Regulation of influenza virus-specific cytotoxic T cell responses by monoclonal antibody to a human T cell differentiation antigen

The results in this report indicate that the OKT3 monoclonal antibody, which is specific for a human T cell differentiation antigen present on 90 to 95% of peripheral T cells, can exert several effects that regulate the generation and expression of human influenza virus-immune cytotoxic T lymphocytes (CTL). The OKT3 antibody, but not OKT1 or OKT11 (which bind to all peripheral T cells), is able to inhibit anti-influenza CTL effector cell activity. An F(ab')2 preparation of OKT3 IgG were as effective as whole IgG for the inhibition of CTL effectors, indicating that the inhibitory activity of the antibody was not a function of the Fc portion of the molecule. OKT3 IgG and OKT3 F(ab')2 fragments (but not OKT4, OKT8, or OKI were able to inhibit the generation of anti-influenza CTL. The culture of human lymphoid cells with OKT3 in the presence or absence of influenza virus induced radioresistant cells that could suppress the CTL response of fresh autologous lymphocytes to influenza. These results suggest that T cell functions can be regulated by signals that are initiated by the binding of antibody to cell surface molecules that may not be related to the T cell antigen-specific receptor(s).     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G₁ protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

Binding of isolated 3T3 surface membranes to growing 3T3 cells and their effect on cell growth

We have quantitated by autoradiography the binding of [¹²⁵I]labeled 3T3 plasma membrane fragments to 3T3 cells growing on the surface of plastic dishes; ie, the same conditions in which these membranes specifically arrest the growth of 3T3 cells early in the G₁ phase of the cell cycle. We have been able to demonstrate that binding of membranes to cells is coincidental with the expression of the growth inhibitory activity of protein(s) present in the membrane fragments. Treatments that reduce binding (heat denaturation of the membranes or culture in the presence of high scrum) also reduce growth inhibitory activity. [¹²⁵I]labeled membranes bound to cells are located primarily on the cell surface (as determined by electron microscope autoradiography) and are exchangeable with unlabeled membranes. We conclude that binding of membranes to cells is necessary but may not be sufficient for the expression of the growth inhibitory activity of these membranes. This approach provides information not only on the average level of binding of membranes to cells, but also provides a quantitative assessment of the variation of the level of membrane to cell binding between different cells in the population.     

The 1A4 molecule (CD27) is involved in T cell activation.

We developed a new mAb, anti-1A4, which recognizes an epitope on the CD27 molecule distinct from those recognized by several known anti-CD27 mAb. Although it has been suggested that the CD27 molecule is a T cell activation Ag, there was little direct evidence that the structure was involved in the T cell activation process. In this study, we showed that anti-1A4 inhibited anti-CD2, anti-CD3, mitogens, or soluble Ag-induced T cell proliferation as well as PWM-driven B cell IgG synthesis. Interestingly, anti-1A4 inhibited IL-2 secretion without affecting IL-2R expression. In addition, pretreatment of T cells with anti-1A4 inhibited the normally sustained intracellular calcium mobilization seen after triggering of T cells via the CD2 or CD3 pathways. Thus, binding of anti-1A4 to the CD27 molecule appears to induce a negative effect on T cell activation. This may be due to either a direct signal to T cells or the blocking of an interaction between T cells and accessory cells or both. These findings support the notion that the CD27 molecule plays an integral role in the process of T cell activation.     

Xenoantisera against a murine T cell tumor product cross-react with immunoglobulin Fab determinants

