Retinoic acid and TGF-ß orchestrate organ-specific programs of tissue residency.

Tissue-resident memory T (TRM) cells are integral to tissue immunity, persisting in diverse anatomical sites where they adhere to a common transcriptional framework. How these cells integrate distinct local cues to adopt the common TRM cell fate remains poorly understood. Here, we show that whereas skin TRM cells strictly require transforming growth factor ß (TGF-ß) for tissue residency, those in other locations utilize the metabolite retinoic acid (RA) to drive an alternative differentiation pathway, directing a TGF-ß-independent tissue residency program in the liver and synergizing with TGF-ß to drive TRM cells in the small intestine. We found that RA was required for the long-term maintenance of intestinal TRM populations, in part by impeding their retrograde migration. Moreover, enhanced RA signaling modulated TRM cell phenotype and function, a phenomenon mirrored in mice with increased microbial diversity. Together, our findings reveal RA as a fundamental component of the host-environment interaction that directs immunosurveillance in tissues.

Divergent molecular networks program functionally distinct CD8+ skin-resident memory T cells.

Skin-resident CD8+ T cells include distinct interferon-γ-producing [tissue-resident memory T type 1 (TRM1)] and interleukin-17 (IL-17)-producing (TRM17) subsets that differentially contribute to immune responses. However, whether these populations use common mechanisms to establish tissue residence is unknown. In this work, we show that TRM1 and TRM17 cells navigate divergent trajectories to acquire tissue residency in the skin. TRM1 cells depend on a T-bet-Hobit-IL-15 axis, whereas TRM17 cells develop independently of these factors. Instead, c-Maf commands a tissue-resident program in TRM17 cells parallel to that induced by Hobit in TRM1 cells, with an ICOS-c-Maf-IL-7 axis pivotal to TRM17 cell commitment. Accordingly, by targeting this pathway, skin TRM17 cells can be ablated without compromising their TRM1 counterparts. Thus, skin-resident T cells rely on distinct molecular circuitries, which can be exploited to strategically modulate local immunity.

Single-cell protein expression profiling resolves circulating and resident memory T cell diversity across tissues and infection contexts.

Memory CD8+ T cells can be broadly divided into circulating (TCIRCM) and tissue-resident memory T (TRM) populations. Despite well-defined migratory and transcriptional differences, the phenotypic and functional delineation of TCIRCM and TRM cells, particularly across tissues, remains elusive. Here, we utilized an antibody screening platform and machine learning prediction pipeline (InfinityFlow) to profile >200 proteins in TCIRCM and TRM cells in solid organs and barrier locations. High-dimensional analyses revealed unappreciated heterogeneity within TCIRCM and TRM cell lineages across nine different organs after either local or systemic murine infection models. Additionally, we demonstrated the relative effectiveness of strategies allowing for the selective ablation of TCIRCM or TRM populations across organs and identified CD55, KLRG1, CXCR6, and CD38 as stable markers for characterizing memory T cell function during inflammation. Together, these data and analytical framework provide an in-depth resource for memory T cell classification in both steady-state and inflammatory conditions.

The RAG1 Ubiquitin Ligase Domain Stimulates Recombination of TCRß and TCRα Genes and Influences Development of αß T Cell Lineages.

RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte Ag receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report in this study that mice with a mutation that inactivates the Rag1 ubiquitin ligase in vitro exhibit decreased rearrangements and altered repertoires of TCRß and TCRα genes in thymocytes and impaired thymocyte developmental transitions that require the assembly and selection of functional TCRß and/or TCRα genes. These Rag1 mutant mice present diminished positive selection and superantigen-mediated negative selection of conventional αß T cells, decreased genesis of invariant NK T lineage αß T cells, and mature CD4+ αß T cells with elevated autoimmune potential. Our findings reveal that the Rag1 ubiquitin ligase domain functions in vivo to stimulate TCRß and TCRα gene recombination and influence differentiation of αß T lineage cells, thereby establishing replete diversity of αß TCRs and populations of αß T cells while restraining generation of potentially autoreactive conventional αß T cells.

Runx3 drives a CD8+ T cell tissue residency program that is absent in CD4+ T cells.

