Fc receptors and human T lymphocytes in frozen-thawed peripheral blood cells

The present study was carried out to investigate the influence of cryopreservation on human T-cell subsets defined by their membrane receptors for Fc IgM (TM) and Fc IgG (TG) and by their membrane antigens. For this purpose isolated T cells, obtained by neuraminidase-treated sheep erythrocyte (E-N) rosetting, and enriched mononuclear cells were cryopreserved using a programmed freezing procedure. A significant decrease of the TM and TG cells was found whereas the proportion of T cells and their subsets determined by monoclonal antibodies seemed not to be influenced. The effectiveness of T-cell separation by E-N rosetting of frozen lymphocytes demonstrated no impairment of the E-receptor binding capacity of T cells. The PHA reactivity of separated T cells was maintained after cryopreservation; however, the spontaneous blastogenesis was reduced significantly. The selective loss of the TM and TG cells seemed to be dependent on the length of the phase transition time; over 90 sec the capacity of the expression of Fc receptors was profoundly affected. Neither an additional 20 hr incubation after hypotonic shock prior to cryopreservation nor incubation after thawing could repair this function of T cells. The data suggest irreversible damage of the Fc receptor expression capacity on the cell membrane as a result of a disturbance of metabolic pathways rather than a preferentially greater sensitivity of these cells to cryopreservation.     

Fc receptors for IgA on human B, and human non-B, non-T lymphocytes.

Recently, receptors for IgA were demonstrated on subpopulations of human T lymphocytes. In this report, TNP-modified ox erythrocytes coated with the IgA myeloma MOPC-315 were used to detect IgA receptor-bearing lymphocytes within the human non T cell lymphocyte population. A mean of 5.3% (range 2.9 to 12.4%) of E-rosette negative human lymphocytes bound IgA-coated indicator cells. Blocking studies with soluble IgA, IgG, and IgM demonstrated that the IgA receptors on the non-T cell populations were separate and distinct from the Fc-receptors for IgG and IgM. Fractionation of the non-T lymphocytes on anti-human (Fab)2 columns into sIg+ and sIg- populations or by rosetting with EAC to provide CRL+ and CRL- populations demonstrated that Fc-IgA receptors were present on a subpopulation of sIg+, CRL+ lymphocytes, and also on sIg- (non-T, non-B) lymphocytes.     

Glucocorticoid receptors in purified subpopulations of human peripheral blood lymphocytes.

On the premise that the differential effects of glucocorticoids on various aspects of the immune response may be mediated by differences in the glucocorticoid receptors in the effector cells, subpopulations of human peripheral blood lymphocytes were examined for these receptors as well as for glucocorticoid responsiveness. Purified T and non-T lymphocytes, when studied by a sensitive whole cell assay technique, contained equivalent amounts of specific glucocorticoid receptor, which, by binding affinity and specificity measurements, were indistinguishable from each other. Furthermore, under in vitro incubation conditions, macromolecular synthesis in both of these cell populations was inhibited by glucocorticoid at concentrations which saturated the receptor sites. It is concluded that the putative differential effects of glucocorticoids on T and non-T lymphocyte-associated functions are probably not mediated by differences in the glucocorticoid receptors in these cell populations.     

Fc receptors on human T lymphocytes. II. Cytotoxic capabilities of human T gamma, T mu, B, and L cells.

Subpopulations of human peripheral blood lymphocytes were prepared by rosetting techniques employing neuraminidase-treated sheep erythrocytes (SRBC_n), sheep erythrocytes coated with IgM and murine complement (EAC′), and bovine erythrocytes coated with IgG and IgM. The isolated subpopulations were tested in assays of natural cytotoxicity (NC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). B cells (SRBC_n?, EAC′+) did not mediate cytotoxicity. L cells (SRBC_n?, EAC′?) mediated NC and ADCC but not MICC. T cells (SRBC_n+) mediated NC, ADCC, and MICC. Separation of T cells into Fc-IgG (Tγ) and Fc-IgM (Tμ) subsets revealed that Tγ cells mediated NC, ADCC, and MICC while Tμ cells mediated only MICC. Thus MICC but not NC or ADCC was solely T-cell mediated. Tγ and L cells were functionally distinguishable in that Tγ cells but not L cells mediated MICC. Tγ cells and Tμ cells differed with regard to NC and ADCC effector function while both subsets mediated MICC.     

