Unconventional Intestinal Intraepithelial Lymphocytes in Health and Disease

Abstract

The intestinal epithelium is constantly exposed to a myriad of antigenic stimuli derived from commensals, food particles and pathogens present in the lumen of the intestines. This complex environment requires a similarly complex immune system capable of preventing exacerbated responses against food particles and commensals, while at the same time eliminating potential pathogens. These functions are accomplished in part by the intraepithelial lymphocyte (IEL) compartment. IELs are a diverse group of immune cells that primarily reside in between intestinal epithelial cells, maintaining an intimate association with these cells. IELs are a diverse population of cells: some of them express a T cell receptor (TCR), while others do not, and within TCR⁺ and TCR⁻ IELs there are many IEL subpopulations that represent different developmental pathways and functions. In this review, we will focus on “unconventional” T cells present in the intestinal epithelium, in particular TCRγδ⁺, TCRαβ⁺CD4⁺CD8αα⁺, and TCRαβ⁺CD8αα⁺ IELs. We will discuss their development and potential functions both in humans and in mice.

I. INTRODUCTION

The small and large intestines are primarily known for their roles in nutrient digestion, absorption, and water retention. Because food is derived from the non-sterile environment outside of the body, the intestines are in constant contact with particles, macro and microorganisms, toxins, and other molecules that may gain access to the lumen of the intestines. Therefore, it is tempting to describe the lumen of the intestines as the “outside inside of the host.” The high burden of antigens in the lumen of the intestines represents an enormous challenge to the host’s immune system, which needs to discriminate between those antigens that may be harmful and require an immediate response, and those that are beneficial and should be tolerated. This critical balance is achieved by a finely tuned mucosal immune response associated with the intestinal epithelium, which comprises a variety of tissues and organs including mesenteric lymph nodes, Peyer’s patches, and lamina propria. Lymphoid cells, such as conventional CD4⁺ and CD8⁺ T cells, B cells, innate lymphoid cells, natural killer (NK) and NKT cells are prominent in these tissues and organs and are involved in different phases of mucosal immune responses.

An important lymphocyte compartment in the intestinal epithelium is represented by cells that reside in intimate association with intestinal epithelial cells (IECs), also known as intraepithelial lymphocytes (IELs).^1–3 The IEL compartment has produced much intrigue since it was first observed by the German physician Ernst Heinrich Weber in 1847, and many fascinating theories about its possible physiological role have been proposed (see the 1977 review by Ferguson).⁴ In terms of size, it is estimated that in the small intestine of mice there is one IEL per 10 IECs, whereas in the colon this ratio drops to one IEL per 40 IECs, indicating that the great majority of IEL is present in the small intestine.⁵ Considering the large surface area of the small and large intestines combined (~ 32 m², according to conservative estimates, although a range of up to 300 m² is reported in the literature⁶), it is easy to realize that the IEL compartment is one of the largest immune sites in the body, and thus, its importance is underscored.

IEL are not a homogeneous population of lymphoid cells. On the contrary, the IEL compartment is comprised of diverse groups of cells with distinct developmental pathways and roles. Because of these features, classifying IEL may appear complicated. A simple way to group these cells is based on the expression (or lack thereof) of a T cell receptor (TCR).² TCR⁺ IELs comprise approximately 80–90% of the total IEL compartment, and because of their large numbers and easier isolation, these cells have historically been more thoroughly studied. TCR⁺ IELs can be further subdivided in TCRαβ⁻ and γδ-expressing cells. Conventional T cells, such as CD4⁺ and CD8αβ⁺ T cells, constitute a significant fraction of the TCRαβ⁺ IEL, and these cells represent antigen experienced cells that were activated outside of the intestines and migrated to the epithelium as effector or memory cells, serving as sentinels against potential antigenic insults. However, a large fraction of TCRαβ⁺ IEL is represented by cells that do not express the CD4 or CD8αβ co-receptors, but instead express CD8αα homodimers. Finally, in mice, ~ 40 to 50% of TCR⁺ IELs correspond to TCRγδ⁺ cells. The remaining proportion of cells is composed of TCR⁻ IELs, which represent a small fraction of the total IEL compartment (~ 10%), and include innate lymphoid cells^7,8 and the newly described IEL family characterized by the intracellular expression of CD3.^9,10

An important distinction between IEL and lymphoid cells in other immune compartments is that a great proportion of TCR⁺ and TCR⁻ IELs express the CD8αα homodimer. For example, a fraction of conventional CD4⁺ and CD8αβ⁺ T cells in the epithelium expresses CD8αα, generating TCRαβ⁺CD4⁺CD8αα⁺ and TCRαβ⁺CD8αβ⁺CD8αα⁺ cells. Expression of CD8αα by conventional CD4⁺ or CD8αβ⁺ T cells is not commonly seen in other immune sites. CD8αα is regarded as a repressor of IEL activation,¹¹ mediated by the interaction with the thymus leukemia (TL) antigen expressed in IEC.^12–14 It is important to note that a human homologue for TL has not been discovered, and therefore how human IEL effector function is regulated is yet to be described.

