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1.  Prolactin and Prolactin Receptor Expression in Rat, Small Intestine, Intraepithelial Lymphocytes During Neonatal Developmen 
Developmental Immunology  2001;8(3-4):319-330.
Intraepithelial lymphocytes (IEL) are specialized T cells found between the epithelial cells of the small intestine. Because of their location, IEL are the first lymphocytes to contact intestinal bacteria and food antigens. In the neonate, IEL may be the first cells of the immune system to interact with milk-borne hormones including prolactin (PRL). PRL, an endocrine hormone abundant in breast milk, interacts with cells through surface receptors. PRL has been shown to function as an immunoregulator and may affect the development of the newborn's immune system. To determine if PRL plays a role in IEL development, small intestine IEL from rats of various ages were examined for the presence of surface prolactin receptor (PRL-R) and several lymphoid markers by flow cytometry. Between birth and 96 days of age about 80% of IEL were found to express PRL-R. These same cells also expressed the mRNA for PRL. Additionally, all of the IEL subpopulations examined were found to express PRL-R. Analysis of the normal development of rat IEL revealed an age related increase in total IEL, CD4 positive cells as well as a peak in interleukin-2 receptor (IL-2R) expression at weaning. In summary, the results indicate that IEL express PRL and PRL-R. In addition, an activation marker, IL-2R, changes in expression during neonatal development.
PMCID: PMC2276076  PMID: 11785680
intestine; intraepithelial; lymphocytes; neonatal development; prolactin; rat; CD4 T cell
2.  Gastrointestinal T Lymphocytes Retain High Potential for Cytokine Responses but Have Severe CD4+ T-Cell Depletion at All Stages of Simian Immunodeficiency Virus Infection Compared to Peripheral Lymphocytes 
Journal of Virology  1998;72(8):6646-6656.
Gastrointestinal complications in human immunodeficiency virus (HIV) infection are indicative of impaired intestinal mucosal immune system. We used simian immunodeficiency virus (SIV)-infected rhesus macaques as an animal model for HIV to determine pathogenic effects of SIV on intestinal T lymphocytes. Intestinal CD4+ T-cell depletion and the potential for cytokine responses were examined during SIV infection and compared with results for lymphocytes from lymph nodes and blood. Flow cytometric analysis demonstrated severe depletion of CD4+CD8− single-positive T cells and CD4+CD8+ double-positive T cells in intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) during primary SIV infection which persisted through the entire course of SIV infection. In contrast, CD4+ T-cell depletion was gradual in peripheral lymph nodes and blood. Flow cytometric analysis of intracellular gamma interferon (IFN-γ) and interleukin-4 (IL-4) production following short-term mitogenic activation revealed that LPL retained same or higher capacity for IFN-γ production in all stages of SIV infection compared to uninfected controls, whereas peripheral blood mononuclear cells displayed a gradual decline. The CD8+ T cells were the major producers of IFN-γ. There was no detectable change in the frequency of IL-4-producing cells in both LPL and peripheral blood mononuclear cells. Thus, severe depletion of CD4+ LPL and IEL in primary SIV infection accompanied by altered cytokine responses may reflect altered T-cell homeostasis in intestinal mucosa. This could be a mechanism of SIV-associated enteropathy and viral pathogenesis. Dynamic changes in intestinal T lymphocytes were not adequately represented in peripheral lymph nodes or blood.
PMCID: PMC109855  PMID: 9658111
3.  GPR18 Controls Reconstitution of Mouse Small Intestine Intraepithelial Lymphocytes following Bone Marrow Transplantation 
PLoS ONE  2015;10(7):e0133854.
Specific G protein coupled receptors (GPRs) regulate the proper positioning, function, and development of immune lineage subsets. Here, we demonstrate that GPR18 regulates the reconstitution of intraepithelial lymphocytes (IELs) of the small intestine following bone marrow transplantation. Through analysis of transcriptional microarray data, we find that GPR18 is highly expressed in IELs, lymphoid progenitors, and mature follicular B cells. To establish the physiological role of this largely uncharacterized GPR, we generated Gpr18-/- mice. Despite high levels of GPR18 expression in specific hematopoietic progenitors, Gpr18-/- mice have no defects in lymphopoiesis or myelopoiesis. Moreover, antibody responses following immunization with hapten-protein conjugates or infection with West Nile virus are normal in Gpr18-/- mice. Steady-state numbers of IELs are also normal in Gpr18-/- mice. However, competitive bone marrow reconstitution experiments demonstrate that GPR18 is cell-intrinsically required for the optimal restoration of small intestine TCRγδ+ and TCRαβ+ CD8αα+ IELs. In contrast, GPR18 is dispensable for the reconstitution of large intestine IELs. Moreover, Gpr18-/- bone marrow reconstitutes small intestine IELs similarly to controls in athymic recipients. Gpr18-/- chimeras show no changes in susceptibility to intestinal insults such as Citrobacter rodentium infections or graft versus host disease. These data reveal highly specific requirements for GPR18 in the development and reconstitution of thymus-derived intestinal IEL subsets in the steady-state and after bone marrow transplantation.
PMCID: PMC4510063  PMID: 26197390
4.  Analysis of the cellular basis of keratinocyte growth factor overexpression in inflammatory bowel disease 
Gut  1999;45(6):848-855.
BACKGROUND—Keratinocyte growth factor (KGF) stimulates gastrointestinal epithelial cells in vivo, and is protective against gastrointestinal injury and colitis. Endogenous KGF is increased in inflammatory bowel disease (IBD), and may be an important mediator of mucosal repair. KGF is expressed by mesenchymal cells and activated intraepithelial lymphocytes (IEL).
