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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Langerak, A. W. Articles by van Dongen, J. J. M. Search for Related Content PubMed PubMed Citation Articles by Langerak, A. W. Articles by van Dongen, J. J. M. Related Collections Immunobiology Neoplasia Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 July 2001, Vol. 98, No. 1, pp. 165-173 IMMUNOBIOLOGY Molecular and flow cytometric analysis of the V repertoire for clonality assessment in mature TCR T-cell proliferations Anton W. Langerak, René van den Beemd, Ingrid L. M. Wolvers-Tettero, Patrick P. C. Boor, Ellen G. van Lochem, Herbert Hooijkaas, and Jacques J. M. van Dongen From the Department of Immunology, University Hospital Rotterdam/Erasmus University Rotterdam, The Netherlands.     Abstract Top Abstract Introduction Patients, materials, and... Results Discussion References Clonality assessment through Southern blot (SB) analysis of TCRB genes or polymerase chain reaction (PCR) analysis of TCRG genes is important for diagnosing suspect mature T-cell proliferations. Clonality assessment through reverse transcription (RT)-PCR analysis of V-C transcripts and flow cytometry with a V antibody panel covering more than 65% of V domains was validated using 28 SB-defined clonal T-cell receptor (TCR)+ T-ALL samples and T-cell lines. Next, the diagnostic applicability of the V RT-PCR and flow cytometric clonality assays was studied in 47 mature T-cell proliferations. Clonal V-C RT-PCR products were detected in all 47 samples, whereas single V domain usage was found in 31 (66%) of 47 patients. The suspect leukemic cell populations in the other 16 patients showed a complete lack of V monoclonal antibody reactivity that was confirmed by molecular data showing the usage of V gene segments not covered by the applied V monoclonal antibodies. Nevertheless, this could be considered indirect evidence for the "clonal" character of these cells. Remarkably, RT-PCR revealed an oligoclonal pattern in addition to dominant V-C products and single V domain expression in many T-LGL proliferations, providing further evidence for the hypothesis raised earlier that T-LGL derive from polyclonal and oligoclonal proliferations of antigen-activated cytotoxic T cells. It is concluded that molecular V analysis serves to assess clonality in suspect T-cell proliferations. However, the faster and cheaper V antibody studies can be used as a powerful screening method for the detection of single V domain expression, followed by molecular studies in patients with more than 20% single V domain expression or large suspect T-cell populations (more than 50%-60%) without V reactivity. (Blood. 2001;98:165-173) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Patients, materials, and... Results Discussion References In striking contrast to the straightforward clonality assessment in most mature B-cell proliferations through single immunoglobulin light chain expression, clonality assessment in suspect T-cell proliferation is not possible through routine immunologic marker analysis. For instance, the T-cell receptor (TCR) expression pattern (TCR or TCR), the unusual CD4/CD8 expression pattern, or the so-called loss of T-cell markers is insufficient to establish the malignant (clonal) character of a suspect T-cell proliferation.1 As a consequence, molecular analysis of TCR gene rearrangements has developed as an important tool for clonality assessment in such proliferations. As with normal T cells in various differentiation stages, TCR genes are rearranged in most T-cell malignancies.2 The presence or absence of identically (clonally) rearranged TCR genes is therefore helpful for distinguishing between (mono)clonal and polyclonal (reactive) T-cell proliferations, respectively. Southern blot (SB) analysis is the classical diagnostic method for clonality assessment. It is highly reliable and in principle can detect every clonal TCR gene rearrangement, provided that optimally positioned probes and appropriate restriction enzymes are used.3-6 Nevertheless, several drawbacks limit the routine application of SB analysis in diagnostic laboratories. SB is labor intensive and time consuming, especially when sequential hybridizations are required; furthermore, relatively large amounts of high-quality DNA are needed for reliable results, which precludes its application on paraffin-embedded samples. Despite these disadvantages, SB-based detection of clonal TCR rearrangements is the gold standard technique by