A microbiota-derived antigen drives CD4+ intraepithelial lymphocyte (CD4IEL) development

CD4⁺ intraepithelial lymphocytes (CD4IEL) are tissue-resident T cells with cytotoxic and regulatory properties; together with peripheral regulatory T cells, they control intestinal inflammation. Recently, Bousbaine and colleagues identified a microbiota-derived conserved antigen that induces CD4IEL differentiation and promotes their regulatory function, attenuating the severity of murine colitis.     

Listeria monocytogenes-induced gamma interferon secretion by intestinal intraepithelial gamma/delta T lymphocytes.

gamma/delta T cells represent a major proportion of intestinal intraepithelial lymphocytes (IEL), and it has been suggested that these IEL serve as a first immune barrier against microbial invasion and that they do so by destroying infected epithelial cells. In the present study, we confirm that both alpha/beta and gamma/delta IEL from naive mice express potent cytotoxicity and produce gamma interferon (IFN-gamma) after T-cell receptor (TCR) engagement by specific monoclonal antibodies (MAb). Intraperitoneal administration of the anti-gamma/delta TCR MAb GL3 caused downregulation of the gamma/delta TCR in IEL, and IEL from gamma/delta TCR-modulated mice failed to express cytotoxic activity and to secrete IFN-gamma after gamma/delta TCR engagement. In contrast, alpha/beta IEL from such mice were still cytolytic and secreted IFN-gamma. Mice were infected orally with virulent Listeria monocytogenes at doses which caused bacterial invasion through the intestinal epithelia. Although alpha/beta and gamma/delta IEL from these mice expressed high cytolytic activities in antibody-redirected killer assays, target cells pulsed with listerial antigens were not lysed. In contrast, IFN-gamma secretion by IEL from L. monocytogenes-infected mice was induced not only by anti-TCR MAb but also by target cells pulsed with listerial antigens, whereas irrelevant antigens, including heat shock protein 60, did not induce IFN-gamma secretion. Furthermore, the number of IFN-gamma-secreting IEL, as assessed by the enzyme-linked immunospot technique, was increased during listeriosis. gamma/delta TCR modulation by GL3 administration abrogated antigen-induced IFN-gamma secretion by IEL from infected mice. These findings suggest that L. monocytogenes induced IFN-gamma secretion by gamma/delta IEL from mice suffering from intestinal L. monocytogenes infection and invasion. Thus, the data provide evidence for a role of IFN-gamma-secreting IEL in local resistance against listeriosis and perhaps other food-borne diseases.     

Lymphokine secretion and proliferation of intraepithelial lymphocytes from murine small intestine.

Compared to other peripheral lymphocytes, intestinal intraepithelial lymphocytes (IEL) have previously been shown to have low proliferative capabilities. However, there are two main populations of IEL in the small intestine of mice. First, there is the thymus-dependent CD3+,Thy-1+ population, most of which expresses the alpha beta T-cell receptor (TcR), and second there is the thymus-independent CD3+,Thy-1- population, most of which expresses the gamma delta TcR. In this study Thy-1-enriched and Thy-1-depleted lymphocytes from murine intestinal epithelium were studied separately for their ability to proliferate and secrete lymphokines in vitro after mitogenic stimulation, after stimulation via the TcR-CD3 complex and after stimulation with the superantigen Staphylococcus aureus B (SEB). Here we show that Thy-1-enriched IEL are not an immunocompromised population of cells but are functionally competent T cells that are capable of proliferation and lymphokine secretion after stimulation with concanavalin A (Con A), phorbol myristate acetate (PMA) and anti-CD3 monoclonal antibody (mAb). Furthermore, Thy-1-enriched IEL proliferate and secrete lymphokines after 'superantigenic' stimulation with SEB. In contrast, the majority of Thy-1-depleted IEL do not proliferate, and secrete only minimal levels of lymphokine to any of the stimuli tested in this study.     

