Cutting Edge Cutting Edge: ␥␦ Intraepithelial Lymphocytes of the Small Intestine Are Not Biased toward Thymic Antigens1 Kirk D. C. Jensen,2 Sunny Shin,2 and Yueh-hsiu Chien3 ␥␦ ⌻ cells, together with ␣ ⌻ cells, are abundantly present in the epithelial layer of the small intestine (IEL) and are essential for the host’s first line of defense. Whether or not ␥␦ IELs, like ␣ IELs, are derived from thymocytes that encounter self-Ags in the thymus is unclear. In this study, we report that a natural population of ␥␦ T cells that are specific for the nonclassical MHC class I molecules T10 and T22 are present in the IEL compartment of mice that do not express T10/T22. Furthermore, the small intestinal homing receptor CCR9 is preferentially expressed on ␥␦ thymocytes that have yet to encounter a ligand, and ␥␦ thymocytes with high affinity for self-ligand are CCR9low. These observations suggest that the Ag-specific repertoire of ␥␦ IELs is not biased toward thymic Ags. Instead, ␥␦ IELs appear suited to respond to novel Ags revealed in pathological settings. The Journal of Immunology, 2009, 182: 7348 –7351. ntraepithelial lymphocytes (IELs)4 associated with the small intestine are represented by both ␣ and ␥␦ 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 ␣ 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 ␥␦ IEL development follows the same rules (3). This is largely based on observations that ␥␦ and ␣ IELs have similar functions and gene expression profiles (4 –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 ␥␦ TCR-ligand recognition in this aspect has been limited to the study of the development of G8 ␥␦
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Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305 Received for publication February 25, 2009. Accepted for publication April 15, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
TCR-transgenic IELs. The G8 ␥␦ TCR is specific for the closely related nonclassical MHC class I 2-microglobulin (2m)-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 ␥␦ IELs, similarly as ␣ IELs, also require ligand recognition in the thymus to develop. However, in another study, T cells from the same line of G8 ␥␦ 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 ␥␦ 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 ␥␦ 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 ␥␦ T cells. When we analyzed the development of T10/T22-specific ␥␦ 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/T22specific ␥␦ T cell repertoire in the periphery (15). Importantly, we have evidence that a considerable population of ␥␦ 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 ␥␦ 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.
Materials and Methods Mice 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 2
K.D.C.J. and S.S. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Yueh-hsiu Chien, 255 Beckman, 279 Campus Drive, Stanford School of Medicine, Stanford, CA 94305. E-mail address:
[email protected]
4 Abbreviations used in this paper: IEL, intestine intraepithelial lymphocyte; B2m⫺/⫺, 2microglobulin deficient; PI, propidium iodide.
1
This work was supported by National Institutes of Health Grants AI33431 and AI062794 (to Y.-h.C.). K.J. was supported by a Stanford Graduate Fellowship and a Cell and Molecular Biology Training Grant from the National Institutes of Health and S.S. was supported by a National Science Foundation Predoctoral Fellowship.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900465
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
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pathogen-free conditions at the Stanford University Animal Facility (Stanford, CA) according to its guidelines.
␥␦ T cell isolation and FACS analysis Abs were purchased from either eBioscience or BD Pharmingen unless otherwise stated. ␥␦ Thymocytes were isolated by positive enrichment with allophycocyanin-labeled GL3 (anti-TCR␦) 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 g/ml CD8␣CT (Caltag Laboratories) to prevent CD8-tetramer interactions, were stained with either 5–7 g/ml PE-labeled T22 tetramer or anti-CD122 (TM-1), together with anti-CD19 Cy5-PE (MB 19-1 or 6D5), anti-TCR Cy5-PE (H57), anti-keyhole limpet hemocyanin Armenian hamster IgG2 Cy-5PE (GL3 isotype), streptavidinCy5-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).