The focus of this review is on “unconventional” TCR⁺ IEL populations, with a particular emphasis on the mouse system, where these cells have been studied extensively. These populations include TCRγδ⁺, TCRαβ⁺CD4⁺CD8αα⁺, and TCRαβ⁺CD8αα⁺CD8αβ⁻CD4⁻ IEL (Fig. 1). Although the TCRαβ⁺CD4⁺CD8αα⁺ IEL population derives from conventional CD4⁺ T cells, its function in the intestinal epithelium appears to be different from that of its precursors. Due to lack of significant studies, TCRαβ⁺CD8αβ⁺CD8αα⁺ IELs will be omitted from this review.

FIG. 1:

Summary of some of the features of the IEL populations reviewed herein

II. TCRγδ⁺ IELS

TCRγδ⁺ cells constitute a minor fraction of the circulating T cell compartment (1–5%), either in the blood or other lymphoid sites.¹⁵ However, the proportion of TCRγδ⁺ cells is significantly higher in barrier sites, such as the intestinal mucosa and the skin. In mice, TCRγδ⁺ cells constitute a large fraction of all lymphoid cells present in the IEL compartment, which depending on the colony, can vary between 40 and 70%.^16,17 Furthermore, > 80% of the total TCRγδ⁺ IELs in mice express CD8αα homodimers, suggesting that these cells are also regulated by the TL-CD8αα interaction.

A. Development

TCRγδ⁺ cells are the first T cells to arise during fetal development; however, as the host develops, the prevalence of TCRγδ⁺ cells decreases as TCRαβ⁺ T cell numbers increase. In adult organisms, TCRγδ⁺ T cells are rare in the circulating immune system, such as in blood, but are predominant in epithelial surfaces such as the skin, intestines, and uterus. Interestingly, in each of these epithelial surfaces TCRγδ⁺ T cells predominantly express in mice a particular variable TCR γ chain segment: Vγ5 in skin, Vγ6 in uterus, and Vγ7 in the intestines.^18–20 In human intestines, TCRγδ⁺ IEL preferentially express the Vγ4 segment.²¹ In mice, the frequency of Vγ7⁺ IEL comprises ~ 40% of the total CD3⁺TCRβ⁻ IEL population 2 days after birth, reaching a plateau of 60–70% at day 4.²² Vγ7⁺ IELs express CD122, TIGIT (an inhibitory co-receptor), Lag3, CD3, and CD8αα.²²

The specific usage of variable TCR γ segments in a precise epithelial environment suggests that the selection of these γ chain fragments is related to organ-specific cues that induce TCRγδ⁺ T cell differentiation, which aligns with their potential roles (see below). In contrast to TCRαβ⁺ T cells, TCRγδ⁺ T cells do not require MCH class I or II for their development,²³ and display normal cellularity in b2-microglobulin deficient mice,²⁴ which excludes TCRγδ⁺ T cells from the requirement of major histocompatibility complex (MHC) class I and related molecules (such as CD1 and MR1) for their selection and/or development.

Most TCRγδ⁺ IELs, in particular Vγ7⁺ cells, develop in nude mice, indicating that although these cells can develop in the thymus, alternative pathways support their development in nude mice.²² In addition, the number of Vγ7⁺ cells in germ-free mice and food antigen-free mice resemble that of conventionally-reared mice,²² indicating that neither the microbiota nor antigens present in food support the generation of Vγ7⁺ cells. Therefore, if the thymus, microbiota, and food antigens do not support the development of TCRγδ⁺ IELs, then this development must depend on interactions with IECs (see below). Although food antigens seem not to be relevant for the development of TCRγδ⁺ IELs, it has been shown that aryl hydrocarbon receptor (Ahr) ligands derived from cruciferous vegetables (such as broccoli and cabbage), sustain Ahr expression and TCRγδ⁺ IEL numbers.²⁵ In addition, Ahr^−/− mice display severely reduced TCRγδ⁺ IEL numbers, indicating the importance of this receptor in TCRγδ⁺ IEL maintenance.²⁵

B. Butyrophilin-Like Molecules Regulate TCRγδ⁺IEL Development

To understand what signal(s) promote the development of TCRγδ⁺ IELs, it is important to consider the environment where they reside. TCRγδ⁺ IELs occupy a niche surrounded by IEC, suggesting that the intimate relationship between these cells should be important for the development of TCRγδ⁺ IEL. Indeed, the selection and upkeep of intraepithelial T cell protein 1 (Skint1), a protein expressed in thymic and skin epithelial cells, proved to select Vγ5 TCRγδ⁺ T cells.^26,27 The expression of Skint1 is restricted to the thymus and skin, so its role in the development of TCRγδ⁺ IEL is questionable. However, the search for genes similar to Skint1 that may be important for the development of TCRγδ⁺ IELs resulted in the identification of the butyrophilin-like gene family (Btnl), which is preferentially expressed in the intestines, in particular in enterocytes.²⁸ In mice, the Btnl family is represented by Btnl1, Btnl2, Btnl4, and Btnl6; whereas in humans is represented by BTNL2, BTNL3, BTNL8, and BTNL9.²⁹ The Btnl family is a member of the Ig superfamily, displaying intracellular, transmembrane IgC and IgV domains, similar in structure to CD80 and PD-L1.²⁸ Some members of the Btnl family are known to form homodimers (e.g., Btnl1–Btnl1) or heterodimers (e.g. Btnl1–Btnl6), the latter with the capacity to stimulate the proliferation of Vγ7⁺ IELs.³⁰ Btln1-Btln6 in mice (BTLN3-BTLN8 in humans) binds directly to the TCRγδ receptor on the “side” of the IgV domain, which does not hinder the recognition of the CDR regions by other molecules that can engage the TCR,^31,32 such as the MHC class Ib molecule T22.³³ These remarkable observations indicate the flexibility of the TCRγδ chains to function as an innate and adaptive receptor.

