In adult tissues, epithelial stem cells exist within distinct residences, each endowing them with exclusive instructions for regenerative fitness under homeostasis and stress. Key components of these 'niches' are immune cells, which classically protect the host against external and internal threats. Whether and how stem cell:immune cell crosstalk contributes to normal tissue biology remains less clear. Here, we discover functional adaptation of resident lymphocytes within two distinct skin stem cell niches and show that through this communication, each niche adjusts to meet diverse tissue demands. In the upper hair follicle, where microbial load is high, T cells express lymphotoxin-β and stimulate adjacent receptor-positive epithelial stem cells to form an immune-competent niche that controls microbial expansion. By contrast, in the epidermis, these T cells produce amphiregulin to maintain continuous stem cell reconstitution of the skin's barrier. Concomitantly, they express the immune checkpoint protein 'LAG-3', which autorestricts lymphocyte numbers, and hence amphiregulin levels, thereby preventing over-proliferative responses. Finally, when epidermal T cells are absent, dermal lymphocytes restore the imbalance by colonizing and adapting to their new niche. Our findings unveil functional specialization and homeostatic resilience of immune-stem cell niches, each tailored to suit the demands of distinct tissue microenvironments.

BACKGROUND AND OBJECTIVE:Globally, Gastric Cancer (GC) ranks as the fifth leading cause of cancer-related deaths. GC is a multifaceted malignancy with diverse etiologies; however, understanding the shared molecular mechanisms can aid in discovering novel targeted therapies for GC. This study has employed a drug repositioning approach to explore new drug candidates for treating GC. METHODS:The human GC cell lines AGS, MKN-45, and KATO-III were treated with different concentrations of dopamine, cabergoline, thioridazine, and entacapone to determine effective doses and IC50 values. In vitro, cytotoxic activity on cancer cell lines was screened based on dose/time using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) was used to measure the mRNA expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Proliferating Cell Nuclear Antigen (PCNA) in each group. The percentage of apoptotic cells was evaluated using Annexin V/PI staining. RESULTS:Dopamine, cabergoline, thioridazine, and entacapone elicited cytotoxic effects on AGS and KATO-III cells in a dose-dependent manner and elevated the percentage of Annexin Vpositive cells, suggesting the occurrence of apoptosis. The expression of Bcl-2 and PCNA was significantly decreased, whereas the expression of Bax was considerably increased in the AGS and KATO-III cells compared to that in the blank group (p < 0.05); however, no similar effect was observed in MKN-45 cells. CONCLUSION:Through in vitro experiments, this study provides evidence that the antipsychotic drugs cabergoline, dopamine, thioridazine, and entacapone can inhibit gastric cancer growth in AGS and KATO-III cells. These findings suggest that these drugs could be repurposed as novel therapeutic agents for the treatment of gastric cancer.

PTEN is well known as a tumor suppressor that inhibits the PI3K/AKT pathway, but the role of its secreted isoform PTEN-Long (PTEN-L) is not fully understood. We developed tools to study PTEN-L independent of PTEN using cancer cell lines engineered to express PTEN-L, a mouse engineered to overexpress Pten-L, and Adeno-Associated Virus (AAV)-Pten-L treated xenograft models. AAV-Pten-L was engineered to be expressed in the liver, where it was efficiently secreted into blood using a strong signal peptide for the liver. Beyond its known ability to inhibit PI3K/AKT signaling, we showed that Pten-L entered tumor cells and macrophages and altered interferon-γ and TGF-β signaling as well as immune cell composition, which triggered the rapid regression of small mouse tumors. Pten-L treatment activated immune cells in the tumor microenvironment, including macrophages, T cells, and NK cells. These changes were associated with enhanced MHC-II and CD80 expression by macrophages and reduced expression of PD-L1 by tumor cells and macrophages. Depletion experiments using anti-CD8 or anti-CD80 antibodies revealed that CD8+ T-cells and antigen-presenting macrophage cells were required for AAV-Pten-L-induced tumor regression. These results demonstrate the capacity of secreted Pten-L to stimulate the innate immune system and attenuate the immune checkpoint ligand PD-L1 in the tumor, which likely together orchestrate an adaptive immune response to inhibit the growth of established tumors. The first and second authors are co-first authors. Jia Xu, Tiphaine Martin, Daniel Lozano-Ojalvo, Andrew Baik, Bruno Giotti, Ashikur Rahaman, Andrew L. Wolfe, Natalie Suhy, Royce Zhou, Zhengxiang He, Kaitlyn Bosch, Madhuri Kalathur, Elias Stratikopoulos, Shen Yao, Ruifang Qiao, Sergio Lira, Emily Gallagher, Joshua Brody, Jordi Ochando, Alexander Tsankov, Katherine Cygnar, Aris Economides, Ramon E. Parsons. Secreted PTEN-long downregulates PI3K signaling and PD-L1 and promotes anti-tumor antigen-presenting cell functions to cause regressions of mouse tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB243.

