Neurofibromin 1 Impairs Natural Killer T-Cell-Dependent Antitumor Immunity against a T-Cell Lymphoma

Neurofibromin 1 (NF1) is a tumor suppressor gene encoding a Ras GTPase that negatively regulates Ras signaling pathways. Mutations in NF1 are linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. In terms of antitumor immunity, CD1d-dependent natural killer T (NKT) cells play an important role in the innate antitumor immune response. Generally, Type-I NKT cells protect (and Type-II NKT cells impair) host antitumor immunity. We have previously shown that CD1d-mediated antigen presentation to NKT cells is regulated by cell signaling pathways. To study whether a haploinsufficiency in NF1 would affect CD1d-dependent activation of NKT cells, we analyzed the NKT-cell population as well as the functional expression of CD1d in Nf1+/− mice. Nf1+/− mice were found to have similar levels of NKT cells as wildtype (WT) littermates. Interestingly, however, reduced CD1d expression was observed in Nf1+/− mice compared with their WT littermates. When inoculated with a T-cell lymphoma in vivo, Nf1+/− mice survived longer than their WT littermates. Furthermore, blocking CD1d in vivo significantly enhanced antitumor activity in WT, but not in Nf1+/− mice. In contrast, a deficiency in Type-I NKT cells increased antitumor activity in Nf1+/− mice, but not in WT littermates. Therefore, these data suggest that normal NF1 expression impairs CD1d-mediated NKT-cell activation and antitumor activity against a T-cell lymphoma.

Neurofibromin 1 (NF1) is a tumor suppressor gene encoding a Ras GTPase that negatively regulates Ras signaling pathways. Mutations in NF1 are linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. In terms of antitumor immunity, CD1d-dependent natural killer T (NKT) cells play an important role in the innate antitumor immune response. Generally, Type-I NKT cells protect (and Type-II NKT cells impair) host antitumor immunity. We have previously shown that CD1dmediated antigen presentation to NKT cells is regulated by cell signaling pathways. To study whether a haploinsufficiency in NF1 would affect CD1d-dependent activation of NKT cells, we analyzed the NKT-cell population as well as the functional expression of CD1d in Nf1 +/− mice. Nf1 +/− mice were found to have similar levels of NKT cells as wildtype (WT) littermates. Interestingly, however, reduced CD1d expression was observed in Nf1 +/− mice compared with their WT littermates. When inoculated with a T-cell lymphoma in vivo, Nf1 +/− mice survived longer than their WT littermates. Furthermore, blocking CD1d in vivo significantly enhanced antitumor activity in WT, but not in Nf1 +/− mice. In contrast, a deficiency in Type-I NKT cells increased antitumor activity in Nf1 +/− mice, but not in WT littermates. Therefore, these data suggest that normal NF1 expression impairs CD1d-mediated NKT-cell activation and antitumor activity against a T-cell lymphoma.
Keywords: neurofibromin 1, cD1d, natural killer T cells, T-cell lymphoma, antitumor immunity inTrODUcTiOn Neurofibromatosis type 1 is an autosomal-dominant disorder caused by a mutation in a tumor suppressor gene encoding the protein neurofibromin 1 (NF1) (1), affecting 1 in 3,500 individuals worldwide. NF1 is a p21 ras (Ras) guanosine triphosphatase (GTP)-activating protein (GAP). It catalyzes the hydrolysis of Ras-GTP, thus negatively regulating multiple Ras-dependent cellular signaling pathways (1). Mutations in NF1 are associated with many diseases, including hematopoietic cancers such as myeloid leukemia and diffuse plexiform neurofibromas (2). Extensive studies from human tissue analyses and mouse models have discovered that loss of heterogyzosity (LOH) of NF1 in Schwann cells and a heterozygous NF1 microenvironment are both important for the formation of neurofibromas (3,4). LOH may also explain the localized formation of tumors in patients with neurofibromatosis type 1 (1).
