Low Percentage of Perforin-Expressing NK Cells during Severe SARS-CoV-2 Infection: Consumption Rather than Primary Deficiency.

Genetic defects in the ability to deliver effective perforin have been reported in patients with hemophagocytic lymphohistiocytosis. We tested the hypothesis that a primary perforin deficiency might also be causal in severe SARS-CoV-2 infection. We recruited 54 volunteers confirmed as being SARS-CoV-2-infected by RT-PCR and admitted to intensive care units or non-intensive care units and age- and sex-matched healthy controls. Compared with healthy controls, the percentage of perforin-expressing CD3-CD56+ NK cells quantified by flow cytometry was low in COVID-19 patients (69.9 ± 17.7 versus 78.6 ± 14.6%, p = 0.026). There was no correlation between the proportions of perforin-positive NK cells and T8 lymphocytes. Moreover, the frequency of NK cells producing perforin was neither linked to disease severity nor predictive of death. Although IL-6 is known to downregulate perforin production in NK cells, we did not find any link between perforin expression and IL-6 plasma level. However, we unveiled a negative correlation between the degranulation marker CD107a and perforin expression in NK cells (r = -0.488, p = 10-4). PRF1 gene expression and the frequency of NK cells harboring perforin were normal in patients 1 y after acute SARS-CoV-2 infection. A primary perforin defect does not seem to be a driver of COVID-19 because NK perforin expression is 1) linked neither to T8 perforin expression nor to disease severity, 2) inversely correlated with NK degranulation, and 3) normalized at distance from acute infection. Thus, the cause of low frequency of perforin-positive NK cells appears, rather, to be consumption.

Immune profiles of pre-frail people living with HIV-1: a prospective longitudinal study.

BACKGROUND: People living with HIV (PLWH) are at risk of frailty, which is predictive for death. As an overactivity of the immune system is thought to fuel frailty, we characterized the immune activation profiles linked to frailty. METHODS: We quantified twenty-seven activation markers in forty-six virological responders (four females and forty-two males; median age, 74 years; median duration of infection, 24 years; median duration of undetectability, 13 years), whose frailty was determined according to the Fried criteria. T cell and NK cell activation was evaluated by flow cytometry, using a panel of cell surface markers. Soluble markers of inflammation, and monocyte activation and endothelial activation were measured by ELISA. The participants' immune activation was profiled by an unsupervised double hierarchical clustering analysis. We used ANOVA p-values to rank immunomarkers most related to Fried score. A Linear Discriminant Analysis (LDA) was performed to link immune activation markers to frailty. RESULTS: 41% of the participants were pre-frail, including 24% with a Fried score of 1, and 17% with a Fried score of 2. ANOVA identified the 14 markers of T cell, monocyte, NK cell, endothelial activation, and inflammation the most linked to Fried 3 classes. The LDA performed with these 14 markers was capable of discriminating volunteers according to their Fried score. Two out of the 5 immune activation profiles revealed by the hierarchical clustering were linked to and predictive of pre-frailty. These two profiles were characterized by a low percentage of CD4 T cells and a high percentage of CD8 T cells, activated CD4 T cells, CD8 T cells, and NK cells, and inflammation. CONCLUSIONS: We identified a particular immune activation profile associated with pre-frailty in PLWH. Profiling participants at risk of developing frailty might help to tailor the screening and prevention of medical complications fueled by loss of robustness. Further studies will indicate whether this frailty signature is specific or not of HIV infection, and whether it also precedes frailty in the general population.

Angiotensin II induces reactive oxygen species, DNA damage, and T-cell apoptosis in severe COVID-19.