The T300A substitution in ATG16L1 associated with Crohn's disease impairs autophagy, yet up to 50% of humans are heterozygous for this allele. Here, we demonstrate that heterozygosity for the analogous substitution in mice (Atg16L1T316A), but not homozygosity, protects against lethal Salmonella enterica Typhimurium infection. One copy of Atg16L1T316A was sufficient to enhance cytokine production through inflammasome activation, which was necessary for protection. In contrast, two copies of Atg16L1T316A inhibited the autophagy-related process of LC3-associated phagocytosis (LAP) and increased susceptibility. Macrophages from human donors heterozygous for ATG16L1T300A displayed elevated inflammasome activation while homozygosity impaired LAP, similar to mice. These results clarify how the T300A substitution impacts ATG16L1 function and suggest it can be beneficial to heterozygous carriers, providing an explanation for its prevalence within the human population.

Abstract Small intestinal epithelial cells (sIECs) form a critical barrier essential for nutrient absorption and host defense. sIECs undergo continuous renewal to maintain their integrity. Phagocytes, such as macrophages, facilitate the clearance of apoptotic cells that enter subepithelial tissues. However, little is known about how sIEC-phagocyte crosstalk maintains barrier integrity. Our study reveals synchrony between sIEC death and the accumulation of a specific subset of macrophages engaged in engulfing dying cells. Notably, we observed a diurnal rhythmicity in the death of sIECs during homeostasis. A fraction of these dying cells enter the subepithelial space, where Tim4+ CD4+ macrophages rhythmically eliminate them. Our findings demonstrate that Tim4+ CD4+ macrophage maintenance depends on dietary vitamin A via macrophage-intrinsic retinoic acid receptor (RAR) signaling. We show that rhythmic Tim4+ CD4+ macrophage accumulation relies on light cues and the macrophage circadian clock. Impaired macrophage RAR signaling disrupts the clearance of dying sIECs, leading to compromised gut barrier integrity and increased bacterial translocation to distant organs. Our work reveals an intricate relationship between sIECs and phagocytes and identifies a crucial role for Tim4+ CD4+ macrophages in maintaining intestinal barrier integrity. Understanding these dynamics could suggest new strategies for maintaining intestinal barrier function during infection and inflammatory disease.

