In-frame Rearrangements Research Articles

New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling

To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin− cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Dδ2-Dδ3 rearrangement occurs at the most immature CD34+CD38−CD1a− stage. TCRB rearrangement starts at the CD34+CD38+CD1a− stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTα) expression pattern show that human TCRβ-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before.

Immunoglobulin light chain gene rearrangements display hierarchy in absence of selection for functionality in precursor-B-ALL.

The general order of the immunoglobulin (Ig) gene rearrangement process in human precursor-B cells is largely known. However, the exact Ig rearrangement patterns reflecting this process, especially those of the Ig light chain genes, are not well established. This requires detailed analysis of the gene configuration of all six IGH, IGK and IGL alleles at the single cell level. As such extensive analyses are difficult to perform in a reliable way within a single normal precursor-B cell, we used 169 precursor-B-ALL (ie six pro-B-ALL, 112 common ALL, and 51 pre-B-ALL) as clonal 'single cell' model system. The Ig gene recombinations show hierarchy starting with IGH gene rearrangements in all cases, followed by IGK rearrangements, IGK deletions and/or IGL rearrangements in 71% of cases. IGK deletions were found in the absence of IGL rearrangements in 34% of cases, which might be explained by the continuous recombinase activity in precursor-B-ALL, resulting in 'end-stage' IGK rearrangements, together with an apparently limited accessibility of the IGL locus. Remarkably, in 5% of cases IGL rearrangements took place in the absence of IGK rearrangements. In addition we found that in-frame IGH rearrangements are not necessarily required for the induction of Ig light chain gene rearrangements and that IGL rearrangements can be induced irrespective of the frame of the accompanying IGK rearrangements. In conclusion, precursor-B-ALL constitute a model system for studying Ig gene rearrangement processes without selection for functionality of the rearrangements or the influence of somatic hypermutations. Nevertheless, the hierarchy of IGH, IGK and IGL rearrrangements is apparent in precursor-B-ALL.

Epstein-Barr virus-positive Hodgkin/Reed-Sternberg-like B cell in non-hodgkin lymphoma: nucleotide sequence of the amplified immunoglobulin heavy-chain variable region gene by the single-cell polymerase chain reaction technique.

We examined nucleotide sequences of Epstein-Barr virus (EBV)-positive Hodgkin/Reed-Sternberg (HRS)-like B cells in a case of diffuse large B-cell lymphoma (DLBCL) and a case of adult T-cell lymphoma (ATL) for single-cell polymerase chain reaction of the immunoglobulin heavy-chain gene variable region (VH gene). HRS-like B cells were scattered in the area irrelevant to the lymphoma infiltrates of DLBCL and in the lymphoma area of ATL. HRS-like B cells were positive for CD20 and CD30 but negative for CD15. EBV presented in HRS-like B cells in both cases but not in any lymphoma cells. VH genes of five HRS-like B cells analyzed in DLBCL were polyclonal and showed in-frame sequences with 0% to 2.8% somatic mutation frequency. In an ATL, VH genes of five HRS-like B cells analyzed were polyclonal and somatically mutated. Four cells carried in-frame rearrangements with 3.5% to 17.7% mutation frequency. One of the VH genes has a one-codon deletion. From the fifth cell, an out-of-frame rearrangement with an insertion and a deletion was obtained. Thus, we showed polyclonal EBV-positive HRS-like B cells in both DLBCL and ATL and that whereas EBV-positive, HRS-like B cells in DLBCL exhibited unmutated and mutated VH gene, those in ATL were found to have a somatically mutated VH gene with/without deletions and/or insertions. The HRS-like B cells may appear because of active EBV infection in a patient who is immunosuppressed from the primary lymphoma.

