T-cell Receptor Delta Gene Rearrangements Research Articles

Prethymic Cytoplasmic CD3 Negative Acute Lymphoblastic Leukemia or Acute Undifferentiated Leukemia: A Case Report

Acute undiffentiated leukemia (AUL) is an acute leukemia with no more than one membrane marker of any given lineage. Blasts often express HLA-DR, CD34, and/or CD38 and may be positive for terminal deoxynucleotidyl transferase (TdT). The expression of CD34, HLA-DR, and CD38 has been shown in pro-T-ALL, although in this case, blasts should also express CD7 and cyCD3. However, some cases of T-ALL without CD3 in the cytoplasm and all TCR chain genes in germ line configuration are reported, features that fit well with a very early hematopoietic cell. We report a case of acute leukemia CD34+/−HLADR+CD7+CD38+cyCD3− in which a diagnosis of AUL was considered. However the blasts were also positive for CD99 and TCR delta gene rearrangement which was found on molecular studies. Therefore a differential diagnosis between AUL and an early cyCD3 negative T-ALL was debated.

T Cell Receptor Gamma and Delta Gene Rearrangements in T-cell Acute Lymphoblastic Leukemia in South India and Quantitation of Minimal Residual Disease

Abstract Background: T cell-Acute Lymphoblastic Leukemia (T-ALL) arises by clonal proliferation of lymphoid precursors arrested at particular stage of differentiation. The incidence of T-ALL in India is 37-43% of ALL. In this study, TCR gamma (TCRG) and TCR delta (TCRD) gene rearrangements were detected in diagnostic samples of ALL. Those clonal rearrangements detected at diagnosis are used as clonal markers for the quantitation of minimal residual disease (MRD) in follow up samples.Patients and Methods: BM/PB from 54 T-ALL patients (34 pediatrics and 20 adults) at diagnosis, treated by MCP 841 protocol were studied. Median age of the patients was 13. The frequency of clonal TCRG and TCRD gene rearrangements were studied by Polymerase Chain Reaction (PCR) coupled with Heteroduplex analysis (HD). Allele Specific Oligos (ASO) was designed by sequencing the junctional region sequence of clonal TCRG and TCRD gene rearrangements. MRD was studied in 4 patients with follow up samples. Diagnostic DNA with almost 100% tumor involvement as standard was serially diluted (50ng to 5ng) in 500ng control DNA to give a final concentration of one leukemic cell in 101 (1 in 101) cells to one leuekemic cell in 105 (1 in 105) cells. The serially diluted diagnostic standard in triplicates was amplified with TaqMan probe for respective TCRG/TCRD gene rearrangements in Real-time PCR along with 500ng of follow up DNA samples at different time points (Unknown) in triplicates. Analysis was done to calculate the amount of leukemic cells in follow up samples. Results: Using PCR-HD analysis, TCRG gene rearrangements were detected in 37 of 54 cases (68.5%) and TCRD gene rearrangements in 16 of 54 cases (29.6%). VgI-Jg1.3/2.3 was more commonly rearranged in 29 cases (53.7%) of T-ALL; VgII-Jg1.3/2.3 in 14 cases (26%). Both VgIII-Jg1.3/2.3 and VgIV-Jg1.3/2.3 rearrangements were detected in 4 cases respectively and VgI-Jg1.1/2.1 was detected in 3 cases. Vd1-Jd1 rearrangement was detected in 9 cases (16.6%); Vd2-Dd3 in 5 cases and Dd2-Dd3 in 4 cases of T-ALL. Both TCRG and TCRD gene rearrangements were detected in 8 cases (14.8%). The junctional region in TCRG rearrangements ranged from 1nucleotide to 11nucleotide (average 7.6 nt) and in TCRD rearrangements ranged from 14 nucleotide to 42 nucleotide (average 27 nt). In patient 1, the initial leukeimia load of 1 (before treatment) was reduced to 3 leukemic cell in 104 cells at the lost follow up. In patient 2, the initial leukemia load was reduced to 3.7 in 104 cells. In Patient 3, the initial leukemia load 1 was reduced to 6 in 102 cells at end of M2 and disease relapsed at M5. In patient 4, the initial leukemia load was reduced to 1.6 leukemic cell in 104 cells. Conclusion: Real-time PCR experiments reached a reproducible sensitivity of detecting one leukemic cell in 104 normal cells. Real-time PCR analysis showed that Patient 3 was not all responded to treatment and it was detectable by Real-time PCR at RI1 stage itself though the disease was clinically evident only at M5 stage of treatment. Thus, monitoring the MRD using Real-time PCR will help to quantitate the accurate amount of residual leukemic load, predate relapse and assess the response to treatment.

