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. 2018 Jan 10;20(1):55-65.
doi: 10.1093/neuonc/nox116.

CD70, a novel target of CAR T-cell therapy for gliomas

Linchun Jin  1   2 , Haitao Ge  2 , Yu Long  1   2 , Changlin Yang  1 , Yifan Emily Chang  1   3 , Luyan Mu  2 , Elias J Sayour  1 , Gabriel De Leon  1   4 , Qiong J Wang  5   6 , James C Yang  5 , Paul S Kubilis  1 , Hongbo Bao  2 , Songsong Xia  2 , Dunyue Lu  2 , Yingjun Kong  7 , Li Hu  2 , Yujiao Shang  7 , Chencheng Jiang  7 , Jing Nie  7 , Shimin Li  7 , Yunhe Gu  8 , Jiahang Sun  9 , Duane A Mitchell  1 , Zhiguo Lin  2 , Jianping Huang  1
Affiliations

CD70, a novel target of CAR T-cell therapy for gliomas

Linchun Jin et al. Neuro Oncol. .

"VSports最新版本" Abstract

Background: Cancer immunotherapy represents a promising treatment approach for malignant gliomas but is hampered by the limited number of ubiquitously expressed tumor antigens and the profoundly immunosuppressive tumor microenvironment. We identified cluster of differentiation (CD)70 as a novel immunosuppressive ligand and glioma target. VSports手机版.

Methods: Normal tissues derived from 52 different organs and primary and recurrent low-grade gliomas (LGGs) and glioblastomas (GBMs) were thoroughly evaluated for CD70 gene and protein expression. The association between CD70 and patients' overall survival and its impact on T-cell death was also evaluated V体育安卓版. Human and mouse CD70-specific chimeric antigen receptors (CARs) were tested respectively against human primary GBMs and murine glioma lines. The antitumor efficacies of these CARs were also examined in orthotopic xenograft and syngeneic models. .

Results: CD70 was not detected in peripheral and brain normal tissues but was constitutively overexpressed by isocitrate dehydrogenase (IDH) wild-type primary LGGs and GBMs in the mesenchymal subgroup and recurrent tumors. CD70 was also associated with poor survival in these subgroups, which may link to its direct involvement in glioma chemokine productions and selective induction of CD8+ T-cell death. To explore the potential for therapeutic targeting of this newly identified immunosuppressive axis in GBM tumors, we demonstrate that both human and mouse CD70-specific CAR T cells recognize primary CD70+ GBM tumors in vitro and mediate the regression of established GBM in xenograft and syngeneic models without illicit effect. V体育ios版.

Conclusion: These studies identify a previously uncharacterized and ubiquitously expressed immunosuppressive ligand CD70 in GBMs that also holds potential for serving as a novel CAR target for cancer immunotherapy in gliomas VSports最新版本. .

Keywords: CD70; chimeric antigen receptors (CARs); gliomas; immunosuppressive ligand; immunotherapy. V体育平台登录.

