Evidence for programmed cell death of self-reactive γδ T cell receptor-positive thymocytes

The negative selection of T cells expressing the γδ T cell antigen receptor (γδ T cells) was studied using transgenic mice expressing a γδ receptor with specificity for an H-2T-linked class I major histocompatibility complex molecule from H-2^b mice. The potentially self-reactive γδ thymocytes in H-2^b/d transgenic mice are larger and have lower levels of γδ T cell receptor expression than γδ thymocytes from H-2^d mice. H-2^b/d γδ thymocytes do not respond to H-2^b antigen-presenting cells, and thus are inactive compared to H-2^d γδ thymocytes. However, the H-2^b/d γδ thymocyte population, but not the H-2^d γδ thymocyte population, undergoes a high rate of programmed cell death when placed in overnight culture. These observations constitute the first direct evidence that self-reactive γδ thymocytes undergo programmed cell death. This in vitro programmed cell death of self-reactive γδ thymocytes may reflect the clonal deletion process that results in a depletion of γδ T cells in the peripheral lymphoid organs of adult H-2^b/d mice. We also present evidence that self-reactive γδ T cells, similarly to αβ T cells, undergo a lesser degree of clonal deletion in neonatal mice compared to adult mice.     

The phenotype of H-2M-deficient mice is dependent on the MHC class II molecules expressed

For a broader view of the role of H-2M as an accessory molecule in antigen presentation, we investigated the degree to which different MHC class II isotypes and alleles depend on H-2M to function in vivo. We generated H-2M-deficient animals expressing E^{k / b} or A^k molecules in addition to the A^b molecules already present in the mutant strain, and compared the ability of the different MHC class II molecules to present antigen at the cell surface for recognition by T cells, and contribute to positive selection of CD4⁺ T cells in the thymus. Biochemical analyses were performed to assess MHC class II maturation, and to determine the peptide content of the molecules. In the absence of H-2M, E^{k / b} molecules containd a more heterogeneous set of class II-associated invariant chain peptides (CLIP) than A^b did, which, unlike A^b -CLIP complexes, were not SDS-stable. Unlike A^b molecules, both E^{k / b} and A^k efficiently presented exogenously added peptides to T cells in the absence of H-2M. In addition, epitopes from some proteins, especially those known to be invariant chain independent, were presented by A^k molecules in the mutant animals. To our surprise, expression of E^{k / b} overcame the positive selection defect observed in H-2M-deficient mice expressing A^b alone. In contrast, A^k expression did not augment positive selection of CD4⁺ T cells in the mutant animals. Some of these findings in vivo contrast significantly with findings from in vitro studies on murine MHC class II molecules in human DM-deficient cell lines.     

Double-positive T cell receptorhigh thymocytes are resistant to peptide/major histocompatibility complex ligand-induced negative selection

To investigate negative selection events during intrathymic ontogeny, we established T cell receptor (TCR)-transgenic mice [N15tg/RAG-2^?/? (H-2^b)] expressing a single TCR specific for vesicular stomatitis virus nuclear octapeptide N_52–59 (VSV8) in the context of the major histocompatibility complex (MHC) class I molecule, K^b. Administration of VSV8 in vivo induced apoptosis in less than 4 h, deleting the majority of immature double-positive (DP) thymocytes by 24 h. In contrast, DP TCR^high as well as single-positive (SP) thymocytes were refractory to this death process. Moreover, DP TCR^high cells differentiated into SP thymocytes in vitro and in vivo, maturing into functional cytotoxic T lymphocytes upon intrathymic transfer to β RAG 2^?/? recipients. Hence, negative selection processes involving MHC-bound peptide ligands are operative only prior to the late DP thymocyte stage in this MHC class I-restricted TCR transgene system.     

Agonist peptide modulates T cell selection thresholds through qualitative and quantitative shifts in CD8 co-receptor expression

Engagement of the TCR is a pivotal step in thymocyte development, ultimately resulting in the survival (positive selection) or loss (negative selection) of developing T cells. The roles of peptides and stromal cell interactions necessary for these selection events, however, are still poorly understood. To investigate the effects of agonist peptide in positive selection, we used a novel cell suspension model for in vitro thymic positive selection in adults. Target thymocytes from H-2Db-restricted TCR transgenic mice, specific to the lymphocytic choriomeningitis virus (LCMV) peptide bred on a non- selecting MHC background (H-2d or TAP-1-/-), were co-cultured with freshly isolated H-2b thymic stromal cells. In the presence of selecting stroma the nominal agonist LCMV peptide induced apoptosis at high concentrations and at low concentrations enhanced the efficiency of positive selection both in numbers of cells 'rescued' and kinetics of appearance of selected single-positive cells. We further illustrate down-modulation of CD8 alpha beta or CD8 beta at high but non-deleting concentrations of agonist peptide. This highlights the ability of the T cell, within the window of positive selection, to modify surface co- receptors both qualitatively and quantitatively in response to increasing avidity TCR-peptide-MHC interactions. The direct consequence of this would be to lower the total signaling events below the threshold for apoptosis induction. Hence if self peptide were not presented in sufficient quantities in the thymus, autoreactive cells may escape deletion and may actually be positively selected.      

