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Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma (GBM) trials. Here, we show that regulatory T (Treg) cells play a key role in GBM resistance to ICBs in experimental gliomas. Targeting glucocorticoid-induced TNFR-related receptor (GITR) in Treg cells using an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, alleviates Treg cell-mediated suppression of anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. The reprogrammed GBM-infiltrating Treg cells express genes associated with a Th1 response signature, produce IFNγ, and acquire cytotoxic activity against GBM tumor cells while losing their suppressive function. αGITR and αPD1 antibodies increase survival benefit in three experimental GBM models, with a fraction of cohorts exhibiting complete tumor eradication and immune memory upon tumor re-challenge. Moreover, αGITR and αPD1 synergize with the standard of care treatment for newly-diagnosed GBM, enhancing the cure rates in these GBM models. Glioblastomas (GBM) are frequently resistant to immune checkpoint blockade therapy. Here the authors show that treatment with an agonistic anti-GITR antibody converts tumor infiltrating regulatory T cells to effector cells, overcoming resistance to PD1 blockade in preclinical models of GBM.

Barrier tissue dysfunction is a fundamental feature of chronic human inflammatory diseases 1 . Specialized subsets of epithelial cells—including secretory and ciliated cells—differentiate from basal stem cells to collectively protect the upper airway 2 – 4 . Allergic inflammation can develop from persistent activation 5 of type 2 immunity 6 in the upper airway, resulting in chronic rhinosinusitis, which ranges in severity from rhinitis to severe nasal polyps 7 . Basal cell hyperplasia is a hallmark of severe disease 7 – 9 , but it is not known how these progenitor cells 2 , 10 , 11 contribute to clinical presentation and barrier tissue dysfunction in humans. Here we profile primary human surgical chronic rhinosinusitis samples (18,036 cells, n  = 12) that span the disease spectrum using Seq-Well for massively parallel single-cell RNA sequencing 12 , report transcriptomes for human respiratory epithelial, immune and stromal cell types and subsets from a type 2 inflammatory disease, and map key mediators. By comparison with nasal scrapings (18,704 cells, n  = 9), we define signatures of core, healthy, inflamed and polyp secretory cells. We reveal marked differences between the epithelial compartments of the non-polyp and polyp cellular ecosystems, identifying and validating a global reduction in cellular diversity of polyps characterized by basal cell hyperplasia, concomitant decreases in glandular cells, and phenotypic shifts in secretory cell antimicrobial expression. We detect an aberrant basal progenitor differentiation trajectory in polyps, and propose cell-intrinsic 13 , epigenetic 14 , 15 and extrinsic factors 11 , 16 , 17 that lock polyp basal cells into this uncommitted state. Finally, we functionally demonstrate that ex vivo cultured basal cells retain intrinsic memory of IL-4/IL-13 exposure, and test the potential for clinical blockade of the IL-4 receptor α-subunit to modify basal and secretory cell states in vivo. Overall, we find that reduced epithelial diversity stemming from functional shifts in basal cells is a key characteristic of type 2 immune-mediated barrier tissue dysfunction. Our results demonstrate that epithelial stem cells may contribute to the persistence of human disease by serving as repositories for allergic memories. Single-cell RNA sequencing is used to characterize cell types in nasal tissues from human patients with chronic rhinosinusitis, revealing a role for tissue stem cells in allergic inflammatory memory.

The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (Combi-GEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas–based knockouts and RNA-interference–based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.

We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function—including Granzyme B—are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology. Existing single-cell RNA-seq methods provide the transcriptome of a cellular phenotype at a single time point. Here, Kimmerling et al . present a microfluidic platform that enables off-chip single-cell RNA-seq after multigenerational lineage tracking under controlled culture conditions.

