Role of intraepithelial lymphocytes in mucosal immune responses of mice experimentally infected with Cryptosporidium parvum

In order to investigate the role of intestinal intraepithelial lymphocytes (IELs) in host defense against Cryptosporidium parvum infection, conventionally bred immunocompetent (ImCT) ICR mice and immunosuppressed (ImSP) littermates were infected orally with 10(6) C. parvum oocysts. Then fecal oocyst excretion, the number and location of IELs, and their T lymphocyte subsets were observed on days 4, 7, 10, 13, 16, and 20 postinfection (PI). Uninfected ImCT and ImSP mice were used as controls. The starting point of oocyst excretion was day 4 PI in both ImCT- and ImSP-infected mice. The highest oocyst excretion occurred on day 7 PI in both groups, though the number of oocysts excreted was 3 times greater in ImSP than in ImCT mice. In ImCT mice, IELs greatly increased in number on days 16 and 20 PI (P < 0.05), but the increase was minimal in ImSP mice. IELs changed their location from the basal area to intermediate and apical areas of villous epithelial cells during the early stage of infection. In ImCT-infected mice, IEL phenotypes also changed; whereas CD4+ cells increased temporarily on day 7 PI (P < 0.05), CD8+ cells increased significantly on days 16 and 20 PI (P < 0.05). The results strongly suggest that IELs play a significant role in host defense against C. parvum infection, with helper T cells initiating control of the infection and cytotoxic T cells eliminating the parasites.     

Rotavirus-specific cytotoxic T lymphocytes appear at the intestinal mucosal surface after rotavirus infection.

The gastrointestinal tract is constantly exposed to a variety of potentially invasive bacteria and viruses. The first line of defense of the host against these pathogens is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IELs), mucus, and secretory immunoglobulins. Little is known about the function, memory, or trafficking of IELs after intestinal infection. We found that IELs obtained 6 days after oral inoculation of mice with the intestinal pathogen rotavirus (simian strain RRV) lysed rotavirus-infected target cells; cytotoxic T lymphocytes (CTLs) were responsible for rotavirus-specific cytotoxic activity. Rotavirus-specific cytotoxic activity by IELs was (i) eliminated by treatment with Thy 1.2-specific immunoglobulin M plus complement, (ii) restricted by proteins encoded at the major histocompatibility complex, and (iii) absent in mock-infected animals. Oral inoculation of mice with RRV also induced rotavirus-specific CTLs in splenic and intestinal lymphocytes (mesenteric lymph nodes, Peyer's patch). Parenteral inoculation induced rotavirus-specific CTLs in splenic, intestinal (IELs, mesenteric lymph nodes, Peyer's patch), and nonintestinal lymphocytes (inguinal nodes). Therefore, presentation of rotavirus to the intestinal mucosal surface was not necessary to induce IELs with virus-specific cytotoxic activity. At 4 weeks after oral or parenteral inoculation of mice with RRV, rotavirus-specific CTL precursors appeared among splenic, Peyer's patch, inguinal, and mesenteric node lymphocytes, but not among IELs. IELs with rotavirus-specific cytotoxic activity may be generated from precursors at a site other than the intestinal mucosal surface. Part of the response of the host to enteric infection may include surveillance and lysis of virus-infected villus epithelial cells by IELs.     

CD8 alpha alpha-mediated intraepithelial lymphocyte snatching of thymic leukemia MHC class Ib molecules in vitro and in vivo

Thymic leukemia (TL) is a MHC class Ib molecule that interacts with CD8alphaalpha homodimers. CD8alphaalpha is abundantly expressed by intraepithelial T lymphocytes (IELs) located in close proximity to TL-expressing intestinal epithelial cells. In this study, we show that CD8alphaalpha(+) IELs "snatch" TL from the plasma membrane of TL-expressing cells and express TL in its proper orientation on their own cell surface. TL snatching is enhanced by cross-linking of IEL TCRs in a phosphatidylinositol kinase-dependent manner, and results in overall alterations to the IEL cell surface detected by enhanced binding of peanut agglutinin lectin. Induction of bowel inflammation results in the presence of TL on IELs, probably via in vivo snatching, providing the initial evidence for the interaction of CD8alphaalpha IELs with intestinal cells.     