T Cell Receptor Gamma and Delta Gene Rearrangements in T-cell Acute Lymphoblastic Leukemia in South India and Quantitation of Minimal Residual Disease

T cell-Acute Lymphoblastic Leukemia (T-ALL) arises by clonal proliferation of lymphoid precursors arrested at particular stage of differentiation. The incidence of T-ALL in India is 37-43% of ALL. In this study, TCR gamma (TCRG) and TCR delta (TCRD) gene rearrangements were detected in diagnostic samples of ALL. Those clonal rearrangements detected at diagnosis are used as clonal markers for the quantitation of minimal residual disease (MRD) in follow up samples.Patients and Methods: BM/PB from 54 T-ALL patients (34 pediatrics and 20 adults) at diagnosis, treated by MCP 841 protocol were studied. Median age of the patients was 13. The frequency of clonal TCRG and TCRD gene rearrangements were studied by Polymerase Chain Reaction (PCR) coupled with Heteroduplex analysis (HD). Allele Specific Oligos (ASO) was designed by sequencing the junctional region sequence of clonal TCRG and TCRD gene rearrangements. MRD was studied in 4 patients with follow up samples. Diagnostic DNA with almost 100% tumor involvement as standard was serially diluted (50ng to 5ng) in 500ng control DNA to give a final concentration of one leukemic cell in 101 (1 in 101) cells to one leuekemic cell in 105 (1 in 105) cells. The serially diluted diagnostic standard in triplicates was amplified with TaqMan probe for respective TCRG/TCRD gene rearrangements in Real-time PCR along with 500ng of follow up DNA samples at different time points (Unknown) in triplicates. Analysis was done to calculate the amount of leukemic cells in follow up samples. Using PCR-HD analysis, TCRG gene rearrangements were detected in 37 of 54 cases (68.5%) and TCRD gene rearrangements in 16 of 54 cases (29.6%). VgI-Jg1.3/2.3 was more commonly rearranged in 29 cases (53.7%) of T-ALL; VgII-Jg1.3/2.3 in 14 cases (26%). Both VgIII-Jg1.3/2.3 and VgIV-Jg1.3/2.3 rearrangements were detected in 4 cases respectively and VgI-Jg1.1/2.1 was detected in 3 cases. Vd1-Jd1 rearrangement was detected in 9 cases (16.6%); Vd2-Dd3 in 5 cases and Dd2-Dd3 in 4 cases of T-ALL. Both TCRG and TCRD gene rearrangements were detected in 8 cases (14.8%). The junctional region in TCRG rearrangements ranged from 1nucleotide to 11nucleotide (average 7.6 nt) and in TCRD rearrangements ranged from 14 nucleotide to 42 nucleotide (average 27 nt). In patient 1, the initial leukeimia load of 1 (before treatment) was reduced to 3 leukemic cell in 104 cells at the lost follow up. In patient 2, the initial leukemia load was reduced to 3.7 in 104 cells. In Patient 3, the initial leukemia load 1 was reduced to 6 in 102 cells at end of M2 and disease relapsed at M5. In patient 4, the initial leukemia load was reduced to 1.6 leukemic cell in 104 cells. Real-time PCR experiments reached a reproducible sensitivity of detecting one leukemic cell in 104 normal cells. Real-time PCR analysis showed that Patient 3 was not all responded to treatment and it was detectable by Real-time PCR at RI1 stage itself though the disease was clinically evident only at M5 stage of treatment. Thus, monitoring the MRD using Real-time PCR will help to quantitate the accurate amount of residual leukemic load, predate relapse and assess the response to treatment.

Diversity of T-cell receptor gene rearrangements in South Indian patients with common acute lymphoblastic leukemia.

