The Preprint Club: A blueprint for community-based peer review: A blueprint for community-based peer review.

Cross-institutional journal clubs focused on preprints are a new approach to community-based peer review and allow ERCs to gain experience.

Single-cell RNA-seq analysis reveals dual sensing of HIV-1 in blood Axl+ dendritic cells.

Sensing of incoming viruses is a pivotal task of dendritic cells (DCs). Human primary blood DCs encompass various subsets that are diverse in their susceptibility and response to HIV-1. The recent identification of the blood Axl+DC subset, endowed with unique capacities to bind, replicate, and transmit HIV-1 prompted us to evaluate its anti-viral response. We demonstrate that HIV-1 induced two main broad and intense transcriptional programs in different Axl+DCs potentially induced by different sensors; an NF-κB-mediated program that led to DC maturation and efficient CD4+ T cell activation, and a program mediated by STAT1/2 that activated type I IFN and ISG responses. These responses were absent from cDC2 exposed to HIV-1 except when viral replication was allowed. Finally, Axl+DCs actively replicating HIV-1 identified by quantification of viral transcripts exhibited a mixed NF-κB/ISG innate response. Our results suggest that the route of HIV-1 entry may dictate different innate sensing pathways by DCs.

Sustained Low Relapse Rate With Highly Variable B-Cell Repopulation Dynamics With Extended Rituximab Dosing Intervals in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: B cell-depleting therapies are highly effective in relapsing-remitting multiple sclerosis (RRMS) but are associated with increased infection risk and blunted humoral vaccination responses. Extension of dosing intervals may mitigate such negative effects, but its consequences on MS disease activity are yet to be ascertained. The objective of this study was to determine clinical and neuroradiologic disease activity, as well as B-cell repopulation dynamics, after implementation of extended rituximab dosing in RRMS. METHODS: We conducted a prospective observational study in a specialized-care, single-center setting, including patients with RRMS participating in the COMBAT-MS and MultipleMS observational drug trials, who had received at least 2 courses of rituximab (median follow-up 4.2 years, range 0.1-8.9 years). Using Cox regression, hazard ratios (HRs) of clinical relapse and/or contrast-enhancing lesions on MRI were calculated in relation to time since last dose of rituximab. RESULTS: A total of 3,904 dose intervals were accumulated in 718 patients and stratified into 4 intervals: <8, ≥8 to 12, ≥12 to 18, and ≥18 months. We identified 24 relapses of which 20 occurred within 8 months since previous infusion and 4 with intervals over 8 months. HRs for relapse when comparing ≥8 to 12, ≥12 to 18, and ≥18 months with <8 months since last dose were 0.28 (95% CI 0.04-2.10), 0.38 (95% CI 0.05-2.94), and 0.89 (95% CI 0.20-4.04), respectively, and thus nonsignificant. Neuroradiologic outcomes mirrored relapse rates. Dynamics of total B-cell reconstitution varied considerably, but median total B-cell counts reached lower level of normal after 12 months and median memory B-cell counts after 16 months. DISCUSSION: In this prospective cohort of rituximab-treated patients with RRMS exposed to extended dosing intervals, we could not detect a relation between clinical or neuroradiologic disease activity and time since last infusion. Total B- and memory B-cell repopulation kinetics varied considerably. These findings, relevant for assessing risk-mitigation strategies with anti-CD20 therapies in RRMS, suggest that relapse risk remains low with extended infusion intervals. Further studies are needed to investigate the relation between B-cell repopulation dynamics and adverse event risks associated with B-cell depletion.

B-cell repopulation dynamics and drug pharmacokinetics impact SARS-CoV-2 vaccine efficacy in anti-CD20-treated multiple sclerosis patients.

