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.     

Expression of IFN-alpha and IFN-gamma receptors on normal human small resting T lymphocytes and large granular lymphocytes

The expression of interferon (IFN) receptors was studied on freshly isolated human lymphocytes from normal donors. Highly enriched populations of small resting T lymphocytes and large granular lymphocytes (LGL) were found to constitutively express high-affinity receptors for IFN-alpha and IFN-gamma. Both types of lymphocytes also had lower-affinity IFN-alpha binding sites. T lymphocytes had a mean of 230 IFN-alpha and 520 IFN-gamma high-affinity receptors per cell, whereas LGL had 520 IFN-alpha and 760 IFN-gamma receptors. However, because LGL were larger than the T lymphocytes, the IFN receptor density was similar on the two types of lymphocytes. The affinity of binding was similar on the two types of normal lymphocytes and on the cultured lymphoblastoid cell line Daudi. The number of IFN receptors per cell and the affinities of the IFN-receptor interactions varied little among the normal donors. Both the freshly isolated normal lymphocytes and the cultured cell line Daudi had separate receptors for type I (alpha and beta) and type II (gamma) IFN. Taken together, our data indicate that two types of freshly isolated normal lymphocytes constitutively express IFN receptors that are similar to those present on the lymphoblastoid cell line Daudi derived from a patient with Burkitt's lymphoma.     

Glucocorticoid receptors in normal human lymphocytes

Glucocorticoid (GC) receptors were studied in intact lymphocytes from 11 donors. GC binding parameters were found to be highly reproducible in repeated experiments with lymphocytes. It was shown that GC receptors in donors' lymphocytes could be distributed into two different classes similarly to the pattern seen in skin fibroblasts. Human lymphocytes are an adequate object for studying genetically determined variability of GC receptors and its clinical importance.     

Altered expression and functional profile of lysophosphatidic acid receptors in mitogen-activated human blood T lymphocytes.

Lysophosphatidic acid (LPA) from platelets and mononuclear phagocytes regulates T cell functions through endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs). Human blood unactivated CD4+ T cells express predominant ly Edg-4 LPA R over marginal levels of Edg-2 LPA R, as assessed by semiquantitative PCR and Western blots. After mitogen activation, the CD4+ T cells express Ed g-2 Rs at approximately one half the level of Edg-4 Rs. Secretion of IL-2 by unactivated Edg-4 R-predominant CD4+ T cells incubated with anti-CD3 plus anti-CD28 antibodies was suppressed significantly and by up to 60% by 10-10 M to 10-6 M LPA, whereas secretion of IL-2 by mitogen-activated Edg-2 R and Edg-4 R codominant CD4+ T cells was enhanced by up to twofold by the same concentrations of LPA. The possibility that the two Edg Rs transduce different LPA signals to CD4+ T cells was supported by findings that IL-2 secretion was inhibited by mouse anti-Edg-4 R monoclonal antibody, but enhanced by mouse anti-Edg-2 R monoclonal antibody. The separate effects of each LPA R were studied in Jurkat T cell transfectants expressing principally human Edg-2 Rs (Jurkat-T-2) or Edg-4 Rs (Jurkat-T-4) and stimulated with anti-CD3 plus phorbol myristate acetate. LPA and anti-Edg-4 R antibody suppressed IL-2 secretion by stimulated Jurkat-T-4 cells, whereas LPA and anti-Edg-2 R antibody enhanced IL-2 secretion by stimulated Jurkat-T-2 cells. Activation-induced alterations in the relative levels of Edg-2 and -4 Rs on CD4+ T cells thus reverse the effects of LPA on T cell receptor-stimulated generation of IL-2.     

Undermethylation of interferon-gamma gene in human T cell lines and normal T lymphocytes.

The relative levels of DNA methylation at CCGG sequences within and around the interferon-gamma (IFN-gamma) gene in normal human tissues and cell lines were examined by Southern blot analysis using isoschizomeric restriction enzymes, HpaII and MspI. On the test of normal tissues, the IFN-gamma gene was undermethylated only in a small population of T lymphocyte, whereas the gene was fully methylated in T cell-depleted lymphocytes and uterus cells. In TCL-Fuj cell line which is a T cell line producing a high level of IFN-gamma spontaneously, the IFN-gamma gene was undermethylated. Moreover, the extent of DNA methylation was inversely correlated to the level of expression of the IFN-gamma gene in several T cell lines including sublines derived from TCL-Fuj cells. However, partial or complete unmethylation at the CCGG sites of IFN-gamma gene was observed in a promyelocytic leukemia cell line and two epithelial cell lines that fail to produce IFN-gamma irrespective of induction. These results suggest that undermethylation of IFN-gamma gene is necessary but not sufficient for its efficient expression.     

