Cell Receptor Gamma Research Articles

Characterisation of T-ALL Cells in Serial NOD/SCID Xenografts.

The treatment of paediatric T lineage ALL has improved considerably in recent years. However, a significant proportion of children continue to fail therapy and intensification of treatment regimens may only lead to an increase in the incidence of adverse risk effects. Therefore, it is necessary to increase our understanding of the early stages of development of this malignancy and its pathogenic mechanisms. In the current study we have established and characterised continuous xenografts from 6 patients with childhood T-ALL as a renewable source of leukaemia cells that retain the essential characteristics of the original disease. Unsorted cells from 6 children diagnosed with T-ALL were inoculated into the lateral tail vein of sublethally irradiated NOD/SCID mice. From two weeks post-inoculation onwards, engraftment levels in the peripheral blood (PB) were monitored by serial tail vein sampling and bone marrow (BM) aspirates were analysed from 4 weeks post inoculation. All animals were sacrificed no later than 10 weeks post inoculation. Human CD45+ cells were first detected 2 weeks after inoculation (1.6–5% CD45+) in animals injected with cells from two of the six patients. By week 4, CD45+ cells were detected in the PB from animals injected with cells from all 6 patients (2–22% CD45+) and higher levels were detected in the BM aspirates (5–52% CD45+). In each case, the levels of human cell engraftment increased with time and by week 10 significant levels of CD45+ cells were detected in NOD/SCID BM (30–99% CD45+). Flow cytometric and FISH analyses confirmed that the engrafted cells had a similar immunophenotype and karyotype as the diagnostic samples, confirming engraftment of leukaemia cells. BM cells harvested from the primary animals were subsequently used in serial transplantation studies to determine whether T-ALL could be transferred into serial recipients. To date, secondary and tertiary transplant experiments have resulted in similar levels of engraftment with no loss of NOD/SCID repopulating potential in all 6 cases. Cells from 2 patients have been used in quaternary, quinary and senary transplants with similar results (57–99% CD45+) and no changes observed in either the rate of engraftment nor the immunophenotype or karyotype of the passaged cells. Molecular analyses were performed on the diagnostic samples and cells from the established xenografts to screen for the presence of unique T cell receptor gamma (TCRG) gene rearrangements, present in approximately 95% of T-ALL patients. The PCR products were subjected to heteroduplex analysis for clonality assessment followed by sequencing of monoclonal products. The exact junctional rearrangement was established by comparison with all known human TCRG germline sequences. Results to date have indicated that the unique rearrangements detected at diagnosis were also found in cells harvested from primary, secondary and tertiary xenografts. Analyses of cells from successive xenografts are ongoing to determine whether the unique genotype remains stable. These findings demonstrate that the immunophenotype, karyotype and genotype of 6 paediatric T-ALL cases were preserved with multiple serial passages in NOD/SCID mice. These continuous xenografts retained the fundamental biological characteristics of the original disease and thus could be a valuable experimental model system to augment our knowledge of the biology and regulation of paediatric T-ALL and a credible tool for the preclinical evaluation of novel agents.

Presence of p16 hypermethylation and Epstein–Barr virus infection in transplant-associated hematolymphoid neoplasm of the skin

Epstein-Barr virus (EBV) is associated with the malignant transformation of B, T, and NK lymphocytes in humans, especially in immunosuppressed individuals. We describe an unusual case confined to the skin in a 39-year-old African American female following a renal transplant. Morphologically and immunophenotypically, the tumor was best classified as a plasmablastic lymphoma; however, the neoplastic population revealed rearrangements of both immunoglobulin heavy chain (IgG) and T cell receptor gamma (TCR-gamma). In situ hybridization demonstrated the presence of Epstein-Barr early RNA species (EBER) in the lymphoma cells, consistent with EBV infection. We have previously demonstrated that EBV-induced reactive oxygen is associated with hypermethylation of the tumor suppressor gene p16 in Burkitt lymphoma, and that p16 hypermethylation is nearly always associated with EBV infection in Burkitt lymphoma. Further studies are needed to determine whether p16 is widely suppressed in immunosuppression-induced lymphoma. In this study, we demonstrated high levels of hypermethylation of the tumor suppressor gene p16, thus supporting the role of EBV as a carcinogen in post-transplant lymphoproliferative disease.

