Pulmonary epithelial T cells induced by viral infection express T cell receptors alpha/beta.

Cells were recovered from the lungs of mice by bronchoalveolar lavage before and after infection with respiratory syncytial (RS) virus. Uninfected mice yielded mostly macrophages, but after RS virus infection lymphocytes also appeared. Recovered cells were stained for CD4, CD8' and either CD3, T cell receptor (TcR) alpha/beta or TcR gamma/delta and analyzed by 3-color flow cytometry. Both the CD4+CD8- and CD4-CD8+ subpopulations stained uniformly for CD3 and TcR alpha/beta, while none stained for TcR gamma/delta. Of the CD4-CD8- cells, about 5% stained for CD3 and TcR alpha/beta during the first week after infection, although this figure increased during the second week. A small and variable fraction of this subset stained for TcR gamma/delta. These results oppose the view that lymphocytes expressing TcR gamma/delta predominate in initial epithelial immune responses to viral infections.     

Subsets of CD3+ (T cell receptor alpha/beta or gamma/delta) and CD3- lymphocytes isolated from normal human gut epithelium display phenotypical features different from their counterparts in peripheral blood

Intestinal intraepithelial lymphocytes (IEL) were studied, after isolation in humans, for their surface antigens with a large variety of monoclonal antibodies. They show peculiar characteristics when compared with peripheral blood lymphocytes and intestinal lamina propria lymphocytes. Although a majority of human intraepithelial lymphocytes (IEL) express an alpha/beta type of T cell receptor (TcR), 13% express a gamma/delta TcR, a percentage which was significantly higher than that found in blood and in lamina propria. In contrast to observations in mice, there was no evidence that normal human TcR gamma/delta+ intestinal IEL might use preferential variable segments of gamma genes. About 10% of human intestinal IEL expressed the alpha chain but not the beta chain of CD8, thus resembling a subset of CD8 alpha+beta- IEL, which was recently described in mice and found to be of thymoindependent origin. In addition, 10% of human IEL had a unique phenotype of immature T cells, as they bore only CD7, but no other T cell or natural killer cell markers. Finally, even the major population of IEL which expressed the usual markers of the T cell lineage (CD3, TcR alpha/beta, CD2, CD4 or CD8 alpha/beta) differed from peripheral blood T lymphocytes by their peculiar expression of surface antigens associated with activation. Indeed, 80% of IEL were CD45R0+, CD45A-, but co-expression of CD11a, CD29 and LFA-3 was inconstant. In addition, 90% of IEL expressed HML-1.     

Functional expression of CD28 on T cell antigen receptor gamma/delta-bearing T lymphocytes

T lymphocytes express a T cell antigen receptor (TcR) complex composed of either an TcR alpha/beta or TcR gamma/delta heterodimer in noncovalent association with the CD3 glycoproteins. CD28, a 44-kDa disulfide-linked homodimer, is present on the surface of the majority of TcR alpha/beta-bearing T lymphocytes. Monoclonal antibodies (mAb) directed against CD28 potentiate activation signals delivered through the CD3/TcR alpha/beta complex. Herein, we demonstrate that CD28 is expressed on approximately 40%-60% of TcR gamma/delta-bearing T lymphocytes in most donors. Anti-CD28 mAb substantially augmented proliferative signals delivered through the TcR gamma/delta, demonstrating the presence of functional CD28 molecules on TcR gamma/delta-bearing T lymphocytes. The majority of TcR gamma/delta+ thymocytes also expressed CD28.     

