CD45-protein tyrosine phosphatase cross-linking inhibits T cell receptor CD3-mediated activation in human T cells

Activation of peripheral blood T cells, and the leukemic T cell line Jurkat, as measured by mobilization of intracellular calcium, by an anti-TCR antibody is blocked by mAb (T191) to the leukocyte common Ag (CD45). T191 also blocked down-regulation of the CD3-TCR complex induced by an anti-CD3 mAb. Vanadate, a phosphotyrosine phosphatase inhibitor, partially blocks the effect of T191 and restored mobilization of intracellular calcium. Assays of the immunoprecipitates of T191 and CD45 from both Jurkat-BM1 and peripheral T cells showed that the immune complexes had intrinsic phosphatase activity. A parallel immunoprecipitate using a mAb (4-10) against HLA class I showed no such activity. Further analysis of the T191 immunocomplex revealed activity against phosphotyrosine, p-nitrophenylphosphate, and [32P-poly-glu-tyr, but not against phosphoserine. Phosphatase activity was inhibited by Vanadate, but not by Zn2+ or F-. These results show that CD45 is a phosphotyrosine phosphatase, and strongly suggest that tyrosine phosphorylation/dephosphorylation is critically involved in activation of T cells through the TCR-CD3 complex.     

Specific CD45 isoforms differentially regulate T cell receptor signaling.

Multiple isoforms of T cell CD45 tyrosine phosphatase are expressed as a result of alternative RNA splicing among extracellular exons. To discern the presence and identity of distinct functions among CD45 isoforms, we compared thymic T cell activation responses by elevating expression of two CD45 isoforms normally found on quiescent T cells. We report that CD45RABC significantly increased CD4+ thymic T cell proliferation in both a mixed lymphocyte reaction and following anti-T cell receptor (TCR) antibody stimulation. Additionally, CD45RABC enhanced Ca2+ mobilization and phosphotyrosine accumulation, and suppressed the inhibitory effect of anti-CD4 antibodies. By contrast, CD45R0 did not enhance TCR signaling or phosphotyrosine levels in CD4+ thymic T cells and required a TCR co-stimulus to augment cellular proliferation. These studies provide genetic evidence that alternative CD45 isoforms are functionally distinct and disclose a unique mechanism by which T cell immunologic responsiveness can be modified.     

Physical associations between CD45 and CD4 or CD8 occur as late activation events in antigen receptor-stimulated human T cells.

Activation of human PBL T cells with solid phase anti-CD3 mAb or during the course of an MLR response gives rise to the association of CD4 or CD8 molecules with the protein tyrosine phosphatase, CD45, on the cell surface. This paired association of cell-surface molecules occurs late in the activation cycle and appears to be dependent upon Ti-CD3-mediated signaling because mitogen-driven activation does not induce formation of the complex. Maximal association occurred 72 to 96 h after exposure to anti-CD3 mAb on both CD4+ and CD8+ T cells. In contrast, association between CD8 and CD45 during an MLR response did not occur until day 6 of a MLR whereas CD4-CD45 association was detected by 72 h of culture. The kinetics of association between CD4 or CD8 and CD45 was measured by fluorescence resonance energy transfer and confirmed by immunoprecipitation of dithiobis succinimidylpropionate or disuccinimidyl suberate cross-linked 125I-labeled resting or activated T cells. The molecules that co-precipitated with either CD4 or CD8 and had an apparent kDa of 180 to 205 could be immunodepleted with anti-CD45 mAb. Furthermore, CD4 or CD8 immunoprecipitates from 96-h activated T cells contained significant levels of protein tyrosine phosphatase activity whereas corresponding immunoprecipitates from resting or recently activated T cells showed little protein tyrosine phosphatase activity. This association may allow CD45 to engage and dephosphorylate lck or another CD4- or CD8-associated substrate in order to reset the receptor complex to receive a new set of stimuli. Our observations suggest that synergistic signaling provided as a consequence of CD4 or CD8 association with the TCR after antigenic stimulation may develop on a different temporal scale than that observed after soluble anti-CD4+ anti-CD3 heteroconjugate antibody cross-linking.     

