Intestinal intraepithelial lymphocytes and lymphoepithelial interactions in the human gastrointestinal mucosa.

Although representing a major immunological apparatus, it is not known how the immune system of the intestinal mucosa differentiates between dietary antigens (resulting in systemic tolerance) and potential pathogens. It is thought that intraepithelial T lymphocytes (IEL) may play a central role in local intestinal immunity and are likely to be important in immunity to gastrointestinal neoplasms and rejection responses to gut allografts. However, the biology of IEL and their unusual immunological microenvironments in the gastrointestinal mucosa are little understood. IEL are predominantly CD8+ TcR alpha beta+ CD3+ T cells which differ from lamina propria and peripheral T cells in many respects. IEL show low expression of CD5, CD6, LFA-1 (CD11a/CD18) and VLA-4, and high expression of HML-1. TcR gamma delta + IEL, although a minority population, are also phenotypically distinct, insofar as they are 50% CD8+, mainly V delta 1+ V gamma 9- and CD4- CD5-. IEL show poor proliferative responses to PHA, anti-CD3 and phorbol ester/calcium ionophore in vitro and have no clear functional role: they neither provide helper nor suppressor functions for Ig synthesis by B cells and do not mediate spontaneous cytotoxicity. However, there is evidence that IEL show preferential activation in response to sheep erythrocytes, presumably signalling via CD2. As normal and inflamed intestinal epithelia do not express ICAM-1, it seems unlikely that the LFA-1/ICAM-1 interaction is of importance to IEL activation. Rather, the CD2 (LFA-2) interaction with LFA-3 expressed by enterocytes may serve both to anchor IEL and to provide an accessory stimulus for activation. Nevertheless, the questions of antigenic specificity and immunological role remain unanswered.     

Immunobead filtration: a novel approach for the isolation and propagation of tumor cells.

We have developed a method to facilitate the isolation and expansion of tumor cells from body fluids and tissue biopsies. Antibody-conjugated magnetic beads (immunobeads) were used to isolate tumor cells from blood, bone marrow, ascitic/pleural fluids, and enzyme-digested tissue biopsies. Filtration of the resulting cell suspension through a 20-micron nylon monofilament filter secured to the base of polystyrene 96-well strips purged the bead-rosetting cell fraction of contaminating normal cells and unbound beads. Tumor cells that bound the magnetic beads were retained on the membrane due to their increased size and concentrated into a small area (0.332 cm2), thus maintaining a high cell density. The filters provided a stable and uniform three-dimensional matrix for cell growth, with a total surface area of 1.42 cm2 available for cell attachment. The filters could be easily removed from the base of the 96-well strips to facilitate handling and transfer between culture vessels. Tumor cells grown on the filters could subsequently be harvested using trypsin/EDTA or left in situ for immunostaining with conventional immunohistochemical procedures. Filter-grown cells have shown extended passage in conventional cell culture in six cases. In two of five cases, the orthotopic implantation of confluent filters that contained approximately 10(4) cells/8 x 8 mm filter successfully produced tumors in nude mice after only 4 weeks. Our new approach may be of value in improving the success rate of generating long-term cultures from previously unproductive sources of tumor cells and thus may yield a greater variety of cell lines/strains for the study of malignant disease.     

Isolation of human small bowel intraepithelial lymphocytes by annexin V-coated magnetic beads

Human intestinal intraepithelial lymphocytes (IEL) are important effector cells of the mucosal immune system and their study is hampered by the difficulty of their isolation. The molecular study of enriched samples of IEL is mandatory in the diagnosis of enteropathy-associated T-cell lymphoma and refractory celiac sprue. In order to isolate human small bowel IEL, we took advantage of the stress that intestinal epithelial cells (IEC) suffer during the conventional initial steps of IEL isolation, which induces their apoptosis but not that of IEL. After cell individualization by dithiothreitol and ethylenediamine tetraacetic acid, two-thirds of human IEC can be stained with Annexin-V due to their surface exposure of phosphatidyl serine, a sign of apoptosis. This percentage increases to 95% after performing a density gradient to enrich for IEL. This allows for the use of Annexin-V-coated magnetic beads, originally designed for the removal of dead cells from cell cultures, to obtain >95% pure, 99% viable and untouched IEL after two rounds of depletion. This simple procedure has proven useful for the isolation of human IEL for functional and molecular studies and can conceivably facilitate the diagnosis of intestinal lymphoid malignancies that rely upon the study of pure IEL preparations.     

