Cancer Immunology, Immunotherapy

, Volume 62, Issue 11, pp 1697–1709 | Cite as

Expanded CD8+ T cells of murine and human CLL are driven into a senescent KLRG1+ effector memory phenotype

  • Joachim Rudolf GöthertEmail author
  • Lewin Eisele
  • Ludger Klein-Hitpass
  • Stefanie Weber
  • Marie-Louise Zesewitz
  • Ludger Sellmann
  • Alexander Röth
  • Hanspeter Pircher
  • Ulrich Dührsen
  • Jan Dürig
Original Article


Altered numbers and functions of T cells have previously been demonstrated in chronic lymphocytic leukemia (CLL) patients. However, dynamics and specific T-cell subset alterations have not been studied in great detail. Therefore, we studied CLL blood lymphocyte subsets of individual patients in a longitudinal manner. Dynamic expansions of blood CD4 + and CD8 + T-cell numbers were consistently associated with a progressively increasing CLL leukemic compartment. Interestingly, the T-cell subset expansion over time was more pronounced in CD38 + CLL. Additionally, we performed gene expression profiling of CD3 + T cells of CLL patients and normal donors. Using gene set enrichment analysis, we found significant enrichment of genes with higher expression in CLL T cells within CD8+ effector memory and terminal effector T-cell gene signatures. In agreement with these data, we observed a marked expansion of phenotypic CD8 + effector memory T cells in CLL by flow cytometry. Moreover, we observed that increments of CD8 + effector memory T cells in human CLL and also mouse CLL (Eμ-TCL1 model) were due to an expansion of the inhibitory killer cell lectin-like receptor G1 (KLRG1) expressing cellular subset. Furthermore, higher plasma levels of the natural KLRG1 ligand E-cadherin were detected in CLL patients compared to normal donor controls. The predominance of KLRG1+ expression within CD8+ T cells in conjunction with increased systemic soluble E-cadherin might significantly contribute to CLL immune dysfunction and might additionally represent an important component of the CLL microenvironment.


Chronic lymphocytic leukemia Gene set enrichment analysis CD8+ T cells KLRG1 



We thank Ute Schmücker and Anja Führer for excellent technical assistance and Brigitte Fischer for help with compiling patient data. We are grateful to Carlo M. Croce for his permission to use Eμ-TCL1 transgenic mice. We thank Günter Fingerle-Rawson for providing Eμ-TCL1 transgenic mice. We are grateful to Peter Horn, Marc Seifert and Ralf Küppers for assistance organizing normal donor blood samples. This work was supported by a grant to J.D. from the Ministerium für Schule, Wissenschaft und Forschung des Landes Nordrhein-Westfalen. J.R.G. receives grant support by the Kompetenznetzwerk Stammzellforschung Nordrhein-Westfalen.

Conflict of interest

The authors declare no competing financial interest.

Supplementary material

262_2013_1473_MOESM1_ESM.pdf (748 kb)
Supplementary material 1 (PDF 747 kb)


