Flagellin-Specific CD4 Cytokine Production in Crohn Disease and Controls Is Limited to a Small Subset of Antigen-Induced CD40L+ T Cells.

Flagellin is an immunodominant Ag in Crohn disease, with many patients showing anti-flagellin Abs. To study the clonality of flagellin-reactive CD4 cells in Crohn patients, we used a common CD154-based enrichment method following short-term Ag exposure to identify Ag-reactive CD4 cells. CD154 expression and cytokine production following Ag exposure compared with negative control responses (no Ag exposure) revealed that only a small fraction of CD154-enriched cells could be defined by Ag-reactive cytokine responses. This was especially true for low-frequency flagellin-reactive CD4 cells compared with polyclonal stimulation or Candida albicans Ag exposure. Moreover, we found that culture conditions used for the assay contributed to background CD40L (CD154) expression in the CD154-enriched CD4 cells. Using a cut-off rule based on flow cytometry results of the negative control CD154-enriched CD4 cells, we could reliably find the fraction of Ag-reactive cells in the CD154-enriched population. Ag-reactive CD4 cytokine production was restricted to CD4 cells with an effector memory phenotype and the highest levels of induced CD154 expression. This has important implications for identifying Ag-specific T cells of interest for single cell cloning, phenotyping, and transcriptomics.

Human Microbiota Flagellins Drive Adaptive Immune Responses in Crohn's Disease.

BACKGROUND AND AIMS: Crohn's disease and ulcerative colitis are characterized by dysregulated adaptive immune responses to the microbiota in genetically susceptible individuals, but the specificity of these responses remains largely undefined. Therefore, we developed a microbiota antigen microarray to characterize microbial antibody reactivity, particularly to human-derived microbiota flagellins, in inflammatory bowel disease. METHODS: Sera from healthy volunteers (n = 87) at the University of Alabama at Birmingham and from patients recruited from the Kirklin Clinic of University of Alabama at Birmingham Hospital, including patients with Crohn's disease (n = 152) and ulcerative colitis (n = 170), were individually probed against microbiota bacterial flagellins of both mouse and human origin and analyzed for IgG and IgA antibody responses. Circulating flagellin-reactive T effector (CD4+CD154+) and T regulatory (CD4+CD137+) cells were isolated and evaluated in selected patients. Resulting adaptive immune responses were compared with corresponding clinical data to determine relevancy to disease behavior. RESULTS: We show that patients with IBD express selective patterns of antibody reactivity to microbiota flagellins. Patients with Crohn's disease, but not patients with ulcerative colitis, display augmented serum IgG to human ileal-localized Lachnospiraceae flagellins, with a subset of patients having high responses to more than 10 flagellins. Elevated responses to CBir1, a mouse Lachnospiraceae flagellin used clinically to diagnose CD, correlated with multi-Lachnospiraceae flagellin reactivity. In this subset of patients with CD, multi-flagellin reactivity was associated with elevated flagellin-specific CD154+CD45RA- T memory cells, a reduced ratio of flagellin-reactive CD4+ T regulatory to T effector cells, and a high frequency of disease complications. CONCLUSIONS: Patients with Crohn's disease display strong adaptive immune response to human-derived Lachnospiraceae flagellins, which may be targeted for prognosis and future personalized therapies.

Human seroreactivity to gut microbiota antigens.

BACKGROUND: Although immune responses directed against antigens from the intestinal microbiota are observed in certain diseases, the normal human adaptive immune response to intestinal microbiota is poorly defined. OBJECTIVE: Our goal was to assess the adaptive immune response to the intestinal microbiota present in 143 healthy adults and compare this response with the response observed in 52 children and their mothers at risk of having allergic disease. METHODS: Human serum was collected from adults and children followed from birth to 7 years of age, and the serum IgG response to a panel of intestinal microbiota antigens was assessed by using a novel protein microarray. RESULTS: Nearly every subject tested, regardless of health status, had serum IgG that recognized a common set of antigens. Seroreactivity to the panel of antigens was significantly lower in atopic adults. Healthy infants expressed the highest level of IgG seroreactivity to intestinal microbiota antigens. This adaptive response developed between 6 and 12 months of age and peaked around 2 years of age. Low IgG responses to certain clusters of microbiota antigens during infancy were associated with allergy development during childhood. CONCLUSIONS: There is an observed perturbation of the adaptive response to antigens from the microbiota in allergic subjects. These perturbations are observable even in childhood, suggesting that optimal stimulation of the adaptive immune system by the microbiota might be needed to prevent certain immune-mediated diseases.