Ras-dependent signaling pathways have been shown to be important for αβ T-cell positive selection (5). Because NF1 is a negative regulatory GAP and highly expressed in leukocytes (6), the absence of NF1 may affect T-cell development. An Nf1 −/− mutation is embryonic lethal (1). Therefore, the method of Nf1 −/− fetal liver reconstitution to immune-deficient mice, such as Rag1 KO mice, has been used to study T-cell development in the absence of NF1 (7). Although an nf1 deficiency in mice increases T-cell numbers in both thymus and spleen, it also causes impaired proliferation of T cells in response to in vitro stimulation (7). Moreover, antigen receptor-induced proliferation is also defective in NF1-deficient peripheral B cells (8), implicating a positive (but unknown) role for NF1 in regulating B and T-cell receptor (TCR)-induced proliferation. An earlier study indicated that NF1 promotes thymocyte positive selection, but has no effect on negative selection (9). Increasing evidence also suggests that NF1 may function in other cellular processes besides negatively regulating Ras function (10). For example, the Sec14-homology domain of NF1 is involved in forming a bipartite lipid-binding module, and possibly binds to cellular glycerophospholipid ligands (11). The loss of NF1 in Drosophila causes a reduction in body size, which is rescued by increasing cAMP protein kinase (PKA) signaling; this suggests that NF1 may also regulate the cAMP signaling pathway in a GAP-independent manner (12).
Natural killer T (NKT) cells express both natural killer (NK) and T-cell markers. Unlike conventional T cells which recognize peptide antigens presented by MHC class I and II molecules, NKT cells are activated by lipid antigens presented by the MHC class I-like molecule, CD1d. CD1d-deficient mice lack NKT cells and NKT-cell development requires positive selection in the thymus, similar to conventional T-cell development (13). Ras/mitogen-activated protein kinase (MAPK) signaling pathways, which are important for αβ T-cell positive selection (5), have also been shown to be critical for NKT-cell development (14). Furthermore, previous work from our laboratory has demonstrated that stimulation of MAPK pathways affects CD1d-mediated antigen presentation (15,16). We have found that activation of the p38 pathway inhibits, whereas activation of ERK pathway increases, CD1d-mediated antigen presentation to NKT cells, likely through regulating the trafficking of CD1d molecules in antigen-presenting cells (15). In line with this, we reported that anthrax toxin inhibits CD1d-mediated antigen presentation by targeting the ERK pathway (16).
Based on TCR usage, NKT cells can be divided into two groups: Type-I (invariant) and Type-II (other CD1d-restricted) NKT cells. Type-I NKT (also called iNKT) cells express an invariant TCR α-chain rearrangement (Vα14Jα18 in mice and Vα24Jα18 in humans) that is associated with β-chains of limited diversity (Vβ8.2, Vβ7, and Vβ2 in mice; Vβ11 in humans). The glycolipid α-galactosylceramide (α-GalCer or PBS57), originally derived from a marine sponge, has been shown to be a specific activator of iNKT cells in a CD1d-dependent manner (17,18). Type-II NKT cells are less well-defined, due to a paucity of ligands identified that are recognized by these NKT cells (19,20). However, by studying CD1d-deficient (lacking both Type-I and Type-II NKT cells) and Jα18-deficient mice (lacking only Type-I NKT cells), it is believed that Type-II NKT cells are similar to T regulatory cells (Tregs) and are mostly immunosuppressive (21). In line with this idea, Type-II NKT cells have been shown to impair tumor immunosurveillance in a CD1d-dependent manner (22).
In the current study, we asked whether NF1, a negative regu lator of Ras/MAPK pathways, impacts CD1d-dependent antitumor activity by NKT cells. Because an Nf1 −/− mutation is embryonic lethal, a haploinsufficient (Nf1 +/− ) mouse model is commonly used for the study of NF1 function in vivo. We analyzed NKT-cell activity as well as the functional expression of CD1d in Nf1 +/− mice, in order to determine whether a haploinsufficiency in NF1 would affect the CD1d/NKT-cell axis in the context of NKT-cell-mediated antitumor activity.

Flow cytometry
Thymocytes and splenocytes were harvested using standard procedures. Liver mononuclear cells (LMNCs) were harvested as described previously (25). To obtain bone marrow-derived dendritic cells (BMDCs), bone marrow cells obtained from mouse femurs and tibias were cultured in the presence of IL-4 (10 ng/mL) and GM-CSF (10 ng/mL) for 7 days. For flow cytometry analyses, single-cell suspensions of all indicated cell types were prepared, and 1 × 10 6 cells were incubated at 4°C for 30 min with various mAb as indicated. The cells were washed three times with HBSS containing 0.1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA). All cells were fixed with 1% paraformaldehyde in PBS and analyzed on a FACSCalibur or LSRII (Becton Dickinson, San Jose, CA, USA).

