TREM2 macrophages drive NK cell paucity and dysfunction in lung cancer.

Natural killer (NK) cells are commonly reduced in human tumors, enabling many to evade surveillance. Here, we sought to identify cues that alter NK cell activity in tumors. We found that, in human lung cancer, the presence of NK cells inversely correlated with that of monocyte-derived macrophages (mo-macs). In a murine model of lung adenocarcinoma, we show that engulfment of tumor debris by mo-macs triggers a pro-tumorigenic program governed by triggering receptor expressed on myeloid cells 2 (TREM2). Genetic deletion of Trem2 rescued NK cell accumulation and enabled an NK cell-mediated regression of lung tumors. TREM2+ mo-macs reduced NK cell activity by modulating interleukin (IL)-18/IL-18BP decoy interactions and IL-15 production. Notably, TREM2 blockade synergized with an NK cell-activating agent to further inhibit tumor growth. Altogether, our findings identify a new axis, in which TREM2+ mo-macs suppress NK cell accumulation and cytolytic activity. Dual targeting of macrophages and NK cells represents a new strategy to boost antitumor immunity.

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.

Immunology of COVID-19: Current State of the Science.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide, igniting an unprecedented effort from the scientific community to understand the biological underpinning of COVID19 pathophysiology. In this Review, we summarize the current state of knowledge of innate and adaptive immune responses elicited by SARS-CoV-2 infection and the immunological pathways that likely contribute to disease severity and death. We also discuss the rationale and clinical outcome of current therapeutic strategies as well as prospective clinical trials to prevent or treat SARS-CoV-2 infection.

CXCR3-CXCL9: It's All in the Tumor.

In this issue of Immunity, Chow et al. (2019) show that the CXCR3-CXCL9 axis is required for reinvigoration of intratumoral CD8+ T cell responses in response to PD-1 blockade and demonstrate that local guidance to dendritic cells, rather than recruitment of T cells into the tumor, underlies the importance of this chemokine axis.

Characteristics and anatomic location of PD-1+TCF1+ stem-like CD8 T cells in chronic viral infection and cancer.

CD8 T cells play an essential role in antitumor immunity and chronic viral infections. Recent findings have delineated the differentiation pathway of CD8 T cells in accordance with the progenitor-progeny relationship of TCF1+ stem-like and Tim-3+TCF1- more differentiated T cells. Here, we investigated the characteristics of stem-like and differentiated CD8 T cells isolated from several murine tumor models and human lung cancer samples in terms of phenotypic and transcriptional features as well as their location compared to virus-specific CD8 T cells in the chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. We found that CD8 tumor-infiltrating lymphocytes (TILs) in both murine and human tumors exhibited overall similar phenotypic and transcriptional characteristics compared to corresponding subsets in the spleen of chronically infected mice. Moreover, stem-like CD8 TILs exclusively responded and produced effector-like progeny CD8 T cells in vivo after antigenic restimulation, confirming their lineage relationship and the proliferative potential of stem-like CD8 TILs. Most importantly, similar to the preferential localization of PD-1+ stem-like CD8 T cells in T cell zones of the spleen during chronic LCMV infection, we found that the PD-1+ stem-like CD8 TILs in lung cancer samples are preferentially located not in the tumor parenchyma but in tertiary lymphoid structures (TLSs). The stem-like CD8 T cells are present in TLSs located within and at the periphery of the tumor, as well as in TLSs closely adjacent to the tumor parenchyma. These findings suggest that TLSs provide a protective niche to support the quiescence and maintenance of stem-like CD8 T cells in the tumor.

Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter.

The germinal center (GC) reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors. The mechanism by which B cells are selected remains unclear, as does the role of the two anatomically defined areas of the GC, light zone (LZ) and dark zone (DZ). We combined a transgenic photoactivatable fluorescent protein tracer with multiphoton laser-scanning microscopy and flow cytometry to examine anatomically defined LZ and DZ B cells and GC selection. We find that B cell division is restricted to the DZ, with a net vector of B cell movement from the DZ to the LZ. The decision to return to the DZ and undergo clonal expansion is controlled by T helper cells in the GC LZ, which discern between LZ B cells based on the amount of antigen captured and presented. Thus, T cell help, and not direct competition for antigen, is the limiting factor in GC selection.

An intra-tumoral niche maintains and differentiates stem-like CD8 T cells.

