George D. Eliopoulos

Specific depletion of alloreactive T cells in HLA‐identical siblings: a method for separating graft‐versus‐host and graft‐versus‐leukaemia reactions

Accumulating evidence indicates that alloreactive donor T cells confer both graft‐versus‐host (GVH) and graft‐versus‐leukaemia (GVL) reactivity following allogeneic bone marrow transplantation. We have developed a method to deplete alloreactive donor T cells with an immunotoxin targeting the α chain of the IL‐2 receptor. In patients with chronic myeloid leukaemia and their HLA‐identical sibling donors, we measured donor helper T‐lymphocyte precursor frequencies (HTLPf) against recipient peripheral blood mononuclear cells (PBMNC; donor versus host), recipient leukaemia cells (donor versus leukaemia) and third‐party PBMNC, before and after the depletion. In seven pairs there was a 4.3‐fold reduction of donor‐versus‐host HTLPf (P = 0.017), without a significant change in the donor frequencies against third party (P = 0.96). In eight further donor–recipient pairs, immunotoxin‐depleted donor versus patient PBMNC HTLPf 4.5‐fold (mean 1/155 000 before and 1/839 000 after depletion, P = 0.007). There was a smaller non‐significant 1.8‐fold reduction in donor‐versus‐leukaemia HTLPf from 1/192 000 to 1/334 000 (P = 0.19). These results suggest that selective T‐cell depletion can significantly deplete donor anti‐host reactivity while conserving antileukaemia reactivity in HLA‐matched donor–recipient pairs.

Increased apoptosis of bone marrow CD34+ cells and impaired function of bone marrow stromal cells in patients with systemic lupus erythematosus

The changes in bone marrow (BM) stem cell reserve and function and stromal cell function in patients with active systemic lupus erythematosus (SLE) were investigated. The study was carried out on seven SLE patients and 28 healthy controls using flow cytometry and in vitro cell culture assays. We found that patients had low CD34+ cells, compared with the control group, reflecting the decrease of both CD34+/CD38− and CD34+/CD38+ cells. Patient CD34+/Fas+ but not CD34−/Fas+ cells were significantly increased. Apoptotic (7AADdim) cells were higher among CD34+/Fas+ than among CD34+/Fas− cells, and individual values of apoptotic CD34+ cells strongly correlated with the number of CD34+/Fas+ cells. These findings are suggestive of a Fas‐mediated apoptosis accounting for the low CD34+ cells in SLE patients. Moreover, we found that patients had low numbers of granulocyte‐macrophage colony‐forming units (CFU‐GM) and erythroid burst‐forming units (BFU‐E), compared with the control group, and that the generation of colony‐forming cells in long‐term BM cultures was significantly reduced. Patient BM stroma failed to support allogeneic progenitor cell growth. In one patient, CD34+ cells were increased, apoptotic CD34+/Fas+ cells were normalized and defective stromal cell function was restored after autologous stem cell transplantation. We concluded that defective haemopoiesis in SLE patients is probably caused, at least in part, to the presence of autoreactive lymphocytes in BM.

Acute and chronic neutropenias. What is new

Abstract. Palmblad J, Papadaki HA, Eliopoulos G (The Karolinska Institute at Huddinge University Hospital, Stockholm, Sweden and University of Crete Medical School, Heraklion, Greece). Acute and chronic neutropenias. What is new? J Intern Med 2001; 250: 476–491.

