The Journal of Immunology

Immune memory responses are rapid and qualitatively distinct from primary responses. They typically develop in the presence of antigen-experienced memory T and B cells and pre-existing antibodies. Although the contribution of T and B cells to recall responses is well defined, the contribution of antibody "memory" and the mechanisms by which pre-existing antibodies modulate the development of germinal center and plasma cell responses is not precisely understood. Here we report on mechanisms that mediate antibody enhancement of germinal center (GC) and plasmablast (PB) compartments, and the parallel process by which they change the affinity threshold for B cell recruitment into immune responses. The data indicate that antibody-mediated enhancement of GC and PB responses is Fc gamma receptor (Fc{gamma}R) dependent and largely complement receptor 1 and 2 (CR1/2) independent. In contrast, the reduction in the affinity threshold for GC entry is independent of both Fc{gamma}Rs and CR1/2. SummaryCipolla et al. show that antibody can modulate immune responses via both Fc gamma receptor dependent and independent mechanisms. These mechanisms influence both the magnitude and composition of the germinal center response.

Regulatory T cells (Tregs) are essential for maintaining immune homeostasis by suppressing excessive activation of effector T cells. Although several mechanisms of Treg-mediated suppression have been described, the molecular signals that contribute to this regulation remain incompletely understood. WNT signaling, best known for its roles in development and tissue homeostasis, has recently emerged as an important regulator of immune function, but its contribution to Treg-mediated immune suppression is largely unknown. Here, we show that Tregs preferentially express multiple canonical WNT ligands, including WNT2B, WNT3, WNT7B, and WNT10B, compared with conventional CD4+ T cells. These WNT proteins were detected intracellularly in Tregs, and WNT2B and WNT3 were actively secreted into culture supernatants. Conventional CD4+ T cells expressed Frizzled receptors capable of sensing these ligands. Pharmacological inhibition of canonical WNT signaling using the antagonist mDKK-1 enhanced CD4+ T cell activation and proliferation and increased pro-inflammatory cytokine expression, while anti-inflammatory IL-10 remained unchanged. Together, these findings identify Tregs as a source of canonical WNT ligands and suggest that Treg-derived WNT signaling contributes to the suppression of effector CD4+ T cell responses. This work reveals a previously underappreciated pathway through which Tregs regulate immune activity and identifies WNT signaling as a potential target for modulating inflammatory immune responses.

Regulatory T (Treg) cells are central mediators of immune tolerance and are generally considered to rely predominantly on mitochondrial metabolism rather than glucose-driven glycolysis. To define the role of glucose metabolism in Treg cells, we investigated the contribution of the hexose transporters GLUT1 and GLUT3. Genetic ablation of GLUT1 in T cells or selectively in Treg cells had minimal impact on Treg cell numbers, phenotype or immune homeostasis, indicating that GLUT1 is largely dispensable in this lineage. By contrast, deletion of GLUT3 in T cells resulted in a marked reduction in Treg cell numbers. However, it remained unclear whether this reduction reflected diminished IL-2 production by GLUT3-deficient conventional T cells or a cell-intrinsic requirement for GLUT3 in Treg cells. To investigate this, we generated mice with Treg cell-specific deletion of GLUT3. These animals developed severe systemic inflammation accompanied by lethal cellular and humoral autoimmunity. Mechanistically, GLUT3-deficient Treg cells exhibited reduced glycolytic activity and mitochondrial respiration, leading to impaired suppressive function and defective effector and follicular Treg cell differentiation. Collectively, our findings demonstrate a non-redundant requirement for GLUT3 in Treg cell metabolic fitness and immune regulation, refining the prevailing view that Treg cells operate largely independently of glucose metabolism. Our data further suggest that therapeutic strategies targeting glucose uptake and glycolysis in autoimmune and inflammatory diseases should account for potential adverse effects on Treg cell-mediated immune tolerance.

