PERSPECTIVES
`
`T I M E L I N E
`
`Jurkat T cells and development of the
`T-cell receptor signalling paradigm
`
`Robert T. Abraham and Arthur Weiss
`
`Twenty years of investigation have yielded
`a detailed view of the signalling machinery
`engaged by T-cell receptors (TCRs). Many
`of the fundamental insights into TCR
`signalling came from studies carried out
`with transformed T-cell lines. Perhaps the
`best known of these model systems is
`the Jurkat leukaemic T-cell line, and here
`we review some of the key advances in
`the field of TCR signalling that were made
`with Jurkat T cells as the host.
`
`By the mid-1980s, T-cell biologists recog-
`nized that a relatively new field, known as
`signal transduction, had direct applications
`to their studies of T-cell activation by anti-
`genic stimuli. Now, after two decades of
`intensive scrutiny, most of the main compo-
`nents in T-cell receptor (TCR) signalling
`have been identified, localized and at least
`partially characterized. During the early days
`of TCR signalling research, immunologists
`had the use of a range of mouse and human
`T-cell lines capable of mounting biologically
`relevant responses to TCR stimulation.
`Prominent members of this legendary group
`were transformed T-cell lines of human (for
`example, HPB-ALL, HuT-78) and mouse
`(EL4, LBRM-33) origins. Although major
`advances stemmed from the use of each of
`these cell lines, arguably the most popular and
`historically significant of this group was the
`human leukaemic T-cell line, Jurkat.
`In this Timeline article, we recount some
`of the many highlights in ‘Jurkat history’,
`which parallels a vast increase in our under-
`standing of T-cell activation (TIMELINE). In
`
`recent years, the lymphocyte signalling field
`has shifted its attention from in vitro experi-
`ments with transformed T cells to in vivo
`studies with genetically altered mice. Our
`goals are to promote a more balanced per-
`spective regarding the relative merits and
`weaknesses of the in vitro model systems, and
`to highlight technological advances that will
`impact strongly on the use of cultured T cells,
`human and mouse alike, as experimental
`tools for signal transduction research.
`
`Emergence of the Jurkat model system
`As is the case today, T-cell biologists in the
`early 1980s focused their research efforts
`mainly on cells of human or mouse origin.
`Arthur Weiss, who was then a postdoctoral
`fellow in John Stobo’s laboratory, was forced
`to study human T cells by default, due to an
`emerging allergy to rodents. To maintain
`human T cells in culture, Weiss needed a
`source of human T-cell growth factors. A pos-
`sible solution to this problem was provided
`by Steven Gillis and James Watson, who were
`screening for transformed human T-cell lines
`that spontaneously or inducibly released large
`quantities of interleukin-2 (IL-2)1. The Jurkat
`cell line was one of several leukaemic cell lines
`obtained for screening purposes from John
`Hansen2,3 at the Fred Hutchinson Cancer
`Research Center, Seattle, USA. Gillis and
`Watson found that Jurkat cells were particu-
`larly robust producers of IL-2 after stimula-
`tion with phytohaemagglutinin (PHA).
`Consequently, Weiss and Stobo obtained the
`cell line from Gillis and Kendall Smith at
`Dartmouth, USA4; however, the cells were
`
`heavily contaminated with mycoplasma. The
`process of curing the cell line of this infection
`yielded the Jurkat E6-1 clone, which eventu-
`ally became the standard Jurkat cell line used
`by many T-cell immunologists.
`Subsequent studies of Jurkat T cells
`revealed that two synergistic signals were
`required for maximal production of IL-2.
`One of these signalling requirements was
`fulfilled by ligation of the TCR with CD3-
`specific antibodies, whereas the ‘second sig-
`nal’ was delivered by phorbol esters (for
`example, phorbol myristate acetate, PMA)5.
`At about the same time, seminal work from
`Ellis Reinherz’s group showed that the
`αβ-TCR heterodimer was associated with
`the CD3 chains (the ζ-chain had not yet
`been discovered)6,7. Although the nature of
`the association between the αβ-TCR and
`CD3 polypeptides remained mysterious, the
`observation that CD3-specific antibodies were
`stimulatory for human T cells prompted spec-
`ulation that the CD3 subunits mediated signal
`transduction across the plasma membrane7.