IMGT/GeneInfo: T cell receptor gamma TRG and delta TRD genes in database give access to all TR potential V(D)J recombinations.

BackgroundAdaptative immune repertoire diversity in vertebrate species is generated by recombination of variable (V), diversity (D) and joining (J) genes in the immunoglobulin (IG) loci of B lymphocytes and in the T cell receptor (TR) loci of T lymphocytes. These V-J and V-D-J gene rearrangements at the DNA level involve recombination signal sequences (RSS). Whereas many data exist, they are scattered in non specialized resources with different nomenclatures (eg. flat files) and are difficult to extract.DescriptionIMGT/GeneInfo is an online information system that provides, through a user-friendly interface, exhaustive information resulting from the complex mechanisms of T cell receptor V-J and V-D-J recombinations. T cells comprise two populations which express the αβ and γδ TR, respectively. The first version of the system dealt with the Homo sapiens and Mus musculus TRA and TRB loci whose gene rearrangements allow the synthesis of the αβ TR chains. In this paper, we present the second version of IMGT/GeneInfo where we complete the database for the Homo sapiens and Mus musculus TRG and TRD loci along with the introduction of a quality control procedure for existing and new data. We also include new functionalities to the four loci analysis, giving, to date, a very informative tool which allows to work on V(D)J genes of all TR loci in both human and mouse species. IMGT/GeneInfo provides more than 59,000 rearrangement combinations with a full gene description which is freely available at .ConclusionIMGT/GeneInfo allows all TR information sequences to be in the same spot, and are now available within two computer-mouse clicks. This is useful for biologists and bioinformaticians for the study of T lymphocyte V(D)J gene rearrangements and their applications in immune response analysis.

Bovine T cell receptor gamma variable and constant genes: combinatorial usage by circulating γδ T cells

Studies here describe expression and sequence of several new bovine T cell receptor gamma (TRG) genes to yield a total of 11 TRG variable (TRGV) genes (in eight subgroups) and six TRG constant (TRGC) genes. Publicly available genomic sequences were annotated to show their placement. Homologous TRG genes in cattle and sheep were assigned, using four accepted criteria. New genes described here include the bovine TRGC6, TRGV2, and TRGV4, homologues of ovine TRGC4, TRGV2, and TRGV4, respectively. The bovine Vgamma7 and BTGV1 clones (previously TRGV4 and TRGV2, respectively) were reassigned to new subgroups TRGV7 and TRGV8, respectively, with approval by the IMGT Nomenclature Committee. Three TRGV subgroups (TRGV5, TRGV6, and TRGV8) were further designated as TRGV5-1 and TRGV5-2, TRGV6-1 and TRGV6-2, and TRGV8-1 and TRGV8-2 because each subgroup is comprised of two mapped genes. The complete sequence of bovine TRGC5 is also reported, for which a limited number of nucleotides was previously available, and shown to be most closely related to ovine TRGC5. Analysis of circulating gammadelta T cells revealed that rearrangement of TRGV genes with TRGC genes is largely dictated by their proximity within one of the six genomic V-J-C cassettes, with all TRG genes expressed by bovine peripheral blood gammadelta T cells. Cattle are useful models for gammadelta T cell biology because they have gammadelta T cells that respond to isopentenylpyrophosphate (IPP) antigens, while mice do not, and some bovine TRGV genes cluster closely with human genes.

Effect of age on the repertoire of cytotoxic memory (CD8+CD45RO+) T cells in peripheral blood: The use of rearranged T cell receptor γ genes as clonal markers

We have established a method to estimate the number of clones in peripheral blood, using rearranged T cell receptor γ genes as clonal markers, selecting cells at random, and establishing the sizes of the clones to which they belong. Clone sizes were quantified by a clone-specific PCR test based on the VNJ junctional sequence, which typically detects 1–2 copies of its target gene. All clones chosen for study were subsequently quantified in blood, and sizes ranged from 3 × 10 − 6 (1 cell in 330,000 CD8+CD45RO+ cells) to 3.5 × 10 − 2 permitting numbers of clones to be estimated from the harmonic mean of clone size. Two independent estimates from a healthy young adult (20–30 years old) gave repertoires of 94,000 and 110,000 clones. Two other healthy young adults gave repertoires of 40,000 and 55,000 clones. Repertoires in four healthy active older (> 75 years old) adults were more variable but generally lower, being 3600, 5500, 14,000 and 97,000 clones, despite enlarged clones making up > 1% of the compartment in the last individual. Overall, young adults had smaller clones ( p = 0.026, non-directional Mann–Whitney U-test). If the human body contains 5 l of blood, clones have 2 × 10 3–1.0 × 10 7 cells in blood. These results confirm a diverse repertoire of rearranged T cell receptor gamma genes. The number of clones thus defined are broadly consistent with other estimates of repertoire, despite differences in marker genes used and subsets of cells studied.

