Role of regulatory T cells and checkpoint inhibition in hepatocellular carcinoma Abstract Immune checkpoint inhibition suggests promising progress for the treatment of advanced hepatocellular carcinoma (HCC). However, the underlying cellular mechanisms remain unclear because liver cancer cells apparently do not upregulate inhibitory checkpoint molecules. Here, we analysed whether regulatory T cells (Tregs) can alternatively trigger checkpoint inhibition pathways in HCC. Using flow cytometry we analysed expression of checkpoint molecules (PD-1, PD-L1, CTLA-4, GITR, Tim-3) on peripheral CD4+CD25+Foxp3+ Tregs and their secretion of inhibitory mediators (IL-10, IL-35, TGF-beta, galectin-9) in 116 individuals (50 patients with HCC, 41 non-tumour bearing liver disease controls, 25 healthy controls). Functional activity of Tregs on T effector cells (IFN-gamma production, cytotoxicity) was characterized in vitro using a lectin-dependent cellular cytotoxicity (LDCC) assay against checkpoint inhibitor-negative P815 target cells. Unlike liver patients without malignancy and healthy controls, the frequency of checkpoint inhibitor-positive Tregs inversely correlated to age of patients with HCC (PD-L1, p = 0.0080; CTLA-4, p = 0.0029) and corresponded to enhanced numbers of Tregs producing IL-10 and IL-35 (p < 0.05 each). Tregs inhibited IFN-gamma secretion and cytotoxicity of CD8+ T cells when added to LDCC against P815 cells. Treg-induced inhibition of IFN-gamma secretion could be partially blocked by neutralizing PD-1 and PD-L1 antibodies specifically in HCC patients. In HCC peripheral Tregs upregulate checkpoint inhibitors and contribute to systemic immune dysfunction and antitumoural activity by several inhibitory pathways, presumably facilitating tumour development at young age. Blocking PD-L1/PD-1 interactions in vitro selectively interfered with inhibitory Treg -T effector cell interactions in the patients with HCC and resulted in improved antitumoural activity also against checkpoint inhibitor-negative tumour cells.

Histone deacetylase inhibition promotes intratumoral CD8 + T-cell responses, sensitizing murine breast tumors to anti-PD1 Abstract Histone deacetylase (HDAC) inhibitors impair tumor cell proliferation and alter gene expression. However, the impact of these changes on anti-tumor immunity is poorly understood. Here, we showed that the class I HDAC inhibitor, entinostat (ENT), promoted the expression of immune-modulatory molecules, including MHCII, costimulatory ligands, and chemokines on murine breast tumor cells in vitro and in vivo. ENT also impaired tumor growth in vivo—an effect that was dependent on both CD8+ T cells and IFNγ. Moreover, ENT promoted intratumoral T-cell clonal expansion and enhanced their functional activity. Importantly, ENT sensitized normally unresponsive tumors to the effects of PD1 blockade, predominantly through increases in T-cell proliferation. Our findings suggest that class I HDAC inhibitors impair tumor growth by enhancing the proliferative and functional capacity of CD8+ T cells and by sensitizing tumor cells to T-cell recognition.

PD-1 + TIGIT + CD8 + T cells are associated with pathogenesis and progression of patients with hepatitis B virus-related hepatocellular carcinoma Abstract Hepatitis B virus-associated hepatocellular carcinoma (HBV-HCC) is usually considered an inflammation-related cancer associated with chronic inflammation triggered by exposure to HBV and tumor antigens. T-cell exhaustion is implicated in immunosuppression of chronic infections and tumors. Although immunotherapies that enhance immune responses by targeting programmed cell death-1(PD-1)/PD-L1 are being applied to malignancies, these treatments have shown limited response rates, suggesting that additional inhibitory receptors are also involved in T-cell exhaustion and tumor outcome. Here, we analyzed peripheral blood samples and found that coexpression of PD-1 and T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) was significantly upregulated on CD4+ and CD8+ T cells from patients with HBV-HCC compared with those from patients with chronic HBV or HBV-liver cirrhosis. Additionally, PD-1+ TIGIT+ CD8+ T-cell populations were elevated in patients with advanced stage and progressed HBV-HCC. Importantly, PD-1+ TIGIT+ CD8+ T-cell populations were negatively correlated with overall survival rate and progression-free survival rates. Moreover, we showed that PD-1+ TIGIT+ CD8+ T cells exhibit features of exhausted T cells, as manifested by excessive activation, high expression of other inhibitory receptors, high susceptibility to apoptosis, decreased capacity for cytokine secretion, and patterns of transcription factor expression consistent with exhaustion. In conclusion, PD-1+ TIGIT+ CD8+ T-cell populations are associated with accelerated disease progression and poor outcomes in HBV-HCC, which might not only have important clinical implications for prognosis but also provide a rationale for new targets in immunotherapy.