BACKGROUND: Lymphopenia is predictive of survival in patients with coronavirus disease 2019 (COVID-19). OBJECTIVE: The aim of this study was to understand the cause of the lymphocyte count drop in severe forms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: Monocytic production of reactive oxygen species (ROSs) and T-cell apoptosis were measured by flow cytometry, DNA damage in PBMCs was measured by immunofluorescence, and angiotensin II (AngII) was measured by ELISA in patients infected with SARS-CoV-2 at admission to an intensive care unit (ICU) (n = 29) or not admitted to an ICU (n = 29) and in age- and sex-matched healthy controls. RESULTS: We showed that the monocytes of certain patients with COVID-19 spontaneously released ROSs able to induce DNA damage and apoptosis in neighboring cells. Of note, high ROS production was predictive of death in ICU patients. Accordingly, in most patients, we observed the presence of DNA damage in up to 50% of their PBMCs and T-cell apoptosis. Moreover, the intensity of this DNA damage was linked to lymphopenia. SARS-CoV-2 is known to induce the internalization of its receptor, angiotensin-converting enzyme 2, which is a protease capable of catabolizing AngII. Accordingly, in certain patients with COVID-19 we observed high plasma levels of AngII. When looking for the stimulus responsible for their monocytic ROS production, we revealed that AngII triggers ROS production by monocytes via angiotensin receptor I. ROSs released by AngII-activated monocytes induced DNA damage and apoptosis in neighboring lymphocytes. CONCLUSION: We conclude that T-cell apoptosis provoked via DNA damage due to the release of monocytic ROSs could play a major role in COVID-19 pathogenesis.

CXCR5/CXCL13 pathway, a key driver for migration of regulatory B10 cells, is defective in patients with rheumatoid arthritis.

OBJECTIVES: Chemokines (CKs) are key players of immune-cell homing and differentiation. CK receptors (CKRs) can be used to define T-cell functional subsets. We aimed to characterize the CKR profile of the regulatory B-cell subset B10+ cells and investigate the CKs involved in their migration and differentiation in healthy donors and patients with RA. METHODS: RNA sequencing and cytometry were used to compare CKR expression between B10+ and B10neg cells. Migration of B10+ and B10neg cells and IL-10 secretion of B cells in response to recombinant CKs or synovial fluid (SF) were assessed. RESULTS: CXCR5 was expressed at a higher level on the B10+ cell surface as compared with other B cells (referred to as B10neg cells). In line with this, its ligand CXCL13 preferentially attracted B10+ cells over B10neg cells. Interestingly, synovial fluid from RA patients contained high levels of CXCL13 and induced strong and preferential migration of B10+ cells. Besides its role in attracting B10+ cells, CXCL13 also promoted IL-10 secretion by B cells. In RA patients, the level of CXCR5 on B-cell surface was reduced. The preferential migration of RA B10+ cells toward CXCL13-rich SF was lost and CXCL13 stimulation triggered less IL-10 secretion than in healthy donors. CONCLUSION: Our results identify that the CXCR5/CXCL13 axis is essential for B10+ cell biology but is defective in RA. Restoring the preferential migration of B10+ within the affected joints to better control inflammation may be part of the therapeutic approach for RA.

Regulatory T cell/Th17 balance in the pathogenesis of paediatric Behçet disease.

OBJECTIVES: Behçet disease (BD) is a chronic systemic inflammatory disorder of unknown aetiology. The aim of this study was to determine the orientation of T cell subpopulations in paediatric BD and more precisely to look for a regulatory T lymphocyte (Treg)/Th17 imbalance. METHODS: T cell subpopulations were analysed by flow cytometry in the peripheral blood of paediatric patients with acute BD (aBD; n = 24), remitting BD (rBD; n = 12) and in healthy controls (HCs; n = 24). Tregs (CD4+CD25hiCD127-/loFoxp3+), activated Tregs (GITR, LAP, CTLA-4 and HLA-DR expression), CD4+ and CD8+ T cells producing IFN-γ (Th1 and Tc1) or IL-17 (Th17 and Tc17) under polyclonal (OKT3/IL-2) or antigenic (Streptococcus sanguis KTH-1 peptides and heat shock protein 60) stimulation were enumerated. RESULTS: Th17 (1.9- and 5.1-fold) and Tc17 (4.0- and 2.0-fold) frequency under mitogenic stimulation was significantly increased in aBD and rBD patients as compared with HCs. Th17 frequency under antigenic stimulation was also higher in patients than in HCs. The percentage and number of Tregs and activated Tregs in patients and in HCs were similar. However, when Tregs were removed, antigen-driven differentiation into Th1 and Th17 was significantly boosted in BD but not in HC CD4+ T cells. CONCLUSION: There is a bias towards Th17 polarization in aBD and rBD in children. Although we did not observe an increase in the number of Tregs in these patients, their Tregs limit CD4+ T cell differentiation into Th1 and Th17 cells. Thus, in paediatric BD, Tregs seem to incompletely counterbalance a Th17 orientation of the Th cell response.

