LGR5 expressing skin fibroblasts define a major cellular hub perturbed in scleroderma.

Systemic sclerosis (scleroderma, SSc) is an incurable autoimmune disease with high morbidity and mortality rates. Here, we conducted a population-scale single-cell genomic analysis of skin and blood samples of 56 healthy controls and 97 SSc patients at different stages of the disease. We found immune compartment dysfunction only in a specific subtype of diffuse SSc patients but global dysregulation of the stromal compartment, particularly in a previously undefined subset of LGR5+-scleroderma-associated fibroblasts (ScAFs). ScAFs are perturbed morphologically and molecularly in SSc patients. Single-cell multiome profiling of stromal cells revealed ScAF-specific markers, pathways, regulatory elements, and transcription factors underlining disease development. Systematic analysis of these molecular features with clinical metadata associates specific ScAF targets with disease pathogenesis and SSc clinical traits. Our high-resolution atlas of the sclerodermatous skin spectrum will enable a paradigm shift in the understanding of SSc disease and facilitate the development of biomarkers and therapeutic strategies.

Coupled scRNA-Seq and Intracellular Protein Activity Reveal an Immunosuppressive Role of TREM2 in Cancer.

Cell function and activity are regulated through integration of signaling, epigenetic, transcriptional, and metabolic pathways. Here, we introduce INs-seq, an integrated technology for massively parallel recording of single-cell RNA sequencing (scRNA-seq) and intracellular protein activity. We demonstrate the broad utility of INs-seq for discovering new immune subsets by profiling different intracellular signatures of immune signaling, transcription factor combinations, and metabolic activity. Comprehensive mapping of Arginase 1-expressing cells within tumor models, a metabolic immune signature of suppressive activity, discovers novel Arg1+ Trem2+ regulatory myeloid (Mreg) cells and identifies markers, metabolic activity, and pathways associated with these cells. Genetic ablation of Trem2 in mice inhibits accumulation of intra-tumoral Mreg cells, leading to a marked decrease in dysfunctional CD8+ T cells and reduced tumor growth. This study establishes INs-seq as a broadly applicable technology for elucidating integrated transcriptional and intra-cellular maps and identifies the molecular signature of myeloid suppressive cells in tumors.

Single-cell immunology: Past, present, and future.

The immune system is a complex, dynamic, and plastic ecosystem composed of multiple cell types that constantly sense and interact with their local microenvironment to protect from infection and maintain homeostasis. For over a century, great efforts and ingenuity have been applied to the characterization of immune cells and their microenvironments, but traditional marker-based and bulk technologies left key questions unanswered. In the past decade, the advent of single-cell genomic approaches has revolutionized our knowledge of the cellular and molecular makeup of the immune system. In this perspective, we outline the past, present, and future applications of single-cell genomics in immunology and discuss how the integration of multiomics at the single-cell level will pave the way for future advances in immunology research and clinical translation.

Multi-tissue single-cell analysis deconstructs the complex programs of mouse natural killer and type 1 innate lymphoid cells in tissues and circulation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are heterogenous innate lymphocytes broadly defined in mice as Lin-NK1.1+NKp46+ cells that express the transcription factor T-BET and produce interferon-γ. The ILC1 definition primarily stems from studies on liver and small intestinal populations. However, NK1.1+NKp46+ cells in the salivary glands, uterus, adipose, and other tissues exhibit nonuniform programs that differ from those of liver or intestinal ILC1s or NK cells. Here, we performed single-cell RNA sequencing on murine NK1.1+NKp46+ cells from blood, spleen, various tissues, and solid tumors. We identified gene expression programs of tissue-specific ILC1s, tissue-specific NK cells, and non-tissue-specific populations in blood, spleen, and other tissues largely corresponding to circulating cells. Moreover, we found that circulating NK cell programs were reshaped in tumor-bearing mice. Core programs of circulating and tumor NK cells paralleled conserved human NK cells signatures, advancing our understanding of the human NK-ILC1 spectrum.

Modeling T cell temporal response to cancer immunotherapy rationalizes development of combinatorial treatment protocols.

Successful immunotherapy relies on triggering complex responses involving T cell dynamics in tumors and the periphery. Characterizing these responses remains challenging using static human single-cell atlases or mouse models. To address this, we developed a framework for in vivo tracking of tumor-specific CD8+ T cells over time and at single-cell resolution. Our tools facilitate the modeling of gene program dynamics in the tumor microenvironment (TME) and the tumor-draining lymph node (tdLN). Using this approach, we characterize two modes of anti-programmed cell death protein 1 (PD-1) activity, decoupling induced differentiation of tumor-specific activated precursor cells from conventional type 1 dendritic cell (cDC1)-dependent proliferation and recruitment to the TME. We demonstrate that combining anti-PD-1 therapy with anti-4-1BB agonist enhances the recruitment and proliferation of activated precursors, resulting in tumor control. These data suggest that effective response to anti-PD-1 therapy is dependent on sufficient influx of activated precursor CD8+ cells to the TME and highlight the importance of understanding system-level dynamics in optimizing immunotherapies.

