Sjögren’s syndrome (SS) is an inflammatory autoimmune disease primarily affecting the exocrine glands; it has a major impact on patients’ lives. The Chinese herbal formula SS-1 is composed of Gan Lu Yin, Sang Ju Yin, and Xuefu Zhuyu decoction, which exerts anti-inflammatory, immunomodulatory, and antifibrotic effects. Our previous study demonstrated that SS-1 alleviates clinical SS. This study aimed to evaluate the efficacy and mechanism of the Chinese herbal formula SS-1 for salivary gland protein-induced experimental Sjögren’s syndrome (ESS). These results showed that ESS treatment with the Chinese herbal formula SS-1 (1500 mg/kg) significantly alleviated the severity of ESS. We found that SS-1 substantially improved saliva flow rates in SS mice and ameliorated lymphocytic infiltrations in submandibular glands. In addition, salivary gland protein-induced SS in mice treated with SS-1 significantly lowered proinflammatory cytokines  in mouse salivary glands and decreased serum anti-M3R autoantibody levels. In addition, we found that CD4+ T cells isolated from SS-1-treated SS mice significantly reduced the percentages of IFN-γ-producing CD4+ T cells (Th1) and IL-17A-producing CD4+ T cells (Th17). Our data show that SS-1 alleviates ESS through anti-inflammatory and immunomodulatory effects, which provides new insight into the clinical treatment of SS. Sjögren’s syndrome (SS) is an inflammatory autoimmune disease affecting primarily the exocrine glands . LymphocH responses in Sjögren’s syndrome  thymidine for 18 h before harvesting. The incorporation of radioactivity was measured in a beta-counter (Beckman Instruments). The IFN-γ and IL-17A production in the cell supernatants was determined by ELISA.For intracellular detection of cytokines, mouse spleen cells were incubated with SG autoantigen for 48 h. GolgiStop  solution was added 6 h before harvesting the cultured cells. The cells were then washed twice in FACS buffer and stained with phycoerythrin-conjugated anti-mouse CD4 . Splenocytes were fixed and subjected to intracellular staining using the Cytofix/Cytoperm Plus Kit  according to the manufacturer’s instructions . FITC-conjugated mAbs specific to murine IFN-γ and IL-17A were purchased from BioLegend . All samples were detected on an Accuri C5 cytometer using C6 Accuri system software .On day 63, the single-cell suspension from the spleen was pressed through sterile stainless steel mesh. After lysis of red blood cells using commercial RBC lysis buffer (Sigma-Aldrich), the cells were washed several times with RPMI 1640 medium and stained with mAbs specific for mouse CD11C, CD80, and CD86 , as well as control isotype-matched antibody. Stained cells were subjected to an Accuri 5 flow cytometer , and the mean fluorescence intensity was calculated using C6 Accuri system software . The cytokine mRNA expression levels of IL-12, IL-23, and IL-4 were examined using real-time PCR.Immature DCs were cultured in the presence or absence of SS-1 extract dilution  for 1 h followed by stimulation with lipopolysaccharide  for 16 h and stained with mAbs specific for mouse CD11C, CD80, and CD86 . After mAb staining, the samples were detected on an Accuri 5 flow cytometer , and the mean fluorescence intensity was calculated using C6 Accuri system software . The cytokines IL-12 and IL-23 in culture supernatants were analyzed using ELISA kits .n = 5). One-way ANOVA with a post hoc Dunnett test was used to compare multiple experimental groups with GraphPad Prism v5.0 software . A p-value of less than 0.05 was considered a significant difference.Data are expressed as the mean ± standard deviation  were determined by qRT-PCR. As shown in The pathogenic mechanisms of SS involve the synergistic actions of anti-SSA and anti-M3R antibodies. The results showed a significant increase in SSA and M3R-specific IgG in the serum collected on days 28, 42, and 63 in all SS mice compared with naïve mice . HoweverTo study the effect of SS-1 on salivary gland protein-specific T cell responses, spleen cells were isolated from different experimental groups and incubated with salivary gland extract for 72 h in vitro . Comparein vivo. On day 63, splenic DCs were purified and analyzed for phenotypic maturation by flow cytometry. As shown in Owing to the extensive capability of DCs to activate naïve T cells, we further evaluated the effect of SS-1 on DC maturation Following the suppression of splenic DC maturation in In this study, we investigated the efficacy and mechanisms of the Chinese herbal formula SS-1 in experimental Sjögren’s syndrome. In SS, the salivary glands are infiltrated and destroyed by inflammatory cells, which further produce cytokines to enhance the damage of glandular epithelium, resulting in reduced exocrine function and increased autoantibody level ,20. In aMost of the infiltrating cells are CD4+ T-cells and, to a lesser degree, antibody-secreting B cells . T cellsRecently, an experimental SS mouse model was established by Lin et al. . Upon imAt present, no disease-modifying therapies are approved to treat SS . Oral glT cells are known to play an important role in SS . Our preVogelsang et al.  also proA previous study showed that SS patients have higher IL-12 mRNA produced by DCs and IL-12 expression in serum than healthy people. However, IL-12 levels in SS patients were not related to anti-SSA or anti-SSB antibodies . During Recent data suggest that glandular hypofunction in SS could result from functional suppression by anti-M3R autoantibodies ,45,46. SSS-1 treatment leads to beneficial therapeutic effects by improving saliva flow rates and lymphocytic infiltration in SG protein-induced ESS due to the regulation of several inflammatory mediators. These mediators include Th1-mediated cytokines and Th17-mediated cytokines. These results indicate that SS-1 treatment may be an effective and safe therapeutic strategy and can be used as an immunomodulator for T cell-mediated autoimmune diseases, including SS.


We show that reactivation of HIV is associated with downregulation of the key H2S producing enzyme cystathionine-γ-lyase (CTH) and reduction in endogenous H2S. Genetic silencing of CTH disrupts redox homeostasis, impairs mitochondrial function, and remodels the transcriptome of latent cells to trigger HIV reactivation. Chemical complementation of CTH activity using a slow-releasing H2S donor, GYY4137, suppressed HIV reactivation and diminished virus replication. Mechanistically, GYY4137 blocked HIV reactivation by inducing the Keap1-Nrf2 pathway, inhibiting NF-κB, and recruiting the epigenetic silencer, YY1, to the HIV promoter. In latently infected CD4+ T cells from ART-suppressed human subjects, GYY4137 in combination with ART prevented viral rebound and improved mitochondrial bioenergetics. Moreover, prolonged exposure to GYY4137 exhibited no adverse influence on proviral content or CD4+ T cell subsets, indicating that diminished viral rebound is due to a loss of transcription rather than a selective loss of infected cells. In summary, this work provides mechanistic insight into H2S-mediated suppression of viral rebound and suggests exploration of H2S donors to maintain HIV in a latent form.A fundamental challenge in human immunodeficiency virus (HIV) eradication is to understand how the virus establishes latency, maintains stable cellular reservoirs, and promotes rebound upon interruption of antiretroviral therapy (ART). Here, we discovered an unexpected role of the ubiquitous gasotransmitter hydrogen sulfide (H Despite advances in antiretroviral therapy (ART), the persistence of latent but replication-competent HIV in cellular reservoirs is a major barrier to cure (2S) modulate antimicrobial activity, inflammatory response, and metabolism of immune cells to influence bacterial and viral infections (2S remained completely unexplored.Human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS), is responsible for 0.6 million deaths and 1.7 million new infections in 2019 , cystathionine-beta-synthase (CBS), and cysteine-aminotransferase (CAT)–3-mercaptopyruvate sulfurtransferase (MPST), respectively , dissociates into HS– and S2– in aqueous solution, and maintains an HS–:H2S ratio of 3:1 at physiological pH , and counteracting systemic inflammation  or indirectly modulates the activity of transcription factors NF-κB and AP-1  and lymphocytic (J1.1 and J-Lat) models of HIV-1 latency (gag transcript in U1 with a low concentration of PMA (5 ng/ml). Treatment of PMA induces detectable expression of gag at 12 hr, which continued to increase for the entire 36 hr duration of the experiment (2S levels using a fluorescent probe 7-azido-4-methylcoumarin (AzMc) that quantitatively detects H2S in living cells . The short hairpin RNA specific for CTH (U1-shCTH) silenced the expression of CTH mRNA and protein by 90% as compared to non-targeting shRNA (U1-shNT) , translation (HIV-1 p24 capsid protein), and release (HIV-1 p24 abundance in the cell supernatant). We found that the low basal expression of HIV-1 gag in U1-shCTH was stimulated by 16-fold upon depletion of CTH (p=0.0018)  . Moreove U1-shNT . In addi U1-shNT , indicat=0.0018) . FurtherU1-shCTH . ConsistU1-shCTH . DeprivaU1-shCTH , indicat-I). These data indicate that CTH and likely CTH-derived H2S supports latency and impede the reactivation of HIV-1.We also depleted CTH levels in J1.1 cells using RNAi and monitored HIV-1 reactivation by assessing intracellular p24 levels under basal conditions. The depletion of CTH triggered HIV-1 reactivation from latency as evident from a sevenfold increase in p24 levels as compared to shNT . A minorFigure 2—figure supplement 1F2S. These include redox signaling , which non-specifically responds to any type of ROS within the cells. Both U1-shNT and U1-shCTH showed comparable levels of cytoplasmic ROS  or high (5 mM) concentrations of GYY4137 using methylene blue colorimetric assay. As a control, we treated U1 cells with 0.3 mM NaHS, which rapidly releases a high amount of H2S. As expected, NaHS treatment rapidly released 90% of H2S within 5 min of treatment  that is known to block HIV-1 reactivation . Pretreareatment . The effreatment . As expereatment . Pretreaanscript . Becauseanscript , we testectively . The addectively . Earlierectively . Therefoectively . Both RNgag transcript as compared to untreated J1.1  of three healthy human donors with HIV-1 (pNL4.3) and monitored HIV-1 replication by measuring the levels of p24 in the supernatant at 3 and 5 days post-infection. The p24 ELISA confirmed a time-dependent increase in viral load, which was uniformly reduced upon pretreatment with a non-toxic concentration of GYY4137 (0.8 mM)  . Overall2S-mediated suppression (U1-GYY+PMA). Consistent with the role of PMA-mediated oxidative stress in preceding HIV-1 reactivation  was upregulated by PMA.To dissect the mechanism of GYY4137-mediated inhibition on HIV-1 reactivation, we examined the expression of 185 genes associated with HIV-1 reactivation using the NanoString nCounter Technology as described above . Expresstivation , expresstivation . Also, te.g., GSH pathway, TXNRD1, HMOX1, GPX4, and SRXN1). Interestingly, while the expression of Nrf2-dependent CTH gene was induced in U1-GYY+PMA, MPST remained unaffected. The Nrf2 activity has been shown to pause HIV-1 infection by inhibiting the insertion of reverse-transcribed viral cDNA into the host chromosome  were repressed in U1-GYY+PMA, indicating that the expression of these enzymes was likely counterbalanced by the elevated expression of other antioxidant systems by H2S. In line with the antagonistic effect of GYY4137 on HIV-1 transcription, the expression of HIV-1 trans-activator (FOS) was downregulated, and an inhibitor of NF-κB signaling (NFKBIA) was induced in U1-GYY+PMA as compared to U1 or U1-PMA. Finally, GYY4137 stimulated the expression of several anti-HIV  and pro-survival factors (CDKN1A and LTBR) that were repressed by PMA  to facilitate repressive chromatin modifications . Overexp2S suppresses HIV-1 reactivation in multiple cell line models of latency, we next studied the ability of GYY4137 to limit virus transcription in primary CD4+ T cells derived from virally suppressed patients. We used a previously established methodology of maintaining CD4+ T cells from infected individuals on ART for a few weeks without any loss of phenotypic characteristics associated with HIV-1 reservoirs (+ T cells from PBMCs of five HIV-1 infected subjects on suppressive ART. The CD4+ T cells were expanded in the presence of interleukin-2 (IL-2), phytohemagglutinin (PHA), and feeder cells (Having shown that Hservoirs . We isoler cells . The exper cells . In thiser cells . We quaner cells . By day er cells .