Human T cell derived, cell-bound complement iC3b is integrally involved in T cell activation

Although the complement system is thought to be mainly involved in innate immunity and in the humoral arm of adaptive responses, evidence implicating that complement impacts T cell responses are accumulating recently. The role of the various activation products of the major complement component C3 were mainly studied so far in animal systems, and investigations regarding the effect of different C3-fragments on human T cells are sparse. Here we show that anti-CD3 activated human T lymphocytes derived from the blood and tonsil of healthy individuals produce C3, and the major cleavage fragment that appears on the T cell surface is iC3b. Based on studies carried out in allogenic system we demonstrate that the T cell membrane bound iC3b binds to the CR3 and probably to CR4 receptors expressed on monocyte-derived dendritic cells, and this interaction leads to significantly enhanced T-cell proliferation. Since neither C3aR and nor C3a binding could be detected on the membrane of anti-CD3 activated T cells, our findings indicate that in humans – in contrast to mice – the C3a peptide is most probably not involved directly in the T cell activation process.     

Covalent binding of C3b to tetanus toxin: influence on uptake/internalization of antigen by antigen-specific and non-specific B cells.

Antigen opsonization by the C3b fragment of complement is a significant event in the modulation of cell-mediated immune response, but its mechanism is still largely unknown. The structural characteristics of C3b allow it to act as a bifunctional ligand between antigen and cells via their membrane C3b receptors. It was thus of interest to study the influence of the covalent link between C3b and antigen on the fixation and internalization of this antigen by antigen-presenting cells. Tetanus toxin (TT) was used as antigen, either free or covalently linked to C3b (TT-C3b). The antigen-presenting cells were TT-specific (4.2) or non-specific (BL15) Epstein-Barr virus (EBV)-transformed B cells. C3b was found to play an important role in antigen fixation and internalization by both antigen-specific and antigen non-specific cells. Covalent binding of C3b on TT (1) permitted fixation and internalization of this antigen by non-specific cells via their complement receptors; (2) enhanced antigen fixation and resulted in cross-linking between membrane immunoglobulins and complement receptors on antigen-specific cells. The consequences of covalent C3b binding to TT were analysed using antigen-specific and antigen-nonspecific cells. In both cases, a net increase in antigen fixation was observed. At the intracellular level, covalent C3b binding to TT resulted in a large TT incorporation in endosomes of nonspecific cells, similar to that observed in antigen-specific cells. Thus, C3b covalently linked to antigen enlarges the array of B-cell types capable of presenting antigen, including non-specific cells.     

MHC class I-mediated exogenous antigen presentation by exosomes secreted from immature and mature bone marrow derived dendritic cells

Exosomes are 50-90 nm vesicles with antigen presenting ability carrying major histocompatibility complex (MHC) class I, class II, abundant co-stimulatory molecules and some tetraspan proteins. Although dendritic cells (DCs) are one of the professional antigen presenting cells capable of presenting exogenous antigens in MHC class I-mediated antigen specific manner (cross-presentation), the cross-presentation ability by exosomes from immature or mature DCs are unknown. Here we show that exosomes released from ovalbumin (OVA) protein-pulsed bone marrow derived dendritic cells (BM-DCs) weakly present the peptide determinants to OVA specific MHC class I-restricted CD8(+) T cell hybridomas. The exosomes secreted by OVA(257-264) peptide- or OVA protein-pulsed mature BM-DCs activated OVA specific MHC class I-restricted T cell hybridomas more efficiently than those from immature BM-DCs. Transporters associated with antigen processing (TAP) deficient mice-derived BM-DCs were also used to examine whether functional TAP activity was required for cross-presentation by exosomes. The exosomes obtained from OVA(257-264) peptide-pulsed BM-DCs derived from TAP(-/-) mice showed a significant antigen presenting ability to OVA specific MHC class I-restricted T cell hybridomas. Altogether, our data indicate that BM-DCs secrete exosomes with weak cross-presentation ability.     

