Beta Chain Gene Rearrangements Research Articles

Distinct T Cell Receptor Gene Repertoires and T Cell Subset Distribution in Peripheral Blood and Bone Marrow of Patients with Multiple Myeloma

Mounting evidence suggests that the T cell repertoire in multiple myeloma (MM) is actively shaped by antigen selection. However, the molecular underpinnings of this observation are not fully understood, while relevant studies have been mostly based on immunophenotypic characterization of T cell subsets, with considerably less information regarding T cell receptor (TR) gene repertoire profiles. Moreover, most available information derives from the analysis of a single tissue compartment [i.e. peripheral blood (PB) or bone marrow (BM)], thus hindering addressing the potential impact of local T cell responses. Here we sought to overcome these limitations through paired analysis of PB and BM aspirates of 20 newly diagnosed patients with MM before the administration of any treatment, from whom we isolated the PB and BM mononuclear cell (PBMC/BMMC) fraction, as well as CD4 + and CD8 + T cells. TR beta (TRB) chain gene rearrangements were RT-PCR amplified on RNA extracted from CD4 + and CD8 + T cells and then subjected to paired-end next generation sequencing (NGS). Raw reads (n=14,369,410 | median 179,618/sample) were processed through a purpose-built bioinformatics pipeline. Productive TRBV-TRBD-TRBJ gene rearrangements were taken into consideration (n=9,212,507 | median 115,156/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 1,005,150 TRΒ distinct clonotypes were assessed (median=12,565/sample). Both CD4 + and CD8 + T cell populations displayed skewed TRBV repertoires (7 TRBV genes and 3 TRBJ genes accounting for about ~40% and ~60% of the repertoire) in both BM and PB. All patients displayed oligoclonal T cell expansions in both CD4 + and CD8 + T cells, albeit these were significantly (p<0.001) more pronounced in CD8 + T cells for both BM and PB samples. That said, oligoclonality was more pronounced in BM vs. PB for both CD4 and CD8 T cells, reaching statistical significance in the former: in particular, the median cumulative frequency of the most expanded clonotypes/sample was ~40% higher in CD4 + BM T cells vs. CD4 + PB T cells (p<0.001). Additionally, a significant shift was noticed in the major clonotype repertoire of CD4 + and CD8 + T cells in both PB and BM. In more detail, the 10 most expanded clonotypes/sample in PB had significantly lower frequency in BM, and, vice versa, the 10 most expanded clonotypes in BM were represented at diminished frequency or were absent in the PB in 12/20 patients. Flow cytometry analysis revealed distinct T cell subset composition in CD4 + or CD8 + T cells, also in different tissue compartments (BM vs. PB). Significant (p<0.001) differences concerned the over-representation of: (i) effector T cells (CD45RA +/CCR7 -, Temra) in CD8 + vs. CD4 + T cells in both BM and PB (median frequencies in BM and PB: 22.5% and 7% for CD8 + T cells; 6% and 1.6% for CD4 + T cells); (ii) CD8 + Temra cells in BM vs. PB (median frequencies of 22.5% vs. 7% in BM and PB, respectively; p<0.001); (iii) CD4 + vs. CD8 + central memory T cells (CD45RO +/CCR7 +, Tcm) in both PB and BM (median frequencies in PB and BM: 42.7% and 49.1% for CD4 + Tcm and 14% and 18.8% for CD8 + Tcm); (iv) CD8 + effector memory T cells (CD45RO +/CCR7 -, Tem) in BM vs. PB (median frequencies of 21.8% vs. 2.7% in BM and PB, respectively); (v) CD8 + naïve T cells (CD45RA +/CCR7 +, Tn) in PB vs. BM (median frequencies of 67.7% vs. 24.2% in PB and BM, respectively). In conclusion, oligoclonal expansions of CD4 + and, particularly, CD8 + T cells in MM that are more pronounced in the BM (at least for CD4 + cells) argue for selection by BM-biased antigens, a claim also supported by the significant differences in the relative frequency of dominant clonotypes in BM vs. PB. The distinctive T cell subset distribution in the BM, characterized by a significant increase of effector at the expense of memory T cells, likely reflects an antigen-driven albeit ineffective immune response.

T Cell Epitope Prediction within the Clonotypic Immunoglobulin Heavy and Light Chains in Patients with Multiple Myeloma