Tissue-resident memory T cells (TRM cells) provide rapid and superior control of localized infections. While the transcription factor Runx3 is a critical regulator of CD8+ T cell tissue residency, its expression is repressed in CD4+ T cells. Here, we show that, as a direct consequence of this Runx3-deficiency, CD4+ TRM cells lacked the transforming growth factor (TGF)-ß-responsive transcriptional network that underpins the tissue residency of epithelial CD8+ TRM cells. While CD4+ TRM cell formation required Runx1, this, along with the modest expression of Runx3 in CD4+ TRM cells, was insufficient to engage the TGF-ß-driven residency program. Ectopic expression of Runx3 in CD4+ T cells incited this TGF-ß-transcriptional network to promote prolonged survival, decreased tissue egress, a microanatomical redistribution towards epithelial layers and enhanced effector functionality. Thus, our results reveal distinct programming of tissue residency in CD8+ and CD4+ TRM cell subsets that is attributable to divergent Runx3 activity.

Familial hemophagocytic lymphohistiocytosis hepatitis is mediated by IFN-γ in a predominantly hepatic-intrinsic manner.

Interferon gamma (IFN-γ) is the main cytokine driving organ dysfunction in Familial Hemophagocytic Lymphohistiocytosis (FHL). Blockade of IFN-γ pathway ameliorates FHL hepatitis, both in animal models and in humans with FHL. Hepatocytes are known to express IFN-γ receptor (IFN-γ-R). However, whether IFN-γ induced hepatitis in FHL is a lymphocyte or liver intrinsic response to the cytokine has yet to be elucidated. Using a IFNgR-/- bone marrow chimeric model, this study showed that non-hematopoietic IFN-γ response is critical for development of FHL hepatitis in LCMV-infected Prf1-/- mice. Lack of hepatic IFN-γ responsiveness results in reduced hepatitis as measured by hepatomegaly, alanine aminotransferase (ALT) levels and abrogated histologic endothelial inflammation. In addition, IFN-γ non-hematopoietic response was critical in activation of lymphocytes by soluble interleukin 2 receptor (sIL-2r) and recruitment of CD8+ effector T lymphocytes (CD8+ CD44hi CD62Llo) (Teff) and inflammatory monocytes. Lastly, non-hematopoietic IFN-γ response results in increased hepatic transcription of type 1 immune response and oxidative stress response pathways, while decreasing transcription of genes involved in extracellular matrix (ECM) production. In summary, these findings demonstrate that there is a hepatic transcriptional response to IFN-γ, likely critical in the pathogenesis of FHL hepatitis and hepatic specific responses could be a therapeutic target in this disorder.

Sphingosine 1-phosphate receptor 5 (S1PR5) regulates the peripheral retention of tissue-resident lymphocytes.

Tissue-resident memory T (TRM) cells provide long-lasting immune protection. One of the key events controlling TRM cell development is the local retention of TRM cell precursors coupled to downregulation of molecules necessary for tissue exit. Sphingosine-1-phosphate receptor 5 (S1PR5) is a migratory receptor with an uncharted function in T cells. Here, we show that S1PR5 plays a critical role in T cell infiltration and emigration from peripheral organs, as well as being specifically downregulated in TRM cells. Consequentially, TRM cell development was selectively impaired upon ectopic expression of S1pr5, whereas loss of S1pr5 enhanced skin TRM cell formation by promoting peripheral T cell sequestration. Importantly, we found that T-bet and ZEB2 were required for S1pr5 induction and that local TGF-ß signaling was necessary to promote coordinated Tbx21, Zeb2, and S1pr5 downregulation. Moreover, S1PR5-mediated control of tissue residency was conserved across innate and adaptive immune compartments. Together, these results identify the T-bet-ZEB2-S1PR5 axis as a previously unappreciated mechanism modulating the generation of tissue-resident lymphocytes.

Discrete tissue microenvironments instruct diversity in resident memory T cell function and plasticity.

Tissue-resident memory T (TRM) cells are non-recirculating cells that exist throughout the body. Although TRM cells in various organs rely on common transcriptional networks to establish tissue residency, location-specific factors adapt these cells to their tissue of lodgment. Here we analyze TRM cell heterogeneity between organs and find that the different environments in which these cells differentiate dictate TRM cell function, durability and malleability. We find that unequal responsiveness to TGFß is a major driver of this diversity. Notably, dampened TGFß signaling results in CD103- TRM cells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFß-responsive CD103+ TRM counterparts. Furthermore, whereas CD103- TRM cells readily modified their phenotype upon relocation, CD103+ TRM cells were comparatively resistant to transdifferentiation. Thus, despite common requirements for TRM cell development, tissue adaptation of these cells confers discrete functional properties such that TRM cells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment.

