Molecular Immunology

In teleost fishes there are three immunoglobulin isotypes named immunoglobulin M (IgM), D (IgD) and T (IgT). IgT has been the last to be described and is considered a teleosts-fish specific isotype. From the recent availability of genome sequences of fishes, an in-depth analysis of Actinopterygii immunoglobulin heavy chain genes was undertaken. With the aid of a bioinformatics pipeline, a machine learning software, CHfinder, was developed that identifies the coding exons of the CH domains of fish immunoglobulins. Using this pipeline, a high number of such sequences were obtained from teleosts and holostean fishes. IgT was found in teleost and holostean fishes that had not been previously described. A phylogenetic analysis reveals that IgT CH1 exons are similar to the IgM CH1. This analysis also demonstrates that the other three domains (CH2, CH3 and CH4) were not generated by recent duplication processes of IgM in Actinopterygii, indicating it is an immunoglobulin with an earlier origin.

SATB1 (Special A-T rich Binding protein 1) is a cell type specific factor involved in chromatin remodelling events that participate in the regulation of the genetic network in developing T cells and neurons. In T cells, SATB1 is a key factor required for lineage commitment, VDJ recombination, development and maturation. In B cells, SATB1 is described as binding to the MARs-E{micro} regions of the IgH locus. Considering that its expression varies during differentiation, the involvement of this factor needed to be clarified in B cells. Using a KO mouse model deleting SATB1 from the pro-B cell stage, we were able to examine the consequences of SATB1 deletion in naive and activated B cell subsets. Our model indicates firstly that SATB1 is not essential for B cell development and the establishment of a broad IgH repertoire. Second, we show that this factor exhibits an ambivalent function in mature B cells, acting sequentially as a positive and negative regulator of Ig gene transcription in naive and activated cells, respectively. Third, our study indicates that the negative regulatory function of SATB1 in B cells extends to the germinal center response in which this factor limits somatic hypermutation of Ig genes. This finding suggests that SATB1 may limit the introduction of unwanted mutations into B cells.

Genes of the major class I and II histocompatibility complex have been extensively studied in mammals. Studies of these antigens in reptiles are very scarce. Here we describe the characteristics of these genes in the suborder Serpentes. We identified the presence of a much larger number of molecules of class I and beta chains of class II than found in mammals. Snakes only have one gene for the class II alpha chain. In these species, class I genes can be classified into two types. Approximately half of the genes lack 10 amino acids in the 1 domain, producing a structural alteration in the interaction region with the T lymphocyte receptor. In the genome of Thamnophis elegans, two haplotypes of an individual were studied revealing a different number and location of class I genes between these haplotypes. The results indicate that in these species, the diversity in the MHC is generated by the presence or absence of genes, independent of the presence of alleles.

Understanding how the evolutionary relationship between immune cells and the blood-brain is important to devise therapeutic strategies that can regulate their critical function. In vertebrates, immune cells follow either a paracellular or transcellular pathway to infiltrate the BBB. In drosophila glial cells form the BBB that regulates the access of immune-like cells to the drosophila brain. However, it is still not known which route immune-like cells follow to infiltrate the drosophila brain. In vertebrates, paracellular migration is dependent on PECAM1, while transcellular migration is dependent on the expression of CAV1. Interestingly drosophila genome lacks both genes. Tre1 superfamily (Tre1, Moody, and Dmel_CG4313) play a diverse role in regulating transepithelial migration in drosophila. However, its evolutionary history and origin are not yet known. We performed phylogenetic analysis, together with HH search, positive selection, and ancestral reconstruction to investigate the Tre1 family Interestingly we found that Tre1 exists in mollusks, insects, ambulacria, and sclaidphora. Moody is shown to be a more ancient protein and it existed since cnidaria emergence and has a homolog (GPCR84) in mammals. The third family member (Dmel_CG4313) only exists in insects. The origin of the family seems to be related to the rhodopsin-like family and in particular family . We found that opsin is the nearest receptor to have a common ancestor with the Tre1 superfamily that seems to have diverged in sponges. We investigated the positive selection of the Tre1 family using PAML. Tre1 seems to have evolved under negative selection, whereas Moody has evolved during positive selection. The sites that we found under positive selection are Likely to play a role in the speciation of function in the case of Moody. We have identified an SH3, in Tre1 and, moody and Dmel_CG4313. Sh3 is known to play a fundamental role in regulating actin movement in a Rho-dependent manner. We suggest that Tre1 could be playing an important role in paracellular diapedesis in drosophila.

