CD81 expressed on human thymocytes mediates integrin activation and interleukin 2-dependent proliferation.

Lymphocyte recognition of antigen by the antigen-specific T cell receptor (TCR) and coreceptor complexes rapidly alters the cell's adhesive properties facilitating high avidity cell-ligand interactions necessary for lymphocyte development and function. Here, we report the expression of CD81 (target of antiproliferative antigen [TAPA]-1) on human thymocytes and the physical association of CD81 with CD4 and CD8 T cell coreceptors. Antibody ligation of CD81 on thymocytes promotes the rapid induction of integrin-mediated cell-cell adhesion via lymphocyte function-associated molecule-1 (LFA-1). Cross-linking CD81 is also shown to be costimulatory with signaling through the TCR/CD3 complex inducing interleukin 2-dependent thymocyte proliferation. These data suggest that a CD81-mediated pathway in thymocytes is involved in the regulation of both cell adhesion and activation.

Infection of human thymocytes by Epstein-Barr virus.

The Epstein-Barr Virus (EBV) causes infectious mononucleosis, and has been strongly associated with certain human cancers. The virus is thought to exclusively bind to B lymphocytes and epithelial cells via receptors (CR2/CD21) that also interact with fragments of the third component of complement (C3). Recent evidence, however, has challenged this belief. We have used two-color immunofluorescence analysis using biotin-conjugated EBV and streptavidin-phycoerythrin along with fluorescein-conjugated anti-T cell antibodies and demonstrated that CD1-positive, CD3-dull (immature) human thymocytes express functional EBV receptors. In four replicate experiments, the binding of EBV to thymocytes ranged between 8 and 18%. This interaction is specific as evidenced by inhibition with nonconjugated virus, anti-CR2 antibodies, aggregated C3, and an antibody to the gp350 viral glycoprotein that the virus uses to bind to CR2. EBV can infect the thymocytes as evaluated by the presence of episomal EBV-DNA in thymocytes that had been incubated with the virus as short as 12 days or as long as 6 weeks. Episomal DNA analysis was performed by Southern blotting with a EBV-DNA probe that hybridizes to the first internal reiteration of the viral DNA. The presence of the EBV genome is also supported by the detection of EBV nuclear antigen 1 in infected thymocytes, assessed by Western blotting with EBV-immune sera. The EBV infection is specific as determined by blocking experiments using anti-CR2 and anti-gp350 antibodies. Finally, virus infection of thymocytes can act synergistically along with interleukin 2 and induce a lymphokine-dependent cellular proliferation. In view of previously reported cases of EBV-positive human T cell lymphomas, the possibility is raised that EBV may be involved in cancers of T lymphocytes that have not been previously appreciated.

1,25-dihydroxyvitamin D3: a novel immunoregulatory hormone.

The hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], at picomolar concentrations, inhibited the growth-promoting lymphokine interleukin-2, which is produced by human T lymphocytes activated in vitro by the mitogen phytohemagglutinin. Other metabolites of vitamin D3 were less effective than 1,25(OH)2D3 in suppressing interleukin-2; their order of potency corresponded to their respective affinity for the 1,25(OH)2D3 receptor, suggesting that the effect on interleukin-2 was mediated by this specific receptor. The proliferation of mitogen-activated lymphocytes was also inhibited by 1,25(OH)2D3. This effect of the hormone became more pronounced at later stages of the culture. These findings demonstrate that 1,25(OH)2D3 is an immunoregulatory hormone.

1,25-dihydroxyvitamin D3 receptors in human leukocytes.

A 1,25-dihydroxyvitamin D3 receptor macromolecule was detected in peripheral mononuclear leukocytes from normal humans. This macromolecule was found to be present in monocytes but absent from normal resting peripheral B and T lymphocytes. However, it was present in established lines of malignant B, T, and non-B, non-T human lymphocytes, as well as in T and B lymphocytes obtained from normal humans and activated in vitro.

