Differences in APOBEC3G Expression in CD4+ T Helper Lymphocyte Subtypes Modulate HIV-1 Infectivity

The cytidine deaminases APOBEC3G and APOBEC3F exert anti–HIV-1 activity that is countered by the HIV-1 vif protein. Based on potential transcription factor binding sites in their putative promoters, we hypothesized that expression of APOBEC3G and APOBEC3F would vary with T helper lymphocyte differentiation. Naive CD4+ T lymphocytes were differentiated to T helper type 1 (Th1) and 2 (Th2) effector cells by expression of transcription factors Tbet and GATA3, respectively, as well as by cytokine polarization. APOBEC3G and APOBEC3F RNA levels, and APOBEC3G protein levels, were higher in Th1 than in Th2 cells. T cell receptor stimulation further increased APOBEC3G and APOBEC3F expression in Tbet- and control-transduced, but not in GATA3-transduced, cells. Neutralizing anti–interferon-γ antibodies reduced both basal and T cell receptor-stimulated APOBEC3G and APOBEC3F expression in Tbet- and control-transduced cells. HIV-1 produced from Th1 cells had more virion APOBEC3G, and decreased infectivity, compared to virions produced from Th2 cells. These differences between Th1- and Th2-produced virions were greater for viruses lacking functional vif, but also seen with vif-positive viruses. Over-expression of APOBEC3G in Th2 cells decreased the infectivity of virions produced from Th2 cells, and reduction of APOBEC3G in Th1 cells increased infectivity of virions produced from Th1 cells, consistent with a causal role for APOBEC3G in the infectivity difference. These results indicate that APOBEC3G and APOBEC3F levels vary physiologically during CD4+ T lymphocyte differentiation, that interferon-γ contributes to this modulation, and that this physiological regulation can cause changes in infectivity of progeny virions, even in the presence of HIV-1 vif

ZNF9 Activation of IRES-Mediated Translation of the Human ODC mRNA Is Decreased in Myotonic Dystrophy Type 2

Peer reviewe

Suppression of HIV-Specific and Allogeneic T Cell Activation by Human Regulatory T Cells Is Dependent on the Strength of Signals

Regulatory T cells (Tregs) suppress immune responses against both self and non-self antigens. Tregs require activation through the T cell receptor (TCR) and IL-2 to exert their suppressive functions. However, how strength of TCR signals modulate the potency of Treg-mediated suppression of antigen-specific T cell activation remain unclear. We found that both strength of TCR signals and ratios of Tregs to target cells, either through superantigen, allogeneic antigens or HIV-specific peptides, modified the suppressive ability of Tregs. While human Tregs were able to mediate suppression in the presence of only autologous antigen-presenting cells, this was much less efficient as compared to when Tregs were activated by allogeneic dendritic cells. In another physiologically relevant system, we show that the strength of peptide stimulation, high frequency of responder CD8+ T cells or presence of high IL-2 can override the suppression of HIV-specific CD8+ T cells by Tregs. These findings suggest that ratios and TCR activation of human Tregs, are important parameters to overcome robust immune responses to pathogens or allogeneic antigens. Modulating the strength of T cell signals and selective enhancement or depletion of antigen-specific Tregs thus may have implications for designing potent vaccines and regulating immune responses during allogeneic transplantation and chronic infections

Allogeneic Tregs are capable of suppressing antigen specific proliferation.

<p>(A) To identify HIV-peptide specific CD8+ T cells PBMC cultures were stained with antibodies for CD3, CD4, CD8, and tetramer specific for HIV peptide, KF11. Plots shown are gated on CD3+ T cells, and tetramer positive cells were also CD8+. (B) Tregs or control T cells were isolated from HIV-negative individuals. PBMC from HIV+ individuals were CFSE labeled and mixed with either HIV-negative allogeneic T or Tregs, and stimulated with 0.2 or 2 ng/ml KF11 HIV peptide. Cells were stained with KF11 Tetramer, and level of proliferation was assessed d5 post activation. Shown is percent suppression by Tregs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002952#pone-0002952-g001" target="_blank">Figure 1</a>. Data is representative of 5 separate experiments.</p

Depletion of Tregs does not result in increased KF11-specific proliferation.

