Gut Intraepithelial Lymphocytes Research Articles

Biological Insights into TCRγδ + and TCRαβ + Intraepithelial Lymphocytes Provided by Serial Analysis of Gene Expression (SAGE)

Intraepithelial lymphocytes (IELs) are abundant, evolutionarily conserved T cells, commonly enriched in T cell receptor (TCR) γδ expression. However, their primary functional potential and constitutive activation state are incompletely understood. To address this, serial analysis of gene expression (SAGE) was applied to murine TCRγδ + and TCRαβ + intestinal IELs directly ex vivo, identifying 15,574 unique transcripts that collectively portray an “activated yet resting,” Th1-skewed, cytolytic, and immunoregulatory phenotype applicable to multiple subsets of gut IELs. Expression of granzymes, Fas ligand, RANTES, prothymosin β4, junB, RGS1, Btg1, and related molecules is high, whereas expression of conventional cytokines and high-affinity cytokine receptors is low. Differentially expressed genes readily identify heterogeneity among TCRαβ + IELs, whereas differences between resident TCRγδ + IELs and TCRαβ + IELs are less obvious.

Reduced Gut Intraepithelial Lymphocytes in VLA1 Null Mice

Very late antigen 1 (VLA1) is an integrin collagen receptor that is expressed by lymphocytes in several disease states. VLA1 blockade has been shown to ameliorate gut disease in experimental graft-versus-host disease. Here we show that in the VLA1 null mouse, which is generally healthy, there is a 50% reduction in gut intraepithelial lymphocytes (IELs) despite an otherwise normal lymphocyte distribution in peripheral blood and lymphoid organs. The γδ to αβ ratios of IELs are unchanged. We also find that IL2-stimulated splenocytes from VLA1 null animals show a deficiency in adhesion to fibrillar and basement membrane collagen as well as reduced proliferation in response to collagen substratum. These results suggest that some, but not all, intraepithelial lymphocytes require VLA1 to survive or proliferate within the gut epithelium or possibly to traverse the basement membrane.

Oligoclonality of Rat Intestinal Intraepithelial T Lymphocytes: Overlapping TCR β-Chain Repertoires in the CD4 Single-Positive and CD4/CD8 Double-Positive Subsets

AbstractPrevious studies in humans and mice have shown that gut intraepithelial lymphocytes (IELs) express oligoclonal TCR β-chain repertoires. These studies have either employed unseparated IEL preparations or focused on the CD8+ subsets. Here, we have analyzed the TCR β-chain repertoire of small intestinal IELs in PVG rats, in sorted CD4+ as well as CD8+ subpopulations, and important differences were noted. CD8αα and CD8αβ single-positive (SP) IELs used most Vβ genes, but relative Vβ usage as determined by quantitative PCR analysis differed markedly between the two subsets and among individual rats. By contrast, CD4+ IELs showed consistent skewing toward Vβ17 and Vβ19; these two genes accounted collectively for more than half the Vβ repertoire in the CD4/CD8 double-positive (DP) subset and were likewise predominant in CD4 SP IELs. Complementarity-determining region 3 length displays and TCR sequencing demonstrated oligoclonal expansions in both the CD4+ and CD8+ IEL subpopulations. These studies also revealed that the CD4 SP and CD4/CD8 DP IEL subsets expressed overlapping β-chain repertoires. In conclusion, our results show that rat TCR-αβ+ IELs of both the CD8+ and CD4+ subpopulations are oligoclonal. The limited Vβ usage and overlapping TCR repertoire expressed by CD4 SP and CD4/CD8 DP cells suggest that these two IEL populations recognize restricted intestinal ligands and are developmentally and functionally related.

An Enhancer That Directs Lineage-Specific Expression of CD8 in Positively Selected Thymocytes and Mature T Cells

Positive selection of CD4+CD8+ T cells to the CD4+CD8− helper and CD4−CD8+ cytotoxic lineages is a multistep process that involves complex regulation of coreceptor gene expression. By analyzing expression of a reporter gene in transgenic mice, we have identified a DNA segment, located between the murine CD8β and CD8α genes, that has enhancer activity restricted to CD8 lineage cells. Remarkably, this enhancer functions in thymocytes undergoing positive selection to the CD4−CD8+ phenotype but not in immature double-positive thymocytes. The enhancer also functions in gut intraepithelial lymphocytes that express CD8α but not CD8β, suggesting that it is specific for CD8α expression. The tight correlation between activation of this enhancer and the final step in positive selection has important implications for understanding the mechanism of lineage commitment in thymocytes.