A further link between innate and adaptive immunity: C3 deposition on antigen-presenting cells enhances the proliferation of antigen-specific T cells

Murine cells of the B lymphoblastoid line A20 and concanavalin A- elicited peritoneal macrophages are shown to activate and fix C3 fragments covalently when incubated in fresh, autologous serum under conditions allowing the initiation of the alternative complement pathway. For the detection of cell-bound C3, cytofluorimetry was performed using FITC-labeled F(ab')2 fragments of anti-mouse C3. Cell- bound C3 fragments are not internalized or shed by the cells under culture conditions for at least two hours. When the antigen-presenting capacity of serum-treated cells was tested using various antigens and experimental systems, augmentation of the proliferation of antigen- specific T cells was found. This enhancing effect was particularly pronounced at suboptimal antigen doses. The elevation of T cell proliferation induced by C3-opsonized antigen-presenting cells (APC) could be abrogated by F(ab')2 fragments of goat anti-mouse C3, suggesting the involvement of C3 receptors expressed by T cells in the process. Using the 7G6 mAb recognizing murine CR1/CR2, the presence of these complement receptors on activated T cells is demonstrated by cytofluorimetry and immunoprecipitation, as well. These results point to the role of C3 bound to acceptor sites on APC in the facilitation of antigen presentation, providing a further link between innate and adaptive immunity.      

Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.

The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed.     

Proliferation of resting B cells is modulated by CR2 and CR1

Absence of the third component of complement, C3, is associated with impaired ability to synthesize antibody, particularly in the presence of limiting antigen [1-9]. The mature B lymphocyte bearing the surface immunoglobulin receptor transduces signals for proliferation and differentiation upon binding of specific antigen. This mature B cell also bears two related membrane proteins, CR2 (the C3d/Epstein-Barr virus receptor) (CD35) [15], which can mediate the binding of ligands to which appropriate cleavage fragments of C3 have become attached [16]. It has been suggested that these receptors play a direct role(s) in B cell activation [17-25]. In light of previous in vivo observations we decided to assess the function of CR2 and CR1 in relation to B cell activation through the membrane IgM receptor. Highly purified splenic B cells were prepared. No contaminating T cells or macrophages were detected by flow cytometric analysis and no proliferative activity was present upon PHA or ConA stimulation of the purified cells. The B cells were separated into low (activated), medium (preactivated) and high density (resting) fractions by Percoll gradient density centrifugation [26]. The responses of the B cell subpopulations to various concentrations of anti mu (DA4.4 monoclonal antibody) [27] were examined for proliferation at 72 h and for IgM/IgG production at 7 days. Low density B cells were maximally stimulated and no concentration of anti-mu was effective in enhancing their responses. High density B cells proliferated to anti-mu in a concentration dependent manner. When substimulatory concentrations of anti-mu were employed, concomitant crosslinking of CR2 (with either of 2 distinct monoclonal antibodies HB-5 [28] or OKB7 [17]) resulted in a 45% enhancement of B cell proliferation above that observed by crosslinking of SIgM alone. In these studies, total IgM and IgG did not increase in the absence of T cells or T cell factors, indicating that terminal differentiation did not occur. In contrast, when a monoclonal antibody to CR1(44D) [29] was employed in an identical experiment, B cell proliferation was completely inhibited. Antibodies to CR2 or CR1 either alone or in crosslinked form did not enhance B cell proliferation. Immune complexes may crosslink the B cell surface in a manner analogous to our model when the immunoglobulin receptor and CR2 are simultaneously engaged. This activation signal may be particularly important in eliciting antibody responses when the quantity of specific antigen or the affinity for antigen is low. The marked inhibition of proliferation induced by CR1 suggests an alternate role for this receptor in modulation of B cell responses.     

CD40L is transferred to antigen‐presenting B cells during delivery of T‐cell help

The delivery of T‐cell help to B cells is antigen‐specific, MHC‐restricted, and CD40L (CD154) dependent. It has been thought that when a T cell recognizes an antigen‐presenting B cell, CD40L expressed on the T‐cell surface engages with CD40 on the surface of B cells as long as the cells remain conjugated. By adding fluorescently labeled anti‐CD40L antibody during overnight incubation of antigen‐presenting B cells with antigen‐specific T cells, we discovered that CD40L does not remain on the surface of the T cell, but it is transferred to and endocytosed by B cells receiving T‐cell help. In the presence of anti‐CD40L antibody, transferred CD40L is nearly absent on bystander B cells that are not presenting antigen, and the bystander cells do not become activated. Because transfer of CD40L to B cells correlates with B‐cell activation, we speculate that persistence of helper T‐cell‐derived CD40L on or in B cells could permit sustained CD40 signaling enabling survival and proliferation of antigen‐presenting B cells following brief interactions with helper T cells in vivo in germinal centers.     

