NKG2A Blockade Potentiates CD8 T Cell Immunity Induced by Cancer Vaccines.

Tumor-infiltrating CD8 T cells were found to frequently express the inhibitory receptor NKG2A, particularly in immune-reactive environments and after therapeutic cancer vaccination. High-dimensional cluster analysis demonstrated that NKG2A marks a unique immune effector subset preferentially co-expressing the tissue-resident CD103 molecule, but not immune checkpoint inhibitors. To examine whether NKG2A represented an adaptive resistance mechanism to cancer vaccination, we blocked the receptor with an antibody and knocked out its ligand Qa-1b, the conserved ortholog of HLA-E, in four mouse tumor models. The impact of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1b axis even in a PD-1 refractory mouse model. NKG2A blockade therapy operated through CD8 T cells, but not NK cells. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines.

Publisher Correction: Actively personalized vaccination trial for newly diagnosed glioblastoma.

The additional author support information was erroneously omitted from the Supplementary Information. This has been corrected online.

Actively personalized vaccination trial for newly diagnosed glioblastoma.

Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors1,2. For treatments using checkpoint inhibitors to be successful, a high mutational load and responses to neoepitopes are thought to be essential3. There is limited intratumoural infiltration of immune cells4 in glioblastoma and these tumours contain only 30-50 non-synonymous mutations5. Exploitation of the full repertoire of tumour antigens-that is, both unmutated antigens and neoepitopes-may offer more effective immunotherapies, especially for tumours with a low mutational load. Here, in the phase I trial GAPVAC-101 of the Glioma Actively Personalized Vaccine Consortium (GAPVAC), we integrated highly individualized vaccinations with both types of tumour antigens into standard care to optimally exploit the limited target space for patients with newly diagnosed glioblastoma. Fifteen patients with glioblastomas positive for human leukocyte antigen (HLA)-A*02:01 or HLA-A*24:02 were treated with a vaccine (APVAC1) derived from a premanufactured library of unmutated antigens followed by treatment with APVAC2, which preferentially targeted neoepitopes. Personalization was based on mutations and analyses of the transcriptomes and immunopeptidomes of the individual tumours. The GAPVAC approach was feasible and vaccines that had poly-ICLC (polyriboinosinic-polyribocytidylic acid-poly-L-lysine carboxymethylcellulose) and granulocyte-macrophage colony-stimulating factor as adjuvants displayed favourable safety and strong immunogenicity. Unmutated APVAC1 antigens elicited sustained responses of central memory CD8+ T cells. APVAC2 induced predominantly CD4+ T cell responses of T helper 1 type against predicted neoepitopes.

Enhanced antitumour immunity following neoadjuvant chemoradiotherapy mediates a favourable prognosis in women with resected pancreatic cancer.

BACKGROUND: This study investigates sex disparities in clinical outcomes and tumour immune profiles in patients with pancreatic ductal adenocarcinoma (PDAC) who underwent upfront resection or resection preceded by gemcitabine-based neoadjuvant chemoradiotherapy (nCRT). METHODS: Patients originated from the PREOPANC randomised controlled trial. Upfront surgery was performed in 82 patients, and 66 received nCRT before resection. The impact of sex on overall survival (OS) was investigated using Cox proportional hazards models. The immunological landscape within the tumour microenvironment (TME) was mapped using transcriptomic and spatial proteomic profiling. RESULTS: The 5-year OS rate differed between the sexes following resection preceded by nCRT, with 43% for women compared with 22% for men. In multivariate analysis, the female sex was a favourable independent prognostic factor for OS only in the nCRT group (HR 0.19; 95% CI 0.07 to 0.52). Multivariate heterogeneous treatment effects analysis revealed a significant interaction between sex and treatment, implying increased nCRT efficacy among women with resected PDAC. The TME of women contained fewer protumoural CD163+MRC1+M2 macrophages than that of men after nCRT, as indicated by transcriptomic and validated using spatial proteomic profiling. CONCLUSION: PDAC tumours of women are more sensitive to gemcitabine-based nCRT, resulting in longer OS after resection compared with men. This may be due to enhanced immunity impeding the infiltration of protumoral M2 macrophages into the TME. Our findings highlight the importance of considering sex disparities and mitigating immunosuppressive macrophage polarisation for personalised PDAC treatment.