Helicobacter pylori infection inhibits phagocyte clearance of apoptotic gastric epithelial cells.

Increased apoptotic death of gastric epithelial cells is a hallmark of Helicobacter pylori infection, and altered epithelial cell turnover is an important contributor to gastric carcinogenesis. To address the fate of apoptotic gastric epithelial cells and their role in H. pylori mucosal disease, we investigated phagocyte clearance of apoptotic gastric epithelial cells in H. pylori infection. Human gastric mononuclear phagocytes were analyzed for their ability to take up apoptotic epithelial cells (AECs) in vivo using immunofluorescence analysis. We then used primary human gastric epithelial cells induced to undergo apoptosis by exposure to live H. pylori to study apoptotic cell uptake by autologous monocyte-derived macrophages. We show that HLA-DR(+) mononuclear phagocytes in human gastric mucosa contain cytokeratin-positive and TUNEL-positive AEC material, indicating that gastric phagocytes are involved in AEC clearance. We further show that H. pylori both increased apoptosis in primary gastric epithelial cells and decreased phagocytosis of the AECs by autologous monocyte-derived macrophages. Reduced macrophage clearance of apoptotic cells was mediated in part by H. pylori-induced macrophage TNF-α, which was expressed at higher levels in H. pylori-infected, compared with uninfected, gastric mucosa. Importantly, we show that H. pylori-infected gastric mucosa contained significantly higher numbers of AECs and higher levels of nonphagocytosed TUNEL-positive apoptotic material, consistent with a defect in apoptotic cell clearance. Thus, as shown in other autoimmune and chronic inflammatory diseases, insufficient phagocyte clearance may contribute to the chronic and self-perpetuating inflammation in human H. pylori infection.

Crohn's patients and healthy infants share immunodominant B cell response to commensal flagellin peptide epitopes.

About half of patients with Crohn's disease (CD) develop selective serum IgG response to flagellin proteins of the Lachnospiraceae family. Here, we identified a dominant B cell peptide epitope in CD, locating in the highly conserved "hinge region" between the D0 and D1 domains at the amino-terminus of Lachnospiraceae flagellins. Serum IgG reactive to this epitope is present at an elevated level in adult CD patients and in pediatric CD patients at diagnosis. Most importantly, high levels of serum IgG to the hinge epitope were found in most infants from 3 different geographic regions (Uganda, Sweden, and the USA) at one year of age. This vigorous homeostatic response decrements with age as it is not present in healthy adults. These data identify a distinct subset of CD patients, united by a shared reactivity to this dominant flagellin epitope that may represent failure of a homeostatic response beginning in infancy.

Suppression of inflammation in ulcerative colitis by interferon-ß-1a is accompanied by inhibition of IL-13 production.

OBJECTIVE: Ulcerative colitis is associated with increased interleukin 13 (IL-13) production by natural killer T cells. Taking advantage of the inhibitory actions of interferon ß on IL-13 expression, this proof-of-concept study aimed to show that decreasing IL-13 production is associated with clinical improvement of ulcerative colitis symptoms. DESIGN: Open-label interventional drug trial. SETTING: Outpatient clinical research hospital. Patients Adult patients with active ulcerative colitis (Short Clinical Colitis Activity Index (SCCAI)≥ 5). Interventions Treatment with 30 µg IM interferon-ß-1a (Avonex) weekly for 12 weeks with 6 month follow-up. MAIN OUTCOME MEASURES: Clinical response was defined as ≥ 3 point drop in the SCCAI for at least two consecutive monitoring visits, and cytokine production was measured in cultured peripheral blood and lamina propria mononuclear cells (LPMC) before and after treatment. RESULTS: 11 of 16 patients were clinical responders, and 4 were in remission (SCCAI ≤ 2) at the end of treatment. Rectal bleeding subscores improved dramatically by week 4 (38% with frank bleeding vs 87% pretreatment). Increased IL-13 production by LPMC T cells fell significantly in clinical responders (690 ± 99 vs 297 ± 58 pg/ml p = 0.015) but was unchanged in non-responders (542 ± 83 vs 510 ± 39 pg/ml). In addition, non-responders had significantly higher production of IL-17 and IL-6 pre-treatment compared to responders. CONCLUSIONS: Interferon-ß-1a induces clinical response and remission in a large subset of patients with ulcerative colitis that is associated with significant inhibition of IL-13 production. In addition, increased IL-17 and IL-6 production is associated with no response to interferon-ß. These data provide a proof-of-concept that IL-13 is an effector cytokine in ulcerative colitis and should be a target for novel therapies.

