Anti-oxidative Amino Acid L-ergothioneine Modulates the Tumor Microenvironment to Facilitate Adjuvant Vaccine Immunotherapy

Abstract

Cancer vaccines consist of a tumor-associated antigen (TAA) and adjuvant. These vaccines induce and activate proliferation of TAA-specific cytotoxic T lymphocytes (CTLs), suppressing tumor growth. The therapeutic efficacy of TAA-specific CTLs depends on the properties of tumor microenvironment. The environments make immunosuppressive by function of regulatory T cells and tumor-associated myeloid cells; thus, regulation of these cells is important for successful cancer immunotherapy. We report here that L-ergothioneine (EGT) with the adjuvant Toll-like receptor 2 (TLR2) ligand modulated suppressive microenvironments to be immune-enhancing. EGT did not augment DC-mediated CTL priming or affect CTL activation in draining lymph node and spleen. However, EGT decreased the immuno-suppressive function of tumor-associated macrophages (TAMs). TLR2 stimulation accompanied with EGT administration downregulated expression of PD-L1, CSF-1R, arginase-1, FAS ligand, and TRAIL in TAMs, reflecting reduction of CTL suppression. An anti-oxidative thiol-thione residue of EGT was essential to dampening CTL suppression. The effect was specific to the thiol-thione residue of EGT because no effect was observed with another anti-oxidant N-acetyl-L-cysteine (NAC). A CTL-suppressive environment made by TLR2 is relieved to be improved by the addition of EGT, which may ameliorate the efficacy of vaccine immunotherapy.

Keywords: L-ergothioneine; cytotoxic T lymphocyte; toll-like receptor; tumor microenvironment; tumor-associated macrophage.

Figure 1

EGT augments tumor-growth retardation induced by cancer vaccine using TLR2/6 ligand. (A) Protocol for this study. (B) WT B6 mice were challenged with LLC-OVA cells (Day 0). Daily i.p. administration of 500 μg EGT was started on day 9 and vaccination (s.c. injection of 15 nmol Pam2CSK4 and 100 μg OVA protein) was performed on day 10 and 14. At day 18, tumors were harvested, and the weight was measured in addition to tumor volume. Data were pooled from two independent experiments with similar results. n = 9–10 mice per group. (C) Tumor growth in sole treatment of EGT. Schedule of EGT administration is shown in (A) Mice were challenged with LLC-OVA cells and i.p. treated with EGT alone or PBS control. At timed intervals, tumor growth was measured. n = 4 mice per group. (D) Mice were challenged with LLC-OVA cells and s.c. treated with OVA, EGT + OVA, Pam2CSK4 or EGT + Pam2CSK4. PBS was used as a control. Tumor growth was chased every other day. n = 4 mice per group. (E) Mice were challenged with EG7 tumor cells and treated with PBS, EGT, Pam2CSK4 + OVA, EGT + Pam2CSK4 + OVA as in (A). Tumor growth were measured. n = 4–5 mice per group. (F) LLC-OVA cells were cultured with 1 or 10 mM EGT for 24 h, and then treated with 50 nM Pam2CSK4. Cell viability was assessed by WST-1 reagent 48 h after Pam2CSK4 treatment. n = 3–4. (G) Anti-CD8β antibody was i.p. injected into LLC-OVA-bearing mice on day 9 and 13 and the treatment described in (A) was performed. n = 4–5 mice per group. *P < 0.05, **P < 0.01. n.s., not significant.

Figure 2

EGT does not increase priming and tumor-infiltration of CTLs. LLC-OVA-implanted WT B6 mice were treated as per Figure 1A. At day 18, tumor, spleen and draining lymph nodes (DLN: inguinal LN) were harvested. Then, population of OVA-tetramer positive cells (A) and total CD8⁺ CD3⁺ cells in tumor (B) were counted by flowcytometry. n = 4–5 mice per group. *P < 0.05. n.s., not significant.

