A review of progress in B cell receptor (BCR) antigen specificity / 细胞与分子免疫学杂志

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.

Structure and function of B-cell linker and its role in the development of B cell-related diseases / 南方医科大学学报

B cell linker (BLNK) is a key linker protein of B cell receptor (BCR) signaling pathway. BLNK participates in the regulation of PLC-γactivity and the activation of Ras pathway through its typical structure and interaction network with other proteins, and is thus widely involved in the regulation of B cell proliferation, differentiation, apoptosis and signal transduction. Furthermore, it is closely related to anaphylactic diseases, multiple sclerosis, chromosomal aneuploidy, aneuglobulinemia, B lymphocytic leukemia and lymphoma. Herein we review the structure and biological function of BLNK and its role in B cell-related diseases. BLNK can cooperate with a series of effective proteins to activate BCR signaling pathway, thereby regulating the development, maturation and function of B cells. The functional mutation of BLNK can destroy the homeostasis of B cells and affect the development and maturation of B cells, which leads to the occurrence of B cell related diseases. A comprehensive understanding of the biological functions of BLNK not only provides insights into the pathogenesis of B cell-related diseases, but also inspires new ideas and helps to find breakthroughs for the treatment of these diseases with BLNK as the therapeutic target.

Traditional Chinese medicine (tumor) prevention and evaluation model based on ultra-early immune response information amplification index naCTL and multidimensional immunoinformatic index TCR/BCR/HLA effective diversity / 中国中药杂志

The best time of tumor intervention is before the formation of tumor. However,due to the limited number of tumor cells,it is difficult to quantify tumor cells and immunity by the current methods available( such as CTC,ct DNA). This affects the tumor prevention in this period,and the in-depth detection,intervention and evaluation of traditional Chinese medicine( TCM)( tumor) prevention. Due to the limitations of the current detection,the evaluation system turns to detect tumor neoantigen-specific CTL( naCTL) that are directly relating to tumor cells and proliferate to high order of magnitudes after activation,and immune repertoire( TCR/BCR/HLA) effective diversity,introduces immune checkpoints,uses information of " disease" in Western medicine and " syndrome" in TCM( prevention),and sets up a multi-dimensional statistical immunity model using a variety of data analysis and related algorithms. This model can amplify the ultra-early information of tumor,indirectly evaluate the quantity and status of tumor cells,and provide quantitative measurement and new evaluation methods for the normalization of immunity and TCM( tumor) prevention. This model is not only one of important evaluation methods for resisting tumor immunity and treating TCM( tumor) prevention,but also will reveal the scientific connotation of TCM syndrome from the perspective of immunology.

TRIAGEM NEONATAL DE IMUNODEFICIÊNCIAS GRAVES COMBINADAS POR MEIO DE TRECS E KRECS: SEGUNDO ESTUDO PILOTO NO BRASIL / NEWBORN SCREENING FOR SEVERE COMBINED IMMUNODEFICIENCIES USING TRECS AND KRECS: SECOND PILOT STUDY IN BRAZIL

RESUMO Objetivo: Validar a quantificação de T-cell receptor excision circles (TRECs) e kappa-deleting recombination circles (KRECs) por reação em cadeia de polimerase (polymerase chain reaction, PCR) em tempo real (qRT-PCR), para triagem neonatal de imunodeficiências primárias que cursam com defeitos nas células T e/ou B no Brasil. Métodos: Amostras de sangue de recém-nascidos (RN) e controles foram coletadas em papel-filtro. O DNA foi extraído e os TRECs e KRECs foram quantificados por reação duplex de qRT-PCR. O valor de corte foi determinado pela análise de Receiver Operating Characteristics Curve, utilizando-se o programa Statistical Package for the Social Sciences (SSPS) (IBM®, Armonk, NY, EUA). Resultados: 6.881 amostras de RN foram analisadas quanto à concentração de TRECs e KRECs. Os valores de TRECs variaram entre 1 e 1.006 TRECs/µL, com média e mediana de 160 e 139 TRECs/µL, respectivamente. Três amostras de pacientes diagnosticados com imunodeficiência grave combinada (severe combined immunodeficiency, SCID) apresentaram valores de TRECs abaixo de 4/µL e um paciente com Síndrome de DiGeorge apresentou TRECs indetectáveis. Os valores de KRECs encontraram-se entre 10 e 1.097 KRECs/µL, com média e mediana de 130 e 108 KRECs/µL, e quatro pacientes com diagnóstico de agamaglobulinemia tiveram resultados abaixo de 4 KRECs/µL. Os valores de corte encontrados foram 15 TRECs/µL e 14 KRECs/µL, e foram estabelecidos de acordo com a análise da Receiver Operating Characteristics Curve, com sensibilidade de 100% para detecção de SCID e agamaglobulinemia, respectivamente. Conclusões: A quantificação de TRECs e KRECs foi capaz de diagnosticar crianças com linfopenias T e/ou B em nosso estudo, validando a técnica e dando o primeiro passo para a implementação da triagem neonatal em grande escala no Brasil.

