Expression of lymphocyte homing receptors and vascular addressins in low-grade gastric B-cell lymphomas of mucosa-associated lymphoid tissue.

In this study, we examined lymphocyte homing receptor and vascular addressin expression in a case of primary gastric B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) with a secondary intestinal spread. We compared the findings with that observed in B cells of normal MALT and MALT acquired as a consequence of Helicobacter pylori-associated gastritis and other low-grade gastric B-cell MALT lymphomas. The neoplastic B cells in the gastric tumor were alpha 4 beta 7-, CD62L+, whereas the intestinal secondary was alpha 4 beta 7+, CD62L-. Incubation of isolated tumor cells from the stomach by H. pylori generated T-cell-dependent proliferation of neoplastic B cells and induced expression of alpha 4 beta 7 integrin similar to the intestinal tumor. These observations indicate that reversal of homing receptor profile in the gastric tumor by antigen specific stimulation may be responsible for secondary intestinal dissemination. In normal stomach and normal MALT, alpha 4 beta 7 and CD62L expression reflected the differentiation of the B cell. Plasma cells were alpha 4 beta 7+, CD62L-, whereas a subset of memory B cells were alpha 4 beta 7-, CD62L+. Homing receptor expression in MALT lymphoma B cells was heterogeneous, however, in line with their memory B-cell phenotype in the majority of cases, the neoplastic B cells were alpha 4 beta 7-, CD62L+. Neoplastic plasma cells were always alpha 4 beta 7+, CD62L-. The venules in normal gastric mucosa expressed mucosal addressin cell adhesion molecule-1 but not peripheral lymph node addressin. In normal MALT, H. pylori-associated follicular gastritis and MALT lymphomas high endothelial venules coexpressed mucosal addressin cell adhesion molecule-1 and peripheral lymph node addressin. These findings suggest expression of lymphocyte homing receptors by B cells and vascular addressins by mucosal venules are similar in normal MALT and MALT lymphomas, and factors controlling normal mucosal B-cell traffic are also operational in MALT lymphomas.     

Expression of the chemokine receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lymphocytes.

Differential expression of adhesion molecules and chemokine receptors has been useful for identification of peripheral blood memory lymphocyte subsets with distinct tissue and microenvironmental tropisms. Expression of CCR4 by circulating memory CD4(+) lymphocytes is associated with cutaneous and other systemic populations while expression of CCR9 is associated with a small intestine-homing subset. CCR5 and CXCR3 are also expressed by discrete memory CD4(+) populations in blood, as well as by tissue-infiltrating lymphocytes from a number of sites. To characterize the similarities and differences among tissue-infiltrating lymphocytes, and to shed light on the specialization of lymphocyte subsets that mediate inflammation and immune surveillance in particular tissues, we have examined the expression of CCR4, CXCR3, and CCR5 on CD4(+) lymphocytes directly isolated from a wide variety of normal and inflamed tissues. Extra-lymphoid tissues contained only memory lymphocytes, many of which were activated (CD69(+)). As predicted by classical studies, skin lymphocytes were enriched in CLA expression whereas intestinal lymphocytes were enriched in alpha(4)beta(7) expression. CCR4 was expressed at high levels by skin-infiltrating lymphocytes, at lower levels by lung and synovial fluid lymphocytes, but never by intestinal lymphocytes. Only the high CCR4 levels characteristic of skin lymphocytes were associated with robust chemotactic and adhesive responses to TARC, consistent with a selective role for CCR4 in skin lymphocyte homing. In contrast, CXCR3 and CCR5 were present on the majority of lymphocytes from each non-lymphoid tissue examined, suggesting that these receptors are unlikely to determine tissue specificity, but rather, may play a wider role in tissue inflammation.     

