Pre-existing inflammatory immune microenvironment predicts the clinical response of vulvar high-grade squamous intraepithelial lesions to therapeutic HPV16 vaccination

Abstract

Background: Vulvar high-grade squamous intraepithelial lesion (vHSIL) is predominantly induced by high-risk human papilloma virus type 16 (HPV16). In two independent trials, therapeutic vaccination against the HPV16 E6 and E7 oncoproteins resulted in objective partial and complete responses (PRs/CRs) in half of the patients with HPV16+ vHSIL at 12-month follow-up. Here, the prevaccination and postvaccination vHSIL immune microenvironment in relation to the vaccine-induced clinical response was investigated.

Methods: Two novel seven-color multiplex immunofluorescence panels to identify T cells (CD3, CD8, Foxp3, Tim3, Tbet, PD-1, DAPI) and myeloid cells (CD14, CD33, CD68, CD163, CD11c, PD-L1, DAPI) were designed and fully optimized for formalin-fixed paraffin-embedded tissue. 29 prevaccination and 24 postvaccination biopsies of patients with vHSIL, and 27 healthy vulva excisions, were stained, scanned with the Vectra multispectral imaging system, and automatically phenotyped and counted using inForm advanced image analysis software.

Results: Healthy vulvar tissue is strongly infiltrated by CD4 and CD8 T cells expressing Tbet and/or PD-1 and CD14+HLA-DR+ inflammatory myeloid cells. The presence of such a coordinated pre-existing proinflammatory microenvironment in HPV16+ vHSIL is associated with CR after vaccination. In partial responders, a disconnection between T cell and CD14+ myeloid cell infiltration was observed, whereas clinical non-responders displayed overall lower immune cell infiltration. Vaccination improved the coordination of local immunity, reflected by increased numbers of CD4+Tbet+ T cells and HLA-DR+CD14+ expressing myeloid cells in patients with a PR or CR, but not in patients with no response. CD8+ T cell infiltration was not increased after vaccination.

Conclusion: A prevaccination inflamed type 1 immune contexture is required for stronger vaccine-induced immune infiltration and is associated with better clinical response. Therapeutic vaccination did not overtly increase immune infiltration of cold lesions.

Keywords: immunotherapy; therapeutic vaccination; tumor microenvironment; vulvar HSIL.

Conflict of interest statement

Competing interests: SHvdB is being named as an inventor on the patent for the use of synthetic long peptides as vaccine for the treatment of HPV-induced diseases, which is exploited by ISA Pharmaceuticals. SHvdB serves as a paid member of the strategy board of ISA Pharmaceuticals. No other potential conflicts of interest relevant to this article were reported.

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Multiplex immunofluorescence staining to detect T cells and myeloid cells. Two multiplex panels were developed to detect T cells and myeloid cells in vulvar tissue. (A) The expression of each of the seven markers for T cells showing CD3, CD8, FoxP3, Tbet, Tim3, PD-1 and DAPI as well as a figure showing all seven markers simultaneously. Arrows indicate examples of analyzed phenotypes. (B) The expression of each of the seven markers for myeloid cells showing CD33, CD68, CD163, PD-L1, CD14, CD11c and DAPI as well as a figure showing all seven markers simultaneously. Arrows indicate examples of analyzed phenotypes.

Figure 2

Healthy vulvar tissue is infiltrated by activated type 1 T cells and several types of myeloid cells while the vulvar high-grade squamous intraepithelial lesion (vHSIL) immune infiltrate is highly heterogenic among different patients. The numbers of intraepithelial and stroma-infiltrating T cell and myeloid cell subtypes, of which the median cell count exceeded the threshold of ≥10 cells/mm2, are presented as cells/mm2 for (A) human papillomavirus-negative healthy vulva (n=27) and (B) vHSIL (n=29) before vaccination. Each dot represents an individual sample, the horizontal bars indicate the median cell counts, and the vertical bars are the 95% CIs.

Figure 3

The tumor microenvironment of complete responders (CR, n=7) displays a similar immune infiltration pattern as found in healthy vulvar tissue. The numbers of intraepithelial and stroma-infiltrating T cell and myeloid cell subtypes, of which the median cell count exceeded the threshold of ≥10 cells/mm2, are presented as cells/mm2 in the (A) prevaccination (n=29) and (B) postvaccination (n=24) vulvar high-grade squamous intraepithelial lesion biopsies of vaccinated patients grouped according to their best clinical response during the 12 months of follow-up, and compared with healthy vulvar tissue (n=27). NR, non-responders (n=12); PR, partial responders (n=10). Statistical differences in the cell types between patient groups are depicted in online supplementary files 6 and 9. (C) Highlighted T cell and myeloid cell subtypes that significantly differed among different response groups, shown as fractional differences in all CD3+ T cells (left) or all CD14+ and CD68+ myeloid cells (right), prevaccination and postvaccination.

Figure 4

Correlation between the absolute numbers of tissue-infiltrating T cells and myeloid cells in the pretreatment and post-treatment vulvar high-grade squamous intraepithelial lesion (vHSIL) biopsies and in healthy vulvar tissue. Non-parametric Spearman r correlation analysis (two tailed) was performed to analyze the coinfiltration of the indicated different immune cell subtypes in the epithelium (E) and stroma (S) and is shown in a heatmap for healthy vulvar tissue (n=27), prevaccination complete responder patients with vHSIL (n=7) and partial responder patients with vHSIL (n=10) as well as postvaccination partial responder patients with vHSIL (n=9).

Figure 5

Immune profile of non-responders (NR), partial responders (PR) and complete responders (CR) bases on the relative infiltration of significantly different immune cell subtypes and human papillomavirus (HPV)-specific T cell reactivity. The strength of the vaccine-induced HPV16-specific T cell response present in peripheral blood mononuclear cells (PBMC) as measured by the production of interferon γ (IFNγ) in the supernatant of PBMC stimulated with six different E6 and E7 peptide pools for clinical NR, PR and CR as determined previously by cytokine bead array is depicted as (A) the mean±SEM of all patients to all six peptide pools tested, before and after two and four vaccinations; and (B) the median of max calculated by taking the median of the highest responses to each peptide pool after two or four vaccinations. Shown is the mean±SEM of all medians per group. (C) and (D) The cell counts of all immune subsets of which the median cell count exceeded the threshold of ≥10 cells/mm2 and the numbers significantly differed between at least two of the patient groups (NR, PR, CR) either before vaccination (C) or after vaccination (D) as well as the median of max of IFNγ production as determined by cytometric bead array are depicted after the data were z-transformed to enable comparison of relative differences.