In human tumor models we tried to identify and clone the TCR of tumor-reactive T cells enriched in mixed lymphocyte tumor-cell cultures (MLTC). In a particular MLTC, we identified a predominant TCR beta chain using the Beta Mark Vbeta Kit, but a corresponding alpha chain could not be amplified via RT-PCR using TRAV-specific forward primers. Therefore, we applied 5RACE to obtain the TCR alpha chain sequences. The 5RACE product revealed an alpha chain that encompassed 89bp of the TRDV1 5UTR, followed by the TRDV1 coding sequence joined in frame to TRAJ24. The ORF reaching from the TRDV1 start codon to the TRAC segment was intact, suggesting a functional TCR. To analyze this MLTC population in greater depth we conducted 10X VDJ sequencing. CellRanger identified the beta chain known from the Beta Mark analysis, but no corresponding alpha chain in the filtered results. The corresponding TRDV-containing TCR alpha chain could, however, be detected in the "all_contig_annotations" files. In a separate project, we performed TCR sequencing of tumor-infiltrating lymphocytes (TILs) in a murine tumor model. Also here, a predominant clonotype contained a TCR alpha chain joining Trdv2-2 in frame to Traj49. Transfection of both TCR cDNAs resulted in cell surface localization of TCR and CD3 as validated by FACS. Tumor recognition of the human, TRDV1-containing TCR could be demonstrated by IFNgamma ELISpot whereas the murine TCR did not recognize a tumor-derived cell line. TRDV-containing alpha chains have been reported in the literature for two HLA I-restricted TCRs against HIV peptides (Ueno et al, Eur J Immunol, 2003). To determine whether such TDRV-containing TCRs are unique events or whether Vdelta segments are commonly incorporated into TCR alpha chains, we queried the NCBI Sequence Read Archive (SRA) for 10X VDJ data and analyzed 21 human and 23 murine datasets. We found that especially TRDV1, Trdv1 and to some extent Trdv2-2 are more commonly incorporated into TCR alpha chains than some TRAV genes, making the TRDV segments a relevant contribution to TCR alpha diversity. For apparently solitary beta chains in 10X VDJ datasets, we suggest to scrutinize the "all_contig_annotations" files as these may contain an accompanying, TRDV-containing alpha chain.

Glycogen synthase kinase 3 (GSK3) is a ubiquitously expressed kinase involved in a myriad of biological processes. Although GSK3 mediated phosphorylation has been shown to induce the degradation of many pro-survival and pro-proliferation factors, cancer cells of different origin show reduced proliferation or survival after GSK3 inhibition. Our current understanding of the role GSK3 plays in normal mature B cells, B cell precursors and transformed B cells is incomplete and does not allow to assess whether GSK3 inhibitors can be used to treat B cell derived malignancies. Here we identify {beta}-catenin as the major factor driving GSK3-inhibition induced changes in B cells. We show that {beta}-catenin accumulation has opposing effects on cell metabolism and survival in mature B cells and B cell precursors. Moreover, we demonstrate that {beta}-catenin destabilizes the commitment to the B cell lineage. In summary, our study identifies {beta}-catenin induced signaling as a factor that can be exploited to limit the survival of malignant B cells.

B cells play a central role in adaptive immune processes, mainly through the production of antibodies. The maturation of the B-cell system with age is poorly studied. We extensively investigated age-related alterations of naive and antigen-experienced B-cell receptor (BCR) repertoires. The most significant changes were observed in the first 10 years of life, and were characterized by altered immunoglobulin gene usage and an increased frequency of mutated antibodies structurally diverging from their germline precursors. Older age was associated with an increased usage of downstream constant region genes and fewer antibodies with self-reactive properties. As mutations accumulated with age, the frequency of germline-encoded self-reactive antibodies decreased, indicating a possible beneficial role of self-reactive B-cells in the developing immune system. Our results suggest a continuous process of change through childhood across a broad range of parameters characterizing BCR repertoires and stress the importance of using well-selected, age-appropriate controls in BCR studies.