Gut intraepithelial lymphocytes induce immunity against Cryptosporidium infection through a mechanism involving gamma interferon production.

Immunological control of infection with cryptosporidia in mice is dependent on CD4+ T cells and the production of gamma interferon (IFN-gamma), but to date, the mucosal T cells which produce IFN-gamma local to the infection have not been characterized. We previously showed that immunity against the gastric parasite Cryptosporidium muris could be adoptively transferred to adult SCID (severe combined immunodeficiency) mice with small intestinal intraepithelial lymphocytes (IEL) from previously infected immunocompetent mice, but only if the donor CD4+ T cells were intact. The present investigation examined whether IFN-gamma was important in the effector mechanisms mediated by immune IEL in SCID mice. The development of resistance against C. muris infection in SCID mice given immune IEL was prevented by treatment with a hamster anti-mouse IFN-gamma-neutralizing monoclonal antibody, but following cessation of antibody treatment, the mice recovered from infection. In further experiments, an enzyme-linked immunospot (ELISPOT) technique was used to compare frequencies of IFN-gamma-producing cells in activated T-cell populations from C. muris-immune and naive donor mice. Stimulation with concanavalin A or a rat anti-mouse CD3 monoclonal antibody resulted in detection of greater numbers of cells producing IFN-gamma from immune than naive IEL populations. Small numbers of IEL from C. muris-immune mice, but not from naive mice, also produced IFN-gamma when cultured with soluble oocyst antigen, but this occurred only if gamma-irradiated spleen cells were cocultured with the immune IEL. These results suggested that IEL were important in the generation of immunity to Cryptosporidium and that one of their crucial functions was to produce IFN-gamma at the site of infection.

CD5- CD8 alpha beta intestinal intraepithelial lymphocytes (IEL) are induced to express CD5 upon antigen-specific activation: CD5- and CD5+ CD8 alpha beta IEL do not represent separate T cell lineages.

We followed alpha beta T cell receptor (TCR) usage in subsets of gut intraepithelial lymphocytes (IEL) in major histocompatibility complex class I-restricted alpha beta TCR-transgenic (tg) mice. The proportion of tg alpha beta TCR+ CD8 alpha beta IEL is reduced compared with CD8+ splenocytes of the same animal, particularly under conventional conditions of maintenance. Further fractionation of CD8 alpha beta IEL according to the expression level of surface CD5 revealed that in conventionally housed animals tg TCR+ CD5- CD8 alpha beta IEL are as frequent as in specific pathogen-free (SPF) mice, whereas tg TCR+ CD5int or, even more pronounced, tg TCR+ CD5hi CD8 alpha beta IEL are greatly diminished when compared with mice kept under SPF conditions. Upon antigen-specific stimulation of CD5- CD8 alpha beta IEL in vitro, CD5 surface expression is up-regulated on a large fraction of cells within 48 h. Up-regulation of CD5 surface expression is further enhanced by the presence of the anti-alpha IEL monoclonal antibody 2E7. This clearly demonstrates that CD5-, and CD5+ CD8 alpha beta IEL cannot be considered as separate T cell lineages.

Adoptive transfer of gut intraepithelial lymphocytes protects against murine infection with Toxoplasma gondii.

Intraepithelial lymphocytes (IEL) of the gut represent a primary immune barrier against infection by orally acquired pathogens. Naturally acquired infection with Toxoplasma gondii induces the proliferation of CD8+ T cells in both the gut and spleen. Gut-derived CD8alpha/beta+ IEL exhibit MHC-restricted cytotoxicity against parasite-infected enterocytes and macrophages. In a murine model, we demonstrate that the adoptive transfer of IEL obtained from inbred mice at day 11 postinfection is able to protect against a virulent challenge in syngenic recipients. In CBA mice, the parasite cyst load within the brain of the recipients receiving primed IEL was reduced by 90%. In BALB/c and C57BL/6 mice, a 50% decrease in mortality was observed following adoptive transfer of primed IEL. To determine the T cell subset responsible for protective immunity, a purified CD8alpha/beta+ IEL population was isolated from infected mice at day 11 postinfection. These cells were able to protect naive mice by adoptive transfer against a lethal parasite challenge. RNA analysis by reverse-transcriptase PCR revealed that primed CD8alpha/beta+ IEL produce significant message for IFN-gamma, an essential cytokine for host protection against toxoplasmosis. Administration of anti-IFN-gamma at the time of adoptive transfer of primed IEL abrogated protection. The adoptive transfer of these protective IEL was not restricted to the Ld class I locus. These data demonstrate that IFN-gamma-producing IEL may be an important primary barrier against acute and perhaps recurrent infection with T. gondii.

