Cutting Edge: TCRαβ⁺ CD8αα⁺ T Cells Are Found in Intestinal Intraepithelial Lymphocytes of Mice That Lack Classical MHC Class I Molecules

Mice

C57BL/6 and 129 strains of mice were obtained from The Jackson Laboratory (Bar Harbor, ME). (C57BL/6 × 129)F₁ mice were bred from the parental strains in the vivarium at the La Jolla Institute of Allergy and Immunology. K^b−/−D^b−/− mice have been described previously (14, 15) and were kindly provided by Dr. J. Forman (University of Texas Southwestern Medical Center, Dallas, TX) with permission of Dr. F. Lemonnier (Institut Pasteur, Paris). The mice were back-crossed three times onto the C57BL/6 background before use. TAP 1^−/− mice on the mixed C57BL/6 × 129 background were bred from stock originally obtained from Dr. L. Van Kaer (Vanderbilt University, Nashville, TN). β₂m^−/− mice on the C57BL/6 × 129 background were bred from stock obtained from Dr. B. Koller (University of North Carolina, Chapel Hill, NC). Dr. M. J. Grusby (Harvard Medical School, Boston) kindly provided mice lacking both the CD1.1 and CD1.2 genes (CD1^−/−) on the mixed BALB/c × 129 background. Mice 6 to 12 wk old of both sexes were used, except for CD1^−/− mice, which were analyzed at 10 mo. All mice were housed under specific pathogen-free conditions in the La Jolla Institute for Allergy and Immunology vivarium.

Flow cytometry

IEL were prepared as described previously (16). IEL or other lymphocytes were suspended in buffer comprised of PBS (pH 7.4) containing 2% (w/v) BSA and 0.02% NaN₃ (w/v). After blocking with the 2.4G2 anti-FcγR mAb (except for the CD16 staining), the cells were stained at 4°C for 30 min with the labeled mAb, then washed and analyzed on a Becton Dickinson FACScan (San Jose, CA) 440 flow cytometer. Lymphocytes were enumerated out of the heterogeneous cell population obtained following IEL preparation by electronic gating, as determined by analysis of FSC and SSC.

The following mAbs were used for phenotypic analysis of lymphocytes: PE- or CyChrome-labeled anti-TCR αβ clone H57-597, FITC-labeled anti-TCR γδ clone GL-3, FITC-labeled anti-CD4 clone GK1.5, FITC- or PE-labeled anti-CD8α clone 53-6.7, PE-labeled anti-CD8β clone 53-5.8, FITC-labeled anti-CD5 clone 53-7.3, FITC-labeled anti-CD103 (integrin α_IEL chain or α_E) clone M290, PE-labeled anti-CD122 (IL2R β-chain) clone TM-b1, PE-labeled anti-CD44 clone IM7, PE-labeled anti-CD16 clone 2.4G2, biotinylated-anti-CD28, and biotinylated-anti-CD90.2 (Thy1.2) clone 53-2.1. All Abs were purchased from PharMingen (San Diego, CA). Stainings involving biotinylated Abs were revealed using streptavidin-tri-color conjugate (Caltag, Burlingame, CA).

TCRαβ⁺ CD8αβ⁺ IEL are absent in K^b−/−D^b−/− mice

IEL populations from different class I-deficient animals were analyzed to evaluate the respective role of MHC class Ia and class Ib molecules in the development of TCRαβ⁺ CD8⁺ IEL. In Fig. 1⇓ is shown representative flow cytometry analysis of IEL populations found in control mice (C57BL/6), three class I-deficient strains, including β₂m^−/− mice, TAP^−/− mice, and K^b−/−D^b−/− mice, as well as CD1^−/− mice. In the absence of the β₂m molecule, the percentage of TCRαβ⁺ cells in the total IEL population is severely decreased (11 ± 2.5% TCRαβ⁺ in β₂m^−/− mice (n = 5) vs 43.4 ± 10.5% in C57BL/6 mice (n = 9)). The decrease is less severe in TAP-deficient animals (27.9 ± 5.2% (n = 6)) and absent in K^b−/−D^b−/− (47.3 ± 11.7% (n = 10)). By contrast, the TCRαβ⁺ CD8⁺ population is markedly decreased in the spleen (<5% vs 30% in C57BL/6) and lymph nodes of the three MHC class Ia knockout mice, but not in CD1^−/− mice (data not shown).

