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The extent to which immune cell phenotypes in the peripheral blood reflect within-tumor immune activity prior to and early in cancer therapy is unclear. To address this question, we studied the population dynamics of tumor and immune cells, and immune phenotypic changes, using clinical tumor and immune cell measurements and single-cell genomic analyses. These samples were serially obtained from a cohort of advanced gastrointestinal cancer patients enrolled in a trial with chemotherapy and immunotherapy. Using an ecological population model, fitted to clinical tumor burden and immune cell abundance data from each patient, we find evidence of a strong tumor-circulating immune cell interaction in responder patients but not in those patients that progress on treatment. Upon initiation of therapy, immune cell abundance increased rapidly in responsive patients, and once the peak level is reached tumor burden decreases, similar to models of predator–prey interactions; these dynamic patterns were absent in nonresponder patients. To interrogate phenotype dynamics of circulating immune cells, we performed single-cell RNA sequencing at serial time points during treatment. These data show that peripheral immune cell phenotypes were linked to the increased strength of patients’ tumor–immune cell interaction, including increased cytotoxic differentiation and strong activation of interferon signaling in peripheral T cells in responder patients. Joint modeling of clinical and genomic data highlights the interactions between tumor and immune cell populations and reveals how variation in patient responsiveness can be explained by differences in peripheral immune cell signaling and differentiation soon after the initiation of immunotherapy.

High-throughput chromosome conformation capture (Hi-C) analyses revealed that the 3D structure of the Neurospora crassa genome is dominated by intra- and interchromosomal links between regions of heterochromatin, especially constitutive heterochromatin. Elimination of trimethylation of lysine 9 on histone H3 (H3K9me3) or its binding partner Heterochromatin Protein 1 (HP1)—both prominent features of constitutive heterochromatin—have little effect on the Hi-C pattern. It remained possible that di- or trimethylation of lysine 27 on histone H3 (H3K27me2/3), which becomes localized in regions of constitutive heterochromatin when H3K9me3 or HP1 are lost, plays a critical role in the 3D structure of the genome. We found that H3K27me2/3, catalyzed by the Polycomb Repressive Complex 2 (PRC2) member SET-7 (SET domain protein-7), does indeed play a prominent role in the Hi-C pattern of WT, but that its presence in regions normally occupied by H3K9me3 is not responsible for maintenance of the genome architecture when H3K9me3 is lost. The Hi-C pattern of a mutant defective in the PRC2 member N. crassa p55 (NPF), which is predominantly required for subtelomeric H3K27me2/3, was equivalent to that of the set-7 deletion strain, suggesting that subtelomeric facultative heterochromatin is paramount for normal chromosome conformation. Both PRC2 mutants showed decreased heterochromatin–heterochromatin contacts and increased euchromatin–heterochromatin contacts. Cytological observations suggested elimination of H3K27me2/3 leads to partial displacement of telomere clusters from the nuclear periphery. Transcriptional profiling of Δdim-5, Δset-7, Δset-7; Δdim-5, and Δnpf strains detailed anticipated changes in gene expression but did not support the idea that global changes in genome architecture, per se, led to altered transcription.

BackgroundThe immune suppressive tumor microenvironment (TME) that inhibits T cell infiltration, survival, and antitumor activity has posed a major challenge for developing effective immunotherapies for solid tumors. Chimeric antigen receptor (CAR)-engineered T cell therapy has shown unprecedented clinical response in treating patients with hematological malignancies, and intense investigation is underway to achieve similar responses with solid tumors. Immunologically cold tumors, including prostate cancers, are often infiltrated with abundant tumor-associated macrophages (TAMs), and infiltration of CD163+ M2 macrophages correlates with tumor progression and poor responses to immunotherapy. However, the impact of TAMs on CAR T cell activity alone and in combination with TME immunomodulators is unclear.MethodsTo model this in vitro, we utilized a novel co-culture system with tumor cells, CAR T cells, and polarized M1 or M2 macrophages from CD14+ peripheral blood mononuclear cells collected from healthy human donors. Tumor cell killing, T cell activation and proliferation, and macrophage phenotypes were evaluated by flow cytometry, cytokine production, RNA sequencing, and functional blockade of signaling pathways using antibodies and small molecule inhibitors. We also evaluated the TME in humanized mice following CAR T cell therapy for validation of our in vitro findings.ResultsWe observed inhibition of CAR T cell activity with the presence of M2 macrophages, but not M1 macrophages, coinciding with a robust induction of programmed death ligand-1 (PD-L1) in M2 macrophages. We observed similar PD-L1 expression in TAMs following CAR T cell therapy in the TME of humanized mice. PD-L1, but not programmed cell death protein-1, blockade in combination with CAR T cell therapy altered phenotypes to more M1-like subsets and led to loss of CD163+ M2 macrophages via interferon-γ signaling, resulting in improved antitumor activity of CAR T cells.ConclusionThis study reveals an alternative mechanism by which the combination of CAR T cells and immune checkpoint blockade modulates the immune landscape of solid tumors to enhance therapeutic efficacy of CAR T cells.

