As a postdoctoral fellow in Mark Davis’s laboratory at Stanford University Medical School, my work focused on TCR repertoire selection in the thymus. At that time—the late 1980s—the first TCR-transgenic lines were being generated and were used to study positive and negative selection (1–3). The overall conclusion from this work, contributed to by many laboratories, was that strong TCR signaling led to thymocyte cell death, whereas weak or moderate TCR signaling led to thymocyte survival and maturation. Yet the biochemical pathways that regulated these alternative cell fates remained unknown.
Leslie J. Berg
As we learned more about T cells, it became clear that TCR signaling was not the only important input into the process of T cell lineage development. Both in the thymus, as well as during mature T cell differentiation following Ag stimulation, signals mediated by costimulatory molecules and cytokines contribute to the ultimate fate of each cell. Nonetheless, more and more data accumulated to indicate that the relative strength of TCR signaling was a key feature in dictating the outcome during T cell development and activation (4).
Headway in understanding this problem required more detailed knowledge of the biochemistry of TCR signaling. In the early 1980s, relatively little was known about T cell signaling. Numerous changes in cell metabolism had been documented as a result of T cell activation, but most of the specific enzymes involved had not been discovered, and the details of how specific signaling proteins were connected in pathways were not known (5). Important breakthroughs were made during the late 1980s and early 1990s that laid the groundwork for all of the subsequent studies. Following the identification of the TCR αβ heterodimer came the discovery of the TCR αβ-association CD3 subunits, the key signaling components of the receptor complex (6, 7). Shortly thereafter, L. Samelson et al. (8) demonstrated that the activated TCR was tyrosine phosphorylated, and J. Imboden and A. Weiss (9–11), among others, showed that TCR stimulation led to phosphoinositide hydrolysis and calcium influx. The first T cell–specific tyrosine kinase, Lck, along with a second src family kinase, Fyn, was then shown to bind to the TCR and to be required for TCR signaling (12, 13). This discovery was followed soon after by the identification of Zap-70, the key enzyme required for all downstream TCR-signaling events (14).
Our own contribution to this field was the cloning of a third T cell tyrosine kinase important for TCR signaling, the Tec kinase ITK (15). We had set out, in the early 1990s, to identify novel T cell tyrosine kinases using degenerate PCR to amplify conserved kinase domain sequences. This effort led to the discovery of a novel Jak family kinase, Jak3, along with ITK. Although Jak3 was found to be essential for cytokine receptor signaling (16), ITK, we then showed, was involved in TCR signaling (17). Over the next several years, the specific biochemical function of ITK was elucidated. Following TCR stimulation, ITK is activated downstream of Lck and Zap-70 and, in turn, phosphorylates and activates phospholipase C-γ1. Phospholipase C-γ1 then generates the two critical second messengers, diacylglycerol and inositol tris-phosphate, leading to calcium mobilization, protein kinase C activation, and activation of the MAPK cascade. As a consequence, when ITK is absent from T cells, these responses are significantly impaired, and T cells exhibit marked reductions in effector functions, such as cytokine production (18).
It came as a surprise to discover that, despite this signaling defect, T cell development in ITK-deficient mice appeared largely intact (18). However, subsequent studies conducted by several laboratories over the ensuing several years demonstrated that T cell development and function were hardly normal in the absence of ITK. Th cell differentiation was impaired (19–21), T cell responses to some classes of pathogens were defective (19, 20, 22), and a closer look at T cell development revealed that the majority of mature cells in the thymus resembled memory T cells, rather than conventional naive CD4+ and CD8+ thymocytes (23, 24).
Following up on this altered T cell development in Itk−/− mice led us to the finding that the memory-phenotype CD8+ T cells found in these mice expressed high levels of the T-box transcription factor, eomesodermin (24). This factor was previously found to be upregulated in bona fide memory CD8+ T cells, and it can promote memory cell development (25). As shown by Kris Hogquist and colleagues (26, 27), excess IL-4 present in Itk−/− mice was required for eomesodermin upregulation by CD8+ thymocytes. However, we reasoned that reduced TCR signaling in the absence of ITK might also be contributing to the development of memory-phenotype, rather than conventional naive, CD8+ T cells.
To test this hypothesis, we performed a gene-expression microarray experiment, focusing on identifying transcription factors that might be regulated by TCR signaling via ITK and, in turn, be regulating eomesodermin expression. This led us to a second transcriptional regulator, IRF4. IRF4, which is not expressed in naive CD8+ T cells, was rapidly induced by TCR stimulation. Furthermore, IRF4 upregulation was dependent on ITK signaling and was induced in a graded manner dependent on the strength of TCR signaling. Thus, the stronger the TCR signal, the more ITK activation occurred; as a consequence, the entire population of CD8+ T cells expressed higher levels of IRF4 as a function of the magnitude of ITK activation. Most importantly, these studies demonstrated that cells expressing little or no IRF4 upregulated high levels of eomesodermin and that this response occurred within 24 h after TCR stimulation (28).
These experiments led us to conclude that ITK is a distinct type of signaling protein, whose function is to provide a graded response in T cells stimulated by different strengths of TCR signaling. This variation in TCR signaling could result from TCRs recognizing MHC/peptide ligands with distinct affinities, as we showed in our in vitro studies, or it could be due to different amounts of Ag encountered by T cells activated in vivo. We conclude that some signaling proteins, like the essential TCR kinase, Zap-70, provide an “on–off” switch to regulate T cell activation. In contrast, we imagine ITK as the mechanism inside a water faucet: turning the handle a small amount produces a trickle of water, whereas cranking the faucet handle all of the way open produces a gushing stream of water. In the case of ITK, the magnitude of ITK activation is not simply regulating how many cells are responding, but instead is producing a distinct gene-expression program and, thus, a distinct lineage of T cell development or differentiation, depending on the strength of TCR stimulation.
Disclosures
The author has no financial conflicts of interest.
- Copyright © 2012 by The American Association of Immunologists, Inc.
References
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