Comparison of T Cell Receptor-Induced Proximal
`Signaling and Downstream Functions in Immortalized
`and Primary T Cells
`
`Rebekah R. Bartelt, Noemi Cruz-Orcutt, Michaela Collins, Jon C. D. Houtman*
`
`Department of Microbiology, University of Iowa, Iowa City, Iowa, United States of America
`
`Abstract
`
`Background: Human T cells play an important role in pathogen clearance, but their aberrant activation is also linked to
`numerous diseases. T cells are activated by the concurrent induction of the T cell receptor (TCR) and one or more
`costimulatory receptors. The characterization of signaling pathways induced by TCR and/or costimulatory receptor
`activation is critical, since these pathways are excellent targets for novel therapies for human disease. Although studies
`using human T cell
`lines have provided substantial
`insight into these signaling pathways, no comprehensive, direct
`comparison of these cell lines to activated peripheral blood T cells (APBTs) has been performed to validate their usefulness
`as a model of primary T cells.
`
`Methodology/PrincipalFindings: We used quantitative biochemical techniques to compare the activation of two widely
`used human T cell lines, Jurkat E6.1 and HuT78 T cells, to APBTs. We found that HuT78 cells were similar to APBTs in
`proximal TCR-mediated signaling events.
`In contrast, Jurkat E6.1 cells had significantly increased site-specific
`phosphorylation of Pyk2, PLCc1, Vav1, and Erk1/Erk2 and substantially more Ca2+ flux compared to HuT78 cells and
`APBTs. In part, these effects appear to be due to an overexpression of Itk in Jurkat E6.1 cells compared to HuT78 cells and
`APBTs. Both cell lines differ from APBTs in the expression and function of costimulatory receptors and in the range of
`cytokines and chemokines released upon TCR and costimulatory receptor activation.
`
`Conclusions/Significance:Both Jurkat E6.1 and HuT78 T cells had distinct similarities and differences compared to APBTs.
`Both cell lines have advantages and disadvantages, which must be taken into account when choosing them as a model T
`cell line.
`
`Citation: Bartelt RR, Cruz-Orcutt N, Collins M, Houtman JCD (2009) Comparison of T Cell Receptor-Induced Proximal Signaling and Downstream Functions in
`Immortalized and Primary T Cells. PLoS ONE 4(5): e5430. doi:10.1371/journal.pone.0005430
`
`Editor: Derya Unutmaz, New York University School of Medicine, United States of America
`
`Received January 23, 2009; Accepted April 9, 2009; Published May 4, 2009
`Copyright: ß 2009 Bartelt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
`unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
`
`Funding: Start-up Funds from the Department of Microbiology at the University of Iowa to JCDH, American Heart Association-Scientist Development Grant
`Number 0830244N to JCDH (www.americanheart.org), American Cancer Society Seed Grant from the Holden Comprehensive Cancer Center at the University of
`Iowa to JCDH, McCord Graduate Fellowship for Wound Healing to RRB. The funders had no role in study design, data collection and analysis, decision to publish,
`or preparation of the manuscript.
`
`Competing Interests: The authors have declared that no competing interests exist.
`
`* E-mail: jon-houtman@uiowa.edu
`
`Introduction
`
`Human T cells control the extent and focus of the adaptive
`immune response to pathogens. T cells are activated by the
`interaction of the cell surface, multi-subunit T cell receptor (TCR)
`with an antigen-bound major histocompatibility complex present
`on an antigen presenting cell [1,2]. In addition to TCR induction,
`T cells also require an activating signal
`from one or more
`costimulatory receptors, such as CD28 or the a4b1 integrin VLA-
`4, to become fully active [1]. Costimulation is critical for the
`specificity of the immune response because it allows T cells to be
`activated only during acute infection. This enables the adaptive
`immune system to mount a response to foreign invaders while
`tolerating its own cells. The mistaken recognition of self leads to
`aberrant T cell activation, resulting in numerous human disease,
`such as autoimmune diseases, cardiovascular disease and allergies/
`asthma [3,4,5]. Signaling pathways
`that are activated by
`TCR and/or costimulatory receptors are good targets for the
`
`development of therapies to these diseases [4,5]. However, before
`effective therapies can be developed, we must
`first better
`understand the intracellular signaling that occurs when a T cell
`is activated.
