Pre-encoded responsiveness to type I interferon in the peripheral immune system defines outcome of PD1 blockade therapy.

Type I interferons (IFN-Is) are central regulators of anti-tumor immunity and responses to immunotherapy, but they also drive the feedback inhibition underlying therapeutic resistance. In the present study, we developed a mass cytometry approach to quantify IFN-I-stimulated protein expression across immune cells and used multi-omics to uncover pre-therapy cellular states encoding responsiveness to inflammation. Analyzing peripheral blood cells from multiple cancer types revealed that differential responsiveness to IFN-Is before anti-programmed cell death protein 1 (PD1) treatment was highly predictive of long-term survival after therapy. Unexpectedly, IFN-I hyporesponsiveness efficiently predicted long-term survival, whereas high responsiveness to IFN-I was strongly associated with treatment failure and diminished survival time. Peripheral IFN-I responsive states were not associated with tumor inflammation, identifying a disconnect between systemic immune potential and 'cold' or 'hot' tumor states. Mechanistically, IFN-I responsiveness was epigenetically imprinted before therapy, poising cells for differential inflammatory responses and dysfunctional T cell effector programs. Thus, we identify physiological cell states with clinical importance that can predict success and long-term survival of PD1-blocking immunotherapy.

Dynamic CD4+ T cell heterogeneity defines subset-specific suppression and PD-L1-blockade-driven functional restoration in chronic infection.

Inhibiting PD-1:PD-L1 signaling has transformed therapeutic immune restoration. CD4+ T cells sustain immunity in chronic infections and cancer, yet little is known about how PD-1 signaling modulates CD4+ helper T (TH) cell responses or the ability to restore CD4+ TH-mediated immunity by checkpoint blockade. We demonstrate that PD-1:PD-L1 specifically suppressed CD4+ TH1 cell amplification, prevents CD4+ TH1 cytokine production and abolishes CD4+ cytotoxic killing capacity during chronic infection in mice. Inhibiting PD-L1 rapidly restored these functions, while simultaneously amplifying and activating TH1-like T regulatory cells, demonstrating a system-wide CD4-TH1 recalibration. This effect coincided with decreased T cell antigen receptor signaling, and re-directed type I interferon (IFN) signaling networks towards dominant IFN-γ-mediated responses. Mechanistically, PD-L1 blockade specifically targeted defined populations with pre-established, but actively suppressed proliferative potential, with limited impact on minimally cycling TCF-1+ follicular helper T cells, despite high PD-1 expression. Thus, CD4+ T cells require unique differentiation and functional states to be targets of PD-L1-directed suppression and therapeutic restoration.

The transcription factor IRF2 drives interferon-mediated CD8+ T cell exhaustion to restrict anti-tumor immunity.

Type I and II interferons (IFNs) stimulate pro-inflammatory programs that are critical for immune activation, but also induce immune-suppressive feedback circuits that impede control of cancer growth. Here, we sought to determine how these opposing programs are differentially induced. We demonstrated that the transcription factor interferon regulatory factor 2 (IRF2) was expressed by many immune cells in the tumor in response to sustained IFN signaling. CD8+ T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor and instead enabled sustained effector functions that promoted long-term tumor control and increased responsiveness to immune checkpoint and adoptive cell therapies. The long-term tumor control by IRF2-deficient CD8+ T cells required continuous integration of both IFN-I and IFN-II signals. Thus, IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a potential target to enhance cancer control.

Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity.

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ+CD8+ T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

Apoptotic cell-induced AhR activity is required for immunological tolerance and suppression of systemic lupus erythematosus in mice and humans.

The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.

Inhibition of the BTK-IDO-mTOR axis promotes differentiation of monocyte-lineage dendritic cells and enhances anti-tumor T cell immunity.

Monocytic-lineage inflammatory Ly6c+CD103+ dendritic cells (DCs) promote antitumor immunity, but these DCs are infrequent in tumors, even upon chemotherapy. Here, we examined how targeting pathways that inhibit the differentiation of inflammatory myeloid cells affect antitumor immunity. Pharmacologic inhibition of Bruton's tyrosine kinase (BTK) and the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) or deletion of Btk or Ido1 allowed robust differentiation of inflammatory Ly6c+CD103+ DCs during chemotherapy, promoting antitumor T cell responses and inhibiting tumor growth. Immature Ly6c+c-kit+ precursor cells had epigenetic profiles similar to conventional DC precursors; deletion of Btk or Ido1 promoted differentiation of these cells. Mechanistically, a BTK-IDO axis inhibited a tryptophan-sensitive differentiation pathway driven by GATOR2 and mTORC1, and disruption of the GATOR2 in monocyte-lineage precursors prevented differentiation into inflammatory DCs in vivo. IDO-expressing DCs and monocytic cells were present across a range of human tumors. Thus, a BTK-IDO axis represses differentiation of inflammatory DCs during chemotherapy, with implications for targeted therapies.

