T Cell Receptor-γ Allele-Specific Selection of Vγ1/Vδ4 Cells in the Intestinal Epithelium

Kalliopi Grigoriadou, Laurent Boucontet and Pablo Pereira
J Immunol October 1, 2002, 169 (7) 3736-3743; DOI: https://doi.org/10.4049/jimmunol.169.7.3736

Abstract

Previous genetic analyses have shown that the relative representation of subsets of γδ intestinal intraepithelial lymphocytes (i-IELs) is influenced by genes linked to the TCRγ, TCRδ, and MHC loci. Here, we have analyzed V-gene use in γδ i-IELs from C57BL/6 (B6) and C57BL/10 (B10) mice and from their F1 and F2 progenies with a larger panel of Vγ- and Vδ-specific mAbs and have shown that the influence of TCRγ-linked genes operates at two levels: one influencing the representation of Vγ1 (or Vγ7) i-IELs and other acting specifically on the Vγ1/Vδ4 i-IEL subset, which represents 3% and 15% of the γδ i-IELs in B6 and B10 mice, respectively. Analysis of mice transgenic for a rearranged Vγ1Jγ4Cγ4 chain of B6 origin demonstrated that the TCRγ-linked genes influencing the representation of the Vγ1/Vδ4 i-IEL subset are the structural genes of TCRγ chains. This influence is allele specific and cell autonomous, as evidenced by the different behavior of Vγ1/Vδ4 cells bearing either parental allele in F1 mice. The representation of Vγ1/Vδ4 cells among γδ thymocytes is similar in B6 and B10 mice, demonstrating that the Vδ4 chain can pair well with both alleles of the Vγ1Jγ4Cγ4 chain and strongly suggesting that a cellular selection mechanism is responsible for the observed differences. The Vγ1-Jγ4 junctional amino acid sequences of B6 Vγ1/Vδ4 i-IELs are diverse but display less variation in length than those found in similar cells from B10 mice, indicating that B6 Vγ1/Vδ4 cells are the target of this cellular selection event.

All throughout its life span, an αβ T cell is submitted to selective pressures based on the specificity of its TCR. During its intrathymic differentiation, a combination of positive and negative selection ensures that a mature T cell can recognize peptide Ags presented by self-MHC molecules without overtly reacting with self-Ags (1, 2, 3, 4). Once in the periphery, naive T cells need continuous interaction with self-MHC molecules to survive and compete with peripheral resident cells (5, 6, 7). Encounter with Ag in the appropriate context will expand and differentiate Ag-specific cells, whereas clearance of the Ag is accompanied by a drastic elimination of the specifically activated cells by apoptosis (8, 9).

Several lines of evidence suggest that γδ T lymphocytes also undergo selection, although the mechanisms remain quite elusive. Studies with mice transgenic (Tg)3 for a TCR-γδ specific for the MHC class Ib molecule T10/T22b have provided evidence for both positive and negative selection of γδ cells (10, 11, 12, 13). However, γδ cells recognizing MHC Ags appear to be the exception rather than the rule. Thus, in studies of mice lacking the β2-microglobulin molecule or MHC class II Ags, the presence at normal levels of γδ cells in various lymphoid organs has rather ruled out a strict and direct role of MHC molecules in the development of γδ cells (14, 15). Furthermore, the frequency of γδ cells recognizing the molecule T10/T22b is ∼0.5% (16).

To analyze the impact of TCR selection in γδ cells and as an important step in the search for endogenous ligands for γδ cells, we (17) and others (18, 19) have used available Vγ- and Vδ-specific mAbs to study the representation of different γδ T cell subsets in different organs and in different strains of mice. In the intestine, the presence of a high frequency of cells expressing the Vδ4 chain correlated with the expression of a gene closely linked to the I-E region of the MHC class II locus (18), although additional experiments with mice Tg for the I-E molecule showed that I-E expression was not sufficient to dictate the Vδ4-high phenotype (20, 21). Similarly, an influence of MHC-linked genes in the repertoire of TCRs expressed by γδ intestinal intraepithelial lymphocytes (i-IELs) was also evident in analyses of recombinant inbred strains generated from C57BL/6 (B6) and DBA/2 founders, although cells not expressing the Vδ4 chain appeared to be the targets of the selective event (17). In these experiments, and in similar experiments performed in the same recombinant inbred strains but analyzing the representation of different γδ T cell subsets among splenic γδ cells (19), the influences of genes linked to the TCRγ and TCRδ loci were also evident.

Although these experiments were interpreted as indicative of cellular selection mechanisms operating on γδ cells, they could not precisely define the cell target of the selection. This was mostly due to the limited number of Vγ and Vδ genes commonly used by γδ cells to form their TCRs and to the fact that only one Vδ-specific Ab was used in those experiments. Thus, an increased or decreased representation of a given subset is always compensated by decreased or increased representation of other subsets, respectively. Therefore, differences in a given subset may result from a selective mechanism operating directly on this subset or may reflect a compensatory mechanism to a selective event acting on a different subset. Furthermore, structural mechanisms rather than cellular selection mechanisms may be at the basis of the influence of the structural genes for TCRγ and δ in the representation of γδ T cell subsets.

To circumvent these problems and to analyze further the role of TCRγ polymorphism in the representation of γδ i-IEL subsets, we have used a larger panel of Vδ-specific mAbs that we have recently produced (22) to analyze the Vγ/Vδ usage by γδ i-IELs from B6 and C57BL/10 (B10) mice and their F1 and F2 progenies. B6 and B10 are genetically related strains of mice (differing by <10% of their genome) (23), which share the same MHC and TCRδ loci but differ in their TCRγ haplotype (24, 25).

