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Optimized immunoglobulin knock-ins using Cas9 reveal peritoneal B cell lineage relationships in vivo

Immunoglobulin (Ig) knock-in mice are valuable tools in basic and translational immunological research. Here we present "Speed-Ig," a rapid Cas9-based method for generating Ig knock-in mouse lines with high on-target integration rates at both heavy and light chain alleles. With standardized target sites and promoter regions, Speed-Ig mice can be used for comparative studies of B cell biology and vaccine optimization in vivo. We used Speed-Ig to create panels of mice with Ig pairs derived from B-1a, B-1b, and B-2 cells. Surprisingly, B-1b and B-2 Ig pairs drove both B-1b and B-2 phenotypes, suggesting a previously unknown lineage relationship between these subsets. We then confirmed the B-1:B-2 relationship with transcription factor reporter lines and through adoptive cell transfer experiments. In summary, our Ig knock-in approach facilitated the discovery of previously unappreciated aspect of innate-like B cell biology.

immunology↗

A comparative analysis of memory B cell and antibody responses against Plasmodium falciparum merozoite surface protein 1 in children and adults from Uganda

Memory B cells (MBCs) and plasma antibodies against Plasmodium falciparum merozoite antigens are important components of the protective immune response against malaria. To gain understanding of how responses against P. falciparum develop in these two arms of the humoral immune system, we evaluated MBC and antibody responses against the most abundant merozoite antigen, merozoite surface protein 1 (MSP1), in individuals from a region in Uganda with high P. falciparum transmission. Our results showed that MSP1-specific B cells in adults with immunological protection against malaria were predominantly IgG+ classical MBCs, while children with incomplete protection mainly harbored IgM+ MSP1-specific classical MBCs. In contrast, anti-MSP1 plasma IgM reactivity was minimal in both children and adults. Instead, both groups showed high plasma IgG reactivity against MSP1 and whole merozoites, with broadening of the response against non-3D7 strains in adults. The antibodies encoded by MSP1-specific IgG+ MBCs carried high levels of amino acid substitutions and recognized relatively conserved epitopes on the highly variable MSP1 protein. Proteomics analysis of MSP119-specific IgG in plasma of an adult revealed a limited repertoire of anti-MSP1 antibodies, most of which were IgG1 or IgG3. Similar to MSP1- specific MBCs, anti-MSP1 IgGs had relatively high levels of amino acid substitutions and their sequences were predominantly found in classical MBCs, not atypical MBCs. Collectively, these results showed evolution of the MSP1-specific humoral immune response with cumulative P. falciparum exposure, with a shift from IgM+ to IgG+ B cell memory, diversification of B cells from germline, and stronger recognition of MSP1 variants by the plasma IgG repertoire.

immunology↗

Longitudinal immune cell profiling in early systemic lupus erythematosus

ObjectiveTo investigate the immune cell profiling and their longitudinal changes in systemic lupus erythematosus (SLE). MethodsWe employed mass cytometry with three different 38-39 marker panels (Immunophenotyping, T cell/monocyte, and B cell) in cryopreserved peripheral blood mononuclear cells (PBMCs) from nine patients with early SLE, 15 patients with established SLE, and 14 non-inflammatory controls. We used machine learning-driven clustering, FlowSOM (Flow Self-Organizing Maps) and dimensional reduction with tSNE (t-distributed Stochastic Neighbor Embedding) to identify unique cell populations in early and established SLE. For the nine early SLE patients, longitudinal mass cytometry analysis was applied to PBMCs at three time points (at enrollment, six months post-enrollment, and one year post-enrollment). Serum samples were also assayed for 65 cytokines by Luminex multiplex assay, and associations between cell types and cytokines/chemokines assessed. ResultsT peripheral helper cells (Tph cells), T follicular helper cells (Tfh cells) and several Ki67+ proliferating subsets (ICOS+ Ki67+ CD8 T cells, Ki67+ regulatory T cells, CD19int Ki67hi plasmablasts, and Ki67hi PU.1hi monocytes) were increased in early SLE. Longitudinal mass cytometry and multiplex serum cytokine assays of samples from early SLE patients revealed that Tfh cells and CXCL10 decreased at one year post-enrollment. CXCL13 correlated positively with several of the expanded cell populations in early SLE. ConclusionsTwo major helper T cell subsets and unique Ki67+ proliferating immune cell subsets were expanded in the early phase of SLE, and the immunologic features characteristic of early SLE evolved over time.

