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The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I–binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2–restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT137–45–specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT137–45, have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.

We have recently described a new form of light chain deposition disease (LCDD) presenting as a severe cystic lung disorder requiring lung transplantation. There was no bone marrow plasma cell proliferation. Because of the absence of disease recurrence after bilateral lung transplantation and of serum-free light chain ratio normalization after the procedure, we hypothesized that monoclonal light chain synthesis occurred within the lung. The aim of this study was to look for the monoclonal B-cell component in 3 patients with cystic lung LCDD. Histologic examination of the explanted lungs showed diffuse nonamyloid κ light chain deposits associated with a mild lymphoid infiltrate composed of aggregates of small CD20+, CD5−, CD10− B lymphocytes reminiscent of bronchus-associated lymphoid tissue. Using polymerase chain reaction (PCR), we identified a dominant B-cell clone in the lung in the 3 studied patients. The clonal expansion of each patient shared an unmutated antigen receptor variable region sequence characterized by the use of IGHV4-34 and IGKV1 subgroups with heavy and light chain CDR3 sequences of more than 80% amino acid identity, a feature evocative of an antigen-driven process. Combined with clinical and biologic data, our results strongly argue for a new antigen-driven primary pulmonary lymphoproliferative disorder.

We report on the characteristics of 21 patients with hepatosplenic gammadelta T-cell lymphoma (HSgammadeltaTCL), an entity recognized since 1994 in the Revised European American Lymphoma (REAL) classification. Median age was 34 years. Patients had splenomegaly (n = 21), hepatomegaly (n = 15), and thrombocytopenia (n = 20). Histopathologic findings were homogeneous and showed the presence of medium-sized lymphoma cells within the sinusoids of splenic red pulp, liver, and bone marrow. Marrow involvement was usually mild but could be demonstrated by phenotyping in all patients. Cells were CD3+CD5-, expressed the gammadelta T-cell receptor, and had a nonactivated cytotoxic cell phenotype (TIA-1+, granzyme B-). Most patients were CD4-/CD8- (16 of 18); CD56+ (15 of 18), expressed the Vdelta1epitope (Vd1+/Vd2-/Vd3-) (9 of 12); and were negative for Epstein-Barr virus (EBV) (18 of 20). Isochromosome arm 7q was documented in 9 of 13 patients. Eight patients had previously undergone kidney transplantation or had a history of systemic lupus, Hodgkin disease, or malaria. Prognosis was poor; median survival time was 16 months, and all but 2 patients ultimately died despite consolidative or salvage high-dose therapy. In conclusion, HSgammadeltaTCL is a disease with distinctive clinical, histopathologic, and phenotypic characteristics. Bone marrow biopsy with combined phenotyping is sufficient for diagnosis, and splenectomy is therefore unwarranted. Current treatment modalities appear to be ineffective in most patients.

It is now widely accepted that polymerase chain reaction (PCR) analysis of cutaneous T-cell clonality is of diagnostic value in cutaneous T-cell lymphomas (CTCLs) and most helpful in the diagnosis of mycosis fungoides (MF). However, the diagnostic and prognostic value of circulating clonal T cells remains unclear. We studied T-cell clonality in the peripheral blood (PB) and the cutaneous lesion, sampled at the same time, in 363 consecutively seen patients with a clinical suspicion of cutaneous lymphoma. Using a PCR technique providing a specific imprint of T-cell clones (PCRγ–denaturing gradient gel electrophoresis), we found that detection of identical circulating and cutaneous T-cell clones was associated with the diagnosis of CTCL (P

Non-Hodgkin's lymphomas are divided into B- and T-cell neoplasms. The existence and the clinical relevance of lymphomas derived from the third lymphocyte lineage, ie, natural killer (NK) cells are still controversial. NK cells are lymphocytes that mediate cytotoxicity without prior sensitization. NK cells also have phenotypic and genotypic characteristics: they express the NK-related antigen CD56, T- cell markers such as CD2 and CD7, but do not express CD5 and T-cell receptor (TCR) proteins, and their TCR locus is not rearranged. Therefore, if NK cell lymphomas exist, they should express some T-cell markers, but not alpha beta or gamma delta TCR proteins. Such lymphomas are actually called TCR silent peripheral T cell lymphomas (PTCL). To detect and characterize NK cell lymphomas, we investigated the immunophenotype and immunogenotype of 35 patients with TCR silent PTCL. The first group included 16 patients with a lymphoma of CD5-CD56+ phenotype, which is identical to normal NK cells. These patients had either a nasal/nasopharyngeal lymphoma (11 cases) or a lymphoma with predominant non-nasal/non-nodal initial involvement (five cases). Eight of the nine cases for which immunogenotypic data were available lacked clonal rearrangement of the TCR gamma genes. Thus, these tumors are likely to be NK cell lymphomas. The second group of 15 cases had a CD5+ phenotype (14 were CD56-, and 1 was CD56+) and clonal rearrangement of TCR gamma genes, indicating that they were true PTCL with unproductive TCR rearrangement. The four remaining cases were CD5- CD56- lymphomas and disclosed either a clonal (two cases) or no clonal (two cases) rearrangements of the TCR gamma genes. Altogether these findings show that CD5-CD56+ so-called “TCR silent PTCL” bear the immunophenotype and immunogenotype of normal NK cells and display peculiar clinical features distinct from true PTCL.

