Intraepithelial Lymphocyte Compartment Research Articles

P981 Risankizumab, but not ustekinumab or vedolizumab, downregulates IL-23-induced mucosal pathogenic Th17 cells in patients with Crohn’s disease

Abstract Background We recently described an interleukin(IL)-23-inducible pathogenic subset of CCR6+CXCR3-CCR5+ mucosal T helper (Th)17 cells (pTh17) infiltrating Crohn’s disease (CD) inflamed mucosa and specifically activated in response to Adherent-Invasive Escherichia Coli. pTh17 cells are functionally and phenotypically distinct from CCR6+CXCR3-CCR5- Th17 cells (cTh17) and from IL-12-induced CCR6+CXCR3+ Th1/17 subset. Here, we investigated pTh17 cells' recirculation into the bloodstream, localization into the intraepithelial lymphocyte (IEL) compartment, and in-vivo modulation by different biological therapies. Methods Adult patients with active CD initiating either vedolizumab as per clinical practice or ustekinumab and risankizumab during the SEQUENCE trial were prospectively recruited at Fondazione IRCCS Cà Granda, Ospedale Policlinico di Milano. In vedolizumab-treated patients, peripheral blood and intestinal biopsies were collected at baseline and at the end of induction. In ustekinumab- and risankizumab-treated patients, intestinal biopsies were collected outside study protocol assessments at baseline and week 24. Endoscopic response was defined as a greater than 50% decrease in the Simple Endoscopic Score from baseline. Peripheral blood mononuclear cells, lamina propria mononuclear cells (LPMC), and IEL were isolated and analyzed through flow cytometry. Results Overall, 34 CD patients were assessed. Demographic and baseline disease characteristics are summarized in Table 1. At baseline, as compared with cTh17, pTh17 cells: 1. are rarely found in the peripheral blood and express low levels of CCR9 and α4β7, 2. express high levels of tissue-resident markers CD69+CD103+ in the mucosa, and 3. infiltrate the IEL layer upon binding of CD103 with E-cadherin. Baseline pTh17 cells were upregulated in patients not achieving vedolizumab-induced endoscopic response. Upon vedolizumab treatment, a selective downregulation of mucosal cTh17 cells but not pTh17 was observed. Remarkably, risankizumab induced a specific downregulation of pTh17 cells, but not cTh17, in both LPMC and IEL layers. Conversely, ustekinumab reduced Th1/17 mucosal subsets, but not pTh17 cells (Figure 1). Conclusion pTh17 cells are largely unable to recirculate through the bloodstream, express markers of gut-tissue residency, and infiltrate the IEL compartment. IL-23 neutralization by risankizumab specifically downregulates mucosal pTh17 cells, as compared with vedolizumab and ustekinumab. Our immunologic results might provide mechanistic clues to vedolizumab latency effect in CD, and potentially explain early signs of risankizumab superiority over ustekinumab in the SEQUENCE study in terms of endoscopic response at 24 weeks.

Epithelial HNF4A shapes the intraepithelial lymphocyte compartment via direct regulation of immune signaling molecules.

Hepatocyte nuclear factor 4 α (HNF4A) is a highly conserved nuclear receptor that has been associated with ulcerative colitis. In mice, HNF4A is indispensable for the maintenance of intestinal homeostasis, yet the underlying mechanisms are poorly characterized. Here, we demonstrate that the expression of HNF4A in intestinal epithelial cells (IECs) is required for the proper development and composition of the intraepithelial lymphocyte (IEL) compartment. HNF4A directly regulates expression of immune signaling molecules including butyrophilin-like (Btnl) 1, Btnl6, H2-T3, and Clec2e that control IEC-IEL crosstalk. HNF4A selectively enhances the expansion of natural IELs that are TCRγδ+ or TCRαβ+CD8αα+ to shape the composition of IEL compartment. In the small intestine, HNF4A cooperates with its paralog HNF4G, to drive expression of immune signaling molecules. Moreover, the HNF4A-BTNL regulatory axis is conserved in human IECs. Collectively, these findings underscore the importance of HNF4A as a conserved transcription factor controlling IEC-IEL crosstalk and suggest that HNF4A maintains intestinal homeostasis through regulation of the IEL compartment.

