Cancer Immunology, Immunotherapy

, Volume 62, Issue 2, pp 359–369 | Cite as

MHC-class I-restricted CD4 T cells: a nanomolar affinity TCR has improved anti-tumor efficacy in vivo compared to the micromolar wild-type TCR

  • Carolina M. Soto
  • Jennifer D. Stone
  • Adam S. Chervin
  • Boris Engels
  • Hans Schreiber
  • Edward J. Roy
  • David M. KranzEmail author
Original article


Clinical studies with immunotherapies for cancer, including adoptive cell transfers of T cells, have shown promising results. It is now widely believed that recruitment of CD4+ helper T cells to the tumor would be favorable, as CD4+ cells play a pivotal role in cytokine secretion as well as promoting the survival, proliferation, and effector functions of tumor-specific CD8+ cytotoxic T lymphocytes. Genetically engineered high-affinity T-cell receptors (TCRs) can be introduced into CD4+ helper T cells to redirect them to recognize MHC-class I-restricted antigens, but it is not clear what affinity of the TCR will be optimal in this approach. Here, we show that CD4+ T cells expressing a high-affinity TCR (nanomolar K d value) against a class I tumor antigen mediated more effective tumor treatment than the wild-type affinity TCR (micromolar K d value). High-affinity TCRs in CD4+ cells resulted in enhanced survival and long-term persistence of effector memory T cells in a melanoma tumor model. The results suggest that TCRs with nanomolar affinity could be advantageous for tumor targeting when expressed in CD4+ T cells.


TCR Adoptive T-cell therapy Tumor targeting Cancer immunotherapy Melanoma 



The authors would like to thank Tom Gajewski for generously providing the B16-SIY murine melanoma cell line. We also thank Melanie Studzinski, Sydney Sherman, and Natalia Wolosowicz for experimental support. This work was supported by a grant from the Melanoma Research Alliance (to D. M. K.), by NIH grant CA097296 (to D. M. K. and H. S.), and funds from an anonymous donor (to EJR). BE was supported by a Research Fellowship of the Deutsche Forschungsgemeinschaft; J. D. S. was supported by the Samuel and Ruth Engelberg/Irvington Institute Fellowship of the Cancer Research Institute.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Morris EC, Tsallios A, Bendle GM, Xue SA, Stauss HJ (2005) A critical role of T cell antigen receptor-transduced MHC class I-restricted helper T cells in tumor protection. Proc Natl Acad Sci USA 102(22):7934–7939 [Epub 2005 May 7920]Google Scholar
  2. 2.
    Schietinger A, Philip M, Liu RB, Schreiber K, Schreiber H (2010) Bystander killing of cancer requires the cooperation of CD4(+) and CD8(+) T cells during the effector phase. J Exp Med 207(11):2469–2477PubMedCrossRefGoogle Scholar
  3. 3.
    Bos R, Sherman LA (2010) CD4+ T-cell help in the tumor milieu is required for recruitment and cytolytic function of CD8+ T lymphocytes. Cancer Res 70(21):8368–8377PubMedCrossRefGoogle Scholar
  4. 4.
    Hung K, Hayashi R, Lafond-Walker A, Lowenstein C, Pardoll D, Levitsky H (1998) The central role of CD4(+) T cells in the antitumor immune response. J Exp Med 188(12):2357–2368PubMedCrossRefGoogle Scholar
  5. 5.
    Surman DR, Dudley ME, Overwijk WW, Restifo NP (2000) Cutting edge: CD4+ T cell control of CD8+ T cell reactivity to a model tumor antigen. J Immunol 164(2):562–565PubMedGoogle Scholar
  6. 6.
    Antony PA, Piccirillo CA, Akpinarli A, Finkelstein SE, Speiss PJ, Surman DR, Palmer DC, Chan CC, Klebanoff CA, Overwijk WW, Rosenberg SA, Restifo NP (2005) CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells. J Immunol 174(5):2591–2601PubMedGoogle Scholar
  7. 7.