  1. 1.
    Chiorazzi N, Rai KR, Ferrarini M (2005) Chronic lymphocytic leukemia. N Engl J Med 352(8):804–815PubMedCrossRefGoogle Scholar
  2. 2.
    Bagnara D, Kaufman MS, Calissano C, Marsilio S, Patten PE, Simone R, Chum P, Yan XJ, Allen SL, Kolitz JE, Baskar S, Rader C, Mellstedt H, Rabbani H, Lee A, Gregersen PK, Rai KR, Chiorazzi N (2011) A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease. Blood 117(20):5463–5472PubMedCrossRefGoogle Scholar
  3. 3.
    Burger JA, Ghia P, Rosenwald A, Caligaris-Cappio F (2009) The microenvironment in mature B-cell malignancies: a target for new treatment strategies. Blood 114(16):3367–3375PubMedCrossRefGoogle Scholar
  4. 4.
    Mellstedt H, Choudhury A (2006) T and B cells in B-chronic lymphocytic leukaemia: faust, Mephistopheles and the pact with the Devil. Cancer Immunol Immunother 55(2):210–220PubMedCrossRefGoogle Scholar
  5. 5.
    Gorgun G, Holderried TA, Zahrieh D, Neuberg D, Gribben JG (2005) Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells. J Clin Invest 115(7):1797–1805PubMedCrossRefGoogle Scholar
  6. 6.
    Ramsay AG, Johnson AJ, Lee AM, Gorgun G, Le Dieu R, Blum W, Byrd JC, Gribben JG (2008) Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug. J Clin Invest 118(7):2427–2437PubMedGoogle Scholar
  7. 7.
    Hofbauer JP, Heyder C, Denk U, Kocher T, Holler C, Trapin D, Asslaber D, Tinhofer I, Greil R, Egle A (2011) Development of CLL in the TCL1 transgenic mouse model is associated with severe skewing of the T-cell compartment homologous to human CLL. Leukemia 25(9):1452–1458PubMedCrossRefGoogle Scholar
  8. 8.
    Gorgun G, Ramsay AG, Holderried TA, Zahrieh D, Le Dieu R, Liu F, Quackenbush J, Croce CM, Gribben JG (2009) Emu-TCL1 mice represent a model for immunotherapeutic reversal of chronic lymphocytic leukemia-induced T-cell dysfunction. Proc Natl Acad Sci USA 106(15):6250–6255PubMedCrossRefGoogle Scholar
  9. 9.
    Roth A, de Beer D, Nuckel H, Sellmann L, Duhrsen U, Durig J, Baerlocher GM (2008) Significantly shorter telomeres in T-cells of patients with ZAP-70+/CD38+ chronic lymphocytic leukaemia. Br J Haematol 143(3):383–386PubMedCrossRefGoogle Scholar
  10. 10.
    Rezvany MR, Jeddi-Tehrani M, Osterborg A, Kimby E, Wigzell H, Mellstedt H (1999) Oligoclonal TCRBV gene usage in B-cell chronic lymphocytic leukemia: major perturbations are preferentially seen within the CD4 T-cell subset. Blood 94(3):1063–1069PubMedGoogle Scholar
  11. 11.
    Rezvany MR, Jeddi-Tehrani M, Wigzell H, Osterborg A, Mellstedt H (2003) Leukemia-associated monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic lymphocytic leukemia. Blood 101(3):1063–1070PubMedCrossRefGoogle Scholar
  12. 12.
    Durig J, Nuckel H, Huttmann A, Kruse E, Holter T, Halfmeyer K, Fuhrer A, Rudolph R, Kalhori N, Nusch A, Deaglio S, Malavasi F, Moroy T, Klein-Hitpass L, Duhrsen U (2003) Expression of ribosomal and translation-associated genes is correlated with a favorable clinical course in chronic lymphocytic leukemia. Blood 101(7):2748–2755PubMedCrossRefGoogle Scholar
  13. 13.
    Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98(9):5116–5121PubMedCrossRefGoogle Scholar
  14. 14.
    Willinger T, Freeman T, Hasegawa H, McMichael AJ, Callan MF (2005) Molecular signatures distinguish human central memory from effector memory CD8 T cell subsets. J Immunol 175(9):5895–5903PubMedGoogle Scholar
