Gene-level alignment of single-cell trajectories.

Single-cell data analysis can infer dynamic changes in cell populations, for example across time, space or in response to perturbation, thus deriving pseudotime trajectories. Current approaches comparing trajectories often use dynamic programming but are limited by assumptions such as the existence of a definitive match. Here we describe Genes2Genes, a Bayesian information-theoretic dynamic programming framework for aligning single-cell trajectories. It is able to capture sequential matches and mismatches of individual genes between a reference and query trajectory, highlighting distinct clusters of alignment patterns. Across both real world and simulated datasets, it accurately inferred alignments and demonstrated its utility in disease cell-state trajectory analysis. In a proof-of-concept application, Genes2Genes revealed that T cells differentiated in vitro match an immature in vivo state while lacking expression of genes associated with TNF signaling. This demonstrates that precise trajectory alignment can pinpoint divergence from the in vivo system, thus guiding the optimization of in vitro culture conditions.

Expression profiling of cerebrospinal fluid identifies dysregulated antiviral mechanisms in multiple sclerosis.

Despite the overwhelming evidence that multiple sclerosis is an autoimmune disease, relatively little is known about the precise nature of the immune dysregulation underlying the development of the disease. Reasoning that the CSF from patients might be enriched for cells relevant in pathogenesis, we have completed a high-resolution single-cell analysis of 96 732 CSF cells collected from 33 patients with multiple sclerosis (n = 48 675) and 48 patients with other neurological diseases (n = 48 057). Completing comprehensive cell type annotation, we identified a rare population of CD8+ T cells, characterized by the upregulation of inhibitory receptors, increased in patients with multiple sclerosis. Applying a Multi-Omics Factor Analysis to these single-cell data further revealed that activity in pathways responsible for controlling inflammatory and type 1 interferon responses are altered in multiple sclerosis in both T cells and myeloid cells. We also undertook a systematic search for expression quantitative trait loci in the CSF cells. Of particular interest were two expression quantitative trait loci in CD8+ T cells that were fine mapped to multiple sclerosis susceptibility variants in the viral control genes ZC3HAV1 (rs10271373) and IFITM2 (rs1059091). Further analysis suggests that these associations likely reflect genetic effects on RNA splicing and cell-type specific gene expression respectively. Collectively, our study suggests that alterations in viral control mechanisms might be important in the development of multiple sclerosis.

Enterotoxins can support CAR T cells against solid tumors.

Responses of solid tumors to chimeric antigen receptor (CAR) T cell therapy are often minimal. This is potentially due to a lack of sustained activation and proliferation of CAR T cells when encountering antigen in a profoundly immunosuppressive tumor microenvironment. In this study, we investigate if inducing an interaction between CAR T cells and antigen-presenting cells (APCs) in lymphoid tissue, away from an immunosuppressive microenvironment, could enhance solid-tumor responses. We combined CAR T cell transfer with the bacterial enterotoxin staphylococcal enterotoxin-B (SEB), which naturally links a proportion of T cell receptor (TCR) Vß subtypes to MHC-II, present on APCs. CAR T cell proliferation and function was significantly enhanced by SEB. Solid tumor-growth inhibition in mice was increased when CAR T cells were administered in combination with SEB. CAR T cell expansion in lymphoid tissue was demonstrated, and inhibition of lymphocyte egress from lymph nodes using FTY720 abrogated the benefit of SEB. We also demonstrate that a bispecific antibody, targeting a c-Myc tag on CAR T cells and cluster of differentiation 40 (CD40), could also enhance CAR T cell activity and mediate increased antitumor activity of CAR T cells. These model systems serve as proof-of-principle that facilitating the interaction of CAR T cells with APCs can enhance their ability to mediate antitumor activity.

A Novel Inhaled Dry-Powder Formulation of Ribavirin Allows for Efficient Lung Delivery in Healthy Participants and Those with Chronic Obstructive Pulmonary Disease in a Phase 1 Study.

