A beneficial tumor microenvironment in oropharyngeal squamous cell carcinoma is characterized by a high T cell and low IL-17(+) cell frequency.

Patients with HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis than patients with non-HPV-induced OPSCC. The role of the immune response in this phenomenon is yet unclear. We studied the number of T cells, regulatory T cells (Tregs), T helper 17 (Th17) cells and IL-17(+) non-T cells (mainly granulocytes) in matched HPV-positive and HPV-negative OPSCC cases (n = 162). Furthermore, the production of IFN-γ and IL-17 by tumor-infiltrating T cells was analyzed. The number of tumor-infiltrating T cells and Tregs was higher in HPV-positive than HPV-negative OPSCC (p < 0.0001). In contrast, HPV-negative OPSCC contained significantly higher numbers of IL-17(+) non-T cells (p < 0.0001). Although a high number of intra-tumoral T cells showed a trend toward improved survival of all OPSCC patients, their prognostic effect in patients with a low number of intra-tumoral IL-17(+) non-T cells was significant with regard to disease-specific (p = 0.033) and disease-free survival (p = 0.012). This suggests that a high frequency of IL-17(+) non-T cells was related to a poor immune response, which was further supported by the observation that a high number of T cells was correlated with improved disease-free survival in the HPV-positive OPSCC (p = 0.008). In addition, we detected a minor Th17 cell population. However, T cells obtained from HPV-positive OPSCC produced significantly more IL-17 than those from HPV-negative tumors (p = 0.006). The improved prognosis of HPV-positive OPSCC is thus correlated with higher numbers of tumor-infiltrating T cells, more active Th17 cells and lower numbers of IL-17(+) non-T cells.

Potential use of lymph node-derived HPV-specific T cells for adoptive cell therapy of cervical cancer.

Adoptive transfer of tumor-specific T cells, expanded from tumor-infiltrating lymphocytes or from peripheral blood, is a promising immunotherapeutic approach for the treatment of cancer. Here, we studied whether the tumor-draining lymph nodes (TDLN) of patients with human papillomavirus (HPV)-induced cervical cancer can be used as a source for ACT. The objectives were to isolate lymph node mononuclear cells (LNMC) from TDLN and optimally expand HPV-specific CD4+ and CD8+ T cells under clinical grade conditions. TDLN were isolated from 11 patients with early-stage cervical cancer during radical surgery. Isolated lymphocytes were expanded in the presence of HPV16 E6 and E7 clinical grade synthetic long peptides and IL-2 for 22 days and then analyzed for HPV16 specificity by proliferation assay, multiparameter flow cytometry and cytokine analysis as well as for CD25 and FoxP3 expression. Stimulation of LNMC resulted in expansion of polyclonal HPV-specific T cells in all patients. On average a 36-fold expansion of a CD4+ and/or CD8+ HPV16-specific T cell population was observed, which maintained its capacity for secondary expansion. The T helper type 1 cytokine IFNγ was produced in all cell cultures and in some cases also the Th2 cytokines IL-10 and IL-5. The procedure was highly reproducible, as evidenced by complete repeats of the stimulation procedures under research and under full good manufacturing practice conditions. In conclusion, TDLN represent a rich source of polyclonal HPV16 E6- and E7-specific T cells, which can be expanded under clinical grade conditions for adoptive immunotherapy in patients with cervical cancer.

M2 macrophages induced by prostaglandin E2 and IL-6 from cervical carcinoma are switched to activated M1 macrophages by CD4+ Th1 cells.

