Progressive changes in composition of lymphocytes in lung tissues from patients with non-small-cell lung cancer

Immune cell infiltration is a common feature of many human solid tumors. Innate and adaptative immune systems contribute to tumor immunosurveillance. We investigated whether tumors evade immune surveillance by inducing states of tolerance and/or through the inability of some immune subpopulations to effectively penetrate tumor nests. Immunohistochemistry and flow cytometry analysis were used to study the composition and distribution of immune subpopulations in samples of peripheral blood, tumor tissue (TT), adjacent tumor tissue (ATT), distant non-tumor tissue (DNTT), cancer nests, cancer stroma, and invasive margin in 61 non-small-cell lung cancer (NSCLC) patients. A significantly higher percentage of T and B cells and significantly lower percentage of NK cells were detected in TT than in DNTT. Memory T cells (CD4+CD45RO+, CD8+CD45RO+) and activated T cells (CD8+DR+) were more prevalent in TT. Alongside this immune activation, the percentage of T cells with immunosuppressive activity was higher in TT than in DNTT. B- cells were practically non-existent in tumor nests and were preferentially located in the invasive margin. The dominant NK cell phenotype in peripheral blood and DNTT was the cytotoxic phenotype (CD56+ CD16+), while the presence of these cells was significantly decreased in ATT and further decreased in TT. Finally, the immunologic response differed between adenocarcinoma and squamous cell carcinoma and according to the tumor differentiation grade. These findings on the infiltration of innate and adaptative immune cells into tumors contribute to a more complete picture of the immune reaction in NSCLC.

San José, California, USA). For multiparametric analysis, a minimum of 4,000 events in lung tissues or 30,000 in peripheral blood in the lymphocyte collection gate (CD45 + versus SSClow) were collected in a FACS CantoTM II analyzer. Data were analyzed using BD FACSDiva Software v.8.01 (BD).
An electronic gate was set for CD45+/SSC low and a minimum of 10, 000 events in this gate were recorded for each sample. An additional electronic gate was made from the lymphocyte gate for T-cells (CD3 + ) and either CD4 + or CD8 + , and the results were expressed as the relative percentage of each marker in the gate for CD4 or CD8 lymphocyte cells. CD4 (and CD8) cells can be classified as effector memory cells by expression of CD45RO. Human regulatory T cell cocktail was used to identify regulatory T cell subsets defined as CD127 low CD25 bright CD4 + . The amount of Treg was expressed as a percentage of total CD4 cells. Other T CD4 or CD8 subsets analyzed were CD39 + CD4 + , and CD39 + CD8 + , CD57 + CD8 + T cells. We also evaluated the presence of mature CD56 + CD16 + , NK cells and subsets. NK cell subpopulations were determined by the selection of CD45+, CD3-, CD20-cells in the lymphocyte gate FSC low /SSC low . The gating strategy adopted offers the ability to detect several subpopulations within the plot: single positive CD16 + , single positive CD56 + and double positive CD16 + CD56 + mature NKcells and low cytolytic CD56 bright , CD16 − NK-cells. Immature NK-cells that express CD161 but not CD16 or CD56 could also be detected.
The percentage lymphocyte infiltration in each tumor was obtained by measuring CD45 + /SSC low cells within the collection gate as a proportion of the total cell count. These data were used to analyze the relationship between the inflammatory infiltration from subsets and clinical-pathological features.

Immunohistochemistry and image analysis
Tissue samples were fixed in 10% neutral formalin and embedded in paraffin; 4 μm-thick sections were taken from the paraffin blocks, mounted on pretreated slices, and stained for CD45, CD20, CD3, CD4 CD8 or CD68., Immunohistochemical staining was carried out using the of UltraVision Quanto (peroxidase) immunohistochemistry detection system (MAD-021881QK/MAD-001881QK Master Diagnóstica) following manufacturer's instructions. In short, paraffin sections were dewaxed and endogenous peroxidase activity blocked in a solution of hydrogen peroxide for 10 min. For antigen retrieval, sections were treated with sodium citrate buffer (pH 6.0). After incubation with the primary antibodies for 16 hours at 4ºC and washing 3 times with PBS for 5 minutes, tissue sections were again incubated for 10 min with the primary antibodies Amplificator (UltraVisionQuanto) and then washed, and incubated with the polymer Quanto (Ultra Vision Quanto) for 10 minutes. DAB was used as chromogen and sections were counterstained with hematoxylin.
No staining was obtained when non-immune serum or PBS was used instead of the primary antibody, confirming the specificity of the latter. All specimens were www.impactjournals.com/oncotarget/

Oncotarget, Supplementary Materials 2016
Progressive changes in composition of lymphocytes in lung tissues from patients with non-small-cell lung cancer