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Targeting convergent pathways prevents the differentiation of tissue-resident memory T cells from multilineage progenitors in the arthritic synovium 2256215
Abstract Introduction Rheumatoid arthritis is an autoimmune disease characterized by chronic, recurrent inflammation of the joints. We identified CD8 resident memory T cells (TRM) in human arthritic joints and demonstrated that TRM remain in the synovium during remission and mediate arthritis flares. However, the developmental origin and differentiation trajectory of TRM in the joints remain unclear. Methods Here, we use two parallel models of synovial TRM development: 1) an antigen-induced arthritis (AIA) murine model, and 2) a human synovial organoid model. Using 10x single-cell RNA sequencing, we discover the progenitor and differentiation pathways of synovial TRM. Antisense oligonucleotides (ASOs) targeting candidate signaling pathways were further applied either to human synovial organoids or through adoptive transfer experiments in the AIA model. Results Using 10x single-cell RNA sequencing, we discovered that synovial TRM derived from both circulating effector memory T cells (TEM) and central memory T cells (TCM) during murine arthritis. We further confirmed that both purified TEM and TCM were discretely differentiated into TRM in human synovial organoids. By integrating RNA sequencing data from both systems, we delineated convergent and divergent differentiation pathways and identified associated transcriptomic changes originating from these distinct progenitor pools. Among the genes expressed in the TRM convergent pathway was the transcription factor, RUNX3. Using ASOs to block RUNX3 expression, we prevented TRM formation along both TEM and TCM differentiation trajectories in human synovial organoids. Consistently, T cells treated with ASOs against Runx3 showed reduced TRM formation in murine arthritis. Conclusion Our results define the trajectory of TRM differentiation in the joint, highlighting these pathways as potential therapeutic targets for modulating chronic arthritis. Funding Source Funding support from NIH/NIAMS K08AR080992, a Joint Biology Consortium microgrant off parent grant NIH/NIAMS P30AR070253, the Rheumatology Research Foundation, the Arthritis National Research Foundation, Boston Children’s Hospital and the Arbuckle Family Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Single-cell and spatial transcriptome profiling reveals innate-adaptive crosstalk in alopecia areata 2251484
Abstract Introduction Alopecia areata (AA) is an autoimmune disorder characterized by immune-mediated attack on hair follicles, resulting in non-scarring hair loss. The collapse of hair follicle (HF) immune privilege (IP) is widely regarded as a central pathogenic mechanism in AA. While CD8+ T cells have been extensively studied in this context, the contribution of macrophages to disease initiation and progression remains less defined. Methods In this study, we investigated the roles of macrophages and CD8+ T cells in shaping immune cell recruitment and local immune dysregulation in AA lesions. Using single-cell transcriptomics, flow cytometry, and immunofluorescence during the development of an AA mouse model, we observed robust expansion and transcriptional reprogramming of CD8+ T cells, accompanied by prominent macrophage infiltration surrounding affected HFs. Results Interrogation of macrophage populations identified two transcriptionally distinct subpopulations: Folr2+ tissue-resident macrophages (TRMacs) and Folr2⁻ monocyte-derived macrophages (MoMacs). Each subset displayed unique gene-expression profiles enriched for pathways involved in T cell activation, leukocyte trafficking, and myeloid differentiation. Integration of spatial transcriptomic and flow cytometry imaging analyses revealed close spatial interactions between macrophage subsets and CD8+ T cells, with macrophage abundance positively correlating with CD8+ T cell infiltration. To assess functional relevance, we depleted macrophages with an antibody directed against colony-stimulating factor 1 receptor (CSF1R) in AA-grafted mice. Macrophage ablation markedly reduced both hair loss severity and the accumulation of cytotoxic NKG2D+ CD8+ T cells in lesional skin. Conclusion Together, these findings support that macrophages play a pivotal role in driving local immune dysregulation in AA. Targeting macrophage subsets may represent a promising therapeutic approach for restoring HF immune privilege and ameliorating disease progression. Funding Source n/a Topic Categories Basic Autoimmunity (BA)
Microglial Remodeling of Cerebrovascular Structure and Function during Respiratory Inflammation 2310075
