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bioRxiv2023

TRPM7-dependent electrical signals drive phagocytic clearance for effective anti-fungal defense

Sentinel phagocytes of the innate immune system have a critical role in detecting and eliminating fungal pathogens. We used patch clamp electrophysiology to explore the electrical signals elicited when macrophages engulf Candida albicans. In the perforated patch configuration, which is least disruptive to intracellular signaling, we detected a composite outwardly rectifying current during the engulfment of C. albicans or zymosan. FTY720, a known inhibitor of ion channel TRPM7, suppressed the current. We then tested the hypothesis that TRPM7 regulates the engulfment and clearance of C. albicans. We found that Trpm7-/- macrophages are highly deficient in the engulfment of C. albicans. Trpm7-/- macrophages initiate phagocytosis of yeast but are defective in sealing the phagocytic cups. While the precise mechanism through which TRPM7 regulates phagosome sealing is not clear, we tested the immunological significance of this discovery using a mouse model of systemic candidiasis. We show that in mice, wherein TRPM7 is deleted selectively in the myeloid cells, infection by C. albicans results in significantly higher lethality, increased colonization of vital organs and increased inflammatory cytokines in the blood. Our study establishes TRPM7 as an ion channel critical for the innate immune responses against fungal pathogens and sets the stage for cell biological studies that define the mechanisms through which TRPM7 regulates phagosome sealing. Significance statementThe worldwide increase in deadly or persistent fungal infections has prompted the research for alternative ways of treatment. We applied the specialized, perforated patch clamp technique to track and identify electrical currents elicited during the detection and engulfment of fungi by macrophages. The ion channel TRPM7 emerged as an important determinant of anti-fungal host defense as its deletion in the murine myeloid cells made the host mice highly susceptible to lethal candidiasis. Ion channels are attractive drug targets whose activation and inhibition can be manipulated with pharmacological therapeutics. This study raises the possibility of enhancing fungal clearance using activators of TRPM7. Such pharmacological strategy may benefit patients of persistent fungal infections that are recalcitrant to anti-fungal drugs.

immunology↗

bioRxiv2023

Sensitization with an allogeneic MHC class I molecule induces anti-tumor immunity in the absence of PD-1 in mice

To investigate the effect of a major histocompatibility complex class I (MHC-I) overexpression to augment immune sensitivity against tumors, we have generated the murine colorectal carcinoma cell line MC38 (with the endogenous H-2b haplotype) overexpressing the allogeneic mouse MHC-I cell surface molecule H-2Kd (MC38 H-2Kd). The tumorigenicity of unmodified parental cells (MC38 PT) and MC38 H-2Kd was tested in vivo by subcutaneous injection into the flank of wild-type (WT) and programmed death-1 (PD-1) knockout (KO) mice in a C57BL/6 (H-2b) genetic background. MC38 PT cells readily formed tumors and grew progressively in both WT and PD-1 KO mice. The speed of MC38 PT tumor growth was slower in PD-1 KO mice than in WT mice. In contrast, MC38 H-2Kd cells showed full sensitivity to rejection by the immune system in both naive WT and PD-1 KO mice, indicated by spontaneous tumor regression. Next, we sought to determine the extent to which H-2Kd-overexpressing tumors could protect the mice against unmodified cancers. PD-1 KO mice were first sensitized with highly immunogenic MC38 H-2Kd cells and then challenged with weakly immunogenic MC38 PT cells. Intriguingly, all PD-1 KO mice gained immunity against the aggressive MC38 tumor and became tumor-free. Sensitizing PD-1 KO mice with growth-arrested (by the pre-treatment with mitomycin C, MMC) and the debris of MC38 H-2Kd tumors also provided full protection against the growth of secondary MC38 PT tumors. Most notably, sensitization with the debris of MC38 H-2Kd cells provided the long-term immunological memory against MC38 PT carcinoma cells. This finding implies that MC38 H-2Kd cells retain highly efficient and durable immunogenicity.

immunology↗

bioRxiv2023

Tumor-Specific CD8+ T Cells from the Bone Marrow Resist Exhaustion and Exhibit Increased Persistence in Tumor-Bearing Hosts as Compared to Tumor Infiltrating Lymphocytes