Low-dose IL-2 in children with recently diagnosed type 1 diabetes: a Phase I/II randomised, double-blind, placebo-controlled, dose-finding study.

AIMS/HYPOTHESIS: Low-dose IL-2 (ld-IL2) selectively activates and expands regulatory T cells (Tregs) and thus has the potential to skew the regulatory/effector T (Treg/Teff) cell balance towards improved regulation. We investigated which low doses of IL-2 would more effectively and safely activate Tregs during a 1 year treatment in children with recently diagnosed type 1 diabetes. METHODS: Dose Finding Study of IL-2 at Ultra-low Dose in Children With Recently Diagnosed Type 1 Diabetes (DF-IL2-Child) was a multicentre, double-blinded, placebo-controlled, dose-finding Phase I/II clinical trial conducted in four centres at university hospitals in France: 24 children (7-14 years old) with type 1 diabetes diagnosed within the previous 3 months were randomly assigned 1:1:1:1 to treatment by a centralised randomisation system, leading to a 7/5/6/6 patient distribution of placebo or IL-2 at doses of 0.125, 0.250 or 0.500 million international units (MIU)/m2, given daily for a 5 day course and then fortnightly for 1 year. A study number was attributed to patients by an investigator unaware of the randomisation list and all participants as well as investigators and staff involved in the study conduct and analyses were blinded to treatments. The primary outcome was change in Tregs, expressed as a percentage of CD4+ T cells at day 5. It pre-specified that a ≥60% increase in Tregs from baseline would identify Treg high responders. RESULTS: There were no serious adverse events. Non-serious adverse events (NSAEs) were transient and mild to moderate. In treated patients vs placebo, the commonest NSAE was injection site reaction (37.9% vs 3.4%), whereas other NSAEs were at the same level (23.3% vs 19.2%). ld-IL2 induced a dose-dependent increase in the mean proportion of Tregs, from 23.9% (95% CI -11.8, 59.6) at the lowest to 77.2% (44.7, 109.8) at the highest dose, which was significantly different from placebo for all dose groups. However, the individual Treg responses to IL-2 were variable and fluctuated over time. Seven patients, all among those treated with the 0.250 and 0.500 MIU m-2 day-1 doses, were Treg high responders. At baseline, they had lower Treg proportions in CD4+ cells than Treg low responders, and serum soluble IL-2 receptor α (sIL-2RA) and vascular endothelial growth factor receptor 2 (VEGFR2) levels predicted the Treg response after the 5 day course. There was no significant change in glycaemic control in any of the dose groups compared with placebo. However, there was an improved maintenance of induced C-peptide production at 1 year in the seven Treg high responders as compared with low responders. CONCLUSIONS/INTERPRETATION: The safety profile at all doses, the dose-dependent effects on Tregs and the observed variability of the Treg response to ld-IL2 in children with newly diagnosed type 1 diabetes call for use of the highest dose in future developments. The better preservation of insulin production in Treg high responders supports the potential of Tregs in regulating autoimmunity in type 1 diabetes, and warrants pursuing the investigation of ld-IL2 for its treatment and prevention. TRIAL REGISTRATION: ClinicalTrials.gov NCT01862120. FUNDING: Assistance Publique-Hôpitaux de Paris, Investissements d'Avenir programme (ANR-11-IDEX-0004-02, LabEx Transimmunom and ANR-16-RHUS-0001, RHU iMAP) and European Research Council Advanced Grant (FP7-IDEAS-ERC-322856, TRiPoD).

Factors of microinflammation in non-diabetic chronic kidney disease: a pilot study.