Long-Term Follow-Up of Patients with T-Large Granular Lymphocyte Leukemia (T-LGL); Experience in a Single Institution.

T-LGL is a clonal indolent lymphoid leukemia characterized by accumulation of large granular T-cells and cytopenias. We have identified 21 patients with T-LGL who were seen at the MD Anderson Cancer Center between 1997 and 2005. The median age at diagnosis was 53.5 years (range 34 to 72) without a male or female predilection. Fourteen patients (67%) were asymptomatic at diagnosis. Five patients (24%) presented with fatigue secondary to anemia; 1 (5%) had symptomatic splenomegaly and 1 presented with a diverticular abscess. Eighty percent of the patients presented with anemia with median hemoglobin of 9.9g/dl (range 7.4 to12.9 g/dl), 47% with neutropenia with a median absolute neutrophil count of 975/ul (range 110 to 5600/ul), and 38% had thrombocytopenia with a median platelets count of 61.5 x 109/l (range18 to 135 x 109/l). Six patients (29%) had splenomegaly, 3 (14.2%) had hepatomegaly and 1 had retroperitoneal lymph node enlargement on CT. During the course of follow-up (median 19 months, range 2–78 months), 11 patient (52%) developed constitutional symptoms. Six patients had recurrent infections including upper respiratory infections, pneumonia, and folliculitis unrelated to initiation of treatment. Associated autoimmune conditions included rheumatoid arthritis in 3 patients (14%) and hemolytic anemia in one patient. Only two patients had cytogenetic abnormalities, both including abnormalities involving chromosome 6. Monoclonal T cell receptor gamma chain gene rearrangements were detected by PCR analysis in 17 of 18 evaluable patients. Immunophenotypic studies revealed 16 patients to be CD4−/CD8+, 3 CD4−/CD8−, 1 CD4+/CD8+, and 1 CD4+/CD8−. CD56 was positive in 1 patient and none expressed CD26. Five patients (24%) have required no therapy, and remain asymptomatic with a median follow up of 67 months (range 5 to 78). Three patients received erythropoietin or G-CSF with some improvement. Nine patients (43%) were treated with cyclosporine, 2 achieving a response, 2 with stable disease, 3 with progression, and 2 were lost follow up. One of the 3 patients with disease progression had splenectomy and 1 was treated with infliximab; both responded. Combination of pentostatin and alemtuzumab was used in 2 patients with no improvement in the cytopenias; therapy was discontinued in both due to worsening of cytopenias and infections. Other treatment modalities included alemtuzumab alone in 1 patient with no improvement, fludarabine in 1 with minor response, combination of fludarabine and cyclophosphamide in 1 with minor response, methotrexate in 2 with no response and splenectomy in 3 with response and no requirements for further intervention. We conclude that T-LGL generally has an indolent course with no need for active therapy; patients with progressive cytopenias or refractory disease may benefit from splenectomy. New treatment modalities are needed.

Comparative analyses of sheep and human TRG joining regions: Evolution of J genes in Bovidae is driven by sequence conservation in their promoters for germline transcription