  15. 15.
    Voehringer D, Koschella M, Pircher H (2002) Lack of proliferative capacity of human effector and memory T cells expressing killer cell lectinlike receptor G1 (KLRG1). Blood 100(10):3698–3702PubMedCrossRefGoogle Scholar
  16. 16.
    Usherwood EJ, Hogan RJ, Crowther G, Surman SL, Hogg TL, Altman JD, Woodland DL (1999) Functionally heterogeneous CD8+ T-cell memory is induced by Sendai virus infection of mice. J Virol 73(9):7278–7286PubMedGoogle Scholar
  17. 17.
    Gattinoni L, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, Yu Z, Finkelstein SE, Theoret MR, Rosenberg SA, Restifo NP (2005) Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells. J Clin Invest 115(6):1616–1626PubMedCrossRefGoogle Scholar
  18. 18.
    Gattinoni L, Zhong XS, Palmer DC, Ji Y, Hinrichs CS, Yu Z, Wrzesinski C, Boni A, Cassard L, Garvin LM, Paulos CM, Muranski P, Restifo NP (2009) Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells. Nat Med 15(7):808–813PubMedCrossRefGoogle Scholar
  19. 19.
    Roberts AD, Ely KH, Woodland DL (2005) Differential contributions of central and effector memory T cells to recall responses. J Exp Med 202(1):123–133PubMedCrossRefGoogle Scholar
  20. 20.
    Cai G, Freeman GJ (2009) The CD160, BTLA, LIGHT/HVEM pathway: a bidirectional switch regulating T-cell activation. Immunol Rev 229(1):244–258PubMedCrossRefGoogle Scholar
  21. 21.
    McNerney ME, Lee KM, Kumar V (2005) 2B4 (CD244) is a non-MHC binding receptor with multiple functions on natural killer cells and CD8+ T cells. Mol Immunol 42(4):489–494PubMedCrossRefGoogle Scholar
  22. 22.
    Kuttruff S, Koch S, Kelp A, Pawelec G, Rammensee HG, Steinle A (2009) NKp80 defines and stimulates a reactive subset of CD8 T cells. Blood 113(2):358–369PubMedCrossRefGoogle Scholar
  23. 23.
    Tsujimura K, Obata Y, Matsudaira Y, Nishida K, Akatsuka Y, Ito Y, Demachi-Okamura A, Kuzushima K, Takahashi T (2006) Characterization of murine CD160+ CD8+ T lymphocytes. Immunol Lett 106(1):48–56PubMedCrossRefGoogle Scholar
  24. 24.
    Arase N, Takeuchi A, Unno M, Hirano S, Yokosuka T, Arase H, Saito T (2005) Heterotypic interaction of CRTAM with Necl2 induces cell adhesion on activated NK cells and CD8+ T cells. Int Immunol 17(9):1227–1237PubMedCrossRefGoogle Scholar
  25. 25.
    Vivier E, Anfossi N (2004) Inhibitory NK-cell receptors on T cells: witness of the past, actors of the future. Nat Rev Immunol 4(3):190–198PubMedCrossRefGoogle Scholar
  26. 26.
    Maecker HT, McCoy JP, Nussenblatt R (2012) Standardizing immunophenotyping for the Human Immunology Project. Nat Rev Immunol 12(3):191–200PubMedGoogle Scholar
  27. 27.
    Masopust D, Vezys V, Marzo AL, Lefrancois L (2001) Preferential localization of effector memory cells in nonlymphoid tissue. Science 291(5512):2413–2417PubMedCrossRefGoogle Scholar
  28. 28.
    Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401(6754):708–712PubMedCrossRefGoogle Scholar
  29. 29.
    Weninger W, Crowley MA, Manjunath N, von Andrian UH (2001) Migratory properties of naive, effector, and memory CD8+ T cells. J Exp Med 194(7):953–966PubMedCrossRefGoogle Scholar
  30. 30.
    Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, Antia R, von Andrian UH, Ahmed R (2003) Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol 4(3):225–234PubMedCrossRefGoogle Scholar
