Ablation of Cbl-b provides protection against transplanted and spontaneous tumors
Jeffrey Y. Chiang, Ihn Kyung Jang, Richard Hodes, Hua Gu
Jeffrey Y. Chiang, Ihn Kyung Jang, Richard Hodes, Hua Gu
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Research Article Oncology

Ablation of Cbl-b provides protection against transplanted and spontaneous tumors

Abstract

A significant challenge to efforts aimed at inducing effective antitumor immune responses is that CD8+ T cells, which play a prominent role in these responses, may be unable to respond to tumors that lack costimulatory signals and that are protected by an immune suppressive environment such as that mediated by TGF-β produced by tumor cells themselves or by infiltrating Tregs, often resulting in tolerance or anergy of tumor-specific T cells. Here we show that the in vitro activation of Cblb–/– CD8+ T cells does not depend on CD28 costimulation and is resistant to TGF-β suppression. In vivo studies further demonstrated that Cblb–/– mice, but not WT controls, efficiently rejected inoculated E.G7 and EL4 lymphomas that did not express B7 ligands and that introduction of the Cblb–/– mutation into tumor-prone ataxia telangiectasia mutated–deficient mice markedly reduced the incidence of spontaneous thymic lymphomas. Immunohistological study showed that E.G7 tumors from Cblb–/– mice contained massively infiltrating CD8+ T cells. Adoptive transfer of purified Cblb–/– CD8+ T cells into E.G7 tumor-bearing mice led to efficient eradication of established tumors. Thus, our data indicate that ablation of Cbl-b can be an efficient strategy for eliciting immune responses against both inoculated and spontaneous tumors.

Authors

Jeffrey Y. Chiang, Ihn Kyung Jang, Richard Hodes, Hua Gu

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Figure 3

Tumor rejection in Cblb–/– mice is mediated by Cblb–/– CD8+ T cells.

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Tumor rejection in Cblb–/– mice is mediated by Cblb–/– CD8+ T cells. ...
(A) Immunohistology of tumor-infiltrating CD8+ cells. Shown are sections of tumors stained with H&E (left) or anti-CD8 antibody (right). CD8+ cells are FITC positive (green). Tumors were from WT and Cblb–/– mice at the seventh or nineteenth day after inoculation. (B) Flow cytometric analysis of tumor infiltrates. Tumor infiltrates were prepared from tumors from WT or Cblb–/– mice. These cells were stained with anti-TCRβ, anti-CD8, anti-CD4, and anti-CD44 antibodies and analyzed on LSR II. Foxp3+ (Treg) cells were identified by intracellular staining with an anti-Foxp3 antibody according to manufacture protocol. Shown at the top left are contour plots of CD8 and TCRβ expression on tumor-infiltrating cells from 1 of 3 independent experiments. The percentages of CD8+ TCRβ+ cells are indicated in the plots. The percentages of CD8+ TCRβ+, CD4+, and Treg infiltrates in total tumor infiltrates are summarized and shown as bars (bottom left). *P < 0.001, **P < 0.005. Histograms show the levels of TCRβ and CD44 expression on the infiltrating CD8+ T cells (right). (C) Eradication of established E.G7 tumors by adoptively transferred Cblb–/– or Cblb–/– OT1 CD8+ T cells. 106 E.G7 cells were inoculated into C57BL/6 mice by s.c. injection. Seven days after the inoculation, 3 × 106 purified WT or Cblb–/– CD8+ T cells (top) or WT OT1 or Cblb–/– OT1 CD8+ T cells (bottom) were transferred into the tumor-bearing mice by i.v. injection. Shown at the left are the tumor volumes at different time points after the CD8+ T cell transfer. Genotypes of the donor cells are indicated on the top of each plot. Results are from 1 of 5 or more independent experiments. The plot in the right panel shows percentages of surviving recipient mice (WT, n = 11; Cblb–/–, n = 13) that received WT or Cblb–/– CD8+ T cells. When the tumor volume reached approximately 5,000 mm3, the mice were euthanized and recorded as dead.