Figure 3

EGT improves CTL functionality in tumor in combination with TLR2/6 ligand. (A–C) LLC-OVA-implanted WT B6 mice were treated as per Figure 1A and tumors were harvested at day 18. (A) Concentration of IFN-γ and TNF-α in tumor was determined by cytometric beads assay (CBA). Data were pooled from two independent experiments resulted in similar profiles. n = 4–10 mice per group. (B) Total tumor cells were re-stimulated with 50 nM of SL8 peptide for 6 h. Brefeldin A was supplied during last 5 h of culture. Then, intracellular expression levels of IFN-γ and TNF-α in CD8⁺ CD3⁺ cells were detected by flowcytometry. Representative data are shown as FACS plot. n = 4–5 mice per group. (C) Cell surface expression of indicated proteins on CD8⁺ CD3⁺ cells were analyzed by flowcytometry. n = 4–5 mice per group. *P < 0.05, **P < 0.01. n.s., not significant.

Figure 4

EGT cancels TAM proliferation induced by TLR2/6 ligand. (A,B) LLC-OVA-implanted WT B6 mice were treated with PBS, EGT, and/or Pam2CSK4 + OVA, as per Figure 1A and tumors were harvested at day 18. In (B), mice were injected with Pam2CSK4 without OVA. Tumor-infiltrating cells were analyzed by flow cytometry to count the number of CD11b⁺ Gr-1^−/low F4/80⁺ cells. Representative data are shown as FACS plot. n = 4–5 mice per group. (C) Sorted tumor-infiltrating F4/80⁺ cells were cultured with or without EGT for 24 h, and then, cells were stimulated with 50 nM of Pam2CSK4. WST-1 assay was performed 72 h after Pam2CSK4 treatment. n = 3. *P < 0.05. n.s., not significant.

Figure 5

EGT converts TAMs into less-suppressive phenotype under TLR2/6 stimulation. (A,B) LLC-OVA-implanted WT B6 mice were treated as shown in Figure 1A and tumors were harvested at day 18. n = 4–5 mice per group. (A) Expression of indicated proteins of CD11b⁺ Gr-1^−/low F4/80⁺ cells were analyzed by flowcytometry. (B) Representative FACS plots of the data in Figure 6A. (B) Sorted tumor-infiltrating F4/80⁺ cells were treated with or without 10 mM of EGT for 24 h, and then, stimulated with 50 nM of Pam2CSK4. Total RNA was collected 4 h after Pam2CSK4 stimulation. n = 3. *P < 0.05, **P < 0.01. n.s., not significant.

Figure 6

EGT relieves TAM-induced decrease of CTL viability/activity depending upon thiol-thione residue. (A) The compound structure of HER, EGT or NAC. (B) F4/80⁺ cells were cultured with/without 10 mM of EGT, HER or NAC for 24 h. Then, CD8⁺ splenocytes were mixed in 1:1 ratio. Promptly, the cells were stimulated with 0.1 μg/mL of αCD3 Ab and 0.25 μg/mL of αCD28 Ab. After 60 h co-culture and Ab stimulation, non-adherent cells were collected. The ratios of the 7AAD⁺ cell and CD44⁺ CD62L⁻ cell populations in CD8⁺ CD3⁺ T cells were determined using flow cytometry. Cells in all conditions were obtained and analyzed at the same time. n = 3. (C) WT B6 mice were challenged with LLC-OVA cells (Day 0). Daily i.p. administration of 350 μg NAC (an equimolecular amount of 500 μg EGT) was started on day 9 and vaccination (s.c. injection of 15 nmol Pam2CSK4 and 100 μg OVA protein) was performed on day 10 and 14. n = 4–5 mice per group. **P < 0.01,***P < 0.001. n.s., not significant.

Figure 7

Summary of this study. (A) Pam2CSK4 and TAA induce DC maturation and CTL priming, which induces tumor cell death. (B) However, TLR2 stimulation increases immunosuppressive TAM proliferation. (C) EGT cancels the proliferation and further renders TAM less immuno-suppressive in combination with TLR2/6 ligand. (D) The EGT function in tumor leads to improved functionality of intratumoral CTLs and enhances therapeutic efficacy of the vaccination.