Specific CD4+ T cell phenotypes associate with bacterial control in people who 'resist' infection with Mycobacterium tuberculosis.

A subset of individuals exposed to Mycobacterium tuberculosis (Mtb) that we refer to as 'resisters' (RSTR) show evidence of IFN-γ- T cell responses to Mtb-specific antigens despite serially negative results on clinical testing. Here we found that Mtb-specific T cells in RSTR were clonally expanded, confirming the priming of adaptive immune responses following Mtb exposure. RSTR CD4+ T cells showed enrichment of TH17 and regulatory T cell-like functional programs compared to Mtb-specific T cells from individuals with latent Mtb infection. Using public datasets, we showed that these TH17 cell-like functional programs were associated with lack of progression to active tuberculosis among South African adolescents with latent Mtb infection and with bacterial control in nonhuman primates. Our findings suggested that RSTR may successfully control Mtb following exposure and immune priming and established a set of T cell biomarkers to facilitate further study of this clinical phenotype.

Personalized Immunomonitoring Uncovers Molecular Networks that Stratify Lupus Patients.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by loss of tolerance to nucleic acids and highly diverse clinical manifestations. To assess its molecular heterogeneity, we longitudinally profiled the blood transcriptome of 158 pediatric patients. Using mixed models accounting for repeated measurements, demographics, treatment, disease activity (DA), and nephritis class, we confirmed a prevalent IFN signature and identified a plasmablast signature as the most robust biomarker of DA. We detected gradual enrichment of neutrophil transcripts during progression to active nephritis and distinct signatures in response to treatment in different nephritis subclasses. Importantly, personalized immunomonitoring uncovered individual correlates of disease activity that enabled patient stratification into seven groups, supported by patient genotypes. Our study uncovers the molecular heterogeneity of SLE and provides an explanation for the failure of clinical trials. This approach may improve trial design and implementation of tailored therapies in genetically and clinically complex autoimmune diseases. PAPERCLIP.

Systems scale interactive exploration reveals quantitative and qualitative differences in response to influenza and pneumococcal vaccines.

Systems immunology approaches were employed to investigate innate and adaptive immune responses to influenza and pneumococcal vaccines. These two non-live vaccines show different magnitudes of transcriptional responses at different time points after vaccination. Software solutions were developed to explore correlates of vaccine efficacy measured as antibody titers at day 28. These enabled a further dissection of transcriptional responses. Thus, the innate response, measured within hours in the peripheral blood, was dominated by an interferon transcriptional signature after influenza vaccination and by an inflammation signature after pneumococcal vaccination. Day 7 plasmablast responses induced by both vaccines was more pronounced after pneumococcal vaccination. Together, these results suggest that comparing global immune responses elicited by different vaccines will be critical to our understanding of the immune mechanisms underpinning successful vaccination.

Antigen-Specific T-Cell Activation Distinguishes between Recent and Remote Tuberculosis Infection.

Rationale: Current diagnostic tests fail to identify individuals at higher risk of progression to tuberculosis disease, such as those with recent Mycobacterium tuberculosis infection, who should be prioritized for targeted preventive treatment. Objectives: To define a blood-based biomarker, measured with a simple flow cytometry assay, that can stratify different stages of tuberculosis infection to infer risk of disease. Methods: South African adolescents were serially tested with QuantiFERON-TB Gold to define recent (QuantiFERON-TB conversion <6 mo) and persistent (QuantiFERON-TB+ for >1 yr) infection. We defined the ΔHLA-DR median fluorescence intensity biomarker as the difference in HLA-DR expression between IFN-γ+ TNF+Mycobacterium tuberculosis-specific T cells and total CD3+ T cells. Biomarker performance was assessed by blinded prediction in untouched test cohorts with recent versus persistent infection or tuberculosis disease and by unblinded analysis of asymptomatic adolescents with tuberculosis infection who remained healthy (nonprogressors) or who progressed to microbiologically confirmed disease (progressors). Measurements and Main Results: In the test cohorts, frequencies of Mycobacterium tuberculosis-specific T cells differentiated between QuantiFERON-TB- (n = 25) and QuantiFERON-TB+ (n = 47) individuals (area under the receiver operating characteristic curve, 0.94; 95% confidence interval, 0.87-1.00). ΔHLA-DR significantly discriminated between recent (n = 20) and persistent (n = 22) QuantiFERON-TB+ (0.91; 0.83-1.00); persistent QuantiFERON-TB+ and newly diagnosed tuberculosis (n = 19; 0.99; 0.96-1.00); and tuberculosis progressors (n = 22) and nonprogressors (n = 34; 0.75; 0.63-0.87). However, ΔHLA-DR median fluorescent intensity could not discriminate between recent QuantiFERON-TB+ and tuberculosis (0.67; 0.50-0.84). Conclusions: The ΔHLA-DR biomarker can identify individuals with recent QuantiFERON-TB conversion and those with disease progression, allowing targeted provision of preventive treatment to those at highest risk of tuberculosis. Further validation studies of this novel immune biomarker in various settings and populations at risk are warranted.

