The Journal of Immunology

This 28-color panel was developed to identify classical and non-classical T lymphocytes in decidual leukocytes and peripheral blood mononuclear cells (PBMC) of pregnant rhesus macaques. By profiling these T lymphocytes, we can investigate how maternal immunity balances tolerance to fetal antigens with protection against vertically transmitted pathogens. The selected markers define memory populations and characterize tissue residency, activation, proliferation, cytotoxicity, trafficking, and exhaustion status. This panel also delineates B lymphocytes and NK cells to confirm expected frequencies. The utility of this panel is aimed at evaluating cellular immune correlates of protection against congenital infections at the maternal-fetal interface and PBMC in rhesus macaques.

Group 2 innate lymphoid cells (ILC2s) regulate type 2 immune responses partly by recruiting eosinophils, but the mechanisms underlying this process remain incompletely understood. Although both ILC2s and type 2 T helper (Th2) cells are capable of expressing IL-5, a cytokine critical for recruiting eosinophils, ILC2s are more potent than Th2 cells in this process. Here, we show that the transcription factor Bhlhe40 promotes GM-CSF production in ILC2s, and Bhlhe40 expression in ILC2s is required for efficient eosinophil recruitment during both papain- and helminth-induced type 2 immune responses. However, Bhlhe40 deficiency in ILC2s had no effect on the production of classical type 2 cytokines, including IL-4, IL-5 and IL-13, despite Bhlhe40 being required for type 2 cytokine production by Th2 cells. Furthermore, in contrast to ILC2s, Th2 cells produced little GM-CSF and administration of GM-CSF rescued eosinophil recruitment in ILC2-specific Bhlhe40-deficient mice. In addition to promoting GM-CSF expression, Bhlhe40 repressed IL-10 production in ILC2s as it did in Th2 cells, particularly during chronic inflammation. Single-cell transcriptomic analyses further supported this regulatory network, and the ChIP-Seq data revealed direct binding of Bhlhe40 to the enhancer and promoter regions within the Il10 and Csf2 loci. Collectively, our findings show that Bhlhe40 exerts distinct gene regulatory functions in ILC2s and Th2 cells, and Bhlhe40 modulates the inflammatory and anti-inflammatory functions of ILC2s by promoting GM-CSF while suppressing IL-10 production.

Antibodies engage both activating Fc Receptors and the inhibitory receptor Fc{gamma}RIIB. Why macrophages need a dedicated inhibitory receptor rather than simply tuning activating receptor signaling is unclear. Using DNA-based chimeric receptors and in silico modeling, we independently controlled activating and inhibitory Fc Receptors. We found that Fc{gamma}RIIB imposed a time limit on macrophage phagocytosis and ERK signaling. The time limit is due to activating Fc Receptors converting PI(4,5)P2 to PI(3,4,5)P3, which is subsequently converted to PI(3,4)P2 by Fc{gamma}RIIB. This leads to a pulse of active signaling, which is sufficient for phagocytosis of small bacteria-sized targets but not phagocytosis of large targets and TNF secretion. Unlike engaging Fc{gamma}RIIB, reducing activating Fc Receptor signaling decreased initiation of phagocytosis, the speed of PI(3,4,5)P3 generation, and the amplitude of ERK signaling. Our results demonstrate that Fc{gamma}RIIB controls the duration of IgG signaling, while the activating Fc Receptors control sensitivity.

Cutaneous leishmaniasis is characterized by chronic inflammatory skin lesions in which CD8{square} T cells exhibit paradoxical functions. While IFN-{gamma}-producing CD8{square} T cells contribute to the development of protective immunity in the draining lymph node, CD8{square} T cells recruited to the infected skin lose their ability to produce IFN-{gamma} and instead acquire cytotoxic functions that exacerbate tissue damage. We previously demonstrated that the hypoxic microenvironment of leishmanial lesions promotes CD8{square} T cell cytotoxicity through induction of Blimp-1. Whether hypoxia also suppresses protective CD8{square} T cell functions, however, is unknown. Here, we show that hypoxia simultaneously suppresses production of the protective cytokines IFN-{gamma} and TNF- while enhancing expression of granzyme B and perforin in activated CD8{square} T cells. In vitro, HIF-1, but not HIF-2, was required for hypoxia-induced expression of granzyme B, perforin, and Blimp-1, whereas suppression of IFN-{gamma} and TNF- occurred independently of HIF signaling, indicating that distinct oxygen-related pathways regulate pathogenic and protective CD8{square} T cell functions. Hypoxia also increased expression of multiple inhibitory receptors on CD8{square} T cells, although lesional CD8{square} T cells lacked expression of the terminal exhaustion-associated transcription factor TOX, suggesting that hypoxia promotes an inhibitory phenotype distinct from terminal exhaustion. Finally, adoptive transfer studies demonstrated that in vivo both HIF-1 and HIF-2 expression in CD8{square} T cells contributed to immunopathology during cutaneous leishmaniasis. Together, these findings identify hypoxia as a key regulator that functionally reprograms CD8{square} T cells by promoting pathogenic cytotoxicity while suppressing protective cytokine production within lesions.