Evidence of specialized leukocyte-vascular homing interactions at the maternal/fetal interface

In normal pregnancy, the maternal immune system fails to reject the fetus or the placenta as an allogeneic graft. We hypothesize that specialized mechanisms of leukocyte recruitment might limit access of circulating maternal immune cells to the maternal/fetal interface. During the critical period of initial trophoblast invasion there is an elegantly orchestrated progression of leukocyte homing events in the decidua basalis, associated with highly regulated expression of vascular addressins and segregation of specialized leukocyte subsets into well-defined decidual microdomains. Neutrophils are limited to the region of necrosis associated with enzymatic digestion at the leading edge of the invading trophoblast, where an almost linear array of maternal blood vessels displays the neutrophil ligand E-selectin. Cells with the phenotype of monocytes but expressing alpha4beta7 integrin are localized in the blood vessels of the specialized "vascular zone", which display the unusual combination of P-selectin (partially associated with platelets) and the alpha4beta7 ligand mucosal vascular addressin-1 (MAdCAM-1). Granulated metrial gland cells (alpha4+beta7-, probably alpha4beta1+) constitute a well-defined cluster positioned in the central decidua basalis around venules prominently expressing the alpha4beta1 ligand VCAM-1 (but not MAdCAM-1). T and B lymphocytes are rare. Our results suggest that selective mechanisms for regulating leukocyte access, associated with microdomain specialization within the decidua basalis, may play a fundamental role in immune regulation during the invasive period of placental development.     

Preferential expression of the mucosal homing receptor integrin alpha 4 beta 7 in gastrointestinal non-Hodgkin's lymphomas.

Recent studies have identified the integrin alpha 4 beta 7 as a mucosal homing receptor that mediates lymphocyte migration to the intestinal mucosa by binding to MAdCAM-1, a vascular recognition molecule (addressin) selectively expressed on mucosal endothelium. In the present study, we have assessed the expression of alpha 4 beta 7 on B- and T-cell non-Hodgkin's lymphomas of different primary localization and on related normal lymphocytes. Among B-lineage lymphomas, expression of alpha 4 beta 7 was present in the majority of cases of malignant lymphomatous polyposis of the intestine and low-grade lymphoma of the mucosa-associated lymphoid tissue/monocytoid B-cell lymphoma and in some cases of precursor B-cell lymphoma. CLL/small lymphocytic lymphoma, (nodal) mantle cell lymphoma, follicular center cell lymphoma, Burkitt's lymphoma, and diffuse large B-cell lymphoma were virtually always alpha 4 beta 7 negative, as was the case when localized in the mucosa-associated lymphoid tissue. The normal B cells of the follicle mantles and part of the B cells of the extrafollicular B-cell compartment of lymphoid tissues expressed moderate levels of alpha 4 beta 7. By contrast, follicular center cells were alpha 4 beta 7 negative. Among T-lineage lymphomas, expression of alpha 4 beta 7 was also strongly related to the primary localization; in mucosal, nodal, and cutaneous T cell lymphomas the percentage of positive cases was 56%, 17%, and 0%, respectively. All cases of precursor T-cell lymphoma were alpha 4 beta 7 negative. High expression of alpha 4 beta 7 was found on a subset of peripheral blood memory T cells as well as on lymphocytes in the intestinal mucosa. We conclude that non-Hodgkin's lymphomas that are related to mucosa-associated B- and T-lymphocyte populations selectively express the mucosal homing receptor alpha 4 beta 7. The presence of this receptor underscores their distinctive character and may play an important role in determining their characteristic mucosal dissemination pattern.     

