Pan-cancer multi-omics analyses reveal crosstalk between the Hippo and immune signaling pathways in the tumor microenvironment.

The Hippo signaling pathway is critical for carcinogenesis. However, the roles of the Hippo signaling pathway in the tumor immune microenvironment have been rarely investigated. This study systematically analyzed the relationship between the Hippo signaling pathway and immune cell infiltration across 32 cancer types. Both bioinformatics analyses and biological experiments revealed that the downstream effector of Hippo signaling YAP1 might inhibit CD8+ T cell infiltration by upregulating the expression of the transcription factor CREB1 in uterine corpus endometrial carcinoma. In addition, esophageal carcinoma (ESCA) patients were classified into three subtypes based on the Hippo-immune gene panel. The subtypes of ESCA had distinct characteristics in immune cell infiltration, immune pathways, and prognosis. Thus, this study also reveals a new classification of the immune subtypes with prognostic characteristics in ESCA.

huARdb: human Antigen Receptor database for interactive clonotype-transcriptome analysis at the single-cell level.

T-cell receptors (TCRs) and B-cell receptors (BCRs) are critical in recognizing antigens and activating the adaptive immune response. Stochastic V(D)J recombination generates massive TCR/BCR repertoire diversity. Single-cell immune profiling with transcriptome analysis allows the high-throughput study of individual TCR/BCR clonotypes and functions under both normal and pathological settings. However, a comprehensive database linking these data is not yet readily available. Here, we present the human Antigen Receptor database (huARdb), a large-scale human single-cell immune profiling database that contains 444 794 high confidence T or B cells (hcT/B cells) with full-length TCR/BCR sequence and transcriptomes from 215 datasets. All datasets were processed in a uniform workflow, including sequence alignment, cell subtype prediction, unsupervised cell clustering, and clonotype definition. We also developed a multi-functional and user-friendly web interface that provides interactive visualization modules for biologists to analyze the transcriptome and TCR/BCR features at the single-cell level. HuARdb is freely available at https://huarc.net/database with functions for data querying, browsing, downloading, and depositing. In conclusion, huARdb is a comprehensive and multi-perspective atlas for human antigen receptors.

A Bayesian phylogenetic hidden Markov model for B cell receptor sequence analysis.

The human body generates a diverse set of high affinity antibodies, the soluble form of B cell receptors (BCRs), that bind to and neutralize invading pathogens. The natural development of BCRs must be understood in order to design vaccines for highly mutable pathogens such as influenza and HIV. BCR diversity is induced by naturally occurring combinatorial "V(D)J" rearrangement, mutation, and selection processes. Most current methods for BCR sequence analysis focus on separately modeling the above processes. Statistical phylogenetic methods are often used to model the mutational dynamics of BCR sequence data, but these techniques do not consider all the complexities associated with B cell diversification such as the V(D)J rearrangement process. In particular, standard phylogenetic approaches assume the DNA bases of the progenitor (or "naive") sequence arise independently and according to the same distribution, ignoring the complexities of V(D)J rearrangement. In this paper, we introduce a novel approach to Bayesian phylogenetic inference for BCR sequences that is based on a phylogenetic hidden Markov model (phylo-HMM). This technique not only integrates a naive rearrangement model with a phylogenetic model for BCR sequence evolution but also naturally accounts for uncertainty in all unobserved variables, including the phylogenetic tree, via posterior distribution sampling.

Adaptive immune responses to SARS-CoV-2 infection in severe versus mild individuals.

The global Coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has affected more than eight million people. There is an urgent need to investigate how the adaptive immunity is established in COVID-19 patients. In this study, we profiled adaptive immune cells of PBMCs from recovered COVID-19 patients with varying disease severity using single-cell RNA and TCR/BCR V(D)J sequencing. The sequencing data revealed SARS-CoV-2-specific shuffling of adaptive immune repertories and COVID-19-induced remodeling of peripheral lymphocytes. Characterization of variations in the peripheral T and B cells from the COVID-19 patients revealed a positive correlation of humoral immune response and T-cell immune memory with disease severity. Sequencing and functional data revealed SARS-CoV-2-specific T-cell immune memory in the convalescent COVID-19 patients. Furthermore, we also identified novel antigens that are responsive in the convalescent patients. Altogether, our study reveals adaptive immune repertories underlying pathogenesis and recovery in severe versus mild COVID-19 patients, providing valuable information for potential vaccine and therapeutic development against SARS-CoV-2 infection.