Complexity of the mouse gut T cell immune system: Identification of two distinct natural killer T cell intraepithelial lineages

Gut thymo-dependent (CD8 alpha + beta + or CD4+) or -independent (CD8 alpha + beta -) intraepithelial lymphocytes (IEL) mediate cytotoxicity following T cell receptor (TCR)-CD3 signaling, but only TCR gamma delta + and alpha beta + thymo-independent IEL show cytotoxicity of natural killer (NK) and antibody-dependent cell-mediated cytotoxicity types. Moreover, TCR alpha beta + and gamma delta + thymo-independent IEL express NK receptors, and may therefore be referred to as NK-TIEL. NK-TIEL cytotoxicity is mediated through perforin, Fas, or both pathways. In contrast to that of other NK cells, this cytotoxicity is not negatively regulated by signals delivered through the recognition of major histocompatibility complex class I molecules. Thus, gut IEL include T cell subsets with unique specificities and functions, ontogenically distinct from other T cell lineages, which may increase the antigenic repertoire diversity of the immune system participating in the defense of the epithelial barrier.

Immunity to Cryptosporidium muris infection in mice is expressed through gut CD4+ intraepithelial lymphocytes

The role of gut intraepithelial lymphocytes (IEL) in immunity to cryptosporidial infection was investigated with a murine infection model involving Cryptosporidium muris. Oocyst shedding was monitored in severe combined immunodeficiency (SCID) mice infected with C. muris following intravenous injection of mesenteric lymph node (MLN) cells or intestinal IEL from BALB/c donor mice which were naive or previously infected with C. muris. SCID mice receiving no lymphoid cells developed chronic infections and excreted large numbers of oocysts until the end of the experiment. SCID mice injected with IEL from immune animals, however, were able to overcome the infection, and furthermore, these animals produced fewer oocysts and recovered sooner than ones which received IEL or MLN cells from naive BALB/c donors. Similar levels of protection were obtained in SCID mice injected with either 2 X 10(6) IEL or MLN cells from immune donor mice. Depletion of CD4+ cells from immune IEL, however, abrogated the ability to transfer immunity to SCID mice, while depletion of CD8+ cells only marginally reduced the protective capacity of immune IEL. Finally, control SCID mice which received no lymphocytes had < or = 1% CD4+ cells in the IEL from the small intestine, whereas the IEL from SCID mice recovered from infection, as a result of injection with immune IEL, contained 15% CD4+ cells. Thus, the ability to control C. muris infection correlated with the presence of the protective CD4+ cells in the gut epithelium.

Extrathymic T cell differentiation

In the mouse, the gut mucosa is a major site of extrathymic differentiation of T cells. Recent data in this past year show that this process differs from the main thymic differentiation pathway not only in its location, but also in its use of costimulatory molecules, signal transduction modules, and mechanisms of repertoire selection. The thymus exerts an influence on the expansion of the extrathymically differentiated gut intraepithelial lymphocytes that appears to be varied in nature, including acting as a source of TCR − progenitors. All gut intraepithelial lymphocytes, whatever their extrathymic or thymic site of differentiation, have common features of activated and specialized cytotoxic cells. Other T cells may differentiate extrathymically, in particular in the liver; these later cells appear to have a very restricted, probably autoreactive repertoire, and also display natural killer cell features.

Gut intraepithelial lymphocyte counts in neonates, infants and children.

Intraepithelial lymphocyte (IEL) counts were histologically assessed in the jejunum, ileum and appendix of 39 neonates (0-28 days), 32 infants (1-9 months) and 13 children (1-9 years). Small intestinal mucosa samples were obtained from 73 autopsies, and from 8 surgical and 3 aspirative biopsies. IEL counts of specimens from the jejunum, ileum and appendix gave similar results in the same patient. The number of IEL counts was significantly lower in neonates for all three segments. The difference between infants and children was more marked in the jejunum than in the ileum, although this was not significant. In the appendix, there was no difference between the different age groups. Our results indicate that postnatal expansion of IEL occurs homogeneously along the gut after the neonatal period.

