Intraepithelial Lymphocyte Population Research Articles

Analysis of intestinal intraepithelial lymphocyte populations in experimental Trichinella spiralis infection of mice

The potential role of intestinal intraepithelial lymphocytes (i-IELs) in the generation of host protective immunity after helminth infection was investigated using the Trichinella spiralis (Owen, 1835)/mouse model. In this study we found a significant rise of TCRgamma(delta)+ i-IELs (P < 0.001) concurrent with the jejunal goblet cells (GC) hyperplasia in T. spiralis-infected C57BL mice on day 4 p.i. However, no direct relationship between the kinetics of the increase in TCRgamma(delta)+ i-IELs and T. spiralis expulsion was observed in infected mice. Taken together, these results implicate that gamma(delta) i-IELs probably perform a unique functions related to the regulation of the GC proliferation accompanying T. spiralis gut infection. As is known, these TCRgamma(delta)+ i-IELs may release mediators or growth factors that in turn influence GC differentiation. With the use of dexamethason (DEX), a potent anti-inflammatory agent which also induces apoptotic cell death in i-IELs, we have confirmed that the expulsion of T. spiralis from the mouse gut is accompanied by an inflammatory response. Indeed, the GC are clearly involved in these phenomena, apparently under the regulation by TCRgamma(delta)+ i-IEL-mediated responses, since DEX abrogated GC proliferation in T. spiralis-infected C57BL mice and subsequently augmented adult worm burden. Our data also show that the rejection of adult worms starts concurrently with a significant increase in TCRalpha(beta)+ and CD8+ i-IELs (P < 0.05 and P < or = 0.01, respectively), namely by day 7 p.i. At the same time, CD4+ cells significantly decreased (P < 0.05) in the intestinal epithelium of T. spiralis-infected, vs uninfected mice. These results may indicate that the TCRalpha(beta)+ and CD8+ i-IELs act as effectors of anti-T spiralis defence reactions. The implications of these findings for the potential role of intestinal intraepithelial CD8+ and TCRalpha(beta)+ cells in the pathogenesis of the intestinal lesions during T. spiralis gut infection are discussed.

The Distribution of Lymphocytes Expressing αβ and γδ T-cell Receptors, and the Expression of Mucosal Addressin Cell Adhesion Molecule-1 in the Canine Intestine

The present study defined the distribution of αβ and γδ T-cell subsets, and the expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the intestinal tract of an outbred population of dogs with no evidence of gastrointestinal disease. A panel of monoclonal antibodies reactive with the canine αβ and γδ T-cell receptors (TCRs) and human MAdCAM-1 was used in a series of immunoperoxidase or immunofluorescence studies. αβ T cells were predominant within the villous lamina propria with a significantly decreasing linear trend from upper villus to crypt (P<0·0001). A proportion of intraepithelial lymphocytes (IELs) also expressed the αβ T-cell receptor, with significantly greater numbers in villous than in crypt epithelium (P<0·0001). However, there were no significant differences in the numbers of either lamina propria or epithelial αβ T cells between different intestinal regions. γδ T cells were rare in the lamina propria, but a prominent γδ IEL population was present and shown by double-colour immunofluorescence studies to be principally of the CD4-CD8α- phenotype. MAdCAM-1 was expressed by endothelial cells in the mucosa, sub-mucosa and muscularis layers of all levels of the intestinal tract. In the mucosa, significantly more MAdCAM-1 positive endothelium was present in regions of crypt than villous lamina propria (P<0·0001), but there were no significant differences between expression in different intestinal regions. The quantitative and qualitative data will enable comparisons of these parameters to be made with those in dogs suffering from inflammatory bowel diseases.

A new isolation method for rat intraepithelial lymphocytes

Intraepithelial lymphocytes (IELs) play critical roles in gut immunity. In mice, γδ T cells are a large component of the IEL population. In the rat, γδ IELs are reportedly much less common, but technical issues suggest that previous analyses should be interpreted cautiously. The study of IELs in rats has been impeded by isolation procedures that are lengthy and complex, leading to small cell yields. For this reason, it is possible that rat IELs analyzed in previous studies have not been representative of the entire IEL compartment. We report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The method is rapid and requires neither enzymatic digestion, nor surgical removal of Peyer's patches, nor vigorous mechanical manipulation of the intestine. The yield of rat IELs using this method is 5- to 10-fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrated that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis revealed five major subsets of IELs based on differential cell surface expression of CD4, CD8, and αβ T cell receptor (TcR). Among the αβTcR − cells was a population of γδ T cells present at levels not previously detected. The isolation of IEL sub-populations using this methodology should facilitate studies of the function of these cells in gut immunity.