Francisella tularensis is a Gram-negative bacterium that causes tularemia, a fatal zoonotic disease. F. tularensis has been used in the bioweapon programs of several countries. Its potential use as a bioterrorism agent led the CDC to classify F. tularensis as a Tier 1 Select Agent. The cytosolic sensor absent in melanoma 2 (Aim2) detects double-stranded DNA in the cytosol of infected cells and subsequently assembles a multiprotein complex known as the inflammasome. Inflammasome activation drives the secretion of IL-1{beta} and IL-18, key proinflammatory cytokines required for controlling F. tularensis infection. Prior studies have shown that F. tularensis actively suppresses Aim2 inflammasome activation; however, the underlying mechanism remains unknown. We hypothesized that F. tularensis suppresses Aim2-mediated responses by modulating the intracellular redox environment. We utilized an F. tularensis mutant lacking OxyR ({Delta}oxyR), a transcriptional regulator that controls the expression of major antioxidant enzymes. Our results show that macrophages infected with the {Delta}oxyR mutant exhibit significantly higher levels of Aim2-dependent Caspase-1 and IL-1{beta} than those infected with wild-type bacteria. The expression of interferon regulatory factor 1 and the guanylate-binding proteins GBP2 and GBP5, upstream signaling components of the Aim2 inflammasome, is markedly higher in {Delta}oxyR-infected macrophages than in controls. These changes were absent in {Delta}oxyR-infected NADPH oxidase-deficient macrophages, which are unable to generate reactive oxygen species. Collectively, these findings demonstrate that macrophage redox environment plays a key role in activating signaling components required for Aim2 inflammasome activation. This work advances our understanding of how F. tularensis-encoded factors subvert host innate immune defenses.

T cells play an essential role in protecting tissues against pathogens and regulating tissue homeostasis. Previous studies highlight that T cells display tissue-specific phenotypic and functional properties, suggesting that T cells adapt to their local environment. However, whether this holds true in inflamed tissues or whether inflammation disrupts any tissue-specific T cell adaptations remains poorly understood. To address this open question, we examined the T cell compartment including conventional CD4 and CD8 T cells, regulatory T cells, gd T cells, and MAIT cells from healthy and inflamed human mucosal tissues. Using high-parameter spectral flow cytometry, we examined phenotype ex vivo and the functional capacity following stimulation, utilizing conventional gating and unsupervised clustering analysis approaches. Overall, we analyzed 65 tissue samples including mild, moderate, and severely inflamed oral gingiva, healthy and inflamed lung, along with healthy and inflamed tissue from the decidual-placental interface. Across these mucosal barrier tissues, we find that tissue location plays a dominant role in shaping the composition, phenotype, and functional capacity of the T cell compartment. Importantly, these tissue-specific adaptations were largely maintained during states of tissue inflammation. This included the ability to exert tissue repair functions, which was preserved across T cell subsets, even in severely inflamed tissues.

Orientia tsutsugamushi (Ot) is an obligately intracellular bacterium that causes scrub typhus, a potentially severe infectious disease characterized by systemic inflammation and multiorgan dysfunction. We recently reported a protective role for IFN-{gamma} signaling in host defense against Ot infection; however, the underlying mechanisms remain obscure. Inducible nitric oxide synthase (iNOS, encoded by Nos2) is a key antimicrobial effector induced downstream of IFN-{gamma} signaling. Here, we used transgenic mouse models to further investigate the biological functions of iNOS. We first revealed the requirement of iNOS for the restriction of Ot growth in cultured bone marrow-derived macrophages. Using an intradermal mouse model, we found that while tissues of Nos2-/- and wild-type mice exhibited comparable bacterial burdens during acute infection phases, Nos2-/- mice developed eschar-like lesions similar to those observed in Ifngr1-/- mice, indicating a critical role for the IFN-{gamma}/iNOS axis in regulating skin pathology in scrub typhus. Notably, Nos2-/- mice displayed impaired bacterial clearance during the recovery phase (day 42), with persistent bacterial burdens in multiple organs accompanied by sustained immune activation and elevated inflammatory responses. Histopathological and biochemical analyses further revealed increased tissue damage and dysregulated physiological homeostasis in Nos2-/- mice during recovery. Mechanistically, iNOS deficiency resulted in heightened myeloid cell activation and prolonged expression of proinflammatory mediators, suggesting a dual contribution of iNOS in both antimicrobial defense and inflammation resolution. Collectively, these findings provide new insight into IFN-{gamma}-mediated defense mechanisms and imply the distinct roles of iNOS during different stages of scrub typhus. Author summaryScrub typhus is a potentially severe infectious disease caused by the bacterium Orientia tsutsugamushi (Ot), which is transmitted to humans through the bite of infected mites. Despite its global impact and expanding geographic distribution, the immune mechanisms that protect against this infection remain incompletely understood. In this study, we examined the role of inducible nitric oxide synthase (iNOS), an immune effector molecule that helps the host control infection. Using mouse models, we found that iNOS plays dual and stage-specific roles during Ot infection. Mice lacking iNOS developed dysregulated immune homeostasis during acute infection and exhibited skin lesions resembling the eschars observed in some patients with scrub typhus. In addition, these mice showed delayed bacterial clearance, prolonged inflammation, and increased tissue damage during the recovery phase. Our findings indicate that iNOS contributes not only to host antimicrobial defense but also to the control of excessive inflammation following infection. These results provide new insight into host defense mechanisms in scrub typhus and may help inform future therapeutic or preventive strategies.

