BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

B cell receptor (BCR) is a key molecule involved in B cell specific recognition and the binding of antigens to produce adaptive humoral immune response. Gene rearrangement and high frequency mutation during B cell differentiation are the main mechanisms of BCR diversification. The enormous diversity and unique molecular structure of BCR determine the diversity and specificity of antigen recognition, shaping complex B cell repertoire with extensive collections of antigen specificities. Therefore, BCR antigen-specific information is vital to understanding the adaptive immune characteristics of different diseases. Our ability to connect BCR repertoire and antigen specificity has been enhanced with the development of B cell related research technologies, such as single cell sorting techniques, high-throughput sequencing (HTS), linking B cell receptor to antigen specificity through sequencing (LIBRA-seq). It could help researchers to better understand humoral immune responses, identify disease pathogenesis, monitor disease progression, design vaccines, and develop therapeutic antibodies and drugs. We summarizes recent studies on antigen-specific BCR of infections, vaccinations, autoimmune diseases and cancer. By analyzing autoantibody sequences of SLE as a case, the identification of autoantigens has become potentially possible due to this characterization.
Receptors, Antigen, B-Cell/metabolism* ; B-Lymphocytes/metabolism* ; Lymphocyte Activation ; High-Throughput Nucleotide Sequencing/methods*

This study aimed at constructing a draft genome of the adult female worm Toxocara canis using next-generation sequencing (NGS) and de novo assembly, as well as to find new genes after annotation using functional genomics tools. Using an NGS machine, we produced DNA read data of T. canis. The de novo assembly of the read data was performed using SOAPdenovo. RNA read data were assembled using Trinity. Structural annotation, homology search, functional annotation, classification of protein domains, and KEGG pathway analysis were carried out. Besides them, recently developed tools such as MAKER, PASA, Evidence Modeler, and Blast2GO were used. The scaffold DNA was obtained, the N50 was 108,950 bp, and the overall length was 341,776,187 bp. The N50 of the transcriptome was 940 bp, and its length was 53,046,952 bp. The GC content of the entire genome was 39.3%. The total number of genes was 20,178, and the total number of protein sequences was 22,358. Of the 22,358 protein sequences, 4,992 were newly observed in T. canis. Following proteins previously unknown were found: E3 ubiquitin-protein ligase cbl-b and antigen T-cell receptor, zeta chain for T-cell and B-cell regulation; endoprotease bli-4 for cuticle metabolism; mucin 12Ea and polymorphic mucin variant C6/1/40r2.1 for mucin production; tropomodulin-family protein and ryanodine receptor calcium release channels for muscle movement. We were able to find new hypothetical polypeptides sequences unique to T. canis, and the findings of this study are capable of serving as a basis for extending our biological understanding of T. canis.
Adult ; B-Lymphocytes ; Base Composition ; Classification ; DNA ; Female ; Genome* ; Genomics ; Humans ; Larva Migrans, Visceral* ; Metabolism ; Mucins ; Peptides ; Protein Structure, Tertiary ; Receptors, Antigen, T-Cell ; RNA ; Ryanodine Receptor Calcium Release Channel ; T-Lymphocytes ; Toxocara canis* ; Toxocara* ; Transcriptome ; Ubiquitin-Protein Ligases

No abstract available.
Antibodies, Monoclonal, Murine-Derived/therapeutic use ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; B-Cell-Specific Activator Protein/metabolism ; Bone Marrow/metabolism/*pathology ; Cyclophosphamide/therapeutic use ; Doxorubicin/therapeutic use ; Endoscopy, Digestive System ; Female ; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor ; Genetic Loci ; Humans ; Liver/metabolism/pathology ; Lymphocytes/cytology/immunology ; Lymphoma, Large B-Cell, Diffuse/complications/*diagnosis/drug therapy ; Lymphoma, T-Cell, Peripheral/complications/*diagnosis/drug therapy ; Middle Aged ; Prednisone/therapeutic use ; Receptors, Antigen, T-Cell, gamma-delta/genetics ; Tomography, X-Ray Computed ; Vincristine/therapeutic use

