Preclinical studies of primary cancer cells are typically done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3 to 10% range, with most tumors in a hypoxic or 1 to 2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anticancer agents is routinely examined at ambient O2. Here, we demonstrate that tumors collected, processed, and propagated at physiologic O2 compared to ambient air display distinct differences in key signaling networks including LGR5/WNT, YAP, and NRF2/KEAP1, nuclear reactive oxygen species, alternative splicing, and sensitivity to targeted therapies. Therefore, evaluating cancer cells under physioxia could more closely recapitulate their physiopathologic status in the in vivo microenvironment.

Tuberculosis (TB) is still a global infectious disease that seriously threatens human beings. The only licensed TB vaccine Bacille Calmette-Guérin (BCG)'s protective efficacy varies significantly among populations and regions. It is very urgent to develop more effective vaccines.
In this study, eleven candidate proteins of Mycobacterium tuberculosis were selected to predict peptides with high-affinity binding capacity for the HLA-DRB1*01:01 molecule. The immunodominant peptides were identified with the enzyme-linked immunospot assay (ELISPOT) and linked in silico to result in a novel polypeptide vaccine in Escherichia coli cells. The vaccine's protective efficacy was evaluated in humanized and wild-type C57BL/6 mice. The potential immune protective mechanisms were explored with Enzyme-linked Immunosorbent Assay (ELISA), flow cytometry, and ELISPOT.
Six immunodominant peptides screened from 50 predicted peptides were used to construct a new polypeptide vaccine named MP3RT. After challenge with M. tuberculosis, the colony-forming units (CFUs), lung lesion area, and the number of inflammatory cells in humanized mice rather than wild-type mice vaccinated with MP3RT were significantly lower than these in mice immunized with PBS. The humanized mice vaccinated with MP3RT revealed significant increases in IFN-γ cytokine production, IFN-γ+ T lymphocytes, CD3+IFN-γ+ T lymphocytes, and the MP3RT-specific IgG antibody.
Taken together, MP3RT is a promising peptides-based TB vaccine characterized by inducing high levels of IFN-γ and CD3+IFN-γ+ T lymphocytes in humanized mice. These new findings will lay a foundation for the development of peptides-based vaccines against TB.
Copyright © 2021 Gong, Liang, Mi, Jia, Xue, Wang, Wang, Zhou, Sun and Wu.

Migration of human plasmablast to the bone marrow is essential for the final differentiation of plasma cells and maintenance of effective humoral immunity. This migration is controlled by CXCL12/CXCR4-mediated activation of the protein kinase AKT. Herein, we show that the CXCL12-induced migration of human plasmablasts is dependent on glucose oxidation. Glucose depletion markedly inhibited plasmablast migration by 67%, and the glucose analog 2-deoxyglucose (2-DG) reduced the migration by 53%; conversely, glutamine depletion did not reduce the migration. CXCL12 boosted the oxygen consumption rate (OCR), and 2-DG treatment significantly reduced the levels of all measured tricarboxylic acid (TCA) cycle intermediates. AKT inhibitors blocked the CXCL12-mediated increase of OCR. CXCL12 enhanced the pyruvate dehydrogenase (PDH) activity by 13.5-fold in an AKT-dependent manner to promote mitochondrial oxidative phosphorylation. The knockdown and inhibition of PDH confirmed its indispensable role in CXCL12-induced migration. Cellular ATP levels fell by 91% upon exposure to 2-DG, and the mitochondrial ATP synthase inhibitor oligomycin inhibited CXCL12-induced migration by 85%. Low ATP levels inhibited the CXCL12-induced activation of AKT and phosphorylation of myosin light chains by 42%, which are required for cell migration. Thus, we have identified a mechanism that controls glucose oxidation via AKT signaling and PDH activation, which supports the migration of plasmablasts. This mechanism can provide insights into the proper development of long-lived plasma cells and is, therefore, essential for optimal humoral immunity. To our knowledge, this study is the first to investigate metabolic mechanisms underlying human plasmablast migration toward CXCL12.

The interplay of immune cells and stem cells in maintaining skin homeostasis and repair is an exciting new frontier in cutaneous biology. With the growing appreciation of the importance of this new crosstalk comes the requirement of methods to interrogate the molecular underpinnings of these leukocyte-stem cell interactions. Here we describe how a combination of FACS, cellular coculture assays, and conditioned media treatments can be utilized to advance our understanding of this emerging area of intercellular communication between immune cells and stem cells.

NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells. It is weakly expressed in resting NK cells but upon target cell stimulation or after incubation with cytokines that enhance NK killing, NKLAM mRNA levels increase and protein is synthesized and is targeted to cytoplasmic granule membranes. We have previously shown that NKLAM plays a role in perforin/granzyme-mediated cytolysis in vitro. To further investigate the function of NKLAM in NK cell-mediated cytotoxicity, we generated, by gene targeting, NKLAM-deficient mice. These mice have normal numbers of NK cells and other lymphoid populations in the spleen. They also have no alterations in NK maturation or NK receptor repertoire. NK cells from NKLAM-deficient and WT mice have comparable amounts of perforin, granzyme B, and lysosomal membrane-associated protein 1 (CD107a) in their cytotoxic granules and comparable levels of granule exocytosis are induced by PMA and calcium ionophore A23187. However, NKLAM-deficient NK cells display significantly less NK cytotoxic activity in vitro than WT NK cells. They also secrete less IFN-gamma upon target cell stimulation, In addition, NKLAM-deficient mice exhibit greater numbers of pulmonary metastases after i.v. injection with B16 melanoma cells. These studies indicate that NKLAM-deficient mice have diminished capacity to control tumor metastases and support the role for NKLAM in NK function both in vitro and in vivo.