Fig. 1: Soluble factors released by CLL cells enhance PD-1 expression on CTLs and suppress their ability to form functional ISs.

A Workflow for CTL generation from CD8⁺ cells purified from buffy coats of healthy donors and their treatment with media conditioned by either healthy B cells or B cells purified from CLL patients. RT-PCR analysis of mRNA (B) and flow cytometric analysis of surface (C) expression of PD-1 in CD8⁺ cells purified from buffy coats, stimulated with anti-CD3/CD28 mAb-coated beads + IL-2 for 48 h (CTLs), in the presence of conditioned media. A representative flow cytometric histogram is shown (n buffy coats from healthy donors = 9, Mann-Whitney Rank Sum test). Immunofluorescence analysis of pTyr (D), F-actin (E), CD3ζ. (F), and PCNT (G) in CTLs activated as in (A-C), mixed with Raji cells (APCs) either unpulsed or pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 15 min at 37 °C. Data are expressed as % of 15-min SAg-specific conjugates harboring staining at the IS ( ≥ 50 cells/sample, n independent experiments = 3, one-way ANOVA test). Representative images (medial optical sections) of the T cell:APC conjugates are shown. Scale bar, 5 μm. Right panels. Relative fluorescence intensity of pTyr (D), F-actin (E), and CD3ζ (F) at the IS (recruitment index, RI; 10 cells/sample, n independent experiments = 3, one-way ANOVA test). G right panel. Measurement of the distance (μm) of the centrosome (PCNT) from the CTL:APC contact in conjugates formed as above (10 cells/sample, n independent experiments = 3, one-way ANOVA test). H Flow cytometric analysis of target cell killing by CTLs cultured for 7 days in conditioned media, using SAg-loaded CFSE-labeled Raji cells as targets at an E:T cell ratio 2.5:1, 5:1 and 10:1. Cells were cocultured for 4 h and stained with propidium iodide prior to processing for flow cytometry. Analyses were carried out gating on CFSE⁺/PI⁺ cells. The histogram shows the percentage (%) of target cells lysed (n ≥ 3, two-way ANOVA test) I. Flow cytometric analysis of degranulation of CTLs cultured for 7 days as in (H), then cocultured with CFSE-stained SAg-loaded Raji cells for 4 h. The histogram shows the percentage (%) of LAMP1⁺ CTLs, measured gating on the CSFE-negative population (n independent experiments ≥ 3, two-way ANOVA test). L Immunofluorescence analysis of pTyr, F-actin, CD3ζ, and PCNT in CTLs activated for 48 h in the presence of conditioned media, mixed with SAg-loaded Raji cells (APCs), and incubated for 15 min at 37 °C. Immediately before mixing the cells, either isotype control (iso Ab) or anti-PD-1 (PD-1 Ab) antibodies were added to CTLs. Data are expressed as % of 15-min SAg-specific conjugates harboring pTyr, F-actin, CD3ζ, and PCNT staining at the IS ( ≥ 50 cells/sample, n independent experiments = 3, one-way ANOVA test). Data are expressed as mean ± SD. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05.