NKT cells suppress EBV-associated tumors

EBV, a first discovered human tumor virus, is considered to be one of the most successful human pathogen. It is involved in the development of many malignancies, such as Hodgkin's lymphoma and nasopharyngeal carcinoma in immunocompromised individuals. Majority of the individuals infected with EBV remain symptom free. Thus, it is believed that EBV has co-evolved with the human immune system to a peaceful co-existence.

It is known that natural killer T cells (NKT) are a special type of T cells that express NK lineage receptors including CD1d. They respond to glycolipid antigen presented by CD1d. CD1d is mainly expressed on antigen presenting cells (APCs, dendritic cells, macrophages and B cells). Thus, NKT cells primarily interact with APCs rather than tissues. Upon activation, these NKT cells release both Th1 and Th2 cytokines. However, it is present not known whether there are subsets of NKT that specialize in Th1 and Th2 cytokine production. Furthermore, how these cytokines lead to a regulated immune response in vivo is not known. Earlier studies have suggested that cytokine profile of NKT cells might be influenced by quality of TCR signal. The quality of TCR signal is in turn dependent on different antigens.

The present paper is from the lab of Dr. Yuling He, Wuhan University School of Medicine, China. In an earlier paper, the authors observed that CD8+ NKT cells drive syngenic T cells into a Th1-bias response to suppress EBV-associated malignancies and that IL-4-biased CD4+ NKT cells do not affect T cell cytotoxicity. In the present work, the authors sought to determine the mechanism of the EBV-induced CD8+ NKT cell response to EBV-associated malignancies.

Source: Xiao W, Li L, Zhou R, Xiao R, Wang Y, Ji X, Wu M, Wang L, Huang W, 

Zheng X, Tan X, Chen L, Xiong T, Xiong J, Jin Y, Tan J, He Y. EBV-induced 

human CD8(+) NKT cells synergise CD4(+) NKT cells suppressing EBV-associated

tumours upon induction of Th1-bias. Cell Mol Immunol. 2009 Oct;6(5):367-79.

PubMed PMID:19887050.

RESULTS:

1. Circulating mDC1cells from patients with EBV-associated malignancies impaired IFN-gamma production of CD8+ NKT cells: In another previous study, the authors observed that the frequencies of total and CD8+ NKT cells in PBMCs from Latent EBV infection (LEI) and control negative subjects (CN) were significantly higher than those from Hodgkin lymphoma (HL) patients and nasopharyngeal carcinoma (NPC) patients. To determine the interaction of CD8+NKT cells with APCs, the authors first determined frequencies and CD1d expression of circulating DCs, which included myeloid dendritic cells 1 and 2 (mDC1 and mDC2) and plasmacytoid dendritic cells (pDC), in PBMCs from NC and LEI subjects , and HL and NPC patients by staining with different markers, BDCA1 for mDC1, BDCA2 for pDC and BDCA3 for mDC2 and CD1d. The frequencies of mDC1 cells were comparable among HL and NPC patients, and LEI and NC subjects. CD1d was found to be predominant expressed on mDC1 cells than mDC2 and pDC cells. The expression level of CD1d was comparable on LEI and NPC subjects and HL and NPC patients. The authors next determined cytokine expression by CD4+ and CD8+ NKT cell lines. These cell lines were generated from PBMC from LEI subjects. Type 1 mDCs were purified from LEI and NC subjects and HL and NPC patients. These mDCs were than co-cultured with CD4+ or CD8+ NKT cell lines in the presence of alphaGalCer or alphaGalCer with CD1d mAb for 1 hr. The mDCs were then re-purified using staining with alpha-GalCer loaded CD1d tetramer. Then, the expression of IFN-gamma and IL-4 was assessed by Q-PCR. Both IFN-gamma and IL-4 were detected in CD4+ and CD8+ NKT cell lines cultured with mDCs from different groups of patients and subjects. The authors observed that both NKT cell lines from LEI and CN subjects highly expressed IFN-gamma upon stimulation with alphaGalCer. Although, not much effect was seen on IL-4 expression among various patients and subjects. Furthermore, using the same set of experiments the authors determined IFN-gamma and IL-4 expression by using ELISA. They observed that prestimulation with alphaGalCer caused significant increase in expression level of IFN-gamma in CD8+ NKT cell lines incubated with PBMCs from LEI and CN than from HL and NPC patients. IL-4 levels were in CD4+ NKT cells cultured with PBMCs from both patients and subjects. These results indicate that circulating mDC1 cells of HL and NPC patients cause a decrease in IFN-gamma production by CD8+ NKT cell line and have no effect on IL-4 production.
   
