Advertisement

Cancer Immunology, Immunotherapy

, Volume 67, Issue 4, pp 525–536 | Cite as

Human c-SRC kinase (CSK) overexpression makes T cells dummy

  • Else Marit Inderberg
  • Nadia Mensali
  • Morten P. Oksvold
  • Lars-Egil Fallang
  • Anne Fåne
  • Gjertrud Skorstad
  • Grethe-Elisabeth Stenvik
  • Cinzia Progida
  • Oddmund Bakke
  • Gunnar Kvalheim
  • June H. Myklebust
  • Sébastien Wälchli
Original Article

Abstract

Adoptive cell therapy with T-cell receptor (TCR)-engineered T cells represents a powerful method to redirect the immune system against tumours. However, although TCR recognition is restricted to a specific peptide–MHC (pMHC) complex, increasing numbers of reports have shown cross-reactivity and off-target effects with severe consequences for the patients. This demands further development of strategies to validate TCR safety prior to clinical use. We reasoned that the desired TCR signalling depends on correct pMHC recognition on the outside and a restricted clustering on the inside of the cell. Since the majority of the adverse events are due to TCR recognition of the wrong target, we tested if blocking the signalling would affect the binding. By over-expressing the c-SRC kinase (CSK), a negative regulator of LCK, in redirected T cells, we showed that peripheral blood T cells inhibited anti-CD3/anti-CD28-induced phosphorylation of ERK, whereas TCR proximal signalling was not affected. Similarly, overexpression of CSK together with a therapeutic TCR prevented pMHC-induced ERK phosphorylation. Downstream effector functions were also almost completely blocked, including pMHC-induced IL-2 release, degranulation and, most importantly, target cell killing. The lack of effector functions contrasted with the unaffected TCR expression, pMHC recognition, and membrane exchange activity (trogocytosis). Therefore, co-expression of CSK with a therapeutic TCR did not compromise target recognition and binding, but rendered T cells incapable of executing their effector functions. Consequently, we named these redirected T cells “dummy T cells” and propose to use them for safety validation of new TCRs prior to therapy.

Keywords

T-cell receptor TCR TCR signalling CSK Immunotherapy 

Abbreviations

APC

Antigen presenting cells

CSK

c-SRC kinase

EuTDA

Europium TDA

FB

Flow buffer

FCS

Fetal calf serum

GFP

Green fluorescent protein

GMP

Good manufacturing practice

HS

Human serum

ITAMs

Immunoreceptor tyrosine-based activation motifs

MAGE-A3

Melanoma-associated antigen 3

MART-1

Melanoma antigen recognized by T cells 1

MFI

Mean fluorescence intensity

PBMC

Peripheral blood mononuclear cells

PFA

Paraformaldehyde

pMHC

Peptide–MHC

RT

Room temperature

SCT

Single-chain trimer

TCR

T-cell receptor

TGFβR2

Transforming growth factor β II

Notes

Acknowledgements

The authors would like to thank the members of the Smeland lab and of the Department of Cellular Therapy for their positive input and support. We are also thankful to Dr. Pierre Dillard for commenting on this manuscript.

Author contributions

EMI, SW, GES, LEF, and JHM conceived and designed the experiments. EMI, NM, MPO, AF, GS, CP, and JHM performed the experiments. SW, OB, GK, and JHM interpreted the data. SW, EMI, NM, and JHM wrote the manuscript and all authors edited the manuscript.

Funding

This work was supported by the Gene Therapy program of the Radium Hospital to Sébastien Wälchli and Anne Fåne, The Norwegian Research Council to Else Marit Inderberg (#244388), and partly supported by an Innovation Grant from Southern and Eastern Norway Regional Health Authority to Nadia Mensali (#13/00367-88).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The study was approved by the Regional Committee for Medical and Health Research Ethics (REC South-East, Norway) (Approval no. 2013/624).

Informed consent

Informed consent from healthy donors was given.

