Characterizing the BCR repertoire during lymphocyte reduction and recovery mediated by cyclophosphamide and granulocyte-macrophage colony-stimulating factor.

Leukopenia is a common manifestation of many diseases, including global outbreak SAS-CoV-2 infection. Granulocyte-macrophage colony-stimulating factor (GM -CSF) has been proved to be effective in promoting lymphocyte regeneration, but adverse immunological effects have also emerged. This study aim to investigate the effect of GM -CSF on BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration. Cyclophosphamide (CTX) and GM -CSF were used to inhibit and stimulate bone marrow hematopoiesis, respectively. High throughput sequencing was applied to detect the characteristics of BCR CDR3 repertoire in controls, CTX group and GM -CSF group. The white blood cells (WBCs) were quickly reduced (P < 0.05) with lymphocytes decreasing causing by CTX, and the WBCs and lymphocytes returned to the level of controls after GM -CSF treatment. The diversity of BCR heavy chain CDR3 repertoire was also significantly decreased in CTX group. Although there is still a big gap from the controls, the diversity was picked up after GM -CSF treatment. The expression of IGHD01-01, IGHD02-14 and IGHJ04-01 with high-frequency usage regularly and significantly changed in three groups, and many genes with low-frequency usage lost in CTX group and did not reappear in GM -CSF group. Moreover, two shared sequences and accounted for the highest proportion in GM -CSF group have been detected in animal model of chronic lymphocytic leukemia. These results revealed that GM -CSF can partially restore changes in the BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration, but it may also lead to rearrangement, proliferation and activation of abnormal B cells, which can provide a basis for further study on the adverse immunological effects and mechanism of GM -CSF treatment.

The Proteolytic Cleavage of Therapeutic Monoclonal Antibody Hinge Region: More Than a Matter of Subclass.

The hinge region of immunoglobulin G (IgG) is involved in C1q and FcγRIIIA-expressing natural killer (NK) cell recruitment. Both heavy chains (HCs) of the hinge region can be cleaved sequentially by several proteases of the tumor/inflammatory/infectious microenvironment, including matrix metalloproteinase 12 (MMP12), or immunoglobulin-degrading enzyme from Streptococcus pyogenes (IdeS), impairing Fc-mediated functions. The cleavage of therapeutic monoclonal antibodies (TmAbs), which are based on a human IgG1, IgG2 or IgG4 structure, has been poorly investigated, although it may represent an escape mechanism to these treatments. Therefore, we used non-reducing SDS-PAGE to compare the cleavage kinetics of five IgG1 TmAbs (trastuzumab, rituximab, cetuximab, infliximab, ipilimumab), one IgG2 TmAb (panitumumab), and two IgG4 TmAbs (nivolumab and pembrolizumab) by MMP12 and IdeS, which were found to cleave the first and second HCs with different kinetics. Panitumumab was more protease-resistant than IgG1 and IgG4 TmAbs. The latter were usually more protease-sensitive, whereas IgG1 TmAbs were usually cleaved with intermediate kinetics. However, we observed intra-subclass variability among IgG4 and IgG1 TmAbs. Nivolumab and pembrolizumab were cleaved similarly by MMP12, whereas pembrolizumab was more IdeS-resistant. Ipilimumab was more IdeS-sensitive and MMP12-resistant than the other IgG1 TmAbs, regardless of G1m allotype. In addition the Fc fragment of IgG1 TmAbs were highly resistant to cleavage by MMP12, whereas their cleavage kinetic by IdeS was very similar to that observed with the intact forms (excluding ipilimumab). Importantly, the cleavage kinetic of ipilimumab Fc fragment by IdeS was superimposable to that of trastuzumab, cetuximab and infliximab Fc fragment, showing that the variability observed for intact ipilimumab is unrelated to its Fc portion. We propose that the variability in the cleavage sensitivity/resistance balance among TmAbs of IgG1 and IgG4 subclasses results partially, from TmAb characteristics related to and/or located in the Fab region. Finally, with ELISA and flow cytometry, we observed that a single cleavage of IgG1 TmAbs greatly decreased their affinity for FcγRIIIA and C1q and their ability to induce FcγRIIIA-dependent functional responses of NK cells. Overall, our results indicate that the cleavage of the hinge region should be considered with TmAbs treatment and in the development of new molecules.

