Over-expression of B7-H1 (PD-L1) molecule in the tumor microenvironment (TME) is a major immune evasion mechanism in some cancer patients and antibody blockade of the B7-H1/PD-1 interaction can normalize compromised immunity without excessive side-effects. Using a genomescale T-cell activity array, we identified Siglec-15 as a critical immune suppressor. While only expressed on some myeloid cells normally, Siglec-15 is broadly upregulated on human cancer cells and tumor-infiltrating myeloid cells, and its expression is mutually exclusive to B7-H1, partially due to its induction by M-CSF and downregulation by IFN-γ. We demonstrate that Siglec-15 suppresses antigen-specific T-cell responses in vitro and in vivo. Genetic ablation or antibody blockade of Siglec-15 amplifies anti-tumor immunity in the TME and inhibits tumor Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Alterations in EGFR, KRAS, and ALK are oncogenic drivers in lung cancer, but how oncogenic signaling influences immunity in the tumor microenvironment is just beginning to be understood. Immunosuppression likely contributes to lung cancer, because drugs that inhibit immune checkpoints like PD-1 and PD-L1 have clinical benefit. Here, we show that activation of the AKT-mTOR pathway tightly regulates PD-L1 expression in vitro and in vivo. Both oncogenic and IFNg-mediated induction of PD-L1 was dependent on mTOR. In human lung adenocarcinomas and squamous cell carcinomas, membranous expression of PD-L1 was significantly associated with mTOR activation. These data suggest that oncogenic activation of the AKT-mTOR pathway promotes immune escape by driving expression of PD-L1, which was confirmed in syngeneic and genetically engineered mouse models of lung cancer where an mTOR inhibitor combined with a PD-1 antibody decreased tumor growth, increased tumor-infiltrating T cells, and decreased regulatory T cells.

Natural killer (NK) cells hold promise for cancer therapy. NK cytotoxicity can be enhanced by expression of chimeric antigen receptors that re-direct specificity toward target cells by engaging cell surface molecules expressed on target cells. We developed a regulatory-compliant, scalable non-viral approach to engineer NK cells to be target-specific based on transfection of mRNA encoding chimeric receptors. Transfection of eGFP mRNA into ex vivo expanded NK cells (N ¼ 5) or purified unstimulated NK cells from peripheral blood (N ¼ 4) resulted in good cell viability with eGFP expression in 85 ± 6% and 86 ± 4%, 24 h after transfection, respectively. An mRNA encoding a receptor directed against CD19 (anti-CD19-BB-z) was also transfected into NK cells efficiently. Ex vivo expanded and purified unstimulated NK cells expressing anti-CD19-BB-z exhibited enhanced cytotoxicity against CD19 þ target cells resulting in X80% lysis of acute lymphoblastic leukemia and B-lineage chronic lymphocytic leukemia cells at effector target ratios lower than 10:1. The target-specific cytotoxicity for anti-CD19-BB-z mRNA-transfected NK cells was observed as early as 3 h after transfection and persisted for up to 3 days. The method described here should facilitate the clinical development of NK-based antigen-targeted immunotherapy for cancer. Cancer Gene Therapy ( IntroductionThe capacity of natural killer (NK) 1 cells to exert cytotoxicity against a variety of cancer cell types makes them an attractive tool for anti-cancer therapy. [2][3][4][5][6][7] Data gathered in the setting of allogeneic hematopoietic stem cell transplantation indicate that donor selection based on the degree of mismatch between expression of killer immunoglobulin-like receptors on donor NK cells and HLA Class I molecules expressed by the patient cells should maximize NK cell killing of target cells, 4,[8][9][10] hence augmenting the efficacy of hematopoietic stem cell transplantation. 6,7,11 In addition, it was reported that the infusion of haploidentical NK cells in a nonmyeloablative transplant setting could produce remissions in patients with acute myeloid leukemia. 5 Although NK cell cytotoxicity has a wide spectrum, some cancer cell types appear less susceptible or refractory to NK cell killing, because of failure to activate NK cells, induction of suppression or both. Among these relatively NKresistant cell types are lymphoid malignancies such as acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (B-CLL) and B-cell non-Hodgkin lymphoma. 3,[12][13][14][15][16] Chimeric antigen receptor has been studied since late 1980s. [17][18][19][20][21][22] It generally contains a single chain variable fragment as the extracellular antigen recognition unit and multiple lymphocyte activation domains as the intracellular activation part. Most work has been focused in arming T cells with this chimeric antigen receptor for antitumor effect 21-23 NK cells transduced with chimeric antigen receptor have also been exploited for anti-tumor effect. Various com...

We evaluated the therapeutic efficacy and mechanisms of action of both mouse and human B7-H4 Immunoglobulin fusion proteins (mB7-H4Ig; hB7-H4Ig) in treating EAE. The present data show that mB7-H4Ig both directly and indirectly (via increasing Treg function) inhibited CD4+ T-cell proliferation and differentiation in both Th1- and Th17-cell promoting conditions while inducing production of IL-10. B7-H4Ig treatment effectively ameliorated progression of both relapsing (R-EAE) and chronic EAE correlating with decreased numbers of activated CD4+ T-cells within the CNS and spleen, and a concurrent increase in number and function of Tregs. The functional requirement for Treg activation in treating EAE was demonstrated by a loss of therapeutic efficacy of hB7-H4Ig in R-EAE following inactivation of Treg function either by anti-CD25 treatment or blockade of IL-10. Significant to the eventual translation of this treatment into clinical practice, hB7-H4Ig similarly inhibited the in vitro differentiation of naïve human CD4+ T-cells in both Th1- and Th17-promoting conditions, while promoting the production of IL-10. B7-H4Ig thus regulates pro-inflammatory T-cell responses by a unique dual mechanism of action and demonstrates significant promise as a therapeutic for autoimmune diseases, including MS.

Electroporation is widely used to transfect and load cells with various molecules. Traditional electroporation using a static mode is typically restricted to volumes less than 1 mL, which limits its use in clinical and industrial bioprocessing applications. Here we report efficient, large volume transfection results by using a scalable-volume electroporation system. Suspended (Jurkat) and adherent cells (10T1/2 and Huh-7) were tested. A large macromolecule, FITC-conjugated dextran (MW=500 kD) was used to measure cell uptake, while a plasmid carrying the gene coding for enhanced green fluorescence protein (eGFP) was used to quantitate the flow electrotransfection efficiency as determined by flow cytometry. The flow electroloading efficiency of FITC-dextran was >90%, while the cell viability was highly maintained (>90%). High flow electrotransfection efficiency (up to 75%) and cell viability (up to 90%) were obtained with processing volumes ranging from 1.5 to 50 mL. No significant difference of electrotransfection efficiency was observed between flow and static electrotransfection. When 50 mL of cell volume was processed and samples collected at different time points during electroporation, the transgene expression and cell viability results were identical. We also demonstrated that DNA plasmid containing EBNA1-OriP elements from Epstein-Barr virus were more efficient in transgene expression than standard plasmid without the elements (at least 500 too 1000-fold increase in expression level). Finally, to examine the feasibility of utilizing flow electrotransfected cells as a gene delivery vehicle, 10T1/2 cells were transfected with a DNA plasmid containing the gene coding for mIL12. mIL12 transfected cells were injected subcutaneously into mice, and produced functional mIL12, as demonstrated by anti-angiogenic activity. This is the first demonstration of efficient, large volume, flow electroporation and the in vivo efficacy of flow electrotransfected cells. This technology may be useful for clinical gene therapy and large-scale bioprocesses.