Improved metastasis-free survival in nonadjuvantly treated postmenopausal breast cancer patients with chemokine receptor 5 del32 frameshift mutations.

The CC-chemokine receptor CCR5 has been associated with cancer progression and metastasis. CCR5 blockers such as Maraviroc are tested in metastatic cancer patients. A mutant allele of CCR5, CCR5-delta32 (CCR5del32), which encodes for a protein with a trans-dominant negative effect on the wildtype protein, is frequently found in populations of northern European origin. We set out to determine if the CCR5del32 genotype is associated with progression of breast cancer. Here, we genotyped 414 breast cancer patients and investigated whether the CCR5 genotype had an association with the likelihood to metastasize within specific subgroups of this cohort. The findings were subsequently confirmed in an independent cohort of 1,017 breast cancer patients. Specifically within the postmenopausal subgroup of the initial cohort (n = 325) individuals carrying the CCR5del32 genotype exhibited a significantly longer metastasis-free survival (MFS, p = 0.038). In an independent cohort, CCR5del32 genotype was confirmed to be associated with prolonged MFS only in postmenopausal patients (n = 579, hazard ratio [HR] = 0.61, 95% confidence interval [95% CI] = 0.38-0.99, p = 0.044), and not in premenopausal patients (n = 438, HR = 1.01, 95% CI = 0.70-1.48, p = 0.94). Our results indicate that CCR5del32 genotype is associated with good prognosis in postmenopausal breast cancer patients. Considering this result, postmenopausal breast cancer patients who are wildtype for CCR5 genotype might benefit from CCR5 blockers, such as Maraviroc.

RANTES production from CD4+ lymphocytes correlates with host genotype and rates of human immunodeficiency virus type 1 disease progression.

Several chemokine and chemokine receptor parameters were measured in peripheral blood mononuclear cells obtained from patients before they became infected with human immunodeficiency virus type 1 (HIV-1). After HIV-1 infection, the parameters were compared with plasma HIV-1 RNA levels and with rates of CD4(+) lymphocyte decline. Patients who were heterozygous for the Delta32CCR5 allele had significantly higher levels of RANTES production from their CD4(+) lymphocytes than did patients who did not carry the Delta32CCR5 allele (P=.01). Higher RANTES production levels from ex vivo-activated CD4(+)-enriched lymphocytes, but not CD8(+) lymphocytes, correlated with lower plasma HIV-1 RNA levels 9-12 months after infection (P= .01) and with slower rates of CD4(+) lymphocyte decline (P= .002). CCR5 expression levels on ex vivo-activated CD4(+) lymphocytes did not correlate with markers of disease progression. These results further support the hypothesis that chemokine production levels are associated with HIV-1 replication in vivo.

N-linked glycosylation of the HIV type-1 gp120 envelope glycoprotein as a major determinant of CCR5 and CXCR4 coreceptor utilization.

The variable V1V2 and V3 regions of the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein (gp120) can influence viral coreceptor usage. To substantiate this we generated isogenic HIV-1 molecularly cloned viruses that were composed of the HxB2 envelope backbone containing the V1V2 and V3 regions from viruses isolated from a patient progressing to disease. We show that the V3 amino acid charge per se had little influence on altering the virus coreceptor phenotype. The V1V2 region and its N-linked glycosylation degree were shown to confer CXCR4 usage and provide the virus with rapid replication kinetics. Loss of an N-linked glycosylation site within the V3 region had a major influence on the virus switching from the R5 to X4 phenotype in a V3 charge-dependent manner. The loss of this V3 N-linked glycosylation site was also linked with the broadening of the coreceptor repertoire to incorporate CCR3. By comparing the amino acid sequences of primary HIV-1 isolates, we identified a strong association between high V3 charge and the loss of this V3 N-linked glycosylation site. These results demonstrate that the N-linked glycosylation pattern of the HIV-1 envelope can strongly influence viral coreceptor utilization and the R5 to X4 switch.

Up-regulation of HIV coreceptors CXCR4 and CCR5 on CD4(+) T cells during human endotoxemia and after stimulation with (myco)bacterial antigens: the role of cytokines.

