Growth inhibition of human T cells by antibodies recognizing the T cell antigen receptor complex

Monoclonal antibodies that bind to the T cell MHC-antigen recognition complex (anti-T3 or anti-Ti) are known to either mimic ligand binding and activate T cells or block ligand binding, leading to an inhibition of T cell activation. In the present experiments, we demonstrate a direct inhibitory effect on the growth of human T cells by anti-T3 or anti-Ti antibodies. The proliferation of human peripheral blood T cells preactivated by exposure to PHA was inhibited in a specific manner by anti-T3. Colony formation in soft agar by REX cells, a leukemic cell line of early T cell phenotype, was completely inhibited by anti-T3 or anti-Ti antibodies, whereas isotype-matched antibodies to a variety of other T cell markers had no effect. Growth of REX cells in suspension culture was not affected by anti-T3 or anti-Ti. A cell line, T3.N1, was established from an agar colony of anti-T3-resistant REX cells. T3.N1 was phenotypically identical to REX except for failure to express any detectable T3 or Ti surface antigen. T3.N1 colony formation in soft agar was not inhibited by anti-T3 or anti-Ti. There was no rise in [Ca2+]i of T3.N1 cells after anti-T3 or anti-Ti exposure. These results indicate that in addition to the well-known positive regulatory effects of ligand binding to the T3/Ti complex, T3/Ti binding can also result in a down-regulatory signal for human T cell growth.     

Orderly expression of B cell antigens during the in vitro differentiation of nonmalignant human pre-B cells

Early human pre-B cells were isolated from fetal bone marrow and induced to differentiate in vitro under the stimulus of phorbol myristic acid or leukocyte-conditioned medium during a 48-hr culture period. Tritiated thymidine culture experiments substantiated that changes in surface marker phenotypes were not the results of outgrowth of subsets responsive to these stimuli. Interestingly, the addition of monoclonal antibodies directed against CALLA resulted in neither proliferation nor differentiation of the fetal lymphoid progenitor cells. Distinct changes in cell surface phenotypes were observed without evidence of cellular enrichment or depletion. The number of CALLA- and TdT-positive cells decreased, whereas the number of B1- and sIgM-positive cells increased. Moreover, a small number of pre-B cells could be driven to a more mature phenotype with the appearance of B2 and sIgG. In contrast, the pan-B B4 antigen did not alter significantly. These changes were even more pronounced when both induction stimuli were present. These studies, and previous studies on the subsets and differentiation of non-T cell acute lymphoblastic leukemias, suggest an orderly acquisition of B cell antigens during the stages of pre-B cell differentiation in man.     

Isolation and characterization of naturally occurring subclasses of human peripheral blood T cells with regulatory functions.

By utilizing naturally occurring autoimmune antibodies from patients with juvenile rheumatoid arthritis, we have isolated and functionally characterized two unique subpopulations of T cells. JRA+ T cells, i.e., those identified by sera from these patients, react poorly in response to allogeneic cells, respond to Con A but not PHA, and do not help in the synthesis and secretion of Ig by B cells. In contrast, JRA- T cells, i.e., those not identified by sera from these patients, respond very well to allogeneic cells, proliferate well in response to PHA but not Con A, and more interestingly, can greatly enhance the secretion of Ig by B cells.     

Cell surface modulation of CD26 by anti-1F7 monoclonal antibody. Analysis of surface expression and human T cell activation

In this paper, we examined in detail the ability of anti-1F7 to modulate 1F7 (CD26) surface expression as well as analyzed the functional relationship between the surface expression of CD3, CD2, and CD26 and human T cell activation. We showed that anti-1F7-induced modulation is an energy-dependent process that occurs via capping and internalization of the Ag-antibody complex. Although the recovery rate for Ag reexpression of 1F7 following optimal modulation is relatively delayed, reexpression of 1F7 is greatly accelerated following phorbol ester treatment. Most importantly, we demonstrated that modulation of the CD26 Ag leads to an enhancement in the proliferative activity of modulated human T cells treated with anti-CD3 or anti-CD2, which is preceded by an enhancement in Ca2+ mobilization. CD26 modulation also led to an increase in anti-CD3- or anti-CD2-mediated T cell clone proliferation. Finally, whereas modulation of the CD26 Ag has an effect on CD3- or CD2-induced T cell activation, modulation of the CD3/TCR complex inhibits the proliferative response of T cells incubated with anti-CD3 plus anti-1F7 or anti-CD2 plus anti-1F7. However, modulation of the CD2 structure does not affect anti-CD3- plus anti-1F7-induced human T cell activation. The above results thus provide additional evidence that the CD26 Ag plays an integral role in the regulation of human T cell activation.     

Triacylglycerol synthesis in isolated fat cells. Evidence that the sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities are dual catalytic functions of a single microsomal enzyme.

The acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (glycerol-P acyltransferase) and acyl-CoA:dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) (DHAP acyltransferase) activities were investigated in vitro in order to evaluate the quantitative contribution of the glycerol-P and DHAP pathways for the synthesis of triacylglycerols in isolated fat cells and to test the hypothesis that these two activities may be dual catalytic functions of a single enzyme. More than 85% of both acyltransferase activities was associated with the microsomal subcellular fraction. The microsomal glycerol-P acyltransferase activity showed an apparent Km of 8 muM for glycerol-P with a Vmax of 15.6 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent Km of 40 muM for DHAP with a Vmax of 9.7 nmol/min/mg. Glycerol-P was a competitive inhibitor (Ki = 7.2 muM) of the DHAP acyltransferase, and DHAP was a competitive inhibitor (Ki = 92 muM) of the glycerol-P acyltransferase. The two acyltransferase activities showed virtual identity in their pH dependence, acyl-CoA chain length dependence, thermolability, and inactivation by N-ethylmaleimide. Trypsin, detergents, collagenase, phospholipases, and various salts and organic solvents also had similar effects on both activities. Taken as a whole, the data strongly suggest that the microsomal glycerol-P and DHAP acyltransferase activities actually represent dual functions of a single enzyme. Calculations based on the above kinetic constants and previously reported glycerol-P and DHAP pools in adipocytes suggest that the in vivo ratio of glycerol-P to DHAP acylation should be greater than 24:1.     

Activation of human thymocytes via the 50KD T11 sheep erythrocyte binding protein induces the expression of interleukin 2 receptors on both T3+ and T3- populations

Recent studies have demonstrated that the 50KD T11 molecule is a surface component of a macrophage-independent alternative pathway of human T cell activation that is unrelated to the T3/Ti antigen-MHC receptor complex. Given the expression of T11 on all human thymocytes, it was of interest to determine whether they could be activated via this pathway. The triggering of T11 by monoclonal antibodies anti-T112 and anti-T113, directed at two unique epitopes on the molecule, induced IL 2 receptor expression on both T3+ and T3- thymocytes but did not induce IL 2 production. Consequently, in contrast to peripheral blood T cells, thymocytes did not proliferate in response to anti-T112 and anti-T113 in the absence of exogenous IL 2. These studies suggest that IL 2 receptor gene activation precedes IL 2 gene activation in T cell development. The ability of the alternative pathway of T cell activation to induce IL 2 receptor expression on T3- thymocytes implies that the T11 molecule may have an important role in early thymocyte ontogeny.     

X-ray reflectivity studies of cPLA2{alpha}-C2 domains adsorbed onto Langmuir monolayers of SOPC

X-ray reflectivity is used to study the interaction of C2 domains of cytosolic phospholipase A(2) (cPLA(2)alpha-C2) with a Langmuir monolayer of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) supported on a buffered aqueous solution containing Ca(2+). The reflectivity is analyzed in terms of the known crystallographic structure of cPLA(2)alpha-C2 domains and a slab model representing the lipid layer to yield an electron density profile of the lipid layer and bound C2 domains. This new method of analysis determines the angular orientation and penetration depth of the cPLA(2)alpha-C2 domains bound to the SOPC monolayer, information not available from the standard slab model analysis of x-ray reflectivity. The best-fit orientation places the protein-bound Ca(2+) ions within 1 A of the lipid phosphate group (with an accuracy of +/-3 A). Hydrophobic residues of the calcium-binding loops CBL1 and CBL3 penetrate deepest into the lipid layer, with a 2 A penetration into the tailgroup region. X-ray measurements with and without the C2 domain indicate that there is a loss of electrons in the headgroup region of the lipid monolayer upon binding of the domains. We suggest that this is due to a loss of water molecules bound to the headgroup. Control experiments with a non-calcium buffer and with domain mutants confirm that the cPLA(2)alpha-C2 binding to the SOPC monolayer is Ca(2+)-dependent and that the hydrophobic residues in the calcium-binding loops are critical for membrane binding. These results indicate that an entropic component (due to water loss) as well as electrostatic and hydrophobic interactions contributes to the binding mechanism.     

Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.     

Studies of in vitro activation and differentiation of human B lymphocytes. I. Phenotypic and functional characterization of the B cell population responding to anti-Ig antibody

Investigation of the activation of splenic B cells by anti-immunoglobulin (Ig) antibody has enabled us to characterize the anti-Ig-responsive B cell and to analyze the phenotypic changes which accompany proliferation and differentiation. The anti-Ig antibody-responsive B cell population was characterized by the expression of high levels of the B2 antigen and represented approximately 40% of splenic B cells. Brisk mitogenesis which peaked at 3 to 4 days was induced by anti-Ig antibody. The proliferative phase was characterized phenotypically by a dramatic decline in B2 antigen expression, with most cells showing no detectable B2 by 4 days post-activation. The other hallmark of this phase was de novo expression of a group of "activation antigens." These included the B cell-restricted antigens B-LAST 1, BB1, and B5, and the T cell-associated interleukin 2 receptor and T12 antigens. Concomitantly, B1, B4, and Ia expression increased, the increase being roughly proportional to the increase in cell size. After day 4, the mitogenic response progressively diminished, while Ig synthesis increased. During this differentiation phase, cell surface antigens again displayed a distinct sequence of changes. The five activation antigens and the B1, B4, and Ia antigens began to decrease. However, two markers, T10 and PCA-1, which are found on plasmacytomas, appeared and their level of expression steadily increased. These changes and the appearance of morphologically identifiable plasma cells required the presence of T cells in this system. T cell supernatants alone induced Ig secretion but did not induce expression of PCA-1 or the appearance of cells with plasma cell morphology. The culture system developed in this study has allowed us to analyze the antigenic changes following activation by anti-Ig antibody. This sequence of changes has not only permitted the identification of antigens which, by their appearance at distinct stages may have an important role in proliferation and differentiation of B cells, but also provides us with the means of studying the function of each antigen.