Immunotherapy is now an integral aspect of cancer therapy. Strategies employing adoptive cell therapy (ACT) have seen the establishment of chimeric antigen receptor (CAR)-T cells using peripheral blood lymphocytes as well as tumor infiltrating lymphocytes (TILs) with significant clinical results. Despite these successes, the limitations of the current strategies are also emerging and novel approaches are needed. The bone marrow (BM) is an immunological niche that houses T cells with specificity for previously encountered antigens, including tumor-associated antigens from certain solid cancers. This study sought to improve our understanding of tumor-specific BM T cells in the context of solid tumors by comparing them with TILs, and to assess whether there is a rationale for using the BM as a source of T cells for ACT against solid malignancies. Herein, we demonstrate that T cells from the BM appear superior to TILs as a source of cells for cellular therapy. Specifically, they possess a memory-enriched phenotype and exhibit improved effector function, greater persistence within a tumor-bearing host, and the capacity for increased tumor infiltration. Taken together, these data provide a foundation for further exploring the BM as a source of tumor-specific T cells for ACT in solid malignancies. Key MessagesO_ST_ABSWhat is already known on this topicC_ST_ABSTIL therapy shows efficacy but significant limitations. T cell quality is an important determinant of responses to cellular immunotherapy. What this study addsT cells from the BM appear superior to TILs in phenotype, transcriptional profile, and function. These differences appear driven by tissue (e.g., bone marrow as compared to tumor). How this study might affect research, practice or policyThe BM could serve as an alternative source of cells for adoptive cellular therapy for solid tumors.

immunology↗

bioRxiv2023

Development and validation of a respiratory syncytial virus multiplex immunoassay

Respiratory syncytial virus (RSV) is one of the leading causes of severe respiratory disease in infants and adults. RSV exists as two subtypes A and B, which co-circulate throughout the season, although one will usually become dominant. While vaccines and monoclonal therapeutic antibodies either are or will shortly become available, correlates of protection remain unclear. For this purpose, we developed an RSV multiplex immunoassay that analyses antibody titers towards the post-F, Nucleoprotein, and a diverse mix of G proteins. Technical and clinical validation showed outstanding performance, while methodological developments enabled identification of the subtype of previous infections through use of the diverse G proteins for approximately 50% of samples. As a proof of concept to show the suitability of the assay in serosurveillance studies, we then evaluated titer decay and age- dependent antibody responses within population cohorts. Overall, the developed assay shows robust performance, is scalable, provides additional information on infection subtype, and is therefore ideally suited to be used in future population cohort studies. ImportanceAlthough respiratory syncytial virus (RSV) is endemic and re-infections are common and harmless to the majority of the population, it is a leading cause of hospitalization in young children, the elderly, or immunocompromised individuals. A better characterization of RSV immunology and spreading dynamics is thus critical for preparedness, especially when interventions aiming to mitigate other diseases (e.g., COVID-19) disturb its endemic cycles. This requires high-throughput information-dense assays. We therefore developed a bead-based multiplex immunoassay that allows measurements of antibodies against multiple RSV antigens simultaneously. We identified antibodies which were strong indicators of previous infection, while others allowed identification of the subtype of the previous infection. The assay itself was shown to be robust and scalable, making it ideal for to keep track of the temporal variation RSV immunity profiles within the population.

immunology↗

bioRxiv2023

Immunotherapy of cytomegalovirus infection by low-dose adoptive transfer of antiviral CD8 T cells relies on substantial post-transfer expansion of central memory cells but not effector-memory cells

