T-cell activation in the intestinal mucosa

Summary:  The vast majority of peripheral T cells exist as resting lymphocytes until a signal for activation has been received. In response to antigen, this activation involves ligation of the T-cell receptor (TCR) and signal transmission through the CD3 complex, which then initiates a cascade of intracellular events that lead to the expression of genes used in T-cell activation. T-cell activation also requires soluble mediators in the form of cytokines and chemokines that regulate the process in both positive and negative ways, and costimulatory signals received in conjunction with TCR/CD3 signaling are important in the activation of T cells. Unlike T cells in other peripheral immune compartments, small and large intestinal intraepithelial lymphocytes (IELs) bear some but not all properties of activated T cells, suggesting that they constitute a large population of 'partially activated' effector cells. Thus, regulation of the IEL activation process must be held in tight check, yet it must be ready to respond to foreign antigen rapidly and effectively. We discuss how costimulatory molecules may hold the key to controlling IEL activation through a multiphase process beginning with cells that have already entered into the early stage of activation.     

T-cell receptor-gamma delta association with lymphocyte populations in sheep intestinal mucosa.

T cells expressing T-cell receptor (TcR)-gamma delta and CD8 represent a significant population in mouse and chicken intra-epithelial lymphocytes (IEL) but represent a minor population in human IEL. We examined the TcR-gamma delta usage and co-expression of CD5, CD4, CD8 and major histocompatibility complex (MHC) class II on isolated sheep IEL and lamina propria lymphocytes (LPL), and compared them with the TcR-gamma delta + cells in peripheral blood, intestinal lymph and jejunal Peyer's patches (PP). There were a number of notable differences. TcR-gamma delta + cells comprised 18% of IEL and 10% of LPL. Among the population of TcR-gamma delta + IEL, 24% were CD8+ and 54% were CD5+, which contrasts with the TcR-gamma delta + cells in blood and intestinal lymph that were universally CD5+ CD4- CD8-. A notable feature of the IEL was the presence of distinct CD8+ and TcR-gamma delta + populations that lacked CD5. Also a high percentage of IEL and LPL were CD2+ and MHC class II+. Analysis of the expression of MHC class II on T-cell subsets, as an indicator of activation, showed that 60-95% of the various IEL and LPL subsets were MHC class II+ compared with only 5-40% in jejunal PP, lymph nodes, spleen and blood. Therefore, it is possible that the circulating TcR-gamma delta + and CD8+ cells that localize in the gut epithelium might become activated and stop the expression of CD5 under the influence of the local microenvironment. These cells appear not to emigrate while still expressing the TcR-gamma delta + (CD8+) CD5- MHC class II+ phenotype. Our data, together with those from other studies, show that there is much heterogeneity in the use of TcR-gamma delta and accessory T-cell molecules by IEL.     

T-cell development in human thymoma.

Human thymoma is derived from thymic epithelial cells and often associated with a large number of cortical thymocytes. Since thymic epithelial cells play key roles in T-cell development in the normal thymus, we hypothesized that the neoplastic epithelial cells of thymoma may support T-cell differentiation. We attempted to reconstitute the T-cell development in vitro by using neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on a proportion of CD4-CD8- cells in thymoma. These CD34+CD4-CD8- cells also expressed both IL-7R alpha-chain and common gamma-chain. Purified CD4-CD8- cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4+CD8+ cells via CD4 single positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. The CD34+CD4-CD8- cells purified from a normal thymus also differentiated to CD4+CD8+ cells in an allogeneic co-culture with the neoplastic epithelial cells of thymoma. In addition, a pleural dissemination from thymoma contained a large amount of cortical thymocytes. These results suggest that the neoplastic epithelial cells retain the function of thymic epithelium and can support T-cell development in thymomas.     

Alterations in RANTES gene expression and T-cell prevalence in intestinal mucosa during pathogenic or nonpathogenic simian immunodeficiency virus infection.