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung condition, causing progressive decline in lung function leading to premature death. Acute exacerbations in COPD patients are predominantly associated with respiratory viruses. Ribavirin is a generic broad-spectrum antiviral agent that could be used for treatment of viral respiratory infections in COPD. Using the Particle Replication In Nonwetting Templates (PRINT) technology, which produces dry-powder particles of uniform shape and size, two new inhaled formulations of ribavirin (ribavirin-PRINT-CFI and ribavirin-PRINT-IP) were developed for efficient delivery to the lung and to minimize bystander exposure. Ribavirin-PRINT-CFI was well tolerated in healthy participants after single dosing and ribavirin-PRINT-IP was well tolerated in healthy and COPD participants after single and repeat dosing. Ribavirin-PRINT-CFI was replaced with ribavirin-PRINT-IP since the latter formulation was found to have improved physicochemical properties and it had a higher ratio of active drug to excipient per unit dose. Ribavirin concentrations were measured in lung epithelial lining fluid in both healthy and COPD participants and achieved target concentrations. Both formulations were rapidly absorbed with approximately dose proportional pharmacokinetics in plasma. Exposure to bystanders was negligible based on both the plasma and airborne ribavirin concentrations with the ribavirin-PRINT-IP formulation. Thus, ribavirin-PRINT-IP allowed for an efficient and convenient delivery of ribavirin to the lungs while minimizing systemic exposure. Further clinical investigations would be required to demonstrate ribavirin-PRINT-IP antiviral characteristics and impact on COPD viral-induced exacerbations. (The clinical trials discussed in this study have been registered at ClinicalTrials.gov under identifiers NCT03243760 and NCT03235726.).

Randomized Trial of Once-Daily Fluticasone Furoate in Children with Inadequately Controlled Asthma.

OBJECTIVE: To evaluate the dose-response, efficacy, and safety of fluticasone furoate (FF; 25 µg, 50 µg, and 100 µg), administered once daily in the evening during a 12-week treatment period to children with inadequately controlled asthma. STUDY DESIGN: This was a Phase IIb, multicenter, stratified, randomized, double-blind, double-dummy, parallel-group, placebo- and active-controlled study in children aged 5-11 years with inadequately controlled asthma. The study comprised a 4-week run-in period, 12-week treatment period, and 1-week follow-up period. Children were randomized to receive either placebo once daily, fluticasone propionate (FP) 100 µg twice daily, FF 25 µg, FF 50 µg, or FF 100 µg each once daily in the evening. Primary endpoint was the mean change from baseline in daily morning peak expiratory flow (PEF) averaged over weeks 1-12. Adverse events (AEs) also were investigated. RESULTS: In total, 593 children were included in the intent-to-treat population. The difference vs placebo in change from baseline daily morning PEF averaged over weeks 1-12 was statistically significant for the FF 25, FF 50, FF 100, and FP 100 groups (18.6 L/min, 19.5 L/min, 12.5 L/min, and 14.0 L/min, respectively; P < .001 for all). The incidence of AEs was greater in the FF groups (32%-36%) than in the placebo group (29%); the most frequent AE was cough. CONCLUSION: FF and FP resulted in significant improvements in morning PEF compared with placebo, suggesting that they are effective treatments for children with inadequately controlled asthma. All treatments were well tolerated; no new safety concerns were identified. TRIAL REGISTRATION: ClinicalTrials.gov:NCT01563029.

Randomised trial of once-daily vilanterol in children with asthma on inhaled corticosteroid therapy.

BACKGROUND: Inhaled corticosteroids (ICS) are effective maintenance treatments for childhood asthma; however, many children remain uncontrolled. Vilanterol (VI) is an inhaled long-acting beta-2 agonist which, in combination with the ICS fluticasone furoate, is being explored as a once-daily treatment for asthma in children. We evaluated the dose-response, efficacy, and safety of once-daily VI (6.25 µg, 12.5 µg and 25 µg) administered in the evening over 4 weeks, on background fluticasone propionate (FP) in children with asthma inadequately controlled on ICS. METHODS: This was a Phase IIb, multicentre, randomised, double-blind, parallel-group, placebo-controlled study in children ages 5-11 years with persistent asthma on ICS and as-needed short-acting beta-agonist. The study comprised a 4-week run-in, 4-week treatment period, and 1-week follow-up. From study start, children replaced their current ICS with open-label FP 100 µg twice daily. Children were randomised to receive placebo, VI 6.25 µg, VI 12.5 µg or VI 25 µg once daily. Primary endpoint was treatment difference between VI 25 and placebo groups in mean change from baseline in evening peak expiratory flow averaged over the 4-week treatment. Secondary endpoints included change from baseline in trough forced expiratory volume in one second (FEV1) at Week 4 and change from baseline in percentage of rescue-free and symptom-free 24-h periods. Safety assessments included incidence of adverse events (AEs) and asthma exacerbations. RESULTS: In total, 456 children comprised the intention-to-treat population. The adjusted treatment difference between VI 25 and placebo groups for the primary endpoint was not statistically significant (p = 0.227) so no statistical inference was made for other VI dose comparisons or other endpoints. No difference in change from baseline in trough FEV1 was observed for any VI treatments versus placebo; however, VI 25 resulted in an additional 0.6 rescue-free days and 0.7 symptom-free days per week versus placebo. The incidence of AEs was slightly higher in the VI groups (28-33 %) versus placebo (22 %). Nine children experienced asthma exacerbations during the treatment period. CONCLUSION: VI plus FP did not result in significant improvements in lung function versus placebo plus FP, but was well tolerated at all doses assessed. TRIAL REGISTRATION: NCT01573767 (ClinicalTrials.gov).