Despite remarkable therapeutic advances in recent years, multiple myeloma (MM) remains incurable, hence representing an unmet clinical need. Therapeutic approaches based on induced T cell anti-tumor immunity towards cancer eradication show promising results in many hematologic malignancies, including multiple myeloma (MM), highlighting the need for comprehensive understanding of the implicated mechanisms. Similar to other mature B cell malignancies, immunoglobulin (IG) gene rearrangements in MM lead to the expression of unique, novel peptide sequences that can be used as neoantigens, conceivably representing a potential attractive target for cancer-specific immune responses. Here we sought to explore this possibility by performing ad hoc prediction of putative T-cell class I/II neoepitopes contained within the MM clonotypic IG gene rearrangements. To that end, we investigated 16 newly diagnosed patients with MM from whom we isolated the peripheral blood and/or bone marrow mononuclear cell (PBMC/BMMC) fraction as well as CD138+ myeloma cells, CD4+ and CD8+ T cells. Clonotypic IG heavy and light chain gene rearrangements were RT-PCR amplified on RNA extracted from the PBMC/BMMC fraction using subgroup-specific leader primers for the IGHV/IGK/LV gene and universal primers annealing to the constant domain (IGHG, IGHM, IGKC, IGLC), in order to produce the full-length V-(D)-J gene rearrangement sequence, plus the start of the constant domain. The corresponding sequences were determined by bidirectional Sanger sequencing. Moreover, T cell receptor beta (TRB) chain gene rearrangements were RT-PCR amplified on RNA extracted from CD4+ and CD8+ T cells and then subjected to paired-end next generation sequencing (NGS). The deduced amino acid sequences of the IG heavy and light chain variable/constant domains were subsequently parsed in peptides and subjected to bioinformatics analysis for the identification of putative T-cell class I/II neoepitopes using the NetMHCpan and NetMHIICpan softwares. The rank score was calculated, considering the 4-digit HLA-A, -B, -C and DRB1 typing for each individual patient. High- and medium-binding peptides (rank score <2%) were selected. Prediction of the binding specificity of T Cell Receptors (TR) to MHC-peptide complexes (pMHCs) was performed by the ERGO-II tool. The rank score was calculated, considering the peptides resulted by NetMHCpan/ NetMHIICpan, the HLA, and the clonotypes for each patient (AUC>0.8). Exact matches to germline and/or proteome databases were excluded. Overall, 757,263 TRΒ distinct clonotypes were assessed (median= 18,244 /sample). All patients displayed oligoclonal T cell expansions in both CD4+ and CD8+ T cells, albeit these were significantly (p<0.001) more pronounced in the latter. Both T cell subpopulations displayed skewed TRBV and TRBJ gene repertoires (7 TRBV genes and 3 TRBJ genes accounting for about 40% and 59% of the repertoire, respectively). Overall, 1,219 predicted neoepitopes were identified. All patients had predicted CD4+ and CD8 + T-cell epitopes within the MM clonotypic IG, either in the heavy chain (16/16 pts, n=459 epitopes) or the light chain (16/16 pts, n=760 epitopes). There was no statistically significant difference in the rank score of peptides involving the complementarity determining region 3 (CDR3) vs. all other IG regions. Interestingly, 998/1,219 (82%) peptides with strong binding affinity resulted from IG gene sequence motifs outside the CDR3. Most of these peptides (853/1,219, 75%) resulted from somatic hypermutations (SHM) across the IG variable domain. Overall, 51,574 clonotypes of CD4+ T cells and 35,751 clonotypes of CD8+ T cells were in silico predicted by ERGO to have strong binding affinity. Relevant to mention, 528 clonotypes with high binding score were found to be shared between CD4+ T cells of all patients and 209 clonotypes between CD8+ T cells of all patients and, thus, were deemed as public. In conclusion, in silico prediction identified a significant number of putative T-cell class I/II neoepitopes contained within the clonotypic IG of MM patients. Many of the identified peptides derive from SHM, implying that the SHM which shapes the MM BcR IG repertoire may produce immunogenic CD4+/CD8+ T cell epitopes. Their actual immunogenicity has to be tested in ex vivo studies, currently underway by our group.

High Throughput Immunoprofiling of Low Count B Lymphocytosis Reveals a Distinct T Cell Receptor Repertoire from Chronic Lymphocytic Leukemia

