Efficiency in Teaching Speaker and Listener Repertoires:Comparing Three Instructional Sequences in Autistic Children

Previous studies have investigated the efficiency in teaching listener and speaker repertoires in children diagnosed with autism spectrum disorder(ASD).Some investigations focused on listener responding by function,feature,and class(LRFFC)and intraverbal by function,feature,and class(FFC).For some children,teaching intraverbal FFC was more efficient because it resulted in a better emergence effect of a related untaught repertoire(LRFFC).For other children,teaching LRFFC along with tacting pictures was more efficient,resulting in a better emergence effect of a related untaught repertoire(intraverbal FFC).In these cases,it is not clear whether the tact increased the efficiency of LRFFC training because a comparison with a condition in which tacts were not required was not conducted.This investigation consisted of a replication with two children diagnosed with ASD.Three instructional sequences were compared:teaching LRFFC-probing intraverbal;teaching LRFFC+tacts-probing intraverbal;teaching intraverbal-probing LRFFC.For a child,all sequences were equally efficient because all related untaught repertoires emerged without errors.However,the acquisition of intraverbals during training occurred with variability.In the case of the second child,the most efficient sequence consisted of teaching intraverbals,resulting in the emergence of LRFFC without errors.In both cases of teaching LRFFC,the emergence of related intraverbals was partial and acquisition of the trained repertoires occurred with variability.The case that did not demand tact responses was slightly more efficient.Data were discussed in the sense that the best instructional sequence may vary from learner to learner.

Vocal repertoire of Azure-winged Magpies(Cyanopica cyanus):A context-associated communication system

Signals within animals’vocal communication are considered functional referential and context-specific.Even in the absence of the context,receivers are expected to acquire the information of calls and respond specificallyWhereas the framework was supported by plenty of evidence,its exhaustivity in describing all animal vocalisations has been questioned.Here,we investigated the vocal repertoire of a cooperatively breeding species,Azure-winged Magpie(Cyanopica cyanus),to present evidence for referential signals.The results showed that Azure-winged Magpies had a relatively large vocal repertoire,consisting of twelve distinct calls.These calls were associated with the context including movement,begging for food,contact,vigilance against predators,etc.However,even the predator-specific alarm calls would induce various responses of receivers.This implies that multiple pieces of information are involved in the vocalisation,which could be utilised by the receiver to select an appropriate response based on the surroundings.Our study gives a detailed description of the context and function of the vocal repertoire in Azure-winged Magpies,laying the foundation for further investigation on the developmental mechanisms of bird vocalisations.This study also suggests that the referential signals of animal vocalisations may not be limited to the context-specific responses of receivers and need to be discussed from a broader perspective.

Human immune repertoire in hepatitis B virus infection

Hepatitis B virus(HBV)infection is a public health threat that affects 257 million people worldwide and can progress to liver cirrhosis,liver failure,and hepatocellular carcinoma.The HBV antigen-induced adaptive immune response plays an important role in HBV clearance.Immune repertoire sequencing(IRS)has been used to investigate the molecular mechanisms behind the immune system,find novel ways to treat HBV infection,and evaluate the genetic responses and immune characteristics of individuals infected by HBV or immunized by HBV vaccine.This review summarizes the human immune repertoire analysis methodology,and the application of the IRS in the prediction of HBV infection progression,treatment,and vaccination.

Human colorectal cancer cells frequently express IgG and display unique Ig repertoire