`An initial event upon TCR activation is the induction of the Src
`family kinases Lck and Fyn, which then phosphorylate the
`immunoreceptor tyrosine-based activation motifs (ITAMs) present
`on several TCR subunits (Figure 1) [1]. The protein tyrosine
`kinase ZAP-70 is recruited to the phosphorylated ITAMs and
`activated upon phosphorylation of tyrosine 319 [1]. Activated Lck,
`Fyn, and ZAP-70 then phosphorylate multiple downstream
`substrates, including linker for activation of T cells (LAT) and
`the tyrosine kinase Pyk2 [6,7,8]. Pyk2 is a member of the Fak
`family of kinases and appears
`to control actin cytoskeletal
`rearrangements that are critical for T cell activation [6]. LAT is
`a hematopoietic-specific adaptor protein that mediates many
`downstream events
`following TCR stimulation. Upon TCR
`activation, LAT is phosphorylated on five conserved tyrosines,
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`Comparison of T Cell Lines
`
`Figure 1. Current model of proximal signaling pathways downstream of TCR activation. TCR activation leads to the induction of
`numerous tyrosine kinases and adaptor proteins. The activation of these signaling molecules leads to morphological changes and alterations in
`transcription that are vital for T cell activation and function. Proteins that are tyrosine phosphorylated upon TCR stimulation are identified with small
`black circles.
`doi:10.1371/journal.pone.0005430.g001
`
`which then bind to several SH2 domain-containing proteins, such
`as the related adaptor proteins Grb2, Grap, and Gads, as well as
`PLC-c1[8]. Once recruited to LAT, PLC-c1 is phosphorylated by
`the Tec family tyrosine kinase Itk at tyrosine 783. This leads to the
`increased ability of PLC-c1 to cleave phosphatidlyinositol
`(4,5)
`bisphosphate into inositol (1,4,5) trisphosphate, which is important
`for Ca2+
`influx, and diacylglycerol, which is important for protein
`kinase C activation [9].
`PLC-c1 and the Grb2 family of adaptor proteins all contain
`SH3 domains that mediate the recruitment of signaling proteins to
`LAT. Once such protein is SLP-76, an adaptor protein that is
`brought to the LAT complex via its interaction with the adaptor
`protein Gads and PLC-c1 (Figure 1) [10]. Upon TCR stimulation,
`SLP-76 is phosphorylated and both Vav1 and Itk bind to these
`phosphorylated tyrosines [10]. Vav1 is a guanine nucleotide
`exchange factor for Rac, a small GTPase that triggers cytoskeletal
`rearrangements downstream of TCR induction [11]. Vav1
`requires the presence of Itk to bind phosphorylated SLP-76,
`although it does not appear that Vav1 and Itk interact directly
`[12]. Similarly, Itk requires binding to phosphorylated SLP-76 to
`maintain its active conformation [13]. The end result of these
`interactions at the LAT complex is the induction of important
`
`downstream functions needed for T cell activation, such as Ca2+
`flux, Erk1/Erk2 activation,
`receptor upregulation and the
`induction of cytokine release.
`Much of our understanding of TCR-mediated signaling was
`discovered using two human T cell lines, the Jurkat E6.1 T cell
`and the HuT78 T cell. The original Jurkat cells were derived from
`a 14-year-old boy with T cell acute lymphoblastic leukemia (T-
`ALL). These cells are thymocytically derived with the character-
`istics of immature thymocytes. The Jurkat E6.1 subclone cell line
`was developed in the 1980s [14]. Sublines of Jurkat E6.1 cells have
`been isolated that are deficient
`in several TCR signaling
`molecules, including stable lines lacking the TCRb chain, Lck,
`LAT, SLP-76, ZAP-70, Vav1, or PLC-c1 [14]. These mutants
`have yielded much information and insight into the function of
`many peripheral TCR signaling molecules. The HuT78 T cell line
`is historically a very important cell line: the H9 subclone of HuT78
`T cells is the cell used to isolate HIV. HuT78 cells were derived
`from a Sezary syndrome patient, and are CD4+, mature,
`cutaneous, lymphoid T cells [15].
