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M L Blue

Publications and source records attributed to M L Blue.

At least 37 records · Page 2Linked to original sources
PubMed1989

Expression of CD1 and class I MHC antigens by human thymocytes.

The acquisition of surface class I MHC molecules is associated with the maturation of thymocytes. Here, surface expression of class I MHC and CD1, which represents a family of MHC-related molecules, was analyzed on various human immature and mature thymocyte subpopulations. Class I expression was inversely related to the expression of CD1. The majority of CD4+ CD8+ cortical type thymocytes expressed low levels of class I MHC Ag, the previously described CD4+ CD8+ thymocyte subpopulation with low CD8 expression exhibited intermediate levels of class I MHC, whereas most of the single positive CD4 and CD8 thymocytes displayed high levels of class I MHC. Biochemical comparison of CD1 and class I showed that thymic class I molecules were post-translationally modified by phosphorylation, whereas CD1 was not phosphorylated. Furthermore, our studies suggested that in addition to CD1/CD8 complexes, thymocytes bear CD8/class I complexes. Chemical cross-linking and peptide mapping studies clearly identified the CD8-associated protein on thymic clones as the class I MHC molecule.

Antigens, CD1↗
PubMed1989

Regulation of CD4 and CD8 surface expression on human thymocyte subpopulations by triggering through CD2 and the CD3-T cell receptor.

Human thymocytes bearing the CD4 and/or CD8 antigens can be fractionated into cells with an immature and more mature phenotype based on their quantitative expression of the CD3 Ag (J. Immunol. 138:3108; J. Immunol. 139:1065). We show that the expression of CD4 and CD8 on thymocyte subpopulations with low CD3 (CD3L) and high CD3 (CD3H) is regulated by activation through the CD2 molecule and perturbation of the CD3-T cell receptor complex (CD3-Ti). Similar to its previously reported effects on peripheral T cells, PMA was able to induce the down-regulation of surface CD4, but not CD8, on thymocyte subpopulations. PMA could induce CD4 and CD8 phosphorylation in both CD3L and CD3H fractions. These results suggest that if changes in phosphorylation represent the mechanism by which CD4 and CD8 are able to transmit signals, this mechanism is operative in both CD3L and CD3H subpopulations. Treatment with anti-T11(2) and anti-T11(3) antibodies (CD2 activation pathway) resulted in partial down-regulation of CD4 but not CD8 surface expression on both CD3L and CD3H thymocytes. Similar treatment had no detectable effect on peripheral T cells. The down-regulation of surface CD4 induced by activation via CD2 could be inhibited by treatment of thymocytes with anti-CD3 antibodies. Treatment of thymocytes with anti-CD3 alone or following CD2 activation induced the selective down-regulation of surface CD8 within 15 minutes. These results suggest that CD2 and CD3-Ti triggering may regulate CD4 and CD8 surface expression on thymocytes. Furthermore, these results suggest that "cross-talk" between the CD2 and CD3-Ti pathway of activation may involve CD4 and CD8 molecules.

Antibodies, Monoclonal↗
PubMed1988

Evidence for specific association between class I major histocompatibility antigens and the CD8 molecules of human suppressor/cytotoxic cells.

Human T lymphocytes, metabolically labeled with 35S-cysteine and 35S-methionine, were reacted with the homobifunctional cross-linking reagent, dithiobis (succinimidyl propionate) (DSP). When detergent lysates from these cells were immunoprecipitated with a monoclonal antibody reactive with the CD8 antigen, a radiolabeled protein of approximately 44 kd was coprecipitated with the CD8 molecule. Immunoprecipitates from detergent lysates prepared without prior chemical cross-linking contained only the 33 kd CD8 molecule. Similar results were obtained when T lymphocytes or a cytotoxic T cell clone (T4T8Cl) were radiolabeled with 32P-orthophosphoric acid. The 44 kd CD8-associated protein was identified as the heavy chain of the class I major histocompatibility antigen by depletion in preclearing experiments with anti-class I MHC antibody and by peptide mapping. Further analyses indicated that the CD8-class I MHC association is due, in part at least, to disulfide bonding, which may be susceptible to cleavage during processing of cell lysates.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Comodulation of CD3 and CD4. Evidence for a specific association between CD4 and approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes.

