Chain Complementarity-determining Region Research Articles

Restricted TcR β chain CDR3 Clonotype Is Associated with Resolved Acute Hepatitis B (AHB) patients with HBsAb

Background: T cells play an important role in the prognosis of HBV infection, and are involved in the seroconversion of a patient from HBsAb negative to HBsAb positive. To compare the T-cell receptor beta chain variable region (TcRBV) complementarity-determining region 3 (CDR3) in subjects with or without HBsAg seroconversion to HBsAb, the TcRBV was determined using high throughput sequencing (HTS). Methods: Clonotype and diversity of CDR3 in peripheral blood mononuclear cells (PBMCs) of subjects with resolved acute hepatitis B (AHB, HBsAb+, and HBsAg-) (n = 5), chronic hepatitis B (CHB, HBsAb-, and HBsAg+) (n = 5), and healthy controls (HC, HBsAb-, and HBsAg-) (n = 3) were determined and analyzed using HTS (MiSeq). Findings: The overlapping rate of CDR3 clones for any two samples in the AHB, CHB, and HC groups were 2.00% (1.74–2.30%), 1.77% (1.43–2.61%), and 1.82% (1.62%–2.12%), respectively; no significant difference was found among the three groups by Kruskal-Wallis H test. In addition, among the top 10 cumulative frequencies of clonotypes, only the frequency of clonotype (TcRBV20-1/TcRBD1/TcRBJ1-2) in the AHB group was lower than that in the HC group (P 0.05). Interpretation: Thus, there are 57 TcRBV clonotypes that may be related to HBsAg seroconversion in AHB subjects, but the diversity of TcRBV CDR3 is not significantly related to the HBsAb positive status. Funding Statement: This work was supported by National Natural Science Foundation of China (81671557), the Zhejiang Provincial Natural Science Foundation of China (LY19H190004), and the National Science and Technology Major Project (2018ZX10101-001). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: This project and protocols involving human subjects were approved by the ethics committee of the First Affiliated Hospital of Zhejiang University. Informed consent was obtained from each subject included in the study.

A stereotyped light chain may shape virus-specific B-cell receptors in HCV-dependent lymphoproliferative disorders

Hepatitis C virus (HCV) causes B-cell lymphoproliferative disorders (LPDs) expressing stereotyped B-cell receptors (BCRs) endowed with rheumatoid factor (RF) activity and putatively recognizing the HCV E2 protein. To further untangle the shaping and function of these BCRs, we analyzed immunoglobulin gene rearrangements of monoclonal B cells from 13 patients with HCV-associated LPDs and correlated their features with the clinical outcomes of antiviral therapy. While only two patients shared a stereotyped heavy-chain complementarity determining region 3 (CDR3) sequence, two kappa chain CDR3 stereotyped sequences accounted for 77% of BCRs. Light chains were enriched in sequences homologous to anti-HCV E2 antibodies compared with heavy chains (7/13 vs. 0/13; p = 0.005). Anti-HCV E2 homology was uniquely associated (7/7 vs. 0/6; p = 0.0006) with a stereotyped CDR3 sequence encoded by IGKV3-20/3D-20 gene(s) accounting for 54% of BCRs. An IGKV3-15/IGKJ1-encoded stereotyped sequence homologous to WA RF accounted for 23% of BCRs. LPDs expressing KCDR3s homologous to anti-HCV E2 antibodies responded more frequently to the eradication of HCV by antiviral therapy (6/6 vs. 1/6; p = 0.015). These findings, although limited by the small sample size, suggest that a stereotyped KCDR3 may predominantly shape anti-HCV specificity of BCRs, possibly providing a signature that may help identifying bona fide HCV-dependent LPDs.

Production and characterization of monoclonal antibodies against Toxoplasma gondii ROP18 with strain-specific reactivity.