Precursor B-Acute Lymphoblastic Leukemia (precursor B-ALL) occurs due to the uncontrolled proliferation of B-lymphoid precursors arrested at a particular stage of B-cell development. Precursor-B-ALL is classified mainly into pro-B-ALL, common-ALL and pre-B-ALL. The Common Acute Lymphoblastic Antigen CD10 is the marker for common-ALL. This study was aimed to examine the diversity of T-cell receptor Gamma (TCRG) and T-cell receptor Delta (TCRD) gene rearrangements in South Indian Common-ALL patients. Clonality of TCRG and TCRD was studied in 52 cases (pediatric=41 and adolescents and young adults=11) of common-ALL. TCRG and TCRD gene rearrangements were amplified by PCR and the clonality was assessed by Heteroduplex analysis of amplified products. In pediatric common-ALL, clonal TCRG and TCRD gene rearrangements were detected in 19 (46.3%) and 18 (43.9%) cases respectively. In adolescents and young adults (AYA), TCRG was rearranged in 8 (72.7%) cases and TCRD was rearranged in 4 (36.3%) cases. In the present study of common-ALL, the frequency of a TCRG rearrangement VII-J1.3/2.3 was significantly high in AYA compared to pediatric (36.3% vs 4.8%; p<0.025). Thus, VII-J1.3/2.3 was highly diverse in AYA compared to pediatric. That shows the difference in biology of the disease between pediatric and AYA in South Indian population. The reason for the high frequency of VII-J1.3/2.3 in AYA of common-ALL in South Indian population in connection with unknown infectious agents or environmental carcinogens needs to be evaluated further.

Heat shock protein induced TCR γδ gene rearrangements in patients with oral cancer

We analyzed the T cell receptor (TCR) gammadelta gene repertoire in peripheral blood and tumor compartment of oral cancer (OC) patients before and after stimulation with heat shock proteins (hsp), which are known ligands for gammadelta T cells. Clonal TCR gamma and delta gene rearrangements in lymphocytes from tumor compartment and peripheral blood were studied using TCR Vgamma and Vdelta gene primers in PCR followed by heteroduplex analysis. Vgamma gene segments derived from VgammaI or VgammaII gene families were most dominantly expressed in peripheral blood lymphocytes (PBL) as compared to tumor infiltrating lymphocytes (TIL) of OC patients. Of the rearranged TCR delta alleles Vdelta1-Jdelta1 and Vdelta2-Jdelta1 gene rearrangements were the most predominant in PBL and TIL of OC patients respectively. Stimulation of gammadelta T cells with hsp 60/70 demonstrated a selective clonal expansion of Vgamma9-Vdelta2 (VgammaII family) subset indicating that, this expanded population of cells could be responsible for eliciting an immune response against oral tumor cells.

Hepatosplenic γδ T-cell lymphoma

To investigate the clinicopathologic characteristics, immunophenotype and TCR gene rearrangements of hepatosplenic T-cell lymphoma in eight Chinese patients. Eight Chinese patients with hepatosplenic gammadelta T-cell lymphomas were studied. Hematoxylin-eosin-stained slides and clinical histories were reviewed. We also carried out immunohistochemical staining for CD3, CD4, CD8, CD20, CD43, CD56, CD79a, UCHL-1, and TCR gammadelta. Rearrangements of TCR gamma and delta chain genes were also studied. The spleens were enlarged and the cut surfaces were homogeneous and red-purple in color without identifiable gross lesions or enlarged hilar lymph nodes. Histologically, lymphoma cells infiltrated the cords of Billroth and often packed the sinuses. Liver biopsy showed lymphoma cell infiltrations in the sinusoids, and three cases showed involvements of the portal tracts. Immunohistochemically lymphoma cells were positive for CD3, CD43, and CD56 in all cases. Four of eight cases were positive for CD8, and all cases were negative for CD4 (6/6). Monoclonal rearrangements of TCR gamma gene were demonstrated by PCR analysis in five out of the eight cases. TCR delta gene rearrangements were detected in six out of the eight cases, which demonstrated single bands on PAGE gel, and the amplification products in two cases were confirmed by sequencing. The clinicopathology of hepatosplenic gammadelta T-cell lymphoma in Chinese patients is similar to what was previously reported except that the splenomegaly is not so massive, and CD8 is positive.

High Clonal Evolution Rate in Relapsing Infant ALL Patients.

