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 2

Proliferation of TCRgagCblb–/– effector cells is independent of exogenous IL-2.

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Proliferation of TCRgagCblb–/– effector cells is independent of exogenou...
TCRgagCblb+/+ and TCRgagCblb–/– cells were cultured with antigen and r-h-IL-2 (25 U/ml) for 3 cycles of stimulation. (A) CD8 and tetramer staining of Cblb+/+ and Cblb–/– effector cells. The percentages of CD8+ tetramer+ cells in the culture (top) and the percentages of CD8+ cells that are also tetramer+ (bottom) are indicated. (B) Histogram overlays of Cblb–/– (black), Cblb+/+ (gray) tetramer+, or naive CD8 cells (filled). Tetramer+Cblb–/– and Cblb+/+ effectors cells were stained for various antigens and analyzed by FACS. (C) Thy1.1+TCRgagCblb+/+ cells were cultured with Thy1.2+TCRgagCblb–/– cells and antigen, stained for CD8 and congenic markers as shown, and analyzed for Stat5 phosphorylation after 24 hours. (D) Effector cells were stimulated with antigen and stained for intracellular IL-2 and IFN-γ. Data is gated on CD8+Thy1.2+ cells. (E) IL-2 production was assessed 24 hours after activation with antigen using a cytometric bead array. *P < 0.05 (t test). (F) CFSE-labeled effector Cblb+/+ (gray line) and Cblb–/– (black line) Thy1.2+ cells were incubated with peptide-pulsed, irradiated Thy1.1+ syngeneic splenocytes with or without r-h-IL-2 (25 U/ml). CFSE dilution was measured on day 4. The numbers in the plots indicate the percentages of proliferating transgenic cells for Cblb+/+ or Cblb–/–, respectively, as indicated by color. Filled histograms indicate no antigen controls. (G) The fold expansion of effector cells 5 days after stimulation with antigen with or without r-h-IL-2 (25 U/ml). Data is representative of 2–3 experiments (A–C, and F) or pooled from 3 independent experiments (D and E).