Human immunodeficiency virus-1 infection protects against a Tc1-to-Tc2 shift in CD8(+) T cells.

Despite the reports of dysfunction of the lytic abilities of CD8(+) T cells during human immunodeficiency virus-1 (HIV-1) disease progression, the effects of infection on the noncytolytic functions of CD8(+) T cells have not been well characterized to date. We examined the effect of HIV-1 infection on the cytokine and chemokine responses of peripheral blood-derived CD8(+) T cells in an in vitro system. Activation of HIV-1-infected CD8(+) T cells with phytohemagglutinin resulted in a 4- to 8-fold increase in the production of macrophage inflammatory protein (MIP)-1α, MIP-1ß, regulated on activation normal T-cell expressed and secreted, and interleukin (IL)-16. Treatment of activated HIV-1-infected CD8(+) T cells with anti-CD3 monoclonal (M) antibody (Ab) and IL-15 induced strong production of interferon-γ (IFN-γ). Treatment of cells with anti-IL-12 MAb and IL-4 to induce a Tc1-to-Tc2 shift resulted in no change in viral production levels or IFN-γ production within the HIV-1-infected CD8(+) T cell population. Initiation of a Tc2-to-Tc1 shift resulted in a 6-fold increase in HIV-1 replication and 2- to 3-fold higher levels of IFN-γ, demonstrating that infection can protect against a Tc1-to-Tc2 shift in CD8(+) T cells.

Comparative efflux of saquinavir, ritonavir and lopinavir from primary human cells.

Therapeutic drug monitoring is an important element in the management of drug treatment in HIV-1 infected patients. We have examined the effect of temperature on the egress of HIV-1 protease inhibitors from primary T lymphocytes to determine optimum conditions to be adopted in the processing of blood samples in order to accurately estimate intracellular or plasma drug concentrations. Peripheral blood mononuclear cells or U937 cells were incubated with radiolabelled saquinavir, ritonavir or lopinavir at a concentration of 1 µM. The cells were washed and resuspended in RPMI medium (without radiolabelled drug) and further incubated at 37°C, room temperature (21°C) or at 4°C. We observed that release of drug ensued upon the removal of cells from bathing media containing drug, with the rate of efflux being slower at 4°C and fastest at 37°C for all the protease inhibitors. There was a more rapid efflux of saquinavir and ritonavir than lopinavir from both cultured monocytic and primary human cells. The rank order of the partition coefficient of the drugs were lopinavir > saquinavir > ritonavir. All factors that may limit optimal estimation of cell-associated drug concentrations must be considered so that intracellular concentrations of drug can be accurately estimated.

Proportion of HIV-1 infected CD8+CD4- T lymphocytes in vivo.

The proportion and significance of HIV-1 infection of CD8+ T-cells was examined in a patient cohort of HIV-1 seropositive (n=28) and seronegative individuals (n=4). It was hypothesized that irrespective of the clinical status of the patients, productively HIV-1 infected CD8+ T-cells would be found and these cells would contribute to the plasma viral load in vivo. Flow cytometric analysis using fluorochrome-conjugated antibodies, RT-PCR analysis using HIV-1(pol) specific primers, and quantification of HIV-1 viral transcripts by ex vivo culture of isolated CD8+ T-cells were employed. In 22 of the 28 patient samples analyzed, a significantly higher proportion of cells with expression of CD8+HIV-1(gag)+ than of CD4+HIV-1(gag)+ T-cells was observed (36.9% +/- 10.0% vs 26.4% +/- 13.1% respectively, p< 0.01). No correlation was observed between absolute CD4 counts, CD8 counts, plasma viral load and CD8+ T cell infection. RT-PCR analysis indicated the presence of HIV-1 transcripts in the ex vivo isolated CD8+ T-cell population. Ex vivo isolated CD8+ T-cells demonstrated productive infection over time. We conclude, with three lines of evidence detecting and measuring HIV-1 infection of CD8+ T-lymphocytes, that this cellular target and reservoir may be central to HIV-1 pathogenesis.

Infection of CD8+CD45RO+ memory T-cells by HIV-1 and their proliferative response.

CD8+ T-cells are involved in controlling HIV-1 infection by eliminating infected cells and secreting soluble factors that inhibit viral replication. To investigate the mechanism and significance of infection of CD8+ T-cells by HIV-1 in vitro, we examined the susceptibility of these cells and their subsets to infection. CD8+ T-cells supported greater levels of replication with T-cell tropic strains of HIV-1, though viral production was lower than that observed in CD4+ T-cells. CD8+ T-cell infection was found to be productive through ELISA, RT-PCR and flow cytometric analyses. In addition, the CD8+CD45RO+ memory T-cell population supported higher levels of HIV-1 replication than CD8+CD45RA+ naïve T-cells. However, infection of CD8+CD45RO+ T-cells did not affect their proliferative response to the majority of mitogens tested. We conclude, with numerous lines of evidence detecting and measuring infection of CD8+ T-cells and their subsets, that this cellular target and potential reservoir may be central to HIV-1 pathogenesis.

Effects of highly active antiretroviral therapy and immune recovery on CD8+ T-cell-mediated inhibition of HIV-1 transcription.

