**** 0.0001. GD3 CAR T cells are cytotoxic toward GD3-expressing focus on cells in vitro. Since TSC lesions overexpress cell surface antigen GD3, we pursued targeting the ganglioside by CAR T cells as a new approach for TSC treatment. single gene give rise to benign tumors in vital organs, associated with constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) (1). Clinical manifestations of TSC vary from skin discolorations to progressive development of tumors in multiple organs, including lungs, as in TSC-associated and sporadic LAM (2). Neurological symptoms include epilepsy and long-term memory impairment (3), TSC-associated neuropsychiatric disorders (4), and autism (5). The tumors are generally benign, growing slowly compared with full-blown cancer. However, tumors develop in any location, causing significant morbidity (6). TSC further predisposes patients to more aggressive tumors, including renal cell carcinoma (7). Thus, there is an unmet need for effective treatment modalities (8). There is currently no cure for TSC, though some treatments can combat symptoms. Rapamycin and derivative mTOR inhibitors have been deployed for patients with TSC. Rapalogs stabilize lung function, reduce skin lesions, and decrease the size of renal angiomyolipomas and lymphatic lesions (9). However, rapalogs are not cytotoxic to tumor cells. Moreover, resulting mTORC2 inhibition is linked to insulin resistance and a risk of type 2 diabetes (10). Treatment is also associated with increased risk of infections (11). These effects can ultimately reduce the quality of life and preclude patients from long-term treatment. Treatment withdrawal causes tumor regrowth in most patients (12). TSC is a monogenic disease (13), and tumor cells with biallelic or mutations generally do not develop further heritable changes in other genes. Compared with hypermutated malignancies, TSC-associated tumors exhibit a predictable phenotype (14). A gene product expressed specifically by cells with mutations may be targetable by immunotherapy. A critical step toward applying immunotherapy to benign tumors will thus be to identify target molecule(s) recognizable by the adaptive immune system, leaving healthy tissues intact. Ganglioside D3 MMP10 (GD3) is a membrane glycosphingolipid expressed during normal brain development and in several malignancies (15). We observed overexpression of GD3 in LAM, wherein mutations in or are responsible for tumor development (16). In LAM, however, multiple options for antigen-specific immune targeting exist as pulmonary lesions express melanogenic enzymes that are immunogenic in vitiligo and melanoma (17, 18). In TSC, the additional challenge remains to identify a target molecule expressed by lesions in every location, while engaging a therapeutic with the potential to overcome immune privilege (19). GD3 expression accelerates tumor growth (20). We propose that GD3 overexpression develops in response to platelet-derived growth factor (PDGF) stimulation of tests assuming equal variance among groups. Where 3 tissue sources were displayed, we performed a 1-way ANOVA (*** 0.001) followed by Holm-?dk multiple comparisons test to compare TSC-affected tissues with controls. In all quantifications, = 3; * 0.05, ** 0.01, *** 0.001. Graphs show individual values and mean Cinchonidine values SD. Since TSC lesions exhibit hyperactivation of mTORC1, we also assessed phospho-S6 expression as a downstream indicator of increased mTORC1 activity to locate the lesions (31). We found that tissues which expressed higher GD3 also had increased phospho-S6 expression, consistent with disease activity in TSC (32) (Figure 1C). Moreover, we also observed colocalization of phospho-S6 and GD3 in tissues obtained from patients diagnosed with TSC (Supplemental Figure 1B), suggesting that GD3 expression is associated with mTORC1 activation in TSC lesional cells (33). Taken together, this analysis demonstrates a greater abundance of GD3 expression in TSC tumor tissues, suggesting the plausibility of a GD3-targeted treatment of TSC. TSC does not cause significant changes in NKT cell infiltration. Since GD3 can be cross-presented by antigen-presenting cells (APCs) and induce iNKT cell responses in a melanoma mouse model (34), we examined NKT cell infiltration in the microenvironment of TSC patient tissues. We also quantified (CD3 expressing) T cells and (CD56 expressing) NK cells while examining (CD3/CD56 double positive) NKT cell and invariant TCR coexpression in tissues from both healthy donors and patients with TSC (= 3). We found similar numbers of CD3+, CD56+, and CD3/CD56 double-positive cells in brain, kidney, skin, and lung Cinchonidine tissues from patients with TSC compared with control tissues (Figure 1D), composed of similar percentages of Cinchonidine iNKT cells as determined by coexpression of the invariant TCR as shown in Supplemental Figure 1C. Despite marked overexpression of GD3 in TSC-derived tissues, the absence of TSC expression does not lead to a significant increase in immune responses to tumor cells. This lack of evidence for a natural immune response to GD3 leads us to believe that introducing an immune response to the ganglioside can help overcome this deficiency to effectively treat TSC and eliminate tumor tissue. Limited NKT infiltration is not due to a lack of CD1d expression in TSC tissues. As a glycolipid antigen, GD3 can be processed and presented by CD1d-expressing APCs and might recruit responding NKT cells. To understand whether tumors in.