Reversal of the T cell immune system reveals the molecular basis for T cell lineage fate determination in the thymus.

T cell specificity and function are linked during development, as MHC-II-specific TCR signals generate CD4 helper T cells and MHC-I-specific TCR signals generate CD8 cytotoxic T cells, but the basis remains uncertain. We now report that switching coreceptor proteins encoded by Cd4 and Cd8 gene loci functionally reverses the T cell immune system, generating CD4 cytotoxic and CD8 helper T cells. Such functional reversal reveals that coreceptor proteins promote the helper-lineage fate when encoded by Cd4, but promote the cytotoxic-lineage fate when encoded in Cd8-regardless of the coreceptor proteins each locus encodes. Thus, T cell lineage fate is determined by cis-regulatory elements in coreceptor gene loci and is not determined by the coreceptor proteins they encode, invalidating coreceptor signal strength as the basis of lineage fate determination. Moreover, we consider that evolution selected the particular coreceptor proteins that Cd4 and Cd8 gene loci encode to avoid generating functionally reversed T cells because they fail to promote protective immunity against environmental pathogens.

The Bone Marrow Protects and Optimizes Immunological Memory during Dietary Restriction.

Mammals evolved in the face of fluctuating food availability. How the immune system adapts to transient nutritional stress remains poorly understood. Here, we show that memory T cells collapsed in secondary lymphoid organs in the context of dietary restriction (DR) but dramatically accumulated within the bone marrow (BM), where they adopted a state associated with energy conservation. This response was coordinated by glucocorticoids and associated with a profound remodeling of the BM compartment, which included an increase in T cell homing factors, erythropoiesis, and adipogenesis. Adipocytes, as well as CXCR4-CXCL12 and S1P-S1P1R interactions, contributed to enhanced T cell accumulation in BM during DR. Memory T cell homing to BM during DR was associated with enhanced protection against infections and tumors. Together, this work uncovers a fundamental host strategy to sustain and optimize immunological memory during nutritional challenges that involved a temporal and spatial reorganization of the memory pool within "safe haven" compartments.

BACH2 enforces the transcriptional and epigenetic programs of stem-like CD8+ T cells.

During chronic infection and cancer, a self-renewing CD8+ T cell subset maintains long-term immunity and is critical to the effectiveness of immunotherapy. These stem-like CD8+ T cells diverge from other CD8+ subsets early after chronic viral infection. However, pathways guarding stem-like CD8+ T cells against terminal exhaustion remain unclear. Here, we show that the gene encoding transcriptional repressor BACH2 is transcriptionally and epigenetically active in stem-like CD8+ T cells but not terminally exhausted cells early after infection. BACH2 overexpression enforced stem-like cell fate, whereas BACH2 deficiency impaired stem-like CD8+ T cell differentiation. Single-cell transcriptomic and epigenomic approaches revealed that BACH2 established the transcriptional and epigenetic programs of stem-like CD8+ T cells. In addition, BACH2 suppressed the molecular program driving terminal exhaustion through transcriptional repression and epigenetic silencing. Thus, our study reveals a new pathway that enforces commitment to stem-like CD8+ lineage and prevents an alternative terminally exhausted cell fate.

Single-cell RNA-seq reveals TOX as a key regulator of CD8+ T cell persistence in chronic infection.

Progenitor-like CD8+ T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including T cell-specific transcription factor 1 (TCF1), but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing the single-cell transcriptomes and epigenetic profiles of CD8+ T cells responding to acute and chronic viral infections, we found that progenitor-like CD8+ T cells became distinct from memory precursor cells before the peak of the T cell response. We discovered a coexpression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursor cells. Moreover, thymocyte selection-associated high mobility group box protein TOX (TOX) promoted the persistence of antiviral CD8+ T cells and was required for the programming of progenitor-like CD8+ T cells. Thus, long-term CD8+ T cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.

Mucosal plasma cells are required to protect the upper airway and brain from infection.

