HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Henderson, S. C. Articles by Bamezai, A. Search for Related Content PubMed PubMed Citation Articles by Henderson, S. C. Articles by Bamezai, A.

CD4⁺ T Cells Mature in the Absence of MHC Class I and Class II Expression in Ly-6A.2 Transgenic Mice¹

S. Christine Henderson^*, Alla Berezovskaya^2,, Andrea English^*, Deborah Palliser^3,, Kenneth L. Rock and Anil Bamezai^4,*

^* Department of Cellular Biology, University of Georgia, Athens, GA 30602; Department of Pathology, Dana-Farber Cancer Institute, Boston, MA 02115; and Department of Pathology, University of Massachusetts Medical Center, Worcester, MA 01655

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The TCRs expressed on T lymphocytes recognize foreign peptides bound to MHC molecules. This reactivity is the basis of specific immune response to the foreign Ag. How such specificities are generated in the thymus is still being debated. Signals generated through TCR upon interaction with self MHC-peptide complexes are critical for maturation of the CD4⁺ helper and CD8⁺ cytotoxic subsets. We have observed maturation of CD4⁺ but not CD8⁺ T cells in Ly-6A.2 transgenic MHC null mice. Since there can be no interactions with MHC molecules in these mice, these CD4⁺ cells must express the T cell repertoire that exists before positive and negative selection. Interestingly, despite an absence of selection by MHC molecules, the CD4⁺ cells that mature recognize MHC molecules at a frequency as high as in CD4⁺ cells in normal mice. These results demonstrate that: 1) the germline sequences encoding TCRs are biased toward reactivity to MHC molecules; and 2) CD4⁺ cells as opposed to CD8⁺ cells have distinct lineage commitment signals. These results also suggest that signals originating from Ly-6 can promote or substitute for signals generated from TCR that are required for positive selection. Moreover, this animal model offers a system to study T cell development in the thymus that can provide insights into mechanisms of lineage commitment in developing T cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The thymus is a unique organ dedicated to the development and maturation of T cells. This is the generative lymphoid organ in which central tolerance is established. Selection of T cells in the thymus is controlled by three major factors: 1) the initial T cell repertoire, which is generated by the rearrangements of V-D-J and V-J gene segments; 2) the nature and concentration of self peptides present in the thymus; and 3) the functional consequence of stimulating developing T cells, i.e., growth stimulation (positive selection) or cell death (negative selection). A body of data indicates the role of self peptides in thymic selection (1, 2). The nature of the T cell repertoire that exists before positive and negative selection is not known. An understanding of this initial T cell repertoire will provide clues to our understanding of the underlying mechanisms of thymic selection.

The immature T cells that differentiate into CD4⁺ helper cells express MHC class II-restricted receptors, whereas those that differentiate into CD8⁺ cytotoxic T cells express MHC class I-restricted T cells. It is generally thought that recognition of the MHC class I molecule by both the TCR and CD8 molecules will provide appropriate signals giving rise to CD8⁺ mature T cells. Likewise, recognition of the class II molecule by TCRs in conjunction with the CD4 molecule will give rise to CD4 helper T cells. The signals that arise from the coreceptor alone are not sufficient for lineage commitment, since cells do not mature in TCR- mutant mice (3). What remains unclear is how the signal delivered by the TCR alone or in conjuction with each of the coreceptors can give rise to two distinct T cell subsets. It also remains unclear at what stage of development the thymocytes commit to CD8 or CD4 lineage and what, if any, additional signals are required for lineage commitment to CD4 and CD8 T cells.

A striking property of the mature T cell repertoire is the high frequency of reactivity to allogeneic MHC molecules (4, 5). Approximately 1 to 10% of mature T cells are alloreactive, and this repertoire overlaps with the Ag-specific T cell repertoire. It has been hypothesized that alloreactive T cells recognize MHC molecules independently of peptides (6). More recently, it was proposed that much of this reactivity is peptide dependent but may not be peptide specific (7). In addition, there are some examples of alloreactive T cells that corecognize specific peptides complexed with allo-MHC (8). It is unclear whether alloreactivity is a consequence of selecting TCRs that recognize self MHC and therefore may be more likely to cross-react with allogenic MHC molecule or if the initial TCR repertoire has an inherent reactivity to the MHC molecule. It is hypothesized that the initial TCR is biased in its reactivity to MHC molecules (9). Some recent studies in which a c-ovalbumin peptide covalently linked to I-A^b was expressed in transgenic mice observed a high frequency of alloreactive T cells (10). More recently, an in vitro system was developed in which maturation of CD4⁺ cells was induced in fetal thymic organ culture (FTOC)⁵ with anti-TCR + anti-CD4 Abs (11). Furthermore, T-T hybridomas were generated from the CD4⁺ thymocytes that mature in FTOC from MHC class I- and II-deficient mice in the presence of anti-TCR + anti-CD4 Abs. Similar frequencies of MHC reactivity were observed in the preselected repertoire and in the mature T cell repertoire in the thymus (11).

We were able to examine the initial T cell repertoire directly based on a chance observation in a Ly-6A.2 transgenic mouse. We have previously generated transgenic mice with the Ly-6A.2 gene under the control of the human CD2 enhancer. The Ly-6A.2 transgene is highly expressed on all T cells in the thymus. This dysregulated expression of Ly-6A.2 causes a substantial but incomplete block in T cell development, which occurs at the stage when Ly-6A.2 expression is normally turned off (12). In the present study, we crossed the Ly-6A.2 transgene with mice that lack MHC class I and class II molecules. In control (nontransgenic) MHC-deficient mice, mature CD4 and CD8 T cells failed to develop as expected, due to an absence of MHC-dependent positive selection (13, 14). Surprisingly, we found that only CD4 T cells, but not CD8 T cells, mature in the absence of MHC molecules in the Ly-6A.2 transgenic mice. The CD4⁺CD8^- T cells that develop in the absence of MHC in Tg⁺MHC^- mice are similar to the CD4⁺ cells that mature in normal mice and represent the initial T cell repertoire that exists before positive and negative selection. In this report, we describe the characteristics of the CD4⁺ cells that mature in the absence of MHC in Ly-6A.2 transgenic mice, and we also determine the reactivity of these cells to MHC molecules to provide direct evidence of MHC reactivity of unselected T cells in an adult mouse. More importantly, these studies indicate that overexpression of Ly-6A.2 can initiate signaling into developing T cells that normally use their Ag-specific ß TCR.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transgenic mice

The Ly-6A.2 transgenic and MHC class I x II-deficient mice that were used in this study have been previously described (13). The mice expressing the Ly-6A.2 transgene were backcrossed to MHC class I^- x II^- mice for six to eight generations.

