Human immunodeficiency virus type 1 (HIV-1) selectively infects cells expressing the CD4 molecule, resulting in substantial quantitative and qualitative defects in CD4+ T lymphocyte function in patients with acquired immunodeficiency syndrome (AIDS). However, only a very small number of cells in the peripheral blood of HIV-1-infected individuals are expressing virus at any given time. Previous studies have demonstrated that in vitro infection of CD4+ T cells with HIV-1 results in downregulation of CD4 expression such that CD4 protein is no longer detectable on the surface of the infected cells. In the present study, highly purified subpopulations of peripheral blood mononuclear cells (PBMCs) from AIDS patients were obtained and purified by fluorescence-automated cell sorting. They were examined with the methodologies of virus isolation by limiting dilution analysis, in situ hybridization, immunofluorescence, and gene amplification. Within PBMCs, HIV-1 was expressed in vivo predominantly in the T cell subpopulation which, in contrast to the in vitro observations, continued to express CD4. The precursor frequency of these HIV-1-expressing cells was about 1/1000 CD4+ T cells. The CD4+ T cell population contained HIV-1 DNA in all HIV-1-infected individuals studied and the frequency in AIDS patients was at least 1/100 cells. This high level of infection may be the primary cause for the progressive decline in number and function of CD4+ T cells in patients with AIDS.

CD4+ T cells of patients with AIDS exhibit a qualitative defect in their ability to respond to soluble antigen while their responses to mitogens remain normal. CD4+ T cells can be broadly divided phenotypically into "naive" [CD45RA+ (2H4+)] and "memory" [CD29+ (4B4+) or CD45RO+ (UCHL1+)] cell subpopulations, which represent distinct maturation stages. To determine the human immunodeficiency virus type 1 (HIV-1) infectability of memory and naive CD4+ T-cell subsets in vitro and to determine the in vivo preference of HIV-1 in these subpopulations, we obtained highly purified CD4+ T-cell subsets from normal and HIV-1-infected individuals and studied them by viral cultivation, quantitative polymerase chain reaction, and functional assays. Polymerase chain reaction studies demonstrated that the memory cell subset of CD4+ T cells is preferentially infected (4- to 10-fold more than naive T cells) by HIV-1 in vitro, and these memory cells are the principal reservoir for HIV-1 within CD4+ T cells obtained from infected individuals. Functional abnormalities attributable to CD4+ T cells in HIV-infected individuals (failure to respond in vitro to soluble antigen or to anti-CD3 monoclonal antibodies) were shown to reside primarily within these memory cells. Thus, the present study suggests that the selective functional defects present in the memory CD4+ T-cell subset of HIV-infected individuals may be a direct result of the preferential infection and consequently greater viral burden within these cells.

When B lymphocytes from normal human peripheral blood were incubated for 1 hour with the retrovirus that causes the acquired immune deficiency syndrome (AIDS), the B cells showed marked proliferation and differentiation. Proliferative responses to the virus peaked on day 4 and appeared to be independent of accessory cells. This finding was repeated with three separate viral isolates, one of which was from a patient from Zaire. The magnitude of the observed responses was comparable to that seen with standard polyclonal B-cell activators. This phenomenon may be at least partially responsible for the polyclonal B-cell activation seen in patients with AIDS.

Individuals infected with HIV frequently develop cytopenias and suppressed hematopoiesis. The role of direct HIV infection of hematopoietic progenitor cells in this process has not been defined. In this study, purified CD34+ bone marrow progenitor cells from 74 Zairian and American patients were studied by both coculture viral isolation and polymerase chain reaction for evidence of HIV infection. A total of 36.5% of Zairian and 14% of American patients had HIV infection of the CD34+ cell subset, with as many as 1 in 500 CD34+ cells infected. Most of the Zairian patients in this study had advanced HIV infection and markedly decreased CD4/CD8 T lymphocyte ratios (mean 0.160 +/- 0.08), and no laboratory value predicted the presence of infection in the CD34+ subset of a given Zairian individual. In contrast, American patients with CD34+ cell infection had total CD4 cells less than 20/mm3 and a greater decrease of the CD4/CD8 T lymphocyte ratio compared to seropositive Americans without CD34+ cell infection (p = 0.003). Hematopoiesis, studied by methylcellulose colony assays, was depressed in all seropositive patients studied with no significant further suppression when CD34+ cells were infected. Thus, CD34+ bone marrow progenitor cells are infected in vivo in a subset of seropositive individuals and may serve as an additional reservoir of virus in HIV-infected individuals.

The present study has investigated the effect of PMA, an inducer of monocyte differentiation, on HIV expression in a chronically infected promonocyte clone. After acute HIV infection of U937 cells, clones that constitutively expressed varying levels of HIV were isolated by limiting dilution. One clone (U1) produced low levels of HIV but was found to increase its production 20-fold after PMA induction, as detected by reverse transcriptase or A capture. Further characterization of U1 indicated that PMA could induce cellular differentiation and maturation in the clone similar to that in uninfected U937 cells. In addition, functional studies revealed that superoxide anion production from the U1 clone was not different from that of uninfected U937 cells. Electron microscopic studies of U1 indicated that PMA induced endocytotic vesicles containing many HIV particles. These studies provide a model at the clonal level to 1) examine latency or chronicity of HIV infection in monocytes and 2) delineate the signals required for conversion to high level viral expression.