Different use of T cell receptor transducing modules in two populations of gut intraepithelial lymphocytes are related to distinct pathways of T cell differentiation.

Most gut intraepithelial cells (IEL) of the mouse are T cells that bear CD8 molecules, present either as alpha-beta chain heterodimers (CD8 beta+) or as alpha chain homodimers (CD8 beta-). All CD8 beta+ IEL bear alpha/beta T cell receptors (TCR); CD8 beta- IEL bear either alpha/beta or gamma/delta TCR and are considered to be a thymus-independent (TI) population, probably arising locally from a small fraction of CD3- IEL containing the recombinant activating gene RAG proteins. Here we report that TI CD8 beta- IEL, whether bearing alpha/beta or gamma/delta TCR, contain, in normal mice, mRNAs for both zeta and Fc epsilon RI gamma chains. These chains are present in their CD3-TCR complexes as homo- or heterodimers. In contrast, only zeta chain mRNA and homodimers are found in gut CD8 alpha/beta+ IEL and in peripheral T lymphocytes. Intestinal CD3- precursor cells contain only gamma chain, and CD3- IL-2R+ thymocyte precursors only zeta chain mRNAs. Only very primitive thymocyte precursors contain detectable gamma chain mRNA, and it thus appears that Fc epsilon RI gamma chain use is switched off at a very early stage during thymocyte differentiation. Thus, T cell differentiation in the gut epithelium differs from that occurring in the thymus, from which CD8 beta+ IEL appear to derive. Use of different TCR transducing modules and CD8 accessory molecules between the TI and the thymus-derived T cell populations provides an explanation for their difference in reactivity to antigenic stimulations and thus in selection of repertoires.

Thymic and extrathymic origins of gut intraepithelial lymphocyte populations in mice.

We have investigated the origin of intraepithelial lymphocytes (IEL) populations in the murine gut, using reconstitution experiments in which the presence of thymus-derived cells of host or donor origin is rigorously controlled: RAG-/- mutant mice which have no T cells, were injected either with the bone marrow (BM) cells of nude mice or with selected peripheral lymph node (LN) T cells of euthymic mice. In thymectomized RAG-/- mice, injection of BM cells from nude mice led, after 2 mo, to the development of a peripheral B cell compartment and to the appearance, in the gut, of IEL bearing homodimeric CD8 alpha chains and either gamma/delta or alpha/beta TCR. In RAG-/- mice with a thymus, a similar injection led to complete lymphoid reconstitution, with the additional appearance in the gut of CD4+, CD8 alpha/beta+ or CD4+CD8 alpha/alpha+ IEL, all bearing alpha/beta TCR. In contrast, injection of LN T cells into these mice reconstituted a gut IEL population made of CD4+, CD8 alpha/beta+, or CD4+ CD8 alpha/alpha+ cells, all bearing alpha/beta TCR; CD8 alpha/alpha+ TCR-gamma/delta+ or alpha/beta+ IEL were not observed. These results demonstrate that the thymus and/or thymic-derived peripheral T cells are absolutely required for the generation of CD4+, CD8 alpha/beta+, and CD4+CD8 alpha/alpha+ IEL, which are thus thymus dependent. In contrast, TCR+ CD8 alpha/alpha+ IEL appear in the absence of the thymus, and thus are thymus independent.

Unique properties of a cytotoxic CD4+CD8+ intraepithelial T-cell line established from the mouse intestinal epithelium.

Growth factor-dependent gut intraepithelial lymphocyte (IEL) cell lines were established from a long-term in vitro culture of BALB/c IEL with syngeneic irradiated spleen cells in the presence of concanavalin A-stimulated spleen supernatant fluids. The cell lines were preferentially consisted of very limited thymoindependent subsets of IEL; i.e., Thy-1+CD5-TCR alpha beta+CD4+CD8 alpha+beta- (double-positive; DP) IEL and Thy-1+CD5-TCR alpha beta+CD4-CD8 alpha+beta- (CD8 single-positive; CD8 SP) IEL. The CD8 SP IEL cell line had cytotoxic activities and was triggered to proliferate by T-cell receptor (TCR)-directed stimuli. The DP IEL cell line expressed high levels of the CD3-TCR alpha beta, exhibited cytotoxic activity in redirected lysis assays, and had perforin in the cytoplasm, indicating the functional maturity of this cell line. However, the DP IEL cell line did not proliferate in response to TCR alpha beta-directed stimuli, which indicated that TCR alpha beta-mediated signalling was able to initiate cytotoxic function but not to induce proliferation of the DP IEL cell line. Although both cell lines were shown to have functional competence, they expressed J11d antigen which marks immaturity in thymocyte differentiation pathways. These results indicate that the established thymoindependent DP and CD8 SP IEL cell lines have unique properties distinct from DP thymocytes and CD8 SP peripheral T cells. Together with a recent report on freshly isolated DP IEL (10), the unique properties of the DP IEL cell line seems to support the notion that DP IEL may undergo a unique maturation process in the gut microenvironment.

