Cutting Edge: γδ Intraepithelial Lymphocytes of the Small Intestine Are Not Biased toward Thymic Antigens¹

Intraepithelial lymphocytes (IELs)⁴ associated with the small intestine are represented by both αβ and γδ T cell lineages with comparable frequencies. These lymphocytes are essential for the host’s defense against pathogens that cross the gut’s mucosal epithelium (1, 2). Although IELs can develop from local precursor cells in surgically or genetically manipulated mouse models, most if not all IELs are of thymic origin in normal animals (reviewed in Ref. 3). In the case of αβ IELs, several reports indicate that these cells develop from thymocyte precursors that are selected on self-agonist peptide/MHCs, and it has been suggested that γδ IEL development follows the same rules (3). This is largely based on observations that γδ and αβ IELs have similar functions and gene expression profiles (4, 5, 6, 7); both populations require the expression of the transcriptional factor TCF-1 to develop (8), and neither population seems to require S1P₁ expression for egress from the thymus and migration to the gut (9).

Direct evidence for γδ TCR-ligand recognition in this aspect has been limited to the study of the development of G8 γδ TCR-transgenic IELs. The G8 γδ TCR is specific for the closely related nonclassical MHC class I β2-microglobulin (β₂m)-associated molecules T10 and T22 (10). It has been reported that adoptively transferred neonatal G8 thymocytes are later found in the IELs of C57BL/6 (B6) mice (11). Because B6 mice express both T10 and T22, this was taken to suggest that γδ IELs, similarly as αβ IELs, also require ligand recognition in the thymus to develop. However, in another study, T cells from the same line of G8 γδ TCR-transgenic mice are abundantly found in the IEL compartment of B₂m^−/− mice, which do not express T10 or T22 on the cell surface (12). Thus, it seems that γδ IELs can develop without encountering ligand.

In the past, we found that T10 and T22 are natural ligands for a sizable population (0.1–1%) of γδ T cells in normal mice (13, 14). We have also developed a T22 tetrameric staining reagent that allowed us to follow and analyze this population of Ag-specific γδ T cells. When we analyzed the development of T10/T22-specific γδ T cells in B6, BALB/c, and B₂m^−/− mice, we found that encountering T10/T22 in the thymus is neither required for nor inhibitory to generate a functional T10/T22-specific γδ T cell repertoire in the periphery (15). Importantly, we have evidence that a considerable population of γδ T cells in the spleen and lymph nodes may not have encountered Ags during development or in the periphery. This represents an important departure from what has been thought previously about γδ T cell development based on the analysis of TCR transgenic mice. Given the anatomical location of IELs below the mucosal epithelium of the small intestine, which separates the outside environment from host’s interior, it is important to know whether or not these cells focus on host-derived “self-Ags” they encounter in the thymus or whether they have the potential to recognize foreign and/or host Ags that are only exposed after infection or injury.

Seven- to 10-wk-old C57BL/6 and B₂m^−/− (C57BL/6) mice were purchased from either The Jackson Laboratory or Taconic and housed under specific pathogen-free conditions at the Stanford University Animal Facility (Stanford, CA) according to its guidelines.

Abs were purchased from either eBioscience or BD Pharmingen unless otherwise stated. γδ Thymocytes were isolated by positive enrichment with allophycocyanin-labeled GL3 (anti-TCRδ) Ab and anti-allophycocyanin microbeads (Miltenyi Biotec) as described previously (15). IELs were obtained using Percoll gradients as described (14) and analyzed without further enrichment. The T22 monomer was folded, biotinylated, and tetramerized as described (13). Enriched cells, having been preincubated with normal hamster and mouse serum, Fc Block (anti-CD16/32), and 4 μg/ml CD8αCT (Caltag Laboratories) to prevent CD8-tetramer interactions, were stained with either 5–7 μg/ml PE-labeled T22 tetramer or anti-CD122 (TM-β1), together with anti-CD19 Cy5-PE (MB 19-1 or 6D5), anti-TCRβ Cy5-PE (H57), anti-keyhole limpet hemocyanin Armenian hamster IgG2κ Cy-5PE (GL3 isotype), streptavidin-Cy5-PE (BD Pharmingen), or Cy5-PE-labeled HLA-A2 tetramers and FITC labeled anti-CCR9 (CDw199). Cells were washed and resuspended in propidium iodide (PI). FACS was performed using an LSR flow cytometer (BD Biosciences) and data were collected with CellQuest software (BD Biosciences). Cells positive for PI and Cy5-PE were excluded from analysis. FACS data was compensated and analyzed using FlowJo software (Tree Star).