Covalent binding of C3b to monoclonal antibodies selectively up-regulates heavy chain epitope recognition by T cells

Protein C3 of the complement system is known for its role in the nonspecific immune response. Covalent binding of C3b to antigen upon complement activation also plays a significant role in specific T cell immune response. C3b-antigen complexes can bind to complement receptors on the antigenpresenting cell, and the C3b antigen link (most often an ester link) remains fairly stable inside the cells. In this study, IgG1,χ and IgG2a,χ murine monoclonal antibodies (mAb) were used as antigens; covalent complexes between mAb and C3b were produced and puritied in vitro from purified proteins; human B cell lines and T cell clones were raised from tumor patients who received mAb injections for cancer therapy or diagnosis. Recognition of epitopes of these mAb by T cell clones when the mAb were processed alone or bound to C3b was compared. IgG or IgG-C3b complexes presented by B cell lines were able to stimulate proliferation of χ light chain-specific T cell clones at similar concentrations. In contrast, IgG-C3b complex recognition by heavy chain-specific T cell clones required 100-fold less IgG-C3b than uncomplexed IgG. As C3b was shown to be covalently bound only to the IgG heavy chains in the complexes, C3b chaperoning is restricted to only the IgG heavy chain and selectively influences intracellular steps of IgG heavy chain processing. This differential modulation of C3b suggests an early dissociation of IgG heavy and light chains in antigenpresenting cells.     

Macrophage-bound C3 fragments as adhesion molecules modulate presentation of exogenous antigens.

The involvement of complement in the response to T cell dependent antigens is generally accepted, however the mechanism has not been clarified. We compared the T cell response in vitro, using antigen-pulsed macrophages from normal and genetically C3-deficient guinea pigs, and show, that C3-fragments fixed covalently to the surface of the antigen-presenting cells are involved in the triggering of responder T cells. Binding of guinea pig C3-specific mAb to oil-elicited, OVA- and PPD-pulsed macrophages of C3D guinea pigs is reduced compared to normal cells, while the expression of Ia antigens is the same. C3-like peptides can be immunoprecipitated only from the lysate of oil-elicited normal cells. These C3-fragments are fixed to the cell-membrane via ester-bonds, since they are released upon treatment with hydroxylamine. In comparison with normal cells, the antigen-presenting capacity of macrophages derived from C3D animals is strongly impaired in cultures containing 10% normal guinea pig serum. A further impairment is observed in cultures with 10% C3D guinea pig serum. Two of the tested C3-specific mAb inhibited antigen-induced T cell proliferation in a dose-dependent manner. Our data point to the importance of C3, as a bivalent molecule, having the capacity to facilitate the cooperation between the antigen-presenting cell and the responder T lymphocytes.     

Mechanism(s) promoting HIV-1 infection of primary unstimulated T lymphocytes in autologous B cell/T cell co-cultures

Resting CD4(+) T cells in the lymphoid tissue (LT) are essential producers of virions at the beginning of HIV infection in vivo. We previously developed a model that allowed in vitro infection of non-prestimulated T lymphocytes in the presence of autologous B lymphocytes and complement. In this study, we try to clarify the mechanism(s) responsible for virus transmission in unstimulated autologous B cell/T cell co-cultures. Ex vivo analyses of patient plasma samples revealed that HIV was opsonized. Flow cytometry showed that opsonized virus preferentially bound to complement receptor (CR)-2 on B lymphocytes in primary B cell/T cell co-cultures. As indicated by cytokine measurements and transwell experiments, soluble factors seemed to play a minor role in enabling infection. Rather, direct interaction between B and T lymphocytes and direct binding of opsonized virus to CR2 on B cells turned out to be essential for productive infection. Antibodies blocking cell-cell adhesion inhibited p24 antigen production. An anti-CR2 antibody blocking C3d-CR2 binding also significantly reduced viral replication. Since the infection of unstimulated T cells by opsonized primary HIV isolates in the presence of B cells was highly efficient independent of the tropism of the virus, this mechanism may be critical in the pathogenesis of HIV.     

Co-stimulation of murine CD4 T cell growth: cooperation between B7 and heat-stable antigen.

The B cell activation antigen B7/BB1 has been shown to co-stimulate growth of human T cells by binding the T cell molecule CD28. In mice, the heat-stable antigen (HSA) has also been shown to act as a co-stimulator for T cell growth. In this study, we have evaluated the contributions of B7 and HSA to the co-stimulatory activity of antigen-presenting cells (APC). Mouse B7 provides co-stimulatory activity for murine CD4 T cells in anti-CD3-induced proliferation. Human CTLA4Ig, a chimeric molecule comprising the extracellular region of CTLA-4 fused to an immunoglobulin C gamma fragment, binds to murine B7. We, therefore, use human CTLA4Ig and the hamster anti-HSA monoclonal antibody 20C9 to analyze the relative contributions of B7 and HSA to the co-stimulatory activity of murine spleen APC. Our data reveal that both murine B7 and HSA are expressed by dendritic cells and by low-density spleen B cells. Either CTLA4Ig alone or anti-HSA alone inhibited CD4 T cell proliferation to anti-CD3 by > 90%, while CTLA4Ig and anti-HSA together were far more efficient in inhibiting clonal expansion of CD4 T cells. These results demonstrate that functionally defined co-stimulation involves at least B7 and HSA and suggest that signals delivered by B7 and HSA synergize in promoting T cell growth.     

