A new surface antigen on intraepithelial lymphocytes in the intestine

A new surface molecule has been discovered on mouse intestinal intraepithelial lymphocytes (IEL) using a rat anti-mouse IEL monoclonal antibody, M290. It was expressed at high levels on nearly all IEL and on a majority of T cells in the gut lamina propria. M290 stained, with lower intensity, a small minority of T cells in other lymphoid tissues. Expression was biased towards the CD8+ subset. Stimulation of peripheral T cells with mitogens did not induce expression of the new antigen but addition of transforming growth factor beta to stimulated T cells had a marked inductive effect. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of IEL surface components precipitated with M290 showed principal bands at 135, 120, 28 and 24 kDa (reduced) and 135, 100, 24 and 21 kDa (nonreduced). Precipitation with antibodies to integrin subunits showed that the new molecular complex was not a member of the beta 1, beta 2, or beta 3 integrin families although all of these were represented on IEL. A 13-amino acid N-terminal sequence obtained from the 120-kDa beta subunit of the antigen prepared from an M290+ T hybridoma (MTC-1) did not show homology with integrins. Pulse-chase studies using MTC-1 cells showed that the 135-kDa alpha subunit was derived from a 147-kDa precursor. The function of this new molecular complex is not yet known.     

Murine intestinal intraepithelial lymphocytes I. Relationship of a novel Thy-1-,Lyt-1-,Lyt-2+, granulated subpopulation to natural killer cells and mast cells

Highly purified populations of intraepithelial lymphocytes (IEL) were obtained from the murine small intestine. We found that 84% of IEL expressed Lyt-2 and that 45-55% possessed the unique phenotype, Thy-1-,Lyt-1-,Lyt-2+. Sixty percent of IEL had granules in their cytoplasm and thus resembled the large granulated lymphocytes associated with natural killer (NK) activity. However, less than 15% of IEL had NK activity in a 6-h assay. This activity resided in a subpopulation of Lyt-2- lymphocytes, leaving a large population of Thy-1-,Lyt-1-,Lyt-2+ granulated cells (45-55% of IEL) with unknown function. Sensitive radioenzymic assays for histamine showed that IEL from healthy CBA mice do not contain this amine. IgE-binding assays revealed that IEL, unlike mast cells, do not carry high-affinity receptors for IgE. Thus, only a small percentage of granulated IEL (less than 15%) possess NK activity, whereas 45-55% of IEL have granules, lack typical characteristics of mast cells, express the unique antigenic phenotype, Thy-1-,Lyt-1-,Lyt-2+ and have unknown function.     

痢疾杆菌口服免疫小鼠肠道相关淋巴组织中CD45R~+和CD45RB~+细胞动态变化的观察

为了解肠道相关淋巴组织（GALT）对痢疾杆菌口服免疫的应答状况,分离了Shigella fiexneri四次免疫后Balb/c 小鼠牌（SP）、肠系膜淋巴结（MLN）、Peyer’s结（PP）、固有膜（LP）和上皮内（IE）淋巴细胞,对末次免疫后1～13d内免疫组与对照组GALT中的CD45R~+和CD45RB~+细胞群作了FACS分析。结果免疫后起初7d IE和LPCD45R~+细胞增加而MLN和PP CD45R~+细胞减少。CD45RB~+LPL在起初4d增加,而CD45RB~+IEL总是低于对照。CD45RB~(lo)/CD45RB~(hi)比例在SP和MLN中几乎无变化,但在PP中先降至最低后上升并超过对照。LP中主要为CD45RBD~(lo),IE中主要为CD45RB~(hi)。这些结果在一定程度上反映了粘膜淋巴细胞CD45表达的组织特异性及在痢疾杆菌免疫后B细胞、记忆细胞和辅助细胞的功能状态和变化趋势。     

Distribution of beta 7 integrins in human intestinal mucosa and organized gut-associated lymphoid tissue.