Some murine monoclonal T lymphoma cells express a surface component that reacts with chicken antisera produced against the Fab fragment of normal mouse IgG. In the present study, we use a solid phase immunoadsorbent consisting of affinity-purified chicken anti-Fab coupled to Sepharose to isolate a product produced by the in vitro T cell line, WEHI-7.1. The affinity-purified T cell surface molecule (IgT) migrated on SDS-PAGE as a single band of approximately 65,000 daltons. The object of these studies was to produce xenoantisera against the purified T cell product cross-reactive with Ig determinants and to characterize the antisera. Rabbits immunized with this purified molecule produced antibodies that reacted with Fab fragments of polyclonal mouse IgG and with the myeloma proteins MOPC-104E and MOPC-41, as detected by enzyme-linked immunosorbent assay (ELISA). This binding was eliminated by adsorption of the antisera with normal polyclonal IgG; however, adsorption with fetuin did not significantly affect the reactivity of the antisera. Radioimmune precipitation assays revealed that the rabbit anti-IgT bound to normal murine spleen and thymus cells; this reactivity was abrogated by adsorption with insolubilized polyclonal IgG. Competition radioimmunoassays demonstrated that detergent extracts of the thymus and the spleen contained material that inhibited the precipitation of MOPC-41; nonlymphoid cells lacked such material. The rabbit anti-IgT serum blocked the binding of antigen by normal T cells; adsorption of the antiserum with polyclonal IgG-Sepharose abrogated this blocking capacity. A solid phase immunoadsorbent prepared from the IgG fraction of the rabbit anti-IgT isolated a single component from formic acid-solubilized mouse thymus. This molecule had an approximate mass of 65,000 to 70,000 daltons. The anti-IgT serum isolated surface IgM and IgD from lactoperoxidase-catalyzed radioiodinated B cells. The anti-IgT serum detected IgM and IgG in mouse serum with the use of immunoelectrophoresis. The anti-IgT immunoadsorbent isolated several components from normal mouse serum, that, when analyzed by SDS-PAGE under reducing conditions, revealed bands corresponding to mu-, gamma-, and light chains as well as components that migrated between mu- and gamma-chains, and another component with an approximate mass of 45,000 daltons. Our results with antibodies to a purified T cell product indicate that a surface component of normal T cells and certain monoclonal T cell tumor lines is serologically related to the Fab fragment of serum Ig and is implicated in the binding of antigen.     

Requirements for T cell activation by OKT3 monoclonal antibody: role of modulation of T3 molecules and interleukin 1

The requirements for activation of human peripheral blood T cells by the mitogenic monoclonal antibody OKT3 were examined. OKT3 binds to a T cell molecule, T3, associated with the T cell antigen receptor and involved in T cell activation. Activation of T cells by OKT3 requires signals provided by accessory cells and is IL 2 dependent. In the presence of accessory cells, OKT3 induces loss of T3 molecules from the cell surface, production of IL 2, expression of IL 2 receptors, and proliferation. Modulation of T3 molecules by OKT3 can be induced in the absence of accessory cells with anti-mouse IgG. These T cells, however, are not induced to express IL 2 receptors or secrete IL 2. The addition of IL 1 induces expression of IL 2 receptors, but does not induce IL 2 secretion or proliferation. Thus, peripheral blood T cells appear to have different requirements for activation compared with antigen-specific T cell clones that can be induced to produce IL 2 when stimulated with OKT3 and IL 1. Expression of IL 2 receptors does not require modulation of T3 molecules, because the binding of OKT3 to T cells in the presence of IL 1 alone is sufficient to induce IL 2 receptor expression. The results suggest that IL 2 secretion depends on cross-linking and modulation of T3 molecules, and additional, as yet undefined, accessory cell signals. The expression of IL 2 receptors and proliferation of T cells can be induced in the absence of these signals when exogenous IL 2 is provided.     

Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes

The antigen receptor molecules on human T lymphocytes are noncovalently associated on the cell surface with the CD3 (T3) molecular complex. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Previous studies have demonstrated that this process requires the participation of monocytes. In the present report, we demonstrate that purified, resting (G0 phase) T cells incubated with monoclonal anti-CD3 antibodies proliferate in response to purified interleukin 2 (IL 2), in a lymphokine dose-dependent fashion. Anti-CD3 antibody or IL 2 alone did not trigger cell division. The effect was specific for anti-CD3 antibodies because monoclonal antibodies reactive with other surface molecules (OKT4, OKT8, L368) were inactive. Furthermore, the same phenomenon was observed when anti-CD3 antibody Leu-4 (IgG1) was incubated with cells of individuals whose monocytes cannot process antibodies of the IgG1 subclass (Leu-4 nonresponders). In addition, both F(ab')2 and Fab fragments of anti-CD3 antibody OKT3 were also capable of rendering T cells receptive to the IL 2 growth signal. These data indicate that neither monocytes nor CD3 receptor cross-linking are required absolutely for resting T cell activation, provided that IL 2 is supplied exogenously. T lymphocytes treated with anti-CD3 antibodies proliferated in response to both purified mitogen-induced and recombinant IL 2. Antibodies to the IL 2 receptor (anti-Tac) inhibited the proliferation. Thus, the most likely mechanism for anti-CD3 antibody-mediated triggering is induction of IL 2 receptors.     

Subcellular localization of prostaglandin-forming cyclooxygenase in Swiss mouse 3T3 fibroblasts by electron microscopic immunocytochemistry

Swiss mouse 3T3 fibroblasts were grown in tissue culture, fixed with lysine-paraformaldehyde-periodate solutions containing 0 to 0.1% Tween 20, and then stained for cyclooxygenase antigenicity using rabbit anti-cyclooxygenase IgG in the peroxidase anti-peroxidase procedure. When examined by light microscopy, those cells fixed in the presence of 0.03 to 0.1% Tween 20 exhibited staining throughout the cytoplasm and around the nucleus but not on the cell surface. No staining occurred when either preimmune IgG or anti-cyclooxygenase IgG adsorbed with purified enzyme was substituted for the immune IgG. Electron microscopic examination of cells treated with fixative containing 0.05% Tween 20 and then stained for cyclooxygenase antigenicity revealed electron-dense deposits on the endoplasmic reticulum and nuclear membrane but not the mitochondrial or plasma membranes. No staining was seen in cells treated with control sera. Agents such as angiotensin II, bradykinin, antidiuretic hormone, and thrombin interact, apparently with the 3T3 cell surface to cause a release of arachidonic acid and prostaglandin E2 formation (Pong, S.S., Hong, S. L., and Levine, L. (1977) J. Biol. Chem. 252, 1408-1413). Our results establish that conversion of arachidonic acid to the prostaglandin endoperoxide precursor of PGE2 actually takes place on the endoplasmic reticulum and the nuclear envelope.     

Effect of T3 modulation on pokeweed mitogen-induced T cell activation: evidence for an alternative pathway of T cell activation

Modulation of the T3 molecule on human T cells with monoclonal anti-T3 antibodies has been shown to result in the disappearance of the T3-Ti complex from the membrane and to preclude subsequent T cell activation by various mitogenic and antigenic stimuli. We have examined the effect of T3 modulation on pokeweed mitogen (PWM)-induced T cell activation. T3 modulation was accomplished by incubating peripheral blood mononuclear cells (PBMC) or mixtures of T cells and non-T cells at 37 degrees C for 18 hr in the presence of UCHT-1, a mouse IgG1 anti-T3 monoclonal antibody. Only donors whose PBMC were unresponsive to the mitogenic activity of this antibody were selected. Although T3 modulation resulted in complete to substantial inhibition of T cell proliferation induced by low PWM concentrations of 5 or 50 ng/ml, it had no effect on T cell proliferation when PWM was added at a concentration of 0.5 and 5 micrograms/ml. The results demonstrate that the higher doses of PWM can induce T cell proliferation via an alternative pathway that does not involve participation of the T3-Ti complex. In contrast, irrespective of the PWM dose added, T3 modulation almost totally inhibited PWM-induced interleukin 2 (IL 2) production. The differential effect of T3 modulation on IL 2 production and on T cell proliferation induced by high doses of PWM suggests that this alternative pathway of T cell proliferation is IL 2 independent. This suggestion was additionally substantiated by the lack of effect of anti-Tac, and anti-IL 2 receptor antibody, on PWM-induced proliferation of T3-modulated T cells. In conclusion our data demonstrate that high doses of PWM can induce T cells to proliferate via an alternative pathway that does not involve perturbation of the T3-Ti complex.     