Crohn's Disease Patients Uniquely Contain Inflammatory Responses to Flagellin in a CD4 Effector Memory Subset.

BACKGROUND: Specific microbial antigens stimulate production of antibodies indicative of the aberrant immune response in Crohn's disease (CD). We tested for T cell reactivity linkage to B cell responses and now report on the prevalence, functionality, and phenotypic differences of flagellin-specific T cells among CD patients, ulcerative colitis (UC) patients, and control subjects and association with clinical features and flagellin seropositivity within CD patients. METHODS: Sera from non-inflammatory bowel disease control subjects, CD patients, and UC patients were probed for antibody reactivity to gut bacterial recombinant flagellin antigens. Peripheral blood mononuclear cells were measured for flagellin antigen (CBir1, A4 Fla2, FlaX) or control (Candida albicans, and CytoStim) reactivity analyzed by flow cytometry for CD154 and cytokine expression on CD4+ T cells. Supernatants from post-flagellin-stimulated and unstimulated cells were used to measure effects on epithelial barrier function. RESULTS: CD patients had a significantly higher percentage of flagellin-specific CD154+ CD4+ cells that have an effector memory T helper 1 and T helper 17 phenotype compared with UC patients and healthy control subjects. There was a positive correlation between the frequency of flagellin-specific CD154+ CD4+ effector memory T cells and serum levels of anti-flagellin immunoglobulin G in the CD patients. In addition, A4 Fla2-reactive T cells from active CD patients produced cytokines that can decrease barrier function in a gut epithelium. CONCLUSIONS: These findings demonstrate a Crohn's-associated flagellin-reactive CD4 cell subset distinct from UC patients and control subjects. There is a link between these cells and flagellin seropositivity. This CD4 cell subset could reflect a particular endophenotype of CD, leading to novel insight into its pathology and treatment.

Crohn's disease patients display inflammatory cytokine responses to flagellin antigens in an expanded effector memory CD4 subset that is not seen in ulcerative colitis or non­inflammatory bowel disease control subjects. These cells correlate with levels of the specific cognate anti-flagellin antibodies.

Mucosa-Associated Microbiota in Barrett's Esophagus, Dysplasia, and Esophageal Adenocarcinoma Differ Similarly Compared With Healthy Controls.

INTRODUCTION: Alterations in the composition of the human gut microbiome and its metabolites have been linked to gut epithelial neoplasia. We hypothesized that differences in mucosa-adherent Barrett's microbiota could link to risk factors, providing risk of progression to neoplasia. METHODS: Paired biopsies from both diseased and nonaffected esophagus (as well as gastric cardia and gastric juice for comparison) from patients with intestinal metaplasia (n = 10), low grade dysplasia (n = 10), high grade dysplasia (n = 10), esophageal adenocarcinoma (n = 12), and controls (n = 10) were processed for mucosa-associated bacteria and analyzed by 16S ribosomal ribonucleic acid V4 gene DNA sequencing. Taxa composition was tested using a generalized linear model based on the negative binomial distribution and the log link functions of the R Bioconductor package edgeR. RESULTS: The microbe composition of paired samples (disease vs nondisease) comparing normal esophagus with intestinal metaplasia, low grade dysplasia, high grade dysplasia, and adenocarcinoma showed significant decreases in the phylum Planctomycetes and the archaean phylum Crenarchaeota (P < 0.05, false discovery rate corrected) in diseased tissue compared with healthy controls and intrasample controls (gastric juice and unaffected mucosa). Genera Siphonobacter, Balneola, Nitrosopumilus, and Planctomyces were significantly decreased (P < 0.05, false discovery rate corrected), representing <10% of the entire genus community. These changes were unaffected by age, tobacco use, or sex for Crenarcha. DISCUSSSION: There are similar significant changes in bacterial genera in Barrett's esophageal mucosa, dysplasia, and adenocarcinoma compared with controls and intrapatient unaffected esophagus. Further work will establish the biologic plausibility of these specific microbes' contributions to protection from or induction of esophageal epithelial dysplasia.

