Consequences for HIV-1 Infection Induced by T Cell-Derived Soluble

Protein Kinase C and NF-κB−Dependent CD4 Downregulation in Macrophages Induced by T Cell-Derived Soluble Factors: Consequences for HIV-1 Infection This information is current as of October 3, 2021. Rui André Saraiva Raposo, David C. Trudgian, Benjamin Thomas, Bonnie van Wilgenburg, Sally A. Cowley and William James J Immunol 2011; 187:748-759; Prepublished online 10 June

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Protein Kinase C and NF-kB–Dependent CD4 Downregulation in Macrophages Induced by T Cell-Derived Soluble Factors: Consequences for HIV-1 Infection

Rui Andre´ Saraiva Raposo,*,† David C. Trudgian,*,‡ Benjamin Thomas,*,‡ Bonnie van Wilgenburg,* Sally A. Cowley,* and William James*

Upon activation, CD4+ T cells release cytokines, chemokines, and other soluble factors that influence the kinetics of HIV-1 replication in macrophages (Mf). In this article, we show that activation of human primary T cells suppresses the early stages of HIV-1 replication in human primary Mf by downregulating the main cellular receptor for the virus CD4. The secreted factors responsible for this effect have a molecular mass greater than conventional cytokines, are independent of Th1 or Th2 polarization, and are not IFN-g, IL-16, RANTES, or macrophage inhibitory factor, as revealed by cytokine array analysis and neutralization assays. CD4 downregulation is entirely posttranslational and involves serine phosphorylation of CD4 and its targeting to an Downloaded from intracellular compartment destined for acidification and degradation. CD4 downregulation is dependent on the activities of both protein kinase C and NF-kB as well as the proteasomes. Using high-resolution liquid chromatography-tandem mass spectrometry analysis in conjugation with label-free protein quantitation software, we found that proteins that promote Mf adherence and spreading, such as attractin, fibronectin, and galectin-3–binding protein, were significantly overrepresented in the activated T cell supernatant fractions. These results reveal the existence of previously unreported anti–HIV-1 proteins, released by activated T cells that downregulate CD4 expression, and are of fundamental importance to understand the kinetics of HIV infection http://www.jimmunol.org/ in vivo. The Journal of Immunology, 2011, 187: 748–759.

D4 is a type 1 transmembrane glycoprotein expressed at expressing cells (7). The primary site of CD4 function is at the the surface of helper and regulatory subsets of T cells, outer cell surface, and several biological and experimental stimuli C monocytes (Mo)/macrophages (Mf), dendritic cells, can trigger CD4 downregulation. Ab-based CD4 cross-linking B cells, eosinophils, megakaryocytes, and mast cells (1–4). In (8), treatment with soluble forms of HIV-1 gp120 (9), exposure T cells, CD4 is well characterized and is known to mediate T cell to gangliosides (10), and phorbol esters (11–15) trigger CD4 development and maturation (5), to stabilize TCR interactions downregulation. However, the mechanisms leading to its down- by guest on October 3, 2021 with peptide–MHC II complexes on APC, and to amplify in- regulation have remained unclear. In myeloid cells, lacking LCK tracellular T cell signal transduction through the constitutive as- expression, CD4 undergoes constitutive endocytosis and recycling sociation with lymphocyte-specific protein tyrosine kinase (LCK) back to the cell surface, and at steady state, ∼40–50% is found (6). CD4 expression levels in cells of myeloid lineage (Mo/Mf inside the cell. LCK expression in T cells confers stability to CD4 and dendritic cells) are 10- to 20-fold less than in T cells (2). CD4 molecules, which remain at the cell surface, and the absence of is also the receptor for IL-16, a chemoattractant to CD4- LCK correlates with enhanced CD4 endocytosis rates (16). CD4 downregulation induced by PMA is a multistep process, which involves initial CD4 serine phosphorylation, thought to increase *Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, † its affinity to clathrin adaptors, increased rates of CD4 endocy- United Kingdom; Graduate Program in Areas of Basic and Applied Biology, Uni- versity of Porto, Porto 4200-465, Portugal; and ‡Central Proteomics Facility, Sir tosis, and altered endosomal sorting and degradation in the lyso- William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United somes (13, 17). Kingdom Since the discovery of HIV-1, many cellular products that inhibit Received for publication November 4, 2010. Accepted for publication May 7, 2011. its replication have been discovered. These are produced by a va- This work was supported by a Ph.D. grant from the Portuguese Foundation for riety of cells from different sources and activation states. One of Science and Technology (SFRH/BD/15903/2005 to R.A.S.R.). S.A.C. is funded by + a Wellcome Trust Career Re-entry fellowship (WT082260). B.v.W. is funded by the these molecules is the CD8 cell antiviral factor (CAF). CAF has Medical Research Council (U.K.) and Edward Penley Abraham trust funds. a molecular mass of 10–50 kDa and lacks identity to conventional Address correspondence and reprint requests to Dr. Rui Andre´ Saraiva Raposo at the ILs, cytokines, and chemokines (reviewed in Ref. 18). The block current address: Division of Experimental Medicine, Department of Medicine, Uni- induced by CAF is at the LTR-driven transcription of viral pro- versity of California, San Francisco, San Francisco, CA 94110. E-mail address: [email protected] teins (19, 20), and albeit, CAF has not yet been identified, it has The online version of this article contains supplemental material. highlighted the existence of naturally occurring leukocyte-derived + soluble factors with anti–HIV-1 activity. Another unidentified Abbreviations used in this article: BafA1, bafilomycin A1; CAF, CD8 cell antiviral factor; FN, fibronectin; GO, Gene Ontology; LCK, lymphocyte-specific protein ty- soluble factor initially described by Verani et al. (21) is the rosine kinase; LC-MS/MS, liquid chromatography-tandem mass spectrometry; macrophage-derived anti–HIV-1 factor (MDAF). MDAF is pro- LMP2, low molecular mass protein 2; Mf, macrophage; MDAF, macrophage- derived anti–HIV-1 factor; MG132, Z-Leu-Leu-Leu-al; MIF, macrophage inhibitory duced by LPS-stimulated Mf (22) and is able to inhibit replication factor; Mo, monocyte; pAb, polyclonal Ab; PKC, protein kinase C; pS, phosphoser- of primary 34 isolates of HIV-1 in both Mf and T cells at the entry ine; qPCR, quantitative PCR; SIn, normalized spectral index; SINQ, Spectral Index level (21). In Mf, MDAF decreases the expression levels of CD4 Normalized Quantitation. and CXCR4, but in T cells, MDAF only decreases CCR5 expres- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 sion levels. MDAF is sensitive to heat and proteinase K treatment www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003678 The Journal of Immunology 749 and is already preformed in Mf. MDAF lacks identity to IL-10, IL- (HIV-1 cDNA sequence between the 59-LTR sequence and the 59 of the 12,IL-16,IFN-g,anda-defensins, and the molecular mechanisms gag gene) as described previously (26–28). To measure multiple rounds of underlying the downregulation of HIV-1’s receptors remain to be HIV-1 infection, Mf were either left untreated or pretreated with supernatants from activated T cells for 18 h prior to infection with HIV-1BaL for fully elucidated, as does its positive identification. 2 h at 37˚C, followed by extensive washing, and cells were overlaid with In common with other laboratories, we found the kinetics of fresh CM+rM-CSF. Supernatant samples of infected cultures were taken at HIV-1 replication was modulated in complex ways by the si- different time intervals over 14 d and stored at 280˚C until use. p24 Ag multaneous presence of Mf and T cells in different ratios and was quantified by ELISA, as described previously (28, 29). activation states (23, 24). To tackle one aspect of this complex Isolation and activation of CD4+ T cells + problem, we studied the effect of activated uninfected CD4 T cell + secretion products on HIV-1–challenged Mf from healthy blood CD4 Th cells were isolated from the CD14-depleted PBMC, by negative selection using MACS (Miltenyi Biotec) according to the manufacturer’s donors. We found that the activation of T cells leads to the release protocol and seeded at 1.0 3 106 cells/ml. Freshly purified CD4+ T cells of soluble factors into culture supernatants, distinct of conven- were .99% CD3+ and .98% CD4+ and anti-CD19 and anti-CD8 staining tional cytokines of both Th1- and Th2-polarized cells that trigger showed little or no contamination with B cells or CD8+ T cells. To induce + internalization and degradation of CD4 glycoprotein, rendering Th1 secretion profile, CD4 T cells were either activated in CM+rIL-2 (70 U/ml) with 1 mg/ml PHA for 5 d or using anti-biotin MACSiBead particles Mf significantly less susceptible to infection. We characterized coupled to biotinylated Abs against human anti-CD2, -CD3, and -CD28 these molecular mechanisms using specific inhibitors to several (Miltenyi Biotec) for 3 d, according to the manufacturer’s instructions. To molecules involved in intracellular signaling and proteolysis. induce Th2 secretion profile, CD4+ T cells were activated using anti-CD2, Using high-resolution liquid chromatography-tandem mass spec- -CD3, and -CD28 beads in CM+rIL-4 (50 ng/ml) with 3 mg/ml mAb IFN-g (clone K3.53) for 3 d. In the experiments regarding the proteomic analysis trometry (LC-MS/MS) analysis in conjunction with label-free +

