Toll-like Receptor Agonist Imiquimod Facilitates Antigen-Specific CD8+ T-cell Accumulation in the Genital Tract Leading to Tumor Control through IFNγ

Abstract

Purpose Imiquimod is a toll-like receptor 7 agonist utilized topically to manage genital warts and basal cell carcinoma. We examine the combination of topical imiquimod with intramuscular administration of CRT/E7, a therapeutic HPV vaccination that comprises a naked DNA vector expressing calreticulin fused to HPV16 E7. Experimental Design Using an orthotopic HPV16 E6/E7+ syngeneic tumor, TC-1, as a model of high-grade cervical/vaginal/vulvar intraepithelial neoplasia, we show that combining CRT/E7 vaccination with cervicovaginal deposition of imiquimod results in synergistic immune-mediated tumor clearance. Results Imiquimod induces cervicovaginal accumulation of activated E7-specific CD8+ T cells elicited by CRT/E7 vaccination. Recruitment was not dependent upon the specificity of the activated CD8+ T cells, but was significantly reduced in mice lacking the IFNγ receptor. Intravaginal imiquimod deposition induced upregulation of CXCL9 and CXCL10 mRNA expression in the genital tract. These chemokines are expressed upon IFNγ receptor activation and attract cells expressing their receptor, CXCR3. In this study, T cells attracted by imiquimod to the cervicovaginal tract expressed CXCR3 as well as the tissue resident memory T cell (Trm) marker CD49a, a mucosal homing integrin. Our results indicate that intramuscular CRT/E7 vaccination in conjunction with intravaginal imiquimod deposition recruits antigen-specific CXCR3+CD8+ T cells to the genital tract. Conclusions Our study has potential clinical relevance because imiquimod is FDA approved for condyloma accuminata and basal cell carcinoma and intramuscular vaccination with pNGVL4a-CRT/E7(detox) is currently undergoing clinical testing, suggesting potential for their synergistic action to induce strong antigen-specific Trm-mediated immune responses and antitumor effects in genital mucosa.

A novel dendritic cell targeting HPV16 E7 synthetic vaccine in combination with PD-L1 blockade elicits therapeutic antitumor immunity in mice

“…To achieve the goal of in vivo DC targeted antigen delivery, the HPV16 E7 long peptide (amino acid [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] containing the dominant T cell epitopes was fused to the single-chain fragment variable (scFv) specific for DEC-205 (aDEC205) using a protein assembly strategy based on the SpyCatcher-SpyTag system as described previously. 26 The SpyTag was genetically fused to the C-terminus of aDEC205 to obtain aDEC205-SpyTag and the E7 long peptide was added to the C-terminus of SpyCatcherDN to obtain Sc-E7, respectively (Fig.…”

“…26 7 d later, the DLNs from the immunized mice were isolated and digested into single-cell suspensions, and then stimulated with 5 mg/mL E7 [49][50][51][52][53][54][55][56][57] peptide in a U-bottom 96-well plate for 6 h. The IFNg producing CD8 C T cells were then determined and analyzed by intracellular staining and flow cytometry. As shown in Fig.…”

“…26 Briefly, the aDEC205-SpyTag fusion protein was expressed by transient transfection in FreeStyle TM 293-F cells and purified on protein A column (GE Healthcare Life Sciences, Pittsburgh, PA, USA). For SpyCatcher-E7 production, the cDNA coding sequence of HPV16 E7 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] was subcloned into the C-terminus of SpyCatcherDN (SpyCatcher with deletion at N-terminus as described previously 26 ) to construct the pDEST14-SpyCatcherDN-E7, then the expression plasmid was transformed into E. coli and the protein was purified by a Ni-NTA agarose column (ComWin Biotech, Beijing, China). For DC targeting vaccine generation, aDEC205-SpyTag (10 mM) and SpyCatcherDN-E7 (or Sc-E7) (15 mM) were mixed in PBS at a molar ratio of 1:1.5 at 4…”

Intravaginal Administration of Fc-Fused IL7 Suppresses the Cervicovaginal Tumor by Recruiting HPV DNA Vaccine-Induced CD8 T Cells

“…In addition, imiquimod facilitates HPV DNA vaccine-induced CD8 T-cell accumulation in the genital tract, leading to tumor control in a mouse cervical cancer model (16). However, in patients, severe local inflammations and ulcerations caused by excessive production of proinflammatory cytokines with repeated topical treatments limit the therapeutic potential and wider adoption (18,19).…”

“…Local administration of toll-like receptor (TLR) 7 and 9 agonists, imiquimod or CpG, after parenteral HPV16 E7 vaccination has been shown to induce accumulation of E7-specific CD8 T cells in the genital tract and regression of genital tumors (16,17). However, in humans, imiquimod use can induce severe adverse events, including, but not limited to, acute and severe local inflammation and ulceration (18,19), while treatment of CpG requires repeated injections due to short-lived efficacy (17).…”