The role of lymphoid cells in antibody-induced suppression of the fourth component of guinea pig complement

Many laboratories have demonstrated that immunoglobulin production by B cells is controlled by networks of interacting lymphocytes and their products. Our laboratory has demonstrated that complement components produced by macrophages are also regulated by networks of interacting cells and humoral factors. Treatment of mice in vivo or guinea pig cells in vitro with anticomponent antibody specifically inhibits synthesis and secretion of the component by macrophages. We have further characterized the cellular basis for in vitro suppression of the fourth component of guinea pig complement. C4 suppression has been accomplished with dispersed spleen cells as well as intact splenic fragments. This facilitated examination of the cells responsible for long-term C4 suppression. The data suggested that C4 suppression required either cell contact or sufficient concentrations of soluble factors. Long-term suppression of C4 depends upon a lymphoid cell contained in the spleen and in lymph nodes but absent or in insufficient concentration in the peritoneum. The lymphocyte that actively maintains suppression was negative for the guinea pig T-cell marker detected by the monoclonal antibody mc8BE6. Therefore, the critical cell is either another T-cell subset or non-T lymphocyte. These data demonstrate that a network of interacting cells analogous to that proposed to regulate antibody synthesis is also involved in regulating some nonlymphoid cell products.     

Antibody-induced suppression and postsuppression stimulation of complement in vitro. III. Long-term C4 suppression is actively maintained by a soluble suppressor factor (FsC4)

Previous work in our laboratory established that individual complement components can be regulated in vivo by administration of specific antibody or immunocompetent cells to newborns and in vitro by administration of specific antibody to cultured peritoneal macrophages or splenic fragments. Antibody-induced suppression of C4 was much longer lasting in cultured guinea pig splenic fragments than in cultured guinea pig peritoneal macrophages, suggesting that splenic fragments contained elements necessary for long-term suppression that were not present in the macrophage monolayers. This publication presents data in support of this concept. Antibody-treated splenic fragments from normal guinea pigs--but not from C4-deficient guinea pigs--elaborated a soluble factor (FsC4) that suppressed C4 production in previously untreated splenic fragments. FsC4 activity was most potent in splenic fragment culture supernatants at those times when intracellular and secreted C4 hemolytic activity and C4 antigen were at their lowest. C4 itself or a fragment of C4 was therefore unlikely to mediate suppression in this system. Residual anti-C4 antibody was ruled out as a mediator of FsC4 activity since it was shown by two independent methods that the amount of anti-C4 antibody carried over with the supernatant was orders of magnitude less than the amount necessary to cause suppression or to neutralize fluid phase C4 in fresh splenic fragment cultures. Preliminary data revealed that FsC4 activity may be mediated by two or more distinct molecular species or may be mediated by a single molecule that exhibits secondary size and charge heterogeneity. The identification of factors that are capable of regulating C4 suggests that, as with immunoglobulins, complement components may be regulated by complex networks of immunocompetent cells and their soluble products.     

Antibody-induced suppression and postsuppression stimulation of complement in vitro. II. Intracellular and extracellular changes in C4 during long-term C4 suppression in guinea pig splenic fragments

Suppression of the synthesis of the fourth component of complement in vitro was originally accomplished by exposing cultured guinea pig peritoneal cells to anti-C4 alloantisera. When guinea pig splenic fragments were used instead of peritoneal cells, equivalent antibody treatment produced C4 suppression of significantly longer duration, lasting weeks instead of days after removal of antibody. As with peritoneal cell monolayers, antibody treatment induced specific suppression of C4 followed by nonspecific stimulation of C4 and other proteins such as C2. Although IgG2 is more readily sequestered by splenic tissue, both IgG1 and IgG2 antibodies were effective in inducing and maintaining suppression. Experiments with radiolabeled antibody demonstrated that a small amount (less than 5%) of the original dose of antibody was retained by the splenic fragments. Because there was no continuous slow release of that antibody, long-term suppression of C4 cannot be explained as a fluid-phase neutralization reaction. Because antibody treatment might induce production of aberrant C4 molecules with no functional activity, C4 antigens was also studied. Tissue culture supernatants were assayed by using an ELISA for C4. In none of these experiments was extracellular C4 antigen detectable immediately after antibody treatment. Extracellular and intracellular C4 were immunoprecipitated from biosynthetically labeled tissue cultures and analyzed by SDS-PAGE. Antibody treatment suppressed intracellular C4 as well as extracellular C4. Although extracellular C4 levels of antibody-treated cultures eventually returned to levels comparable to untreated cultures, intracellular C4 levels of treated fragments remained lower than controls for the full period of observation (22 days). Therefore, a short (4-day) exposure to anti-C4 antibody induced long-term effects that profoundly altered regulation of C4 synthesis and secretion by cultured splenic macrophages.     