MR1-Independent Activation of Human Mucosal-Associated Invariant T Cells by Mycobacteria.

Tuberculosis (TB) is the leading cause of mortality from a single infectious agent, Mycobacterium tuberculosis Relevant immune targets of the partially efficacious TB vaccine bacille Calmette-Guérin (BCG) remain poorly defined. Mucosal-associated invariant T (MAIT) cells are MHC-related protein 1 (MR1)-restricted T cells, which are reactive against M. tuberculosis, and underexplored as potential TB vaccine targets. We sought to determine whether BCG vaccination activated mycobacteria-specific MAIT cell responses in humans. We analyzed whole blood samples from M. tuberculosis-infected South African adults who were revaccinated with BCG after a six-month course of isoniazid preventative therapy. In vitro BCG stimulation potently induced IFN-γ expression by phenotypic (CD8+CD26+CD161+) MAIT cells, which constituted the majority (75%) of BCG-reactive IFN-γ-producing CD8+ T cells. BCG revaccination transiently expanded peripheral blood frequencies of BCG-reactive IFN-γ+ MAIT cells, which returned to baseline frequencies a year following vaccination. In another cohort of healthy adults who received BCG at birth, 53% of mycobacteria-reactive-activated CD8 T cells expressed CDR3α TCRs, previously reported as MAIT TCRs, expressing the canonical TRAV1-2-TRAJ33 MAIT TCRα rearrangement. CD26 and CD161 coexpression correlated with TRAV1-2+CD161+ phenotype more accurately in CD8+ than CD4-CD8- MAIT cells. Interestingly, BCG-induced IFN-γ expression by MAIT cells in vitro was mediated by the innate cytokines IL-12 and IL-18 more than MR1-induced TCR signaling, suggesting TCR-independent activation. Collectively, the data suggest that activation of blood MAIT cells by innate inflammatory cytokines is a major mechanism of responsiveness to vaccination with whole cell vaccines against TB or in vitro stimulation with mycobacteria (Clinical trial registration: NCT01119521).

[Lupus erythematosus-a clinico-pathological heterogeneous disease]. / Lupus erythematodes ­ ein klinisch-pathologisch heterogenes Krankheitsbild.

Lupus erythematosus (LE) is an autoimmune disorder where immune tolerance towards nucleic acids is lost and a hyperactivated type I interferon system drives chronic immune activation. Typically, signs, symptoms, and clinical disease course are very variable between patients. Cutaneous LE can be associated with or precede systemic involvement or be limited to the skin, necessitating careful examination and follow-up of patients. LE skin disease includes a wide range of manifestations and precise classification for clinical studies is challenging. In this review article we discuss common and rare manifestations of cutaneous lupus with its clinical presentation and histopathological characteristics.

Differences in antibody responses between trivalent inactivated influenza vaccine and live attenuated influenza vaccine correlate with the kinetics and magnitude of interferon signaling in children.

BACKGROUND: Live attenuated influenza vaccine (LAIV) and trivalent inactivated influenza vaccine (TIV) are effective for prevention of influenza virus infection in children, but the mechanisms associated with protection are not well defined. METHODS: We analyzed the differences in B-cell responses and transcriptional profiles in children aged 6 months to 14 years immunized with these 2 vaccines. RESULTS: LAIV elicited a significant increase in naive, memory, and transitional B cells on day 30 after vaccination, whereas TIV elicited an increased number of plasmablasts on day 7. Antibody titers against the 3 vaccine strains (H1N1, H3N2, and B) were significantly higher in the TIV group and correlated with number of antibody-secreting cells. Both vaccines induced overexpression of interferon (IFN)-signaling genes but with different kinetics. TIV induced expression of IFN genes on day 1 after vaccination in all age groups, and LAIV induced expression of IFN genes on day 7 after vaccination but only in children <5 years old. IFN-related genes overexpressed in both vaccinated groups correlated with H3N2 antibody titers. CONCLUSIONS: These results suggest that LAIV and TIV induced significantly different B-cell responses in vaccinated children. Early induction of IFN appears to be important for development of antibody responses.

