Intramuscular therapeutic vaccination targeting HPV16 induces T cell responses that localize in mucosal lesions

Abstract

About 25% of high-grade cervical intraepithelial neoplasias (CIN2/3) caused by human papillomavirus serotype 16 (HPV16) undergo complete spontaneous regression. However, to date, therapeutic vaccination strategies for HPV disease have yielded limited success when measured by their ability to induce robust peripheral blood T cell responses to vaccine antigen. We report marked immunologic changes in the target lesion microenvironment after intramuscular therapeutic vaccination targeting HPV16 E6/E7 antigens, in subjects with CIN2/3 who had modest detectable responses in circulating T lymphocytes. Histologic and molecular changes, including markedly (average threefold) increased intensity of CD8(+) T cell infiltrates in both the stromal and epithelial compartments, suggest an effector response to vaccination. Postvaccination cervical tissue immune infiltrates included organized tertiary lymphoid-like structures in the stroma subjacent to residual intraepithelial lesions and, unlike infiltrates in unvaccinated lesions, showed evidence of proliferation induced by recognition of cognate antigen. At a molecular level, these histologic changes in the stroma were characterized by increased expression of genes associated with immune activation (CXCR3) and effector function (Tbet and IFNβ), and were also associated with an immunologic signature in the overlying dysplastic epithelium. High-throughput T cell receptor sequencing of unmanipulated specimens identified clonal expansions in the tissue that were not readily detectable in peripheral blood. Together, these findings indicate that peripheral therapeutic vaccination to HPV antigens can induce a robust tissue-localized effector immune response, and that analyses of immune responses at sites of antigen are likely to be much more informative than analyses of cells that remain in the circulation.

Figures

Fig. 1. Intramuscular immunization with DNAE7 prime, followed by recombinant vaccinia (rVacE6E7) boost, induces HPV16 E6 and E7–specific TH1 immune responses in the peripheral blood

(A) Cellular immune responses in unfractionated PBMCs, quantified by IFN-γ ELISpot assays. All subjects received two intramuscular DNA vaccinations at weeks 0 and 4. Dose escalation of recombinant vaccinia (rVacE6E7) boost vaccination at week 8; subjects received either 1.6 × 105 PFU (DDV1), 1.6 × 106 PFU (DDV2), or 1.6 × 107 PFU (DDV3). Results are expressed as SFUs per 106 PBMCs. (B and C) Multiparametric flow cytometry was used to detect HPV16- and HPV18-specific production of IFN-γ. Bar graphs summarizing ICS and flow cytometry analyses of PBMC response to HPV16 and HPV18 peptides: (B) CD8+IFN-γ+ and (C) CD4+IFN-γ+. Subjects with complete histologic regression at week 15 are designated with a bar below the subject ID.

Fig. 2. Tissue CD8+ T cell infiltrates in the target lesion increase after vaccination

(A) Representative immunohistochemical (IHC) staining for CD8 in lesional tissue before (left column) and after (right column) vaccination (patient 3009). (B) These infiltrates are Tbet+. (C) In contrast, the intensity of Foxp3+ infiltrates does not change substantially. (D) Bar graphs depicting quantitated CD8+ and Foxp3+ infiltrates, and the ratio of CD8/Foxp3+ cells in epithelium (e) and stroma (s) of CIN3, before and after vaccination, in all study subjects. Data from bar graphs are means of 3 to 10 regions of interest (ROIs) quantitated per tissue compartment per subject. Error bars show SEM. P < 0.05, **P < 0.01, Wilcoxon signed rank test. Scale bars, 50 μm.