Abstract Introduction Microglia are brain-resident immune cells that have valence on virtually all areas of the cerebral landscape, one locus of particular intrigue being the vasculature. Previous work has detailed robust microglia-vascular interactions in physiology. However, how these interactions are altered during inflammation, particularly respiratory inflammation, remains poorly understood. Here, we demonstrate that respiratory inflammation elicits marked changes in microglial spatial organization and vascular interactions. Methods To simulate inflammation, we used intratracheal lipopolysaccharide (IT-LPS) injection. To evaluate microglial response, we quantified capillary-associated microglia (CAM) density, expression of activation and homeostatic markers (CD68 and P2RY12), and microglial emergence, clearance, and migration, a process that can be broadly defined as landscape rearrangement. To assess vascular structure and function, we measured capillary diameter, cerebral blood flow (CBF), tight junction integrity, and blood-brain barrier (BBB) permeability. Ongoing work aims to assess the contribution of microglia to inflammation-associated vascular changes using the Csf1rFIRE mouse model, in which microglia fail to develop. Results IT-LPS treatment induced a robust response, evidenced by transient weight loss in treated mice. We observed increased levels of landscape rearrangement and greater densities of CAM. Despite observing no difference in P2RY12 expression, IT-LPS treated mice produced a higher density of CD68+ microglia, consistent with a more phagocytic phenotype. These changes coincide with modulations in the vasculature, including reduced capillary diameter and impaired CBF. Further, tight junction integrity was perturbed and BBB permeability increased, indicating compromised vascular structure and function. Conclusion By studying these interactions, we hope to uncover novel relationships between microglia and the vasculature relevant for brain function in various pathologies. Funding Source Nucleate, DojoGrants Research Fellowship Echols Scholars Program, Ingrassia Family Research Grant University of Virginia, Harrison Undergraduate Research Award Topic Categories Neuroimmunology (NEUR)
Aging releases the brakes on the T follicular helper cell — B cell axis in murine atherosclerosis 2256916
Abstract Introduction Aging is the most dominant risk factor for atherosclerotic cardiovascular disease (ASCVD), which is characterized by the build-up of lipids and immune cells in the arterial wall. Adaptive immune responses play a detrimental role in atherosclerosis development. Studies in young mice have shown that follicular T helper (Tfh) cell responses in secondary lymphoid organs promote B cell differentiation and drive high-affinity antibody production, aggravating atherosclerotic plaque formation. In this study, we aimed to address age-associated changes in the Tfh-B cell axis in atherosclerotic mice. Methods Using flow cytometry we analyzed Tfh and antibody-secreting cells of young (5 months), middle-aged (12 months) and aged (22 months) Ldlr-/- mice. Age-associated changes in Tfh and B cells were further characterized by scRNAseq and scTCR/BCRseq of spleens from young (5 months) and aged (22 months) Ldlr-/- mice. Total and oxidation-specific epitope (OSE) antibody levels were measured in serum. Results Aging was associated with expansion of Tfh (3.8-fold) and Tfr cells (2.9-fold) in lymphoid organs of Ldlr-/- mice. The disproportionate increase in Tfh cells led to a significantly elevated Tfh/Tfr ratio in aged mice. Similarly, scRNAseq revealed age-associated clonal expansion of splenic Tfh cells expressing Pdcd1, Cxcr5, and Bcl6, alongside enhanced expression of Tfh differentiation markers (Ascl2, Il21r), Tfh—B cell interaction genes (Cd40lg), and senescence- (Spp1) and exhaustion-associated (Tigit, Ctla4 and Lag3) genes. In line with these findings, we observed a > 3-fold increase in splenic plasmablast and plasma cells, accompanied by a significant increase in total and OSE-specific IgA, anti-inflammatory IgM, and proinflammatory IgG2b and IgG2c serum levels in aged Ldlr-/- mice. Conclusion In conclusion, aging causes overactivation of the Tfh-B cell axis in atherosclerotic mice. Targeting the Tfh-B cell axis could represent a promising therapeutic strategy to combat atherosclerosis. Funding Source Dutch Heart Foundation, EIC Pathfinder B-specific, AtheroNeth Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Optimized arenavirus vectors with tumor-tropic mutations as new class of amplificators of anti-tumoral T cell responses 2259607
Abstract Introduction Recent understandings of anti-tumoral immune mechanisms reveal a critical role for strong and sustainable activation of CD8+ T cells for successful immunotherapy. Recently, we showed that the non-cytopathic lymphocytic choriomeningitis virus (LCMV) induces strong anti-tumor responses without directly killing tumor cells but by activation of CD8+ T cells. Here, we aim to generate an optimized arenavirus based on LCMV for application in cancer patients and its potential benefit with combination therapies. Methods We performed multiple passaging of the wild-type strain LCMV-WE in several murine and human cancer cells. The occurring mutations were identified and their role tested in vitro and in vivo in attenuated reassortant LCMV. Results We identified several mutations, which showed accelerated propagation in cancer cells leading to an attenuated reassortant arenavirus strain with accelerated entry in a broad spectrum of human cancer cells. This strain exhibited strong anti-tumoral efficacy in a variety of tumor models with minimal replication in healthy tissues and no severe disease symptoms