immunology↗

STAT1 Gain-of-Function Variants Drive Altered T Cell Prevalence, Metabolism, and Heightened IL-6 Sensitivity

Patients with Signal Transducer and Activator of Transcription 1 (STAT1) gain-of-function (GOF) pathogenic variants exhibit susceptibility to infections, autoimmunity, and cancer due to enhanced or prolonged STAT1 phosphorylation following cytokine stimulation. While interferons (IFNs) are canonical STAT1 activators, other cytokines that may also contribute to pathology in STAT1 GOF patients have been less well defined. Here we analyzed the immune profiles and cytokine responses of two patients with heterozygous GOF mutations in the STAT1 coiled-coil domain. A systems immunology approach revealed major changes in the T cell compartment and minor changes in the B cells, NK cells, and myeloid cells. Both patients with STAT1 GOF differed from healthy individuals in the abundance and phenotype of effector memory, Th17, and Treg populations. STAT1 GOF T cells displayed a pattern of increased activation and had elevated markers of glycolysis and lipid oxidation. Hypersensitivity of T cells to IL-6 was observed with intense, sustained STAT1 phosphorylation in memory T cell populations that exceeded that induced by IFNs. Together, these results show a role for STAT1 in T cell metabolism and suggest that IL-6 may play a critical role to promote T cell memory formation and activation in patients with STAT1 GOF.

immunology↗

Characterization of SARS-CoV-2 public CD4+ αβ T cell clonotypes through reverse epitope discovery

The amount of scientific data and level of public sharing produced as a consequence of the COVID-19 pandemic, as well as the speed at which these data were produced, far exceeds any previous effort against a specific disease condition. This unprecedented situation allows for development and application of new research approaches. One of the major technical hurdles in immunology is the characterization of HLA-antigen-T cell receptor (TCR) specificities. Most approaches aim to identify reactive T cells starting from known antigens using functional assays. However, the need for a reverse approach identifying the antigen specificity of orphan TCRs is increasing. Utilizing large public single-cell gene expression and TCR datasets, we identified highly public CD4+ T cell responses to SARS-CoV-2, covering >75% of the analysed population. We performed an integrative meta-analysis to deeply characterize these clonotypes by TCR sequence, gene expression, HLA-restriction, and antigen-specificity, identifying strong and public CD4+ immunodominant responses with confirmed specificity. CD4+ COVID-enriched clonotypes show T follicular helper functional features, while clonotypes depleted in SARS-CoV-2 individuals preferentially had a central memory phenotype. In total we identify more than 1200 highly public CD4+ T cell clonotypes reactive to SARS-CoV-2. TCR similarity analysis showed six prominent TCR clusters, for which we predicted both HLA-restriction and cognate SARS-CoV-2 immunodominant epitopes. To validate our predictions we used an independent cohort of TCR repertoires before and after vaccination with ChAdOx1, a replication-deficient simian adenovirus-vectored vaccine, encoding the SARS-CoV-2 spike protein. We find statistically significant enrichment of the predicted spike-reactive TCRs after vaccination with ChAdOx1, while the frequency of TCRs specific to other SARS-CoV-2 proteins remains stable. Thus, the CD4-associated TCR repertoire differentiates vaccination from natural infection. In conclusion, our study presents a novel reverse epitope discovery approach that can be used to infer HLA- and antigen-specificity of orphan TCRs in any context, such as viral infections, antitumor immune responses, or autoimmune disease. HighlightsO_LIIdentification of highly public CD4+ T cell responses to SARS-CoV-2 C_LIO_LISystematic prediction of exact immunogenic HLA class II epitopes for CD4+ T cell response C_LIO_LIMethodological framework for reverse epitope discovery, which can be applied to other disease contexts and may provide essential insights for future studies and clinical applications C_LI O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

immunology↗

A recombinant SARS-CoV-2 RBD antigen expressed in insect cells elicits immunogenicity and confirms safety in animal models