Monocytes attracted by tumor-induced chronic inflammation differentiate to APCs, the type of which depends on cues in the local tumor milieu. In this work, we studied the influence of human cervical cancer cells on monocyte differentiation and showed that the majority of cancer cells either hampered monocyte to dendritic cell differentiation or skewed their differentiation toward M2-like macrophages. Blocking studies revealed that M2 differentiation was caused by tumor-produced PGE(2) and IL-6. TGF-ß, IL-10, VEGF, and macrophage colony-stimulating factor did not play a role. Notably, these CD14(+)CD163(+) M2 macrophages were also detected in situ. Activation of cancer cell-induced M2-like macrophages by several TLR-agonists revealed that compared with dendritic cells, these M2 macrophages displayed a tolerogenic phenotype reflected by a lower expression of costimulatory molecules, an altered balance in IL-12p70 and IL-10 production, and a poor capacity to stimulate T cell proliferation and IFN-γ production. Notably, upon cognate interaction with Th1 cells, these tumor-induced M2 macrophages could be switched to activated M1-like macrophages that expressed high levels of costimulatory molecules, produced high amounts of IL-12 and low amounts of IL-10, and acquired the lymphoid homing marker CCR7. The effects of the interaction between M2 macrophages and Th1 cells could partially be mimicked by activation of these APCs via CD40 in the presence of IFN-γ. Our data on the presence, induction, and plasticity of tumor-induced tolerogenic APCs in cervical cancer suggest that tumor-infiltrated Th1 cells can stimulate a tumor-rejecting environment by switching M2 macrophages to classical proinflammatory M1 macrophages.

Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment.

Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don't respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/ß2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.

Influenza matrix 1-specific human CD4+ FOXP3+ and FOXP3(-) regulatory T cells can be detected long after viral clearance.

Control and termination of infection with Influenza A virus is associated with increased IL-10 production in mouse models. Notably, IL-10 can be produced by Treg. Therefore, we investigated whether the population of IL-10-producing influenza-specific CD4(+) T cells comprised Treg as they are potent suppressors of the adaptive immune response. Influenza-specific IL-10-producing T cells were detected in all human donors displaying influenza-specific immunity. Isolation of Matrix 1 protein-specific IL-10-producing T-cell clones revealed that a substantial proportion of these T-cell clones displayed the capacity to suppress effector cells, functionally identifying them as Treg. Both FOXP3(+) and FOXP3(-) CD4(+) Treg were isolated and all were able to exert their suppressive capacity when stimulated with cognate antigen, including influenza virus-infected cells. In vitro suppression was not mediated by IL-10 but involved interference with the IL-2 axis. The isolated Treg suppressed amongst others the IL-2 production of influenza-specific T-helper cells as well as partially prevented the upregulation of the high-affinity IL-2 receptor on CD8 effector cells. So far the induction of virus-specific Treg has only been studied in the context of chronic viral infections. This study demonstrates that virus-specific Treg can also be induced by viruses that are rapidly cleared in humans.

Intratumoral HPV16-Specific T Cells Constitute a Type I-Oriented Tumor Microenvironment to Improve Survival in HPV16-Driven Oropharyngeal Cancer.

Purpose: Human papillomavirus (HPV)-associated oropharyngeal squamous cell cancer (OPSCC) has a much better prognosis than HPV-negative OPSCC, and this is linked to dense tumor immune infiltration. As the viral antigens may trigger potent immunity, we studied the relationship between the presence of intratumoral HPV-specific T-cell responses, the immune contexture in the tumor microenvironment, and clinical outcome.Experimental Design: To this purpose, an in-depth analysis of tumor-infiltrating immune cells in a prospective cohort of 97 patients with HPV16-positive and HPV16-negative OPSCC was performed using functional T-cell assays, mass cytometry (CyTOF), flow cytometry, and fluorescent immunostaining of tumor tissues. Key findings were validated in a cohort of 75 patients with HPV16-positive OPSCC present in the publicly available The Cancer Genome Atlas database.Results: In 64% of the HPV16-positive tumors, type I HPV16-specific T cells were present. Their presence was not only strongly related to a better overall survival, a smaller tumor size, and less lymph node metastases but also to a type I-oriented tumor microenvironment, including high numbers of activated CD161+ T cells, CD103+ tissue-resident T cells, dendritic cells (DC), and DC-like macrophages.Conclusions: The viral antigens trigger a tumor-specific T-cell response that shapes a favorable immune contexture for the response to standard therapy. Hence, reinforcement of HPV16-specific T-cell reactivity is expected to boost this process. Clin Cancer Res; 24(3); 634-47. ©2017 AACRSee related commentary by Laban and Hoffmann, p. 505.