Concurrent infections in patients with human immunodeficiency virus (HIV) infection stimulate HIV replication. Chemokine receptors CXCR4 and CCR5 can act as HIV coreceptors. The authors hypothesized that concurrent infection increases the HIV load through up-regulation of CXCR4 and CCR5. Using experimental endotoxemia as a model of infection, changes in HIV coreceptor expression were assessed in 8 subjects injected with lipopolysaccharide (LPS, 4 ng/kg). The expression of CXCR4 and CCR5 on CD4(+) T cells was increased 2- to 4-fold, 4 to 6 hours after LPS injection. In whole blood in vitro, LPS induced a time- and dose-dependent increase in the expression of CXCR4 and CCR5 on CD4(+) T cells. Similar changes were observed after stimulation with cell wall components of Mycobacterium tuberculosis (lipoarabinnomannan) or Staphylococcus aureus (lipoteichoic acid), or with staphylococcal enterotoxin B. LPS increased viral infectivity of CD4-enriched peripheral blood mononuclear cells (PBMCs) with a T-tropic HIV strain. In contrast, M-tropic virus infectivity was reduced, possibly because of elevated levels of the CCR5 ligand cytokines RANTES and MIP-1beta. LPS-stimulated up-regulation of CXCR4 and CCR5 in vitro was inhibited by anti-TNF and anti-IFN gamma. Incubation with recombinant TNF or IFN gamma mimicked the LPS effect. Anti-interleukin 10 (anti-IL-10) reduced CCR5 expression, without influencing CXCR4. In accordance, rIL-10 induced up-regulation of CCR5, but not of CXCR4. Intercurrent infections during HIV infection may up-regulate CXCR4 and CCR5 on CD4(+) T cells, at least in part via the action of cytokines. Such infections may favor selectivity of HIV for CD4(+) T cells expressing CXCR4. (Blood. 2000;96:2649-2654)

HIV-1 infectability of CD4+ lymphocytes with relation to beta-chemokines and the CCR5 coreceptor.

CD4+ lymphocytes exhibit variable permissiveness to the replication of HIV-1. A cohort of sexually-exposed-yet-uninfected individuals were previously shown to have CD4+ lymphocytes refractory for M-tropic viral replication. In particular, two individuals from this population whose CD4+ lymphocytes exhibited complete resistance to M-tropic viral replication were later shown to be homozygous for a 32bp (delta32) deletion in the gene encoding for CCR5. In screening diverse populations, HIV-1 infected individuals heterozygous for the delta32 allele were statistically favored in their disease course to harbor lower viral loads and exhibit slower rates of CD4+ cell loss when compared to control CCR5 wild-type individuals. Further comparative analysis between individuals in the exposed but uninfected cohort who demonstrated intermediate levels of in vitro viral replication and CD4+ lymphocytes isolated from uninfected delta32 heterozygous individuals indicate that reduced levels of in vitro M-tropic replication are a CCR5-related phenomenon: CD4+ lymphocytes from these individuals were more sensitive to the HIV-1 blocking effects of recombinant chemokines, displayed lower CCR5 cell surface expression levels and a proportionate increase in the production of RANTES when compared to CD4+ lymphocytes from control individuals. These results suggest that the CCR5 phenotype is important in determining the replicative capacity of M-tropic HIV-1 in vitro. The implications of these results with relation to HIV-1 transmission and disease progression are discussed.

Chemokine receptor allelic polymorphisms: relationships to HIV resistance and disease progression.

It is now well established that an array of CC and CXC chemokine receptors, in association with the CD4 molecule, can interact with the HIV-1 gp120 protein to facilitate viral fusion. A 32bp deletion in the CC chemokine receptor CCR5, the major M-tropic viral co-receptor, provides considerable protection against HIV-1 transmission and has been associated with a delay in disease progression. The effects of the Delta32 allele appear to be mediated through the phenotype of CCR5 expression as opposed to genotype. Here we discuss the potential effects that the Delta32 allele and other polymorphisms in the chemokine receptor repertoire may have on both HIV-1 transmission and disease progression.

Reduced HIV-1 infectability of CD4+ lymphocytes from exposed-uninfected individuals: association with low expression of CCR5 and high production of beta-chemokines.