Tumour-infiltrating lymphocytes are associated with a survival benefit in several tumour types and with the response to immunotherapy1-8. However, the reason some tumours have high CD8 T cell infiltration while others do not remains unclear. Here we investigate the requirements for maintaining a CD8 T cell response against human cancer. We find that CD8 T cells within tumours consist of distinct populations of terminally differentiated and stem-like cells. On proliferation, stem-like CD8 T cells give rise to more terminally differentiated, effector-molecule-expressing daughter cells. For many T cells to infiltrate the tumour, it is critical that this effector differentiation process occur. In addition, we show that these stem-like T cells reside in dense antigen-presenting-cell niches within the tumour, and that tumours that fail to form these structures are not extensively infiltrated by T cells. Patients with progressive disease lack these immune niches, suggesting that niche breakdown may be a key mechanism of immune escape.

Antibody effector functions mediated by Fcγ-receptors are compromised during persistent viral infection.

T cell dysfunction is well documented during chronic viral infections but little is known about functional abnormalities in humoral immunity. Here we report that mice persistently infected with lymphocytic choriomeningitis virus (LCMV) exhibit a severe defect in Fcγ-receptor (FcγR)-mediated antibody effector functions. Using transgenic mice expressing human CD20, we found that chronic LCMV infection impaired the depletion of B cells with rituximab, an anti-CD20 antibody widely used for the treatment of B cell lymphomas. In addition, FcγR-dependent activation of dendritic cells by agonistic anti-CD40 antibody was compromised in chronically infected mice. These defects were due to viral antigen-antibody complexes and not the chronic infection per se, because FcγR-mediated effector functions were normal in persistently infected mice that lacked LCMV-specific antibodies. Our findings have implications for the therapeutic use of antibodies and suggest that high levels of pre-existing immune complexes could limit the effectiveness of antibody therapy in humans.

CD8 T Cell Exhaustion in Chronic Infection and Cancer: Opportunities for Interventions.

Antigen-specific CD8 T cells are central to the control of chronic infections and cancer, but persistent antigen stimulation results in T cell exhaustion. Exhausted CD8 T cells have decreased effector function and proliferative capacity, partly caused by overexpression of inhibitory receptors such as programmed cell death (PD)-1. Blockade of the PD-1 pathway has opened a new therapeutic avenue for reinvigorating T cell responses, with positive outcomes especially for patients with cancer. Other strategies to restore function in exhausted CD8 T cells are currently under evaluation-many in combination with PD-1-targeted therapy. Exhausted CD8 T cells comprise heterogeneous cell populations with unique differentiation and functional states. A subset of stem cell-like PD-1+ CD8 T cells responsible for the proliferative burst after PD-1 therapy has been recently described. A greater understanding of T cell exhaustion is imperative to establish rational immunotherapeutic interventions.

Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1-targeted therapy in lung cancer patients.

Exhausted T cells in chronic infections and cancer have sustained expression of the inhibitory receptor programmed cell death 1 (PD-1). Therapies that block the PD-1 pathway have shown promising clinical results in a significant number of advanced-stage cancer patients. Nonetheless, a better understanding of the immunological responses induced by PD-1 blockade in cancer patients is lacking. Identification of predictive biomarkers is a priority in the field, but whether peripheral blood analysis can provide biomarkers to monitor or predict patients' responses to treatment remains to be resolved. In this study, we analyzed longitudinal blood samples from advanced stage non-small cell lung cancer (NSCLC) patients (n = 29) receiving PD-1-targeted therapies. We detected an increase in Ki-67+ PD-1+ CD8 T cells following therapy in ∼70% of patients, and most responses were induced after the first or second treatment cycle. This T-cell activation was not indiscriminate because we observed only minimal effects on EBV-specific CD8 T cells, suggesting that responding cells may be tumor specific. These proliferating CD8 T cells had an effector-like phenotype (HLA-DR+, CD38+, Bcl-2lo), expressed costimulatory molecules (CD28, CD27, ICOS), and had high levels of PD-1 and coexpression of CTLA-4. We found that 70% of patients with disease progression had either a delayed or absent PD-1+ CD8 T-cell response, whereas 80% of patients with clinical benefit exhibited PD-1+ CD8 T-cell responses within 4 wk of treatment initiation. Our results suggest that peripheral blood analysis may provide valuable insights into NSCLC patients' responses to PD-1-targeted therapies.

Immune checkpoint inhibitors in advanced non-small cell lung cancer.

