Heterogeneity of human natural killer cell populations.

Natural killing (NK) in human donors was determined by the ability of peripheral blood subpopulations to lyse the myeloid target, K562, in a 2 to 4 hr ⁵¹Cr release assay. The most active cell was a non-T cell which passed through nylon columns (representing 10 to 25% of column passed cells). A second column passed cell population, with characteristics of T lymphocytes (75 to 90% of column passed cells), was also capable of mediating natural killing. Non-T cells which were retained by the nylon columns (45 to 55% of adherent cells) lacked NK activity. However, nylon adherent T cells (45 to 55% of adherent cells) were consistently active in NK assays, illustrating an important subset of NK effector cell often overlooked. Both column passed and adherent T cells were further separated according to their ability to bind IgG or IgM immune complexes, showing that those mediating NK have receptors for IgG (Tγ+) but not for IgM (Tμ+).     

Heterogeneity of respiratory dendritic cell subsets and lymphocyte populations in inbred mouse strains

Background

Inbred mouse strains are used in different models of respiratory diseases but the variation of critical respiratory leukocyte subpopulations across different strains is unknown.

Methods

By using multiparameter flow cytometry we have quantitated respiratory leukocyte subsets including dendritic cells subpopulations, macrophages, classical T and B cells, natural killer cells, γδTCR+ T cells and lineage-negative leukocytes in the five most common inbred mouse strains BALB/c, C57BL/6, DBA/2, 129SV and C3H. To minimize confounding environmental factors, age-matched animals were received from the same provider and were housed under identical specific-pathogen-free conditions.

Results

Results revealed significant strain differences with respect to respiratory neutrophils (p=0.005; up to 1.4 fold differences versus C57BL/6 mice), eosinophils (p=0.029; up to 2.7 fold), certain dendritic cell subsets (p≤0.0003; up to 3.4 fold), T (p<0.001; up to 1.6 fold) and B lymphocyte subsets (p=0.005; up to 0.4 fold), γδ T lymphocytes (p=0.003; up to 1.6 fold), natural killer cells (p<0.0001; up to 0.6 fold) and lineage-negative innate leukocytes (p≤0.007; up to 3.6 fold). In contrast, total respiratory leukocytes, macrophages, total dendritic cells and bronchoalveolar lavage leukocytes did not differ significantly. Stimulation of respiratory leukocytes via Toll-like receptor 4 and 9 as well as CD3/CD28 revealed significant strain differences of TNF-α and IL-10 production.

Conclusion

Our study demonstrates significant strain heterogeneity of respiratory leukocyte subsets that may impact respiratory immunity in different disease models. Additionally, the results may help identification of optimal strains for purification of rare respiratory leukocyte subsets for ex vivo analyses.     

Heterogeneity and function of human B lymphocytes

B lymphocytes represent an important arm of the immune system. Besides their main function of providing antibodies protecting against pathogens, they also exert some regulatory functions, in particular for secondary lymphoid tissue differentiation. Human B cells can be divided in various subsets representing different maturation stages and different pathways of humoral immune responses. Na?ve IgMlow IgDhigh CD27- B cells can participate in T-cell dependent immune responses leading to germinal center formation in follicles of secondary lymphoid organs. Interactions with follicular helper T cells, a recently identified CD185+ T cell population providing help to follicular B cell, involve costimulatory molecules including CD40, CD27, CD278 and SAP-recruiting receptors. B cell interaction with follicular helper T cells represents a critical step controlling the generation of plama cells that ultimately produce high affinity, somatically mutated, class-switched antibodies or of their memory B cell counterpart (identified as CD27+ Ig switched or IgMonly B cells). IgMhigh IgDlow CD27+ B cells are a puzzling population apparently specialized in T-independent responses to bacterial capsular polysaccharides. The extra-follicular, probably antigen-independent, differentiation pathway of these cells, allowing pre-immune repertoire diversification by somatic hypermutation, is not yet characterized. However, circulating IgMhigh IgDlow CD27+ B cells are similar to splenic marginal zone B cells. In addition to these subsets, minor populations can also be identified in peripheral blood, such as transitional B cells and plasma blasts. All together, deciphering human B cell heterogeneity provides tools for investigations of humoral immunodeficiencies and auto-immune diseases, that will in return shed more light on B cell biology.     

B lymphocyte regulation of human hematopoiesis

Epstein Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) were derived from seven different individuals. The ability of BLCL supernatants to stimulate hematopoietic colony formation in vitro was tested in a conventional stem cell assay system. Supernatants promoted the growth of single (GM, E, MK) as well as multi-lineage (GEMM) colonies in bone marrow cultures. Our results indicate that EBV-transformed B lymphocytes produce cytokines that affect in vitro stem cell proliferation and differentiation. These studies demonstrate the regulatory potential of activated B lymphocytes in human hematopoiesis.     