Generation of A5 and D8 ␥␦ TCR-expressing Jurkat cells Individual tetramer-positive ␥␦ IELs from B2m⫺/⫺ mice (A5 and D8) were isolated by FACS and rearranged TCR␥ and TCR␦ 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 Tetramer decay analysis was performed as described (14). Briefly, 25 nM T22 tetramer and GL3 FITC were used to stain ␥␦ TCR Jurkat transfectants for 45 min at room temperature. Cells were washed and resuspended in FACS buffer (2% FCS in PBS) with 1 g/ml PI and 200 g/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 ␥␦ TCR expression, as determined by staining with GL3.
Results and Discussion Encountering ligand in the thymus is not required for the development of ␥␦ IELs
To determine whether interaction with thymic ligand is a prerequisite for the development of ␥␦ IELs, we first used a fluorescently labeled T22 tetramer to analyze the frequency of T10/T22-specific ␥␦ 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 ␥␦ IELs cells in B2m⫺/⫺ and B6 mice. This result is consistent with earlier observations that although the number of ␣ IELs in B2m⫺/⫺ mice is drastically reduced, that of ␥␦ 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 TCR␦ CDR3 region as those previously reported for T10/T22-specific ␥␦ T cells (14) and express mostly V␥7, as ␥␦ IELs do in general (data not shown). T10/T22-specific ␥␦ 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 ␥␦ IEL clones derived from B2m⫺/⫺ mice, A5 and D8, in TCR⫺/⫺ Jurkat cells to reconstitute these TCR specificities. Using a tetramer decay assay (14), we compared the T22 tetramer-binding half-lives, t1/2, of A5 and D8 (Fig. 2A) to a panel of T10/T22specific, ␥␦ TCR-expressing Jurkat cells. We found that the A5 TCR exhibits the longest t1/2 of T22 tetramer binding. In con-
FIGURE 1. T10/T22-specific ␥␦ T cells in the IEL compartments of B2m⫺/⫺ and B6 mice. A, Two representative FACS plots of ␥␦ IELs from B2m⫺/⫺ (C57BL/6) (top panels) and C57BL/6 mice (bottom panels) stained with T22 tetramer are shown (n ⬎ 8). The number within the gate indicates the percentage of tetramer-positive cells among total ␥␦ T cells. B, The relative mean fluorescence intensity (MFI) and the total number of T22 tetramer positive ␥␦ IELs from B6 and B2m⫺/⫺ mice are plotted. Each symbol represents the result of one mouse analyzed on the same day as the other mice using the same FACS instrument settings. Horizontal bars represent the average MFI or absolute number of the tetramer-positive cells from each strain of mice. The p values were calculated with Student’s t test.
trast, the D8 TCR has one of the shortest t1/2 (Fig. 2, B and C). This result, together with what we observed previously (15), indicates that T10/T22-specific ␥␦ 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. Ag naive ␥␦ thymocytes express high levels of the gut homing receptor CCR9
The chemokine CCL25 is selectively expressed by epithelial cells of the small intestine (17, 18). Its receptor, CCR9, has been identified on ␣ T cells that that can home to the gut (19). In CCR9⫺/⫺ mice, ␥␦ 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 ␥␦ T cells. Consistent with the idea that encountering a thymic ligand is not required for the development of ␥␦ IELs, tetramer-positive thymocytes in B2m⫺/⫺ mice are mostly CCR9high (Fig. 3A). Furthermore, up-regulation of the IL2/IL-15 receptor common -chain (CD122) has been used
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FIGURE 2. T10/T22-specific ␥␦ IELs from B2m⫺/⫺ mice exhibit a broad range of T22 binding kinetics. A, The CDR3 ␥ and ␦ sequences of two representative T22-specific ␥␦ IELs from B2m⫺/⫺ mice, A5 and D8, as well as the corresponding regions of the G8 ␥␦ TCR. The consensus T10/T22 Ag recognition determinant encoded by the tryptophan (W) and the Glu-Gly-Tyr-GluLeu (EGYEL) amino acids within the TCR␦ CDR3 is underlined. B, T22 tetramer decay kinetics for the A5, D8, and G8 TCR-expressing cell lines. Representative decay plots depict the natural logarithm of the normalized fluorescence vs time after T22 monomer addition for the A5 (Œ), D8 (f), and G8 (〫) TCR-expressing J.RT3-T3.5 cell lines stained with the T22 tetramer. Plots are representative of at least three independent experiments. C, The t1/2 values for ␥␦ TCR:T22 tetramer binding. This summary of t1/2 values was obtained from tetramer decay analysis of T10/T22-specific, ␥␦ TCR-expressing cell lines generated under similar conditions. The mouse origin and average t1/2 (in minutes) are listed. An asterisk (ⴱ) indicates previously reported t1/2 values (14).
