Identification of anti-thrombin antibodies in the antiphospholipid syndrome that interfere with the inactivation of thrombin by antithrombin.

The combined presence of anti-phospholipid (PL) Ab, including lupus anticoagulants (LAC) and/or anticardiolipin Ab (aCL), and thrombosis is recognized as the antiphospholipid syndrome (APS). LAC are detected as an inhibitory effect on PL-restricted in vitro blood coagulation tests, and are comprised mainly of Ab against beta(2) glycoprotein I and prothrombin (PT). Recently, anti-PT Ab (aPT) were found to be associated with thrombosis by some investigators, although this is not confirmed by others. Considering that aPT are heterogeneous in patients and that PT is converted into thrombin, we hypothesize that certain aPT in patients may bind to thrombin, and that some of such anti-thrombin Ab may interfere with thrombin-antithrombin (AT) interaction and thus reduce the AT inactivation of thrombin. To test this hypothesis, we searched for anti-thrombin Ab in APS patients and then studied those found for their effects on the AT inactivation of thrombin. The results revealed that most, but not all, aPT-positive patient plasma samples contained anti-thrombin Ab. To study the functional significance of these Ab, we identified six patient-derived mAb that bound to both PT and thrombin. Of these mAb, three could reduce the AT inactivation of thrombin, whereas others had minimal effect. These findings indicate that some aPT in patients react with thrombin, and that some of such anti-thrombin Ab could inhibit feedback regulation of thrombin. Because the latter anti-thrombin Ab are likely to promote clotting, it will be important to develop specific assays for such Ab and study their roles in thrombosis in APS patients.

Isolation, characterization and sequence analysis of five IgG monoclonal anti-beta 2-glycoprotein-1 and anti-prothrombin antigen-binding fragments generated by phage display.

We have isolated five monoclonal IgG anti-beta 2-glycoprotein-1 (anti-beta 2G-1) and anti-prothrombin Fab from a patient with autoantibodies to oxidized low-density lipoproteins by phage display method. Analysis of their binding specificity revealed that all three beta 2GP-1-enriched mAbs (B14, B22, B27) reacted with beta 2GP-1 while both prothrombin-isolated mAbs (P11 and P13) reacted with prothrombin. Intriguingly, mAb P11 reacted with beta 2GP-1 and prothrombin and showed comparable binding affinity to both Ags, with Kd values of 1.6 x 10-6 M for beta 2GP-1 vs 3.2 x 10-6 M for prothrombin. This clone may thus, define a hitherto unknown shared epitope between beta 2GP-1 and prothrombin. Sequence analysis of all five clones showed significant mutations of the expressed genes. One rearranged V-D-J segment was repeatedly employed by three clones (mAbs B22, B27, and P13). However, all three clones used different L chains. Of note, the pairing of VH6-D-J with the L5-Vk1 L chain in mAb P13 resulted in the loss of binding to beta 2GP-1 and specific reactivity to prothrombin. Together, these data suggest that while the VH6-D-J chain may be important in the binding to beta 2GP-1, pairing with certain L chains may influence this binding. These data are the first human IgG anti-beta 2GP-1 and anti-prothrombin sequences reported; both represent the major subsets of antiphospholipid Abs present in antiphospholipid syndrome patients.

Isolation of an IgG monoclonal anti-dnaJ antibody from an immunoglobulin combinatorial library from a patient with rheumatoid arthritis.

OBJECTIVE: Previously, we showed that rheumatoid arthritis (RA) had both antibodies and T cells specific for the QKRAA-encompassing Escherichia coli dnaJ protein. These findings suggest that the bacteria induced anti-dnaJ responses may cross react with the human homolog of bacterial dnaJ in the joint, resulting in tissue damage. METHODS: We used the combinatorial library technique to isolate and characterize an IgG monoclonal anti-dnaJ antibody (designated CG1) from the blood of a patient with RA. RESULTS: Sequence analysis of CG1 revealed that its heavy and light chain V regions were respectively most homologous to the 3d279d VH4 and the O18 Vk1 genes. Interestingly, 3d279d is frequently expressed by B cells stimulated with staphylococcal enterotoxin; and O18 is the main gene employed by the Vk1 IgG antibodies against Haemophilus influenzae. CONCLUSION: The combinatorial immunoglobulin library method represents an interesting model of how to approach the isolation and characterization of antibody-like reagents in the elucidation of autoantigens in RA.

Immunological and molecular analysis of three monoclonal lupus anticoagulant antibodies from a patient with systemic lupus erythematosus.

Antiphospholipid antibodies (APA), including lupus anticoagulants (LAC; as detected by in vitro blood clotting tests) and anti-cardiolipin antibodies (ACA; as assayed by solid-phase immunoassay), are strongly associated with recurrent thrombosis, thrombocytopenia, and recurrent fetal loss in some patients with systemic lupus erythematosus (SLE). The combined presence of APA and these clinical manifestations is termed antiphospholipid syndrome (APS). LAC and ACA comprise heterogeneous and somewhat overlapping autoantibody subsets. To date, it is unclear what degree of heterogeneity is present in an individual patient and between patients. To begin to address these issues, we generated three monoclonal LAC antibodies from a patient with SLE and APS. These antibodies were studied for their binding specificities and variable (V) region nucleotide sequences. All three LAC were unreactive with DNA, cardiolipin or other phospholipids. Sequence analysis of these antibodies revealed extensive overlap in their Ig V genes with anti-DNA antibodies and other autoantibodies characteristic of lupus. These data provide the first V gene sequence information on a group of SLE-derived LAC without ACA activity, representative of a similar subset of LAC found in patients with APS.

