The Investigation Of The Role Of Vav1 In The Suppressive Function Of Indoleamine-2,3-dioxygenase On The Activity Of Tumor Infiltrating T Lymphocytes

Purpose:This study intendes to investigate the relationship between the signal transducerVavl and the activity of tumor infiltrating T lymphocytes (TIL-T).Indoleamine-2,3-dioxygenase (IDO), a tryptophan catabolic enzyme, plays animportant role in immune escape through suppressing T-cell function. Since Vavlsignaling pathway regulates T cell homeostasis, this study was designed to test thehypothesis that IDO induces T-cell immunosuppression through inhibiting Vavlsignaling.Materials and Methods:Firstly, we investigate the expression of Vavl gene in TIL-T. Then theexpression of IDO in the tumors is detected by immunohistochemistry (IHC).Chinese hamster ovary (CHO) cells were stably transfected with human IDO(CHO/IDO). The transcription and expression of IDO gene were detected by RT-PCRand Western blot analysis, respectively. The enzyme activity of IDO was measuredusing Hitachi amino acid automatic analyzer. CD3~+ T cells were isolated from humanperipheral blood monouclear cells and sorted by CD3~+ microbeads in MACS. Afterco-culture of CHO/IDO cells with T cells in the presence or absence of an anti-CD3antibody which can activate T cell receptor (TCR) and/or 1-methyl-L-tryptophan(1-MT) which can inhibit IDO activity, T cell proliferation and apoptosis weredetermined. T cell total RNA and cellular protein samples were isolated for detectingVavl gene and protein expression and activate state as well as the activation of thedownstream signal transducers of Vavl by realtime-PCR and immunoprecipitation orimmunoblotting.Results:T cells isolated from some patients have low response to the stimuli, and can notproliferate normally. Moreover, the level of Vavl expression in these TIL-T is lowerthan the T cells isolated from PBMC.The positive rate of IDO expression is 27.3% in our experiment. The IDOexpression in tumor tissues is consistent with that in metastatic lymph nodes. In the IDO positive tissues, the levels of Vavl in TIL-T are lower than normal(P＜0.05).The expression of IDO in transfected CHO cells was identified by RT-PCR andWestern blotting. And the IDO transgenic CHO cells yielded a high enzyme activityand resulted in complete depletion of tryptophan from the culture medium. We foundthat IDO produced by these IDO-expressing CHO cells significantly inhibitedinterleukin (IL)-2 expression and proliferative response in T cells and increased theapoptosis of T cells. IDO suppressed Vavl mRNA and protein production in T cells.Furthermore, IDO inhibited TCR activation-induced Vavl phosphorylation, whichrepresents Vavl's activation state and the activation of the downstream signaltransducers of Vavl in T cells. These effects on T-cells induced by co-culture ofCHO/IDO can be were attenuated by 1-MT.Conclusions:The activities of TIL-T correlate with the expression of Vavl. Vavl expressionis defective in T cells in the microinviroment of some tumors.The low expression and activity of Vavl lead to the deficiency of IL-2 synthesis,and then T cells cannot proliferation normally in respond to stimuli.The inhibitory effects of IDO on T cell immune responses may be throughdown-regulating of Vavl protein expression and activation. These studies provideinsights into the mechanisms of immune escape induced by IDO and the therapeuticapplication of IDO inhibitors for cancer treatments.