Basis for defective responses of rheumatoid arthritis synovial fluid lymphocytes to anti-CD3 (T3) antibodies.

Synovial fluid mononuclear cells (SFMC) from patients with active rheumatoid arthritis characteristically respond poorly to mitogens. In this study, mitogenic antibodies reactive with the CD3(T3) antigen on human T lymphocytes were used to analyze the basis for the deficiency. OKT3-induced proliferation and release of interleukin 1 (IL-1) and interleukin 2 (IL-2) from SFMC were depressed in all patients. Purified IL-1 or recombinant IL-2 restored proliferative responses in SFMC and increased IL-2 receptor density. Exogenous IL-1 also enhanced IL-2 release. Fractionation of SFMC supernatants on phosphocellulose columns revealed the presence of IL-1 and a potent IL-1 inhibitor. The monocyte-derived IL-1 inhibitor blocked IL-1-dependent responses of normal peripheral blood lymphocytes to OKT3, but had no effect on IL-2-dependent events. These results suggest that IL-1 inhibitor(s) in SFMC impair(s) OKT3-induced mitogenesis by interfering with the effects of IL-1 on T lymphocytes. The net result is deficient IL-2 secretion, IL-2 receptor expression, and impaired cellular proliferation. This novel inhibitory circuit provides a rational explanation for the diminished function of synovial fluid T lymphocytes in rheumatoid arthritis patients.

1 alpha,25-Dihydroxyvitamin D3 receptors in human thymic and tonsillar lymphocytes.

In vitro activated human peripheral blood lymphocytes possess the receptor protein for 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In the present study we have examined whether activated lymphocytes that occur in vivo in human thymuses and tonsils also possess receptors for 1,25(OH)2D3. Freshly isolated lymphocyte preparations, from five separate surgical specimens of thymus and tonsil, were depleted of monocytes and examined, before and after fractionation on a density gradient of Percoll, for [3H] 1,25(OH)2D3 binding by means of sucrose density gradient sedimentation, by saturation analysis of the binding, and by DNA-cellulose chromatography. The state of activation of the lymphocyte preparations was determined using [3H] thymidine incorporation, DNA and RNA quantitation (using acridine orange), and by determining the presence or absence of markers of activation (interleukin-2 receptor, transferrin receptor, and HLA-DR molecules). In both the thymic and the tonsillar lymphocyte preparations we detected a 1,25(OH)2D3-binding molecule possessing sedimentation coefficient of 3.3 S and dissociation constant of 10(-10) M as well as DNA binding capability. In thymic lymphocytes, the 1,25(OH)2D3 receptor concentration correlated positively with the number of lymphocytes expressing the transferrin receptor (r = 0.84; p less than 0.05). In addition, in both thymic and tonsillar lymphocytes the concentration of 1,25(OH)2D3 receptors correlated positively with the number of cells in the G1a phase of the cell cycle (r = 0.79, p less than 0.01, and r = 0.88, p less than 0.001 for thymic and tonsillar lymphocytes, respectively). In contrast, the 1,25(OH)2D3 receptor concentration in these preparations did not correlate with the rate of cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

The antiproliferative effect of calcitriol on human peripheral blood mononuclear cells.

