Natural Antiviral Factors Expressed by CD8+T Cells and Other Immune System Cells

HIV感染的自然历史在不同的个体中是高度异质性的，从稳定的无症状状态到快速的疾病演变。HIV疾病进展速度的一个主要决定因素是体内病毒复制水平，这是由参与免疫和炎症反应的各种细胞产生的细胞因子和趋化因子的复杂网络调节的。1995年，艾滋病毒和趋化因子领域的意外相遇极大地促进了我们对艾滋病发病机制的理解，为开发有效的预防和治疗艾滋病毒的措施开辟了新的前景。所有已知的hiv抑制趋化因子通过识别和阻断在易感细胞表面表达的特定病毒共受体而起作用。因此，RANTES, MIP-1&#945；和MIP-1&#946；选择性地抑制使用CCR5作为辅助受体的HIV分离物，而SDF-1选择性地抑制使用CXCR4的HIV分离物。由于其固有的抗病毒特性，这些趋化因子的类似物或功能模拟物目前正在开发作为抗hiv治疗药物或杀微生物剂。

The natural history of HIV infection is highly heterogeneous in different individuals, spanning from a steady asymptomatic condition to a rapid disease evolution. A major determinant of the pace of HIV disease progression is the in vivo level of viral replication, which is regulated by a complex network of cytokines and chemokines produced by various cells involved in immunologic and inflammatory responses. The unexpected encounter, in 1995, between the fields of HIV and chemokines has dramatically advanced our understanding of AIDS pathogenesis, opening new perspectives for the development of effective prophylactic and therapeutic measures against HIV. All known HIV-suppressive chemokines act by recognizing and blocking specific viral coreceptors expressed on the surface of susceptible cells. Thus, RANTES, MIP-1&#945; and MIP-1&#946; selectively inhibit HIV isolates that use CCR5 as a coreceptor, while SDF-1 selectively inhibits those using CXCR4. Owing to their inherent antiviral properties, analogues or functional mimics of these chemokines are currently under development as anti-HIV therapeutics or microbicides. 1) Identification of CXCL4 as a novel HIV-suppressive chemokine. Chemokines are produced by various cells that are activated in the course of immunologic and inflammatory responses, including platelets, which are specialized anucleated cells that store in their &#945;-granules large amounts of CXCL4 and CCL5/RANTES, as well as the CXCL7/NAP-2 precursor, &#946;-thromboglobulin. We examined the effects of CXCL4, the most abundant protein contained within the platelet &#945;-granules, on HIV infection. We found that treatment with either recombinant or native human CXCL4 causes a dose-dependent inhibition of HIV infection in different experimental systems including primary CD4+ T cells and macrophages, as well as neoplastic CD4+ T-cell lines and the MAGI assay. Of note, at variance with other HIV-suppressive chemokines, CXCL4 is active on a broad spectrum of HIV isolates, with similar potency against viruses that use CCR5 and those that use CXCR4 as coreceptors. Analysis of a panel of primary HIV isolates documented different sensitivities to CXCL4, with some isolates displaying a lack of sensitivity to CXCL4 at the concentrations used in our studies (half-maximal inhibitory concentration IC50 range: <25 to >600 nM). However, the sensitivity to CXCL4-mediated inhibition was not correlated with the coreceptor usage phenotype, the genetic subtype or the in vitro adaptation history of such isolates. These results identified CXCL4 as a novel HIV-suppressive chemokine with an atypically broad spectrum of antiviral activity. 2) Characterization of the mechanism of antiviral action of CXCL4. CXCL4 is unique among chemokines because many of its biological activities are not apparently mediated by activation of a specific chemokine receptor. Although a splice variant of CXCR3, designated CXCR3b, was shown to bind CXCL4 with high affinity, its physiological relevance remains uncertain. Our initial approach toward elucidating the mechanism of anti-HIV action of CXCL4 was to identify which step in the viral replication cycle is inhibited by this chemokine. Kinetic experiments using the MAGI assay demonstrated that CXCL4 blocks an early step in the viral replication cycle. Thus, we used an HIV entry assay to demonstrate that CXCL4 is a potent inhibitor not only of viral entry but also of virion attachment to the surface of susceptible cells. Of note, inhibition of entry of different HIV isolates correlated with their sensitivity to CXCL4-mediated inhibition in infection assays. Experiments of receptor modulation and binding competition ruled out a direct interaction between CXCL4 and the major HIV receptor molecules, i.e., CD4 and the coreceptors. Thus, we switched our attention toward the viral envelope. Using a virion-capture assay, we found that CXCL4 immobilized on beads efficiently and specifically binds to intact HIV virions irrespective of their coreceptors-usage phenotype. Further tests demonstrated that CXCL4 is able to co-immunoprecipitate the gp120 envelope glycoprotein from the surface of chronically infected cells, thus documenting a direct interaction between CXCL4 and native gp120. By virion-capture competition using a panel of gp120-specific mAbs, we were able to map the CXCL4-binding site on gp120 to a region that is in close proximity to, but not overlapping with the CD4-binding domain. Altogether, these results demonstrated that CXCL4 inhibits the HIV attachment and entry steps through a novel mechanism that involves direct binding to the viral envelope rather than to the cellular coreceptors. 3) Correlation between CXCL4 levels and clinical/immunological parameters of disease progression in HIV-infected subjects. CXCL4 is primarily produced by megakaryocytes and platelets, and is promptly released by platelets upon activation. Although its primary function is to promote blood coagulation, CXCL4 has multiple, seemingly unrelated, activities including blockade of angiogenesis, activation of immune cells and, as we discovered, inhibition of HIV. Of note, a variety of platelet abnormalities have been described in patients with HIV infection, whose severity correlates with the progression of the immunodeficiency. As a first step toward elucidating the clinical relevance of CXCL4 as an endogenous HIV-suppressive factor, we measured the serum levels of CXCL4 in a cohort of HIV-infected subjects (n = 279) selected to represent different clinical stages of HIV infection. Linear regression analysis showed that serum CXCL4 concentrations were positively correlated with peripheral blood CD4+ T-cell counts (p = 0.0008), CD8+ T-cell counts (p <0.0001) and platelet counts (p <0.0001), and negatively correlated with levels of HIV plasma viremia (p = 0.0156) and C-reactive protein (p = 0.0001). In multivariate regression analysis, the ranks of CXCL4 were found to be independently associated with the CD4+ T-cell ranks (p = 0.0016). These data are compatible with a potential in vivo protective effect of CXCL4. However, it has to be emphasized that the serum levels of CXCL4 do not represent actual circulating levels of the chemokine, but rather the chemokine reservoir harbored by circulating platelets, indicating that subjects with less advanced HIV disease are endowed with a larger storage pool of CXCL4. Although we cannot exclude that the true plasmatic levels of CXCL4 may also be elevated in the early stages of HIV-1 infection, the current technology does not permit to obtain reliable measurements without the interference of contaminating platelets. Additional studies will be important to elucidate the role of CXCL4 in the natural history of HIV disease.