CXCR3 expression in regulatory T cells drives interactions with type I dendritic cells in tumors to restrict CD8+ T cell antitumor immunity

Abstract

Infiltration of regulatory T (Treg) cells, an immunosuppressive population of CD4⁺ T cells, into solid cancers represents a barrier to cancer immunotherapy. Chemokine receptors are critical for Treg cell recruitment and cell-cell interactions in inflamed tissues, including cancer, and thus are an ideal therapeutic target. Here, we show in multiple cancer models that CXCR3⁺ Treg cells were increased in tumors compared with lymphoid tissues, exhibited an activated phenotype, and interacted preferentially with CXCL9-producing BATF3⁺ dendritic cells (DCs). Genetic ablation of CXCR3 in Treg cells disrupted DC1-Treg cell interactions and concomitantly increased DC-CD8⁺ T cell interactions. Mechanistically, CXCR3 ablation in Treg cells increased tumor antigen-specific cross-presentation by DC1s, increasing CD8⁺ T cell priming and reactivation in tumors. This ultimately impaired tumor progression, especially in combination with anti-PD-1 checkpoint blockade immunotherapy. Overall, CXCR3 is shown to be a critical chemokine receptor for Treg cell accumulation and immune suppression in tumors.

Keywords: CXCR3; Tregs; cancer; checkpoint blockade; cross presentation; dendritic cells; immunotherapy; regulatory T cells.

Figure 1.. CXCR3⁺ Treg cells are generated in response to tumors.

(A) Flow cytometric analysis of FOXP3 versus CXCR3 expression in live CD45⁺CD4⁺ T cells in tumor draining lymph node (dLN) and tumor (MC38). (B-D) Quantification of perecent CXCR3⁺ T cells in peripheral lymph node (pLN), dLN and tumor in Treg cells (B), eCD4⁺ (CD4⁺Foxp3⁻, C), and CD8⁺ T cells (D) on day 15 post MC38 tumor inoculation (n=16 pooled from four independent experiments). (E) Time course of Treg cell (Foxp3⁺CD25⁺/Live CD45⁺CD4⁺, left panel) and CXCR3⁺ Treg cell (right panel) frequency over the course of tumor growth in dLN (light purple) and tumor (dark purple) (n=3–4 mice/group, representative of two independent experiments). (F-G) Left panel: Flow cytometric staining for CD44 versus CXCR3 in FOXP3⁺CD25⁺ of Live CD45⁺ cells in dLN (F) and MC38 tumors (G). Right panels: Quantification of specified proteins in CD44⁻ (black), CD44⁺CXCR3⁻ (blue), and CD44⁺CXCR3⁺ (red) Treg cells (n=4–5 mice pooled from two independent experiments). Data represents mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 from two-way ANOVA followed by Dunnet’s multiple comparisons test. Also see Figure S1.

FIGURE 2.. Treg-specific CXCR3-deficiency impedes cancer.

(A) Left: genotype of female mice depicting the allele configuration of Foxp3 and Cxcr3 on the X chromosomes. Right: Schematic depicting the resulting Treg phenotypes from this genotype due to random X inactivation before and after DT treatment. Complementary flow cytometric analysis of CXCR3 versus Foxp3 GFP reporters in Live CD45⁺CD4⁺ cells from dLN before and after DT treatment is shown below schematic. (B) Experimental strategy to deplete Foxp3^DTR-GFP-expressing Treg cells in female mice with DT. (C-E) Left: genotype of female mice and representative flow cytometric analysis of CXCR3 versus Foxp3 GFP reporters in Live CD45⁺CD4⁺ cells from tumors with DT treatment. Right: tumor growth curves of MC38, EL4, and 9464D tumors in indicated mice treated with PBS or DT (n=7–8 mice/group, representative of three independent experiments for MC38 and two independent experiments for EL4 and 9464D). Results from Foxp3^DTR-GFP;Cxcr3⁺/Foxp3^TM2-GFP;Cxcr3^KO mice that generate Cxcr3^KO Treg cells with DT (C). Results from Foxp3^DTR-GFP;Cxcr3⁺/Foxp3^TM2-GFP;Cxcr3⁺ mice that maintain Cxcr3⁺ Treg cells with DT (D). Results from Foxp3^DTR-GFP;Cxcr3^KO/Foxp3^TM2-GFP;Cxcr3⁺ mice that maintain Cxcr3⁺ Treg cells with DT (E). Data represents mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and not significant (n.s.) from two-way ANOVA with Sidak’s multiple comparisons. Also see Figure S2.

