Terminal Deoxynucleotidyl Transferase Expression Research Articles

Immunoreactivity of MIC2 (CD99) and Terminal Deoxynucleotidyl Transferase in Bone Marrow Clot and Core Specimens of Acute Myeloid Leukemias and Myelodysplastic Syndromes

MIC2 ("thymus leukemia") antigen has been shown to be expressed by T cells and monocytes, as well as B cells and granulocyte-lineage cells. It is most intensely expressed by the most immature thymus T-lineage cells and is more intensely expressed by CD34-positive/CD33-positive myeloid cells (compared to more mature myeloid cells) and the earliest CD34-positive/CD10-positive B-cell precursor cells (compared to cells of later B-cell precursor stages). CD99 (MIC2) is characteristically expressed in precursor B- and T-cell lymphoblastic lymphomas/leukemias, as well as in Ewing sarcoma/primitive neuroectodermal tumors (ES/PNET). It has also been shown to be expressed in a few terminal deoxynucleotidyl transferase (TdT)-positive myeloid processes, but has been uniformly negative in TdT-negative myeloid processes. A more recent study showed that 43% of acute myeloid leukemias (AMLs) and 55% of chloromas express CD99, concluding that CD99 is commonly expressed in AML and rarely seen in myeloproliferative disorders, myelodysplastic syndromes, or normal bone marrow. Although this study speculated that MIC2 expression was probably not limited to TdT-positive AML, there was no comparison with TdT reactivity in this study. Since AML and high-grade myelodysplastic syndrome may occasionally be difficult to distinguish morphologically from acute lymphoblastic leukemia and ES/ PNET, we undertook a study to analyze MIC2 expression in conjunction with TdT reactivity in distinguishing AML or high-grade myelodysplastic syndrome from acute lymphoblastic leukemia and ES/PNET. We studied bone marrow core and clot paraffin specimens from AML (classified according to criteria of the World Health Organization; n = 49), myelodysplastic syndromes (n = 4), precursor B-cell acute lymphoblastic leukemia (n = 4), ES/PNET (n = 1), and normal bone marrow (n = 3) with MIC2 (CD99) and TdT immunohistochemistry. Overall, CD99 was expressed in 24 (49%) of 49 AML cases, including all (11/11) TdT-positive cases. CD99 was expressed in all subtypes of AML except M5. Myelodysplastic syndromes and normal bone marrow specimens were uniformly CD99 negative. Expression of TdT was limited to a subset of AML-M0, -M1, -M2, and -M4, and AML with multilineage dysplasia. In contrast to a previous study, CD99 expression was not restricted to TdT-positive hematologic proliferations. In particular, the CD99-positive M3 and M7 AMLs were TdT negative. An M5 AML may likely be excluded based on a uniform TdT-negative/CD99-negative immunophenotype. In addition, in our experience, CD99 should be routinely evaluated on bone marrow clots, owing to decreased reactivity or loss of reactivity in rapid decalcifying (RDO) solution-decalcified specimens.

Evaluation of CD7 and terminal deoxynucleotidyl transferase (TdT) expression in CD34+ myeloblasts from patients with myelodysplastic syndrome

There is an emerging use of flow cytometry to evaluate patients with myelodysplastic syndrome (MDS). We have studied CD7 and TdT expression in the CD34+ myeloid blast cell population in 55 bone marrow samples of patients with MDS. CD7 and/or TdT were detected in 38 out of 55 patients (69%). CD7 expression was not related to other bad prognosis data but conversely, we found an association between TdT+ CD34 myeloblasts and high-risk MDS patients according to the International Prognostic Scoring System. Therefore, CD7 and TdT may help to establish the diagnosis of MDS and, TdT expression also seems to be a useful marker in distinguishing risk groups.

Expression of the plasmacytoid dendritic cell marker BDCA-2 supports a spectrum of maturation among CD4+ CD56+ hematodermic neoplasms

