The HIV latency reversing agent HODHBt inhibits the phosphatases PTPN1 and PTPN2

Nonreceptor tyrosine phosphatases (NTPs) play an important role in regulating protein phosphorylation and have been proposed as attractive therapeutic targets for cancer and metabolic diseases. We have previously identified that 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhanced STAT activation upon cytokine stimulation, leading to increased reactivation of latent HIV and effector functions of NK and CD8 T cells. Here, we demonstrate that HODHBt interacted with and inhibited the NTPs PTPN1 and PTPN2 through a mixed inhibition mechanism. We also confirm that PTPN1 and PTPN2 specifically controlled the phosphorylation of different STATs. The small molecule ABBV-CLS-484 (AC-484) is an active site inhibitor of PTPN1 and PTPN2 currently in clinical trials for advanced solid tumors. We compared AC-484 and HODHBt and found similar effects on STAT5 and immune activation, albeit with different mechanisms of action leading to varying effects on latency reversal. Our studies provide the first specific evidence to our knowledge that enhancing STAT phosphorylation via inhibition of PTPN1 and PTPN2 is an effective tool against HIV.

Sex as a biological variable.

All experiments using primary human PBMCs use cells isolated from both male and female donors.

CETSA-MS

CETSA-MS was performed at Pelago Biosciences, Sweden.

Sample matrix. Pooled human PBMCs were purchased from 3H Biomedical. Cells where thawed the day before the experiment in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin (PS) (all from Thermo Fisher Scientific) and cultured at 37°C and 5% CO₂. For the experiment, the cells were pelleted, washed with HBSS (Thermo Fisher Scientific), and pelleted again. Cell viability was measured with trypan blue exclusion, and cells with a viability above 90% were used for the experiment.

K562 cells were obtained from ATCC (CCL-243). They were cultured at 37°C and 5% CO₂ in RPMI 1640 medium with 10% FBS and 1% PS (all from Thermo Fisher Scientific). For the experiment, the cells were pelleted, washed with HBSS (Thermo Fisher Scientific), and pelleted again. Cell viability was measured with trypan blue exclusion, and cells with a viability above 90% were used for the experiment.

For both cell types, cell pellets were resuspended in CETSA buffer (20 mM HEPES, 138 mM NaCl, 1 mM MgCl₂, 5 mM KCl, 2 mM CaCl₂ [pH 7.4]) at a density of 40 × 10⁶ cells/mL and used as the 2× matrix solution.

Compounds. HODHBt was purchased from Bio-techne (catalog 6994) and stored at –20°C.

Compressed CETSA-MS experiment. PBMCs were divided into 8 aliquots each and mixed with an equal volume of either one of the 7 test compound concentrations or control at 2× final concentration in the experimental buffer. The resulting final concentrations of the compound were 1, 3, 10, 30, 100, 300, and 1,000 μM; 0.1% DMSO was used as a vehicle control. For K562, only the 1000 µM concentration of HODHBt and the 0.1% DMSO vehicle control were done in quadruplicates. Incubations were performed for 60 minutes at 37°C.

Each of the 8 treated samples (8 concentration points) was divided into 24 aliquots (12 temperature points, 2 replicates) that were all subjected to a heat challenge. After heating, all temperature points for each test condition were pooled to generate 8 × 2 individual (compressed) samples. In addition, nonheated samples were processed alongside the experiment in a single replicate and used to distinguish between changes in thermal stability and changes in protein abundance caused by the treatment.

Precipitated proteins were pelleted by centrifugation (30,000g), and supernatants constituting the soluble protein fraction were kept for further analysis.

Protein digestion and labeling. Equal amounts of total protein from each soluble fraction were subjected to reduction and denaturation, followed by alkylation with chloroacetamide. Proteins were subsequently digested with endoproteinase Lys-C and trypsin.

After complete digestion had been confirmed by nano–liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), samples were labeled with 16-plex Tandem Mass Tag reagents (TMTPro, Thermo Fisher Scientific) according to the manufacturer protocol.