CD4+ T cell activation and concomitant mTOR metabolic inhibition can ablate microbiota-specific memory cells and prevent colitis.

Microbiota-reactive CD4+ T memory (TM) cells are generated during intestinal infections and inflammation, and can revert to pathogenic CD4+ T effector (TE) cells, resulting in chronicity of inflammatory bowel disease (IBD). Unlike TE cells, TM cells have a low rate of metabolism unless they are activated by reencountering cognate antigen. Here, we show that the combination of cell activation and metabolic checkpoint inhibition (CAMCI), by targeting key metabolic regulators mTORC and AMPK, resulted in cell death and anergy, but enhanced the induction of the regulatory subset. Parenteral application of this treatment with a synthetic peptide containing multiple flagellin T cell epitopes (MEP1) and metabolic inhibition successfully prevented the development of CD4+ T cell-driven colitis. Microbiota-specific CD4+ T cells, especially the pathogenic TE subsets, were decreased 10-fold in the intestinal lamina propria. Furthermore, using the CAMCI strategy, we were able to prevent antigen-specific TM cell formation upon initial antigen encounter, and ablate existing TM cells upon reactivation in mice, leading to an altered transcriptome in the remaining CD4+ T cells after ablation. Microbiota flagellin-specific CD4+ T cells from patients with Crohn's disease were ablated in a similar manner after CAMCI in vitro, with half of the antigen-specific T cells undergoing cell death. These results indicate that parenteral activation of microbiota-specific CD4+ T cells with concomitant metabolic inhibition is an effective way to ablate pathogenic CD4+ TM cells and to induce T regulatory (Treg) cells that provide antigen-specific and bystander suppression, supporting a potential immunotherapy to prevent or ameliorate IBD.

Multiple endoscopic biopsies in research subjects: safety results from a National Institutes of Health series.

BACKGROUND: Routine endoscopic mucosal biopsies are generally considered safe. However, the outcomes of performing large numbers of biopsies in subjects enrolled in research protocols have not been reported. OBJECTIVE: Our purpose was to assess the safety of taking numerous mucosal biopsy specimens during endoscopic procedures (eg, >20/endoscopic procedure) in research subjects. DESIGN: Single-center retrospective chart review. SETTING: Research hospital: National Institutes of Health (NIH) Clinical Center. PATIENTS: Volunteers who underwent research protocol endoscopies with large numbers of biopsies during 2001 to 2008 at the NIH. MAIN OUTCOME MEASUREMENTS: Charts were reviewed for the occurrence of procedure-related major/minor complications. RESULTS: A total of 253 research endoscopies were performed on 133 patients: 169 colonoscopies, 64 sigmoidoscopies, and 20 upper endoscopies. A total of 9,661 biopsy specimens were obtained for research and histopathologic examination (mean 38.2 +/- 15.6 per procedure). No major complications were identified. Minor complications occurred with 13 (5.1%) lower endoscopic procedures and included self-limited bleeding (4), pain (5), or both (4). There was no statistically significant association between the number of biopsies, type of procedure, location of research biopsies, operator, polypectomy, or the use of nonsteroidal anti-inflammatory drugs and the risk of complications. LIMITATIONS: Retrospective design, modest sample size. CONCLUSIONS: This is the first report on the safety of performing large numbers of endoscopic biopsies in research subjects. This practice is well tolerated and appears to have no more than minimal risk without appreciably increasing the risk of otherwise routine endoscopy.

Anti-interleukin-12 antibody for active Crohn's disease.

