DEVELOPMENTAL REGULATION OF T CELL ANTIGEN RECEPTOR EXPRESSION AND FUNCTION

DAVID L. WIEST, Ph.D., Associate Member

The specific recognition and destruction of target cells (e.g., tumors) by cytotoxic T lymphocytes is mediated by a structure on the T cell surface known as the T cell antigen receptor (TCR) complex, which upon encountering its cognate ligand transduces activation signals to the T cell cytoplasm. Since the ligand-binding and signaling capabilities of the TCR reside on distinct polypeptide subunits (TCRab subunits bind antigen while CD3gde and z subunits transduce signals), it is essential for TCR function that these distinct subunits only be expressed on the cell surface as part of a complete complex. Surface expression of only complete TCR complexes is ensured by tight control of both subunit assembly in the endoplasmic reticulum (ER) and the subsequent movement of those complexes through the network of membrane-bound organelles termed the "secretory apparatus".

TCR complexes are not only vital for the function of mature T lymphocytes, but are also responsible for directing the maturation of immature T cell precursors in the thymus. T cell maturation can be subdivided into two transitions (Figure 1). During the first transition, immature precursors attempt to construct the antigen binding subunits of the TCR (first b and then a) by recombining individual gene segments into a single coding unit. If recombination of the gene segments comprising the TCRb locus is unsuccessful, the precursors die. Successful recombination of the TCRb locus promotes survival by activating an isoform of the TCR called the pre-TCR complex. Pre-TCR signals drive proliferative expansion, maturation of T cell precursors from the CD4^-CD8^- (double negative or DN) to the CD4⁺CD8⁺ (double positive or DP) stage of development, and rearrangement of the TCRa locus. DP thymocytes that have productively rearranged both of their TCRb and TCRa loci are eligible to attempt the second transition, during which precursor survival is determined by the specificity of the TCR complex they express. Cells bearing "harmful" TCRs, such as those mediating autoimmune diseases, are eliminated, whereas cells bearing "useful" TCR complexes, such as those specific for invading pathogens or tumor cells, mature and become immunocompetent. The screening process that determines if a TCR is useful remains poorly understood, but is quite sensitive to the levels of TCR expressed. Indeed, fluctuations in TCR surface density adversely affect the outcome of selection events.

Our laboratory is currently investigating the way(s) in which thymocyte development is controlled by antigen receptors. Specifically, we seek to: 1) understand how pre-TCR complexes are used to alert developing thymocytes that TCRb rearrangement has been productive; 2) identify the gene products induced by pre-TCR signaling that propel development of DN thymocytes to the DP stage; and, 3) understand how controlling the efficiency of subunit assembly within the ER maintains abTCR surface expression on DP thymocytes at precisely the correct level required for normal selection.

Previously, we reported that primary thymocytes express "conventional pre-TCR complexes" comprising disulfide linked pTa-TCRb heterodimers associated with CD3gde and TCRz. We now have evidence that immature thymocytes express an additional isoform. This isoform, the pTa-X complex, is not recognized by anti-TCRb antibodies (Ab) and is biochemically distinct from conventional pre-TCR complexes in that: 1) although not recognized by anti-TCRb Ab, it comprises pTa-TCRb dimers associated with CD3ge and TCRz, but not CD3d; 2) the pTa-TCRb dimers from the pTa-X complex exhibit a larger relative molecular mass (M_r) than do the pTa-TCRb dimers found in conventional pre-TCRs; and, 3) it is expressed on some immature thymic lymphomas but not others, suggesting the possibility that the pTa-X complex may be developmentally regulated. We are currently investigating this possibility by assessing the distribution of pTa-X complexes among subpopulations of developing thymocytes. Additionally, we have been and continue working toward understanding why the TCR b polypeptide of pTa-X complexes has a larger M_r (than those of conventional pre-TCRs) and why they are not recognized by anti-TCRb Ab. The larger M_r is due to extensive modification of pTa with O-linked glycan moieties--carbohydrates appended to serine or threonine residues. These extensive O-linked modifications correlate with, and may be responsible for, the lack of recognition by anti-TCRb Ab. Experiments are in progress to test whether preventing addition of O-linked modifications can restore Ab recognition. In view of their lack of recognition by anti-TCRb Ab, we hypothesize that the potential physiologic significance of the pTa-X complexes, which account for half the pre-TCR complexes expressed by immature thymocytes in vivo, may be their inability to be engaged by potential extracellular ligands. Consequently, they may represent "silent" forms of the pre-TCR which are unable to be triggered and may even interfere with pre-TCR signaling.