as an indicator of self-ligand recognition for ␣ thymocytes (22) and ␥␦ thymocytes (15, 23). Interestingly, we found that ␥␦ thymocytes, which express high levels of CCR9, are CD122low and, conversely, those that express high levels of CD122 are CCR9low (Fig. 3B). Not surprisingly, ␥␦ IELs are also CD122low (Fig. 3C). These results suggest that ␥␦ 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 ␥␦ thymocytes in B6 mice are CCR9low (Fig. 3A). 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 ␥␦ 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. 1B). These results indicate that although self-reactivity is present within the IEL compartment, there is bias against a
CUTTING EDGE: ␥␦ IEL REPERTOIRE DEVELOPMENT
FIGURE 3. High-affinity, T10/T22-specific ␥␦ thymocytes in ligand-expressing mice are predominately CCR9low. A, Representative FACS plots of the CCR9 expression on high tetramer staining intensity (bold line histogram), intermediate tetramer staining intensity (normal line histogram), and T22 tetramer-positive and tetramer-negative (dashed histogram) ␥␦ thymocytes from B6 and B2m⫺/⫺ mice. The gates are drawn diagonally to normalize for relative TCR expression (n ⫽ 3). B, A representative FACS plot of CD122 and CCR9 expression on ␥␦ thymocytes from B6 mice (n ⫽ 3). Numbers specify the percentage of ␥␦ thymocytes within the indicated gate. C, Representative histogram analysis of CD122 (bold line) expression on ␥␦ IELs from B6 mice (n ⫽ 12). Rat IgG2b, isotype Ab staining is also plotted (shaded histogram).
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. ␥␦ TCRs have the highest possible numbers of different sequences at the CDR3 junctions (24). Although the T10/T22 recognition determinant is located on the TCR␦ 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 ␥␦ T cell intestinal homing is selected by ␥␦ TCR affinity to its ligand. ␥␦ IELs are uniquely suited to act as a “first line of defense”
The majority of ␣ and ␥␦ IELs express CD8␣␣, have similar transcriptional profiles (5–7) and also have similar functions as
The Journal of Immunology
those of cytotoxic cells (4). Although ␣ IELs are selected on self-agonist peptide/MHC interactions in the thymus (reviewed in Ref. 25), our present results indicate that ␥␦ 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 ␣ IELs fluctuates wildly in response to microbial colonization, but under the same experimental conditions ␥␦ T cells in the intestinal epithelium seem to vary little (26 –29). This suggests that thymic output rather than peripheral cues, including ligand expression, may play a greater role in establishing the ␥␦ IEL repertoire. This aspect is reminiscent of how the splenic ␥␦ 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 ␥␦ T cell repertoire to respond to Ags that the host encounters for the first time. ␥␦ T cells recognize Ags directly without the requirement for Ag being processed and presented by specialized APCs (10). Furthermore, ␥␦ T cells can be activated by TCR triggering alone, without prior Ag-specific priming (15). ␥␦ 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” ␥␦ IELs are uniquely suited to act as a “first line of defense,” complementing the function of the self-peptide/MHC-specific repertoire of ␣ IELs.
Acknowledgments 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.
Disclosures The authors have no financial conflict of interest.
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