Epithelial HNF4A shapes the intraepithelial lymphocyte compartment via direct regulation of immune signaling molecules

Abstract Hepatocyte Nuclear Factor 4A (HNF4A) is a highly conserved orphan nuclear receptor that has been associated with ulcerative colitis. Mice lacking Hnf4a specifically in the intestinal epithelial cells (IECs) are more susceptible to DSS-induced colitis and develop spontaneous colonic inflammation with aging. However, the mechanisms how HNF4A maintains intestinal homeostasis and protects against colitis remain unclear. Here we demonstrate that epithelial HNF4A shapes the intraepithelial lymphocyte (IEL) compartment by selectively promoting the development of natural IEL subsets that are TCRgd+ or TCRab+CD8aa+. Mechanistically, HNF4A serves as a transcription factor that directly regulates epithelial expression of immune signaling molecules involved in the IEC-IEL crosstalk. Those molecules include the butyrophilin-like (Btnl) molecules Btnl1 and Btnl6. Further, while HNF4A is stably expressed along the intestinal tract, these molecules are more abundant in the small intestine, which correlates with the distribution of natural IELs. We propose HNF4A paralog, HNF4G, which is restricted to the small intestine, cooperates with HNF4A in driving expression of signaling molecules and accounts for the geographic specificity. Moreover, HNF4A-BTNL regulatory axis is conserved in Caco2 cells of human colorectal origin. Collectively, our findings underscore the importance of HNF4A as a conserved transcription factor controlling IEC-IEL crosstalk and suggest that HNF4A maintains intestinal homeostasis through regulation of the IEL compartment.

Heterogeneity of human intestinal intraepithelial T cells and their abnormal distribution in Crohn’s disease revealed at high resolution

Abstract Crohn’s disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Recurrent/relapsing CD and resistance to medical treatments result in complications requiring surgery. High-dimensional single-cell profiling approaches, such as scRNA-seq and mass cytometry, have been recently performed on intestinal specimens from patients with IBD and controls. However, most of these studies have analyzed whole mucosal biopsies or the lamina propria (LP) compartment, while few have addressed the intraepithelial lymphocytes (IEL) compartment. Here, we profiled T cells purified from the IEL and LP from terminal ileum resections of adult severe CD cases by single cell technologies. Our study defined a vast heterogeneity of T cell lineages in the IEL compartment. IEL included, among others, unique γδT cell subsets: NKp30+γδ T cells expressing RORγt, which produced IL-26 upon NKp30 engagement and a subset expressing PDGFD and CSF1, which may act on epithelial cells, IEL ILC1s, and macrophages, respectively. We have also observed long-lived memory TCF7+CD8+ T cells expressing DC chemoattractants and TFH subsets that may respond to distinct glutathione-conjugated lipids. CD IEL showed a significant increase of activated TH17, coupled with decreased CD8+ T cells, γδT cells, TFH, and Treg. Conversely, the LP showed increased CD8+ T cells and reduced CD4+ T cells with a relative increase of TH17 over Treg/TFH. Results provide an unbiased view of diversity of cell lineages and their functional states in the intestinal mucosa of controls and CD and identify an altered spatial distribution of T cell subsets between the IEL and the LP compartments.

