Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity (original) (raw)
Genetic origins of EO Abs. Preliminary data documented that each of the EO B-cell hybridomas expressed an IgM-κ Ab (data not shown). Using a strategy designed to enable the unbiased cloning of the vast majority of rearrangements among VH-μ and Vκ genes (Table 1), RNA was extracted first from the EO6 cell line, the best characterized of the POVPC-specific EO Ab–expressing lines. In four of seven separate PCR reactions, each of which targeted different VH FR1 sequences, amplimers of the expected size (∼380 bp) were obtained. After independent cloning and DNA sequence determinations, each of the cloned amplimers was found to represent the same unique in-frame VH-μ gene rearrangement, derived from the S107 family. This gene rearrangement was 100% identical to a previously reported nonmutated, functional, canonical VHS107.1-DFL16.1-JH1 rearrangement that is also expressed in the T(EPC)15 IgA-κ plasmacytoma (10) (Figure 1). Amplimers of the expected size (∼350 bp) were obtained in two of six separate Vκ gene amplification reactions of cDNA from EO6 cells (Table 1). Sequence determination of plasmids containing these independently cloned inserts yielded the same in-frame nonmutated, canonical Vκ22-Jκ5 rearrangement that was also previously reported for the T(EPC)15 plasmacytoma.
The DNA sequences and VDJ splice sites of the somatic VH rearrangements expressed in the EO6 B-cell hybridoma and the T(EPC)15 plasmacytoma, and their relationship to the most homologous germ-line (GL) VH, DH, and JH gene segments. Sequence analysis for the 372 encoding nucleotides of the EO6 VH rearrangement did not reveal a single nucleotide variation suggestive of somatic hypermutation. The canonical T15/EO6 VDJ rearrangement has been described as an archetype for primary homology-directed recombination, which more frequently occurs during B-lymphogenesis in the fetal liver. CDR: complementarity-determining region.
To independently confirm the genetic origin of the VH gene expressed by the EO6 cell line, separate RT-PCR reactions were performed using primers specific to unique upstream leader-specific sequences that can discriminate between the V1 gene and the V11/13 functional genes of the S107 family. Amplification was obtained only for the V1-specific primer, and subsequent sequence analysis of the cloned product confirmed the exact nonmutated Ab gene sequence identified in the earlier VH gene cloning studies.
Equivalent studies were also performed with cDNA from three other OxLDL-reactive POVPC-specific cell lines. As with EO6, DNA sequence analysis of the VH and VL genes of the EO3, EO4, and EO7 cell lines indicated that they were each 100% homologous to the classical T15 Ab. Figure 2 shows the deduced amino acid sequences for these Abs; the Ab gene usage for each of these EO cell lines and their binding reactivities (6) are compiled in Table 2.
Reduced amino acid sequence alignment of EO autoantibodies with the classic anti-PC Ab T15. (a) Variable region of Ig light-chain sequences of EO autoantibodies are aligned against Vκ22/Jκ5 gene rearrangement. (b) Variable regions of heavy-chain sequences of EO autoantibodies are aligned against S107.1/DFL16.1/JH1 rearrangement. Both VH and VL show 100% homology to the germline genes of T15.
Summary of gene usage and immunological properties of T15 and autoantibodies from apoE-deficient mice
EO autoantibodies are recognized by T15-specific anti-idiotypes. To characterize the VH and VL regions expressed in the different EO Abs and determine their relationship to the classic T15 Ab, we tested the recognition of these Abs by three well-characterized T15-specific anti-idiotypic Abs. These were T139.2, directed against the unique T15-specific Vκ22-Jκ5 light chain (11); Tc54.6, directed against the unique T15-specific VHS107.1 usage (11, 12); and AB1-2, directed against a conformational determinant requiring both the heavy- and light-chain CDR3 regions of T15 (13). As shown in Table 2, each of the POVPC-specific, anti-OxLDL Abs for which the VH and VL region sequences were determined (EO3, EO4, EO6, and EO7) also expressed the T15 idiotype (i.e., they are recognized by each of three anti-idiotypic Abs). In addition, three other POVPC-specific autoantibodies, EO1, EO2, and EO5 (6), were also found to express the T15 idiotype (data not shown). In contrast, EO12 and EO14, which bind MDA-LDL (3), did not express any of the T15 markers.