Identification of a novel population of CD8 alpha+ beta- bone marrow-derived dendritic epidermal cells. This unique population is subsequently outnumbered by thymus-independent expansion of the CD8 alpha+ beta+ dendritic epidermal cells.

Maturation of some T cell populations has been suggested to occur in epithelial tissues. The CD8 molecule, which is expressed as heterodimers made of alpha/beta-chains on virtually all peripheral T cells, is preferentially expressed as homodimers made only of alpha-chains on a subset of CD8+ mouse gut intraepithelial lymphocytes, which arise from bone marrow (BM) precursors in a thymus-independent mechanism. This unique population of CD8 alpha+ beta- T cells, however, has not been identified in other lymphoid tissues in normal adult mice. Because our previous study demonstrated that reconstitution of thymectomized irradiated mice with T cell-depleted BM cells resulted in the appearance of CD8+, TCR-alpha beta+ dendritic epidermal cells (DEC) and suggested that a proportion of the BM-derived DEC may mature within the epidermis, we asked whether the unique phenotype of CD8 alpha+ beta- could be detected among the CD8+ BM-derived DEC. In this report for the first time we identified this unique population of CD8 alpha+ beta- cells among the DEC. Although this population comprised the predominant fraction of the BM-derived DEC in the early post-transfer period (2 to 3 mo), gradual shift from the CD8 alpha+ beta+ phenotypes occurred during the late post-transfer period (4 to 6 mo) independently of the presence of the thymus. Kinetic studies on the BM-derived DEC revealed that this phenotypic shift could be caused by the subsequent expansion of the CD8 alpha+ beta+ DEC. In contrast, the CD8 alpha+ beta- population was the major subset of the BM-derived intraepithelial lymphocytes throughout the entire observation period and the phenotypic shift with age was never observed. These results indicate that CD8 alpha+ beta- cells are preferentially detected on T cells that home to the epithelial tissues and that in the epidermis, but not in the gut epithelia, the subsequent expansion of the CD8 alpha+ beta+ DEC would dilute the high number of the CD8 alpha+ beta- cells.

Gut intraepithelial lymphocytes and gastrointestinal diseases

Intraepithelial lymphocytes interspersed between intestinal epithelial cells form a large population of T cells, mainly CD8+, close to the intestinal lumen. Together with lamina propria T cells and plasma cells, they represent the effector limb of the gut-associated lymphoid system. Recent data highlighting their original properties and their interactions with epithelial cells are reviewed; their contributions to immune defenses and gastrointestinal diseases in humans are discussed. Current Opinion in Gastroenterology 1993, 9:953-961

T cell development in mice lacking the CD3-zeta/eta gene.

The CD3-zeta and CD3-eta polypeptides are two of the components of the T cell antigen receptor (TCR) which contribute to its efficient cell surface expression and account for part of its transducing capability. CD3-zeta and CD3-eta result from the alternative splicing of a single gene designated CD3-zeta/eta. To evaluate the role of these subunits during T cell development, we have produced mice with a disrupted CD3-zeta/eta gene. The analysis of thymocyte populations from the CD3-zeta/eta-/- homozygous mutant mice revealed that they have a profound reduction in the surface levels of TCR complexes and that the products of the CD3-zeta/eta gene appear to be needed for the efficient generation and/or survival of CD4+CD8+ thymocytes. Despite the almost total absence of mature single positive thymocytes, the lymph nodes from zeta/eta-/- mice were found to contain unusual CD4+CD8- and CD4-CD8+ single positive cells which were CD3-. In contrast to the situation observed in the thymus, the thymus-independent gut intraepithelial lymphocytes present in zeta/eta-/- mice do express TCR complexes on their surface and these are associated with Fc epsilon RI gamma homodimers. These results establish an essential role for the CD3-zeta/eta gene products during intrathymic T cell differentiation and further emphasize the difference between conventional T cells and thymus-independent gut intraepithelial lymphocytes.