BACKGROUND There is growing evidence proving that many human carcinomas, including colon cancer, can overexpress immunoglobulin(Ig); the non B cancer cell-derived Ig usually displayed unique V(D)J rearrangement pattern that are distinct from B cell-derived Ig. Especially, the cancer-derived Ig plays important roles in cancer initiation, progression, and metastasis. However, it still remains unclear if the colon cancer-derived Ig can display unique V(D)J pattern and sequencing, which can be used as novel target for colon cancer therapy.AIM To investigate the Ig repertoire features expressed in human colon cancer cells.METHODS Seven cancerous tissue samples of colon adenocarcinoma and corresponding noncancerous tissue samples were sorted by fluorescence-activated cell sorting using epithelial cell adhesion molecule as a marker for epithelial cells. Ig repertoire sequencing was used to analyze the expression profiles of all 5 classes of Ig heavy chains(IgH) and the Ig repertoire in colon cancer cells and corresponding normal epithelial cells.RESULTS We found that all 5 IgH classes can be expressed in both colon cancer cells and normal epithelial cells. Surprisingly, unlike the normal colonic epithelial cells that expressed 5 Ig classes, our results suggested that cancer cells most prominently express IgG. Next, we found that the usage of Ig in cancer cells caused the expression of some unique Ig repertoires compared to normal cells. Some VH segments, such as VH3-7, have been used in cancer cells, and VH3-74 was frequently present in normal epithelial cells. Moreover, compared to the normal cell-derived Ig, most cancer cell-derived Ig showed unique VHDJH patterns.Importantly, even if the same VHDJH pattern was seen in cancer cells and normal cells, cancer cell-derived IgH always displayed distinct hypermutation hot points.CONCLUSION We found that colon cancer cells could frequently express IgG and unique IgH repertoires, which may be involved in carcinogenesis of colon cancer. The unique IgH repertoire has the potential to be used as a novel target in immune therapy for colon cancer.

Targeted Genotyping of a Whole-Gene Repertoire by an Ultrahigh-Multiplex and Flexible HD-Marker Approach

Targeted genotyping is an extremely powerful approach for the detection of known genetic variations that are biologically or clinically important.However,for non-model organisms,large-scale target geno-typing in a cost-effective manner remains a major challenge.To address this issue,we present an ultrahigh-multiplex,in-solution probe array-based high-throughput diverse marker genotyping(HD-Marker)approach that is capable of targeted genotyping of up to 86000 loci,with coverage of the whole gene repertoire,in what is a 27-fold and six-fold multiplex increase in comparison with the conventional Illumina GoldenGate and original HD-Marker assays,respectively.We perform extensive analyses of var-ious ultrahigh-multiplex levels of HD-Marker(30 k-plex,56 k-plex,and 86 k-plex)and show the power and excellent performance of the proposed method with an extremely high capture rate(about 96%)and genotyping accuracy(about 96%).With great advantages in terms of cost(as low as 0.0006 USD per geno-type)and high technical flexibility,HD-Marker is a highly efficient and powerful tool with broad appli-cation potential for genetic,ecological,and evolutionary studies of non-model organisms.

T-cell and B-cell repertoire diversity are selectively skewed in children with idiopathic nephrotic syndrome revealed by high-throughput sequencing

Background Clinical studies suggest that the dysfunction of T cells and B cells may play an essential role in the pathogenesis of idiopathic nephrotic syndrome(INS),but laboratory evidence is lacking.Therefore,this study explored T-cell receptor(TCR)and B-cell receptor(BCR)profiling in children with idiopathic nephrotic syndrome.Methods High-throughput sequencing technology was used to profile the TCR and BCR repertoires in children with INS.Peripheral blood was collected from ten INS patients,including five vinculin autoantibody-positive patients and five vin-culin autoantibody-negative patients,before and after treatment.TCR and BCR libraries were constructed by 5'-RACE and sequenced by a DNBSEQ-T7 sequencer,and sequence analyses were performed using ReSeqTools,FastP,MiXCR,and VDJtools.Results The TRA(T-cell receptorα),TRG(T-cell receptor y),and IGH(immunoglobulin heavy chain)repertoires of the INS group were occupied by highly abundant clonotypes,whereas small clonotypes occupied the healthy group,especially TRA.A significant increase in the Shannon-Weaver index was observed for the TRA and TRG repertoires after treatment in vinculin autoantibody-negative patients,but a significant increase in the IGH repertoire after treatment was observed in vinculin autoantibody-positive patients.The frequency of some V-J pairs was significantly enriched in steroid-sensitive nephrotic syndrome patients.The usage frequency of the V and J genes was skewed in patients,which seemed not related to immunosuppressive therapy.However,after effective treatment,dynamic changes in the size of the individual clonotype were observed.Conclusion T-cell and B-cell immunity contribute to the pathogenesis of different INSs.