Development in higher organisms requires selective gene silencing, directed in part by di-/trimethylation of lysine 27 on histone H3 (H3K27me2/3). Knowledge of the cues that control formation of such repressive Polycomb domains is extremely limited. We exploited natural and engineered chromosomal rearrangements in the fungus Neurospora crassa to elucidate the control of H3K27me2/3. Analyses of H3K27me2/3 in strains bearing chromosomal rearrangements revealed both position-dependent and position-independent facultative heterochromatin. We found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive, subtelomeric H3K27me2/3. We ascertained that such telomere-proximal facultative heterochromatin requires native telomere repeats and found that a short array of ectopic telomere repeats, (TTAGGG)17, can induce a large domain (~225 kb) of H3K27me2/3. This provides an example of a cis-acting sequence that directs H3K27 methylation. Our findings provide new insight into the relationship between genome organization and control of heterochromatin formation.

Despite recent therapeutic advances, metastatic castration-resistant prostate cancer (mCRPC) remains lethal. Chimeric antigen receptor (CAR) T cell therapies have demonstrated durable remissions in hematological malignancies. We report results from a phase 1, first-in-human study of prostate stem cell antigen (PSCA)-directed CAR T cells in men with mCRPC. The starting dose level (DL) was 100 million (M) CAR T cells without lymphodepletion (LD), followed by incorporation of LD. The primary end points were safety and dose-limiting toxicities (DLTs). No DLTs were observed at DL1, with a DLT of grade 3 cystitis encountered at DL2, resulting in addition of a new cohort using a reduced LD regimen + 100 M CAR T cells (DL3). No DLTs were observed in DL3. Cytokine release syndrome of grade 1 or 2 occurred in 5 of 14 treated patients. Prostate-specific antigen declines (>30%) occurred in 4 of 14 patients, as well as radiographic improvements. Dynamic changes indicating activation of peripheral blood endogenous and CAR T cell subsets, TCR repertoire diversity and changes in the tumor immune microenvironment were observed in a subset of patients. Limited persistence of CAR T cells was observed beyond 28 days post-infusion. These results support future clinical studies to optimize dosing and combination strategies to improve durable therapeutic outcomes. ClinicalTrials.gov identifier NCT03873805 . A phase 1 trial of PSCA-targeting CAR T cell therapy in patients with PSCA-positive, metastatic castration-resistant prostate cancer demonstrates that the treatment, using a protocol with reduced lymphodepletion, is safe and shows preliminary clinical activity.