Western Blot analysis
Thymocytes and splenocytes were lysed, separated on a 10% SDS-PAGE gel and then transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Billerica, MA, USA). The blot was then probed with phospho-JNK1/2 or ERK1/2-specific antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA), and developed using chemiluminescence prior to exposure on film. The same membrane was then stripped and reprobed with total JNK1/2-or ERK1/2-specific antibodies (Cell Signaling Technology Inc.). Images were quantified using ImageJ (1.37v; National Institutes of Health, Bethesda, MD, USA).
statistics Graphs were generated and statistics calculated using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). The mean of triplicates of a representative assay is shown with error bars representing the SEM, using Student's t-test analyses. For the statistical analysis of survival rate, the log-rank test was performed. A p-value < 0.05 was considered significant. resUlTs increased JnK and erK activation in Nf1 +/− Mice Previous reports have shown increased Ras-GTP levels in unstimulated thymocytes from Nf1 +/− mice when compared with WT mice (7). In our study, we also observed elevated ERK phosphorylation in splenocytes and thymocytes when they were stimulated with Phorbol 12-myristate 13-acetate ( Figure 1A; Figures  comparable inKT-cell Population in WT and Nf1 +/− Mice Previous studies have suggested that an NF1 deficiency increases the number of immature and mature conventional T cells in vivo, but reduces cell proliferation in response to TCR and IL-2 stimulation in vitro (7). NF1 promotes thymocyte positive selection, a process that is also required for NKT-cell development (9,28). To determine if an NF1 deficiency affects iNKT-cell development, we compared the iNKT-cell populations in thymus, spleen, and liver from Nf1 +/− mice to those from WT littermates. We found there were comparable levels of iNKT cells in WT and Nf1 +/− mice (Figure 2), suggesting that a haploinsufficiency in NF1 has a minimal effect on NKT-cell development and their numbers in the periphery.
lower cD1d expression on BMDcs from Nf1 +/− Mice Although a haploinsufficiency in NF1 did not seem to affect iNKT-cell development, we found that BMDCs from Nf1 +/− mice expressed lower levels of CD1d (but similar amounts of  MHC class I and II) on the cell surface compared with WT BMDCs (Figure 3A; Figure S1 in Supplementary Material). Furthermore, similar to the reduced CD1d expression observed in Nf1 +/− BMDCs, there was also a significant decrease in the splenic B220 + CD21 hi CD23 int CD1d hi population in Nf1 +/− mice (Figures 3B,C). These cells express a high level of CD21 and low to intermediate levels of CD23, suggesting they are marginal zone B (MZB) cells. We also analyzed DCs (MHC II + CD11c + ) and macrophages (MHC II + F4/80 + ) for CD1d expression, but there were no differences between WT and Nf1 +/− mice (data not shown). Although BMDCs from Nf1 +/− mice expressed less CD1d on their surface, they were similar to WT BMDCs in their ability to activate both Type-I and Type-II NKT-cell hybridomas ( Figure 3D). Interestingly, thymocytes from both WT and Nf1 +/− mice express similar levels of CD1d and have a comparable ability in stimulating NKT cells ( Figure S8 in    LMNCs from Nf1 +/− mice were more responsive to CD1dmediated antigen presentation than those from their WT littermates (Figures 4A,B). The addition of an anti-CD1d antibody blocked the activation of iNKT cells (Figure 4C), demonstrating that the NKT-cell activation was CD1d-specific. In contrast to antigen-specific activation, when LMNCs from Nf1 +/− mice were stimulated with anti-CD3 and anti-CD28 antibodies, they secreted a similar level of cytokines as their WT littermates ( Figure S2 in Supplementary Material). Thus, these data demonstrate that iNKT cells (but not conventional T cells) from Nf1 +/− mice are more activated than those from WT littermates upon exogenous lipid Ag stimulation in vitro.
To determine whether the NF1 haploinsufficiency would affect NKT-cell function in vivo, we injected the iNKT-cell ligand α-GalCer to Nf1 +/− mice and their WT littermates. At different time points, sera were harvested and circulating IL-4 and IFNγ were measured. Nf1 +/− mice produced similar levels of these cytokines as their WT littermates ( Figure S3 in Supplementary Material). Therefore, these results suggest that iNKT cells are functionally normal in Nf1 +/− mice.

