Intraepithelial Lymphocytes In Mice Research Articles

Osteopontin provides a survival signal for intestinal intraepithelial lymphocytes in mice

Abstract Intraepithelial lymphocytes (IEL) are a diverse immune population residing in between intestinal epithelial cells, which possess important roles during mucosal immune responses. The IEL family comprises cells such as TCRαb+CD4+, TCRαb+CD8+, TCRgd+, TCRab+CD8aa+, iCD8a and iCD3+, among others. Osteopontin (OPN), a pleiotropic cytokine encoded by the Spp-1 gene, is well known for its function in tissue remodeling, modulation of Th1 responses development, maintenance of Th17 function, and homeostasis of NK cells. Herein, we investigated the impact of OPN in the IEL compartment. We compared the cell numbers of IEL, lamina propria (LP) lymphocytes and splenocytes from Spp-1−/− and wild-type (WT) mice. OPN deficiency reduced the number of TCRγδ+, TCRβ+CD4+, TCRβ+CD8α+ and TCRβ+CD4+CD8α+ IEL, but had no effect on LP and spleen lymphocytes. In in vitro studies, IEL from WT mice cultured in the presence of rOPN showed better survival rates than IEL incubated alone. The increased survival was blunted to control levels when CD44, a receptor for OPN, was blocked with anti-CD44 antibody. When total T cells (including Tregs) from WT splenocytes were transferred into Spp-1−/−Rag-2−/− and Rag2−/− mice, we observed that although OPN deficiency did not affect donor-derived IEL reconstitution at day 7 post-transfer, it decreased the number of CD4+ and CD4+CD8α+ IEL at day 28 post-transfer. As expected, Rag-2−/− mice remained healthy throught the experiment; however, Spp-1−/−Rag-2−/− recipient mice presented a remarkably loss of body weight and colon inflammation. Together, our results indicate that OPN is a critical survival signal for IEL via CD44, and that OPN-deficiency promotes development of colitis even in the presence of Tregs.

Human intraepithelial lymphocytes.

The location of intraepithelial lymphocytes (IEL) between epithelial cells, their effector memory, cytolytic and inflammatory phenotype positions them to kill infected epithelial cells and protect the intestine against pathogens. Human TCRαβ+CD8αβ+ IEL have the dual capacity to recognize modified self via natural killer (NK) receptors (autoreactivity) as well as foreign antigen via the T cell receptor (TCR), which is accomplished in mouse by two cell subsets, the naturally occurring TCRαβ+CD8αα+ and adaptively induced TCRαβ+CD8αβ+ IEL subsets, respectively. The private/oligoclonal nature of the TCR repertoire of both human and mouse IEL suggests local environmental factors dictate the specificity of IEL responses. The line between sensing of foreign antigens and autoreactivity is blurred for IEL in celiac disease, where recognition of stress ligands by induced activating NK receptors in conjunction with inflammatory signals such as IL-15 can result in low-affinity TCR/non-cognate antigen and NK receptor/stress ligand interactions triggering destruction of intestinal epithelial cells.

In vivo blockade of T cell development reveals alternative pathways for generation of intraepithelial lymphocytes in mice

Intraepithelial lymphocytes (IELs) are resident cells localized within the intestinal epithelia and play an important role in regulating gut inflammations and host defense against pathogens. CD8α+ TCRαβ+ IELs are heterogeneous populations that are generated from T cell precursors including CD4− CD8α− double-negative (DN) cells and CD4+ CD8α+ double-positive (DP) cells. However, developmental pathways of TCRαβ+ IELs remained unclear. To gain insight into the mechanisms, we generated mice (Bcl11bΔDN2 mice) that lack thymic precursors (DN CD5+ TCRβ+ cells) for CD4− CD8αα+ TCRαβ+ IELs. Unexpectedly, we found that, in the absence of the precursors in thymi of Bcl11bΔDN2 mice, CD4− CD8αα+ TCRαβ+ IELs were still present in the intestine though the number was reduced. Adoptive transfer experiment showed that their precursors were highly enriched in CD8α+ TCRβ− thymocytes. The CD4− CD8αα+ TCRαβ+ IELs in Bcl11bΔDN2 mice are distinguished by Thy1.2 expression and are indeed present in WT mice. Taken together, our study reveal a novel developmental pathway for CD8αα+ TCRαβ+ IELs.