BACKGROUND AND PURPOSE: Recent findings document a blunted humoral response to SARS-CoV-2 vaccination in patients on anti-CD20 treatment. Although most patients develop a cellular response, it is still important to identify predictors of seroconversion to optimize vaccine responses. METHODS: We determined antibody responses after SARS-CoV-2 vaccination in a real-world cohort of multiple sclerosis patients (n = 94) treated with anti-CD20, mainly rituximab, with variable treatment duration (median = 2.9, range = 0.4-9.6 years) and time from last anti-CD20 infusion to vaccination (median = 190, range = 60-1032 days). RESULTS: We find that presence of B cells and/or rituximab in blood predict seroconversion better than time since last infusion. Using multiple logistic regression, presence of >0.5% B cells increased probability of seroconversion with an odds ratio (OR) of 5.0 (95% confidence interval [CI] = 1.0-28.1, p = 0.055), whereas the corresponding OR for ≥6 months since last infusion was 1.45 (95% CI = 0.20-10.15, p = 0.705). In contrast, detectable rituximab levels were negatively associated with seroconversion (OR = 0.05, 95% CI = 0.002-0.392, p = 0.012). Furthermore, naïve and memory IgG+ B cells correlated with antibody levels. Although retreatment with rituximab at 4 weeks or more after booster depleted spike-specific B cells, it did not noticeably affect the rate of decline in antibody titers. Interferon-γ and/or interleukin-13 T-cell responses to the spike S1 domain were observed in most patients, but with no correlation to spike antibody levels. CONCLUSIONS: These findings are relevant for providing individualized guidance to patients and planning of vaccination schemes, in turn optimizing benefit-risk with anti-CD20.

Correlation between leukocyte phenotypes and prognosis of amyotrophic lateral sclerosis.

The prognostic role of immune cells in amyotrophic lateral sclerosis (ALS) remains undetermined. Therefore, we conducted a longitudinal cohort study including 288 ALS patients with up to 5-year follow-up during 2015-2020 recruited at the only tertiary referral center for ALS in Stockholm, Sweden, and measured the levels of differential leukocytes and lymphocyte subpopulations. The primary outcome was risk of death after diagnosis of ALS and the secondary outcomes included functional status and disease progression rate. Cox model was used to evaluate the associations between leukocytes and risk of death. Generalized estimating equation model was used to assess the correlation between leukocytes and functional status and disease progression rate. We found that leukocytes, neutrophils, and monocytes increased gradually over time since diagnosis and were negatively correlated with functional status, but not associated with risk of death or disease progression rate. For lymphocyte subpopulations, NK cells (HR= 0.61, 95% CI = [0.42-0.88] per SD increase) and Th2-diffrentiated CD4+ central memory T cells (HR= 0.64, 95% CI = [0.48-0.85] per SD increase) were negatively associated with risk of death, while CD4+ effector memory cells re-expressing CD45RA (EMRA) T cells (HR= 1.39, 95% CI = [1.01-1.92] per SD increase) and CD8+ T cells (HR= 1.38, 95% CI = [1.03-1.86] per SD increase) were positively associated with risk of death. None of the lymphocyte subpopulations was correlated with functional status or disease progression rate. Our findings suggest a dual role of immune cells in ALS prognosis, where neutrophils and monocytes primarily reflect functional status whereas NK cells and different T lymphocyte populations act as prognostic markers for survival.

Development of humoral and cellular immunological memory against SARS-CoV-2 despite B cell depleting treatment in multiple sclerosis.

B cell depleting therapies (BCDTs) are widely used as immunomodulating agents for autoimmune diseases such as multiple sclerosis. Their possible impact on development of immunity to severe acute respiratory syndrome virus-2 (SARS-CoV-2) has raised concerns with the coronavirus disease 2019 (COVID-19) pandemic. We here evaluated the frequency of COVID-19-like symptoms and determined immunological responses in participants of an observational trial comprising several multiple sclerosis disease modulatory drugs (COMBAT-MS; NCT03193866) and in eleven patients after vaccination, with a focus on BCDT. Almost all seropositive and 17.9% of seronegative patients on BCDT, enriched for a history of COVID-19-like symptoms, developed anti-SARS-CoV-2 T cell memory, and T cells displayed functional similarity to controls producing IFN-γ and TNF. Following vaccination, vaccine-specific humoral memory was impaired, while all patients developed a specific T cell response. These results indicate that BCDTs do not abrogate SARS-CoV-2 cellular memory and provide a possible explanation as to why the majority of patients on BCDTs recover from COVID-19.

HIV-2-Infected Macrophages Produce and Accumulate Poorly Infectious Viral Particles.