Mice deficient in Btnl1 present severe deficiency in Vγ7⁺ IELs, whereas the numbers of Vγ7⁻, TCRβ⁺CD8αα⁺, TCRβ⁺CD8αβ⁺, and TCR⁺CD8α⁻ IEL remain the same or are increased, possibly as a compensatory mechanism.²² However, using an elegant experimental approach where Btnl1 expression was temporarily re-established in the intestinal epithelium, especially during the first weeks of life, an increase in mature Vγ7⁺CD122^hi IEL was observed in adult mice.²² Therefore, Btnl1’s role in TCRγδ⁺ IEL maturation and expansion varies depending on the age of the host. Interestingly, although Btnl1 appears to be essential for the maturation of Vγ7⁺ IEL, global or IEC-targeted Btnl6 deficiency only results in partial reduction of this IEL population.³⁴ In addition, temporal depletion of Btnl1, 4, 6 in adult mice showed that once Vγ7⁺ IELs are mature, their prevalence in the intestinal epithelium does not require sustained expression of the Btnl family of proteins.³²

C. TCRγδ⁺ IEL Role in the Intestinal Epithelium

Since their discovery, the role of TCRγδ⁺ IELs in the intestinal epithelium has been an area of intensive research. Earlier reports using global TCRδ-deficient mice (Tcrd^−/−) indicated that these mice displayed reduced intestinal crypt cell numbers and decreased generation of IEC.³⁵ Indeed, TCRγδ⁺ IELs have been proven to have an important role in protecting and promoting IEC growth and turnover.³⁶ The role of TCRγδ⁺ IELs in intestinal homeostasis was also evident during intestinal infection with the apicomplexan parasite Eimeria vermiformis, where Tcrg^−/− mice displayed increased intestinal blood loss and villus tip damage, indicating a role for TCRγδ⁺ IELs in regulating the pathologic consequences of the response against E. vermiformis.³⁷

The role of TCRγδ⁺ IELs has been extensively studied in the mouse model of intestinal epithelial injury caused by dextran sulfate sodium (DSS). Intestinal epithelial injury with DSS results in increased localized recruitment of keratinocyte growth factor–producing TCRγδ⁺ IELs to the damaged sites. In addition, Tcrg^−/− mice exposed to DSS are more susceptible to DSS-induced injury and displayed delayed tissue repair.^38–40 These reports clearly show that TCRγδ⁺ IEL are a key population of cells involved in tissue repair. Moreover, TCRγδ⁺ IEL in mice undergoing DSS-mediated tissue injury express augmented levels of immuno-modulatory and antibacterial factors, such as KC, CXCL-9, interleukin (IL)-1β, MIP2α, RegIIIγ, and lysozyme, indicating that in addition to tissue repair, TCRγδ⁺ IEL also promote an anti-microbial response to prevent pathogen penetration of the epithelium.⁴¹

TCRγδ⁺ IEL are also involved in immune responses against certain pathogens. For example, during Toxoplasma gondii infection, TCRγδ⁺ IELs synergize with TCRαβ⁺CD8αα⁺ IELs to confer long-term immunity against this parasite.⁴² Moreover, in the absence of TCRγδ⁺ IELs, the intestinal barrier function is compromised, resulting in Toxoplasma, as well as Salmonella typhimurium, epithelial translocation.⁴³

As mentioned above, TCRγδ⁺ IELs express Reg3g, which is upregulated upon encounter with specific bacteria groups of the intestinal microbiota, implying that TCRγδ⁺ IELs have a critical role controlling pathobionts from causing tissue injury.⁴⁴ Expression of Reg3g by TCRγδ⁺ IELs depends on the activity of MyD88 by IECs,⁴⁴ indicating an important crosstalk between TCRγδ⁺ IELs and IECs (see below).

TCRγδ⁺ IELs are also important during intestinal worm infections. In the case of Nippostrongylus brasiliensis, TCRγδ⁺ IELs are required to initiate a rapid expulsion of the adult worms from the intestine, which would reduce egg production and subsequent parasite spreading.⁴⁵ Protection against this parasite is potentially mediated by TCRγδ⁺ IEL-derived IL-13.⁴⁵ Consistent with their role in tissue protection and repair, absence of TCRγδ⁺ IELs during infection with N. brasiliensis results in more severe pathology.⁴⁵

TCRγδ⁺ IELs have also been involved in the establishment and maintenance of oral tolerance, because depletion of these cells either by monoclonal antibodies or genetic means abolishes tolerance to ovalbumin and results in failure to produce IL-10.^46,47 However, the mechanism involved in maintenance of oral tolerance by TCRγδ⁺ IELs is not fully understood.

