Frequency Of Cells Research Articles

KLF4 enhances transplantation-induced hematopoiesis by inhibiting TLRs and non-canonical NFκB signaling at a steady state.

The transcription factor Krüppel-like factor 4 (KLF4) acts as a transcriptional activator and repressor. KLF4 plays a role in various cellular processes, including the dedifferentiation of somatic cells into induced pluripotent stem cells. Although it has been shown to enhance self-renewal in embryonic and leukemia stem cells, its role in adult hematopoietic stem cells (HSC) remains underexplored. We demonstrate that conditional deletion of the Klf4 gene in hematopoietic cells led to an increased frequency of immunophenotypic hematopoietic stem cells (HSCs) in the bone marrow, along with a normal distribution of lymphoid and myeloid progenitor cells. Non-competitive bone marrow transplants showed normal engraftment and multi-lineage reconstitution, except for monocytes and T cells. However, the loss of KLF4 hindered hematological reconstitution in competitive serial bone marrow transplants, highlighting a critical role for KLF4 in stress-induced hematopoiesis. Transcriptome analysis revealed an upregulation of NFκB2 and toll-like receptors (e.g., TLR4) in Klf4-null HSCs during homeostasis. Flow cytometry and immunoblot analysis confirmed the increased cell surface expression of TLR4 and the activation of NFκB2 in HSCs under homeostatic conditions, whereas NFκB2 expression drops after radiation compared to steady-state levels. Our findings suggest that the constitutive activation of the TLR4-NFκB2 pathway inhibits the ability of HSCs to regenerate blood after transplantation in cytoablated bone marrow.

Abstract TP350: The Effect of Type 2 Diabetes on the Transcriptome of Immune Cells in the Bone Marrow in Response to Ischemic Stroke

Type 2 diabetes mellitus (T2DM) induces chronic inflammation, which can worsen the secondary injury following ischemic stroke. Yet, whether and how T2DM-induced inflammation affects the production of bone marrow-derived immune cells and their transcriptome remains largely unexplored. Hypothesis: T2DM biases the immune system to a proinflammatory phenotype in the bone marrow, increases the transmigration of myeloid cells and compounds the secondary injury caused by stroke. Methods: We induced ischemic stroke by distal occlusion of the middle cerebral artery (MCAO) in T2DM mouse model db/db mice and normoglycemic control db/+ mice. Three days post-stroke, bone marrow (BM) cells and tissue were subjected to single-cell RNA sequencing (scRNAseq) and GeoMx digital spatial profiling (DSP). Myeloid cell populations were quantified by Fluorescent cell activated sorting (FACS). We used the NicheNet tool to analyze the cell-cell communication between BM populations. Results: We detected an increased frequency of hematopoietic precursor and myeloid progenitor cells in the BM of T2DM mice compared to control mice via FACS, although CD11b (+) cells were significantly increased only in control and not in T2DM mice after stroke. GeoMx DSP found that the upregulation of cd47, s100a9, cd177, pecam1 genes in both CD115 and Ly6G expressing cells were more pronounced in T2DM BM compared to control BM after stroke. Pathway enrichment analysis pointed to an increased activation of signals for myeloid cell activation and migration in T2DM stroke BM compared to control stroke BM, relative to their respective non-stroke counterpart. NicheNet integrated with single-cell RNA sequencing data corroborated with a differential increase of ccr2 and ccr3 interactions with a number of chemokine ligands in the BM of T2DM stroke mice compared to control stroke mice. Conclusion: our data suggest that T2DM enhances myeloid cell migration and chemotaxis after stroke. Ongoing studies will identify the tissue compartments to which increased myeloid cells transmigrate in T2DM mice after stroke and the effect on stroke outcomes.