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VSports app下载 - Figures

Fig. 1
Fig. 1
Overexpression of CD70 on primary and recurrent gliomas, but not on tumor infiltrating T cells. (A–B) Differential CD70 gene expression in normal brain, primary and recurrent LGG, and GBM. The gene expression level was defined as RSEM (RNA-seq by expectation-maximization), and patient’s informations were culled from TCGA RNA-seq datasets. (C) Protein level of CD70 was evaluated by immunohistochemistry using tumors from primary LGGs (n = 41), GBMs (n = 44), and normal brains (n = 7). (D) CD70 protein expression between primary and recurrent GBMs. Tumors from 7 paired GBMs before and after recurrence were evaluated. (E) The CD70 positivity within each CD70-expressing primary GBM. (F) Determination of CD70 expression on tumor and T cells. Seven CD70+ GBMs were co-stained with fluorescent conjugated CD3 (green) and CD70 (red) antibodies; 4′,6′-diamidino-2-phenylindole was used for nuclear staining. Representative pictures (with an enlarged overlaid area) from one tumor are shown. Mann–Whitney U test was used.
Fig. 2
Fig. 2
CD70 gene expression is inversely correlated with overall survival in patients with primary gliomas. (A–B) CD70 expression among different subgroups of LGGs and GBMs. (C–F) Predicted median overall survival decreases with increasing CD70 expression in primary LGGs, IDH wild-type LGGs, GBMs, and GBMs in the mesenchymal subgroup (CD70 RNA-seq expression was considered as a continuous predictor of survival, adjusted for gender and age in all subgroup models using Cox proportional hazards (PH) regression; further adjustment was made for tumor subtype in the GBM subgroup model). (G) Summary of differential median overall survival between CD70 high and low groups defined by mean values of CD70 RSEM in LGGs and GBMs (subgroup median survival estimates adjusted for age, gender and GBM tumor subtype via Cox PH regression).
Fig. 3
Fig. 3
CD70 involved in cytokine/chemokine productions in GBM. (A–B) CD70 overexpression influences the cytokine/chemokine pathway in a primary GBM line. Triplicated RNA samples from pGBM#3 (CD70+) were transduced with CD70 (Over-exp) or lentiviral vector control (blank), confirmed by gene (fragments per kilobase of transcript per million mapped reads, FPKM) and protein expression (flow cytometry). Gene enrichment of the upregulated gene list (Over-exp vs blank) was performed by PANTHER-Pathway; a volcano plot and the top-ranked pathways are shown. (C) Primary GBM lines secrete various chemokines. Culture supernatants from the primary lines were collected 8 days after the tumors were seeded, and chemokine array was performed. (D–G) Gene expression changes between overexpression and silence of CD70 in pGBM#3 were evaluated by real-time quantitative PCR. (H) IL-8 secretion was measured by enzyme-linked immunosorbent assay. Unpaired t-test was used.
Fig. 4
Fig. 4
CD70 is associated with T-cell infiltration and CD8+ T-cell death in GBM. (A) Relative higher numbers of infiltrating CD3+ T cells in CD70+ compared with CD70− tumors. Paraffin-embedded GBM tumor samples described in Fig. 1 were analyzed by immunohistochemistry for CD70 and CD3, respectively, using serial slides. (B) CD70 predominantly induces CD8+ T-cell death after engaging tumor cells. CD70-manipulated pGBM#3 cells were cocultured with 3 allogeneic PBMCs obtained from healthy donors. Fluorescence activated cell sorting (FACS) analysis was performed 14 days after the coculture (left). The cell death of T-cell subsets was quantitated by propidium iodide (PI) staining for both CD4+ and CD8+ T cells (right). These results were representative of 4 individual experiments. Mann–Whitney test was used to assess statistical significance. (C) The ratio of CD4+ to CD8+ T cells among the cocultured T cells. FACS analysis from (B) was also evaluated by CD4+ to CD8+ ratio, and the gating strategy is shown in the upper panel. Paired t-test was used to assess statistical significance.
Fig. 5
Fig. 5
Tumor recognitions of gliomas by human and mouse CD70 CAR T cells. (A) Human CAR construct and its transduction efficiency in human T cells. The transduction efficiency was measured by tdTomato positive cells and the frequency of CD4+/CD8+ T cells 4 days posttransduction are shown. (B) GBM targets for hCAR T cells. GBM lines naturally expressing various levels of CD70 were tested for the CD70 surface expression by FACS. (C–D) Recognition of these lines by hCAR T cells. The recognitions were measured by IFN-γ release and cytotoxic killing assay. (E–G) Manipulation of tumor CD70 expression alters the CAR T cells recognition. The tumor lines were cocultured with manipulated GBM lines by either overexpressing or silencing CD70. (H) Irradiation enhances tumor CD70 expression and CAR T-cell recognition. The U87 line was irradiated with 6 Gy, CD70 expression was determined by FACS on day 3 and then cocultured with CAR T cells. (I) Transduction of mCD70 CAR to mouse T cells. Construct of mouse CD70-specific CAR (mCD70-CAR), transduction efficiency determined by tdTomato and frequency of CD4+ /CD8+ T cells in the mCAR T cells 4 days posttransduction are shown. (J–M) CD70-overexpressed murine glioma lines were recognized by mCD70-CAR T cells. The tumors (104) were cocultured with (104) mCD70-CAR T cells or vector/NT control T cells, respectively. For the killing assay, various ratios of effector to target (E:T) were respectively cocultured. Unpaired t-test was used.
Fig. 6
Fig. 6
Antitumor response of CD70 CAR T cells against CD70+ gliomas in vivo. (A) Antitumor response of hCAR T cells in human xenograft glioma model. CD70+ (U87.Luc, 5 × 104/mouse) tumor-bearing (confirmed by imaging 6 days after tumor inoculation) mice were adoptively transferred through tail-vein injection with NT or various doses (105–107/mouse, 8/group) of hCAR T cells 7 days after the tumor implantation. (B–C) A complete response and prolonged survival induced by mCAR T cells in syngeneic glioma models. Two models were used: groups of 6–8 weeks C57BL/6J mice (10/group) were intracranially inoculated with 1 × 105 KR70 tumor cells derived from a tumor clone, designated as KR70-C, and then adoptively transferred (1 × 107) NT or mCAR T cells on days 5 and 7 post tumor implantation. Luciferase imaging was carried out 10 and 17 days after the tumor implantation. The same experiment described in (B) was performed, except the inoculated tumor cells were derived from bulk tumors, KR70-B. All experiments were repeated at least 2 times. Mann–Whitney test and the log-rank test were used.
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