The aggregation of a specific class of lymphocyte surface molecules results in patching, capping, and surface modulation of the aggregated ligand. Both CD4, an associative recognition structure found on helper T lymphocytes, and CD3, a component of the T cell receptor complex, are members of this functional subgroup. When 125I-labeled monoclonal antibodies reactive with either CD4 (19Thy 5D7) or CD3 (RW24B6) were bound to T lymphocytes, the subsequent addition of goat anti-mouse Ig resulted in their rapid, temperature-dependent internalization. Whereas the binding of 125I-19Thy 5D7 (anti-CD4) was inhibited by greater than 90% in the presence of unlabeled 19Thy 5D7, no inhibition occurred in the presence of unlabeled antibody reactive with CD3 (RW28C8). We took advantage of the fact that these antibodies were of different isotypes (19Thy 5D7:IgG2a; RW28C8:IgGl) to determine whether the internalization of CD3 induced the comodulation of CD4. T lymphocytes preincubated with 125I-19Thy5D7 (anti-CD4) and unlabeled RA28C8 (anti-CD3) were treated with goat anti-mouse IgGl under conditions shown to quantitatively internalize CD3. After 1 h at 37 degrees C, T lymphocytes had internalized 10.5 +/- 2.6% (n = 3) of their antibody-bound cell surface CD4. After similar incubations with media alone or with goat anti-mouse IgGl in the absence of prebound RW28C8 (anti-CD3), no internalization of CD4 could be detected. Control antibodies reactive with CD45R (2H4, IgGl) also failed to induce the internalization of CD4. Similar results were obtained by using a helper T cell clone (T4C1) that internalized 9.6 +/- 2.8% (n = 3) of its antibody-bound cell surface CD4 in response to CD3 modulation. In a reciprocal experiment, 125I-anti-CD3 (RW24B6, IgG2b) was preincubated with T4Cl cells together with unlabeled anti-CD4 (12T4D11, IgG1) prior to the addition of goat anti-mouse IgGl. The quantitative modulation of CD4 induced the co-internalization of 4.6 +/- 0.6% (n = 3) of cell surface CD3. These results suggest that approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes are specifically associated with CD4. Furthermore, our results suggest that an average of two CD4 molecules associate with each CD3:T cell receptor complex.

Antibodies, Monoclonal↗
PubMed1988

Regulation of T cell clone function via CD4 and CD8 molecules. Anti-CD4 can mediate two distinct inhibitory activities.

The functional effects resulting from CD4 and CD8 perturbation were analyzed by using a CD4+CD8+ clone and anti-CD4 and anti-CD8 monoclonal antibodies. Perturbation of CD8, but not CD4, by soluble antibody resulted in the inhibition of CD3-T cell receptor (CD3-Ti) triggering as determined by flow cytometric measurements of intracellular free Ca2+ concentrations. In addition, the CD3-T cell receptor-mediated cytotoxic function of the CD4+CD8+ clone was inhibited by anti-CD8, but not by anti-CD4. These results suggest that CD8, but not CD4, was functionally associated with CD3-Ti on the CD4+CD8+ clone. Although CD4 perturbation did not affect CD3-Ti-mediated activities, it resulted in the inhibition of the interleukin 2-dependent proliferation of this clone. Perturbation of CD8 did not affect the interleukin 2 dependent proliferation of the CD4+CD8+ clone. On the other hand, CD4 molecules of another CD4+CD8- clone unlike those of the CD4+CD8+ clone, were clearly linked to T cell receptor function. These results indicate that CD4 perturbation can result in two distinct regulatory activities; one involves the regulation of CD3-T cell receptor function, whereas the other is not directly associated with CD3-T cell antigen receptor function. The data are also consistent with the notion that CD4 and CD8 do not merely function as recognition and adhesion elements for accessory cell major histocompatibility complex molecules, but have a direct role in the regulation of T cell activation.

Antibodies, Monoclonal↗
PubMed1988

Crosslinking CD3 with CD2 using sepharose-immobilized antibodies enhances T lymphocyte proliferation.

