American Society of Hematology

Figure 2

$Figure 2. PD-1 expression in T cells of HL patients and functional analysis of PD-1–PD-L signaling. (A) Histograms showing PD-1 expression in tumor-infiltrating T cells of 3 HL patients. CD4+ and CD8+ T cells in bulk HL cell suspensions were analyzed for PD-1. MFI indicates mean fluorescent intensity. (B) Proportion of PD-1+ and CD4+CD25+ cells in peripheral blood T cells of HL and B-NHL patients compared with those of healthy volunteers. Patients' characteristics are shown in Table 1. One HL patient was not analyzed for CD4+CD25+ cells. MC indicates mixed cellularity; NC, nodular sclerosis; LR, lymphocyte rich; MALT, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue; HBV, hepatitis B virus infection; HCV, hepatitis C virus infection; and MAC, mycobacterium avium complex infection. Patients classified as “during treatment” include those who are undergoing chemotherapy or radiotherapy. (C) Proportion of PD-1+ cells, Foxp3+ cells, and those positive for both PD-1 and Foxp3, in peripheral CD4+ T cells of 9 tumor-bearing HL patients. Representative dot-plot data of 2 patients: one had low and the other relatively high Foxp3 expression (Table 2). (D) Blockade of PD-Ls restores IFN-γ–producing function of HL-infiltrating T cells. Bulk tumor cell suspensions of 2 HL patients (left) and 1 DLBCL patient (right) were cultured with blocking antibodies, and IFN-γ levels in the supernatants were measured as described in “Methods.” Samples were analyzed in duplex. Error bars indicate standard deviation of duplicate measurements. (E) Blockade of PD-Ls inhibits tyrosine phosphorylation of SHP-2. After bulk HL cell suspensions were cultured with PD-L blocking antibodies or isotype control antibody, cell lysates were immunoprecipitated with anti–SHP-2 antibody and its tyrosine phosphorylation was examined by immunoblotting with PY20 antibody (top panel). The blot was subsequently reprobed with anti–SHP-2 antibody (bottom panel). The position of SHP-2 is indicated by arrowheads. Asterisks denote the immunoglobulin heavy chain. (F) Blockade of PD-Ls revive mainly the IFN-γ–producing function of CD4+ T cells. T-cell subsets were depleted or enriched from HL cell suspensions by MACS. IFN-γ production by each cell fraction was evaluated as in panel D.$

PD-1 expression in T cells of HL patients and functional analysis of PD-1–PD-L signaling. (A) Histograms showing PD-1 expression in tumor-infiltrating T cells of 3 HL patients. CD4⁺ and CD8⁺ T cells in bulk HL cell suspensions were analyzed for PD-1. MFI indicates mean fluorescent intensity. (B) Proportion of PD-1⁺ and CD4⁺CD25⁺ cells in peripheral blood T cells of HL and B-NHL patients compared with those of healthy volunteers. Patients' characteristics are shown in Table 1. One HL patient was not analyzed for CD4⁺CD25⁺ cells. MC indicates mixed cellularity; NC, nodular sclerosis; LR, lymphocyte rich; MALT, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue; HBV, hepatitis B virus infection; HCV, hepatitis C virus infection; and MAC, mycobacterium avium complex infection. Patients classified as “during treatment” include those who are undergoing chemotherapy or radiotherapy. (C) Proportion of PD-1⁺ cells, Foxp3⁺ cells, and those positive for both PD-1 and Foxp3, in peripheral CD4⁺ T cells of 9 tumor-bearing HL patients. Representative dot-plot data of 2 patients: one had low and the other relatively high Foxp3 expression (Table 2). (D) Blockade of PD-Ls restores IFN-γ–producing function of HL-infiltrating T cells. Bulk tumor cell suspensions of 2 HL patients (left) and 1 DLBCL patient (right) were cultured with blocking antibodies, and IFN-γ levels in the supernatants were measured as described in “Methods.” Samples were analyzed in duplex. Error bars indicate standard deviation of duplicate measurements. (E) Blockade of PD-Ls inhibits tyrosine phosphorylation of SHP-2. After bulk HL cell suspensions were cultured with PD-L blocking antibodies or isotype control antibody, cell lysates were immunoprecipitated with anti–SHP-2 antibody and its tyrosine phosphorylation was examined by immunoblotting with PY20 antibody (top panel). The blot was subsequently reprobed with anti–SHP-2 antibody (bottom panel). The position of SHP-2 is indicated by arrowheads. Asterisks denote the immunoglobulin heavy chain. (F) Blockade of PD-Ls revive mainly the IFN-γ–producing function of CD4⁺ T cells. T-cell subsets were depleted or enriched from HL cell suspensions by MACS. IFN-γ production by each cell fraction was evaluated as in panel D.

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