Regulation of B-cell function and expression of CD11c, T-bet, and FcRL5 in response to different activation signals.

CD11c, FcRL5, or T-bet are commonly expressed by B cells expanding during inflammation, where they can make up >30% of mature B cells. However, the association between the proteins and differentiation and function in the host response remains largely unclear. We have assessed the co-expression of CD11c, T-bet, and FcRL5 in an in vitro B-cell culture system to determine how stimulation via the BCR, toll-like receptor 9 (TLR9), and different cytokines influence CD11c, T-bet, and FcRL5 expression. We observed different expression dynamics for all markers, but a largely overlapping regulation of CD11c and FcRL5 in response to BCR and TLR9 activation, while T-bet was strongly dependent on IFN-γ signaling. Investigating plasma cell differentiation and APC functions, there was no association between marker expression and antibody secretion or T-cell help. Rather the functions were associated with TLR9-signalling and B-cell-derived IL-6 production, respectively. These results suggest that the expression of CD11c, FcRL5, and T-bet and plasma cell differentiation and improved APC functions occur in parallel and are regulated by similar activation signals, but they are not interdependent.

Disease-specific plasma protein profiles in patients with fever after traveling to tropical areas.

Fever is common among individuals seeking healthcare after traveling to tropical regions. Despite the association with potentially severe disease, the etiology is often not determined. Plasma protein patterns can be informative to understand the host response to infection and can potentially indicate the pathogen causing the disease. In this study, we measured 49 proteins in the plasma of 124 patients with fever after travel to tropical or subtropical regions. The patients had confirmed diagnoses of either malaria, dengue fever, influenza, bacterial respiratory tract infection, or bacterial gastroenteritis, representing the most common etiologies. We used multivariate and machine learning methods to identify combinations of proteins that contributed to distinguishing infected patients from healthy controls, and each other. Malaria displayed the most unique protein signature, indicating a strong immunoregulatory response with high levels of IL10, sTNFRI and II, and sCD25 but low levels of sCD40L. In contrast, bacterial gastroenteritis had high levels of sCD40L, APRIL, and IFN-γ, while dengue was the only infection with elevated IFN-α2. These results suggest that characterization of the inflammatory profile of individuals with fever can help to identify disease-specific host responses, which in turn can be used to guide future research on diagnostic strategies and therapeutic interventions.

Diagnostic markers reflecting dysregulation of the host response in the transition to tuberculosis disease.

Sustained control of Mycobacterium tuberculosis infection without evidence of disease is based on a finely tuned balance between pro- and anti-inflammatory responses. Loss of this balance leads to tuberculosis (TB) disease, in which exacerbated myeloid and neutrophil activation is common. Proteomic and transcriptomic assessment of the host response can detect increasing immune activation associated with TB disease progression several months before clinical disease. Future diagnostic methods based on measuring host response biomarkers that are able to detect this dysregulation could therefore be valuable in the early detection of TB disease progression.

Correction: Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0274415.].

A genomic platform for surveillance and antigen discovery in Plasmodium spp. using long-read amplicon sequencing.

Many vaccine candidate proteins in the malaria parasite Plasmodium falciparum are under strong immunological pressure and confer antigenic diversity. We present a sequencing and data analysis platform for the genomic surveillance of the insertion or deletion (indel)-rich antigens merozoite surface protein 1 (MSP1), MSP2, glutamate-rich protein (GLURP), and CSP from P. falciparum using long-read circular consensus sequencing (CCS) in multiclonal malaria isolates. Our platform uses 40 PCR primers per gene to asymmetrically barcode and identify multiclonal infections in pools of up to 384 samples. With msp2, we validated the method using 235 mock infections combining 10 synthetic variants at different concentrations and infection complexities. We applied this strategy to P. falciparum isolates from a longitudinal cohort in Tanzania. Finally, we constructed an analysis pipeline that streamlines the processing and interpretation of epidemiological and antigenic diversity data from demultiplexed FASTQ files. This platform can be easily adapted to other polymorphic antigens of interest in Plasmodium or any other human pathogen.

