Phenotypic characterization of CD8(+)NKT cells

We describe a novel CD8(+)NKT cell population expressing TCRalpha /beta or TCRgamma /delta. These CD8(+)NKT cells were prominent in the liver, and except for the thymus, virtually absent in other lymphoid organs. CD8(+)NKT cells expressed activation markers and comprised a high proportion of Ly49(+) cells. The development of the majority of CD8(+)NKT cells expressing TCRalpha /beta, but not TCRgamma /delta, depended on classical MHC class I. No CD8(+)NKT cells were detectable in young athymic mice, whereas the cells expressing TCRgamma /delta, but not TCRalpha /beta, appeared randomly in aged athymic mice. CD8(+)NK1(+) TCRalpha /beta cells showed polyclonal TCRVbeta usage and were virtually devoid of TCRValpha14. CD8(+)NK1(+) TCRgamma /delta cells predominantly expressed TCRVgamma1, 2 and 4, and Vdelta4, 5, 6 and 7. CD8(+)NKT cells, in particular those expressing TCRgamma /delta, were a major population in early life. IFN-gamma, but not IL-4, was induced in CD8(+)NKT cells by in vitro stimulation, independent of the TCRalpha /beta or TCRgamma /delta lineage. Hence, these cells represent a unique, though heterogeneous T cell population that shares markers with, but is distinct from, both conventional NKT cells and conventional CD8(+) T cells, and that may play a role in immune regulation.     

Anti-ergotypic T cells in naïve rats

T regulatory cells play an important role in regulating T cell responses. Anti-ergotypic T cells are a subset of regulatory T cells that proliferate in response to activation markers, ergotopes, expressed on activated, and not on resting syngeneic T cells. Here we report the presence of anti-ergotypic T cells in lymph nodes, spleens and thymuses of naive rats. The development of anti-ergotypic T cells appeared to be independent of antigen priming, as thymocytes from one-day old rats exhibited significant anti-ergotypic proliferative responses. The anti-ergotypic T cells were found to be of the CD8+ phenotype, and included both TCRalpha/beta+ and TCRgamma/delta+ T cells. The TCRgamma/delta+ anti-ergotypic T cells secreted IFNgamma and TNFalpha in response to activated T cells; the TCRalpha/beta+ T cells proliferated but did not secret detectable cytokines. We found that the interaction between the anti-ergotypic T cells and stimulator T cells required cell-to-cell contact between the T cells. Professional APCs were not needed. The response of the TCRalpha/beta+CD8+ anti-ergotypic T cells was MHC-I restricted and B7-CD28 dependent; the response of the TCRgamma/delta+ anti-ergotypic T cells was B7-CD28 dependent, but was not inhibited by antibodies to classical MHC-I or MHC-II molecules. The existence of anti-ergotypic T cells in naive animals suggests that these cells might have a role in the regulation and maintenance of the immune system.     

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.     

The role of Notch and IL-7 signaling in early thymocyte proliferation and differentiation

We have analyzed the roles of Notch and IL-7 signaling in the proliferation and differentiation of mouse progenitor thymocyte subpopulations cultured on Notch delta-like-1 ligand-expressing OP9 stromal cells. Using bulk and limiting dilution cultures, we show that DN1 and DN2 cells require both Notch and IL-7 signaling for efficient proliferation and differentiation into cytoplasmic TCRbeta and surface TCRalpha/beta and TCRgamma/delta expressing T cells. Selection for cytoplasmic TCRbeta-positive cells is dependent on preTalpha expression. Both gamma/delta and alpha/beta TCR expressing T cells arising in culture can be efficiently stimulated by anti-CD3 cross-linking, suggesting that they might be functional. The differentiation of adult, but not fetal, DN1 and DN2 thymocytes into CD4 and/or CD8 expressing cells is inhibited by IL-7. Finally, efficient proliferation and differentiation of DN3 cells requires Notch signaling and preTCR expression, but is independent of IL-7.     