Intraepithelial, lamina propria and Peyer's patch lymphocytes of the rat small intestine: isolation and characterization in terms of immunoglobulin markers and receptors for monoclonal antibodies.

Methods have been determined for the isolation, purification and subsequent characterization of separate populations of rat intestinal lymphoid cells, namely intraepithelial (IEL), lamina propria (LPL) and Peyer's patch lymphocytes (PPL). Dissociation of the epithelium from the basement membrane with subsequent release of IEL was achieved by citrate buffer incubation followed by vortex agitation. LPL were released from the remaining tissue by scraping, and PPL were similarly obtained. Some preparations of lamina propria were further subjected to collagenase digestion. After filtration and density gradient centrifugation, average yields of 220 x 10(4) IEL, 54 x 10(4) LPL and 220 x 10(4) PPL per gram of gut were obtained. Immunofluorescence characterization demonstrated that cells bearing the MRC OX8 (T-suppressor) marker predominated in IE1 (73%) and were present in lower concentrations in LPL (26%) and PPL (6%). Cells with the W3/25 (T-helper) marker accounted for a small proportion of each of the lymphocyte preparations. IE1 were unusual in containing a population of cells which were negative for the W3/13 marker for T cells, but were MRC OX8 positive. B lymphocytes were present in PPL (55%) and LPL (31%), but were virtually absent in IEL (less than 1%). Few plasma cells were observed. The techniques described will allow functional investigations to be made and lead to a better understanding of mucosal immunity.     

Isolation of plasma membranes from rat C6 glioma cells cultivated on microcarriers.

We have studied the feasibility of rat C6 glioma cell cultivation on microcarrier beads and the isolation of their plasma membranes from the beads. Cells were cultivated on Cytodex-1 microcarrier beads and the plasma membranes were subsequently isolated from confluent cell monolayers on the beads. This approach yielded approximately 4 x 10(6) cells/ml in a 1 L spinner vessel. Enzymatic assays indicated an 18-fold enrichment of plasma membranes isolated from the beads with minor contamination by other cell organelles. Assay for IGF-I receptor binding capacity revealed that 70% of the total receptor binding capacity could be recovered in the plasma membrane fraction isolated from the beads as compared with the receptor binding capacity of intact cells, demonstrating the functional integrity of the isolated membranes. Electron microscopy and immunofluorescence analysis indicated that the isolated plasma membranes were highly homogeneous with the majority exposing the cytoplasmic surface. Our procedure of C6 glioma cell cultivation on microcarriers and subsequent plasma membrane isolation, provides large quantities of homogeneous and metabolically active membranes which can be used to study receptor-mediated effects on cell proliferation and differentiation.     

Gamma delta T cell receptor-positive cells of the human gastrointestinal mucosa: occurrence and V region gene expression in Heliobacter pylori-associated gastritis, coeliac disease and inflammatory bowel disease.

T cells expressing the gamma delta heterodimer of the T cell receptor (TCR) were studied with respect to their occurrence and expression of gamma delta TCR variable region (V) genes in the normal gastrointestinal mucosa and in a variety of inflammatory conditions. In controls, gamma delta TCR+ cells were a minority population confined to the epithelial compartment of stomach, small bowel and colonic mucosae. Unlike in the periphery, gastro-intestinal gamma delta TCR+ intraepithelial lymphocytes (IEL) were mainly V delta 1+ (89.98 +/- 17.70%); few were V delta 2+ (6.04 +/- 13.8%) or V gamma 9+ (11.38 +/- 10.73%). All gamma delta TCR+ IEL were CD5low; nearly half were CD8+ and the remainder were CD4-CD8- 'double negatives'. There was no significant change from normal in percentages of gamma delta TCR+ IEL in H. pylori-associated gastritis, Crohn's disease and ulcerative colitis. However, in coeliac disease, gamma delta TCR+ IEL were elevated from 2.54% (+/- 1.71) in controls to 29.6% (+/- 16.1) in untreated patients (P less than 0.001) and 18.5% (+/- 7.2) in treated patients (P less than 0.001) and more were CD4-CD8-. Otherwise, gamma delta TCR+ IEL phenotypes were little changed: the majority remained V delta 1+V delta 2-V gamma 9- and all were CD5low. These data suggest that increased gamma delta TCR+ IEL are not a generalized response to intestinal inflammation or to stress proteins, although the typical V delta 1+V delta 2-V gamma 9- CD5low phenotype is retained.     