Monocyte infiltration is an independent positive prognostic biomarker in vulvar squamous cell carcinoma.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) arises after an HPV infection or the mutation of p53 or other driver genes and is treated by mutilating surgery and/or (chemo) radiation, with limited success and high morbidity. In-depth information on the immunological make up of VSCC is pivotal to assess whether immunotherapy may form an alternative treatment. METHODS: A total of 104 patient samples, comprising healthy vulva (n = 27) and VSCC (n = 77), were analyzed. Multispectral immunofluorescence (15 markers) was used to study both the myeloid and lymphoid immune cell composition, and this was linked to differences in transcriptomics (NanoString nCounter, 1258 genes) and in survival (Kaplan-Meier analyses). RESULTS: Healthy vulva and VSCC are both well infiltrated but with different subpopulations of lymphoid and myeloid cells. In contrast to the lymphoid cell infiltrate, the density and composition of the myeloid cell infiltrate strongly differed per VSCC molecular subtype. A relative strong infiltration with epithelial monocytes (HLADR-CD11c-CD14+CD68-CD163-CD33-) was prognostic for improved survival, independent of T cell infiltration, disease stage or molecular subtype. A strong infiltration with T cells and/or monocytes was associated with drastic superior survival: 5-year survival > 90% when either one is high, versus 40% when both are low (p < 0.001). CONCLUSION: A hot myeloid and/or lymphoid infiltrate predicts excellent survival in VSCC. Based on the response of similarly high-infiltrated other tumor types, we have started to explore the potential of neoadjuvant checkpoint blockade in VSCC.

APOLLO: neo-adjuvant pembrolizumab for primary vulvar squamous cell carcinoma-a multicenter, single-arm, phase II, clinical proof-of-concept study.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) is a rare cancer for which the cornerstone of treatment is surgery with high complication rates. The unmet need is a less radical and more effective treatment for VSCC. PRIMARY OBJECTIVES: To investigate the impact of mono-immunotherapy pembrolizumab as neoadjuvant treatment for primary resectable VSCC patients. STUDY HYPOTHESIS: Some primary VSCC patients display a specific immune profile which is associated with better survival. In other tumors, this profile is associated with a better response to programmed cell death protein 1 (PD-1) checkpoint blockade which may reinvigorate tumor-specific T cells. This potentially results in a reduced tumor load and less radical surgery and/or adjuvant treatment in patients with this immune profile. TRIAL DESIGN: This is an investigator-initiated, prospective, single arm, multicenter, phase II clinical trial. INCLUSION CRITERIA: Patients with VSCC clinical stage International Federation of Gynecology and Obstetrics (FIGO) I-III (2021) eligible for primary surgery, with at least one measurable lesion of at least one dimension ≥10 mm in the largest diameter, are included in this study. MAIN EXCLUSION CRITERIA: Patients not suitable for surgery and/or previously treated with immunomodulatory agents, and/or who suffer from comorbidities that may interfere with PD-1 blockade, are excluded from the study. ENDPOINTS: The clinical efficacy of neoadjuvant pembrolizumab in VSCC is measured by an objective change in tumor size according to the Response Evaluation Criteria In Solid Tumors version 1.1 (RECIST 1.1) and documented by calipers using standardized digital photography with a reference ruler. In addition, the activation, proliferation, and migration of T cells in the tumor will be studied. The secondary endpoints are pathological complete responses at the time of surgery, feasibility, and safety. SAMPLE SIZE: 40 patients with FIGO I-III (2021) primary VSCC will be enrolled. ESTIMATED DATES FOR COMPLETING ACCRUAL AND PRESENTING RESULTS: The intervention phase started in July 2023 and will continue until July 2025. The expected completion of the entire study is July 2026. TRIAL REGISTRATION NUMBER: NCT05761132.

The common HLA class I-restricted tumor-infiltrating T cell response in HPV16-induced cancer.