Gamma delta T cells expressing CD8 or CD4low appear early in murine foetal thymus development.

Three-colour flow cytometry was used to study the distribution of TCR gamma delta+ cells among CD4+CD8-, CD4-CD8+, CD4+CD8+, and CD4-CD8- cell populations during thymic development. Thymocytes were obtained either directly from embryos at different stages of gestation (ex vivo) or from organ cultures maintained in vitro. In both cases, TCR gamma delta+ cells were found predominantly among the double negative (CD4-CD8-) and CD8 single positive subsets. These cells were actively dividing as demonstrated by 7 amino actinomycin D (7AAD) labelling. A small population of TCR gamma delta+ cells expressing low levels of CD4 was identified early and transiently (days 15-18) during development, but this subset was rare in the adult thymus. In newborn mice, adult mice, and late during organ culture, TCR gamma delta+ cells were found mainly within the CD4-CD8- compartment of thymocytes, although a minor population of CD8+ cells (5-10%) bearing gamma delta receptor was routinely observed. In contrast, few gamma delta cells were contained among the CD4+CD8+ subset at any timepoint studied. These data highlight differences between the ontogeny of alpha beta and gamma delta cells in the thymus, and suggest that a CD4+CD8+ intermediate may not be a requisite for the intrathymic differentiation of murine gamma delta T cells.     

Membranes of activated CD4+ T cells expressing T cell receptor (TcR) alpha beta or TcR gamma delta induce IgE synthesis by human B cells in the presence of interleukin-4.

In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact-mediated signal provided by activated T cell receptor (TcR) gamma delta+, CD4+ and TcR alpha beta+, CD4+ T cell clones and interleukin (IL)-4. The signal provided by these T cell clones was antigen nonspecific, indicating that the TcR alpha beta/CD3 or TcR gamma delta/CD3 complexes were not involved in these T-B cell interactions. Activated TcR alpha beta+, CD8+, and TcR gamma delta+, CD4-CD8-, or resting CD4+ T cell clones were ineffective. Intact TcR alpha beta+ or TcR gamma delta+, CD4+ T cell clones could be replaced by plasma membrane-enriched fractions isolated from these activated CD4+ T cell clones. In contrast, membranes isolated from resting TcR alpha beta+, CD4+, TcR gamma delta+, CD4+ T cell clones or an Epstein-Barr virus (EBV)-transformed B cell line (EBV-LCL) failed to provide the costimulatory signal that, in addition to IL-4, is required for induction of IgE synthesis. As described for intact CD4+ T cells, CD4+ T cell membranes induced purified surface IgM+ B cells to switch to IgG4- and IgE- but not to IgA-producing cells, excluding the possibility of a preferential outgrowth of IgG4- and IgE-committed B cells. The membrane activity was inhibited by protease or heat treatment. Induction of IgE synthesis by B cells co-cultured with both TcR alpha beta+, CD4+ and TcR gamma delta+, CD4+ T cell clones and membrane preparations of these cells was blocked by anti-class II major histocompatibility complex (MHC) monoclonal antibodies (mAb), whereas various anti-CD4 mAb had differential blocking effects. Murine L cells, or EBV-LCL transfected with CD4 could not replace CD4+ T cell clones. These results indicate that, although CD4 and class II MHC antigens are required for productive CD4+ T cell clone-B cell interactions, an additional signal, provided by a membrane associated (glyco)protein that is induced by activation of both TcR alpha beta and TcR gamma delta, CD4+ T cells, is needed for induction of IgE production in the presence of IL-4.     

Sequential development of intraepithelial gamma delta and alpha beta T lymphocytes expressing CD8 alpha beta in neonatal rat intestine: requirement for the thymus.