Mounting evidence supports the notion that T cells are crucial for the survival and expansion of the malignant clone in chronic lymphocytic leukemia (CLL). Recently, NGS immunoprofiling of the T cell receptor (TR) gene repertoire in CLL revealed “disease-specific” TR clonotypes shared by different patients that persisted and further expanded over time, suggesting that antigen drive likely underlies T-cell expansions in CLL. CLL-like Monoclonal B-cell Lymphocytosis (MBL) is characterized by the presence of CLL-like clonal B cells in lower numbers compared to CLL with no other evidence of disease. MBL is distinguished into high and low count (HC-MBL and LC-MBL, respectively). HC-MBL may evolve to CLL requiring treatment and is considered a pre-leukemic condition, while LC-MBL has an unclear, if any, link to CLL, posing a particular conundrum, especially considering that despite sharing CLL-associated genomic aberrations it displays a distinct immunoglobulin gene repertoire. The precise mechanisms underlying MBL onset and/or progression into overt CLL remain unknown. Arguably, these could entail the acquisition of particular genetic lesions and/or extrinsic signals delivered to the clonal B cells, including those emanating from T cells. Considering the above, here we aimed at characterizing T cells in the early steps of CLL ontogeny by detailed immunoprofiling of the TR gene repertoire in LC-MBL, a thus far unexplored area.The study group comprised 43 individuals with CLL-like (CD5+) LC-MBL and 7 individuals with non CLL-like LC-MBL (atypical CLL-like and CD5- LC-MBL). The presence of a LC-MBL population was confirmed by a standardized flow cytometry protocol. TR beta chain (TRB) gene rearrangements were RT-PCR amplified with the use of the BIOMED 2 protocol. Sequencing libraries were prepared with the TruSeq DNA LT Kit and run on the MiSeq Sequencer using the MiSeq Reagent Kit v3 (500 cycles). Raw NGS reads were subjected to data filtering based on read length and quality. Filtered-in sequences were submitted to IMGT/HighV-QUEST and an in-house bioinformatics pipeline for clonotype computation and repertoire characterization.Overall, 9,385,319 productive filtered-in TRBV-TRBD-TRBJ rearrangement sequences were evaluated corresponding to an average of 32,205 different (unique) clonotypes/sample (clonotype: same TRBV gene and CDR3 amino acid sequence). Skewing of the TRBV repertoire was identified in both LC-MBL categories. In specific, the 4/5 most frequently utilized TRBV genes were identical between the 2 categories (TRBV29-1, TRBV12-3, TRBV19, TRBV6-5) and cumulatively accounted for a virtually identical part of the respective repertoire (28.7% versus 28.6%). Comparison to our published data from CLL revealed similarities regarding TRBV gene usage, particularly for CLL-like LC-MBL. Oligoclonality was evident in both MBL categories with the 10 most frequent clonotypes accounting for 21.3% of the total repertoire in CLL-like LC-MBL and 20.3% in non CLL-like LC-MBL, again not differing significantly from CLL (26.3%). All the above 3 entities were clearly more oligoclonal in comparison to 2 healthy control samples from aged-matched used as a reference. Cluster analysis among the 10 most frequent clonotypes of all LC-MBL and CLL samples revealed infrequent matches between: (i) different CLL-like LC-MBL samples (24/430, 0.055%); (ii) CLL-like and non CLL-like LC-MBL (8/500, 0.016%); and, (iii) CLL-like LC-MBL and CLL (2/680, 0.002%).Altogether, these findings indicate that, similar to CLL, antigen drive likely underlies T-cell expansions also in LC-MBL. However, despite similar TRBV gene usage and degree of oligoclonality, scant shared clonotypes were identified, thus indicating that the antigenic stimuli and/or immune processes shaping the TR profiles in LC-MBL and CLL are distinct. DisclosuresAgathangelidis:Gilead Sciences: Research Funding. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Novartis SA: Research Funding; Abbvie: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding. Ghia:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Pharmaceuticals: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria.

High-Throughput T Cell Receptor (TR) Repertoire Analysis of Virus-Specific T Cells: Implications for T Cell Immunotherapy and Viral Infection Risk Stratification

▪Viral infections, mainly by cytomegalovirus (CMV), Epstein Barr virus (EBV) and polyomavirus type I (BKV), are major causes of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). As effective immune responses against human viruses rely on an armamentarium of T-cell receptor (TR) repertoire capable of recognizing a broad range of antigenic peptides of those pathogens, reconstitution of antiviral immunity, either by spontaneous generation of endogenous virus-specific T cells (VSTs) or by adoptive immunotherapy with VSTs, plays a critical role to fight infections.We here evaluated the diversity and clonality of TR repertoire of functional tri-virus-specific T cell products generated from immunocompetent donors (n=10) and compared their TR gene repertoire to that of peripheral blood mononuclear cells (PBMCs) from patients who had undergone allo-HSCT (n=5). To generate tri-VSTs, PBMCs derived from 15-20ml of peripheral blood of normal donors, were exposed to EBV, CMV and BKV overlapping peptides and cultured in the presence of interleukin 4 (IL-4) and IL-7 for 10 days in G-rex bioreactors. Specificity of donor-derived VSTs and patient-derived PBMCs was measured by IFN-γElispot. TR diversity was investigated by next-generation sequencing on a MiSeq Sequencer, after amplification of TR beta chain gene rearrangements by RT-PCR with the BIOMED-2 protocol. Raw NGS reads were filtered based on their length and quality and the filtered-in sequences were submitted to IMGT/HighVQUEST. Metadata analysis and clonotype computation were performed using a validated in-house bioinformatics platform. As clonotype we defined sequences carrying the same TRBV gene and identical CDR3 amino acid sequence.Tri-VSTs provided 947,298 productive TRBV-TRBD-TRBJ rearrangements and a polyclonal and highly diverse TR gene repertoire, consisting of a total of 169,502 unique clonotypes (average: 16,950/sample, range 4,057-45,602), 64,971 (38.3%) of which were expanded (corresponding to more than one sequence). In terms of clonality, the mean relative frequency of the major clonotype in all tri-VSTs was 12.6% (range 3.3-29.2%). Interestingly, among tri-VST cell lines, 637 clonotypes were shared (present in >2/10 samples), 80 were highly shared (present in >3/10 samples) while 7 were present in 6-8 different VST lines and largely expanded, accounting for up to 29.2% of all sequences.Importantly, there were 65 of 96 major VST clonotypes shared, thus suggesting that they were potentially associated with recognition of the targeted viruses. Given that 4/10 VSTs cell lines were not specific for CMV, while being EBV-and BKV-specific, dominant TRs in those 4 cell lines can potentially be associated with EBV- or BKV-activity. By searching a public database of TR clonotypes with known reactivity against EBV and/or CMV (ShugayM, Nucleic Acids Research, 2018), we found 8 shared EBV-specific and 4 shared CMV-specific clonotypes among our VSTs and the 499 public clonotypes. When we compared the produced VSTs with PBMCs from 3 allo-grafted patients with circulating CMV-, BKV- and EBV-specific T cells and previous viral reactivation, we detected 163 shared clonotypes. Likewise, we observed 21 and 23 shared clonotypes in similar frequencies, between VSTs and PBMCs from 2 patients with CMV- or BKV-specific T cell immunity. These data identify clones that potentially expand in vivo and protect patients from viral infections.Overall, our findings reveal high levels of TR clonality in cell lines enriched for T cells reactive against EBV and/or CMV and/or BKV and provide insights into the TR repertoire of ex vivo- or endogenously-generated VSTs. Our approach may help to identify optimal TRs for immunotherapy as well as TRs which can be used as a tool for risk stratification of viral infections. DisclosuresAgathangelidis:Gilead: Research Funding. Gemenetzi:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Abbvie: Research Funding; Janssen: Honoraria, Research Funding.

