Human TH17 cells are long-lived effector memory cells

Abstract

T helper 17 (TH17) cells have been shown to contribute to multiple disease systems. However, the functional phenotype and survival pattern of TH17 cells as well as the underlying mechanisms that control TH17 cells have been poorly investigated in humans, significantly hampering the clinical targeting of these cells. Here, we studied human TH17 cells in the pathological microenvironments of graft-versus-host disease, ulcerative colitis, and cancer; TH17 cell numbers were increased in the chronic phase of these diseases. Human TH17 cells phenotypically resembled terminally differentiated memory T cells but were distinct from central memory, exhausted, and senescent T cells. Despite their phenotypic markers of terminal differentiation, TH17 cells mediated and promoted long-term antitumor immunity in in vivo adoptive transfer experiments. Furthermore, TH17 cells had a high capacity for proliferative self-renewal, potent persistence, and apoptotic resistance in vivo, as well as plasticity-converting into other types of TH cells. These cells expressed a relatively specific gene signature including abundant antiapoptotic genes. We found that hypoxia-inducible factor-1α and Notch collaboratively controlled key antiapoptosis Bcl-2 family gene expression and function in TH17 cells. Together, these data indicate that human TH17 cells may be a long-lived proliferating effector memory T cell population with unique genetic and functional characteristics. Targeting TH17-associated signaling pathway would be therapeutically meaningful for treating patients with autoimmune disease and advanced tumor.

Fig. 1

High amounts of T_H17 cells are found in sites of chronic diseases. Case numbers: blood (31), oral mucosa (6), spleen (5), tonsil (6), colon cancer (21), and colitis (12). (A) High numbers of IL-17⁺ cells were detected in oral mucosa tissues in patients with GVHD. Consecutive sections of oral mucosa biopsies were stained for IL-17 (brown) or CD3 (red). P < 0.001, acute versus chronic. (B) High percentages of T_H17 cells were found in colitic colon and colon cancer tissues. Tissue single cells were stained for T_H17 cell markers, analyzed by flow cytometry, and gated on CD3⁺ cells. (C and D) CD4⁺ and T_H17 cells were found in colitic colon tissues. Colon tissues were stained for CD3 (green) (C). Absolute numbers of CD4⁺ and T_H17 cells were quantified based on flow cytometry analysis. Results are expressed as the absolute numbers of CD4⁺ or T_H17 per microgram tissue ± SEM (D). P < 0.001, colitic lesion (empty bars) versus adjacent tissue (filled bars). (E) Colon tissues were stained for CD3 and IL-17, and analyzed with a fluorescence microscope. Red, CD3; green, IL-17. DAPI, 4′,6-diamidino-2-phenylindole. (F to H) T_H17 cells were found in different organs. Single cells were stained for T_H17 cell markers and analyzed by flow cytometry. P < 0.001, tonsil and spleen as compared with blood.

Fig. 2

Primary T_H17 cells exhibit a terminally differentiated phenotype but mediate potent antitumor immunity. (A to D) Tissue or blood cells were stained for the indicated markers and cytokines and analyzed by flow cytometry. Results are expressed as the percentage of the specific subset of CD4⁺ T cells. n = 10 to 25. (A) T_H17 cells were in the CD45RO⁺CD45RA⁻ memory T subset. (B) Relationship between T_H17 cells and expression of CCR7 and CD62L. Three CD4⁺ T cell subsets were sorted and stained for IL-17. (C) Relationship between T_H17 cells and expression of CD57, KLRG-1, and PD-1. (D) Expression of the described markers on primary T_H0, T_H1, and T_H17 cells. P < 0.001, for CD28 and CD95 expression on T_H17 as compared with T_H1 and T_H0 cells, and P < 0.05, for CD127 and CD27 expression on T_H17 as compared with T_H1 and T_H0 cells. (E to H) Effector cytokine profile of primary T_H17 cells in different tissues gated on CD4⁺ T cells. (I) T_H17 cells mediate and promote tumor regression in vivo. Ovarian cancer–bearing NSG mice (16, 17) received PBS, T_H17, and/or CD8⁺ T cells. Mean ± SEM of tumor volumes are shown (n = 4 to 6 mice per group). *P < 0.05.