GPR18 is required for a normal CD8αα intestinal intraepithelial lymphocyte compartment.

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.

Effect of restraint stress on the population of intestinal intraepithelial lymphocytes in mice

The impact of restraint stress on the intestinal immune system, particularly on intestinal intraepithelial lymphocytes (i-IEL), has not been described in detail. Thus, the purpose of this study was to assess the effects of restraint stress, including those produced by increases in glucocorticoids and catecholamines, on the population of i-IEL. Mice were exposed to 1 or 4 h restraint stress for 4 day, and the number of IEL in the mucosa of the proximal small intestine was determined by immunohistochemistry. The effects of restraint were also analyzed in mice submitted to different procedures: adrenalectomy, chemical sympathectomy, and treatment with a glucocorticoid antagonist (RU486), dexamethasone, and epinephrine. The main findings were that: (1) chronic restraint-stress reduced the i-IEl population in the small intestine; (2) adrenalectomy, treatment with RU-486 and chemical sympathectomy decreased the number of γδ, CD4+ and CD8+ T cells in non-stressed groups; (3) dexamethasone reduced the number of γδ and CD8+ T cells, and (4) epinephrine reduced the number of γδ, CD4+ and CD8+ T cells. These results demonstrated that restraint stress decreased the number of i-IEL in the proximal small intestine of mice, mainly by the combined action of higher concentrations of catecholamines and glucocorticoids, and that lower concentrations of glucocorticoids and catecholamines in unstressed mice preserved the population of i-IEL.

Late Postnatal Expansion of Self-reactive CD8αα+ Intestinal Intraepithelial Lymphocytes in Mice*

The intestinal epithelium is unique in that it harbors auto-reactive T cells largely absent from the peripheral TCR repertoire in normal mice. Intestinal intraepithelial lymphocytes (IEL) expressing self-reactive TCR are mostly CD8αα+ cells in adult H-Y TCR RAG−/− male mice homozygous for the restricting MHC I allele, H-2Db. By contrast, in male mice heterozygous for the restricting and non-restricting MHC I allele, H-2Dd (MHC F1, H-2Db/d), IEL are composed of CD8αβ and CD8αα+ T cells. Here we demonstrate that IEL in the immediate postnatal period of MHC homozygous male mice were mostly CD8− T cells, while IEL in MHC F1 male mice were CD8− and CD8αβ+ T cells. Regardless of the MHC I configuration and the ability to support positive selection of CD8αβ+ cells in the thymus, the expansion of CD8αα+ IEL was a late postnatal event that followed a reduction in CD8− IEL. Furthermore, although in vivo treatment with the specific peptide antigen resulted in an earlier accumulation of activated IEL, the expansion of CD8αα+ IEL remained inefficient until late in postnatal life. Finally, as CD8− IEL stimulated with TCR agonists in vitro, acquired expression of CD8αα, we propose that CD8αα+ IEL derive from CD8− IEL intermediates. Whether CD8− IEL are CD8αβ-lineage cells that escape deletion in the thymus or are T cells targeted to the intestine from the thymus because of the early and high level TCR transgene expression in this model, is not clear. The signals required for the expansion of CD8αα+ IEL are however, incomplete in the immediate postnatal intestine. Determining the factors required for the expansion or retention of CD8αα+ IEL bearing high affinity, self-specific TCR will further elucidate the in vivo role of these T cells in intestinal homeostasis and perhaps, autoimmunity.

Diurnal changes in intraepithelial lymphocytes (IELs) in the small intestine of mice.