Accumulation of CD4+CD7- T cells in inflammatory skin lesions: evidence for preferential adhesion to vascular endothelial cells

The CD7- subset of CD4+ memory T cells reflects a stable differentiation state of post-thymic helper T cells and represents a small subpopulation in circulating blood. We here demonstrate that CD7- T cells preferentially accumulate in skin lesions under chronic inflammatory conditions irrespective of the particular disease. As adhesion to vascular endothelial cells (EC) is required for migration of circulating lymphocytes into tissues, we analysed the adherence of purified subsets of CD4+ memory T cells to endothelial cells in vitro. Compared with CD4+CD7+ T cells, cells of the CD4+CD7- subset preferentially adhere to EC, which is moreover increased after prestimulation of EC with tumour necrosis factor-alpha (TNF-alpha). Stimulated EC increase expression of intercellular adhesion molecule-1 (CD54) and E-selectin (CD62E), the ligand of which, cutaneous lymphocyte-related antigen (CLA), is highly expressed in CD4+CD7- T cells but not in CD4+CD7+ T cells. LFA-1 is expressed in a bimodal distribution on CD4+CD7- T cells in contrast to CD4+CD7+ cells, whereas VLA-1, VLA-3, and VLA-5 are nearly similarly expressed in both T cell subsets. Our results imply that the preferred adherence of CD4+CD7- memory T cells to vascular EC, which is increased after long-term EC stimulation with TNF-alpha, is likely to facilitate their accumulation in various inflammatory skin lesions.     

Tonsillar B cells do not express PSGL-1, but a significant fraction displays the cutaneous lymphocyte antigen and exhibits effective E- and P-selectin ligand activity

Skin-homing T cells are defined by the expression of the cutaneous lymphocyte-associated antigen (CLA) which enables the cells to selectively bind to vascular endothelial E-selectin close to sites of cutaneous inflammation, an initial step in the effective extravasation from blood into the inflamed tissue. Essentially all CLA on T cells decorates the backbone of the P-selectin glycoprotein ligand-1 (PSGL-1). In this study we show that human peripheral blood B cells (PBBC) and tonsillar B cells (TBC) do not display PSGL-1 in fluorescence-activated cell sorter analysis using different murine monoclonal antibodies and polyclonal rabbit anti-PSGL-1 antiserum. A significant population of TBC, however, expresses a HECA-452-reactive epitope. These cells represent nonactivated IgM(+)/IgG(-) mature B lymphocytes. Up to 50% of the TBC in a given preparation strongly bind to E- and up to 79% to P-selectin. The shear stress resistance in a parallel-plate flow chamber system was high. Neuraminidase treatment of TBC totally and O-sialoglycoprotein endopeptidase partially diminished HECA-452 reactivity and reduced E- but not P-selectin ligand activities. Mocarhagin had no effect in the assays. The data suggest a different ligand for P-selectin and a distinct glycoprotein carrier for the E-selectin ligand as compared to T cells or other leukocytes. Adhesion to P-selectin, however, still required sulfation of the ligand for function. Western blots of TBC cell lysates detected a >240-kD HECA-452-reactive material that was resistant to reducing conditions. Anti-PSGL-1 did not reveal immunoreactive material in these cell lysates. B cell activation did neither significantly change HECA positivity nor induce PSGL-1 expression. Cultured, activated TBC, however, maintained expression of the integrin alpha4beta7. Human peripheral blood B cells had similar cell surface characteristics to TBC. Our observations suggest that several adhesion molecules may be involved in B cell homing which include CLA, the P-selectin ligand, and structures such as alpha4beta7.     

Differential expression of adhesion molecules and chemokines between nasal and small intestinal mucosae: implications for T- and sIgA+ B-lymphocyte recruitment

Nasal and small intestinal mucosae are the first sites of contact with infectious agents and the sites of T-cell-mediated and secreted immunoglobulin A (IgA)-mediated defences against pathogens. We investigated the factors controlling the infiltration of CD3(+) T lymphocytes and surface IgA(+) (sIgA(+)) B lymphocytes into swine epithelium and lamina propria (LP) within and between these two mucosal effector sites. Vascular addressins, vascular cell adhesion molecule 1 and mucosal addressin cell adhesion molecule-1 were reciprocally expressed in both mucosae. Strong expression of alpha(4)beta(1) relative to alpha(4)beta(7) was characteristic of CD3(+) T cells in nasal mucosa LP and epithelium and of sIgA(+) cells in nasal mucosa epithelium. The same profile was observed on corresponding blood cells. Conversely, higher levels of integrins beta(7) and alpha(4)beta(7) than alpha(4)beta(1) were characteristic of CD3(+) T cells and sIgA(+) cells in the small intestine. However, about 40% of the LP-activated sIgA(+) cells displayed sIgA(high), integrin alpha(4) and integrin alpha(4) expression. Whereas CCL19, CXCL12, CCL21 and CCL28 messenger RNAs were similarly expressed in both mucosae, CCL25 messenger RNA was only expressed in the small intestine. Thus, the nasal and small intestine mucosae represent separate compartments for infiltration by CD3(+) T cells and sIgA(+) effector cells, with the exception of a population of small intestine activated sIgA(+) cells, which may gain access to both mucosae.     