The emergence of immune checkpoint inhibitors for the treatment of cancer has led to major changes to the therapeutic landscape of lung cancer. Improvements in overall survival relative to standard chemotherapy have been observed in the first-line and second-line therapy settings for patients with advanced non-small cell lung cancer (NSCLC) who are treated with immune checkpoint inhibitors. Consequently, every patient with advanced-stage NSCLC is now a candidate for immune checkpoint inhibitor therapy. However, it is clear that the benefit from therapy is not universal, and identification of biomarkers to select therapy has assumed importance. In addition to programmed cell death receptor ligand 1 expression, both tissue-based and blood-based markers are under evaluation to select patients. In an era of increasing costs of care and potential for toxicities related to immune checkpoint inhibition, proper patient selection is critical to the optimal use of this new class of agents. In addition, development of novel combination approaches has also emerged as an important way to improve the efficacy of immune checkpoint inhibition. Studies in earlier stages of NSCLC are already underway with the hope of improving the cure rate. In this article, the authors review the current landscape of immune checkpoint inhibitors in the treatment of advanced NSCLC. Cancer 2018;124:248-61. © 2017 American Cancer Society.

Comparison of the toxicity profile of PD-1 versus PD-L1 inhibitors in non-small cell lung cancer: A systematic analysis of the literature.

BACKGROUND: Monoclonal antibodies against programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) are effective therapies in patients with non-small cell lung cancer (NSCLC). Herein, the authors performed a systematic review investigating differences in the toxicities of PD-1 and PD-L1 inhibitors. METHODS: An electronic literature search was performed of public databases (MEDLINE, Excerpta Medica dataBASE [EMBASE], and Cochrane) and conference proceedings for trials using PD-1 inhibitors (nivolumab and pembrolizumab) and PD-L1 inhibitors (atezolizumab, durvalumab, and avelumab) in patients with NSCLC. A formal systematic analysis was conducted with Comprehensive Meta-Analysis software (version 2.2). Clinical and demographic characteristics, response, and toxicity data were compared between both groups. RESULTS: A total of 23 studies reported between 2013 and 2016 were eligible for analysis. The total number of patients evaluated for toxicities was 3284 patients in the PD-1 group and 2460 patients in the PD-L1 group. The baseline patient characteristics of the 2 groups were similar, although there was a trend toward increased squamous histology in the group treated with PD-L1 (32% vs 25%; P = .6). There was no difference in response rate noted between PD-1 (19%) and PD-L1 (18.6%) inhibitors (P = .17). The incidence of overall adverse events (AEs) was comparable between the PD-1 and PD-L1 inhibitors (64% [95% confidence interval (95% CI), 63%-66%] vs 66% [95% CI, 65%-69%]; P = .8). Fatigue was the most frequently reported AE with both classes of drugs. Patients treated with PD-1 inhibitors were found to have a slightly increased rate of immune-related AEs (16% [95% CI, 14%-17%] vs 11% [95% CI, 10%-13%]; P = .07) and pneumonitis (4% [95% CI, 3%-5%] vs 2% [95% CI, 1%-3%]; P = .01) compared with patients who received PD-L1 inhibitors. CONCLUSIONS: In this systematic review involving 5744 patients with NSCLC, the toxicity and efficacy profiles of PD-1 and PD-L1 inhibitors appear to be similar. Cancer 2018;124:271-7. © 2017 American Cancer Society.

T cell receptor sequencing of activated CD8 T cells in the blood identifies tumor-infiltrating clones that expand after PD-1 therapy and radiation in a melanoma patient.

PD-1-targeted therapy has dramatically changed advanced cancer treatment. However, many questions remain, including specificity of T cells activated by PD-1 therapy and how peripheral blood analysis correlates to effects at tumor sites. In this study, we utilized TCR sequencing to dissect the composition of peripheral blood CD8 T cells activated upon therapy, comparing it with tumor-infiltrating lymphocytes. We report on a nonagenarian melanoma patient who showed a prominent increase in peripheral blood Ki-67 + CD8 T cells following brain stereotactic radiation and anti-PD-1 immunotherapy. Proliferating CD8 T cells exhibited an effector-like phenotype with expression of CD38, HLA-DR and Granzyme B, as well as expression of the positive costimulatory molecules CD28 and CD27. TCR sequencing of peripheral blood CD8 T cells revealed a highly oligoclonal repertoire at baseline with one clonotype accounting for 30%. However, the majority of dominant clones-including a previously identified cytomegalovirus-reactive clone-did not expand following treatment. In contrast, expanding clones were present at low frequencies in the peripheral blood but were enriched in a previously resected liver metastasis. The patient has so far remained recurrence-free for 36 months, and several CD8 T cell clones that expanded after treatment were maintained at elevated levels for at least 8 months. Our data show that even in a nonagenarian individual with oligoclonal expansion of CD8 T cells, we can identify activation of tumor-infiltrating CD8 T cell clones in peripheral blood following anti-PD-1-based immunotherapies.