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.

Phenotypic comparison of ileal intraepithelial lymphocyte populations of suckling and weaned calves

Ileal intraepithelial lymphocyte (IEL) suspensions from suckling calves (1–3 weeks old) and weaned calves (3–6 months old) were phenotyped to determine whether there were differences in the lymphocyte populations consistent with postnatal maturation of the mucosal immune system. Flow cytometric comparisons of IEL from the two age groups revealed the presence of significantly larger proportions of CD4 + T lymphocytes and CD8 + T cells in the weaned animals. In contrast, there was a significantly larger proportion of B-B2 + IEL in the suckling calves. Freshly isolated IEL from both groups of calves expressed mRNA for TNF-α and IFN-γ, but not IL-4 or IL-10. The B-B2 + IEL population was more closely examined by flow cytometry. These cells co-expressed IgM and CD21. However, they did not express IgA, IgG1, nor any of several additional leukocyte differentiation molecules. Immunohistochemical data confirmed the presence of IgM + lymphocytes, and the paucity of IgA + and IgG1 + lymphocytes in suckling calf ileum. However, substantial numbers of IgA + and IgG1 + cells were observed in weaned calf ileum. Together, the data are consistent with ongoing postnatal maturation of the gut mucosal immune system.

Bovine T cell responses to Cryptosporidium parvum infection

To better understand the immune mechanisms important for clearing of the primary infection and the subsequent development of resistance to Cryptosporidium parvum infection, several groups have recently characterised changes within the lymphoid cell population of the intestinal mucosa and associated lymphoid tissue in calves with cryptosporidiosis. In naive animals, infection results in a significant increase in the number of CD4 + and CD8 + T cells present within the intraepithelial lymphocyte population, lamina propria and Peyer’s patch of the ileum. This is accompanied by a rapid and transient increase in the number of γ\\ δ T cells present within the intestinal villi. In response to a challenge infection in immune calves, there is a substantial increase in the number of CD4 + T cells present in the Peyer’s patch of the ileum and a specific localisation of CD8 + T cells to the epithelium of the intestinal villi. Together, these data demonstrate that C. parvum elicits a strong cell-mediated response following both primary and secondary infections in calves, and that CD8 + T cells may play an important role in the bovine immune response to C. parvum infection.

Local Ileal Cytokine Responses in Cattle during a Primary Infection with Cryptosporidium parvum

In the present study, localized changes in cytokine transcription profiles were examined in neonatal calves following a primary infection with Cryptosporidium parvum, using competitive reverse transcriptase polymerase chain reaction (RT-PCR). Total RNA was prepared from ileocecal lymph nodes (LN), lamina propria lymphocytes (LPL), and intraepithelial lymphocytes (IEL) isolated from neonatal calves 7 days after C. parvum infection. Competitive RT-PCR performed on cDNA samples containing internal cytokine gene competitor molecules showed increases in the levels of interferon-gamma and interleukin-12 (IL-12) (P40) mRNA in both LPL and IEL populations but not in the draining LN. In addition, the levels of mRNA of the newly identified growth factor IL-15 decreased in the IEL of the infected animals. No consistent differences were seen in any of the cell populations when the samples were analyzed for IL-10 and levels of mRNA for IL-2 and IL-4 were low and highly variable in both infected and control groups in all 3 lymphocyte populations.

Invited Review/Controversial Issue: Can Duodenal Mucosa Appear Normal in Gluten-Sensitive Enteropathy (Celiac Disease)?

