Slow Turnover of HIV-1 Receptors on Quiescent CD4⁺ T Cells Causes Prolonged Surface Retention of gp120 Immune Complexes <i>In Vivo</i>

<div><p>Peripheral blood CD4⁺ T cells in HIV-1⁺ patients are coated with Ig. However, the causes and consequences of the presence of Ig⁺ CD4⁺ T cells remain unknown. Previous studies have demonstrated the rapid turnover of viral receptors (VRs) on lymphoma and tumor cells. The present study investigates the turnover of VRs on peripheral quiescent CD4⁺ T cells (qCD4s), which are the most abundant peripheral blood CD4⁺ T cells. Utilizing pharmacological and immunological approaches, we found that the turnover of VRs on qCD4s is extremely slow. As a result, exposure to gp120 or HIV-1 virions <i>in vitro</i> causes gp120 to remain on the surface for a long period of time. It requires approximately three days for cell-bound gp120 on the surface to be reduced by 50%. In the presence of patient serum, gp120 forms surface immune complexes (ICs) that are also retained for a long time. Indeed, when examining the percentages of Ig⁺ CD4⁺ T cells at different stages of HIV-1 infection, approximately 70% of peripheral resting CD4⁺ T cells (rCD4s) were coated with surface VRs bound to slow-turnover gp120-Ig. The levels of circulating ICs in patient serum were insufficient to form surface ICs on qCD4s, suggesting that surface ICs on qCD4s require much higher concentrations of HIV-1 exposure such as might be found in lymph nodes. In the presence of macrophages, Ig⁺ CD4⁺ T cells generated <i>in vitro</i> or directly isolated from HIV-1⁺ patients were ultimately phagocytosed. Similarly, the frequencies and percentages of Ig⁺ rCD4s were significantly increased in an HIV-1⁺ patient after splenectomy, indicating that Ig⁺ rCD4s might be removed from circulation and that non-neutralizing anti-envelope antibodies could play a detrimental role in HIV-1 pathogenesis. These findings provide novel insights for vaccine development and a rationale for using Ig⁺ rCD4 levels as an independent clinical marker.</p></div

Schematic figure summarizes the causes and consequences of sIC⁺ rCD4s.

<p>rCD4s continuously travel between the blood stream and LNs over a period of approximately 1 d. Because a large proportion of HIV-1 is produced in the LNs, the target T cells that migrate to the LNs are exposed to high concentrations of HIV-1, gp120, or ICs as well as anti-env Abs. Prolonged retention of gp120-VR complexes on rCD4s causes the retention of sICs in a manner that reflects the levels of HIV-1 exposure in the LNs. sIC⁺ rCD4s are removed from circulation through ADCP or ADCC by macrophages or NK cells, respectively. The sIC⁺ rCD4s that are not removed from circulation remigrate to the LNs to be exposed to a high concentration of gp120/HIV-1. The percentages and amounts of sICs on rCD4s in the blood reflect a balance of five factors, namely, the levels of virus production in lymphoid tissues, the levels of anti-env Abs, the turnover dynamics of sICs on rCD4s, the duration of repeated exposure by continuous migration to the lymphoid organs, and the levels of immunological elimination of sIC⁺ rCD4s.</p

sICs of IgG or IgM on purified rCD4s from HIV-1⁺ Pts is molecularly linked to surface CD4 and shows slow turnover.

<p>(a) Summary of the percentages of IgG⁺ rCD4s or IgM⁺ rCD4s in healthy individuals (<i>H</i>), acute HIV-1⁺ individuals (<i>A</i>), and chronic pre-symptomatic HIV-1⁺ individuals (<i>C</i>) before, after 3 yrs of complete suppression of VL (<50 copies/ml) with ART (Treated), or untreated for 3 yrs (Untreated). Bars, SD. (b) Representative FACS of IgG expression on rCD4s from HIV-1⁺ Pts. (c) Anti-IgG Ab immunoblotting of purified HIV-1⁺ Pt rCD4 lysates. For the comparison 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. (d) Three-dimensional reconstitution confocal micrographs of Igs (Qdot655, green) and CD4 (Cy2, red) in rCD4s from an HIV-1⁺ Pt. (e, f) Representative time course of FACS (e) and calculated half-life of sICs (f) in purified rCD4s from an HIV-1⁺ Pt. Bar, SD. (g) Percentage of Ig⁺ cells in purified HIV-1⁺ Pt rCD4s without (none) or with 10 min of 0.05% trypsinization (Tryp). (h) Percentage of Ig⁺ cells in purified HIV-1⁺ Pt rCD4s before (0 h), after 58 h of culture (58 h), or 58 h of culture with exposure to HIV-1⁺ Pt serum (58 h+Pt-serum). (i) 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 discontinued therapy after substantial suppression of VLs (left panels). HIV-1 RNA levels in two other patients were suppressed to undetectable levels for approximately 2 yr with ART (right panels). (j, k) Summary of the percentages (k) and representative FACS (j) of Igs on purified HIV-1⁺ Pt rCD4s before and after 6 h of PMA (0.3 ng/ml) exposure. (l) Fluorescence and DIC images of purified HIV-1⁺ Pt rCD4s that were stained with anti-Ig Abs (Cy2, green) and goat polyclonal anti-CD4 (Cy3, red) before and after 6 h of PMA exposure. Data in d and l are representative of five independent experiments.</p