The T300A substitution in ATG16L1 associated with Crohn’s Disease impairs autophagy, yet up to 50% of humans are heterozygous for this allele. Here we demonstrate that heterozygosity for the analogous substitution in mice ( Atg16L1T316A ), but not homozygosity, protects against lethal Salmonella enterica Typhimurium infection. One copy of Atg16L1T316A was sufficient to enhance cytokine production through inflammasome activation, which was necessary for protection. In contrast, two copies of Atg16L1T316A inhibited the autophagy-related process of LC3-associated phagocytosis (LAP) and increased susceptibility. Macrophages from human donors heterozygous for ATG16L1T300A displayed elevated inflammasome activation while homozygosity impaired LAP, similar to mice. These results clarify how the T300A substitution impacts ATG16L1 function and suggest it can be beneficial to heterozygous carriers, providing an explanation for its prevalence.One-Sentence Summary Heterozygosity of Crohn’s diseases risk variant ATG16L1 T300A confers protection against bacterial infections.### Competing Interest StatementKC has received research support from Pfizer, Takeda, Pacific Biosciences, Genentech, and Abbvie; consulted for or received an honoraria from Puretech Health, Genentech, and Abbvie; and is an inventor on U.S. patent 10,722,600 and provisional patent 62/935,035 and 63/157,225.

BACKGROUND & AIMS:Patients with inflammatory bowel disease (IBD) frequently develop extraintestinal manifestations (EIMs) that contribute substantially to morbidity. We assembled the largest multicohort data set to date to investigate the clinical, serologic, and genetic factors associated with EIM complications in IBD. METHODS:Data were available in 12,083 unrelated European ancestry IBD cases with presence or absence of EIMs (eg, ankylosing spondylitis [ankylosing spondylitis and sacroiliitis], primary sclerosing cholangitis [PSC], peripheral arthritis, and skin and ocular manifestations) across 4 cohorts (Cedars-Sinai Medical Center, National Institute for Diabetes and Digestive and Kidney Diseases IBD Genetics Consortium, Sinai Helmsley Alliance for Research Excellence Consortium, and Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn's Disease cohort). Clinical and serologic parameters were analyzed by means of univariable and multivariable regression analyses using a mixed-effects model. Within-case logistic regression was performed to assess genetic associations. RESULTS:Most EIMs occurred more commonly in female subjects (overall EIM: P = 9.0E-05, odds ratio [OR], 1.2; 95% CI, 1.1-1.4), with CD (especially colonic disease location; P = 9.8E-09, OR, 1.7; 95% CI, 1.4-2.0), and in subjects who required surgery (both CD and UC; P = 3.6E-19, OR, 1.7; 95% CI, 1.5-1.9). Smoking increased risk of EIMs except for PSC, where there was a "protective" effect. Multiple serologic associations were observed, including with PSC (anti-nuclear cytoplasmic antibody; IgG and IgA, anti-Saccharomyces cerevisiae antibodies; and anti-flagellin) and any EIM (anti-nuclear cytoplasmic antibody; IgG and IgA, anti-Saccharomyces cerevisiae antibodies; and anti-Pseudomonas fluorescens-associated sequence). We identified genome-wide significant associations within major histocompatibility complex (ankylosing spondylitis and sacroiliitis, P = 1.4E-15; OR, 2.5; 95% CI, 2.0-3.1; PSC, P = 2.7E-10; OR, 2.8; 95% CI, 2.0-3.8; ocular, P = 2E-08, OR, 3.6; 95% CI, 2.3-5.6; and overall EIM, P = 8.4E-09; OR, 2.2; 95% CI, 1.7-2.9) and CPEB4 (skin, P = 2.7E-08; OR, 1.5; 95% CI, 1.3-1.8). Genetic associations implicated tumor necrosis factor, JAK-STAT, and IL6 as potential targets for EIMs. Contrary to previous reports, only 2% of our subjects had multiple EIMs and most co-occurrences were negatively correlated. CONCLUSIONS:We have identified demographic, clinical, and genetic associations with EIMs that revealed underlying mechanisms and implicated novel and existing drug targets-important steps toward a more personalized approach to IBD management.

