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

Abstract

Immunotherapy of vulvar high-grade squamous intraepithelial lesion (vHSIL) is investigated as an alternative for surgery, because of high comorbidity and risk of recurrence. Limited evidence exists on the role and composition of the immune microenvironment in current immunotherapeutic approaches for vHSIL. The vHSIL of 29 patients biopsied before treatment with imiquimod were analyzed by two multiplex seven-color immunofluorescence panels to investigate the pre-existing T-cell and myeloid cell composition in relation to treatment response. The samples were scanned with the Vectra multispectral imaging system. Cells were automatically phenotyped and counted with inForm advanced image analysis software. Cell counts and composition were compared to that of vHSIL patients before therapeutic vaccination (n = 29) and to healthy vulva (n = 27). Our data show that the immune microenvironment of complete responders (CR) to imiquimod resembled the coordinated infiltration with type 1 CD4⁺ and CD8⁺ T cells and CD14⁺ inflammatory myeloid cells also found in healthy vulva. However, more CD8⁺ T cells and FoxP3⁺ regulatory T cells were present in CR. The lesions of partial responders (PR) lacked such a coordinated response and displayed an impaired influx of CD14⁺ inflammatory myeloid cells. Importantly, complete responses after imiquimod or therapeutic vaccination showed the same dependency on a pre-existing coordinated type 1 T-cell and CD14⁺ myeloid cell infiltration. In conclusion, a good clinical outcome after two different forms of immunotherapy for vHSIL is associated with the presence of a primary inflammatory process resulting in the coordinated influx of several types of immune cells which is then amplified.

Keywords: TLR7; imiquimod; immune microenvironment; therapeutic vaccine; vulvar HSIL.

FIGURE 1

Multiplex immunofluorescence to detect T cells and myeloid cells in vHSIL. A, Composite image including all individual markers of the seven‐color T‐cell panel staining, consisting of the markers CD3, CD8, FoxP3, Tbet, Tim3, PD‐1 and DAPI. B, Composite image including all individual markers of the seven‐color myeloid cell panel staining, consisting of the markers CD33, CD68, CD163, PD‐L1, CD14, CD11c and DAPI

FIGURE 2

The pre‐imiquimod TME differs between the different response groups. A, The numbers of intraepithelial and stromal infiltrating T‐cell and myeloid cell subtypes are presented as cells/mm² epithelium and cells/mm² stroma in the pre‐imiquimod vHSIL biopsies of 29 patients. Each dot represents an individual sample, the horizontal bars indicate the median cell counts and the vertical bars are the 95% confidence intervals. B, T‐cell and C, myeloid‐cell infiltrate of the pre‐imiquimod vHSIL patients when grouped according to response (PR, CR), and compared to healthy vulvar tissue (n = 27), presented as median cells/mm² epithelium and median cells/mm² stroma. A threshold of a median cell count ≥10 cells/mm² in at least one group (NR, PR, CR or healthy vulva) was applied to study the changes in biologically common phenotypes. CR, complete responders (n = 14); PR, partial responders (n = 12)

FIGURE 3

Correlation between the absolute numbers of tissue infiltrating T cells and myeloid cells in the pre‐imiquimod vHSIL biopsies of partial responders and complete responders. Nonparametric Spearman r correlation analysis (two‐tailed) was performed to analyze the co‐infiltration of the indicated different immune cell subtypes in the epithelium (E) and stroma (S), and is shown in a heatmap for vHSIL partial responders and complete responders to topical imiquimod treatment. Red indicates a strong positive correlation between the two cell phenotypes, blue a strong negative correlation, the green squares indicate the most striking differences between the partial and complete responders. A threshold of a median cell count ≥10 cells/mm² in at least one group (NR, PR, CR or healthy vulva) was applied to study the changes in biologically common phenotypes. CR, complete responders (n = 14); PR, partial responders (n = 12)

FIGURE 4

Fractional differences between the pre‐imiquimod and pre‐vaccination immune microenvironment in vHSIL, divided based on clinical response to the respective therapy. A, Fractional composition of the T cells and myeloid cells in the pre‐imiquimod vHSIL cohort, grouped according to response (NR: n = 3, PR: n = 12, CR: n = 14), and compared to healthy vulva (n = 27). B, Fractional composition of the T cells and myeloid cells in the pretherapeutic vaccination vHSIL cohort, grouped according to response (NR: n = 12, PR: n = 10, CR: n = 7), and compared to healthy vulva (n = 27)

FIGURE 5

Summary of complete responder's vHSIL immune microenvironment in the context of the mode of action of imiquimod and therapeutic vaccination. The vHSIL immune microenvironment of complete responders to imiquimod and therapeutic HPV16 synthetic long peptide (SLP) vaccination comprises a coordinated infiltration with type 1 (Tbet⁺) CD4⁺ and CD8⁺ T cells as well as CD14⁺ inflammatory myeloid cells. Imiquimod stimulates CD14⁺ myeloid cells to produce pro‐inflammatory cytokines which activate and attract the type 1 T cells. ¹³ Whereas, therapeutic vaccination stimulates the type 1 T‐cell response, which in turn produces cytokines that also stimulate the influx of the lesion by CD14⁺ myeloid cells. ⁹ This figure was created using adapted images of Servier Medical Art, licensed under a creative commons attribution 3.0 unported license