Bispecific anti-idiotype/anti-CD3 antibody therapy of murine B cell lymphoma.

Retargeting of T cells by bispecific IgG which binds to both CD3 and a tumor-associated Ag can induce T cell lysis of target cells irrespective of TCR specificity. The current studies were designed to further explore the efficacy and specificity of bispecific IgG-directed therapy in an immunocompetent animal model, and to evaluate the mechanisms responsible for bispecific IgG-directed inhibition of tumor cell growth by using the 38C13 murine lymphoma system. In vitro, proliferation of activated T cells in the presence of bispecific IgG was increased when the relevant, but not the irrelevant target cells were present. Bispecific IgG specifically induced activated T cell mediated lysis of cells expressing the target Ag, but not of cells expressing an irrelevant Ag, even when the irrelevant cells were in the same cell mixture, indicating contact between target cells and T cells plays a major role in bispecific IgG-mediated lysis. Bispecific IgG was less effective than anti-Id at inducing target cell lysis when peritoneal macrophages were used as effectors, suggesting bispecific IgG Fc is not responsible for cytotoxicity in this system. In vivo, bispecific IgG was significantly superior to anti-Id, anti-CD3, or a combination of anti-Id and anti-CD3 in preventing tumor growth in immunocompetent mice inoculated with syngeneic lymphoma. Phenotypic evaluation of tumors that emerged despite therapy indicated bispecific IgG selects for the emergence of Id variant lymphoma cells. In separate studies, 38C13 tumor inocula containing cells recognized by the therapeutic antibody were supplemented with a small number of 38C13 cells which expressed a distinct Id not recognized by the therapeutic antibody. Untreated mice inoculated with this mixture developed tumors containing cells of both phenotypes, whereas tumors emerging from mice treated with bispecific IgG contained only cells expressing the nonreactive Id. These studies demonstrate bispecific IgG-directed lysis is therapeutically superior to monospecific anti-Id therapy in the 38C13 tumor model, and that tumor lysis is mediated largely by cell-cell contact. As with other forms of anti-Id based therapy, Id variants can emerge as resistant cell populations after bispecific IgG therapy.     

Accessory cell-T cell interactions involved in anti-CD3-induced T4 and T8 cell proliferation: analysis with monoclonal antibodies

The effect of monoclonal antibodies (Mab) directed at T cell and accessory cell (AC) surface molecules on OKT3-induced T4 and T8 cell proliferation was examined. Mab directed at nonpolymorphic class I (W6/32, MB40.5) and class II (L243) major histocompatibility complex (MHC)-encoded gene products, an epitope common to LFA-1, CR3, and the p150, 95 molecule (60.3), and a heterodimer present on monocytes (M phi) and activated T cells (4F2) inhibited M phi-supported OKT3-induced proliferation of both T4 and T8 cells. Moreover, an Mab directed at the CD4 molecule (66.1) inhibited OKT3-induced T4 but not T8 cell proliferation, whereas an Mab directed at the CD8 molecule (OKT8) inhibited T8 but not T4 cell responses. With the exception of 66.1, each inhibited OKT3-induced T cell proliferation when added as late as 15 hr after the initiation of culture. Inhibition could not be explained by competition for Fc receptors on the AC. A variety of other Mab including OKT11 and those directed at other HLA-DR and DQ determinants were not inhibitory. The inhibitory Mab were found to diminish T4 cell IL 2 production and IL 2 receptor expression. Consequently, IL 2 reversed some but not all of the Mab-mediated inhibition of T cell proliferation. In contrast to the effects noted with M phi-supported responses, 60.3 and 66.1 but neither L243 nor 4F2 inhibited OKT3-induced T4 cell proliferation supported by Ia- or IFN-gamma-treated Ia+ endothelial cells. None of the Mab tested inhibited T cell proliferation induced by the AC-independent stimuli OKT3 and phorbol myristate acetate (PMA) or calcium ionophore and PMA in the presence or absence of added AC. The data therefore suggest that the Mab inhibit OKT3-induced activation of T4 and T8 cells by preventing necessary interactions between AC and T cell surface proteins. Moreover, the results suggest that different arrays of interaction molecules are involved in OKT3-induced T cell proliferation depending on the nature of the AC and the responding T cell subset.     