P-1859. Immunity against Acute Human Babesiosis and Lyme Disease

Abstract Background Babesiosis,a disease caused by Babesia microti (Bm) and transmitted by Ixodes scapularis ticks, has significant morbidity in older and immunocompromised hosts. The natural host response against Bm has not been well-studied. Co-infection with Borrelia burgdorferi (Bb), the etiology of Lyme disease (LD) transmitted by the same deer tick, may impact clinical severity. We aimed to define the immune response to acute human babesiosis and LD in an endemic region in NY, with the hypothesis that specialized innate lymphocytes act early to control primary Bm infection.Figure 1.TNFα cytokine profiling for paired acute and 1-month post-treatment cases of babesiosis, Lyme Disease, and Co-infection.n=6/group. Statistical comparison by Kruskal-Wallis test *p<0.05 Methods Serum and Peripheral Blood Mononuclear Cells (PBMCs) were isolated from a well-characterized human cohort study: acute hospitalized study volunteers with confirmed Bm by peripheral blood microscopy and PCR (n=13), Bb infection by CDC criteria (n=5) or Bm/Bb co-infection (n=5) and compared to paired samples 1-month post-infection or healthy controls (n=12) collected during 2021-2023 at Stony Brook University Hospital, Long Island, NY. PBMCs from babesiosis cases were analyzed by flow cytometry and serum cytokine profiling was performed in mono/co-infected cases to define immune responses over time.Figure 2.IL27 cytokine profiling for paired acute and 1-month post-treatment cases of babesiosis, Lyme Disease, and Co-infection.n=6/group. Statistical comparison by Kruskal-Wallis test *p<0.05 Results Acute monoinfected babesiosis cases demonstrated inflammatory signaling through TNFα and IL27 while IL6, IL21, and IL10 were elevated in Bm and Bb mono/co-infection. Cytokine signatures trended toward resolution one-month post-Bm infection, but were persistently elevated during LD. We also observed persistently elevated CXCL10 and TNFα in LD. Flow cytometry of babesiosis cases vs controls revealed expansion of activated innate lymphocyte subsets including CD8α+ γδ T cells and Natural Killer (NK) cells. In contrast, innate T cells called MAIT cells were activated, but depleted from blood. There was no difference in the frequency of B cells, conventional CD4+/CD8+ T cells, or regulatory T cells during infection.Figure 3.The frequency and activation of γδ T cells in paired acute and 1-month post-treatment cases of babesiosis.n=12 healthy, n=13 babesiosis; Comparisons by Kruskal-Wallis test *p<0.05 **p<0.005 Conclusion This study identifies distinct inflammatory pathways underlying acute human babesiosis and LD and highlights early immune responses of innate lymphocytes during acute Bm infection. Ongoing studies will define the immune mechanisms that control primary Bm infection and will determine how this immunity is modulated by Bb co-infection.Figure 4.MAIT cell frequency and activation in paired acute and 1-month post treatment cases of babesiosis.n=12 healthy, n=13 babesiosis; Comparisons by Kruskal-Wallis test *p<0.05 Disclosures All Authors: No reported disclosures

Recirculating regulatory Tcells mediate thymic regeneration through amphiregulin following damage.

Thymic injury associated with disease or cancer treatment reduces Tcell production and makes patients more vulnerable to infections and cancers. Here, we examined the role of regulatory T (Treg) cells on thymic regeneration. Treg cell frequencies increased in the thymus in various acute injury models. Depletion of Treg cells impaired thymic regeneration, impacting both the thymocyte compartment and the stromal cell compartment; adoptive transfer of Treg cells enhanced regeneration. Expansion of circulating Treg cells, as opposed to that of tissue resident or recent thymic emigrants, explained this increase, as seen using parabiotic and adoptive transfer models. Single-cell analyses of recirculating Treg cells revealed expression of various regenerative factors, including the cytokine amphiregulin. Deletion of amphiregulin in these Treg cells impaired regeneration in the injured thymus. We identified an analogous population of CD39+ICOS+ Treg cells in the human thymus. Our findings point to potential therapeutic avenues to address aging- and treatment-induced immunosuppression.

Biophysical modeling and experimental analysis of the dynamics of C. elegans body-wall muscle cells.

This study combines experimental techniques and mathematical modeling to investigate the dynamics of C. elegans body-wall muscle cells. Specifically, by conducting voltage clamp and mutant experiments, we identify key ion channels, particularly the L-type voltage-gated calcium channel (EGL-19) and potassium channels (SHK-1, SLO-2), which are crucial for generating action potentials. We develop Hodgkin-Huxley-based models for these channels and integrate them to capture the cells' electrical activity. To ensure the model accurately reflects cellular responses under depolarizing currents, we develop a parallel simulation-based inference method for determining the model's free parameters. This method performs rapid parallel sampling across high-dimensional parameter spaces, fitting the model to the responses of muscle cells to specific stimuli and yielding accurate parameter estimates. We validate our model by comparing its predictions against cellular responses to various current stimuli in experiments and show that our approach effectively determines suitable parameters for accurately modeling the dynamics in mutant cases. Additionally, we discover an optimal response frequency in body-wall muscle cells, which corresponds to a burst firing mode rather than regular firing mode. Our work provides the first experimentally constrained and biophysically detailed muscle cell model of C. elegans, and our analytical framework combined with robust and efficient parametric estimation method can be extended to model construction in other species.