CD45 inhibits CD40L-induced microglial activation via negative regulation of the Src/p44/42 MAPK pathway

It has been reported that ligation of CD40 with CD40 ligand (CD40L) results in microglial activation as evidenced by p44/42 mitogen-activated protein kinase (MAPK) dependent tumor necrosis factor alpha (TNF-alpha) production. Previous studies have shown that CD45, a functional transmembrane protein-tyrosine phosphatase, is constitutively expressed at moderate levels on microglial cells and this expression is greatly elevated on activated microglia. To investigate the possibility that CD45 might modulate CD40L-induced microglial activation, we treated primary cultured microglial cells with CD40L and anti-CD45 antibody. Data show that cross-linking of CD45 markedly inhibits CD40L-induced activity of the Src family kinases Lck and Lyn. Further, co-treatment of microglia with CD40L and anti-CD45 antibody results in significant inhibition of microglial TNF-alpha production through inhibition of p44/42 MAPK activity, a downstream signaling event resulting from Src activation. Accordingly, primary cultured microglial cells from mice deficient in CD45 demonstrate hyper-responsiveness to ligation of CD40, as evidenced by increased p44/42 MAPK activation and TNF-alpha production. Taken together, these results show that CD45 plays a novel role in suppressing CD40L-induced microglial activation via negative regulation of the Src/p44/42 MAPK cascade.     

Molecular mechanisms for Kv1.3 potassium channel current inhibition by CD3/CD28 stimulation in Jurkat T cells

In T lymphocyte, activation of Kv1.3 channel, the major voltage-dependent K⁺ channel, is an essential step for cell proliferation in immune responses. Here, effects of anti-CD3 and anti-CD28 antibodies on Kv1.3 current were examined in three types of human T lymphocyte derived cell lines, Jurkat E6-1, p56lck-kinase deficient mutant JCaM.1, and CD45-phosphatase deficient mutant J45.01. Kv1.3 current was partly reduced by CD3 stimulation and more strongly by addition of anti-CD28 antibody in E6-1. In JCaM.1, Kv1.3 current responses to anti-CD28/CD3 antibodies were similar to those in E6-1. In J45.01, CD3 stimulation partly inhibited Kv1.3 current, but the additive reduction by CD28 stimulation was not significant. The inhibition of tyrosine phosphatase in E6-1 abolished the additional inhibition by anti-CD28 antibody in a similar manner as in J45.01. In conclusion, the stimulation of CD28 in addition to CD3 strongly inhibits Kv1.3 current and this additive inhibition is mediated by CD45 activation.     

Role of the CD45 (T-200) molecule in anti-CD3-triggered T cell-mediated cytotoxicity

NK-depleted human peripheral blood lymphocytes can be modulated with anti-CD3 to kill certain targets during 3-hr cytotoxicity assays. When triggered by anti-CD3 antibody, these effector T cells killed only NK-sensitive targets, such as K562 and HEL 92.1.7, and NK-resistant targets, such as Daudi, whose killing is inhibited by anti-CD45 (T-200) monoclonal antibodies, such as 13.3. NK-sensitive targets, MOLT-4, U266/AF10, Jurkat, and CCFR-CEM, and 10 NK-resistant cell lines, including Raji, IM-9, U698, U937, and GM-1056, whose killing is not inhibited by anti-CD45 monoclonal antibodies, were not killed by alpha-CD3-T effectors, suggesting that the CD45 molecule may be involved in the killing process. Anti-CD3-triggered T cell killing of target cells was inhibited greater than 95% by the monoclonal antibody 13.3. This inhibition of cytotoxicity by 13.3 was not due to competition of this IgG1 antibody for Fc receptor binding site on the target cell, since the IgG1 monoclonal antibody anti-beta 2-microglobulin did not block cytotoxicity. Single cell assays and calcium pulse assays showed that CD45 is involved in a postbinding, pre-calcium-dependent stage, similar to that shown for NK cytotoxicity. There was a relative shift of importance of different epitopes of CD45 in anti-CD3-T cytotoxicity compared to NK cytotoxicity. Anti-CD45 antibodies which bind to the C terminus end of the molecule played a more important role in anti-CD3-T cytotoxicity than NK cytotoxicity. Thus, a subset of T cells exists that exhibits anti-CD3-triggered non-MHC-restricted killing of certain NK-sensitive and NK-resistant targets in association with a CD45 molecule which is functionally different from the NK CD45 molecule.     