ABSTRACT Objective: To validate the quantification of T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) by real-time polymerase chain reaction (qRT-PCR) for newborn screening of primary immunodeficiencies with defects in T and/or B cells in Brazil. Methods: Blood samples from newborns and controls were collected on filter paper. DNA was extracted and TRECs, and KRECs were quantified by a duplex real-time PCR. The cutoff values were determined by receiver operating characteristic curve analysis using SPSS software (IBM®, Armonk, NY, USA). Results: Around 6,881 samples from newborns were collected and TRECs and KRECs were quantified. The TRECs values ranged between 1 and 1,006 TRECs/µL, with mean and median of 160 and 139 TRECs/µL, respectively. Three samples from patients with severe combined immunodeficiency (SCID) showed TRECs below 4/µL and a patient with DiGeorge syndrome showed undetectable TRECs. KRECs values ranged from 10 to 1,097 KRECs/µL, with mean and median of 130 and 108 KRECs/µL. Four patients with agammaglobulinemia had results below 4 KRECs/µL. The cutoff values were 15 TRECs/µL and 14 KRECs/µL and were established according to the receiver operating characteristic curve analysis, with 100% sensitivity for SCID and agammaglobulinemia detection, respectively. Conclusions: Quantification of TRECs and KRECs was able to diagnose children with T- and/or B-cell lymphopenia in our study, which validated the technique in Brazil and enabled us to implement the newborn screening program for SCID and agammaglobulinemia.

Targeting B-cell receptor signaling: Changing the treatment landscape of B-cell Lymphoma.

Introduced in 1998, the anti-CD20 monoclonal antibody rituximab, with its unique mechanism of action, was the first agent to improve survival in patients with B-cell lymphoma (BCL) treated with chemotherapy. Laboratory investigation of the B-cell receptor signaling pathway identified the critical nature of this pathway for normal B-cell development, survival and proliferation. Further investigation showed that lymphoma cell lines were also dependent upon this pathway and hence small molecule inhibitors of critical proteins in the pathway were synthesized and shown to be cytotoxic. Subsequent translation to the clinic has shown impressive activity in some types of B-cell lymphoma. The aim of this article is to provide an overview of the constituents of the BCR signaling pathway, to illustrate how addiction to this pathway is critical for survival of some BCL, and to summarize the clinical experience with novel small molecule inhibitors of specific proteins in the BCR pathway. We speculate that combination of these agents with newer drugs, each with a unique mechanism of action might lead to improved therapy and the eventual elimination of standard chemotherapy from our therapeutic arsenal.

Recent Advances of Researches on Expression, Function and Regulation of CD22 / 中国实验血液学杂志

CD22 is a type I transmembrane protein expressed on most mature B lymphocyte, and plays a significant role in signal transduction pathways. CD22 acts as a co-receptor of the B-cell receptor (BCR) that inhibits the BCR signaling by antigen-receptor interaction. The phosphorylation of CD22 can be triggered by cross-linking of CD22 with the BCR through antigen, then predominantly triggers the dephosphorylation and inactivation of downstream proteins and inhibit the BCR signaling. Autoimmune disease could be caused by the abnormal expression or dysfunction of CD22 which interrupts BCR signaling and then influences the quantity and function of B cells. The further study of the function and regulation of CD22 would help us understanding the pathogenesis of autoimmune disease and setting theoretical basis for its targeting treatment. In this article, the structure and expression of CD22, the ligands of CD22, the regulation of BCR and transmenbrane signaling, the effect of CD22 on B cells, and CD22 and autoimmune diseases were reviewed.

