BACKGROUND: In this study, we comprehensively profiled the T-cell receptor (TCR) repertoire of the tumor and adjacent normal tissue in patients with HBV-associated hepatocellular carcinoma (HCC) and determined the baseline characteristics and clinical significance of TCR.
METHODS: High-throughput sequencing was used to determine the profile of complementarity-determining region 3 (CDR3) of the TCR-β chain variable (TRBV) in the tumor and normal tissue samples of 14 HCC patients. At the same time, TRBV diversity and differences in expression between tumor and normal tissues were investigated. The cumulative frequency of top 100 CDR3 (CF100), clonality, and Shannon entropy as indices to evaluate diversity, RESULTS: The diversity of TRBV CDR3 showed no significant difference between tumor and normal tissues. Of the 58 V gene segments in TRBV, TRBV16 and TRBV7-6 had a significantly higher frequency in the tumor group than in the normal group (p < 0.05). The frequency of 14 J gene segments showed no significant difference between tumor and normal tissues. In contrast, the frequency of 22 TRBVx/BJx combinations was significantly higher in the tumor than in the normal tissue. In addition, the length and type of TRBV CDR3 were similar in tumor and normal tissues, and a Gaussian distribution was observed in both groups.
CONCLUSIONS: This study provided a large amount of information about the TCR lineage in HBV-associated HCC, laying the foundation for further research. In addition, the fact that the immune repertoire (TRBV CDR3) hardly differs between tumor and adjacent normal tissue provides a new clue for exploring the mechanism of the liver as an organ with immune privileges.

Artificial Intelligence (AI) techniques have made great advances in assisting antibody design. However, antibody design still heavily relies on isolating antigen-specific antibodies from serum, which is a resource-intensive and time-consuming process. To address this issue, we propose a Pre-trained Antibody generative large Language Model (PALM-H3) for the de novo generation of artificial antibodies heavy chain complementarity-determining region 3 (CDRH3) with desired antigen-binding specificity, reducing the reliance on natural antibodies. We also build a high-precision model antigen-antibody binder (A2binder) that pairs antigen epitope sequences with antibody sequences to predict binding specificity and affinity. PALM-H3-generated antibodies exhibit binding ability to SARS-CoV-2 antigens, including the emerging XBB variant, as confirmed through in-silico analysis and in-vitro assays. The in-vitro assays validate that PALM-H3-generated antibodies achieve high binding affinity and potent neutralization capability against spike proteins of SARS-CoV-2 wild-type, Alpha, Delta, and the emerging XBB variant. Meanwhile, A2binder demonstrates exceptional predictive performance on binding specificity for various epitopes and variants. Furthermore, by incorporating the attention mechanism inherent in the Roformer architecture into the PALM-H3 model, we improve its interpretability, providing crucial insights into the fundamental principles of antibody design.

UNASSIGNED: Humanization is typically adopted to reduce the immunogenicity of murine antibodies generated by hybridoma technology when used in humans.
UNASSIGNED: Two different strategies of antibody humanization are popularly employed, including \"complementarity determining region (CDR) grafting\" and \"framework (FR) shuffling\" to humanize a murine antibody against human programmed death-1 (PD-1), XM PD1. In CDR-grafting humanization, the CDRs of XM PD-1, were grafted into the human FR regions with high homology to the murine FR counterparts, and back mutations of key residues were performed to retain the antigen-binding affinities. While in FR-shuffling humanization, a combinatorial library of the six murine CDRs in-frame of XM PD-1 was constructed to a pool of human germline FRs for high-throughput screening for the most favorable variants. We evaluated many aspects which were important during antibody development of the molecules obtained by the two methods, including antibody purity, thermal stability, binding efficacy, predicted humanness, and immunogenicity, along with T cell epitope prediction for the humanized antibodies.
UNASSIGNED: While the ideal molecule was not achieved through CDR grafting in this particular instance, FR-shuffling proved successful in identifying a suitable candidate. The study highlights FR-shuffling as an effective complementary approach that potentially increases the success rate of antibody humanization. It is particularly noted for its accessibility to those with a biological rather than a computational background.
UNASSIGNED: The insights from this comparison are intended to assist other researchers in selecting appropriate humanization strategies for drug development, contributing to broader application and understanding in the field.

