Faculty Bibliography

To study the in vivo role of IL-4-expressing cells, we developed a strategy to tag these cells, by generating mice in which one IL-4 allele was replaced with a cDNA encoding the human CD2 (huCD2) cell-surface molecule. Expression of the huCD2 reporter was, like IL-4, restricted to the appropriately polarized T helper 2 cells. However, most of the cells expressed only the IL-4 or the targeted allele. Analysis of the frequency of monoallelic versus biallelic expression suggests that the activation of each individual allele is regulated by a stochastic process whose probability can be augmented by increasing the strength of signal delivered through the TCR. Allele-specific activation may be a general feature of cytokine regulation that contributes to the functional diversity within T helper cell subpopulations

Infection with attenuated simian immunodeficiency virus (SIV) in rhesus macaques has been shown to raise antibodies capable of neutralizing an animal challenge stock of primary SIVmac251 in CEMx174 cells that correlate with resistance to infection after experimental challenge with this virulent virus (M. S. Wyand, K. H. Manson, M. Garcia-Moll, D. C. Montefiori, and R. C. Desrosiers, J. Virol. 70:3724-3733, 1996). Here we show that these neutralizing antibodies are not detected in human and rhesus peripheral blood mononuclear cells (PBMC). In addition, neutralization of primary SIVmac251 in human and rhesus PBMC was rarely detected with plasma samples from a similar group of animals that had been infected either with SIVmac239Deltanef for 1.5 years or with SIVmac239Delta3 for 3.2 years, although low-level neutralization was detected in CEMx174 cells. Potent neutralization was detected in CEMx174 cells when the latter plasma samples were assessed with laboratory-adapted SIVmac251. In contrast to primary SIVmac251, laboratory-adapted SIVmac251 did not replicate in human and rhesus PBMC despite its ability to utilize CCR5, Bonzo/STRL33, and BOB/gpr15 as coreceptors for virus entry. These results illustrate the importance of virus passage history and the choice of indicator cells for making assessments of neutralizing antibodies to lentiviruses such as SIV. They also demonstrate that primary SIVmac251 is less sensitive to neutralization in human and rhesus PBMC than it is in established cell lines. Results obtained in PBMC did not support a role for neutralizing antibodies as a mechanism of protection in animals immunized with attenuated SIV and challenged with primary SIVmac251

We and others have recently identified a CD8 locus enhancer (E8) that directs expression in mature CD8 single-positive thymocytes and peripheral CD8+ T cells and in extrathymically derived intestinal intraepithelial lymphocytes (IEL). In this study, we show that deletion of E8, by homologous recombination results in reduced CD8alphaalpha homodimer expression on IEL. Since CD8 expression on thymus-derived T cells was normal, other enhancers regulate CD8 expression in these cells. By exploiting a transgenic reporter expression assay, we identified three additional enhancers that directed expression in diverse thymocyte subsets and mature T cells but not in CD8alphaalpha+ IEL. The results suggest that CD8alpha expression is primarily regulated by E8, in IEL and by the novel enhancers in the thymus-dependent lineages