Quantitative isolation of mouse and human intestinal intraepithelial lymphocytes by elutriation centrifugation

Intestinal intraepithelial lymphocytes (IEL) are specialized subsets of T cells with distinct functional capacities. While some IEL subsets are circulating, others such as CD8αα TCRαβ IEL are believed to represent non-circulating resident T cell subsets [Sim, G.K., Intraepithelial lymphocytes and the immune system. Adv. Immunol., 1995. 58: 297-343.]. Current methods to obtain enriched preparations of intraepithelial lymphocytes are mostly based on Percoll density gradient or magnetic bead-based technologies [Lundqvist, C., et al., Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. J. Immunol. Methods, 1992. 152(2): 253-263.]. However, these techniques are hampered by a generally low yield of isolated cells, and potential artifacts due to the interference of the isolation procedure with subsequent functional assays, in particular, when antibodies against cell surface markers are required.Here we describe a new method for obtaining relatively pure populations of intestinal IEL (55-75%) at a high yield (> 85%) by elutriation centrifugation. This technique is equally suited for the isolation and enrichment of intraepithelial lymphocytes of both mouse and human origin. Time requirements for fractionating cell suspensions by elutriation centrifugation are comparable to Percoll-, or MACS-based isolation procedures. Hence, the substantially higher yield and the consistent robust enrichment for intraepithelial lymphocytes, together with the gentle treatment of the cells during elutriation that does not interfere with subsequent functional assays, are important aspects that are in favor of using this elegant technology to obtain unmanipulated, unbiased populations of intestinal intraepithelial lymphocytes, and, if desired, also of pure epithelial cells.     

Intestinal intraepithelial lymphocytes prevent pathogen-driven inflammation and regulate the Smad/T-bet pathway of lamina propria CD4+ T cells

Intraepithelial lymphocytes (IEL) play a key role in gut homeostasis and are critical effector cells preventing the inflammatory intestinal lesions induced in mice following oral infection with Toxoplasma gondii. In this intestinal inflammatory model, CD4(+) T lymphocytes from the lamina propria (LP) synergize with the infected enterocytes to secrete pro-inflammatory chemokines and cytokines. In this study, we assessed the mechanisms accounting for the ability of IEL to modulate the inflammatory activity of these cells. Adoptive transfer of IEL purified from wild-type mice, or CD154-,CD95L- or IL-10-deficient mice infected with T. gondii completely impairs the development of the lethal ileitis in recipient mice orally infected with T. gondii.Compared with unprimed IEL isolated from naive mice, the CD8 alpha beta TCR alpha beta subset of primed IEL, isolated from T. gondii-infected mice, secretes increased amount of TGF-beta. IEL interact with the LP CD4(+) T lymphocytes, down-regulate their production of inflammatory cytokines such as IFN-gamma and reduce their proliferative activity. These effects are linked to the secretion of TGF-beta and are correlated with a shift in the balance between Smad7/T-bet down-regulation and Smad2/Smad3 up-regulation in LP CD4(+) T lymphocytes.     

T lymphocytes of the human colonic mucosa: functional and phenotypic analysis.