Intraepithelial lymphocytes expressing the {gamma}{delta} T cell receptor ({gamma}{delta} IEL) continuously survey the intestinal epithelium to promote mucosal host defense. Although {gamma}{delta} IELs migrate in and out of the lateral intercellular space (LIS) between adjacent enterocytes, the molecular mechanisms governing their migratory behavior are incompletely understood. Based on the known role of CD47, or integrin associated protein (IAP), in mediating neutrophil transepithelial migration, we investigated whether CD47 expression reflects a conserved mechanism regulating {gamma}{delta} IEL surveillance behavior. Here, we report that conditional CD47 deletion on intestinal epithelial cells or {gamma}{delta} T cells had no effect on IEL composition. Using intravital imaging, we identified complementary roles for CD47 on {gamma}{delta} IELs and epithelial cells, with epithelial CD47 restricting {gamma}{delta} IEL motility and {gamma}{delta} T-cell-derived CD47 promoting cell migration. Further investigation revealed that both CD47 and CD18 contribute to {gamma}{delta} IEL surveillance behavior, although CD47 regulates {gamma}{delta} IEL migration in a CD18-independent manner.

Invariant natural killer T (iNKT) cells require mitochondrial metabolism for terminal effector development. We found iNKT cells express elevated levels of proteins regulating mitochondrial membrane dynamics, and identified Opa1, but not Drp1, as selectively required for iNKT cell differentiation. OPA1 deficiency disrupted mitochondrial cristae organization, reduced mitochondrial membrane potential, increased mitochondrial mass, and altered calcium homeostasis in iNKT cells. Bulk and single-cell transcriptomic analyses revealed impaired TCR-responsive gene expression, activation of mitochondrial stress adaptation and integrated stress response, enhanced glycolysis, and retention of immature differentiation features. SCENITH analysis demonstrated increased glycolytic dependence; LDHA became required in Opa1-deficient iNKT cells while dispensable for normal iNKT development, indicating compensatory glycolytic adaptation. EGTA in thymic organ culture partially restored NKT1 marker expression in Opa1-deficient cells. Co-deletion of Drp1 improved mitochondrial morphology, TCR signaling, stress and metabolic adaptation, and partially rescued iNKT cell differentiation, demonstrating that balanced mitochondrial dynamics coordinate mitochondrial function and terminal effector development.

Macrophages in the synovial lining are critical for the maintenance of healthy tissue while also contributing to the pathogenesis of rheumatoid arthritis (RA). However, the field currently lacks a unifying characterization of synovial macrophage heterogeneity across steady-state and inflammation. Here, we defined 4 transcriptionally distinct populations of synovial macrophages CX3CR1+MHCII- (lining); CX3CR1-MHCII- (interstitial/sublining); CX3CR1-MHCII+ (monocyte-derived); and CX3CR1+MHCII+ (infiltrating). The MHCII-populations are long-lived and derived from embryonic precursors regardless of localization while the MHCII+ populations differentiate from bone marrow (BM) progenitors dependent on CCR2. We identified conserved activation pathways between acute and chronic mouse models of inflammatory arthritis as well as novel arthritis-associated subpopulations. During peak inflammation, the influx of BM-derived cells was associated with upregulation of monocyte-related genes with concurrent down-regulation of tissue-resident genes. Our results provide a unifying schema for describing synovial macrophages across conditions and pave the way for future studies in modulating transcriptional activity in rheumatoid arthritis.