Amelioration of Murine Macrophage Activation Syndrome by Monomethyl Fumarate in Both a Heme Oxygenase 1-Dependent and Heme Oxygenase 1-Independent Manner.

OBJECTIVE: Macrophage activation syndrome (MAS) is characterized by increased serum levels of ferritin and heme oxygenase 1 (HO-1), and yet no known function is ascribed to these molecules in MAS. Because HO-1 is antiinflammatory, we hypothesized that pharmacologic activation of HO-1 could ameliorate MAS disease activity. Dimethyl fumarate (DMF), a treatment approved by the US Food and Drug Administration for multiple sclerosis, activates HO-1. Monomethyl fumarate (MMF) is the active metabolite of DMF. We therefore evaluated whether MMF could elicit HO-1-dependent therapeutic improvements in a murine model of MAS. METHODS: We induced MAS by repeated activation of Toll-like receptor 9 (TLR-9) in wild-type and myeloid-specific HO-1-deficient mice. MMF was administered twice daily to test its efficacy. We assessed organ weights, serum cytokine levels, histologic features of the spleen and liver tissue, and complete blood cell counts to evaluate disease activity. Statistical testing was performed using Student's t-test or by 2-way analysis of variance as appropriate. RESULTS: The presence of HO-1 was required for the majority of TLR-9-induced interleukin-10 (IL-10). IL-10 production in TLR-9-induced MAS was found to correlate with the myeloid-HO-1 gene dose in myeloid cells (P < 0.001). MMF treatment increased the levels of HO-1 in splenic macrophages by ~2-fold (P < 0.01), increased serum levels of IL-10 in an HO-1-dependent manner in mice with TLR-9-induced MAS (P < 0.005), and improved multiple disease parameters in both an HO-1-dependent and HO-1-independent manner. CONCLUSION: TLR-9-induced production of IL-10 is regulated by HO-1 activity both in vitro and in vivo. Therapeutic enhancement of the HO-1/IL-10 axis in a murine model was able to significantly ameliorate MAS disease activity. These results suggest that HO-1 may be viable as a MAS therapeutic target, and treatment with DMF and MMF should be considered in future investigations of MAS therapy.

A Spontaneous RAG1 Nonsense Mutation Unveils Naturally Occurring N-Terminal Truncated RAG1 Isoforms.

The RAG1 and RAG2 proteins are essential for the assembly of Ag receptor genes in the process known as VDJ recombination, allowing for an immense diversity of lymphocyte Ag receptors. Congruent with their importance, RAG1 and RAG2 have been a focus of intense study for decades. To date, RAG1 has been studied as a single isoform; however, our identification of a spontaneous nonsense mutation in the 5' region of the mouse Rag1 gene lead us to discover N-truncated RAG1 isoforms made from internal translation initiation. Mice homozygous for the RAG1 nonsense mutation only express N-truncated RAG1 isoforms and have defects in Ag receptor rearrangement similar to human Omenn syndrome patients with truncating 5' RAG1 frameshift mutations. We show that the N-truncated RAG1 isoforms are derived from internal translation initiation start sites. Given the seemingly inactivating Rag1 mutation, it is striking that homozygous mutant mice do not have the expected SCID. We propose that evolution has garnered RAG1 and other important genes with the ability to form truncated proteins via internal translation to minimize the deleterious effects of 5' nonsense mutations. This mechanism of internal translation initiation is particularly important to consider when interpreting nonsense or frameshift mutations in whole-genome sequencing, as such mutations may not lead to loss of protein.

Genetic Deficiency of Interferon-γ Reveals Interferon-γ-Independent Manifestations of Murine Hemophagocytic Lymphohistiocytosis.