The availability of genomic clones representative of the T cell receptor gamma ( TRG1@ and TRG2@) ovine loci enabled us to compare the germline genomic organization and nucleotide diversity of joining ( J) segments and reconstruct their evolutionary history by phylogenetic analysis of cattle, sheep and human expressed sequences. Expression profiling (RT-PCR data) in fetus and adult indicated that only the ovine J genes in which two or more of the key sequence features, such as recombination signal sequences (RSS), 3′ splice sites, and core sequences, are missing or severely altered fail to be transcribed. Comparative genomic examination of the two human with the six sheep germline transcription promoters located at 5′ of the relevant constant ( C)-distal J segments showed a strong conservation of the redundant STAT consensus motifs, indicating that TRG1@ and TRG2@ loci are under the influence of IL-7 and STAT signalling. These findings support the phylogenetic analysis of human and Bovidae (cattle and sheep) that revealed a different grouping pattern of C-distal compared to C-proximal J segments. Likewise, the phylogenetic behaviour of either C-distal and C-proximal J segments is in accordance with the Bovidae TRG clusters evolution. Comparison of sheep and human structures of recombination signal sequences (RSS) has highlighted a greater conservation in sheep 12 RSS rather than 23 RSS thus suggesting that the initial recruitment of recombination activating genes (RAG) products requires at least one relatively high-affinity RSS per recombination event.

Characterization of feline T cell receptor gamma ( TCRG) variable region genes for the molecular diagnosis of feline intestinal T cell lymphoma

A diagnosis of intestinal lymphoma is currently made on the basis of clinical and morphologic criteria. This can prove problematic for many reasons that include inadequate sample size, the coexistence of lymphoma and inflammation, and the inability to assess architectural integrity of all tissue compartments in biopsy specimens obtained endoscopically. The detection of a clonal population of cells in a lymphoproliferative lesion represents an important criterion for the diagnosis of neoplasia, but this has not been assessed in feline intestinal lymphoma. T cell receptor gamma ( TCRG) gene rearrangement analysis using polymerase chain reaction (PCR) is a methodology that can be used to detect clonality in T cell populations. The basis of this assay depends on the assessment of the junctional diversity that results from rearrangement of TCRG V (variable) and J (joining) gene segments. Feline TCRG transcripts from normal small intestine and spleen were obtained using a rapid amplification of cDNA ends (5′RACE) method. Limited diversity of TCRG V and J gene segments was observed. The high degree of sequence homology in the TCRG V and J gene segments was exploited to develop a PCR test for the assessment of TCRG V–J junctional diversity and hence clonality determination of T cell populations in cats. Molecular clonality determination was applied to feline intestinal lymphoplasmacytic inflammatory bowel disease (IBD) (9 cats), and transmural and mucosal T cell lymphoma (28 cats). Clonal rearrangement of the TCRG V–J junction was detected in 22 of 28 intestinal T cell lymphomas, and oligoclonality was detected in 3 intestinal T cell lymphomas. This contrasted with the detection of polyclonal rearrangement in normal intestinal tissues (3 cats) and in lymphoplasmacytic IBD (9 cats). It is proposed that assessment of TCRG V–J junctional diversity for the detection of clonality represents an important adjunctive tool for the diagnosis of T cell lymphoma in the cat.

A rare case of the cutaneous form of adult T-cell leukaemia/lymphoma: assessment of remission by PCR for clonal T-cell receptor gamma gene rearrangements in an electron beam-irradiated cutaneous lesion

Adult T-cell leukaemia/lymphoma is a lymphoproliferative disorder aetiologically associated with human T-cell lymphotropic virus type I infection. A cutaneous lesion often develops in the disease, and in rare cases, is even the only manifestation. Here we report a rare case of 'cutaneous' adult T-cell leukaemia/lymphoma with neither atypical cells in the peripheral blood nor lymph node involvement. All nodular lesions were completely eliminated after local electron beam irradiation (20 Gy/nodule in total). To evaluate whether or not there were residual lymphoma cells in the skin, we performed PCR to detect clonal T cell receptor gamma gene rearrangements. The sample from the nodule before irradiation showed evidence of a rearranged band, which was not detected at the same site after treatment nor in any peripheral blood. The findings suggest that this procedure is useful for the evaluation of therapeutic effects and the early detection of lymphoma recurrence.

Concurrent Mycosis Fungoides and Precursor B Cell Lymphoblastic Lymphoma in a 6‐Year‐Old Child

We report a 6-year-old boy with mycosis fungoides on the dorsal surface of his left hand, a diagnosis supported by positive T cell receptor gamma gene rearrangement findings. Precursor B cell lymphoblastic lymphoma of the left orbit developed subsequently. He was treated with chemotherapy and remained under control.