The Mycobacterium tuberculosis 71‐kDa heat‐shock protein induces proliferation and cytokine secretion by murine gut intraepithelial lymphocytes

Murine intraepithelial lymphocytes (IEL) respond poorly to T cell mitogens and to monoclonal antibody stimulation of T cell receptor (TCR)- and CD3- associated molecules. In contrast, we found that a soluble extract of Mycobacterium tuberculosis (Mtb), but not purified protein derivative of tuberculin, induced significant proliferative responses in IEL cultures. The active component was apparently a heat shock protein (HSP), since recombinant 71-kDa HSP from Mtb induced IEL to proliferate, while 65-kDa HSP from M. bovis and M. leprae did not. Both alpha/beta and gamma/delta TCR-enriched IEL gave proliferative responses to 71-kDa HSP. Further, culture supernatants from IEL stimulated with 71-kDa HSP contained elevated levels of interleukin-(IL)-3/granulocyte-macrophage colony-stimulating factor, interferon-gamma and IL-6, but not IL-2, IL-4, IL-5 or transforming growth factor-beta. Finally, several IEL T cell clones have been maintained for up to 6 weeks, when stimulated with 71-kDa HSP, IL-2 and feeder cells. Our results show that the 71-kDa HSP of Mtb induces IEL T cells to divide and to secrete cytokines and this may offer a model for cloning and study of IEL T cells in vitro.

A kinetic study on the deletion of thymic, peripheral, and gut-associated V beta 6+ T cells in an Mls-1b BALB/c colony infected with an exogenous mouse mammary tumor virus.

Mls-1a expression results in the deletion of T cells bearing V beta 6 chains of the TCR. However, V beta 6+ T cells are also deleted in Mls-1b BALB/c mice that have been infected with an exogenous mouse mammary tumor virus (swiss mice) via maternal milk intake, and whose open reading frame region is markedly similar to that of the provirus Mtv-7. In this report we describe the kinetics of V beta 6+ T cell deletion in the thymus, spleen, lymph nodes, and gut-associated lymphoid populations of these BALB/c mice from the early weeks of life to 6 mo of age. Deletion of V beta 6+ T cells within the CD4+ T cell population was more obvious in the thymus than in the spleen at 8 wk of age. However, the earliest incidence of deletion was observed in the gut intraepithelial lymphocyte population at 5 wk of age. Furthermore Mtv-7 (SW) transcripts were only found in the gut in the first wk of life, whereafter they could be detected in the thymus, spleen, and lymph nodes. This report suggests that after entering the intestinal tract of host mice, mouse mammary tumor virus (swiss mice) is subsequently transferred to the thymus and peripheral lymphoid organs resulting in the deletion of CD4+V beta 6+ T cells in that order.

Tissue distribution of Mtv-7-like exogenous retroviral transcripts and clonal deletion of V beta 6+ T cells in Mls-1b BALB/c mice.

We have previously described an Mls-1a-like clonal deletion of mature CD4+ T cells which express V beta 6 and V beta 8.1 chains of the TCR in half of the mice of a BALB/c, Mls-1b colony (BALB/c IC). This occurs in the absence of the Mtv-7 provirus which is responsible for the clonal deletion in Mls-1a mice. We developed a polymerase chain reaction assay in order to study the presence of retroviral transcripts homologous to the viral superantigen gene (vSAG) of Mtv-7 and Mtv-6 in various tissues. Mtv-7 homologous transcripts were present in the mammary glands of lactating BALB/c IC mice and in the thymuses and/or spleens of BALB/c IC virgin mice with deletion of V beta 6+ lymph node T cells, and not in BALB/c IC with normal V beta 6 expression. These results indicate that this BALB/c colony is infected with an exogenous mouse mammary tumour virus (MMTV) whose vSAG is similar to Mtv-7, as recently reported. Thymectomies performed at 4-5 weeks of age (at least 4 weeks before detection of clonal deletion), did not affect the occurrence of clonal deletion in peripheral lymph nodes when tested 20 weeks later. This suggests that clonal deletion can be achieved without further intrathymic contact with the antigen. Since MMTV is transmitted through milk and is likely to be present in the gut, we evaluated the percentage of V beta 6+CD4+ T cells within the gut intraepithelial lymphocyte (IEL) population. Mice with normal V beta 6 expression in lymph nodes may show partial deletion of V beta 6+CD4+ IEL.(ABSTRACT TRUNCATED AT 250 WORDS)