in immune compromised murine model systems. Dissecting the anti-tumoral mechanism revealed that tumor-specific T cells were expanded, recruited to tumors and differentiated into potent effector cells. Furthermore, this virus strain amplified the anti-tumoral activity of a BiTE (Trp1/CD3) and combination with CAR- T cells to increase anti-tumor efficacy. In non-human primates, treatment with this strain led to increase in virus-mediated cytokine levels as well as T cells in the blood by maintaining a safe application. Conclusion In conclusion, by using the biological principle of mutation and selection, we developed an arenavirus-based immune therapy safely applied in preclinical model systems and GLP studies as well as harboring a unique mode of action and anti-tumoral efficacy. Combination with T cell-directed therapies including BITEs and CAR-T cells resulted in strong synergistic anti-tumoral responses. Funding Source Abalos Therapeutics; BMBF KMUi Topic Categories Translational and Interventional Immunology (TI)
People living with HIV have increased CD4+ T cell metabolic activity and reduced influenza-reactive cellular responses to vaccination 2260148
Abstract Introduction While antiretroviral therapy has improved health outcomes for people living with HIV (PLWH), individuals are more likely to experience severe influenza infection and infection-related complications. Methods This study investigated basal immunometabolic function and influenza vaccine-induced antibody and T cell responses in 17 PLWH on antiretroviral therapy and 17 individuals living without HIV (PLWoH). Results We found no significant differences in serum hemagglutination inhibition antibody titers against influenza A (H1N1, H3N2) or influenza B (B-Victoria, B-Yamagata) antigens between PLWH and PLWoH pre- and post-vaccination, nor in the fold-change of titers. Only PLWoH had significantly increased frequencies of influenza-reactive CD4+ T cells in response to influenza vaccination. Naive and memory CD4+ T cells from PLWH exhibited higher basal metabolic activity compared to PLWoH. CD4+ T cell activation and metabolic activity in PLWH were largely independent of HIV viral load. Conclusion These findings suggest that while PLWH can mount humoral responses to influenza vaccination, increased CD4+ T cell activation and altered metabolism of CD4+ T cells may influence cellular immune responses to vaccination. This highlights how the adaptive immune system in PLWH is affected even when HIV replication is controlled. Funding Source NIH R01 AI142095 Topic Categories Viral Immunology (VIR)
Decoding Single-Cell Protein Interactomes with the Proximity Network Assay 2309978
Abstract Introduction Proteins on the cell surface do not act in isolation, rather, their spatial organization and physical interactions are key determinants of the cellular function. Methods Using DNA-barcoded antibodies and proximity-dependent ligation, PNA simultaneously measures the abundance, clustering, and colocalization of 155 surface proteins, generating nanoscale surface maps comprising ∼50,000 molecular positions per cell without the use of optics. Results This spatially resolved readout enables systematic analysis of membrane protein networks across thousands of cells. We demonstrate the utility of PNA by identifying the proxiome of the CD19 CAR receptor at steady state and revealing dynamic proteomic remodeling during tumor cell encounter, including key phenomena such as trogocytosis and cell—cell conjugate formation. By integrating spatial context with multiplex protein profiling at scale, PNA provides a powerful platform for protein interactomics, biomarker discovery, and mechanistic insights across immunology, oncology, and cell therapy research. Conclusion By integrating spatial context with multiplex protein profiling at scale, PNA provides a powerful platform for protein interactomics, biomarker discovery, and mechanistic insights across immunology, oncology, and cell therapy research. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)
Aging Narrows Human B1-like Cell Repertoire and Impairs Defense Against Encapsulated Pathogens 2256562
Abstract Introduction B1-like B cells are an innate-like subset of human B cells that produce natural antibodies essential for early defense against encapsulated bacterial pathogens such as Streptococcus pneumoniae. A diverse B1-like cell repertoire is critical for robust immune responses, and reduced repertoire diversity has been implicated in the age-associated decline of innate immunity. However, the impact of aging on human B1-like cell frequency and repertoire diversity remains poorly defined. Methods In this study, we examined B1-like B cells from healthy younger and older adults using multicolor flow cytometry and single-cell B cell receptor (BCR) repertoire analysis using semi-nested RT-PCR. Results Our results revealed a significant age-associated decline in circulating B1-like B cells, accompanied by a marked reduction in phosphorylcholine (PC)-specific B1-like cells–an antigenic determinant commonly present on bacterial and self-lipids. Repertoire profiling demonstrated a substantial loss of diversity in older individuals, characterized by reduced clonal expansion and