Cutting Edge: TCR-ß Selection Is Required at the CD4+CD8+ Stage of Human T Cell Development.

T cell development is predicated on the successful rearrangement of the TCR gene loci, which encode for Ag-specific receptors. Recombination-activating gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T cell development. In this study, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCR ß-chain in mediating ß-selection during human T cell development. In stark contrast to mice, human RAG2-KO T lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRß rearrangements. Nonetheless, RAG2-KO DPs retrovirally transduced to express a rearranged TCR ß-chain showed increased survival and proliferation as compared with control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRß- and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide important insights as to the distinct requirement for the TCR ß-chain during human T cell development.

Evidence for a role of RUNX1 as recombinase cofactor for TCRß rearrangements and pathological deletions in ETV6-RUNX1 ALL.

T-cell receptor gene beta (TCRß) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knock-out mouse model and demonstrate by deep TCRß sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRß rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRß gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRß gene rearrangements in T-ALL samples harboring heterozygous Runx1 mutations. Comparable to the Runx1+/- mouse model, heterozygous Runx1 mutations in T-ALL patients displayed no detectable impact on TCRß rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions.

Immune and Circulating Tumor DNA Profiling After Radiation Treatment for Oligometastatic Non-Small Cell Lung Cancer: Translational Correlatives from a Mature Randomized Phase II Trial.

PURPOSE: NCT01725165 was a phase II prospective trial in which patients with non-small cell lung cancer were randomized to local consolidative therapy (LCT) versus maintenance therapy or observation (MT/O). METHODS AND MATERIALS: Peripheral blood from patients enrolled on NCT01725165 were labeled as (1) baseline, (2) early follow-up (FU) if obtained in the first or second FU evaluation (6-18 weeks), and (3) late FU if obtained in the third to sixth FU evaluations (22-50 weeks). All patients who underwent LCT and were included in this analysis received radiation. Among 49 randomized patients, 21 patients underwent T cell CDR3 variable region sequencing using immunoSEQ, 31 patients underwent circulating tumor DNA (ctDNA) analysis using next-generation sequencing with a 1021 cancer gene panel, and cytokine concentration was assayed in 19 patients using enzyme-linked immunosorbent assay. All analyses were exploratory and not corrected for multiple testing. RESULTS: No associations were identified between baseline T cell repertoire and ctDNA metrics with patient outcomes. Among baseline cytokines, interleukin 1α was the only cytokine associated with both overall survival (hazard ratio, 0.02; 95% confidence interval, 0.1-0.5; P = .0006) and progression-free survival (hazard ratio, 0.5; 95% confidence interval, 0.2-0.9; P = .03). At early FU, LCT was associated with decreased ctDNA burden, including lower number of detected mutations (median, 2 [interquartile range {IQR}, 1-6] vs 6 [IQR, 4-18]) and decreased average variable allele frequency (VAF; median, 0.006 [IQR, 0.003-0.010] vs 0.011 [IQR, 0.007-0.014]) compared with MT/O. Among 6 patients with serial ctDNA analysis, a rise in ctDNA detected mutation burden preceded clinical progression by 6.7 months. At early FU, LCT was associated with changes in T cell clonality that suggested oligoclonal expansion specifically increased T cell clonality (median, 0.15 [IQR, 0.12-0.24] vs 0.10 [IQR, 0.05-0.13]) and frequency of top 10 clones (median, 0.14 [IQR, 0.06-0.18] vs 0.21[IQR, 0.19-0.28]). CONCLUSION: LCT was associated with decreased ctDNA burden and oligoclonal expansion at early FU timepoints. Baseline interleukin 1α was associated with improved patient outcomes.

CD8-positive pseudolymphoma in lues maligna and human immunodeficiency virus with monoclonal T-cell receptor-beta rearrangement.

A 42-year-old Caucasian man suffered from disseminated plaques and ulcerated nodules for 6 weeks. He had weight loss and generalized lymphadenopathy. Underlying diseases were not known up till then. Based on a skin biopsy, the diagnosis of CD8-positive cutaneous T-cell lymphoma, type mycosis fungoides was made in a pathological reference center for lymphoma. A reproducible T-cell receptor (TCR)-beta rearrangement was detectable. Before starting therapy, a new biopsy was taken and the previous diagnosis was re-evaluated taking clinical images and symptoms into account. Based on both, the diagnosis of a CD8+ pseudolymphoma in lues maligna and human immunodeficiency virus was made. We highlight histopathologic clues for the correct diagnosis, and we emphasize the indispensability of clinical-pathological correlation. Furthermore, we discuss the differential diagnosis of CD8+ lymphoproliferative disorders.