    Novy P, Quigley M, Huang X, Yang Y (2007) CD4 T cells are required for CD8 T cell survival during both primary and memory recall responses. J Immunol 179(12):8243–8251PubMedGoogle Scholar
  8. 8.
    Wang LX, Shu S, Disis ML, Plautz GE (2007) Adoptive transfer of tumor-primed, in vitro-activated, CD4+ T effector cells (TEs) combined with CD8+ TEs provides intratumoral TE proliferation and synergistic antitumor response. Blood 109(11):4865–4876 [Epub 2007 Feb 4866]Google Scholar
  9. 9.
    Qin Z, Blankenstein T (2000) CD4+ T cell–mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN gamma receptor expression by nonhematopoietic cells. Immunity 12(6):677–686PubMedCrossRefGoogle Scholar
  10. 10.
    Cohen PA, Peng L, Plautz GE, Kim JA, Weng DE, Shu S (2000) CD4+ T cells in adoptive immunotherapy and the indirect mechanism of tumor rejection. Crit Rev Immunol 20(1):17–56PubMedCrossRefGoogle Scholar
  11. 11.
    Quezada SA, Simpson TR, Peggs KS, Merghoub T, Vider J, Fan X, Blasberg R, Yagita H, Muranski P, Antony PA, Restifo NP, Allison JP (2010) Tumor-reactive CD4(+) T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts. J Exp Med 207(3):637–650PubMedCrossRefGoogle Scholar
  12. 12.
    Xie Y, Akpinarli A, Maris C, Hipkiss EL, Lane M, Kwon EK, Muranski P, Restifo NP, Antony PA (2010) Naive tumor-specific CD4(+) T cells differentiated in vivo eradicate established melanoma. J Exp Med 207(3):651–667PubMedCrossRefGoogle Scholar
  13. 13.
    Fuertes MB, Kacha AK, Kline J, Woo SR, Kranz DM, Murphy KM, Gajewski TF (2011) Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8{alpha}+ dendritic cells. J Exp Med 208(10):2005–2016PubMedCrossRefGoogle Scholar
  14. 14.
    Harlin H, Meng Y, Peterson AC, Zha Y, Tretiakova M, Slingluff C, McKee M, Gajewski TF (2009) Chemokine expression in melanoma metastases associated with CD8+ T-cell recruitment. Cancer Res 69(7):3077–3085PubMedCrossRefGoogle Scholar
  15. 15.
    Holler PD, Holman PO, Shusta EV, O’Herrin S, Wittrup KD, Kranz DM (2000) In vitro evolution of a T cell receptor with high affinity for peptide/MHC. Proc Natl Acad Sci USA 97(10):5387–5392PubMedCrossRefGoogle Scholar
  16. 16.
    Holler PD, Kranz DM (2003) Quantitative analysis of the contribution of TCR/pepMHC affinity and CD8 to T cell activation. Immunity 18:255–264PubMedCrossRefGoogle Scholar
  17. 17.
    Li Y, Moysey R, Molloy PE, Vuidepot AL, Mahon T, Baston E, Dunn S, Liddy N, Jacob J, Jakobsen BK, Boulter JM (2005) Directed evolution of human T-cell receptors with picomolar affinities by phage display. Nat Biotechnol 23(3):349–354PubMedCrossRefGoogle Scholar
  18. 18.
    Zhao Y, Bennett AD, Zheng Z, Wang QJ, Robbins PF, Yu LY, Li Y, Molloy PE, Dunn SM, Jakobsen BK, Rosenberg SA, Morgan RA (2007) High-affinity TCRs generated by phage display provide CD4+ T cells with the ability to recognize and kill tumor cell lines. J Immunol 179(9):5845–5854PubMedGoogle Scholar
  19. 19.