While blood antibodies mediate protective immunity in most organs, whether they protect nasal surfaces in the upper airway is unclear. Using multiple viral infection models in mice, we found that blood-borne antibodies could not defend the olfactory epithelium. Despite high serum antibody titers, pathogens infected nasal turbinates, and neurotropic microbes invaded the brain. Using passive antibody transfers and parabiosis, we identified a restrictive blood-endothelial barrier that excluded circulating antibodies from the olfactory mucosa. Plasma cell depletions demonstrated that plasma cells must reside within olfactory tissue to achieve sterilizing immunity. Antibody blockade and genetically deficient models revealed that this local immunity required CD4+ T cells and CXCR3. Many vaccine adjuvants failed to generate olfactory plasma cells, but mucosal immunizations established humoral protection of the olfactory surface. Our identification of a blood-olfactory barrier and the requirement for tissue-derived antibody has implications for vaccinology, respiratory and CNS pathogen transmission, and B cell fate decisions.

Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

Chemokine guidance of central memory T cells is critical for antiviral recall responses in lymph nodes.

A defining feature of vertebrate immunity is the acquisition of immunological memory, which confers enhanced protection against pathogens by mechanisms that are incompletely understood. Here, we compared responses by virus-specific naive T cells (T(N)) and central memory T cells (T(CM)) to viral antigen challenge in lymph nodes (LNs). In steady-state LNs, both T cell subsets localized in the deep T cell area and interacted similarly with antigen-presenting dendritic cells. However, upon entry of lymph-borne virus, only T(CM) relocalized rapidly and efficiently toward the outermost LN regions in the medullary, interfollicular, and subcapsular areas where viral infection was initially confined. This rapid peripheralization was coordinated by a cascade of cytokines and chemokines, particularly ligands for T(CM)-expressed CXCR3. Consequently, in vivo recall responses to viral infection by CXCR3-deficient T(CM) were markedly compromised, indicating that early antigen detection afforded by intranodal chemokine guidance of T(CM) is essential for efficient antiviral memory.

Protective human antibodies against a conserved epitope in pre- and postfusion influenza hemagglutinin.

Phylogenetically and antigenically distinct influenza A and B viruses (IAV and IBV) circulate in human populations, causing widespread morbidity. Antibodies (Abs) that bind epitopes conserved in both IAV and IBV hemagglutinins (HAs) could protect against disease by diverse virus subtypes. Only one reported HA Ab, isolated from a combinatorial display library, protects against both IAV and IBV. Thus, there has been so far no information on the likelihood of finding naturally occurring human Abs that bind HAs of diverse IAV subtypes and IBV lineages. We have now recovered from several unrelated human donors five clonal Abs that bind a conserved epitope preferentially exposed in the postfusion conformation of IAV and IVB HA2. These Abs lack neutralizing activity in vitro but in mice provide strong, IgG subtype-dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines.

Enolase inhibitors as therapeutic leads for Naegleria fowleri infection.

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amoebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50 = 0.14 ± 0.04 µM) that was toxic to trophozoites (EC50 = 0.21 ± 0.02 µM) while the reported CC50 was >300 µM. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the end of the one-week observation, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. However, intranasal HEX delivery was not curative as brains of six of the eight survivors were positive for amoebae. These findings suggest that HEX requires further evaluation to develop as a lead for treatment of PAM.

The Chemokine Receptor CX3CR1 Defines Three Antigen-Experienced CD8 T Cell Subsets with Distinct Roles in Immune Surveillance and Homeostasis.