The rhoptry kinase 18 of Toxoplasma gondii (TgROP18) has been identified as a key virulence factor that allows the parasite to escape from host immune defences and promotes its proliferation in host cells. Although much research is focused on the interaction between host cells and TgROP18, the development of monoclonal antibodies (mAbs) against TgROP18 has not been reported till date. To produce mAbs targeting TgROP18, two hybridomas secreting mAbs against TgROP18, designated as A1 and T2, were generated using cell fusion technology. The subtypes of the A1 and T2 mAbs were identified as IgG3 λ and IgM κ, and peptide scanning revealed that the core sequences of the antigenic epitopes were 180LRAQRRRSELVFE192 and 351NYFLLMMRAEADM363, respectively. The T2 mAb specifically reacted with both T. gondii type I and Chinese I, but not with T. gondii type II, Plasmodium falciparum or Schistosoma japonicum. Finally, the sequences of heavy chain and light chain complementarity-determining regions of T2 were amplified, cloned and characterized, making the modification of the mAb feasible in the future. The development of mAbs against TgROP18 would aid the investigation of the molecular mechanisms underlying the modulation of host cellular functions by TgROP18, and in the development of strategies to diagnose and treat Toxoplasmosis.

High-throughput sequencing reveals the diversity of TCR β chain CDR3 repertoire in patients with severe acne

Acne is a common chronic inflammatory skin disease, and the inflammation immune response runs through all stages of acne lesions. In this study, we use a combination of multiplex-PCR and high-throughput sequencing technologies to analyze T cell receptor β chain CDR3 (complementarity-determining region 3) in peripheral blood isolated from severe acne patients. Once compared with healthy controls, we propose to identify acne-relevant CDR3 peptides. Our results reveal that the diversity of T cell receptor β chain (TRB) CDR3 sequences in the peripheral blood of the severe acne vulgaris (SA) group differed from that of the control group. In addition, we find 10 TRB CDR3 sequences, amino acid sequences and V-J combinations with significantly different expressions between the SA group and the non-acne (NA) group (P < 0.0001). These findings may contribute to a better understanding of the role of immunity in the pathogenesis of acne and may serve as biomarkers for evaluating risk or prognosis of severe acne disease in future.

A novel mass spectrometric-based proteomic approach challenges the current paradigm on cryoglobulin typing in the diagnostic laboratory

Cryoglobulins precipitate out of serum at temperatures lower than 37°C and can have serious clinical manifestations. They may be associated with haematological malignancies, chronic infections and autoimmune diseases. Traditional typing of cryoglobulins use the Brouet classification, depending on the presence or not of rheumatoid factor activity: type I (monoclonal immunoglobulin with no rheumatoid factor), type II (mixed cryoglobulin with monoclonal rheumatoid factor) and type III (mixed cryoglobulin with polyclonal rheumatoid factor). Current methods used to type cryoglobulins include electrophoresis and immunofixation. We describe a novel, streamlined workflow of cryoglobulin analysis using mass spectrometry (MS) to characterise the proteomic signatures [heavy chain complementarity-determining region 3 (HCDR3) regions] of cryoglobulins. This provides an extension to traditional immunochemical methods of analysis and offers increased sensitivity; monitoring capacity by HCDR3 regions at the amino acid level; and clinical applications. Outstandingly, MS analysis offers predictive value by detecting the presence of pathogenic clonotypes before the onset of clinical manifestations. We highlight its applicability and benefits through clinical cases, demonstrating the greater ability to molecularly characterise cryoprecipitates in a similar turn-around time to current methodology. Work to establish clinically relevant libraries of antibody clonotypes is currently underway.

Long-Range Conformational Changes in Monoclonal Antibodies Revealed Using FPOP-LC-MS/MS.