Immunotherapies targeting truly tumor-specific targets focus on the expansion and activation of T cells against neoantigens or oncogenic viruses. One target is the human papilloma virus type 16 (HPV16), responsible for several anogenital cancers and oropharyngeal carcinomas. Spontaneous and vaccine-induced HPV-specific T cells have been associated with better clinical outcome. However, the epitopes and restriction elements to which these T cells respond remained elusive. To identify CD8+ T cell epitopes in cultures of tumor infiltrating lymphocytes, we here used multimers and/or a functional screening platform exploiting single HLA class I allele-engineered antigen presenting cells. This resulted in the detection of 20 CD8+ T cell responses to 11 different endogenously processed HLA-peptide combinations within 12 HPV16-induced tumors. Specific HLA-peptide combinations dominated the response in patients expressing these HLA alleles. T cell receptors (TCRs) reactive to seven different HLA class I-restricted peptides could be isolated and analysis revealed tumor reactivity for five of the six TCRs analyzed. The tumor reactive TCRs to these dominant HLA class I peptide combinations can potentially be used to engineer tumor-specific T cells for adoptive cell transfer approaches to treat HPV16-induced cancers.

Immunotherapeutic Potential of TGF-ß Inhibition and Oncolytic Viruses.

In cancer immunotherapy, a patient's own immune system is harnessed against cancer. Immune checkpoint inhibitors release the brakes on tumor-reactive T cells and, therefore, are particularly effective in treating certain immune-infiltrated solid tumors. By contrast, solid tumors with immune-silent profiles show limited efficacy of checkpoint blockers due to several barriers. Recent discoveries highlight transforming growth factor-ß (TGF-ß)-induced immune exclusion and a lack of immunogenicity as examples of these barriers. In this review, we summarize preclinical and clinical evidence that illustrates how the inhibition of TGF-ß signaling and the use of oncolytic viruses (OVs) can increase the efficacy of immunotherapy, and discuss the promise and challenges of combining these approaches with immune checkpoint blockade.

NKG2A is a late immune checkpoint on CD8 T cells and marks repeated stimulation and cell division.

The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to block NKG2A are currently tested in several efficacy trials for different tumor indications, it is important to characterize the NKG2A+ CD8 T cell population in the context of other inhibitory receptors. Here we used a well-controlled culture system to study the kinetics of inhibitory receptor expression. Naïve mouse CD8 T cells were synchronously and repeatedly activated by artificial antigen presenting cells in the presence of the homeostatic cytokine IL-7. The results revealed NKG2A as a late inhibitory receptor, expressed after repeated cognate antigen stimulations. In contrast, the expression of PD-1, TIGIT and LAG-3 was rapidly induced, hours after first contact and subsequently down regulated during each resting phase. This late, but stable expression kinetics of NKG2A was most similar to that of TIM-3 and CD39. Importantly, single-cell transcriptomics of human tumor-infiltrating lymphocytes (TILs) showed indeed that these receptors were often coexpressed by the same CD8 T cell cluster. Furthermore, NKG2A expression was associated with cell division and was promoted by TGF-ß in vitro, although TGF-ß signaling was not necessary in a mouse tumor model in vivo. In summary, our data show that PD-1 reflects recent TCR triggering, but that NKG2A is induced after repeated antigen stimulations and represents a late inhibitory receptor. Together with TIM-3 and CD39, NKG2A might thus mark actively dividing tumor-specific TILs.

Cross-presentation of a TAP-independent signal peptide induces CD8 T immunity to escaped cancers but necessitates anchor replacement.

Cancer cells frequently display defects in their antigen-processing pathway and thereby evade CD8 T cell immunity. We described a novel category of cancer antigens, named TEIPP, that emerge on cancers with functional loss of the peptide pump TAP. TEIPPs are non-mutated neoantigens despite their 'self' origin by virtue of their absence on normal tissues. Here, we describe the development of a synthetic long peptide (SLP) vaccine for the most immunogenic TEIPP antigen identified thus far, derived from the TAP-independent LRPAP1 signal sequence. LRPAP121-30-specific CD8 T cells were present in blood of all tested healthy donors as well as patients with non-small cell lung adenocarcinoma. SLPs with natural flanking, however, failed to be cross-presented by monocyte-derived dendritic cells. Since the C-terminus of LRPAP121-30 is an unconventional and weakly binding serine (S), we investigated if replacement of this anchor would result in efficient cross-presentation. Exchange into a valine (V) resulted in higher HLA-A2 binding affinity and enhanced T cell stimulation. Importantly, CD8 T cells isolated using the V-variant were able to bind tetramers with the natural S-variant and respond to TAP-deficient cancer cells. A functional screen with an array of N-terminal and C-terminal extended SLPs pointed at the 24-mer V-SLP, elongated at the N-terminus, as most optimal vaccine candidate. This SLP was efficiently cross-presented and consistently induced a strong polyclonal LRPAP121-30-specific CD8 T cells from the endogenous T cell repertoire. Thus, we designed a TEIPP SLP vaccine from the LRPAP1 signal sequence ready for validation in clinical trials.