`Despite being highly useful for several reasons, such as mutant
`sublines, high rates of transfection, and ease of growth, Jurkat E6.1
`cells have several known abnormalities. These include deficiencies
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`in PTEN and SHIP, lipid phosphatases that regulate phospho-
`inositide-3 kinase (PI3K) function [16,17]. The lack of PTEN and
`SHIP leads to several irregularities including constitutive expres-
`sion of AKT, increased levels of phosphorylated phosphoinositide
`lipids, and the constitutive association of Itk with the plasma
`membrane [17,18]. HuT78 cells also have deficiencies; they have
`abnormal c-myc function and over express Bcl-xL, suggesting
`problems with anti-apoptotic pathways in these cells [19,20].
`Although all cell lines have inherent problems, there are many
`reasons to use cell
`lines as model systems for studying the
`activation and function of human T cells. Cell lines are useful
`because they offer an unlimited, genetically manipulatable and
`widely accessible supply of experimental material that can be
`stored long-term and grown at a low cost. Using continuous cell
`lines also reduces the human burden and ethical considerations of
`experimentation and substantially reduces donor
`to donor
`variation. It is important however, that the pros and cons of a
`specific cell
`line are appreciated to allow for interpretation of
`experiments using these cell
`lines. Surprisingly,
`there are no
`previous studies that directly compare TCR and/or costimulatory-
`mediated signaling and function in the human T cell
`lines to
`human activated peripheral blood T cells (APBTs). Therefore, we
`chose to compare the Jurkat E6.1 T cells and HuT78 T cells to
`APBTs in an effort to determine the conduciveness of using these
`T cell lines as a model for peripheral TCR signaling and function.
`We found that there was no noteworthy difference among the
`three cell
`lines when examining early signaling events such as
`ZAP-70, LAT, and SLP-76 phosphorylation. However, there were
`significant differences in downstream signaling events, costimula-
`tory receptor expression and activation and in the cytokines and
`chemokines released upon TCR induction. Advantages and
`disadvantages exist for both cell lines, which must be considered
`during the experimental design process.
`
`Results
`
`In order to compare proximal TCR-mediated signaling events
`in Jurkat E6.1 T cells, HuT78 T cells, and APBTs, equal cell
`numbers were activated with maximal doses of stimulatory TCR
`antibodies. The samples were then analyzed by immunoblotting
`using phosphospecific and pan antibodies to specific signaling
`molecules. We calculated the ratio of phosphorylated protein to
`total protein for each specific protein using quantified densities
`from the immunoblots. By using this method,
`the total
`phosphorylation per protein of a specific signaling molecule for
`each cell type was determined, regardless of the size of the cell or
`the concentration of protein present in each cell. Thus, the relative
`extent of activation of key signaling molecules was compared
`between each cell type.
`
`Jurkat E6.1 T cells, HuT78 T cells, and APBTs had
`significant differences in the phosphorylation of the
`tyrosine kinases ZAP-70, but not the adaptor proteins
`LAT and SLP-76
`Before the relative activation of critical signaling molecules can
`be assessed, the surface expression of the TCR ab chain in the
`various T cell
`lines must be determined. This information is
`needed because differences in the surface expression of the TCR
`ab chain in Jurkat E6.1 T cells, HuT78 T cells and APBTs may
`result in variations in receptor-mediated signaling events. To this
`end, the surface expression of the TCR ab chain in the Jurkat
`E6.1 T cells, HuT78 T cells and APBTs was examined by flow
`cytometry. The mean fluorescent intensity (MFI) of the TCR ab
`chain expression for three separate experiments was 78+/219 for
`
`Comparison of T Cell Lines
`
`Jurkat E6.1 T cells, 29+/29 for HuT78 T cells and 60+/28 for
`the APBTs (Figure 2 and data not shown). It was consistently
`observed that HuT78 T cells had significantly less TCR ab chain
`expression than Jurkat E6.1 T cells and APBTs, whereas there was
`no significant difference in the expression of TCR ab chain
`between the Jurkat E6.1 T cells and APBTs (Figure 2). Thus,
`theHuT78 T cells have significantly less TCR surface expression
`than the Jurkat E6.1 T cell and APBTs. However, whether this
`difference leads
`to alterations
`in the extent of
`intracellular
`signaling is unknown.