COVID-19 pandemic has accelerated the development of vaccines against its etiologic agent, SARS-CoV-2. However, the emergence of new variants of the virus requires new immunization strategies in addition to the current vaccines approved for human administration. In the present report, the immunological and safety evaluation in mice and hamsters of a subunit vaccine based on the RBD sub-domain with two adjuvants of oil origin is described. The RBD protein was expressed in insect cells and purified by chromatography until >95% purity. The protein was shown to have the appropriate folding as determined by ELISA and flow cytometry binding assays to its receptor, as well as by its detection by hamster immune anti-S1 sera under non-reducing conditions. In immunization assays in mice and hamsters, the purified RBD formulated with adjuvants based on oil-water emulsifications and squalene was able to stimulate specific neutralizing antibodies and confirm the secretion of IFN-{gamma} after stimulating spleen cells with the purified RBD. The vaccine candidate was shown to be safe, as demonstrated by the histopathological analysis in lungs, liver and kidney. These results demonstrate the potential of the purified RBD administered with adjuvants through an intramuscular route, to be evaluated in a challenge against SARS-CoV-2 and determine its ability to confer protection against infection.

immunology↗

COVID-19 lung disease shares driver AT2 cytopathic features with Idiopathic pulmonary fibrosis

BackgroundIn the aftermath of Covid-19, some patients develop a fibrotic lung disease, i.e., post-COVID-19 lung disease (PCLD), for which we currently lack insights into pathogenesis, disease models, or treatment options. MethodUsing an AI-guided approach, we analyzed > 1000 human lung transcriptomic datasets associated with various lung conditions using two viral pandemic signatures (ViP and sViP) and one covid lung-derived signature. Upon identifying similarities between COVID-19 and idiopathic pulmonary fibrosis (IPF), we subsequently dissected the basis for such similarity from molecular, cytopathic, and immunologic perspectives using a panel of IPF-specific gene signatures, alongside signatures of alveolar type II (AT2) cytopathies and of prognostic monocyte-driven processes that are known drivers of IPF. Transcriptome-derived findings were used to construct protein-protein interaction (PPI) network to identify the major triggers of AT2 dysfunction. Key findings were validated in hamster and human adult lung organoid (ALO) pre-clinical models of COVID-19 using immunohistochemistry and qPCR. FindingsCOVID-19 resembles IPF at a fundamental level; it recapitulates the gene expression patterns (ViP and IPF signatures), cytokine storm (IL15-centric), and the AT2 cytopathic changes, e.g., injury, DNA damage, arrest in a transient, damage-induced progenitor state, and senescence-associated secretory phenotype (SASP). These immunocytopathic features were induced in pre-clinical COVID models (ALO and hamster) and reversed with effective anti-CoV-2 therapeutics in hamsters. PPI-network analyses pinpointed ER stress as one of the shared early triggers of both diseases, and IHC studies validated the same in the lungs of deceased subjects with COVID-19 and SARS-CoV-2-challenged hamster lungs. Lungs from tg-mice, in which ER stress is induced specifically in the AT2 cells, faithfully recapitulate the host immune response and alveolar cytopathic changes that are induced by SARS-CoV-2. InterpretationLike IPF, COVID-19 may be driven by injury-induced ER stress that culminates into progenitor state arrest and SASP in AT2 cells. The ViP signatures in monocytes may be key determinants of prognosis. The insights, signatures, disease models identified here are likely to spur the development of therapies for patients with IPF and other fibrotic interstitial lung diseases. FundingThis work was supported by the National Institutes for Health grants R01-GM138385 and AI155696 and funding from the Tobacco-Related disease Research Program (R01RG3780). One Sentence SummarySevere COVID-19 triggers cellular processes seen in fibrosing Interstitial Lung Disease RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSIn its aftermath, the COVID-19 pandemic has left many survivors, almost a third of those who recovered, with a mysterious long-haul form of the disease which culminates in a fibrotic form of interstitial lung disease (post-COVID-19 ILD). Post-COVID-19 ILD remains a largely unknown entity. Currently, we lack insights into the core cytopathic features that drive this condition. Added value of this studyUsing an AI-guided approach, which involves the use of sets of gene signatures, protein-protein network analysis, and a hamster model of COVID-19, we have revealed here that COVID-19 -lung fibrosis resembles IPF, the most common form of ILD, at a fundamental level--showing similar gene expression patterns in the lungs and blood, and dysfunctional AT2 processes (ER stress, telomere instability, progenitor cell arrest, and senescence). These findings are insightful because AT2 cells are known to contain an elegant quality control network to respond to intrinsic or extrinsic stress; a failure of such quality control results in diverse cellular phenotypes, of which ER stress appears to be a point of convergence, which appears to be sufficient to drive downstream fibrotic remodeling in the lung. Implications of all the available evidenceBecause unbiased computational methods identified the shared fundamental aspects of gene expression and cellular processes between COVID-19 and IPF, the impact of our findings is likely to go beyond COVID-19 or any viral pandemic. The insights, tools (disease models, gene signatures, and biomarkers), and mechanisms identified here are likely to spur the development of therapies for patients with IPF and, other fibrotic interstitial lung diseases, all of whom have limited or no treatment options. To dissect the validated prognostic biomarkers to assess and track the risk of pulmonary fibrosis and develop therapeutics to halt fibrogenic progression.