To study the regulation of androgen receptor (AR) expression in human prostate cancer LNCaP cells by triptolide (TP) and the possible mechanism, by using qRT-PCR and Western blot, the AR mRNA and protein levels in TP treated LNCaP cells were detected, and the AR protein level in TP and NF-κB inhibitor treated LNCaP cells was also detected; a series of pGL3-AR promoter reporter gene vectors were built using restriction-free cloning method, and the vectors were employed to investigate the effects of TP on the transcriptional activity of AR promoter in LNCaP cells; the upstream proteins which may play regulatory roles were detected using western blot assay. After treated LNCaP cells with TP for 48 h, AR mRNA and protein expressions decreased with increasing TP concentration. The expression of AR target gene PART1 and prostate specific antigen (PSA) was also downregulated by TP treatment; a series of pGL3-AR promoter reporter vectors were constructed and validated by sequencing and luciferase activity; the results of dual luciferase reporter assay showed that TP downregulated AR at the transcriptional level; PI3K/AKT/NF-κB pathway which is associated with AR promoter activity was drowregulated by TP. In conclusion, our results demonstrated that the transcriptional activity of AR in LNCAP cells was downregulated by TP, and PI3K/AKT/NF-κB pathway may be involved in the regulation mechanism.
Cell Line, Tumor ; Diterpenes ; pharmacology ; Down-Regulation ; Epoxy Compounds ; pharmacology ; Genetic Vectors ; Humans ; Male ; NF-kappa B ; antagonists & inhibitors ; Phenanthrenes ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Promoter Regions, Genetic ; Prostate-Specific Antigen ; metabolism ; Prostatic Neoplasms ; metabolism ; RNA, Messenger ; Receptors, Androgen ; metabolism ; Signal Transduction ; Transcriptional Activation

<b>OBJECTIVEb>To compare the expression differences of breast cancer resistance protein(BCRP/ABCG2) and P-glycoprotein(P-gp) in breast cancer tissue before chemotherapy and in residual breast cancer tissue, and to explore its correlation with breast cancer stem cells.

<b>METHODSb>Immunohistochemistry was used to detect the expression of ABCG2, P-gp, and breast cancer stem cells(BCSCs) markers(CD44 and CD24) in breast cancer tissue before chemotherapy and residual breast cancer tissue after chemotherapy. Immunofluorescence was applied for determination of the CD44 and CD24 protein expressions of BCSCs microspheres cells. The monoclone-forming ability of BCSCs microspheres cells was detected by limited dilution assay. The expressions of ABCG2, P-gp, CD44, and CD24 proteins were detected by Western blot.

<b>RESULTSb>Compared with those in breast cancer tissue before chemotherapy, the expression levels of ABCG2 and P-gp were positively correlated with the expression level of CD44 protein(Χ(2)=41.34, r=0.83;Χ(2)=22.81, r=0.61) in residual breast cancer tissue after chemotherapy;meanwhile, they were negatively correlated with the expression of CD24 protein(Χ(2)=-21.25, r=0.72;Χ(2)=-17.26, r=0.65) (all P<0.05) .The diameter of BCSCs microspheres were increased significantly after chemotherapy.The content of BCSCs increased by about 2.5 times after chemotherapy.The expressions of ABCG2, P-gp and CD44 proteins significantly increased and that of CD24 protein significantly declined(P<0.05) .

<b>CONCLUSIONb>Chemotherapy endows residual breast cancer tissue with cancer stem cells-like features, leading to multidrug resistance of breast cancer.

ATP Binding Cassette Transporter, Sub-Family B ; ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; metabolism ; ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Adult ; Aged ; Breast Neoplasms ; drug therapy ; metabolism ; CD24 Antigen ; metabolism ; Cell Culture Techniques ; Drug Resistance, Neoplasm ; Female ; Humans ; Hyaluronan Receptors ; metabolism ; Middle Aged ; Neoplasm Proteins ; metabolism ; Neoplasm, Residual ; Neoplastic Stem Cells ; cytology ; metabolism

<b>OBJECTIVEb>To identify the immunohistochemical patterns of follicular dendritic cell (FDC) meshwork and Ki-67 labeling index in small B-cell lymphomas (SBLs) and their significance in differential diagnosis.

<b>METHODSb>Sixty-eight cases of SBLs were included collected from November 2008 to June 2012. The patterns of FDCs and Ki-67 expression were studied on paraffin sections by CD21, CD23 and Ki-67 immunohistochemistry. The characteristic staining patterns of FDCs and Ki-67 expression among different SBLs were analyzed statistically.

<b>RESULTSb>The age of SBL patients ranged from 28 to 85 years with a mean of 55.2 years. The male to female ratio was 1.2:1. Fifty-five cases involved only lymph nodes (80.9%), and the remaining cases involved multiple extra-nodal sites. Histological classification of the cases was made according to the 2008 WHO lymphoma classification criteria: 22 were low-grade follicular lymphomas (FLs, including grade 1 and grade 2), 19 marginal zone lymphomas (MZLs), 17 mantle cell lymphomas (MCLs), and 10 chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLLs). FDC meshwork limited to the central part of neoplastic follicles was characteristic for FL (90.9%, 20/22). The germinal center FDC meshwork was destroyed primarily at periphery in MZL (14/19). The absence or scattered FDC clusters were typical of SLL/ CLL. Irregular FDC was seen in 7/17 of MCL, while 7/17 MCL displayed FDC pattern similar to that of CLL/SLLs. The pattern of FDCs was a significantly diagnostic feature in distinguishing the four types of SBLs (P < 0.01). Ki-67 was also a statistically significant parameter (P < 0.05) with decreasing labeling index as the following: MCL, FL, SLL and MZL. Ki-67 showed scattered pattern in germinal centers with loss of polarity in FLs. MZL presented uniformly scattered positive pattern in interfollicullar areas. Ki-67 staining was uniform in MCL, but its labeling index varied from 5% to 90%. The Ki-67 index was higher in the morphological "proliferation centers" of all CLL/SLLs.