Human Papillomavirus Downregulates the Expression of IFITM1 and RIPK3 to Escape from IFNγ- and TNFα-Mediated Antiproliferative Effects and Necroptosis.

The clearance of a high-risk human papillomavirus (hrHPV) infection takes time and requires the local presence of a strong type 1 cytokine T cell response, suggesting that hrHPV has evolved mechanisms to resist this immune attack. Using an unique system for non, newly, and persistent hrHPV infection, we show that hrHPV infection renders keratinocytes (KCs) resistant to the antiproliferative- and necroptosis-inducing effects of IFNγ and TNFα. HrHPV-impaired necroptosis was associated with the upregulation of several methyltransferases, including EZH2, and the downregulation of RIPK3 expression. Restoration of RIPK3 expression using the global histone methyltransferase inhibitor 3-deazaneplanocin increased necroptosis in hrHPV-positive KCs. Simultaneously, hrHPV effectively inhibited IFNγ/TNFα-mediated arrest of cell growth at the S-phase by downregulating IFITM1 already at 48 h after hrHPV infection, followed by an impaired increase in the expression of the antiproliferative gene RARRES1 and a decrease of the proliferative gene PCNA. Knockdown of IFITM1 in uninfected KCs confirmed its role on RARRES1 and its antiproliferative effects. Thus, our study reveals how hrHPV deregulates two pathways involved in cell death and growth regulation to withstand immune-mediated control of hrHPV-infected cells.

TLR2 ligand-synthetic long peptide conjugates effectively stimulate tumor-draining lymph node T cells of cervical cancer patients.

The potency of human papillomavirus type 16 (HPV16)-encoded synthetic long peptides (SLP), conjugated to an optimized Toll-like receptor 2 ligand (TLR2-L), was assessed in ex vivo activation of HPV16+ cancer patient-derived T cells. Two highly immunogenic SLP sequences derived from the oncogenic E6 protein of HPV16 were selected and conjugated to a Pam3CSK4-based TLR2-L under GMP conditions. Both conjugates were able to mature human DCs in vitro and to activate human skin-derived antigen-presenting cells (APCs) upon intradermal injection in an ex vivo skin model, associated with induction of a favorable chemokine profile to attract and activate T cells. The conjugated SLPs were efficiently processed by APCs, since HPV16-specific CD4+ and CD8+ T-cell clones isolated from HPV16+ cervical tumors proliferated in response to both conjugates. The TLR2-L SLP conjugates significantly enhanced ex vivo T helper type 1 T-cell activation in cell suspensions obtained from tumor-draining lymph nodes (LN) resected during hysterectomy of HPV16+ cervical cancer patients. These results show that TLR2-L SLP conjugates can activate circulating or LN-derived tumor-specific T cells, a promising outcome for studying these two conjugates in a phase I/II clinical safety and immunogenicity trial.

The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation.

High-risk human papillomaviruses (hrHPVs) infect keratinocytes and successfully evade host immunity despite the fact that keratinocytes are well equipped to respond to innate and adaptive immune signals. Using non-infected and freshly established or persistent hrHPV-infected keratinocytes we show that hrHPV impairs the acetylation of NFκB/RelA K310 in keratinocytes. As a consequence, keratinocytes display a decreased pro-inflammatory cytokine production and immune cell attraction in response to stimuli of the innate or adaptive immune pathways. HPV accomplishes this by augmenting the expression of interferon-related developmental regulator 1 (IFRD1) in an EGFR-dependent manner. Restoration of NFκB/RelA acetylation by IFRD1 shRNA, cetuximab treatment or the HDAC1/3 inhibitor entinostat increases basal and induced cytokine expression. Similar observations are made in IFRD1-overexpressing HPV-induced cancer cells. Thus, our study reveals an EGFR-IFRD1-mediated viral immune evasion mechanism, which can also be exploited by cancer cells.

A phase 1/2 study combining gemcitabine, Pegintron and p53 SLP vaccine in patients with platinum-resistant ovarian cancer.