Low affinity binding of an LFA-3/IgG1 fusion protein to CD2+ T cells is independent of cell activation

Quantitative analysis of binding of the bivalent recombinant soluble fusion protein, LFA-3/IgG1, shows that the fusion protein binds to human CD2+ PBLs primarily through low affinity (KD approximately 140 microM) but also through high avidity (90 nM) interactions. The concentration dependence for LFA-3/IgG1 PBL binding took the form of two overlapping bell-shaped curves separated by a clear and reproducible minimum. This was accounted for in part by minor heterogeneity in the LFA-3/IgG1 preparations, and potentially by the ability of the ligand to bind to both CD2 and Fc receptors (FcR), best evidenced by the distinct binding properties of the fusion protein to NK and T cells. The low affinity LFA-3/ IgG1 binding to T cells is consistent with binding to CD2 only, and is in agreement with the low affinity reported for interactions between soluble forms of LFA-3 and CD2 by surface plasmon resonance technology. Moreover, as the low affinity determinations are similar for CD2 on resting and activated T cells, although the CD2 molecule has been reported to be altered to reveal new epitopes upon T cell activation, the binding data argue against multiple cell activation-dependent affinity states of CD2 for LFA-3 binding. This is distinct from that observed with other adhesion partners, and suggests that the different adhesion pathways utilize distinct mechanisms to mediate cell adhesion.     

Partial characterization of a growth-inhibiting protein in 3T3 cell plasma membranes

A plasma membrane-enriched fraction from 3T3 cells has been detergent-solubilized, and the supernatant of this solubilization was reconstituted into liposomes using soybean lecithin. When these vesicles were added to actively growing cells, cell growth rates were inhibited to levels that were comparable to those observed with the original plasma membranes (at least 50% of maximum growth rates). Liposomes without proteins, or liposomes containing proteins from SV3T3 plasma membranes did not significantly inhibit growth of 3T3 cells. Treatment of the reconstituted vesicles with urea or high concentrations of salt did not eliminate the growth-inhibiting properties of these reconstituted membranes. These results indicate that the specific growth-inhibiting factor in 3T3 cell plasma membranes is a membrane protein that has significant non-polar interactions with the membrane bilayer.     

An efficient method for introducing macromolecules into living cells

The hemagglutinin (HA) of influenza virus was used to obtain efficient and rapid bulk delivery of antibodies and horseradish peroxidase (HRP) into the cytoplasm of living tissue culture cells. By exploiting HA's efficient cell surface expression, its high affinity for erythrocytes, and its acid-dependent membrane fusion activity, a novel delivery method was developed. The approach is unique in that the mediator of both binding and fusion (the HA) is present on the surfaces of the target cells. A recently developed 3T3 cell line which permanently expresses HA, Madin-Darby canine kidney cells infected with influenza virus, and CV-1 cells infected with a simian virus 40 vector carrying the HA gene were used as recipient cells. Protein-loaded erythrocytes were bound to the HA on the cell surface and a brief drop in pH to 5.0 was used to trigger HA's fusion activity and hence delivery. About 3 to 8 erythrocytes fused per 3T3 and CV-1 cell, respectively, and 75-95% of the cells received IgG or HRP. Quantitative analysis showed that 1.8 X 10(8) molecules of HRP and 1.4 X 10(7) IgG molecules were delivered per CV-1 cell and 6.2 X 10(7) HRP molecules per 3T3 cell. Cell viability, as judged by methionine incorporation into protein and cell growth and division, was not impaired. Electron and fluorescence microscopy showed that the fused erythrocyte membranes remained as discrete domains in the cell's plasma membrane. The method is simple, reliable, and nonlytic. The ability to simultaneously and rapidly deliver impermeable substances into large numbers of cells will permit biochemical analysis of the fate and effect of a variety of delivered molecules.     

