PAX5 haploinsufficiency induced CD8+ T cells dysfunction or exhaustion by high expression of immune inhibitory-related molecules.

PURPOSE: PAX5 haploinsufficiency promoting tumorigenesis is related to immune escape. But the mechanisms of PAX5 mutations inducing tumor immune escape have not been clarified. Our aim was to study how PAX5 haploinsufficiency influences effector CD8 + T cells in tumor microenvironment. METHODS: We estimated the proportions of 22 immune cell types and the expressions of immune inhibitory-related molecules based on gene expression profiles (GEPs) from children's B- acute lymphoblastic leukemia(B-ALL) with PAX5 mutations by CIBERSORT, an established algorithm. We constructed the PAX5 haplodeletion A20 cell lines, built allografted A20 tumor models and evaluated the effect of PAX5 haplodeletion on immune inhibitory-related molecules in the tumor microenvironment (TME). RESULTS: Our results indicated the percentages of T cells in bone marrow of children's B-ALL with PAX5 mutations were not statistically different from that in bone marrow of B-ALL without PAX5 mutations, except for T follicular helper (Tfh) cells. But a variety of up-regulated immune inhibitory-related molecules in bone marrow of children's B- ALL with PAX5 mutations were identified. By different approaches, we found that several immune inhibitory-related molecules of CD8+ T cells in TME of PAX5 haplodeletion clones such as TIM3, NR4A1 and BATF, were increased significantly compared with that of PAX5 wild type control. The IFN-ɤ of CD8+ T cells in TME of PAX5 haplodeletion tumors was decreased significantly compared with that of PAX5 wild type control. CONCLUSION: Our study showed that PAX5 haploinsufficiency induced CD8+ T cells dysfunction or exhaustion by high expression of TIM3, NR4A1 and BATF in the CD8+ T cells of TME.

Pax5 regulates B cell immunity by promoting PI3K signaling via PTEN down-regulation.

The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving Pten-targeting microRNAs. Additional PTEN loss in Pten,Pax5 double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.

B Lymphocyte Specification Is Preceded by Extensive Epigenetic Priming in Multipotent Progenitors.

B lymphocyte development is dependent on the interplay between the chromatin landscape and lineage-specific transcription factors. It has been suggested that B lineage commitment is associated with major changes in the nuclear chromatin environment, proposing a critical role for lineage-specific transcription factors in the formation of the epigenetic landscape. In this report, we have used chromosome conformation capture in combination with assay for transposase-accessible chromatin sequencing analysis to enable highly efficient annotation of both proximal and distal transcriptional control elements to genes activated in B lineage specification in mice. A large majority of these genes were annotated to at least one regulatory element with an accessible chromatin configuration in multipotent progenitors. Furthermore, the majority of binding sites for the key regulators of B lineage specification, EBF1 and PAX5, occurred in already accessible regions. EBF1 did, however, cause a dynamic change in assay for transposase-accessible chromatin accessibility and was critical for an increase in distal promoter-enhancer interactions. Our data unravel an extensive epigenetic priming at regulatory elements annotated to lineage-restricted genes and provide insight into the interplay between the epigenetic landscape and transcription factors in cell specification.

Establishment and characterization of a porcine B cell lymphoma cell line.

The lack of available, well characterized, established, domestic porcine cell lines hinders the advancement of porcine cellular immunology. A case of multicentric lymphoma was diagnosed in a market weight pig at the time of slaughter. Affected lymph nodes and spleen were collected and used for single cell isolation and analysis. Cell lines were established by 3 rounds of limiting dilution from splenic and subiliac lymph node lymphomas. Surface marker staining identified the cells as CD21+, CD79a+, CD20+, PAX5+, and CD3- and cells were grown and easily passaged in cell culture. Transcriptome analysis was carried out to further characterize these rapidly proliferating cells validating the initial cytometric findings, confirming their identity as B cell lymphomas, and suggesting that they arose from germinal center centroblasts with aberrant control of BCL6 expression. Functional analysis identified the cells as being involved in cancer, cell movement, cell survival, and apoptosis. These new porcine B cell lymphoma cell lines will be a valuable resource for more in-depth cellular investigations into the porcine immune system and cancer, as well as providing a potential tool for the growth of lymphotropic viruses of pigs and humans.