Activation of lymphocytes leads to the expression of receptors for the calcitropic hormone calcitriol [1,25(OH)2D3], and calcitriol is a potent inhibitor of interleukin-2 (IL-2) and of lymphocyte proliferation. We used peripheral blood mononuclear cells (PBM) activated in vitro with phytohemagglutinin to study 1) the relationship between 1,25(OH)2D3 receptor expression, IL-2 production, and 1,25(OH)2D3-induced inhibition of PBM proliferation in connection with the cell cycle; 2) the effect of 1,25(OH)2D3 on PBM activation and on the expression of activation-related molecules including the IL-2 receptor, and 3) the role of calcium in the antiproliferative effect of the hormone. 1,25(OH)2D3 receptor expression occurred when PBM entered the G1a phase of the cell cycle. The concentration of the receptor protein reached a peak at G1b and declined during the S phase. 1,25(OH)2D3 inhibited cell proliferation by blocking PBM at the G1a-G1b border. The antiproliferative effect of calcitriol was not caused by hormonal interference with the calcium-dependent activation process nor with the expression of activation-related molecules including the IL-2 receptor. Moreover, this effect was not influenced by extracellular calcium, suggesting that the hormonal action cannot be due to calcium translocation. These findings support the contention that 1,25(OH)2D3-induced inhibition of PBM proliferation is mediated through selective inhibition of IL-2 production.

Inhibition of interleukin-1 production by 1,25-dihydroxyvitamin D3.

The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], inhibits the proliferation of T lymphocytes and production of growth-promoting factors (including interleukin-2) (IL2) in CTLL2 murine cells. In this study, we investigated the role of monocytes in this hormone-mediated inhibitory effect, by testing the effects of 1,25-(OH)2D3 on the ability of the mitogenic lectin phytohemagglutinin (PHA) to induce T cell activation in either a monocyte-dependent or phorbol myristate acetate (PMA)-driven (monocyte-independent) system. The results indicate that proliferation of T cells and production of growth-promoting factors are inhibited by 1,25-(OH)2D3 only in the monocyte-dependent system. Preincubation of monocytes with 1,25-(OH)2D3 for various periods of time and subsequent removal of the hormone resulted in inhibition of the PHA-driven proliferation of T cells. Preincubation for 2 h resulted in 20% inhibition, while preincubation for 36 h reduced proliferation to 50% of the control value [no 1,25-(OH)2D3 exposure]. These data suggested that monocytes are important participants in 1,25-(OH)2D3-mediated events. Therefore, we tested the effects of the hormone on the production of IL1, a monocyte-derived product thought to be involved in the induction of IL2 release and the subsequent development of the T cell proliferative response. 1,25-(OH)2D3 inhibited the production of both extracellular and cell-associated immunoreactive IL1 alpha and IL1 beta. Indomethacin, a prostaglandin synthetase inhibitor, did not alter the inhibitory properties of 1,25-(OH)2D3, suggesting that prostaglandins are not responsible for the inhibitory phenomenon. We conclude that part of the ability of 1,25-(OH)2D3 to inhibit T cell proliferation may be due to direct effects on monocytes by down-regulating IL-1 production. However, it is unlikely that the immunoregulatory properties of 1,25-(OH)2D3 on T cells are mediated solely through monocytes, and it is possible that the hormone also exerts its influence directly on T cells.

Fractionation of human lymphocyte subpopulations on immunoglobulin coated Petri dishes.

This report describes a simple solid-phase technique for the positive selection of lymphocytes labeled with fluoresceinated antibodies. B lymphocytes were labeled with fluoresceinated anti-human Ig or monoclonal anti-human Ia (L243), and then were bound to plastic culture dishes coated with affinity purified goat anti-fluorescein specific antibody. Bound cells were eluted at 37 degrees C with 1 mM fluorescein-L-lysine phosphate-buffered saline. Functionally the eluted Ig positive cells responded to pokeweed mitogen (PWM) by in vitro secretion of IgM, as measured by radioimmunoassay of culture supernatants. The secretion of IgM was dependent on the addition of T lymphocytes. Moreover, the isolated B cells were functionally receptive to 'help' and 'suppression' by T cells with and without Fc receptors for IgG respectively. T cell subsets were fractionated on plastic culture dishes coated with heat aggregated rabbit or human IgG. the non-bound cells (enriched T(gamma-)) provided collaborative 'help' in the PWM induced IgM secretion response by human B lymphocytes. The bound cells (enriched T(gamma+)) eluted with 0.01 M EDTA in phosphate-buffered saline, suppressed IgM secretion. This method can be adapted to fractionate subsets of lymphocytes for which a fluoresceinated antibody is available. For routine functional studies, the isolation of cell types with conventional or monoclonal antibodies does not require the use of a fluorescence activated cell sorter, but only an antifluorescein labeled Petri dish. In conclusion, a rapid solid-phase technique enables us to prepare enriched populations of functionally active lymphocytes.