AIMS—To investigate the relative contributions of these cellular sources of KGF expression in IBD.
METHODS—IELs and lamina propria lymphocytes (LPL) were isolated from inflamed and uninflamed IBD tissues. mRNA expression was determined by ribonuclease protection assay. In situ hybridisation was combined with immunohistochemistry to determine whether KGF mRNA was expressed by specific cell types in vivo.
RESULTS—Low levels of KGF mRNA expression were detected in three of five IEL samples derived from inflamed tissue, but not in two IEL samples from uninflamed tissue. No KGF expression was detected in LPLs from either inflamed or uninflamed tissue. In contrast, KGF was expressed by primary cultures of human intestinal fibroblasts, and was induced by treatment with interleukin 1.
CONCLUSIONS—The major source of KGF expression in IBD was lamina propria cells of non-immune origin, most likely fibroblasts and/or smooth muscle cells. Compared with these cell types, relatively little KGF synthesis was associated with IELs in inflamed IBD tissue.

Keywords: fibroblasts; intestinal inflammation; intraepithelial lymphocytes; keratinocyte growth factor; lamina propria lymphocytes
PMCID: PMC1727748  PMID: 10562583
5.  Development of lntraepithelial Cells in the Porcine Small Intestine 
Developmental Immunology  2001;8(2):147-158.
The number, phenotype, localisation and development of intraepithelial lymphocytes (IEL) from duodenum (Du) and ileum (Il) were studied by immunohistochemistry (IHC) and light and electron microscopy in unweaned (0–7 weeks old) and six months-old pigs. Developmental changes at birth showed that 38% of the total lymphocytes in the villi were IEL, mainly of the CD2+CD4-CD8- double negative (DN) phenotype. That proportion rose to over 50% at week 5 after birth, resembling adult proportion, although still with fewer cells than in adult pigs. CD4+ cells appeared relatively early in life although they were confined to the lamina propria (LP) and CD8+ cells were found only in low numbers. In the villi of adult animals, almost half of the total number of lymphocytes were IEL (49% Du, 52% Il). Over half of these IEL (52% Du, 53% Il) showed the CD2+CD4-CD8+ phenotype and were localized at the epithelium's basement membrane. Numerous (43% Du, 42% Il) DN IEL were found grouped at the enterocyte nucleus level and relatively few (5% in Du and Il) granular IEL were found apically in the epithelium. These proportions were homogeneously maintained along the villi's tip, middle and bottom, suggesting that the IEL may have their origin in the LP. Therefore, the IEL compartment in the porcine intestine develops slowly with age and is actually composed by a heterogeneous population of cells (null, DN and CD8+). These results may explain the increased susceptibility of young animals to disease during the lactation period and should be taken into account when functional studies are carried out with IEL. The quantitative results of this paper established a model for studies on the effect of age, diet, normal flora, infection and oral immunization on the IEL of the gut.
PMCID: PMC2276065  PMID: 11589310
IEL; immune development; lymphocyte; mucosal immunology; small intestine; swine
6.  Intraepithelial and lamina propria lymphocytes show distinct patterns of apoptosis whereas both populations are active in Fas based cytotoxicity in coeliac disease 
Gut  2001;49(3):380-386.
BACKGROUND—Lamina propria (LPLs) and intraepithelial (IELs) lymphocytes are markedly increased in coeliac mucosa, and are thought to play a crucial role in the generation of villous atrophy in coeliac disease (CD). However, the mechanisms by which they mediate the killing of enterocytes in this condition are still poorly characterised.
AIM—We investigated Fas mediated cytotoxicity and apoptosis of both LPLs and IELs, isolated from 10 untreated coeliac patients, 10 coeliac patients on a gluten free diet, and 10 biopsied controls.
METHODS—Fas and Fas ligand expression were assessed by flow cytometry and immunocytochemistry. Lymphocyte cytotoxicity against Fas expressing Jurkat cells was determined by the Jam test. The effect of the antagonist ZB4 anti-Fas antibody on apoptotic activity exerted by coeliac lymphocytes against enterocytes was analysed. Lymphocyte apoptosis was assessed by oligonucleosome ELISA.
RESULTS—LPLs and IELs showed increased apoptotic activity and higher levels of Fas ligand expression in untreated CD compared with treated CD patients and controls. Enterocyte apoptosis observed after coculturing coeliac lymphocytes and enterocytes in the presence of ZB4 antibody was reduced. In active CD, LPLs manifested increased apoptosis whereas IELs showed decreased apoptosis.
CONCLUSIONS—Our results support the involvement of the Fas/Fas ligand system in CD associated enterocyte apoptosis. Increased LPL apoptosis is likely to downregulate mucosal inflammation whereas decreased IEL apoptosis could be responsible for autoimmune and malignant complications of CD.

Keywords: apoptosis; coeliac disease; cytotoxicity assay; Fas/Fas ligand system; intraepithelial lymphocytes; lamina propria lymphocytes
PMCID: PMC1728419  PMID: 11511560
7.  Evaluation of the immunoregulatory activity of intraepithelial lymphocytes in a mouse model of chronic intestinal inflammation 
International Immunology  2010;22(12):927-939.