which to validate other methods for clonality assessment in suspect T-cell proliferations. Today, polymerase chain reaction (PCR) analysis of TCR gamma (TCRG) genes is most widely applied; the relatively restricted combinatorial repertoire of TCRG genes limits the number of required PCR primers. However, this limited repertoire also results in high background amplification of similar rearrangements in normal T cells, thereby reducing the sensitivity of the assay. Other approaches focus on analysis of TCRB genes or TCRB gene products (RNA, proteins, or both), exploiting the high diversity in V gene usage for clonality studies. PCR-based analysis of the V repertoire mostly concerns reverse transcription (RT)-PCR amplification of V-C transcripts to limit the number of primers needed to cover the many involved gene segments.7 With the recent availability of a panel of V antibodies that covers more than 65% of all V domains,8 flow cytometric analysis of the V repertoire now promises to be a relevant alternative approach for molecular clonality studies.9,10 This is especially true because reference values have been determined in healthy controls for the available individual V antibodies.10 Here we present 2 different approaches of V repertoire analysis: V-C RT-PCR and flow cytometry with V antibodies. After validation on SB-defined clonal TCR+ T-cell acute lymphoblastic leukemia (T-ALL) samples and a series of T-cell lines, the diagnostic applicability of the 2 methods was tested in a series of 47 mature T-cell proliferations. The feasibility and applications of both V clonality assays are discussed.     Patients, materials, and methods Top Abstract Introduction Patients, materials, and... Results Discussion References Patients, cell samples, and cell lines Peripheral blood (PB) samples were collected from patients (n = 47) showing persistent mature TCR+ T-cell proliferation, including T-cell chronic lymphocytic leukemia (T-CLL), T-cell large granular lymphocyte leukemia (T-LGL), T-cell non-Hodgkin lymphoma (T-NHL), T-cell prolymphocytic leukemia (T-PLL), and mycosis fungoides/Sézary syndrome (MF/SS). Mononuclear cells (MNCs) were isolated by Ficoll-Hypaque (density, 1.077 g/mL; Pharmacia, Uppsala, Sweden) density centrifugation and used for immunophenotyping and V analysis and for DNA and RNA isolation. TCR+ or cytoplasmic TCR+ (CyTCR+) T-cell lines (n = 12) and MNC fractions of PB or bone marrow (BM) samples from patients with TCR+ T-ALL (n = 16) were used as reference samples. Immunophenotyping and analysis of V domains of expressed TCR chains MNCs were analyzed for cell membrane expression of T-cell markers and for expression of the HLA-DR antigen (L243), cytoplasmic TCR (F1), TCR (BMA031) and TCR (11F2) with the following monoclonal antibodies (mAbs): CD2 (Leu-5b), CD3 (Leu-4), CD4 (Leu-3a), CD5 (Leu-1), CD7 (3A1), CD8 (Leu-2a), CD16 (Leu-11c), CD56 (Leu-19), and CD57 (Leu-7). The mAbs of the Leu series, as well as L243 and 11F2, were obtained from Becton Dickinson (San Jose, CA), the 3A1 hybridoma was obtained from ATCC (Rockville, MD), F1 from T-cell Sciences (Needham, MA), and BMA031 from Immunotech (Marseilles, France). Immunofluorescence stainings were performed as described and evaluated on a FACScan flow cytometer using CellQuest software (Becton Dickinson).10 The samples were studied in more detail for V domain expression using a panel of carefully selected V mAbs (Table 1).10 Data acquisition and analysis of the V immunofluorescence stainings in T-ALL samples and T-cell lines were performed in double stainings using unconjugated V mAbs, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated goat antimouse immunoglobulin (CLB, Amsterdam, The Netherlands) and subsequently normal mouse serum to block free antigen binding sites of the second-step reagent, after which CD3-PE (Leu-4) was added.10 In T-cell lines with cytoplasmic TCR expression (F1 reactivity), cells were permeabilized before V staining. For the mature TCR+ T-cell proliferations, the V mAbs were used in combination with CD3-PerCP (Leu-4) and occasionally CD4-PE (Leu-3a) or CD8-PE (Leu-2a) to further define the immunophenotype of the T cells.10 The V antibodies were from Immunotech (Marseilles, France), T-cell Sciences (Needham, MA), and T-cell Diagnostics (Cambridge, MA) (Table 1 for the origin of the V antibodies). In addition, several samples were studied by means of 6 mixtures of clustered V antibodies as a fast and easy method for the identification of single V domain expression, as