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MHC Class I Allele Dosage Alters CD8 Expression by Intestinal Intraepithelial Lymphocytes¹

Bradley S. Podd^2,*, Caroline Åberg^2,*, Kimberly L. Kudla^*, Lataya Keene^*, Erin Tobias^* and Victoria Camerini^3,*,

^* Department of Pediatrics and Beirne B. Carter Center for Immunology, University of Virginia Health Sciences Center, Charlottesville, VA 22908

	Abstract

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The development of TCR ⁺, CD8⁺ intestinal intraepithelial lymphocytes (IEL) is dependent on MHC class I molecules expressed in the thymus, while some CD8⁺ IEL may arise independently of MHC class I. We examined the influence of MHC I allele dosage on the development CD8⁺ T cells in RAG 2^-/- mice expressing the H-2D^b-restricted transgenic TCR specific for the male, Smcy-derived H-Y Ag (H-Y TCR). IEL in male mice heterozygous for the restricting (H-2D^b) and nonrestricting (H-2D^d) MHC class I alleles (MHC F₁) were composed of a mixture of CD8⁺ and CD8⁺ T cells, while T cells in the spleen were mostly CD8⁺. This was unlike IEL in male mice homozygous for H-2D^b, which had predominantly CD8⁺ IEL and few mostly CD8^- T cells in the spleen. Our results demonstrate that deletion of CD8⁺ cells in H-Y TCR male mice is dependent on two copies of H-2D^b, whereas the generation of CD8⁺ IEL requires only one copy. The existence of CD8⁺ and CD8⁺ IEL in MHC F₁ mice suggests that their generation is not mutually exclusive in cells with identical TCR. Furthermore, our data imply that the level of the restricting MHC class I allele determines a threshold for conventional CD8⁺ T cell selection in the thymus of H-Y TCR-transgenic mice, whereas the development of CD8⁺ IEL is dependent on, but less sensitive to, this MHC class I allele.

	Introduction

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The majority of CD8⁺ T cells found in the peripheral circulation and secondary lymphoid organs express the CD8 heterodimer, whereas CD8⁺ intestinal intraepithelial lymphocytes (IEL)⁴ express CD8 as an heterodimer or as an homodimer (1, 2). CD8⁺ IEL, like their CD8⁺ T cell counterparts in the spleen and peripheral circulation, express the TCR and arise following positive selection by self-MHC class I molecules expressed in thymus (1, 3). By contrast CD8, a form of CD8 that is almost never found on T cells outside the intestine, is expressed on TCR ⁺ and TCR ⁺ IEL (1, 2). The development and selection of these IEL, however, are less defined and are probably distinct from those of CD8⁺ IEL. For example, while CD8⁺ IEL are dependent on MHC class I for their development, CD8⁺ IEL persist despite targeted deletion of the MHC class I K and D locus genes (4, 5, 6). Interestingly, while CD8⁺ IEL expressing the TCR are present in ₂-microglobulin knockout mice (₂m^-/-), those expressing the TCR are markedly reduced. These results suggest that TCR ⁺, CD8⁺ IEL require a ₂m-dependent, but nonclassical, MHC I molecule (MHC class Ib) for some aspect of their development or differentiation in the thymus, gut, or some other site in the periphery (7, 8, 9). As the enrichment of CD8⁺ IEL in certain mouse strains correlates with the level of Qa-2 expression, this ₂m-dependent MHC class Ib molecule, or other related MHC molecules may be required for the development and differentiation of CD8⁺ IEL (10, 11, 12).

Although numerous mouse models have demonstrated that some CD8⁺ IEL develop independently of conventional MHC class I molecules, the IEL generated in these mice may encompass different TCR repertoires subject to varying selective pressures during development and differentiation in the thymus, gut, or periphery. TCR-transgenic mice, however, have been widely used to more precisely define the development of IEL. The H-2D^b MHC class I-restricted TCR-transgenic mouse, whose TCR is reactive to a male peptide derived from the Y-linked gene, Smcy (H-Y TCR), has been used widely used to define the selection of CD8⁺ T cells in the thymus and gut. In this model, positive selection of CD8⁺ T cells occurs in the thymus of female mice, which lack the agonist ligand, and negative selection of these T cells occurs in male mice, which express the agonist ligand (13, 14). Therefore, female mice have CD8⁺ T cells in the lymph node and spleen, whereas male mice lack these T cells due to deletion in the thymus (13, 15, 16, 17). Despite positive selection of CD8⁺ cells in the female thymus, few CD8⁺ T cells are present among IEL; in fact, the majority of IEL are CD8^-CD8^- T cells. By contrast, while male mice delete CD8⁺ T cells in the thymus and therefore virtually lack CD8⁺ T cells in the spleen and gut, a large population of CD8⁺ IEL is present (15, 16).

