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The Journal of Immunology, 2005, 175: 2495-2500.
Copyright © 2005 by The American Association of Immunologists

Lymphoid Differentiation Pathways Can Be Traced by TCR {delta} Rearrangements1

Eva Fronková2,*,{dagger}, Ondrej Krejcí2,3,*,{dagger},{ddagger}, Tomás Kalina*,{ddagger}, Ondrej Horváth§, Jan Trka*,{dagger} and Ondrej Hrusák4,*,{ddagger}

* Childhood Leukemia Investigation Prague, Czech Republic; {dagger} Department of Pediatric Hematology/Oncology and {ddagger} Department of Immunology, Charles University 2nd Medical School, Prague, Czech Republic; and § Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TCR gene rearrangement generates diversity of T lymphocytes by V(D)J recombination. Ig genes are rearranged in B cells using the same enzyme machinery. TCRD (TCR {delta}) genes are frequently incompletely rearranged in B precursor leukemias and recently were found in a significant portion of physiological B lymphocytes. Incomplete TCRD rearrangements (V-D) thus serve as natural indicators of previous V(D)J recombinase activity. Functional V(D)J recombinase has recently been found in murine NK precursors. We tested whether physiological NK cells and other leukocyte subpopulations contained TCR rearrangements in humans. This would provide evidence that V(D)J recombinase was active in the ancestry cells and suggest common pathways among the positive cell types. TCRD were rearranged in 3.2–36% of NK cells but not in nonlymphoid leukocytes. The previously known phenomenon of TCRD transcription in NK cells is a possible mechanism that maintains the chromatin open at the TCRD locus. In comparison, TCRG rearrangements were frequent in T cells, low to negative in B and NK cells, and negative in nonlymphoid cells, suggesting a tighter control of TCRG. Levels of TCRD rearrangements were similar among the B lymphocyte subsets (B1-B2, naive-memory). In conclusion, human NK cells pass through a differentiation step with active V(D)J recombinase similar to T and B lymphocytes and unlike nonlymphoid leukocytes. This contradicts recent challenges to the concept of separate lymphoid and myeloid differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Physiological B, T{alpha}{beta}, and T{gamma}{delta} lymphocytes express the IgH + IgL (Ig H and L chains), TCR{alpha}{beta} or TCR{gamma}{delta} molecules, respectively. This concept was conserved throughout the last 450 million years of vertebrate development (1). The diversity of an adaptive immune response originates from the rearrangements of multiple gene loci (called V, D, and J) coding for Ig and TCR (2). The general organization of the V, D, and J gene loci as well as the enzyme machinery (including RAG-1, RAG-2, and nonhomologous end-joining DNA repair enzymes) involved in their rearrangement are similar among various TCR and IG genes (2). The regulatory mechanisms that allow V(D)J rearrangement of the appropriate genes include chromatin accessibility of either TCR or IG genes (3). However, the events driving the precursor cell toward rearrangement of TCR vs IG genes are not yet completely understood. These events appear to be connected to the fundamental T vs B lineage decision. In this respect, a previously accepted concept proposed that B cells exclusively rearrange IG genes, but not TCR genes, and vice versa for T cells (2). Leukemia cells from B and NK lineages break this rule by rearranging TCR genes in most cases (4). The most frequently rearranged TCR genes in B precursor leukemias are TCRG and TCRD (59 and 55%, respectively), followed by TCRB (35%) (4, 5). TCRD rearrangements have been reported in NK leukemia (6). Frequent TCR rearrangements in B precursor leukemias led to several hypotheses linking the inadequate regulation of recombinase activity to the malignant process (4, 7, 8, 9).

