In 2000, the importance of iNKT cells in tumor immunosurveillance and initiation of the antitumor immune response was demonstrated using a carcinogen-induced tumor model in mice that had various lymphocyte subsets knocked out by gene targeting or depletion (68). Both CD28 and CD40 are needed to spur an effective antitumor immune response after CAGL114 -GalCer injection (29, 30, 69). positively and negatively influence iNKT cell activation and function and skew the immune response (Signal 2). This study will review the background of iNKT cells and their co-stimulatory requirements for general function and in antitumor immunity. We will explore the impact of monoclonal antibody administration for both blocking inhibitory pathways and engaging stimulatory pathways on iNKT cell-mediated antitumor immunity. This review will highlight the incorporation of co-stimulatory molecules in antitumor dendritic cell vaccine strategies. The use of co-stimulatory intracellular signaling domains in chimeric antigen receptor-iNKT therapy will be assessed. Finally, we will explore the influence of innate-like receptors and modification of immunosuppressive cytokines (Signal 3) on cancer immunotherapy. (39). Further studies are needed to address these disparities found in the literature to determine the effects of GITR on iNKT cell activation. OX40 (CD134), a TNFRSF member, is expressed on iNKT cells and interacts with OX40L on APCs but the outcome of this interaction is debated. In the pancreas, the OX40:OX40L interaction between iNKT cells and plasmacytoid DCs during LCMV infection, tested using neutralizing antibodies, induces IFN-/ production by the DCs and dampens the adaptive immune response to avoid tissue damage (40). By contrast, stimulation of OX40 with an agonistic monoclonal antibody on liver-resident iNKT cells results in caspase-1-dependent pyroptosis and release of inflammatory cytokines that cause tissue injury (41). In a tumor model, iNKT cell expansion and IFN- production are enhanced by upregulation of OX40L on DCs (42). OX40 is stereotypically thought of as a stimulatory co-receptor, but its role in iNKT cell responses is unclear and may be tissue specific. CD155, a member of the immunoglobulin superfamily, is expressed on iNKT cells and interacts with CD226, CD96, and TIGIT. CD155 blockade or knockout increases NKT1 cells and decreases MI-1061 both NKT2 and NKT17 cell generation during development in Balb/c MI-1061 and C57BL/6 mice (43). Its effect on iNKT cell activation and cytokine production has not been published. There are three different T cell immunoglobulin mucin (TIM) receptors expressed by iNKT cells (TIM-1, 3, and 4), and they have differing effects on iNKT cell activation. TIM-1 engagement on iNKT cells by monoclonal antibodies suppresses Th1 responses but enhances Th2 responses in both and models (44). Conversely, TIM-1 engagement by phosphatidylserinea marker of apoptosisenhances iNKT cell MI-1061 activation, proliferation, and cytokine production (45). In a nonalcoholic fatty liver disease model, TIM-3 is shown to control liver-resident iNKT cell homeostasis with direct TIM-3 signaling inducing apoptosis and indirect signaling from IL-15, produced by TIM-3 stimulated Kupffer cells, promoting iNKT cell proliferation (46). TIM-3 is classically an inhibitory receptor and is upregulated on human iNKT cells in chronic viral infections (47). TIM-4 is expressed but does not have an effect on iNKT cell development or function (48). The effects of TIM-1 and TIM-3 need to be further assessed in iNKT cell biology. B and T lymphocyte attenuator (BTLA), a member of the CD28 family that interacts with B7-H4, is an inhibitory co-receptor that is expressed on iNKT cells. Thus far, it has only been examined in ConA-induced hepatitis with both studies demonstrating that BTLA knockout increases iNKT cell cytokine production and exacerbates hepatitis (49, 50), indicating an inhibitory role of BTLA in iNKT cell function. Although these results align with the role of BTLA in conventional T cells, more research is needed to assess the role of BTLA in other immune models. Lymphocyte activation gene (LAG)-3, a member of the immunoglobulin superfamily that interacts with MHC class II, is induced on iNKT cells after activation. It has an inhibitory affect with overexpression resulting in inhibition of proliferation due to cell cycle arrest (51). LAG-3 is upregulated on human iNKT cells in chronic viral infection and is associated with decreased cytokine production (52). These inhibitory effects are consistent with the effects of LAG-3 in conventional T cells. Programmed death (PD)-1, a member of the CD28 family, is constitutively expressed on iNKT cells.