Fig. 3. In vaccinated patients, the cervix is infiltrated by activated effector memory T cells with potent effector functions

(A to D) T cells were isolated from the cervix of healthy controls (normal, n = 5), patients with untreated CIN2/3 (n = 7), and patients with CIN2/3 after vaccination (n = 4). The percent of CD4 versus CD8 T cells (A), the CD4/CD8 ratio (B), the percent Foxp3+ Treg (C), and the CD8/Foxp3+ ratio (D) are shown. The CD4/CD8 ratio tended to increase in untreated CIN and normalize in vaccinated patients. The CD8/Foxp3+ ratio was lowest in untreated CIN and tended to increase after vaccination, although only the normal versus CIN2/3 group reached statistical significance. Data from bar graphs are means of cases analyzed. Error bars show SEM. P < 0.05, Wilcoxon rank sum test. (E) Representative histograms of surface phenotype and cytokine production of T cells from vaccinated patients (n = 4). Surface phenotype stains were performed on unstimulated cells, and cytokine analysis stains were performed after stimulation with phorbol 12-myristate 13-acetate and ionomycin. Most of the T cells from patients after vaccination were memory CD45RO+ T cells, many expressed the activation marker CD69, most lacked the central memory markers L-selectin/CCR7, and all expressed the gut-tropic homing receptor α4β7 integrin. Most of both CD4 and CD8 T cells produced IFN-γ, and a significant subset of CD4 T cells expressed interleukin-17 (IL-17) and/or IL-22. All histograms are gated to show CD3+ T cells; the gating strategy and representative isotype controls are included in fig. S3.

Fig. 4. Postvaccination lymphoid neogenesis in target lesions

(A) Hematoxylin and eosin–stained sections demonstrate organized lymphoid structures that are localized to the stroma immediately subjacent to residual dysplastic epithelium (top row, ×64 magnification; second row, ×160 magnification). Inflammatory infiltrates are accompanied by high endothelial venule–like vessels (triangle points, black dotted outline), access to dysplastic epithelium (white dotted outline), and lesional epithelial apoptosis (fat arrows). (B) TLSs in postvaccination stroma express Ki67, central CD20 (a pan-B cell marker), CD3 (a pan-T cell marker), and peripheral lymph node addressin (PNAd), which identifies vascular endothelium in high endothelial venules. (C) Representative IHC of Ki67 before (left) and after (right) vaccination, and bar graph summarizing quantitative image analysis of Ki67+ in lesional stroma before and after vaccination. Data from bar graphs are means of 3 to 10 ROIs quantitated per tissue compartment for each subject. Error bars show SEM. **P < 0.01, Wilcoxon signed rank test. Scale bars, 50 μm.

Fig. 5. Postvaccination stromal TLSs are associated with a functional signature

Laser capture microdissection of cervical mucosal epithelium and subjacent stroma from normal (three), CIN2/3 (three), and vaccinated CIN2/3 (three) cases. (A) In lesional epithelium overlying TLSs, expression of CD8 and CXCR3 is increased in postvaccination tissue. (B) Transcripts for IFN-β are increased in the stromal compartment of vaccinated CIN2/3. Data from scatter plots are means of duplicate per case; error bars show SEM. *P < 0.05, P < 0.01, Wilcoxon rank sum test. Dotted lines indicate the threshold of sensitivity of detection. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 6. Diversity and overlap of tissue and peripheral blood TCRs

(A) The diversity of TCR in the blood greatly exceeds that in postvaccination tissue (n = 3). (B) TCRs that are shared between the tissue and blood of individual subjects who have been vaccinated comprise a greater fraction of tissue TCRs than peripheral blood TCRs. (C) Heat maps depicting the frequency of shared TCRs in tissue and peripheral blood of three vaccinated (*) subjects (3007, 3009, and 3062) and one unvaccinated subject (0207CR), all of whom had HPV16+ CIN2/3. (D) Heat maps depicting the frequency of shared TCRs in tissue and blood samples of two vaccinated subjects with shared human leukocyte antigen (HLA) alleles (3009 and 3062) and shared TCRs in tissue and blood samples of an unvaccinated (0207) and a vaccinated (3009) subject who shared HLA alleles.