Solid-phase selection of human T lymphocyte subpopulations using monoclonal antibodies.

This report describes a novel solid-phase technique for the positive selection of human T lymphocyte subsets labeled by indirect immunofluorescence with monoclonal anti-bodies. Fluorescein labeled normal human T cells or a T cell line were fractionated on plastic culture dishes precoated with affinity chromatography purified anti-fluorescein antibodies. Cell binding was specific for fluorescein, and was both time and temperature dependent. Bound cells were eluted at 37 degrees C with fluorescein-L-lysine. The eluted cells were enriched with highly viable and functional human T cell subsets. Thus Leu3 monoclonal antibody selected cells were shown to provide helper activity in the pokeweed mitogen induced IgM and IgG immunoglobulin secretory response of autologous B cells. The Leu2 antibody selected T cells suppressed both IgM and IgG secretory responses. In addition, studies with the monoclonal antibody 1G11, which binds to an antigen expressed on acute lymphocytic leukemia (T-ALL) cells and the T-ALL derived cell line RPMI-8402, demonstrated that this solid-phase technique can be used to select for cells which are present at low frequencies in a mixed population. It thus provides a simple and reproducible means for the preparative isolation of lymphocyte subsets associated with autoimmune and neoplastic disease for functional and biochemical analysis.

Interactions of 1,25-dihydroxyvitamin D3 and the immune system.

A series of recent discoveries indicate that the hormonal form of vitamin D3, namely, 1,25(OH)2D3 plays a role in the regulation of the immune system. Cells of the monocyte/macrophage lineage possess receptors for 1,25(OH)2D3 regardless of their activation stage; cells of the lymphoid lineage also express these receptors but only at certain stages of their differentiation pathway and upon activation. Further, 1,25(OH)2D3 promotes the differentiation of monocyte precursors towards monocyte/macrophages and enhances monocyte function in antigen presentation. In addition 1,25(OH)2D3 is a potent inhibitor of interleukin-2 (IL-2) and suppresses effector functions of both T and B lymphocytes via IL-2-dependent as well as via IL-2-independent mechanisms. The theoretical and clinical implications of these discoveries are discussed.

The role of the T3 molecular complex on human T lymphocyte-mediated cytotoxicity.

The above overview of the experimental data clearly indicates that the T3 molecular complex is intimately involved in T cell activation. The precise role of the T3 complex in the activation process, however, is not clearly understood. The surface association of the T3 complex with the antigen receptor, along with the ability of antibodies to these molecules to render T cells receptive to IL2, reveal a possible mechanism by which specific antigen initiates T cell activation and growth. However, it would be difficult to reconcile this specific effect of anti-T3 antibodies with their effect on CTL function. Since the T3 complex is not a specific marker of any particular effector T cell population, but it is found on all T lymphocytes, we favor the hypothesis that the complex is involved in a more fundamental step of T cell activation, and we believe that triggering of the 'lethal hit', expression of IL2 receptors, and secretion of IL2 are mere manifestations of this basic process. The natural ligand of the antigen receptor is obviously the specific antigen. However, the natural ligand of the T3 complex is unknown. Possibly, its natural ligand is the antigen receptor itself after it has interacted with antigen. A simple scenario, then of the early events of T cell activation would include antigen recognition and binding, followed by an interaction between the antigen receptor and the T3 complex which then activates or allows expression of specific pathways depending on the particular effector population involved. Thus, the inhibition of CTL function by anti-T3 antibodies could be explained by interference with the antigen receptor-T3 complex interaction following target cell recognition. This interaction may be the event that signals the initiation of the 'lethal hit' process.