One step toward understanding the way in which pre-TCR signaling is triggered in vivo was to establish an in vitro model system that will allow us to study pre-TCR signaling and to identify downstream molecular targets of pre-TCR signals, particularly those targets that are required for executing the differentiation program. To this end, we have established such a system, the thymic lymphoma, scid.adh, which exhibits the potential to differentiate in vitro when exposed to stimuli (e.g., TAC:CD3e engagement; see below) that mimic pre-TCR signaling in vivo. Scid.adh arose spontaneously in a mouse bearing the scid mutation, which prevents the recombination events necessary to generate the antigen binding subunits of the TCR complex, TCRa and TCRb. Consequently, the phenotype of scid.adh is similar to that expected for immature thymocytes poised to receive a pre-TCR signal, i.e., CD44^-CD25⁺CD4^-CD8^-. Moreover, stimulation by Ab crosslinking of the TAC:CD3e signaling chimera (CD3e signaling domain fused to the exodomain of the human interleukin-2 receptor a subunit, TAC) induces changes in gene expression known to be induced by pre-TCR signaling, including: 1) upregulation of CD2, CD5, CD27, and CD28 surface markers as well as molecular markers such as the germline transcripts from the TCRa constant region and the transcription factor Egr-1; and, 2) downregulation of CD25 as well as the molecular markers pre-Ta and the recombinase activating genes (RAG) 1 and RAG2. Taken together, these results indicate that stimuli that mimic pre-TCR signaling can induce scid.adh to execute at least some of the steps associated with the DN to DP transition. Consequently, scid.adh has the potential to serve as a useful tool with which to investigate the regulation of pre-TCR function as well as to identify its downstream molecular targets.

Most cell surface receptor complexes are activated by ligand engagement, for which Ab-stimulation frequently serves as an effective surrogate. However, this does not appear to be true for the pre-TCR complex. Ab- engagement of surface pre-TCR complexes in vivo fails to promote maturation of thymocytes to the DP stage, instead arresting their development. Moreover, removal of the potential ligand-binding exodomains of TCRb and pTa does not abrogate the ability of the pre-TCR to support development of thymocytes to the DP stage. Taken together, these data suggest that ligand engagement of surface pre-TCR complexes may not be responsible for pre-TCR activation in vivo and that transmembrane domain-mediated preTCR complex assembly may be sufficient. Two ligand-independent models of pre-TCR triggering have been proposed. The first posits that assembly of TCRb with the remaining pre-TCR subunits and transport to the cell surface are sufficient to trigger pre-TCR signaling, even in the absence of ligand engagement. The second suggests that surface expression of pre-TCR complexes may not be necessary and that pre-TCR signaling is triggered internally, while the pre-TCR is en route to the cell surface. These models are currently unresolved. We have begun testing these models in the scid.adh cell line and our data parallels what has been shown in normal thymocytes in vivo. Indeed, while TAC:CD3e-mediated stimulation of scid.adh induces the battery of gene expression changes described above (which we term in vitro maturation), stimulation of surface pre-TCR complexes with anti-TCRb Ab fails to do so. For example, flow cytometric analysis revealed that while anti-TCRb stimulation of scid.adh is able to upregulate CD5 expression, indicating that a signal was transduced, that signal was unable to achieve significant downregulation of the differentiation marker CD25, one of the earliest events know to follow pre-TCR signaling (Figure 2). Thus, as is true for normal thymocytes in vivo, Ab-engagement of surface pre-TCR complexes is unable to induce in vitro maturation of scid.adh. Experiments are currently in progress to determine why Ab-stimulation of surface pre-TCR complexes is unable to drive the in vitro maturation of scid.adh.