A recent study showed that just one point mutation F33 to Y in the complementarity-determining region 1 of heavy chain (H-CDR1) could lead to the auto-antibody losing its DNA binding ability. However, the potential molecular mechanisms have not been well elucidated. In this study, we investigated how the antibody lost the DNA binding ability caused by mutation F33 to Y in the H-CDR1. We found that the electrostatic force was not the primary driving force for the interaction between anti-DNA antibodies and the antigen single strand DNA (ssDNA), and that the H-CDR2 largely contributed to the binding of antigen ssDNA, even larger than H-CDR1. The H-F33Y mutation could increase the hydrogen-bond interaction but impair the pi-pi stacking interaction between the antibody and ssDNA. We further found that F33H, W98H and Y95L in the wiletype antibody could form the stable pi-pi stacking interaction with the nucleotide bases of ssDNA. However, the Y33 in mutant could not form the parallel sandwich pi-pi stacking interaction with the ssDNA. To further confirm the importance of pi-pi stacking, the wildtype antibody and the mutants (F33YH, F33AH, W98AH and Y95AL) were experimentally expressed in CHO cells and purified, and the results from ELISA clearly showed that all the mutants lost the ssDNA binding ability. Taken together, our findings may not only deepen the understanding of the underlying interaction mechanism between autoantibody and antigen, but also broad implications in the field of antibody engineer.

Accurate prediction of the structurally diverse complementarity determining region heavy chain 3 (CDR-H3) loop structure remains a primary and long-standing challenge for antibody modeling. Here, we present the H3-OPT toolkit for predicting the 3D structures of monoclonal antibodies and nanobodies. H3-OPT combines the strengths of AlphaFold2 with a pre-trained protein language model and provides a 2.24 Å average RMSDCα between predicted and experimentally determined CDR-H3 loops, thus outperforming other current computational methods in our non-redundant high-quality dataset. The model was validated by experimentally solving three structures of anti-VEGF nanobodies predicted by H3-OPT. We examined the potential applications of H3-OPT through analyzing antibody surface properties and antibody-antigen interactions. This structural prediction tool can be used to optimize antibody-antigen binding and engineer therapeutic antibodies with biophysical properties for specialized drug administration route.

BACKGROUND: Uremia-associated immunodeficiency, mainly characterized by T cell dysfunction, exists in patients on maintenance hemodialysis (MHD) and promotes systemic inflammation. However, T cell senescence, one of the causes of T cell dysfunction, has not been clearly revealed yet. In this cross-sectional research, we aimed to study the manifestation of T cell premature senescence in MHD patients and further investigate the associated clinical factors.
METHODS: 76 MHD patients including 33 patients with cardiovascular diseases (CVD) and 28 patients with arteriovenous fistula (AVF) event history were enrolled in this study. Complementarity determining region 3 (CDR3) of T cell receptor (TCR) was analyzed by immune repertoire sequencing (IR-Seq). CD28- T cell subsets and expression of senescence marker p16 and p21 genes were detected by multicolor flow cytometry and RT-qPCR, respectively.
RESULTS: MHD patients had significantly decreased TCR diversity (P < 0.001), increased CDR3 clone proliferation (P = 0.001) and a left-skewed CDR3 length distribution. The proportion of CD4 + CD28- T cells increased in MHD patients (P = 0.014) and showed a negative correlation with TCR diversity (P = 0.001). p16 but not p21 expression in T cells was up-regulated in MHD patients (P = 0.039). Patients with CVD exhibited increased expression of p16 and p21 genes (P = 0.010 and 0.004, respectively), and patients with AVF events showed further TCR diversity and evenness reduction (P = 0.002 and 0.017, respectively) compared to patients without the comorbidities. Moreover, age, average convection volume, total cholesterol, high-density lipoprotein cholesterol and transferrin saturation were associated with TCR diversity or CD4 + CD28- T cell proportion (P < 0.05).
CONCLUSIONS: MHD patients undergo T cell premature senescence characterized by significant TCR diversity reduction and repertoire skew, as well as accumulation of the CD4 + CD28- subset and up-regulation of p16 gene. Patients with CVD or AVF events show higher level of immunosenescence. Furthermore, T cell senescence in MHD patients is associated with blood cholesterol and uremic toxin retention, suggesting potential intervention strategies in the future.

The thymus-derived lymphocytes of jawed vertebrates have four T-cell receptor (TCR) chains that play a significant role in immunity. As chickens have commercial value, their immune systems require a great deal of attention. Local chicken breeds are an essential part of poultry genetic resources in China. Here, we used high-throughput sequencing to analyze the TCRα and TCRβ repertoires and their relative expression levels in the native chicken breeds Baier Buff, Longyou Partridge, Xiaoshan, and Xianju. We found that TCR Vα and TCR Vβ were expressed and included 17, 19, 17, and six segments of the Vα2, Vα3, Vβ1, and Vβ2 subgroups, respectively. V-J pairing was biased; Jα11 was utilized by nearly all Vα segments and was the most commonly used. Breed-specific V segments and V-J pairings were detected as well. The results of the principal coordinate analysis (PCoA) as well as the V-J pairing and CDR3 diversity analyses suggested that the four local chicken breeds did not significantly differ in terms of TCR diversity. Hence, they expressed not significant differentiation, and they are rich genetic resources for the development and utilization of immune-related poultry breeding.