CD8 alpha-deficient mice are highly susceptible to 5-fluorouracil-induced lethality

Intestinal intraepithelial lymphocytes (i-IEL) expressing CD8 alpha are located in the intestine and may confer protection against invasion of intestinal microflora. We found that mice rendered deficient in CD8 alpha molecules by homologous recombination were susceptible to 5-fluorouracil (5-FU)-induced lethality accompanied by translocation of members of the enterobacteria. The number of i-IEL was greatly reduced on day 6 after 5-FU administration in both CD8 alpha(+/-) mice and CD8 alpha(-/-) mice, whereas the recovery of the level of i-IEL thereafter was significantly impaired in CD8 alpha(-/-) mice compared with that in CD8 alpha(+/-) mice. The ability of i-IEL to produce gamma interferon in response to immobilized T-cell receptor (TCR) alpha beta or TCR gamma delta monoclonal antibodies was significantly lower in CD8 alpha(-/-) mice than in CD8 alpha(+/-) mice. Transfer of CD8(+) i-IEL conferred significant protection against 5-FU-induced lethality in CD8 alpha(-/-) mice. The results suggest that CD8(+) i-IEL play an important role in protection against 5-FU-induced lethality with translocation of Enterobacteriaceae.     

Phenotype of intraepithelial lymphocytes in euthymic and athymic mice: implications for differentiation of cells bearing a CD3-associated gamma delta T cell receptor

This study was carried out to determine the exact phenotype of intraepithelial lymphocytes (IEL) in euthymic and athymic nude mice. The phenotype of IEL in euthymic and athymic mice is mainly CD3+CD8+. However, based on Thy-1- and CD3-associated receptor expression we can subdivide the CD3CD8 population into different subpopulations in euthymic and athymic mice. In euthymic and athymic mice several CD3CD8 populations can be defined. One population expressing Thy-1 and the T cell receptor (TcR) alpha beta is absent in athymic mice. Two other CD3+CD8+ populations can be detected in euthymic and athymic mice. Based on Northern blot and flow cytometric analysis we have to conclude that these populations express the CD3-associated TcR gamma delta. One of the TcR gamma delta-expressing populations also expresses Thy-1 at low surface density. This is in contrast to the CD3CD8 population expressing the TcR alpha beta, which expresses Thy-1 at high surface density. There are also, however, especially in athymic nude mice, significant numbers of CD3-CD8+ cells present with the same localization as IEL. The function of these cells is yet unknown. Using a probe for the delta chain we have shown that IEL preferentially express 2-kb mRNA, while nearly no delta chain 1.7-kb mRNA is expressed by these cells. This is in contrast to delta mRNA in thymocytes. Equal quantities of the 1.7- and 2.0-kb delta chain mRNA species were found in RNA isolated from thymocytes. The results imply that CD3+CD8+ intestinal IEL expressing the CD3-associated TcR gamma delta can differentiate in absence of the thymus and represent a thymus-independent lineage of cells bearing this receptor.     

Selective expression of CD8 alpha (Ly-2) subunit on activated thymic gamma/delta cells

Although most cells of the T cell receptor (TcR) gamma/delta lineage are CD4-CD8-, some gamma/delta cells express CD8. We show here that mouse thymic gamma/delta cells express CD8 following in vitro activation by concanavalin A. Staining with monoclonal and polyclonal antibodies to the CD8 alpha (Ly-2) and CD8 beta (Ly-3) subunits indicates that only CD8 alpha is expressed by these activated TcR gamma/delta cells. Furthermore, immunoprecipitation and reduced/nonreduced gel analysis reveals that thymic gamma/delta cells express CD8 alpha as a homodimer. In contrast, similarly activated TcR alpha/beta cells express a conventional CD8 alpha/CD8 beta heterodimer.     

Patterns of membrane TcR alpha beta and TcR gamma delta chain expression by normal blood CD4+CD8-, CD4-CD8+, CD4-CD8dim+ and CD4-CD8- lymphocytes.

