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Noncanonical binding of Lck to CD3ε promotes TCR signaling and CAR function

Nature Immunology volume 21, pages 902–913 (2020)Cite this article

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An Author Correction to this article was published on 19 November 2020

This article has been updated

Abstract

Initiation of T cell antigen receptor (TCR) signaling involves phosphorylation of CD3 cytoplasmic tails by the tyrosine kinase Lck. How Lck is recruited to the TCR to initiate signaling is not well known. We report a previously unknown binding motif in the CD3ε cytoplasmic tail that interacts in a noncanonical mode with the Lck SH3 domain: the receptor kinase (RK) motif. The RK motif is accessible only upon TCR ligation, demonstrating how ligand binding leads to Lck recruitment. Binding of the Lck SH3 domain to the exposed RK motif resulted in local augmentation of Lck activity, CD3 phosphorylation, T cell activation and thymocyte development. Introducing the RK motif into a well-characterized 41BB-based chimeric antigen receptor enhanced its antitumor function in vitro and in vivo. Our findings underscore how a better understanding of the functioning of the TCR might promote rational improvement of chimeric antigen receptor design for the treatment of cancer.

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Fig. 1: Stimulation of the TCR increases TCR–Lck proximity.
Fig. 2: The SH3 domain of Lck mediates TCR binding.
Fig. 3: Noncanonical binding of SH3(Lck) to nonphosphorylated CD3ε.
Fig. 4: The activity of Lck in the vicinity of the TCR increases upon TCR stimulation.
Fig. 5: The RK motif is important for TCR activation.
Fig. 6: The CD3ε RK motif is required for thymic T cell development.
Fig. 7: The CD3ε RK motif enhances in vitro tumor cell killing by CAR T cells.
Fig. 8: The CD3ε RK motif enhances in vivo tumor control by CAR T cells.

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Data availability

The authors declare that all relevant data supporting the findings of this study are available within the paper and its supplementary information files.

Change history

  • 19 November 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank K. Fehrenbach, A. Buschky, L. Herr, S. Pathan-Chhatbar and R. Heissmann for technical assistance and K. Astrahantseff, K. Schachtrup, Y. Kulathu and H. van Santen for critical reading of the manuscript. We also thank B. Alarcón for providing reagents and S. Andrade for scientific discussion. This study was supported by the German Research Foundation (DFG) through BIOSS - EXC294 and CIBSS - EXC 2189 to W.W.S., M.K. and S.M.; SFB1160 (P5 to S.M.); SFB850 (C10 to S.M.); SFB854 (B19 to W.W.S.); and SFB1381 (A9 to W.W.S.). The work of P.M. is financially supported by DFG through GU1225/3-1. O.S.Y., N.M.W., A.M. and S.M.B. were supported by DFG through GSC-4 (Spemann Graduate School). F.A.H. was partially supported by the Baden Württemberg Stiftung (Eliteprogramm to S.M.). R.M.V.C. was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 721358.

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Author notes

  1. These authors jointly supervised this work: Wolfgang W. Schamel, Susana Minguet.

Authors and Affiliations

  1. Faculty of Biology, Albert-Ludwigs-University of Freiburg, Freiburg, Germany

    Frederike A. Hartl, Esmeralda Beck-Garcìa, Nadine M. Woessner, Lea J. Flachsmann, Rubí M.-H. Velasco Cárdenas, Simon M. Brandl, Gina J. Fiala, Anna Morath, O. Sascha Yousefi, Julia Zimmermann, Nico Hoefflin, Maja Köhn, Wolfgang W. Schamel & Susana Minguet

  2. Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany

    Frederike A. Hartl, Nadine M. Woessner, Rubí M.-H. Velasco Cárdenas, Simon M. Brandl, Gina J. Fiala, Anna Morath, O. Sascha Yousefi, Nico Hoefflin, Maja Köhn, Robert Zeiser, Wolfgang W. Schamel & Susana Minguet

  3. Center of Chronic Immunodeficiency CCI, University Clinics and Medical Faculty, Freiburg, Germany

    Frederike A. Hartl, Wolfgang W. Schamel & Susana Minguet

  4. Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-University Freiburg, Freiburg, Germany