T lymphocyte proliferation can be triggered through interactions with either CD3:Ti, the target of antigen-specific activation, or CD2, the target of an antigen-independent activation pathway. Sepharose-immobilized antibody reactive with CD3 was used to aggregate the T cell receptor complex resulting in T lymphocyte activation. When CD3 was simultaneously crosslinked with CD2 using Sepharose beads coupled to antibodies directed at both determinants, T cell proliferation was markedly enhanced (stimulation index = 8- to 11-fold). A smaller enhancement was induced when CD3 was crosslinked with several other functionally relevant T cell surface molecules. The relative mitogenic potency of the accessory molecules tested was CD2 greater than CD4 greater than CD8 greater than 2H4. Little or no increased proliferation resulted from crosslinking CD3 with class I or class II major histocompatibility antigens. The added proliferation induced by CD3: CD2 crosslinking did not occur in the presence of soluble antibodies directed against CD2. Human thymocytes, the majority of which express both CD3 and CD2, were similarly activated by Sepharose-immobilized antibodies. Our results suggest that specific interactions between T cell surface molecules may play a role in the regulation of lymphocyte activation.

Antibodies, Monoclonal↗
PubMed1987

Phosphorylation of CD4 and CD8 molecules following T cell triggering.

CD4 and CD8 molecules have been implicated in the regulation of T cell activation. In the present study, CD4 and CD8 were modified by increased phosphorylation when T cell clones or T cells were either exposed to phorbol-12-myristate- 13-acetate or were triggered via the CD3-T cell receptor complex. Activation of T cells through the CD2 sheep erythrocyte binding protein, using anti-T11(2) and -T11(3) antibodies, also resulted in CD4 and CD8 phosphorylation. These findings suggest that signals derived from two different receptor pathways can converge and result in similar molecular modifications of CD4 and CD8. Furthermore, phorbol myristate acetate treatment or activation via the CD2 pathway induced phosphorylation of the CD4 and CD8 molecules of thymocytes, suggesting that these molecules may be functional in thymus. Together, our findings indicate that CD4 and CD8 phosphorylation is a consequence of T cell triggering, and suggest that CD4 and CD8 phosphorylation may represent a molecular signaling mechanism among the CD3-T cell receptor complex, CD2, CD4, and CD8.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Identification and isolation of a T4+T8+ cell with high T3 expression in human thymus: a possible late intermediate in thymocyte differentiation.

By using sensitive three-color fluorescence flow cytometric techniques, we were able to identify a T4+T8+ thymocyte with high T3 surface density (T3H) representing 4 to 9% of thymocytes. To characterize the T3HT4+T8+ cell, thymic subpopulations with high T3 surface density (T3H) and lower T3 density (T3L/T3-) were compared with regard to T6 expression. The T3H subpopulation was characterized by lower numbers of T6+ cells and reduced levels of T6 antigen density, whereas the T3L/T3- population was greater than 90% T6+ and expressed this antigen at high cell surface density. In addition, T3H fractions appeared to possess higher levels of nuclear activation with respect to the T3L/T3- population as indicated by increased log 90 degrees scatter profiles. These results suggest that thymocytes with high T3 surface expression are not only more differentiated, but also more activated than the majority of the thymic population. The T3HT4+T8+ fraction could be distinguished from T4+T8+ thymocytes with lower T3 density not only by an increased log 90 degrees scatter profile, but also by the presence of T4+T8+ cells with reduced levels of T8 surface antigen. Our results indicate that T4+T8+ thymocytes with high T3 surface density are a distinct subpopulation and may represent the immediate precursors of the phenotypically more mature T3HT4+T8- and T3HT8+T4- subpopulations found in human thymus.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Cross-linking of T3 (CD3) with T4 (CD4) enhances the proliferation of resting T lymphocytes.

Monoclonal antibodies reactive with defined T lymphocyte surface antigens were covalently coupled to protein A-Sepharose beads using the bifunctional imidoester, dimethyl pimelimidate. Sepharose-immobilized antibody reactive with T3 induced the proliferation of resting T lymphocytes in the presence of either recombinant interleukin 2 or phorbol myristate acetate. When monoclonal antibodies reactive with T3 and T4 were coupled to the same Sepharose bead (hereafter designated Sepharose (T3:T4)), proliferation was enhanced an average of three-fold. Similarly prepared Sepharose beads coupled to anti-T3 and anti-T8 also enhanced proliferation over that observed with anti-T3 alone. Sepharose (T3:T4) similarly increased the proliferation of T4+ lymphocytes and a T4+ clone but failed to enhance the proliferation of T8+ lymphocytes. The increased proliferation of T4+ lymphocytes resulted from a preferential activation of the T4+2H4- helper population over the T4+2H4+ suppressor-inducer population. The enhanced proliferation induced by Sepharose (T3:T4) could be completely inhibited by soluble anti-T4. These results suggest that perturbation of T3 may be a minimal signal for T cell activation and that the assembly of a multimeric complex including T3 and T4 may be required for optimal T cell activation.

