Antigen Receptor Gene Rearrangement Research Articles

Application of Polymerase Chain Reaction to Analysis of Antigen Receptor Rearrangements to Support Endoscopic Diagnosis of Canine Alimentary Lymphoma

We evaluated the usefulness of polymerase chain reaction for antigen receptor gene rearrangement analysis (PARR) of endoscopic biopsy specimens for diagnosis of canine alimentary lymphoma. Two endoscopic biopsy specimens were obtained from each lesion in 78 dogs with gastrointestinal symptoms. One specimen was histopathologically examined by a pathologist, and the other was analyzed by PARR. All samples were categorized into three groups [lymphoma (n=4), adenocarcinoma (n=5) and enteritis groups (n=69)] based on the histopathological diagnosis. In the lymphoma group, one case was IgH major-positive, and three cases were TCRgamma-positive, representing clonal expansion of B- and T-cells, respectively. PARR produced negative results for all cases in the adenocarcinoma group. In the enteritis group, six cases were TCRgamma-positive. Two of the six TCRgamma-positive enteritis cases were cytologically diagnosed as lymphoma by fine needle aspiration during a laparotomy. In the enteritis group, the survival times were compared between the TCRgamma-positive and TCRgamma-negative cases. The overall survival time of the TCRgamma-positive enteritis cases was significantly shorter than that of the TCRgamma-negative enteritis cases according to a log-rank test (p<0.0001). With regard to other factors, such as age, clinical signs and the serum albumin concentration, there were no significant differences between the TCRgamma-positive and TCRgamma-negative enteritis cases. In conclusion, PARR is capable of detecting alimentary lymphoma and latent alimentary lymphoma, which cannot be histopathologically diagnosed using endoscopic biopsy specimens. Furthermore, a TCRgamma-positive result in PARR may imply a poor prognosis.

Sensitivity for the Detection of a Clonally Rearranged Antigen Receptor Gene in Endoscopically Obtained Biopsy Specimens from Canine Alimentary Lymphoma

Canine alimentary lymphoma is currently diagnosed on the basis of findings of cytological or histopathological examination. However, it is often difficult to histopathologically distinguish alimentary lymphoma from lymphocytic-plasmacytic enteritis. Recently, the application of polymerase chain reaction for antigen receptor gene rearrangement (PARR) has been reported. In the present study, we assess the sensitivity of PARR analysis in diagnosing canine alimentary lymphoma using endoscopically obtained biopsy specimens from 12 dogs that were histopathologically diagnosed as having lymphoma. The sensitivity of PARR analysis in diagnosing alimentary lymphoma was found to be 66.7%, which was lower than that of other lymphoid malignancies. A combination of histipathological examination and findings of PARR analysis may improve the diagnostic accuracy.

Use of Formalin-Fixed Paraffin-Embedded Tissue and Single-Strand Conformation Polymorphism Analysis for Polymerase Chain Reaction of Antigen Receptor Rearrangements in Dogs

PCR for antigen receptor gene rearrangement analysis (PARR) is a new diagnostic method for lymphoid neoplasia. In PARR using formalin-fixed paraffin-embedded tissues (PARR-FFPE), control DNA amplification was successful in only three of five samples. The formalin fixation times of the three samples were shorter than those of the others. Analysis of the formalin fixation time and DNA amplification controls suggested that a formalin fixation time of less than one week is appropriate. Additionally, application of single strand conformation polymorphism (SSCP) for PARR provided clearer results than conventional PARR in 16 unfixed tissues and three FFPE tissues. These results show that PARR-FFPE is viable for tissues with an appropriate formalin fixation time and that application of FFPE and SSCP for PARR are useful for diagnosis and retrospective study of canine lymphoid neoplasia.