Infections induce pathogen-specific T cell differentiation into diverse effectors (Teff) that give rise to memory (Tmem) subsets. The cell-fate decisions and lineage relationships that underlie these transitions are poorly understood. Here, we found that the chemokine receptor CX3CR1 identifies three distinct CD8+ Teff and Tmem subsets. Classical central (Tcm) and effector memory (Tem) cells and their corresponding Teff precursors were CX3CR1- and CX3CR1high, respectively. Viral infection also induced a numerically stable CX3CR1int subset that represented ∼15% of blood-borne Tmem cells. CX3CR1int Tmem cells underwent more frequent homeostatic divisions than other Tmem subsets and not only self-renewed, but also contributed to the expanding CX3CR1- Tcm pool. Both Tcm and CX3CR1int cells homed to lymph nodes, but CX3CR1int cells, and not Tem cells, predominantly surveyed peripheral tissues. As CX3CR1int Tmem cells present unique phenotypic, homeostatic, and migratory properties, we designate this subset peripheral memory (tpm) cells and propose that tpm cells are chiefly responsible for the global surveillance of non-lymphoid tissues.

Therapeutic glycan-specific antibody binding mediates protection during primary amoebic meningoencephalitis.

Naegleria fowleri (N. fowleri) infection via the upper respiratory tract causes a fatal CNS disease known as primary amoebic meningoencephalitis (PAM). The robust in vivo immune response to N. fowleri infection underlies the immunopathology that characterizes the disease. However, little is known about why this pathogen evades immune control. Infections occur in seemingly healthy individuals and effective clinical options are lacking, thus a nearly 98% fatality rate. It is unclear how or if host factors may contribute to susceptibility or disease exacerbation, yet mechanistic studies of the in vivo immune response and disease progression are hampered by a lack of tools. In this study, we have generated monoclonal antibodies to N. fowleri surface antigens and shown them to be excellent tools for studying the in vivo immune response. We also identified one monoclonal, 2B6, with potent inherent anti-amoebastatic activity in vitro. This antibody is also able to therapeutically prolong host survival in vivo and furthermore, recombinant antibodies with an isotype more capable of directing immune effector activity further improved survival when given therapeutically. Thus, we report the generation of a novel monoclonal antibody to N. fowleri that can enhance beneficial immune functions, even when given therapeutically during disease. We believe this provides evidence for the potential of therapeutic antibody treatments in PAM.IMPORTANCENaegleria fowleri (N. fowleri) is a free-living amoeba that is found ubiquitously in warm freshwater. While human exposure is common, it rarely results in pathogenesis. However, when N. fowleri gains access to the upper airway, specifically the olfactory mucosa, infection leads to a lethal disease known as primary amoebic meningoencephalitis (PAM). As a free-living amoeba, N. fowleri does not need a mammalian host; indeed, it can be accurately described as an accidental opportunistic pathogen. While most opportunistic infections occur in humans who are immunocompromised, there are no reported immune dysfunctions associated with N. fowleri infection. Therefore, the basis for N. fowleri opportunism is not known, and the reasons why some humans develop PAM while others do not are simply not well understood. It is reasonable to speculate that local or acute immune failures, potentially even a lack of prior adaptive immunity, are related to disease susceptibility. Careful immune profiling and characterization of the in vivo immune response to N. fowleri in a mammalian host are desperately needed to understand which host factors are critical to defense, and how these responses might be compromised in a way that results in lethal infection. To identify genes and pathways that provide resistance against in vivo N. fowleri infection, we generated surface reactive monoclonal antibodies (Abs) that provide rapid amoeba detection and quantification in vivo. Interestingly, N. fowleri binding Abs have been readily detected in the serum and saliva of humans and animals suggesting that non-lethal exposure drives a humoral immune response against the amoeba. Yet, how Abs might interact with Naegleria in vivo or contribute to preventing lethal infection is not well understood. In this study, we have generated and characterized a monoclonal antibody (Ab), Clone 2B6, that recognizes a glycosylated surface antigen present in cultured in vitro N. fowleri as well as mouse passaged N. fowleri. When clone 2B6 binds to N. fowleri, it inhibits amoeba motility and feeding behavior, leading to strong growth inhibition. Mice treated systemically and intracerebrally with Ab displayed a delayed disease onset and prolonged survival. In addition, we found that enhancing immune-directed effector activity via antibody isotype could further enhance survival without obvious immunopathogenic side effects. These findings show the potential for antibody treatment as an additional therapeutic to those used currently in PAM.