`Upon engagement of the TCR with a specific peptide-MHC
`complex,
`the tyrosine kinases ZAP-70 is phosphorylated and
`activated by the Src family kinases Lck and Fyn [1]. Therefore, it
`was of interest to determine if there were differences in the TCR-
`induced phosphorylation of ZAP-70 among Jurkat E6.1 T cells,
`HuT78 T cells and APBTs. Phosphorylation of ZAP-70 at tyrosine
`residue 319 is
`required for
`its activation and subsequent
`phosphorylation of
`the adaptor molecules LAT and SLP-76
`[21,22]. The phosphorylation of this tyrosine is an excellent read-
`out for enzymatically active ZAP-70. As seen in Figure 3, the
`relative levels of
`tyrosine 319 phosphorylation were similar
`between HuT78 T cells and APBTs. However,
`there was a
`significant and reproducible 2–3 fold decrease in the site-specific
`phosphorylation of ZAP-70 in Jurkat E6.1 T cells (Figure 3B). This
`suggests that Jurkat E6.1 T cells have reduced levels of TCR-
`induced ZAP-70 activation and potentially have altered down-
`stream signaling.
`We next examined the phosphorylation of signaling proteins
`downstream of ZAP-70 to see if the diminished phosphorylation of
`this important early kinase translated to later differences in TCR-
`mediated signaling. ZAP-70 is the known kinase for the adaptor
`proteins LAT and SLP-76 [23]. The phosphorylation of LAT at
`tyrosine 191 is essential
`for the formation of multi-protein
`signaling complexes that
`transmit
`the signal
`from the TCR
`complex to downstream effectors [7,8]. When the relative levels of
`LAT tyrosine 191 phosphorylation were examined, no differences
`
`Figure 2. HuT78 T cells have less TCR surface expression than
`Jurkat E6.1 T cells and APBTs. The surface expression of the TCR a/b
`chain on Jurkat E6.1 T cells, HuT78 T cells, and APBTs was assessed by
`flow cytometry. A representative plot of the TCR surface expression of
`these cell lines is shown. The isotype control for each cell had a mean
`fluorescent intensity of ,8. The experiment was repeated three times.
`doi:10.1371/journal.pone.0005430.g002
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`Comparison of T Cell Lines
`
`Figure 3. Jurkat E6.1 T cells exhibit decreased ZAP-70 Y319 phosphorylation compared to HuT78 T cells and APBTs. (A) Three
`samples of Jurkat E6.1 T cells, HuT78 T cells, and APBTs were stimulated and the cellular proteins were separated by SDS-PAGE. The site-specific
`phosphorylation and expression of ZAP-70 was assessed by immunoblotting using ZAP-70 pY319 (top) and pan-ZAP-70 (bottom) antibodies. (B) The
`immunoblots were analyzed by densitometry and the ratio of the intensity of the phosphospecific ZAP-70 band to the pan-ZAP-70 band for each cell
`line was averaged. Letters represents significant differences of p,0.05.
`doi:10.1371/journal.pone.0005430.g003
`
`were observed between Jurkat E6.1 and APBT samples or between
`HuT78 and APBT samples (Figure 4A and 4B). However, there
`was a small but significant difference in the phosphorylation of
`LAT tyrosine 191 between Jurkat E6.1 and HuT78 T cell lines
`(Figure 4A and 4B). These data show that the phosphorylation of
`LAT is not substantially different among the cell lines, indicating
`the binding capacity of LAT in each cell is similar.
`Another important adaptor protein for functions downstream of
`TCR activation is SLP-76. This protein is phosphorylated on three
`tyrosine residues, including tyrosine 128. Phosphorylated SLP-76
`is essential
`for bringing Vav1 and Itk to the LAT signaling
`complex [10], which is vital for linking proximal TCR signaling to
`events such as calcium influx and actin cytoskeletal rearrange-
`ment. As seen for LAT, no difference was seen among the two cell
`lines and APBTs in the phosphorylation of SLP-76 tyrosine 128
`(Figure 4C and 4D), suggesting that SLP-76 has a similar signaling
`capacity in each type of cell. Together, these data indicate that the
`reduced levels of TCR a/b chain surface expression in the HuT78
`T cells compared to other T cell lines and the decrease in ZAP-70
`phosphorylation observed in Jurkat E6.1 T cells compared to
`HuT78 T cells and APBTs does not translate into differences in
`the activation of signaling proteins immediately downstream of
`ZAP-70.