From simple to complex: Protein-based biomarker discovery in tuberculosis.

Tuberculosis (TB) is a deadly infectious disease that affects millions of people globally. TB proteomics signature discovery has been a rapidly growing area of research that aims to identify protein biomarkers for the early detection, diagnosis, and treatment monitoring of TB. In this review, we have highlighted recent advances in this field and how it is moving from the study of single proteins to high-throughput profiling and from only using proteomics to include additional types of data in multi-omics studies. We have further covered the different sample types and experimental technologies used in TB proteomics signature discovery, focusing on studies of HIV-negative adults. The published signatures were defined as either coming from hypothesis-based protein targeting or from unbiased discovery approaches. The methodological approaches influenced the type of proteins identified and were associated with the circulating protein abundance. However, both approaches largely identified proteins involved in similar biological pathways, including acute-phase responses and T-helper type 1 and type 17 responses. By analysing the frequency of proteins in the different signatures, we could also highlight potential robust biomarker candidates. Finally, we discuss the potential value of integration of multi-omics data and the importance of control cohorts and signature validation.

Delayed generation of functional virus-specific circulating T follicular helper cells correlates with severe COVID-19.

Effective humoral immune responses require well-orchestrated B and T follicular helper (Tfh) cell interactions. Whether these interactions are impaired and associated with COVID-19 disease severity is unclear. Here, longitudinal blood samples across COVID-19 disease severity are analysed. We find that during acute infection SARS-CoV-2-specific circulating Tfh (cTfh) cells expand with disease severity. SARS-CoV-2-specific cTfh cell frequencies correlate with plasmablast frequencies and SARS-CoV-2 antibody titers, avidity and neutralization. Furthermore, cTfh cells but not other memory CD4 T cells, from severe patients better induce plasmablast differentiation and antibody production compared to cTfh cells from mild patients. However, virus-specific cTfh cell development is delayed in patients that display or later develop severe disease compared to those with mild disease, which correlates with delayed induction of high-avidity neutralizing antibodies. Our study suggests that impaired generation of functional virus-specific cTfh cells delays high-quality antibody production at an early stage, potentially enabling progression to severe disease.

Archaic humans have contributed to large-scale variation in modern human T cell receptor genes.

Human T cell receptors (TCRs) are critical for mediating immune responses to pathogens and tumors and regulating self-antigen recognition. Yet, variations in the genes encoding TCRs remain insufficiently defined. Detailed analysis of expressed TCR alpha, beta, gamma, and delta genes in 45 donors from four human populations-African, East Asian, South Asian, and European-revealed 175 additional TCR variable and junctional alleles. Most of these contained coding changes and were present at widely differing frequencies in the populations, a finding confirmed using DNA samples from the 1000 Genomes Project. Importantly, we identified three Neanderthal-derived, introgressed TCR regions including a highly divergent TRGV4 variant, which mediated altered butyrophilin-like molecule 3 (BTNL3) ligand reactivity and was frequent in all modern Eurasian population groups. Our results demonstrate remarkable variation in TCR genes in both individuals and populations, providing a strong incentive for including allelic variation in studies of TCR function in human biology.

A protein signature associated with active tuberculosis identified by plasma profiling and network-based analysis.

Annually, approximately 10 million people are diagnosed with active tuberculosis (TB), and 1.4 million die of the disease. If left untreated, each person with active TB will infect 10-15 new individuals. The lack of non-sputum-based diagnostic tests leads to delayed diagnoses of active pulmonary TB cases, contributing to continued disease transmission. In this exploratory study, we aimed to identify biomarkers associated with active TB. We assessed the plasma levels of 92 proteins associated with inflammation in individuals with active TB (n = 20), latent TB (n = 14), or healthy controls (n = 10). Using co-expression network analysis, we identified one module of proteins with strong association with active TB. We removed proteins from the module that had low abundance or were associated with non-TB diseases in published transcriptomic datasets, resulting in a 12-protein plasma signature that was highly enriched in individuals with pulmonary and extrapulmonary TB and was further associated with disease severity.

