Human tonsil CD8 T_FR inhibit T_FH and GC B cell function.

<p>Tonsil cells were sorted to isolate CD8 T_FR, CD8 conv, CD3+CD8-CXCR5+ T_FH, and CD19+CD38+ GC B cells and X4- or R5-spinoculated. Cells were then co-cultured at indicated ratios for 2 days and analyzed. (A) IL-21 production by T_FH with increasing (left to right) number of CD8 T_FR (n = 4). (B) Representative examples from X4- and R5-spinoculations showing IL-21 production by T_FH alone, 1:1 with CD8 T_FR, 1:1 with CD8 T_FR and anti-Tim3 antibody (500 ng/μl; right panels), and 1:1 with CD8 T_FR and an isotype control antibody (500 ng/μl). (C) Results from a total of 6 tonsils (isotype n = 3) as described in B. (D) IgG production in X4-spinoculated cultures with 2.5 μg/mL CpG-B stimulation in CD8 T_FR, T_FH, and B cell co-cultures as measured by ELISA. All co-cultures are 1:1 (n = 7). Statistical significance was determined by non-parametric Wilcoxon matched-pairs tests (B) or one-way ANOVA (Friedman test, C) and is displayed as * = p<0.05, ** = p<0.01 and *** = p<0.001.</p

Most human tonsil follicular homing CD8 T cells are CD8 T_FR.

<p>Disaggregated tonsil cell cultures were mock-spinoculated or spinoculated with X4- or R5-tropic HIV and cultured for 2 days (n = 6). (A) Of the viable CD3+CD8+ population expressing the follicular phenotype CXCR5+CCR7-, the percent CD44^hi (CD8 T_FR) and all other CD3+CD8+ (CD8 conv) in mock- and HIV-spinoculated cultures is shown. (B) The percent CCR7 expression on CD8 T_FR (red) and CD8 conv (blue) compared to an FMO control (black). Graphs depict median and range. Statistical significance was determined by non-parametric one-way ANOVA (Friedman test) and is displayed as * = p<0.05.</p

CD8 T_FR suppress IL-21 production in T_FH via Tim-3 and induce apoptosis via HLA-E in SIV-infected rhesus macaques.

<p>Disaggregated cells from lymphoid tissues of SIV-infected rhesus macaques (n = 2) were sorted for T_FH and CD8 T_FR and co-cultured at a 1:1 ratio for 2 days with or without HLA-E blocking antibody and analyzed by flow cytometry. (A) Flow gating showing the percent T_FH producing IL-21, and (B) percent T_FH expressing Annexin-V (n = 2).</p

CD8 T_FR induces T_FH apoptosis via HLA-E.

<p>Human tonsil cells were sorted to isolate CD8 T_FR, CD8 conv, and T_FH, spinoculated with R5-tropic HIV, and cultured for 2 days. (A) Representative flow plots showing the percent of Annexin-V+ T_FH in co-culture at 1:1 ratio with CD8 T_FR or CD8 conv 2 days after R5-spinoculatoin. (B) Results from a total of 6 tonsil for mock-, X4-, and R5-spinoculation (isotype n = 3) in A. Co-cultures were also performed with HLA-E blocking antibody or isotype controls (500 ng/μl). (C) Number of T_FH per microliter on day 0 and day 2 when cultured alone (circle, triangle), 1:1 with CD8 T_FR (square), or 1:1 with conventional CD8 T cells (upside down triangle) (n = 6). Statistical significance was determined by Wilcoxon matched-pairs tests and is displayed as * = p<0.05 and ** = p<0.01.</p

Most rhesus macaque CXCR5+CCR7- CD8 T cells are CD8 T_FR.

<p>Disaggregated cells from lymph node and spleen of SIV-uninfected (n = 6) and SIV-infected (n = 6) rhesus macaques were analyzed by flow cytometry. (A) Percentages of CD8 T_FR and CD8 conv from all CD3+CD8+CXCR5+CCR7- are shown for SIV-uninfected and SIV-infected animals. (B) The percent CCR7+ cells in CD8 T_FR and CD8 conv cell populations(C) Representative images of rhesus macaque spleen tissue staining. CD20 staining was performed to determine follicular (F) and extrafollicular (EF) regions. (D) The percentage of CXCR5+ CD8 T cells in follicular and extrafollicular regions as determined from images similar to C. Left plot (white bars) show uninfected rhesus macaques, right plot (black bars) show chronically infected rhesus macaques. LN = lymph node, Mes = mesenteric, Ing = inguinal, Ax = axial, and Sp = spleen. Graphs depict median and range. Statistical significance was determined by non-parametric one-way ANOVA (Friedman test) and is displayed as *** = p<0.001.</p

Suppression of Foxo1 Activity and Down-Modulation of CD62L (L-Selectin) in HIV-1 Infected Resting CD4 T Cells

<div><p>HIV-1 hijacks and disrupts many processes in the cells it infects in order to suppress antiviral immunity and to facilitate its replication. Resting CD4 T cells are important early targets of HIV-1 infection in which HIV-1 must overcome intrinsic barriers to viral replication. Although resting CD4 T cells are refractory to infection <i>in vitro</i>, local environmental factors within lymphoid and mucosal tissues such as cytokines facilitate viral replication while maintaining the resting state. These factors can be utilized <i>in vitro</i> to study HIV-1 replication in resting CD4 T cells. <i>In vivo</i>, the migration of resting naïve and central memory T cells into lymphoid tissues is dependent upon expression of CD62L (L-selectin), a receptor that is subsequently down-modulated following T cell activation. CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation. Here we report that HIV-1 down-modulates CD62L in productively infected naïve and memory resting CD4 T cells while suppressing Foxo1 activity and the expression of KLF2 mRNA. Partial T cell activation was further evident as an increase in CD69 expression. Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7rα, Myc, CCR5, Fam65b, S1P₁ (EDG1), CD52, Cyclin D2 and p21^CIP1, indicating a profound reprogramming of these cells. The Foxo1 inhibitor AS1842856 accelerated <i>de novo</i> viral gene expression and the sequella of infection, supporting the notion that HIV-1 suppression of Foxo1 activity may be a strategy to promote replication in resting CD4 T cells. As Foxo1 is an investigative cancer therapy target, the development of Foxo1 interventions may assist the quest to specifically suppress or activate HIV-1 replication <i>in vivo</i>.</p></div

CD62L down-modulation is not the result of apoptosis, virus contact or protease cleavage, but is influenced by PI3K.