`
`Insights into calcium signalling
`During the same time period, Weiss adopted
`the somatic-cell genetic strategy pioneered
`by Marcus Nabholz8, to generate a set of
`TCR-negative Jurkat sublines that he used to
`investigate whether the αβ-TCR heterodimer
`and the CD3 complex were independently
`expressed by T cells. His studies, carried out
`in part as a collaboration with Pam Ohashi
`in Tak Mak’s laboratory, indicated that cell-
`surface expression of the αβ-TCR het-
`erodimer and CD3 polypeptides required
`that these molecules be co-expressed9,10. The
`observation that these Jurkat sublines no
`longer responded to PHA indicated that the
`response to this mitogenic lectin was depen-
`dent on cell-surface TCR expression11. By con-
`trast, the TCR-negative cells could respond
`to a pharmacological ‘cocktail’, indicating
`that cell-surface TCR expression could be
`bypassed. Exposure of these cells, as well as
`normal Jurkat cells, to the combination of a
`calcium ionophore (for example, ionomycin)
`
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`P E R S P E C T I V E S
`
`Timeline | Contributions of Jurkat T cells to characterizing the T-cell receptor signalling pathway
`
`Identification
`of the Jurkat
`cell line
`
`Discovery that TCR
`ligation triggers
`intracellular Ca2+
`mobilization
`
`Observation that TCR
`stimulation triggers
`protein tyrosine
`phosphorylation
`
`Characterization of ZAP70
`
`Identification of PLC-γ1
`as a TCR-linked signalling
`enzyme
`
`Characterization of
`LCK expression defect
`in J.CAM1 cells
`
`Identification
`of ZAP70-
`deficient
`Jurkat cells
`
`Characterization
`of CARMA1 in
`Jurkat cells
`
`1980
`
`1984
`
`1987
`
`1990
`
`1991
`
`1992
`
`1998
`
`2002
`
`Discovery that
`Jurkat cells
`release IL-2
`when stimulated
`with PHA
`
`Use of TCR/CD3-
`specific antibodies
`as stimuli for IL-2
`production by
`Jurkat cells
`
`Generation of
`J.CaMI–3 mutants
`
`Reports that
`protein tyrosine
`phosphorylation
`is required for
`TCR signalling
`
`Identification of ITAMs as
`signal-transducing motifs
`in the CD3 cytoplasmic
`domain
`
`Characterization of LAT-
`deficient Jurkat cells
`
`IL-2, interleukin-2; ITAM, immunoreceptor tyrosine-based activation motif; LAT, linker for activation of T cells; PHA, phytohaemagglutinin; PLC-γ1, phospholipase C-γ1; TCR, T-cell receptor;
`ZAP70, ζ-chain-associated protein of 70kD.
`
`and a phorbol ester (for example, PMA)
`induced a robust activation response, as mea-
`sured by IL-2 production. Shortly thereafter,
`other investigators demonstrated that this
`combination triggered both IL-2 release and
`cell-cycle progression in normal resting T-cell
`populations12,13. Interestingly, TCR ligands
`and Ca2+ ionophores behaved as interchange-
`able activating stimuli for Jurkat cells, and
`both agents acted synergistically with phorbol
`esters14. Based on these results, Weiss and
`John Imboden (also a postdoctoral fellow in
`the Stobo laboratory) surmised that TCR
`stimulation provoked an increase in the
`intracellular free Ca2+ concentration in Jurkat
`cells. At the same time, Imboden’s wife,
`Dolores Shoback, was using a newly devel-
`oped fluorescent dye, Quin-2, to monitor
`changes in intracellular Ca2+ concentration in
`parathyroid hormone-stimulated cells15.
`Quin-2 had been developed by Roger Tsien,
`who had already established its usefulness in
`measuring the intracellular Ca2+ concentra-
`tion in lymphoid cells16. With Shoback’s
`assistance, Imboden and Weiss demonstrated
`that antibody- or PHA-dependent TCR
`stimulation triggered a rapid increase in
`intracellular Ca2+ concentration in TCR-
`positive Jurkat cells, but failed to do so in
`the TCR-negative somatic mutants9,17,18.
`These studies showed that the TCR func-
`tioned as a Ca2+-mobilizing transmembrane
`receptor during T-cell activation.