Cytokine synthesis and apoptosis by intestinal intraepithelial lymphocytes: Signaling of high density αβ T cell receptor+ and γδ T cell receptor+ T cells via T cell receptor-CD3 complex results in interferon-γ and interleukin-5 production,while low density T cells undergo DNA fragmentation

To study the biological consequences of cytokine production and apoptosis by intraepithelial lymphocytes (IEL), we have studied these characteristics in both the high and low density CD3⁺ IEL populations. Stimulation of low- or high-density CD3⁺ IEL via the T cell receptor (TCR)-CD3 complex using monoclonal anti-CD3, anti-αβ TCR or anti-γδ TCR antibodies resulted in opposing effects. In one case, a significant number of the high-density CD3⁺ T cells entered cell cycle from the resting stage (DNA replication was observed) and anti-TCR-CD3 treatment enhanced the numbers of interferon-γ and interleukin-5 spot-forming cells in this cell fraction. In contrast, when the low-density αβ TCR⁺ or γδ TCR⁺ T cells were activated via the TCR-CD3 complex, DNA fragmentation was observed. These results demonstrated that the activation signals transduced via the TCR-CD3 complex resulted in their entry into the cell cycle and subsequent interferon-γ and interleukin-5 production in the high-density IEL T cell subset. However, identical signals induced apoptosis in the majority of the low-density fraction of CD3⁺ IEL.     

Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level. In the present study the response of purified CD4⁺ and CD8⁺ T lymphocytes to stimulation with anti-TCR monoclonal antibody (the first signal) in combination with anti-CD4 or anti-CD8, anti-CD2 and anti-CD28 MoAbs (the costimulatory signals) was investigated. Both CD4⁺ and CD8⁺ T cells from the patients showed significantly reduced IL-2 release following stimulation via TCR and costimulation via CD4 or CD8 and CD2, respectively. However, normal IL-2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker, CD69, after TCR + CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL-2 and IL-4 release were impaired in CD4⁺ lymphocytes following TCR + CD28 stimulation. In addition, stimulation via TCR + CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule, or restricted to cells producing Thl-type cytokines such as IL-2, and is expressed in both CD4⁺ and CD8⁺T cell subsets.     

Differential effects of chlorination of bacteria on their capacity to generate NO, TNF-alpha and IL-6 in macrophages.

The phenotype and function of murine intestinal intraepithelial lymphocytes (IEL) was studied in a Percoll-fractionated preparation that consisted of low-density cells which migrated to the 40-50% Percoll interface (IEL-40), medium-density cells which migrated to the 50-55% interface (IEL-50), and high-density cells which migrated to the 55-70% interface (IEL-55). IEL-40 and IEL-50 cells, the subsets phenotypically most similar to mature IEL, consisted of CD3+ T cells that included CD4- CD8+ and CD4+ CD8+ cells; CD4+ CD8- cells were present only in the IEL-50 fraction. T-cell receptor (TcR)alpha beta and TcR gamma delta cells were present in both IEL-40 and IEL-50 fractions. In contrast, most IEL-55 were CD3-, heat-stable antigen (HSA)+ cells that were not B cells; some IEL-55 cells were CD3lo HSA- or CD3lo HSA+ suggesting that IEL-55 are immature T cells. TcR alpha beta but not TcR gamma delta was expressed in the IEL-55 fraction. All three IEL fractions consisted of both CD8 alpha alpha and CD8 alpha beta cells. There was considerable functional heterogeneity between the three IEL fractions such that CD3-induced proliferation was greatest for IEL-50 cells and least for IEL-55 cells; that activity correlated with the proportion of Thy-1+ cells within the fractions. Both IEL-40 and IEL-50 fractions contained activated cytotoxic T lymphocytes (CTL) that were 8-16-fold more lytic than IEL-55 cells. That IEL-55 cells may be precursors of some IEL-40 and IEL-50 cells was demonstrated by a shift in cell density and by an increase in proportions of cells expressing markers of IEL-40 and IEL-50 cells following in vitro stimulation via CD3. The relevance of these findings to differences in functional activities reported for murine IEL is discussed.     