    Nadine M. Woessner, Simon M. Brandl, Anna Morath & O. Sascha Yousefi

  5. Department of Medicine I, Medical Center, University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Sanaz Taromi & Robert Zeiser

  6. Faculty of Medical and Life Sciences, Furtwangen University, Campus VS-Schwenningen, Furtwangen, Germany

    Sanaz Taromi

  7. Pharmaceutical Bioinformatics, Albert-Ludwigs-University of Freiburg, Freiburg, Germany

    Pankaj Mishra & Stefan Günther

  8. Biopolymers, University of Bayreuth, Bayreuth, Germany

    Birgitta M. Wöhrl & Kristian Schweimer

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  1. Frederike A. Hartl
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Contributions

F.A.H. and E.B.G. performed most experiments. L.J.F. and N.M.W. performed PLA. G.J.F., S.T. and R.Z. participated in the in vivo experiments. O.S.Y., A.M. and J.Z. helped with cloning, sequence analyses and in the experiments with the Lck reporter. R.M.V.C., S.M.B. and N.M.W. contributed to the CAR experiments. S.M.B. performed the CRISPR/Cas9 gene editing. K.S. and B.W. performed the NMR studies. P.M. and S.G. performed the molecular modeling. M.K. and N.H. synthesized the inhibitory ZAP-70 peptide. W.W.S. and S.M. designed the study and wrote the paper.

Corresponding author

Correspondence to Susana Minguet.

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Competing interests

F.A.H., W.W.S. and S.M. are listed as inventors on a patent application filed by the University of Freiburg (PCT/EP2020/058075) related to the use of the RK motif in immunotherapy against cancer. W.W.S. receives consulting income from TCR2 therapeutics.

Additional information

Peer review information Jamie D. K. Wilson and Ioana Visan were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 The in situ proximity ligation assay demonstrates spatial proximity of the TCR and Lck.

a, Scheme of the proximity ligation assay (PLA), a red fluorescent dot indicates a distance of smaller than 80 nm between CD3δ and Lck. b, Technical PLA controls in Jurkat cells stimulated with the anti-hCD3ε antibody OKT3 at 37 °C for 5 min. PLA was performed with both primary antibodies (left), with only the anti-CD3δ primary antibody (middle) or with only the anti-Lck primary antibody (right). In all cases both secondary antibodies were used. The quantification of one representative experiment was analyzed using unpaired Student’s t test (bar diagram). c, PLA between the TCR (CD3δ) and Lck in Jurkat cells expressing surface TCR and Lck (left), Jurkat cells with reduced CD3ε expression by shRNA and thus lacking surface TCR (middle) and JCaM1.6 cells lacking Lck expression (right). Quantification of one representative experiment is shown (bar diagram). Mean values ± s.e.m. are shown. ***P < 0.001. d, Flow cytometry using specific anti-CD4 and anti-CD8 antibodies was used to demonstrate no detectable expression of these coreceptors in Jurkat T cells. In vitro expanded primary human PBMCs (blue) were used as positive control.

Extended Data Fig. 2 The SH3 domain of Lck binds to the CD3ε cytoplasmic tail.

a, Sequence of the murine CD3-derived peptides used in the study. b, A pull-down (PD) assay using glutathione beads bound to GST (-) or to GST-fusion proteins containing either the Lck SH2 domain (SH2) or both Lck SH2 and SH3 domains (SH2-SH3) was performed. These beads were incubated with the indicated biotinylated peptides as shown in (a) (PP refers to doubly phosphorylated ITAMs). Immunoblotting was performed using streptavidin-HRPO and anti-GST antibodies. c, Flow cytometric analysis in Jurkat T cells retrovirally transduced with scrambled or CD3ε-specific shRNAs before and after FACS sorting of GFP+ cells. The new Jurkat T cell variants were named H8 (scrambled shRNA) and H8εsh (CD3ε-specific shRNA). d, Immunoprecipitation of surface TCRs from H8 and H8εsh cells and immunoblot analysis (a long and a short exposure for CD3ε are shown). e, Flow cytometric analysis of murine (mCD3ε) and endogenous human CD3ε (hCD3ε) expression in H8εsh cells reconstituted with murine CD3εWT, CD3εPRSΔ or CD3εK76T using specific antibodies (145-2C11 and UCHT1, respectively). Murine 2B4 and human Jurkat T cells were used as controls. f, Cells as in e were left untreated (-) or stimulated for 5 min at 37 °C with 5 μg/ml anti-mCD3ε antibody. A PD using Nck SH3.1-beads detects TCRs that have switched to the active conformation and thereby exposed the PRS in CD3ε.