Differences in conformational dynamics between two full-length monoclonal antibodies have been probed in detail using Fast Photochemical Oxidation of Proteins (FPOP) followed by proteolysis and LC-ESI-MS/MS analyses. FPOP uses hydroxyl radical labeling to probe the surface-accessible regions of proteins and has the advantage that the resulting covalent modifications are irreversible, thus permitting optimal downstream analysis. Despite the two monoclonal antibodies (mAbs) differing by only three amino acids in the heavy chain complementarity determining regions (CDRs), one mAb, MEDI1912-WFL, has been shown to undergo reversible self-association at high concentrations and exhibited poor pharmacokinetic properties in vivo, properties which are markedly improved in the variant, MEDI1912-STT. Identifying the differences in oxidative labeling between the two antibodies at residue level revealed long-range effects which provide a key insight into their conformational differences. Specifically, the amino acid mutations in the CDR region of the heavy chain resulted in significantly different labeling patterns at the interfaces of the CL-CH1 and CH1-CH2 domains, with the nonaggregating variant undergoing up to four times more labeling in this region than the aggregation prone variant, thus suggesting a change in the structure and orientation of the CL-CH1 interface. The wealth of FPOP and LC-MS data obtained enabled the study of the LC elution properties of FPOP-oxidized peptides. Some oxidized amino acids, specifically histidine and lysine, were noted to have unique effects on the retention time of the peptide, offering the promise of using such an analysis as an aid to MS/MS in assigning oxidation sites.

A generalized HIV vaccine design strategy for priming of broadly neutralizing antibody responses.

Vaccine induction of broadly neutralizing antibodies (bnAbs) to HIV remains a major challenge. Germline-targeting immunogens hold promise for initiating the induction of certain bnAb classes; yet for most bnAbs, a strong dependence on antibody heavy chain complementarity-determining region 3 (HCDR3) is a major barrier. Exploiting ultradeep human antibody sequencing data, we identified a diverse set of potential antibody precursors for a bnAb with dominant HCDR3 contacts. We then developed HIV envelope trimer-based immunogens that primed responses from rare bnAb-precursor B cells in a mouse model and bound a range of potential bnAb-precursor human naïve B cells in ex vivo screens. Our repertoire-guided germline-targeting approach provides a framework for priming the induction of many HIV bnAbs and could be applied to most HCDR3-dominant antibodies from other pathogens.

Immunoglobulin somatic hypermutation has clinical impact in DLBCL and potential implications for immune checkpoint blockade and neoantigen-based immunotherapies

BackgroundDiffuse large B-cell lymphoma (DLBCL) harbors somatic hypermutation (SHM) in the immunoglobulin heavy chain and light chain variable region genes, IGHV and IGK/LV. Recent studies have revealed that IGV SHM creates neoantigens that activate T-cell responses against B-cell lymphoma.MethodsTo determine the clinical relevance of IGV SHM in DLBCL treated with standard immunochemotherapy, we performed next-generation sequencing of the immunoglobulin variable regions and complementarity determining region 3 (CDR3) for 378 patients with de novo DLBCL. The prognostic effects of IGV SHM and ongoing SHM or intra-clonal heterogeneity were analyzed in the training (192 patients), validation (186 patients), and overall DLBCL cohorts. To gain mechanistic insight, we analyzed the predicted IG-derived neoantigens’ immunogenicity potential, determined by the major histocompatibility complex-binding affinity and the frequency-of-occurrence of T cell-exposed motifs (TCEMs) in a TCEM repertoire derived from human proteome, microbiome, and pathogen databases. Furthermore, IGV SHM was correlated with molecular characteristics of DLBCL and PD-1/L1 expression in the tumor microenvironment assessed by fluorescent multiplex immunohistochemistry.ResultsSHM was commonly found in IGHV and less frequently in IGK/LV. High levels of clonal IGHV SHM (SHMhigh) were associated with prolonged overall survival in DLBCL patients, particularly those without BCL2 or MYC translocation. In contrast, long heavy chain CDR3 length, the presence of IGHV ongoing SHM in DLBCL, and high clonal IGK/LV SHM in germinal center B-cell–like (GCB)-DLBCL were associated with poor prognosis. These prognostic effects were significant in both the training and validation sets. By prediction, the SHMhigh groups harbored more potentially immune-stimulatory neoantigens with high binding affinity and rare TCEMs. PD-1/L1 expression in CD8+ T cells was significantly lower in IGHV SHMhigh than in SHMlow patients with activated B-cell–like DLBCL, whereas PD-1 expression in CD4+ T cells and PD-L1 expression in natural killer cells were higher in IGK/LV SHMhigh than in SHMlow patients with GCB-DLBCL. PD-L1/L2 (9p24.1) amplification was associated with high IGHV SHM and ongoing SHM.ConclusionsThese results show for the first time that IGV SHMhigh and ongoing SHM have prognostic effects in DLBCL and potential implications for PD-1/PD-L1 blockade and neoantigen-based immunotherapies.