The origin of CD8⁺ IEL in male H-Y TCR-transgenic mice is not well defined. It has been proposed that post-thymic T cells with low levels of CD8 (CD8^low) escape deletion in the thymus and are expanded in the gut as CD8⁺ IEL in the presence of the nominal Ag recognized by this transgenic TCR (17). It has also been proposed, however, that TCR ⁺, CD8^-, or CD8^low T cells result from the premature expression of and H-Y TCR transgenes in the TCR lineage, cells known to be enriched within the compartments of the gut (18, 19). Whether the generation of CD8⁺ IEL in H-Y TCR transgenic mice is driven by premature expression of the TCR transgenes or reflects the postthymic differentiation or generation of CD8^- T cells in the gut is still a matter of controversy.

In the present study, we used a genetic approach to understand more fully the role of conventional MHC class I molecules in the development of CD8⁺ IEL. We assessed whether alterations in MHC class I allele dosage skewed the development of CD8⁺ and CD8⁺ IEL in the H-2D^b-restricted H-Y TCR-transgenic mouse line. Our results imply that deletion of conventional CD8⁺ cells is tightly constrained by signals generated via peptide/MHC class I in the thymus, while generation of CD8⁺ IEL, perhaps locally within the gut, is less dependent on these signals. Furthermore, our studies demonstrate that positive selection of CD8⁺ T cells does not abrogate the development of CD8⁺ IEL. Taken together, our results suggest that the differentiation of CD8⁺ IEL may involve signals that are overlapping with CD8⁺ IEL, but are not wholly dependent on MHC class I.

	Materials and Methods

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Mice

Female and male SJL.B6 (H-2D^b/b) and B10.D2 recombinase-activating gene 2 (RAG2^-/-) (H-2D^d/d) mouse strains homozygous for the H-Y TCR transgene (SJL.B6 H-Y TCR RAG2^-/- and B10.D2 H-Y RAG2^-/-, respectively) were obtained from Taconic Farms (Tarrytown, NY) (20, 21). H-Y TCR, RAG2^-/-, H-2D^b/b mice were also obtained from Dr. M. Vacchio (National Institutes of Health, Bethesda, MD). Intercrosses of either non-TCR-transgenic RAG-deficient male or female SJL.B6 RAG2^-/- (H-2D^b/b) or B10.D2 RAG2^-/- (H-2D^d/d) mice with either H-Y TCR RAG2^-/--transgenic mouse strain noted above were used in the generation of F₁ mice under a vendor breeding agreement (Taconic Farms) (22). Mice generated from either cross had one chromosomal complement of the H-Y TCR and transgenes and were heterozygous for the restricting (H-2D^b) and nonrestricting (H-2D^d) MHC class I alleles (H-2D^b/d; MHC F₁). In some cases, F₁ mice were generated from a cross between male or female C57BL/10 RAG2^-/- (H-2D^b/b) mice expressing the H-Y TCR transgene with non-TCR-transgenic male or female B10.D2 RAG2^-/- (H-2D^d/d; Taconic Farms). These mice were similar to the MHC F₁ mice described above, were heterozygous for the MHC class I alleles, H-2D^b/d, and had one chromosomal complement of the H-Y TCR transgenes.

F₂ mice were generated by intercrossing F₁ mice derived as noted above. N2 generation mice were derived from MHC F₁ mice backcrossed with SJL.B6 RAG2 ^-/- (H-2D^b/b) mice. F₂ and N₂ mice were selected for carriage of the TCR transgene by PCR typing. The primers used to identify the HY TCR transgene that spanned the CDR3 H-Y transgene V8.2-D1-J2.3 region were (in 5'–3' orientation) 5'-GACATTGAGCTGTAATCAGAC (forward) and 5'-ACAGCGTTTCTGCACTGTTATCACC (reverse) (17). N₂ and F₂ mice harboring the TCR transgene were further typed for MHC class I haplotype by mAb staining of lymphocyte populations (see below).

Mice were housed and bred in a laminar flow barrier facility at the University of Virginia Health Sciences Center vivarium (Charlottesville, VA) under specific pathogen-free conditions. Pregnant mice were identified and monitored daily until delivery. The day of birth was identified as day 0 of life, and individual mice were examined between 4 and 16 wk of age. The animal care and use committee at the University of Virginia approved all animal protocols and procedures.