LC-MS/MS analysis. For each TMT16-plex set, peptides were separated by multidimensional chromatography, and high-resolution MS/MS data was acquired with an Orbitrap Exploris 240 mass spectrometer coupled to a Dionex Ultimate3000 nanoLC system (both from Thermo Fisher Scientific).

Protein identification and quantification. Protein identification was performed by database search against 95,607 human protein sequences in Uniprot (UP000005640, download date: 2019-02-21) using the Sequest HT algorithm as implemented in the ProteomeDiscoverer 2.5 software package. Data were recalibrated using the recalibration function in PD2.5, and the final search tolerance setting included a mass accuracy of 10 ppm and 50 mDa for precursor and fragment ions, respectively. A maximum of 2 missed cleavage sites was allowed using fully tryptic cleavage enzyme specificity (lysine [K], arginine [R], no proline [P]). Dynamic modifications included oxidation of methionine, and deamidation of asparagine and glutamine. Dynamic modification of protein N-termini by acetylation was also allowed. Carbamidomethylation of Cysteine, TMTpro modification of Lysine, and peptide N-termini were set as static modifications.

For protein identification, validation was done at the peptide-spectrum-match (PSM) level using the following acceptance criteria; 1 % FDR determined by Percolator scoring based on q value, rank 1 peptides only (54).

For quantification, a maximum coisolation of 50% was allowed. Reporter ion integration was done at 20 ppm tolerance, and the integration result was verified by manual inspection to ensure the tolerance setting was applicable. For individual spectra, an average reporter ion signal/noise ratio of > 20 was required. Furthermore, shared peptide sequences were not used for quantification.

Compressed CETSA MS data processing and ranking. Protein intensities were normalized, ensuring the same total ion current in each quantification channel. Intensity values were then log₂ transformed and aligned between treatments and replicates, so each protein has the same mean intensity in all treatments and replicates.

The fold changes of any given protein across the concentration range was quantified by using the vehicle condition as the reference (i.e., a constant value of 1). Fold changes were also transformed to log₂ values, to achieve a normal distribution around 0. Processed data were uploaded to Pelago data portal.

To estimate effect size (amplitude) and P value (significance) of the protein hits, the individual protein concentration-response curve was fitted using the following formula:

where logI indicates log₂-transformed protein intensity; C indicates log₁₀-transformed compound concentration; and A, B, C_M, and scale indicate curve parameters for the fit. Using the values from the model fit, the effective concentration corresponding to 50% of maximal signal is estimated: pEC_M* = −C_M. Apparent pEC_M* values should be used with caution, given the short incubation time and the specific experiment design.

Significance of the effect for each protein was assessed using 2-way ANOVA F test using the model fitted with formula above. Benjamini-Hochberg correction was applied to F test derived P values to adjust for multiple comparison.

