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PDF file - 379K, Dose response curves of human PDA cell lines treated with either MEK inhibitor GSK1120212, AKT inhibitor GSK690693, or both together

XLS file - 41K, Cell lines used in this study and their genotype

PDF file - 194K, RAF and MEK co inhibition

XLS file - 65K, Interaction indicies of MEK inhibitor GSK1120212, AKT inhibitor GSK690693 in PDA cell lines

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PDF file - 331K, MEK inhibitor PD325901 inhibits MEK in vivo

PDF file - 578K, Characterization of a new, syngeneic, orthotopic mouse model of PDA

Tables 1 and 2. Table 1: List of antibodies used in this study. Table 2: List of mRNA sequences assessed by digital molecular barcoding using the nCounter

Supplementary Video C. VTLM single cell killing of HEK293 cells by HERV-K env-specific CAR+ T cells

Purpose: The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma.Experimental Design: Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis. HERV-K env–specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env–specific CAR+ T cells were expanded ex vivo on activating and propagating cells (AaPC) and characterized for CAR expression and specificity. This includes evaluating the HERV-K–specific CAR+ T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model.Results: We detected HERV-K env protein on melanoma but not in normal tissues. After electroporation of T cells and selection on HERV-K+ AaPC, more than 95% of genetically modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env+ tumor targets in an antigen-specific manner. Even though there is apparent shedding of this TAA from tumor cells that can be recognized by HERV-K env–specific CAR+ T cells, we observed a significant antitumor effect.Conclusions: Adoptive cellular immunotherapy with HERV-K env–specific CAR+ T cells represents a clinically appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors. Clin Cancer Res; 21(14); 3241–51. ©2015 AACR.

Purpose: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved.Experimental Design: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing.Results: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1negVδ2neg subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1negVδ2neg>Vδ2) of survival of mice with ovarian cancer xenografts.Conclusions: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans. Clin Cancer Res; 20(22); 5708–19. ©2014 AACR.

PDF file - 1405KB, Supplementary Materials and Methods. Supplementary Figures - (1) Tumor cell line culture conditions and media formulations (2) Co-culture conditions for gamma/delta T cells and designer aAPC (3) Intracellular cytokine production, Luminex, and neutralizing antibody cytolysis assays (4) Lentivirus packaging and transduction of CAOV3 cells Contents of the Supplemental Data include figures: (1) An example of the gating strategy for gamma/delta T-cell analyses (2) Schematic of DNA plasmid pLVU3G-effLuc-T2A-mKateS158A used to co-express enhanced firefly luciferase (effLuc) and mKate (3) Expression of activation markers CD38 and CD95 on propagated gamma/delta T cells (4) aAPC developed for co-culture with gamma/delta T cells to determine the impact of introduced co-stimulatory molecules (5) Expansion of UCB-derived gamma/delta T cells on aAPC with IL-2 and IL-21 (6) Surface expression of TCRdelta1 and TCRdelta2 chains on gamma/delta T cells derived from PBMC prior to propagation (7) Abundance of Vdelta and Vgamma mRNA species in gamma/delta T cells prior to ex vivo numeric expansion (8) Surface expression of TCRdelta1 and TCRdelta2 chains on gamma/delta T cells derived from UCB and propagated on aAPC with IL-2 and IL-21 (9) mRNA expression of shared Valpha/Vdelta alleles in gamma/delta T cells separated and then propagated on aAPC, IL-2, and IL-21 (10) Abundance of mRNA species coding for Vgamma chains in gamma/delta T-cell subsets (11) In vitro lysis of tumor cell line panel by polyclonal gamma/delta T cells (12) Specific lysis of hematological and solid tumor cells by Vdelta T-cell subsets (13) Immunophenotypes associated with PBMC-derived Vdelta T-cell subsets expanded on aAPC/IL-2/IL-21. Supplemental Table 1 - details the antibodies used in this study, where they were purchased, and their hybridoma clone.

Supplementary Video D. Physical health of mice group with tumor and without HERV-K env-specific CAR+ T-cell treatment.

Supplementary figure 3 and 4. Figure S3: Generation and specificity of 6H5mAx. Figure. S4: Generation of EL4-HERV-K and specificity of HERV-K env-specific CAR+ T cells.

Supplementary materials

Supplementary figure 1 and 2. Figure S1: HERV-K env expression on primary tumor and normal human tissues. Figure. S2: Phenotype and mRNA profile of HERV-K env-specific CAR+ T cells.

Supplementary figure 5. Generation and specificity of HERV-K env-specific CAR+ ffLuc+ T cells in vitro and in vivo.

Supplementary Video E. Physical health of mice group with tumor and with HERV-K env-specific CAR+ T-cell treatment.

BALB/c thymocytes can be divided into three distinct subsets according to the expression of a ligand for the NK activation receptor NKG2D (NKG2D-L) and the expression of MHC class I (MHC-I). The first subset (MHC-Imid/NKG2D-Lhigh or "N+") is predominately CD4+CD8+ double positive (DP), comprises approximately 35% of thymocytes in a 6-8-week-old adult and contains uncommitted cells that have neither undergone selection nor are committed to death by neglect. The second subset (MHC-Ilow/NKG2D-Llow or "M-"), also mostly DP cells, comprises approximately 50% of thymocytes and consists of cells committed to death by apoptosis, likely due to neglect. By contrast, the third subset (MHC-Ihigh/NKG2D-Llow or "M+") is largely single positive (SP), represents approximately 15% of thymocytes and mostly contains more mature cells that have undergone successful positive selection. The major advantage of the analysis is that it splits DP cells into two subpopulations, one committed to death by apoptosis and the other subjected to selection. The analysis also suggests that NKG2D-L may play a role in thymocyte development.