Monoclonal antibodies to common epitopes of the human alpha/beta T-cell receptor preferentially activate CD45RA+ T-cells.

The murine monoclonal antibody BMA 031 (IgG2b) is directed to a monomorphic epitope on the human alpha/beta T-cell receptor. In contrast to anti-CD3 antibodies of the IgG2b isotype, BMA 031 is able to induce a proliferative response in T-cells from IgG2b low responders. This response occurs independently of cross-linking conditions indicating that the mode of activation differs from stimulation by the anti-CD3 antibody OKT3 (IgG2a) which strictly depends on cross-linking conditions. to further characterize the stimulatory potential of the two antibodies we studied the lymphocyte subsets responsive to stimulation by BMA 031 and OKT3. In CD45RA+ cells both antibodies exhibited similar effects. They induced weak expression of the 55-kDa chain of the interleukin-2 receptor (CD25), virtually no interleukin-2 secretion, but nevertheless strong proliferation. In CD45R0+ cells OKT3 and BMA 031 showed markedly different effects. OKT3 stimulated strong CD25 expression, strong interleukin-2 production, and marked proliferation. In contrast, CD45R0+ cells stimulated by BMA 031 showed only weak CD25 expression but neither interleukin-2 production nor proliferation. These data suggest that CD45RA+ and CD45R0+ cells differ in their capability to produce interleukin-2 upon stimulation via the CD3/T-cell receptor complex and also in the requirement for interleukin-2 to mount a proliferative response. The differential effect of OKT3 and BMA 031 in CD45R0+ cells probably results from the failure of BMA 031 to trigger interleukin-2 production which may be a consequence of its inability to induce CD3/T-cell receptor cross-linking in IgG2b low responders BMA 031 is therefore a useful tool for the selective activation of CD45RA+ cells in these individuals.     

The functionally distinct subpopulations of human CD4+ helper/inducer T lymphocytes defined by anti-CD45R antibodies derive sequentially from a differentiation pathway that is regulated by activation-dependent post-thymic differentiation

The CD4+ helper/inducer T cell population is comprised of functionally distinct subsets identifiable by the HB11 (anti-CD45R) mAb. We have previously shown that the cells that provide help for antibody production express the CD4+CD45R- phenotype. In contrast, CD4+ CD45R+ cells have minimal, if any, helper cell functions; rather, these cells function as inducers of Ts cell activity. The lineal relationship of these phenotypically and functionally distinct CD4+ subsets is unknown. In the present studies, we have examined the hypothesis that the CD4+ subpopulations identifiable with anti-CD45R antibodies represent "virgin" or "memory" T cells sequentially derived from a common differentiation pathway but differing in their relative maturation. When freshly purified cells were tested, CD4+ CD45R+ cells had no Th cell function. However, after in vitro activation with PHA and propagation in IL-2, CD4+CD45R+ cells acquired the ability to provide help for antibody production. Moreover, this functional acquisition by these cells was accompanied by their conversion to the CD4+CD45R- phenotype. Analyses of the activation, growth kinetics, and functional dose-response characteristics of CD4+CD45R+ and CD4+CD45R- cells demonstrated that our findings did not result from the selective growth of CD4+ CD45R- cells contaminating the CD4+CD45R+ preparations. Thus, these data demonstrate that the "helper" and "suppressor-inducer" subsets of CD4+ cells identified by anti-CD45R antibodies are not comprised of fully mature, phenotypically and functionally stable T cells. Rather, these CD4+ subsets appear to represent cells at different maturational stages of an activation-dependent, post-thymic differentiation pathway.     

