Human cytokine-induced killer cells have enhanced in vitro cytolytic activity via non-viral interleukin-2 gene transfer

Modulation of the immune system by genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Interleukin-2 (IL-2) is a crucial cytokine which induces potent antitumor response. Cytokine-induced killer cells (CIK) have been described as highly efficient cytotoxic effector cells capable of lysing tumor cell targets and are capable of recognizing these cells in a non-MHC restricted fashion. Dendritic cells (DC) are the major antigen presenting cells. This study evaluated the antitumor effect of CIK cells which were non-virally transfected with IL-2 and co-cultured with pulsed and unpulsed DC. Human CIK cells generated from peripheral blood were transfected in vitro with plasmid encoding for the human IL-2. Transfection involved a combination of electrical parameters and a specific solution to deliver plasmid directly to the cell nucleus by using the Nucleofector(® )electroporation system. Nucleofection resulted in the production of IL-2 with a mean of 478.5 pg/10(6 )cells (range of 107.6–1079.3 pg /10(6 )cells/24 h) compared to mock transfected CIK cells (31 pg/10(6 )cells) (P = 0.05). After co-culturing with DC their functional ability was assessed in vitro by a cytotoxicity assay. On comparison with non-transfected CIK cells co-cultured with DCs (36.5 ± 5.3 %), transfected CIK cells co-cultured with DC had a significantly higher lytic activity of 58.5 ± 3.2% (P = 0.03) against Dan G cells, a human pancreatic carcinoma cell line

Case Report Sclerosing Sertoli Cell Tumor of the Testis: Case Report and Review of the Literature

ABSTRAC

Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein

Accumulation of myeloid-derived suppressor cells (MDSCs) associated with inhibition of dendritic cell (DC) differentiation is one of the major immunological abnormalities in cancer and leads to suppression of antitumor immune responses. The molecular mechanism of this phenomenon remains unclear. We report here that STAT3-inducible up-regulation of the myeloid-related protein S100A9 enhances MDSC production in cancer. Mice lacking this protein mounted potent antitumor immune responses and rejected implanted tumors. This effect was reversed by administration of wild-type MDSCs from tumor-bearing mice to S100A9-null mice. Overexpression of S100A9 in cultured embryonic stem cells or transgenic mice inhibited the differentiation of DCs and macrophages and induced accumulation of MDSCs. This study demonstrates that tumor-induced up-regulation of S100A9 protein is critically important for accumulation of MDSCs and reveals a novel molecular mechanism of immunological abnormalities in cancer

MyD88-dependent expansion of an immature GR-1+CD11b+ population induces T cell suppression and Th2 polarization in sepsis

Polymicrobial sepsis alters the adaptive immune response and induces T cell suppression and Th2 immune polarization. We identify a GR-1+CD11b+ population whose numbers dramatically increase and remain elevated in the spleen, lymph nodes, and bone marrow during polymicrobial sepsis. Phenotypically, these cells are heterogeneous, immature, predominantly myeloid progenitors that express interleukin 10 and several other cytokines and chemokines. Splenic GR-1+ cells effectively suppress antigen-specific CD8+ T cell interferon (IFN) γ production but only modestly suppress antigen-specific and nonspecific CD4+ T cell proliferation. GR-1+ cell depletion in vivo prevents both the sepsis-induced augmentation of Th2 cell–dependent and depression of Th1 cell–dependent antibody production. Signaling through MyD88, but not Toll-like receptor 4, TIR domain–containing adaptor-inducing IFN-β, or the IFN-α/β receptor, is required for complete GR-1+CD11b+ expansion. GR-1+CD11b+ cells contribute to sepsis-induced T cell suppression and preferential Th2 polarization

Lipid accumulation and dendritic cell dysfunction in cancer

Professional antigen presenting cells, dendritic cells (DC) are responsible for initiation and maintenance of immune responses. Here, we report that a substantial proportion of DCs in tumor-bearing mice and cancer patients have increased levels of triglycerides. Lipid accumulation in DCs was caused by increased uptake of extracellular lipids due to up-regulation of scavenger receptor A. DCs with high lipid content were not able to effectively stimulate allogeneic T cells or present tumor-associated antigens. DCs with high and normal lipid levels did not differ in expression of MHC and co-stimulatory molecules. However, lipid-laden DCs had reduced capacity to process antigens. Pharmacological normalization of lipid levels in DCs with an inhibitor of acetyl-CoA carboxylase restored the functional activity of DCs and substantially enhanced the effects of a cancer vaccine. These findings support the regulation of immune responses in cancer by manipulation of lipid levels in DCs

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The Arabidopsis aminopeptidase-P (AtAPP1) is a regulator of auxin signaling.