Cutaneous Langerhans Cell Sarcoma Associated with Mediastinal T‐Cell Acute Lymphoblastic Lymphoma: Clonally Related Neoplasms

An 8‐year‐old male presented with several 1–4 cm, indurated, brownish‐red nodules with superficial, punctate hemorrhagic crusts localized to the groin, which developed over several weeks. An initial biopsy was consistent with Langerhans cell histiocytosis. A work up, including a chest and abdominal CT scan demonstrated a large mediastinal mass which was diagnosed as precursor T cell lymphoblastic lymphoma. Treatment with a chemotherapeutic protocol resulted in shrinkage of both the mediastinal and cutaneous lesions. Late in the course of treatment the cutaneous lesions rapidly recurred and spread to various sites. A second skin biopsy showed an extensive diffuse infiltrate of histiocytic‐appearing cells extending from the papillary dermis into the superficial subcutis. The cells had markedly pleomorphic, vesicular nuclei and abundant amphophilic cytoplasm. Numerous mitotic figures were present. The neoplastic cells were S‐100 protein and CD1a positive by immunohistochemistry. The histopathologic diagnosis was Langerhans cell sarcoma. Gene rearrangement studies demonstrated clonal rearrangement of the T cell receptor gamma gene in specimens from both the mediastinal mass and one of the recurrent cutaneous lesions. The sizes of the PCR products were identical demonstrating a clonal relationship between the two neoplasms.

Analysis of Lymphocyte Monoclonality in Patients with Sjögren's Syndrome Associated with Lymphoproliferative Disorder or Lymphoma.

Purpose: Among 450 Sjögren's syndrome (SS) patients in our hospital, we investigated 27 cases of malignant lymphoma (ML). In many reports, the risk of developing ML is higher, for example 43.8 times greater in patients with SS than in a comparable normal population in Kassan, et al, and 47.8 times greater in our Cases. Besides MALT lymphoma, other types of nodal lymphomas have been observed in patients with SS. To clarify the relationship between lymphocyte monoclonality and the development of the lymphomas in SS, we performed a clonality analysis of the tissues from salivary glands and lymphoproliferative organs.Method. Clonality analysis was applied to lymphocytes in SS-related lymphoproliferative tissues from 3 patients who had lymphoproliferative disorder and were observed sequentially. We also compared the clonality between minor salivary gland and lymphoma tissues from 6 SS patients with nodal lymphoma. PCR amplification, cloning and sequencing were performed to identify immunoglobulin heavy chain complementarity-determining region 3 (IgH-CDR3) and T cell receptor-gamma (TCR-γ) gene rearrangement.Results. In the 3 patients with sequential observation, B cell proliferation progressed from polyclonal or oligoclonal into monoclonal expansion in the salivary gland or extraglandular tissues. In one of these patients, lymphoma development was seen in the salivary gland. In all of the 6 SS patients with nodal lymphomas, the B cell clone in the salivary glands were different from those in the lymphoma tissues.Conclusion. In patients with SS, there were two patterns of lymphoma progression. One was the multi-step process in which polyclonal B cell expansion evolves into monoclonal disease in extra-nodal lymphomas in salivary or lacrimal glands. The other was the independent clonality between salivary glands and lymphomas in patients with nodal lymphomas, and SS may play a role in the development of nodal lymphoma. Considering that the microenvironments of lymph nodes and salivary glands are different, the mechanism of the development of the extra-nodal lymphomas and the nodal lymphomas may be different.

Pathologic diagnosis and subtyping of lymphoma in bone marrow biopsies using histologic examination, immunohistochemistry and gene rearrangement studies

