Antiviral CD8+ T-cell immune responses are impaired by cigarette smoke and in COPD.

BACKGROUND: Virus infections drive COPD exacerbations and progression. Antiviral immunity centres on the activation of virus-specific CD8+ T-cells by viral epitopes presented on major histocompatibility complex (MHC) class I molecules of infected cells. These epitopes are generated by the immunoproteasome, a specialised intracellular protein degradation machine, which is induced by antiviral cytokines in infected cells. METHODS: We analysed the effects of cigarette smoke on cytokine- and virus-mediated induction of the immunoproteasome in vitro, ex vivo and in vivo using RNA and Western blot analyses. CD8+ T-cell activation was determined in co-culture assays with cigarette smoke-exposed influenza A virus (IAV)-infected cells. Mass-spectrometry-based analysis of MHC class I-bound peptides uncovered the effects of cigarette smoke on inflammatory antigen presentation in lung cells. IAV-specific CD8+ T-cell numbers were determined in patients' peripheral blood using tetramer technology. RESULTS: Cigarette smoke impaired the induction of the immunoproteasome by cytokine signalling and viral infection in lung cells in vitro, ex vivo and in vivo. In addition, cigarette smoke altered the peptide repertoire of antigens presented on MHC class I molecules under inflammatory conditions. Importantly, MHC class I-mediated activation of IAV-specific CD8+ T-cells was dampened by cigarette smoke. COPD patients exhibited reduced numbers of circulating IAV-specific CD8+ T-cells compared to healthy controls and asthmatics. CONCLUSION: Our data indicate that cigarette smoke interferes with MHC class I antigen generation and presentation and thereby contributes to impaired activation of CD8+ T-cells upon virus infection. This adds important mechanistic insight on how cigarette smoke mediates increased susceptibility of smokers and COPD patients to viral infections.

Dendritic Cells or Macrophages? The Microenvironment of Human Clear Cell Renal Cell Carcinoma Imprints a Mosaic Myeloid Subtype Associated with Patient Survival.

Since their initial description by Elie Metchnikoff, phagocytes have sparked interest in a variety of biologic disciplines. These important cells perform central functions in tissue repair and immune activation as well as tolerance. Myeloid cells can be immunoinhibitory, particularly in the tumor microenvironment, where their presence is generally associated with poor patient prognosis. These cells are highly adaptable and plastic, and can be modulated to perform desired functions such as antitumor activity, if key programming molecules can be identified. Human clear cell renal cell carcinoma (ccRCC) is considered immunogenic; yet checkpoint blockades that target T cell dysfunction have shown limited clinical efficacy, suggesting additional layers of immunoinhibition. We previously described "enriched-in-renal cell carcinoma" (erc) DCs that were often found in tight contact with dysfunctional T cells. Using transcriptional profiling and flow cytometry, we describe here that ercDCs represent a mosaic cell type within the macrophage continuum co-expressing M1 and M2 markers. The polarization state reflects tissue-specific signals that are characteristic of RCC and renal tissue homeostasis. ErcDCs are tissue-resident with increasing prevalence related to tumor grade. Accordingly, a high ercDC score predicted poor patient survival. Within the profile, therapeutic targets (VSIG4, NRP1, GPNMB) were identified with promise to improve immunotherapy.

Identification of EZH2 as Cancer Stem Cell Marker in Clear Cell Renal Cell Carcinoma and the Anti-Tumor Effect of Epigallocatechin-3-Gallate (EGCG).