Fig. 3

T_H17 cells have functional and genetic stem cell–like features. (A to D) T_H17 cells gave rise to other T_H cells. Primary T_H17 cells were stimulated with the cytokine milieu for T_reg (A and B) or T_H1 (C and D) induction for 3 days and analyzed by flow cytometry. Results are expressed as the percentage of Foxp3⁺ (B) or IFN-g⁺ cells (D) in T_H17 cells. n = 5. *P < 0.05. (E and F) IL-17⁺Foxp3⁺(E) and IL-17⁺IFN-γ⁺(F) T cells in cancers. Colon (n = 9) and ovarian cancer (n = 25) T cells were analyzed by flow cytometry. (G and H) T_H17 cells had superior in vivo persistence. HLA-A2⁺ T_H subsets were mixed with HLA-A2⁻IL-17⁻CD4⁺ T cells and transfused to NSG mice. On day 5, human T cells were analyzed. (I) The percentage of changes between the initial and the recovered ratios of HLA-A2⁺ and HLA-A2⁻ cells was calculated. Three paired donors. *P < 0.001. (J) Representative histogram shows different T subsets (HLA-A2⁺) in spleen. (I to K) T_H17 cells expressed stem cell genes. SuperArray was performed in primary T_H17 and controls (I). Selective stem cell genes were quantified (J and K). n = 8. P < 0.05.

Fig. 4

T_H17 cells have increased proliferative capacity. (A to C) T_H17 cells had potent expansion capacity. Primary (A and B) and polarized T_H subsets (C) were stimulated with TCR for 3 (A and C) or 14 (B) days. Cell proliferation/expansion was detected by [³H]thymidine incorporation (A), CFSE dilution (B), or increased cell numbers (C). n = 3. *P < 0.05. (D and E) Cytokines stimulated T_H17 expansion. Primary HLA-A2⁺ T_H17 and HLA-A2⁻IL-17⁻ control T cells were separately cultured (D) or initially mixed and cocultured (E) for 3 days with IL-7 plus IL-15. The absolute cell numbers (D) or the percentage (E) of T_H17 cells was determined by flow cytometry. n = 3. *P < 0.05. (F) T_H17 cells were in S phase. NSG mice received T_H17 or control cells and BrdU. Cell cycling phase was analyzed on day 5 by flow cytometry to determine BrdU⁺ human T cells in mouse spleen. n = 2. (G) T_H17 cells expressed increased Ki67 expression in blood and colon cancer. Eight to 10 donors. P < 0.05, T_H17 as compared with T_H1/T_H0. (H to K) T_H17 cells expressed different amounts of cell cycling genes. Expression of multiple cyclin genes (H and I) and CDK inhibitors (J and K) was quantified in primary (H and J) and polarized T_H17 cells (I and K). n = 8. *P < 0.05.

Fig. 5

T_H17 cells are resistant to apoptosis. (A) T_H17 cells did not express caspase 3. The expression of caspase 3 in T cells was analyzed by flow cytometry. n = 4. (B) T_H17 cells were resistant to apoptosis induced by TCR activation. T subsets were stimulated with anti-CD3, and cell apoptosis was analyzed with annexin V staining. n = 5. P < 0.01, T_H17 as compared with T_H1/T_H2 on day 19. (C) T_H17 cells were resistant to apoptosis induced by in vitro cisplatin. Primary T_H17 and control cells were treated with cisplatin for 48 hours. Annexin V⁺ cells were analyzed. n = 4. P < 0.01. (D to F) Chemotherapy increased IL-17 production. Ovarian cancer patients received one cycle of cisplatin therapy. Blood mononuclear cells were activated, and cytokines were measured in the supernatants with enzyme-linked immunosorbent assay (ELISA). n = 6. P < 0.001. (G) Chemotherapy reduced IL-6 but not IL-17 production in ovarian cancer ascites. Ovarian cancer patient was treated with cisplatin. Cytokines were measured in the ascites fluid by ELISA. Left scale shows IL-17 (filled circles), and right scale shows IL-6 (empty circles). (H and I) T_H17 cells spontaneously expressed high levels of BCL2 family genes. Real-time PCR was performed in primary T_H17 and control cells for BCL2 (H) and BCLXL (I). n = 5. *P < 0.001.

