An Atlas of Human Regulatory T Helper-like Cells Reveals Features of Th2-like Tregs that Support a Tumorigenic Environment

Abstract

Regulatory T cells (Tregs) play a pivotal role in maintaining immunological tolerance, but they can also play a detrimental role by preventing antitumor responses. Here, we characterized T helper (Th)-like Treg subsets to further delineate their biological function and tissue distribution, focusing on their possible contribution to disease states. RNA sequencing and functional assays revealed that Th2-like Tregs displayed higher viability and autocrine interleukin-2 (IL-2)-mediated activation than other subsets. Th2-like Tregs were preferentially found in tissues rather than circulation and exhibited the highest migratory capacity toward chemokines enriched at tumor sites. These cellular responses led us to hypothesize that this subset could play a role in maintaining a tumorigenic environment. Concurrently, Th2-like Tregs were enriched specifically in malignant tissues from patients with melanoma and colorectal cancer compared to healthy tissue. Overall, our results suggest that Th2-like Tregs may contribute to a tumorigenic environment due to their increased cell survival, higher migratory capacity, and selective T-effector suppressive ability.

Keywords: T helper-like regulatory cells; chemokine receptor; immunoregulation; tumor immunity; tumor immunology.

Graphical abstract

Figure 1

Identification of Four Th-like Tregs Based on CXCR3, CCR4, and CCR6 Expression (A) CCR4, CXCR3, and CCR6 expression was analyzed in memory CD4⁺CD25^hiCD127^lowCD45RA⁻ Tregs. Four Th-like lineages were identified in the circulation: Th2, Th17, Th1, and Th1/17-like Tregs. (B) FoxP3 expression between Teff and Th-like Treg subsets. For (A) and (B), data are presented as mean ± SEM (n = 8) using independent values (RM one-way ANOVA with Tukey’s test). (C and D) Principal-component analysis (C) and volcano plots (D) showing ANOVA of RNA-seq data obtained from activated Th-like Treg subsets. Thick vertical lines indicate 1.5-fold change threshold (n = 3, using independent values clustered with ellipsoids). (E) Heatmap showing upregulation of Th-lineage genes between Th-like Treg subsets using Partek software. (F and G) Protein expression of GATA3, RORγτ, and T-bet in FoxP3⁺ Treg subsets (F) and absolute values of cytokine production by activated Th-like Treg subsets (G) (n = 4, mean ± SEM using independent values, RM one-way ANOVA with Tukey’s test). For all statistical tests, ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05 were considered significant.

Figure 2

Th2-like Tregs Exhibit Higher Viability, Activation, and JAK-STAT Signaling Pathway Than Other Treg Subsets (A) Total percentages of live and blasting cells between Th-like Tregs 72 hr post-TCR activation in the absence of IL-2 (n = 10, mean ± SEM using independent values, RM one-way ANOVA with Tukey’s test). (B) Distribution of dead, apoptotic, and live cells between Th-like Tregs after TCR activation (n = 5). (C and D) The percentage of live and blasting cells was analyzed in Th-like Treg subsets activated in the presence of neutralizing antibodies for IL-2, IL-4, IFN-γ, and IL-17 (all at 10 μg/mL) (C) or 250 U/ml exogenous IL-2 (D) (n = 4, mean ± SEM using bar charts, RM two-way ANOVA with Tukey’s test). (E) STAT5 signaling and p53 expression was measured in Th-like Tregs 16 hr post-TCR activation in the presence or absence of IL-2 (250 U/mL) (n = 4, mean ± SEM using bar charts, RM two-way ANOVA with Sidak’s test). (F) Heatmap showing upregulation of JAK-STAT, TCR signaling pathway and pro and anti-apoptotic genes between Th-like Treg subsets using Partek software and the KEGG database. For all statistical tests, ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05 were considered significant.