Antibodies, Monoclonal↗
PubMed1987

Activation of immature cortical thymocytes through the T11 sheep erythrocyte binding protein.

Two major pathways, the T cell receptor and the T11 alternate pathway, allow for T cell activation. In the human thymus, the T cell antigen receptor complex is reduced or absent on immature thymocytes, whereas the T11 glycoprotein is present at high cell surface density on all thymocytes. To determine whether activation through the T11 pathway induces similar or different changes in mature and immature thymocytes, we fractionated thymocytes according to their surface expression of the T3-T cell receptor (T3/Ti) complex. We report that two populations, one with high and one with low T3/Ti expression, can be activated through the T11 pathway to undergo nuclear activation and express IL 2 receptors. Moreover, in the absence of accessory cells, only the most mature population, expressing high T3 density, could be induced to proliferate, whereas the subset representing immature cortical thymocytes required accessory cells for proliferation. These findings suggest that the cellular microenvironment may have a critical role in regulating the activation of immature cortical thymocytes and that this cell population may not represent "nonfunctional" dead end cells, but rather a valid intermediate in human thymic differentiation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Development of natural killer cells in human thymocyte culture: regulation by accessory cells.

In vitro culture of human thymocytes resulted in the development of cells with natural killer (NK) activity and the acquisition of a pan-NK antigen (NKH1) by a large number of thymocytes. The ability to kill the NK-sensitive target, K562, was restricted to thymocytes expressing the NKH1 antigen. All NKH1+ thymocytes displayed a mature T cell phenotype, T3+T11+T8+T4-. Both the acquisition of NK activity and the development of cells with the NKH1+ phenotype could be suppressed by culturing thymocytes in the presence of adherent mononuclear cells. These results suggest that adherent accessory cells have the ability to regulate the development of T cell lineage NK cells.

Antibodies, Monoclonal↗
PubMed1987

Characterization of the T3+T4+T8+ thymocyte intermediate in vitro.

Sensitive two-color fluorescence staining and cell-sorting techniques were used to isolate a T4+T8+ thymocyte subpopulation with high T3 density from human thymocyte cultures. Previously, this population was shown to give rise to both T4+T8- and T8+T4- thymocytes. In the present study, this T3+T4+T8 population was shown to be functionally as well as phenotypically distinct from either T8+T4- cells or T4+T8- cells present in the same culture. The T3+T4+T8+ cell had intermediate cytotoxic capacities relative to T8+T4- and T4+T8- thymocyte fractions. The proliferative capacity of the T4+T8+ population although less than that of the T8+T4- subset exceeded the proliferative response of the T4+T8- population. The time of appearance of large numbers of T3+T4+T8+ cells in culture as well as functional properties exhibited by T3+T4+T8+ cells are consistent with the notion that the T3+T4+T8+ cell represents an activated intermediate in thymocyte differentiation. The T3+T4+T8+ thymocyte may be an important intermediate in in vivo as well as in in vitro thymic differentiation. Moreover, the analysis of its functional properties may contribute to an understanding of functional responses exhibited by the most mature (T3+) population isolated from human thymus.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Secondary immune amplification following live poliovirus immunization in humans.

Eight subjects inoculated orally with live attenuated poliovirus were investigated to study the effects of live virus infection on human T-cell responses. Proliferation to poliovirus and unrelated recall antigens were measured serially over a 3-week period. Five of eight subjects inoculated demonstrated a clear anamnestic response to poliovirus, but three did not. Only the five subjects demonstrating an anamnestic response to poliovirus were found to have augmented secondary immune responses to two unrelated recall antigens (tetanus toxoid and reovirus) and in the autologous mixed lymphocyte response (AMLR). No consistent changes were found in circulating T-cell surface activation antigens whether or not the subjects responded to poliovirus. These studies suggest that an asymptomatic poliovirus infection associated with immunization in humans can induce nonspecific secondary immune amplification as measured by in vitro T-cell proliferative response. This amplification pathway is a potential mechanism for immune responses against antigens other than those of the infecting virus.