CD3−CD8+ intestinal intraepithelial lymphocytes (IEL) and the extrathymic development of IEL

Present evidence suggests that a majority of murine CD3⁺ intraepithelial intestinal lymphocytes (IEL) are extrathymically derived T cells and that these extrathymically derived IEL phenotypically express the CD8 homodimer (CD8αα). Recently, CD3^? IEL have been reported to express the recombination activating gene (RAG-1), suggesting that precursors to extrathymically derived CD3⁺CD8⁺αα IEL exist on the intestinal epithelium. To study in detail whether these CD3^? IEL can develop into CD3⁺CD8⁺αα IEL, we analyzed the CD3^? IEL subset and found that it can be separated into two subsets, namely CD3^?CD8^? and CD3^?CD8⁺ IEL. We show that (1) CD3^?CD8^? IEL are mostly small, non-granular and phenotypically Pgp-1⁺ IL-2R⁺ B220^?, while CD3^?CD8⁺ IEL are mostly large, granular and phenotypically Pgp-1^? IL-2R⁺ B220⁺, (2) CD3^?-CD8⁺ IEL express the RAG-1 gene, and (3) CD3^?CD8^?, CD3^?CD8⁺ and CD3⁺CD8⁺αα IEL, respectively, appear sequentially in normal ontogeny and in bone marrow-reconstituted thymectomized radiation chimeras. In the latter, virtually all CD3⁺CD8⁺αα IEL expressed the γδ T cell receptor (TCR), but not the αβ TCR. From this and what is presently known about T cell development, we propose that CD3^?CD8⁺ IEL are an intermediate in extrathymic IEL development and that the development of extrathymically derived IEL occurs at the intestinal epithelium from CD3^?CD8^? to CD3^?CD8⁺ to CD3⁺(γδ TCR)CD8⁺αα.     

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.     

Differential effect on TCR:CD3 stimulation of a 90-kD glycoprotein (gp90/Mac-2BP), a member of the scavenger receptor cysteine-rich domain protein family

We studied the effects of a 90-kD glycoprotein (gp90/Mac-2BP) belonging to the scavenger receptor family, present in normal serum and at increased levels in inflammatory disease and cancer patients, on some T cell function parameters. Whereas the lymphocyte proliferative response to non-specific mitogens such as phytohaemagglutinin (PHA) and concanavalin A (Con A), but not pokeweed mitogen (PWM), was strongly reduced, probably due to the lectin-binding properties of gp90/Mac-2BP, the response to T cell receptor (TCR) agonists such as superantigens and allogeneic cells was potentiated. When lymphocytes were stimulated with different anti-TCR:CD3 MoAbs, both in soluble and solid-phase form, gp90/Mac-2BP was able to down-regulate the proliferative response to anti-CD3 MoAb, whereas the response to anti-TCR αβ MoAb was enhanced. A similar differential effect was observed when a MoAb against CD5 (another member of the scavenger receptor superfamily) was added to anti-CD3 or anti-TCR-stimulated cells; anti-CD5 MoAb strongly down-modulated the CD3-mediated response, whereas its presence in culture was associated with potentiation of the response to TCR αβ agonists. gp90/Mac-2BP was able per se to up-regulate Ca²⁺ levels in freshly isolated lymphocytes; moreover, its presence in culture was associated with increased Ca²⁺ mobilization following stimulation with anti-TCR αβ, but not anti-CD3 MoAb. These data indicate that gp90/Mac-2BP could be able to influence some immune responses, possibly through multiple homologous interactions with other members of the scavenger receptor family; moreover, our findings suggest that signalling through the different components of the TCR:CD3 complex may follow distinct activation pathways into the cells.     

