Peripheral gene signatures reveal distinct cancer patient immunotypes with therapeutic implications for autologous DC-based vaccines.

Dendritic cells (DCs) have received considerable attention as potential targets for the development of novel cancer immunotherapies. However, the clinical efficacy of DC-based vaccines remains suboptimal, largely reflecting local and systemic immunosuppression at baseline. An autologous DC-based vaccine (DCVAC) has recently been shown to improve progression-free survival and overall survival in randomized clinical trials enrolling patients with lung cancer (SLU01, NCT02470468) or ovarian carcinoma (SOV01, NCT02107937), but not metastatic castration-resistant prostate cancer (SP005, NCT02111577), despite a good safety profile across all cohorts. We performed biomolecular and cytofluorometric analyses on peripheral blood samples collected prior to immunotherapy from 1000 patients enrolled in these trials, with the objective of identifying immunological biomarkers that may improve the clinical management of DCVAC-treated patients. Gene signatures reflecting adaptive immunity and T cell activation were associated with favorable disease outcomes and responses to DCVAC in patients with prostate and lung cancer, but not ovarian carcinoma. By contrast, the clinical benefits of DCVAC were more pronounced among patients with ovarian carcinoma exhibiting reduced expression of T cell-associated genes, especially those linked to TH2-like signature and immunosuppressive regulatory T (TREG) cells. Clinical responses to DCVAC were accompanied by signs of antitumor immunity in the peripheral blood. Our findings suggest that circulating signatures of antitumor immunity may provide a useful tool for monitoring the potency of autologous DC-based immunotherapy.

An Autologous Dendritic Cell Vaccine Promotes Anticancer Immunity in Patients with Ovarian Cancer with Low Mutational Burden and Cold Tumors.

PURPOSE: The successful implementation of immune checkpoint inhibitors (ICI) in the clinical management of various solid tumors has raised considerable expectations for patients with epithelial ovarian carcinoma (EOC). However, EOC is poorly responsive to ICIs due to immunologic features including limited tumor mutational burden (TMB) and poor lymphocytic infiltration. An autologous dendritic cell (DC)-based vaccine (DCVAC) has recently been shown to be safe and to significantly improve progression-free survival (PFS) in a randomized phase II clinical trial enrolling patients with EOC (SOV01, NCT02107937). PATIENTS AND METHODS: We harnessed sequencing, flow cytometry, multispectral immunofluorescence microscopy, and IHC to analyze (pretreatment) tumor and (pretreatment and posttreatment) peripheral blood samples from 82 patients enrolled in SOV01, with the aim of identifying immunologic biomarkers that would improve the clinical management of patients with EOC treated with DCVAC. RESULTS: Although higher-than-median TMB and abundant CD8+ T-cell infiltration were associated with superior clinical benefits in patients with EOC receiving standard-of-care chemotherapy, the same did not hold true in women receiving DCVAC. Conversely, superior clinical responses to DCVAC were observed in patients with lower-than-median TMB and scarce CD8+ T-cell infiltration. Such responses were accompanied by signs of improved effector functions and tumor-specific cytotoxicity in the peripheral blood. CONCLUSIONS: Our findings suggest that while patients with highly infiltrated, "hot" EOCs benefit from chemotherapy, women with "cold" EOCs may instead require DC-based vaccination to jumpstart clinically relevant anticancer immune responses.

CD4+ T Cells of Prostate Cancer Patients Have Decreased Immune Responses to Antigens Derived From SARS-CoV-2 Spike Glycoprotein.

The adaptive immune response to severe acute respiratory coronavirus 2 (SARS-CoV-2) is important for vaccine development and in the recovery from coronavirus disease 2019 (COVID-19). Men and cancer patients have been reported to be at higher risks of contracting the virus and developing the more severe forms of COVID-19. Prostate cancer (PCa) may be associated with both of these risks. We show that CD4+ T cells of SARS-CoV-2-unexposed patients with hormone-refractory (HR) metastatic PCa had decreased CD4+ T cell immune responses to antigens from SARS-CoV-2 spike glycoprotein but not from the spiked glycoprotein of the 'common cold'-associated human coronavirus 229E (HCoV-229E) as compared with healthy male volunteers who responded comparably to both HCoV-229E- and SARS-CoV-2-derived antigens. Moreover, the HCoV-229E spike glycoprotein antigen-elicited CD4+ T cell immune responses cross-reacted with the SARS-CoV-2 spiked glycoprotein antigens. PCa patients may have impaired responses to the vaccination, and the cross-reactivity can mediate antibody-dependent enhancement (ADE) of COVID-19. These findings highlight the potential for increased vulnerability of PCa patients to COVID-19.

M2-like macrophages dictate clinically relevant immunosuppression in metastatic ovarian cancer.