A recent report indicates that TCRγδ⁺ IELs may possess an important role during nutritional shifts. When mice are fed a diet rich in carbohydrates, IEC switch their transcriptional program to properly digest and absorb carbohydrates.⁴⁸ This switch is mediated by TCRγδ⁺ IELs, which migrate from the villi to the crypts, where they inhibit production of IL-22 by innate lymphoid cells.^48,49 How TCRγδ⁺ IELs respond to nutritional cues remains to be elucidated. TCRγδ⁺, as well as other IELs such as TCRαβ⁺CD8αα⁺ cells, express integrin β7, which helps these cells to home to the intestinal epithelium.⁵⁰ Interestingly, Itgb7^−/− (which encodes for integrin β7) mice, which are deficient in TCRγδ⁺ and other IELs, are resistant to obesity, hypertension, and atherosclerosis.⁵¹ GLP-1, an enteroendocrine-derived incretin, exerts glucose control and mediates various beneficial metabolic effects.⁵² TCRγδ⁺ IELs express the GLP-1 receptor, which serves as a sink for GLP-1, resulting in increased energy uptake that may lead to obesity, diabetes, and other diseases.⁵¹ Thus, TCRγδ⁺ IELs, and most likely other GLP-1R⁺ IELs, serve as gatekeepers that modulate the energy intake of the host.

TCRγδ⁺ IEL mobility has been an area of exciting research in the past years. Elegant studies using intravital multiphoton microscopy have shown that TCRγδ⁺ IELs are very motile and rapidly migrate between IECs and the basement membrane of the epithelium.^53–55 TCRγδ⁺ IEL motility appears to be a mechanism for these IELs to interact with IECs in steady-state conditions, probably picking up signals derived from the intestinal lumen. Indeed, in germ-free mice, TCRγδ⁺ IELs display decreased IEC scanning compared with conventional animals, suggesting that the microbiota promotes TCRγδ⁺ IEL mobility.⁵⁴ Interestingly, during infection with Salmonella and Toxoplasma, TCRγδ⁺ IEL “squeeze” further in between IEC in a process that the authors call “flossing.”⁵⁴ Flossing is more evident in areas of the epithelium with higher pathogen burden, suggesting that TCRγδ⁺ IEL modified their motility behavior to respond to infections. Scanning and flossing are dependent on Myd88 expression by IEC, since in its absence these processes are significantly decreased.⁵⁴ In addition, TCRγδ⁺ IEL motility in the intestinal epithelium is mediated by contact with enterocytes via homotypic interactions between occludins.⁵⁵

In individuals with inflammatory bowel disease, increased severity is highly associated with abundant numbers of TCRγδ⁺ IELs.^56,57 Similar observations have been reported in patients with celiac disease.⁵⁸ However, in celiac disease, the increase in TCRγδ⁺ IELs also correlates with a deep reshaping of the TCRγδ⁺ IEL repertoire. For example, healthy humans present a semi-invariant Vγ4+Vδ1⁺ and Vγ4⁺Vδ1⁻ IEL populations that recognize BTNL3/8 and are poised in epithelium expressing cytotoxicity receptors to eliminate virus-infected or malignant cells. However, in celiac disease individuals, the Vγ4⁺Vδ1⁺ IEL are permanently lost and are supplanted by gluten sensitive Vδ1⁺ IELs that no longer respond to BTNL3/8.⁵⁹

Whether TCRγδ⁺ IELs possess a detrimental or favorable function in these diseases is yet to be determined. However, considering their observed functions in the mouse model, it is likely that TCRγδ⁺ IELs in humans also protect and restore the intestinal mucosa after injury.

D. Summary

Because of their abundance in the mouse intestinal epithelium, TCRγδ⁺ IELs have gained much attention since their discovery, and due to exquisite tools available for their study, many aspects of their biology have been successfully interrogated. As we have discussed throughout this section, TCRγδ⁺ IELs constitute an important population in the intestinal epithelium, which maintains tissue homeostasis and guards against pathogens.

III. TCRαβ⁺CD4⁺CD8αα⁺ IEL

For several years, it was considered that outside of developing thymocytes, human and mouse TCRαβ⁺CD4⁺ cells did not co-express the CD8 co-receptor. However, when multicolor fluorescence flow cytometry became available, it was discovered that the IEL compartments of naïve mice of different backgrounds consistently harbor TCRαβ⁺CD4⁺CD8⁺ cells,^60,61 and later it was demonstrated that, contrary to what is observed in the thymus, TCRαβ⁺CD4⁺CD8⁺ cells in the intestines almost exclusively express CD8αα homodimers instead of the conventional CD8αβ co-receptor.⁶² Here, we present how these intriguing cells develop and their potential role in mucosal immune responses.