<p>(A) PBMC from HIV+ individuals were stained with antibodies directed against CD4 and CD25. CD4+CD25^hi cells were removed from the PBMC by sorting CD4+CD25^hi T cells using FACS. Total or CD4⁺CD25^hi depleted, or PBMC were then CFSE labeled and stimulated with KF11 peptide. Cells were stained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002952#pone-0002952-g003" target="_blank">Figure 3A</a>, and percentage of KF11-specific CD8⁺ T cells that proliferated is shown for 3 representative donors. In conditions that CD4⁺CD25^hi cells were added back to cultures, they were added at a ratio of at least 3∶1 CD4⁺CD25^hi to labeled PBMC. (B) Treg, T_N or T_M cells were isolated from purified CD4⁺ T cells from HIV+ individual, based on expression of CD45RO and CD25. T_N cells from the same donor were CFSE labeled, and unlabeled T_N, T_M or Tregs were added at a ratio of 1∶1 target to effector T cells. Cultures were stimulated with SEB pulsed DCs, and fixed on day 4–5 post activation. Histogram of CFSE labeled target cells are displayed, and the percentage of cells that have proliferated as shown in gates. Representative is one out of three experiments.</p

Strength of TCR activation and Treg numbers modulates level of suppression.

<p>CD4+ naïve T cells were isolated and CFSE labeled. Unlabeled control T_M or Tregs were added to the cultures at the target to Treg ratio as indicated. Cells were stimulated with 2 different concentrations of SEB, or in the presence of IL-2. Shown is percent suppression by the Tregs when normalized to T_M or T_N control. The % suppression on Y-axis is calculated as follows: We first determined the percent of CFSE labeled target cells dividing in response to the stimuli alone. We then determined the proliferative percentage of cells in the presence of Tregs or control effector T cells. The percent suppression was then calculated by percent reduction in proliferation of the target cells in the presence of effectors as compared to target cells alone. As an example, in one experiment 40% of the CFSE labeled T cells divided in response to SEB stimulation. In the presence of Tregs the same cells divided only 4%. Thus, we represent this as 90% suppression. Data shown are representative of three replicate experiments.</p

Treg mediated suppression of allogeneic T cell responses.

<p>(A) Experimental setup allogeneic vs. autologous stimulation of CFSE labeled target cells (T_N), and Tregs. (B) Conditions were designed as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002952#pone-0002952-g002" target="_blank">Figure 2A</a>. Shown is percent suppression by the Tregs normalized to control T cells. Each point represents an individual experiment. in total 8 experiments separate experiments were performed. P values are shown above the brackets as calculated by Student's t Test.</p

Cell ratios and IL-2 effect Treg suppression of HIV specific proliferation.

<p>(A) Tregs or control T_N cells were isolated from HIV-negative individuals. PBMC from HIV+ were CFSE labeled and stimulated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002952#pone-0002952-g003" target="_blank">Figure 3B</a>. Tregs or control T cells were added at the ratio indicated. Shown is percent suppression by Tregs as described (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002952#pone-0002952-g001" target="_blank">Figure 1</a> legend). (B) HIV-positive PBMC was stimulated with KF11 peptide for 5 days in the presence of allogeneic Tregs and increasing concentrations of IL-2. Data is representative of three separate experiments.</p

Th2 cells express lower levels of hA3G and hA3F than Th1.

<p>Naïve CD4+ T cells were derived to either a Th1 or Th2 phenotype using cytokines as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000292#s4" target="_blank">Materials and Methods</a>. The cells were then stained for intracellular cytokine production or surface markers (A) to confirm differentiation. Cytoplasmic RNA was isolated from the cells and used for qRT-PCR to determine the level of hA3G (B) or hA3F (C) mRNA. Error bars represent media and interquartile range (*p = 0.0039). <i>In vitro</i> cytokine-derived Th1 or Th2 cells were also lysed and subjected to Western Blotting with a hA3G specific antibody and levels of expression were quantified using a LICOR Odyssey system. A representative blot is shown (D) as well as the compilation of 8 individual donors (E) with quantities expressed as quantified intensity of hA3G bands per quantified intensity of Beta-Actin bands of the same lane. Error bars represent median and interquartile range (*p = 0.0078).</p

Increased Infectivity of HIV-1 produced from Th2 cells compared to Th1 cells.

<p><i>vif</i>-competent (+) or <i>vif</i>-deleted (−) HIV-1(NL4-3) was used to infect cultures of Th1 and Th2 cells as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000292#s4" target="_blank">Materials and Methods</a>. Infectivity of the virions produced was determined by infection of the TZM-bl indicator cell line and determination of luciferase activity. An example from an individual donor is shown (A). The experiment was repeated on a total of seven donors with both the <i>vif</i>-competent (B) and <i>vif</i>-deleted virus(C). Error bars represent median and interquartile range of difference in infectivity of virions produced from Th1 cells versus Th2 cells for vif-positive virus (B; *p = .031) and vif-negative virus (C; **p = .016). To test for correlation, individual donor cells' levels of hA3G protein expression were plotted against infectivity of the <i>vif</i>-competent and <i>vif</i>-deleted virions produced from that individual's Th1 cells (D) and Th2 cells (E).</p