B cell development and VDJ rearrangement in the fetal pig

Hematopoietic activity of the swine has been documented in three phases during fetal ontogeny. The hematopoietic system develops first in the yolk sac, then in fetal liver and finally in the bone marrow. Using flow cytometry (FCM) and molecular biological techniques we show that B-cell lymphogenesis and the appearance of B cells follows a pattern. First, VDJ rearrangement occurs at the 20th day of gestation (DG20) in the yolk sac at a time when light chain transcription is absent. Next, B-cell lymphogenesis is detected at DG30 in the fetal liver. Thereafter, bone marrow becomes the major B lymphopoietic organ (DG45). In yolk sac and fetal liver, more than 90% of the VDJ rearrangements were in-frame but expression of μ heavy chain could not be clearly detected by FCM. However, cells with a putative phenotype of B-cell precursors are present. These cells express high levels of MHC class II (SLA-DR) and low levels of CD2 and CD25. CDR3 length analysis (spectratyping) indicates that the heavy chain repertoire is oligoclonal at this time with large inter-animal variations. Consistent with our earlier reports, fetal VDJ rearrangements are not mutated and there is no evidence for an age-dependent increase in TdT activity or a change in V H and D H usage from those used by B-cells formed in the yolk sac or fetal liver. However, our findings indicate major differences in the regulatory environment and/or selective pressures in yolk sac and fetal liver versus bone marrow. In contrast with the yolk sac and fetal liver, the proportion of in-frame VDJ rearrangements in the bone marrow correspond to a value indicative of random recombination.

Thymocyte maturation: selection for in-frame TCR alpha-chain rearrangement is followed by selection for shorter TCR beta-chain complementarity-determining region 3.

Thymocyte maturation consists of a number of stages, the goal of which is the production of functioning T cells that respond to foreign antigenic peptides using their clonotypic receptors. Selection of a productively rearranged TCR beta-chain is the first stage in the process and occurs at the double-negative to double-positive (DP) transition. Later maturation stages are based on changes in markers such as CD5, CD69, or IL-7R. A stage in which a-chains are selected has also been identified using beta-chain transgenic mice. Here we identify two additional selection stages in human thymocytes based on characteristics of the TCR. alpha selection is measured directly by identification of in-frame rearrangements and is associated with the appearance of CD3 on the DP thymocyte surface. The next stage has not yet been described and involves selection of thymocytes that express shorter TCR beta-chain complementarity-determining region 3 (CDR3). This stage is associated with the acquisition of high levels of CDR3 by DP cells and the transition to SP thymocytes. The extent of CDR3 length selection observed is a function of the TCR V and J genes. We propose that CDR3 length selection is based on recognition of the MHC. Thus, there exist limitations on the allowable length of that portion of the TCR most intimately in contact with MHC and peptide. This may be a physical representation of positive selection.

Molecular features responsible for the absence of immunoglobulin heavy chain protein synthesis in an IgH− subgroup of multiple myeloma

AbstractThis study involved 12 patients with multiple myeloma (MM), in whom malignant plasma cells did not contain immunoglobulin heavy chain (IgH) protein chains. Southern blot analysis revealed monoallelic Jh gene rearrangements in 10 patients, biallelic rearrangement in 1 patient, and biallelic deletion of the Jh and Cμ regions in 1 patient. Heteroduplex polymerase chain reaction analysis enabled the identification and sequencing of 9 clonal Jhgene rearrangements. Only 4 of the joinings were complete Vh-(D)-Jhrearrangements, including 3 in-frame rearrangements with evidence of somatic hypermutation. Five rearrangements concerned incomplete Dh-Jh joinings, mainly associated with deletion of the other allele. Curiously, in at least 1 of these 5 cases the second allele seemed to be in germline configuration, whereas the in-frame Vκ-Jκgene rearrangements contained somatic mutations. The configuration of the IGH genes was further investigated by use of Ch probes. In 5 patients the rearrangements in the Jh and Ch regions were not concordant, probably caused by illegitimate IGH class switch recombination (chromosomal translocations to 14q32.3). These data indicate that in many IgH− MM patients illegitimateIGH class switch rearrangement or illegitimate deletion of the functional Vh-(Dh)-Jhallele are responsible for IgH negativity. For example, the exclusive presence ofDh-Jhrearrangements in combination with mutated IGK genes can only be explained in terms of normal B-cell development, if the second (functional) IGH allele is deleted, which was probably the case in most patients. Therefore, defects at the DNA level are responsible for the lack of IgH protein production in most IgH− MM patients.

