Human monocyte spreading induced by activated factor B of the complement alternative pathway: differential effects of Fab' and F(ab')2 antibody fragments directed to C5, C6, and C7

Human peripheral blood mononuclear phagocytes are induced by activated Factor B (Bb) of the complement alternative pathway to undergo morphological shape changes in vitro which have been described as "spreading." The spreading reaction induced by Bb has previously been shown to depend upon the enzymatic activity of Bb and to be inhibited by Fab' antibody fragments directed to C5 (but not anti-C3 Fab'). The possibility that Bb may exert its effect on monocytes by initiating assembly of terminal complement complexes comprised of C5b, 6, 7, C5b-8, or C5b-9 was addressed in the present study. The effects were tested of Fab' and F(ab')2 antibody fragments directed to C5, C6, C7, and C8 and to neoantigens expressed in the assembling terminal complement complexes on the monocyte spreading reaction induced by Bb. Differential effects of monovalent Fab' and divalent F(ab')2 antibody fragments were observed. Anti-C5, C6, and C7 Fab' were found to inhibit the spreading reaction induced by Bb in an immunologically specific manner. Divalent F(ab')2 fragments directed to these same proteins (but not to C3, C4, C8, or C9) induced monocyte spreading in the complete absence of Bb or other recognized inducing agents. Monocyte spreading induced by hybridoma immunoglobulin (Ig) directed to C5 and C7 was found to be correlated with the binding of 10(6) molecules Ig per cell. These findings support the notion that C5, C6, and C7 (or an analogous system of cellular proteins) are associated with the surface of human peripheral blood monocytes and that these proteins may play a role in certain reactions by which mononuclear phagocytes are induced to altered states of cellular physiology.     

Altered surface distribution of both C3b receptors and Fc receptors on neutrophils induced by anti-C3b receptor or aggregated IgG

Human neutrophils to which monospecific Fab' or F(ab')2 anti-C3b receptor had been bound at 0 degrees C were incubated for timed intervals at temperatures ranging from 0 degrees C to 37 degrees C, after which the cells were labeled with TRITC -conjugated second antibody. Neutrophils bearing Fab' anti-C3b receptor and incubated for up to 30 min at 37 degrees C, and cells bearing F(ab')2 anti-C3b receptor and incubated at 0 degrees C, exhibited diffusely distributed punctate clusters of receptors. Neutrophils bearing the bivalent anti-receptor and incubated at 30 degrees C or 37 degrees C for 5 min had redistributed C3b receptors into caps and patches that were associated with subplasmalemmal accumulations of myosin. The redistribution of cross-linked C3b receptors was inhibited by pretreatment of the neutrophils with either cytochalasin D or chlorpromazine. On approximately one-half of the cells demonstrating capped C3b receptors there was a corresponding redistribution of Fc receptors, as demonstrated by subsequent binding of FITC-aggregated IgG (FITC agg-IgG). In contrast, capping of C3b receptors did not alter the diffuse distribution of HLA-A on these cells. Cross-linking of Fc receptors on neutrophils by FITC agg-IgG also induced temperature-dependent capping of these receptors that was inhibited by cytochalasin D and chlorpromazine. In approximately one-half of the cells demonstrating capped Fc receptors, subsequent labeling of C3b receptors revealed a similar redistribution of these receptors. Thus, the neutrophil responds to cross-linking of either C3b receptors or Fc receptors by a cytoskeletal-dependent rearrangement of both receptors that causes their overlapping topographic distribution, demonstrating a form of cooperative interaction between these two types of receptors that are involved in the phagocytic reactions of these cells.     

Inhibition of the mixed lymphocyte culture by peritoneal exudate cells.

Inhibition of mixed lymphocyte cultures (MLC) by macrophages and by supernatants of short term cultured macrophages was assessed by incorporation of ³H-thymidine (TdRH3) and also by blast cell counts and by determination of cellmediated lympholysis. Peritoneal exudate cells (PEC) induced by thioglycollate, at concentrations >10%, inhibited all three parameters of MLC. Lower concentrations of PEC, and supernatants from cultured PEC, inhibited TdRH3 incorporation, but had no significant effect on blast cell counts or on generation of cytotoxic effector cells. Inhibition by the supernatants could be reversed by dialysis or by use of low specific activity TdRH3. These data indicate that macrophages can inhibit proliferative responses in MLC, but that this must be carefully distinguished from selective inhibition of TdRH3 incorporation.     