Fig. 6

HIF-1α regulates T_H17 cell apoptosis and persistence. (A and B) T_H17 cells expressed HIF1A. HIF1A expression was quantified by real-time PCR in primary T_H17 (A) and polarized T cell subsets (B). n = 5 to 8. *P < 0.05. (C to E) HIF-1 blockade reduced T_H17 persistence in vivo. Primary HLA-A2⁺ T_H17 cells and HLA-A2⁻IL-17⁻CD4⁺ T cells were mixed and pretreated with PBS, DMSO, and the HIF-1 inhibitor echinomycin for 48 hours and transfused to NSG mice. Human T cells were stained and analyzed for HLA-A2 and human CD5 (C), and human HIF1A (D) and IL17A (E) were quantified in the spleen. n = 5. (F and G) HIF-1 blockade increased T_H17 apoptosis in vivo. Primary T_H17 cells were pretreated with HIF-1 inhibitor or DMSO and transferred to NSG mice. After 2 days, annexin V expression (F) and B_CL2 gene expression (G) were analyzed in human T cells in mouse spleen. n = 6. P < 0.05. (H) HIF-1 blockade increased T_H17 apoptosis in vitro. Enriched primary T_H17 cells were transfected with lentiviral vector encoding shHIF-1α and then activated with TCR for 3 days. T_H17 and T_H1 cell numbers were counted, and the percentage of cell loss was calculated. n = 3. P < 0.05. (I to K) HIF-1 blockade increased T_H17 apoptosis induced by chemotherapy. Primary T_H17 cells were transfected with lentiviral vector encoding shHIF-1 or scramble and cultured with cisplatin for 48 hours. Cell apoptosis was analyzed with annexin V expression (I). BCL2 (J) and BCLXL (K) genes were quantified. n = 7. P < 0.01.

Fig. 7

Notch and Bcl-2 family regulate the stem cell-like feature of the T_H17 cells. (A) Notch bound to and activated the Bcl-2 promoter. Black boxes show Notch binding sites in the BCL2 promoter (upper panel). Human embryonic kidney (HEK) 293 cells were cotransfected with the hBCL2-EGFP (enhanced green fluorescent protein) promoter with the indicated plasmids for 38 hours. The intensity of EGFP was measured with flow cytometry. LTR, long terminal repeat; TSS, transcription start site. (B and C) Notch blockade suppressed BCL2 and BCLXL expression. Primary T cells were treated with Notch inhibitor for 24 hours. BCL2 (B) and BCLXL (C) expression was quantified by real-time PCR. n = 3. P < 0.05. (D) Notch blockade increased T_H17 cell apoptosis. Primary T_H17 cells were cultured with the Notch inhibitor for 3 days. Annexin V expression was analyzed by flow cytometry. n = 3. (E) Notch activation increased BCL2 expression on T_H17 cells. Primary T_H17 cells were transfected with Notch-IC. After 24 hours, Bcl-2 expression was analyzed by flow cytometry. n = 3. (F) HIF blockade reduced Notch signaling gene expression in T_H17 cells. Primary T_H17 cells were transfected with shHIF-1α. After 24 hours, Notch signaling gene expression was quantified. n = 3. (G) Notch activation rescued T_H17 cell apoptosis induced by HIF blockade. Primary T_H17 cells were transfected with lentiviral vectors encoding shHIF-1α and/or Notch-IC. After 24 hours, annexin V expression was analyzed by flow cytometry. n = 3.