Immunoglobulin kappa light-chain V, J, and C gene sequences of the owl monkey Aotus nancymaae.

Sequences of Aotus nancymaae immunoglobulin kappa light-chain rearrangements were analyzed after reverse transcription polymerase chain reaction. Among 22 in-frame rearrangements analyzed, 12 IGKV genes belonging to the families 1, 2, or 3 were identified. Aotus counterparts for all five human IGKJ genes were found. The identity of the deduced human and Aotus amino acid sequences was between 83% and 92% for junctional regions and 74% for the constant region. Sequence comparisons between rearrangements indicated that somatic mutations, the addition of nongermline-encoded nucleotides, and exonuclease trimming contribute to the generation of diversity of Aotus immunoglobulin kappa chains. The high identity of Aotus and human IGK genes is comparable to that of T-cell receptor genes and further supports the proposal to use the Aotus Plasmodium falciparum infection model for the evaluation of malaria vaccine candidates.

Molecular features responsible for the absence of immunoglobulin heavy chain protein synthesis in an IgH− subgroup of multiple myeloma

This study involved 12 patients with multiple myeloma (MM), in whom malignant plasma cells did not contain immunoglobulin heavy chain (IgH) protein chains. Southern blot analysis revealed monoallelic J(H) gene rearrangements in 10 patients, biallelic rearrangement in 1 patient, and biallelic deletion of the J(H) and C(micro) regions in 1 patient. Heteroduplex polymerase chain reaction analysis enabled the identification and sequencing of 9 clonal J(H) gene rearrangements. Only 4 of the joinings were complete V(H)-(D)-J(H) rearrangements, including 3 in-frame rearrangements with evidence of somatic hypermutation. Five rearrangements concerned incomplete D(H)-J(H) joinings, mainly associated with deletion of the other allele. Curiously, in at least 1 of these 5 cases the second allele seemed to be in germline configuration, whereas the in-frame V(kappa)-J(kappa) gene rearrangements contained somatic mutations. The configuration of the IGH genes was further investigated by use of C(H) probes. In 5 patients the rearrangements in the J(H) and C(H) regions were not concordant, probably caused by illegitimate IGH class switch recombination (chromosomal translocations to 14q32. 3). These data indicate that in many IgH(-) MM patients illegitimate IGH class switch rearrangement or illegitimate deletion of the functional V(H)-(D(H))-J(H) allele are responsible for IgH negativity. For example, the exclusive presence of D(H)-J(H) rearrangements in combination with mutated IGK genes can only be explained in terms of normal B-cell development, if the second (functional) IGH allele is deleted, which was probably the case in most patients. Therefore, defects at the DNA level are responsible for the lack of IgH protein production in most IgH(-) MM patients.

Unexpected rearrangement and expression of the immunoglobulin lambda1 locus in scid mice.

In severe combined immunodeficient (scid) mice, V(D)J recombination is severely impaired due to a recessive mutation (scid). Thus, we were surprised to find in this study that Vlambda1-Jlambda1 rearrangement is routinely detectable in scid fetal liver, adult bone marrow, and spleen in the apparent absence of completed VH-DJH and Vkappa-Jkappa rearrangements. Particularly surprising, we found the level of Vlambda1-Jlambda1 rearrangement in scid fetal liver to be comparable to that in fetal liver of wild-type mice. The majority of scid Vlambda1-Jlambda1 rearrangements contained abnormal deletions at the VJ junction, consistent with the known effect of scid. However, approximately 15% of Vlambda1-Jlambda1 rearrangements lacked abnormal deletions. Productive lambda1 transcripts resulting from in-frame rearrangements were readily detectable in scid adult bone marrow and spleen, consistent with our ability to detect lambda1-expressing cells by flow cytometry in the spleens of bcl-2-transgenic scid mice. Strikingly, lambda1 transcripts from individual scid mice often showed VJ junctional sequences with the same recurring palindromic (P) additions of three, four, or five nucleotides. To account for these findings, we suggest that (a) nonhomologous end joining of Vlambda1 and Jlambda1 coding ends in fetal B lineage cells may not be (severely) impaired by scid; (b) recurring P additions in scid lambda1 transcripts may reflect certain molecular constraints imposed by scid on the resolution of Vlambda1 and Jlambda1 hairpin coding ends; and (c), scid lymphocytes with productively rearranged Vlambda1 and Jlambda1 elements may differentiate into recombinase-inactive cells and emigrate from bone marrow to spleen.

