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Cross-Positive Selection of Thymocytes Expressing a Single TCR by Multiple Major Histocompatibility Complex Molecules of Both Classes: Implications for CD4⁺ versus CD8⁺ Lineage Commitment¹

Koji Eshima^*, Harumi Suzuki and Nobukata Shinohara^2,*

^* Department of Immunology, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan; and Department of Microbiology and Immunology, Yamaguchi University School of Medicine Ube, Yamaguchi, Japan

	Abstract

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This study has investigated the cross-reactivity upon thymic selection of thymocytes expressing transgenic TCR derived from a murine CD8⁺ CTL clone. The Id^high+ cells in this transgenic mouse had been previously shown to mature through positive selection by class I MHC, D^q or L^q molecule. By investigating on various strains, we found that the transgenic TCR cross-reacts with three different MHCs, resulting in positive or negative selection. Interestingly, in the TCR-transgenic mice of H-2^q background, mature Id^high+ T cells appeared among both CD4⁺ and CD8⁺ subsets in periphery, even in the absence of RAG-2 gene. When examined on ₂-microglobulin^–/– background, CD4⁺, but not CD8⁺, Id^high+ T cells developed, suggesting that maturation of CD8⁺ and CD4⁺ Id^high+ cells was MHC class I (D^q/L^q) and class II (I-A^q) dependent, respectively. These results indicated that this TCR-transgenic mouse of H-2^q background contains both classes of selecting MHC ligands for the transgenic TCR simultaneously. Further genetic analyses altering the gene dosage and combinations of selecting MHCs suggested novel asymmetric effects of class I and class II MHC on the positive selection of thymocytes. Implications of these observations in CD4⁺/CD8⁺ lineage commitment are discussed.

	Introduction

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The T cell clones are selected for compatibility to self Ag-presenting molecules (MHC molecules) after completing the rearrangement of TCR structural genes in the thymus. During this process, the choice of the coreceptor to be expressed is also made according to the class of the selecting MHC molecules. Analyses of a number of TCR-transgenic (Tg)³ mouse lines and of mice deficient for MHC molecules clearly showed that CD4/CD8 double-positive (DP) cells expressing class I MHC-responsive TCR differentiate into CD8⁺ T cells, while class II MHC-restricted DP cells differentiate into CD4⁺ lineage. Recently, it was revealed that a zinc finger transcription factor Th-POK/cKrox plays a key role in the CD4⁺ vs CD8⁺ lineage commitment (1, 2). It still remains to be precisely clarified, however, how DP thymocytes undergoing positive selection could judge the class of MHC that they recognize, to differentiate into the appropriate lineages (for review, see Refs.3 and 4).

Considering the size of the allelic heterogeneity of MHC molecules observed among wild mice, the chance of successful positive selection appears extremely low, even in the case with the full possibilities, i.e., heterozygotes of two full (I-E-positive) haplotypes. Nevertheless, it is also quite possible that a single TCR can find multiple different MHC molecules as compatible partners. In fact, T cell clones specific for a protein Ag presented by a self MHC molecule frequently show cross-reactivity to allogeneic MHC (+ peptide) (5, 6, 7, 8, 9). TCR may well show similar cross-reactivity (multiple specificity) at the level of positive selection in which lower affinity interaction is crucial. Such cross-reactions would minimize the wastefulness of the selection. The cross-reactivity in thymic selection can be best shown in TCR-Tg mice. In this study, an example of positive selection of a single TCR by multiple different MHC molecules (cross-positive selection) is shown. The cross-positive selection in this case involves even MHC molecules of different classes. This unique system provided us with an opportunity to verify several models proposed to date to explain CD4⁺ vs CD8⁺ lineage commitment.

To account for the mechanism by which MHC class specificity of TCR determines the lineage fate, two models were initially proposed: the instructive model and the stochastic/selective model (3, 4). The instructive model proposes that coreceptor molecules that assist with TCR in MHC recognition transduce signals that determine the lineage. Namely, recognition of class I MHC by TCR and CD8 delivers the signal(s) to shut off the CD4 gene and to initiate the differentiation into CD8⁺ T cells, while TCR and CD4 coengagement by class II MHC sends signal(s) to silent CD8 gene and to induce CD4⁺ lineage differentiation. In contrast, the stochastic/selective model postulates two-step differentiation of DP thymocytes. In this model, TCR-signaled DP cells differentiate into either CD4⁺ or CD8⁺ lineage in a stochastic way, following which only the cells with matched combinations of TCR specificity for MHC class and coreceptor expression would be filtered out by receiving the survival signals to mature.