Figure 3.. Enhanced CD8 tumor immunity with Cxcr3^KO Treg cells.

(A-B) Quantification of total intratumoral CD8⁺ T cells from Foxp3^DTR-GFP;Cxcr3⁺/Foxp3^TM2-GFP; Cxcr3^KO female mice harboring Cxcr3⁺ (+PBS, black) or Cxcr3^KO (+DT, red) Treg cells in MC38 tumors (day 28) (n=6–8 per group, data pooled from two independent experiments). (B) Tumor-specific p15E-reactive CD8⁺ T cells were measured from indicated organs (day 15, MC38) (n=4–5 mice per group, data representative of two independent experiments). Mice without tumors (naïve) served as a negative control for p15E/K^b tetramer stain (grey). Representative flow cytometric analysis of intratumoral pE15 tetramer stain in PBS and DT treated mice (right panel). (C) Growth of MC38 tumors in control females (+PBS, black), control females treated with anti-CD8 depleting antibody (+PBS +α-CD8, green), Cxcr3^KO Treg cell females (+DT, red), or Cxcr3^KO Treg cell females treated with an anti-CD8 depleting antibody (+DT +α-CD8, blue) (n=8 mice per group, representative of two independent experiments). Representative flow cytometric analysis of CD4⁺ and CD8⁺ cells in spleens of control or anti-CD8 treated groups (right panel). (D) IFN-γ⁺TNFa⁺CD8⁺ of Live CD45⁺ in tumors (day 15, MC38) from indicated mice after no stimulation (no stim) or stimulation with PMA and Ionomycin (P+I) ex vivo (n=4–5 per group). (E) PD-1 expression on intratumoral CD8⁺ T cells (day 28, MC38) (data pooled from three independent experiments). (F) Growth of MC38 tumors in mice with Cxcr3⁺ (+PBS, black) or Cxcr3^KO Treg cells (+DT, red), or treated with anti-PD-1 in mice with Cxcr3⁺ (+α-PD-1, purple) or Cxcr3^KO Treg cells (+DT+α-PD-1, green) (n=6–7 mice per group). Data represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 from two-way ANOVA followed by Dunnet’s multiple comparisons test, two-way ANOVA with Sidak’s multiple comparisons, or student’s t tests. Tumor growth assessed using multiple regression analyses. Also see Figure S3.

Figure 4.. CXCR3 is required for Treg accumulation and activation in tumors.

(A-B) Quantification of intratumoral Treg cell frequency or their PD-1 and CTLA-4 expression from Foxp3^DTR-GFP;Cxcr3⁺/Foxp3^TM2-GFP;Cxcr3^KO female mice harboring Cxcr3⁺ (+PBS, black) or Cxcr3^KO (+DT, red) Treg cells at day 15 (A) or day 28 (B) of MC38 tumor progression (n=7 mice per group, pooled from two independent experiments). (C) Schematic describing the generation of CXCR3⁺ and Cxcr3ΔTregcells by in vitro differentiation, fluorescent dye-labeling, and co-transfer into MC38 tumor-bearing mice for localization analysis. (D) Analysis of Treg cells for CXCR3 and T-BET expression prior to adoptive transfer. (E) Representative flow cytometry plots from dLN and MC38 tumors for transferred Cxcr3⁺ and Cxcr3^KO Treg cells. (F) Absolute number of Cxcr3⁺ (black) or Cxcr3^KO Treg cells (red) cells recovered. Lines connect Treg cells collected from the same mouse (data pooled from three independent experiments). Data represents mean ± SEM; *p < 0.05, **p < 0.01 by unpaired (or paired analysis in F) Student’s t tests. Also see Figure S4.