Heterogeneity of the human phosphocholine-specific B cell repertoire

We have utilized a highly efficient method of culturing small numbers of Epstein Barr virus (EBV)-infected cells to analyze the heterogeneity of human antibodies specific for phosphocholine (PC). Lymphocytes from peripheral blood or tonsils of individuals who had no evidence of recent pneumococcal infection were infected with EBV and cultured at limiting dilution. After correction for the cloning efficiency, between 1/1500 and 1/10,000 B cells produced specific anti-PC antibodies by our criteria. Examination of the heterogeneity of these antibodies revealed that most individuals had an overwhelming predominance of anti-PC antibodies with kappa-light chain. Fine specificity analysis of 39 monoclonal anti-PC antibodies demonstrated that the IgM antibodies examined displayed significant binding site diversity, whereas the IgA PC-specific clones were much less heterogeneous. In general, the human anti-PC antibodies had a much higher relative affinity (Krel) for choline and glycerophosphocholine than the murine antibody families. Through examination of the human PC-specific B cell repertoire we have drawn some interesting parallels with the well-defined murine clonotype families and have begun to dissect the human response to this naturally occurring antigenic determinant.     

Heterogeneity of macrophage populations in human lymphoid tissue and peripheral blood

Human lymphoid tissue and peripheral blood leukocytes were stained with six monoclonal antibodies directed against monocyte/macrophage populations. The staining pattern described by each of these monoclonal reagents was compared with the distribution of morphologically distinguishable tissue macrophages. The results show that there exists considerable heterogeneity of tissue macrophages based on the expression of surface and/or cytoplasmic antigens; furthermore, the distribution of cells bearing particular antigenic determinants is associated with distinct regions in normal lymphoid tissue. Double staining methods demonstrated that these antibodies bind to different, as well as to identical, macrophage populations. OKM-1 antibody binds predominantly to sinus histiocytes and tingible body macrophages. The Leu M-1 reagent stains interdigitating reticulum cells, while the KiM-4 antibody labels follicular dendritic cells. Leu M-3 antibody identifies cells predominantly in the germinal center, and histiocytes lining the sinuses. Both CM-1 and BRL-M.1 appear to stain tissue macrophages distributed throughout the lymphoid tissue.     

Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.     

Age-dependent changes in B lymphocyte lineage cell populations of autoimmune-prone BXSB mice

BXSB mice, a recently developed autoimmune strain, develop a human lupus-like disease with B cell hyperplasia in peripheral lymphoid organs. Unlike other experimental models of autoimmunity and human lupus, BXSB male mice manifest accelerated autoimmune phenomena through the influence of a Y chromosome-linked enhancing factor. The present studies were performed to investigate the features of B lymphopoiesis in BXSB mice and to determine whether differences exist between BXSB males and females in this respect. B lineage cell populations in the marrow of BXSB mice were identified phenotypically by studying the cytoplasmic mu-heavy chains of IgM (c mu), and functionally by their ability to acquire clonability and sIg in short-term liquid cultures. Male BXSB mice became deficient in both the precursors of functional B cells and c mu + pre-B cells by the age of 8 to 12 wk. This followed a transient increase in this population, which peaked when the mice were 2 to 4 wk old. In females, substantial numbers of functional B cell precursors and c mu + cells were maintained until more than 4 mo of age. Cells lacking Ig but bearing a B lineage cell antigen (14.8) were elevated in numbers in both BXSB males and females until 16 wk of age when compared to normal strains of mice. At the time pre-B cells and functional B precursors were elevated in numbers, some sIg- cells were shown to form colonies in mitogen-stimulated semisolid agar cultures without a period of preculture. Most of these sIg- cells seemed to bear the B lineage cell antigen (14.8). They were independent of both G-10 adherent regulatory cells and Thy-1+ cells for their colony formation. These results indicate that B lymphocyte formation may be maintained in a hyperactive state in BXSB females, whereas males become deficient in B cell precursors very early in life. This early decline might be related to the accelerated development of autoimmune disease in BXSB mice. Bone marrow transplantation studies showed that these unusual characteristics of B lymphopoiesis were reciprocally transferable with unseparated bone marrow cells between BXSB males and females. This finding indicates that sex hormones are not a critical variable in abnormal B lymphocyte formation in this strain, and that the premature deficiency of immediate B precursors in males may be regulated by a genetic factor(s) located on the Y chromosome.     