An appraisal of Fc receptors on human peripheral blood B and L lymphocytes.

Human circulating lymphocytes with easily detectable surface immunoglobulin have been divided into two populations, B cells and L cells. This second population lacks membrane-incorporated Ig, but has a receptor for membrane-labile cytophilic IgG. In this study purified B and L lymphocytes were examined for Fc receptors that bind aggregated IgG and IgG complexed to erythrocytes. Purified lymphocyte populations were prepared by nylon columns and by negative selection with rosetting techniques. L lymphocytes bound aggregated guinea pig and human IgG, and formed rosettes with human erythrocytes sensitized with Ripley IgG (EA). Treatment of L lymphocytes with trypsin had no effect on the receptors for IgG. B lymphocytes did not bind EA and attachment of aggregated IgG was variable; up to one-third of these cells fixed aggregated human IgG to the cell membrane. Trypsin treatment abolished binding of Agg-IgG to B cells in sharp contrast to its effect on L cells. Furthermore, double-label immunofluorescence studies showed that cells with both membrane-incorporated Ig and receptors for aggregated guinea pig IgG were rare. These studies indicate that human peripheral blood B lymphocytes lack a high affinity, trypsin-resistant Fc receptor that is present on L lymphocytes.     

Stabilization of methionine-enkephalin in human and rat blood

Methods of preventing the degradation of 3H-methionine-enkephalin (3H-ME) in human blood both at 37 degrees C and under conditions of immediate cooling were examined. We found that, contrary to previous suggestions, use of aprotinin (with or without immediate cooling) was ineffective in preventing the degradation of 3H-ME in blood. Thus, previous reports on the circulating levels of ME which relied on such procedures to stabilize the ME may have reported artifactually low values. However, we found that citric acid effectively prevents 3H-ME breakdown in both human and rat blood. Thus, we propose the use of citric acid, mixed with blood immediately upon collection, as an effective method for the stabilization of ME in blood.     

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.     

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.     

Immature T lymphocytes in human neonatal blood

Thirty-two cord blood samples taken after caesarean section or vaginal delivery and concurrent venous blood samples obtained from normal adult controls were evaluated using monoclonal antibodies. The percentage of circulating pan-T-cell+ lymphocytes was significantly lower in cord blood (46%) compared with adult controls (72%). In the cord cells, 22% showed reactivity with the common thymocyte antigen compared with less than 1% in adult controls. The helper:suppressor ratio was lower in cord blood (1.71) compared with 1.98 for adult blood. These figures reflect a unique population (12%) of immature T cells in cord blood that coexpress helper and suppressor phenotypes. These features are not found in adult blood. These double-labeling studies characterized a previously undescribed blood T-cell phenotype which correlates negatively with gestational age (R = -0.93). These studies reveal the presence of an immature population of T cells in normal human neonatal blood that exhibit the phenotype characteristic of normal developing thymocytes.     

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.     

A receptor for IgA on human T lymphocytes.

Receptors for IgA antibody-antigen complexes were demonstrated on 2 to 18% (mean 6.7%) of human peripheral blood T cells. The proportion of cells bearing detectable IgA receptors was low in freshly prepared T cells and increased in number after 18 to 24 hr of culture similar to the time course of appearance of the Tmu receptor. These T receptors were shown to be distinctly different from Fc-IgM and Fc-IgG receptors on T cells by blocking studies with purified IgA, IgG, and IgM.     

Unmasking by antimetabolites of receptors for sheep red blood cells on human lymphocytes

The action of antimetabolites (puromycin, cycloheximide) and cold was studied in the human rosette system. We found that the number of detectable receptors for sheep red blood cells on peripheral blood lymphocytes was increased in presence of some concentration of these drugs. A similar finding was noted when the blood lymphocytes were left at 4 °C. The possibility that both cold and antimetabolites, by modifying the cell membrane mobility, increase the receptor affinity and thus the number of detectable receptors is discussed. Another attractive possibility is also presented. We propose that the unmasking effect by antimetabolites is due to inhibition of protein synthesis which is necessary to better express the receptors for sheep red blood cells on human lymphocytes. This concept of decreased protein synthesis affecting the expression of surface receptors may be a more general phenomenon.