Downregulated Expression of Ly-6-ThB on Developing T Cells Marks CD4+CD8+ Subset Undergoing Selection in the Thymus

Interaction of TCRs on CD4+CD8+ immature T cell with MHC-peptide complexes on stromal cells is required for positive and negative selection in the thymus. Identification and characterization of a subpopulation of CD4+CD8+ thymocytes undergoing selection in the thymus will aid in understanding the mechanisms underlying lineage commitment and thymic selection. Herein, we describe the expression of Ly-6 ThB on developing thymocytes. The majority of CD4+CD8+ thymocytes express Ly-6 ThB at high levels. Its expression is downregulated in a subset of CD4+CD8+ thymocytes as well as in mature CD4+CD8- and CD4-CD8+ T cells. More importantly, interaction of TCR/coreceptor with the self-MHC-peptide contributes to the downregulation of ThB expression on developing thymocytes. These findings indicate that downregulation of ThB on CD4+CD8+ thymocytes identifies a unique subset (CD4+CD8+ThBneg–low) of thymocytes that has received the initial signals for thymic selection but have not yet downregulated the CD4 and CD8 cell surface expression. In addition, these results also indicate that a high frequency (Ÿ20–40%) of CD4+CD8+ immature thymocytes receive these initial signals during thymic selection.


INTRODUCTION
Development of thymocytes and their selection occurs in discreet steps in the thymus. It has become increasingly clear that various cell surface proteins on T lymphocytes interacting with their ligands on the stromal cells are critical for these processes (1). A key set of proteins involved in the thymic development is the TCR/CD3 complex and antigen coreceptors (CD4 and CD8) (2,3). For example, expression of TCR-beta (pre-TCR complex) is critical for CD4-CD8to CD4+CD8 / cell development (2,3).
Expression of TCR-alpha-beta is critical for further development into either CD4/CD8 helper T cells or CD4-CD8 / cytotoxic T cells (3)(4)(5). The underlying mechanisms involved in these developmental proc-108 JUSTIN T. REESE et al. esses and lineage commitment to CD4 + helper or CD8 + cytotoxic T cell are unclear. The identification and analysis of a subset within CD4+CD8 / thymocytes that have received initial signals through their TCR's will aid in providing insights into the underlying mechanisms for T cell selection as well as lineage commitment.
ThB is a member of the Ly-6 multigene family expressed on thymocytes, B cells, and keratinocytes (6)(7)(8)(9). Similar to other known members of the Ly-6 multigene family, ThB is 10-12 kDa in molecular mass and linked to the cell surface by a GPI-anchor (10,11). Although the biological function of the ThB molecule is unknown, recent reports indicate that the human homologue of ThB (E48) has cell-cell adhesion function in keratinocytes (9). These latter observations are consistent with the cell adhesion properties reported for other members of the Ly-6 gene family (12)(13)(14).
A distinct feature of some Ly-6 proteins is their regulated expression during the development of hematopoietic cells (11,15). For example, Ly-6A.2 is expressed on stem cells (16,17) as well as on the a CD44 / subset of CD4-CD8-CD3-T cells in the thymus (18). Ly-6A.2 is absent on the the majority of immature thymocytes including the CD4+CD8 / subset but reexpressed on mature T cells in the thymus and spleen (19). In this regard, approximately 50-80% of thymocytes express ThB, but expression of this molecule at different developmental stages dring thymic development is unknown.
Herein, we present evidence that Ly-6 ThB expression is tightly regulated during thymic development. Interaction of antigen receptor and CD4/CD8 with self-MHC-peptide contributes to the downregulation of ThB on CD4+CD8 / thymocytes. These results indicate that ThB neg-lw CD4/CD8 + T cells are composed of a subset of thymocytes that have received initial signals through the antigen receptor and coreceptor but have not yet downregulated their coreceptor. In addition, this study also indicates that a high frequency of developing CD4/CD8 / thymocytes express an antigen receptor that has bias towards reactivity to self-MHC-peptide complexes.

RESULTS
Expression of ThB on T cell subsets in the thymus and the periphery We sought to examine ThB expression in BALB/c mice for each of the four thymic subsets defined by anti-CD4 and anti-CD8 antibodies ( Figure 1A).
