Arenavirus Induced CCL5 Expression Causes NK Cell-Mediated Melanoma Regression

Abstract

Immune activation within the tumor microenvironment is one promising approach to induce tumor regression. Certain viruses including oncolytic viruses such as the herpes simplex virus (HSV) and non-oncolytic viruses such as the lymphocytic choriomeningitis virus (LCMV) are potent tools to induce tumor-specific immune activation. However, not all tumor types respond to viro- and/or immunotherapy and mechanisms accounting for such differences remain to be defined. In our current investigation, we used the non-cytopathic LCMV in different human melanoma models and found that melanoma cell lines produced high levels of CCL5 in response to immunotherapy. In vivo, robust CCL5 production in LCMV infected Ma-Mel-86a tumor bearing mice led to recruitment of NK cells and fast tumor regression. Lack of NK cells or CCL5 abolished the anti-tumoral effects of immunotherapy. In conclusion, we identified CCL5 and NK cell-mediated cytotoxicity as new factors influencing melanoma regression during virotherapy.

Keywords: CCL5; LCMV; NK cells; arenavirus; immunotherapy; innate immunity; melanoma; virotherapy.

Figure 1

Human Ma-Mel-86a melanoma cell line produces CCL5 after LCMV infection. NOD/SCID mice were injected with 2 × 10⁶ melanoma cells subcutaneously in the left flank and once the tumor diameter was about 5 mm, mice were infected with 2 × 10⁶ PFU LCMV WE intratumorally or left untreated. Tumor growth of Ma-Mel-86a (A; n = 4–5), Ma-Mel-86c (B; n = 3), and Ma-Mel-51 (C; n = 4) was monitored at the indicated time points.

Figure 2

Human Ma-Mel-86a melanoma cell line produces CCL5 after LCMV infection. Ma-Mel-51 and Ma-Mel-86a cells were cultured in a 24 well plate at a density of 2 × 10⁵ cells/well and infected with LCMV WE (multiplicity of infection [MOI] 1). (A) Expression of CCL5 in the culture supernatant was assessed by ELISA (n = 6). (B) Expression of LCMV nucleoprotein (green) was stained and analyzed by fluorescent microscopy (n = 4). (C) Expression of different chemokines was checked by qRT-PCR (n = 6). (D) Expression of CCL5 (red) was stained and analyzed by fluorescent microscopy (n = 3). Data are shown as mean ± s.e.m. Significant differences between the two groups were detected by unpaired two-tailed t-tests and are indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3

CCL5 is produced in Ma-Mel-86a derived tumors in vivo upon infection with LCMV. NOD/SCID mice were injected with 2 × 10⁶ Ma-Mel-86a or Ma-Mel-51 cells subcutaneously in the left flank and once the tumor diameter was about 5 mm, they were infected with 2 × 10⁶ PFU LCMV WE intratumorally or left untreated. qRT-PCR for chemokine expression in the tumor was analyzed on day 10 (A; n = 4). The expression data for each gene and cell line was normalized to the corresponding control measurement. Mice were sacrificed on day 5 and day 12 after LCMV intratumoral injection and the expression of CCL5 (red) was analyzed by immunofluorescence (B; n = 4). Data are shown as mean ± s.e.m. and analyzed by unpaired Student's t-test. *P < 0.05.

Figure 4

CCL5 is important for anti-tumoral activity. (A) 2 × 10⁶ Ma-Mel-86a melanoma cells were injected subcutaneously in the flank of NOD/SCID mice. One group was treated with Maraviroc (50 mg/kg body weight/mice), another with the combination of Maraviroc and 2 × 10⁶ PFU LCMV WE, the third group treated with LCMV only and the last group was left untreated. Tumor growth was followed as indicated (n = 3). (B,C) 2 × 10⁶ B16-Ova empty vector or B16-Ova-CCL5 cells, overexpressing CCL5, were injected subcutaneously in the flank of C57B6 mice. Once the tumor diameter was about 5 mm, they were infected with 2 × 10⁶ PFU LCMV WE intratumorally or left untreated. Tumor growth (B) and survival (C) were monitored (n = 4).

Figure 5

CCL5 induces NK cell mediated cytotoxicity. (A) NOD/SCID mice were injected with 2 × 10⁶ Ma-Mel-86a or Ma-Mel-51 cells subcutaneously in the left flank. Once the tumor diameter was about 5 mm, they were infected with 2 × 10⁶ PFU LCMV WE intratumorally or left untreated. Mice were sacrificed on day 4 and day 10 after LCMV WE injection and infiltration of NK cells (blue) was analyzed by immunofluorescence (n = 4). (B) NOD/SCID (n = 4) or NSG (n = 5) mice were injected with 2 × 10⁶ Ma-Mel-86a cells subcutaneously in the left flank. Tumor growth was followed. (C) NSG mice were injected with 2 × 10⁶ Ma-Mel-86a cells subcutaneously in the left flank and once the tumor diameter was about 5 mm, they were treated with 2 × 10⁶ PFU LCMV WE intratumorally or left untreated. Tumor growth was followed (n = 4). (D) NOD/SCID and NSG mice were injected with 2 × 10⁶ Ma-Mel-51 cells subcutaneously in the left flank and left untreated. Tumor growth was followed (n = 4).