Flow cytometry

Cells were stained for immunofluorescence as described previously (15). One x 10⁶ thymocytes, Tris-NH₄Cl-treated spleen cells or lymphocytes purified from peripheral blood were incubated with various Abs followed by appropriate fluorochrome-conjugated second-step reagents. Cells (5 or 10 x 10³) were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Reagents used for this analysis were: phycoerythrin-conjugated anti-CD4 (YTS 191.1.2, Life Technologies), FITC-conjugated anti-CD8 (53-6.7, Life Technologies), FITC-anti-heat-stable Ag (HSA; PharMingen, San Diego, CA), biotin anti-Vß, biotin-anti-CD44, biotin-anti-CD40 ligand, biotin-anti-Ly-6A/E, biotin-anti-TCR-ß (PharMingen), anti-Ly-6A.2 (3E7) (15), anti-Ly-6A.2 (3A7) (16), streptavidin-Red 613 and streptavidin-Red 670 (Life Technologies, Arlington Heights, IL), and goat anti-rat IgG-FITC (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Anti-IA^d/b/q (M5/114) and anti-class I (M1/42) were obtained from American Type Culture Collection, Manassas, VA.

T-T hybridomas

T cells from spleen were cultured with anti-CD3 Abs, and after 2 days of incubation the blasts were separated by Ficoll-Hypaque separation. The CD4⁺ blast cells were obtained by panning on anti-mouse Ig-coated plates after incubating the cells with anti-CD4 (GK1.5). Purification of cells by panning usually gave 80 to 90% pure cell population in our hands. The CD4⁺ cells were fused to BW5147 ß-negative (17), and hypoxanthine-aminopterin-thymidine (HAT)-resistant cells were selected per standard protocols. Plating efficiency was >80%.

Cell culture

Microcultures were set up as described before (18) in 96-well flat-bottom plates (Corning Glass, Corning, New York) in a final volume of 200 µl of culture medium consisting of RPMI 1640 supplemented with 20 mM HEPES, 2 mM L-glutamine, 1 mM nonessential amino acids (Irvine Scientific, Irvine, CA), 10% heat-inactivated FCS (Sigma Chemicals, St. Louis, MO), 0.25 µg/ml of fungibact (Life Technologies), and 5 x 10^-5 M 2-ME; or in some experiments, cultures were set up in 24-well plates (Corning Glass) in a final volume of 1 ml in the presence or absence of PMA and calcium ionophore (Sigma Chemicals). The precise culture conditions are given in the applicable figure legend.

Reactivity to MHC was examined by culturing 5 x 10⁵ or 1 x 10⁵ hybrid cells with 5 x 10⁵ gamma-irradiated splenic stimulator cells in a 200-µl culture medium. After 24 h, 100 µl from these cultures was harvested and evaluated for IL-2 content by incubating it with the IL-2-dependent cell line HT-2.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Appearance of CD4⁺CD8^- T cells in the thymus of Ly-6A.2Tg⁺MHC^- mice

Ly-6A.2Tg⁺ mice were bred with MHC class I^- and class II^- double deficient mice (in which the Aß^b gene and ß₂-microglobulin were disrupted by homologous recombination), and their transgenic progeny were backcrossed with MHC I^- x II^- mutant mice for several generations. The progeny obtained from these breedings that lacked the expression of MHC class I and II molecules, with or without expression of the Ly-6A.2 transgene, were used for analysis. As shown in Table I, the total number of cells recovered from the thymi of Tg⁺MHC^- mice was similar to the number of cells observed in Tg⁺MHC⁺ mice and is markedly reduced compared with Tg^-MHC⁺ mice. The Tg⁺MHC^- animals also have a reduction in the development of T cells beyond the CD4^-CD8^- stage as compared with Tg⁺MHC⁺ mice. These observations indicate that interactions of TCRs with MHC molecules at the CD4⁺CD8⁺ cell stage does not contribute to the block in T cell development in Ly-6A.2 transgenic mice.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Analysis of T cell subsets in the thymus of Ly-6A.2 transgenic MHC- mice. Thymocytes from 4- to 8-wk-old normal (Ly-6A.2Tg-MHC+), Ly-6A.2Tg-MHC-, Ly-6A.2Tg+MHC+, and Ly-6A.2Tg+MHC- mice were examined for expression of CD4, CD8, and TCR-ß as described in Materials and Methods. A, Shows expression of CD4 and CD8 on different subsets that appear in the thymi of these mice. The number above box R1 indicates the percentage of mature CD4+CD8- cells. B, CD4+CD8- thymocytes (R1 box in upper four panels) were analyzed for the expression of TCR-ß (lower four panels). The y-axis indicates that TCR-ß expression is shown only from a CD4+CD8- subset.