immunology↗

The impact of murine cytomegalovirus (mCMV) route and age at infection upon virus spread, immune responses and the establishment of latency

Cytomegalovirus (CMV) is a ubiquitous human virus, which establishes a characteristic lifetime infection in its host. Murine CMV (mCMV) is a widely-used infection model that has been employed to investigate the nature and extent of CMVs contribution to inflammatory, immunological, and health disturbances in humans. In an effort to assess the role of route and age in modeling hCMV infection in mice, we have performed a comparative analysis of two common experimental modes of infection (intraperitoneal and intranasal) at two different clinically relevant ages (4 weeks, or prepubescent childhood equivalent, and 12 weeks, or young postpubescent adult). We found that while both routes of infection led to similar early viral loads, differential activation of several parameters of innate immune function were observed. In particular, younger, prepubescent mice exhibited the strongest NK activation in the blood in response to i.p. infection, with this trend holding true in NK cells expressing the mCMV-specific receptor Ly49H. Moreover, i.p. infected animals accumulated a larger amount of anti-mCMV IgG and experienced a greater expansion of both acute and latent phase CD8+ T cells. This was especially true for young postpubescent mice, further illustrating a distinction in the bloodborne immune response across not only infection routes, but also ages. These results may be important in the understanding of how a more physiologically applicable model of CMV influences immunity, inflammation, and health over the lifespan.

immunology↗

SARS-CoV-2-specific T cells in unexposed adults display broad trafficking potential and cross-react with commensal antigens

The baseline composition of T cells directly impacts later response to a pathogen, but the complexity of precursor states remains poorly defined. Here we examined the baseline state of SARS-CoV-2 specific T cells in unexposed individuals. SARS-CoV-2 specific CD4+ T cells were identified in pre-pandemic blood samples by class II peptide-MHC tetramer staining and enrichment. Our data revealed a substantial number of SARS-CoV-2 specific T cells that expressed memory phenotype markers, including memory cells with gut homing receptors. T cell clones generated from tetramer-labeled cells cross-reacted with bacterial peptides and responded to stool lysates in a MHC-dependent manner. Integrated phenotypic analyses revealed additional precursor diversity that included T cells with distinct polarized states and trafficking potential to other barrier tissues. Our findings illustrate a complex pre-existing memory pool poised for immunologic challenges and implicate non-infectious stimuli from commensal colonization as a factor that shapes pre-existing immunity. One Sentence SummaryPre-existing immunity to SARS-CoV-2 contains a complex pool of precursor lymphocytes that include differentiated cells with broad tissue tropism and the potential to cross-react with commensal antigens.

immunology↗

Clinical implementation of single-cell RNA sequencing using liver fine needle aspirate tissue sampling and centralized processing captures compartment specific immuno-diversity

Blood samples are frequently collected in human studies of the immune system but poorly represent tissue-resident immunity. Understanding the immunopathogenesis of tissue-restricted diseases, such as chronic hepatitis B, necessitates direct investigation of local immune responses. We developed a workflow that enables frequent, minimally invasive collection of liver fine-needle aspirates in multi-site international studies and centralized single-cell RNA sequencing data generation using the Seq-Well S3 picowell-based technology. All immunological cell types were captured, including liver macrophages, and showed distinct compartmentalization and transcriptional profiles, providing a systematic assessment of the capabilities and limitations of peripheral blood samples when investigating tissue-restricted diseases. The ability to electively sample the liver of chronic viral hepatitis patients and generate high-resolution data will enable multi-site clinical studies to power fundamental and therapeutic discovery.

immunology↗

Neutralization Fingerprinting Technology for Characterizing Polyclonal Antibody Responses to Dengue Vaccines