Characterization and TCR variable region gene use of mouse resident nasal gammadelta T lymphocytes

Tissue-resident gammadelta T lymphocytes, such as dendritic epidermal T cells, intestinal intraepithelial lymphocytes (IEL), and resident pulmonary lymphocytes, are known to support local tissue homeostasis and host defense. Inhaled antigens, toxins, and microorganisms first interact with the immune system through contact with the nasal mucosa. Herein, we characterized two populations of resident nasal lymphocytes (RNL) that are present in the nasal mucosa: nasal IEL (nIEL) and nasal lamina propria lymphocytes (nLPL). gammadelta TCR+ and alphabeta TCR+ nIEL and nLPL were detected by immunofluorescent staining. Mononuclear cells (5-15%) were CD3+ RNL by FACS analysis. Among the CD3+ RNL, 20-30% were GL3+ gammadelta T cells, which were double-negative for CD4 and CD8 and predominantly expressed a Vgamma4/Vdelta1 TCR. These results demonstrate that RNL might be crucial for the host defense and tissue homeostasis in the nasal mucosa.     

Regional variation in the proliferative rate and lifespan of alpha beta TCR+ and gamma delta TCR+ intraepithelial lymphocytes in the murine small intestine.

Using double staining for T-cell receptor (TCR) and 5-bromo-2'-deoxyuridine (BRdU) we have examined the proliferation rates and lifespan of murine intraepithelial lymphocytes (IEL's) in vivo. After a 24-hr pulse of BRdU the number of labelled alpha beta TCR+ IEL was significantly higher in the ileum than the duodenum. In contrast, incorporation of BRdU into gamma delta TCR+ IEL was significantly higher in the duodenum than the ileum. This regional variation was also seen after a 4-hr pulse of BRdU indicating that the differences probably reflect local rates of proliferation in the epithelium. Over a 6-day labelling period, the accumulation of labelled alpha beta TCR+ and gamma delta TCR+ IEL was linear, which allowed IEL lifespan to be calculated. There was considerable variation between groups of mice but the 50% population renewal time for alpha beta TCR+ IEL was 12-36 days in the duodenum and 9-11 days in the ileum, and for gamma delta TCR+ IEL was 12-21 days in the duodenum and 26-100 days in the ileum. The incorporation of BRdU into V beta 8+ IEL showed the same regional variation as alpha beta TCR+ IEL and the V delta 4 population behaved like the total gamma delta TCR+ IEL population. In contrast V beta 11+, potentially self-reactive IEL, showed a regional pattern of labelling like gamma delta TCR+ IEL. Incorporation of BRdU into both alpha beta TCR+ and gamma delta TCR+ IEL in germ-free mice was very low and did not show marked regional variation. alpha beta TCR+ and gamma delta TCR+ IEL from both proximal and distal bowel were cytotoxic. Therefore alpha beta TCR+ and gamma delta TCR+ IEL show different rates of division in different sections of the gut, perhaps reflecting responses to different antigens. Both alpha beta TCR+ and gamma delta TCR+ IEL reside in the epithelium for weeks during which time the gut epithelial population will have been renewed many times.     

Increased division of alpha beta TCR+ and gamma delta TCR+ intestinal intraepithelial lymphocytes after oral administration of cholera toxin.

Cholera toxin (CT) or its subunits were given orally to mice and division of intestinal intraepithelial lymphocytes (IEL) in vivo measured by double immunofluorescence using 5-bromo-2'-deoxyuridine (BRdU) and membrane alpha beta T-cell receptors (TCR) or gamma delta TCR staining in frozen sections. Cholera toxin (10 micrograms) produced a two- to eightfold-increase in the uptake of BRdU in alpha beta TCR+ IEL in the duodenum and a two-to fivefold increase in gamma delta TCR IEL in the ileum. Increased uptake of BRdU was also seen after a dose of 100 micrograms of CT but this dose was also associated with the loss of alpha beta TCR+ IEL and gamma delta TCR+ IEL in the duodenum. CT-A and CT-B subunit produced increased BRdU incorporation by alpha beta TCR in the duodenum and by gamma delta TCR IEL in the ileum. Cholera toxin therefore appears to be mitogenic for IEL probably due to an indirect mechanism.     