Immunological hyporesponsiveness in tuberculosis: The role of mycobacterial glycolipids.

Glycolipids constitute a major part of the cell envelope of Mycobacterium tuberculosis (Mtb). They are potent immunomodulatory molecules recognized by several immune receptors like pattern recognition receptors such as TLR2, DC-SIGN and Dectin-2 on antigen-presenting cells and by T cell receptors on T lymphocytes. The Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic relatives, phosphatidylinositol mannosides (PIMs) and lipomannan (LM), as well as other Mtb glycolipids, such as phenolic glycolipids and sulfoglycolipids have the ability to modulate the immune response, stimulating or inhibiting a pro-inflammatory response. We explore here the downmodulating effect of Mtb glycolipids. A great proportion of the studies used in vitro approaches although in vivo infection with Mtb might also lead to a dampening of myeloid cell and T cell responses to Mtb glycolipids. This dampened response has been explored ex vivo with immune cells from peripheral blood from Mtb-infected individuals and in mouse models of infection. In addition to the dampening of the immune response caused by Mtb glycolipids, we discuss the hyporesponse to Mtb glycolipids caused by prolonged Mtb infection and/or exposure to Mtb antigens. Hyporesponse to LAM has been observed in myeloid cells from individuals with active and latent tuberculosis (TB). For some myeloid subsets, this effect is stronger in latent versus active TB. Since the immune response in individuals with latent TB represents a more protective profile compared to the one in patients with active TB, this suggests that downmodulation of myeloid cell functions by Mtb glycolipids may be beneficial for the host and protect against active TB disease. The mechanisms of this downmodulation, including tolerance through epigenetic modifications, are only partly explored.

Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis.

Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1-4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12-80%) and specificities (14-100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.

Alternative B Cell Differentiation During Infection and Inflammation.

Long-term protective immunity to infectious disease depends on cell-mediated and humoral immune responses. Induction of a strong humoral response relies on efficient B cell activation and differentiation to long-lived plasma cells and memory B cells. For many viral or bacterial infections, a single encounter is sufficient to induce such responses. In malaria, the induction of long-term immunity can take years of pathogen exposure to develop, if it occurs at all. This repeated pathogen exposure and suboptimal immune response coincide with the expansion of a subset of B cells, often termed atypical memory B cells. This subset is present at low levels in healthy individuals as well but it is observed to expand in an inflammatory context during acute and chronic infection, autoimmune diseases or certain immunodeficiencies. Therefore, it has been proposed that this subset is exhausted, dysfunctional, or potentially autoreactive, but its actual role has remained elusive. Recent reports have provided new information regarding both heterogeneity and expansion of these cells, in addition to indications on their potential role during normal immune responses to infection or vaccination. These new insights encourage us to rethink how and why they are generated and better understand their role in our complex immune system. In this review, we will focus on recent advances in our understanding of these enigmatic cells and highlight the remaining gaps that need to be filled.

Preserved Mucosal-Associated Invariant T Cells in the Cervical Mucosa of HIV-Infected Women with Dominant Expression of the TRAV1-2-TRAJ20 T Cell Receptor α-Chain.