High-Throughput T Cell Receptor Sequencing Reveals Differential Immune Repertoires in Autoimmune Thyroid Disease

Background: Autoimmune thyroid diseases (AITDs) are chronic autoimmune diseases specific to thyroid and mainly include Graves' diseases (GD) and Hashimoto' thyroiditis (HT). The adaptive immunoreactivity of CD4+ T cells plays a crucial role in the pathogenesis of AITDs, but very little has been known about its changes in disease status. Methods: We collected peripheral CD4+ T cells from 12 GD patients, including 6 newly diagnosed GD (NGD) and 6 refractory GD (RGD) patients, 6 HT patients and 6 healthy controls, and examined the gene expression profiles of T cells receptor (TCR) β chain complementarity determining region 3 (CDR3) using high-throughput sequencing. Results: Some TCR genes were expressed more preferentially in AITDs group than in the healthy control group, including TRBV15 (P=0.001), TRBV4-2 (P=0.003), TRBV9 (P=0.007), TRBV3-2 (P=0.012), TRBV7-8 (P=0.015), TRBV25-1 (P=0.019), TRBV12-4 (P=0.019) and TRBV27 (P=0.02) in GD patients as well as TRBV29-1 (P=0.004), TRBV12-4 (P=0.004), TRBV6-5 (P=0.011), TRBV7-2 (P=0.012), TRBV27 (P=0.012), TRBV9 (P=0.031) and TRBV4-2 (P=0.032) in HT patients. Moreover, subgroup analysis of GD showed that the difference in the TCR spectrum between the normal group and NGD was not obvious, but a large number of differential genes appeared in the RGD group. Conclusion: TCR spectrum has changed in patients with AITDs with many genes being upregulated. Moreover, this difference is not apparent in GD patients at the initial stage, but as the disease progresses, the differences in TCR profiles became more pronounced. Funding: The present work was supported by grants from the Science and Technology Development Fund of Pudong New District Minsheng Scientific Research (Medical and Health) Project (No. PKJ2018-Y39) and the National Natural Science Foundation of China (Nos. 81873636 and 81670722). Declaration of Interest: The authors declare that they have no conflict of interest. Ethical Approval: The study was approved by the Ethics Committee of Zhoupu Hospital. All subjects were informed of the present research project and signed written informed consent.

Somatic hypermutation to counter a globally rare viral immunotype drove off-track antibodies in the CAP256-VRC26 HIV-1 V2-directed bNAb lineage.

Previously we have described the V2-directed CAP256-VRC26 lineage that includes broadly neutralizing antibodies (bNAbs) that neutralize globally diverse strains of HIV. We also identified highly mutated “off-track” lineage members that share high sequence identity to broad members but lack breadth. Here, we defined the mutations that limit the breadth of these antibodies and the probability of their emergence. Mutants and chimeras between two pairs of closely related antibodies were generated: CAP256.04 and CAP256.25 (30% and 63% breadth, respectively) and CAP256.20 and CAP256.27 (2% and 59% breadth). Antibodies were tested against 14 heterologous HIV-1 viruses and select mutants to assess breadth and epitope specificity. A single R100rA mutation in the third heavy chain complementarity-determining region (CDRH3) introduced breadth into CAP256.04, but all three CAP256.25 heavy chain CDRs were required for potency. In contrast, in the CAP256.20/27 chimeras, replacing only the CDRH3 of CAP256.20 with that of CAP256.27 completely recapitulated breadth and potency, likely through the introduction of three charge-reducing mutations. In this individual, the mutations that limited the breadth of the off-track antibodies were predicted to occur with a higher probability than those in the naturally paired bNAbs, suggesting a low barrier to the evolution of the off-track phenotype. Mapping studies to determine the viral immunotypes (or epitope variants) that selected off-track antibodies indicated that unlike broader lineage members, CAP256.20 preferentially neutralized viruses containing 169Q. This suggests that this globally rare immunotype, which was common in donor CAP256, drove the off-track phenotype. These data show that affinity maturation to counter globally rare viral immunotypes can drive antibodies within a broad lineage along multiple pathways towards strain-specificity. Defining developmental pathways towards and away from breadth may facilitate the selection of immunogens that elicit bNAbs and minimize off-track antibodies.