Distinct Signals Mediate Maturation and Allelic Exclusion in Lymphocyte Progenitors

Successful in-frame rearrangement of immunoglobulin heavy chain genes or T cell antigen receptor (TCR) β chain genes in lymphocyte progenitors results in formation of pre-BCR and pre-TCR complexes. These complexes signal progenitor cells to mature, expand in cell number, and suppress further rearrangements at the immunoglobulin heavy chain or TCRβ chain loci, thereby ensuring allelic exclusion. We used transgenic expression of a constitutively active form of c-Raf-1 (Raf-CAAX) to demonstrate that activation of the Map kinase pathway can stimulate both maturation and expansion of B and T lymphocytes, even in the absence of pre-TCR or pre-BCR formation. However, the same Raf signal did not mediate allelic exclusion. We conclude that maturation of lymphocyte progenitors and allelic exclusion require distinct signals.

Molecular Analysis of Single B Cells From T-Cell–Rich B-Cell Lymphoma Shows the Derivation of the Tumor Cells From Mutating Germinal Center B Cells and Exemplifies Means by Which Immunoglobulin Genes Are Modified in Germinal Center B Cells

T-cell–rich B-cell lymphoma (TCRBCL) belongs to the group of diffuse large cell lymphomas (DLL). It is characterized by a small number of tumor B cells among a major population of nonmalignant polyclonal T cells. To identify the developmental stage of the tumor progenitor cells, we micromanipulated the putative neoplastic large CD20+ cells from TCRBCLs and amplified and sequenced immunoglobulin (Ig) V gene rearrangements from individual cells. In six cases, clonal Ig heavy, as well as light chain, gene rearrangements were amplified from the isolated B cells. All six cases harbored somatically mutated V gene rearrangements with an average mutation frequency of 15.5% for heavy (VH) and 5.9% for light (VL) chains and intraclonal diversity based on somatic mutation. These findings identify germinal center (GC) B cells as the precursors of the transformed B cells in TCRBCL. The study also exemplifies various means how Ig gene rearrangements can be modified by GC B cells or their malignant counterparts in TCRBCL: In one case, the tumor precursor may have switched from κ to λ light chain expression after acquiring a crippling mutation within the initially functional κ light chain gene. In another case, the tumor cells harbor two in-frame VH gene rearrangements, one of which was rendered nonfunctional by somatic mutation. Either the tumor cell precursor entered the GC with two potentially functional in-frame rearrangements or the second VHDHJHrearrangement occurred in the GC after the initial in-frame rearrangement was inactivated by somatic mutation. Finally, in each of the six cases, at least one cell contained two (or more) copies of a clonal Ig gene rearrangement with sequence variations between these copies. The presence of sequence variants for V region genes within single B cells has so far not been observed in any other normal or transformed B lymphocyte. Fluorescence in situ hybridization (FISH) points to a generalized polyploidy of the tumor cells.