of the T cell supernatant fractions, CD4 T cells were activated as de- Downloaded from protein quantitation software, we found that proteins reported to scribed above but in OpTmizer T cell expansion serum-free media (Invi- promote Mf adherence and spreading, such as attractin, ﬁbro- trogen), supplemented with 2 mM L-glutamine (PAA), 100 U/ml penicillin nectin (FN), and galectin-3–binding protein, were signiﬁcantly (PAA), and 100 mg/ml streptomycin (PAA). overrepresented in the activated T cell supernatant fractions with Preparation of conditioned and cytokine-neutralized CD4 downregulating and anti–HIV-1 activities. supernatants from activated CD4+ T cells

+ Materials and Methods Conditioned cell-free supernatants of activated CD4 T cells were col- http://www.jimmunol.org/ lected, ﬁltered (0.45 mm; Millipore), and stored at 280˚C until used. For Abs and reagents dose- and temperature-dependent studies, T cell supernatants were diluted mAbs used were as follows: anti–CD4-FITC (clone RPA-T4; BD Phar- in fresh CM or heated at 56 or 100˚C for 30 min. Supernatants were , , , , , , mingen), anti–CCR5-FITC (clone CTC5; R&D Systems), and anti– fractionated into 3-, 5-, 10-, 30-, 50-, 100-, and 50–100-kDa CXCR4-PE (clone 44717.111; R&D Systems) and matching isotype molecular mass fractions using Amicon centrifugal ﬁlter devices (Milli- controls (R&D Systems), anti-CD4 (clone 34915; R&D Systems), and pore), according to the manufacturer’s protocol. The obtained fractions were further diluted and tested for activity. Neat supernatants from acti- anti–IFN-g (clone K3.53; R&D Systems; ED50 = 0.06–0.3 mg/ml in the presence of 5 ng/ml IFN-g). Polyclonal Abs (pAb) used were as follows: vated T cells were neutralized of IFN-g with 2 mg/ml mAb against IFN-g (clone K3.53), neutralized of IL-16 with 2 mg/ml pAb against IL-16, rabbit anti–IL-16 (PeproTech; ED50 = 0.07–0.012 mg/ml in the presence of neutralized of RANTES with 5 mg/ml pAb against RANTES, and neu-

4.20 ng/ml IL-16), rabbit anti-RANTES (PeproTech; ED50 = 5–7 mg/ml in by guest on October 3, 2021 the presence of 100 ng/ml RANTES), chicken anti-macrophage inhibitory tralized of MIF with 10 mg/ml pAb against MIF or a mixture of appro- factor (MIF) (Lifespan Biosciences), rabbit anti-proteasome subunit 20S priate control Ig incubated for 1 h at room temperature. low molecular mass protein 2 (LMP2) C-terminal (Abcam), rabbit anti- GAPDH (R&D Systems), and rabbit anti-phosphoserine (pS) conjugates Flow cytometry (Millipore). rIL-2, rIL-4, and rM-CSF were purchased from R&D Systems The expression of CD4, CCR5, and CXCR4 was determined by direct and PHA from Sigma-Aldrich. Inhibitor of proteasomal activity Z-Leu- + immunofluorescence 18 h posttreatment with supernatants from activated Leu-Leu-al (MG132), inhibitor of vacuolar type H -ATPases bafilomycin T cells. Mf in staining buffer (10 mg/ml human IgG [Sigma-Aldrich], 1% A1 (BafA1), and inhibitor of protein synthesis and secretion brefeldin A (5 FCS, and 0.01% NaN3) were incubated with 5 mg/ml specific mAbs or mg/ml) were purchased from Sigma-Aldrich. Inhibitor of NF-kB activation matched isotype controls on ice for 30–45 min. For intracellular staining, Bay11-7082 and the inhibitor of protein kinase C (PKC) activity Go¨6976 cells were fixed, permeabilized with 0.2% saponin (Sigma-Aldrich), and were purchased from Calbiochem, dissolved in DMSO (Sigma-Aldrich), stained. The percentage of positive cells and mean fluorescence intensities and used at described concentrations. Recombinant fragment corresponding (MFI) were analyzed by FACSCalibur (BD Biosciences) with 15,000– to aa 1–398 of human CD4 (Abcam) was used a concentration of 10 mg/ml. 20,000 gated events collected, and data were processed using FlowJo Isolation of monocytes and HIV-1 infections (version 7.2.4). Protein expression levels were determined by dividing the geometrical MFI of the Ab staining over the MFI of the isotype control. Adult human blood was obtained from anonymous donors through the U.K. National Blood Service and tested negative for HIV-1, hepatitis B/C, and Immunofluorescence microscopy syphilis. Local Institutional Review Board approval was sought for this Untreated or T cell supernatant-treated Mf for 18 h were washed and fixed work from Oxford University’s Central University Research Ethics for 60 min, followed by quenching in ammonium chloride for 20 min at Committee, and we were informed that specific ethical approval was un- room temperature. Cells were permeabilized, washed, and stained for necessary for this study. PBMC were isolated using Ficoll-Plaque Plus (GE LMP2. Primary Ab was detected using Alexa Fluor 555 goat anti-rabbit Healthcare) by density gradient centrifugation from heparinized buffy IgG (Molecular Probes). Coverslips were mounted on slides with coats. Monocytes were isolated by CD14-positive selection using MACS VectaShield-DAPI Mounting medium (Vector Laboratories) and analyzed (Miltenyi Biotec), according to the manufacturer’s instructions, and seeded at room temperature using a noninverted Pascal LSM8 laser-scanning in RPMI 1640-10% FCS (PAA), 2 mM L-glutamine (PAA), 100 U/ml confocal microscope linked to Pascal software (Zeiss). Images were ac- penicillin (PAA), and 100 mg/ml streptomycin (PAA) (complete medium quired using a 363 oil immersion objective (1.4 aperture) and processed [CM]), supplemented with 50 ng/ml rM-CSF for 7 d. For quantitative PCR using Adobe Photoshop. (qPCR), Mf were either left untreated or pretreated with supernatants from activated T cells for 18 h prior to infection with DNase-treated HIV- Quantitative real-time PCR analysis of CD4 1BaL R5-tropic strain [AIDS Research and Reference Reagent Program, National Institutes Health (25)] by spinoculation for 90 min at 37˚C. Viral Total cellular RNA of untreated Mf or Mf treated for 18 h with T super- inoculum was removed, cells were washed and overlaid with fresh CM natants was isolated using the RNeasy Blood mini kit (Qiagen), according +rM-CSF, and infections were left to proceed for 28 h. DNA was extracted to the manufacturer’s recommendations. cDNA was produced using the using a DNeasy Blood and Tissue Kit (Qiagen), according to the manu- Ambion RETROscript kit (Qiagen), according to the manufacturer’s pro- facturer’s instructions, and qPCR to measure early stages of HIV-1 repli- tocol. qPCR were performed using the SYBR Green detection system and cation were carried out to detect late-stage reverse transcription products primers against CD4 (described in Ref. 30) and b-actin (Eurogentec primer 750 CD4 DOWNREGULATION IN Mf mix). Data were collected and analyzed using an OpticonMonitor (version panel A detection biotinylated Ab mixture (1 in 100) at room temperature. 2.03). CD4 levels were normalized to the corresponding b-actin levels T cell supernatants were added to the membranes and incubated overnight at according to the protocol described in Ref. 31. Results were normalized to 1, 4˚C. Membranes were washed and incubated with HRP-conjugated strep- with 1 being defined as CD4 expression in control Mf. tavidin (1 in 2000) at room temperature for 30 min, developed using chemiluminescence-type solution, and exposed to x-ray film for 5–10 min. Quantitative western blotting Detection of endotoxin Untreated Mf or Mf treated for 18 h with T cell supernatants were lysed in ice-cold lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 1% [v/v] Endotoxin levels were measured in supernatants from activated or control n-dodecyl b-D-maltoside [Sigma-Aldrich]), 13 protease inhibitor mixture unactivated T cells using a granulocyte CD62L-shedding assay, following [Roche], and phosphatase inhibitor mixture 2 [1:100; Sigma-Aldrich]). the method described by Ref. 36 in which granulocytes respond to endo- Western blots were carried out, and membranes were scanned using Od- toxin by cleavage of the ectodomain of surface CD62L. In brief, hepa- yssey (LI-COR). For CD4 phosphorylation, Mf were washed free of rinized whole blood was incubated with an equal volume of supernatant or media, detached, and treated with concentrated, molecular mass-frac- known endotoxin standards (Escherichia coli O55:B5 endotoxin; Lonza) tionated T cell supernatants (1:5 dilution) for 0, 5, 10, 90, and 360 min. for 1 h at 37˚C, then an R-PE–conjugated mouse Ab against CD62L Cells were spun and lysed, and Western blots were performed to detect (Serotec) was added at a final concentration of 1:40 for an additional hour CD4 serine phosphorylation. at 4˚C. Erythrocytes in the samples were subsequently lysed with FACS Lysing Solution (BD Biosciences) for 5 min, remaining leukocytes were Cell viability assays washed with PBS, and then analyzed using a FACSCalibur (BD Bio- sciences), with CellQuest acquisition software and FlowJo (version 7.6) The effect of the pharmacological inhibitors on Mf viability was evaluated analysis software. Granulocytes were gated according to their high for- using the MTS assay (Promega), according to the manufacturer’s protocol. ward/side scatter characteristics, and gates were then established for this Background absorbance readings from reagent and media alone were population to determine CD62L-positive versus CD62L-negative gran- deducted, and the values were expressed as a percentage of the untreated