Regulation of cytolytic activity in CD3- and CD3+ killer cell clones by monoclonal antibodies (anti-CD16, anti-CD2, anti-CD3) depends on subclass specificity of target cell IgG-FcR

Anti-CD3 MAb can inhibit MHC-restricted cytolytic activity of CD3+ mature cytotoxic T cells. In particular effector-target cell combinations, however, anti-CD3 MAb enhance or induce cytolysis by cross-linking CD3+ effector and IgG-FcR+ target cells. Virtually all natural killer (NK) cells or NK cell-derived clones are CD3-4-8- but do express CD2 and CD16 (IgG-FcR) antigens. We have studied how these cell surface molecules are involved in the regulation of cytolytic activities. The addition of anti-CD2 MAb to effector and target cells was found to induce conjugate formation of the IgG-FcR+ target cells with the effector cell and nonspecific cytolysis of, for instance, the P815 mouse mastocytoma cells. Enhancement or induction of conjugate formation and cytolysis of IgG-FcR+, P815, U937, and Daudi cells was also accomplished by using anti-CD16 MAb (e.g., Leu-11c (B73.1) or CLB Fc-gran 1 (VD2) MAb). Some human and mouse tumor cell lines (K562, P815, and U937) appear to express distinct types of IgG-FcR, showing different affinities for distinct subclasses of MAb (e.g., IgG1, IgG2a), but another line (Daudi) expresses only one type of IgG-FcR preferentially binding IgG1 MAb. Here we demonstrate that IgG-FcR on the effector cells can act as activation sites because anti-CD3 as well as anti-CD16 MAb of IgG1 and IgG2a subclasses can induce lytic activity of target cells bearing the relevant IgG-FcR. These data demonstrate that induction of conjugate formation and cytolysis by MAb occur when the target cells bear IgG-FcR with "specificity" for those MAb. Thus, besides via CD3, cytolytic activity by mature T and NK cells also can be induced via the CD2 and CD16 antigens on these cells.     

Functional studies on B cell hybridomas with B cell surface antigens. I. Effects of anti-immunoglobulin antibodies on proliferation and differentiation

B cell hybridomas with Ia and IgM molecules on the cell membrane were treated with either purified goat anti-mouse mu antibody (anti-mu) or monoclonal rat anti-mouse IgM antibody (anti-IgM). The spontaneous uptake of [3H] thymidine by these cells was markedly inhibited by both reagents. These hybrid cells could be induced to differentiate into IgM-secreting cells in the presence of these reagents at high frequency. Furthermore, the induction of IgM secretion by B cell hybridomas treated with these antibodies was completely T cell independent, and cell division was not required for the differentiative response to anti-mu. In addition, F(ab')2 fragments of anti-mu showed more effects on proliferation and differentiation of these cells than intact anti-mu. Interestingly, TH2.54, a subline of B cell hybridomas, could generate IgG2a production as well as IgM when incubated with anti-mu. These findings suggest very strongly that the interaction of either goat anti-mu or monoclonal rat anti-IgM with surface IgM molecules on the cell membrane of the B cell hybridomas inhibits in vitro spontaneous proliferation, and results in providing signals for differentiation into Ig-secreting cells without T cell factors.     

Induction of T cell-dependent B cell differentiation by anti-CD3 monoclonal antibodies