Intraepithelial lymphocytes (IELs) represent the first line of lymphocyte defense against the intestinal bacteria. Although previous studies have demonstrated a protective role of IELs in the development of colitis, the data supporting a regulatory role for IELs are limited. The objective of this study was to examine the suppressive activity of IELs in vitro and in vivo using a mouse model of chronic small and large bowel inflammation. Adoptive transfer of CD8α+ IELs isolated from small intestines of wild-type (WT) mice into TCR βxδ-deficient (TCR βxδ−/−) recipients did not prevent or delay the onset of the disease induced by WT CD4+CD45RBhigh T cells. On the contrary, we observed a more rapid onset of wasting and clinical signs of intestinal inflammation when compared with animals injected with CD4+CD45RBhigh T cells alone. Histopathological scores of small and large bowel did not differ significantly between the two groups. Transfer of IELs alone did not produce any pathological changes. Real-time PCR analysis of intestinal tissues showed up-regulation of message for Th1- and macrophage-derived cytokines in colon and small bowel. Using Foxp3-GFP reporter mice, we were unable to detect any Foxp3+ cells within the CD8α+ IELs but did find a small population of Foxp3+CD4+ IELs in the small and large bowel. Using in vitro suppression assay, we found that neither TCRαβ+CD8αα+, TCRαβ+CD8αβ+ nor TCRγδ+CD8αα+ IELs were capable of suppressing CD4+ T-cell proliferation. Taken together, our data do not support an immunoregulatory role for CD8α+ IELs in a mouse model of small and large bowel inflammation.
PMCID: PMC3031346  PMID: 21071622
adaptive immune system; animal models; cytokines; inflammation; inflammatory bowel disease; intraepithelial lymphocytes; TCRβxδ-deficient mice
8.  Spontaneous secretion of interferon γ and interleukin 4 by human intraepithelial and lamina propria gut lymphocytes 
Gut  1998;42(5):643-649.
Background—Cytokines secreted by intestinal T lymphocytes probably play a critical role in regulation of the gut associated immune responses. 
Aims—To quantify interferon γ (IFN-γ) and interleukin 4 (IL-4) secreting cells (SC) among human intraepithelial (IEL) and lamina propria (LPL) lymphocytes from the duodenum and right colon in non-pathological situations and in the absence of in vitro stimulation. 
Patients—Duodenal and right colonic biopsy specimens were obtained from patients with no inflammation of the intestinal mucosa. 
Methods—Intraepithelial and lamina propria cell suspensions were assayed for numbers of cells spontaneously secreting IFN-γ and IL-4 by a two site reverse enzyme linked immunospot technique (ELISPOT). 
Results—The relatively high proportion of duodenal lymphocytes spontaneously secreting IFN-γ (IEL 3.6%; LPL 1.9%) and IL-4 (IEL 1.3%; LPL 0.7%) contrasted with the very low numbers of spontaneously IFN-γ SC and the absence of spontaneously IL-4 SC among peripheral blood mononuclear cells. In the basal state, both IFN-γ and IL-4 were mainly produced by CD4+ cells. Within the colon, only 0.2% of IEL and LPL secreted IFN-γ in the basal state, and 0.1% secreted IL-4. 
Conclusions—Compared with peripheral lymphocytes substantial proportions of intestinal epithelial and lamina propria lymphocytes spontaneously secrete IFN-γ and/or IL-4. These cytokines are probably involved in the normal homoeostasis of the human intestinal mucosa. Disturbances in their secretion could play a role in the pathogenesis of gastrointestinal diseases. 

Keywords: intestinal lymphocytes; ELISPOT; interferon γ; interleukin 4
PMCID: PMC1727108  PMID: 9659157
9.  Differential Roles of Segmented Filamentous Bacteria and Clostridia in Development of the Intestinal Immune System 
Infection and Immunity  1999;67(7):3504-3511.
The presence of microflora in the digestive tract promotes the development of the intestinal immune system. In this study, to evaluate the roles of two types of indigenous microbe, segmented filamentous bacteria (SFB) and clostridia, whose habitats are the small and large intestines, respectively, in this immunological development, we analyzed three kinds of gnotobiotic mice contaminated with SFB, clostridia, and both SFB and clostridia, respectively, in comparison with germfree (GF) or conventionalized (Cvd) mice associated with specific-pathogen-free flora. In the small intestine, the number of αβ T-cell receptor-bearing intraepithelial lymphocytes (αβIEL) increased in SFB-associated mice (SFB-mice) but not in clostridium-associated mice (Clost-mice). There was no great difference in Vβ usage among GF mice, Cvd mice, and these gnotobiotic mice, although the association with SFB decreased the proportion of Vβ6+ cells in CD8β− subsets to some extent, compared to that in GF mice. The expression of major histocompatibility complex class II molecules on the epithelial cells was observed in SFB-mice but not in Clost-mice. On the other hand, in the large intestine, the ratio of the number of CD4− CD8+ cells to that of CD4+ CD8− cells in αβIEL increased in Clost-mice but not in SFB-mice. On association with both SFB and clostridia, the numbers and phenotypes of IEL in the small and large intestines changed to become similar to those in Cvd mice. In particular, the ratio of the number of CD8αβ+ cells to that of CD8αα+ cells in αβIEL, unusually elevated in the small intestines of SFB-mice, decreased to the level in Cvd mice on contamination with both SFB and clostridia. The number of immunoglobulin A (IgA)-producing cells in the lamina propria was more elevated in SFB-mice than in Clost-mice, not only in the ileum but also in the colon. The number of IgA-producing cells in the colons of Clost-mice was a little increased compared to that in GF mice. Taken together, SFB and clostridia promoted the development of both IEL and IgA-producing cells in the small intestine and that of only IEL in the large intestine, respectively, suggesting the occurrence of compartmentalization of the immunological responses to the indigenous bacteria between the small and large intestines.
