Chris D. Platsoucas

Immunomodulation of human leukocytes by staphylococcal enterotoxin A: Augmentation of natural killer cells and induction of suppressor cells

Staphylococcal enterotoxin A (SEA), a protein isolated from culture supernatants of Staphylococcus aureus, is a potent T-cell mitogen and an inducer of interferon-gamma (IFN-gamma). We report here that SEA exhibits a number of significant in vitro immunomodulatory functions. In vitro treatment of human peripheral blood monocyte-depleted lymphocytes with SEA resulted in significant augmentation of their natural killer cytotoxicity against target cells from hemopoietic (K562, Daudi) or solid (melanoma, lung, colon) human tumor cell lines. SEA was found to be more effective than interferons-alpha (natural or Escherichia coli-derived) in augmenting natural killer (NK) cytotoxicity of peripheral blood lymphocytes. Studies on the kinetics of the augmentation revealed a significant increase of NK within 3 hr of in vitro treatment with SEA at 37 degrees C. A neutralizing monoclonal antibody specific for human IFN-gamma did not affect the augmentation of natural killer cytotoxicity by SEA, suggesting that SEA augmented natural killer cytotoxicity primarily by a mechanism not involving induction of interferon-gamma. Furthermore, in vitro treatment with SEA resulted in significant augmentation of antibody-dependent cell-mediated cytotoxicity and of natural killer-like cytotoxicity, generated in mixed lymphocyte culture, against the K562 targets. Induction of suppressor cells to proliferative responses of autologous or allogeneic mononuclear cells to phytohemagglutinin (PHA) or to allogeneic cells in mixed lymphocyte culture was observed after in vitro treatment of peripheral blood mononuclear leukocytes with SEA for 24 or 48 hr at 37 degrees C. In addition, the presence of SEA in mixed lymphocyte cultures (MLC) resulted in significant inhibition of the generation of specific T-cell-mediated cytotoxicity in MLC. These results suggest that SEA, which may be involved in S. aureus infections and in treatment with extracorporeal perfusion systems over S. aureus columns, can regulate a number of significant lymphoid functions.

B cells from a patient with chronic lymphocytic leukemia bind to sheep erythrocytes: An immunological analysis

Abstract Binding of sheep erythrocytes to chronic lymphocytic leukemia cells with B-cell properties was observed in a patient with chronic lymphocytic leukemia (CLL). Monoclonal surface immunoglobulin on the surface of B cells probably binds sheep erythrocytes through Forssman antigen. The B-cell nature of the CLL cells was confirmed by lack of cytotoxicity of anti-T cell serum for the lymphocytes of this patient, by rosette formation with mouse red blood cells, and by resynthesis of monoclonal surface immunoglobulin after trypsinization.

Immunoglobulin synthesis and secretion by leukemic B cells from patients with chronic lymphocytic leukemia.

We investigated the ability of purified E-rosette negative largely leukemic B cells from 15 patients with B-cell chronic lymphocytic leukemia (CLL) to synthesize and secrete IgM, IgA and IgG spontaneously or in the presence of purified autologous or allogeneic T4 cells from normal donors, in PWM-induced differentiation system. We observed moderate but significant IgM synthesis and secretion (19.7 +/- 8.9 micrograms/dl, n = 5) by leukemic B cells alone in 5 of 15 patients examined. These IgM concentrations were significantly higher (p less than 0.005) than those produced by purified E-rosette negative cells from normal donors (4.3 +/- 4.5 micrograms/dl; n = 6) in the absence of T cells. Purified E-rosette negative leukemic B cells alone from patients with CLL did not produce IgA or IgG. Addition of purified autologous or allogeneic T4 cells from normal donors resulted in significant increase of IgM production by leukemic B cells from certain patients or initiated IgM secretion in others. However, these IgM levels (73.9 +/- 56.6 micrograms/dl) were significantly lower (p less than 0.003) to those produced by mixtures of T4 cells and B cells form normal donors (211.6 +/- 58.0 micrograms/dl, n = 6). Addition of purified autologous or allogeneic T4 cells from normal donors to purified largely leukemic B cells from patients with CLL resulted in production of very small amounts of IgA in 4 of 15 patients (10.6 +/- 6.3 micrograms/dl vs 154.7 +/- 35.8 micrograms/dl produced by T4 and B cells from normal donors; n = 6), but did not support IgG synthesis and secretion. Purified T4 cells from certain patients with CLL exhibit defective helper function to immunoglobulin production by E-rosette negative cells from normal donors.

T-cell imbalances in patients with multiple myeloma: an analysis by monoclonal antibodies.