Background Human immunity relies on the coordinated responses of many cellular subsets and functional states. Inter-individual variations in cellular composition and communication could thus potentially alter host protection. Here, we explore this hypothesis by applying single-cell RNA-sequencing to examine viral responses among the dendritic cells (DCs) of three elite controllers (ECs) of HIV-1 infection. Results To overcome the potentially confounding effects of donor-to-donor variability, we present a generally applicable computational framework for identifying reproducible patterns in gene expression across donors who share a unifying classification. Applying it, we discover a highly functional antiviral DC state in ECs whose fractional abundance after in vitro exposure to HIV-1 correlates with higher CD4 + T cell counts and lower HIV-1 viral loads, and that effectively primes polyfunctional T cell responses in vitro. By integrating information from existing genomic databases into our reproducibility-based analysis, we identify and validate select immunomodulators that increase the fractional abundance of this state in primary peripheral blood mononuclear cells from healthy individuals in vitro. Conclusions Overall, our results demonstrate how single-cell approaches can reveal previously unappreciated, yet important, immune behaviors and empower rational frameworks for modulating systems-level immune responses that may prove therapeutically and prophylactically useful.

Mucosal associated invariant T (MAIT) cells are a class of innate-like T cells that utilize a semi-invariant αβ T cell receptor to recognize small molecule ligands produced by bacteria and fungi. Despite growing evidence that immune cells at mucosal surfaces are often phenotypically and functionally distinct from those in the peripheral circulation, knowledge about the characteristics of MAIT cells at the lung mucosal surface, the site of exposure to respiratory pathogens, is limited. HIV infection has been shown to have a profound effect on the number and function of MAIT cells in the peripheral blood, but its effect on lung mucosal MAIT cells is unknown. We examined the phenotypic, functional, and transcriptomic features of major histocompatibility complex (MHC) class I-related (MR1)-restricted MAIT cells from the peripheral blood and bronchoalveolar compartments of otherwise healthy individuals with latent Mycobacterium tuberculosis ( Mtb ) infection who were either HIV uninfected or HIV infected. Peripheral blood MAIT cells consistently co-expressed typical MAIT cell surface markers CD161 and CD26 in HIV-negative individuals, while paired bronchoalveolar MAIT cells displayed heterogenous expression of these markers. Bronchoalveolar MAIT cells produced lower levels of pro-inflammatory cytokine IFN-γ and expressed higher levels of co-inhibitory markers PD-1 and TIM-3 than peripheral MAIT cells. HIV infection resulted in decreased frequencies and pro-inflammatory function of peripheral blood MAIT cells, while in the bronchoalveolar compartment MAIT cell frequency was decreased but phenotype and function were not significantly altered. Single-cell transcriptomic analysis demonstrated greater heterogeneity among bronchoalveolar compared to peripheral blood MAIT cells and suggested a distinct subset in the bronchoalveolar compartment. The transcriptional features of this bronchoalveolar subset were associated with MAIT cell tissue repair functions. In summary, we found previously undescribed phenotypic and transcriptional heterogeneity of bronchoalveolar MAIT cells in HIV-negative people. In HIV infection, we found numeric depletion of MAIT cells in both anatomical compartments but preservation of the novel phenotypic and transcriptional features of bronchoalveolar MAIT cells.