Preparation of lymphocyte populations

Mucosal lymphocytes were prepared from the small intestine of individual mice using our previously published procedure (23). Briefly, the small intestine was dissected from the mesentery and washed in RPMI 1640 (Life Technologies, Grand Island, NY). The intestine was cut longitudinally, and the contents were removed before it was cut into 0.5-cm pieces. IEL were released from the epithelium during shaking in calcium- and magnesium-free HBSS (Life Technologies) supplemented with 1 mM DTT (Sigma, St. Louis, MO) three times for 20 min each time at 250 rpm. Mononuclear cells collected from the epithelial layer were filtered through stainless steel mesh and enriched on a discontinuous 20/40/70% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ) gradient at 900 x g for 20 min. The resultant cell populations were washed in RPMI 1640 with 10% FCS and analyzed as outlined below.

Mononuclear cells were prepared from the thymus and spleen following mechanical disruption of the capsules with a glass pestle on stainless steel mesh or between frosted glass slides. Cell suspensions of splenocytes were depleted of RBC by hypotonic lysis in distilled water as we have described previously (24). The resultant mononuclear cells from the spleen and thymus were suspended in PBS mAb staining buffer containing 5% FCS and 0.02% NaN₃ (both from Sigma) and stored at 4°C until use as noted below.

Antibody staining and flow cytometric analysis of lymphocyte populations

Mononuclear cell populations were suspended at a concentration of 1 x 10⁶ cells/ml in PBS mAb staining buffer. Four-color immunofluorescence cell surface labeling was conducted by adding the optimal concentration of mAb either directly conjugated to FITC, PE, allophycocyanin, biotin, or unconjugated primary mAb as necessary to cell suspensions at 4°C with incubation for 20–30 min. FcR blocking with anti-CD16/CD32 added to a final concentration of 5.0 µg/10⁶ cells was used in all cases where cell populations were incubated with unconjugated primary mAb and in some cases with directly conjugated mAb. After incubation with primary mAb, the samples were washed three times in PBS with 0.02% NaN₃ at 4°C. The fluorochrome-conjugated secondary reagents Tricolor or PE-Cy 7-conjugated streptavidin (Caltag Laboratories, South San Francisco, CA) in PBS mAb staining buffer were used to detect biotinylated primary mAb (see below), while anti-mouse IgG1 FITC (A85-1) was added to detect mouse anti-clonotypic TCR (T3.70) (14). The secondary reactions were incubated for 20–30 min and washed as described above for primary mAb staining reactions. After mAb staining and washing, all samples were fixed in PBS with 0.02% sodium azide with 1% paraformaldehyde and stored at 4°C until analysis by flow cytometry (both from Sigma). The mAb reagents used in this study were anti-CD3 (145-2C11) FITC or allophycocyanin, anti-CD4 (YTS 191.1) FITC or PE, anti-CD8 (53-6.7) PE or allophycocyanin, anti-CD8 (53-5.8) FITC, anti-CD16/CD32 (2.4G2) purified, anti-TCR (H57-597) FITC, anti-TCR V8.1,8.2 (MR5-2) PE or biotin, anti-CD45 (30-F1) PE, anti-MHC H-2D^d (34-2-12) FITC or biotin, and anti-MHC H-2D^b (28-14-8) PE or biotin (all from BD PharMingen, San Diego, CA). Purified anti-clonotypic TCR mAb (T3.70) was a gift from Dr. H. Cheroutre (La Jolla Institute for Allergy and Immunology, San Diego, CA) (14).

Mononuclear cell suspensions were analyzed on a FACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose, CA) in the Beirne Carter Center Flow Cytometry Core Facility (University of Virginia). Forward angle and side angle light scatter settings were used to distinguish lymphocytes from epithelial cells based on the size and granularity of these populations. Between 5,000 and 30,000 events gated on the forward and side angle light scatter properties were acquired, and the data were analyzed using CellQuest software (BD Immunocytometry Systems). Single and multiple parameter analyses using dot plots and histograms with corresponding statistics were used. The number of T cells present among IEL and spleen was calculated as we have described previously (24). Briefly, the total cell yield was determined by light microscopy and corrected for the percentage of mononuclear cells gated by forward and side angle light scatter and for the expression of CD45 and CD3 as determined by flow cytometry.

Statistical analysis

The computer software program StatView (Abacus Concepts, Berkeley, CA) was used to calculate the SD between experimental samples. Differences between more than two mean values were determined with Student’s two-tailed t test using the analysis features of this program. We considered p < 0.05 to be statistically significant.