Cytomegaloviruses (CMVs) are host species-specific in their replication. It is a hallmark of all CMVs that productive primary infection is controlled by concerted innate and adaptive immune responses in the immunocompetent host. As a result, the infection usually passes without overt clinical symptoms and develops into latent infection, referred to as latency. During latency, the virus is maintained in a non-replicative state from which it can reactivate to productive infection under conditions of waning immune surveillance. In contrast, infection of an immunocompromised host causes CMV disease with viral multiple-organ histopathology resulting in organ failure. Primary or reactivated CMV infection of hematopoietic cell transplantation (HCT) recipients in a "window of risk" between therapeutic hematoablative leukemia therapy and immune system reconstitution remains a clinical challenge. Studies in the mouse model of experimental HCT and infection with murine CMV (mCMV), followed by clinical trials in HCT patients with human CMV (hCMV) reactivation, have revealed a protective function of virus-specific CD8 T cells upon adoptive cell transfer (AT). Memory CD8 T cells derived from latently infected hosts are a favored source for immunotherapy by AT. Strikingly low numbers of these cells were found to prevent CMV disease, suggesting either an immediate effector function of few transferred cells or a clonal expansion generating high numbers of effector cells. In the murine model, the memory population consists of resting central memory T cells (TCM), as well as of conventional effector-memory T cells (cTEM) and inflationary effector-memory T cells (iTEM). iTEM, increase in numbers over time in the latently infected host, a phenomenon known as memory inflation (MI). They thus appeared to be a promising source for use in immunotherapy. However, we show here that iTEM contribute little to the control of infection after AT, which rests almost exclusively on a superior proliferation potential of TCM. Author SummaryImmunotherapy of reactivated cytomegalovirus infection in immunocompromised HCT recipients by adoptive transfer (AT) of antiviral CD8 T cells is the last resort to fight virus variants that have acquired resistance to standard antiviral drugs. Provision of cell numbers high enough for clearance of productive infection remains a logistical limitation for AT to become clinical routine. Although use of donor memory CD8 T cells has become the standard in clinical AT, little is known about the relative antiviral efficacies of memory CD8 T-cell activation subsets, such as central memory cells (TCM) and different populations of effector-memory cells (TEM). A reliable quantitative comparison of the antiviral efficacies of memory CD8 T-cell subsets is precluded in clinical investigation, because independent cohorts of AT donors and AT recipients unavoidably differ in many genetical, immunological, and virological variables. Therefore, this is a question for which a preclinical animal model is predestined. We show here in the well-established mouse model of low-dose AT that CMV infection is by far most efficiently controlled by virus-specific TCM, based on a superior potential to proliferate even in extra-lymphoid tissue to prevent virus spread. For clinical AT, our data provide an argument to favor transfer of sorted TCM rather than TEM.

immunology↗

bioRxiv2023

Tick hemocytes have pleiotropic roles in microbial infection and arthropod fitness

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here, we describe immune cells or hemocytes from the clinically relevant tick Ixodes scapularis using bulk and single cell RNA sequencing combined with depletion via clodronate liposomes, RNA interference, Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa) and RNA-fluorescence in situ hybridization (FISH). We observe molecular alterations in hemocytes upon tick infestation of mammals and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We predict distinct hemocyte lineages and reveal clusters exhibiting defined signatures for immunity, metabolism, and proliferation during hematophagy. Furthermore, we perform a mechanistic characterization of two I. scapularis hemocyte markers: hemocytin and astakine. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, which impacts blood feeding and molting behavior of ticks. Hemocytin specifically affects the c-Jun N-terminal kinase (JNK) signaling pathway, whereas astakine alters hemocyte proliferation in I. scapularis. Altogether, we uncover the heterogeneity and pleiotropic roles of hemocytes in ticks and provide a valuable resource for comparative biology in arthropods.

immunology↗

bioRxiv2023

Increased pulmonary monocyte infiltration and attenuated phagocytosis defines perinatal dysfunction of innate immunity in Cystic Fibrosis

In Cystic Fibrosis (CF) patients, cycles of infection and inflammation lead to fatal lung damage. While diminished mucus clearance is restored by highly effective CFTR modulator therapy, inflammation and infection persist in treated patients, suggesting alternative mechanisms may contribute to impaired immunity in CF. Here, we made use of a CF pig model to investigate the innate immune system at birth, before the onset of infection. We observed a substantial change in the composition of tissue resident immune cells towards emergency myelopoiesis, accompanied by increased infiltration of monocytes into CF lungs. A more immature status in the transcriptome profile of classical monocytes in CF pigs and preschool children with CF correlated with reduced phagocytic capacity, confirming a congenital and translationally conserved aberration of the immune system in CF. The lack of CFTR expression in circulating monocytes indicates an indirect etiology of these effects and suggests that additional immunological treatments are necessary for CF patients. One Sentence SummaryIncreased infiltration of lung tissue by monomyeloid cells and their impaired phagocytic potential cause dysfunctional imprinting of mucosal immunity in Cystic Fibrosis airways at birth and suggest early and specific treatment of the innate immune system in patients.