Characteristics of rabbit hapten-specific and germline-based BCR repertoires following repeated immunization

The rabbit is well known for producing diverse antibodies against various antigens including small molecules such as drugs and toxins,due to a robust immune response.Elucidating how hapten repeated immunization shapes the rabbit B cell receptor(BCR)repertoire is crucial to understanding rabbit immune response to small molecules and assisting rare antibody discovery/engineering.In this study,we enriched and sequenced chloramphenicol(CAP)-specific rabbit B cells following repeated immunization,and analyzed both CAP-specific repertoires combined with the structure and affinity features of V1S69/V1S37 germline-based BCRs.The length of rabbit complementarity-determining region 3 of heavy chain(CDRH3)increased after hapten immunization.Repeated immunization significantly reduced the diversity of CAP-specific rabbit BCR clonotypes,and changed the frequency of VDJ usage and the type of V(D)J recombination.The average number of mutations among VL is notably higher than that of VH genes in rabbits,however,they are both not changed along with repeated immunization.Moreover,repeated immunization resulted in an increase surface charge and a decrease in solvent accessible surface area,leading to improvement in the stability of the most abundant V1S69/V1S37 germline-based BCR,along with an affinity increase from an IC50 of 898.2 ng mL^(−1)at the 1st immunization to 4.16 ng mL^(−1)at the 6th immunization.The study provides a benchmark for rabbit repertoire-scale analyses and offers a method for antibody discovery of small molecules.

Characterization of heavy-chain antibody gene repertoires in Bactrian camels

Camelids are the only mammals that can produce functional heavy-chain antibodies(HCAbs).Although HCAbs were discovered over 30 years ago,the antibody gene repertoire of Bactrian camels remains largely underexplored.To characterize the diversity of variable genes of HCAbs(VHHs),germline and rearranged VHH repertoires are constructed.Phylogenetics analysis shows that all camelid VHH genes are derived from a common ancestor and the nucleotide diversity of VHHs is similar across all camelid species.While species-specific hallmark sites are identified,the non-canonical cysteines specific to VHHs are distinct in Bactrian camels and dromedaries compared with alpacas.Though low divergence at the germline repertoire between wild and domestic Bactrian camels,higher expression of VHHs is observed in some wild Bactrian camels than that of domestic ones.This study not only adds our understanding of VHH repertoire diversity across camelids,but also provides useful resources for HCAb engineering.

Antibody upstream sequence diversity and its biological implications revealed by repertoire sequencing

The sequence upstream of the antibody variable region(antibody upstream sequence[AUS])consists of a 5′untranslated region(5′UTR)and a preceding leader region.The sequence variations in AUS affect antibody engineering and PCR based antibody quantification and may also be implicated in mRNA transcription and translation.However,the diversity of AUSs remains elusive.Using 5′rapid amplification of cDNA ends and high-throughput antibody repertoire sequencing technique,we acquired full-length AUSs for human,rhesus macaque,cynomolgus macaque,mouse,and rat.We designed a bioinformatics pipeline and identified 3307 unique AUSs,corresponding to 3026 and 1457 unique sequences for 5′UTR and leader region,respectively.Comparative analysis indicated that 928(63.69%)leader sequences are novel relative to those recorded in the international ImMunoGeneTics information system.Evolutionarily,leader sequences are more conserved than 5′UTR and seem to coevolve with their downstream V genes.Besides,single-nucleotide polymorphisms are position dependent for leader regions and may contribute to the functional reversal of the downstream V genes.Finally,the AUGs in AUSs were found to have little impact on gene expression.Taken together,our findings can facilitate primer design for capturing antibodies efficiently and provide a valuable resource for antibody engineering and molecule-level antibody studies.