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FIGURE 1.

Phenotypic characterization of small intestine IEL of C57BL/6 and various class I-deficient mice. Three-color stainings for TCRαβ, CD4 and CD8α, or TCRαβ, CD8α and CD8β were conducted. TCRαβ staining is shown in the top row. Analysis of CD4, CD8 expression (middle and bottom rows) was done after gating on the TCRαβ⁺ cells. Numbers represent the percentages of fluorescence-positive cells in corresponding area. The data shown are representative from one of nine mice for C57BL/6, ten mice for K^b−/−D^b−/−, four mice for CD1^−/−, four mice for β₂m^−/−, and six mice for TAP^−/−.

In the intestine of control animals, five different TCRαβ⁺ populations are detected, DN, CD4⁺CD8αα⁺ (DP), CD4⁺ single positive, CD8αβ⁺ single positive, and CD8αα⁺ single positive, as previously reported (17, 18). While TAP^−/− animals contain the same five populations, in both β₂m^−/− and K^b−/−D^b−/− mice, the TCRαβ⁺ CD8αβ⁺ cells are missing (Fig. 1⇑). The relative contribution or percentage of the different TCR αβ⁺ populations among total IEL was evaluated by flow cytometry (Fig. 2⇓). No significant difference can be detected for the TCRαβ⁺ CD4 single positive and DN cells between the three class Ia-deficient animals (Fig. 2⇓). A significant percentage of CD8αβ⁺ IEL can still be detected in TAP^−/− mice (2.2 ± 0.9%) but not in β₂m^−/− mice (0.2 ± 0.2%), although this population is greatly decreased when compared with C57BL/6 mice (15.9 ± 6.3%). The MHC class Ia-deficient K^b−/−D^b−/− mice are similar to the β₂m^−/− mice with virtually no TCRαβ⁺ CD8αβ⁺ cells present in the intestine (1.0 ± 0.7%) compared with control animals (p = 0.00001). Moreover, the total cell number of TCRαβ⁺ CD8αβ⁺ IEL in K^b−/−D^b−/− mice is reduced ∼15 times compared with the wild type (see Table I⇓).

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FIGURE 2.

TCRαβ⁺ IEL subsets in C57BL/6, K^b−/−D^b−/−, CD1^−/−, β₂m^−/−, and TAP^−/−. IEL were incubated with anti-TCRαβ-CyChrome, anti-CD4-FITC, and anti-CD8α-PE, or with anti-TCRαβ-CyChrome, anti-CD8α-FITC, and anti-CD8β-PE Abs. The CD4, CD8 expression phenotype in each animal was determined after gating on TCRαβ⁺ cells. The true percentage of CD8αα single-positive cells was calculated by subtracting the percentage of TCRαβ⁺ CD4/CD8 DP cells (all of these are CD8αα⁺) from the percentage of TCRαβ⁺ CD8αα⁺ obtained in the CD8α vs CD8β staining. The percentage of cells with each type of coreceptor among total IEL for each animal was determined by multiplying the percentage of cells among IEL that are TCRαβ⁺ (a shown in the top row of Fig. 1⇑) by the percentage of TCRαβ⁺ cells expressing each type of coreceptor (as shown in the lower rows of Fig. 1⇑). The results are the mean (±SD) percentage derived from the same number of mice described in Fig. 1⇑. Statistical comparisons were conducted using Student’s two-tailed t test. ∗, p = 0.00001; ∗∗, p = 0.0028; and ∗∗∗, p = 0.0457 when K^b−/−D^b−/− mice are compared with C57BL/6.