Dengue is a major public health threat. There are four serotypes of dengue virus (DENV), therefore efforts are focused on development of safe and effective tetravalent DENV vaccines. While neutralizing antibodies contribute to protective immunity, there are still important gaps in understanding of immune responses elicited by dengue infection and vaccination, including defining immune correlates of protection. To that end, here we present a computational modeling framework for evaluating the specificities of neutralizing antibodies elicited to tetravalent DENV vaccines, based on the concept of antibody-virus neutralization fingerprints. We developed and applied this framework to samples from clinical studies of TAK-003, a tetravalent vaccine candidate currently in phase 3 trials, to characterize the effect of prior dengue infection (baseline) on the specificities of vaccine-elicited antibody responses. Our results suggested a similarity of neutralizing antibody specificities in baseline-seronegative individuals. In contrast, amplification of pre-existing neutralizing antibody specificities was predicted for baseline-seropositive individuals, thus quantifying the role of immunologic imprinting in driving antibody responses to DENV vaccines. The analysis framework proposed here can apply to studies of sequential dengue infections and other tetravalent DENV vaccines and can contribute to understanding dengue immune correlates of protection to help guide further vaccine development and optimization.

immunology↗

Minimal cross-over between mutations associated with Omicron variant of SARS-CoV-2 and CD8+ T cell epitopes identified in COVID-19 convalescent individuals

There is a growing concern that ongoing evolution of SARS-CoV-2 could lead to variants of concern (VOC) that are capable of avoiding some or all of the multi-faceted immune response generated by both prior infection or vaccination, with the recently described B.1.1.529 (Omicron) VOC being of particular interest. Peripheral blood mononuclear cell samples from PCR-confirmed, recovered COVID-19 convalescent patients (n=30) infected with SARS-CoV-2 in the United States collected in April and May 2020 who possessed at least one or more of six different HLA haplotypes were selected for examination of their anti-SARS-CoV-2 CD8+ T-cell responses using a multiplexed peptide-MHC tetramer staining approach. This analysis examined if the previously identified viral epitopes targeted by CD8+ T-cells in these individuals (n=52 distinct epitopes) are mutated in the newly described Omicron VOC (n=50 mutations). Within this population, only one low-prevalence epitope from the Spike protein restricted to two HLA alleles and found in 2/30 (7%) individuals contained a single amino acid change associated with the Omicron VOC. These data suggest that virtually all individuals with existing anti-SARS-CoV-2 CD8+ T-cell responses should recognize the Omicron VOC, and that SARS-CoV-2 has not evolved extensive T-cell escape mutations at this time. ImportanceThe newly identified Omicron variant of concern contains more mutations than any of the previous variants described to date. In addition, many of the mutations associated with the Omicron variant are found in areas that are likely bound by neutralizing antibodies, suggesting that the first line of immunological defense against COVID-19 may be compromised. However, both natural infection and vaccination develop T-cell based responses, in addition to antibodies. This study examined if the parts of the virus, or epitopes, targeted by the CD8+ T-cell response in thirty individuals who recovered from COVID-19 in 2020 were mutated in the Omicron variant. Only one of 52 epitopes identified in this population contained an amino acid that was mutated in Omicron. These data suggest that the T-cell immune response in previously infected, and most likely vaccinated individuals, should still be effective against Omicron.

immunology↗

In-depth characterization of the serum antibody epitope repertoire in inflammatory bowel disease using phage-displayed immunoprecipitation sequencing

Inflammatory bowel diseases (IBD), e.g. Crohns disease (CD) and ulcerative colitis (UC), are chronic immune-mediated inflammatory diseases. A comprehensive overview of an IBD-specific antibody epitope repertoire is, however, lacking. We leveraged a high-throughput phage-displayed immunoprecipitation sequencing (PhIP-seq) workflow to identify antibodies against 344,000 antimicrobial, immune and food antigens in 497 IBD patients as compared to 1,326 controls. IBD was characterized by 373 differentially abundant antibodies (202 overrepresented and 171 underrepresented), with 17% shared by both IBDs, 55% unique to CD and 28% unique to UC. Antibodies against bacterial flagellins dominated in CD and were associated with ileal involvement, fibrostenotic disease and anti-Saccharomyces cerevisiae antibody positivity, but not with fecal microbiome composition. Antibody epitope repertoires accurately discriminated CD from controls (AUC=0.89), and similar discrimination was achieved when using only ten antibodies (AUC=0.87). IBD patients thus show a distinct antibody repertoire against selected peptides, allowing patient stratification and discovery of immunological targets.