Materials and Methods

Mice

C57BL/6JIco (B6), BALB/cByJIco, 129/SvPasIco, CBA/JIco, DBA/2Jico, and FVB/Nico were obtained from Iffa Credo (L’Abresle, France). C57BL/10SnJ (B10), C57BL/10J, and B10.D2/nSnJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). C3H/HePas, A/J, DBA/1J, CBA/N, and NOD/Lt were obtained from the Pasteur Institute (Paris, France). AKR/OlaHsd, NZB/OlaHsd, NZW/OlaHsd, SJL/JHanHsd, SWR/OlaHsd, and 129/OlaHsd were obtained from Harlan (Gannat, France). B6 mice Tg for a rearranged Vγ1Jγ4Cγ4 chain have been previously described (26, 27). (B10 × B6)F1 hybrid mice (B10B6F1) and B10B6F2 mice were produced in our animal facilities. All animals were used between 6 and 12 wk of age.

Cell preparation and cultures

Single-cell suspensions were prepared from thymus and inguinal and axillary lymph nodes (LNs) according to standard procedures. CD4−CD8− double negative (DN) thymocytes were prepared by complement-mediated killing as described (28), and i-IELs were prepared as described (29). To expand γδ cells, total LN cells (5 × 106 cells/ml) from individual mice were cultured in 24-well plates previously coated with 10 μg/ml anti-Cδ mAb (3A10) in 2 ml of either DMEM or RPMI 1640 with Glutamax-1 (Life Technologies, Gaithersburg, MD) supplemented with sodium pyruvate, 5 × 10−5 M 2-ME, nonessential amino acids, and antibiotics (all from Life Technologies), 10% FCS (Boeringer Mannheim, Meylan, Germany) and 100 U/ml of mouse rIL-2. After 3 days cells were further expanded for 2–4 days in flasks in the presence of rIL-2.

Abs, staining, and cell sorting

Anti-CD4 (RL.174), anti-CD8 (HO 2.2), anti-Cδ (3A10), anti-Vγ1 (2.11), anti-Vδ6.3/4 (clone 9D3) (27), anti-Vγ7 (F2.67), anti-Vδ6B (F4.22; Vδ6B refers to the Vd6 genes that have >90% identity at the nucleotide level with the previously described pλ12) (30), and anti-Vδ5 (F45.145) were prepared and used as described (22). These Vδ Abs were described by Pereira et al. (22). The 7C10 mAb was obtained in the same fusion as 9D3 (27) and its specificity was partially described by Azuara and Pereira (31). PE- and biotin-labeled anti-Vδ4 (GL2) and PE-labeled anti-Vδ6.2/6.3 (8F4H7B7) were purchased from BD PharMingen (San Diego, CA).

Cells (105–106) were incubated in staining buffer (PBS, 3% FCS, 0.1% NaN3) with the indicated labeled mAbs for 30 min on ice and were washed twice. When biotin-conjugated Abs were used, the cells were further incubated with PE-labeled streptavidin (Southern Biotechnology, Birmingham, AL) or allophycocyanin-labeled streptavidin (BD PharMingen) for another 15 min on ice. After two washes, cells were analyzed using a FACScan or a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). Dead cells were gated out by their forward and side scatter profiles and by their staining with propidium iodide. Data were analyzed using the CellQuest program.

For FACS sorting, i-IELs and DN thymocytes were prepared as above, incubated with the appropriate mAb as described above, and sorted in a FACStarPlus (BD Biosciences). Purity of the sorted populations was >98%.

PCR, cloning, and sequencing

Total cellular RNA from FACS-sorted cells was extracted with RNA-B (Bioprobe Systems, Montreuil, France). cDNA was synthesized as described by Azuara et al. (28). The sequences of the Vγ1-, Cγ-, Vδ4-, Cδ-, and FAM-labeled Jδ1 primers are described by Azuara et al. (28). The sequence of the FAM-labeled Jγ4 primer used was CAAATATCTTGACCCTGA and that of the Cγ4 was CTTTCCAATACACCCTTA. PCR and run-off reactions were performed as in Refs. 28 and 32 . The TOPO TA cloning kit (Invitrogen, San Diego, CA) was used for cloning and the ABI PRISM dye terminator cycle sequencing ready reaction kit (PerkinElmer, Wellesley, MA) was used for sequencing according to the manufacturer’s instructions. The Cγ primer was used to sequence the Vγ1-Jγ4 junctions.

Genetic and statistical analyses

Male and female B10B6F2 mice (n = 83) were separated into three groups according to their TCRγ haplotype as defined by the anti-Vγ1 and 7C10 mAbs. Mice having all of their Vγ1+ cells stained by the 7C10 mAb were defined as having both TCRγ loci of B10 haplotype. Mice in which a fraction of the Vγ1+ cells were stained by the 7C10 mAb were considered to have inherited one TCRγ allele from the parental B10 strain and the other from the B6 strain. Mice whose Vγ1+ cells did not stain with the 7C10 mAb were defined as having inherited both TCRγ alleles from the parental B6 strain. The TCRγ haplotype thus defined was confirmed by the analyses of the same F2 mice with the D13 Mit3 marker. This marker is polymorphic between the B6 and the B10 strains (23) and maps at the same genetic distance from the centromere as the TCRγ locus. Consistent with this predicted genetic localization, only one recombinant between the D13 Mit3 marker and the TCRγ haplotype defined by the 7C10 mAb was found among the 83 F2 animals tested. These data provided genetic evidence indicating that the gene encoding the protein recognized by the 7C10 mAb is located in the vicinity of the TCRγ locus. Polymorphism at the D13 Mit3 marker was analyzed by PCR as described (31). For each of these three groups the mean values ± SD of the percentages of each γδ i-IEL subset were calculated and compared by ANOVA followed by unpaired Student’s t tests using the StatView program.

