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Background: Amplivant is a molecularly optimized Toll-like receptor 2 ligand that can be covalently conjugated to tumor peptide antigens. In preclinical models, amplivant-adjuvanted synthetic long peptides (SLPs) strongly enhanced antigen presentation by dendritic cells, T cell priming and induction of effective antitumor responses. The current study is a first-in-human trial to investigate safety and immunogenicity of amplivant conjugated to human papillomavirus (HPV) 16-SLP., Methods: A dose escalation phase I vaccination trial was performed in 25 patients treated for HPV16 positive (pre-)malignant lesions. Amplivant was conjugated to two SLPs derived from the two most immunodominant regions of the HPV16 E6 oncoprotein. The vaccine, containing a mix of these two conjugates in watery solution without any other formulation, was injected intradermally three times with a 3-week interval in four dose groups (1, 5, 20 or 50 µg per conjugated peptide). Safety data were collected during the study. Peptide-specific T cell immune responses were determined in blood samples taken before, during and after vaccination using complementary immunological assays., Results: Toxicity after three amplivant-conjugated HPV16-SLP vaccinations was limited to grade 1 or 2, observed as predominantly mild skin inflammation at the vaccination site and sometimes mild flu-like symptoms. Adverse events varied from none in the lowest dose group to mild/moderate vaccine-related inflammation in all patients and flu-like symptoms in three out of seven patients in the highest dose group, after at least one injection. In the lowest dose group, vaccine-induced T cell responses were observed in the blood of three out of six vaccinated persons. In the highest dose group, all patients displayed a strong HPV16-specific T cell response after vaccination. These HPV16-specific T cell responses lasted until the end of the trial., Conclusions: Amplivant-conjugated SLPs can safely be used as an intradermal therapeutic vaccine to induce robust HPV16-specific T cell immunity in patients previously treated for HPV16 positive (pre-) malignancies. Increased vaccine dose was associated with a higher number of mild adverse events and with stronger systemic T cell immunity., Trial Registration Numbers: NCT02821494 and 2014-000658-12., Competing Interests: Competing interests: FO, CJMM, DVF and GAvdM are inventors of a patent application related to the work in this article entitled 'Adjuvant compound', with publication number WO 2013/051936 and filing date October 4, 2012. CJMM and WJK receive a salary from ISA Pharmaceuticals BV and are in possession of ISA stock appreciation rights and are inventors on patents that are licensed to or owned by ISA Pharmaceuticals BV, dealing with synthetic long peptide vaccines. SHB is named as an inventor on the patent for the use of synthetic long peptides as vaccine. SHB serves as a paid member of the strategy board of ISA Pharmaceuticals and received honoraria as a consultant for PCI Biotech, IO Biotech and DC prime., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Expression of killer cell lectin-like receptor B1 ( KLRB1 ), the gene encoding the cell surface molecule CD161, is associated with favorable prognosis in many cancers. CD161 is expressed by several lymphocyte populations, but its role and regulation on tumor-specific CD4+ T cells is unknown., Methods: We examined the clinical impact of CD4+CD161+ T cells in human papillomavirus (HPV)16+ oropharyngeal squamous cell carcinoma (OPSCC), analyzed their contribution in a cohort of therapeutically vaccinated patients and used HPV16-specific CD4+CD161+ tumor-infiltrating lymphocytes and T cell clones for in-depth mechanistic studies., Results: Central and effector memory CD4+ T cells express CD161, but only CD4+CD161+ effector memory T cells (Tem) are associated with improved survival in OPSCC. Therapeutic vaccination activates and expands type 1 cytokine-producing CD4+CD161+ effector T cells. The expression of CD161 is dynamic and follows a pattern opposite of the checkpoint molecules PD1 and CD39. CD161 did not function as an immune checkpoint molecule as demonstrated using multiple experimental approaches using antibodies to block CD161 and gene editing to knockout CD161 expression. Single-cell transcriptomics revealed KLRB1 expression in many T cell clusters suggesting differences in their activation. Indeed, CD4+CD161+ effector cells specifically expressed the transcriptional transactivator SOX4, known to enhance T cell receptor (TCR) signaling via CD3ε. Consistent with this observation, CD4+CD161+ cells respond more vigorously to limiting amounts of cognate antigen in presence of interleukin (IL)-12 and IL-18 compared to their CD161- counterparts. The expression of CD161/ KLRB1 and SOX4 was downregulated upon TCR stimulation and this effect was boosted by transforming growth factor (TGF)β1., Conclusion: High levels of CD4+CD161+ Tem are associated with improved survival and our data show that CD161 is dynamically regulated by cell intrinsic and extrinsic factors. CD161 expressing CD4+ T cells rapidly respond to