We examined the human immunodeficiency virus type 1 infectability of CD4+ lymphocytes isolated from CCR5 wild-type individuals, individuals heterozygous for the delta32 allele of CCR5, and HIV-1-exposed but uninfected (EU) individuals who had CD4+ lymphocytes refractory to M-tropic viral replication. None of the EU individuals were found to be heterozygous for the delta32 allele. The CD4+ lymphocytes isolated from CCR5/delta32 and EU individuals were less infectable with an M-tropic viral isolate of HIV-1 than CCR5/CCR5 control individuals but were equally as infectable with a T-tropic viral isolate. The restriction to M-tropic viral isolate replication did not associate with any profound genotypic change in the CCR5 gene. CD4+ lymphocytes from CCR5/delta32 and CCR5/CCR5 EU individuals were more sensitive to the HIV-inhibitory effects of the recombinant beta-chemokines RANTES, MIP-1alpha, and MIP-1beta than were CD4+ lymphocytes from CCR5/CCR5 control individuals. CD4+ lymphocytes from EU individuals also showed increased sensitivity to recombinant beta-chemokines and low surface expression of CCR5. A phenotype of low CCR5 expression and high secretion of beta-chemokines is associated with reduced infectability of cells by M-tropic HIV-1. This phenotype may also be associated with protection against sexual transmission of HIV-1.

The HIV type 1 coreceptor CCR5 and its role in viral transmission and disease progression.

The purified CD4+ lymphocytes of a group of highly exposed but HIV-1-uninfected individuals were determined to be less susceptible to infection with multiple non-syncytium-inducing (NSI) primary isolates of HIV-1 than were CD4+ lymphocytes from nonexposed control individuals. This relative resistance to HIV-1 infection did not extend to T cell line-adapted or syncytium-inducing (SI) primary viral isolates, was restricted by the envelope glycoprotein, and was associated with an increased production of the C-C chemokines RANTES, MIP-1alpha, and MIP-1beta. The block to replication in CD4+ lymphocytes from two exposed-uninfected subjects was at the point of entry, as was the block imposed by the recombinant C-C chemokines RANTES, MIP-1alpha, and MIP-1beta. Resistance to infection and the high production of beta chemokines were characteristic of every CD4+ lymphocyte clone from the exposed-uninfected subjects. We have now identified the mechanism underlying this in vitro and in vivo resistance to infection: These individuals have inherited a homozygous 32-bp nucleotide deletion (delta32) within the gene encoding the coreceptor for primary NSI isolates of HIV-1 (CCR5). This deletion encodes a severely truncated and unstable protein that is not expressed on the cell surface. This allele is common in the Caucasian population, with a frequency of 0.0808, but is not found in people of African or Asian ancestry. To determine its role in HIV-1 transmission and disease progression, we analyzed the CCR5 genotype of 1252 homosexual men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). No infected participant was found to be homozygous for the delta32 allele whereas 3.6% of at-risk but uninfected Caucasian participants were homozygous, showing the highly protective role of this genotype against sexual acquisition of HIV-1. No evidence was found to suggest that heterozygotes were protected against HIV-1 infection, but a limited protective role against disease progression was noted. The delta32 allele of CCR5 is therefore an important host factor in HIV-1 transmission and pathogenesis.

Genetic subtype-independent inhibition of human immunodeficiency virus type 1 replication by CC and CXC chemokines.

We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1alpha against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4+ T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1beta, MIP-1alpha. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1beta, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1beta, MIP-1alpha.

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

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.

Macrophages and CD4+ T lymphocytes from two multiply exposed, uninfected individuals resist infection with primary non-syncytium-inducing isolates of human immunodeficiency virus type 1.

Despite multiple, high-risk sexual exposures, some individuals remain uninfected with human immunodeficiency virus type 1 (HIV-1). CD4+ lymphocytes from these individuals are less susceptible to infection in vitro with some strains of HIV-1, suggesting that the phenotype of the virus may influence its ability to interact with certain CD4+ cells. In the present study, we examined the susceptibility of CD4+ T lymphocytes and macrophages from two exposed uninfected individuals (EU2 and EU3) to infection with a panel of biologically cloned isolates of HIV-1 having either a non-syncytium-inducing (NSI) or a syncytium-inducing (SI) phenotype. Our results indicate that CD4+ T lymphocytes from EU2 and EU3 are resistant to infection with NSI isolates of HIV-1 but are susceptible to infection with primary SI isolates. In addition, we found that macrophages from EU2 and EU3 are resistant to infection with both NSI and SI isolates. The latter finding was confirmed by using several uncloned NSI and SI isolates obtained from patients during acute HIV-1 infection. In further experiments, env clones encoding glycoproteins characteristic of NSI or SI viruses were used in single-cycle infectivity assays to evaluate infection of CD4+ lymphocytes and macrophages from EU2 and EU3. Consistent with our previous results, we found that macrophages from these individuals are resistant to infection with NSI and SI env-pseudotyped viruses, while CD4+ T lymphocytes are resistant to NSI, but not SI, pseudotyped viruses. Overall, our results demonstrate that CD4+ cells from two exposed uninfected individuals resist infection in vitro with primary, macrophage-tropic, NSI isolates of HIV-1, which is the predominant viral phenotype found following HIV-1 transmission. Furthermore, infection with NSI isolates was blocked in both CD4+ T lymphocytes and macrophages from these individuals, suggesting that there may be a common mechanism for resistance in both cell types.