PMCID: PMC116537  PMID: 10377132
10.  Influence of beta 2-microglobulin expression on gamma interferon secretion and target cell lysis by intraepithelial lymphocytes during intestinal Listeria monocytogenes infection. 
Infection and Immunity  1996;64(2):569-575.
Numerous microbial pathogens, including Listeria monocytogenes, enter the host through the intestine. Although relatively little is known about the biological functions of intestinal intraepithelial lymphocytes (i-IEL), they are generally considered a first line of defense against intestinal infections. In the mouse, the vast majority of i-IEL express the CD8 coreceptor either as a CD8 alpha/alpha homodimer or as a CD8 alpha/beta heterodimer. The CD8 receptor of T-cell receptor TcR gamma/delta i-IEL is exclusively homodimeric, whereas the CD8-expressing TcR alpha/beta i-IEL segregate into equal fractions of CD8 alpha/alpha and CD8 alpha/beta cells. We infected beta 2-microglobulin (beta 2m)+/- mice (possessing all i-IEL populations) and beta 2m -/- mutant mice (lacking all CD8 alpha/beta + i-IEL and having few CD8 alpha/alpha + TcR alpha/beta i-IEL) with L. monocytogenes per os and determined their biological functions after TcR ligation with monoclonal antibodies. Cytolytic activities of TcR alpha/beta and TcR gamma/delta i-IEL from beta 2m +/- mice were not influenced by intestinal listeriosis. Cytolytic activities of TcR alpha/beta i-IEL were impaired in uninfected beta 2m -/- mice, but this reduction was reestablished as a consequence of intestinal listeriosis. Frequencies of gamma interferon (IFN-gamma)-producing TcR alpha/beta i-IEL in uninfected beta 2m -/- mice were reduced, compared with that in their heterozygous controls. Equally low frequencies of IFN-gamma-producing TcR gamma/delta i-IEL in beta 2M +/- and beta 2m-/- mutants were found. Listeriosis increased frequencies of INF-gamma-producing TcR alpha/beta and TcR gamma/delta i-IEL in both mouse strains. Most remarkably, the proportion of IFN-gamma-producing TcR gamma/delta i-IEL was elevated 10-fold in listeria-infected beta 2M -/- mice. Our findings show that the beta 2m-independent CD8 beta- i-IEL expressing either TcR alpha/beta or TcR gamma/delta are stimulated by intestinal listeriosis independent of regional beta 2m expression. We conclude that the three major CD8+ i-IEL populations are stimulated by intestinal listeriosis and that CD8 beta- i-IEL compensate for the total lack of CD8 beta+ i-IEL in beta 2M -/- mutant mice. Hence, in contrast to the peripheral immune system, which crucially depends on CD8 alpha/beta + TcR alpha/beta lymphocytes, the mucosal immune system can rely on additional lymphocytes expressing the CD8 alpha/alpha homodimer.
PMCID: PMC173803  PMID: 8550209
11.  Diversion of intestinal flow decreases the numbers of interleukin 4 secreting and interferon γ secreting T lymphocytes in small bowel mucosa 
Gut  2000;46(1):40-45.
BACKGROUND/AIMS—The intestinal immune system faces large amounts of antigens, and its regulation is tightly balanced by cytokines. In this study, the effect of intestinal flow diversion on spontaneous secretion of interleukin (IL)-4 and interferon (IFN)- γ was analysed.
METHODS—Eight patients (two with Crohn's disease, four with ulcerative colitis, and two with previous colon cancer) carrying a double lumen small bowel stoma after a total colectomy procedure were included in the study. For each patient, eight biopsy samples were taken endoscopically from both the diverted and non-diverted part of the small bowel. Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) were isolated separately and assayed for numbers of cells spontaneously secreting IL-4 and/or IFN-γ by an ELISPOT technique.
RESULTS—Compared with the non-diverted mucosa, a significant decrease in the number of spontaneously IFN-γ secreting CD3 lymphocytes was observed in the diverted small bowel mucosa among both IELs (p = 0.008) and LPLs (p = 0.007). The same results, although less significant, were obtained for IL-4, especially in LPLs (p = 0.01).
CONCLUSION—The intestinal content influences the spontaneous secretion of IFN-γ and IL-4 by intestinal lymphocytes. These results could help to elucidate the anti-inflammatory role of split ileostomy in patients suffering from inflammatory bowel diseases.

Keywords: intestine; T lymphocytes; mucosa; interleukin-4; interferon-γ; ELISPOT
PMCID: PMC1727786  PMID: 10601053
12.  Human colonic intraepithelial and lamina proprial lymphocytes: cytotoxicity in vitro and the potential effects of the isolation method on their functional properties. 
Gut  1981;22(3):177-186.
Colonic mucosal lymphoid cells, selectively enriched for intraepithelial (IEL) or lamina proprial lymphocytes (LPL), were isolated by sequential EDTA-collagenase treatment of resected human colons. Cytotoxic activities of colonic and peripheral blood lymphoid cells (PBL) were tested in three different assays, using chicken erythrocytes (CRBC) and Chang cells as targets. Antibody-dependent cell-mediated cytotoxicity (ADCC) and PHA-induced cytotoxicity (MICC) for both targets were shown by all the isolates of PBL, as was spontaneous cell-mediated cytotoxicity (SCMC) for Chang cells. However, no SCMC or ADCC for Chang cells was found with LPL, and IEL showed minimal or no activity in either assay. PBL, LPL and IEL demonstrated MICC for Chang cells but, contrasting with PBL and LPL, IEL showed no MCC for CRBC. No significant differences were found between the cytotoxic capabilities of colonic lymphoid cells from patients with inflammatory bowel disease and those from patients with other colonic diseases. Importantly, control studies with PBL showed that SCMC for Chang cells and ADCC for CRBC and Chang cells were reduced by collagenase treatment used in the isolation, of LPL. Also, SCMC for Chang cells was reduced by the treatment of PBL with EDTA. In contrast, neither EDTA nor collagenase reduced MICC for CRBC or Chang cells. Both forms of treatment induced variable degrees of cell losses in the PBL. By analogy, it can be implied that the isolation of intestinal mononuclear cells using EDTA and collagenase may influence some of their cytotoxic activities in vitro. This raises an important caveat in the interpretation of such studies.
