Acute Stress Regulates Sex-Related Molecular Responses in the Human Jejunal Mucosa: Implications for Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) is a prevalent gastrointestinal disorder linked to intestinal barrier dysfunction and life stress. We have previously reported that female sex per se determines an increased susceptibility to intestinal barrier dysfunction after cold pain stress (CPS). We aimed to identify sex-related molecular differences in response to CPS in healthy subjects to understand the origin of sex bias predominance in IBS. In 13 healthy males and 21 females, two consecutive jejunal biopsies were obtained using Watson's capsule, at baseline, and ninety minutes after CPS. Total mucosal RNA and protein were isolated from jejunal biopsies. Expression of genes related to epithelial barrier (CLDN1, CLDN2, OCLN, ZO-1, and ZO-3), mast cell (MC) activation (TPSAB1, SERPINA1), and the glucocorticoid receptor (NR3C1) were analyzed using RT-qPCR. NR3C1, ZO-1 and OCLN protein expression were evaluated through immunohistochemistry and western blot, and mucosal inflammation through MC, lymphocyte, and eosinophil numbering. Autonomic, hormonal, and psychological responses to CPS were monitored. We found an increase in jejunal MCs, a reduced CLDN1 and OCLN expression, and an increased CLDN2 and SERPINA1 expression 90 min after CPS. We also found a significant decrease in ZO-1, OCLN, and NR3C1 gene expression, and a decrease in OCLN protein expression only in females, when compared to males. CPS induced a significant increase in blood pressure, plasma cortisol and ACTH, and subjective stress perception in all participants. Specific and independent sex-related molecular responses in epithelial barrier regulation are unraveled by acute stress in the jejunum of healthy subjects and may partially explain female predominance in IBS.

Acute psychological stress increases paracellular permeability and modulates immune activity in rectal mucosa of healthy volunteers.

BACKGROUND: Psychological stress and increased permeability are implicated as contributing factors in the initiation and worsening of gastrointestinal diseases. A link between stress and intestinal permeability has been shown in animal models as well as in human small intestine, but stress effects on the human colorectal mucosal barrier has not been reported. OBJECTIVE: To investigate the potential effects of acute psychological stress on colorectal mucosal barrier function and to explore stress-induced molecular events in the rectal mucosa under healthy conditions. METHODS: Endoscopic biopsies were taken from the rectosigmoid region of healthy volunteers, who had been subjected to dichotomous listening stress and after a control session, respectively. Paracellular and transcellular permeability were assessed in modified Ussing chambers. RNA expression (microarray technology confirmed by quantitative real-time polymerase chain reaction) and biological pathway analysis were used to investigate the local mucosal response to acute stress. RESULTS: Dichotomous listening stress induced a subjective and objective stress response, and significantly increased paracellular but not transcellular permeability. We also identified a stress-induced reduction in RNA expression of genes related to immune cell activation and maturation (CR2, CD20, TCLA1, BANK1, CD22, FDCSP), signaling molecules of homing of immune cells to the gut (chemokines: CCL21, CXCL13, and CCL19, and receptors: CCR7, CXCR5), and innate immunity (DUOX2). Eight of the 10 top down-regulated genes are directly involved in B cell activation, signaling and migration. The systemic stress response correlated positively with paracellular permeability and negatively with DUOX2 expression. CONCLUSION: Dichotomous listening stress increases paracellular permeability and modulates immune cell activity in the rectal mucosa. Further studies are warranted to identify the primary mechanisms of stress-mediated reduction of mucosal defensive activity and barrier dysfunction, and their potential implications for gastrointestinal disorders.

Mucosal Plasma Cell Activation and Proximity to Nerve Fibres Are Associated with Glycocalyx Reduction in Diarrhoea-Predominant Irritable Bowel Syndrome: Jejunal Barrier Alterations Underlying Clinical Manifestations.