Neurodegenerative diseases (ND) are characterized by progressive loss of neuronal function. Mechanisms of ND pathogenesis are incompletely understood, hampering the development of effective therapies. Langer-hans cell histiocytosis (LCH) is an inflammatory neoplastic disorder caused by hematopoietic progenitors ex-pressing mitogen-activated protein kinase (MAPK)-activating mutations that differentiate into senescent myeloid cells that drive lesion formation. Some individuals with LCH subsequently develop progressive and incurable neurodegeneration (LCH-ND). Here, we showed that LCH-ND was caused by myeloid cells that were clonal with peripheral LCH cells. Circulating BRAFV600E+ myeloid cells caused the breakdown of the blood-brain barrier (BBB), enhancing migration into the brain parenchyma where they differentiated into senescent, inflammatory CD11a+ macrophages that accumulated in the brainstem and cerebellum. Blocking MAPK activity and senescence programs reduced peripheral inflammation, brain parenchymal infil-tration, neuroinflammation, neuronal damage and improved neurological outcome in preclinical LCH-ND. MAPK activation and senescence programs in circulating myeloid cells represent targetable mechanisms of LCH-ND.

Introduction: Gastric cancer is one of the common causes of cancer-related death in the world. Neurotransmitters have recently been related to the proliferation of cancer cells, but the role of neurotransmitters in the progression of gastric cancer is still unexplored. The cross-talk between the nervous system and immune cells through serotonin and its receptors in the tumor microenvironment can impact tumor progress. Our purpose is to expose probable changes in serotonin receptors, acetylcholinesterase, and monoamine oxidase A gene expression in gastric cancer. Methods: Transcript of serotonin receptors (5-HTR2A, 5-HTR2B, 5-HTR3A, 5-HTR7) and monoamine oxidase A genes in the peripheral blood mononuclear cells (40 patients and 40 control) and tissue (21 tumors and 21 normal adjacent tissues) were assessed. The gene expression was analyzed by quantitative real-time PCR using suitable primers. Statistical analysis was performed using appropriate software (REST, Prism). Results: Significantly higher amounts of 5-HTR2A, 5-HTR2B, 5-HTR3A, 5-HTR7, and acetylcholinesterase gene transcripts were found in the peripheral blood of gastric cancer patients compared with healthy individuals. The expression of 5-HTR2B and 5-HTR3A genes was significantly higher (p = 0.0250, p = 0.0005, respectively) and the acetylcholinesterase gene was lower in the tissue of patients (p = 0.0119) compared with adjacent normal tissue. Conclusion: This study highlights the role of serotonin receptors in gastric cancer that might have suggestions for the development of novel therapeutics and defensive approaches that target factors associated with the link between the nervous system, cancer cells, and the tumor microenvironment.

Neurodegenerative diseases (ND) are characterized by progressive loss of neuronal function. Mechanisms of ND pathogenesis are incompletely understood, hampering the development of effective therapies. Langerhans cell histiocytosis (LCH) is an inflammatory neoplastic disorder caused by hematopoietic progenitors expressing MAPK activating mutations that differentiate into senescent myeloid cells that drive lesion formation. Some patients with LCH subsequently develop progressive and incurable neurodegeneration (LCH-ND). Here, we show that LCH-ND is caused by myeloid cells that are clonal with peripheral LCH cells. We discovered that circulating BRAF V600E + myeloid cells cause the breakdown of the blood-brain barrier (BBB), enhancing migration into the brain parenchyma where they differentiate into senescent, inflammatory CD11a + macrophages that accumulate in the brainstem and cerebellum. Blocking MAPK activity and senescence programs reduced parenchymal infiltration, neuroinflammation, neuronal damage and improved neurological outcome in preclinical LCH-ND. MAPK activation and senescence programs in circulating myeloid cells represent novel and targetable mechanisms of ND.