Characterization of Distinct T Cell Receptor Repertoires in Tumor and Distant Non-tumor Tissues from Lung Cancer Patients

T cells and T cell receptors(TCRs)play pivotal roles in adaptive immune responses against tumors.The development of next-generation sequencing technologies has enabled the analysis of the TCRb repertoire usage.Given the scarce investigations on the TCR repertoire in lung cancer tissues,in this study,we analyzed TCRb repertoires in lung cancer tissues and the matched distant non-tumor lung tissues(normal lung tissues)from 15 lung cancer patients.Based on our results,the general distribution of T cell clones was similar between cancer tissues and normal lung tissues;however,the proportion of highly expanded clones was significantly higher in normal lung tissues than in cancer tissues(0.021%±0.002%vs.0.016%±0.001%,P=0.0054,Wilcoxon signed rank test).In addition,a significantly higher TCR diversity was observed in cancer tissues than in normal lung tissues(431.37±305.96 vs.166.20±101.58,P=0.0075,Mann-Whitney U test).Moreover,younger patients had a significantly higher TCR diversity than older patients(640.7±295.3 vs.291.8±233.6,P=0.036,Mann-Whitney U test),and the higher TCR diversity in tumors was significantly associated with worse cancer outcomes.Thus,we provided a comprehensive comparison of the TCR repertoires between cancer tissues and matched normal lung tissues and demonstrated the presence of distinct T cell immune microenvironments in lung cancer patients.

Characteristics of the TCR Vβ repertoire in imatinib-resistant chronic myeloid leukemia patients with ABL mutations

Diversity in the T cell receptor(TCR) repertoire provides a miniature defense ability for the T cell immune system that may be related to tumor initiation and progression. Understanding the T cell immune status of leukemia patients is critical for establishing specific immunotherapies. Previous studies have reported abnormal TCR repertoires and clonally expanded TCR V? T cells in chronic myeloid leukemia in chronic phase(CP-CML). In this study, we investigated the distribution and clonality of the TCR V? repertoire in 4 cases with imatinib-resistant CML in blast crisis(BC-CML) with abelson murine leukemia viral oncogene homolog 1(ABL1) kinase domain mutations(KDMs). Examination of TCR V? expression and clonality was performed by reverse transcription-polymerase chain reaction(RT-PCR) and Gene Scan analysis. Significantly skewed TCR V? repertoires were observed in BC-CML patients with different KDMs, and 4 to 8 oligoclonally expanded TCR V? subfamilies could be identified in each sample. Intriguingly, a relatively highly expanded V?9 clone with the same length as complementarity-determining region 3(CDR3)(139 bp) was found in all three CML patients in lymphoid blast crisis(LBC-CML) who had different KDMs, but the clone was not detected in the only CML patient in myeloid blast crisis(MBC-CML). In conclusion, restricted TCR V? repertoire expression and decreased clone complexity was a general phenomenon observed in the BC-CML patients with different KDMs, indicating the T-cell immunodeficiency of these patients. In addition, clonally expanded V?9 T cell clones may indicate a specific immune response to leukemia-associated antigens in LBC-CML patients.

Changes in the T-cell receptor Vβ gene repertoire after allogeneic hematopoietic stem cell transplantation