	Results

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Development and expansion of CD8⁺ IEL requires at least one copy of the restricting MHC class I allele, H-2D^b, in H-Y TCR-transgenic male mice

The phenotype of IEL in H-Y TCR-transgenic mice has been previously documented, although the findings have not always been in full agreement (15, 16, 17). Therefore, before examining the development of CD8⁺ IEL subsets in H-Y TCR-transgenic mice bred to be heterozygous for the restricting (H-2D^b) and nonrestricting MHC class I alleles (H-2D^d), we wanted to confirm previous findings in this mouse line on the RAG2^-/- background (17). We examined H-Y TCR-transgenic mice homozygous for H-2D^b and homozygous for H-2D^d MHC class I alleles. More than 90% of CD45⁺ mononuclear cells isolated from the intestinal epithelium of H-2D^b mice were CD3⁺ and expressed both transgenic and TCR chains (Fig. 1, A and B). By contrast, <10% of CD45⁺ mononuclear cells isolated from the intestinal epithelium of H-2D^d mice were CD3⁺ and expressed the clonotypic TCR chains (Fig. 1, C and D). When CD3⁺ IEL from each mouse strain were examined for CD8 and CD8 chain expression, 74% of IEL from the H-2D^b homozygous mice were CD8⁺, while only 7% were CD8⁺ and 19% were CD8^- (Fig. 1B). This was in contrast to IEL prepared from mice homozygous for H-2D^d, which were largely CD8^- T cells (Fig. 1D).

View larger version (33K): [in this window] [in a new window]   FIGURE 1. The development of CD8+ IEL in male H-Y TCR-transgenic mice requires the restricting MHC class I allele, H-2Db. IEL were isolated from male H-Y TCR-transgenic mice homozygous for H-2Db (A and B) and H-2Dd (C and D). Flow cytometry was used to determine the expression of CD3 by CD45+ IEL (A and C). Anti-CD3, anti-T3.70 (followed by anti-mouse IgG1), and anti-V8.2 mAb were used to determine the expression of the TCR and transgenes by CD3+ IEL, and anti-CD8 and anti-CD8 mAb were used to determine the coreceptor expression by CD3+ IEL (B and D). Single-fluorescence histogram gating on CD45+ cells demonstrated the percentage of CD3+ IEL in each mouse strain (A and C). The expression of H-Y TCR - and -chain transgenes and CD8 and CD8 coreceptors are shown in representative two-color dot-plot analysis on IEL electronically gated on CD3 and CD45 expression (B and C). The percentage of CD3+CD45+ cells in each histogram and the percentage of cells in the relevant quadrants of the dot plots are shown in the upper right corners. Data are representative of a minimum of three experiments and represent one experiment from data presented in Fig. 4E.

View larger version (10K): [in this window] [in a new window]   FIGURE 2. One copy of H-2Db is required for the expansion of IEL in H-Y TCR-transgenic male mice. IEL were prepared from male and female H-Y TCR-transgenic mice on the MHC backgrounds shown. The number of CD3+CD45+ T cells in each subset was calculated by multiplying the total number of viable cells, as determined by light microscopy, by the percentage of CD45+, CD3+ cells, as determined by flow cytometry. Each symbol represents the analysis of cells from a single experiment, which was comprised of a single mouse. n = 7 for male H-2Db/b, n = 11 for male H-2Db/d, n = 4 for male H-2Dd/d, and n = 4 female H-2Db/d. *, p < 0.05 when male IEL H-2Db/d were compared with male H-2Dd/d or female H-2Db/d; , p = ns when male IEL H-2Db/d compared with male H-2Db/b.

H-Y TCR-transgenic male mice heterozygous for the MHC class I alleles, H-2D^b and H-2D^d contain CD8⁺ and CD8⁺ IEL

We next generated mice that were heterozygous for the restricting H-2D^b and nonrestricting H-2D^d MHC class I alleles and were designated MHC F₁ mice. A minor population of CD3⁺ IEL was present among CD45⁺ cells isolated from H-Y TCR-transgenic H-2D^d homozygous mice, while no CD3 expression, as expected, was detected among CD45⁺ cells isolated from the intestinal epithelium of the non-TCR-transgenic (RAG-deficient) H-2D^b mice (Fig. 3, A and B). CD45⁺ mononuclear cells isolated from the intestinal epithelium of male MHC F₁ progeny derived from this cross were largely CD3⁺ and nearly all expressed the clonotypic TCR - and -chains (Fig. 3, C and D). Similar results were obtained with IEL in MHC F₁ mice generated by crossing nontransgenic C57BL/10 RAG2^-/- mice with B10.D2 H-Y TCR RAG2^-/- mice (data not shown). The number of IEL isolated from MHC F₁ male mice was not statistically different from the number of IEL isolated from male mice homozygous for H-2D^b (Fig. 2). These results indicate that one copy of H-2D^b is required for the development and expansion of CD3⁺ IEL in male H-Y TCR-transgenic mice.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. One copy of the restricting MHC I class I allele, H-2Db, is sufficient for the development of IEL in H-Y TCR-transgenic male mice, but hemizygosity of this allele alters CD8 isoform expression by these T cells. CD45+CD3+ IEL were electronically gated and shown in single-fluorescence histograms for H-Y TCR-transgenic male mice on the nonrestricting MHC background (A), in non-H-Y TCR-transgenic mice on the restricting MHC background (B), and in MHC F1 male progeny (C). IEL isolated from male MHC F1 progeny were analyzed for the expression of the clonotypic TCR - and -chains (D) and the expression of the CD8 and CD8 coreceptors (E). CD8 isoform expression by IEL isolated from H-Y TCR-transgenic mice on the restricting MHC background is shown for comparison in F. The percentage of cells in each quadrant is shown in the upper right corner. The data are representative of a minimum of seven experiments derived from the analysis of individual mice.