Irritable bowel syndrome (IBS) is a disorder of brain-gut interaction characterised by abdominal pain and changes in bowel habits. In the diarrhoea subtype (IBS-D), altered epithelial barrier and mucosal immune activation are associated with clinical manifestations. We aimed to further evaluate plasma cells and epithelial integrity to gain understanding of IBS-D pathophysiology. One mucosal jejunal biopsy and one stool sample were obtained from healthy controls and IBS-D patients. Gastrointestinal symptoms, stress, and depression scores were recorded. In the jejunal mucosa, RNAseq and gene set enrichment analyses were performed. A morphometric analysis by electron microscopy quantified plasma cell activation and proximity to enteric nerves and glycocalyx thickness. Immunoglobulins concentration was assessed in the stool. IBS-D patients showed differential expression of humoral pathways compared to controls. Activation and proximity of plasma cells to nerves and IgG concentration were also higher in IBS-D. Glycocalyx thickness was lower in IBS-D compared to controls, and this reduction correlated with plasma cell activation, proximity to nerves, and clinical symptoms. These results support humoral activity and loss of epithelial integrity as important contributors to gut dysfunction and clinical manifestations in IBS-D. Additional studies are needed to identify the triggers of these alterations to better define IBS-D pathophysiology.

The Role of Purported Mucoprotectants in Dealing with Irritable Bowel Syndrome, Functional Diarrhea, and Other Chronic Diarrheal Disorders in Adults.

Chronic diarrhea is a frequent presenting symptom, both in primary care medicine and in specialized gastroenterology units. It is estimated that more than 5% of the global population suffers from chronic diarrhea. and that about 40% of these subjects are older than 60 years. The clinician is frequently faced with the need to decide which is the best therapeutic approach for these patients. While the origin of chronic diarrhea is diverse, impairment of intestinal barrier function, dysbiosis. and mucosal micro-inflammation are being increasingly recognized as underlying phenomena characterizing a variety of chronic diarrheal diseases. In addition to current pharmacological therapies, there is growing interest in alternative products such as mucoprotectants, which form a mucoadhesive film over the epithelium to reduce and protect against the development of altered intestinal permeability, dysbiosis, and mucosal micro-inflammation. This manuscript focuses on chronic diarrhea in adults, and we will review recent evidence on the ability of these natural compounds to improve symptoms associated with chronic diarrhea and to exert protective effects for the intestinal barrier.

Overexpression of corticotropin-releasing factor in intestinal mucosal eosinophils is associated with clinical severity in Diarrhea-Predominant Irritable Bowel Syndrome.

Corticotropin-releasing factor (CRF) has been identified in intestinal mucosal eosinophils and associated with psychological stress and gut dysfunction. Irritable bowel syndrome (IBS) is commonly characterized by altered intestinal motility, immune activation, and increased gut barrier permeability along with heightened susceptibility to psychosocial stress. Despite intensive research, the role of mucosal eosinophils in stress-associated gut dysfunction remains uncertain. In this study, we evaluated eosinophil activation profile and CRF content in the jejunal mucosa of diarrhea-predominant IBS (IBS-D) and healthy controls (HC) by gene/protein expression and transmission electron microscopy. We also explored the association between intestinal eosinophil CRF and chronic stress, and the potential mechanisms underlying the stress response by assessing eosinophil response to neuropeptides. We found that mucosal eosinophils displayed higher degranulation profile in IBS-D as compared to HC, with increased content of CRF in the cytoplasmic granules, which significantly correlated with IBS clinical severity, life stress background and depression. Eosinophils responded to substance P and carbachol by increasing secretory activity and CRF synthesis and release, without promoting pro-inflammatory activity, a profile similar to that found in mucosal eosinophils from IBS-D. Collectively, our results suggest that intestinal mucosal eosinophils are potential contributors to stress-mediated gut dysfunction through CRF production and release.

Increased Colonic Epithelial Permeability and Mucosal Eosinophilia in Ulcerative Colitis in Remission Compared With Irritable Bowel Syndrome and Health.