DEVELOPMENTAL CONTROL OF TCR ASSEMBLY. WIEST, RHODES

The antigen-driven selection events that cause immature DP thymocytes to mature to the CD4 or CD8 single positive stage are critically dependent upon precise control of the level of TCR expressed on the surface of pre-selection DP thymocytes. DP thymocytes synthesize as much of each TCR subunit as do mature T cells, yet thymocytes express ~10-fold fewer abTCR complexes on the cell surface. Consequently, a post-translational regulatory mechanism must be responsible for limiting TCR expression in the immature thymocytes. We have shown previously that this form of regulation targets the process of TCR subunit assembly in the ER. In particular, it is the TCRa and/or TCRz subunits that appear to be most directly affected. We seek to determine if TCRa, TCRz, or both are involved in limiting TCR assembly and to determine the molecular basis for this regulation. By using gene-targeted mice in which one allele of TCRz is ablated (decreasing z expression 2-fold), we have shown that a two-fold decrease in TCRz expression causes a proportional reduction in the number of surface TCR complexes. This observation was particularly surprising considering only ~1/10 of newly synthesized z molecules are actually assembled into complete TCR complexes. These results suggest that, despite the fact that most nascent TCRz molecules are not assembled into the TCR complex, TCR assembly is extremely sensitive to the amount of the TCRz that is made. Our laboratory has generated several cell lines with which to study this phenomenon. Experiments are currently in progress to overexpress TCRz and TCRa in these cell lines to examine if overexpression is able to compensate for the inefficient TCR assembly phenotype. Once the subunit(s) responsible for limiting TCR assembly is identified, we plan to employ a mutagenesis strategy to identify the protein domains important in this process. Curiously, we recently observed a protein that associates with TCRz only in immature thymocytes and we are in the process of isolating it and investigating its potential role in TCR assembly.

DAVÉ, V.P., ALLMAN, D., WIEST, D.L., KAPPES, D.J. Limiting TCR expression leads to quantitative but not qualitative changes in thymic selection. J. Immunol. (in press).

DAVÉ, V.P., KEEFE, R., BERGER, M.A., DRBAL, K., PUNT, J.A., WIEST, D.L., ALARCON, B., KAPPES, D.J. Altered functional responsiveness of thymocyte subsets from CD3d-deficient mice to TCR/CD3 engagement. Int. Immunol. 10:1481-1490, 1998.

FULLER-ESPIE, S., TOWLER, P.H., WIEST, D.L., TIETJEN, I., SPAIN, L.M. Transmembrane polar residues of tcr b chain are required for signal transduction. Int. Immunol. 10:923-933, 1998.

TROP, S., STEFF, A.-M., DENIS, F., WIEST, D.L., HUGO, P. The connecting peptide of pTa dictates weak association of the pre-TCR with the TCR-z subunit. Eur. J. Immunol. (in press).

WASSERMAN, R., LI, Y.-S., SHINTON, S.A., CARMACK, C.E., MANSER, T., WIEST, D.L., HAYAKAWA, K., HARDY, R.R. Contrasting responses by fetal and adult B cell precursors to immunoglobulin heavy chain-surrogate light chain assembly. J. Exp. Med. 187:259-264, 1998.

WIEST, D.L., CARLETON, M.O. Protein synthesis and intracellular sorting. In Hematology: Basic Principles and Practice, Third Edition, edited by P. McGlave. Churchill Livingstone Inc., New York (in press).

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998