The etiology of such abnormalities as lymphocytosis, neutropenia, and arthropathy diagnosed either by a rheumatologist or a hematologist often remains obscure. These clinical findings may be associated with the presence of circulating T-cell large granular lymphocytes (T-LGLs) 123. LGL disorders comprise a spectrum of polyclonal, oligoclonal, or monoclonal expansions 4, which arise mostly from mature, activated cytotoxic T lymphocytes (T-LGL) CD3⁺/CD8⁺/CD57⁺/CD16⁺ and less often from natural killer cells (NK-LGL) CD3-/CD2⁺/CD56⁺/CD16⁺ 5. Clinically pronounced monoclonal proliferation of T-LGLs with bone marrow and spleen infiltration is diagnosed as T-LGL leukemia, a rare, indolent, chronic disorder with characteristic features such as mild lymphocytosis, neutropenia, and anemia. They may be autoimmune by nature or result from a T-cell-mediated suppressor effect on hemopoesis 67. The T-LGL leukemia diagnosis is confirmed by monoclonal T-cell receptor (TCR) gene rearrangement detected in abnormal CD3⁺/CD57⁺ cell populations 56. An interesting feature of T-LGL leukemia is its strong association with a number of autoimmune disorders and immunological abnormalities, most common in patients with rheumatoid arthritis (RA) (30% of patients), which usually precedes or develops concurrently with the hematological process 8910. Patients with T-LGL leukemia and accompanying RA closely resemble patients with Felty syndrome (FS) in clinical presentation: neutropenia, RA, variable splenomegaly, and immunogenetic findings such as a high prevalence of HLA-DR4 1112. Moreover, monoclonal T-LGL lymphocytosis may be found in up to one third of FS patients 11131415. Burks and Loughran 7 suggest that these two entities represent variants of the same clinicopathologic process. The aim of the present study was to perform an extensive clinical, histopathological, flow cytometric as well as genetic evaluation of 21 patients with T-LGL lymphocytosis associated with inflammatory arthropathy or with no arthritis symptoms. Our results demonstrate that patients with RA and neutropenia represent a continuous spectrum of T-LGL proliferations although monoclonal expansions are observed most frequently. The histopathological pattern and immunophenotype of the bone marrow infiltration as well as molecular characteristics were similar in T-LGL leukemia patients with and without arthritis.

A group of 21 patients with lymphocytosis and neutropenia, including several with arthropathy and splenomegaly, was enrolled in this study. Written informed consent was obtained from all of the patients, and the study was approved by the local bioethical committee of the Institute of Hematology and Transfusion Medicine in Warsaw. Complete blood count with manual differential analysis of blood cells was performed in all cases. Blood smears stained with May-Grünwald-Giemsa were examined for the presence of large granular lymphocytes.

Features of articular disease were defined in terms of duration and diagnosis (American Rheumatism Association [ARA] criteria for diagnosis of RA) 16. In some patients, tests were done for rheumatoid factor (RF) (nephelometry), anticyclic citrullinated peptide (CCP) antibodies and anticardiolipin antibodies (aCLs) (enzyme-linked immunosorbent assay, ELISA), antinuclear antibodies (ANAs) (Hep2 cells), and cytoplasmic and perinuclear antineutrophil cytoplasmic antibodies (ELISA), depending on the clinical presentation of the patient.

Trephine biopsies of all 21 patients were histopathologically examined. They were fixed in Oxford fixative, routinely processed, and stained with hematoxylin and eosin. Immunohistochemical studies were done (EnVision™ Detection Systems) (Dako Denmark A/S, Glostrup, Denmark) (DAKO) using the following mono- and polyclonal antibodies: CD3, myeloperoxydase, hemoglobin (polyclonal), CD20 (L26), CD8 (C8/144B) (DAKO) and CD4 (4B12), CD57 (NK-1), and granzyme B (11F1) (Novocastra, now part of Leica Microsystems, Wetzlar, Germany). Positive and negative controls were included.