Hepatitis B virus markers and lymphocyte populations in the asymptomatic multiple-drug addict

The authors have studied in particular hepatitis B virus markers and the ratio of OKT4+/OKT8+ cells in two subgroups of 52 deprived asymptomatic drug addicts. In the group 1 (deprived for 19 days on an average), 52% of the subjects showed a ratio of OKT4+/OKT8+ less than 1, whereas in the group 2 (deprived for 30 months on an average), this percentage is only of 25%. HBV markers were present in 90% of the subjects in each group. We would like to point out the high frequency of the anti-HBc positivity without other markers in these two groups, respectively 20% in the group 1 and 32% in the group 2. These results emphasize the interest in screening systematically this marker in all blood donors.     

Heterogeneity of peripheral blood monocyte populations in human immunodeficiency virus-1 seropositive patients

Abstract Monocytes from human immunodeficiency virus (HIV) patients have an increased heterogeneity of phenotype and function. In a study of 120 HIV patients we have demonstrated that they have normal monocyte differential counts but that with progression of the disease an increasing proportion of monocytes show phenotypic and functional evidence for activation or maturation. A proportion of the monocytes are larger, with increased expression of CD11b, HLA-DR, CD45 and CD16. Concomitantly there was increased expression of TNF-α, high constitutive synthesis of PGE2 and high plasma IL-6 levels. This suggested that there exist a more dynamic situation of recuitment, activation and maturation of peripheral blood monocytes driven by HIV infection which results in a broader phenotypic profile.     

The Ba fragment of complement factor B inhibits human B lymphocyte proliferation

Normal human B lymphocyte function is finely regulated by both positive and negative signals at each stage of activation, proliferation, and differentiation. Activation signals include antigen and surface Ig cross-linking agents such as anti-mu or anti-delta. Signals inducing proliferation include IL-2, high m.w.-B cell growth factor (BCGF), and low m.w.-BCGF. IL-2 as well as IL-6 and other partially characterized B cell differentiation factors can induce terminal differentiation of proliferating B cells into Ig-secreting plasma cells. Various C components have been described to regulate B cell function including Bb that enhances proliferation, C5a that enhances Ig production, and C3a that inhibits Ig production. In our study, we examined the ability of the factor B cleavage fragment Ba to influence human B cell function. Ba did not affect the activation of resting B cells but inhibited the proliferation of activated B cells stimulated with either high m.w.-BCGF or low m.w.-BCGF. The inhibition occurred with doses of Ba as low as 1 microgram/ml (29 nM). Ba was found to bind to activated human B lymphocytes in a saturable manner with an apparent K of approximately 25 nM and an apparent Bmax of 56,000 sites/cell. A peptide made of the carboxy terminal 10 amino acids of Ba (GHGPGEQQKR), was also found to inhibit growth factor induced proliferation of activated B cells but at an ID50 of approximately 5 microM. Finally, Ba was found to inhibit the terminal differentiation of Staphylococcus aweus Cowan-activated B cells stimulated with B cell differentiation factors but not Ig secretion by the partially differentiated EBV-transformed cell line SKW.6. Thus, concentrations of Ba achievable in vivo at sites of active inflammation were found to act on human B lymphocytes by inhibiting their proliferation. This may act to limit the immune response to a specific antigenic challenge.     

Separation and characterization of human T G and T M lymphocyte populations

During the last few years a number of experimental evidences have shown the presence of Fc receptors for IgG or IgM on the membrane of human T cells. These two different receptors are detectable and mutually exclusive on distinct cell populations named respectively TG, TM and T "null" (which lack detectable receptors). Studies on the functional activities of these cells have shown that TM and TG lymphocytes play an antitetical role in regulating B cell response, TM exerting an "helper" activity on the differentiation of B lymphocytes while TG having a "suppressor" one. The aim of this study has been to determine the values of these two subpopulations in a group of twenty control subjects. Our results have shown that TG constitute 10%, whereas TM represent 40% of the total T cells. After EA-G rosetting, the purification of this subpopulation on a density gradient has shown an enrichment of more than 90% in TG cells, while TM contaminate this fraction for less than 4%. The purity of the fraction containing TM has been evaluated using the localization of alpha-naphthyl acetate esterase activity, which has shown that more than 88% of the cells in this fraction are positive for this enzyme.     

Transmembrane ion conductance in human B lymphocyte activation.

