Activated iNKT cells enhance the anti-tumor effect of antigen specific CD8 T cells on mesothelin-expressing salivary gland cancer

Abstract

Background: Salivary gland cancers are not sensitive to conventional radiotherapy or chemotherapy regimens. Therefore, the development of a new treatment strategy is of critical importance for improving the prognosis. We examined the expression of mesothelin molecules in salivary gland cancers and the efficacy of adoptive cell therapy based on mesothelin-specific chimeric antigen receptor transduced T cells.

Methods: The expression of mesothelin molecule was studied in salivary gland cancer samples obtained from 16 patients as well as a salivary gland cancer cell line (A-253) and five other cell lines. The activation of mesothelin-specific chimeric antigen receptor-expressing CD8 T cells after stimulation with mesothelin and the effects of invariant natural killer T cells on this activation were evaluated.

Results: Mesothelin was detected in the A-253 cells and the surgical specimens except for the case of squamous cell carcinoma to various degrees. Following stimulation with mesothelin expressing cancer cells, chimeric antigen receptor T cells were dose-dependently activated; this activation was enhanced by co-culture with invariant natural killer T cells and subsequently abrogated by treatment with anti-interferon-γ antibodies. Furthermore, the cytotoxicity of chimeric antigen receptor T cells against various cancer cells was further augmented by invariant natural killer T cells.

Conclusions: The use of adoptive transfer with mesothelin-specific chimeric antigen receptor-expressing CD8 T cells against salivary gland cancers is an effective therapy and invariant natural killer T cells are expected to be used in adjuvant treatment for T cell-based immunotherapy.

Keywords: Adoptive immunotherapy; Chimeric antigen receptor; Cytotoxic T lymphocyte; Natural kiiler T-cells; Salivary gland cancer.

Fig. 1

MSLN expressions in the surgical samples obtained from the patients with various types of SGC and the various cancer cell lines. a-h Immunohistochemical images of mucoepidermoid carcinoma (a, case 12 and d, case 5), adenocarcinoma (b, case 13), salivary duct carcinoma (c, case 16 and f, case 15) and adenoid cystic carcinoma (e, case 11) are shown. Positive control tumor (g) and negative control pleomorphic adenoma (h, case 17) are also shown. The red and blue colors indicate MSLN and DAPI. i Histograms of MSLN expression on the various cancer cell lines analyzed by flow cytometry are shown. Gray shadows indicate isotype matched control. J RT-PCR analysis of MSLN were performed with case 12 (a), case 13 (b), case 11 (e) and case 15 (f). The MSLN-transduced or wild type K-562 cells were used for positive and negative control

Fig. 2

Preparation of the anti-MSLN lentiviral vector engineered with CD8 T cells and highly purified iNKT cells. a Schematic representation of MSLN-binding CARs. For a binding-control CAR, a truncated TCRζ domain was utilized. b The expression levels of the SS1 scFv fusion proteins on human primary CD8 T cells were examined using flow cytometry. The thin and bold lines indicate SS1-Δζ and SS1–28-BBζ, respectively. c iNKT cells were purified from human PBMCs stimulated with αGalCer for eight days

Fig. 3

CD107a mobilization assay of SS1 CAR-expressing human CD8 T cells after stimulation of the various cancer cell lines with or without the adjuvant activated iNKT cells. The engineered T cells were incubated for 3.5 h after stimulation and analyzed. CD8⁺CAR⁺7AAD⁻ cells are shown

Fig. 4

Cellular proliferation of SS-1 CAR-transduced CD8 T cells. CAR-expressing CD8 T cells were stained with PKH26 and cultured for 72 h in the presence of M108, A-253, IMC3 and FaDu tumor cells. a A histogram of the PKH26 expression of CD8⁺CAR⁺Vα24⁻7AAD⁻ cells and (b) proliferative index (T(x)) are shown

Fig. 5

Synergistically augmented cytotoxicity of the CAR-redirected CD8 T cells and activated iNKT cells against the cancer cells expressing MSLN molecules at various intensities. Four-hour chromium-51 release assays against four kinds of target cells, MSLN-transduced K562 (a), A-253 (b), IMC-3 (c) and FaDu (d) cells, were performed. The effector cells were SS1-Δζ or SS1–28-BBζ and/or iNKT cells stimulated with αGalCer. Statistically significant differences are indicated by asterisks and daggers: *, P < 0.05; **, P < 0.01=