Increased interferon I signaling, DNA damage response and evidence of T-cell exhaustion in a patient with combined interferonopathy (Aicardi-Goutières Syndrome, AGS) and cohesinopathy (Cornelia de Lange Syndrome, CdLS)

BackgroundType I interferonopathies including Aicardi-Goutiéres Syndrome (AGS) represent a heterogeneous group of clinical phenotypes. Herein, we present a Case with combined AGS and Cornelia de Lange Syndrome (CdLS)—a cohesinopathy—with comprehensive analysis of the immune and genomic abnormalities.Case and methodsA 20-year old man presented with chilblain lesions and resorption of distal phalanges of fingers and toes, somatic and psychomotor retardation, microcephaly, synophrys, hearing losing and other aberrancies consistent with the phenotype of CdLS. We used whole exome sequencing to genetically map the associated mutations and performed transcriptome profiling and enrichment analysis in CD14+ monocytes of the patient and immune phenotyping by mass cytometry (CyToF), comparing to healthy individuals and lupus patients as disease controls. DNA damage response was assayed by confocal microscopy in the peripheral blood of this patient.ResultsNext generation exome sequencing confirmed a homozygous SAMHD1 gene mutation and a hemizygous non-synonymous mutation on SMC1A gene, responsible for the AGS and CdLS, respectively. Transcriptome profiling of CD14+ monocytes of the patient showed enrichment of type I IFN signaling and enhanced DNA damage response pathway. Broad immune phenotype of the peripheral blood of the patient revealed absence of activated T cell populations, increased frequency of NK cells and plasmablasts and enhanced granulocytic lineage. Further analysis suggested activation of the ATM branch of DNA damage response and increased apoptosis in the periphery of the patient.ConclusionsA rare case of a patient bearing two genetic lesions (responsible for AGS/CdLS syndromes) exhibits distinctive features of genomic damage and interferon responses. Immune phenotype revealed granulocytic skewing and absence of activated T cells compatible with chronic antigenic stimulation and/or homing of these cells at sites of inflammation.

Estrogen deficiency promotes neurodegeneration in female hemi-parkinsonian mice: The role of regulatory T cells.

Circulating levels of the female hormone estrogen has been associated with the development of Parkinson's disease (PD), although the underlying mechanism remains unclear. Immune homeostasis mediated by peripheral regulatory T cells (Treg) is a crucial factor in PD. The aim of this study was to explore the effects of estrogen deficiency on neuroinflammation and neurodegeneration in a rodent model of PD, with particular reference to Treg. Estrogen deficiency was established in a mouse model by bilateral ovariectomy (OVX). PD was modeled by the injection of LPS into the striatum. Motor performance was assessed in each experimental group. Dopaminergic degeneration was evaluated using tyrosine-hydroxylase (Th) immunohistochemical staining of the substantia nigra (SN) and striatum. Dopamine and dopamine metabolite levels in the striatum were also evaluated, together with the infiltration of CD4 T cells into the SN. Neuroinflammation was assessed by evaluating the mRNA level of microglial and M1/M2 phenotype markers, as well as the abundance of pro-inflammatory cytokines in the midbrain. The frequency of peripheral Treg cells was evaluated using flow cytometry. OVX prior to LPS injection markedly aggravated neurodegeneration, neuroinflammation, motor performance, and CD4 T cell infiltration compared to the LPS-only group. Estradiol treatment or activation of the G protein-coupled estrogen receptor (GPER) in OVX mice prior to LPS injection induced functional improvement and reduced the levels of neurodegeneration, neuroinflammation, and CD4 T cell infiltration. OVX prior to LPS injection also led to a decreased frequency of Treg compared to the LPS-only group. Moreover, estradiol treatment or GPER activation of OVX mice prior to LPS injection significantly increased the Treg frequency. Antibody-mediated depletion of Treg after GPER activation counteracted the ability of GPER to alleviate neurodegeneration, CD4 T cell infiltration, the release of pro-inflammatory cytokines, and motor performance in the PD model. Estrogen deficiency can disrupt Treg-mediated immune homeostasis and thus further aggravate microglial inflammation and dopaminergic degeneration in PD model. The effects of estrogen on Treg may be partially mediated by GPER signaling, and thus GPER is a promising target for PD, especially in estrogen-deficient women.

Specific types of male infertility are correlated with T cell exhaustion or senescence signatures

The association between male infertility and health status has yet to be unraveled. Here, by combining multiparameter phenotyping and scRNA-seq, we delineate the immune status of infertile men both at the semen and systemic levels. We first observe that young infertile men have a pro-inflammatory milieu with increased frequency of myeloid cells and inflammatory mediators in the seminal fluid and the peripheral blood, which are immune alterations typically observed in healthy elderly men. Transcriptomic profiling confirms the upregulation of genes associated with the interferon-gamma and -alpha responses in peripheral blood T cells of infertile men with oligo-astheno-teratozoospermia or non-obstructive azoospermia, with distinct T cell signatures of exhaustion and senescence discriminating the two infertile conditions. These findings provide evidence that subtypes of male infertility are characterized by specific immune signatures and unravel the potential link between infertility and the risk of developing secondary diseases.

TIGIT-expression on natural killer cell subsets is an early indicator of alleviating liver inflammation following bulevirtide treatment in chronic hepatitis D.