Lymphoid tissues are an important HIV reservoir site that persists in the face of antiretroviral therapy and natural immunity. Targeting these reservoirs by harnessing the antiviral activity of local tissue-resident memory (T RM ) CD8 + T-cells is of great interest, but limited data exist on T RM -like cells within lymph nodes of people living with HIV (PLWH). Here, we studied tonsil CD8 + T-cells obtained from PLWH and uninfected controls from South Africa. We show that these cells are preferentially located outside the germinal centers (GCs), the main reservoir site for HIV, and display a low cytolytic and a transcriptionally T RM -like profile distinct from blood CD8 + T-cells. In PLWH, CD8 + T RM -like cells are expanded and adopt a more cytolytic, activated, and exhausted phenotype not reversed by antiretroviral therapy (ART). This phenotype was enhanced in HIV-specific CD8 + T-cells from tonsils compared to matched blood suggesting a higher antigen burden in tonsils. Single-cell transcriptional and clonotype resolution showed that these HIV-specific CD8 + T-cells in the tonsils express heterogeneous signatures of T-cell activation, clonal expansion, and exhaustion ex-vivo. Interestingly, this signature was absent in a natural HIV controller, who expressed lower PD-1 and CXCR5 levels and reduced transcriptional evidence of T-cell activation, exhaustion, and cytolytic activity. These data provide important insights into lymphoid tissue-derived HIV-specific CD8 + T RM -like phenotypes in settings of HIV remission and highlight their potential for immunotherapy and targeting of the HIV reservoirs.

Background The Lentivirus human immunodeficiency virus (HIV) causes chronic inflammation and AIDS in humans, with variable rates of disease progression between individuals driven by both host and viral factors. Similarly, simian lentiviruses vary in their pathogenicity based on characteristics of both the host species and the virus strain, yet the immune underpinnings that drive differential Lentivirus pathogenicity remain incompletely understood. Methods We profile immune responses in a unique model of differential lentiviral pathogenicity where pig-tailed macaques are infected with highly genetically similar variants of SIV that differ in virulence. We apply longitudinal single-cell transcriptomics to this cohort, along with single-cell resolution cell-cell communication techniques, to understand the immune mechanisms underlying lentiviral pathogenicity. Results Compared to a minimally pathogenic lentiviral variant, infection with a highly pathogenic variant results in a more delayed, broad, and sustained activation of inflammatory pathways, including an extensive global interferon signature. Conversely, individual cells infected with highly pathogenic Lentivirus upregulated fewer interferon-stimulated genes at a lower magnitude, indicating that highly pathogenic Lentivirus has evolved to partially escape from interferon responses. Further, we identify CXCL10 and CXCL16 as important molecular drivers of inflammatory pathways specifically in response to highly pathogenic Lentivirus infection. Immune responses to highly pathogenic Lentivirus infection are characterized by amplifying regulatory circuits of pro-inflammatory cytokines with dense longitudinal connectivity. Conclusions Our work presents a model of lentiviral pathogenicity where failures in early viral control mechanisms lead to delayed, sustained, and amplifying pro-inflammatory circuits, which in turn drives disease progression.

Broadly neutralizing antibodies (bnAbs) against HIV-1 are an effective means of preventing transmission. To better understand the mechanisms by which HIV-specific bnAbs naturally develop, we investigated blood and lymphoid tissue in pediatric infection, since potent bnAbs develop with greater frequency in children than adults. As in adults, the frequency of circulating effector T-follicular helper cells (T ) in HIV infected, treatment naïve children correlates with neutralization breadth. However, major differences between children and adults were also observed both in circulation, and in a small number of tonsil samples. In children, T cells are significantly more abundant, both in blood and in lymphoid tissue germinal centers, than in adults. Second, HIV-specific T cells are more frequent in pediatric than in adult lymphoid tissue and secrete the signature cytokine IL-21, which HIV-infected adults do not. Third, the enrichment of IL-21-secreting HIV-specific T in pediatric lymphoid tissue is accompanied by increased T regulation via more abundant regulatory follicular T-cells and HIV-specific CXCR5+ CD8 T-cells compared to adults. The relationship between regulation and neutralization breadth is also observed in the pediatric PBMC samples and correlates with neutralization breadth. Matching neutralization data from lymphoid tissue samples is not available. However, the distinction between infected children and adults in the magnitude, quality and regulation of HIV-specific T responses is consistent with the superior ability of children to develop high-frequency, potent bnAbs. These findings suggest the possibility that the optimal timing for next generation vaccine strategies designed to induce high-frequency, potent bnAbs to prevent HIV infection in adults would be in childhood.

To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.