Reagents

Human recombinant IL-2 (rIL-2) and rIL-15 were obtained via the BRB/NCI Preclinical Repository. Human αIL-12 (catalog 500-P154G), αIL-4 (catalog 500-P24), TGF-β (catalog 100-21), rIL12 (catalog 200-12), rIL-4 (catalog 200-04), rIL-21 (catalog 200-21), rIL-7 (catalog 200-07), and rhIFN-β (catalog 300-02BC) were purchased from Peprotech. Human rIFN-α2 (catalog NBP2-34971) was purchased from Novus Biologicals. Human rIFN-γ (catalog 570206) was purchased from BioLegend. Raltegravir (catalog HRP-11680) and Nelfinavir (catalog ARP-4621) were purchased from the NIH HIV Reagent Program. Fluorogenic PTP1B catalytic domain assay kit (catalog 79764) and recombinant GST-tag TC-PTP (PTPN2, 30013) were purchased from BPSBioscience. CRISPR GFP-Cas9, PTPN1 and PTPN2 crRNA, and tracrRNA were purchased from IDT. AC-484 (catalog HY-145923) was purchased from MedChem Express. HEK-Blue CLR selection cocktail (catalog h-csm), Puromycin (catalog ant-pr-1), Blasticidin (catalog ant-bl-1), Zeocin (catalog ant-zn-1), and QUANTI-Blue solution (catalog rep-qbs2) purchased from Invivogen. CytoTox 96 nonradioactive cytotoxicity assay (catalog G1780) was purchased from Promega. CorPlex Cytokine panel kit was ordered from Quanterix (catalog 85-0329). Antibodies were purchased from BioLegend (PE aSTAT5 phospho [Y694], 936903; FITC αCD4, 300506; PerCPCy5.5 αCD56, 362506; APC-Cy7 αCD69, 310914), eBioscience (eF450 fixable viability dye), BD Horizon (BV786 αCD3, 563918; human FC block, 564220), Thermo Fisher Scientific (PE αCD8, 12-0086-42), Beckman Coulter (FITC KC57, 6604665), Cell Signaling Technologies (PTP1B, 5311S; TC-PTP [TC45], 58935S; CRKL, 38710S; STAT, 94205S; RPL7A, 2415S; pSTAT5 [Y694], 9359S; pSTAT1[Y701], 7649S; STAT1, 9177S; pSTAT3 [Y705], 9145S; STAT3, 9139S; pSTAT2, 4441P; STAT2, 4594S; pSTAT4, 4134S; STAT4, 2653S; pSTAT6, 9361S; STAT6, 9362S), Proteintech (PTPN2 polyclonal, 11214-1-AP), Sigma Aldrich (β-actin [AC-15], A5441), and Jackson ImmunoResearch (αRabbit secondary antibody, 111-035-046; αMouse secondary antibody, 115-0350146).

K562 cells were a gift from Katherine Chiappinelli (George Washington University, Washington, DC, USA).

Cell line culture. K562 were cultured in complete RPMI.

CETSA. For the CETSA experiments, the protocol was adapted from Jafari et al. (10). Naive CD4 T cells were isolated from donor PBMCs by negative selection and activated using αCD3/CD28 beads (Dynal/Invitrogen) for 72 hours as previously described (5). The cells were then expanded for a further 7 days in the presence of 30 IU/mL IL-2 in RPMI supplemented with 1% L-glutamine, 10% fetal bovine serum (FBS), and 1% PS (complete RPMI). On day 10, the cells were washed with PBS and resuspended in PBS supplemented with protease inhibitor cocktail (cOmplete, Roche) and phosphatase inhibitor cocktail (phosSTOP, Roche) at a concentration of 20 × 10⁶ cells/mL. Cell suspensions were lysed by freeze-thaw 3 times in liquid nitrogen, and the lysate fractions were separated from debris by centrifugation at 20,000g for 20 minutes at 4°C. Cell lysates were treated with 100 μM HODHBt or the inactive control HBt and incubated at 25°C for 30 minutes. Samples were then separated into 7 × 50 μL aliquots and heated at increasing temperatures for 3 minutes using a thermocycler (MultiGene OptiMAX). The samples were spun down at 15,000 rpm for 10 minutes to remove precipitates and analyzed by Western blot. The following antibodies were used at the noted concentrations: STAT5 (1:1,000), pSTAT5 (1:1,000), CRKL (1:1,000), PTPN1 (1:500), PTPN2 (1:1,000), and β-actin (1:5,000). Band intensities were quantified and plotted as a function of temperature to generate the melting curves of each protein/treatment combination.

For the purified protein CETSAs, PTPN1 catalytic domain was purified as described below. TC-PTP (PTPN2) catalytic domain protein was purchased from BPS Bioscience (catalog 30013). Protein was diluted to equal 62.5 ng in 100 μL PBS supplemented with protease and phosphatase inhibitors. The protein dilutions were treated with DMSO, 100 μM HODHBt, or the inactive control HBt and incubated at 25°C for 30 minutes. Samples were then split into 2 × 50 μL aliquots and heated at 55°C for 3 minutes using a thermocycler. The samples were spun down at 15,000 rpm for 10 minutes to remove precipitates and analyzed by Western blot. The following antibodies were used at the noted concentrations: PTPN1 (1:1,000) and PTPN2 (Proteintech, 1:1,000). Band intensities were quantified and plotted as a function of temperature compared with the unheated control.