Individual tetramer-positive γδ IELs from B₂m^−/− mice (A5 and D8) were isolated by FACS and rearranged TCRγ and TCRδ loci were amplified from cDNA and sequenced as described (14). Full-length TCR chains were subcloned and retrovirally introduced into the Jurkat cell line J.RT3-T3.5 (a gift from A. Weiss, University of California, San Francisco, CA) as described (14).

Tetramer decay analysis was performed as described (14). Briefly, 25 nM T22 tetramer and GL3 FITC were used to stain γδ TCR Jurkat transfectants for 45 min at room temperature. Cells were washed and resuspended in FACS buffer (2% FCS in PBS) with 1 μg/ml PI and 200 μg/ml T22 monomer. The monomer concentration at which the tetramer decay was concentration independent was determined before the actual tetramer decay assays. At various time points, cells were resuspended and analyzed by FACS. PI-positive cells and those with low side scatter were excluded from analysis. The tetramer decay data were normalized to the level of γδ TCR expression, as determined by staining with GL3.

To determine whether interaction with thymic ligand is a prerequisite for the development of γδ IELs, we first used a fluorescently labeled T22 tetramer to analyze the frequency of T10/T22-specific γδ IELs in C57BL/6 and B₂m^−/− mice. B6 mice express both T10 and T22, and B₂m^−/− mice express neither molecule on the cell surface. As shown in Fig. 1, we observed similar numbers of T10/T22-specific γδ IELs cells in B₂m^−/− and B6 mice. This result is consistent with earlier observations that although the number of αβ IELs in B₂m^−/− mice is drastically reduced, that of γδ IELs are generally B₂m independent (16). Single-cell TCR analysis showed that regardless of the murine genetic background, T10/T22-specific IELs have TCRs with the same T10/T22 recognition motif (W-SEGYEL) in the TCRδ CDR3 region as those previously reported for T10/T22-specific γδ T cells (14) and express mostly Vγ7, as γδ IELs do in general (data not shown).

T10/T22-specific γδ IELs from B₂m^−/− mice also showed a spectrum of affinity to T22 as evaluated by tetramer-staining intensity (Fig. 1). To test this more rigorously, we expressed TCR heterodimers of two different T10/T22-specific γδ IEL clones derived from B₂m^−/− mice, A5 and D8, in TCRβ^−/− Jurkat cells to reconstitute these TCR specificities. Using a tetramer decay assay (14), we compared the T22 tetramer-binding half-lives, t_1/2, of A5 and D8 (Fig. 2,A) to a panel of T10/T22-specific, γδ TCR-expressing Jurkat cells. We found that the A5 TCR exhibits the longest t_1/2 of T22 tetramer binding. In contrast, the D8 TCR has one of the shortest t_1/2 (Fig. 2, B and C). This result, together with what we observed previously (15), indicates that T10/T22-specific γδ T cells are present in all lymphoid organs and in the small intestine in B₂m^−/− mice and exhibit a broad spectrum of affinity to T10/T22.

