A Vaccination Regimen Incorporating the Synergistic Combination of CpG-Containing Oligodeoxynucleotides and IL-2 Can Elicit CD8+ T Cell Responses That Effect Anti-Tumor Immunity from T Cell Repertoires Undergoing Reconstitution by Homeostatic Peripheral Expansion after BMT.
Authors: Kochenderfer James , Chien Christopher , Simpson Jessica , Gress Ronald 
Published: April 2020

Abstract
Development of CD8+ T cell responses targeting tumor-associated antigens after autologous stem cell transplants might eradicate residual tumor cells and decrease relapse rates. Because thymic function dramatically decreases with increasing age, CD8+ T cell reconstitution in the first few months after autologous stem cell transplant in middle-aged patients is primarily the result of homeostatic peripheral expansion (HPE) of mature T cells contained in the reinfused cells. To study antigen-specific T cell responses during HPE, we performed syngeneic bone marrow transplants (BMT) on mice that had been thymectomized at 4–6 weeks of age and then vaccinated the mice against a self-antigen. Tyrosinase-related-protein-2 (TRP-2) is a protein that expressed by normal melanocytes and the poorly immunogenic B16 melanoma. We vaccinated mice with a regimen consisting of an epitope from TRP-2 (TRP-2180–188) mixed with CpG-containing oligodeoxynucleotides (CpG ODN) in incomplete Freund’s adjuvant on days 14, 17, 20, and 28 after BMT. Interleukin-2 (IL-2) was administered on days 21–23 and days 29–32 after BMT. When TRP-2180–188-specific CD8+ T cell responses were measured on day 33 after BMT by ex vivo peptide stimulation of splenocytes followed by intracellular cytokine staining for interferon gamma, 9.1% of CD8+ T cells were specific for TRP-2180–188, and a mean absolute number of 2.3x106 TRP-2180–188-specific CD3+CD8+ splenocytes were detected in mice that received vaccination regimens including CpG ODN and IL-2. In contrast, when we administered the same regimen with control injections instead of IL-2, 4.0% of CD3+CD8+ splenocytes were specific for TRP-2180–188 (P&lt;0.05 IL-2 versus control injections), and a mean of only 0.2x106 TRP-2180–188-specific CD3+CD8+ splenocytes were detected (P&lt;0.01 IL-2 versus control injections). When mice were vaccinated with TRP-2180–188 without CpG ODN and IL-2 was administered, 1.4% of CD3+CD8+ splenocytes were specific for TRP-2180–188 (P&lt;0.001 CpG ODN versus no CpG ODN), and 0.2x106 CD3+CD8+ splenocytes were TRP-2180–188-specific (P&lt;0.001 CpG ODN versus no CpG ODN). To test the in vivo anti-tumor efficacy of the vaccine-elicited CD8+ T cells, we challenged mice subcutaneously on day 14 after BMT with B16 tumor cells and on the same day initiated vaccination with the regimen including TRP-2180–188+CpG ODN and IL-2 described above. As a negative control, we treated a second group of mice identically except that a control peptide, OVA257–264, was substituted for TRP-2180–188. Survival was enhanced in TRP-2180–188-vaccinated mice compared to OVA257–264-vaccinated mice (P=0.0007), and tumor growth was inhibited. The mean tumor size 25 days after tumor injection was 17.9 mm2 in TRP-2180–188-vaccinated mice versus 71.3 mm2 in OVA257–264-vaccinated mice (P=0.0009). Depletion of CD8+ T cells abrogated this epitope-specific anti-tumor effect. This is the first report to demonstrate that CD8+ T cells specific for a self-antigen and capable of effecting in vivo anti-tumor immunity can be elicited by vaccination from T cell repertoires undergoing reconstitution by HPE after BMT. The number of TRP-2180–188-specific CD8+ T cells elicited by vaccination after BMT was increased 10-fold by synergism between CpG ODN and IL-2.