Increasing popularity of serologic and permeability studies in patients investigated for possible celiac disease has resulted in a growing awareness among clinicians that currently accepted histologic criteria for diagnosis of celiac disease lack sensitivity. The patients who improve on a gluten-free diet with abnormal findings from serologic and permeability studies may have normal or “almost” normal villous/crypt morphology. The population of intraepithelial lymphocytes once extensively discussed in the literature may after all prove useful in identifying the individuals who may benefit from dietary gluten withdrawal. A recently outlined spectrum of the mucosal pattern (M. N. Marsh), which may be seen in the gluten-sensitive enteropathy is valuable in assessing histomorphology of small bowel biopsies from the patients investigated for celiac disease.

Intraepithelial lymphocytes: origins, distribution, and function.

Intraepithelial lymphocytes (IEL) are associated with the intestinal tract, respiratory tract, genitourinary tract epithelium, and the skin and are the first immune system cells to encounter pathogens that have invaded an epithelial surface. IEL are predominantly T cells (CD3+) with CD8+ cells predominating at most, but not all, sites. Both TCR alphabeta+ and TCR gammadelta+ cells are found within IEL populations and an increasing body of evidence suggests that some IEL may arise extrathymically. The presence within intestinal IEL of cells expressing potentially self-reactive TCR suggests that T cell selection within epithelia may differ from thymic T cell selection although recent evidence suggests that these cells may in fact be nonresponsive. IEL exhibit various cytotoxic activities including alloreactive and virus-specific CTL activity, NK activity and spontaneous cytotoxicity, activities consistent with an immune surveillance or first line of defence role. IEL also appear activated in vivo and secrete a variety of cytokines. Subsets of IEL have been shown to provide B cell help, to play a role in the maintenance of oral tolerance and to regulate epithelial cell function. In this review the morphology, distribution and phenotype of IEL, the potential for extrathymic development and possible functions of this unique lymphoid population are discussed.

Cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns

Twenty-six experimentally infected calves were monitored daily for oocyst excretion. All began excreting oocysts 3–6 days p.i. Most calves ( n=23) excreted oocysts for 6–9 days, with a daily range from 4 × 10 2 to 4.15 × 10 7 oocystsg −1 of faeces. Over half the calves excreted peak numbers of oocysts 6–8 days p.i. Diarrhoea, observed intermittently beginning as early as day 3 p.i., lasted 4–16 days and varied greatly in severity from calf to calf. In a second study, nine of 18 calves were orally inoculated with 5 × 10 6 oocysts between birth and 2 days of age and nine remained uninfected. Monoclonal antibodies for cell surface markers indicated substantial increases in CD4 + and CD8 + T cells in the intraepithelial lymphocyte population of the ilea of infected calves at 7–9 days of age. RT-PCR demonstrated increases in mRNA for interleukin-12 and interferon-γ that correlated with increases in both CD4 + and CD8 + intraepithelial lymphocyte cells. Increased mRNA for interleukin-12 and interferon-γ from lamina propria lymphocytes correlated with increased numbers of CD8 + cells. No changes were found in interleukin-2, interleukin-4 or interleukin-10 mRNA levels. However, interleukin-15 mRNA, possibly from epithelial cells contaminating intraepithelial lymphocytes, was decreased in infected calves and had a negative correlation with increases in CD4 + and CD8 + cells. No differences were detected in mRNA levels for cytokines from lymph node lymphocytes.

Spermine induces maturation of the immature intestinal immune system in neonatal mice.

In mice, immunological adaptation of the gut to microbial and nutritional antigens occurs at weaning in parallel with biochemical and morphological maturation. Because oral administration of spermine to neonatal rats has been shown to induce biochemical and morphological maturation, we investigated whether spermine also affects maturation of the mucosal immune system. Swiss mice 7, 12, and 27 days old were given spermine orally (0.5 mumol/g body weight) during 3 days. Intestinal length was measured, and lactase and sucrase activities were determined. The phenotype of intraepithelial and lamina propria lymphocytes was assessed by FACS analysis using markers for CD3, TCR alpha beta, TCR gamma delta, CD4, CD8 alpha, CD8 beta, CD5, CD18, CD54, and CD49d. Similar to what occurs during natural development, spermine treatment of neonatal mice increased intestinal length, decreased lactase activity, and increased sucrase activity. The percentage of intraepithelial lymphocytes expressing TCR alpha beta, CD4, CD5, and CD54, as well as the levels of expression of these antigens, increased after spermine treatment on day 12, similarly to natural maturation. The increase in expression of CD3, TCR gamma delta, CD18, and CD49d did not reach statistical significance. No effect was observed on CD8 expression. The phenotype of lamina propria lymphocytes was not affected. Spermine administration to 7- and 27-day-old mice had no effect on the phenotype of either intraepithelial or lamina propria lymphocytes. Oral spermine administration to neonatal mice induced, in parallel with biochemical maturation, precocious maturation of the murine intestinal immune system and particularly affected differentiation of the intraepithelial lymphocyte population.

