Alterations in the epithelial stem cell compartment could contribute to permanent changes in the mucosa of patients with ulcerative colitis.

OBJECTIVE: UC is a chronic inflammatory disease of the colonic mucosa. Growing evidence supports a role for epithelial cell defects in driving pathology. Moreover, long-lasting changes in the epithelial barrier have been reported in quiescent UC. Our aim was to investigate whether epithelial cell defects could originate from changes in the epithelial compartment imprinted by the disease. DESIGN: Epithelial organoid cultures (EpOCs) were expanded ex vivo from the intestinal crypts of non-IBD controls and patients with UC. EpOCs were induced to differentiate (d-EpOCs), and the total RNA was extracted for microarray and quantitative real-time PCR (qPCR) analyses. Whole intestinal samples were used to determine mRNA expression by qPCR, or protein localisation by immunostaining. RESULTS: EpOCs from patients with UC maintained self-renewal potential and the capability to give rise to differentiated epithelial cell lineages comparable with control EpOCs. Nonetheless, a group of genes was differentially regulated in the EpOCs and d-EpOCs of patients with UC, including genes associated with antimicrobial defence (ie, LYZ, PLA2G2A), with secretory (ie, ZG16, CLCA1) and absorptive (ie, AQP8, MUC12) functions, and with a gastric phenotype (ie, ANXA10, CLDN18 and LYZ). A high rate of concordance was found in the expression profiles of the organoid cultures and whole colonic tissues from patients with UC. CONCLUSIONS: Permanent changes in the colonic epithelium of patients with UC could be promoted by alterations imprinted in the stem cell compartment. These changes may contribute to perpetuation of the disease.

Commensal-Specific CD4(+) Cells From Patients With Crohn's Disease Have a T-Helper 17 Inflammatory Profile.

BACKGROUND & AIMS: Crohn's disease (CD) has been associated with an altered immune response to commensal microbiota, mostly based on increased seroreactivity to microbial proteins. Although T cells are believed to contribute to the development of CD, little is known about the antigens involved. We investigated the antigen-specificity of T cells isolated from patients with CD. METHODS: We isolated peripheral blood mononuclear cells from 65 patients with CD and 45 healthy individuals (controls). We investigated T-cell reactivity to commensal microbial antigens using proliferation assays (based on thymidine incorporation and carboxyfluorescein succinimidyl ester dilution). Gene expression patterns were determined using microarray and real-time polymerase chain reaction analyses. Cytokines, chemokines, and antibodies were measured by enzyme-linked immunosorbent assay, flow cytometry, or multiplex cytokine assays. Intestinal crypts were obtained from surgical resection specimens of 7 individuals without inflammatory bowel disease. We examined the effects of commensal-specific CD4(+) T cells on primary intestinal epithelial cells from these samples. RESULTS: The bacterial proteins FlaX, A4-fla2, and YidX increased proliferation of CD4(+) T cells isolated from peripheral blood of patients with CD compared with controls. In blood samples from controls, CD4(+) T cells specific for FlaX, A4-fla2, or YidX had a T-helper (Th)1 phenotype; a larger proportion of CD4(+) T cells specific for these proteins in patients with CD had a Th17 phenotype or produced Th1 and Th17 cytokines. When supernatants collected from commensal-specific CD4(+) T cells from patients with CD were applied to healthy intestinal epithelial cells, the epithelial cells increased the expression of the chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL8 and the CC chemokine ligand 20 (CCL20). CONCLUSIONS: A larger proportion of commensal-specific CD4(+) T cells from patients with CD have a Th17 phenotype or produce Th1 and Th17 cytokines, compared with T cells from controls; this might contribute to intestinal inflammation in patients with CD. These cells might be targeted for treatment of CD. The transcriptional data of commensal-specific CD4(+) T cells from healthy individuals and CD patients have been deposited in the Gene Expression Omnibus at the National Center for Biotechnology Information (accession no: GSE70469).

Transcriptional analysis of the intestinal mucosa of patients with ulcerative colitis in remission reveals lasting epithelial cell alterations.

OBJECTIVE: Ulcerative colitis (UC) is a chronic condition characterised by the relapsing inflammation despite previous endoscopic and histological healing. Our objective was to identify the molecular signature associated with UC remission. DESIGN: We performed whole-genome transcriptional analysis of colonic biopsies from patients with histologically active and inactive UC, and non-inflammatory bowel disease (non-IBD) controls. Real-time reverse transcriptase-PCR and immunostaining were used for validating selected genes in independent cohorts of patients. RESULTS: Microarray analysis (n=43) demonstrates that UC patients in remission present an intestinal transcriptional signature that significantly differs from that of non-IBD controls and active patients. Fifty-four selected genes were validated in an independent cohort of patients (n=30). Twenty-nine of these genes were significantly regulated in UC-in-remission subjects compared with non-IBD controls, including a large number of epithelial cell-expressed genes such as REG4, S100P, SERPINB5, SLC16A1, DEFB1, AQP3 and AQP8, which modulate epithelial cell growth, sensitivity to apoptosis and immune function. Expression of inflammation-related genes such as REG1A and IL8 returned to control levels during remission. REG4, S100P, SERPINB5 and REG1A protein expression was confirmed by immunohistochemistry (n=23). CONCLUSIONS: Analysis of the gene signature associated with remission allowed us to unravel pathways permanently deregulated in UC despite histological recovery. Given the strong link between the regulation of some of these genes and the growth and dissemination of gastrointestinal cancers, we believe their aberrant expression in UC may provide a mechanism for epithelial hyper-proliferation and, in the context of malignant transformation, for tumour growth.