The role of a mutant CCR5 allele in HIV-1 transmission and disease progression.

A 32-nucleotide deletion (delta 32) within the beta-chemokine receptor 5 (CCR5) gene has been described in subjects who remain uninfected despite extensive exposure to HIV-1. This allele was found to be common in the Caucasian population with a frequency of 0.0808, but was not found in people of African or Asian ancestry. To determine its role in HIV-1 transmission and disease progression, we analyzed the CCRS genotype of 1252 homosexual men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). No infected participant was found to be homozygous for the delta 32 allele, whereas 3.6% of at-risk but uninfected Caucasian participants were homozygous, showing the highly protective role of this genotype against sexual acquisition of HIV-1. No evidence was found to suggest that heterozygotes were protected against HIV-1 infection, but a limited protective role against disease progression was noted. The delta 32 allele of CCR5 is therefore an important host factor in HIV-1 transmission and pathogenesis.

Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection.

Rare individuals have been multiply exposed to HIV-1 but remain uninfected. The CD4+ T-cells of two of these individuals, designated EU2 and EU3, are highly resistant in vitro to the entry of primary macrophagetropic virus but are readily infectable with transformed T-cell line adapted viruses. We report here on the genetic basis of this resistance. We found that EU2 and EU3 have a homozygous defect in CKR-5, the gene encoding the recently described coreceptor for primary HIV-1 isolates. These individuals appear to have inherited a defective CKR-5 allele that contains an internal 32 base pair deletion. The encoded protein is severely truncated and cannot be detected at the cell surface. Surprisingly, this defect has no obvious phenotype in the affected individuals. Thus, a CKR-5 allele present in the human population appears to protect homozygous individuals from sexual transmission of HIV-1. Heterozygous individuals are quite common (approximately 20%) in some populations. These findings indicate the importance of CKR-5 in HIV-1 transmission and suggest that targeting the HIV-1-CKR-5 interaction may provide a means of preventing or slowing disease progression.

HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.

The beta-chemokines MIP-1alpha, MIP-1beta and RANTES inhibit infection of CD4+ T cells by primary, non-syncytium-inducing (NSI) HIV-1 strains at the virus entry stage, and also block env-mediated cell-cell membrane fusion. CD4+ T cells from some HIV-1-exposed uninfected individuals cannot fuse with NSI HIV-1 strains and secrete high levels of beta-chemokines. Expression of the beta-chemokine receptor CC-CKR-5 in CD4+, non-permissive human and non-human cells renders them susceptible to infection by NSI strains, and allows env-mediated membrane fusion. CC-CKR-5 is a second receptor for NSI primary viruses.

Relative resistance to HIV-1 infection of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual exposure.

Some individuals remain uninfected with human immunodeficiency virus type-1 (HIV-1) despite multiple high-risk sexual exposures. We studied a cohort of 25 subjects with histories of multiple high-risk sexual exposures to HIV-1 and found that their CD8+ lymphocytes had greater anti-HIV-1 activity than did CD8+ lymphocytes from nonexposed controls. Further studies indicated that their purified CD4+ lymphocytes were less susceptible to infection with multiple primary isolates of HIV-1 than were CD4+ lymphocytes from the nonexposed controls. This relative resistance to HIV-1 infection did not extend to T-cell line-adapted strains, was restricted by the envelope glycoprotein, was not explained by the cell surface density of CD4 molecules, but was associated with the activity of the C-C chemokines RANTES, MIP-1alpha, and MIP-1beta. This relative resistance of CD4+ lymphocytes may contribute to protection from HIV-1 in multiply exposed persons.

Differential antibody isotype reactivity to specific antigens in human lymphatic filariasis: gp15/400 preferentially induces immunoglobulin E (IgE), IgG4, and IgG2.

