sICs on Resting CD4+ T Cells from HIV-1infected Subjects show Similar Kinetics to gp120-Igs We attempted to clarify whether sIC+ rCD4s in the peripheral blood of HIV+ Pts were also caused by cell-bound gp120. To this end, we first studied the dynamics of sICs in rCD4s purified from HIV+ Pts and sought to determine whether the dynamics are similar to Ig-gp120-VRs. The estimated mean duration of a 50% Dynamics of Immune Complexes on Resting T Cells 6 Dynamics of Immune Complexes on Resting T Cells comparison PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19650784 of IgG binding levels, MFI values of IgG on rCD4s of the lysate samples are denoted above. IgG, positive IgG control; H, rCD4 lysate from an HIV-1-seronegative healthy donor. Three-dimensional reconstitution confocal micrographs of Igs and CD4 in rCD4s from an HIV-1+ Pt. Representative time course of FACS and calculated half-life of sICs in purified rCD4s from an HIV-1+ Pt. Bar, SD. Percentage of Ig+ cells in purified HIV-1+ Pt rCD4s without or with 10 min of 0.05% trypsinization. Percentage of Ig+ cells in purified HIV-1+ Pt rCD4s before, after 58 h of culture, or 58 h of culture with exposure to HIV-1+ Pt serum. Changes in percentages of IgM+ or IgG+ rCD4s in blood, plasma VL, and CD4 lymphocyte counts during ART in the four HIV-1+ Pts. Two patients MedChemExpress Halofuginone discontinued therapy after substantial suppression of VLs. HIV-1 RNA levels in two other patients were suppressed to undetectable levels for approximately 2 yr with ART. Summary of the percentages and representative FACS of Igs on purified HIV-1+ Pt rCD4s before and after 6 h of PMA exposure. Fluorescence and DIC images of purified HIV-1+ Pt rCD4s that were stained with anti-Ig Abs and goat polyclonal anti-CD4 before and after 6 h of PMA exposure. Data in d and l are representative of five independent experiments. doi:10.1371/journal.pone.0086479.g002 reduction in sICs in purified peripheral rCD4s from six patients was 21.7665.62 h . rCD4s should contain a certain number of cells in stages beyond G0. Therefore, the turnover of VRs and/or sICs in purified rCD4s may be much faster than in qCD4s; taking this into account, the calculated halflife of sICs on the patients’ rCD4s roughly matched the turnover of sICs in qCD4s. More importantly, trypsin treatment to remove trypsin-sensitive cell surface molecules significantly reduced the level of sICs. Similarly, once sIC levels were reduced in rCD4s, the levels were not restored by exposing the cells to Pt serum, suggesting that rCD4s from HIV-1+ Pts either do not allow attachment of cICs or do not express surface epitopes for auto-Abs in Pt serum. Collectively, sICs on the patients’ rCD4s consisted of cell-bound molecules with similar kinetics to gp120-Igs. change in frequency of IgG+ and IgM+ rCD4s in blood was relatively slow compared with the change in plasma VLs, the frequencies in peripheral blood correlated to plasma VLs. Therefore, these results collectively indicate that at least cell-bound HIV-1 or related molecules are involved in the formation of sICs on rCD4s in vivo. Interestingly, the percentage of both IgG+ and IgM+ rCD4s appears to be inversely correlated to the number of CD4+ T cells in peripheral blood. sICs are Attached to Surface CD4 on Resting CD4+ T Cells from HIV-1-infected Subjects Next, we investigated whether colocalized sICs and CD4 were molecularly linked. To examine this possibility, rCD4s purified from HIV-1+ Pts were exposed to phorbol myristate acetate to induce 
CD4 internalization and determine whether sICs cou