The inappropriate activation or suppression of T cells can be implicated in nearly all human diseases, including cancer, heart disease, multiple sclerosis, asthma, allergies, diabetes, and many others 
. Early signaling pathways induced by both the TCR and/or costimulatory receptors are excellent targets for therapies to these diseases. Although a great deal is known about the induction of signaling by these receptors, much more must be learned before therapies can be effectively developed to target these pathways. T cell lines are an integral part of the research of T cell signaling due to their ease of use, low cost, and reduced human burden. However, not all cell lines are equally useful as a model system of human T cells. In this paper, we investigated differences in peripheral TCR signaling, cytokine release and costimulatory receptor function and expression among the Jurkat E6.1 T cell line, the HuT78 T cell line, and APBTs.
To examine the relative extent of activation of key signaling molecules in Jurkat E6.1 T cells, HuT78 T cells, and APBTs, we used a previously described method for quantifying the amount of phosphorylation per protein present in an activated T cell 
. By using this method, the total phosphorylation per protein of a specific signaling molecule for each cell type was determined and compared, regardless of the total amount of protein present. This is important since there were substantial differences between the T cell lines and APBTS in the expression of several of the examined signaling proteins, including ZAP-70, Pyk2 and Vav1. By normalizing the phosphorylation level of each protein to the absolute amount expressed in each cell type, we were able to eliminate potential artifacts due to differences in expression levels.
The first signaling event to occur upon TCR activation is the stimulation of the Src family kinases Lck and Fyn 
. We investigated the phosphorylation of Lck and Fyn at the activating tyrosine residue 394 and 420, respectively, and found that these sites had constitutive phosphorylation in Jurkat E6.1 T cells, but moderate TCR-inducible phosphorylation in HuT78s and APBTs (data not shown). However, the low level of inducible phosphorylation of Lck and Fyn made the immunoblots difficult to quantify using the described method. These differences in the regulation of Lck and Fyn did not appear to matter since only a slight decrease was seen in the phosphorylation of tyrosine 319 on the tyrosine kinase ZAP-70 in Jurkat E6.1 T cells compared to HuT78 T cells and APBTs (). This difference in ZAP-70 activation did not translate into changes in the phosphorylation of the ZAP-70 substrates, LAT and SLP-76 (). Together, these results indicate that although there are small differences in events immediately following TCR activation, the relative levels of activated LAT and SLP-76 in each cell type remains the same.
The similarities between Jurkat E6.1 T cells, HuT78 T cells and APBTs ended at signaling events downstream of LAT and SLP-76. Jurkat E6.1 T cells have deficiencies in the expression of lipid phosphatases PTEN and SHIP, leading to the constitutive activation of AKT, a serine-threonine kinase important for cell survival, and other downstream signaling molecules 
. In addition to this defect, we observed three new defects in Jurkat E6.1 T cells compared to APBTs. First, Jurkat E6.1 cells have a significant increase in the relative phosphorylation of PLCγ1 tyrosine 783 compared to HuT78 T cells and APBTs (). In contrast, HuT78 T cells had reduced levels of PLCγ1 tyrosine 775 phosphorylation compared to Jurkat E6.1 and APBTs (). Interestingly, these data indicate these two sites on PLCγ1 may have differential regulation by intracellular kinases and phosphatases. The end result of alterations in PLCγ1 phosphorylation in the various cell lines was that Jurkat E6.1 T cells exhibited a nearly 7×-fold greater TCR-induced Ca2+
flux than HuT78 T cells and APBTs (). In addition, Jurkat E6.1 T cells also had increased levels of site-specific phosphorylation of Vav1 and Erk1/Erk2 ( and ).
The reason for the distorted phosphorylation of PLCγ1 and Vav1 does not appear to be due to deficiencies in the expression of PTEN and SHIP, since we have recently shown that inhibition of PI3K does not alter the site specific phosphorylation of these proteins (NCO and JCDH, manuscript submitted). The hyperphosphorylation of Erk1/Erk2 and the increased levels of TCR-induced Ca2+
influx could, in part, be due to decreased expression of PTEN and SHIP, because PI3K signaling is needed for the optimal activation of these events in human T cells (NCO and JCDH, manuscript submitted). However, a probable mechanism for this distorted signaling appears to involve Itk, which had both higher levels of expression and phosphorylation at tyrosine 511 in Jurkat E6.1 T cells than in HuT78 T cells and APBTs (). There was some variation in the Itk expression levels in various sublines of Jurkat E6.1 T cells, with most expressing high levels of Itk (data not shown). This indicates that genetic drift in the various sublines of this T cell line leads to differences in the expression of intracellular signaling proteins. Importantly, this is the first time that the over-expression of Itk, an crucial molecule at the intersection of TCR and costimulatory receptor-mediated signaling in T cells 
, has been observed in Jurkat E6.1 cells.
