Fig. 4: HIV-induced activation of p53 pathway and KLF2 leads to MYC downregulation and loss of proliferation markers.
From: HIV infection reprogrammes CD4+ T cells for quiescence and entry into proviral latency
a, RIT-induced p53 activation leads to slower proliferation in CD4+ T cells from 2 healthy donors (with 2–3 independent replicates per donor) incubated in proliferation media. The statistical values were based on two-tailed t-tests. For the 48 h timepoint, t-statistic was t(5) = −2.71, 95% confidence interval (CI): [−1.326, −0.037] and Cohen’s d = 1.11 (large effect). Here and in panels f–h, values shown are the mean ± s.d. of biological replicates. b, Flow cytometry analysis indicates a strong downregulation of proliferation markers in cells treated with the p53 agonist RITA for 96 h, despite the presence of high IL-2 in the media. Cells treated with vehicle for 96 h or with quiescence-inducing cytokines for 3 weeks (left and right panels, respectively) are included as negative and positive controls. c, Pre-treatment of CD4+ T cells from 3 healthy donors with p53 inhibitor pifithrin-α partially inhibits HIV-mediated loss of proliferation markers cyclin D3 and Ki67 in primary CD4+ T cells. In the boxplots: the central line, lower and upper edges of the box correspond to the median, first (Q1) and third (Q3) quartiles, respectively, which in this plot are identical to the data points; the whiskers extend to the most extreme data points within 1.5× the interquartile range (IQR). For the comparisons of uninfected versus +HIV vehicle-treated and +HIV vehicle versus pifithrin-treated samples, two-tailed Student’s t-test was used, paired by donor, with the t-statistics of t(2) = 9.03 and t(2) = −5.86, Cohen’s dz= 5.21 and 3.38 (both large), and 95% CIs = [14.70, 41.44] and [2.61, 17.07] percentage points, respectively. d, p53 pathway activation in isolation leads to downregulation of MYC targets and some additional proliferative pathways that are also negatively enriched in HIV-infected cells. For this panel and i, plus and minus signs at the bottom indicate positive or negative enrichment. e, KLF2 is among transcripts showing the strongest negative correlation with MYC in the main effector cell polarized subtypes. f, RT–qPCR analysis of KLF2 level in vector or HIV-infected primary human CD4+ T cells from 2 healthy donors (with 3 PCR replicates per donor) in the presence or absence of Raltegravir (labelled here as Ralteg) confirms the induction of KLF2 after HIV infection and its dependence on proviral integration. g,h, Knockdown of KLF2 prevents entry into quiescence in CD4+ T cells from 2 healthy donors. Proliferating cells were treated with 2 siRNAs against KLF2 (siKLF2) or a non-targeting control siRNA and were incubated in quiescence-inducing media. Cell number (g) or the fraction of cells positive for both cyclins D3 and B1 (h) was measured at indicated timepoints. i, KLF2 knockdown results in upregulation of pathways associated with proliferation, including mTORC1 signalling and HIF-1α-regulated genes (hypoxia pathway). j, Pre-treatment with Simvastatin, a pharmaceutical drug shown to upregulate KLF2, enhances HIV-induced loss of proliferation markers in primary CD4+ T cells. The circular gate marks the location of proliferating cells, with cells inside and outside this gate shown as blue and red dots, respectively. k, Unbiased shRNA screen results point to KLF2 as a gene critical for maintenance of cellular quiescence and proviral latency. Similarly, the negative enrichment of MYC points to its critical role in the maintenance of the cellular proliferative state and/or proviral transcriptional activity. shRNA screen hits are shown as circles, with the Y axis indicating relative enrichment. A p53-induced negative regulator of cellular proliferation, CDKN1A/p21, is also enriched.