After intensive assessments of these two models, two related models have been currently proposed, i.e., the strength of signal model and the kinetic signaling model. The strength of signal model proposes that the strength or duration of the TCR signal dictates the fates of DP cells; stronger/longer signal would direct the precursor cells into CD4⁺ T cells, whereas weaker/shorter signal would drive them into CD8⁺ lineage (10, 11, 12, 13, 14, 15, 16, 17, 18). This model is supported by the fact that Lck, a src family tyrosine kinase that plays crucial roles in TCR-mediated signal transduction, is associated with CD4 at a much larger proportion than with CD8 molecule (19, 20). This fact may enable the MHC class II recognition by TCR and CD4 to trigger stronger signals than by TCR plus CD8. The kinetic signaling model proposes that the CD4⁺/CD8⁺ lineage choice would be influenced by the differential regulations in coreceptor gene expression. This is based on the observation that TCR-signaled DP cells terminate the CD8 gene expression transiently, which drives most of the positively selected cells into CD4⁺/CD8^low cells (21, 22, 23, 24, 25, 26). At this stage, sustained TCR signal would be sent into class II MHC-restricted T cells and they become CD4 single-positive (SP) cells. In contrast, TCR signal may be ceased in class I-restricted DP cells by the loss of CD8, which would lead to Runx3-mediated CD8 reactivation and CD4 silencing, and consequently to CD8⁺ lineage differentiation (27, 28).

Each of these models predicts different fates of the DP thymocytes expressing TCR that are responsive to both class I and class II MHC simultaneously in positive selection. The instructive model predicts that if TCR on DP thymocyte reacts with both classes of MHC, those cells would lose both of the coreceptor expressions and become CD4/CD8 double-negative T cells. From the stochastic/selective model, it is expected that both CD4⁺ and CD8⁺ subsets would mature in a mutually independent manner (without being affected with each other). In contrast, both the strength of signal model and the kinetic signaling model predict that those dual class-restricted DP cells would differentiate into CD4⁺, rather than CD8⁺ lineage.

In this study, we show that the DP cells expressing a single Tg TCR could develop into both CD4⁺ and CD8⁺ lineage when both classes of selecting MHCs are present in thymus. However, development of CD4⁺ lineage was always dominant over CD8 lineage. In addition, by the use of mice with various H-2 haplotypes and of ₂-microglobulin (₂m)-deficient mice, it was indicated that the selecting class II MHC was inhibitory to CD8⁺ lineage differentiation, whereas the selecting class I MHC facilitated the maturation of CD4⁺ lineage. These results did not perfectly match to the prediction from either model described above, suggesting that some modification(s) is required to be made to the models.

	Materials and Methods

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Mice

The TCR^QM11-Tg mouse was generated previously (29), and the progenies were typed by staining peripheral blood T cells using anti-Id Ab (29). B10.QBR (H-2^bq4), B10.S (H-2^s), and B10.D2 (H-2^d) mice were purchased from SLC; DBA/1 (H-2^q) and C57BL/6 (H-2^b) mice were obtained from Charles River Laboratories; and NOD/Shi (H-2^g7) and C3H/HeN (H-2^k) mice were from CLEA Japan. The H-2^b mice deficient for RAG-2 (30) or ₂m (31) were kindly provided by Dr. M. Shimamura (Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan). All mice used in this study were maintained in specific pathogen-free facility of Kitasato University School of Medicine. The experimental procedure was approved by the Animal Experimentation and Ethics Committee of the Kitasato University School of Medicine, and all animal experiments were conducted following the guidelines of the committee.

Reagents

Abs used in this study were as follows: FITC-labeled mAbs against CD4 (GK1.5), CD8 (53.6-7), CD69 (H1.2F3), and I-A^b (AF6-120.1); PE-labeled Abs to CD4, CD8, and CD154 (MR1); and PE-Cy5-labeled streptavidin were purchased from BD Pharmingen. Biotinylated anti-TCR^QM11 Id Ab and FITC-labeled polyclonal rabbit Ab reactive to mouse Ig (but not to rat Ig) were prepared in our laboratory. Anti-D^q/L^q Ab (30-5-7, mouse IgG2a (32)) and anti-CD3 Ab (145-2C11, hamster IgG) were prepared as ascitic form. PMA and the calcium ionophore ionomycin were purchased from Sigma-Aldrich.