Extended Data Fig. 3 The RK motif is a conserved motif in the CD3ε cytoplasmic tail.

a, The CD3ε cytoplasmic tail sequences from different vertebrate species were retrieved from UniprotKB. The following conserved motifs are highlighted: the proline-rich sequence (PRS; blue), the identified RK motif (green) and the ITAM (underlined). The RK motif is evolutionarily conserved from lobe-finned fishes to mammals. b, Schematic of the relative frequencies of the corresponding amino acids at that position among different species from a as reflected by the height of symbols. Sequence logo was generated with Weblogo Berkeley. c, Flow cytometry analysis of transduced mCD3ε and endogenous hCD3ε expression levels in H8εsh cells reconstituted by lentiviral transduction with the following murine CD3ε variants: WT, mutation in the RK motif (RKAA), double mutation in the RK motif and PRS (DOM) or a mutation with reduced capability to switch to the active TCR conformation (K76T). Murine 2B4 and human Jurkat T cells were used as controls. d, PD using Nck SH3.1-beads and cell lysates of H8εsh cells reconstituted with mCD3ε variants as indicated. Cells were either left untreated (-) or incubated for 2 h on ice with 5 μg/ml of the anti-mCD3ε antibody (+) to stabilize the TCR in its active conformation and in the presence of 10 μM PP2, to avoid phosphorylation. Lysates and bead-purified proteins were analyzed by immunoblotting. One representative experiment is shown (left panel). Relative signals (ζ/GST) on stimulated cells were normalized to its corresponding unstimulated sample (Uns) and statistical analysis was performed using unpaired Student’s t test on pooled data from six independent experiments. Mean values ± s.e.m. are indicated (right panel). *P < 0.05; **P < 0.01; NS, not significant. e, The affinity of the WT and RKAA mutant CD3ε peptides to the Lck SH3 domain was calculated by adding these unlabeled peptides stepwise to 15N labeled Lck SH3 while performing NMR.

Extended Data Fig. 4 Characterization of the reporter for Lck activity at the TCR.

a, Flow cytometric analysis of ζ and ζ-Reporter expression on the surface of the ζ-, or ζ-Reporter-reconstituted or control MA5.8 cell lines using an anti-mCD3ε antibody. b, Immunoprecipitation (IP) and immunoblotting of surface TCRs using anti-mCD3ε (145-2C11) in ζ-, or ζ-Reporter-reconstituted or control MA5.8 cells. c, Scheme depicting the alternative ζ-Reporters designed to confirm constitutive exposure of the kinase-dead kinase domain of Lck within the reporter. The CD3ε proline rich sequence (PRS) was fused to the C-terminus of the ζ-Reporter. Different lengths of the CD3ε PRS were used, either containing 21 or 11 amino acids, resulting in ζ-Rep-εPRS21 or ζ-Rep-εPRS11. d and e, The expression of the ζ-Rep-εPRS constructs in MA5.8 cells was investigated as in b. f, PD assay using Nck SH3.1-beads and immunoblotting in unstimulated lysates of cells containing the ζ-Rep-εPRS reporters. The quantification of two independent experiments (mean values ± s.e.m.) is shown in the lower panel. Arrows indicated sequential PD to avoid saturation of the Nck(SH3.1)-beads. g, MA5.8 cells containing the ζ-Reporter were stimulated with plate-bound anti-mCD3ε antibodies (10 μg/ml) for 20 hours in the presence of DMSO, control peptide or a peptide inhibiting the catalytic activity of ZAP-70 (ZAP70inh). Cells were stained and measured by flow cytometry to determine the percent of CD69-positive cells. Mean values ± s.e.m. and statistical analysis using one-way ANOVA between treatments with control peptide or ZAP70inh are shown. ****P < 0.0001. h, Immunoprecipitation (IP) and immunoblotting of surface TCRs. MA5.8 cells containing the ζ-Reporter and knock out for ZAP-70 using the CRISPR/Cas9 technology were either left untreated (-) or stimulated (+) at 37 °C. One representative experiment is shown.

