Costimulation of CD4+ T cells via CD28 modulates human immunodeficiency virus type 1 infection and replication in vitro.

Stimulation of human immunodeficiency virus type 1 (HIV-1)-infected donor peripheral blood mononuclear cells (PBMCs) via the TCR-CD3 complex induces HIV-1 production in vitro (Zarling JM, et al.: Nature [London] 1990;347:92; Haffar OK, et al.: J Virol 1992;66:4279; Moran PM, et al.: AIDS Res Hum Retroviruses 1993;9:455). However, in addition to the primary stimulatory signal delivered through the TCR-CD3 complex, optimal T cell activation requires secondary or costimulatory signals delivered via various T cell accessory proteins (Alton A, et al.: Adv Immunol 1990;48:227). In this article we explore the role of costimulation of T cells via CD28 in HIV-1 replication. Ligation of CD28 with either a CD28-specific MAb or by coculture of PBMCs with Chinese hamster ovary (CHO) cell lines stably expressing either of the CD28 counterreceptors, B7-1 (CD80) or B7-2 (CD86), concomitant with stimulation via CD3, results in increased virus replication compared to stimulation via CD3 alone. CD28 ligation also augments de novo infection of CD3-stimulated seronegative donor PBMCs with cell-free virus. Increased virus replication following CD28 ligation is not solely attributed to increased levels of endogenous IL-2, because addition of an anti-IL-2-neutralizing antibody only partially inhibits the response. In contrast, interfering with the interaction between CD28 and its counterreceptors on antigen-presenting cells (APCs) using CTLA4Ig effectively inhibits virus replication. At high concentrations CTLA4Ig also reduces cell proliferation. These in vitro results suggest that CD28 plays a central role in HIV-1 replication and that interfering with the CD28 costimulatory pathway may modify the course of HIV-1 infection.

Kinetics of infected cell appearance as a determinant of number of human immunodeficiency virus-1 infectious units.

In order to optimize detection of human immunodeficiency virus-1 (HIV-1)-infected cells, the temporal appearance of virus antigens in newly infected H9 cell cultures was examined. Analyses were accomplished by indirect immunofluorescence labeling with each of 10 monoclonal antibodies and evaluation by flow cytometry. Of the antibodies examined, those specific for HIV-1 capsid protein p24, matrix protein p17, or their precursor molecule p55 allowed the earliest and most sensitive detection in infected cells fixed to allow detection of intracellular antigen. Discrimination of infected cells from uninfected cells was much less sensitive when three antibodies specific for HIV-1 glycoproteins were used to detect intracellular or cell surface antigen. In several experiments involving the time course of infection, we observed no differences in cell numbers between infected and uninfected H9 cultures initiated at identical cell concentrations. We hypothesized that it might be possible to quantitate infectious HIV-1 virions from the kinetics of infected cell appearance. Straight-line relationships between the log p24-positive cells and the time after infection were observed. These quantitative observations were employed to calculate the number of infectious units originally added to the culture that were capable of infecting H9 cells. The production of infectious virus, but not of cytopathic effects, was required. The results of this novel approach to the titration of infectious HIV-1 particles agreed well with those from median cell culture infective dose determination. This method could be employed with other infectious agents for which detection of cell-associated antigens is possible in cell cultures not destroyed by infection.

TCR-independent CD28-mediated gene expression in peripheral blood lymphocytes from donors chronically infected with HIV-1.

Complete activation of peripheral blood T cells requires both T-cell receptor (TCR) stimulation and CD28 costimulation. Signalling pathways associated specifically with CD28 are not well understood, however, because ligation of CD28 in the absence of TCR stimulation does not give rise to cellular responses in normal cells. In peripheral blood lymphocytes (PBL) from donors chronically infected with human immunodeficiency virus-1 (HIV-1), CD28 can induce viral replication through an alternative pathway that does not require TCR ligation. We have exploited this observation to study CD28-mediated signal transduction using reverse transcriptase-mediated polymerase chain reaction (RT-PCR) to amplify viral RNA. Independent ligation of CD28 on donor PBL induced expression of the HIV-1 tat gene but not the interleukin-2 (IL-2) gene. Viral induction did not occur following pretreatment of cells with actinomycin D, suggesting it was mediated through transcriptional activation of the viral long terminal repeat (LTR). tat was induced in the presence of the protein kinase C inhibitor H-7, but was inhibited by cyclosporin A. Our results demonstrate that CD28 is linked directly to specific signalling pathways leading to de novo induction of genes in PBL.