Tumor-associated macrophages expressing galectin-9 identify immunoevasive subtype muscle-invasive bladder cancer with poor prognosis but favorable adjuvant chemotherapeutic response Abstract Purpose Tumor-associated macrophages (TAMs) exist as heterogeneous subsets and have dichotomous roles in cancer-immune evasion. This study aims to assess the clinical effects of Galectin-9+ tumor-associated macrophages (Gal-9+TAMs) in muscle-invasive bladder cancer (MIBC). Experimental design We identified Gal-9+TAMs by immunohistochemistry (IHC) analysis of a tumor microarray (TMA) (n = 141) from the Zhongshan Hospital and by flow cytometric analysis of tumor specimens (n = 20) from the Shanghai Cancer Center. The survival benefit of platinum-based chemotherapy in this subpopulation was evaluated. The effect of the tumor-immune microenvironment with different percentages of Gal-9+TAMs was explored. Results The frequency of Gal-9+TAMs increased with tumor stage and grade. Gal-9+TAMs predicted poor overall survival (OS) and recurrence-free survival (RFS) and were better than Gal-9−TAMs and TAMs to discriminate prognostic groups. In univariate and multivariate Cox regression analyses, patients with high percentages of Gal-9+TAMs showed the prominent survival benefit after receiving adjuvant chemotherapy (ACT). High Gal-9+TAM infiltration correlated with increasing numbers of regulatory T cells (Tregs) and mast cells and decreasing numbers of CD8+T and dendritic cells (DCs). Dense infiltration of Gal-9+TAMs was related to reduced cytotoxic molecules, enhanced immune checkpoints or immunosuppressive cytokines expressed by immune cells, as well as active proliferation of tumor cells. Additionally, the subpopulation accumulated was strongly associated with PD-1+TIM-3+CD8+T cells. Conclusions Gal-9+TAMs predicted OS and RFS and response to ACT in MIBC patients. High Gal-9+TAMs were associated with a pro-tumor immune contexture concomitant with T cell exhaustion.

ISG15 pathway knockdown reverses pancreatic cancer cell transformation and decreases murine pancreatic tumor growth via downregulation of PDL-1 expression Abstract Interferon-stimulated gene 15 (ISG15) is a 15 kDa protein induced by type I interferons (IFN-α and IFN-β) and is a member of the ubiquitin-like superfamily of proteins. The ISG15 pathway is highly expressed in various malignancies, including pancreatic ductal adenocarcinoma (PDAC), suggesting a potential role of the ISG15 pathway (free ISG15 and ISG15 conjugates) in pancreatic carcinogenesis. However, very little is known about how the ISG15 pathway may contribute to pancreatic tumorigenesis. In the current study, we demonstrate that ISG15 pathway knockdown reverses the KRAS-associated phenotypes of PDAC cells such as increased proliferation and colony formation. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated ISG15 knockdown decreased tumor programmed death ligand-1 (PDL-1) expression leading to increased number of CD8+ tumor-infiltrating lymphocytes and decreased pancreatic tumor growth. In addition, the syngeneic subcutaneous mouse model revealed that knocking down the ISG15 pathway significantly decreased the rate of tumor incidence and increased the survival rate. Interestingly, the ISG15 knockdown-mediated PDL-1 downregulation in pancreatic tumors increased the efficacy of anti-programmed cell death protein-1 (PD-1) treatment. ISG15 knockdown in combination with anti-PD-1 treatment synergistically increased the number of CD8+ tumor-infiltrating lymphocytes. Additionally, ISG15 knockdown alone significantly decreased the number of tumor-infiltrating regulatory T cells (Tregs) compared to wild type tumors treated with anti-PD-1 antibody. Overall, these findings suggest that strategies to target the ISG15 pathway by itself or in combination with immunotherapy may lead to improved survival for patients diagnosed with PDAC.

Tumor genetic alterations and features of the immune microenvironment drive myelodysplastic syndrome escape and progression Abstract The transformation and progression of myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (sAML) involve genetic, epigenetic, and microenvironmental factors. Driver mutations have emerged as valuable markers for defining risk groups and as candidates for targeted treatment approaches in MDS. It is also evident that the risk of transformation to sAML is increased by evasion of adaptive immune surveillance. This study was designed to explore the immune microenvironment, immunogenic tumor-intrinsic mechanisms (HLA and PD-L1 expression), and tumor genetic features (somatic mutations and altered karyotypes) in MDS patients and to determine their influence on the progression of the disease. We detected major alterations of the immune microenvironment in MDS patients, with a reduced count of CD4+ T cells, a more frequent presence of markers related to T cell exhaustion, a more frequent presence of myeloid-derived suppressor cells (MDSCs), and changes in the functional phenotype of NK cells. HLA Class I (HLA-I) expression was normally expressed in CD34+ blasts and during myeloid differentiation. Only two out of thirty-six patients with homozygosity for HLA-C groups acquired complete copy-neutral loss of heterozygosity in the HLA region. PD-L1 expression on the leukemic clone was also increased in MDS patients. Finally, no interplay was observed between the anti-tumor immune microenvironment and mutational genomic features. In summary, extrinsic and intrinsic immunological factors might severely impair immune surveillance and contribute to clonal immune escape. Genomic alterations appear to make an independent contribution to the clonal evolution and progression of MDS.

