In colorectal cancer mast cells contribute to systemic regulatory T-cell dysfunction

Abstract

T-regulatory cells (Treg) and mast cells (MC) are abundant in colorectal cancer (CRC) tumors. Interaction between the two is known to promote immune suppression or loss of Treg functions and autoimmunity. Here, we demonstrate that in both human CRC and murine polyposis the outcome of this interaction is the generation of potently immune suppressive but proinflammatory Treg (DeltaTreg). These Treg shut down IL10, gain potential to express IL17, and switch from suppressing to promoting MC expansion and degranulation. This change is also brought about by direct coculture of MC and Treg, or culture of Treg in medium containing IL6 and IL2. IL6 deficiency in the bone marrow of mice susceptible to polyposis eliminated IL17 production by the polyp infiltrating Treg, but did not significantly affect the growth of polyps or the generation of proinflammatory Treg. IL6-deficient MC could generate proinflammatory Treg. Thus, MC induce Treg to switch function and escalate inflammation in CRC without losing T-cell-suppressive properties. IL6 and IL17 are not needed in this process.

Fig. 1.

Proinflammatory cytokines are elevated in CRC tumors. Cytokine levels in fresh tumor lysates from CRC patients were determined by ELISA; n = 11 in triplicate, P < 0.0003.

Fig. 2.

Foxp3⁺ T cells accumulate in human CRC tumors. Total number and frequency of CD4⁺ (A) or Foxp3⁺ (B) cells per total number of cells in each 200× field of vision. Total: CD4, margin 121 ± 6 cells vs. tumor 119 ± 6 cells; Foxp3, margin 17 ± 2 cells vs. tumor 37 ± 2 cells, P = 0.0001; Frequency: CD4, margin 15 ± 0.8% vs. tumor 12.2 ± 0.6%, P = 0.009; Foxp3, margin 2.2 ± 0.2% vs. tumor 3.8 ± 0.3%, P = 0.0001, two-tailed unpaired t test with Welch’s correction; n = 13, three to five fields per slide. (C) Frequency of Foxp3⁺ cells to CD4⁺ cells determined from serial section stainings, margin 13.7 ± 1.1% vs. tumor 29.9 ± 1.6%, P = 0.0001. (D) Representative FACS plot of tumor-derived MNC gated on CD4 and analyzed for Foxp3 and CD25. (E) Compiled frequencies of CD4⁺CD25⁺Foxp3⁺ cells and (F) CD4⁺CD25⁺Foxp3⁻ tumor-infiltrating T cells; n = 9 patients. P values: (E) P = 0.0063 for margin 5.3 ± 1.2% vs. tumor 10.3 ± 1.5%, P = 0.0452 for tumor vs. blood 4.8 ± 1.9%; (F) not significant.

Fig. 3.

Increased MC infiltrate in CRC tumors. Total number and frequency of CAE⁺ (A) or tryptase⁺ (B) cells per total number of cells in each 200× field of vision. Total number: CAE, margin 8 ± 1 cells vs. tumor 22 ± 1 cells, P = 0.0001; tryptase, margin 17 ± 1 cells vs. tumor 30 ± 2 cells, P = 0.0001. Frequency: CAE, margin 1.7 ± 0.2% vs. tumor 7.4 ± 0.6%, P = 0.0001. (C) Frequency of CD11b⁺ (Upper, margin 12.3 ± 1.1% vs. tumor 18.6 ± 1.6, P = 0.0013) and Mac⁺ (Lower, margin 7.8 ± 0.6 vs. tumor 12.1 ± 1.1%, P = 0.0022) myeloid cells in CRC. For all, n = 13, three to five fields per slide, two-tailed unpaired t test with Welch's correction.

Fig. 4.

Treg from CRC patients are proinflammatory. (A) Treg from healthy donors PB and CRC PB, tumor, and margin inhibit T-cell proliferation; n = 2 for PB and n = 1 for tissue, tested in triplicate. (B) Compiled values of % LAD2-MC degranulation sensitized (IgE) and cross-linked with anti-(IgE+Ag = 40.1 ± 3.2%) incubated with CD4 effector (Teff) or CD4⁺CD25+ (Treg) T cells. Healthy donors Treg vs. healthy donors Teff, 34.4 ± 2.8% vs. 45.3 ± 3.3%, P = 0.0003; healthy donors Treg vs. CRC PB, 34.4 ± 2.8% vs. 48.8 ± 4.6%, P = 0.0354; healthy donors Treg vs. CRC tumor, 34.4 ± 2.8% vs. 50.4 ± 2.2%, P = 0.0179; healthy donors Treg vs. CRC margin, 34.4 ± 2.8% vs. 47.1 ± 3%, P = 0.0185; n = 5, in duplicate.

Fig. 5.