Results

Representation of different γδ i-IEL subsets in B6, B10, B10B6F1, and B10B6F2 mice

To estimate the relative proportion of different Vγ/Vδ i-IEL subsets in B6 and B10 mice, we performed three-color immunofluorescence analysis with mAbs recognizing the Cδ chain together with available mAbs specific for different Vγ (Vγ1 and Vγ7) and Vδ (Vδ4, Vδ5, Vδ6.3, and Vδ6B) chains (22). The Vγ1 and Vγ7 mAbs recognize >85%, whereas the four Vδ-specific Abs recognize ∼70% of the γδ i-IELs in B6 and B10 mice. A representative staining experiment of B6 and B10 γδ i-IELs is shown in Fig. 1⇓, whereas the relative proportions of each Vγ/Vδ subset defined by these mAbs in B6 (n = 32), B10 (n = 24), and B10B6F1 (n = 18) mice are compared in Fig. 2⇓, A–C. Similarly to DBA/2 γδ i-IELs (17), B10 γδ i-IELs contain a relatively high proportion (>15%) of Vγ1/Vδ4 cells, whereas the same cell subset represents only ∼2% of the γδ i-IELs in B6 mice (Fig. 2⇓B, top left panel). This different representation of the Vγ1/Vδ4 subset between the B6 and B10 mouse strains is likely to result from two mechanistically different events. One is a Vδ-independent event influencing the relative representation of cells bearing different Vγ chains in a strain-specific manner. The existence of such an event is supported by the fact that the representations of the Vγ1/Vδ5 and of the Vγ1/Vδ6 subsets are also higher in B10 than in B6 mice (Fig. 2⇓B, top panels). Accordingly, B10 mice contain a higher proportion of Vγ1+ i-IELs than do B6 mice (Fig. 2⇓A). The second event operates specifically on Vγ1/Vδ4 cells and is readily evident when the representation of the different Vγ1/Vδ subsets in the parental strains are calculated as a fraction of the total Vγ1+ i-IELs present in each strain (thus minimizing the differences in the representation of the Vγ1/Vδ4 subset due to the Vδ-independent mechanism previously defined). Thus, whereas the representation of the Vγ1/Vδ5 and the Vγ1/Vδ6 subsets among Vγ1+ i-IELs in B6 and B10 mice are comparable, that of the Vγ1/Vδ4 subset is threefold higher in B10 than in B6 mice (Fig. 2⇓C, top panels).

FIGURE 1.
FIGURE 1.

FACS profiles of γδ i-IELs stained with different Vγ- and Vδ-specific Abs. i-IELs from B6 (top panels) and B10 mice (bottom panels) were stained with the indicated FITC-labeled anti-Vγ Abs, PE-labeled anti-Cδ Ab, and biotin-labeled anti-Vδ Abs followed by allophycocyanin-labeled streptavidin and were analyzed in a FACSCalibur flow cytometer. Dot plots represent the indicated Vγ- and Vδ-specific staining among electronically gated γδ+ cells. Numbers indicate percent of positive cells in each quadrant.

FIGURE 2.
FIGURE 2.

Representation of different γδ T cell subsets in i-IELs from B6, B10, B10B6F1, and B10B6F2 mice. i-IELs from B6 (n = 32) and B10 (n = 24) mouse strains, B10B6F1 hybrids (n = 18), or B10B6F2 progeny (n = 83) either were incubated with FITC-labeled anti-Vγ and biotin-labeled anti-Cδ or anti-Vδ mAbs followed by streptavidin-PE and analyzed in a FACScan or were labeled as described in Fig. 1⇑ and analyzed in a FACSCalibur. A and D, Fraction of γδ+ i-IELs expressing the Vγ1 or the Vγ7 chains in the indicated strains (A) or in F2 animals separated on the basis of their TCRγ haplotype (D). B and E, Fraction of γδ+ i-IELs expressing the Vγ1 (top) or the Vγ7 chain (bottom) together with the indicated Vδ chains. C and F, Fraction of Vγ1+ (top) or Vγ7+ (bottom) i-IELs expressing the indicated Vδ chains. Results are shown as the mean ± SEM (A–C) or as the mean ± SD of the F2 animals grouped according to whether they have inherited their TCRγ locus from the B6 parental strain (B6), the B10 parental strain (B10), or both (F1) (D–F). Statistical analyses were performed by ANOVA followed by Student’s t tests. ∗∗∗, p < 0.0001; ∗∗, 0.0001 < p < 0.001; ∗, 0.001 < p < 0.01.

For both phenotypic traits (levels of Vγ1 i-IELs and of Vγ1/Vδ4 cells), the frequencies observed in B10B6F1 animals were intermediate between the two parental strains (Fig. 2⇑), indicating that nonrecessive B10 genes are responsible for high Vγ1 and Vγ1/Vδ4 phenotypes. Furthermore, both phenotypic traits are genetically controlled by an element(s) physically linked to the TCRγ locus, as indicated by the correlation between the levels of Vγ1 and Vγ1/Vδ4 cells and the inheritance of the TCRγ haplotype observed in B10B6F2 (n = 83) mice (Fig. 2⇑, D–F). Thus, F2 animals that inherited two B10 alleles, two B6 alleles, or one allele from each parental strain at the TCRγ locus show levels of Vγ1 and Vγ1/Vδ4 i-IELs similar to those found in B10, B6, and B10B6F1 mice, respectively (Fig. 2⇑).

