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 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).