Interactions of Tissue-Resident T Cells.

Resident memory T cells (TRM) are non-circulating cells that play a critical role in protection from local infections and cancers. Flow cytometric and transcriptional analyses of these cells have defined their distinct phenotypes; imaging allows study of their morphology, localization, and interactions within tissues. Here, we describe commonly used methods to generate cutaneous CD8+ TRM and to prepare skin samples for analysis, including staining of cryostat sections, epidermal sheets, and tissue whole mounts.

Virus-Specific T Cells From Cryopreserved Blood During an Emergent Virus Outbreak for a Potential Off-the-Shelf Therapy.

During the first outbreak of an emergent virus, methods need to be developed to rapidly establish suitable therapies for patients with high risk of severe disease caused by the pathogen. Considering the importance of the T-cell response in controlling viral infections, adoptive cell therapy with virus-specific T cells has been used as a safe and effective antiviral prophylaxis and treatment for immunocompromised patients. The main objective of this study was to establish an effective and safe method to cryostore whole blood as starting material and to adapt a T-cell activation and expansion protocol to generate an off-the-shelf antiviral therapeutic option. Additionally, we studied how memory T-cell phenotype, clonality based on T-cell receptor, and antigen specificity could condition characteristics of the final expanded T-cell product. Twenty-nine healthy blood donors were selected from a database of convalescent plasma donors with a confirmed history of SARS-CoV-2 infection. Blood was processed using a fully automated, clinical-grade, and 2-step closed system. Eight cryopreserved bags were advanced to the second phase of the protocol to obtain purified mononucleated cells. We adapted the T-cell activation and expansion protocol, without specialized antigen-presenting cells or presenting molecular structures, in a G-Rex culture system with IL-2, IL-7, and IL-15 cytokine stimulation. The adapted protocol successfully activated and expanded virus-specific T cells to generate a T-cell therapeutic product. We observed no major impact of post-symptom onset time of donation on the initial memory T-cell phenotype or clonotypes resulting in minor differences in the final expanded T-cell product. We showed that antigen competition in the expansion of T-cell clones affected the T-cell clonality based on the T-cell receptor ß repertoire. We demonstrated that good manufacturing practice of blood preprocessing and cryopreserving is a successful procedure to obtain an initial cell source able to activate and expand without a specialized antigen-presenting agent. Our 2-step blood processing allowed recruitment of the cell donors independently of the expansion cell protocol timing, facilitating donor, staff, and facility needs. Moreover, the resulting virus-specific T cells could be also banked for further use, notably maintaining viability and antigen specificity after cryopreservation.

Discipline in Stages: Regulating CD8+ Resident Memory T Cells.

Resident memory CD8+ T (TRM) cells are a lymphocyte lineage distinct from circulating memory CD8+ T cells. TRM lodge within peripheral tissues and secondary lymphoid organs where they provide rapid, local protection from pathogens and control tumor growth. However, dysregulation of CD8+ TRM formation and/or activation may contribute to the pathogenesis of autoimmune diseases. Intrinsic mechanisms, including transcriptional networks and inhibitory checkpoint receptors control TRM differentiation and response. Additionally, extrinsic stimuli such as cytokines, cognate antigen, fatty acids, and damage signals regulate TRM formation, maintenance, and expansion. In this review, we will summarize knowledge of CD8+ TRM generation and highlight mechanisms that regulate the persistence and responses of heterogeneous TRM populations in different tissues and distinct microenvironments.

CD49a Regulates Cutaneous Resident Memory CD8+ T Cell Persistence and Response.

CD8+ tissue-resident memory T cells (TRM) persist at sites of previous infection, where they provide rapid local protection against pathogen challenge. CD8+ TRM expressing the α1 chain (CD49a) of integrin VLA-1 have been identified within sites of resolved skin infection and in vitiligo lesions. We demonstrate that CD49a is expressed early following T cell activation in vivo, and TGF-ß and IL-12 induce CD49a expression by CD8+ T cells in vitro. Despite this rapid expression, CD49a is not required for the generation of a primary CD8+ T cell response to cutaneous herpes simplex virus (HSV) infection, migration of CD8+ T cells across the epidermal basement membrane, or positioning of TRM within basal epidermis. Rather, CD49a supports CD8+ TRM persistence within skin, regulates epidermal CD8+ TRM dendritic extensions, and increases the frequency of IFN-γ+ CD8+ TRM following local antigen challenge. Our results suggest that CD49a promotes optimal cutaneous CD8+ TRM-mediated immunity.

Migratory DCs activate TGF-ß to precondition naïve CD8+ T cells for tissue-resident memory fate.

Epithelial resident memory T (eTRM) cells serve as sentinels in barrier tissues to guard against previously encountered pathogens. How eTRM cells are generated has important implications for efforts to elicit their formation through vaccination or prevent it in autoimmune disease. Here, we show that during immune homeostasis, the cytokine transforming growth factor ß (TGF-ß) epigenetically conditions resting naïve CD8+ T cells and prepares them for the formation of eTRM cells in a mouse model of skin vaccination. Naïve T cell conditioning occurs in lymph nodes (LNs), but not in the spleen, through major histocompatibility complex class I-dependent interactions with peripheral tissue-derived migratory dendritic cells (DCs) and depends on DC expression of TGF-ß-activating αV integrins. Thus, the preimmune T cell repertoire is actively conditioned for a specialized memory differentiation fate through signals restricted to LNs.