In the normal thymus, mature T cells up-regulate their expression of TCR after being positively selected. Therefore, to determine whether the CD4⁺CD8^- thymocytes in Ly-6A.2 transgenic, MHC^- mice are phenotypically mature, we examined their expression of TCR. Thymocytes were stained with fluorochrome-conjugated mAbs directed against CD4, CD8, and TCR molecules in three-color immunofluorescence. As shown in Figure 1B, the CD4⁺ thymocytes that appear in Tg⁺MHC^- thymi have up-regulated the surface expression of TCR. Furthermore, this increase in the expression of TCR is similar to that seen in CD4⁺ thymocytes from normal mice. Very few cells showing TCR^{intermediate/high} were observed in Tg^-MHC^- mice, which is consistent with other published reports (Fig. 1A and 13 . Our results indicate that TCR expression in CD4⁺CD8^- cells from normal mice was similar to that in cells that mature in Ly-6A.2 transgenic mice in the absence of MHC.

Down-regulation of HSA on CD4⁺CD8^-TCR^high thymocytes in Tg⁺MHC^- mice

Another molecule expressed on the cell surface of thymocytes in which down-regulation correlates with cells undergoing positive selection is the HSA. This molecule is highly expressed on CD4⁺CD8⁺TCR^{low/intermediate} thymocytes, but its expression is low on the majority of TCR^high thymocytes that are also either CD4⁺CD8^- or CD4^-CD8⁺ (21, 22). Therefore, we examined the expression of HSA in normal (Tg^-MHC⁺), Tg^-MHC^-, and Tg⁺MHC^- mice by staining with anti-TCR-ß and anti-HSA Abs. Figure 2 shows that the expression of HSA is down-regulated in TCR^high cells in the Tg⁺MHC^- thymus. This reduction in HSA expression was similar to that observed in TCR^high cells in the normal (MHC⁺) thymus (Fig. 2). Moreover, the high level of HSA expression on CD4⁺CD8⁺ thymocytes from Tg⁺MHC^-, Tg^-MHC^- animals is comparable. These results suggest that the Ly-6A.2 overexpressed on CD4⁺CD8⁺ thymocytes delivers a signal(s) that is MHC+ peptide independent but results in the selection of CD4⁺CD8^-TCR^highHSA^low mature thymocytes.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Analysis of TCR-ß and HSA expression on thymocytes. T cells from the thymus of 4- to 8-wk-old normal (Ly-6A.2Tg-MHC+), Ly-6A.2Tg-MHC-, and Ly-6A.2Tg+MHC- mice were analyzed for the expression of TCR-ß and HSA.

We next examined whether the positively selected CD4⁺CD8^- cells are exported from the thymus and populate the peripheral lymphoid organs. We analyzed the spleen and lymph nodes of 4- to 8-wk-old mice for the expression of CD4 and CD8 molecules. Figure 3A shows that CD4⁺CD8^- T cells account for 50.2% (range, 35–60% in different experiments) of cells in lymph nodes of Tg⁺MHC^- mice and for 49.6% (range, 50–70%) of lymph node cells in normal MHC⁺ mice. In contrast, this subpopulation made up only 8.4% (range, 4–8%) of lymph node cells in mice lacking MHC class I and II molecules. Moreover, the level of expression of CD4 on lymph node T cells from Tg⁺MHC^- and control (Tg^-MHC⁺) mice was comparable but higher than that observed in Tg^-MHC^- mice. The level of expression of the TCR on CD4⁺CD8^- cells from Tg⁺MHC^- lymph node and spleen was also similar to that on CD4⁺CD8^- cells in normal mice (data not shown). These results indicate that the T cells that mature in Tg⁺MHC^- mice migrate to the periphery and accumulate in lymph nodes (Fig. 3A) as well as in the spleen (data not shown). In contrast, very few CD4^-CD8⁺ cells were detected in the spleen and lymph nodes of Tg⁺MHC^- and Tg^-MHC^- mice, which is consistent with the observation that there are few of these cells in the thymi of mice lacking MHC molecules. These observations reinforce our conclusion that overexpression of Ly-6A.2 results in the maturation of CD4⁺ and not CD8⁺ cells in the absence of MHC molecules.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Analysis of T cell subsets in the lymph node of Ly-6A.2Tg+MHC- mice. Cells from the lymph nodes of 4- to 8-wk-old normal (Ly-6A.2Tg-MHC+), Ly-6A.2Tg-MHC-, and Ly-6A.2Tg+MHC- mice were analyzed for the expression of CD4, CD8, and CD44 as described in Materials and Methods. A, Shows the expression of CD4 and CD8 on lymph node cells. The numbers above each box represent the percentage of mature CD4+CD8- cells that appear in the lymph node. B, Expression of CD44 on CD4+CD8- (boxed in A) lymph node cells. The number above the bar represents percentage of cells that express higher levels of CD44.

It has been reported previously that a majority of mature naive, CD4⁺CD8^- lymph node T cells from normal mice express low levels of the CD44 molecule (14). Therefore, we examined the expression of CD44 molecules on CD4⁺ cells in lymph nodes obtained from Tg⁺MHC^- mice. Figure 3B shows that in these mice a majority of CD4⁺ lymph node cells were CD44^low. Their expression of CD44 molecules is similar to that on CD4⁺ T cells from lymph nodes of normal mice. In contrast, the small number of CD4⁺CD8^- cells in class I and II double-deficient mice express high levels of CD44 (Fig. 3), as reported earlier (23). These results indicate that CD4⁺CD8^- cells in Tg⁺MHC^- mice have a mature phenotype.