Previous studies in congenitally athymic nude rats have suggested that the thymus is important for the development of intestinal T cells. Here we have examined the effect of the nude mutation on intraepithelial lymphocyte (IEL) development from the perinatal period. By immunohistochemistry it was shown that CD3(-)CD8 alpha alpha + putative IEL precursors colonized the epithelium of both normal and athymic neonatal rats. Mature T cells, however, did not develop in athymic neonates. In normal rats, gamma delta T cells were present at birth and alpha beta T cells appeared within 8 days of postnatal life. At this age, the composition and relative number of intraepithelial T cells were similar to that in normal adult rats, with the exception that most neonatal T-cell receptor-gamma delta + and -alpha beta + IEL expressed CD8 beta. By contrast, extrathymic T-cell maturation in the gut of congenitally athymic rats occurred slowly, as CD3+ IEL did not appear until 4-6 months of age. These intraepithelial T cells displayed variable phenotypes and appeared to be induced by environmental antigens as they were not found in isolator-kept old nudes. In conclusion, the present results indicate that the major colonization of the gut epithelium with gamma delta and alpha beta T cells expressing CD8 alpha beta takes place perinatally and requires the presence of the thymus. The developmental relationship between these neonatal T cells and more immature CD3- CD8 alpha alpha +/- IEL remains elusive.     

An accessory role of TCRgammadelta (+) cells in the exacerbation of inflammatory bowel disease in TCRalpha mutant mice

T cell receptor alpha mutant (TCRalpha (-/-)) mice, which spontaneously develop colitis under conventional conditions, did not show any signs of colitis under germ-free conditions, leaving TCRalpha (-)beta (+) cells (beta (dim) cells) and TCRgamma delta (+) cells much reduced. Moreover, TCRalpha (-/-) mice with alymphoplastic mutation (aly/aly TCRalpha (-/-) mice), which lack Peyer's patches and peripheral lymph nodes, did not suffer from colitis. While both beta (dim) cells and TCRgamma delta (+) cells were present in the colons of aly/aly TCRalpha (-/-) mice and aly/+ TCRalpha (-/-) mice, cytotoxicity of colonic TCRgamma delta (+) cells in aly/aly TCRalpha (-/-) mice was almost abolished. Transfer of TCRgamma delta (+) cells from TCRalpha (-/-) mice into scid/scid mice or aly/aly TCRalpha (-/-) mice could not induce colitis, but injection of anti-TCRdelta mAb into TCRalpha (-/-) mice prevented colitis from developing. Finally, TCRalpha (-/-) mice expressing transgenic (Tg) KN6-TCRgamma delta hardly developed colitis, accompanied by colonization of non-cytotoxic Tg TCRgamma delta (+) cells in their colonic mucosa. These results demonstrate that intestinal resident TCRgamma delta (+) cells may be involved in the exacerbation of inflammatory bowel disease in TCRalpha (-/-) mice.     

Evidence for the early recruitment of T-cell receptor gamma delta+ T cells during rat listeriosis.

We have previously reported that heat-shock protein (hsp) 60-reactive T-cell receptor (TCR)gamma delta+ T cells appear in the peritoneal cavity during the early stage of infection with Listeria monocytogenes in mice. In this study, we examined the kinetics of TCR gamma delta+ T cells during listeriosis in F344 rats by flow cytometry using a V65 monoclonal antibody (mAb) directed to a constant determinant of rat TCR gamma delta chains. TCR gamma delta+ T cells significantly increased in the peritoneal cavity on day 6 and then decreased by day 10 after infection, in parallel with the kinetics of hsp60 expression in the peritoneal macrophages during listeriosis in F344 rats. Most of the early appearing TCR gamma delta+ T cells were of the CD4- CD8 alpha beta+ CD5+ lymphocyte function-associated antigen (LFA)-1 alpha high CD45RC- interleukin-2 receptor (IL-2R) alpha- phenotype, although a significant fraction of the TCR gamma delta+ T cells expressed CD8 alpha only. The increase in TCR gamma delta+ T cells during listeriosis was prominent in F1 (F344 x Lewis) rats but only marginal in Lewis rats, which was correlated with the expression level of hsp 60 in the peritoneal macrophages. The peritoneal TCR gamma delta+ T cells in naive F344 rats appeared to proliferate significantly in response to recombinant hsp 60 (rhsp 60) derived from Mycobacterium bovis bacillus Calmette-Guérin (BCG). These results imply that the early appearance of hsp 60-reactive TCR gamma delta+ T cells during listerial infection can be generalized across species.     