Neoantigen landscape dynamics during human melanoma-T cell interactions.

Recognition of neoantigens that are formed as a consequence of DNA damage is likely to form a major driving force behind the clinical activity of cancer immunotherapies such as T-cell checkpoint blockade and adoptive T-cell therapy. Therefore, strategies to selectively enhance T-cell reactivity against genetically defined neoantigens are currently under development. In mouse models, T-cell pressure can sculpt the antigenicity of tumours, resulting in the emergence of tumours that lack defined mutant antigens. However, whether the T-cell-recognized neoantigen repertoire in human cancers is constant over time is unclear. Here we analyse the stability of neoantigen-specific T-cell responses and the antigens they recognize in two patients with stage IV melanoma treated by adoptive T-cell transfer. The T-cell-recognized neoantigens can be selectively lost from the tumour cell population, either by overall reduced expression of the genes or loss of the mutant alleles. Notably, loss of expression of T-cell-recognized neoantigens was accompanied by development of neoantigen-specific T-cell reactivity in tumour-infiltrating lymphocytes. These data demonstrate the dynamic interactions between cancer cells and T cells, which suggest that T cells mediate neoantigen immunoediting, and indicate that the therapeutic induction of broad neoantigen-specific T-cell responses should be used to avoid tumour resistance.

Correlates of immune and clinical activity of novel cancer vaccines.

Cancer vaccines are solely meant to amplify the pool of type 1 cytokine oriented CD4+ and CD8+ T cells that recognize tumor antigen and ultimately foster control and destruction of a growing tumor. They are not designed to deal with all aspects of immune ignorance, exclusion, suppression and escape that are generally in place in patients with cancer and may prevent the T cells to enter the tumor or to exert their effector function. This simple fact prompted for a reappraisal of the many recent trials in which therapeutic cancer vaccines have been examined as monotherapy. In this review, I focus on trials examining therapeutic cancer vaccines at different stages of existing disease. The analysis of vaccine-induced immune responses and clinical activity of therapeutic cancer vaccines revealed four levels of evidence for vaccine efficacy. The lowest levels, reflect the many trials in which the strength of the tumor-reactive T cell response of vaccinated patients is associated with better clinical outcome or change in tumor marker. The highest levels indicate occasional regressions of tumors and metastases after vaccination or reflect a stronger clinical impact of vaccine in a randomized trial. A whole series of trials in which vaccine-induced tumor immunity correlates with the clinical impact of cancer vaccines in premalignant diseases, settings of low tumor burden or tumor regressions in patients with cancer, form an attest to the fact that cancer vaccines work. While the current number of true clinical responders in each cancer trial is too low for firm conclusions on immune correlates of clinical reactivity in cancer, extrapolation of the results from vaccinated patients with pre-cancers suggest a requirement of broad type 1 T cell reactivity.

Regulatory T Cell Depletion Using a CRISPR Fc-Optimized CD25 Antibody.

Regulatory T cells (Tregs) are major drivers behind immunosuppressive mechanisms and present a major hurdle for cancer therapy. Tregs are characterized by a high expression of CD25, which is a potentially valuable target for Treg depletion to alleviate immune suppression. The preclinical anti-CD25 (αCD25) antibody, clone PC-61, has met with modest anti-tumor activity due to its capacity to clear Tregs from the circulation and lymph nodes, but not those that reside in the tumor. The optimization of the Fc domain of this antibody clone has been shown to enhance the intratumoral Treg depletion capacity. Here, we generated a stable cell line that produced optimized recombinant Treg-depleting antibodies. A genome engineering strategy in which CRISPR-Cas9 was combined with homology-directed repair (CRISPR-HDR) was utilized to optimize the Fc domain of the hybridoma PC-61 for effector functions by switching it from its original rat IgG1 to a mouse IgG2a isotype. In a syngeneic tumor mouse model, the resulting αCD25-m2a (mouse IgG2a isotype) antibody mediated the effective depletion of tumor-resident Tregs, leading to a high effector T cell (Teff) to Treg ratio. Moreover, a combination of αCD25-m2a and an αPD-L1 treatment augmented tumor eradication in mice, demonstrating the potential for αCD25 as a cancer immunotherapy.