BACKGROUND: Crohn's disease is associated with excess cytokine activity mediated by type 1 helper T (Th1) cells. Interleukin-12 is a key cytokine that initiates Th1-mediated inflammatory responses. METHODS: This double-blind trial evaluated the safety and efficacy of a human monoclonal antibody against interleukin-12 (anti-interleukin-12) in 79 patients with active Crohn's disease. Patients were randomly assigned to receive seven weekly subcutaneous injections of 1 mg or 3 mg of anti-interleukin-12 per kilogram of body weight or placebo, with either a four-week interval between the first and second injection (Cohort 1) or no interruption between the two injections (Cohort 2). Safety was the primary end point, and the rates of clinical response (defined by a reduction in the score for the Crohn's Disease Activity Index [CDAI] of at least 100 points) and remission (defined by a CDAI score of 150 or less) were secondary end points. RESULTS: Seven weeks of uninterrupted treatment with 3 mg of anti-interleukin-12 per kilogram resulted in higher response rates than did placebo administration (75 percent vs. 25 percent, P=0.03). At 18 weeks of follow-up, the difference in response rates was no longer significant (69 percent vs. 25 percent, P=0.08). Differences in remission rates between the group given 3 mg of anti-interleukin-12 per kilogram and the placebo group in Cohort 2 were not significant at either the end of treatment or the end of follow-up (38 percent and 0 percent, respectively, at both times; P=0.07). There were no significant differences in response rates among the groups in Cohort 1. The rates of adverse events among patients receiving anti-interleukin-12 were similar to those among patients given placebo, except for a higher rate of local reactions at injection sites in the former group. Decreases in the secretion of interleukin-12, interferon-gamma, and tumor necrosis factor alpha by mononuclear cells of the colonic lamina propria accompanied clinical improvement in patients receiving anti-interleukin-12. CONCLUSIONS: Treatment with a monoclonal antibody against interleukin-12 may induce clinical responses and remissions in patients with active Crohn's disease. This treatment is associated with decreases in Th1-mediated inflammatory cytokines at the site of disease.

Both IL-12p70 and IL-23 are synthesized during active Crohn's disease and are down-regulated by treatment with anti-IL-12 p40 monoclonal antibody.

BACKGROUND: Interleukin (IL)-12p70 and IL-23 are key T helper-1 (TH1) cytokines that drive the inflammation seen in numerous models of intestinal inflammation. These molecules contain an identical p40 chain that is bound to a p35 chain in IL-12 and a p19 chain in IL-23, making both potentially susceptible to modulation by an anti-IL-12p40 monoclonal antibody (mAb). METHODS: In the present study, we sought to determine whether active inflammation in Crohn's disease (CD) is associated with the increased synthesis of both of these cytokines and whether patients treated with an anti-IL-12p40 mAb down-regulate IL-23 as well as IL-12p70 as previous reported. RESULTS: To this end we initially determined that IL-12p70 secretion by control and CD antigen-presenting cells (macrophages) in lamina propria mononuclear populations is optimized by stimulation with CD40L and interferon-gamma. In subsequent studies using these stimulation conditions we found that patients with CD manifested both increased IL-12p70 and IL-23 secretion before anti-IL-12p40 mAb treatment and normal levels of secretion of these cytokines following cessation of treatment. Antigen-presenting cells in lamina propria mononuclear cells from ulcerative colitis patients, in contrast, produced only baseline levels of IL-23. Finally, we found that IL-23-induced T cell production of IL-17 and IL-6 are also greatly reduced after antibody treatment. The latter data are parallel to those from previous studies showing that anti-IL-12p40 down-regulates IFN-gamma and tumor necrosis factor-alpha secretion. CONCLUSIONS: We conclude that CD but not ulcerative colitis is associated with high levels of both IL-12p70 and IL-23 secretion as well as the secretion of downstream effector cytokines, and that this cytokine production is down-regulated following administration of IL-12p40 mAb.

Peptide YY as a growth factor for intestinal epithelium.

Peptide YY is an abundant distal gut hormone that may play a significant role in intestinal epithelial proliferation. Gut epithelial cells express specific receptors for PYY, PYY induces proliferation in intestinal cells in vivo and in vitro, and the Y1 receptor subtype couples to mitogenic signaling pathways. In addition to proposed physiologic effects on gut mucosal maintenance, PYY proliferative effects may be hypothesized to contribute to pathophysiologic consequences of stimulated growth.

Body Mass Index Is Associated With Mucosal Disease in Crohn's: Results of a Case-Control Study.

BACKGROUND: Recent studies have suggested that increased body mass index (BMI) may have an adverse effect on treatment outcomes and natural history in Crohn's disease (CD). We aimed to test the hypothesis that CD patients with higher BMI would be more likely than those with lower BMI to have persistent active mucosal disease. METHODS: We designed a case-control study. Sample population comprised CD patients with active disease at the beginning of observation. At the end of observation, cases had persistent active mucosal disease and controls had entered remission. With multivariable logistic regression models, we evaluated the effect of baseline BMI as a continuous variable and a categorical variable on persistent active mucosal disease. RESULTS: We analyzed data from 104 patients (36 cases and 68 controls). In a model containing BMI as a continuous variable, higher BMI was significantly associated with persistent active mucosal disease (odds ratio (OR) = 1.09 per unit increase; 95% confidence interval (CI), 1.02 - 1.17; P = 0.012). In a model containing BMI as a categorical variable, obese patients were 2.7 times more likely to have persistent active mucosal disease compared to non-obese patients (OR = 2.72; 95% CI, 1.00 - 7.35; P = 0.049). CONCLUSION: Excessive weight measured both quantitatively as BMI and categorically as obesity in CD patients is associated with persistent active mucosal disease.