Studies of Fc gamma receptors of human B lymphocytes: phospholipase A2 activity of Fc gamma receptors

The presence of phospholipase A2 activity within human B cell Fc gamma receptors was investigated. Lysate produced by detergent treatment of chronic lymphocytic leukemia cells that had 1% of the cells surface radioiodinated was subjected to affinity chromatography by using either rac-1-(9-carboxynonyl)-2-hexadecylglycero-3-phosphorylcholine-Sepharose (PC-Sepharose) or heat-aggregated human IgG-Sepharose 4B conjugate (IgG-Sepharose). The materials eluted from both adsorbants by ethylenediaminetetraacetate- or urea-containing buffer were further purified by gel filtration and isoelectric focusing in the presence of 6 M urea. Both isolated PC- and IgG-binding materials were homogeneous, when judged by gel filtration and isoelectric focusing, and had identical isoelectric points (pI = 6.5), peptide maps, and amino acid compositions. Furthermore, both preparations catalyzed equally the hydrolysis of phosphatidylcholine to release fatty acid from the 2 position. Optimal enzymatic activity depended on the presence of Ca2+, was maximal at pH 9.5, and was augmented by Fc gamma fragments. Both preparations specifically bound to the Fc portion of IgG and inhibited human antibody-coated erythrocyte rosette formation by peripheral mononuclear cells. Our data thus demonstrate the identity of PC- and IgG-binding materials and suggest that a functional activity of the human B cell Fc gamma receptor is the generation of phospholipase A2 activity within the plasma membrane.     

Shared idiotypes of human peripheral blood B and T lymphocytes.

In a patient with an IgG lambda monoclonal serum component possessing anti-streptolysin O activity, we have demonstrated peripheral blood B and T lymphocytes with shared or similar idiotypes. The idiotypic T lymphocyte membrane structure was capable of binding the specific antigen (SLO). After radioiodination and subsequent detergent solubilization of the same T cell population, immunoprecipitation of the lysate by employing anti-idiotypic antibodies, resulted in the isolation of a polypeptide chain with a m.w. of 70,000 on SDS polyacrylamide gels under reducing conditions. The polypeptide expressed no isotypic immunoglobulin markers. Internal labeling experiments indicated that this membrane structure was actively synthesized by the T lymphocytes.     

Subpopulations of human peripheral blood lymphocytes.

Multiple staining protocols have been developed for the classification of subpopulations of human peripheral blood lymphocytes. Of the non-T (E^?) cells, roughly half (10–20% PBL) have receptors for complement components as detected with complement-coated zymosan particles, but do not show Fc receptors as detected with Ripley IgG-coated human RBC. The other half are C^?, Fc⁺, with a small percentage possessing both receptors. The C⁺, Fc^? cells can be subdivided into cells which are IgM⁺ (75%) or IgM^?. Cells with Fc receptors detected with aggregated IgG were IgM⁺.     

Activation of Fc receptor-bearing lymphocytes by immune complexes. I. Stimulation of lymphokine production by nonadherent human peripheral blood lymphocytes

Activation of human peripheral blood lymphocytes by incubation with particulate immune complexes or aggregated human gamma-globulin was studied by measuring the release of leukocyte migration inhibitory factor (LIF) activity. LIF-active supernatants were consistently produced when nonadherent lymphocytes containing less than 1% surface immunoglobulin-bearing cells and less than 0.2% nonspecific esterase-positive monocytes were incubated in the presence of RBC sensitized with rabbit or human antibodies or with pooled heat-aggregated human gamma-globulin. This immune complex-induced lymphokine production (ICLP) was dependent on the presence of cells bearing receptors for the Fc portion of IgG (Fc gamma). ICLP could not be demonstrated with lymphocyte preparations enriched for B cells even though the latter showed vigorous LIF production in the presence of complement-sensitized erythrocytes. ICLP was dependent on the concentration of lymphocytes and of stimulant as well as on the duration of coincubation, and it required active metabolic processes and RNA and protein synthesis but not DNA synthesis. Ca++ but not Mg++ was obligatory. ICLP by non-B Fc gamma receptor-bearing lymphocytes may play a role in antibody-dependent protective inflammation and immunologic injury phenomena, which is similar to that of lymphokine release by antigen-activated T cells in delayed hypersensitivity responses.     

