Cancer Immunology, Immunotherapy

, Volume 57, Issue 8, pp 1151–1160 | Cite as

CD40 ligation in vivo can induce T cell independent antitumor effects even against immunogenic tumors

  • Alexander L. Rakhmilevich
  • Ilia N. Buhtoiarov
  • Miroslav Malkovsky
  • Paul M. Sondel
Original Article


Antitumor effects of CD40 ligation appear to involve distinct antitumor effector cells in different experimental models. In this study, we tested whether T cells were required for antitumor effects of agonistic anti-CD40 mAb (αCD40) against immunogenic versus poorly immunogenic tumors. Treatment of mice bearing poorly immunogenic B16 melanoma and its more immunogenic variant, B16-hsp72.1, with αCD40 resulted in a similar level of tumor growth suppression. Depletion of T cells did not reduce the antitumor effects in these 2 tumor models. To generate antitumor T cell responses, C57BL/6 mice were immunized with irradiated B16-hsp72.1. Treatment of these vaccinated mice challenged with a high dose of B16-hsp72.1 tumor cells with αCD40 induced tumor growth suppression, which was reduced by T-cell depletion, demonstrating that T cells were involved in the antitumor effect of αCD40. However, immunized mice depleted of T cells and treated with αCD40 were still able to suppress tumor growth as compared to tumor growth in immunized, T cell-depleted mice not treated with αCD40, suggesting that T cells were not required for the antitumor effect of αCD40. To confirm a lack of correlation between tumor immunogenicity and T-cell requirement in antitumor effects of CD40 ligation, we found that αCD40 induced tumor growth suppression in nude and SCID/beige mice bearing highly immunogenic tumors such as Meth A sarcoma, suggesting that macrophages may play a role. Indeed, both poorly immunogenic and highly immunogenic tumors were sensitive to in vitro growth inhibition by macrophages from αCD40-treated mice. Taken together, our results indicate that antitumor effects induced by αCD40, even against immunogenic tumors, can be observed in the absence of T cells and may involve macrophages.


Tumor Immunity T cells Monocytes/macrophages Anti-CD40 mAb 



CD40 ligand


Anti-CD40 monoclonal antibody



Peritoneal exudate cells


Immunoglobulin G





The authors thank Drs. Jackie Hank, Jacek Gan, and Hillary Lum for helpful discussions.