ulocyte populations. All standards and samples were tested against three Downloaded from control cells absorbance. independent blood donors. ProteoMiner enrichment Statistical analysis The large dynamic range of protein abundance in the concentrated 50–100- Statistical analysis was performed by paired t test using GraphPad Prism kDa T cell supernatant fractions was reduced using the ProteoMiner (version 5.01). Asterisks indicate the p values as follows: *p = 0.05–0.01, kit (Bio-Rad), in accordance with the manufacturer’s instructions. For **p = 0.01–0.001, ***p , 0.001, and p . 0.05, NS. Significance refers to downstream mass spectrometry analysis, enriched samples were reduced difference from the controls, unless otherwise stated; n refers to the http://www.jimmunol.org/ and loaded onto NuPAGE 4–12% Bis-Tris precast gels (Invitrogen). number of blood donors tested. Coomassie-stained gel lanes were cut into 10 equal pieces, digested with trypsin, and analyzed by LC-MS/MS using label-free software. Results Protein quantitation and identification using label-free Soluble factors released from activated T cells prevent Spectral Index Normalized Quantitation and MaxQuant infection of Mf by HIV-1 and downregulate CD4 expression ProteoMiner-treated unactivated 50–100 kDa and activated 50–100-kDa The capacity of conditioned activated T cell supernatants to block fractions of T cell supernatants were in gel-digested and analyzed by LC- MS/MS using an Orbitrap mass spectrometer (Thermo Scientific) coupled HIV-1 infection of Mf was investigated by p24 ELISA quantifi- to a U3000 nano-HPLC system (Dionex). Each sample was injected in cation over 14 d. HIV-1 infection of Mf pretreated for 18 h by guest on October 3, 2021 triplicate using a 120-min LC gradient and a data-dependent acquisition showed slower kinetic rates of infection and reduced p24 pro- method in which 2+, 3+, and 4+ charged species were selected for frag- duction when compared with infected and untreated Mf (Fig. 1A). mentation. Fold changes in protein abundance between unactivated and activated samples of the T cell supernatants was estimated using two label- By qPCR analysis, Mf pretreated for 18 h with T cell super- free quantitation methods. MaxQuant Label Free (version 1.0.13.13) (32, natants contained lower levels of HIV-1 reverse transcription 33) calculates the intensity under the reconstructed ion chromatograms for products (Fig. 1B), indicating that the block in viral replication is individual peptides and compares between samples to estimate protein at an early stage. Viral replication was unaffected by the reagents abundance changes (http://www.maxquant.org). Spectral Index Normal- (PHA or IL-2) used to activate the T cells (Fig. 1B). ized Quantitation (SINQ) (D.C. Trudgian, manuscript in preparation) is an in-house developed label-free quantitation tool, based on the accepted By qPCR analysis, we showed that the early stages of HIV-1 method normalized spectral index (SIn) (34). The tool is incorporated into were affected. Therefore, we investigated whether treatment Oxford University’s Central Proteomics Facility Pipeline (35) and allows with the T cell supernatants had any effect on protein expression the quantification of relative protein abundances between different samples levels of HIV-1’s receptor and coreceptors. No statistically sig- and the absolute amount of protein within a sample to be estimated in an nificant change was observed in the MFI levels of coreceptors automated manner from the calculated SIn. Data were searched using the search engines Mascot, Open Mass Spectrometry Search Algorithm, and CXCR4 and CCR5, compared with untreated control Mf (Fig. X!Tandem against International Protein Index Human database for the 1C). CD4 expression levels at the surface of treated Mf were SINQ calculation and using Mascot for only the MaxQuant calculation. reduced 2-fold compared with untreated Mf (p = 0.0074; n =8) The precursor mass tolerance was set at 20 ppm and the MS/MS mass (Fig. 1C), and the percentage of Mf expressing surface CD4 was accuracy at 0.5 Da. Data were statistically analyzed, and false-discovery , rate was determined using iProphet. Proteins identified at ,1% false- reduced by 60% (p 0.001; n = 10) (Fig. 1D). In addition, a 2- to discovery rate with two or more unique peptides were imported to Pro- 3-fold reduction in total CD4 expression (surface and in- teinCenter software (version 3.3.2; Proxeon) and filtered for extracellular tracellular) was observed in treated Mf (Fig. 1D, gray bars). Mf proteins using a Gene Ontology (GO) filter. Protein identifications are treated with cell culture supernatants from unactivated and acti- listed in Supplemental Tables I and II. Fold changes in relative protein abundance were estimated by submitting data to the SINQ software tool vated T cells in the presence of brefeldin A (inhibitor of protein for automated calculation of the SIn. Technical triplicates were used for synthesis and secretion) did not exhibit altered CD4 levels, sug- each label free analysis and the same data were used for both MaxQuant gesting the factors responsible for CD4 downregulation are de- and SINQ label-free analysis. The mean relative protein abundance ratios pendent on the de novo protein synthesis and secretion induced by between the activated and the unactivated T cell supernatant fraction were cellular activation of T cells (Fig. 1D). calculated and plotted onto a graph. To investigate the kinetics of CD4 downregulation, we de- Human proteome profiler cytokine arrays termined the expression levels of the receptor after the addition of Human cytokine array panel A kits were used according to the manufacturer’s the T cell supernatants over a period of 24 h. After an initial lag of protocol (R&D Systems). Although membranes were blocked for 1 h, cell- 4 h, CD4 expression levels were reduced by 2-fold after 6 h of free supernatants from CD4+ T cells were incubated with cytokine array treatment, as observed in the drop of MFI levels, and downregu- The Journal of Immunology 751 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 1. Soluble factors released by activated T cells protect HIV-1 infection in Mf and reduce CD4 protein expression levels. A,Mf were pretreated with supernatants from anti-CD2–, anti-CD3–, and anti-CD28–activated T cells (T cell activated T cell supernatant [Sup], gray line) for 18 h or left untreated (control, black line), prior to infection with HIV-1BaL. Supernatant samples of infected cultures were harvested at different time intervals, and p24 levels were quantified by ELISA (means 6 SEM, n = 3). B, Quantification of HIV-1 reverse transcripts. Mf were pretreated with supernatants from the following: IL-2/PHA–activated T cells; anti-CD2–, anti-CD3–, and anti-CD28–activated T cells; unconditioned media supplemented with IL-2; unconditioned media supplemented with IL-2/PHA; or left untreated (control, black bar) for 18 h, prior to infection with HIV-1BaL. Cells were harvested 28 h postinfection, and real-time qPCR were carried out, as described in Materials and Methods (means + SD error bars, n = 4). C, Surface receptor levels. Mf were treated with supernatants from activated T cells (gray bars) or left untreated (control, black bars) for 18 h. Receptor levels were determined by flow cytometry. See Materials and Methods for settings (means + SD error bars; CD4 control: n = 6; CXCR4 control: n = 9; CCR5 control: n = 6; CD4 T cell Sup: n = 8; CXCR4 T cell Sup: n = 7; CCR5 T cell Sup: n = 6). D, Percentage of Mf expressing surface (black bars) and total (gray bars) CD4. Mf were treated with supernatants from the following: IL-2/PHA–activated T cells; anti-CD2–, anti-CD3–, and anti-CD28–activated T cells; unactivated T cells; activated T cells in the presence of 5 mg/ml brefeldin A; or left untreated for 18 h, followed by flow cytometry (means + SD error bars, n = 4). E, Kinetics of CD4 downregulation. Mf were treated with supernatants from anti-CD2–, anti-CD3–, and anti-CD28–activated T cells for 0, 2, 4, 6, 18, and 24 h (T cell Sup, gray line) or left untreated (control, black line), followed by flow cytometry analysis of total CD4 expression levels (MFI means 6 SEM, n = 3). F, CD4 downregulation is mediated by a soluble factor of 50–100 kDa. Size fractionation of activated T cell supernatants was performed as described in Materials and Methods.Mf were treated with unfractionated (neat) or T cell supernatant size fractions (,3, ,5, ,10, ,30, ,50, ,100, and 50–100 kDa) or left untreated (control, black bar) for 18 h, followed by flow cytometry analysis of CD4 surface expression levels (means + SD error bars, n = 3). G, Anti– HIV-1 activity is present in the 50–100-kDa size fraction. Mf were pretreated with unfractionated (neat) or T cell supernatant size fractions (,30, ,50,