References

  1. 1.
    Comrie WA, Burkhardt JK (2016) Action and traction: cytoskeletal control of receptor triggering at the immunological synapse. Front Immunol 7:68.  https://doi.org/10.3389/fimmu.2016.00068 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Werlen G, Palmer E (2002) The T-cell receptor signalosome: a dynamic structure with expanding complexity. Curr Opin Immunol 14(3):299–305CrossRefPubMedGoogle Scholar
  3. 3.
    Malissen B, Bongrand P (2015) Early T cell activation: integrating biochemical, structural, and biophysical cues. Annu Rev Immunol 33:539–561.  https://doi.org/10.1146/annurev-immunol-032414-112158 CrossRefPubMedGoogle Scholar
  4. 4.
    Chakraborty AK, Weiss A (2014) Insights into the initiation of TCR signaling. Nat Immunol 15(9):798–807.  https://doi.org/10.1038/ni.2940 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Chen L, Flies DB (2013) Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol 13(4):227–242.  https://doi.org/10.1038/nri3405 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Chow LM, Fournel M, Davidson D, Veillette A (1993) Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk. Nature 365(6442):156–160.  https://doi.org/10.1038/365156a0 CrossRefPubMedGoogle Scholar
  7. 7.
    Okada M (2012) Regulation of the SRC family kinases by Csk. Int J Biol Sci 8(10):1385–1397.  https://doi.org/10.7150/ijbs.5141 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Vang T, Liu WH, Delacroix L, Wu S, Vasile S, Dahl R, Yang L, Musumeci L, Francis D, Landskron J, Tasken K, Tremblay ML, Lie BA, Page R, Mustelin T, Rahmouni S, Rickert RC, Tautz L (2012) LYP inhibits T-cell activation when dissociated from CSK. Nat Chem Biol 8(5):437–446.  https://doi.org/10.1038/nchembio.916 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Schoenborn JR, Tan YX, Zhang C, Shokat KM, Weiss A (2011) Feedback circuits monitor and adjust basal Lck-dependent events in T cell receptor signaling. Sci Signal 4(190):ra59.  https://doi.org/10.1126/scisignal.2001893 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Tan YX, Manz BN, Freedman TS, Zhang C, Shokat KM, Weiss A (2014) Inhibition of the kinase Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full TCR signaling. Nat Immunol 15(2):186–194.  https://doi.org/10.1038/ni.2772 CrossRefPubMedGoogle Scholar
  11. 11.
    Wu CY, Rupp LJ, Roybal KT, Lim WA (2015) Synthetic biology approaches to engineer T cells. Curr Opin Immunol 35:123–130.  https://doi.org/10.1016/j.coi.2015.06.015 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Palmer DC, Guittard GC, Franco Z, Crompton JG, Eil RL, Patel SJ, Ji Y, Van Panhuys N, Klebanoff CA, Sukumar M, Clever D, Chichura A, Roychoudhuri R, Varma R, Wang E, Gattinoni L, Marincola FM, Balagopalan L, Samelson LE, Restifo NP (2015) Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance. J Exp Med 212(12):2095–2113.  https://doi.org/10.1084/jem.20150304 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Daniel-Meshulam I, Horovitz-Fried M, Cohen CJ (2013) Enhanced antitumor activity mediated by human 4-1BB-engineered T cells. Int J Cancer 133(12):2903–2913.  https://doi.org/10.1002/ijc.28320 PubMedGoogle Scholar
  14. 14.
    Weichsel R, Dix C, Wooldridge L, Clement M, Fenton-May A, Sewell AK, Zezula J, Greiner E, Gostick E, Price DA, Einsele H, Seggewiss R (2008) Profound inhibition of antigen-specific T-cell effector functions by dasatinib. Clin Cancer Res 14(8):2484–2491.  https://doi.org/10.1158/1078-0432.CCR-07-4393 CrossRefPubMedGoogle Scholar
  15. 15.