`These findings provoked considerable
`interest in the intermediate events that coupled
`TCR stimulation to this abrupt increase in
`intracellular Ca2+ concentration. Ongoing
`studies in non-lymphoid cell types indicated
`that, for many transmembrane receptors, an
`important pathway of intracellular signalling
`
`involved the activation of phospholipase C
`(PLC), which catalysed the hydrolysis of
`membrane phosphatidylinositol-4,5-bispho-
`sphate (PIP2) to the Ca2+-mobilizing second
`messenger, inositol-1,4,5-trisphosphate (IP3).
`Accordingly, Imboden examined the effect of
`TCR crosslinking on phosphoinositide break-
`down in Jurkat T cells, and found that TCR
`stimulation provoked the hydrolysis of
`phosphoinositides to inositol phosphates,
`indicating that TCR ligation activated a PLC
`isoform17.
`Inspired by his mentor’s earlier foray
`into somatic-cell genetics, Mark Goldsmith,
`who was Weiss’s first graduate student,
`devised a mutant selection scheme aimed at
`isolating Jurkat cells that were resistant to
`killing by chronic exposure to PHA. Given
`that the cellular response to PHA was highly
`TCR dependent, Goldsmith predicted that
`this strategy would yield Jurkat-derived
`clones bearing defects in the TCR signalling
`pathway. At about the same time, Tsien19
`unveiled another Ca2+ indicator dye, Indo-1,
`the fluorescent properties of which were
`compatible with use on the flow cytometer,
`and therefore of particular interest to
`immunologists. Goldsmith was able to com-
`bine his live–dead selection protocol with a
`secondary, Indo-1-based cell-sorting step
`aimed at the selection of mutant Jurkat cells
`that failed to elicit increased intracellular
`Ca2+ concentrations in response to TCR
`crosslinking. To avoid the isolation of receptor-
`negative Jurkat mutants, he alternately
`sorted for cells that did not flux calcium and
`for those that retained TCR expression. This
`two-step sorting procedure yielded three inde-
`pendent Jurkat-derived clones, designated
`J.CaM1–3 (REFS 20–22).
`
`To determine the specificity of these muta-
`tions for the TCR-coupled signalling
`machinery, Goldsmith carried out an elegant
`set of experiments in which the J.CaM cell
`lines were transfected with a cDNA encoding
`the human type 1 muscarinic receptor
`(HM1)21,23. This receptor signals through the
`activation of a heterotrimeric G protein, and
`is not normally expressed by T cells. Notably,
`when transfected with HM1, Jurkat cells, as
`well as the J.CaM sublines, responded to car-
`bachol (a HM1 agonist) with a marked
`increase in intracellular Ca2+ concentration.
`These results confirmed the idea that TCR
`crosslinking triggered intracellular Ca2+
`mobilization through a mechanism that
`was biochemically distinct from that used
`by G-protein-coupled receptors. Prompted
`by Imboden’s finding that TCR crosslinking
`stimulated phosphoinositide hydrolysis in
`Jurkat cells17, Goldsmith examined his J.CaM
`cell lines for evidence of defects in PLC acti-
`vation, and found that TCR-induced pro-
`duction of inositol phosphate was uniformly
`suppressed in these cells.
`
`Protein tyrosine kinase signalling
`Early protein tyrosine kinase studies. By
`1990, a growing number of immunologists
`were focused on protein tyrosine phospho-
`rylation as a proximal signalling event
`elicited by TCR ligation. If correct, this
`would indicate that the TCR-signalling
`mechanism resembled that used by mem-
`bers of the receptor tyrosine kinase family,
`the most-studied members of which were
`the platelet-derived growth factor (PDGF)
`and epidermal growth factor (EGF) recep-
`tors. The only problem with this model was
`that the TCR, unlike the PDGF and EGF
`
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`

`receptors, lacked a cytoplasmic domain bear-
`ing intrinsic protein tyrosine kinase (PTK)
`activity. This conundrum could be resolved
`if the TCRs were to recruit a membrane-
`associated or cytoplasmic PTK that was
`extrinsic to the receptor itself. As is almost
`always the case, this idea was initiated by
`earlier research findings, which indicated
`that T cells were richly endowed with non-
`receptor PTKs and their antagonists, the
`protein tyrosine phosphatases (PTPases).
`In 1985, Jamey Marth, a postdoctoral fel-
`low in Roger Perlmutter’s laboratory, dis-
`covered a T-cell-specific SRC family PTK,
`known as LCK, which was destined to leave
`a major imprint on the TCR signalling para-
`digm that evolved during the 1990s24.