Cytotoxic T cells in chronic idiopathic neutropenia express restricted antigen receptors.

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by female predominance and mostly uncomplicated course. Crucial to CIN pathophysiology is the presence of activated T lymphocytes with myelosuppressive properties in both peripheral blood (PB) and bone marrow (BM). We systematically profiled the T cell receptor beta chain (TRB) gene repertoire in CD8+ cells of 34 CIN patients through subcloning/Sanger sequencing analysis of TRBV-TRBD-TRBJ gene rearrangements. Remarkable repertoire skewing and oligoclonality were observed, along with shared clonotypes between different patients, alluding to antigen selection. Cross-comparison of our sequence dataset with public TRB sequence databases revealed that CIN may rarely share common immunogenetic features with other entities, however, the CIN TRB repertoire is largely disease-biased. Overall, these findings suggest that CIN may be driven by long-term exposure to a restricted set of specific CIN-associated antigens.

Clinical Experience With Modified, Single-Tube T-Cell Receptor Vß Flow Cytometry Analysis for T-Cell Clonality.

OBJECTIVES: Analysis of T-cell clonality using the IO Test Beta Mark (Beckman Coulter, Fullerton, CA) flow cytometry assay is cumbersome owing to the required eight-part analyses that limit its clinical utility. Here, we describe a simple modification that may improve its clinical utility. METHODS: By simply combining all T-cell receptor (TCR) Vß antibodies into one reagent tube, we show that T-cell repertoire analysis can be efficiently performed with an increased potential to closely link the aberrant immunophenotype of a neoplastic T-cell clone to its apparent clonal TCR Vß restriction. We review our clinical experience and include flow cytometry sorting studies in conjunction with TRG gene rearrangement analysis to demonstrate the feasibility of this approach. RESULTS: We show in representative cases that this modified approach permits evaluation of TCR Vß clonality in T-cell neoplasms without a priori knowledge of the TCR Vß isoform restriction and that this approach is suitable for samples containing a minor population of neoplastic T cells, hypocellular samples such as skin biopsy specimens and body fluids, and fine-needle aspirations. We estimate a false-positive rate for identifying a clonal population for this approach to be less than 6.2%. CONCLUSIONS: Our modified approach of TCR Vß analysis could improve routine clinical evaluation for abnormal T-cell populations by flow cytometry.

Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor ß (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient.

Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor ß-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.

A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRß locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications.

Distinct morphologic, phenotypic, and clinical-course characteristics of indolent peripheral T-cell lymphoma.

Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) consists of a heterogeneous group of lymphomas. Patients generally show an aggressive clinical course and very poor outcome. Although the 2008 World Health Organization classification of PTCL-NOS includes 3 variants, low-grade lymphoma is not included. Of 277 PTCL-NOS cases recorded in our consultation files, we examined the clinicopathologic characteristics of 10 patients with T-cell lymphomas composed of small-sized cells with slight nuclear atypia. Eight patients showed extranodal involvement (5 patients, spleen; 3 patients, thyroid), and 5 patients were at clinical stage I or II. Histologically, all samples presented diffuse infiltrate of small lymphoid cells, with few mitotic figures. Immunohistologically, all samples were positive for CD3, and CD20 was detected in 5 samples. All samples showed a low Ki-67 labeling index (mean, 1.05%), and 7 samples were positive for central memory T-cell markers. Clonal T-cell receptor γ chain and/or α-ß chain gene rearrangements were detected in all 10 patients. Five patients received chemotherapy, whereas for 3 patients, treatment consisted only of observation following surgical resection of the spleen or thyroid. Nine patients were alive at a median follow-up time of 19.5 months, whereas 1 patient died of an unrelated disease. The present study strongly indicates that T-cell lymphoma with small-sized lymphoma cells and a low Ki-67 labeling index is a distinct variant. Recognition of this novel lymphoma subtype, which should not be defined merely as PTCL-NOS, should be seriously considered.

