Cells mediating specific in vitro cytotoxicity. II. Probable autonomy of thymus-processed lymphocytes (T cells) for the killing of allogeneic target cells.

In order to investigate whether only T cells are involved in a cell-mediated cytotoxic system in vitro, we tested the cytotoxicity of immune killing cell populations as deprived as possible of B cells. Educated thymus cells, immune spleen cells purified by filtration through a column of beads coated with antimouse Ig antiserum, and finally educated thymus cells further purified by filtration through such a column fully retained their specific cytotoxic activity. This very strongly suggests that only T cells are involved in the killing of target cells by allogeneic immune cells in vitro, in this system. Receptor-bearing cells involved in killing in the present system are thus very probably T cells. This point was further strengthened by the demonstration of specific adsorption, on the relevant monolayers, of each of the three above mentioned killing cell populations.

T-cell response to B-cells and Epstein-Barr virus antigens in infectious mononucleosis.

After Epstein-Barr virus (EBV) infection in vivo, B-cells with latent virus infection persist indefinitely through life. These cells grow in vitro on explanation and can be established as immortal B-cell lines. To reconcile the unlimited growth potential in vitro with the maintenance of a low proportion of B-cells infected by EBV in vivo, a strict in vivo control mechanism has to be postulated. Certain aspects of this control are apparent when the primary infection is followed by infectious mononucleosis. This is characterized by lymphocytosis and the presence of activated T-cells. The T-cell proliferation is probably the manifestation of the immune response against EBV antigens. However, the reaction of T-cells upon encounter of B-blasts is also likely to contribute to the events. At present, it is difficult to detect an EBV-specific component in the action of the T-cells in the acute phase of mononucleosis exerted on B-cells. However, for the clinical course of the disease the activation of T-cells is important. The activated T-cells may control and also eliminate the B-cells infected by EBV. In addition to the immunity which develops during the disease, th immunoregulatory mechanism is likely to have a role in the inhibition of B-cell proliferation.

Cell-mediated immune reactions in three patients with malignant lymphoproliferative diseases in remission and abnormally high Epstein-Barr virus antibody titers.

Two patients with Hodgkin's disease in remission and one chronic lymphatic leukemia patient with extraordinarily high anti-Epstein-Barr virus (EBV) (viral capsid antigen) antibody titers (greater than 10,000) were selected to study a spectrum of cell-mediated immune responses, including natural killer, interferon-boosted killer, antibody-dependent lymphocytotoxicity, and T-cell-mediated reactions. The purpose was to compare these reactions in patients with immunosuppression and a high EBV load who can hold their EBV-carrying cells under control with the corresponding reactions in patients with EBV-carrying lymphoproliferative disease. In contrast to the latter group, the three patients of the present study showed a less profound and less general suppression of the immune responses. Multiple effector mechanisms probably safeguard against the proliferation of EBV-transformed B-cells. Clinically manifest EBV-carrying lymphoproliferative disease occurs only in very severe immunodeficiencies effecting multiple effectors.

Use of cryopreserved lymphocytes for the indirect leukocyte migration inhibition assay.

Production of leukocyte migration inhibitory factor (LIF) by fresh and cryopreserved lymphocytes from the same donors was detected by indirect leukocyte migration inhibition (LMI) assay. The same results were obtained when fresh and frozen lymphocytes were tested in parallel. This indicates that cryopreservation does not impair the ability of lymphocytes to produce LIF.

The EBV-carrying, beta2M/HLA deficient Burkitt lymphoma line Daudi is sensitive to EBV-specific killer T-cells of mononucleosis patients.

The beta2-microglobulin/HLA deficient Burkitt lymphoma line Daudi was tested for sensitivity to EBV-specific cytotoxicity mediated by natural killer (NK)-depleted T-cells from acute mononucleosis patients. While the Daudi line was not as sensitive as the reference EBV-genome-positive target line, it was clearly sensitive in the majority of cases. This would speak against a major role of syngeneic restriction in this system.

EBV-related cytotoxicity of Fc receptor negative T lymphocytes separated from the blood of infectious mononucleosis patients.

Epstein-Barr virus (EBV) related specific killing was demonstrated previously in the T cell enriched subpopulations of blood lymphocytes of IM patients. In the present work we demonstrate that the cytotoxic cells belong to the Fc negative T subset. T cells in the blood of IM patients can be divided in 2 categories, 1 Fc positive with non discriminative cytotoxicity and the other, Fc negative with selective cytotoxicity against EBV genome carrying lymphoblastoid cell lines.