Binding of C-reactive protein to the pneumococcal capsule or cell wall results in differential localization of C3 and stimulation of phagocytosis

C-reactive protein (CRP) is a serum protein that shows rapid increases of as much as 1000-fold in concentration in response to infection, traumatic injury, or inflammation. CRP reacts with the phosphocholine moiety of pneumococcal cell wall C-polysaccharide, and this reaction can lead to complement activation in vitro and protection against pneumococcal infection in vivo. We have previously studied the chemiluminescence response of human neutrophils to Streptococcus pneumoniae as a measure of in vitro opsonophagocytosis by CRP and complement. CRP in the presence of complement was an effective opsonin for S. pneumoniae serotype 27 (Pn27), but not for serotypes 3 or 6. Because Pn27 differs from most serotypes of S. pneumoniae in containing phosphocholine in its capsular polysaccharide, we have determined the sites of CRP and C3 fixation to Pn27 and S. pneumoniae serotype 4 (Pn4), and related these to the ability of CRP and complement to opsonize these serotypes in vitro. By using a chemiluminescence (CL) assay to measure opsonophagocytosis, CRP was shown to enhance the response of human neutrophils and monocytes to Pn27 in the presence of normal human serum. The CL response of neutrophils and monocytes to Pn4 was not affected by the addition of CRP to serum. The addition of anti-capsular antibody to Pn4 and Pn27 enhanced the CL responses of both neutrophils and monocytes to both bacteria. The localization of bound CRP and C3 on Pn4 and Pn27 was determined by immunoelectron microscopy. CRP bound to Pn4 only in the cell wall region and C3 was located in this area whether or not CRP was present. Anti-capsular antibody deposited C3 in the capsule of Pn4. In contrast, Pn27 bound CRP throughout the capsule and cell wall areas. C3 was deposited in the cell wall region of Pn27 by serum alone and in the cell wall region and capsule when CRP or anti-capsular antibody was present. Because C3 fixation to the capsule was consistently associated with enhanced responses by phagocytic cells, it appears that the site of CRP binding and subsequent complement activation may be critical in the opsonophagocytosis of S. pneumoniae. These findings extend the correlation between capsular C3 and opsonization to a nonimmune system. By using CRP and different pneumococcal serotypes we have shown that the same molecules that are effective in the stimulation of phagocytic cells when bound to the capsule are not effective when bound to the cell wall.     

Role of CR2 in the human adult and neonatal in vitro antibody response to type 4 pneumococcal polysaccharide.

A number of studies have indicated that the complement receptor type 2 (CR2), which is the receptor for C3d, a degradation fragment of the complement component C3, regulates B lymphocyte activation and growth. Early reports have described that C3 regulates T cell-dependent (TD) antibody responses. The involvement of CR2 in the antibody response to T cell-independent type 2(TI-2) antigens was investigated because neonatal B cells, which are unresponsive to TI-2 antigens both in vivo and in vitro, express a significantly decreased level of CR2 as compared to B cells of adult donors. We utilized type 4 pneumococcal polysaccharide (PS4) as a model TI-2 antigen. In order to study the relationship between CR2 and the response to PS4, B cells were costimulated with PS4 and monoclonal antibodies (MAb) to CR2. HB5 and OKB7 anti-CR2 monoclonal antibodies enhanced the in vitro response of adult B cells to PS4, as measured in a PS4-specific spot-forming cell assay. Neonatal B cells could only be induced to respond to PS4 using high concentrations of OKB7 anti-CR2 MAb. The 8-mercaptoguanosine (8MGuo), an agent that can overcome the in vitro unresponsiveness to PS4 of neonatal B cells, increased CR2 expression on adult and neonatal B cells. Furthermore, 8MGuo synergizes strongly with anti-CR2 antibodies in augmenting the anti-PS4 antibody response. Data presented in this report provide evidence of CR2 involvement in the antibody response to PS4 and that the neonatal B cell unresponsiveness to TI-2 antigens may be due to the decreased expression of CR2.     