Chronic Inflammation Permanently Reshapes Tissue-Resident Immunity in Celiac Disease

Tissue-resident lymphocytes play a key role in immune surveillance, but it remains unclear how these inherently stable cell populations respond to chronic inflammation. In the setting of celiac disease (CeD), where exposure to dietary antigen can be controlled, gluten-induced inflammation triggered a profound depletion of naturally occurring Vγ4+/Vδ1+ intraepithelial lymphocytes (IELs) with innate cytolytic properties and specificity forthe butyrophilin-like (BTNL) molecules BTNL3/BTNL8. Creation of a new niche with reduced expression of BTNL8 and loss of Vγ4+/Vδ1+ IELs was accompanied by the expansion of gluten-sensitive, interferon-γ-producing Vδ1+ IELs bearing Tcell receptors (TCRs) with a shared non-germline-encoded motif that failed to recognize BTNL3/BTNL8. Exclusion of dietary gluten restored BTNL8 expression but was insufficient to reconstitute the physiological Vγ4+/Vδ1+ subset among TCRγδ+ IELs. Collectively, these data show that chronic inflammation permanently reconfigures the tissue-resident TCRγδ+ IEL compartment in CeD. VIDEO ABSTRACT.

G-Protein Coupled Receptor 18 Contributes to Establishment of the CD8 Effector T Cell Compartment.

The requirements for effector and memory CD8 T cell development are incompletely understood. Recent work has revealed a role for G-protein coupled receptor 18 (GPR18) in establishment of the intestinal CD8αα intraepithelial lymphocyte compartment. Here, we report that GPR18 is also functionally expressed in conventional CD8αβ T cells. When the receptor is lacking, mice develop fewer CD8+ KLRG1+ Granzyme B+ effector-memory cells. Bone marrow chimera studies show that the GPR18 requirement is CD8 T cell intrinsic. GPR18 is not required for T-bet expression in KLRG1+ CD8 T cells. Gene transduction experiments confirm the functional activity of GPR18 in CD8 T cells. In summary, we describe a novel GPCR requirement for establishment or maintenance of the CD8 KLRG1+ effector-memory T cell compartment. These findings have implications for methods to augment CD8 effector cell numbers.

Commensal metabolite indol-3-propionic acid promotes gut barrier function by regulating IL-22 production during intestinal inflammatory conditions

Abstract Microbiota-derived metabolites provide signals to maintain homeostasis of the epithelial cell layer in the intestinal mucosa that in turn shape immune responses to bacterial pathogens. In contrast, disruption of these signals can result in dysregulated responses to microbiota that cause inflammatory bowel diseases (IBD). Here we tested the hypothesis that a metabolite produced via tryptophan metabolism by commensal bacteria, Indol-3-propionic acid (IPA), promotes immune homeostasis and gut barrier function in the intestinal mucosa during inflammatory conditions and prevents IBD. We investigated the effect of IPA on the intestinal mucosa using a model of colitis caused by oral infection with Citrobacter rodentium (Crod). Remarkably, we observed that IPA treatment results in protection of mice from colitis. Our studies show that IPA treatment results in increased expression of IL-22 by intraepithelial lymphocytes (IELs) of the small intestine (S). In fact, most of the IL-22 observed at 4 days following oral Crod infection is produced by IELs from small intestines. Since we have previously shown that IPA is a potential ligand of the nuclear receptor pregnane X receptor (PXR), which contributes to the maintenance of the intestinal barrier function, we further investigated if IPA protects by activating PXR or other pathways in the gut mucosa. Indeed we discovered an increase in the absolute numbers of IL-17A and IFNγ producing lymphocytes from the IEL compartment of the colon by the IPA treatment and this effect was dependent on PXR expression. In conclusion, we believe the discovery of metabolites like IPA that promotes the repair of the intestinal epithelial cell layer can provide new strategies to prevent and treat IBD.

Analysis of Immune Cells from Human Mammary Ductal Epithelial Organoids Reveals Vδ2+ T Cells That Efficiently Target Breast Carcinoma Cells in the Presence of Bisphosphonate.