The aim of the study was to develop a new therapeutic strategy to target cancer stem cells (CSCs) in clear cell renal cell carcinoma (ccRCC) and to identify typical CSC markers to improve therapy effectiveness. It was found that the corrected-mRNA expression-based stemness index was upregulated in kidney renal clear cell carcinoma (KIRC) tissues compared to non-tumor tissue and increased with higher tumor stage and grade. EZH2 was identified as a CSC marker and prognosis factor for KIRC patients. The expression of EZH2 was associated with several activated tumor-infiltrating immune cells. High expression of EZH2 was enriched in immune-related pathways, low expression was related to several metabolic pathways. Epigallocatechin-3-gallate (EGCG) was identified as the most potent suppressor of EZH2, was able to inhibit viability, migration, and invasion, and to increase the apoptosis rate of ccRCC CSCs. KIF11, VEGF, and MMP2 were identified as predictive EGCG target genes, suggesting a potential mechanism of how EZH2 might regulate invasiveness and migration. The percentages of FoxP3+ Treg cells in the peripheral blood mononuclear cells of ccRCC patients decreased significantly when cultured with spheres pretreated with EGCG plus sunitinib compared to spheres without treatment. Our findings provide new insights into the treatment options of ccRCC based on targeting CSCs.

CD206+ tumor-associated macrophages cross-present tumor antigen and drive antitumor immunity.

In many solid cancers, tumor-associated macrophages (TAM) represent the predominant myeloid cell population. Antigen (Ag) cross-presentation leading to tumor Ag-directed cytotoxic CD8+ T cell responses is crucial for antitumor immunity. However, the role of recruited monocyte-derived macrophages, including TAM, as potential cross-presenting cells is not well understood. Here, we show that primary human as well as mouse CD206+ macrophages are effective in functional cross-presentation of soluble self-Ag and non-self-Ag, including tumor-associated Ag (TAA), as well as viral Ag. To confirm the presence of cross-presenting TAM in vivo, we performed phenotypic and functional analysis of TAM from B16-F10 and CT26 syngeneic tumor models and have identified CD11b+F4/80hiCD206+ TAM to effectively cross-present TAA. We show that CD11b+CD206+ TAM represent the dominant tumor-infiltrating myeloid cell population, expressing a unique cell surface repertoire, promoting Ag cross-presentation and Ag-specific CD8+ T cell activation comparable with cross-presenting CLEC9A+ DCs (cDC1). The presence of cross-presenting CD206+ TAM is associated with reduced tumor burden in mouse syngeneic tumor models and with improved overall survival in cutaneous melanoma patients. Therefore, the demonstration of effective Ag cross-presentation capabilities of CD206+ TAM, including their clinical relevance, expands our understanding of TAM phenotypic diversity and functional versatility.

T-Cells Expressing a Highly Potent PRAME-Specific T-Cell Receptor in Combination with a Chimeric PD1-41BB Co-Stimulatory Receptor Show a Favorable Preclinical Safety Profile and Strong Anti-Tumor Reactivity.

The hostile tumor microenvironment (TME) is a major challenge for the treatment of solid tumors with T-cell receptor (TCR)-modified T-cells (TCR-Ts), as it negatively influences T-cell efficacy, fitness, and persistence. These negative influences are caused, among others, by the inhibitory checkpoint PD-1/PD-L1 axis. The Preferentially Expressed Antigen in Melanoma (PRAME) is a highly relevant cancer/testis antigen for TCR-T immunotherapy due to broad expression in multiple solid cancer indications. A TCR with high specificity and sensitivity for PRAME was isolated from non-tolerized T-cell repertoires and introduced into T-cells alongside a chimeric PD1-41BB receptor, consisting of the natural extracellular domain of PD-1 and the intracellular signaling domain of 4-1BB, turning an inhibitory pathway into a T-cell co-stimulatory pathway. The addition of PD1-41BB to CD8+ T-cells expressing the transgenic PRAME-TCR enhanced IFN-γ secretion, improved cytotoxic capacity, and prevented exhaustion upon repetitive re-challenge with tumor cells in vitro without altering the in vitro safety profile. Furthermore, a single dose of TCR-Ts co-expressing PD1-41BB was sufficient to clear a hard-to-treat melanoma xenograft in a mouse model, whereas TCR-Ts without PD1-41BB could not eradicate the PD-L1-positive tumors. This cutting-edge strategy supports development efforts to provide more effective TCR-T immunotherapies for the treatment of solid tumors.

Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy.

Activation of co-stimulatory pathways in cytotoxic T lymphocytes expressing chimeric antigen receptors (CARs) have proven to boost effector activity, tumor rejection and long-term T cell persistence. When using antigen-specific T cell receptors (TCR) instead of CARs, the lack of co-stimulatory signals hampers robust antitumoral response, hence limiting clinical efficacy. In solid tumors, tumor stroma poses an additional hurdle through hindrance of infiltration and active inhibition. Our project aimed at generating chimeric co-stimulatory switch proteins (CSP) consisting of intracellular co-stimulatory domains (ICD) fused to extracellular protein domains (ECD) for which ligands are expressed in solid tumors. The ECD of CD40L was selected for combination with the ICD from the CD28 protein. With this approach, it was expected to not only provide co-stimulation and strengthen the TCR signaling, but also, through the CD40L ECD, facilitate the activation of tumor-resident antigen-presenting cells (APCs), modulate activation of tumor endothelium and induce TCR-MHC independent apoptotic effect on tumor cells. Since CD28 and CD40L belong to different classes of transmembrane proteins (type I and type II, respectively), creating a chimeric protein presented a structural and functional challenge. We present solutions to this challenge describing different CSP formats that were successfully expressed in human T cells along with an antigen-specific TCR. The level of surface expression of the CSPs depended on their distinct design and the state of T cell activation. In particular, CSPs were upregulated by TCR stimulation and downregulated following interaction with CD40 on target cells. Ligation of the CSP in the context of TCR-stimulation modulated intracellular signaling cascades and led to improved TCR-induced cytokine secretion and cytotoxicity. Moreover, the CD40L ECD exhibited activity as evidenced by effective maturation and activation of B cells and DCs. CD40L:CD28 CSPs are a new type of switch proteins designed to exert dual beneficial antitumor effect by acting directly on the gene-modified T cells and simultaneously on tumor cells and tumor-supporting cells of the TME. The observed effects suggest that they constitute a promising tool to be included in the engineering process of T cells to endow them with complementary features for improved performance in the tumor milieu.

Checkpoint Inhibition in Bladder Cancer: Clinical Expectations, Current Evidence, and Proposal of Future Strategies Based on a Tumor-Specific Immunobiological Approach.

In contrast with other strategies, immunotherapy is the only treatment aimed at empowering the immune system to increase the response against tumor growth. Immunotherapy has a role in the treatment of bladder cancer (BC) due to these tumors' high tumor mutational burden (TMB) and mostly prominent immune infiltrate. The therapy or combination has to be adjusted to the tumor's immunobiology. Recently, a new class of immunotherapeutic agents, immune checkpoint inhibitors (ICI), has shown potential in increasing treatment chances for patients with genitourinary cancers, improving their oncological outcomes. The clinical efficacy of ICI has been shown in both the first-line treatment of cisplatin-ineligible patients, with programmed death ligand 1 (PD-L1)-positive tumors (atezolizumab, pembrolizumab), and in second-line settings, for progression after platinum-based chemotherapy (atezolizumab, pembrolizumab, and nivolumab for FDA and EMA; durvalumab and avelumab for FDA alone). Predicting the response to ICI is important since only a subset of patients undergoing ICI therapy develop a concrete and lasting response. Most of the patients require a different therapy or therapy combination to achieve tumor control. The cancer immunity cycle provides a conceptual framework to assist therapy selection. Biomarkers to predict response to ICI must identify where the cancer immunity cycle is disrupted. We reviewed the current knowledge on ICI treatment in BC, going from basic science to current data and available clinical evidence. Secondly, a critical analysis of published data is provided, and an original panel of biomarkers able to predict response to ICI treatment, based on tumor-specific immune profiling, is proposed.

Immune infiltrates in patients with localised high-risk soft tissue sarcoma treated with neoadjuvant chemotherapy without or with regional hyperthermia: A translational research program of the EORTC 62961-ESHO 95 randomised clinical trial.