Induction of human TCR gamma delta + and TCR gamma delta-CD2+CD3- double negative lymphocytes by bacterial stimulation

When human blood mononuclear cells (MNC) were incubated with heat-killed bacteria, proliferation of MNC was observed 5 days after stimulation, showing a peak on day 7. Interestingly, the bioassay of the culture supernatant and Northern blot analysis of mRNA demonstrated that no IL-2 production was associated with these proliferative responses. The induced lymphoblasts consisted predominantly of TCR gamma delta + (22.4 +/- 9.3%) and TCR gamma delta-CD2+CD3-(33.2 +/- 11.8%) double negative lymphocytes (n = 10), which were initially minor populations (less than 10%) in freshly isolated MNC. The prominent induction of TCR gamma delta + cells was confirmed by Northern blot analysis. TCR gamma delta + cells induced by bacterial stimulation seemed to generate from lymphocytes lacking the apparent expression of gamma delta TCR. The inducing capability for double negative cells is present in a large number of species of bacteria, especially Gram-positive bacteria. Gel filtration analysis of ultrasonicated filtrates of Staphylococcus aureus and Streptococcus pyogenes revealed that a substance with an Mr of 25-26 kd could be substituted for whole bacterial particles in the cell proliferative responses. In contrast to the purified protein derivative (PPD)-induced response, the response described here was inducible in the cord blood of neonates who had not yet been exposed to the corresponding bacterial infection. The physicochemical properties of the sonicated filtrates were different from those of PPD. These results suggested that the present phenomenon may be nonspecific, polyclonal (or oligoclonal) activation of TCR gamma delta + and TCR gamma delta -CD2+CD3- cells by bacterial stimulation.     

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.     

Expression of the alpha/beta and gamma/delta T-cell receptors in 57 cases of peripheral T-cell lymphomas. Identification of a subset of gamma/delta T-cell lymphomas.

Fifty-seven cases of peripheral T-cell lymphoma were studied for cell expression of the T-cell receptor (TCR) chains, using monoclonal antibodies specific for the beta chain (beta F1) of the alpha/beta TCR, and for the delta chain (anti-TCR delta-1) of the gamma/delta TCR. Three different patterns were demonstrated: in 39 cases (69%), the phenotype (CD3+beta F1+TCR delta-1-) was that of most normal T cells. A second pattern was found on six cases (10%), which were of CD3+beta F1-TCR delta-1+ phenotype, and in which DNA analysis showed a clonal rearrangement of the delta locus in the five cases studied. It is suggested that these cases are the neoplastic counterpart of the small subpopulation of normal T cells that express gamma delta receptor. It is of considerable interest that these gamma delta lymphomas had unusual clinicopathologic presentations, as one case corresponded to a lethal midline granuloma and the five others to hepatosplenic lymphomas with a sinusal/sinusoidal infiltration in spleen, marrow, and liver. The fact that the distribution of the neoplastic gamma delta cells in the splenic red pulp resembles that of normal gamma delta cells reinforces the concept of a preferential homing of gamma delta T cells to this tissue. A third pattern (CD3 +/- beta F1-TCR delta-1-) was seen in 12 cases (21%), in which, by contrast to normal post-thymic T cells, no evidence of either alpha beta or gamma delta T cell receptor was found.     