Nf1 +/− Mice Bearing rMa/s Tumors surviving longer Than WT Mice
Because we observed decreased CD1d expression but increased iNKT-cell activity in vitro in Nf1 +/− mice, it was important to determine what the impact of a haploinsufficiency of NF1 would be on CD1d-dependent antitumor activity. Previous reports have suggested that Nf1 +/− mice are predisposed to developing multiple cancers after 1 year of age, and thus have a shorter life span compared with WT mice (1). To address this question, we inoculated Nf1 +/− mice and their WT littermates with the RMA/S T cell lymphoma transfected with an empty vector or the murine cd1d1 cDNA (23). They were then observed for tumor incidence and survival rate. Surprisingly, Nf1 +/− mice had a better survival rate and longer median survival time (MST) than their WT littermates when they were challenged with either CD1d-positive or CD1d-negative RMA/S tumor cells although, in this experiment, the difference between Nf1 +/− and WT mice was not statistically significant ( Figure S4 in Supplementary Material). Thus, in terms of survival, the antitumor activity in Nf1 +/− mice exceeds that of their WT littermates.
Blocking cD1d In Vivo enhancing antitumor activity in WT But not Nf1 +/− Mice Our previous studies have shown enhanced survival in CD1ddeficient mice when they were inoculated with RMA/S T-cell lymphoma cells (23). It was possible that the reduced CD1d expression in Nf1 +/− mice altered host antitumor activity. To test this hypothesis, Nf1 +/− and WT mice were treated with an anti-CD1d antibody or isotype control at various times before and after they were inoculated with RMA/S-CD1d cells. Blocking CD1d expression by a CD1d-specific antibody significantly enhanced antitumor activity in WT mice. The CD1d-specific antibody treatment in Nf1 +/− mice also increased the survival rate of tumor-bearing mice. Although reproducible, the difference was not statistically significant in this experiment (Figures 5A-C). In a parallel experiment, CD1d was also genetically deleted from Nf1 +/− mice by back-crossing Nf1 +/− mice to CD1d1 −/− mice. Thus, Nf1 +/− , CD1d1 −/− , Nf1 +/− /CD1d1 −/− , and WT mice were inoculated with RMA/S-CD1d cells, WT mice had the lowest survival rate among these four different strains of mice (Figures 5D,E). As was observed when CD1d was blocked by antibody in vivo, deleting CD1d genetically from WT (but not Nf1 +/− ) mice significantly enhanced their survival rate ( Figure 5F). Therefore, reduced CD1d expression in Nf1 +/− mice very likely contributes to host antitumor activity in this model system.

DiscUssiOn
Neurofibromatosis type 1 is a disease caused by mutations in the NF1 gene, a negative regulator of the Ras signaling pathway. Elevated Ras/ERK activation has been reported in Nf1 +/− mice and reconstituted NF1 −/− mice, as well as cells from NF1 patients (7,30). Hyperactivation of the Ras/ERK pathway was also confirmed in the current study by the detection of increased phospho-ERK in the thymus and spleen of Nf1 +/− mice. Although hyperactivation of the Ras/ERK pathway has been reported to be associated with a defect in NKT-cell development (31), we did not observe any defect in NKT-cell development in Nf1 +/− mice. Instead, our work has demonstrated that NKT cells from Nf1 +/− mice actually have enhanced CD1d-dependent activation, compared with those from their WT littermates. We observed similar levels of circulating cytokines in Nf1 +/− mice after in vivo treatment with α-GalCer, even though APCs from Nf1 +/− mice expressed lower levels of CD1d compared with their WT littermates. This may be explained by the increased responsiveness of NKT cells in vitro.
On the other hand, the reduced CD1d expression found in BMDCs from Nf1 +/− mice suggests that NF1 positively regulates CD1d expression. It is worthwhile to point out that this effect might be due to in vitro cultures, as we did not observe a difference in CD1d expression in splenic DCs. It is well-known that NF1 is a negative regulator of the Ras/MAPK pathway (1). We not only observed hyperactivation of the Ras/ERK pathway but also detected elevated Ras/JNK pathway activation in the thymus and spleen of Nf1 +/− mice. Daginakatte et al. (32) also reported increased JNK activation in Nf1 +/− microglia cells, but not Nf1 −/− astrocytes, which likely contributed to the increased proliferation of Nf1 +/− microglia cells in that study. Consistent with our findings, they also did not observe increased p38 activation in these tissues (32). Our results are particularly interesting because we found that blocking the JNK pathway increases (and activation of JNK decreases) CD1d-mediated antigen presentation (Liu et al., manuscript in preparation). In contrast, we previously reported that elevated ERK activation enhances CD1d-mediated antigen presentation during a viral infection by regulating intracellular CD1d trafficking (15). Thus, it is very likely that the reduction of CD1d expression in Nf1 +/− APCs is an outcome of the combined regulation of multiple signaling pathways as a consequence of the NF1 haploinsufficiency. It is worthwhile to mention that we did not observe any difference in CD1d recycling ( Figure S5 in Supplementary Material) or CD1d distribution by confocal microscopy between BMDCs from Nf1 +/− or WT littermates (data not shown). This suggests that the reduced CD1d expression on the cell surface of Nf1 +/− APCs is not due to modified CD1d intracellular trafficking caused by an NF1 haploinsufficiency. Marginal zone B cells, which express a high level of CD1d, are reduced in Nf1 +/− mice. Moreover, it is known that Notch2 is indispensable to MZB development (33). A recent report suggests that Notch is the effector of NF1 in neurological tissue (34). Thus, a haploinsufficiency of NF1 may affect Notch2 and thereby alter MZB development. However, NF1 may have other unknown functions, not just as a Ras-GAP. A 2,839-aminoacid protein, NF1 contains two major functional domains: a Ras-GAP-related domain (Ras-GRD) and a Sec14-interactive domain. The Sec14-interactive domain is involved in forming a bipartite lipid-binding module and possibly binds to a cellular glycerophospholipid ligand (11). Further investigations are needed to determine how NF1 regulates CD1d expression.