  1. 1.
    Bennett SRM, Carbone FR, Karamalis F, Flavell RA, Miller JFA, Heath WR (1998) Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478–480PubMedCrossRefGoogle Scholar
  2. 2.
    Buhtoiarov IN, Lum HD, Berke G, Paulnock DM, Sondel PM, Rakhmilevich AL (2005) CD40 ligation induces antitumor reactivity of murine macrophages via an IFN gamma-dependent mechanism. J Immunol 174:6013–6022PubMedGoogle Scholar
  3. 3.
    Buhtoiarov IN, Lum HD, Berke G, Sondel PM, Rakhmilevich AL (2006) Synergistic activation of macrophages via CD40 and TLR9 results in T cell independent antitumor effects. J Immunol 176:309–318PubMedGoogle Scholar
  4. 4.
    Campbell AC, Hersey P, MacLennan IC, Kay HE, Pike MC (1973) Immunosuppressive consequences of radiotherapy and chemotherapy in patients with acute lymphoblastic leukaemia. BMJ 2:385–388PubMedCrossRefGoogle Scholar
  5. 5.
    DeKruyff RH., Gieni RS, Umetsu DT (1997) Antigen-driven but not lipopolysaccharide-driven IL-12 production in macrophages requires triggering of CD40. J Immunol 158:359–366PubMedGoogle Scholar
  6. 6.
    Diehl L, Boer AT, Schoenberger SP, Voort E, Schumacher TNM, Melief CJM, Offringa R, Toes REM (1999) CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments antitumor vaccine efficacy. Nat Med 5:774–779PubMedCrossRefGoogle Scholar
  7. 7.
    French RR, Chan HTC, Tutt AL, Glennie MJ (1999) CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat Med 5:548–553PubMedCrossRefGoogle Scholar
  8. 8.
    Gruber TA, Skelton DC, Kohn DB (2002) Requirement for NK cells in CD40 ligand-mediated rejection of Philadelphia chromosome-positive acute lymphoblastic leukemia cells. J Immunol 168:73–80PubMedGoogle Scholar
  9. 9.
    Hirano A, Longo DL, Taub DD, Ferris DK, Young LS, Eliopoulos AG, Agathanggelou A, Cullen N, Macartney J, Fanslow WC, Murphy WJ (1999) Inhibition of human breast carcinoma growth by a soluble recombinant human CD40 ligand. Blood 93:2999–3007PubMedGoogle Scholar
  10. 10.
    Hunig T (1983) T-cell function and specificity in athymic mice. Immunol Today 4:84–87CrossRefGoogle Scholar
  11. 11.
    Imaizumi K, Kawabe T, Ichiyama S, Kikutani H, Yagita H, Shimokata K, Hasegawa Y (1999) Enhancement of tumoricidal activity of alveolar macrophages via CD40–CD40 ligand interaction. Am J Physiol 277: L49–L57PubMedGoogle Scholar
  12. 12.
    Kedl RM, Jordan M, Potter T, Kappler J, Marrack P, Dow S (2001) CD40 stimulation accelerates deletion of tumor-specific CD8(+) T cells in the absence of tumor-antigen vaccination. Proc Natl Acad Sci USA 98:10811–10816PubMedCrossRefGoogle Scholar
  13. 13.
    Leoprechting VA, Bruggen VDP, Pahl HL, Aruffo A, Simon JC (1999) Stimulation of CD40 on immunogenic human malignant melanomas augments their cytotoxic T lymphocyte-mediated lysis and induces apoptosis. Cancer Res 59:1287–1294Google Scholar
  14. 14.
    Lum HD, Buhtoiarov IN, Schmidt BE, Berke G, Paulnock DM, Sondel PM, Rakhmilevich AL (2006) In vivo CD40 ligation can induce T cell-independent antitumor effects that involve macrophages. J Leuk Biol 79:1181–1192CrossRefGoogle Scholar
  15. 15.
    Lum HD, Buhtoiarov IN, Schmidt BE, Berke G, Paulnock DM, Sondel PM, Rakhmilevich AL (2006) Tumoristatic effects of anti-CD40 mAb-activated macrophages involve nitric oxide and tumor-necrosis factor-α. Immunol 118:261–270CrossRefGoogle Scholar
  16. 16.
    Mackey MF, Gunn JR, Ting P, Kikutani H, Dranoff G, Noelle RJ, Barth RJ (1997) Protective immunity induced by tumor vaccines requires interaction between CD40 and its ligand, CD154. Cancer Res 57:2569–2574PubMedGoogle Scholar
  17. 17.
    Nakajima A, Kodama T, Morimoto S, Azuma M, Takeda K, Oshima H, Yoshino S, Yagita H, Okumura K (1998) Antitumor effect of CD40 ligand: elicitation of local and systemic antitumor responses by IL-12 and B7. J Immunol 161:1901–1907PubMedGoogle Scholar
  18. 18.
    North RJ, Bursuker I. (1984) Generation and decay of the immune response to a progressive fibrosarcoma. I. Ly-1 + 2- suppressor T cells down-regulate the generation of Ly-1–2+ effector T cells. J Exp Med 159:1295–1311PubMedCrossRefGoogle Scholar
  19. 19.
    O’Sullivan B, Thomas R (2003) CD40 and dendritic cell function. Crit Rev Immunol 23:83–107PubMedCrossRefGoogle Scholar