Molecular Analysis of Single B Cells From T-Cell–Rich B-Cell Lymphoma Shows the Derivation of the Tumor Cells From Mutating Germinal Center B Cells and Exemplifies Means by Which Immunoglobulin Genes Are Modified in Germinal Center B Cells

AbstractT-cell–rich B-cell lymphoma (TCRBCL) belongs to the group of diffuse large cell lymphomas (DLL). It is characterized by a small number of tumor B cells among a major population of nonmalignant polyclonal T cells. To identify the developmental stage of the tumor progenitor cells, we micromanipulated the putative neoplastic large CD20+ cells from TCRBCLs and amplified and sequenced immunoglobulin (Ig) V gene rearrangements from individual cells. In six cases, clonal Ig heavy, as well as light chain, gene rearrangements were amplified from the isolated B cells. All six cases harbored somatically mutated V gene rearrangements with an average mutation frequency of 15.5% for heavy (VH) and 5.9% for light (VL) chains and intraclonal diversity based on somatic mutation. These findings identify germinal center (GC) B cells as the precursors of the transformed B cells in TCRBCL. The study also exemplifies various means how Ig gene rearrangements can be modified by GC B cells or their malignant counterparts in TCRBCL: In one case, the tumor precursor may have switched from κ to λ light chain expression after acquiring a crippling mutation within the initially functional κ light chain gene. In another case, the tumor cells harbor two in-frame VH gene rearrangements, one of which was rendered nonfunctional by somatic mutation. Either the tumor cell precursor entered the GC with two potentially functional in-frame rearrangements or the second VHDHJHrearrangement occurred in the GC after the initial in-frame rearrangement was inactivated by somatic mutation. Finally, in each of the six cases, at least one cell contained two (or more) copies of a clonal Ig gene rearrangement with sequence variations between these copies. The presence of sequence variants for V region genes within single B cells has so far not been observed in any other normal or transformed B lymphocyte. Fluorescence in situ hybridization (FISH) points to a generalized polyploidy of the tumor cells.

A Limited Role for β-Selection During γδ T Cell Development

AbstractT cells belong to two distinct lineages expressing either αβ or γδ TCR. During αβ T cell development, it is clearly established that productive rearrangement at the TCR β locus in immature precursor cells leads to the expression of a pre-TCR complex. Signaling through the pre-TCR results in the selective proliferation and maturation of TCR β+ cells, a process that is known as β-selection. However, the potential role of β-selection during γδ T cell development is controversial. Whereas PCR-RFLP and sequencing techniques have provided evidence for a bias toward in-frame VDJβ rearrangements in γδ cells (consistent with β-selection), γδ cells apparently develop normally in mice that are unable to assemble a pre-TCR complex due to a deficiency in TCR β or pTα genes. In this report, we have directly addressed the physiologic significance of β-selection during γδ cell development in normal mice by quantitating intracellular TCR β protein in γδ cells and correlating its presence with cell cycle status. Our results indicate that β-selection plays a significant (although limited) role in γδ cell development by selectively amplifying a minor subset of γδ precursor cells with productively rearranged TCR β genes.

Alternative splicing of rearranged T cell receptor delta sequences to the constant region of the alpha locus.

The T cell receptor (TCR) alpha/delta locus is composed of a common, shared set of variable (V) and distinct diversity (D), joining (J), and constant (C) genes. It has been recognized for several years that transcripts of the rearranged VDJdelta or VJalpha genes are spliced to the Cdelta or Calpha genes, respectively, encoding distinct TCR delta and alpha proteins. Herein, we describe the discovery of a splicing variation that allows the assembled VDJdelta genes to be fused with the Calpha gene. This variation is prominent in TCRdelta gene-deficient mice but is also detectable in wild-type mice. Furthermore, we show that several in-frame VDJdelta rearrangements in TCRdelta gene-deficient mice are strikingly underrepresented, suggesting that the alternative transcripts, with protein coding capacity, influence the development of alphabeta thymocytes. In-frame TCRgamma gene rearrangements do not appear underrepresented, indicating that the effect is not mediated by the gamma chain. Instead, indirect evidence supports the hypothesis that the delta/alpha chimeric protein acts in conjunction with the TCRbeta chain. These results have implications for the transcriptional control of the TCRalpha/delta locus and provide a novel insight into the distinct functional capacities of the TCR alpha and delta proteins during thymocyte development.