BACKGROUND: The EORTC 62961-ESHO 95 randomised trial showed improved long-term survival of patients with high-risk soft-tissue sarcoma by adding regional hyperthermia to neoadjuvant chemotherapy. We hypothesised that immune infiltrate of patients treated with neoadjuvant therapy associate with clinical outcome. METHODS: Tumour infiltrating lymphocytes (TILs) and CD8, FOXP3, PD-1, and PD-L1 were evaluated in sequential biopsies of patients after four cycles of therapy. RESULTS: From a subgroup of 109 patients who had been randomised between July 1997 and November 2006 to neoadjuvant chemotherapy (53 patients) or neoadjuvant chemotherapy with regional hyperthermia (56 patients), 137 biopsies were obtained. TILs increased in paired second biopsies independent of treatment allocation (p < 0.001). FOXP3 regulatory T cells decreased (p = 0.002), and PD-L1 expression of tumours became undetectable. In the multivariate analysis, post-treatment high TILs correlated to LPFS (HR: 0.34; 95% CI 0.15-0.75; p = 0.008) and DFS (HR: 0.38; 95% CI 0.17-0.82; p = 0.015). In comparing post-treatment immune infiltrate between treatment arms, tumour response was associated with neoadjuvant chemotherapy with regional hyperthermia (p = 0.013) and high TILs (p = 0.064). High CD8 cell infiltration was associated with improved LPFS (HR: 0.27; 95% CI 0.09-0.79; Log-rank p = 0.011) and DFS (HR: 0.25; 95% CI 0.09-0.73; Log-rank p = 0.006). Improved survival at 10 years was associated with immune infiltrate after neoadjuvant chemotherapy with regional hyperthermia. CONCLUSION: Preoperative therapy re-programs a non-inflamed tumour at baseline into an inflamed tumour. The post-treatment immune infiltrate became predictive for clinical outcomes. The combination with regional hyperthermia primes the tumour microenvironment, enabling enhanced anti-tumour immune activity in high-risk soft tissue sarcomas. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00003052.

Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function.

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissues characterized by scant T cell infiltration and predominance of immunosuppressive myeloid cells. Given the important roles of extracellular vesicles (EVs) in cancer-host crosstalk, we hypothesized that EVs secreted by EwS tumors target myeloid cells and promote immunosuppressive phenotypes. Here, EVs were purified from EwS and fibroblast cell lines and exhibited characteristics of small EVs, including size (100-170 nm) and exosome markers CD63, CD81, and TSG101. Treatment of healthy donor-derived CD33+ and CD14+ myeloid cells with EwS EVs but not with fibroblast EVs induced pro-inflammatory cytokine release, including IL-6, IL-8, and TNF. Furthermore, EwS EVs impaired differentiation of these cells towards monocytic-derived dendritic cells (moDCs), as evidenced by reduced expression of co-stimulatory molecules CD80, CD86 and HLA-DR. Whole transcriptome analysis revealed activation of gene expression programs associated with immunosuppressive phenotypes and pro-inflammatory responses. Functionally, moDCs differentiated in the presence of EwS EVs inhibited CD4+ and CD8+ T cell proliferation as well as IFNγ release, while inducing secretion of IL-10 and IL-6. Therefore, EwS EVs may promote a local and systemic pro-inflammatory environment and weaken adaptive immunity by impairing the differentiation and function of antigen-presenting cells.

Vγ9Vδ2 T Cells Concurrently Kill Cancer Cells and Cross-Present Tumor Antigens.