Two alternative integrins involved in mucosal homing (alpha 4 beta 7) or epithelial retention (alpha E beta 7) of lymphocytes were examined in the human gut. The distribution of the beta 7 subunit [monoclonal antibody (mAb) M301] was bimodal in that it was strongly expressed by alpha E beta 7 + cells but weakly by alpha 4 beta 7 + cells. More than 90% of intraepithelial lymphocytes (IEL), including the minor subsets of CD4+, T-cell receptor (TCR) gamma/delta +, and CD3- cells, expressed alpha E beta 7 as did most lamina propria CD8+ (88%) and a fraction (36%) of CD4+ lymphocytes. Conversely, B-lineage cells (CD19+) and macrophages (CD68+) were negative. In gut-associated lymphoid tissue (GALT: Peyer's patches and appendix) only a few (< 5%) cells were positive for alpha E beta 7 (confined to CD8+ lymphocytes and CD11c+ putative dendritic cells). A relatively small fraction of IEL (30-50%) expressed alpha 4 beta 7 (mAb Act-1), while most (70%) lamina propria T and B lymphocytes, blasts, plasma cells and macrophages were positive. In GALT, T lymphocytes expressed similar levels of alpha 4 beta 7 as in the lamina propria whereas relatively few B lymphocytes (< 50%) were positive. Isolated lamina propria CD8+, CD4+, CD19+, and CD38+ cells contained mRNA for alpha 4 and the former three subsets as well as appendix CD8+ cells also for beta 7 while only lamina propria CD8+ cells had mRNA for alpha E. Together, the results suggested that alpha E beta 7 and alpha 4 beta 7 are differentially regulated in inductive sites and effector sites of the human gut. Because lymphoid cells at both sites expressed mainly alpha 4 beta 7, this integrin may be a homing receptor on memory and effector cells bound for lamina propria as well as on naive lymphocytes extravasating in GALT. Conversely, because alpha E beta 7 was mainly expressed by CD8+ cells in epithelium and lamina propria, it was probably induced after extravasation, in agreement with the observation that IEL and a fraction of lamina propria T lymphocytes (mainly CD8+ cells) generally expressed higher levels of beta 7 than most CD4+ and B cells. Also a subset of putative dendritic cells located near the follicle-associated epithelium of GALT expressed alpha E beta 7, perhaps reflecting epithelial interaction during primary immune responses.     

A monoclonal antibody (HML-1) defining a novel membrane molecule present on human intestinal lymphocytes

A monoclonal antibody, HML-1, was produced by fusion of NSI myeloma cells with spleen cells of a mouse immunized with isolated human intestinal intraepithelial lymphocytes (IEL). Immunofluorescence studies of isolated cells, as well as immunoperoxidase staining of tissue sections, indicated that HML-1 labeled all the various subsets of human intestinal IEL, approximately 40% of lamina propria T cells, 30% mesenteric lymphoblasts and some lymphocytes in other mucosae, particularly IEL. Conversely, it revealed only rare cells in all other lymphoid compartments. Analysis by polyacrylamide gel gradient electrophoresis showed that HML-1 precipitated two major noncovalently bound components of approximate mol. masses of 105 and 150 kDa from human IEL. HML-1 thus defines a novel human membrane antigen present on a subpopulation of lymphocytes preferentially associated with epithelia, and particularly with the intestinal epithelium. The characteristics of this human antigen are very similar to those of an antigen we had previously described in the rat. The possible functional role of this novel class of lymphocyte membrane antigens as well as the nature of the mechanism that triggers their expression remain to be elucidated.     

Expansion of alpha beta T-cell receptor-bearing intestinal intraepithelial lymphocytes after microbial colonization in germ-free mice and its independence from thymus.