RANTES, a beta-chemokine, can suppress human immunodeficiency virus (HIV) as well as simian immunodeficiency virus (SIV) infections in T-lymphocyte cultures in vitro. However, the association of RANTES levels in peripheral blood with viral loads and disease outcome in HIV infection has been inconclusive. SIV-infected rhesus macaques were evaluated to determine whether RANTES gene expression correlated with suppression of viral infection in intestinal lymphoid tissues. Intestinal tissues were obtained from rhesus macaques infected with either pathogenic or nonpathogenic SIVmac variants at various stages of infection (primary acute, asymptomatic, and terminal). We examined the level of SIV infection (in situ hybridization), RANTES expression (quantitative competitive RT-PCR), and T-cell counts (immunohistochemistry). The most pronounced increase in RANTES gene expression in intestinal tissues was observed in primary SIV infection, which correlated with the pathogenicity of the infecting virus and not the tissue viral loads. Our results demonstrated that in contrast to the occurrence of viral suppression by RANTES in vitro, there was no direct correlation between high RANTES gene expression and suppression of viral loads in intestinal lymphoid tissues. Thus RANTES expression in the gut lymphoid tissue may not be a correlate for viral suppression. However, RANTES gene expression in primary SIV infection may be part of early host immune response to viral infection.     

The lack of direct effects of a monoclonal antibody against murine T-cell suppressor factor on murine embryo development in vitro.

In previous studies, we reported that the injection of monoclonal antibody 14-30, specific for a T-cell suppressor factor (TSF), into mice during early stages of pregnancy could decrease the percentage of females that maintained pregnancy. In addition, further work has demonstrated the presence of an immunoreactive protein in fetal and maternal tissues with physiochemical properties similar to TSF. However, one alternate explanation for the antipregnancy effects of the injections of monoclonal antibody, not related to a specific role for TSF in early pregnancy, is the possibility of direct effects upon the embryo or embryonic antigens that prevent continued embryonic development. In the present studies, early preimplantation embryos were incubated with the monoclonal antibody 14-30 specific for TSF and the subsequent development of the embryos examined. The results of these studies demonstrate that monoclonal antibody, which has been shown to bind T-cell suppressor factor and has antipregnancy effects when injected in vivo, does not interfere with development of preimplantation or implantation stage mouse embryos in vitro.     

人小肠黏膜防御素5 mRNA的表达特点及意义

目的 检测人小肠黏膜防御素5基因(Alpha defensin 5，DEFA5)的表达，为从分子水平上探讨小肠黏膜抗菌防御机制提供实验依据。方法 从2例新鲜尸体和2例右半结肠癌患者的回肠部位取小肠黏膜提取总RNA，通过RT-PCR扩增出DEFA5的两个外显子序列，比较其个体间的差异；沿小肠轴间隔30cm取新鲜尸体小肠黏膜提取总RNA，仍通过RT-PCR扩增编码HD-5的阅读框序列，比较HD-5沿肠轴的表达趋势。结果 DEFA5在个体间的表达是一致的，通过RT-PCR获得的外显子序列大小为451bp，且碱基序列与GenBank中的DEFA5标准序列相一致；通过PCR获得的编码HD-5的阅读框序列为303bp，其中，酶切位点12bp，保护性碱基6bp，终止子3bp，编码HD-5的序列282bp(94AA)；沿着空肠到回肠的肠轴方向，DEFA5在空回肠黏膜均有表达，至回肠末端最强，并有逐渐增强的趋势。结论 DEFA5基因的表达在个体问是保守的，而且沿着空肠到回肠的肠轴方向在mRNA水平的表达有逐渐增加的趋势，提示HD-5作为天然免疫物质在小肠黏膜屏障功能的维护中起着十分重要的作用。此外，本实验获得的HD-5阅读框序列为进一步重组表达HD-5奠定了基础。     

T-cell receptor expression in the T-cell malignancies.