Flow cytometry

Single cell suspension was prepared from spleens or thymi using frosted slide glasses (Matsunami Glass). Flow cytometric analyses were performed, as described previously (33). Briefly, one million cells were stained in and washed with ice-cold HBSS containing 0.5% BSA and 0.02% NaN₃. Secondary staining was performed in the same manner. Stained cells after washing were examined by flow cytometric analyses on FACSCalibur (BD Biosciences). Data were acquired and analyzed on CellQuest software.

Development and maintenance of T cell lines

The CD4⁺ and CD8⁺ T cell lines (designated as 4Rq11 and 8Rq11, respectively) were established from peripheral blood of H-2^q RAG-2^–/– TCR^QM11-Tg mouse by stimulating peripheral blood T cells with irradiated splenocytes of C3H/HeN mouse after depleting CD8⁺ or CD4⁺ cells, respectively, by Abs and rabbit complement. The cell lines were maintained by biweekly stimulation with C3H/HeN splenocytes, in DMEM supplemented with 10% FCS and 2.5% conditioned medium prepared from 48-h culture of Con A-stimulated LEW rat splenocytes.

Cell-mediated cytotoxicity assay

Cytolytic activity of T cell lines was performed using anti-CD3 mAb, 2C11 and FcR⁺ tumors, P815 (H-2^d, mastocytoma, Fas^–), and A20.2J (H-2^d, B lymphoma, Fas⁺) as targets. The target cells were labeled with 100 µCi of Na₂⁵¹CrO₄ for 1 h at 37°C in 10% CO₂ incubator. Washed target cells (2500 cells/well) were incubated with 40,000 effector T cells in round-bottom 96-well plate (Falcon) in the presence of gradually diluted ascitic form of 2C11 for 6 h in CO₂ incubator at 37°C. After incubation, supernatants were harvested, and the radioactivities in the supernatants were measured by gamma counter. Specific lysis was calculated as follows: percent specific release = 100 x (experimental release – spontaneous release)/(maximal release – spontaneous release). Spontaneous release or maximum release was determined from wells with target cells alone or wells in which target cells were lysed with 1% Nonidet P-40, respectively. Assays were performed at triplicates.

HT-2 assay

Forty thousand 4Rq11 or 8Rq11 were cultured with 8 x 10⁵ irradiated (20 Gy) splenocytes from C3H/HeN or C57BL/6 mouse in a microtiter plate for 14 h. The amount of the cytokines released in the supernatants was examined for the ability to support the growth of HT-2 cells, as described previously (33). Briefly, 1 x 10⁴ HT-2 cells were cultured in the presence of the serially diluted supernatants for 24 h, and [³H]thymidine incorporation for the last 8 h was measured.

Bone marrow chimera

The H-2^q mice of ₂m^–/– or ₂m^+/– were obtained by using DBA/1 mouse (H-2^q) and H-2^b ₂m^–/– mouse. The recipient mice were irradiated (8 Gy) using the x-ray irradiator MBR-1505R (Hitachi Medico) with a filter (Cu, 0.5 mm; Al, 2 mm). Fifteen million bone marrow cells from H-2^b RAG-2^–/– TCR^QM11-Tg mouse were injected i.v. 20 h after irradiation. The reconstituted mice were analyzed 2 mo later.

	Results

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Description of the TCR^QM11-Tg mouse

In a previous study, we created a Tg mouse bearing genomic TCR - and -chain genes from a CD8⁺, I-A^k-specific allogeneic CTL clone, QM11, which was established from a B10.QBR mouse (H-2^bq4: K^b, I-A^b, D^q/L^q) (29). Using highly specific anti-Id Ab, it was demonstrated that this clone (Id^high+ cells) had been positively selected by D^q/L^q molecule to differentiate into CD8⁺ cells. As shown in Fig. 1, Id^high+ cells matured into CD8⁺ subset (but not into CD4⁺ cells) in periphery of Tg mouse of H-2^bq4. In contrast, Id^high+ T cells were not observed either in CD8⁺ or CD4⁺ subset in B6 (H-2^b) background, which differs from B10.QBR (H-2^bq4) only at the H-2 D (L) region. The MHCs of H-2^b haplotype do not appear to have any effects on the selection of Id^high+ cells (29), which was further supported by the observation that the thymi from ₂m-deficient and -sufficient H-2^b Tg mice contained similar percentage and number of DP cells (data not shown). The Id^high+ cells are the ones that have been positively selected via TCR^QM11, and Id^low/– cells have been shown to be V-double expressers selected by virtue of other pairs of TCR (29). Interestingly, the number of CD8⁺ Id^high+ cells was larger in H-2^bq4 Tg mouse than in H-2^bxbq4 (B6 x B10.QBR)F₁ mouse (Fig. 1). This may indicate the gene dosage effect of D^q/L^q molecule on the efficiency of positive selection for CD8⁺ Id^high+ cells.