Karyopherin alpha 1 is a putative substrate of the RAG1 ubiquitin ligase

The RAG1 recombinase, which participates in DNA manipulation during rearrangement of antigen receptor genes in developing immune cells, possesses ubiquitin ligase activity. The nuclear transport protein karyopherin alpha 1 (KPNA1) binds to RAG1 upstream of its ubiquitin ligase domain, but this interaction is not required for nuclear localization of RAG1. We found that the isolated ubiquitin ligase domain of RAG1 (amino acids 218–389) promoted ubiquitylation of purified KPNA1. While RAG1 auto-ubiquitylation is dependent on the ubiquitin conjugating enzyme CDC34, ubiquitylation of KPNA1 was best supported by UbcH2/Rad6 and UbcH5a. Ubiquitylation of KPNA1 required the lysine/arginine-rich region spanning RAG1 amino acids 218–263 upstream of the RAG1 ubiquitin ligase domain, but RAG1 was still able to undergo auto-ubiquitylation in this region even in the presence of KPNA1. This is the first putative substrate identified for the RAG1 ubiquitin ligase, and to our knowledge it is the first reported case of ubiquitylation of KPNA1.

Large anaplastic spinal B‐cell lymphoma in a cat

A 5-year-old female spayed domestic shorthair cat was presented for evaluation of tetraparesis. The neurologic lesion was localized to the cervical spinal segment (C1-C6). A left axillary mass was identified, and the results of fine needle aspiration cytology indicated malignant round cell neoplasia of possible histiocytic origin. The cells were large, had marked anisocytosis and anisokaryosis, occasional bi- and multinucleation, and cytoplasmic vacuolation. Euthanasia was performed due to the poor prognosis associated with severe, progressive neurologic signs and a malignant neoplasm. Postmortem examination revealed spinal cord compression and an extradural mass at the C1-C2 spinal segment, with neoplastic cells in the adjacent vertebral bodies, surrounding skeletal muscle, left axillary lymph node, and bone marrow from the right femur. The initial histologic diagnosis was anaplastic sarcoma, but immunohistochemical results indicated the cells were CD20+ and CD45R+ and CD3-, compatible with a diagnosis of B-cell lymphoma. CD79a staining was nonspecific and uninterpretable. Weak to moderate CD18 positivity and E-cadherin positivity were also observed. Clonality of the B-cell population could not be demonstrated using PCR testing for antigen receptor gene rearrangement. To the authors' knowledge, this is the first reported case of a feline spinal anaplastic B-cell lymphoma exhibiting bi- and multinucleated cells. The prognostic significance of this cell morphology and immunophenotype is unknown.

Erythrophagocytic low‐grade extranodal T‐cell lymphoma in a cat

A 13-year-old male castrated domestic shorthair cat was presented to the referring veterinarian with a 2-month history of weight loss and lethargy. Splenomegaly, hepatomegaly, nonregenerative anemia, neutropenia, and hyperbilirubinemia were noted. Results of testing for feline immunodeficiency virus, feline leukemia virus, Toxoplasma gondii, and Mycoplasma sp. were negative. On cytologic examination of aspirates from the enlarged spleen and liver, a population of erythrophagocytic round cells was observed. Splenectomy and a liver biopsy were done which revealed a population of CD3+/CD79a- erythrophagocytic mononuclear round cells localized in the hepatic and splenic sinusoids. T-cell PARR (PCR for antigen receptor gene rearrangements) analysis of bone marrow and spleen demonstrated a single band indicative of a clonal proliferation of T cells. Based on the marked splenomegaly, sinusoidal infiltration, lack of lymphadenopathy, and results of cytology, PARR, and immunophenotyping, a diagnosis of low-grade extranodal T-cell lymphoma was made. The cat was treated with chlorambucil and prednisolone; clinical and laboratory abnormalities resolved and the cat has remained clinically normal for 2.5 years. To our knowledge, this report documents the first case of an erythrophagocytic T-cell lymphoma in a cat. The clinicopathologic findings were suggestive of hepatosplenic T-cell lymphoma, a neoplasm described previously only in humans and dogs.