Enriched CD4+CD8-/CD4-CD8-, CD4-CD8+/CD4-CD8- and CD4-CD8- cell suspensions were prepared from normal peripheral blood by selective immunomagnetic depletion of monoclonal antibody-defined lymphocyte populations. Subsequent examination of these modified cell fractions by two-colour flow cytometry provided a means of determining the expression of membrane T-cell receptor (TcR)alpha beta and TcR gamma delta chains by both major (CD4+ and CD8+) and minor (CD3+CD4-CD8dim+ and CD3+CD4-CD8-) lymphocyte subpopulations. Normal CD4+CD8- lymphocytes were almost invariably (greater than 99%) TcR alpha beta+, whereas lymphocytes expressing membrane CD8, which could be further subdivided according to differences in fluorescent staining intensity into CD3+CD4-CD8+, CD3+CD4-CD8dim+ and CD3-CD4-CD8dim+ components, were characterized by distinct differences in patterns of TcR chain expression. In contrast to CD3+CD4-CD8+ cells, which were predominantly (99%) TcR alpha beta+, CD3+CD4-CD8dim+ lymphocytes showed a significant proportion (33%) of TcR gamma delta+ cells (natural killer-associated CD3-CD4-CD8dim+ cells were uniformly TcR-). The highest proportion (62%) of TcR gamma delta+ cells was associated with the CD3+CD4-CD8- fraction, but these studies also revealed that a significant minority of this population was TcR alpha beta+. Despite some evidence for normal inter-individual variation, further analysis of membrane CD8 fluorescent intensities confirmed clear differential relationships for TcR alpha beta and TcR gamma delta chain expression.     

Phenotypical and Functional Characterization of Small Intestinal TcRγδ+ T Cells in Coeliac Disease

Increased numbers of TcR gamma delta + T cells are present in the small intestinal epithelium of patients with coeliac disease (CoD). Their function, however, is unknown. In order to facilitate detailed functional studies, intestinal gamma delta T cells have been isolated from small intestinal biopsies of patients with CoD (n = 18) and controls (n = 14). As expected, increased numbers of V delta 1+ TcR gamma delta + T cells were detected in freshly isolated intraepithelial cell suspensions (IEL) from CoD patients. Also, in the in vitro expanded IEL T-cell populations from CoD patients the numbers of V delta 1+ TcR gamma delta + T cells were increased compared with similar cell cultures from control patients. From IEL cultures derived from six CoD patients, 107 T-cell clones were generated by limiting dilution and analysed. Sixty of these clones were either CD4 or CD8 positive TcR alpha beta + clones. The remaining 47 clones expressed the TcR gamma delta. Further phenotypical analysis of the gamma delta T-cell clones indicated that the TcR gamma delta + T-cell population in the small intestinal epithelium of CoD patients is heterogeneous: four TcR gamma delta phenotypes could be detected and, although the majority of the TcR gamma delta + T cells were CD4 CD8, gamma delta T-cell clones expressing either a CD8 alpha alpha homodimer, a CD8 alpha beta heterodimer or CD4 were also identified. In contrast to the TCR alpha beta + IEL, most TcR gamma delta + IEL were CD5 negative. Furthermore, biochemical analysis indicated that the increase in V delta 1+ gamma delta T cells in the small intestinal epithelium of CoD patients was not the result of a monoclonal expansion. The small intestinal epithelium-derived gamma delta T-cell clones were functional in vitro since the majority of these clones were able to lyse target cell lines such as K562. Molt4 and Daudi. These novel findings therefore indicate that the gamma delta T cells in the small intestine of CoD patients represent a heterogeneous population and that such cells are functional in vitro. The isolation and the in vitro propagation and cloning of these cells may open new avenues for the study of the putative immune mechanisms leading to coeliac disease.     

Identification and characterization of rat T cell subpopulations expressing T cell receptors alpha/beta and gamma/delta