BACKGROUND: The relationships between digestive bacterial translocation, uremic toxins, oxidative stress and microinflammation in a population of chronic kidney disease (CKD) patients without metabolic nor inflammatory disease are unknown. METHODS: Bacterial translocation, uremic toxins, oxidative stress, and inflammation were assessed by measuring plasma levels of 16S ribosomal DNA (16S rDNA), p-cresyl sulfate (PCS), indoxyl sulfate (IS), indole acetic acid (IAA), F2-isoprostanes, hsCRP and receptor I of TNFα (RITNFα) in patients without metabolic nor inflammatory disease. 44 patients with CKD from stage IIIB to V and 14 controls with normal kidney function were included from the nephrology outpatients. 11 patients under hemodialysis (HD) were also included. Correlations between each factor and microinflammation markers were studied. RESULTS: 16S rDNA levels were not increased in CKD patients compared to controls but were decreased in HD compared to non-HD stage V patients (4.7 (3.9-5.3) vs 8.6 (5.9-9.7) copies/µl, p = 0.002). IS, PCS and IAA levels increased in HD compared to controls (106.3 (73.3-130.4) vs 3.17 (2.4-5.1) µmol/l, p < 0.0001 for IS; 174.2 (125-227.5) vs 23.7 (13.9-52.6) µmol/l, p = 0.006 for PCS; and 3.7 (2.6-4.6) vs 1.3 (1.0-1.9) µmol/l, p = 0.0002 for IAA). Urea increased in non-HD stage V patients compared to controls (27.6 (22.7-30.9) vs 5.4 (4.8-6.4) mmol/l, p < 0.0001) and was similar in HD and in non-HD stage V (19.3 (14.0-24.0) vs 27.6 (22.7-30.9) mmol/l, p = 0.7). RITNFα levels increased in HD patients compared to controls (12.6 (9.6-13.3) vs 1.1 (1.0-1.4) ng/ml, p < 0.0001); hsCRP levels increased in non-HD stage V patients compared to controls (2.9 (1.4-8.5) vs 0.8 (0.5-1.7) mg/l, p = 0.01) and remained stable in HD patients (2.9 (1.4-8.5) vs 5.1 (0.9-11.5) mg/l, p = 1). F2-isoprostanes did not differ in CKD patients compared to controls. Among uremic toxins, IS and urea were correlated to RITNFα (r = 0.8, p < 0.0001 for both). PCS, IS and urea were higher in patients with hsCRP≧5 mg/l (p = 0.01, 0.04 and 0.001 respectively). 16S rDNA, F2-isoprostanes were not correlated to microinflammation markers in our study. CONCLUSIONS: In CKD patients without any associated metabolic nor inflammatory disease, only PCS, IS, and urea were correlated with microinflammation. Bacterial translocation was decreased in patients under HD and was not correlated to microinflammation.

The two human CXCR4 isoforms display different HIV receptor activities: consequences for the emergence of X4 strains.

CXCR4 is a chemokine receptor that plays key roles with its specific ligand, CXCL12, in stem cell homing and immune trafficking. It is also used as a coreceptor by some HIV-1 strains (X4 strains), whereas other strains (R5 strains) use an alternative coreceptor, CCR5. X4 strains mainly emerge at late stages of the infection and are linked to disease progression. Two isoforms of this coreceptor have been described in humans: CXCR4-A and CXCR4-B, corresponding to an unspliced and a spliced mRNA, respectively. In this study, we show that CXCR4-B, but not CXCR4-A, mediates an efficient HIV-1 X4 entry and productive infection. Yet, the chemotactic activity of CXCL12 on both isoforms was similar. Furthermore, HIV-R5 infection favored CXCR4-B expression over that of CXCR4-A. In vitro infection with an R5 strain increased CXCR4-B/CXCR4-A mRNA ratio in PBMCs, and this ratio correlated with HIV RNA plasma level in R5-infected individuals. In addition, the presence of the CXCR4-B isoform favored R5 to X4 switch more efficiently than did CXCR4-A in vitro. Hence, the predominance of CXCR4-B over CXCR4-A expression in PBMCs was linked to the ability of circulating HIV-1 strains to use CXCR4, as determined by genotyping. These data suggest that R5 to X4 switch could be favored by R5 infection-induced overexpression of CXCR4-B. Finally, we achieved a specific small interfering RNA-mediated knockdown of CXCR4-B. This represents a proof of concept for a possible gene-therapeutic approach aimed at blocking the HIV coreceptor activity of CXCR4 without knocking down its chemotactic activity.