The CD4⁺CD8^-TCR^high cells from Ly-6A.2 Tg⁺MHC^- mice up-regulate the CD40 ligand upon stimulation

CD4⁺CD8^- T cells that are selected by MHC class II molecules help B cell function in normal mice. The helper activity is mediated in part by the interaction of the CD40 molecule (expressed on B cells) with the CD40 ligand (expressed on activated CD4⁺CD8^- T cells) (24). Since the Ly-6A.2 transgene supports positive selection of CD4⁺CD8^- cells, we next sought to investigate whether these lymphocytes cells have the ability to express the ligand for CD40. The CD40 ligand is expressed preferentially on CD4⁺CD8^- T cells on stimulation with PMA and calcium ionophore (23). Lymphoid cells from the spleen (Fig. 4) and lymph nodes (data not shown) of Tg⁺MHC^- mice were exposed to the combination of PMA and calcium ionophore for 4 to 5 h and analyzed for the expression of CD4, CD8, and CD40 ligand. Figure 4 shows that a majority of CD4⁺CD8^- (Fig. 4B) and not CD4^-CD8⁺ (Fig. 4A) splenic cells from Ly-6A.2 transgenic MHC^- mice expressed the CD40 ligand. Similar expression was also observed on CD4⁺CD8^- cells from the spleen (Fig. 4B) and lymph nodes (data not shown) of normal mice. In contrast, the CD40 ligand was not up-regulated on the small number of CD4⁺CD8^- cells that are observed in Tg^-MHC^- lymph nodes. These results indicate that the CD4⁺CD8^- cells that selectively mature in the Ly-6A.2 transgenic and MHC^- thymus respond to stimulation in a manner similar to normal CD4⁺CD8^- Th cells and that they are functionally mature.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Expression of CD40 ligand on spleen cells from Ly-6A.2Tg+MHC- mice. Tris-NH4Cl-treated splenic cells from 4- to 8-wk-old normal (Ly-6A.2Tg-MHC+), Ly-6A.2Tg-MHC-, and Ly-6A.2Tg+MHC- mice were cultured with a combination of PMA (20 ng/ml) and calcium ionophore (0.25 µg/ml) for 4 to 5 h, then analyzed for the expression of CD4, CD8, and CD40 ligand (gp39) molecules as described in Materials and Methods. The CD4-CD8+ (A) and CD4+CD8- (B) cells were gated and examined for the expression of CD40 ligand.

Another property of mature T cells selected in the normal thymus is their responsiveness to stimulation through the TCR/CD3 complex by proliferation. In contrast, immature CD4⁺CD8⁺ thymocytes do not proliferate under similar conditions (25). To determine whether the Tg⁺ CD4⁺CD8^- thymocytes that have matured in the absence of MHC molecules are immunoresponsive, we examined whether they responded to stimulation with anti-CD3 Abs. As shown in Figure 5, thymocytes from Tg⁺MHC^- mice proliferated under these culture conditions. Similar results were obtained when Con A was used as mitogen (data not shown). The magnitude of these responses was even greater than that observed with Tg^-MHC⁺ thymocytes (data not shown). In contrast, proliferation was not observed in cultures with Tg^-MHC^- thymocytes (Fig. 5). These results further indicate that T cells that mature in Ly-6A.2 transgenic mice in the absence of MHC molecules are functionally competent.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Responsiveness of thymocytes from Ly-6A.2Tg+MHC- mice to anti-CD3 stimulation. Microcultures were prepared with 5 x 105 thymocytes from 4- to 8-wk-old Ly-6A.2Tg+MHC+, Ly-6A.2Tg-MHC- (closed squares), Ly-6A.2+MHC- (closed circles) in the presence or absence of anti-CD3 Ab supernatant at dilutions indicated. After 2 days, the proliferation was quantitated by measuring the incorporation of [3H]thymidine. The data displayed are a representative experiment; error bars show intra-assay variation.

A body of data indicates that negative and positive selection in the thymus occurs at the CD4⁺CD8⁺ cell stage, and therefore the Vß repertoire of most CD4⁺CD8⁺ cells is an unselected one. To examine whether the Vß repertoire is unselected, we compared the Vß repertoires of CD4⁺ that mature in Ly-6A.2Tg⁺MHC^- mice with the double-positive (DP) thymocytes from the same mouse. Figure 6 shows that these two repertoires are similar. Moreover, this Vß repertoire is also similar to one observed in DP thymocytes from the Tg^-MHC^- mice. This later observation supports the idea that the CD4⁺ cells that mature in Ly-6Tg⁺ mice in the absence of MHC is unaltered. Analysis of the Vß (Vß3, -5, -8, -9, -10, and -11) repertoire in the CD4⁺ and CD4⁺CD8⁺ subsets in the C57BL/6 parent mice indicated no differences except positive selection of Vß8 CD4⁺ T cells (data not shown). The absence of clonal deletion may be due to the lack of I-E expression in C57BL/6, which is known to present MTV-8, -9, and -17, resulting in efficient deletion of specific Vß-expressing T cells (26, 27).

View larger version (36K): [in this window] [in a new window]   FIGURE 6. ß T cell repertoire in Ly-6A.2Tg+MHC- mice. Thymocytes from Tg+MHC- and Tg-MHC- mice were stained with anti-CD4--phycoerythrin, anti-CD8--FITC, and anti-Vß (Vß3, -5, -8, -9, -10b, and -11)-biotin (PharMingen) followed by streptavidin-Red 613 in a three-color immunofluorescence. Vß expression on gated CD4+CD8+ (DP) and CD4+ single positive (SP) cells from Ly-6Tg+MHC- (three experiments) and on DP cells from Tg-MHC- (two experiments) mice is represented.