Most strains of human immunodeficiency virus type 1 (HIV-1) which have only been carried in vitro in peripheral blood mononuclear cells (primary isolates) can be neutralized by antibodies, but their sensitivity to neutralization varies considerably. To study the parameters that contribute to the differential neutralization sensitivity of primary HIV-1 isolates, we developed a neutralization assay with a panel of genetically engineered cell lines (GHOST cells) that express CD4, one of eight chemokine receptors which function as HIV-1 coreceptors, and a Tat-dependent green fluorescent protein reporter cassette which permits the evaluation and quantitation of HIV-1 infection by flow cytometry. All 21 primary isolates from several clades could grow in the various GHOST cell lines, and their use of one or more coreceptors could easily be defined by flow cytometric analysis. Ten of these primary isolates, three that were CXCR4 (X4)-tropic, three that were CCR5 (R5)-tropic, and four that were dual- or polytropic were chosen for study of their sensitivity to neutralization by human monoclonal and polyclonal antibodies. Viruses from the X4-tropic category of viruses were first tested since they have generally been considered to be particularly neutralization sensitive. It was found that the X4-tropic virus group contained both neutralization-sensitive and neutralization-resistant viruses. Similar results were obtained with R5-tropic viruses and with dual- or polytropic viruses. Within each category of viruses, neutralization sensitivity and resistance could be observed. Therefore, sensitivity to neutralization appears to be the consequence of factors that influence the antibody-virus interaction and its sequelae rather than coreceptor usage. Neutralization of various viruses by the V3-specific monoclonal antibody, 447-52D, was shown to be dependent not only on the presence of the relevant epitope but also on its presentation. An epitope within the envelope of a particular virus is not sufficient to render a virus sensitive to neutralization by an antibody that recognizes that epitope. Moreover, conformation-dependent factors may overcome the need for absolute fidelity in the match between an antibody and its core epitope, permitting sufficient affinity between the viral envelope protein and the antibody to neutralize the virus. The studies indicate that the neutralization sensitivity of HIV-1 primary isolates is a consequence of the complex interaction between virus, antibody, and target cell

HIV-1 Tat activates transcription through binding to human cyclin T1, a regulatory subunit of the TAK/P-TEFb CTD kinase complex. Here we show that the cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo. A Tat:TAR recognition motif (TRM) was identified at the carboxy-terminal edge of the cyclin domain, and we show that hCycT1 can interact simultaneously with Tat and CDK9 on TAR RNA in vitro. Alanine-scanning mutagenesis of the hCycT1 TRM identified residues that are critical for the interaction with Tat and others that are required specifically for binding of the complex to TAR RNA. Interestingly, we find that the interaction between Tat and hCycT1 requires zinc as well as essential cysteine residues in both proteins. Cloning and characterization of the murine CycT1 protein revealed that it lacks a critical cysteine residue (C261) and forms a weak, zinc-independent complex with HIV-1 Tat that greatly reduces binding to TAR RNA. A point mutation in mCycT1 (Y261C) restores high-affinity, zinc-dependent binding to Tat and TAR in vitro, and rescues Tat transactivation in vivo. Although overexpression of hCycT1 in NIH3T3 cells strongly enhances transcription from an integrated proviral promoter, we find that this fails to overcome all blocks to productive HIV-1 infection in murine cells

The chemokine receptor CCR5 plays a key role in the CD4-dependent entry of human and simian immunodeficiency viruses into target cells. We have mapped the interaction sites on CCR5 for a number of novel anti-CCR5 monoclonal antibodies and have used these to study the role of the CCR5 N-terminal ectodomain in viral entry and to demonstrate differential CCR5 epitope expression on different cell types. Deletions of the CCR5 amino terminal domain or substitution with equivalent regions from other chemokine receptors did not affect cell surface expression or reactivity with loop-specific antibodies, suggesting that the loop regions remained conformationally intact. Exchanges of the amino terminal segment of CCR5 with the equivalent domains of CCR1, CCR2, and CXCR4 did not significantly affect infection with virus pseudotyped with envelope glycoproteins (Envs) from HIV-2 and SIV, but substitution with the CXCR4 sequence abrogated entry mediated by Env from HIV-1. In contrast, deletion of the amino terminus abrogated CCR5 receptor activity for all viral Envs examined. These data indicate that the amino terminus of CCR5 has an essential role in entry mediated by diverse viral Envs but that the sequence requirements are more relaxed for the HIV-2 and SIV Envs compared to the HIV-1 Env examined. This suggests that different viral Envs make distinct and specific interactions with the amino terminus of CCR5. Viral Env utilization of CCR5 expressed on 293-T cells does not always correlate with the cellular tropism of the virus, and one possible explanation is that Env-accessible interaction sites on CCR5 differ on different cell types. We therefore analyzed binding of several anti-CCR5 monoclonal antibodies to cell lines and primary cells that express this chemokine receptor and found that whereas all antibodies bound to CCR5-transfected 293T cells, several did not bind to PBMC. The results suggest that CCR5 undergoes cell type specific structural modifications which may affect interaction with different HIV and SIV envelope glycoproteins.