Improved clonality analysis based on immunoglobulin kappa locus for canine cutaneous plasmacytoma.

Sensitivity of clonality analysis based on immunoglobulin heavy chain (IGH) in canine cutaneous plasmacytoma is lower than that in diffuse large B cell lymphoma (DLBCL) because of somatic hypermutation occurring at the IGH locus. Therefore, this study aimed to improve the sensitivity of clonality analysis for canine cutaneous plasmacytoma. To achieve this, clonality analysis based on the immunoglobulin kappa chain (IGK) locus was established. Sensitivity and specificity were examined in genomic DNA extracted from formalin-fixed paraffin-embedded sections of cutaneous plasmacytomas, DLBCLs, and lymph nodes without lymphoma. Forward primers were designed based on the IGKV genes, and reverse primers were designed based on the IGKJ genes and kappa deleting element (Kde). Analysis using IGKV and IGKJ primers demonstrated clonality in 24 of 29 cutaneous plasmacytomas (82.8%), while analysis with primers for IGKV and Kde showed clonality in 16 of 29 cases (55.2%). In DLBCL, the IGKV and IGKJ primer set yielded clonality in 18 of 23 cases (78.3%), and the IGKV and Kde primer set yielded 9 of 23 cases (39.1%). No clonal results were obtained from 23 lymph nodes without lymphoma. Sensitivity of the IGKV and IGKJ primer set was significantly higher than that of the IGH primers reported previously. Thus, clonality analysis based on the IGK locus can be utilized for canine B cell tumors. In conclusion, clonality testing based on IGH and IGK may be beneficial as an adjunct tool for diagnosis of canine B cell tumors including cutaneous plasmacytoma.

Combined Influence of B-Cell Receptor Rearrangement and Somatic Hypermutation on B-Cell Class-Switch Fate in Health and in Chronic Lymphocytic Leukemia.

A diverse B-cell receptor (BCR) repertoire is required to bind a wide range of antigens. BCRs are generated through genetic recombination and can be diversified through somatic hypermutation (SHM) or class-switch recombination (CSR). Patterns of repertoire diversity can vary substantially between different health conditions. We use isotype-resolved BCR sequencing to compare B-cell evolution and class-switch fate in healthy individuals and in patients with chronic lymphocytic leukemia (CLL). We show that the patterns of SHM and CSR in B-cells from healthy individuals are distinct from CLL. We identify distinct properties of clonal expansion that lead to the generation of antibodies of different classes in healthy, malignant, and non-malignant CLL BCR repertoires. We further demonstrate that BCR diversity is affected by relationships between antibody variable and constant regions leading to isotype-specific signatures of variable gene usage. This study provides powerful insights into the mechanisms underlying the evolution of the adaptive immune responses in health and their aberration during disease.

Epigenetic modifications of the VH region after DJH recombination in Pro-B cells.

The variable region of murine immunoglobulin heavy chain (Igh) is assembled by sequential DH -JH and VH -DJH recombination. The accessibility of the Igh locus determines the order of rearrangement. Because of the large number of VH genes and the lack of a suitable model, the epigenetic modifications of VH genes after DJH recombination have not previously been characterized. Here, we employed two v-Abl pro-B cell lines, in which the Igh locus is in germline and DJH -recombined configurations, respectively. The DJH junction displays the characteristics of a recombination centre, such as high levels of activation-associated histone modifications and recombination-activating gene protein (RAG) binding in DJH -rearranged pro-B cells, which extend the recombination centre model proposed for the germline Igh locus. The different domains of the VH region have distinct epigenetic characteristics after DJH recombination. Distal VH genes have higher levels of active histone modifications, germline transcription and Pax5 binding, and good quality recombination signal sequences. Proximal VH genes are relatively close to the DJH recombination centre, which partially compensates for the low levels of the above active epigenetic modifications. DJH recombination centre might serve as a cis-acting element to regulate the accessibility of the VH region. Furthermore, we demonstrate that RAG weakly binds to functional VH genes, which is the first detailed assessment of RAG dynamic binding to VH genes. We provide a way for VH -DJH recombination in which the VH gene is brought into close proximity with the DJH recombination centre for RAG binding by a Pax5-dependent chromosomal compaction event, and held in this position for subsequent cleavage and VH -DJH joining.