GPR65 has been shown to be a critical regulator of Th17 cell pathogenicity. Loss of GPR65 in mice results in a decrease in Th17 cells and reduced susceptibility to a mouse model of multiple sclerosis. The CREB/CRTC2 pathway has emerged as an important regulator of immune function. We have previously shown that the CREB/CRTC2 pathway modulates autoimmune disease by promoting differentiation of Th17 cells. In this study we performed RNA-seq to identify Th17 genes regulated by the CREB/CRTC2 pathway. Our RNA-seq analysis led us to uncover the first mechanism of regulation of the orphan receptor GPR65 by the CREB/CRTC2 pathway. We show that GPR65 is a target of the CREB/CRTC2 pathway through expression studies and chromatin immunoprecipitation. In addition, we show that targeting GPR65 with small molecules alters the expression of IL-17A. Understanding the regulation of GPR65 will be crucial in developing small molecules to treat patients with Th17 cell-mediated disorders.

Junctional adhesion molecule-like (JAML) is an adhesion molecule known to promote T cell activation and T cell-mediated tumor rejection. In the current study, we show that JAML expression is enriched on mouse intratumoral NK cells compared with splenic NK cells. JAML+ NK cells were associated with tissue residency and co-expressed the immune checkpoints PD-1 and LAG3. JAML expression could be induced on splenic NK cells by IL-2 and further enhanced by IL-21. JAML levels were inversely correlated with inhibitory signaling, as NK cells expressing self-recognizing Ly49 receptors had reduced JAML expression, suggesting regulation of JAML expression by MHC class I molecules. Interaction with the JAML ligand CXADR also reduced JAML surface expression, indicating that tumor-mediated membrane stripping may represent a mechanism of immunoediting. Although JAML RNA transcripts were detectable in human NK cells, JAML protein was found only intracellularly. Together, these findings identify the JAML-CXADR interaction as a potential regulatory pathway in NK cell-mediated killing of tumors.

In germinal centers, activated B cells modify their antigen receptors through somatic hypermutation (SHM), followed by antigenic selection that favors expansion of high affinity B cells. The affinity maturation process is critical for development of broadly neutralizing antibodies (bnAbs) against the human immunodeficiency virus-1 (HIV-1). BnAbs have been isolated from some people living with HIV-1. Because these antibodies target conserved epitopes of the HIV-1 Envelope (Env) protein, they inhibit a broad spectrum of viruses. Eliciting bnAbs by vaccination is a top priority for HIV-1 prevention, but reproducing the lengthy maturation of bnAbs is a major challenge. The problem is typified by VRC01 class antibodies, which recognize the CD4 binding site of HIV-1 Env protein. To reach the CD4 binding site, antibodies need to navigate through adjacent glycans. Accommodating the glycans requires multiple SHMs in germinal center (GC) B cells, including infrequent events. For this reason, VRC01 vaccine development often stalls at this point. We have generated a mouse model aimed at providing a potential solution for navigating this vaccine design impediment. To this end, we made a mouse model that expresses a stalled VRC01 intermediate conditionally in GC B cells. This system has three advantages: 1) direct expression of the intermediate obviates prior immunization steps, thereby shortening the immunization scheme; 2) the conditional expression system bypasses tolerance control checkpoints that sometimes delete B cells expressing bnAbs; 3) the intermediate responds to immunization in GCs, the physiological site of affinity maturation. With this model, we established an immunization method to mature the VRC01 intermediate into heterologous neutralizing antibodies against viruses with a native glycan shield. Since high mutation load is common among bnAbs, the germinal center conditional expression system could provide a general tool for boost immunogen design to overcome roadblocks in the maturation pathway. Author summaryIn response to antigenic stimulation, cognate B cells become activated and form germinal centers in lymphoid tissues. Germinal center B cells modify their antigen receptors through somatic hypermutation (SHM) of immunoglobulin variable region gene exons, with antigen selecting for high affinity B cells by providing survival advantage. This mechanism accounts for antibody affinity maturaton over the gradual course of an immune response. Affinity maturation is critical for generating potent, neutralizing antibodies against diverse strains of the human immunodeficiency virus-1 (HIV-1). These broadly neutralizing antibodies (bnAbs) are heavily mutated, reflecting lengthy affinity maturation over years of chronic infection. Recapitulating the affinity maturation process is a major challenge for bnAb induction by vaccination. In immunization experiments, bnAb development often stalls at rate limiting steps that involve infrequent, but functionally important, mutational events. Overcoming such obstacles requires boost immunogens that can stimulate the stalled B cells to acquire the requisite mutations. To this end, we recapitulated the maturation arrest of a bnAb lineage by expressing a stalled antibody in mouse germinal center B cells. Using this mouse model, we developed boost immunization conditions that advanced the antibody maturation beyond a roadblock to attain neutralizing activities against heterogenous viruses.