The CD34+CD38LowCD19+ Candidate Leukemic Stem Cell Phenotype Revisited: Useful for Flow MRD Monitoring?

The assessment of minimal residual disease during therapy is a powerful prognostic marker in childhood acute lymphoblastic leukemia but current techniques, whether molecular analysis of antigen receptor gene rearrangements or flow cytometric analysis of aberrant immunophenotypes, track the blast population(s) that predominate(s) at diagnosis. Recent evidence from diagnostic TEL/AML1 positive cases (Hong et al, 2008) suggests that the candidate leukemic stem cell population may be characterised by the immunophenotype CD34+CD38LowCD19+ and thus the identification and quantitation of this cell population at diagnosis and during therapy may have greater clinical significance.Cytogenetic subgroupNumber with candidate populationNumber without candidate populationTEL/AML181High Hyperdiploid65Other1516Therefore, we have sought this proposed leukemic stem cell population in 51 consecutive diagnostic precursor-B acute lymphoblastic leukaemia samples using multi-parametric flow cytometry. Patient blasts were stained with the antibodies CD34PerCP, CD38FITC and CD19APC and 50,000 events acquired on a FACSCalibur. The postulated cancer stem cell population, CD34+CD38LowCD19+, was found in 57% of patients (29 of 51). Correlation with major cytogenetic subgroups showed that 89% (8 of 9) of TEL/AML1 positive cases had evidence of the population, compared to 55% (6 of 11) of high hyperdiploid and 48% (15 of 31) of the remainder (see Table). There was no evidence of the population in ‘normal marrow’ samples which were from children in the latter stages of therapy for ALL i.e. end of treatment bone marrow samples (n=4). We also examined bone marrow samples taken 28 days after the start of treatment in a cohort of patients scoring positive for the candidate population at diagnosis (n=11) and found evidence for persistence of the cells in 4 of them.CD38 under-expression, in relation to normal B cell precursors, is a common feature of ALL blasts and classification of CD38 expression levels revealed that 92% (24/26) of these under-expressers unsurprisingly housed the population because their blasts were also CD34+ and CD19+. Most importantly, since 43% of the diagnostic samples analysed show no evidence of this population (or it exists below the limits of detection of the assay i.e.<0.1% ), it seems unlikely that this represents a definitive cancer stem cell population in childhood ALL per se and would, therefore, not be appropriate as a generic marker of minimal residual leukemic stem cells.

Activation of 12/23-RSS-Dependent RAG Cleavage by hSWI/SNF Complex in the Absence of Transcription

Maintenance of genomic integrity during antigen receptor gene rearrangements requires (1) regulated access of the V(D)J recombinase to specific loci and (2) generation of double-strand DNA breaks only after recognition of a pair of matched recombination signal sequences (RSSs). Here we recapitulate both key aspects of regulated recombinase accessibility in a cell-free system using plasmid substrates assembled into chromatin. We show that recruitment of the SWI/SNF chromatin-remodeling complex to both RSSs increases coupled cleavage by RAG1 and RAG2 proteins. SWI/SNF functions by altering local chromatin structure in the absence of RNA polymerase II-dependent transcription or histone modifications. These observations demonstrate a direct role for cis-sequence-regulated local chromatin remodeling in RAG1/2-dependent initiation of V(D)J recombination.

Role of minimal residual disease evaluation in leukemia therapy

In patients with acute leukemia, measurements of minimal residual disease (MRD) provide unique information on response to treatment. The most useful currently available MRD assays are polymerase chain reaction amplification of fusion transcripts and rearranged antigen-receptor genes and flow cytometric detection of aberrant immunophenotypes. Many studies in children and adults with acute lymphoblastic leukemia and acute myeloid leukemia have demonstrated a strong association between the presence of MRD and risk of relapse. Therefore, the use of information on MRD to adjust the intensity of therapy holds great potential for improving long-term clinical outcome. This article discusses the methodologies available for productive MRD testing and the clinical significance of the results.