We studied 55 patients (50 women, 5 men) with severe facial pain and biopsy-proven neuralgia-inducing cavitational osteonecrosis (NICO) of the alveolar bone of the jaws. Our aim was to assess the pathophysiologic contributions to NICO of anticardiolipin antibodies (aCLA), thrombophilia (increased tendency to intravascular thrombi), and hypofibrinolysis (reduced ability to lyse thrombi). Of the 55 patients, 43 (78%) had one or more tests positive for thrombophilia or hypofibrinolysis (or both), and only 12 (22%) were normal. Eighteen of 55 (33%) patients had high aCLA (>2 SD above mean value for control subjects); immunoglobulin G (IgG) ( p = 0.01) and immunoglobulin A (IgA)( p = 0.001) levels were higher in patients than in controls. The distribution of elevated aCLA immunoglobulin classes among patients was as follows: IgG alone, 5 (9%); IgA alone, 7 (13%); and IgM alone, 3 (5%). Three patients (5%) had high levels of both IgG and IgA aCLA. Other defects of the thrombotic or fibrinolytic systems in the 55 patients included high lipoprotein(a) in 36% (vs 20% in control subjects ( p = 0.03)), low stimulated tissue plasminogen activator activity (tPA-Fx) in 22% (vs 7% in control subjects ( p = 0.08)), high plasminogen activator inhibitor activity (PAI-Fx) in 18% (vs 8% in control subjects ( p = 0.03)), resistance to activated protein C in 16% (vs 0% in control subjects ( p = 0.007)), low antigenic protein C in 4% (vs 0% in control subjects ( p > 0.2)), and low antigenic protein S in 4% (vs 0% in control subjects ( p > 0.2)). Anticardiolipin antibodies and other defects of the thrombotic and fibrinolytic systems appear to be common, potentially reversible pathogenetic risk factors associated with osteonecrosis of the jaw.

MHC class I family members serve multiple functions beyond antigen presentation. We provide insight into the structure, expression and function of the Mill subfamily. This family includes two surface glycoproteins, Mill1 and Mill2. Protein sequences for Mill1 and Mill2 are most highly related to the NKG2D ligands, MICA and MICB, but neither of them bound to NKG2D. Computer-based protein modelling indicated that hereditary haemochromatosis protein (HFE), a molecule involved in iron uptake, was most similar. Mill1 and Mill2 were observed on cycling thymocytes, proliferating smooth muscle cells and fibroblasts. Using soluble Mill proteins, we found evidence for a soluble ligand in serum. Like HFE, the Mill family may be involved in nutrient metabolism. Skin was one of the only three organs found to express transcripts for both Mill1 and Mill2. Addition of antibodies specific for Mill2 to wounded skin enhanced healing. Our results suggest a role for the Mill proteins in cellular metabolism, with possible therapeutic significance.

ULBPs are human ligands for NKG2D, an activating receptor expressed on natural killer (NK) cells, NK1.1+ T cells, and T cells. ULBPs are expressed by a variety of leukemias, carcinomas, melanomas, and tumor cell lines. ULBP expression correlates with improved survival in cancer patients, however, the nature of the immune response that ULBPs elicit is not well understood. We report that ectopic expression of ULBP1 or ULBP2 on murine EL4 or RMA tumor cells elicits potent antitumor responses in syngeneic C57BL/6 and SCID mice. Although binding of ULBP3 to murine NKG2D could not be demonstrated in vitro, ULBP3 can also stimulate antitumor responses, suggesting that ULBP3 binds to murine NKG2D or possibly another receptor in vivo. ULBP expression was found to recruit NK cells, NK1.1+ T cells, and T cells to the tumor. IL-15 was found to strongly enhance the immune response directed against ULBP-expressing tumors. Tumors can evade NKG2D immunity by down-regulating expression of NKG2D. Our data suggest that IL-15 may be useful for overcoming this tumor-evasion strategy. Together, these results demonstrate that ULBP expression can elicit a potent immune response and suggest that ULBPs, alone or in combination with IL-15, can be exploited for antitumor therapy.

T cells cultured for 3 h with antigen-presenting cells (APCs) stimulated syngeneic IL-2-activated NK cells as measured via a standard chromium-release assay. Discrete caps containing both TCR and MHC-I had formed on the surface of these activated T cells. When conjugates were formed between NK cells and these activated T cells, >80% of the contact sites were in the MHC-I d i m region outside the TCR-MHC-I cap. Stimulation with phorbol myristate acetate plus lonomycin, which bypasses the need for cell surface events during activation, did not induce either cap formation or NK cell activation. Further, the addition of the protein transport inhibitor Brefeldin A did not block activation of NK cells. MHC-I is the major inhibitory ligand recognized by NK cells. One possible mechanism for the activation of NK cells by TCR-MHC-I-capped T cells is that aggregation of MHC-I into one region leaves the remaining T cell surface denuded of ligands for NK-inhibitory receptors. As a test of this hypothesis, we aggregated MHC-I on T cells with plate-bound anti-MHC-I mAb. This treatment conferred upon the T cells the capacity to activate NK cells, suggesting that MHC-I clustering could contribute to the observed phenomenon.