Notch3 drives development and progression of cholangiocarcinoma.

The prognosis of cholangiocarcinoma (CC) is dismal. Notch has been identified as a potential driver; forced exogenous overexpression of Notch1 in hepatocytes results in the formation of biliary tumors. In human disease, however, it is unknown which components of the endogenously signaling pathway are required for tumorigenesis, how these orchestrate cancer, and how they can be targeted for therapy. Here we characterize Notch in human-resected CC, a toxin-driven model in rats, and a transgenic mouse model in which p53 deletion is targeted to biliary epithelia and CC induced using the hepatocarcinogen thioacetamide. We find that across species, the atypical receptor NOTCH3 is differentially overexpressed; it is progressively up-regulated with disease development and promotes tumor cell survival via activation of PI3k-Akt. We use genetic KO studies to show that tumor growth significantly attenuates after Notch3 deletion and demonstrate signaling occurs via a noncanonical pathway independent of the mediator of classical Notch, Recombinant Signal Binding Protein for Immunoglobulin Kappa J Region (RBPJ). These data present an opportunity in this aggressive cancer to selectively target Notch, bypassing toxicities known to be RBPJ dependent.

Novel Monoclonal Antibodies for Studies of Human and Rhesus Macaque Secretory Component and Human J-Chain.

Immunoglobulin A (IgA) antibodies exist in monomeric, dimeric, and secretory forms. Dimerization of IgA depends on a 15-kD polypeptide termed "joining (J) chain," which is also part of the binding site for an epithelial glycoprotein called "secretory component (SC)," whether this after apical cleavage on secretory epithelia is ligand bound in secretory IgA (SIgA) or in a free form. Uncleaved membrane SC, also called the "polymeric Ig receptor," is thus crucial for transcytotic export of SIgA to mucosal surfaces, where it interacts with and modulates commensal bacteria and mediates protective immune responses against exogenous pathogens. To evaluate different forms of IgA, we have produced mouse monoclonal antibodies (MAbs) against human J-chain and free SC. We found that J-chain MAb 9A8 and SC MAb 9H7 identified human dimeric IgA and SIgA in enzyme-linked immunoassay and western blot analysis, as well as functioning in immunohistochemistry to identify cytoplasmic IgA of intestinal lamina propria plasmablasts/plasma cells and crypt epithelium of distal human intestine. Finally, we demonstrated that SC MAb 9H7 cross-reacted with rhesus macaque SIgA. These novel reagents should be of use in the study of the biology of various forms of IgA in humans and SIgA in macaques, as well as in monitoring the production and/or isolation of these forms of IgA.

Different spectra of recurrent gene mutations in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors.

We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobulin genes, i.e. #1, #6, #8 and #59 (22-34%), often in association with trisomy 12, and was significantly different (P<0.001) to the frequency observed in subset #2 (4%, aggressive disease, variable somatic hypermutation status) and subset #4 (1%, indolent disease, mutated immunoglobulin genes). Interestingly, subsets harboring a high frequency of NOTCH1 mutations were found to carry few (if any) SF3B1 mutations. This starkly contrasts with subsets #2 and #3 where, despite their immunogenetic differences, SF3B1 mutations occurred in 45% and 46% of cases, respectively. In addition, mutations within TP53, whilst enriched in subset #1 (16%), were rare in subsets #2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s).

Analysis of celiac disease autoreactive gut plasma cells and their corresponding memory compartment in peripheral blood using high-throughput sequencing.