MSI-H colorectal cancers preferentially retain and expand intraepithelial lymphocytes rather than peripherally derived CD8<Superscript>+</Superscript> T cells

The healthy colorectal mucosa contains many resident intraepithelial lymphocytes (IELs) consisting of partially activated yet hyporesponsive CD8⁺ T cells. A predominant feature of colorectal cancers (CRCs) characterized by high levels of microsatellite instability (MSI-H) is heavy infiltration by an intraepithelial population of tumor infiltrating lymphocytes (iTILs). While it has been assumed that these iTILs originate from tumor infiltration by peripheral CD8⁺ effector T cells, their origin remains unknown. In light of the phenotypic and functional differences exhibited by IELs and peripheral T cells, elucidation of the precursor population of iTILs in MSI-H CRCs could clarify the role played by these lymphocytes in tumor progression. The aim of the present study was to investigate whether MSI-H CRCs interact differently with IEL- versus peripherally-derived CD8⁺ T cells. Using a Transwell assay system to mimic basolateral infiltration of tumor cells by lymphocytes, T cell migration, retention, proliferation and phenotypic alterations were investigated. Results indicate that MSI-H CRCs preferentially retain and expand IEL-derived cells to a greater degree than their microsatellite stable (MSS) counterparts. While MSI-H CRCs also retained more peripherally derived T cells, this number was considerably less than that from the IEL population. While interaction of IELs with either CRC type led to baseline lymphocyte activation, MSS CRCs induced upregulation of additional activation markers on retained IELs compared to MSI-H CRCs. These results suggest that the abundant iTILs present in MSI-H CRCs result from expansion of the preexisting mucosal IEL population and imply a limited prognostic role for iTILs in MSI-H CRC.     

Upregulation of ICOS on CD43+ CD4+ murine small intestinal intraepithelial lymphocytes during acute reovirus infection

Murine intestinal intraepithelial lymphocytes (IELs) can be classified according to expression of a CD43 glycoform recognized by the S7 monoclonal antibody. In this study, we examined the response of S7+ and S7- IELs in mice during acute reovirus serotype 3 (Dearing strain) infection, which was confirmed by virus-specific real-time PCR. In vivo proliferation increased significantly for both S7- and S7+ IELs on day 4 post-infection as determined by BrdU incorporation; however, expression of the inducible costimulatory (ICOS) molecule, which peaked on day 7 post-infection, was upregulated on S7+ CD4+ T cells, most of which were CD4+8- IELs. In vitro ICOS stimulation by syngeneic peritoneal macrophages induced IFN-gamma secretion from IELs from day 7 infected mice, and was suppressed by treatment with anti-ICOS mAb. Additionally, IFN-gamma mRNA increased in CD4+ IELs on day 6 post-infection. These findings indicate that S7- and S7+ IELs are differentially mobilized during the immune response to reovirus infection; that the regulated expression of ICOS is associated with S7+ IELs; and that stimulation of IELs through ICOS enhances IFN-gamma synthesis during infection.     

Multiple levels of activation of murine CD8(+) intraepithelial lymphocytes defined by OX40 (CD134) expression: effects on cell-mediated cytotoxicity, IFN-gamma, and IL-10 regulation.