Identification of multiple potent neutralizing and non-neutralizing antibodies against Epstein-Barr virus gp350 protein with potential for clinical application and as reagents for mapping immunodominant epitopes

Prevention of Epstein-Barr virus (EBV) infection has focused on generating neutralizing antibodies (nAbs) targeting the major envelope glycoprotein gp350/220 (gp350). In this study, we generated 23 hybridomas producing gp350-specific antibodies. We compared the candidate gp350-specific antibodies to the well-characterized nAb 72A1 by: (1) testing their ability to detect gp350 using enzyme-linked immunosorbent assay, flow cytometry, and immunoblot; (2) sequencing their heavy and light chain complementarity-determining regions (CDRs); (3) measuring the ability of each monoclonal antibody (mAb) to neutralize EBV infection in vitro; and (4) mapping the gp350 amino acids bound by the mAbs using competitive cell and linear peptide binding assays. We performed sequence analysis to identify 15 mAbs with CDR regions unique from those of murine 72A1 (m72A1). We observed antigen binding competition between biotinylated m72A1, serially diluted unlabeled gp350 nAbs (HB1, HB5, HB11, HB20), and our recently humanized 72A1, but not gp350 non-nAb (HB17) or anti-KSHV gH/gL antibody.

Composition and diversity analysis of the B-cell receptor immunoglobulin heavy chain complementarity-determining region 3 repertoire in patients with acute rejection after kidney transplantation using high-throughput sequencing.

The aim of the present study was to assess the genetic diversity of the B-cell receptor (BCR) in kidney transplant recipients with acute rejection. A total of three patients with acute rejection after kidney transplantation were examined by performing a composition and diversity analysis of the BCR immunoglobulin heavy chain (IGH) complementarity-determining region 3 (H-CDR3) repertoire. The peripheral blood mononuclear cells of patients were collected at 1 day prior to (Pre1), as well as 1 day (Post1) and 7 days (Post7) after the transplantation, and DNA was extracted. High-throughput sequencing technology was applied to determine the BCR repertoire. Raw sequences in FASTQ format were analyzed with the Basic Local Alignment Search Tool. The diversity of the BCR repertoire was assessed by calculating Shannon entropy, Simpson's diversity index, the Gini coefficient and highly expanded clone distributions. The diversity of the BCR repertoire at Pre1 was greater than that at Post1 or Post7. The diversity of the BCR repertoire was the lowest at Post1 and increased at Post7 but failed to reach the pre-transplantation levels. Patients exhibited the loss of seven IGH variable (IGHV)3 family genes, while five new genes were expressed at a low frequency. Furthermore, five IGHV-IGH joining (IGHJ) gene pairings, including IGHJ6-IGHV3-11, were detected in the patients. Up- and downregulated genes were assessed by calculating the expression frequencies of the IGH diversity and IGHV gene families at Post1 and Post7. The results of the H-CDR3 length distribution and H-CDR3 amino acid (AA) usage analyses indicated that in Case 1 and 2, the AA length was similar at mostly 14–18 AA, while that in Case 3 was relatively stable at 12–16 AA. In conclusion, the present results illustrate the diversity of H-CDR3 in patients with acute rejection after kidney transplantation may provide novel ideas, methods and means of monitoring and analyzing the immune status of patients under physiological and pathological conditions.

Selective Tryptophan Oxidation of Monoclonal Antibodies: Oxidative Stress and Modeling Prediction.