We observed increased antitumor activity in Nf1 +/− mice compared with their WT littermates. Although the differences were not always statistically significant, Nf1 +/− mice consistently survived longer than their WT littermates. Treatment with a CD1d-specific mAb has shown to protect mice from tumor metastasis by several groups (22,35,36). It has been suggested that the CD1d mAb may block the activation of "immunosuppressive" Type-II NKT cells (22). Crosslinking CD1d by a specific mAb can also activate antigen-presenting cells, such as DCs, to produce the proinflammatory cytokines IL-12 and IFN-γ (35,36). Reduced CD1d expression in Nf1 +/− mice may be somewhat like CD1d-deficient mice (or CD1d mAb-treated mice), in that there is a dysfunction in (or reduced activity of) "immunosuppressive" Type-I NKT cells; this could explain the increased antitumor activity observed in Nf1 +/− mice. It is also consistent with the findings that blocking CD1d in vivo enhanced antitumor activity in WT but Nf1 +/− mice. In the current study, WT and Jα18 −/− mice died at similar rates and CD1d1 −/− mice survived longer than WT mice. The results suggest that Type-I NKT cells have little impact on antitumor activity whereas Type-I NKT cells are immunosuppressive in this model system (Figure 7). Nf1 +/− /Jα18 −/− mice survived much longer than Nf1 +/− littermates, suggesting that NF1-haploinsufficient Type-I NKT cells, although demonstrating increased activity in vitro, suppressed antitumor activity in vivo.
Type-I NKT cells can directly destroy tumor cells, especially those expressing CD1d on their surface, by performing cytolysis via perforin, granzyme B, Fas ligand (FasL), and TRAIL (37). Type-I NKT cells can also suppress the function of myeloid-derived suppressor cells (MDSC) and suppressive IL-10-producing neutrophils, to enhance antitumor immunity (20). Type-I NKT cells are capable of producing both Th1 and Th2 cytokines (38). The avidity and stability of antigen/TCR complex determines the type of cytokine production. Strong antigen/TCR interaction causes FigUre 7 | A working model illustrating that neurofibromin 1 (NF1) plays distinct roles in regulating the antitumor activity of Type-I and Type-II NKT cells in vivo. NF1 reduces the immunosuppressive activity of Type-I NKT cells, as NF1-haploinsufficient Type-I NKT cells suppress antitumor immunity. In contrast, NF1 expression upregulates CD1d levels and enhances the immunosuppressive activity of Type-II NKT cells. In line with this, a haploinsufficiency in NF1 causes a reduction in CD1d expression and decreases the immunosuppressive activity of Type-II NKT cells, augmenting antitumor immunity.
NKT cells to produce Th1 cytokines, whereas weak antigen/ TCR interaction results in Th2 cytokines from NKT cells (39). Th1-biased and IFNγ-producing Type-I NKT cells greatly boost antitumor immunity (20). On the other hand, Type-I NKT cells have been reported to be immunosuppressive by supporting Tregs and/or suppressing tumor-specific CD8 + T cells (40,41). In the current study, the reduced CD1d expression observed in Nf1 +/− mice may further cause Type-I NKT cells to become Th2-biased and thereby suppress the antitumor activity of CTL and NK cells.
In conclusion, NF1-haploinsufficient Type-I NKT cells are more immunosuppressive compared with WT Type-I NKT cells, through a currently unknown mechanism.