Regional specialization in the mucosal immune system: primed cells do not always home along the same track

According to the current paradigm of lymphocyte trafficking, primed B and T cells extravasate in the intestinal lamina propria chiefly by means of the mucosal homing receptor alpha4beta7, which interacts with the vascular addressin MAdCAM-1. However, as discussed here, this mechanism cannot explain the preferential homing of B cells with a high level of J-chain expression to mucosal effector sites outside the gut.     

Topographic distribution of homing receptors on B and T cells in human gut-associated lymphoid tissue: relation of L-selectin and integrin alpha 4 beta 7 to naive and memory phenotypes.

In mice, integrin alpha 4 beta 7 is the main receptor used by lymphocytes that home to the Peyer's patches, although L-selectin contributes to the initial interaction with high endothelial venules. Less is known about the expression and function of these adhesion molecules in humans. The distribution of L-selectin and alpha 4 beta 7 on various B- and T-cell subsets was examined in human Peyer's patches (n = 8) and appendix (n = 4), collectively called gut-associated lymphoid tissue. Multicolor immunophenotyping was performed on cryosections, and dispersed cells were examined by flow cytometry. In cryosections, CD45RA+ T cells around and within interfollicular high endothelial venules, as well as surface (s)IgD+ B lymphocytes in the follicle mantles, often expressed abundant L-selectin but only intermediate levels of alpha 4 beta 7. CD45RO+ T cells and sIgD- B cells expressed higher levels of alpha 4 beta 7 and were often located near putative efferent lymphatics; only a small fraction (< 20%) of such memory cells expressed L-selectin. By flow cytometry, considerably more T than B lymphocytes co-expressed L-selectin and alpha 4 beta 7 (40% versus 25% and 67% versus 39%, respectively). In samples with many L-selectin+ cells (> 30%), more of these lymphocytes co-expressed alpha 4 beta 7 than in samples with few L-selectin+ cells. Because L-selectin and alpha 4 beta 7 were co-expressed on lymphocytes located near high endothelial venules, and because such co-expression was relatively common when many L-selectin+ cells were present, both of these molecules might participate in homing to human gut-associated lymphoid tissue. Such homing is probably most pronounced for T lymphocytes that were found to express L-selectin and alpha 4 beta 7 more often than B lymphocytes. The selective and relatively high expression of alpha 4 beta 7 on memory cells located near efferent lymphatics indicated a different migratory capacity; after exit from gut-associated lymphoid tissue, such stimulated cells might home mainly to mucosal effector sites.     

Evaluation of circulating intestinally committed memory B cells in children vaccinated with attenuated human rotavirus vaccine