TRBV kinetics and its association with HLA disparity and aGVHD following allogeneic hematopoietic stem cell transplantation.

INTRODUCTION: The relative expression of T cell receptor (TCR) beta variable (TRBV) and TCR diversity was compared between recipients receiving human leukocyte antigen (HLA)-mismatched transplants and those receiving HLA-matched transplants, using granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells and bone marrow as grafts. METHODS: The kinetics of the relative expression of TRBV family members were analyzed using real-time quantitative PCR. Additionally, the association of TRBV clonotype with acute graft-versus-host disease (aGVHD) was determined by cloning and sequence analysis of the CDR3 region. RESULTS: The TCR diversity in recipients receiving HLA-mismatched transplants was significantly lower than in those receiving HLA-matched transplants at 1 month and 2 months after hematopoietic stem cell transplantation (HSCT) (both P < 0.05). However, these differences disappeared 3 months after transplantation. The relative expression of TRBV27 (n = 7 recipients) at the onset of aGVHD was higher than in corresponding donors (P = 0.025), but no significant differences were observed between recipients lacking aGVHD and their donors at serial time points after HSCT. CONCLUSION: Our results suggest that HLA disparity may affect the relative expression of TRBV in the early phase after transplantation, and TRBV27 may be associated with the onset of aGVHD.

Extensive molecular mapping of TCRα/δ- and TCRß-involved chromosomal translocations reveals distinct mechanisms of oncogene activation in T-ALL.

Chromosomal translocations involving the TCR loci represent one of the most recurrent oncogenic hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) and are generally believed to result from illegitimate V(D)J recombination events. However, molecular characterization and evaluation of the extent of recombinase involvement at the TCR-oncogene junction has not been fully evaluated. In the present study, screening for TCRß and TCRα/δ translocations by FISH and ligation-mediated PCR in 280 T-ALLs allowed the identification of 4 previously unreported TCR-translocated oncogene partners: GNAG, LEF1, NKX2-4, and IL2RB. Molecular mapping of genomic junctions from TCR translocations showed that the majority of oncogenic partner breakpoints are not recombinase mediated and that the regulatory elements predominantly used to drive oncogene expression differ markedly in TCRß (which are exclusively enhancer driven) and TCRα/δ (which use an enhancer-independent cryptic internal promoter) translocations. Our data also imply that oncogene activation takes place at a very immature stage of thymic development, when Dδ2-Dδ3/Dδ3-Jδ1 and Dß-Jß rearrangements occur, whereas the bulk leukemic maturation arrest occurs at a much later (cortical) stage. These observations have implications for T-ALL therapy, because the preleukemic early thymic clonogenic population needs to be eradicated and its disappearance monitored.

Manual microdissection technique in a case of subcutaneous panniculitis-like T-cell lymphoma: a case report and review.

Demonstration of T-cell receptor gene monoclonality often plays an important role in the diagnosis of T-cell lymphoma. When a test to detect monoclonality is performed on whole tissue sections, the presence of a reactive lymphocyte population may reduce sensitivity. This may be especially true for early or borderline cases of lymphoma. Microdissection techniques may be utilized to more readily identify a clonal population of lymphocytes. Subcutaneous panniculitis-like T-cell lymphoma represents a cutaneous lymphoid neoplasm whose clinical course may vary from an indolent, waxing and waning course to an aggressive course resulting in death. We report the first case of a microdissection technique used to facilitate diagnosing a case of subcutaneous panniculitis-like T-cell lympoma.

TCRß clonality improves diagnostic yield of TCRγ clonality in refractory celiac disease.