Background We distinguished graft-versus-host disease (GVHD) from graft-versus-leukemia (GVL) effects and to investigate the distribution of T-cell receptor (TCR) Vβ gene repertoire in individuals with leukemia before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods Peripheral blood mononuclear cells (PBMC) were obtained from 10 normal individuals, 8 donors and 11 patients with leukemia before and after transplantation. Polymerase chain reaction (PCR) amplification of complementarity-determining region 3 (CDR3) of 24 TCR Vβ genes was used to examine serial samples of PBMC. The PCR products were further analyzed by genescan to evaluate clonality of T cells.Results The 24 TCR Vβ gene repertoire displayed highly diverse and polyclonal spectratypes in all normal individuals and 4 of 8 donors. Anoth er 4 donors expressed part of the 24 TCR Vβ subfamily and 1 donor had oligoclonality. The expressions of the 24 TCR Vβ subfamilies were skewed and restric ted in 11 leukemia patients before and after transplantation. Some absences of 24 TCR Vβ subfamily expression were quite similar between the recipients pro-transplantation and related donors. The number of subfamilies expressed increased over time post-transplantation, but the restricted expressions of the subfamily could last 6-30 months after transplantation. All patients with GVHD and some without GVHD exhibited T cell clonal expansion. The expansive T cell clone was distributed in Vβ 2-3, 16-17, 18-19, 21 and Vβ 23 in patients with GVHD and in Vβ 7, 9, 16 and 19 in patients without GVHD. One patient with syngeneic-HSCT (syn-HSCT) had Vβ 15 and 16 T cell expansion after transplantation. One patient displayed Vβ 18 T cell expansion after donor lymphocyte infusion (DLI).Conclusions Normal individuals express the entire 24 TCR Vβ ge ne repertoire and have polyclonal distribution. However, the TCR Vβ gene repertoire is only partially expressed in some donors. The TCR Vβ gene repertoire is restrictedly expressed in a skew fashion in patients with leukemia before and after transplantation. The number of TCR Vβ gene subfamilies increases over time post- transplantation. GVHD and GVL effects may induce the proliferation of T cell clones.Clinical GVL response may be distinguished from GVHD alloreactivity through the host MHC antigen.

Characterization of human αβTCR repertoire and discovery of D-D fusion in TCRβ chains

The characterization of the human T-cell receptor （TCR） repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we ana- lyzed the diversity and complexity of both the TCRa and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene （TRAV42/DV1）. Moreover, we uncovered the presence of tandem TRBD （TRB D gene） usage in -2% of the productive human TCRβ CDR3 sequences.

Profiling the pattern of the human T-cell receptorγδcomplementary determinant region 3 repertoire in patients with lung carcinoma via high-throughput sequencing analysis

γδT cells function as sentinels in early host responses to infections and malignancies.Specifically,γδT cells recognize tumor-associated stress antigens via T-cell receptor(TCR)γδand play important roles in the antitumor immune response.In this study,we characterized the pattern of the human TCRγδcomplementary determinant region 3(CDR3)repertoire in patients with lung carcinoma(LC)via high-throughput sequencing.The results showed that the diversity of CDR3δwas significantly reduced,and that of CDR3γwas unchanged in LC patients compared with healthy individuals;in addition,LC patients shared significantly more CDR3δsequences with each other than healthy individuals.The CDR3 length distribution and N-addition length distribution did not significantly differ between LC patients and healthy individuals.In addition,the CDR3 repertoire tended to use more Vδ2 and fewer Vδ1 germline gene fragments among LC patients.Moreover,we found a combination of four TCRγδrepertoire features that focus on CDR3δand can be used as a biomarker for LC diagnosis.Our research suggests that the TCRγδCDR3 repertoire changed in LC patients due to the antitumor immune response byγδT cells in vivo,and these changes primarily focus on the amplification of certain tumor-specific CDR3δclones among patients.This study demonstrates the role ofγδT cells from the TCRγδCDR3 repertoire in tumor immunity and lays the foundation for elucidating the mechanism underlying the function ofγδT cells in antitumor immunity.

T-cell receptor repertoire analysis for the diagnosis and treatment of solid tumor: A methodology and clinical applications

T cells,which are involved in adaptive immunity,are essential in the elimination of tumor cells.Mature T cells can specifically recognize the antigen on the major histocompatibility complex(MHC)molecule through T-cell receptors(TCR).The unique rearrangement mechanisms during T-cell maturation provide great diversity to TCR,ensuring specific recognition between T cells and antigens.Thus,TCR repertoire analysis occupied an important position in T-cell regarding research.Nowadays,next-generation sequencing technology allows the simultaneous detection of TCR sequences with high throughput,and several evaluation indexes facilitate the measure of TCR repertoire.Based on this new methodology,discoveries are made across a range of tumor types.Results have shed light on the TCR repertoire differences between cancer patients and healthy control as well as between individual’s lesions,paracancer,and peripheral blood samples.The potential of TCR repertoire as a biomarker for immunotherapy efficacy is also widely studied as TCR repertoire represents different baseline within individuals and shows dynamic change during treatment.Accurate delineation of the T-cell repertoire can further the understanding of the immune system response to tumorigenesis.Still,existing researches are insufficient to clarify the specific clinical implications of TCR dynamic change and the definite role of TCR repertoire diversity during the treatment process.The results of some studies are even contrary.In this article,we reviewed TCR rearrangement mechanisms and analysis methods.Recent progress of TCR sequencing technology in tumor research is also discussed.In conclusion,intensive studies over an extended range of cancer types and a broadened group of subjects should be carried to solidify the TCR repertoire’s position as an immunotherapy biomarker.

