Journal: Oncotarget

Article Title: PD1 is expressed on exhausted T cells as well as virus specific memory CD8+ T cells in the bone marrow of myeloma patients

doi: 10.18632/oncotarget.25882

Figure Lengend Snippet: (A, B, C) Frequency of EOMES high Tbet low of PD1+CD8+ T cells. Bone marrow cells were surface stained with anti CD3, CD4, CD8 and PD1 followed by intranuclear staining with anti-Tbet and anti-EOMES, or isotype controls . Gates were set on isotype controls. Figure shows % EOMES high Tbet low (A) and Tbet high EOMES low (B) of PD1+CD8+ T cells in patients with low plasma cell percentage (≤ 10%) or high plasma cell percentage (>10 % PC). (C) % EOMES high Tbet low and Tbet high EOMES low of PD1 expressing CD8 T cells in patients with high tumor load of 40% and above. (D-G) Cytotoxic activity against autologous myeloma cells. Purified CD8+ T cells from the bone marrow of patients were co-cultured with anti CD3/CD28 beads (D) or purified, autologous myeloma cells (E) or the T cells alone (F). For the last 4 hours of the culture period, anti CD8, PD1 and CD107a or isotype controls were added as described in the Materials and Method section. Figures show the staining of isotype control (dashed lines) and CD107a (solid lines) on one representative sample of three positive . (G) Pooled data for cytotoxic activity (% CD107a expression) of CD8+ T cells co-cultured with autologous myeloma cells. (H, I) Proliferation of CD8+PD1+ T cells in response to autologous APCs. CFSE labeled CD3+PD1+ cells from the bone marrow of two myeloma patients (1,2) were cultured with (H) or without (I) autologous, adherent cells in the presence of recombinant IL2 as described. (J) PD1+ EBV specific CD8+ T cells in the bone marrow of myeloma patients. PD1+ cells enriched from the bone marrow was cultured with autologous, adherent cells as described in Materials and Methods in the presence of EBV specific peptides. After 10 days of culture the cells were seeded onto IFNγ ELIspot strips together with differentiated DC and EBV peptides (filled bar), without peptides (open bar) or with anti CD3 antibody (striped bar). Spots were developed after 24 hrs. Figure shows representative data from one of the 6 EBV positive patients tested. Data from all the 6 patients are shown in Table .

Article Snippet: The cells were then added to the autologous, adherent cells and incubated in RPMI (10% human serum), 20 U/mL rIL2 (R&D systems 202-IL-010) for 6 days.

Techniques: Staining, Clinical Proteomics, Expressing, Activity Assay, Purification, Cell Culture, Control, Labeling, Recombinant, Enzyme-linked Immunospot