Extracorporeal photopheresis (ECP) in patients with steroid-dependent Crohn's disease: an open-label, multicenter, prospective trial.

BACKGROUND: Extracorporeal photopheresis (ECP) involves ex vivo leukocyte treatment with methoxsalen and UVA light to generate a tolerogenic response. A previous trial demonstrated that ECP permits corticosteroid withdrawal in steroid-dependent Crohn's disease (CD) patients who were in clinical remission. We studied the effect of ECP on steroid withdrawal in steroid-dependent CD. METHODS: Patients with CD for ≥ 6 months, in remission at baseline while on steroids, but who had failed at ≥ 1 steroid withdrawal were included. Patients received two ECP treatments every 2 weeks for the 24-week steroid tapering period and underwent steroid-tapering. Patients completing steroid tapering could receive maintenance ECP (two treatments/week) every month for 24 weeks. RESULTS: Thirty-one patients (Crohn's Disease Activity Index [CDAI] score 91; Inflammatory Bowel Disease Questionnaire [IBDQ] 172.5) were enrolled (baseline corticosteroid dose, 20 mg/day); 65% were refractory to/intolerant of anti-tumor necrosis factor (TNF) agents or immunosuppressants. After 24 weeks of ECP, 7 of 31 (22.6%) patients discontinued steroids while maintaining a CDAI of <150. At week 24, the steroid dose for the remaining patients on corticosteroids was 10 mg (P < 0.003 vs. baseline) with a CDAI of 110 and an IBDQ of 179. Following maintenance treatment, three patients remained in steroid-free remission. The 10 patients in the study and receiving ECP at week 48 had a steroid dose of 3.5 mg with a CDAI of 40 and an IBDQ of 188. CONCLUSIONS: ECP permitted discontinuation or reduction of steroids in a population of refractory steroid-dependent CD patients. ECP may be useful in permitting steroid withdrawal in selected steroid-dependent CD patients. Ideally, these results need to be confirmed in a "sham-controlled clinical trial.

Remestemcel-L: human mesenchymal stem cells as an emerging therapy for Crohn's disease.

INTRODUCTION: Human mesenchymal stem cells (Prochymal brand of remestemcel-L) have been developed for experimental use in Crohn's disease and other conditions. Mesenchymal stems cells (MSCs) have been shown to inhibit inflammatory responses of innate and adaptive immune cells as well as have reparative effects on inflamed tissues. Promising preliminary therapeutic results of MSCs on gastrointestinal graft-versus-host disease have lead to Phase III trials for active Crohn's disease. AREAS COVERED: This review examines the discovery and characterization of mesenchymal stem cells, their immune effects, their use in animal models of disease, the production and administration of remestemcel-L and the published results of trials of remestemcel-L and alternative MSCs in Crohn's disease. EXPERT OPINION: The Prochymal brand of remestemcel-L represents the successful pharmaceutical development of mesenchymal stem cells for potential therapy in human disease. While preliminary results show promise of this therapy in terms of efficacy and safety, robust trials confirming efficacy results, explanation of the mechanism of response and estimates of effect compared to other biologics and immunosuppressants will be needed before this is an approved and widely accepted therapy.

The effect of intermittent IL-2 therapy on CD4 T cells in the gut in HIV-1-infected patients.

We sought to determine the effects of interleukin-2 administered in combination with antiretroviral therapy (ART) on CD4+ T cells in the gut. Lymphocytes from whole blood, colon, and terminal ileum of HIV-infected adults treated with interleukin-2 and ART or ART alone were examined. There were no differences between groups in the proportion of CD4+ T cells or in expression of CD25 or Ki67 by CD4+ T cells in the gut. Although IL-2 administration leads to expansion of peripheral blood CD4+ T cells, there is no alteration in the proportion or activation of CD4+ T cells in the gut mucosa.