Distinct INKT Cell Responses Against Different Target Cells And Their Therapeutic Potential In T-cell Lymphomas

iNKT cells are innate-like T cells that possess both T and NK cell surface markers.They recognize lipid antigens presented by CD1 d and serve as a bridge between innate and adaptive immunity.After activation,iNKT cells respond quickly to be among the first responders in the scene.iNKT cells can exert direct cytotoxicity against tumor cells in a predominant perforin-and granzyme B-dependent manner,with additional TNF-? and Fas/Fas L-mediated killing.They upregulate co-stimulating receptors and rapidly produce a cytokine ‘storm' upon activation which subsequently activates other immune cells to play indirect anti-tumor function.The anti-tumor responses endow iNKT cells with therapeutic potential for cancer.However,the characteristics of human iNKT cells and their responses against malignant T cells in human remain elusive.In this study,a panel of antigen presenting cells and tumor cells were selected as stimulators or targets,including primary T cells,primary B cells,primary monocytes,malignant T cell line(Jurkat),B-lymphoblastoid cell(721.221),myeloid leukemia cell(K562),and solid tumor cells(PC-3 and Hep G2).Distinct cytokine/cytotoxicity responses of iNKT cells against different targets were investigated.Moreover,CD1 d levels in T-cell lymphomas and iNKT cell function in the patients were evaluated to explore the therapeutic potential of iNKT cells in T-cell lymphomas.1.Enrichment of human iNKT cells and varied iNKT cell responses against different antigen presenting cellsPBMCs were separated from peripheral blood samples of healthy donors,and iNKT cells were expanded by ?-Gal Cer stimulation.Enrichment of iNKT cells were achieved through FACS or MACS sorting with PBS57/CD1 d tetramer.Primary iNKT cells and expanded iNKT cells were stimulated with T cells,B cells or monocytes and analyzed for cytokine production by Cytometric Bead Array.Expanded iNKT cells exhibited enhanced cytokine production than primary iNKT cells.Both B cells and monocytes were able to stimulate them to secret significant levels of IFN-? and TNF-?.Monocytes were the most potent activators for human iNKT cells and they were responsible for ?-Gal Cer-induced iNKT cell expansion in peripheral blood mononuclear cells.The low levels of IL-4 and IL-10 suggested the Th1-biased response of human iNKT cells.It was worthy to note that neither primary nor ?-Gal Cer expanded iNKT cells produced detectable cytokines in response to T cells.Collectively,our results suggested the differential cytokine production of human iNKT cells in response to different antigen presenting cells.2.Human iNKT cells exhibited differential cytokine/cytotoxicity responses against different tumor cellsIn this section,a panel of tumor cells were selected as targets,including Tlymphoma cells(Jurkat),B-lymphoblastioid cells(721.221),myeloid leukemia cells,and solid tumor cells(PC-3 and Hep G2).Among those cells,only Jurkat cells had detectable CD1 d expression on the cell surface.To minimize the effects of differential CD1 d levels,all cells used here were transfected with human CD1 d.The enriched human iNKT cells secreted proper amounts of IFN-? and TNF-? against the solid tumor cells and some hematopoietic cells(721.221 and K562).Meanwhile,human iNKT cells exhibited extensive but varied cytotoxicity to different targets.The cytotoxicity of iNKT cells against Jurkat-CD1 d cells was strongest and relied mainly on the Fasinduced cell apoptosis,while the cytotoxicity of iNKT cells against other cells were mainly relied on perforin and granzyme B pathway.All the responses were CD1 d dependent as iNKT cells show no or lower response against CD1 d untransfectants or CD1 d blocked Jurkat cells.Collectively,the results indicated the differential cytokine/cytotoxicity responses of human iNKT cells against different targets.In Jurkat cell case,human iNKT cells showed more intensive cytotoxicity and less efficient cytokine production.3.The functional status of iNKT cells and its therapeutic potential in T cell lymphomaBlocking of Fas/Fas L interaction by anti-Fas or anti-Fas L antibody significantly increased the antitumor cytokine production and inhibited the cytotoxicity of iNKT cells stimulated by Jurkat cells,suggesting Fas/Fas L interaction was responsible for the imbalanced cytokine/cytotoxicity responses.Exogenous IL-2 helped to increase the IFN-? production and retained the strong cytotoxicity of iNKT cells against Jurkat cells.After analyzing CD1 d expression in T-cell lymphoma biopsies,we found elevated CD1 d expression in lymphoma tissues.Moreover,co-localization of Fas and CD1 d was also occasionally found.However,defects in frequency and function of circulating iNKT cells were observed in the patients,which could be partly rescued by exogenous IL-2.Collectively,the Fas/Fas L-dependent aberrant iNKT cell responses and the reversibility of the defects suggest the distinct iNKT cell manipulation of CD1d-and Fas-bearing T cell malignancies.Conclusion:Our results highlight differential iNKT cell responses against different targets and the imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells.Moreover,elevated CD1 d and Fas levels in T-cell lymphomas as well as defects in iNKT cells were found in the patients.The Fas/Fas L-dependent aberrant iNKT cell responses and the reversibility of the defects suggest the distinct iNKT cell manipulation in T cell malignancies.Original points in this study:1.iNKT cell exhibited imbalanced cytokine/cytotoxicity responses against Jurkat cells,which was CD1 d dependent and relied mostly on Fas/Fas L interaction.2.Elevated CD1 d and Fas levels as well as defects in iNKT cells of T-cell lymphoma patients were found.Exogenous IL-2 helped to rescue the defects of iNKT cells from the patients.