CD8 isoform expression among IEL segregates with the MHC haplotype in H-Y TCR-transgenic mice

The H-Y TCR-transgenic mice used in the generation of MHC F₁ mice have several copies of and TCR transgenes integrated on the same chromosome and are carried in the homozygous state (25). To evaluate whether CD8 isoform expression in MHC F₁ mice was due to quantitative differences in the level of TCR expression in these mice, we examined the expression of transgenic TCR V and TCR V levels in MHC F₁ and MHC homozygous mouse lines by flow cytometry. We found that the level of TCR transgenes in MHC F₁ mice was similar to that in the H-Y TCR MHC homozygous mouse lines (Figs. 1D, 3D, and 4, A and B). Furthermore, TCR and transgene levels were similar in the H-Y TCR-transgenic line obtained from Dr. Melanie Vacchio, a line maintained by crossing H-2D^b-nontransgenic RAG2^-/- mice with H-Y TCR-transgenic RAG2^-/- mice H-2D^b (data not shown). These results suggest that the level of TCR transgene expression does not differ significantly between MHC F₁ and MHC class I homozygous H-Y TCR-transgenic mice and therefore is an unlikely cause of the retention of CD8⁺ T cells in male MHC F₁ mice.

To determine whether CD8 isoform usage segregated with the MHC haplotype we generated additional mouse lines that were either heterozygous or homozygous for H-2D^b. F₂ mice were generated by intercrossing MHC F₁ mice and N2 mice were generated by backcrossing MHC F₁ mice with non-H-Y RAG2^-/- (H-2D^b mice). MHC F₁ mice were generated using male mice from either the B10.D2 or SJL.B6 strain; therefore, a different strain serves as the Y chromosome donor. IEL derived from F₂ and N₂ mice demonstrated again that CD8 isoform expression segregated with the MHC haplotype. In the example shown in Fig. 4, 59% of CD3⁺ IEL in N₂ MHC heterozygous mice were CD8⁺, while 40% of IEL were CD8⁺ and 1% of IEL were CD8^-CD8^- (Fig. 4C). By contrast, IEL in N₂ mice homozygous for the selecting MHC class I alleles were largely CD8⁺ IEL (Fig. 4D). The increase in CD8⁺ IEL in MHC F₁ mice was also seen in F₂ mice that were heterozygous for the restricting and nonrestricting MHC alleles (Fig. 4E). A large number of experiments derived from individual mice were pooled and compared with H-2D^b homozygous mice and illustrate the same trend (Fig. 4E). In the example shown, CD8⁺ IEL were increased in MHC heterozygous mice compared with mice homozygous for the restricting MHC (Fig. 4E). Additionally, the strain of the male mouse (Y chromosome) used to generate MHC F₁ mice did not alter theappearance of CD8⁺ T cells (data not shown). Collectively, these data indicate that the appearance of CD8⁺ IEL segregated with the heterozygous configuration of the MHC class I alleles, H-2D^b and H-2D^d.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. CD8 isoform expression by IEL segregates with the MHC haplotype in male H-Y TCR-transgenic mice. The level of transgenic TCR -chain expression by CD3+ IEL was determined following mAb staining of IEL derived from MHC F1 mice (A) and H-Y TCR-transgenic mice homozygous for H-2Db (B). The geometric mean fluorescence of TCR V8.2 expression by CD3+ IEL is shown in the upper left corner of each histogram. CD8 isoform expression by CD3+ IEL isolated from a N2 generation H-Y TCR-transgenic mice heterozygous for the restricting and nonrestricting MHC class I alleles (C) and mice homozygous for the restricting MHC class I alleles (D) were determined as noted in Fig. 3. The percentage of cells in the relevant quadrants on the dot plots is shown in the upper right corner. The data are representative of a minimum of four experiments derived from the analysis of individual mice. E, Summary data for coreceptor expression by CD3+ IEL derived from H-Y TCR-transgenic mouse lines as a function of their H-2D genotype is shown. CD8+, CD8+ or CD8- IEL populations were determined my three- and four-color flow cytometry are indicated by the graph legend. *, p < 0.05 compared with H-2Db homozygous mice; , p < 0.05 compared with MHC F1 mice. Data were derived from a minimum of three individual experiments, each derived from the analysis of individual mice.