ObjectivePerianal Crohn's disease (pCD) occurs in up to 40% of patients with CD and is associated with poor quality of life, limited treatment responses and poorly understood aetiology. We performed a genetic association study comparing CD subjects with and without perianal disease and subsequently performed functional follow-up studies for a pCD associated SNP in Complement Factor B (CFB). DesignImmunochip-based meta-analysis on 4056 pCD and 11 088 patients with CD from three independent cohorts was performed. Serological and clinical variables were analysed by regression analyses. Risk allele of rs4151651 was introduced into human CFB plasmid by site-directed mutagenesis. Binding of recombinant G252 or S252 CFB to C3b and its cleavage was determined in cell-free assays. Macrophage phagocytosis in presence of recombinant CFB or serum from CFB risk, or protective CD or healthy subjects was assessed by flow cytometry. ResultsPerianal complications were associated with colonic involvement, OmpC and ASCA serology, and serology quartile sum score. We identified a genetic association for pCD (rs4151651), a non-synonymous SNP (G252S) in CFB, in all three cohorts. Recombinant S252 CFB had reduced binding to C3b, its cleavage was impaired, and complement-driven phagocytosis and cytokine secretion were reduced compared with G252 CFB. Serine 252 generates a de novo glycosylation site in CFB. Serum from homozygous risk patients displayed significantly decreased macrophage phagocytosis compared with non-risk serum. ConclusionpCD-associated rs4151651 in CFB is a loss-of-function mutation that impairs its cleavage, activation of alternative complement pathway, and pathogen phagocytosis thus implicating the alternative complement pathway and CFB in pCD aetiology.

Chronic stress underlies the etiology of both major depressive disorder (MDD) and irritable bowel syndrome (IBS), two highly prevalent and debilitating conditions with high rates of co-morbidity. However, it is not fully understood how the brain and gut bi-directionally communicate during stress to impact intestinal homeostasis and stress-relevant behaviours. Using the chronic social defeat stress (CSDS) model, we find that stressed mice display greater intestinal permeability and circulating levels of the endotoxin lipopolysaccharide (LPS) compared to unstressed control (CON) mice. Interestingly, the microbiota in the colon also exhibit elevated LPS biosynthesis gene expression following CSDS. Additionally, CSDS triggers an increase in pro-inflammatory colonic IFNγ + Th1 cells and a decrease in IL4 + Th2 cells compared to CON mice, and this gut inflammation contributes to stress-induced intestinal barrier permeability and social avoidance behaviour. We next investigated the role of enteric neurons and identified that noradrenergic dopamine beta-hydroxylase (DBH) + neurons in the colon are activated by CSDS, and that their ablation protects against gut pathophysiology and disturbances in social behaviour. Retrograde tracing from the colon identified a population of corticotropin-releasing hormone-expressing (CRH + ) neurons in the paraventricular nucleus of the hypothalamus (PVH) that innervate the colon and are activated by stress. Chemogenetically activating these PVH CRH + neurons is sufficient to induce gut inflammation, barrier permeability, and social avoidance behaviour, while inhibiting these cells prevents these effects following exposure to CSDS. Thus, we define a stress-activated brain-to-gut circuit that confers colonic inflammation, leading to impaired intestinal barrier function, and consequent behavioural deficits.

Myeloid cells comprise the majority of immune cells in tumors, contributing to tumor growth and therapeutic resistance. Incomplete understanding of myeloid cells response to tumor driver mutation and therapeutic intervention impedes effective therapeutic design. Here, by leveraging CRISPR/Cas9-based genome editing, we generate a mouse model that is deficient of all monocyte chemoattractant proteins. Using this strain, we effectively abolish monocyte infiltration in genetically engineered murine models of de novo glioblastoma (GBM) and hepatocellular carcinoma (HCC), which show differential enrichment patterns for monocytes and neutrophils. Eliminating monocyte chemoattraction in monocyte enriched PDGFB -driven GBM invokes a compensatory neutrophil influx, while having no effect on Nf1 -silenced GBM model. Single-cell RNA sequencing reveals that intratumoral neutrophils promote proneural-to-mesenchymal transition and increase hypoxia in PDGFB -driven GBM. We further demonstrate neutrophil-derived TNF-a directly drives mesenchymal transition in PDGFB -driven primary GBM cells. Genetic or pharmacological inhibiting neutrophils in HCC or monocyte-deficient PDGFB -driven and Nf1 -silenced GBM models extend the survival of tumor-bearing mice. Our findings demonstrate tumor-type and genotype dependent infiltration and function of monocytes and neutrophils and highlight the importance of targeting them simultaneously for cancer treatments.