A large proportion of intestinal intraepithelial lymphocytes (IEL) comprises alpha beta and gamma delta T-cell receptor (TcR)-bearing T cells. The numbers of alpha beta and gamma delta IEL are reported to be very different between germ-free and conventional microbial conditions. In this study, we investigated the kinetics of both types of TcR-bearing cells after microbial colonization in germ-free mice and the influence of thymus deprivation on IEL populations during the microbial association process. Immediately after association with microbes in germ-free animals, the number of alpha beta TcR-bearing IEL gradually increased. Fourteen days after microbial association the number of alpha beta IEL equalled that of gamma delta TcR-bearing IEL. Approximately 1 month after microbial association, the number of alpha beta IEL was several times greater than that of gamma delta IEL, having almost reached the level in conventional mice reared in a conventional animal room after birth. On the other hand the number of gamma delta IEL hardly changed throughout this microbial association process. Two-colour analysis involving anti-alpha beta TcR and anti-Lyt-2 or Lyt-3 antibodies showed that the major fraction of IEL that increased after microbial association comprised alpha beta TcR-bearing T cells expressing CD8 antigen composed of a homodimer of alpha-chains, which was not detected in other gut associated-lymphoid tissues (GALT) such as Peyer's patch, mesenteric lymph node and lamina propria tissue. The number of alpha beta T cells in these GALT increased within 1 week more quickly than that of IEL. The increase in alpha beta IEL after microbial association was not prevented by thymectomy. These results strongly suggest that the progenitors of alpha beta TcR-bearing IEL expand outside the thymus in response to microbial colonization in germ-free mice.     

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.     

Rapid killing of murine lymph node T blasts by intestinal intraepithelial lymphocytes in vitro

We examined the effect of murine intestinal intraepithelial lymphocytes (IEL) on the proliferation of murine lymph node T cells (LN-T) in vitro. An IEL fraction prevented the proliferation of LN-T stimulated with antigen and X-irradiated spleen cells, or with anti-CD3 monoclonal antibodies (mAb). Concanavalin A-activated LN-T were less sensitive. Such an inhibitory activity was recovered from a CD8-depleted population by panning of bulk IEL using anti-CD8α mAb. This population of BALB/c IEL showed less granzyme A activity, and its surface markers were positive for CD8 (～4%), CD3 (80–90%), CD4 (2–6%), αβTcR (45–70%), and γδTcR (4–9%). Asialo-GM1 and Thy1.2 were variably expressed, but interleukin-2 (IL-2) receptor-α and FcγR were not. By contrast, no cytotoxicity against YAC-1 was detected in a CD8-depleted IEL population by a 6-h ⁵¹Cr-release assay. Although IEL from severe-combined immunodeficient mice lacking CD4, CD8 and TcR, but expressing IL-2 receptor, showed cytotoxicity against YAC-1, their inhibitory activity against LN-T was almost the same as that by IEL from BALB/c mice. When LN-T blasts (>75% CD4⁺) activated with anti-CD3 were treated with CD8-depleted IEL, intact cellular DNA of the T blasts disappeared within 1 h with increased amounts of small-sized DNA. These results suggest that CD8^? IEL directly and nonspecifically kill lymph node CD4⁺ T blasts and possibly down-regulate TcR-mediated proliferation of peripheral T cells in the gut epithelium.     

Comprehensive phenotypic analysis of the gut intra-epithelial lymphocyte compartment: perturbations induced by acute reovirus 1/L infection of the gastrointestinal tract

Intestinal intra-epithelial lymphocytes (IELs) form a highly specialized lymphoid compartment. IELs consist primarily of T cells that are dispersed as single cells within the epithelial cell layer that surrounds the intestinal lumen. These lymphocytes along with lamina propria lymphocytes are considered to play an important role in the regulation of immune responses. IELs are heterogeneous with regard to phenotype, and they contain sub-populations with diverse functions. In our most recent study, we found that intra-duodenal inoculation of mice with reovirus serotype 1/strain Lang (reovirus 1/L) induced expression of both germinal center and T cell antigen and CD11c on IELs suggesting these cells to be the recently stimulated cells in gut mucosal tissue. We also demonstrated that IELs from these mice when cultured in vitro in the presence of reovirus 1/L-pulsed antigen-presenting cells generated reovirus 1/L-specific MHC-restricted CTL whose function was mediated utilizing perforin, Fas-FasL and TRAIL mechanisms. This present study provides a comprehensive analysis of the diverse subsets of IELs, which function with other mucosal cells to provide a strong, protective immunity in a highly regulated fashion inside the microenvironment of the intestinal epithelium. We demonstrated that the IEL population contains both thymus-dependent (TD) and thymus-independent (TI) lymphocytes in mice and that a complex phenotype is present when sub-populations are analyzed for TCR, Thy-1, CD4, CD8 and B220 expression in a comprehensive manner. In reovirus 1/L-inoculated mice, we found a decrease in the TI population and an increase in the TD population characterized by significant alterations in various sub-populations. This increase was largely due to an increase in CD4(+), CD8(+) and CD4/CD8 double-positive sub-populations of TD IELs. Intracellular cytokine analysis demonstrated induction of IFN-gamma and an increase in effector/cytotoxic CD8 and CD4 cells after reovirus 1/L infection. These results suggest that TD IELs may play an important role in the clearance of reovirus 1/L infection from gut.     