Persistence of memory T lymphocytes, in the apparent absence of antigen, is a hallmark of immune memory and key to adaptive immunity to recurrent infections. The signaling pathways ensuring survival and quiescence of the memory T cells are largely enigmatic. Here we show, by inhibition in vivo, that persistence of surface CD69+KLF2-tissue-resident memory T cells of murine bone marrow and spleen is blocked by antibodies to the integrins VLA-4 and LFA-1, connecting the memory T cells to VCAM1 and ICAM1 of stromal cells. Persistence requires the PI3K/AKT signaling pathway, since it is blocked by Wortmannin, and it involves PI3K-dependent survival genes. Surface CD69-KLF2+ memory T cells of the bone marrow are also dependent on integrin-mediated contact to stromal cells. Their persistence critically depends on the NF-kB pathway, their PI3K signaling pathway is not relevant. Blocking Jak1 and 3 of the interleukin-7 and -15 signaling pathways does affect memory T cells of the spleen, but not those of the bone marrow. Thus, tissue-resident KLF2+ and KLF2-memory T cells, and memory T cells of spleen and bone marrow, use different signaling pathways, adapting them to their respective tissues and reflecting an unexpected heterogeneity in the molecular mechanisms of persistence.

Many genetic variants associated with increased type 1 diabetes (T1D) risk are located within the SKAP2 gene; however, the mechanisms by which these variants confer disease risk remain unclear. SKAP2 encodes an adapter protein that functions within the integrin signaling pathway and is found at the highest levels in myeloid leukocytes. We recently identified a de novo gain-of-function SKAP2 mutation in an individual with T1D, leading to hyperactive integrin signaling in myeloid cells. To dissect the mechanisms by which this mutation may lead to T1D, we generated a knock-in mouse line containing the orthologous p.G153R substitution in mouse SKAP2 on the diabetes-prone nonobese diabetic (NOD) genetic background. Both female and male SKAP2G153R/G153R mice developed accelerated T1D. The SKAP2G153R/G153R mice also exhibited a unique spectrum of autoantibodies, leading to immune-complex nephritis. Accelerated infiltration of pancreatic islets by myeloid cells, B lymphocytes, and activated T cells was observed in SKAP2G153R/G153R mice. Single-cell RNA sequencing demonstrated a type 1 IFN{gamma}-driven inflammatory program within the pancreatic islets of SKAP2G153R/G153R mice. Dendritic cells from SKAP2G153R/G153R mice demonstrated increased antigen-presenting capacity, characterized by enhanced adhesion to T cells during immune synapse formation. Macrophages and neutrophils from SKAP2G153R/G153R mice also showed increased integrin signaling responses, with neutrophils expressing high levels of activated {beta}2 integrins on the cell surface. When backcrossed onto the C57BL/6J genetic background, the SKAP2G153R/G153R mice developed spontaneous autoantibody formation and exhibited accelerated autoimmunity, including nephritis, in the pristane-induced model of autoimmune disease. These findings demonstrate that dysregulation of leukocyte integrin signaling, through alterations in SKAP2, may increase the genetic risk for autoimmunity and T1D.