`
`Jurkat E6.1 T cells have hyperphosphorylated PLC-c1 and
`exhibited exaggerated Ca2+ signaling compared to
`HuT78 T cells and APBTs
`Next, differences in several important signaling events down-
`stream of LAT and SLP-76 were characterized in Jurkat E6.1 T
`cells, HuT78 T cells, and APBTs. PLC-c1 activation is important
`for both intracellular calcium flux and protein kinase C activation
`[1,8]. Tyrosine 783 on PLC-c1 is phosphorylated by the Tec
`for PLC-c1 activation and
`family kinase Itk and is essential
`
`enzymatic function [13,24,25]. Therefore, the phosphorylation of
`PLC-c1 tyrosine 783 was characterized to determine if there was a
`difference in the phosphorylation of this important residue among
`the T cell lines. Surprisingly, a 10-fold increase in the relative
`phosphorylation of PLC-c1 tyrosine 783 was observed in the
`Jurkat E6.1 T cell
`line, compared to the HuT78 T cells and
`APBTs (Figure 5A and 5B). In contrast, no difference in the site-
`specific phosphorylation of PLC-c1 was
`found between the
`HuT78 T cells and APBTs (Figure 5A and 5B). This suggests
`that PLCc1 is substantially more activated in Jurkat E6.1 T cells
`compared to HuT78 T cells or APBTs.
`The phosphorylation of tyrosine 775 on PLCc1 is also known to
`be critical for TCR-induced Ca2+
`influx and transcription factor
`activation [26]. Therefore, we examined whether the TCR-
`mediated phosphorylation of this site is also increased in Jurkat
`E6.1 T cells compared to other T cell lines. Interestingly, Jurkat
`E6.1 T cells and APBTs had no detectable difference in the TCR-
`induced phosphorylation of tyrosine 775 on PLCc1 (Figure 5C
`and 5D). In contrast, HuT78 T cells had significantly less
`phosphorylation of this site compared to both Jurkat E6.1 T cells
`and APBTs
`(Figure 5C and 5D). This
`indicates
`that
`the
`phosphorylation of tyrosines 775 and 783 on PLCc1, which are
`both required for optimal TCR-induced function of this protein,
`may be differentially regulated.
`The observation that PLCc1 Y783 is hyperphosphorylated in
`Jurkat E6.1 T cells compared to HuT78 T cells and APBTs lead us
`to further examine the kinetics of PLC-c1 phosphorylation. This
`was done in order to determine if
`the large differences in
`phosphorylation were seen not only in the amount of TCR-
`induced phosphorylation of PLC-c1 tyrosine 783 at a single
`timepoint, but also in the timing and duration of this phosphor-
`ylation event. A substantial difference was seen in the kinetics of
`phosphorylation of PLC-c1 tyrosine 783 between Jurkat E6.1 T
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`Comparison of T Cell Lines
`
`Figure 4. Jurkat E6.1 T cells, HuT78 T cells, and APBTs have similar levels of LAT and SLP-76 phosphorylation. (A) Three samples of
`Jurkat E6.1 T cells, HuT78 T cells, and APBTs were stimulated and the cellular proteins were separated by SDS-PAGE. The site-specific phosphorylation
`and expression of LAT was assessed by immunoblotting using LAT pY191 (top) and pan-LAT (bottom) antibodies. (B) The immunoblots were analyzed
`by densitometry and the ratio of the intensity of the phosphospecific LAT band to the pan-LAT band for each cell line was averaged. Letters
`represents significant differences of p,0.01. (C) Three samples of Jurkat E6.1 T cells, HuT78 T cells, and APBTs were stimulated and the cellular
`proteins were separated by SDS-PAGE. The site-specific phosphorylation and expression of SLP-76 was assessed by immunoblotting using SLP-76
`pY128 (top) and pan-SLP-76 (bottom) antibodies. (D) The immunoblots were analyzed by densitometry and the ratio of the intensity of the
`phosphospecific SLP-76 band to the pan-SLP-76 band for each cell line was averaged. There were no significant differences between cell lines and
`p.0.25.