Figure 5.. CXCR3 expression on Tregs facilitates CD11c⁺ cell interaction in tumors.

(A) Genotype of female mice and schematic depicting the resulting Treg phenotypes after DT treatment. Mice were treated with DT beginning 7 days prior to tumor inoculation and throughout tumor progression. (B-C) Representative confocal microscopy images from WT (B) and KO (C) mice as described in (A) at 15 days of tumor progression showing MC38-CFP (blue), CD11c-YFP (yellow), Foxp3^TM2-GFP (green), and CD8⁺ (red) cells. Arrowheads point to cell-cell interactions between Tregs and CD11c⁺ cells (representative of 60–70 images analyzed from 3 mice per group pooled from 3 independent experiment). Scale bar = 50 μm. (D-F) High magnification examples of counted cell-cell interactions between Treg-CD11c cells (D, E) and CD8-CD11c cells (E, F). Scale bar = 15 μm. (G-H) Total numbers of Tregs, CD11c cells (G) or CD8⁺ cells (H) were counted per field at 20X magnification. Cell-cell interactions were examined at higher magnification and confirmed through multiple z-stacks. Cell-cell interactions were normalized based on the number of Tregs (G) or CD8⁺ cells (H) per field to yield a probability of interaction per Treg or CD8⁺ cell (representative of 60–70 images analyzed from 3 mice per group and pooled from 3 independent experiments). Data represents mean ± SEM; *p < 0.05, **p < 0.01 ***p < 0.001 by Student’s t test.

Figure 6.. Type 1 DCs are required for the preferential localization of CXCR3⁺ Tregs in tumors.