Autoreactive IgA plasma cells (PCs) specific for the enzyme transglutaminase 2 (TG2) are abundant in the small intestine of patients with active celiac disease (CD), and their number drops in patients treated by dietary gluten elimination. Little is known about their characteristics and their role in the disease. In this study, using high-throughput sequencing of the IgH V region (IGHV) genes, we have studied features of TG2-specific PCs and their related B cell clones in peripheral blood. We found that TG2-specific PCs from both untreated and treated patients have acquired lower number of somatic hypermutation and used focused IGHV repertoire with overrepresentation of the IGHV3-48, IGHV4-59, IGHV5-10-1, and IGHV5-51 gene segments. Furthermore, these PCs were clonally expanded and showed signs of affinity maturation. Lineage trees demonstrated shared clones between gut PCs and blood memory B cells, primarily IgAs. Some trees also involved IgG cells, suggesting that anti-TG2 IgA and IgG responses are related. Similarly to TG2-specific PCs, clonally related memory IgA B cells of blood showed lower mutation rates with biased usage of IGHV3-48 and IGHV5-51. Such memory cells were rare in peripheral blood, yet detectable in most patients assessed by production of anti-TG2 Abs in vitro following stimulation of cells from patients who had been on a long-term gluten-free diet. Thus, the Ab response to TG2 in CD, while maintaining its IGHV gene usage, is dynamically regulated in response to gluten exposure with a low degree of maintenance at both PC and memory B cell levels in patients in remission.

Comparative study of IgA VH 3 gene usage in healthy TST(-) and TST(+) population exposed to tuberculosis: deep sequencing analysis.

The acquired immune response against tuberculosis is commonly associated with T-cell responses with little known about the role of B cells or antibodies. There have been suggestions that B cells and humoral immunity can modulate the immune response to Mycobacterium tuberculosis. However, the mechanisms involving B-cell responses in M. tuberculosis are not fully understood, in particular the antibody gene preferences. We hypothesized that a preferential use of V genes can be seen associated with resistance to infection mainly in the IgA isotype, which is of prominent importance for infection by pathogens via the mucosal route. We studied healthy individuals with long-term exposure to tuberculosis, infected (TST(+) ) and uninfected TST(-) ) with M. tuberculosis. From a total of 22 V genes analysed, the TST(-) population preferred the VH 3-23 and Vκ1 genes. The VH 3-23 genes were subsequently subjected to 454 amplicon sequencing. The TST(-) population showed a higher frequency of the D3-10 segment compared with the D3-22 segment for the TST(+) population. The J segment usage pattern was similar for both populations with J4 segment being used the most. A preferential pairing of J4 segments to D3-3 was seen for the TST(-) population. The antibodyome difference between both populations suggests a preference for antibodies with VH 3-23, D3-3, JH 4 gene usage by the TST(-) population that could be associated with resistance to infection with M. tuberculosis.

Neuroblastoma killing properties of Vδ2 and Vδ2-negative γδT cells following expansion by artificial antigen-presenting cells.

PURPOSE: The majority of circulating human γδT lymphocytes are of the Vγ9Vδ2 lineage, and have T-cell receptor (TCR) specificity for nonpeptide phosphoantigens. Previous attempts to stimulate and expand these cells have therefore focused on stimulation using ligands of the Vγ9Vδ2 receptor, whereas relatively little is known about variant blood γδT subsets and their potential role in cancer immunotherapy. EXPERIMENTAL DESIGN: To expand the full repertoire of γδT without bias toward specific TCRs, we made use of artificial antigen-presenting cells loaded with an anti γδTCR antibody that promoted unbiased expansion of the γδT repertoire. Expanded cells from adult blood donors were sorted into 3 populations expressing respectively Vδ2 TCR chains (Vδ2(+)), Vδ1 chains (Vδ1(+)), and TCR of other δ chain subtypes (Vδ1(neg)Vδ2(neg)). RESULTS: Both freshly isolated and expanded cells showed heterogeneity of differentiation markers, with a less differentiated phenotype in the Vδ1 and Vδ1(neg)Vδ2(neg) populations. Expanded cells were largely of an effector memory phenotype, although there were higher numbers of less differentiated cells in the Vδ1(+) and Vδ1(neg)Vδ2(neg) populations. Using neuroblastoma tumor cells and the anti-GD2 therapeutic mAb ch14.18 as a model system, all three populations showed clinically relevant cytotoxicity. Although killing by expanded Vδ2 cells was predominantly antibody dependent and proportionate to upregulated CD16, Vδ1 cells killed by antibody-independent mechanisms. CONCLUSIONS: In conclusion, we have demonstrated that polyclonal-expanded populations of γδT cells are capable of both antibody-dependent and -independent effector functions in neuroblastoma.