CD8⁺ T cells are more numerous and CD8^-CD8^- T cells are less abundant among IEL and in the spleen of MHC F₁ H-Y TCR-transgenic mice

The restoration of CD8⁺ T cells among IEL in MHC F₁ mice suggested that CD8 lineage cells were positively selected in the thymus and expanded in the periphery of MHC F₁ mice. We hypothesized that MHC F₁ mice would, therefore, have a reduction in CD8^lowlow or CD8^- T cells among IEL and in the spleen if these cells were generated during deletion in thymus or tolerance in the periphery. We found that while up to 33% of IEL, on the average, were CD8^- in H-2D^b homozygous male mice, MHC F₁ mice had no >5% of these T cells, a difference that was statistically significant when a large number of mice from individual experiments were compared (Fig. 4E). The reduction in CD8^- IEL was also seen in the individual FACS dataplots shown in Fig. 3, E and F, and in Fig. 4, C and D. Similar findings are seen among T cells in the spleen of these mice. In the example shown in Fig. 5A, 80% of T cells in the spleen of MHC F₁ mice were CD8⁺, and 13% of T cells were CD8^-. By contrast, 95% of T cells in H-2D^b homozygous mice were CD8^-, and 3% of T cells had lower levels of CD8 and CD8 (Fig. 5A). These results are consistent with the increased generation or reduced destruction and survival of CD8⁺ T cells in MHC F₁ mice compared with male mice homozygous for H-2D^b.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. CD8+ T cells are more numerous in the spleen and CD4+ CD8+ thymocytes are more abundant in the thymus of MHC F1 mice than in mice homozygous for the restricting MHC, H-2Db. The expression of CD8 and CD8 was determined by multicolor flow cytometry of cells derived from the spleen by electronically gating on CD45+CD3+ T cells (A). Thymocytes were gated on CD45 and the expression of CD4 and CD8 coreceptor expression was determined (B). H-2Db homozygous (A, upper panel, and B), and MHC F1 mice (A, lower panel, and C) were analyzed. A, Few T cells in the spleen of MHC H-2Db mice (A, top panel) are CD8+ or expressed lower levels of CD8 and than did T cells in the spleen of MHC F1 mice (A, lower panel). B, The expression of CD4 and the CD8 coreceptor by total cells in thymus of H-2Db homozygous male mice was markedly reduced compared with MHC F1 male mice (C). The percentage of cells in relevant quadrants is shown in the upper right corners of the FACS dot plots. These data are representative of at least three experiments derived from the analysis of individual mice between 1 and 3 mo of age.

Positive selection in female mice is not altered by MHC class I allele dosage in H-Y TCR-transgenic mice

Our findings in MHC F₁ male mice prompted us to evaluate whether MHC class I allele dosage altered T cell development in female H-2D^b homozygous H-Y TCR-transgenic mice. Female mice are known to have few CD8⁺ IEL despite positive selection of this transgenic TCR in the thymus of H-2D^b female mice (13). Although we identified differences between T cells in the thymus, spleen, and intestine of MHC F₁ male mice, the phenotype of T cells in MHC F₁ female mice was similar to that in female H-2D^b homozygous mice. In the example shown, the majority of T cells among IEL were CD8^- among both MHC F₁ and H-2D^b female mice (63 and 57%, respectively; Fig. 6). In addition, the number and phenotype of IEL in female mice were similar to what has been described previously for H-2D^b homozygous mice on the RAG2^-/- background (Fig. 6) (19). The thymus of MHC F₁ and H-2D^b homozygous mice was composed of CD4⁺CD8⁺ double-positive, CD8⁺ single-positive, and CD4^-CD8^-, double-negative cells consistent with selection to the CD8⁺ T cell lineage in these mice (data not shown). These data imply that one copy of H-2D^b is sufficient to mediate positive selection of CD8⁺ T cells in the thymus of female mice, although CD8⁺ and CD8⁺ T cells remain poorly represented among IEL.