`Independent reports from Andre Veillette
`and Joseph Bolen, and Chris Rudd and
`Stuart Schlossman, provided evidence that
`LCK contributed to antigen-dependent T-cell
`activation by transmitting tyrosine-phospho-
`rylation-dependent signals from the CD4
`and CD8 coreceptors25,26. In the meantime, a
`research team that included Larry Samelson
`and Richard Klausner discovered that TCR
`crosslinking provoked the tyrosine phos-
`phorylation of low molecular mass (20–25
`kD), receptor-associated polypeptides that
`were eventually identified as the CD3-ζ
`subunits27,28. Michael Reth formalized the
`link between inducible tyrosine phosphory-
`lation of the CD3-ζζ polypeptides and TCR
`signalling with the prediction that the cyto-
`plasmic domains of these receptor subunits
`contained immunoreceptor tyrosine-based
`activation motifs (ITAMs)29. The model that
`ITAM phosphorylation allows the activated
`TCR to recruit specific signalling proteins
`subsequently received support from studies
`carried out by Brian Seed, Weiss, Klausner,
`Bernard Malissen and others. These investi-
`gators fused the extracellular and trans-
`membrane domains of receptors, such as
`CD4 and CD8, to the CD3-ε or the CD3-ζ
`cytoplasmic regions30–35. When ectopically
`expressed in Jurkat cells or other trans-
`formed haematopoietic-cell lines, these
`chimeric proteins sensitized the cells to acti-
`vation by antibodies specific for their extra-
`cellular domains. These findings, together
`with results from many other laboratories,
`confirmed that the CD3-ε, -γ, -δ and -ζ sub-
`units were the ‘business end’ of the TCR with
`respect to transmembrane signalling, and that
`the ITAMs were centrally involved in linking
`the cell-surface receptor to the intracellular
`signalling machinery in T cells.
`As a result of the initial observations, the
`stage was clearly set for a flurry of reports
`showing that TCR stimulation provoked the
`
`tyrosine phosphorylation of many intracellular
`proteins in Jurkat T cells and other T-cell
`lines36–44. Based on the pharmacological evi-
`dence that these phosphorylation events were
`crucial for T-cell activation37,43, many laborato-
`ries joined the race to identify the substrates for
`these TCR-associated PTKs. These studies led
`to the conclusion that two SRC family PTKs,
`LCK and FYN, were proximate effectors of the
`first wave of protein tyrosine phosphorylation
`events triggered by TCR stimulation36–44. As
`discussed later, however, SRC family kinases
`were not the only class of PTK recruited to the
`TCR during T-cell activation.
`
`TCR-associated PTKs and their substrates. In
`the early 1990s, the identification of proteins
`(phosphorylated or otherwise) from whole
`cell extracts or immunoprecipitates was a
`considerably more challenging undertaking
`than it is today. The limited sensitivities of the
`analytical technologies available at this time
`imposed a high premium on the preparation
`of large numbers of homogeneously respon-
`sive T cells as the starting point for experi-
`ments, and not surprisingly, Jurkat cells were a
`popular choice among investigators who
`wished to identify TCR substrates. The
`emphasis on protein biochemistry also created
`a demand for T-cell activating agents that trig-
`gered maximal phosphorylation responses in
`Jurkat cells. Several groups took advantage of a
`pharmacological mode of T-cell activation
`that bypassed the requirement for ligation of
`cell-surface TCRs. Reports from several labo-
`ratories had shown that a mixture of hydrogen
`peroxide and sodium orthovanadate, termed
`pervanadate, was a powerful activator of
`protein tyrosine phosphorylation in both
`Jurkat and normal T cells45,46. With a pool of
`appropriately activated Jurkat cells available,
`investigators had several options regarding
`the substrate purification strategy. Two basic
`approaches, which we termed the ‘pull-down’
`and the ‘who’s home?’ assays, proved especially
`popular during the rush to harvest phospho-
`proteins from activated T-cell extracts. Several
`important signalling proteins emerged from
`the pull-down experiments. An example of
`paramount importance is the tyrosine kinase
`ζ-chain-associated protein of 70kD (ZAP70),
`which was isolated by Andy Chan (another
`member of the Weiss laboratory) from acti-
`vated Jurkat T-cell extracts47. Subsequent
`studies thoroughly documented the pivotal
`roles of ZAP70 in the tyrosine phosphoryla-
`tion of numerous proteins, including PLC-γ1,
`linker for activation of T cells (LAT), SH2-
`domain-containing leukocyte protein of 76 kD
`(SLP76) and VAV1, during TCR-dependent
`T-cell activation48–50. Using a strategy now
`
`P E R S P E C T I V E S
`
`Jurkat E6 cells
`
`Mutagenesis
`
`Stimulation
`
`Selection:
`Cell death, reporter-gene
`activation and Ca2+ signalling
`
`Isolation of TCR+ clones
`
`Immunoblotting
`using phospho-
`tyrosine-specific
`antibody
`
`Ca2+ signalling
`
`Immunoblotting
`for candidate
`signalling proteins
`
`Figure 1 | Generalized protocol for the
`selection and analysis of Jurkat-derived T-cell
`receptor (TCR)-signalling mutants. Jurkat E6
`cells are mutagenized by ionizing radiation, the
`point mutagen ethylmethanesulphonate (EMS)
`or the frameshift mutagen ICR-191. The cells
`are subjected to a high-throughput selection
`procedure, typically involving resistance to a
`death-inducing stimulus (for example, TCR
`ligation) or collection of non-responsive cells by
`fluorescence-activated cell sorting (FACS). Several
`cycles of selection are generally required to yield
`the necessary enrichment of non-responsive cells.
