Synergism between CpG ODN and IL-2 Leads to Massive CD8+ T Cell Responses Against a Self-Antigen and Epitope-Specific Anti-Tumor Immunity.
Authors: Kochenderfer Jim , Chien Chris , Gress Ronald 
Published: October 2019

Abstract
Oligodeoxynucleotides with CpG motifs (CpG ODN) enhance vaccine-elicited T cell responses and might improve the efficacy of anti-cancer vaccines. B16F1 is a poorly immunogenic murine melanoma that expresses tyrosinase related protein-2 (TRP-2), which is also expressed by normal melanocytes and is subject to self-tolerance. Amino acids 180–188 of TRP-2 (TRP-2180–188) form an immunogenic MHC class I-presented epitope. We injected mice subcutaneously with B16F1 and then administered a vaccine containing TRP-2180–188+CpG ODN in IFA (incomplete Freund’s adjuvant) on days 0, 3, 6, and 14 after tumor injection. As a negative control, we vaccinated a second group of mice with the OVA257–264 peptide+CpG ODN in IFA. TRP-2180–188+CpG ODN vaccination did not cause epitope-specific inhibition of B16F1 growth. In an attempt to increase anti-tumor efficacy, we added low-dose IL-2 on days 7–10 and 15–18 after tumor injection to the vaccination regimen described above. With the addition of IL-2, epitope-specific inhibition of tumor growth occurred (day 19 tumor size: 21 mm2 with TRP-2180–188 vaccination versus 93 mm2 with OVA257–264 vaccination, P=0.002). Because tumor growth inhibition was epitope-specific, we hypothesized that it was due to an IL-2-mediated increase in CD8+ T cell responses against TRP-2180–188. To test this hypothesis, we gave TRP-2180–188+CpG ODN vaccines and low-dose IL-2 to mice, and then we measured TRP-2180–188-specific CD8+ T cell responses by ex vivo peptide stimulation of splenocytes for six hours followed by intracellular cytokine staining for interferon-γ (IFNγ). When mice were vaccinated with TRP-2180–188+CpG ODN and given IL-2, a mean of 18.3% of CD3+CD8+ splenocytes produced IFNγ after ex vivo peptide stimulation with TRP-2180–188, but only 0.1% of CD3+CD8+ splenocytes produced IFNγ in response to the negative control peptide OVA257–264; therefore, the mean TRP-2180–188-specific CD8+ T cell response was 18.2%. The same vaccine regimen given with control PBS injections instead of IL-2 elicited a mean TRP-2180–188-specific response of only 1.0% of CD3+CD8+ splenocytes (P<0.001, IL-2 versus no IL-2). Mice that received TRP-2180–188+CpG ODN vaccinations and IL-2 had a mean absolute number of 5.6x106 TRP-2180–188-specific CD3+CD8+ splenocytes. Mice vaccinated identically but not receiving IL-2 had only 0.1x106 TRP-2180–188-specific CD3+CD8+ splenocytes (P<0.001 IL-2 versus no IL-2). Vaccines containing TRP-2180–188 without CpG ODN given with low-dose IL-2 elicited responses in which a mean of 2.8% of CD3+CD8+ splenocytes and an absolute number of 0.5x106 CD3+CD8+ splenocytes were specific for TRP-2180–188 (P<0.001 for percentage and absolute number, IL-2+CpG ODN versus IL-2 without CpG ODN). Tumor-bearing mice generated TRP-2180–188-specific CD8+ T cell responses only when vaccinated with TRP-2180–188 despite the presence of B16F1 tumors expressing the TRP-2 protein. TRP-2180–188+CpG ODN vaccines protected mice from B16F1 challenge when B16F1 was injected 5 days after the last dose of vaccine only when IL-2 was also administered. When we vaccinated mice prophylactically, survival was higher in TRP-2180–188-vaccinated mice than in OVA257–264-vaccinated mice (P=0.0005), and 3/13 TRP-2180–188-vaccinated mice developed vitiligo that was consistent with autoimmunity against melanocytes. This is the first report of synergism between IL-2 and CpG ODN. This synergism causes a striking increase in vaccine-elicited CD8+ T cell responses and leads to self-epitope-specific anti-tumor immunity.