diminished light-chain gene coherence. In contrast, younger donors exhibited a broader, more polyclonal B1-like repertoire with frequent PC-specific clones linked to natural protective antibodies. These findings indicate that aging induces both quantitative and qualitative impairments in the B1-like B cell compartment, leading to “holes” in the natural antibody repertoire and weakened defense against encapsulated bacteria. Conclusion Together, these results identify the age-related loss of B1-like cell diversity as a key mechanism underlying impaired humoral protection in older adults and highlight the need for strategies to preserve or restore this critical B cell subset to enhance resistance to pneumococcal and other bacterial infections in aging populations. Funding Source NIH Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Host Immune Composition Complexity Shape Murine IFN-γ ELISpot Readouts 2251432
Abstract Introduction ELISpot enables highly sensitive counting of cytokine-secreting cells at single-cell resolution; however, it remains unclear how the host’s immune experience interacts with assay parameters to dictate readouts. We established a mouse strain and microbial experience-tuned workflow for murine IFN-γ ELISpot through joint optimization of cell number, stimulant dose, and capture time with intracellular cytokine staining (ICS) validation. Methods Peripheral blood and splenocytes were harvested from inbred and outbred specific-pathogen free (SPF), or -pathogen experienced (SPExp) mice. Using an in-house IFN-γ ELISpot, we titrated cell input, anti-CD3/CD28 mAb concentration, and incubation time (1-4 or 22 hours). Plates were imaged on an ImmunoSpot S6 Entry (CTL), and spot counts and sizes were quantified. In parallel, IFN-γ production was assessed by ICS and flow cytometry (4-hour incubation with BfA added in the last hour). Results Across SPF mice, spot counts exhibited sigmoidal responses to the number of cells seeded, stimulation dose, and incubation time; the broadest dynamic range was at 4 × 10^5 cells with a 4-hour capture. In SPExp cohorts, just 2 × 10^4 cells maximized signal, and the optimal window shifted with days post last infection. PBMC ELISpot mirrored spleen longitudinally. Spot number aligned best with Ag-experienced CD8+ frequency and IFN-γ GMFI, with additional IFNγ contribution from naïve CD8+, CD4+, and NK cells. Conclusion Thus, titrating cells seeded and capture time to immune cell composition context improves accuracy. SPExp mice reach maximal dynamic range at low inputs, enabling minimally invasive longitudinal PBMC ELISpot. Across cohorts, ELISpot signal aligns with the product of Ag-experienced CD8 frequency and per-cell IFN-γ output by ICS, offering a quantitative bridge between frequency and function. Funding Source NIH T32 GM007337, UI Stead Family Children’s Hospital Discretionary Fund, NIH K12 Child Health Research Career Development Award (CHRCDA) Topic Categories Cytokines and Chemokines and their Receptors (CCR)
Immune checkpoint blockade enhances CD4 effector function and enables control of persistent viral infection 2310195
Abstract Introduction T cell exhaustion, a defining hallmark of chronic viral infections and cancer, is characterized by the progressive loss of effector function and sustained expression of inhibitory receptors. Although immune checkpoint blockade (ICB) therapies, such as PD-1 inhibition, can transiently reinvigorate exhausted CD8+ T cell subsets, their efficacy is limited by the small pool of self-renewing, progenitor-like TCF1+ CXCR5+ CD8+ T cells. We previously demonstrated that B cell—derived IL-27 signaling is required for the expansion of this progenitor-like population during chronic infection. Using an IL-27 receptor—deficient mouse model, we uncover a previously unrecognized mechanism by which PD-1 inhibition mediates viral clearance and enhanced humoral responses. Methods IL-27 receptor—deficient mice were infected with LCMV clone 13 and treated with PD-1—blocking antibodies at later stages post-infection. Following completion of antibody treatment, immune responses were analyzed using flow cytometry to characterize T cell and B cell subsets. Antiviral antibody responses and viral clearance were evaluated using serological assays, sequencing approaches, and focus-forming assay. Results PD-1 inhibition promoted viral clearance during the persistent phase without inducing the immunopathology typically observed with early anti—PD-1 treatment. This was associated with increased IL-21 production by T follicular helper (Tfh) cells, leading to augmented cytotoxic and humoral antiviral responses. Conclusion Our findings reveal a previously unrecognized and novel, IL-27—independent CD4+ T cell axis through which PD-1 checkpoint blockade restores antiviral immunity during persistent viral infection. These results redefine the mechanistic landscape of checkpoint immunotherapy, highlighting CD4+ T cells as critical effectors in controlling persistent viral infections. Funding Source N/A Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
CD40+ MHC-II+ astrocytes promote Th17 cell-driven CNS autoimmunity 2257206
