CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro - PubMed (original) (raw)

CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro

L Wu et al. J Exp Med. 1997.

Abstract

Chemokine receptors serve as coreceptors for HIV entry into CD4+ cells. Their expression is thought to determine the tropism of viral strains for different cell types, and also to influence susceptibility to infection and rates of disease progression. Of the chemokine receptors, CCR5 is the most important for viral transmission, since CCR5 is the principal receptor for primary, macrophage-tropic viruses, and individuals homozygous for a defective CCR5 allele (delta32/delta32) are highly resistant to infection with HIV-1. In this study, CCR5-specific mAbs were generated using transfectants expressing high levels of CCR5. The specificity of these mAbs was confirmed using a broad panel of chemokine receptor transfectants, and by their non-reactivity with T cells from delta32/delta32 individuals. CCR5 showed a distinct pattern of expression, being abundant on long-term activated, IL-2-stimulated T cells, on a subset of effector/memory T cells in blood, and on tissue macrophages. A comparison of normal and CCR5 delta32 heterozygotes revealed markedly reduced expression of CCR5 on T cells from the heterozygotes. There was considerable individual to individual variability in the expression of CCR5 on blood T cells, that related to factors other than CCR5 genotype. Low expression of CCR5 correlated with the reduced infectability of T cells with macrophage-tropic HIV-1, in vitro. Anti-CCR5 mAbs inhibited the infection of PBMC by macrophage-tropic HIV-1 in vitro, but did not inhibit infection by T cell-tropic virus. Anti-CCR5 mAbs were poor inhibitors of chemokine binding, indicating that HIV-1 and ligands bind to separate, but overlapping regions of CCR5. These results illustrate many of the important biological features of CCR5, and demonstrate the feasibility of blocking macrophage-tropic HIV-1 entry into cells with an anti-CCR5 reagent.

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Figures

Figure 1

Identification of CCR5-specific mAbs. (A) mAb 3A9 staining of various L1.2 transfectants. Stable L1.2 transfectants expressing either CCR1, CCR2b, CCR3, CCR4, CCR5, CXCR1 (IL-8 RA), CXCR2 (IL-8RB), CXCR3, and CXCR4 (fusin/Lestr) were stained with anti-CCR5 mAb 3A9. Negative control staining for all the L1.2 transfectants (not shown) resembled the staining shown for 3A9 on CCR1 L1.2 cells. (B) Lymphoblast as well as small lymphocyte staining within the paracortical region of mediastinal lymph node. (C) macrophage staining within the medullary region of a mediastinal lymph node. Photomicrographs (B) ×400; (C) ×500.

Figure 2

CCR5 expression on various populations of blood lymphocytes. A two-color staining protocol was used to assess for expression of CCR5 (_x_-axis in all plots) and the T cell subset markers CD4 and CD8, the naive/memory markers CD45RO and CD45RA, the activation markers CD26 and CD25, as well as L-selectin, CD95 (Fas) and CD56. The subset marker staining (_y_-axis) is indicated for each plot. Quadrants were set according to the staining of control mAbs. The staining was representative of eight donors analyzed.

Figure 3

Expression of CCR5 on T cells from normal (+/+), heterozygous (+/Δ32), and Δ32 homozygous (Δ32/Δ32) individuals. (A) Identification of +/+, +/Δ32, and Δ32/Δ32 individuals by PCR. Genomic DNA was isolated from PBMC of selected blood donors. PCR reactions were carried out using a set of 5′- and 3′- primers as described in Materials and Methods, and the reaction products were run on a 4% Nusieve GTG agarose gel and DNA bands stained by ethidium bromide. Under these conditions, a 174-bp band was detected for a +/+ individual (donor 5), a 142-bp band for Δ32 homozygous individuals (donors 1 and 2), and both 172- and 142-bp bands for heterozygous (+/Δ32) individuals (donors 3 and 4). Lane M shows the molecular weight markers. (B) Assessment of CCR5 expression on blood lymphocytes from +/+, +/Δ32, and Δ32/Δ32 individuals. Lymphocytes from the three types of individuals were stained with the anti-CCR5 mAb 3A9, and analyzed on the FACS®. Dot plots show fluorescence intensity (_y_-axis) and forward scatter (cell size, _x_-axis). The horizontal line in each plot indicates the point above which cells were considered positive, based on isotypematched control staining. This level of staining in all three plots resembled the staining of 3A9 shown for the Δ32/Δ32 individual. The staining profiles shown were representative of over 35 analyzed for +/+ individuals, 11 for +/Δ32 individuals, and 4 for Δ32/Δ32 individuals, although variability was observed (see below). (C ) Staining of anti-CD3–activated, rhIL-2–stimulated T cells from +/+, +/Δ32 and Δ32/Δ32 individuals. PBMC were activated with anti-CD3 mAb, and maintained in rhIL-2 for 21 d. Cell size (forward light scatter) is shown on the _x_-axis, and staining with mAb 3A9 on the _y_-axis.

Figure 4

Heterogeneity of CCR5 expression. Blood lymphocytes from 36 +/+ individuals, 11 +/Δ32 individuals, and 4 Δ32/Δ32 individuals were assessed for percentage CCR5 positive (3A9 staining) cells (left), and MFI of the positive subset (right). Each point represents the value obtained for a single individual. Staining was performed using mAb 3A9, and FACS® analysis. To calculate MFI, the positively stained subset was gated, according to control staining, and MFI was calculated using CellQuest software.

Figure 5

CCR5 surface expression correlates with macrophage-tropic HIV infectability. PBMC from 8 subjects were tested for CCR5 genotype by PCR. Genotypes are represented by (+) for a wild-type allele and (−) for a Δ32 allele. PBMC were stimulated with anti-CD3 antibody and carried for 16 d in the presence of rhIL-2. CD8+ cells were depleted using immunomagnetic beads. Expression of CCR5 or CXCR4 on the surface of CD4+ cells was determined by dual staining with anti-CD4 antibody and mAb 3A9 (CCR5) or 12G5 (CXCR4), respectively, on a FacsCaliber® (Becton Dickinson). Enriched CD4+ cells were inoculated at 2 × 105 cells per well with 600 TCID50 of JR-CSF (a) or R3H (b) and p24 production was measured on days 4, 7, and 11. Based on the growth rates of the two isolates in vitro, day 4 and day 11 p24 results for JR-CSF and R3H, respectively, are shown (dark bars) in comparison to percentage of cells expressing specific coreceptors (stippled bars).

Figure 6

Inhibition of macrophage-tropic HIV-1 infection of PBMC by anti-CCR5 mAb. 7-d PHA-activated CD4+ cells from two CCR5 wild-type homozygotes (LW4 and LW5) were inoculated with 600 TCID of JR-CSF (a) or R3H (b) alone (▪) or in the presence of 100 μg/ml of CCR5 mAb 3A9 (•) or 200 ng each of recombinant RANTES, MIP-1α, and MIP-1β (▵). HIV-1 p24 production was measured over the course of 9 d. To determine the sensitivity of JR-CSF to inhibition by 3A9, the amount of viral inhibition was calculated when serial fivefold dilutions of mAb 3A9 were added to the PBMC at the time of virus inoculation (c). 50% inhibitory doses (ID50) on LW4 (▪) and LW5 (♦) PBMC were 0.5 and 2.3 μg/ml. 90% inhibitory doses (ID90) were 17.2 and 15.3 μg/ml.

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CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro - PubMed (original) (raw)