Intrathymic δ Selection Events in γδ Cell Development

The major pathway of γδ cell development is shown to be regulated by in-frame rearrangements at the T cell receptor (TCR) δ locus. Such “δ selection” occurs at or around the same point in thymocyte development as selection for in-frame rearrangements at the TCRβ locus. However, there are at least two major differences with β selection: first, δ selection commonly involves selection on the cognate TCR chain, γ, suggesting that there is no “preTγ” chain of major biological significance; second, most γδ-selected thymocytes differentiate rather than proliferate. Nonetheless, some δ selection events seemingly facilitate thymocyte expansion, similar to αβ T cell development. In these cases, TCRγ selection is less obvious. Furthermore, the capacity of individual γ chains to facilitate γδ selection is shown to vary with developmental age. The results further clarify early T cell development at the β selection/δ selection stage and place clear constraints on models of cell fate determination.

Notch Activity Influences the αβ versus γδ T Cell Lineage Decision

The choice between the αβ or γδ T cell fates is influenced by the production of functional, in-frame rearrangements of the TCR genes, but the mechanism that controls the lineage choice is not known. Here, we show that T cells that are heterozygous for a mutation of the Notch1 gene are more likely to develop as γδ T cells than as αβ T cells, implying that reduced Notch activity favors the γδ T cell fate over the αβ T cell fate. A constitutively activated form of Notch produces a reciprocal phenotype and induces thymocytes that have functional γδTCR gene rearrangements to adopt the αβ T cell fate. Our data indicate that Notch acts together with the newly formed T cell antigen receptor to direct the αβ versus γδ T cell lineage decision.

TCR gene recombination and alpha beta-gamma delta lineage divergence: productive TCR-beta rearrangement is neither exclusive nor preclusive of gamma delta cell development.

Two types of T lymphocytes can be generated intrathymically, distinguishable by either TCR-gamma delta or -alpha beta surface expression. Regulation of the intrathymic divergence of these cells is unresolved, at least in part because thymically derived gamma delta cells have rarely been studied. We used quantitative Southern blotting together with PCR-based cloning/sequencing and restriction fragment length polymorphism to analyze TCR-alpha and -beta gene recombination in thymically derived gamma delta cells. We found that TCR-beta gene recombination is a frequent occurrence in thymic gamma delta cells. Furthermore, not only do complete (V-D-J) TCR-beta gene rearrangements occur in thymic gamma delta cells, but the frequency of in-frame rearrangements is greater than would be predicted based upon random occurrence. In contrast, we show that thymically derived gamma delta cells do not make detectable rearrangements of the TCR-alpha locus. These studies clearly demarcate a point for alpha beta vs gamma delta commitment in the thymus, after TCR-beta but before TCR-alpha gene recombination. Further, while our data support gamma delta lineage commitment as a consequence of successful TCR-gamma and -delta gene rearrangement, we do not find support for a competitive model of lineage commitment, since productive TCR-beta gene rearrangement does not necessarily relegate cells to the alpha beta lineage.

Mitogens, superantigens, and nominal antigens elicit distinctive patterns of TCRB CDR3 diversity

The third complementarity-determining region (CDR3) is the only nongermline-encoded hypervariable region of the T cell receptor β (TCRB) chain, and it is the region that has been predicted to confer fine specificity of the TCR for peptide-MHC complexes. For this reason analysis of TCRB CDR3 heterogeneity may provide insight into immune mechanisms operative in infectious and autoimmune diseases. PBMC stimulated with either mitogen (PHA), superantigen (TSST-1), or nominal antigen (tetanus toxoid) have been compared with unstimulated PBMC using a two-dimensional approach. Analysis of the expressed TCRBV gene repertoire CDR3 length profile coupled with SSCP methodology enabled the discrimination of sequences with the same CDR3 length. For both freshly isolated and PHA- stimulated PBMC, a normally distributed spectrum of CDR3 lengths (five or more products) was observed. These products differed by 3 bp (1 amino acid) due to the strict requirement for in-frame rearrangements in the CDR3 region of TCR. By contrast, tetanus toxoidstimulated PBMC had restricted profiles for most TCRBV families after as few as 7 days of incubation. The oligoclonal nature of samples showing CDR3 length restriction was revealed by SSCP analysis and confirmed by sequence determination. Superantigen stimulation resulted in unique patterns of diversity, which included polyclonal expansion of specific TCRBV families as well as oligoclonal expansion of most other TCRBV families. These data reveal complex yet distinct patterns of TCR diversity in response to different T cell activation stimuli.