Non-immune chronic idiopathic neutropenia of adult: an overview

Abstract: There is strong evidence that non‐immune chronic idiopathic neutropenia of adult is a cytokine‐mediated syndrome characterized by (a) neutropenia of varying degree associated with a low number of lineage‐specific CD34+ cells and increased production of inhibitors of hematopoiesis, including transforming growth factor‐β1 and tumor necrosis factor‐α; (b) lymphopenia due to selective loss of primed/memory T‐cells and NK cells; (c) increased splenic volume on ultrasonography in 48.1% of patients; (d) osteopenia and/or osteoporosis in 60.0% of patients; (e) anemia, mostly of the type of anemia of chronic disease, in 15.6% of patients; (f) features of chronic antigenic stimulation, including increased proportion of bone marrow plasma cells, increased serum levels of IgG1 and/or IgA, increased frequency of monoclonal gammopathy of undetermined significance, increased frequency of antinuclear antibodies with specific reactivity, and increased serum levels of circulating immune complexes; and (g) increased concentrations of a variety of macrophage‐derived pro‐inflammatory cytokines and chemokines capable of affecting bone metabolism, bone marrow function, and leukocyte trafficking. All these findings are suggestive of the existence of an unrecognized low‐grade chronic inflammatory process which may be involved in the pathogenesis of the disorder. Neutropenia in these patients is probably the result of a combination of at least three factors, reduced neutrophil production in bone marrow, enhanced neutrophil extravasation, and increased sequestration and/or extravasation of neutrophils into the spleen.

Activated T‐lymphocytes with myelosuppressive properties in patients with chronic idiopathic neutropenia

To characterize the cellular components responsible for the impaired granulopoiesis in chronic idiopathic neutropenia (CIN), we investigated the origin of the proapoptotic cytokine producing cells in the bone marrow (BM) microenvironment of CIN patients. We found that the interferon gamma (IFNγ) and/or Fas‐ligand expressing cells in patient BM mononuclear cells and long‐term BM culture stroma cells were the CD3+ T‐lymphocytes but not the CD14+ monocytes/macrophages. The percentage of activated T‐lymphocytes was increased in patients’ BM as indicated by the proportions of human leucocyte antigen (HLA)‐DR+, CD25+, CD38+, CD69+ and Fas+ cells within the CD3+ fraction. Intracellular IFNγ expression was higher in the BM than peripheral blood of the patients and was associated with increased BM T‐lymphocyte numbers. In crossover experiments, patient CD3+ T‐lymphocytes conferred autologous and allogeneic haemopoietic progenitor cell colony inhibition. Patients’ T‐cell receptor repertoire and polymerase chain reaction analysis did not reveal any clonal T‐lymphocyte expansion, suggesting the absence of a direct, antigen‐driven recognition of CD34+ myeloid progenitor cells by patient T‐lymphocytes. We conclude that CIN patients have increased number of activated T‐lymphocytes in the BM, probably in the setting of a localized polyclonal immune reaction and that these cells confer an inhibitory effect on myelopoiesis through myelosuppressive cytokines including Fas‐ligand and IFNγ.

Serum levels of soluble IL-6 receptor in multiple myeloma as indicator of disease activity.

Serum soluble interleukin-6 receptor (sIL-6R) concentrations were measured in 50 patients with plasma cell dyscrasias using a commercially available immunoenzymatic assay kit. There were 40 patients with multiple myeloma (MM), 5 patients with monoclonal gammopathy of undetermined significance (MGUS), 3 patients with solitary plasmacytoma (SPC), 1 patient with chronic myelogenous leukaemia and multiple myeloma (CML/MM), and 1 patient with plasma cell leukaemia (PCL). We found that serum sIL-6R concentrations were higher in MM patients (62.53 +/- 38.85 ng/ml) than in 20 normal volunteers studied (36.75 +/- 13.79 ng/ml) (p < 0.01). The cut-off value of 65 ng/ml seen in 2 of our controls was arbitrarily taken as the upper limit of the control range for serum sIL-6R; according to this criterion, 14 patients with MM (35%), 1 patient with SPC, the unique patient with CML + MM, and the unique patient with PCL had elevated concentrations of the receptor. Patients with MGUS had normal sIL-6R values. In MM patients, serum sIL-6R levels correlated with the clinical phase of the disease: they were elevated in patients with early or late active disease and ranged within normal limits in patients with plateau-phase disease (p < 0.001). Thirteen of 27 patients with active MM had elevated serum sIL-6R values, i.e. 48.1%, but only 1 out of 13 patients with disease in the plateau phase, i.e. 7.7% (p < 0.05). Furthermore, in the entire group of MM patients, serum sIL-6R levels correlated with the concentrations of serum beta 2-microglobulin, (p < 0.02), CRP (p < 0.01), ferritin (p < 0.01) and LDH (p < 0.01), while they did not correlate with disease stage, haemoglobin levels, proportion of marrow myeloma cells, the values of serum IL-6, the levels of serum albumin, or the grade of bone lesions. We conclude that elevated serum sIL-6R levels should be related to the growth of myeloma cells and suggest that serum sIL-6R concentrations may be used as an indicator of disease activity.