Previous studies have shown that CD8 beta plays a role in both enhancing CD8 alpha-associated Lck kinase activity and promoting the development of CD8-lineage T cells. To examine the role of this enhancement in the maturation of CD8-lineage cells, we assessed CD8 alpha-associated Lck kinase activity in both T cell hybridomas and thymocytes of mice expressing CD8 beta mutations known to impair CD8 T cell development. Lack of CD8 beta expression or expression of a cytoplasmic domain-deleted CD8 beta resulted in a severalfold reduction in CD8 alpha-associated Lck kinase activity compared with that observed with cells expressing wild-type CD8 beta chain. This analysis indicated a critical role for the cytoplasmic domain of CD8 beta in the regulation of CD8 alpha-associated Lck activity. Decreased CD8 alpha-associated Lck activity observed with the various CD8 beta mutations also correlated with diminished in vivo cellular tyrosine phosphorylation. In addition, analysis of CD8 beta mutant mice (CD8 beta-/- or cytoplasmic domain-deleted CD8 beta transgenic) indicated that the degree of reduction in CD8 alpha-associated Lck activity associated with each mutation correlated with the severity of developmental impairment. These results support the importance of CD8 beta-mediated enhancement of CD8 alpha-associated Lck kinase activity in the differentiation of CD8 single-positive thymocytes

Entry of primate lentiviruses into target cells has recently been shown to depend upon the interaction of the viral envelope glycoprotein with CD4 and one or more members of the G protein-coupled receptor (GPCR) family of transmembrane proteins. In vivo, the transmission of HIV-1 infection generally requires viral strains that utilise chemokine recep- tor CCR5, and these strains prevail during the early course of infection. Strains isolated later, in the course of progression to immunodeficiency, are often CXCR4-tropic or are dual tropic for both chemokine receptors. SIV isolates also use CCR5 but are only rarely specific for CXCR4. Instead, SIVs use two orphan members of the GPCR family, named Bonzo/STRL33/TYMSTR and BOB/GPR15. Strains of HIV-2, which are closely related to the SIVs, also often utilise CXCR4, CCR5, BOB and/or Bonzo. Additional GPCR family members have also been shown to be utilised by various strains of HIV and SIV, albeit less efficiently and less frequently. Here we discuss the potential relationship between receptor specificity and viral pathogenesis as well as efforts to develop animal model systems to study the mechanism of disease progression.

HIV-1 uses chemokine coreceptors for cell entry. CXCR4 is the major coreceptor for T-cell-line-adapted isolates and CCR5 for non-T-cell-line-adapted isolates. This study investigated if coreceptor usage differs between genetic subtypes of HIV-1. Eighty-one primary isolates representing nine different genetic subtypes (A-J, except I) were tested on U87.CD4 glioma cells stably expressing chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4. Coreceptor usage was compared to biological phenotype of the isolates (rapid/high, syncytium-inducing or slow/low, non-syncytium-inducing) and to clinical and immunological status of the study subjects. CXCR4 usage was perfectly correlated to the biological phenotype for all subtypes; all of 26 isolates with rapid/high phenotype and none of 55 isolates with slow/low phenotype could infect the CXCR4 expressing cell line. Importantly, the CXCR4-positive, rapid/high phenotype was underrepresented among subtype C isolates. Furthermore, dual tropism for CXCR4 and CCR5 was not found among subtype D isolates. Uni- and multivariate analyses indicated that these subtype-specific differences in coreceptor usage were not due to differences in clinical status, CD4 counts, or treatment. This study shows that CXCR4 usage determines the biological phenotype for all subtypes, but that there appear to exist subtype-dependent differences in frequency of usage of certain coreceptors. This opens up the possibility that genetic subtypes may differ in important biological properties such as virulence, tissue tropism, and transmissibility