Next, we found that Pyk2 was hyperphosphorylated in TCR-stimulated Jurkat E6.1 T cells compared to HuT78 T cells and APBTs (). Again, this is the first description of the increased phosphorylation of Pyk2 in Jurkat E6.1 T cells. The mechanism for the increased phosphorylation of Pyk2 is unknown, but does not appear to be due to alterations in the expression of PTP-PEST. Pyk2 is important since it integrates receptor signals controlling actin cytoskeletal rearrangement 
. The increased relative phosphorylation of Pyk2, in conjunction with the augmented phosphorylation of Vav1 (), suggests that the actin cytoskeletal rearrangement in Jurkat E6.1 T cells may be altered compared to HuT78 T cells and APBTs.
Finally, Jurkat E6.1 T cells release substantially fewer cytokines and chemokines upon TCR activation than HuT78 T cells and APBTs (). However, the amount of IL-2 and IL-8 released by Jurkat E6.1 T cells is substantially greater than the levels of these hormones produced by APBTs. These differences are likely due to the thymocytic origin of the Jurkat E6.1 T cells and the fact that the cells were selected for their ability to produce large amounts of IL-2 
Although this study suggests that Jurkat E6.1 T cells differ in signaling from APBTs, they do have advantages (). The cells are incredibly resilient and easy to grow and maintain. There are also many useful mutant lines of Jurkat E6.1 T cells, each lacking a specific molecule in the peripheral TCR signaling pathway. Jurkat E6.1 T cells are remarkably easy to transfect, with transfection efficiencies routinely reaching 80–90%. Finally, a hidden advantage pointed to in this study is that they have exaggerated signaling, making changes much easier to detect. Even with the increased proximal signaling, studies using Jurkat E6.1 are likely valid since many of the discoveries and observations initially found in Jurkat E6.1 T cells have been subsequently shown in APBTs. Together, the data show that although the Jurkat E6.1 T cell line has substantially altered TCR-induced signaling, this cell line can still be a useful model for TCR signaling if deficiencies and problems are taken into account and the results are confirmed in APBTs.
Phosphorylation per protein in Jurkat E6.1 and HuT78 cell lines as compared to APBTs.
Conversely, this study points to the many similarities between HuT78 T cells and APBTs. One significant difference in the relative levels of early TCR-induced signaling events between HuT78 T cells and APBTs was the reduced TCR-induced phosphorylation of Pyk2 in HuT78 T cells (). This is likely due to the expression of PTP-PEST, a known phosphatase for Pyk2, in HuT78 T cells and not activated APBTs. However, this difference may be less important than it seems, since a recent study has shown that PTP-PEST is expressed in naïve primary T cells and not activated T cells, the subtype of APBTs used for these studies 
. In addition, HuT78 T cells produce a greater number of cytokines and chemokines upon TCR stimulation than Jurkat E6.1 T cells. Finally, HuT78 T cell can express several costimulatory receptors found in activated T cells, allowing investigation of these costimulatory pathways in a model T cell line. HuT78 T cells also have disadvantages, however. They do not express the costimulatory receptors CD2 or CD28 (), and are unable to be co-stimulated by fibronectin, suggesting that although VLA-4 is expressed, it may not be functional in these cells (). In addition, HuT78 cells have elevated levels of IL-2 production without costimulation as compared to APBTs (). HuT78 T cells also have abnormal c-myc function and over express Bcl-xL, which indicates possible problems with the anti-apoptotic pathway in these cells 
. All of this information must be kept in mind when choosing to use HuT78 T cells as a model system for T cell biology.
The results of this study indicate that HuT78 T cells are similar to APBTs in peripheral TCR signaling, whereas Jurkat E6.1 T cells exhibit exaggerated signaling (). In addition, there were differences in cytokine release and costimulatory receptor expression and function between the various cell lines. These results are significant in that, for the first time, a direct comparison of commonly used cell lines and APBTs has been performed. Though it is clear that APBTs are the ideal cells to use for examining TCR signaling, this is not always feasible. Both Jurkat E6.1 T cells and HuT78 T cells have advantages and disadvantages, but HuT78 T cells are clearly the most like APBTs in the phosphorylation of important peripheral TCR signaling molecules and cytokine release. The relative advantages and disadvantages of each cell line must be taken into account when choosing a model cell line for individual experiments.