    Okoye I, Wang L, Pallmer K, Richter K, Ichimura T, Haas R, Crouse J, Choi O, Heathcote D, Lovo E, Mauro C, Abdi R, Oxenius A, Rutschmann S, Ashton-Rickardt PG (2015) T cell metabolism. The protein LEM promotes CD8(+) T cell immunity through effects on mitochondrial respiration. Science 348(6238):995–1001.  https://doi.org/10.1126/science.aaa7516 CrossRefPubMedGoogle Scholar
  16. 16.
    Ebert PJR, Cheung J, Yang Y, McNamara E, Hong R, Moskalenko M, Gould SE, Maecker H, Irving BA, Kim JM, Belvin M, Mellman I (2016) MAP kinase inhibition promotes T cell and anti-tumor activity in combination with PD-L1 checkpoint blockade. Immunity 44(3):609–621.  https://doi.org/10.1016/j.immuni.2016.01.024 CrossRefPubMedGoogle Scholar
  17. 17.
    Eil R, Vodnala SK, Clever D, Klebanoff CA, Sukumar M, Pan JH, Palmer DC, Gros A, Yamamoto TN, Patel SJ, Guittard GC, Yu Z, Carbonaro V, Okkenhaug K, Schrump DS, Linehan WM, Roychoudhuri R, Restifo NP (2016) Ionic immune suppression within the tumour microenvironment limits T cell effector function. Nature 537(7621):539–543.  https://doi.org/10.1038/nature19364 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hinrichs CS, Restifo NP (2013) Reassessing target antigens for adoptive T-cell therapy. Nat Biotechnol 31(11):999–1008.  https://doi.org/10.1038/nbt.2725 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Jones BS, Lamb LS, Goldman F, Di Stasi A (2014) Improving the safety of cell therapy products by suicide gene transfer. Front Pharmacol 5:254.  https://doi.org/10.3389/fphar.2014.00254 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Roybal KT, Rupp LJ, Morsut L, Walker WJ, McNally KA, Park JS, Lim WA (2016) Precision tumor recognition by T cells with combinatorial antigen-sensing circuits. Cell 164(4):770–779.  https://doi.org/10.1016/j.cell.2016.01.011 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Vang T, Abrahamsen H, Myklebust S, Enserink J, Prydz H, Mustelin T, Amarzguioui M, Tasken K (2004) Knockdown of C-terminal Src kinase by siRNA-mediated RNA interference augments T cell receptor signaling in mature T cells. Eur J Immunol 34(8):2191–2199.  https://doi.org/10.1002/eji.200425036 CrossRefPubMedGoogle Scholar
  22. 22.
    Heemskerk MH, Hoogeboom M, de Paus RA, Kester MG, van der Hoorn MA, Goulmy E, Willemze R, Falkenburg JH (2003) Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region. Blood 102(10):3530–3540CrossRefPubMedGoogle Scholar
  23. 23.
    Walchli S, Kumari S, Fallang LE, Sand KM, Yang W, Landsverk OJ, Bakke O, Olweus J, Gregers TF (2014) Invariant chain as a vehicle to load antigenic peptides on human MHC class I for cytotoxic T-cell activation. Eur J Immunol 44(3):774–784.  https://doi.org/10.1002/eji.201343671 CrossRefPubMedGoogle Scholar
  24. 24.
    Walchli S, Loset GA, Kumari S, Johansen JN, Yang W, Sandlie I, Olweus J (2011) A practical approach to T-cell receptor cloning and expression. PLoS One 6(11):e27930CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Inderberg EM, Walchli S, Myhre MR, Trachsel S, Almasbak H, Kvalheim G, Gaudernack G (2017) T cell therapy targeting a public neoantigen in microsatellite instable colon cancer reduces in vivo tumor growth. Oncoimmunology 6(4):e1302631.  https://doi.org/10.1080/2162402X.2017.1302631 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Walseng E, Walchli S, Fallang LE, Yang W, Vefferstad A, Areffard A, Olweus J (2015) Soluble T-cell receptors produced in human cells for targeted delivery. PLoS One 10(4):e0119559.  https://doi.org/10.1371/journal.pone.0119559 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Almasbak H, Rian E, Hoel HJ, Pule M, Walchli S, Kvalheim G, Gaudernack G, Rasmussen AM (2011) Transiently redirected T cells for adoptive transfer. Cytotherapy 13(5):629–640.  https://doi.org/10.3109/14653249.2010.542461 CrossRefPubMedGoogle Scholar