A significant proportion of HIV-2-infected patients exhibit natural virological control that is generally absent from HIV-1-infected patients. Along with CD4+ T cells, HIV-1 targets macrophages which may contribute to viral spreading and the latent reservoir. We have studied the relationship between macrophages and HIV-2, focusing on post-entry steps. HIV-2-infected monocyte-derived macrophages (MDMs) produced substantial amounts of viral particles that were largely harbored intracellularly. New viruses assembled at the limiting membrane of internal compartments similar to virus-containing compartments (VCCs) described for HIV-1. VCCs from MDMs infected with either virus shared protein composition and morphology. Strikingly, HIV-2 Gag was mostly absent from the cytosol and almost exclusively localized to the VCCs, whereas HIV-1 Gag was distributed in both locations. Ultrastructural analyses of HIV-2-infected MDMs revealed the presence of numerous VCCs containing both immature and mature particles in the lumen. HIV-2 particles produced de novo by MDMs were poorly infectious in reporter cells and in transmission to activated T cells through a process that appeared independent of BST2 restriction. Rather than being involved in viral spreading, HIV-2-infected macrophages may represent a cell-associated source of viral antigens that can participate in the immune control of HIV-2 infection.

Constitutive Siglec-1 expression confers susceptibility to HIV-1 infection of human dendritic cell precursors.

The human dendritic cell (DC) lineage has recently been unraveled by high-dimensional mapping, revealing the existence of a discrete new population of blood circulating DC precursors (pre-DCs). Whether this new DC population possesses specific functional features as compared to the other blood DC subset upon pathogen encounter remained to be evaluated. A unique feature of pre-DCs among blood DCs is their constitutive expression of the viral adhesion receptor Siglec-1. Here, we show that pre-DCs, but not other blood DC subsets, are susceptible to infection by HIV-1 in a Siglec-1-dependent manner. Siglec-1 mediates pre-DC infection of CCR5- and CXCR4-tropic strains. Infection of pre-DCs is further enhanced in the presence of HIV-2/SIVmac Vpx, indicating that Siglec-1 does not counteract restriction factors such as SAMHD1. Instead, Siglec-1 promotes attachment and fusion of viral particles. HIV-1-infected pre-DCs produce new infectious viral particles that accumulate in intracellular compartments reminiscent of the virus-containing compartment of macrophages. Pre-DC activation by toll-like receptor (TLR) ligands induces an antiviral state that inhibits HIV-1 fusion and infection, but Siglec-1 remains functional and mediates replication-independent transfer of HIV-1 to activated primary T lymphocytes. Altogether, Siglec-1-mediated susceptibility to HIV-1 infection of pre-DCs constitutes a unique functional feature that might represent a preferential relationship of this emerging cell type with viruses.

Proliferative memory SAMHD1low CD4+ T cells harbour high levels of HIV-1 with compartmentalized viral populations.

We previously reported the presence of memory CD4+ T cells that express low levels of SAMHD1 (SAMHD1low) in peripheral blood and lymph nodes from both HIV-1 infected and uninfected individuals. These cells are enriched in Th17 and Tfh subsets, two populations known to be preferentially targeted by HIV-1. Here we investigated whether SAMHD1low CD4+ T-cells harbour replication-competent virus and compartimentalized HIV-1 genomes. We sorted memory CD4+CD45RO+SAMHD1low, CD4+CD45RO+SAMHD1+ and naive CD4+CD45RO-SAMHD1+ cells from HIV-1-infected patients on anti-retroviral therapy (c-ART) and performed HIV-1 DNA quantification, ultra-deep-sequencing of partial env (C2/V3) sequences and phenotypic characterization of the cells. We show that SAMHD1low cells include novel Th17 CCR6+ subsets that lack CXCR3 and CCR4 (CCR6+DN). There is a decrease of the % of Th17 in SAMHD1low compartment in infected compared to uninfected individuals (41% vs 55%, p<0.05), whereas the % of CCR6+DN increases (7.95% vs 3.8%, p<0.05). Moreover, in HIV-1 infected patients, memory SAMHD1low cells harbour high levels of HIV-1 DNA compared to memory SAMHD1+ cells (4.5 vs 3.8 log/106cells, respectively, p<0.001), while naïve SAMHD1+ showed significantly lower levels (3.1 log/106cells, p<0.0001). Importantly, we show that SAMHD1low cells contain p24-producing cells. Moreover, phylogenetic analyses revealed well-segregated HIV-1 DNA populations with compartmentalization between SAMHD1low and SAMHD1+ memory cells, and limited viral exchange. As expected, the % of Ki67+ cells was significantly higher in SAMHD1low compared to SAMHD1+ cells. There was positive association between levels of HIV-1 DNA and Ki67+ in memory SAMHD1low cells, but not in memory and naïve SAMHD1+ CD4+ T-cells. Altogether, these data suggest that proliferative memory SAMHD1low cells contribute to viral persistence.