Molecular characterization and transcriptional expression of a B cell transcription factor Pax5 in Nile tilapia (Oreochromis niloticus).

Pax5 (Paired Box 5), a nuclear transcription factor expressed in B cell specifically, is a key regulator for B cell activation. In this study, we cloned and identified a Pax5 gene (OnPax5) from Nile tilapia (Oreochromis niloticus), which has an open reading frame of 1278 bp, encoding deduced amino acid sequence of 425 residues. OnPax5 contains a conserved DNA-binding domain encoding the paired box, an octapeptide, a homeobox homology region, a transactivation and a repressor domain. OnPax5 is constitutively expressed in various analyzed tissues of tilapia, with a relatively high expression in lymphoid organs, including spleen (SPL), anterior kidney (AK), and thymus. What's more, OnPax5 is highly expressed in leukocytes especially in IgM+ lymphocytes sorted from peripheral blood (PBL), SPL and AK. When stimulated with lipopolysaccharide (LPS) in vivo, OnPax5 expression was significantly up-regulated in PBL, SPL and AK. Upon stimulation with LPS, pokeweed mitogen and mouse anti-OnIgM monoclonal antibody in vitro, the expression of OnPax5 was also significantly up-regulated in leukocytes from SPL and AK. Taken together, Pax5, the B cell lineage specific activator factor, might get involved in B cell activation in Nile tilapia.

Tumor CD73/A2aR adenosine immunosuppressive axis and tumor-infiltrating lymphocytes in diffuse large B-cell lymphoma: correlations with clinicopathological characteristics and clinical outcome.

Novel immune checkpoint blockades, including those targeting CD73 and A2aR, are being evaluated in malignancies in clinical trials. Here, we investigated the expression of CD73 and A2aR as well as tumor-infiltrating lymphocytes (TILs), and analyzed their correlations with clinicopathological characteristics and survival in diffuse large B-cell lymphoma (DLBCL). We found that CD73 expression on tumor cells, rather than the total protein and gene levels of CD73, was associated with survival. Patients with CD73+ /Pax-5+ (median survival, 57.8 months; 95% CI, 46.4-69.3) experienced significantly poorer outcomes than those with CD73- /Pax-5+ (median survival, 73.5 months; 95% CI, 65.9-81.2). Additionally, A2aR expression on both total TILs and CD8+ TILs was correlated with survival. Patients with A2aR+ TILs (median survival, 53.3 months; 95% CI, 40.6-66.0) had a significantly shorter survival time than patients with A2aR- TILs (median survival, 74.5 months; 95% CI, 67.5-81.5). Spearman's rank test showed that CD73 expression on tumor cells was positively correlated with A2aR expression on TILs (R = 0.395, p = 0.001). We further found that patients could be more precisely stratified through the combination of CD73 tumor cell expression and A2aR TILs expression, and patients with CD73+ /Pax-5+ and A2aR+ TILs experienced the worst outcome. We also revealed that patients with CD73+ /Pax-5+ and low CD8+ TILs or low absolute lymphocyte counts had unfavorable outcomes. Overall, our findings uncovered that patients with CD73+ on tumor cells as well as A2aR+ on TILs or low CD8+ TILs exhibited inferior survival, supporting potential combination strategies using CD73/A2aR immunosuppressive blockades as treatment options for DLBCL patients.

CD30, CD15, CD50, and PAX5 Expressions as Diagnostic Markers for Hodgkin Lymphoma (HL) and Systemic Anaplastic Large Cell Lymphoma (sALCL).