The γδ T cells are a subpopulation of T cells that are abundantly found in the skin and mucous membranes. Their reactivity to self-antigens and ability to secrete various cytokines make them a key component in psoriasis development. Although the correlation between the immune repertoire (IR) of γδ T-cell receptors and the occurrence and severity of psoriasis remains incompletely explored, high-throughput sequencing of γδ T cells has led to a deeper understanding of IR in psoriasis. This study investigated the differences between γδ T cells in patients with psoriasis and healthy controls. The γδ T cells were identified via immunofluorescence staining and a correlation analysis was performed according to the psoriasis area and severity index (PASI) scores. The IR sequencing method was used to detect IR in the γδ T-cell receptors. The findings demonstrated more skin γδ T cells in patients with psoriasis, which were positively correlated with the PASI score. There were subtle differences in most variable (V), diversity (D) and joining (J) gene segments and VJ/VDJ combination segments between patients with psoriasis and healthy controls. However, a higher diversity of complementarity-determining region 3 (CDR3) was observed in patients with psoriasis. In summary, the IR of skin γδ T cells was significantly altered in patients with psoriasis, and the diversity in the cell\'s CDR3 population is a promising biomarker for assessment of psoriasis severity.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western adults, although the incidence of CLL is relatively low in Asian populations. However, with the aging population, the incidence of CLL is increasing in China. The interaction between CLL cells and the microenvironment plays a crucial role in the recognition of antigens by the B-cell receptor immunoglobulin (BCR IG). The mutational status of the immunoglobulin heavy variable region (IGHV) is a classical prognostic marker for CLL. Over 40% of CLL patients exhibit biased usage of IGHV and highly similar amino acid sequences in the heavy complementarity-determining region 3 (HCDR3), known as the BCR stereotypy. Different subgroups of stereotyped BCR exhibit distinct biological and clinical features. Among them, subset #2 with mutated IGHV and poor prognosis, as well as the subset #8 with a high risk of Richter transformation, have been recommended by the European Research Initiative on CLL to be included in clinical reports on IGHV mutational status. This review summarizes the definition, distribution, biological characteristics, and clinical significance of clonality patterns of the BCR in CLL.
慢性淋巴细胞白血病（CLL）是西方成人最常见的白血病，尽管CLL在亚洲人种中发病率偏低，但随着人口老龄化，CLL发病率在我国呈上升趋势。CLL细胞与微环境的交互中，B细胞受体免疫球蛋白（B-cell receptor immunoglobulin，BCR IG）对抗原的识别起重要作用，免疫球蛋白重链可变区（IG heavy variable region，IGHV）的突变状态更是判断CLL预后的经典标志物。超过40%的CLL患者间存在IGHV的偏向性使用和高度相似的重链可变区互补决定区3（heavy complementarity-determining region 3，HCDR3）氨基酸片段，此现象称为同型模式BCR。不同亚群的同型模式BCR存在独特的生物学和临床特征。其中IGHV有突变但预后差的#2亚群和发生Richter转化风险高的#8亚群被欧洲CLL研究启动组推荐纳入IGHV突变状态的临床报告之中。本文综述CLL中同型模式BCR的定义、分布、生物学特征和临床意义。.

Tumor-infiltrating lymphocytes (TILs) play a key role in regulating the host immune response and shaping tumor microenvironment. It has been previously shown that T cell infiltration in penile tumors was associated with clinical outcomes. However, few studies have reported the T cell receptor (TCR) repertoire in patients with penile cancer. In the present study, we evaluated the TCR repertoires in tumor and adjacent normal tissues from 22 patients with penile squamous cell carcinoma (PSCC). Analysis of the T cell receptor beta-variable (TRBV) and joining (TRBJ) genes usage and analysis of complementarity determining region 3 (CDR3) length distribution did not show significant differences between tumor and matched normal tissues. Moreover, analysis of the median Jaccard index indicated a limited overlap of TCR repertoire between these groups. Compared with normal tissues, a significantly lower diversity and higher clonality of TCR repertoire was observed in tumor samples, which was associated with clinical characteristics. Further analysis of transcriptional profiles demonstrated that tumor samples with high clonality showed increased expression of genes associated with CD8 + T cells. In addition, we analyzed the TCR repertoire of CD4 + T cells and CD8 + T cells isolated from tumor tissues. We identified that expanded clonotypes were predominantly in the CD8 + T cell compartment, which presented with an exhausted phenotype. Overall, we comprehensively compared TCR repertoire between penile tumor and normal tissues and demonstrated the presence of distinct T cell immune microenvironments in patients with PSCC.