BACKGROUND: Mucosa-associated invariant T (MAIT) cells are innate-like T cells with specialized antimicrobial functions. Circulating MAIT cells are depleted in chronic human immunodeficiency virus (HIV) infection, but studies examining this effect in peripheral tissues, such as the female genital tract, are lacking. METHODS: Flow cytometry was used to investigate circulating MAIT cells in a cohort of HIV-seropositive (HIV+) and HIV-seronegative (HIV-) female sex workers (FSWs), and HIV- lower-risk women (LRW). In situ staining and quantitative polymerase chain reaction were performed to explore the phenotype of MAIT cells residing in paired cervicovaginal tissue. The cervicovaginal microbiome was assessed by means of 16S ribosomal RNA gene sequencing. RESULTS: MAIT cells in the HIV+ FSW group were low in frequency in the circulation but preserved in the ectocervix. MAIT cell T-cell receptor gene segment usage differed between the HIV+ and HIV- FSW groups. The TRAV1-2-TRAJ20 transcript was the most highly expressed MAIT TRAJ gene detected in the ectocervix in the HIV+ FSW group. MAIT TRAVJ usage was not associated with specific genera in the vaginal microbiome. CONCLUSIONS: MAIT cells residing in the ectocervix are numerically preserved irrespective of HIV infection status and displayed dominant expression of TRAV1-2-TRAJ20. These findings have implications for understanding the role of cervical MAIT cells in health and disease.

Systems analysis shows a role of cytophilic antibodies in shaping innate tolerance to malaria.

Natural immunity to malaria develops over time with repeated malaria episodes, but protection against severe malaria and immune regulation limiting immunopathology, called tolerance, develops more rapidly. Here, we comprehensively profile the blood immune system in patients, with or without prior malaria exposure, over 1 year after acute symptomatic Plasmodium falciparum malaria. Using a data-driven analysis approach to describe the immune landscape over time, we show that a dampened inflammatory response is associated with reduced γδ T cell expansion, early expansion of CD16+ monocytes, and parasite-specific antibodies of IgG1 and IgG3 isotypes. This also coincided with reduced parasitemia and duration of hospitalization. Our data indicate that antibody-mediated phagocytosis during the blood stage infection leads to lower parasitemia and less inflammatory response with reduced γδ T cell expansion. This enhanced control and reduced inflammation points to a potential mechanism on how tolerance is established following repeated malaria exposure.

Plasmodium falciparum-Specific Memory B-Cell and Antibody Responses Are Associated With Immunity in Children Living in an Endemic Area of Kenya.

Identifying the mechanism of naturally acquired immunity against Plasmodium falciparum malaria could contribute to the design of effective malaria vaccines. Using a recently developed multiplexed FluoroSpot assay, we assessed cross-sectional pre-existing memory B-cells (MBCs) and antibody responses against six well known P. falciparum antigens (MSP-119, MSP-2 (3D7), MSP-2 (FC27), MSP-3, AMA-1 and CSP) and measured their associations with previous infections and time to clinical malaria in the ensuing malaria season in Kenyan children. These children were under active weekly surveillance for malaria as part of a long-term longitudinal malaria immunology cohort study, where they are recruited from birth. After performing Cox regression analysis, we found that children with a breadth of three or more antigen-specific MBC or antibody responses at the baseline had a reduced risk for malaria in the ensuing P. falciparum transmission season. Specifically, MBC responses against AMA-1, MSP-2 (3D7) and MSP-3, as well as antibody responses to MSP-2 (3D7) and MSP-3 were prospectively associated with a reduced risk for malaria. The magnitude or breadth of MBC responses were however not correlated with the cumulative number of malaria episodes since birth. We conclude that increased breadth for merozoite antigen-specific MBC and antibody responses is associated with protection against malaria.

Distinct kinetics of antibodies to 111 Plasmodium falciparum proteins identifies markers of recent malaria exposure.

Strengthening malaria surveillance is a key intervention needed to reduce the global disease burden. Reliable serological markers of recent malaria exposure could improve current surveillance methods by allowing for accurate estimates of infection incidence from limited data. We studied the IgG antibody response to 111 Plasmodium falciparum proteins in 65 adult travellers followed longitudinally after a natural malaria infection in complete absence of re-exposure. We identified a combination of five serological markers that detect exposure within the previous three months with >80% sensitivity and specificity. Using mathematical modelling, we examined the antibody kinetics and determined that responses informative of recent exposure display several distinct characteristics: rapid initial boosting and decay, less inter-individual variation in response kinetics, and minimal persistence over time. Such serological exposure markers could be incorporated into routine malaria surveillance to guide efforts for malaria control and elimination.