Twenty-eight cases with T-cell neoplasms (10 with T-cell acute lymphoblastic leukemia [T-ALL], 10 with T-lymphoblastic lymphoma, and 8 with peripheral T-cell lymphomas) and 2 cases with reactive lymph nodes were immunohistochemically stained with monoclonal antibodies beta F1, delta TCS1, and WT31; beta F1 antibody recognizes the beta-subunit of T-cell receptor (TCR), whereas delta TCS1 and WT31 recognize the delta- and alpha beta-subunits of TCR, respectively. Five cases with T-ALL, four with T-lymphoblastic lymphoma (T-LL), and seven with peripheral T-cell lymphomas were positive for beta F1. None showed positive reactivity for delta TCS1. One case with T-LL and four cases with peripheral T-cell lymphomas were positive for WT31. Of the nine cases positive for beta F1 among T-ALLs and T-LLs, six were also positive for CD1 (OKT6), whereas six of seven positive cases for CD1 were positive for beta F1. The authors therefore suggest that TCR beta is expressed in the immature T-cells just earlier than or around the same stage of differentiation as those expressing CD1. The authors' immuno-electron microscopy study revealed that positive reactivity for beta F1 was localized predominantly in the cytoplasm of the neoplastic cells in the cases with T-ALL, T-LL and peripheral T-cell lymphomas, and in the cytoplasm of the reactive T-cells. However, it was not localized on the surface membrane. In contrast, positive reactivity for WT31 was localized on the surface membrane of the neoplastic and reactive T-cells. Only half of the cases of peripheral T-cell lymphomas showed positive reactivity for WT31. The authors consider that it may not be a very useful antibody for the detection of TCR alpha beta on the T-cell neoplasms using frozen tissue sections.     

Expression of T-cell receptors TcR1 (gamma/delta) and TcR2 (alpha/beta) in the human intestinal mucosa.

Cryostat sections of normal human adult gastrointestinal mucosae were studied by double-label immunofluorescence with antibodies to CD3, CD4, CD8, CD5 and CD6, in parallel with antibodies beta F1 and TCR delta 1 against beta-chains and delta-chains of the T-cell receptor (TcR) types TcR2 (alpha/beta) and TcR1 (gamma/delta), respectively. Virtually no TcR1+ were found within the lamina propria. In the epithelial compartment, TcR1+ cells were infrequent: in the small bowel, congruent to 2% of T cells were TcR1+. In the colonic epithelium, the percentage of T cells expressing gamma/delta-chains was higher, with a mean value approximating 15-20%, although this apparently large percentage increase compared with small bowel reflects in part a much lower density of colonic IEL, as absolute numbers of TCR delta 1+ cells were comparable. Of the TcR1+ population, about half were CD4- CD8-, 'double negatives' and the remainder were CD8+. TcR1+ cells were also CD5- CD6-, irrespective of expression of CD8. No CD4+ cells expressing TcR1 were observed: essentially all CD4+ cells were beta F1+, with some variability of labelling intensity. Approximately 30-50% of the CD8+ subset expressed the beta F1 antigen strongly. However, in the remaining TcR1- CD8+ cells, which were all of the CD5- CD6- phenotype, expression of the beta F1 antigen was only detectable when streptavidin and biotin conjugates were used for amplification of labelling. Thus, the CD8+ CD5- subset, a prominent population of the epithelial compartment of the small bowel, was either TcR2dull in the majority or TcR1+ in a minority. Our data imply that gamma/delta TcR1 cells may be actively excluded from intestinal lamina propria, and that any preferential localization that does occur is limited and is rather a feature of the colonic mucosa, rather than the small bowel.     