Human immunodeficiency virus type 1 infection of CD4+ T cells down-regulates the expression of CD28: effect on T cell activation and cytokine production.

Infection with human immunodeficiency virus type 1 (HIV-1) results in dysregulation of normal T cell function. To study the effects of HIV-1 at the cellular level, primary T cell lines were generated by alloantigen stimulation of CD4+ T cells collected from peripheral blood of HIV-1-infected donors. Using Epstein-Barr virus-infected B lymphocytes (EBV-LCL) as a source of alloantigen, the T cell lines were expanded in vitro for 7 weeks. Uninfected T cell lines were cultured in parallel. Virus was inducible from the infected lines with stimulation, and complete infection was achieved after 4-7 weeks depending on the line. The down-modulation of CD28 expression correlated with virus replication and spread. Furthermore, CD28 mRNA was not inducible in the infected lines after stimulation with alloantigen. Loss of CD28 correlated with reduced responsiveness to costimulation with a monoclonal antibody to CD28 following similar engagement of the CD3 protein. In contrast, activation with alloantigen was not affected. HIV-1 infection and down-modulation of CD28 did not alter the relative levels of IL-2, IFN-gamma, and IL-4 mRNA. Production of the various cytokine mRNAs following alloantigen stimulation was inhibited by CTLA4Ig and thus remained under the regulation of CD80 and CD86 expressed on the EBV-LCL. Taken together, our data suggest that dysregulation of normal T cell function associated with HIV-1 infection may result in part form the loss of CD28 expression.

IL-7 up-regulates HIV-1 replication in naturally infected peripheral blood mononuclear cells.

We have investigated the effect of exogenous IL-7 on replication of HIV-1 in PBMCs isolated from asymptomatic, chronically infected donors. We show that IL-7 induced virus replication and increased proviral DNA levels in CD8- PBMC cultures. IL-7 also increased the levels of doubly spliced HIV-1 tat RNA in these cultures. In comparison, IL-2 induced lower levels of virus production than IL-7, but had a more pronounced effect on cell proliferation. The IL-7-mediated increase in virus replication was not inhibited by neutralizing mAbs to IL-1 beta, IL-2, IL-6, or TNF-alpha, and was only partially dependent on ligation of the T cell accessory molecule CD28. CD8+ cells inhibited the increase in viral replication following IL-7 stimulation, but did not prevent virus replication following ligation of CD3 in the presence of IL-7. The data shows that IL-7 regulates HIV-1 replication in naturally infected PBMCs.

HIV-specific humoral and cellular immunity in rabbits vaccinated with recombinant human immunodeficiency virus-like gag-env particles.

Recombinant human immunodeficiency virus type-1 (HIV-1)-like gag-env particles produced in mammalian cells were inoculated into two New Zealand white rabbits. In parallel, two control rabbits were inoculated with the homologous HIV-1 virions inactivated by ultra violet light (uv) and psoralen treatments. The humoral and cellular immune responses to HIV-1 were evaluated for both groups of animals. Recombinant particles elicited humoral immunity that was specific for all the viral structural proteins. The antibodies recognized both denatured and nondenatured proteins. Moreover, the sera neutralized the in vitro infectivity of the homologous virus in CEM cells. Importantly, the recombinant particles also generated a T helper response by priming with the HIV proteins. Similar results were observed with inactivated virus immunization. Therefore, our results suggest that the recombinant HIV-like particles elicit functional humoral immunity as well as cellular immunity and represent a novel vaccine candidate for AIDS.

Costimulation of T-cell activation and virus production by B7 antigen on activated CD4+ T cells from human immunodeficiency virus type 1-infected donors.