The representation of the Vγ7/Vδ i-IEL subsets in the two parental strains is not exactly the reciprocal of that of the Vγ1/Vδ subsets, suggesting that additional “selective” events operate specifically on Vγ7/Vδ cells (likely Vγ7/Vδ6B and/or Vγ7/Vδ4 subsets) (Fig. 2⇑, B and C, lower panels). The representation of both subsets in B10B6F1 animals is intermediate between B6 and B10 mice, similar to that of the Vγ1/Vδ subsets. In contrast, their representation is not influenced by an element(s) linked to the TCRγ locus, further indicating that their differences result from different selective events (Fig. 2⇑, E and F, lower panels). Such selective events appear to be specific to the B10 substrain used in these experiments (C57BL/10SnJ) because the representation of the Vγ7/Vδ subsets in C57BL/10J mice was comparable to those found in B6 mice (data not shown). The mechanisms responsible for the different representation of Vγ7/Vδ subsets between B10 and B6 were not studied further.

Altogether, these experiments extend previous observations on the genetic influence of an element(s) closely linked to the TCRγ locus in the representation of γδ i-IEL subsets (17). They demonstrate that this influence operates at two different levels: one influencing the representation of Vγ1 (or Vγ7) i-IELs and the other acting specifically on the Vγ1/Vδ4 i-IEL subset. The following experiments were aimed at characterizing the selective mechanisms responsible for the different representation of the Vγ1/Vδ4 i-IEL subset in B6 and B10 mice.

Evidence for Vγ1 allele-dependent selection of the Vγ1/Vδ4 i-IEL subset

Obvious candidate genes that could influence the representation of the Vγ1/Vδ4 i-IEL subset are the structural genes of TCRγ chains. If that were the case, the different levels of Vγ1/Vδ4 i-IELs observed in B6 and B10 mice must relate to polymorphism at the regions coding for their Vγ1Jγ4Cγ4 chains, implying that the B6 and B10 Vγ1Jγ4Cγ4 alleles must behave differently with regard to whatever mechanism is responsible for high or low levels of Vγ1/Vδ4 i-IELs. Alternatively, the representation of the Vγ1Vδ4 i-IEL subset may be influenced by a gene(s) unrelated to the structural genes of TCRγ chains but closely linked to them. The analysis of the Vδ repertoire expressed by Vγ1+ i-IELs isolated from B6 and B10B6F1 mice Tg for a rearranged Vγ1Jγ4Cγ4 chain of B6 origin (Tg-γ) may distinguish between these two alternative possibilities, because the vast majority of the γδ cells in these mice express the transgenic chain (Ref. 31 and data not shown). Thus, if the former hypothesis is correct, the levels of Vδ4+ i-IELs among Tg+ i-IELs would be expected to be similar in B6 and in B10B6F1 Tg-γ mice, and it should compare well with the fraction of Vδ4+ cells among Vγ1+ i-IELs found in normal B6 mice. In contrast, if the latter hypothesis is correct, the levels of Vδ4+ i-IELs among Tg+ cells would differ in B6 and B10B6F1 Tg-γ mice and should compare well with the fraction of Vδ4+ cells among Vγ1+ i-IELs found in normal B6 and B10B6F1 mice, respectively. As shown in Fig. 3⇓, the Vγ1/Vδ4 subset represents ∼9% of the Vγ1+ i-IELs in B6 and B10B6F1 Tg-γ mice as well as in wild-type B6 mice, whereas the same population represents >20% of the Vγ1+ i-IELs in non-Tg B10B6F1 mice. These results strongly suggest that the allelic form of the Vγ1Jγ4Cγ4 chain dictates the representation of the Vγ1Vδ4 subset among γδ i-IELs in these strains.

FIGURE 3.
FIGURE 3.

Levels of Vγ1/Vδ4 i-IELs in wild-type and Vγ1Jγ4Cγ4 Tg-γ animals. i-IELs from the indicated mouse strains were stained with FITC-labeled anti-Vγ1 and biotin-labeled anti-Vδ4 mAb followed by streptavidin-PE and were analyzed in FACScan. Data are shown as the fraction of Vγ1+ cells expressing the Vδ4 chain and represent the mean ± SEM of 4–32 animals analyzed individually. Data from B6, B10, and B10B6F1 mice are the same as in Fig. 2⇑ and are shown for comparison. Levels of Vδ4+ cells among Vγ1+ i-IELs in B10B6F1 Tg mice are significantly different (p < 0.0001) from those found in wild-type B10B6F1 mice but are not significantly different (p > 0.05) from those obtained in wild-type or B6 Tg-γ mice.

The previous statement would predict that the intermediate phenotype observed in F1 mice results from a different and independent behavior of the Vγ1Jγ4Cγ4 chains encoded in the parental TCRγ haplotypes. We have recently produced a mAb (7C10) that appears to recognize the Vγ1Jγ4Cγ4 chain present on the TCRγa haplotype (i.e., B10) but not the Vγ1Jγ4Cγ4 chain present in any other TCRγ haplotype tested, including the B6 TCRγb haplotype (Ref. 31 and Fig. 4⇓). To further characterize the specificity of this mAb, we isolated seven Vγ1-bearing hybridomas (four 7C10+ and three 7C10−) originated from (B6 × DBA/2)F1 thymocytes and sequenced their functional Vγ1Cγ4 chains. All four 7C10+ hybridomas expressed a functionally rearranged Vγ1Cγ4 chain of the parental DBA/2 allele, whereas the three 7C10− hybridomas expressed a Vγ1Cγ4 chain of the parental B6 allele (p < 0.02, according to a Spearman rank correlation coefficient test with the null hypothesis that the two variables, reactivity to the 7C10 mAb and expression of the Vγ1a allele, are independent of each other).