Age-associated increase in number of CD4+CD8+ intestinal intraepithelial lymphocytes in rats.

A significant number of CD4+CD8+ T cells were detected in intestinal intraepithelial lymphocytes (IEL) of various strains of rats including Wistar, WKA, BN, LEW and F344. The site of the CD4+CD8+ population in IEL increased with age in all strains we examined. Most IEL bearing CD8 expressed no CD5 antigen in young rats, while all CD4+CD8+ IEL and some of CD8+ IEL in aged rats were of CD5+CD45RB- phenotype. In germ-free Wistar rats, age-associated increase in the number of CD4+CD8+CD5+ IEL was not evident, indicating that stimulation by the intestinal microflora was important for expansion of the CD4+CD8+CD5+CD45RB- IEL. Aged athymic F344 nude rats contained appreciable numbers of CD4+ IEL and CD8+ IEL but few CD4+CD8+ IEL, suggesting that the CD4+CD8+ IEL may be derived from thymus-dependent populations. Unlike a majority of CD4+CD8+ thymocytes bearing a low intensity of CD3/T cell receptor (TcR) alpha/beta, the CD4+CD8+ T cells in IEL expressed a high intensity of CD3/TcR alpha/beta on their surface. The CD4+CD8+ IEL appear to contribute to the spontaneous proliferation of the IEL in aged rats as assessed by tritiated thymidine incorporation after in vitro culture with medium only. These results suggest that with aging a unique CD4+CD8+ IEL may expand at a local site of the intestine under the influence of intestinal microflora and may contribute to the first line of defense against various pathogens in the epithelium.     

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.     

Altered distribution of mucosal NK cells during HIV infection

The human gut mucosa is a major site of human immunodeficiency virus (HIV) infection and infection-associated pathogenesis. Increasing evidence shows that natural killer (NK) cells have an important role in control of HIV infection, but the mechanism(s) by which they mediate antiviral activity in the gut is unclear. Here, we show that two distinct subsets of NK cells exist in the gut, one localized to intraepithelial spaces (intraepithelial lymphocytes, IELs) and the other to the lamina propria (LP). The frequency of both subsets of NK cells was reduced in chronic infection, whereas IEL NK cells remained stable in spontaneous controllers with protective killer immunoglobulin-like receptor/human leukocyte antigen genotypes. Both IEL and LP NK cells were significantly expanded in immunological non-responsive patients, who incompletely recovered CD4+ T cells on highly active antiretroviral therapy (HAART). These data suggest that both IEL and LP NK cells may expand in the gut in an effort to compensate for compromised CD4+ T-cell recovery, but that only IEL NK cells may be involved in providing durable control of HIV in the gut.     

Functional properties of lymphocytes isolated from murine small intestinal epithelium

Intraepithelial lymphocytes (IEL) were isolated from murine small intestine using a modification of previously published procedures. Analysis of IEL by immunofluorescence using monoclonal antibodies showed they were predominantly Lyt-2+ cells, with relatively few B cells or macrophages present. IEL cultured at sufficiently high cell densities proliferated in response to concanavalin A (Con A), phytohaemagglutin (PHA) and lipopolysaccharide (LPS). IEL were also capable of recognizing alloantigens in an in vivo graft-versus-host assay and in an in vitro mixed lymphocyte reaction. These studies therefore confirm that murine IEL contain cells with immunologic properties characteristic of typical T lymphocytes.     