PMCID: PMC1419515  PMID: 6262195
13.  Non-equilibrium and differential function between intraepithelial and lamina propria virus-specific TCRαβ+ CD8αβ + T cells in the small intestinal mucosa 
Mucosal immunology  2009;2(5):450-461.
The gastrointestinal mucosa regularly encounters commensal and pathogenic microbiota. Gut mucosal lymphocytes consist of two phenotypically different populations residing in the intestinal intraepithelial (IEL) compartment and lamina propria (LP). Little is known about compositional and functional differences of antigen-specific T cells from these mucosal compartments after mucosal infection, or the degree of trafficking between them. We here studied the B8R20 – 27-specific CD8 T-cell response in LP and IEL compartments after intrarectal immunization with modified vaccinia virus Ankara (MVA). CD8+ T cells in the IEL compartment had much lower avidity than in the LP or spleen during acute and memory phases. Surprisingly, the TCR Vβ-chain distribution of antigen-specific T cells and the length of the CDR3 region of the dominant Vβ genes showed substantial dissimilarities between IEL and LP antigen-specific CD8αβ T cells in individual mice, increasing with time. We show functional and compositional differences between these mucosal compartments during the effector and memory phases of the immune response, indicating limited crosstalk and microenvironmental differences between the IEL, LP, and spleen. The restricted migration of cells from each of these mucosal compartments could partly account for a founder effect we observed in the IEL TCRαβ CD8αβ epitope-specific repertoire that might impact protective efficacy.
PMCID: PMC3224999  PMID: 19571797
14.  Acute mucosal pathogenesis of feline immunodeficiency virus is independent of viral dose in vaginally infected cats 
Retrovirology  2010;7:2.
The mucosal pathogenesis of HIV has been shown to be an important feature of infection and disease progression. HIV-1 infection causes depletion of intestinal lamina propria CD4+ T cells (LPL), therefore, intestinal CD4+ T cell preservation may be a useful correlate of protection in evaluating vaccine candidates. Vaccine studies employing the cat/FIV and macaque/SIV models frequently use high doses of parenterally administered challenge virus to ensure high plasma viremia in control animals. However, it is unclear if loss of mucosal T cells would occur regardless of initial viral inoculum dose. The objective of this study was to determine the acute effect of viral dose on mucosal leukocytes and associated innate and adaptive immune responses.
Cats were vaginally inoculated with a high, middle or low dose of cell-associated and cell-free FIV. PBMC, serum and plasma were assessed every two weeks with tissues assessed eight weeks following infection. We found that irrespective of mucosally administered viral dose, FIV infection was induced in all cats. However, viremia was present in only half of the cats, and viral dose was unrelated to the development of viremia. Importantly, regardless of viral dose, all cats experienced significant losses of intestinal CD4+ LPL and CD8+ intraepithelial lymphocytes (IEL). Innate immune responses by CD56+CD3- NK cells correlated with aviremia and apparent occult infection but did not protect mucosal T cells. CD4+ and CD8+ T cells in viremic cats were more likely to produce cytokines in response to Gag stimulation, whereas aviremic cats T cells tended to produce cytokines in response to Env stimulation. However, while cell-mediated immune responses in aviremic cats may have helped reduce viral replication, they could not be correlated to the levels of viremia. Robust production of anti-FIV antibodies was positively correlated with the magnitude of viremia.
Our results indicate that mucosal immune pathogenesis could be used as a rapid indicator of vaccine success or failure when combined with a physiologically relevant low dose mucosal challenge. We also show that innate immune responses may play an important role in controlling viral replication following acute mucosal infection, which has not been previously identified.
PMCID: PMC2835650  PMID: 20085648
15.  Dietary nucleotides increase the mucosal IgA response and the secretion of transforming growth factor β from intestinal epithelial cells in mice 
Cytotechnology  2002;40(1-3):49-58.
We have investigated the influence of dietary nucleotides on the intestinal immune system in ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic mice (OVA-TCR Tg mice). When mice were supplied with water supplemented with 2% OVA ad libitum, the faecal OVA-specific immunoglobulin A (IgA) level significantly increased in those fed a nucleotide-supplemented diet (NT(+) diet) compared with those fed a nucleotide-free control diet (NT(–) diet). In the NT(+) diet-fed mice, secretion of transforming growth factor β (TGF-β), which is an isotype-specific switch factor for IgA, from intestinal epithelial cells (IECs) was significantly increased. Furthermore, an increased proportion of intestinal intraepithelial lymphocytes (IELs) bearing γδ TCR (TCRγδ+ IELs) and increased secretion from IECs of interleukin 7 (IL-7), which is essential for the development of TCRγδ+ IELs, were also observed in OVA-TCR-Tg mice fed the NT(+) diet, as we previously demonstrated using BALB/c mice (Nagafuchi et al., Biosci. Biotechnol. Biochem. 64: 1459-65 (2000)). Considering that TCRγδ+ T cells and TGF-β are important for an induction of the mucosal IgA response, our results suggest that dietary nucleotides augment the mucosal OVA-specific IgA response by increasing the secretion of TGF-β from IECs and the proportion of TCRγδ+ IELs.