The human Vγ9Vδ2 T cell is a unique cell type that holds great potential in immunotherapy of cancer. In particular, the therapeutic potential of this cell type in adoptive cell therapy (ACT) has gained interest. In this regard optimization of in vitro expansion methods and functional characterization is desirable. We show that Vγ9Vδ2 T cells, expanded in vitro with zoledronic acid (Zometa or ZOL) and Interleukin-2 (IL-2), are efficient cancer cell killers with a trend towards increased killing efficacy after prolonged expansion time. Thus, Vγ9Vδ2 T cells expanded for 25 days in vitro killed prostate cancer cells more efficiently than Vγ9Vδ2 T cells expanded for 9 days. These data are supported by phenotype characteristics, showing increased expression of CD56 and NKG2D over time, reaching above 90% positive cells after 25 days of expansion. At the early stage of expansion, we demonstrate that Vγ9Vδ2 T cells are capable of cross-presenting tumor antigens. In this regard, our data show that Vγ9Vδ2 T cells can take up tumor-associated antigens (TAA) gp100, MART-1 and MAGE-A3 - either as long peptide or recombinant protein - and then present TAA-derived peptides on the cell surface in the context of HLA class I molecules, demonstrated by their recognition as targets by peptide-specific CD8 T cells. Importantly, we show that cross-presentation is impaired by the proteasome inhibitor lactacystin. In conclusion, our data indicate that Vγ9Vδ2 T cells are broadly tumor-specific killers with the additional ability to cross-present MHC class I-restricted peptides, thereby inducing or supporting tumor-specific αßTCR CD8 T cell responses. The dual functionality is dynamic during in vitro expansion, yet, both functions are of interest to explore in ACT for cancer therapy.

T-cell engager antibodies enable T cells to control HBV infection and to target HBsAg-positive hepatoma in mice.

BACKGROUND & AIMS: Current antiviral therapies control but rarely eliminate HBV, leaving chronic HBV carriers at risk of developing hepatocellular carcinoma (HCC). Lacking or dysfunctional virus-specific adaptive immunity prevents control of HBV and allows the virus to persist. Restoring antiviral T-cell immunity could lead to HBV elimination and cure of chronically infected patients. METHODS: We constructed bispecific T-cell engager antibodies that are designed to induce antiviral immunity through simultaneous binding of HBV envelope proteins (HBVenv) on infected hepatocytes and CD3 or CD28 on T cells. T-cell engager antibodies were employed in co-cultures with healthy donor lymphocytes and HBV-infected target cells. Activation of the T-cell response was determined by detection of pro-inflammatory cytokines, effector function (by cytotoxicity) and antiviral effects. To study in vivo efficacy, immune-deficient mice were transplanted with HBVenv-positive and -negative hepatoma cells. RESULTS: The 2 T-cell engager antibodies synergistically activated T cells to become polyfunctional effectors that in turn elicited potent antiviral effects by killing infected cells and in addition controlled HBV via non-cytolytic, cytokine-mediated antiviral mechanisms. In vivo in mice, the antibodies attracted T cells specifically to the tumors expressing HBVenv resulting in T-cell activation, tumor infiltration and reduction of tumor burden. CONCLUSION: This study demonstrates that the administration of HBVenv-targeting T-cell engager antibodies facilitates a robust T-cell redirection towards HBV-positive target cells and provides a feasible and promising approach for the treatment of chronic viral hepatitis and HBV-associated HCC. LAY SUMMARY: T-cell engager antibodies are an interesting, novel therapeutic tool to restore immunity in patients with chronic hepatitis B. As bispecific antibodies, they bind envelope proteins on the surface of the hepatitis B virus (HBV) and CD3 or CD28 on T cells. This way, they induce a potent antiviral and cytotoxic T-cell response that leads to the elimination of HBV-positive cells. These bispecific T-cell engager antibodies are exciting therapeutic candidates for chronic hepatitis B and HBV-associated hepatocellular carcinoma.

Tumor Lactic Acidosis: Protecting Tumor by Inhibiting Cytotoxic Activity Through Motility Arrest and Bioenergetic Silencing.