CD4⁺ T cells from Tg⁺MHC^- mice show high reactivity to many MHC molecules

To determine whether the preselected T cells are predisposed to recognize MHC molecules, we first tested the reactivity of these cells to irradiated spleen cells derived from H-2^b, H-2^k mice. Lymph node cells from Tg⁺MHC^- mice show high levels of alloreactivity (response to APC from CBA mice) as well as reactivity to self MHC (I-A^b-expressing APC, the same as in the MHC mutant mice) (Fig. 7). These responses are blocked by the M5/114 (anti-I-A^b, -I-A^d, -I-E^d, -I-E^k) Ab, and no response is elicited by APC from mice that lack MHC molecules (Fig. 7, right panel) nor is this reactivity blocked by irrelevant isotype matched Ab (data not shown). Reactivity of the CD4⁺ cells that mature independently of MHC was also observed with APC derived from mice of the H-2^d, H-2^s, and H-2^q haplotype, albeit to varying degrees (data not shown). These data demonstrate that lymph node cells from Ly-6Tg⁺MHC^- mice respond to MHC molecules. Similar results were also obtained with T cells from the thymi of these mice (data not shown). These results suggest that the preselected repertoire is evolutionarily biased to recognize MHC molecules.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. Reactivity of CD4+ T cells from Tg+MHC- mice to MHC molecules. Microcultures with 5 x 105 lymph node cells from C57BL/6 (H-2b) or CBA/J (H-2k), or Ly-6Tg- MHC- or Ly-6Tg+ MHC- mice were set up with 2.5 x 106 irradiated spleen cells with (white) or without (dark) anti-MHC class II Ab (M5/114). Cells were pulsed with [3H]thymidine and harvested after 5 days of culture. The data are expressed as arithmetic mean cpm of [3H]thymidine incorporated.

Figure 7 indicates that CD4⁺ cells that mature in the absence of MHC molecules in Ly-6A.2 transgenic mice show reactivity to a number of MHC molecules. To test the frequency of this reactivity, we stimulated T cells from Ly-6Tg⁺MHC^-, Ly-6Tg^-MHC⁺ (C57BL/6) mice with soluble anti-CD3 Ab and purified CD4⁺ cells and fused them with TCR--negative TCR-ß-negative BW5147 (17). Hybridomas that tested positive for CD4 expression were further tested for reactivity with different MHC. Each hybridoma was stimulated with APCs from three different strains. An average of 12 to 15% of the T cells from Ly-6Tg⁺MHC^- mice reacted to each of the MHC-expressing APCs tested (Table II). In contrast, 10 to 12% of the T cells from Tg^-MHC⁺ mice reacted with two different MHC tested, whereas about 8% of the hybrids from Ly-6A.2Tg⁺MHC^- mice reacted with self MHC (I-A^b) and about 4% of the hybrids from the normal mice reacted with self MHC. These results indicate that TCRs on CD4⁺ cells that mature in the absence of MHC in Ly-6A.2 transgenic mice recognize many MHC molecules tested at high frequencies.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study was originally undertaken to understand the mechanisms involved in the inhibition of T cell development in Ly-6A.2 transgenic mice. To that end, we conclude that TCR interactions with the MHC molecules do not contribute to this block in development (Table I). These observations are not surprising because we now know that the blockade of maturation in the Ly-6A.2 transgenic mice occurs at a stage of development when the TCR is not expressed (12). However, this was not known when these experiments were initiated. Nevertheless, these experiments led us to the unexpected observation that ectopic expression of Ly-6A.2 molecule on CD4⁺CD8⁺ thymocytes allowed maturation of CD4⁺CD8^- cells independently of MHC-peptide complexes. This later observation provides another reason that Ly-6A.2 may be extinguished on CD4⁺CD8⁺ cells. These results raise the possibility that another Ly-6 family member, which is endogenously expressed on CD4⁺CD8⁺ cells, might play a role in positive selection.

The CD4⁺CD8^- cells that appear in the thymus and periphery of Tg⁺MHC^- mice appear to be similar to the CD4⁺ mature cells by a number of phenotypic criteria. They have down-regulated CD8 and HSA molecules and up-regulated TCR proteins. The CD4⁺CD8^- cells that mature in the thymi of Ly-6A.2⁺MHC^- mice also express the CD40 ligand on cell stimulation and proliferate upon cross-linking of the TCR/CD3 complex. The proliferative responses of Tg⁺MHC^- thymocytes were similar to the response of Tg⁺MHC⁺ thymocytes (data not shown) and were, in all cases, not less than those observed for thymocytes from normal (Tg^-MHC⁺) mice. In contrast, the thymocytes from the Tg^-MHC^- do not proliferate after cross-linking of their TCRs, which is consistent with a previous report (14). Furthermore, the CD4⁺CD8^- T cell emigrate from the thymus to peripheral lymphoid organs.

Previous reports have demonstrated that overexpression of CD8 can promote the selection of CD4⁺ cells in the absence of class II molecules (23), and Bcl-2 expression allows maturation of CD8⁺ cells in the absence of a class I molecule (29). More recently, preferential maturation of CD8 cells in Notch 1 transgenic mice was observed (30). The Notch-mediated maturation of CD8⁺ cells was not observed in the absence of both class I and II molecules. The Ly-6 transgenic mouse is the first example that we are aware of in which CD4⁺ cells appear in the absence of both MHC class I and II molecules and therefore provides a unique opportunity to study thymic selection.

Why does overexpression of the Ly-6A.2 transgene result in maturation of CD4⁺ cells?

Maturation of CD4⁺ cells in the absence of MHC expression is surprising. Our experiments also indicate that CD4⁺ cells do not mature in Ly-6A.2Tg⁺ MHC^- mice if the expression is low (fivefold; data not shown). It is possible that this maturation is driven by the interaction of overexpressed Ly-6A.2 on thymocytes with its ligand in the thymus. This notion is consistent with our recent observation that indicates the presence of intrathymic Ly-6A.2 ligand (31). This interaction may either result in signals that mimic the TCR signaling or use the TCR/CD3 complex to signal into the cell. The rationale for this hypothesis is as follows. First, cross-linking Ly-6A.2 proteins on T cells with Abs causes cell activation and secretion of cytokines (16). Second, Ly-6A.2 binds to several key protein tyrosine kinases, e.g., p56^lck and p59^fyn, that are critical for signaling through TCR (32). Third, mutations or antisense oligonucleotides that decrease Ly-6A.2 expression also diminish T cell responsiveness (33, 34, 35). Reciprocally, a loss of TCR/CD3 expression also results in a lack of immune responsiveness through Ly-6A.2 (36, 37). More recently, Ly-6A null mice have been generated and have altered the proliferative responses of mature T cells (38). In these mice, lack of Ly-6A.2 expression results in higher proliferation of splenic T cells to anti-CD3 Abs, mitogens, and alloantigens. Taken together, these results indicate that Ly-6A.2 is able to regulate signaling negatively or positively through the TCR/CD3 complex. Why Ly-6A.2 expression has opposite effects on signaling through the TCR/CD3 is unclear.