Homotypic interactions mediated by Slamf1 and Slamf6 receptors control NKT cell lineage development

Commitment to the T and natural killer T (NKT) cell lineages is determined during alphabeta T cell receptor (TCR)-mediated interactions of common precursors with ligand-expressing cells in the thymus. Whereas mainstream thymocyte precursors recognize major histocompatibility complex (MHC) ligands expressed by stromal cells, NKT cell precursors interact with CD1d ligands expressed by cortical thymocytes. Here, we demonstrated that such homotypic T-T interactions generated "second signals" mediated by the cooperative engagement of the homophilic receptors Slamf1 (SLAM) and Slamf6 (Ly108) and the downstream recruitment of the adaptor SLAM-associated protein (SAP) and the Src kinase Fyn, which are essential for the lineage expansion and differentiation of the NKT cell lineage. These receptor interactions were required during TCR engagement and therefore only occurred when selecting ligands were presented by thymocytes rather than epithelial cells, which do not express Slamf6 or Slamf1. Thus, the topography of NKT cell ligand recognition determines the availability of a cosignaling pathway that is essential for NKT cell lineage development.     

Location-specific regulation of transgenic Ly49A receptors by major histocompatibility complex class I molecules

Inhibitory receptors expressed on natural killer (NK) cells and T cells specific for major histocompatibility complex (MHC) class I are believed to prevent these cells from responding to normal self tissues. To understand the regulation and function of Ly49 receptor molecules in vivo, we used the CD2 promoter to target Ly49A expression to all thymocytes, T cells, and NK cells. In animals expressing its MHC class I ligand, H-2D^d or H-2D^k, there was a large decrease in the expression of Ly49A on thymocytes, peripheral T cells, and NK1.1⁺ cells. The extent of the down-regulation of Ly49A was dependent on the expression of the MHC ligand for Ly49A and on the site where the cells were located. The level of expression of endogenous Ly49A was similarly found to be dependent upon the organ where the cells resided. Data from bone marrow chimeras indicated that most cell types may regulate Ly49A expression, but the efficacy to regulate receptor expression may vary depending on the cell type.     

Positive and Negative Selection in Transgenic Mice Expressing a T-Cell Receptor Specific for Influenza Nucleoprotein and Endogenous Superantigen

A transgenic mouse was generated expressing on most (>80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8⁺ and recognizes αα366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class ,MHC D^b (Townsend et al., 1986). The receptor utilizes the Vβ11 and Vα4 gene segments for the β chain and α chain, respectively (Palmer et al., 1989). The usage of Vβ11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2^b, IE^-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2^k and BALB/c H-2^d) and the endogenous Mtv ligands. Positive selection of CD8⁺ T cells expressing the Vβ11 is seen in C57BL/10 transgenic mice (H-2^b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period of ^{in vitro} culture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8⁺ peripheral T-cells in these (H-2^k or H-2^d) F5 mice are predominantly Vβ11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2^d scid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired.     

The T/B cell interaction involved in induction of the mouse IgG2ah suppression is restricted by major histocompatibility complex class I,but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2a^b suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igh^a haplotype, when neonatally transferred into histocompatible Igh^a/b heterozygotes, are able to induce a specific and total suppression of the IgG2a^b allotype. Nevertheless, only transfer of IgG2a^b-primed Igh^a T splenocytes induces this suppression in Igh^b/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8⁺ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4⁺CD8⁺ T cell cooperation and operated via the recognition by the involved TCR of Cγ2a^b-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Igh^b mice genetically deficient for MHC class I (β2-microglobulin^%, or β2m^%) or class II (I-Aβ^%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2a^b T cells transferred into Igh^b H-2^b I-Aβ^% mice carry out the suppression process normally, while in Igh^b H-2^b β2m^% recipients, their suppression induction capacity is significantly inhibited. Moreover, the Cγ2a^b 103–118 peptide, identified as the sole Cγ2a^b-derived peptide able to amplify the anti-IgG2a^b T cell reactivity in Igh^a H-2^b mice, is also able to stabilize the H-2D^b, but not the H-2K^b class I molecules at the surface of RMA-S (TAP2^?, H-2^b) cells. These results indicate that, despite the CD4⁺/CD8⁺ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2a^b+ B cells for suppression establishment.     