Intracellular degradation of the complement C3b/C4b receptor in the absence of ligand

Human neutrophils (PMN) respond to various soluble stimuli by translocating intracellular complement C3b/C4b receptors (CR1) to the cell surface. Ligand-independent internalization of surface CR1 has been demonstrated previously, but the fate of total cellular CR1 during PMN stimulation has not been determined. In order to study the fate of CR1 during neutrophil activation, we have employed a unique approach for the quantitative analysis of intracellular antigens which allows simultaneous measurement of total cellular and surface membrane antigen pools. Stimulation of isolated PMN with N-formyl-Met-Leu-Phe or ionomycin resulted in a mean 7-fold increase in surface CR1 expression within 15 min. Total cellular CR1 decreased by as much as 45% within 15 min, with loss continuing for up to 1 h. Inclusion of NH4Cl during PMN stimulation inhibited the loss of total CR1 without affecting surface CR1 expression. Addition of phenylmethylsulfonyl fluoride inhibited loss of total CR1 and enhanced the stimulus-induced increases in surface CR1. These data suggest that intracellular degradation of CR1 occurs during stimulation of PMN and may involve proteolysis in an acidic intracellular compartment. Since our experiments were done with isolated PMN in the absence of serum and complement components, this degradation occurred in the absence of C3b, the ligand for CR1. To our knowledge, ligand-independent degradation of a cell surface receptor has not been previously detected.     

Site of action of a soluble immune response suppressor (SIRS) produced by concanavalin A-activated spleen cells.

The cellular site of action of SIRS, a soluble immune response suppressor released by Con A-activated spleen cells which suppresses antibody responses to heterologous erythrocytes by murine spleen cells in vitro, was investigated. Exposure of spleen cells to SIRS for 2 hr at 37 degrees C or 1 hr at 4 degrees C was sufficient to suppress 5-day antibody responses in vitro. Similar exposure of splenic or peritoneal exudate macrophages to SIRS also suppressed antibody responses by untreated splenic lymphoid cells; exposure of splenic lymphoid cells to SIRS was without effect. SIRS did not act via T cells which might have contaminated the macrophage preparations. SIRS-mediated suppression could be partially overcome by an excess of normal peritoneal exudate macrophages, but not by an excess of T or B cells. These data indicate that the target cell of SIRS activity is the macrophage. The results are discussed in the context of macrophage functions that could be affected by SIRS.     

Interactions of human complement with virus particles containing the Nipah virus glycoproteins

Complement is an innate immune response system that most animal viruses encounter during natural infections. We have tested the role of human complement in the neutralization of virus particles harboring the Nipah virus (NiV) glycoproteins. A luciferase-expressing vesicular stomatitis virus (VSV) pseudotype that contained the NiV fusion (F) and attachment (G) glycoproteins (NiVpp) showed dose- and time-dependent activation of human complement through the alternative pathway. In contrast to our findings with other paramyxoviruses, normal human serum (NHS) alone did not neutralize NiVpp infectivity in vitro, and electron microscopy demonstrated no significant deposition of complement component C3 on particles. This lack of NiVpp neutralization by NHS was not due to a global inhibition of complement pathways, since complement was found to significantly enhance neutralization by antibodies specific for the NiV F and G glycoproteins. Complement components C4 and C1q were necessary but not sufficient by themselves for the enhancement of antibody neutralization. Human complement also enhanced NiVpp neutralization by a soluble version of the NiV receptor EphrinB2, and this depended on components in the classical pathway. The ability of complement to enhance neutralization fell into one of two profiles: (i) anti-F monoclonal antibodies showed enhancement only at high and not low antibody concentrations, and (ii) anti-G monoclonal antibodies and EphrinB2 showed enhancement at both high and very low levels of antibody (e.g., 3.1 ng) or EphrinB2 (e.g., 2.5 ng). Together, these data establish the importance of human complement in the neutralization of particles containing the NiV glycoproteins and will help guide the design of more effective therapeutics that harness the potency of complement pathways.     