,100, and 50–100 kDa) or left untreated (control, black bar) for 18 h prior to infection with HIV-1BaL. Cells were harvested 28 h postinfection, and real- time qPCR were carried out as described in Materials and Methods (means + SD error bars, n = 4). H, Anti–HIV-1 activity is dose dependent and (I) 752 CD4 DOWNREGULATION IN Mf lation was maximal by 18 h (Fig. 1E). The reduced levels of CD4 of CD4 expression levels in treated Mf showed that both MG132 expression in treated Mf correlate with the low susceptibility to and BafA1, when added individually, significantly inhibited CD4 infection by HIV-1 at the viral entry stage (Fig. 1A,1B). downregulation by .20% (p = 0.0013; n = 4 and p = 0.0020; n = Size fractionation and concentration of the T cell supernatants 4, respectively), and the decrease in the expression levels of CD4 were performed using different molecular mass cutoff filters. The was completely blocked when both inhibitors were added together resultant ,3-, ,5-, ,10-, ,30-, and ,50-kDa fractions were (Fig. 2D, left panel). Using quantitative Western blotting analysis, shown to have no effect on the level of CD4 expressed by Mf, we confirmed the protection of CD4 by MG132 and BafA1 (Fig. but the ,100- and 50–100-kDa fractions resulted in a significant 2D, right panel). Taken together, these data suggest that CD4 decrease in the levels of CD4 (p = 0.0301; n = 3) (Fig. 1F). In degradation induced by components in the T cell supernatants is addition, the ,30- and ,50-kDa fractions did not affect HIV-1 dependent on both the proteasomes and acidic compartments. replication, whereas the ,100- and 50–100-kDa fractions resulted Treatment of Mf with both inhibitors in unconditioned media had in a significant block of the early stages on HIV-1 replication by no effect on the number of cells expressing CD4 (Supplemental 86 and 87%, respectively (p , 0.001; n = 4) (Fig. 1G). The block Fig. 1A) and did not affect cell viability (Supplemental Fig. 1B). on the early stages of HIV-1 replication induced by the ,100- and Because CD4 degradation induced by the T cell supernatants was 50–100-kDa fractions correlates with low levels of CD4 in treated dependent on the proteasome, we investigated the cellular distri- Mf. Anti–HIV-1 activity induced by the T cell supernatants was bution of LMP2, a catalytic active subunit of the proteasome, by shown to be dose dependent (Fig. 1H) and temperature sensitive immunofluorescence microscopy and LMP2 expression levels by (Fig. 1I). This suggests that the T cell-derived soluble factors quantitative Western blotting 18 h posttreatment. In control un- responsible for CD4 downregulation and anti–HIV-1 activity are treated Mf, LMP2 is located in clusters in the perinuclear region heat sensitive and with a molecular mass greater than that of of the cell and inside the nucleus, as demonstrated previously (37, Downloaded from conventional known cytokines and chemokines. 38). In contrast, LMP2 in treated Mf appears to be dispersed throughout the cytosol (Fig. 2E, upper panel). Using quantitative The mechanism of CD4 downregulation Western blotting analysis, we detected an increase in the expres- The decrease in the expression levels of cellular CD4 protein in sion levels of LMP2 in treated Mf. We also detected the ap- treated Mf prompted us to examine whether this effect could be pearance of a higher molecular mass band, reported to be an un- observed at the transcriptional level. We showed by Western blot processed or posttranslationally modified intermediate of LMP2 http://www.jimmunol.org/ that total cellular CD4 levels were reduced by 2-fold in treated (pre-LMP2) (37). Eighteen-hour treatment with T cell super- Mf (p , 0.001; n = 10) (Fig. 2A, left panel) and, using quanti- natants in the presence of MG132 and BafA1 restored LMP2 tative real-time qPCR analysis, that the levels of CD4 transcript expression levels back to control levels and led to the disappear- remained unaffected by treatment with T cell supernatants ance of the pre-LMP2 form (Fig. 2E, lower panel). The increased (p = 0.1102; n = 3) (Fig. 2A, right panel). This shows that the levels of LMP2 expression coincide with the increased proteaso- observed effect is due to a posttranslational modification of CD4. mal activity and degradation of CD4 in treated Mf. To investigate how stable and for how long CD4 downregulation The genes for TAP1 and LMP2 are adjacent in the human ge- was maintained in treated Mf, we determined the recovery of CD4 nome and are expressed divergently from a shared bidirectional by guest on October 3, 2021 expression levels after the T cell supernatants were removed from promoter, which is under the regulatory control of NF-kB (39). the cells by flow cytometry. Twenty-four and 48 h after the T cell Therefore, we assessed whether inhibiting NF-kB activity had any supernatants were removed, CD4 expression levels were still effect on proteasomal activity through an effect on LMP2 ex- significantly reduced by 2- and 1.5-fold (Fig. 2B). Eight days after pression and hence restored CD4 levels in treated Mf. Bay11- T cell supernatants were removed, CD4 expression levels were 7082, an anti-inflammatory agent that selectively and irreversibly fully restored, and no significant difference was observed when inhibits IkBa phosphorylation, preventing the activation of NF-kB compared with control untreated Mf (Fig. 2B). These data in- (40) was used in our assays. The results showed that CD4 deg- dicate that CD4 downregulation induced by the T cell supernatants radation induced by the T cell supernatants 18 h posttreatment was can be fully reverted after 1 wk. inhibited by increasing, nonlethal concentrations of Bay11-7082, Because serine phosphorylation has been reported to be involved reaching statistical significance at 5 mg/ml (p = 0.0027; n =3) in early posttranslational modification and downregulation mecha- with 30% increase in the percentage of Mf expressing CD4 (Fig. nisms of CD4 (11, 14, 15), we used quantitative Western blotting to 2F). Bay11-7082 had no statistically significant effect on CD4 detect any increase in CD4 serine phosphorylation after treatment levels in unconditioned media-treated Mf (Supplemental Fig. 1C) with concentrated 50–100-kDa T cell supernatant fractions. Anal- or affected cell viability (Supplemental Fig. 1D). ysis of the individual CD4 and pS band intensities showed an increase It has been demonstrated that both HIV-1 infection and phorbol in the ratio of pS/CD4 as early as 5 min after treatment (Fig. 2C). esters induce serine phosphorylation and degradation of CD4. To investigate whether the underlying mechanisms leading to This process is accompanied by an increase in intracellular cal- CD4 downregulation involved the protein degradation pathways in cium, 1,2-diacylglycerol, and inositol 1,4,5-triphosphate concen- Mf, we tested whether inhibitors of these pathways would restore trations and suggested to be dependent on PKC activity (41–44). its cellular abundance. CD4 expression levels were determined by We investigated whether T cell supernatant-induced CD4 degra- flow cytometry and quantitative Western blotting after 18 h dation was dependent on the activity of PKC. We used Go¨6976, a posttreatment with T cell supernatants in the presence of nonlethal cell-permeable, reversible, and ATP-competitive inhibitor of PKC concentrations of MG132 (proteasomal inhibitor) and/or BafA1 activity (45, 46). The presence of PKC inhibitor blocked CD4 (inhibitor of vacuolar type H+-ATPases). Flow cytometry analysis degradation in 18-h treated Mf at the lowest concentration tested