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.     

Development of T lymphocytes in the nasal-associated lymphoid tissue (NALT) from growing Wistar rats

The aim of the present report was to study the development of several T-lymphocyte subsets in the nasal-associated lymphoid tissue (NALT) of growing Wistar rats. CD5+ and CD4+ lymphocytes gradually increased with age. A predominance of CD8alpha+ over CD4+ T cells was found from 7 to 45 days but from 45 to 60 days of age T helper cells outnumbered the cytotoxic subpopulation. The majority of CD8+ T lymphocytes expressed the heterodimeric isoform. The most relevant findings by immunohistochemistry are: (1) the predominance of TCRgammadelta+ and CD8alpha+ cells at 7 days postpartum over all the other T-cell subpopulations; and (2) that TCRgammadelta+ outnumbered TCRalphabeta+ T cells from 7 to 45 days postpartum whereas alphabeta T cells predominated in 45- and 60-day-old rats. Besides, cytometric studies have shown that the percentages of TCRgammadelta+, CD8alpha+, as well as the population coexpressing both phenotypes (TCRgammadelta+CD8alpha+), were significantly higher in rats at 7 days postpartum when compared to 60 day-old rats. In the present study, the finding of a high number of gammadelta+ and CD8+ T cells early in NALT development may indicate the importance of these subpopulations in the protection of the nasal mucosa in suckling and weaning Wistar rats.     

Presence of murine fetal liver cells capable of being induced to differentiate in vitro into T cell receptor-positive cells.

Mouse fetal liver cells were analyzed for the surface expression of T cell markers. Fetal liver cells prepared from mouse embryos at 14.5 days of gestation contained a small number of CD4+ cells (1.4%), but virtually no cells positive for any other T cell markers such as CD8, CD3 and T cell receptor (TcR). When a fetal liver cell suspension prepared from BALB/c(male) x AKR(female) F1 embryos at 14.5 days of gestation was cultured in medium supplemented with culture supernatants of both WEHI-3 and concanavalin A-stimulated rat spleen cells, TcR alpha beta+ and CD4+ cells were generated, whereas CD8+ and TcR gamma delta+ cells were hardly detectable. Most of TcR alpha beta+ and CD4+ cells were H-2d+, thus clearly showing their fetal origin. Treatment with anti-CD4, anti-CD3 or anti-TcR alpha beta antibodies plus complement or electronic sorting to remove cells expressing these markers failed to inhibit the generation of T cell marker-positive cells following culture in vitro. On the other hand, depletion of Thy-1.2+ cells reduced their generation. These findings indicate the presence of some progenitor T cells in fetal liver with the Thy-1+, CD3-, CD4-, CD8-, TcR- phenotype, which can be induced to differentiate into TcR alpha beta+ cells in the presence of specific humoral supplements without the influence of the thymus.     

Participation of leukocyte function-associated antigen-1 and NK cells in the homing of thymic CD8+NKT cells to the liver

A distinct CD8+NKT cell population expressing TCRalpha/beta or TCRgamma/delta has been identified in liver and thymus. We wondered whether cell adhesion molecules play a role in the homing of CD8+NKT cells to the liver. The number of liver CD8+NKT cells was markedly reduced in leukocyte function-associated antigen (LFA)-1-/- mice compared with wild-type (WT) mice. The reduction was restricted to the liver only and no measurable alterations were found in other organs. In the liver of SCID mice reconstituted with thymocytes from LFA-1-/- or WT mice, the number of donor-derived CD8+NKT cells was comparable and the vast majority of these cells expressed TCRalpha/beta. In a reciprocal radiation thymocyte reconstitution system with LFA-1-/- and WT mice, LFA-1 expressed on liver cells other than CD8+NKT cells appeared to be required for the homing of thymic CD8+NKT cells to the liver. The accumulation of donor thymocyte-derived CD8+NKT cells in the liver of SCID mice was severely impaired by in vivo depletion of NK cells, but not of Kupffer cells. These results not only indicate that thymus provides a source for CD8+NKT cells expressing TCRalpha/beta in the liver, but also suggest that LFA-1 expressed on NK cells is involved in the homing of thymic CD8+NKT cells to the liver.     