PMCID: PMC3449534  PMID: 19003104
dietary nucleotides; IgA response; intestinal epithelial cells; intraepithelial lymphocytes; T-cell receptor γδ+ T cells; transforming growth factor β
16.  Impairment of intestinal intraepithelial lymphocytes in Id2 deficient mice 
Gut  2004;53(4):480-486.
Background: Id2, an inhibitor of basic helix-loop-helix transcription factors, regulates cell differentiation. Id2−/− mice exhibit a variety of phenotypes in the immune system.
Aims: In this study we investigated whether Id2 plays a role in intestinal intraepithelial lymphocytes (IELs), which constitute the main defence against pathogens in the intestinal tract.
Methods: Flow cytometry and bone marrow transplantation were used to analyse and characterise subsets of IELs of Id2−/− mice. Gene expression was analysed by real-time polymerase chain reaction. Intestinal barrier function was evaluated by treating mice with 5-fluorouracil (5-FU).
Results: Among the four members of the Id gene family, Id2 was selectively expressed in all T cell subsets in the small intestinal IELs. Id2−/− mice showed alteration in the proportions of T cell subsets and a substantial reduction in the number of IELs, especially those of the CD4+ and CD8αβ+ T cell subsets, indicating a more pronounced effect on thymus derived IELs. Expression of αE integrin was reduced in CD4+ and CD8αβ+ T cell subsets in IELs of Id2−/− mice. IELs isolated from C57BL/6 mice reconstituted with Id2−/− bone marrow cells showed a similar phenotype to that of Id2−/− mice, indicating that the defects are intrinsic to bone marrow derived cells. Expression of genes encoding intestinal epithelial cell derived cytokines was reduced in Id2−/− mice. The 5-FU treatment revealed impaired intestinal barrier function of Id2−/− mice.
Conclusions: The Id2 gene is essential for constituting the intestinal mucosal barrier, particularly with respect to IELs. Id2 null mutant mice may provide a good experimental model for studying the ontogeny of IELs and intestinal inflammation and infection.
PMCID: PMC1774007  PMID: 15016739
Id2 mice; intestinal intraepithelial lymphocytes; lamina propria lymphocytes; mucosal immunity; small intestine
17.  Development and Maintenance of the Gut-Associated Lymphoid Tissue (Galt): the Roles of Enteric Bacteria and Viruses 
Developmental Immunology  1998;6(1-2):13-18.
GALT can be subdivided into several compartments: (a) Peyer's patches (PP); (b) lamina propria (LP); and (c) intraepithelial leukocyte (IEL) spaces. The B-cell follicles of PP are quiescent in neonatal and germ-free (GF) adult mice. Germinal centers (GC), including sIgA+ blasts, appear in the B follicles of formerly GF adult mice about 10-14 days after monoassociation with various gut commensal bacteria. The GC wax and wane over about a 3-week period, although the bacterial colonizers remain in the gut at high density. Neonatal mice, born of conventionally reared (CV), immunocompetent mothers, display GC reactions in PP postweaning, although pups of SCID mothers display precocious GC reactions at about 14 days of life. Normally, gut colonization of neonates with segmented filamentous bacteria (SFB) leads to explosive development of IgA plasmablasts in LP shortly after weaning. Commensal gut bacteria and the immunocompetency of mothers also appears to control the rate of accumulation of primary B cells from “virgin” B cells in neonates.
Enteric reovirus infection by the oral route can cause the activation of CD8+ T cells in the interfollicular regions of PP and the appearance of virus-specific precursor cytotoxic T lymphocytes (pCTL) in the IEL spaces. Such oral stimulation can also lead to “activation” of both CTL and natural killer (NK) cells in the IEL spaces. More normally, colonization of the gut with SFB also leads to similar activations of NK cells and “constitutively” cytotoxic T cells.
PMCID: PMC2276005  PMID: 9716901
Enteric viruses; gut-associated lymphoid tissue (GALT); gut commensal bacteria; IgA responses in gut; intraepithelial leukocytes; Peyer's patches
18.  GPR18 is required for a normal CD8αα intestinal intraepithelial lymphocyte compartment 
The Journal of Experimental Medicine  2014;211(12):2351-2359.
G protein–coupled receptor 18 is required for accumulation of CD8α+ intraepithelial lymphocytes in the intestine.
Intraepithelial lymphocytes (IELs) play an important role in maintaining the physiology of the small intestine. The majority of mouse IELs express CD8αα and are either γδ or αβ T cells. Although the development and homing of CD8αα IELs have been studied in some detail, the factors controlling their homeostasis and positioning are incompletely understood. Here we demonstrate that G protein–coupled receptor 18 (GPR18) is abundantly expressed in CD8αα IELs and that mice lacking this orphan receptor have reduced numbers of γδT IELs. Mixed bone marrow chimera experiments reveal a markedly reduced contribution of GPR18-deficient cells to the CD8αα IEL compartment and a reduction in the CD8αβ T cell subset. These defects could be rescued by transduction with a GPR18-expressing retrovirus. The GPR18-deficient γδT IELs that remained in mixed chimeras had elevated Thy1, and there were less granzyme B+ and Vγ7+ cells, indicating a greater reduction in effector-type cells. Flow cytometric analysis indicated GPR18 deficiency more strongly affected the CD8αα cells in the intraepithelial compared with the adjacent lamina propria compartment. These findings establish a requirement for GPR18 in CD8αα and CD8αβ IELs, and we suggest the receptor has a role in augmenting the accumulation of CD8 T cells in the intraepithelial versus lamina propria compartment.
