Preparative separation of human B and T lymphocytes by free flow electrophoresis

An electrophoretic method for the quantitative separation of human B and T lymphocytes in a carrier-free system is presented. The method is based on the fact that B and T lymphocytes show marked overlap in their size and density characteristics, but differ sufficiently in surface charge to be separable by electrophoresis. The technique is performed in phosphate-buffered saline and appears to be especially suitable for the enrichment of nonstimulated, functionally intact lymphocytes which can be directly used for further immunological or biochemical studies.     

Chromosomal proteins in human B and T lymphocytes.

Histones and non-histone chromosomal proteins were characterized in B and T human lymphocytes by means of polyacrylamide disc gel electrophoresis. It was found that while histones do not present appreciable differences in the two examined populations, non-histone chromosomal proteins exhibit distinct electrophoretic profiles. Low molecular weight proteins predominate in B lymphocytes whereas high and intermediate proteins are largely represented in T lymphocytes. The latter proteins may be related to the capability of these resting cells to proliferate under appropriate antigenic stimuli.     

Uridine labeling of human lymphocytes: Differential uptake by T and B cells

Human peripheral blood lymphocytes were labeled with [³H]uridine and separated into immunoglobulin (Ig)-positive-enriched and Ig-negative populations by rosetting and Ficoll sedimentation. The Ig-negative (T cell-rich) fraction was found to be more heavily labeled than the B cell-enriched population, in agreement with previous results in rats. Combined autoradiography and rosetting confirmed the differential uridine labeling of T and B cells. Incorporation of cytidine and adenosine by T and B cell-enriched populations showed similar but less dramatic differential labeling.     

Survival of human T and B lymphocytes after X-irradiation.

The survival of unstimulated human T and B lymphocytes after X-irradiation in vitro was measured by Trypan Blue dye exclusion over a period of four days. B cell numbers were observed to decline rapidly even after relatively low doses, but T cell numbers fell much more slowly. A comparison of the percentage survival 96 hours after irradiation shows that in this system T cells are between approximately 2 and 5 times more resistant than B cells. Data for interphase death after 48 hours are compared with cytogenetic data for interphase loss of PHA-stimulated human lymphocytes and are shown to be in broad agreement at radiation doses below 400 rad. It is suggested that at higher doses mitotic delay may be increasingly important leading to selection of non-irradiated cells at 48 hours.     

Differentiation of human T and B peripheral blood lymphocytes by high-resolution cell image analysis

Automated cell image analysis of light and electron microscopic pictures was used for differentiation of nonlabeled lymphocytes in blood smears and in smears of purified lymphocyte suspensions. The percentages of T and B lymphocytes were determined by a two-step rosette assay with sheep red blood cells (T cells) and an immunofluorescence assay with FITC-labeled antihuman globulin (B cells). Images from 1,400 Feulgen-stained and 12,000 Pappenheim-stained cells were analyzed. Various classification methods allowed two lymphocyte subpopulations to be discriminated at the light and electron microscopic levels on the basis of different visual and subvisual morphologic features. As found by immunologic methods, morphologically determined subpopulations corresponded to T and non-T cells, with no further differentiation of non-T cells into B or null cells possible. The results allow the conclusion that there are morphologic differences between human T and non-T cells, with the differences distinguishable from individual variations as well as from alterations induced by sample preparation.     

Mouse hepatitis virus 3 replication in T and B lymphocytes correlate with viral pathogenicity