Developing strategies to enhance cancer prevention is a paramount goal, particularly given recent concerns about surgical treatment of preinvasive states such as ductal carcinoma in situ. Promoting effective immunosurveillance by leukocytes that scan for nascent neoplastic transformations represents a potential means to achieve this goal. Because most breast cancers arise within the ductal epithelium, enhancing protective immunosurveillance will likely necessitate targeting one or more of the distinctive lymphocyte types found in these sites under normal conditions. Here, we have characterized the intraepithelial lymphocyte compartment of non-cancerous human breast tissue and identified a subset of T lymphocytes that can be pharmacologically targeted to enhance their responses to breast cancer cells. Specifically, Vδ2(+) γδ T cells were consistently present in preparations of mammary ductal epithelial organoids and they proliferated in response to zoledronic acid, an aminobisphosphonate drug. Vδ2(+) T cells from breast ductal organoids produced the antitumor cytokine IFNγ and efficiently killed bisphosphonate-pulsed breast carcinoma cells. These findings demonstrate the potential for exploiting the ability of Vδ2(+) γδ T cells to respond to FDA-approved bisphosphonate drugs as a novel immunotherapeutic approach to inhibit the outgrowth of breast cancers.

Increased Intraepithelial Vα24 Invariant NKT Cells in the Celiac Duodenum.

Celiac Disease (CD) is an interferon (IFN)γ-mediated duodenal hypersensitivity to wheat gluten occurring in genetically predisposed individuals. Gluten-free diet (GFD) leads to a complete remission of the disease. Vα24-restricted invariant NKT (iNKT) cells are important to maintain immune homeostasis in the gut mucosa because of their unique capacity to rapidly produce large quantities of both T-helper (Th)1 and Th2 cytokines upon stimulation. We studied the presence of these cells in the CD duodenum. Duodenal biopsies were obtained from 45 untreated-CD patients (uCD), 15 Gluten Free Diet-CD patients (GFD-CD), 44 non-inflamed non-CD controls (C-controls) and 15 inflamed non-CD controls (I-controls). Two populations from Spain and Argentina were recruited. Messenger RNA (mRNA) expression of Vα24-Jα18 (invariant TCRα chain of human iNKT cells), IFNγ and intracellular transcription factor Forkhead Box P3 (Foxp3), and flow cytometry intraepithelial lymphocyte (IEL) profile were determined. Both uCD and GFD-CD patients had higher Vα24-Jα18 mRNA levels than non-CD controls (I and C-controls). The expression of Vα24-Jα18 correlated with Marsh score for the severity of mucosal lesion and also with increased mRNA IFNγ levels. uCD and GFD-CD patients had decreased mRNA expression of FoxP3 but increased expression of Vα24-Jα18, which revealed a CD-like molecular profile. Increased numbers of iNKT cells were confirmed by flow cytometry within the intraepithelial lymphocyte compartment of uCD and GFD-CD patients and correlated with Vα24-Jα18 mRNA expression. In conclusion, we have found an increased number of iNKT cells in the duodenum from both uCD and GFD-CD patients, irrespective of the mucosal status. A CD-like molecular profile, defined by an increased mRNA expression of Vα24-Jα18 together with a decreased expression of FoxP3, may represent a pro-inflammatory signature of the CD duodenum.

The composition and differentiation potential of the duodenal intraepithelial innate lymphocyte compartment is altered in coeliac disease

ObjectiveCoeliac disease (CD), a gluten-induced enteropathy, alters the composition and function of duodenal intraepithelial T cells. The intestine also harbours four types of CD3-negative intraepithelial lymphocytes (IELs) with largely unknown...