H3N2 influenza virus infection induces broadly reactive hemagglutinin stalk antibodies in humans and mice.

Broadly neutralizing antibodies directed against the conserved stalk domain of the viral hemagglutinin have attracted increasing attention in recent years. However, only a limited number of stalk antibodies directed against group 2 influenza hemagglutinins have been isolated so far. Also, little is known about the general level of induction of these antibodies by influenza virus vaccination or infection. To characterize the anti-stalk humoral response in the mouse model as well as in humans, chimeric hemagglutinin constructs previously developed in our group were employed in serological assays. Whereas influenza virus infection induced high titers of stalk-reactive antibodies, immunization with inactivated influenza virus vaccines failed to do so in the mouse model. Analysis of serum samples collected from human individuals who were infected by influenza viruses also revealed the induction of stalk-reactive antibodies. Finally, we show that the hemagglutinin stalk-directed antibodies induced in mice and humans have broad reactivity and neutralizing activity in vitro and in vivo. The results of the study point toward the existence of highly conserved epitopes in the stalk domains of group 2 hemagglutinins, which can be targeted for the development of a universal influenza virus vaccine in humans.

T cell receptor repertoires associated with control and disease progression following Mycobacterium tuberculosis infection.

Antigen-specific, MHC-restricted αß T cells are necessary for protective immunity against Mycobacterium tuberculosis, but the ability to broadly study these responses has been limited. In the present study, we used single-cell and bulk T cell receptor (TCR) sequencing and the GLIPH2 algorithm to analyze M. tuberculosis-specific sequences in two longitudinal cohorts, comprising 166 individuals with M. tuberculosis infection who progressed to either tuberculosis (n = 48) or controlled infection (n = 118). We found 24 T cell groups with similar TCR-ß sequences, predicted by GLIPH2 to have common TCR specificities, which were associated with control of infection (n = 17), and others that were associated with progression to disease (n = 7). Using a genome-wide M. tuberculosis antigen screen, we identified peptides targeted by T cell similarity groups enriched either in controllers or in progressors. We propose that antigens recognized by T cell similarity groups associated with control of infection can be considered as high-priority targets for future vaccine development.

Distribution and maturation of skin dendritic cell subsets in two forms of cutaneous T-cell lymphoma: mycosis fungoides and Sézary syndrome.

Dendritic cells (DCs) critically regulate immune responses and the "immune-surveillance" of tumours. This study retrospectively analysed the distribution and maturation status of DC-subsets in T-cell lymphoma of the skin. Mycosis fungoides and Sézary syndrome (n = 25) were investigated immunohistochemically for DC subsets, based on C-type lectin receptor expression: Langerhans' cells (langerin/CD207+, DEC-205/CD205+), dermal DCs (DC-SIGN/CD209+, CD205+) and plasmacytoid DC (BDCA-2/CD303+). Maturation status was assessed by double-labelling for CD83 and CD208/DC-LAMP. DCs were interspersed between the neoplastic infiltrate, and a marked increase in numbers of all three subsets was noted, DC-SIGN+ dermal DCs constituting the majority. Substantial numbers of plasmacytoid DCs were consistently observed. Most DCs in epidermis and dermis were phenotypically immature. Amongst the relatively few mature DCs in the dermis, langerin+ cells predominated. There was a positive correlation between the histological intensity of the tumour infiltrate and DC numbers. It is possible that mature DCs reflect ongoing anti-tumour immune responses, and immature DCs the induction of tumour tolerance.

Disease characteristics and serological responses in patients with differing severity of COVID-19 infection: A longitudinal cohort study in Dhaka, Bangladesh.