Intravenous (i.v.) infusions of Ig concentrates are an effective but expensive treatment for patients with autoimmune thrombocytopenic purpura (AITP). The optimal treatment protocol and the long-term results are uncertain, and the precise mechanism by which the platelet count increases is poorly understood. Twenty adult patients with chronic AITP were enrolled in a prospective study to compare the respective efficacy of two high-dose IVIgG induction regimens (1 g v 2 g/kg body weight) and the long-term effect of six 1 g/kg body weight i.v. IgG reinfusions. An initial response was observed in all 18 evaluable patients: the platelet count increased to a mean value of 251 x 10(9)/L (range 72 to 836 x 10(9)/L) and the mean pretreatment platelet count was multiplied by 14.6. No difference in efficiency was observed between the two i.v. IgG dosages. The degree of the platelet count increment correlated in both groups with the increase in the clearance of antibody-coated red blood cells, measured by an isotopic method, but not with the serum IgG elevation. Treatment was considered to have failed in 11 patients, 90 days after the last i.v. IgG reinfusion (D90), because the platelet counts were comparable with pretreatment values. In contrast, a complete response was observed at D90 in five patients (mean platelet count: 184 x 10(9)/L; range: 150 to 250 x 10(9)/L) and a partial response at D90 was obtained in the remaining two patients (platelet counts: 70 and 104 x 10(9)/L). Five of the 7 responders at D90 kept a platelet count above 50 x 10(9)/L during the entire follow-up period (mean 33 months; range: 5 to 66) with no further treatment; unfortunately, no clinical or biologic criteria were found to be predictive of the long-term response. This study shows that an i.v. IgG infusion regimen of 1 g/kg body weight could safely replace the classical 2 g/kg body weight dosage, at least in patients who do not have life-threatening thrombocytopenia. Moreover, repeated i.v. IgG reinfusion could be an alternative for AITP patients in whom splenectomy is contraindicated.

Two leukemia patients, refractory to chemotherapy, were treated with T101-ricin A-chain immunotoxin (T101 IT). Patient 1 (T-ALL) received a single 13.5 mg dose of T101 IT IV (12-hour infusion). Patient 2 (B-CLL) was treated with a daily 25 mg dose of T101 IT IV (two-hour infusion) over three consecutive days. Patient 2 also received 300 mg of chloroquine IM on days two and three as enhancer. In vivo binding of T101 IT was demonstrated by FACS analysis using either an antimouse Ig- FITC or anti-A-chain-FITC antibodies. Following IT therapy, the expression of T65 antigen on target cells dropped to 50% and 20% of pretreatment levels, respectively. In patient 1, circulating blast cells remained unsaturated during therapy while in patient 2, cells were fully saturated for four to six hours following each infusion. Pharmacokinetic studies showed a rapid clearance of T101 IT after IV administration. Antimouse and anti-A-chain antibodies could not be detected. There were no treatment-related adverse effects. In patient 1 a rapid but transient decrease of target cells was observed, possibly related to the administration of the antibody part of T101 IT. In contrast, patient 2 showed a 40% reduction of the lymphocyte count, which remained stable over a period of 2 weeks. Such a clinical benefit following IT therapy in patient 2 could be ascribed to the absence of circulating free antigen and the complete saturation of target cells.

The cellular distribution of immunoglobulins in human malignant and normal B cells was investigated by immunoelectron microscopy by direct incubation of fixed cells with electron microscopy by direct incubation of fixed cells with peroxidase-coupled antibody. These conjugates penetrated into the cell, resulting in the simultaneous detection of surface and cytoplasmic immunoglobulins. The latter were seen as specific intracisternal staining of the perinuclear space and endoplasmic reticulum and occasionally of the Golgi complex. Plasma cells were frequently characterized by a heterogeneity of reactivity of the endoplasmic reticulum. Minute amounts of cytoplasmic immunoglobulin were demonstrated in cells without developed secretory organelles, such as lymphoma cells and lymphocytes from chronic lymphocytic leukemia (CLL). The method allowed us to define several subsets of cells according to the expression of surface and cytoplasmic immunoglobulins and thus to determine the stage of maturation of cells involved in monoclonal proliferation.

The nature of cells present in the blood, marrow, and spleen of patients with hairy cell leukemia is largely debated. These cells have been tentatively categorized on the basis of either monocytic or lymphocytic markers, and the accumulating data points to the fact that they share some characteristics of both cell types. Although hairy cells are known to lack myeloperoxidase-positive granules, present in normal human monocytes, we investigated the possible presence of other peroxidase activities differing from the granule-bound myeloperoxidase. The study was carried out with several methods based on the incubation of fixed and unfixed cells in the presence of diaminobenzidine and hydrogen peroxide. A peroxidase activity was found in hairy cells, located always in the endoplasmic reticulum but not in the Golgi apparatus or in any granule. By its cytochemical characteristics it appears to be closely related to that of tissue macrophages, activated blood monocytes, and other nonlymphocytic hematopoietic cells. This peroxidase is not found in lymphocytes with B or T phenotypes.

The phenotype of fresh and cultured leukemic cells from patients with hairy cell leukemia was studied using a panel of monoclonal antibodies in addition to the detection of peroxidase activity under electron microscopy. In fresh samples, the leukemic cells from 11 patients displayed predominantly a B phenotype, as judged by their reactivity with the B1 monoclonal antibody and surface immunoglobulin expression. Ultrastructural peroxidase activity, characteristic of hairy cells, was observed in all cases studied. When hairy cells were cultured in the presence of phytohemagglutinin and irradiated T cells, their phenotype converted from surface Ig+, B1+, OKT3-, OKT11- to surface Ig-, B1+, OKT3-, OKT11+. In contrast, the peroxidase activity remained unchanged. Some hairy cells were also OKM1+, but no conclusion could be made about the MO2 antigen, a more specific marker of monocytes. The variability of the phenotype in vivo and in vitro indicates that reliable markers are required for identifying hairy cells. When studied together, the staining by B1 monoclonal antibody and the ultrastructural detection of peroxidase, enable the identification of hairy cells with certainty.