PTPN1 protein purification. PTPN1 protein was provided by Heidi Schubert and Chris Hill (University of Utah, Salt Lake City, Utah, USA). A total of 2 L of His-TEV-PTP1B (catalytic domain: 1-301; Addgene, 102719) in BL21(DE3)RIL (Agilent) cells was grown in Luria broth at 37°C until they reached an OD600 of 0.6 and then cooled, induced to a final concentration of 0.4 mM IPTG (Invitrogen) and grown overnight at 18°C. The cell pellet was resuspended in 80 mL of lysis buffer (20 mM Tris [pH 7.5], 40 mM imidazole, 300 mM NaCl, 10% glycerol) with protease inhibitors leupeptin (Roche), aprotinin, and pepstatin (both from GoldBio). The sample was sonicated prior to a high-speed spin to pellet the insoluble fraction. The soluble supernatant was incubated with 5 mL equilibrated Qiagen NiNTA resin for 30 seconds prior to washing the resin with an additional 100 mL of lysis buffer. The salt concentration was reduced to 100 mM prior to elution (20 mM Tris [pH 7.5], 250 mM imidazole, 100 mM NaCl, 10% glycerol). The protein was dialyzed against 50 mM Tris (pH 8.0). In total, 500 mM NaCl, 1 mM DTT, and homemade TEV protease were added overnight. A s200 SEC column (GE Healthcare) was run in 20 mM Tris (pH 7.5), 50 mM NaCl, and 1 mM DTT to finish the preparation. The protein was concentrated to 7–9 mg/mL and stored at –80°C.

PTPN1/PTPN2 catalytic domain inhibition assay. HODHBt inhibition (IC₅₀) of PTPN1 enzymatic activity was measured using the fluorogenic PTP1B (catalytic domain) assay kit (BPS Bioscience, 79764) following manufacturer’s instructions. It is designed to measure inhibition of enzyme catalyzation of dephosphorylation of fluorogenic substrate. For PTPN2, the catalytic domain of TC-PTP (PTPN2) was used instead of PTP1B (PTPN1).

The mode of inhibition of HODHBt was determined by adding a final concentration of 0.4 pg/mL of each enzyme to varying concentrations of PTP substrate and HODHBt. The fluorescence signal was measured every 15 seconds for 30 minutes by spectrophotometer using an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Data were analyzed using GraphPad 9.0 software and Michaelis-Mention equation fit.

Genome editing via CRISPR/Cas9. Predesigned guide RNAs for PTPN1 (5′-ACCACAACGGGCCCGTGCTC-3′) and PTPN2 (5′-GCACTACAGTGGATCACCGC-3′) were obtained from IDT. Protocol for transfection by electroporation with the Neon from IDT was followed. Briefly, ribonucleoproteins (RNPs) for PTPN1 and PTPN2 were prepared by first incubating 200 μM Alt-R-CRISPR-Cas9 crRNA (IDT) and Alt-R-CRISPR-Cas9 tracrRNA (IDT) (final duplex concentration of 44 μM) at 95°C for 5 minutes. The guide RNA duplexes were then combined with Alt-R Cas9 GFP (final concentrations 22 pmol and 18 pmol) and incubated at room temperature for 10–20 minutes as per the manufacturer’s instructions. The RNPs were then transfected into K562 cells (0.25 × 10⁶ per reaction) by electroporation. For the PTPN1 + PTPN2 dual condition, equal volumes of PTPN1 and PTPN2 RNP were transfected into the cells via electroporation (Neon). Forty-eight hours later, 0.5 × 10⁶ K562 cells were collected and stained for pSTAT5. The rest were collected for gDNA isolation and KO efficiency determination (described below).