B cell development requires the ordered rearrangement of Ig genes encoding H and L chain proteins that assemble into BCRs or Abs capable of recognizing specific Ags. Ig{kappa} rearrangement is promoted by chromatin accessibility and by relative abundance of RAG1/2 proteins. Expression of the E26-transformation-specific (ETS) transcription factor Spi-C is activated in response to double-stranded DNA breaks (DSBs) in small pre-B cells to negatively regulate pre-BCR signaling and Ig{kappa} rearrangement. However, it is not clear if Spi-C regulates Ig{kappa} rearrangement through transcription or by controlling RAG expression. In this study, we investigated the mechanism of Spi-C negative regulation of Ig{kappa} light chain rearrangement. Using an inducible expression system in a pre-B cell line, we found that Spi-C negatively regulated Ig{kappa} rearrangement, Ig{kappa} transcript levels, and Rag1 transcript levels. We found that Ig{kappa} and Rag1 transcript levels were increased in small pre-B cells from Spic-/- mice. In contrast, Ig{kappa} and Rag1 transcript levels were activated by PU.1 and were decreased in small pre-B cells from PU.1-deficient mice. Using chromatin immunoprecipitation analysis, we identified an interaction site for PU.1 and Spi-C located in the Rag1 promoter region. These results suggest that Spi-C and PU.1 counterregulate Ig{kappa} transcription and Rag1 transcription to effect Ig{kappa} recombination in small pre-B cells.

CD20 is a B cell-specific four-helix transmembrane protein and a prominent target of therapeutic anti-CD20 antibodies. CD20 is localized within a membrane nanocluster harboring the IgD class B cell antigen receptor (IgD-BCR) where it functions as a gatekeeper for the resting state of naive B lymphocytes. How CD20 exerts its gatekeeper function is not yet known. Using Ramos and human peripheral blood B cells, we show here that the serine/threonine kinase PKC{delta}, constitutively phosphorylates serine residues at the cytosolic tails of CD20. The phosphorylated CD20 becomes a target for 14-3-3 adaptor proteins that link CD20 to the RhoA GDP/GTP exchange factor GEF-H1 and the microtubule (MT) network controlling the stability of the IgD-BCR nanocluster. The binding of anti-CD20 antibodies results in MT disassembly and the replacement of the GEF-H1/CD20 complex by a RhoA-GTP/ROCK1/CD20 complex which drives actomyosin contractility. Our study suggests that CD20 not only maintains the resting state, but also orchestrates the MT/actin switch in active B lymphocytes. This could have implications for treatment with anti-CD20 antibodies and may help to optimize therapeutic protocols. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/656301v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1cfe15aorg.highwire.dtl.DTLVardef@d3ee14org.highwire.dtl.DTLVardef@1b375eforg.highwire.dtl.DTLVardef@103c9dd_HPS_FORMAT_FIGEXP M_FIG C_FIG

The cytokine Interleukin 1 (IL-1) is an evolutionary innovation of vertebrates. Fish and amphibia have one IL1 gene, while mammals have two copies of IL1, IL1A and IL1B, with distinct expression patterns and differences in their proteolytic activation. Our current understanding of the evolutionary history of IL-1 is mainly based on phylogenetic analyses, but this approach provides no information on potentially different functions of IL-1 homologs, and it remains unclear which biological activities identified for IL-1 and IL-1{beta} in mammals are present in lower vertebrates. Here, we use in vitro and in vivo experimental models to examine the expression patterns and cleavage of IL-1 proteins from various species. We found that IL-1 in the teleost medaka shares the transcriptional patterns of mammalian IL-1, and its processing also resembles that of mammalian IL-1, which is sensitive to cysteine protease inhibitors specific for the calpain and cathepsin families. By contrast, IL-1 proteins in reptiles also include biological properties of IL-1{beta}. Therefore, we propose that duplication of the ancestral IL1 gene led to segregation of expression patterns and protein processing that characterizes the two extant forms of IL-1 in mammals.