Staphylococcus aureus persistently colonises the nasal tissue (NT) of a significant proportion of the population. The long-lasting impact that asymptomatic S. aureus exposure has on immune memory at colonised barrier sites is incompletely understood, potentially impacting vaccine responsiveness in a pre-exposed population. Tissue resident memory (TRM) cells are long-lived T-cells which remain poised at barrier sites for localised reactivation following antigen exposure. This study demonstrates an increase in NT CD4+ and {gamma}{delta}+ TRM cells in response to S. aureus colonisation, which undergo expansion and IL-17 production upon secondary S. aureus exposure. Interestingly, these cells were also capable of non-specific reactivation, with IL-17+ TRM cells in S. aureus colonised mice enhancing protection against K. pneumoniae infection. Ex-vivo data suggest that non-specific CD4+ TRM cell re-activation is pro-inflammatory cytokine dependent, but antigen independent. Overall, these findings demonstrate that S. aureus nasal colonisation shapes long-lasting TRM cell responses in the NT, which have the potential for non-specific bystander reactivation during subsequent heterologous infection.

Maintaining brain homeostasis is crucial for proper function of the central nervous system and has traditionally been attributed to neuronal and glial interactions. However, recent research highlights the essential role of brain-resident immune cells in this process. Our study characterizes brain-specific CD8+ T cells and elucidates their significant contribution to brain homeostasis and behavior. We identified a distinct population of CD8+ T cells that infiltrates the brain during early development, undergoes clonal expansion, and acquires effector memory-like characteristics through interactions with microglia. Notably, the absence of these cells results in hyperactivation of neuronal activity and abnormal behaviors, due to loss of regulation of interferon-gamma (IFN-{gamma}) secreted by CD8+ T cells. Our findings demonstrate that IFN-{gamma} secreting brain-specific CD8+ T cells are crucial for maintaining the physiological level of neuronal excitability and normal behavioral patterns. This study provides novel insights into neuroimmune interactions, emphasizing the critical role of CD8+ T cells in sustaining brain function and behavior.

Allograft inflammatory factor-1 (AIF1) is a scaffold protein expressed predominantly in myeloid antigen-presenting cells (APCs) and associated with inflammatory disease. Although genetic studies have linked AIF1 loci to immune traits, its causal role in physiological immune responses remains poorly defined. We examined AIF1 deficiency using a conditional knockout model with deletion of AIF1 in hematopoietic cells (AIF1-cKO) during development and challenged mice with Listeria monocytogenes. AIF1 loss impaired bacterial clearance and diminished inflammatory responses, indicating reduced immune readiness during infection. AIF1-cKO mice exhibited impaired expansion of antigen-specific CD4+ and CD8+ T cells, accompanied by regulatory-associated phenotypic changes. These defects were associated with reduced cDC1 frequencies and transcriptional and phenotypic remodeling of splenic macrophages toward a less inflammatory state. Single-cell RNA sequencing revealed transcriptional alterations across multiple myeloid and lymphoid compartments despite AIF1 expression being largely restricted to myeloid APC, indicating broader immune remodeling. Increased Tgfbr1 expression was a recurrent feature across several immune populations. Consistent with this finding, AIF1-deficient cells displayed enhanced TGF{beta} responsiveness, while Tgfbr1 silencing partially restored inflammatory responses and T cell priming ex vivo. These findings establish AIF1 as a regulator of immune competence that promotes effective innate and adaptive immune responses.

Ubiquitin-dependent regulation of NF-{kappa}B signaling is essential for B-cell homeostasis and fate decisions, yet the contribution of specific deubiquitinating enzymes remains incompletely defined. OTUB1, a lysine-48-specific deubiquitinase, has been broadly implicated in immune regulation, including control of NF-{kappa}B signaling and prevention of immune hyperactivation. Previous studies have demonstrated that B cell-specific deletion of OTUB1 leads to B cell hyperplasia, increased antibody production, and lupus-like autoimmunity, highlighting its importance in maintaining B cell tolerance and immune homeostasis. Using B cell-specific OTUB1-deficient mice, we show that loss of OTUB1 leads to a marked expansion of marginal zone (MZ) B cells and their precursor populations in the spleen, accompanied by an activated phenotype and enhanced proliferative responses, particularly upon CD40 stimulation. OTUB1 deficiency results in altered CD40-induced NF-{kappa}B signaling, characterized by enhanced I{kappa}B degradation and increased nuclear accumulation of p50-containing NF-{kappa}B complexes, despite reduced RelA stability. Mechanistically, we show that OTUB1 interacts with RelA, restricting its lysine-48-linked ubiquitination and proteasomal degradation, thereby stabilizing this key transcription factor. Collectively, these findings identify RelA as a novel OTUB1 target and uncover an additional layer of ubiquitin-dependent control of NF-{kappa}B signaling that governs splenic B-cell homeostasis and marginal zone B-cell development.