Shared epitopes of avian immunoglobulin light chains.

Like all jawed vertebrates, birds (Aves) also produce antibodies i.e. immunoglobulins (Igs) as a defence mechanism against pathogens. Their Igs are composed of two identical heavy (H) and light (L) chains which are of lambda isotype. The L chain consists of variable (VL), joining (JL) and constant (CL) region. Using enzyme immunoassays (EIA) and two monoclonal antibodies (mAbs) (3C10 and CH31) to chicken L chain, we analysed their cross-reactivity with sera from 33 avian species belonging to nine different orders. Among Galliformes tested, mAbs 3C10 and CH31 reacted with L chains of chicken, turkey, four genera of pheasants, tragopan and peafowl, but not with sera of grey partridge, quail and Japanese quail. Immunoglobulins of guinea-fowl reacted only with mAb 3C10. Both mAbs reacted also with the L chain of Eurasian griffon (order Falconiformes) and domestic sparrow (order Passeriformes). Sera from six other orders of Aves did not react with either of the two mAbs. EIA using mAbs 3C10 and CH31 enabled detection of antibodies to major avian pathogens in sera of chickens, turkeys, pheasants, peafowl, Eurasian griffon and guinea-fowl (only with mAb 3C10). The N-terminal amino acid sequence of pheasant L chain (19 residues) was identical to that of chicken. Sequences of genes encoding the L chain constant regions of pheasants, turkey and partridge were determined and deposited in the public database (GenBank accession numbers: FJ 649651, FJ 649652 and FJ 649653, respectively). Among them, amino acid sequence of pheasants is the most similar to that of chicken (97% similarity), whereas those of turkey and partridge have greater similarity to each other (89%) than to any other avian L chain sequence. The characteristic deletion of two amino acids which is present in the L chain constant region in Galliformes has been most likely introduced to their L chain after their divergence from Anseriformes.

Noncore RAG1 regions promote Vß rearrangements and αß T cell development by overcoming inherent inefficiency of Vß recombination signal sequences.

The RAG proteins are comprised of core endonuclease domains and noncore regions that modulate endonuclease activity. Mutation or deletion of noncore RAG regions in humans causes immunodeficiency and altered TCR repertoire, and mice expressing core but not full-length Rag1 (Rag1(C/C)) or Rag2 (Rag2(C/C)) exhibit lymphopenia, reflecting impaired V(D)J recombination and lymphocyte development. Rag1(C/C) mice display reduced D-to-J and V-to-DJ rearrangements of TCRß and IgH loci, whereas Rag2(C/C) mice show decreased V-to-DJ rearrangements and altered Vß/VH repertoire. Because Vßs/VHs only recombine to DJ complexes, the Rag1(C/C) phenotype could reflect roles for noncore RAG1 regions in promoting recombination during only the D-to-J step or during both steps. In this study, we demonstrate that a preassembled TCRß gene, but not a preassembled DßJß complex or the prosurvival BCL2 protein, completely rescues αß T cell development in Rag1(C/C) mice. We find that Rag1(C/C) mice exhibit altered Vß utilization in Vß-to-DJß rearrangements, increased usage of 3'Jα gene segments in Vα-to-Jα rearrangements, and abnormal changes in Vß repertoire during αß TCR selection. Inefficient Vß/VH recombination signal sequences (RSSs) have been hypothesized to cause impaired V-to-DJ recombination on the background of a defective recombinase as in core-Rag mice. We show that replacement of the Vß14 RSS with a more efficient RSS increases Vß14 recombination and rescues αß T cell development in Rag1(C/C) mice. Our data indicate that noncore RAG1 regions establish a diverse TCR repertoire by overcoming Vß RSS inefficiency to promote Vß recombination and αß T cell development, and by modulating TCRß and TCRα gene segment utilization.

