Abstract
How T cells respond with extraordinary sensitivity to minute amounts of agonist peptide and major histocompatibility complex (pMHC) molecules on the surface of antigen-presenting cells bearing large numbers of endogenous pMHC molecules is not understood. Here we present evidence that CD4 affects the responsiveness of T helper cells by controlling spatial localization of the tyrosine kinase Lck in the synapse. This finding, as well as further in silico and in vitro experiments, led us to develop a molecular model in which endogenous and agonist pMHC molecules act cooperatively to amplify T cell receptor signaling. At the same time, activation due to endogenous pMHC molecules alone is inhibited. A key feature is that the binding of agonist pMHC molecules to the T cell receptor results in CD4-mediated spatial localization of Lck, which in turn enables endogenous pMHC molecules to trigger many T cell receptors. We also discuss broader implications for T cell biology, including thymic selection, diversity of the repertoire of self pMHC molecules and serial triggering.
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Acknowledgements
We thank M. Krogsgaard for providing TCR binding data before publication. Supported by the National Institutes of Health, Howard Hughes Medical Institute, Helen Hay Whitney Foundation (Q.J.L.), QB3 Institute at UC Berkeley (S.Y.Q.), National Science Foundation (A.R.D.) and Burroughs Wellcome Fund (A.R.D.).
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Supplementary information
Supplementary Fig. 1
Schematic representation of the enzymatic reactions leading up to activation of downstream substrates by ZAP70. P-ase represents a phosphatase that can carry out dephosphorylation reactions. Dashed box: Reduced enzymatic modification scheme that exhibits similar qualitative behavior to the full cascade14. (PDF 17 kb)
Supplementary Fig. 2
Schematic indicating the various types of binding and unbinding reactions included in the computer simulations. M is either type of pMHC (ag/MHC or en/MHC), T is TCR regardless of phosphorylation state (TCR or TCRp), C is CD4 and L is Lck. A solid line indicates an existing non-covalent association. CD4 and Lck remain together at all times. The number in parentheses in the header on each category is the number of reactions in each direction to which each schematic corresponds; it is 2nM+nT, where nM is the number of pMHC and nT is the number of TCR on one side of a diagram. The indices of rate constants refer to the number of reactions in Table S1. The asterisks on TCR-pMHC dissociation reactions indicate that the rate can be either k2 or the en/MHC off-rate, which is varied. (PDF 267 kb)
Supplementary Fig. 3
Fraction of trials (in silico experiments) with sustained calcium. (a) Cooperative model in which two pMHC, CD4, and Lck can be spatially localized (Fig.5). (b) Non-cooperative model in which endogenous and agonist pMHC behave autonomously. enMHC and agMHC denote endogenous and agonist pMHC, respectively. The plots range from the fraction of trials with sustained calcium being 0 (red) to 1 (violet); the contour spacing is 0.1. The criterion for sustained calcium is given in Fig. 6 of the main text; each point was calculated from 20 independent trials. The parameters for the binding and dissociation rates of agonist pMHC to TCR are taken from Grakoui et al. 4. (PDF 218 kb)
Supplementary Fig. 4
TCR phosphorylation. (a) Logarithm (base 10) of the average total number of phosphorylated TCR (nP), and (b) ratio of nP obtained in simulations with both antigenic and endogenous pMHC (nPa+e) to the sum of those obtained in separate simulations with the corresponding number of antigenic (nPa) and endogenous (nPe) pMHC [r = nPa+e/(nPa + nPe)] for the cooperative model in which two pMHC can be spatially localized with CD4 (Lck). (c) and (d) Same as (a) and (b) for the non-cooperative model in which endogenous and agonist pMHC behave autonomously. (e) and (f) Same as (a) and (b) for a model in which CD4 binding to pMHC is blocked. Each point was calculated from 20 independent trials. (PDF 378 kb)
Supplementary Fig. 5
Analysis of the effects of distributing endogenous pMHC off-rates over a range. (a) The degree of darkness measures the ratio of the number of TCR phosphorylated in simulations in which the endogenous pMHC off-rates were uniformly distributed in 11 groups that spanned the specified range centered on 100 s−1 (nPrange) to that obtained in simulations in which all endogenous pMHC off-rates were 100 s−1 (nPmean) [r = nPrange/nPmean]. For example, a range of 50 s−1 means that the endogenous pMHC off-rates varied from 75 s−1 to 125 s−1 in steps of 5 s−1. (b) Same as (a) but for integrated Ca+2. Each point was calculated from 20 independent trials. The basic message is that the qualitative behavior does not change upon having a mixture of endogenous pMHC molecules. (PDF 290 kb)
Supplementary Fig. 6
The situation if pMHC dimers are prevalent on APC surfaces (the zig-zag line indicates association). (a) With high probability, pMHC dimers will be hetrodimers of agonist and endogenous pMHC because the latter are in abundance. (b) TCR binding to agonist pMHC leads to recruitment and binding of CD4. This also leads to the spatial localization of Lck in a complex that is functionally identical to 5.i.c. (c) TCR binds to an endogenous pMHC, and can be triggered in spite of small half life, because Lck is "ready and waiting". (PDF 365 kb)
Supplementary Note
Master Equations Corresponding to Eqs. 1-3. (PDF 68 kb)
Supplementary Video 1
The accumulation of Lck in the synapse when the T cell is in contact with two K5 peptides. Multiple channel 3-D time lapse microscopic images were recorded at 30s intervals. Top left: DIC image; top right: radiometric Fura (340nm/380nm) image indicating the calcium concentration inside the T cell; bottom right: en-face view of K5 peptide signal (PE channel) in the synapse; bottom left: the best focal plane of Lck-GFP. (MOV 576 kb)
Supplementary Video 2
Lck accumulation in the synapse when the T cell is in contact with six K5 peptides. Multiple channel 3-D time lapse microscopic images were recorded at 30s intervals. Top left: DIC image; top right: radiometric Fura (340nm/380nm) image indicating calcium concentration inside the T cell; bottom right: en-face view of K5 peptide signal (PE channel) in the synapse; bottom left: best focal plane of Lck-GFP. (MOV 735 kb)
Supplementary Video 3
Lck accumulation in the synapse when the T cell is in contact with six K5 peptides and also in the presence of anti-CD4 antibody. Multiple channel 3-D time lapse microscopic images were recorded at 30s intervals. Top left: DIC image; top right: radiometric Fura (340nm/380nm) image indicating calcium concentration inside the T cell; bottom right: en-face view of K5 peptide signal (PE channel) in the synapse; bottom left: best focal plane of Lck-GFP. (MOV 760 kb)
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Li, QJ., Dinner, A., Qi, S. et al. CD4 enhances T cell sensitivity to antigen by coordinating Lck accumulation at the immunological synapse. Nat Immunol 5, 791–799 (2004). https://doi.org/10.1038/ni1095
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DOI: https://doi.org/10.1038/ni1095
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