An Immunometabolic Shift Modulates Cytotoxic Lymphocyte Activation During Melanoma Progression in TRPA1 Channel Null Mice

Abstract

Melanoma skin cancer is extremely aggressive with increasing incidence and mortality. Among the emerging therapeutic targets in the treatment of cancer, the family of transient receptor potential channels (TRPs) has been reported as a possible pharmacological target. Specifically, the ankyrin subfamily, representing TRPA1 channels, can act as a pro-inflammatory hub. These channels have already been implicated in the control of intracellular metabolism in several cell models, but little is known about their role in immune cells, and how it could affect tumor progression in a process known as immune surveillance. Here, we investigated the participation of the TRPA1 channel in the immune response against melanoma tumor progression in a mouse model. Using Trpa1 ^+/+ and Trpa1 ^-/- animals, we evaluated tumor progression using murine B16-F10 cells and assessed isolated CD8+ T cells for respiratory and cytotoxic functions. Tumor growth was significantly reduced in Trpa1 ^-/- animals. We observed an increase in the frequency of circulating lymphocytes. Using a dataset of CD8+ T cells isolated from metastatic melanoma patients, we found that TRPA1 reduction correlates with several immunological pathways. Naïve CD8+ T cells from Trpa1 ^+/+ and Trpa1 ^-/- animals showed different mitochondrial respiration and glycolysis profiles. However, under CD3/CD28 costimulatory conditions, the absence of TRPA1 led to an even more extensive metabolic shift, probably linked to a greater in vitro killling ability of Trpa1 ^-/- CD8+ T cells. Therefore, these data demonstrate an unprecedented role of TRPA1 channel in the metabolism control of the immune system cells during carcinogenesis.

Keywords: CD8+ T cells; TRPA1 channel; immunometabolism; melanoma; metabolic shift.

Figure 1

Evaluation of body weight, food intake, and tumor volume of Trpa1 ^+/+ or Trpa1 ^-/- mice. Values are shown as mean (n = 13 for Trpa1 ^+/+ and n=18 for Trpa1 ^-/-) ± SD. All temporal analyses were carried out using Two-Way ANOVA followed by Bonferroni post-test. Tumor weight was calculated using unpaired Student’s t-test. (A) Animal weight; (B) Food intake; (C) Tumor volume; (D) Tumor weight. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant, at each time point between genotypes.

Figure 2

Blood analysis of Trpa1 ^+/+ or Trpa1 ^-/- mice on the 22^nd day after B16-F10 cell inoculation. (A) White blood cells; (B, C) Absolute number and percentage of lymphocytes; (D, E) Absolute number and percentage of monocytes; (F, G) Absolute number and percentage of granulocytes; (H) Red blood cells; (I) Hemoglobin; (J) Representative percentage of lymphocytes, granulocytes, and monocytes. Values are shown as mean (n = 3 for Trpa1 ^+/+ and n=4 for Trpa1 ^-/-) ± SD. *p < 0.05; **p < 0.01; ns, not significant. Statistical analyses were performed by Student’s t-test between the genotypes.

Figure 3

Evaluation of tumor-associated macrophages (TAM) from Trpa1^+/+ or Trpa1^-/- mice. (A) Gating strategy for the definition of TAM populations; (B) Analysis of frequency of TAM in tumors. Subpopulations of M1 and M2 TAM were analyzed. Values are presented as the mean (n = 5) ± SD of the frequency (%) in each group. ns, not significant.

Figure 4

(A) Analysis of transcriptome data of T CD8+ cells sorted from human patient tumors. We used GSEA to compare the changes in gene expression induced by CD3 activation in TRPA1 low and high cells. FDR-adjusted p values < 0.05 were considered statistically significant. IL-2 and Stat1 NES: -2,05246 and FDR: 1,00e-06; Oxidative phosphorylation NES: -1,9378266 and FDR 1,00e-06; IF gamma response NES: -1,8219428 and FDR 4,22e-04; IF alpha response NES: -1,6389698 and FDR 3,92e-03; (B) Gating strategy for the definition of CD8+ T cell populations and analysis of frequency of infiltrating CD8 T cells in tumors from Trpa1^+/+or Trpa1^-/- mice. (C, D) Bulk tumor mRNA isolation and qRT-PCR analysis 22 days after subcutaneous inoculation of B16-F10 cell inoculation; (C) Perforin mRNA relative levels; (D) Granzyme mRNA relative levels. Statistical analyses were performed by Student’s t-test. Values are presented as the mean (n = 9) ± SD of the frequency (%) in each group. *p < 0.05; ***p < 0.001.

Figure 5

Metabolic parameters from spleen-derived Trpa1 ^+/+ or Trpa1 ^-/- T CD8 lymphocytes. (A) Basal unstimulated levels of oxygen consumption rate (OCR) of freshly isolated T CD8 cells; (B) OCR levels of T CD8 exposed to a cocktail with ionomycin and PMA compared to the basal rate; (C) Area under the curves of B; (D) Extracellular acidification rates (ECAR) for both non-stimulated and stimulated cells from the two groups. Values are presented as the mean (n = 8) ± SD of each group. Statistical analyses were performed by Student’s t-test in C and by One Way ANOVA followed by Tukey in (D) Each well contained 100,000 cells. *p < 0.05; **p < 0.01 between genotypes. Difference between the different conditions within the same genotype is represented by letters a ≠ b, p < 0.05.

Figure 6

Mitochondrial metabolic evaluation from spleen-derived Trpa1^+/+ or Trpa1^-/- T CD8 lymphocytes under CD3/CD28 activation. (A) Traces obtained from unstimulated` Trpa1^+/+ or Trpa1^-/- T CD8 cells after injections of oligomycin (ATP synthase inhibitor), CCCP (uncoupler) and antimycin A plus rotenone (Complex III and I inhibitors); (B) Same as in A but cells were stimulated for 30 min using immobilized CD3/CD28; (C) Extracellular acidification rate (ECAR) from unstimulated and stimulated cells; (D) Basal respiration; (E) ATP-linked oxygen consumption rate; (F) Maximal oxygen consumption rate; (G) Spare capacity (difference between maximal and basal respiration); (H) Proton-leak linked oxygen consumption rate; (I) Non-mitochondrial respiration (this part is subtracted from all the other respiration rates shown in D–H). Values are presented as the mean (n = 8) ± SD of each group. *p < 0.05; **p < 0.01. One-Way ANOVA analysis followed by Tukey was performed to evaluate differences between conditions and genotypes.

Figure 7

Killing capacity of CD8⁺ cells from Trpa1 ^+/+ and Trpa1^-/- mice co-cultured with B16-F10 cells. A and B) Representative assessment of B16-F10 cell killing by CD8⁺ T cells from Trpa1^+/+ (A) and Trpa1^-/- (B) mice under non stimulated conditions. C and D) Representative assessment of B16-F10 cell killing by CD8⁺ T cells from Trpa1 ^+/+ (C) and Trpa1 ^-/- (D) mice under CD3/CD28 stimulation. (E) Normalized frequency of B16-F10 cell death by CD8⁺ T cells from Trpa1 ^+/+ and Trpa1^-/- mice; values are presented as the mean (n = 3 and 6 for unstimulated and stimulated group, respectively) ± SD. Statistical analyses were performed by Student’s t-test. **p < 0.01, ns, not significant.