PURPOSE: Preclinical tumor models show that chemotherapy has immune modulatory properties which can be exploited in the context of immunotherapy. The purpose of this study was to determine the feasibility and immunogenicity of combinations of such an immunomodulatory chemotherapeutic agent with immunotherapy, p53 synthetic long peptide (SLP) vaccine and Pegintron (IFN-α) in patients with platinum-resistant p53-positive epithelial ovarian cancer (EOC). EXPERIMENTAL DESIGN: This is a phase 1/2 trial in which patients sequential 6 cycles of gemcitabine (1000 mg/kg2 iv; n = 3), gemcitabine with Pegintron before and after the first gemcitabine cycle (Pegintron 1 µg/kg sc; n = 6), and gemcitabine and Pegintron combined with p53 SLP vaccine (0.3 mg/peptide, 9 peptides; n = 6). At baseline, 22 days after the 2nd and 6th cycle, blood was collected for immunomonitoring. Toxicity, CA-125, and radiologic response were evaluated after 3 and 6 cycles of chemotherapy. RESULTS: None of the patients enrolled experienced dose-limiting toxicity. Predominant grade 3/4 toxicities were nausea/vomiting and dyspnea. Grade 1/2 toxicities consisted of fatigue (78%) and Pegintron-related flu-like symptoms (72%). Gemcitabine reduced myeloid-derived suppressor cells (p = 0.0005) and increased immune-supportive M1 macrophages (p = 0.04). Combination of gemcitabine and Pegintron stimulated higher frequencies of circulating proliferating CD4+ and CD8+ T-cells but not regulatory T-cells. All vaccinated patients showed strong vaccine-induced p53-specific T-cell responses. CONCLUSION: Combination of gemcitabine, the immune modulator Pegintron and therapeutic peptide vaccination is a viable approach in the development of combined chemo-immunotherapeutic regimens to treat cancer.

CD40-mediated amplification of local immunity by epithelial cells is impaired by HPV.

The interaction between the transmembrane glycoprotein surface receptor CD40 expressed by skin epithelial cells (ECs) and its T-cell-expressed ligand CD154 was suggested to exacerbate inflammatory skin diseases. However, the full spectrum of CD40-mediated effects by ECs underlying this observation is unknown. Therefore, changes in gene expression after CD40 ligation of ECs were studied by microarrays. CD40-mediated activation for 2 hours stimulated the expression of a coordinated network of immune-involved genes strongly interconnected by IL8 and TNF, whereas after 24 hours anti-proliferative and anti-apoptotic genes were upregulated. CD40 ligation was associated with the production of chemokines and the attraction of lymphocytes and myeloid cells from peripheral blood mononuclear cells (PBMCs). Thus, CD40-mediated activation of ECs resulted in a highly coordinated response of genes required for the local development and sustainment of adaptive immune responses. The importance of this process was confirmed by a study on the effects of human papilloma virus (HPV) infection to the EC's response to CD40 ligation. HPV infection clearly attenuated the magnitude of the response to CD40 ligation and the EC's capacity to attract PBMCs. The fact that HPV attenuates CD40 signaling in ECs indicates the importance of the CD40-CD154 immune pathway in boosting cellular immunity within epithelia.

Chemotherapy alters monocyte differentiation to favor generation of cancer-supporting M2 macrophages in the tumor microenvironment.