HIV-1/gp120 remains on the surface of qCD4s for a long period of time due to slow VR turnover.

<p>(<b>a, b</b>) Time course of surface VR expression (<b>a</b>) and representative FACS of CXCR4/CCR5 expression on qCD4s (<b>b</b>) following a variety of activation stimuli. (<b>c</b>) The effect of BFA (10 µg/ml), cycloheximide (50 µg/ml) and ActD (20 µg/ml) on the surface expression of CXCR4 (left) and CD4 (right) on qCD4s. (<b>d, e</b>) Confocal micrographs of CD4, CXCR4, and gp120 in qCD4s that were exposed or not exposed to the indicated strain of gp120 or HIV-1 before (<b>d</b>) or after (<b>e</b>) 16 h of anti-CD3 Ab exposure. qCD4s with (<b>e</b>) or without (<b>d</b>) permeabilization were stained with anti-CD4 goat polyclonal Abs (Cy3, red), anti-CXCR4 mouse mAbs (Qdot 655, blue), and anti-gp120 rabbit antiserum (Cy2, green). (<b>f</b>) Time course of cell-bound gp120, sICs (left panel), or surface CD4 expression (right panel) on gp120_IIIB-pulsed or untreated qCD4s. The gp120_IIIB-pulsed qCD4s were further incubated with HIV-1⁺ Pt serum (Pt-serum) to form sICs or untreated and cultured in the absence or presence of anti-CD3 Abs. The effect of T22 pre-exposure on cell-bound gp120_IIIB in anti-CD3 Ab stimulation (αCD3+T22) was also examined. (<b>g</b>) Time course of cell-bound gp120, sICs, or surface CD4 expression on HIV-1_Lai (Lai)<b>-</b> (<b>left</b>), HIV-1_BaL (BaL)<b>-</b>, or clinical isolate (Clinical)<b>-</b> (<b>right</b>) pulsed qCD4s. HIV-1-pulsed qCD4s were further incubated with HIV-1⁺ Pt serum (<b>Pt-serum</b>) to form sICs or untreated and cultured in the absence or presence of anti-CD3 Abs. The effect of enfuvirtide (<b>Enf</b>) exposure was also examined. (<b>h</b>) The amount and location of cell-bound gp120 in gp120_IIIB-pulsed qCD4s that were cultured in the absence or presence of anti-CD3 Abs were assessed by confocal microscopy (<b>upper</b>) or by western blotting (<b>bottom</b>). The lower numbers indicate the value by densitometry. (<b>i</b>) Time course of chemotaxis inhibition on gp120_IIIB-, gp120_BaL- (<b>upper</b>), or HIV-1_Lai- (<b>bottom</b>) pulsed qCD4s. Chemotaxis of gp120- or HIV-1-pulsed or non-pulsed qCD4s toward the indicated chemokines was evaluated using a transwell assay. Bars, SD. The data here are representative of at least three independent experiments.</p

rCD4s from HIV-1⁺ Pts are coated with gp120.

<p>(<b>a</b>) Representative FACS data from rCD4s purified from healthy controls (H1, H2) or chronic asymptomatic patients (Pt-f2r1, Pt-f2r2) stained with Leu3a and CD4v4 (numbers in FACS plots indicate percentages of MFIs of Leu3a/CD4v4). (<b>b</b>) Summary of results of percentages of MFIs of Leu3a/CD4v4 in purified rCD4s from healthy controls (H) and chronic asymptomatic patients (C; CD4 counts: 420±84.6 (± SD); IgG⁺ rCD4s: 75.5±12.6% (± SD)). (<b>c, d</b>) Detection of cell-bound gp120 on rCD4s in a patient with low anti-gp120 Ab levels. (<b>c</b>) Western blot test results for the HIV-1⁺ Pt (NDA-01) at initial admission and three months after. N, negative control; Pt, patient serum; Po, positive control. HIV-1 infection was defined as detectable amounts of plasma HIV-1 RNA (1.5×10⁵ copies/ml at initial admission), a positive antibody test (HIV1/2 ELISA), and low CD4⁺ T cell counts (38 cells/µl). Plasma HIV-1 env and gag region sequences revealed that the patient was infected with a clade B HIV-1. (<b>d</b>) FACS data from rCD4s stained with anti-IgG (upper left), anti-IgM (upper right), anti-gp120 (rabbit anti-gp120 antiserum) (lower left), or purified IgG from pooled serum from HIV-1⁺ Pts (lower right).</p

sICs on qCD4s trigger Fc-mediated effector systems.