Despite the fact that the cell surface receptor HVEM (TNFRSF14) appears to be implicated in the development and progression of B-cell lymphomas, its specific role in these tumours is still unclear. On the one hand, HVEM over-expression is related to worse prognosis in some types of B-cell lymphoma and other solid tumours. On the other hand, most mutations of HVEM in B-cell lymphomas are thought to promote tumour growth through the loss of function. Here, we used a CRISPR-Cas9 system to study the effect of HVEM loss on gene expression in a murine model of A20 B-cell lymphoma (belonging to the diffuse large B-cell lymphoma group). We show that loss of HVEM does not affect the doubling rate of A20 tumour cells in culture, but leads to a decrease in BTLA expression. HVEM-deficient A20 cells do not present a different pattern of metastatic dissemination to lymphoid organs compared with unmodified A20 cells. However, we observed a significant expansion of endogenous B-cells as a result of A20 tumour implantation in the thymus. Although we found no differences in the dissemination or progression of HVEM-deficient A20 cells, our results reveal that loss of HVEM alters the leukocyte recruitment capacity of A20 cells in hepatic tumour nodules at the intermediate stage of tumour development, which may be of relevance as a mechanism of immune evasion.

Marginal zone (MZ) B cells are considered to be innate-like immune cells, and follicular (FO) B cells are considered to be adaptive immune B cells. Yet, the proliferative response of MZ and FO B cells to different innate stimuli remains unclear. Here, we investigated cell growth, division, and death to determine the collective proliferative response of MZ and FO B cells in response to innate stimuli, LPS and CpG. We show that the growth rate of FO B cells is higher than that of MZ B cells in response to CpG, though MZ B cells acquire a higher mass at division, whereas both the growth rate and mass at division for MZ and FO B cells remain similar in response to LPS stimulation. We show that MZ B cells divide faster and induce a higher cRel expression in response to both CpG and LPS stimulation than FO B cells. A higher proportion of MZ B cells enter first division in response to LPS stimulation, not in response to CpG stimulation, than FO B cells. Interestingly, CpG stimulation, not LPS stimulation, leads to higher cell death in MZ B cells than FO B cells. In response to LPS stimulation, MZ B cells show a higher cell number at early time and a reduced/ similar cell number at late time, whereas in response to CpG stimulation, MZ B cells show a lower cell number at both early and late time. Our study suggests that LPS and CpG stimulation impact cell growth, division, and death differently, which in turn regulate different proliferative responses of MZ and FO B cells. Thus, our study offers a new perspective that different innate stimuli regulate different features of proliferative responses.

T and B cells are the two arms of the adaptive immune system that mediate cellular and humoral immunity, respectively, using highly diverse repertoires of antigen receptors. T cells recognize antigens using the T cell receptor (TCR), whereas B cells use the B cell receptor (BCR or surface immunoglobulin). Complementary determining regions (CDR3) of TCRs and BCRs are randomly generated through somatic VDJ recombination and nucleotide deletions and insertions at the V-D and D-J junctions. Contrary to this paradigm, here we describe two networks of millions of TCR{beta} and IGH clonotypes that are made from only two CDR3 sequences and associated with more 63 diseases. The TCR{beta} network members bore either the prototypic signature CDR3 sequence (CASSPGTEAFF), its N-terminal VD motif (CASSPGT) recombined with various J{beta} segments (CASSPGT-J{beta}x) or its DJ{beta} motif recombined with various V{beta} (V{beta}x-PGTEAFF). The BCR network members exhibit one signature CDR3 sequence (CARx1-4DTAMVYYFYDW) made from an invariant DJH motif (DTAMVYYFDYW) combined with various VH genes. The prototypes of the two networks are apparently teleogically related as they were dually expressed on the rare population of dual expresser (DE) lymphocytes and molecular dynamic simulations show that they were interacting partners. We conclude that members of the two networks represent a core set of evolutionary-conserved primordial antigen receptors that play fundamental roles in host defense and autoimmune diseases.