Direct evidence for a functional role of HLA-DRB1 and -DRB3 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T lymphocytes

The contribution of the HLA-DRB1, -B3, and -B5 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T cells isolated from an atopic individual (HLA-DRw12, DR7; DRw52b) with perennial rhinitis was investigated. Using a panel of histocompatible and histoincompatible accessory cells, the restriction specificity obtained for a long term T cell suggested that a component of the dust mite reactive repertoire recognized antigen in association with DRB3 gene products. Oligonucleotide DNA typing of the presenting cell panel demonstrated a correlation between the DRw52b allele and T cell responsiveness. Murine fibroblasts expressing DRw52b, but not DRw52a or -c molecules, presented antigen to both the T cell line and cloned T cells (DE26) derived from the line, indicating that the supertypic specificity DRw52b was able to restrict recognition of dust mite antigens. Additional T cell clones (DE9 and DE41) also isolated from the line were restricted by the products of the B1 gene locus (DRw12B1) as determined by murine fibroblasts transfected with the appropriate HLA-DR genes. Clone DE9 was degenerate in its restriction specificity, also recognizing dust mite presented by accessory cells expressing the DR2 subtypes. Presentation by fibroblasts transfected with DRw12B1, DR2Dw2B5 genes and EBV-transformed B cell lines expressing DR2DW21B1 and -B5 indicated that the functional site restricting recognition may be associated with residues 70 and 71 of the DR beta chain helical wall of the antigen combining site. Furthermore, we have recently demonstrated that both T cell clones DE9 and DE26 induce allergen dependent IgE synthesis in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum regulates the expression of complement receptor 2 on human B cell lines

Complement receptor 2 (CR2) participates in the regulation of B cell responses to antigen. In this study we report that treatment of IM-9 B lymphoblastoid cells or Raji Burkitt's lymphoma cells with 10% heat-inactivated fetal bovine serum for 24 hr increased both the CR2 mRNA level and CR2 surface protein expression more than twofold. No change in the CR2 expression level was observed if cells were cultured in serum-free medium. The CD 19 mRNA level decreased after 24 hr independently of the presence of serum. The serum-stimulated increase in CR2 expression was not due to changes in the proliferative capacity of the cells and could not be mimicked by various cytokines. However, IFN-γ as well as OKB7, a CR2-specific monoclonal antibody, blocked the serum-induced increase in CR2 expression at the mRNA level. Our data show for the first time that factors in serum induce the expression of the CR2 gene and that signals initiated by IFN-γ and OKB7 interfere with the serum-induced changes. Because stimuli that alter CR2 expression can influence the extent of the B cell response to antigen-C3d complexes, serum factors may play a role in regulating the responsiveness of B cells.     

Dual overlapping peptides recognized by insulin peptide B:9-23 T cell receptor AV13S3 T cell clones of the NOD mouse

T cells isolated from islets of non-obese diabetic (NOD) mice are enriched for insulin-reactive cells. The great majority of these T cells recognize insulin B chain peptide (B:9-23). B:9-23 reactive T cell clones are diabetogenic and show a dramatic TCR alpha -chain restriction (predominant AV13S3). We have studied the reactivity of five different B:9-23 reactive T cell clones to truncated peptides and alanine substituted analogues of B:9-23. Amongst these AV13S3 T cell clones, one reacted with peptide B:9-16 and four with B:13-23. The two peptides have in common only four amino acids (B:13-16; EALY). Having defined minimal peptide epitopes, we evaluated a mutant insulin sequence (B:13 glutamine) which retains metabolic activity. As predicted, this single amino acid change abrogated T cell reactivity. In addition, we have created a modified I-A(g7)gene with the B:9-23 peptide covalently linked to I-A(g7). Antigen presenting cells transfected with this construct were excellent presenting cells for all clones studied. The definition of dual peptide motifs and creation of bioactive covalent I-A(g7)-B:9-23 should facilitate studies of the pathogenic significance and antigen recognition by B:9-23 reactive diabetogenic T cells.     