CD4+CD56+ hematodermic neoplasms are rare, aggressive hematopoietic malignancies usually presenting with cutaneous masses followed by a leukemic phase. The blastic morphology, CD56 expression and lack of definitive myeloid or T-cell markers initially resulted in assignment of this tumor to the NK-cell lineage. Accumulating evidence now suggests that these neoplasms represent malignant counterparts to the plasmacytoid dendritic cell. BDCA-2 is a cell surface protein whose expression is restricted to human plasmacytoid dendritic cells, in a differentiation stage-specific manner. In the current study, we assessed expression of BDCA-2 in CD4+CD56+ hematodermic neoplasms using a new antibody reagent we developed for use in fixed tissue sections. In 10 of 19 cases of CD4+CD56+ hematodermic neoplasm, BDCA-2 immunoreactivity was detected, whereas no expression was observed in NK-lineage tumors (0 of six). Interestingly, expression of terminal deoxynucleotidyl transferase, a marker of immaturity/blast stage, was significantly and negatively correlated with BDCA-2 in CD4+CD56+ hematodermic neoplasms whereas a positive correlation was observed between BDCA-2 and CD7. These findings demonstrate that BDCA-2 is expressed predominantly in the CD7+ subset of hematodermic neoplasms, and similar to non-neoplastic plasmacytoid dendritic cells, expression indicates a relatively more mature differentiation state. Clinical follow-up data confirm the aggressiveness of these tumors and suggests that BDCA-2 immunoreactivity, as identified here, may herald a significant reduction in survival.

Immunohistologic Identification and Quantification of Human Bone Marrow Hematopoietic Stem Cells.

A recent experimental and clinical observation on the use of bone marrow-derived stem/progenitor cells (BM-SC) to treat non-hematologic degenerative diseases has renovated the long-standing knowledge of their biology and function. However, the paradoxical lack of histological studies limits the identification of SC within the human BM, constraining their purification and further obscuring the sites where SC fate is decided. In an attempt to identify human hematopoietic BM-SC and their relationships with the tissue environment, bone marrow biopsies from 31 subjects without BM involvement by hemo-lymphoproliferative diseases were analysed with stringent morphometric criteria for the quantification of cells expressing CD117 (c-kit) and CD34, as candidate SC markers, and/or CD45, CD3, CD20, myeloperoxidase (MPOX), CD68, Glycophorin A (GlyA) and Factor VIII (vWF), as lineage commitment. Immunofluorescence combined with confocal examination was also performed to allow the simultaneous detection of multiple surface/cytoplasmic antigens together with transcription factors. Hematopoietic cells constituted 54% of the tissue among which MPOX, GlyA and vWF positive cells represented 43.8%, 13.5% and 0.06% of marrow cells, respectively. The frequency of CD34pos cells was 93.5±43.6 each 105 total nucleated cells; c-kit involved 2.7±2.6/105 cells and c-kit-CD34 co-expression was present in 1.14±0.98/105cells. When the incidence of CD45 was evaluated among these two cell populations only 15% of c-kitpos and 49% of CD34pos cells showed hematopoietic lineage commitment, indicating the presence in the human bone marrow of undifferentiated SC. The analysis of the expression of Terminal Deoxynucleotidyl Transferase (TdT) in hematopoietic cells demonstrated that 10% of c-kitpos, 20% of CD34pos and 0.62% of CD45pos cells possessed the nuclear transcription factor. The distribution of BM-SC was predominantly scattered throughout the tissue although interstitial and paratrabecular clusters were detected and are currently under intense scrutiny in search of BM-SC sites of activation. In a subset of patients, the histological analysis was complemented by the cytofluorimetric estimation of the same BM cells populations. The results of this study may contribute to the improvement in the isolation and characterization of BM-SC aimed to the identification of their niches and to an appropriate clinical application in the field of regenerative medicine.

Presence of somatic hypermutation and activation‐induced cytidine deaminase in acute lymphoblastic leukemia L2 with t(14;18)(q32;q21)

Acute lymphoblastic leukemia (ALL) with L2 (FAB) morphology has rarely been reported to show t(14;18)(q32;q21). We aimed to delineate the stage at which this type of ALL is derived in B-lineage differentiation. The somatic hypermutation (SHM) of the variable region of immunoglobulin heavy chain (IgV(H)) gene and the expression of terminal deoxynucleotidyl transferase (TdT), recombination-activating gene 1 and 2 (RAG-1 and -2), and activation-induced cytidine deaminase (AID) were investigated in three cell lines and two fresh samples, including a pair of matched fresh and cell line cells. TdT, RAG-1, and RAG-2 were variably expressed. AID was expressed in four of five samples. SHM of the IgV(H) gene was found in all samples with high average frequency (11.84%) comparable with that in follicular lymphoma. Ongoing mutation was seen in two fresh samples. As AID and SHM are generally regarded as properties exhibited by mature B cells, the presence of AID and SHM in this study seems to be incompatible with the general understanding of the early stage derivation of ALL in B-lineage differentiation. The results here give some insight into the relationship between disease type (ALL or lymphoma) and derivation stage, the overlapping of the early stage phenotype and the mature genomic characteristics, and the probable relationship between the mechanism of the occurrence of t(14;18)(q32;q21) and the machinery causing SHM.