temperature sensitive. Mf were pretreated with undiluted (100%) or diluted (50–5%) of the original concentration of T cell supernatants for 18 h and pretreated with heat inactivated (56 or 100˚C for 30 min) T cell supernatants or left untreated (control, black bars) for 18 h, prior to infection with HIV-1BaL. Cells were harvested 28 h postinfection, and real-time qPCR were carried out as described in Materials and Methods (means + SD error bars, n = 4). Signiﬁcance is compared with control, unless otherwise indicated. *p = 0.05–0.01, **p , 0.01–0.001, ***p , 0.001. The Journal of Immunology 753 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 2. CD4 downregulation is a posttranslational mechanism that involves the proteasomes and acidic compartments and is dependent on PKC and NF-kB activities. A, CD4 downregulation is posttranslational. Mf were treated with supernatants from activated T cells for 18 h (gray bars) or left untreated (control, black bars), CD4 protein expression levels were determined by quantitative Western blotting analysis (n = 10, left panel), and CD4 mRNA levels were determined by quantitative RT-PCR (n =3,right panel), as described in Materials and Methods. B, CD4 expression levels remain reduced up to 8 d after removal of the T cell supernatants. Mf were treated with supernatants from activated T cells for 18 h (gray bars) or left untreated (control, black bars), washed, and left in culture with fresh media for 24 h, 48 h, and 8 d. At the indicated time points, cells were used for flow cytometry analysis of total CD4 expression levels (means + SD error bars, n =5)C, Downregulation mechanism involves CD4 serine phosphorylation. CD4 pS levels in Mf were determined by quantitative Western blotting analysis after treatment with concentrated size fraction of 50–100 kDa for 0, 5, 10, 90, and 360 min. Figure depicts histograms corresponding to CD4 and pS stains. CD4 and pS pixel intensity bands and pS/CD4 ratios are shown. D, CD4 downregulation involves the proteasomes and acidic compartments. The percentage of Mf expressing total CD4 after 18 h treatment with T cell supernatants in the presence of 5 mM MG132 and/or 100 nM BafA1 (n = 4) was determined by flow cytometry (left panel) or by quantitative Western blotting analysis (right panel). CD4 and GAPDH pixel intensities are depicted. E, Cellular localization of LMP2 in control (top panel) and T cell supernatant-treated (lower panel)Mf was conducted by immunofluorescence microscopy 18 h posttreatment with supernatants from activated T cells. LMP2 expression levels in Mf were determined by quantitative Western blotting analysis 18 h posttreatment with supernatants from activated T cells in the presence or absence of 5 mM MG132 and 100 nM BafA1. F, CD4 downregulation is dependent on the activities of NF-kB and (G) PKC. The percentage of Mf expressing total CD4 after 18 h treatment with T cell supernatants in the presence or absence of Bay11-7082 (n = 3) and in the presence or absence of Go¨6976 (n = 3) was determined by flow cytometry. Bars represent means + SD error bars. **p , 0.01–0.001, ***p , 0.001.