T Cell Repertoire Diversity Is Decreased in Type 1 Diabetes Patients

Type 1 diabetes mellitus （T1D） is an immune-mediated disease. The autoreactive T cells in T1D patients attack and destroy their own pancreatic cells. In order to systematically investigate the potential autoreactive T cell receptors （TCRs）, we used a high-throughput immune repertoire sequencing technique to profile the spectrum of TCRs in individual T1D patients and controls. We sequenced the T cell repertoire of nine T1D patients, four type 2 diabetes （T2D） patients,and six nondiabetic controls. The diversity of the T cell repertoire in T1D patients was significantly decreased in comparison with T2D patients （P = 7.0E-08 for CD4＋ T cells, P = 1.4E-04 for CD8＋ T cells） and nondiabetic controls （P = 2.7E-09 for CD4＋ T cells, P = 7.6E-06 for CD8 ＋ T cells）. Moreover, T1D patients had significantly more highly-expanded T cell clones than T2D patients （P = 5.2E-06 for CD4＋ T cells, P = 1.9E-07 for CD8＋ T cells） and nondiabetic controls （P = 1.7E-07 for CD4＋ T cells, P =Y3E-03 for CD8＋ T cells）. Furthermore, we iden- tified a group of highly-expanded T cell receptor clones that are shared by more than two TID patients. Although further validation in larger cohorts is needed, our data suggest that T cell receptor diversity measurements may become a valuable tool in investigating diabetes, such as using the diversity as an index to distinguish different types of diabetes.

Can rarefaction be used to estimate song repertoire size in birds？

Song repertoire size is the number of distinct syllables, phrases, or song types produced by an individual or population. Repertoire size estimation is particularly difficult for species that produce highly variable songs and those that produce many song types. Estimating repertoire size is important for ecological and evolutionary studies of speciation, studies of sexual selection, as well as studies of how species may adapt their songs to various acoustic environments. There are several methods to estimate repertoire size, however prior studies discovered that all but a full numerical count of song types might have substantial inaccuracies associated with them. We evaluated a somewhat novel approach to estimate repertoire size--rarefaction; a technique ecologists use to measure species diversity on individual and population levels. Using the syllables within American robins＇ Turdus migratorius repertoire, we compared the most commonly used techniques of estimating repertoires to the results of a rarefaction analysis. American robins have elaborate and unique songs with few syllables shared between individuals, and there is no evidence that robins mimic their neighbors. Thus, they are an ideal system in which to compare techniques. We found that the rarefaction technique results resembled that of the numerical count, and were better than two alternative methods （behavioral accumulation curves, and capture-recapture） to estimate syllable repertoire size. Future estimates of repertoire size, particularly in vocally complex species, may benefit from using rarefaction techniques when numerical counts are unable to be performed [Current Zoology 57 （3）： 300-306, 2011].