The involvement of OX40 (CD134) in the activation of CD8(+) intestinal intraepithelial lymphocytes (IELs) has been studied using freshly isolated IELs and in vitro CD3-stimulated IELs. Although freshly isolated CD8(+) IELs exhibited properties of activated T cells (CD69 expression and ex vivo cytotoxicity), virtually all CD8(+) IELs from normal mice were devoid of other activation-associated properties, including a lack of expression of OX40 and the ligand for OX40 (OX40L) and an absence of intracellular IFN-gamma staining. However, OX40 and OX40L expression were rapidly up-regulated on CD8 IELs following CD3 stimulation, indicating that both markers on IELs reflect activation-dependent events. Unlike IELs, activated lymph node T cells did not express OX40L, thus indicating that OX40-OX40L communication in the intestinal epithelium is part of a novel CD8 network. Functionally, OX40 expression was exclusively associated with IELs with active intracellular IFN-gamma synthesis and markedly enhanced cell-mediated cytotoxicity. However, OX40 costimulation during CD3-mediated activation significantly suppressed IL-10 synthesis by IELs, whereas blockade of OX40-OX40L by anti-OX40L mAb markedly increased IL-10 production. These findings indicate that: 1) resident CD69(+)OX40(-) IELs constitute a population of partially activated T cells poised for rapid delivery of effector activity, 2) OX40 and OX40L expression defines IELs that have undergone recent immune activation, 3) OX40(+) IELs are significantly more efficient CTL than are OX40(-) IELs, and 4) the local OX40/OX40L system plays a critical role in regulating the magnitude of cytokine responses in the gut epithelium.     

Induction of a rapid and strong antigen-specific intraepithelial lymphocyte response during oral Encephalitozoon cuniculi infection

Encephalitozoon cuniculi continues to pose a problem for immunocompromised patients. Previous studies from our laboratory have elucidated the importance of the CD8(+) T cell subset in the protection against systemic parasite infection. There have been no studies related to the mucosal immunity induced against this orally acquired pathogen. In the present study, the immune response generated in the gut after oral E. cuniculi infection was evaluated. An early and rapid increase of the intraepithelial lymphocyte (IEL) population of orally infected animals was observed. This increase in the IEL population started as early as day 3 and peaked at day 7 postinfection with persistent elevation thereafter. At day 7 postinfection, IELs expressed strong cytokine messages (IFN-gamma and IL-10) and were highly cytotoxic for parasite-infected syngeneic macrophages. At an E:T ratio of 80:1, these cells were able to cause >60% Ag-specific target cell lysis. A significant increase in the CD8alphaalpha subset of IEL in response to an oral E. cuniculi infection was observed. To the best of our knowledge, such an early expansion of an IEL population exhibiting strong ex vivo cytotoxicity has not been reported with infectious models. These data suggest that IELs act as important barriers for multiplication of this organism leading to the successful resolution of infection. The protective role of IELs may be due both to their inflammatory (IFN-gamma production and cytotoxic response) as well as immunoregulatory (IL-10 production) properties.     

Recovery from chronic rotavirus infection in mice with severe combined immunodeficiency: virus clearance mediated by adoptive transfer of immune CD8+ T lymphocytes.

Severe combined immunodeficient (SCID) mice lack both functional T and B cells. These mice develop chronic rotavirus infection following an oral inoculation with the epizootic diarrhea of infant mice (EDIM) rotavirus. Reconstitution of rotavirus-infected SCID mice with T lymphocytes from immunocompetent mice allows an evaluation of a role of T-cell-mediated immunity in clearing chronic rotavirus infection. Complete rotavirus clearance was demonstrated in C.B-17/scid mice 7 to 9 days after the transfer of immune CD8+ splenic T lymphocytes from histocompatible BALB/c mice previously immunized intraperitoneally with the EDIM-w strain of murine rotavirus. The virus clearance mediated by T-cell transfer was restricted to H-2d-bearing T cells and occurred in the absence of rotavirus-specific antibody as determined by enzyme-linked immunosorbent assay, neutralization, immunohistochemistry, and radioimmunoprecipitation. Temporary clearance of rotavirus was observed after the transfer of immune CD8+ T cells isolated from the intestinal mucosa (intraepithelial lymphocytes [IELs]) or the spleens of BALB/c mice previously infected with EDIM by the oral route. Chronic virus shedding was transiently eliminated 7 to 11 days after spleen cell transfer and 11 to 12 days after IEL transfer. However, recurrence of rotavirus infection was detected 1 to 8 days later in all but one SCID recipient receiving cells from orally immunized donors. The viral clearance was mediated by IELs that were both Thy1+ and CD8+. These data demonstrated that the clearance of chronic rotavirus infection in SCID mice can be mediated by immune CD8+ T lymphocytes and that this clearance can occur in the absence of virus-specific antibodies.     