Inhibition of acute passive transfer experimental autoimmune myasthenia gravis with Fab antibody to complement C6

The role of the lytic complement C5b-9 membrane attack complex (MAC) in acute passive transfer experimental autoimmune myasthenia gravis (EAMG) produced in rats was investigated by in vivo inhibition of MAC formation with anti-C6 Fab. Anti-C6 Fab totally inhibited in vitro serum hemolytic activity, but did not consume or inhibit early complement pathways. Injection of rats with 0.12 mg/ml anti-C6 Fab reduced serum C6 to 8% and inhibited the muscle weakness, electrophysiologic abnormalities and loss of acetylcholine receptor (AChR) associated with acute EAMG. This level of C6 inhibition reduced the total serum complement hemolytic activity to 29% of normal but did not reduce the serum levels of complement components C3, C5, or C7. Treatment of rats with lower amounts of anti-C6 Fab (0.08 mg/ml) also inhibited clinical and electrophysiologic signs of EAMG, however, the lower amount of anti-C6 did not prevent the loss of muscle AChR. Both the higher and the lower amount of anti-C6 Fab inhibited the accumulation of macrophages at muscle motor end-plates. The inhibition by anti-C6 indicates that muscle weakness and electrophysiologic abnormalities associated with EAMG are dependent on the complement MAC, and that muscle weakness results from tissue injury in addition to loss of muscle membrane and AChR.     

Differential production of interferon and lymphotoxin by human tonsil lymphocytes.

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [³H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [³H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.     

Characterization of guinea pig mitogenic factors. I. Evidence that antigen-induced mitogenic factor and mitogenic factor from mixed leukocyte cultures are distinct molecular entities.

Mitogenic factor from BCG-sensitized cells stimulated with antigen (PPD) was found to have a m.w. between 20 and 25,000 daltons and an isoelectric point of about 7.5. The blastogenic activity of this factor was not affected by L-fucose or heating at 56 degrees C for up to 1 hr. Mitogenic factor obtained from supernatants of allogeneic cell mixtures (MLC-MF) on the other hand, had a m.w. at 15 to 18,000 daltons and an isoelectric point of 6.5. The blastogenic activity of MLC-MF was inhibited by 0.1 M L-fucose. The factor was stable at 56 degrees C for 1 hr. An antibody prepared against MLC-MF inhibited the MLC reaction as well as the activity of MLC-MF on non-committed cells. This antibody, however, did not affect the response of lymphocytes to PHA or PPD and had no suppressive effect on PPD-MF. The antibody was not cytotoxic and its suppressive activity in the MLC response could not be absorbed out by lymphoid cells indicating that it is probably directed against a lymphocyte activation product (MLC-MF) rather than membrane antigens. The chemical and immunologic differences exhibited by PPD-MF and MLC-MF indicate that these two lymphokines are distinct molecular entities.     

Binding and complement activation by C-reactive protein via the collagen-like region of C1q and inhibition of these reactions by monoclonal antibodies to C-reactive protein and C1q

Ligand-complexed C-reactive protein (CRP), like aggregated or complexed IgG, can react with C1q and activate the classical C pathway. Whereas IgG is known to bind to the globular region and not to the collagen-like region (CLR) of C1q, the site of interaction of C1q with CRP has not been defined. CRP-trimers were prepared by cross-linking and found to bind to C1q and to activate the C system. Heat-aggregated IgG (Agg-IgG) did not block the binding of CRP-trimers to C1q, nor did CRP-trimers block binding of Agg-IgG to C1q, suggesting that CRP and IgG bind at different sites. ELISA and Western blot analysis showed that CRP-trimers bound to the CLR, whereas Agg-IgG bound only to the globular region; similarly, anti-CLR mAb inhibited binding of CRP-trimers to C1q whereas anti-globular region mAb did not. Reactivity with CRP-trimers as well as with Agg-IgG was retained after reduction/alkylation and SDS treatment of C1q. A group of 22 anti-CRP mAb directed against at least six distinct native-CRP epitopes and eight distinct neo-CRP epitopes was tested for ability to inhibit the CRP-CLR interaction; one mAb, anti-native CRP mAb 8D8, with strong inhibitory activity was identified. Fab' of 8D8 blocked binding of CRP-trimers to intact C1q as well as CLR, and also inhibited CRP (CRP-trimers and CRP-protamine complexes) induced C activation, but had no effect on C1q binding or C activation by Agg-IgG. These results indicate that a conformation-determined region on CRP binds to a sequence-determined region on the CLR of C1q in an interaction which leads to C activation. Anti-CRP and anti-C1q mAb that specifically inhibit this interaction are described.     

