Defining cellular population dynamics at single-cell resolution during prostate cancer progression.

Advanced prostate malignancies are a leading cause of cancer-related deaths in men, in large part due to our incomplete understanding of cellular drivers of disease progression. We investigate prostate cancer cell dynamics at single-cell resolution from disease onset to the development of androgen independence in an in vivo murine model. We observe an expansion of a castration-resistant intermediate luminal cell type that correlates with treatment resistance and poor prognosis in human patients. Moreover, transformed epithelial cells and associated fibroblasts create a microenvironment conducive to pro-tumorigenic immune infiltration, which is partially androgen responsive. Androgen-independent prostate cancer leads to significant diversification of intermediate luminal cell populations characterized by a range of androgen signaling activity, which is inversely correlated with proliferation and mRNA translation. Accordingly, distinct epithelial populations are exquisitely sensitive to translation inhibition, which leads to epithelial cell death, loss of pro-tumorigenic signaling, and decreased tumor heterogeneity. Our findings reveal a complex tumor environment largely dominated by castration-resistant luminal cells and immunosuppressive infiltrates.

Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations.

The Fbw7 ubiquitin ligase targets many proteins for proteasomal degradation, which include oncogenic transcription factors (TFs) (e.g., c-Myc, c-Jun, and Notch). Fbw7 is a tumor suppressor and tumors often contain mutations in FBXW7, the gene that encodes Fbw7. The complexity of its substrate network has obscured the mechanisms of Fbw7-associated tumorigenesis, yet this understanding is needed for developing therapies. We used an integrated approach employing RNA-Seq and high-resolution mapping (cleavage under target and release using nuclease) of histone modifications and TF occupancy (c-Jun and c-Myc) to examine the combinatorial effects of misregulated Fbw7 substrates in colorectal cancer (CRC) cells with engineered tumor-associated FBXW7 null or missense mutations. Both Fbw7 mutations caused widespread transcriptional changes associated with active chromatin and altered TF occupancy: some were common to both Fbw7 mutant cell lines, whereas others were mutation specific. We identified loci where both Jun and Myc were coregulated by Fbw7, suggesting that substrates may have synergistic effects. One coregulated gene was CIITA, the master regulator of MHC Class II gene expression. Fbw7 loss increased MHC Class II expression and Fbw7 mutations were correlated with increased CIITA expression in TCGA colorectal tumors and cell lines, which may have immunotherapeutic implications for Fbw7-associated cancers. Analogous studies in neural stem cells in which FBXW7 had been acutely deleted closely mirrored the results in CRC cells. Gene set enrichment analyses revealed Fbw7-associated pathways that were conserved across both cell types that may reflect fundamental Fbw7 functions. These analyses provide a framework for understanding normal and neoplastic context-specific Fbw7 functions.

Immunogenic Chemotherapy Enhances Recruitment of CAR-T Cells to Lung Tumors and Improves Antitumor Efficacy when Combined with Checkpoint Blockade.

Adoptive therapy using chimeric antigen receptor-modified T cells (CAR-T cells) is effective in hematologic but not epithelial malignancies, which cause the greatest mortality. In breast and lung cancer patients, CAR-T cells targeting the tumor-associated antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) infiltrate tumors poorly and become dysfunctional. To test strategies for enhancing efficacy, we adapted the KrasLSL-G12D/+;p53f/f autochthonous model of lung adenocarcinoma to express the CAR target ROR1. Murine ROR1 CAR-T cells transferred after lymphodepletion with cyclophosphamide (Cy) transiently control tumor growth but infiltrate tumors poorly and lose function, similar to what is seen in patients. Adding oxaliplatin (Ox) to the lymphodepletion regimen activates tumor macrophages to express T-cell-recruiting chemokines, resulting in improved CAR-T cell infiltration, remodeling of the tumor microenvironment, and increased tumor sensitivity to anti-PD-L1. Combination therapy with Ox/Cy and anti-PD-L1 synergistically improves CAR-T cell-mediated tumor control and survival, providing a strategy to improve CAR-T cell efficacy in the clinic.

Publisher Correction: Orchestrating single-cell analysis with Bioconductor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Orchestrating single-cell analysis with Bioconductor.

Recent technological advancements have enabled the profiling of a large number of genome-wide features in individual cells. However, single-cell data present unique challenges that require the development of specialized methods and software infrastructure to successfully derive biological insights. The Bioconductor project has rapidly grown to meet these demands, hosting community-developed open-source software distributed as R packages. Featuring state-of-the-art computational methods, standardized data infrastructure and interactive data visualization tools, we present an overview and online book (https://osca.bioconductor.org) of single-cell methods for prospective users.

ZEB1, ZEB2, and the miR-200 family form a counterregulatory network to regulate CD8+ T cell fates.

