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

Abstract

BackgroundVulvar 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.MethodsTwo 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.ResultsHealthy 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.ConclusionA 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.

CD163+cytokine-producing cDC2 stimulate intratumoral type 1 T cell responses in HPV16-induced oropharyngeal cancer

“…Notably, we have shown before that approximately 99% of the CD3 + CD8 – T cells express CD4. 23 As the reactivity of the TILs against the tumor-specific viral oncoproteins E6 and E7 of HPV16 was already known, 3 8 9 we were able to assess whether these CD14 ‒ CD33 ‒ CD163 + cells preferentially accumulated in tumors with a concomitant tumor-specific type 1 T cell reaction. Indeed, higher numbers of intraepithelial and stromal CD14 ‒ CD33 ‒ CD163 + cells were found in OPSCC containing HPV16-specific TIL (HPV16 + IR + ) when compared with the group of OPSCC in which no intratumoral HPV16-specific TILs (HPV16 + IR - ) were detected ( figure 1D and online supplementary figure 2 ).…”

Lack of myeloid cell infiltration as an acquired resistance strategy to immunotherapy

“…The absence of a coordinated CD8 + T cell and inflammatory myeloid cell infiltration was also found to act as primary resistance mechanism in patients with HPV16-induced premalignant lesions. 20 …”

“…Formalin-fixed paraffin-embedded biopsies of 24 HPV16 + vulvar high-grade squamous intraepithelial lesions (vHSIL) patients, treated with an experimental vaccine containing 13 SLPs covering the entire amino acid sequence of HPV16 oncoproteins E6 and E7, were cut in 4 um thick sections. These samples were stained with two multiplex immunofluorescence panels, 20 one for T cells and one for myeloid cells. The slides were then scanned with the Vectra multispectral imaging system (PerkinElmer), and cells were, after manual training, automatically phenotyped and counted with inForm advanced image analysis software (PerkinElmer).…”

“…To validate these findings in the human setting, we analyzed the immune microenvironment of patients with HPV16induced vHSIL, after their systemic HPV16-specific T-cell response had been amplified by HPV16 SLP vaccination (n=24). 20 Interestingly, clinical responders (defined as≥50% lesion regression 12 months after vaccination) displayed significantly higher infiltration Open access with CD14 + CD68 +/-CD163inflammatory macrophages after vaccination when compared with non-responders ( figure 3D). This influx of CD14 + inflammatory macrophages coincided with significant T-cell infiltration, primarily type I cytokine-producing (Tbet + ) T cells (figure 3D).…”

A pre‐existing coordinated inflammatory microenvironment is associated with complete response of vulvar high‐grade squamous intraepithelial lesions to different forms of immunotherapy

“…Notably, most studies identified immune cells at the single marker level, and some at a double or triple marker level. This provides only limited insights in the complexity and functional status of the many different immune cells that together compose the immune microenvironment, when compared to the multispectral panels that can be used nowadays 9 …”