抗CD20嵌合抗体片段F(ab′)2突变体在大肠杆菌中的高效分泌表达

构建抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,研究其在大肠杆菌中的高效表达及其表达产物的生物学活性。采用PCR法构建抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,并用双脱氧终止法测定DNA序列 ;采用 19L发酵罐高密度发酵抗CD20嵌合抗体片段F(ab′)₂ 突变体 ,采用亲和色谱和分子筛色谱法纯化表达产物 ,并用SDS-PAGE和薄层激光扫描鉴定纯化产物 ;采用活细胞间接免疫荧光法测定纯化产物与靶细胞的结合活性 ;MTT法测定纯化产物对Raji细胞的生长抑制作用 ,并研究其作用机理。DNA序列测定结果表明 ,抗CD20嵌合抗体片段F(ab′)₂ 突变体已成功构建 ,表达可溶性产物的产量达 360mg L ,具有与Raji细胞 (CD20⁺)结合的活性 ,并抑制Raji细胞的生长 ,其作用机理为诱导Raji细胞凋亡。此突变体有望成为治疗非何杰金氏B细胞淋巴瘤的药物。     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Inhibition of secondary in vitro antibody responses by the third component of complement

Purified human C3 was found to inhibit rat in vitro secondary antibody responses. Fifty percent inhibition of antibody-forming cell development occurred with C3 concentrations of 26 micrograms/ml. This decrease was not the result of a general toxicity or a shift in the antibody response kinetics. Using cell mixing experiments, we could not detect a C3-induced suppressor lymphocyte or macrophage. C3 was active when added to culture early (day 0 or 1 or during a 24-hr antigen prepulse) or late (day 3, 5, or 7)--the early addition being more suppressive. Regardless of the addition time, there was a characteristic 48- to 72-hr lag before the inhibitory effect was manifested. C3 could inhibit antibody-forming cell development after stimulation with the thymus-independent antigens, trinitrophenyl-Brucella abortus and dinitrophenyl-Ficoll, as well as the thymus-dependent antigens, dinitrophenyl-bovine gamma-globulin and chicken gamma-globulin suggesting that C3 was not selective for B memory cell subpopulations. Further characterization of our C3 preparation indicated that the majority of the suppressive activity resided in a small m.w. protein resembling the C3a fragment of C3. Human C3a preparations generated either by trypsin cleavage or zymosan activation of C3 were also tested in our antibody response system and were able to inhibit antibody-forming cell development. These data implicate C3 cleavage products as negative regulators of antibody formation.     

Comparison of immune responsiveness in mice after single or multiple donor-specific transfusions

Immune responsiveness was compared in B6AF1 mice after one, two, three, or four donor-specific DBA/2 blood transfusions (DST). Ten days after the last transfusion, the spleen cells of transfused mice were assayed for direct lymphocyte-mediated cytotoxicity, for the ability to respond in mixed lymphocyte culture (MLC) and cell-mediated lymphocytotoxic (CML) assays to DBA/2 and C3H/He antigens, and for the ability to inhibit the MLC and CML response of normal B6AF1 to DBA/2 and C3H/He antigens. Immune responsiveness was also tested in B6AF1 2 to 80 days after a single DBA/2 DST. The MLC response of transfused mice was specifically suppressed to the blood donor after both single and multiple transfusions. The CML response to DBA/2 was suppressed after a single DST, but returned to normal after multiple transfusions. Spleen cells from transfused mice did not inhibit the MLC response of normal B6AF1 mice to DBA/2 or C3H/He antigens after one or two transfusions regardless of time tested, but were able to inhibit the response to both stimulators after three or more transfusions. The MLC response remained specifically suppressed to the blood donor for as long as 80 days after a single DST, while the CML response was suppressed up to 50 days after transfusion, but had returned to normal by 80 days.     

Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: suppression of IL 2 production by anti-p50 monoclonal antibodies

Monoclonal antibody 9.6 is specific for a 50 kd T cell surface protein (p50) associated with the sheep erythrocyte (E)-receptor on human T lymphocytes. This antibody interferes with many T cell functions. We have examined the effect of antibody 9.6 on lymphocyte proliferation and interleukin 2 (IL 2) production triggered by mitogens, soluble antigens, and alloantigens to elucidate the mechanism(s) of its immunosuppressive action. At concentrations as low as 50 ng/ml, 9.6 suppressed lymphocyte proliferation and the elaboration of IL 2 by T cells stimulated by PHA, alloantigens, or low concentrations of the phorbol ester TPA (less than or equal to ng/ml). Furthermore, in cultures stimulated by a combination of PHA plus TPA, 9.6 did not inhibit the acquisition of IL 2 receptors but inhibited proliferation and IL 2 production. Immunoaffinity-purified IL 2 completely restored lymphocyte proliferation in cultures inhibited by 9.6. Studies of kinetics of inhibition by 9.6 showed that this antibody inhibited lymphocyte proliferation induced by PHA, alloantigen, and PPD even when added at 24, 48, and 72 hr, respectively, after the initiation of these cultures, suggesting that 9.6 does not block lectin binding or antigen recognition by T cells and that it can inhibit lymphocyte proliferation even after cells have undergone one or more rounds of cell division. A dose-response analysis of lymphocyte proliferation induced by PHA or by TPA demonstrated that the degree of inhibition by 9.6 decreased with increasing concentrations of these mitogens. Antibody 9.6 did not inhibit lymphocyte response induced by optimal concentrations of PHA (50 to 100 micrograms/ml; PHA-M) but inhibited proliferation of maximally induced lymphocytes by using a synergistic combination of low concentrations of PHA (5 micrograms/ml, PHA-M) plus TPA (1 ng/ml). Taken together, these findings indicate that 1) 9.6 inhibits lymphocyte proliferation by affecting IL 2 production, 2) 9.6 does not inhibit the acquisition of 9.6 receptors induced by a synergistic combination of PHA plus TPA, and 3) p50 molecules may be involved in multiple pathways of T cell activation.     

Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-kinase is thought to regulate the phosphorylation state of the substrates including myosin light chain (MLC), ERM (ezrin/radixin/moesin) family proteins and adducin by their direct phosphorylation and by the inactivation of myosin phosphatase. Here we identified the sites of phosphorylation of MBS by Rho-kinase as Thr-697, Ser-854 and several residues, and prepared antibody that specifically recognized MBS phosphorylated at Ser-854. We found by use of this antibody that the stimulation of MDCK epithelial cells with tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF) induced the phosphorylation of MBS at Ser-854 under the conditions in which membrane ruffling and cell migration were induced. Pretreatment of the cells with Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with Rho functions, or Rho-kinase inhibitors inhibited the TPA- or HGF-induced MBS phosphorylation. The TPA stimulation enhanced the immunoreactivity of phosphorylated MBS in the cytoplasm and membrane ruffling area of MDCK cells. In migrating MDCK cells, phosphorylated MBS as well as phosphorylated MLC at Ser-19 were localized in the leading edge and posterior region. Phosphorylated MBS was localized on actin stress fibers in REF52 fibroblasts. The microinjection of C3 or dominant negative Rho-kinase disrupted stress fibers and weakened the accumulation of phosphorylated MBS in REF52 cells. During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulated at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 was increased. Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates including MLC and ERM family proteins in vivo in a cooperative manner.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Inhibition of stimulation in murine mixed lymphocyte cultures with an alloantiserum directed against a shared Ia determinant.

Pools of high titered alloantisera were raised by immunizing (B10.A/SgSn X A/WySn)F1 mice with C57BL/10Sn(B10) spleen cells. This serum (F1 anti-B10), when added to one-way mixed lymphocyte cultures (MLC), inhibited stimulation of B10.A splenic responders by both B10 and B10.D2/nSn irradiated, splenic stimulators. The B10 stimulation was suppressed approximately 85% whereas the mean suppression of B10.D2 stimulation was approximately 60%. In the ofrmer case, the serum contained antibodies reactive with multiple major histocompatibility complex determinants on the stimulator cells. In the latter case, the cytoxic reactivity of the serum was directed principally against an I region-associated determinant Ia.8) shared by B10 and B10.D2 and coded for by a gene(s) in the I-A subregion. The magnitude of the suppression of the response to B10.D2 cells (60%) was similar to the reduction in stimulation observed when the Ia.8 difference was eliminated genetically by using (B10 X B10.A)F1 responder cells against irradiated B10.D2 stimulators. Ihhibition of MLC by this antiserum was a function of reactivity with stimulator and not responder cells. Although some pools of F1 anti-B10 antiserum produced partial inhibition of the responder cell in a B10.D2 vs B10.Ax MLC combination, the results were inconsistent and not correlated with the anti-Ia.8 cytotoxicity titers. In addition, an F1 anti-B10 antiserum pool, which consistently failed to inhibit the responder cell, nevertheless inhibited both irradiated B10.D2 and (B10.A X B10.D2)F1 cells from stimulating B10.A responder cells. However, this same antiserum did not inhibit stimulation of B10.D2 responder cells by the (B10.A X B10.D2)F1 stimulators. Thus, the binding of antibodies to the non-stimulating antigens on the F1 stimulator cell did not interfere with the capacity of the appropriate stimulating antigens to cause stimulation. All of these results are consistent with the hypothesis that Ia allo-antigens are the major stimulating determinants of I region-associated MLC reactions.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. III. Effect of priming on precursor frequencies.