CD8+ T Cell Priming in Established Chronic Viral Infection Preferentially Directs Differentiation of Memory-like Cells for Sustained Immunity.

CD8+ T cell exhaustion impedes control of chronic viral infection; yet how new T cell responses are mounted during chronic infection is unclear. Unlike T cells primed at the onset of infection that rapidly differentiate into effectors and exhaust, we demonstrate that virus-specific CD8+ T cells primed after establishment of chronic LCMV infection preferentially generate memory-like transcription factor TCF1+ cells that were transcriptionally and proteomically distinct, less exhausted, and more responsive to immunotherapy. Mechanistically, adaptations of antigen-presenting cells and diminished T cell signaling intensity promoted differentiation of the memory-like subset at the expense of rapid effector cell differentiation, which was now highly dependent on IL-21-mediated CD4+ T cell help for its functional generation. Chronic viral infection similarly redirected de novo differentiation of tumor-specific CD8+ T cells, ultimately preventing cancer control. Thus, targeting these T cell stimulatory pathways could enable strategies to control chronic infection, tumors, and enhance immunotherapeutic efficacy.

Reactive oxygen species modulate macrophage immunosuppressive phenotype through the up-regulation of PD-L1.

The combination of immune checkpoint blockade with chemotherapy is currently under investigation as a promising strategy for the treatment of triple negative breast cancer (TNBC). Tumor-associated macrophages (TAMs) are the most prominent component of the breast cancer microenvironment because they influence tumor progression and the response to therapies. Here we show that macrophages acquire an immunosuppressive phenotype and increase the expression of programmed death ligand-1 (PD-L1) when treated with reactive oxygen species (ROS) inducers such as the glutathione synthesis inhibitor, buthionine sulphoximine (BSO), and paclitaxel. Mechanistically, these agents cause accumulation of ROS that in turn activate NF-κB signaling to promote PD-L1 transcription and the release of immunosuppressive chemokines. Systemic in vivo administration of paclitaxel promotes PD-L1 accumulation on the surface of TAMS in a mouse model of TNBC, consistent with in vitro results. Combinatorial treatment with paclitaxel and an anti-mouse PD-L1 blocking antibody significantly improved the therapeutic efficacy of paclitaxel by reducing tumor burden and increasing the number of tumor-associated cytotoxic T cells. Our results provide a strong rationale for the use of anti-PD-L1 blockade in the treatment of TNBC patients. Furthermore, interrogation of chemotherapy-induced PD-L1 expression in TAMs is warranted to define appropriate patient selection in the use of PD-L1 blockade.

The Aryl Hydrocarbon Receptor: Connecting Immunity to the Microenvironment.

The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor and transcription factor activated through cognate ligand binding. It is an important factor in immunity and tissue homeostasis, and structurally diverse compounds from the environment, diet, microbiome, and host metabolism can induce AhR activity. Emerging evidence suggests that AhR is a key sensor allowing immune cells to adapt to environmental conditions and changes in AhR activity have been associated with autoimmune disorders and cancer. Furthermore, AhR agonists or antagonists can impact immune disease outcomes identifying AhR as a potentially actionable target for immunotherapy. In this review, we describe known ligands stimulating AhR activity, downstream proinflammatory and suppressive mechanisms potentiated by AhR, and how this understanding is being applied to immunopathology to help control disease outcomes.

Peroxisome Proliferator-Activated Receptor-δ Acts within Peripheral Myeloid Cells to Limit Th Cell Priming during Experimental Autoimmune Encephalomyelitis.

