Regeneration and tolerance factor prevents bystander T-cell death associated with human immunodeficiency virus infection - PubMed (original) (raw)

Regeneration and tolerance factor prevents bystander T-cell death associated with human immunodeficiency virus infection

Richard A Derks et al. Clin Diagn Lab Immunol. 2004 Sep.

Abstract

Human immunodeficiency virus (HIV) infection is characterized by a depletion of T cells. This depletion is caused both by the virus-induced death of infected T cells and by the death of uninfected cells (bystander depletion) by a mechanism which is largely uncharacterized. Regeneration and tolerance factor (RTF) is a subunit of the vacuolar ATPase and a protein that is involved with activation and apoptosis. Anti-RTF antibodies mediate apoptosis in T lymphocytes. When anti-RTF was added to lymphocytes from an HIV-positive individual, they underwent larger amounts of apoptosis than cells taken from healthy controls. When lymphocytes were examined by Western blotting, those from HIV-positive individuals exhibited increased levels of expression of the 50-kDa protein (P < 0.001). A 70-kDa protein was the predominant form of RTF in uninfected control lymphocytes, being expressed in 100% of individuals studied. The expression of the 50-kDa protein in HIV-positive individuals correlated with decreased absolute CD4 counts with a sensitivity of 92% and a positive predictive value of 86%. When uninfected lymphocytes were stimulated with anti-CD3 and anti-CD28, no RTF was detected during early stimulation but a 50-kDa protein was expressed during late stimulation. When the susceptibilities of the lymphocytes to anti-RTF-induced apoptosis were measured, they correlated with the size of the RTF protein expressed. The cells were not susceptible to apoptosis when the 70-kDa RTF was present but were susceptible when the 50-kDa RTF was present. We propose that the increase in the levels of the 50-kDa RTF on cells from HIV-positive individuals is important in preventing the cell from undergoing apoptosis.

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Figures

FIG. 1.

FIG. 1.

Anti-RTF antibody-induced increased apoptosis in lymphocytes from HIV-positive individuals compared to those from healthy control individuals. Lymphocytes from seven HIV-positive individuals and seven healthy donors (uninfected) were incubated for 24 h either with the isotype control (Iso) or with monoclonal (anti-RTF) antibody 2C1. These were then analyzed for apoptosis by incubating them with annexin V-FITC and determination of the level of annexin V binding by flow cytometry on a Facscalibur (BD) flow cytometer. All values at the top indicate the percentage of annexin V binding for each population. The difference between the healthy and HIV-positive populations was judged to be significant if the P value was <0.001 by Student's t test.

FIG. 2.

FIG. 2.

(A) Western blot. RTF is expressed in two different forms in HIV-positive and HIV-negative individuals. Lymphocytes from HIV-negative individuals (lane 1) and HIV-positive individuals (lanes 2 and 3) were tested by Western blotting and probed with monoclonal antibody 2C1. Top row, 70-kDa RTF protein; bottom row, 50-kDa RTF protein. The results are representative of those for lymphocytes from HIV-negative individuals and HIV-positive individuals that express RTF (lane 3) and that do not express RTF at detectable levels (lane 2). (B) The percentage of the 50-kDa RTF protein is increased in lymphocytes from HIV-positive individuals. The results are the percentages of the 50-kDa RTF protein expressed by nine HIV-positive individuals and seven HIV-negative individuals, as measured by desitometry. Means are presented with standard errors. The results were deemed to be significant if the P value was <0.001 by Student's t test.

FIG. 3.

FIG. 3.

The amount of the 50-kDa RTF protein correlates with decreased absolute CD4 counts. The amount of the 50-kDa RTF protein was measured from Western blots with monoclonal antibody 2C1 by densitometry to generate the percentage of the 50-kDa protein expressed, and the result was plotted against the absolute CD4 counts. The graph is divided to represent counts of >400 and <400 cells and RTF positive and RTF negative (above and below the horizontal line, respectively).

FIG. 4.

FIG. 4.

(A) Western blotting. RTF is expressed in two different forms in unactivated and activated (CD3 and CD28) lymphocytes. Lymphocytes from a healthy individual were stimulated with plate-bound CD3 and CD28 and harvested at the indicated times (in hours), tested by Western blotting, and probed with monoclonal antibody 2C1. Top row, proteins of 70 kDa; bottom row, proteins of 50 kDa. Each column represents a stimulation time of 0, 18, 48, and 72 h, respectively. (B) RT-PCR. RTF mRNA expression precedes 50-kDa protein expression. The RT-PCR products of PBMCs were stimulated with anti-CD3 antibody. Top row, RTF message; bottom row, β-actin message. Stimulation times of 0, 18, 48, and 72 h are indicated at the top. (C) Susceptibility to anti-RTF-induced apoptosis during stimulation. Lymphocytes were stimulated (CD3 and CD28) for 18 to 72 h and incubated with monoclonal antibody 2C1. Apoptosis, by way of annexin V binding, was then measured by flow cytometry. The x axis represents the times that the lymphocytes were stimulated before the addition of 2C1. The y axis represents the changes in the levels of annexin V binding between the populations incubated with 2C1 and those incubated with the isotype control.

FIG. 5.

FIG. 5.

Surface ATPase activity is inhibited by anti-RTF. PBMCs with anti-RTF antibody or isotype control antibody were examined for surface ATPase activity by adding [γ-32P]ATP and measuring the release of 32Pi. The y axis represents surface ATPase activity (measured in counts of 32Pi per minute). Error bars represent the standard error of the mean.

FIG. 6.

FIG. 6.

Anti-RTF and ATP generate more apoptosis together than either one does alone. Top row, flow cytometry histograms of J774A1 macrophages incubated with ATP or anti-RTF, or both; bottom row, J774A1 cells incubated with ATP, anti-RTF, and different concentrations of ATPase, as indicated. The x axis represents annexin V staining. The means of four independent experiments with standard errors are given for each panel.

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