Peripheral lymphoid organs of the rat were investigated for the presence of lymphocytes that expressed the pan-T cell markers CD5 and OX-52 but not the T cell receptor (TcR) alpha/beta. Two such populations were identified: 2% to 4% of lymphocytes in adult spleen, lymph nodes and peripheral blood are CD5+ TcR alpha/beta- and express the OX-52 antigen at the same density as TcR alpha/beta+ T-cells. About 90% of these cells are CD8+. A second population is CD5-, CD8+ and OX-52low. Radioimmunoprecipitation from digitonin lysates of surface-labeled cells with an anti-CD3 antiserum showed that the CD5+, but not the CD5- population of TcR alpha/beta- cells expresses a CD3-associated disulfide-linked cell surface molecule of about 100 kDa apparent mol. mass. Upon reduction, one major band, migrating with 48 kDa was observed. A band of the same size was obtained with an anti-human delta chain peptide antiserum, indicating that the CD3-associated non-TcR alpha/beta molecule is the rat TcR gamma/delta. Functional assays showed that most, if not all natural killer (NK) cell activity is present in the CD5(-)-OX-52low population. Reactivity to foreign major histocompatibility complex (MHC) antigens in mixed lymphocyte reaction was exclusively found in TcR alpha/beta+ splenic T cells. It is concluded that rat gamma/delta T cells in the spleen do not contain a high frequency of cells with specificity for foreign MHC antigens. The seeding of the periphery with alpha/beta and the presumptive gamma/delta T cells was followed from birth. Most prominently in the spleen, alpha/beta T cells reached adult levels much later than gamma/delta T cells. Taken together, these findings demonstrate the expression of the TcR gamma/delta on a minor population of peripheral rat T cells with the predominant phenotype CD4-CD8+ that has no NK cell activity when freshly isolated and does not contain a high frequency of alloreactive cells.     

Development of CD8 alpha/beta + TCR alpha beta intestinal intraepithelial lymphocytes in athymic nu/nu mice and participation in regional immune responses.

On the basis of the CD8 coreceptor expression, T-cell receptor (TCR)alpha beta-bearing intestinal intraepithelial lymphocytes (i-IEL) segregate into two populations. The CD8 alpha alpha + TCR alpha beta i-IEL develop thymus independently, whereas the CD8 alpha beta + TCR alpha beta i-IEL are generally considered to be thymus dependent. Flow cytometry analysis revealed a distinct population of CD8 alpha beta + TCR alpha beta i-IEL in individual athymic nu/nu mice. The i-IEL encompassing CD8 alpha beta + TCR alpha beta cells expressed potent cytolytic and interferon-gamma-producing activities. These findings demonstrate that CD8 alpha beta + TCR alpha beta i-IEL can develop in nu/nu mice independently from a functional thymus and suggest that these cells, directly or indirectly, perform biological functions in the gut.     

Characterization of T-cell receptor gamma delta T cells appearing at the early phase of murine Listeria monocytogenes infection.

It has been shown that T-cell receptor (TcR) gamma delta + CD4- CD8- T cells increase in number and have an important role in early protection in murine Listeria monocytogenes infection. In this report, to characterize further the phenotype of the gamma delta T cells in listeriosis, we analysed V region gene usage and in vitro antigen recognition of the TcR gamma delta T cells in the peritoneal cavity of mice at the early phase after i.p. infection with a sublethal dose of L. monocytogenes. The gamma delta T cells predominantly expressed V delta 6 which has been reported to be expressed by TcR gamma delta-bearing foetal thymocyte hybridomas specific to mycobacterial and self heat-shock protein (hsp) 60. These early appearing CD3+ CD4- CD8- T cells in Listeria-infected mice, which were reported to be TcR gamma delta T cells, increased in proportion and in size by in vitro stimulation with recombinant hsp 60 from Mycobacterium bovis and purified protein derivative from M. tuberculosis but not by stimulation with heat-killed L. monocytogenes. A 65,000 MW molecule was detected in the lysate of viable L. monocytogenes but not in the lysate of heat-killed L. monocytogenes by a monoclonal antibody (mAb) raised against mycobacterial hsp 60. These results suggest that the V delta 6-bearing peripheral gamma delta T cells are activated by recognizing listerial hsp 60 expressed by viable L. monocytogenes. The hsp 60-reactive V delta 6-bearing T cells may have an important role in protection against L. monocytogenes and other parasites that express hsp 60 at high level.     

In vivo induction of gamma/delta T cells with highly potent and selective anti-tumor cytotoxicity.