BACKGROUND: the expression of CD30, CD15, CD50, and PAX5 are used to help in confirming diagnosis of HL and sALCL; however data on the proportion of these markers have not been available. The study was aimed to identify the proportion of CD30, CD15, CD50 and PAX5 expressions and characteristics of patients with HL and sALCL at Dharmais National Cancer Center Hospital between 2005 and 2015. METHODS: a retrospective observational study was conducted using data from medical records and histopathological results of HL and sALCL adult patients who sought treatment at the hospital between 2005 and 2015. Immunohistochemistry (IHC) examinations were performed and data on the proportion of positive CD30, CD15, CD50, and PAX5 expressions were analyzed descriptively. RESULTS: a total of 45 patients were recruited in this study, with the majority (42 patients, 93.3%) were HL patients and only 6.7% were sALCL patients. The median age of HL patients was younger than sALCL patients; 35 (18-72 years old) versus 54 (49-61 years old). Moreover, the immunohistochemistry examination demonstrated that the positive CD15, CD30, CD50, and PAX5 expressions were found respectively in 37.5%, 88.9%, 31.2%, and 31.2% patients with HL; while in patients with sALCL, in spite of their small sample size, positive CD30, CD15, CD50 and PAX5 expressions were found in 100%; 66,7%; 50%; and 50%, respectively. Overall, CD15, CD50, and PAX5 positive expressions were found in 39.5%, 32.4%, and 32.4% patients who had HL and sALCL; while positive expression of CD30 was found in 89.5% of them. CONCLUSION: present study shows that almost 90% patients have positive CD30 expression;  while the positive expressions of CD15, CD50, and PAX5 are found in less than 40% patients. It indicates that CD30 is an important diagnostic marker for HL and sALCL and it may improve treatment strategy.

The correlation between Pax5 deletion and patients survival in Iranian children with precursor B-cell acute lymphocytic leukemia.

Despite advances in treatment, children with acute lymphoblastic leukemia (ALL) still experience drug resistance and relapse. Several gene mutations are involved in the onset of this disease and resistance to therapy. The present study examines the incidence of IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, JAK2, and Xp22.33 gene deletions/duplications associated with pediatric ALL in Iran and investigates the possible effect of these mutations on drug resistance. Three-year disease-free survival (3DFS) was evaluated for children diagnosed with Philadelphia negative precursor-B-cell ALL hospitalized at Sayed-al-Shohada Hospital, Isfahan-Iran, from January 2009 until December 2012. DNA was extracted from bone marrow slides, and ALL correlated gene deletions and duplications were measured using Multiplex Ligation-dependent Probe Amplification (MLPA) method. The correlation between gene mutations and 3DFS was then assessed. Among the nine aforementioned investigated genes, 63% of samples showed at least one gene mutation. At least two concomitant genomic mutations were observed in 42% of samples. Pax5 deletion was the most prevalent gene mutation observed in 45% of cases, and showed significant negative impact on response to treatment. CDKN2A/B (9p21.3) gene deletion, and ETV6 (12p13.2) gene duplication also demonstrated negative effect on patient survival and contributed to a worse prognosis if concomitant with Pax5 gene deletion. ALL patients with one of the gene deletions including Pax5  and CDKN2A/B (9p21.3) or ETV6 (12p13.2) gene duplication are classified as high-risk patients and need more intensified protocols of treatment to improve their chance of survival.

Kidins220 and tumour development: Insights into a complexity of cross-talk among signalling pathways (Review).

The mechanistic complexes of kinase D-interacting substrate of 220 kDa/ankyrin repeat-rich membrane spanning (Kidins220/ARMS) bind and integrate a variety of cellular cues to mediate neuronal activities such as neuronal differentiation, survival, and cytoskeleton remodelling by interacting with a variety of binding partners. Accumulated evidence has also indicated its role in the regulation of vascular development. Mice with Kidins220 knockdown phenotypically present with cardiovascular abnormalities. Kidins220 also contributes to immunomodulation in combination with B cells and T cells. Moreover, emerging evidence has revealed that this protein regulates many crucial cellular processes and thus has been implicated in an increasing number of malignancies. Here, we review recent advances in our understanding of Kidins220 and its role in cancer development. Further investigation is warranted to shed light on the role played by Kidins220 in the dynamic arrangement of the cytoskeleton and epithelial-mesenchymal transition, and its implication in tumourigenesis and cancer progression.

A cyclin D1-positive diffuse large B-cell lymphoma of germinal center B-cell-like subtype in the right tonsil: A rare case report.