immunology↗

bioRxiv2023

Multi-omic profiling of pathogen-stimulated primary immune cells

We performed long-read transcriptome and proteome profiling of pathogen-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors to discover new transcript and protein isoforms expressed during immune responses to diverse pathogens. Long-read transcriptome profiling reveals novel sequences and isoform switching induced upon pathogen stimulation, including transcripts that are difficult to detect using traditional short-read sequencing. Widespread loss of intron retention occurs as a common result of all pathogen stimulations. We highlight novel transcripts of NFKB1 and CASP1 that may indicate novel immunological mechanisms. RNA expression differences did not result in differences in the amounts of secreted proteins. Clustering analysis of secreted proteins revealed a correlation between chemokine (receptor) expression on the RNA and protein levels in C. albicans- and Poly(I:C)-stimulated PBMCs. Isoform aware long-read sequencing of pathogen-stimulated immune cells highlights the potential of these methods to identify novel transcripts, revealing a more complex transcriptome landscape than previously appreciated.

immunology↗

bioRxiv2023

A monoclonal antibody collection for C. difficile typing

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in adults. Various C. difficile strains circulate currently, associated with different outcomes and antibiotic resistance profiles. However, most studies still focus on the reference strain 630 that does not circulate anymore, partly due to the lack of immunological tools to study current clinically important C. difficile PCR ribotypes. Herein, we immunized mice expressing human variable antibody genes with the Low Molecular Weight (LMW) subunit of the surface layer protein SlpA from various C. difficile strains. Monoclonal antibodies purified from hybridomas bound LMW with high-affinity and whole bacteria from current C. difficile ribotypes with different cross-specificities. This first collection of anti-C. difficile mAbs represent valuable tools for basic and clinical research.

immunology↗

bioRxiv2023

Unveiling the enigma of Brain-resident immune cells

The immune system has been extensively studied in traditional immune hubs like the spleen and lymph nodes. However, recent advances in immunology highlight unique immune cell characteristics across anatomical compartments. In this study, we challenged conventional thinking by uncovering distinct immune cell populations within the brain parenchyma, separate from those in the blood, meninges, and choroid plexus, with unique transcriptional profiles. Brain-resident immune cells are not derived from maternal immune cells, and age-related changes, with an increase in CD8+ T cells in aged mice, are noted. Alzheimers disease (AD) alters microglias interaction with brain-resident immune cells, emphasizing immune-brain dynamics. Furthermore, we reveal dynamic immune cell interactions and essential cytokine roles in brain homeostasis, with stable cytokine expression but emerging signaling pathways in AD. In summary, this study advances our understanding of brain-resident immune cells in both normal and pathological conditions.

immunology↗

bioRxiv2023

Membrane positioning across antigen-induced synaptic contacts tunes CAR-T cell signaling and effector responses

Tumor antigen recognition by chimeric antigen receptors (CAR) triggers phosphorylation of their cytoplasmic portions resulting in CAR-T cell activation. We and others have shown that immunoreceptor triggering depends on the formation of close synaptic contacts, determined by the span of immunoreceptor-ligand complexes, from which large inhibitory phosphatases such as CD45 are sterically excluded. Here, we show, varying CAR-antigen complex span, that CAR-T cell activation depends on a formation of close contacts with target cells. CAR-antigen complexes with a span of 4 immunoglobulin superfamily (IgSF) domains maximize CAR-T cell activation, closely matching the span of endogenous TCR-pMHC complexes. Longer CAR-antigen complexes precipitously reduced triggering and cytokine production, but notably, anti-tumor cytotoxicity was largely preserved due to a [~]10-fold lower signaling threshold for mobilization of cytolytic effector function. Increased intermembrane spacing disrupted short-spanned PD-1-PD- L1 interactions, reducing CAR-T cell exhaustion. Together, our results show that membrane positioning across the immunological synapse can be engineered to generate CAR-T cells with clinically desirable functional profiles in vitro and in vivo.

immunology↗

bioRxiv2023

Multimodal profiling of peripheral blood identifies proliferating circulating effector CD4+ T cells as predictors for response to integrin α4β7-blocking therapy in patients with inflammatory bowel disease

Despite the success of biological therapies in inflammatory bowel disease (IBD), patient management remains challenging due to a lack of therapy response predictors. Here we prospectively sampled two cohorts of IBD patient cohorts receiving the anti-integrin 4{beta}7 antibody vedolizumab. Samples were subjected to mass cytometry, single-cell RNA sequencing, single-cell V(D)J sequencing, serum proteomics, and multidimensional flow cytometry to comprehensively assess vedolizumab-induced immunological changes in the peripheral blood and their potential associations with treatment response. Vedolizumab induced changes in the abundance of both circulating innate and adaptive immune cell compartments and modified the T cell receptor diversity of circulating gut-homing CD4+ memory T cells. Through integration of multimodal parameters and machine learning, we identify that pretreatment activated proliferating CD4+ memory T cell abundance is associated with treatment failure, independent of clinical variables, thereby providing a reliable predictive classifier with significant implications for the personalized management of IBD patients.