Three monoclonal antibodies (mAb) recognizing the CD3 (T3) surface complex each induced B cell differentiation (as measured by PFC generation) in cultures containing T + non-T cells. Irradiation of the T cells before culture usually augmented the PFC response. An IgG2a mAb (454) induced PFC in all donors tested, whereas two IgG1 mAb (147 and 446) induced PFC in only 80% of the donors tested. This heterogeneity in PFC response to IgG1 anti-CD3 mAb strictly paralleled the heterogeneity in proliferative response to IgG1 anti-CD3 mAb and was governed by cells within the non-T population. In IgG1 anti-CD3 high responders (HR), all anti-CD3 mAb tested induced Tac expression. In IgG1 anti-CD3 low responders (LR), mAb 454 induced Tac expression, but mAb 147 did not. However, when the cultures were supplemented with exogenous interleukin 2, Tac expression and PFC generation in response to mAb 147 was similar to the response to mAb 454 in both HR and LR. The addition of anti-Tac to the cultures partially inhibited anti-CD3-induced PFC generation. These studies indicate that anti-CD3 mAb can lead to B cell differentiation under appropriate experimental conditions and may be valuable in studying polyclonal T cell-dependent B cell differentiation in normal and disease states.     

Histidine-rich glycoprotein blocks T cell rosette formation and modulates both T cell activation and immunoregulation

Histidine-rich glycoprotein (HRGP) is a plasma and platelet protein with undefined function in vivo. It has been reported to inhibit rosette formation between murine T cells and erythrocytes. We have shown that HRGP binds specifically to human T lymphocytes but not sheep erythrocytes and have demonstrated a 56-kDa HRGP-binding protein on the T cell surface which is distinct from the CD2 sheep erythrocyte receptor. We have now investigated whether HRGP can inhibit human T cell-sheep erythrocyte rosette formation and whether HRGP can modulate T cell activation. HRGP at physiologic concentrations specifically inhibited rosette formation between human T lymphocytes and sheep erythrocytes. HRGP suppressed proliferation of antigen receptor (CD3)-triggered T cells induced by interleukin 2; this suppression was specifically reversed by prior incubation of HRGP with affinity-purified anti-HRGP IgG. Addition of HRGP 12-24 h after CD3 triggering no longer suppressed T cell proliferation, suggesting HRGP suppressed T cell division by interfering with one or more early events in the process of T cell activation. Human serum (containing 100-150 micrograms/ml HRGP) was also capable of suppressing T cell proliferation; serum which had been immunodepleted of HRGP no longer inhibited T cell proliferation. Furthermore, HRGP inhibited interleukin 2 receptor expression on activated T cells, causing decreased T cell interferon-gamma release and altered T cell-dependent inhibition of erythropoiesis. HRGP is thus capable of modulating T cell activation and T cell immunoregulation; HRGP may function as a natural suppressive regulator of human T lymphocyte activation.     

Osmotic stress and the freeze-thaw cycle cause shedding of Fc and C3b receptors by human polymorphonuclear leukocytes

A major problem in the cryopreservation of human polymorphonuclear leukocytes (PMN) is the loss of phagocytic function in cryopreserved cells. This is not a problem with cryopreserved monocytes. To study the reasons for this difference in detail, PMN and monocytes were either osmotically stressed in hypertonic media or were frozen to various temperatures. Cells were then returned to conditions of physiologic osmolarity and temperature. All cells remained viable. However, the ability of PMN to phagocytize bacteria and to bind sheep erythrocytes (E) opsonized with IgG, C3b, or C3bi decreased sharply after exposure to media of 600 mOsM or greater and after freezing to -1.5 degrees C. In contrast, monocytes were unaffected until a concentration of 1500 mOsM or a freezing temperature of -5 degrees C was exceeded. To determine whether the functional losses of surface receptor activity in PMN resulted from a loss of receptors from the membranes or from inactivation or internalization of receptors, opsonized E were incubated in the supernatants from stressed PMN. On subsequent incubation with healthy PMN, these E made fewer rosettes than control opsonized E. The inhibitory effect of the supernatants on rosetting of IgG-sensitized E could be removed by preincubation with IgG bound to Sepharose 4B. Immunoprecipitation of C3b and C3bi receptors from surface-iodinated, osmotically stressed, and control PMN suggested that about 50% of cell surface complement receptors were lost from the cell surface during osmotic stress. These experiments suggest that receptors for IgG and C3 are extruded from PMN cell membranes as a result of hyperosmotic stress, which is associated with the freeze-thaw cycle. This may be an early event in the functional damage done to PMN during attempts at cryopreservation.