Precise identification of cell states altered in disease using healthy single-cell references.

Joint analysis of single-cell genomics data from diseased tissues and a healthy reference can reveal altered cell states. We investigate whether integrated collections of data from healthy individuals (cell atlases) are suitable references for disease-state identification and whether matched control samples are needed to minimize false discoveries. We demonstrate that using a reference atlas for latent space learning followed by differential analysis against matched controls leads to improved identification of disease-associated cells, especially with multiple perturbed cell types. Additionally, when an atlas is available, reducing control sample numbers does not increase false discovery rates. Jointly analyzing data from a COVID-19 cohort and a blood cell atlas, we improve detection of infection-related cell states linked to distinct clinical severities. Similarly, we studied disease states in pulmonary fibrosis using a healthy lung atlas, characterizing two distinct aberrant basal states. Our analysis provides guidelines for designing disease cohort studies and optimizing cell atlas use.

A spatially resolved atlas of the human lung characterizes a gland-associated immune niche.

Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org ). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new 'gland-associated immune niche' has implications for respiratory health.

Early human lung immune cell development and its role in epithelial cell fate.

Studies of human lung development have focused on epithelial and mesenchymal cell types and function, but much less is known about the developing lung immune cells, even though the airways are a major site of mucosal immunity after birth. An unanswered question is whether tissue-resident immune cells play a role in shaping the tissue as it develops in utero. Here, we profiled human embryonic and fetal lung immune cells using scRNA-seq, smFISH, and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including innate lymphoid cells, natural killer cells, myeloid cells, and lineage progenitors. By the canalicular stage, we detected naive T lymphocytes expressing high levels of cytotoxicity genes and the presence of mature B lymphocytes, including B-1 cells. Our analysis suggests that fetal lungs provide a niche for full B cell maturation. Given the presence and diversity of immune cells during development, we also investigated their possible effect on epithelial maturation. We found that IL-1ß drives epithelial progenitor exit from self-renewal and differentiation to basal cells in vitro. In vivo, IL-1ß-producing myeloid cells were found throughout the lung and adjacent to epithelial tips, suggesting that immune cells may direct human lung epithelial development.

An integrated cell atlas of the lung in health and disease.

Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1+ profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas.

Cross-talk between tumors at anatomically distinct sites.

Cancer tissue is not homogenous, and individual metastases at different anatomical locations can differ from the primary tumor and from one another in both their morphology and cellular composition, even within an individual patient. Tumors are composed of cancer cells and a range of other cell types, which, together with a variety of secreted molecules, collectively comprise the tumor microenvironment (TME). Cells of the TME can communicate with each other and with distant tissues in a form of molecular cross-talk to influence their growth and function. Cross-talk between cancer cells and local immune cells is well described and can lead to the induction of local immunosuppression. Recently, it has become apparent that tumors located remotely from each other, can engage in cross-talk that can influence their responsiveness to various therapies, including immunotherapy. In this article, we review studies that describe how tumors systemically communicate with distant tissues through motile cells, extracellular vesicles, and secreted molecules that can affect their function. In addition, we summarize evidence from mouse studies and the clinic that indicate an ability of some tumors to influence the progression and therapeutic responses of other tumors in different anatomical locations.

Understanding T cell phenotype for the design of effective chimeric antigen receptor T cell therapies.

Rapid advances in immunotherapy have identified adoptive cell transfer as one of the most promising approaches for the treatment of cancers. Large numbers of cancer reactive T lymphocytes can be generated ex vivo from patient blood by genetic modification to express chimeric antigen receptors (CAR) specific for tumor-associated antigens. CAR T cells can respond strongly against cancer cells, and adoptive transferred CAR T cells can induce dramatic responses against certain types of cancers. The ability of T cells to respond against disease depends on their ability to localize to sites, persist and exert functions, often in an immunosuppressive microenvironment, and these abilities are reflected in their phenotypes. There is currently intense interest in generating CAR T cells possessing the ideal phenotypes to confer optimal antitumor activity. In this article, we review T cell phenotypes for trafficking, persistence and function, and discuss how culture conditions and genetic makeups can be manipulated to achieve the ideal phenotypes for antitumor activities.