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

B cell maintenance of subcapsular sinus macrophages protects against a fatal viral infection independent of adaptive immunity.

Neutralizing antibodies have been thought to be required for protection against acutely cytopathic viruses, such as the neurotropic vesicular stomatitis virus (VSV). Utilizing mice that possess B cells but lack antibodies, we show here that survival upon subcutaneous (s.c.) VSV challenge was independent of neutralizing antibody production or cell-mediated adaptive immunity. However, B cells were absolutely required to provide lymphotoxin (LT) α1ß2, which maintained a protective subcapsular sinus (SCS) macrophage phenotype within virus draining lymph nodes (LNs). Macrophages within the SCS of B cell-deficient LNs, or of mice that lack LTα1ß2 selectively in B cells, displayed an aberrant phenotype, failed to replicate VSV, and therefore did not produce type I interferons, which were required to prevent fatal VSV invasion of intranodal nerves. Thus, although B cells are essential for survival during VSV infection, their contribution involves the provision of innate differentiation and maintenance signals to macrophages, rather than adaptive immune mechanisms.

Endothelial heparan sulfate controls chemokine presentation in recruitment of lymphocytes and dendritic cells to lymph nodes.

Heparan sulfate can bind several adhesion molecules involved in lymphocyte trafficking. However, the in vivo function of endothelial heparan sulfate in lymphocyte homing and stimulation of the immune response has not been elucidated. Here, we generated mutant mice deficient in the enzyme Ext1, which is required for heparan sulfate synthesis, in a Tek-dependent and inducible manner. Chemokine presentation was diminished in the mutant mice, causing the lack of appropriate integrin-mediated adhesion, and resulted in a marked decrease in lymphocyte sticking to high endothelial venules and in recruitment of resident dendritic cells through lymphatic vessels to the lymph nodes. As a consequence, mutant mice displayed a severe impairment in lymphocyte homing and a compromised contact hypersensitivity response. By contrast, lymphocyte rolling was increased because of loss of electrostatic repulsion by heparan sulfate. These results demonstrate critical roles of endothelial heparan sulfate in immune surveillance and immune response generation.

The great balancing act: regulation and fate of antiviral T-cell interactions.

The fate of T lymphocytes revolves around a continuous stream of interactions between the T-cell receptor (TCR) and peptide-major histocompatibility complex (MHC) molecules. Beginning in the thymus and continuing into the periphery, these interactions, refined by accessory molecules, direct the expansion, differentiation, and function of T-cell subsets. The cellular context of T-cell engagement with antigen-presenting cells, either in lymphoid or non-lymphoid tissues, plays an important role in determining how these cells respond to antigen encounters. CD8(+) T cells are essential for clearance of a lymphocytic choriomeningitis virus (LCMV) infection, but the virus can present a number of unique challenges that antiviral T cells must overcome. Peripheral LCMV infection can lead to rapid cytolytic clearance or chronic viral persistence; central nervous system infection can result in T-cell-dependent fatal meningitis or an asymptomatic carrier state amenable to immunotherapeutic clearance. These diverse outcomes all depend on interactions that require TCR engagement of cognate peptide-MHC complexes. In this review, we explore the diversity in antiviral T-cell behaviors resulting from TCR engagement, beginning with an overview of the immunological synapse and progressing to regulators of TCR signaling that shape the delicate balance between immunopathology and viral clearance.

Subcapsular sinus macrophages prevent CNS invasion on peripheral infection with a neurotropic virus.