Abstract Introduction Astrocyte subsets contribute to the pathology of multiple neurologic disorders, including the T cell driven autoimmune disease of the central nervous system (CNS) multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). However, little is known about the functional interactions of astrocytes with CD4+ T cells. Methods We used rabies barcode interaction detection followed by sequencing (RABID-seq), in combination with single-cell RNA sequencing, in vitro co-culture systems, and cell-specific in vivo CRISPR—Cas9-based genetic perturbation studies to identify and define a subset of astrocytes that interact with T cells during EAE. Furthermore, We harnessed universal Labeling Immune Partnerships by SorTagging Intercellular Contacts (uLIPSTIC) to analyze astrocyte-interacting CD4+ T cells. Results We identified a subset of astrocytes that expresses CD40 and MHC-II, which promotes CNS-targeting T cell autoimmunity. By harnessing uLIPSTIC to analyze astrocyte-interacting CD4+ T cells, we found that direct astrocyte-CD4+ T cell interactions promote Th17 responses in EAE. Moreover, the genetic inactivation of CD40+ MHC-II+ antigen presenting astrocytes ameliorates EAE. In addition, by applying in vivo subproteomic approaches and AlphaFold-Multimer predictions, we established that CD40 activation in astrocytes by CD40L expressed by T cells induces the accumulation of PLIN4-positive lipid droplets (LDs), which provide acetyl-CoA to boost acetylation-driven NF-kB activation and antigen presentation. Finally, we detected CD40+ MHC-II+ LD+ astrocytes in CNS with MS by snRNA-seq and immunohistochemistry. Conclusion In summary, these studies define an astrocyte subset that promotes Th17-driven CNS autoimmunity. These findings may guide novel therapeutic approaches for MS and other neurologic diseases. Funding Source Human Frontier Science Program long-term postdoctoral fellowship Topic Categories Neuroimmunology (NEUR)
Expanding the Antigen Targeting of CAR T Cells via a Universal Bispecific T cell Engager 2259699
Abstract Introduction Although CAR T cell therapy has shown remarkable clinical effects in the treatment of hematological cancers, the application of CAR T cells to treat solid tumors remains ineffective. Bispecific T cell Engagers (BiTE) are chimeric proteins capable of retargeting T cell receptors (TCR) to specific tumor antigens. CAR T cells expressing BiTEs have demonstrated superior effects against solid tumors in mouse models and early human clinical trials. Methods Here, we have engineered T cells to secrete a universal BiTE termed, “SNAP-BiTE”. Consisting of a fusion of the SNAPtag enzyme to an anti-CD3 scFv, this protein covalently assembles with co-administered antibody “adaptors” containing a benzylguanine (BG) motif, thus allowing for post-translational and user-defined antigen targeting. We engineered human T cells to secrete SNAP-BiTE and measured activity in vitro in tumor cell coincubation assays by flow cytometry. Results Primary human T cells were engineered via a retroviral vector to secrete SNAP-BiTE. Co-incubating these cells with tumor cells and various BG-conjugated antibody adaptors (trastuzumab, rituximab, cetuximab, and mirvetuximab) led to antigen specific T cell activation and target cell lysis. Activity was responsive to the dose of antibody adaptor. In addition, multiple adaptors could be used to simultaneously assemble BiTEs targeting multiple antigens and lyse multiple populations of target cells. Importantly, SNAP-BiTE expressed from engineered T cells also engaged and robustly activated BiTE(-) T cells, amplifying anti-tumor activity. Finally, SNAP-BiTE could be efficiently expressed by CAR T cells to amplify and broaden their antigen targeting. Conclusion SNAP-BiTE is a powerful approach to program engineered and endogenous T cell activity against multiple user-specified tumor antigens for improved anti-tumor cell therapy function. In vivo testing of CAR T cells secreting SNAP-BiTE in human tumor xenograft mouse models is underway. Funding Source NIH T32EB00102, NIH 1R01GM142007, NIH 1R01CA290866 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
An allogeneic CD19 targeted Engineered Treg approach for treating B cell mediated autoimmune diseases 2260315
Abstract Introduction CD19 CAR T cells have demonstrated promising efficacy in SLE by deeply depleting B cells, but these autologous cell therapies are expensive to deploy, come with significant toxicity risks, and lack the ability to control additional immune cell functions driving autoreactive B cell generation and lupus pathogenesis. Methods We developed GNTI-350, an allogeneic hypoimmune CD19 CAR—engineered regulatory T cell (CAR19 EngTreg) therapy, which leverages stable FOXP3 expression, a CD19 CAR for selective targeting, and a rapamycin-inducible IL-2 signaling complex to sustain Treg function. Results Persistence of GNTI-350 is enabled by Immune Evasion Engineering (IEE), a hypoimmune technology that allows MHC-deficient allogeneic cell therapies to evade host NK cell clearance. Human CAR19 EngTregs expressed canonical Treg markers, suppressed T cell proliferation, blocked B cell differentiation into plasma cells, and depleted CD19+ B cells in humanized mice with minimal inflammatory cytokine release versus conventional CD19 CAR T cells. In the SLE123 mouse model, CAR19 mEngTregs persisted >4 months, lowered autoantibodies, improved kidney pathology, and reduced the levels of T follicular helper (Tfh) and germinal center (GC) B cells - potentially showing a reinforcement of T follicular regulatory (Tfr) cell function. Conclusion These data show that GNTI-350 can safely achieve a durable immune reset and may provide superior therapeutic benefit over CAR-T approaches to patients with B cell—driven autoimmune diseases. Funding Source Venture capital, NIH Topic Categories Therapeutic Approaches to Autoimmunity (THER)