In vitro positive selection of alpha beta TCR transgenic thymocytes by a conditionally immortalized cortical epithelial clone

Development of mature CD4 and CD8 single-positive T cells requires a process known as positive selection, which depends on the specific recognition of self-peptide-MHC complexes on thymic stromal cells by immature CD4+CD8+ thymocytes. We have used an in vitro reaggregate system to study the positive selection of thymocytes by conditionally immortalized thymic epithelial clones. Thymocytes from mice transgenic for the F5 alpha beta TCR, specific for a peptide from the influenza nucleoprotein in the context of H-2Db, are positively selected in the H- 2b MHC background, but fail to mature in mice expressing the H-2q haplotype. Development of embryonic day 15 F5 H-2q transgenic thymocytes was followed in reaggregate cultures supplemented with H-2b- expressing epithelial clones. A conditionally immortalized cortical epithelial clone, derived from H-2Kb-tsA58 transgenic mice, was found to be as efficient as freshly isolated thymic stromal cells in positively selecting CD8 transgenic thymocytes. In contrast, an H-2b- expressing kidney epithelial clone did not augment positive selection above background levels, implying that the effect of the thymic epithelial clone was not merely the presentation of selecting MHC molecules. Mature transgenic thymocytes generated in reaggregate cultures were able to differentiate into functionally competent cytotoxic T cells. This model provides an important in vitro system for the detailed study of the specific molecular interactions leading to positive selection of developing thymocytes.      

Effect of an inhibitor of DNA methylation on T cells. II. 5-azacytidine induces self-reactivity in antigen-specific T4 cells

During T-cell maturation, thymocytes interact with thymic stromal major histocompatibility complex (MHC) determinants and thymic hormones, and proliferate, apparently in response to MHC gene products, in the absence of antigen. The maturing thymocytes also express a series of cell surface molecules, at one stage coexpressing T4, T6, and T8. Mature T cells express either T4 or T8, lack T6, bear the T3-Ti receptor complex on the cell surface, and require antigen in addition to MHC determinants to proliferate. To study whether DNA methylation may be involved in regulating phenotypic and functional changes observed during thymocyte maturation, cloned, T4⁺ Interleukin-2 dependent, antigen-specific T cells were treated with an inhibitor of DNA methylation, 5-azacytidine (5-azaC). The 5-azaC treated cells lost the requirement for antigen and could be activated by autologous macrophages alone. Anti-class II and anti-T3, but not anti-class I monoclonal antibodies, inhibited activation of 5-azaC treated T4⁺ cells by macrophages, implying that the T3-Ti receptor complex may be recognizing class II MHC molecules without antigen. No changes in T3 and T4 expression were noted, and neither T8 nor T6 was induced.     

The influence of invariant chain on the positive selection of single T cell receptor specificities

The appearance of peptide-loaded major histocompatibility complex (MHC) class II molecules at the cell surface depends critically on the invariant chain (Ii). We have studied the influence of Ii on the positive selection of CD4⁺ T cells, mediated by class II molecules expressed on thymic stromal cells. Invariant chain-deficient mice (Ii°) were crossed with different T cell receptor (TcR) transgenic strains and the emergence of mature CD4 single-positive thymocytes measured in Ii°/TcR transgenic offspring. Positive selection was nearly absent in Ii°/2B4 mice, which display receptors specific for a moth cytochrome c (MCC) peptide in the context of E^k. In addition, no T cell response was elicited when nontransgenic Ii° animals were injected with this peptide, even though antigenpresenting cells (APC) from such mice were perfectly capable of presenting it, suggesting that selection of the entire anti-MCC 88-103 repertoire depends on Ii. Positive selection also appeared strongly reduced in another line of Ii°/TcR transgenic mice (Ii°/BDC2.5). However, in sharp contrast, a third line (Ii°/3A9) exhibited almost normal positive selection of thymocytes displaying the transgene-encoded receptor. These thymocytes were exported to the periphery; peripheral T cells could respond normally to the appropriate peptide in vitro. The most likely interpretation of these findings is that selection of most CD4⁺ T cells depends on MHC class II complexes loaded with peptide in an Ii-dependent pathway, but some can be selected on class II complexes that are either loaded along an alternative, Ii-independent, route or are empty. This is consistent with the involvement of peptide in positive selection of CD4⁺ T cells, for which there exists little prior evidence.     

