Inhibitory receptor expression by Melan-A specific CD8 T-cells depending on vaccination.

<p>(A) Co-expression of KLRG-1, TIM-3, PD-1 and CD160, and of LAG-3, BTLA, 2B4 and CTLA-4 by Melan-A specific CD8 T-cells. Blood samples from healthy donors (HD) or from patients before immunotherapy (before vacc.) or after peptide+IFA vaccination with or without CpG-ODN 7909 were enriched for CD8 T-cells using magnetic beads. Melan-A-specific CD8 T-cells were identified by staining with CD8-specific antibody and tetramer. Positivity for inhibitory receptors was defined respective to isotype controls. n = 4 for HD; n = 3 for before vacc.; n = 9 for after vaccination without CpG-ODN and n = 11 for after vaccination with CpG-ODN. Colors of the pie arcs depict the expression of individual inhibitory receptors, while the color in the pie depicts the number of co-expressed inhibitory receptors. Co-expression was analyzed with SPICE 5.2. p-values of the permutation test are shown in tables next to the corresponding pie charts. (B) Hierarchical clustering based on co-expression data of the eight inhibitory receptors shown in A, including the four differentiation subsets (N, CM, EM, EMRA) of total CD8 T-cells. (C) Mean expression and SD of four inhibitory receptors upregulated on Melan-A-specific T-cells with vaccination. Data from HD and from patients before vaccination were pooled for the group without vaccination (no vacc.). n = 7 for no vacc.; n = 9 for vaccination with CpG-ODN.</p

Expression of ligands of inhibitory receptors in melanoma metastases and by melanoma cell lines.

<p>(A,B) Paraffin-embedded tumor sections from 16 to 18 tumors were stained by immunohistochemistry for seven inhibitory receptors and CD8. (A) Representative stainings (magnification ×200) for each ligand investigated. (B) Summary of immunohistochemical stainings represented as percent of positive samples. Low (<10%), intermediate (int; 10–50%) and high (>50%) expression is indicated in a color scale. infilt: infiltration of CD8 T-cells in tumor cell nests; sec: secreted i.e. intra- and extracellular presence of galectin-9. (C) Summary of expression by melanoma cell lines on the surface or intracellular (ic) as percent of positive cell lines.</p

Expression profiles of inhibitory receptors with differentiation.

<p>(A) CD8 T-cell subsets were defined depending on expression of CCR7 and CD45RA, namely naive (N), central memory (CM), effector memory (EM) and effector memory RA⁺ (EMRA) cells. Gates used for inhibitory receptor analysis are shown in the four quadrants. (B) Mean values of inhibitory receptor expression in relation to the differentiation status. Individual values are shown in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030852#pone.0030852.s001" target="_blank">Figure S1B</a></i>. n = 31 for “staining 1” (KLRG-1, TIM-3, PD-1 and CD160); n = 21 for “staining 2” (LAG-3, BTLA, 2B4 and CTLA-4); four samples of staining 1 were from healthy donors, the remaining from melanoma patients. (C) Co-expression of KLRG-1, TIM-3, PD-1 and CD160 (staining 1) and of LAG-3, BTLA, 2B4 and CTLA-4 (staining 2). Colors of the pie arcs depict the expression of individual inhibitory receptors, while the color in the pie depicts the number of co-expressed inhibitory receptors. p-values of the permutation test are shown in tables next to the corresponding pie charts. Co-expression was analyzed with SPICE 5.2.</p

Schematic representation of inhibitory receptor co-expression according to differentiation status and physical location.

<p>Naive cells express BTLA and TIM-3. After peptide vaccination, Melan-A specific T-cells upregulate KLRG-1, 2B4, TIM-3 and PD-1, while they downregulate BTLA. Total CD8 T-cells upregulate similar inhibitory receptors, but less PD-1 and TIM-3. They also express CD160, which is not expressed by tumor-specific T-cells. In TILN, both total CD8 T-cells, which are to a large extent tumor-specific, and Melan-A specific T-cells downregulate KLRG-1 (and in total CD8 T-cells CD160) and concomitantly express more PD-1, LAG-3, TIM-3 and CTLA-4.</p

Expression of inhibitory receptors by CD8 T-cells derived from blood and tumor-infiltrated lymph nodes (TILNs).

<p>(A) Co-expression analysis of total CD8 T-cells. Colors of the pie arcs depict the expression of individual inhibitory receptors, while the color in the pie depicts the number of co-expressed inhibitory receptors. Co-expression was analyzed with SPICE 5.2. n = 9/8 (TILN) and 31/17 (blood) for staining 1 and staining 2 respectively. (B) Melan-A- (red) and EBV- (black) specific CD8 T-cells. Positivity for the inhibitory receptor was defined respective to isotype controls. Blood samples were from patients vaccinated either with CpG-ODN or without CpG-ODN. n = 20/21 (blood; Melan-A/EBV) and n = 9/6 (TILN; Melan-A/EBV) for staining 1; n = 23/24 (blood; Melan-A/EBV) and n = 8/5 (TILN; Melan-A/EBV) for staining 2 except BTLA, and n = 8/7 (TILN; Melan-A/EBV) for BTLA.</p

Expression of inhibitory receptors on self/tumor-specific T-cells.

<p>Expression of inhibitory receptors by Melan-A, NY-ESO-1 and MAGE-A10-specific T-cells from a representative patient (LAU 1169). CD8 T-cells were enriched using magnetic bead sorting. Melan-A- (black), NY-ESO-1- (green) and MAGE-A10- (blue) specific CD8 T-cells were identified by tetramer staining as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030852#s2" target="_blank">Materials and Methods</a> section. An isotype control (grey) is shown as reference.</p