High frequencies of CD5+TcR alpha/beta- T cells were induced in the peritoneal cavity of rats immunized with syngeneic W439 lymphoma cells. These TcR alpha/beta- cells expressed TcR delta mRNA as analyzed by the polymerase chain reaction technique. The delta + (TcR gamma/delta +) T cells were of the CD2+, CD3+, CD4-, CD8+, CD45RB+ phenotype and showed stronger anti-tumor cytotoxicity compared to the TcR alpha/beta + T cells. The cytotoxic effects of both alpha/beta and gamma/delta T cells were selective for the W439 lymphoma cells and were not directed to other syngeneic tumors, natural killer targets and syngeneic or allogeneic normal cells. T cells, including both alpha/beta and gamma/delta cells, were induced when WF rats were immunized with allogeneic BN spleen cells. In this case the gamma/delta T cells showed allo-selective cytotoxicity, although weaker compared to the TcR alpha/beta + T cells. The gamma/delta T cells, induced by immunization with either W439 cells or BN spleen cells, were selective for the immunogen used and had no effect on irrelevant target cells, indicating that these effector cells were not activated by a shared gamma/delta T cell-related superantigen. Since highly potent tumor-selective gamma/delta cytotoxic T lymphocytes could be induced by syngeneic lymphoma cells, we suggest a role for gamma/delta T cells in the defense against certain types of tumors.     

T Lymphocytes in Human Gut Epithelium Preferentially Express the α/β Antigen Receptor and are often CD45/UCHL1-Positive

A revived interest in intraepithelial lymphocytes (IEL) has been elicited by several recent reports suggesting that murine and avian intestinal epithelium contains mainly CD3+CD8+ cells expressing the gamma/delta T-cell receptor (TcR) for antigen; this contrasts with systemically distributed T cells which preferentially employ the TcR alpha/beta. An anatomical dichotomy in the distribution of these two T-cell lineages has hence been proposed. Here we report that this concept does not hold true in man. In situ studies with monoclonal TcR-framework antibodies showed that most (70-90%) human intestinal IEL (which are mainly CD3+CD8+) expressed TcR alpha/beta. Moreover, almost half of the intraepithelial CD3+ cells were positive for the smallest (180 kDa) CD45 molecule (UCHL1); this probably reflected that they are antigen-primed and thus represent traditional CD3+CD8+ alpha/beta+ memory T cells.     

Intraepithelial TcR alpha/beta+ lymphocytes express CD45RO more often than the TcR gamma/delta+ counterparts in coeliac disease.

Expression of CD45RO on intraepithelial lymphocytes (IEL) bearing the T-cell receptor (TcR) alpha/beta or gamma/delta was studied in situ by three-colour immunofluorescence on jejunal tissue sections from 21 patients with coeliac disease and eight controls. CD45RA-TcR alpha/beta+ IEL expressed CD45RO significantly more often (75%) than the preferentially expanded TcR gamma/delta+ counterpart (59%). Triple staining for CD3, CD4/8 and CD45RA or CD45RB revealed that all CD3 + 4 - 8 - IEL (taken to be TcR gamma/delta+) expressed CD45RB and none were CD45RA. CD45RO positivity was of the same magnitude (66%) on the predominating monoclonal antibody delta TCS1-reactive fraction of TcR gamma/delta+ cells as on the remainder of the TcR gamma/delta+ subset. These results suggest that gluten exposition in patients with coeliac disease leads to accumulation of CD45RA-, putative antigen-primed memory cells of both TcR phenotypes. The less marked CD45RO expression within the preferentially expanded TcR gamma/delta+ subset of IEL may be of particular biological interest.     