FIGURE 4.
FIGURE 4.

The 7C10 mAb recognizes the Vγ1Jγ4Cγ4 chain present in the TCRγa haplotype. DN thymocytes from B10 (TCRγa; see Refs. 24 , 25 , and 33 for TCRγ haplotypes), B6 (TCRγb), AKR (TCRγc), and 129/Sv (TCRγe) were stained with PE-labeled anti-Cδ, FITC-labeled 7C10, and biotin-labeled anti-Vγ1 (2.11) followed by allophycocyanin-labeled streptavidin and were analyzed in a FACSCalibur. Dot plots represent 7C10 and Vγ1 staining among electronically gated γδ+ thymocytes. Numbers represent percent of cells in each quadrant. Other TCRγa strains tested and positive for 7C10 were C57BL/10J, B10.D2, C3H/He, CBA/J, CBA/N, DBA/1, and DBA/2. Also, NOD/Lt mice were positive for 7C10. Tested 7C10-negative strains were BALB/c, SJL, SWR, NZW, NZB (TCRγb), A/J, and 129/Ola (TCRγe) and FVB (unknown TCRγ haplotype).

With this mAb we separated the Vγ1a- (Vγ1+7C10+) and the Vγ1b-expressing (Vγ1+7C10−) i-IELs from B10B6F1 animals, analyzed independently their TCRδ repertoires, and compared them with those found in Vγ1 i-IELs present in B10 and B6 mice. The results of these analyses are shown in Fig. 5⇓. In B10B6F1 mice, different levels of Vδ4+ cells among Vγ1+7C10+ and Vγ1+7C10− i-IELs were readily evident, whereas the levels of Vδ5+ or Vδ6+ cells among Vγ1+7C10+ and Vγ1+7C10− i-IELs were comparable. Interestingly, similar levels of Vδ4+ cells were found among Vγ1+7C10+ i-IELs in B10B6F1 and B10 mice on one hand and among the Vγ1+7C10− cells in B10B6F1 and B6 mice on the other.

FIGURE 5.
FIGURE 5.

Vδ chain expression among Vγ1+7C10+ and Vγ1+7C10− i-IELs from B10B6F1 mice. i-IELs from B10B6F1 (n = 7) were stained with FITC-labeled 7C10, PE-labeled anti-Vδ, and biotin-labeled anti-Vγ1 mAbs followed by allophycocyanin-labeled streptavidin and were analyzed in a FACSCalibur. Results represent the fraction of Vγ1+ cells bearing the indicated Vδ chains among cells expressing the B10 Vγ1Jγ4Cγ4 chain (Vγ1+7C10+ cells, top panels) or the B6 Vγ1Jγ4Cγ4 chain (Vγ1+7C10− cells, bottom panels). Data from B10 (7C10+) and B6 (7C10−) Vγ1+ i-IELs are the same as in Fig. 2⇑ and are shown for comparison. Data are shown as mean ± SEM.

Altogether, these results demonstrate that polymorphism at the regions coding for the Vγ1Jγ4Cγ4 chain influence the representation of the Vγ1/Vδ4 i-IEL subset and, consequently, strongly suggest that the TCRγ-linked gene influencing this phenotypic trait is the structural gene encoding the Vγ1Jγ4Cγ4 chain.

Sequence polymorphism at the Vγ1Jγ4Cγ4 chains of B6 and B10 mice

Nucleotide sequences of the B10 and B6 Vγ1Jγ4Cγ4 chains obtained in our laboratory were identical with those previously published (34, 35). They differ by two nucleotides, which result in two amino acid substitutions: an Ala to Glu in the V-domain (position 12) and a Lys to Glu in the C-domain (position 157), with the B6 Vγ1Jγ4Cγ4 chain containing Glu at these positions. In a homology-based modeling of the B6 Vγ1Jγ4Cγ4 chain, these two polymorphic residues between the B6 and the B10 Vγ1Jγ4Cγ4 alleles are predicted to be solvent exposed but far away from any complementarity-determining region (CDR). Thus, Glu12 is predicted to lie near the peptide loop that separates the V and the C domains, whereas Glu157 is located within the contact area between the V and C domains (Fig. 6⇓).

FIGURE 6.
FIGURE 6.

Schematic representation of the B6 Vγ1Jγ4Cγ4 chain. A model of the B6 Vγ1Jγ4Cγ4 chain based on homology with the human Vγ9/Vδ2 TCR was obtained from SwissModel (http://www.expasy.ch/swissmod/SWISS-MODEL.html). The side chains of the two residues polymorphic between the B6 and the B10 Vγ1Jγ4Cγ4 chains are shown as atom representations.

The Vδ4 chain can pair equally well with Vγ1a and Vγ1b chains

The different representation of the Vγ1/Vδ4 i-IEL subset in B6 and B10 mice and its dependence on the Vγ1Jγ4Cγ4 allele could be easily explained without the need for invoking a cellular selection mechanism if the Vδ4 chain could not pair correctly with the B6 Vγ1Jγ4Cγ4 chain. Although the fact that ∼2% of γδ i-IELs in B6 mice express the Vγ1/Vδ4 TCR seems incompatible with this hypothesis (see Fig. 2⇑), it could be argued that this low fraction of Vγ1/Vδ4 i-IELs may result from the expansion of even rarer Vγ1+Vδ4+ cells. Therefore, we sought to analyze the levels of Vγ1/Vδ4 cells in a situation in which mature γδ cell expansion is limited, which is usually the case in the organs where the cells develop. Because the origin of the γδ i-IEL is still a controversial matter, we decided to analyze this issue in the thymus. As shown in Fig. 7⇓, B6, B10, or B10B6F1 γδ thymocytes contain similar levels (∼8%) of Vγ1/Vδ4 cells, strongly suggesting that the Vδ4 chain can pair well with both alleles of the Vγ1Jγ4Cγ4 chain.