Social and ecological complexity is associated with gestural repertoire size of wild chimpanzees

Increasing our understanding of primate gestural communication can provide new insights into language evolution.A key question in primate communication is the association between the social relationships of primates and their repertoire of gestures.Such analyses can reveal how primates use their repertoire of gestural communication to maintain their networks of family and friends,much as humans use language to maintain their social networks.In this study we examined the association between the repertoire of gestures(overall,manual and bodily gestures,and gestures of different modalities)and social bonds(presence of reciprocated grooming),coordinated behaviors(travel,resting,co-feeding),and the complexity of ecology(e.g.noise,illumination)and sociality(party size,audience),in wild East African chimpanzees(Pan troglodytes schweinfurthii).A larger repertoire size of manual,visual gestures was associated with the presence of a relationship based on reciprocated grooming and increases in social complexity.A smaller repertoire of manual tactile gestures occurred when the relationship was based on reciprocated grooming.A smaller repertoire of bodily gestures occurred between partners who jointly traveled for longer.Whereas gesture repertoire size was associated with social complexity,complex ecology also influenced repertoire size.The evolution of a large repertoire of manual,visual gestures may have been a key factor that enabled larger social groups to emerge during evolution.Thus,the evolution of the larger brains in hominins may have co-occurred with an increase in the cognitive complexity underpinning gestural communication and this,in turn,may have enabled hominins to live in more complex social groups.

Characterization of human IgM and IgG repertoires in individuals with chronic HIV-1 infection

Advancements in high-throughput sequencing(HTS)of antibody repertoires(Ig-Seq)have unprecedentedly improved our ability to characterize the antibody repertoires on a large scale.However,currently,only a few studies explored the influence of chronic HIV-1 infection on human antibody repertoires and many of them reached contradictory conclusions,possibly limited by inadequate sequencing depth and throughput.To better understand how HIV-1 infection would impact humoral immune system,in this study,we systematically analyzed the differences between the IgM(HIV-IgM)and IgG(HIV-IgG)heavy chain repertoires of HIV-1 infected patients,as well as between antibody repertoires of HIV-1 patients and healthy donors(HH).Notably,the public unique clones accounted for only a negligible proportion between the HIV-IgM and HIV-IgG repertoires libraries,and the diversity of unique clones in HIV-IgG remarkably reduced.In aspect of somatic mutation rates of CDR1 and CDR2,the HIV-IgG repertoire was higher than HIV-IgM.Besides,the average length of CDR3 region in HIV-IgM was significant longer than that in the HH repertoire,presumably caused by the great number of novel VDJ rearrangement patterns,especially a massive use of IGHJ6.Moreover,some of the B cell clonotypes had numerous clones,and somatic variants were detected within the clonotype lineage in HIV-IgG,indicating HIV-1 neutralizing activities.The in-depth characterization of HIV-IgG and HIV-IgM repertoires enriches our knowledge in the profound effect of HIV-1 infection on human antibody repertoires and may have practical value for the discovery of therapeutic antibodies.

Next-generation sequencing and single-cell RT-PCR reveal a distinct variable gene usage of porcine antibody repertoire following PEDV vaccination

Porcine epidemic diarrhea virus(PEDV)is the most common diarrhea-causing pathogen in newborn piglets.The clarifications of the overall antibody repertoire and antigen-specific antibody repertoire are essential to provide important insights into the B-cell response and reshape new vaccines.Here,we applied next-generation sequencing(NGS)technology to investigate immunoglobulin(Ig)variable(V)gene segment usage of swine B-cells from peripheral blood lymphocytes(PBL)and mesenteric lymph node(MLN)cells following PEDV vaccination.We identified the transcripts of all functional Ig V-genes in antibody repertoire.IgHV1 S2,IgKV1-11,and IgLV3-4 were the most prevalent gene segments for heavy,kappa,and lambda chains,respectively,in PBL and MLN.Unlike previous studies,IgKV1,instead of IgKV2,and IgLV3,instead of IgLV8,were the prevalent Ig V-gene families for kappa and lambda light chains,respectively.We further examined the antibody repertoire of PEDV spike-specific B cells by single-cell RT-PCR.In contrast to the overall antibody repertoire,Ig V-gene segments of PEDV spike-specific B cells preferentially adopted IgHV1-4 and IgHV1-14 for heavy chain,IgKV1-11 for kappa chain,and IgLV3-3 for lambda chain.These results represent a comprehensive analysis to characterize the Ig V-gene segment usage in the overall and PEDV spike-specific antibody repertoire in PBL and MLN.