Differential contribution of Fas- and perforin-mediated mechanisms to the cell-mediated cytotoxic activity of naive and in vivo-primed intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IELs) are known to exert strong constitutive cytotoxic activity. In the present study we compared the Ag-specific cytotoxic activity and the effector mechanisms involved in non-Ag-primed, naive and in in vivo-primed IELs and splenic CD8 T cells. Ex vivo isolated naive CD8alphaalpha TCRalphabeta IELs, CD8alphabeta IELs, and splenocytes from lymphocytic choriomeningitis virus (LCMV)-specific TCR transgenic mice exert Ag-specific cytotoxic activity in a long-term, but not in a short-term, cytotoxicity assay. This cytotoxic activity is mainly Fas-Fas ligand mediated and is significantly reduced in the presence of 20 microg/ml Fas-Fcgamma1 fusion protein. Both CD8alphabeta IELs and CD8alphabeta splenocytes isolated from LCMV-infected C57BL/6 mice exert potent perforin-dependent cell-mediated cytotoxicity. CD8alphaalpha TCRalphabeta IELs from LCMV-infected animals, however, show only minimal Ag-specific cytotoxicity. The potent cytotoxic activity of in vivo activated CD8alphabeta IELs is not affected by the addition of Fas-Fcgamma1. Nevertheless CD8alphabeta IELs from LCMV-infected perforin-deficient mice exert Ag-specific cytotoxicity in a short-term cytotoxicity assay, and this cytotoxicity is almost completely blocked by the addition of Fas-Fcgamma1. These results demonstrate that naive CD8alphabeta IELs exert Ag-specific, Fas-Fas ligand-mediated, constitutive cytotoxic activity in a long-term cytotoxicity assay, whereas primed CD8alphabeta IELs primarily use the perforin-dependent exocytosis pathway to exert their potent cytotoxic activity. Furthermore, these results clearly illustrate the requirement for Ag-specific determination of IEL-mediated cytotoxicity, because the elevated, but variable, frequencies of memory-type T cells in this compartment may lead to ambiguous results when polyclonal activation or redirected assays are used.     

Characterization of intraepithelial lymphocytes in human endometrium

Intraepithelial lymphocytes (IELs) were characterized and quantitated in normal non-pregnant endometrium and in early pregnancy decidua using H & E and phloxine tartrazine stains and a panel of monoclonal antibodies in an indirect immunoperoxidase technique. The relative numbers of granulated and non-granulated IELs varied according to menstrual cycle stage and in early pregnancy all IELs appeared to be granulated. There was a higher surface:gland ratio for IELs in proliferative endometrium compared with late secretory phase and early pregnancy endometrium. In proliferative endometrium most IELs were T cells, predominantly of the CD8 + subset. In first trimester decidua, higher numbers of CD56 + cells were observed, in keeping with the increased proportion of granulated IELs. IEL populations in human endometrium vary according to menstrual cycle stage and endometrial IELs appear to show phenotypic differences compared with IELs in the human gastrointestinal tract.     

Memory and distribution of virus-specific cytotoxic T lymphocytes (CTLs) and CTL precursors after rotavirus infection.

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.     

Abnormal T cell development in CD3-zeta-/- mutant mice and identification of a novel T cell population in the intestine.