Serum very long-chain fatty acid-containing lipids predict response to immune checkpoint inhibitors in urological cancers Abstract Checkpoint inhibitors (CPI) have significantly changed the therapeutic landscape of oncology. We adopted a non-invasive metabolomic approach to understand immunotherapy response and failure in 28 urological cancer patients. In total, 134 metabolites were quantified in patient sera before the first, second, and third CPI doses. Modeling the association between metabolites and CPI response and patient characteristics revealed that one predictive metabolite class  (n = 9/10) were very long-chain fatty acid-containing lipids (VLCFA-containing lipids). The best predictive performance was achieved through a multivariate model, including age and a centroid of VLCFA-containing lipids prior to first immunotherapy (sensitivity: 0.850, specificity: 0.825, ROC: 0.935). We hypothesize that the association of VLCFA-containing lipids with CPI response is based on enhanced peroxisome signaling in T cells, which results in a switch to fatty acid catabolism. Beyond use as a novel predictive non-invasive biomarker, we envision that nutritional supplementation with VLCFA-containing lipids might serve as an immuno sensitizer.

Predominance of M2 macrophages in gliomas leads to the suppression of local and systemic immunity Abstract Glioblastoma is a highly prevalent and aggressive form of primary brain tumor. It represents approximately 56% of all the newly diagnosed gliomas. Macrophages are one of the major constituents of tumor-infiltrating immune cells in the human gliomas. The role of immunosuppressive macrophages is very well documented in correlation with the poor prognosis of patients suffering from breast, prostate, bladder and cervical cancers. The current study highlights the correlation between the tumor-associated macrophage phenotypes and glioma progression. We observed an increase in the pool of M2 macrophages in high-grade gliomas, as confirmed by their CD68 and CD163 double-positive phenotype. In contrast, less M1 macrophages were noticed in high-grade gliomas, as evidenced by the down-regulation in the expression of CCL3 marker. In addition, we observed that higher gene expression ratio of CD163/CCL3 is associated with glioma progression. The Kaplan–Meier survival plots indicate that glioma patients with lower expression of M2c marker (CD163), and higher expression of M1 marker (CCL3) had better survival. Furthermore, we examined the systemic immune response in the peripheral blood and noted a predominance of M2 macrophages, myeloid-derived suppressor cells and PD-1+ CD4 T cells in glioma patients. Thus, the study indicates a high gene expression ratio of CD163/CCL3 in high-grade gliomas as compared to low-grade gliomas and significantly elevated frequency of M2 macrophages and PD-1+ CD4 T cells in the blood of tumor patients. These parameters could be used as an indicator of the early diagnosis and prognosis of the disease.

Preclinical development of T-cell receptor-engineered T-cell therapy targeting the 5T4 tumor antigen on renal cell carcinoma Abstract 5T4 (trophoblast glycoprotein, TPBG) is a transmembrane tumor antigen expressed on more than 90% of primary renal cell carcinomas (RCC) and a wide range of human carcinomas but not on most somatic adult tissues. The favorable expression pattern has encouraged the development and clinical testing of 5T4-targeted antibody and vaccine therapies. 5T4 also represents a compelling and unexplored target for T-cell receptor (TCR)-engineered T-cell therapy. Our group has previously isolated high-avidity CD8+ T-cell clones specific for an HLA-A2-restricted 5T4 epitope (residues 17–25; 5T4p17). In this report, targeted single-cell RNA sequencing was performed on 5T4p17-specific T-cell clones to sequence the highly variable complementarity-determining region 3 (CDR3) of T-cell receptor α chain (TRA) and β chain (TRB) genes. Full-length TRA and TRB sequences were cloned into lentiviral vectors and transduced into CD8+ T-cells from healthy donors. Redirected effector T-cell function against 5T4p17 was measured by cytotoxicity and cytokine release assays. Seven unique TRA-TRB pairs were identified. All seven TCRs exhibited high expression on CD8+ T-cells with transduction efficiencies from 59 to 89%. TCR-transduced CD8+ T-cells demonstrated redirected cytotoxicity and cytokine release in response to 5T4p17 on target-cells and killed 5T4+/HLA-A2+ kidney-, breast-, and colorectal-tumor cell lines as well as primary RCC tumor cells in vitro. TCR-transduced CD8+ T-cells also detected presentation of 5T4p17 in TAP1/2-deficient T2 target-cells. TCR-transduced T-cells redirected to recognize the 5T4p17 epitope from a broadly shared tumor antigen are of interest for future testing as a cellular immunotherapy strategy for HLA-A2+ subjects with 5T4+ tumors.

Antisense targeting of CD47 enhances human cytotoxic T-cell activity and increases survival of mice bearing B16 melanoma when combined with anti-CTLA4 and tumor irradiation Abstract Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47 m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.