Abstract The extent that immune cell phenotypes in the peripheral blood reflect within-tumor immune activity prior to and early in cancer therapy is unclear. To address this question, we studied the population dynamics of tumor and immune cells, and immune phenotypic changes, using clinical tumor and immune cell measurements and single cell genomic analyses. These samples were serially obtained from a cohort of advanced gastrointestinal cancer patients enrolled on a trial with chemotherapy and immunotherapy. Using an ecological population model, fitted to clinical tumor burden and immune cell abundance data from each patient, we find evidence of a strong tumor-circulating immune cell interaction in responder patients, but not those patients that progress on treatment. Upon initiation of therapy, immune cell abundance increased rapidly in responsive patients, and once the peak level is reached, tumor burden decreases, similar to models of predator-prey interactions; these dynamic patterns were absent in non-responder patients. To interrogate phenotype dynamics of circulating immune cells, we performed single cell RNA sequencing at serial time points during treatment. These data show that peripheral immune cell phenotypes were linked to the increased strength of patients’ tumor-immune cell interaction, including increased cytotoxic differentiation and strong activation of interferon signaling in peripheral T-cells in responder patients. Joint modeling of clinical and genomic data highlights the interactions between tumor and immune cell populations and reveals how variation in patient responsiveness can be explained by differences in peripheral immune cell signaling and differentiation soon after the initiation of immunotherapy. One sentence summary Peripheral immune cell differentiation and signaling, upon initiation of immunotherapy, reflects tumor attacking ability and patient response. Significance statement The evolution of peripheral immune cell abundance and signaling over time, as well as how these immune cells interact with the tumor, may impact a cancer patient’s response to therapy. By developing an ecological population model, we provide evidence of a dynamic predator-prey like relationship between circulating immune cell abundance and tumor size in patients that respond to immunotherapy. This relationship is not found either in patients that are non-responsive to immunotherapy or during chemotherapy. Single cell RNA-sequencing (scRNAseq) of serial peripheral blood samples from patients show that the strength of tumor-immune cell interactions is reflected in T-cells interferon activation and differentiation early in treatment. Thus, circulating immune cell dynamics reflect a tumor’s response to immunotherapy.

Development in higher organisms requires selective gene silencing, directed in part by di-/tri-methylation of lysine 27 on histone H3 (H3K27me2/3). Knowledge of the cues that control formation of such repressive Polycomb domains is extremely limited. We exploited natural and engineered chromosomal rearrangements in the fungus Neurospora crassa to elucidate the control of H3K27me2/3. Analyses of H3K27me2/3 in strains bearing chromosomal rearrangements revealed both position-dependent and position-independent facultative heterochromatin. We found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive, subtelomeric H3K27me2/3. We ascertained that such telomere-proximal facultative heterochromatin requires native telomere repeats and found that a short array of ectopic telomere repeats, (TTAGGG)17, can induce a large domain (~225 kb) of H3K27me2/3. This provides an example of a cis-acting sequence that directs H3K27 methylation. Our findings provide new insight into the relationship between genome organization and control of heterochromatin formation.

Background: The immune suppressive tumor microenvironment (TME) that inhibits T cell infiltration, survival, and anti-tumor activity has posed a major challenge for developing effective immunotherapies for solid tumors. Chimeric antigen receptor (CAR)-engineered T cell therapy has shown unprecedented clinical response in treating patients with hematological malignancies, and intense investigation is underway to achieve similar responses with solid tumors. Immunologically cold tumors, including prostate cancers, are often infiltrated with abundant tumor-associated macrophages (TAMs), and infiltration of CD163+ M2 macrophages correlates with tumor progression and poor responses to immunotherapy. However, the impact of TAMs on CAR T cell activity alone and in combination with TME immunomodulators is unclear. Methods: To model this in vitro, we utilized a novel co-culture system with tumor cells, CAR T cells, and polarized M1 or M2 macrophages from CD14+ PBMCs collected from healthy human donors. Tumor cell killing, T cell activation and proliferation, and macrophage phenotypes were evaluated by flow cytometry, cytokine production, RNA sequencing, and functional blockade of signaling pathways using antibodies and small molecule inhibitors. We also evaluated the TME in humanized mice following CAR T cell therapy for validation of our in vitro findings. Results: We observed inhibition of CAR T cell activity with the presence of M2 macrophages, but not M1 macrophages, coinciding with a robust induction of PD-L1 in M2 macrophages. We observed similar PD-L1 expression in TAMs following CAR T cell therapy in the TME of humanized mice. PD-L1, but not PD-1, blockade in combination with CAR T cell therapy altered phenotypes to more M1-like subsets and led to loss of CD163+ M2 macrophages via IFNγ signaling, resulting in improved anti-tumor activity of CAR T cells. Conclusion: This study reveals an alternative mechanism by which the combination of CAR T cells and immune checkpoint blockade modulates the immune landscape of solid tumors to enhance therapeutic efficacy of CAR T cells. Competing Interest Statement S.J.P. and S.J.F. are scientific advisors to and receive royalties from Mustang Bio. S.J.P. is a scientific advisor and/or receives royalties from Imugene Ltd., Bayer, and Adicet Bio. All other authors declare that they have no competing interests.