Normal human colonic lymphocyte populations were isolated for both phenotypic analysis by double-label immunofluorescence and assessment for regulatory effects on Ig production by co-culture with responder cells from colonic mucosa and peripheral blood. Mean CD4:CD8 ratios for colonic intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) were comparable to values obtained from tissue sections. IEL alone did not produce Ig in vitro and were without effect on Ig production when co-cultured with LPL. However, T-enriched LPL had a marked helper effect for T-depleted LPL. Maximal help was for IgA production, increasing with numbers of T-enriched cells. In colonic LPL T-depleted and T-enriched co-cultures, pokeweed mitogen (PWM) had no significant effect. By contrast, in co-cultures of T-enriched and T-depleted peripheral blood mononuclear cells, Ig production was PWM-dependent. In all experiments with colonic mucosal responder cells, IgG production was low. The effects of unfractionated colonic biopsy lymphocytes on T-depleted peripheral blood mononuclear cells were additive for IgM production and synergistic for IgA synthesis, although almost no IgG was produced. Moreover, PWM had helper effects for IgM, but was suppressive for IgA production. These data suggest that colonic mucosal regulatory cells reside in the lamina propria, and predominantly provide help for IgA and IgM synthesis. The data further suggest the existence of a pre-stimulated IgA-specific T helper cell population.     

Ex Vivo Expansion and Th1/Tc1 Maturation of Umbilical Cord Blood T Cells by CD3/CD28 Costimulation

One major limitation of UCBT is the lack of donor cells available for posttransplantation donor leukocyte infusions (DLI) to boost immunity or induce graft-versus-leukemia (GVL) activity. Starting from a ∼5% fraction of a UCB graft, we report the feasibility and biological characteristics of ex vivo expansion of frozen/thawed CB T cells by anti-CD3 and anti-CD28 antibody–coated Dynal beads in the presence of interleukin (IL)-2. We postulated that while undergoing expansion, UCB T cells may mature toward a Th1/Tc1 phenotype and acquire the potential for cytotoxicity. Whereas an almost 2-log expansion also led to the acquisition of IL-12Rα and an increase in Th1 characteristics, postexpansion lymphocytes produced less interferon-γ, tumor necrosis factor-α, and granzyme B; stored almost no perforin; and lacked cytotoxicity against allogeneic targets. Collectively, these suggest relative safety from acute/hyperacute graft-versus-host disease. CD8⁺ T cells expanded preferentially, whereas a higher rate of apoptosis in CD4⁺ T cells also promoted an inverted CD4/CD8 ratio. Most expanded T cells retained expression of CD27, CD28, and L-selectin but down-regulated CCR-7. In summary, UCB T cell proliferation sustained by CD3/CD28 co-stimulatory beads and IL-2 can lead to clinically relevant doses of DLI from a very small fraction of the UCB graft, although future strategies to reduce apoptosis may enhance their clinical potential.     

Human intestinal lamina propria and intraepithelial lymphocytes express receptors specific for chemokines induced by inflammation

To determine which chemokine receptors might be involved in T lymphocyte localization to the intestinal mucosa, we examined receptor expression on human intestinal lamina propria lymphocytes (LPL), intraepithelial lymphocytes (IEL) and CD45RO+beta7hi gut homing peripheral blood lymphocytes (PBL). Virtually all LPL and IEL expressed CXCR3 and CCR5, receptors that have been associated with Th1(Tc1)/Th0 lymphocytes, while CCR3 and CCR4, receptors associated with Th2 (Tc2)lymphocytes, CCR7, CXCR1 and CXCR2 were not expressed. CXCR3 and CCR5 receptors were functional, as LPL and IEL migrated to their respective ligands I-TAC and RANTES. In addition, most alphaEbeta7- LPL and IEL expressed high levels of CCR2. While the majority of CD45RO(-)beta7hi PBL also expressed CXCR3 and CCR5, a proportion of these cells were CXCR3- and/or CCR5- and some expressed CCR4 and/or CCR7, indicating that lymphocytes recruited to the intestinal mucosa represent a subset of these cells. In summary, our results show that LPL and IEL within the normal intestine express a specific and similar array of chemokine receptors whose ligands are constitutively expressed in the intestinal mucosa and whose expression is up-regulated during intestinal inflammation. These results support the view that CXCR3, CCR5 and CCR2 may play an important role in lymphocyte localization within the intestinal mucosa.     