Expression of EBV/C3d receptors on T cells: biological significance.

There is overwhelming evidence that a single polypeptide serves as a receptor for both the Epstein-Barr Virus (EBV), and for certain enzymatic fragments of C3. This receptor, termed CR2 (CD21), is known to be expressed on the surfaces of B cells, and a large body of evidence suggests that CR2, or related structures, are also expressed on cells of the T lineage. Here, Constantine Tsoukas and John Lambris review the studies of CR2 expression in T cells and offer some speculation on its possible biological significance.

Regulation of expression of interleukin 2 receptors upon triggering of the TCR-CD3 complex on human T lymphocytes.

Monoclonal antibodies reactive with CD3 molecular complex can induce antigen-associated early biochemical changes in purified, monocyte-depleted resting T cell populations and synergize with interleukin 2 (IL2) in the induction of T-cell proliferation. Interleukin 2 mediates its effects via two receptor molecules of apparent 70-75 kD (p70/p75) and 50-55 kD (p50/55) molecular weights respectively. Using radioactive IL2 and bi-functional cross-linking chemistry, we are able to determine that incubation of purified, monocyte-depleted, resting T cells with anti-CD3 (OKT3) antibody induces a significant and selective increase in the expression of p70/75 IL2 receptors from their low constitutively expressed levels. This event occurs in the complete absence of cellular proliferation. Although IL2 also causes the upregulation of p70/75 molecules, it is the synergistic action of both antibody and lymphokine which is needed for the induction of significant amounts of the p50/55 IL2 receptors and the concomitant cellular proliferation. The effect of anti-CD3 on p70/75 receptor expression is specific, as determined by the inability of a non-related (anti-CD2) monoclonal antibody of the same subclass (IgG2a) to induce a similar effect. The Ca++ ionophore ionomycin, under conditions that cause significant intracellular Ca++ influx cannot by itself mediate upregulation of IL2 receptor expression in T cells. Since anti-CD3 itself can induce intracellular Ca++ increase in purified T cells, the finding with the ionophore suggests that the intracellular Ca++ accumulation alone cannot account for the IL2 receptor molecular events described here. Addition of PMA induces both p70/75 and p50/55 IL2 receptor upregulation, as well as IL2-dependent proliferation. Although resting T cells constitutively express p70/75 receptors, under our experimental conditions and with the concentration of IL2 used, these molecules cannot transduce the lymphokine signal efficiently. Thus, in a physiologic context, a simple interpretation of our data could be that upon interaction of the TCR/CD3 with antigen a selective upregulation of p70/75 IL2 receptors renders them competent of not only binding the lymphokine, but also transducing its signal. The latter event leads to the expression of p50/55 receptors and subsequent proliferation. Whether an increase in the numbers of these receptors is all that is needed or additional events are necessary merits further investigation.

Expression of CR2/EBV receptors on human thymocytes detected by monoclonal antibodies.

The biologic effects of the third component of complement, C3, are mediated via receptors which specifically bind the enzymatic degradation products resulting from the cleavage of C3. One of the products, C3d, has been associated with binding to the second complement receptor CR2 (CD21). This receptor, which is identical to the receptor for Epstein-Barr virus (EBV), has been primarily found on cells of the B lineage, but not on mature T cells or other cells of erythroid or myeloid lineages. In the present investigation, we report the presence of CR2 on human thymocytes. Indirect immunofluorescence analysis employing monoclonal anti-CR2 antibodies revealed a range of thymocyte reactivity from 15% to 63% in thirteen experiments using cells of different donors. Reactivity was always greater with the monoclonal anti-CR2 (CD21) antibody HB-5 than with two other antibodies which recognize distinct epitopes on the CR2 molecule. Two-color immunofluorescence analysis indicated that the brightest of the HB-5-stained thymocytes also reacted with the monoclonal anti-CD1 antibody T6 (immature thymocyte marker) while some of the duller HB-5-staining cells reacted with the monoclonal anti-CD3 antibody Leu-4 (mature thymocyte marker). Immunoprecipitation of CR2 on thymocytes with antibody HB-5 and polyacrylamide gel electrophoretic analysis revealed a protein of 145 kDa molecular mass which is consistent with the size of CR2 found on B lymphocytes. These findings raise several questions regarding the biologic role of CR2-EBV receptor on cells of the T lineage.