Fine-Needle Aspiration Cytology and Immunocytochemistry of Abdominal Non-Hodgkin's Lymphomas.

The cytomorphology and immunologic characteristics of cells obtained by fine-needle aspiration biopsy of 34 consecutive patients with abdominal lymphomas were analyzed. Nineteen patients had no previous diagnosis, while 15 had previously known or suspected lymphomas. On cytology 21 high-grade and 13 low-intermediate-grade lymphomas were diagnosed. Immunologic characterization of aspirated cells identified one T-cell and 33 B-cell neoplasms. A monoclonal light chain expression was detected in 27 of the B-cell lymphomas. The results were in good agreement with those from histologic (n = 19) and immunohistochemical (n = 5) evaluations. The value and accuracy of fine-needle aspiration cytology in conjunction with immunocytochemistry are detailed.

Cladribine for untreated or early low-grade non-Hodgkin's lymphoma.

PATIENTS AND METHODS: Forty-four patients, with low-grade non-Hodgkin's lymphoma (LG-NHL) were included in a phase II study between June 1993 and May 1995 and treated with cladribine (CdA) 0.12 mg/kg as a 2 h i.v. infusion daily x 5, repeated after 28 days for up to 6 courses. Thirty-four patients were previously untreated and 10 had progressive disease after initial response to limited chlorambucil treatment. Five patients had also received involved field radiotherapy. Eight patients had mantle cell lymphomas, 22 follicle centre lymphomas, 5 lymphoplasmacytoid lymphomas, 4 small cell lymphocytic lymphomas, 4 marginal zone B-cell lymphomas and I had unclassified low-grade NHL. The response rate was 64%, with 11 (25%) CR and 17 (39%) PR while 5 (11%) patients progressed during treatment. The response rate was similar in previously treated and untreated patients. The median number of CdA courses delivered was 3 (1-6) in non-responding patients and 6 (2-6) in responders. Median survival from inclusion was not reached with a median follow-up of 40 months. The median time to progression was 7 mo for all patients, 25+ mo for CR and 16 mo for PR patients. Toxicity was sometimes severe with 2 treatment related deaths, one infectious related and one due to a mucocutaneous syndrome and pulmonary microembolism. In addition, 5 grade 3 or 4 infectious episodes were seen. Seven patients experienced grade 3 or 4 thrombocytopenia and 20 had grade 3 or 4 neutropenia. We conclude that the majority of patients with low-grade non-Hodgkin's lymphoma respond to CdA but that the adverse effects may be severe.

Growth fraction in non-Hodgkin's lymphomas and reactive lymphadenitis determined by Ki-67 monoclonal antibody in fine-needle aspirates.

The fraction of proliferation cells was analysed in fine needle aspirates from a series of 448 non-Hodgkin's lymphomas and 199 reactive hyperplasias using an immunoperoxidase staining with monoclonal antibody Ki-67. There was a good correlation between proliferation fraction and cytologic assignment to high and low grade lymphomas. Thus high grade lymphomas had a high median percentage of Ki-67 positive cells with a figure of 82.1 for lymphoblastic, 60.0 for immunoblastic, and 59.7 for centroblastic lymphomas. For low grade lymphomas the figures were 17.1 and 11.1 percent for centroblastic/centrocytic and CLL/immunocytoma, respectively. The fraction of proliferation cells in reactive lymphadenitis varied between 1-50% with a median of 11.5%. Analysis of Ki-67 positivity can accordingly not be used to differentiate benign from neoplastic proliferations. Within all lymphoma subgroups but lymphoblastic lymphoma, there was a marked variation in fraction of Ki-67 positive cells, which resulted in a certain overlap between high and low grade lymphomas. The results show that cells procured through fine-needle aspiration can be used to analyse the fraction of proliferating cells which contributes information about the growth rate of the individual tumours that can not be obtained through cytologic classification.

Diagnosis of lymphoma, leukemia, and metastatic tumor involvement of the cerebrospinal fluid by cytology and immunocytochemistry.

