ANOVA for A and unpaired, two-tailed t test for B and C; *, P = 0.05; **, P = 0.01; ***, P = 0.001. Scatter plots in all panels represent mean ± SEM.

Fig 3: TCF1+and TCF1-Tregs Have Distinct Transcriptomes(A) Principle-component analysis (PCA) of the transcriptomes of the R1, R2, and R3 Treg subsets as defined in Figure 1.(B) Volcano plots showing the comparisons among the three Treg subsets. The T cell activation gene signature (red open circles) was superimposed onto each plot. The numbers of T cell activation signature genes that were over- or under-represented in each comparison and the p values (by ?2 test) were shown.(C and D) Heatmaps (row normalized) depicting the expression of the selected transcription factor (C) and key functional molecules (D) in the R1, R2, and R3 Treg subsets. Genes were ranked and clustered according to their correlation to Tcf7 (C) or Icos (D). r2, correlation coefficient; min, lowest expression in each row; max, highest expression in each row.(E and F) Flow cytometric analysis of surface molecule: ICOS, CD103, and KLRG1 (E) and transcription factors: IRF4 and Blimp1 (F) in the R1, R2, and R3 Treg subsets.(G) Graphic summary illustrating the revised model of the Treg pool in peripheral lymphoid organs.The data are representative of one experiment with n = 2 replicates for each Treg subsets (A–D) or n = 3 independent experiments (E and F). Shown are mean ± SEM (E and F). p values in (B) were calculated using the chi-square (?2) test. p values in (E) and (F) were calculated using the two-tailed unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., non-significant.See also Figure S2 and Tables S1, S2, and S3.

Fig 4: IRE1a inhibition attenuates CT26 tumor growth in conjunction with enhanced MHC-I expression on tumor DCs and CD8+ T cell recruitment and activation. Mice were inoculated s.c. with CT26 cells, grouped out 7 d later, and treated with vehicle or G9668 (p.o., 250 mg/kg, BID). (A) Growth trajectories of CT26 tumors in vehicle- and G9668-treated animals over 17 d (n = 15). (B–G) Flow cytometry analysis of tumor-infiltrating DCs and T cells from mice treated for 7 d. (B–D) Quantification of tumor-infiltrating total DCs (B) and cDC1s (C) and characterization of cDC1 expression of MHC-I and CD59 (D) by flow cytometry (n = 9 for vehicle-treated group and n = 10 for G9668-treated group). Total DCs were characterized as F4/80low, CD11c+, and MHC-IIhigh, while cDC1s were characterized as CD103+ XCR1+ CD11b-. (E–G) Measurement of tumor-infiltrating CD8+ T cell abundance (E), expression of indicated activation markers (F; n = 9 for vehicle-treated group and n = 10 for G9668-treated group), and binding of GP70 tetramers (G; n = 6 for vehicle and 8 for G9668 group). Analysis was performed using one-way ANOVA for A and unpaired, two-tailed t-test for B–G; *, P = 0.05; **, P = 0.01; ***, P = 0.001; ****, P = 0.0001. Scatter plots in all panels represent mean ± SEM.

Fig 5: IRE1a activation depletes MHC-I heavy-chain mRNAs via RIDD. (A) Consensus stem-loop endomotifs for RIDD recognition in murine MHC-I heavy-chain H-2K, H-2D, and H-2L mRNA sequences. (B) Purified recombinant IRE1a kinase-RNase (KR) protein was incubated with RNA transcripts of H-2K, H-2D, and H-2L, and G9668 (10 µM), followed by agarose gel electrophoresis to determine transcript integrity. (C and D) BMDCs were treated with actinomycin D (2 µg/ml) to block de novo transcription and pulsed with ovalbumin (500 µg/ml) for indicated time points (C) or with G9668 (3 µM) for 8 h (D). Indicated transcripts were analyzed by RT-qPCR. (E) C57BL/6 mice were injected with Flt3L-Fc (i.v., 10 mg/kg) and 8 d later were treated with G9668 (p.o., 250 mg/kg, BID) for 24 h in combination with a single injection of DEC-OVA (i.v., 2.5 mg/kg) 2.5 h before sacrifice. Viable splenic DCs (CD11c+ MHC-IIhigh F4/80-) were gated and sorted cDC1 (CD103+ XCR1+ CD11b-) subpopulation cells were analyzed by RT-qPCR for indicated transcript levels. Statistical analysis was performed using unpaired, two-tailed t test; *, P = 0.05; ***, P = 0.001. Image in B represents three similar experiments, and molecular weights represent base pairs. Bar graphs in C and D represent mean ± SD from three independent biological repeats; bar graphs in E represent mean ± SD from two independent technical repeats. Source data are available for this figure: SourceData F4.