View larger version (52K): [in this window] [in a new window]   FIGURE 1. Positive selection of Idhigh+ cells by H-2Dq/Lq molecule. Peripheral blood leukocytes from TCRQM11-Tg or non-Tg mice of indicated H-2 haplotypes were stained with FITC-labeled anti-CD8 Ab, PE-labeled anti-CD4 Ab, and biotinylated anti-Id Ab, followed by PE-Cy5-labeled streptavidin. Stained cells were analyzed by three-color flow cytometry. Id expressions on CD8+ and CD4+ T cells are shown as histograms (right panels).

To investigate the cross-reactivity of this Tg TCR to other MHC molecules in thymic selection, we crossed this Tg mice to various strains of laboratory mice and examined the maturation of Id^high+ cells. As shown in Fig. 2A, in H-2^bxd background, Id^high+ cells differentiated into CD8⁺ subset. This population was not observed in ₂m-deficient H-2^bxd mouse (data not shown), nor in H-2^bxg7 mouse, which shares H-2K^d, but not H-2D^d (/L^d) molecule with H-2^bxd mouse, suggesting that D^d or L^d is the selecting element of H-2^d.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Cross-reaction of Tg thymocytes to class I MHC of H-2d and H-2s in thymic selection. A, PBL from TCRQM11-Tg mice of (C57BL/6 x B10.D2)F1 (H-2bxd) or (C57BL/6 x NOD)F1 (H-2bxg7) background were stained with Abs to CD4, CD8, or TCRQM11 Id. Id expressions on CD8+ or CD4+ cells are shown. Shaded histograms indicate negative staining. B, Single cell suspension of thymocytes from TCR-Tg (right panels) or non-Tg (left panels) deficient (lower panels) or sufficient (upper panels) for 2m was stained with Abs to CD4, CD8, or Id, and expression of CD4 and CD8 is shown. The average number of total thymocytes (±SD, n = 3–4) was also shown on each panel.

TCR^QM11 could cross-react with class II MHC in thymic positive selection to generate mature CD4⁺, Id^high+ cells

Among various strains, it was found that, on H-2^q background, Id^high+ cells differentiated into CD4⁺ subset (Fig. 3). On this background, Id^high+ cells were also found in CD8⁺ population, although the number and the proportion of mature CD8⁺ Id^high+ cells in thymus or spleen were always smaller than those of mature CD4⁺ Id^high+ cells (data not shown). The maturation of CD8⁺ Id^high+ cells was most likely due to positive selection by the originally determined selecting class I MHC, D^q/L^q. The maturation of CD4⁺ Id^high+ cells was presumed to be dependent on I-A^q, because this molecule is the only class II MHC in H-2^q mouse. To examine this possibility, the TCR-Tg mice were crossed with ₂m-deficient mice to obtain H-2^q, ₂m^–/– TCR-Tg mice, and the maturation of Id^high+ cells in these mice was investigated. As shown in Fig. 4A, CD8⁺ Id^high+ cells disappeared in ₂m^–/– TCR-Tg mouse, while CD4⁺ Id^high+ cells could still mature in the absence of ₂m. These results indicate that, in H-2^q TCR-Tg mouse, both class I and class II MHC mediate positive selection, resulting in maturation of CD8⁺ Id^high+ and CD4⁺ Id^high+ T cells. Interestingly, however, the generation of CD4⁺ Id^high+ cells in ₂m^–/– mice appeared to be impaired as compared with in ₂m^+/– background (Fig. 4B), suggesting that class I MHC could contribute to efficient maturation of CD4⁺ Id^high+ cells when selecting class II MHC molecules coexist.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Maturation of Idhigh+ cells in both CD8+ and CD4+ subsets in mouse of H-2q haplotype. Id expression on CD8+ or CD4+ cells in peripheral blood of TCRQM11-Tg mice of indicated haplotypes was analyzed. Shaded histograms indicate negative staining.