Essential functions for ID proteins at multiple checkpoints in invariant NKT cell development.

Invariant NKT (iNKT) cells display characteristics of both adaptive and innate lymphoid cells (ILCs). Like other ILCs, iNKT cells constitutively express ID proteins, which antagonize the E protein transcription factors that are essential for adaptive lymphocyte development. However, unlike ILCs, ID2 is not essential for thymic iNKT cell development. In this study, we demonstrated that ID2 and ID3 redundantly promoted iNKT cell lineage specification involving the induction of the signature transcription factor PLZF and that ID3 was critical for development of TBET-dependent NKT1 cells. In contrast, both ID2 and ID3 limited iNKT cell numbers by enforcing the postselection checkpoint in conventional thymocytes. Therefore, iNKT cells show both adaptive and innate-like requirements for ID proteins at distinct checkpoints during iNKT cell development.

A study of the mechanism of the chaperone-like function of an scFv of human creatine kinase by computer simulation.

A new application of antibodies is to use them as macromolecular chaperones. Protein antigens usually have multiple epitopes, thus, there may be a plurality of antibodies binding to one antigen. However, not all antibodies that bind to one antigen could act as a chaperone. Experiments show that some screened anti-human creatine kinase single chain antibodies (scFV) could assist in the folding and stabilizing of the enzyme, while others could not. We built the model of the single chain antibody (scFv-A4) that increased the stability of human creatine kinase (HCK) by the homology modeling method. Epitopes of human creatine kinase were predicted by computer and then the binding of scFv-A4 and HCK was modeled with computer. The calculation results were further combined with the peptide array membrane experiment results to obtain reliable models for the scFv-A4-HCK complex. Based on the above study we gave an explanation about how scFv-A4 could act as a macromolecular chaperone assisting the folding of HCK. This study provides an approach for predicting antigen-antibody binding mode and also a useful theoretical guidance for the study of antibodies' chaperone-like function.

The restricted DH gene reading frame usage in the expressed human antibody repertoire is selected based upon its amino acid content.

The Ab repertoire is not uniform. Some variable, diversity, and joining genes are used more frequently than others. Nonuniform usage can result from the rearrangement process, or from selection. To study how the Ab repertoire is selected, we analyzed one part of diversity generation that cannot be driven by the rearrangement mechanism: the reading frame usage of DH genes. We have used two high-throughput sequencing methodologies, multiple subjects and advanced algorithms to measure the DH reading frame usage in the human Ab repertoire. In most DH genes, a single reading frame is used predominantly, and inverted reading frames are practically never observed. The choice of a single DH reading frame is not limited to a single position of the DH gene. Rather, each DH gene participates in rearrangements of differing CDR3 lengths, restricted to multiples of three. In nonproductive rearrangements, there is practically no reading frame bias, but there is still a striking absence of inversions. Biases in DH reading frame usage are more pronounced, but also exhibit greater interindividual variation, in IgG(+) and IgA(+) than in IgM(+) B cells. These results suggest that there are two developmental checkpoints of DH reading frame selection. The first occurs during VDJ recombination, when inverted DH genes are usually avoided. The second checkpoint occurs after rearrangement, once the BCR is expressed. The second checkpoint implies that DH reading frames are subjected to differential selection. Following these checkpoints, clonal selection induces a host-specific DH reading frame usage bias.

Shaping of human germline IgH repertoires revealed by deep sequencing.