+ T cells with the protein kinase C (PKC) activator, prostratin, in the absence of any treatment. The activation of viral transcription was measured 24 hr later by RT-qPCR. The removal of ART uniformly resulted in a viral reactivation by prostratin in CD4+ T cells of HIV1 patients  and quiescence (CD127) marker. As expected, CD38 expression increased, and CD127 expression decreased at days 7 and 14 during the activation phase, followed by a gradual reversal of the pattern during the resting phase at days 21 and 28 , central memory (TCM), transitional memory (TTM), and effector memory (TEM) is affected by the GYY4137 in our ex vivo expansion model. We observed that the CD4+ T cells that responded to ex vivo activation were mainly composed of TTM and TEM in our patient samples  to quiescence phase (21–28 days)  . The addulations .+ T cells immediately isolated from the patient’s PBMCs (ex vivo) and the expanded cells treated with ART+ GYY4137 or ART alone for the entire duration of the experiment  as T cell activation stimuli  treatment of ART and ART+GYY4137 were stimulated by prostratin or left unstimulated and subjected to mitochondrial flux analysis. The unstimulated cells from both ART and ART+GYY4147 cultures did not show any difference in OCR , and was nearly comparable to unstimulated cells  in the reduced state on respiratory complex I leading to mitoROS generation by incomplete reduction of O2   . Also, Hpoptosis , which ipoptosis . Finallyachinery . Importaachinery .2S sufficiency and HIV-1 restriction factors  and negative correlation with HIV-1 trans-activators , consistent with an interrelationship between cell metabolism and HIV-1 latency . Both mitochondrial dysfunction and oxidative stress are major complications associated with long-term exposure to ART and contribute to inflammation and organ damage  invariably results in the emergence of resistant mutants . In this2S gas in modulating HIV-1 latency and reactivation. First, we examined the differential expression of enzymes involved in H2S biogenesis and levels of H2S upon HIV-1 latency and reactivation. Next, we genetically silenced the expression of the main H2S producing gene CTH and showed its importance in maintaining HIV-1 latency. We performed detailed mechanistic studies on understanding the role of CTH in regulating cellular antioxidant response, mitochondrial respiration, ROS generation to maintain HIV-1 latency. Finally, pharmacological donor of H2S (GYY4137) was used to reliably increase endogenous H2S levels and to study its consequence on HIV-1 latency and rebound. Primary CD4+ T cells derived from ART-treated HIV-1 infected patients were used to assess the effect of GYY4137 in modulating HIV-1 rebound by improving mitochondrial bioenergetics and mitigating redox stress.The primary objective of this study was to understand the role H+ T lymphocytic cell line Jurkat, HEK293T were procured from ATCC, Manassas, VA. The chronically infected U1, J1.1, ACH-2, J-Lat 6.3, J-Lat 10.6, and TZM-bl cell lines were obtained through NIH HIV Reagent Program, Division of AIDS, NIAID, NIH, USA. All cell lines are verified to be mycoplasma free by EZdetect PCR Kit for Mycoplasma Detection (HIMEDIA). The cell lines were cultured in RPMI 1640 (Cell Clone) supplemented with L-glutamine (2 mM), 10% fetal bovine serum , penicillin (100 units/ml), streptomycin (100 μg/ml) at 37°C and 5% CO2. HEK293T and TZM-bl cells were cultured in Dulbecco’s modified Eagle’s medium (Cell Clone) supplemented with 10% FBS.The human pro-monocytic cell line U937, CD4Sodium hydrosulfide (NaHS), morpholin-4-ium 4-methoxphenyl(morpholino) phosphinodithioate dichloromethane complex (GYY4137), Phorbol-12-myristate-13-acetate (PMA), N-acetyl cysteine (NAC), Prostratin, DL-Propargylglycine (PAG), and L-Cysteine were purchased from Sigma-Aldrich. Recombinant human TNF-α was purchased from InvivoGen. The antiretroviral drugs efavirenz, zidovudine, raltegravir, and lamivudine were obtained through the NIH HIV Reagent Program.5 cells/ml) were stimulated with PMA (5 ng/ml) or TNF-α (100 ng/ml) for the time indicated in the figure legends. HIV-1 reactivation was determined by intracellular gag RT-qPCR or p24 estimation in supernatant by HIV-1 p24 ELISA . J-Lat 6.3 cells were stimulated with Prostratin (2.5 μM) for 24 hr and HIV-1 reactivation was assessed by estimating GFP+ cells  using BD FACSVerse flow cytometer (BD Biosciences). The data were analyzed using FACSuite software (BD Biosciences).Latently infected U1, J1.1, and J-Lat 10.6 , according to the manufacturer’s protocol. RNA (500 ng) was reverse transcribed to cDNA , subjected to quantitative real-time PCR , and performed using the Bio-Rad C1000 real-time PCR system. HIV-1 reactivation was assessed using gene-specific primers . The expTotal cell lysates of PMA-treated and untreated U1, J1.1, U937, and Jurkat cell lines were prepared using radioimmunoprecipitation (RIPA) lysis buffer , 1× phosphatase inhibitor cocktail [Sigma-Aldrich]). After incubation on ice for 20 min, the lysates were centrifuged at 12,000 rpm, 4°C for 15 min. Clarified supernatant was taken and total protein concentration was determined by Bicinchoninic Acid Assay . Total protein extracts were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were probed with an anti-CBS (EPR8579), CTH (ab151769), MPST (ab154514), and anti-HIV-1 p24 (ab9071) from Abcam; Nrf2 (CST-12721), Keap1 (CST-4678), NF-κB p65 (CST-6956), phospho-NF-κB p65 (Ser536) (CST-3033), YY1 (CST-63227), and GAPDH (CST-97166) from Cell Signaling Technologies, Inc; and anti-rabbit IgG (CST-7074) and anti-mouse IgG (CST-7076) were used a secondary antibody. Proteins were detected by ECL and visualized by chemiluminescence  using the Bio-Rad Chemidoc Imaging system . For membrane reprobing, stripping buffer was used  for 20 min at 55°C. After extensive washing with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (Sigma-Aldrich), membrane was blocked and reincubated with desired antibodies.2S levels of U1 and U937 cells were detected using H2S specific fluorescent probe as described  and NaOH (3%) (1:1 ratio) to trap H2S for 30 min. The reaction was terminated using 10% trichloroacetic acid solution. Following this, reactants were incubated with 20 mM N,N-dimethylphenylendiamine (NNDPD) in 7.2 M HCl and 30 mM FeCl3 in 1.2 M HCl for 30 min and absorbance was measured at 670 nm. The concentration of H2S was determined by plotting absorbance on a standard curve generated using NaHS .HFor generating CBS and CTH knockdown in U1 and J1.1 cells, we used validated pooled gene-specific shRNAs from the RNAi Consortium (TRC) library , washed with PBS followed by fixation and permeabilization using a fixation and permeabilization kit (eBiosciences). Permeabilized cells were then incubated with 50 μl of 1:100 dilution of phycoerythrin (PE)-conjugated mouse anti-p24 monoclonal antibody  for 30 min at room temperature. After incubation, the cells were washed two times and the fluorescence of stained samples was acquired using BD FACSVerse flow cytometer (BD Biosciences). The data were analyzed using FACSuite software (BD Biosciences).7 cells) were harvested and lysed by sonication in assay buffer on ice. Lysates were clarified by centrifugation at 12,000 rpm, 4°C for 10 min. Then, 10 μl of sample was added to each well and incubated with enzyme mix and detection reagent as per the manufacturer’s instruction. The assay plate was read using fluorescence plate reader at Ex/Em=365/450 nm in kinetic mode at room temperature. The concentration of cysteine in sample was calculated from standard curve.The quantification of cysteine was determined using Cysteine assay kit (Sigma-Aldrich). U1-shNT and U1-shCTH cells (105 cells/ml) were cultured in complete RPMI 1640 medium containing 208.16 μM L-cystine (as cysteine source) and 100 μM L-methionine (denoted as [+Cys]) or RPMI 1640 medium without L-methionine and L-cystine  supplemented with 100 μM L-methionine  (denoted as cysteine-free RPMI [−Cys]), 10% dialyzed FBS (Gibco), penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were harvested at indicated time points and viral reactivation was analyzed by gag RT-qPCR.U1 cells  according to the manufacturer’s instructions. RNA concentration and purity were measured using a Nanodrop spectrophotometer , bioanalyzers systems , and Qubit assays (Thermo Fisher Scientific). An nCounter gene expression assay was performed according to the manufacturer’s protocol. The assay utilized a custom-made NanoString codeset designed to measure 185 genes, including six house-keeping genes . This cu+ T cells) were seeded at a density of 104–105 per well in a Seahorse flux analyzer plate precoated with Cell-Tak (Corning). Cells were incubated for 1 hr in a non-CO2 incubator at 37°C before loading the plate in the seahorse analyzer. To assess mitochondrial respiration, three OCR measurements were performed without any inhibitor in XF assay media to measure basal respiration, followed by sequential addition of oligomycin (1 μM), an ATP synthase inhibitor (complex V), and three OCR measurements to determine ATP-linked OCR and proton leakage. Next, cyanide-4-(trifluoromethoxy)phenylhydrazone , was injected to determine the maximal respiration rate and the SRC. Finally, rotenone (0.5 μM) and antimycin A (0.5 μM), inhibitors of NADH dehydrogenase (complex I) and cytochrome c - oxidoreductase (complex III), respectively, were injected to completely shut down the ETC to analyze nmOCR. Seahorse data were normalized to total amount of protein (μg) and mitochondrial respiration parameters were analyzed using Wave Desktop 2.6 software (Agilent Technologies).OCRs were measured using a Seahorse XFp extracellular flux analyzer (Agilent Technologies) as per the manufacturer’s instructions. Briefly, cells (U1 or