Identification of αβ and γδ T Cell Receptor-Positive Cells

Two lineages of T lymphocytes bearing the CD3 antigen can be defined on the basis of the nature of the heterodimeric receptor chain (alpha beta or gamma delta T cell receptor (TCR) expressed. Precise identification of alpha beta and gamma delta TCR+ cells is essential when studying the tissue distribution and function of these different T cells. In immunofluorescence studies gamma delta TCR+ cells have been identified as CD3+WT-31- or CD3+CD4-CD8- cells. However, this may not be the optimal procedure because gamma delta TCR+ cells are weakly WT-31+, and some are CD8+. The aim of this study was to evaluate a panel of monoclonal antibodies (MoAb) directed against different chains of the TCR-T3 complex for a more precise identification of alpha beta+ and gamma delta TCR+ cells in flow cytometric studies. We found that the MoAb anti-Ti-gamma A and delta-TCS-1, recognizing the TCR-gamma and the TCR-delta chain respectively, only reacted with a subpopulation of gamma delta TCR+ cells, whereas another TCR-delta chain recognizing MoAb anti-TCR-delta 1 reacted with all gamma delta TCR+ cells. All MoAb reported to belong to the CD3 group reacted with both alpha beta TCR+ and gamma delta TCR+ cells as expected. Our results indicate that all gamma delta TCR+ cells can be identified with the MoAb anti-TCR-delta 1. Because no MoAb recognizing the TCR-alpha or TCR-beta chains at the cell surface of intact cells are yet available, we suggest that alpha beta TCR+ cells could be identified as CD3+ anti-TCR-delta 1-cells.     

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.     

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.     

Differential thymus dependence of rat CD8 isoform expression.

Expression of the rat CD8 molecule was studied using five novel monoclonal antibodies (mAb), four of which are specific for the V-like domain of CD8 alpha, whereas one reacts either with the beta chain or with a determinant only expressed on the CD8 alpha/beta heterodimer. mAb to both chains effectively blocked purified lymph node CD8 T cells in mixed lymphocyte reaction and in cell-mediated cytotoxicity. Flow cytometric analysis showed that CD8 T cells from lymph nodes or spleen of normal rats almost exclusively express the alpha/beta isoform, regardless of the T cell receptor isotype (alpha/beta or gamma/delta). In contrast, natural killer (NK) cells carry only CD8 alpha chains. This CD8 alpha + beta - phenotype was also prominent among CD8 T cells from athymic rats and from intestinal epithelium of normal rats. CD8 alpha homodimers can also be expressed as a result of activation, as shown by analysis of CD4 CD8 double-positive T cells obtained from highly purified lymph node CD4 T cells by in vitrok stimulation. Such CD4+CD8 alpha + beta - cells also represent a major subset among adult intestinal intraepithelial lymphocytes (IEL), suggesting local activation. Taken together, the difference in CD8 isoform expression among T cells from athymic rats, NK cells, and gut IEL versus CD8 T cells from peripheral lymphatic organs of euthymic animals suggests that like in mice, expression of the CD8 heterodimer is more dependent on intrathymic maturation than that of the homodimer. Since the more stringent thymus dependence of CD8 alpha + beta + T cells may be due to a requirement for thymic selection on self major histocompatibility complex class I antigens, the virtually exclusive CD8 alpha + beta + phenotype of peripheral rat gamma/delta T cells could mean that antigen recognition by this subset is also restricted by MHC class I molecules.