INTRODUCTION: Cyclin D1-positive tumor cells are commonly found in mantle cell lymphoma but they are very rare in diffuse large B-cell lymphoma. CLINICAL FINDINGS/PATIENT CONCERNS: Here we present a rare case of cyclin D1-positive diffuse large B-cell lymphoma in the right tonsil of a 50-year-old man. Computed tomographic imaging detected a mass, about 2.5 cm × 1.8 cm in size, in the left side of the oropharynx. DIAGNOSES: Microscopically, the tumor cells were located under the pharyngeal mucosa and diffusely arranged. The tumor cells were large, with marked nuclear atypia. On performing immunohistochemistry, the tumor cells showed diffuse positive staining for CD10, CD20, cyclin D1, and Pax-5, and negative staining for CD3, CD15, CD30, CD56, and CK. Bcl-6 and Mum-1 expression were observed in 60% and 80% of tumor cells, respectively. The tumor Ki67 index was about 60%. Based on these findings, The tumor was diagnosed as a rare cyclin D1-positive diffuse large B-cell lymphoma rather than a mantle cell lymphoma. CONCLUSION: Cyclin D1-positive large B-cell lymphoma is rare, but as large B-cell lymphoma is a common type of lymphoma, cyclin D1-positive large B-cell lymphoma should be considered a major possibility during differential diagnosis, including in the tonsils.

Human population-specific gene expression and transcriptional network modification with polymorphic transposable elements.

Transposable element (TE) derived sequences are known to contribute to the regulation of the human genome. The majority of known TE-derived regulatory sequences correspond to relatively ancient insertions, which are fixed across human populations. The extent to which human genetic variation caused by recent TE activity leads to regulatory polymorphisms among populations has yet to be thoroughly explored. In this study, we searched for associations between polymorphic TE (polyTE) loci and human gene expression levels using an expression quantitative trait loci (eQTL) approach. We compared locus-specific polyTE insertion genotypes to B cell gene expression levels among 445 individuals from 5 human populations. Numerous human polyTE loci correspond to both cis and trans eQTL, and their regulatory effects are directly related to cell type-specific function in the immune system. PolyTE loci are associated with differences in expression between European and African population groups, and a single polyTE loci is indirectly associated with the expression of numerous genes via the regulation of the B cell-specific transcription factor PAX5. The polyTE-gene expression associations we found indicate that human TE genetic variation can have important phenotypic consequences. Our results reveal that TE-eQTL are involved in population-specific gene regulation as well as transcriptional network modification.

Regulated localization of an AID complex with E2A, PAX5 and IRF4 at the Igh locus.

Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates somatic hypermutation (SH) and class switch recombination (CSR) by deaminating cytosine to uracil. The targeting of AID and therefore SH and CSR to Ig genes is a central process of the immune system, but the trans-acting factors mediating the specific targeting have remained elusive. Here we show that defective calmodulin inhibition of the transcription factor E2A after activation of the B cell receptor (BCR) leads to reduced BCR, IL4 plus CD40 ligand stimulated CSR to IgE and instead CSR to other Ig classes. AID that initiates CSR is shown to be in a complex with the transcription factors E2A, PAX5 and IRF4 on key sequences of the Igh locus. Calmodulin shows proximity with each of them after BCR stimulation. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks these reductions. AID binds directly to the bHLH domain of E2A and to the PD domain of PAX5. E2A, AID, PAX5 and IRF4 are components of a CSR complex that is redistributed on the Igh locus by BCR signaling through calmodulin binding.

PAX5 promotes pre-B cell proliferation by regulating the expression of pre-B cell receptor and its downstream signaling.

PAX5 is indispensable for the commitment of early lymphoid progenitors to the B cell lineage as well as for the development of B cells. Although previous studies have indicated that the Pax5-conditional-knockout mouse exhibited dedifferentiation of mature B cell and the development of aggressive lymphomas, the changes of Pax5 gene expressions in pre-B cells have not been analyzed. To understand the functional importance of Pax5 gene in the proliferation and survival of pre-B cells, we established a Pax5-knockdown model using 70Z/3 pre-B cell line. Pax5 knockdown 70Z/3 cells (70Z/3-KD cells) showed down-regulations of pre-BCR compounds such as CD19, BLNK, Id2 and λ5. The signaling via pre-BCRs was significantly diminished in the 70Z/3-KD cells, and this alteration was normalized by restored Pax5 gene expression. Loss of PAX5 reduced the growth rates in the 70Z/3-KD cells, compared to the mock cells. Meanwhile, the proliferation of pre-B cells was reduced by the knockdown of Pax5 gene. Moreover, further examinations showed that PAX5 was also activated in B cell acute lymphoblastic leukemia (B-ALL) as a cell proliferation enhancer. These findings suggested that pax5 is critically important for the proliferation and survival of pre-B cells.

Estrogen receptor signal in regulation of B cell activation during diverse immune responses.