A Histone Deacetylase Inhibitor, Panobinostat, Enhances Chimeric Antigen Receptor T-cell Antitumor Effect Against Pancreatic Cancer.

PURPOSE: In this article, we describe a combination chimeric antigen receptor (CAR) T-cell therapy that eradicated the majority of tumors in two immunocompetent murine pancreatic cancer models and a human pancreatic cancer xenograft model. EXPERIMENTAL DESIGN: We used a dual-specific murine CAR T cell that expresses a CAR against the Her2 tumor antigen, and a T-cell receptor (TCR) specific for gp100. As gp100 is also known as pMEL, the dual-specific CAR T cells are thus denoted as CARaMEL cells. A vaccine containing live vaccinia virus coding a gp100 minigene (VV-gp100) was administered to the recipient mice to stimulate CARaMEL cells. The treatment also included the histone deacetylase inhibitor panobinostat (Pano). RESULTS: The combination treatment enabled significant suppression of Her2+ pancreatic cancers leading to the eradication of the majority of the tumors. Besides inducing cancer cell apoptosis, Pano enhanced CAR T-cell gene accessibility and promoted CAR T-cell differentiation into central memory cells. To test the translational potential of this approach, we established a method to transduce human T cells with an anti-Her2 CAR and a gp100-TCR. The exposure of the human T cells to Pano promoted a T-cell central memory phenotype and the combination treatment of human CARaMEL cells and Pano eradicated human pancreatic cancer xenografts in mice. CONCLUSIONS: We propose that patients with pancreatic cancer could be treated using a scheme that contains dual-specific CAR T cells, a vaccine that activates the dual-specific CAR T cells through their TCR, and the administration of Pano.

Combined BRAF, MEK, and CDK4/6 Inhibition Depletes Intratumoral Immune-Potentiating Myeloid Populations in Melanoma.

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a-/-Pten-/- melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.

MAIT cells regulate NK cell-mediated tumor immunity.

The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.

Primary and metastatic breast tumors cross-talk to influence immunotherapy responses.

The presence of a tumor can alter host immunity systematically. The immune-tumor interaction in one site may impact the local immune microenvironment in distal tissues through the circulation, and therefore influence the efficacy of immunotherapies to distant metastases. Improved understanding of the immune-tumor interactions during immunotherapy treatment in a metastatic setting may enhance the efficacy of current immunotherapies. Here we investigate the response to αPD-1/αCTLA4 and trimAb (αDR5, α4-1BB, αCD40) of 67NR murine breast tumors grown simultaneously in the mammary fat pad (MFP) and lung, a common site of breast cancer metastasis, and compared to tumors grown in isolation. Lung tumors present in isolation were resistant to both therapies. However, in MFP and lung tumor-bearing mice, the presence of a MFP tumor could increase lung tumor response to immunotherapy and decrease the number of lung metastases, leading to complete eradication of lung tumors in a proportion of mice. The MFP tumor influence on lung metastases was mediated by CD8+ T cells, as CD8+ T cell depletion abolished the difference in lung metastases. Furthermore, mice with concomitant MFP and lung tumors had increased tumor specific, effector CD8+ T cells infiltration in the lungs. Thus, we propose a model where tumors in an immunogenic location can give rise to systemic anti-tumor CD8+ T cell responses that could be utilized to target metastatic tumors. These results highlight the requirement for clinical consideration of cross-talk between primary and metastatic tumors for effective immunotherapy for cancers otherwise resistant to immunotherapy.

Enhancing chimeric antigen receptor T-cell immunotherapy against cancer using a nanoemulsion-based vaccine targeting cross-presenting dendritic cells.