The EBI2 receptor is coexpressed with CCR5 in CD4+ T cells and boosts HIV-1 R5 replication.

OBJECTIVE: CCR5, a G protein-coupled receptor (GPCR), is used by most HIV strains as a coreceptor. In this study, we looked for other GPCRs able to modify HIV-1 infection. DESIGN: We analyzed the effects of one GPCR coexpressed with CCR5, EBI2, on HIV-1 replicative cycle. METHODS: We identified GPCRs expressed in primary CD4+CCR5+ T cells by multi-RT-qPCR. We studied GPCR dimerization by FRET technology. Cell lines expressing EBI2 were established by transduction with HIV vectors. HIV-1 entry was quantified with virions harboring ß-lactamase fused to the viral protein vpr, early and late HIV-1 transcriptions by qPCR, NFkB nuclear activation by immunofluorescence and transfection, and viral production by measuring p24 concentration in culture supernatant by ELISA. RESULTS: We showed that EBI2 is naturally expressed in primary CD4+CCR5+ T cells, and that CCR5 and EBI2 heterodimerize. We observed that this coexpression reduced viral entry by 50%. The amount of HIV reverse transcripts was similar in cells expressing or not EBI2. Finally, the presence of EBI2 induced the translocation of NFkB and activated HIV-1 genome expression. Globally, the result was a drastic HIV-1 R5, but not X4, overproduction in EBI2-transduced cells. CONCLUSIONS: EBI2 expression in CD4+CCR5+ cells boosts HIV-1 R5 productive infection. As the natural ligand for EBI2 is present in blood and lymphoid tissues, the constant EBI2 activation might increase HIV replication in CD4+ T cells. It might be of interest to test the effect of EBI2 antagonists on the residual viral production persisting in patients aviremic under treatment.

Regulatory T and B cells in pediatric Henoch-Schönlein purpura: friends or foes?

BACKGROUND AND OBJECTIVES: Henoch-Schönlein purpura (HSP) is the most common immunoglobulin A-mediated systemic vasculitis in childhood. We studied immune dysregulation in HSP by analyzing regulatory T (Treg), T helper 3 (Th3), and regulatory B cell (Breg) subpopulations that might intervene in immune activation, IgA production, and HSP clinical manifestations. METHODS: This prospective study included 3 groups of children: 30 HSP on acute phase, 30 HSP on remission, and 40 healthy controls (HCs) matched on age. Treg, Breg, and Th3 were analyzed by flow cytometry. Serum immunoglobulin and cytokine levels were quantified by ELISA and Luminex. RESULTS: Treg frequencies were higher in acute HSP than in remitting HSP and HCs (6.53% [4.24; 9.21] vs. 4.33% [3.6; 5.66], p = 0.002, and vs. 4.45% [3.01; 6.6], p = 0.003, respectively). Activated Th3 cells (FoxP3 + Th3 cells) tend to be more abundant in HSP than in HCs (78.43% [50.62; 80.84] vs. 43.30% [40.20; 49.32], p = 0.135). Serum IgA, IL-17, and latency-associated peptide (a marker of the anti-inflammatory cytokine TGF-beta production) were significantly and inflammatory cytokines TNF-alpha, IL-1-beta, and IL-6 were non-significantly higher in HSP than HCs. Bregs were identical between the groups, but, in patients with renal impairment, Breg percentage was lower compared to those without. Treg removal in PBMC culture resulted in an increase in IgA production in HSP proving a negative regulatory role of Tregs on IgA production. CONCLUSIONS: In pediatric HSP, immune activation persists in spite of an increase in Th3 and Tregs. Th3 could be involved in IgA hyperproduction, inefficiently downregulated by Tregs. Lack of Bregs appears linked to renal impairment.