2. EBV induced CD8+ NKT cells promote a Th1 biased response that suppresses EBV-associated tumor cells in vitro: The authors observed in one of their early study that CD4+ NKT cells in PBMCs from HL and NPC patients and LEI and CN subjects produced similar low levels of IFN-gamma and high levels of IL-4, IL-13, IL-10 and TGF-beta1. In contrast, CD8+ NKT cells from control subjects produced expressed high levels of IFN-gamma and IL-2 but low levels of IL-4, IL-13, IL-10, and TGF-beta1. They also found that CD8+ NKT cells from LEI and CN subjects efficiently killed alpha-GalCer loaded EBV-associated HL and NPC tumor cell lines but not non-tumor cells. CD4+ from various patients and subjects either did not show such killing or showed very weak toxicity to both tumor and non-tumor cells. In the same study, the authors also observed that EBV-induced human NKT cells rapidly responded to EBV-associated tumor cells by secreting cytotoxic cytokines such as IL-10 and IFN-gamma. In the present study, the authors tried to verify whether this killing was specific, the authors first demonstrated present of EBV through the use of southern blot and QPCR in these cell lines. Thus, EBV-genes, LMP1, EBNA1, BZLF1, BALF2, and RAZ were examined in human EBV-associated tumor cell lines and non-tumor cell lines. The authors were able to detect these viral genes and their mRNA transcripts in tumor cell lines but not in non-tumor cell lines. The next task was to determine the mechanism by which NKT cells inhibit EBV-associated malignancies in vivo. Thus, the authors established hu-thym-SCID chimaeras. These chimaeras were implanted with human Hodgkin´s-derived EBV-associated B-cell lymphoma cell lines (LBC) or NPC. The animals were sacrificed 12 weeks post tumor implantation. Various organs including thyms, liver spleen and peripheral blood were collected and single cell suspensions were prepared. These cells were stimulated with alphaGalCer and cytokine expression (IL-4, IL-10 and IFN-gamma) was determined, using cytokine Ab, CD4 Ab, CD8 Ab and CD1d tetramer. The authors observed that a large number of CD8+ NKT cells expressed IFN-gamma and IL-2, but few cells expressed IL-4, IL-10 and IL-13. In contrast, CD4+ NKT cells expressed moderate levels of IL-4, IL-10 and IL-13 and low levels of IFN-gamma.
   
3. EBV- induced CD4+ NKT cells synergized with CD8+ NKT cells to promote a Th1 bias response against EBV-associated tumors: In their earlier work, the authors observed some kind of synergy between EBV-exposed thymic CD4+ and CD8+ NKT cells to suppress EBV-associated malignancies and prolong animal survival. To further investigate the mechanism of this synergistic effect, the SCID mice were adoptively transferred iv with different combinations of immune cells (CD4+/CD8+ NKT cells or both and CD3+CD56-Cd161- T cells) purified from EBV-exposed hum-thym-SCID chimaeras. After, three days these chimaeras were re-challenged with EBV. The scientists observed that in chimaeras transferred with CD4+ NKT cell alone, the re-challenge with EBV caused inhibition in proliferation of CD3+ T cell in comparison to chimaeras transferred with CD8+ NKT cell alone. Interesting, the chimaeras transferred with both CD4+ and CD8+ NKT cells showed a vigorous proliferation of CD3+ T cells and a high frequency of IFN-gamma producing cells.
   

To be continued……………….