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Positive selection of H-Y TCR-transgenic IEL in female mice is not altered by MHC class I gene dosage. The expression of CD8 and CD8 by T cells among IEL (A) and in the spleen (B) of female H-Y TCR MHC F1 mice (left panels, A and B) female H-2Db homozygous mice (right panels, A and B). The expression of CD8 and CD8 was determined following electronic gating on CD45+CD3+ cells in each compartment. The majority of IEL on both MHC backgrounds were CD8-, while T cells in the spleen were predominantly CD8+, expressing high levels of CD8 and (B). The percentage of T cells present in the relevant quadrants is shown in the upper right corners. The data are representative of a minimum of three experiments derived from the analysis of individual mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our data support a central and a peripheral mechanism for the generation and expansion of CD8⁺ T cells in MHC F₁ male mice. First, the thymus of MHC F₁ mice had a marked increase in thymocytes known to be progenitors of CD8⁺ T cells, namely, CD4⁺CD8⁺ double-positive cells and CD8 single-positive cells expressing high levels of the CD8 and CD8 chains and TCR and transgenes (Fig. 5 and data not shown). Cells in the thymus of H-2D^b homozygous mice were, however, almost exclusively CD4^-CD8^- double-negative cells with lower levels of CD8 and the TCR transgenes, consistent with negative selection (Fig. 5 and data not shown). Taken together, these results suggest that CD8⁺ T cells found among IEL and in the spleen were positively selected in the thymus of MHC F₁ male mice. By contrast to IEL in male mice, T cells in the spleen and among IEL in female MHC F₁ mice were similar in number and phenotype to those in female mice homozygous for H-2D^b. These data imply that the window of positive selection in the thymus is functionally larger than the window of negative selection; the latter is drastically altered in MHC F₁ male mice. Second, the selective pressure to down-regulate CD8 and expression by T cells in the periphery of MHC F₁ mice may be less than that in MHC homozygous mice, hence accounting for the preservation of this T cell subset despite the potential autospecificity in MHC F₁ male mice. In support of this we found fewer CD8^- and CD8^lo T cells among IEL and in the spleen of MHC F₁ male mice compared with H-2D^b homozygous mice. It can be inferred, however, that lower levels of CD8 and CD8 expression by CD8⁺ T cells in the periphery of MHC F₁ male mice compared with female mice may reflect a negative influence on CD8 coreceptor expression in male mice (Figs. 5A and 6B). Notably, MHC F₁ male mice, like H-Y TCR-transgenic mice homozygous for the restricting MHC, have no evidence of inflammatory disease in the gut or elsewhere (17). CD8⁺ T cells were, therefore, not autoreactive by virtue of their development in the thymus and their presence in peripheral organs replete in the cognate Ag. Although CD8^- and CD8^low T cells may derive from either central or peripheral mechanisms, as discussed above, these T cells may derive following the early expression of the H-Y TCR transgenes during ontogeny (18, 19). This pathway could certainly explain the presence of largely CD8^- T cells in H-Y TCR-transgenic mice on the nonrestricting MHC background (H-2D^d) and in female H-Y TCR-transgenic mice (18, 19). The reduction of CD8^- and CD8^low T cells that we documented in MHC F₁ mice, however, suggests that CD8^- and CD8^low T cells in male mice homozygous for the restricting MHC normally derive from MHC class I-restricted negative selection in the thymus or following their generation in the periphery. The reduction in these populations in MHC F₁ mice would not be expected if their generation was solely due to events independent of MHC-restricted selection in the thymus. We cannot exclude, however, that events secondary to this TCR-transgenic model account for the aberrant generation of T cells in the H-Y TCR-transgenic mouse line. Taken together, our data suggest that CD8⁺ lineage T cells are selected in the thymus of MHC F₁ mice, while in H-2D^b homozygous mice this T cell lineage is efficiently deleted in the thymus and perhaps in the periphery.

The mechanism by which CD8⁺ T cells are generated in MHC F₁ mice was not directly elucidated by our studies. We determined, however, that the generation of CD8⁺ T cells was dependent on at least one copy of the selecting MHC class I allele, H-2D^b, regardless of whether F₂ or N₂ H-Y TCR generation male mouse lines were examined. Furthermore, CD8⁺ T cells among IEL and splenocytes were not dependent on the strain of male mouse (SJL.B6, B10.D2, or C57BL/10) used to generate the MHC F₁ mouse lines, suggesting that possible differences in the Smcy-derived peptide were not responsible for our results. This is in contrast to 5F TCR-transgenic mice expressing either a high or low affinity ligand for this TCR. Mice expressing the high affinity ligand had mostly CD8⁺ and CD8^- IEL, similar to H-Y TCR-transgenic male mice homozygous for H-2D^b, whereas mice expressing a low affinity antagonist ligand had both CD8⁺ and CD8⁺ IEL, similar to the MHC F₁ mice in our study (26). However, unlike MHC F₁ mice, CD8⁺ T cells were absent in the spleen of 5F TCR-transgenic mice, suggesting that these T cells were not conventionally derived in the thymus (26). By contrast, our data support the hypothesis that CD8⁺ T cells in MHC F₁ male mice were derived via a conventional thymus-dependent pathway and that these T cells established residence within the spleen and within the intestinal epithelium.