Our recent study has shown that incomplete TCRD rearrangements that are indistinguishable on the molecular level from those observed in typical B precursor acute lymphoblastoid leukemia (ALL)5 lymphoblasts could be found in B cells of all normal subjects tested (10). Thus, the "cross-lineage" rearrangements in leukemia may be facilitated by a physiological situation in which developing B lymphoid cells allow incomplete TCRD rearrangements. In fact, end-point PCR results of Steenbergen et al. (11) in the mid-1990s already indicated the presence of TCRD rearrangements in healthy peripheral B cells. No quantitative data were available until the real-time quantitative (RQ-)PCR results, which confirmed that TCRD are present in up to 16% of the alleles of purified B cells in normal human subjects (10). These findings suggest that rearrangements in TCRD genes can be used as naturally occurring markers of recombinase activity also in other human cell types. Therefore, we used this approach to analyze incomplete TCRD rearrangements in all major subsets of human peripheral blood leukocytes. This approach shows which cells have passed through a developmental step with active recombinase and may add important information to the recent reappraisal of leukocyte differentiation pathways (12). The situation, however, appears to be more complicated with other TCR and IG genes, which are rearranged later in the respective lineage differentiation (13) and different strength of regulation applies to them (14). To test whether TCRG genes rearrange in the "improper" lineage with the same frequency (as one might speculate due to their high frequency in B-lineage ALL), we adopted a similar RQ-PCR system for the detection of TCRG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and donors

Cells were isolated from peripheral blood of healthy donors by Ficoll-Paque (density, 1.077 g/ml; Pharmacia) density centrifugation. DNA from cell lines HT29 (colon carcinoma), BT474 (breast carcinoma), HeLa, and AC (XX cells from amniocentesis) served as a negative control. Peripheral blood from a patient with a complete DiGeorge syndrome containing <0.1% CD3pos cells (of lymphocytes) was used for B and NK cell sorting. Manipulation with volunteer donor and patient cells was performed after obtaining an informed consent and in compliance with the institutional ethical board.

Cell separation and DNA isolation

Sorting of NK cells. Cells were stained with a combination of FITC-labeled anti-CD3 and either PE-labeled anti-CD56 (samples 1 and 2) or pooled PE-labeled anti-CD56 and anti-CD16 (Immunotech). CD3neg(CD16)CD56pos cells were sorted either on FACSVantageSE cell sorter (samples 1–5) or on FACS Aria (samples 6 and 7) (both from BD Biosciences). The purity of sorted NK cells was verified by reanalysis of the samples on the cell sorter (total purity: samples 1–5, >99% NK cells; samples 6 and 7, 96% NK cells; all samples, 0.0–0.1% CD3pos cells in sorted NK fraction).

Sorting of B cell subsets. Cells were stained with PE-labeled anti-CD19 (Diatec) combined with a FITC-labeled mAb against either CD5 (Serotec) or CD27 (BD Biosciences). In addition, biotinylated mAbs against CD3 and CD56 (Serotec) were added followed by a streptavidin-tricolor (Caltag Laboratories) staining. Propidium iodide was added to exclude dead cells. All B cells were sorted as CD19posCD3negCD56negpropidium iodideneg and the subsets were sorted as: B1, CD5pos; B2, CD5neg, memory CD27pos, and naive CD27neg. The purity of sorted B fractions was >97.8%, in all except one sample (one B2 sorted sample was 96.5% pure, containing 3.3% events negative for all Ags assessed). Possible CD3pos contamination ranged from 0.0 to 0.38%, median 0.0%.

Sorting of T cells. T cells were sorted along with the sorting of total B cells (10) (five specimens) as CD3posCD19neg or in combination with the NK sorting of specimens 6 and 7 (described above) as CD3posCD16,56neg cells. Granulocytes were obtained by magnetic depletion of CD3pos and CD19pos cells (Miltenyi Biotec) from granulocyte-erythrocyte fractions after Ficoll-Paque density centrifugation. The purity was >99% of CD3negCD19neg cells. Monocytes were obtained by repeated magnetic separation of CD14pos cells (Miltenyi Biotec). The purity was >94% of CD14pos and >98% of CD3negCD19neg cells, CD3pos cells ranging from 0.16 to 0.52%, median 0.38% per sorted population. Genomic DNA from sorted cells was isolated by a QIAamp DNA Blood micro Kit or Mini kit (both from Qiagen). DNA was stored in –20°C before processing.