Staphylococcal enterotoxin B induces potent cytotoxic activity by intraepithelial lymphocytes

In food poisoning, Staphylococcus aureus secretes staphylococcal enterotoxin B (SEB), a superantigen that causes intense T-cell proliferation and cytotoxicity. The effects of SEB on lytic activity by human intestinal intraepithelial lymphocytes (IEL) were investigated. Jejunal IEL, from morbidly obese individuals undergoing gastric bypass operations, were tested for SEB-induced cytotoxicity against C1R B-lymphoblastoid cells, HT-29 adenocarcinoma cells, or CD1d-transfected cells using the 51Cr-release assay. Fas and Fas ligand expression were detected by immunofluorescence and flow cytometry and soluble ligand by enzyme-linked immunosorbent assay (ELISA). In the presence of SEB, IEL became potently cytotoxic against C1R cells and interferon-gamma (IFN-gamma)-precultured HT-29 cells, causing 55+/-10% and 31+/-6% lysis, respectively, greater than that by phytohaemagglutinin (PHA)-, interleukin-2 (IL-2)-, or anti-T-cell receptor (TCR)-activated IEL. SEB-stimulated peripheral blood (PB) CD8+ T cells lysed similar numbers of C1R cells but fewer HT-29 cells (53+/-13% and 8+/-5%, respectively). IEL killing of C1R cells involved interaction of major histocompatibility complex (MHC) class II with TCR, CD2 with CD58, and CD11a with CD54, and was perforin mediated. SEB-induced IEL lysis of HT-29 cells, in contrast, was caused by an unknown target cell structure, not MHC class II or CD1d, and resulted from a combination of perforin and Fas-mediated events. The potent cytotoxic activities of IEL promoted by SEB utilize two different mechanisms, depending on the surface receptors expressed by the target cells.     

Cytotoxic activity against tumour cells mediated by intermediate TCR cells in the liver and spleen.

Morphological and phenotypic characterization in previous studies has indicated that intermediate (int) T-cell receptor (TCR) cells or T natural killer (TNK) cells may stand at an intermediate position between NK cells and high TCR cells of thymic origin in phylogenetic development. In this study, a functional study on cytotoxic activity against various tumour targets was performed in each purified subset. When a negative selection method entailing in vivo injection of anti-asialo GM, antibody or anti-interleukin (IL)-2R beta monoclonal antibody (mAb) was applied, IL-2R beta 1 CD3 NK cells were found to have the highest NK activity while IL-2R beta 1 int CD3 (or TCR) cells had a lower level of the NK activity. High CD3 cells (freshly isolated) did not have any such activity. Sorting experiments further revealed that the NK function mediated by int CD3 cells was augmented when they were exposed to anti-CD3 mAb. anti-TCR alpha beta, or anti-TCR-delta mAb. This phenomenon was not observed in NK cells and high CD3 cells. More importantly, when anti-CD3 mAb (or anti-TCR mAb) was added to the assay culture, int CD3 cells became cytotoxic against even NK-resistant tumour (Fc gamma R-. Fas+) targets. Liver mononuclear cells or int CD3 cells exposed to anti-CD3 mAb for 6 hr showed an elevated level of perforin in their cytoplasms. The present results suggest that int CD3 cells are usually non-cytotoxic against various tumours but become functional after being stimulated via the TCR CD3 complex.     

Effect of hypertonicity and monensin on CD3/TCR surface expression in human T cells

Pretreatment of T lymphocytes with anti-CD3 or anti-TCR mAb results in the disappearance of the T cell receptor complex (TCR/CD3) from the cell surface. The mechanisms of this down-regulation have not been fully elucidated. We demonstrate here that the modulation of the CD3/TCR complex can be completely inhibited by hypertonic medium, a condition known to block receptor-mediated endocytosis. Consequently, the sequestration of the CD3/TCR complex associated to relevant mAb in acid vesicles did not occur under hypertonicity condition. Moreover, monensin, which is known to interfere with receptor recycling, was found to amplify the CD3/TCR modulation induced by specific mAb and decrease the uptake of 125I-labeled anti-CD3/TCR mAb by T cells. We therefore propose that mAb-CD3/TCR complexes are internalized via coated pits by a receptor-mediated endocytosis and then transported to acid compartments. MAb are degraded in lysosomes during this process, whereas CD3/TCR molecules recycle back to cell surface.