Extended Data Fig. 5 The RK motif is important for ligand-induced recruitment of Lck to the TCR at 37 °C.

Proximity ligation assay between the TCR (CD3δ) and Lck was done as in Fig. 1. The indicated H8εsh cell variants were either left unstimulated (Uns), stimulated with 5 μg/ml of anti-mCD3ε antibody (145-2C11) or with 0.1 mM pervanadate (PerV) at 37 °C for 5 min. Data from five independent experiments were pooled and statistical analysis compared anti-mCD3ε stimulated H8εsh-CD3εWT cells and each of the H8εsh cell variants (grey bars) using unpaired Student’s t test. Mean values ± s.e.m. are shown; *P < 0.05; **P < 0.01; NS, not significant.

Extended Data Fig. 6 The CD3ε RK motif is required for thymic T cell development.

a, Flow cytometric analysis of GFP expression levels in hematopoietic stem cells (HSC) from CD3ε-/- CD45.2+ donor mice transduced with lentiviruses encoding either mCD3εWT or mCD3εRKAA and IRES-GFP (black histograms). Non-transduced (NT) cells served as a control (grey histograms). b, Flow cytometric analysis of CD4 and CD8 surface expression on thymocytes isolated 6 weeks after injection of transduced CD3ε-/- CD45.2+ HSC into irradiated Rag2-/- recipient mice. GFP+ cells were gated and T cell development analyzed. As reference the thymus of a CD3ε-/- CD45.2+ mouse was analyzed (most right panel) c, The percent and d, the total number of thymocytes in the populations indicated was determined (DP: CD4+CD8+; DN: CD4−CD8−; CD4: CD4+CD8−; CD8: CD4−CD8+). e, Flow cytometric analysis of CD44 and CD25 surface expression on DN thymocytes. The DN populations were subdivided into DN1 to DN4 populations: DN1 (CD44+CD25−); DN2 (CD44+CD25+); DN3 (CD44−CD25+); and DN4 (CD44−CD25−). Representative dot plots are shown. Mean values ± s.e.m. of 9 mice are shown. Statistical analysis was performed using unpaired Student’s t-test. *P < 0.05; **P < 0.01; NS, not significant.

Extended Data Fig. 7 The CD3ε RK motif enhances tumor killing by CAR-T cells.