It is interesting to note that signaling through Ly-6A.2 results in the maturation of only CD4⁺ cells and not CD8⁺ cells. One possibility is that Ly-6A.2 molecules provide a signal that favors the maturation of CD4⁺ cells as opposed to CD8 cells. Alternatively, this signaling may simply allow progression down the default pathway of maturation for the maturation of CD4⁺ cells (39). This interpretation is consistent with the recent observation that maturation of CD8 cells needs additional lineage-specific signals, which may be delivered by the activated Notch-1 molecule upon its interaction with its ligand (30). We propose that the signals to activate the Notch or its ligand or other CD8 lineage-specific signals are not delivered in Ly-6A.2Tg⁺MHC^- mice. We hope that additional experiments with Ly-6A.2Tg⁺MHC^- mice will provide insights into the mechanism of lineage commitment.

The mature CD4 T cell repertoire truly represents the initial T cell repertoire

Since overexpression of Ly-6A.2 results in the maturation of CD4⁺ cells in the absence of MHC class I and II molecules, it is likely that these cells represent the initial unselected T cell repertoire. This contention is supported by the observation that the Vß repertoire of the CD4⁺ cells and CD4⁺CD8⁺ cells from the Ly-6A.2Tg⁺MHC^- mice are similar. Furthermore, this similarity extends to the repertoire of the CD4⁺CD8⁺ DP cells from Tg^-MHC^- mice.

It is very unlikely that CD4⁺ cells selected in the thymus of Tg⁺MHC^- mice are selected by H2-O, an atypical MHC class II molecule, for the following reasons: 1) H-2O is primarily expressed intracellularly (40); 2) Abs to I-A and I-E block the reactivity of these cells to I-A- and I-E-expressing APCs, and these Abs are not known to cross-react with H2-O (L. Karlsson, unpublished observations). Therefore, these results strongly suggest that CD4⁺ cells are not selected by the H-2O protein. It is also unlikely that CD4⁺ cells are selected by the class I heavy chain on the surface of cells in b₂-microglobulin knock-out mice, since the reactivity of these cells to MHC is inhibited by anti-class II Abs. Taken together, these observations strongly argue that CD4⁺ cells that mature in the absence of MHC class I and II molecules represent the T cell repertoire that exists before thymic selection.

MHC reactivity of the initial T cell repertoire

The phenomenon of alloreactivity is not well understood. Our results suggest that a higher frequency of alloreactive T cells exists before thymic selection than previously predicted. Approximately 12 to 15% of the hybrids from the Ly-6A.2Tg⁺MHC^- mice reacted with different MHC molecules. This is similar to the 10 to 12% of the hybrids from the normal mice that showed this reactivity. These results would suggest that alloreactivity is inherent in the preselected repertoire, and it is not a consequence of selection of the receptor that reacts with self MHC and peptide. These results are consistent with the recent observations that have analyzed the reactivity of CD4⁺ cells that mature in the FTOC of MHC^- mice in the presence of a combination of anti-TCR and anti-CD4 Abs (11) or by bispecific CD3/CD4 Abs (41). The percentage of reactivity to H-2^b was lower than with H-2^d and H-2^k and probably reflects reactivity to only I-A molecules (since I-E is not expressed). Approximately 4% of the hybrids derived from C57BL/6 mice reacted with APCs from the same mouse strain (Table II). This reactivity may reflect T cells that respond to syngeneic APCs, described as a phenomenon of syngeneic MLR (SMLR) (42, 43, 44).

It has become increasingly clear that negative selection plays a crucial role in shaping the T cell repertoire. The degree to which the clonal deletion may impact the T cell repertoire varies from 5 to 50% (13, 44). CD4⁺ cells that mature in the Ly-6A.2 transgenic mice react with syngeneic stimulators at a higher frequency, indicating that the T cell repertoire in these mice will include T cells that are normally deleted in the the thymus. Analysis of peripheral repertoire in Ly-6A.2Tg⁺MHC^- mice will provide clues about the nature of self-reactive T cells that normally do not exit the thymus.

In summary, our results indicate that CD4⁺ cells that appear in the thymi of Ly-6A.2 transgenic mice in the absence of MHC molecules are phenotypically like mature T cells. These cells represent the preselected T cell repertoire that has high reactivity to MHC molecules. These later results indicate that the initial, preselected T cell repertoire has an evolutionary bias in its reactivity to MHC protein.

	Acknowledgments

 
We thank Drs. Laurie Glimcher and Michael Grusby for providing MHC class I x II-deficient mice. We also thank Dr. Laurie Glimcher for critically reading the manuscript and Dr. B. J. Fowlkes for discussion.

	Footnotes

 
¹ This work was supported by the University of Georgia Research Foundation (A.B.) and National Institutes of Health Grant GM 38515 (K.L.R.). A.B. was the recipient of an investigator award from the Arthritis Foundation.

² Present address: Division of Hematology and Oncology, Dana-Farber Cancer Institute, Boston, MA 02115.

³ Present address: Department of Immunology, Saint Mary’s Hospital Medical School, Norfolk Place, London W2 1P6, U.K.

⁴ Address correspondence and reprint requests to Dr. Anil Bamezai, 615 Biologic Sciences Bldg., University of Georgia, Athens, GA 30602. E-mail address:

⁵ Abbreviations used in this paper: FTOC, fetal thymic organ culture; Tg, transgenic; HSA, heat-stable Ag; DP, double positive.