B- and T-cell Development Both Involve Activity of the Unfolded Protein Response Pathway

The unfolded protein response (UPR) signaling pathway regulates the functional capacity of the endoplasmic reticulum for protein folding. Beyond a role for UPR signaling during terminal differentiation of mature B cells to antibody-secreting plasma cells, the status or importance of UPR signaling during hematopoiesis has not been explored, due in part to difficulties in isolating sufficient quantities of cells at developmentally intermediate stages required for biochemical analysis. Following reconstitution of irradiated mice with hematopoietic cells carrying a fluorescent UPR reporter construct, we found that IRE1 nuclease activity for XBP1 splicing is active at early stages of T- and B-lymphocyte differentiation: in bone marrow pro-B cells and in CD4(+)CD8(+) double positive thymic T cells. IRE1 was not active in B cells at later stages. In T cells, IRE activity was not detected in the more mature CD4(+) T-cell population but was active in the CD8(+) cytotoxic T-cell population. Multiple signals are likely to be involved in activating IRE1 during lymphocyte differentiation, including rearrangement of antigen receptor genes. Our results show that reporter-transduced hematopoietic stem cells provide a quick and easy means to identify UPR signaling component activation in physiological settings.

Characterization of the Roles of Rac1 and Rac2 GTPases in Lymphocyte Development

This chapter describes methods for the analysis of B and T lymphocyte development in mice deficient in Rac1 and/or Rac2 GTPases. The development of both B and T cells is critically dependent on transition through checkpoints monitoring for correct rearrangement of antigen receptor genes. Progression through these checkpoints depends on signaling from the antigen receptors. In addition, signals from cytokine, chemokine, Notch, and death receptors play important roles in the survival, proliferation, and migration of developing lymphocytes. Analysis of these processes in mice deficient in these GTPases can illuminate their roles in transducing signals from these different receptors.

Extrathymic rearrangement of αβT-lymphocyte antigen receptor genes during pregnancy

The existence of alphabetaT-lymphocyte differentiation processes have been demonstrated in mouse peripheral lymphoid organs during pregnancy. Study of pregnant Swiss mice has shown that the development of the second half of gestation is accompanied by expression of RAG-1 recombinase mRNA and unrearranged TCR alpha-chain (pre-TCRalpha) preferentially in T-lymphocytes of lymph nodes involved in uterine drainage (para-aortal lymph nodes), and to a lesser extent in other lymph nodes (mainly from axillary lymph nodes). The data suggest that during pregnancy the differentiation of alphabetaT lymphocytes may occur not only in central (thymus) but also in peripheral lymphoid organs.

Quantitative analysis of minimal residual disease predicts relapse in children with B-lineage acute lymphoblastic leukemia in DFCI ALL Consortium Protocol 95-01

AbstractIn a prospective trial in 284 children with B-lineage acute lymphoblastic leukemia (ALL), we assessed the clinical utility of real-time quantitative polymerase chain reaction analysis of antigen receptor gene rearrangements for detection of minimal residual disease (MRD) to identify children at high risk of relapse. At the end of induction therapy, the 5-year risk of relapse was 5% in 176 children with no detectable MRD and 44% in 108 children with detectable MRD (P < .001), with a linear association of the level of MRD and subsequent relapse. Recursive partitioning and clinical characteristics identified that the optimal cutoff level of MRD to predict outcome was 10−3. The 5-year risk of relapse was 12% for children with MRD less than one leukemia cell per 103 normal cells (low MRD) but 72% for children with MRD levels greater than this level (high MRD) (P < .001) and children with high MRD had a 10.5-fold greater risk of relapse. Based upon these results we have altered our treatment regimen for children with B-lineage ALL and children with MRD levels greater than or equal to 10−3 at the end of 4 weeks of multiagent induction chemotherapy now receive intensified treatment to attempt to decrease their risk of subsequent relapse.