S1: Unaltered tumor burden in colons of ACKR2-/-ApcMin/+ mice. S2: Delayed onset of tumor development in the small intestine of ACKR2-/-ApcMin/+ mice. S3. Decreased inflammatory markers in the distal intestine tumors of ACKR2-/-ApcMin/+ mice. S4. Elevated mast cell infiltration into microadenomas in ACKR2-/-ApcMin/+ mice. S5. Identification of BMMC. S6. Relative expression levels of chemokine receptors in BMMC. S7. Dinitrophenyl (DNP) induced calcium release in BMMC. S8. Analysis of CCR2 and CCR5 expression in immune cells from ACKR2-/- mice. S9. Uptake and MHC-1 mediated presentation of peptide antigens by mast cells. S10. Mast cell infiltration into ACKR2-/- tumors is not dependent on T cells. Supplemental Table 1: List of primers used for mouse genotyping. Supplemental Table 2: List of mouse Real Time PCR primer sequences.

PDF file - 1052K, S1. CX3CL1 staining in mammary glands of WT and WT and CX3CL1+ mice. S2. Staining of CX3CR1 in tumors and mammary glands of Tg-neu mice. S3. Effects of Ad-CX3CL1or Ad-LacZ injection in tumor proliferation, apoptosisand angiogenesis. S4. CX3CL1 overexpression increases breast carcinogenesis in Tg-neu mice. S5. CX3CL1 or CX3CR1 mRNA expression in breast cancer cell lines with distinct metastatic potential. S6. CX3CL1 expression in a panel of breast cancer cell lines. S7. CX3CL1 induces EMT in breast cancer cells. S8. Lack of lymphoid metastases in Ad-CX3CL1-injected Tg-neu mice. S9. CX3CL1 induces transactivation of the EGF pathway. S10. ErbB ligand expression in T47D cells. S11. CX3CL1 deficiency does not affect the structure and composition of the mammary gland in Tg-neu mice. S12. Hemizygous CX3CL1 mRNA expression in T-neu-CX3CL1+/-. Supplemental Table 1. Media used to culture the cancer cell lines. Supplemental Table 2. Primers and annealing conditions used for PCR.

Abstract Background Vedolizumab (VDZ) is a frontline drug for Ulcerative colitis (UC) and Crohn’s disease (CD) that targets integrin α4β7, a gut-homing receptor. Despite significant use, the mechanism(s) of action (MOA) of VDZ remain unclear. Methods Peripheral blood mononuclear cells (PBMC) from UC patients (n=43) and paired intestinal biopsies in a subset (n=12) were examined at week 0 (pre-infusion) and 14, by multiparameter flow cytometry (FC). TNF inhibitor (TNFi)-treated UC patients served as controls. Drug MOA was further examined in detail in murine models using single-cell RNA-seq, FC, conventional and immunofluorescent microscopy (IF). Photoconvertible (Kikumi) mice were used to study cellular trafficking after anti-α4β7 (DATK32) antibody (mAb) administration. Two distinct UC cohorts (n=42 and n=21 respectively) were used to correlate GI immune alterations following VDZ with mucosal healing. Results A significant decrease of colonic and ileal naïve B and T cells was noted in VDZ- but not in TNFi-treated patients (Fig 1A), while total T cell-frequencies were unchanged. Circulating gut-homing plasmablasts (β7+) were significantly decreased post-VDZ. Peyer’s patches (PP) in anti-α4β7-treated mice showed a rapid loss of cellularity associated with decreased follicular naïve B cells (Fig 1B). Single-cell RNA-seq also demonstrated a significant loss of follicular B cells, including a unique population of epithelium-associated B cells following anti-α4β7 mAb. In Kikumi mice, anti-α4β7 mAb impaired non-photoconverted follicular B cells and T cells into photoconverted PPs (Fig 1C) demonstrating that loss of PP cellularity was due to impaired cellular ingress. In VDZ-treated (but not TNFi-treated) UC patients, lymphoid aggregate (LA) size was significantly reduced in treatment responders compared to non-responders (defined by absence of histological inflammation). A distinct validation cohort further confirmed that reduced lymphoid aggregate size was associated with response to VDZ-therapy (Fig 1D). Conclusion VDZ is associated with impaired ingress of naïve B and T cells, resulting in attrition of intestinal LA. Reduced LA size is associated with VDZ response. Immune inductive site targeting represents a novel MOA of VDZ in patients with UC.

