Lineage-Specific Expression of Human Immunodeficiency Virus (HIV) Receptor/Coreceptors in Differentiating Hematopoietic Precursors: Correlation With Susceptibility to T- and M-Tropic HIV and Chemokine-Mediated HIV Resistance by C. Chelucci, I. Casella, M. Federico, U. Testa, G. Macioce, E. Pelosi, R. Guerriero, G. Mariani, A. Giampaolo, H.J. Hassan, and C. Peschle Blood Volume 94(5):1590-1600 September 1, 1999 ©1999 by American Society of Hematology

Representative results of CXCR4 and CCR5 mRNA expression analyzed by RT-PCR in quiescent HPCs after purification (A) and during in vitro megakaryocytic (Mk), monocytic (Mo), granulocytic (G), or erythroid (E) differentiation (B). Representative results of CXCR4 and CCR5 mRNA expression analyzed by RT-PCR in quiescent HPCs after purification (A) and during in vitro megakaryocytic (Mk), monocytic (Mo), granulocytic (G), or erythroid (E) differentiation (B). In this experiment, HPCs were 97% CD34+ as evaluated by flow cytometry and 95% clonogenetic progenitors. Total RNA from CEM cell line (CXCR4) and monocytes (CCR5) and from UT-7 cell line was used as positive and negative controls, respectively. RT-PCR samples were normalized for GAPDH. C. Chelucci et al. Blood 1999;94:1590-1600 ©1999 by American Society of Hematology

Flow cytometric analyses of CXCR4 and CCR5 on 97% CD34+ HPCs Flow cytometric analyses of CXCR4 and CCR5 on 97% CD34+ HPCs. (A) Unstained control for evaluation of HPC physical parameters. Flow cytometric analyses of CXCR4 and CCR5 on 97% CD34+ HPCs. (A) Unstained control for evaluation of HPC physical parameters. (B) HPCs stained with PE- or FITC-conjugated control antibodies. Cells were double-labeled with an FITC-conjugated anti-CD34 along with CXCR4-PE MoAb (C) or CD34-PE along with anti–CCR5-FITC MoAb (clone 45502.111) (D) as described in Materials and Methods. C. Chelucci et al. Blood 1999;94:1590-1600 ©1999 by American Society of Hematology

Flow cytometric analysis of CXCR4 along megakaryocytic (Mk), monocytic (Mo), granulocytic (G), and erythroid (E) HPC differentiation. Flow cytometric analysis of CXCR4 along megakaryocytic (Mk), monocytic (Mo), granulocytic (G), and erythroid (E) HPC differentiation. Cells were collected for analysis at the indicated days of culture and double-labeled with PE-conjugated anti-CXCR4 MoAb along with the specific cell-lineage FITC-conjugated MoAbs (-CD61, -CD14, -CD15, -Glyc-A). Representative results from 1 of 3 independent experiments are shown. C. Chelucci et al. Blood 1999;94:1590-1600 ©1999 by American Society of Hematology

Flow cytometric analysis of CCR5 during megakaryocytic (Mk), monocytic (Mo), granulocytic (G), and erythroid (E) differentiation of HPCs. Cells were collected for analysis at the indicated days of culture and double-labeled with PE-conjugated anti-CCR5 MoAb... Flow cytometric analysis of CCR5 during megakaryocytic (Mk), monocytic (Mo), granulocytic (G), and erythroid (E) differentiation of HPCs. Cells were collected for analysis at the indicated days of culture and double-labeled with PE-conjugated anti-CCR5 MoAb (clone 2D7) along with the specific FITC-conjugated cell lineage MoAbs. Representative results from 1 of 3 independent experiments are shown. C. Chelucci et al. Blood 1999;94:1590-1600 ©1999 by American Society of Hematology

Immunofluorescence labeling of CXCR4 and CCR5 in normal monocytes and neutrophilic granulocytes isolated from PB. (A and D) Staining using matched control antibodies. Immunofluorescence labeling of CXCR4 and CCR5 in normal monocytes and neutrophilic granulocytes isolated from PB. (A and D) Staining using matched control antibodies. Monocytes were stained with either PE-conjugated CXCR4 (B) or CCR5 (C) MoAb along with CD14-FITC MoAb; neutrophils were labeled with either PE-conjugated CXCR4 (E) or CCR5 (F) MoAb along with CD15-FITC MoAb. C. Chelucci et al. Blood 1999;94:1590-1600 ©1999 by American Society of Hematology

Percentage of CD4+ cells during HPC (▵), monocytic (○), megakaryocytic (•), granulocytic (□), and erythroid (▪) differentiation (mean ± SEM values from 4 separate experiments). Percentage of CD4+ cells during HPC (▵), monocytic (○), megakaryocytic (•), granulocytic (□), and erythroid (▪) differentiation (mean ± SEM values from 4 separate experiments). Cells from unilineage culture, collected at the indicated days, were labeled with PE-conjugated Leu3A MoAb and analyzed by FACS for fluorescence intensity. C. Chelucci et al. Blood 1999;94:1590-1600 ©1999 by American Society of Hematology

Inhibition of T-tropic HIV infection of Mk precursors by SDF-1. Inhibition of T-tropic HIV infection of Mk precursors by SDF-1. Day 5 Mk culture cells were treated or not with 1 μg/mL SDF-1 and then inoculated with 0.1 m.o.i of NL4-3. HIV p24 production was measured through 9 days postinfection (p.i.) as described in Materials and Methods. (A) Percentage of p24 level decrease in SDF-1 treated from untreated (control values) Mks (mean ± SEM values from 3 independent experiments). (B) Infection inhibition results from a representative experiment. (▩), NL4-3; (▨), NL4-3 + SDF-1. C. Chelucci et al. Blood 1999;94:1590-1600 ©1999 by American Society of Hematology

Inhibition of M-tropic (A) and T-tropic (B) HIV infection of day 5 Mo precursors. Inhibition of M-tropic (A) and T-tropic (B) HIV infection of day 5 Mo precursors. Cells were treated or not with a cocktail of MIP-1, MIP-1β, and RANTES or SDF-1, and then inoculated with either BaL or NL4-3 HIV strains, respectively (see Materials and Methods). HIV p24 production was measured for 14 days postinfection (p.i.). Representative results from 1 of 3 independent experiments are shown. (□), BaL; (▪), BaL + MIP-1, MIP-1β, RANTES; (▩), NL4-3; (▨), NL4-3 + SDF-1. C. Chelucci et al. Blood 1999;94:1590-1600 ©1999 by American Society of Hematology