We propose that the signals driving negative selection are reduced in the thymus of MHC F₁ mice, as the probability of interaction between the cognate Ag in the context of the restricting MHC class I molecule, H-2D^b with its TCR is reduced by virtue of the haploid state. Therefore, an individual cell is less likely to receive a negative signal or receives a cumulative signal that is not sufficient to drive negative selection, thereby allowing the paradoxical selection of CD8⁺ lineage cells in the thymus of MHC F₁ male mice. The generation of CD8⁺ lineage cells in MHC F₁ male mice can be explained by the quantitative affinity model of TCR selection in the thymus (26, 27, 28). One model to show the relationship between signal strength and selection bias in the thymus is depicted in Fig. 7. A high avidity signal normally generated in H-Y TCR-transgenic male mice homozygous for the restricting MHC, H-2D^b, is sufficient to trigger negative selection and deletion of a majority of CD8⁺ lineage cells in the thymus. By contrast, mice heterozygous for the restricting MHC allele, H-2D^b, generate a signal below the threshold required for negative selection, but sufficient to allow positive selection of CD8⁺ lineage cells in the thymus. In fact, several TCR-transgenic models demonstrate that selection bias in the thymus is directly proportional to the density of the relevant MHC proteins (29, 30, 31). In the 2C TCR-transgenic model the level of the restricting MHC was varied using transgenic constructs expressing H-2L^d, which is normally deleting in the wild-type 2C TCR-transgenic mice. Mice expressing lower levels of this allele allowed positive selection, while higher levels promoted negative selection of CD8⁺ lineage cells in the thymus (31). It is possible, however, that H-2D^b in the haploid state would not support the positive selection of CD8⁺ T cells, in which case the nonrestricting MHC class I molecule, H-2D^d, or some other MHC molecule would provide an accessory signal. H-2D^d in the homozygous state is evidently not sufficient to support the development of CD8⁺ T cells, as these mice have few T cells, most of which are CD8^-. Studies to determine the specificity of alternative MHC class I alleles and nonclassical MHC molecules in our model are currently underway.

View larger version (14K): [in this window] [in a new window]   FIGURE 7. MHC class I allele dosage disrupts negative selection in the thymus and contributes to the development of CD8+ T cells in male H-Y TCR-transgenic mice. The relationship between cumulative signal strength and selection bias in the thymus with respect to the development of CD8+ T cells is shown. The presence of one or two copies of the restricting MHC class I allele H-2Db results in the generation of intrinsically different signals to the developing thymocyte. In the case of two copies of the restricting MHC class I allele H-2Db, a strong signal is generated, and negative selection occurs. By contrast, one copy of the selecting MHC class I allele generates a lower signal and allows positive selection to occur in male mice. Altered signal strength, therefore, allows positive selection of CD8+ T cells in the thymus, resulting in the restoration of CD8+ T cells among IEL and in the spleen of MHC F1 male mice. Figure adapted from Smyth et al. (34 ).

In summary, our data support a crucial role for the level of MHC class I expression as a control point in T cell development in the thymus and gut. The negative selection of conventional T cells in the thymus has a higher threshold than does positive selection of CD8⁺ T cells or the generation of CD8⁺ IEL. Variations in the level of MHC during ontogeny, particularly in cases where the level of cognate Ag is constant, alterations in MHC expression in response to cytokines or in the context of inflammation may be important in TCR repertoire selection in the thymus or in the generation of CD8⁺ T cells in the gut. Model systems such as ours may provide significant insight into the differentiation Ags responsible for the generation of CD8⁺ and CD8⁺ IEL and in elucidating their function in immune homeostasis in the gut.

	Acknowledgments

 
We thank Dr. David Camerini (University of Virginia) and Dr. Mitchell Kronenberg (La Jolla Institute for Allergy and Immunology) for helpful suggestions and critical reading of this manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI44990-01 (to V.C.).

² B.S.P. and C.. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Victoria Camerini, Department of Pediatrics, University of Virginia Health Sciences Center, Jordan Hall, Room 2215, 1300 Jefferson Park Avenue, Charlottesville, VA 22908. E-mail address: vc3p{at}virginia.edu

⁴ Abbreviations used in this paper: IEL, intestinal intraepithelial lymphocyte; ₂m, ₂-microglobulin; H-Y TCR, TCR-transgenic mouse line reactive to H-Y Ag; MHC F₁, H-Y TCR H-2D^b/b x H-2D^d/d; RAG, recombination-activating gene.

Received for publication April 12, 2001. Accepted for publication June 26, 2001.

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