Primary CD47 cells using sonication in MES (2-(N-morpholino) ethanesulfonic acid) buffer. Lysates were clarified by centrifugation and total protein concentration was estimated using BCA assay. Total glutathione, reduced glutathione (GSH), and oxidized glutathione (GSSG) were measured using the glutathione assay kit  according to the manufacturer’s instructions. To measure ROS, cells were loaded with 10 μM of CM-H2DCFDA  or 5 μM of MitoSOX Red  for 30 min at 37°C and exposed to H2O2 (100 μM) or antimycin A (2 μM) or PMA (5 ng/ml) or left untreated at 37°C and 5% CO2. The fluorescence of stained samples was acquired using BD FACSVerse flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (BD Biosciences).Total cell lysate was prepared from 10g, room temperature), and filtered through a 0.45-μm-pore-size membrane filter  to clear cell debris. Virus was concentrated using 5× PEG-it (System Biosciences) as per the manufacturer’s protocol and virus pellet obtained was aliquoted in opti-MEM and stored at –80°C. Viral titration was done using HIV-1 reporter cell line, TZM-bl (NIH HIV Reagent Program) as described earlier , according to the manufacturer’s protocol, in HEK293T cells using HIV-1 NL4-3 DNA . The medium was replaced with fresh medium at 6 hr post-transfection, and supernatants were collected after 60 hr, centrifuged  for 6 hr or left untreated and then stimulated with PMA (30 ng/ml) for 4 hr. Cells were fixed using 1% formaldehyde, neutralized with glycine, and harvested in ice-cold PBS. Cells were lysed and nuclei were sheared by sonication using Bioruptor Pico (Diagenode Inc) to obtain DNA fragments of 200–500 nucleotides. The clarified lysates were immunoprecipitated using ChIP-grade anti-NF-κB p65 (CST-6956) or anti-YY1 (CST-63227), or normal rabbit IgG (as a negative control) for overnight at 4°C followed by incubation with ChIP-grade protein G magnetic beads at 4°C for 4 hr. Chromatin was eluted from protein G beads, reverse-cross linked, and DNA was column purified. Quantitative genomic PCR was done by SYBR green-based real-time PCR using primers spanning the ER, RBE I, and RBE III regions on HIV-1 LTR. The GAPDH gene was used as the reference gene to see non-specific binding. Total input (10%) was used to normalize equal amount of chromatin taken across the samples. The relative proportion of co-immunoprecipitated DNA fragments were determined with the help of threshold cycle (CT) values for each qPCR product using the equation (100×2[CT(input-3.32)- CT(IP)]). The data obtained were represented as fold enrichment normalized to IgG background for each IP reaction.The chromatin immunoprecipitation (ChIP) assays were performed using SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads)  according to the manufacturer’s instructions. Briefly, 10+ T cells purification. The CD4+ T cells were purified from 50×106 PBMCs using EasySep Human CD4+ T cell isolation kit (STEMCELL Technologies).PBMCs were collected from five HIV-1 seropositive subjects on stable suppressive ART and three healthy HIV-seronegative donors . All sub+ T cells were cultured for 3 days after isolation in RPMI 1640 supplemented with 10% FBS, 100 U/ml interleukin-2 , and activated by adding the Dynabeads Human T-Activator CD3/CD28 using a bead/cell ratio of 1:2. Post-activation CD4+ T cells were pre-treated with 800 μM GYY4137 for 6 hr, and infected with HIV-NL4.3 (1 ng p24/ 106 cells) by spinoculation at 1000×g for 90 min at 32°C. Following infection, cells were washed and cultured in the presence or absence of 800 μM GYY4137 in complete media containing 100 U/ml IL-2. To quantify the virion release, supernatant was harvested from infected cells and centrifuged at 400×g for 10 min and virus concentration was estimated by HIV-1 p24 ELISA  according to the manufacturer’s instruction.Primary CD4+ T cells from five ART suppressed, aviremic HIV-1 infected donors were cultured at 37°C in a 5% (v/v) CO2 humidified atmosphere in RPMI 1640 medium (Gibco) with GlutaMAX, HEPES, 100 U/ml IL-2, 1 μg/ml Remel phytohemagglutinin (PHA) (Thermo Fisher Scientific), gamma-irradiated feeder PBMCs  and either ART alone  or ART +100 μM GYY4137. After 7 days, CD4+ T cells were cultured only with ART alone or ART+GYY4137 and 100 U/ml IL-2. For stimulation experiments on day 28, ART and ART+GYY4137 were washed off and 1×106 cells were treated with 1 µM prostratin for 24 hr.Primary CD4+ T cells (1×106) from ART or ART+GYY4137 treated groups were harvested to isolate total RNA using Qiagen RNAeasy isolation kit and 200 ng of total RNA was reverse transcribed . Reverse transcribed cDNA was diluted tenfold and amplified using primers against HIV-1 LTRs and seminested—PCR was performed using primers and probe listed in 6 cells were lysed  at 55°C for 16 hr followed by inactivation at 95°C for 5 min. Digested product was used as a template to set up the first PCR with Taq polymerase (NEB), 1× Taq buffer, dNTPs, HIV-1 and CD3 primers for 12 cycles. The second-round amplification was done using seminested PCR strategy wherein tenfold dilution of first round PCR product was used as a template, HIV-1, and CD3 primers/probes, Taqman Fast Advance master mix (Applied Biosystems) using SetupOnePlus Real-time PCR system (Applied Biosystems). DNA isolated from ACH2 cells that contain a single copy of HIV-1 per cell was used to obtain standard curve.CD4+ T cells derived from HIV-1 infected patients were stained for surface markers using monoclonal antibodies: CD4-BUV395 (SK3), CD45RA-APC-H7 (HI100), CD27-BV785 (O323), CCR7-Alexa 647 (G043H7), CD38-PE-Cy5 (HIT2), and CD127-PerCP-Cy5.5 (eBioRDR5). Additionally, cells were stained with Live/Dead fixable Aqua dead cell stain or AviD (Invitrogen) to exclude dead cells from the analysis as per the manufacturer’s instructions. PBMCs from HIV positiveART-treated patients were incubated with ART or ART in combination with 100 μM GYY4137 and either left unstimulated or stimulated with either CMVpp65 (JPT Technologies) or anti-CD3/anti-CD28 beads (at a bead:cell ratio of 0.25:1) for 18 hr. Cells were stained with antibodies for analysis of cell surface markers CD3-BV570 (UCHT1), CD4-FITC (RPA-T4), CD8-PerCP-Cy5.5 (SK1), CD25-BV421 (M-A251), CD69-PE (L78), and intracellular cytokines IFNγ-Alexa Fluor 700 (B27) and IL-2-APC (MQ1-17H12). Stained samples were run on BD FACSAria Fusion flow cytometer  and data were analyzed with FlowJo version 9.9.6 and v10 .CD4+ T cell lines cultured for 14 days in the presence of ART or ART in combination with 100 μM GYY4137 were washed and rested overnight. Cells were labeled with 0.5 μM cell trace violet (CTV) (Invitrogen) and incubated at 37°C for 7 min in the dark, after which CTV uptake was stopped by addition of 5 ml ice-cold complete RPMI media. Cells were pelleted down washed once with complete RPMI and used in the cell proliferation assay. CTV-labeled cells were seeded at a density of 0.5×106/well and stimulated with either 1.7 μg/ml CMVpp65 (JPT Technologies), anti-CD3/anti-CD28 T cell activator beads at a bead:cell ratio of 0.25:1 (Thermo Fisher Scientific) or 100 ng/ml SEB (Sigma-Aldrich). After 3 days, IL-2 (20 U/ml) supplemented medium was added to the cells and cultured for another 2 days. Proliferation was measured by CTV dilution after 5 days by flow cytometry. In some experiments, after 5 days of culture CTV-labeled cells were pelleted and stained with antibodies to CD3, CD4, and CD25 and CD25 expression was analyzed by flow cytometry.PHA/IL-2 expanded CD4t-test (in which two groups are compared) and one-way or two-way ANOVA  were used. For flow cytometry experiments, data are represented as means ± SEM and p-value between paired samples was determined by Wilcoxon matched-pairs signed-rank test. Analysis of NanoString data was performed using the nSolver platform. Differences in p-values <0.05 and fold change >1.5 were considered significant.All statistical analyses were performed using GraphPad Prism software for Macintosh (version 9.0.0). The data values are indicated as mean ± S.D. For statistical analysis Student’s  2S) inhibits HIV replication and reactivation by a variety of mechanisms including inhibition of NF-κB and recruitment of the epigenetic silencer, YY1, to the HIV promoter. They further report that H2S helps in maintaining mitochondrial bioenergetics and redox homeostasis. Altogether, the study provides novel insights into the mechanisms underlying HIV transcription and reactivation.Pal and colleagues show that hydrogen sulfide  is best known for it smell of rotten eggs and its high toxicity due to interference with oxygen transport. Recent studies suggest, however, that H2S is involved in various physiological and pathological processes and might exert beneficial therapeutic effects at very low doses. Thus, H2S-releasing compounds are considered for the treatment of e.g. cardiovascular diseases. In the present study, Pal et al. report that H2S also plays an important role in reactivation of HIV from latency. The first show that HIV reactivation reduces the levels of endogenous H2S in cell lines. They further show that suppression of the H2S producing enzyme CTH enhances HIV reactivation and modulates the expression of cellular genes associated with apoptosis and mitochondrial function. The authors also provide evidence that endogenous production of H2S or release from small molecule H2S donors allows U1 and Jurkat cells to maintain normal redox homeostasis and mitochondrial bioenergetics and suggest that this promotes HIV-1 latency. Finally, they provide evidence that the H2S donor GYY4137 modulates Nrf2, NF-κB, and YY1 pathways and suppresses HIV reactivation in latently infected T cells from HIV-infected individuals. The study addresses an important topic from a different angle. It is comprehensive and provides insights into the mechanisms underlying the effect of H2S on HIV latency. In addition, the impact of H2S donors on reactivation of HIV is of significant interest. Limitations of the study are that potential risks and problems associated with H2S as therapeutic agent are not addressed. H2S is highly toxic; thus, dosage will be a major challenge. Treatment would not only affect cells harboring latent HIV but all cells and the H2S-mediated mechanisms proposed to suppress HIV reactivation, such as inhibition of NF-κB and altered metabolism, would be expected to cause side-effects. Finally, the authors propose to combine H2S donors with ART to suppress HIV reactivation. However, effective ART usually prevents viral replication with high efficiency. Thus, rebound after treatment interruption (and not under ART) is the problem. In the end, a functional cure only makes sense if it doesn't need to be combined with ART and does not require daily treatment.1. Problems of dosing and side-effects of H2S donors should be critically discussed. The advantage of combining an H2S donor with ART did not become clear to me since the latter very reliably prevents viral replication.