Th1 cells are viewed as a cornerstone of immunity to fungi and other intracellular pathogens. Despite the widely accepted role of Th1 cells in antifungal resistance, the development of protective strategies harnessing them is stunted by a limited understanding of how best to promote their development. We and others have reported a requisite role for Th17 cells in resistance to fungi. We have long been puzzled about how to reconcile seminal roles for both Th1 and Th17 subsets. Here we report that Th17 cells convert into polyfunctional Th1 cells producing multiple cytokines, including IFN-{gamma}, TNF and GM-CSF when we used adjuvant formulations that include glucopyranosyl lipid adjuvant (GLA) to enhance antifungal immunity. GLA induced plastic Th17 cells that convert into polyfunctional Th1 memory cells.

The aberrant inflammation that characterizes severe COVID-19 is incompletely understood. Given the persistence of SARS-Cov-2 RNA and nucleocapsid protein (N) and the presence of anti-N antibody during the course of severe infection, we investigated the role of RNA-containing immune complexes (ICs) in driving inflammation. We found that ICs consisting of SARS-CoV-2 RNA, N, and anti-N IgG1 stimulate primary human monocytes in vitro to produce inflammatory cytokines and chemokines in a manner dependent on Fc{gamma} receptors and partially dependent on toll-like receptor-8. In addition, the inflammatory response induced in monocytes by RNA-containing ICs caused endothelial dysfunction in vascularized micro-organs. Using nasopharyngeal samples from SARS-CoV-2-infected individuals, SARS-CoV-2 RNA and N were captured by anti-N monoclonal antibody in the absence of lysing reagents, indicating that SARS-CoV-2 RNA and N complexes are present outside of virions and cells. Finally, we found that during an early wave of COVID-19, the anti-N IgG:IgM ratio predicted severe clinical outcomes, consistent with a role for inflammatory, IgG-mediated phagocytic clearance of nucleic acid-containing ICs in SARS-CoV-2 pathogenesis, perhaps mitigated by non-inflammatory, IgM-mediated clearance. We conclude that RNA-containing ICs may play a role in the pathogenesis of severe COVID-19. Since all pathogenic viruses encode nucleic acid-binding proteins, such as N, and these proteins often elicit an antibody response, inflammatory clearance of nucleic acid-containing ICs may also contribute to disease severity in other viral infections.

The outcome of a Plasmodium infection depends on the timely regulation of the robust pro-inflammatory response required to eliminate the parasite, but this response can cause tissue damage if not properly controlled. IL-10 is an important regulatory cytokine that prevents immunopathology during many Plasmodium infections; however, this protection comes at the expense of less effective parasite control. This is illustrated by infection with P. yoelii, in which mice exhibit a lower parasite load in the absence of IL-10. However, the immune components that limit parasite burden in the absence of IL-10 remain poorly understood. Abolishing IL-10 led to a predicted increase in TH1 polarization and higher production of IL-12 and IFN-{gamma}. However, the enhanced production of these cytokines did not explain the improved parasite control seen in Il10-/- mice. Loss of IL-10 signaling reduced the accumulation of germinal center B cells and plasmablasts in the spleen, indicating a role for IL-10 in supporting the humoral response. However, although B cells are essential for survival, they do not play a critical role in early parasite control in IL-10-deficient mice. Moreover, Il10-/-mice lacking IFN-{gamma} and B cells can limit early parasite expansion, suggesting that IL-10 suppresses host-protective pathways beyond the functions of B cells and IFN-{gamma} in parasite control.