To assess the value of histologic examination, immunohistochemistry and gene rearrangement studies in the diagnosis and subtyping of lymphoma with bone marrow involvement (BMI). Sixty-two formalin fixed, paraffin embedded bone marrow biopsy specimens were studied. Immunohistochemical and immunoglobulin heavy chain (IgH) and T-cell receptor gene rearrangement studies were performed in each case. Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) demonstrated mainly and interstitial infiltration by dysplastic lymphocytes, with intertrabecular nodular arrangement or in dispersion. Sometimes, pseudofollicles may be noted. A predominantly para- or intertrabecular infiltration by nodules of lymphoma cells was characteristic of follicle center cell lymphoma (FCL) cases. In most lymphoplasmacytoid lymphoma (LPL) cases, there was infiltration by small lymphocytes and plasma cells between bony trabeculae. In marginal zone cell lymphoma (MZL), vague inter- or para-trabecular nodules of polymorphic lymphoma cells with clear cytoplasm might be noted. Small to medium-sized dysplastic lymphocytes, with absence of paraimmunoblasts or pseudofollicles, were the most frequent findings in mantle cell lymphoma (MCL). Hairy cell leukemia (HCL) might be identified by the presence of distinct cell membrane and abundant clear cytoplasm, resulting in a "fried-egg" appearance. Tumor cells with large nuclei and eosinophilic nucleoli were characteristically seen in lymphomatosis diffusa (Hodgkin's disease, HD). In T-cell non-Hodgkin lymphoma with BMI, dispersed or clusters of intertrabecular neoplastic lymphoid cells with clear cytoplasm and gyriform nuclei were often observed. In diffuse large B-cell lymphoma (DLBL), the tumor cells were large and isolated or arranged in diffuse pattern. Immunohistochemically, a panel of markers, including CD3 CD20, and CD79 are valuable for the differential diagnosis of T- and B-cell lymphomas. The neoplastic cells in MCL were cyclin D1- and CD5-positive, while BCL2- and CD10-positivity was characteristic for FCL. CLL/SLL cells might be stained with CD5 and CD23, in addition to CD20 and CD79. CD25 expression might be noted in HCL: the positivity for CD15, CD30 and fascin suggests HD. There was a higher positivity rate for IgH gene rearrangement in CLL/SLL, LPL MZL and DLBL (80%, 60%, 66.7%, 70% respectively) and for T- cell receptor gamma gene rearrangement in T-cell lymphoma (66.7%). A combination of histopathology, immunohistochemistry and IgH / T-cell receptor gamma gene rearrangement studies may be of aid to the diagnosis and subtyping of lymphoma with BMI, especially if there is only a small number of tumor cells present in the specimen.

T cell receptor gamma ( TCRG) gene rearrangements in Brazilian children with acute lymphoblastic leukemia: analysis and implications for the study of minimal residual disease

The high frequency of T cell receptor gamma ( TCRG) gene rearrangements in both B-lineage and T cell acute lymphoblastic leukemia (ALL), its easy detection and the lower incidence of oligoclonality make this gene one of the main target for the detection of minimal residual disease by PCR in childhood ALL. We analyzed the frequency and type of TCRG rearrangements in DNA samples obtained from the bone marrow of 102 Brazilian children at diagnosis using PCR and automatic sequencing. TCRG rearrangements were found in 69% of patients with B-lineage ALL and in 94% of patients with T cell ALL. In contrast to other studies, rearrangements involving the Vγ9 segment reported to be uncommon were the most frequent both in B-lineage and T cell ALL and involved 49/109 (45%) of the rearranged alleles. This fact should be considered when standardizing consensus primers for the study of minimal residual disease in different populations.

T Cell Receptor-γ Gene Analysis of CD30+ Large Atypical Individual Cells in CD30+ Large Primary Cutaneous T Cell Lymphomas

The hallmark of primary cutaneous CD30+ large T cell lymphoma are large lymphoid tumor cells, at least 75% of which, by definition, must be positive for CD30. The relatively benign clinical course of this lymphoma type has been explained with CD30-induced apoptosis, on the assumption that expression of CD30 defines the tumor clone; however, this hypothesis has not been tested on the molecular level to date. In this study we analyzed CD30+ cells in four patients with primary cutaneous CD30+ large T cell lymphoma by single cell polymerase chain reaction of T cell receptor-gamma genes followed by sequencing. Here, we demonstrate that most of the large CD30+ atypical cells possessed identical T cell receptor-gamma gene rearrangements, indicative of clonal proliferation. Nevertheless, polyclonally rearranged T cells were present in all CD30+ samples studied. In addition, one patient showed a second clone in a separate biopsy and three of four patients showed chromosomal imbalances as revealed by comparative genomic hybridization. Taken together, our data suggest that the CD30+ population in primary cutaneous CD30+ large T cell lymphoma indeed contains the tumor clone, thus providing molecular support for a link between clinical course and CD30-related signaling. Importantly, however, CD30 expression does not define the tumor clone as bystander T cells, as well as occasional additional clones, are also present in this population.