Immune reconstitution under antiretroviral therapy: the new challenge in HIV-1 infection.

Although highly active antiretroviral therapy has enabled constant progress in reducing HIV-1 replication, in some patients who are "aviremic" during treatment, the problem of insufficient immune restoration remains, and this exposes them to the risk of immune deficiency-associated pathologies. Various mechanisms may combine and account for this impaired immunologic response to treatment. A first possible mechanism is immune activation, which may be because of residual HIV production, microbial translocation, co-infections, immunosenescence, or lymphopenia per se. A second mechanism is ongoing HIV replication. Finally, deficient thymus output, sex, and genetic polymorphism influencing apoptosis may impair immune reconstitution. In this review we will discuss the tools at our disposal to identify the various mechanisms at work in a given patient and the specific therapeutic strategies we could propose based on this etiologic diagnosis.

Low levels of peripheral blood activated and senescent T cells characterize people with HIV-1-associated neurocognitive disorders.

Background: HIV infection induces a 75% increase in the risk of developing neurocognitive impairment (NCI), which has been linked to immune activation. We therefore looked for immune activation markers correlating with NCI. Method: Sixty-five people aged 55-70 years living with controlled HIV-1 infection were enrolled in the study and their neurocognitive ability was assessed according to the Frascati criteria. Fifty-nine markers of T4 cell, T8 cell, NK cell, and monocyte activation, inflammation and endothelial activation were measured in their peripheral blood. White matter hyperintensities (WMH) were identified by magnetic resonance imaging. Double hierarchical clustering was performed for the activation markers and 240 patients including the 65 whose neurocognitive performance had been evaluated. Results: Thirty-eight percent of volunteers presented NCI. Twenty-four percent of them were asymptomatic and fourteen percent had a mild disorder. Strikingly, activated (HLA-DR+) as well as senescent (CD57+CD28-CD27±) T4 cells and T8 cells were less prevalent in the peripheral blood of participants with NCI than in participants without the disorder. Accordingly, the percentage of HLA-DR+ T4 cells was lower in volunteers with periventricular and deep WMH. The double hierarchical clustering unveiled six different immune activation profiles. The neurocognitive performances of participants with two of these six profiles were poor. Here again, these two profiles were characterized by a low level of T4 and T8 cell activation and senescence. Conclusion: Our observation of low circulating levels of activated and senescent T cells in HIV-1 patients with NCI raises the interesting hypothesis that these lymphocytes may be recruited into the central nervous system.

T4 apoptosis in the acute phase of SARS-CoV-2 infection predicts long COVID.

Background: As about 10% of patients with COVID-19 present sequelae, it is important to better understand the physiopathology of so-called long COVID. Method: To this aim, we recruited 29 patients hospitalized for SARS-CoV-2 infection and, by Luminex®, quantified 19 soluble factors in their plasma and in the supernatant of their peripheral blood mononuclear cells, including inflammatory and anti-inflammatory cytokines and chemokines, Th1/Th2/Th17 cytokines, and endothelium activation markers. We also measured their T4, T8 and NK differentiation, activation, exhaustion and senescence, T cell apoptosis, and monocyte subpopulations by flow cytometry. We compared these markers between participants who developed long COVID or not one year later. Results: None of these markers was predictive for sequelae, except programmed T4 cell death. T4 lymphocytes from participants who later presented long COVID were more apoptotic in culture than those of sequelae-free participants at Month 12 (36.9 ± 14.7 vs. 24.2 ± 9.0%, p = 0.016). Conclusions: Our observation raises the hypothesis that T4 cell death during the acute phase of SARS-CoV-2 infection might pave the way for long COVID. Mechanistically, T4 lymphopenia might favor phenomena that could cause sequelae, including SARS-CoV-2 persistence, reactivation of other viruses, autoimmunity and immune dysregulation. In this scenario, inhibiting T cell apoptosis, for instance, by caspase inhibitors, could prevent long COVID.

Repetitive mRNA vaccination is required to improve the quality of broad-spectrum anti-SARS-CoV-2 antibodies in the absence of CXCL13.

