Interleukin-7 regulates CD127 expression and promotes CD8+ T cell activity in patients with primary cutaneous melanoma

Abstract

Background: Interleukin (IL)-7 signaling through CD127 is impaired in lymphocytes in cancers and chronic infections, resulting in CD8⁺ T cell exhaustion. The mechanisms underlying CD8⁺ T cell responses to IL-7 in melanoma remain not completely elucidated. We previously showed reduced IL-7 level in melanoma patients. Thus, the aim of this study was to investigate the effect of IL-7 regulation on CD127 expression and CD8⁺ T cell responses in melanoma.

Methods: Healthy controls and primary cutaneous melanoma patients were enrolled. Membrane-bound CD127 (mCD127) expression on CD8⁺ T cells was determined by flow cytometry. Soluble CD127 (sCD127) protein level was measured by ELISA. Total CD127 and sCD127 mRNA level was measured by real-time PCR. CD8⁺ T cells were stimulated with recombinant human IL-7, along with signaling pathway inhibitors. CD8⁺ T cells were co-cultured with melanoma cell line, and the cytotoxicity of CD8⁺ T cells was assessed by measurement of lactate dehydrogenase expression.

Results: Plasma sCD127 was lower in melanoma patients compared with controls. The percentage of CD8⁺ T cells expressing mCD127 was higher, while sCD127 mRNA level was lower in peripheral and tumor-infiltrating CD8⁺ T cells from melanoma patients. There was no significant difference of total CD127 mRNA expression in CD8⁺ T cells between groups. IL-7 stimulation enhanced total CD127 and sCD127 mRNA expression and sCD127 release by CD8⁺ T cells. However, mCD127 mRNA expression on CD8⁺ T cells was not affected. This process was mainly mediated by phosphatidylinositol 3-kinase (PI3K) pathway. CD8⁺ T cells from melanoma patients exhibited decreased cytotoxicity. IL-7 stimulation promoted CD8⁺ T cell cytotoxicity, while inhibition of PI3K dampened IL-7-induced elevation of CD8⁺ T cell cytotoxicity.

Conclusion: The current data suggested that insufficient IL-7 secretion might contribute to CD8⁺ T cell exhaustion and CD127 dysregulation in patients with primary cutaneous melanoma.

Keywords: CD127; CD8+ T lymphocytes; Immune regulation; Interleukin-7; Melanoma.

Fig. 1

Membrane-bound CD127 (mCD127) on CD8⁺ T cells was higher, while soluble (sCD127) was lower in patients with primary cutaneous melanoma. Peripheral blood mononuclear cells were isolated from all enrolled subjects (38 patients with primary cutaneous melanoma and 22 healthy controls), while tissue-infiltrating lymphocytes were isolated from tumor and para-tumor tissues of 14 melanoma patients with surgical operation. Cells were stained with anti-CD8 and anti-CD127, and were analyzed by flow cytometry. a The representative flow cytometry analyses for peripheral blood mononuclear cells and tissue-infiltrating lymphocytes are shown. Live lymphocytes were gated according to forward scatter (FSC) and side scatter (SSC). mCD127 expression within CD8⁺ T cells were assessed. b The percentage of peripheral CD8⁺ T cells expressing mCD127 was elevated in melanoma patients compared with controls. The percentage of tissue-infiltrating CD8⁺ T cells expressing mCD127 was also increased in tumor tissues compared with para-tumor tissues. CD8⁺ T cells were purified from peripheral bloods and tissue-infiltrating lymphocytes. Total CD127 mRNA and sCD127 mRNA variant in CD8⁺ T cells was measured by real-time PCR. c Total CD127 mRNA level in peripheral CD8⁺ T cells was comparable between melanoma patients and controls. Total CD127 mRNA level in tissue-infiltrating CD8⁺ T cells was also comparable between tumor tissues and para-tumor tissues. d sCD127 mRNA level in peripheral CD8⁺ T cells was lower in melanoma patients compared with controls. sCD127 mRNA level in tissue-infiltrating CD8⁺ T cells was also lower in tumor tissues compared with para-tumor tissues. e sCD127 expression in the plasma was measured by ELISA. Plasma sCD127 level was lower in melanoma patients compared with controls. Individual level of each subject is shown. Statistical analysis was performed using Student’s t test