Identification of amplified clonal T cell populations in the blood of patients with chronic graft-versus-host disease: positive correlation with response to photopheresis.

Chronic graft-versus-host disease (cGVHD) is a multiorgan disorder with skin manifestations resembling scleroderma. Since photopheresis, a treatment that induces an anticlonotypic immune response, has proven to be effective in both cutaneous T cell lymphomas with circulating clonal T cells and in cGVHD, we have searched for circulating clonal T cell populations in patients with cGVHD, and determined whether T cell clonality in the blood is associated with therapeutic response. We screened blood samples from 27 patients after HLA-matched allogeneic bone marrow transplantation (allo-BMT), 10 without cGVHD and 17 with extensive cGVHD, for clonal T cell receptor gamma (TCR gamma) gene rearrangements using fluorescent-based polymerase chain reaction (PCR) and automated high-resolution capillary electrophoresis. Amplified populations of clonal T cells with unique TCR gamma gene rearrangements were found in six of 10 (60%) allo-BMT patients without cGVHD and 13 of 17 (76.5%) allo-BMT patients with cGVHD (P = 0.41), as compared to none of 10 (0%) healthy controls. Twelve patients with cGVHD were treated by photopheresis, and the presence of amplified populations of clonal T cells was found to be associated with a cutaneous response to photopheresis, as eight of eight (100%) clone-positive vs none of four (0%) clone-negative patients experienced a clinically significant cutaneous response to treatment (P = 0.001). Our findings suggest that patients with cGVHD that have detectable expanded clonal T cell populations in their peripheral blood, may be more likely to respond to treatment by photopheresis.

Combined analysis of T cell receptor gamma and immunoglobulin heavy chain gene rearrangements at the single-cell level in lymphomas with dual genotype.

By prospectively studying immunoglobulin heavy chain gene (IgH) and T cell receptor gamma (TCRgamma) gene rearrangements in 398 lymphoma cases, a dual genotype was observed in 13% of B cell and 11% of T cell lymphomas. According to histological subtype, the highest incidence was observed for mantle cell lymphomas (32%) and lymphoplasmacytic lymphoma (21%) among B cell lymphomas, and for angioimmunoblastic lymphoma (AILT) (46%) and Sézary syndrome (SS) (50%) among T cell lymphomas. To determine whether the dual genotype corresponds to the presence of two distinct monoclonal populations or to the presence of both rearrangements within the same lymphoma cells, single-cell microdissection was used after immunohistochemistry and a single-cell combined IgH and TCRgamma gene analysis was designed after a whole-genome amplification step. This protocol was applied to the study of two nodal B cell lymphomas (one diffuse large B cell lymphoma and one mantle cell lymphoma) and two cutaneous T cell lymphomas (one AILT and one SS). Two cases (SS and mantle cell lymphoma) were true bigenotypic lymphomas, as both IgH and TCRgamma monoclonal rearrangements were detected in the same cells. Conversely, in the diffuse large B cell lymphoma and AILT cases, large CD22+ single cells exhibited only the monoclonal IgH rearrangement but not the TCRgamma gene that was detected in CD3+ single cells. Such an approach allows the identification of true bigenotypic lymphoma among dual genotypic lymphoma. Specific genetic alterations may be further amplified from microdissected cryopreserved material, such as the t(11;14) breakpoint detected in bigenotypic B cells of the mantle cell lymphoma case.

T cell receptor gamma gene rearrangements as targets for detection of minimal residual disease in acute lymphoblastic leukemia by real-time quantitative PCR analysis.