Bulevirtide (BLV) is a novel and the only approved treatment option for patients with chronic hepatitis D (CHD). BLV alleviates liver inflammation already early during treatment when only minor HDV RNA changes are observed. We hypothesized that BLV-treatment may influence immune cells in CHD patients and performed a high-resolution analysis of natural killer (NK) cells before and during BLV-therapy. BLV-treated CHD patients (n=20) from a single-center cohort were longitudinally analyzed for clinical, molecular, and virological parameters. Peripheral blood mononuclear cells (PBMCs) were studied at baseline (BL), therapy weeks 3 (TW3) and 48 (TW48) by spectral flow cytometry. Healthy donors (HD), chronic hepatitis C (CHC) patients after direct-acting antivirals (DAA)-treatment and patients with chronic hepatitis B (CHB) were used as controls. Overall NK cell frequencies remained stable during BLV-treatment. However, biochemical responders (BR) showed distinct NK cell immunophenotypic features before and during therapy. TIGIT-expression increased on CD56dim and CD56bright NK cells during the course of BLV-treatment and inversely correlated with alanine aminotransferase (ALT) levels in CHD but not CHC or CHB patients. High frequencies of TIGIT- CD57+ CD56dim NK cells at BL and low levels during therapy were indicative of a biochemical response. We here suggest that lacking the expression of the immune checkpoint inhibitor TIGIT on NK cell subtypes may be a hallmark of liver inflammation in HDV infection. BLV-therapy is associated with a reappearance of TIGIT on these cells, which may be one mechanism of why liver enzymes rapidly improve during therapy.

Potential therapeutic effect of dimethyl fumarate on Treg/Th17 cell imbalance in biliary atresia.

The imbalance between Tregs and proinflammatory Th17 cells in children with biliary atresia (BA) causes immune damage to cholangiocytes. Dimethyl fumarate (DMF), an immunomodulatory drug, regulates the Treg/Th17 balance in diseases like multiple sclerosis (MS). This study explores DMF's effect on Treg/Th17 balance in BA and its potential mechanism. The differential gene expression profiles in liver of BA and choledochal cyst (CC) patients were analyzed by single-cell RNA sequencing (scRNA-seq). Treg and Th17 cell frequencies in BA hilar lymph nodes (LNs) were determined by flow cytometry. CD3+ T cells were isolated from BA hilar LNs and treated with DMF in vitro to observe their differentiation. The effects of DMF were evaluated on BA mouse model, and enzyme-linked immunosorbent assay to measure biochemical markers and cytokine profiles. The Treg/Th17 ratio in the liver was determined by flow cytometry. Nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream antioxidant genes solute carrier family 7 member 1 (Slc7a11), heme oxygenase - 1 (Hmox1) was validated by q-PCR and Western blot. ScRNA-seq showed CD4+ T cells in BA liver were enriched in antioxidant pathways. The Treg/Th17 ratio in BA hilar LNs was significantly reduced compared to CC. In vitro, DMF promoted Treg differentiation and inhibited Th17 differentiation. In vivo, the Treg/Th17 ratio increased in the liver of the DMF 40 mg/kg group. In the 40 mg/kg DMF group, interleukin-17 A (IL-17 A) expression decreased as seen in pathological staining. DMF increased Nrf2, Hmox1, Slc7a11 mRNA and protein levels in DMF 40 mg/kg group. There is a Treg/Th17 imbalance in BA patients' hilar LNs, which DMF can restore in vitro. DMF improves the survival rate of BA mice and corrects the Treg/Th17 imbalance, possibly via the Nrf2/antioxidant response elements (ARE) pathway.

P0064 Investigating a pathogenic role for IL-18 in Inflammatory Bowel Disease beyond the classical type 1 immunity paradigm

Abstract Background Crohn’s disease (CD) and ulcerative colitis (UC) are intestinal diseases driven by chronic reactivation of memory T helper (Thm) 1-like and Th17m cells, respectively. Disease remission remains difficult to achieve and maintain due to inter-patient heterogeneity in disease severity and underlying immune responses. Moreover, variations in the pathogenic Thm responses, the treatment target, are understudied. The proinflammatory cytokine IL-18 enhances IL-12-induced IFN-γ production by Th1 cells, as seen in Th1-driven inflammatory diseases. However, its potential pathogenic role in immune-mediated diseases, like CD and UC, is unclear. Upon investigating immune responses in therapy-naïve pediatric CD and UC patients, we detected increased IL-18 plasma concentrations that were correctable by therapy. We hypothesized that IL-18 plays a role in both CD and UC by stimulating Th1m but also Th17m cells, worsening disease. Methods We examined therapy-naïve pediatric CD and UC patient plasma proteomics (92 proteins, Proximity Extension Assay technology; CD: n=85, UC: n =31, control: n=25), circulating gut-homing Thm populations, transcriptomes of intestinal biopsies (CD: n=57, UC: n =21, control: n=12), and clinical disease parameters. Further, we assessed cytokine responses by healthy donor-derived Th1m and Th17m cells after IL-18 and IL-12 stimulation in vitro. Results Plasma IL-18 concentrations correlated to concentrations of Th1-associated proteins (IFN-γ, CXCL9, CXCL11, CXCL10, PDL1) in CD and UC. Interestingly, in UC patients, IL-18 concentrations also correlated to Th17-associated proteins (IL-12B, IL-17A, IL-24). In agreement, preliminary in vitro data showed that IL-18 + IL-12 stimulation increases IL-17A- and IFN-γ-secreting cell frequencies in CCR6+ Th17m cells. In CD, plasma IL-18 concentrations correlated to frequencies of circulating gut-homing CCR6+CXCR3+CCR4+ Th17m, a pathogenic population known to acquire Th1 features and IFN-γ production. Ileal and colonic mRNA expression of IL-18 pathway genes associated with degree of histological inflammation. Higher IL-18 plasma concentrations at diagnosis associated with moderate to severe disease and worse endoscopic scores in both CD and UC and greater disease extent in UC, suggesting a relationship between IL-18 and disease severity. In line with its preferential production in the ileum, plasma IL-18 concentrations were significantly higher in CD patients with ileal involvement compared to CD patients with colonic disease. Conclusion We identify IL-18 as a potential enhancer of both Th1-like and Th17-driven inflammation. High plasma IL-18 is detectable in subgroups of CD and UC patients, raising the question whether IL-18 acts on a specific disease-associated Thm cell population.