Transcriptional Response to IL-7 by Immature Human Thymocytes Demonstrates Specific Effects on Differentiation as Well as Survival Maintenance and Proliferation.

Cytokine signaling has been thought to have either instructive effects on hematopoietic cell differentiation, or only survival and proliferation effects on cells whose differentiation is programmed by mechanisms other than cytokine signaling. Thymopoiesis is a useful model for examining the question of how cytokines affect lymphohematopoietic development because it is tightly regulated with well-defined developmental stages, and loss of thymopoiesis is not lethal. IL-7 is a cytokine that is known to be essential for normal thymocyte development as demonstrated by low thymocyte numbers in IL-7-deficient and IL-7R-deficient mice. To determine whether IL-7 has direct effects on human thymocyte differentiation or only acts a survival/proliferation factor, changes in gene expression in CD3-CD4-CD8-(”triple-negative”, TN) human thymocytes were analyzed after in vitro stimulation with IL-7. TN thymocytes were enriched by negative selection with anti-CD3, -CD4, -CD8 immunomagnetic columns, followed by FACS purification, factor starvation for 12–16 hours, and stimulation with IL-7. The RNA was extracted, amplified by in vitro transcription, labelled, and hybridized on Affymetrix U133 microarrays. Analyses of 10 paired samples of unstimulated and IL-7 stimulated cells revealed 56 genes which were up-regulated and 22 genes which were down-regulated greater than 1.25 fold (p<0.05). Confirmatory quantitative RT-PCR demonstrated significant data compression, e.g., signal changes of 1.25 fold by array corresponded to 8–20 fold differences by qRT-PCR. Many of the up-regulated genes were not lymphoid- or thymocyte-specific and were known previously, e.g., anti-apoptotic (Bcl-2) or cell cycle regulatory (pim-1 cyclin D2). There was a pattern of changes in gene expression which would be expected to down-modulate IL-7R signaling - decreased IL-7R itself, increased SOCS family genes, decreased PI-3K p85α. There were also alterations in genes specifically expressed during lymphocyte or thymocyte differentiation. Mal, which encodes a T lymphoid specific proteolipid tetraspannin that is thought to be a necessary component of lipid rafts containing the T cell receptor (TCR) for antigen, was up-regulated 4-fold. On the other hand, IL-7 down-regulated expression of terminal deoxynucleotidyl transferase (Tdt). IL-7 appeared to regulate cytokine production and responsiveness. There was altered expression of secreted protein and recptor genes that would alter the interactions of TN thymocytes with their microenvironment, e.g., increased P-selectin, IL-4R, frizzled 10, and decreased TGFβ 1, EGF. The complex changes in gene expression induced by IL-7 in TN cells suggest a direct instructive role for IL-7 signaling in the tight regulation of the different stages of thymic differentiation, rather than only regulation of survival, maintenance and/or proliferation.

Nonpositive Terminal Deoxynucleotidyl Transferase in Pediatric Precursor B-Lymphoblastic Leukemia

Terminal deoxynucleotidyl transferase (TdT) is a unique intranuclear DNA polymerase that catalyzes the template-independent addition of deoxynucleotides to the 3'-hydroxyl terminus of oligonucleotide primers. The expression of TdT is restricted to lymphoid precursors. It is a useful marker in distinguishing acute lymphoblastic leukemia (ALL) from mature lymphoid neoplasms. Although TdT– T-cell ALL has been reported in the literature rarely, the frequency and significance of TdT-nonpositive (TdTnp) B-cell ALL have not been examined extensively. We reviewed the immunophenotypes of 186 new cases of pediatric B-cell ALL and found 5 TdTnp cases (2.7%). They showed significantly higher frequencies of a WBC count of more than 50,000/μL (>50.0 × 109/L), CD10–, CD34–, and MLL gene rearrangement compared with those in TdT+ cases (3/5 [60%] vs 27/181 [14.9%], P = .03; 3/5 [60%] vs 11/181 [6.1%], P = .003; 4/5 [80%] vs 24/179 [13.4%], P = .002; 3/5 [60%] vs 9/181 [5.0%], P = .0019; respectively). These results indicate that nonpositive TdT does not rule out a diagnosis of ALL and suggest that TdTnp B-cell ALL might be associated with CD10– and CD34– disease, a high WBC count, and MLL gene rearrangement.

