Abrogating Cbl-b in effector CD8+ T cells improves the efficacy of adoptive therapy of leukemia in mice
Ingunn M. Stromnes, … , Hua Gu, Philip D. Greenberg
Ingunn M. Stromnes, … , Hua Gu, Philip D. Greenberg
Published September 20, 2010
Citation Information: J Clin Invest. 2010;120(10):3722-3734. https://doi.org/10.1172/JCI41991.
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Research Article Hematology

Abrogating Cbl-b in effector CD8+ T cells improves the efficacy of adoptive therapy of leukemia in mice

Abstract

The clinical use of adoptive immunotherapy with tumor-reactive T cells to treat established cancers is limited in part by the poor in vivo survival and function of the transferred T cells. Although administration of exogenous cytokines such as IL-2 can promote T cell survival, such strategies have many nonspecific activities and are often associated with toxicity. We show here that abrogating expression of Casitas B-lineage lymphoma b (Cbl-b), a negative regulator of lymphocyte activation, in tumor-reactive CD8+ T cells expanded ex vivo increased the efficacy of adoptive immunotherapy of disseminated leukemia in mice. Mechanistically, Cbl-b abrogation bypassed the requirement for exogenous IL-2 administration for tumor eradication in vivo. In addition, CD8+ T cells lacking Cbl-b demonstrated a lower threshold for activation, better survival following target recognition and stimulation, and enhanced proliferative responses as a result of both IL-2–dependent and –independent pathways. Importantly, siRNA knockdown of Cbl-b in human CD8+CD28– effector T cell clones similarly restored IL-2 production and proliferation following target recognition independent of exogenous IL-2, enhanced IFN-γ production, and increased target avidity. Thus, abrogating Cbl-b expression in effector T cells may improve the efficacy of adoptive therapy of some human malignancies.

Authors

Ingunn M. Stromnes, Joseph N. Blattman, Xiaoxia Tan, Sara Jeevanjee, Hua Gu, Philip D. Greenberg

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Figure 7

Targeting Cbl-b expression by siRNA in human effector CD8+CD28– T cell clones restores T cell function.

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Targeting Cbl-b expression by siRNA in human effector CD8+CD28– T cell c...
(A) Human CD8+CD28– CMV-specific effector T cell clones (MT-3) were transduced with 1 out of 3 Cbl-b–specific siRNAs (Cbl-b1, Cbl-b2, or Cbl-b3), and their efficiency in reducing Cbl-b protein levels relative to Actin was measured by Western blot. (B) Parental and siRNA-transduced MT-3 cell clones were incubated with CMVpp65 peptide-pulsed APCs, and the percentage of IFN-γ–positive clones was determined by intracellular cytokine staining and normalized to the percentage of the maximum response. (C) MT-3 clones transduced with Cbl-b2 siRNA, and control clones (vector only) were incubated with T2 cells (HLA-A2+) with or without CMVpp65. After 24 hours, IL-2 secretion was quantified by a cytobead array. (D) IL-2 secretion by parental and transduced MT-3 clones after antigen stimulation. As a positive control, MT-3 clones were also transduced with CD28 as described previously (21). (E) The efficiency of Cbl-b2 siRNA in knocking down Cbl-b relative to Actin in human CD8+CD28– MelanA-specific clones (SK-111) was determined by Western blot. (F) Parental or Cbl-b2 siRNA-transduced SK-111 cells were incubated with antigen-negative (Ag–) HLA-A2+ 375 (MelanA26–35–) or antigen-positive 526 (MelanA26–35+) melanoma, and IFN-γ production was assessed after 24 hours. (G) IL-2 production by parental and Cbl-b2 siRNA-transduced SK-111 T cells was measured 24 hours after stimulation with T2 cells with or without MelanA26–35 peptide. (H) The proliferation of Cbl-b2 siRNA-transduced and parental SK-111 T cells in response to antigen with or without r-h-IL-2 (50 U/ml) was measured by 3H-thymidine incorporation.