We have previously shown that supplementation of culture media with human serum instead of fetal bovine serum (FBS) increases HIV-1 production in infected TZM-bl cells, mainly by augmentation of viral transcription through activation of members of the AP-1 family . However, certain cytokines are able to modulate the in-vivo and in-vitro expression of CD4 of primary cells, thus directly influencing HIV-1 entry efficiency [2–4]. We therefore aimed to study the effect of human serum on the receptor and co-receptor expression in these cells.
Using flow cytometry, we found that increasing amounts of pooled human serum from healthy donors, in comparison to FBS, directly correlated with a higher intensity of CD4. We found a 40% increase between those cells cultured with 5% or 20% human serum. In contrast, human serum gave a decrease in CXCR4 expression. The levels of CCR5 varied between experiments with a general decrease at higher human serum concentrations in six out nine separate observations. In the three other experiments, no change was observed, possibly due to the limited amounts of CCR5 in these cells (Fig. 1a).
The CD4 steady state levels were not affected by human serum over time (Fig. 1b) and they were not dependent on de novo synthesis as the increase with human serum versus FBS was seen also after pretreating the cells with 50 μmol/l cycloheximide (Fig. 1c). This suggested an effect of human serum on the stabilization of the receptor at the cell surface or a role on its recycling .
We then tested Sup T1 cells (T-cell derived) in which, unlike TZM-bl cells, the CD4 molecule is stabilized at the cell surface by p56lck . Increasing amounts of human serum or FBS did not alter the CD4 levels in this cell line. However, after treatment with cycloheximide, the CD4 levels were constantly 10–20% higher in those Sup T1 cells that were cultured with 5% or 20% human serum than with FBS (three separate observations; P < 0.05). In these cells, the translation inhibitor treatment might have allowed for the detection of small influences on the receptor's recycling that otherwise would be masked by the stable levels of an anchored CD4. To study whether this difference was indeed due to an effect on CD4 recycling, we treated the Sup T1 cells with 100 nmol/l of phorbol 12, 13dibutyrate (PDBu), which induces receptor internalization. The cells cultured with 20% human serum had a faster recovery of CD4 to baseline levels as compared to those with 20% FBS or serum-free media (Fig. 1d). This was also found for the transferrin receptor (TfR), which is known to undergo continuous recycling (Fig. 1e) . Although this molecule is recycled much faster to the cell surface (<30 min) than the CD4 receptor (>120 min to reach baseline levels) , we also found a dose-dependent effect on recycling by human serum.
In infected HIV-1 cells, the nef protein is known to downmodulate CD4 by promoting its internalization to early endosomes followed by degradation by the lysosomes [9,10]. By transfecting TZM-bl cells with an HIV-1 subtype C-nef encoding plasmid, we were able to confirm the impact on recycling as the number of cells detected by flow cytometry expressing baseline levels of CD4 was consistently higher in those samples cultured with human serum than in those with FBS.
As higher receptor levels promote viral infectivity and viral spread, we tested whether an increase in CD4 expression after culture with human serum would result in a higher yield of syncytia. We developed a flow cytometry-based method in which TZM-bl cells were precultured for 48 h with 10% FBS or 10% human serum and thereafter co-cultured with chronically infected ACH-2 cells stained with carboxyfluorescein succinimidyl ester (Invitrogen, Carlsbad, California, USA). Fusion of the two cell populations would result in transfer of the intracellular dye from the infected cells to the TZM-bl cells. We found that, in comparison with the cells cultured in 10% FBS, those cultured with 10% human serum always gave a 20–30% increase in syncytia formation (five separate observations; P < 0.05).
Our findings illustrate effects on the cells that are underestimated by common cell culture methods. Moreover, they highlight the importance of having proper controls when performing studies on viral pathogenicity and neutralization to avoid unintended bias in the interpretation of the results. We have not yet explored the reason for the negative effect that human serum had on the surface expression of the co-receptor CXCR4 and possibly also CCR5.
Conflicts of interest
There are no conflicts of interest.
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