A. Development

TCRαβ⁺CD4⁺ and TCRαβ⁺CD8αβ⁺ IELs are descendants of conventional T cells, which mature in the thymus and are MHC class II and I restricted, respectively. In the thymus, T cell precursors undergo positive and negative selection, yielding mature naïve single-positive CD4⁺ or CD8⁺ T cells. Thymic T cell fate is driven by dynamic interactions between transcription factors. The expression of runt-related transcription factor 3 (Runx3) and the zinc-finger transcription factor MAZR (encoded by the Patz1 gene) controls CD8⁺ T cell development from double-positive precursors, while CD4⁺ T cell development is primarily controlled by the T helper-inducing POZ/Kruppel-like factor, ThPOK (encoded by the Zbtb7b gene).^63–68 After development in the thymus, T cells migrate to the peripheral lymphoid organs to encounter foreign antigens and differentiate into effector and memory cells. Those cells primed in immune compartments associated with the intestinal mucosa, such as the mesenteric lymph nodes and Peyer’s patches, may migrate into the IEL compartment where they reside as conventional effector and memory T cells.

A fraction of activated TCRαβ⁺CD4⁺ IELs acquire CD8αα cell surface expression upon homing to the intestinal epithelium, becoming TCRαβ⁺CD4⁺CD8αα⁺ IELs or double-positive IELs (DP IEL). DP IELs were first discovered in mice in 1990 and account for up to 50% of murine TCRβ⁺CD4⁺ IELs.⁶¹

Similar to other CD4⁺ T cells, the TCR is important for the initial thymic development of TCRαβ⁺CD4⁺CD8αα⁺ IELs. Once in the IEL compartment, DP IELs present a restricted or oligoclonal TCR repertoire.⁶⁹ In recent years, it has become apparent that regulatory T cells may function as a reservoir of DP IEL,⁷⁰ a concept later confirmed by Hidde Ploegh and collaborators. This group used somatic cell nuclear transfer to generate a transnuclear mouse line that carries a TCR cloned from the nucleus of a regulatory T cell isolated from the mesenteric lymph nodes. Interestingly, they observed that in the mesenteric lymph nodes of these mice, cells derived from the transnuclear clone developed into regulatory T cells, but in the intestines, these cells gave rise to TCRαβ⁺CD4⁺CD8αα⁺ IELs independently of Foxp3 expression.⁷¹ These and other reports indicate that DP IELs may share epitope recognition with conventional regulatory T cells.⁷² However, contrary to regulatory T cells that require TCR recognition for their maintenance, once DP cells are present in the IEL compartment, their persistence in the epithelium is not affected by the absence of their cognate antigen.⁷³

The transition of CD4⁺ T cells migrating from the mesenteric lymph nodes into the lamina propria or the IEL compartment proceeds in a stepwise manner. For example, regulatory T cells moving into the epithelium first shut down their regulatory T cell programming and then acquire an epithelial-specific transcription profile, characterized by the expression of CD8αα, granzyme B, and other molecules.⁷⁴ Although not empirically proven, due to their anatomical proximity, it has been assumed that the lamina propria feeds the IEL compartment with CD4⁺ T cells. However, Mucida’s group recently showed that the lamina propria and IEL CD4⁺ T cell precursors diverge from cells derived from the mesenteric lymph nodes, and that the IEL CD4⁺ T cell precursors acquired a pre-IEL phenotype prior to populating the epithelium.⁷⁴

B. Role of Transcription Factors in the Development of TCRαβ⁺CD4⁺CD8αα⁺ IELs

The development of DP IELs from conventional TCRαβ⁺CD4⁺ T cells is controlled by the upregulation and downregulation of different transcription factors. In the intestinal epithelium, CD4⁺ IEL trigger a CD8⁺ T lineage programing through upregulation of the transcription factors Runx3 and T-bet, and suppression of ThPOK; T-bet being essential for inducing Runx3 expression.^75–77 Using adoptive transfer of naïve ThPOK^GFP+CD4⁺ T cells into immune-deficient Rag1^−/− mice, Mucida et al. demonstrated that CD4⁺ helper T cells lose Zbtb7b expression as they become effector cells in the intestine, while gaining the expression of CD8αα.⁷⁷ Using elegant experiments with knock-in reporter mice, this group also proved that upregulation of Runx3 on TCRβ⁺CD4⁺ T cells was associated with acquisition of CD8αα expression in intestinal tissues.⁷⁷

C. Role of Cytokines in the Development of TCRαβ⁺CD4⁺CD8αα⁺ IELs

The intestinal microenvironment provides external stimuli such as transforming growth factor-β (TGF-β), retinoic acid (RA), IL-27, and interferon (IFN)-γ, which promote the expression of CD8αα.^76–79 Using in vitro and in vivo approaches, the role of TGF-β and/or RA on the modulation of Zbtb7b and Runx3 expression by CD4⁺ T cells was evaluated. In vitro culture of naïve CD4⁺ T cells either with TGF-β alone or TGF-β plus RA induced Runx3 and CD8αα expression while inhibiting the expression of ThPOK.⁷⁶ Mice with conditional TGF-β receptor-deficiency in T cells maintained the expression of ThPOK by CD4⁺ IEL but displayed significant reduction in the frequency of DP IEL.⁷⁶ In addition, in vitro stimulation of naïve CD4⁺ T cells with TGF-β, RA and either IFN-γ or IL-27 upregulated T-bet and Runx3, and downregulated ThPOK leading to rapid acquisition of CD8αα cell surface expression. Either Tbx21 or Runx3-defecient CD4⁺ T cells showed a drastic reduction in differentiation into DP IELs following adoptive transfer into Rag1^−/− mice. These results highlight both the synergistic and complementary effects of T-bet and Runx3 in regulating the differentiation of CD4⁺ T cells into DP IELs.