Steroid Responsive T-Cell Mediated Pure Red Cell Aplasia Following Allogeneic Hematopoietic Stem Cell Transplantation

Pure red cell aplasia (PRCA) is an unusual complication following allogeneic hematopoietic stem cell transplantation (HSCT), the true incidence of which is not known. Almost all cases reported in the literature describe major ABO-incompatibility between the recipient and the donor as the major risk factor for this complication. Delayed recovery of reticulocyte counts and hypoplasia of erythroblasts in the marrow is attributed to incompatible hemagglutinins in recipients. There are reports of successful treatment of PRCA using different strategies include rapid tapering of calcineurin inhibitors, corticosteroids, donor lymphocyte infusion, rituximab, and/or plasma exchange. Additional causes of PRCA following allogeneic HSCT include parvovirus B19 infection and graft-versus-host disease (GVHD). The pathogenesis of PRCA in parvovirus infection is thought to be viral-mediated suppression of erythroid precursors in the bone marrow and this is due to the tropism of this virus for erythroid progenitor cells. Nonmyeloablative and reduced intensity conditioning regimens have been reported to have an increased risk of PRCA due to persistence of recipient lymphocytes. Here we report two cases of PRCA following allogeneic HSCT from major ABO mismatched donors. Both cases were correlated with the presence of a T-cell clone in the peripheral blood and bone marrow, and there was complete resolution of anemia with a short course of steroid treatment, as well as disappearance of the T-cell clone. Patient # 1 – A 24 year old female of Mediterranean descent underwent an HLA- 8/10 matched and major ABO-mismatched sibling donor bone marrow transplant for Sickle-Beta Thalassemia. She received a myeloablative but reduced toxicity conditioning regimen consisting of busulfan 16mg/kg, fludarabine 140mg/m2, cyclophosphamide 105mg/kg, and alemtuzumab 52mg/m2. Her immediate post-transplant course was relatively benign and significant for a mild CMV colitis which responded with resolution after treatment with anti-viral therapy. Her red cell transfusion needs had significantly decreased to every four weeks by day 130. However, at day 146 from HSCT, she started requiring red cell transfusions every two weeks, with a drop in the reticulocyte count to 0.6%. No antibodies were detected, and the patient was negative for parvovirus B19 and did not have any evidence of GVHD. However, her bone marrow showed an marked erythroid hypoplasia, and was positive for a T-cell clone with gamma chain gene rearrangement in the T-cell receptor. Due to a new onset of transfusion need, she was treated with a short course of steroids with an excellent response and disappearance of the T-cell clone from peripheral blood. She remained on immunosuppression during this entire period with a calcineurin inhibitor. Patient# 2 – A 10 year old Hispanic girl underwent a 10/10 HLA-matched and major ABO-mismatced sibling donor bone marrow transplant for idiopathic acquired severe aplastic anemia. Her preparative regimen consisted of 200 mg/kg of cyclophosphamide and 16mg/kg of rabbit-anti-thymocyte globulin. Her immediate post-transplant course was complicated by E.coli bacteremia. She had prompt engraftment and became transfusion independent on day 86. A cyclosporine taper was initiated at day 100 but held at day 119 when anemia was first noted. Peripheral blood showed a T-cell clone with gamma and beta chain gene rearrangement. She was started on a moderate dose of steroids with a good response, and has been tapered down to physiologic replacement dose of steroids with normal Hb and transfusion independence. With the resolution of anemia, the gamma chain gene rearrangement is not seen in the T-cell clonality assay, however, the beta chain gene rearrangement persists. To our knowledge, this is the first report of a T-cell mediated PRCA following allogeneic HSCT. Moreover, the PRCA was steroid responsive and not associated with GVHD in both the patients. Disclosures:No relevant conflicts of interest to declare.

Cytotoxic peripheral T cell lymphoma arising in a patient with nodular lymphocyte predominant Hodgkin lymphoma: a case report

In this paper, we describe a case of nodular lymphocyte predominant Hodgkin lymphoma with the subsequent development of a peripheral T cell lymphoma. This case is unusual in that the sheets of atypical and small to intermediate-sized T cells in the diffuse component were CD8 positive and expressed cytotoxic proteins. The diagnosis of peripheral T cell lymphoma was supported by the demonstration of a clonal T cell receptor beta chain gene rearrangement by Southern blot analysis. Peripheral T cell lymphoma with a cytotoxic phenotype is a rare entity with an aggressive clinical behavior. As such, this report emphasizes the need to consider a diagnosis of coexisting peripheral T cell lymphoma in cases of nodular lymphocyte predominant Hodgkin lymphoma with atypical features, such as few or poorly defined B cell macronodules and diffuse T cell areas. The examination of both T cell receptor gamma and beta chain gene rearrangements should be performed to confirm such cases.