BACKGROUND: Barrier dysfunction is recognized as a pathogenic factor in ulcerative colitis (UC) and irritable bowel syndrome (IBS), but it is unclear to what extent the factors related to barrier dysfunction are disease-specific. The aim of this study was to compare these aspects in UC patients in remission, IBS patients, and healthy controls (HCs). METHODS: Colonic biopsies were collected from 13 patients with UC in remission, 15 patients with IBS-mixed, and 15 HCs. Ulcerative colitis patients had recently been treated for relapse, and biopsies were taken from earlier inflamed areas. Biopsies were mounted in Ussing chambers for measurements of intestinal paracellular permeability to 51chromium (Cr)-ethylenediaminetetraacetic acid (EDTA). In addition, biopsies were analyzed for mast cells and eosinophils by histological procedures, and plasma tumor necrosis factor (TNF)-α was assessed by ELISA. RESULTS: Ussing chamber experiments revealed an increased 51Cr-EDTA permeability in UC and IBS (P < 0.05). The 51Cr-EDTA permeability was higher in UC compared with IBS (P < 0.005). There were increased numbers of mucosal mast cells and eosinophils in UC and IBS and more eosinophils in UC compared with IBS (P < 0.05). Also, increased extracellular granule content was found in UC compared with HCs (P < 0.05). The 51Cr-EDTA permeability correlated significantly with eosinophils in all groups. Plasma TNF-α concentration was higher in UC compared with IBS and HCs (P < 0.0005). CONCLUSIONS: Results indicate a more permeable intestinal epithelium in inactive UC and IBS compared with HCs. Ulcerative colitis patients, even during remission, demonstrate a leakier barrier compared with IBS. Both eosinophil numbers and activation state might be involved in the increased barrier function seen in UC patients in remission.

Intestinal Mucosal Mast Cells: Key Modulators of Barrier Function and Homeostasis.

The gastrointestinal tract harbours the largest population of mast cells in the body; this highly specialised leukocyte cell type is able to adapt its phenotype and function to the microenvironment in which it resides. Mast cells react to external and internal stimuli thanks to the variety of receptors they express, and carry out effector and regulatory tasks by means of the mediators of different natures they produce. Mast cells are fundamental elements of the intestinal barrier as they regulate epithelial function and integrity, modulate both innate and adaptive mucosal immunity, and maintain neuro-immune interactions, which are key to functioning of the gut. Disruption of the intestinal barrier is associated with increased passage of luminal antigens into the mucosa, which further facilitates mucosal mast cell activation, inflammatory responses, and altered mast cell⁻enteric nerve interaction. Despite intensive research showing gut dysfunction to be associated with increased intestinal permeability and mucosal mast cell activation, the specific mechanisms linking mast cell activity with altered intestinal barrier in human disease remain unclear. This review describes the role played by mast cells in control of the intestinal mucosal barrier and their contribution to digestive diseases.

Toll-like receptors-mediated pathways activate inflammatory responses in the esophageal mucosa of adult eosinophilic esophagitis.

OBJECTIVES: Esophageal microbiota and regulation of adaptive immunity are increasingly being investigated in eosinophilic esophagitis (EoE). Toll-like receptors (TLRs) play a central role in the initiation and maintenance of innate immune activity. Our objective was to characterize the esophageal and duodenal innate immune response in EoE and its modulation by dietary therapy. METHODS: Esophageal and duodenal biopsy samples were collected from 10 adults with untreated EoE, before and after effective treatment with a six-food elimination diet (SFED), and 10 controls with normal esophagus. In all cases, bacterial load (by mRNA expression of 16S), TLRs, mucins, transcription factors, interleukins, components of the NKG2D system, and innate immunity effectors were assessed by qPCR. Protein expression of TLRs were also determined by immunofluorescence. RESULTS: Bacterial load and TLR1, TLR2, TLR4, and TLR9 were overexpressed on biopsies with active EoE compared with controls. Muc1 and Muc5B genes were downregulated while Muc4 was overexpressed. Upregulation of MyD88 and NFκB was found together with IL-1ß, IL-6, IL-8, and IL-10 mediators and PER-1, iNOS, and GRZA effectors. NG-K2D components (KLRK1, IL-15, MICB) were also upregulated. In all cases, changes in active EoE were normalized following SFED and mucosal healing. Duodenal samples also showed increased expressions of TLR-1, TLR-2, and TLR-4, but not 16S or any other mediators nor effectors of inflammation. CONCLUSIONS: Esophageal TLR-dependent signaling pathways in EoE support the potential implication of microbiota and the innate immune system in the pathogenesis of this disease.

Epithelial immunity: priming defensive responses in the intestinal mucosa.