Complex multi-tissue regeneration capacity varies across vertebrates. Mammals are amongst the least regenerative species, while salamanders can regenerate complex tissues such as limbs and tails throughout life. Previous studies have shown that innate and adaptive immune cells are present during salamander limb regeneration. While innate immune cells have been shown to promote limb regeneration, it is unknown whether adaptive immunity is responsive to amputation or plays a role in appendage regeneration. Here we show that during limb regeneration in axolotls, the immune response is characterized by a coordinated immunoregulatory signature including the downregulation of antigen presentation, cytokine secretion, and T cell activation. To test the role of adaptive immune cells in regeneration, we generated Recombination activating gene 1 deficient (Rag1-/-) newts. Rag1-/- newts lack antigen receptor recombination and show a marked reduction of adaptive immune cells. We find that Rag1-/- newts do not reject allografts, confirming their functional immunodeficiency. Finally, we demonstrate that both larval and adult newts regenerate appendages in the absence of adaptive immunity. Our work demonstrates that the adaptive arm of the immune system is not required for appendage regeneration and establishes an important model for novel experimental approaches in comparative immunology and regenerative biology.

RNA-binding proteins are key players in determining the fate of mRNA. One such RNA binding protein, Fragile X messenger ribonucleoprotein (FMRP), has an established role in RNA transcription, metabolism, translation, and degradation in the brain and reproductive system. Although FMRP is expressed in immune cells, little is known about how FMRP influences immune cell mRNA transcript outcomes. Here, we show that macrophage infection with the intracellular pathogen Listeria monocytogenes induces FMRP translocation from the cytoplasm to the nucleus. We show that infected macrophages lacking FMRP have impaired Il6 induction in response to L. monocytogenes infection. Finally, we show that macrophages lacking FMRP have increased susceptibility to inflammatory cell death. Together, these data implicate FMRP in modulating proinflammatory gene expression during bacterial infection.

The variable regions of antibody heavy chains (HCs) and light chains (LCs) are assembled by V(D)J recombination in progenitor B cells to generate an immense repertoire of primary B cell receptors (BCRs), the precursors of affinity-matured antibodies secreted in response to antigen stimulation. The complementarity determining region (CDR) 1, 2 and 3 of antibodies are the principal antigen contact sites, with CDR3 being highly diverse due to V(D)J junctional diversification by terminal deoxynucleotidyl transferase (TdT). The HC CDR3 (CDR H3) plays a prominent role in broadly neutralizing antibodies (bnAbs) against the human immunodeficiency virus-1 (HIV-1). BnAbs overcome the genetic heterogeneity of HIV-1 by recognizing conserved epitopes on the HIV-1 Envelope (Env) protein. Reaching these targets requires long CDR H3s that penetrate through the glycan shield or other structural hindrances on the Env protein. The shortage of human antibodies with such long CDR H3s poses a challenge for bnAb elicitation by vaccination. To aid immunogen design, we generated six mouse models for inducing bnAbs against particular HIV-1 Env epitopes. In each mouse model, we integrated the human HC VH, D, JH segments and LC VL, JL segments of a bnAb lineage into the mouse HC and LC loci, with each set engineered to undergo V(D)J recombination and to generate diverse human HC and LC variable regions. Combined action of V(D)J recombination and TdT- mediated junctional diversification in developing B cells generated a range of human variable region exons for a given bnAb lineage that contained highly diverse CDR3s in each mouse model. Moreover, these repertoires contained humanized antibodies that had bnAb-like long CDR H3s that could potentially serve as bnAb precursors. Therefore, these mouse models can be used to test whether immunogens can induce bnAbs from rare and diverse precursors in a complex antibody repertoire. Author summaryThe human immunodeficiency virus-1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS). An efficacious HIV-1 vaccine is needed to control the AIDS pandemic. However, in multiple clinical trials, vaccine candidates failed to confer protection against HIV-1 infection. The lack of efficacy is mainly due to the enormous heterogeneity of HIV-1 strains in human circulation. A breakthrough in the field has been the identification of broadly neutralizing antibodies (bnAbs) in a small fraction of HIV-1 infected patients. Because these antibodies recognize conserved targets on different HIV-1 strains, they can inhibit a wide spectrum of viruses. Eliciting HIV-1 bnAbs is a top priority for vaccine development. For this endeavor, a major difficulty is that most bnAbs have unusual properties. To induce bnAbs, vaccines must be highly selective for rare human antibodies that can develop into bnAbs. To facilitate this effort, we have generated a panel of mouse models that can produce potential precursors for major types of HIV-1 bnAbs. We engineered mouse models to produce diverse precursors in complex antibody repertoires, which mimic the challenging condition in human vaccination. These mouse models can be used to assess and optimize vaccine candidates at the preclinical stage.

