1 Neuroscience Group, Weatherall Institute for Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK 2 National Institute of Arthritis, Musculoskeletal and Skin Diseases, NIH, Bethesda, MD 20892, USA 3 Immunobiology, National Institute of Biological Standards and Control, Potters Bar, Hertfordshire EN6 3QG, UK 4 Present address: Department of Thoracic Surgery, Osaka University Graduate School of Medicine,E12-2 Yamadaoka, Suita, Osaka 565-0871, Japan 5 Present address: FIRS laboratories, Llanishen, Cardiff CF14 5DU, UK 6 Present address: Department of Clinical Neurology, Peoples Hospital, Laizhou, 261400, Shandong, China
The first two authors contributed equally to this workCorrespondence to: N. Willcox; E-mail: nick.willcox{at}imm.ox.ac.uk
Transmitting editor: D. T. Fearon
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Abstract |
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Keywords: anti-cytokine autoantibody, dendritic cell, human anti-IFN- antibody, human anti-IL-12 antibody, paraneoplastic autoimmunity, tumor immunity
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Introduction |
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Paraneoplastic autoimmunity is more common in patients with thymomas (25). These are epithelial neoplasms (4,5) found in 10% of myasthenia gravis (MG) patients and can usually be removed surgically. They often resemble disorganized thymic cortex (5,6), typically generating abundant maturing thymocytes (6,7) and exporting many of their progeny to the periphery (7,8). Around 30% of all thymoma patients develop MG (25). In addition to the characteristic pathogenic autoantibodies to the muscle ACh receptor (AChR), they usually have others to various intracellular muscle proteins, including actin, myosin (9,10) and especially titin (11), although their pathogenicity is debatable. The association between MG and thymomas has long been a puzzle. It is not even clear whether they actively autoimmunize Th cells against muscle epitopes (3,5,8) or merely fail to tolerize the T cells they export against self-antigens (12).
The anti-AChR antibodies in MG almost exclusively recognize the extracellular AChR conformation (13,14). Since B cells are rare and intact AChR undetectable in thymomas (15), we suggested that only Th are primed there against linear AChR epitopes (3) by professional antigen-presenting cells (APC) (3,16); also that AChR-specific B cells are subsequently activated in the periphery, perhaps explaining the occasional onset of MG long after a thymoma has been removed (3,10,14).
By contrast, in early-onset MG (EOMG) patients, lymph node-like infiltrates are regularly seen in the thymic medulla (3,4,14). They resemble those seen in the target organs in other autoimmune diseases (1719) and are probably provoked by the muscle-like myoid cells (20) that express AChR (21,22). The co-localizing germinal centers and the T cell areas contain terminally differentiated (radio-resistant) plasma cells (23) that spontaneously synthesize anti-AChR (24,25) and are a rich source for cloning combinatorial antibodies (26,27). Thus, B cells reacting to AChR have clearly been selectively pre-activated in vivo, whereas those specific for extrinsic antigens produced antibodies only in the presence of pokeweed mitogen (PWM) (25,28), a T cell-dependent B cell stimulant that concomitantly reduces anti-AChR production by plasma cells (22,24).
Such lymph node-type infiltrates are often seen in the uninvolved thymic remnant adjacent to an MG patients thymoma (4), whereas, in the thymoma itself, myoid cells are rare (4,15) and infiltrates/anti-AChR synthesis are uncommon/undetectable (46,29).
Unexpectedly, high-titer neutralizing autoantibodies are found against IFN- in >70% of MG/thymoma cases at diagnosis and against IL-12 in
50% (3032). These autoantibodies show an intriguingly close relationship with these tumors, also occurring in
30% of thymoma cases without MG (31). Moreover, they often increase when thymomas recur (3032), suggesting more direct autoimmunization in thymomas against the cytokines than against AChR.
Seeking footprints of such in situ autoimmunization, we have compared production of autoantibodies against AChR, titin, IFN- and IL-12 by cultured thymoma cells, and cloned combinatorial Fabs to check the specificity and clonal origins of the plasma cells detected.