(Fig. 2G) and had no effect on CD4 in control cells (Supplemental infection. In contrast, M2-activated Mf by the addition of exog- Fig. 1E). Go¨6976 did not affect cell viability (Supplemental Fig. enous IL-4 and showed no signiﬁcant effect on either receptor 1F). This suggests that CD4 degradation induced by the T cell expression or HIV-1 DNA levels. supernatants is dependent upon PKC activity. To test whether CD4 downregulation induced by T cell supernatants was due to an indirect effect through the modulation of CD4 downregulation is independent of the secretion of Mf activation state, we investigated the protein content of super- conventional Th1 and Th2 cytokines from polarized T cells natants once T cells were polarized into Th1 and Th2 phenotypes Cassol et al. (47) reported that activation of Mf through the using proteomic cytokine arrays (Fig. 3A, membrane coordinates). classical M1 pathway by the addition of exogenous IFN-g and Unstimulated CD4+ T cells released IL-2 and the MIF. Th1- TNF-a downregulates CD4 expression and is refractory to HIV-1 polarized T cells released IFN-g, TNF-a, GM-CSF, RANTES, 754 CD4 DOWNREGULATION IN Mf Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 3. IFN-g, IL-16, RANTES, and MIF are not responsible for CD4 downregulation induced by the T cell supernatants. A, Human proteome proﬁler cytokine array panel A membrane coordinates containing 36 different anticytokines printed in duplicate (see details in Materials and Methods). Representative membranes of day 3 supernatants from unactivated T cells; day 3 anti-CD2–, anti-CD3–, and anti-CD28–activated T cells (Th1 Sup); day 3 anti-CD2–, anti-CD3–, and anti-CD28–activated T cells in the presence of IL-4 and anti–IFN-g–neutralizing Abs (Th2 Sup); and 50–100-kDa fraction of day 3 anti-CD2–, anti-CD3–, and anti-CD28–activated T cells. B, CD4 downregulation is independent of Th1- and Th2-released cytokines by polarized T cells. The percentage of Mf expressing total CD4 after 18 h treatment with Th1 or Th2 supernatants from polarized T cells (n = 3). C, CD4 downregulation is not because of IFN-g, IL-16, RANTES, or MIF. Percentage of Mf expressing total CD4 after 18 h treatment with T cell supernatants neutralized for 1 h with 2 mg/ml mAb against IFN-g Ab (clone K3.53) (n = 3), 2 mg/ml pAb against IL-16, 5 mg/ml pAb against RANTES, and 10 mg/ml pAb against MIF (n = 3). D, Active factors in the T cell supernatants do not interact with surface CD4. The percentage of Mf expressing total CD4 after 18 h treatment with T cell supernatants preincubated for 1 h in the presence of 10 mg/ml rCD4 (n = 3). Bars represent means + SD error bars. The signiﬁcance is compared with control, unless otherwise indicated. **p , 0.01–0.001. PC, positive control.

MIF, CCL3, CCL4 and low amounts of IL-17 and IL-13. Th2- The supernatants used for cytokine profiling were tested for polarized T cells (rIL-4 and anti–IFN-g–blocking Ab) released IL- their capacity to decrease CD4 expression levels in Mf. When 5, IL-8, IL-13, and IL-17, but no detectable levels of IFN-g were assayed for activity, Th2-derived supernatants resulted in a 30% detected. Because we had evidence that a component in the 50– decrease of CD4 levels (p = 0.0088; n = 3) and Th1 produced 100-kDa fractions reduced the amount of CD4 and efficiently a 50% decrease (p = 0.0012; n = 3) (Fig. 3B). This indicates that blocked HIV-1 infection in treataed-Mf, we investigated the cy- CD4 downregulation and anti–HIV-1 activity cannot be attributed tokine profile of this fraction and found it contained none of the to any of the soluble factors detectable using the arrays, and are molecules that could be detected using the array (Fig. 3A). unlikely to be a consequence of Mf polarization. The Journal of Immunology 755

IFN-g, IL-16, RANTES, MIF, and endotoxin are not supernatants, we used unstimulated T cell supernatant fractions responsible for CD4 downregulation induced by the T cell as a negative control. CD4+ T cells were activated for 3 d in supernatants OpTmizer T cell expansion serum-free media, and alternatively, + Even though the total molecular mass of IFN-g homodimer is ∼40 CD4 T cells were left unstimulated for 3 d. At day 3, culture kDa and so below the threshold of the 50–100-kDa fraction, and supernatants were harvested, filtered, and size fractionated through the finding that supernatants from Th2-polarized cells lacking 50- and 100-kDa centrifugal filters. The dynamic range of protein IFN-g induced CD4 downregulation, it could still be possible that abundance was reduced prior to LC-MS/MS analysis using Proteo- IFN-g was responsible for the observation we reported. To address Miner. this, we neutralized any IFN-g in the supernatants of activated A representative Coomassie-stained gel is shown in Supple- T cells and found that CD4 levels remained significantly reduced mental Fig. 3B. Supplemental Table I lists the proteins identified in neutralized supernatants (Fig. 3C), compared with controls in the unactivated T cell supernatant fraction after GO-Slim ex- (p = 0.0048; n = 4). tracellular component filtering. Abundant serum proteins such as IL-16 selectively decreases CD4 at the protein and transcrip- albumin, transthyretin, lactotransferrin, afamin, many classes of tional levels in Mf but not in T cells or dendritic cells (30). Al- SERPINs, and complement-related proteins were identified with though IL-16 was not detected by cytokine profiling analysis of large numbers of peptides. Proteins reported to be membrane as- Th1 culture supernatants or the 50–100-kDa fraction, and the sociated or cell derived and secreted were also detected including finding that CD4 transcriptional levels were unaffected, we still vitamin D-binding protein, N-acetylmuramoyl-L-alanine amidase investigated whether neutralizing any IL-16 present in the culture (50), fibulin-1 (51), and matrix metalloproteinase-9. Supplemental Table II lists the proteins identified in the activated T cell super- supernatants would restore CD4 expression. Mf treated with Downloaded from IL-16 neutralized supernatants from activated T cells contained natant fraction after GO-Slim extracellular component filtering. reduced levels of CD4 expression, compared with control (p = Attractin (dipeptidylpeptidase-L, “L” from lymphocytes), reported 0.0068; n = 3) (Fig. 3C). We also tested whether the presence of to be expressed upon activation of T cells and targeted to the RANTES and MIF in the supernatants could contribute to CD4 membrane, followed by the release of the secreted form (52–54), downregulation using neutralizing Abs and found no restoration in was identified with 13 unique peptides. Galectin-3–binding pro- CD4 expression levels (Fig. 3C). Altogether, these data indicate tein (Mac-2–binding protein) promotes integrin-mediated cell http://www.jimmunol.org/ that IFN-g, IL-16, RANTES, and MIF do not account for the adhesion and stimulates host defense against viruses, and tumor decrease in CD4 expression levels induced by the T cell super- cells (55, 56) and FN involved in cell adhesion, cell motility, natants. opsonization, wound healing, and maintenance of cell shape were We then tested whether proteins in the T cell supernatant would uniquely identified only in the activated T cell supernatant fraction directly bound to surface CD4, thereby inducing its internalization (57). and degradation. Preincubation of the T cell supernatants with high Relative protein abundance in unactivated and activated T cell concentrations of a recombinant protein fragment corresponding supernatant fractions were determined using label-free proteomic to the first 398 aa of human CD4 still induced CD4 downregula- software, SINQ and MaxQuant, and the relative protein abundance ratios are shown in Fig. 4. Albumin, apolipoproteins D and H, tion (Fig. 3D). by guest on October 3, 2021 Endotoxin analysis of supernatants from activated or control hemopexin, complement-related protein C3, transthyretin, and unactivated T cells was performed using a granulocyte CD62L- antithrombin-III (SERPINC1) have ratios close to 1, indicating shedding assay to rule out the possibility that the observed CD4 that the protein abundances between the two fractions did not downregulation was due to high endotoxin levels in the super- change. In contrast, AMBP protein, haptoglobin, and a-2-HS- natants. This assay was chosen because it is compatible with tis- glycoprotein were 10-fold more abundant in the activated T cell . sue culture samples containing FCS, in contrast to commercially fraction, and attractin was 40-fold more abundant in the acti- available recombinant Limulus Factor C/fluorogenic substrate vated fraction. FN, QSOX1, biotinidase, fetuin-B, and galectin-3– endotoxin assay kits. Supplemental Fig. 2A shows that endotoxin binding protein were only found in the activated fractions. levels in T cell supernatants (which were concurrently validated as active regarding macrophage CD4 downregulation) (Supplemental Fig. 2B) were not significantly different from unactivated T cell Discussion supernatants (p = 0.53) or from complete unconditioned medium In this study, we demonstrate that conditioned supernatants, in or the blank standard (p = 0.82; two-way ANOVA; df = 16), and which T cells were stimulated, reduce the capacity of HIV-1 to all were below the level of detection of this assay, which was 0.01 replicate and decrease the expression levels of CD4 in treated Mf. EU/ml endotoxin. These data indicate that endotoxin is not re- The proteins responsible for decreasing the levels of CD4 are sponsible for CD4 downregulation in Mf treated with activated dependent on de novo synthesis and secretion triggered by T cell T cell supernatants. activation. CD4 downregulation in treated Mf is entirely posttranslational, involves rapid serine phosphorylation of CD4, and is a complex process, dependent on vacuolar acidification and pro- Proteomic analysis of the T cell supernatant fraction and teasomal activity. In fact, when proteasomal or vacuolar acidifi- label-free quantitation cation-dependent pathways were individually blocked in treated The large dynamic range resulting from highly abundant albumin Mf, CD4 expression levels were only partially restored, sug- peptides and other cell culturing conditions related contaminants gesting that both pathways are required for full CD4 degra- made detection of secreted proteins problematic. To reduce the dation. Proteomic-based identification of CD4-interacting proteins protein dynamic range present in the concentrated T cell super- in Mf (58) detected an E3 ubiquitin ligase to be associated with natant fractions, we used ProteoMiner enrichment kit (48, 49). CD4 in the presence of MG132, strengthening the involvement of Supplemental Fig. 3A shows the capacity of ProteoMiner to the proteasomes in induced CD4 degradation. We were unable to deplete highly abundant contaminants from the supernatant frac- detect ubiquitin-modified CD4 in treated Mf (data not shown), tions. To perform a comparative analysis of the proteins differ- which might be due to the low expression of CD4 in these cells. In entially expressed in the active fraction of the activated T cell treated Mf, LMP2-containing proteasomes are redistributed from 756 CD4 DOWNREGULATION IN Mf