Infection with the human immunodeficiency virus type 1 (HIV-1) requires T-cell activation. Recent studies have shown that interactions of the T-lymphocyte receptors CD28 and CTLA-4 with their counter receptor, B7, on antigen-presenting cells are required for optimal T-cell activation. Here we show that HIV-1 infection is associated with decreased expression of CD28 and increased expression of B7 on CD4+ T-cell lines generated from seropositive donors by alloantigen stimulation. Loss of CD28 expression was not seen on CD4+ T-cell lines from seronegative donors, but up-regulation of B7 expression was observed upon more prolonged culture. Both T-cell proliferation and interleukin 2 mRNA accumulation in HIV-1-infected cultures required costimulation with exogenous B7 because these events were blocked by CTLA4Ig, a soluble form of CTLA-4 that binds B7 with high avidity. In contrast, levels of HIV-1 RNA were not affected by CTLA4Ig, indicating that regulation of virus transcription in these cultures did not depend upon CD28-B7 engagement. Infected T cells could present alloantigen to fresh, uninfected CD4+ T cells, leading to increased proliferation and virus spread to the activated cells. Both of these events were blocked by CTLA4Ig. Thus, chronic activation of HIV-1-infected CD4+ T cells reduces expression of CD28 and increases expression of B7, thereby enabling these T cells to become antigen-presenting cells for uninfected CD4+ T cells; this might be another mechanism for HIV-1 transmission via T-cell-T-cell contact.

CNI-H0294, a nuclear importation inhibitor of the human immunodeficiency virus type 1 genome, abrogates virus replication in infected activated peripheral blood mononuclear cells.

Active nuclear importation of the human immunodeficiency virus (HIV) type 1 (HIV-1) preintegration complex (PIC) is required for the productive infection of nondividing cells, but it is believed to be dispensable for the infection of proliferating cells, such as activated T lymphocytes. To investigate this question, we exploited the properties of the small arylene bis (methyl ketone) compound CNI-H0294. We have previously shown that this compound associated with the HIV-1 matrix protein nuclear localization sequence and blocked binding of the HIV-1 PIC to yeast karyopherin alpha. CNI-H0294 abrogated nuclear importation of the HIV-1 genome in macrophages and effectively inhibited infection of nondividing cells. In this study we demonstrate that CNI-H0294 inhibits binding of the HIV-1 PIC to human karyopherin alpha and reduces nuclear importation of the viral genome in primary peripheral blood mononuclear cells (PBMCs). We also demonstrate that CNI-H0294 inhibits acute infection of PBMC cultures in vitro with a primary isolate of HIV-1 and reduces virus replication and virus load in cultures of endogenously infected PBMCs from seropositive individuals. Thus, as for infection of nondividing, terminally differentiated macrophages, HIV-1 uses active nuclear importation of the virus genome to infect activated CD4+ T cells. These results support nuclear importation as a novel target and CNI-H0294 and its derivatives as novel compounds for therapeutic intervention in HIV infection and AIDS.

Inhibition of virus production in peripheral blood mononuclear cells from human immunodeficiency virus (HIV) type 1-seropositive donors by treatment with recombinant HIV-like particles.

We have previously reported on the assembly of recombinant human immunodeficiency virus (HIV)-like particles that contain gag structural proteins and present env glycoproteins gp120 and gp41 on their surfaces (O. Haffar,. J. Garriques, B. Travis, P. Moran, J. Zarling, and S.-L. Hu, J. Virol. 64:2653-2659, 1990). On the basis of their structures, we hypothesized that the recombinant particles would interfere with virus infection and tested our hypothesis in vitro by using peripheral blood mononuclear cells (PBMC) from HIV type 1-seropositive donors. Addition of the recombinant particles to PBMC concomitant with stimulation by anti-CD3 inhibited virus production, as determined by reduced levels of p24 in the culture supernatants. This inhibition of p24 production correlated with lower levels of cell-associated viral DNA. Several lines of evidence suggested that the recombinant particles exerted their antiviral effects primarily by inhibiting virus production from latently infected cells and not by inhibiting subsequent virus spread. Importantly, CD4+ T-cell stimulation by specific antigen or by anti-CD3 was not inhibited by treatment with the recombinant particles. This apparent selective inhibition of virus replication in infected PBMC represents a novel property of the recombinant HIV-like particles.