Macrophage migration inhibitory factor activates antigen-presenting dendritic cells and induces inflammatory cytokines in ulcerative colitis

The level of macrophage migration inhibitory factor (MIF) and the functions of dendritic cells (DC) are up-regulated in the peripheral blood, and the numbers of MIF-expressing cells and mature DC are increased at the colonic mucosa from patients with ulcerative colitis (UC). However, a functional relationship between MIF and DC, and the role of MIF in the pathogenesis of UC, are not clear. In this study, we showed that a pure population of peripheral blood DC is a new and still unknown source of MIF. DC from UC patients produced significantly higher levels of MIF (17 x 5 +/- 9 x 8 ng/ml, n = 10) compared with patients with Crohns disease (CD) (4 x 6 +/- 2 x 5 ng/ml, n = 5, P< 0 x 01) and control subjects (5 x 0 +/- 2 x 6 ng/ml, n = 10, P< 0 x 01). A double immunofluorescence study revealed the expression of MIF by CD83-positive mature DC at the colonic mucosa from UC patients. Blood DC treated with high amounts of MIF (500 ng/ml) showed a significantly higher stimulatory capacity (43287 +/- 5998 CPM, n = 5) in an allogenic mixed leucocyte reaction compared with untreated DC (27528 +/- 8823 CPM, n = 5, P< 0 x 05). Study of intracellular cytokine expression showed that MIF induced significant levels of interleukin (IL)-1 and IL-8 in monocytes and DC from UC and CD patients. These results showing the capacity of MIF to induce increased functional capacity of DC, and to produce IL-1 and IL-8 from monocytes and DC, indicate a role of MIF in the induction and/or perpetuation of the inflammatory environment in UC.     

Adhesion molecules utilized in binding of intraepithelial lymphocytes to human enterocytes

The expression of adhesion molecules by human duodenal intraepithelial lymphocytes (IEL) was examined by two-color flow cytometry. Resting IEL expressed LFA-1, HML-1, CD44. Stimulation with phytohemagglutinin (PHA) resulted in down-regulation of expression of these molecules with induction of expression of ICAM-1 and VLA-4. VLA-4 expression was also found on non-activated IEL from patients with celiac disease. In addition, IEL expressed an antigen recognized by a novel monoclonal antibody D2.1. The molecular mass of D2.1 is heterogenous: 82 kDa in peripheral blood lymphocytes and 44 kDa in an IEL line. Expression of this antigen was also up-regulated by PHA. To determine the involvement of these antigens in binding of IEL to human enterocytes, we developed a system based on adherence of an IEL cell line to the I407 fetal intestinal cell line. Monoclonal antibodies VLA-4, D2.1 and to a lesser extent ICAM-1 blocked adherence of IEL to I407 cells. These data suggest that VLA-4 and D2.1 may be involved in adherence of IEL to human enterocytes or secreted matrix molecules in vivo.     

Bovine gut-associated lymphoid tissue--morphologic and functional studies. I. Isolation and characterization of leukocytes from the epithelium and lamina propria of bovine small intestine

A successful technique for the isolation of highly pure suspensions of viable leukocytes from the small intestine of cattle is described. Procedures ranging from mechanical mincing to enzymatic digestion of tissues were compared. The most reliable and reproducible procedure was the sequential treatment of tissues with dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA) in calcium-magnesium-free salt solutions, and collagenase. Two populations of mucosal leukocytes were obtained from the small intestine. One population was derived from within the epithelium (intraepithelial leukocytes, IEL), the second from within the lamina propria (lamina propria leukocytes, LPL). At least 2 X 10(6) viable leukocytes were obtainable from each square centimeter of the intestinal mucosa from either the epithelium or lamina propria. Erythrocyte rosetting and immunofluorescence characterization with conventional antisera and monoclonal antibodies (MAB) demonstrated that IEL were predominantly T cells (60%), with relatively few B cells present (10%), while LPL contained relatively high numbers of B cells (28%) and a reasonable percentage of T cells (45%). Both cell populations proliferate in response to stimulation with T and B cell mitogens. Addition of the thiol compound, 2-mercaptoethanol (2-ME) strongly augmented the mitogenic response of both cell isolates. Human recombinant interleukin-2 (hr-IL-2) in the presence or absence of additional stimuli was found to be able to induce the proliferation of both cell types. These results demonstrate that functional leukocytes can be isolated from the small intestine of cattle, and that they can maintain their responsiveness to both T and B cell mitogens and to exogenous cloned IL-2.     