BACKGROUND: Refractory celiac disease (RCD) is a preneoplastic condition as many patients develop an enteropathy-type T-cell lymphoma, a mature T-cell receptor α-ß lymphoma arising in the gut with an ominous outcome. Recently, research focused on a population of intraepithelial intestinal lymphocytes expressing the same lymphoma T-cell receptor variable region (V)γ, as shown by polymerase chain reaction (PCR) analysis and sequencing. Meanwhile, the Biomedicine and Health-2 Concerted Action has made available standardized, highly specific, and sensitive PCR assays not only for Vγ but also for Vß. GOALS: We verified whether analyzing both rearrangements in duodenal biopsies from RCD patients increases the diagnostic accuracy of this method. STUDY: Duodenal biopsies were analyzed from 15 RCD patients, 21 negative controls, and 2 positive controls (enteropathy-type T-cell lymphoma complicating celiac disease). Multiplex clonality analyses were performed according to the Biomedicine and Health-2 protocols. PCR products were cloned and sequenced. RESULTS: Monoclonal rearrangements were found in 5/15 samples from patients with RCD (both rearrangements in 2 cases, Vß only in 2, and only 1 solitary Vγ clonality). Monoclonality was found in 4/8 of the RCD patients who subsequently died, whereas only 1/7 of the patients still alive presented a monoclonal rearrangement. Positive controls revealed both monoclonal rearrangements; rearrangements were not detected in 20 of 21 negative controls. Sequencing of the amplified fragments confirmed the results. CONCLUSIONS: The combined analysis of both rearrangements allowed recognition of monoclonal populations in otherwise negative patients, with detection rates from 20% (Vγ only) to 33% (Vγ and Vß), thus raising the likelihood of early identification of RCD patients at high risk of death.

Flow cytometric immunophenotypic assessment of T-cell clonality by vß repertoire analysis in fine-needle aspirates and cerebrospinal fluid.

Flow cytometric T-cell receptor V(ß) repertoire analysis (TCR-V(ß)-R) is a sensitive method to detect T-cell clonality; however, its implementation in low-cellularity specimens has not been established. We developed a strategy to use TCR-V(ß)-R in cerebrospinal fluid (CSF) and fine-needle aspirate (FNA) specimens. Initially, full TCR-V(ß)-R was evaluated in diagnostic/screening specimens from 8 patients with T-cell neoplasia to determine tumor-specific TCR-V(ß) protein expression. Subsequently, an abbreviated, patient-specific TCR-V(ß)-R evaluation was performed in 17 paucicellular specimens from the patients (8 CSF, 9 FNA) for staging and monitoring of minimal residual disease (MRD). A single cocktail containing 3 anti-V(ß) antibodies (1 tumor-specific and 2 negative controls) in combination with other antibodies chosen to help gate on atypical T cells is highly sensitive and specific for detecting low-level neoplastic T-cell involvement in paucicellular specimens. This TCR-V(ß)-R strategy is valuable in staging and evaluating MRD in patients with T-cell non-Hodgkin lymphoma.

Traumatic ulcerative granuloma with stromal eosinophilia: report of a case and literature review.

Traumatic eosinophilic granuloma with stromal eosinophilia is a rare entity that affects the oral mucosa and has a controversial etiologic pathogenesis. Histologically, these lesions are characterized by a dense and deeply infiltrative lymphoproliferation showing epitheliotropic characteristics and massive eosinophilia. Frequently, a population of mitotically active, atypical mononuclear cells can be noted. This report describes a case of traumatic eosinophilic granuloma with stromal eosinophilia in the floor of the mouth of an 88-year-old man. The phenotypic and genotypic profiles of the inflammatory infiltrate and large atypical mononuclear cells, using immunohistochemical and polymerase chain reaction-based molecular analysis, were analyzed.

Epstein-Barr virus-positive T-cell lymphoma cells having chromosome 22q11.2 deletion: an autopsy report of DiGeorge syndrome.

Reported herein was the first autopsy case of Epstein-Barr virus-associated T-cell lymphoma in a 25-year-old man with DiGeorge syndrome. Systemic lymph nodes demonstrated diffuse encasement by large lymphoma cells positive for CD45, CD2, CD3, CD5, CD7, CD8, TIA1, and granzyme B, accompanied with marked hemophagocytosis. Almost 100% of lymphoma cells were both EBER- and LMP-1-positive, and EBNA2-negative. The rearrangement of T-cell receptor ß gene was proved by polymerase chain reaction. Clinical and pathologic features coincided with Epstein-Barr virus-associated T/NK-cell lymphoproliferative disorder preceded by chronic active Epstein-Barr virus infection. A fluorescence in situ hybridization using paraffin-embedded tissues demonstrated a mosaic chromosome 22q11.2 deletion with both host cardiac myocytes and lymphoma cells, suggesting that Epstein-Barr virus-associated T-cell lymphoma was associated with and derived from the cells carrying the chromosomal abnormality. Furthermore, the lymphomagenesis of our case correlated with defect of cellular immunity in DiGeorge syndrome.