Carbohydrate specificity of IgM autoantibodies to CD45 in Systemic Lupus Erythematosus

Patients with SLE develop IgM autoantibodies to different isoforms of CD45, the major surface membrane protein tyrosine phosphatase on lymphocytes and other nucleated hemopoietic cells. Because such autoantibodies could have a potential role in the development of immune dysfunction in this disorder, we performed a series of experiments to characterize their antigenic specificity further. Blots of recombinantE. coli fusion proteins encoded by exons 3–7 of the p220 and p180 isoforms were uniformly non-reactive with SLE IgM, suggesting that anti-CD45 autoantibodies in SLE are directed against conformational and/or carbohydrate epitopes, rather than linear polypeptide epitopes. This issue was examined further using chemically and enzymatically modified CD45 purified from T cells by lectin affinity chromatography as substrates. Treatment of CD45 with 25 mM sodium-m-periodate, sufficient to abrogate binding to various lectins, abolished the reactivity with SLE anti-CD45 autoantibodies. On the other hand, digestion of CD45 with neuraminidase enhanced the binding of anti-CD45 autoantibodies from some of the SLE sera. This result probably reflects decreased steric hindrance or charge repulsion because the binding of mouse monoclonal antibodies directed against linear polypeptide epitopes of CD45 was similarly enhanced. Digestion of CD45 with N-glycosidase F had no effect on autoantibody staining. Taken together, these data suggest that IgM anti-CD45 autoantibodies in SLE recognize non-sialylated carbohydrate determinants in the highly O-glycosylated polymorphic domains of CD45.Abbreviations SLE systemic lupus erythematosus - SBA soybean agglutinin - RCAI Ricinus communis agglutinin - SNL Sambucus nigra lectin - MBP maltose binding protein - mAb monoclonal antibody - WGA wheat germ agglutinin     

Localization of immunocompetent cells in the human epididymis

Ultrastructural and immunohistochemical (anti-CD3, CD20, CD45RO, CD68 antibodies) studies of human epididymis were performed. In electron microscopy, lymphocytes and macrophages were observed between epididymal epithelial cells and in the inerstitial tissue. However, immunohistochemical reactions with anti-CD3, CD45RO and CD68 antibodies were positive mainly in cells located the interstitial tissue. The reaction with anti-CD20 antibody was positive neither in epithelium, nor in the interstitial tissue.     

High levels of CD45 are coordinately expressed with CD4 and CD8 on avian thymocytes.

In this paper we describe the avian homolog of mammalian CD45. We show that this Ag is expressed on all leukocytes but not on erythroid cells or their immediate precursors. Immunoprecipitations demonstrated that B lineage cells from the bursa of Fabricius expressed a higher molecular mass variant (215 kDa) than did T lineage cells from the thymus (190 kDa), and crucially, these high molecular mass molecules had intrinsic phosphotyrosine phosphatase activity characteristic of mammalian CD45. We show that levels of CD45 expression as detected by mAb LT40 in the avian thymus are heterogeneous and further that mAb LT40 can deplete all phosphotyrosine phosphatase activity from thymocyte membrane preparations. Therefore total levels of CD45 are heterogeneous among avian thymocytes. Specifically, 87 to 89% of thymocytes expressed fourfold higher levels of surface CD45 (CD45hi) than the remaining 11 to 13% (CD45lo). The CD45lo population contained exclusively thymocytes with the phenotype CD3-4-8lo, characteristic of the immediate precursors to the CD3-4+8+ thymic population which are CD45hi. The shift from low to high levels of surface CD45 expression therefore occurred at the same stage as the transition from CD4-8lo to CD4+8+ and before the expression of CD3. The protein tyrosine kinase activity associated with CD4 and CD8 (p56lck) and the phosphatase activity of CD45 have been implicated elsewhere in jointly regulating peripheral T cell signal transduction and subsequent cellular responses. The coordinated expression of high levels of CD45 with both CD4 and CD8 in the avian thymus supports the possibility that these molecules may function together in regulating thymocyte growth and/or differentiation.     