Analysis of T-Cell Receptor Gene Rearrangement for Predicting Clinical Outcome in Patients With Cutaneous T-Cell Lymphoma

To extend previous observations regarding the prognostic value of analyzing lymph node DNA from patients with cutaneous T-cell lymphoma for the presence of a monoclonal T-cell population by Southern blot vs polymerase chain reaction (PCR) methods. Inception cohort study from 1982 to 1998. Recruitment of new patients ended in 1994. A tertiary care referral center in Seattle, Wash. Patients Fifty-five uniformly staged patients with the diagnosis of cutaneous T-cell lymphoma who underwent a lymph node biopsy, 21 with clinically abnormal nodes and 34 with normal nodes. Interventions Lymph nodes were evaluated for T-cell receptor (TCR) gamma-chain gene rearrangement by 2 PCR methods: capillary electrophoresis and denaturing gradient gel electrophoresis. The same lymph nodes were evaluated by Southern blot analysis for TCR beta-chain gene rearrangement and examined histopathologically on the basis of the National Cancer Institute lymph node classification system. Patients were observed clinically for a mean of 9.5 years. Skin stage, clinical lymph node examination, lymph node histologic examination, Southern blot analysis, and PCR analyses were evaluated as potential prognostic predictors by univariate and multivariate analyses. The statistical association of TCR analysis and clinical outcome was determined among all patients. Hazard ratios (HRs) by Cox proportional hazards regression analysis were used to estimate the risk of a poor clinical outcome. Cumulative survival rates were analyzed by the Kaplan-Meier method. A skin stage of T3 (tumors) or T4 (erythroderma) was the most powerful predictor of a poor clinical outcome (HR, 31.3 vs T1; P<.001). Patients with detectable TCR gamma-chain gene rearrangement in lymph node DNA by PCR also were more likely to have a poor outcome (HR, 5.1; P<.001), but it was a less powerful predictor than skin stage. Even when the skin stage, presence or absence of lymphadenopathy, and histologic lymph node score were known for the patient, Southern blot analysis still added to prediction of a poor outcome (HR, 9.3; P = .007), whereas PCR provided no statistically significant additional information on outcome. Detection of a monoclonal T-cell population by PCR in lymph nodes of patients with cutaneous T-cell lymphoma does not enhance prediction of clinical outcome and probability of survival beyond what can be determined from clinical examination and histologic lymph node scores. Skin stage and the presence or absence of lymphadenopathy remain the most important determinants of clinical outcome.

Prevalence of mycosis fungoides and its association with EBV and HTLV-1 in Pakistanian patients.

Mycosis fungoides (MF) is an indolent T cell lymphoma that is distinguished from other lymphomas by its initial appearance on the skin. The histologic diagnosis of MF may be difficult because there is significant overlap in the histologic features of neoplastic T-cell infiltrates and inflammatory dermatoses. This T-cell neoplasm commonly occurs in a mixed, reactive background and can show only a subtle degree of cytologic atypia, rendering histologic diagnosis difficult. In this study MF constituted 0.86% of all non-Hodgkin s lymphoma (NHL) both T and B, as compared to the Western studies which have reported 0.5% prevalence for MF of all NHL. Polymerase chain reaction (PCR) technique was used to assess T-cell clonality in paraffin-embedded skin biopsies clinically and pathologically suspicious for early MF. Out of the 14 cases diagnosed as MF, amplifiable DNA was isolated from 6 cases, which were further studied for T-cell receptor (TcR) beta, gamma, and delta chain gene rearrangements. Clonal product was seen in 4 out of 6 cases for beta, gamma, and delta TcR chain genes. Association for Epstein Barr virus (EBV) was observed in 3 out of 6 cases (50%) of MF. Although these 3 cases were positive for EBV by PCR, but were negative by in-situ hybridization (ISH). No heterogeneity was noted in these 3 cases of MF for BamHI E, K, N, and Z regions of EBV. All six cases were negative for HTLV-1 (tax region) by PCR. It was concluded that the prevalence of MF in Pakistani population is comparable to the Western data, and that EBV association to MF cases was higher than in Western studies.

Maintenance and characterization of an Epstein Barr virus-infected CD56-negative T cell lymphoma.

T cell lymphoma carrying Epstein Barr virus (EBV+TL) is very rare among Western countries while it is much more common among Japanese. Here we report an EBV+TL which has been maintained for years by the use of mice with severe combined immune deficiency (SCID) mice. Lymphoma was obtained from a 55‐year‐old male suffering from oculomotor nerve palsy and lymphadenopathy. A small piece of biopsied tumor was transplanted into SCID mice and the lymphoma has been maintained for over 3 years with passages every 2–3 weeks. The maintained lymphoma, termed as TMS24, and the original lymphoma cells showed identical phenotype and genotype, including diffuse medium‐sized cell morphology lacking granules, suppressor/cytotoxic immunophenotype and identical T cell receptor β‐chain gene rearrangement mode. Further, both were shown to carry an identical EBV clone in terms of the number of terminal repeats and the latency II‐type restricted gene expression profile. Cytogenetically, TMS24 retained two characteristic chromosomal translocations of t(l;18)(q32;q21) and t(6;12)(p21;q24). Since only one cell line with such characters has been reported previously, TMS24 should be useful for detailed analysis of EBV+TL.