`Clonal cell populations are then generated and
`re-screened to ensure that selected clones retain
`the defective phenotype. To define the molecular
`basis of the TCR-signalling defect, the selected
`sublines are profiled for TCR-dependent Ca2+
`responses, protein tyrosine phosphorylation
`patterns and expression of candidate signalling
`proteins by immunoblotting.
`
`known as immunoproteomics, several research
`groups captured phosphoproteins from
`Jurkat T-cell extracts with immobilized phos-
`photyrosine-specific antibodies. In another
`milestone study, Weiguo Zhang, a postdoc-
`toral fellow in Samelson’s laboratory, used
`this technique to isolate LAT — a plasma-
`membrane-localized adaptor protein that
`nucleates a multiprotein signalling complex
`during TCR ligation49,51.
`The who’s home? assay for PTK substrate
`identification was more biased than the pull-
`down assays, but was relatively quick and easy
`to carry out. Simply put, whole-cell extracts or
`
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`P E R S P E C T I V E S
`
`Table 1 | Panel of Jurkat-derived TCR-signalling mutants
`Cell line
`Selection
`Genetic defect
`TCR-α negative
`JRT-T3.1
`TCR expression
`TCR-β negative
`JRT3-T3.5
`TCR expression
`Ca2+ mobilization
`LCK negative
`J.CaM1
`Ca2+ mobilization
`LAT negative
`J.CaM2
`J.CaM3
`Ca2+ mobilization
`N.D.
`J45.01
`CD45 expression
`CD45 negative
`J14
`CD69 expression
`SLP76 negative
`Ca2+ mobilization
`ZAP70 negative
`P116
`ANJ3
`Ca2+ mobilization
`LAT deficient
`PLC-γ1 deficient
`Ca2+ mobilization
`P98
`J.γ1
`PLC-γ1 negative
`Ca2+ mobilization
`G4
`Ca2+ mobilization
`LCK negative
`
`References
`11
`11
`20,57
`22,58
`20
`40
`59
`60
`64
`63
`63
`(R.T.A. et al.,
`unpublished
`observations)
`80
`VAV1 deficient
`Specific gene targeting
`J.Vav1
`NF-κB activation
`68
`CARMA negative
`JPM50.6
`Jurkat cells were randomly mutagenized with chemicals or ionizing radiation, and were selected according
`to the phenotypic parameter indicated in the second column. The underlying genetic defect is indicated in
`the third column. The term ‘negative’ indicates that no protein is detectable by immunoblotting; ‘deficient’
`indicates that a low level (>10% of wild-type) is present in the mutant cell line. LAT, linker for activation of
`T cells; N.D., not determined; NF-κB, nuclear factor-κB; PLC-γ1, phospholipase C-γ1; SLP76, SRC-
`homology 2 (SH2)-domain-containing leukocyte protein of 76 kD; TCR, T-cell receptor; ZAP70, ζ-chain-
`associated protein of 70kD.