Quantitative distribution of Ig-containing cells in the normal human intestinal mucosa

The Ig-containing cell population of human intestinal mucosa (jejunum and ileum) has been quantitatively studied in 15 normal healthy adult men, by means of the peroxidase-antiperoxidase (PAP) method. The resolution obtained using the PAP method improves that obtained by fluorescence microscopy methods. The results obtained revealed that the number of Ig-containing cells progressively decreases from the basal portion of mucosa to the tip of the microvilli. Most Ig-containing cells were plasma cells; however, stained precursor cells (immunoblasts) were also observed in the human intestinal mucosa. The epithelial lining of the intestinal mucosa was also stained by anti IgA, anti IgG, anti IgM and anti IgE antibodies. Plasma cells were not seen to cross the epithelium. The absolute values, expressed as the number of Ig-containing cells per "mucosal tissue unit" (a 6-micron-thick and 500 micron wide block of tissue, including the mucosa at full height from the muscularis mucosa), and the relative percentages of the different Ig-containing cell population were the following: IgA: 86.71 +/- 15.58 cells (48.52%); IgG: 52.30 +/- 19.01 cells (29.6%); IgM: 21.44 +/- 8.23 cells (12.0%); IgE: 14.70 +/- 6.60 cells (8.22%), and IgD: 3.54 +/- 1.05 cells (1.9%). The number of lambda chains (76.29 +/- 24.38; 56.07%) were slightly more abundant than that of chi chains (59.77 +/- 11.93; 43.92%). The differences between the mean values of different Ig-classes were significant, and the differences between lambda and chi chains were also significant.     

T-cell receptor expression in intestinal intra-epithelial lymphocyte subpopulations of normal and athymic mice.

Intra-epithelial lymphocytes (IEL) in murine small intestine were analysed for the presence of cell-surface antigens and T-cell receptor allotype in normal and athymic BALB/c mice by immunoperoxidase histochemistry on frozen sections and immunofluorescence on isolated IEL. In frozen sections, IEL of normal mice were 97.7% CD45+, 93.5% CD3+, 46.2% Thy-1+, 91.1% CD8+, 10.7% CD4+ and 21.1% KJ16+ (V beta 8.1 and 8.2). FACS analysis of isolated IEL confirmed the level of KJ16 expression and also demonstrated that 25% of IEL were F23.1+ (V beta 8.1-8.3). Immunofluorescent double-staining revealed a skewed distribution of T-cell receptor (TcR) expression on Thy-1+ and Thy-1- IEL. KJ16 and F23.1 were expressed on 25.9% and 32.7% of Thy-1+ IEL, respectively; however, the frequency of V beta 8 expression was diminished on Thy-1- IEL (4.1% KJ16+ and 12.1% F23.1+). IEL are present in athymic mice, but at reduced levels. In frozen sections these cells were 91.9% CD45+, 69.5% CD3+, less than 1% Thy-1+, 83.6% CD8+, less than 1% CD4+ and less than 1% KJ16+. Thus it appears that in normal mice there may be two distinct lineages of IEL, a thymus-dependent Thy-1+ population which utilizes the alpha beta T-cell receptor and a thymus-independent Thy-1- population (represented in athymic mice), which may possibly utilize the alternative gamma delta TcR.     

Variation of human intestinal gamma-glutamyl transpeptidase in ontogenetic development.

Activity of gamma-glutamyl transpeptidase in human intestines was measured against alpha-naphthylamide and 12 gamma-glutamyl amino acids and peptides as substrate. Distinctly altered activity was found to accompany ontogenetic development. The ratio of the transpeptidase activity tested against monoglutamyl substrates in the intestines of 7-month fetuses, newborns and adults was 15:1:4, whereas the ratio of gamma-glutamyl cyclotransferase activities in the same age groups was 1-0:1-2:1-6. Distinct differences were found in resistance to heating, sensitivity to L-serine plus borate, and other effectors, and electrophoretic mobility, between fetal gamma-glutamyl transpeptidase and the enzyme from adults, which supports the hypothesis of existence of two forms of the enzyme in the human intestines. The results suggest involvement of gamma-glutamyl transpeptidase in the pathomechanism of celiakia in children.     