<b>CONCLUSIONb>Immunohistochemical staining patterns of FDC meshworks and Ki-67 labeling index offer a significant discriminatory power in the differential diagnoses among SBLs.

Adult ; Aged ; Aged, 80 and over ; Antigens, CD20 ; metabolism ; Dendritic Cells, Follicular ; pathology ; Diagnosis, Differential ; Female ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; metabolism ; Leukemia, Lymphocytic, Chronic, B-Cell ; metabolism ; pathology ; Lymphoma, B-Cell ; classification ; metabolism ; pathology ; Lymphoma, B-Cell, Marginal Zone ; metabolism ; pathology ; Lymphoma, Follicular ; metabolism ; pathology ; Lymphoma, Mantle-Cell ; metabolism ; pathology ; Lymphoma, Non-Hodgkin ; metabolism ; pathology ; Male ; Middle Aged ; Receptors, Complement 3d ; metabolism ; Receptors, IgE ; metabolism ; Retrospective Studies

The paracaspase MALT1 is essential for the activation of NF-κB in response to T cell receptor (TCR) stimulation. It recruits downstream TRAF6 and activates the E3 ligase activity of TRAF6 to polyubiquitinate several targets, which ultimately leads to NF-κB activation. Here we identified ubiquitin-specific protease 2a (USP2a) as a MALT1-associated protein by biochemical affinity purification. Endogenous USP2a constitutively interacted with TRAF6, but dynamically interacted with MALT1 and CARMA1 in a stimulation-dependent manner. RNA interference (RNAi)-mediated silencing of USP2a attenuated TCR-induced NF-κB activation and production of interleukin-2 (IL-2). In addition, the ubiquitination of MALT1 and TRAF6 were both suppressed by USP2a knockdown. By knockdown and reconstitution assays, we found that USP2a mediated the interaction between MALT1 and TRAF6 in a catalytic activity-dependent manner. Furthermore, USP2a deSUMOylated TRAF6. Our findings implicate that USP2a plays an important role in TCR signaling by deSUMOylating TRAF6 and mediating TRAF6-MALT1 interaction.
Caspases ; metabolism ; Endopeptidases ; deficiency ; genetics ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Interleukin-2 ; biosynthesis ; Jurkat Cells ; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ; NF-kappa B ; metabolism ; Neoplasm Proteins ; metabolism ; Receptors, Antigen, T-Cell ; metabolism ; Signal Transduction ; Sumoylation ; TNF Receptor-Associated Factor 6 ; metabolism

Adolescent ; Antigens, CD20 ; metabolism ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Burkitt Lymphoma ; drug therapy ; metabolism ; pathology ; surgery ; Child ; Child, Preschool ; Cyclophosphamide ; therapeutic use ; DNA-Binding Proteins ; metabolism ; Doxorubicin ; therapeutic use ; Female ; Follow-Up Studies ; Genes, myc ; Humans ; Hyaluronan Receptors ; metabolism ; Ki-67 Antigen ; metabolism ; Lymphoma, Large B-Cell, Diffuse ; drug therapy ; genetics ; metabolism ; pathology ; surgery ; Male ; Neoplasm Staging ; Neprilysin ; metabolism ; Peritoneal Neoplasms ; drug therapy ; genetics ; metabolism ; pathology ; surgery ; Prednisone ; therapeutic use ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins c-bcl-6 ; Stomach Neoplasms ; drug therapy ; genetics ; metabolism ; pathology ; surgery ; Translocation, Genetic ; Vincristine ; therapeutic use

B lymphocyte cell senses and acquires foreign antigens through clonal distributed B cell receptors (BCRs) expressed on the surface of plasma membrane. The presentation formats of antigens are quite diverse. Based on their Brownian diffusion mobility, there are three forms: free mobile soluble antigens, lateral mobile membrane bound antigens, and fixed immobile antigens. Here, using high resolution high speed live cell imaging approaches, we provide evidence that BCR microclusters are formed on the surface of B cells shortly after B cell's encountering of antigens with each format of motion features. Through high speed live cell imaging, we determine that these BCR microclusters show dynamic growth feature and by doing so function as the basic platforms for B cells to acquire the antigens. We propose that the formation and dynamic growth of BCR microcluster is a universal mechanism for B cell to response to antigens with diverse motion features.
Adaptive Immunity ; Animals ; Antigens ; immunology ; metabolism ; B-Lymphocytes ; immunology ; metabolism ; Cell Membrane ; metabolism ; Cells, Cultured ; Humans ; Mice ; Mice, Transgenic ; Microscopy, Fluorescence ; Protein Transport ; immunology ; Receptors, Antigen, B-Cell ; metabolism ; Single-Cell Analysis ; Time-Lapse Imaging