In contrast, Nf1 +/− /Jα18 −/− mice survived much longer than their Jα18 −/− littermates, indicating that Type-II NKT cells in Nf1 +/− mice are not as immunosuppressive as WT Type-II NKT cells. Nf1 +/− /Jα18 −/− mice survived at a similar rate as Nf1 +/− /CD1 −/− mice, further confirming that Type-II NKT cells in Nf1 +/− mice are not immunosuppressive. Our work suggests that NF1 is required for the immunosuppressive activity of Type-II NKT cells. The reduced CD1d expression in Nf1 +/− mice may functionally alter Type-II NKT cells, moving from suppressing to enhancing antitumor activity.
A recent publication has suggested that Tregs are also important in the balance of antitumor activity involving Type-I/Type-II NKT cells (29). We did not observe any changes in Tregs in our studies (data not shown). Further studies are needed to investigate tumor immunosurveillance by Type-I/Type-II NKT cells as well as Tregs in Nf1 +/− mice.
One question raised from our tumor challenge study is the identity of the effector cells that are responsible for removing the tumor cells in vivo. Because the RMA/S cell line is Tap-2 deficient, they express a very low level of MHC I molecules on their surface (42). Thus, the effector cells for eliminating RMA/S cells are unlikely to be CD8 + cytotoxic T cells. NKT cells have also been shown to exhibit cytotoxicity activity against CD1d + cells (23). However, because Nf1 +/− mice are more resistant to both CD1d + and CD1d − RMA/S cells, it is unlikely that the sole effector cells are NKT cells. Another population of cytotoxic cells that could play a role here are NK cells. RMA/S cells are highly susceptible to NK cell-mediated lysis (43). We speculate that Type-I and Type-II NKT cells may impact the function of NK cells in the RMA/S tumor model (Figure 7). On the other hand, the cytolytic activity of NK cells is also regulated by many signaling pathways (44). It has been reported that NK cells from vav-1 (a GEF)-deficient mice have reduced cytotoxicity (45), suggesting that NK cell activity may be impacted by changes in Ras/MAPK pathways. However, in the current study, we did not observe increased cytotoxicity by NK cells in Nf1 +/− mice ( Figure S6 in Supplementary Material). Of further interest, a recent report suggests that the inoculation of mice with RMA/S cells causes NK cell anergy and escape from antitumor immunity (46). Importantly, NK cell anergy only occurs in the tumor proximal environment and is likely due to impaired ERK activation downstream of activating receptors on NK cells (46). It is possible that NK cells in Nf1 +/− mice may be compensated for by reduced ERK phosphorylation and rescue of MAPK/ERK signaling in the tumor microenvironment; thus, Nf1 +/− mice would exhibit an increase in antitumor immunity and have enhanced survival. Further studies will be focused on how NF1 regulates the antitumor activity of NKT and NK cells in Nf1 +/− mice.
Neurofibromas are derived from a broad range of cells, including hyperproliferative Schwann cells, fibroblasts, mast cells and perineural cells (47). Loss of heterogyzosity of NF1 in Schwann cells and a heterozygous NF1 microenvironment are both important for the formation of neurofibromas (3). Schwann cells have been shown to express CD1d and can activate NKT cells to secrete anti-inflammatory cytokines (48). We speculate that the absence of NF1 in Schwann cells from NF1 patients may cause a deficiency in CD1d expression. It would be interesting to study the tumor immunosurveillance activity of Type-I and Type-II NKT cells within the neurofibroma microenvironment, where Schwann cells are NF1deficient. Further studies are necessary to understand the role of the CD1d/NKT-cell axis in NF1-dependent disease progression.
In summary, we have found reduced CD1d expression but increased antitumor activity in a haploinsufficiency model of NF1. This is likely due to reduced immunosuppressive activity by Type-II NKT cells, rather than by an increase in antitumor activity by Type-I NKT cells. The results support the hypothesis that NF1 regulates CD1d-mediated NKT-cell activation and consequent antitumor activity (Figure 7). Future work will focus on investigating how NF1 may regulate the antitumor activity of NKT cells. Our study may therefore provide mechanistic support to target NF1 to improve CD1d/NKT-cell-based immunetherapy.

eThics sTaTeMenT
All animal procedures were approved by the Indiana University School of Medicine's Institutional Animal Care and Use Committee.
aUThOr cOnTriBUTiOns RB and JL contributed to the concept and design of the paper. JL, RG, MK, and GR contributed to the acquisition of data. JL, RG,