Long-term immunity depends partly on the establishment of memory CD8+ T cells. We identified a counterregulatory network between the homologous transcription factors ZEB1 and ZEB2 and the miR-200 microRNA family, which modulates effector CD8+ T cell fates. Unexpectedly, Zeb1 and Zeb2 had reciprocal expression patterns and were functionally uncoupled in CD8+ T cells. ZEB2 promoted terminal differentiation, whereas ZEB1 was critical for memory T cell survival and function. Interestingly, the transforming growth factor ß (TGF-ß) and miR-200 family members, which counterregulate the coordinated expression of Zeb1 and Zeb2 during the epithelial-to-mesenchymal transition, inversely regulated Zeb1 and Zeb2 expression in CD8+ T cells. TGF-ß induced and sustained Zeb1 expression in maturing memory CD8+ T cells. Meanwhile, both TGF-ß and miR-200 family members selectively inhibited Zeb2. Additionally, the miR-200 family was necessary for optimal memory CD8+ T cell formation. These data outline a previously unknown genetic pathway in CD8+ T cells that controls effector and memory cell fate decisions.

Myeloid-targeted immunotherapies act in synergy to induce inflammation and antitumor immunity.

Eliciting effective antitumor immune responses in patients who fail checkpoint inhibitor therapy is a critical challenge in cancer immunotherapy, and in such patients, tumor-associated myeloid cells and macrophages (TAMs) are promising therapeutic targets. We demonstrate in an autochthonous, poorly immunogenic mouse model of melanoma that combination therapy with an agonistic anti-CD40 mAb and CSF-1R inhibitor potently suppressed tumor growth. Microwell assays to measure multiplex protein secretion by single cells identified that untreated tumors have distinct TAM subpopulations secreting MMP9 or cosecreting CCL17/22, characteristic of an M2-like state. Combination therapy reduced the frequency of these subsets, while simultaneously inducing a separate polyfunctional inflammatory TAM subset cosecreting TNF-α, IL-6, and IL-12. Tumor suppression by this combined therapy was partially dependent on T cells, and on TNF-α and IFN-γ. Together, this study demonstrates the potential for targeting TAMs to convert a "cold" into an "inflamed" tumor microenvironment capable of eliciting protective T cell responses.

Interleukin-10 from CD4+ follicular regulatory T cells promotes the germinal center response.

CD4+ follicular regulatory T (Tfr) cells suppress B cell responses through modulation of follicular helper T (Tfh) cells and germinal center (GC) development. We found that Tfr cells can also promote the GC response through provision of interleukin-10 (IL-10) after acute infection with lymphocytic choriomeningitis virus (LCMV). Sensing of IL-10 by B cells was necessary for optimal development of the GC response. GC B cells formed in the absence of Treg cell-derived IL-10 displayed an altered dark zone state and decreased expression of the transcription factor Forkhead box protein 1 (FOXO1). IL-10 promoted nuclear translocation of FOXO1 in activated B cells. These data indicate that Tfr cells play a multifaceted role in the fine-tuning of the GC response and identify IL-10 as an important mediator by which Tfr cells support the GC reaction.

Polycomb Repressive Complex 2-Mediated Chromatin Repression Guides Effector CD8+ T Cell Terminal Differentiation and Loss of Multipotency.

Understanding immunological memory formation depends on elucidating how multipotent memory precursor (MP) cells maintain developmental plasticity and longevity to provide long-term immunity while other effector cells develop into terminally differentiated effector (TE) cells with limited survival. Profiling active (H3K27ac) and repressed (H3K27me3) chromatin in naive, MP, and TE CD8+ T cells during viral infection revealed increased H3K27me3 deposition at numerous pro-memory and pro-survival genes in TE relative to MP cells, indicative of fate restriction, but permissive chromatin at both pro-memory and pro-effector genes in MP cells, indicative of multipotency. Polycomb repressive complex 2 deficiency impaired clonal expansion and TE cell differentiation, but minimally impacted CD8+ memory T cell maturation. Abundant H3K27me3 deposition at pro-memory genes occurred late during TE cell development, probably from diminished transcription factor FOXO1 expression. These results outline a temporal model for loss of memory cell potential through selective epigenetic silencing of pro-memory genes in effector T cells.

NK Cell Responses Redefine Immunological Memory.

Immunological memory has traditionally been regarded as a unique trait of the adaptive immune system. Nevertheless, there is evidence of immunological memory in lower organisms and invertebrates, which lack an adaptive immune system. Despite their innate ability to rapidly produce effector cytokines and kill virally infected or transformed cells, NK cells also exhibit adaptive characteristics such as clonal expansion, longevity, self-renewal, and robust recall responses to antigenic or nonantigenic stimuli. In this review, we highlight the intracellular and extracellular requirements for memory NK cell generation and describe the emerging evidence for memory precursor NK cells and their derivation.

IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium.

A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell-cell interactions of circulating leukocytes with IL-1- or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17-induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17-activated PCs, but not ECs, induce pertussis toxin-sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1ß, and IL-8. Furthermore, IL-17-activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17-treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17-activated PCs can modulate neutrophil functions within the perivascular tissue space.

Autoreactive T Cells from Patients with Myasthenia Gravis Are Characterized by Elevated IL-17, IFN-γ, and GM-CSF and Diminished IL-10 Production.

Myasthenia gravis (MG) is a prototypical autoimmune disease that is among the few for which the target Ag and the pathogenic autoantibodies are clearly defined. The pathology of the disease is affected by autoantibodies directed toward the acetylcholine receptor (AChR). Mature, Ag-experienced B cells rely on the action of Th cells to produce these pathogenic Abs. The phenotype of the MG Ag-reactive T cell compartment is not well defined; thus, we sought to determine whether such cells exhibit both a proinflammatory and a pathogenic phenotype. A novel T cell library assay that affords multiparameter interrogation of rare Ag-reactive CD4(+) T cells was applied. Proliferation and cytokine production in response to both AChR and control Ags were measured from 3120 T cell libraries derived from 11 MG patients and paired healthy control subjects. The frequency of CCR6(+) memory T cells from MG patients proliferating in response to AChR-derived peptides was significantly higher than that of healthy control subjects. Production of both IFN-γ and IL-17, in response to AChR, was also restricted to the CCR6(+) memory T cell compartment in the MG cohort, indicating a proinflammatory phenotype. These T cells also included an elevated expression of GM-CSF and absence of IL-10 expression, indicating a proinflammatory and pathogenic phenotype. This component of the autoimmune response in MG is of particular importance when considering the durability of MG treatment strategies that eliminate B cells, because the autoreactive T cells could renew autoimmunity in the reconstituted B cell compartment with ensuing clinical manifestations.

The transcription factors ZEB2 and T-bet cooperate to program cytotoxic T cell terminal differentiation in response to LCMV viral infection.

The transcription factor T-bet is critical for cytotoxic T lymphocyte (CTL) differentiation, but it is unclear how it operates in a graded manner in the formation of both terminal effector and memory precursor cells during viral infection. We find that, at high concentrations, T-bet induced expression of Zeb2 mRNA, which then triggered CTLs to adopt terminally differentiated states. ZEB2 and T-bet cooperate to switch on a terminal CTL differentiation program, while simultaneously repressing genes necessary for central memory CTL development. Chromatin immunoprecipitation sequencing showed that a large proportion of these genes were bound by T-bet, and this binding was altered by ZEB2 deficiency. Furthermore, T-bet overexpression could not fully bypass ZEB2 function. Thus, the coordinated actions of T-bet and ZEB2 outline a novel genetic pathway that forces commitment of CTLs to terminal differentiation, thereby restricting their memory cell potential.

Production of IL-10 by CD4(+) regulatory T cells during the resolution of infection promotes the maturation of memory CD8(+) T cells.

Memory CD8(+) T cells are critical for host defense upon reexposure to intracellular pathogens. We found that interleukin 10 (IL-10) derived from CD4(+) regulatory T cells (Treg cells) was necessary for the maturation of memory CD8(+) T cells following acute infection with lymphocytic choriomeningitis virus (LCMV). Treg cell-derived IL-10 was most important during the resolution phase, calming inflammation and the activation state of dendritic cells. Adoptive transfer of IL-10-sufficient Treg cells during the resolution phase 'restored' the maturation of memory CD8(+) T cells in IL-10-deficient mice. Our data indicate that Treg cell-derived IL-10 is needed to insulate CD8(+) T cells from inflammatory signals, and reveal that the resolution phase of infection is a critical period that influences the quality and function of developing memory CD8(+) T cells.

IL-7-Induced Glycerol Transport and TAG Synthesis Promotes Memory CD8+ T Cell Longevity.

Memory T cells are critical for long-term immunity against reinfection and require interleukin-7 (IL-7), but the mechanisms by which IL-7 controls memory T cell survival, particularly metabolic fitness, remain elusive. We discover that IL-7 induces expression of the glycerol channel aquaporin 9 (AQP9) in virus-specific memory CD8+ T cells, but not naive cells, and that AQP9 is vitally required for their long-term survival. AQP9 deficiency impairs glycerol import into memory CD8+ T cells for fatty acid esterification and triglyceride (TAG) synthesis and storage. These defects can be rescued by ectopic expression of TAG synthases, which restores lipid stores and memory T cell survival. Finally, we find that TAG synthesis is a central component of IL-7-mediated survival of human and mouse memory CD8+T cells. This study uncovers the metabolic mechanisms by which IL-7 tailors the metabolism of memory T cells to promote their longevity and fast response to rechallenge.