Immunoglobulin (Ig) and T-cell Receptor (TcR) gene rearrangements are used as patient-specific PCR targets for MRD detection in ALL. However, oligoclonality was reported in childhood ALL, and in those cases, the uncertainty of which clone is going to emerge at relapse may give rise to false negative MRD results. In particular, oligoclonality is a peculiar characteristic of Infant ALL (less than 1 year of age at diagnosis). So far, the prognostic relevance of MRD monitoring in Infant ALL has not been defined, and the assessment of oligoclonality and stability of PCR markers may have a profound impact in MRD monitoring. We successfully studied by PCR the frequency and stability of the currently used rearrangements of the Ig Heavy (IgH), Ig Kappa deleting element (IgK-Kde), TcR delta (TcRD), and TcR gamma (TcRG) gene rearrangements in 42/43 Infant ALL patients prospectively enrolled in Italy in the Interfant-99 International ALL protocol. Pro-B, common and pre-B ALL were 29 (67%), 8 (19%), and 6 (14%), respectively. Sixty-three percent of cases were prednisone good responders (PGR), 70% were MLL-rearranged, 44% were aged less than 6 months, and 21% of cases had more than 300.000 wbc at diagnosis. Overall, rearrangements of the IgH, IgK, TcRD, and TcRG genes were found in 91, 21, 40, and 21% of Infant ALL, respectively. The pattern of Ig/TcR rearrangements in immunophenotypic subgroups of Infant ALLs has been compared to the one of older children prospectively enrolled in Italy into the AIEOP-BFM ALL2000 protocol (n=649). While the overall frequency of IgH and TcRD are similar in the two age groups, IgK and TcRG are less rearranged in Infant cases, mainly due to the low percentage in the pro-B group (10% versus 41% for IgK VK-Kde, and 17% vs 36% for TcRG). In particular, none of the 29 pro-B cases showed rearrangement of the IgK intron. Sixteen Infant relapsed so far (37%), and 12 of them were successfully analyzed for Ig/TcR rearrangement at diagnosis and relapse. Ten were early (less than 18 months from diagnosis), and 2 were late relapses. None of the patients showed an identical Ig/TcR pattern at diagnosis and relapse; 7 (58%) had at least one, and 1 had at least 2 PCR targets preserved, while 4 cases (33%) presented completely different markers at relapse. This distribution was independent on age at diagnosis, site and time of relapse. Interestingly, we observed an increase of complete-IgH (from 10 alleles in 7 patients to 23 alleles in 11) and TcRG (from 4 alleles in 2 patients to14 alleles in 8) rearrangements at relapse. The stability of Ig/TcR markers was as it follows: IgH, 6/26; IgK, 3/5; TcRD, 1/9 and TcRG, 0/4. In conclusion, the oligoclonality feature of Infant ALL may potentially hamper the MRD predictivity in Infant ALL. An appropriate identification and selection of Ig/TcR MRD-PCR targets in Infant ALL is a crucial premise for obtaining clinically relevant MRD data and for preventing false-negative MRD results. Ig Kappa may be a preferential marker for MRD studies in Infant ALL, although its use is limited by the low frequency of the rearrangement. The leukemia-specific MLL genomic breakpoint may potentially overcome these limitations and improve the MRD analysis.

TCR delta gene rearrangements revealed by fine structure of the recombination junction in mice.

The standard products of V(D)J recombination of immunoglobulin and T cell receptor genes are two kinds of DNA junction, a coding joint and a signal joint. TCR delta V-D and D-D signal joints in adult mouse thymocytes were sequenced following PCR amplification. We observed differential nucleotide insertions at the V delta-D delta signal joints, depending on the V delta and D delta gene usage in the developmental stage. Nucleotide insertions at the V delta-D delta 1 signal joints were less frequent for the V delta 4, 5 genes preferentially utilized in adult thymocytes than for the V delta 3, 6 genes, infrequently rearranged to D delta 1. In addition to standard signal joints, unexpectedly, novel nonstandard products, "replacement joints" of D delta 1 substituted downstream by the recombination signal sequence of V delta were also found. However, no D delta 2-associated replacement joints other than V delta 5 were found. The other replacement joints of D delta 1-D delta 2 recombination were also observed. The mutation in TCR beta gene affected the frequency of nucleotide insertions at the V delta-D delta signal joints and inhibited the formation of replacement joint. Recombination mechanism generating the replacement joint and the possible role of TCR beta in up-regulation of TCR delta gene rearrangements are discussed.