a, Scheme depicting the vectors encoding for the 41BB-based and US Food and Drug Administration-approved ζCAR (top), and the two novel CARs designed in this study. The promoter used was human EF1α. ECD, extracellular domain, hinge region; TM, transmembrane region; ICD, intracellular domain; GFP, green fluorescent protein b, Flow cytometric analysis of surface CAR expression levels in human expanded T cells expressing the ζCAR, εRKζCAR or εRKAAζCAR. Analysis was done using paired one-way ANOVA test, each dot represent an independent donor (n = 9). c, Anti-tumor activity (specific killing activity against Daudi Burkitt-lymphoma cells) of human primary T cells transduced with the 41BB-based and US Food and Drug Administration-approved ζCAR was assayed by three standard methods: 51Cr release, a FACS-based assay and a luciferase-based assay. Statistical analysis was performed using unpaired Student’s t-test. d, Specific killing activity against CD19-expressing Daudi Burkitt-lymphoma cells of human T cells transduced with the lentiviral vector coding for the ζCAR at different multiplicities of infection (MOI) correlates with the level of surface expression of the ζCAR fitting a symmetrical sigmoidal curve (n = 24 points analyzed, R square = 0.91, black dots and black curve). The specific killing activity of human T cells transduced with lentiviral vectors coding for εRKζCAR and εRKAAζCAR at a MOI = 4 are shown as bars. e, Degranulation, measured by CD107α appearance on the cell surface, of CAR T cells upon 3 h incubation without and with CD19-expressing Nalm6 cells. Representative results of 2 independent experiments with 2 independent donors are shown. f, Phenotype of CAR T cells at day 8 after CAR transduction. The percentage of naïve (GFP+CD3+CD45RA+CD27+), central memory (TCM, GFP+CD3+CD45RA-CD27+), effector memory (TEM, GFP+CD3+CD45RA-CD27−) and terminally differentiated CD45RA (TEMRA, GFP+CD3+CD45RA+CD27−) populations was quantified. Representative results of 3 independent experiments with 3 independent donors are shown. g, CAR T cells were incubated for the indicated times in the presence or absence of CD19-expressing Nalm6 tumor cells. Expression of the indicated markers of activation was assayed by flow cytometry. Effective activation for a given marker was calculated by dividing the percentage of positive CAR T cells cultured with Nalm6 cells by the percentage of positive cells cultured alone. Mean values of triplicates ± s.e.m. and P values calculated using one-way ANOVA are indicated. Representative results of 2 independent experiments with 2 independent donors are shown. h, Technical PLA controls in Jurkat T cells genetically disrupted for ζ (JKζKO) and expressing the ζCAR. The cells were stimulated with Fc-CD19 at 4 °C, in the presence of PP2 for 120 min. PLA was performed with both primary antibodies (left), with only the anti-ζ primary antibody (middle) or with only the anti-Lck primary antibody (right). In all cases both secondary antibodies were used. The quantification of one representative experiment was analyzed using one-way ANOVA test. Mean values ± s.e.m. are shown. i, Biological PLA controls in Jurkat T cells expressing surface TCR and Lck (left), Jurkat T cells genetically disrupted for ζ (JKζKO) (middle) and Jurkat T cells genetically disrupted for Lck (JKLckKO) (right). The PLA was performed between the TCR (ζ) and Lck at 4 °C, in the presence of PP2 for 120 min. Quantification of one representative experiment is shown. Statistical analysis was performed as in h. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant.

Extended Data Fig. 8 The CD3ε RK motif enhances CAR-Nck proximity.

a, Technical PLA controls in Jurkat T cells genetically disrupted for ζ (JKζKO) expressing the ζCAR. The cells were stimulated with Fc-CD19 at 37 °C for 5 min. PLA was performed with both primary antibodies (left), with only the anti-ζ primary antibody (middle) or with only the anti-Nck primary antibody (right). In all cases both secondary antibodies were used. The quantification of one representative experiment was analyzed using ANOVA test. Mean values ± s.e.m. are shown. b, Biological PLA controls in Jurkat T cells expressing surface TCR and Nck (left), Jurkat T cells genetically disrupted for ζ (JKζKO) (middle) and Jurkat T cells silenced for expression of Nck (JKshNck1/2) (right). The PLA was performed between the TCR (ζ) and Nck at 37 °C. Quantification of one representative experiment is shown. Statistical analysis was performed as in a. c, JKζKO cells were used to lentivirally express the CARs as indicated or were transduced with an empty vector (Mock). PLA between the CAR (ζ) and Nck in cells either left unstimulated (Uns) or stimulated with recombinant Fc-CD19 for 5 min. Data are representative of 3 independent experiments. Statistical analysis was performed using unpaired one-way ANOVA test. Mean values ± s.e.m. are shown. ****P < 0.0001.

Extended Data Fig. 9 The CD3ε RK motif enhances in vivo tumor control by CAR T cells.

Log-rank Mantel-Cox survival test of Nalm6-bearing mice treated 3 days after tumor cell inoculation (5 × 105 Nalm6 cells i.v.) with 3 × 106 (left, n = 4 mice) or 1.5 ×106 (right, n = 8 mice) CAR-expressing human T cells. NT, non-transduced T cells; ζCAR, ζCAR-transduced T cells. **P < 0.01, NS, not significant.

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Hartl, F.A., Beck-Garcìa, E., Woessner, N.M. et al. Noncanonical binding of Lck to CD3ε promotes TCR signaling and CAR function. Nat Immunol 21, 902–913 (2020). https://doi.org/10.1038/s41590-020-0732-3

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