+ T cells. However, all mechanistic data were obtained in cell lines that may not faithfully represent the status of latently HIV infected cells in vivo. Ideally, some key finding should be confirmed in primary cells. Otherwise this limitation should be mentioned. Similarly, the cell line used harbor copies of T cell line adapted HIV-1 strains that may differ from proviruses representing the latent HIV reservoirs in vivo.2. The study is comprehensive and it is appreciated that the authors analyzed the impact of an H2S donor in primary CD43. The authors propose to repurpose H2S donors for clinical treatment. Are such agents already used in the clinic or currently just examined in preclinical animal models?4. The authors state that H2S "bolsters the anti-HIV potential of immune cells". This conclusion does not seem to be fully warranted based on the data presented. Thus, the statement should be cautioned or further substantiated.Reviewer #2:While the idea of H2S acting as a regulator of HIV-1 latency is interesting, there is a long list of inconsistencies that hamper the enthusiasm for this manuscript.Much of the cited literature that is used to make the case for their hypothesis is very old and actually refers to active HIV infection and patient studies prior to ART. Also, the literature they cite regarding the role of H2S as an antimicrobial agent seem to be limited to tuberculosis infection.The choice of the latently infected model cell lines is rather unfortunate. There are much better defined models out there these days than J1.1 or U1 cells, such as the J-LAT cells from the Verdin lab or the various reporter cell lines generated by Levy and co-workers. In particularly, U1 cells should not be considered as latently infected, as the virus has a defect in the Tat/TAR axis and is mostly just transcriptionally attenuated. It is unclear why the authors only use J-LAT cells for one of the last experiments.It is further unclear why the authors perform most of the experiments using U1 cells, which are considered promonocytic, but in the end seek to demonstrate the influence of H2S on latent HIV-1 infection in CD4 T cells. Performing all experiments in J1.1 or better J-LAT cells would have seemed more intuitive.The authors suggest that H2S production would control latent HIV-1 infection and reactivation. Regarding the idea that CBS, CTH or possibly MPST would control latent infection as a function of their ability to produce H2S from different sources, there are several questions. First, if H2S is the primary factor, why would the presence of e.g. MPST no compensate for the reduction of CTH? Second, why would J1.1 and U1 cells both host latent HIV-1 infection events, however, their CDB/CTH/MPST composition is completely different? Third, natural variations in CTH expression caused by culture over time are larger than variations caused by PMA activation.Also, the statement that H2S production as exerted per loss of CTH would control reactivation is not supported by the kinetic data. In latently HIV-1 infected T cell lines or monocytic cell lines, PMA-mediated HIV-1 reactivation at the protein level is usually almost complete after 24 hours, but at this time point the difference between e.g. CTH levels only begins to appear in U1 cells.The data for J1.1. are even less convincing.Figure 2F. PMA is known to induce an oxidative stress response, however, in the experiments the data suggest that PMA results in a downregulated oxidative stress response. Maybe the authors could explain this discrepancy with the literature. In fact, both shRNA transductions, scr and CTH-specific seem to result in a lower PMA response.Given that the others in subsequent experiments use GYY4137, which is supposed to mimic the increased release of H2S, the authors should have definitely included experiments in which they would overexpress CTH, e.g. by retroviral transduction. Specifically in U1 cells, which seemingly do not express CBS, overexpression of CBS should also result in a suppressed phenotype.Figure 4F: The authors need to explain how they can measure a 4-fold gag RNA expression change in untreated cells. Also, according to Figure 4A, 300 µM GYY produces much less H2S than 5mM, yet the suppressive effect of 300 µM GYY is much higher?Initially, the authors argue "that the depletion of CTH could contribute to redox imbalance and mitochondrial dysfunction to promote HIV-1 reactivation"(p. 9). Less CTH would suggest less produced H2S. However, later on in the manuscript they demonstrate that addition of a H2S source (GYY4137) results in the suppression of HIV-1 replication and supposedly HIV-1 reactivation. This is somewhat confusing.CTH, or for that matter CBS or MPST do not only produce H2S, however, they also are part of other metabolic pathways. It would have been interesting and important to study how these metabolic pathways were affected by the genetic manipulations and also how the increased presence of H2S (GYY4137) would affect the metabolic activity of these enzymes or their expression.H2S has been reported to cause NFkB inhibition by sulfhydration of p65; as such, the findings here are not particularly novel or surprising. Also, H2S induced sulfhydration is rather not targeted to a specific protein, let alone a HIV protein, making this approach a very unlikely alternative to current ART forms.The description of the primary T cell model used to generate the data in Figure 6 is slightly misleading. Also, the idea of this model was originally to demonstrate that "block and lock" by didehydro-cortistatin is possible. In this application, the authors did not investigate whether GYY4137 would actually induce a HIV "block and lock" over an extended period of time.The authors claim that the major conclusion of their study is that HIV-1 reactivation is coupled to depletion of endogenous H2S, which is associated with dysfunctional mitochondrial bioenergetics, in particular suppressed OXPHOS, GSH/GSSG imbalance, and elevated mitoROS. However, the authors never provide evidence that endogenous H2S is altered in latently HIV-1 infected cells . By the end of the manuscript, the authors have not provided clear evidence that the effects of e.g. CTH deletion would be mediated by the production of H2S, and not by another function of the enzyme. Similarly, the inability of stimuli to trigger efficient HIV-1 reactivation following the provision of unnaturally high levels of H2S is not surprising given reports on the effect of GYY4137 as anti-inflammatory agent and suppressor NF-κB activation. Unless the authors were to demonstrate a true "block and lock" effect by GYY4137 the data will likely have limited impact on the HIV cure field.eLife. Your letter of appeal has been considered by a Senior Editor and a Reviewing editor, and we are prepared to consider a revised submission with no guarantees of acceptance.Thank you for choosing to send your work entitled "Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis" for consideration at Upon careful consideration of your letter and after discussion with reviewers, we have the following recommendations.We recognize that there are several strong aspects of the work presented in your manuscript however, the possible clinical implications of the work are overstated. In addition, further clarification is required in terms of the molecular mechanism proposed. We are willing to consider a revised manuscript based on the following provisors:1. Regarding the conclusion that the reactivation of HIV-2 associated with CTH knockdown must be due to an absence of H2S: This conclusion is problematic as CTH has other functions as well. In addition, other proteins can produce H2S. The addition of H2S in the form of GYY4137, and associated inhibition of HIV-1 reactivation, does not bring further clarity as the concentrations of H2S used are much higher than anything the cells could produce by themselves. As CTH catalyses the last step in the trans-sulfuration pathway from methionine to cysteine, an important experiment would be to demonstrate that CHT KO is specific for H2S production and does not affect cysteine levels. Also demonstrating that cysteine deprivation does not lead to HIV-1 reactivation due to a generalised stress response would be important.2. Cell cultures produce so little H2S that it cannot be measured. As such, in the experiments, addition of GYY4137 produce completely unphysiological H2S concentrations. Given that protein sulfurylation affects thousands of proteins, the addition of GYY4137 is not a biologically relevant reproduction of the presence of CTH, as the experimental system may be associated proteome-wide activity changes. H2S donors are mostly considered for cancer and anti-inflammatory treatments, because they seem to be immunosuppressive. To translate these findings towards clinical relevance for a cure of latent HIV-1 infection, you would have to show that lack of reactivation is not associated with a lack of T cell response. For example, you could demonstrate that T cells, after being cultured in GYY4137 containing medium, still respond to anti-CD3 stimulation with IL-2 secretion or CD25 upregulation. A better assay would be that the extended presence of GYY4137 does not abrogate the ability of T cells to proliferate upon antigen recognition (CMV pp65 should be a good antigen in combination with CFSE stains).3. Clinical Relevance: Unless GYY4137 produces a "lock" state of latent HIV-1 infection events, or selectively kills latently HIV-1 infected T cells, there is no clinical relevance for a cure. No one will exchange ART for an immuno-suppressive experimental treatment, that already in vitro is not sufficiently potent to completely block reactivation. We suggest you remove any focus in the discussion on possible clinical implications and approach presenting this study from a fundamental research perspective as you do have some compelling findings. [Editors’ note: The authors appealed the original decision. What follows is the authors’ response to the first round of review.]Reviewer #1:[…] 1. Problems of dosing and side-effects of H2S donors should be critically discussed. The advantage of combining an H2S donor with ART did not become clear to me since the latter very reliably prevents viral replication.2S donors in the revised manuscript . The higher concentration of H2S is associated with toxicity and side effects (2), whereas lower micro molar concentrations are beneficial (3). The absorption and metabolism are reasonably well characterized for exogenously administered high doses of H2S . The sulfide oxidation pathways is involved in oxidation of excess H2S to thiosulphate and sulphate, which are cleared from the body via the urine in free or conjugated form (6). We assessed cytotoxicity of H2S donor (GYY4137) in all cell lines and primary CD4+ T cell model and selected non-toxic concentrations of GYY4137 to perform assays. As suggested by the editors, we have toned down the clinical potential of H2S donor in the revised manuscript.We appreciate the comment. We appropriately discussed dosing and the side effects of H+ T cells. However, all mechanistic data were obtained in cell lines that may not faithfully represent the status of latently HIV infected cells in vivo. Ideally, some key finding should be confirmed in primary cells. Otherwise this limitation should be mentioned. Similarly, the cell line used harbor copies of T cell line adapted HIV-1 strains that may differ from proviruses representing the latent HIV reservoirs in vivo.2. The