Function of C3 in a humoral response: iC3b/C3dg bound to an immune complex generated with natural antibody and a primary antigen promotes antigen uptake and the expression of co-stimulatory molecules by all B cells,but only stimulates immunoglobulin synthesis by antigen-specific B cells

Previous studies have shown that an optimal humoral response to a primary protein antigen requires C3 and CR2 (CD21). Sera from non-immunized donors contain natural IgM and IgG antibodies to the primary antigen keyhole limpet haemocyanin (KLH), and these have been previously shown to form immune complexes (IC) that activate the classical pathway of C, fixing iC3b/C3dg onto the KLH antigen. Such KLH IC bind to CR2 on KLH-non-specific B lymphocytes, resulting in antigen processing and MHC class II-dependent presentation to KLH-specific helper T cells. KLH IC also induce B lymphocytes to express the CD80 co-stimulatory molecule via simultaneous CR2 ligation with C3 and FcγRII (CD32) stimulation by IgG natural antibody. The current study demonstrated that KLH IC ligation to either CR2 or FcγRII resulted in activation of a second co-stimulatory molecule, LFA-1 (CD11a, CD18). The possibility of polyclonal B cell stimulation by the presentation of KLH-iC3b/C3dg by antigen-non-specific B cells was excluded by demonstration that in vitro cultivation of peripheral blood mononuclear cells (PBMC) with KLH-iC3b/C3dg elicited only anti-KLH, and did not stimulate synthesis of antibodies to hepatitis C virus (HCV) or tetanus toxoid (TT). Of greatest significance, a specific anti-KLH response was only detectable in cultures stimulated with KLH-iC3b/C3dg and not in cultures stimulated with KLH alone or KLH-IgG. Thus, iC3b/C3dg that was bound to a primary protein antigen enhanced recognition and specific immunoglobulin synthesis by antigen-specific B cells, even though the antigen was taken up and processed via CR2 by both antigen-specific and non-specific B cells.     

Expression and role of Fc- and complement-receptors on human dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells, which take up pathogens/foreign structures in peripheral tissues, then migrate to secondary lymphoid organs where they initiate adaptive immune responses by activating naive T-cells. In the early phase of antigen uptake pattern recognition receptors (including mannose-, scavenger- and toll-like receptors) that recognize pathogen-associated molecular patterns play an important role. Later receptors binding opsonized antigen are also involved in phagocytosis. These cell membrane molecules include various Fc-receptors, recognizing different isotypes of antibodies and various complement-receptors, such as CR3, CR4 and the C1q-binding complex of calreticulin and CD91. Here we aim to summarize how these immunecomplex binding receptors are involved in the initiation of DC maturation, and how they influence antigen presentation as well as some additional functions of these cells.     

CR3 (CD11b, CD18): a phagocyte and NK cell membrane receptor with multiple ligand specificities and functions.

The C3 receptor CR3 is expressed on phagocytic cells, minor subsets of B and T cells, and natural killer (NK) cells. It has important functions both as an adhesion molecule and a membrane receptor mediating recognition of diverse ligands such as intercellular adhesion molecule-1 (ICAM-1) and fixed iC3b. The receptor is capable of undergoing an activation event that regulates both its specificity for various ligands and its ability to mediate phagocytosis or extracellular cytotoxicity. Certain bacteria express carbohydrates or lipopolysaccharides (LPS) that can bind to and activate CR3, allowing the receptor to assume its activated state. Soluble beta-glucan derived from the yeast Saccharomyces cerevisiae is a particularly potent stimulator of CR3, and produces an activated state of the receptor that permits neutrophil phagocytosis of iC3b-coated erythrocytes or NK, cell cytotoxicity of iC3b-coated tumour cells, that are normally resistant to NK cells.     

Effector or target cell selection mediated by C3 bridges

Potential effector cells (including stimulated lymphocytes and cultured monocytes) and potential target cells of NK and AK type cytotoxic reactions (including several lymphoblastoid cell lines) cleave the third complement component (C3). As a result of expression of C3bA sites such cells are able to bind covalently the activated C3b through its metastable binding site and thereby become "armed" by the C3b. This permits C3b-bridge formation between these cells and CR1-bearing cells. The "effector selection" (i.e. when C3b is bound covalently to potential target cells) or "target selection" (when C3b is covalently bound to C3bA sites on potential effector cells) mediated by C3b bridges results in enhanced killing capacity. Macrophages activate and bind C3b as well; but the covalent binding of C3b by these cells inhibits Fc receptor mediated ADCC type killing.