Proteogenomics for the Comprehensive Analysis of Human Cellular and Serum Antibody Repertoires.

The vast repertoire of immunoglobulins produced by the immune system is a consequence of the huge amount of antigens to which we are exposed every day. The diversity of these immunoglobulins is due to different mechanisms (including VDJ recombination, somatic hypermutation, and antigen selection). Understanding how the immune system is capable of generating this diversity and which are the molecular bases of the composition of immunoglobulins are key challenges in the immunological field. During the last decades, several techniques have emerged as promising strategies to achieve these goals, but it is their combination which appears to be the fruitful solution for increasing the knowledge about human cellular and serum antibody repertoires.In this chapter, we address the diverse strategies focused on the analysis of immunoglobulin repertoires as well as the characterization of the genomic and peptide sequences. Moreover, the advantages of combining various -omics approaches are discussed through review different published studies, showing the benefits in clinical areas.

Estimate of within population incremental selection through branch imbalance in lineage trees.

Incremental selection within a population, defined as limited fitness changes following mutation, is an important aspect of many evolutionary processes. Strongly advantageous or deleterious mutations are detected using the synonymous to non-synonymous mutations ratio. However, there are currently no precise methods to estimate incremental selection. We here provide for the first time such a detailed method and show its precision in multiple cases of micro-evolution. The proposed method is a novel mixed lineage tree/sequence based method to detect within population selection as defined by the effect of mutations on the average number of offspring. Specifically, we propose to measure the log of the ratio between the number of leaves in lineage trees branches following synonymous and non-synonymous mutations. The method requires a high enough number of sequences, and a large enough number of independent mutations. It assumes that all mutations are independent events. It does not require of a baseline model and is practically not affected by sampling biases. We show the method's wide applicability by testing it on multiple cases of micro-evolution. We show that it can detect genes and inter-genic regions using the selection rate and detect selection pressures in viral proteins and in the immune response to pathogens.

The role of BCR isotype in B-cell development and activation.

The development and function of B lymphocytes critically depend on the non-germline B-cell antigen receptor (BCR). In addition to the diverse antigen-recognition regions, whose coding sequences are generated by the somatic DNA rearrangement, the variety of the constant domains of the Heavy Chain (HC) portion contributes to the multiplicity of the BCR types. The functions of particular classes of the HC, particularly in the context of the membrane BCR, are not completely understood. The expression of the various classes of the HC correlates with the distinct stages of B-cell development, types of B-cell subsets, and their effector functions. In this chapter, we summarize and discuss the accumulated knowledge on the role of the µ, δ, and γ HC isotypes of the conventional and precursor BCR in B-cell differentiation, selection, and engagement with (auto)antigens.

Qualifying high-throughput immune repertoire sequencing.

Diversity of B and T cell receptors, achieved by gene recombination and somatic hypermutation, allows the immune system for recognition and targeted reaction against various threats. Next-generation sequencing for assessment of a cell's gene composition and variation makes deep analysis of one individual's immune spectrum feasible. An easy to apply but detailed analysis and visualization strategy is necessary to process all sequences generated. We performed sequencing utilizing the 454 system for CLL and control samples, utilized the IMGT database and applied the presented analysis tools. With the applied protocol, malignant clones are found and characterized, mutational status compared to germline identity is elaborated in detail showing that the CLL mutation status is not as monoclonal as generally thought. On the other hand, this strategy is not solely applicable to the 454 sequencing system but can easily be transferred to any other next-generation sequencing platform.

IgG-switched CLL has a distinct immunogenetic signature from the common MD variant: ontogenetic implications.