High Dimensional Immune Profiling Reveals Different Response Patterns in Active and Latent Tuberculosis Following Stimulation With Mycobacterial Glycolipids.

Upon infection with Mycobacterium tuberculosis (Mtb) the host immune response might clear the bacteria, control its growth leading to latent tuberculosis (LTB), or fail to control its growth resulting in active TB (ATB). There is however no clear understanding of the features underlying a more or less effective response. Mtb glycolipids are abundant in the bacterial cell envelope and modulate the immune response to Mtb, but the patterns of response to glycolipids are still underexplored. To identify the CD45+ leukocyte activation landscape induced by Mtb glycolipids in peripheral blood of ATB and LTB, we performed a detailed assessment of the immune response of PBMCs to the Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic precursor phosphatidyl-inositol mannoside (PIM), and purified-protein derivate (PPD). At 24 h of stimulation, cell profiling and secretome analysis was done using mass cytometry and high-multiplex immunoassay. PIM induced a diverse cytokine response, mainly affecting antigen-presenting cells to produce both pro-inflammatory and anti-inflammatory cytokines, but not IFN-γ, contrasting with PPD that was a strong inducer of IFN-γ. The effect of PIM on the antigen-presenting cells was partly TLR2-dependent. Expansion of monocyte subsets in response to PIM or LAM was reduced primarily in LTB as compared to healthy controls, suggesting a hyporesponsive/tolerance pattern derived from Mtb infection.

Generation of plasma cells and CD27-IgD- B cells during hantavirus infection is associated with distinct pathological findings.

OBJECTIVE: Human hantavirus infections can cause haemorrhagic fever with renal syndrome (HFRS). The pathogenic mechanisms are not fully understood, nor if they affect the humoral immune system. The objective of this study was to investigate humoral immune responses to hantavirus infection and to correlate them to the typical features of HFRS: thrombocytopenia and transient kidney dysfunction. METHODS: We performed a comprehensive characterisation of longitudinal antiviral B-cell responses of 26 hantavirus patients and combined this with paired clinical data. In addition, we measured extracellular adenosine triphosphate (ATP) and its breakdown products in circulation and performed in vitro stimulations to address its effect on B cells. RESULTS: We found that thrombocytopenia was correlated to an elevated frequency of plasmablasts in circulation. In contrast, kidney dysfunction was indicative of an accumulation of CD27-IgD- B cells and CD27-/low plasmablasts. Finally, we provide evidence that high levels of extracellular ATP and matrix metalloproteinase 8 can contribute to shedding of CD27 during human hantavirus infection. CONCLUSION: Our findings demonstrate that thrombocytopenia and kidney dysfunction associate with distinctly different effects on the humoral immune system. Moreover, hantavirus-infected individuals have significantly elevated levels of extracellular ATP in circulation.

Positive selection of IgG+ over IgM+ B cells in the germinal center reaction.

Positive selection of high-affinity B cells within germinal centers (GCs) drives affinity maturation of antibody responses. Here, we examined the mechanism underlying the parallel transition from immunoglobulin M (IgM) to IgG. Early GCs contained mostly unswitched IgM+ B cells; IgG+ B cells subsequently increased in frequency, dominating GC responses 14-21 days after antigen challenge. Somatic hypermutation and generation of high-affinity clones occurred with equal efficiency among IgM+ and IgG+ GC B cells, and inactivation of Ig class-switch recombination did not prevent depletion of IgM+ GC B cells. Instead, high-affinity IgG+ GC B cells outcompeted high-affinity IgM+ GC B cells via a selective advantage associated with IgG antigen receptor structure but independent of the extended cytoplasmic tail. Thus, two parallel forms of GC B-cell-positive selection, based on antigen receptor variable and constant regions, respectively, operate in tandem to ensure high-affinity IgG antibodies predominate in mature serum antibody responses.