BACKGROUND: COVID-19 caused by SARS-CoV-2 ranges from asymptomatic to severe disease and can cause fatal and devastating outcome in many cases. In this study, we have compared the clinical, biochemical and immunological parameters across the different disease spectrum of COVID-19 in Bangladeshi patients. METHODOLOGY/PRINCIPAL FINDINGS: This longitudinal study was conducted in two COVID-19 hospitals and also around the community in Dhaka city in Bangladesh between November 2020 to March 2021. A total of 100 patients with COVID-19 infection were enrolled and classified into asymptomatic, mild, moderate and severe cases (n = 25/group). In addition, thirty age and sex matched healthy participants were enrolled and 21 were analyzed as controls based on exclusion criteria. After enrollment (study day1), follow-up visits were conducted on day 7, 14 and 28 for the cases. Older age, male gender and co-morbid conditions were the risk factors for severe COVID-19 disease. Those with moderate and severe cases of infection had low lymphocyte counts, high neutrophil counts along with a higher neutrophil-lymphocyte ratio (NLR) at enrollment; this decreased to normal range within 42 days after the onset of symptom. At enrollment, D-dimer, CRP and ferritin levels were elevated among moderate and severe cases. The mild, moderate, and severe cases were seropositive for IgG antibody by day 14 after enrollment. Moderate and severe cases showed significantly higher IgM and IgG levels of antibodies to SARS-CoV-2 compared to mild and asymptomatic cases. CONCLUSION/SIGNIFICANCE: We report on the clinical, biochemical, and hematological parameters associated with the different severity of COVID-19 infection. We also show different profile of antibody response against SARS-CoV-2 in relation to disease severity, especially in those with moderate and severe disease manifestations compared to the mild and asymptomatic infection.

Vi-Vaccinations Induce Heterogeneous Plasma Cell Responses That Associate With Protection From Typhoid Fever.

Vi-polysaccharide conjugate vaccines are efficacious against cases of typhoid fever; however, an absolute correlate of protection is not established. In this study, we investigated the leukocyte response to a Vi-tetanus toxoid conjugate vaccine (Vi-TT) in comparison with a plain polysaccharide vaccine (Vi-PS) in healthy adults subsequently challenged with Salmonella Typhi. Immunological responses and their association with challenge outcome was assessed by mass cytometry and Vi-ELISpot assay. Immunization induced significant expansion of plasma cells in both vaccines with modest T follicular helper cell responses detectable after Vi-TT only. The Vi-specific IgG and IgM B cell response was considerably greater in magnitude in Vi-TT recipients. Intriguingly, a significant increase in a subset of IgA+ plasma cells expressing mucosal migratory markers α4ß7 and CCR10 was observed in both vaccine groups, suggesting a gut-tropic, mucosal response is induced by Vi-vaccination. The total plasma cell response was significantly associated with protection against typhoid fever in Vi-TT vaccinees but not Vi-PS. IgA+ plasma cells were not significantly associated with protection for either vaccine, although a trend is seen for Vi-PS. Conversely, the IgA- fraction of the plasma cell response was only associated with protection in Vi-TT. In summary, these data indicate that a phenotypically heterogeneous response including both gut-homing and systemic antibody secreting cells may be critical for protection induced by Vi-TT vaccination.

Mass Cytometry Defines Virus-Specific CD4+ T Cells in Influenza Vaccination.

The antiviral response to influenza virus is complex and multifaceted, involving many immune cell subsets. There is an urgent need to understand the role of CD4+ T cells, which orchestrate an effective antiviral response, to improve vaccine design strategies. In this study, we analyzed PBMCs from human participants immunized with influenza vaccine, using high-dimensional single-cell proteomic immune profiling by mass cytometry. Data were analyzed using a novel clustering algorithm, denoised ragged pruning, to define possible influenza virus-specific clusters of CD4+ T cells. Denoised ragged pruning identified six clusters of cells. Among these, one cluster (Cluster 3) was found to increase in abundance following stimulation with influenza virus peptide ex vivo. A separate cluster (Cluster 4) was found to expand in abundance between days 0 and 7 postvaccination, indicating that it is vaccine responsive. We examined the expression profiles of all six clusters to characterize their lineage, functionality, and possible role in the response to influenza vaccine. Clusters 3 and 4 consisted of effector memory cells, with high CD154 expression. Cluster 3 expressed cytokines like IL-2, IFN-γ, and TNF-α, whereas Cluster 4 expressed IL-17. Interestingly, some participants had low abundance of Clusters 3 and 4, whereas others had higher abundance of one of these clusters compared with the other. Taken together, we present an approach for identifying novel influenza virus-reactive CD4+ T cell subsets, a method that could help advance understanding of the immune response to influenza, predict responsiveness to vaccines, and aid in better vaccine design.

VoPo leverages cellular heterogeneity for predictive modeling of single-cell data.

High-throughput single-cell analysis technologies produce an abundance of data that is critical for profiling the heterogeneity of cellular systems. We introduce VoPo (https://github.com/stanleyn/VoPo), a machine learning algorithm for predictive modeling and comprehensive visualization of the heterogeneity captured in large single-cell datasets. In three mass cytometry datasets, with the largest measuring hundreds of millions of cells over hundreds of samples, VoPo defines phenotypically and functionally homogeneous cell populations. VoPo further outperforms state-of-the-art machine learning algorithms in classification tasks, and identified immune-correlates of clinically-relevant parameters.