Oxidation of tryptophan not only generates heterogeneity of a therapeutic monoclonal antibody (mAb) but also can be a potential critical quality attribute (CQA) of the product. In this study, mAbs A-C of IgG1 and IgG4 (immunoglobulin G, IgG) isotypes with oxidized tryptophan (Trp) residues were selectively generated by incubating the mAbs with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) in formulations containing l-methionine. The site-specific oxidation of tryptophan residues were confirmed by liquid chromatography coupled with mass spectrometry (LC-MS) studies. The site of oxidation was identified to be a conserved tryptophan residue in the heavy chain complementarity determining region 3 (CDR3) of mAbs A and B with no significant oxidation found on other tryptophan residues including those in close proximity to CDR3. For mAb C, all tryptophan residues including one in the heavy chain CDR1 and a tryptophan in close proximity to heavy chain CDR3 were not susceptible to oxidation. For all three mAbs, the structure and tryptophan oxidation relationship was further studied by computational modeling of the variable domain of the antibodies (variable fragment, Fv). The computational modeling provided a structural understanding at the molecular level to the tryptophan oxidation, where high solvent accessibility is a prerequisite for heavy chain CDR3 tryptophan oxidation. However, higher oxidation susceptibility of tryptophan in heavy chain CDR3 did not linearly correlate to higher solvent accessibility, suggesting that other factors including side-chain orientation and/or surrounding structure elements around the heavy chain CDR3 may also be involved. Through this study, we demonstrate that a selective oxidation system, together with computational modeling, can be an important tool to identify potential CQAs of a therapeutic mAb such as tryptophan oxidation liabilities during the mAb's development.

Plant-derived chimeric antibodies inhibit the invasion of human fibroblasts by Toxoplasma gondii.

The parasite Toxoplasma gondii causes an opportunistic infection, that is, particularly severe in immunocompromised patients, infants, and neonates. Current antiparasitic drugs are teratogenic and cause hypersensitivity-based toxic side effects especially during prolonged treatment. Furthermore, the recent emergence of drug-resistant toxoplasmosis has reduced the therapeutic impact of such drugs. In an effort to develop recombinant antibodies as a therapeutic alternative, a panel of affinity-matured, T. gondii tachyzoite-specific single-chain variable fragment (scFv) antibodies was selected by phage display and bioinformatic analysis. Further affinity optimization was attempted by introducing point mutations at hotspots within light chain complementarity-determining region 2. This strategy yielded four mutated scFv sequences and a parental scFv that were used to produce five mouse–human chimeric IgGs in Nicotiana benthamiana plants, with yields of 33–72 mg/kg of plant tissue. Immunological analysis confirmed the specific binding of these plant-derived antibodies to T. gondii tachyzoites, and in vitro efficacy was demonstrated by their ability to inhibit the invasion of human fibroblasts and impair parasite infectivity. These novel recombinant antibodies could therefore be suitable for the development of plant-derived immunotherapeutic interventions against toxoplasmosis.

Abstract CT010: Pharmacodynamic (PD) changes in tumor RNA expression and the peripheral blood T cell receptor (TCR) repertoire in a phase I study of OX40 agonistic monoclonal antibody (mAb) PF-04518600 (PF-8600)