Assessing a role for enhancer-blocking activity in gene regulation within the murine T-cell receptor alpha/delta locus.

Although situated close together within the T-cell receptor (TCR) alpha/delta locus, TCR delta and TCR alpha gene segments are controlled by two developmental stage-specific enhancers and are activated according to distinct developmental programmes. We previously used a stable transfection colony assay to identify an enhancer-blocking element, blocking element alpha/delta-1 (BEAD-1), between the TCR delta and alpha gene segments of the human TCR alpha/delta locus. We hypothesized that enhancer-blocking by BEAD-1 might be required to prevent the TCR delta enhancer from activating TCR alpha gene segment transcription and rearrangement at the double negative stage of thymocyte development. Here, we used a transfection approach to define partial enhancer-blocking activity in an analogous position of the murine TCR alpha/delta locus. To test the functional significance of this activity in vivo, we used gene targeting to delete the region from the endogenous locus. We found no perturbation of TCR delta and TCR alpha gene expression and rearrangement on targeted alleles, indicating that enhancer-blocking activity in this region is not required to maintain the developmentally distinct activation profiles of the two genes. We suggest that appropriate regulation may be achieved as a result of intrinsic biases in enhancer-promoter interactions or a developmental stage specificity to promoter function that is distinct from any additional specificity imposed by the enhancers themselves.

Unusual T-cell receptor-delta gene rearrangement patterns revealed by screening of a large series of childhood acute lymphoblastic leukaemia by multiplex polymerase chain reaction.

Rearrangements of the T-cell receptor (TCR) and immunoglobulin genes are considered as useful clonal markers in lymphoproliferative disorders of B- and T-cell lineage, and are frequently used for the detection of minimal residual disease (MRD). In this paper, we report on the unexpected results of an extensive analysis of TCR-delta chain gene rearrangement frequencies and patterns in leukaemic bone marrow DNA samples collected from 438 children with initial (n = 112) or relapsed (n = 326) acute lymphoblastic leukaemia (ALL). By applying a previously described multiplex polymerase chain reaction, the overall incidence of non-deleted TCR-delta gene rearrangements in ALL was 47% (206/438), 52% in initial ALL (58/112) and 45% in relapsed ALL (148/326). As expected, the majority of B-cell precursor (BCP) ALL had incomplete Vdelta2-Ddelta3 or Ddelta2-Ddelta3 TCR-delta gene rearrangements, whereas most T-ALL showed complete rearrangements of the TCR-delta gene locus (Vdelta1-Jdelta1, Vdelta2-Jdelta1, Vdelta3-Jdelta1). However, unexpectedly, 5/206 rearranged TCR-delta alleles in BCP-ALL showed a complete Vdelta-(Ddelta)-Jdelta gene rearrangement pattern, and 3/31 T-ALL had an incomplete recombination. Theoretically, complete TCR-delta gene rearrangements should not occur in cells other than T-lymphocytes and have only been reported once previously in BCP-ALL. The data contribute to the discussion about the reliable screening for clonal markers in ALL.

Ig Heavy Chain Gene Rearrangements in T-Cell Acute Lymphoblastic Leukemia Exhibit Predominant Dh6-19 and Dh7-27 Gene Usage, Can Result in Complete V-D-J Rearrangements, and Are Rare in T-Cell Receptor β Lineage