KO efficiency analysis of CRISPR/Cas9-edited K562 cells. Genomic DNA (gDNA) was obtained from cell pellets by resuspending in 50 μL Quick Extract DNA Extraction Solution (Lucigen, QE0905T) and following the extraction program as per the manufacturer’s instructions. Genomic DNA (2 μL) was then PCR amplified (50 μL total reaction volume) using the following primers: PTPN1 Forward (5′-CTATACCACATGGCCTGACTTT-3′), PTPN1 Reverse (5′-GAGTCTCAGGTACGCCTTTATTAG-3′), PTPN2 Forward (5′-ACTGCCAGTGGAAGCAAT-3′), PTPN2 Reverse (5′-TTTGGAGTCCCTGAATCACC-3′). KO efficiency was measured using the T7endonuclease I from NEB (catalog M0302S) and following manufacturer instructions. Analysis was performed using the GelAnalyzer 19.1 software and T7EI beta tool from Horizon Discovery.

Primary cell model of latency. Naive CD4 T cells were isolated via negative selection from PBMCs obtained from HIV^– donors. Cultured Tcm cells were generated and infected as previously described (5, 55, 56). Naive CD4 T cells were isolated from PBMCs from HIV^– donors by negative selection (STEMCELL Technologies, 19555) and activated at 0.5 × 10⁶ cells/mL with αCD3/CD28 Dynabeads (1:1 bead/cell ratio) in the presence of 1 μg/mL αIL-4, 2 μg/mL αIL-12, and 10 ng/mL TGF-β for 72 hours. Dynabeads were removed on day 3, and cells were subsequently expanded in RPMI supplemented with 1% L-glutamine, 10% FBS, and 1% penicillin/streptomycin (complete RPMI) with 30 IU/mL IL-2 before being infected on day 7 via spinoculation with the X4-tropic virus NL4-3. Levels of intracellular p24 were assessed 72 hours later (day 10) by flow cytometry prior to the infected cells being crowded in 96-well round-bottom plates to facilitate spread of infection (100,000 cells/well). On day 13, the cells were uncrowded and plated in the presence of an ART cocktail (1 μM raltegravir + 0.5 μM nelfinavir) and 30 IU/mL IL-2, and p24 levels were again measured by flow cytometry. Ninety-six hours later (day 17), the CD4⁺ cells were sorted from the infected cultures by positive selection (Dynabeads CD4 Positive Isolation Kit, Thermo Fisher Scientific, 11331D), and p24 levels were measured before and after sort. The CD4⁺ cells were then resuspended at 1 × 10⁶ cells/mL and plated with reactivation conditions for a further 48 hours; reactivation was measured by p24 stain on day 19.

Flow cytometry. Flow cytometry was used to measure the levels of STAT5 phosphorylation in the CRISPR/Cas9-edited K562 cells and total CD4 T cells, as well as immune activation in PBMCs. Between 0.3 × 10⁶ and 0.5 × 10⁶ cells for each condition were collected and washed with PBS before resuspension in 100 μL FACS buffer (PBS + 2% FBS) with 0.1 μL viability dye (eBioscience Fixable Viability Dye eFluor 450, Thermo Fisher Scientific, 65-0863-18). For immune activation flow cytometry, PBMCs were incubated with Fc block (564220, BD Biosciences) prior to staining with viability dye. Cells were incubated for 10 minutes at 4°C before being washed with 500 μL FACS buffer. Cells were then resuspended in 100 μL prewarmed Fix Buffer I (BD Bioscience, 557870) and incubated at 37°C for 10 minutes. Cells were washed with 500 μL FACS buffer, resuspended in precooled Perm Buffer III (BD Bioscience, 558050), and incubated on ice for 30 minutes. Cells were washed with 500 μL FACS buffer, resuspended with 100 μL FACS buffer + 2.5μL pSTAT5(Y694)-PE (BioLegend, 936903), and incubated for 1 hour at RT in the dark. Cells were washed with 500 μL FACS buffer and resuspended in 200 μL PBS/2% PFA and kept in the dark prior to analysis on a BD LSR Fortessa X20 flow cytometer with FACSDiva software (Becton Dickinson). Data were analyzed using FlowJo (TreeStar Inc.).