`doi:10.1371/journal.pone.0005430.g004
`
`cells, and HuT78 T cells. HuT78 T cells reached maximal
`phosphorylation at 1 minute post-TCR stimulation, followed by a
`steady decrease in phosphorylation over 30 minutes (Figure 6A).
`APBTs and HuT78 T cells had similar TCR-induced phosphor-
`ylation kinetics of PLC-c1 Y783 (data not shown). In contrast, the
`Jurkat E6.1 T cell
`line reached maximal phosphorylation at
`2 minutes and the phosphorylation of tyrosine 783 decreased only
`
`slightly over the course of 30 minutes (Figure 6A). This indicates
`that Jurkat E6.1 T cell have not only greater relative PLC-c1
`tyrosine 783 phosphorylation than HuT78 T cells and APBTs, but
`that they also sustain that level of hyperphosphorylation for a
`much longer time.
`The TCR-induced influx of Ca2+
`into human T cells is
`controlled by PLC-c1 activation. The influx of Ca2+
`is important
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`Comparison of T Cell Lines
`
`Figure 5. T cell lines have differences in PLCc1 phosphorylation. (A) Three samples of Jurkat E6.1 T cells, HuT78 T cells, and APBTs were
`stimulated and the cellular proteins were separated by SDS-PAGE. The site-specific phosphorylation and expression of PLCc1 was assessed by
`immunoblotting using PLCc1 pY783 (top) and pan-PLCc1 (bottom) antibodies. (B) The immunoblots were analyzed by densitometry and the ratio of
`the intensity of the phosphospecific PLC-c1 band to the pan-PLC-c1 band for each cell line was averaged. Letters represents significant differences of
`p,0.0005. (C) Three samples of Jurkat E6.1 T cells, HuT78 T cells, and APBTs were stimulated and the cellular proteins were separated by SDS-PAGE.
`The site-specific phosphorylation and expression of PLCc1 was assessed by immunoblotting using PLCc1 pY775 (top) and pan-PLCc1 (bottom)
`antibodies. (D) The immunoblots were analyzed by densitometry and the ratio of the intensity of the phosphospecific PLC-c1 band to the pan-PLC-c1
`band for each cell line was averaged. Letters represents significant differences of p,0.03.
`doi:10.1371/journal.pone.0005430.g005
`
`for many effectors functions in T cells, including NFAT activation
`and cytokine and chemokine production [1,27]. Due to its
`importance in downstream T cell functions, we examined whether
`the hyperphosphorylation of PLC-c1 in Jurkat E6.1 T cells lead to
`alterations in the levels of TCR-induced Ca2+
`influx in the T cell
`lines and APBTs. To this end, we utilized a quantitative, real-time
`measurement method for intracellular Ca2+
`developed by Tsien
`
`and coworkers [28]. Using this method, it was observed that both
`showed TCR inducible Ca2+
`influx
`cell
`lines and APBTs
`(Figure 6B). Jurkat E6.1 T cells exhibited approximately 7–10-
`fold higher Ca2+
`flux immediately following stimulation than
`HuT78 T cells or APBTs, as well as a higher level and longer time
`of sustained Ca2+
`(Figure 6B). HuT78 T cells also showed higher
`levels of initial Ca2+
`signaling than APBTs, although they returned
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`Comparison of T Cell Lines
`
`Figure 6. Jurkat E6.1 T cells have increased PLCc1 function compared to HuT78 T cells and APBTs. (A) Jurkat E6.1 T cells and HuT78 T
`cells were stimulated over a 309 time course and the cellular proteins were separated by SDS-PAGE. The site-specific phosphorylation and expression
`of PLCc1 and the expression of actin was assessed by immunoblotting using PLCc1 pY783 (top) and pan-PLCc1 (bottom) antibodies. (B) TCR-induced
`calcium flux was measured quantitatively as described above. Traces for Jurkat E6.1 T cells, HuT78 T cells and APBTs are shown.
`doi:10.1371/journal.pone.0005430.g006
`
`to similar levels as APBTs much more quickly than Jurkat E6.1
`T cells (Figure 6B). These data indicate that
`the increased
`phosphorylation of PLC-c1 tyrosine 783 in Jurkat E6.1 T cells also
`translated into an amplification in Ca2+
`flux, as compared to
`HuT78 and APBT cells.