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Methods |
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Autoantibody assays
The radio-immunoassays (RIA) for serum antibodies used [125I]-bungarotoxin (
-BuTx)human AChR (2427), or [125I]IFN-
, [125I]IL-12p70 or [125I]titin (30-kDa recombinant fragment; DLD Diagnostika, Hamburg) (32). Culture supernatants (75 µl) were incubated overnight at 20°C with the labeled antigen (at least 5000 c.p.m.) before immunoprecipitation (plus normal serum as carrier) and meticulous washing [to ensure backgrounds below
300 c.p.m. (24)]. With Fabs, we used an anti-human Fab as the precipitating antibody (26,27). Results were expressed as the percentage of the available c.p.m. precipitated (after subtraction of the background with cycloheximide).
For Western blotting, 20 µg of IFN- plus 20 µg BSA was electrophoresed in acrylamide gels and blotted onto nitrocellulose strips, which were blocked overnight in 4% milk powder and then incubated in the various Fab or serum samples (all at 1:200 in 4% milk powder) for 1 h at 20°C. After extensive washing, the strips were probed with horseradish peroxidase-conjugated anti-Fab or anti-IgG antibodies (Dako, Ely, UK) at 1:10,000 or 1:2000 and processed for enhanced chemiluminescence (ECL; Amersham Pharmacia, Little Chalfont, UK).
Neutralization of the anti-viral activity of human IFN- was assayed as described elsewhere (31). In brief, dilutions of Fabs or sera were pre-incubated with 25 Laboratory Units of each IFN-
preparation at 37°C for 1 h, before incubation with 2D9 cells for 24 h at 37°C. The antibody/IFN-
mixture was then replaced with a challenge dose of encephalomyocarditis virus; after 24 h, the wells were stained with amido blue black and absorbance read at 620 nm.
Combinatorial Fabs
Cases 11 and 23 were selected because of their high serum titers against IFN-, IL-12, AChR and titin. Before starting the work shown in Fig. 1, we had already noted strong spontaneous anti-AChR production by cells from case 23s remnant (but not his thymoma), so mRNA was isolated from frozen remnant cells; with case 11, we had already pooled equal numbers of cells from thymoma and remnant, thus using all the remnant cells to prepare RNA before any were cultured.
We prepared cDNA, amplified VHCH1 and VC
segments, and co-ligated them to yield combinatorial libraries, which we then expressed in
phage, as previously described (26,27). From each unamplified combinatorial library, we screened
2.5 x 105 plaques with 125I-iodinated IFN-
2, IL-12, titin or
-BuTx-labeled fetal AChR on colony lifts (see Fig. 2a), after optimizing conditions on dot-blots with the donors sera. Positive plaques were picked, cloned to homogeneity, and rescued into phagemid for sequencing and expression (26,27), as were several control Fabs that were negative in all screens. The GenBank accession numbers of the positive Fabs are AY173081AY173112.
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Results |
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For both thymomas and thymic remnants, most of the anti-AChR antibody producers (with and without PWM) had been pre-treated with corticosteroids (Fig. 1a and b, solid symbols), which preferentially deplete immature thymocytes, and enrich B and plasma cells (6). In the producers, serum anti-AChR titers were sometimes modest. As expected (6,10,22,29), there were many non-producers despite high serum titers, including all peripheral blood lymphocyte samples (Table 2).
Production in culture of antibodies against titin
We found minimal anti-titin antibody production in 14 of 15 of the MG thymoma cultures and in all nine remnants tested (despite high serum titers in six cases). It reached 22% only with one thymoma (with PWM; case 8, who had low/medium serum titers against titin, IFN- and IL-12). It was marginal in three other thymomas and three remnants (36.5%), again only with PWM (not shown).
Spontaneous anti-IFN- and anti-IL-12 antibody production by thymoma cells
In surprising contrast with anti-AChR, we noted striking spontaneous antibody production against both cytokines in thymoma as well as remnant cultures (Fig. 1c and d; see Supplementary Data, available at International Immunology online); its frequent inhibition by PWM again implicates plasma cells (22,24). Furthermore, spontaneous anti-IFN- production was seen in significantly more seropositive cases than for anti-AChR (Table 2, footnote a), including both recurrences (cases 14 and 15). The broadly similar binding levels and prevalences of antibodies against IFN-
and AChR in cultures from thymic remnants (and from thymomas with PWM) indicate comparable assay sensitivities.