DOP075 Tissue fibrosis modulates interleukin (IL)-13 signaling in intestinal epithelial cells

Abstract Background Inflammatory Bowel Diseases (IBDs) are characterized by chronic inflammation of the intestinal tissue and persistent disruption of the intestinal epithelial barrier. A common and dangerous complication of chronic inflammation during IBD is fibrosis, a mechanical stiffening of the intestinal tissue [1,2]. Previous work suggests that immune cell activity, for example Interleukin (IL)-13-mediated macrophage activation, can be mechanosensitive [3,4]. However, it is not currently well understood how fibrosis affects the inflammatory response in intestinal epithelial cells. Methods To address how tissue stiffness impacts intestinal inflammation, we studied the effects of inflammatory cytokine signaling in intestinal epithelial cells using a recently developed intestinal organoid monolayer model. We cultured intestinal organoid monolayers on soft (0.5kPa), intermediate (5kPa) or stiff (15kPa) hydrogels to mimic physiological or fibrotic conditions and studied the cellular response to IL-13, an inflammatory cytokine implicated in IBD and loss of barrier integrity. To investigate the impacts of substrate stiffness and IL-13 signaling on epithelial cell function, we combined high-resolution live microscopy, quantitative image analysis using AI-based models and biophysical measurements. Results We found that substrate mechanosensing and IL-13 signaling interact synergistically to modulate intestinal epithelial stem cell differentiation, leading to a dramatic increase in the frequency of secretory cells. Furthermore, organoids cultured on stiff substrates no longer respond to IL-13 treatment, suggesting that fibrotic conditions may promote an inflamed-like state even in the absence of inflammatory cytokines. To further investigate cross-talk between substrate mechanics and IL-13 signalling, we inhibited downstream IL-13 signalling and cell contractility. We found that both the stiffness- and IL-13-induced phenotypes could be rescued by either inhibiting myosin-2 activity or blocking downstream IL-13 signalling, further indicating a co-dependence and synergy of substrate stiffness and IL-13. Further experiments implicate the relocalization of myosin-2 to the cell cortex and increased nuclear accumulation of the mechanosensitive protein YAP in mediating this response. Experiments using mouse models of colitis further support a role for YAP in this process. Conclusion Taken together, this work reveals how tissue mechanics modulate the inflammatory response in the intestinal epithelium. Future translational work based on these findings will inspire new approaches to mitigate the effects of fibrosis during IBD.

DOP095 IL-23-activated cells as response biomarkers for risankizumab in Crohn’s disease patients