RQ-PCR

RQ-PCR for TCRD was performed as described previously (10) in the LightCycler rapid thermal cycler system (Roche Diagnostics). RQ- PCR for TCRG was performed in the iCycler IQ thermal cycler system (Bio-Rad). Forward V{gamma}I, V{gamma}II, and V{gamma}IV family-specific primers were described previously (15). For V{gamma}I-J{gamma} and V{gamma}II-J{gamma} rearrangements, the J{gamma} 1.3/2.3–3' reverse primer and TJ{gamma}1.3/2.3 germline TaqMan probe are designed to recognize both the J{gamma}1.3 and J{gamma}2.3 gene segments (16). V{gamma}IV-J{gamma} rearrangements were detected in the analogous manner using the consensus forward V{gamma}IV primer, J{gamma}1.1/2.1 reverse primer, and TJ{gamma}1.1/2.1 TaqMan probe (16). PCR amplification was conducted in 1x reaction buffer (20 mmol/L Tris-HCl (pH 8.4), 50 mmol/L KCl, and 3.0 mmol MgCl2) containing 200 µmol/L of each dNTP, 0.25 µmol/L, 0.5 µmol/L, and 0.5 µmol/L of V{gamma}I,V{gamma}II and V{gamma}IV forward primers, respectively, 0.5 µmol/L of reverse primer, 0.2 µmol/L of the probe, BSA in a final concentration of 0.04%, and 1 U of Platinum TaqDNA polymerase (Invitrogen Life Technologies) in a final reaction volume of 25 µl. For each PCR, 1 µl of DNA at a concentration of 0.01–0.2 µg/µl was used. The cycling conditions were as follows: initial denaturing at 94°C for 5 min, 50 cycles of denaturing at 94°C for 15 s, and annealing-extension at 64°C for 1 min. {beta}2-microglobulin housekeeping gene ({beta}2m) was used to normalize the DNA concentration and quality. The system for quantification of {beta}2m was described previously (17). Quantification was performed with iCycler iQ Optical System software, version 3.0a.

The starting concentration of template was measured against a dilution series of positive control DNA in buffered water. The REH cell line served as a positive control for the V{delta}2-D{delta}3, V{gamma}I-J{gamma}1.3/2.3, and VgII-Jg1.3/2.3 rearrangements. For the D{delta}2-D{delta}3 and V{gamma}IV-J{gamma}1.1./2.1 systems, patient samples bearing monoclonal rearrangement were used as a reference. Each sample was run in duplicate and the mean value was used for further analysis. The sensitivity of all RQ-PCR systems was better than 3 x 10–4, representing approximately four to eight rearranged alleles per reaction. The amount of rearranged alleles presented here is a ratio between the concentration of rearranged genes and {beta}2m. Any non-T specimen was considered positive if the specific TCR signal exceeded the sensitivity threshold, given as the frequency of alleles of possible T cell contaminants. PAGE analysis of PCR products was performed on 5% polyacrylamide gel (Sigma-Aldrich).

GeneScan analysis was performed using the VD2 primer labeled at its 5' end with FAM. Aliquots of PCR products (1 µl) were mixed with 10 µl of Hi-Di formamide and 0.5 µl of the GeneScan-500 [ROX] internal size standard (Applied Biosystems). The products were separated on 3100 POP-6 polymer (Applied Biosystems) and analyzed by 310 GeneScan 3.7 software (Applied Biosystems).