Expression of human lactotransferrin receptors in phytohemagglutinin-stimulated human peripheral blood lymphocytes. Isolation of the receptors by antiligand-affinity chromatography

In the resting rate, the human peripheral blood lymphocytes did not show detectable surface and intracellular receptors for human lactotransferrin. However, both types of lactotransferrin receptors were expressed during stimulation of lymphocytes with phytohemagglutinin. The appearance of receptors was time-dependent and the number of receptors reached a plateau after at least two days of mitogen stimulation. These results suggest that the presence of surface receptors on mitogen-stimulated lymphocytes is not consecutive to a modification of subcellular distribution but to an induction of biosynthesis of the receptors. As measured by incorporation of [3H]thymidine into DNA, addition of human lactotransferrin in a serum-free medium increased the proliferative activity of phytohemagglutinin-stimulated lymphocytes. Optimal enhancement of [3H]thymidine incorporation was obtained by adding 30% iron-saturated lactotransferrin at a concentration of 0.17 microM. Therefore, the role of lactotransferrin in the response of lymphocytes to mitogen stimulation appears to be similar to that previously described for serotransferrin. The lactotransferrin receptor was visualized using 125I-labeled lactotransferrin on nitrocellulose paper after electroblotting of the Triton X-100 extract of the phytohemagglutinin-stimulated lymphocytes as two protein bands of 100 and 110 kDa molecular mass. Purification of the lactotransferrin receptor from the Triton-X-100-soluble extract of stimulated lymphocytes was performed by antiligand-affinity chromatography. The binding of lactotransferrin to the purified receptors was reversible and dependent on concentration and pH.     

Comparison of the Fc receptors for IgE on human lymphocytes and monocytes

Fc receptors for IgE (Fc epsilon R) on human peripheral blood lymphocytes and monocytes and cultured lymphoblastoid and macrophage-like cell lines were compared with respect to: 1) binding affinity for radiolabeled IgE, 2) inhibition of IgE-specific rosette formation and inhibition of binding of radiolabeled IgE by an antiserum raised against Fc epsilon R isolated from a lymphoblastoid cell line, and 3) m.w. of radiolabeled cell surface proteins precipitated with the anti-Fc epsilon R serum. Scatchard analysis of 125I-IgE binding to lymphocytes, monocytes, and their corresponding cell lines showed biphasic binding curves with all cell types, from which 2 binding affinities were calculated to be KA = 6.2 +/- 1.1 and 2.0 +/- 0.5 x 10(7) M-1. The anti-Fc epsilon R serum inhibited both IgE rosette formation and binding of radiolabeled IgE by lymphocytes and monocytes but did not inhibit IgE rosettes formed by basophils. The inhibitory activity of the anti-Fc epsilon R serum could be absorbed with Fc epsilon R(+) but not with Fc epsilon R(-) cell lines. The anti-Fc epsilon R serum precipitated 2 peptides having m.w. of approximately 47,000 and 23,000 daltons from lysates of both cell surface-labeled lymphocyte and macrophage cell lines. These data indicate that Fc epsilon R on normal lymphocytes and monocytes, as well as on cultured lymphoblastoid and macrophage-like cells, are related structurally, since they share antigenic determinants, bind IgE with a similar affinity, and have similar m.w. However, they differ in all 3 parameters from Fc epsilon R on basophilic granulocytes.     

Mitogenic receptors on human peripheral blood lymphocytes: the interaction of Phaseolus vulgaris erythroagglutinating phytohemagglutinin and anti-thymocyte globulin on the human peripheral blood lymphocyte membrane.

A horse anti-human thymocyte antibody (ATG) obtained from the Upjohn Company was shown to stimulate DNA synthesis in human lymphocytes with a time course and magnitude of radioactive thymidine uptake comparable to that seen with phytohemagglutinin (E-PHA) and concanavalin A (Con A). Low mitogenic or nonmitogenic concentrations of intact ATG or its Fab fragments inhibited E-PHA-induced mitogenesis, whereas the response to Con A was unaffected. Competitive binding studies with ATG and E-PHA revealed mutual inhibition of binding to lymphocytes suggesting that E-PHA and the ATG share a common receptor site on the cell surface. ATG binding was unaffected by Con A. From the analysis of the binding data and the inhibition of mitogenesis, it appears that at least part of the E-PHA response in human lymphocytes involves receptors that are not acted on by Con A.     