The proportion of Th1 cells, which prevail in gut mucosa, is decreased in inflammatory bowel syndrome

T lymphocytes and their cytokines have an important role in the regulation of immune responses in the gut and in the pathogenesis of intestinal inflammation such as in Crohn's disease. The aim of this study was to analyse the Th1/Th2 cytokine profile (IFN-gamma, IL-2, IL-4 and IL-10) in intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) in Crohn's disease (CD) and ulcerative colitis (UC) in relation to healthy controls (C). Colonic and ileal biopsy specimens were obtained from controls (n = 13) and patients with CD (n = 32). Colonic biopsies were obtained from patients with UC (n = 11). Intracytoplasmic IFN-gamma, IL-2, IL-4 and IL-10 were determined by flow cytometry after PMA-ionomycin stimulation in IEL and LPL. In colonic LPL, a significant proportional decrease of IFN-gamma and IL-2 producing CD3+ cells was observed in patients with CD and UC compared to controls. In ileal LPL, a similar tendency was found although differences were not significant. In IEL no differences in cytokine profiles could be observed. Flow cytometric analysis of intracytoplasmic cytokines at single cell level showed a proportional decrease of IFN-gamma and IL-2 producing T cells in colonic lamina propria in patients with inflammatory bowel disease.     

Regional Specialization of Intraepithelial T Cells in the Murine Small and Large Intestine

We investigated intraepithelial T cells from the small intestine, SI (jejunum, ileum) and the large intestine, LI (colon) of euthymic (BALB/c, H–2^d; C. B–17 +/+, H–2^d; C57BL/6, H–2^b) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/xid) mice. From individual euthymic and athymic mice, 7 × 10⁶ intraepithelial lymphocytes (IEL) per mouse were isolated from the SI. Ten–fold lower numbers of IEL were obtained from the LI epithelium (4 × 10⁵ IEL per mouse). Thymus–dependent and -independent T cells represented > 80% of SI–IEL but the fraction of T cells was reduced from 20% to 40% in LI–IEL. In euthymic mice, αβ T cells predominated in SI–IEL and in particular in LI–IEL populations, while SI–IEL and LI-IEL populations of athymic mice contained predominantly αβ T cells. The intraepithelial T cell subset distribution was different in SI versus LI: mainly CD8⁺ T cells were present in the SI, but a large CD4⁺ T cell subset was present in the LI.‘Double positive’ CD4⁺ CD8α⁺ T cells were present mainly in the SI epithelium but were rare in the LI epithelium. In euthymic as well as athymic mice, T cells expressing the homodimeric CD8αβ isoform predominated in the SI epithelium, while T cells expressing the heterodimeric CD8αβ isoform predominated in the LI epithelium. LI–derived TCRα⁺ IEL displayed the CD2⁺ CD28⁺ LPAM–1/2^? M290⁺ phenotype, and a fraction of them expressed the L–selectin LECAM–1. In contrast, a large fraction of TCRα⁺ SI-IEL was CD2^? CD28^? LPAM–1/2^? M290⁺ and LECAM–1^?. RAG–1/2 expression was detectable by RT–PCR in IEL from the SI but not the LI. Striking differences in phenotype were thus apparent between thymus–dependent and thymus–independent T cells in the epithelial layer of the jejunum/ileum and the colon of the mouse.     

An improved method for isolating intraepithelial lymphocytes (IELs) from the murine small intestine with consistently high purity

Methods for obtaining preparation of intestinal intraepithelial lymphocytes (IELs) present special challenges for immunologists due to difficulties in recovering IELs devoid of contaminating enterocytes. Although high-purity preparations can be achieved using techniques such as flow cytometric or magnetic-activated cell sorting, those methods may not be feasible on a routine basis and may result in low overall cell recoveries. Thus, most procedures today rely on density gradient centrifugation as a means of separating IEL and non-hematopoietic cells; however, the purity of IELs from those preparations can vary considerably. Here, we describe a modification of an IEL purification technique that uses two sequential Percoll gradients rather than one gradient in the purification scheme. This alteration consistently results in 80-85% IEL purity in cell preparations. Moreover, it requires no additional reagents, has no adverse effect on the phenotypic composition of recovered IELs or on the cell viability, and adds minimal additional time to the isolation protocol. It is expected that this procedure will have practical benefit as a means of isolating IELs with high purity on a routine basis that can be used for in vivo or in vitro studies of IEL function.     