Immunophenotyping of peripheral blood lymphocytes was performed in 15 patients with a three-color FACScalibur cytometer (flow cytometry, FCM) (Becton Dickinson, San Jose, CA, USA) (BD) and analyzed by CellQuest software (BD). Lymphocytes were treated with monoclonal antibodies against CD45 and HLA-DR; pan-B antigen: CD19 (BD); pan-T antigens: CD3 (DAKO), CD2, CD4, CD5, CD7, CD8, CD43, TCRαβ, and TCRγδ (BD); and NK-specific markers: CD16 (DAKO), CD56 (BD), CD57 (Sigma-Aldrich, St. Louis, MO, USA), and human IL-2 Rα receptor CD25 (BD). Isotype controls were used.

TCR genes as well as immunoglobulin heavy-chain (IGH) and kappa (IGK) and lambda (IGL) light-chain gene rearrangements were tested in 19 patients following the BIOMED-2 protocol 17. DNA was isolated from blood/bone marrow mononuclear cells with the column method (Qiagen, Hilden, Germany) after Ficoll separation. TCRBV-TCRJ gene rearrangements were tested using 23 forward and 9 reverse primers (V_β-J_β1, J_β2) and 23 forward and 4 reverse primers for regions V_β-J_β2 and 2 forward and 13 reverse primers for regions D_β1, D_β2-J_β. TCRG gene rearrangements were tested using 2 forward and 2 reverse primers for regions V_γ If, V_γ10-J_γ and 2 forward and 2 reverse primers for regions coding V_γ9, V_γ11-J_γ. TCRD gene rearrangements were tested using 7 forward and 5 reverse primers for regions coding V_δ, D_δ2-J_δ, D_δ3. The IGH gene rearrangement test consisted of three multiplex polymerase chain reaction (PCR) tubes with 27 forward and 5 reverse primers, IGK tests consisted of 2 multiplex PCR tubes with 13 forward and 3 reverse primers, and the IGL test consisted of 1 multiplex PCR tube with 6 forward and 2 reverse primers. PCR products underwent heteroduplex analysis (95°C for 5 minutes and 4°C for 60 minutes) and were separated using electrophoresis on polyacrylamide gel and visualized by ethidium bromide. Cytogenetic studies on bone marrow aspirate samples of 7 patients were performed using a G-banding technique, and the results were analyzed according to International System for Human Cytogenetic Nomenclature (1995). The T-LGL leukemia diagnosis was made according to the World Health Organization (WHO) classification 6 in cases with monoclonal LGL lymphocytosis CD3⁺/CD57⁺/TCRαβ⁺/γδ⁺ of more than 6 months in duration. Cases with circulating LGLs of greater than 2 × 10⁹/L in peripheral blood as well as patients with leucopenia and smaller expansions of LGL were included. The diagnosis of T-LGL lymphocytosis in 21 patients was based on blood and bone marrow tests, including immunophenotypic and molecular studies.

The pathogenic relationship between RA and various T-LGL proliferations that are a spectrum of disorders from reactive expansion of 'normal' CD8⁺ cytotoxic T lymphocytes through chronic oligoclonal or monoclonal LGL lymphocytosis to clinically overt T-LGL leukemia is unclear 4. This association is considered rare, perhaps due to underdiagnosis of T-LGL proliferations as the cause of neutropenia in patients with RA 3. We have evaluated data of 21 T-LGL lymphocytosis patients and identified a relatively high proportion of patients with arthropathy, also reported by some authors 3101920. Nine patients had RA and neutropenia and some also had splenomegaly and were diagnosed as FS, whereas 2 other patients showed milder non-erosive unclassified arthritis. Our FS patients presented a spectrum of T-LGL expansions.