SUMMARY: : To date, the relation between the CD8 antiviral factor (CAF) and clinical indicators of disease progression in HIV-1 infection (CD4 T-cell counts and viral load [VL]) is inconclusive. Particularly, the effect of antiretroviral therapy and immune recovery on CAF production remains unclear. Using a transient transfection assay and a reporter gene activated by the HIV-1 long terminal repeat (LTR), we analyzed CAF production in CD8 T cells of HIV-1-positive individuals divided into 3 groups: patients on protease inhibitor (PI)-based therapy, patients on nonnucleoside reverse transcriptase inhibitor (NNRTI)-based therapy, and patients receiving no therapy. We found that within the untreated group, CAF activity inversely correlated with VL and high CAF was associated with lower VLs over a period of 0.5 to 3 years. Furthermore, patients who were drug-naive demonstrated significantly higher CAF than untreated patients who had previously undergone antiretroviral therapy. CAF activity in treated patients was similar to CAF in drug-naive patients and higher than in off-treatment patients. There seemed to be a trend toward higher CAF in patients on NNRTI-based therapy compared with those on PI-based therapy. These results suggest that immune recovery after highly active antiretroviral therapy (HAART) contributes to the normalization of CAF levels in HIV-1-positive individuals. Furthermore, we have distinguished between CD8 T-cell-mediated suppression of HIV-1 replication and gene transcription.

Inhibition of HIV-1 entry into cells.

Treatments for HIV-1 include drugs which act to inhibit specific steps in the virus life cycle such as reverse transcription and viral maturation. In 1995 breakthroughs were made in our understanding of the entry of HIV-1 into cells. HIV-1 was shown to use, in addition to the CD4 receptor, chemokine co-receptors, primarily CCR5 and CXCR4, for entry into CD4+ T cells and macrophages. These discoveries have provided another target for the treatment of HIV-1 infection. Drugs developed to block HIV-1 entry include CD4 receptor inhibitors, chemokine receptor inhibitors and inhibitors of attachment and membrane fusion. These drugs may add a further treatment for HIV-1 infection along with protease inhibitors and reverse transcription inhibitors. Several of the entry inhibitors are currently being used in clinical trials and demonstrate efficacy in vivo. In this review, the entry blocking drugs that have recently been patented, their mode of action in inhibiting HIV-1 and their efficacy in clinical trials will be discussed.

Identification of mutations in proviral long terminal repeats of HIV type 1-infected subjects naive to drug therapy.

Human immunodeficiency virus type 1 (HIV-1) proviral DNA sequences in the 5' long terminal repeat (LTR) were examined among 28 drug-naive individuals. Twenty-four subjects had highly conserved LTR sequences, however, more significant changes were observed in the remaining four LTR sequences. These included a 9-bp deletion preceding the NF kappa B elements and a duplication of the RBF-2 motif. A higher overall frequency of mutations within the LTR occurred within NFAT-1 and Sp-1 sequences. Importantly, a novel 16-bp deletion was found in the distal NFAT-1 site.

CD8+ T-cells: function and response to HIV infection.

CD8+ T-cells are a critical component of the cellular immune response and they play an important role in the control of viral infection. During HIV infection, CD8+ T-cells are able to recognize infected cells through an MHC-I dependent process and are able to lyse cells harboring viral infection by the secretion of perforin and granzymes. These cytotoxic T-lymphocytes (CTL) can also eliminate virally infected cells through the engagement of death-inducing ligands expressed by CD8+ T-cells with death receptors on the surface of the infected cell. In addition, CD8+ CTL secrete soluble factors such as beta-chemokines and the CD8+ antiviral factor (CAF) that suppress viral binding and transcription, respectively. In order for HIV to survive the pressures placed upon it by the immune system, the virus has adopted numerous strategies to evade the CD8+ T-cell response. The high mutation rate of HIV has allowed the virus to escape CD8+ T-cell recognition in addition to its ability to down-regulate surface MHC-I expression from infected cells. Also, by altering the pattern of cytokine production and engagement of cellular receptors, HIV disrupts proper CD8+ T-cell signaling. The resultant improper T-cell receptor (TcR) stimulation creates an anergic state in these cells. By affecting the function of CD4+ T-cells and antigen presenting cells that are required for proper CD8+ T-cell maturation, HIV is able to decrease the circulating pool of effector and memory CD8+ T-cells that are able to combat viral infection. The end result is the aberration of CD8+ T-cell function.

Production of CD8+ T cell nonlytic suppressive factors by CD28, CD38, and HLA-DR subpopulations.

HIV infection may be modified by CD8(+) T cells by the production of nonlytic antiviral factors. To determine subpopulations that mediate nonlytic, antiviral activity, we examined the production of beta chemokines and of CD8 antiviral factor (CAF) by different subsets, using CD8(+) cells derived from 24 HIV-1-infected and 25 uninfected individuals. Subjects with CD8(+) cell counts greater than 200/microl produced increased levels of MIP-1alpha by CD8(+)CD28(+), CD8(+)CD38(-), and CD8(+)HLA-DR(+) subsets as compared with uninfected controls. CD8(+)CD38(-) cells produced higher levels of MIP-1beta and RANTES. CAF production was increased by CD8(+)CD38(+) and CD8(+)HLA-DR(+) cells of HIV-infected individuals as compared with uninfected controls. Chemokine production was increased by cells that do not express activation markers, whereas CAF activity was increased by cells expressing CD38 or HLA-DR. These findings shed light on CD8(+) T cell noncytotoxic antiviral factor production during HIV infection.