Approximately 40% of the CD4-CD8subset that comprise 2-4% of total thymocytes express high levels of ThB on their surface (DN/R4). A majority of CD4/CD8 + thymocytes (62%) expresses high levels of ThB (DP/R2). In contrast, 93% of CD4/CD8 , and 93% of CD4-CD8/thymocytes are ThB negative-lw (Figure 1 A&B). Similar results were obtained with a number of strains of mice including, C57BL/6, SWR, AKR, SJL (data not shown). One notable exception was the higher expression of ThB as well as higher numbers of ThB expressing thymocytes in C57BL/6 mice compared to BALB/c mice, which is consistent with previous report (25). Approximately 80% of the CD4/CD8 + thymocytes from C57BL/6 mice express higher levels of ThB as compared to about 60% this subset in BALB/c mice. Mature CD4 / and CD8 / T cell subsets in the periphery (lymph node and spleen) do not show surface expression of ThB (data not shown) as previously described (8). These data indicate that ThB expression is downregulated during development of immature CD4+CD8 / to mature CD4+CD8 and CD4-CD8 / cells. These results also indicate regulated expression of ThB within the CD4+CD8 / subset of thymocytes and identify novel ThB lw and ThB high subpopulations with in CD4+CD8 / subset. Error bars indicate standard error gated on either DP thymocytes or CD4 + thymocytes that have partially or completely downregulated the CD8 coreceptor. Figure 2 shows that the expression of ThB is lowest in CD4/CD8 T cells and intermediate in CD4+CD8 lw and highest in CD4/CD8 + T cells in the thymus. Similarly CD41WCD8 / T cells also expressed lower levels of ThB (Figure2) and CD4-CD8 / showed the lowest expression like CD4/CD8 helper subset (data not shown). These results strongly suggest that as thymocytes mature and lose CD4 or CD8 coreceptors, there is a coordinate loss of ThB.
Maturation of CD4 + helper and CD8 + cytotoxic T cells from CD4/CD8 + immature T cell involves upregulation of TCR/CD3, CD5 proteins and downmodulation of CD4 or CD8 molecules. To further examine the expression of ThB on developing T cells we analyzed BALB/c thymocytes using anti-ThB and anti-CD5 (Figure 3 A&B). Majority of the thymocytes that show higher expression of CD5 have downregulated ThB expression (65.1+/-8%). A subset of thymocytes (28.3+/-12%) with higher expression of CD5 also have high expression of ThB. These suggest that with in CD5 + thym,ocytes both ThB high and ThB lw subpopulations exist. Similar analysis using anti-ThB and anti-CD3 antibodies indicated that majority of CD3 high (approximately 16.5%) (mature subset) expressed lower levels of ThB. Interestingly, about 25% of CD3 neg-intermidiate (immature subset) cells show downregulation of ThB. This analysis indicates that as T cells become mature (i.e., upregulation of CD5 and TCR) they lose ThB expression from the cell surface. Taken together our data strongly suggests that downregulation of ThB expression correlates with the maturity of developing T cells in the thymus.
Regulation of ThB surface expression by interaction of antigen receptor and co-receptor with self-MHC-peptide complexes The progression of CD4+CD8 + thymocytes to the mature CD4+CD8 or CD4-CD8 + thymocytes require ligation of the TCR and the CD4 or CD8 coreceptor. We hypothesized that interaction of the antigen receptor with self-MHC results in the downregulation of ThB expression on CD4/CD8 + thymocytes. To examine this hypothesis we determined the expression of ThB in the AND TCR transgenic mice that express the transgene either on a selecting (H-2b, B10) or non-selecting (H-2 d B10.D2) background (21). The expression of ThB was lower on thymocytes from AND-H-2 b mice than on the T cells from AND-H-2 d mice ( Figure 4A). As reported previously, we also observed a higher percentage of CD4 / ceils in AND transgenic mice on H-2b(Selecting) as compared to H-2 d (non.-Selecting) background (21). Further analysis of the ThBneg, ThB lw and ThB high subsets in AND transgenic mice on selecting and non-selecting genetic backgrounds indicated that almost all the ThB neg thymocytes are either CD4 + mature or CD4CD8immature T cells ( Figure 4B). In contrast, the majority of ThB high thymocytes belonged to the CD4+CD8 + subset. Interestingly, the ThB lw thymocytes included both immature CD4+CD8 + thymocytes as well as mature CD4+and CD8 / T cells. Moreover, a highe r percentage of ThB high cells were observed in AND transgenic mice under non-selecting (24%) than the selecting (9.7%) background. This was consistent with observation that higher percentages of ThB lw T cells were observed under selecting (60%) than non-selecting (49%) backgrounds. In addition a majority of T cells maturing under "non-selecting" (RAG/) backgound also downregulate the expression of ThB and therefore indicate that non-selecting background does provide weak positive slection. These findings suggest that the expression of ThB correlates with the positive selection of T cells in TCR transgenic mice and these results are consistent with what we observe in normal non-transgenic mice ( Figure 1).