Control of Mycobacterium tuberculosis (Mtb) infection requires CD4+ T cell recognition of infected macrophages. However, T cells also colocalize with non-infected macrophages in granulomas. We investigated whether these bystander macrophages present Mtb antigens and shape human CD4+ T cell responses. Using ex vivo co-culture systems, non-infected monocyte-derived macrophages (MDMs) were exposed to Mtb-infected MDMs or infection-conditioned supernatants before co-incubation with autologous memory CD4+ T cells from individuals with latent Mtb infection (LTBI). Bystander macrophages activated memory CD4+ T cells through MHC-II-dependent antigen presentation. Single-cell T cell receptor (TCR) sequencing and TCR-transduced reporter cell lines identified Mtb-specific clonotypes recognizing both infected and bystander macrophages, as well as clonotypes preferentially recognizing infected cells. Strikingly, a subset of TCRs recognized infected but not bystander macrophages. Antigen transfer occurred through soluble Mtb proteins rather than extracellular vesicles. Compared with responses to infected macrophages, bystander macrophages induced attenuated effector responses. These findings reveal antigen-specific recognition of bystander macrophages and suggest that antigens preferentially presented by infected cells may inform TB vaccine design.

B cell signaling is required for germinal center (GC) selection and synergizes with T cell-help leading to differentiation into effector cells and a protective antibody response. Here, we studied the relationship between B cell signaling and the selection of functional mutations in a humanized mouse model of a CD4 binding-site specific HIV-1 broadly neutralizing antibody precursor. While BCR signaling increased with antigen affinity, immunization-induced frequency of a functional mutation was inversely related to affinity and favored a gain in association rate. Antigen-specific GC B cells and key mutation frequency were higher in the mid-affinity (0.5 - 5M) than in either the higher or lower affinity group, and were consistent with the significantly higher serum neutralization titers in the mid-range group. Our studies show that BCR signaling imposes boundaries (upper/lower) for selection of antibody functional mutations and support a "Goldilocks Zone" model that defines the favored BCR signaling strength for GC selection.

Unconventional T cells recognize non-peptide antigens presented by molecules other than the major histocompatibility complex (MHC) proteins. Among unconventional T cells, natural killer T (NKT) cells, which recognize CD1d-lipid complexes, mucosal-associated invariant T (MAIT) cells, which recognize MR1-metabolite complexes, and {gamma}{delta} T cells, are thoroughly studied. CD1b presents self- and mycobacterial lipids to relatively understudied T cell subsets. Like MAIT cells and type I NKT cells, CD1b-specific cells include subpopulations with conserved TCRs. Consequent to their recognition of a nearly monomorphic antigen-presenting molecule, CD1b-specific T cells might share innate-like features with MAIT, type I NKT, and {gamma}{delta} T cells. Due to their low frequency in the peripheral blood, CD1a-, CD1b-, and CD1c-specific T cells have been studied predominantly as in vitro-expanded clones, so even basic information about their native ex vivo immunophenotypes is lacking. Here, we sort and transcriptionally profile ex vivo two T cell populations that recognize CD1b presenting mycobacterial mycolipids and compare them with conventional CD4 and CD8 T cells, {gamma}{delta} T cells, NK cells, MAIT cells, and NKT cells. We show that both the invariant TCR-expressing CD4+, CD1b-GMM-specific germline encoded mycolyl-reactive (GEM) T cells, as well as the diverse TCR-expressing CD1b-GMM-specific T cells, are transcriptionally closer to conventional T cells than to the innate-like T cell populations {gamma}{delta}, MAIT and type I NKT cells. Thus, despite their recognition of non-polymorphic antigen presenting molecules, CD1b-specific T cells show adaptive rather than innate-like transcriptional features.