described by Van den Beemd et al.10 These 6 mixtures consisted of: MIX1, V3 (CH92) and V5.1 (IMMU157); MIX2, V8.1/8.2 (56C5.2) V12.2 (VER2.32), and V17 (E17.5F3); MIX3, V2 (MPB2/D5), V16 (TAMAYA1.2), and V23 (HUT78.7/AF23); MIX4, V6.1 (CRI304.3), V7.1 (Zoé), V9.1 (FIN9), V11.1 (C21), and V14 (CAS1.1.3); MIX5, V13.6 (JU74.3), V18 (BA62.6), V20 (ELL1.4), V21.3 (IG125), and V22 (IMMU546); and MIX6, V5.2/5.3 (4H11), V6.7 (OT145), and V13.1/13.3 (BAM13).10                                View this table: [in this window] [in a new window]   Table 1. V antibodies and their reactivity in healthy controls DNA and RNA isolation and cDNA synthesis DNA was isolated from frozen MNCs and cell lines as described earlier.3 Total RNA was isolated from all samples using RNAzol (Tel-Test, Friendswood, TX). After oligo dT annealing for 3 minutes at 85°C, 2 µg total RNA was subsequently reverse transcribed in 40 µL volumes for 1 hour at 41°C using Superscript II RT enzyme (Life Technologies, Paisley, United Kingdom) in the presence of dNTPs and RNAguard (Amersham Pharmacia Biotech, Uppsala, Sweden). Southern blot analysis Southern blot (SB) analysis of the TCRB genes was performed as described.3 The 32P-labeled TCRBJ1, TCRBJ2, and TCRBC genomic DNA probes (DAKO, Carpinteria, CA) were used in subsequent hybridizations of EcoRI- and HindIII-digested DNA to determine the rearrangement status of the T-cell lines and patient samples.5 PCR amplification Oligonucleotide primers used for amplification of V-C transcripts are given in Table 2. Most V family-specific primers were adapted from those published by Gorski et al,7 but several primers were added to maximize recognition of V gene segments within a given family and to minimize cross-annealing to other V gene families at the 3' primer ends. The C primer was also adapted from Gorski et al.7 The quality of the studied cDNA samples was determined through RT-PCR analysis of the ubiquitously expressed ABL gene. PCR amplification of the TCRB genes of the cDNA samples was performed in multiple tubes (n = 31), each containing one of the V family primers and the C primer (Table 2). Reactions were performed in 20 µL volumes, containing one fortieth (1 µL) of the cDNA reaction mixture, 2.5 pmol V family primer, 2.5 pmol C primer, 0.2 mM dNTPs, 1.5 mM MgCl2, and 0.2 U AmpliTaq DNA polymerase in reaction buffer II (Applied Biosystems, Foster City, CA). PCR reaction conditions for the Perkin-Elmer 480 thermal cycler (Applied Biosystems) were initial denaturation of 3 minutes at 94°C, followed by 35 to 40 cycles of 1 minute at 94°C, 1 minute at 60°C, 2 minutes at 72°C, and a final extension step of 10 minutes at 72°C.                                View this table: [in this window] [in a new window]   Table 2. V family primers and C primer Heteroduplex analysis After amplification, half (10 µL) the PCR mixtures were loaded on 1% agarose gels to evaluate PCR product formation with the various V-C primer combinations. PCR products were visualized by ethidium bromide using UV light. The other 10 µL PCR reaction mixture was subjected to heteroduplex analysis to discriminate between monoclonal and polyclonal PCR products.11 In short, heteroduplex analysis consisted of 5' denaturation at 94°C immediately followed by 60' renaturation at 4°C before electrophoresis on 6% nondenaturing polyacrylamide gels (polyacrylamide:bisacrylamide 29:1) in 0.5× TBE buffer.11 Ethidium bromide-stained homoduplex or heteroduplex PCR products were visualized with UV light. Sequencing If heteroduplex analysis showed PCR products to be clonal, the homoduplexes were excised from the gel and eluted as described.12 Eluted products were either directly sequenced or reamplified in a second-step PCR reaction using the same primers as in the initial reaction. Sequencing was performed on the ABI377 fluorescence sequencer (Applied Biosystems) using the dye terminator cycle sequencing kit and AmpliTaq FS (Applied Biosystems).12 Assignment of V, D, J, and C gene segments and reading frames of the involved TCRB gene rearrangements was performed as described.13,14     Results Top Abstract Introduction Patients, materials, and... Results Discussion References Molecular and flow cytometric V analysis in T-cell lines To study the feasibility of V analysis in clonal T-cell populations, we used a panel of 12 T-cell lines (Table 3). Although only 4 of these cell lines showed TCR membrane expression, all had cytoplasmic expression of TCR chains. SB analysis revealed clonal TCRB gene rearrangements in