Incorporation of decay-accelerating factor into the baculovirus envelope generates complement-resistant gene transfer vectors

Baculovirus vectors are an efficient means to deliver genes into hepatocytes in vitro. In experiments that exclude components of the complement system, gene transfer is facilitated. Therefore, the complement system has been defined to represent a potent primary barrier to direct application of baculoviruses in vivo. Here we have genetically manipulated baculoviruses so that the complement-regulatory protein human decay- accelerating factor (DAF) is incorporated into the viral envelope. We found that this modification protected baculovirus vectors against complement-mediated inactivation. Complement-resistant baculovirus vectors were additionally analyzed by immunoblotting and electron microscopy, showing the extent of envelope-incorporated DAF and shape of complement-resistant baculoviruses after exposure to complement. This modified baculovirus vector allowed for an enhanced gene transfer into complement-sufficient neonatal rats in vivo, and thus represents a step in the development of improved alternative viral vectors for gene therapy.     

The mechanisms of inhibition of human IL 2 production. II. PGE2 induction of suppressor T lymphocytes

In vitro and in vivo experiments indicate that the production of interleukin 2 (IL 2) by T lymphocytes is critical for the development of the effector phase of immunity. Complex cellular interactions are involved for the induction of IL 2 production. We have shown in a previous study that in humans monocytes can transmit opposite signals to the IL 2-producing cells. In addition to the positive signal delivered through the release of interleukin 1, human monocytes can deliver a negative signal through the release of prostaglandin E2 (PGE2). This monokine, known to activate suppressor mechanisms in several systems, was shown to inhibit IL 2 production. The data presented in this paper show that this PGE2-dependent inhibition is strictly dependent upon the presence of radiosensitive T cells in the culture, suggesting that PGE2 induces the activation of suppressor T cells modulating IL 2 production. Kinetics experiments indicate that these suppressor cells are radiosensitive during their induction phase but become radioresistant after 18 hr of incubation in the presence of PGE2. Successful in vitro induction of suppressor cells by incubation of enriched T cells with PGE2 was decisive for the analysis of the phenomenon. The induced suppressors were capable of inhibiting IL 2 production by fresh autologous T cells as well as inhibiting PHA proliferative response by these cells. A quantitative evaluation of IL 2 receptors on PGE2-treated cells has indicated that this absorption capacity was similar to the capacity of PBL known to express a low number of IL 2 receptors, thus excluding a suppression via absorption or competition for IL 2. No detectable killing of IL 2-producing cells by PGE2-induced suppressors was observed. The OKT4 and OKT8 phenotype of suppressor cells was examined. T cells were purified at two stages of differentiation before or after induction by PGE2 in vitro treatment. We conclude from these experiments that PGE2 activates suppressor cells among precursors segregating predominantly with the OKT8 subset and fewer cells with the OKT4 subset. After differentiation, however, the suppressor cells segregate with the OKT8 subset only. Such results were obtained by using positive selection (cellular affinity columns) and negative selection (monoclonal antibodies plus complement).     

Some mechanisms involved in the prostaglandin E2 immunosuppressive effect in (CBA x C57BL)F1 mice in vivo

The cellular mechanisms involved in the immunosuppressive effect of prostaglandin E2 (PGE2) multiple injections into (CBA x C57Bl)F1 mice in vivo have been studied. PGE2 injection increases the induction of specific T-suppressors. In addition, there is a decrease in macrophage phagocytic activity and in the phagocytosis index, apparently mediated by Fc gamma receptors (Fc gamma R) and not by the macrophage complement receptor (C3R). The induction of antibody synthesis by using "immune" macrophages injected into a syngeneic recipient results in considerable decrease in the accumulation of antibody-forming cells if the macrophage donor has been pretreated with exogenous PGE2 in comparison with untreated controls. These cellular mechanisms are possibly one part of the diverse way in which PGE2 exerts an immunosuppressive effect in vivo and contributes to humoral immune response suppression.     