<p>(<b>a–e</b>) Autologous macrophages phagocytose qCD4s with sICs. Orange-CMTMR-labeled macrophages (red) cocultured with CFSE-labeled autologous qCD4s (green) exposed to the indicated concentrations of gp120_IIIB, HIV-1_Lai, or medium followed by incubation with HI patient serum (Pt-serum), non-HI patient serum, or medium for 1 h before coculture. (<b>a–c</b>) Confocal micrographs of representative data (<b>a</b>) and summary of phagocytosis assays (<b>b, c</b>) shown as percentages of macrophages containing 1–2, 3–5, or >6 qCD4s. The numbers in (<b>a</b>) denote percentages of macrophages containing at least one qCD4; inset shows a macrophage containing >10 qCD4s. (<b>d</b>) Time course of TUNEL assay on sIC⁺ qCD4s that were phagocytosed by macrophages. Confocal images of macrophages (arrowheads, red), TUNEL⁺ (green) and cell nuclei (Topro-3, blue). The numbers indicate the percentage of TUNEL⁺ phagocytosed qCD4s/total phagocytosed qCD4s. (<b>e</b>) Summary of the time course of phagocytosis assays. (<b>f</b>) Summary of apoptotic qCD4s in the NK cell-mediated ADCC assay. CFSE-labeled NK cells incubated with autologous qCD4s (2∶1), which were exposed to the indicated concentrations of gp120_IIIB, gp120_BaL, HIV-1_Lai, HIV-1_BaL, or medium. (G) Summary of effects of IL-2 (50 ng/ml) or IL-15 (20 ng/ml) treatment on NK cell-mediated ADCC. Bars, SD. The data presented here are representative of at least three independent experiments.</p

The characteristics of ESBL-producing <i>E. coli</i> positive/negative groups in travelers to India.

1)<p>Student’s t-test, ²⁾ Fisher’s exact test.</p><p>ESBL = extended-spectrum β-lactamase; SD = standard deviation; VFR = visiting friends and relatives;</p

The characteristics of ESBL-producing <i>E. coli</i> positive/negative groups.

1)<p>Student’s t-test, ²⁾ Fisher’s exact test.</p><p>ESBL = extended-spectrum β-lactamase; SD = standard deviation; VFR = visiting friends and relatives;</p

Epidemiology of Extended-Spectrum β-Lactamase Producing <i>Escherichia coli</i> in the Stools of Returning Japanese Travelers, and the Risk Factors for Colonization

<div><p>Objective</p><p>Travel overseas has recently been considered a risk factor for colonization with drug-resistant bacteria. The purpose of this study was to establish the epidemiology and risk factors associated with the acquisition of drug-resistant bacteria by Japanese travelers.</p><p>Methods</p><p>Between October 2011 and September 2012, we screened the stools of 68 Japanese returning travelers for extended-spectrum β-lactamase (ESBL) producing <i>Escherichia coli</i>. All specimens were sampled for clinical reasons. Based on the results, the participants were divided into an ESBL-producing <i>E. coli</i> positive group (18 cases; 26%) and an ESBL-producing <i>E. coli</i> negative group (50 cases; 74%), and a case-control study was performed. Microbiological analyses of ESBL-producing strains, including susceptibility tests, screening tests for metallo-β-lactamase, polymerase chain reaction amplification and sequencing of <i>bla</i>_CTX-M genes, multilocus sequence typing, and whole genome sequencing, were also conducted.</p><p>Results</p><p>In a univariate comparison, travel to India was a risk factor (Odds Ratio 13.6, 95% Confidence Interval 3.0–75.0, p<0.0001). There were no statistical differences in the characteristics of the travel, such as backpacking, purpose of travel, interval between travel return and sampling stool, and duration of travel. Although 10 of 13 analyzed strains (77%) produced CTX-M-15, no ST131 clone was detected.</p><p>Conclusion</p><p>We must be aware of the possibilities of acquiring ESBL-producing <i>E. coli</i> during travel in order to prevent the spread of these bacteria not only in Japan but globally.</p></div