In a double blind trial, 319 fully immunized children received two doses of either placebo or 10(6.7) focus-forming units of the attenuated RIX4414 human rotavirus (RV) vaccine ("all-in-one" formulation). Plasma RV-specific IgA (RV IgA), stool RV IgA, and circulating total and RV memory B cells (CD19+ IgD+/- CD27+) with an intestinal homing phenotype (alpha4beta7+ CCR9+/-) were measured, after the first and second doses, as potential correlates of protection. After the first and/or second dose, 54% of vaccinees and 13% of placebo recipients had plasma RV IgA. Before vaccination, most (95%) of the children (of both study groups) were breast-fed and had stool RV IgA (68.64%). Coproconversion (4-fold increase) after the first and/or second dose was observed in 32.7% of vaccinees and 17.4% of placebo recipients. No significant difference was seen when comparing the frequencies of any subset of memory B cells between vaccinees and placebo recipients. Statistically significant weak correlations were found between plasma RV IgA titers and coproconversion, and several subsets of memory B cells. The vaccine provided 74.8% protection (95% confidence interval, 30.93-92.62) against any RV gastroenteritis and 100% protection (95% confidence interval, 14.53-100) against severe RV gastroenteritis. When vaccinees and placebo recipients were considered together, a correlation was found between protection from disease and plasma RV IgA measured after dose 2 and RV memory (IgD- CD27+ alpha4beta7+ CCR9+) circulating B cells measured after dose 1. However, the correlation coefficients for both tests were low (<0.2), suggesting that other factors are important in explaining protection from disease.     

Quantitative and functional characteristics of intestinal-homing memory T cells: analysis of Crohn's disease patients and healthy controls

Circulating memory T cells can be subdivided on the basis of beta7 integrin expression. The beta7+ population contains cells primed in the intestine capable of homing back to the gut. We hypothesized that cytokine production by beta7+ memory T cells reflects the specialized mucosal compartment in which they were primed. Flow cytometry of whole blood was used to assess numbers of beta7+ (beta7hi and beta7int) and beta7- memory T cells and their production of Th1 and regulatory cytokines in healthy controls and Crohn's disease patients. In controls, beta7+ and beta7- memory T cells displayed a similar qualitative profile of cytokine production but the beta7+ population was enriched for cytokine-producing effector cells. In addition, the beta7hi population contained more cytokine-producing cells than the beta7int population, suggesting a gradient of cytokine production based on beta7 integrin expression. In active Crohn's disease, there was altered expression of beta7 integrin with a decrease in intestinal-homing memory T cells and an increase in systemic memory T cells. Furthermore, there was a selective loss of IL-10 and increase in TGF-beta in both beta7+ and beta7- memory T cell subsets which may contribute to the pathogenesis of the inflammatory process in Crohn's disease.     

Distribution and phenotype of rotavirus-specific B cells induced during the antigen-driven primary response to 2/6 virus-like particles administered by the intrarectal and the intranasal routes

Selection of mucosal sites is an important step in mucosal vaccine development. The intrarectal (IR) route represents an alternative to the oral route of immunization; nevertheless, immune responses induced by this route are not well defined. Here, we studied the early primary B cell response (induction, homing, and phenotype) induced by IR immunization with rotavirus (RV)-2/6 virus-like particles (VLP). Using flow cytometry, we traced RV-specific B cells in different lymphoid tissues and analyzed the expression of alpha4beta7 and CCR9, which are important receptors for homing to the gut, as well as CD5, a marker expressed by B1-a cells, which are a major source of natural antibodies. We observed a massive, specific B cell response in rectal follicles, lumbar, and mesenteric lymph nodes but not in Peyer's patches or cervical lymph nodes. A minority of cells expressed alpha4beta7, suggesting a probable lack of migration to the gut, whereas CCR9 and CD5 were expressed by 30-50% and 30-75% of specific B cells, respectively. Then, we compared the intranasal route of immunization and observed similar B cell frequency and phenotype but in respiratory lymphoid tissues. These results confirm the high compartmentalization of B cell responses within the mucosal system. They show that CCR9 expression, conversely to alpha4beta7, is not restricted to B cells induced in the gut. Finally, an important part of the RV-specific B cell response induced at the mucosal level during the primary response to VLP is most likely a result of B1-a cells.     