Binding of EO6/T15 to OxLDL and PC. T15 is a classic Ab to PC, the “headgroup” moiety [-PO4-(CH2)2-N-(CH3)3] of many phospholipids, including PAPC. Because the genetic and idiotypic analyses indicated that these EO autoantibodies to OxLDL used VH and VL regions identical to those of T15, we systematically compared the antigen-binding properties of T15 (an IgA) with a representative EO Ab, EO6 (an IgM). As expected, EO6 bound to Cu-OxLDL and POVPC-BSA, but not to native LDL or MDA-LDL (6) (Figure 3a). As reported previously, T15 bound to its known antigen, PC, derivatized onto the carrier proteins BSA and KLH (data not shown) (14). Like EO6, T15 also bound to Cu-OxLDL and POVPC-BSA, but not to native LDL or MDA-LDL (Figure 3b). In addition, with reactivity similar to that of T15, EO6 bound to the PC conjugates PC-BSA and PC-KLH. Neither EO6 nor T15 bound to the native, unoxidized phospholipid PAPC (data not shown).
Binding of EO6 and T15 to oxidation-specific epitopes of LDL and PC. The indicated antigens were plated on microtiter wells at the indicated concentrations overnight at 4°C. EO6 (a) or T15 (b) was added at 5 μg/mL followed by the AP-conjugated goat anti-mouse IgM (for EO6) or IgA (for T15) secondary Abs. The amount of bound Abs was expressed as RLU/100 ms. Nat LDL, native LDL.
To further investigate the fine binding specificity of EO6 to PC, a competition immunoassay was performed. Figure 4 shows that the binding of EO6 to Cu-OxLDL was totally inhibited by soluble PC (PC-Cl) and by PC-KLH. In analogous experiments, the binding of T15 to Cu-OxLDL was also inhibited by PC-Cl in a dose-dependent manner (data not shown). In control experiments, the binding of another oxidation-specific IgM monoclonal autoantibody, EO14, to its antigen, MDA-LDL, was unaffected by PC-Cl (data not shown).
Inhibition of EO6 binding to Cu-OxLDL by PC, as sodium salt (PC-Cl) or PC-KLH conjugate. Cu-OxLDL (10 μg/mL) was plated on microtiter wells overnight at 4°C. EO6 (10 μg/mL) was added to wells in the absence or presence of the indicated concentrations of competitors, and the amount of bound EO6 was detected by AP-conjugated goat anti-mouse IgM. The amount of bound EO6 was expressed as the percent of EO6 binding to Cu-OxLDL in the absence of competitor. Inset: Inhibition of EO6 binding to Cu-OxLDL by both EO6 and T15. Cu-OxLDL (10 μg/mL) was coated on microtiter wells overnight at 4°C. Biotinylated EO6 (10 μg/mL) was added to microtiter wells in the absence or presence of the indicated concentrations of competitors. The amount of biotinylated EO6 bound to Cu-OxLDL was then detected by AP-conjugated NeutrAvidin®. The amount of biotinylated EO6 bound to the antigen in the absence of competitor was expressed as a percentage of the control. Nonspecific mouse IgA was used as isotype control for T15.
During the oxidation of LDL, a large number of reactive lipid peroxidation products are generated that can serve as epitopes for Ab recognition. Therefore, we directly tested the ability of T15 to compete with biotinylated EO6 for binding to OxLDL. The inset in Figure 4 demonstrates that T15 competed effectively, whereas a control murine IgA did not. This suggests that these two mAb’s are directed against an immunologically closely related (if not identical) epitope on OxLDL. In addition, we demonstrated that the binding of EO6 to PC-KLH was inhibited by both PC-KLH and Cu-OxLDL in a dose-dependent manner (data not shown). In addition, the demonstration that the anti-idiotype T139.2 competed very effectively with EO6 for binding to PC-KLH provided further confirmation that the binding of EO6 to PC uses the same antigen-binding site used by the classic T15 Ab (data not shown).
EO6 and T15 bind to bacterial C-PS. PC is the immunodominant determinant in the teichoic acid–containing C-PS of Streptococcus pneumoniae (15), and both EO6 and T15 bound to C-PS, whereas EO14 (an MDA-LDL–specific Ab) did not (Figure 5a). Furthermore, the binding of both EO6 and T15 to OxLDL was dose-dependently inhibited by the addition of C-PS (Figure 5b). These data indicate that both EO6 and T15 recognize a common epitope present on OxLDL as well as on C-PS.
Binding of EO6 and T15 to pneumococcal C-PS. (a) C-PS was plated on microtiter wells at the indicated concentrations overnight at 4°C. Each Ab (5 μg/mL) was added to the plate and detected by AP-conjugated secondary Abs (goat anti-mouse IgM for EO6 and EO14, goat anti-mouse IgA for T15). The amount of bound Ab was expressed as RLU/100 ms. (b) Cu-OxLDL or MDA-LDL (10 μg/mL) was plated as antigen, and the indicated Ab was added to microtiter wells in the absence or presence of the indicated concentrations of C-PS. Bound Ab was detected by AP-conjugated secondary Abs (goat anti-mouse IgM for EO6 and EO14, goat anti-mouse IgA for T15), and was expressed as percent control of Ab binding to its antigen without competitor.