To understand better how selection processes balance the benefits of Ig repertoire diversity with the risks of autoreactivity and nonfunctionality of highly variable IgH CDR3s, we collected millions of rearranged germline IgH CDR3 sequences by deep sequencing of DNA from mature human naive B cells purified from four individuals and analyzed the data with computational methods. Long HCDR3 regions, often components of HIV-neutralizing Abs, appear to derive not only from incorporation of long D genes and insertion of large N regions but also by usage of multiple D gene segments in tandem. However, comparison of productive and out-of-frame IgH rearrangements revealed a selection bias against long HCDR3 loops, suggesting these may be disproportionately either poorly functional or autoreactive. Our data suggest that developmental selection removes HCDR3 loops containing patches of hydrophobicity, which are commonly found in some auto-antibodies, and at least 69% of the initial productive IgH rearrangements are removed from the repertoire during B cell development. Additionally, we have demonstrated the potential utility of this new technology for vaccine development with the identification in all four individuals of related candidate germline IgH precursors of the HIV-neutralizing Ab 4E10.

Skewed primary Igκ repertoire and V-J joining in C57BL/6 mice: implications for recombination accessibility and receptor editing.

Previous estimates of the diversity of the mouse Ab repertoire have been based on fragmentary data as a result of many technical limitations, in particular, the many samples necessary to provide adequate coverage. In this study, we used 5'-coding end amplification of Igκ mRNAs from bone marrow, splenic, and lymph node B cells of C57BL/6 mice combined with amplicon pyrosequencing to assess the functional and nonfunctional Vκ repertoire. To evaluate the potential effects of receptor editing, we also compared V/J associations and usage in bone marrows of mouse mutants under constitutive negative selection or an altered ability to undergo secondary recombination. To focus on preimmune B cells, our cell sorting strategy excluded memory B cells and plasma cells. Analysis of ~90 Mbp, representing >250,000 individual transcripts from 59 mice, revealed that 101 distinct functional Vκ genes are used but at frequencies ranging from ~0.001 to ~10%. Usage of seven Vκ genes made up >40% of the repertoire. A small class of transcripts from apparently nonfunctional Vκ genes was found, as were occasional transcripts from several apparently functional genes that carry aberrant recombination signals. Of 404 potential V-J combinations (101 Vκs × 4 Jκs), 398 (98.5%) were found at least once in our sample. For most Vκ transcripts, all Jκs were used, but V-J association biases were common. Usage patterns were remarkably stable in different selective conditions. Overall, the primary κ repertoire is highly skewed by preferred rearrangements, limiting Ab diversity, but potentially facilitating receptor editing.

PI3Kß plays a critical role in neutrophil activation by immune complexes.

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the ß isoform of phosphoinositide 3-kinase (PI3Kß) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kß alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kß and PI3Kδ, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3Kß by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B4. Coincident activation by a tyrosine kinase-coupled receptor (FcγR) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the ß isoform of PI3K. PI3Kß-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kß and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kß as a potential therapeutic target in inflammatory disease.

T-cell receptor γ chain variable and joining region genes of subgroup 1 are clonally rearranged in feline B- and T-cell lymphoma.

Lymphoma is one of the most common tumours of domestic cats. Microscopically, it can be challenging to distinguish lymphoma (monoclonal lymphoid proliferation) from reactive lymphoid hyperplasia (polyclonal lymphoid proliferation). Molecular methods that characterize cellular clonality can overcome this diagnostic challenge; however, it is essential to know which variable and joining region variants are utilized by neoplastic lymphocytes before a sensitive and specific assay can be developed. The present study describes a polymerase chain reaction assay that allows for complete sequencing of clonally recombined T-cell receptor (TCR) γ chain genes from formalin-fixed and paraffin wax-embedded samples of feline lymphoma. The variable (V) and joining (J) region variants of the TCR γ chain were characterized in 50 feline lymphomas. Amplification and sequencing with primers directed against conserved framework regions 1, 2 and 4 of the TCR γ chain identified clonal rearrangement in 68% of T-cell lymphomas and 22% of B-cell lymphomas. The distribution of TCR variants present in B- and T-cell lymphomas was similar and included V region variants 1 and 3, and J region variants 1.2, 1.3 and 1.5. V region variants 2 and 4 were not identified in either tumour type. Some feline B-cell lymphomas had a clonally rearranged TCR, a finding reported in human, but not canine, B-cell lymphoma.