D. Role of the Microbiota and Other Factors in the Development of TCRαβ⁺CD4⁺CD8αα⁺ IELs

The anatomical location of IELs between the lumen of the intestines and the sterile environment under the basolateral region of the epithelium suggests that these cells may depend on the microbiota for their development and/or maintenance. For example, DP IELs develop poorly in the intestines of germ-free mice.^70,75,80 Specifically, Lactobacillus reuteri is responsible for the development of DP IELs through generation of indole derivatives of dietary tryptophan, such as indole-3-lactic acid, which activates the Ahr in CD4⁺ T cells and leads to the downregulation of Zbtb7b.⁸⁰ In vitro culture of naïve CD4⁺ T cells with supernatants of L. reuteri grown in tryptophan-containing medium and TGF-β induced CD4⁺CD8αα⁺ T cell differentiation similar to the AhR agonist 2,3,7,8-tetrachlorodibenzodioxin. Mice with specific deletion of Ahr in T cells had a drastic reduction in the frequency of DP IELs compared to wild-type littermates.⁸⁰ Other studies have shown that both IFN-γ and the microbiota promoted ileum epithelial cells to express MHC class II and programed death-ligand 1 (PD-L1), which support DP IEL programming.⁸¹ PD-1/PD-L1 signaling induces CD8αα acquisition by CD4⁺ IELs by suppressing Zbtb7b expression.⁸¹

Maintenance of DP IELs in the intestinal epithelium is mediated in part by the phosphoprotein osteopontin. Osteopontin is a pleiotropic protein with various critical roles in many physiological pathways. A recent report from our laboratory has shown that mice with a global deficiency in the gene encoding for osteopontin (Spp1) display severe reduction in the frequencies and cellularity of DP IELs during steady state conditions.⁸² We have also shown in in vitro assays that survival of DP IELs is mediated by osteopontin interacting with cell surface CD44 present in DP IELs, and most likely, osteopontin is produced by innate CD8αα cells.⁸²

Another factor that impacts the development of DP IELs is the vitamin D receptor (VDR). Mice lacking VDR present DP IEL deficiency, and the remaining TCRαβ⁺CD4⁺CD8αα⁺ cells display low CCR9 expression levels, which may affect precursor homing into the intestinal epithelium.⁸³

Finally, the cell adhesion class I-restricted T-cell associated molecule (Crtam) expressed by activated CD4⁺ T cells and DP IELs and binds to Cadm1 expressed in CD103⁺ dendritic cells, appears to be an essential molecule involved in residency and maintenance of DP IELs. For example, Crtam^−/− mice present lower levels of DP IELs, indicating that the Crtam-Cadm1 interaction is important for DP IEL development.⁸⁴

E. TCRαβ⁺CD4⁺CD8αα⁺ IEL Role in the Intestinal Epithelium

DP IELs are a special subpopulation of TCRαβ⁺CD4⁺ IELs that perform dual functions including immunosuppression and cytotoxicity. Together DP IELs and the regulatory Foxp3⁺CD4⁺ T cells in the lamina propria promote immunosuppressive functions and intestinal tolerance to food antigens.⁷⁰ This study further showed that lamina propria T regulatory cells migrate into the epithelial layer where they acquire CD8αα and lose Foxp3 expression but maintain their immunoregulatory functions.⁷⁰

The immunosuppressive function of DP IELs was initially described in adoptive transfer experiments, in which in vitro differentiated Th2 cells were adoptively transferred into Rag2^−/− mice.⁸⁵ Following adoptive transfer and reconstitution of intestinal epithelium, Th2 cells, but surprisingly not Th1 cells, were able to gain CD8αα expression. A subsequent transfer of isolated Th2-derived DP IELs protected Rag2^−/− recipient mice from colitis induced by Th1 cells in IL-10-dependent manner.⁸⁵

As a result of losing ThPOK and gaining Runx3 and CD8αα expression, DP IELs display cytolytic functions similar to cytotoxic CD8⁺ T cells.^75,86 Following adoptive transfer of naïve CD4⁺ T cells, DP IELs express cytolytic molecules associated with NK cells and mature CD8⁺ CTL which include granzyme B, perforin, IFN-γ, CD2 family member CD244 (2B4), and MHC I-restricted T-cell associated molecule (CRTAM).⁷⁵ In addition, upon repeated stimulation with ovalbumin in the presence of IL-15, DP IELs display cytotoxic-like features.⁷⁵

In patients with chronic intestinal inflammation such as celiac and inflammatory bowel diseases, a reduction in the frequency of DP IELs was observed, implying an immunosuppressive function of DP IELs in humans.^87,88 Moreover, celiac disease patients display a high frequency of granzyme B⁺CD4⁺CD8α⁺ IELs in duodenal biopsies. In the absence of IL-10 signaling, mice fed on gluten presented increased frequency and numbers of DP IELs and TCRβ⁺CD4⁺ IELs. These results highlighted the critical role of IL-10 in preventing CD4⁺ IEL accumulation and protecting against gluten-induced epithelial damage.⁸⁹