Nuclear estrogen receptors modulate regulatory T cell suppressive function 2309981
Abstract Introduction Autoimmune diseases are driven by an intricate combination of genetic and epigenetic factors, but the specific mechanisms underlying the etiologies of these disorders are not fully understood. 17β-estradiol (estrogen, E2) is a steroid sex hormone with potent immunoregulatory functions that contributes to various autoimmune diseases. E2 signaling through the nuclear estrogen receptors alpha and beta (ERα and ERβ) have clear immunomodulatory effects, with ER+ generally promoting and ERβ generally restraining inflammation. Previous work from our lab demonstrated that ERβ-specific signaling is reduced in inflamed mucosal tissues and T cells isolated from female Crohn’s disease patients, similar to previous reports in lupus patients, supporting the premise that ERβ functions to negatively regulate inflammation. Methods Our current study investigates the functional roles of ERβ in regulatory T cells (Tregs), in which we hypothesized that ERβ promotes Treg suppressive function. To test this, we performed ex vivo Treg suppression assays by co-culturing WT effector T cells (Teffs) with WT, ERα-KO, or ERβ-KO Tregs with or without E2. We also tested Treg suppression in vivo using a T cell transfer of colitis model. Immunodeficient Rag2-KO mice were co-injected with WT Teffs (CD4+CD25-) and WT, ERα-KO, or ERβ-KO Tregs (CD4+CD25+) to test the ability of Tregs to prevent colitis development. Results Our results show that E2 treatment reduces ex vivo suppressive ability of WT and ERβ-KO Tregs, as proliferation of Teffs is enhanced in these groups. In line with this, ERβ-KO Tregs are less suppressive in vivo. Rag2-KO recipients of ERβ-KO Tregs display more severe disease compared to recipients of WT Tregs, including stunted weight gain and increased histological inflammation. Conclusion These findings show that deletion of ERβ results in impaired Treg suppression, thus supporting a critical role for ERβ in promoting Treg function both ex vivo and in vivo. Funding Source NIH/NIDDK Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
GLUT1 in Memory CD4+ T-cells Correlates with Sustained Antibody Responses and Polyfunctionality Post-RSV Vaccination in Immunocompromised Individuals 2250589
Abstract Introduction The immunogenicity of respiratory syncytial virus (RSV) vaccination in immunocompromised individuals is poorly understood due to limited published data in this population, specifically on cellular responses to novel RSV vaccines. To address this gap, T-cell responses were evaluated in immunocompromised individuals who received an approved RSV prefusion F (preF) protein subunit vaccine. Methods In a national prospective observational cohort, 44 immunocompromised individuals reported receiving either adjuvanted RSVPreF3 (AREXVY™) or unadjuvanted RSVpreF (ABRYSVO™). The cohort was predominantly solid organ transplant recipients but also included participants with autoimmune conditions on immunosuppressants. T-cell responses, RSV preF antibodies, and RSV neutralizing antibodies were assessed at baseline, early (2 or 4 weeks), and late (12 weeks) post-vaccination using flow cytometry following RSV-A2 fusion protein peptide pool stimulation, ELISA, and live-virus neutralization, respectively. Results Post-vaccination, there were significant increases in RSV-specific memory CD4+ T-cells producing IFN-γ, IL-2, and TNF, and increases in polyfunctional CD4+ T-cells. Both vaccines, without significant differences, increased CD4+ T-cell polyfunctionality. A subset of individuals did not have an increase in CD4+ T-cell responses, demonstrating the need to investigate predictors of response. GLUT1 (Glucose transporter 1) is a marker of T-cell metabolic activation and fitness. In cytokine-producing memory CD4+ T-cells, GLUT1 expression correlated with polyfunctionality, RSV F-specific IgG titers, and neutralizing antibody titers (NT50) at the late but not the early time point. Conclusion These findings suggest that sustained T-cell metabolic fitness, as indicated by GLUT1, may be linked to the durability of both cellular and humoral immune responses. Understanding the role of GLUT1 in shaping immune responses may provide novel methods for optimizing vaccine strategies for immunocompromised individuals. Funding Source National Institutes of Health (NIH) grants 1R01AI190359, 3U01AI138897-04S1, and 3UM1AI109565 Topic Categories Vaccines and Immunotherapy (VAC)
Treatment Responses Alter Myeloid Activation in BRAF-Mutant Melanoma 2252856