suboptimal antigen stimulation suggesting that CD161, similar to SOX4, is involved in the amplification of TCR signals in CD4+ T cells., Competing Interests: Competing interests: GS reports personal fees from Pieris Pharmaceuticals GmbH outside the submitted work. FF has received honoraria for lecturing at the ESMO Virtual Advanced Course on Biomarkers for Precision Medicine 2021. PtD reports grants from Oncode Institute and Cancer Genomics Center Netherlands (CGC.NL). VMB reports funding from Centre National de la Recherche Scientifique, French Government (National Research Agency, ANR) through the 'Investments for the Future' programs LABEX SIGNALIFE ANR-11-LABX-0028 and IDEX UCAJedi ANR-15-IDEX-01, Cancéropole PACA and Fondation ARC pour la recherche sur le Cancer. SHvdB reports grants from IO Biotech and DC Prime for immunemonitoring of trials and personal consulting fees from ISA Pharmaceuticals, PCI Biotech, DC Prime, CHDR consultancy services and AGLAIA outside the submitted work. The other authors declare no conflict of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Background: A profound insight into the immune landscape of vulvar squamous cell carcinoma (VSCC) is lacking. Here, an in-depth interrogation of T cell infiltration, local immune contexture, signaling pathways and checkpoint molecule expression was performed in early-stage and late-stage VSCC., Methods: The type, location, and infiltration pattern of T cells were studied in 109 patients with primary VSCC FIGO stage I-III. RNA expression of genes involved in immune oncology and oncogenic signaling pathways was analyzed in 40 VSCC, matched for prognostic clinicopathological variables, analyzed for HPV and p53 status, and selected based on T cell infiltration., Results: High intraepithelial infiltration with CD4 or CD8 T cells was associated with longer overall and recurrence-free survival and formed an independent prognostic factor, outperforming molecular subtype and stage of the disease. Strong T cell infiltrated VSCC displayed a coordinated immune response reflected by a positive association between T cells and different lymphocyte and myeloid cell subsets. The expression of genes involved in the migration of T cells and myeloid cells, T cell activation and costimulation, interferon (IFN)-γ signaling, cytotoxicity and apoptosis was higher than in low infiltrated tumors. An active immune signaling profile was observed in all inflamed, part of the altered-excluded and not in altered-immunosuppressed or deserted VSCC. While several checkpoint molecules were overexpressed, only PD-L1 expression displayed discriminatory ability and clinical usefulness. High PD-L1 expression was detected in all inflamed and ~60% of the altered-excluded VSCC., Conclusion: An active immune signaling profile is present in 35% of primary FIGO I-III VSCCs, suggesting potential responsiveness to neoadjuvant PD-1/PD-L1 immunotherapy., Competing Interests: Competing interests: The authors (KEK, SJS, MIEvP and SHvdB) have filed a patent relating to the treatment of vulvar squamous cell carcinoma (VSCC) and methods for identifying subjects with VSCC who are likely to benefit from such therapy., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: 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.)

Background: Vulvar squamous cell carcinoma (VSCC) has been suggested to consist of three subtypes; HPV-positive, HPV-negative mutated TP53 or HPV-negative TP53 wildtype, with different clinical courses. To analyze the immune infiltrate in these molecular subtypes and its impact on clinical outcome, an in-depth study of the tumor immune microenvironment was performed., Methods: Sixty-five patients with invasive VSCC matched for age, FIGO stage and treatment modality, were grouped according to the presence of HPV and p53 protein expression status. Archived tissues were analyzed for intraepithelial and stromal expression of CD3, CD8, Foxp3, PD-1, and pan-keratin in randomly selected areas using immunofluorescence. Additional phenotyping of T cells was performed ex-vivo on VSCC (n = 14) and blood samples by flow cytometry. Healthy vulvar samples and blood served as controls., Results: Based on T-cell infiltration patterns about half of the VSCC were classified as inflamed or altered-excluded while one-third was immune-deserted. High intraepithelial helper T cell infiltration was observed in 78% of the HPV-induced VSCC, 60% of the HPVnegVSCC/p53wildtype and 40% of the HPVnegVSCC with abnormal p53 expression. A high intraepithelial infiltration with activated (CD3 + PD-1 + ), specifically helper T cells (CD3 + CD8 - Foxp3 - ), was associated with a longer recurrence-free period and overall survival, irrespective of HPV and p53 status. Flow cytometry confirmed the tumor-specific presence of activated (CD4 + PD-1 ++ CD161 - CD38 + HLA-DR + and CD8 + CD103 + CD161 - NKG2A +/- PD1 ++ CD38 ++ HLA-DR + ) effector memory T cells., Conclusion: This is the first study demonstrating an association between intraepithelial T cells and clinical outcome in VSCC. Our data suggest that abnormal p53 expressing VSCCs mostly are cold tumors whereas HPV-driven VSCCs are strongly T-cell infiltrated.