As the largest interface between the outside and internal milieu, the intestinal epithelium constitutes the first structural component facing potential luminal threats to homeostasis. This single-cell layer is the epicenter of a tightly regulated communication network between external and internal factors that converge to prime defensive responses aimed at limiting antigen penetration and the maintenance of intestinal barrier function. The defensive role developed by intestinal epithelial cells (IEC) relies largely on the variety of receptors they express at both extracellular (apical and basolateral) and intracellular compartments, and the capacity of IEC to communicate with immune and nervous systems. IEC recognize pathogen-associated molecules by innate receptors that promote the production of mucus, antimicrobial substances, and immune mediators. Epithelial cells are key to oral tolerance maintenance and also participate in adaptive immunity through the expression of immunoglobulin (Ig) receptors and by promoting local Ig class switch recombination. In IEC, different types of antigens can be sensed by multiple immune receptors that share signaling pathways to assure effective responses. Regulated defensive activity maintains intestinal homeostasis, whereas a breakdown in the control of epithelial immunity can increase the intestinal passage of luminal content and microbial invasion, leading to inflammation and tissue damage. In this review, we provide an updated overview of the type of immune receptors present in the human intestinal epithelium and the responses generated to promote effective barrier function and maintain mucosal homeostasis.

Downregulation of mucosal mast cell activation and immune response in diarrhoea-irritable bowel syndrome by oral disodium cromoglycate: A pilot study.

BACKGROUND AND GOAL: Diarrhoea-predominant irritable bowel syndrome (IBS-D) exhibits intestinal innate immune and mucosal mast cell (MC) activation. MC stabilisers have been shown to improve IBS symptoms but the mechanism is unclear. Our primary aim was to investigate the effect of oral disodium cromoglycate (DSCG) on jejunal MC activation and specific innate immune signalling pathways in IBS-D, and secondarily, its potential clinical benefit. STUDY: Mucosal MC activation (by ultrastructural changes, tryptase release and gene expression) and innate immune signalling (by protein and gene expression) were quantified in jejunal biopsies from healthy (HS; n = 16) and IBS-D subjects after six months of either treatment with DSCG (600 mg/day, IBS-D-DSCG group; n = 18) or without treatment (IBS-D-NT group; n = 25). All IBS-D patients recorded abdominal pain and bowel habits at baseline and in the last 10 days prior to jejunal sampling. RESULTS: IBS-D-NT exhibited significant MC activation and over-expression of immune-related genes as compared to HS, whereas in IBS-D-DSCG MC activity and gene expression were similar to HS. Furthermore, DSCG significantly reduced abdominal pain and improved stool consistency. CONCLUSION: Oral DSCG modulates mucosal immune activity and improves gut symptoms in IBS-D patients. Future placebo-controlled clinical trials are needed for confirmation of clinical benefit of DSCG for IBS-D.

miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea.

OBJECTIVE: Micro-RNAs (miRNAs) play a crucial role in controlling intestinal epithelial barrier function partly by modulating the expression of tight junction (TJ) proteins. We have previously shown differential messenger RNA (mRNA) expression correlated with ultrastructural abnormalities of the epithelial barrier in patients with diarrhoea-predominant IBS (IBS-D). However, the participation of miRNAs in these differential mRNA-associated findings remains to be established. Our aims were (1) to identify miRNAs differentially expressed in the small bowel mucosa of patients with IBS-D and (2) to explore putative target genes specifically involved in epithelial barrier function that are controlled by specific dysregulated IBS-D miRNAs. DESIGN: Healthy controls and patients meeting Rome III IBS-D criteria were studied. Intestinal tissue samples were analysed to identify potential candidates by: (a) miRNA-mRNA profiling; (b) miRNA-mRNA pairing analysis to assess the co-expression profile of miRNA-mRNA pairs; (c) pathway analysis and upstream regulator identification; (d) miRNA and target mRNA validation. Candidate miRNA-mRNA pairs were functionally assessed in intestinal epithelial cells. RESULTS: IBS-D samples showed distinct miRNA and mRNA profiles compared with healthy controls. TJ signalling was associated with the IBS-D transcriptional profile. Further validation of selected genes showed consistent upregulation in 75% of genes involved in epithelial barrier function. Bioinformatic analysis of putative miRNA binding sites identified hsa-miR-125b-5p and hsa-miR-16 as regulating expression of the TJ genes CGN (cingulin) and CLDN2 (claudin-2), respectively. Consistently, protein expression of CGN and CLDN2 was upregulated in IBS-D, while the respective targeting miRNAs were downregulated. In addition, bowel dysfunction, perceived stress and depression and number of mast cells correlated with the expression of hsa-miR-125b-5p and hsa-miR-16 and their respective target proteins. CONCLUSIONS: Modulation of the intestinal epithelial barrier function in IBS-D involves both transcriptional and post-transcriptional mechanisms. These molecular mechanisms include miRNAs as master regulators in controlling the expression of TJ proteins and are associated with major clinical symptoms.