T15 Abs recognize apoptotic but not normal endothelial cells. We have previously shown that EO6 bound to apoptotic cells and inhibited their phagocytosis by macrophages (7). To test whether this was a common feature of all T15 Abs regardless of isotype, we examined the capacity of the classic IgA T15 to bind to apoptotic PAECs by flow cytometric analysis. In these studies, serum deprivation was used to induce apoptosis in PAECs. As illustrated in Figure 6, T15 preferentially bound to PAECs that were in advanced stages of apoptosis (Figure 6c), but not to normal cells or those in very early stages of apoptosis (Figure 6b).
T15 IgA recognizes apoptotic cells. FACS® analysis of T15 Ab and control IgA (C-IgA; nonspecific monoclonal) binding to apoptotic PAECs. (a) Apoptosis-induced PAECs were gated into two populations according to PI intensity and FSC as described (7). Region 2 (R2): normal cells and cells in very early apoptosis. Region 1 (R1): cells with dim and bright PI staining (apoptotic). (b) Binding of control IgA and T15 to R2 cells. (c) Binding of control IgA and T15 to R1 cells.
Abs with the T15 idiotype are present in atherosclerotic lesions. We previously demonstrated that EO6 immunostains OxLDL-associated determinants in atherosclerotic lesions from rabbits and humans (3). We have also shown that autoantibodies to OxLDL are present in lesions, in part complexed to OxLDL (16). To determine whether some of the anti-OxLDL Abs deposited in the atherosclerotic lesions of mice consist of the EO6 and/or T15 idiotype, we immunostained advanced lesions from cholesterol-fed LDLR–/– mice for the presence of Abs expressing the T15/EO6 idiotype. Figure 7a demonstrates the marked deposition of Ig in murine lesions, as reported previously (9). In Figure 7b, an adjacent section was stained with the biotinylated AB1-2 anti-T15 idiotype, which yielded the same pattern of staining depicted for total Ig deposition. An adjacent section stained with an equivalent concentration of a biotinylated isotype control did not reveal staining (Figure 7c). In aortic sections from other diseased mice, specific staining with this anti-idiotypic reagent was also demonstrated, although the extent of T15 immunostaining varied in different atherosclerotic lesions (not shown). Because staining with AB1-2 recognizes a T15-specific idiotope that is independent of the associated isotype or isotypes (e.g., IgA, IgM, or IgG), these studies cannot distinguish the isotypes of the deposited T15 idiotype–bearing Ig’s. Nevertheless, these studies document that T15 and EO6 Abs are specifically deposited in lesions of murine atherosclerosis.
Presence of T15 idiotypic Abs in atherosclerotic lesions. Atherosclerotic segments of aorta of LDLR–/– mice were prepared and stained as described in Methods. Epitopes recognized are indicated by a red color; the nuclei are counterstained with methyl green. (a) Section immunostained with a combined anti-mouse IgG/IgM antisera, indicating the presence of Ig in lesion. (b) An adjacent section immunostained with biotinylated AB1-2 (anti-T15 idiotype). A similar pattern demonstrates that some Ig deposited in the lesion represents Abs of the T15 idiotype. (c) An adjacent section stained with biotinylated nonimmune IgG was free of specific staining.
OxLDL- and POVPC-reactive EO cell lines display highly restricted clonal heterogeneity. To evaluate their clonal heterogeneity, a PCR-based strategy was developed to characterize the genomic VH rearrangements in OxLDL- and POVPC-reactive EO cell lines, using conditions wherein nonrearranged genomic templates do not yield an amplimer product (see Methods). In previous studies, we demonstrated that EO6 cells expressed transcripts for the productive T15 VH gene (S107.1-DFL16.1-JH1) and also a nonproductive, frame-shifted S107.13-DSP2.2-JH3 rearrangement. Confirming these findings, amplification of the genomic DNA of EO6 cells yielded two bands, with a larger, 1,500-bp product shown by DNA sequence analysis to represent the productive T15 VH gene and flanking sequence, and a smaller 800-bp product that represents the nonproductive S107 rearrangement on another allele. Significantly, the other OxLDL- or POVPC-specific cell lines, EO3, EO4, and EO7, also yielded only bands of these exact same sizes, which were shown to be identical by sequence analysis and Hin_dIII_ and Bst_NI_ fingerprint patterns (data not shown). Notably, amplification of the genomic DNA of the fusion partner used to create these cell lines yielded only the smaller (800-bp) band, indicating that in the EO cell lines this band was a remnant of the fusion event, and not a marker of the rescued B cells. These data provide compelling evidence that each of these OxLDL- or POVPC-reactive EO hybridomas are genetically identical, likely arising from the same clonal set of B lymphocytes that are specific for an oxidation-associated neodeterminant.