Refining the diagnosis of T-cell large granular lymphocytic leukemia by combining distinct patterns of antigen expression with T-cell clonality studies.

T-cell large granular lymphocytic (LGL) leukemia is a complex diagnosis, requiring persistent clonal expansions of LGLs, and cytopenias. Often the diagnosis is unclear as non-clonal expansions of LGLs commonly occur in reactive conditions. To better understand T-LGL leukemia, we performed a comprehensive clinicopathologic analysis of 85 patients with LGL expansions. Interestingly, distinct CD8+(dim)/CD57+ populations, seen by flow cytometry, were significantly associated with clonal T-LGL leukemia (P < 0.001) as well as neutropenia (median absolute neutrophil count (ANC) 1.45 vs 3.19 × 10(9)/l; P = 0.0017). Furthermore, cases with distinct CD8+(dim)/CD57+ populations and monoclonal T cells had even lower ANCs (median ANC 1.41 × 10(9)/l; P = 0.001) compared with cases without these dual criteria. Additionally, complete or partial loss of CD5 expression was independently associated with clonal T-LGL leukemia (P<0.001) and neutropenia (median ANC 1.41 vs 2.70 × 10(9)/l; P = 0.002). This study describes specific immunophenotypic parameters to better define clonal cases of T-LGL leukemia associated with significant neutropenia.

Measurement of absolute T cell receptor rearrangement diversity.

T cell receptor (TCR) diversity is critical for adaptive immunity. Existing methods for measuring such diversity are qualitative, expensive, and/or of uncertain accuracy. Here, we describe a method and associated reagents for estimating the absolute number of unique TCR Vß rearrangements present in a given number of cells or volume of blood. Compared to next generation sequencing, this method is rapid, reproducible, and affordable. Diversity of a sample is calculated based on three independent measurements of one Vß-Jß family of TCR rearrangements at a time. The percentage of receptors using the given Vß gene is determined by flow cytometric analysis of T cells stained with anti-Vß family antibodies. The percentage of receptors using the Vß gene in combination with the chosen Jß gene is determined by quantitative PCR. Finally, the absolute clonal diversity of the Vß-Jß family is determined with the AmpliCot method of DNA hybridization kinetics, by interpolation relative to PCR standards of known sequence diversity. These three component measurements are reproducible and linear. Using titrations of known numbers of input cells, we show that the TCR diversity estimates obtained by this approach approximate expected values within a two-fold error, have a coefficient of variation of 20%, and yield similar results when different Vß-Jß pairs are chosen. The ability to obtain accurate measurements of the total number of different TCR gene rearrangements in a cell sample should be useful for basic studies of the adaptive immune system as well as in clinical studies of conditions such as HIV disease, transplantation, aging, and congenital immunodeficiencies.

Churg-Strauss syndrome associated with elevated levels of serum interleukin-5 and T cell receptor-Cß gene rearrangement.

A 58-year-old woman was diagnosed with Churg-Strauss syndrome (CSS) based on the symptoms of bronchial asthma, eosinophilia, mononeuritis multiplex and histological examination of the right sural nerve. Prior to treatment, the serum interleukin (IL)-5 level was high, and rearrangement of the T cell receptor (TCR) gene was identified. This is the first report of TCR gene rearrangement in a patient with CSS. The expanded T cell clone may be responsible for the overproduction of IL-5. Further studies are warranted to disclose a prevalence of TCR gene rearrangement in CSS patients and its pathophysiological roles in the development of this disease.

CD8(+) T-cell lymphoproliferative disorder associated with Epstein-Barr virus in a patient with rheumatoid arthritis during methotrexate therapy.

A 75-year-old woman with rheumatoid arthritis (RA) who was receiving methotrexate (MTX) therapy developed Epstein-Barr virus (EBV)-associated CD8(+) T-cell lymphoproliferative disorder (LPD) and meningoencephalitis. She was successfully treated with acyclovir and corticosteroids plus MTX cessation. T-cell LPD is relatively rare in RA patients receiving MTX. To our knowledge, this is the first report of CD8(+) T-cell LPD with EBV genome occurring during MTX therapy for RA. EBV infection should be carefully monitored to assess severe EBV-associated complications.