<p><b>A.</b> Cells infected with for 5 days, then were analyzed for GFP, CD62L and Annexin V surface expression, and permeability to 7-AAD. Data are representative of >5 independent experiments. <b>B.</b> Contact with HIV-1 virions does not affect CD62L surface expression. Cells were treated with the reverse transcriptase inhibitor efavirenz (EFV) (2 µg/ml) and infected by spinoculation or spinoculated without virus (Mock). Surface CD62L expression was analyzed at the indicated times after infection. A positive control infection was performed without EFV as indicated. EFV completely prevented the appearance of GFP+ cells (not shown). <b>C.</b> The PI3K and mTORC2 inhibitors LY294002 and PI-103 suppress HIV-1-induced CD62L down-modulation. Infected cells were treated with LY294002 (5 µM), PI-103 (5 µM) or a carrier control (DMSO) 1 day and again 4 hours prior to staining for CD62L. Middle graph: Data from 3 independent experiments with different cell donors. Y axis = the mean fluorescence intensity of CD62L staining (MFI) for each condition expressed as a percentage of the CD62L MFI of the GFP-negative cells. Right: GFP expression is modestly suppressed by LY294002 and PI-103 (MFI = 1761 and 1471 respectively, vs.1981 for DMSO control). LY294002 and PI-103 treatments were statistically different (p<0.01) from their respective DMSO controls by a T(x) population comparison test <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110719#pone.0110719-Roederer1" target="_blank">[134]</a>. <b>D.</b> Metalloprotease inhibitor (MTPI) TAPI-1 (50 µM) does not inhibit HIV-1-induced CD62L surface down-modulation. Left: Control experiment demonstrating inhibition of PMA-induced CD62L downmodulation by MTPI. Cells were treated with PMA with and without MTPI and analyzed one hour later. Right: Cells infected with HIV-1 GFP reporter virus were treated with MTPI 1 day and again 3 hours prior to staining. Data are representative of 2 independent experiments.</p

Foxo1 inhibitor AS1842856 accelerates HIV-1 expression.

<p>AS1842856 (AS) was applied to cells 1 day before and 1 day after infection with single round GFP reporter virus. CD45RO- naïve T cells are shown. <b>A.</b> GFP and CD62L expression in infected naïve CD4+ T cells on the indicated day after infection. Donor A is shown. <b>B.</b> The number of GFP+ cells on the indicated day after infection. Data are average and SD of the two donors. <b>C.</b> Mean fluorescence intensity (MFI) of GFP in the GFP+ cells. Data are average and SD of the two donors. <b>D.</b> Fold increase in the number and the MFI of GFP+ cells, and total GFP fluorescence in the cells calculated as the product of the number of GFP+ cells multiplied by their MFI. Data are average and SD of the two donors. <b>E.</b> Near-full length reverse transcripts on day 2 after infection for one representative cell donor. Data are averages and SD for replicate PCR reactions for each donor. <b>F.</b> HIV-1 fully spliced RNA presented relative to day 1 DMSO condition. Data are averages and SD for replicate PCR reactions for each donor. <b>G.</b> Virus production measured by genomic RNA in culture medium. Data are averages and SD for replicate PCR reactions for each donor. <b>H.</b> CD62L MFI on the indicated day. Data are MFI of GFP+ cells divided by background staining. Data are average and SD of the two donors. <b>I.</b> CD69 MFI on day 7 post infection on naïve and memory resting CD4+ T cells for donor A.</p

Down-modulation of CD62L and upregulation of CD69 and on productively infected naïve and memory resting CD4 T cells.

<p><b>A.</b> Resting IL-7 treated CD4+ T cells were infected with the GFP reporter virus NLENG1, restricted to a single round of infection by treatment with the protease inhibitor indinavir (2 µM). Flow analysis was carried out on day 5 post infection on cells with and without activation by αCD3/CD28 beads on day 3 p.i. Grey: isotype-matched IgG control antibody staining. Blue: GFP-negative cells. Green: GFP+ cells. Data are representative of 4 independent experiments. <b>B.</b> Fold change in CD62L and CD69 expression on naïve and memory CD4+ resting T cells in A. With on exception (*) all values were statistically different (p<0.01) from the negative control by a T(x) population comparison test <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110719#pone.0110719-Roederer1" target="_blank">[134]</a>. <b>C.</b> Failure of GFP+ cells proliferate demonstrates that CD62L-low cells are not produced by preferential expansion of an existing CD62L-low population. Experiment shown is representative of >5 similar experiments. <b>D.</b> IL-4 supports HIV-1-induced down-modulation of CD62L on both naïve and memory resting CD4+ T cells. Experiment shown is representative of >3 similar experiments. <b>E.</b> CD62L down-modulation on HIV-1 infected naïve tonsil T cells 7 days after infection. Similar results were observed on CD45R0+cells (not shown). Experiment shown is representative of 2 similar experiments.</p