In several cases (e.g. case 4), thymoma cells spontaneously produced much more anti-IFN- (and anti-IL-12; Supplementary Data) than those from the remnants (Fig. 1c and d), whereas anti-AChR production was usually lower by both. Moreover, the anti-IFN-
production was substantially radioresistant, confirming the contribution by plasma cells in one thymoma (broken lines in Fig. 1c); even though very few viable cells remained in these cultures by day 20, their anti-IFN-
levels were still well above background. In contrast, in peripheral blood lymphocytes, we found resting memory cells only (Fig. 1e and Supplementary Data).
No remnant was found in four elderly/recurrent cases. Eight of the other 13 remnants produced anti-IFN- spontaneously, some again showing plasma cell behavior (Fig. 1d and Table 2). Importantly, not all the producers were taking corticosteroids (open symbols in Fig. 1 and Supplementary Data); when not, total cell yields were 4- to 10-fold higher from thymomas than remnants and thus their total antibody productivities were
20-fold greater (not shown).
In general, results were very similar for anti-IL-12p70 (Supplementary Data), although fewer cases were positive (Table 2). Again, autoantibody production in culture was detected only in cases seropositive against IL-12, supporting its specificity, as with the other antigens (Table 2).
Notably, two thymomas (thymomas 6 and 7) gave surprising PWM responses against IFN- despite low serum titers, as noted previously for anti-AChR in a patient whose serum titer later rose
100-fold (10); similarly, one remnant (remnant 9) spontaneously produced anti-IL-12 unexpectedly well. All three had been pre-treated with corticosteroids.
Specificities of combinatorial Fabs in RIA, Western blotting and neutralization assays
We cloned Fabs from thymus/thymoma cells from two patients with high serum titers against all four antigens. VHCH1 and VC
genes were recombined from cDNA libraries and expressed in
phage (26,27). Even after careful screening of
2.5 x 105
phage plaques from each of these unamplified combinatorial libraries, none proved to be specific for IL-12 or titin and only one for AChR (D anti-AChR), despite both donors high serum titers. In contrast, the same screening had yielded >20 distinct AChR-specific Fabs from three EOMG thymi (26,27).
Screening similarly with [125I]IFN- (Fig. 2a), we isolated nine specific Fabs from the thymoma-plus-remnant of case 11 (M1M14) and six from 23s remnant (D1D6) (Fig. 2 and Table 3). All 15 Fabs bound IFN-
significantly in RIA, but each immunoprecipitated only 1570% of the available [125I]IFN-
, even with saturating amounts of Fab (Fig. 2b and Table 3); evidently, they recognized only a subset of the labeled IFN-
molecules. Interestingly, however, both donors sera precipitated >90% of the total IFN-
. So did each donors Fabs when pooled; collectively, therefore, they represent the full range of reactivity. The completely negative RIA results with all of these Fabs against [125I]
-BuTxAChR confirm their IFN-
specificity (not shown).
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We tested four Fabs for neutralization of the anti-viral activity of the 12 human IFN- subtypes. The donors sera had high titers against all subtypes, whereas the Fabs titers were far lower and their profiles much more restricted (Fig. 3). Surprisingly, although originally screened against IFN-
2, Fabs M4, D4 and D5 neutralized IFN-
1 better, whereas M13 neutralized only IFN-
2, 4, 5 and 21 (Fig. 3). When high- and medium- or low-titer Fabs were paired, we saw no sign of either synergy or interference in neutralization of IFN-
1, 2, 4 or 21 (not shown).
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Seven of the IFN--specific Fabsderived from either donorhad the commonly used VH3-23, paired in six of them with the very prevalent V
O2/12 gene, both being highly mutated in each Fab (Table 3, upper). This combination was seen in five of the six Fabs that bound IFN-
most strongly, four of which derived from patient 23s thymic remnant (D). The V
s in these four were clearly related, with identical CDR3s and six shared coding mutations (d in Table 3), plus another three to five unique ones. Their VHs form two pairs, each again having identical CDR3s and 1115 shared coding mutations (b and c in Table 3) plus two to five unique ones. These results clearly indicate ongoing antigen-driven clonal evolution in this patients thymic remnant.
A fifth strongly binding Fab (M4), from the other patient (patient 11), showed intriguing resemblances; all five share not only a Lys72 Asn replacement but also CDR3s with identical lengths and very similar amino acid compositions despite using different DHJH genes (Table 3, lower). Moreover, in their V
CDR1s, M4 has a Ser31
Asn substitution, while the others have the same change at position 30.