Mucosal pathobiology and molecular signature of epithelial barrier dysfunction in the small intestine in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is one of the most prevalent gastrointestinal disorders in developed countries. Its etiology remains unknown; however, a common finding, regardless of IBS subtype, is the presence of altered intestinal barrier. In fact, signaling and location of cell-to-cell adhesion proteins, in connection with increased immune activity, seem abnormal in the intestinal epithelium of IBS patients. Despite that most research is performed on distal segments of the intestine, altered permeability has been reported in both, the small and the large bowel of all IBS subtypes. The small intestine carries out digestion and nutrient absorption and is also the site where the majority of immune responses to luminal antigens takes place. In fact, the upper intestine is more exposed to environmental antigens than the colon and is also a site of symptom generation. Recent studies have revealed small intestinal structural alterations of the epithelial barrier and mucosal immune activation in association with intestinal dysfunction, suggesting the commitment of the intestine as a whole in the pathogenesis of IBS. This review summarizes the most recent findings on mucosal barrier alterations and its relationship to symptoms arising from the small intestine in IBS, including epithelial structural abnormalities, mucosal immune activation, and microbial dysbiosis, further supporting the hypothesis of an organic origin of IBS.

Increased humoral immunity in the jejunum of diarrhoea-predominant irritable bowel syndrome associated with clinical manifestations.

BACKGROUND AND AIMS: Altered intestinal barrier is associated with immune activation and clinical symptoms in diarrhoea-predominant IBS (IBS-D). Increased mucosal antigen load may induce specific responses; however, local antibody production and its contribution to IBS aetiopathogenesis remain undefined. This study evaluated the role of humoral activity in IBS-D. METHODS: A single mucosal jejunal biopsy, luminal content and blood were obtained from healthy volunteers (H; n=30) and IBS-D (n=49; Rome III criteria) participants. Intraepithelial lymphocytes, mast cells, B lymphocytes and plasma cells were studied by imaging techniques. Differential gene expression and pathway analysis were assessed by microarray and PCR techniques. Blood and luminal immunoglobulins (Igs) were quantified. Gastrointestinal symptoms, respiratory atopy and stress and depression were also recorded. RESULTS: Patients with IBS-D showed a higher number and activation of mucosal B lymphocytes and plasma cells (p<0.05). Mast cell density was increased in patients with IBS-D (non-atopic) and in close proximity to plasma cells (p<0.05). Microarray profiling identified differential humoral activity in IBS-D, involving proliferation and activation of B lymphocytes and Igs production (p<0.001). Mucosal humoral activity was higher in IBS-D, with upregulation of germline transcripts and Ig genes (1.3-fold-1.7-fold increase; p<0.05), and increased IgG(+) cells and luminal IgG compared with H (p<0.05), with no differences in blood. Biological markers of humoral activity correlated positively with bowel movements, stool form and depression. CONCLUSIONS: Enhanced small bowel humoral immunity is a distinctive feature of IBS-D. Mucosal Ig production contributes to local inflammation and clinical manifestations in IBS-D.

The jejunum of diarrhea-predominant irritable bowel syndrome shows molecular alterations in the tight junction signaling pathway that are associated with mucosal pathobiology and clinical manifestations.