View larger version (36K): [in this window] [in a new window]   FIGURE 4. Maturation of CD4+, but not CD8+, Idhigh+ cells in H-2q Tg mice deficient for 2m. A, Single cell suspension of thymocytes from H-2q TCR-Tg mice of 2m+/– (upper panels) or 2m–/– (lower panels) was stained with Abs to CD4, CD8, or Id. The expression of CD8 and CD4 on total (left panels) or Idhigh+ (right panels) is shown. B, The numbers of CD4 SP and CD8 SP Idhigh+ thymocytes from H-2q Tg 2m+/– (top, n = 5) or 2m–/– (bottom, n = 6) mice are shown.

Although T cells expressing only Tg TCR were readily identified as Id^high+ cells and distinguished from Id^low+ or Id^– cells that matured independently of Tg TCR, we confirmed that cells expressing a single TCR could truly differentiate into both CD4⁺ and CD8⁺ cells in TCR^QM11-Tg mice of H-2^q background. The TCR-Tg mice were crossed with RAG-2-deficient mice, and the T cell differentiation was investigated in H-2^q RAG-2^–/– TCR-Tg mice. As shown in Fig. 5, both CD4 and CD8 SP subsets were observed as Id^high+ cells in the thymi and in periphery of these mice. Once again, the number and the proportion of CD4⁺ cells were larger than those of CD8⁺ cells. Despite that the CD4/CD8 ratio varied among individuals, CD4⁺ population was always dominant. This variation appeared independent of their sex or age (Fig. 5B, and data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Differentiation of T cells in RAG-2-deficient TCRQM11-Tg mice. A, Thymocytes from RAG-2-deficient Tg mice of indicated H-2 haplotypes were analyzed for the expression of CD4, CD8, and Id. There were no significant differences in total thymocyte number among these mice. Id expression of CD4 SP or CD8 SP thymocytes from H-2q mouse is shown as histograms. The shaded histograms show negative staining. B, PBL from H-2q were stained with Abs to CD4, CD8, or Id. The Id expression (left histogram) and CD4/CD8 expression on Id+ PBL (left bottom) are shown. The ratio of CD4+/CD8+ Id+ cells in PBL from H-2q RAG-2–/– Tg mice (5–9 wk old) is shown in the right graph.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Functional characteristics of CD4+ and CD8+ cells matured in H-2q, RAG-2–/–, TCRQM11-Tg mouse. A, CD8+ (8Rq11, circle) and CD4+ (4Rq11, triangle) T cell lines were established from a H-2q RAG-2-deficient Tg mouse, and their cytolytic activities were examined using FcR+ target cells and anti-CD3 Ab, 2C11 (diluted ascites). Cytotoxic activity on Fas-negative P815 (open symbols) and on Fas-positive A20.2J (closed symbols) was shown as percent specific 51Cr release. E:T ratio was 16. B, The lymphokine production by 8Rq11 (circle, dashed line) or 4Rq11 (triangle, solid line) was examined with IL-2/IL-4-dependent HT-2 cells. Shown were proliferative responses of HT-2 cells in the presence of diluted culture supernatants of the T cell lines stimulated with C3H (expressing I-Ak, for which TCRQM11 is specific) or B6 splenocytes. C, PBLs from H-2q, RAG-2–/–, TCRQM11-Tg, or wild-type mouse were cultured in the presence (PMA/IM) or absence (medium) of PMA (10 ng/ml) and ionomycin (1 µg/ml). After 8-h culture, the cells were harvested, and the expression of CD154 (upper panels) or CD69 (lower panels) on CD4+ and CD8+ T cells was examined.

Cross talk of selecting class I and class II MHC signals in positive selection of Id^high+ cells

The observations described above have shown that D^q/L^q and I-A^q are the selecting MHCs for TCR^QM11, and therefore that H-2^q mouse has both class I and class II, and H-2^bq4 mouse has only class I-selecting MHC molecules for TCR^QM11. Using these mice as well as the neutral H-2^b mice, we next evaluated the effect of the density of selecting ligands on the lineage commitment of Id^high+ cells. From the strength of signal model, it was predicted that the alteration of total ligand density would influence the ratio of CD4⁺/CD8⁺ Id^high+ cells. We thus expected that in H-2^bxq mice, which have half densities of selecting MHCs of both classes as compared with H-2^q mice, the differentiation of CD8⁺ Id^high+ cells might be promoted even in the presence of selecting class II MHC. However, in H-2^bxq mice, the differentiation efficiency of Id^high+ cells into both CD8⁺ and CD4⁺ subsets were similarly decreased (Figs. 7 and 8A). Interestingly, in H-2^bq4xq mice, as compared with H-2^bxq mice, the hemizygous increase of selecting class I ligand enhanced the generation of not only CD8⁺ Id^high+ cells, but also of CD4⁺ cells, indicating that the selecting class I ligand could contribute to positive selection of CD4⁺ T cells. This was consistent with the observation in Fig. 4, in which it was shown that in the absence of ₂m, the efficiency of positive selection of CD4⁺ Id^high+ T cells was decreased.