Fifty-five cerebrospinal fluid (CSF) specimens from 42 patients with suspected meningeal tumor involvement were reviewed. Cytology in conjunction with immunocytochemistry identified 26 CSF specimens as malignant. There were fifteen cases of lymphoma, four cases of leukemia, two cases of carcinoma, and two cases of melanoma. A monoclonal light chain expression was demonstrated in nine out of eleven B cell lymphomas. The three T-cell lymphomas all expressed pan T markers (CD 3) and two the T-helper antigen (CD 4). One patient had meningeal involvement of a true histiocytic lymphoma which was identified by its large atypical cells which were positive for alpha-1-anti-trypsin and muramidase. In four patients with a primary diagnosis of acute lymphoblastic leukemia, CSF involvement was confirmed by the demonstration of blasts with CD 10 (cALLA) or light chain restriction. Epithelial or melanocytic markers were demonstrated on the tumor cells in CSF from the remaining four patients. In 29 CSF specimens a diagnosis of reactive lymphocytosis was made using cytomorphology which mostly was characterized by macrophages mixed with small mature lymphoid cells. Immunologic evaluation showed that these mature cells were CD 10 negative T-cells and only few specimens contained polyclonal B-cells. The subsequent clinical course of these patients showed no evidence of CNS malignancy. It is concluded that cytology should be used in conjunction with immunocytochemistry to accurately evaluate CSF specimens from patients with possible malignant meningitis.

Fine-needle aspiration cytology and immunocytochemistry of Hodgkin's disease, suppurative type.

We describe 3 cases of Hodgkin's disease (HD) of unusual suppurative type, which were diagnosed on fine-needle aspirates. The smears were dominated by neutrophils, macrophages, and cellular debris. Only a few large, atypical cells of Hodgkin and Reed-Sternberg type were observed. The differential diagnoses of such smears include infectious mononucleosis, tuberculosis, metastatic lymph node involvement, non-Hodgkin's large-cell anaplastic Ki-1-positive lymphomas, T-cell-rich B-cell lymphomas, and peripheral T-cell lymphomas of mixed type. Immunocytochemistry identified the large atypical cells as CD 30 (BerH2)-positive and negative for CD 45 (LCA) in cytospin material from 2 patients, which allowed a conclusive diagnosis of HD.

Fine-needle aspiration cytology and immunocytochemistry of soft-tissue extramedullary plasma-cell neoplasms.

This study presents 19 patients with extramedullary plasma-cell tumors diagnosed by fine-needle aspiration (FNA) cytology together with immunocytochemistry. Eight patients had primary extramedullary plasmacytoma, while 11 patients had tumors secondary to myeloma. The most common localization was soft tissue (9 cases), followed by lymph nodes (5), scalp (3), and oral and nasal mucosa (2). All FNA smears were cellular, and 12 cases showed dissociated monomorphic plasma cells. Seven cases showed a dominance of immature bare nuclei, which made then difficult to diagnose conclusively using cytomorphology only. Immunocytochemistry demonstrated monoclonal expression of light immunoglobulin chains in all cases which, together with demonstration of CD 38 positivity and cytomorphology, allowed a conclusive diagnosis of plasmacytoma.

Cytomorphology and immunocytochemistry of fine needle aspirates from blastic non-Hodgkin's lymphomas.

Fine needle aspirates from 54 consecutive patients with primary or recurrent blastic (high-grade malignant) non-Hodgkin's lymphomas (NHLs) were analyzed by cytomorphology and immunocytochemistry. The cytologic diagnoses induced follicular center-cell-derived (centroblastic or anaplastic centrocytic) lymphoma (31 cases), immunoblastic lymphoma (11 cases), lymphoblastic lymphoma (9 cases) and histiocytic lymphoma (3 cases). Immunocytochemistry showed a B-cell phenotype of the neoplastic lymphocytes in all lymphoblastic lymphomas, 29 follicle center-cell lymphomas and 4 immunoblastic lymphomas. Four of the immunoblastic lymphomas were of T-cell origin while one case was not evaluable due to necrosis. A histiocytic origin was confirmed in two of the three cases that had a cytologic diagnosis of histiocytic lymphoma; the third case was shown by immunocytochemistry to be a true Ki-1-positive large cell lymphoma. Histologic and immunohistochemical analysis were performed on surgical biopsies from 18 patients. The results were in agreement with those on the fine needle aspiration (FNA) material in 14 cases. Three lymphomas could be phenotyped on aspirated material while marker studies on excised material were inconclusive. One lymph node aspirate contained mostly necrotic cells, which were unsatisfactory for adequate immunocytochemistry. However, sections from a removed tonsil from the same patient could be used for conclusive histology and phenotyping. In conclusion, the high diagnostic accuracy of combined cytomorphologic and immunocytochemical assessment of FNA samples validates the use of the technique in the diagnostic work-up of blastic (high-grade malignant) NHLs. In fact, the diagnostic accuracy seems so high that the technique can safely be used in the final diagnosis of blastic NHLs.