`
`immunoprecipitated proteins from activated
`Jurkat cells were immunoblotted with phos-
`photyrosine-specific antibodies in a search
`for immunoreactive bands with molecular
`masses corresponding to those of known sig-
`nalling proteins. One of the many successes
`of this approach was the identification of a
`~150 kD PTK substrate in T cells, known as
`PLC-γ1 (REFS 52–54). The recognition that
`TCR stimulation triggered the tyrosine phos-
`phorylation and activation of PLC-γ1 helped
`to elucidate the events leading to phospho-
`inositide breakdown and intracellular Ca2+
`mobilization in activated T cells. With the
`large number of phospho-specific antibody
`reagents now available, combined with the
`power of protein database screening, the use
`of antibody-capture techniques for the iden-
`tification of protein kinase substrates is by no
`means passé55.
`
`TCR-signalling mutants
`By the mid-1990s, the list of candidate effectors
`of TCR signalling was growing exponentially,
`and so was the need to understand how each of
`these proteins contributed to the T-cell activa-
`tion response. Functional questions were best
`addressed with genetic approaches, and once
`again, Jurkat T cells stepped to the fore as ‘ideal’
`hosts for gene-transfer-based experiments.
`A proven strategy for the generation
`of genetically altered Jurkat T-cell lines
`involved genome-wide mutagenesis — the
`
`mutant-selection protocol used earlier by
`Goldsmith and Weiss56 (FIG. 1). As the list of
`candidate signalling proteins in T cells
`grew, so did the need for mutant T-cell lines
`that lacked known components of the TCR-
`signalling machinery. An early example of
`this approach was provided by Gary Koretzky,
`who was a postdoctoral fellow in the Weiss
`laboratory. Koretzky used the cell sorter to
`isolate CD45-negative variants that sponta-
`neously arose from the leukaemic T-cell line,
`HPB-ALL41, and from mutagenized Jurkat
`cells40. The CD45-deficient cells had marked
`defects in TCR-dependent PTK activation
`and phosphoinositide breakdown, and clearly
`defined a positive role for PTPase activity in
`early signalling from the TCR.
`Armed with new knowledge regarding
`TCR-linked signalling proteins, another
`member of the Weiss laboratory returned to
`study an ‘old’ Jurkat mutant and made an
`important discovery. David Straus hypothe-
`sized that a ‘missing’ 56 kD phosphoprotein
`in the J.CaM1 cell line20 might be LCK. His
`guess proved correct, and the markedly
`defective TCR signalling phenotype dis-
`played by J.CaM1 cells indicated that LCK
`occupied a receptor-proximal position in the
`cytoplasmic signalling cascade initiated by
`TCR crosslinking57. The J.CaM1 cell line
`quickly became a mainstay for studies of
`LCK regulation and function in activated
`T cells. Several years later, the TCR signalling
`
`defect in the J.CaM2 cell line was causally
`linked to loss of expression of the LAT adaptor
`protein22,58.
`As the TCR signalling field entered the
`mid-1990s, several laboratories continued to
`subject randomly mutagenized Jurkat T-cell
`populations to increasingly diverse selec-
`tion protocols. Deborah Yablonski, working
`in the Weiss laboratory, screened mutage-
`nized Jurkat populations for cells that failed
`to express the activation marker CD69 in
`response to TCR stimulation, and isolated a
`mutant clone that lacked the crucial adaptor
`protein, SLP76 (REF. 59). Brandi Williams and
`Brenda Irvin, two graduate students in Robert
`Abraham’s laboratory, undertook a large-scale
`effort to isolate Jurkat somatic mutants that
`failed to increase intracellular Ca2+ concentra-
`tions after exposure to pervanadate. Their
`work yielded several important additions to
`the existing series of Jurkat somatic mutants
`(TABLE 1). The most widely heralded member
`of this group is the P116 cell line, which lacks
`expression of ZAP70 (REF. 60). Serendipitously,
`the parental Jurkat E6 cell line was negative
`for expression of SYK61, and so, P116 cells
`were amenable to complementation experi-
`ments with wild-type or mutated versions of
`either ZAP70 or SYK. These cells remain a
`popular tool for TCR-signalling research, as
`indicated by their recent application in
`studies of the function of ZAP70 in activa-
`tion-associated cytoskeletal rearrangements
`in T cells62. The same pervanadate-based
`selection protocol yielded several additional
`Jurkat somatic mutants, including the G4
`(LCK deficient), ANJ3 (LAT deficient), and,
`most recently, the P98 (reduced PLC-γ1
`expression) and J.γ1 (PLC-γ1 deficient) cell
`lines63,64.