Human B lymphocytes were examined to determine whether transmembrane ion conductance plays a role in cell activation. Mitogens (anti-human IgM F(ab')2 fragment (anti-mu) and PMA) were used to stimulate B lymphocytes. Mitogen-induced DNA synthesis was inhibited by tetraethylammonium-Cl (TEA), 4-aminopyridine (4AP), verapamil, and diltiazem in a dose-dependent manner. This inhibition was not due to reduction in cell viability as determined by trypan blue exclusion. Mitogen-induced increases in RNA synthesis were partially inhibited by TEA and 4AP and were more completely inhibited by verapamil and diltiazem. Mitogen-induced cell volume increases were not affected by TEA or 4AP but were completely inhibited by verapamil and diltiazem. B lymphocytes stimulated with anti-mu expressed G1 phase cell surface antigens in the presence of TEA or 4AP, but failed to do so in the presence of verapamil or diltiazem. Substitution of PMA for anti-mu as the mitogen did not alter the effects of TEA or 4AP. However, verapamil inhibited PMA-induced expression of G1 phase cell surface markers although diltiazem did not. The patch clamp technique was used to directly examine plasma membrane ionic currents in whole-cell, cell-attached, and inside-out patch configurations. Activation of B lymphocytes with either anti-mu or the Ca2+ ionophore, A23187, inhibited opening of one type of channel in cell-attached patches. In inside-out patches, this channel type conducted current when the bath [Ca2+] was low (6 X 10(-8) M) but failed to conduct current when the bath [Ca2+] was increased above 1 X 10(-6) M. The results of these experiments are consistent with the hypothesis that activation of B lymphocytes induces alterations in plasma membrane ion conductance. Single channel studies suggest that activation induced increases in [Ca2+]i may directly inhibit a specific set of plasma membrane ion channels as one mechanism by which transmembrane ion flux is altered.     

Heterogeneity of lipoprotein B

Combined very low and low density lipoproteins were derived from human plasma by polyanion precipitation and the low density lipoprotein fraction (density 1.027–1.050 g/ml) was isolated by sequential ultracentrifugation. When this fraction was applied to Sepharose column chromatography, three lipoproteins were eluted. The first and third peaks were minor components while the second peak represented the bulk of LDL. Further chromatographic and electrophoretic studies indicated that the component representing the second peak was heterogeneous. This component was subsequently delipidated at pH 4 in a quaternary biphasic solvent system. The apoproteins remained soluble after delipidation and were treated with various deaggregating agents. On column isoelectric focusing in the presence of 4 M urea the apoproteins banded as broad overlapping peaks between pH 3 and 7. When hexanol was added to the system, distinct apoprotein subfractions were resolved.     

Isolation and electrophoretic characteristics of 3 lymphocyte populations of normal human blood]

Cell electrophoresis allows separation of normal human blood lymphocytes into two main groups which are a function of their relative rates of migration, with regard to the reference speed (1 mum.sec.-1V-1.cm): the lymphocytes which have a greater mobility than this value seem to be T-lymphocytes (80,1 per cent for 42 healthy adults); on the contrary, B-lymphocytes have an inferior mobility (19,9 per cent). Two known methods are used for the selection of the lymphoid populations: spontaneous rosetting with sheep's red blood cells, which are characteristic of T lymphocytes, and adherence to nylon wool columns, which is dominant in the case of B-lymphocytes. This method confirms the fact that T-lymphocytes have a rapid migration and B-lymphocytes a slow migration. We have isolated a third population, having neither the T markers, nor the B markers. It has a very homogeneous migration, centered on the two classes 1,05 and 1,10 mum.sec.-1.V.-1.cm.     

Lectin histochemistry of the mast cell: Heterogeneity of rodent and human mast cell populations

Summary Mast cell granules contain a variety of N-linked saccharides. Heterogeneity of the expression of these saccharides in mast cells was studied in rodent and human tissues which were so selected as to contain all the mast cell subsets previously identified using other criteria. Dermal and intestinal mucosal mast cells were stained with lectins using an avidin-biotin system. It was found that dermal and subepidermal mast cells in the rat and mouse, and mucosal and dermal human mast cells showed very similar lectin binding properties to each other, with a fine variation in the inlensity of staining with certain lectins. Rat mucosal mast cells, however, showed a distinctive pattern of lectin binding which was not seen in mast cells from any other tissue studied. The chemical basis of the differences seen were deduced and the possible significance of these structural variations is discussed.     

Surface marker characterization of EBV target cells in normal blood and tonsil B lymphocyte populations

Human FACS-sorted B lymphocyte subpopulations were investigated for their susceptibility to immortalization by Epstein Barr virus (EBV). Only B cells reacting with the monoclonal antibody B2 were immortalized, whereas cells reacting with anti-human IgG or the monoclonal antibody BB2 were not responding. Cells positive or negative for IgM, IgD, Burkitt's lymphoma antigen (BLA), BB1, and HB2 were all transformed by EBV.