Abstract Background The interleukin (IL)-23 signaling is a vital pathway in Crohn’s disease (CD). IL-23 activates T-helper (Th) 17 cells, IL-17-secreting CD8 T (Tc)17 cells, γδ T cells, natural killer (NK) T cells, and group 3 innate lymphoid cells (ILC3) to secrete inflammatory cytokines including IL-17, IFN-γ, and TNF-α. Risankizumab is the first selective IL-23 antagonist approved for moderate-to-severe CD; however, no biomarker currently exists to predict response to this medication. Here, we characterized the phenotypes of IL-23-activated cells that predict response to risankizumab in CD patients. Methods Adult patients with active ileal/colonic CD were included (n = 28). Blood samples were collected before and after initiation of risankizumab. Peripheral blood mononuclear cells (PBMC) were isolated and cryopreserved for batch analysis. After stimulation with IL-23, PBMCs were stained using a pre-optimized and validated antibody panel for 37 lineage markers and cytokines for CyTOF. Cell clusters were resolved using UMAP (Figure 1), opt-SNE and FlowJo. Patients’ responses were evaluated 12-40 weeks after initiation of risankizumab based on clinical symptoms (e.g., CDAI) and endoscopic/radiographic assessment. Results Among the IL-23-activated cells, increased frequency of NKT cells was identified in the peripheral blood of non-responders before initiation of risankizumab (not shown). In pre-risankizumab PBMCs, we identified significantly elevated TNF-α in Th17 cells, Tc17 cells, NKT cells, and MAIT cells in non-responders vs. responders (Figure 2A). Similarly, in PBMCs 4 weeks after 1st risankizumab infusion, Tc17 cells, γδ T cells, NKT cells, and MAIT cells in non-responders produced significantly more TNF-α compared to those cells from responders. In addition, we observed increased IL-17A in multiple IL-23-activated cells in non-responders 4 weeks after starting risankizumab (Figure 2B). Peripheral and mucosal IL-23-activated cells from later non-responders before and 4 weeks after treatment spontaneously secreted TNF-α and IL-17A without stimulation. A similar correlation between therapeutic response and expression of TNF-α or IL-17A was not observed in ustekinumab, an anti-IL-12/IL-23 antibody (not shown). Conclusion CyTOF of patients’ PBMCs before and after starting risankizumab revealed previously unknown molecular signatures, especially augmented TNF-a production in multiple IL-23-activated cells, which predict non-response to risankizumab in CD patients. Those findings identified a novel mechanism of anti-IL-23 resistance, may help identify response biomarkers for IL-23 antagonists and provide a rationale for anti-IL-23/anti-TNF combination therapy in a subset of patients with refractory CD.

P0179 Spatial immune profiling of Crohn’s Disease fistula carcinomas - Defining a distinct cancer subtype

Abstract Background Fistula formation is a common and debilitating complication of Crohn’s disease (CD). Although rare, CD-associated fistula carcinomas present significant diagnostic and prognostic challenges. This study aims to identify disease-specific immune cell subsets in CD-associated fistula carcinomas. Methods The study analyzed tissue samples from 10 CD patients with fistula carcinomas, 7 patients with CD-associated fistulas, and 6 patients with sporadic colorectal cancer (CRC). In the tumor samples, the main tumor, infiltration front, and non-involved areas were included. Using a 36-marker panel, the immune landscape was characterized through imaging mass cytometry. The samples were processed, stained, and analyzed for immune cell compositions, cell-cell interactions, and spatial microenvironments. Results The immune infiltrate observed in fistula carcinomas displayed similarities to both CD fistulas and CRC. Fistula-carcinoma samples exhibited elevated levels of neutrophils, B cells, and CD163high macrophages. CRC main tumor samples exhibited a higher presence of intraepithelial CD8+ lymphocytes and CD163low macrophages. The highest levels of Cleaved Caspase-3 were observed in CRC main tumor samples, which exhibited a positive correlation with CD163low macrophages and cytotoxic T cells. Conversely, a negative correlation was observed between cleaved Caspase-3 and cytotoxic T cells in fistula-carcinoma main tumor samples. The analysis of cellular microenvironments and dimensionality reduction clustering based on immune cell frequencies revealed that fistula carcinomas exhibit a unique combination of immune cell characteristics derived from both CD fistulas and CRC. Conclusion The immune landscape of CD-associated fistula carcinomas exhibits characteristics of both CD fistulas and CRC, indicating a complex pathogenesis driven by chronic inflammation. These findings suggest that fistula carcinomas constitute a distinct cancer subtype, necessitating further research to develop targeted therapeutic strategies.

P0126 Single-cell analysis reveals enrichment of mucosal Th17-polarized T cells in Acute Severe Ulcerative Colitis

Abstract Background Acute severe ulcerative colitis (ASUC), a severe and potentially life-threatening manifestation of ulcerative colitis (UC) affects up to 25% of patients over their lifetime and requires urgent medical intervention. T cell subsets and myeloid cells play a critical role in the pathogenesis of mucosal inflammation. In this study, we aimed to perform an unbiased analysis of mucosal immune cell populations in patients with ASUC and to identify ASUC-specific cell type alterations. Methods Ten adult ASUC patients (defined by the Truelove and Witts criteria), 7 patients with active UC (not fulfilling ASUC criteria), and 5 normal volunteers (NV) were recruited (Fig. 1A). Intestinal mononuclear cells were isolated from endoscopically obtained intestinal biopsies and analyzed by single-cell RNA-sequencing (scRNA-seq). Linear mixed-effects models were applied to compare lineage-normalized cell type fractions across groups, while adjusting for relevant covariates including age, sex, and body mass index. Results Unsupervised clustering of 127,795 cells, expressing 38,224 unique genes, identified 45 distinct cell types (Fig. 1B). Among the salient findings specific to lymphoid cells, re-clustering of the T cell/NK cell compartment identified 9 cell subsets (Fig. 1C-D). Notably, TH17 cells (RORA, CTSH, CXCR6, and CCL20) and cycling TH17 cells (MKI67, PCLAF, SMC4, NUSAP1, and RRM2) were significantly enriched in patients with ASUC compared to those with active UC (adjusted P=0.019 and P=0.027, respectively) and NV (adjusted P<0.001 for both) (Fig. 1E-F). Among myeloid cells, of the 11 delineated subsets, patients with ASUC showed a significant reduction in lineage-normalized fractions of type 2 conventional dendritic cells (cDC2s) (CD1C, FCER1A) relative to both active UC and NV (adjusted P=0.017 for both) (Fig. 1G). Additionally, a cluster of CD14+ monocytes (CD14, FCN1, and LYZ) was significantly elevated in patients with ASUC compared to NV (adjusted P=0.032). Conclusion Elevated TH17 cell frequencies distinguish ASUC from UC. While further investigations, including protein-level validation of transcriptomic data and an assessment of the impact of cellular perturbation on treatment outcome, are ongoing, these findings shed light on a potentially underappreciated role for TH17-polarized T cells in ASUC.