study is comprehensive and it is appreciated that the authors analyzed the impact of an H2S donor in primary CD4+ T cells derived from latent HIV-1 infected patients. Consistent with the cell line data, GYY4137 maintains mitochondrial bioenergetics (OXPHOS), suppresses mitochondrial reactive oxygen species (ROS), and induces the expression of the Nrf2-dependent antioxidant genes . In the revised manuscript, we further extended these observations and showed that the effect of GYY4137 is not due to the impaired CD4+ T cells activation .We appreciate the reviewer’s concern. For the reasons outlined by the reviewer, we examined the effect of GYY4137 on the CD4+ T cells from healthy donors and assessed the influence of GYY4137 on viral replication. Similar to findings in cell lines, GYY4137 diminished HIV-1 replication in primary CD4+ T cells without affecting cellular viability .To further allay the reviewer’s concern, we isolated primary CD43. The authors propose to repurpose H2S donors for clinical treatment. Are such agents already used in the clinic or currently just examined in preclinical animal models?2S donors are in human clinical trials. A list of donors and the progress made in clinical trials are indicated in Several H4. The authors state that H2S "bolsters the anti-HIV potential of immune cells". This conclusion does not seem to be fully warranted based on the data presented. Thus, the statement should be cautioned or further substantiated.We have rephrased the sentence to reflect our data showing the ability of GYY4137 to reduce the chances of viral rebound.Reviewer #2:While the idea of H2S acting as a regulator of HIV-1 latency is interesting, there is a long list of inconsistencies that hamper the enthusiasm for this manuscript.Much of the cited literature that is used to make the case for their hypothesis is very old and actually refers to active HIV infection and patient studies prior to ART. Also, the literature they cite regarding the role of H2S as an antimicrobial agent seem to be limited to tuberculosis infection.2S is comprehensively examined in the context of tuberculosis. Given the close association of TB with HIV, we thought our study is very timely and essential. However, we would like to point out that the references showing the effect of H2S on infection caused by respiratory viruses are included in the manuscript (7-9). Further, recent findings showing the influence of H2S in the context of SARS-CoV2 infection are also included in the revised manuscript .We have revised the list of literature and included more relevant references post-ART era. Recently, the antimicrobial role of HThe choice of the latently infected model cell lines is rather unfortunate. There are much better defined models out there these days than J1.1 or U1 cells, such as the J-LAT cells from the Verdin lab or the various reporter cell lines generated by Levy and co-workers. In particularly, U1 cells should not be considered as latently infected, as the virus has a defect in the Tat/TAR axis and is mostly just transcriptionally attenuated. It is unclear why the authors only use J-LAT cells for one of the last experiments.cbs, cth, and mpst transcripts . Additionally, we have performed several other mechanistic experiments in J-LAT cells to validate the data generated in U1 (see response below).As suggested by the reviewer, we have generated new data using J-LAT cells in the revised manuscript. First, we confirmed that PMA-mediated HIV-1 reactivation in J-LAT cells is associated with the down-regulation of It is further unclear why the authors perform most of the experiments using U1 cells, which are considered promonocytic, but in the end seek to demonstrate the influence of H2S on latent HIV-1 infection in CD4 T cells. Performing all experiments in J1.1 or better J-LAT cells would have seemed more intuitive.2S biogenesis counteracts HIV-1 reactivation.The choice of U1 was based on our earlier studies showing that U1 cells uniformly recapitulate the association of redox-based mechanisms and mitochondrial bioenergetics with HIV-latency and reactivation (10-12). We have validated key findings of U1 cells in J1.1 and J-Lat cell lines. We genetically and chemically silenced the expression of CTH in J-Lat cells and examined the effect on HIV-1 reactivation. Consistent with U1 and J1.1, genetic silencing of CTH using CTH-specific shRNA (shCTH) reactivated HIV-1 in J-Lat . Supporting this, pre-treatment of J-Lat with non-toxic concentrations of a well-established CTH inhibitor, propargylglycine (PAG) further stimulated PMA-induced HIV-1 reactivation . Altogether, using various cell line models of HIV-1 latency, we confirmed that endogenous HThe authors suggest that H2S production would control latent HIV-1 infection and reactivation. Regarding the idea that CBS, CTH or possibly MPST would control latent infection as a function of their ability to produce H2S from different sources, there are several questions. First, if H2S is the primary factor, why would the presence of e.g. MPST no compensate for the reduction of CTH? Second, why would J1.1 and U1 cells both host latent HIV-1 infection events, however, their CDB/CTH/MPST composition is completely different? Third, natural variations in CTH expression caused by culture over time are larger than variations caused by PMA activation.2S in the sulfur network. CBS and CTH reside in the cytoplasm, whereas MPST is mainly involved in cysteine catabolism and is mitochondrial localized. The lack of compensation of CTH by MPST could be due to the compartmentalization of their activities. Furthermore, CTH and CBS activities are regulated by diverse metabolites, including heme, S-adenosyl methionine (SAM), and nitric oxide/carbon monoxide (NO/CO). In contrast, MPST activity responds to cysteine availability. How substrates/cofactors availability and enzyme choices are regulated in the cellular milieu of J1.1 and U1 is an interesting question for future experimentation.These questions are important and complex. CBS, CTH, and MPST produce H2S biogenesis and cellular physiology (13). Some of these factors are likely responsible for differential expression of CBS, CTH, and MPST in J1.1 and U1 cells. Regardless of these concerns, viral reactivation uniformly reduces the expression of CTH in U1, J1.1, and J-Lat. While we cannot completely rule out natural variations in CTH expression over prolonged culturing, in our experimental setup CTH remained stably expressed and consistently showed down-regulation upon PMA treatment as compared to untreated conditions.Moreover, the tissue-specific expression/activity of CBS and CTH dictates their relative contributions in HAlso, the statement that H2S production as exerted per loss of CTH would control reactivation is not supported by the kinetic data. In latently HIV-1 infected T cell lines or monocytic cell lines, PMA-mediated HIV-1 reactivation at the protein level is usually almost complete after 24 hours, but at this time point the difference between e.g. CTH levels only begins to appear in U1 cells.The data for J1.1. are even less convincing.2S. We have not made any claims that depletion of CTH precedes HIV reactivation. However, our CTH knockdown data clearly showed that diminished expression of CTH reactivates HIV-1 in the absence of PMA, which is consistent with our hypothesis that H2S production is likely to be a critical host component for maintaining viral latency.We have performed the kinetics of p24 production and CTH in U1 cells. We showed that the levels of p24 gradually increased from 6 h and kept on increasing till the last time point, i.e., 36 h post-PMA-treatment . The p24 ELISA detected a similar kinetics of p24 increase in the cell supernatant . The CTH levels show reduction at 24 h and 36 h. Based on these data, we report that HIV-1 reactivation is associated with diminished biogenesis of endogenous HFigure 2F. PMA is known to induce an oxidative stress response, however, in the experiments the data suggest that PMA results in a downregulated oxidative stress response. Maybe the authors could explain this discrepancy with the literature. In fact, both shRNA transductions, scr and CTH-specific seem to result in a lower PMA response.In our experiment, PMA treatment for 24 h results in down-regulation of oxidative stress genes. However, the effect of PMA on the oxidative stress responsive genes is time-dependent. In our earlier publication, we showed that 12 h PMA treatment induces oxidative stress responsive genes in U1 cells (12), whereas at 24 h, the expression of genes is down-regulated (10). Genetic silencing of CTH resulted in elevated mitochondrial ROS and GSH imbalance, which is in line with a further decrease in the expression of oxidative stress responsive genes as compared to PMA alone. As a consequence, PMA-treatment of U1-shCTH induced HIV-1 reactivation, which supersedes that stimulated by PMA or shCTH alone.Given that the others in subsequent experiments use GYY4137, which is supposed to mimic the increased release of H2S, the authors should have definitely included experiments in which they would overexpress CTH, e.g. by retroviral transduction. Specifically in U1 cells, which seemingly do not express CBS, overexpression of CBS should also result in a suppressed phenotype.2S levels using GY44137. Treatment with GYY4137 suppressed HIV reactivation in multiple cell lines and primary CD4+ T cells. As suggested by the reviewer, overexpression of CTH could be another strategy to validate these findings. However, since the transsulfuration pathway and active methyl cycle are interconnected and share metabolic intermediates , overexpression of CTH could disturb this balance and may lead to metabolic paralysis. Owing to these potential limitations, we used a slow releasing H2S donor (GYY4137) to chemically complement CTH deficiency during HIV reactivation. We thank the reviewer for this comment.We have explored the role of elevated HFigure 4F: The authors need to explain how they can measure a 4-fold gag RNA expression change in untreated cells. Also, according to Figure 4A, 300 µM GYY produces much less H2S than 5mM, yet the suppressive effect of 300 µM GYY is much higher?2S have profound and distinct effects on cellular physiology . One possibility is that the high concentration of H2S induces mitochondrial sulfide oxidation pathway to avert toxicity. This might modulate mitochondrial activity and ROS, resulting in the suppression of GYY4137 effect. Consistent with this, higher concentrations of H2S have been shown to cause pro-oxidant effects, DNA damage and genotoxicity . We have discussed these possibilities in the revised manuscript The four-fold-expression in untreated cells is likely due to leaky control of viral transcription in J1.1 cells (14 16). However, to avoid confusion, we have replotted the results by normalizing the data generated upon PMA mediated HIV reactivation with the PMA untreated cells in the revised manuscript . The suppressive effect of GYY4137 at the lower concentration is intriguing but consistent with the findings that high and low concentrations of HInitially, the authors argue "that the depletion of CTH could contribute to redox imbalance and mitochondrial dysfunction