Viral pathogenicity may be regulated by host defense mechanisms at the virus-immune cell interaction level. The immune system plays an important role in the outcome of acute disease induced by the mouse hepatitis virus type 3 (MHV3) virus. The lymphoid cells act as effectors in the virus elimination as well as targets for viral replication. In order to demonstrate a correlation between MHV3 pathogenicity and viral replication in lymphocytes, genetically-determined resistant A/J and susceptible C57BL/6 mice were infected with pathogenic (L2-MHV3) or nonpathogenic (YAC-MHV3) viral strains. Pathogenicity and histopathologic studies have revealed that lymphoid organs such as thymus and spleen, showed injuries or atrophy in susceptible mice infected with L2-MHV3. No histopathologic lesions in the lymphoid organs occurred in C57BL/6 mice infected with YAC-MHV3 or A/J mice infected with both viruses. The mechanisms involved in the lymphoid injuries were studied regarding viral replication in the lymphoid organs and cells in infected mice. Results indicate that cell depletion in lymphoid organs is caused by a complete viral replication in lymphoid cells. Thy1.2+ and surface IgM+ lymphoid cells from susceptible C57BL/6 mice infected with L2-MHV3 were permissive to viral replication and to subsequent cell lysis. No cell lysis, however, occurred in lymphoid cells from C57BL/6 mice infected with YAC-MHV3 and A/J mice infected with both virus strains. In vitro studies, with purified T and B cell populations were performed to determine the mechanism effecting susceptibility or resistance to viral-induced cell lysis occurring in such cells. A blockade, probably occurring at the viral RNA polymerase activity level, prevents viral replication in resistant cells between the stages of fixation of the virus at the cell-surface receptor and the viral protein translation. These experiments indicate that an intrinsic virus-specific resistant mechanism occurs in lymphoid cells that plays a major role in the viral pathogenicity.     

Immortalization of human T lymphocytes by oncogenes

Immortalized human T cell lines were established by cotransfecting c-Ha-ras and c-myc oncogenes to lymph node lymphocytes. The cell lines kept growing for 3 months after establishment without a decrease in growth rate. The cells did not require interleukin-2(IL-2) for their growth, but addition of IL-2 stimulated the growth of these cells. Flow cytometric analysis revealed that these cells were T cells expressing CD4 or CD8 antigens. A CD4 positive (CD4⁺) cell line produced IL-6, indicating that the cell line belongs to helper T cells. The CD8 positive (CD8⁺) cell line possessed cytotoxicity to tumor cells, indicating that the cell line were killer T cells. Both cell lines were able to proliferate in serum-free medium indefinitely.     

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.     

Activation of human T lymphocytes by bryostatin

The immunologic effects of bryostatin (Bryo), a PKC activator with antineoplastic activity, were assessed and compared to PMA. Bryo induced IL-2R expression on CD4+ and CD8+ human T lymphocytes with a dose response comparable to PMA. However, Bryo induced only a marginal proliferative response as compared with the vigorous response induced by PMA. Bryo mediated functional receptor expression because the proliferative response was enhanced by addition of rIL-2. Furthermore, the proliferative response was inhibited by the relatively specific Ca+, phospholipid-dependent protein kinase (PKC) inhibitor, H-7, indicating a role of PKC in Bryo-induced activation. Addition of the calcium ionophore, ionomycin, to Bryo-stimulated lymphocytes resulted in the production and secretion of IL-2 with a concomitant proliferative response. This effect of the calcium ionophore could be inhibited by cyclosporine with identical results obtained in PMA-stimulated cultures. A most intriguing finding was that Bryo could effectively antagonize PMA-induced T cell proliferation. Although this mechanism of inhibition is unclear, a discussion with respect to differential effects on potential intracellular PKC isoforms is provided. These studies indicated that Bryo has potent immunopotentiating properties that share some similar effects of the phorbol ester, PMA, but offers the additional property of modulating other phorbol ester effects on proliferation.     

Prolactin receptors on human T and B lymphocytes: antagonism of prolactin binding by cyclosporine

Prolactin (PRL) receptors have been identified recently on human peripheral blood mononuclear cells (MNC) and may be involved in the regulation of cell-mediated immunity. Cyclosporine (CsA), an immunosuppressive cyclic endecapeptide utilized to prolong graft survival in human organ transplant patients, affects PRL binding to MNC. At concentrations of CsA from 10(-10) through 10(-8) M, the amount of PRL bound to MNC markedly increased to ca. 400% of controls, whereas CsA concentrations of 10(-6) and 10(-5) M totally inhibited PRL binding to lymphocytes. The ability of low concentrations of CsA to enhance PRL binding was temperature-dependent and did not occur when binding assays were conducted at 4 degrees C. PRL displaced [3H]CsA from lymphocytes with ca. 50% displacement at 10(-9) M PRL and total displacement at concentrations of 10(-7), 10(-6), and 10(-5) M. Growth hormone did not displace [3H]CsA in similar experiments. CsA also did not alter the binding of a beta-receptor antagonist to MNC, again suggesting that CsA was specific in its antagonism of PRL binding. A CsA analog with no immunosuppressive action, cyclosporin H, did not alter PRL binding to MNC. Furthermore, PRL receptors were demonstrated on four cell lines of human and mouse origin. Finally, PRL receptors were identified on purified populations of T and B lymphocytes isolated from human spleens, and CsA again inhibited PRL binding at concentrations of 10(-7) and 10(-6) M. The presence of PRL receptors on T and B lymphocytes suggests that PRL may be involved in the regulation of humoral and cell-mediated immunity, and that one effect of CsA on immune function may be its ability to inhibit the effects of PRL action on these lymphocytes.     