Recent advances in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) was largely considered to be a disease of slow progression, standard treatment with Chlorambucil and having almost similar prognosis. With the introduction of molecular methods for understanding the disease pathophysiology in CLL there has been a remarkable change in the approach towards the disease. The variation in B-cell receptor response and immunoglobulin heavy chain variable region (IGHV) mutation, genetic aberration and defect in apoptosis and proliferation has had an impact on therapy initiation and prognosis. Early diagnosis of molecular variant is therefore necessary in CLL.

The growth of B cell receptor microcluster is a universal response of B cells encountering antigens with different motion features

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.

Effect of proline rich domain of an RNA-binding protein Sam68 in cell growth process, death and B cell signal transduction / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Sam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in RNA metabolism, signal transduction regulation of cell growth and cell proliferation in DT40 cell line.</p><p><b>METHODS</b>By using gene targeting method, we isolated a mutation form of Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. Southern, Northern, and Western blot, phosphorylation and flow-cytometric analyses were performed to investigate the Sam68 functions.</p><p><b>RESULTS</b>A slower growth rate (2.1 hours growth elongation) and longer S phase (1.7 hours elongation) was observed in the Sam68 mutant cells. Serum depletion resulted in increased amounts of dead cells, and expansion of S phase in mutant cells. Upon B cell cross-linking, the maximal level of tyrosine phosphorylation on BLNK was observed to be significantly lower in mutant cells.</p><p><b>CONCLUSIONS</b>The proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.</p>

Receptor selection and B cell immune tolerance / 生物医学工程学杂志

The immune system of mammalian has developed sophisticated mechanism to deal with diverse unknown antigens by random rearrangement of V, D and J antigen gene fragments. The immune self-tolerance is the mechanism to avoid self-destruction by the gene rearrangement generated autoreactive lymphocytes. Until recently it was believed that autoreactive lymphocytes are either deleted or rendered unable to respond by the supposed cell or clone selection mechanism. However, recent findings from a number of laboratories suggest instead of cell selection but receptor selection plays an essential role in immune self-tolerance. Receptor selection is carried out by secondary or nested rearrangement of antigen receptor gene fragments, and can occur at different stages of lymphocyte differentiation. Furthermore, secondary rearrangement of receptor gene also plays an important role in shaping immune response after the interaction of receptor with antigen by altering its specificity.

Characteristic analysis of acute leukemia immunophenotyping in 180 cases by flow cytometry / 中国实验血液学杂志

To study the acute Leukemia immunophenotype and its diagnosis value, three color direct immunofluorescece staining methods of flow cytometry and immunophenotype of antibody integration system were used for detection of 180 cases of acute leukemia. The results showed that patients with ALL expressed lymphocyte antigen, and 49.4% patients with ALL accompanied myeloid antigen; all patients with AML expressed myeloid antigen, and 43.2% patients with AML accompanied lymphocyte antigen. In conclusion, leukemia immunophenotyping by three-color direct immunofluore staining methods could define some particular types of leukemia with an important value in diagnosis, treatment and predicting prognosis of acute leukemia.

Detection of cytoplasmic antigens by flow cytometry and its implication for leukemia immunophenotyping / 中国实验血液学杂志

The purpose of this study was to optimize a fixation procedure for detection of cytoplasmic antigens by flow cytometry(FCM) and to evaluate the effect of intracellular CD3, CD22, CD79a and myeloperoxidase(MPO) in lineage assignment. Four kinds of fixation procedure and three or four color direct immunofluorescence staining were used to permeate cell membrane and label cell surface and intracellular antigens by means of FCM. Results showed that percentage of cytoplasmic antigens positive cells was the highest and cell scatter and fluorescence intensity of CD45 were not changed after using of FACS permeabilization solution. MPO protein was positive in 16/18 acute myeloid leukemia(AML) patients. 4 cases of T cell-acute lymphoblastic leukemia (T-ALL) cases were positive for cytoplasmic CD3(c CD3) but surface CD3 was negative. c CD22 was only detected in 9/13 of B-ALL and cCD79a was positive in 5/5 B-ALL. 18/38 cases of acute leukemia were expressed in more than one lineage marker, 8/21 cases of acute non-lymphocytic leukemia(ANLL) were CD7 positive. 7/17 cases of acute lymphocytic leukemia (ALL) expressed CD13. After further cytoplasmic antigen detection, one was considered to be a T/myeloid biphenotypic leukemia, another one was diagnosed as biclonal or mixed leukemia. The results suggest that intracellular CD3,CD22,CD79a and MPO are lineage specific markers, they are very important for biphenotypic and biclonal/mixed acute leukemia identification