PMCID: PMC4235638  PMID: 25348153
19.  Small Intestinal Intraepithelial Lymphocytes Expressing CD8 and T Cell Receptor γδ Are Involved in Bacterial Clearance during Salmonella enterica Serovar Typhimurium Infection 
Infection and Immunity  2012;80(2):565-574.
The intestinal immune system is crucial for the maintenance of mucosal homeostasis and has evolved under the dual pressure of protecting the host from pathogenic infection and coexisting with the dense and diverse commensal organisms in the lumen. Intestinal intraepithelial lymphocytes (iIELs) are the first element of the host T cell compartment available to respond to oral infection by pathogens. This study demonstrated that oral infection by Salmonella enterica serovar Typhimurium promoted the expansion of iIELs, particularly CD8+ TCRγδ+ IELs, enhanced expression of NKG2D on iIELs, increased expression of MULT1, and decreased expression of Qa-1 by intestinal epithelial cells (IECs), leading to activation of, particularly, CD8+ TCRγδ+ iIELs and cytolytic activity against S. Typhimurium-infected IECs. Blockade of NKG2D recognition or depletion of TCRγδ+ cells using a depleting monoclonal antibody significantly attenuated the clearance of S. Typhimurium in the intestine and other tissues. This study suggests that iIELs, particularly CD8+ TCRγδ+ iIELs, play important roles in the detection of pathogenic bacteria and eradication of infected epithelial cells and, thus, provide protection against invading pathogens. These data further our understanding of the mechanisms by which the immune system of the intestinal mucosa discriminates between pathogenic and commensal organisms.
PMCID: PMC3264294  PMID: 22144492
20.  Small intestinal CD8+TCRγδ+NKG2A+ intraepithelial lymphocytes have attributes of regulatory cells in patients with celiac disease  
Intraepithelial lymphocytes (IELs) bearing the γδ TCR are more abundant in the small intestinal mucosa of patients with celiac disease (CD) compared with healthy individuals. However, their role in disease pathogenesis is not well understood. Here, we investigated the functional attributes of TCRγδ+ IELs isolated from intestinal biopsies of patients with either active celiac disease (ACD) or those on a gluten-free diet (GFD). We found that compared with individuals with ACD, individuals on GFD have a higher frequency of CD8+TCRγδ+ IELs that express the inhibitory NK receptor NKG2A and intracellular TGF-β1. TCR triggering as well as cross-linking of NKG2A increased both TGF-β1 intracellular expression and secretion in vitro. Coculture of sorted TCRγδ+NKG2A+ IELs, IL-15–stimulated TCRαβ+ IELs, and HLA-E+ enterocytes resulted in a decreased percentage of cytotoxic CD8+TCRαβ+ IELs expressing intracellular IFN-γ and granzyme-B and surface NKG2D. This inhibition was partially abrogated by blocking either TGF-β alone or both NKG2A and HLA-E. Thus, our data indicate that suppression was at least partially mediated by TGF-β secretion as a result of engagement of NKG2A with its ligand, HLA-E, on enterocytes and/or TCRαβ+ IELs. These findings demonstrate that human small intestinal CD8+TCRγδ+ IELs may have regulatory potential in celiac disease.
PMCID: PMC2117760  PMID: 18064301
21.  Sphingosine 1-phosphate dependence in the regulation of lymphocyte trafficking to the gut epithelium 
The Journal of Experimental Medicine  2007;204(10):2335-2348.
It is well established that intraepithelial T lymphocytes (IELs) are derived from conventional single-positive (SP) thymocytes, as well as unconventional double-negative (DN) thymocytes and CD103+CD8αβ recent thymic emigrants (RTEs). We show that IELs can be divided into two groups according to their dependency on sphingosine 1-phosphate (S1P) for trafficking into the intestines. CD4 or CD8αβ naive lymphocytes originating from SP thymocytes express high levels of type 1 S1P receptor (S1P1), and their preferential migration into the large intestine is regulated by S1P. In contrast, RTEs migrate exclusively into the small intestine, whereas DN thymic IEL precursors expressing either TCRαβ or TCRγδ migrate into both the small and large intestines. S1P does not play a role in the migration pathways of these unconventional thymic IEL precursors. Thus, down-regulation of S1P1 expression or disruption of the S1P gradient halted conventional CD4 or CD8αβ IEL trafficking into the intestines, but did not affect the trafficking of unconventional thymic IEL precursors. These data are the first to demonstrate that a lipid-mediated system discriminates IELs originating from conventional and unconventional thymic precursors.
PMCID: PMC2118459  PMID: 17875673
22.  Characteristics of natural killer cells in the murine intestinal epithelium and lamina propria 
The Journal of Experimental Medicine  1982;155(6):1785-1796.
