Colin P. Worman

Chronic lymphocytic leukemic (CLL) cells secrete multispecific autoantibodies.

A subset of B cells expressing the CD5 marker, a 67 KD molecule, has been implicated in the pathogenesis of autoimmune disease. To study the immunoglobulin repertoire of CD5+ B cells we investigated chronic lymphocytic leukemic (CLL) cells, since the majority of the malignant clones express CD5. CLL were induced to secrete their IgM in vitro by phorbol 12-myristate 13-acetate (PMA) and the supernatants screened for binding to a panel of autoantigens. Twelve out of 14 CLL clones were autoreactive, binding to Fc of IgG, ssDNA, dsDNA, histones, cardiolipin, or cytoskeletal components. Many also bound to more than one antigen tested for, showing multispecificity. Our data suggest that a high proportion of CD5+ B cells are programmed to secrete multispecific autoantibodies.

Interferon is effective in hairy-cell leukaemia

Seventeen patients with hairy‐cell leukaemia (HCL) and peripheral cytopenias were given human lymphoblastoid interferon (Wellferon), 3 megaunits daily or 6 megaunits on alternate days intramuscularly, for 4–24 weeks. Twelve of the patients had undergone splenectomy, three had no palpable spleen and had therefore not been offered surgery, and two patients with substantial splenomegaly were given interferon (IFN) as treatment of first choice.

Recombinant interferon alfa 2 a in the treatment of patients with early stage B chronic lymphocytic leukaemia

Summary. 18 patients with early stage, previously untreated H‐CLL were given interferon alfa (IFNα) 2a, 3 MU thrice weekly, subcutaneously. The peripheral lymphocyte count decreased in all patients. Response was delayed in three patients until they had received a median of 5 months therapy, one of whom had an initial transient increase in lymphocytes. Two patients normalized their blood lymphocyte counts, but neither achieved complete remission (CR). Responses were transient in eight patients lasting a median of 5 months (3–21). Binding anti‐IFNa antibodies were present in 9/17 patients tested (53%). Low titre binding antibodies (< 533 IBU/ml) were not associated with LHR, but high titre antibodies (>4401 IBU/ml) were. Two of 12 patients assessed had a > 3 g/1 increase in baseline serum IgG levels during IFNα therapy, one of whom reverted to pretreatment levels in association with LHR. Haematological toxicity was moderate, other than in two patients, one of whom developed autoimmune haemolytic anaemia and the other thrombo‐cytopenia. We conclude that IFNα lowers the lymphocyte count in early stage CLL, that the response may be delayed and that anti‐IFNcc antibodies may play a role in a proportion of those in whom the response is transient.

HAIRY‐CELL LEUKAEMIA

If recognition in the form of an international meeting can be taken as indicating that a subject has come of age, then hairy-cell leukaemia (HCL) recently reached this milestone at the approximate age of 2 5. The first International Workshop on HCL was held in October 198 3 at the University of Chicago, not very far from Columbus, Ohio, where the disease was first’ definitively recognized in 19 5 8 under the name of leukaemic reticuloendotheliosis (Bouroncle et al, 19 5 8 ) . The Workshop involved the presentation of papers by some 2 5 participants from throughout the world, all with a particular and often long-standing interest in different aspects of HCL. As a result, most aspects of the disease were considered and discussed and the proceedings have just appeared as two handsome issues of Seminars in Oncology (December 1984). Dr Harvey Golomb (University of Chicago) and his organizing committee are to be congratulated on making the meeting possible. It therefore seems timely to use this Annotation briefly to review the disease, particularly emphasizing points that have emerged since the disease was last comprehensively reviewed in 1980 (Cawley et al, 1980a).

An indirect rosette technique for the identification and separation of human lymphocyte populations by monoclonal antibodies: A comparison with immunof

A simple indirect rosette technique is described which allows the rapid enumeration, morphological identification and separation of monoclonal-antibody (MoAb)-defined human lymphocyte populations. The method is based on the binding to MoAb-stained cells of marker ox red blood cells coated with anti-mouse immunoglobulin. When compared with indirect immunofluorescence staining assessed by microscopy, the rosette method is quicker, shows greater sensitivity and is less subjective. Purification of helper (OKT4), suppressor (OKT8) or B-cell (BA1) subpopulations by positive or negative enrichment of rosetted cells gives purity and recoveries comparable with that of the fluorescence-activated cell sorter (FACS). Such purification is readily performed without loss of viability or function and is more easily done sterilely than with the FACS.

Production of tumour-derived suppressor factor in patients with acute myeloid leukaemia.

In an attempt to investigate the underlying cause of impaired cellular cytotoxic functions in patients with acute myeloid leukaemia and the relative ineffectiveness of immunotherapy with recombinant interleukin 2 (IL-2), normal donor lymphocytes were incubated in AML sera and in supernatant of myeloblasts. There was significant inhibition of both the natural killer activity and the lectin dependent cellular cytotoxicity of the normal donor lymphocytes compared to when incubation took place in autologous or normal allogeneic sera or marrow supernatant. This inhibition was time-related and partially reversible by washing of the normal lymphocytes immediately before the cytotoxicity assay. The suppressor factor, however, did not inhibit the IL-2 induced lymphocyte proliferation or affect the cytotoxicity-linked cytoplasmic serine esterase expression in the normal lymphocytes. This suppressor phenomenon was of myeloblast origin. Chronic exposure to the tumour-derived suppressor factor may be responsible for the impaired cellular cytotoxic functions observed in patients with acute myeloid leukaemia. It may also suppress the in vivo cytotoxic functions of IL-2 activated lymphocytes in patients treated with recombinant IL-2, hence leading to the disappointing results of immunotherapy so often encountered in clinical setting.