    Robbins PF, Li YF, El-Gamil M, Zhao Y, Wargo JA, Zheng Z, Xu H, Morgan RA, Feldman SA, Johnson LA, Bennett AD, Dunn SM, Mahon TM, Jakobsen BK, Rosenberg SA (2008) Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions. J Immunol 180(9):6116–6131PubMedGoogle Scholar
  20. 20.
    Chervin AS, Stone JD, Holler PD, Bai A, Chen J, Eisen HN, Kranz DM (2009) The impact of TCR-binding properties and antigen presentation format on T cell responsiveness. J Immunol 183(2):1166–1178PubMedCrossRefGoogle Scholar
  21. 21.
    Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, Rosenberg SA (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314(5796):126–129PubMedCrossRefGoogle Scholar
  22. 22.
    Johnson LA, Morgan RA, Dudley ME, Cassard L, Yang JC, Hughes MS, Kammula US, Royal RE, Sherry RM, Wunderlich JR, Lee CC, Restifo NP, Schwarz SL, Cogdill AP, Bishop RJ, Kim H, Brewer CC, Rudy SF, VanWaes C, Davis JL, Mathur A, Ripley RT, Nathan DA, Laurencot CM, Rosenberg SA (2009) Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 114(3):535–546PubMedCrossRefGoogle Scholar
  23. 23.
    Gattinoni L, Powell DJ Jr, Rosenberg SA, Restifo NP (2006) Adoptive immunotherapy for cancer: building on success. Nat Rev Immunol 6(5):383–393PubMedCrossRefGoogle Scholar
  24. 24.
    Kammertoens T, Blankenstein T (2009) Making and circumventing tolerance to cancer. Eur J Immunol 39(9):2345–2353PubMedCrossRefGoogle Scholar
  25. 25.
    Schmitt TM, Ragnarsson GB, Greenberg PD (2009) T cell receptor gene therapy for cancer. Hum Gene Ther 20(11):1240–1248PubMedCrossRefGoogle Scholar
  26. 26.
    Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL, Kammula US, Hughes MS, Restifo NP, Raffeld M, Lee CC, Levy CL, Li YF, El-Gamil M, Schwarz SL, Laurencot C, Rosenberg SA (2011) Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 29(7):917–924PubMedCrossRefGoogle Scholar
  27. 27.
    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):6548–6559PubMedGoogle Scholar
  28. 28.
    Borbulevych OY, Santhanagopolan SM, Hossain M, Baker BM (2011) TCRs used in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens via distinct mechanisms. J Immunol 187(5):2453–2463PubMedCrossRefGoogle Scholar
  29. 29.
    Corse E, Gottschalk RA, Krogsgaard M, Allison JP (2010) Attenuated T cell responses to a high-potency ligand in vivo. PLoS Biol 8(9):1–12Google Scholar
  30. 30.
    Engels B, Chervin AS, Sant AJ, Kranz DM, Schreiber H (2012) Long-term persistence of CD4(+) but rapid disappearance of CD8(+) T cells expressing an MHC class I-restricted TCR of nanomolar affinity. Mol Ther 20(3):652–660PubMedCrossRefGoogle Scholar
  31. 31.
    Holler PD, Chlewicki LK, Kranz DM (2003) TCRs with high affinity for foreign pMHC show self-reactivity. Nat Immunol 4(1):55–62PubMedCrossRefGoogle Scholar
  32. 32.
    Zhang B, Bowerman NA, Salama JK, Schmidt H, Spiotto MT, Schietinger A, Yu P, Fu YX, Weichselbaum RR, Rowley DA, Kranz DM, Schreiber H (2007) Induced sensitization of tumor stroma leads to eradication of established cancer by T cells. J Exp Med 204(1):49–55PubMedCrossRefGoogle Scholar
  33. 33.
    Mumberg D, Monach PA, Wanderling S, Philip M, Toledano AY, Schreiber RD, Schreiber H (1999) CD4(+) T cells eliminate MHC class II-negative cancer cells in vivo by indirect effects of IFN-gamma. Proc Natl Acad Sci USA 96(15):8633–8638PubMedCrossRefGoogle Scholar
  34. 34.