It is difficult to differentiate between T-LGL leukemia and reactive T-LGL proliferations, especially in autoimmune diseases. Molecular studies may establish the clonal nature of T cells but not in all cases confirm the diagnosis of leukemia as oligoclonal/monoclonal proliferations of T-LGL may occur in natural or pathologic immune responses to strong antigens in autoimmune diseases or viral infections such as Epstein-Barr virus and HIV 21. A monoclonal population of CD8⁺, CD57⁺ T cells was found both in neutropenic and non-neutropenic patients with RA as well as in healthy, mostly elderly individuals 11222324. All these cases represent a benign monoclonal T-LGL proliferation rather than true T-LGL leukemia because they are clinically asymptomatic. Moreover, diagnostic criteria of T-LGL leukemia in the context of the LGL population volume are still a subject of discussion. Monoclonal LGL lymphocytosis of greater than 2 to 5 × 10⁹/L is required for the diagnosis of T-LGL leukemia according to the WHO classification 6. However, in patients with monoclonal T-LGL lymphocytosis and leucopenia, this criterion is not useful. Expansion of LGLs of less than 0.5 × 10⁹/L represents reactive lymphocytosis 6, but there are also borderline cases with T-LGL oligoclonal or monoclonal lymphocytosis values ranging from 0.5 to 2 × 10⁹/L. For such cases, Dhodapkar and colleagues 10 suggested the term 'T cell clonopathy of undetermined significance' or 'monoclonal clonopathy of unclear significance'. However, Semenzato and colleagues 25 described 9 patients with chronic monoclonal T-LGL lymphocytosis of 0.5 to 2 × 10⁹/L with clinical and laboratory features typical for T-LGL leukemia. Thus, they pointed out that, for T-LGL leukemia diagnosis, the LGL count by itself is not critical but a comprehensive analysis of clinical, immunopathological, and molecular data is necessary. However, the border between leukemic and reactive clonal expansions of T-LGLs is narrow because clinical and hematologic characteristics of T-LGL leukemia are far from typically malignant 9.

In our group of patients with arthritis, 9 presented symptoms of FS. Three patients (patients 2, 4, and 5) fulfilled the WHO criteria for T-LGL leukemia diagnosis with absolute LGL lymphocytosis of greater than 2 × 10⁹/L and monoclonal rearrangement of TCRB, TCRG, or TCRD genes. In the bone marrow, they had interstitial and intrasinusoidal linear infiltration of T cells expressing CD3, CD8, CD57, and granzyme B described by Morice and colleagues 26 as a typical pattern for T-LGL leukemia. Intrasinusoidal infiltration seems to indicate a leukemic nature of the disease and may occur in other subtypes of leukemia/lymphoma 27. Reactive lymphoid nodules, reported by Osuji and colleagues 28 to be frequent in T-LGL leukemia, were also present. In FCM, T-LGL leukemia cells revealed abnormal expression of CD5, CD7, and CD43 antigen, which corresponds to the findings of Lundell and colleagues 29. The other 6 patients with FS showed relative LGL lymphocytosis but had absolute LGL lymphocytosis of less than 2 × 10⁹/L due to leucopenia.

In three of those 6 patients (patients 1, 3, and 6) with LGL lymphocytosis ranging from 0.9 to 1.4 × 10⁹/L, a clearly monoclonal rearrangement of TCRB and TCRG genes as well as typical for T-LGL leukemia immunophenotype were found. However, interstitial infiltration of the bone marrow was most common. In one patient, FCM showed abnormal expression of one T-cell antigen. The question is whether these cases should be diagnosed as true T-LGL leukemia or as a T-cell clonopathy of unclear significance associated with FS.

Our patient 7 displayed features of monoclonal transformation of polyclonal LGLs. In PCR analysis, apart from monoclonal TCRD gene rearrangement, a weak monoclonal product in the V_γ9, V_γ11-J_γ region among polyclonal background was found. Abundant interstitial infiltrates of T cells were observed in the bone marrow, but without intrasinusoidal localization. This case seems to support the hypothesis that T-LGL leukemia may develop from polyclonal or oligoclonal expansions 30 and correlates with the report of Langerak and colleagues 31, who found a single dominant and several weak additional gene products in β-chain variable region (V_β) transcripts in numerous T-LGL leukemia cases.