Highly purified populations of lymphocytes were obtained from the murine intestinal mucosa using EDTA-collagenase isolation procedures in combination with discontinuous density centrifugation. Intraepithelial lymphocytes (IEL) were separated from lamina propria lymphocytes (LPL) and, within these two populations, fractions enriched or depleted in gut granular lymphocytes (gGL) were obtained. Using these cells in cytotoxic assays, it was shown that both IEL and LPL possess natural killer (NK) activity, and this was associated with gGL. The major effector cells of gut NK activity appeared to be Thy-1.2+, Lyt-1.1-, and Lyt-2.1-. The susceptibility of gut NK cells to anti-Thy-1.2 plus complement (C) was significantly higher than that of splenic NK cells. In contrast, anti-asialo GM1 and anti-NK-1.2 plus C only slightly affected the gut NK activity. Thus, the phenotype of the gut NK cells appears to be different from the splenic one and provides further evidence for NK heterogeneity and establishes the compartmentalization of one NK subpopulation. Beige mice, deficient in splenic NK activity, also had very low gut NK activity. W/Wv mice, which lack mast cell precursors, had normal numbers of gGL and diminished, but still present, gut and splenic NK activity. This deficiency did not segregate with the genes responsible for the basic hemopoietic stem cell defect, and these results argue against a close ontogenetic relationship between IEL, gGL, and intestinal mucosal mast cells. The relevance of these observations to the cell lineage of the effector cell of gut NK activity is discussed.
PMCID: PMC2186713  PMID: 7077224
23.  Memory and distribution of virus-specific cytotoxic T lymphocytes (CTLs) and CTL precursors after rotavirus infection. 
Journal of Virology  1991;65(3):1318-1324.
The gastrointestinal tract is constantly exposed to a variety of potentially invasive bacteria, viruses, and parasites. The first line of defense against these pathogens is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IELs), mucus, and secretory immunoglobulins. In addition, the intestine is a rich source of lymphocytes located within Peyer's patches and the lamina propria. Little is known about the function, memory, trafficking, or origin of intestinal T lymphocytes after intestinal infection. We studied the murine cytotoxic T-lymphocyte (CTL) response to the intestinal pathogen rotavirus (simian strain RRV). Adult mice were inoculated orally or via the hind footpad with RRV; virus-specific cytotoxic activities in intestinal and nonintestinal lymphocyte populations were determined by 51Cr release assays. In addition, virus-specific CTL precursor (CTLp) frequencies were determined by limiting-dilution analysis. IELs containing rotavirus-specific cytotoxic activity were detected after oral but not footpad inoculation and expressed alpha/beta but not gamma/delta cell surface protein; virus-specific CTLs did not appear to arise from CTLp among IELs. In addition, the site at which RRV was presented to the immune system determined the site at which RRV-specific CTLp first appeared. Frequencies of rotavirus-specific CTLp detected in Peyer's patches were 25- to 30-fold greater after oral than after footpad inoculation. However, regardless of the route of inoculation, rotavirus-specific CTLp were distributed throughout the lymphoid system 21 days after infection. Implications of these findings for vaccine design are discussed.
PMCID: PMC239907  PMID: 1847457
24.  Trans-presentation of IL-15 by intestinal epithelial cells drives development of CD8αα IELs1 
IL-15 is crucial for the development of intestinal intraepithelial lymphocytes (IEL) and delivery is mediated by a unique mechanism known as trans-presentation. Parenchymal cells have a major role in the trans-presentation of IL-15 to IELs, but the specific identity of this cell type is unknown. To investigate whether the intestinal epithelial cells (IEC) are the parenchymal cell type involved, a mouse model that expresses IL-15Rα exclusively by the IECs (Villin/IL-15Rα Tg) was generated. Exclusive expression of IL-15Rα by the IECs restored all the deficiencies in the CD8αα+TCRαβ+and CD8αα+TCRγδ+ subsets that exist in the absence of IL-15Rα. Interestingly, most of the IEL recovery was due to the preferential increase in Thy1lo IELs, which compose a majority of the IEL population. The differentiation of Thy1hiCD4−CD8− thymocytes into Thy1−CD8αα IELs was found to require IL-15Rα expression specifically by IECs and thus, provides evidence that differentiation of Thy1lo IELs is one function of trans-presentation of IL-15 in the intestines. In addition to effects in IEL differentiation, trans-presentation of IL-15 by IECs also resulted in an increase in IEL numbers that was accompanied by increases in Bcl-2, but not proliferation. Collectively, this study demonstrates that trans-presentation of IL-15 by IECs alone is completely sufficient to direct the IL-15-mediated development of CD8αα+ T cell populations within the IEL compartment, which now includes a newly identified role of IL-15 in the differentiation of Thy1lo IELs.
PMCID: PMC2706935  PMID: 19553528
T cells; cytokine receptors; cell differentiation; mucosa; thymus
25.  Synergy Between Intraepithelial Lymphocytes and Lamina Propria T Cells Drives Intestinal Inflammation During Infection 
Mucosal immunology  2011;4(6):658-670.
Oral infection of C57BL/6 mice with Toxoplasma gondii triggers severe necrosis in the ileum within 7–10 days of infection. Lesion development is mediated by Th-1 cytokines, CD4+ T cells, and sub-epithelial bacterial translocation. As such, these features share similarity to Crohn’s disease. Recently, we uncovered a role for intraepithelial lymphocytes (IEL) in mediating pathology after Toxoplasma infection. We show here that αβ and not γδ T cell IELs mediate intestinal damage. By adoptive transfer of mucosal T cells into naive Rag1−/− mice, we demonstrate that IEL do not function alone to cause inflammatory lesions, but act with CD4+ T lymphocytes from the lamina propria. Furthermore, recipient mice pretreated with broad-spectrum antibiotics to eliminate intestinal flora resisted intestinal disease after transfer of IEL and lamina propria lymphocytes. Our data provide valuable new insight into mechanisms of intestinal inflammation, findings that have important implications for understanding human inflammatory bowel disease.
PMCID: PMC3196813  PMID: 21796113

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