Citations

Abstract Introduction Alzheimer’s disease (AD) is characterized by pathologies including amyloid, tau, neurodegeneration reflected in established blood biomarkers and track with clinical changes. However, the association between peripheral cell-specific signatures and AD-related phenotypes remain poorly characterized. Methods We analyzed bulk blood transcriptome data from the Mayo Clinic Study of Aging (MCSA) and Emory University Vascular study. We used BayesPrism, CIBERSORTx, and an in-house pipeline CNNreg to deconvolute these data and obtain peripheral cell proportions. Cell type specific transcripts were estimated with BayesPrism. We compared cell proportions between cases (AD/MCI) and controls. Association analysis was performed between cell type specific transcripts and AD-related phenotypes including diagnosis and cognition. Meta-analysis of transcripts associations and gene ontology analysis were conducted to assess the enriched pathways for significant genes. Results In MCSA, B and CD4+ T cells proportions are significantly lower while that of myeloid cells higher in cases. The Emory cohort had similar trends. We identified transcripts associated with AD-related phenotypes in CD4+ T and CD8+ T cells. In CD4+ T cells, transcripts downregulated in case are enriched in response to stimulus pathway, whereas those upregulated in negative regulation of immune response. Downregulation of CD4+ T genes enriched in extracellular matrix disassembly and epigenetic regulation and upregulation of those in protein localization associate with better cognition. In CD8+T cells, genes pertinent to vascular development were downregulated while those to metabolic processes were upregulated in cases. Downregulation of CD8+T genes involved in lipid transportation and upregulation of those in mitochondria associate with better cognition. Conclusion We identified peripheral cellular transcriptional changes associated with AD/MCI phenotypes and involved in important biological pathways, revealing potential disease mechanisms in AD. Funding Source RF1 AG051504 Topic Categories Neuroimmunology (NEUR)

Abstract Introduction Primary sclerosing cholangitis (PSC) is an immune-mediated chronic fibrotic liver disease with no effective medical therapies. We showed that bile duct epithelial cells, called cholangiocytes, express IL-17C, a cytokine that modulates immune responses. Mucosal-associated invariant T cells (MAITs) have been implicated in fibrosis, but their mechanistic role in PSC remains unknown. SPARC (secreted protein acidic and rich in cysteine) is an extracellular matrix protein whose role in hepatic fibrosis is unexplored. Methods Flow cytometry, RNAscope, ELISA, RNA sequencing, immunofluorescence, immunocytochemistry, spheroid culture. Results Flow cytometry of bile duct brushings from PSC patients (n = 8) revealed that 41.32% of T cells were MAITs, indicating their proximity to cholangiocytes within the epithelial-immune niche. MAITs co-cultured with PSC patients-derived cholangiocytes showed increased activation than with normal human cholangiocytes (p = 0.018, n = 4). RNAscope on human livers identified increased IL17C expression in PSC cholangiocytes compared to controls (p = 0.064, n = 3), and IL-17C levels were elevated in PSC bile (p = 0.035, n = 25). Surprisingly, the receptor of IL-17C is enriched in MAITs in PSC (re-analysis of single cell-RNA seq, PMID: 33774059). Bulk RNA sequencing of IL-17C-treated peripheral blood MAITs revealed upregulation of T cell activation markers and pathways associated with hepatic fibrosis. Moreover, IL-17C-treated MAITs increased SPARC secretion (p = 0.025, n = 7), an effect attenuated by IL-17C blockade. Consistently, conditioned media from IL-17C—treated MAITs activated hepatic fibroblasts (HF), evidenced by increased PDGFRβ expression (p = 0.048, n = 6). SPARC directly promoted HF activation, marked by increased α-SMA (p = 0.039, n = 5). Co-culture of primary human HF, MAITs and cholangiocytes in the presence of IL-17C enhanced HF activation. Conclusion IL-17C drives MAITs toward a profibrogenic phenotype in PSC, promoting HF activation through SPARC. Funding Source C-SIG P30DK084567 Topic Categories Immune Mechanisms of Human Disease (HUM)