BACKGROUND: The immunological microenvironment of primary high-grade serous carcinomas (HGSCs) has a major impact on disease outcome. Conversely, little is known on the microenvironment of metastatic HGSCs and its potential influence on patient survival. Here, we explore the clinical relevance of the immunological configuration of HGSC metastases. METHODS: RNA sequencing was employed on 24 paired primary tumor microenvironment (P-TME) and metastatic tumor microenvironment (M-TME) chemotherapy-naive HGSC samples. Immunohistochemistry was used to evaluate infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ (lysosomal-associated membrane protein 3) dendritic cells (DCs), NKp46+ (natural killer) cells and CD68+CD163+ M2-like tumor-associated macrophages (TAMs), abundance of PD-1+ (programmed cell death 1), LAG-3+ (lymphocyte-activating gene 3) cells, and PD-L1 (programmed death ligand 1) expression in 80 samples. Flow cytometry was used for functional assessments on freshly resected HGSC samples. RESULTS: 1468 genes were differentially expressed in the P-TME versus M-TME of HGSCs, the latter displaying signatures of extracellular matrix remodeling and immune infiltration. M-TME infiltration by immune effector cells had little impact on patient survival. Accordingly, M-TME-infiltrating T cells were functionally impaired, but not upon checkpoint activation. Conversely, cytokine signaling in favor of M2-like TAMs activity appeared to underlie inhibited immunity in the M-TME and poor disease outcome. CONCLUSIONS: Immunosuppressive M2-like TAM infiltrating metastatic sites limit clinically relevant immune responses against HGSCs.

Gene expression profiling of circulating tumor cells and peripheral blood mononuclear cells from breast cancer patients.

Circulating tumor cells (CTCs) are cancer cells that are released from a tumor into the bloodstream. The presence of CTCs in peripheral blood has been associated with metastasis formation in patients with breast cancer. Therefore, the molecular characterization of CTCs may improve diagnostics and support treatment decisions. We performed gene expression profiling to evaluate the enriched CTCs and peripheral blood mononuclear cells (PBMCs) of breast cancer patients using an expression panel of 55 breast cancer-associated genes. The study revealed several significantly differentially expressed genes in the CTC-positive samples, including a few that were exclusively expressed in these cells. However, the expression of these genes was barely detectable in the PBMC samples. Some genes were differentially expressed in PBMCs, and the expression of these genes was correlated with tumor grade and the formation of metastasis. In this study, we have shown that the enriched CTCs of breast cancer patients overexpress genes involved in proteolytic degradation of the extracellular matrix (ECM) as well as genes that play important roles in the epithelial-mesenchymal transition (EMT) process that may occur in these cells.

High hydrostatic pressure induces immunogenic cell death in human tumor cells.

Recent studies have identified molecular events characteristic of immunogenic cell death (ICD), including surface exposure of calreticulin (CRT), the heat shock proteins HSP70 and HSP90, the release of high-mobility group box protein 1 (HMGB1) and the release of ATP from dying cells. We investigated the potential of high hydrostatic pressure (HHP) to induce ICD in human tumor cells. HHP induced the rapid expression of HSP70, HSP90 and CRT on the cell surface. HHP also induced the release of HMGB1 and ATP. The interaction of dendritic cells (DCs) with HHP-treated tumor cells led to a more rapid rate of DC phagocytosis, upregulation of CD83, CD86 and HLA-DR and the release of interleukin IL-6, IL-12p70 and TNF-α. DCs pulsed with tumor cells killed by HHP induced high numbers of tumor-specific T cells. DCs pulsed with HHP-treated tumor cells also induced the lowest number of regulatory T cells. In addition, we found that the key features of the endoplasmic reticulum stress-mediated apoptotic pathway, such as reactive oxygen species production, phosphorylation of the translation initiation factor eIF2α and activation of caspase-8, were activated by HHP treatment. Therefore, HHP acts as a reliable and potent inducer of ICD in human tumor cells.

The δ subunit of RNA polymerase is required for rapid changes in gene expression and competitive fitness of the cell.

RNA polymerase (RNAP) is an extensively studied multisubunit enzyme required for transcription of DNA into RNA, yet the δ subunit of RNAP remains an enigmatic protein whose physiological roles have not been fully elucidated. Here, we identify a novel, so far unrecognized function of δ from Bacillus subtilis. We demonstrate that δ affects the regulation of RNAP by the concentration of the initiating nucleoside triphosphate ([iNTP]), an important mechanism crucial for rapid changes in gene expression in response to environmental changes. Consequently, we demonstrate that δ is essential for cell survival when facing a competing strain in a changing environment. Hence, although δ is not essential per se, it is vital for the cell's ability to rapidly adapt and survive in nature. Finally, we show that two other proteins, GreA and YdeB, previously implicated to affect regulation of RNAP by [iNTP] in other organisms, do not have this function in B. subtilis.

