T-cell receptor gene rearrangements as markers of lineage and clonality in T-cell neoplasms.

Ig gene rearrangements represent markers of lineage, clonality, and differentiation of B cells, allowing a molecular diagnosis and immunogenotypic classification of B-cell neoplasms. We sought to apply a similar approach to the study of T-cell populations by analyzing rearrangements of the T-cell receptor beta-chain (T beta) gene. Our analysis, by Southern blotting hybridization using T beta-specific probes of DNAs from polyclonal T cells and from 12 T-cell tumors, indicates that T beta gene rearrangement patterns can be used as markers of (i) lineage, allowing the identification of polyclonal T-cell populations, and (ii) clonality, allowing the detection of monoclonal T-cell tumors. In addition, our data indicate that T beta gene rearrangements represent early and general markers of T-cell differentiation since they are detectable in histologically different tumors at all stages of T-cell development. The ability to determine lineage, clonality, and stage of differentiation has significant implications for future experimental and clinical studies on normal and neoplastic T cells.     

T-cell receptor gene rearrangements and the diagnosis of human T-cell neoplasms

The rearranging antigen receptor genes of lymphoid cells serve as unique clonal markers of lymphoid neoplasms. Gene rearrangement analysis is a highly sensitive and reproducible tool which is useful in the diagnosis and classification of malignant lymphoma/leukemia. Although clonality can often be determined among B cell neoplasms by virtue of immunoglobulin isotype analysis, no such phenotypic marker of clonality exists for T cells. Therefore, clonality of T lymphoproliferative processes is most readily determined by rearrangement analysis of the T cell antigen receptor genes. The alpha, beta, gamma, and delta genes of the T cell receptor gene family encode heterodimeric surface antigen receptors and undergo rearrangement early in T cell differentiation. Identification of rearrangement of T cell antigen receptor genes provides valuable diagnostic information concerning cellular lineage, clonality and classification of T cell neoplasms. This molecular approach is applicable to the diagnosis of occult disease, relapse, and resolution of diagnostic dilemmas in any type of tissue sample including fluids and needle aspirations.     

N-terminal truncated human RAG1 proteins can direct T-cell receptor but not immunoglobulin gene rearrangements

The proteins encoded by RAG1 and RAG2 can initiate gene recombination by site-specific cleavage of DNA in immunoglobulin and T-cell receptor (TCR) loci. We identified a new homozygous RAG1 gene mutation (631delT) that leads to a premature stop codon in the 5' part of the RAG1 gene. The patient carrying this 631delT RAG1 gene mutation died at the age of 5 weeks from an Omenn syndrome-like T(+)/B(- )severe combined immunodeficiency disease. The high number of blood T-lymphocytes (55 x 10(6)/mL) showed an almost polyclonal TCR gene rearrangement repertoire not of maternal origin. In contrast, B-lymphocytes and immunoglobulin gene rearrangements were hardly detectable. We showed that the 631delT RAG1 gene can give rise to an N-terminal truncated RAG1 protein, using an internal AUG codon as the translation start site. Consistent with the V(D)J recombination in T cells, this N-terminal truncated RAG1 protein was active in a plasmid V(D)J recombination assay. Apparently, the N-terminal truncated RAG1 protein can recombine TCR genes but not immunoglobulin genes. We conclude that the N-terminus of the RAG1 protein is specifically involved in immunoglobulin gene rearrangements.     

Clonal immunoglobulin heavy chain and T-cell receptor γ gene rearrangements in primary gastric lymphoma

AIM:To study the diagnostic value of immunoglobulin heavy chain(IgH)and T-cell receptorγ (TCR-γ)gene monoclonal rearrangements in primary gastric lymphoma(PGL).METHODS:A total of 48 patients with suspected PGL at our hospital were prospectively enrolled in this study from January 2009 to December 2011.The patients were divided into three groups(a PGL group,a gastric linitis plastica group,and a benign gastric ulcer group)based on the pathological results(gastric mucosal specimens obtained by endoscopy or surgery)and follow-up.Endoscopic ultrasonography(EUS)and EUSguided biopsy were performed in all the patients.The tissue specimens were used for histopathological examination and for IgH and TCR-γ gene rearrangement polymerase chain reaction analyses.RESULTS:EUS and EUS-guided biopsy were successfully performed in all 48 patients.In the PGL group(n=21),monoclonal IgH gene rearrangements were detected in 14(66.7%)patients.A positive result for each set of primers was found in 12(57.1%),8(38.1%),and 4(19.0%)cases using FR1/JH,FR2/JH,and FR3/JH primers,respectively.Overall,12(75%)patients with mucosal-associated lymphoid tissue lymphoma(n=16)and 2(40%)patients with diffuse large B-cell lymphoma(n=5)were positive for monoclonal IgH gene rearrangements.No patients in the gastric linitis plastica group(n=17)and only one(10%)patient in the benign gastric ulcer group(n=10)were positive for a monoclonal IgH gene rearrangement.No TCRgene monoclonal rearrangements were detected.The sensitivity of monoclonal IgH gene rearrangements was 66.7%for a PGL diagnosis,and the specificity was96.4%.In the PGL group,8(100%)patients with stage IIE PGL(n=8)and 6(46.1%)patients with stage IE PGL(n=13)were positive for monoclonal IgH gene rearrangements.CONCLUSION:IgH gene rearrangements may be associated with PGL staging and may be useful for the diagnosis of PGL and for differentiating between PGL and gastric linitis plastica.     