Lymph nodes (LNs) capture microorganisms that breach the body's external barriers and enter draining lymphatics, limiting the systemic spread of pathogens. Recent work has shown that CD11b(+)CD169(+) macrophages, which populate the subcapsular sinus (SCS) of LNs, are critical for the clearance of viruses from the lymph and for initiating antiviral humoral immune responses. Here we show, using vesicular stomatitis virus (VSV), a relative of rabies virus transmitted by insect bites, that SCS macrophages perform a third vital function: they prevent lymph-borne neurotropic viruses from infecting the central nervous system (CNS). On local depletion of LN macrophages, about 60% of mice developed ascending paralysis and died 7-10 days after subcutaneous infection with a small dose of VSV, whereas macrophage-sufficient animals remained asymptomatic and cleared the virus. VSV gained access to the nervous system through peripheral nerves in macrophage-depleted LNs. In contrast, within macrophage-sufficient LNs VSV replicated preferentially in SCS macrophages but not in adjacent nerves. Removal of SCS macrophages did not compromise adaptive immune responses against VSV, but decreased type I interferon (IFN-I) production within infected LNs. VSV-infected macrophages recruited IFN-I-producing plasmacytoid dendritic cells to the SCS and in addition were a major source of IFN-I themselves. Experiments in bone marrow chimaeric mice revealed that IFN-I must act on both haematopoietic and stromal compartments, including the intranodal nerves, to prevent lethal infection with VSV. These results identify SCS macrophages as crucial gatekeepers to the CNS that prevent fatal viral invasion of the nervous system on peripheral infection.

Mannose receptor 1 mediates cellular uptake and endosomal delivery of CpG-motif containing oligodeoxynucleotides.

Recognition of microbial components is critical for activation of TLRs, subsequent innate immune signaling, and directing adaptive immune responses. The DNA sensor TLR9 traffics from the endoplasmic reticulum to endolysosomal compartments where it is cleaved by resident proteases to generate a competent receptor. Activation of TLR9 by CpG-motif containing oligodeoxynucleotides (CpG ODNs) is preceded by agonist endocytosis and delivery into the endolysosomes. The events that dictate this process remain largely unknown; furthermore, it is unclear whether the receptors involved in mediating uptake of exogenous DNA are conserved for both naturally derived pathogenic DNA and synthetic ODNs. In this study, we report that peritoneal macrophages from a wild-derived inbred mouse strain, MOLF/Ei, are hyporesponsive to CpG ODN but are fully responsive to bacterial DNA, thus implying that microbial recognition is not fully recapitulated by a synthetic analog. To identify the gene responsible for the CpG ODN defect, we have performed genome-wide linkage analysis. Using N2 backcross mice, we mapped the trait with high resolution to a single locus containing Mrc1 as the gene conferring the trait. We show that mannose receptor 1 (MRC1; CD206) is involved in CpG ODN uptake and trafficking in wild-derived MOLF/Ei peritoneal macrophages. Furthermore, we show that other strains of wild-derived mice also require MRC1 for CpG-induced cytokine responses. These findings reveal novel functions for MRC1 and demonstrate that wild-derived mice are important and indispensable model for understanding naturally occurring regulators of inflammatory responses in innate immune pathways.

Olfactory immunology: the missing piece in airway and CNS defence.

The olfactory mucosa is a component of the nasal airway that mediates the sense of smell. Recent studies point to an important role for the olfactory mucosa as a barrier to both respiratory pathogens and to neuroinvasive pathogens that hijack the olfactory nerve and invade the CNS. In particular, the COVID-19 pandemic has demonstrated that the olfactory mucosa is an integral part of a heterogeneous nasal mucosal barrier critical to upper airway immunity. However, our insufficient knowledge of olfactory mucosal immunity hinders attempts to protect this tissue from infection and other diseases. This Review summarizes the state of olfactory immunology by highlighting the unique immunologically relevant anatomy of the olfactory mucosa, describing what is known of olfactory immune cells, and considering the impact of common infectious diseases and inflammatory disorders at this site. We will offer our perspective on the future of the field and the many unresolved questions pertaining to olfactory immunity.