`
`Jurkat E6.1 T cells have hyperphosphorylated Pyk2 and
`Vav1 compared to HuT78 T cells and APBTs
`The intracellular tyrosine kinase Pyk2 is critical for integrating
`receptor mediated signals that control the rearrangement of the
`actin cytoskeleton [6]. The levels of the enzymatic activity of Pyk2
`is controlled by the phosphorylation of
`tyrosine 580 in the
`activation loop of
`the kinase domain [6]. Therefore,
`the
`phosphorylation of this site can be used as a marker for the
`induction of the kinase activity of Pyk2. To investigate the relative
`activation of Pyk2 in Jurkat E6.1 T cells, HuT78 T cells, and
`APBTs, the cells were stimulated for 1 minute (Jurkat E6.1 cells) or
`30 seconds (HuT78 T cells and APBTs), which corresponded to
`the maximal activation time of Pyk2 in these cell types (M. Collins
`and J.C.D. Houtman, unpublished results). Interestingly,
`the
`relative level of Pyk2 tyrosine 580 phosphorylation was signifi-
`cantly different between Jurkat E6.1 T cells, HuT78 T cells and
`APBTs, with Jurkat E6.1 T cells having the highest relative level of
`Pyk2 tyrosine 580 phosphorylation and HuT78 T cells having the
`lowest (Figure 7A and Figure 5B). These experiments show that
`the enzymatic activity of Pyk2 is different in the individual T cell
`lines, suggesting that there are differences in the ability of the TCR
`to control the actin cytoskeleton between Jurkat E6.1 T cells,
`HuT78 T cells and APBTs.
`
`the
`is
`these differences
`One potential mechanism for
`differential expression of PTP-PEST, a tyrosine phosphatase
`known to dephosphorylate Pyk2 in T cells [29]. To investigate
`this possibility, the expression of PTP-PEST in the three T cell
`lines was characterized. Unfortunately, the expression of PTP-
`PEST was not detectable in cell
`lysates
`(data not
`shown).
`Therefore, PTP-PEST was immunoprecipitated from unstimu-
`lated Jurkat E6.1 T cells, HuT78 T cells and APBTs. As seen in
`Figure 7C, HuT78 T cells express PTP-PEST, whereas Jurkat
`E6.1 T cells and APBTs have no detectable expression of this
`phosphatase. These results agree with a recent study that also
`observed the Jurkat E6.1 T cells and APBTs do not express PTP-
`PEST [30]. These data suggest that the expression of PTP-PEST
`in HuT78 T cells results in reduced levels of Pyk2 phosphory-
`lation compared to APBTs. In contrast, the significant differences
`in the TCR-induced phosphorylation of Pyk2 in Jurkat E6.1 T
`cells and APBTs are not due to differential expression of this
`phosphatase.
`Vav1 is a guanine nucleotide exchange factor for the small
`GTPase Rac1 and is important
`for receptor-mediated actin
`cytoskeletal rearrangement in human T cells [11]. An obligate step
`in the enzymatic activation of Vav1 is the phosphorylation of
`tyrosine 174 [11]. In contrast to Pyk2, Vav1 tyrosine 174 had a
`moderate 2-fold hyperphosphorylation in Jurkat E6.1 T cells
`compared to both HuT78 T cells and APBTs (Figure 8A and
`Figure 6B). These data suggest that Vav1 is hyperactivated in
`Jurkat E6.1 T cells, which, in conjunction with the hyperpho-
`sphorylation of Pyk2, indicates a potential distortion of cytoskeletal
`rearrangement in these cells.
`
`PLoS ONE | www.plosone.org
`
`7
`
`May 2009 | Volume 4 |
`
`Issue 5 | e5430
`
`UPenn Ex. 2075
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`Comparison of T Cell Lines
`
`Figure 7. Jurkat E6.1 T cells, HuT78 T cells, and APBTs have different levels of Pyk2 Y580 phosphorylation. (A) Three samples of Jurkat
`E6.1 T cells, HuT78 T cells, and APBTs were stimulated and the cellular proteins were separated by SDS-PAGE. The site-specific phosphorylation and
`expression of Pyk2 was assessed by immunoblotting using Pyk2 pY580 (top) and pan-Pyk2 (bottom) antibodies. (B) The immunoblots were analyzed
`by densitometry and the ratio of the intensity of the phosphospecific Pyk2 band to the pan-Pyk2 band for each cell line was averaged. Letters
`represents significant differences of p,0.05. (C) PTP-PEST was immunoprecipitated for Jurkat E6.1 T cells, HuT78 T cells and APBTs. The expression of
`PTP-PEST in the different T cells was assessed by immunoblotting.