Human polyclonal anti-hepatitis B surface antigen immunoglobulin reduces the frequency of acute rejection after liver transplantation for chronic hepatitis B

BACKGROUND: Use of polyclonal anti-hepatitis B surface antigen immunoglobulin (HBIg) has been shown to reduce hepatitis B virus (HBV) recurrence after liver transplantation (LT) and to decrease the frequency of acute cellular rejection (ACR). However, the protective role of HBIg against ACR remains controversial, since HBV infection has been also associated with a lower incidence of ACR. AIM: To assess the relationship between HBIg immunoprophylaxis and the incidence of rejection after LT. METHODS: 260 patients (158 males, 43 + or - 14 years old) submitted to LT were retrospectively evaluated and divided into three groups, according to the presence of HBsAg and the use of HBIg. Group I was comprised of HBsAg-positive patients (n = 12) that received HBIg for more than 6 months. Group II was comprised of HBsAg-positive patients that historically have not received HBIg or have been treated irregularly for less than 3 months (n = 10). Group III was composed of 238 HBsAg-negative subjects that have not received HBIg. RESULTS: HBIg-treated patients (group I) had significantly less ACR episodes, when compared to group II and III. No differences between groups II and III were observed. CONCLUSIONS: Long-term HBIg administration contributes independently to reduce the number of ACR episodes after LT

Obtention of rabies antigen through BHK21 cells adhered to microcarriers

Foram produzidos quatro lotes de antigeno rabico a partir de suspensoes de virus resultantes de celulas BHK21 infectadas, aderidas a microcarregadores do tipo Cytodex 1 e cultivadas em biorreator. Em paralelo foi utilizada a metodologia de producao de virus rabico com celulas BHK21 em monocamadas, contidas em garrafas de 350cm2. Os resultados encontrados demonstraram que os titulos infectantes foram de 10 elevado a 6.69 DL50/mL para as suspensoes virais obtidas em garrafas e 10 elevado a 7.28 DL50/mL para as do biorreator. Os volumes das suspensoes virais colhidas foram, em media de 11.900 mL por lote do biorreator e 800 mL por garrafa. Com o antigeno produzido no biorreator foram imunizados 10 cavalos. As medias dos titulos de anticorpos anti-rabicos encontrados nos soros destes animais foram de 240 e 212 UI/mL, respectivamente apos a base e o primeiro reforco. Atraves da infeccao de celulas BHK21 aderidas, a microcarregadores e cultivadas em biorreator, pode-se obter antigeno rabico em larga escala e com titulos infectantes satisfatorios

Anticuerpos y receptores de células B / Antibodies and B cell receptors

Las inmunoglobulinas o anticuerpos son glicoproteínas que se encuentran en la superficie de los linfocitos B y son secretadas por las células plasmáticas, linfocitos B terminalmente diferenciados, en respuesta a un antígeno y, como tal, representan la inmunidad humoral de los vertebrados. Existen 5 formas o isotipos principales: IgG, IgM, IgA, IgD e IgE. Las Igs presentan una estructura básica que posee dos cadenas pesadas (H) idénticas y dos cadenas livianas (L) idénticas, unidas entre sí por puentes disulfuro e interacciones no covalentes. Ambos tipos de cadenas presentan un patrón estructural que consiste de segmentos o dominios de 110 aminoácidos. El análisis de su secuencia de aminoácidos revela la existencia de un dominio variable (V) hacia el extremo aminoterminal y varios dominios constantes (C) hacia el extremo carboxilo terminal. Las cadenas pesadas también poseen un dominio variable (VL) y uno constante (CL). Las cadenas pesadas también poseen un dominio variable (VH) pero tienen 3 ó 4 dominios constantes (CH). Los dominios variables de mabas cadenas contienen zonas de alta variabilidad no contiguas en la secuencia de aminoácidos, son las denominadas regiones hipervariables o CRD (determinantes de complementariedad) y son las principales responsables de la diversidad de los anticuerpos...