  31. 31.
    Blackburn SD, Shin H, Haining WN, Zou T, Workman CJ, Polley A, Betts MR, Freeman GJ, Vignali DA, Wherry EJ (2009) Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol 10(1):29–37PubMedCrossRefGoogle Scholar
  32. 32.
    Henson SM, Franzese O, Macaulay R, Libri V, Azevedo RI, Kiani-Alikhan S, Plunkett FJ, Masters JE, Jackson S, Griffiths SJ, Pircher HP, Soares MV, Akbar AN (2009) KLRG1 signaling induces defective Akt (ser473) phosphorylation and proliferative dysfunction of highly differentiated CD8+ T cells. Blood 113(26):6619–6628PubMedCrossRefGoogle Scholar
  33. 33.
    Voehringer D, Blaser C, Brawand P, Raulet DH, Hanke T, Pircher H (2001) Viral infections induce abundant numbers of senescent CD8 T cells. J Immunol 167(9):4838–4843PubMedGoogle Scholar
  34. 34.
    Heffner M, Fearon DT (2007) Loss of T cell receptor-induced Bmi-1 in the KLRG1+ senescent CD8+ T lymphocyte. Proc Natl Acad Sci USA 104(33):13414–13419PubMedCrossRefGoogle Scholar
  35. 35.
    Streeck H, Kwon DS, Pyo A, Flanders M, Chevalier MF, Law K, Julg B, Trocha K, Jolin JS, Anahtar MN, Lian J, Toth I, Brumme Z, Chang JJ, Caron T, Rodig SJ, Milner DA Jr, Piechoka-Trocha A, Kaufmann DE, Walker BD, Altfeld M (2011) Epithelial adhesion molecules can inhibit HIV-1-specific CD8+ T-cell functions. Blood 117(19):5112–5122PubMedCrossRefGoogle Scholar
  36. 36.
    Sarkar S, Kalia V, Haining WN, Konieczny BT, Subramaniam S, Ahmed R (2008) Functional and genomic profiling of effector CD8 T cell subsets with distinct memory fates. J Exp Med 205(3):625–640PubMedCrossRefGoogle Scholar
  37. 37.
    Bichi R, Shinton SA, Martin ES, Koval A, Calin GA, Cesari R, Russo G, Hardy RR, Croce CM (2002) Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression. Proc Natl Acad Sci USA 99(10):6955–6960PubMedCrossRefGoogle Scholar
  38. 38.
    Kiaii S, Kokhaei P, Mozaffari F, Rossmann E, Pak F, Moshfegh A, Palma M, Hansson L, Mashayekhi K, Hojjat-Farsangi M, Osterborg A, Choudhury A, Mellstedt H (2013) T cells from indolent CLL patients prevent apoptosis of leukemic B cells in vitro and have altered gene expression profile. Cancer Immunol Immunother 62(1):51–63 Google Scholar
  39. 39.
    Junevik K, Werlenius O, Hasselblom S, Jacobsson S, Nilsson-Ehle H, Andersson PO (2007) The expression of NK cell inhibitory receptors on cytotoxic T cells in B-cell chronic lymphocytic leukaemia (B-CLL). Ann Hematol 86(2):89–94PubMedCrossRefGoogle Scholar
  40. 40.
    Klein U, Tu Y, Stolovitzky GA, Mattioli M, Cattoretti G, Husson H, Freedman A, Inghirami G, Cro L, Baldini L, Neri A, Califano A, La-Favera R (2001) Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells. J Exp Med 194(11):1625–1638PubMedCrossRefGoogle Scholar
  41. 41.
    Huttmann A, Klein-Hitpass L, Thomale J, Deenen R, Carpinteiro A, Nuckel H, Ebeling P, Fuhrer A, Edelmann J, Sellmann L, Duhrsen U, Durig J (2006) Gene expression signatures separate B-cell chronic lymphocytic leukaemia prognostic subgroups defined by ZAP-70 and CD38 expression status. Leukemia 20(10):1774–1782PubMedCrossRefGoogle Scholar
  42. 42.
    Welte S, Kuttruff S, Waldhauer I, Steinle A (2006) Mutual activation of natural killer cells and monocytes mediated by NKp80-AICL interaction. Nat Immunol 7(12):1334–1342PubMedCrossRefGoogle Scholar