Rare detection of phenotypically immature lymphocytes in Hashimoto thyroiditis and rheumatoid arthritis

It has been suggested that recombination activating gene (RAG)-dependent revision of the immunoglobulin genes in germinal centres may contribute to local production of autoantibodies in Hashimoto thyroiditis (HT) and rheumatoid arthritis (RA). To test this hypothesis we examined HT and RA tissues for expression of RAG and terminal deoxynucleotidyl transferase (TdT) in situ. Paraffin-embedded tissues from 19 HT patients and from 20 RA patients were subjected to immunohistochemistry using TdT-specific antibodies. Expression of the RAGs was studied by in situ hybridisation. Tonsil sections were used as a control. Expression of TdT and RAGs was detected in extrafollicular lymphocytes in control tonsil sections. By contrast, only rare TdT-expressing cells were identified in 11 of 19 HT and in 2 of 20 RA samples. Germinal centre B-cells were consistently TdT- and RAG-negative. These results suggest that local RAG-dependent receptor revision in germinal centres is unlikely to contribute to production of autoantibodies in HT and RA. The presence of TdT-positive extrafollicular cells may represent an influx of immature cells in the context of chronic immune stimulation.

Terminal deoxynucleotidyl transferase is down-regulated by AP-1-like regulatory elements in human lymphoid cells.

Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyses the incorporation of deoxyribonucleotides into the 3'-hydroxyl end of DNA templates and is thought to increase junctional diversity of antigen receptor genes. TdT is expressed only on immature lymphocytes and acute lymphoblastic leukaemia cells and its transcriptional expression is tightly regulated. We had previously found that protein kinase C (PKC) activation down-regulates TdT expression. PKC-activation induces the synthesis of the Fos and Jun proteins, known as the major components of activation protein 1 (AP-1) transcriptional factor implicated in transcriptional control. Here we report the identification of several DNA-protein interactions within the TdT promoter region in non-TdT expressing human cells. Sequence analysis revealed the presence of a putative AP-1-like DNA-binding site, suggesting that AP-1 may play a relevant role in TdT transcriptional regulation. Using a different source of nuclear extracts and the AP-1-TdT motif as a probe we identified several DNA-protein retarded complexes in electrophoretic mobility shift assays. Super-band shifting analysis using an antibody against c-Jun protein confirmed that the main interaction is produced by a nuclear factor that belongs to the AP-1 family transcription factors. Our findings suggest that the TdT gene expression is down-regulated, at least in part, through AP-1-like transcription factors.

Rare detection of phenotypically immature lymphocytes in Hashimoto thyroiditis and rheumatoid arthritis

It has been suggested that recombination activating gene (RAG)-dependent revision of the immunoglobulin genes in germinal centres may contribute to local production of autoantibodies in Hashimoto thyroiditis (HT) and rheumatoid arthritis (RA). To test this hypothesis we examined HT and RA tissues for expression of RAG and terminal deoxynucleotidyl transferase (TdT) in situ. Paraffin-embedded tissues from 19 HT patients and from 20 RA patients were subjected to immunohistochemistry using TdT-specific antibodies. Expression of the RAGs was studied by in situ hybridisation. Tonsil sections were used as a control. Expression of TdT and RAGs was detected in extrafollicular lymphocytes in control tonsil sections. By contrast, only rare TdT-expressing cells were identified in 11 of 19 HT and in 2 of 20 RA samples. Germinal centre B-cells were consistently TdT- and RAG-negative. These results suggest that local RAG-dependent receptor revision in germinal centres is unlikely to contribute to production of autoantibodies in HT and RA. The presence of TdT-positive extrafollicular cells may represent an influx of immature cells in the context of chronic immune stimulation.