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis.

Summary:Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.

Expression of the Tpl2/Cot oncogene in human T-cell neoplasias.

BackgroundTpl2/Cot oncogene has been identified in murine T-cell lymphomas as a target of MoMuLV insertion. Animal and tissue culture studies have shown that Tpl2/Cot is involved in interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) production by T-cells contributing to T-cell proliferation. In the present report we examined a series of 12 adult patients with various T-cell malignancies, all with predominant leukemic expression in the periphery, for the expression of Tpl2/Cot oncogene in order to determine a possible involvement of Tpl2/Cot in the pathogenesis of these neoplasms.ResultsOur results showed that Tpl2/Cot was overexpressed in all four patients with Large Granular Lymphocyte proliferative disorders (LGL-PDs) but in none of the remaining eight patients with other T-cell neoplasias. Interestingly, three of the LGL-PD patients displayed neutropenia, one in association with sarcoidosis. Serum TNF-α levels were increased in all Tpl2/Cot overexpressing patients while serum IL-2 was undetectable in all subjects studied. Genomic DNA analysis revealed no DNA amplification at the Tpl2/Cot locus in any of the samples analyzed.ConclusionsWe conclude that Tpl2/Cot, a gene extensively studied in animal and tissue culture T-cell models may be also involved in the development of human LGL-PD and may have a role in the pathogenesis of immune manifestations associated with these diseases. This is the first report implicating Tpl2/Cot in human T-cell neoplasias and provides a novel molecular event in the development of LGL-PDs.

Systemic mastocytosis: a rare cause of noncirrhotic portal hypertension simulating autoimmune cholangitis—report of four cases

Four patients with systemic mastocytosis, two men and two women, are presented. Three of them (patients 1, 2, and 4) developed portal hypertension and ascites without histological evidence of cirrhosis in liver biopsy. The remaining patient (patient 3) had severe bone lesions with multiple vertebral fractures. None of the patients had skin or lymph node involvement. Two patients (patients 1 and 2) died 12 and 9 months after diagnosis with acute nonlymphocytic leukemia and overt mastocytic leukemia, respectively, while the other two (patients 3 and 4) are alive 58 and 14 months after diagnosis. Treatment with hydroxyurea or cytosine arabinoside had not any beneficial effect in two patients, while a substantial amelioration of back pain had been obtained by local irradiation and recombinant human interferon-a-2b administration in one patient (patient 3). All patients had laboratory findings compatible with autoimmune cholangitis. We concluded that systemic mastocytosis is a rare cause of noncirrhotic portal hypertention often simulating autoimmune cholangitis and leading to the erroneous diagnosis of liver cirrhosis. Diagnosis is based on the presence of mast cells in Giemsa-stained liver histological sections, and it may be confirmed by immunohistochemical detection of tryptase in the cytoplasm of these abnormally proliferating cells.

Epstein-Barr Virus-Associated High-Grade Anaplastic Plasmacytoma in a Renal Transplant Patient

Allograft transplant patients have an increased risk of developing polyclonal or monoclonal lymphoproliferative disorders, but high-grade anaplastic plasmacytomas are extremely rare in these patients. We present a renal transplant patient who developed multiple extramedullary high-grade anaplastic plasmacytomas in the oral cavity, the left maxillary antrum, the scalp, the thigh and the upper abdominal wall with no evidence of diffuse bone marrow infiltration. Epstein-Barr virus (EBV) mRNA transcripts were detected within the myeloma cells by in situ hybridization using EBER 1–2 probes. Following discontinuation of immunosuppression applied, the patient was treated with a cyclophosphamide-prednisone regimen followed by local irradiation, and a complete remission was achieved within four weeks. We concluded that EBV-associated high-grade anaplastic plasmacytomas constitute one more type of post-transplant lymphoproliferative disorder, and that despite their characterization as highly malignant neoplasms, their clinical behavior is not always aggressive.