PURPOSE: Immunoglobulin G-switched chronic lymphocytic leukemia (G-CLL) is a rare variant of CLL, whose origin and ontogenetic relationship to the common IgM/IgD (MD-CLL) variant remains undefined. Here, we sought for clues about the ontogeny of G-CLL versus MD-CLL by profiling the relevant IG gene repertoires. EXPERIMENTAL DESIGN: Using purpose-built bioinformatics methods, we performed detailed immunogenetic profiling of a multinational CLL cohort comprising 1,256 cases, of which 1,087 and 169 expressed IG mu/delta and gamma heavy chains, respectively. RESULTS: G-CLL has a highly skewed IG gene repertoire that is distinct from MD-CLL, especially in terms of (i) overuse of the IGHV4-34 and IGHV4-39 genes and (ii) differential somatic hypermutation (SHM) load. Repertoire differences were also found when comparing subgroups with similar SHM status and were mainly attributed to the exclusive representation in G-CLL of two major subsets with quasi-identical (stereotyped) B-cell receptors. These subsets, namely #4 (IGHV4-34/IGKV2-30) and #8 (IGHV4-39/IGKV1(D)-39), were found to display sharply contrasting SHM and clinical behavior. CONCLUSIONS: G-CLL exhibits an overall distinct immunogenetic signature from MD-CLL, prompting speculations about distinct ontogenetic derivation and/or immune triggering. The reasons underlying the differential regulation of SHM among G-CLL cases remain to be elucidated.

Using synthetic templates to design an unbiased multiplex PCR assay.

T and B cell receptor loci undergo combinatorial rearrangement, generating a diverse immune receptor repertoire, which is vital for recognition of potential antigens. Here we use a multiplex PCR with a mixture of primers targeting the rearranged variable and joining segments to capture receptor diversity. Differential hybridization kinetics can introduce significant amplification biases that alter the composition of sequence libraries prepared by multiplex PCR. Using a synthetic immune receptor repertoire, we identify and minimize such biases and computationally remove residual bias after sequencing. We apply this method to a multiplex T cell receptor gamma sequencing assay. To demonstrate accuracy in a biological setting, we apply the method to monitor minimal residual disease in acute lymphoblastic leukaemia patients. A similar methodology can be extended to any adaptive immune locus.

Association of protein kinase C-delta with the B cell antigen receptor complex.

Protein kinase C (PKC)-delta is a diacylglycerol-dependent, calcium-independent novel PKC isoform and has been demonstrated to exert negative regulatory functions in B lymphocytes as well as in mast cells. Whereas in mast cells PKC-delta functionally interacts with the high-affinity receptor for IgE, FcepsilonR1, no such association has been described for the B cell antigen receptor (BCR). In this report, for the first time, we demonstrate the interaction of PKC-delta with different classes of BCR by means of affinity purification and native protein complex analysis. Using a C-terminally truncated Ig-alpha as well as non-phosphorylated and phosphorylated peptides representing C-terminal regions of Ig-alpha, the dependence of this BCR/PKC-delta interaction on tyrosine-phosphorylated Ig-alpha is shown. Finally, splenocytes from PKC-delta-deficient mice are found to exert reduced phosphorylation of PKD (a.k.a. PKC-mu) in response to BCR engagement, suggesting the early, membrane-proximal activation of an attenuating kinase complex including PKC-delta and PKD.

Receptor revision of immunoglobulin heavy chain variable region genes in normal human B lymphocytes.

Contrary to the general precepts of the clonal selection theory, several recent studies have provided evidence for the secondary rearrangement of immunoglobulin (Ig) genes in peripheral lymphoid tissues. These analyses typically used transgenic mouse models and have only detected secondary recombination of Ig light chain genes. Although Ig heavy chain variable region (V(H)) genes encode a substantial element of antibody combining site specificity, there is scant evidence for V(H) gene rearrangement in the periphery, leaving the physiological importance of peripheral recombination questionable. The extensive somatic mutations and clonality of the IgD(+)Strictly-IgM(-)CD38(+) human tonsillar B cell subpopulation have now allowed detection of the first clear examples of receptor revision of human V(H) genes. The revised VDJ genes contain "hybrid" V(H) gene segments consisting of portions from two separate germline V(H) genes, a phenomenon previously only detected due to the pressures of a transgenic system.