FIGURE 7.
FIGURE 7.

Representation of the Vγ1/Vδ4 subset in the thymus of B10, B6, and B10B6F1 mice. DN thymocytes from B10 (n = 7), B6 (n = 8), and B10B6F1 (n = 10) mice were stained with FITC-labeled anti-Vγ1, PE-labeled anti-Vδ4, and biotin-labeled anti-Cδ Abs followed by allophycocyanin-labeled streptavidin and were analyzed in a FACScan. Results are shown as the fraction of Vγ1+Vδ4+ cells among electronically gated δ-positive cells. Data are shown as the mean ± SEM.

B6 Vγ1/Vδ4 i-IELs are the target of the selective event

Lack of pairing constraints for the B6 Vγ1 and Vδ4 chains makes it likely that cellular selection events are responsible for the differences in the representation of Vγ1/Vδ4 i-IELs in B6 and B10 mice and for the strong overrepresentation of the B10 allele of the Vγ1Jγ4Cγ4 chain among Vγ1/Vδ4 i-IELs in B10B6F1 animals (>90% of the Vγ1/Vδ4 i-IELs in B10B6F1 animals expressed the Vγ1a allele, whereas the same allele was expressed in ∼65% of the Vγ1+Vδ4− i-IELs; data not shown). However, the experiments presented here are compatible with an increased expansion and/or survival of Vγ1/Vδ4 i-IELs in B10 mice as well as with a specific deletion or decreased survival of Vγ1/Vδ4 i-IELs in B6 mice. Although intermediate levels of αβ or γδ cell populations expressing defined TCR V-gene segments in F1 mice have been currently used as suggestive of positive selection of the cells studied (19, 36, 37), the fact that the Vγ1/Vδ4 cells expressing distinct Vγ1Jγ4Cγ4 alleles are submitted to different selective pressures precludes such a conclusion.

The target cell at which selection operates and, consequently, the nature of the selection may be inferred from the analyses of the junctional diversity of the Vγ1 and Vδ4 chains expressed by the Vγ1/Vδ4 i-IEL subsets present in B6 and B10 mice. These analyses can be performed at the population level by studying the distributions of the length of the CDR3 in TCRγ and TCRδ junctions (38). In polyclonal populations, CDR3 lengths distribute in a Gaussian-like curve with a well-defined number of peaks that is characteristic of each TCR chain, whereas such Gaussian distribution is lost in less diverse populations.

As shown in Fig. 8⇓ (left panels), the profiles of the distribution of CDR3 lengths observed on Vδ4-(Dδ)-Jδ1 junctions expressed by sorted Vγ1+Vδ4+ i-IELs isolated from B6 and B10 mice are quite similar. Both contain ∼12 defined peaks differing in length by three nucleotides, thus corresponding to in-frame rearrangements. These profiles are typical of polyclonal TCRδ junctions. In contrast, CDR3 length distributions of Vγ1-Jγ4 junctions expressed by the same sorted populations in both mouse strains were clearly different (Fig. 8⇓, right panels). In B10 mice, the typical profile of polyclonal Vγ-Jγ junctions was evident, with three to five defined peaks forming a Gaussian-type curve and centered on a CDR3 of 9 aa in length (39). In contrast, the profile obtained from sorted Vγ1+Vδ4+ i-IELs from B6 mice was not Gaussian and showed a prominent peak at a CDR3 length of 10 aa. This profile was unique to the Vγ1+Vδ4+ i-IEL subset, as indicated by the Gaussian-type profile centered on a CDR3 of 9 aa obtained from sorted Vγ1+ i-IELs from B6 mice (which contains >97% Vγ1+Vδ4− cells).

FIGURE 8.
FIGURE 8.

CDR3 length distribution analyses of Vγ1-Jγ4 and Vδ4-(D)-Jδ1 rearrangements expressed by different i-IEL subsets from B6 and B10 mice. Total RNA from sorted Vγ1+Vδ4+ and Vγ1+Vδ4− i-IELs from pools of eight B6 and B10 mice or from total Vγ1+ i-IELs of B6 mice were amplified by RT-PCR with either Vδ4- and Cδ-specific primers or with Vγ1- and Cγ4-specific primers. An aliquot of each amplification product was submitted to a run-off reaction with either a FAM-labeled Jδ1 primer or a FAM-labeled Jγ4 primer, and the final products were resolved in an automated sequencer. Plot shows the profiles of fluorescence intensity vs the length of the fragments of Vδ4-Jδ1 (left panels) or Vγ1-Jγ4 (right panels) from one representative experiment. At least seven independent amplifications were performed with different amounts of cDNAs and/or different primers with similar results. The prominence of the peak at a CDR3 length of 10 aa in the Vγ1-Jγ4 junctions present in Vγ1+Vδ4+ i-IELs from B6 mice was also observed in similar analyses performed on Vγ1+Vδ4+ i-IELs sorted from two individual B6 mice. CDR3 length was defined as in Ref. 39 and represents the number of amino acid residues between the second Cys present in all V segments and the Gly-Lys-Gly (or Ala-Lys-Gly) motif present in all J segments minus four.

From these experiments we conclude that B6 Vγ1/Vδ4 i-IELs, rather than the B10 Vγ1/Vδ4 cells, are the target of the cellular selection event described here. Because B6 mice contain very low levels of Vγ1/Vδ4 i-IELs, these experiments suggest that the selective event results in the reduction of the Vγ1/Vδ4 subset among B6 i-IELs. Such a selective event appears to spare a fraction of Vγ1/Vδ4 cells that are enriched in Vγ-Jγ junctions with relatively long CDR3.