View larger version (53K): [in this window] [in a new window]   FIGURE 7. Id expression on CD4 SP and CD8 SP thymocytes matured in various H-2 circumstances. Thymocytes from Tg mice on indicated H-2 background were analyzed for the expression of CD4, CD8, and Id. The histograms show the Id expression after gating for CD4 SP or CD8 SP cells. The scales for y-axes (relative cell number) are same in all histograms. The shaded histograms indicate negative staining. The total thymocyte numbers were similar among the mice on these backgrounds at similar age.

View larger version (29K): [in this window] [in a new window]   FIGURE 8. Effect of the density and combination of selecting MHC ligands on the differentiation of Idhigh+ cells. A, The percentages of CD4+ Idhigh+ cells () and CD8+ Idhigh+ cells () in peripheral blood T cells in the mice of indicated H-2 haplotypes were analyzed. B, Shown are the absolute numbers of CD4 SP and CD8 SP Idhigh+ cells in the thymi from the mice of indicated H-2 haplotypes, which were from breeding of male and female H-2bq4xq TCR-Tg mice.

	Discussion

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In the present study, the detailed analyses of TCR^QM11-Tg mice provided us with several important implications for the issues on thymic selection. 1) A single TCR on DP thymocytes could interact with multiple MHC molecules, irrespective of their classes, at thymic selection. 2) When both classes of selecting MHCs are present simultaneously, DP cells could differentiate into both CD4⁺ and CD8⁺ lineage. 3) In the positive selection of dual class-responsive TCR, the effect of two classes of selecting MHCs was asymmetric. Namely, the selecting MHC class I was critical for generating CD8⁺ cells, but also was supportive for CD4⁺ differentiation. In contrast, selecting class II MHC was mandatory for CD4⁺ lineage differentiation, but was inhibitory for CD8⁺ differentiation.

The interaction of a single TCR with multiple MHC upon thymic selection

The analyses of TCR^QM11-Tg mice have revealed an example of cross-selection of a single TCR by multiple MHC products. In this Tg system, Id^high+ cells were positively selected by D^q/L^q, D^d/L^d, and surprisingly I-A^q. Also, H-2^s class I (this study) and I-A^k (29) have been shown to function as negative selecting elements. Thus, there does not appear to be a clear border between class I and class II MHC products in terms of reactivity with TCR^QM11. At this point, it is not clear whether or not such borderless cross-reactivity is unique to TCR^QM11, as the Tg TCR was derived from a CD8⁺ CTL clone specific for allo-class II MHC (29). However, we tend to speculate that such cross-reactivity might well be the case in general. Accumulated information on crystal structure of TCR/peptide/MHC complexes suggested that there are no conserved contact sites found on these molecules, despite the roughly similar docking orientation. In addition, basic structures of both classes of MHC proteins are very similar, except for the bound peptides being more exposed from the groove in class II MHC than in class I MHC (for reviews, see Refs.36, 37, 38).

Thus, it is quite likely that class I and class II MHC molecules actually look alike to TCR. In fact, there are no critical (although some) differences in V/V usage between class I MHC-restricted and class II MHC-restricted TCRs (39, 40). If this is the case, the coreceptor may well primarily determine the class restriction. Because mature T cells express exclusively one species of coreceptor molecules, such borderless cross-reactions ought not to be frequently observed. At the DP stage of thymocytes, however, T cell clones may well find multiple MHC products as their selecting elements in a borderless manner, owing to their hermaphroditic expression of both species of coreceptors. Very recently, Huseby et al. (41) reported that many clones derived from the mice in which negative selection is limited were very cross-reactive with allo-MHCs, and some of them even showed the reactivities against both classes of MHCs. These results indicate that preselected DP cells contain a significant portion of cells with dual class-responsive TCR. Thus, the process of positive selection of usable clones in the thymus may not be as wasteful as it appears.