The role of signalling through oestrogen receptors (ERs) in the regulation of B cell activation is an area of growing importance not only in terms protective immunity but also in the determination of the mechanisms of the onset of autoimmune disorders and cancers. The mode of signalling action of this single chain nuclear receptor protein molecule depends on its ability to bind to the promoters of Pax5, HOXC4 and apolipoprotein B RNA-editing enzyme activation-induced cytidine deaminase (AID) genes. ER-mediated transcriptional regulation induces class switch recombination of the immunoglobulin heavy chain variable (VH) to DH-JH genes and somatic hypermutation in developing B cells. The mode of action of ER is associated with BCR-signal pathways that involve the regulator proteins BAFF and APRIL. Additionally, the plasma membrane-bound G protein-coupled oestrogen receptor-1 (GEPR1) directs diverse cell signalling events in B cells that involve the MAPK pathways. These signals are immensely important during progenitor and precursor B cell activation. We have focused our goals on the medicinal aspects of ER-signalling mechanisms and their effects on polyclonal B cell activation.

A robust in vivo model for B cell precursor acute lymphoblastic leukemia.

B cell precursor acute lymphoblastic leukemia (BCP ALL) is the most common malignancy in children. While treatments have improved remarkably over the past four decades, resistant disease and late effects that result from cytotoxic chemotherapy remain serious problems for individuals with BCP ALL. Improved genetic tools have led to the discovery of numerous somatic mutations associated with BCP ALL, leading to a framework for the genetic classification of BCP ALL. In this issue of the JCI, Duque-Afonso et al. develop an accurate in vivo model for BCP ALL that recapitulates the key features of human disease, including acquired mutations in genes encoding PAX5 and components of the JAK/STAT pathway. The authors further show, as proof of principle, that this model can be used to evaluate the efficacy of drugs designed to target specific acquired mutations in patients with BCP ALL.

B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: a shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals.

Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1ß, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF(+) B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM(+) mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative to susceptible trout.

Utility of PAX8 mouse monoclonal antibody in the diagnosis of thyroid, thymic, pleural and lung tumours: a comparison with polyclonal PAX8 antibody.

AIMS: The purpose of this study was to compare the immunohistochemical staining profiles of PAX8-polyclonal, PAX8-monoclonal, PAX5-monoclonal and PAX6-monoclonal antibodies in several histological types of primary thoracic and thyroid tumours. In addition, we analysed PAX8 mRNA expression by using in-situ hybridization. METHODS AND RESULTS: We compared polyclonal PAX8 and monoclonal PAX8, PAX5 and PAX6 antibodies in 962 samples (687 lung carcinomas, 40 malignant pleural mesotheliomas, 138 thymic tumours and 97 thyroid tumours) using the tissue microarray technique. Among thyroid tumours, the monoclonal and polyclonal PAX8 antibodies showed a high positive rate (98.0%). Of 167 polyclonal PAX8 antibody-positive tumours, except for thyroid tumours, 54 cases tested positive for PAX5 and/or PAX6 (31 lung carcinomas and 23 thymic tumours). No PAX8 mRNA expression was detected using RNAscope (in-situ hybridization technique) other than in thyroid tumours. A portion of polyclonal PAX8 antibody-positive tumours showed cross-reactivity for PAX5 or PAX6 protein. CONCLUSIONS: Monoclonal PAX8 antibody showed high specificity to thyroid tumours and was superior to the polyclonal antibody.

Allelic exclusion of IgH through inhibition of E2A in a VDJ recombination complex.

A key feature of the immune system is the paradigm that one lymphocyte has only one Ag specificity that can be selected for or against. This requires that only one of the alleles of genes for AgR chains is made functional. However, the molecular mechanism of this allelic exclusion has been an enigma. In this study, we show that B lymphocytes with E2A that cannot be inhibited by calmodulin are dramatically defective in allelic exclusion of the IgH locus. Furthermore, we provide data supporting that E2A, PAX5, and the RAGs are in a VDJ recombination complex bound to key sequences on the Igh gene. We show that pre-BCR activation releases the VDJ recombination complex through calmodulin binding to E2A. We also show that pre-BCR signaling downregulates several components of the recombination machinery, including RAG1, RAG2, and PAX5, through calmodulin inhibition of E2A.