Abstract Introduction Cancer immunotherapy has transformed the treatment landscape by leveraging the immune system to eradicate malignant cells. Among immune effectors, CD8+ T cells play a pivotal role in tumor clearance through recognition and destruction of cancer cells. To effectively counter the rapid proliferation and continuous evolution of cancer, robust effector function of cytotoxic CD8+ T cells is essential. The expression of effector genes in CD8+ T cells is tightly regulated, ensuring that cytotoxic molecules are expressed only at appropriate times and in relevant contexts. We previously reported that upregulation of the gene NKG7 following immunotherapy is critical for clinical response and demonstrated that NKG7 expression is both necessary and sufficient for T cell cytotoxicity. However, the mechanisms governing NKG7 expression in CD8+ T cells remain poorly defined. Methods We performed a cytokine screen to identify potential drivers of NKG7 expression at both the transcriptional and protein levels in cell lines and healthy donor peripheral blood mononuclear cells (PBMC). We employed quantitative RT-PCR, flow cytometry, and Western blotting to determine and validate the expression level of NKG7 and relevant signal molecules. We validated the signal pathways identified in single cell RNAseq data analysis in PD-1 therapy-responsive CD8+ T cells. Results In PBMC derived CD8+ T cells, we found that IL-2 increases NKG7 mRNA levels, correlating with a dose dependent increase in NKG7 protein expression. IL-2—induced NKG7 expression is associated with elevated levels of T-bet, a transcription factor essential for effector T cell differentiation. Consistent with this, scRNA-seq analysis revealed enrichment of the IL-2 signaling pathway in CD8+ T cells responsive to anti—PD-1 therapy. Conclusion Our results suggest that NKG7 is upregulated by IL-2 and T-bet signaling. Due to the critical role of this signaling axis during effector CD8+ T cell differentiation, NKG7 expression may be determined during this phase. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Introduction The ability of the adaptive immune system to have memory of previous pathogen encounters is the basis for vaccination as one of the most successful medical interventions. With increasing age, the ability to develop protective immunity after vaccination declines. Differentiation into T memory cells is at least in part determined during the early cell divisions after activation when cells undergo assymmetric cell division (ACD). Fate decisions depend on the uneven distribution of signaling molecules and transcription factors with accumulation of mTORC1 activity biasing for an effector and TCF1 for a memory state. We explored whether ACD of naïve CD4 T cells is impaired in older adults. Methods We used human primary cells from young and older adults. Human naive CD4 T cells were isolated and cultured in RPMI, 10% FBS and antibiotics on plates coated with anti-CD3 Ab, anti-CD28 Ab and huICAM1 for 3 days. We used transfection by electroporation to modify gene expression. Read out systems included confocal microscopy, flow cytometry and lysosome function assays. Results were compared by t-test with values of p < 0.05 considered significant. Results Naive CD4 T cell responses from older adults is characterized by increased activation of the AKT-mTORC1 pathway and reduced ACD of mTORC1 activity. Increased RICTOR expression resulting in increased mTORC2 activity in naive CD4 T cells from older individuals accounted for the activation of the AKT-mTORC1 pathway. AKT inhibition or silencing of RICTOR restores ACD of mTORC1 activity as well as of TCF1. Conversely, ACD was also improved through inducing WNT signaling by GSK3β inhibition irrespective of RICTOR expression. Conclusion Our data show that increased RICTOR expression in naïve T cells from older adults at least in part accounts for defective memory cell generation. Silencing of RICTOR or downstream AKT inhibition can enforce both mTORC1 ACD and TCF1 ACD suggesting that mTORC2 is a promising target to improve vaccine responses. Funding Source n/a Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Abstract Introduction Microglia