Mechanisms of binding of cutaneous lymphocyte-associated antigen-positive and αeβ7-positive lymphocytes to oral and skin keratinocytes

Intraepithelial lymphocytes (IEL) utilize the integrin αeβ7 on their surface to bind to E-cadherin on epithelial cells in the gut and breast. In oral mucosa and skin IEL express αeβ7 and the cutaneous lymphocyte-associated antigen (CLA) but the mechanisms of adhesion of these subsets to keratinocytes are unknown. Levels of αeβ7 and CLA were up-regulated on peripheral blood lymphocytes (PBL) by transforming growth factor-β (TGF-β) and interleukin-12 (IL-12), respectively, and both groups of lymphocytes adhered onto oral and skin keratinocytes. Adhesion of IL-12-activated PBL was totally abolished by anti-lymphocyte-associated function antigen type 1 (anti-LFA-1) antibodies but was unaffected by anti-αeβ7 antibodies indicating that adhesion of the CLA-positive subset is mediated via LFA-1 interaction with intercellular adhesion molecule-1 (ICAM-1). Adhesion of TGF-β-activated PBL to E-cadherin-positive oral and skin keratinocytes was partially inhibited by anti-αeβ7 antibodies but was unaffected by the blocking antibody E4.6 against E-cadherin which detects the binding site for αeβ7-positive lymphocytes in breast and gut epithelium. TGF-β-activated PBL also bound to an E-cadherin-negative oral keratinocyte cell line and adhesion was inhibited by anti-αeβ7 antibodies. These results strongly suggest that in oral epithelium and epidermis αeβ7-positive lymphocytes do not bind to E-cadherin and there may be a novel second ligand for the αeβ7 integrin.     

Gamma/delta T cells and the diagnosis of coeliac disease.

Gamma/delta T cells are increased in the gut epithelium of patients with coeliac disease compared with normal controls. The aim of this study was to determine whether the increase in gamma delta intraepithelial lymphocytes (IEL) is specific for coeliac disease, in which case it could be of diagnostic importance. Biopsies were obtained from children with no intestinal disease, coeliac disease, cow-milk-sensitive enteropathy/post-enteritis syndrome (CMSE PES) and miscellaneous other enteropathies (n = 67). Intraepithelial CD3+ and gamma delta T cells were identified in frozen sections using peroxidase immunohistochemistry. In normal biopsies there were 0-7 gamma delta IEL/100 cells in the epithelium. In untreated coeliac patients this increased to 9-22 gamma delta IEL/100 cells in the epithelium (P = 0.000004). Of 27 patients with morphologic intestinal damage which was not due to coeliac disease, four with CMSE/PES had gamma delta IEL/100 cells in the epithelium in the same range as the patients with coeliac disease. Of these, two had high densities of CD3+ IEL in the epithelium and were indistinguishable from patients with untreated coeliac disease. The other two could be excluded as possible coeliacs because their CD3+ IEL/100 epithelial cells were in the normal range. Thus an increase in gamma delta IEL is not specific for coeliac disease. However, enumeration of both of gamma delta IEL and CD3+ IEL densities will be useful in the exclusion of coeliac disease as a diagnosis in some children.     