F. Summary

DP IELs are a unique population of TCR⁺ IELs that originate from conventional CD4⁺ T cells migrating to the intestinal epithelium after activation. DP IELs express CD8αα on their surface by a mechanism mediated by down and up regulation of different transcription factors (Zbtb7b, and Runx3 and Tbx21, respectively). In addition, the development of DP IELs is tightly controlled by the gut microenvironment, which provides cytokines and signals derived from commensals. DP IELs play dual functions in intestinal mucosal immunity, which include immunoregulatory functions as well as cytotoxic activities.

IV. TCRαβ⁺CD8αα⁺ IELS

TCRαβ⁺CD8αα⁺ IELs (CD8αα IELs) are a subset of unconventional******l IELs comprising approximately one-third of the TCRαβ⁺ IEL population in mice.⁹⁰ These cells are characterized by the expression of CD8αα homodimers and a significant lack of a traditional CD8αβ or CD4 co-receptor. However, these cells express high levels of NK cell receptors, including those of the Ly49 family,⁹¹ and their signaling components, including molecules with activating domains, such as DAP12.⁹²

The TCR repertoire of these cells is oligoclonal,⁶⁹ and can be either MHC class I or II restricted, frequently to unconventional class I molecules.⁹³ b2 microglobulin-deficient (B2m^−/−) mice, which lack all classical and nonclassical MHC class I molecules, have a notable reduction in CD8αα IEL,^94,95 while mice singly deficient in various MHC class I molecules (TL, H2-K^b, H2-D^b, CD1d, Qa-2) display little to no reduction in this cell type.^14,95–97

A. Development

An important question in the field (and the object of much controversy^98,99 has been the site of development of CD8αα IELs. Original evidence suggested that CD8αα IELs may arise in a thymic-independent mechanism, as athymic mice contained intestinal CD8αα IELs,⁹⁸ and the CD8αα pool in wild-type mice contained numerous self-reactive and “forbidden” TCR clones, indicating that these cells did not undergo typical thymic negative selection.¹⁰⁰ The proposed site for the development of CD8αα IELs were small clusters of cells present at the base of the crypts, known as cryptopatches. Cells present in cryptopatches express the Lin⁻c-kit⁺IL-7Rα⁺ phenotype associated with common lymphoid progenitors.^101,102 However, later work determined that while some vestigial mechanisms lead to CD8αα IEL production in athymic mice, such as in the cryptopatches, normally these cells develop from the pool of double-negative thymocytes, and undergo agonist selection in the thymus, similarly to the positive selection process undergone by conventional TCRαβ⁺CD8αβ⁺ T cells.¹⁰³

The thymic double-negative precursors of CD8αα IELs have been characterized by several different classifications.^104,105 Perhaps the most adopted description is within the thymic TCRb⁺CD5⁺CD122+H-2K+CD4⁻CD8⁻ population between “Type A” IEL precursors (IELp), which are PD-1⁺T-bet⁻, and “Type B” IELp, which are PD-1⁻T-bet⁺. Differences have been described between these types in localization (Type A IELp in the thymic cortex, Type B in the thymic medulla), MHC restriction (Type A to classical and nonclassical MHC class I molecules, Type B IELp to both MHC class II and nonclassical MHC class I molecules), and antigen specificity (Type A to self-antigens, Type B to non-self-antigens). There is no clear product-precursor relationship between the subsets, and both can give rise to CD8αα IELs upon adoptive transfer.¹⁰⁶

After undergoing agonist selection, progenitor cells exit the thymus in an S1P-dependent manner.¹⁰⁷ Expression of the CD8αα homodimer is gained in the periphery, a process under the control of enhancers at the CD8 locus (E8_I).¹⁰⁸ TGF-β is an important signal inducing the expression of CD8αα, and mice lacking TGF-β or its downstream signaling molecule Smad3 have defective generation of CD8αα IELs.⁷⁸ IL-15 is an additional signal promoting CD8αα IEL homeostasis in the intestines. While IL-15 is not required for the survival or maturation of IELp,¹⁰⁹ it is required for the maintenance and/or differentiation of CD8αα IELs in the intestinal niche.¹¹⁰ One of the roles of IL-15 in this cell type is to promote the upregulation of T-bet,¹⁰⁹ an important transcription factor for CD8αα IEL cell fate. The major source of IL-15 in the gut is IECs.¹¹¹

B. TCRαβ⁺CD8αα⁺ IEL Role in the Intestinal Epithelium

The role of CD8αα IELs is incompletely understood. They appear to play a role in regulation of immune responses, but unlike Treg cells, CD8αα IELs have not been shown to conclusively regulate immune responses in many disease states and these cells have low expression of the Treg-characterizing transcription factor Foxp3.⁹² CD8αα IELs also show some similarity to NKT cells in that they express a wide variety of activating and inhibitory NK receptors.⁹² Compared with TCRαβ⁺CD8αβ⁺ IELs, CD8αα IELs have enhanced expression of genes involved in immune regulation, including Tgfb3 and Lag3.⁹²