Indium-labelled human gut-derived T cells from healthy subjects with strong in vitro adhesion to MAdCAM-1 show no detectable homing to the gut in vivo

Integrin alpha4beta 7 is the principal gut-homing receptor, and it is assumed that expression of this specific integrin directs lymphocytes to the gut in vivo. Adoptive cellular immunotherapy against inflammatory bowel disease (IBD) may depend on the expression of integrin alpha4beta 7 to accomplish local delivery of intravenously injected regulatory T cells in inflamed gut mucosa. The present study aimed to investigate whether in vitro expanded human T cells from the colonic mucosa maintain integrin expression, show in vitro adhesion and retain in vivo gut-homing properties during cultivation. Whole colonic biopsies from healthy subjects were cultured in the presence of interleukin-2 (IL-2) and IL-4. The integrin expression of the cultured T cells was determined by flow cytometry and in vitro adhesion was assessed in a mucosal addressin cell adhesion molecule 1 (MAdCAM-1) adhesion assay. We studied the homing pattern after autologous infusion of 3 x 10(8 111)Indium ((111)In)-labelled T cells in five healthy subjects using scintigraphic imaging. The cultured CD4(+)CD45RO(+) gut-derived T cells express higher levels of integrin alpha4beta 7 than peripheral blood lymphocytes (PBLs) and show strong adhesion to MAdCAM-1 in vitro, even after (111)In-labelling. Scintigraphic imaging, however, showed no gut-homing in vivo. After prolonged transit through the lungs, the T cells migrated preferentially to the spleen, liver and bone marrow. In conclusion, it is feasible to infuse autologous T cells cultured from the gut mucosa, which may be of interest in adoptive immunotherapy. Despite high expression of the gut-homing integrin alpha4beta 7 and adhesion to MAdCAM-1 in vitro, evaluation by (111)In-scintigraphy demonstrated no gut-homing in healthy individuals.     

A rapid method for isolating murine intestine intraepithelial lymphocytes with high yield and purity.

Because murine intestine intraepithelial lymphocytes (IEL) are dispersed throughout the intestine epithelium, it is important that IEL extraction procedures result in lymphocyte preparations of sufficient purity for use in in vitro and in vivo experimental systems. Here, we describe an improved technique for isolating murine IEL consisting of a single 30 min extraction followed by multiple nylon wool filtrations and centrifugation through Percoll. This procedure yields a preparation of IEL with high overall recovery and purity yet takes only 2-2.5 h. Evaluation of individual steps in the extraction process indicated that nylon wool filtration, in particular multiple filtrations, and Percoll fractionation both were important for achieving highly-enriched IEL populations by removal of enterocytes and cellular debris, and demonstrated that multiple nylon wool filtration improved the overall IEL recovery. This procedure has several advantages for studies of murine IEL in that the resultant IEL population is ideal for phenotypic, functional, or molecular analyses. Moreover, this technique is effective for isolating IEL on a single-animal basis, thereby permitting analyses of IEL from individual mice rather than as pooled IEL obtained from several animals.     

Reliable isolation of human immunodeficiency virus from cultures of naturally infected CD4+ T cells

A procedure for reliable and reproducible isolation of human immunodeficiency virus (HIV) from cultures of CD4+ T cells from healthy HIV seropositive individuals is described. Using immunomagnetic cell separation techniques, CD4+ T cells were positively selected from blood or peripheral blood mononuclear cells from 34 HIV infected individuals in CDC group II. The cells were stimulated with beads coated with a monoclonal antibody specific for the T cell receptor alpha/beta dimer and cultured in medium containing recombinant IL 2. HIV was isolated from 100% of the 41 cultures from 34 individuals. As this culture system allows reproducible isolation of HIV from cultures of naturally infected CD4+ T cells in the absence of other autologous or allogeneic cells, it may provide a good test system for the study of factors affecting the replication of HIV at low multiplicities of infection.