In vivo T-lymphocyte activation and transient reduction of viral replication in macaques infected with simian immunodeficiency virus

While it is well established that cellular activation can increase human immunodeficiency virus (HIV) replication in T lymphocytes, it is also clear that both activated CD8+ and CD4+ T lymphocytes mediate anti-HIV activity. To assess the relative importance of these contrary effects on HIV replication in vivo, we evaluated the consequences of Mycobacterium bovis BCG and staphylococcal enterotoxin B (SEB) inoculation in vivo in rhesus monkeys chronically infected with simian immunodeficiency virus of macaques (SIVmac). BCG inoculation induced as much as a 2.5-log reduction of plasma and intracellular SIV RNA in SIVmac-infected monkeys. This down-regulation of virus replication persisted as long as 4 weeks after BCG inoculation. Similarly, SEB injection resulted in up to a 3-log decrease in plasma and intracellular SIV RNA in SIVmac-infected macaques. Interestingly, the short-term reduction of viremia in these monkeys correlated with the peak in vivo production of SEB- and BCG-induced cytokine responses. However, no long-term clinical benefit was observed in the SIVmac-infected macaques. These studies provide in vivo evidence that potent T-cell stimulation driven by antigens other than the virus itself can, under some circumstances, mediate short-term reduction of viremia in AIDS virus-infected individuals.     

Complement reversal of immunosuppression induced with plasma of rats infected with Trypanosoma brucei rhodesiense

Suppression of antibody production by splenic lymphocytes from rats immunized with sheep red blood cells (SRBC) after incubation with plasma from rats infected with Trypanosoma brucei rhodesiense was confirmed. Suppressive activity became evident in plasma after the sixth day of infection and was manifested by reduction in the number of hemolytic Jerne plaques produced by the treated cells. The activity was temporally associated with increased amounts of soluble immune complex (SIC) reduced titers of lytic complement, elevated titers of immunoconglutinin (IK) and anemia. Treatment of suppressive plasma with hemolysin sensitized SRBC alexinated with horse complement to reduce IK did not reduce suppressive activity, and the activity appeared to have been enhanced when the plasma was heated to inactivate the remaining complement (C'). When fresh rat C' was added to the treated cells, the suppression was largely, though not completely, reversed. Treatment of spleen cells with SIC prepared in vitro from bovine serum albumin (BSA) and rabbit antiBSA also suppressed the plaque forming capacity of the cells. Complexes of BSA-antiBSA-C' and complexes of BSA-antiBSA-C'-IK were equally suppressive. Again, addition of fresh C' to cells treated with these complexes largely, though not completely, reversed the suppressive effect on the cells. From the results it is suggested that immunosuppression associated with experimental T. b. rhodesiense infection may be in part a suppression of the capacity of induced lymphocytes to produce antibody. It is possible that the suppression was mediated by SIC present in the plasma of the infected rats and this effect was probably enhanced by reduced levels of complement in the suppressive plasma.     

Down-regulation of human complement factor H sensitizes non-small cell lung cancer cells to complement attack and reduces in vivo tumor growth

Malignant cells are often resistant to complement activation through the enhanced expression of complement inhibitors. In this work, we examined the protective role of factor H, CD46, CD55, and CD59 in two non-small cell lung cancer cell lines, H1264 and A549, upon activation of the classical pathway of complement. Complement was activated with polyclonal Abs raised against each cell line. After blocking factor H activity with a neutralizing Ab, C3 deposition and C5a release were more efficient. Besides, a combined inhibition of factor H and CD59 significantly increased complement-mediated lysis. CD46 and CD55 did not show any effect in the control of complement activation. Factor H expression was knockdown on A549 cells using small interfering RNA. In vivo growth of factor H-deficient cells in athymic mice was significantly reduced. C3 immunocytochemistry on explanted xenografts showed an enhanced activation of complement in these cells. Besides, when mice were depleted of complement with cobra venom factor, growth was recovered, providing further evidence that complement was important in the reduction of in vivo growth. In conclusion, we show that expression of the complement inhibitor factor H by lung cancer cells can prevent complement activation and improve tumor development in vivo. This may have important consequences in the efficiency of complement-mediated immunotherapies.     