The stem-cell zone of the small intestinal epithelium. I. Evidence from paneth cells in the adult mouse

Stem cells in the small intestinal epithelium are known to differentiate into columnar, mucous, enteroendocrine, and Paneth cells. However, the site of initiation of stem-cell differentiation has been unknown. To approach this problem we determined the site of stem-cell differentiation along the Paneth cell line, using light microscopic morphometry and radioautography. The smallest Paneth cells containing the smallest granules were in positions 6 and 7, while the largest ones containing the largest granules were in positions 1 and 2 at the base of the crypt. Paneth cell death was less prevalent above position 3 than it was in position 1. Since cell size, granule size, and cell death are indicators of Paneth cell age, it was deduced that there is a gradient of Paneth cell age in the crypt base, with the oldest Paneth cells at the bottom, and the youngest at the top. After single injection or continuous infusion of ³H-thymidine, the first labeled Paneth cells to appear were the highest Paneth cells in their crypt column. Later, labeled Paneth cells became more prevalent in lower positions, and, eventually, appeared in position 1. The size of granules in labeled Paneth cells increased with time. It was concluded that Paneth cells originate in position 5 or above and then migrate downward. These results are consistent with a stem-cell zone hypothesis, which proposes that stem cells in positions 1–4 receive no inducement to differentiate. Only those stem cells that migrate up out of the stem-cell zone into position 5 will be induced and then begin to differentiate.     

Lymphocyte subpopulations in the human small intestine. The findings in normal mucosa and in the mucosa of patients with adult coeliac disease

Lymphocyte subpopulations in human small intestinal mucosa have been studied using an immunofluorescence technique on tissue sections. In the normal intestine, the majority of intraepithelial lymphocytes (IEL) were of suppressor-cytotoxic phenotype (HuTLA+ UCHTI+ OKT8+ OKT4-; 84%). Only one-third of these OKT8+IEL reacted with anti-Leu-1, and antibody directed towards a 67,000 dalton antigen found on peripheral blood T cells. IEL failed to express the activation antigen, Tac, and also lacked detectable C3b receptor (C3RTO5-). The remaining T IEL, as well as the predominant lamina propria T lymphocytes (LPL), were OKT4+ OKT8-, helper type T cells. Most of the lamina propria OKT8+ cells were also Leu-1-. In patients with adult coeliac disease, the proportions of OKT8+ and OKT4+ lymphocytes in the epithelium were not altered. However, the proportion of OKT8+ Leu-1+TIEL was significantly increased (56 vs 32%; P less than 0.02). IEL were also HLA-DR-, Tac- and C3RTO5-. The proportion of OKT8+ cells in the lamina propria was slightly, but significantly, increased (40 vs 32%; P less than 0.005). Mucosal findings in treated patients did not differ from normal. Lymphocytes with the phenotype of natural killer cells (HNK-1) were rarely found in normal or diseased mucosa. No alterations in the proportions of circulating T lymphocytes or their subsets were found in patients with coeliac disease. These findings illustrate the heterogeneity of lymphocyte subpopulations in normal and in diseased small intestinal mucosa. The changes found in adult coeliac disease may reflect the increased traffic of IEL into the epithelium.     

Endocrine cells in the intestinal metaplasia of gastric mucosa.

Sections of gastric mucosa removed during surgery for cancer or peptic ulcer and containing regions of intestinal metaplasia were studied by the immunofluorescence technique using several antiserums against intestinal hormones. Endocrine cells such as cells containing somatostatin, glicentin (gut GLI-I), motilin, and probably cholecystokinin were found within metaplastic intestinal epithelium while secretin and neurotensin, which are present in the normal intestinal mucosa, were not detected in metaplastic epithelium. The endocrine-cell population present in the intestinal metaplasia resembles that found in the cryptal region of the normal small intestine, a finding in accordance with the fact that intestinal metaplasia of gastric mucosa usually reproduces structural and histochemical characteristics of small intestinal crypts.