Fig. 2

Recombinant human IL-7 stimulation did not affect mCD127 expression on CD8⁺ T cells, but induced sCD127 release in patients with primary cutaneous melanoma and controls. 10⁴ of peripheral CD8⁺ T cells (nineteen patients with primary cutaneous melanoma and nine healthy controls) and tissue-infiltrating CD8⁺ T cells (tumor and para-tumor tissues of seven melanoma patients) were stimulated with recombinant human IL-7 (10 ng/mL) for 48 h in the presence of anti-CD3/CD28. Control cells were cultured with anti-CD3/CD28 only. Cells and supernatants were harvested. a mCD127 expression on CD8⁺ T cells was investigated by flow cytometry, and the representative flow dots are shown. b There was no significant difference of the percentage of peripheral CD8⁺ T cells expressing mCD127 between cells with and without IL-7 stimulation in either melanoma patients or controls. c There was no remarkable difference of the percentage of tissue-infiltrating CD8⁺ T cells expressing mCD127 between cells with and without IL-7 stimulation in either tumor or para-tumor tissues. d IL-7 stimulation enhanced total CD127 mRNA level in peripheral CD8⁺ T cells in both melanoma patients and controls. e IL-7 stimulation enhanced total CD127 mRNA level in tissue-infiltrating CD8⁺ T cells in both tumor tissues and para-tumor tissues. f IL-7 stimulation promoted sCD127 mRNA level in peripheral CD8⁺ T cells in both melanoma patients and controls. g IL-7 stimulation promoted sCD127 mRNA level in tissue-infiltrating CD8⁺ T cells in both tumor tissues and para-tumor tissues. h IL-7 stimulation promoted sCD127 secretion by peripheral CD8⁺ T cells in both melanoma patients and controls. i IL-7 stimulation promoted sCD127 secretion by tissue-infiltrating CD8⁺ T cells in both tumor tissues and para-tumor tissues. Individual level of each subject is shown. Statistical analysis was performed using Student’s t test

Fig. 3

Influence of JAK, STAT5, and PI3K inhibitor to IL-7-mediated CD127 expression in CD8⁺ T cells in patients with primary cutaneous melanoma. 10⁴ of purified peripheral CD8⁺ T cells from fourteen patient with primary cutaneous melanoma were stimulated with recombinant human IL-7 (10 ng/mL) for 48 h in the presence of anti-CD3/CD28, along with either JAK inhibitor (10 µmol/L), STAT5 inhibitor (250 µmol/L), or PI3K inhibitor (25 µmol/L). Control cells were cultured with anti-CD3/CD28 only. Cells and supernatants were harvested. Total CD127 mRNA and sCD127 mRNA level in CD8⁺ T cells were measured by real-time PCR. a IL-7 stimulation promoted total CD127 mRNA expression in CD8⁺ T cells. Either JAK inhibitor or STAT5 inhibitor did not affect IL-7-mediated elevation of total CD127 mRNA expression, while PI3K inhibitor abrogated the effect of IL-7 on total CD127 mRNA induction in CD8⁺ T cells. b IL-7 stimulation also enhanced sCD127 mRNA level in CD8⁺ T cells. Either JAK inhibitor or STAT5 inhibitor did not influence IL-7-induced elevation of sCD127 mRNA expression, while PI3K inhibitor abrogated the effect of IL-7 on sCD127 mRNA induction in CD8⁺ T cells. c sCD127 level in the supernatants was measured by ELISA. IL-7 stimulation promoted sCD127 level in the cultured supernatant of CD8⁺ T cells. Either JAK inhibitor or STAT5 inhibitor did not influence IL-7-induced sCD127 secretion, while PI3K inhibitor abrogated the effect of IL-7 on sCD127 production by CD8⁺ T cells. Individual level of each subject is shown. Statistical analysis was performed using one-way ANOVA and LSD-t test

Fig. 4

Influence of PI3K inhibitor to IL-7-mediated CD8⁺ T cell cytotoxicity in patients with primary cutaneous melanoma. Purified peripheral CD8⁺ T cells (nine HLA-A02 restricted patient with primary cutaneous melanoma and six HLA-A02 restricted healthy individuals) and tissue-infiltrating CD8⁺ T cells (tumor and para-tumor tissues of six HLA-A02 restricted melanoma patients) were stimulated with recombinant human IL-7 (10 ng/mL) and PI3K inhibitor (25 µmol/L) for 48 h in the presence of anti-CD3/CD28. Cells were washed twice, and 10⁴ of stimulated CD8⁺ T cells were co-cultured with 10⁵ of SK-MEL-5 cells for another 48 h. The percentage of target cell death was calculated by measurement of LDH level in the supernatants. IFN-γ and TNF-α level in the supernatants was measured by ELISA. Inhibition of PI3K dampened the cytotoxicity of a peripheral CD8⁺ T cells from melanoma patients and controls, as well as b tissue-infiltrating CD8⁺ T cells from tumor tissues and para-tumor tissues. Inhibition of PI3K inhibitor suppressed IFN-γ secretion by c peripheral CD8⁺ T cells from melanoma patients and controls, as well as d tissue-infiltrating CD8⁺ T cells from tumor tissues and para-tumor tissues. Inhibition of PI3K inhibitor also suppressed TNF-α secretion by e peripheral CD8⁺ T cells from melanoma patients and controls, as well as f tissue-infiltrating CD8⁺ T cells from tumor tissues and para-tumor tissues. Individual level of each subject is shown. Statistical analysis was performed using one-way ANOVA and LSD-t test