The T cell antigen receptor (TCR)-associated invariable membrane proteins (CD3-gamma, -delta, -epsilon and -zeta) are critical to the assembly and cell surface expression of the TCR/CD3 complex and to signal transduction upon engagement of TCR with antigen. Disruption of the CD3-zeta gene by homologous recombination resulted in a structurally abnormal thymus which primarily contained CD4- CD8- and TCR/CD3very lowCD4+CD8+ cells. Spleen and lymph nodes of CD3-zeta-/- mutant mice contained a normal number and ratio of CD4+ and CD8+ single positive cells that were TCR/CD3very low. These splenocytes did not respond to antibody cross-linking or mitogenic triggering. The V beta genes of CD4-CD8- and CD4+CD8+ thymocytes and splenic T cells were productively rearranged. These data demonstrated that (i) in the absence of the CD3-zeta chain, the CD4- CD8- thymocytes could differentiate to CD4+CD8+ TCR/CD3very low thymocytes, (ii) that thymic selection might have occurred, (iii) but that the transition to CD4+CD8- and CD4-CD8+ cells took place at a very low rate. Most strikingly, intraepithelial lymphocytes (IELs) isolated from the small intestine or the colon expressed normal levels of TCR/CD3 complexes on their surface which contained Fc epsilon RI gamma homodimers. In contrast to CD3-zeta containing IELs, these cells failed to proliferate after triggering with antibody cross-linking or mitogen. In comparison to thymus-derived peripheral T cells in the spleen and lymph nodes, the preferential expression of normal levels of TCR/CD3 in intestinal IELs suggested they mature via an independent extrathymic pathway.     

ART2, a T cell surface mono-ADP-ribosyltransferase, generates extracellular poly(ADP-ribose)

NAD functions in multiple aspects of cellular metabolism and signaling through enzymes that covalently transfer ADP-ribose from NAD to acceptor proteins, thereby altering their function. NAD is a substrate for two enzyme families, mono-ADP-ribosyltransferases (mARTs) and poly(ADP-ribose) polymerases (PARPs), that covalently transfer an ADP-ribose monomer or polymer, respectively, to acceptor proteins. ART2, a mART, is a phenotypic marker of immunoregulatory cells found on the surface of T lymphocytes, including intestinal intraepithelial lymphocytes (IELs). We have shown that the auto-ADP-ribosylation of the ART2.2 allelic protein is multimeric. Our backbone structural alignment of ART2 (two alleles of the rat art2 gene have been reported, for simplicity, the ART2.2 protein investigated in this study will be referred to as ART2) and PARP suggested that multimeric auto-ADP-ribosylation of ART2 may represent an ADP-ribose polymer, rather than multiple sites of mono-ADP-ribosylation. To investigate this, we used highly purified recombinant ART2 and demonstrated that ART2 catalyzes the formation of an ADP-ribose polymer by sequencing gel and by HPLC and MS/MS mass spectrometry identification of PR-AMP, a breakdown product specific to poly(ADP-ribose). Furthermore, we identified the site of ADP-ribose polymer attachment on ART2 as Arg-185, an arginine in a crucial loop of its catalytic core. We found that endogenous ART2 on IELs undergoes multimeric auto-ADP-ribosylation more efficiently than ART2 on peripheral T cells, suggesting that these distinct lymphocyte populations differ in their ART2 surface topology. Furthermore, ART2.2 IELs are more resistant to NAD-induced cell death than ART2.1 IELs that do not have multimeric auto-ADP-ribosylation activity. The data suggest that capability of polymerizing ADP-ribose may not be unique to PARPs and that poly(ADP-ribosylation), an established nuclear activity, may occur extracellularly and modulate cell function.     

Regional variations in the distributions of small intestinal intraepithelial lymphocytes (IELs) in BALB/c +/+, nu/+, and nu/nu mice

Regional variations in intraepithelial lymphocytes (IELs) in the small intestine were examined in BALB/c +/+, nu/+, and nu/nu mice. The small intestine was obtained from 11- to 12-week-old mice and divided equally into three (proximal, middle, and distal) parts. The IELs were isolated from each part of the intestine, and the total numbers of IELs in nu/+ and nu/nu mice were about a fifth of those in +/+ mice. Regional variations in the distribution of the IEL alphabeta, but not the gammadelta T-cell subset were found by use of flow cytometry in +/+ and nu/+ mice. On the other hand, such differences were not found in nu/nu mice, suggesting that thymus-independent development of T cells is not different among regions. Different local expansion of thymus-dependent alphabeta T cells may cause the regional variations seen in the distribution of alphabeta T cell IELs in +/+ and nu/+ mice.     