`The application of somatic-cell genetics
`to Jurkat cells also offered some unexpected
`insights into other receptor-mediated sig-
`nalling pathways. For example, the LCK-
`deficient J.CaM1 cells and ZAP70-deficient
`P116 cells allowed Andrew Larner and co-
`workers65 to define new roles for these PTKs
`in the anti-proliferative effect of interferon-α
`in T cells. Adrian Ting and Seed used a syn-
`thetic reporter gene to isolate Jurkat T-cell
`lines that failed to activate nuclear factor-κB
`(NF-κB) in response to stimulation with
`tumour-necrosis factor (TNF)66. This effort
`produced one Jurkat mutant that lacked the
`TNF-receptor-associated protein kinase, RIP,
`and a second that failed to express the
`inhibitor of NF-κB kinase (IKK) signalsome
`component, IKK-γ 67. Although efforts to
`derive additional Jurkat somatic mutants are
`winding down in most laboratories, impor-
`tant publications continue to appear in the
`
`304 | APRIL 2004 | VOLUME 4
`
`www.nature.com/reviews/immunol
`
`© 2004 Nature Publishing Group
`
`UPenn Ex. 2074
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`P E R S P E C T I V E S
`
`in vivo model systems. First, the production of
`transgenic and knockout mice requires con-
`siderable time and expense; therefore, mouse
`models are simply not a feasible first choice
`for many laboratories. Second, germline gene
`
`disruptions that involve key signalling proteins
`frequently lead to blocks in T-cell develop-
`ment. Although such developmental blocks
`provide important information in their own
`right, investigations into the function of the
`
`APC
`
`MHC class II
`
`CD4
`
`LCK
`
`RAS
`
`CD3ζ
`
`P P
`
`ZAP70
`
`TCR
`
`CD3ε
`and δ
`
`FYN
`
`P
`GRB2
`
`SOS
`
`LAT
`
`P
`
`P
`
`C -γ 1
`
`S
`
`L
`
`P
`
`7
`
`6
`
`S
`C γ 1
`D
`
`A
`
`L
`
`G
`
`P
`
`L
`
`P
`
`P
`
`IP3
`
`DAG
`
`RAS-
`GRP
`
`Intracellular Ca2+
`
`PKC-θ
`
`MAPK
`
`T cell
`
`GDP
`
`1
`
`V
`
`A
`
`V
`
`P
`
`P
`NCK
`
`PA
`K1
`
`GTP
`
`RH O
`
`Cytoskeletal
`rearrangement
`
`IL-2 gene expression
`Cell-cycle entry
`T-cell effector functions
`
`Figure 2 | Proximal signalling complexes and downstream responses induced by T-cell receptor
`(TCR) ligation. A model outlining our current knowledge of TCR and linker for activation of T cells (LAT)
`signalling complexes is illustrated. Following TCR ligation, SRC-family protein tyrosine kinases (PTKs) —
`for example, LCK and FYN — are activated, resulting in phosphorylation of CD3 modules of the TCR
`complex and activation of SYK-family PTKs —for example, ζ-chain-associated protein of 70kD (ZAP70).
`Activated ZAP70 phosphorylates LAT and SLP76 (SRC-homology 2 (SH2)-domain-containing leukocyte
`protein of 76 kD). Tyrosine-phosphorylated LAT then recruits several SH2-domain-containing proteins,
`including growth factor receptor-bound protein 2 (GRB2), GRB2-related adaptor protein (GADS) and
`phospholipase C-γ1 (PLC-γ1). Through its constitutive association with GADS, SLP76 is also recruited to
`LAT following TCR stimulation. Evidence indicates that SLP76 also constitutively associates with the SH3
`domain of PLC-γ1. Activation of PLC-γ1 results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate
`to inositol 3,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 production leads to increases of intracellular
`free Ca2+ concentration, whereas DAG can activate both protein kinase C-θ (PKC-θ) and RAS guanyl
`nucleotide-releasing protein (RASGRP). Phosphorylated LAT also recruits the SH2 domain of GRB2,
`and therefore, the GRB2-associated RAS guanosine nucleotide-exchange factor (GEF), son-of-sevenless
`(SOS), thereby providing an additional possible mechanism of RAS activation through LAT. Tyrosine-
`phosphorylated SLP76 also associates with the RHO-family GEF, VAV1, and the adaptor protein, NCK.