  28. 28.
    Myklebust JH, Irish JM, Brody J, Czerwinski DK, Houot R, Kohrt HE, Timmerman J, Said J, Green MR, Delabie J, Kolstad A, Alizadeh AA, Levy R (2013) High PD-1 expression and suppressed cytokine signaling distinguish T cells infiltrating follicular lymphoma tumors from peripheral T cells. Blood 121(8):1367–1376.  https://doi.org/10.1182/blood-2012-04-421826 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    de Felipe P, Luke GA, Hughes LE, Gani D, Halpin C, Ryan MD (2006) E unum pluribus: multiple proteins from a self-processing polyprotein. Trends Biotechnol 24(2):68–75CrossRefPubMedGoogle Scholar
  30. 30.
    Johnson LA, Heemskerk B, Powell DJ Jr, Cohen CJ, Morgan RA, Dudley ME, Robbins PF, Rosenberg SA (2006) Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes. J Immunol 177(9):6548–6559CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Yu YY, Netuschil N, Lybarger L, Connolly JM, Hansen TH (2002) Cutting edge: single-chain trimers of MHC class I molecules form stable structures that potently stimulate antigen-specific T cells and B cells. J Immunol 168(7):3145–3149CrossRefPubMedGoogle Scholar
  32. 32.
    Lissina A, Ladell K, Skowera A, Clement M, Edwards E, Seggewiss R, van den Berg HA, Gostick E, Gallagher K, Jones E, Melenhorst JJ, Godkin AJ, Peakman M, Price DA, Sewell AK, Wooldridge L (2009) Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers. J Immunol Methods 340(1):11–24.  https://doi.org/10.1016/j.jim.2008.09.014 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Davis DM (2007) Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response. Nat Rev Immunol 7(3):238–243.  https://doi.org/10.1038/nri2020 CrossRefPubMedGoogle Scholar
  34. 34.
    Hudrisier D, Riond J, Mazarguil H, Gairin JE, Joly E (2001) Cutting edge: CTLs rapidly capture membrane fragments from target cells in a TCR signaling-dependent manner. J Immunol 166(6):3645–3649CrossRefPubMedGoogle Scholar
  35. 35.
    Osborne DG, Wetzel SA (2012) Trogocytosis results in sustained intracellular signaling in CD4(+) T cells. J Immunol 189(10):4728–4739.  https://doi.org/10.4049/jimmunol.1201507 CrossRefPubMedGoogle Scholar
  36. 36.
    Aucher A, Magdeleine E, Joly E, Hudrisier D (2008) Capture of plasma membrane fragments from target cells by trogocytosis requires signaling in T cells but not in B cells. Blood 111(12):5621–5628.  https://doi.org/10.1182/blood-2008-01-134155 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Irish JM, Kotecha N, Nolan GP (2006) Mapping normal and cancer cell signalling networks: towards single-cell proteomics. Nat Rev Cancer 6(2):146–155.  https://doi.org/10.1038/nrc1804 CrossRefPubMedGoogle Scholar
  38. 38.
    Wolchinsky R, Hod-Marco M, Oved K, Shen-Orr SS, Bendall SC, Nolan GP, Reiter Y (2014) Antigen-dependent integration of opposing proximal TCR-signaling cascades determines the functional fate of T lymphocytes. J Immunol 192(5):2109–2119.  https://doi.org/10.4049/jimmunol.1301142 CrossRefPubMedGoogle Scholar
  39. 39.
    Adams CL, Grierson AM, Mowat AM, Harnett MM, Garside P (2004) Differences in the kinetics, amplitude, and localization of ERK activation in anergy and priming revealed at the level of individual primary T cells by laser scanning cytometry. J Immunol 173(3):1579–1586CrossRefPubMedGoogle Scholar
  40. 40.
    Schmid DA, Irving MB, Posevitz V, Hebeisen M, Posevitz-Fejfar A, Sarria JC, Gomez-Eerland R, Thome M, Schumacher TN, Romero P, Speiser DE, Zoete V, Michielin O, Rufer N Evidence for a TCR affinity threshold delimiting maximal CD8 T cell function. J Immunol 184(9):4936–4946Google Scholar