The Mode and Duration of Anti-CD28 Costimulation Determine Resistance to Infection by Macrophage-Tropic Strains of Human Immunodeficiency Virus Type 1 In Vitro

We have investigated the ability of anti-CD28 antibody costimulation to induce resistance to macrophage (M)-tropic strains of human immunodeficiency virus type 1 (HIV-1) in vitro. Our results confirm the observations of Levine et al. (15) that stimulation of CD4 T cells with anti-CD3/anti-CD28 antibodies coimmobilized on magnetic beads renders the cells resistant to infection by M-tropic strains of HIV-1. The resistance was strongest when the beads were left in the cultures throughout the experiment. In contrast, stimulation of CD4 T cells with the same antibodies immobilized on the surface of plastic culture dishes failed to induce resistance and resulted in high levels of p24 production. This was true even if the cells were passaged continuously on freshly coated plates. If the beads were removed after initial stimulation, p24 production increased over time and produced a result intermediate to the other forms of stimulation. For beads-in, beads-out, and one-time plate stimulated cultures, resistance to infection correlated with down-regulation of CCR5 expression at the cell surface and with increased production of beta-chemokines. However, cultures of CD4 T cells continuously passaged on anti-CD3/anti-CD28-coated plates produced large amounts of p24 despite decreased levels of CCR5 expression and increasing production of beta-chemokines. Expression of the T-cell activation markers CD25 and CD69 and production of gamma interferon further supported the differences in plate versus bead stimulation. Our results explain the apparent contradiction between the ability of anti-CD28 antibody costimulation to induce resistance to HIV infection when presented on magnetic beads and the increased ability to recover virus from the cells of HIV-positive donors who are on highly active antiretroviral therapy when cells are stimulated by anti-CD3/anti-CD28 immobilized on plastic dishes.     

Monoclonal antibodies to CD45 modify LPS-induced arachidonic acid metabolism in macrophages

Signaling by lipopolysaccharide (LPS) through CD14 involves the activation of protein tyrosine kinases of the src family and leads to cytokine production and activation of arachidonic acid metabolism in macrophages. CD45 protein tyrosine phosphatase (PTPase) might play a role in modulating the response through this pathway. Although a critical role in regulation of T-cell signaling for CD45 has been demonstrated, little is known about its role in macrophages. Monoclonal antibodies to CD45 and F(ab')(2) fragments of the monoclonal antibody enhanced the response of differentiated THP-1 monocytic cells to LPS for the release of radiolabeled arachidonic acid metabolites, prostaglandin E(2), and tumor necrosis factor alpha. The enhancing effect of anti-CD45 mAbs was shown to occur primarily through CD14-dependent signaling by performing the experiments under conditions favoring that pathway. Further, LPS may be able to alter the enzymatic activity of CD45, as shown by Western blots of CD45 immunoprecipitates in which LPS caused a transient change in the phosphorylation state of CD45. We conclude that CD45 appears to play a role in LPS-induced responses through the CD14 pathway, possibly through its PTPase activity.     

Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb

 In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3⁺ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fcγ receptor (FcγRI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab′)₂, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18⁺ ovarian tumor cell line), was also compared in the presence of a bispecific antibody (OC/TR, anti-CD3 × MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 ×antitumoral bispecific antibodies. Received: 6 December 1995 / Accepted: 4 June 1996     

Cytochalasins enhance the proliferation of CD4 cells through the CD3-Ti antigen receptor complex or the CD2 molecule through an effect on early events of activation.

Cytochalasins are known to inhibit or enhance the proliferation of T cells induced by mitogens in a concentration-dependent fashion. To clarify the mechanism by which cytochalasins enhance T cell proliferation, we examined which activation pathways and events in signal transduction were affected by cytochalasins. We also examined subsets of CD4 cells for a preferential response to cytochalasins. Cytochalasins enhanced the proliferation of CD4 cells induced by optimal doses of anti-CD3 antibody or suboptimal doses of anti-CD2 antibodies. Cytochalasins, at low concentrations, enhanced the rise in intracellular Ca2+ and production of IP3 in CD4 cells activated by anti-CD2 or CD3 antibodies. Cytochalasins also enhanced the modulation of CD3 induced by anti-CD3 antibody. These results suggest that cytochalasins enhance the proliferation of CD4 cells by affecting early events in signal transduction after activation through the CD3-Ti Ag-receptor complex or CD2 molecule. At the doses used, cytochalasins appear to interact with cytochalasin-binding sites in the cell membrane. Cytochalasins predominantly enhanced CD3-mediated proliferation in the CD29-subset of CD4 cells.     

