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.

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.

Immunolocalization in the epidermis.

Immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment in the epidermis thereby providing evidence as to their functionality. This chapter therefore describes a dependable method for immunolocalization within the epidermis.