Two patients (patients 10 and 11) with no rearrangement of TCR genes may serve as examples of autoimmune-disease-related, polyclonal, reactive, chronic proliferation of LGL lymphocytes. In these two cases, the pattern of bone marrow infiltration and FCM analysis were consistent with reactive T-LGL lymphocytosis 2629. T-LGLs were dispersed in the bone marrow and they did not lose any pan-T-cell antigens in FCM.

As in other reports 29, FCM analysis of all of our cases with clonal rearrangement of TCR genes showed normal reactive peripheral blood CD57⁺ T lymphocytes with no pan-T-cell antigen abnormalities in addition to a population of CD57⁺ T-LGL leukemia cells. It is conceivable that this population of cells may represent precursors of T-LGL leukemia.

The clinical course of 9 patients with FS, irrespective of T-LGL absolute count and their clonality, was similar, with neutropenia being the most common presentation, and remained indolent for 1 to 7 years of follow-up. They were treated with immunomodulatory agents exclusively, and 2 patients had partial hematologic responses.

The described group of patients represents a spectrum of T-LGL proliferations and supports the hypothesis that monoclonal expansion of LGLs with features of T-LGL leukemia may result from the transformation of initially stable and benign polyclonal or oligoclonal proliferation of these lymphocytes 30. This proliferation may be a consequence of the disregulated reaction of the immune system to viral or auto-antigens associated with autoimmune processes 3032. There are similarities in immunogenetic profile between T-LGL leukemia and autoimmune diseases 32. Moreover, molecular analysis revealed common motifs in the TCRB genes in T-LGL proliferations, suggesting a potential role of antigenic stimulation in the clonal evolution of the disease 333435. Unfortunately, the triggering antigens and genetic events that cause neoplastic transformation remain unknown. Bowman and colleagues 11 examined two groups of patients with FS, without and with clonal proliferation of T-LGLs, but no continuous distributions of T-LGLs in these two groups were observed. Recently, Langerak and colleagues 4 and Sandberg and colleagues 36 have shown a continuous spectrum of T-LGL proliferations using sensitive PCR techniques.

It is worth emphasizing that, in 4 of our patients, IGKV and IGLV gene rearrangements were detected in PCR analysis. Trephine biopsy examination showed a nearly normal number of B lymphocytes dispersed and/or localized in lymphoid nodules with the pattern and phenotype indicative of their reactive nature. This phenomenon most probably is due to chronic antigen stimulation in autoimmune disease 37. It can also correspond to crosslineage light-chain gene rearrangements. None of our patients developed a B-cell lymphoma during follow-up.

Most of our patients had long-lasting RA at the time of diagnosis of T-LGL leukemia, but 2 patients showed milder non-erosive unclassified arthritis resembling RA. Similar findings had been reported by Snowden and colleagues 3. Interestingly, these 2 patients had similar clinical features: T-LGL absolute count of less than 2 × 10⁹/L due to leucopenia, severe neutropenia, recurrent infections, skin lesions, splenomegaly, and monoclonal rearrangements of TCRB genes. Bone marrow infiltration was typical for T-LGL leukemia, but T-LGL cells had the unusual phenotype (CD4⁺). In one of these patients, arthropathy appeared after 17 years of hematological abnormalities, which indicates that T-LGL expansion may also precede inflammatory arthropathy and may be responsible for the development of immunological abnormalities 8. TCRαβ⁺/CD4⁺ T-LGL lymphocytosis is a rare subgroup of monoclonal LGL lymphoproliferations and usually is characteristic of a more indolent clinical course 35.