Signals regulating CD8αα IELs in the intestinal niche are complex. The vitamin D receptor and Ahr have both been implicated in CD8αα IEL homeostasis, as the CD8αα IEL compartment is significantly reduced in mice lacking Ahr,²⁵ or the vitamin D receptor. In particular, the vitamin D receptor appears to play a role in the proper homing of CD8αα IEL precursors to the gut.⁸³ The microbiota and food antigens also play a role in CD8αα IELs homeostasis, in contrast to what is seen regarding TCRγδ⁺ IELs, as a significant decrease in CD8αα IELs is seen in food-antigen free and germ-free mice.¹¹² Furthermore, mice deficient in Nod2 have a substantial reduction in CD8αα IELs, but normal numbers of thymic IELp, indicating that the importance of microbial sensing is specific to the intestinal niche.¹¹³ Additional evidence comes from studies showing that the diversity of the CD8αα IEL compartment changes upon exposure to microbial antigens in the gut, in a mechanism that may potentially involve generation of CD8αα IELs from CD4⁺ IELs.⁷² Even though many CD8αα IELs have self-reactive TCRs, these cells do not appear to break tolerance in the intestines and cause inflammation.¹¹⁴ This is likely due to the function of the CD8αα homodimer as a co-repressor, preventing activation of the self-reactive TCRs upon antigen stimulation.¹¹ Providing evidence for a potential regulatory function, CD8αα IELs prevented disease development in T-cell-deficient hosts in the mouse adoptive transfer model of colitis when adoptively transferred with CD4⁺CD45RB^hi T cells.¹¹⁵

C. Humans vs. Mice

While the CD8αα IEL subset constitutes a significant fraction of mouse IELs, it is still debated whether this subset is present in humans. Some have suggested that the TCRαβ⁺CD4⁺CD8αα⁺ IEL subset found in humans is analogous in function to the CD8αα IELs found in mice. Alternatively, in human development, a population of TCRαβ⁺CD8αβ⁺ cells are PD-1⁺CD8αα⁺, indicating that these cells have recently undergone agonist selection, similar to CD8αα IELp in mice. These cells display an innate-like gene expression profile and functional phenotype and may also represent the precursors to a CD8αα IEL-like population in humans.¹¹⁶ Due to their co-expression of CD8αα and CD8αβ, these cells may have been overlooked in previous studies of the human IEL compartment. Further analysis of these human IEL subsets is needed to determine their phenotypic and functional similarity to the mouse CD8αα IEL subset.

A significant difference between human and mouse CD8αα-expressing IELs is that while the interaction between CD8αα and TL is critical for the function of CD8αα IELs in mice, this interaction has not yet been described in any human IEL population. It is interesting to speculate on whether a similar interaction exists between human IELs and a homolog of TL, or whether human CD8αα may interact with entirely different binding partners. A difference in CD8αα binding specificity may help explain the poorly understood functional significance of CD8αα-expressing IELs in humans.

Correlations of CD8αα IEL prevalence or function with human health and disease have been limited given the lack of consensus on their existence. While it is well known that humans with intestinal diseases, including IBD, display abnormal IEL compartments, the exact role of CD8αα IELs in pathogenesis or prevention of disease is unclear. The high level of redundancy in the human IEL compartment, as well as the phenotypic plasticity of certain subsets, for example, the upregulation of activating NK receptors on CD8αβ⁺ cells during certain disease states, such as celiac disease,¹¹⁷ may render the presence of a CD8αα IEL population in humans unnecessary.

D. Summary

As a significant proportion of the mouse IEL compartment, CD8αα IELs have been extensively studied, and much has been learned in recent years about their development, regulation, and function. However, the enigmatic features of this subset, as well as their debated presence in humans, means much is left to be discovered regarding these cells. While the exact role of CD8αα IELs in specific immune responses remains to be seen, it is clear that CD8αα IELs play an important role in innate defense and protection within the intestinal niche.

V. CONCLUDING REMARKS

The term “unconventional” refers to the historical classification of lymphoid cells, in which those cells that were first described in the spleen or lymph nodes represent “conventional” T cells. However, as we have discussed in this review, TCR⁺ IELs are specialized lymphoid cells with “conventional” roles tailored for the environment of the intestines. Some of these functions are shared with “conventional” T cells, whereas others are unique functions related to intestinal homeostasis. Moreover, we have discussed that some IELs are directly derived from “conventional” CD4⁺ T cells, such as DP IELs, indicating that these cells represent a subset of peripheral CD4⁺ T cells that migrated to the intestinal epithelium. Other cells, like TCRγδ⁺ IELs share many features with similar cells present in other immune sites, such as the skin or spleen. Whereas murine TCRαβ⁺CD8αα⁺ IELs represent a peculiar lymphoid population without an obvious counterpart in humans, and with still unclarified roles in the intestinal mucosa (Fig. 1). Thus, we hope that this review promotes a better understanding of these IEL populations and highlights that these and other IELs are conventional immune cells with specific roles for the environment of the intestinal epithelium.