To investigate whether, besides their CDR3 length, other structural features were apparent in the Vγ-Jγ junctions present in the B6 Vγ1/Vδ4 i-IELs, their expressed Vγ1-Jγ4 rearrangements were cloned and sequenced. Consistent with the population analyses shown above, 11 of 26 clones (42.3%) contained a CDR3 of 10 aa in length. Their junctional nucleotide sequences are shown in Fig. 9⇓A, whereas their predicted amino acid sequences are shown in Fig. 9⇓B. Most nucleotide and amino acid sequences were unique. The frequent presence of Ile at position 4 of the CDR3 is likely due to the usual presence of the last codon of the Vγ1 (ATA) in these junctions. Similarly, that of Ser at position 6 results from the presence of the first codon of the Jγ4 (TCA) in the junctions. Finally, the overrepresentation of Gly and Arg at position 5 is mainly due to the presence in the junctions of the P nucleotides of the Jγ4 segment (GA). Comparable frequencies of the same amino acids at these positions were also found in Vγ1-Jγ4 junctions of the same CDR3 length isolated from B10 Vγ1/Vδ4 i-IELs or from B6 Vγ1 i-IELs (data not shown).

FIGURE 9.
FIGURE 9.

Junctional sequences of Vγ1Jγ4Cγ4 chains expressed by the Vγ1/Vδ4 i-IEL subset of B6 mice. Total RNA from sorted Vγ1+Vδ4+ i-IELs from a pool of eight B6 mice was amplified as described in the legend of Fig. 8⇑, and the PCR products were cloned and sequenced as described in Materials and Methods. A, Nucleotide sequences of Vγ1-Jγ4 junctions with a CDR3 length of 10 aa (see Fig. 8⇑) expressed by Vγ1+Vδ4+ i-IELs of B6 origin. B, Predicted amino acid sequences corresponding to the nucleotide sequences shown in A. Dashes denote identity.

Tissue-specific selection of Vγ1/Vδ4 cells

To investigate whether similar selective events also operate in other peripheral sites, we evaluated and compared the representation of the Vγ1+ and the Vγ1/Vδ4 populations among LN γδ cells in B6, B10, and B10B6F1 mice (Fig. 10⇓A). The representation of both cell populations among LN γδ cells was significantly lower in B6 than in B10 mice, although the differences were less pronounced than those previously observed among γδ i-IELs. Thus, Vγ1+ cells represented 62% and 42% of the LN γδ cells in B10 and B6 mice, respectively (Fig. 10⇓A, left panel), whereas less than a twofold difference was observed in the representation of the Vγ1/Vδ4 subset in these mouse strains (Fig. 10⇓A, middle panel). B10B6F1 animals displayed intermediate levels of these two populations, and analyses of B10B6F2 mice showed that the different representation of these γδ populations is influenced by TCRγ-linked genes (data not shown). The representation of Vδ4+ cells among Vγ1+ LN cells in both strains of mice was also significantly different, although these difference appeared too small to be of major biological significance (Vδ4+ cells represented 32% and 26% of the Vγ1+ cells in B10 and B6 mice, respectively; Fig. 10⇓A, right panel). Taken together, these data indicate that most of the differences observed in the representation of the Vγ1/Vδ4 LN subset between these two strains of mice can be attributed to the Vδ-independent selective event. Consistent with this interpretation, the CDR3 length distribution of the Vγ1-Jγ4 junctions present in Vγ1/Vδ4 LN cells in B6 mice contains a predominant peak at a CDR3 of 9 aa (Fig. 10⇓B), although a slight increase in the frequency of junctions with a CDR3 of 10 aa was noticed in some mice (data not shown). Altogether, these results suggest that the Vδ-dependent selection mechanism operating on Vγ1/Vδ4 cells is more prevalent in, if not confined to, the intestinal epithelium.

FIGURE 10.
FIGURE 10.

Selective events operating on LN γδ cells. A, LN cells from the indicated strains were expanded in vitro as detailed in Materials and Methods and stained as indicated in Fig. 2⇑. Data are shown as the mean ± SEM of five to ten animals analyzed individually for each strain. Statistical analyses were performed by ANOVA followed by Student’s t tests (∗∗∗, p < 0.0001; ∗∗, 0.0001 < p < 0.001; ∗, 0.001 < p < 0.01). B, Total RNA from sorted Vγ1+Vδ4+ and Vγ1+Vδ4− LN cells from a pool of four B6 mice were amplified with Vγ1- and Cγ4-specific primers. An aliquot of each amplification product was submitted to a run-off reaction with a FAM-labeled Jγ4 primer, and the final products were resolved in an automated sequencer. Plot shows the profiles of fluorescence intensity vs the length of the fragments.