Tumor-infiltrating Treg from CRC lack IL10 but produce IL17. (A) Representative FACS plots of cytokine production by live CD4⁺CD25⁺Foxp3⁺ cells isolated from CRC patients and healthy donor PB. (B–D) Compiled frequencies among total MNC of IL10-producing T cells derived from CRC patients or healthy donors; n = 5. CD4⁺CD25⁺Foxp3⁺, for margin 5.9 ± 1.9% vs. tumor 2 ± 0.7%, P = 0.0418; for CRC PB 2.2 ± 0.6% vs. healthy donors PB 5.7 ± 0.7%, P = 0.0022; CD4⁺CD25⁺Foxp3⁻, CRC PB 1.1 ± 0.3% vs. HD PB 4.3 ± 0.9, P = 0.0481; CD4⁺CD25⁻Foxp3⁺ not significant. (E–G) Compiled frequencies of T cells expressing IL17; n = 9; CD4⁺CD25⁺Foxp3⁺, margin 4 ± 1.3% vs. tumor 9.9 ± 2.5%, P = 0.0050; CD4⁺CD25⁺Foxp3⁻, not significant; CD4⁺CD25⁻Foxp3⁺, margin 3.8 ± 1.6% vs. tumor 10.5 ± 3.3%, P = 0.0179.

Fig. 6.

Treg become proinflammatory in the presence of IL6. Typical FACS plot of cytokine production (A) or pSTAT3 (B) expression by Treg from healthy donors PB untreated or treated with IL6 in complete medium containing IL2 for 5 days, n = 3. (C) Compiled values of % LAD2-MC degranulation and impact of Treg from healthy donors untreated or pretreated with IL6; 34.4 ± 2.8% vs. 47.8 ± 4.9%, P = 0.0025, n = 4 in duplicate. (D) Treg cultured for 5 days with or without IL6 assayed for suppressing CD4 T cell proliferation; n = 2 in duplicate.

Fig. 7.

MC and IL6 alter murine Treg functions. (A) Typical FACS plots of intracellular cytokines in Treg derived from wt spleen cultured without or with BMMC for 3 or 5 days. Live cells were gated on CD4⁺CD25⁺Foxp3⁺, n = 5. (B) Compiled frequencies of % pSTAT3 expressing Treg, cultured alone (white bar), with IL6 (striped bar), or with BMMC (gray bar; 1:1), striped compared with white, 6.6 ± 0.6% vs. 2.7 ± 0.4%, P = 0.0106; gray compared with white, 9.1 ± 0.6% vs. 2.7 ± 0.4%, P = 0.003; n = 3). (C) MC degranulation with no Treg (black 31.2 ± 1.3%), with Treg (white 18.8 ± 1.8%, P = 0.0035), or with IL6 pretreated Treg (striped 25.9 ± 1.7%, P = 0.0046); n = 3. (D) Suppression of T cell proliferation by untreated or IL6 pretreated Treg, n = 2 in triplicate. (E) BMMC degranulation assessed in absence (IgE+Ag) or presence of Treg from healthy mice (wt) or polyp-ridden mice (APC^Δ468, P = 0.0007 compared with wt Treg and compared with IgE+Ag, P = 0.0427), n = 3 in duplicate. (F) Frequency of MCp among total MNCs isolated from the intestine of Rag^−/− mice in absence of Treg (black bar 1,339 ± 58 MCp) or on inclusion of Treg from spleen and MLN of healthy mice (white, wt 838 ± 28MCp) or polyp-ridden mice (gray, APC^Δ468 2300 ± 119 MCp); n = 3 in duplicate, P = 0.0001.

Fig. 8.

Functional alteration of Treg by MC is independent of IL6 and IL17. (A) Typical FACS plot of IL17 production by polyp-infiltrating Treg from mice reconstituted with wt BM or IL6^−/− BM. Total MNC were isolated from microdissected polyps, stimulated with anti-CD3/CD28 for 3 days and then stained for analysis. Cells were gated for CD4⁺CD25⁺. (B) Compiled % of IL17 producing Foxp3⁺ cells; wt BM 6 ± 1.5%, IL6^−/− BM 1.8 ± 0.2%, n = 3, P = 0.0236, one-tailed unpaired t test. (C) IL17 ELISA of serum; white, IL6^−/− BM (127 ± 12.9 pg/mL) compared with black, wt BM (213.3 ± 17.1pg/mL), P = 0.0007; gray, control healthy wt mouse (64.3 ± 7.8 pg/mL) compared with polyp-ridden with wt BM P = 0.0001, and compared with polyp-ridden IL6^−/− BM mice P = 0.0.003. (D) wt Treg inhibited BMMC degranulation (control vs. IgE+Ag; 19.5 ± 1.2% vs. 30.9 ± 1.3%, P = 0.0001), whereas Treg from poly-ridden mice with wt (30 ± 1.9%) or IL6^−/− BM (29.8 ± 1.7%) did not; P = 0.0036 wt BM compared with control wt Treg; P = 0.0150 IL6^−/− BM compared with control wt Treg. (E) After 5 days in culture, wt B6 Treg inhibit BMMC degranulation (no MC compared with IgG+Ag; white = 16.8 ± 1.8% vs. black = 31.2 ± 1.3%, P = 0.0040), whereas preincubation for 5 days with BMMC (gray 26.2 ± 2%, P = 0.0212) or IL6^−/− BMMC (striped 22.9 ± 1.7%, P = 0.0282) did not; n = 3 mice, in duplicate.