Dendritic cells (DCs) are central orchestrators of antiviral immunity but can be exploited by HIV-1 to facilitate systemic dissemination. HIV-1 recognition by CD169 triggers massive endocytosis (MEND)-related internalization of viral particles into sac-like compartments in monocyte-derived dendritic cells (MDDCs), thereby promoting trans-infection of CD4+ T cells. However, MDDCS represent only one DC subpopulation, and whether other physiologically relevant DC subsets exhibit similar behavior remains unclear. Moreover, endogenous CD169 expression and viral capture have not been fully characterized in cDC1, DC2, or pDCs, while remain unknown in DC3. We therefore investigated the contribution of these DC subsets to CD169-mediated HIV-1 capture and subsequent viral dissemination. We used flow cytometry to assess CD169 expression and viral capture across DC subsets differentiated from CD34 hematopoietic stem and progenitor cells (HSPC) obtained from bone marrow (BM) and cord blood (CB), two sources that differ in cellular maturity. Confocal microscopy was used to evaluate sac-like compartment formation following immune activation with IFN- or LPS and treatment with MEND inhibitors. Trans-infection capacity was quantified by measuring luciferase activity in TZM-bl reporter cells. We identified cDC1, DC2, DC3, moDC (similar to MDDCs), and pDC subsets following the differentiation of CD34 HSPC from both CB and BM. Among these populations, DC2, DC3, and moDC displayed high CD169 expression, which was significantly upregulated by IFN- or LPS stimulation. BM-derived DCs exhibited higher basal CD169 expression than CB-derived DCs, whereas stimulation induced greater CD169 upregulation in CB-derived DCs. DC2, DC3, and moDC efficiently captured HIV-1 particles in cultures derived from both sources, and viral uptake was further enhanced by immune activation. Viral internalization into sac-like compartments depended on CD169 binding and MEND-related mechanisms, ultimately facilitating HIV-1 transfer to target cells. DC2 and DC3 are key dendritic cell subsets that support CD169 dependent HIV-1 capture and are associated with MEND-driven internalization into sac-like compartments and subsequent viral transfer to target cells, as was previously described for moDCs. We further observed distinct basal and inducible CD169 expression profiles in BM- and CB-derived DCs, consistent with source-dependent maturation states. These results identify DC2 and DC3 as key cellular contributors to HIV-1 dissemination.

Depletion of B cells in multiple sclerosis (MS) is beneficial yet there is no defined pathogenic B cell subset in MS. Here, we demonstrate that T-bet+ memory B cells are rare in control human brain specimens but are found in MS lesions. Transfer of murine T-bet+ memory B cells into mice with ongoing experimental autoimmune encephalomyelitis (EAE), a model of MS, worsened their disability and demyelination. Microglia/macrophage density increased in central nervous system parenchyma even though B cells mostly remained in barriers. In culture, secreted factors from T-bet+ memory B cells promoted macrophage migration. IFN-{gamma} produced by T-bet+ memory B cells activated microglia to secrete chemokines that further enabled macrophage migration. Consistent with their age-associated elevation, conditional deletion of T-bet in B cells lowered EAE severity in aged but not young mice. We define T-bet+ memory B cells as pathogenic in EAE and MS through their IFN-{gamma}-facilitated interactions with microglia/macrophages.

While professional antigen-presenting cells drive adaptive immunity, atypical cell types can fulfill this role in the bone marrow. Megakaryocytes (MKs) are canonically recognized for platelet production, but recent studies indicate functional heterogeneity and immune potential. We found that ~20% of bone marrow MKs express Major Histocompatibility Complex (MHC) II and co-stimulatory receptors CD80, CD86, CD40, and CD83. These MKs process and present antigen to activate T cells ex vivo in an MHC II-dependent manner. MK/T cell interactions induced TGF-{beta}1 secretion and promoted induced Treg differentiation. Prior stimulation of MKs with LPS or Poly I:C was associated with modest Th1-associated CD4+ T cell responses, including IFN-{gamma} and TNF- production, without robust Th17 differentiation. Immunopeptidomics of the murine MK MHC II receptor confirmed occupancy by exogenous peptides, suggesting in vivo functionality. Using a murine model with MK-targeted deletion of MHC II (Pf4-MHC{Delta}/{Delta}), we observed altered TLR signaling and reduced bone marrow TGF-{beta}1. Together, these findings identify MHC II+ MKs as noncanonical antigen-presenting cells with the potential to modulate CD4 T cell responses as part of the immune regulation of the bone marrow niche.