Figure 3

Th-like Tregs Suppress Cell Division of Th-like Teffs without Preference for Lineage Counterparts (A) Representative dot plots of Th-like Teffs. Th2, Th17, Th1, and Th1/17 were identified from memory Teff CCR4⁺ cells. (B) Representative histograms and total percentages of divided Cell Trace Violet⁺ Th-like Teff subsets (1 × 10⁵) stimulated with anti-CD3/CD28 beads at a 40:1 (cell/bead) ratio for 4 days (n = 5, mean ± SEM using bar chart and independent values, RM one-way ANOVA with Tukey’s multiple comparison test). (C and D) Representative histograms (C) and division (Div.) index (D) were obtained from suppression assays between memory Teff or Th-like Teff and Th-like Treg subsets. Teffs (1 × 10⁵) alone or with autologous Tregs (0.5 × 10⁵) were activated with anti-CD3/CD28 beads at a 40:1 (cell/bead) ratio for 4 days. The data are presented as division index obtained from FlowJo analysis (n = 6, mean ± SEM using bar charts, RM two-way ANOVA with Tukey’s test). (E) Absolute values of IL-4, IFN-γ, IL-17, and IL-10 obtained from supernatants after 4 days of suppression assays (n = 6, mean ± SD using bars, RM Two-way ANOVA with Tukey’s test). For all statistical tests, ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05 were considered significant.

Figure 4

Th2-like Tregs Exhibit Higher Chemotaxis toward CCL17/22 Than Other Th-like Tregs and Th2-like Teffs (A and B) Volcano plots showing RNA-seq data obtained from activated Th-like Treg subsets (A), and heatmaps showing upregulation of leukocyte transendothelial migration, chemokines, and chemokine receptors genes between Th-like Treg subsets using Partek software and the KEGG database (B). (C) Representative dot plots and percentage of migrated memory Teffs and Tregs. Memory Teffs (5 × 10⁴) and memory Tregs (5 × 10⁴) were placed in the top chamber of a 5-μm-pore Transwell filter system with ICAM (1 μg/mL). Bottom chambers were filled with media only; CCL17/22 (0.5 μg/mL), CCL20 (0.5 μg/mL), or CXCL10 (0.5 μg/mL); or a combination of all of them. The percentage of migration for each subset was calculated as (number of cells in the bottom chamber after 1 hr × 100)/initial number of cells in the top chamber. (D and E) Representative dot plots and percentage of migration between Th-like Treg subsets (D) and between CCR4⁺ Th-like Teff and Th-like Tregs (E) (n = 6, mean ± SEM using bar charts, RM two-way ANOVA with Sidak’s test). For all statistical tests, ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05 were considered significant.

Figure 5

Distribution of Th-like Teff and Treg Subsets in Health and Malignancy (A) Representative plots of chemokine receptor expression in Th-like Tregs obtained from tissues. (B) Pie charts and total percentages of Th-like Tregs and Th-like Tregs Teff in tissues and peripheral blood (mean ± SEM using boxplots, RM two-way ANOVA with Sidak’s test). Thymus = 6, spleen = 8, liver perfusates = 6, healthy skin = 5, skin with cancer = 4, healthy colon = 6, colon with cancer = 5, peripheral blood from healthy donors = 8, peripheral blood from patients with skin cancer = 10, and peripheral blood from patients with colon cancer = 5. (C and D) Treg/Teff ratio (C) and tissue distribution of Th-like cells between healthy individuals and patients with skin or colon cancer (D). In (C), data are presented as mean ± SEM using individual values (one-way ANOVA with Dunnett’s test). In (D), data are presented as mean ± SEM using boxplots (RM two-way ANOVA with Sidak’s multiple comparison test). (E) Representative plots and total percentages of Th2-like Tregs obtained from patients with colorectal cancer. Specimens obtained from tumor sites were compared with samples obtained from distant areas to the tumor (n = 4, independent values, two-tailed t test). ^∗p < 0.05 was considered significant. For all statistical tests, ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05 were considered significant.

Figure 6

Disease Pathway Analysis of RNA-Seq Data Obtained from Th-like Treg Subsets (A and B) Heatmap showing upregulation of Th-like Treg genes in disease pathways (A) and previously reported upregulated and downregulated genes by tumor infiltrating Tregs (De Simone et al., 2016, Plitas et al., 2016) using Partek software and KEGG database (B). (C) CCR8 expression in Th-like Treg subsets (n = 5, boxplot using minimum to maximum, ordinary one-way ANOVA, with Holm-Sidak’s test). ^∗∗p < 0.01 and ^∗p < 0.05 were considered significant.