Human cord blood CD34+Pax-5+ B-cell progenitors: single-cell analyses of their gene expression profiles

Circulating CD34(+) cells are used in reparative medicine as a stem cell source, but they contain cells already committed to different lineages. Many think that B-cell progenitors (BCPs) are confined to bone marrow (BM) niches until they differentiate into B cells and that they do not circulate in blood. The prevailing convention is that BCP transit a CD34(+)CD19(-)10(+) early-B-->CD34(+)CD19(+)CD10(+) B-cell progenitor (pro-B)-->CD34(-)CD19(+)CD10(+) B-cell precursor (pre-B) differentiation pathway within BM. However, populations of CD34(+)CD10(+) and CD34(+)CD19(+) cells circulate in adult peripheral blood and neonatal umbilical cord blood (CB) that are operationally taken as BCPs on the basis of their phenotypes, although they have not been submitted to a systematic characterization of their gene expression profiles. Here, conventional CD34(+)CD19(+)CD10(+) and novel CD34(+)CD19(+)CD10(-) BCP populations are characterized in CB by single-cell sorting and multiplex analyses of gene expression patterns. Circulating BCP are Pax-5(+) cells that span the early-B, pro-B, and pre-B developmental stages, defined by the profiles of rearranged V-D-J(H), CD79, VpreB, recombination activating gene (RAG), and terminal deoxynucleotidyl transferase (TdT) expression. Contrary to the expectation, circulating CD34(+)CD19(-)CD10(+) cells are essentially devoid of Pax-5(+) BCP. Interestingly, the novel CD34(+)CD19(+)CD10(-) BCP appears to be the normal counterpart of circulating preleukemic BCPs that undergo chromosomal translocations in utero months or years before their promotion into infant acute lymphoblastic B-cell leukemia after secondary postnatal mutations. The results underscore the power of single-cell analyses to characterize the gene expression profiles in a minor population of rare cells, which has broad implications in biomedicine.

Substantial N diversity is generated in T cell receptor alpha genes at birth despite low levels of terminal deoxynucleotidyl transferase expression in mouse thymus.

N region diversity in antigen receptors is a developmentally regulated process in B and T lymphocytes, which correlates with the differential expression of terminal deoxynucleotidyl transferase (TdT). To precisely determine the onset of TdT gene activation during T cell differentiation and thymic ontogeny, TdT expression was directly detected at the cellular level by in situ hybridization and TdT function was assessed by analyzing the distribution of N additions in alpha and beta TCR genes at early stages of development. Even though TdT transcripts were undetectable at birth, substantial N additions were observed in ValphaJalpha junctions and 3 days later in VbetaDbetaJbeta junctions, indicating that TdT expression could be induced in immature thymocytes much earlier than expected. Indeed low TdT expression level was found in TN3/4 and DP from fetal day 17, suggesting that the onset of TdT expression occurs simultaneously in both populations and may depend on microenvironmental cues. Moreover significant increase in the proportion of thymocytes expressing high levels of TdT mRNA during the first week after birth without a similar increase in the level of N diversity suggests that TdT expression and TdT function in the generation of N diversity are not strictly correlated.

The development of primary and secondary lymphoid tissues in the nurse shark Ginglymostoma cirratum: B-cell zones precede dendritic cell immigration and T-cell zone formation during ontogeny of the spleen.

Secondary lymphoid tissue and immunoglobulin (Ig) production in mammals is not fully developed at birth, requiring time postnatally to attain all features required for adaptive immune responses. The immune system of newborn sharks - the oldest vertebrate group having adaptive immunity - also displays immature characteristics such as low serum IgM concentration and high levels of IgM1gj, an innate-like Ig. Primary and secondary lymphoid tissues in sharks and other cartilaginous fish were identified previously, but their cellular organization was not examined in detail. In this study of nurse shark lymphoid tissue, we demonstrate that the adult spleen contains well-defined, highly vascularized white pulp (WP) areas, composed of a central T-cell zone containing a major histocompatibility complex (MHC) class II+ dendritic cell (DC) network and a small number of Ig+ secretory cells, surrounded by smaller zones of surface Ig+ (sIg+) B cells. In neonates, splenic WPs are exclusively B-cell zones containing sIgM+-MHC class IIlow B cells; thus compartmentalized areas with T cells and DCs, as well as surface Ig novel antigen receptor (sIgNAR)-expressing B cells are absent at birth. Not until the pups are 5 months old do these WP areas become adult-like; concomitantly, sIgNAR+ B cells are readily detectable, indicating that this Ig class requires a 'mature immune-responsive environment'. The epigonal organ is the major site of neonatal B lymphopoiesis, based on the presence of developing B cells and recombination-activating gene 1 (RAG1)/terminal deoxynucleotidyl transferase (TdT) expression, indicative of antigen receptor rearrangement; such expression persists into adult life, whereas the spleen has negligible lymphopoietic activity. In adults but not neonates, many secretory B cells reside in the epigonal organ, suggesting, like in mammals, that B cells home to this primary lymphoid tissue after activation in other areas of the body.