  43. 43.
    Powell DJ Jr, Rosenberg SA (2004) Phenotypic and functional maturation of tumor antigen-reactive CD8+ T lymphocytes in patients undergoing multiple course peptide vaccination. J Immunother 27(1):36–47PubMedCrossRefGoogle Scholar
  44. 44.
    Champagne P, Ogg GS, King AS, Knabenhans C, Ellefsen K, Nobile M, Appay V, Rizzardi GP, Fleury S, Lipp M, Forster R, Rowland-Jones S, Sekaly RP, McMichael AJ, Pantaleo G (2001) Skewed maturation of memory HIV-specific CD8 T lymphocytes. Nature 410(6824):106–111PubMedCrossRefGoogle Scholar
  45. 45.
    Schreeder DM, Pan J, Li FJ, Vivier E, Davis RS (2008) FCRL6 distinguishes mature cytotoxic lymphocytes and is upregulated in patients with B-cell chronic lymphocytic leukemia. Eur J Immunol 38(11):3159–3166PubMedCrossRefGoogle Scholar
  46. 46.
    Tinhofer I, Weiss L, Gassner F, Rubenzer G, Holler C, Greil R (2009) Difference in the relative distribution of CD4+ T-cell subsets in B-CLL with mutated and unmutated immunoglobulin (Ig) VH genes: implication for the course of disease. J Immunother 32(3):302–309PubMedCrossRefGoogle Scholar
  47. 47.
    Grundemann C, Schwartzkopff S, Koschella M, Schweier O, Peters C, Voehringer D, Pircher H (2010) The NK receptor KLRG1 is dispensable for virus-induced NK and CD8+ T-cell differentiation and function in vivo. Eur J Immunol 40(5):1303–1314PubMedCrossRefGoogle Scholar
  48. 48.
    Hofmann M, Schweier O, Pircher H (2012) Different inhibitory capacities of human and mouse KLRG1 are linked to distinct disulfide-mediated oligomerizations. Eur J Immunol 42(9):2484–2490PubMedCrossRefGoogle Scholar
  49. 49.
    Li Y, Hofmann M, Wang Q, Teng L, Chlewicki LK, Pircher H, Mariuzza RA (2009) Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition. Immunity 31(1):35–46PubMedCrossRefGoogle Scholar
  50. 50.
    Mackus WJ, Frakking FN, Grummels A, Gamadia LE, De Bree GJ, Hamann D, van Lier RA, van Oers MH (2003) Expansion of CMV-specific CD8+CD45RA+CD27 T cells in B-cell chronic lymphocytic leukemia. Blood 102(3):1057–1063PubMedCrossRefGoogle Scholar
  51. 51.
    Steininger C, Rassenti LZ, Vanura K, Eigenberger K, Jager U, Kipps TJ, Mannhalter C, Stilgenbauer S, Popow-Kraupp T (2009) Relative seroprevalence of human herpes viruses in patients with chronic lymphocytic leukaemia. Eur J Clin Invest 39(6):497–506PubMedCrossRefGoogle Scholar
  52. 52.
    Nunes C, Wong R, Mason M, Fegan C, Man S, Pepper C (2012) Expansion of a CD8+PD-1+ replicative senescence phenotype in early stage CLL patients is associated with inverted CD4:CD8 ratios and disease progression. Clin Cancer Res 18(3):678–687PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Joachim Rudolf Göthert
    • 1
    Email author
  • Lewin Eisele
    • 1
  • Ludger Klein-Hitpass
    • 2
  • Stefanie Weber
    • 1
  • Marie-Louise Zesewitz
    • 1
  • Ludger Sellmann
    • 1
  • Alexander Röth
    • 1
  • Hanspeter Pircher
    • 3
  • Ulrich Dührsen
    • 1
  • Jan Dürig
    • 1
  1. 1.Department of Hematology, West German Cancer Center (WTZ)University Hospital EssenEssenGermany
  2. 2.Institute of Cell BiologyUniversity Hospital EssenEssenGermany
  3. 3.Department of Immunology, Institute of Medical Microbiology and HygieneUniversity of FreiburgFreiburgGermany

Personalised recommendations