Several studies have shown that quantitative detection of minimal residual disease (MRD) predicts clinical outcome in childhood acute lymphoblastic leukemia (ALL). In this report we investigated the applicablility of T cell receptor gamma (TCRG) gene rearrangements as targets for MRD detection by real-time quantitative PCR analysis. Seventeen children with precursor-B-ALL and 15 children with T-ALL were included in this study. Using an allele-specific (ASO) forward primer in combination with germline Jgamma reverse primers and Jgamma TaqMan probes, a reproducible sensitivity of < or =10(-4) (defined by strict criteria) was obtained in only four out of 19 (21%) TCRG gene rearrangements in precursor-B-ALL patients and in 10 out of 15 (67%) TCRG gene rearrangements in T-ALL patients. The main reason for not obtaining a reproducible sensitivity of < or =10(-4) in approximately 60% of cases was the non-specific amplification of TCRG gene rearrangements in normal T-lymphocytes. A maximal sensitivity of < or =10(-4) (defined by less strict criteria) was obtained in 42% of TCRG gene rearrangements in precursor-B-ALL patients. The number of inserted nucleotides was significantly higher in T-ALL (mean: 8.5) as compared to precursor-B-ALL (mean: 6.8) and appeared to be the most important predictor for reaching a reproducible sensitivity < or =10(-4). The usage of a touchdown PCR or the usage of an ASO reverse primer in combination with Vgamma member forward primers and TaqMan probes did not clearly improve the overall results. Nevertheless, RQ-PCR analysis of TCRG gene rearrangements in follow-up samples obtained from 12 ALL patients showed the applicability of this method for MRD detection. We conclude that RQ-PCR analysis of TCRG gene rearrangements can be used for the detection of MRD, but that sensitivities might be limited due to non-specific amplification. This method is applicable in the majority of T-ALL patients and in almost half of precursor-B-ALL patients, particularly when used as second-choice target for confirmation of the MRD results obtained via the first-choice target.

Lymphoid Tissues from Patients with Infectious Mononucleosis Lack Monoclonal B and T Cells

In typical cases of infectious mononucleosis (IM), lymphoid tissue is rarely submitted for pathological examination. When lymphoid tissues from IM cases are examined, the histological appearance of IM may be difficult to distinguish from malignant lymphoma. The purpose of this study was to address the utility of clinical molecular assays for T and B cell clonality in distinguishing IM from lymphoid malignancy. DNA was recovered from paraffin-embedded archival lymphoid tissues of 18 cases of IM and 13 control cases representing other reactive lymphoid hyperplasias. T cell receptor gamma (TCR-gamma) and immunoglobulin heavy chain (IgH) gene rearrangements were assayed using our standard clinical polymerase chain reaction procedures targeting each of the four functional variable (V) families and the three joining (J) families of the TCR-gamma gene, and framework III of the IgH gene, respectively. In 17 of 18 cases of IM, no monoclonal T or B cell populations were detectable. One case, the only spleen specimen in the study, had an oligoclonal pattern of TCR-gamma rearrangements. The control cases representing other reactive hyperplasias also lacked monoclonality. The assays used were sensitive to clonal populations as small as 5% of cells. In this case series, no monoclonal lymphoid populations were identified in any case of IM. This finding suggests that molecular studies are useful in distinguishing IM from lymphoid neoplasms.

Lymphocyte development in neonatal and adult c-Kit-deficient (c-KitW/W) mice.

Hematopoietic stem cells and lymphocyte progenitors express the receptor tyrosine kinase c-Kit. In fetal and neonatal life, c-Kit plays a redundant role in T, and no apparant role in B cell development. In neonatal mice deficient for both c-Kit and the common gamma chain (gammac), a component of the interleukin-7 (IL-7) receptor, the thymus is alymphoid, and therefore lacks T cell receptor (TCR) beta, gamma, and delta rearrangements. Thus, a critical role for c-Kit in T cell development around birth is well established. More recently, it has become possible to examine the impact of c-Kit deficiency under conditions of steady state lymphopoiesis in adult life. Such analysis has been made possible by the identification of a viable adult c-Kit-deficient (c-KitW/W) variant, termed the Vickid mouse. The Vickid mouse arose by outcrossing c-KitW-bearing mice of the WB strain, in which lack of c-Kit is lethal, to a mixed genetic background. In adult Vickid mice, mainstream alphabeta TCR+ thymocyte development, and B cell development in the bone marrow are severely c-Kit-dependent with progressive age. Analysis of other pathways of developing T cells, i.e. CD4-CD8- (double neagative [DN]) alphabeta TCR+ and DN gammadelta TCR+ thymocytes revealed that the development of both lineages is also severely affected by lack of c-Kit. However, numbers of gammadelta TCR+ T cells decline before numbers of alphabeta TCR+ T cells in the thymus. In contrast to T and B cell development, generation of NK cells is not affected in adult c-KitW/W mice.