Surface Ig on B lymphocytes from cattle and sheep.

IgD, first demonstrated in humans, has been unequivocally shown to exist in primates and rodents. In addition to IgM a second unique membrane isotype, generally considered to be IgD, has been demonstrated in a number of other species, including dogs and chickens. Because of its assumed widespread presence, it is widely accepted that IgD is phylogenetically conserved and therefore functionally important in B cell maturation. In the present paper, we could not demonstrate IgD on bovine B cells derived from peripheral blood, lymph nodes, spleen and fetal spleen by precipitation with anti-light chain antibodies. This lack of detectable IgD was confirmed in peripheral blood B cells of sheep, and raises questions on the requirement for IgD in cell differentiation and Ig secretion. At present it is not clear whether cattle (and sheep) are an exception in this context. Reports of the presence of IgD in different species are largely based on the assumption that non-IgM surface Ig is most likely IgD. Our data question this extrapolation and stress the need for further isotype characterization of the surface Ig in different species. Lack of surface IgD has been observed in human and mouse B-1 cells, most of which express the surface marker CD5. The possibility that all bovine B cells belong to the B-1 lineage is discussed.

Function of B-cell antigen receptor of different classes.

Most mature B lymphocytes co-express two classes of antigen receptor, IgM and IgD. The differences in the signal transduction from the 2 receptors are still a matter of controversy. We have analysed B-cell lines expressing IgM or IgD antigen receptors with the same antigen specificity. Cross-linking of these receptors with either antigen or class-specific antibodies results in the activation of protein tyrosine kinases and the phosphorylation of the same substrate proteins. The kinetics and intensity of phosphorylation, however, were quite different between the 2 receptors when they were cross-linked by antigen. In membrane IgM-expressing cells, the substrate phosphorylation reached a maximum already after 1 min and diminished after 60 min whereas in the membrane IgD-expressing cells, the substrate phosphorylation increases further over time, reached its maximum at 60 min and persisted longer than 240 min after exposure to antigen. Recently prolonged signaling has been found to be responsible for signaling differences between tyrosine kinase receptors using otherwise similar signaling routes. Thus, the duration of a signal may be an important biological feature of signal transducing cascades.

[Immunoglobulin G subclass distribution of anti-intercellular substance antibodies in pemphigus]. / Distribution en sous-classes d'immunoglobulines G des anticorps anti-substance intercellulaire du pemphigus.

INTRODUCTION: The purpose of this study was to analyze the IgG subclass distribution of pemphigus anti-epithelial cell surface (ECS) antibodies and to determine whether it differs according to clinical features. MATERIALS AND METHODS: 25 skin biopsies and 16 serum samples, obtained from 27 cases of pemphigus, were analyzed by direct and indirect IF staining, with mice anti-human IgG subclasses monoclonal antibodies. RESULTS: IgG1 deposits were observed in 21 of 25, IgG2 in 2, IgG3 in 0, and IgG4 in the 25 biopsies. IgG1 anti-ECS anti-ECS antibodies were detected in all 16 sera, IgG2 in 1, IgG3 in 1, and IgG4 in 15 sera. The anti-ECS IgG subclass distribution does not differ according to the clinical parameters studied. DISCUSSION: The isotypic restriction to IgG1 and IgG4 subclasses, observed in this study, is similar to previously reported results. The heterogenous distribution and the small number of the studied samples did not allow to put in evidence a correlation with the clinical parameters.

IgG+, CD5+ human chronic lymphocytic leukemia B cells. Production of IgG antibodies that exhibit diminished autoreactivity and IgG subclass skewing.