Abstract Introduction: PF-8600 is a fully human IgG2 agonistic mAb against the tumor necrosis factor superfamily receptor OX40. OX40 improves T cell survival, proliferation, and activation and may enhance anti-tumor immunity. In an ongoing phase I study (NCT02315066) of 52 patients (pts) with melanoma, hepatocellular carcinoma, head and neck carcinoma, or renal cell carcinoma treated with PF-8600 monotherapy in dose escalation, 2 pts had best overall response of partial response (PR) and 27 of stable disease. PF-8600 was well-tolerated at all doses. Peripheral blood flow cytometry had previously shown increased proliferation and activation of CD4 central memory T cells at certain dose levels, suggesting a PD effect. RNAseq analysis of tumor biopsy samples and TCR sequencing of peripheral blood were used to further support proof of mechanism. Methods: Biopsy samples at baseline and wk 6 were collected from 5 dose cohorts [0.1 (4), 0.3 (3), 1.5 (4), 3.0 (3) and 10.0 (2), dose in mg/kg (n)]. Biopsy tissue was analyzed by RNAseq and gene ranking-based gene set enrichment analysis (FGSEA) to identify immune pathways potentially up-regulated by OX40. CD4/CD8 cells were isolated from peripheral blood samples from 4 dose cohorts (0.1, 0.3, 1.5, and 3.0 mg/kg). DNA was extracted for sequencing of the TCR-β chain complementarity-determining region 3 (CDR3). Results: In a combined analysis of samples from pts dosed with ≥1.5 mg/kg, the top 3 gene sets showing enrichment at wk 6 of therapy were associated with inflammatory response, interferon-γ response and allograft rejection. These gene sets were identical to the top 3 most enriched in tumor from a syngeneic mouse tumor model exposed to a murine OX40 agonist. This pattern of enrichment was not observed if samples from lower doses were included in the analysis. TCR sequencing revealed clonal expansion of CD4/CD8 T cells at all dose levels at wk 6 [CD4: mean = 8 expanded clones per 100,000 clones (range = 1 - 80), CD8: mean = 56 (range = 1 - 500)]. The 2 patients with PR had among the lowest numbers of expanded CD4 and CD8 clones (CD4: 4 and 2; CD8: 7 and 4). Conclusion: Enrichment of gene sets associated with immune activation in tumor tissue from patients dosed with PF-8600 provides evidence supporting an active, immunomodulatory mechanism. Peripheral CD4/CD8 T cell populations exhibited clonal expansion in response to dosing with PF-8600 at all dose levels further suggesting a PD effect. However, clinical response did not necessarily correlate with a high number of expanded T cell clones, suggesting that clinical response to OX-40 agonism may be driven by the expansion of select anti-tumor T cell clones rather than a broad expansion of T cell clones in the peripheral blood. The phase I study will continue to evaluate PD changes in the tumor and peripheral blood in dose-expansion cohorts of PF-8600 ± utomilumab. Citation Format: Adi Diab, Omid Hamid, John A. Thompson, Willeke Ros, Fredericus A. L. M. Eskens, Toshihiko Doi, Siwen Hu-Lieskovan, Hua Long, Tenshang Joh, Shoba Potluri, Xiao Wang, Catherine Fleener, Carrie Turich Taylor, Bishu J. Ganguli, Jeffrey Chou, Anthony B. El-Khoueiry. Pharmacodynamic (PD) changes in tumor RNA expression and the peripheral blood T cell receptor (TCR) repertoire in a phase I study of OX40 agonistic monoclonal antibody (mAb) PF-04518600 (PF-8600) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT010.

Association of the characteristics of B- and T-cell repertoires with papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Complementarity-determining region 3 (CDR3) of B-cell receptors (BCRs) and T-cell receptors are the major site of antigen recognition, which determines a unique clone type, and are considered to be the representative of the disease. In the present study, high-throughput sequencing was used to analyze the association of characteristics of the BCR immunoglobulin heavy chain (IGH) and the T-cell receptor β chain (TRB) CDR3 genes in PTC and corresponding pericarcinous tissues from patients. A difference of CDR3 length distributions of total IGH CDR3 sequences between the two groups was revealed. IGHV3-11/IGHJ6, TRBV2/TRBJ1-2 and TRBV2/TRBJ1-1 may be biomarkers for the development of PTC. Furthermore, it was revealed that the extent of the common clonotype expressions at the amino acid level was slightly higher compared with the nucleotide level. The Shannon entropy demonstrated a diversity reduction in PTC compared with the pericarcinous group, and the highly expended clone (HEC) expression of PTC was higher compared with that of the corresponding pericarcinous group. Additionally, the highest clone frequency percentage of IGH and TRB was at 0.1–1.0% degree of expansion, as HEC expression was higher in PTC compared with the matched group. There was no shared clone of HECs in the two groups either at the amino acid level or at the nucleotide expression level. The differential expression of CDR3 sequences of PTC have been identified in the present study. Further research is required for assessing the immune repertoire size, diversity, cloning tracking and finding public clones of T-cell and B-cell populations in the development of PTC.

High-Throughput Sequencing of the Expressed Torafugu (Takifugu rubripes) Antibody Sequences Distinguishes IgM and IgT Repertoires and Reveals Evidence of Convergent Evolution.