Detection of BLT substrate-specific proteases in individual human peripheral blood leucocytes and bone marrow cells: Application of the method to the classification of leukaemia

A trypsin-like serine esterase (SE) is known to be present in cultured cells with cytolytic potential. The distribution pattern of this enzyme in haematological cells and body tissues has been assessed using a method which permits rapid identification of individual cells. Cells and tissue sections were fixed and immersed in the substrate N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT)/Fast Blue BB chromogen solution. To identify the phenotype of SE+ cells the cytochemical stain was followed by the application of monoclonal antibody and alkaline phosphatase-anti-alkaline phosphatase (APAAP) complex immunocytochemical procedures. CD8+ and CD57+ lymphocytes showed SE+ granules. Neutrophil granulocytes and progenitors other than undifferentiated myeloblasts developed a dense stain while eosinophils were negative. 35% of monocytes showed positivity mainly in the area of nuclear indentation. Tumour-infiltrating SE+ lymphocytes could also be demonstrated with this method.

CYTOCHEMISTRY OF HUMAN LYMPHOCYTE SUBPOPULATIONS DELINEATED BY MONOCLONAL ANTIBODIES

Recently, Armitage ~t ul ( 1982) reported cytochemical differences between human lymphoid subsets defined by monoclonal antibodies and purified using fluorescence activated cell sorter (FACS). The authors reached the conclusions that dot-staining for Acid Phosphatase (AcP) and Non Specific Esterase (NSE) were markers for the so-called ‘true’ T-cells defined by IJCHT1 monoclonal antibody. Moreover, they reported an increase of positive cells in helper/inducer T-cells (Leu 3a + cells) and slightly lower values in suppressor/cytotoxic T-cells (l,eu2a+ cells). IJsing quite different technology and monoclonal antibodies, we obtained similar results (Bernard & Dufer, 1982). The identification of cell surface antigens was performed using a specific monoclonal antibody rosetting technique (Bernard et ul, 1982), including incubation of lymphoid cells with monoclonal antibodies from mouse hybridomas and subsequent treatment with ox erythrocytes coated with affinity purified rabbit anti-mouse Ig antibody. The rosette suspension obtained was cytocentrifuged and stained for cytochemical detection of AcP and NSE. It should be pointed out that this cytochemical analysis does not require any isolation procedure of the lymphoid subsets and thus avoids contamination by unwanted cells. We used monoclonal antibodies of the OK series and results are summarized in Table I. It appears that dot-like NSE reactivity is a marker of the majority ofT-cells (T3 +, T4+, T8 ’ cells). This staining pattern is quite uncommon in non-T-cells (T3 cells), in immature T-cells (T6 + cells), in HLA-DR antigens bearing cells (I1 + cells) or in cells carrying the M 1 antigen. As compared with total T cells (T3+ cells), the number of positive cells was higher in

In vivo and in vitro primed lymphocytes. Correlation of cytochemically detected BLT-specific lymphoid serine protease with cytotoxic activity.

We describe the validation of a cytochemical method to detect a cytolytic cell-specific lymphoid serine protease which can be upregulated during viral infection and allogeneic stimulation. The cytolytic cell specificity was ascertained by demonstrating a high correlation between BLT substrate-specific serine protease (SP) activity and cytotoxicity of in vivo and in vitro stimulated lymphocytes. The presence of SP in peripheral blood lymphocytes was compared with their capacity to kill K562 targets in a lectin-dependent cytotoxicity assay. The correlation coefficient was 0.92 and 0.93 at E:T ratios 10:1 and 20:1 respectively. In allogeneic mixed lymphocyte cultures an increase of SP activity in effector lymphocytes was paralleled by an augmentation of cytotoxic capacity towards stimulator target cells. SP+ granules showed intracellular polarization to the effector/target cell interface during conjugate formation. These results together with previous studies suggest that this method provides a sensitive assay which predicts the cytolytic potential present in a lymphocyte population.

Lymphocyte activation and serine-esterase induction following recombinant interleukin-2 infusion for lymphomas and acute leukaemias

SummaryIn this study we investigated the pattern of T lymphocyte changes in 16 adult patients with acute myeloid leukaemia (8), non-Hodgkins lymphoma (4) and Hodgkins disease (4) treated with continuous infusion of recombinant interleukin-2 (rIL-2). Effects indicative of lymphocyte activation occurred even prior to any rIL-2 therapy in these patients, being most prominent in patients with active diseases. Following each course of cytokine therapy, there were further changes in these parameters. Significant rebound lymphocytoses occurred with a concomitant increase in the cytotoxic functions and induction of the cytotoxicity-linked cytoplasmic serine esterase. Hence, both the natural killer and lectin-dependent cellular cytotoxicity activities were up-regulated. There were also increases in the serum sIL-2 receptor, sCD4 and sCD8 levels. More CD3+ lymphocytes, especially cells bearing CD4, were also recruited to the pool of potential effector cells, as demonstrated by the greater proportion of cells expressing the cytoplasmic serine esterase.