IL-21 signaling modulates the differentiation, distribution, and abundance of CD8 T cell subsets (LYM7P.616)

Abstract The activation of naïve CD8 T cells typically results in the formation of effector (TE) cells as well as memory cells that are retained over time. Memory CD8 T cells can be further subdivided into central memory (TCM), effector memory (TEM) and tissue-resident memory (TRM) subsets, which cooperate to confer immunological protection. Using adoptive transfer studies and mixed bone marrow chimeras in which CD8 T cells either do or do not express the IL-21 receptor (IL-21R), we discovered that under homeostatic or lymphopenic conditions IL-21 acts directly on CD8 T cells to favor the accumulation of TE/TEM populations. The inability to perceive IL-21 signals resulted in reduced accumulation of CD8 T cells in various tissues including the small intestine intraepithelial lymphocyte compartment. The reduced fraction of IL-21R-/- CD8 TRM cells in the small intestine also displayed markedly lower levels of the cytolytic effector molecule granzyme B. Notably, IL-21 signaling was associated with the expression of the chemokine receptor CX3CR1 and the integrin α4β7 on circulating CD8 T cells in the mixed bone marrow chimeras. Acute lymphocytic choriomeningitis virus infection overcame the necessity for IL-21 to establish CD8 TE/TEM and TRM subsets. Overall, this study demonstrates that IL-21 modulates the differentiation, accumulation, and function of effector-phenotype CD8 T cells in a context-dependent manner.

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.

Constitutive induction of intestinal Tc17 cells in the absence of hematopoietic cell‐specific MHC class II expression

The enteric pathogen Citrobacter rodentium induces a mucosal IL-17 response in CD4(+) Thelper (Th17) cells that is dependent on the Nod-like receptors Nod1 and Nod2. Here, we sought to determine whether this early Th17 response required antigen presentation by major histocompatibility complex class II (MHCII) for full induction. At early phases of C. rodentium infection, we observed that the intestinal mucosal Th17 response was fully blunted in irradiated mice reconstituted with MHCII-deficient (MHCII(-/-) →WT) hematopoietic cells. Surprisingly, we also observed a substantial increase in the relative frequency of IL-17(+) CD8(+) CD4(-) TCR-β(+) cells (Tc17 cells) and FOXP3(+) CD8(+) CD4(-) TCR-β(+) cells in the lamina propria and intraepithelial lymphocyte compartment of MHCII(-/-) →WT mice compared with that in WT→WT counterparts. Moreover, MHCII(-/-) →WT mice displayed increased susceptibility, increased bacterial translocation to deeper organs, and more severe colonic histopathology after infection with C. rodentium. Finally, a similar phenotype was observed in mice deficient for CIITA, a transcriptional regulator of MHCII expression. Together, these results indicate that MHCII is required to mount early mucosal Th17 responses to an enteric pathogen, and that MHCII regulates the induction of atypical CD8(+) T-cell subsets, such as Tc17 cells and FOXP3(+) CD8(+) cells, in vivo.

Distinct LLO-specific CD4 T cells infiltrate the intestinal mucosa following oral Listeria monocytogenes infection. (49.16)

Abstract We analyzed the mucosal CD4 T cell response to oral Listeria monocytogenes (LM) infection. Early after infection, a subset of blood-borne LLO-specific CD4 T cells expressed α4β7. Subsequently, antigen-specific CD4 T cells accumulated in the lamina propria and intraepithelial lymphocyte compartment where they were retained long-term. These data suggested that priming in the mucosal lymphoid tissues generated mucosa-seeking CD4 T cells with high memory potential. While naïve CD4 T cells contained both Ly6Chigh/CD27high and Ly6Clow/CD27high subsets, most primary and memory mucosal LLO-specific CD4 T cells were Ly6Clow/CD27low. In contrast, LLO-specific CD4 T cells in the spleen and MLN were comprised of four subsets based on CD27 and Ly6C expression. Upon challenge, mucosal CD4 T cells phenotypically resembled those in the primary infection, except for the appearance of a PD-1+ subset. Mucosal CD27low Ly6Clow and splenic CD27lowLy6Clow or high CD4 T cells either produced both IFN-γ and IL-2 or IFNγ alone and little IL-17 was detected. These results indicated that antigen-specific intestinal effector and memory CD4 T cells were phenotypically and functionally distinct from their lymphoid counterparts. Furthermore, the distinct phenotype of mucosal memory CD4 T cells implied that these cells may not recirculate. The generation of mucosal memory CD4 T cells in response to oral infection suggested a potential protective role for these cells in maintaining barrier infection.