Lymphatic filarial infection in humans is associated with a strong skewing of the immune response towards the TH2 arm, with prominent interleukin 4-producing cells and elevated levels of immunoglobulin G4 (IgG4) and IgE antibodies in peripheral blood. To determine how such a generalized TH2 imbalance governs responses to individual parasite antigens, the profiles of isotypes of antibodies to two recombinant proteins of Brugia spp. were studied. One molecule was the C-terminal portion of the filarial heat shock protein 70 (Bpa-26), representative of a cytoplasmic protein, and the second antigen was a single unit of the tandem repeats of a Brugia polypeptide (BpL-4), a secreted product which is prominently exposed to the immune system. Serum samples from 146 individuals resident in areas in which brugian filariasis is endemic were used, and it was found that whereas the levels of IgG1 and IgG3 responses to both Bpa-26 and BpL-4 were high, IgG4 and IgE antibodies to only BpL-4, not to Bpa-26, were prominent. Thus, an antigen which is chronically exposed to the immune system elicited a TH2-dependent isotype switch, as manifested by increased IgG4 and IgE responses. Moreover, IgG4 and IgE responses to BpL-4 showed a strong negative association, suggesting that mediators other than interleukin 4 must be responsible for such differential regulation of these two isotypes. When the data were analyzed as a function of clinical status, a striking association between elevated levels of IgG3 antibodies to Bpa-26 and manifestation of chronic obstructive disease was found; elephantiasis patients showed significantly higher levels of IgG3 antibodies to Bpa-26 than microfilaremics and asymptomatic amicrofilaremics. This indicates that an imbalance of isotypes of antibodies to particular filarial antigens might play a role in the pathogenesis of chronic disease.

Primary structure of and immunoglobulin E response to the repeat subunit of gp15/400 from human lymphatic filarial parasites.

We have isolated and sequenced clones encoding the repeated subunit of the surface-associated glycoprotein gp15/400 from the two nematode species predominantly responsible for lymphatic filariasis in humans: Brugia malayi and Wuchereria bancrofti. The amino acid sequence of the 15-kDa subunit, derived from the nucleotide sequence of the gene fragment from B. malayi, is identical to that previously reported for B. pahangi, whereas the derived W. bancrofti protein sequence differs in only 7 of 132 residues. The identity of the protein in the two Brugia species allowed us to use a recombinant from B. pahangi to examine the serological response of adult Indonesian subjects infected with B. malayi. The polymerase chain reaction-amplified subunit was expressed in Escherichia coli via the pDS56/RBS11 plasmid and purified by nickel-chelating chromatography. A significant proportion of individuals produced antigen-specific immunoglobulin E (IgE). This was most pronounced in the individuals with elephantiasis, with 14 of 15 showing elevated titers and a mean of 3.2 ng of specific IgE ml-1. Only 2 of 15 microfilaremic individuals possessed elevated titers of specific IgE, with a mean of 0.045 ng ml-1 for the group as a whole. Asymptomatic amicrofilaremic residents showed approximately equal numbers of responders (defined as having a value in the radioimmunoassay greater than two standard deviations above controls) and nonresponders, with a group mean of 1.2 ng of antigen-specific IgE ml-1.

T cell responsiveness correlates differentially with antibody isotype levels in clinical and asymptomatic filariasis.

To establish the relationships among T and B cell responses, active infection, and clinical manifestations in lymphatic filariasis, filarial-specific lymphocyte proliferation, IgG antibody isotypes, and IgE levels were determined in an exposed population: 31 asymptomatic amicrofilaremics, 43 microfilaremics, 12 symptomatic amicrofilaremics, and 52 elephantiasis patients. Lymphocyte proliferation was higher in elephantiasis patients and asymptomatic amicrofilaremics than in microfilaremics (P < .004). A proportion of asymptomatic amicrofilaremics (32%), elephantiasis patients (37%), and symptomatic amicrofilaremics (58%) showed antigen-specific lymphocyte unresponsiveness, and lymphocyte proliferation to filarial antigens correlated negatively with specific IgG4 levels (rho = -0.315, P < .001). As elevated specific IgG4 is an indicator of active infection, it is argued that active infection may result in lymphocyte hyporesponsiveness irrespective of clinical category. Of those with elevated specific IgE levels and high T cell proliferative responses, 70% had elephantiasis, suggesting these factors have a role in pathology. However, the existence of a proportion of elephantiasis patients with low anti-filarial IgE and T cell unresponsiveness to filarial antigens suggests that elephantiasis can be caused by distinct processes.