Clement C. S. Hsu

Lymphocytes bearing B- and T-cell markers in patient with lymphosarcoma cell leukemia.

Abstract Sequential studies over a period of 8 mo of a patient with lymphosarcoma cell leukemia and a peripheral blood leukocyte count ranging from 200,000 to 700,000/mm3 indicated that from 51% to 90% of the peripheral blood lymphocytes formed E rosettes with sheep erythrocytes and, therefore, were thymus-derived T cells. With T-cell-specific antiserum, 72% of the lymphocytes were shown to be T cells. On initial examination, 15% of the lymphocytes also were found to carry surface immunoglobulin (Ig), a marker for bone marrow-derived B cells. The percentage of Ig-bearing cells increased progressively to 92% over the ensuing 4 mo. Surface Ig was found to consist of monoclonal IgM-lambda and by partial removal of cell surface proteins with trypsin, more cells could be stained with fluoresceinated anti-Ig. Incubation of the patients plasma with normal and leukemic lymphocytes did not increase the percentages of cells with surface Igs. During the next 4 mo, various percentages of the cells were also found to carry IgG (50–78%), IgA (23–36%), IgD (5–93%), and IgE (5–28%). It is postulated that the patient has a T-cell leukemia, during the course of which inert B-cell genes controlling synthesis of Igs were activated.

Inhibition of lymphocyte reactivity in vitro by autologous polymorphonuclear cells (PMN)

Abstract The influence of autologous polymorphonuclear cells (PMN) on lymphocyte reactivity was investigated by monitoring the uptake of tritiated thymidine by unstimulated, phytohemagglutinin (PHA)-stimulated, and fetuin-stimulated lymphocytes in vitro . Addition of PMN at PMN-to-lymphocyte ratios (P:L) of 0.5 to 2.0 progressively inhibited lymphocyte reactivity. Soluble extracts, obtained by sonication and ultracentrifugation (100,000 g for 90 min) of PMN, also inhibited lymphocytes. The PMN-derived inhibitor(s) is noncytotoxic, heat labile (56 °C for 60 min), resistant to freeze-thawing (20 cycles), and appears to be nondialyzable. Inhibition was more marked when the factor was added at the initiation of lymphocyte cultures than when added with the tritiated thymidine 24 hr prior to cell harvest. Thus thymidine released by PMN which diluted the radiolabeled nucleotide and degradation of the tritiated thymidine did not explain these results. Lymphocytes incubated for 3 days in the medium containing the inhibitor reacted normally to PHA following washing, indicating that inhibition was reversible. These results suggest that a PMN-derived lymphocyte inhibitor(s) may modulate lymphocyte-mediated immune reactivity.

Peripheral Blood Lymphocyte Responses to Phytohemagglutinin and Pokeweed Mitogen during Pregnancy

Summary Peripheral blood lymphocytes from women in late gestation were cultured in an allogeneic reference plasma and autologous plasma. Lymphocytes cultured with autologous plasma exhibited markedly low response to both pokeweed mitogen (PWM, a B- and T-cell stimulant) and phytohemagglutinin (PHA, a predominant T-cell stimulant). When cultured with the reference plasma, lymphocytes from pregnant women showed a markedly impaired response to PWM whereas their responses to PHA was essentially the same as that of age-matched clinically well nonpregnant control women. The findings may suggest B-lymphocyte alterations during pregnancy in addition to plasma lymphocyte inhibitors. Dissociation of plasma inhibitory effects on lymphocyte responses to PHA and PWM was also noted. This study was supported by the Otho S. A. Sprague Memorial Institute and by a grant from G. D. Searle & Co. The author is indebted to Dr. Paul Urnes, Dept. of Obstetrics and Gynecology, Northwestern Memorial Hospital, Chicago, for his vital contribution to this study, to Dr. Philip Y. Paterson for his advice and review of the manuscript and Miss Amy E. Sedory for her technical assistance.

Coexpression of multiple immunoglobulin (Ig) heavy chain classes on human leukemic B lymphocytes (B cells)

Abstract Ig determinants on leukemic B cells from 15 chronic lymphocytic leukemia (CLL) and 4 lymphosarcoma cell leukemia (LSL) patients were studied using goat antisera to individual heavy or light chains and a polyvalent goat anti-Ig. In 16 of 19 cases only one type of light chain was detected on the leukemic cells. Heavy chains on the cells varied from a single mu to combinations of up to four classes. Mu chain was the principal heavy chain on leukemic cells in 17 of 19 cases, followed, in the order of frequency, by delta ( 6 19 ), gamma ( 5 19 ), alpha ( 3 19 ), and epsilon ( 1 19 ) chains. To investigate the intrinsic or extrinsic origin of the surface Ig determinants, leukemic cells were cultured for 3 days after preexisting surface Ig had been labeled with the respective fluorescent antisera. The prelabeled Ig molecules were shed from cell surfaces by Day 3 of culture. Ig determinants reexpressed on the cell surface during the in vitro incubation were identified by restaining the antibody-prelabeled cells. With this technique, nearly all heavy chain determinants on leukemic B cells were proven to be generated from within the cell. Lack of a consistent association among antiserum reactivities and the absence of staining of cells by at least two of the seven goat antisera excluded the potential pitfalls of antiserum cross reactivities or nonspecific attachment of antisera as the cause of cell staining. The high proportion of cells carrying heavy-chain determinants indicated that the heavy chains were coexpressed on the same leukemic B cells. Serial studies in a single patient revealed appearance of endogenous alpha and delta chains on cells previously expressing gamma and mu chains. Our study demonstrates that a leukemic B cell can simultanously express multiple heavy-chain classes in different combinations, and that some of these heavy chains are generated during the course of the illness.

Comparative Inhibitory Effects of Serum on Lymphocyte Responses to Mitogenic Stimulation

The effects of sera from pregnant women, human umbilical cords and nonpregnant healthy control individuals on human lymphocyte responses to phytohemagglutinin (PHA), pokeweed mitogen (PWM) and allogeneic lymphocytes (MLR) are described. Maternal sera invariably inhibited lymphocyte responses to PHA. In MLR and PWM-stimulated lymphocyte responses, however, some maternal sera had pronounced inhibitory effect while other maternal sera had none. Similar dissociation of inhibitory effects of lymphocyte responses to PHA and to PWM or in MLR was noted in some cord sera but not in the control sera we studied. The observations may reflect different serum factors impinging upon different lymphocyte subpopulations responsive to different mitogens. Preliminary attempts to relate the serum inhibitory effects to various serum components revealed that inhibition of MLR appeared to correlate with increases in alpha-2-globulins and beta-globulins. In contrast, inhibition of PHA responses was not similarly related to any one or two specific serum constituents.