Interleukin-6-mediated resistance to immunotherapy is linked to impaired myeloid cell function.

High serum levels of interleukin-6 (IL-6) correlate with poor prognosis and chemotherapy resistance in several cancers. The underlying mechanisms and its effects on immunotherapy are largely unknown. To address this, we developed a human papillomavirus type 16 (HPV16)-associated tumor model expressing IL-6 to investigate the impact of tumor-expressed IL-6 during cisplatin chemotherapy and HPV16 synthetic long peptide vaccination as immunotherapy. The effects of tumor-produced IL-6 on tumor growth, survival and the tumor microenvironment were analyzed. Our data demonstrated that tumor-produced IL-6 conferred resistance to cisplatin and therapeutic vaccination. This was not caused by a changed in vitro or in vivo growth rate of tumor cells, or a changed sensitivity of tumor cells to chemotherapy or T-cell-mediated killing. Furthermore, no overt differences in the frequencies of tumor-infiltrating subsets of T cells or CD11b+ myeloid cells were observed. IL-6, however, affected the systemic and local function of myeloid cells, reflected by a strong reduction of major histocompatibility complex (MHC) class II expression on all major myeloid cell subtypes. Resistance to both therapies was associated with a changed intratumoral influx of MHC class II+ myeloid cells toward myeloid cells with no or lower MHC class II expression. Importantly, while these IL-6-mediated effects provided resistance to the immunotherapy and chemotherapy as single therapies, their combination still successfully mediated tumor control. In conclusion, IL-6-mediated therapy resistance is caused by an extrinsic mechanism involving an impaired function of intratumoral myeloid cells. The fact that resistance can be overcome by combination therapies provides direction to more effective therapies for cancer.

Simplified Monopalmitoyl Toll-like Receptor 2 Ligand Mini-UPam for Self-Adjuvanting Neoantigen-Based Synthetic Cancer Vaccines.

Synthetic vaccines, based on antigenic peptides that comprise MHC-I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll-like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam3 CysSK4 . A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block pre-prepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.

Epitope Selection for HLA-DQ2 Presentation: Implications for Celiac Disease and Viral Defense.

We have reported that the major histocompatibility molecule HLA-DQ2 (DQA1*05:01/DQB1*02:01) (DQ2) is relatively resistant to HLA-DM (DM), a peptide exchange catalyst for MHC class II. In this study, we analyzed the role of DQ2/DM interaction in the generation of DQ2-restricted gliadin epitopes, relevant to celiac disease, or DQ2-restricted viral epitopes, relevant to host defense. We used paired human APC, differing in DM expression (DMnull versus DMhigh) or differing by expression of wild-type DQ2, versus a DM-susceptible, DQ2 point mutant DQ2α+53G. The APC pairs were compared for their ability to stimulate human CD4+ T cell clones. Despite higher DQ2 levels, DMhigh APC attenuated T cell responses compared with DMnull APC after intracellular generation of four tested gliadin epitopes. DMhigh APC expressing the DQ2α+53G mutant further suppressed these gliadin-mediated responses. The gliadin epitopes were found to have moderate affinity for DQ2, and even lower affinity for the DQ2 mutant, consistent with DM suppression of their presentation. In contrast, DMhigh APC significantly promoted the presentation of DQ2-restricted epitopes derived intracellularly from inactivated HSV type 2, influenza hemagglutinin, and human papillomavirus E7 protein. When extracellular peptide epitopes were used as Ag, the DQ2 surface levels and peptide affinity were the major regulators of T cell responses. The differential effect of DM on stimulation of the two groups of T cell clones implies differences in DQ2 presentation pathways associated with nonpathogen- and pathogen-derived Ags in vivo.

Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Further, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.

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

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.