immunology↗

bioRxiv2023

Inflammatory Profiles Induced following Intranasal Vaccination with Ricin Toxin-Immune Complexes

Ricin toxin (RT), a ribosome-inactivating protein derived from castor beans (Ricinus communis), is lethal in extremely small doses via inhalation, due in part to pulmonary inflammation triggeredby the toxin. While no licensed countermeasures for RT exists, a myriad of toxin-neutralizing monoclonal antibodies (mAbs) against RTs enzymatic (RTA) and binding (RTB) subunits have been described, including PB10 (anti-RTA) and SylH3 (anti-RTB). We recently demonstrated that a single administration of PB10 and SylH3 as an RT immune complex (RIC) to mice by the intranasal route functions as a potent immunogen, stimulating the rapid onset of high titer toxin-neutralizing antibodies that persist for months. In this report, we demonstrate that the de novo antibody response following RICs exposure is wholly dependent on CD4+ T cell help, as treatment of mice with an anti-CD4 monoclonal antibody abrogated the onset of RT-specific antibodies. To investigate other immunological factors associated with RICs, we examined the early local and systemic inflammatory cytokine and chemokine responses associated with intranasal RT and RIC administration. We report that RICs stimulate an inflammatory profile in the bronchoalveolar lavage fluids (BALF) and serum similar to that elicited by RT itself, but markedly reduced in magnitude. Of the 32 analytes examined, the most pronounced were IL-6, KC (CXCL1), G-CSF, and GM-CSF. To evaluate the role of IL-6 in driving the de novo onset of RT antibodies, groups of mice were treated (or not) with an IL-6 neutralizing monoclonal antibody prior to intranasal RICs challenge. While the monoclonal antibody treatment successfully depleted circulating IL-6 to levels below detection, it had no impact on the kinetics or magnitude of RICs-induced RT-specific antibody response. Thus, our results suggest that other cytokines/chemokines triggered by RICs are involved in influencing long lasting durable antibody responses to RT.

immunology↗

bioRxiv2023

Innate immune memory after brain injury drives inflammatory cardiac dysfunction

The enormous medical burden of stroke is not only due to the brain injury itself and the acute systemic effects, but is largely determined by chronic comorbidities that develop secondarily after stroke. We hypothesized that the high rate of comorbidity developing after a stroke might have a shared immunological cause, however, the chronic effects of brain injury on systemic immunity have so far been barely investigated. Here, we identified myeloid innate immune memory as a cause of remote organ dysfunction after stroke. Using single-cell sequencing, we identified persistent pro-inflammatory transcriptomic changes in resident monocytes/macrophages in multiple organs one month after experimental ischemic brain injury, which was particularly abundant in the heart and associated with the development of cardiac fibrosis and diastolic dysfunction. A similar phenotype was seen in myocardial autopsy samples from stroke versus control patients. We observed chronic functional changes in myeloid hematopoiesis driven by post-stroke IL-1{beta}-mediated epigenetic changes. These alterations could be transplanted to naive recipient mice and were sufficient to induce cardiac dysfunction. By effectively blocking the trafficking of pro-inflammatory monocytes from the bone marrow to the heart using a dual CCR2/5 inhibitor, we successfully prevented post-stroke cardiac dysfunction. This approach holds promising potential as a novel immune-targeted secondary prevention therapy. We anticipate that the epigenetic immune reprogramming mechanisms detailed here for the brain-heart axis could be generalized to provide a novel framework for explaining the development of various comorbidities after acute tissue injury in remote organs.

immunology↗

bioRxiv2023

Time- and lineage-resolved transcriptional profiling uncovers gene expression programs and clonal relationships that underlie human T lineage specification