γδ T cells play a protective role during infection with Nippostrongylus brasiliensis by promoting goblet cell function in the small intestine

The intestinal epithelium is rich in γδ T cells and the gut is a site of residence for a wide variety of pathogens, including nematodes. Although CD4+ T-cell receptor (TCR) -αβ+ T helper type 2 T cells are essential for the expulsion of intestinal nematodes, little information is available on the function of γδ T cells in this type of infection. Here, we demonstrate two major functions of γδ T cells as a potently protective T-cell population against Nippostrongylus brasiliensis infection using γδ T-cell-deficient (TCR-δ(-/-) ) mice. First, γδ T cells are required to initiate rapid expulsion of adult worms from the intestine and to limit egg production. Second, γδ T cells prevent the pathological intestinal damage associated with nematode infection, evident by increased clinical disease and more severe microscopic lesions in infected TCR-δ(-/-) mice. γδ T-cell deficiency led to delayed goblet cell hyperplasia in association with reduced expression of phosphorylated STAT6, MUC2, Trefoil factor-3 (TFF3) and T helper type 2 cytokines including interleukin-13 (IL-13). TCR-δ(-/-) mice also produced more interferon-γ than wild-type mice. Within the intraepithelial lymphocyte compartment, γδ T cells produced IL-13. Adoptive transfer of γδ T cells or administration of recombinant IL-13 to TCR-δ(-/-) mice successfully reduced the egg production by N. brasiliensis. Collectively, these data provide strong evidence that γδ T cells play an important role in controlling infection with intestinal nematodes and limiting infection-induced pathology.

Intestinal Epithelial Cells Modulate CD4 T Cell Responses via the Thymus Leukemia Antigen

The intestinal epithelium is comprised of a monolayer of intestinal epithelial cells (IEC), which provide, among other functions, a physical barrier between the high Ag content of the intestinal lumen and the sterile environment beyond the epithelium. IEC express a nonclassical MHC class I molecule known as the thymus leukemia (TL) Ag. TL is known to interact with CD8αα-expressing cells, which are abundant in the intestinal intraepithelial lymphocyte compartment. In this report, we provide evidence indicating that expression of TL by IEC modulates the cytokine profile of CD4(+) T cells favoring IL-17 production. We show in an adoptive transfer model of colitis that donor-derived cells become more pathogenic when TL is expressed on IEC in recipient animals. Moreover, TL(+)IEC promote development of IL-17-mediated responses capable of protecting mice from Citrobacter rodentium infection. We also show that modulation of IL-17-mediated responses by TL(+)IEC is controlled by the expression of CD8α on CD4(+) T cells. Overall, our results provide evidence for an important interaction between IEC and CD4(+) T cells via TL, which modulates mucosal immune responses.

E-cadherin promotes accumulation of a unique memory CD8 T-cell population in murine salivary glands

The salivary glands are important effector sites for IgA-mediated humoral immunity to protect oral surfaces. Within murine submandibular glands (SMG), we identified a memory CD8 T-cell population that exhibited a unique cell-surface phenotype distinct from memory CD8 T cells in spleen but similar to memory T cells resident in the intraepithelial lymphocyte compartment of the intestinal mucosa. In mice immune to lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus(VSV), virus-specific memory CD8 T cells with this unusual phenotype were present in SMG at remarkably high frequencies. LCMV-specific memory CD8 T cells in SMG showed potent functional activities in vivo, including cytokine-induced bystander proliferation, antigen-triggered IFNγ production, and viral clearance. Adoptive transfer experiments further revealed that the capacity to accumulate in SMG decreased during CD8 T-cell differentiation and that SMG CD8 T cells were poorly replenished from the circulation, indicating that they were tissue-resident. Moreover, they preferentially relocalized within their tissue of origin after adoptive transfer and antigen rechallenge, thus revealing an imprinted differentiation status. Accumulation of memory CD8 T cells within SMG did not require local antigen presentation but was promoted by the epithelial differentiation molecule E-cadherin intrinsically expressed by these CD8 T cells. This finding extends the epithelial-restricted function of E-cadherin to an impact on lymphocyte accumulation within epithelial tissues.