Adoptive T cell therapy (ACT) is highly effective in the treatment of hematologic malignancies, but shows limited success in solid tumors. Inactivation of T cells in the tumor milieu is a major hurdle to a wider application of ACT. Cytotoxicity is the most relevant activity for tumor eradication. Here, we document that cytotoxic T cells (CTL) in lactic acidosis exhibited strongly reduced tumor cell killing, which could be compensated partly by increasing the CTL to tumor cell ratio. Lactic acid intervened at multiple steps of the killing process. Lactic acid repressed the number of CTL that performed lytic granule exocytosis (degranulation) in tumor cell co-culture, and, additionally impaired the quality of the response, as judged by the reduced intensity of degranulation and lower secretion of cytotoxins (perforin, granzyme B, granzyme A). CTL in lactic acid switched to a low bioenergetic profile with an inability to metabolize glucose efficiently. They responded to anti-CD3 stimulation poorly with less extracellular acidification rate (ECAR). This might explain their repressed granule exocytosis activity. Using live cell imaging, we show that CTL in lactic acid have reduced motility, resulting in lower field coverage. Many CTL in lactic acidosis did not make contact with tumor cells; however, those which made contact, adhered to the tumor cell much longer than a CTL in normal medium. Reduced motility together with prolonged contact duration hinders serial killing, a defining feature of killing potency, but also locally confines cytotoxic activity, which helps to reduce the risk of collateral organ damage. These activities define lactic acid as a major signaling molecule able to orchestrate the spatial distribution of CTL inside inflamed tissue, such as cancer, as well as moderating their functional response. Lactic acid intervention and strategies to improve T cell metabolic fitness hold promise to improve the clinical efficacy of T cell-based cancer immunotherapy.

Co-Regulation of Immune Checkpoint PD-L1 with Interferon-Gamma Signaling is Associated with a Survival Benefit in Renal Cell Cancer.

BACKGROUND: Programmed death ligand (PD-L1)-based immune checkpoint blockade therapy for metastatic renal cell carcinoma (RCC) achieves significant response rates in a subgroup of patients. The relevance of PD-L1 gene regulation for disease outcome is not clear. OBJECTIVE: To evaluate PD-L1 expression and its dependence on interferon-γ (IFN-γ) in RCC cell lines and tissues in relation to disease outcome. METHODS AND PATIENTS: Regulation of PD-L1-mRNA and PD-L1 protein was studied in cell lines from clear cell RCC (ccRCC) and papillary RCC (pRCC) by quantitative RT-PCR and Western-blot analysis. PD-L1-mRNA correlation and gene-set enrichment analysis (GSEA) of the IFN-γ pathway were conducted with RNA-Seq from ccRCC, pRCC, and skin cutaneous melanoma (SKCM) tissue. In addition, patient overall survival (OS) and disease-free survival (DFS) (cBioPortal for Cancer Genomics) were considered. RESULTS: In ccRCC-like cell lines, PD-L1 was induced by canonical IFN-γ signaling, whereas in a pRCC-like cell line, PD-L1 was refractory towards IFN-γ signaling. In ccRCC and SKCM tissues, GSEA revealed significant IFN-γ pathway activation in tissue samples with high PD-L1-mRNA levels. This was not observed in pRCC tissue. ccRCC and SKMC patients with low PD-L1-mRNA levels had significantly shorter OS and DFS than those with high PD-L1-mRNA levels. In pRCC patients, no significant difference in OS and DFS with regard to PD-L1-mRNA tissue levels was obvious. CONCLUSIONS: The findings suggest that ccRCC and pRCC differ with respect to PD-L1 regulation by IFN-γ-signaling. High PD-L1-mRNA levels in tumor tissues with a positive IFN-γ signature favorably affect OS and DFS.

A comparative view on the expression patterns of PD-L1 and PD-1 in soft tissue sarcomas.

