Universal recording of immune cell interactions in vivo.

Immune cells rely on transient physical interactions with other immune and non-immune populations to regulate their function1. To study these 'kiss-and-run' interactions directly in vivo, we previously developed LIPSTIC (labelling immune partnerships by SorTagging intercellular contacts)2, an approach that uses enzymatic transfer of a labelled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ T helper cells and antigen-presenting cells, however. Here we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the steady-state cellular partners of regulatory T cells and identify germinal centre-resident T follicular helper cells on the basis of their ability to interact cognately with germinal centre B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalogue of the immune populations that physically interact with intestinal epithelial cells at the steady state and profile the evolution of the interactome of lymphocytic choriomeningitis virus-specific CD8+ T cells in multiple organs following systemic infection. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.

Lessons for the Next Generation of Scientists from the Second Annual Arthur and Sandra Irving Cancer Immunology Symposium.

The Arthur and Sandra Irving Cancer Immunology Symposium has been created as a platform for established cancer immunologists to mentor trainees and young investigators as they launch their research career in the field. By sharing their different paths to success, the senior faculty mentors provide an invaluable resource to support the development of the next generation of leaders in the cancer immunology community. This Commentary describes some of the key topics that were discussed during the 2022 symposium: scientific and career trajectory, leadership, mentoring, collaborations, and publishing. For each of these topics, established investigators discussed the elements that facilitate success in these areas as well as mistakes that can hinder progress. Herein, we outline the critical points raised in these discussions for establishing a successful independent research career. These points are highly relevant for the broader scientific community.

Genetic tracing reveals transcription factor Foxp3-dependent and Foxp3-independent functionality of peripherally induced Treg cells.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are an essential suppressive T cell lineage of dual origin: Foxp3 induction in thymocytes and mature CD4+ T cells gives rise to thymic (tTreg) and peripheral (pTreg) Treg cells, respectively. While tTreg cells suppress autoimmunity, pTreg cells enforce tolerance to food and commensal microbiota. However, the role of Foxp3 in pTreg cells and the mechanisms supporting their differentiation remain poorly understood. Here, we used genetic tracing to identify microbiota-induced pTreg cells and found that many of their distinguishing features were Foxp3 independent. Lineage-committed, microbiota-dependent pTreg-like cells persisted in the colon in the absence of Foxp3. While Foxp3 was critical for the suppression of a Th17 cell program, colitis, and mastocytosis, pTreg cells suppressed colonic effector T cell expansion in a Foxp3-independent manner. Thus, Foxp3 and the tolerogenic signals that precede and promote its expression independently confer distinct facets of pTreg functionality.

Ectopic activation of the miR-200c-EpCAM axis enhances antitumor T cell responses in models of adoptive cell therapy.

Adoptive T cell therapy (ACT) is a promising strategy for treating cancer, but it often fails because of cell intrinsic regulatory programs that limit the degree or duration of T cell function. In this study, we found that ectopic expression of microRNA-200c (miR-200c) markedly enhanced the antitumor activity of CD8+ cytotoxic T lymphocytes (CTLs) during ACT in multiple mouse models. CTLs transduced with miR-200c exhibited reduced apoptosis during engraftment and enhanced in vivo persistence, accompanied by up-regulation of the transcriptional regulator T cell factor 1 (TCF1) and the inflammatory cytokine tumor necrosis factor (TNF). miR-200c elicited these changes by suppressing the transcription factor Zeb1 and thereby inducing genes characteristic of epithelial cells. Overexpression of one of these genes, Epcam, was sufficient to augment therapeutic T cell responses against both solid and liquid tumors. These results identify the miR-200c­EpCAM axis as an avenue for improving ACT and demonstrate that select genetic perturbations can produce phenotypically distinct T cells with advantageous therapeutic properties.

A unified atlas of CD8 T cell dysfunctional states in cancer and infection.

CD8 T cells play an essential role in defense against viral and bacterial infections and in tumor immunity. Deciphering T cell loss of functionality is complicated by the conspicuous heterogeneity of CD8 T cell states described across experimental and clinical settings. By carrying out a unified analysis of over 300 assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) experiments from 12 studies of CD8 T cells in cancer and infection, we defined a shared differentiation trajectory toward dysfunction and its underlying transcriptional drivers and revealed a universal early bifurcation of functional and dysfunctional T cell states across models. Experimental dissection of acute and chronic viral infection using single-cell ATAC (scATAC)-seq and allele-specific single-cell RNA (scRNA)-seq identified state-specific drivers and captured the emergence of similar TCF1+ progenitor-like populations at an early branch point, at which functional and dysfunctional T cells diverge. Our atlas of CD8 T cell states will facilitate mechanistic studies of T cell immunity and translational efforts.

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down.

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity.

Dendritic cells (DCs) play a critical role in orchestrating adaptive immune responses due to their unique ability to initiate T cell responses and direct their differentiation into effector lineages. Classical DCs have been divided into two subsets, cDC1 and cDC2, based on phenotypic markers and their distinct abilities to prime CD8 and CD4 T cells. While the transcriptional regulation of the cDC1 subset has been well characterized, cDC2 development and function remain poorly understood. By combining transcriptional and chromatin analyses with genetic reporter expression, we identified two principal cDC2 lineages defined by distinct developmental pathways and transcriptional regulators, including T-bet and RORγt, two key transcription factors known to define innate and adaptive lymphocyte subsets. These novel cDC2 lineages were characterized by distinct metabolic and functional programs. Extending our findings to humans revealed conserved DC heterogeneity and the presence of the newly defined cDC2 subsets in human cancer.

Cancer Immunosurveillance by Tissue-Resident Innate Lymphoid Cells and Innate-like T Cells.

Malignancy can be suppressed by the immune system in a process termed immunosurveillance. However, to what extent immunosurveillance occurs in spontaneous cancers and the composition of participating cell types remains obscure. Here, we show that cell transformation triggers a tissue-resident lymphocyte response in oncogene-induced murine cancer models. Non-circulating cytotoxic lymphocytes, derived from innate, T cell receptor (TCR)αß, and TCRγδ lineages, expand in early tumors. Characterized by high expression of NK1.1, CD49a, and CD103, these cells share a gene-expression signature distinct from those of conventional NK cells, T cells, and invariant NKT cells. Generation of these lymphocytes is dependent on the cytokine IL-15, but not the transcription factor Nfil3 that is required for the differentiation of tumor-infiltrating NK cells, and IL-15 deficiency, but not Nfil3 deficiency, results in accelerated tumor growth. These findings reveal a tumor-elicited immunosurveillance mechanism that engages unconventional type-1-like innate lymphoid cells and type 1 innate-like T cells.