The chemokine CCL25 is selectively expressed by epithelial cells of the small intestine (17, 18). Its receptor, CCR9, has been identified on αβ T cells that that can home to the gut (19). In CCR9^−/− mice, γδ T cells are significantly reduced in the IEL compartment (20, 21), suggesting that the expression of CCR9 is also important for the gut homing of γδ T cells. Consistent with the idea that encountering a thymic ligand is not required for the development of γδ IELs, tetramer-positive thymocytes in B₂m^−/− mice are mostly CCR9^high (Fig. 3,A). Furthermore, up-regulation of the IL-2/IL-15 receptor common β-chain (CD122) has been used as an indicator of self-ligand recognition for αβ thymocytes (22) and γδ thymocytes (15, 23). Interestingly, we found that γδ thymocytes, which express high levels of CCR9, are CD122^low and, conversely, those that express high levels of CD122 are CCR9^low (Fig. 3,B). Not surprisingly, γδ IELs are also CD122^low (Fig. 3 C). These results suggest that γδ thymocytes that have not encountered a thymic ligand may have a greater potential to home to the gut.

Consistent with this supposition, we find that T10/T22-specific γδ thymocytes in B6 mice are CCR9^low (Fig. 3,A). In particular, cells with the highest tetramer staining intensity correlated with the lowest degree of CCR9 expression when compared with their intermediate tetramer-staining or tetramer-negative γδ thymocyte counterparts. In addition, when we enumerated T22 tetramer-positive cells and compared the mean fluorescence intensities of this population within the IEL compartments of B6 and B₂m^−/− mice, we found that the T22 tetramer-positive population had significantly higher tetramer staining intensities in B₂m^−/− mice as compared with B6 mice (Fig. 1 B). These results indicate that although self-reactivity is present within the IEL compartment, there is bias against a high-affinity, self-reactive repertoire presumably and at least in part by the regulation of CCR9 surface expression during thymic development.

In this context, the hallmark of the adaptive immune system is that the diversity created by somatic gene recombination not only broadens the Ag-specific repertoire but also creates receptors with different Ag-binding affinities, such that cells with the best fit for Ag recognition have the potential of being selected for optimum immune functions. γδ TCRs have the highest possible numbers of different sequences at the CDR3 junctions (24). Although the T10/T22 recognition determinant is located on the TCRδ CDR3 region generated by VDJ recombination, much of the diversity at the CDR3 junctions confers T10/T22-specific TCRs with different affinities to the Ag (14). Our observation in this study provides an example indicating that γδ T cell intestinal homing is selected by γδ TCR affinity to its ligand.

The majority of αβ and γδ IELs express CD8αα, have similar transcriptional profiles (5, 6, 7) and also have similar functions as those of cytotoxic cells (4). Although αβ IELs are selected on self-agonist peptide/MHC interactions in the thymus (reviewed in Ref. 25), our present results indicate that γδ IELs focus on Ags that they have not encountered in the thymus. Interestingly, in several experimental systems it has been noted that the number of αβ IELs fluctuates wildly in response to microbial colonization, but under the same experimental conditions γδ T cells in the intestinal epithelium seem to vary little (26, 27, 28, 29). This suggests that thymic output rather than peripheral cues, including ligand expression, may play a greater role in establishing the γδ IEL repertoire. This aspect is reminiscent of how the splenic γδ T cell repertoire is shaped. It is largely made of recent thymic immigrants, rapidly turns over (30), and is not biased toward self-reactivity or “Ag-experienced” cells (15). These characteristics seem to ensure that Ag naive T cells are maintained in the periphery, which would allow the γδ T cell repertoire to respond to Ags that the host encounters for the first time.

γδ T cells recognize Ags directly without the requirement for Ag being processed and presented by specialized APCs (10). Furthermore, γδ T cells can be activated by TCR triggering alone, without prior Ag-specific priming (15). γδ IELs might be ideally suited to mount responses triggered by the direct recognition of pathogens or host Ags that are not expressed under normal physiological conditions but are induced or exposed by stress, infection, or injury. Thus, the “Ag naive” γδ IELs are uniquely suited to act as a “first line of defense,” complementing the function of the self-peptide/MHC-specific repertoire of αβ IELs.

We thank E. Adams and K.C. Garcia for supplying the T22 monomer for the tetramer decay assays and M. M. Davis for critically reading the manuscript.

The authors have no financial conflict of interest.