Absence of immunoglobulin and T-cell receptor gene rearrangements in myelodysplastic syndromes and acute nonlymphocytic leukemias

The arrangement of the immunoglobulin and T-cell receptor genes has been analysed in 72 cases of primary myelodysplastic syndrome and 17 cases of acute nonlymphocytic leukemia. DNA was extracted from bone marrow aspirates, digested with at least two restriction enzymes, and hybridised with probes for the joining region of the immunoglobulin heavy chain gene, the constant region of the T-cell receptor beta chain gene, and the joining region of the T-cell receptor gamma chain gene. All cases showed germline arrangements of the immunoglobulin and the T cell receptor genes. Thus, true interlineage infidelity, myeloid to lymphoid, is a rare occurrence in myelodysplasia and in myeloid leukemias.     

T-cell subsets and suppressor cells in human bone marrow.

To characterize immune suppressive and hematopoietic features of enriched subsets of human marrow cells, we separated these cells on Percoll density gradients. CD4+ and CD8+ T cells (CD3+) were enriched in the high-density marrow cell fractions and reduced in low-density fractions. CD4-CD8- (CD3+) T cells expressing the alpha beta T-cell antigen receptor were at least 10 times less numerous than the CD4+ and CD8+ T cells in all fractions. Purified populations of the CD4-CD8- alpha beta + T cells obtained by flow cytometry suppressed the mixed leukocyte reaction (MLR). Another population of suppressor cells that expressed neither T-cell (CD3) nor natural killer cell (CD16) surface markers was also identified. The latter cells had the phenotypic and functional characteristics of "natural suppressor" cells. Suppressor cell activity was enriched in the low-density fractions along with hematopoietic progenitors (colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid). The progenitor and suppressor cell activities were depleted in high-density fractions. The latter fractions made vigorous responses in the MLR. The low-density fractions, which accounted for less than 10% of the input marrow cells, suppressed the MLR and did not respond. Further evaluation of the low-density fractions may be of value in allogeneic bone marrow transplantation due to the reduction of CD4+ and CD8+ T cells and the enrichment of hematopoietic progenitors as well as immune suppressor cells that may inhibit graft-versus-host disease.     

Concomitant T-cell receptor alpha and delta gene rearrangements in individual T-cell precursors.

A debate has recently surfaced concerning the degree of precommitment attained by alpha beta and gamma delta T-cell precursors prior to T-cell receptor (TCR) gene rearrangement. It has been suggested that precursors may be precommitted to rearrange either alpha or delta genes, but not both, thus giving rise to alpha beta- and gamma delta-producing T cells, respectively. Alternatively, the precursors may be flexible with regard to potential TCR gene rearrangements. To address this controversy, the gene rearrangements among a group of T-cell hybridomas from fetal, newborn, and early postnatal mouse thymi were examined. Six probes spanning the delta and alpha loci were used in Southern blot analyses to characterize the rearrangements which occurred on homologous chromosomes in each cell. Although homologous chromosomes often rearranged in synchrony within the alpha locus, a number of hybridomas were found which had retained a delta rearrangement on one chromosome and an alpha rearrangement on the second. Results show that a precommitment by T cells to rearrange delta or alpha genes in a mutually exclusive manner is not an absolute feature of mouse thymocyte development.     