Activated CD3−CD16+ Natural Killer Cells Express a Subset of the Lymphokine Genes Induced in Activated αβ+ and γω+ T cells

In this study we analysed the potential of highly purified polyclonal TcR alpha beta+, TcR gamma delta + and CD3- NK cells, to produce lymphokines in response to mitogenic stimulation. RNA hybridizations were performed to detect with high sensitivity the induction of multiple lymphokine genes. Upon stimulation with lectin and phorbol ester TcR gamma delta + lymphocytes expressed the same set of lymphokine genes as the TcR alpha beta + lymphocytes expressed the same set of lymphokine genes as the TcR alpha beta + lymphocytes, which included IL-2, -3, -4, -5, GM-CSF, TNF alpha and beta, IFN gamma. In contrast, a more limited set of lymphokine genes (GM-CSF, TNF alpha and beta, IFN gamma) was induced in activated CD3- NK cells, thus indicating that this subpopulation of cells may display different regulatory functions, with respect to CD3+ T lymphocytes.     

Selective death of T cell receptor gamma/delta+ intraepithelial lymphocytes by apoptosis

In mice, the majority of T cells expressing the gamma/delta T cell receptor (TcR) are found at mucosal surfaces, especially the intestinal epithelium. Here we show that in vitro, the majority of TcR gamma/delta+ intraepithelial lymphocytes, but not TcR alpha/beta+ intraepithelial lymphocytes, undergo rapid and selective programmed cell death by apoptosis.     

Intraepithelial T Cells of the TcRγ/δ+CD8− and Vδ1/Jδ1+ Phenotypes are Increased in Coeliac Disease

Expression of the gamma/delta T-cell receptor (TcR) for antigen on CD3+ intraepithelial lymphocytes (IEL) was studied in situ by two-colour immunofluorescence on jejunal tissue sections from 24 patients with coeliac disease and 17 controls. The proportion of intraepithelial TcR gamma/delta+ cells was significantly increased (P less than 0.002) in untreated (median 20%, range 11-53%) as well as in treated (gluten-free diet) coeliac disease (median 23%, range 16-55%) compared with controls (median 2%, range 0-39%). Although TcR alpha/beta+ IEL dominated both in controls and coeliac disease, T cells expressing the TcR gamma/delta were preferentially located within the epithelium rather than in the lamina propria. Paired staining for TcR gamma/delta and CD8 revealed that most (approximately 90%) intraepithelial TcR gamma/delta+ lymphocytes in coeliac disease were CD8-. A remarkably large fraction (median 67%, range 58-94%) of intraepithelial TcR gamma/delta+ cells expressed the V delta 1/J delta 1-encoded epitope revealed by monoclonal antibody delta TCS1. Our results suggested that increase of the intraepithelial TcR gamma/delta+ CD8- subset of T cells is particularly related to coeliac disease.     

Characterization of T cell receptors on resident murine dendritic epidermal T cells

Cell lines derived from Thy-1+ dendritic epidermal cells (Thy-1+DEC) display a marked heterogeneity in T cell receptor (TcR) expression including CD3-associated alpha/beta, C gamma 1/delta or C gamma 2/delta and C gamma 4/delta TcR. In order to investigate whether this heterogeneity is primarily imposed by in vitro culture conditions or, alternatively, is already present within the epidermis, we studied TcR expression by Thy-1+DEC in situ and on freshly isolated epidermal cell suspensions greatly enriched for Thy-1+DEC. Immunolabeling experiments showed that resident Thy-1+DEC are CD45+, Thy-1+, CD3+, TcR V beta 8-, CD5-, CD4- CD8-, CD25- lymphocytes. Immunoprecipitation of lysates from 125I-surface-labeled Thy-1+DEC-enriched epidermal cells with anti-CD3 epsilon, anti-C gamma 1,2,3 and anti-C gamma 4 antibodies, respectively, and subsequent analysis of the precipitates by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed only CD3-associated 35 kDa/45 kDa gamma/delta heterodimers. The demonstration of TcR heterodimers on resident Thy-1+DEC strongly implies that these cells are functional T cells. The selective expression of C gamma 1/delta (C57BL/6) and C gamma 1/delta or C gamma 2/delta (C3H/He) TcR makes these cells useful for the study of gamma/delta TcR function.