FIGURE 4. Proteomic and relative protein abundance analysis of the concentrated T cell supernatant fraction. SINQ and MaxQuant label-free quantitative analysis of the relative protein abundance in the activated T cell supernatant fraction over the unstimulated supernatant fraction. Box lists the uniquely identi- ﬁed proteins in the activated fractions and the number of unique peptides (parentheses). Downloaded from

the nucleus to the cytosol, and LMP2 expression levels are up- A link among PKC activity, CD4 phosphorylation, and the http://www.jimmunol.org/ regulated. Both redistribution to the cytosol and upregulation of capacity of HIV-1 to infect target cells has been established, and LMP2-containing proteasomes favor the interaction of newly in- concentrations of PKC inhibitors that effectively block PKC ac- ternalized CD4 molecules with the proteasomes and hence in- tivity also block HIV-1 replication (45). PMA, which induces CD4 creased rates of CD4 degradation. We have also shown that de- internalization and degradation, has been linked to the activation gradation of intracellular CD4 in treated Mf was dependent on of PKC and CD4 phosphorylation (59). Our data have shown that NF-kB activation. LMP2 expression, being dependent on NF-kB treatment of Mf with concentrated active fractions of the T cell activation (39), was upregulated in treated Mf and restored back supernatants result in CD4 serine phosphorylation, which prom- to normal levels in the presence of MG132 and BafA1. MG132 pted us to investigate the involvement of PKC in this process. blocks the activity of the proteasomes and reduces IkB degrada- CD4 degradation was sensitive to the PKC inhibitor Go¨6976, in- by guest on October 3, 2021 tion, essential for the activation of NF-kB. Taken together, our dicating a role for PKC in induced CD4 downregulation. In vitro data suggest that CD4 degradation is mediated by an NF-kB– work using pharmacological inhibitors and genetic manipulation dependent upregulation of LMP2. It is also clear that these path- of PKC gene expression identiﬁed NF-kB as a downstream target. ways are induced by components in the T cell supernatants rather In vivo work has also associated the loss of PKC activity with than constitutively active in the Mf, promoting effective degra- defects in the regulation of NF-kB target genes (Refs. 60 and 61 dation of CD4. and reviewed in Ref. 62). Activation of NF-kB is dependent on the

FIGURE 5. Proposed mechanism of CD4 internalization and degradation induced by T cell-derived soluble factors. T cell activation triggers the de novo synthesis and secretion of soluble factors including integrin binding molecules. Engagement to integrin receptors induces the intracellular activation of PKC and serine phosphorylation of CD4. Phos- phorylated CD4 is internalized and targeted for proteolytic degradation in intracellular acidic compartments. In parallel, PKC activation leads to the downstream phosphorylation of IkBa, which disassembles from NF-kB, allowing its nuclear translocation and activation of NF-kB– dependent genes, including LMP2, a catalytic active subunit of the proteasome. Newly internalized CD4 is proteolytic degraded by cytosolic proteasomes. The Journal of Immunology 757 appropriate degradation of its inhibitory interaction partner, IkB, Ag) and promotes cell adhesion and spreading in a process me- after phosphorylation by the IkB kinase (62). In B cells, PKC diated by b integrins (55). activity is necessary for the activation of NF-kB through the Using high-resolution LC-MS/MS analysis, we found that phosphorylation of IkB kinase (63), and in the monocytic cell line proteins that promote Mf adherence and spreading, such as U937, PKC activity is necessary and sufficient for IkB phos- attractin, fibronectin, and galectin-3–binding protein, were sig- phorylation/degradation and NF-kB nuclear translocation (64). nificantly overrepresented in the activated T cell supernatant In our experiments, inhibition of both activities of PKC and NF- fractions. Potentially, their promotion of Mf adherence and kB blocked CD4 degradation in treated Mf, suggesting that genes spreading may allow better antigenic presentation to T cells. The under the regulatory control of NF-kB, such as the proteasomal engagement of these proteins to their surface receptors triggers subunit LMP2, play a role in this pathway. intracellular activation of signaling molecules, in particular PKC. Released products from both Th1- and Th2-polarized T cells In fact, PKC has been implicated in integrin-mediated events, induced CD4 downregulation in Mf and the concentrated 50–100- including focal adhesion formation, cell spreading, cell migration, kDa fraction, which we showed had potent anti–HIV-1 activity and cytoskeletal rearrangements. Treatment of many cell types and induced CD4 downregulation, contained no detectable levels with phorbol esters directly stimulates the activation of PKC, of cytokines and chemokines. Nevertheless, the use of a combi- promoting adhesion, spreading, and migration on extracellular nation of neutralizing Ab against likely candidates such as IFN-g matrices (72, 73). By contrast, inhibition of PKC activity blocks and IL-16 led to the conclusion that these cytokines do not ac- cell adhesion, cell spreading (74), and cell migration (75). count for the decreased levels of CD4 induced by the T cell We propose that activation of PKC initiates a cascade of supernatants. We have also demonstrated that induced CD4 events, including CD4 phosphorylation, downstream activation downregulation was not a consequence of the direct bounding of of NF-kB, and transcription of NF-kB–dependent genes, such as Downloaded from components in the T cell supernatants with CD4 at the surface the LMP2, promoting CD4 internalization and further degra- of the Mf. In addition, we showed that endotoxin cannot be dation (Fig. 5). Our data show strong evidence for the influence accounted for in the CD4 downregulation phenotype because the of T cells in mediating Mf susceptibility to HIV-1. T cell-derived detected levels of endotoxin in both control and T cell supernatant soluble factors have the capacity to induce CD4 internalization samples were indistinguishable and extremely low (,0.01 EU/ and degradation in Mf, which render the cells refractory to in- ml). fection by HIV-1. http://www.jimmunol.org/ Last, we applied a combination of different proteomic-based Our findings are of crucial importance for the understanding of techniques to identify the proteins present in the 50–100-kDa Mo/Mf biology as well as the immunopathogenesis of HIV, and active fraction from the activated T cell supernatants. The use of the proteins identified in our study could have implications as ProteoMiner bead technology allowed the better separation and potential antiviral therapies. resolution of least abundant proteins, which would otherwise be undetected by LC-MS/MS analysis. As we had initially shown Acknowledgments using the proteomic cytokine array, the 50–100-kDa active frac- We thank Dr. Gemma Carter, Dr. Michael (Kenny) Moore, and Dr. Claudia tion was free of conventional cytokines and chemokines. Brockmeyer for helpful discussions and Gabriela Ridlova for helping with by guest on October 3, 2021 Antithrombin-III (SERPINC1) has been shown in a previous the mass spectrometry analysis. We also thank Dr. Fernando Martinez for study attempting to identify CAF as having anti–HIV-1 properties advice on performing the CD62L endotoxin assay. (65). We detected SERPIN1 in the supernatants, but because its relative abundance remained unchanged between the unactivated Disclosures and activated fractions, it cannot account for the observed effects. The authors have no financial conflicts of interest. Attractin (dipeptidylpeptidase-L) was the most upregulated protein by activation of T cells, and its abundance increased .40- References fold. 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SUPPLEMENTAL TABLES Table SI: Proteins identified in the unactivated T cell supernatant fraction.