Positive selection in the presence of both classes of selecting MHC: asymmetric effects of two classes of selecting MHC on the positive selection of dual class-responsive T cells

In the TCR^QM11-Tg mouse of H-2^q background, in which both class I- and class II-selecting MHC molecules were available, maturation of Id^high+ cells was observed in both CD4⁺ and CD8⁺ subsets. The development of CD4⁺ lineage was observed in H-2^q, ₂m^–/– background, in which I-A^q may be the only MHC molecule, showing that the maturation of CD4⁺ Id^high+ cells was indeed I-A^q dependent. This was further confirmed by the analyses of thymocytes from bone marrow chimeras created using H-2^q ₂m^–/– mice as recipients of bone marrows from H-2^b RAG2^–/– TCR-Tg mice. Again, only CD4 SP, but not CD8 SP thymocytes, were found in these mice (our unpublished observation).

However, I-A^q molecule itself may not be a good selector of TCR^QM11-expressing cells. In the H-2^q ₂m^–/– mice, differentiation of CD4⁺ Id^high+ cells was much less efficient when compared with that in class I-sufficient mice. This suggests that the selecting class I MHC could enhance the efficiency of class II-dependent differentiation into CD4⁺ cells (Fig. 4). A similar observation was made in the comparison of peripheral Id^high+ cells between H-2^bxq (heterozygous for both I-A^q and D^q/L^q) and H-2^bq4xq (heterozygous I-A^q and homozygous D^q/L^q) mice (Figs. 7 and 8). On the contrary, such inefficient selector, I-A^q molecule was apparently inhibitory to the generation of mature CD8⁺ Id^high+ cells, as shown in Figs. 7 and 8.

These asymmetric properties of class I and class II MHC in thymic positive selection have become evident for the first time in the system of dual class-restricted TCR-Tg mouse, albeit the precise mechanisms have remained unknown. However, these mechanisms, in addition to other mechanisms (25, 39), could underlie the higher proportion of CD4⁺ T cells than CD8⁺ T cells in normal animals of normal conditions. It is also possible that these mechanisms may contribute to explain the influence of class II MHC on the differentiation of some pathogenic CD8⁺ T cells (42, 43), as recently suggested by Logunova et al. (44).

Although cross-reactivities of other Tg TCRs upon thymic selection have not been investigated so intensively, TCRs responsive to both classes of MHC might exist also in normal animal more frequently than generally appreciated. Some T cell clones were in fact demonstrated to show specificity to both classes of MHC molecules (41, 42, 43, 44, 45, 46, 47). When positively selected, those cells with TCRs of dual class specificity should tend to differentiate into CD4⁺ T cells rather than CD8⁺ T cells, as observed in the present study. It is expected, however, that some of those cells should emerge in CD8⁺ lineage more efficiently when the recognition of class II MHC is absent, or is hindered by the absence of CD4 molecules. In fact, Shimizu and Takeda (48) reported that CD8⁺ T cells from MHC class II-deficient mouse frequently respond to syngeneic as well as allogeneic class II MHC. Interestingly, the authors also reported that they could not detect reciprocal reactivity, i.e., the reactivity of CD4⁺ T cells from ₂m^–/– mouse to class I MHC. These findings argue that DP cells indeed include a significant portion of cells bearing TCR responsive to both classes of MHC, and that those cells could be much more apparent in class II MHC^–/– than in ₂m^–/– mice.

With regard to the CD4-deficient mice, Tyznik et al. (49) and Pearce et al. (50) demonstrated that class II-restricted CD8⁺ T cells were readily detectable after primary bacterial or viral infections in CD4^–/– mice. In these studies, the generation of class II-restricted CD8⁺ T cells was interpreted as a result of misdirection of class II MHC-restricted thymocytes (into CD8⁺ lineage) due to decreased affinity of TCR/MHC (+ peptide) interaction by the absence of CD4. This was based on the previous interesting report by Matechak et al. (11) showing that in some Tg mice bearing class II-restricted TCR, T cells differentiate into CD8⁺ T cells in CD4-deficient background. From these observations, the authors have proposed that the lineage fate would be determined by the strength of the TCR signal. Our present study has raised another possibility that those Tg TCR could possibly be restricted with both class I and class II MHCs. In fact, it was shown that, in class II MHC-restricted AND TCR-Tg mouse, which generates a large number of CD8⁺ T cells in the absence of CD4, a small population of CD8⁺ T cells existed even in the presence of CD4 and in the absence of endogenous TCR -chain (11). This situation is reminiscent of H-2^q TCR^QM11-Tg RAG2^–/– mouse in which both CD4⁺ and CD8⁺ subsets could mature into periphery with the predominant maturation of CD4⁺ subset, and the removal of the selecting class II ligand results in optimal CD8 differentiation.