`doi:10.1371/journal.pone.0005430.g007
`
`Itk, the common link between PLC-c1 and Vav1, has
`increased expression in Jurkat E6.1 T cells
`The hyperphosphorylation of both Vav1 and PLC-c1 at
`important tyrosine residues, as well as exaggerated Ca2+
`flux, in
`Jurkat E6.1 T cells led us to examine Itk, the common link among
`these events. Itk requires Vav1 to bind SLP-76, an event necessary
`for Itk to phosphorylate PLC-c1 Y783 [12]. Phosphorylation of
`tyrosine residue 511 is required for the activation of Itk [31],
`therefore the phosphorylation of this site serves as a marker for the
`induction of Itk kinase activity. Unfortunately, we were unable to
`directly detect any TCR-induced site-specific phosphorylation of
`Itk in stimulated T cell lysates (data not shown). Consequently, Itk
`immunoprecipitations were performed to examine differences in
`the phosphorylation of Itk tyrosine 511. Interestingly, there was
`observable TCR-induced phosphorylation of Itk tyrosine 511in
`Jurkat E6.1 T cells, but little detectable phosphorylation of this site
`
`in HuT78 T cells and APBTs (Figure 9A and Figure 7B). In
`addition, there was an increase in the amount of immunoprecip-
`itated Itk in Jurkat E6.1 T cells compared to HuT78 T cells and
`APBTs (Figure 9A), suggesting Jurkat E6.1 cells have increased
`expression of Itk. To more directly compare expression of Itk in
`the T cell lines, the levels of Itk in an equal cell number of Jurkat
`E6.1 T cells and HuT78 T cells, and APBTs was measured by
`immunoblotting. Jurkat E6.1 T cells had at least a 2-fold greater
`Itk expression than HuT78 T cells (Figure 9C). Jurkat E6.1 T cells
`from different laboratories had differences in the relative levels of
`Itk, with most having increased levels of this protein. This suggests
`that there are variations in the relative levels of signaling proteins
`in individual sublines of the Jurkat E6.1 T cells. Collectively, these
`data indicate that the increased expression of Itk in Jurkat E6.1 T
`cells compared to HuT78 T cells and APBTs is linked to
`exaggerated TCR-mediated signaling.
`
`PLoS ONE | www.plosone.org
`
`8
`
`May 2009 | Volume 4 |
`
`Issue 5 | e5430
`
`UPenn Ex. 2075
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`Comparison of T Cell Lines
`
`Figure 8. Jurkat E6.1 T cells exhibit hyperphosphorylated of Vav1 Y174 compared to HuT78 T cells and APBTs. (A) Three samples of
`Jurkat E6.1 T cells, HuT78 T cells, and APBTs were stimulated and the cellular proteins were separated by SDS-PAGE. The site-specific phosphorylation
`and expression of Vav1 was assessed by immunoblotting using Vav1 pY174 (top) and pan-Vav1 (bottom) antibodies. (B) The immunoblots were
`analyzed by densitometry and the ratio of the intensity of the phosphospecific Vav1 band to the pan-Vav1 band for each cell line was averaged.
`Letters represents significant differences of p,0.005.
`doi:10.1371/journal.pone.0005430.g008
`
`Jurkat E6.1 T cells have hyperphosphorylated Erk1 and
`Erk2 compared to HuT78 T cells and APBTs
`for the
`The MAP kinases Erk1 and Erk2 are important
`regulation of the expression and function of the transcription
`factor AP-1 [1]. The activation of these kinases is downstream of
`LAT phosphorylation, Vav1 activation, and Ca2+
`influx [1]. The
`phosphorylation of Erk1 and Erk2 on threonine 202 and tyrosine
`204 in the activation loop of their kinase domain is required for full
`enzymatic activity of these proteins. Thus, the dual phosphoryla-
`tion of these sites is a measure of the enzymat