CD56(+) TdT(+) blastic natural killer cell tumor of the skin: a primitive systemic malignancy related to myelomonocytic leukemia.

An unusual cutaneous tumor that has blastic morphology and coexpresses CD56 and terminal deoxynucleotidyl transferase (TdT) has been recently recognized and termed blastic natural killer cell lymphoma. The authors identified seven cases of such CD56(+)TdT(+) blastic tumors presenting in skin at their institution. The authors correlated clinical course with histomorphology and immunophenotype. All 7 patients (6 men, 1 woman, 52-85 years) presented with rapidly growing, frequently multiple cutaneous nodules. All patients had low level bone marrow involvement at diagnosis and frequently had lymph node involvement. Tumor cells were of intermediate size with irregular nuclear contours, fine chromatin, and indistinct small nucleoli. The expression of TdT varied between 5% and over 90% of the neoplastic cell population. Tumor cells were negative for surface CD3, CD5, and CD20 in all cases, but some patients showed expression of CD2 (three out of five), cytoplasmic CD3 (two out of seven), CD4 (six out of seven), and CD16 (three out of seven). Molecular studies showed absence of T-cell receptor gene rearrangements in all cases. All seven patients had rapid progression of disease, and six patients have died of their disease or complications. Three patients developed progressively increasing numbers of bone marrow blasts that had a myeloid immunophenotype and were negative for TdT and CD56. Two patients met criteria for acute myeloid leukemia at 11 and 22 months after presentation, respectively. CD56(+) TdT(+) blastic tumor presenting in skin is a systemic malignancy likely of primitive/undifferentiated hematopoietic origin. Patients might subsequently develop tumors of myeloid or myelomonocytic phenotype, indistinguishable from acute myelogenous leukemia.

Expression of the recombination-activating genes in extrafollicular lymphocytes but no apparent reinduction in germinal center reactions in human tonsils

AbstractV(D)J recombination in lymphocytes is mediated by 2 recombination-activating genes, RAG1 and RAG2,which are expressed during lymphocyte development in bone marrow and thymus. Prompted by studies reporting re-expression of the RAGs in germinal center B cells, the expression of RAGs and terminal deoxynucleotidyl transferase (TdT) in human lymphoid tissues was examined using in situ hybridization and immunohistochemistry, respectively. Here it is shown that RAGs and TdT are not reinduced in germinal center reactions. However, RAG+/TdT+ cells are frequently present in extrafollicular areas of tonsils mainly at the boundary between lymphoid tissue and fibrous scaffold. Phenotypic analyses suggest that these cells are B cells. Finally, it is shown that RAG+/TdT+ cells are found more frequently in tonsils than in other peripheral lymphoid tissues. This may reflect an increased influx of RAG+/TdT+ cells as a result of higher antigenic stimulation at this site. Alternatively, this observation may indicate that the tonsils are an additional site of lymphocyte ontogeny.

B-cell precursors differentiated from cord blood CD34+ cells are more immature than those derived from granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells.

Umbilical cord blood (CB) has been widely used instead of bone marrow (BM) and peripheral blood (PB) for stem cell transplantation (SCT). However, problems of sustained immunodeficiency after CB transplantation remain to be resolved. To elucidate the mechanism of immunodeficiency, we compared the characteristics of B cells differentiated in vitro from CD34+ cells of CB with those of PB. Purified CD34+ cells from CB and PB were cultured on murine stroma cell-line MS-5 with stem cell factor and granulocyte colony-stimulating factor for 6 weeks. The B-cell precursors (pre-B cells) that differentiated in this culture system, were analysed as to their immunoglobulin heavy chain (IgH) variable region gene repertoire and the expression of B-cell differentiation-related genes. CD10+ CD19+ pre-B cells were differentiated from both PB and CB. Although the usages of IgH gene segments in pre-B cells differentiated from CB and PB were similar, the N region was significantly shorter in CB-derived than PB-derived cells. Productive rearrangements were significantly fewer in cells of CB than PB in the third week. Among a number of B-cell differentiation-related genes, the terminal deoxynucleotidyl transferase (TdT) gene was not expressed in CB-derived cells during the culture. These results indicated that immature features of pre-B cells from CB, such as lack of TdT expression, and a short N region and few productive rearrangements in the IgH gene, might cause the delay in mature B-cell production.

