Contributions of the T cell receptor-associated CD3gamma-ITAM to thymocyte selection.

The immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 chains associated with the T cell receptor (TCR) are crucial for TCR signaling. To probe the role of the CD3gamma-ITAM in T cell development, we created knock-in mice in which the CD3gamma chain of the TCR complex is replaced by a mutant signaling-deficient CD3gamma chain, lacking the CD3gamma-ITAM. This mutation results in considerable impairment in positive selection in the polyclonal TCR repertoire. When CD3gamma-deltaITAM mice are crossed to mice expressing transgenic F5 TCRs, their thymocytes are completely unable to perform positive selection in vivo in response to intrathymic ligands. Also, the in vitro positive selection response of double-positive (DP) thymocytes with F5-CD3gamma-deltaITAM mutant receptors to their agonist ligand and many of its variants is severely impaired or abrogated. Yet, the binding and dissociation constants of agonist ligands for the F5 receptor are not affected by the CD3gamma-deltaITAM mutation. Furthermore, DP thymocytes with mutant receptors can respond to agonist ligand with normal antigen sensitivity and to normal levels, as shown by their ability to induce CD69 up-regulation, TCR down-regulation, negative selection, and ZAP70 and c-Jun NH2-terminal kinase activation. In sharp contrast, induction of extracellular signal-regulated kinase (ERK) activation and linker for activation of T cells (LAT) phosphorylation are severely impaired in these cells. Together, these findings underscore that intrinsic properties of the TCR-CD3 complex regulate selection at the DP checkpoint. More importantly, this analysis provides the first direct genetic evidence for a role of the CD3gamma-ITAM in TCR-driven thymocyte selection.

A robust automated method to analyze rodent motion during fear conditioning.

A central question in the study of LTP has been to determine what role it plays in memory formation and storage. One valuable form of learning for addressing this issue is associative fear conditioning. In this paradigm an animal learns to associate a tone and shock, such that subsequent presentation of a tone evokes a fear response (freezing behavior). Recent studies indicate that overlapping cellular processes underlie fear conditioning and LTP. The fear response has generally been scored manually which is both labor-intensive and subject to potential artifacts such as inconsistent or biased results. Here we describe a simple automated method that provides unbiased and rapid analysis of animal motion. We show that measured motion, in units termed significant motion pixels (SMPs), is both linear and robust over a wide range of animal speeds and detection thresholds and scores freezing in a quantitatively similar manner to trained human observers. By comparing the frequency distribution of motion during baseline periods and to the response to fox urine (which causes unconditioned fear), we suggest that freezing and non-freezing are distinct behaviors. Finally, we show how this algorithm can be applied to a fear conditioning paradigm yielding information on long and short-term associative memory as well as habituation. This automated analysis of fear conditioning will permit a more rapid and accurate assessment of the role of LTP in memory.

Gene transfer of MHC-restricted receptors.

Adoptive therapy with allogeneic or tumor-specific T-cells has shown substantial clinical effects for several human tumors, but the widespread application of this strategy remains a daunting task. The antigen specificity of T-lymphocytes is solely determined by the T-cell receptor (TCR) alpha and beta chains. Consequently, genetic transfer of TCR chains may form an alternative and potentially appealing strategy to impose a desirable tumor-antigen specificity onto cytotoxic or helper T-cell populations. In this strategy, autologous or donor-derived T-cell populations are equipped with a TCR of defined reactivity in short-term ex vivo cultures, and re-infusion of the redirected cells is used to supply T-cell reactivity against defined tumor-specific antigens. We have previously described the genetic introduction of T-cell receptor genes into peripheral T-cells in mouse model systems. Here we discuss the requirements for the successful genetic modification of murine T-lymphocytes and the subsequent use of such genetically modified cells in in vivo models.

Generation of T cell help through a MHC class I-restricted TCR.

CD4+ T cells that are activated by a MHC class II/peptide encounter can induce maturation of APCs and promote cytotoxic CD8+ T cell responses. Unfortunately, the number of well-defined tumor-specific CD4+ T cell epitopes that can be exploited for adoptive immunotherapy is limited. To determine whether Th cell responses can be generated by redirecting CD4+ T cells to MHC class I ligands, we have introduced MHC class I-restricted TCRs into postthymic murine CD4+ T cells and examined CD4+ T cell activation and helper function in vitro and in vivo. These experiments indicate that Ag-specific CD4+ T cell help can be induced by the engagement of MHC class I-restricted TCRs in peripheral CD4+ T cells but that it is highly dependent on the coreceptor function of the CD8beta-chain. The ability to generate Th cell immunity by infusion of MHC class I-restricted Th cells may prove useful for the induction of tumor-specific T cell immunity in cases where MHC class II-associated epitopes are lacking.

Specificity and affinity motifs for Grb2 SH2-ligand interactions.

Protein-protein interactions are often mediated by the recognition of short continuous amino acid stretches on target proteins by specific binding domains. Affinity-based selection strategies have successfully been used to define recognition motifs for a large series of such protein domains. However, in many biological systems specificity of interaction may be of equal or greater importance than affinity. To address this issue we have developed a peptide library screening technology that can be used to directly define ligands for protein domains based on both affinity and specificity of interaction. We demonstrate the value of this approach by the selection of peptide ligands that are either highly specific for the Grb2 Src homology 2 (SH2) domain or that are cross-reactive between a group of related SH2 domains. Examination of previously identified physiological ligands for the Grb2 SH2 domain suggests that for these ligands regulation of the specificity of interaction may be an important factor for in vivo ligand selection.

The impact of self-tolerance on the polyclonal CD8+ T cell repertoire.

TCRs possess considerable cross-reactivity toward structurally related Ags. Because the signaling threshold for negative selection is lower than that required for activation of mature T cells, the question arises as to which extent thymic deletion of self-specific T cells affects T cell responsiveness toward foreign peptides. In this study we show, in three different mouse models systems, that the polyclonal CD8(+) T cell repertoire has a marked ability to react against the majority of Ags related to self despite self-tolerance, even in cases where self and foreign differ only marginally at a single TCR-contact residue. Thus, while individual T cells are markedly cross-reactive, the ability to distinguish between closely related Ags is introduced at the polyclonal T cell level.