Soft tissue sarcomas (STSs) are heterogeneous cancers associated with poor prognosis due to high rates of local recurrence and metastasis. The programmed death receptor ligand 1 (PD-L1) is expressed in several cancers. PD-L1 interacts with its receptor, PD-1, on the surface of tumor-infiltrating lymphocytes (TILs), thereby attenuating anti-cancer immune response. Immune checkpoint inhibitors targeting this interaction have been established as effective anti-cancer drugs. However, studies on the PD-L1 and PD-1 expression status in STS are commonly limited by small sample size, analysis of single STS subtypes, or lack of combinatorial marker assessment. To overcome these limitations, we evaluated the expression patterns of intratumoral PD-L1, the number of TILs, their PD-1 expression, and associations with clinicopathological parameters in a large and comprehensive cohort of 225 samples comprising six STS subtypes. We found that nearly all STS subtypes showed PD-L1 expression on the tumor cells, albeit with a broad range of positivity across subtypes (50% angiosarcomas to 3% synovial sarcomas). Co-expression and correlation analyses uncovered that PD-L1 expression was associated with more PD-1-positive TILs (P < 0.001), higher tumor grading (P = 0.016), and worse patients' 5-year overall survival (P = 0.028). The results were in line with several publications on single STS subtypes, especially when comparing findings for STS with low and high mutational burden. In sum, the substantial portion of PD-L1 positivity, the co-occurrence of PD-1-positive TILs, and the association of PD-L1 with unfavorable clinical outcome provide rationales for immune checkpoint inhibition in patients with PD-L1-positive STS.

Systemic antitumor effect by regional hyperthermia combined with low-dose chemotherapy and immunologic correlates in an adolescent patient with rhabdomyosarcoma - a case report.

Introduction: An abscopal effect is a clinical observation whereby a local treatment is associated with regression of metastatic cancer at a site distant from the primary location of treatment. Here, we describe the clinical systemic effect induced by regional hyperthermia combined with low-dose chemotherapy and provide immunologic correlates.Case presentation: A 15-year-old patient had been diagnosed with alveolar rhabdomyosarcoma (ARMS). All previous treatment options failed in the patient including haploidentical stem cell transplantation and donor lymphocyte infusion. The patient presented with local and metastatic disease, and upon admission, underwent regional hyperthermia combined with low-dose chemotherapy. Immediately following therapy severe skin reactions were observed. Skin biopsies revealed an intraepithelial lymphocytic infiltration dominated by CD3+/CD8+ T cells with a regular network of dendritic cells. Clinical images compared before and during sequential treatment cycles showed complete metabolic response of the local tumor for more than 10 months of therapy. In addition, metastases completely regressed although they were not direct targets of regional hyperthermia. The systemic effect was associated with enhanced frequency of NK cells and T cells expressing the lectin-like natural-killer group 2 D activating receptor (NKG2D), an increase of the CD56bright subset of NK cells, as well as an increase of effector/memory and effector CD8+ and CD4+ T cells in the blood while the percentage of CD25+FOXP3+ regulatory T cells declined.Conclusions: Regional hyperthermia combined with low-dose chemotherapy had the potential to create a systemic effect which was associated with activation of NK cells and T cells.

Collagen density regulates the activity of tumor-infiltrating T cells.