Administration, Oral↗
PubMed1987

Human thymocyte subpopulations: maturational stages defined by the expression of the T3-T cell receptor complex.

Using sensitive fluorescence flow-cytometric techniques, human thymocyte subpopulations were fractionated according to their surface expression of the T3-T cell receptor (T3-Ti) complex. Two major subpopulations, one expressing low (immature subpopulation) and one high T3 antigen surface density (mature fraction) were characterized in detail with respect to surface antigen expression, right-angle scatter and proliferative capacity. Thymocyte subpopulations were activated through the T11 molecule (alternate pathway) and compared with regard to interleukin-2 (IL-2) receptor expression, changes in right-angle scatter and 3H-thymidine incorporation. We report that both populations could be activated through the T11 pathway to undergo nuclear activation and express IL-2 receptors. Moreover, in the absence of accessory cells, only the most mature population, expressing high T3 density, could be induced to proliferate, whereas immature cortical thymocytes required accessory cells for proliferation. These findings suggest that the cellular microenvironment may have a critical role in regulating the activation of immature cortical thymocytes.

Antigen-Presenting Cells↗
PubMed1986

Biosynthesis and surface expression of T8 by peripheral blood T4+ cells in vitro.

Biosynthetic labeling with 35S-methionine and 35S-cysteine of isolated T4+ cells from Con A-activated T cells demonstrated that the T8 antigen was synthesized by activated T4+ cells. Two-color fluorescence analysis of the activated T cell population from which the T4+ fraction was obtained showed that both T4+T8- and T4+T8+ cells were present. The T8 antigen that was immunoprecipitated by monoclonal anti-T8 from activated T4+ cells migrated with an electrophoretic mobility corresponding to an m.w. of approximately 33,000, a previously reported m.w. value for T8 antigen. Con A activation of highly purified peripheral T4+T8- and T8+T4- subsets indicated that both T4+T8- and T8+T4- cells can give rise to T4+T8+ cells. However, substantial T4, T8 coexpression by T4+T8- cells required a signal from T8+T4- cells which could be supplied by incubating T4+T8- cells with irradiated T8+ cells or the supernatant from Con A-activated T8+T4- cells. The generation of T4+T8+ cells from a subset of T4+T8- T cells may be an important mechanism in immune activation and/or the further differentiation of peripheral T4+ cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Human thymocyte maturation in vitro: a flow cytometric analysis.

Using an in vitro culture system, light scatter analyses, and two-color flow cytometry, we provide evidence that the interleukin-2 (IL-2) and transferrin receptors can be induced within 48 hr on nonproliferating immature thymocytes. The thymocytes (greater than 35%) that expressed the transferrin and IL-2 receptors demonstrated nuclear activation as measured by log 90 degrees light scatter analysis. Increases in antigen-receptor-associated T3-antigen expression followed transferrin and IL-2-receptor induction and occurred on maximally activated T4+T8+ thymocytes on Day 3 of culture. Maximal T3 expression did not occur until Days 5-7 and paralleled loss of T4, T8 coexpression, suggesting an association between a mature T3-Ti antigen receptor complex and a mature T4, T8 phenotype.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Discrete stages of human thymocyte activation and maturation in vitro: correlation between phenotype and function.

Using light scatter, flow cytometric and functional analyses, three major stages in the in vitro activation of human thymocytes were defined. The earliest stage (day 0-2) was characterized by the induction of the interleukin 2 (IL2) and transferrin receptors, the T cell lineage specific Ta1 antigen, and increased reactivity with anti-T8 antibody. At this time, major changes in nuclear morphology but not cell size were observed. Early-stage thymocytes were immature with regard to T3 and cytotoxic capacities. The second stage (day 3-4) of in vitro culture was distinguished by loss of T6, maximal activation (both in cell size and nuclear morphology) and maximal expression of both transferrin and IL2 receptors. At this stage, nearly all thymocytes expressed T3, 30-70% of thymocytes were T4+T8+, and functionally, only small increases in cytotoxic capacities were observed. The third stage of maturation (day 5-7) represented thymocytes with reduced levels of activation as measured by forward and right angle light scatter analysis and declining IL2 and transferrin receptor expression. However, these thymocytes exhibited high levels of T3 antigen density, loss of T4, T8 coexpression and pronounced cytotoxic and detectable inducer function capabilities.

Antibodies, Monoclonal↗