Dynamics of T-cell infiltration during the course of ovarian cancer: the gradual shift from a Th17 effector cell response to a predominant infiltration by regulatory T-cells.

The type of immune cells that are present within the tumor microenvironment can play a crucial role in the survival of patients. However, little is known about the dynamics of the tumor-infiltrating immune cells during disease progression. We studied the immune cells that infiltrated the tumor tissues of ovarian cancer patients at different stages of disease. The early stages of development of ovarian carcinomas were characterized by a strong Th17 immune response, whereas in stage II patients, recruitment of high numbers of Th1 cells was observed. In disseminated tumors (Stages III-IV), we detected a dominant population of Helios(+) activated regulatory T cells (Tregs) along with high numbers of monocytes/macrophages and myeloid dendritic cells (mDCs). Tumor-infiltrating Tregs had markedly lower expression of CCR4 than circulating Tregs, and the numbers of tumor-infiltrating Tregs significantly correlated with the levels of CCL22 in ovarian tumor cell culture supernatants, suggesting their recruitment via a CCR4/CCL22 interaction. CCL22 was mainly produced by tumor cells, monocytes/macrophages and mDCs in the primary ovarian tumors, and its expression markedly increased in response to IFNγ. Taken together, the specific recruitment of Tregs, probably triggered by inflammatory stimuli, leads to a significant immune suppression in the advanced stages of ovarian cancer.

Rapid changes in gene expression: DNA determinants of promoter regulation by the concentration of the transcription initiating NTP in Bacillus subtilis.

In bacteria, rapid changes in gene expression can be achieved by affecting the activity of RNA polymerase with small molecule effectors during transcription initiation. An important small molecule effector is the initiating nucleoside triphosphate (iNTP). At some promoters, an increasing iNTP concentration stimulates promoter activity, while a decreasing concentration has the opposite effect. Ribosomal RNA (rRNA) promoters from Gram-positive Bacillus subtilis are regulated by the concentration of their iNTP. Yet, the sequences of these promoters do not emulate the sequence characteristics of [iNTP]-regulated rRNA promoters of Gram-negative Escherichia coli. Here, we identified the 3'-promoter region, corresponding to the transcription bubble, as key for B. subtilis rRNA promoter regulation via the concentration of the iNTP. Within this region, the conserved -5T (3 bp downstream from the -10 hexamer) is required for this regulation. Moreover, we identified a second class of [iNTP]-regulated promoters in B. subtilis where the sequence determinants are not limited to the transcription bubble region. Overall, it seems that various sequence combinations can result in promoter regulation by [iNTP] in B. subtilis. Finally, this study demonstrates how the same type of regulation can be achieved with strikingly different promoter sequences in phylogenetically distant species.

The N-terminal region is crucial for the thermostability of the G-domain of Bacillus stearothermophilus EF-Tu.

Bacterial elongation factor Tu (EF-Tu) is a model monomeric G protein composed of three covalently linked domains. Previously, we evaluated the contributions of individual domains to the thermostability of EF-Tu from the thermophilic bacterium Bacillus stearothermophilus. We showed that domain 1 (G-domain) sets up the basal level of thermostability for the whole protein. Here we chose to locate the thermostability determinants distinguishing the thermophilic domain 1 from a mesophilic domain 1. By an approach of systematically swapping protein regions differing between G-domains from mesophilic Bacillus subtilis and thermophilic B. stearothermophilus, we demonstrate that a small portion of the protein, the N-terminal 12 amino acid residues, plays a key role in the thermostability of this domain. We suggest that the thermostabilizing effect of the N-terminal region could be mediated by stabilizing the functionally important effector region. Finally, we demonstrate that the effect of the N-terminal region is significant also for the thermostability of the full-length EF-Tu.

YbxF, a protein associated with exponential-phase ribosomes in Bacillus subtilis.

The ybxF gene is a member of the streptomycin operon in a wide range of gram-positive bacteria. In Bacillus subtilis, it codes for a small basic protein (82 amino acids, pI 9.51) of unknown function. We demonstrate that, in B. subtilis, YbxF localizes to the ribosome, primarily to the 50S subunit, with dependence on growth phase. Based on three-dimensional structures of YbxF generated by homology modeling, we identified helix 2 as important for the interaction with the ribosome. Subsequent mutational analysis of helix 2 revealed Lys24 as crucial for the interaction. Neither the B. subtilis ybxF gene nor its paralogue, the ymxC gene, is essential, as shown by probing DeltaybxF, DeltaymxC, or DeltaybxF DeltaymxC double deletion strains in several functional assays.