Discussion

Previous experiments analyzing V-gene use by γδ i-IELs or splenocytes with a limited number of Vγ- and Vδ-specific mAbs have shown that genes closely linked to the TCRγ, TCRδ, and MHC loci influence the representation of γδ cell subsets (17, 18, 19). These experiments provided indirect evidence suggesting that peripheral γδ cells may be submitted to cellular selection mechanisms. However, neither the selective mechanisms nor the target cell of the selective events could be unambiguously defined in those experiments. By analyzing V-gene use in γδ i-IELs from B6 and B10 mouse strains and in their F1 and F2 progenies with a large panel of Vγ- and Vδ-specific mAbs, we have shown that the linkage to the TCRγ locus of the representation of γδ i-IEL subsets results from two mechanistically different events possibly acting on two distinct target cells. The first event influences the representation of cells expressing different Vγ chains independently of the δ chain and is evident not only in the different representation of cells expressing the Vγ1 or the Vγ7 chains among i-IELs (Fig. 2⇑), but also in the representation of cells expressing the Vγ1 or the Vγ4 chains among γδ thymocytes (data not shown) and LN cells (Fig. 10⇑). Such an event may be responsible, at least in part, for the observed influence of the TCRγ locus in the representation of Vγ4−Vδ4+ splenocytes (likely representing Vγ1/Vδ4 cells) in crosses between B6 and DBA/2 parental strains (19). A mechanistically simple hypothesis to explain these results postulates that rearrangements involving the Jγ1 or the Jγ4 gene segments occur at different frequencies in γδ precursors from different mouse strains. This possibility is currently under investigation. Alternatively, a Vγ-dependent, Vδ-independent selective mechanism can also be invoked.

The second event is likely a cellular selection event acting specifically on Vγ1/Vδ4 i-IELs, and it has two major consequences. First, animals carrying the TCRγb haplotype contain a reduced proportion of Vγ1/Vδ4 i-IELs. Second, the remaining Vγ1/Vδ4-bearing i-IELs in TCRγb mice contain Vγ1Jγ4Cγ4 chains that are selectively enriched for CDR3 lengths longer by 1 aa than the prevalent size of Vγ1Jγ4Cγ4 chains observed in other Vγ1-bearing cells in the same mice. Our experiment excluded structural pairing constraints as a possible mechanism to explain the low abundance of Vγ1/Vδ4 in the intestinal epithelium of B6 mice, because Vγ1/Vδ4 cells expressing a very diverse repertoire of TCRs are present in similar numbers in the thymus and in the periphery of B6 and B10 mice. There are at least three possible explanations for these observations. First, Vγ1b/Vδ4 i-IELs may be actively deleted. Alternatively, Vγ1a/Vδ4 i-IELs may be truly positively selected by an endogenous ligand, whereas Vγ1b/Vδ4 i-IELs fail to react with the same ligand and are passively deleted. Finally, Vγ1a/Vδ4 i-IELs but not Vγ1b/Vδ4 i-IELs may be expanded in situ via specific Ag stimulation. Analysis of germ-free mice and mice deficient in molecules involved in death and survival of lymphocytes (currently underway) may help to clarify this issue. Whatever the mechanism, our results indicate a differential reactivity of Vγ1a/Vδ4 and Vγ1b/Vδ4 i-IELs that has a physiological consequence.

The different reactivities of the Vγ1a/Vδ4 and the Vγ1b/Vδ4 i-IELs, as well as the overrepresentation of Vγ1 chains containing CDR3s of 10 aa in length in the Vγ1/Vδ4 i-IEL subset in B6 mice must relate to polymorphisms at the coding region of the Vγ1Jγ4Cγ4 chain. Based on the sequence identity and on the expected structural homology of the V and C domains of the mouse Vγ1Jγ4Cγ4 chain with the Vγ9Cγ chain of the recently crystallized human receptor TCR Vγ9Vδ2 (40), a model of the Vγ1Jγ4Cγ4 chain was simulated (Fig. 6⇑). In this model, the two polymorphic residues between the B6 and the B10 Vγ1Jγ4Cγ4 alleles are disposed far away from any CDR. Given that the human Vγ9Cγ chain and the mouse Vγ1Cγ4 share 47% identity at the amino acid level, it is very likely that the model predicts accurately the position of these residues in the mouse Vγ1Cγ4 chain. How polymorphism at any of these two residues affects CDR3 length in B6 Vγ1/Vδ4 i-IELs is not readily evident from these analyses. Given their predicted localization, the possibility that they exert a direct effect in the structure of the CDR3 appears unlikely. However, because their side chains are predicted to be exposed to the solvent, they could participate in the overall recognition through a putative interaction with a distinct site of the ligand.

The fact that the Vγ1-Jγ4 junctions present in the Vγ1/Vδ4 i-IELs of B6 mice are enriched in relatively long but highly diverse in-sequence CDR3 suggests that selection operates more on the overall CDR3 structure than on their primary amino acid sequence. This suggests that germline-encoded elements present in the Ag-binding site of the Vγ1Vδ4 TCR are determinant in ligand recognition and specificity, as has been proposed for the recognition of prenyl pyrophosphates by human Vγ9Vδ2 cells (40, 41). This may be a general feature of ligand recognition by γδ T cells, which often correlates with V and/or J segment usage but allows a high degree of diversity at the V to J junction (42, 43, 44, 45). The experiments presented here should provide a good experimental model to analyze γδ T cell reactivity in vivo. Unraveling the specificity of γδ cells, their mechanisms of Ag recognition, and the consequences of such recognition may be the keys to the understanding of their unique functions.

Acknowledgments

We thank P. Alzari and G. Bentley for their help in the construction of the structural model of the Vγ1Jγ4Cγ4 chain.

Footnotes

  • ↵1 This work was supported by institutional grants and by grants from the Association pour la Recherche sur le Cancer. K.G. was supported by a fellowship from the Fondation pour la Recherche Médicale.

  • ↵2 Address correspondence and reprint requests to Dr. Pablo Pereira, Unité du Développement des Lymphocytes, Centre National de la Recherche Scientifique, Unité de Recherche Associée 1961, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris cedex 15, France. E-mail address: ppereira{at}pasteur.fr

  • ↵3 Abbreviations used in this paper: Tg, transgenic; i-IEL, intestinal intraepithelial lymphocyte; LN, lymph node; DN, double negative; CDR, complementarity-determining region.

  • Received April 22, 2002.
  • Accepted August 1, 2002.

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