Sequence and diversity of T-cell receptor beta-chain V and J genes of the owl monkey Aotus nancymaae.

The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and has therefore been recommended by the World Health Organization as a model for the evaluation of malaria vaccine candidates. Recently, we have shown that Aotus TCRVA genes and TCRJA segments exhibit a high degree of similarity to human counterparts. In the present report we used reverse transcription polymerase chain reaction to analyze the sequences of A. nancymaae TCR beta-chain gene rearrangements. Alignment with human sequences and phylogenetic comparison identified 18 distinct Aotus TCRBV genes homologous to the human TCRBV gene families 2, 4, 5, 6, 7, 9, 12, 15, 24, and 28. Multiple Aotus genes were found in the TCRBV4, 5, 6, and 7 families. Some of these TCRBV genes aligned best to the same human gene and thus do not seem to have separate human homologues. Amino acid sequences of the Aotus TCRBV genes were 77 to 90% identical to their closest human counterparts. Ten distinct Aotus TCRBJ segments homologous to the human segments J1-1, J1-2, J1-4, J1-5, J1-6, J2-1, J2-2, J2-3, J2-4, J2-5 were found. In some cases the amino acid sequences of Aotus and human TCRBJ segments were completely identical. A comparison of the proportion of synonymous and non-synonymous substitutions in Aotus vs human beta-chain-encoding genes revealed a dominance of synonymous substitutions in TCRBJ segments and of nonsynonymous substitutions in TCRBV segments. Dominance of nonsynonymous substitutions was more pronounced in TCRBV CDR1 and CDR2 regions than in the framework regions. No evidence for the emergence of new TCRBJ segments or TCRBV families was found. These results confirm that the TCR repertoire in primates is remarkably stable and support the concept of using Aotus monkeys as an infection model for the evaluation of future subunit vaccine candidates.

Regulation of Vbeta germline transcription in RAG-deficient mice by the CD3epsilon-mediated signals: implication of Vbeta transcriptional regulation in TCR beta allelic exclusion.

During the thymic development of alphabeta lineage T cells, maturation of the CD4- CD8- double-negative (DN) cells into the CD4+ CD8+ double-positive cells is accompanied by the induction of TCR beta allelic exclusion. Recent studies have shown that these events are regulated by the signals through the pre-TCR complex which consists of the TCR beta, pre-TCR alpha and CD3 components. The Vbeta germline transcripts are detected prior to the TCR beta chain gene rearrangements in the DN thymocytes. To examine the effects of the pre-TCR-mediated signals on Vbeta germline transcription, we analyzed thymocytes from RAG-2-deficient mice treated with anti-CD3epsilon antibody. The germline transcripts of all Vbeta we examined, except for Vbeta14, were down-regulated by the anti-CD3epsilon antibody treatment. These data indicate that the regulation of Vbeta germline transcription by the signals through the pre-TCR complex may reflect the modulation of Vbeta accessibility to the VDJ recombinase, which contributes to TCR beta allelic exclusion.

Analysis of T-cell receptor gene rearrangement in lymph nodes of patients with mycosis fungoides. Prognostic implications.

To determine the prognostic value of analyzing lymph node (LN) DNA from patients with mycosis fungoides for the presence of a monoclonal T-cell population. Inception cohort study. A tertiary care referral center in Seattle, Wash. Fifty-five uniformly staged patients with the diagnosis of mycosis fungoides and who had a lymph node biopsy, 21 with clinically abnormal nodes and 34 with normal nodes. Lymph nodes were evaluated by Southern blot analysis for T-cell receptor beta-chain (TCRB) gene rearrangement and by histopathologic examination for the LN classification using the National Cancer Institute system. Patients were observed clinically for a mean (+/- SD) of 4.7 +/- 3.4 years. Patients with detectable TCRB gene rearrangement in lymph node DNA had an increased likelihood of a poor clinical outcome and a decreased probability of survival (P < .001 for both) compared with patients with the TCRB germline. Although patients with clinically enlarged nodes were more likely to have the TCRB gene rearranged, those with normal nodes and the TCRB gene rearranged also had a poor clinical outcome and a decreased probability of survival. Similar to those with the TCRB gene rearranged, most patients with advanced histopathologic changes (LN3 and LN4) had a poor prognosis. The presence of a rearranged TCRB gene, however, correctly predicted some patients with intermediate LN scores (LN2) who had a poor clinical outcome. Detection of a monoclonal T-cell population, as demonstrated by a rearranged TCRB gene on Southern blot analysis, in LNs of patients with mycosis fungoides is predictive of a poor clinical outcome and a reduced probability of survival. Lymph node TCRB gene analysis provides additional prognostic information for patients with mycosis fungoides with intermediate LN histopathology.

Productive T-cell receptor beta-chain gene rearrangement: coincident regulation of cell cycle and clonality during development in vivo.