all cell lines. In 6 of the T-cell lines, 2 clear V-C RT-PCR products were found, and in the other 6 only one was found (Table 3). The V-C products of the various cell lines contained V gene segments from many distinct V families, with a slight predominance of V2-C products (n = 3). Remarkably, in most identified transcripts, J2 region gene segments were used, which fits with the predominance of V-J2 gene rearrangements in immature T-cell malignancies.5 Further sequencing of the clonal products revealed that all except one of the cell lines showed a single in-frame V-C RT-PCR product (Table 3). In cell line KT-1, both an in-frame V18-C and an in-frame V15-C transcript were found, whereas in the other 5 cell lines with 2 clonal V-C products only one appeared in-frame.                                View this table: [in this window] [in a new window]   Table 3. V usage in T-cell lines and T-ALL samples as determined by molecular analysis and mAb reactivity Flow cytometric V analysis using a panel of V family antibodies (Table 1) showed reactivity with one of the V mAbs from the panel in 10 of the 12 cell lines, with a specificity profile that completely correlated with the presence of the identified in-frame V-C transcript (Table 3). Cell line KT-1, having in-frame V15-C and V18-C transcripts, reacted with the V18 antibody. However, double (membrane or cytoplasmic) V expression could not be confirmed or excluded in this cell line because V15-specific antibodies were not available. In 2 cell lines, no reactivity was observed with any of the V mAbs of the panel, despite cytoplasmic TCR chain (F1 mAb) detection. In the RPMI 8402 cell line, this could easily be explained by the lack of a V24-specific antibody, but for the DND-41 cell line the appropriate V18 antibody was present in the panel. The fact that DND-41 expresses a TCR rather than an TCR might, however, hamper proper detection of the recognized epitope of the V18 domain expressed in this cell line. Molecular and flow cytometric V analysis in T-ALL samples In addition to the 12 T-cell lines, we studied 16 TCR+ T-ALL for their V expression profile using molecular techniques and flow cytometric analysis (Table 3). V-C RT-PCR heteroduplex analysis revealed a single clonal in-frame V-C transcript in 6 of the 16 T-ALL samples. The remaining 10 samples had at least 2 clonal V-C transcripts; in 2 cases, even 3 and 4 clonal V-C transcripts were present, suggestive of subclone formation with a minor clone (less than 10%) not detectable by SB (Table 3) but readily detectable by RT-PCR. In contrast to the low frequency of bi-allelic in-frame V-C products in T-cell lines, 6 of 10 T-ALL samples were found to contain double in-frame V-C transcripts (Table 3). The identified V-C products in the T-ALL represented V segments from many V families, with a predominance of V3-C products (n = 5). Gene segments of the J1 region were identified in 17 V-C products, whereas in 12 products, J2 segments could be found. Reactivity with one of the V mAbs was seen in 8 of the 16 T-ALL samples. In all 8 samples, the V antibody reactivity pattern was completely in line with the results of the molecular V analysis, confirming the single (in-frame) transcript in most of these 8 samples. Although samples T077 and T140 with 2 in-frame V-C transcripts might have shown double V protein expression, this did not occur, as evidenced by the single V8 (T077) and V3 (T140) membrane expression on these cells. Potential double V expression in a few other cases (T012, T044, T145) remained undetected because of the lack of the relevant V mAbs for both in-frame alleles in the panel. In 8 T-ALL samples, no reactivity of the entire T-ALL cell population with any of the V antibodies from the panel was seen, despite the clear presence of a TCR molecule on the membrane. In virtually all instances, this nonreactivity can most probably be attributed to expression of the V-C product for which no V mAb is available (eg, V4 in T015 or V24 in T012) or might be caused by a family member not covered by the available V family antibody (eg, members of the V5, V6, and V7 families). The precise members of each V gene family could not be recognized in our study because the position of our V primers is close to the 3' end of the segments, thereby resulting in PCR products that do not include sufficient sequence information for V member identification. Sample T139 with in-frame V3-C and V11-C products is remarkable in that the cells should have been recognized with either the