Myeloid Cell Interaction with HIV: A Complex Relationship.

Cells of the myeloid lineage, particularly macrophages, serve as primary hosts for HIV in vivo, along with CD4 T lymphocytes. Macrophages are present in virtually every tissue of the organism, including locations with negligible T cell colonization, such as the brain, where HIV-mediated inflammation may lead to pathological sequelae. Moreover, infected macrophages are present in multiple other tissues. Recent evidence obtained in humanized mice and macaque models highlighted the capacity of macrophages to sustain HIV replication in vivo in the absence of T cells. Combined with the known resistance of the macrophage to the cytopathic effects of HIV infection, such data bring a renewed interest in this cell type both as a vehicle for viral spread as well as a viral reservoir. While our understanding of key processes of HIV infection of macrophages is far from complete, recent years have nevertheless brought important insight into the uniqueness of the macrophage infection. Productive infection of macrophages by HIV can occur by different routes including from phagocytosis of infected T cells. In macrophages, HIV assembles and buds into a peculiar plasma membrane-connected compartment that preexists to the infection. While the function of such compartment remains elusive, it supposedly allows for the persistence of infectious viral particles over extended periods of time and may play a role on viral transmission. As cells of the innate immune system, macrophages have the capacity to detect and respond to viral components. Recent data suggest that such sensing may occur at multiple steps of the viral cycle and impact subsequent viral spread. We aim to provide an overview of the HIV-macrophage interaction along the multiple stages of the viral life cycle, extending when pertinent such observations to additional myeloid cell types such as dendritic cells or blood monocytes.

Mapping the human DC lineage through the integration of high-dimensional techniques.

Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises two main functionally specialized lineages, whose origins and differentiation pathways remain incompletely defined. Here, we combine two high-dimensional technologies-single-cell messenger RNA sequencing (scmRNAseq) and cytometry by time-of-flight (CyTOF)-to identify human blood CD123+CD33+CD45RA+ DC precursors (pre-DC). Pre-DC share surface markers with plasmacytoid DC (pDC) but have distinct functional properties that were previously attributed to pDC. Tracing the differentiation of DC from the bone marrow to the peripheral blood revealed that the pre-DC compartment contains distinct lineage-committed subpopulations, including one early uncommitted CD123high pre-DC subset and two CD45RA+CD123low lineage-committed subsets exhibiting functional differences. The discovery of multiple committed pre-DC populations opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting.

From dendritic cells to B cells dysfunctions during HIV-1 infection: T follicular helper cells at the crossroads.

T follicular helper (Tfh) cells are essential for B-cell differentiation and the subsequent antibody responses. Their numbers and functions are altered during human and simian immunodeficiency virus (HIV/SIV) infections. In lymphoid tissues, Tfh cells are present in germinal centre, where they are the main source of replicative HIV-1 and represent a major reservoir. Paradoxically, Tfh cell numbers are increased in chronically infected individuals. Understanding the fate of Tfh cells in the course of HIV-1 infection is essential for the design of efficient strategies toward a protective HIV vaccine or a cure. The purpose of this review is to summarize the recent advance in our understanding of Tfh cell dynamics during HIV/SIV infection. In particular, to explore the possible causes of their expansion in lymphoid tissues by discussing the impact of HIV-1 infection on dendritic cells, to identify the molecular players rendering Tfh cells highly susceptible to HIV-1 infection, and to consider the contribution of regulatory follicular T cells in shaping Tfh cell functions.

Decreased HIV-specific T-regulatory responses are associated with effective DC-vaccine induced immunity.