In vivo role of lymphocyte subpopulations in the control of virus excretion and mucosal antibody responses of cattle infected with rotavirus.

T-cell control of primary rotavirus infection and mucosal antibody responses to rotavirus was studied with monoclonal antibodies (MAb) to deplete gnotobiotic calves of CD4+, CD8+, BoWC1+, or both CD4+ and CD8+ lymphocytes prior to infection with rotavirus. Injection of these MAb produced specific reductions in circulating and tissue lymphocyte subpopulations. Following infection, control calves developed fecal immunoglobulin M (IgM) and IgA antibodies and serum IgM and IgG1 antibodies; there was no IgG2 antibody produced. Anti-CD4-treated calves had reduced fecal and serum antibody responses to rotavirus compared with control calves. The IgM response was less affected than the other isotypes. Calves concurrently injected with MAb to CD4 and CD8 had antibody responses similar to those of calves injected with anti-CD4 antibody alone. No effect on serum or fecal antibody levels was seen when MAb to CD8 or BoWC1 were injected alone. Virus excretion was significantly increased in calves depleted of CD8+ cells. Depletion of CD4+ cells or BoWC1+ cells had no effect on virus excretion. Calves depleted of both CD4+ and CD8+ cells excreted amounts of virus similar to those of calves depleted of CD8+ cells alone. Onset and duration of virus excretion were not affected by any of the MAb treatments. We conclude that a CD8+ cell population is involved in limiting primary rotavirus infection, while CD4+ or BoWC1+ (gamma/delta+ TcR) lymphocytes are not. Furthermore, CD4+ lymphocytes (but not CD8+ or BoWC1+ lymphocytes) were shown to be important in the generation of mucosal, as well as systemic, antibody responses.     

CD4 molecules are associated with the antigen receptor complex on activated but not resting T cells

To explore the relationship between CD4 and CD3/Ti on the T cell surface, we have studied a panel of Ag-specific Th cell lines and clones, as well as resting and mitogen-activated CD4+ cells. Our results show that exposure of Th cells to their specific antigenic stimuli, but not to irrelevant stimuli, induced the rapid disappearance of approximately 20 to 35% of CD3 and CD4 molecules. The modulation of these molecules was detected in less than 1 h, became maximal at 12 h, and recovered thereafter in parallel. Treatment of Th cells with anti-CD4 antibody prevented Ag-induced modulation of CD3, and treatment with anti-CD3 blocked modulation of CD4. In the absence of Ag, treatment of these cells with an antibody (WT-31) directed at a conformational determinant within CD3/Ti or with the combination of anti-CD3 antibody and goat anti-mouse Ig, also resulted in significant modulation of CD4. Similar treatment of PHA-activated CD4+ T cells with anti-CD3/Ti antibodies also induced CD4 modulation; however, the same antibodies failed to affect CD4 expression on fresh resting T cells. These results indicate that on activated, but not resting T cells, CD4 molecules can be physically associated with CD3/Ti. We postulate that this association is essential for efficient Th cell activation, and further that the ability of anti-CD4 antibodies to inhibit helper functions is due to their prevention of CD4-CD3/Ti interaction on the T cell surface.     

