Short-term LPS-modulated DCs inoculation interferes with CIA progression when loaded with CII.

Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.

Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. Escherichia coli is considered the workhorse for this purpose, given its fast growth rate and feasible manipulation. However, bacterial inclusion body formation remains a challenge for further protein purification. We analyzed and optimized the expression conditions for three different proteins: an anti-MICA scFv, MICA, and p19 subunit of IL-23. We used a response surface methodology based on a three-level Box-Behnken design, which included three factors: post-induction temperature, post-induction time and IPTG concentration. Comparing this information with soluble protein data in a principal component analysis revealed that insoluble and soluble proteins have different optimal conditions for post-induction temperature, post-induction time, IPTG concentration and in amino acid sequence features. Finally, we optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays. These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Further studies using the system proposed in this study may lead to the identification of optimal environmental factors for a given protein sequence, favoring the acceleration of bioprocess development and structural studies.

Natural-killer group 2, member D (NKG2D) binds to a variety of ligands, including the major histocompatibility complex (MHC) class I chain-related proteins (MIC) and UL16-binding proteins (ULBP). It is regarded as a co-activating receptor on NK cells, having an important role in the cell-mediated immune response to tumours. We studied the influence of interleukin (IL)-10 on the regulation of MIC and ULBP expression on melanoma cells, and its effect on the cytotoxic function of NK cells in vitro.Here, we show that, in the presence of IL-10, FMS mel and BL mel cell lines decreased MICA and ULBP2 surface expression, whereas MHC class I did not change substantially on the cell surface. MICA mRNA levels decreased in IL-10-treated FMS and IL-10-transduced BL cell lines. Interestingly, we observed that MICB surface expression and its mRNA levels increased upon IL-10 treatment in a melanoma cell line. These changes in NKG2D ligands surface expression patterns owing to IL-10 treatment resulted in an effect on lysis susceptibility mediated by lymphocyte-activated killer cells, as tumour cell lines that displayed a higher decrease of MICA on their surface had lower levels of lysis. In addition, expression of CD107a was downregulated on the surface of NK cells following stimulation with IL-10-treated FMS cells. Our results suggest a novel function for IL-10 in the modulation of NKG2D ligand expression and in the control of cytotoxicity mediated by NKG2D/NKG2D ligand axis. Keywords: immune evasion; interleukin 10; melanoma cells; natural killer cell; NKG2D ligands Natural-killer group 2, member D (NKG2D) is a co-activating receptor expressed on NK cells. 1 It has also been identified on gd T, ab CD8 + T cells and in NKT cells in human beings. 2 NKG2D binds to a variety of ligands that resemble the major histocompatibility complex (MHC) class I proteins. [3][4][5] In human beings, the UL16-binding proteins (ULBP), 6,7 also denominated retinoic acid early transcript 1, 8 and MHC class I chain-related proteins A and B (MICA and MICB, respectively) 3,4 have been described. Ligands for NKG2D (NKG2DL) are rarely detectable on the surface of healthy cells, but they can be upregulated by cellular malignant transformation, viral infection and cellular stress, among other stimuli, such as chemotherapeutic drugs. [8][9][10][11] NKG2D and its ligands have a critical role in tumour immune surveillance. 5,12 In fact, NKG2D-deficient mice are defective in establishing a response against spontaneous malignancies. 13 In addition, there is evidence of an early induction of NKG2DL surface expression during spontaneous tumour genesis. 14 However, during the immunoediting process, tumour cells can interfere with NKG2D-mediated activity by different mechanisms, such as expression of tumourderived NKG2DL by exosomes and release of soluble forms of NKG2DL by metalloproteases or alternative splicing. 2,15-18 These molecules interact directly with NK cells and CD8 + T cells in an NKG2D-dependent manner, and strongly reduce the NKG2D-mediated cytotoxi...