Abstract Introduction Melanoma is responsible for 80% of skin cancer-related deaths. Approximately 50% of melanoma patients carry the BRAFV600E mutation, which promotes tumor growth. While the use of BRAF inhibitors (BRAFi) has improved outcomes, acquired resistance is a persistent clinical challenge and the impact of BRAFi resistance on the immune tumor microenvironment (iTME) is incompletely understood. We have shown that the addition of the synthetic triterpenoid CDDO-Me to the BRAFi PLX4720 arrested resistance and significantly reduced tumor burden, while CDDO-Me as a single agent or in combination with BRAFi prior to resistance was ineffective. Thus, we hypothesize that this disparity in treatment efficacy may be attributable to temporally regulated BRAFi treatment-induced changes to the iTME. Methods Drug-induced changes in the iTME pre and post-resistance were characterized in tumors of engrafted transgenic BRAF/Pten mice using scRNA-seq, flow cytometry, and ELISA. T cell proliferation assays were used to assess functional changes in tumor-conditioned myeloid cells. Results BRAFi treatment attenuated immunosuppressive myeloid activation pre-resistance, while BRAFi resistance was associated with enhanced tumor-associated macrophage (TAM) recruitment and immunosuppressive myeloid subset activation. However, the addition of CDDO-Me to BRAFi at resistance reversed these outcomes and rescued the ability of TAMs to induce T cell proliferation. Conclusion The iTME changes with tumor progression and responds dynamically to BRAFi treatment and resistance. This work establishes for the first time that BRAFi promotes myeloid-mediated induction of T cell activation, which is lost at resistance but can be rescued with the addition of CDDO-Me. Because BRAFi resistance and iTME immunosuppression is reversed by CDDO-Me treatment, these results provide the foundation for its potential use in combination therapies for melanoma. Funding Source Dartmouth Health Cancer Center Developmental Funding (Prouty Pilot Grant) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Intranasal SARS-CoV-2 vaccine boosting alters immune imprinting and B cell repertoire 2331448
Abstract Introduction Immune imprinting is a phenomenon in which prior antigen exposure limits variant-specific de novo immune responses to subsequent related antigens. The serum response to Omicron boosters in mice and human are strongly imprinted by prior vaccines encoding SARS-CoV-2 Wuhan-1 spike (e.g., mRNA-1273). Strategies including changes of dosage and interval have had limited success in enhancing variant-specific immunity. Intranasal (i.n.) vaccination is a promising approach that elicits mucosal and systemic immunity. Here we analysed the response of variant boosters delivered i.m. or i.n. after mRNA-1273 priming at the serum, cellular, and B cell repertoire levels to understand how boosting routes modulate the imprinting effect. Methods C57BL/6 mice were primed i.m. with mRNA-1273 and heterologously boosted i.m. or i.n. with a chimpanzee adenoviral-vectored vaccine (ChAd-SARS-CoV-2-S) against XBB.1.5 spike. Serum and bronchoalveolar lavage fluid (BALF) were analysed for cross-reactive and strain-specific spike-binding IgG/IgA. Neutralizing activity was assessed using pseudovirus neutralization assays. Cross-reactive and strain-specific spike-binding memory B cells (MBCs) in draining lymph nodes (dLNs) were quantified. Ongoing studies are using single-cell BCR sequencing to analyse plasmablast clonotypes, with selected ones expressed as mAbs for functional and epitope analyses. Results Intranasal boosting with ChAd-SARS-CoV-2-S against XBB.1.5 spike induced higher fractions of XBB.1.5-specific serum IgG and BALF IgA with greater neutralizing activity. Intranasal vaccination increased XBB.1.5-specific MBCs in dLNs, indicating a route-dependent shift in B cell specificity. Conclusion Intranasal boosting promotes greater variant-specific response at both the serum and cellular levels than i.m. boosting. Ongoing B cell repertoire and mAb analyses will provide mechanistic insight into how vaccination route reshapes clonal selection and maturation, informing rational vaccination design. Funding Source Moderna and NIH R01AI157155 Topic Categories Vaccines and Immunotherapy (VAC)
Cellular ancestry determines plasma cell diversity 2257317
Abstract Introduction Plasma cells (PCs) secrete antibodies with diverse specificities and isotypes. PCs also exhibit differences in cellular states, antibody output, and longevity. While these features are often correlated with residence tissue, the processes that lead to more durable plasma cell responses remain unclear. Our hypothesis is that cellular ancestry contributes to PC diversity. Methods We chart transcriptional and clonal diversity in human bone marrow PCs (BMPCs), circulating memory B cells (MBCs), and adenoidal germinal center B cells (GCBCs) at the single-cell level to investigate human PC heterogeneity. Using a combination of genetic and pharmacological approaches with single-cell transcriptomic read-outs, we further address our hypothesis in mice. Results We identified various cellular and immune processes that determine human BMPC heterogeneity, primarily linked to NF-kB signaling, migration/adhesion, MHC-II expression, and metabolism. Surface CD19 