We have extended our studies using several TCR transgenic mice: (1) 5C.C7 TCR transgene (22) expressed on selecting (B10) and non-selecting (B10.A) genetic backgrounds ( Figure 5). 2. DOll TCR transgenic that recognizes the c-ovalbumin peptide in the context of the MHC class II molecule I-A d (20). 3 Figure 7A). The downregulation of ThB was specific because expression of HSA was not altered in these mice ( Figure 7B). This expression is not altered in MHC-deficeint mice injected with saline (controls) ( Figure 7A) as well as anti-CD4 + isotype control antibody (data not shown). Moreover the downmodulation of ThB was not observed on day 2 after the injection of anti-CD3 + anti-CD4 antibody (data not shown). Injection of antibody did not alter the cell numbers recovered from the thymus (8.6x107 in saline injected Vs lxl08in anti-CD3+anti-CD4 injected mice), therefore indicating that anti-CD3 and anti-CD4 antibodies did not induce cell death in ThBhigh subset ( Figure 7A). These results suggests that ligation of TCR and CD4 proteins is required for downregulation of ThB expression.

DISCUSSION
An interesting feature of the products of the Ly-6 locus is their regulated expression during develop-.ment (11,26). Ly-6A.2, one of the better-studied members of this gene family, shows tightly regulated expression during thymic development. Dysregulating the expression of Ly-6A.2 on T cells in the thymus induces a block in development at the CD4-CD8cell stage, where normal expression of Ly-6A.2 is lost (18). These observations indicate that regulated expression of Ly-6A.2 is important for normal T cell development (18). The data in this report indicates that ThB also shows regulated expression on developing T cells in the thymus that is distinct and regulated at different stages of thymic development than Ly-6A.2. High ThB expression is observed on some CD4-CD8-, on the majority of CD4/CD8 + thymocytes and its expression is lost on mature CD4+CD8 and CD4-CD8 / T cells. Similarly, lower expression of ThB also correlates with the maturity of developing T cells in the thymus as defined by CD3 and CD5 expression. Interestingly, the expression of CD5 on CD4/CD8 + thymocytes is not biphasic (27) where as expression of ThB does dissect CD4+CD8+ subset into two subsets (Figure 1). These results indicate that ThB is a unique cell surface marker on developing T cells whose expression further dissects the CD5 expressing developing T cells in the thymus.  gested that the expression of CD3, HSA and CD69 can define the differentiation stage of thymocytes within the DP subset. DP CD3 hi thymocytes are thought to arise from DP CD3 int thymocytes (28). HSA is highly expressed on the majority of DP thymocytes and is down regulated in the majority of mature T cells in the medulla (29). Moreover, up regulation of CD69 seems to correlate with the maturation of DP thymocytes to the SP cell stage (30). Only 10% of CD4+CD8 / thymocytes upregulate CD69 and audition for T-cell antigen receptor mediated positive selection (30,31). The level of ThB can be used to further dissect this subset of thymocytes. Therefore ThB expression defines unique subset with in the T cell subset (CD4/CD8+) in the thymus of normal mice known to undergo thymic selection. Isolation and characterization of this subset will provide further insights into T cell development in the thymus.
Although it is noteworthy that the expression of a number of Ly-6 molecules fluctuate during T cell development in the thymus, how this regulation occurs is unknown. Here we describe, that one factor which contributes to the regulation of Ly-6 ThB is These results suggest that antigen receptor and coreceptor ligation is necessary for downmodulation of ThB. It is possible that additional proteins expressed on CD4/CD8 / T cells including ThB as well as other cell-autonomous mechanisms may also contribute to the downmodulation of ThB.