La inmunoperoxidasa en el estudio de las trombocitopenias inmunológicas / Immunoperoxidase in the studies of immune thrombocytopenias

The usefulness of different techniques to measure platelet bound IgG has been reviewed by George(16). We present here the results obtained with a technique designed to measure membrane bound IgG employing an anti-human IgG labeled with peroxidase and using O-dianisidine-H2O2 to reveal the enzymatic activity(17). We studied 152 patients with chronic autoimmune thrombocytopenic (ATP) including 120 adults and 32 children (age below 15 years old), diagnosed by exclusion of diseases that may be associated with thrombocytopenic purpura of either immune or non-immune mechanisms. Besides, 79 patients with thrombocytopenia related to other diseases were also evaluated. The normal values in 215 controls were 188 +/- 4 IgG molec/platelet (mean +/- SE), while in the whole population of chronic ATP the results were 4714 +/- 344, p < 0.001. In pediatric cases the results had a tendency to values higher than in adults. A negative correlation was found between the number of platelets and the amount of bound IgG, r = 0.41 p < 0.001. IgG bound platelets were also increased in treated patients at relapse. The percent of normal IgG bound platelet was 4,5 percent in patients with a platelet count below 50.000/microliter and 39 percent in those with normal platelet number. Patients with secondary thrombocytopenia had elevated IgG/platelet while the values were normal in patients with thrombocytopenia of unknown etiology. We conclude that the immunoperoxidase technique is useful to establish the immunologic nature of thrombocytopenia.

La inmunología en lactantes y preescolares en riesgo nutricional / Immunologic assessment of children at risk of undernutrition

La desnutrición, con su alta prevalencia de procesos infecciosos, origina cambios en el sistema inmune. Se realizó la evaluación del estado nutricional e inmunológica en 55 niños lactantes y preescolares que presentaron diarrea (ED) o infección respiratoria (IR) y se comparó con un grupo control. Se determinaron linfocitos T (LT) por rosetas y B (LB) por inmunoglobulina de superficie, a niveles de inmunoglobulinas A, G y M, y fracciones 3 y 4 de complemento por inmunodifusión radial. LT y LB en controles sanos fue de 52,3 ñ 7,9 y 20,1 ñ 6,4 respectivamente; en los enfermos fueron bajos IgA y C3 fue para EDy mayores y significativos en IR. La asociación patología desnutrición disminuyó los LT y LB y la fracción 3 de complemento. En los desnutridos las poblaciones de linfocitos aumentaron después e una semana de terapia específica y nutricional. Hubo relación entre severidad del daño nutricional y cambios inmunológicos

Actividad inmunoadyuvante del Staphylococcus aureus: efecto sobre la respuesta humoral y el sistema fagocítico mononuclear en ratas / Inmunoadyuvant activity of staphylococcus aureus:effect on humoral response and mononuclear phagocytic system in rats

Se estudió el efecto del S. aureus y de varios tipos celulares sobre: a) producción de anticuerpos (hemaglutininas) y b) estimulación del Sistema Fagocítico Mononuclear (S.F.M.). Ratas adultas, endocriadas,recibieron:I) células de exudado peritoneal (CEP); II) células del bazo (CB); III) células de bazo no-adherentes (CBN-A); IV) células de exudado peritoneal normal (CEPN); V) células de bazo normal (CBN); VI) células de bazo normal no-adherentes (CBN N-A); VII) S. aureas s.c. y VIII) (-). I, II y III proveían de ratas inoculadas 7 días antes con S. aureus y IV,V y VI de animales normales. Se sensibilizó con glóbulos rojos de carnero al 50 por ciento. Se determinó el título de hemaglutininas séricas totales y con 2 mercapto etanol en la Respuesta primaria y en la Respuesta secundaria, posterior al segundo inóculo. Se estimó peso y se practicó un estudio anatomopatológico del hígado y bazo de los animales tratados con S. aureus y testigos. Se hallaron diferencias significativas en el título de hemaglutininas en todos los días estudiados (Respuesta primaria y secundaria)-p<0,0l;p<0,05-entre los grupos I,II,III,VII vs. IV,V,VI y VIII. Se pesó el hígado y bazo (corregido/peso del animal) en ratas inoculadas con S. aureus (1) y sin tratamiento o control (2). Los valores fueron significativamente mayores en (1) (p<0,001). Hígado: a) 5,746 Ð 0,67; b) 3,609 Ð 0,456. Bazo: a)0,5698 Ð 0,067; b) 0,325 Ð 0,05. Las modificaciones anatomopatológicas en hígado y bazo se observaron con mayor frecuencia e intensidad en los animales tratados que en los testigos (p<0,01). Se confirma el efecto adyuvante del S. aureus, ya que produce aumento de peso e importantes modificaciones anatomopatológicas del órganos del S.F.M., eleva la respuesta humoral a un antígeno no relacionado y estimula la memoria inmunológica