OBJECTIVES: Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells is a form of cancer immunotherapy that has achieved remarkable efficacy in patients with some haematological cancers. However, challenges remain in CAR T-cell treatment of solid tumours because of tumour-mediated immunosuppression. METHODS: We have demonstrated that CAR T-cell stimulation through T-cell receptors (TCRs) in vivo can generate durable responses against solid tumours in a variety of murine models. Since Clec9A-targeting tailored nanoemulsion (Clec9A-TNE) vaccine enhances antitumour immune responses through selective activation of Clec9A+ cross-presenting dendritic cells (DCs), we hypothesised that Clec9A-TNE could prime DCs for antigen presentation to CAR T cells through TCRs and thus improve CAR T-cell responses against solid tumours. To test this hypothesis, we used CAR T cells expressing transgenic TCRs specific for ovalbumin (OVA) peptides SIINFEKL (CAROTI) or OVA323-339 (CAROTII). RESULTS: We demonstrated that the Clec9A-TNEs encapsulating full-length recombinant OVA protein (OVA-Clec9A-TNE) improved CAROT T-cell proliferation and inflammatory cytokine secretion in vitro. Combined treatment using the OVA-Clec9A-TNE and CAROT cells resulted in durable responses and some rejections of tumours in immunocompetent mice. Tumour regression was accompanied by enhanced CAROT cell proliferation and infiltration into the tumours. CONCLUSION: Our study presents Clec9A-TNE as a prospective avenue to enhance CAR T-cell efficacy for solid cancers.

Novel combination immunotherapy for pancreatic cancer: potent anti-tumor effects with CD40 agonist and interleukin-15 treatment.

OBJECTIVES: With the poorest 5-year survival of all cancers, improving treatment for pancreatic cancer is one of the biggest challenges in cancer research. We sought to explore the potential of combining both priming and activation of the immune system. To achieve this, we combined a CD40 agonist with interleukin-15 and tested its potential in pancreatic cancer. METHODS: Response to this combination regimen was assessed in pancreatic ductal adenocarcinoma mouse models, and a thorough analysis of the tumor microenvironment was performed. RESULTS: We demonstrated profound reduction in tumor growth and increased survival of mice with the majority of mice being cured when both agents were combined, including an unprecedented 8-fold dose reduction of CD40 agonist without losing any efficacy. RNAseq analysis showed involvement of natural killer (NK) cell- and T-cell-mediated anti-tumor responses and the importance of antigen-presenting cell pathways. This combination resulted in enhanced infiltration of tumors by both T cells and NK cells, as well as a striking increase in the ratio of CD8+ T cells over Tregs. We also observed a significant increase in numbers of dendritic cells (DCs) in tumor-draining lymph nodes, particularly CD103+ DCs with cross-presentation potential. A critical role for CD8+ T cells and involvement of NK cells in the anti-tumor effect was highlighted. Importantly, strong immune memory was established, with an increase in memory CD8+ T cells only when both interleukin-15 and the CD40 agonist were combined. CONCLUSION: These novel preclinical data support initiation of a first-in-human clinical trial with this combination immunotherapy strategy in pancreatic cancer.

Macrophage-Derived CXCL9 and CXCL10 Are Required for Antitumor Immune Responses Following Immune Checkpoint Blockade.

PURPOSE: Response rates to immune checkpoint blockade (ICB; anti-PD-1/anti-CTLA-4) correlate with the extent of tumor immune infiltrate, but the mechanisms underlying the recruitment of T cells following therapy are poorly characterized. A greater understanding of these processes may see the development of therapeutic interventions that enhance T-cell recruitment and, consequently, improved patient outcomes. We therefore investigated the chemokines essential for immune cell recruitment and subsequent therapeutic efficacy of these immunotherapies. EXPERIMENTAL DESIGN: The chemokines upregulated by dual PD-1/CTLA-4 blockade were assessed using NanoString-based analysis with results confirmed at the protein level by flow cytometry and cytometric bead array. Blocking/neutralizing antibodies confirmed the requirement for key chemokines/cytokines and immune effector cells. Results were confirmed in patients treated with immune checkpoint inhibitors using single-cell RNA-sequencing (RNA-seq) and paired survival analyses. RESULTS: The CXCR3 ligands, CXCL9 and CXCL10, were significantly upregulated following dual PD-1/CTLA-4 blockade and both CD8+ T-cell infiltration and therapeutic efficacy were CXCR3 dependent. In both murine models and patients undergoing immunotherapy, macrophages were the predominant source of CXCL9 and their depletion abrogated CD8+ T-cell infiltration and the therapeutic efficacy of dual ICB. Single-cell RNA-seq analysis of patient tumor-infiltrating lymphocytes (TIL) revealed that CXCL9/10/11 was predominantly expressed by macrophages following ICB and we identified a distinct macrophage signature that was associated with positive responses to ICB. CONCLUSIONS: These data underline the fundamental importance of macrophage-derived CXCR3 ligands for the therapeutic efficacy of ICB and highlight the potential of manipulating this axis to enhance patient responses.