High prevalence of 'Borrelia-like' organisms in skin biopsies of sarcoidosis patients from Western Austria.

BACKGROUND: Sarcoidosis is a multisystem granulomatous disorder of unknown aetiology with frequent skin involvement. OBJECTIVE: To investigate the frequency of 'Borrelia-like' organisms in cutaneous sarcoidosis (CS) by focus-floating microscopy (FFM). METHODS: Retrospective analysis of 38 CS specimens by immunohistochemistry with polyclonal anti-Borrelia antibody and assessment by FFM. RESULTS: Specimens of 34.2% CS were positive for 'Borrelia-like' organisms by FFM. Usually single spirochetes, rarely pairs or small clusters of bacteria were observed between collagen bundles or at the periphery of granulomas. Polymerase chain reaction (PCR) was performed in addition in 11 cases and was negative in all biopsies. Samples of erythema migrans served as positive controls: 92.3% of 39 samples were positive by FFM, but only 46.6% gave positive results by PCR. Of 61 negative controls only one specimen was falsely positive by immunohistochemistry. CONCLUSION: Detection of 'Borrelia-like' organisms by FFM in tissue sections of CS underlines the possibility that such microorganism maybe involved in the pathogenesis of some cases of CS.

Longitudinal profiling of human blood transcriptome in healthy and lupus pregnancy.

Systemic lupus erythematosus carries an increased risk of pregnancy complications, including preeclampsia and fetal adverse outcomes. To identify the underlying molecular mechanisms, we longitudinally profiled the blood transcriptome of 92 lupus patients and 43 healthy women during pregnancy and postpartum and performed multicolor flow cytometry in a subset of them. We also profiled 25 healthy women undergoing assisted reproductive technology to monitor transcriptional changes around embryo implantation. Sustained down-regulation of multiple immune signatures, including interferon and plasma cells, was observed during healthy pregnancy. These changes appeared early after embryo implantation and were mirrored in uncomplicated lupus pregnancies. Patients with preeclampsia displayed early up-regulation of neutrophil signatures that correlated with expansion of immature neutrophils. Lupus pregnancies with fetal complications carried the highest interferon and plasma cell signatures as well as activated CD4+ T cell counts. Thus, blood immunomonitoring reveals that both healthy and uncomplicated lupus pregnancies exhibit early and sustained transcriptional modulation of lupus-related signatures, and a lack thereof associates with adverse outcomes.

Investigation of the role of typhoid toxin in acute typhoid fever in a human challenge model.

Salmonella Typhi is a human host-restricted pathogen that is responsible for typhoid fever in approximately 10.9 million people annually1. The typhoid toxin is postulated to have a central role in disease pathogenesis, the establishment of chronic infection and human host restriction2-6. However, its precise role in typhoid disease in humans is not fully defined. We studied the role of typhoid toxin in acute infection using a randomized, double-blind S. Typhi human challenge model7. Forty healthy volunteers were randomized (1:1) to oral challenge with 104 colony-forming units of wild-type or an isogenic typhoid toxin deletion mutant (TN) of S. Typhi. We observed no significant difference in the rate of typhoid infection (fever ≥38 °C for ≥12 h and/or S. Typhi bacteremia) between participants challenged with wild-type or TN S. Typhi (15 out of 21 (71%) versus 15 out of 19 (79%); P = 0.58). The duration of bacteremia was significantly longer in participants challenged with the TN strain compared with wild-type (47.6 hours (28.9-97.0) versus 30.3(3.6-49.4); P ≤ 0.001). The clinical syndrome was otherwise indistinguishable between wild-type and TN groups. These data suggest that the typhoid toxin is not required for infection and the development of early typhoid fever symptoms within the context of a human challenge model. Further clinical data are required to assess the role of typhoid toxin in severe disease or the establishment of bacterial carriage.

Comparison of CyTOF assays across sites: Results of a six-center pilot study.

For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments. We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center. Data were analyzed at the primary center, including investigating the effects of two normalization methods. All six sites performed similarly, with CVs for both Frequency of Parent and median signal intensity (MSI) values<30%. Increased background was seen when using the premixed antibody cocktail aliquots at each site, suggesting that cocktails are best made fresh. Both normalization methods tested performed adequately for normalizing MSI values between centers. Clustering algorithms revealed slight differences between the prestained and the sites-stained samples, due mostly to the increased background of a few antibodies. Therefore, we believe that multicenter mass cytometry assays are feasible.