immunology↗

Reconstitution of T cell immunity against EBV in the immunocompromised host by adoptive transfer of peptide-stimulated T cells after allogeneic stem cell transplantation

Reconstitution of T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by peptide stimulation and adoptively transferred them to a patient with angioimmunoblastic T-cell lymphoma (AITL), who had developed persisting high titers of EBV concomitant to relapse after transplantation. T cell receptor beta (TCR{beta}) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse. Author summaryA characteristic feature of all herpesviruses is their persistence in the hosts body after primary infection. Hence, the hosts immune system is confronted with the problem to control these viruses life-long. Well-known representative of the herpesvirus group are the classic Herpes-Simplex Virus (HSV-1) and Varicella Zoster Virus (VZV, causing chicken pox); a less known representative is Epstein Barr Virus (EBV, causing mononucleosis). When the immune system is severely compromised, for example after stem cell transplantation from a foreign (allogeneic) donor, these viruses can reappear, as they are already in the hosts body. Especially EBV cause life-threatening complications after stem cell transplantation and only reinforcement of the hosts immune system can reestablish viral control. Here we show that ex vivo manufactured EBV-specific T cells can reestablish long-term control of EBV and that these cells persist in the hosts body over months. These results give us a better understanding of viral immune reconstitution post-transplant and of clinically-relevant T cell populations against EBV.

immunology↗

Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information

The study and manipulation of T cell receptors (TCRs) is central to multiple fields across basic and translational immunology research. Produced by V(D)J recombination, TCRs are often only recorded in the literature and data repositories as a combination of their V and J gene symbols, plus their hypervariable CDR3 amino acid sequence. However, numerous applications require full-length coding nucleotide sequences. Here we present Stitchr, a software tool developed to specifically address this limitation. Given minimal V/J/CDR3 information, Stitchr produces complete coding sequences representing a fully spliced TCR cDNA. Due to its modular design, Stitchr can be used for TCR engineering using either published germline or novel/modified variable and constant region sequences. Sequences produced by Stitchr were validated by synthesizing and transducing TCR sequences into Jurkat cells, recapitulating the expected antigen specificity of the parental TCR. Using a companion script, Thimble, we demonstrate that Stitchr can process a million TCRs in under ten minutes using a standard desktop personal computer. By systemizing the production and modification of TCR sequences, we propose that Stitchr will increase the speed, repeatability, and reproducibility of TCR research. Stitchr is available on GitHub.

immunology↗

Interferon lambda signals in maternal tissues to exert protective and pathologic effects in a gestational-stage dependent manner

Interferon lambda (IFN-{lambda}, type III IFN) is constitutively secreted from human placental cells in culture and reduces Zika virus (ZIKV) transplacental transmission in mice. However, the roles of IFN-{lambda} during healthy pregnancy and in restricting congenital infection remain unclear. Here we used mice lacking the IFN-{lambda} receptor (Ifnlr1-/-) to generate pregnancies lacking either maternal or fetal IFN-{lambda} responsiveness and found that the antiviral effect of IFN-{lambda} resulted from signaling exclusively in maternal tissues. This protective effect depended on gestational stage, as infection earlier in pregnancy (E7 rather than E9) resulted in enhanced transplacental transmission of ZIKV. In Ifnar1-/- dams, which sustain robust ZIKV infection, maternal IFN-{lambda} signaling caused fetal resorption and intrauterine growth restriction. Pregnancy pathology elicited by poly(I:C) treatment also was mediated by maternal IFN-{lambda} signaling, specifically in maternal leukocytes, and also occurred in a gestational stage-dependent manner. These findings identify an unexpected effect of IFN-{lambda} signaling specifically in maternal (rather than placental or fetal) tissues, which is distinct from the pathogenic effects of IFN-{beta} (type I IFN) during pregnancy. These results highlight the complexity of immune signaling at the maternal-fetal interface, where disparate outcomes can result from signaling at different gestational stages. IMPORTANCEPregnancy is an immunologically complex situation, which must balance protecting the fetus from maternal pathogens with preventing maternal immune rejection of non-self fetal and placental tissue. Cytokines, such as interferon lambda (IFN-{lambda}), contribute to antiviral immunity at the maternal-fetal interface. We found in a mouse model of congenital Zika virus infection that IFN-{lambda} can have either a protective antiviral effect or cause immune-mediated pathology, depending on the stage of gestation when IFN-{lambda} signaling occurs. Remarkably, both the protective and pathologic effects of IFN-{lambda} occurred through signaling exclusively in maternal immune cells, rather than in fetal or placental tissues, or in other maternal cell types, identifying a new role for IFN-{lambda} at the maternal-fetal interface.