Anti-tumor antibody produced by human tumor-infiltrating and peripheral blood B lymphocytes

Cell suspensions from 69 human tumor biopsies and malignant effusions depleted of infiltrating T cells were incubated for 10–14 days with mitomycin-C-treated cells of the transformed T cell line MOT as feeder cells. B lymphocytes proliferated and differentiated as indicated by immunoglobulin (Ig) seerction in the culture supernatants (B cell expansion). Ig was present in culture supernatants of tumor cell suspensions incubated without MOT feeder cells (non-expanded cells), but the addition of MOT feeder cells to these cultures invariably resulted in a significant increase in Ig concentration. While IgG, IgA. and IgM isotypes were all detected in supernatants of both expanded- and nonexpanded tumor cell suspensions, the increase in total Ig induced by MOT feeder cells was mainly due to an increase in IgG. Peripheral blood B lymphocytes (PBBL) from 15 cancer patients and 4 healthy individuals were also successfully expanded by the same method. In these it was shown that IgA was the predominant Ig isotype. Using a modified enzyme-linked immunosorbent assay, IgG of 25/36 expansions from tumor cell suspensions showed reactivity with autologous tumor targets, and that from 10/13 expansions reacted with allogeneic tumor targets of the same histological diagnosis. No reactivity was found against tumor targets of different histology. IgG of 4/10 expansions of PBBL from cancer patients showed reactivity with allogeneic tumor targets of the same histology, while no reactivity was demonstrated against tumor targets of different histology. IgG of expanded PBBL from healthy individuals showed no reactivity against tumor targets. This method allows detailed study of the specific humoral antitumor immune response of intratumoral and peripheral blood B lymphocytes in cancer.Work supported by grants from the Share and Concern Foundations and grant CA MOPP from the National Institutes of Health, C.J.A.P. is a visiting scientist from the University of Nijmegen, Department of Medical Oncology, Nijmegen, The Netherlands, and is supported by a Fulbright Senior Research Grant and grants from the Dutch Cancer Society and the Regional Cancer Center of the East Netherlands (IKO). J.A.M.B. is a visiting scientist from the University of Sao Paolo, Department of Immunology, Brazil, and is supported by grant 90/1844-4 from the FAPESP     

Electron microscopic morphometric analysis of human T and B peripheral blood lymphocytes

Using the system of morphometric analysis described in this paper, human peripheral blood T and B lymphocytes, labeled with specific surface markers, can be compared on different analytical levels. They show differences in their surface and the eccentricity of cells, in the relative surfaces occupied by peripheral and central condensed chromatin, in the average surface of the central chromatin clumps and in the number of perichromatin granules per nuclear surface. The morphometric analysis reveals the importance of examining the nuclear and the surface parameters in the characterization of lymphocytes, confirming that a detailed analysis of the nuclear characteristics can contribute to the identification of T and B lymphocytes by transmission electron microscopy.     

Studies on nucleoli in rosetting T and B lymphocytes of the human peripheral blood

The incidence of various nucleolar types was studied in human rosetting lymphocytes to provide an information on nucleolar types present in T and B lymphocytes of the peripheral blood. The results clearly demonstrate that both T and B lymphocytes of the peripheral blood mostly contain ring shaped nucleoli ("resting nucleoli") and less frequently other nucleolar types such as nucleoli with nucleolonemata or compact nucleoli ("active nucleoli") and micronucleoli ("inactive nucleoli"). Since all known nucleolar types and particularly micronucleoli may be observed in both T and B lymphocytes, nucleoli in these cells cannot indicate the type or origin of these cells but simply the state of the nucleolar RNA synthesis.     

Fc gamma-receptor-bearing, non-B lymphocytes in human peripheral blood: cytophilic immunoglobulin binds almost exclusively to large granular lymphocytes

Cytophilic IgG (CYT-Ig) has previously been reported to bind to both the "TG" (E+, Fc gamma R+) and "L" (E-, Fc gamma R+) subsets of non-B lymphocytes in human peripheral blood. Present investigations show that IgG-binding cells, as detected by a sensitive antiglobulin rosetting reaction, are contained almost entirely within the large granular lymphocyte (LGL) subpopulation, and that fewer than 5% of other non-B lymphocytes acquire IgG from serum. Cell membrane-bound IgG sterically blocks the reaction of LGL with sheep red blood cells and therefore influences the proportions of these cells characterized as TG (E+) or L (E-) lymphocytes. Although the majority of TG lymphocytes are LGL, a further subpopulation of E+, Fc gamma R+ cells are detectable under particular test conditions. Unlike LGL, these lymphocytes do not react with rabbit IgG-coated ox RBC (EAG) in saline, but will form EAG rosettes when the reaction is enhanced in the presence of Ficoll. These Fc gamma R+ cells are mostly of typical small-lymphocyte morphology and do not bind detectable amounts of CYT-Ig, nor do they express the monoclonal antibody-defined VEP 13 determinant associated with Fc gamma R on LGL.