To analyze reactivated cells, cells were stained for CD4, viability, and intracellular p24-Gag as previously described (55).

SEAP and cytotoxicity assays. HEK-Blue IL-2/15 cells, HEK-Blue IFN-α/β cells, and HEK-Blue IFN-γ cells were purchased from Invivogen. Cells were maintained in DMEM supplemented with 10% (v/v) heat-inactivated FBS, 1% penicillin, and 1% streptomycin (complete DMEM). Cells were selected with complete DMEM + 30 μg/mL blasticidin and 100 μg/mL zeocin. Cells were maintained in complete DMEM with 1× HEK-Blue CLR selection cocktail, and 1 μg/mL puromycin.

To evaluate the ability of HODHBt to enhance transcriptional activity of STAT1 and STAT2, HEK-Blue IFN-α/β cells and HEK-Blue IFN-γ cells were plated at 50,000 cells/well in a 96-well flat-bottom plate for 24 hours prior to treatment to facilitate adherence. Cells were treated in sextuplet for each HODHBt and IFN concentration for 24 hours. After 24 hours of treatment, plates were spun down at 15,000g for 5 minutes before 20 μL of each well was transferred to a fresh 96-well flat-bottom plate. Then, 180 μL of prepared fresh Quanti-Blue solution was added to each well, and plates were incubated at 37°C for 2 hours. Secreted embryonic alkaline phosphatase (SEAP) levels were measured using a spectrophotometer at 640 nm. For toxicity evaluation, 50 μL of each well was transferred to a fresh 96-well flat-bottom plate. Next, 50 μL of prepared CytoTox 96 reagent was added to each well, and the plates were incubated at room temperature in the dark for 30 minutes. Finally, 50 μL of stop solution was added to each well, and the absorbance was measured using a spectrophotometer at 490 nm.

To evaluate the transcriptional activity of AC-484 and HODHBt, HEK-Blue IL-2/15 cells were used following the same protocol as detailed above. Cells were treated in sextuplet for each compound and IL-15 concentration for 24 hours.

Western blotting. K562 cells were treated with the indicated conditions for 24 hours. Primary total CD4 T cells were isolated from PBMCs by negative selection and treated with indicated conditions for 24 hours. Cells were then washed with PBS and lysed with NETN extract buffer comprised of 100 mM NaCl, 20 mM Tris-Cl (pH 8), 0.5 mM EDTA, 0.5% Nonidet P-40, protease inhibitor cocktail (cOmplete, Roche), and phosphatase inhibitor cocktail (phosSTOP, Roche) for 30 minutes on ice. Lysates were purified by centrifugation at 13,200g for 10 minutes at 4°C, and proteins were visualized on SDS-PAGE. All primary antibodies were used at 1:1,000 concentrations except for β-actin (1:10,000). Secondary anti-rabbit and anti-mouse antibodies were used at a 1:10,000 dilution.

Primary cell pSTAT2 assay. Naive CD4 T cells were isolated from PBMCs from HIV^– donors by negative selection (STEMCELL Technologies, 19555) and activated at 0.5 × 10⁶ cells/mL with αCD3/CD28 Dynabeads (1:1 bead/cell ratio) in the presence of 1 μg/mL αIL-4, 2 μg/mL αIL-12, and 10 ng/mL for 72 hours. Cells were expanded in the presence of 30 IU/mL IL-2 for 48 additional hours before being treated with 100 μM HODHBt, 10 ng/mL IFN-α, or 100 μM HODHBt + 10 ng/mL IFN-α for 24 hours. pSTAT2 levels were measured by flow cytometry (Cell Signaling Technologies).

