The presence of metastases in mediastinal lymph nodes (LNs) is essential for planning lung cancer treatment and assessing anticancer immune responses. The aim of the study was to assess LNs for the presence of neoplastic cells and evaluate lung cancer-selected antigen expression. LN aspirates were obtained during an EBUS/TBNA procedure. The cells were analyzed using a hematological analyzer and flow cytometry. It was possible to indicate the presence of cells characterized by high fluorescence connected with high metabolic activity using a hematological analyzer and to determine their non-hematopoietic origin using flow cytometry. Using these methods together, we detected very quickly a high proportion of cancer cells in LNs. We noticed that it was possible to determine a high expression of EpCAM, TTF-1, Ki67, cytokeratin, HER, and differences between non-small-cell (NSCLC) and small-cell lung cancer (SCLC) for the antigens MUC-1, CD56, HLA-DR, CD39, CD184, PD-L1, PD-L2 and CTLA-4 on tumor cells. We report, for the first time, that the detection of tumor cells in LNs with the expression of specific antigens is easy to evaluate using a hematological analyzer and flow cytometry in EBUS/TBNA samples. Such precise characteristics of non-hematopoietic cells in LNs may be of great diagnostic importance in the detection of micrometastases.

Brain tumor–initiating cells (BTICs) drive glioblastoma growth through not fully understood mechanisms. Here, we found that about 8% of cells within the human glioblastoma microenvironment coexpress programmed cell death 1 (PD-1) and BTIC marker. Gain- or loss-of-function studies revealed that tumor-intrinsic PD-1 promoted proliferation and self-renewal of BTICs. Phosphorylation of tyrosines within the cytoplasmic tail of PD-1 recruited Src homology 2–containing phosphatase 2 and activated the nuclear factor kB in BTICs. Notably, the tumor-intrinsic promoting effects of PD-1 did not require programmed cell death ligand 1(PD-L1) ligation; thus, the therapeutic antibodies inhibiting PD-1/PD-L1 interaction could not overcome the growth advantage of PD-1 in BTICs. Last, BTIC-intrinsic PD-1 accelerated intracranial tumor growth, and this occurred in mice lacking T and B cells. These findings point to a critical role for PD-1 in BTICs and uncover a nonimmune resistance mechanism of patients with glioblastoma to PD-1– or PD-L1–blocking therapies.

Specific B-cell subsets can regulate T-cell immune responses, and are termed regulatory B cells (Breg). The majority of Breg cells described in mouse and man have been identified by IL-10 production and are known to suppress allergy and autoimmunity. However, Breg cell mediated immune suppression, independent of IL-10, also occurs. Here we show that Breg cells play a critical role in regulating humoral immunity mediated by CD4(+)CXCR5(+)PD-1(+) follicular helper T cells, and can suppress inflammation in autoimmune disease through elevated expression of PD-L1. We have also identified that these B cells are resistant to αCD20 B-cell depletion. This work describes how Breg cells are critical in humoral homoeostasis and may have implications for the regulation of autoimmune diseases.