Autoreactivity by design: innate B and T lymphocytes

Innate B and T lymphocytes are a subset of lymphocytes that express a restricted set of semi-invariant, germ-line-encoded, autoreactive antigen receptors. Although they have long been set apart from mainstream immunological thought, they now seem to represent a distinct immune-recognition strategy that targets conserved stress-induced self-structures, rather than variable foreign antigens. Innate lymphocytes regulate a range of infectious, tumour and autoimmune conditions. New studies have shed light on the principles and mechanisms that drive their unique development and function, and show their resemblance to another subset of innate lymphocytes, the natural killer cells.     

Difference in polyamine transport in human B and T lymphocytes.

Preparations enriched in human blood B lymphocytes are able to take up polyamines efficiently. Uptake by T cells is barely detectable. Human non-circulating B cells (from tonsils) have a much lower ability to take up polyamines, as do mixed populations of bovine lymph nodes. B cells contain a higher amount of endogenous polyamines and show higher ornithine decarboxylase activity than T cells.     

Class II MHC self-antigen presentation in human B and T lymphocytes

Human CD4(+) T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4(+) T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.     

Sister chromatid exchange in highly purified human B and T lymphocytes

Summary Sister chromatid exchange (SCE) rates were determined in human peripheral blood B and T lymphocyte populations highly purified by immunologic methods. The purified populations were supplemented with -irradiated unseparated autologous mononuclear cells to restore helper-functions and stimulated with pokeweed mitogen (PWM) and phytohemagglutinin (PHA), respectively. Measured at the different peaks of proliferation after identical bromodeoxyuridine (BrdU) incubation times, T lymphocytes showed significantly higher SCE frequencies than B lymphocytes. In both populations, different proliferation kinetics and a different minimal BrdU concentration for sister chromatid differentiation (SCD) were observed.     

Mitogen induction of deoxyuridine triphosphatase activity in human T and B lymphocytes

Deoxyuridine triphosphatase (dUTPase; deoxyuridine diphosphohydrolase; EC 3.6.1.23) activity during mitogen stimulation was investigated in human T-cell and B-cell enriched mononuclear leucocyte fractions as well as in a mixed lymphocyte population. The dUTPase activity was very low in the resting peripheral blood lymphocytes. A remarkable enhancement of enzymatic activity was observed when cells were stimulated with different mitogens; T-cells and non-separated lymphocytes with phytohaemagglutinin, and the B-cell enriched fraction with pokeweed mitogen. There was a positive correlation between dUTPase activity and the enhancement of macromolecule synthesis (protein and RNA). In particular, a highly significant correlation was observed between dUTPase activity and DNA synthesis in the three human lymphocyte populations studied. This supports the view that the enzyme dUTPase may have a significant role in cellular proliferation. The physiological role of the enzyme is discussed.     

Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase.

Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 microM acyclovir [9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells contained multiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not required for cell transformation and the initial amplification of the viral genome.     

Studies on the electrophoretic separability of B and T human lymphocytes.

Cell electrophoresis experiments were performed at physiological, ionic strength on Hypaque-Ficoll separated normal human peripheral blood lymphocytes. The initial lymphocyte preparations averaged 72% T cells and 22% B cells as estimated by resetting techniques. A bimodal electrophoretic distribution of fast T cells and slow B cells such as has been repeatedly shown to exist for experimental animals could not be demonstrated. Neither B- nor T-enriched populations showed any correlation between mobility and degree of enrichment. A strong positive correlation was found between the non-rosetting cells and a very slow electrophoretic subpopulation produced during the B enrichment procedure. It was also found that considerable variability in lymphocyte mobilities existed from person to person and even in the same individual sampled over several months; these observations imply that the pooling of electrophoretic data can be misleading. The importance of these findings lies in the growing interest in relating lymphocyte electrophoresis to disease processes and immunological rejection phenomena.