are the resident immune cells of the central nervous system, classically studied for neuroinflammation. A specialized subset, vessel-associated microglia (VAM), whose somata directly contact capillaries, constitutes ∼30% of microglia in the naïve brain. Only recently have VAM been recognized as regulators of brain circulation, influencing blood—brain barrier (BBB) integrity and cerebral blood flow, yet their role in pathological vascular remodeling remains unclear. To address this gap, we used glioma models in which peritumoral capillaries undergo extensive vascular remodeling and BBB dysfunction, providing an in vivo platform to probe capillary vulnerability and vascular stability. Methods We combined pharmacological microglial ablation via CSF1R inhibitors with longitudinal in vivo two-photon imaging to visualize microglia—capillary interactions. Behavioral testing, survival analysis, T2-weighted MRI, and immunostaining were performed in parallel. Results Over half of the microglia-depleted mice developed acute, lethal paralysis by 15 days, whereas control tumor-bearing mice remained asymptomatic and survived 30 days. MRI revealed large intracranial hemorrhages and severe edema, and two-photon imaging showed nodular, abnormal vasculature at paralysis onset. The peritumoral region contained dense capillary networks with a marked increase in VAMs (>60%). Genetic lineage tracing confirmed these cells were microglia, exhibiting elongated, vessel-wrapping morphology, also observed following single-vessel photothrombosis and consistent with a response to capillary injury. Conclusion Our findings identify VAMs as critical neurovascular stabilizers that preserve peritumoral capillary integrity and prevent lethal hemorrhage in glioma, highlighting an essential immune role in brain vascular homeostasis. Notably, intracranial hemorrhage has repeatedly emerged across clinical trials of CSF1R inhibitors, raising important safety considerations for CSF1R-targeted therapies in the diseased brain. Funding Source AHA postdoc fellowship (26POST1560008) (J.Z.) Topic Categories Neuroimmunology (NEUR)

Abstract Introduction Here we demonstrate that respiratory infection with Francisella tularensis induces the formation of induced bronchus-associated lymphoid tissue (iBALT) as a protective mechanism against pulmonary tularemia-like disease in mice. While semi-invariant natural killer T (NKT) cells and mucosal-associated invariant T (MAIT) cells are abundant at the lung mucosa, their roles in infectious diseases remain poorly understood. Pulmonary tularemia, the most severe form of tularemia, arises from respiratory infection with F. tularensis. Using C57BL/6 mice infected with the live vaccine strain (LVS) of F. tularensis to model tularemia-like disease, we previously showed that NKT cells exacerbate disease, as NKT-deficient CD1d⁻/⁻ mice were protected. Notably, protected CD1d⁻/⁻ mice developed iBALT structures in the lungs, which were absent in C57BL/6 mice. Methods Molecular immunology methods. Results Respiratory LVS infection in CD1d⁻/⁻ mice induced a robust expansion of IL-17-producing MAIT17 cells and a decrease in regulatory T cells in the lungs compared to C57BL/6 mice. Single-cell RNA sequencing revealed a gene signature in MAIT cells linked to factors that drive iBALT formation. Supporting this notion, adoptive transfer of LVS-activated lung MAIT cells from infected CD1d⁻/⁻ mice conferred protection against tularemia-like disease in RAG2⁻/⁻ recipients, while splenic MAIT cells did not despite the fact that both populations homed to the lungs after transfer. Lung-derived MAIT cells were enriched in IL-17-producing MAIT17 cells, whereas splenic MAIT cells predominantly consisted of interferon-gamma-producing MAIT1 cells. Last, IL-17 was identified as critical for iBALT development during respiratory LVS infection in CD1d⁻/⁻ mice. Conclusion In summary, our findings establish MAIT cells as central mediators of IL-17-dependent protection against pulmonary tularemia-like disease and underscore the potential protective role of iBALT in disease resistance. Funding Source NIH AI137082, VA Merit BX001610, VA SRCS BX004595 Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Abstract