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A cell line-based bioassay of non β-chemokine-related HIV suppressive activity

Braun, Joséphineac; Beretta, Albertobc

Research Letters

aINSERM U445, ICGM, Hôpital Cochin, 27 rue du Fbg. St Jaques, 75014 Paris, France; bDisease Clinic, St Raffaele Hospital, via Stamira D'Ancona 20, 20127 Milan, Italy; and cCIRBS, Hôpital St Joseph, 185 rue Raymond Losserand, 75014 Paris, France.

Sponsorship: This work was supported by ANRS (Agence Nationale de Recherche sur le SIDA) and SIDACTION, France.

Received: 23 March 2000;

revised: 8 September 2000; accepted: 12 September 2000.

CD8 T cells suppress HIV replication in cultured CD4 T cells without any apparent killing of infected cells [1]. This CD8 antiviral activity is usually detected as a reduction of HIV production by CD4 T cells cultured with CD8 T cells [2], and is mediated by a soluble factor (CD8 antiviral factor; CAF) detectable in trans-well cultures [3]. Higher levels of CAF have been associated with higher CD4 cell counts and better clinical status [4–6], and are detectable in the blood of patients with primary HIV infection a long time before neutralizing antibodies [7]. Similarly, in children born to seropositive mothers, CAF appears before cytotoxic T lymphocytes (CTL) and is associated with lower viral load and favourable early survival [8,9].

CAF can be measured on naturally or exogenously infected CD4 T cells cultured in the presence of autologous or heterologous CD8 T cells or supernatants from activated CD8 cells [1]. A one-fourth dilution of supernatant is the highest level permissible for the detection of a 50% suppression of viral replication. This has been taken as evidence that CAF is present in very low amounts in CD8 cell supernatants. Clearly, the low sensitivity of this type of assay represents a major technical pitfall in the characterization of CAF. On the other hand, CD8/CD4 cell co-culture assays do not discriminate between CAF and other CD8 T cell functions such as direct cytotoxicity. In addition, because all standard CAF assays are based on the use of peripheral blood T cells as indicator cells, results are difficult to standardize as a result of donor to donor variations.

To address these problems we developed a new assay that directly measures CAF on a human glioma-derived cell line, which is stably transfected with CD4, CXCR4 and a HIV-1 long terminal repeat-Lac-Z construct inducible by the HIV-1 transactivator Tat (U373MG-CD4-CXCR4) [10]. Suppression of replication of both T cell line adapted and primary X4 viruses by CD8 cell supernatants can be measured by the addition of a chromogenic substrate.

For our experiments we selected two non-infected non-exposed control individuals (donor d#3 and d#6) who displayed different levels of CAF as measured in two different peripheral blood mononuclear cell-based assays. The first compared the levels of X4 virus replication in cultures depleted or not of CD8 cells. The second assay measured the suppression of one T cell line adapted and two primary X4 viruses by supernatants of purified CD8 cells co-stimulated with CD3/CD28 antibodies. On the basis of these assays d#3 and d#6 were classified as ‘low’ and ‘high’ CAF producers, respectively.

CD8 cell supernatants from d#3 and d#6, collected at days 3, 6 and 10 were then titrated on U373MG-CD4-CXCR4 cells infected with NL4-3 or the two primary X4 viruses (215 and 248) and β-galactosidase activity was measured 48 h later. The percentage reduction of viral replication in the presence of CD8 supernatant was calculated as 100 − {100/(OD infected cells − OD uninfected cells) × (OD infected cells with CD8 cell supernatant − OD uninfected cells)}. The titration curves are shown in Fig. 1. The CAF titres on the three viruses were determined by regression analysis as the dilution giving 50% inhibition of infection (50% inhibitory titre; IT50). Maximum suppressive activity was detected at day 6 after stimulation for d#6 (1/234 to 1/818) and at day 3 for d#3 (1/47 to 1/170). The difference between the two donors was consistent and reproducible at each day of supernatant collection for the three viruses tested (Fig. 1, right panels).

Fig. 1.

Fig. 1.

We ruled out the involvement of β-chemokines in the suppressive activity because all experiments were performed using X4 viruses, and the preincubation of supernatants with neutralizing antibodies specific for macrophage-inflammatory proteins 1α, 1β, and RANTES (regulated upon activation: normal T cell expressed/secreted) did not modify the inhibitory effect. Furthermore, we obtained a maximum 20% reduction of HIV replication with a stromal cell-derived factor 1α concentration as high as 1 μM, indicating that this chemokine cannot account for the suppressive activity detected in our assay. Finally, in all our experiments, CD8 cell supernatants were produced in the presence of a neutralizing polyclonal anti-IFNα antibody, thereby excluding the involvement of this cytokine.

CAF is elevated in individuals who remain seronegative despite repetitive exposures to HIV (exposed uninfected). As a preliminary test of the potential clinical applications of the cell line-based assay, we titrated supernatants derived from CD8 cells of 11 exposed-uninfected individuals and determined their IT50 against NL4-3. Ten out of 11 exposed-uninfected individuals tested displayed IT50, which were equivalent or higher than that of the ‘high’ CAF producer, d#6. One exposed-uninfected individual displayed an IT50 similar to that of the ‘low’ CAF producer, d#3.

The cell line-based assay thus allows a rapid assessment of CAF in supernatants of CD8 cells, and may be of value for evaluating the CD8 cell response of individuals in different clinical conditions.

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The U373MG-CD4-CXCR4 cell line was a kind gift of Dr Marc Alizon.

Joséphine Braunac

Alberto Berettabc

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