Cloning and DNA sequencing

Cloning of PCR products was performed with a TOPO TA Cloning kit (Invitrogen Life Technologies). Plasmid DNA from 45 single colonies (2–3 colonies per sorted population) was isolated using a JETPREP Plasmid Miniprep kit (GENOMED). The presence of the V{delta}2-D{delta}3 insert was verified by PCR using primers identical to those used in the RQ-PCR analysis. Forty colonies with a clear monoclonal band on PAGE were selected for sequencing. Sequencing was performed in the ABI PRISM 310 Genetic Analyzer with a BigDye Primer v3.0 Sequencing kit (Applied Biosystems).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TCRD gene loci rearrange in NK cells

RQ-PCR analysis of purified CD3negCD56pos cells of two different donors showed a clear positivity of the rearranged TCRD gene, which corresponded to 4.5 and 12.4% of the alleles, respectively. Similarly, a subsequent analysis of CD3negCD56,16pos cells from five different donors showed rearrangement V{delta}2-D{delta}3 in 3.2–36% of the alleles and D{delta}2-D{delta}3 in 0.13–3.0% of the alleles (Fig. 1). In addition, we analyzed sorted B and NK cells of a patient with a complete DiGeorge syndrome for presence of rearranged V{delta}2-D{delta}3 alleles; they were present in 24 and 32%, respectively. No T cell contamination (0 among >4200 acquired events) was detected in the sorted specimens. No amplification signal was detected in the non-lymphoid DNA samples (granulocytes and monocytes) during the entire RQ-PCR analysis. The PCR products were reanalyzed by PAGE. In all blood NK cell specimens, the products contained oligoclonal to polyclonal DNA (Fig. 2). Both positive controls, REH lymphoblastic cell line, and a sample of an ALL patient with one TCRD-rearranged allele, showed a clear monoclonal band. The fact that the patterns of PCR products differ among the donors virtually excludes false positivity due to the contamination by PCR products. It should be noted that our system detects only an incomplete TCRD rearrangement since rearrangement of any J{delta} locus to the TCRD{delta}3 would prevent binding of the reverse primer (18). Therefore, incomplete rearrangements of TCRD are present in peripheral NK cells. Along with our previous finding that both B and T lymphocytes contain the TCRD rearrangement, we now show that all major lymphoid lineages, unlike other cells investigated, contain rearranged TCRD genes. NK cells thus, similarly as T and B lymphocytes, pass through a stage with an active V(D)J recombinase apparatus.



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  		FIGURE 1. Percentage of TCRD rearranged alleles in sorted cells. Frequency of V{delta}2-D{delta}3 (•) and D{delta}2-D{delta}3 ({circ}) rearranged alleles in B cell subsets, including B1 (CD5pos), B2 (CD5neg), naive (CD27neg) and memory (CD27pos), T cells (CD3pos), NK cells (sorted either as CD3negCD56pos or CD3negCD56,16pos), monocytes, and granulocytes. The percentage is derived from a dilution curve of a positive control (100%) bearing monoclonal rearrangement. REH cell line (V{delta}2-D{delta}3 rearrangement) and primary lymphoblasts from a patient with ALL (D{delta}2-D{delta}3 rearrangement) were used as a positive control.

 


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  		FIGURE 2. PAGE analysis of V{delta}2-D{delta}3 rearrangements. Peripheral NK lymphocyte samples are shown in lanes 1–7. Positive control REH cell line is shown (+). MW indicates 100-bp molecular marker.

 
Size of the PCR products corresponds with the expected base pair length

To address the size of the PCR products, we performed a capillary electrophoresis under denaturing conditions. Specific PCR products are expected to have approximately the same size (300–350 bp in our system). As shown in Fig. 3, the sizes of the products fit to the expected range in all of the lymphocytic subsets tested as well as in the control leukemic cell line. Patterns of the amplified PCR products vary among different aliquots of the same sample, suggesting different unique sequences are amplified in each individual PCR. The calculated numbers of rearranged alleles range from 60 to 300/aliquot. Therefore, the interaliquot differences can be viewed as a consequence of random amplification.



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  		FIGURE 3. GeneScan electrophoretograms from ABI 310 Genetic Analyzer. VD2-DD3 PCR products from CD3pos (a and b), CD3negCD56pos (c and d), CD3negCD16,56pos (e and f), CD19pos5pos (g and h), CD19posCD5neg (i and j), CD19posCD27pos (k and l), and CD19posCD27neg (m and n); duplicates of each sorted subpopulation are always acquired from the same donor. A 324-bp REH cell line monoclonal product (100x diluted) is shown in o, the HeLa cell line sample did not show any amplification (p).