Influence of glycosylphosphatidylinositol-linked H-2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)-linked H-2D^d molecule. The GPI-linked H-2D^d molecule is recognized by H-2D^d-specific antibodies and cytotoxic T lymphocytes. Expression of the GPI-linked H-2D^d molecule on H-2^b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H-2^b, H-2D^d) from rejection by NK cells. In addition, NK cells from mice expressing the GPI-linked H-2D^d molecule as a transgene were able to kill nontransgenic H-2^b lymphoblast target cells. The GPI-linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.     

T cell receptor targeting to thymic cortical epithelial cells in vivo induces survival,activation and differentiation of immature thymocytes

We report that targeting of T cell receptors (TcR) to non-major histocompatibility complex (MHC) molecules on thymic cortical epithelial cells by hybrid antibodies in vivo and in fetal thymic organ cultures results in phenotypic and functional differentiation of thymocytes. A single pulse with hybrid antibodies rescues immature, CD4/8 double-positive thymocytes from their programmed death in vivo, induces expression of the early activation antigen CD69 followed by TcR up-regulation, concomitant down-regulation of CD8 or CD4 and their conversion to functional mature T cells by day 3. This temporal sequence of maturation only affects small thymocytes without co-induction of blastogenesis. TcR targeting to MHC class II-positive epithelial cells predominantly induces CD4-positive T cells. This generation of CD4 single-positive T cells occurs also in MHC class II-deficient mice and thus is independent of CD4-MHC class II interactions. Moreover, in the presence of a specific deleting antigen (Mls 1^a),TcR targeting results in transient activation of immature thymocytes, however, not in subsequent TcR (Vβ6) up-regulation and development of single-positive T cells. Our findings imply that TcR cross-linking to cortical epithelial cells is sufficient to confer a differentiation signal to immature thymocytes. Futhermore, this approach distinguishes two independent TcR-mediated intrathymic events: activation and subsequent deletion of the same thymocyte subset.     

Allogeneic recognition of class I molecules: Anti-H-2Ld repertoire of H-2b mice includes T cells recognizing mutant class II H-2b (Abm12) molecules

Two major histocompatibility complex (MHC) class I-reactive T cell clones derived from H-2^b mice, generated against the allogeneic L^d molecule, were found to recognize the H-2^b class II mutant A^bm12 molecule as well. In addition, these clones also recognize the class II A^s molecule, and display a class II-dependent reactivity to staphylococcal enterotoxin B. Neither the class I nor the class II alloreactivities of the clones were found to be dependent on other MHC molecules. Both clones express CD4+CD8^? phenotypes. The CD4 molecule appears to be involved in their class II reactivity, while little or no role for CD4 could be detected in the class I reactivity. This is the first report of a class I/class II cross-reactivity being mediated by CD4⁺ T cells. The structural basis for this cross-reactivity is discussed.     

Tolerance induction as a multi-step process

Tolerant T cells are characterized by their partial or full resistance to activation by antigen. We investigated whether tolerant T cells were still receptive to further tolerogenic signals. T cells expressing a transgenic T cell receptor (TCR) specific for the major histocompatibility complex (MHC) class I molecule K^b were deleted in mice carrying K^b but not in mice expressing the mutant K^b-molecule K^bm1 [TCR (H-2^{bm1 × k}) mice]. These T cells were tolerant in vivo but could be activated in vitro by the K^b antigen. This in vitro reactivity was abolished after the tolerant T cells encountered K^b-positive cells that had been intravenously injected. Furthermore, in TCR (H-2^{bm1 × k}), mice expressing K^b only on hepatocytes, no T lymphocytes bearing the transgenic TCR could be found in the periphery, indicating that the additional contact with K^b on hepatocytes led to deletion of the tolerant T cells. These findings demonstrate that tolerance induction can be a multi-step process.     

Generation of mature T cell populations in the thymus: CD4 or CD8 down-regulation occurs at different stages of thymocyte differentiation

We have studied the differentiation and repertoire selection during the maturation of CD4⁺CD8⁺ (DP) thymocytes into CD4⁺CD8^- (CD4SP) and CD8⁺CD4^- (CD8SP) T cells, in normal mice, mice transgenic for T cell receptor (TcR)-αβ restricted by either class I or class II major histocompatibility (MHC), and in mice deficient in class I or class II MHC expression. Our data suggest that mature CD4 and CD8 T cells derive from different pathways of T cell differentiation in the thymus. Thus, interaction of DP thymocytes with MHC class II leads to the immediate down-regulation of CD8, which occurs simultaneously with an increase in TcR expression; DPTcR^loHSA^hi thymocytes mature into a CD4⁺CD8^lo TcR^hiHSA^hi intermediate population. This cell population generates CD4SP thymocytes, the majority of which are still HSA^hi. In contrast, interaction with MHC class I induces the up-regulation of TcR, which precedes the down-regulation of CD4; DPTcR^lo generate DPTcR^hi thymocytes, the majority of which are the committed precursors of CD8SP cells. Further differentiation results in CD4 down-regulation and the transition from DPTcR^hi into CD8⁺CD4^lo TcR^hiHSA^lo and CD8SPTcR^hiHSA^- T cells. Since down-regulation of CD4 and CD8 occurs at different stages of thymocyte differentiation, our results do not support a stochastic/selective model of lineage commitment in the thymus.     