Rearranged IGH genes were detected by Southern blotting in 22% of 118 cases of T-cell acute lymphoblastic leukemia (ALL) and involved monoallelic and biallelic rearrangements in 69% (18/26) and 31% (8/26) of these cases, respectively. IGH gene rearrangements were found in 19% (13/69) of CD3− T-ALL and in 50% of TCRγδ+ T-ALL (12/24), whereas only a single TCRβ+ T-ALL (1/25) displayed a monoallelicIGH gene rearrangement. The association with the T-cell receptor (TCR) phenotype was further supported by the striking relationship between IGH and TCR delta (TCRD) gene rearrangements, ie, 32% of T-ALL (23/72) with monoallelic or biallelicTCRD gene rearrangements had IGH gene rearrangements, whereas only 1 of 26 T-ALL with biallelic TCRD gene deletions contained a monoallelic IGH gene rearrangement. Heteroduplex polymerase chain reaction (PCR) analysis with Vh and Dh family-specific primers in combination with a Jhconsensus primer showed a total of 39 clonal products, representing 7 (18%) Vh-(Dh-)Jh joinings and 32 (82%) Dh-Jh rearrangements. Whereas the usage of Vh gene segments was seemingly random, preferential usage of Dh6-19 (45%) and Dh7-27 (21%) gene segments was observed. Although the Jh4 and Jh6 gene segments were used most frequently (33% and 21%, respectively), a significant proportion of joinings (28%) used the most upstream Jh1 and Jh2 gene segments, which are rarely used in precursor-B-ALL and normal B cells (1% to 4%). In conclusion, the high frequency of incomplete Dh-Jh rearrangements, the frequent usage of the more downstream Dh6-19 and Dh7-27 gene segments, and the most upstream Jh1 and Jh2 gene segments suggests a predominance of immature IGH rearrangements in immature (non-TCRβ+) T-ALL as a result of continuing V(D)J recombinase activity. More mature β-lineage T-ALL with biallelic TCRD gene deletions apparently have switched off their recombination machinery and are less prone to cross-lineageIGH gene rearrangements. The combined results indicate thatIGH gene rearrangements in T-ALL are postoncogenic processes, which are absent in T-ALL with deleted TCRD genes and completed TCR alpha (TCRA) gene rearrangements.

Clinical relevance of T-cell receptor delta gene rearrangements in childhood B-precursor cell acute lymphoblastic leukaemia.

The large majority of childhood B-precursor cell acute lymphoblastic leukaemia cases present IgH and TCRdelta gene rearrangements. These rearrangements have been widely used as specific markers for monitoring minimal residual disease. However, their prognostic value still remains unclear. In order to determine whether IgH and TCRdelta gene rearrangements have any influence on relapse and event-free survival (EFS), we analysed the clinical impact of these genetic characteristics in 51 B-precursor acute lymphoblastic leukaemia patients. 46/51 patients (90.2%) showed IgH gene rearrangements by Southern blot and/or polymerase chain reaction (PCR) analysis. No statistically significant associations were found between IgH gene rearrangement pattern and age, sex, WBC count, immunophenotype, risk factor, relapse or EFS. 27/41 patients (66%) showed Vdelta2Ddelta3 recombination by Southern blot and/or PCR analysis. At a median follow-up of 53 months the estimated 5-year EFS probability was 78 +/- 3% for the whole group. The EFS probability among patients with a Vdelta2Ddelta3 recombination pattern in the TCRdelta locus was 90 +/- 3%, whereas for patients without Vdelta2Ddelta3 recombination was 39 +/- 13% (P < 0.005). IgH rearrangement patterns do not appear to influence relapse or EFS probability. However, TCRdelta gene rearrangement patterns have a relevant impact on the relapse rate and the EFS probability. Patients with Vdelta2Ddelta3 recombination have better clinical outcome than patients without this recombination, independent of any other prognostic factors.

Multiplex PCR for simultaneous detection of the most frequent T cell receptor-delta gene rearrangements in childhood ALL.

A rapid and simple multiplex polymerase chain reaction (PCR) is described that is capable of identifying the six most frequent rearrangements of the T cell receptor (TCR)-delta gene segments in childhood acute lymphoblastic leukemia (ALL). The PCR products amplified in a single reaction are of different size for each TCR-delta gene rearrangement. Therefore, they are readily and unambiguously distinguished after agarose gel electrophoresis and assigned to a specific V-D-J gene rearrangement. There is no need for labor-intensive and time-consuming Southern blot hybridization or nested PCR. To evaluate the multiplex assay we chose 45 DNA samples of childhood ALL analyzed beforehand for TCR-delta gene rearrangements by Southern blot and single PCR of which 30 showed TCR-delta gene rearrangements. The multiplex PCR results corresponded to the Southern blot and single PCR analyses. The described multiplex PCR enables the detection of clonal markers in about 50% of patients in order to monitor minimal residual disease (MRD) in prospective studies with a high turnover of samples.

Alpha beta lineage-committed thymocytes can be rescued by the gamma delta T cell receptor (TCR) in the absence of TCR beta chain.