The role of regulatory T cells (Tregs) in vaccination has been poorly investigated. We have reported that vaccination with ex vivo-generated dendritic-cells (DC) loaded with HIV-lipopeptides (LIPO-5-DC vaccine) in HIV-infected patients was well tolerated and highly immunogenic. These responses and their relation to viral replication following analytical treatment interruption (ATI) were variable. Here, we investigated whether the presence of HIV-specific Tregs might explain these differences. Co-expression of CD25, CD134, CD39 and FoxP3 was used to delineate both antigen-specific Tregs and effectors T cells (Teffs). Median LIPO-5 specific-CD25+CD134+ polyfunctional T cells increased from 0.1% (IQR 0-0.3) before vaccination (week -4) to 2.1% (IQR 1.1-3.9) at week 16 following 4 immunizations (p=0.001) and were inversely correlated with maximum viral load following ATI (r=-0.77, p=0.001). Vaccinees who displayed lower levels of HIV-specific CD4+CD134+CD25+CD39+FoxP3+ Tregs responded better to the LIPO-5-DC vaccine. After vaccination, the frequency of HIV-specific Tregs decreased (from 69.3 at week -4 to 31.7% at week 16) and inversely correlated with HIV-specific IFN-γ-producing cells (r=-0.64, p=0.002). We show that therapeutic immunization skewed the HIV-specific response from regulatory to effector phenotype which impacts on the magnitude of viral replication following ATI.

Low SAMHD1 expression following T-cell activation and proliferation renders CD4+ T cells susceptible to HIV-1.

OBJECTIVES: HIV-1 replication depends on the state of cell activation and division. It is established that SAMHD1 restricts HIV-1 infection of resting CD4 T cells. The modulation of SAMHD1 expression during T-cell activation and proliferation, however, remains unclear, as well as a role for SAMHD1 during HIV-1 pathogenesis. METHODS: SAMHD1 expression was assessed in CD4 T cells after their activation and in-vitro HIV-1 infection. We performed phenotype analyzes using flow cytometry on CD4 T cells from peripheral blood and lymph nodes from cohorts of HIV-1-infected individuals under antiretroviral treatment or not, and controls. RESULTS: We show that SAMHD1 expression decreased during CD4 T-cell proliferation in association with an increased susceptibility to in-vitro HIV-1 infection. Additionally, circulating memory CD4 T cells are enriched in cells with low levels of SAMHD1. These SAMHD1 cells are highly differentiated, exhibit a large proportion of Ki67 cycling cells and are enriched in T-helper 17 cells. Importantly, memory SAMHD1 cells were depleted from peripheral blood of HIV-infected individuals. We also found that follicular helper T cells present in secondary lymphoid organs lacked the expression of SAMHD1, which was accompanied by a higher susceptibility to HIV-1 infection in vitro. CONCLUSION: We demonstrate that SAMHD1 expression is decreased during CD4 T-cell activation and proliferation. Also, CD4 T-cell subsets known to be more susceptible to HIV-1 infection, for example, T-helper 17 and follicular helper T cells, display lower levels of SAMHD1. These results pin point a role for SAMHD1 expression in HIV-1 infection and the concomitant depletion of CD4 T cells.

A highly relevant and efficient single step method for simultaneous depletion and isolation of human regulatory T cells in a clinical setting.

Regulatory T cells (Tregs) are pivotal in preventing autoimmunity. They play a major but still ambiguous role in cancer and viral infections. Functional studies of human Tregs are often hampered by numerous technical difficulties arising from imperfections in isolating and depleting protocols, together with the usual low cell number available from clinical samples. We standardized a simple procedure (Single Step Method, SSM), based on magnetic beads technology, in which both depletion and isolation of human Tregs with high purities are simultaneously achieved. SSM is suitable when using low cell numbers either fresh or frozen from both patients and healthy individuals. It allows simultaneous Tregs isolation and depletion that can be used for further functional work to monitor suppressive function of isolated Tregs (in vitro suppression assay) and also effector IFN-γ responses of Tregs-depleted cell fraction (OX40 assay). To our knowledge, there is no accurate standardized method for Tregs isolation and depletion in a clinical context. SSM could thus be used and easily standardized across different laboratories.

Role of miR-155 in the regulation of lymphocyte immune function and disease.

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression within cells. One particular miRNA, miR-155, is highly expressed within lymphocytes (both B and T cells) and mediates a number of important roles. These include shaping the transcriptome of lymphoid cells that control diverse biological functions vital in adaptive immunity. The use of mice engineered to be deficient in miR-155, as well as the identification of endogenous targets of miR-155 in T cells by transcriptome-wide analysis, has helped to unravel the crucial role that this miRNA plays in fine tuning the regulation of lymphocyte subsets such as B cells, CD8(+) and CD4(+) T cells ranging from T helper type 1 (Th1), Th2, Th17 and regulatory T cells. In this review, we summarize what we have learned about miR-155 in the regulation of lymphocyte responses at the cellular and molecular levels and in particular, we focus on the recent findings showing that miR-155 shapes the balance between tolerance and immunity.