Introduction Cutaneous lichen planus (cLP) is a T cell—mediated skin disease in which cytotoxic CD8+ T cells attack basal keratinocytes at the dermo-epidermal junction. While studies have characterized T cell subsets in lesional tissue, circulating immune signatures in cLP are incompletely defined. We performed deep immunophenotyping of circulating T-cell subsets to map systemic alterations in cLP patients vs healthy controls. Methods We included 12 treatment-refractory cLP patients (mean age 63.6 ± 13.6 years) and 2 healthy controls (37.5 ± 10.6 years). Whole blood was profiled for 27 markers by flow cytometry. Sequential gating: singlets → live → CD3+ T cells → CD4+/CD8+/γδ; FOXP3+ Tregs were defined within CD4+. Frequencies were expressed as % of the immediate parent population. Group comparisons used two-tailed exact Mann—Whitney tests with Hodges—Lehmann median differences. Results Across frozen specimens, cLP patients exhibited broad T-cell activation. Proliferation increased in CD4 (Ki67+, p < 0.05), CD8 (6.785% vs 0.885%; Δ=5.90; p < 0.05), and Tregs (83.0% vs 46.8%; Δ=34.1; p < 0.05). Intracellular CTLA-4 was higher on CD4 (11.07% vs 2.615%; Δ=8.45; p < 0.05), CD8 (21.05% vs 9.30%; Δ=11.6; p < 0.05), and γδ T cells (47.20% vs 24.45%; Δ=22.75; p < 0.05). CXCL13+ cells expanded in CD4 (28.20% vs 12.45%; Δ=15.5; p < 0.05), CD8 (18.05% vs 3.595%; Δ=14.46; p < 0.05), γδ (2.805% vs 0.465%; Δ=2.285; p < 0.05), and Tregs (7.56% vs 1.17%; Δ=6.39; p < 0.05). Tregs were TBET-high (78.75% vs 61.55%; Δ=17.3; p < 0.05); GITR+ Tregs trended up (p = 0.1978). Conclusion cLP blood displays a CXCL13+, proliferative T-cell signature with increased intracellular CTLA-4 across CD4, CD8, γδ, and Treg compartments. CXCL13 elevation aligns with lesion-level literature implicating CXCL13+ CD8+ T cells that are suppressed in JAK-STAT therapy responders. Tregs exhibit a GITR upregulated, Th1-like phenotype that suggests loss of suppressive capacity, warranting further investigation on whether Treg dysfunction plays a causative role in cLP. Funding Source N/A Topic Categories Immune Mechanisms of Human Disease (HUM)

Abstract Introduction It is critical for the immune system to destroy cells harboring intracellular infection to remove pathogen burden. This function is widely attributed to cytotoxic T cells, which can physically engage with their targets through the T cell receptor complex, releasing cytotoxic granules, resulting in a specific death of infected targets. However, effector cytokines tumor necrosis factor-α (TNF) and interferon-γ (IFNγ), secreted by T cells, are themselves cytotoxic in combination to a degree. While TNF has appreciated cell death function through the TNF receptor 1 (TNFR1) signaling pathway, no clear cytotoxic function is attributed to IFNγ. Because of the diffusible nature of these cytokines, it is possible for T cells to elicit cytotoxicity without requiring direct contact with their targets, termed paracrine killing. Whether paracrine killing occurs during viral infection and how it is regulated is unclear. Paracrine killing poses issues of both sensitivity to these cytokines in that high concentrations of these cytokines are needed to affect target cells, as well as specificity in discriminating infected from uninfected host cells. Accounting for both issues, we hypothesize that paracrine killing is elicited through pathogen restriction whereby only infected cells are susceptible to death. Methods Analysis was performed in vitro using the Incucyte live cell imaging system and Western blots with fibroblast targets and a VSV infection model and OT1 T cells were leveraged ex vivo. Results TNF and IFNγ protect cells against the cytopathic effect of the virus. TNF specifically synergizes with VSV resulting in death of the infected cells and the two cytokines have distinct roles that work in concert. We demonstrate that TNF and IFNγ released by activated T cells suppress viral infection of nearby cells in a paracrine manner. Conclusion Here we provide insight and context into how distinct cytokines function in a cooperative manner for immune protection during an infection. Funding Source Mayo Clinic Graduate School of Biomedical Sciences Topic Categories Viral Immunology (VIR)