Intestinal dendritic cells increase T cell expression of alpha4beta7 integrin

The integrin alpha4beta7 binds to MAdCAM-1 and contributes to homing of lymphocytes to gut and other mucosal tissues. In humans, the alpha4beta7(hi) subset of circulating memory cells appears to have been primed in mucosal tissues. The factors that determine whether alpha4beta7(lo) naive cells become alpha4beta(hi) or alpha4beta7(-) cells upon differentiation are poorly understood but could include an influence of the activating antigen-presenting cell. To address this point, the induction of alpha4beta7 following activation of mouse cells with the APC-dependent stimulus soluble anti-CD3 has been examined. Almost all mouse T cells freshly isolated from mesenteric lymph nodes (MLN) and peripheral (PLN; axillary, brachial and inguinal) lymph nodes stained only weakly for alpha4beta7 but a subpopulation became alpha4beta7(hi) upon activation with anti-CD3 in a cell cycle- and accessory cell-dependent manner. A small proportion (approximately 1.5 %) of the starting cells gave rise to alpha4beta7(hi) cells after culture. A higher proportion of alpha4beta7(hi) cells were generated in MLN than PLN cultures. Peyer's patch cultures gave intermediate values. In crossover experiments, MLN dendritic cells (DC) induced higher proportions and numbers of alpha4beta7(hi) cells than PLN DC irrespective of the source of T cells. Therefore, in addition to their other immunoregulatory roles, DC have the potential to shape immune responses by influencing the homing of the lymphocytes they activate.     

Distribution and phenotype of murine rotavirus-specific B cells induced by intranasal immunization with 2/6 virus-like particles

Virus-like particles containing the rotavirus (RV) internal proteins VP2 and VP6 (2/6-VLP) have been shown to induce serum and fecal antibodies as well as protection in mice after intranasal administration with a mutant of E. coli toxin, LT-R192G. To better understand the origin of fecal IgA induced by this protocol, we studied the RV-specific B cell response in systemic and mucosal lymphoid tissues using a flow cytometry assay that allows quantification and phenotypic characterization of RV-specific B lymphocytes. We also assessed the RV-specific antibody-secreting cells in the spleen and intestinal lamina propria (ILP). A remarkably high frequency of RV-specific B cells was found in the respiratory lymphoid tissues and spleen, of which only a minority expressed the alpha4beta7 integrin (intestinal homing receptor). In contrast, but in accordance with alpha4beta7 expression at the induction site, a very low response was observed in intestinal lymphoid tissues (mesenteric lymph nodes and ILP), which did not increase after a second immunization. Thus, intranasal immunization with a nonreplicating antigen does not induce an important number of RV-specific B cells with an intestinal homing profile.     

Skin-homing CLA+ T cells and regulatory CD25+ T cells represent major subsets of human peripheral blood memory T cells migrating in response to CCL1/I-309

Functionally distinct T cell subsets exhibit specific chemokine receptor profiles that regulate their tissue localization. Here, we show that human peripheral blood CD4(+) and CD8(+) cutaneous (CLA(+)), but not intestinal memory (integrin beta(7) (+)) nor IL-4-producing T cells, represent major subpopulations of circulating T cells that specifically migrate in response to the chemokine I-309/CCL1 by virtue of CCR8 expression. Expression of CCR8 is markedly up-regulated upon activation and in vitro culture of human CLA(+) T cells, suggesting the involvement of CCR8 in localization of cutaneous memory T cells to the skin. Interestingly, amongst circulating memory CD4(+)CD45RO(+) T cells, chemotactic responsiveness to CCL1 is restricted to cells expressing CD25 and/or CLA surface markers for regulatory T cells (Treg) and skin-homing T cells and maximal responsiveness is observed on CLA(+)CD25(+)T cells. Such pattern of CCL1 responsiveness suggests that the CCR8/CCL1 axis may regulate trafficking of cutaneous Treg and memory T cells into the skin.     