Productive gene rearrangement at the T-cell receptor (TCR) beta-chain locus facilitates formation of the "pre-TCR," a molecular complex that is important for the subsequent development of alpha beta T cells. The transition of thymocytes from a population of cells undergoing TCRbeta chain genes to a population enriched in cells with productively rearranged TCRbeta chain genes is known as "beta selection." This is the first point in alpha beta T-cell development at which the products of an activated TCR locus define cell phenotype. Toward an understanding of these events, this study has focused on a set of thymocytes defined by cell surface phenotype as HSA+ CD44low CD25+, in which the bulk of TCRbeta gene rearrangement occurs. The analysis of this set, presented here, allows its novel subdivision into two subsets that are respectively strong candidates for cells immediately prior to and immediately following TCRbeta selection. Cells that have passed beta selection differ from the preceding cells by several criteria, including hyperphosphorylation of Rb, increased expression of cyclins A and B, down-regulation of p27, increased CDK2 activity, an induction of cdc2 activity, and progression through DNA synthesis. Consistent with these changes being attributable to productive TCRbeta chain gene rearrangement, the identified "beta-selected" subset is not detected in mutant mice that cannot assemble a pre-TCR. Interestingly, there is a coincident selective and transient down-regulation of the protein RAG2, on which TCR gene rearrangement obligatorily depends. Together, these findings demonstrate that productive TCR gene rearrangement is associated with events that can ensure thymocyte expansion and monoclonality.

The Clonal Nature of Circulating Sezary Cells

To determine if circulating Sezary cells can be classified as reactive or neoplastic based on the ability to detect the presence or absence of clonal T-cell receptor beta chain (TCR-beta) gene rearrangements by Southern blot analysis, we evaluated the peripheral blood of 25 patients: 11 patients with Sezary syndrome (SS), 11 with benign inflammatory dermatoses (BID), and three normal controls. Three of 11 patients with SS, with Sezary counts ranging from 14% to 52%, did not demonstrate any clonal TCR-beta gene rearrangements in the peripheral blood, despite a TCR-beta rearrangement by Southern blot analysis in the skin. Ten of 11 BID patients and all normal controls showed no evidence of a TCR-beta gene rearrangement in the peripheral blood. However, one patient with psoriasis demonstrated a TCR-beta gene rearrangement in the peripheral blood. The TCR-beta gene rearrangement detected in this patient, confirmed with polymerase chain reaction (PCR) amplification of the TCR-gamma gene rearrangement, did not correlate with the presence of circulating Sezary cells or the increased risk of neoplasia. Our results indicate that circulating Sezary cells may be monoclonal (neoplastic) or polyclonal (reactive), as defined by TCR gene rearrangement studies. Circulating Sezary cells in SS may be reactive in nature and not accurately reflect the actual tumor burden in the peripheral blood. The presence of circulating Sezary cells or the presence of a clone of cells defined by TCR-beta gene rearrangement in the peripheral blood is not limited to neoplastic disease processes.

Characterization of T Cells Immortalized by Tax1 of Human T-Cell Leukemia Virus Type 1

Peripheral blood T cells were immortalized in vitro by introduction of the Tax1 gene of human T-cell leukemia virus type 1 (HTLV-1) with a retroviral vector and were characterized for transformation-associated markers. Long-term observation showed that these Tax1-immortalized T cells eventually exhibited very similar features that were characteristic of HTLV-1-immortalized T cells, ie, increased expression of egr-1, c-fos, IL-2R alpha, and Lyn and decreased expression of Lck and cell-surface CD3 antigen. Among these changes, an increase in the expression of Lyn and a decrease in the expression of Lck and cell-surface CD3 antigen were observed only in Tax1-immortalized T cells after long-term culture. The expression level of Tax1 protein did not differ significantly between early and late passage of cells, and the cellular clonality was found to be the same by the analysis of the retroviral vector integration site and the T-cell receptor beta-chain gene rearrangement pattern. These changes in the expression of Lyn, Lck, and cell-surface CD3 antigen probably resulted from indirect effects of Tax1 that appeared after extended culture.

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.

Thymic selection events mediated by the pre-TCR do not depend upon a limiting ligand.

Thymocyte differentiation requires the production of a functional TCR, the culmination of a carefully orchestrated series of events in which TCR beta chain gene rearrangement precedes that of TCR alpha genes. The product of a successful rearrangement of the TCR beta locus associates with an invariant protein in immature thymocytes to form the 'pre-TCR' complex, which is required for allelic exclusion at the TCR beta locus, the expression of CD4 and CD8 co-receptors, and the clonal expansion of immature thymocytes. The pivotal role for the beta chain protein during early thymocyte development led us to investigate the relative differentiation efficiency within the same thymus of cells which do and cells which do not possess productive TCR gene rearrangements. Using mixed radiation bone marrow chimeras to establish an in vivo competition between TCR beta transgenic (Tg) and non-Tg bone marrow cells, we show that the prior productive rearrangement of a TCR beta chain gene only subtly enhances the efficiency of intrathymic differentiation. Further, we have compared the relative differentiation efficiency of TCR alpha beta and TCR beta Tg cells within the mixed chimera system by altering the proportion of TCR Tg bone marrow cells in the reconstituting inoculum. As expected, Tg cells carrying both alpha and beta chains of a selectable TCR are developmentally hindered compared with their non-Tg counterparts by the lack of ample numbers of intrathymic positively selecting ligands or niches. In contrast, parallel experiments using TCR beta Tg bone marrow cells demonstrate that the early selection events mediated by the pre-TCR do not similarly depend upon a ligand present in limiting quantities.