In TLR7-driven macrophage activation syndrome (MAS), inflammatory hemophagocytes (iHPCs) differentiate from Ly6CHI monocytes, phagocytose red blood cells and promote disease, including anemia and thrombocytopenia. We demonstrate here that IRF5 is required for iHPC differentiation and MAS in TLR7-overexpressing (TLR7.1) mice. Both constitutive and myeloid-specific Irf5 deletion reduced iHPCs and improved anemia, thrombocytopenia and survival. Furthermore, therapeutic inhibition of IRF5 ameliorated MAS features and reduced splenic and circulating iHPCs. While cell-intrinsic IRF5 expression was required for iHPC differentiation, it was not required for TLR7.1 Ly6CHI monocyte differentiation and monocyte transcriptional programs. We further show that the transcriptome and chromatin landscape changed dramatically as iHPCs differentiated from TLR7.1 Ly6CHI monocytes. Many transcriptional programs gained in iHPCs were enriched in genes associated with IRF5-binding accessible chromatin regions, including those associated with NF-kB signaling, cytokine and chemokine production, and complement activation. Our data suggest that IRF5 collaborates with other transcription factor families, including NF-kB, ETS and AP1 members, to regulate iHPC gene programs. Together, our findings demonstrate that expression of IRF5 in myeloid cells is critical for MAS, for iHPC differentiation, and acts broadly across iHPC-specific gene programs in TLR7-driven inflammation.

The cell-intrinsic complement system has emerged as an important orchestrator of a variety of cell-physiological processes, with complement components interacting with intracellular effector systems to regulate cellular responses to pathogens or noxious stimuli. For instance, intracellular C5 signaling through a mitochondrial C5a receptor (C5aR1) controls IL-1{beta} production in human monocytes and macrophages. Here, we investigated whether cell-intrinsic C3 similarly regulates inflammatory responses in macrophages. In LPS-stimulated C3 knockout THP-1-derived macrophages, interferon (IFN)-{beta} production was increased, accompanied by elevated expression of interferon-stimulated genes and enhanced secretion of IFN-induced cytokines and chemokines. C3-deficient cells showed increased phosphorylation of IRF3 at Ser396 and a stabilization of the interaction between IRF3 and TBK1, along with enhanced IRF3 dimerization and nuclear translocation. TBK1 phosphorylation was unaffected, indicating that C3 limits IRF3-TBK1 complex formation rather than upstream TBK1 activation. Small-molecule inhibitors of complement factors B and D restored full-length C3 abundance in LPS-stimulated primary human macrophages, consistent with inhibition of the C3 convertase. It also reduced LPS-induced IFN-{beta} production in primary human macrophages and THP-1 cells, suggesting that full-length, uncleaved C3 suppresses IFN-{beta} production. Collectively, these findings identify cell-intrinsic C3 as a suppressor of IFN-{beta} production in human macrophages, highlighting the importance of the cell-intrinsic complement system in fine-tuning inflammatory responses to pathogens.

The MRTFs (myocardin-related transcription factors) are essential for effective cytoskeletal function during differentiation and development. To investigate the role played by the MRTFs in T cell development and function we inactivated Mrtfb in Mrtfa-null mice at the DN2 stage (MrtfabKOT animals). Positive selection, and development of MrtfabKOT regulatory (Treg), {gamma}{delta}, and NK T cells proceeded normally. Thymic egress was impaired, however, with mature T cells accumulating at the cortico-medullary junction, and in vitro MrtfabKOT thymocytes exhibited impaired cytoskeletal responses to S1P and migratory cytokines. MrtfabKOT mice were lymphopenic, with defective homeostatic proliferation and T cell homing to peripheral lymphoid organs. Although thymocyte negative selection was unaffected, MrtfabKOT animals develop autoimmune phenotypes in lacrimal gland and intestinal mucosa. Using the experimental colitis model, we show that these phenotypes reflect functional deficits in MrtfabKOT Treg cells. Our findings show that MRTF-SRF signalling plays an unanticipated role in thymocyte egress and immune tolerance, and reveal a role of the MRTFs in Treg function.