A role for lipid bodies in the cross-presentation of phagocytosed antigens by MHC class I in dendritic cells.

Dendritic cells (DCs) have the striking ability to cross-present exogenous antigens in association with major histocompatibility complex (MHC) class I to CD8(+) T cells. However, the intracellular pathways underlying cross-presentation remain ill defined. Current models involve cytosolic proteolysis of antigens by the proteasome and peptide import into endoplasmic reticulum (ER) or phagosomal lumen by the transporters associated with antigen processing (TAP1 and TAP2). Here, we show that DCs expressed an ER-resident 47 kDa immune-related GTPase, Igtp (Irgm3). Igtp resides on ER and lipid body (LB) membranes where it binds the LB coat component ADFP. Inactivation of genes encoding for either Igtp or ADFP led to defects in LB formation in DCs and severely impaired cross-presentation of phagocytosed antigens to CD8(+) T cells but not antigen presentation to CD4(+) T cells. We thus define a new role for LB organelles in regulating cross-presentation of exogenous antigens to CD8(+) T lymphocytes in DCs.

Cardiac allograft rejection as a complication of PD-1 checkpoint blockade for cancer immunotherapy: a case report.

INTRODUCTION: The increased availability of immunotherapeutic agents for the treatment of a wide array of cancer in the general oncology practice setting will reveal rare and unique toxicities. MATERIALS AND METHODS: The mechanism of cardiac allograft rejection in the context of PD-1 antibody therapy was explored in a patient with cutaneous squamous cell cancer complicating long-standing cardiac allograft. Immune cell infiltrate in the myocardium and peripheral blood lymphocyte repertoire were assessed using myocardial biopsy and temporal analysis of peripheral blood samples. The efficacy of high-intensity immunosuppression to reverse graft rejection was explored. RESULTS: Endomyocardial biopsy showed acute moderate diffuse cellular rejection with a predominant population of CD3+, CD8+ and CD4+ infiltrating lymphocytes; peripheral blood circulating lymphocytes showed a high frequency of proliferating and activated CD8+ T cells expressing PD-1 compared to a normal control. There was no difference in the activation and proliferation of CD4+ T cells compared to a normal control. Cardiac function improved following high-intensity immunosuppression and patient survived for up to 7 months after discontinuation of nivolumab. CONCLUSIONS: Immune checkpoint inhibitors should be avoided in allograft recipients but high-intensity immunosuppression is effective to salvage allograft rejection induced by these agents.

Antigen-specific CD4 T-cell help rescues exhausted CD8 T cells during chronic viral infection.

CD4 T cells play a critical role in regulating CD8 T-cell responses during chronic viral infection. Several studies in animal models and humans have shown that the absence of CD4 T-cell help results in severe dysfunction of virus-specific CD8 T cells. However, whether function can be restored in already exhausted CD8 T cells by providing CD4 T-cell help at a later time remains unexplored. In this study, we used a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection to address this question. Adoptive transfer of LCMV-specific CD4 T cells into chronically infected mice restored proliferation and cytokine production by exhausted virus-specific CD8 T cells and reduced viral burden. Although the transferred CD4 T cells were able to enhance function in exhausted CD8 T cells, these CD4 T cells expressed high levels of the programmed cell death (PD)-1 inhibitory receptor. Blockade of the PD-1 pathway increased the ability of transferred LCMV-specific CD4 T cells to produce effector cytokines, improved rescue of exhausted CD8 T cells, and resulted in a striking reduction in viral load. These results suggest that CD4 T-cell immunotherapy alone or in conjunction with blockade of inhibitory receptors may be a promising approach for treating CD8 T-cell dysfunction in chronic infections and cancer.

Sustained CD28 costimulation is required for self-renewal and differentiation of TCF-1+ PD-1+ CD8 T cells.