CD4+ T cells are key components of adaptive immunity. The cell differentiation equips CD4+ T cells with new functions. However, the effect of cell differentiation on T cell receptor (TCR) repertoire is not investigated. Here, we examined the features of TCR beta (TCRB) repertoire of the top clones within naive, memory and regular T cell (Treg) subsets: repertoire structure, gene usage, length distribution and sequence composition. First, we found that memory subsets and Treg would be discriminated from naive by the features of TCRB repertoire. Second, we found that the correlations between the features of memory subsets and naive were positively related to differentiation levels of memory subsets. Third, we found that public clones presented a reduced proportion and a skewed sequence composition in differentiated subsets. Furthermore, we found that public clones led naive to recognize a broader spectrum of antigens than other subsets. Our findings suggest that TCRB repertoire of CD4+ T cell subsets is skewed in a differentiation-depended manner. Our findings show that the variations of public clones contribute to these changes. Our findings indicate that the reduce of public clones in differentiation trim the antigen specificity of CD4+ T cells. The study unveils the physiological effect of memory formation and facilitates the selection of proper CD4+ subset for cellular therapy.

The Major Histocompatibility Complex class I (MHC-I) molecules present antigenic peptides (AP) to CD8+ T cells for self versus non-self recognition. Loading of AP on MHC-I takes place in the endoplasmic reticulum (ER), upon shuttling of cytoplasmic AP substrates to the ER. Understanding of this process has been influenced by the view that MHC-I antigens are produced from the proteasomal degradation of cellular proteins. Recent observations on the intronic and untranslated region-derived peptides as well as on the non-AUG translation products presented on the MHC-I open the possibility that antigenic peptides can derive from pre-spliced mRNAs translated in the nuclear compartment. In this brief report, we show that a fraction of human MHC-I molecules (human leukocyte antigens type A, HLA-A) is present in the nuclei of cells, in the proximity of histone H2B. With this finding, we hope to initiate a new direction of research on the nuclear role of MHC-I and ask whether the loading of antigens can take place in the nuclear compartment.

Follicular lymphoma (FL) is the most common indolent form of non-Hodgkin lymphoma arising from malignant germinal center (GC) B-cells. The genetic hallmark that leads to the development of FL is the t(14:18) which occurs early in the bone marrow during B cell development, thereby placing the anti-apoptotic BCL2 gene under the direct control of the transcriptional enhancers in 3 of immunoglobulin heavy chain locus (IgH 3RR) and leading to the constitutive expression of the BCL2 protein. To assess the impact of the BCL2 deregulation on B-cell fate and try to reproduce FL development in mice, two models were designed: the Ig{kappa}-BCL2 (Knock in of the BCL2 in the light chain Ig kappa locus) and the 3RR-BCL2 (Transgene containing BCL2 and a micro-3RR), both containing the full BCL2 promoter region.

Studies in a mouse model revealed Mycobacterium tuberculosis (Mtb) with a deletion of rel, regulator of the stringent response, could not establish a persistent infection. Studies in cattle with a Mycobacterium. a. paratuberculosis rel deletion mutant revealed inability to establish a persistent infection was associated with development of CD8 cytotoxic T cells (CTL) that kill intracellular bacteria. Further comparative studies ex vivo with Mbv Calmette-Guerin (BCG) and a BCG rel deletion mutant revealed no clear difference in development of CTL in vitro. As reported, a study of the recall response was conducted with cattle vaccinated with either BCG or with BCGrel, to determine if information could be obtained that would show how gene products under control of rel interfere with the CTL response to mycobacterial pathogens in vivo. The study revealed the CTL response elicited by vaccination with BCG was impaired, in comparison with the response elicited by BCGrel. Comparative analysis of the recall response ex vivo revealed the functional impairment was not associated with the timing of appearance of the recall response, expression of IFN-{gamma}, TNF-, IL-17, or IL-22, or molecules that mediate intracellular killing. Further studies are needed to determine how CD8 CTL functional activity is modulated in vivo by gene products regulated by rel.