to promote HIV-1 reactivation"(p. 9). Less CTH would suggest less produced H2S. However, later on in the manuscript they demonstrate that addition of a H2S source (GYY4137) results in the suppression of HIV-1 replication and supposedly HIV-1 reactivation. This is somewhat confusing.2S by diminished expression of CTH (U1-shCTH) resulted in higher mitochondrial ROS and GSH/GSSG imbalance. Both of these alterations are known to reactivate HIV-1 and promote replication . The addition of GYY4137 chemically compensated for the diminished expression of CTH, and prevented HIV-1 reactivation in U1-shCTH. These events are expected to suppress HIV-1 replication and reactivation. We have made this distinction clear in the revised manuscript .We show that depletion of endogenous HCTH, or for that matter CBS or MPST do not only produce H2S, however, they also are part of other metabolic pathways. It would have been interesting and important to study how these metabolic pathways were affected by the genetic manipulations and also how the increased presence of H2S (GYY4137) would affect the metabolic activity of these enzymes or their expression.2S generating steps and two cysteine-producing reactions, the modulation of CTH by HIV is likely to have a widespread influence on transsulfuration pathway and active methyl cycle intermediates. Our future strategies are to generate a comprehensive understanding of sulfur metabolism underlying HIV latency and reactivation. These experiments require multiple biochemical and genetic technologies with appropriate controls. We hope that the reviewer would agree with our views that these experiments should be a part of future investigation. We thank the reviewer for this comment.We fully agree with the reviewer. In fact, our NanoString data show that upon CTH knockdown (U1-shCTH), MPST levels were down-regulated and CBS remained undetectable . Additionally, GYY4137 treatment induced the expression of CTH but not MPST upon PMA addition . We have incorporated these findings in the revised manuscript . Given that CBS and CTH catalyzed at least eight HH2S has been reported to cause NFkB inhibition by sulfhydration of p65; as such, the findings here are not particularly novel or surprising. Also, H2S induced sulfhydration is rather not targeted to a specific protein, let alone a HIV protein, making this approach a very unlikely alternative to current ART forms.2S exerts its influence on HIV latency. Recent studies point towards the importance of the Nrf2-Keap1 axis in sustaining HIV-latency (20). Our data suggest an important role for Nrf2-Keap1 signaling in mediating the influence of H2S on HIV latency. Additionally, recruitment of an epigenetic silencer YY1 is also affected by H2S. Interestingly, YY1 activity is modulated by redox signaling (21), suggesting H2S could be an important regulator of YY1 activity in HIV-infected cells. We have so far, no evidence for viral proteins targeted by H2S. However, experiments to examine global S-persulfidation of host and HIV protein are ongoing in the laboratory to fill this knowledge gap. Lastly, our findings raise the possibility of exploring H2S donors with the current ART  for reducing virus reactivation. We have tone down the clinical relevance of our findings.We believe that NF-κB inhibition is not the only mechanism by which HThe description of the primary T cell model used to generate the data in Figure 6 is slightly misleading. Also, the idea of this model was originally to demonstrate that "block and lock" by didehydro-cortistatin is possible. In this application, the authors did not investigate whether GYY4137 would actually induce a HIV "block and lock" over an extended period of time.2S in blocking HIV rebound. Our results indicate the future possibility of investigating GYY4137 to lock HIV in deep latency for an extended period of time. However, comprehensive investigation would require long-term experiments and samples from multiple HIV subjects. In the current pandemic times with overburdened Indian clinical settings, we cannot plan these experiments. However, we hope our data form a solid foundation for HIV researchers to perform extended “block and lock” studies using H2S donors.As suggested by the reviewer, we have cited the didehydro-cortistatin studies as the basis of our strategy. Our idea was to adapt the primary T cell model to begin understanding the role of HThe authors claim that the major conclusion of their study is that HIV-1 reactivation is coupled to depletion of endogenous H2S, which is associated with dysfunctional mitochondrial bioenergetics, in particular suppressed OXPHOS, GSH/GSSG imbalance, and elevated mitoROS. However, the authors never provide evidence that endogenous H2S is altered in latently HIV-1 infected cells . By the end of the manuscript, the authors have not provided clear evidence that the effects of e.g. CTH deletion would be mediated by the production of H2S, and not by another function of the enzyme. Similarly, the inability of stimuli to trigger efficient HIV-1 reactivation following the provision of unnaturally high levels of H2S is not surprising given reports on the effect of GYY4137 as anti-inflammatory agent and suppressor NF-κB activation. Unless the authors were to demonstrate a true "block and lock" effect by GYY4137 the data will likely have limited impact on the HIV cure field.2S levels in the latently infected primary cells due to the assay's sensitivity and the insufficient number of cells latently infected with HIV-1. However, in the revised manuscript we have clearly shown that cysteine levels are not affected by CTH depletion and cysteine deprivation does not reactivate HIV-1. These results indicate that the effects of CTH depletion are likely mediated by H2S. This is consistent with our data showing that GYY4137 specifically complement CTH deficiency and blocks HIV-1 reactivation in U1-shCTH. Further, we carried in-depth investigation to show that the effect of GYY4137 is not due to impaired activation of CD4+ T cells.It's difficult to measure H2S production, we cannot rule out the effect of other metabolites in this process. However, we think that this is outside the scope of the present study. 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(2020) Alterations of redox and iron metabolism accompany the development of HIV latency. EMBO J 39, e10220921. Bonavida, B., and Baritaki, S. (2012) Inhibition of Epithelial-to-Mesenchymal Transition (EMT) in Cancer by Nitric Oxide: Pivotal Roles of Nitrosylation of NF-kappaB, YY1 and Snail. For Immunopathol Dis Therap 3, 125-133[Editors’ note: what follows is the authors’ response to the second round of review.]Upon careful consideration of your letter and after discussion with reviewers, we have the following recommendations.We recognize that there are several strong aspects of the work presented in your manuscript however, the possible clinical implications of the work are overstated. In addition, further clarification is required in terms of the molecular mechanism proposed. We are willing to consider a revised manuscript based on the following provisors:1. Regarding the conclusion that the reactivation of HIV-2 associated with CTH knockdown must be due to an absence of H2S: This conclusion is problematic as CTH has other functions as well. In addition, other proteins can produce H2S. The addition of H2S in the form of GYY4137, and associated inhibition of HIV-1 reactivation, does not bring further clarity as the concentrations of H2S used are much higher than anything the cells could produce by themselves. As CTH catalyses the last step in the trans-sulfuration pathway from methionine to cysteine, an important experiment would be to demonstrate that CHT KO is specific for H2S production and does not affect cysteine levels. Also demonstrating that cysteine deprivation does not lead to HIV-1 reactivation due to a generalised stress response would be important.gag transcript was measured at 6 h, 12 h, and 24 h. The expression of gag transcript was not significantly affected upon cysteine depletion, suggesting that HIV reactivation is not stimulated by deprivation of cysteine .We thank the editors for raising these concerns. To address these comments, we measured cysteine levels in cell lysates of CTH knock down U1 cells (U1-shCTH) and compared it with the control cells (U1-shNT). The cysteine levels remain comparable in the lysates derived from both the cell lines, suggesting that CTH depletion does not affect cysteine levels . The data also indicate that other routes for cysteine biosynthesis  likely compensate for CTH depletion in U1 cells. As suggested by the reviewer, we next assessed if cysteine deprivation reactivates HIV. To deprive cells for cysteine, we used RPMI medium lacking cysteine and supplemented with dialyzed FBS  as per the published protocol (1). The U1 cells were cultured in complete RPMI medium or cysteine-free RPMI medium and 2. Cell cultures produce so little H2S that it cannot be measured. As such, in the experiments, addition of GYY4137 produce completely unphysiological H2S concentrations. Given that protein sulfurylation affects thousands of proteins, the addition of GYY4137 is not a biologically relevant reproduction of the presence of CTH, as the experimental system may be associated proteome-wide activity changes. H2S donors are mostly considered for cancer and anti-inflammatory treatments, because they seem to be immunosuppressive. To translate these findings towards clinical relevance for a cure of latent HIV-1 infection, you would have to show that lack of reactivation is not associated with a lack of T cell response. For example, you could demonstrate that T cells, after being cultured in GYY4137 containing medium, still respond to anti-CD3 stimulation with IL-2 secretion or CD25 upregulation. A better assay would be that the extended presence of GYY4137 does not abrogate the ability of T cells to proliferate upon antigen recognition (CMV pp65 should be a good antigen in combination with CFSE stains).