Spermine Induces Maturation of the Immature Intestinal Immune System in Neonatal Mice

Background:In mice, immunological adaptation of the gut to microbial and nutritional antigens occurs at weaning in parallel with biochemical and morphological maturation. Because oral administration of spermine to neonatal rats has been shown to induce biochemical and morphological maturation, we investigated whether spermine also affects maturation of the mucosal immune system.Methods:Swiss mice 7, 12, and 27 days old were given spermine orally (0.5 μmol/g body weight) during 3 days. Intestinal length was measured, and lactase and sucrase activities were determined. The phenotype of intraepithelial and lamina propria lymphocytes was assessed by FACS analysis using markers for CD3, TCRαβ, TCRγδ, CD4, CD8α, CD8β, CD5, CD18, CD54, and CD49d.Results:Similar to what occurs during natural development, spermine treatment of neonatal mice increased intestinal length, decreased lactase activity, and increased sucrase activity. The percentage of intraepithelial lymphocytes expressing TCRαβ, CD4, CD5, and CD54, as well as the levels of expression of these antigens, increased after spermine treatment on day 12, similarly to natural maturation. The increase in expression of CD3, TCRγδ, CD18, and CD49d did not reach statistical significance. No effect was observed on CD8 expression. The phenotype of lamina propria lymphocytes was not affected. Spermine administration to 7‐ and 27‐day‐old mice had no effect on the phenotype of either intraepithelial or lamina propria lymphocytes.Conclusions:Oral spermine administration to neonatal mice induced, in parallel with biochemical maturation, precocious maturation of the murine intestinal immune system and particularly affected differentiation of the intraepithelial lymphocyte population.

Age-dependent intestinal lymphoproliferative disorder due to stem cell factor receptor deficiency: parameters in small and large intestine.

Signaling through c-Kit/stem cell factor (SCF) is crucial for normal development of erythroid and myeloid hematopoietic precursors and of melanocytes and germ cells. While peripheral lymphoid populations of W/Wv and SI/SId mice appear normal, we demonstrated that the intraepithelial lymphocyte (IEL) populations of small (SI) and large (LI) intestine were significantly affected. IEL populations of young W/Wv animals were indistinguishable from those of their control littermates, but an age-dependent decrease in SI and LI TCRgamma delta IEL occurred in c-Kit mutant mice. In SI, but not in LI, this diminution was accompanied by gross expansion of TCRalpha beta IEL that resulted in significantly increased IEL:epithelial cell ratios in c-Kit mutant mice. Bromodeoxyuridine labeling studies revealed that the increase in cell numbers was due to lymphoproliferation that occurred in situ. Interestingly, TCRgamma delta IEL expressed cell surface c-Kit, while the expanding population of TCRalpha beta IEL did not. Analysis of radiation bone marrow chimeras demonstrated that the dysregulation required either disruption of stromal cell SCF or IEL c-Kit and showed that the effect on IEL or their precursors was not due to other changes in the intestinal microenvironment. Lamina propria T cell populations in these mice were unaffected, reinforcing the idea that the developmental requirements of these gut-resident lymphocyte populations are distinct. Overall, the results demonstrated that the development of intestinal TCRgamma delta IEL, regardless of location, shares common requirements for SCF, while SI and LI TCRalpha beta IEL may develop along distinct pathways. Possible mechanisms for the loss of proliferative regulation in gut T cells in c-Kit/SCF deficiency are discussed.

Activation of intestinal intraepithelial T lymphocytes in calves infected with Cryptosporidium parvum.