Commitment of the alpha beta and gamma delta T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCR gamma delta-transgenic or TCR beta knockout mice, both of which are unable to generate TCR alpha beta-positive T cells, develop phenotypically alpha beta-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCR beta protein, the gamma delta TCR can promote the development of alpha beta-like thymocytes, which, however, do not expand significantly and do not mature into gamma delta T cells. These results show that commitment to the alpha beta lineage can be determined independently of the isotype of the TCR, and suggest that alpha beta versus gamma delta T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCR gamma and delta gene rearrangements on alpha beta T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.

TCR-delta gene rearrangement and selection during fetal thymocyte development.

TCR delta-chain gene rearrangements were analyzed at different stages of thymic ontogeny. The VDJ delta junctional sequences of the dominantly expressed TCR delta-chain V7 subfamily are highly conserved in fetal and neonatal thymocytes. They are equally conserved in C delta-targeted mice lacking cell surface expression of gamma delta receptors, indicating evolutionary selection acting at the level of DNA rearrangement. Yet, in C delta-mutant mice, the frequency of in-frame transcripts is reduced to 61%, from 88% in wild-type mice, suggesting cellular selection of this DV7-overexpressing gamma delta thymocyte subset. In contrast, in genomic DNA rearrangements, no difference was found in the frequency of productive rearrangements between mutant (26%) and wild-type mice (31%). This in-frame rate, characteristic of random rearrangements, indicates that positive selection is not required for thymic maturation of gamma delta T cells. The results are discussed with regard to models of alpha beta/gamma delta lineage commitment.

Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells.

The presence of T and NK cells in the human fetal liver and the fact that fetal liver hemopoietic progenitor cells develop into T and NK cells suggest a role for the fetal liver compartment in T and NK cell development. In this work, we show that the capacity of fetal liver progenitors to develop into T cells, in a human/mouse fetal thymic organ culture system, is restricted to an immature subset of CD34+ CD38- cells. No T cell-committed precursors are contained within the more differentiated CD34+ CD38+ population. This conclusion is supported by the observations that no TCR-delta gene rearrangements and no pre-TCR-alpha expression can be detected in this population. However, NK cells were derived from CD34+ CD38- and CD34+ CD38+ fetal liver cells cultured in the presence of IL-15, IL-7, and Flt-3 ligand. Eighty to ninety percent of cells arising from the CD34+ CD38+ population expressed the NK cell-associated markers CD56, CD16, CD94, and NKR-P1A. Several subpopulations of NK cell precursors were identified by differential expression of these receptors. Based on the detection of populations with a similar antigenic profile in freshly isolated fetal liver cells, we propose a model of NK cell differentiation. Collectively, our findings suggest that CD34+ cells differentiate into NK cells, but not into mature T cells, in the human fetal liver.

Detection of T-cell receptor delta gene rearrangement in T-cell malignancies by clonal specific polymerase chain reaction and its application to detect minimal residual disease.

A clonal-specific polymerase chain reaction technique to detect T-cell receptor delta gene rearrangement in acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL) was evaluated. It was applied to detect minimal residual disease. A sensitive and specific technique to detect minimal residual disease for T-cell malignancies was explored. Southern analysis and polymerase chain reaction (PCR) were used to detect the rearranged V-D-J segment of T-cell receptor delta (TCR delta) gene from malignant cell specimens of patients with leukemia and lymphoma of T-cell lineage. The PCR product was sequenced and from the DNA sequences of the V-D-J region, a 3' anti-sense primer was designed and synthesized for clonal specific PCR (CS-PCR). T-cell receptor delta (TCR-delta) gene rearrangement was studied in 40 cases of acute leukaemia and lymphoma of T-cell lineage at diagnosis. Using Southern analysis, the positive rates were 28 and 32% for the 18 T-lymphoma and 22 T-ALL, respectively. A one stage Polymerase Chain Reaction (PCR) technique was used to detect the rearrangement in Southern positive cases and the PCR positive rates were 80 and 86%, respectively. The PCR technique had a sensitivity of 0.1%. Serial follow-up marrow specimens were available from 4 T-ALL patients following chemotherapy for monitoring of minimal residual disease. Their PCR products were DNA sequenced. A 3' primer was designed for each case for a clonal specific (CS) PCR. The technique had a sensitivity of 0.003%. It was applied to detect minimal residual disease in serial follow-up marrow samples. The first patient had persistent negative CS-PCR results and enjoyed continuous remission for more than 3 years. The second patient with negative one stage PCR but positive CS-PCR results had eventual relapse of leukaemia. The other two patients never achieved a morphological remission. These preliminary results appeared to support the usefulness of these PCR techniques in detecting minimal residual disease and predicting relapses for ALL. However, further clinical correlation in larger populations of patients is necessary.