Implications for CD4⁺ vs CD8⁺ lineage commitment

Although the exact frequency of dual class-restricted T cells awaits further investigations, the TCR-Tg system, in which DP thymocytes expressing the Tg TCR can recognize both classes of MHC, provided us with an opportunity to evaluate several models of the mechanism of CD4⁺/CD8⁺ lineage choice. Importantly, this could be done by simple genetic analyses of Tg mice without further artificial techniques.

At present, our results appear most consistent with the notion that CD4⁺ vs CD8⁺ lineage choice was determined by the duration of TCR signals into DP thymocytes in such a way as proposed in the kinetic signaling model (4, 24, 25), and supported by numbers of studies (12, 15, 18, 28, 51, 52). Brugnera et al. (24) showed that TCR signaling in DP thymocytes responding to intrathymic MHC ligands decreases the CD8 gene expression before the lineage commitment, regardless of the ligand MHC classes. When DP thymocytes react with both classes of intrathymic MHC molecules, they would once lose their CD8 expression. In many of them, their differentiation into CD8⁺ lineage would be inhibited by the persistent engagement of class II MHC by TCR and CD4 on them. This may be the reason that selecting class II MHC was inhibitory to class I-dependent CD8⁺ differentiation of dual class-restricted T cells. Regarding the ability of selecting class I ligand to increase the efficiency of CD4⁺ differentiation, it could be because class I ligand-induced signal helped to augment the number of thymocytes undergoing positive selection (that are subject to the lineage determination), thereby increasing the number of both lineages of mature T cells. This hypothesis was supported by the observation that the proportion of CD69⁺ cells among DP thymocytes was larger in H-2^bq4xq mice as compared in H-2^bxq mice when examined on RAG2-deficient background (our unpublished data).

One concern would be that the kinetic signaling model, in theory, does not allow the differentiation of DP thymocytes expressing the dual class-restricted TCRs into CD8⁺ lineage cells, which were constantly observed in periphery of H-2^q TCR^QM11-Tg mice as a significant population. We are currently thinking two possibilities that are not mutually exclusive. First, it is possible that not all DP cells may be able to be selected to mature even when they express TCRs that could potentiate the positive selection, because the positive selection may be somewhat an inefficient process mediated by low avidity interactions between TCRs and MHC ligands (53). It could thus be possible that even in the presence of both classes of selecting MHCs, TCRs (and CD4) on some DP cells might not be engaged by class II ligands. Some of such DP cells would be induced to apoptosis before maturation, but some of them could be rescued by the class I MHC-mediated positive selection to differentiate into CD8⁺ lineage. The second possibility is that among thymic stroma cells that are capable of mediating positive selection, there might be cells with very low or no expression of class II MHC molecules, or cells with low/no expression of CD83, which was shown to be critical in CD4 (but not CD8) lineage differentiation (54). Those cells, if they exist, would function as specific CD8-selector cells, although the presence of those cells may not be apparent in a normal condition. Thus, it is possible that the DP thymocytes expressing dual class-restricted TCR may differentiate into CD8⁺ lineage when they encounter such stroma cells for thymic positive selection.

Further studies are required to investigate these possibilities, and some of them are currently being addressed.

	Acknowledgments

 
We cordially thank Drs. T. Saito (RIKEN Research Center for Allergy and Immunology) and M. Takahashi and Y. Takagaki (Kitasato University) for the encouraging support. We also thank Dr. M. Shimamura for providing RAG-2^–/– and ₂m^–/– mice.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
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.

¹ This study was supported by Parents’ Association Grant of Kitasato University School of Medicine, Kitasato University Research Grant for Young Researchers, and the Hi-tech Research Center Grant from the Ministry of Education, Science, and Culture of Japan.

² Address correspondence and reprint requests to Dr. Nobukata Shinohara, Department of Immunology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. E-mail address: nobu{at}med.kitasato-u.ac.jp

³ Abbreviations used in this paper: Tg, transgenic; ₂m, ₂-microglobulin; DP, double positive; SP, single positive.

Received for publication August 25, 2005. Accepted for publication November 18, 2005.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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