Enhancement of the function of neutrophil type-1 and type-3 complement receptors by human leukocyte inhibitory factor (LIF)

The lymphokine leukocyte inhibitory factor (LIF) has previously been documented to enhance several neutrophil (PMN) functions, including stimulated chemotaxis and superoxide generation, phagocytosis and adherence of opsonized targets, and antibody-dependent cellular cytotoxicity. The present studies were designed to investigate the effects of LIF on PMN function mediated by the complement components C3b and C3bi. LIF induced a dose-dependent increase in superoxide production generated by opsonized zymosan (up to 97.1 +/- 31.4% at 16 U LIF/ml; P less than 0.01). While neither control nor LIF-treated PMN were capable of inducing phagocytosis of either C3b- or C3bi-opsonized sheep erythrocytes (E) directly, exposure to LIF caused a significant (P less than 0.05) increase in their adherence to E (137.4 and 59.4%, respectively). Specificity for complement receptor function was confirmed by the ability of anti-CR1 antibody to block adherence of LIF-treated PMN to EAC3b (77.0% inhibition) and anti-CR3 antibody to block adherence to EAC3bi (70.2% inhibition). Increased C3b and C3bi function may have been due, at least in part, to increased expression of their respective surface membrane receptors. Thus, using indirect immunofluorescence, LIF induced a 38.2% increase in fluorescence of the anti-CR1 antibody and a 96.1% increase in anti-CR3 binding. These studies describe an additional mechanism through which LIF may have an important pro-inflammatory role in vivo.     

Dendritic cell synthesis of C3 is required for full T cell activation and development of a Th1 phenotype

Previous studies have found that deficiency of complement component C3 is associated with reduced T cell responses in several disease models including viral infection, autoimmune disease, and transplantation. However, the underlying mechanism is unclear. In this study, we demonstrate that dendritic cells (DCs) are able to synthesize C3 and this synthesis is required for the capacity of DCs to stimulate alloreactive T cell responses in vitro and in vivo. Compared with C3-producing DCs, C3-nonproducing DCs exhibit reduced potency to stimulate an alloreactive T cell response, favor the polarization of CD4(+) T cells toward Th2 phenotype, and have regulatory T cell-driving capacity. In addition, priming mice with C3-deficient DCs compared with wild-type DCs led to delayed skin allograft rejection. Our findings that nonproduction of C3 by DCs significantly reduced T cell stimulation and impaired allograft rejection provide a potentially important explanation of how C3-deficient mice develop reduced T cell responses and of how C3-deficient donor kidney is protected from T cell-mediated graft rejection.     

Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.     

Comparison of GK1.5 and chimeric rat/mouse GK1.5 anti-CD4 antibodies for prolongation of skin allograft survival and suppression of alloantibody production in mice.

GK1.5 is a rat mAb that recognizes the mouse CD4 Ag. It has been shown to deplete CD4+ cells in vivo and to be immunosuppressive. To evaluate the effect of the C region of this antibody in achieving cell depletion, chimeric antibodies, each having the rat GK1.5 V regions and one of the four mouse IgG C region isotypes, were compared with the native rat antibody. The chimeric antibodies and the native antibody were tested for their ability to mediate in vitro C-dependent cytotoxicity, in vivo cell depletion, and prolongation of allogeneic skin graft survival and suppression of alloantibody production. In vitro C-dependent cytotoxicity assays revealed that rat IgG2b and the chimeric antibodies containing mouse IgG2a, mouse IgG2b, and mouse IgG3 were effective in lysing CD4+ lymphocytes whereas mouse IgG1 was ineffective. In vivo studies of CD4+ cell depletion showed that mouse IgG2a, rat IgG2b, and mouse IgG2b were effective isotypes, mouse IgG1 was less effective, and mouse IgG3 did not deplete CD4+ cells. A correlation was found between the ability of an isotype to deplete CD4+ cells in vivo and its ability to prolong the survival of skin allografts and to suppress alloantibody production. The nondepleting mouse IgG3 was ineffective in these assays. Overall the most effective mouse isotype was IgG2a which was as effective as rat IgG2b. These results indicate 1) that syngeneic isotypes of mAb can cause cell depletion and consequently the prolongation of allograft rejection and suppression of alloantibody production; 2) that not all isotypes are equally effective; and 3) that the ability of a given isotype to deplete cells in vivo does not correlate with its ability to mediate C-dependent lysis in vitro. Our results are consistent with the hypothesis that in vivo depletion of cells is mediated by opsonization and binding through the FcR.