immunology↗

Differential recognition and cytokine induction by the peptidorhamnomannan from Sporothrix brasiliensis and S. schenckii

Sporotrichosis is a deep mycosis caused by dimorphic species of the genus Sporothrix, with differences in pathogenicity between S. schenckii and S. brasiliensis species. Recently, it was discovered that the cell wall peptidorhamnomannan (PRM) of Sporothrix spp. is a pathogen associated molecular pattern (PAMP). Interestingly, S. brasiliensis PRM has additional unknown rhamnose residues. We hypothesize that the structural differences of Sporothrix spp PRMs impact the hosts immune response and may explain the severity of sporotrichosis caused by S. brasiliensis. Here we demonstrate that S. brasiliensis yeasts and its PRM (S.b PRM) induced a strong inflammatory response in human PBMCs, with high production of TNF-, IL-6 and IL-1{beta} and induction of T-helper cytokines IFN-{gamma}, IL-17 and IL-22. In contrast, S. schenckii yeasts and its PRM induced higher concentrations of interleukin-1 receptor antagonist (IL-1Ra), which resulted in low production of T-helper cytokines such as IL-17 and IL-22. CR3 and dectin-1 were required for cytokine induction by both PRMs, while TLR2 and TLR4 were required for the response of S.s PRM and S.b PRM, respectively. IL-1{beta} and IL-1 production induced by S. brasiliensis yeasts and S.b PRM were dependent on inflammasome and caspase-1 activation. S. schenckii and S.s PRM were able to induce IL-1{beta} independent of ROS. In conclusion, these findings improve our understanding of the pathogenesis of Sporothrix spp. by reporting differences of immunological responses induced by S. schenckii and S. brasiliensis. The study also opens the gateway for novel treatment strategies targeting local inflammation and tissue destruction induced by S. brasiliensis infection through IL-1 inhibition.

immunology↗

Cytotoxic T lymphocytes targeting a conserved SARS-CoV-2 spike epitope are efficient serial killers

Understanding the cellular immune response to infections, cancers and vaccines lags behind the investigation of humoral responses. While neutralizing antibody responses wane over time, the ability of T cells to recognize viruses including SARS-CoV-2 is instrumental to providing long-term immunity. Although T-cell receptor (TCR) repertoire screening can provide insights into the skewing of a T-cell response elicited upon vaccination or infection, they unfortunately provide no assessment into the functional capacity of T cells or their ability to eliminate virally infected targets. We have used time-lapse imaging microscopy in nanowell grids (TIMING) to integrate the migration of individual T cells with analysis of effector functions including cytokine secretion and cytotoxicity. Machine learning is then applied to study thousands of videos of dynamic interactions as T cells with specificity for SARS-CoV-2 eliminate targets bearing spike protein as a surrogate for viral infection. Our data provide the first direct evidence that cytotoxic T lymphocytes from a convalescent patient targeting an epitope conserved across all known variants of concern (VoC) are serial killers capable of eliminating multiple infected targets. These data have implications for development of vaccines to provide broad and sustained cellular immunity and for the recovery and monitoring of individuals who have been exposed to SARS-CoV-2. Multidisciplinary abstractWe present an imaging platform that uses artificial intelligence (AI) to track thousands of individual cell-cell interactions within nanowell arrays. We apply this platform to quantify how the T cell component of adaptive immunity responds to infections. Our results show that T cells specific for a conserved epitope within the SARS-CoV-2 spike protein are serial killers that can rapidly eliminate virally infected targets. The ability to map the functional capacity of T cells and their ability to kill infected cells provides fundamental insights into the immunology of vaccines and recovery from infections. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/477535v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@3e3eaborg.highwire.dtl.DTLVardef@844cacorg.highwire.dtl.DTLVardef@1c5ecfdorg.highwire.dtl.DTLVardef@14a0b44_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