  41. 41.
    Nika K, Soldani C, Salek M, Paster W, Gray A, Etzensperger R, Fugger L, Polzella P, Cerundolo V, Dushek O, Hofer T, Viola A, Acuto O (2010) Constitutively active Lck kinase in T cells drives antigen receptor signal transduction. Immunity 32(6):766–777.  https://doi.org/10.1016/j.immuni.2010.05.011 CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Wei P, Wong WW, Park JS, Corcoran EE, Peisajovich SG, Onuffer JJ, Weiss A, Lim WA (2012) Bacterial virulence proteins as tools to rewire kinase pathways in yeast and immune cells. Nature 488(7411):384–388.  https://doi.org/10.1038/nature11259 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Linette GP, Stadtmauer EA, Maus MV, Rapoport AP, Levine BL, Emery L, Litzky L, Bagg A, Carreno BM, Cimino PJ, Binder-Scholl GK, Smethurst DP, Gerry AB, Pumphrey NJ, Bennett AD, Brewer JE, Dukes J, Harper J, Tayton-Martin HK, Jakobsen BK, Hassan NJ, Kalos M, June CH (2013) Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma. Blood 122(6):863–871.  https://doi.org/10.1182/blood-2013-03-490565 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Cameron BJ, Gerry AB, Dukes J, Harper JV, Kannan V, Bianchi FC, Grand F, Brewer JE, Gupta M, Plesa G, Bossi G, Vuidepot A, Powlesland AS, Legg A, Adams KJ, Bennett AD, Pumphrey NJ, Williams DD, Binder-Scholl G, Kulikovskaya I, Levine BL, Riley JL, Varela-Rohena A, Stadtmauer EA, Rapoport AP, Linette GP, June CH, Hassan NJ, Kalos M, Jakobsen BK (2013) Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells. Sci Transl Med 5(197):197ra103.  https://doi.org/10.1126/scitranslmed.3006034 CrossRefPubMedGoogle Scholar
  45. 45.
    Depontieu FR, Qian J, Zarling AL, McMiller TL, Salay TM, Norris A, English AM, Shabanowitz J, Engelhard VH, Hunt DF, Topalian SL (2009) Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy. Proc Natl Acad Sci USA 106(29):12073–12078.  https://doi.org/10.1073/pnas.0903852106 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Piepenbrink KH, Blevins SJ, Scott DR, Baker BM (2013) The basis for limited specificity and MHC restriction in a T cell receptor interface. Nat Commun 4:1948.  https://doi.org/10.1038/ncomms2948 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Sewell AK (2012) Why must T cells be cross-reactive? Nat Rev Immunol 12(9):669–677.  https://doi.org/10.1038/nri3279 CrossRefPubMedGoogle Scholar
  48. 48.
    Almasbak H, Lundby M, Rasmussen AM (2010) Non-MHC-dependent redirected T cells against tumor cells. Methods Mol Biol 629:453–493.  https://doi.org/10.1007/978-1-60761-657-3_28 PubMedGoogle Scholar
  49. 49.
    Kenderian SS, Ruella M, Shestova O, Klichinsky M, Aikawa V, Morrissette JJ, Scholler J, Song D, Porter DL, Carroll M, June CH, Gill S (2015) CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia. Leukemia 29(8):1637–1647.  https://doi.org/10.1038/leu.2015.52 CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Liu Z, Li Z (2014) Molecular imaging in tracking tumor-specific cytotoxic T lymphocytes (CTLs). Theranostics 4(10):990–1001.  https://doi.org/10.7150/thno.9268 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Section for Cellular Therapy, Department for Cancer TreatmentOslo University Hospital-RadiumhospitaletOsloNorway
  2. 2.Department of BiosciencesUniversity of OsloOsloNorway
  3. 3.Centre for Immune RegulationUniversity of OsloOsloNorway
  4. 4.Section for Cancer Immunology, Institute for Cancer ResearchOslo University Hospital-RadiumhospitaletOsloNorway
  5. 5.Centre for Cancer BiomedicineUniversity of OsloOsloNorway
  6. 6.Gilead Sciences ASOsloNorway
  7. 7.Montebello Diagnostics ASOsloNorway

Personalised recommendations