Detection of clonal T-cell receptor gamma-chain gene rearrangements in Reed-Sternberg cells of classic Hodgkin disease

Recent molecular single-cell studies have shown that in approximately 95% of cases, Reed-Sternberg cells of classic Hodgkin disease (HD) are derived from B cells of germinal center origin. Attempts to determine the cellular nature of the remaining cases have so far failed. To clarify whether they are derived from T cells, this study examined 791 single CD30(+) Hodgkin and Reed-Sternberg (HRS) cells from 13 T-cell marker-positive cases and from 6 cases with null-cell phenotype for rearranged T-cell receptor-gamma (TCR-gamma) genes by single copy polymerase chain reaction. Monoclonally rearranged TCR-gamma genes were detectable in 2 of the 13 classic HD cases with T-cell marker-positive HRS cells, with none detectable in the null-cell cases. Eight of the T-cell marker-positive cases and all 6 null-cell cases were also studied for rearrangements of immunoglobulin genes. Six of the 8 T-cell marker-positive cases harbored clonal immunoglobulin gene rearrangements. The 2 cases without rearranged immunoglobulin genes were those that contained clonal TCR-gamma rearrangements and lacked expression of the B-cell-specific activator protein. From these findings we conclude that cases of classic HD with T-cell-derived HRS cells definitely exist, although their overall incidence at 1% to 2% is very low. Even within the T-cell marker-positive cases only a minority (15%) were derived from T cells. The majority (85%) originated from B cells, indicating that the T-cell antigens expressed by HRS cells are, in contrast to those expressed in non-Hodgkin lymphoma, not lineage specific.     

TCR gene rearrangements and expression of the pre-T cell receptor complex during human T-cell differentiation

Recent studies have identified several populations of progenitor cells in the human thymus. The hematopoietic precursor activity of these populations has been determined. The most primitive human thymocytes express high levels of CD34 and lack CD1a. These cells acquire CD1a and differentiate into CD4(+)CD8(+) through CD3(-)CD4(+)CD8(-) and CD3(-)CD4(+) CD8alpha+beta- intermediate populations. The status of gene rearrangements in the various TCR loci, in particular of TCRdelta and TCRgamma, has not been analyzed in detail. In the present study we have determined the status of TCR gene rearrangements of early human postnatal thymocyte subpopulations by Southern blot analysis. Our results indicate that TCRdelta rearrangements initiate in CD34(+)CD1a- cells preceding those in the TCRgamma and TCRbeta loci that commence in CD34(+)CD1a+ cells. Furthermore, we have examined at which cellular stage TCRbeta selection occurs in humans. We analyzed expression of cytoplasmic TCRbeta and cell-surface CD3 on thymocytes that lack a mature TCRalphabeta. In addition, we overexpressed a constitutive-active mutant of p56(lckF505) by retrovirus-mediated gene transfer in sequential stages of T-cell development and analyzed the effect in a fetal thymic organ culture system. Evidence is presented that TCRbeta selection in humans is initiated at the transition of the CD3(-)CD4(+)CD8(-) into the CD4(+)CD8alpha+beta- stage.     

Assessment of proliferation during maturation of the B lymphoid lineage in normal human bone marrow

Different stages of B lymphoid maturation were identified in normal bone marrow using multiple cell surface markers. The proliferation status of each of these maturational stages was determined by simultaneous quantitative DNA analysis on a flow cytometer. The technique used to quantify these parameters preserved the cell surface immunofluorescence, the light scattering properties and the stoichiometric binding to DNA. The proliferating cells were confined to a distinct population of cells expressing CD10. The number of proliferating cells in these populations was relatively constant among 12 separate bone marrow samples. The data suggest that the timing and rate of proliferation of cells within a single lineage may be a preprogrammed aspect of normal maturation.     

Malignant histiocytosis with T cell receptor and immunoglobulin gene rearrangements]

A case of malignant histiocytosis with rearrangements of both T-cell receptor and immunoglobulin genes. The patient was a 69 year-old woman suffering from high fever, which was unresponsive to the administration of various antibiotics and steroids for more than two weeks. Laboratory findings on admission revealed disseminated intravascular coagulopathy and liver dysfunction. The bone marrow examination showed an increased number of giant cells. Some of the giant cells had phagocytosis of various blood cells and were cytochemically stained with non-specific esterase, but not with myeloperoxidase and PAS. Immunohistochemical study revealed that alpha 1-antitrypsin alpha 1-antichymotrypsin, lysozyme and CD15 were all detected in the cytoplasm of some giant cells while CD30 was not detected. Of interest was the rearrangements of the T-cell receptor, Ig heavy chain and kappa chain genes on bone marrow mononuclear cells demonstrated by Southern blot analysis.     

Developmental hierarchy of immunoglobulin gene rearrangements in human leukemic pre-B-cells.