PROTEIN GENE UNIPROT NUMBER OF iPROPHET % PROTEIN NAME NAME ACCESSION UNIQUE PEPTIDES PROBABILITY COVERAGE Serum albumin ALB P02768 177 1 86.70 Transthyretin TTR P02766 43 1 73.47 Lactotransferrin LTF P02788 38 1 62.16 Hemopexin HPX P02790 28 1 59.09 Ceruloplasmin CP P00450 28 1 39.06 Alpha-2-HS-glycoprotein AHSG P02765 22 1 44.57 Haptoglobin HP P00738 20 1 53.33 Afamin AFM P43652 18 1 35.73 Alpha-1B-glycoprotein A1BG P04217 14 1 44.44 Vitamin D-binding protein GC P02774 12 1 31.01 Lysozyme C LYZ P61626 11 1 39.86 Antithrombin-III SERPINC1 P01008 10 1 33.41 Beta-2-glycoprotein 1 APOH P02749 10 1 46.96 Retinol-binding protein 4 RBP4 P02753 9 1 51.74 Apolipoprotein D APOD P05090 8 1 38.10 N-acetylmuramoyl-L-alanine PGLYRP2 Q96PD5 7 1 22.22 amidase Fibulin-1 FBLN1 P23142 6 1 12.66 Serotransferrin TF P02787 5 1 12.75 Gelsolin GSN P06396 5 1 9.08 Matrix metalloproteinase-9 MMP9 P14780 4 1 6.65 Complement C3 C3 P01024 4 1 3.85 Sex hormone-binding globulin SHBG P04278 4 1 18.91 Plasma protease C1 inhibitor SERPING1 P05155 4 1 10.60 Complement C4-A C4A P0C0L4 4 1 3.21 Thyroxine-binding globulin SERPINA7 P05543 4 1 15.18 Leukocyte elastase inhibitor SERPINB1 P30740 3 1 8.97 Neutrophil collagenase MMP8 P22894 3 1 11.78 Alpha-1-antitrypsin SERPINA1 P01009 3 1 11.96 Neutrophil elastase ELANE P08246 3 1 26.97 Eosinophil cationic protein RNASE3 P12724 3 1 26.88 Inter-alpha-trypsin inhibitor heavy ITIH3 Q06033 3 1 3.82 chain Azurocidin AZU1 P20160 3 1 10.36 Protein S100-A9 S100A9 P06702 3 0.9916 26.32 Putative phospholipase B-like 1 PLBD1 Q6P4A8 2 1 5.24 Periostin POSTN Q15063 2 1 4.90 Alpha-1-acid glycoprotein 1 ORM1 P02763 2 1 11.94 Attractin ATRN O75882 2 0.9999 1.61 Protein AMBP AMBP P02760 2 1 9.38 ______Table SI shows protein and gene name, Uniprot accession number, total number of unique peptides identified for each protein, iProphet probability calculated from the false discovery rate and the percentage of protein coverage.

Table SII: Proteins identified in the activated T cell supernatant fraction.

GENE UNIPROT NUMBER OF iPROPHET % PROTEIN PROTEIN NAME NAME ACCESSION UNIQUE PEPTIDES PROBABILITY COVERAGE Serum albumin ALB P02768 188 1 84.89 Transthyretin TTR P02766 59 1 76.19 Hemopexin HPX P02790 48 1 70.78 Ceruloplasmin CP P00450 38 1 45.26 Afamin AFM P43652 30 1 43.91 Alpha-2-HS-glycoprotein AHSG B7Z8Q2 28 1 35.33 Haptoglobin HP P00738 23 1 59.65 Lactotransferrin LTF P02788 23 1 43.04 Alpha-1B-glycoprotein A1BG P04217 22 1 48.28 N-acetylmuramoyl-L-alanine PGLYRP2 Q96PD5 15 1 36.81 amidase Antithrombin-III SERPINC1 P01008 14 1 43.97 Attractin ATRN O75882 13 1 11.06 Beta-2-glycoprotein 1 APOH P02749 13 1 55.36 Alpha-1-Antichymotrypsin SERPINA3 P01011 11 1 33.48 Vitamin D-binding protein GC P02774 9 1 33.76 Fibronectin FN1 P02751 9 1 5.53 Thyroxine-binding globulin SERPINA7 P05543 9 1 32.53 Retinol-binding protein 4 RBP4 P02753 7 1 41.29 Fibulin-1 FBLN1 P23142 6 1 12.66 Serotransferrin TF P02787 6 1 15.04 Plasma protease C1 inhibitor SERPING1 P05155 6 1 16.40 Protein AMBP AMBP P02760 6 1 26.42 Complement C3 C3 P01024 5 1 4.45 Apolipoprotein D APOD P05090 5 1 31.22 Sulfhydryl oxidase 1 QSOX1 O00391 5 1 9.64 Alpha-1-antitrypsin SERPINA1 P01009 4 1 16.03 Gelsolin GSN P06396 4 1 6.78 Inter-alpha-trypsin inhibitor ITIH3 Q06033 4 1 4.94 heavy Alpha-1-acid glycoprotein 2 ORM2 P19652 3 0.99985 16.92 Periostin POSTN Q15063 3 1 6.82 Alpha-1-acid glycoprotein 1 ORM1 P02763 3 0.99985 15.42 Biotinidase BTD P43251 3 1 7.92 Complement C1r C1RL Q9NZP8 3 1 4.93 Angiotensinogen AGT P01019 3 1 10.93 Corticosteroid-binding globulin SERPINA6 P08185 2 0.99945 6.91 Fetuin-B FETUB Q9UGM5 2 1 7.59 Galectin-3-binding protein LGALS3BP Q08380 2 1 4.62 Complement factor B C2 B4E1Z4 2 0.9999 2.13 Complement C4-A C4A P0C0L4 2 0.9997 1.66 ______Table SII shows protein and gene name, Uniprot accession number, total number of unique peptides identified for each protein, iProphet probability calculated from the false discovery rate and the percentage of protein coverage.