The objective of this study was to identify disease-related changes in lymphocyte populations within ileal mucosae of calves with cryptosporidiosis. Groups of five neonatal calves were orally infected at 3 days of age with 10(8) oocysts and maintained in enteric-pathogen-free conditions until clinical disease was established or until the animals had recovered from disease. Age-matched uninfected calves were used for comparison. Ileal mucosal lymphocytes were collected, quantitated, and phenotyped to determine whether changes in lymphocyte composition occurred in infected animals. We observed significantly larger numbers of intraepithelial CD8+ T lymphocytes in ileal mucosae from acutely infected calves compared with those from control animals. In addition, a proportion of intraepithelial CD4+ T cells from acutely infected calves coexpressed CD25, whereas there was an absence of coexpressed CD25 on CD4+ T cells from control calves. Ex vivo reverse transcriptase PCR of RNA from intraepithelial lymphocytes from control calves showed a cytokine expression pattern consisting of tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma), while intraepithelial lymphocytes from calves with cryptosporidiosis expressed IFN-gamma but not TNF-alpha. Together, the results indicate that changes occur in the ileal intraepithelial lymphocyte population coincidently with Cryptosporidium parvum-induced enteric disease.

TAP-independent selection of CD8+ intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IEL) are mostly CD8 single positive T cells. IEL with a TCR-alpha(beta) that are CD8 single positive are absent from beta(2)-microglobulin (beta(2)m)-deficient mice, consistent with the idea that these IEL, like other TCR-alpha(beta)+, CD8+ T cells, require class I molecules for positive selection. In contrast, here we show that substantial numbers of TCR-alpha(beta)+, CD8 single positive IEL are present in mice deficient for the transporter associated with Ag processing 1 (TAP 1) gene, although T cells with this phenotype are absent from thymus, spleen, and lymph nodes of these same mice. The majority of TCR-alpha(beta)+, CD8 single positive IEL in TAP-deficient mice expresses CD8 molecules composed of alpha(alpha) homodimers and they express a diverse set of V(beta) gene segments. In addition, the number of TCR-alpha(beta)+, CD4/CD8 double positive IEL is decreased in beta(2)m-deficient mice but not in TAP-deficient mice. The dependence of the two TCR-alpha(beta)+ IEL populations that express CD8alpha(alpha) homodimers on beta(2)m as opposed to TAP molecules is striking. It suggests that TAP-independent but beta(2)m-requiring nonclassical class I molecules expressed by cells in the intestine, such as the thymus leukemia Ag and CD1, could play a pivotal role in the development and/or the accumulation of major subpopulations of TCR-alpha(beta)+ IEL.

Phenotypic and functional assessment of intraepithelial lymphocytes (IEL) isolated from rat colon and small bowel

We have compared the proliferative potential of IELs isolated from rat colon (CIEL) and small bowel (SBIEL), and compared this with that observed using spleen lymphocytes. Unless additional irradiated spleen cells were added as a source of accessory cells, both IEL populations show poor proliferation in response to Con A stimulation. The CD4/CD8 ratio in spleen, SBIEL and CIEL was markedly different (3:1, 1:3, and 1:1, respectively). Cells expressing surface markers characteristic of macrophages were not routinely found in SBIELs. Both IEL preparations inhibited spleen cell proliferation in response to Con A or immobilized anti-CD3, and produced a soluble factor(s) capable of causing similar inhibition. For CIEL this inhibition was dependent upon a proliferation-independent but cell-cell contact dependent event.

Antiviral cytotoxic T lymphocytes in vaginal mucosa of simian immunodeficiency virus-infected rhesus macaques.

The mucosal immune system of the female reproductive tract is of central importance for protection against sexually transmitted diseases, including HIV; however, this arm of the immune system remains poorly understood. Antiviral CTL responses never have been documented in the genital tract and the role of CTL in this anatomic site is unknown. In this study, CD8+ intraepithelial lymphocytes (IEL) in the vaginas of six simian immunodeficiency virus (SIV)-infected female rhesus macaques were identified by immunohistochemistry to be CD2+ and TCR beta-chain+. In addition, the majority of CD8+ IEL contained TIA-1+ cytoplasmic granules that are associated with CTL activity. CD8+ T cells were isolated from the vaginal epithelium and submucosa and amplified by limiting dilution in the presence of feeder cells. SIV p55gag and/or gp160env-specific lysis was detected in cultures of vaginal epithelial but not submucosal CD8+ T lymphocytes. Estimated SIV-specific precursor CTL frequencies were higher in the vaginal CD8+ IEL population of chronically infected monkeys than in the same cells from acutely infected monkeys or a naive control monkey. These results provide the first demonstration that antiviral CTL are present in the vaginal epithelium, and suggest that a vaccine may be able to generate anti-HIV CTL in the genital mucosa.