    Zhang B, Karrison T, Rowley DA, Schreiber H (2008) IFN-gamma- and TNF-dependent bystander eradication of antigen-loss variants in established mouse cancers. J Clin Invest 118(4):1398–1404PubMedCrossRefGoogle Scholar
  35. 35.
    DuPage M, Cheung AF, Mazumdar C, Winslow MM, Bronson R, Schmidt LM, Crowley D, Chen J, Jacks T (2011) Endogenous T cell responses to antigens expressed in lung adenocarcinomas delay malignant tumor progression. Cancer Cell 19(1):72–85PubMedCrossRefGoogle Scholar
  36. 36.
    de Witte MA, Jorritsma A, Kaiser A, van den Boom MD, Dokter M, Bendle GM, Haanen JB, Schumacher TN (2008) Requirements for effective antitumor responses of TCR transduced T cells. J Immunol 181(7):5128–5136PubMedGoogle Scholar
  37. 37.
    Huster KM, Busch V, Schiemann M, Linkemann K, Kerksiek KM, Wagner H, Busch DH (2004) Selective expression of IL-7 receptor on memory T cells identifies early CD40L-dependent generation of distinct CD8+ memory T cell subsets. Proc Natl Acad Sci USA 101(15):5610–5615PubMedCrossRefGoogle Scholar
  38. 38.
    Powell DJ Jr, Dudley ME, Robbins PF, Rosenberg SA (2005) Transition of late-stage effector T cells to CD27+ CD28+ tumor-reactive effector memory T cells in humans after adoptive cell transfer therapy. Blood 105(1):241–250PubMedCrossRefGoogle Scholar
  39. 39.
    Bachmann MF, Wolint P, Schwarz K, Jager P, Oxenius A (2005) Functional properties and lineage relationship of CD8+ T cell subsets identified by expression of IL-7 receptor alpha and CD62L. J Immunol 175(7):4686–4696PubMedGoogle Scholar
  40. 40.
    Wen FT, Thisted RA, Rowley DA, Schreiber H (2012) A systematic analysis of experimental immunotherapies on tumors differing in size and duration of growth. OncoImmunology 1(2):172–178PubMedCrossRefGoogle Scholar
  41. 41.
    Finkelstein SE, Heimann DM, Klebanoff CA, Antony PA, Gattinoni L, Hinrichs CS, Hwang LN, Palmer DC, Spiess PJ, Surman DR, Wrzesiniski C, Yu Z, Rosenberg SA, Restifo NP (2004) Bedside to bench and back again: how animal models are guiding the development of new immunotherapies for cancer. J Leukoc Biol 76(2):333–337PubMedCrossRefGoogle Scholar
  42. 42.
    Borbulevych OY, Insaidoo FK, Baxter TK, Powell DJ Jr, Johnson LA, Restifo NP, Baker BM (2007) Structures of MART-126/27-35 Peptide/HLA-A2 complexes reveal a remarkable disconnect between antigen structural homology and T cell recognition. J Mol Biol 372(5):1123–1136PubMedCrossRefGoogle Scholar
  43. 43.
    Kline J, Zhang L, Battaglia L, Cohen KS, Gajewski TF (2012) Cellular and molecular requirements for rejection of B16 melanoma in the setting of regulatory T cell depletion and homeostatic proliferation. J Immunol 188(6):2630–2642PubMedCrossRefGoogle Scholar
  44. 44.
    Schuler T, Blankenstein T (2003) Cutting edge: CD8+ effector T cells reject tumors by direct antigen recognition but indirect action on host cells. J Immunol 170(9):4427–4431PubMedGoogle Scholar
  45. 45.
    Hunder NN, Wallen H, Cao J, Hendricks DW, Reilly JZ, Rodmyre R, Jungbluth A, Gnjatic S, Thompson JA, Yee C (2008) Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N Engl J Med 358(25):2698–2703PubMedCrossRefGoogle Scholar
  46. 46.
