Higher expression of KCNK10 (TREK-2) K+ channels and their functional upregulation by lipopolysaccharide treatment in mouse peritoneal B1a cells.

Innate-like CD5+ B1a cells localized in serous cavities are activated by innate stimuli, such as lipopolysaccharide (LPS), leading to T cell-independent antibody responses. Although ion channels play crucial roles in the homeostasis and activation of immune cells, the electrophysiological properties of B1a cells have not been investigated to date. Previously, in the mouse B cell lymphoma cells, we found that the voltage-independent two-pore-domain potassium (K2P) channels generate a negative membrane potential and drive Ca2+ influx. Here, we newly compared the expression and activities of K2P channels in mouse splenic follicular B (FoB), marginal zone B (MZB), and peritoneal B1a cells. Next-generation sequencing analysis showed higher levels of transcripts for TREK-2 and TWIK-2 in B1a cells than those in FoB or MZB cells. Electrophysiological analysis, using patch clamp technique, revealed higher activity of TREK-2 with the characteristic large unitary conductance (~ 250 pS) in B1a than that in FoB or MZB cells. TREK-2 activity was further increased by LPS treatment (>2 h), which was more prominent in B1a than that in MZB or FoB cells. The cytosolic Ca2+ concentration of B cells was decreased by high-K+-induced depolarization (ΔRKCl (%)), suggesting the basal Ca2+ influx to be driven by negative membrane potential. The LPS treatment significantly increased the ΔRKCl (%) in B1a, though not in FoB and MZB cells. Our study was the first to compare the K2P channels in mouse primary B cell subsets, elucidating the functional upregulation of TREK-2 and augmentation of Ca2+ influx by the stimulation of Toll-like receptor 4 in B1a cells.

Preferential Infiltration of Unique Vγ9Jγ2-Vδ2 T Cells Into Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is clinically highly aggressive as a result of evolutionary dynamics induced by cross-talk between cancer cells and a heterogeneous group of immune cells in tumor microenvironment. The brain harbors limited numbers of immune cells with few lymphocytes and macrophages; thus, innate-like lymphocytes, such as γδ T cells, have important roles in antitumor immunity. Here, we characterized GBM-infiltrating γδ T cells, which may have roles in regulating the GBM tumor microenvironment and cancer cell gene expression. V(D)J repertoires of tumor-infiltrating and blood-circulating γδ T cells from four patients were analyzed by next-generation sequencing-based T-cell receptor (TCR) sequencing in addition to mutation and immune profiles in four GBM cases. In all tumor tissues, abundant innate and effector/memory lymphocytes were detected, accompanied by large numbers of tumor-associated macrophages and closely located tumor-infiltrating γδ T cells, which appear to have anti-tumor activity. The immune-related gene expression analysis using the TCGA database showed that the signature gene expression extent of γδ T cells were more associated with those of cytotoxic T and Th1 cells and M1 macrophages than those of Th2 cells and M2 macrophages. Although the most abundant γδ T cells were Vγ9Vδ2 T cells in both tumor tissues and blood, the repertoire of intratumoral Vγ9Vδ2 T cells was distinct from that of peripheral blood Vγ9Vδ2 T cells and was dominated by Vγ9Jγ2 sequences, not by canonical Vγ9JγP sequences that are mostly commonly found in blood γδ T cells. Collectively, unique GBM-specific TCR clonotypes were identified by comparing TCR repertoires of peripheral blood and intra-tumoral γδ T cells. These findings will be helpful for the elucidation of tumor-specific antigens and development of anticancer immunotherapies using tumor-infiltrating γδ T cells.

Serosal Cavities Contain Two Populations of Innate-like integrin α4highCD4+ T Cells, Integrin α4ß1+α6ß1+α4ß7- and α4ß1+α6ß1-α4ß7+ Cells.

We previously reported peritoneal innate-like integrin α4 (CD49d)highCD4+ T cells that provided help for B-1a cells. Here we analyzed the expression of various integrin chains on the peritoneal and pleural integrin α4highCD4+ T cells and investigated the functional heterogeneity of the subpopulations based on the integrin expression. Pleural cavity contained a lower ratio of integrin α4highCD4+ T cells to integrin α4lowCD4+ T cells than peritoneal cavity, but the pleural integrin α4highCD4+ T cells have the same characteristics of the peritoneal integrin α4highCD4+ T cells. Most of integrin α4highCD4+ T cells were integrin ß1highß7-, but a minor population of integrin α4highCD4+ T cells was integrin ß1+ß7+. Interestingly, the integrin α4highß1highß7- CD4+ T cells expressed high levels of integrin α4ß1 and α6ß1, whereas integrin α4highß1+ß7+ CD4+ T cells expressed high levels of integrin α4ß1 and α4ß7, suggesting an alternative expression of integrin α6ß1 or α4ß7 in combination with α4ß1 in respective major and minor populations of integrin α4highCD4+ T cells. The minor population, integrin α4highß1+ß7+ CD4+ T cells, were different from the integrin α4highß1highß7- CD4+ T cells in that they secreted a smaller amount of Th1 cytokines upon stimulation and expressed lower levels of Th1-related chemokine receptors CCR5 and CXCR3 than the integrin α4highß1highß7- CD4+ T cells. In summary, the innate-like integrin α4highCD4+ T cells could be divided into 2 populations, integrin α4ß1+α6ß1+α4ß7- and α4ß1+α6ß1-α4ß7+ cells. The functional significance of serosal integrin α4ß7+ CD4+ T cells needed to be investigated especially in view of mucosal immunity.

Early Development in the Peritoneal Cavity of CD49dhigh Th1 Memory Phenotype CD4+ T Cells with Enhanced B Cell Helper Activity.

The Th cells that regulate peritoneal B-1 cell functions have not yet been well characterized. To address this question, we investigated peritoneal CD4(+) T cells, observed a high frequency of the conjugates of B-CD4(+) T cells in the peritoneal cavity, and identified a population of CD49d(high)CD4(+) T cells that constituted about half of all CD4(+) T cells in the peritoneal cavity, but were rarely found in other compartments. Peritoneal CD49d(high)CD4(+) T cells were CD44(high)CD62L(low); expressed integrin α4ß1 and CXCR3; and rapidly secreted IFN-γ, TNF-α, and IL-2, showing features of proinflammatory Th1 cells. Peritoneal CD49d(high)CD4(+) T cells developed spontaneously, were detected at the age of 12 d, and showed stem cell-like properties. Their development was observed in mice deficient for signaling lymphocytic activation molecule-associated protein, but not in athymic nude mice and mice lacking in expression of MHC class II on thymic epithelial cells. Peritoneal CD49d(high)CD4(+) T cells were more resistant to irradiation and more sensitive to NAD-induced cell death than CD49d(low)CD4(+) T cells. Notably, peritoneal CD49d(high)CD4(+) T cells also showed some characteristics of follicular Th cells, such as the expression of programmed cell death 1, ICOS, IL-21, and CXCR5. Moreover, peritoneal CD49d(high)CD4(+) T cells enhanced the secretion of IgM Abs by B-1a cells and IgG Abs by splenic B cells. These data suggest that peritoneal CD49d(high)CD4(+) T cells may be innate-like CD4(+) T cells, which develop early and have a dual capacity to support both humoral and cellular immunity.