`A trimolecular complex between SLP76, VAV1 and NCK-associated p21-activated kinase 1 (PAK1) has been
`proposed as a potential mechanism for SLP76 regulation of actin cytoskeletal rearrangements following
`TCR stimulation. APC, antigen-presenting cell; IL-2, interleukin-2; MAPK, mitogen-activated protein kinase.
`
`literature. A noteworthy example involved
`the selection of Jurkat somatic mutants with
`defects in TCR-mediated NF-κB activation68.
`One of the resulting mutant cell lines failed to
`express CARMA1, a scaffolding protein
`required for the coupling of TCR ligation to
`activation of the IKK signalsome. More
`recently, Jeroen Roose, a postdoctoral fellow in
`the Weiss laboratory, collaborated with Patrick
`Brown’s laboratory on expression array analy-
`ses of some of the mutant Jurkat lines. These
`studies revealed that TCR-mediated signal-
`transduction pathways, surprisingly, are not
`silent in unstimulated Jurkat cells, but instead
`are tonically active and suppress the expression
`of a set of genes, including the recombinase-
`activating genes (RAGs)69.
`
`Problems with Jurkat cells
`As transgenic and knockout mouse models
`increased in popularity, some T-cell biolo-
`gists questioned the physiological relevance
`of in vitro experiments carried out with
`Jurkat and other transformed T-cell lines.
`The issue came to a head in 2000, when
`Jurkat cells were shown to be defective in the
`expression of two lipid phosphatases, PTEN
`(phosphatase and tensin homologue) and
`SHIP (SH2-domain-containing inositol
`polyphosphate 5’ phosphatase)70–72. The
`biological implications of SHIP deficiency
`in T cells are poorly understood, but the
`absence of PTEN raised some immediate
`concerns. Loss of PTEN leads to constitu-
`tive activation of the phosphatidylinositol
`3-kinase (PI3K)-signalling pathway, which
`includes the protein serine-threonine kinase,
`AKT, and the PTK IL-2-inducible T-cell kinase
`(ITK) in Jurkat cells73. It remains unclear,
`however, to what extent the abnormal PTEN
`status of Jurkat cells alters their response to
`TCR stimulation, as the importance of PI3K
`in the early phase of TCR signalling is still
`poorly understood74.
`
`T-cell signalling in vivo and in vitro
`In the twenty-first century, immunologists
`have at their disposal a powerful array of
`in vivo model systems, together with sophisti-
`cated technologies (for example, cell imaging,
`gene microarrays and mass spectrometry)
`that allow both molecular and system-wide
`views of the T-cell activation process. The
`limitations of long-term cell lines such as
`Jurkat are now widely acknowledged, and the
`relative advantages of mouse models for stud-
`ies of T-cell development and function are
`well documented75. Although cultured T-cell
`lines, including Jurkat, may have lost some of
`their earlier appeal, they still have several
`noteworthy strengths compared with the
`
`NATURE REVIEWS | IMMUNOLOGY
`
`VOLUME 4 | APRIL 2004 | 3 0 5
`
`© 2004 Nature Publishing Group
`
`UPenn Ex. 2074
`Miltenyi v. UPenn
`IPR2022-00855
`
`

`

`Conclusion
`The signalling machinery engaged by the
`TCR is now understood in exquisite detail49
`(FIG. 2). Many of the signalling proteins that
`are depicted in the increasingly tangled dia-
`grams of the TCR-signalling cascade were
`first identified and characterized in vitro in
`long-term cultured T-cell lines. Now, trans-
`genic and gene-targeted mice are providing
`fabulous insights into the relationship
`between signal output from the TCR and
`normal T-cell physiology. In the midst of all
`of the excitement surrounding the mouse
`models, long-term cultured T-cell lines
`might seem like relics from a bygone era of
`immunological research. However, a data-
`base search reveals that, although the use of
`Jurkat cells may have peaked in the late
`1990s, hundreds of published reports con-
`tinued to cite this cell line during the past
`year (FIG. 3). We hope that this Timeline article
`has provided both veterans and newcomers
`alike with an historical perspective of TCR
`signalling, together with a new (or renewed)
`sense of appreciation for the numerous con-
`tributions of the Jurkat cell line to this field
`during the past two decades. In recent
`years, these cells have taken a few knocks
`from the immunology com