Red blood cell (RBC) alloimmunization is a clinically significant complication in transfused patients whose immunological determinants remain incompletely understood. Type I interferon (IFN-I) signaling drives RBC alloimmunization in murine models, and systemic lupus erythematosus (SLE) is characterized by constitutive IFN-I hyperactivation alongside elevated alloimmunization rates. We analyzed three publicly available SLE RNA-seq cohorts (GSE72509, GSE112087, GSE122459; whole blood and PBMC; total n = 150 SLE) in a pre-specified discovery-replication-validation design. A 14-gene IFN-I signature score was computed per sample; differential expression, gene set enrichment analysis, and Spearman correlation were performed independently per cohort. IFN-I scores were significantly elevated in SLE versus healthy controls in all three cohorts (p < 0.01 each). IFN-high SLE patients showed 665 differentially expressed genes, with enrichment of alloimmunization-associated and plasmablast differentiation gene sets confirmed by GSEA. The alloimmunization signature score correlated significantly with IFN-I score across all three independent cohorts ({rho} = +0.77, +0.51, +0.60; all FDR q < 0.05); Tfh differentiation showed no association in any cohort. To our knowledge, this represents the first human transcriptomic evidence that IFN-I pathway activity in SLE is coupled to alloimmunization-associated immune programs in vivo. These findings identify IFN-I score as a candidate biomarker of alloimmunization susceptibility in SLE and provide translational rationale for prospective studies incorporating transfusion outcome data.

Polymorphonuclear neutrophils (PMNs) serve as frontline defenders against injury and infection, eliminating pathogens and initiating mucosal tissue repair. However, excessive PMN transepithelial migration (TEpM) contributes to chronic mucosal inflammatory disorders, including inflammatory bowel disease. PMN pro-inflammatory and pro-repair functions are regulated by incompletely defined signaling cascades involving kinases and phosphatases. Here, we determined how the protein tyrosine phosphatase CD45/PTPRC regulates PMN trafficking and effector functions in the gut. Pharmacologic inhibition of CD45 significantly reduced PMN colonic TEpM in vitro and in vivo and decreased intestinal PMN trafficking was observed in transgenic mice with PMN-specific deletion of CD45 (MRP8-Cre;Cd45fl/fl). Beyond limiting TEpM, CD45 depletion impaired key antimicrobial functions, including degranulation and phagocytosis, indicating broader effects on PMN effector activity. Importantly, recovery from dextran sodium sulfate (DSS)-induced colitis and biopsy-induced colonic wounding was delayed in MRP8-Cre;Cd45fl/fl mice, linking altered PMN function to defective mucosal healing. Mechanistically, CD45 depletion reduced surface expression of the {beta}2 integrin CD11b/CD18 and inactivated the Src family kinase member Lyn. Together, data highlight a novel CD45-CD11b-Lyn signaling axis that regulates PMN trafficking and effector functions in the intestine and identify CD45 as a promising target for modulating PMN function to promote mucosal tissue repair.

High-parameter flow cytometry is essential for dissecting the intricate landscape of T-cell diversity. In this study, we directly compare conventional flow cytometry (CFC) and spectral flow cytometry (SFC) for high-dimensional T-cell phenotyping, assessing how spectral detection and panel-design strategies influence analytical performance. Using peripheral blood mononuclear cells from healthy donors stained with both an established (v1) and an optimized (v2) fluorochrome-labelled antibody panel, and analyzed through manual gating and unsupervised approaches, we found that CFC reliably identified major T-cell subsets. However, spectral acquisition consistently delivered clear technical advantages, including improved signal-to-noise ratios, higher staining index values, and superior resolution of low-intensity and co-expressed markers. These improvements translated into more sharply delineated multidimensional clusters and a markedly enhanced resolution of T-cell differentiation states. Moreover, the optimized spectral panel enhanced the unsupervised detection of rare populations, such as cytotoxic CD4 T-cells (PD-1GZMB). However, despite the overall increase in data quality achieved with SFC, the selection of antibody clones may influence the measured frequencies of the identified populations. Finally, SFC - particularly when coupled with rational panel optimization and the use of advanced fluorophores - consistently delivers superior, higher-quality measurements and improved multidimensional resolution, thereby substantially enhancing the robustness and sensitivity of high-parameter T-cell phenotyping for comprehensive immunological studies.