Peroxisome proliferator-activated receptor (PPAR)-δ is a fatty acid-activated transcription factor that regulates metabolic homeostasis, cell growth, and differentiation. Previously, we reported that mice with a global deficiency of PPAR-δ develop an exacerbated course of experimental autoimmune encephalomyelitis (EAE), highlighting a role for this nuclear receptor in limiting the development of CNS inflammation. However, the cell-specific contribution of PPAR-δ to the more severe CNS inflammatory response remained unclear. In this study, we studied the specific involvement of PPAR-δ in myeloid cells during EAE using mice that had Cre-mediated excision of floxed Ppard driven by the lysozyme M (LysM) promoter (LysM Cre :Ppard fl/fl). We observed that LysM Cre :Ppard fl/fl mice were more susceptible to EAE and developed a more severe course of this disease compared with Ppard fl/fl controls. The more severe EAE in LysM Cre :Ppard fl/fl mice was associated with an increased accumulation of pathogenic CD4+ T cells in the CNS and enhanced myelin-specific Th1 and Th17 responses in the periphery. Adoptive transfer EAE studies linked this EAE phenotype in LysM Cre :Ppard fl/fl mice to heightened Th responses. Furthermore, studies using an in vitro CD11b+ cell:Th cell coculture system revealed that CD11b+CD11c+ dendritic cells (DC) from LysM Cre :Ppard fl/fl mice had a heightened capacity to prime myelin oligodendrocyte glycoprotein (MOG)-specific Th cells compared with Ppard fl/fl counterparts; the effects of DC on Th1 cytokine production were mediated through production of the IL-12p40 homodimer. These studies revealed a role for PPAR-δ in DC in limiting Th cell priming during EAE.

Type I Interferon in Chronic Virus Infection and Cancer.

Type I interferons (IFN-Is) are emerging as key drivers of inflammation and immunosuppression in chronic infection. Control of these infections requires IFN-I signaling; however, prolonged IFN-I signaling can lead to immune dysfunction. IFN-Is are also emerging as double-edged swords in cancer, providing necessary inflammatory signals, while initiating feedback suppression in both immune and cancer cells. Here, we review the proinflammatory and suppressive mechanisms potentiated by IFN-Is during chronic virus infections and discuss the similar, newly emerging dichotomy in cancer. We then discuss how this understanding is leading to new therapeutic concepts and immunotherapy combinations. We propose that, by modulating the immune response at its foundation, it may be possible to widely reshape immunity to control these chronic diseases.

Apoptotic cell responses in the splenic marginal zone: a paradigm for immunologic reactions to apoptotic antigens with implications for autoimmunity.

Apoptotic cells drive innate regulatory responses that result in tolerogenic immunity. This is a critical aspect of cell physiology as apoptotic cells expose potentially dangerous nuclear antigens on the surface in apoptotic blebs, and failure in their recognition, phagocytosis, or destruction can cause dramatic autoimmunity in experimental models and is linked to development and progression of systemic pathology in human. The marginal zone is a specialized splenic environment that serves as a transitional site from circulation to peripheral lymphoid structures. The marginal zone serves a key role in trapping of particulates and initiation of innate responses against systemic microbial pathogens. However in recent years, it has become clear the marginal zone is also important for initiation of immune tolerance to apoptotic cells, driving a coordinated response involving multiple phagocyte and lymphocyte subsets. Recent reports linking defects in splenic macrophage function to systemic lupus erythematosus in a manner analogous to marginal zone macrophages in lupus-prone mice provide an impetus to better understand the mechanistic basis of the apoptotic cell response in the marginal zone and its general applicability to apoptotic cell-driven tolerance at other tissue sites. In this review, we discuss immune responses to apoptotic cells in the spleen in general and the marginal zone in particular, the relationship of these responses to autoimmune disease, and comparisons to apoptotic cell immunity in humans.

Selective Memory to Apoptotic Cell-Derived Self-Antigens with Implications for Systemic Lupus Erythematosus Development.

Autoimmune diseases are characterized by pathogenic immune responses to self-antigens. In systemic lupus erythematosus (SLE), many self-antigens are found in apoptotic cells (ACs), and defects in removal of ACs from the body are linked to a risk for developing SLE. This includes pathological memory that gives rise to disease flares. In this study, we investigated how memory to AC-derived self-antigens develops and the contribution of self-memory to the development of lupus-related pathology. Multiple injections of ACs without adjuvant into wild-type mice induce a transient primary autoimmune response without apparent anti-nuclear Ab reactivity or kidney pathology. Interestingly, as the transient Ab response reached baseline, a single boost injection fully recalled the immune response to ACs, and this memory response was furthermore transferable into naive mice. Additionally, the memory response contains elements of pathogenicity, accompanied by selective memory to selective Ags. Thus, we provide evidence for a selective self-memory that underlies progression of the response to self-antigens with implications for SLE development therapy.

The amino acid sensor GCN2 inhibits inflammatory responses to apoptotic cells promoting tolerance and suppressing systemic autoimmunity.

