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

Wu L, Paxton WA, Kassam N, Ruffing N, Rottman JB, Sullivan N, Choe H, Sodroski J, Newman W, Koup RA, Mackay CR - J. Exp. Med. (1997)

Bottom Line: A comparison of normal and CCR5 delta32 heterozygotes revealed markedly reduced expression of CCR5 on T cells from the heterozygotes.Low expression of CCR5 correlated with the reduced infectability of T cells with macrophage-tropic HIV-1, in vitro.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.

View Article: PubMed Central - PubMed

Affiliation: LeukoSite, Inc., Cambridge, Massachusetts 02142, USA.

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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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.
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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.

Mentions: The CCR5 mutant allele occurs at a frequency of 0.092 in the Caucasian population (35, 36). Individuals homozygous or heterozygous for the CCR5 mutant allele were identified by screening individuals at low and high risk for HIV-1 infection, using PCR. Fig. 3 A shows the PCR pattern for the three types of individuals: wild-type homozygous (+/+), heterozygous (+/Δ32), and homozygous for the deletion (Δ32/Δ32). A representative immunofluorescent staining of blood lymphocytes from each type of individual is shown in Fig. 3 B. An interesting finding was the very weak expression of CCR5 on +/Δ32 individuals. As expected, lymphocytes from Δ32/Δ32 individuals were CCR5 negative.


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

Wu L, Paxton WA, Kassam N, Ruffing N, Rottman JB, Sullivan N, Choe H, Sodroski J, Newman W, Koup RA, Mackay CR - J. Exp. Med. (1997)

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.
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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.
Mentions: The CCR5 mutant allele occurs at a frequency of 0.092 in the Caucasian population (35, 36). Individuals homozygous or heterozygous for the CCR5 mutant allele were identified by screening individuals at low and high risk for HIV-1 infection, using PCR. Fig. 3 A shows the PCR pattern for the three types of individuals: wild-type homozygous (+/+), heterozygous (+/Δ32), and homozygous for the deletion (Δ32/Δ32). A representative immunofluorescent staining of blood lymphocytes from each type of individual is shown in Fig. 3 B. An interesting finding was the very weak expression of CCR5 on +/Δ32 individuals. As expected, lymphocytes from Δ32/Δ32 individuals were CCR5 negative.

Bottom Line: A comparison of normal and CCR5 delta32 heterozygotes revealed markedly reduced expression of CCR5 on T cells from the heterozygotes.Low expression of CCR5 correlated with the reduced infectability of T cells with macrophage-tropic HIV-1, in vitro.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.

View Article: PubMed Central - PubMed

Affiliation: LeukoSite, Inc., Cambridge, Massachusetts 02142, USA.

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.

Show MeSH
Related in: MedlinePlus