B cells, which are critical for intestinal homeostasis, remain understudied in ulcerative colitis (UC). In this study, we recruited three cohorts of patients with UC (primary cohort, n = 145; validation cohort 1, n = 664; and validation cohort 2, n = 143) to comprehensively define the landscape of B cells during UC-associated intestinal inflammation. Using single-cell RNA sequencing, single-cell IgH gene sequencing and protein-level validation, we mapped the compositional, transcriptional and clonotypic landscape of mucosal and circulating B cells. We found major perturbations within the mucosal B cell compartment, including an expansion of naive B cells and IgG+ plasma cells with curtailed diversity and maturation. Furthermore, we isolated an auto-reactive plasma cell clone targeting integrin αvβ6 from inflamed UC intestines. We also identified a subset of intestinal CXCL13-expressing TFH-like T peripheral helper cells that were associated with the pathogenic B cell response. Finally, across all three cohorts, we confirmed that changes in intestinal humoral immunity are reflected in circulation by the expansion of gut-homing plasmablasts that correlates with disease activity and predicts disease complications. Our data demonstrate a highly dysregulated B cell response in UC and highlight a potential role of B cells in disease pathogenesis. Multi-modal profiling reveals major alterations in colonic B cells in patients with ulcerative colitis, including reduced clonal diversity of plasma cells, and suggests that circulating gut-homing plasmablasts could serve as a biomarker for disease activity.

Myeloid cells comprise the majority of immune cells in tumors, where their content and composition is determined by tumor type and driver mutation. While these cells are essential for shaping the tumor microenvironment, promoting tumor growth, and contributing to therapeutic resistance, targeting tumor-associated myeloid cells, including bone-marrow-derived monocytes and neutrophils, has not been successful in the clinics. Monocyte chemoattractant protein (MCP) family, comprising of Ccl2, Ccl7, Ccl8, Ccl12, are essential for monocytes trafficking to the tumor sites. To eliminate monocyte recruitment, we leveraged CRISPR/Cas-9 based gene editing tool to generate a mouse strain that is devoid of all MCP genes, which we termed quadruple MCP knockout (qMCP-/-). Using these mice in combination with genetically engineered mouse models (GEMM) of glioblastoma (GBM), we abolished tumor monocyte infiltration. Due to the functional redundancy of MCP family members, we show that targeting individual MCP genes leads to compensation by other MCPs. In contrast, when all MCPs are genetically deleted and monocyte recruitment is abolished, neutrophil infiltration ensues. Single-cell RNA sequencing revealed that intratumoral neutrophils promoted proneural-to-mesenchymal transition in GBM, and supported tumor aggression by facilitating hypoxia response via TNF production. Remarkably, pharmacologic or genetic interventions that suppress both monocytes and neutrophil infiltration improve the survival of GBM-bearing mice. Taken together, our findings establish that specific subsets of myeloid cells can influence the dynamism of tumor microenvironment, and they emphasize the importance of targeting both monocytes and neutrophils simultaneously for effective GBM immunotherapy.