Abstract Introduction Bone marrow (BM) is the primary immune organ responsible for stem cell maintenance. Neurotropic infections elicit sequestration of T cells within the BM. Changes of the BM niche during brain viral infections are unknown. Intracranial infection with the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV) in C57BL/6 mouse is cleared by 30 days due to generation of an antigen-specific CD8 T cell response. Methods We used brain viral infection and injury models to test T cell responses against brain antigens in the bone marrow. Results We determined that 1-10% of CD8 T cells present in the sternal, femoral, and cranial BM were virus antigen-specific 7 days post intracranial infection. Over 70% of these CD8 T cells were tissue resident. We determined antigen-specific CD8 T cells were generated in the BM within 4 days of infection. Continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen-specific CD8 T cells in any BM compartment. CD8 T cells in the BM established durable memory populations and were reactivated upon antigen reencounter. In the memory phase (200 days post brain TMEV infection), bone marrow resident CD8 T cell could be reactivated upon cognate peptide delivery. Such reactivation of antigen specific memory CD8 T cells caused an increase in lineage-, Sca-1+, ckit + (LSK) cells in the BM in a CD8 dependent manner. Similarly, injection of the model antigen AF488-conjugated OVA mixed with Poly IC into the brain recapitulated antigen specific CD8 T cell generation within the bone marrow and further demonstrated the antigen presenting cell involved in the response to be a CD103 expressing myeloid DC. We conclude that viral infections and inflammatory stimulation in the brain induce in situ effector and memory T cell responses within the BM which is associated with significant stem cell dysregulation. Conclusion Our data paves the way for crucial studies of BM resident antigen-specific CD8 T cells in health and disease. Funding Source NIH K99/R00 Topic Categories Viral Immunology (VIR)

Abstract Introduction Screening individuals with latent tuberculosis infection (LTBI) from regions with varying TB endemicity is critical for global TB control. Immune responses to TB differ due to variations in exposure, immune adaptations, and environmental factors. This study evaluated antigen-specific immune responses in untreated LTBI individuals from low- and high-TB-endemic settings. Methods Peripheral blood mononuclear cells from untreated LTBI individuals from low-endemic (USA) and high-TB-endemic settings (Mexico) were stimulated with PPD, MTB300, ESAT-6/CFP-10 (RD1) peptides, mycobacterial glycopeptidolipids (GPL), and controls. We compare flow cytometric immune responses of T-cell intracellular cytokines and activation markers using non-parametric statistical methods. Results We studied age-matched, untreated LTBI individuals who tested IGRA(+)/TST(+) from low-TB-endemic (n = 18) and 18 healthcare workers from a high-TB-endemic settings. IGRA results showed no statistical differences across groups. The frequency of CD3+CD8+IFN-γ+HLA-DR+ and total CD3+TNF-α+ T cells after RD1 stimulation was significantly higher in the high-TB-endemic group (P < 0.05). Additionally, we observed significant increased frequencies in CD3/CD4+TNF-α+HLA-DR+ and CD8+TNF-α+HLA-DR+ T cells stimulated by GPL in the same group, probably associated with nontuberculous mycobacteria (NTM) exposures. Our findings suggest possible recent TB and NTM exposures in healthcare workers from a high-TB-endemic setting, resulted in increased antigen-specific adaptive TNF-α and IFN-γ producing T cells, which are important for pathogen control and immunity. Conclusion We found significant differences in antigen-specific cell immunity, including HLA-DR activated T-cells, a marker of recent antigen encounter, in individuals from a high-TB-endemic settings. These findings underscore the importance of settings-specific immune biomarkers for improving LTBI diagnostics, clinical management, and vaccination trials. Funding Source National Institute of Allergy and Infectious Diseases at the National Institutes of Health Topic Categories Immune Mechanisms of Human Disease (HUM)