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Determines the Functional Fate of T
Proximal TCR-Signaling Cascades
Antigen-Dependent Integration of Opposing
Shen-Orr, Sean C. Bendall, Garry P. Nolan and Yoram
Ron Wolchinsky, Moran Hod-Marco, Kfir Oved, Shai S.
2014; 192:2109-2119; Prepublished online 31
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The Journal of Immunology
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The Journal of Immunology
Antigen-Dependent Integration of Opposing Proximal
TCR-Signaling Cascades Determines the Functional Fate
of T Lymphocytes
Ron Wolchinsky,* Moran Hod-Marco,* Kfir Oved,* Shai S. Shen-Orr,†Sean C. Bendall,‡
Garry P. Nolan,‡and Yoram Reiter*
T cell anergy is a key tolerance mechanism to mitigate unwanted T cell activation against self by rendering lymphocytes
functionally inactive following Ag encounter. Ag plays an important role in anergy induction where high supraoptimal doses
lead to the unresponsive phenotype. How T cells “measure” Ag dose and how this determines functional output to a given antigenic
dose remain unclear. Using multiparametric phospho-flow and mass cytometry, we measured the intracellular phosphorylation-
dependent signaling events at a single-cell resolution and studied the phosphorylation levels of key proximal human TCR activation-
and inhibition-signaling molecules. We show that the intracellular balance and signal integration between these opposing signaling
cascades serve as the molecular switch gauging Ag dose. An Ag density of 100 peptide–MHC complexes/cell was found to be the
transition point between dominant activation and inhibition cascades, whereas higher Ag doses induced an anergic functional state.
Finally, the neutralization of key inhibitory molecules reversed T cell unresponsiveness and enabled maximal T cell functions, even in
the presence of very high Ag doses. This mechanism permits T cells to make integrated “measurements” of Ag dose that determine
subsequent functional outcomes. The Journal of Immunology, 2014, 192: 2109–2119.
Two processes prevent these effector cells from performing self-
reactivity: a thymic selection process that eliminates autoreactive
clones by negative selection (1) and a well-regulated multistage
priming process that is necessary for the maturation of naive cells into
effector CTLs (2–4). CTLs that have successfully surmounted these
regulatory barriers elicit their response as effectors and differentiate
into memory cells (5, 6). No subsequent regulatory restrictions have
been reported for these properly primed and activated CTLs.
Anergy is a hyporesponsive state described in lymphocytes, in
which a cell ceases to proliferate and to secrete crucial proinflam-
matory cytokines. Anergy has been categorized further into several
states differ in the types of cytokines inhibited, the requirement
for Ag persistence, and the ability of the cells to recover from this
hyporesponsive state (8). In addition, the various anergic states
differ in the biochemical pathways responsible for their onset and
ytotoxic T lymphocytes act as the effector arm of cell-
mediated immune response to kill self cells that are vir-
ally infected or have undergone malignant transformation.
maintenance; clonal anergy is initiated by blocking the Ras-MAPK
pathway, whereas a defect in ZAP70 phosphorylation was shown to
be involved in the peripheral anergy state (8).
Induction of T cell anergy was shown to be influenced by
peptide–MHC (pMHC) levels in mice (9) and human T cells (10).
High levels of pMHC on target cells generated unresponsiveness
by enhancing TCR downregulation. This induction of T cell anergy
using a supraoptimal Ag dose also was an extremely rapid process,
because preincubation with peptide for as little as 30 min was
sufficient to induce a complete unresponsive state (11).
We showedthat primary humanCTLs are alsosubjected to pMHC
dose-dependent anergy (12), whereupon exceeding a threshold
∼100 pMHC-I–specific complexes, a significant decrease in CTL
proliferation, killing, and cytokine secretion was observed, as were
alterations in the expression of key immunological synapse surface
molecules. Significant changes in the gene-expression signatures in
these CTLs exposed to supraoptimal pMHC doses also occurred,
indicating an anergy-like phenotype in these memory CTLs (12).
A significantamount of workhasbeenpublishedon the triggering
complexes required for initiation of T cell responses (13). However,
much less attention has been paid to the effect of high Ag doses and
their role in T cell function. For example, the molecular mechanisms
underlying the switch from activation and optimal activity to anergy
that is induced by Ag doses remain unknown.
In the current study, we explored the molecular mechanisms by
which CTLs distinguish between low-to-optimal and supraoptimal
levels of presented peptide–MHC class I (pMHC-I) complexes. We
asked how T cells sense Ag dose and make the decision to become
anergic after exposure to high Ag dose. We show that Ag-induced
hyporesponsiveness is calcium independent and is distinct from
currently described anergy molecular mechanisms. By evaluating
the differential phosphorylation of 21 proteins involved in TCR sig-
naling at low, optimal, and high pMHC-I Ag doses using phospho-
flow and mass cytometry, we show that the intracellular bal-
ance and signal integration between opposing activation- and
*Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel;
†Department of Immunology, The Bruce Rappaport Faculty of Medicine, Technion-Israel
Institute of Technology, Haifa 32000, Israel; and‡Baxter Laboratory in Stem Cell Biology,
Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305
Received for publication April 30, 2013. Accepted for publication December 23,
This work was supported by Research Grant 781/11 from the Israel Science Foundation
(to Y.R.). S.S.S.-O. is a Taub fellow and is supported in part by Israel Science Foundation
Address correspondence and reprint requests to Prof. Yoram Reiter, Technion-Israel
Institute of Technology, Technion City, Room 333, Haifa 32000, Israel. E-mail ad-
The online version of this article contains supplemental material.
Abbreviations used in this article: CyTOF, cytometry time-of-flight; MFI, median
fluorescence intensity; pMHC, peptide–MHC; pMHC-I, peptide–MHC class I; SGG,
by guest on December 24, 2015
nonmonotonic effector function and a corresponding functional
phenotype of these CTLs (i.e., potent killing of target cells at
optimal Ag doses and hyporesponsiveness at a high dose of
pMHC-I complexes on target cells). Thus, we suggest that TCRs
can sense Ag dose through the intracellular balance and signal
integration between activatory- and inhibitory-related signals.
This sensing machinery dictates the final functional output of
these CTLs, whether optimal effector functions or hypores-
The authors have no financial conflicts of interest.
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The Journal of Immunology2119
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