Previous studies indicate that addition of anti-CD3 and anti-CD4 antibodies to fetal thymi from MHC class I-IImice results in maturation of CD4 + T cells and downregulates the expression of HSA from HSA laigh to HSA lw therefore suggesting that these cells have undergone positive selection. In contrast, our studies in which anti-CD3 and anti-CD4 antibodies are injected in vivo does not result in maturation of CD4 / helper T cells. Moreover, the expression of HSA also remains unaltered in these injected mice. These different results may be attributed to the different experimental systems used (i.e. fetal thymic organ culture Vs injection into the mice) or differences in the amount of antibodies reaching thymocytes under these different experimental conditions. .Interestingly, injection of anti-CD3 and anti-CD4 antibodies in MHC class I-IImice cause downregulation of ThB expression on CD4+CD8 + thymocytes.
These later observations may suggest that anti-CD3+anti-CD4 antibodies do not provide sufficient ligation for full positive selection to proceed but may initiate some initial events in this process (i.e. downregulation of ThB expression). These later results are consistent with other data that downregulation of ThB requires interaction of TCR and co-receptor with their ligand.
Previously published data have demonstrated two patterns of ThB expression (25). Thymocytes from C57BL/6 mice show higher ThB expression, in comparison with thymocytes from BALB/c mice which are known to show lower levels of ThB. Our observations are consistent with these published results since only about 80% of CD4/CD8 / thymocytes express high levels of ThB in C57BL/6 mice as compared to 50-60% in BALB/c mouse. These differences do not arise from expression of different alleles in these two mouse strains (32). The genetic basis of these differences is not known. The genetic variation of ThB expression has not been mapped to H-2 locus and therefore lack of I-E expression in C57BL/6 is unlikely to influence ThB expression (6). These results suggest that ligation of TCR and CD4 molecules are one of many factors that may contribute to downregulation of ThB expression. The CD4+CD8 / subset of thymocytes represents a critical stage where MHC restriction, lineage commitment and thymic selection occur. The underlying mechanisms for these processes are largely unknown. Identification of the transition phases within the CD4/CD8 + subset that are dependent on the interaction of TCR/coreceptor with MHC may aid in the isolation and analysis of cells auditioning for thymic selection. Our results also indicate that this interaction regulates the expression of ThB at the DP cell stage and we are able to identify a unique CD4+CD8+ThB lw subset in the thymus. The CD4+CD8+ThB lw cells constitute about 20-40% of the total thymocyte pool. Based on the data from non-transgenic, TCR-transgenic and MHC null mice, CD4+CD8+ThB lw cells seems to have received initial signals of thymic selection. Our results are unable to determine whether CD4/CD8 + thymocytes down-regulating expression of Ly-6-ThB are undergoing or destined to be negatively or positively selected. Given previous reports that all the superantigen-reactive TCR-Vf5 beating T cells are included and present in the CD4+CD8 / subset but absent in the mature CD4 + or CD8 / subsets (33) suggests that downmodulation of ThB occurs prior to disappearance of cells by negative selection and may be the result of first and initial interaction of TCR/coreceptor with self-MHC+peptide as previously proposed (34). This data also suggests that a.high percentage of DP thymocytes that have rearranged their TCR react with self-MHC-peptide complexes that results in downmodulation of ThB expression. These results are consistent with other recent observations that T cell repertoire that exists prior to thymic selection is highly biased in its reactivity to MHC molecules (35)(36)(37). The data presented in here are consistent with our previous estimates that indicate that this reactivity is atleast in the range of 10-15% (35). In addition, TCR-alpha gene loci are reported to undergo secondary gene rearrangement that would increase the possibility of generating TCRs with reactivity to self-MHC and peptide complexes (38).
The biological significance of downregulation of ThB is unclear. Considering the expression pattern of ThB (this manuscript) and adhesion properties of human homologue of ThB (E48) (9) as well as other Ly-6 proteins (12)(13)(14), we hypothesize that one of the functions of ThB is to hold thymocytes in the appropriate thymic region where thymic selection occurs. Therefore, downregulation of ThB will aid in de-adhesion and movement of maturing T cells towards the medulla. Alternatively it is also possible that ThB participates in positive or negative selection which might alter signaling threshold on T cells undergoing selection. This possibility is consistent with signaling property of Ly-6 proteins (11). The generation of ThB transgenic mice and null mutants will provide better insight into this phenomenon.