V3 or the V11 mAb, both of which belong to a single-member gene family. However, for reasons that remain unclear, this did not occur. One possible explanation might be that the actual epitope in either the V3 or the V11 chain was modified or influenced by the junctional region or by the involved J segment. V repertoire analysis for clonality assessment in mature T-cell proliferations Although the results of the V analysis in T-cell lines and T-ALL samples showed that the clonal cell population could easily be identified by V mAb reactivity or by lack of reactivity with any of the V mAbs in the panel, the relevant diagnostic application of V gene analysis in daily practice concerns the analysis of postthymic mature lymphoproliferations of the T-cell lineage. For this reason we chose to study the applicability of V repertoire analysis in a large series of 47 mature TCR T-cell proliferations (including T-NHL, T-CLL, T-PLL, and T-LGL) that, in the past decade, had been sent to our laboratory with a strong suspicion of clonality (Table 4). All samples were indeed found to contain clonal TCRB rearrangements in SB analysis. Next, the samples were subjected to V-C RT-PCR, followed by heteroduplex analysis to confirm or exclude the clonal character of the obtained RT-PCR products. This revealed the presence of dominant clonal V-C transcripts in all 42 samples from which RNA could be isolated. In 26 cases only a single transcript was detectable, whereas in 14 cases double V-C products were seen (Table 4). Remarkably, in 2 samples (93-067 and 96-019), 3 dominant clonal products were apparent. SB analysis of case 96-019 already suggested more than 2 rearranged alleles, but in case 93-067 this was not anticipated from the SB pattern because no additional bands were apparent. Further sequencing in the latter case revealed that the 3 PCR products contained distinct junctional regions, excluding cross-annealing of primers as an explanation for the occurrence of multiple bands.                                View this table: [in this window] [in a new window]   Table 4. V analysis for clonality assessment in mature T-cell proliferations To compare the molecular and flow cytometric V results in these T-cell proliferations, we sequenced the V-C RT-PCR products. Unfortunately, sequence analysis could not be performed in the 5 samples from which RNA was lacking. In-frame V-C transcripts were indeed identified in all 26 evaluable cases that showed only a single RT-PCR product (Table 4). Remarkably, in most (at least 9 of 16) sequenced cases with multiple V-C products, more than one (distinct) in-frame V-C transcript was found. Flow cytometric analysis of the V repertoire was performed in all 47 cases. Most of the studied samples (n = 31) appeared to have a restricted V reactivity pattern, with predominance of a single V mAb reactivity (exemplified in Figure 1). Although this restricted reactivity concerned many different V specificities, V2 (n = 5) and V3 (n = 4) were observed more frequently than others. The latter may not be too surprising given the relatively high frequency of, especially, V2+ TCR cells in healthy controls (Table 1 contains median values).10 In the other 16 cases, the complete lack of reactivity of the suspect leukemic cell population with any of the individual V mAbs or the 6 V mixtures of the panel10 was considered indirect evidence for the "clonal" character of these cells (Figure 2). View larger version (40K): [in this window] [in a new window]   Figure 1. Flow cytometric V analysis in patients with mature T-cell proliferations compared with healthy controls. Using V8.1/8.2, V13.1/13.3, and V22 (FITC-labeled) double immunofluorescence stainings with CD3-PerCP, only small percentages of CD3+/V+ cells can be identified in healthy controls (upper panel), whereas similar double stainings in samples 93-027, 93-067, and 96-049 enable identification of large T-cell populations with single V8.1/8.2, V13.1/13.3, and V22 expression, respectively (lower panel). View larger version (34K): [in this window] [in a new window]   Figure 2. Flow cytometric V repertoire analysis using V mAb mixtures in mature T-cell proliferation patients. (A) In healthy controls, 60% to 65% of CD3+ cells are recognized in double immunofluorescence stainings with 6 different V mAbs mixtures in combination with CD3-PerCP.10 (B) Using comparable stainings in T-CLL patient 98-086, 99% of CD3+ cells are recognized by V mAbs mix 1, whereas all other 5 mixes only recognize less than 1% of all CD3+ cells. This indicates the presence of a large, presumably clonal CD3+ T-cell population with single V expression. (C) Double immunofluorescence stainings with the 6 V mAbs mixtures resulted in less than 5% CD3+/V+ cells in T-CLL patient 98-002, suggesting the presence of a large, presumably clonal CD3+ T-cell population with single V expression not recognized by any of the V mAbs in the current panel. Comparison of the data from molecular and flow cytometric analyses revealed complete concordance between the identified in-frame V-C transcript and the expressed V domain of the TCR chain in 26 cases (Table 4). Remarkably, in case 95-121, we identified V6.7- and V13.1/13.3-positive cells, which is in line with the presence of the corresponding in-frame V-C transcripts in this sample and the presence of 2 clones as found by SB analysis and immunophenotyping. In addition, in sample 91-030, 2 populations (V5.2/5.3 and V22) were found, but here no clear evidence was detected for further immunophenotypical heterogeneity or for clonal V22-C transcripts, suggesting that the V22-positive population concerned either a small subclone or a polyclonal V22+ cell population. The latter is more likely because a monoclonal subclone of 10% V22+ T-cells should have been identified in PCR heteroduplex analysis; in contrast, only polyclonal (heteroduplex) V22-C PCR products were observed in sample 91-030. In 16 cases no single V reactivity of the clonal cell population was observed, despite the presence of clonal V-C RT-PCR products in 15 of these samples (one was not studied through RT-PCR). Close examination revealed that in all 15 cases, the clonal transcripts contained gene segments derived from multimember V families (V5, V6, V7, and V13) known to be incompletely covered by the respective mAbs in the current panel. The position of the various primers for these V families of gene segments, however, did not allow for a detailed analysis into the exact gene member that was used. Nevertheless, even in these cases without V mAb reactivity, the molecular and flow cytometric results were not discordant. V oligoclonality in mature T-cell proliferations Although in all analyzed mature T-cell proliferations 1 or 2 dominant V-C RT-PCR products could be identified, in several samples a whole array of additional V-C products of variable, but mostly weak, intensity were found, next to the dominant clonal V-C product(s), as exemplified in Figure 3. Close examination of these oligoclonal samples disclosed that virtually all were diagnosed as T-LGL leukemias, whereas only a few concerned patients with T-CLL or Sézary syndrome. Despite their oligoclonal character, in all cases a dominant clonal cell population was observed, as evidenced by the flow cytometric data. View larger version (46K): [in this window] [in a new window]   Figure 3. V-C RT-PCR heteroduplex analysis. After reverse transcription of total RNA of T-LGL patient 86-041, cDNA was PCR amplified using V family primers in combination with a C primer. On heteroduplex analysis, 2 major clonal products (V1-C and V7-C) belonging to the dominant clone were observed in addition to several weaker clonal V-C products. These latter products represented rearranged alleles of small subclones that were not identified with flow cytometric V analysis.     Discussion Top Abstract Introduction Patients, materials, and... Results Discussion References V repertoire analysis for identification of clonal cell populations Clonality assessment of mature T-cell proliferations has long been performed by means of SB analysis. Although SB analysis is highly reliable owing to an almost complete lack of false-positive and false-negative results, it is time consuming and labor intensive, and it requires relatively large amounts of high-quality DNA. The latter is generally unavailable in paraffin-embedded tissues or small tissue biopsies. For this reason, PCR analysis of TCRG gene rearrangements has been used as an alternative strategy.15-17 However, the relatively restricted diversity of TCRG gene rearrangements and the resultant high-background amplification of similar rearrangements in normal T cells limits the potential of this approach. Because the TCRB recombination diversity is essentially larger than that of TCRG genes, analysis of the V repertoire of TCRB genes has been put forward as a diagnostic strategy for clonality studies in suspect T-cell proliferations. This approach can also be used to study the actual TCRB repertoire in other disease states with