OBJECTIVES: Diarrhea-predominant irritable bowel syndrome (IBS-D) patients show altered epithelial permeability and mucosal micro-inflammation in both proximal and distal regions of the intestine. The objective of this study was to determine the molecular events and mechanisms and the clinical role of upper small intestinal alterations. METHODS: Clinical assessment and a jejunal biopsy was obtained in IBS-D patients and healthy subjects. Routine histology and immunohistochemistry was performed in all participants to assess the number of mast cells (MCs) and intraepithelial lymphocytes. RNA in tissue samples was isolated to identify genes showing consistent differential expression by microarray analysis followed by pathway and network analysis in order to identify the biological functions of the differentially expressed genes in IBS-D. Gene and protein expression of tight junction (TJ) components was also assessed by quantitative real-time polymerase chain reaction and confocal microscopy to evaluate the pathways identified by gene expression analysis. RESULTS: The analysis reveals a strong association between the transcript signature of the jejunal mucosa of IBS-D and intestinal permeability, MC biology, and TJ signaling. The expression of zonula occludens 1 (ZO-1) was reduced in IBS-D at both gene and protein level, with protein redistribution from the TJ to the cytoplasm. Remarkably, our analysis disclosed significant correlation between ZO proteins, MC activation, and clinical symptoms. CONCLUSIONS: IBS-D manifestations are linked to molecular alterations involving MC-related dysregulation of TJ functioning in the jejunal mucosa.

Chronic psychosocial stress induces reversible mitochondrial damage and corticotropin-releasing factor receptor type-1 upregulation in the rat intestine and IBS-like gut dysfunction.

The association between psychological and environmental stress with functional gastrointestinal disorders, especially irritable bowel syndrome (IBS), is well established. However, the underlying pathogenic mechanisms remain unknown. We aimed to probe chronic psychosocial stress as a primary inducer of intestinal dysfunction and investigate corticotropin-releasing factor (CRF) signaling and mitochondrial damage as key contributors to the stress-mediated effects. Wistar-Kyoto rats were submitted to crowding stress (CS; 8 rats/cage) or sham-crowding stress (SC; 2 rats/cage) for up to 15 consecutive days. Hypothalamic-pituitary-adrenal (HPA) axis activity was evaluated. Intestinal tissues were obtained 1h, 1, 7, or 30 days after stress exposure, to assess neutrophil infiltration, epithelial ion transport, mitochondrial function, and CRF receptors expression. Colonic response to CRF (10 µg/kg i.p.) and hyperalgesia were evaluated after ending stress exposure. Chronic psychosocial stress activated HPA axis and induced reversible intestinal mucosal inflammation. Epithelial permeability and conductance were increased in CS rats, effect that lasted for up to 7 days after stress cessation. Visceral hypersensitivity persisted for up to 30 days post stress. Abnormal colonic response to exogenous CRF lasted for up to 7 days after stress. Mitochondrial activity was disturbed throughout the intestine, although mitochondrial response to CRF was preserved. Colonic expression of CRF receptor type-1 was increased in CS rats, and negatively correlated with body weight gain. In conclusion, chronic psychosocial stress triggers reversible inflammation, persistent epithelial dysfunction, and colonic hyperalgesia. These findings support crowding stress as a suitable animal model to unravel the complex pathophysiology underlying to common human intestinal stress-related disorders, such as IBS.

Developmental changes in intraepithelial T lymphocytes and NK cells in the small intestine of neonatal rats.

The main objective of this study was to characterize developmental changes in small intestinal intraepithelial lymphocyte (IEL) subpopulations during the suckling period, thus contributing to the understanding of the development of diffuse gut-associated lymphoid tissue (GALT) and to the identification of early mechanisms that protect the neonate from the first contact with diet and gut microbial antigens. The study was performed by double labeling and flow cytometry in IEL isolated from the proximal and distal small intestine of 1- to 21-d-old Lewis rats. During the suckling period, intraepithelial natural killer (NK) cells changed from a typical systemic phenotype, CD8+, to a specific intestinal phenotype, CD8-. Analysis of CD8+ IEL revealed a progressive increase in the relative number of CD8+ IEL co-expressing TCRalphabeta, cells associated with acquired immunity, whereas the percentage of CD8+ cells expressing the NK receptor, i.e. cells committed to innate immunity, decreased. At weaning, IEL maturity was still not achieved, as revealed by a phenotypic pattern that differed from that of adult rats. Thus, late after weaning, the regulatory CD8+CD4+ T IEL population appeared and the NK population declined. In summary, the intestinal intraepithelial compartment undergoes changes in its lymphocyte composition associated with the first ingestion of food. These changes are focused on a relatively high proportion of NK cells during the suckling period, and after weaning, an expansion of the regulatory CD8+CD4+ T cells.