Arabidopsis APP1 is a membrane-associated soluble aminopeptidase with activity against N-terminal Arg\u3eLys\u3eLeu=Pro\u3eTyr residues. APP1 is a single copy gene and was originally isolated by its weak affinity for the auxin transport inhibitor 1-Naphthylphthalamic acid (NPA). A role in wounding responses was suggested when AtAPP1 was co-purified with proline rich proteins (PRPs) and arabinogalactan protein (AGPs) that accumulate in the cell wall in response to physical damage (Fowler et al., 1999; Murphy et al., 2002) and with the recent findings that excess use of Alanap (NPA) leading to a weakened defense response and susceptibility resulting in mature watermelon vine decline (MWVD) (Makam et al., 2005). AtAPP1 enzymatic activity is inhibited by bestatin, apstatin (a APP specific inhibitor), but activated by NPA consistent with its isolation. AtAPP1 also exhibits sensitivity to metal chelators (EDTA, EGTA) and to both leucine aminopeptidase and serine protease inhibitors. Enzyme kinetic analysis suggest an allosteric catalytic mechanisms for AtAPP1. A very unique character of AtAPP1 is its PPlase activity. This activity is involved in isomerization of cis to trans forms of prolyl residue, which is the rate limiting step for processing of proline residues in biologically important peptides. APP1 expression is induced by auxin, wounding, and jasmonic acid. APP1 exhibits affinity for WPP peptide motifs and binds poly-arg containing dynorphin A like motifs with relatively high affinity in vivo and in vitro. Mutations in such conserved di-prolyl motif stabilizes the AUX/IAAs which are central to auxin signal transduction. Events leading to such stability and factors involved in this process is still unknown. In addition, AtAPP1 has the auxin responsive TGTCTC element in its promoter and the affinity to these motifs (di-prolyl and poly-arg) suggest a crucial role for AtAPP1 in auxin responses. Results are presented here illustrating AtPP1 modulation of auxin responses through its enzymatic, mutational and interaction analysis

Renal arginine metabolism

Kidneys of normal animals remove citrulline from blood and convert it, stoichiometrically to arginine. This citrulline arises from the intestinal metabolism of glutamine. This intestinal-renal pathway constitutes the major endogenous source of arginine. The objectives of the present studies are to determine the location of arginine synthesis in kidney and its response to citrulline concentrations in vitro and in vivo and to different arginine or protein intakes in rats. -- Investigations on the localization of enzymes of arginine synthesis (argininosuccinate synthetase and argininosuccinate lyase) and of breakdown (arginase and ornithine aminotransferase), revealed that the enzymes of arginine synthesis are exclusively present in the cytosol of the cells of the proximal convoluted tubule and that of arginine degradation are enriched in other kidney regions. -- Arginine synthesis from citrulline in isolated kidney cortical tubules was found to be highly sensitive to citrulline concentrations in the physiological plasma range (0.06 mM), suggesting that renal arginine synthesis in vivo could be regulated by circulating citrulline levels. Thus, in studies on renal arginine synthesis in vivo, it was found that kidneys of rats infused with citrulline (saline-infused, as controls) responded to the elevated plasma citrulline levels by increasing its uptake and producing increased quantities of arginine. -- In studies with rats fed different levels of arginine (0.0, 0.5, 2.0%) or protein (5, 12 and 50%) for 1 week, it was found that the renal uptake of citrulline and release of arginine and also circulating citrulline levels were similar in all these animals. This suggested that renal arginine synthesis is independent of dietary arginine or protein intake. The results suggest that availability of citrulline is a limiting factor for renal arginine synthesis in rats

Células supresoras derivadas de mieloides como reguladoras del sistema inmunológico

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that expand during cancer, inflammation and infection, and that have a remarkable ability to suppress T-cell responses. These cells constitute a unique component of the immune system that regulates immune responses in healthy individuals and in the context of various diseases. In this Review, we discuss the origin, mechanisms of expansion and suppressive functions of MDSCs, as well as the potential to target these cells for therapeutic benefit