B-cell antigen receptor (BCR) or antibody diversity arises from somatic recombination of immunoglobulin (Ig) gene segments and is concentrated within the Ig heavy (H) chain complementarity-determining region 3 (CDR-H3). We performed high-throughput sequencing of the expressed antibody heavy-chain repertoire from adult torafugu. We found that torafugu use between 70 and 82% of all possible V (variable), D (diversity), and J (joining) gene segment combinations and that they share a similar frequency distribution of these VDJ combinations. The CDR-H3 sequence repertoire observed in individuals is biased with the preferential use of a small number of VDJ, dominated by sequences containing inserted nucleotides. We uncovered the common CDR-H3 amino-acid (aa) sequences shared by individuals. Common CDR-H3 sequences feature highly convergent nucleic-acid recombination compared with private ones. Finally, we observed differences in repertoires between IgM and IgT, including the unequal usage frequencies of V gene segment and the biased number of nucleotide insertion/deletion at VDJ junction regions that leads to distinct distributions of CDR-H3 lengths.

Characteristics of IgH-CDR3 repertoire of peripheral B cells in a patient with primary biliary cholangitis: a preliminary study using high-throughput sequencing

Objective: To analyze the characteristics of immunoglobulin heavy chain complementarity-determining region (IgH-CDR3) repertoire of peripheral B cells in a patient with primary biliary cholangitis (PBC) and to investigate the diversity of the immune system. Methods: Arm-PCR was used to amplify the IgH-CDR3 region of circulating B cells isolated from a PBC patient, and high-throughput sequencing was used to analyze the amplified product. The characteristics of immune repertoire were analyzed by bioinformatics. Results: In total, 329219 sequence reads were generated from the sample, with 325540 total CDR3 sequences and 72774 distinct CDR3 sequences, and the D50 of IGH-CDR3 was 7.7. The dominant CDR3 length of the sample was 45 nt (9.6%); the N addition with the highest frequency ranged from 13 to 14 nt (5.25%); the J trimming with the highest frequency was 0 nt (12.7%); the three most frequent V alleles were V4-59 (9.5%), V3-23 (8.1%), and V1-69 (6.4%). Conclusion: The diversity of IgH-CDR3 repertoire is relatively low in this patient with PBC, with several B-cell clonal expansions. The specificity needs to be further verified after increasing the sample size.

HIV Envelope Glycoform Heterogeneity and Localized Diversity Govern the Initiation and Maturation of a V2 Apex Broadly Neutralizing Antibody Lineage

SUMMARYUnderstanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germ-line B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design.

Dynamic Perturbations of CD4 and CD8 T Cell Receptor Repertoires in Chronic Hepatitis B Patients upon Oral Antiviral Therapy.

Long-term treatment with nucleos(t)ide analogs (NUCs) can improve the antiviral T cell response in chronic hepatitis B (CHB) patients. Whether and to what extent the T cell response is improved by NUCs in the early stage leading to hepatitis B e antigen (HBeAg) seroconversion remain to be clarified. A total of 22 CHB patients undergoing 2-year telbivudine-based therapy were enrolled, including 10 exhibiting a complete response (CR) and 12 exhibiting a non-complete response (NCR) according to HBeAg seroconversion at week 52. Peripheral CD4+ and CD8+ T cells were sorted at baseline, weeks 12, and 24. The T cell receptor β chain (TCRβ) complementarity-determining region 3 was analyzed by unbiased high-throughput sequencing. Compared with NCR group, patients in CR group had a much lower percentage of persistent clonotypes (P < 0.001) but remarkably higher percentages of new and expanded clonotypes (P < 0.05) between any two time points for both CD4 and CD8 subsets. The CD4 T cells exhibited a stronger response than CD8 population in the patients. The number of new and expanded clonotypes was inversely associated with the decline of viral antigen. In conclusion, NUC-based therapy induces a broad and vigorous T cell response with rapid decline of antigenemia during the early stage of treatment. A broad T cell expansion is crucial for HBeAg seroconversion. Our findings suggest that the potent suppression of hepatitis B virus replication by NUC monotherapy complemented with additional immunomodulatory strategies may increase the likelihood of a functional cure for CHB in the future.