Analysis of homology-directed recombination in VDJ junctions from cytoplasmic Ig- pre-B cells of newborn mice.

We previously showed that most VD and DJ gene combinations from newborn surface Ig- pre-B cells had one to three predominant junctions, all of which occurred at the sites of short sequence homologies between the two coding ends. Because the majority of sequences that are present in pre-B cells are in-frame, however, the possibility existed that the frequency of occurrence of predominant IgH junctions was skewed by proliferation of pre-B cells with productive rearrangements. In this study, we analyzed cytoplasmic Ig- pre-B cells, because these cells should not yet be subject to such selection. Two-thirds of the rearrangements from this population in the adult were out-of-frame, suggesting that these rearrangements are unbiased. In newborn cIg- pre-B cells, DJ junctions still showed the same predominant sequences as sIg- pre-B cells, but there was less use of predominant junctions in VD junctions for three of four different VH genes analyzed. For those three VH genes, an average of 30% of the sequences were in-frame. When only the in-frame rearrangements from these cIg- newborn cells were analyzed, frequencies of predominant VD junctions were comparable to those in sIg- pre-B cells. For sequences using the VHS107/V11 gene, however, 67% of the junctions were created at the site of the same dinucleotide in the V gene, and as a result, 73% of the sequences were in-frame. Thus homology-directed recombination does not initially produce as much junctional homogeneity as anticipated in all VD combinations, although it is a frequently used mechanism in the early fetal/neonatal gene rearrangements.

Influence of the V(D)J recombination mechanism on the formation of the primary T and B cell repertoires.

T and B cells exploit the mechanism of V(D)J recombination to make diverse or very restricted repertoires at varying times during ontogeny. Fetal repertoires are limited since there are no N nucleotides. Also, if short sequence homologies are present near the coding ends, junctions are preferentially made at that site. For gamma delta TCR, and to a lesser extent for Ig, this results in a very homogeneous population of junctions early in ontogeny. alpha beta TCR, however, have a paucity of homologous stretches, and maintain junctional diversity in the newborn. In both newborns and adults, some coding ends show very restricted nucleotide deletion, while others show heterogeneous and extensive deletion. It appears that the sequences of the coding ends have been selected through evolution as a mechanism to control repertoire formation.

The in vivo effects of opioid peptides on the murine immune response.

We have previously reported that met-enkephalin has dual immunomodulatory properties in vitro. We have continued this investigation using an in vivo system. In this study, Alzet miniosmotic pumps were loaded with either met-enkephalin, DTLET or FK 33-824 and were surgically implanted into BAF1/J mice. Twenty-four hours after pump implantation, mice were challenged with sub-optimal, optimal or supraoptimal immunizing doses of antigen. The immune response was assessed 4 or 5 days after primary immunization. FK 33-824, a met-enkephalin analogue, had no effect on the response of mice challenged with a suboptimal antigen dose. However, FK 33-824, at a pump concentration of 10(-3) M, suppressed the response against optimal challenge doses of antigen. At a pump concentration of 10(-8) M, FK 33-824 suppressed, enhanced or had no effect on the supraoptimal antigen dose-induced immune response. The suppressive effect of FK 33-824 in mice immunized with either optimal or supraoptimal doses of antigen was blocked by naloxone. Met-enkephalin and its delta opioid receptor specific analogue, DTLET, had no effect on the immune response to optimal antigen immunization. These results indicate that FK 33-824 has in vivo immunomodulatory activity and provide evidence that opioid peptides may either upregulate or downregulate the in vivo immune response depending on the strength of the response.

Role of homology-directed recombination: predominantly productive rearrangements of Vh81X in newborns but not in adults.

In the neonate, Ig V-D-J junctions often occur at regions of short sequence homology, resulting in one to two predominant junctional sequences for most V-D and D-J recombinations. We have proposed that this mechanism of homology-directed recombination may play a role in the non-random usage of VH genes observed in fetal and neonatal life, since use of the short homologies at V-D junctions would preferentially make productive rearrangements for the overutilized 7183 and Q52 VH genes, and would make predominantly non-productive rearrangements for the underutilized VHJ558 gene family. Here we test this hypothesis for the 81X gene from the VH7183 family. Since pre-B cells which have rearranged the 81X gene do not appear to undergo the normal clonal proliferation before light chain rearrangement, analysis of the percentage of productive versus non-productive rearrangements for this VH gene is not skewed by the expansion of pre-B cells with productively rearranged IgH alleles. If V-D-J rearrangements were random, one would predict that only one-third of the rearrangements would be in-frame. This is close to what we observed for the 81X gene in adult bone marrow. In contrast, we show that 62% of all 81X rearrangements in fetal/newborn pre-B cells were productive. Forty-one percent of all the neonatal pre-B sequences containing DFL16 or DSP2 used homology-directed recombination to create the predominantly observed V-D junctional sequences, and 93% of those sequences were productive. This is consistent with our hypothesis that the mechanism of homology-directed recombination would result in an increased proportion of productive 81X rearrangements in the newborn. Therefore, we suggest that in fetal and neonatal life, when N regions are lacking, VH7183 and VHQ52 genes are more likely to undergo productive rearrangements than other VH families and thus are much more likely to contribute to the early B cell repertoire.