During persistent antigen stimulation, such as in chronic infections and cancer, CD8 T cells differentiate into a hypofunctional programmed death protein 1-positive (PD-1+) exhausted state. Exhausted CD8 T cell responses are maintained by precursors (Tpex) that express the transcription factor T cell factor 1 (TCF-1) and high levels of the costimulatory molecule CD28. Here, we demonstrate that sustained CD28 costimulation is required for maintenance of antiviral T cells during chronic infection. Low-level CD28 engagement preserved mitochondrial fitness and self-renewal of Tpex, whereas stronger CD28 signaling enhanced glycolysis and promoted Tpex differentiation into TCF-1neg exhausted CD8 T cells (Tex). Furthermore, enhanced differentiation by CD28 engagement did not reduce the Tpex pool. Together, these findings demonstrate that continuous CD28 engagement is needed to sustain PD-1+ CD8 T cells and suggest that increasing CD28 signaling promotes Tpex differentiation into more functional effector-like Tex, possibly without compromising long-term responses.

MacroH2A restricts inflammatory gene expression in melanoma cancer-associated fibroblasts by coordinating chromatin looping.

MacroH2A has established tumour suppressive functions in melanoma and other cancers, but an unappreciated role in the tumour microenvironment. Using an autochthonous, immunocompetent mouse model of melanoma, we demonstrate that mice devoid of macroH2A variants exhibit increased tumour burden compared with wild-type counterparts. MacroH2A-deficient tumours accumulate immunosuppressive monocytes and are depleted of functional cytotoxic T cells, characteristics consistent with a compromised anti-tumour response. Single cell and spatial transcriptomics identify increased dedifferentiation along the neural crest lineage of the tumour compartment and increased frequency and activation of cancer-associated fibroblasts following macroH2A loss. Mechanistically, macroH2A-deficient cancer-associated fibroblasts display increased myeloid chemoattractant activity as a consequence of hyperinducible expression of inflammatory genes, which is enforced by increased chromatin looping of their promoters to enhancers that gain H3K27ac. In summary, we reveal a tumour suppressive role for macroH2A variants through the regulation of chromatin architecture in the tumour stroma with potential implications for human melanoma.

Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses.

Resistance to several prevalent infectious diseases requires both cellular and humoral immune responses. T cell immunity is initiated by mature dendritic cells (DCs) in lymphoid organs, whereas humoral responses to most antigens require further collaboration between primed, antigen-specific helper T cells and naive or memory B cells. To determine whether antigens delivered to DCs in lymphoid organs induce T cell help for antibody responses, we targeted a carrier protein, ovalbumin (OVA), to DCs in the presence of a maturation stimulus and assayed for antibodies to a hapten, (4-hydroxy-3-nitrophenyl) acetyl (NP), after boosting with OVA-NP. A single DC-targeted immunization elicited long-lived T cell helper responses to the carrier protein, leading to large numbers of antibody-secreting cells and high titers of high-affinity antihapten immunoglobulin Gs. Small doses of DC-targeted OVA induced higher titers and a broader spectrum of anti-NP antibody isotypes than large doses of OVA in alum adjuvant. Similar results were obtained when the circumsporozoite protein of Plasmodium yoelii was delivered to DCs. We conclude that antigen targeting to DCs combined with a maturation stimulus produces broad-based and long-lived T cell help for humoral immune responses.

Route of antigen uptake differentially impacts presentation by dendritic cells and activated monocytes.

Dendritic cells (DCs), which maintain tolerance and orchestrate T cell immune responses, comprise a heterogeneous group of cells. For example, in the steady state, murine spleen contains pre-DC-derived CD8(+) and CD8(-) conventional DCs. During inflammation, monocytes become activated and acquire some DC-like features, such as expression of CD11c and MHC class II. Although each of these cell types can present Ag, the relative efficiency of processing and presentation after Ag capture by different routes has not yet been systematically compared. To this end, we administered OVA to various conventional DCs and activated monocytes by receptor-mediated endocytosis, pinocytosis, or phagocytosis and measured internalization and presentation to MHC class I- and MHC class II-restricted T cells. We find that CD8(-) DCs are more efficient than any other type of APC tested in terms of presenting Ag to MHC class II-restricted T cells, irrespective of the route of Ag capture. In contrast, both subsets of splenic DCs are highly effective in cross-presenting Ags to CD8(+) T cells. DCs and activated monocytes cross-presented Ags delivered by DEC205-mediated endocytosis and pinocytosis. However, DCs differ from activated monocytes in that the latter are several orders of magnitude less efficient in presenting Ags captured by phagocytosis to CD8(+) or CD4(+) T cells. We conclude that DCs derived from pre-DCs differ from monocyte-derived cells in that DCs process and present Ags efficiently irrespective of the route of Ag capture. Our observations have significant implications for understanding initiation of immune responses and vaccination strategies targeting DCs and activated monocytes.