There is evidence that the initial interaction between HIV-1 and the host that is essential for infection is the specific binding of the viral envelope glycoprotein, gp120, to the CD4 molecule found on certain T cells and monocytes. Most individuals infected with HIV develop antibodies against the gp120 protein. Although in vitro treatment of CD4+ T cells with mAb to a specific epitope of the CD4 molecule (T4a) blocks virus binding, syncytia formation, and infectivity, it is unclear if antibodies to gp120 from an infected individual that can inhibit the binding of gp120 to CD4 is in any way related to the clinical course of disease. Our present study characterizes the binding of 125I-labeled rgp120 to CD4+ cells, and describes an assay system that measures a potentially relevant form of immunity to HIV infection, i.e., the blocking of HIV binding to CD4+ cells. Optimal binding conditions included a 2-h incubation at 22 degrees C, 4 x 10(6) CD4+ cells, and 1 nM gp120. The dissociation constant (KD) for gp120 binding to cell surface CD4 was 5 nM, and was inhibited by soluble CD4 and by mAb to T4a but not to T3 or T4. For the binding inhibition assay, negative controls included healthy seronegatives, seronegatives with connective tissue diseases, patients with HTLV-1 disease, and patients infected with HIV-2. In studying over 100 sera, the assay was highly sensitive (98%) and specific (100%). The majority of HIV+ sera could inhibit binding at dilutions of 1/100 to 1/1000. No correlation was noted between binding inhibition (BI) titer in this assay and clinical stage of HIV infection. In addition, there was no correlation between BI titer and HIV neutralizing activity. The BI titer was correlated with the titer of anti-gp160 (r = 0.63) and the titer of anti-gp120 (r = 0.52) antibodies determined by Western blot dilution. As with neutralizing antibodies and other forms of immune response to HIV, it is unclear what role antibody blocking of HIV binding to CD4+ cells may play in active immunity to HIV in infected individuals. This activity may prove to have some value in protection against initial HIV infection and, thus, the assay may be of use in monitoring vaccine trials.

Several reports implicate Langerhans cells of skin as susceptible targets, reservoirs, and vectors for transmission of HIV: 1) numbers of Langerhans cells in skin of HIV-infected patients were decreased about 50% of that in control skin; 2) as many as 30% of Langerhans cells in the skin of HIV-infected patients were morphologically abnormal; 3) viral particles typical for HIV were identified in or around 2 to 5% of these cells; and 4) infectious HIV was isolated from skin biopsies of infected patients. These results were consistent with similar observations of HIV-infected macrophages in such tissues as brain, lung, and lymph node. Despite these findings, other investigators find no evidence for virus infection in the epidermis of HIV-infected patients by any of several immunohistochemical or ultrastructural criteria. To address this controversy, we obtained skin from 28 HIV-seropositive subjects at various clinical stages by full thickness biopsy or suction blister. Samples were analyzed by transmission electron microscopy for presence of HIV virions, by immunofluorescent staining for viral proteins, by in situ hybridization for HIV-specific mRNA, by polymerase chain reaction amplification of virus-specific DNA, and by direct virus isolation by coculture of epidermis onto monocyte target cells. By any of these techniques, demonstration of HIV in the epidermis of infected patients was equivocal and even then, infrequent. In contrast, viral DNA was detected from the dermis of the same skin samples (26 of 28 samples). Moreover, the number and morphology of Langerhans cells in skin of infected patients were within normal limits, regardless of stage of disease. These studies in toto suggest that a role for Langerhans cells as a principal viral reservoir or vector of transmission is highly unlikely.

In this study, we have investigated the basic requirements for HIV-1 infection of CD8+ lymphocytes in vitro. Unfractionated PBL obtained from healthy HIV-1 seronegative donors were activated with PHA and infected in vitro with HIV-1LAV. Based on immunofluorescent labeling, the vast majority of cells (85 to 97%) surviving peak virus replication belonged to the CD8+ subset and only a small percentage (0.5 to 1.5%) were CD4+. Amplification of HIV-1 proviral sequences by polymerase chain reaction performed on the sorted surviving CD8+ cells demonstrated that CD8+ cells harbored HIV-1 proviral DNA. In addition, stimulation of these HIV-1-infected, CD8(+)-sorted cells either with PHA or anti-CD2 mAb resulted in induction of virus replication, as measured by reverse transcriptase activity. Electron microscopic analysis of CD8+ cells chronically infected with HIV-1 and stimulated with PHA showed typical virions budding from, and associated with, the surface of cells immunolabeled with gold beads directed toward the CD8 molecule. Infection of CD8+ cells with HIV-1 occurred only when CD4+ cells were present in the PHA-activated lymphocyte population exposed to HIV-1 at the beginning of the culture or when sorted CD8+CD4- lymphocytes were cocultured with autologous sorted CD8-CD4+ cells that had been previously infected with HIV-1. Coculture of these cells with PHA-blasts and incubation of their supernatants with a CD4+ cell line showed that these chronically infected CD8+ cells could spread HIV-1 infection to uninfected CD4+ cells after stimulation with PHA or anti-CD2 mAb. Therefore, these results suggest that the minimal requirement for in vitro infection of CD3+CD8+CD4- lymphocytes is the presence of infected CD4+ cells and that infected CD8+ T lymphocytes can further spread the infection to CD4+ cells.