Diurnal changes in small intestinal intraepithelial lymphocytes (IELs) were examined in 8- to 10-week-old BALB/cA male mice. The ratio of T cell subsets expressing CD8 alpha alpha homodimer/CD8 alpha beta heterodimer was found to be higher in the dark period than that in the light period. Increased expression of Thy-1.2 on gamma delta T cells was also observed in the light period. No significant changes were found in other subsets. This is the first report to document diurnal changes in the small intestinal IELs in mice.

Hormone Regulation of Immune Homeostasis: Local or Long Distance?

The endocrine system and the immune system consist of dynamic biological processes involved on the one hand in the regulation of a complex array of metabolic and physiologic activities, and on the other hand in protection against infection and disease. Evidence for bidirectional functional involvement of immune–endocrine interactions can be seen at many levels, including codependence during critical stages of development, the complementary use of shared molecular mediators and receptors by both systems, and the integrated participation of the immune system and the endocrine system in resistance or susceptibility to disease. Moreover, recent findings—principally derived from studies of intestinal intraepithelial lymphocytes in mice—suggest that immune–endocrine interactions are essential for the proper development of intestinal T cells, and indicate that this most likely occurs via a local network of hormone synthesis and utilization. In the present article, these findings will be discussed in the context of immune–endocrine collaboration, with particular attention given to the involvement of the thymus in this process, and a hypothesis will be proposed which suggests that the homeostatic balance between health and disease is largely driven by local rather than systemic hormonal regulatory events.

Effects of a Nonapeptide Thymic Hormone on Intestinal Intraepithelial Lymphocytes in Mice Following Administration of 5-Fluorouracil

A significant fraction of murine small intestinal intraepithelial lymphocytes (i-IELs) maturate in local sites outside the thymus. However, there is evidence suggesting that extrathymic differentiation of i-IELs is still influenced by the thymus or thymus-derived factors. Facteur thymique serique (FTS), a nonapeptide thymic hormone, is involved in several aspects of intra- and extrathymic T cell differentiationin vivo.In this study, we investigated the effects of FTS on the kinetics of i-IELs in mice following a single administration of 5-fluorouracil (5-FU). FTS treatment significantly accelerated the recovery in cell number of i-IELs after administration of 5-FU. Flow cytometric analysis revealed that this accelerated recovery was mainly due to a rapid increase in CD8αα+i-IELs. Similar findings were also evident in adult thymectomized (ATX) mice, indicating that FTS treatment caused a rapid recovery of CD8αα+i-IELs following 5-FU administration in the absence of a functional thymus. Furthermore, expression levels of the mRNAs for interleukin-2, interferon-γ, and transforming growth factor β1in the i-IELs were augmented by FTS treatment. Notably, FTS treatment protected mice from 5-FU-induced lethal toxicity, accompanied with an inhibition of the translocation ofEnterobactericeae.These results suggest that FTS has an important function in the extrathymic maturation and activation of i-IELs in the small intestine following 5-FU administration, which may contribute at least partly to the protection against 5-FU-induced lethal toxicity.

Immunoregulatory functions for murine intraepithelial lymphocytes: gamma/delta T cell receptor-positive (TCR+) T cells abrogate oral tolerance, while alpha/beta TCR+ T cells provide B cell help.