Some TCRαβ⁺ CD8αα⁺ IEL are not dependent upon MHC class Ia molecules

The percentage of TCRαβ⁺ CD8αα⁺ cells is greatly decreased in β₂m^−/− mice (1.8 ± 0.6%) and moderately decreased in TAP^−/− (4.4 ± 2.5%) when compared with control animals (10.9 ± 4.0%) (Fig. 2⇑). By contrast, in K^b−/−D^b−/− animals the CD8αα⁺ population is present (Fig. 1⇑), and its percentage among total IEL (21 ± 7.7%) is increased when compared with C57BL/6 mice (p = 0.0028) (Fig. 2⇑). Furthermore, the total cell number of TCRαβ⁺ CD8αα⁺ is increased three times when compared with C56BL/6 mice and 35 times when compared with β₂m^−/− or TAP^−/− mice (see Table I⇑). Hence, it appears that at least some TCRαβ⁺ CD8αα⁺ IEL are not dependent upon the MHC class Ia molecules K and D, and that the number of such cells may be negatively regulated by classical class I expression.

The difference between K^b−/−D^b−/− and β₂m^−/− mice suggests that the TCRαβ⁺ CD8αα⁺ population requires a class Ib molecule(s). Because CD1 has been reported to be expressed in the intestinal epithelium, we used CD1^−/− mice to test for a possible role for this class I molecule. The five populations of TCRαβ⁺ IEL detected in control animals were also present in CD1-deficient mice (Fig. 1⇑), and the percentages of the different TCRαβ⁺ populations among total IEL found in CD1^−/− were not significantly different from the ones found in control animals (Fig. 2⇑).

In agreement with our earlier results (8), we found that the DP IEL are greatly decreased in β₂m^−/− mice, but they are not decreased in either TAP 1^−/− or K^b−/−D^b−/− mice (Fig. 2⇑). While these data suggest a possible role for class I molecules other than K or D either in the induction or maintenance of DP IEL, this population tends to be quite variable, and it should be noted that other studies have reported only a small decrease (6) or even an increase in DP IEL in β₂m^−/− mice (7).

Phenotypic analysis of TCRαβ⁺ CD8αα⁺ IEL in K^b−/−D^b−/− mice

The expression of the homodimeric form of the CD8 molecule has been shown to define a highly unusual subset of T cells that fail to express markers normally found on mature T cells, including CD28, CD5, and CD90 (Thy1.2) (17, 19, 20). They have been described to express the activation marker CD44 as well as CD103 (21), the α subunit of a mucosal-specific integrin (22). TCRαβ⁺ CD8αα⁺ from the K^b−/−D^b−/− mice are CD5⁻ CD28⁻ and CD90⁻, while they express CD103, CD44, CD16, and low levels of CD122 (Fig. 3⇓), suggesting that the population of CD8αα⁺ IEL present in the class Ia-deficient mice is similar to the population found in normal mice.

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FIGURE 3.

Phenotypic characterization of TCRαβ⁺ CD8αα⁺ IEL in C57BL/6 and K^b−/−D^b−/− mice. Three-color staining using anti-TCRαβ⁺, anti-CD4 together with anti-CD8β, and either anti-CD5, anti-CD103, anti-CD122, anti-CD44, anti-CD16, anti-CD28, or anti-CD90 mAbs. Phenotypic analysis of TCRαβ⁺ CD8αα⁺ IEL was achieved by gating on cells positive for TCRαβ⁺ but negative for CD4 and CD8β (profile with black fill). Such a gate contains mainly CD8αα and a minority of DN (<3%) cells. Profiles with white fill represent negative controls for the CD103, CD122, CD44, CD16, and CD28 stainings, while they correspond to the positive control TCRαβ⁺ CD4⁺ CD8β⁺ gated cells for the stainings with anti-CD5 and anti-CD90 mAbs. These results are representative of two mice in each group with similar results.