OBJECTIVE: Familial hemophagocytic lymphohistiocytosis (FHLH) is a complex cytokine storm syndrome caused by genetic abnormalities rendering CD8+ T cells and natural killer cells incapable of cytolytic killing. In murine models of FHLH, interferon-γ (IFNγ) produced by CD8+ T cells has been identified as a critical mediator of disease, and an IFNγ-blocking antibody (emapalumab) has recently been approved by the Food and Drug Administration. However, development of hemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) in patients who are genetically unresponsive to IFNγ questions the absolute necessity of IFNγ in driving disease. This study was undertaken to determine the necessity of IFNγ in driving HLH. METHODS: IFNγ-/- Prf1-/- mice were infected with lymphocytic choriomeningitis virus (LCMV), and HLH immunopathologic features, including survival, weight loss, cytopenias, cytokine profiles, and immune cell phenotypes, were assessed. Mixed bone marrow chimeras were created to determine the immune cell-intrinsic role of IFNγ receptor signaling. CD8+ T cell depletion and interleukin-33 (IL-33)/ST2 blockade were performed using monoclonal antibodies. RESULTS: LCMV infection of IFNγ-/- Prf1-/- mice resulted in severe HLH-like disease. CD8+ T cells and the IL-33/ST2 axis remained essential mediators of disease; however, IFNγ-independent HLH immunopathology correlated with a 10-15-fold increase in neutrophilia (P < 0.001) and an altered cytokine milieu dominated by IL-6, IL-1ß, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05). Furthermore, IFNγ regulated CD8+ T cell expression of GM-CSF and neutrophil survival. CONCLUSION: IFNγ is not necessary for the development of fulminant HLH, requiring physicians to consider case-by-case treatment strategies. Use of therapies that target upstream activators of CD8+ T cells, such as IL-33/ST2 signaling, may be more universally applicable treatment options that ameliorate both IFNγ-dependent and -independent manifestations of HLH/MAS.

Disruption of IL-33 Signaling Limits Early CD8+ T Cell Effector Function Leading to Exhaustion in Murine Hemophagocytic Lymphohistiocytosis.

Danger signals mediated through ST2, the interleukin-33 (IL-33) receptor, amplify CD8+ T cell-mediated inflammation in the murine model of familial hemophagocytic lymphohistiocytosis type 2 (FHL2), and blockade of ST2 provides a potential therapeutic strategy in this disease. However, the long-term effects of disrupting IL-33/ST2 signaling on the CD8+ T cell compartment are unknown. Here, we examined the evolution of the T cell response in murine FHL type 2 in the absence of ST2 signaling and found that CD8+ T cells gradually undergo exhaustion, similar to a related nonfatal FHL model. ST2 inhibition indirectly promotes CD8+ T cell exhaustion, and in contrast to other forms of FHL, reversal of exhaustion does not affect mortality. Disruption of IL-33 signaling exerts a more significant impact on the CD8+ T cell compartment early in the course of disease by intrinsically limiting CD8+ T cell proliferative and cytokine production capacity. Our data thus suggest that while ST2 blockade ultimately enables the development of CD8+ T cell exhaustion in late-stage murine FHL2, exhaustion is merely an effect, rather than the cause, of extended survival in these mice. The acute impact of ST2 inhibition on both the quantity and quality of the effector CD8+ T cell response more likely underlies the protective benefits of this treatment. This study provides evidence that redefines the relationship between CD8+ T cell exhaustion and mortality in murine FHL and supports the therapeutic use of ST2 blockade during the acute stage of disease.

CD8 T Cell Memory Increases Immunopathology in the Perforin-Deficient Model of Hemophagocytic Lymphohistiocytosis Secondary to TNF-α.

Familial hemophagocytic lymphohistiocytosis 2 (FHL2) is a cytokine storm syndrome characterized by immune hyperactivation with viral infection due to a CD8 T cell cytotoxic killing defect secondary to a perforin deficiency. As most studies of FHL2 mice have used pathogen naïve animals, the effects of immune memory on FHL2 are understudied. We utilized an immunization model of the perforin-deficient mouse to study the effects of immune memory on FHL2. Prior CD8 T cell specific antigen exposure leads to enhanced HLH disease with increased morbidity and decreased time to mortality. Enhanced disease is associated with altered cytokine production and T cell proliferation. Response to IFNγ blockade is reduced and TNFα gains a pathogenic role, while blockade of the IL-33 receptor ST2 remains effective. These results suggest that pre-existing immune memory may worsen outcome and alter treatment response for FHL2 patients who may not be naïve to their immune triggers.