Transcription factor KLF2 is associated with the dysfunctional status of NK cells and the prognosis of pediatric B-ALL patients.

Natural killer cells, an important component of the innate immune system, can directly recognize and lyse virally infected or transformed cells. However, NK cells fail to restrain the growth of malignancies, such as B-cell acute lymphoblastic leukemia (B-ALL). The molecular genetics of NK cells in the B-ALL bone marrow microenvironment and the mechanisms underlying the inhibited function of NK cells at the single-cell level remain largely elusive. In this study, we studied the frequency and absolute number of NK cells in peripheral blood samples collected from 43 healthy volunteers and 104 pediatric B-ALL patients diagnosed at Hunan Children's Hospital. We also analyzed published single-cell RNA sequencing (scRNAseq) data from B-ALL and normal bone marrow samples using unsupervised clustering. Our findings were further validated using bulk transcriptomic data and clinical data from a cohort of 139 B-ALL bone marrow samples. We found that the frequency and number of NK cells were significantly decreased in the bone marrow and peripheral blood of B-ALL patients. In-depth analysis of scRNAseq data identified 12 NK cell clusters. Among them, the C2 cluster, which is present in healthy bone marrow but reduced in B-ALL bone marrow, displays overexpression of a transcription factor KLF2 and a significant downregulation of the "leukocyte proliferation" pathway. Furthermore, we found that the expression of KLF2 in B-ALL at diagnosis was positively correlated with the percentage of leukemia cells and the positive rate of minimal residual disease (MRD), indicating that KLF2 is a marker of poor prognosis. There are dramatic differences at the single-cell level in the transcriptomics of NK cells between healthy donors and B-ALL patients. A transcription factor, KLF2, which is enriched in the C2 cluster of NK cells, has been suggested to regulate the proliferation of NK cells and is associated with poor prognosis of pediatric B-ALL.

Functional validation of a novel STAT3 'variant of unknown significance' identifies a new case of STAT3 GOF syndrome and reveals broad immune cell defects.

Signal transducer and activator of transcription 3 (STAT3) orchestrates crucial immune responses through its pleiotropic functions as a transcription factor. Patients with germline monoallelic dominant negative or hypermorphic STAT3 variants, who present with immunodeficiency and/or immune dysregulation, have revealed the importance of balanced STAT3 signaling in lymphocyte differentiation and function, and immune homeostasis. Here, we report a novel missense variant of unknown significance in the DNA-binding domain of STAT3 in a patient who experienced hypogammaglobulinemia, lymphadenopathy, hepatosplenomegaly, immune thrombocytopenia, eczema, and enteropathy over a 35-year period. In vitro demonstration of prolonged STAT3 activation due to delayed dephosphorylation, and enhanced transcriptional activity, confirmed this to be a novel pathogenic STAT3 gain-of-function variant. Peripheral blood lymphocytes from this patient, and patients with confirmed STAT3 Gain-of-function Syndrome, were collected to investigate mechanisms of disease pathogenesis. B cell dysregulation was evidenced by a loss of class-switched memory B cells and a significantly expanded CD19hiCD21lo B cell population, likely influenced by a skewed CXCR3+ TFH population. Interestingly, unlike STAT3 dominant negative variants, cytokine secretion by activated peripheral blood STAT3 GOF CD4+ T cells and frequencies of Treg cells were intact, suggesting CD4+ T cell dysregulation likely occurs at sites of disease rather than the periphery. This study provides an in-depth case study in confirming a STAT3 gain-of-function variant and identifies lymphocyte dysregulation in the peripheral blood of patients with STAT3 gain-of-function syndrome. Identifying cellular biomarkers of disease provides a flow cytometric-based screen to guide validation of additional novel STAT3 gain-of-function variants as well as provide insights into putative mechanisms of disease pathogenesis.