T cells develop from multi-potent hematopoietic progenitors in the thymus and provide adaptive protection against pathogens and cancer. However, the emergence of human T cell-competent blood progenitors, and their subsequent specification to the T lineage, has been challenging to capture in real time. Here, we leveraged a pluripotent stem cell differentiation system to understand the transcriptional dynamics and cell fate restriction events that underlie this critical developmental process. Time-resolved single cell RNA sequencing revealed that cell-cycle exit, downregulation of the multipotent hematopoietic program, and upregulation of >90 lineage-associated transcription factors all occur within a highly co-ordinated and narrow developmental window. Computational gene-regulatory network inference elucidated the transcriptional logic of T lineage specification, uncovering an important role for YBX1. We mapped the differentiation cell fate hierarchy using transcribed lineage barcoding and mathematical trajectory inference and discovered that mast and myeloid potential bifurcate from each other early in haematopoiesis, upstream of T lineage restriction. Collectively, our analyses provide a quantitative, time-resolved model of human T cell specification with relevance for regenerative medicine and developmental immunology.

immunology↗

bioRxiv2023

Single-cell RNA sequencing unveils the hidden powers of zebrafish kidney for generating both hematopoiesis and adaptive antiviral immunity

The vertebrate kidneys play two evolutionary conserved roles in waste excretion and osmoregulation. Besides, the kidney of fish is considered as a functional ortholog of mammalian bone marrow that serves as a hematopoietic hub for generating blood cell lineages and immunological responses. However, knowledge about the properties of kidney hematopoietic cells, and the functionality of kidney in fish immune systems remain to be elucidated. To this end, our present study generated a comprehensive atlas with 59 hematopoietic stem/progenitor cell (HSPC) and immune-cell types from zebrafish kidney via single-cell transcriptome profiling analysis. These populations included almost all known cells associated with innate and adaptive immunity, and displayed differential responses to viral infection, indicating their diverse functional roles in antiviral immunity. Remarkably, HSPCs were found to have extensive reactivities to viral infection, and the trained immunity can be effectively induced in certain HSPCs. In addition, the antigen-stimulated adaptive immunity can be fully generated in kidney, suggesting kidney acting as a secondary lymphoid organ. These results indicated that fish kidney is a dual-functional entity with functionalities of both primary and secondary lymphoid organs. Our findings illustrated the unique features of fish immune system, and highlighted the multifaced biology of kidney in ancient vertebrates.

immunology↗

bioRxiv2023

Mapping lung hematopoietic progenitors: Developmental kinetics and response to Influenza A viral infection

The bone marrow is a specialised niche responsible for the maintenance of hematopoietic stem and progenitor cells during homeostasis and inflammation. Recent studies however have extended this essential role to the extramedullary and extravascular lung microenvironment. Here, we provide further evidence for a reservoir of hematopoietic stem and progenitor cells within the lung from embryonic day 18.5 until adulthood. These lung progenitors display distinct microenvironment-specific developmental kinetics compared to their bone marrow counterparts, exemplified by a rapid shift from a common myeloid to megakaryocyte-erythrocyte progenitor dominated niche with increasing age. In adult mice, Influenza A viral infection results in a transient reduction in multipotent progenitors within the lungs, with a parallel increase in downstream granulocyte-macrophage progenitors and dendritic cell populations associated with acute viral infections. Our findings suggest lung hematopoietic progenitors play a role in re-establishing immunological homeostasis in the respiratory mucosa, which may have significant clinical implications for maintaining pulmonary health following inflammatory perturbation.

immunology↗

bioRxiv2023

Ultrasensitive allele inference from immune repertoire sequencing data with MiXCR

Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), has been shown to be of critical importance for immune responses to pathogens and vaccines. In recent years, B cell and T cell receptor repertoire sequencing (Rep-Seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci in different populations. Here we present a novel algorithm for extra-sensitive and specific variable (V) and joining (J) gene allele inference and genotyping allowing reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput genotyping and novel allele discovery from a wide variety of existing datasets. The developed algorithm is a part of the MiXCR software (https://mixcr.com) and can be incorporated into any pipeline utilizing upstream processing with MiXCR. We demonstrate the accuracy of this approach using Rep-Seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) Rep-Seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA; TRB) Rep-Seq dataset, representing 134 individuals. This allowed us to assess the genetic diversity of genes within the IGH, TRA and TRB loci in different populations and demonstrate the connection between antibody repertoire gene usage and the number of allelic variants present in the population. Finally we established a database of allelic variants of V and J genes inferred from Rep-Seq data and their population frequencies with free public access at https://vdj.online.

immunology↗