Efficient apoptotic cell clearance and induction of immunologic tolerance is a critical mechanism preventing autoimmunity and associated pathology. Our laboratory has reported that apoptotic cells induce tolerance by a mechanism dependent on the tryptophan catabolizing enzyme indoleamine 2,3 dioxygenase 1 (IDO1) in splenic macrophages (MΦ). The metabolic-stress sensing protein kinase GCN2 is a primary downstream effector of IDO1; thus, we tested its role in apoptotic cell-driven immune suppression. In vitro, expression of IDO1 in MΦs significantly enhanced apoptotic cell-driven IL-10 and suppressed IL-12 production in a GCN2-dependent mechanism. Suppression of IL-12 protein production was due to attenuation of IL-12 mRNA association with polyribosomes inhibiting translation while IL-10 mRNA association with polyribosomes was not affected. In vivo, apoptotic cell challenge drove a rapid, GCN2-dependent stress response in splenic MΦs with increased IL-10 and TGF-ß production, whereas myeloid-specific deletion of GCN2 abrogated regulatory cytokine production with provocation of inflammatory T-cell responses to apoptotic cell antigens and failure of long-tolerance induction. Consistent with a role in prevention of apoptotic cell driven autoreactivity, myeloid deletion of GCN2 in lupus-prone mice resulted in increased immune cell activation, humoral autoimmunity, renal pathology, and mortality. In contrast, activation of GCN2 with an agonist significantly reduced anti-DNA autoantibodies and protected mice from disease. Thus, this study implicates a key role for GCN2 signals in regulating the tolerogenic response to apoptotic cells and limiting autoimmunity.

B Cell-Intrinsic IDO1 Regulates Humoral Immunity to T Cell-Independent Antigens.

Humoral responses to nonproteinaceous Ags (i.e., T cell independent [TI]) are a key component of the early response to bacterial and viral infection and a critical driver of systemic autoimmunity. However, mechanisms that regulate TI humoral immunity are poorly defined. In this study, we report that B cell-intrinsic induction of the tryptophan-catabolizing enzyme IDO1 is a key mechanism limiting TI Ab responses. When Ido1(-/-) mice were immunized with TI Ags, there was a significant increase in Ab titers and formation of extrafollicular Ab-secreting cells compared with controls. This effect was specific to TI Ags, as Ido1 disruption did not affect Ig production after immunization with protein Ags. The effect of IDO1 abrogation was confined to the B cell compartment, as adoptive transfer of Ido1(-/-) B cells to B cell-deficient mice was sufficient to replicate increased TI responses observed in Ido1(-/-) mice. Moreover, in vitro activation with TLR ligands or BCR crosslinking rapidly induced Ido1 expression and activity in purified B cells, and Ido1(-/-) B cells displayed enhanced proliferation and cell survival associated with increased Ig and cytokine production compared with wild-type B cells. Thus, our results demonstrate a novel, B cell-intrinsic, role for IDO1 as a regulator of humoral immunity that has implications for both vaccine design and prevention of autoimmunity.

Amino acid metabolism inhibits antibody-driven kidney injury by inducing autophagy.

Inflammatory kidney disease is a major clinical problem that can result in end-stage renal failure. In this article, we show that Ab-mediated inflammatory kidney injury and renal disease in a mouse nephrotoxic serum nephritis model was inhibited by amino acid metabolism and a protective autophagic response. The metabolic signal was driven by IFN-γ-mediated induction of indoleamine 2,3-dioxygenase 1 (IDO1) enzyme activity with subsequent activation of a stress response dependent on the eIF2α kinase general control nonderepressible 2 (GCN2). Activation of GCN2 suppressed proinflammatory cytokine production in glomeruli and reduced macrophage recruitment to the kidney during the incipient stage of Ab-induced glomerular inflammation. Further, inhibition of autophagy or genetic ablation of Ido1 or Gcn2 converted Ab-induced, self-limiting nephritis to fatal end-stage renal disease. Conversely, increasing kidney IDO1 activity or treating mice with a GCN2 agonist induced autophagy and protected mice from nephritic kidney damage. Finally, kidney tissue from patients with Ab-driven nephropathy showed increased IDO1 abundance and stress gene expression. Thus, these findings support the hypothesis that the IDO-GCN2 pathway in glomerular stromal cells is a critical negative feedback mechanism that limits inflammatory renal pathologic changes by inducing autophagy.

Marginal zone CD169+ macrophages coordinate apoptotic cell-driven cellular recruitment and tolerance.