Uneven colonization of the lymphoid periphery by T cells that undergo early TCR{alpha} rearrangements.

A sparse population of thymocytes undergoes TCRalpha gene rearrangement early in development, before the double-positive stage. The potential of these cells to contribute to the peripheral T cell pool is unknown. To examine the peripheral T cell compartment expressing a repertoire biased to early TCR gene rearrangements, we developed a mouse model in which TCRalpha rearrangements are restricted to the double-negative stage of thymocyte development. These mice carry floxed RAG2 alleles and a Cre transgene driven by the CD4 promoter. As expected, conventional T cell development is compromised in such Cre(+) RAG2(fl/fl) mice, and the TCRalphabeta(+) T cells that develop are limited in their TCRalpha repertoire, preferentially using early rearranging Valpha genes. In the gut, the Thy-1(+)TCRalphabeta(+) intraepithelial lymphocyte (IEL) compartment is surprisingly intact, whereas the Thy-1(-)TCRalphabeta(+) subset is almost completely absent. Thus, T cells expressing a TCRalpha repertoire that is the product of early gene rearrangements can preferentially populate distinct IEL compartments. Despite this capacity, Cre(+) RAG2(fl/fl) T cell progenitors cannot compete with wild-type T cell progenitors in mixed bone marrow chimeras, suggesting that in normal mice, there is only a small contribution to the peripheral T cell pool by cells that have undergone early TCRalpha rearrangements. In the absence of wild-type competitors, aggressive homeostatic proliferation in the IEL compartment can promote a relatively normal Thy-1(+) TCRalphabeta(+) T cell pool from the limited population derived from Cre(+) RAG2(fl/fl) progenitors.

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

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

Phenotypic and Functional Similarity of Gut Intraepithelial and Systemic T Cells in a Teleost Fish

Gut-associated lymphocytes were described in fish, but their involvement in immune responses is still unknown. In rainbow trout, intraepithelial lymphocytes (IELs) are scattered between gut epithelial cells, but neither Peyer's patches nor mesenteric lymph nodes were identified. Rainbow trout IELs contain mainly T cells, because they expressed transcripts of T cell marker homologs of CD8, CD4, CD28, CD3epsilon, TCRzeta, TCRgamma, and TCRbeta and lacked IgM. However, trout IELs did not show specific homing to the gut mucosa, which in mammals defines IELs as a distinctive mucosal population. A detailed analysis of the TCRbeta repertoire of rainbow trout IELs was performed in both naive and virus-infected animals. TCRbeta transcripts of rainbow trout IELs were highly diverse and polyclonal in adult naive individuals, in sharp contrast with the restricted diversity of IEL oligoclonal repertoires described in birds and mammals. Significant modifications of the trout IEL TCRbeta repertoire were observed after a systemic infection with a fish rhabdovirus and were especially marked for Vbeta4-bearing receptors as previously reported for spleen cells. Thus, we could not find any specific properties of the trout IEL TCRbeta repertoire compared with the spleen and pronephros TCRbeta repertoire, which questions the reality of a distinct IEL compartment in teleosts. Our findings suggest that a highly diversified alphabeta TauCR repertoire is maintained in fish IELs in the absence of Peyer's patches and mesenteric lymph nodes, whereas the restricted diversity of mouse alphabeta IELs is attributed to multiple cycles of activation and recirculation, allowing a progressive narrowing of the repertoire.