Current therapy of gynecologic malignancies consists of platinum-containing chemotherapy. Resistance to therapy is associated with increased levels of interleukin (IL)-6 and prostaglandin E2 (PGE(2)), 2 inflammatory mediators known to skew differentiation of monocytes to tumor-promoting M2 macrophages. We investigated the impact of cisplatin and carboplatin on 10 different cervical and ovarian cancer cell lines as well as on the ability of the tumor cells to affect the differentiation and function of cocultured monocytes in vitro. Treatment with cisplatin or carboplatin increased the potency of tumor cell lines to induce IL-10-producing M2 macrophages, which displayed increased levels of activated STAT3 due to tumor-produced IL-6 as well as decreased levels of activated STAT1 and STAT6 related to the PGE(2) production of tumor cells. Blockade of canonical NF-κB signaling showed that the effect of the chemotherapy was abrogated, preventing the subsequent increased production of PGE(2) and/or IL-6 by the tumor cell lines. Treatment with the COX-inhibitor indomethacin and/or the clinical monoclonal antibody against interleukin-6 receptor (IL-6R), tocilizumab, prevented M2-differentiation. Importantly, no correlation existed between the production of PGE(2) or IL-6 by cancer cells and their resistance to chemotherapy-induced cell death, indicating that other mechanisms underlie the reported chemoresistance of tumors producing these factors. Our data suggest that a chemotherapy-mediated increase in tumor-promoting M2 macrophages may form an indirect mechanism for chemoresistance. Hence, concomitant therapy with COX inhibitors and/or IL-6R antibodies might increase the clinical effect of platinum-based chemotherapy in otherwise resistant tumors.

Hsp70 expression and induction as a readout for detection of immune modulatory components in food.

Stress proteins such as heat shock proteins (Hsps) are up-regulated in cells in response to various forms of stress, like thermal and oxidative stress and inflammation. Hsps prevent cellular damage and increase immunoregulation by the activation of anti-inflammatory T-cells. Decreased capacity for stress-induced Hsp expression is associated with immune disorders. Thus, therapeutic boosting Hsp expression might restore or enhance cellular stress resistance and immunoregulation. Especially food- or herb-derived phytonutrients may be attractive compounds to restore optimal Hsp expression in response to stress. In the present study, we explored three readout systems to monitor Hsp70 expression in a manner relevant for the immune system and evaluated novel Hsp co-inducers. First, intracellular staining and analysis by flow cytometry was used to detect stress and/or dietary compound induced Hsp70 expression in multiple rodent cell types efficiently. This system was used to screen a panel of food-derived extracts with potent anti-oxidant capacity. This strategy yielded the identity of several new enhancers of stress-induced Hsp70 expression, among them carvacrol, found in thyme and oregano. Second, CD4(+) T-cell hybridomas were generated that specifically recognized an immunodominant Hsp70 peptide. These hybridomas were used to show that carvacrol enhanced Hsp70 levels increased T-cell activation. Third, we generated a DNAJB1-luc-O23 reporter cell line to show that carvacrol increased the transcriptional activation of a heat shock promoter in the presence of arsenite. These assay systems are generally applicable to identify compounds that affect the Hsp level in cells of the immune system.

An unexpectedly large polyclonal repertoire of HPV-specific T cells is poised for action in patients with cervical cancer.

The diversity and extent of the local tumor-specific T-cell response in a given individual is largely unknown. We have performed an in-depth study of the local T-cell repertoire in a selected group of patients with cervical cancer, by systematic analyses of the proportion, breadth, and polarization of human papillomavirus (HPV) E6/E7-specific T cells within the total population of tumor-infiltrating lymphocytes (TIL) and tumor-draining lymph node cells (TDLNC). Isolated T cells were stimulated with sets of overlapping E6 and E7 peptides and analyzed by multiparameter flow cytometry with respect to activation, cytokine production, and T-cell receptor Vbeta usage. HPV-specific CD4+ and CD8+ T-cell responses were detected in TIL and TDLNC and their relative contribution varied between <1% and 66% of all T cells. In general, these HPV-specific responses were surprisingly broad, aimed at multiple E6 and E7 epitopes and involved multiple dominant and subdominant T-cell receptor Vbetas per single peptide-epitope. In most patients, only few IFNgamma-producing T cells were found and the amount of IFNgamma produced was low, suggesting that these are poised T cells, rendered functionally inactive within the tumor environment. Importantly, stimulation of the TIL and TDLNC with cognate antigen in the presence of commonly used Toll-like receptor ligands significantly enhanced the effector T-cell function. In conclusion, our study suggests that within a given patient with HPV-specific immunity many different tumor-specific CD4+ and CD8+ T cells are locally present and poised for action. This vast existing local T-cell population is awaiting proper stimulation and can be exploited for the immunotherapy of cancer.