BACKGROUND: Tumor progression is accompanied by dramatic remodeling of the surrounding extracellular matrix leading to the formation of a tumor-specific ECM, which is often more collagen-rich and of increased stiffness. The altered ECM of the tumor supports cancer growth and metastasis, but it is unknown if this effect involves modulation of T cell activity. To investigate if a high-density tumor-specific ECM could influence the ability of T cells to kill cancer cells, we here studied how T cells respond to 3D culture in different collagen densities. METHODS: T cells cultured in 3D conditions surrounded by a high or low collagen density were imaged using confocal fluorescent microscopy. The effects of the different collagen densities on T cell proliferation, survival, and differentiation were examined using flow cytometry. Cancer cell proliferation in similar 3D conditions was also measured. Triple-negative breast cancer specimens were analyzed for the number of infiltrating CD8+ T cells and for the collagen density. Whole-transcriptome analyses were applied to investigate in detail the effects of collagen density on T cells. Computational analyses were used to identify transcription factors involved in the collagen density-induced gene regulation. Observed changes were confirmed by qRT-PCR analysis. RESULTS: T cell proliferation was significantly reduced in a high-density matrix compared to a low-density matrix and prolonged culture in a high-density matrix led to a higher ratio of CD4+ to CD8+ T cells. The proliferation of cancer cells was unaffected by the surrounding collagen-density. Consistently, we observed a reduction in the number of infiltrating CD8+ T-cells in mammary tumors with high collagen-density indicating that collagen-density has a role in regulating T cell abundance in human breast cancer. Whole-transcriptome analysis of 3D-cultured T cells revealed that a high-density matrix induces downregulation of cytotoxic activity markers and upregulation of regulatory T cell markers. These transcriptional changes were predicted to involve autocrine TGF-ß signaling and they were accompanied by an impaired ability of tumor-infiltrating T cells to kill autologous cancer cells. CONCLUSIONS: Our study identifies a new immune modulatory mechanism, which could be essential for suppression of T cell activity in the tumor microenvironment.

Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells.

Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.

Chimeric PD-1:28 Receptor Upgrades Low-Avidity T cells and Restores Effector Function of Tumor-Infiltrating Lymphocytes for Adoptive Cell Therapy.

Inherent intermediate- to low-affinity T-cell receptors (TCR) that develop during the natural course of immune responses may not allow sufficient activation for tumor elimination, making the majority of T cells suboptimal for adoptive T-cell therapy (ATT). TCR affinity enhancement has been implemented to provide stronger T-cell activity but carries the risk of creating undesired cross-reactivity leading to potential serious adverse effects in clinical application. We demonstrate here that engineering of low-avidity T cells recognizing a naturally processed and presented tumor-associated antigen with a chimeric PD-1:28 receptor increases effector function to levels seen with high-avidity T cells of identical specificity. Upgrading the function of low-avidity T cells without changing the TCR affinity will allow a large arsenal of low-avidity T cells previously thought to be therapeutically inefficient to be considered for ATT. PD-1:28 engineering reinstated Th1 function in tumor-infiltrating lymphocytes that had been functionally disabled in the human renal cell carcinoma environment without unleashing undesired Th2 cytokines or IL10. Involved mechanisms may be correlated to restoration of ERK and AKT signaling pathways. In mouse tumor models of ATT, PD-1:28 engineering enabled low-avidity T cells to proliferate stronger and prevented PD-L1 upregulation and Th2 polarization in the tumor milieu. Engineered T cells combined with checkpoint blockade secreted significantly more IFNγ compared with T cells without PD-1:28, suggesting a beneficial combination with checkpoint blockade therapy or other therapeutic strategies. Altogether, the supportive effects of PD-1:28 engineering on T-cell function make it an attractive tool for ATT. Cancer Res; 77(13); 3577-90. ©2017 AACR.

Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma.

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it's expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies.

DGK-α: A Checkpoint in Cancer-Mediated Immuno-Inhibition and Target for Immunotherapy.

Immunotherapy is moving to the forefront of cancer treatments owing to impressive durable responses achieved with checkpoint blockade antibodies and adoptive T-cell therapy. Still, improvements are necessary since, overall, only a small percentage of patients benefit from current therapies. Here, I summarize evidence that DGK-α may represent an immunological checkpoint suppressing the activity of cytotoxic immunocytes in the tumor microenvironment. DGK-inhibitors can restore the antitumor function of tumor-suppressed adaptive and innate cytotoxic immunocytes. The activity of DGK-inhibitors lays downstream of current checkpoint blockade antibodies. Thus, synergistic effects are expected from combination strategies. Moreover, DGK-inhibitors may permit a double-strike attack on tumor cells as DGK-inhibition may not only re-instate immunological tumor attack but also may harm tumor cells directly by interfering with oncogenic survival pathways. Together, DGK-inhibitors have very promising characteristics and may be beneficially included into the armamentarium of cancer immunotherapeutics.