We have used a special class of human acute lymphocytic leukemias, the common "non-T/non-B" cell type, to define a hierarchy of genetic rearrangements that occur during the earliest stages of B-cell maturation. This has allowed us to identify intermediate cells predicted by a hierarchial model in which immunoglobulin heavy chain variable region gene formation precedes that of light chain and in which kappa light chain gene formation precedes that of lambda. The model emphasizes the flexible nature of immunoglobulin gene recombination that not infrequently produces aberrant or null genes that are phenotypically excluded from expression. Remaining alleles or isotypic genes can then be utilized as "spares" undergoing recombination until a valid gene is formed. Significantly, the excluded allele or isotype is frequently deleted from the genome. In addition to defining a pathway of genetic maturation, this analysis provides a powerful means to further classify cases of non-T/non-B-cell acute lymphocytic leukemia, most of which seem to reside at early stages along the B-cell pathway of differentiation.     

Heteroduplex analysis of T-cell receptor gamma gene rearrangements for diagnosis and monitoring of cutaneous T-cell lymphomas

The possibility to detect markers of T-cell clonality at the T-cell receptor (TCR) beta and gamma loci in skin biopsy samples has proven to be helpful for the often difficult clinical and immunohistochemical diagnosis of cutaneous T-cell lymphoma (CTCL). However, particularly at the early stage of the neoplastic infiltration, an emerging clonal pattern at Southern may be obscured by the germline TCR configuration of the predominant dermal and epidermal cell component. Additionally, multiple TCR gamma rearranged bands of variable intensity are often observed, either in the presence or in the absence of a major clone. To overcome these difficulties, we have investigated the T-lymphocyte clonality in selected patients with variable signs of CTCL by means of heteroduplex analysis of the amplified TCR gamma VJ junctions, separated in nondenaturing polyacrylamide gel. This technique has several advantages over standard Southern blot because it is simple, rapid, not radioactive, and likely more sensitive than other polymerase chain reaction-based procedures. In particular, the cases with uncertain or contradictory TCR beta and gamma patterns were solved by the heteroduplex analysis, showing homoduplex or heteroduplex bands of clonal nature. The direct sequence of the VJ junctions, easily obtained from the homoduplex or heteroduplex bands, allowed us to confirm the same clonal marker in two apparently different skin lesions and in different biopsy samples obtained from the same patients, either at the same or different time points, thus emphasizing the utility of this method in monitoring CTCL clinical progression.     

An in situ study of CD34(+) cells in human fetal bone marrow

The purpose of this study was to characterize the spatial distribution, number and size of CD34(+) cells in fetal bone marrow. Thin sections of normal fetal bone marrow from lumbar vertebrae were stained using CD34 antibody QBend/10. Sections were used under light microscopy with various eyepiece graticules to make measurements of CD34(+) cells in situ. Results showed that at mid- and late gestation, approximately 2% and 0.5% of fetal bone marrow cells were CD34(+) respectively. The mean distance of CD34(+) cells from the nearest trabecular bone surface was 61 +/- 4 and 46 +/- 4 microm, respectively, for mid- and late gestation. The mean distance to the nearest neighbour was 46 +/- 5 and 105 +/- 15 microm, and the mean distance to the nearest blood vessel was 13 +/- 1 and 17 +/- 2 microm respectively. The concentration of CD34(+) cells in the peripheral region was 6.5 times greater than that at the centre of the sections. Overall, the percentage number of CD34(+) cells decreased with gestational age. The cellular and nuclear diameters of CD34(+) cells remained unchanged throughout mid- and late gestation at 5.4 +/- 0.1 and 3.8 +/- 0.1 microm respectively. This information will be used to calculate the natural background alpha-radiation dose to haemopoietic stem cells.     

Dual rearrangement of immunoglobulin and T-cell receptor gene in a case of T-cell hairy-cell leukemia.

We report a case of T-cell hairy-cell leukemia with a dual rearrangement of Ig- and T-cell receptor genes. The cytochemical, transmission electron microscopy, and surface antigens data (CD3+, CD8+, CD11+, HLA-DR+, CD19-, CD20-) were consistent with a T-cell hairy-cell leukemia. Molecular analysis according to Southern revealed a dual rearrangement of immunoglobulin heavy-chain (JH) and T-cell receptor beta (TcR beta) chain genes. Our findings suggest that the coexistence of JH and TcR gene rearrangements, frequently detected in acute leukemia, may also be observed in hematologic malignancies derived from more differentiated cells.