 
Sequencing confirms TCRD rearrangements

We have cloned RQ-PCR V{delta}2-D{delta}3 products in Escherichia coli to obtain monoclonal copies of incompletely rearranged TCRD locus genes suitable for sequencing. Plasmid DNA from 40 single colonies was sequenced. Two colonies revealed an unspecific signal and sequences of all others are shown in Table I. In six observations, two colonies representing PCR products from the same specimen had identical sequences. Capillary electrophoresis performed at the end stage of PCR revealed that a limited number of products dominated the reaction. It is therefore not unlikely to observe the same PCR product to be cloned in different bacterial colonies. All 13 different colonies selected from 7 peripheral NK cell samples contained a sequence of the V{delta}2-D{delta}3 rearrangement. Nucleotides (1–9 bp) were inserted in each sequence. Deletions of nucleotides from the 3' end of the V segment or the 5' end of the D segment were present each time in 12 clones.


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  		Table I. Sequence of TCRD V{delta}2-D{delta}3 rearrangements in NK, T, B1, and B2 cellsa

 
No difference in TCRD rearrangement pattern among B cell subsets

We have previously found V{delta}2-D{delta}3 and D{delta}2-D{delta}3 rearrangements in 8.3–15% and 6.5–16% alleles of human adult peripheral B lymphocytes, respectively (10). Our next aim was to investigate whether B1 and B2 subsets of B lymphocytes contain different proportions of the TCRD rearrangement, which would suggest a different regulation of the rearrangement activity within these two lineages and/or interactions between TCRD rearrangements and B1/B2 decision making. With respect to the IG rearrangement, there are differences between the B1 and B2 repertoires, although less prominent than previously thought (19). However, we found no difference among the percentage of cells with TCRD rearrangements between B1 and B2 subsets (V{delta}2-D{delta}3, 6.9–21%, 5.3–11%; D{delta}2-D{delta}3, 0.9–3%, 0.6–1.6% in B1 and B2 cells, respectively; Fig. 1). We then asked whether the process of affinity maturation influenced the percentage of B cells with cross-lineage TCRD rearrangements. We found no differences between the naive (CD27neg) and memory (CD27pos) B cells regarding the percentage of cells with TCRD rearrangements (V{delta}2-D{delta}3, 5–11% and 2.6–16%; D{delta}2-D{delta}3, 0.6–1.4% and 0.6–1.2% in naive and memory B cells, respectively). B cells bearing the TCRD rearrangement are thus functional B lymphocytes capable of maturation upon Ag stimulation, not defective cells arrested in a naive form. There is no obvious difference in the number of inserted and deleted nucleotides between the investigated subsets (Table I).

Low level of TCRG rearrangements in B subsets and NK cells

We further examined whether some rearrangements of TCRG ({gamma}) are also present in sorted cell populations. We analyzed rearrangements in V{gamma}I, V{gamma}II, and V{gamma}IV families using family-specific forward primers and reverse primers located in the respective J{gamma} segments. In all sorted T lymphocytes, a high percentage of rearranged TCRG alleles was present (Table II). Clearly, TCRG is often rearranged in both alleles, as described previously (20). In contrast, B cell subpopulations contained only low levels of rearranged TCRG alleles. Similarly, the level of TCRG rearranged alleles in sorted NK cells was low to negative (Table II). As in the case of TCRD, PAGE analysis confirmed oligoclonal to polyclonal rearrangements in positive samples, whereas all T cell samples showed a polyclonal smear (data not shown). We found no such rearrangements in granulocytes, monocytes, and non-blood cells.