high T-cell activity, such as autoimmune diseases,18-22 immunodeficiencies,23-25 and alloreactivity in patients who have undergone transplantation.26-28 Until recently, this type of analysis mainly concerned PCR-based assays.18,21,22,26,29,30 However, a much faster and more quantitative analysis of the TCR repertoire is now possible through the use of mAbs directed against the V domains of TCR molecules, which cover 65% to 70% of V domains in blood T lymphocytes of children and adults.10,31-34 We performed parallel molecular and flow cytometric V analyses in a series of SB-defined TCR T-ALL samples and T-cell lines to validate both approaches for the detection of clonality. Indeed, clonal V-C transcripts could easily be detected in all T-ALL samples and T-cell lines. Moreover, the corresponding V mAb reactivity patterns also indicated the presence of large, single V domain ("clonal") cell populations in these cases, indicating that both methods are suitable for detecting aberrant T-cell proliferations. However, patients with T-ALL generally do not have diagnostic dilemmas for which V analysis is required because thymus derived T-ALL is relatively easy to diagnose using mAbs against the nuclear enzyme TdT and various CD antigens. Given that the main diagnostic problems generally concern suspect mature (postthymic) T-cell proliferations, we studied a series of 47 such mature T-cell proliferations, proven to be clonal based on SB analysis. RT-PCR heteroduplex analysis of V-C transcripts in all 42 analyzed cases confirmed the (mono)clonal character of the studied cell samples. Flow cytometric V repertoire analysis of the suspect mature T-cell proliferations identified single V domain expression in 31 (66%) of 47 cases. Together with the single V reactivity in 10 of 12 T-cell lines and in 8 of 16 T-ALL samples, this means that 49 (65%) of 75 cases were picked up through flow cytometric V repertoire analysis. In 16 of the 47 cases showing extensive T-cell proliferation, the combined V antibody reactivity covered less than 20% of all CD3+ T cells (instead of approximately 65%), which is indirect proof for the presence of a large T-cell population with single, though unidentified, V domain expression (Table 4). Flow cytometric V repertoire analysis can thus be used as a (quantitative) screening method for the detection of large, aberrant T-cell populations with single V domain expression. To define single V expression, the mean normal V values should be used, also taking into account the differences in the use of particular V domains between CD4+ and CD8+ T-cell populations and observations of a more restricted V usage of especially CD8 T lymphocytes in the elderly.10,35-39 Taken together, this means that we consider T-cell expansions aberrant if they concern more than the mean V value plus 3 SD (generally more than 20%) of the peripheral blood T cells or if the suspect population exceeds 2.0 × 109/L. The arbitrary cut-off of 2.0 × 109/L is considered an important criterion for diagnosing T-cell LGL leukemia,40 though even lower absolute counts might sometimes occur. Use of a 6-tube test kit with V mAbs mixtures, each covering maximally 10% to 15% of the V repertoire (Figure 2), further enables the screening for V repertoire restrictions.10 Meanwhile, an attractive commercial 8-tube kit (IO Test Beta Mark) has become available (Immunotech/Beckman Coulter, Marseilles, France); in each of the 8 tubes, 3 distinctly labeled V antibodies are present that not only allow detection of single V expression but also direct identification of the involved V domain or family. The results in this study suggest that detection of a T-cell population with restricted V usage in principle implies clonality of the involved proliferation. The fact that detection of single V expression is not necessarily equal to clonality is illustrated by, for example, case 91-030 showing a small V22+ population of approximately 10% that was not found to be clonal in molecular analysis. Although this expanded V22+ population was smaller than 20%, we think that in doubtful cases it generally remains important to confirm the presumed clonality by amplification of the involved V-C product. In case of large V unreactive T-cell populations, the clonal character should be established by PCR using a complete set of V family primers or by other molecular approaches, such as PCR analysis of TCRG genes or SB analysis of TCRB genes. The detection limits of both Southern blot analysis (5%-10%) and PCR-based assays (1%-5%) are superior over flow cytometric analysis of the V repertoire, which has a sensitivity of 20% for single V domain expression because of the background of normal V usage. Nevertheless, this sensitivity of the latter method might be improved if additional immunophenotypic markers are included to detect V usage in combination with a specific T-cell phenotype such as CD4, CD8, CD56, and CD57.1 Finally, a major advantage of flow cytometric V analysis over PCR-based assays is that once established and confirmed, the V-restricted T-cell population can easily and quantitatively be monitored in combination with other markers during and after therapy, using age-dependent reference values for comparison.10,41 Oligoclonality of T-LGL proliferations During analysis of the mature T-cell proliferations, we observed that especially in many T-LGL samples, multiple weak additional products were seen next to 1 or 2 V-C transcripts belonging to the immunophenotypically dominant clone. Limited sequencing of these clonal products did not show similar V gene segments or junctional region sequences. This observation provides further evidence for the hypothesis raised earlier, which is that T-LGL derive from polyclonal or oligoclonal proliferations of antigen-activated cytotoxic T-cells and that, in some situations, transformation or dysregulation of growth or apoptosis results in T-LGL leukemia showing a more restricted and dominant V usage (expressed as percentage MNCs) and a raised absolute cell count of abnormal cells.42-45 Furthermore, the generally indolent course of this type of T-cell proliferation and the relatively lower white blood cell counts (compared to T-CLL and T-PLL proliferations) are also in line with a pretransformation state of oligoclonal proliferations of activated T-cells. Monoreactivity in T-ALL and mature T-cell populations Remarkably, in many T-ALL (6 of 10) and mature T-cell proliferations (at least 9 of 16) with bi-allelic TCRB gene rearrangements and, to a lesser extent, in T-cell lines (1 of 6 cases), double in-frame transcripts were observed. In theory this could lead to double V expression in particular samples. However, in virtually all cases that could be evaluated, the clonal T-cell population reacted with only one of the V mAbs; monoreactivity cannot be proven formally in a few samples because of the lack of appropriate V antibodies in the panel. In case 95-121, the 2 identified in-frame V-C transcripts appeared to be derived from 2 distinct T-cell populations, given the results of flow cytometric analysis. Taken together, the data strongly suggest that in cases with double in-frame V-C products, monospecificity of the TCR is guaranteed by regulation at the level of translation of TCR chains or by preferential pairing of one TCR chain with the involved TCR chain. We conclude from our data that flow cytometric V repertoire analysis is a fast and relatively cheap alternative tool that can be used as a powerful screening method in patients with suspect T-cell proliferations. Formally, however, clonality as deduced from single V domain expression (more than 20% of cells) or from the lack of reactivity of a large (more than 50% to 60%) TCR+ T-cell population would still need proof by molecular assays. Nevertheless, flow cytometric analysis has the additional advantages of looking within T-cell subsets and precise quantitation of the V+ cells. An additional interesting application is the flow cytometric monitoring of V+ leukemic cells during and after therapy, once a single V expression has been determined.     Acknowledgments We thank Prof dr R. Benner for support and Ms J. Boon for secretarial assistance. We also thank the following clinicians and scientists for submitting samples from patients with suspect mature T-cell proliferations: H. J. Adriaansen, P. B. Berendes, J. W. Gratama, E. Harthoorn-Lasthuizen, C. van der Heul, J. van Helden, J. C. Kluin-Nelemans, P. J. Lugtenburg, C. Lynas, L. Marcelis, E. Moreau, P. Sonneveld, W. Slieker, J. W. Smit, H. Storm, P. Vandenberghe, M. B. van `t Veer, and G. Verhoef.     Footnotes Submitted November 8, 2000; accepted March 9, 2001. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Anton W. Langerak, Department of Immunology, University Hospital Rotterdam, Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands; e-mail:langerak{at}immu.fgg.eur.nl.     References Top Abstract Introduction Patients, materials, and... Results Discussion References 1. 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