An in vitro model of T cell receptor revision in mature human CD8+ T cells

V(D)J recombination is a mechanism peculiar to the somatic rearrangement of antigen receptor genes. It requires both expression of the RAG-1 and RAG-2 recombinases and accessibility of the substrate to its recombinase and post-cleavage/DNA repair stage. TCR revision is a genetic correction mechanism that changes T cell specificity by re-activating V(D)J recombination in peripheral T cells. This process is now well described in both normal or pathological murine and human settings. Many of its features, such as the question of whether it occurs in truly mature T cells, remain to be elucidated. Its occurrence in human CD8+ T cells is also an open question.We have therefore established an in vitro model of TCR revision in mature human CD8+ T cells to determine whether down-regulation of the TCR/CD3 complex from the cell surface in the presence of IL7 as a factor favouring chromatin remodelling initiates a TCR revision pathway. Only mature CD8+ T cells carrying already-formed antigen receptors were used. CD8+ T cells treated with anti-CD3 and IL7 showed rearrangement intermediates and expressed new Vβ-chains on their surface. Investigation of the molecular pathway thus induced disclosed up-regulation of the RAG-2 transcript, but absence of the ‘canonical’ RAG-1 mRNA. A surprising finding was the demonstration of alternative splice forms of this mRNA, already expressed in untreated CD8+ T cells, encoding for the full-length RAG-1 protein, which was increased three-fold in the treated cells.All the V(D)J requirements were thus fulfilled when mature human CD8+ T cells were stimulated with anti-CD3 and IL7. Induction of TCR revision in vitro in mature T cells is an easily controllable system that could be employed in further studies to elucidate the molecular pathways involved in secondary V(D)J rearrangements in peripheral cells.

Relapse in children with acute lymphoblastic leukemia involving selection of a preexisting drug-resistant subclone

AbstractRelapse following remission induction chemotherapy remains a barrier to survival in approximately 20% of children suffering from acute lymphoblastic leukemia (ALL). To investigate the mechanism of relapse, 27 matched diagnosis and relapse ALL samples were analyzed for clonal populations using polymerase chain reaction (PCR)–based detection of multiple antigen receptor gene rearrangements. These clonal markers revealed the emergence of apparently new populations at relapse in 13 patients. More sensitive clone-specific PCR revealed that, in 8 cases, these “relapse clones” were present at diagnosis and a significant relationship existed between presence of the relapse clone at diagnosis and time to first relapse (P < .007). Furthermore, in cases where the relapse clone could be quantified, time to first relapse was dependent on the amount of the relapse clone at diagnosis (r = −0.84; P = .018). This observation, together with demonstrated differential chemosensitivity between subclones at diagnosis, argues against therapy-induced acquired resistance as the mechanism of relapse in the informative patients. Instead these data indicate that relapse in ALL patients may commonly involve selection of a minor intrinsically resistant subclone that is undetectable by routine PCR-based methods. Relapse prediction may be improved with strategies to detect minor potentially resistant subclones early during treatment, hence allowing intensification of therapy.

Regionalized combinatorial diversity provides an evolutionary link between innate and adaptive immunity (90.7)

Abstract Adaptive immunity emerged in jawed vertebrates exhibiting the ability to express somatically rearranged antigen receptor genes with combinatorial binding sites consisting of residues from each of the paired v-type (variable) Ig domains (i.e., VαVβ). The origins of contemporary antigen receptors have been described by a model in which an ancestral Ig gene diversified to form polymorphic variants consisting of an antigen binding domain linked to an effector module (i.e., VC). The high degree of clustering of polymorphic residues at the combining site between v-type Ig domains throughout evolution of adaptive immunity suggests that this feature confers a powerful advantage in creating a diverse repertoire of antigen receptors. To address the origins of adaptive immunity, we solved the crystal structure of VCBP3 D1 and D2 from the cephalochordate Branchiostoma floridae, which has characteristics of a primordial antigen receptor: a high degree of germline encoded polymorphism and paired v-type Ig domains with polymorphism clustered at the combining site. In addition to finding combinatorial recognition in a jawless invertebrate, we solved the structure of a primordial V domain to 1.15 Å, a level of refinement that permits new insight into the atomic basis for Ig domain organization and stability.