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  		Table II. Percentage of rearranged TCRG alleles in sorted cellsa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Incomplete rearrangements of TCRD are present in peripheral NK cells

Presented data show that NK cells as well as T and B lymphocytes contain incomplete TCRD rearrangements. This complies with the recent data by Borghesi et al. (21), which proved high RAG2 expression in murine NK precursors; the cells indeed did rearrange an artificial recombinase target. In addition, TdTpos cells do develop also into NK cells (22). Furthermore, 3 of 62 splenic murine NK cells contained D-JH joins when analyzed by single-cell PCR (21). To date, there is no known physiological recombinase target in NK lineage and this is the first report showing a natural outcome of RAG gene activity in the form of V(D)J rearrangements in human NK cells. Early studies could not find TCRD rearrangements in NK cells by Southern blot (23, 24). The incomplete VD re-arrangements that are described possibly evaded the detection by Southern blot, because <5% of rearranged alleles are likely to remain undetected unless they represent a single clone. Because there is no evidence showing the expression of immunoreceptor genes by NK cells, only incomplete V(D)J rearrangements are likely to be present in NK cells. A possible explanation of the observed data is that the final decision for NK lineage commitment takes place in developmental stages, in which some V(D)J rearrangements are allowed. One can speculate whether the definitive NK lineage decision prevents completion of V(D)J rearrangement or whether the complete V(D)J rearrangement is the principal factor that determines the lineage decision towards B, T{alpha}{beta}, or T{gamma}{delta} lineages.

Mature NK cells do not contain RAG-1 activity (24), indicating that the TCRD rearrangements occur early during differentiation. Complying with this, the TCRD rearrangements in the NK cells appear to have occurred at early stages when TdT is active, since N nucleotides are inserted. In addition, NK cells are known to transcribe the TCRD locus. The resulting transcripts vary in length (0.8–2.3 kb), transcribe the J and C regions of TCRD, and have an unknown function. The analysis of their sequence locates the transcription initiation site immediately downstream of the D{delta}3 gene segment. Therefore, the VD rearrangements described in this article would not affect the size of these transcripts. Germline transcription of the VDJ regions is important for opening the chromatin before the V(D)J rearrangements (25). To our knowledge, TCRD transcripts have not been studied in NK cell precursors. A possible link between TCRD transcripts and the V(D)J rearrangements should be addressed in appropriate differentiation stages.

TCR{gamma} and TCR{delta}: relationships between biological importance and cross-lineage rearrangements

There are multiple checkpoints on which the rearrangements and expression of immunoreceptor genes are regulated. One of the key regulatory mechanisms is chromatin accessibility, which is driven mostly by cis-acting transcriptional elements allowing germline transcription, sequence-specific transcription factors, and chromatin structure changes (14). Complex regulatory mechanisms cause various loci to be rearranged at different stages of cell differentiation, TCRD being among the earliest, and also that some loci are nonrandomly preferred for recombination (14). Recently, murine B cell precursors have been shown to express functional V(D)J recombinase, even before the chromatin of IGH genes opens for its activity (26). The immunoreceptor genes with open chromatin that is rearranged by such recombinase activity would result in findings that are described here and previously (10).

Our data showing V{gamma}I-J{gamma}1.3 rearrangements as the most frequent among T cells (followed by V{gamma}II and V{gamma}IV) are in line with the observed frequency of V{gamma} rearrangements in ALL, showing 56, 18, and 1% of childhood ALL cases with V{gamma}I, II, and IV rearrangements, respectively (4). However, the TCRG genes are rearranged much less frequently than TCRD in healthy non-T cells and in some cases even below the detection limit of the method used in this study. The hypothesis that TCRD rearrangements in leukemic cells just represent a physiologically occurring phenomenon cannot be generalized and certainly is not true for immunoreceptor genes under tighter regulation. Presented data only show that TCRG rearrangements do occur in normal B cells. The reason why they are so frequent in B precursor ALL may be explained by one of the previously posed hypotheses: protracted V(D)J recombinase activity could lead to rearrangements of the "improper loci" (7); leukemic cells could develop from those B cells that fail to die by apoptosis (4); and unregulated V(D)J recombinase could also take place in the malignant transformation itself (9). Alternatively, cells with rearranged TCRG could be prone to the malignant transformation. Another hypothesis, which in the authors’ opinion does not have much support from immunophenotyping or other recent studies, proposed that malignant cells could derive from a yet undetermined B/T precursor (8).