Tolerance to apoptotic cells is essential to prevent inflammatory pathology. Though innate responses are critical for immune suppression, our understanding of early innate immunity driven by apoptosis is lacking. Herein we report apoptotic cells induce expression of the chemokine CCL22 in splenic metallophillic macrophages, which is critical for tolerance. Systemic challenge with apoptotic cells induced rapid production of CCL22 in CD169(+) (metallophillic) macrophages, resulting in accumulation and activation of FoxP3(+) Tregs and CD11c(+) dendritic cells, an effect that could be inhibited by antagonizing CCL22-driven chemotaxis. This mechanism was essential for suppression after apoptotic cell challenge, because neutralizing CCL22 or its receptor, reducing Treg numbers, or blocking effector mechanisms abrogated splenic TGF-ß and IL-10 induction; this promoted a shift to proinflammatory cytokines associated with a failure to suppress T cells. Similarly, CCR4 inhibition blocked long-term, apoptotic cell-induced tolerance to allografts. Finally, CCR4 inhibition resulted in a systemic breakdown of tolerance to self after apoptotic cell injection with rapid increases in anti-dsDNA IgG and immune complex deposition. Thus, the data demonstrate CCL22-dependent chemotaxis is a key early innate response required for apoptotic cell-induced suppression, implicating a previously unknown mechanism of macrophage-dependent coordination of early events leading to stable tolerance.

Amino acid catabolism: a pivotal regulator of innate and adaptive immunity.

Enhanced amino acid catabolism is a common response to inflammation, but the immunologic significance of altered amino acid consumption remains unclear. The finding that tryptophan catabolism helped maintain fetal tolerance during pregnancy provided novel insights into the significance of amino acid metabolism in controlling immunity. Recent advances in identifying molecular pathways that enhance amino acid catabolism and downstream mechanisms that affect immune cells in response to inflammatory cues support the notion that amino acid catabolism regulates innate and adaptive immune cells in pathologic settings. Cells expressing enzymes that degrade amino acids modulate antigen-presenting cell and lymphocyte functions and reveal critical roles for amino acid- and catabolite-sensing pathways in controlling gene expression, functions, and survival of immune cells. Basal amino acid catabolism may contribute to immune homeostasis that prevents autoimmunity, whereas elevated amino acid catalytic activity may reinforce immune suppression to promote tumorigenesis and persistence of some pathogens that cause chronic infections. For these reasons, there is considerable interest in generating novel drugs that inhibit or induce amino acid consumption and target downstream molecular pathways that control immunity. In this review, we summarize recent developments and highlight novel concepts and key outstanding questions in this active research field.

Cytosolic DNA sensing via the stimulator of interferon genes adaptor: Yin and Yang of immune responses to DNA.

DNA is immunogenic and many cells express cytosolic DNA sensors that activate the stimulator of interferon genes (STING) adaptor to trigger interferon type I (IFN-ß) release, a potent immune activator. DNA sensing to induce IFN-ß triggers host immunity to pathogens but constitutive DNA sensing can induce sustained IFN-ß release that incites autoimmunity. Here, we focus on cytosolic DNA sensing via the STING/IFN-ß pathway that regulates immune responses. Recent studies reveal that cytosolic DNA sensing via the STING/IFN-ß pathway induces indoleamine 2,3 dioxygenase (IDO), which catabolizes tryptophan to suppress effector and helper T-cell responses and activate Foxp3-lineage CD4(+) regulatory T (Treg) cells. During homeostasis, and in some inflammatory settings, specialized innate immune cells in the spleen and lymph nodes may ingest and sense cytosolic DNA to reinforce tolerance that prevents autoimmunity. However, malignancies and pathogens may exploit DNA-induced regulatory responses to suppress natural and vaccine-induced immunity to malignant and infected cells. In this review, we discuss the biologic significance of regulatory responses to DNA and novel approaches to exploit DNA-induced immune responses for therapeutic benefit. The ability of DNA to drive tolerogenic or immunogenic responses highlights the need to evaluate immune responses to DNA in physiologic settings relevant to disease progression or therapy.

Cutting edge: DNA sensing via the STING adaptor in myeloid dendritic cells induces potent tolerogenic responses.

Cytosolic DNA sensing via the stimulator of IFN genes (STING) adaptor incites autoimmunity by inducing type I IFN (IFN-αß). In this study, we show that DNA is also sensed via STING to suppress immunity by inducing IDO. STING gene ablation abolished IFN-αß and IDO induction by dendritic cells (DCs) after DNA nanoparticle (DNP) treatment. Marginal zone macrophages, some DCs, and myeloid cells ingested DNPs, but CD11b(+) DCs were the only cells to express IFN-ß, whereas CD11b(+) non-DCs were major IL-1ß producers. STING ablation also abolished DNP-induced regulatory responses by DCs and regulatory T cells, and hallmark regulatory responses to apoptotic cells were also abrogated. Moreover, systemic cyclic diguanylate monophosphate treatment to activate STING induced selective IFN-ß expression by CD11b(+) DCs and suppressed Th1 responses to immunization. Thus, previously unrecognized functional diversity among physiologic innate immune cells regarding DNA sensing via STING is pivotal in driving immune responses to DNA.