The effect of specific priming with alloantigens on the frequency of cytolytic T lymphocyte precursors (CTL-P) has been investigated. Alloimmune lymphoid cells were obtained from the spleen of C57BL/6 (H-2b) mice primed with DBA/2 (H-2d) tumor cells or from 14-day unidirectional mixed leukocyte cultures (C57BL/6 anti-DBA/2). CTL-P frequencies directed against H-2d alloantigens were estimated by limiting dilution analysis in a sensitive micro MLC system. Under these conditions, an apparent increase of 3 to 4-fold in CTL-P frequency was observed in alloimmune (as compared with normal) C57BL/6 spleen cells. Evidence was obtained suggesting that this increase was specific for the priming alloantigens. A much greater increase in CTL-P frequency (25 to 100-fold) was observed after alloimmunization of C57BL/6 spleen cells in unidirectional MLC. Under the latter conditions, 5 to 20% of the surviving splenic MLC cells could be identified operationally as CTL-P. A similar enrichment in CTL-P frequency was obtained when lymph node, peripheral blood, or thymus cells were cultured for 14 days in MLC. These studies provide direct evidence that the pool of specific CTL-P can be expanded after alloimmunization. Furthermore, the very high frequencies observed after in vitro priming indicate that this system should be particularly useful for future studies of the progeny of individual CTL-P.     

Effect of activated lymphocytes on the regulation of hematopoiesis: suppression of in vitro granulopoiesis by OKT8+Ia+T cells induced by alloantigen stimulation

We studied the effects of alloantigen-stimulated lymphocytes in the regulation of hematopoiesis. Alloantigen-stimulated lymphocytes were harvested on days 2 to 3, days 6 to 7, or days 9 to 10 of MLC and were tested for their effects on granulocyte/macrophage progenitor cells (CFU-C). Dose-dependent suppression of CFU-C was observed when alloantigen-stimulated lymphocytes from days 6 to 7 and days 9 to 10 MLC were added to the cultures of autologous or allogeneic bone marrow cells for CFU-C assays. Suppressive activity was detected in the T cell fraction but not in the non-T cell fraction. For further characterization of these CFU-C/suppressor cells, alloantigen-stimulated lymphocytes were treated with radiation (2000 rad) or with monoclonal antibodies against T cell subsets and complement (C) before culture. Suppressive activity was completely abolished by treatment with OKT8 or OKIa1 antibodies and C whereas suppression was retained after radiation treatment. These observations suggest that CFU-C/suppressor cells can be induced by alloantigen stimulation in MLC and that they are radioresistant OKT8+ and Ia+ T cells.     

The complement SC5b-9 complex mediates cell adhesion through a vitronectin receptor.

Adhesion of cells to the terminal complement complex of C5b through C9 containing the serum S-protein (SC5b-9) was investigated using a microtiter plate attachment assay with L8 myoblast indicator cells. The skeletal muscle-derived L8 myoblasts bound and spread on substratum coated with SC5b-9, and with the vitronectin/S-protein component of SC5b-9. The myoblasts did not adhere to substratum coated with collagen, laminin, or fibronectin. The cell attachment was blocked by antibody to vitronectin/S-protein, whereas antibody to the other components C5, C6, C7, C8, or C9 had minimal effect. The cells were not bound to free vitronectin because attachment activity was removed by adsorption with an anti-C6 antibody column. The L8 cell attachment was dependent on divalent cations, was blocked by synthetic peptides containing the amino acid sequence Arg-Gly-Asp, and was inhibited by antivitronectin receptor antibody. These results indicate that cells adhere to the SC5b-9 complex through interaction of the vitronectin component with an integrin vitronectin receptor. Cell attachment to terminal C complexes could be used for leukocyte adherence and migration during inflammation, and also for attachment of tissue cells during regeneration after disease or traumatic injury.