Several questions exist regarding CD5+ B cells. These include the ability of these cells, as compared to CD5- B cells, to undergo an Ig isotype class switch, the subclasses utilized, and the effects that switching may have on antigen binding. To address these issues, ten patients with chronic lymphocytic leukemia (CLL) whose CD5+ leukemic B cell clones produced IgG were studied. Monoclonal IgG was collected from PMA-stimulated CLL cells and from heterohybridomas constructed with these cells, and then analyzed for IgG subclass utilization, autoreactivity, and DNA idiotype expression. The monoclonal B cells from 80% of the CLL patients produced IgG1 and those from 20% produced IgG3. None produced IgG2. In contrast to the known autoreactivity of IgM-producing CD5+ CLL cells (> 50% autoreactive), none of these IgG antibodies reacted significantly with the autoantigens tested. However, three did react significantly with autoantigen after artificially increasing antibody valency by crosslinking. Whereas five of the IgG molecules expressed a cross reactive idiotypic (CRI) marker characteristic of non-mutated kappa anti-DNA antibodies, three expressed a CRI displayed primarily on mutated IgG anti-DNA antibodies. Thus, some CD5+ human B cells can undergo an isotype class switch that for these CLL cells is biased against IgG2 and in favor of the IgG1 and IgG3. In their native state the IgG molecules secreted by these isotype-switched CD5+ cells have diminished autoreactivity, as compared to IgM-producing CLL cells. Since some of the IgG antibodies could be made auto- and poly-reactive by increasing antigen-binding valency, while others expressed idiotypic markers of mutated antibodies, certain of these CD5+ B cells probably utilize non-mutated Ig V genes coding for polyreactive antibodies, whereas others may use genes that have undergone somatic mutation and that code for more restricted specificities. Therefore, both valency and VH gene mutation may account for the diminished autoreactivity of these CD5+ B cell-derived IgG antibodies.

Blood lymphocyte characteristics as predictors of prognosis in chronic lymphocytic leukemia of B-cell type.

The prognostic information of blood lymphocyte characteristics and clinical findings was assessed in 62 patients with chronic lymphocytic leukemia of B cell type. Bivariate and multivariate survival analyses were performed using age, Rai stage, surface membrane immunoglobulin (smIg) isotype pattern of the leukemic clone, total lymphocyte counts, numbers of proliferating lymphocytes and T cell subpopulations. Rai stages III and IV, high numbers of blood lymphocytes in S-phase (S+) and sm mu isotype were found to be partly independent factors predicting short therapy-free survival. Patients with a sm mu+ leukemic cell clone had a shorter therapy-free and total survival compared to those with sm mu+/delta+ and sm delta+ leukemic cells. Moreover, patients with high numbers of blood S+ lymphocytes had a shorter therapy-free and total survival compared to those with few S+ cells. These prognostic variables were valid also in patients with a low tumour burden (Rai stages 0, I and II) and may thus be of clinical importance as a guideline for therapeutic intervention.

Prognostic significance of immunologic phenotype in hairy cell leukemia: does it exist?

Of 94 hairy cell leukemia (HCL) patients studied for immunologic phenotype of their hairy cells, 89 patients had B cell markers and five patients were both surface immunoglobulin (slg) negative and E rosette negative. Forty of the 77 cases that had the heavy chains individually determined had IgG only (52.0%); 23 others had IgG in addition to other Ig heavy chains. Seventy-nine patients had monoclonal light chains; 65 with kappa chain and 14 with lambda chain. The only significant difference with respect to survival among the various slg groups occurred between the kappa chain and the lambda chain groups. Within the first 46 months after diagnosis of HCL, 20 deaths occurred among the 65 kappa chain patients, whereas the first and only death among the lambda chain patients occurred at 68 months after diagnosis. The only clinical or laboratory parameter that was significantly different between these two immunologic subgroups was the incidence of infections. Among the lambda chain patients, an infectious complication rate of 28.6% was observed subsequent to the diagnosis of HCL, whereas this rate was 68.8% in kappa chain patients (P = .005). The survival of lambda patients was found to exceed that of the kappa patients by the generalized Wilcoxon test (P = .03). However, when the log rank test was used, no significant difference was detected (P = .13).