The Neonatal Development of Intraepithelial and Lamina Propria Lymphocytes in the Murine Small Intestine

During early neonatal life, important changes occur in the gut. The intestine is challenged by both milk and a microbial flora. Later on, at weaning, the diet of mice changes from milk to pelleted food leading to changes in microbial contents. This period seems essential for a complete development of the mucosal immune system. We investigated the development of both intraepithelial (IEL) and lamina propria lymphocytes (LPL), from day 5, and every 5 days, up to day 30 after birth. IEL and LPL were isolated from the small intestine and the phenotype was assessed by FACS analyses, using antibodies for detection of T-cell markers CD3, TCRαβ, TCRγδ, CD4, CD8α, CD8β, CD5, CD18, CD54, and CD49d. Our data show a clear increase in the number of LPL just before weaning, while the number of IEL increased after day 15. A more mature pattern of membrane antigen expression of both IEL and LPL was observed at weaning. The adhesion molecules CD18, CD54, and CD49d, essential for cellular communication of lymphocytes, showed an expression peak at weaning. In conclusion, the mouse mucosal immune system develops during the first 3 weeks of neonatal life leading to the formation of a more mature immune system at weaning.     

Intestinal T lymphocytes of different rat strains in immunotoxicity

In order to study the intestinal mucosal immune cells, with emphasis on single T lymphocytes, an inventory was made of single and organized lymphocytes in the epithelium and lamina propria of the small intestines of untreated Wistar, Fischer 344, and Lewis rats. The single and organized lymphocytes were examined microscopically. In addition, the single lymphocytes in the epithelium (IEL) and lamina propria (LPL) were analyzed by flow cytometry. Next, the use of flow cytometry analysis was explored to detect changes in the IEL T-lymphocyte population in subacute oral studies with the immunomodulating agents azathioprine and hexachlorobenzene. Untreated random-bred Wistar rats exhibited a large interindividual variability in IEL composition, while the variability was small in inbred Fischer 344 and Lewis rats. The explorative study with the 2 model immunomodulating compounds demonstrated that hexachlorobenzene increased the number of intraepithelial T lymphocytes with CD8+ phenotype at the cost of T cells with CD4+ phenotype in Lewis rats. Azathioprine did not induce distinct effects on the percentages of IEL. The data indicate that the intraepithelial lymphocytes in the intestines are a potential target for orally administered immunomodulating compounds and should therefore receive more attention in toxicologic pathology studies.     

Identification and characterization of lymphoid precursors in the murine intestinal epithelium.

Although in vivo evidence supports a role for the murine intestinal epithelium in the extrathymic generation of certain intraepithelial T lymphocytes (IEL), no intraepithelial cells with in vitro lymphoid progenitor potential have yet been demonstrated. Using reaggregate fetal thymic organ culture techniques, we show that a subset of CD3(-) cells isolated from the intestinal epithelium of young mice is capable of generating T cells (alpha beta and gamma delta) and NK1.1(+) cells in vitro. A novel IEL subset bearing a low level of CD45 was identified and found to comprise cells expressing highly immature lymphoid markers including CD34, c-kit, CD122, CD127 and high levels of CD16 and CD44. This subset represents 20-30% of intraepithelial CD45(+) cells from 4-week-old wild-type and nude mouse strains and contains cells with in vitro T cell differentiation capacity. The identification of such an early pluripotent precursor phenotype within the intestinal epithelium implies that the potential for T cell generation exists at this site, and suggests that extrathymic T cell generation may occur within the epithelium itself.     

T cell receptor-bearing cells among rat intestinal intraepithelial lymphocytes are mainly alpha/beta+ and are thymus dependent

Rearrangement of both the beta and gamma chain T cell receptor (TcR) genes was detected in intestinal intraepithelial lymphocytes (IEL) from normal euthymic rats. Flow cytometric analyses showed that about 73% of the IEL were CD3+ (1F4) and that 67% were TcR alpha/beta+ (R73). About 5% of the IEL were found to be CD3+, TcR alpha/beta- in double-labeling experiments suggesting that a small fraction of IEL in the rat express the alternative TcR gamma/delta. More than 70% of the IEL were granular implying that many CD3+ IEL are granular. In IEL from athymic nude rats no rearrangement of either the TcR beta or gamma chain genes or surface expression of CD3 or TcR alpha/beta was detected despite the fact that about 95% of the cells were granular and morphologically similar to those in normal rats. Taken together our data suggest that the majority of IEL in the rat express the conventional TcR alpha/beta and that TcR-bearing cells in the gut epithelium are thymus dependent.