Interestingly, patient 11s Fabs included another closely related pair of VH3-23 sequences (M2 and M12), with four shared (e in Table 3) and two or three unique mutations; they had an identical 11-amino-acid insert in their CDR2 regions, lengthening it to 28 residues, that might result from duplication and mutation of part of the CDR2. They were weak binders, although M2 also used a mutated 02/12 V.
Of the remaining eight Fabs, only M13 bound and neutralized strongly; five of them used VH4 and three used VH1 genes. The frequently recurring V 02/12 showed very variable numbers of mutations that did not correlate clearly with binding (as in M12 and M13); however, it included numerous coding substitutions in the one anti-AChR Fab of case 23, as did the VH3-48.
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Discussion |
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As reviewed in the Introduction, there is very similar pre-activation of a mature plasma cell response against AChR in the hyperplastic EOMG thymus and it correlates well with the presence of native AChR-expressing myoid cells. The closely analogous responses to the cytokines shown here likewise demonstrate prior activation, but now in the thymoma itself. By extension, they strongly imply (i) the presence of native IFN- and/or IL-12 molecules recognizable by specific B cells, and thus (ii) that some cell type(s) are actively autoimmunizing against these cytokines in both primary and recurrent thymomas. Therefore, (iii) thymomas constitute a dangerous autoimmunogenic microenvironment and do not simply fail to tolerize the T cells they generate. Not only can these antibodies affect Th1 polarization (33), they must also hold unique clues to autoimmunizing cell types in paraneoplastic MG (see below).
The resulting responses against these cytokines may well involve the germinal centers noted within 20% of MG thymomas (4,6), which could readily explain the ongoing clonal diversification evident in some of the M Fabs. The contrastingly minimal spontaneous production of anti-AChR fits well with the absence of the native receptor and myoid cells in thymomas (15), and with the proposed priming there only of Th against linear AChR epitopes (3,16,20).
The autoantibodies produced
Our success in isolating combinatorial Fabs correlates well with the easier detection of plasma cells specific for IFN- than the other three antigens (Fig. 1 and Table 2). It also confirms the apparent specificity of the antibodies produced in culture (Fig. 1 and Supplementary Data), as the Fabs showed no detectable cross-reactivity between IFN-
and AChR. Moreover, they must have high affinities for the native molecules; even as monomers, they bound these antigens at low molarities (<109 M) and neutralized the anti-viral activity of some IFN-
subtypes (Fig. 3). Their preference for native IFN-
is shown by the stronger binding by several Fabs in RIA than in Western blots (Table 3, lower).
When pooled, the IFN--specific Fabs appeared representative of the repertoire in these donors sera. However, each Fab bound only a subset of the labeled IFN-
, or neutralized only certain subtypes, implying recognition of distinct epitopes, perhaps differing in their sensitivity to iodination or to neutralization. [Differences in binding kinetics are unlikely because of the almost constant binding by each Fab over
10-fold concentration range (Fig. 2)]. These samples are clearly a valuable source for cloning a range of human anti-IFN-
and anti-IL-12 antibodies, especially in view of their specificity for the native molecules, and of the available mix of memory B cells and pre-activated plasma cells. That might help to define IFN-
epitopes in more detail.
The VH3-23 and V 02/12 genes are widely used [e.g. (23,26,27)]; their co-occurrence in both the present cases, especially in five of their six strongest-binding Fabs, suggests that they may be natural partners in situ. The clear evidence of ongoing clonal evolutionin both VH and V
in case 23s thymic remnant strongly implies an antigen-driven response in the germinal centers previously noted in this sample (6). With the other donor, the evidence is less compelling because of the greater diversity among his (M) Fabs, which include one clonally related pair. They could well derive from his thymoma, which strongly produced anti-IFN-
in culture (Fig. 1c; 11); interestingly, four of the five unmutated V
sequences are from this case, suggesting a higher proportion of naive or recently primed clones.
Clues to potential autoimmunizing cell types in thymomas
Our findings predict an autoimmunizing cell type in thymomas that is (i) potently immunogenic, (ii) expresses IL-12 and/or a broad range of IFN- subtypes in their native form, and (iii) can activate both Th and B cells, (iv) apparently leading to antigen-driven antibody diversification.