CLONAL EXPANSION OF Vβ8+ T LYMPHOCYTES IN CHRONIC IMMUNE THROMBOCYTOPENIC PURPURA. • 892

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune hematologic disorder characterized by the presence of antiplatelet antibodies which lead to accelerated platelet clearance by the reticuloendothelial system. Although the platelet antibodies are produced by autoreactive B lymphocytes, there is also evidence to implicate T lymphocytes in the pathogenesis of this autoimmune disorder. We identified two unrelated males with chronic ITP who had highly elevated numbers of circulating Vβ8+ peripheral blood T lymphocytes documented on multiple occasions. Patient 1 had 8-12% Vβ8+ T lymphocytes, predominantly CD4+ (normal 2-4%), while patient 2 had 15-35% Vβ8+ T lymphocytes, almost exclusively CD8+. Total RNA was isolated from peripheral blood lymphocytes, and used for RT-PCR analysis and nucleotide sequencing of Vβ8-(D)-J-C gene rearrangements: For patient 1, 8 of 19 transcripts had an identical in-frame Vβ8.2-Dβ1.1-Jβ1.2-Cβ1 gene rearrangement, indicating a clonal expansion. Similarly, patient 2 had 8 of 23 mRNA transcripts with a Vβ8.3-Dβ1.1-Jβ1.2-Cβ1 in-frame rearrangement, and 7 of 23 with an in-frame Vβ8.2-Jβ1.1-Cβ1 rearrangement. Other Vβ8-(D)-J-C mRNA transcripts from these patients, as well as those from controls, demonstrated unique rearrangements using random combinations of N, Dβ, and Jβ segments. Patient 1 underwent splenectomy with resolution of his thrombocytopenia, along with normalization of the% peripheral blood Vβ8+ T lymphocytes. RT-PCR analysis of peripheral blood lymphocyte cytokine expression demonstrated elevated amounts of TNF-α, IL-2, andγ-IFN mRNA which decreased after splenectomy. Histologic staining of the splenic tissue revealed Vβ8+ T lymphocytes within the germinal centers, and RT-PCR identified the clonal Vβ8 sequence within the spleen. These results provide strong evidence that clonal T lymphocyte populations are present in chronic ITP, and are important in the pathogenesis of this autoimmune disorder.

T cell receptor gene recombination patterns and mechanisms: cell death, rescue, and T cell production.

The antigen-specific receptors of T and B lymphocytes are generated by somatic recombination between noncontiguous gene segments encoding the variable portions of these molecules. The semirandom nature of this process, while desirable for the generation of diversity, has been thought to exact a high price in terms of sterile (out-of-frame) products. Historically, the majority of T lymphocytes generated in mammals were thought to be useless, either because they generated such sterile rearrangements or because the receptors generated did not appropriately recognize self-molecules (i.e., positive and negative selection). In the studies described here, we characterize the onset of T cell receptor (TCR) alpha and beta chain gene rearrangements and quantitate their progression throughout T cell development. The results show that T cell production efficiency is enhanced through (a) rearrangement of TCR-beta chain genes early during T cell development, with selective expansion of those cells possessing in-frame rearrangements; (b) deletion of sterile rearrangements at the TCR-alpha chain locus through ordered (proximal to distal) sequential recombination; and (c) modification of nonselectable alpha/beta heterodimer specificities through generation and expression of new TCR-alpha chains. In addition, we demonstrate strict correlations between successful TCR-beta gene rearrangement, the onset of TCR-alpha gene rearrangement, rapid cell division, and programmed cell death, which together serve to maintain cell turnover and homeostasis during T cell development.