Since the initial spread of severe acute respiratory syndrome coronavirus 2 infection, several viral variants have emerged and represent a major challenge for immune control, particularly in the context of vaccination. We evaluated the quantity, quality, and persistence of immunoglobulin G (IgG) and IgA in individuals who received two or three doses of messenger RNA (mRNA) vaccines, compared with previously infected vaccinated individuals. We show that three doses of mRNA vaccine were required to match the humoral responses of preinfected vaccinees. Given the importance of antibody-dependent cell-mediated immunity against viral infections, we also measured the capacity of IgG to recognize spike variants expressed on the cell surface and found that cross-reactivity was also strongly improved by repeated vaccination. Last, we report low levels of CXCL13, a surrogate marker of germinal center activation and formation, in vaccinees both after two and three doses compared with preinfected individuals, providing a potential explanation for the short duration and low quality of Ig induced.

The intensity of immune activation is linked to the level of CCR5 expression in human immunodeficiency virus type 1-infected persons.

Immune activation is a main driver of AIDS- and non-AIDS-linked morbidities in the course of HIV-1 infection. As CCR5, the main HIV-1 co-receptor, is not only a chemokine receptor but also a co-activation molecule expressed at the surface of T cells, it could be directly involved in this immune activation. To test this hypothesis, we measured by flow cytometry the mean number of CCR5 molecules at the surface of non-activated CD4(+) T cells (CCR5 density), which determines the intensity of CCR5 signalling, and the percentage of CD8(+) T cells over-expressing CD38 (CD38 expression), a major marker of immune activation, in the blood of 67 HIV-1-infected, non-treated individuals. CCR5 density was correlated with CD38 expression independently of viral load (P=0.016). CCR5 density remained unchanged after highly active anti-retroviral therapy (HAART) introduction or cessation, whereas CD38 expression decreased and increased, respectively. Moreover, pre-therapeutic CCR5 density was highly predictive (r=0.736, P<10(-4) ) of residual CD38 over-expression after 9 months of HAART. Hence, CCR5 might play an immunological role in HIV-1 infection as a driver of immune activation. This could explain why CCR5 antagonists may have an inhibitory effect on immune activation.

NK Cells Acquire CCR5 and CXCR4 by Trogocytosis in People Living with HIV-1.

NK cells play a major role in the antiviral immune response, including against HIV-1. HIV-1 patients have impaired NK cell activity with a decrease in CD56dim NK cells and an increase in the CD56-CD16+ subset, and recently it has been proposed that a population of CD56+NKG2C+KIR+CD57+ cells represents antiviral memory NK cells. Antiretroviral therapy (ART) partly restores the functional activity of this lymphocyte lineage. NK cells when interacting with their targets can gain antigens from them by the process of trogocytosis. Here we show that NK cells can obtain CCR5 and CXCR4, but barely CD4, from T cell lines by trogocytosis in vitro. By UMAP (Uniform Manifold Approximation and Projection), we show that aviremic HIV-1 patients have unique NK cell clusters that include cells expressing CCR5, NKG2C and KIRs, but lack CD57 expression. Viremic patients have a larger proportion of CXCR4+ and CCR5+ NK cells than healthy donors (HD) and this is largely increased in CD107+ cells, suggesting a link between degranulation and trogocytosis. In agreement, UMAP identified a specific NK cell cluster in viremic HIV-1 patients, which contains most of the CD107a+, CCR5+ and CXCR4+ cells. However, this cluster lacks NKG2C expression. Therefore, NK cells can gain CCR5 and CXCR4 by trogocytosis, which depends on degranulation.

Inflammatory Markers after Switching to a Dual Drug Regimen in HIV-Infected Subjects: A Two-Year Follow-Up.