+ T cell lines for 14 days in presence of ART or ART in combination with 100 μM GYY4137. Cells were then labelled with 0.5 μM cell trace violet (CTV) and stimulated with either CMV, anti-CD3/anti-CD28 beads (at a bead: cell ratio of 1:0.25) or Staphylococcal enterotoxin B . After three days, IL-2 (20 U/ml) supplemented medium was added to the cells and cultured for another two days. Proliferation was measured by CTV dilution and CD25 expression using flow cytometry after 5 days of culture . We included activation with SEB, as it is a known superantigen that can trigger polyclonal T cell activation by binding to the T cell receptor at the variable region of the β-chain (relevant to our system). Hence, in these long-term polyclonal cultures where frequencies of T cells specific to any given antigen e.g., CMV may hugely vary between samples or CMV-specific cells may not be detected as shown by our data, we chose to more directly test CD4+ T cell survival and proliferative capacity using SEB and CD3/28 beads as stimuli. The data demonstrate that long-term culture of cells in the presence of GYY4137 does not reduce or interfere with the capacity of the cells to respond to activation by CD3/28 or in response to SEB .This is an excellent recommendation. Accordingly, we cultured PHA/IL-2 expanded CD4+ T cell or CD8+ T cell response to CMV peptide stimulation as measured by IFNγ expression or in the capacity of CD4+ and CD8+ T cells to express CD25 or CD69 in response to either CD3/28 or CMV stimulation . Collectively, these findings demonstrate that CD4+ T cell function in both short-term and long-term cultures is preserved in the presence of GYY4137.To extend the suggestion of the reviewer further, we conducted another series of experiments to test the impact of GYY4137 in a more physiologically relevant short term T cell activation assay. Here PBMCs from HIV+ ART-treated patients were incubated with ART or ART in combination with 100 μM GYY4137 for 18 hr. Cells were analyzed for surface expression of CD25 and CD69 and intracellular staining of IFNγ by flow cytometry. The comprehensive gating strategy and representative flow cytometry plots are depicted in Figure 6—figure supplement 3A-3B of the revised manuscript. The data generated demonstrate no difference between GYY4137-treated versus untreated cultures in terms of CD43. Clinical Relevance: Unless GYY4137 produces a "lock" state of latent HIV-1 infection events, or selectively kills latently HIV-1 infected T cells, there is no clinical relevance for a cure. No one will exchange ART for an immuno-suppressive experimental treatment, that already in vitro is not sufficiently potent to completely block reactivation. We suggest you remove any focus in the discussion on possible clinical implications and approach presenting this study from a fundamental research perspective as you do have some compelling findings.We agree with the reviewer. We have removed the statement indicating possible clinical implications of these findings.


Colorectal cancer (CRC) is estimated to be one of the most common cancers and the leading cause of cancer-related death worldwide. SOX9 is commonly overexpressed in CRC and participates in drug resistance. In addition, DNA damage repair confers resistance to anticancer drugs. However, the correlation between DNA damage repair and high SOX9 expression is still unclear. In this study, we aimed to investigate the function and the specific underlying mechanism of the SOX9-dependent DNA damage repair pathway in CRC.in vivo.The expression levels of SOX9 and MMS22L in CRC were examined by immunohistochemistry (IHC) and TCGA analysis. RNA sequencing was conducted in RKO SOX9-deficient cells and RKO shControl cells. Mechanistic studies were performed in CRC cells by modulating SOX9 and MMS22L expression, and we evaluated drug sensitivity and DNA damage repair signaling events. In addition, we investigated the effect of oxaliplatin in tumors with SOX9 overexpression and low expression of MMS22L via regulation of MMS22L expression and participation in DNA double-strand break repair. SOX9 was upregulated and formed a complex with MMS22L, which promoted the nuclear translocation of MMS22L upon oxaliplatin treatment. Moreover, the xenograft assay results showed that oxaliplatin abrogated tumor growth from cells with MMS22L downregulation in mice.Our study showed that SOX9 has a higher expression level in CRC tissues than in normal tissues and predicts poor prognosis in CRC patients. Overexpression and knockdown of SOX9 were associated with the efficacy of oxaliplatin. In addition, SOX9 activity was enriched in the DNA damage repair pathway In CRC, activation of the SOX9-MMS22L-dependent DNA damage pathway is a core pathway regulating oxaliplatin sensitivity. Targeting this pathway in oxaliplatin-resistant CRC cells is a promising therapeutic option. Colorectal cancer (CRC) is one of the most common cancers and the leading cause of cancer-related death worldwide . CRC patSOX9 is a transcription factor that has been reported to be linked with stem cell maintenance and is commonly overexpressed in solid tumors . High exin vitro  and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 100 U/ml penicillin-streptomycin. The culture conditions were a cell incubator with a temperature of 37°C and 5% carbon dioxide. Oxaliplatin was purchased from Selleck Chemical .CRC tumor and adjacent normal tissue slices were obtained from the Department of Pathology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, China, with approval from the Institutional Ethics Committee. All patients signed the informed consent form, and clinical data are provided in 6 cells/well) were infected with lentivirus. Stably transduced cells were selected and enriched with 4 μg/ml puromycin . The sequences of the shRNA and the primers used to amplify the full-length sequence of human SOX9 are listed below:The CMV-MCS-3FLAG-SV40-Neomycin lentiviral Vector expressing full-length human SOX9 (NM_000346.4) and the U6-MCS-Ubiquitin-cherry-IRES-puromycin shSOX9 lentiviral Vector were purchased from GeneChem . The lentiviral packaging procedure was performed as described previously . RKO andSOX9-RNAi: forward: 5′-CCGGCTCCACCTTCACCTACAT GAACTCGAGTTCATGTAGGTGAAGGTGGAGTTTTTG-3′, reverse: 5′-GATCCAAAAACTCCACCTTCACCTACATGA ACTCGAGTTCATGTAGGTGAAGGTGGAG-3′.SOX9-control: forward: 5′-CCGGTTCTCCGAACGTGTCA CGTCTCGAGTTCATGTAGGTGAAGGTGGAGTTTTTG-3′, reverse: 5′-GATCCAAAAATTCTCCGAACGTGTCACGTC TCGAGTTCATGTAGGTGAAGGTGGAG-3′.MMS22L-RNAi: forward: 5′-CCGGCCAACTCTTCAAGGA AACTAACTCGAGTTAGTTTCCTTGAAGAGTTGGTTT TTG-3′, reverse: 5′-AATTCAAAAACCAACTCTTCAAGG AAACTAACTCGAGTTAGTTTCCTTGAAGAGTTGG-3′.MMS22L-control: forward: 5′-CCGGTTCTCCGAACGTGT CACGTCTCGAGTTAGTTTCCTTGAAGAGTTGGTTTTTG-3′, reverse: 5′-AATTCAAAAATTCTCCGAACGTGTCACGTC TCGAGTTAGTTTCCTTGAAGAGTTGG-3′.The primers used to amplify the full-length sequence of human SOX9 were as follows: forward: 5′-GAGGAAGTCG GTGAAGAACGG-3′, reverse: 5′-CTGAGCGGGGTTCATG TAGG-3′.TM 2500 or Illumina HiSeqTM X Ten) by Haplox . The differentially expressed genes (DEGs) were identified using Cuffdiff with q value < 0.05 and fold change >2 as the criteria. Gene set enrichment analysis (GSEA) was performed on the software.broadinstitute.org/gsea website. Volcano plots, pathway enrichment analysis visualizations and heatmaps were generated with the R program. All details were described in our previous work  and RKO shSOX9 cells. An RNA library was constructed using a TruSeq Stranded mRNA LT Sample Prep Kit  and subjected to 125 bp/150 bp paired-end sequencing on an Illumina platform  according to the manufacturer’s instructions, and protein was obtained as described above. The samples were separated by SDS-PAGE, and proteins were transferred to Polyvinylidene Difluoride (PVDF) membranes . After blocking in 5% milk-PBST for 1 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight and with secondary antibodies at room temperature for 1 h. Positive bands were visualized by enhanced chemiluminescence (ECL) . Information on the primary antibodies is provided in Total protein was obtained by the following steps. Cancer cells were lysed using radioimmunoprecipitation assay buffer :Phenylmethylsulfonyl Fluoride  = 100:1 on ice for 30 min. Samples were centrifuged at 15,000 × Total RNA was extracted from cancer cells using TRIzol Reagent , and quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a SYBR Prime Script RT-PCR kit  in a CFX Connect Real-Time System . GAPDH was used as the internal control. The primer sequences are listed as follows:SOX9: forward: 5′-AGGAAGCTCGCGGACCAGTAC-3′, reverse: 5′-GGTGGTCCTTCTTGTGCTGCAC-3′; GAPDH: forward: 5′-GTTCGTCATGGGTGTGAAC-3′, reverse: 5′-ATGGCATGGACTGTGGTCAT-3′; MMS22L: forward: 5′- ATGTGAGCGGGAATCTCTT-3′, reverse: 5′-GAAACACT TGGGGTTCGTC-3′.4 cells per well) in a 24-well plate. 12 h after plating, cells were exposed to oxaliplatin at the indicated doses for 4 h. Then, cells were fixed with 4% paraformaldehyde and permeabilized with PBS containing 0.3% Triton X-100. After blocking with goat serum, cells were incubated with primary antibodies at 4°C overnight. Then, cells were incubated with fluorescent secondary antibodies  (1:400) at room temperature for 30 min. Nuclei were stained with DAPI. A Nikon Eclipse Ti-S fluorescence microscope  was used to acquire images. The paraffin sections required antigen repair prior to the above procedure.Cells were seeded on coverslips  was added to each well prior to incubation at 37°C for 2 h. Optical density was measured based on the absorbance at a wavelength of 450 nm.CRC cells were seeded in 96-well plates at a density of 2 × 10CRC cells were treated with 5 μM oxaliplatin. According to the manufacturer’s instructions, the early apoptosis rate was analyzed by the Annexin-V staining method using a PE Annexin-V Apoptosis Detection Kit  and a FITC Annexin-V Apoptosis Detection Kit . We used FlowJo v.10 software for data analysis.in vivo, 4-week-old BALB/c nude mice  were subcutaneously inoculated with RKO (5 × 106 cells/100 μl PBS) cells stably transfected with Vector+shCtrl, oeSOX9+shCtrl, Vector+shMMS22L, oeSOX9+shMMS22L plasmids. Mice were intraperitoneally injected with oxaliplatin (7.5 mg/kg) every 2 days for 10 days and sacrificed on Day 28 (2). A portion of the xenograft tumors in the mice was taken for IHC staining. All animal experiments were performed in compliance with the guidelines of the Animal Ethics Committee of the Affiliated Tumor Hospital of Guangxi Medical University.To evaluate the role of the SOX9-MMS22L axis n Day 28 . The tumt-tests were used to determine the level of significance. All data used in this study satisfied the assumptions of the statistical tests. All quantitative data are shown as the mean ± S.D. or mean ± S.E.M. values as indicated. P < 0.05 was considered statistically significant. All experiments were carried out at least three times.Statistical analysis was performed with GraphPad Prism 7.0 . One-way ANOVA or two-tailed unpaired high CRC patients showed shorter overall survival, disease-specific survival and progression-free survival times than SOX9low patients in the TCGA database  (in vivo and that knocking down MMS22L reverses this effect.To further demonstrate the effect of SOX9 on CRC cells 5 mg/kg) . After 25 mg/kg) . IHC sta5 mg/kg) . TherefoIn vitro studies suggest a role for SOX9 in regulating cell differentiation in the intestinal epithelium (Cancer cells aquire drug resistance through different mechanisms , includiithelium . A transithelium . In humaithelium , similarMMS22L is mainly localized in the nucleus and has been reported to be critical for protecting against DSBs formation in S-phase cells by dealing with replication fork stalling/collapse events . FurtherIn this manuscript, through overexpression or knockdown of SOX9 in CRC cells and a series of subsequent experiments, we elucidated that SOX9 can affect the sensitivity of CRC cells to oxaliplatin and preliminarily explored the mechanism of this phenomenon. However, there was a relative absence of SOX9 expression in drug-resistant CRC cells compared with wild-type cells. Therefore, we will screen oxaliplatin-resistant CRC cells in our next study and continue to perform more in-depth research. In conclusion, our study confirmed that SOX9, a transcription factor, is involved in the maintenance of stemness and the induction of drug resistance in CRC cells. Furthermore, we revealed a mechanism by which SOX9 regulates the expression and nuclear translocation of MMS22L and oxaliplatin resistance in CRC cells and provided a new focus for solving the problem of oxaliplatin resistance in CRC patients.PRJNA707264.The data presented in the study are deposited in the SRA repository, accession number The animal study was reviewed and approved by the Ethics Committee of Affiliated Tumor Hospital of Guangxi Medical University. All authors have consented to publication of the results presented in this manuscript.YL, HW, and TL performed the experiments. YL, HW, TL, QL, ZM, YS, FL, ML, XP, and JL provided technical support. CX, WT, XP, and JL provided critical comments. YL, HW, TL, CX, WT, XP, and JL analyzed the data, designed the experiments, and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intravenous immunoglobulin (IVIG) showed its therapeutic efficacy on Kawasaki disease (KD). However, the mechanisms by which it reduces systemic inflammation are not completely understood. Dendritic cells (DCs) and T cells play critical roles in the pathogenic processes of immune disorders. Assessing the quantity of DC subsets and T cells and identifying functional molecules present on these cells, which provide information about KD, in the peripheral blood may provide new insights into the mechanisms of immunoglobulin therapy.