The Ebola virus glycoprotein (EBOV)-GP is extensively glycosylated. Its expression induces a physical alteration of surface adhesion molecules, which causes cell rounding and detachment of the infected cells. This phenomenon likely plays a crucial role in viral pathogenicity. In this study, we show that such morphological changes are cell line-dependent as well as dependent on the surface proteins that interact with EBOV-GP in cis and trans. We have generated data showing that natural killer (NK) cell receptors (NCRs: NKp44 and NKp46), selectins (CD62E/P/L) and inhibitory Siglecs function as receptors for Ebola-GP and human papilloma virus (HPV-L1). We used HEK293 cells transfected with Ebola-GP and recombinant fusion proteins containing the extracellular domain of each of these receptors linked to the Fc of human IgG1, which showed significant differences in their virus-binding behavior compared to HEK293 cells transfected with empty vector. Further, to demonstrate that EBOV-GP is a ligand for NKp44 and other NK-receptors, and to investigate their role in immune escape, we also used human HEK-293, HeLa- and hamster CHO-GP-transfectants. Our data show that the NK receptors NKp44 and NKp46 play a key role in recognizing EBOV (Ebolavirus) and strongly suggest that other inhibitory (Siglec-7, Siglec-5) and non-inhibitory homing receptors (P-Selectin, L-Selectin, E-Selectin, and DC-SIGNR/DC-SIGN) take part in the interaction with virus particles. In addition, we show that NKp44, and NKp46, Siglec-7, and -5, and P-, L-, E-selectins as well as of and DC-SIGNR/DC-SIGN bind to the artificial viral envelope of a lentiviral vector that contains EBOV-GP. Altogether we prove that NCRs and a range of other inhibitory and activating receptors can interact with viral envelope/capsid proteins and that such interaction could play an important role in the elimination of virus infected cells. Our findings could be used to develop new strategies for prevention and treatment of infections by these viruses. Author summaryThe innate immune system is able to recognize specifically certain virus components. Here we show that activating NK-cell receptors (NKp44, and NKp46) are involved in such interaction by using HEK293 and CHOK1 cells transfected with the Ebola virus glycoprotein (EBOV-GP) and by binding studies with purified EBOV-GP. In detail, we have found moderate to strong affinity of Siglecs (Siglec-7, and -5), selectins (P-, L-, E-Selectin) and DC-SIGNR/DC-SIGN to purified EBOV-GP, and to cells transfected with EBOV-GP as well as to the envelope of a lentiviral vector carrying the EBOV-GP. Our findings show that NKp44, and NKp46, Siglec-7, and -5, as well as P-and L-selectins have a strong affinity to EBOV-G.

Rab8a is a small GTPase with a wide range of reported functions in different cell types, including vesicle recycling, vesicle traffic to cilia, cell ruffling, migration, neurite outgrowth, Toll-like receptor signalling and T cell receptor docking at the immune synapse. However, the role of Rab8a in B lymphocytes has not been described to date. Here, using a conditional B cell-specific Rab8a knockout mouse model, we investigate the role of Rab8a both in vivo and in vitro. Rab8a KO mice present enhanced antibody responses to both T-dependent and T-independent immunisations. Rab8a KO cells showed normal BCR trafficking and antigen processing and presentation but however, increased class-switch recombination. While the early BCR signalling responses, such as proximal kinase activation and calcium-flux, were normal, the signalling via AKT and ERK1/2 was decreased. We propose that the lack of Rab8a alters cellular signalling leading to enhanced antibody responses and increased class-switch recombination potentially via downmodulation of the PI3K/AKT/mTOR pathway.

Cetaceans correspond to mammals that have returned to the marine environment. Adaptive changes are very significant with the conversion of the limbs into flippers. It is studied the changes that have occurred in immunoglobulins, MHC class I and II and T cell receptors genes. Constant regions of immunoglobulins are similar to those of the rest of mammals. An exception is the IgD gene, which is composed of three CH domains but CH1 similar to CH1 of immunoglobulin M. In the IGHV locus, it exist a decrease in the number of VH genes with the absence of genes within Clan I. The number of V{lambda} genes is greater than that of V{kappa}. In the genes for T lymphocyte receptors, it exists a decrease in the number of V genes with loss of significant clades and subclades. In V{beta} and V{gamma}, there is also the loss of clades. These declines of V, V{beta} and V{gamma} are not present Artiodactyla, and they are specific to Cetaceans. In MHC present tree evolutive lines of class I genes. These species have DQ, DR, DO and DM genes, but they are no present DP genes.