Cholera toxin inhibits resting human T cell activation via a cAMP-independent pathway.

The catalytic subunit of cholera toxin (CT) can chemically modify the alpha polypeptides of certain G-binding proteins and thus alter their function. In order to study the involvement of CT-sensitive G proteins in T cell activation, we have utilized CT in an in vitro system in which purified, resting human peripheral T cells are activated by anti-CD3 antibodies and rIL-2. Perturbation of the TCR/CD3 molecular complex by anti-CD3 antibodies causes changes in membrane phospholipids and induces a rise in cytoplasmic Ca2+. These events, however, are insufficient to allow progression into cellular proliferation and addition of IL-2 is required. Under these conditions, treatment of cells with a low concentration of CT (2 ng/ml) causes a significant inhibition of the anti-CD3-induced calcium event as well as the anti-CD3 plus IL-2-stimulated proliferation. Under our experimental conditions, inhibition of both proliferation and intracellular Ca2+ elevation by CT requires the involvement of the TCR/CD3 complex. This is supported by the observation that the toxin does not inhibit either the proliferation triggered by ionomycin and PMA or the Ca2+ influx induced by the ionophore. These data suggest that in TCR/CD3-mediated T cell activation CT acts at a point between TCR/CD3 perturbation and the generation of intracellular Ca2+. In view of the ability of CT to activate the alpha subunit of the G protein that stimulates adenyl cyclase (G alpha s), it is possible that the effect of CT on T cells is secondary to intracellular elevation of cAMP. However, measurement of cAMP levels both early after CT addition and at later time points, when proliferation is maximal, reveals lack of cyclic nucleotide accumulation. The presented data are consistent with the interpretation that the CT-mediated inhibition is caused by the modification of a G-binding protein that is either directly or indirectly associated with triggering of T cells via the TCR/CD3 molecular complex. The data also suggest that this protein is not G alpha s and it probably represents an as yet unidentified moiety or one of the several G proteins that have been recently described as regulators of phospholipase C activation.

EBV induces proliferation of immature human thymocytes in an IL-2-mediated response.

The receptor for EBV, CD21 is expressed on a population of immature human thymocytes and facilitates infection of these cells by EBV. Thymocytes infected by EBV become responsive to exogenous rIL-2- or CD2-mediated stimulation in vitro. To address whether such costimulation may be provided by thymic presenting cells and to study the cellular effects of EBV infection, the present work utilizes thymocyte cultures containing autologous thymic presenting cells. In the presence of these presenting cells, EBV induces proliferation of thymocytes. EBV infection promotes the formation of adhesions between two populations of cells in an APC responder fashion, and separation of these two populations abrogates the proliferative response to EBV. The response is mediated by IL-2 because Ab blocking of the IL-2R inhibits proliferation as does cyclosporin A. EBV promotes an expansion in the number of CD4+8+ thymocytes, and the proliferating population is vulnerable to TCR/CD3-generated signals, indicating that the responding cells are phenotypically and functionally immature. Finally, addition of exogenous IL-2 to EBV-exposed thymocytes promotes a second wave of proliferation. Phenotypic characterization of the EBV-induced, IL-2-responding cells shows them to express reduced levels of CD1 and a transitional CD4(high)8(low) phenotype. These data characterize the cellular response to EBV infection in thymocytes and may offer insight into EBV-associated T lineage malignancies and autoimmune disorders.