American Society of Hematology

Figure 2.

$Figure 2. CCR5 modulation on surface of CD4+ lymphocytes. CCR5 expression was evaluated by 2D7 Mab indirect binding, and relative surface expression was evaluated as the percentage of CCR5 expression on CD4+ cells. (A) CCR5 down-regulation in response to presence of 200 ng Abs for 48 hours. Total serum Abs were purified on Sepharose column and quantified by ELISA. Ig fractions were then affinity-purified on the relative CCR5 peptide and incubated with CD4+ lymphocytes. From the top: cells were not incubated with Igs; total Igs from healthy control; RANTES (50 nM); CCR5/peptide 3-specific Ig from LTNPs no. 22, no. 21, and no. 20. The data are representative of 2 experiments performed. (B) Kinetic of CCR5 down-regulation by 200 ng CCR5/peptide 3–specific Ig from LTNP no. 20. Total serum Abs were affinity-purified on Sepharose column and quantified by ELISA. Ig fractions were then affinity-purified on the relative CCR5 peptide and incubated with CD4+ lymphocytes for 1, 12, 24, and 48 hours before analysis. As positive controls, cells were not incubated with Igs, and as negative control cells were incubated with Igs from HCs. The data are representative of 3 experiments performed. (C) CCR5 expression on CD4+ lymphocytes from some LTNP patients carrying (no. 22, no. 20, no. 152) or not carrying (L01, M02, and C03) anti-CCR5 Abs. CD4+ lymphocytes from LTNPs no. 22, no. 20, and no. 152 were cultured for a further 7 days in the absence of anti-CCR5 Abs. The data are representative of 2 experiments performed. Error bars represent SDs of 3 replicates per each data point. (D) Susceptibility of infection by either HIV no. 36 (R5) or HIV no. 45 (R5, X4, R3) on CD4+ lymphocytes from some LTNPs carrying (no. 22, no. 20, no. 152) or not carrying (L01, M02, and C03) anti-CCR5 Abs. The data are representative of 2 experiments performed. Error bars represent SDs of 3 replicates per each data point. (E) Abs to first loop of CCR5 induce receptor endocytosis by clathrin-dependent pathway. Serum Igs from 2 LTNPs carrying CCR5 specific Abs were affinity-purified on Sepharose column and quantified by ELISA. Ig fractions were then affinity-purified on the relative CCR5 peptide (peptide 3) and incubated on CCR5-transfected cells for 48 hours in the presence or not of specific chemicals. All experiments were repeated twice, and SDs are shown.$

CCR5 modulation on surface of CD4⁺ lymphocytes. CCR5 expression was evaluated by 2D7 Mab indirect binding, and relative surface expression was evaluated as the percentage of CCR5 expression on CD4⁺ cells. (A) CCR5 down-regulation in response to presence of 200 ng Abs for 48 hours. Total serum Abs were purified on Sepharose column and quantified by ELISA. Ig fractions were then affinity-purified on the relative CCR5 peptide and incubated with CD4⁺ lymphocytes. From the top: cells were not incubated with Igs; total Igs from healthy control; RANTES (50 nM); CCR5/peptide 3-specific Ig from LTNPs no. 22, no. 21, and no. 20. The data are representative of 2 experiments performed. (B) Kinetic of CCR5 down-regulation by 200 ng CCR5/peptide 3–specific Ig from LTNP no. 20. Total serum Abs were affinity-purified on Sepharose column and quantified by ELISA. Ig fractions were then affinity-purified on the relative CCR5 peptide and incubated with CD4⁺ lymphocytes for 1, 12, 24, and 48 hours before analysis. As positive controls, cells were not incubated with Igs, and as negative control cells were incubated with Igs from HCs. The data are representative of 3 experiments performed. (C) CCR5 expression on CD4⁺ lymphocytes from some LTNP patients carrying (no. 22, no. 20, no. 152) or not carrying (L01, M02, and C03) anti-CCR5 Abs. CD4⁺ lymphocytes from LTNPs no. 22, no. 20, and no. 152 were cultured for a further 7 days in the absence of anti-CCR5 Abs. The data are representative of 2 experiments performed. Error bars represent SDs of 3 replicates per each data point. (D) Susceptibility of infection by either HIV no. 36 (R5) or HIV no. 45 (R5, X4, R3) on CD4⁺ lymphocytes from some LTNPs carrying (no. 22, no. 20, no. 152) or not carrying (L01, M02, and C03) anti-CCR5 Abs. The data are representative of 2 experiments performed. Error bars represent SDs of 3 replicates per each data point. (E) Abs to first loop of CCR5 induce receptor endocytosis by clathrin-dependent pathway. Serum Igs from 2 LTNPs carrying CCR5 specific Abs were affinity-purified on Sepharose column and quantified by ELISA. Ig fractions were then affinity-purified on the relative CCR5 peptide (peptide 3) and incubated on CCR5-transfected cells for 48 hours in the presence or not of specific chemicals. All experiments were repeated twice, and SDs are shown.

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