    Dudley ME, Wunderlich JR, Yang JC, Sherry RM, Topalian SL, Restifo NP, Royal RE, Kammula U, White DE, Mavroukakis SA, Rogers LJ, Gracia GJ, Jones SA, Mangiameli DP, Pelletier MM, Gea-Banacloche J, Robinson MR, Berman DM, Filie AC, Abati A, Rosenberg SA (2005) Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol 23(10):2346–2357PubMedCrossRefGoogle Scholar
  47. 47.
    Kaluza KM, Thompson JM, Kottke TJ, Flynn Gilmer HC, Knutson DL, Vile RG (2012) Adoptive T cell therapy promotes the emergence of genomically altered tumor escape variants. Int J Cancer 131(4):844–854PubMedCrossRefGoogle Scholar
  48. 48.
    Spiotto MT, Rowley DA, Schreiber H (2004) Bystander elimination of antigen loss variants in established tumors. Nat Med 10(3):294–298PubMedCrossRefGoogle Scholar
  49. 49.
    Zhang B, Zhang Y, Bowerman NA, Schietinger A, Fu YX, Kranz DM, Rowley DA, Schreiber H (2008) Equilibrium between host and cancer caused by effector T cells killing tumor stroma. Cancer Res 68(5):1563–1571PubMedCrossRefGoogle Scholar
  50. 50.
    Weinhold M, Sommermeyer D, Uckert W, Blankenstein T (2007) Dual T cell receptor expressing CD8+ T cells with tumor- and self-specificity can inhibit tumor growth without causing severe autoimmunity. J Immunol 179(8):5534–5542PubMedGoogle Scholar
  51. 51.
    Bendle GM, Linnemann C, Hooijkaas AI, Bies L, de Witte MA, Jorritsma A, Kaiser AD, Pouw N, Debets R, Kieback E, Uckert W, Song JY, Haanen JB, Schumacher TN (2010) Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy. Nat Med 16(5):565–570 (561p following 570)PubMedCrossRefGoogle Scholar
  52. 52.
    Schodin BA, Tsomides TJ, Kranz DM (1996) Correlation between the number of T cell receptors required for T cell activation and TCR-ligand affinity. Immunity 5:137–146PubMedCrossRefGoogle Scholar
  53. 53.
    Brusko TM, Koya RC, Zhu S, Lee MR, Putnam AL, McClymont SA, Nishimura MI, Han S, Chang LJ, Atkinson MA, Ribas A, Bluestone JA (2010) Human antigen-specific regulatory T cells generated by T cell receptor gene transfer. PLoS One 5(7):e11726PubMedCrossRefGoogle Scholar
  54. 54.
    Spiotto MT, Yu P, Rowley DA, Nishimura MI, Meredith SC, Gajewski TF, Fu YX, Schreiber H (2002) Increasing tumor antigen expression overcomes “ignorance” to solid tumors via crosspresentation by bone marrow-derived stromal cells. Immunity 17(6):737–747PubMedCrossRefGoogle Scholar
  55. 55.
    Blank C, Brown I, Peterson AC, Spiotto M, Iwai Y, Honjo T, Gajewski TF (2004) PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic CD8+ T cells. Cancer Res 64(3):1140–1145PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Carolina M. Soto
    • 1
  • Jennifer D. Stone
    • 2
  • Adam S. Chervin
    • 2
  • Boris Engels
    • 3
  • Hans Schreiber
    • 3
  • Edward J. Roy
    • 1
  • David M. Kranz
    • 2
    Email author
  1. 1.Neuroscience ProgramUniversity of IllinoisUrbanaUSA
  2. 2.Department of BiochemistryUniversity of IllinoisUrbanaUSA
  3. 3.Department of Pathology and Committee on ImmunologyThe University of ChicagoChicagoUSA

Personalised recommendations