Validation of the intra-day and inter-day assays.

<p>Validation of the intra-day and inter-day assays.</p

Mean (±SD) plasma concentration–time profiles of 3MBM and 4MBM administered to 12 Sprague–Dawley rats.

<p>Mean (±SD) plasma concentration–time profiles of 3MBM and 4MBM administered to 12 Sprague–Dawley rats.</p

Precursor ion and product ion spectra of 3MBM (a), 4MBM (b) and PPD (c).

<p>Precursor ion and product ion spectra of 3MBM (a), 4MBM (b) and PPD (c).</p

Chemical structures of MT (a), PPD (b), 3MBM (c) and 4MBM (d).

<p>Chemical structures of MT (a), PPD (b), 3MBM (c) and 4MBM (d).</p

PK parameters of 3MBM and 4MBM in rat plasma (n = 6, mean ± SD) after oral administration of 3MBM and 4MBM.

<p>PK parameters of 3MBM and 4MBM in rat plasma (n = 6, mean ± SD) after oral administration of 3MBM and 4MBM.</p

Differences in TCR-Vβ Repertoire and Effector Phenotype between Tumor Infiltrating Lymphocytes and Peripheral Blood Lymphocytes Increase with Age

<div><p>Tumor infiltrating lymphocytes (TIL) reflect the host's anti-tumor immune response, and can be a valuable predictor of prognosis. However, many properties of TIL are not fully understood. In the present study, TCR-Vβ repertoires of cancer patients were primarily analyzed by flow cytometry. Abnormally expressed TCR-Vβ subfamilies were generally found in both TIL and peripheral blood lymphocytes (PBL) of each patient. Of note, increased patient age was associated with increasingly biased TCR-Vβ repertoire in TIL but not in PBL, and the dispersion degree of the differences of TCR-Vβ subfamilies between TIL and PBL correlated positively with age (<i>P</i> = 0.007). Utilizing immunoscope analysis, we identified the age-related reduction in TCR-Vβ diversity, but polyclonal pattern was predominant in significantly expanded TCR-Vβ subfamilies. In addition, we found that older patients possessed a decreased ratio of CD8⁺CD62L⁺ non-effector cells in TIL compared to PBL, implying age-related increase of CD8⁺CD62L⁻ effector cells in TIL. The colocalization analysis of CD8 and CD3, however, suggested the suppressed activity of these effector cells in tumor microenvironment. These findings further elucidate the properties of TIL, showing an increasing difference between TIL and PBL with age, which may provide insight for the development of effective immunotherapies for cancer patients of different ages.</p></div

Analysis of clonal expansion in TIL of 5 lung cancer patients.

<p>Seven TCR subfamilies (BV3, BV7, BV8, BV13, BV16, BV17, and BV21) overexpressed in TIL of five lung cancer patients were selected to show the CDR length distribution. Decreased numbers and/or biased distribution of CDR3 peaks with increased age of patients were shown. TCRBV21, which did not display remarkable overexpression by FCM detection, but showed oligoclonal/monoclonal expansion pattern in 3 out of 5 TIL specimens, was selected as the control. The numbers in brackets are the ages of patients. The arrows point to monoclonal expansion peaks. The triangle (▴) means overexpression, and inverted-triangle (▾) means underexpression, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102327#pone-0102327-g001" target="_blank">Figure 1</a>.</p

TCR repertoire in TIL and PBL of cancer patients.

<p>The TIL or peripheral blood lymphocytes were stained with anti-CD3 and 24 anti-TCRBV antibodies from a TCR Vβ Repertoire Kit. After lysing erythrocytes, washing, and fixing, TIL or PBL were subjected to FCM analysis. For a given TCRBV subfamily expression, any value higher or lower than the mean of the healthy control group, plus or minus 3 SD, was considered abnormal expression (higher labeled with triangle ▴; lower labeled with inverted-triangle ▾). PL indicates patient with lung cancer, PC indicates patient with colon cancer, PH indicates patient with liver cancer.</p

Colocalization analysis of CD3 and CD8 on TIL of patients and on PBL of healthy controls.

<p>TIL of cancer patients and PBL of healthy controls, either freshly isolated (<b>A</b>) or stimulated (<b>B</b>) with 1000 U/ml IFN-γ (at day 0), 50 ng/ml OKT3 (at day 1) and 500 U/ml IL-2 (from day 1 onward to day 6), were separately incubated with mAbs, CD8-FITC and CD3-PC5. The cells were observed with a confocal laser scanning microscope. The colocalization of CD3 and CD8 was analyzed, and colocalization index (r) was calculated by FV10-ASW2.1 software. For each group, 30 different cells from 3 individual were used for calculating the colocalization index (<b>C</b>). *<i>P</i><0.05; **<i>P</i><0.01, unpaired 2-tailed Student's <i>t</i> test.</p

Changed T cell subsets.

<p>The freshly isolated TIL or peripheral blood was stained with directly conjugated mouse mAbs against human antigens, CD45RO-ECD/CD8-FITC, or CD3-PC5/CD8-FITC/CD62L-PE. The ratios of percentage of CD8⁺CD62L⁺ subset (gated CD3⁺ cells) in TIL to that in coupled PBL (<b>A</b>) and percentage of CD45RO⁺CD8⁺ subset in TIL to that in coupled PBL (<b>B</b>) were matched with the ages of patients. The coupled percentages of CD8⁺CD62L⁺ subset (gated CD3⁺ cells) in TIL and PBL (<b>C</b>) and CD45RO⁺CD8⁺ subset in TIL and PBL (<b>D</b>) were matched with the ages of patients. The data for PBL of patient PC2 (age 44) is not available.</p