Regulation of T cell receptor delta gene rearrangement by c-Myb.

Developmental activation of VDJ recombination at the T cell receptor (TCR) delta locus is controlled by an intronic transcriptional enhancer (E delta). Transcriptional activation by E delta is dependent on c-Myb. To determine whether c-Myb plays a role in the activation of TCR-delta gene rearrangement, we compared VDJ recombination in transgenic mice carrying two versions of a human TCR-delta gene minilocus recombination substrate. One includes a wild-type E delta, whereas the other carries an E delta with a mutation that abrogates c-Myb binding. We demonstrate that an intact Myb binding site is necessary for efficient rearrangement of the minilocus substrate, suggesting that c-Myb plays a crucial role in activating VDJ recombination at the endogenous TCR-delta locus.

Heterogeneity of the T-Cell Receptor 8 Gene Indicating Subclone Formation in Acute Precursor B-Cell Leukemias

Precursor B-cell acute lymphoblastic leukemias (B-ALLs) have been shown to be oligoclonal at the Ig heavy-chain (IgH) gene level in up to 40% of cases by Southern blot hybridization. In contrast, oligoclonality as deduced from diversity of T-cell receptor (TcR)-delta gene rearrangements of the immature types (ie, V delta 2-D delta 3, D delta 2-D delta 3) has not been reported, so far. We detected oligoclonality characterized by the coexistence of different junctional regions of identical V delta 2-D delta 3 rearrangements in four childhood precursor B-ALLs. No variation was found in the IgH gene status. Therefore, we define these populations as subclones. Two leukemias displayed the variants in an unequal proportion. In the other two leukemias, for which similar quantities of the coexisting rearrangements were detected, single cell-nuclei polymerase chain reaction (PCR) showed two separate leukemic populations. Subclone formation could not be demonstrated by Southern blot hybridization, but was detectable after PCR amplification of the V delta 2-D delta 3 rearrangement and separation by polyacrylamide gel electrophoresis. The variants arose independently from each other, as deduced from their individual sequences. Using subclone-specific oligonucleotides for hybridization to amplified DNA obtained at diagnosis and during follow-up from bone marrow samples, we demonstrate, (1) specificity of all subclone-deduced probes, (2) that one residual leukemic cell can be detected in 10(4) to 10(5) normal mononuclear cells in a semiquantitative assay, and (3) that none of the subclones persisted after induction therapy. We propose that in a leukemic cell population, TcR-delta gene diversity arises after rearrangements of the IgH genes resulting in apparent clonality at the IgH gene level. However, cells are oligoclonal, if the TcR-delta gene rearrangements are considered. As various subclones may respond differently to chemotherapy, they may hamper the detection of minimal residual disease. Therefore, we use all subclone-specific oligonucleotides for hybridization to amplified DNA from follow-up samples.

TCR delta gene rearrangements in acute myeloid leukemia with T-lymphoid antigen expression.

In this review we present our data concerning T-cell receptor (TCR) delta gene rearrangements in acute myeloid leukemia with coexpression of T-lymphoid features (CD2/CD4/CD7; Ly+ AML). We found a correlation between TCR delta gene rearrangements and coexpression of these T-lymphoid features. Ten of 66 Ly+ AML and only one of 44 AML cases without this coexpression exhibited TCR delta gene rearrangements (p = .028). In contrast, no correlation was observed between terminal deoxynucleotidyl transferase (TdT) expression and the occurrence of TCR delta gene rearrangements in AML. Rearrangements were found in two of 25 AML with and seven of 71 AML cases without TdT expression. Interestingly, nucleotide sequencing of junctional sites revealed up to 36 N-nucleotides in cases without or with only weak TdT expression indicating downregulation of TdT expression after the TCR rearrangement took place. Complete V delta 1J delta 1 and incomplete D delta 2J delta 1 gene rearrangements were observed most frequently in Ly+ AML. These recombination patterns were similar to patterns observed in acute T-lymphoblastic leukemia with coexpression of myeloid features (My+ T-ALL) suggesting transformation of a common myeloid/T-lymphoid progenitor cell in these cases.