Rotavirus-specific B cells induced by recent infection in adults and children predominantly express the intestinal homing receptor alpha4beta7

In vivo replication of rotaviruses is generally limited to enterocytes. Because of this restriction, most blood circulating rotavirus-specific B cells are hypothesized to originate in Peyer's patches and should express the intestinal homing receptor alpha4beta7. To test this hypothesis in humans, we used a flow cytometry assay that identifies antigen-activated (IgD-) B cells (CD19+) that express surface rotavirus-specific immunoglobulin. With this assay we could detect rotavirus-specific B cells in both children and adults with an acute rotavirus (RV) infection. Staining with an anti-alpha4beta7 monoclonal antibody, we could determine that B cells that express rotavirus-specific surface immunoglobulin predominantly express alpha4beta7. The response of rotavirus-specific antibody-secreting cells in the peripheral blood of children and adults with acute rotavirus infection was also studied by ELISPOT. The antibody-secreting cells of children were mainly of the IgM isotype, while the antibody-secreting cells of adults were predominantly of the IgA and IgG isotype. alpha4beta7+ and alpha4beta7- subsets of peripheral blood mononuclear cells were purified using paramagnetic beads and then tested in the ELISPOT assay. Rotavirus-specific antibody-secreting cells were predominantly present in the alpha4beta7+ subpopulation. The flow cytometry assay we have described will permit future studies to characterize the phenotype of virus-specific B cells and could be useful in the study of the immunogenicity and protective efficacy of RV vaccines and the identification of markers of protective immunity.     

Alpha4beta1 and alpha4beta7 CD4 T cell numbers increase and CLA CD4 T cell numbers decrease in systemic sclerosis

We studied the expression of adhesion molecules affecting recirculation and homing on peripheral blood CD4(+) T cells of patients with systemic sclerosis (SSc), in order to evaluate whether the distribution of tissue targeted subsets could reflect the participation of internal organs or the extent of cutaneous involvement [i.e. limited cutaneous (lc) and diffuse cutaneous (dc)]. Peripheral blood mononuclear cells (PBMC) from 51 patients with SSc and 19 sex- and age-matched controls were investigated by cytofluorimetric analysis for lymphocyte subpopulations carrying the following surface molecules: CD3, CD4, CLA, alpha4beta7 and alpha4beta1. Standard routine biochemistry and clinical examinations were also performed in all patients. We found that both alpha4beta1(+) and alpha4beta7(+) cells within the CD4(+) T cell population were significantly increased, while CLA(+) CD4(+) T cells were significantly reduced in SSc, compared to healthy donors. Significantly lower absolute numbers of alpha4beta7(+) cells were found in lc- compared to dc-SSc. Patients with oesophageal involvement had high numbers of alpha4beta7(+) cells, while those with nephritis also showed low levels of CLA(+) cells. Lung involvement was related directly to alpha4beta1(+) cell numbers and inversely to alpha4beta7(+) CD4 cell numbers. Taken together, our findings demonstrate that distinct CD4(+) T cell populations with selective homing properties show changes from normal distribution in SSc, and such changes are related to clinical expression and organ involvement in the course of the disease.     

Distribution of beta 7 integrins in human intestinal mucosa and organized gut-associated lymphoid tissue.