Maximum immunobioactivity of murine small intestinal intraepithelial lymphocytes resides in a subpopulation of CD43+ T cells

CD43 has been linked to many function-associated T cell activities. Using mAbs that recognize two different CD43 determinants, we show that, although mouse small intestinal intraepithelial lymphocytes (IELs) expressed the CD43 core molecule reactive with mAb R2/60, only about one-half of the total IELs-including some but not all of the TCRalphabeta and TCRgammadelta cells-expressed the CD43 S7(-) reactive determinant. CD43 S7(+) IELs secreted more IL-2, IL-4, IL-10, IL-17, and IFN-gamma following anti-CD3 stimulation, and were >4-fold more cytotoxic in fresh isolates and >16-fold more cytotoxic after anti-CD3 stimulation, than S7(-) IELs. S7(+) but not S7(-) IELs from the ileum of IL-10(-/-) mice spontaneously produced IFN-gamma. In vivo BrdU uptake by IELs in non-Ag-primed mice was greatest in the S7(+) population, indicating that significantly more S7(+) IELs than S7(-) IELs undergo cell expansion under normal homeostatic conditions. DNA microarray analyses showed that S7(+) IELs expressed higher levels of genes associated with activated T cells, whereas S7(-) IELs expressed genes used in the regulation of NK cells. These findings define two functionally distinct populations of IELs based on CD43 expression independent of TCR class, and they identify a subset of IELs that may serve as a target to better control intestinal inflammation.     

Glutamine supplementation increases Th1-cytokine responses in murine intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IELs) are major effector cells in the gut mucosal immune system, and are phenotypically distinct from thymic and peripheral T cells. Although nutritional supplementation with glutamine affects the intestinal immune response, it remains unclear whether this is a direct effect via the IEL-derived cytokines. This study examined changes in IEL-derived cytokine production following treatment with glutamine in vitro. Murine IELs were purified and activated with PMA plus ionomycin, and then cultured in the presence of various glutamine concentrations. IEL-derived cytokines were measured using a cytometric bead array (CBA) system, and IEL subsets were analyzed by flow cytometry. Treatment with glutamine increased the production of IL-2 and IFN-gamma from IELs in the presence of PMA plus ionomycin, but had no effect on TNFalpha, IL-4, or IL-5 production. Treatment with alanine or glucose had no regulatory effect on IEL-derived cytokines. Glutamine therefore had a direct effect on the production of selected IEL-derived Th1-cytokines, and enteral supplementation with glutamine may influence the intestinal immune responses mediated by IELs.     

Activation of intra-epithelial lymphocytes; their morphology,marker expression and ultimate fate

Intraepithelial lymphocytes (IELs) have been considered to play a key role in the defense system of the small intestine. Its mechanism has not been made sufficiently clear. Studies on IELs have been extremely limited to functions of αβ T-cell receptor (αβTCR) IELs (αβ-IELs). Since, in the mouse duodenum and jejunum, γδ-IELs consist 75 % of IELs, it thus would be inappropriate to argue the mechanism without extensive discussions over the functions of γδ-IELs. In previous studies, we found that the anti-CD3 monoclonal antibody (mAb) injection induced DNA fragmentation in intestinal epithelial cells (IECs) and DNA repair immediately after, that these responses were reproduced by anti-γδTCR mAb not by anti-αβTCR mAb and that the DNA fragmentation was induced by Granzyme B secreted by IELs, totally independent of Perforin. To further explore the functions of IELs in situ, we undertook experiments exclusively focused on IELs, on their changes and ultimate fate after the stimulation in mouse in vivo system. The current study demonstrated that the injected anti-CD3 mAb bound to CD3 on IELs, that the mAb activated γδ-IELs, leading to their degranulation, that changes occurred irreversibly in IELs and finally that activated IELs died in situ. γδ-IELs could be considered to respond to various stimulations most likely without the need of accessory cells (“always ready for rapid response”), to die in situ (“disposable”) and thus to respond to the stimulation only once (“a one-shot responder”). These characteristics of γδ-IELs are important to further elucidate the functions of γδ-IELs in the intestinal defense system.