Fc glycoengineering of a PD-L1 antibody harnesses Fcγ receptors for increased antitumor efficacy.

FDA-approved anti-PD-L1 monoclonal antibodies (mAbs) bear the IgG1 isotype, whose scaffolds are either wild-type (e.g., avelumab) or Fc-mutated and lacking Fcγ receptor (FcγR) engagement (e.g., atezolizumab). It is unknown whether variation in the ability of the IgG1 Fc region to engage FcγRs renders mAbs with superior therapeutic activity. In this study, we used humanized FcγR mice to study the contribution of FcγR signaling to the antitumor activity of human anti-PD-L1 mAbs and to identify an optimal human IgG scaffold for PD-L1 mAbs. We observed similar antitumor efficacy and comparable tumor immune responses in mice treated with anti-PD-L1 mAbs with wild-type and Fc-mutated IgG scaffolds. However, in vivo antitumor activity of the wild-type anti-PD-L1 mAb avelumab was enhanced by combination treatment with an FcγRIIB-blocking antibody, which was co-administered to overcome the suppressor function of FcγRIIB in the tumor microenvironment (TME). We performed Fc glycoengineering to remove the fucose subunit from the Fc-attached glycan of avelumab to enhance its binding to the activating FcγRIIIA. Treatment with the Fc-afucosylated version of avelumab also enhanced antitumor activity and induced stronger antitumor immune responses compared with the parental IgG. The enhanced effect by afucosylated PD-L1 antibody was dependent on neutrophils and associated with decreased frequencies of PD-L1+ myeloid cells and increased infiltration of T cells in the TME. Our data reveal that the current design of FDA-approved anti-PD-L1 mAbs does not optimally harness FcγR pathways and suggest two strategies to enhance FcγR engagement to optimize anti-PD-L1 immunotherapy.

Spatial and Temporal Mapping of Breast Cancer Lung Metastases Identify TREM2 Macrophages as Regulators of the Metastatic Boundary.

Cancer mortality primarily stems from metastatic recurrence, emphasizing the urgent need for developing effective metastasis-targeted immunotherapies. To better understand the cellular and molecular events shaping metastatic niches, we used a spontaneous breast cancer lung metastasis model to create a single-cell atlas spanning different metastatic stages and regions. We found that premetastatic lungs are infiltrated by inflammatory neutrophils and monocytes, followed by the accumulation of suppressive macrophages with the emergence of metastases. Spatial profiling revealed that metastasis-associated immune cells were present in the metastasis core, with the exception of TREM2+ regulatory macrophages uniquely enriched at the metastatic invasive margin, consistent across both murine models and human patient samples. These regulatory macrophages (Mreg) contribute to the formation of an immune-suppressive niche, cloaking tumor cells from immune surveillance. Our study provides a compendium of immune cell dynamics across metastatic stages and niches, informing the development of metastasis-targeting immunotherapies. SIGNIFICANCE: Temporal and spatial single-cell analysis of metastasis stages revealed new players in modulating immune surveillance and suppression. Our study highlights distinct populations of TREM2 macrophages as modulators of the microenvironment in metastasis, and as the key immune determinant defining metastatic niches, pointing to myeloid checkpoints to improve therapeutic strategies. This article is featured in Selected Articles from This Issue, p. 2489.

Anti-CTLA-4 antibodies drive myeloid activation and reprogram the tumor microenvironment through FcγR engagement and type I interferon signaling.

Despite the clinical success of checkpoint inhibitors, a substantial gap still exists in our understanding of their mechanism of action. While antibodies to cytotoxic T lymphocyte-associated protein-4 (CTLA-4) were developed to block inhibitory signals in T cells, several recent studies have demonstrated that Fcγ receptor (FcγR)-dependent depletion of regulatory T cells (Treg) is critical for antitumor activity. Here, using single-cell RNA sequencing, we dissect the impact of anti-CTLA-4-blocking, Treg cell-depleting and FcR-engaging activity on the immune response within tumors. We observed a rapid remodeling of the innate immune landscape as early as 24 h after treatment. Using genetic Treg cell ablation models, we show that immune remodeling was not driven solely by Treg cell depletion or CTLA-4 blockade but mainly through FcγR engagement, downstream activation of type I interferon signaling and reduction of suppressive macrophages. Our findings indicate that FcγR engagement and innate immune remodeling are involved in successful anti-CTLA-4 treatment, supporting the development of optimized immunotherapy agents bearing these features.