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 1

Cbl-b regulates IL-2 production and proliferation of naive TCRgag cells.

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Cbl-b regulates IL-2 production and proliferation of naive TCRgag cells....
(A) CD8α and H-2Db/gag tetramer staining of splenocytes isolated from naive TCRgagCblb+/+ and TCRgagCblb–/– mice. The numbers in the plots indicate the percentages of CD8+ tetramer+ cells per spleen (top) and the percentages of CD8+ T cells in the spleen that bind tetramer (bottom). (B) Naive CD8+ tetramer+Cblb+/+ and Cblb–/– cells were analyzed for CD44, CD62L, CD25, and CD69 expression using flow cytometry. (C) TCRgagCblb+/+ and TCRgagCblb–/– cells were stimulated with peptide-pulsed splenocytes (5 μg/ml), and cytokine production was assessed after 24 hours by a cytobead array. *P < 0.05 (t test). (D) CD8+ TCRgagCblb+/+ and TCRgagCblb–/– mice were stimulated as in C, and the percentage of IL-2–secreting transgenic cells was determined by intracellular cytokine staining after 5 hours. *P < 0.005 (t test). (E) MFI was quantified by gating on the IL-2–secreting population as shown in the representative FACS plot. (F) CFSE-labeled TCRgagCblb+/+ (gray line) and TCRgagCblb–/– (black line) CD8 cells were incubated with irradiated, congenic (Thy1.1+) splenocytes pulsed with gag peptide. r-h-IL-2 (25 U/ml) was added to otherwise identical wells (bottom row). FACS plots were gated on Thy1.2+ CD8+ T cells on day 3. Filled histograms indicate no antigen. Data is either representative of 2–3 experiments (A, B, E, and F) or pooled from 3 independent experiments (C and D).