Two alternative integrins involved in mucosal homing (alpha 4 beta 7) or epithelial retention (alpha E beta 7) of lymphocytes were examined in the human gut. The distribution of the beta 7 subunit [monoclonal antibody (mAb) M301] was bimodal in that it was strongly expressed by alpha E beta 7 + cells but weakly by alpha 4 beta 7 + cells. More than 90% of intraepithelial lymphocytes (IEL), including the minor subsets of CD4+, T-cell receptor (TCR) gamma/delta +, and CD3- cells, expressed alpha E beta 7 as did most lamina propria CD8+ (88%) and a fraction (36%) of CD4+ lymphocytes. Conversely, B-lineage cells (CD19+) and macrophages (CD68+) were negative. In gut-associated lymphoid tissue (GALT: Peyer's patches and appendix) only a few (< 5%) cells were positive for alpha E beta 7 (confined to CD8+ lymphocytes and CD11c+ putative dendritic cells). A relatively small fraction of IEL (30-50%) expressed alpha 4 beta 7 (mAb Act-1), while most (70%) lamina propria T and B lymphocytes, blasts, plasma cells and macrophages were positive. In GALT, T lymphocytes expressed similar levels of alpha 4 beta 7 as in the lamina propria whereas relatively few B lymphocytes (< 50%) were positive. Isolated lamina propria CD8+, CD4+, CD19+, and CD38+ cells contained mRNA for alpha 4 and the former three subsets as well as appendix CD8+ cells also for beta 7 while only lamina propria CD8+ cells had mRNA for alpha E. Together, the results suggested that alpha E beta 7 and alpha 4 beta 7 are differentially regulated in inductive sites and effector sites of the human gut. Because lymphoid cells at both sites expressed mainly alpha 4 beta 7, this integrin may be a homing receptor on memory and effector cells bound for lamina propria as well as on naive lymphocytes extravasating in GALT. Conversely, because alpha E beta 7 was mainly expressed by CD8+ cells in epithelium and lamina propria, it was probably induced after extravasation, in agreement with the observation that IEL and a fraction of lamina propria T lymphocytes (mainly CD8+ cells) generally expressed higher levels of beta 7 than most CD4+ and B cells. Also a subset of putative dendritic cells located near the follicle-associated epithelium of GALT expressed alpha E beta 7, perhaps reflecting epithelial interaction during primary immune responses.     

Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells

Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4(+) and CD8(+) T cells secreting INFgamma, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals. In children with acute RV infection, these T cells were barely or not detectable. In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFgamma-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection. Using microbead-enriched CD4(+) and CD8(+) T cell subsets, IFNgamma-secreting RV-specific CD8(+) but not CD4(+) T cells were detected in recently infected children. Using the same approach, both CD4(+) and CD8(+) RV-specific T cells were detected in healthy adults. Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFgamma-secreting CD4(+) T cells from adult volunteers preferentially express the intestinal homing receptor alpha4beta7, but not the peripheral lymph node homing receptor L-selectin. In contrast, CMV-specific INFgamma-secreting CD4(+) T cells preferentially express L-selectin but not alpha4beta7. These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFgamma is independent of the expression of these homing receptors.     

Skin-homing T cells in human cutaneous allergic inflammation

The cutaneous lymphocyte-associated antigen (CLA) is a carbohydrate epitope present on memory/effector T cells that infiltrate inflamed skin. E-selectin is the ligand for CLA and is induced under inflammation on endothelial cells. CLA was originally postulated as a phenotype marker for skin-associated T cells. We studied the specific in vitro response to skinassociated allergens of CLA+and CLA-CD45RO+T cells in atopic dermatitis (AD) and contact dermatitis (CD), which represent two well-characterized T cell-mediated cutaneous allergic inflammations. Whereas CLA+T cells from AD patients preferentially responded to house dust mite (HDM) and CLA+T cells from nickel CD patients showed an increased response to nickel, CLA-T cells showed very little response in both cases. In contrast, tetanus toxoid, a systemically acting antigen, induced a proliferative response in both CLA+and CLA-cells. Interestingly the response to HDM in patients with asthma±AD was preferentially found in the CLA-subset indicating the involvement of different homing receptors for mucosal tissues. Moreover, CLA+T cells showed enhanced migration through activated human umbilical vein endothelial cell monolayers compared to CLA-T cells. The CLA binding to E-selectin is initially responsible for the extravasation that also involves VLA-4/VCAM-1 and LFA-1/ ICAM-1 interactions. We have recently identified IL-8 as an endothelial cell-derived chemokine and the IL-8 receptor type B which control CLA+T cell migration. Such a CLA-mediated migration would localize memory/effector T cells that respond to antigens and reach the body through inflamed skin. Our data support the existence of a regionalization of the immune system and in particular of the skin immune system. It may allow an efficient distribution of the immune defense to different sites of the body.