Detection and Characterization of Circulating Tumor Cells in Colorectal Cancer Patients via Epithelial-Mesenchymal Transition Markers.

Background/Objectives: Liquid biopsy methods have gained prominence as minimally invasive tools to improve cancer treatment outcomes. Circulating tumor cells (CTCs) offer valuable insights into both primary and metastatic lesions. However, validating the CTC test results requires confirmation that the detected cells originate from cancer tissue. While studies have identified CTCs in colorectal cancer (CRC) patients using molecular markers, simultaneous validation of their cancer tissue origin remains unexplored. Methods: This study introduces a simple approach to detect adenomatous polyposis coli (APC) gene abnormalities alongside established CTC markers using a molecular imaging flow cytometer (MI-FCM). Given that APC gene abnormalities occur in 60-70% of CRC patients, their detection serves as strong evidence of cancer origin. Results: Our method achieved 92% concordance with DNA sequence analysis of tumor-derived cells. In a proof-of-concept study using 5 mL of whole blood from CRC patients, we observed a high frequency of cells exhibiting APC abnormalities, cytokeratin (CK), and vimentin (Vim) expression. Extending the study to 80 CRC patients across pathological stages I-IV confirmed CK and Vim as valid CTC markers. Three distinct cell populations were identified in blood: CK+/Vim-, CK+/Vim+, and CK-/Vim+. CTC number and frequency increased progressively with cancer stage. Conclusions: This is the first report demonstrating CK and Vim as effective markers for direct CTC detection in CRC patients. Our findings provide evidence-based validation of CTC markers, offering new insights and advancing approaches for patient care.

CD4+FOXP3Exon2+ regulatory T cell frequency predicts breast cancer prognosis and survival.

CD4+FOXP3+ regulatory T cells (Tregs) suppress immune responses to tumors, and their accumulation in the tumor microenvironment (TME) correlates with poor clinical outcome in several cancers, including breast cancer (BC). However, the properties of intratumoral Tregs remain largely unknown. Here, we found that a functionally distinct subpopulation of Tregs, expressing the FOXP3 Exon2 splicing variants, is prominent in patients with hormone receptor-positive BC with poor prognosis. Notably, a comprehensive examination of the TCGA validated FOXP3E2 as an independent prognostic marker in all other BC subtypes. We found that FOXP3E2 expression underlies BCs with defective mismatch repair and a stem-like signature and highlights pathways involved in tumor survival. Last, we found that the TME induces FOXP3E2 through the CXCL12/CXCR4 axis and confirmed the higher immunosuppressive capacity of FOXP3E2+ Tregs derived from patients with BC. Our study suggests that FOXP3E2+ Tregs might be used as an independent biomarker to predict BC prognosis and survival and to develop super-targeted immunotherapies.

Evaluating the Diagnostic Value of Lymphocyte Subsets in Bronchoalveolar Lavage Fluid and Peripheral Blood Across Various Diffuse Interstitial Lung Disease Subtypes.

Diffuse interstitial lung diseases (ILD) include conditions with identifiable causes such as chronic eosinophilic pneumonia (CEP), sarcoidosis (SAR), chronic hypersensitivity pneumonitis (CHP), and connective tissue disease-associated interstitial pneumonia (CTD), as well as idiopathic interstitial pneumonia (IIP) of unknown origin. In non-IIP diffuse lung diseases, bronchoalveolar lavage (BAL) fluid appearance is diagnostic. This study examines lymphocyte subsets in BAL fluid and peripheral blood of 56 patients with diffuse ILD, excluding idiopathic pulmonary fibrosis (IPF), who underwent BAL for diagnostic purposes. Patients were classified into CEP, SAR, CHP, CTD, and IIP groups, and clinical data, BAL cell analysis, and peripheral blood mononuclear cell analysis were compared. Eosinophils and type 3 innate lymphocytes (ILC3s) were significantly increased in the BAL fluid of the CEP group. Receiver operating characteristic curve analysis identified eosinophils ≥ 8% in BAL cells and ILC3s ≥ 0.0176% in the BAL lymphocyte fraction as thresholds distinguishing CEP. SAR patients exhibited significantly elevated CD4/CD8 ratios in the BAL fluid, with a ratio of 3.95 or higher and type 1 innate lymphoid cell frequency ≥ 0.254% as differentiation markers. High Th1 cell frequency (≥17.4%) in BAL lymphocytes in IIP, elevated serum KL-6 (≥2081 U/mL) and SP-D (≥261 ng/mL) in CHP, and increased BAL neutrophils (≥2.0%) or a low CD4/CD8 ratio (≤1.2) in CTD serve as distinguishing markers for each ILD. Excluding CEP and SAR, CD4+ T cell frequencies, including Th1, Th17, and Treg cells in peripheral blood, may differentiate IIP, CHP, and CTD.