Received for publication December 10, 1997. Accepted for publication March 3, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Sha, W. C., C. A. Nelson, R. D. Newberry, J. K. Pullen, L. R. Pease, J. H. Russell, D. Y. Loh. 1990. Positive selection of transgenic receptor-bearing thymocytes by K^b antigen is altered by K^b mutations that involve peptide binding. Proc. Natl. Acad. Sci. USA 87:6186.[Abstract/Free Full Text]
2. Nikolic-Zugic, J., M. J. Bevan. 1990. Functional and phenotypic delineation of two subsets of CD4 single positive cells in the thymus. Int. Immunol. 2:135.[Abstract/Free Full Text]
3. Mombaerts, P., A. R. Clarke, M. A. Rudnicki, J. Iacomini, S. Itohara, J. J. Lafaille, L. Wang, Y. Ichikawa, R. Jaenisch, M. L. Hooper. 1992. Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages. Nature 360:225.[Medline]
4. Fischer-Lindahl, K., D. Wilson. 1977. Histocompatibility antigen-activated cytotoxic T lymphocytes. II. Estimates of the frequency and specificity of precursors. J. Exp. Med. 145:508.[Abstract/Free Full Text]
5. Matzinger, P., M. J. Bevan. 1977. Hypothesis: why do so many lymphocytes respond to major histocompatibility antigens?. Cell. Immunol. 29:1.5.
6. Bevan, M.. 1984. High determinant density may explain the phenomenon of alloreactivity. Immunol. Today 5:128.
7. Bluestone, J. A., A. Kaliyaperumal, S. Jameson, S. Miller, R. Dick. 1993. Peptide-induced changes in class I heavy chains alter allorecognition. J. Immunol. 151:3943.[Abstract]
8. Elliott, T. J., H. N. Eisen. 1990. Cytotoxic T lymphocytes recognize a reconstituted class I histocompatibility antigen (HLA-A2) as an allogeneic target molecule. Proc. Natl. Acad. Sci. USA 87:5213.[Abstract/Free Full Text]
9. Jerne, N. K.. 1971. The somatic generation of immune recognition. Eur. J. Immunol. 1:1.[Medline]
10. Ignatowicz, L., J. Kappler, P. Marrack. 1996. The repertoire of T-cells shaped by a single MHC/peptide ligand. Cell 84:521.[Medline]
11. Zerrahn, J., W. Held, D. H. Raulet. 1997. The MHC reactivity of the T cell repertoire prior to positive and negative selection. Cell 88:627.[Medline]
12. Bamezai, A., D. Palliser, A. Berezovskaya, J. McGrew, K. Higgins, E. Lacy, K. L. Rock. 1995. Regulated expression of Ly-6A.2 is important for normal T cell development. J. Immunol. 154:4233.[Abstract]
13. Grusby, M. J., R. S. Johnson, V. E. Papaioannou, L. H. Glimcher. 1991. Depletion of CD4⁺ T cells in major histocompatibility complex class II-deficient mice. Science 253:1417.[Abstract/Free Full Text]
14. Cosgrove, D., D. Gray, A. Dierich, J. Kaufman, M. Lemerur, C. Benoist, D. Mathis. 1991. Mice lacking MHC class II molecules. Cell 66:1051.[Medline]
15. Bamezai, A. H. Reiser, K. L. Rock. 1988. T cell receptor/CD3 negative variants are unresponsive to stimulation through the Ly-6 encoded molecule, TAP. J. Immunol. 141:1423.[Abstract]
16. Rock, K. L., E. T. H. Yeh, C. F. Gramm, S. I. Haber, H. Reiser, B. Benacerraf. 1986. TAP, a novel T cell activating protein involved in the stimulation of MHC-restricted T lymphocytes. J. Exp. Med. 163:315.[Abstract/Free Full Text]
17. White, J., M. Blackman, J. Bill, J. Kappler, P. Marrack, D. P. Gold.. 1989. Two better cell lines for making hybridomas expressing specific T cell receptors. J. Immunol. 143:1822.[Abstract]
18. Bamezai, A., V. Goldmacher, K. L. Rock. 1991. Internalization of glycosylphosphatidylinositol (GPI)-anchored lymphocyte proteins. II. GPI-anchored transmembrane molecules internalize through distinct pathways. Eur. J. Immunol. 22:15.
19. Crump, A. L., M. J. Grusby, L. H. Glimcher, H. Cantor. 1993. Thymocyte development major histocompatibility complex-deficient mice: evidence for stochastic commitment to the CD4 and CD8 lineages. Proc. Natl. Acad. Sci. USA 90:10739.[Abstract/Free Full Text]
20. Chan, S. H., C. Benoist, D. Mathis. 1993. A challenge to the instructive model of positive selection. Immunol. Rev. 135:119.[Medline]
21. Bruce, J., F. W. Symington, T. J. McKearn, J. Sprent. 1981. A monoclonal antibody discriminating subsets of T and B cells. J. Immunol. 127:2496.[Abstract]
22. Crispe, I. N., M. J. Bevan. 1987. Expression and functional significance of the J11d marker in mouse thymocytes. J. Immunol. 138:2013.[Abstract]
23. Robey, E., A. Itano, W. C. Fanslow, B. J. Fowlkes. 1994. Constitutive CD8 expression allows inefficient maturation of CD4⁺ helper T cells in class II major histocompatibility complex mutant mice. J. Exp. Med. 179:1997.[Abstract/Free Full Text]
24. Laman, J. D., E. Claassen, R. J. Noelle. 1994. Immunodeficiency due to a faulty interaction between T cells and B cells. Curr. Opin. Immunol. 6:636.[Medline]
25. Havran, W. L., M. Poenie, J. Kimura, R. Tsien, A. Weiss, J. P. Allison. 1987. Expression and function of the CD3-antigen receptor on murine CD4⁺8⁺ thymocytes. Nature 330:170.[Medline]