Functionally distinct human T-lymphocyte subpopulations have recently been defined by monoclonal antibodies, recognizing cell surface differentiation antigens expressed as distinct populations of T cells. We determined the proportions and absolute numbers of T-cell subsets in 24 patients with multiple myeloma. These patients exhibited in the peripheral blood significantly decreased proportions of T3-positive cells (63.13±17.6 vs 79.70±7.07% of the normal controls;P<0.0005), significantly decreased proportions of T4-positive cells (39.13±13.01 vs 54.55±5.30% of the normal controls;P<0.0005), and moderately increased proportions of T8-positive cells (37.21±13.70 vs 29.90±4.87% of the normal controls;P<0.029). Furthermore, we observed in these patients significantly decreased absolute numbers of peripheral blood lymphocytes (1240.00±577.80 vs 2185.70±955.43 of the normal controls;P<0.001), T3-positive (950.05±590.23 vs 1732.25±771.60;P<0.0005), and T4-positive cells (434.04±155.95 vs 1182.50±490.96;P<0.0005). In contrast, the absolute numbers of T8-positive cells were normal (584.22±456.17 vs 662.15±369.20 of the normal controls; NS). These imbalances resulted in significantly decreased values (P<0.0005) of the ratio of the T4/T8 phenotypes (1.19±0.59 vs 1.86±0.29 of the controls). Untreated patients (13 of 24) exhibited significantly decreased proportions of T3-positive (P<0.003) and T4-positive (P<0.001) cells and increased proportions of T8-positive cells (P<0.007). Absolute numbers of T4-positive cells were significantly decreased (P<0.007), and those of T3-positive cells were moderately decreased (P<0.04). In contrast, absolute numbers of WBC, lymphocytes, and T8-positive cells were within the normal range. Treated patients (11 of 24) exhibited significantly decreased proportions of T3-positive (P<0.0005) and T4-positive (P<0.0005) cells and normal proportions of T8-positive cells. Decreased absolute numbers of peripheral blood lymphocytes (P<0.002), T3-positive cells (P<0.0005), and T4-positive cells (P<0.0005) were found in these patients. The absolute numbers of T8-positive cells were within the normal range. These results demonstrate significant T-cell imbalances in patients with multiple myeloma. The significance of these findings is discussed.

Separation of human lymphocyte subpopulations by density gradient electrophoresis: I. Different mobilities of T (Tμ, Tα) and B lymphocytes from human tonsils

Abstract T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.

Differential Effects of Human Alpha and Gamma Interferons on Mixed Lymphocyte Culture and on T-Cell-Mediated Cytotoxicity

We compared the effects of natural and recombinant (r) alpha (IFN-α) and gamma (IFN-γ) interferons on the proliferative responses of human peripheral blood mononuclear cells to mitogens and allogeneic cells in mixed lymphocyte culture (MLC) and on the generation of specific T-cell-mediated cytotoxicity. In 14 of 19 donors, natural IFN-γ and rIFN-γ had no significant effect on the proliferative responses to mitogens or allogeneic cells in MLC, even at very high IFN-γ concentrations (10,000 U/ml). In the remaining 5 donors, a statistically significant (p 2 significantly inhibited (p 2. Additionally, we compared the ability of human rIFN-α subtypes to inhibit proliferative responses to allogeneic cells in MLC. rIFN-α2, rIFN-α4, and rIFNα7 displayed the most potent inhibitory activity of allogeneic responses and were active at concentrations as low as 0.3–0.6 ng/ml. The rIFN-α2/α1 hybrid molecule was also capable of significantly inhibiting proliferative responses in MLC at concentrations of 0.6 ng/ml or higher. rIFN-α1 and rIFN-δ4α1 inhibited at considerably higher concentrations (30 ng/ml or higher).

ANALYSIS OF HUMAN NATURAL KILLER CELLS BY MONOCLONAL ANTIBODIES

Publisher Summary This chapter reviews a study of human natural killer (NK) cells by monoclonal antibodies. In this study, OKT3, OKT4, OKT8, OKM1, OKT11a, and OKT10 monoclonal antibodies and Clone 9.6 and αIa (Clone 7.2) monoclonal antibodies were obtained. Monocyte-depleted human peripheral blood lymphocytes were used in the study. Cells to be separated using the fluorescence-activated cell sorter were incubated with the appropriate monoclonal antibody (0.25 μg/l × 106 cells) for 30 min at 4°C, and labeled with an FITC-conjugated F(ab1)2 fragment of goat antiserum to mouse Ig. The cells were separated in the FACS IV (Becton–Dickinson). Antibody and complement treatment (rabbit complement, Pel Freeze, Little Rock, Ark.) was carried out by a two-step protocol. A representative experiment of the natural-killer cytotoxicity mediated by 9.6-positive, OKT11a-positive or OKM1-positive cells, purified by the fluorescence activated cell sorter, is shown in the chapter. Statistically significant differences in the NK cytotoxicity mediated by purified 9.6-positive and OKT111a-positive cells were not observed. The results provided evidence suggesting the existence of subpopulations of NK cells.