Patients with T-LGL leukemia with or without arthropathy share many similarities. Arthritis and T-LGL leukemia patients more often had leucopenia and splenomegaly and a higher incidence of specific autoantibodies as compared with those without arthropathy. The two groups showed a similar histopathological pattern and immunophenotype of bone marrow infiltration. They also had similar molecular and genetic characteristics with the most frequent rearrangements of V_β-J_β1, J_β2 and V_γ If V_γ10-J regions of TCR genes and normal karyotype. In other studies, FCM analysis of the V_β TCR repertoire in T-LGL leukemia cases did not reveal the preferential use of any specific V_β gene 293038. Davey and colleagues 20 reported that rearrangement of V_β-6 genes occurred only in patients with T-LGL leukemias associated with RA, although no unique patterns of junctional sequence rearrangement were seen for patients with T-LGL leukemia with and without arthritis.

Cytopenias (especially neutropenia) that appear in the course of RA-associated T-LGL lymphocytosis are the result of humoral and cellular immune mechanisms 7. Immune complexes or antibodies to neutrophil antigens may induce apoptosis of neutrophils in patients with FS 39. Compensatory granulocytic hyperplasia with left-shifted maturation is observed in bone marrow as the consequence of excessive peripheral destruction of mature granulocytes 7. In the described group of patients, only two with FS (one with polyclonal LGL lymphocytosis, the other with T-LGL leukemia) had hypercellular bone marrow with increased granulocyte precursor count and left-shifted maturation, but no antyneutrophil antibodies were present. The majority of our patients with T-LGL leukemia with and without RA showed a decreased or normal number of granulocyte precursor count in the bone marrow with left-shifted maturation. There is a hypothesis that T-LGL leukemia cells may inhibit proliferation and differentiation of granulocytes in the mechanisms of Fas-mediated apoptosis of myeloid precursors and this could be the explanation of granulocyte hypoplasia and paucity of mature forms in the bone marrow 7. Effector cytotoxic T cells as well as T-LGL leukemia cells express Fas ligand, a member of the tumor necrosis factor family, which can bind Fas and initiate apoptosis in the target cells through this pathway 40. It is known that progenitor cells (CD34⁺) in the bone marrow may express Fas in response to tumor necrosis factor-alpha (TNF-α) and interferon-gamma (INF-γ) in vitro 41 and cytotoxic T lymphocytes in T-LGL leukemia may spontaneously produce INF-γ and TNF-α after stimulation 42.

RA and neutropenia patients represented a continuous spectrum of T-LGL proliferations, although monoclonal expansions were most frequently observed. Arthritis and T-LGL leukemia patients more often had leucopenia and splenomegaly and a higher incidence of specific autoantibodies as compared with those without arthropathy. The histopathological pattern and immunophenotype of bone marrow infiltration, molecular and genetic characteristics, as well as indolent clinical course were similar in T-LGL leukemia patients, with and without arthritis. Further studies involving patients with RA and T-LGL proliferations may provide important data for a better understanding of RA pathogenesis.

aCL = anticardiolipin antibody; ANA = antinuclear antibody; ARA = American Rheumatism Association; BD = Becton Dickinson, San Jose, CA, USA; CCP = anticyclic citrullinated peptide (antibody); CSA = cyclosporine A; ELISA = enzyme-linked immunosorbent assay; FCM = flow cytometry; FS = Felty syndrome; G-CSF = granulocyte-colony stimulating factor; IGH = immunoglobulin heavy chain; IGK = immunoglobulin kappa; IGKV = immunoglobulin kappa variable; IGL = immunoglobulin lambda; IGLV = immunoglobulin lambda variable; INF-γ = interferon-gamma; LGL = large granular lymphocyte; MTX = methotrexate; NK = natural killer; PCR = polymerase chain reaction; PR = partial response; RA = rheumatoid arthritis; RF = rheumatoid factor; TCR = T-cell receptor; T-LGL = T-cell large granular lymphocyte; TNF-α = tumor necrosis factor-alpha; WHO = World Health Organization.

The authors declare that they have no competing interests.

MP-S conceived of the study, carried out histopathological studies, analyzed data, and wrote the manuscript. GR carried out the flow cytometry studies. MM and HMŁ carried out the molecular genetic studies. KM and RR analyzed clinical data of patients. KW and RM critically reviewed the manuscript. All authors read and approved the final manuscript.