Primary cell pSTAT5 time course. Total CD4 T cells were isolated via negative isolation from PBMCs from HIV^– donors. Cells were then pretreated with 100 μM HODHBt, 10 μM AC-484, and 5 μM AC-484 for 2 hours prior to the addition of 30 IU/mL IL-2. The 1-hour time point sample was taken and stained for pSTAT5 1 hour after the addition of IL-2. The 24-hour and 48-hour time points were stained at the respective time after IL-2 addition. For the IL-15 samples, cells were pretreated with 100 μM HODHBt or 10 μM AC-484 for 2 hours prior to the addition of 100 ng/mL IL-15. Samples were stained for pSTAT5 48 hours after the addition of IL-15, and flow cytometry analysis was performed as described above.

PBMC immune activation and cytokine analysis. PBMCs from HIV^– donors were pretreated at 3 × 10⁶/mL for 2 hours with 100 μM HODHBt, 10 μM AC-484, 1 μM AC-484, and 500 nM AC-484; then IL-15 was added at 1 ng/mL and incubated for 48 hours. Cells were collected and stained for flow cytometry analysis, and supernatants were frozen at –20°C.

Frozen supernatants were thawed, and assay was performed according to manufacturer protocol. Ten cytokines were measured using Quanterix SP-X Corplex Cytokine Panel (IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-22, TNF-α).

Statistics. Statistical analyses were performed using GraphPad Prism 9.0 software. The statistical analysis used is indicated in each figure legend. P < 0.05 was considered statistically significant. Two-way ANOVA F test using Benjamini-Hochberg correction to derived P values to adjust for multiple comparisons were used, as well as Wilcoxon matched-pairs signed-rank test, Dunnett’s multiple-comparison test, and Tukey’s multiple-comparison test.

Study approval. Volunteers 17 years and older at the Gulf Coast Regional Blood Center served as blood participants. WBC concentrates (buffy coat) prepared from a single unit of whole blood by centrifugation were purchased.

Data availability. Values for data points shown in graphs are provided in the Supporting Data Values file. All additional data are provided in the supplement.

AB and JNH conceptualized the work. JNH, TDZ, CL, MS, DCC, and AB designed methodology, and experiments were carried out by CSH, JT, JNH, TDZ, WW, DCC, MS, CL, ER, and EKM. AB acquired funding and performed project administration. AB, RBJ, and NSS supervised experimental output and manuscript preparation. JNH and AB wrote the original manuscript, and JNH, NSS, TDZ, and AB reviewed and edited the final manuscript.

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View Supporting data values

We would like to thank Xabier Arias-Moreno for his insightful comments while preparing this manuscript. Additionally, we would like to thank Heidi Schubert for her consultation and expert advice while preparing this manuscript. This work was funded in part by NIH grant R21/R33 AI116212 (to AB), NIH grant R56 AI145683 (to AB), and NIH grant UM1AI164565 (RBJ, AB). EKM was supported by T32CA247756 from the National Cancer Institute. CSH was supported by T32AI158105 from the National Institute of Allergy and Infectious Diseases. This research has been facilitated by the services and resources provided by District of Columbia, Center for AIDS research, an NIH funded program (AI117970), which is supported by the following NIH cofunding and participating institutes and centers: NIAID, NCI, NICJD, NHLBI, NIDA, NINH, NIA, FIC, NIGGIS, NDDK, and OAR. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Address correspondence to: Alberto Bosque, Department of Microbiology, Immunology and Tropical Medicine, School of Medicine & Health Sciences, George Washington University, 2300 I Street, NW, Ross Hall 620, Washington, DC 20037, USA. Email: abosque@gwu.edu.

Conflict of interest: AB has a patent application on the use of HODHBt and other benzotriazine derivatives to enhance immune responses and a patent on the use of HODHBt and other benzotriazine derivatives as latency reversing agents (patent application no. PCT/US19/39336 and patent no. US9730928B2).

Copyright: © 2024, Howard et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License.

Reference information: JCI Insight. 2024;9(18):e179680.https://doi.org/10.1172/jci.insight.179680.

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