Abrogating Cbl-b in effector CD8+ T cells improves the efficacy of adoptive therapy of leukemia in mice
Ingunn M. Stromnes, Joseph N. Blattman, Xiaoxia Tan, Sara Jeevanjee, Hua Gu, Philip D. Greenberg
Ingunn M. Stromnes, Joseph N. Blattman, Xiaoxia Tan, Sara Jeevanjee, Hua Gu, Philip D. Greenberg
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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.