Tumor mutational load, CD8+ T cells, expression of PD-L1 and HLA class I to guide immunotherapy decisions in NSCLC patients.

OBJECTIVES: A minority of NSCLC patients benefit from anti-PD1 immune checkpoint inhibitors. A rational combination of biomarkers is needed. The objective was to determine the predictive value of tumor mutational load (TML), CD8+ T cell infiltration, HLA class-I and PD-L1 expression in the tumor. MATERIALS AND METHODS: Metastatic NSCLC patients were prospectively included in an immune-monitoring trial (NTR7015) between April 2016-August 2017, retrospectively analyzed in FFPE tissue for TML (NGS: 409 cancer-related-genes) and by IHC staining to score PD-L1, CD8+ T cell infiltration, HLA class-I. PFS (RECISTv1.1) and OS were analyzed by Kaplan-Meier methodology. RESULTS: 30 patients with adenocarcinoma (67%) or squamous cell carcinoma (33%) were included. High TML was associated with better PFS (p = 0.004) and OS (p = 0.025). Interaction analyses revealed that patients with both high TML and high total CD8+ T cell infiltrate (p = 0.023) or no loss of HLA class-I (p = 0.026), patients with high total CD8+ T cell infiltrate and no loss of HLA class-I (p = 0.041) or patients with both high PD-L1 and high TML (p = 0.003) or no loss of HLA class-I (p = 0.032) were significantly associated with better PFS. Unsupervised cluster analysis based on these markers revealed three sub-clusters, of which cluster-1A was overrepresented by patients with progressive disease (15 out of 16), with significant effect on PFS (p = 0.007). CONCLUSION: This proof-of-concept study suggests that a combination of PD-L1 expression, TML, CD8+ T cell infiltration and HLA class-I functions as a better predictive biomarker for response to anti-PD-1 immunotherapy. Consequently, refinement of this set of biomarkers and validation in a larger set of patients is warranted.

Tissue-Specific Gene Expression during Productive Human Papillomavirus 16 Infection of Cervical, Foreskin, and Tonsil Epithelium.

Epidemiological data confirm a much higher incidence of high-risk human papillomavirus 16 (HPV16)-mediated carcinogenesis of the cervical epithelium than for other target sites. In order to elucidate tissue-specific responses to virus infection, we compared gene expression changes induced by productive HPV16 infection of cervical, foreskin, and tonsil organotypic rafts. These rafts closely mimic persistent HPV16 infection, long before carcinogenesis sets in. The total number of gene expression changes varied considerably across the tissue types, with only 32 genes being regulated in common. Among them, we confirmed the Kelch-like family protein KLHL35 and the laminin-5 complex to be upregulated and downregulated, respectively, in all the three tissues. HPV16 infection induces upregulation of genes involved in cell cycle control, cell division, mitosis, DNA replication, and DNA damage repair in all the three tissues, indicative of a hyperproliferative environment. In the cervical and tonsil epithelium, we observe significant downregulation of genes involved in epidermis development, keratinocyte differentiation, and extracellular matrix organization. On the other hand, in HPV16-positive foreskin (HPV16 foreskin) tissue, several genes involved in interferon-mediated innate immunity, cytokine signaling, and cellular defenses were downregulated. Furthermore, pathway analysis and experimental validations identified important cellular pathways like STAT1 and transforming growth factor ß (TGF-ß) to be differentially regulated among the three tissue types. The differential modulation of important cellular pathways like TGF-ß1 and STAT1 can explain the sensitivity of tissues to HPV cancer progression.IMPORTANCE Although the high-risk human papillomavirus 16 infects anogenital and oropharyngeal sites, the cervical epithelium has a unique vulnerability to progression of cancer. Host responses during persistent infection and preneoplastic stages can shape the outcome of cancer progression in a tissue-dependent manner. Our study for the first time reports differential regulation of critical cellular functions and signaling pathways during productive HPV16 infection of cervical, foreskin, and tonsil tissues. While the virus induces hyperproliferation in infected cells, it downregulates epithelial differentiation, epidermal development, and innate immune responses, according to the tissue type. Modulation of these biological functions can determine virus fitness and pathogenesis and illuminate key cellular mechanisms that the virus employs to establish persistence and finally initiate disease progression.