  20. 20.
    Payer E, Strohal R, Kutil R, Elbe A, Stingl G (1992) Demonstration of a CD3+ lymphocyte subset in the epidermis of athymic nude mice. Evidence for T cell receptor diversity. J Immunol 149:413–420PubMedGoogle Scholar
  21. 21.
    Ridge JP, Rosa FD, Matzinger P (1998) A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393:474–478PubMedCrossRefGoogle Scholar
  22. 22.
    Rolink A, Melchers F, Andersson J (1996) The SCID but not the RAG-2 gene product is required for 5 mu-S epsilon heavy chain class switching. Immunity 5:319–330PubMedCrossRefGoogle Scholar
  23. 23.
    Schoenberger SP, Toes REM, van der Voort EIH, Offringa R, Melief CJM (1998) T-cell help for cytotoxic T lymphocytes is mediated by CD40–CD40L interactions. Nature 393:480–483PubMedCrossRefGoogle Scholar
  24. 24.
    Solomayer EF, Feuerer M, Bai L, Umansky V, Beckhove P, Meyberg GC, Bastert G, Schirrmacher V, Diel IJ (2003) Influence of adjuvant hormone therapy and chemotherapy on the immune system analysed in the bone marrow of patients with breast cancer. Clin Cancer Res 9:174–180PubMedGoogle Scholar
  25. 25.
    Sotomayor EM, Borrello I, Tubb E, Rattis FM, Bien H, Lu Z, Fein S, Schoenberger S, Levitsky H (1999) Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40. Nat Med 5:780–787PubMedCrossRefGoogle Scholar
  26. 26.
    Stumbles PA, Himbeck R, Frelinger JA, Collins EJ, Lake RA, Robinson BW (2004) Cutting edge: tumor-specific CTL are constitutively cross-armed in draining lymph nodes and transiently disseminate to mediate tumor regression following systemic CD40 activation. J Immunol 173:5923–5928PubMedGoogle Scholar
  27. 27.
    Todryk SM, Tutt AL, Green MH, Smallwood JA, Halanek N, Dalgleish AG, Glennie MJ (2001) CD40 ligation for immunotherapy of solid tumours. J Immunol Meth 248:139–147CrossRefGoogle Scholar
  28. 28.
    Turner JG, Rakhmilevich AL, Burdelya L, Neal Z, Imboden M, Sondel PM, Yu H (2001) Anti-CD40 antibody induces antitumor and anti-metastatic effects: role of natural killer cells. J Immunol 166:89–94PubMedGoogle Scholar
  29. 29.
    Tutt AL, O’Brien L, Hussain A, Crowther GR, French RR, Glennie MJ (2002) T cell immunity to lymphoma following treatment with anti-CD40 monoclonal antibody. J Immunol 168:2720–2728PubMedGoogle Scholar
  30. 30.
    van Mierlo GJ, den Boer AT, Medema JP, van der Voort EI, Fransen MF, Offringa R, Melief CJ, Toes RE (2002) CD40 stimulation leads to effective therapy of CD40(-) tumors through induction of strong systemic cytotoxic T lymphocyte immunity. Proc Natl Acad Sci USA 99:5561–5566PubMedCrossRefGoogle Scholar
  31. 31.
    Vesosky B, Hurwitz AA (2003) Modulation of costimulation to enhance tumor immunity. Cancer Immunol Immunother 52:663–669PubMedCrossRefGoogle Scholar
  32. 32.
    Vonderheide RH, Dutcher JP, Anderson JE, Eckhardt SG, Stephans KF, Razvillas B, Garl S, Butine MD, Perry VP, Armitage RJ, Ghalie R, Caron DA, Gribben JG (2001) Phase I study of recombinant human CD40 ligand in cancer patients. J Clinic Oncol 19:3280–3287Google Scholar
  33. 33.
    Vonderheide RH, Flaherty KT, Khalil M, Stumacher MS, Bajor DL, Hutnick NA, Sullivan P, Mahany JJ, Gallagher M, Kramer A, Green SJ, O’Dwyer PJ, Running KL, Huhn RD, Antonia SJ (2007) Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody. J Clinic Oncol 25:876–883CrossRefGoogle Scholar
  34. 34.
    Wells AD, Rai SK, Salvato MS, Band H, Malkovsky M (1998) Hsp72-mediated augmentation of MHC class I surface expression and endogenous antigen presentation. Int Immunol 10:609–617PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Alexander L. Rakhmilevich
    • 1
    • 4
  • Ilia N. Buhtoiarov
    • 1
    • 4
  • Miroslav Malkovsky
    • 2
    • 4
  • Paul M. Sondel
    • 1
    • 3
    • 4
  1. 1.The Department of Human OncologyUniversity of Wisconsin, K4/413 Clinical Science CenterMadisonUSA
  2. 2.Department of Medical Microbiology and ImmunologyUniversity of Wisconsin, K4/448 Clinical Science CenterMadisonUSA
  3. 3.Department of PediatricsUniversity of Wisconsin, K4/448 Clinical Science CenterMadisonUSA
  4. 4.The UW Comprehensive Cancer Center University of Wisconsin, K4/448 Clinical Science CenterMadisonUSA

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