expression, somatic hypermutation (SHM) patterns, and clonal structure suggest that BMPCs show diverse cellular ancestry - lower SHM associates to an MBC direct ancestor, while higher SHM is linked to a GCBC direct ancestor. We further characterize the clonal overlap between BMPCs, circulating MBCs, and adenoidal GCBCs, and link it to BMPC cellular states, clonotypes, and surface CD19 expression. Using a hapten immunization mouse model, we show that MBC-derived splenic PCs exhibit increased NF-kB and ELL2-associated gene signatures, reminiscent of long-lived human BMPC gene signatures. Ongoing studies address whether MBC-derived PCs preferentially migrate to the bone marrow or have a longer lifespan than GCBC-derived PCs. Conclusion These findings highlight cellular ancestry as a previously overlooked determinant of PC heterogeneity and suggest that MBC differentiation could lead to more durable PC responses. This finding could be leveraged to induce longer-lasting humoral immunity by designing vaccination strategies that elicit MBC-derived PCs. Funding Source FASI, Sanofi, NIH Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Endoplasmic reticulum stress as a determinant of Mycobacterium tuberculosis survival in alveolar macrophages 2259865
Abstract Introduction Alveolar macrophages (AMs) are the first innate immune cells to encounter Mycobacterium tuberculosis (M.tb) following natural infection. AMs are potent phagocytes, and they are permissive to M.tb survival, replication, and dissemination. Endoplasmic reticulum stress (ERS) is triggered by the accumulation of misfolded/unfolded proteins during cellular stress, and ERS-mediated apoptosis plays a critical role in eliminating intracellular pathogens. While ERS-induced apoptosis in M1 macrophages can effectively clear M.tb, in M2 macrophages it can favor bacterial persistence. AMs exhibit unique molecular and metabolic features, expressing a hybrid M1/M2 phenotype. However, the role of ERS in M.tb infected AMs and its impact on bacterial survival remain poorly understood. Methods In this study, alveolar macrophage-like (AML) cells derived from peripheral blood mononuclear cells were infected with M.tb to characterize the ERS response over time. We analyzed the expression of key ERS-related genes and proteins following infection with either live M.tb or its virulence factors, HBHA and ESAT-6. Additionally, ERS was induced in AML cells to assess its influence on bacterial survival. Results AML cells displayed distinct ERS signatures upon infection, with significant differences in gene and protein expression compared to ERS-induced and virulence factor only controls. Notably, cells without ERS harbored higher viable bacterial counts. Conclusion The permissive nature of AMs to M.tb could be in part due to the down-regulation of ERS and related apoptosis. The results of our study highlight a potential protective role of ERS in restricting M.tb survival. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
Cytokine Programming Combined with 4-1BB Enrichment Enhances Ovarian TIL Expansion and Function 2260346
Abstract Introduction Ovarian cancer remains the most lethal gynecologic malignancy and the fifth leading cause of cancer-related deaths in women worldwide. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) has shown durable efficacy in melanoma. In ovarian cancer, higher TIL levels correlate with improved survival; however, clinical translation of TIL-based ACT remains limited by challenges in expanding functional, tumor-specific T cells ex vivo. Methods Tumor tissue from 12 patients with stage III/IV high-grade epithelial ovarian carcinoma was used for TIL expansion, including matched pre- and post-chemotherapy samples. During priming, a cytokine cocktail promoted Th1/17 and Tc1/Tc17 polarization, enhancing effector differentiation while maintaining stemness. After five days, CD137+ (4-1BB+) T cells were enriched and expanded in G-Rex bioreactors with irradiated feeders, anti-CD3, and low-dose IL-2. TILs were evaluated by flow cytometry, CD107a degranulation, IFN-γ ELISA, and scRNA-TCR-seq, compared to the NCI high-dose IL-2 protocol. Results Our method generated 15-20 billion TILs within 30 days, with >95% viability and ∼2,500-fold expansion. 4-1BB+ enrichment reduced expression of exhaustion markers PD-1 and CD38 and enhanced tumor reactivity, marked by increased IFN-γ secretion with autologous tumor and ovarian cancer lines. scRNA-seq revealed more cytotoxic CD8+ and fewer CD4+ Th2 cells compared to the NCI protocol. Transcriptomic profiling also showed CD8+ T cells with reduced metabolic activity and altered chromatin remodeling, consistent with persistence and transcriptional plasticity. TCR sequencing indicated that bulk tumor and NCI-expanded TILs were diverse with many singletons, while 4-1BB+ cells were oligoclonal and dominated by hyperexpanded CD8+ effectors. Conclusion Cytokine programming combined with 4-1BB -based enrichment enables robust expansion of metabolically reprogrammed, tumor-specific TILs, presenting a promising ACT strategy for ovarian cancer. Funding Source NIH-HL-007260R01 CA250458, R01 CA236379, GREX Grant Program Topic Categories Translational and Interventional Immunology (TI)