Requirements for DNA hairpin formation by RAG1/2

The rearrangement of antigen receptor genes is initiated by double-strand breaks catalyzed by the RAG1/2 complex at the junctions of recombination signal sequences and coding segments. As with some "cut-and-paste" transposases, such as Tn5 and Hermes, a DNA hairpin is formed at one end of the break via a nicked intermediate. By using abasic DNA substrates, we show that different base positions are important for the two steps of cleavage. Removal of one base in the coding flank enhances hairpin formation, bypassing a requirement for a paired complex of two signal sequences. Rescue by abasic substrates is consistent with a base-flip mechanism seen in the crystal structure of the Tn5 postcleavage complex and may mimic the DNA changes on paired complex formation. We have searched for a tryptophan residue in RAG1 that would be the functional equivalent of W298 in Tn5, which stabilizes the DNA interaction by stacking the flipped base on the indole ring. A W956A mutation in RAG1 had an inhibitory effect on both nicking and hairpin stages that could be rescued by abasic substrates. W956 is therefore a likely candidate for interacting with this base during hairpin formation.

Key factors in the organized chaos of early T cell development

A fundamental issue in T cell development is what controls whether a thymocyte differentiates into a gammadelta T cell or an alphabeta T cell, each defined by their distinct T cell receptor. Most likely, lessons learned in studying that issue will also provide insight into how the thymus produces T cell subsets with distinct functional and regulatory potentials. Here we review recent experiments, focusing on three factors that regulate thymocyte differentiation up to and including the expression of the first products of antigen receptor gene rearrangements. Those factors are the archetypal developmental regulator Notch, intrinsic signals emanating from antigen-receptor complexes, and trans conditioning, which reflects communication between different subsets of thymocytes. We also review new findings on the positive selection of gammadelta T cells and on extrathymic T cell development.

A Case of Atypical Canine Lymphoma with Oral Mass and Multiple Osteolysis

An 8-year-old female Golden Retriever had an oral mass and lameness. Multiple osteolysis of the systemic skeleton without monoclonal gammopathy was shown on electrophoresis of serum and urine samples. Cytological and histopathological examinations of the oral mass revealed atypical polymorphic cells similar to myeloid cells, and bone marrow aspiration indicated that these abnormal cells also might have invaded the bone marrow. These cells were negative to peroxidase and non-specific esterase staining, and clonal expansion of B lymphocytes could be detected by polymerase chain reaction (PCR) assay for antigen receptor gene rearrangement. The case was diagnosed as atypical lymphoma and treated by multi-drug chemotherapy. On the 142nd day after the first admission, the case had remission and the oral mass and multiple osteolysis were improved.

Target DNA Structure Plays a Critical Role in RAG Transposition

Antigen receptor gene rearrangements are initiated by the RAG1/2 protein complex, which recognizes specific DNA sequences termed RSS (recombination signal sequences). The RAG recombinase can also catalyze transposition: integration of a DNA segment bounded by RSS into an unrelated DNA target. For reasons that remain poorly understood, such events occur readily in vitro, but are rarely detected in vivo. Previous work showed that non-B DNA structures, particularly hairpins, stimulate transposition. Here we show that the sequence of the four nucleotides at a hairpin tip modulates transposition efficiency over a surprisingly wide (>100-fold) range. Some hairpin targets stimulate extraordinarily efficient transposition (up to 15%); one serves as a potent and specific transposition inhibitor, blocking capture of targets and destabilizing preformed target capture complexes. These findings suggest novel regulatory possibilities and may provide insight into the activities of other transposases.