Separation of human bone marrow cell populations by density gradient electrophoresis: Differential mobilities of myeloid (CFU-C), monocytoid, and lymphoid cells

Abstract Human bone marrow cells from normal donors (purified by centrifugation on Ficoll-Hypaque density cushion) were separated by density gradient electrophoresis, on the basis of their surface charge. The separated cell fractions were analyzed by morphology, surface markers, and functional tests. Morphological examination revealed that immature cells of myeloid and erythroid lineage were significantly enriched in the high-mobility fractions, whereas mature cells in general, e.g., small lymphocytes, monocytes and granulocytes (few) were highly enriched in the low-mobility fractions. Ripley rosette-forming cells, as well as complement receptor and Fc (IgG)-receptor-bearing lymphocytes were relatively enriched in the high-mobility fractions, although they were present in small proportions and in the rest of the fractions. E-rosette-forming cells were enriched in the intermediate- and low-mobility fractions. T cells with Fc receptors for IgM (Tμ cells), determined by a double-rosetting technique, were found to be enriched in the intermediate-mobility fractions. In contrast T cells with Fc receptors for IgG (Tγ cells) were significantly enriched in the low-mobility fractions. Cells in the intermediate-mobility fractions (Tμ enriched) responded by proliferation to mitogens and to allogeneic cells in mixed lymphocyte culture. Cells in the very high or very low (Tγ enriched) mobility fractions responded poorly. Colony-forming units in vitro (CFU-C), determined using either the human leukocyte feeder or the placental CSA culture systems, were found to be significantly enriched in the high-mobility fractions. Low-mobility fractions were devoid of CFU-C. These studies demonstrate that density gradient electrophoresis can be successfully applied for the separation of human bone marrow cells of different lineage and studies of their functional properties.

Augmentation of natural killer cytotoxicity by alpha or gamma natural and recombinant interferons and interferon inducers. Effect of monocytes

We investigated the effect of human peripheral blood monocytes on the augmentation of natural killer cytotoxicity by alpha or gamma natural and recombinant interferons (IFN) and certain interferon inducers. We observed that: (1) in the majority of the donors examined (75%) human peripheral blood monocytes do not affect natural killer cytotoxicity, determined by a 4-hour chromium-51 release assay, against target cells from hemopoietic human tumor cell lines. (2) Monocytes are not required and do not affect the augmentation of natural killer cytotoxicity by Escherichia coli-derived IFN-gamma, natural human IFN-gamma, E. Coli-derived IFN-alpha 2 or natural human IFN-alpha. E. Coli-derived IFN-gamma and natural human IFN-gamma have been reported to activate monocyte cytotoxicity determined in 72-hour assay. (3) Monocytes are not required for the augmentation of natural killer cytotoxicity against target cells from hemopoietic tumor cell lines by polyinosinic acid-polycytidylic acid or staphylococcal enterotoxin A.

AUGMENTATION OF HUMAN NATURAL KILLER CYTOTOXICITY BY ALPHA-INTERFERON AND INDUCERS OF GAMMA-INTERFERON. ANALYSIS BY MONOCLONAL ANTIBODIES

Publisher Summary This chapter discusses an analysis of the augmentation of human natural killer (NK) cells by IFN-α and inducers of gamma interferon, using monoclonal antibodies to lymphoid and monocytoid cell populations. Treatment of the cells with monoclonal antibody and complement resulted in complete depletion of the population of interest, as judged by immunofluorescence analysis. In vitro treatment of purified OKT11a-positive, clone 9.6-positive, or OKMl-positive cells with human leukocyte interferon resulted in significant augmentation of their NK cytotoxicity. It is possible that IFN-α induces the differentiation of pre-NK cells to NK effector cells. Additional studies are in progress using combinations of monoclonal antibodies to identify the NK cell populations that are augmented by the modifiers. Treatment with the OKT11a monoclonal antibody and complement significantly decreased the augmentation of NK cytotoxicity by subsequent treatment with IFN-α. It was observed that the OKT3 monoclonal antibody in the absence of complement augments the NK and antibody-dependent cell-mediated cytotoxicity of monocyte-depleted peripheral blood lymphocytes from certain donors.