Induction of calcium flux and enhancement of cytolytic activity in natural killer cells by cross-linking of the sheep erythrocyte binding protein (CD2) and the Fc-receptor (CD16)

Binding of the anti-cluster of differentiation (CD) 2 monoclonal antibody 9-1 causes an increase in the concentration of cytoplasmic-free calcium ([Ca2+]i) in cultured CD3-/CD16+ natural killer (NK) cells. This response did not occur in cultured CD3+/CD16- cytotoxic T lymphocytes (CTL). Anti-CD16 antibodies could partially block the calcium response when NK cells were stimulated with intact antibody 9-1, and antigen-binding fragment F(ab')2 of antibody 9-1 did not produce a calcium response. Thus an interaction of the 9-1 antibody with CD16 Fc receptors was required for the functional effect. The dual interaction of antibody 9-1 with both CD2 and CD16 was demonstrated by comodulation experiments. The cytolytic activity of cultured NK cells was increased by antibody 9-1 but not by F(ab')2 fragments of antibody 9-1. The enhanced lytic activity was blocked by anti-CD16 antibody, anti-CD18 antibody, and anti-CD2 antibodies that do not block the binding of antibody 9-1. This pattern was distinct from antibody-dependent cell-mediated cytotoxicity which was blocked only by the anti-CD16 antibody. Thus antibody 9-1 enhanced cytotoxicity by activating effector cells. There was no enhancement of lytic activity when F(ab')2 of antibody 9-1 were cross-linked with a polyclonal antiglobulin, even though [Ca2+]i was increased. These results show that induction of a [Ca2+]i response is not sufficient to enhance lytic activity in NK cells, and suggest that signals delivered through CD16 are necessary.     

Importance of immunoglobulin isotype in therapy of experimental autoimmune encephalomyelitis with monoclonal anti-CD4 antibody

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease mediated by CD4+ T cells. Prior studies have established that monoclonal anti-CD4 antibodies can reverse EAE. To determine whether immunoglobulin isotype plays a role in the therapy of EAE with anti-CD4 antibody, an isotype switch variant family of the mouse IgG1 anti-rat CD4 antibody W3/25 was isolated with the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a W3/25 isotype variants all had identical binding capacities for rat CD4+ T cells. Although all three W3/25 isotypes showed some beneficial effects in the amelioration of EAE, the IgG1 and IgG2a W3/25 antibodies were superior to the IgG2b W3/25 in the treatment of EAE. Multiparameter fluorescence-activated cell sorter analysis of T cell subpopulations from treated rats showed that none of the antibodies of the W3/25 isotype switch variant family substantially depleted CD4+ target cells in vivo. These experiments demonstrate that immunoglobulin isotype is important in the monoclonal antibody therapy of autoimmune disease. They indicate that therapy of EAE may be successful without a major depletion of CD4+ lymphocytes. Immunotherapy may be optimized by selecting an appropriate isotype of a monoclonal antibody.     

Differential activation requirements for virgin and memory T cells

Most studies of the activation requirements for T cells have used either T cell lines or populations of normal T cells that consist of a mixture of virgin and Ag-primed T cells. These two subpopulations of T cells can now be distinguished in humans by their reactivity with mAb. The anti-CD45R antibody HB10 identifies virgin T cells (T degrees) that are non-reactive to recall Ag and relatively poor at providing help for B cell differentiation. Conversely, memory T cells (T') that can react to recall Ag and enhance Ig production are non-reactive with anti-CD45R, but can be identified with the UCHL1 antibody. We have used these antibodies to separate the T degrees and T' populations and examine their activation requirements. On activation CD45R+ cells rapidly began to lose the CD45R Ag and express the UCHL1 Ag in increased amounts, whereas the UCHL1+ cells retained this phenotype. Both populations responded to PHA in the presence of monocytes, but when triggered by an antibody to CD3 only the T' cells were induced to express IL-2R, produce IL-2, and to proliferate. The T degrees population of cells remained relatively quiescent by all of these parameters. However, anti-CD3 stimulation conditioned the T degrees cells for IL-2 responsiveness, inasmuch as the addition of rIL-2 resulted in significant IL-2R expression and proliferation. When the CD4+ T degrees and CD4+ T' subpopulations were isolated and examined in the same assays similar results were obtained. The data indicate that fundamental differences exist in the triggering requirements for T degrees and T' cells.