Interestingly, V{delta}2pos T{gamma}{delta} cells represent probably the first lymphocyte subpopulation that matures in human ontogeny after birth (27). Although the crucial target Ags (comprising low molecular mass nonprotein molecules (1)) for these V{delta}2pos cells are yet to be specified, their high biological significance has been envisaged (27). Unlike T{alpha}{beta} cells, the crucial role of T{gamma}{delta} lymphocytes is in local immunity, with no or limited possibility of positive selection leading to clonal expansion (28, 29). Therefore, it seems important to keep the V{delta}2 locus accessible for recombination machinery. Recent study shows that a principle of allelic exclusion is applied to the human TCRD gene at the level of completed rearrangement (30). Analogously, regulatory mechanisms that prevent the ectopic TCR{delta} expression are utilized only downstream of the V to D rearrangement, leading to the absence of completed VDJ{delta} rearrangements in non-T cells (10).

Developmental perspective

The presented data collectively shows that TCRD rearrangements can be used to track progeny cells of precursors with an active V(D)J recombinase. Obviously, both presented data and our previous findings (10) show that only every tenth cell in each population appears to have the V{delta}2D{delta}3 rearrangement. Therefore, a sufficient number of cells need to be sorted to obtain quantifiable RQ-PCR data. This method showed that leukocyte lineages that are traditionally considered to be derived from a common lymphoid progenitor (except for lymphoid dendritic cells, which were not tested) pass through a stage with active V(D)J recombinase, whereas myeloid/monocytic cells do not. For the first time, human NK cells were shown to rearrange any V(D)J, which complements recent findings of IG rearrangements in mice (21).

Separate lymphoid and myeloid development stemming from the respective common progenitors have been included in the hematology textbooks for many years. Katsura (12) has recently challenged this concept, showing that sorted mouse fetal cells rarely give rise to B and T lineages in fetal thymic organ culture, whereas bi-potent B myeloid or T myeloid precursors could be identified. The authors interpreted this as an indication that B and T lineages first separate from each other and only then each of these lineages separates from myeloid fate. Other studies do not support this view, characterizing progenitors of lymphoid developmental potential by multiple approaches, including gene expression profiling (31, 32). The refusal of the common lymphoid progenitor (CLP) has been contested from several viewpoints, including possible in vitro cellular interactions that may inhibit CLP function in the model (33). Alternatively, a cell committing to the T lineage in this clonal assay may inhibit neighboring cells to become B cells.

Presented TCRD rearrangement data expand expression profiling studies identifying RAG2 as one of the genes discriminating CLP (32). Based on these data, all lymphoid cell lines pass through a stage with an active VDJ complex in ancestry, and TCRD rearrangements serve as a suitable marker of commitment to lymphoid lineage. Moreover, our data strongly support the existence of distinct lymphoid and myeloid progenitors.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Projects MSM0021620813, IGA NE7430-3, GAUK 62/2004 and NR8269-3/2005. Back

2 E.F. and O.K. contributed equally to this work. Back

3 Current address: Cincinnati Children’s Hospital Medical Center, OH. Back

4 Address correspondence and reprint requests to Dr. Ondrej Hrusak, Childhood Leukemia Investigation Prague, Department of Immunology, Vuvalu 84, 150 06 Praha 5, Czech Republic. E-mail address: Ondrej.Hrusak{at}lfmotol.cuni.cz Back

5 Abbreviations used in this paper: ALL, acute lymphoblastic leukemia; RQ, real-time quantitative; {beta}2m, {beta}2-microglobulin; CLP, common lymphoid progenitor; pos, positive; neg, negative. Back

Received for publication August 31, 2004. Accepted for publication June 1, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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