+ T cells in peripheral blood were analyzed through flow cytometry.In total, 54 patients with KD and 27 age-matched healthy controls (HCs) were included in this study. The number, percentage, and phenotype of DC subsets and CD4+ T cells than HCs. Human leucocyte antigen-DR (HLA-DR) expression was reduced on CD1c+ myeloid DCs (CD1c+ mDCs), whereas that on plasmacytoid DCs (pDCs) did not change significantly. Both pDCs and CD1c+ mDCs displayed significantly reduced expression of co-stimulatory molecules, including CD40, CD86. pDCs and CD1c+ mDCs presented an immature or tolerant phenotype in acute stages of KD. Number of circulating pDC and CD1c+ mDC significantly inversely correlated with plasma interleukin-6 (IL-6) levels in KD patients pre-IVIG treatment. No significant differences were found concerning the DC subsets and CD4+ T cells in patients with KD with and without coronary artery lesions. Importantly, these altered quantity and phenotypes on DC subsets and CD4+ T cells were restored to a great extent post-IVIG treatment. T helper (Th) subsets including Th1 and Th2 among CD4+ T cells did not show alteration pre- and post-IVIG treatment, although the Th1-related cytokine IFN-γ level in plasma increased dramatically in patients with KD pre-IVIG treatment.Patients with KD exhibited fewer peripheral DC subsets and CD4+ mDCs presented an immature or tolerant phenotype in acute stages of KD, IVIG treatment restored the quantity and functional molecules of DCs and CD4+ T cells to distinct levels in vivo, indicating the involvement of DCs and CD4+ T cells in the inflammation in KD. The findings provide insights into the immunomodulatory actions of IVIG in KD.pDCs and CD1c Kawasaki disease (KD) is a febrile systemic vasculitis that predominately affects children less than 5 years of age and is the leading cause of acquired cardiac disorders in children . Increas+ T cells in KD. Systemic studies on DCs and T cells are warranted to gain insights into the role of these immune cells in the KD pathogenesis and IVIG treatment.Intravenous immunoglobulin (IVIG) treatment remains the most effective therapy currently for KD. Although the underlying mechanisms are not fully elucidated, it is well-accepted that IVIG cures patients by down-regulating inflammatory responses, which further protects the vascular system and myocardium from immune-mediated damage. Diverse mechanisms have been suggested to explain the anti-inflammatory activity of IVIG, including the neutralization of microbial toxins or cytokines , suppres+ mDC, expression of the inhibitory receptors on CD4+ T cells and helper T cell subsets in patients suffering from KD and the changes of these parameters over the course of IVIG treatment.In this study, we determined the frequency, number, phenotype of peripheral pDC and CD1cA total of 54 with KD admitted to the Department of Cardiology, Children’s Hospital of Soochow University, from June 2020 to June 2021 participated in this study. All the participants conformed to the diagnostic criteria revised by AHA Kawasaki Disease Guideline . All pat+ and Mg2+. After the samples were washed, peripheral blood leukocytes were resuspended in PBS containing 2.5% fetal bovine serum (FBS) and incubated at 4°C for 30 min in the dark with the following fluorochrome-conjugated monoclonal antibodies including surface CD3-FITC (SK7), CD14-FITC (HCD14), CD15-FITC (HI98), CD16-FITC (3G8), CD19-FITC (HIB19), CD20-FITC (2H7), CD56-FITC (MEM-188), HLA-DR-PE/CY7 (L243), CD123-APC(6H6), CD1c-PE(L161), CD86-PE/CY7(IT2.2), CD40-BV421(5C3), CD4-BV421 (RPA-T4), Tim3-APC (F38-2F2), TIGIT-PE (A15153G), and PD-1-BV510 (NAT105).All the samples were washed and analyzed using the Attune NxTflow cytometer (LifeTechnology). Approximately 3 × 106 and 1 × 106 of peripheral blood leukocytes were used to analyze DCs and CD4+ T cells by flow cytometry, respectively. All antibodies were purchased from BioLegend  or BD Biosciences . Staining was performed as previously described  viability staining. The absolute number of CD1c+ mDCs and pDCs was calculated from the peripheral blood leukocytes count multiplied by the proportion of each subset within peripheral blood leukocytes per milliliter, as determined by flow cytometric analysis.Peripheral blood (2 mL) was collected from both healthy controls (HCs) and patients at two time points: before IVIG treatment (pre-IVIG) and 3 days after completing initial IVIG treatment (post-IVIG). Peripheral blood leukocytes were isolated from EDTA-blood through red blood cell lysis within 4 h. Briefly, red blood cells (RBCs) were lysed using human erythrocyte lysing solution (BioLegend), and samples were washed twice with phosphate-buffered saline (PBS) without Ca22 in the presence of 5 ng/mL phorbol myristate acetate (PMA), 0.5 μg/mL ionomycin, and Golgiplug . Cells were washed and stained with anti-CD4 at 4°C for 30 min in the dark. After washed twice, cells were fixed with lysing buffer (BD Biosciences), permeabilized with permeabilization solution (BD Biosciences), and were then incubated with antibodies against IFN-γ and IL-4 (BD Biosciences) for intracellular staining, according to the manufacturer’s instructions. Th1 and Th2 cells were identified as CD4+IFN-γ+ and CD4+IL-4+, respectively. Dead cells were excluded using a fixable live/dead dye (Invitrogen). The data were analyzed using FlowJo v10.4 software .For the detection of intracellular cytokines by flow cytometry, peripheral blood leukocytes were cultured in 10% fetal calf serum in RPMI-1640 and incubated for 5 h at 37°C under 5% COThe Human Th1/Th2/Th17 Kit (BD Biosciences) was used to measure plasma cytokine levels. IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17A, and IL-10 levels were assessed. Data were analyzed using CBA software. The individual cytokine concentrations were indicated by their fluorescence intensities. The concentrations of all the cytokines were reported in picogram per milliliter.P value of < 0.05 was considered significant.Statistical analysis was performed using IBM SPSS Statistics 26.0. All data are expressed as median (interquartile range). For comparison between patients with KD and HCs, Mann-Whitney U-test was used. The Wilcoxon signed-rank test for paired samples was used for comparing the patients before and after IVIG treatment. The significance of difference in sex distribution in patients and controls was analyzed using the chi-square test. Correlations were analyzed using the Spearman correlation test. A A total of 54 patients with KD and 27 healthy controls were recruited based on our inclusion and exclusion criteria. Their characteristics are shown in + mDCs among pan-DCs was significantly decreased in patients with KD pre-IVIG compared with those in healthy controls  in the two DC subsets in the peripheral circulation of patients with KD and HCs. These molecules are critical for DCs to elicit adaptive immune responses.P = 0.1822, P = 0.3130, P < 0.0001, P < 0.0001, P = 0.0010, P = 0.0170, P = 0.0006, P = 0.0091, P = 0.5288, P = 0.5158, Although no significant significance was observed between pre-and post-IVIG treatment in patients with KD and HCs in terms of pDCs, the MFI of the antigen presenting molecule HLA-DR exhibited a decreasing trend  signal. Production of intracellular IFN-γ and IL-4, which are the representative factors of Th1 and Th2, respectively, in CD4+ T cells, was analyzed in the peripheral blood of patients with KD and HCs. The gating strategy for determining Th1 and Th2 cells is shown in + T cells . IL-17A, IFN-γ, TNF-α, IL-6, IL-10, and IL-2 levels were higher in patients with KD pre-IVIG than in HCs (P = 0.0183)    . The number and proportion of circulating pDCs and CD1c+ mDCs were not significantly different in KD-CAL and KD-NCAL groups  and HLA-DR staining, the makers of DC staining in our study are consistent with those of previous report  and HLA-DR. Burns et al.  and Th2 (IL-4-producing CD4+ T cells) cells were identified by intracellular cytokine staining in our study. These experimental differences, as well as other methodological differences may account for the discrepancy. The function of T cells is regulated by inhibitory receptors such as PD-1, TIGIT, and TIM-3  post-IVIG therapy; and according to the data on the plasma cytokines, the level of immunosuppressive molecule IL-10 was significantly decreased post-IVIG therapy. Thus, we speculate that the restoration of the cell number and phenotype of CD4+ T cells is involved in the recovery of the immune system of the patients post- IVIG therapy to a certain degree. To the best of our knowledge, this study is the first to describe the variation in inhibitory receptor expression on CD4+ T cells in KD. These results demonstrated that IVIG induced multiple phenotypic and functional changes in DC subsets and CD4+ T cells, mainly by promoting the resolution of inflammation, in patients with KD. The specific mechanisms leading to these effects should be further investigated.The mechanisms of action of IVIG in KD have been studied extensively; however, the role of DCs and CD4ed by Fc . In thisactivity . IVIG caactivity . The rea 1 cells , 40. Non 1 cells . Accordi+ mDC in patients with KD pre-IVIG treatment. A previous study by Dawicki et al. demonstrated that IL-6 act to selectively promote the accumulation of DC subsets into an inflamed lymph node in mouse model in response to bacterial peptidoglycan , the Natural Science Foundation of Jiangsu Province (BK20190053), the Medical Science Program of Jiangsu Province (H2019002), the Talent Engineering Project of Jiangsu Province (BRA2018393), the Science and Technology Project of Jiangsu Province (BE2019672), the Suzhou Clinical Medicine Expert Project (GSWS2019015), the Science and Technology Program of Suzhou (SYS2018067), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX20_2725).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.