Enumeration of lymphocyte subpopulations by immunofluorescent staining of whole blood smears.

A simple and rapid technique for the enumeration of lymphocyte subpopulations by immunofluorescent staining of blood smears is described. The extremely small volumes of blood required make the technique particularly suitable for samples from paediatric patients. Results compare closely with conventional staining of lymphocytes in suspension.

Cyclosporin A directly inhibits human B-cell proliferation by more than a single mechanism.

Cyclosporin A (CsA) is a potent immunosuppressive agent that inhibits T-cell proliferation and lymphokine production. There is less information on the direct effect of CsA on B-cells. We investigated the proliferative responses of human tonsillar B-lymphocytes to a "T dependent" mitogen, pokeweed mitogen (PWM), and to a "T independent" mitogen, Staphylococcus aureus (SA). Both responses were strongly inhibited by CsA. Nonspecific cytotoxicity was ruled out, and the inhibition was not reversed by adding IL1, IL2, or BCGF individually or in combination. Maximal inhibition of the PWM response occurred when CsA was added early in the culture period. Cyclosporin A added 18 hours after the start of culture was less effective, and adding CsA after 36 hours resulted in only minimal inhibition. However, with SA as mitogen, addition after 36 hours still affected substantial inhibition. These results, on the time of action and resistance to reversal by exogenous growth factors, suggest that CsA can directly inhibit human B-cells by a mechanism similar to its action on T-lymphocytes, blocking an early event critical to entry into cell cycle, but an additional mechanism of inhibition later in the cell cycle may also operate when the proliferative signal is provided by the T-independent mitogen SA.

Interleukins and inhibitors in human lymphocyte regulation.

It seems clear, from studies in many laboratories, that both IL-1 and IL-2 play an important role in lymphocyte activation and are thus significant in the initiation and amplification of an optimal immune response. These conclusions have been extended to human lymphocytes, and preliminary data with macrophages and T-cells from alveolar spaces suggest that the interleukins are also important in vivo. A number of details remain to be worked out concerning these complex cellular and humoral interactions involved in triggering the immune response, and the increasing availability of purified reagents should help to speed the clarification of remaining issues. To date, much less work has been done with inhibitory factors produced by macrophages and lymphocytes than with the stimulatory interleukins. The preliminary studies by our laboratory and by others suggest that some suppressive agents may act through interference with the IL-1-IL-2 pathway and that others may be interleukin independent. Such investigations are only beginning, however, and much more investigation is needed to dissect in detail the mechanisms by which immunologically nonspecific inhibitory factors may contribute to the regulation of early steps in the immune response.

Correlation between Ig-synthesis patterns and lymphoma classification.

This study examines the link between free immunoglobulin (Ig) light-chain (LC) secretion and the developmental stage of the neoplastic B-cell of origin in B-cell lymphomas. The Kiel developmental scheme for lymphoma classification has been used to define the tumour-cell populations. Twenty-four B-cell lymphomas have been studied. In the small-lymphocytic lymphoma group, secreted Ig consisted of LC exclusively or in excess over heavy-chain (HC). Lymphomas of follicular-centre-cell origin, considered to be from cells further along the normal B-cell differentiation pathway, can be divided into centroblasts or centrocytes according to their histological appearance in tissue sections. Centroblastic lymphomas exhibited strong surface IgM or IgG expression, and the secretion of whole Ig was higher than by cells from the small-lymphocytic lymphomas. Synthesis of HC and LC was balanced in these cultures, both intracellularly and in secreted material. The centrocytic lymphomas comprised a functionally more heterogeneous group, the SIg varied in intensity and was of surface IgM, D or G. Likewise Ig synthesis was variable in quantity and composition, some cases secreting LC exclusively while others, including the cases expressing SIgG, secreted balanced HC and LC. In the Kiel classification centrocytes are considered to be more mature than centroblasts. Our data suggest that centrocytic lymphomas are heterogeneous in origin, some preceding and others following centroblasts in the B-cell maturation sequence. These data are discussed in relation to current concepts of B-cell maturation and lymphoma histology.

Hairy cell leukaemia occurring with an unrelated paraproteinaemia. A biochemical and immuno-electron microscopic study.

A detailed study is described of a case of hairy cell leukaemia, presenting with a serum paraprotein of an immunoglobulin (Ig) class different from that synthesised by the neoplastic cells. The case was unusual in its association with leukaemic arthropathy but ultrastructurally the hairy cells were typical. By immunofluorenscence and immuno-electron microscopy the neoplastic cells expressed IgA lambda both on the cell surface and intracellularly in the rough endoplasmic reticulum, perinuclear space and Golgi apparatus. No Ig was observed in the ribosomal-lamellae complexes. These cells also synthesised and secreted Ig of class A lambda in culture. However the serum paraprotein was of class IgA chi and could not be attributed to an abnormal population of plasma cells in the bone marrow. There was no other evidence for myeloma and the IgA chi paraproteinaemia appeared to be benign, apparently unrelated to the neoplastic proliferation of hairy cells.

Free immunoglobulin light chain synthesis by human foetal liver and cord blood lymphocytes.

Immunoglobulin (Ig) synthesis by immature B lymphocytes from human foetal liver and cord blood has been investigated. Seven out of fifteen preparations of foetal liver cells and eight out of eleven cord bloods synthesized Ig detectable by biosynthetic labelling. All cultures of foetal lymphocytes with detectable Ig synthesis secreted free light chain. Two of these cases also synthesized free mu heavy chain. Cord blood lymphocyte synthesis patterns were variable ranging from free light chain as the only secreted Ig product to balanced synthesis of heavy and light chains. No cord blood cultures synthesized detectable free mu chains. Free Ig-light-chain synthesis appears to be associated with normal immature B lymphocytes and the results are discussed in relation to the B-cell differentiation pathway.

Induction of balanced immunoglobulin chain synthesis in free light chain-producing lymphocytes by mitogen stimulation.

Neoplastic cells from 2 cases of CLL synthesized and secreted excess free Ig light chain in culture, confirmed by precipitation with anti-idiotypic antibody. Small and medium sized normal human spleen cell subpopulations, staining predominantly for surface IgM and D, also synthesized and secreted excess free light chain. PWM stimulation induced balanced synthesis of heavy and light chains in CLL and normal spleen subpopulations after 6 days in culture, accompanied in spleen but not CLL by the appearance of mature plasma cells. These data demonstrate that normal cell counterparts of neoplastic lymphoid synthesis patterns can be identified in spleen. Furthermore, the synthesis pattern alteration after PWM stimulation suggests a relationship between free light chain synthesis and B cell immaturity. The failure of CLL cells to develop into detectable plasma cells suggests a restricted maturation response to mitogen compared with normal spleen.

Immunoglobulin synthesis by neoplastic B lymphocytes: free light chain synthesis as a marker of B cell differentiation.

Immunoglobulin synthesis has been investigated in 55 cases of B lymphocytic disease. Free light chain was the predominant product of Ig synthesis and the sole secreted Ig product from neoplasms lacking surface Ig or expressing sparse surface IgM as detected by fluorescent-labeled antibody. In contrast, neoplastic cells expressing strong surface IgM or IgG synthesized and secreted balanced amounts of heavy and light chain. Intermediate patterns of synthesis were shown by neoplastic cells expressing IgM with IgD. These patterns of synthesis occurred irrespective of the disease entity and are discussed in relation to models of B cell maturation.