Objective: Immunadapt is a study evaluating the impact of combination antiretroviral treatment (cART) simplification on immune activation. We previously showed that switching to dual therapies could be associated six months later with macrophage activation. Followup continued up to 24 months after treatment simplification. Materials and Methods: Immunadapt is a prospective single arm study of successfully treated subjects simplifying cART from triple to dual regimens. Before cART change, at 6 months, and between 18 and 24 months following the switch, we measured IP-10, MCP-1, soluble CD14 (sCD14), soluble CD163 (sCD163), and lipopolysaccharide binding protein. Patients were stratified according to lower or greater likelihood of immune activation (CD4 nadir < 200, previous AIDS-defining event or very-low-level viremia during follow-up). Variables were compared using matched Wilcoxon tests. Results: From April 2019 to September 2021, 14 subjects were included (mean age 60 years, 12 men, 26 years since HIV infection, CD4 nadir 302 cells/mm3, 18 years on cART, 53 months on last cART). Twenty-one months following the switch, all but one subject maintained their viral load < 50 cp/mL. One subject had two viral blips. For the entire population, the sCD163 values increased significantly from baseline (+36%, p = 0.003) and from 6 months after the switch. The other markers did not change. After 6 months, the sCD163 increase was more pronounced in subjects with greater likelihood of immune activation (+53% vs. +19%, p = 0.026) Conclusions: cART simplification to dual therapy was associated with macrophage activation despite successful virological control after almost two years' follow-up. This was more pronounced in those at risk of immune activation.

A Non-Invasive Neonatal Signature Predicts Later Development of Atopic Diseases.

Background: Preterm birth is a major cause of morbidity and mortality in infants and children. Non-invasive methods for screening the neonatal immune status are lacking. Archaea, a prokaryotic life domain, comprise methanogenic species that are part of the neonatal human microbiota and contribute to early immune imprinting. However, they have not yet been characterized in preterm neonates. Objective: To characterize the gut immunological and methanogenic Archaeal (MA) signature in preterm neonates, using the presence or absence of atopic conditions at the age of one year as a clinical endpoint. Methods: Meconium and stool were collected from preterm neonates and used to develop a standardized stool preparation method for the assessment of mediators and cytokines and characterize the qPCR kinetics of gut MA. Analysis addressed the relationship between immunological biomarkers, Archaea abundance, and atopic disease at age one. Results: Immunoglobulin E, tryptase, calprotectin, EDN, cytokines, and MA were detectable in the meconium and later samples. Atopic conditions at age of one year were positively associated with neonatal EDN, IL-1ß, IL-10, IL-6, and MA abundance. The latter was negatively associated with neonatal EDN, IL-1ß, and IL-6. Conclusions: We report a non-invasive method for establishing a gut immunological and Archaeal signature in preterm neonates, predictive of atopic diseases at the age of one year.

Low perforin expression in CD8+ T lymphocytes during the acute phase of severe SARS-CoV-2 infection predicts long COVID.

T cell cytotoxicity plays a major role in antiviral immunity. Anti-SARS-CoV-2 immunity may determine acute disease severity, but also the potential persistence of symptoms (long COVID). We therefore measured the expression of perforin, a cytotoxic mediator, in T cells of patients recently hospitalized for SARS-CoV-2 infection. We recruited 54 volunteers confirmed as being SARS-CoV-2-infected by RT-PCR and admitted to Intensive Care Units (ICUs) or non-ICU, and 29 age- and sex-matched healthy controls (HCs). Amounts of intracellular perforin and granzyme-B, as well as cell surface expression of the degranulation marker CD107A were determined by flow cytometry. The levels of 15 cytokines in plasma were measured by Luminex. The frequency of perforin-positive T4 cells and T8 cells was higher in patients than in HCs (9.9 ± 10.1% versus 4.6 ± 6.4%, p = 0.006 and 46.7 ± 20.6% vs 33.3 ± 18.8%, p = 0.004, respectively). Perforin expression was neither correlated with clinical and biological markers of disease severity nor predictive of death. By contrast, the percentage of perforin-positive T8 cells in the acute phase of the disease predicted the onset of long COVID one year later. A low T8 cytotoxicity in the first days of SARS-CoV-2 infection might favor virus replication and persistence, autoimmunity, and/or reactivation of other viruses such as Epstein-Barr virus or cytomegalovirus, paving the way for long COVID. Under this hypothesis, boosting T cell cytotoxicity during the acute phase of the infection could prevent delayed sequelae.