Fibroblastic reticular cells enhance T cell metabolism and survival via epigenetic remodeling.

Lymph node fibroblastic reticular cells (FRCs) respond to signals from activated T cells by releasing nitric oxide, which inhibits T cell proliferation and restricts the size of the expanding T cell pool. Whether interactions with FRCs also support the function or differentiation of activated CD8+ T cells is not known. Here we report that encounters with FRCs enhanced cytokine production and remodeled chromatin accessibility in newly activated CD8+ T cells via interleukin-6. These epigenetic changes facilitated metabolic reprogramming and amplified the activity of pro-survival pathways through differential transcription factor activity. Accordingly, FRC conditioning significantly enhanced the persistence of virus-specific CD8+ T cells in vivo and augmented their differentiation into tissue-resident memory T cells. Our study demonstrates that FRCs play a role beyond restricting T cell expansion-they can also shape the fate and function of CD8+ T cells.

Author Correction: Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade.

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

Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade.

T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8+ tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8+ TILs include a subpopulation of 'progenitor exhausted' cells that retain polyfunctionality, persist long term and differentiate into 'terminally exhausted' TILs. Consequently, progenitor exhausted CD8+ TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8+ T cells might be an important component of improving the response to checkpoint blockade.

Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade.

Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition and tumour inflammation, respectively. Loss of ADAR1 overcomes resistance to PD-1 checkpoint blockade caused by inactivation of antigen presentation by tumour cells. Thus, effective anti-tumour immunity is constrained by inhibitory checkpoints such as ADAR1 that limit the sensing of innate ligands. The induction of sufficient inflammation in tumours that are sensitized to interferon can bypass the therapeutic requirement for CD8+ T cell recognition of cancer cells and may provide a general strategy to overcome immunotherapy resistance.

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

Fibroblastic reticular cells: organization and regulation of the T lymphocyte life cycle.

The connective tissue of any organ in the body is generally referred to as stroma. This complex network is commonly composed of leukocytes, extracellular matrix components, mesenchymal cells, and a collection of nerves, blood, and lymphoid vessels. Once viewed primarily as a structural entity, stromal cells of mesenchymal origin are now being intensely examined for their ability to directly regulate various components of immune cell function. There is particular interest in the ability of stromal cells to influence the homeostasis, activation, and proliferation of T lymphocytes. One example of this regulation occurs in the lymph node, where fibroblastic reticular cells support the maintenance of naive T cells, induce Ag-specific tolerance, and restrict the expansion of newly activated T cells. In an effort to highlight the varied immunoregulatory properties of fibroblastic reticular cells, we reviewed the most recent advances in this field and provide some insights into potential future directions.

A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system.

Therapies that target the function of immune cells have significant clinical efficacy in diseases such as cancer and autoimmunity. Although functional genomics has accelerated therapeutic target discovery in cancer, its use in primary immune cells is limited because vector delivery is inefficient and can perturb cell states. Here we describe CHIME: CHimeric IMmune Editing, a CRISPR-Cas9 bone marrow delivery system to rapidly evaluate gene function in innate and adaptive immune cells in vivo without ex vivo manipulation of these mature lineages. This approach enables efficient deletion of genes of interest in major immune lineages without altering their development or function. We use this approach to perform an in vivo pooled genetic screen and identify Ptpn2 as a negative regulator of CD8+ T cell-mediated responses to LCMV Clone 13 viral infection. These findings indicate that this genetic platform can enable rapid target discovery through pooled screening in immune cells in vivo.

The epigenetic landscape of T cell exhaustion.

Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8+ T cells and show that it is distinct from functional memory CD8+ T cells. Exhausted CD8+ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8+ T cells.

Stable inhibitory activity of regulatory T cells requires the transcription factor Helios.

The maintenance of immune homeostasis requires regulatory T cells (T(regs)). Given their intrinsic self-reactivity, T(regs) must stably maintain a suppressive phenotype to avoid autoimmunity. We report that impaired expression of the transcription factor (TF) Helios by FoxP3(+) CD4 and Qa-1-restricted CD8 T(regs) results in defective regulatory activity and autoimmunity in mice. Helios-deficient T(regs) develop an unstable phenotype during inflammatory responses characterized by reduced FoxP3 expression and increased effector cytokine expression secondary to diminished activation of the STAT5 pathway. CD8 T(regs) also require Helios-dependent STAT5 activation for survival and to prevent terminal T cell differentiation. The definition of Helios as a key transcription factor that stabilizes T(regs) in the face of inflammatory responses provides a genetic explanation for a core property of T(regs).

Lymph node fibroblastic reticular cell transplants show robust therapeutic efficacy in high-mortality murine sepsis.

Sepsis is an aggressive inflammatory syndrome and a global health burden estimated to kill 7.3 million people annually. Single-target molecular therapies have not addressed the multiple disease pathways triggered by septic injury. Cell therapies might offer a broader set of mechanisms of action that benefit complex, multifocal disease processes. We describe a population of immune-specialized myofibroblasts derived from lymph node tissue, termed fibroblastic reticular cells (FRCs). Because FRCs have an immunoregulatory function in lymph nodes, we hypothesized that ex vivo-expanded FRCs would control inflammation when administered therapeutically. Indeed, a single injection of ex vivo-expanded allogeneic FRCs reduced mortality in mouse models of sepsis when administered at early or late time points after septic onset. Mice treated with FRCs exhibited lower local and systemic concentrations of proinflammatory cytokines and reduced bacteremia. When administered 4 hours after induction of lipopolysaccharide endotoxemia, or cecal ligation and puncture (CLP) sepsis in mice, FRCs reduced deaths by at least 70%. When administered late in disease (16 hours after CLP), FRCs still conveyed a robust survival advantage (44% survival compared to 0% for controls). FRC therapy was dependent on the metabolic activity of nitric oxide synthase 2 (NOS2) as the primary molecular mechanism of drug action in the mice. Together, these data describe a new anti-inflammatory cell type and provide preclinical evidence for therapeutic efficacy in severe sepsis that warrants further translational study.