26. Dyson, P. J., A. M. Knight, S. Fairchild, E. Simpson, K. Tomonari. 1991. Genes encoding ligands for deletion of V_ß11 T cells cosegregate with mammary tumor virus genomes. Nature 349:531.[Medline]
27. Woodland, D., M. P. Happ, J. Bill, E. Palmer. 1990. Requirement for cotolerogenic gene products in the clonal deletion of I-E reactive T cells. Science 247:964.[Abstract/Free Full Text]
28. Ohteki, T., H. R. MacDonald. 1994. Major histocompatibility complex class I related molecules control the development of CD4⁺8^- and CD4^-8^- subsets of natural killer 1.1⁺ T cell receptor-alpha/beta⁺ cells in the liver of mice. J. Exp. Med. 180:699.[Abstract/Free Full Text]
29. Linette, G. P., M. J. Grusby, S. M. Hedrick, T. H. Hansen, L. H. Glimcher, S. J. Korsmeyer. 1994. Bcl-2 is upregulated at the CD4⁺CD8⁺ stage during positive selection and promotes thymocyte differentiation at several control points. Immunity 1:197.[Medline]
30. Robey, E., D. Chang, A. Itano, D. Cado, H. Alexander, D. Lans, G. Weinmaster, P. Salmon. 1996. An activated form of Notch influences the choice between CD4 and CD8 T cell lineages. Cell 87:483.[Medline]
31. Bamezai, A., K. L. Rock. 1995. Overexpressed Ly-6A.2 mediates cell-cell adhesion by binding its ligand expressed on lyphoid cells. Proc. Natl. Acad. Sci. USA 92:4294.[Abstract/Free Full Text]
32. Stefanova, I., V. Horejsi, I. Ansotegui, W. Knapp, H. Stockinger. 1991. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science 254:1016.[Abstract/Free Full Text]
33. Yeh, E. T. H., H. Reiser, A. Bamezai, K. L. Rock. 1988. TAP transcription and phosphatidylinositol linkage mutants are defective in activation through the T cell receptor. Cell 52:665.[Medline]
34. Flood, P. M., J. P. Dougherty, Y. Ron. 1990. Inhibition of Ly-6A antigen expression prevents T cell activation. J. Exp. Med. 172:115.[Abstract/Free Full Text]
35. Lee, S. K., B. Su, S. E. Maher, A. L. Bothwell. 1994. Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity. EMBO J. 13:2167.[Medline]
36. Bamezai, A., H. Reiser, K. L. Rock. 1988. T cell receptor/CD3 negative variants are unresponsive to stimulation through the Ly-6 encoded molecule, TAP. J. Immunol. 141:1423.
37. Sussman, J. J., T. Saito, E. M. Shevach, R. N. Germain, J. D. Ashwell. 1988. Thy-1 and Ly-6-mediated lymphokine production and growth inhibition of a T cell hybridoma requires coexpression of the T cell antigen receptor complex. J. Immunol. 140:2520.[Abstract]
38. Stanford, W. L., S. Haque, R. Alexander, Xuemei Liu, A. M. Latour, R. H. Snodgrass, B. H. Koller, P. M. Flood. 1997. Altered proliferative response by T lymphocytes of Ly-6A (Sca-1) null mice. J. Exp. Med. 186:705.[Abstract/Free Full Text]
39. Suzuki, H., J. A. Punt, L. G. Granger, A. Singer. 1995. Asymmetric signaling requirements for thymocyte commitment to the CD4⁺ versus CD8⁺ T cell lineages: a new perspective on thymic commitment and selection. Immunity 2:413.[Medline]
40. Karlsson, L., C. D. Surh, J. Sprent, P. A. Peterson. 1992. An unusual class II molecule. Immunol. Today 13:469.[Medline]
41. Merkenschlager, M., D. Graf, M. Lovatt, U. Bommhardt, R. Zamoyska, A. G. Fisher. 1997. How many thymocytes audition for selection?. J. Exp. Med. 186:1149.[Abstract/Free Full Text]
42. Smith, J. B., R. D. Pasternak. 1978. Syngeneic mixed lymphocyte reaction in mice: strain distribution, kinetics, participating cells, and absence in NZB mice. J. Immunol. 121:1889.[Abstract/Free Full Text]
43. Johnson, E. D., C. J. Krco, C. S. David. 1982. Ia antigens are the stimulating determinants in the syngeneic mixed lymphocyte reaction. Transplantation 33:552.[Medline]
44. Nussenzweig, M. C., R. M. Steinman. 1980. Contribution of dendritic cells to stimulation of the murine syngeneic mixed leukocyte reaction. J. Exp. Med. 151:1196.[Abstract/Free Full Text]

This article has been cited by other articles:

				J.-I. Rodriguez-Barbosa, Y. Zhao, G. Zhao, A. Ezquerra, and M. Sykes Murine CD4 T Cells Selected in a Highly Disparate Xenogeneic Porcine Thymus Graft Do Not Show Rapid Decay in the Absence of Selecting MHC in the Periphery J. Immunol., December 15, 2002; 169(12): 6697 - 6710. [Abstract] [Full Text] [PDF]

				S. C. Henderson, M. M. Kamdar, and A. Bamezai Ly-6A.2 Expression Regulates Antigen-Specific CD4+ T Cell Proliferation and Cytokine Production J. Immunol., January 1, 2002; 168(1): 118 - 126. [Abstract] [Full Text] [PDF]

				D. L. Pflugh, S. E. Maher, and A. L. M. Bothwell Ly-6I, a New Member of the Murine Ly-6 Superfamily with a Distinct Pattern of Expression J. Immunol., July 1, 2000; 165(1): 313 - 321. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Henderson, S. C. Articles by Bamezai, A. Search for Related Content PubMed PubMed Citation Articles by Henderson, S. C. Articles by Bamezai, A.