Differential reactivity of residual CD8+ T lymphocytes in TAP1 and β2-microglobulin mutant mice

TAP1 -/- and β2-microglobulin (β2m) -/- mice (H-2^b background) express very low levels of major histocompatibility complex (MHC) class I molecules on the cell surface. Consequently these mice have low numbers of mature CD8⁺ T lymphocytes. However, TAP1 -/- mice have significantly higher numbers of CD8⁺ T cells than β2m -/- mice. Alloreactive CD8⁺ cytotoxic T lymphocyte (CTL) responses were also stronger in TAP1 -/- mice than in β2m -/- mice. Alloreactive CTL generated in TAP1 -/- and β2m -/- mice cross-react with H-2^b-expressing cells. Surprisingly, such cross-reactivity was stronger with alloreactive CTL from β2m -/- mice than with similar cells from TAP1 -/- mice. The β2m -/- mice also responded more strongly when primed with and tested against cells expressing normal levels of H-2^b MHC class I molecules. Such H-2^b-reactive CD8⁺ CTL from β2m -/- mice but not from TAP1 -/- mice also reacted with TAP1 -/- and TAP2-deficient RMA-S cells. In contrast, H-2^b-reactive CD8⁺ CTL from neither β2m -/- mice nor TAP1 -/- mice killed β2m -/- cells. In line with these results, β2m -/- mice also responded when primed and tested against TAP1 -/- cells. We conclude that the reactivity of residual CD8⁺ T cells differs between TAP1 -/- and β2m -/- mice. The MHC class I-deficient phenotype of TAP1 -/- and β2m -/- mice is not equivalent: class I expression differs between the two mouse lines with regard to quality as well as quantity. We propose that the differences observed in numbers of CD8⁺ T cells, their ability to react with alloantigens and their cross-reactivity with normal H-2^b class I are caused by differences in the expression of MHC class I ligands on selecting cells in the thymus.     

Efficient presentation of endogenous superantigen by H-2Aq

Endogenous superantigens encoded by mouse mammary tumor viruses associate with MHC class II and interact with T cells bearing particular Vβ gene segments. H-2E is more efficient at presentation than H-2A, indeed A^q has not been shown to be capable of presenting endogenous superantigens. Atypically, the superantigen vSAG-3 encoded by Mtv-3 is presented efficiently in non-obese diabetic (H-2^g7) mice by H-2A; we have examined the independent contributions of vSAG-3 and A^g7 to this process. A^g7 was not found to have a more general ability to efficiently present endogenous superantigens other than Mtv-3. Examination of Mtv-3-mediated thymic deletion of Vβ 3⁺ thymocytes in the presence of H-2^q additionally demonstrated the efficient presentation of vSAG-3 by A^q. Interaction of vSAG-3 with A^q and A^g7 is likely to reflect the unique sequence of Mtv-3 within the second polymorphic region previously implicated in MHC class II binding. The demonstration that mouse endogenous superantigens can be presented by a wider range of MHC haplotypes than previously thought is further evidence for their immunological impact on the mouse population.     

Lineage fate alteration of thymocytes developing in an MHC environment containing MHC/peptide ligands with antagonist properties.

A18 TCR transgenic thymocytes which are H-2E(k) restricted and normally selected into the CD4 lineage, are exclusively selected into the CD8 lineage in an H-2(q) MHC background. CD8 T cell selection in the H-2(q) background is far more efficient than default selection of A18 CD8 cells on a CD4(-/-) H-2E(k +) background. This suggests the involvement of special selecting ligands. Analogues of the cognate peptide for A18 with antagonist properties for the A18 TCR have previously been shown to effect a lineage diversion from CD4 to CD8 in fetal thymic organ cultures and intriguingly the MHC(q) background contains unidentified natural MHC class II ligands which similarly show antagonist properties for the A18 TCR. Despite the presence of these unidentified MHC class II ligands in the H-2(q) background and their potential influence on developing A18 thymocytes, however, MHC class I molecules were essential for thymic selection of A18 CD8 T cells.