Arrested rearrangement of TCR V beta genes in thymocytes from children with X-linked severe combined immunodeficiency disease.

Human X-linked severe combined immunodeficiency disease (SCID) is an immunodeficiency disorder in which T cell development is arrested in the thymic cortex. B lymphocytes in children with X-linked SCID seem to differentiate normally. X-linked SCID is associated with a mutation in the gene that encodes the IL-2R gamma-chain. Because TCR-beta gene recombination is a pivotal initial event in T lymphocyte ontogeny within the thymus, we hypothesized that a failure to express normal IL-2R gamma could lead to impaired TCR-beta gene recombination in early thymic development. PCR was used to determine the status of TCR-beta gene-segment rearrangements in thymic DNA that had been obtained from children with X-linked SCID. The initial step in TCR-beta gene rearrangement, that of D beta to J beta recombination, was readily detected in all thymus samples from children with X-linked SCID; in contrast, V beta to DJ beta gene rearrangements were undetectable in the same samples. Both D beta to J beta and V beta to DJ beta TCR genes were rearranged in the thymic tissues obtained from immunologically normal children. We conclude that TCR beta-chain gene rearrangement is arrested in children with X-linked SCID. Our results suggest a causative relationship between the failure of TCR beta-chain gene rearrangements to proceed beyond DJ beta rearrangements and the production of a nonfunctional IL-2R gamma-chain.

EXPRESSION OF SUPERANTIGEN-LIKE SPECIFICITIES ON MURINE SJL/J-B LYMPHOMAS - ANTITUMOR V-BETA-17A+ T-LYMPHOCYTES USE A DIVERSE SET OF T-CELL RECEPTOR V-ALPHA-CHAIN GENE-SEQUENCES

Previously we have demonstrated that the tumor infiltrating lymphocytes found in SJL/J mice with reticulum cell sarcomas (RCS) are CD4+ T cells which express T cell receptor (TCR) beta chain molecules comprised principally of Vbeta17a encoded variable gene segments. Herein we report both the cellular and molecular characterization of a panel of twenty RCS-specific T cell hybridomas derived from tumor infiltrating T cells of the mesenteric lymph nodes of an SJL/J mouse bearing the transplanted RCS LA-12 tumor. We determined by flow cytometry that all of the RCS reactive T cell hybridomas expressed TCR containing Vbeta17a encoded variable gene segments. Moreover, most of these T cell hybridomas (17 of 20) co-expressed the CD4 molecule, as did the tumor infiltrating T cells from which they were derived. Using Southern blot analysis of beta chain gene rearrangements. we determined that the predominant Vbeta17a gene usage seen in these hybridomas was not the result of the expansion, either in vivo or in vitro, of a single Vbeta17a+ RCS-reactive T cell clone. The hybridization patterns of hybridoma DNA, as revealed by a Jbeta2 probe, were dissimilar and indicated that multiple unique Vbeta17a+ T cells participate in the RCS response. Furthermore, using Northern blot analysis, we were able to determine that the T cell hybridomas used a varied set of TCR Valpha chain gene segments. The T cell hybridomas, for which the Valpha chain gene segments were determined, used Valpha chains comprised of either Valpha1, Valpha4 or Valpha8 family gene segments. Based on these findings we conclude that Valpha chain usage, if not random, is disparate in response to RCS tumors. Furthermore, these data suggest that the important reactivity for the RCS tumor antigen(s) lies with the beta chain (Vbeta17a) but not with the alpha chain used by the T cell. This pattern of alpha and beta chain utilization is consistent with reported TCR usage patterns exhibited by T cells in response to superantigens - where the beta chain and not the a chain dictates antigenic responsiveness. Therefore, our current findings suggest that the tumor antigen(s) presented by this RCS tumor is similar in nature to that of a superantigen.

Double-negative (CD4- CD8-) T cells from adult T-cell leukemia patients also have poor expression of the T-cell receptor alpha beta/CD3 complex

We present four patients with adult T-cell leukemia (ATL) derived from a novel T-cell subset (CD4-, CD8- [double-negative, DN], T-cell receptor [TCR] alpha beta+). In the ATL cells of these patients, neither gene nor surface expression of CD4 and CD8 antigens was detected. Clinical and laboratory data showed no difference between DN-ATL and CD4+ATL patients. In contrast to typical CD4+ATL cells, DN-ATL cells were shown to express the protein and messenger RNA (mRNA) for S100 beta in immunocytochemical assay and the reverse-transcription polymerase chain reaction assay. The mean fluorescence intensity of the TCR/CD3 complex was extremely low in all four DN-ATL patients as well as in typical CD4+ ATL. All four patients had TCR beta and gamma chain gene rearrangements, with deletion of TCR delta chain gene and mRNA expression for TCR alpha, beta, and CD3 delta but not for TCR gamma and delta chain genes. Thus, CD4- CD8- TCR alpha beta T cells are also a target for human T-cell lymphotrophic virus type I-induced leukemogenesis. In addition, expression of the TCR alpha beta/CD3 complex on the DN-ATL cells was further diminished by the addition of anti-CD3 or anti-TCR alpha beta monoclonal antibody. These results suggest that the decreased expression of the TCR alpha beta/CD3 complex by ATL cells plays a key role in the development of ATL, irrespective of CD4 expression.