Terminal deoxynucleotidyl transferase deficiency decreases autoimmune disease in MRL-Fas(lpr) mice.

The neonatal Ab and TCR repertoires are much less diverse, and also very different from, the adult repertoires due to the delayed onset of terminal deoxynucleotidyl transferase (TdT) expression in ontogeny. TdT adds nontemplated N nucleotides to the junctions of Igs and TCRs, and thus its absence removes one of the major components of junctional diversity in complementarity-determining region 3 (CDR3). We have generated TdT-deficient MRL/lpr, Fas-deficient (MRL-Fas(lpr)) mice, and show that they have an increased lifespan, decreased incidence of skin lesions, and much lower serum levels of anti-dsDNA, anti-chromatin, and IgM rheumatoid factors. The generalized hypergammaglobulinemia characteristic of MRL-Fas(lpr) mice is also greatly reduced, as is the percentage of CD4(-)CD8(-)B220(+) (double-negative) T cells. IgG deposits in the kidney are significantly reduced, although evidence of renal disease is present in many mice at 6 mo. CDR3 regions of both IgH and TCR from peripheral lymphocytes of MRL-Fas(lpr) mice are shorter in the absence of TdT, and there is a paucity of arginines in the IgH CDR3 regions of the MRL-Fas(lpr) TdT(-/-) mice. Because the amelioration of symptoms is so widespread, it is likely that the absence of N regions has more of an affect than merely decreasing the precursor frequency of anti-dsDNA B cells. Hence, either the T or B cell repertoires, or more likely both, require N region diversity to produce the full spectrum of autoimmune lupus disease.

Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase.

V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.

Expression of recombination-activating genes and terminal deoxynucleotidyl transferase and secondary rearrangement of immunoglobulin kappa light chains in rheumatoid arthritis synovial tissue.

Lymphocytic infiltrates in rheumatoid arthritis (RA) synovium often resemble lymphoid follicles and contain clonally related Ig transcripts, suggesting in situ antigen-dependent B cell selection. Recent reports have shown expression of recombination-activating genes (RAGs) and concurrent secondary rearrangement of Ig genes in normal peripheral lymphoid organs (receptor revision). We sought to determine if RAG-mediated receptor revision of Ig kappa light chains occurs in B cells within the RA synovium. Because we previously reported enhanced N-region addition at V(L)-J(L) joins in clonally expanded light-chain transcripts from RA synovium, we also sought expression of terminal deoxynucleotidyl transferase (TdT), which is normally expressed only in B cell precursors or immature B cells. Reverse transcription-polymerase chain reaction (PCR) was used to detect RAG and TdT transcripts from unselected and B cell-enriched synovial and peripheral blood mononuclear cells obtained from 12 RA patients. Activity of RAG protein was sought using ligation-mediated PCR to detect recombination intermediates, and immunohistochemistry was performed to identify RAG+ cells within synovia. We found evidence of RAG-mediated secondary Ig kappa light chain rearrangements in about one-third of RA synovia. TdT expression was found in several samples, but did not correlate with RAG expression. RAG-mediated secondary Ig rearrangements of kappa light chains may contribute to the local production of antibodies to autoantigens (e.g., rheumatoid factor) or exogenous antigens, or it may represent a failed attempt at immune tolerance. TdT expression suggests the presence of immature B cells in RA synovia. These findings have important implications for the local generation of antibodies in RA and other chronic inflammatory diseases.

Evidence that terminal deoxynucleotidyltransferase expression plays a role in Ig heavy chain gene segment utilization.

TdT is a nuclear enzyme that catalyzes the addition of random nucleotides at Ig and TCR V(D)J junctions. In this paper we analyze human IgH rearrangements generated from transgenic minilocus mice in the presence or absence of TdT. In the absence of TdT, the pseudo-VH gene segment present in the minilocus is rearranged dramatically more frequently. Additionally, JH6 gene segment utilization is increased as well as the number of rearrangements involving only VH and JH gene segments. Thus, the recombination of IgH gene segments that are flanked by 23-nt spacer recombination signal sequences may be influenced by TdT expression. Extensive analysis indicates that these changes are independent of antigenic selection and cannot be explained by homology-mediated recombination. Thus, the role played by TdT may be more extensive than previously thought.