Past work has shown that a subset of effector T cells with unique characteristics could abrogate hapten- or antigen-induced tolerance, and the reconstitution of this immune response has been termed contrasuppression. We have studied contrasuppression in a model of oral tolerance (OT) in which adoptively transferred antigen-specific T contrasuppressor (Tcs) cells reverse OT and result in antibody responses to the eliciting antigen. In the present study, we show that murine intraepithelial lymphocytes (IELs) from mice orally immunized with sheep red blood cells (SRBC) contain T cells that exhibit Tcs cell activity. This effect was mediated by CD3+ gamma/delta T cell receptor-positive (TCR+), but not alpha/beta TCR+ T cells, and gamma/delta TCR+ Tcs cells were associated with both the CD4-,CD8+ and CD4-,CD8- (double-negative) IEL fractions. The CD4-,CD8+ gamma/delta TCR+ IELs were further separated into Vicia villosa-adherent and -nonadherent fractions. Adoptive transfer of V. villosa-adherent gamma/delta TCR+ T cells to mice with OT to SRBC resulted in splenic IgA, IgM, and IgG subclass anti-SRBC responses, while V. villosa-nonadherent gamma/delta TCR+ T cells were without activity. The gamma/delta TCR+ IELs did not support in vitro antibody responses in B cell cultures, while alpha/beta TCR+ IELs were effective T helper cells. Further, cytokine production by the gamma/delta TCR+ IELs was examined, and the gamma/delta TCR+ V. villosa-adherent fraction, which possessed contrasuppressor function, contained low levels of IL-5 mRNA and small numbers of IL-5-producing cells when compared with alpha/beta TCR+ IELs and V. villosa-nonadherent gamma/delta TCR+ IELs. Our results now show that mouse IELs contain two distinct types of T cells that function in the immune response, e.g., alpha/beta TCR+ T cells that produce IL-5 and function as helper cells, and gamma/delta TCR+ T cells that restore antibody responses in mice that had been orally tolerized with antigen.

Anti-CD3-or PHA-induced cytotoxicity in human intraepithelial and lamina propria lymphocytes

Morphological data in humans and rodents and functional data of intraepithelial lymphocytes of mice support the idea that cytotoxic cells are a significant population of the human mucosa. Previously it was shown that human IEL have no spontaneous cell-mediated cytotoxicity and that human LPL cells have anti-CD3-mediated cytotoxicity. We confirm that most individuals have anti-CD3- or PHA-mediated cytotoxicity of LPL. In IEL we do not find cytotoxic function in short-term assays. There is no difference between patients with colon cancer and inflammatory bowel disease.

Presence of intestinal intraepithelial lymphocytes in mice with severe combined immunodeficiency disease

The murine intestinal epithelium contains a heterogeneous population of intraepithelial leukocytes (IEL) most of which are granulated, Thy-1-CD5-CD8+. In order to assess the lineage relationship of this subgroup of IEL to peripheral T cells, we examined IEL in mice with the severe combined immunodeficiency (scid/scid) mutation, which lack T and B cells in peripheral lymphoid tissues. Electron and light microscopy showed that the intestine from scid/scid mice had granulated IEL similar to IEL in normal C.B-17 mice. Flow cytometry of isolated IEL stained with monoclonal antibodies against Thy-1, CD3, CD4, CD5 and CD8 showed that scid/scid mice IEL contained cells with the Thy-1-CD4-CD5-CD8+ phenotype. Immunohistochemical staining of IEL in tissue sections with antibodies to Thy-1 and CD8 confirmed that the Thy-1-CD8+ cells were in the intestinal epithelium. These scid/scid IEL also lacked CD3 expression and mRNA for the V gamma 7 V region gene of the gamma T cell receptor. We conclude that scid/scid mice contain precursors for IEL that can differentiate into a granulated Thy-1-CD5-CD8+ IEL in the intestine. The absence of CD8+ peripheral T cells in these mice suggests that these IEL differ from classical T cells in their ability to differentiate and express CD8 and do not require T cell receptor expression for their localization to the intestine.

Limiting dilution analysis of functional T cells in the gut mucosa.

The function, nature and derivation of the lymphoid cells which are found in the gut epithelium remains controversial. In man, there is little doubt that virtually all intraepithelial lymphocytes (IEL) are of the T cell lineage because most of them are recognized by monoclonal antibodies specific for the T3 antigen complex associated with the T cell receptor (1,2). Functional data on these cells is, however, completely lacking. In rodents, the situation is much less clear because IEL have not been tested for the expression of T cell receptor related antigens and because of a body of evidence that many IEL are thymus-independent, contain granules and are Thy-1- (3–6). Indeed, we have recently shown by limiting dilution analysis that 1 in about 170 of normal mouse IEL are mast cell precursors, and that the frequency can reach as high as 1 in 60 if the mice are worm infected (7).