Development of TCRγδ iIELs

Interest has focused recently on the development and function of T and B lymphocytes in the mucosal surface, particularly on a population of intestinal intraepithelial lymphocytes (iIELs) that develop by thymic and extrathymic pathways in the mucosa (for review see ref 1). Our approach to studying TCRγδ iIEL development has been to utilize G8 transgenic (Tg) mice expressing an antigen-specific, developmentally-regulated, γδTCR. These mice have provided a useful tool to dissect T-cell development of intestinal TCRγδ cells. In this review, we summarize our findings of studies using this model system to dissect the complex events that regulate TCRγδ iIEL development and functional activity. The data suggest a maturational scheme for TCRγδ iIELs wherein the majority of TCRγδ iIELs migrate to the gut in an immature state where they develop in response to TCR ligation. As TCRγδ iIELs develop, surface and functional phenotypes change in a discernable pattern. These findings provide an important first step in analysing TCRγδ development, maturation and differentiation in normal animals

Accumulation of donor-specific cytotoxic T cells in intestinal lymphoid tissues following intestinal transplantation

Utilizing a rat model of semiallogeneic intestinal transplantation, recipients were evaluated for accumulation of donor-specific cytotoxic T cells in spleen, mesenteric lymph nodes, Peyer's patches, lamina propria, and intraepithelial lymphocytes using limiting dilution analysis. Naive animals exhibited a low frequency of cytotoxic T cells in spleen and mesenteric lymph nodes with minimal activity detected in Peyer's patches and intraepithelial lymphocytes, but not detectable activity in lamina propria. Orthotopic intestinal transplantation resulted in significant increases in cytotoxic T-cell activity in recipient Peyer's patches as early as Day 6 and by Day 8 in spleen, mesenteric lymph nodes, lamina propria and intraepithelial lymphocyte populations. Graft lamina propria and intraepithelial lymphocytes exhibited significant cytotoxic T-cell activity as early as 4 days following transplant. The highest donor-specific cytotoxic T-cell activity was observed in graft intraepithelial lymphocytes on Day 8 posttransplant. These studies demonstrate rapid expansion of donor-specific cytotoxic T cells which migrate to the graft site within 4 days after semiallogeneic intestinal transplantation.

Toxoplasma gondii oral infection induces specific cytotoxic CD8 alpha/beta+ Thy-1+ gut intraepithelial lymphocytes, lytic for parasite-infected enterocytes.

The first line of defense of the host after primary infection by Toxoplasma gondii is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IEL) (mainly of CD8 phenotype), mucus, and secretory Igs. T. gondii cysts were administered orally to CBA/J mice to determine whether parasite-specific cytotoxic IEL can be elicited within the intestine. We found that oral infection led to an increase in the relative percentages of the CD8 beta + and Thy-1+ IEL populations between day 9 and day 13 after infection. At these times, T. gondii-primed, but not control mice, generated parasite-specific cytotoxic effector IEL for toxoplasma-infected macrophages. This cytotoxic activity was genetically restricted. By using magnetically activated cell sorting, the effector IEL were shown to be CD8 alpha/beta + Thy-1+ T lymphocytes and FACScan analysis revealed that they mainly express TCR-alpha beta. A significant level of cytotoxicity was also observed against T. gondii-infected epithelial cells from the MODE-K cell line, suggesting that parasite-specific IEL may ensure epithelial integrity by rapidly killing infected enterocytes. Finally, toxoplasma stimulation in vitro led to IFN-gamma production by T. gondii-primed IEL. Taken together, these data suggest that antigen-specific IEL, bearing the CD8 alpha/beta + Thy-1+, TCR-alpha beta + phenotype, can act directly as effector cells through a specific CTL activity at the intestinal level and may indirectly activate other mucosal effector mechanisms through IFN-gamma secretion.