(A-D) Representative confocal microscopy images from Foxp3^DTR-GFP;Cxcr3⁺/Foxp3^TM2-GFP; Cxcr3⁺;CD11c-YFP mice treated with DT and analyzed at 15 days of tumor progression showing CD11c-YFP (yellow) and Foxp3^TM2-GFP (green) (A), anti-CXCL10 and MC38-CFP (B), anti-CXCL9 and MC38-CFP (C), and a composite image (D). Arrowheads point to Tregs (green), CXCL9⁺ (red), and CXCL10⁺ (white) cells. Scale bar = 20 μm. (E) Correlation between the number of Foxp3^TM2-GFP-expressing CXCR3⁺ Tregs (blue) from DT treated Foxp3^DTR-GFP; Cxcr3⁺/Foxp3^TM2-GFP;Cxcr3⁺;CD11c-YFP mice or Foxp3^TM2-GFP-expressing CXCR3^KO Tregs (red) from DT treated Foxp3^DTR-GFP;Cxcr3⁺/Foxp3^TM2-GFP;Cxcr3^KO;CD11c-YFP mice versus CXCL9⁺CD11c⁺ cells per field at 20X magnification in MC38 tumors at day 15 of tumor progression (representative of 50–60 images analyzed from 3 mice per group and pooled from 3 independent experiments). (F) MC38 tumors from DT-treated mice bearing CXCR3⁺ or CXCR3^KO Tregs were harvested at day 15 of tumor progression and CXCL9 and CXCL10 were quantified by ELISA (n= 6–7 mice per group, pooled from two independent experiments). (G) MC38 tumors from WT or Batf3^KO mice were tested for CXCL9 and CXCL10 levels as in (G) (n= 3–4 mice per group, pooled from two independent experiments). (H) Representative confocal microscopy images from Foxp3^DTR-GFP;Cxcr3⁺/Foxp3^TM2-GFP;Cxcr3⁺ mice with CXCR3⁺ Tregs at 15 days of tumor progression showing MC38-CFP (blue), anti-XCR1 (yellow), Foxp3^TM2-GFP (green), and anti-CXCL9⁺ (red) cells. Scale bar = 5 μm. (I-J) Fraction of CXCL9⁺ cells that are XCR1⁺ (CXCL9⁺XCR1⁺/CXCL9⁺) assessed by immunofluorescence (I). Total Tregs (J) and XCR1-Treg interactions (K) were examined at higher magnification and confirmed through multiple z-stacks. Cell-cell interactions were normalized based on the number of Tregs (K) per field to yield a probability of interaction per Treg. Cells were counted per field at 20X magnification (representative of 22–24 images analyzed from 4 mice per group). (L) MC38 tumors from WT (black), Batf3^KO (grey), or WT mice treated with PBS (black), anti-CD8 (blue), or anti-IFN-γ (green) were tested for CXCL9 by ELISA (L, n= 4 mice per group). (M-N) Flow cytometric analysis for intracellular CXCL9 protein in DC1 and DC2 from MC38 tumors in mice as described in (L) and quantified in (N, n= 4 mice per group). (O) Flow cytometric analysis for intracellular CXCL9 protein in DC1 and DC2 from MC38 tumors in mice with CXCR3⁺ (green) or CXCR3^KO (red) Tregs after treatment with DT or in Batf3^KO (grey) mice and quantified (n=3–4 mice per group). (P) Schematic describing the generation of CXCR3⁺ and CXCR3^KO Tregs by in vitro differentiation, fluorescent dye-labeling, and co-transfer into MC38 tumor-bearing C57BL/6 wildtype or Batf3^KO mice for localization analysis after 24 hours. (Q) Absolute number of CXCR3⁺ (black filled) or CXCR3^KO (red filled) Tregs recovered in dLN and tumor 24 hours after transfer into C57BL/6 wildtype mice (WT) or number of CXCR3⁺ (black hatched) or CXCR3^KO (red hatched) Tregs recovered after transfer to Batf3^KO mice. Lines connect CXCR3⁺ and CXCR3^KO Tregs collected from the same mouse (n=3 mice per genotype/experiment and all data pooled from three independent experiments). Data represents mean ± SEM; *p < 0.05, **p < 0.01 by Student’s t test. For correlation in (F), a simple linear regression was employed and slopes between regressions were compared.

Figure 7.. Lack of CXCR3⁺ Treg cells boosts cross-presentation in tumors.

(A) Experimental setup to assess tumor antigen cross-presentation by analyzing SIIN/H-2K^b presentation on antigen presenting cells (25D1.16 antibody) and CD8⁺ SIIN/K^b-tetramer⁺ T cell responses to ovalbumin expressed from β2m-deficient tumors. (B-E) Representative flow cytometric analysis (B) and quantification (C-E) of the frequencies of intratumoral SIIN/K^b-tetramer-specific CD8⁺ T cells in indicated mice (day 15–20, MC38) (C-D, n=5–6 mice per group, data pooled from three independent experiments; E, n=3–4 mice per group, data representative of two independent experiments). (F-G) Representative flow cytometric analysis (F) and quantification (G) of SIIN/H-2K^b presentation on intratumoral DC1 (XCR1⁺CD103⁺) (left) and DC2 (right) using 25D1.16 from MC38-β2m-deficient tumors that do no express OVA (OVA-negative, black) or OVA-expressing tumors in Foxp3^DTR-GFPCxcr3^KO/Foxp3^TM2-GFP;Cxcr3⁺ (CXCR3⁺, green) and Foxp3^DTR-GFPCxcr3⁺/Foxp3^TM2-GFP;Cxcr3^KO mice treated with DT (CXCR3^KO, red) or PBS (CXCR3⁺, grey) (n=4–5 mice per DT-treated group pooled from two experiments). Data represents mean ± SEM; *p < 0.05, **p < 0.01 from one-way ANOVA followed by Dunnet’s multiple comparisons test. Also see Figure S7.