Normally, after appropriate stimulation, both IFN- and IL-12 are produced mainly by dendritic cell (DC) subsets (3436). In the thymus, DC, NK cells and thymocytes can all be generated from a common progenitor (37). As thymomas often generate thymocytes in vast excess (6,7), there could be parallel aberrations in DC differentiation or behavior. Moreover, since certain DC can also prime naive B cells (38,39), DC subsets in thymomas seem particularly plausible autosensitizing agents. They are further implicated both by the coincident responses to IL-12p70and its inducible p40 chainas well as to IFN-
in its native form, and by the much more broadly reactive antibodies to most IFN-
subtypes than seen after IFN-
therapy (A. Meager et al., in preparation). By contrast, MG/thymoma cases rarely have autoantibodies against IFN-ß (31), which is mainly produced by fibroblasts. The neoplastic epithelial cells may well contribute too, although their APC activity is weak (40); in addition, titin is expressed by thymoma epithelial cells (rather than DC) (41), but we failed to detect spontaneous antibody production against it. Furthermore, since the patients anti-cytokine autoantibodies do not obviously correlate with the very variable thymoma histology (3032), the DC there may be a more plausible common factor, as we are now investigating. In view of their hemopoietic origins, they also seem likelier candidates for maintaining these responses (often for years) after thymomectomy (31), and possibly even for provoking the autoimmune bone marrow aplasias that associate with
5% of thymomas (25). Finally, IFN-
DC interactions have recently also been implicated in immunopathogenesis in systemic lupus erythematosus (42).
With the AChR, many groups have reported expression of isolated subunits by thymic epithelial cells, whether in normal thymus (43), in EOMG hyperplasia (44) or in thymomas (45,46), but the intact receptor has not been detected in thymomas (15). We have therefore hypothesized that linear AChR epitopes from the hyperplastic or neoplastic epithelial cells initially autoimmunize Th (perhaps with help from DC), whereas the eventual priming of B cells against the native AChR conformation depends on thymic myoid cells in EOMG (20) and possibly also in thymic remnants (see below). With IFN- and IL-12, by contrast, we now propose that both Th and B cells could be primed in the thymoma against these simpler molecules by a cell type such as DC. These suggestions can readily explain the observed pattern of prior activation in the thymomas of plasma cell responses to IFN-
and/or IL-12, but not to AChR. Identifying this cell type may give valuable clues to Th autoimmunization against AChR and possibly also to the even more puzzling mechanisms in some late-onset MG patients who show similar serology with no thymic tumor or hyperplasia, but only atrophy.
Contributions from thymic remnants versus thymomas
Whereas the response patterns in thymic remnants were broadly similar to AChR, IFN- and IL-12, spontaneous antibody production was much more striking in the thymomas against the cytokines than against AChR (Fig. 1a and c and Table 2); even when AChR-specific B cells were present there, they showed little sign of prior activation (Fig. 1a) (10,29). Several other findings also implicate the thymomas in the responses to IFN-
and IL-12 even more deeply than the remnantswhere total antibody productivity was often much lower. In general, these serum antibodies co-occur particularly with thymomas, above all after their recurrence (3032); by then, there is usually no thymic remnantas must also apply in many elderly cases with primary thymomas. In contrast, these antibodies are not found in EOMG, where the thymus is even more consistently hyperplastic than in the remnants.
The prior activation of similar responses in the remnants might reflect either migration of DC from thymomas or the trapping of circulating IFN- or IL-12 in germinal centers after forming complexes with autoantibodies, just as occurs normally with many extraneous antigens (47). The parallel responses in remnants to AChR are probably provoked by the AChR+ myoid cells, as in EOMG (20); interestingly, the one AChR-specific Fab was derived from a remnant and preferred the fetal AChR isoform that is expressed by thymic myoid cells (21). We suspect that B cells must also be primed elsewhereespecially in elderly MG/thymoma cases with no remaining thymus or myoid cells. If they are mainly induced in muscle lymphorrhages, that could explain the minimal anti-titin responses in thymomas. Alternatively, that antibody assay may merely be less sensitive.
Recirculating memory B cells in thymomas
Previously, anti-AChR production was noted only with cells from remnants (6,22,29). The unexpected responses here (with PWM) to AChR in occasional thymomas might paradoxically reflect the more frequent pre-treatment with corticosteroids (13 of 24 cases) and so might the surprisingly high productivity of anti-cytokine antibodies despite low serum titers in some cases. As well as enriching such rare cell types as B and plasma cells (6), this treatment seems to reduce serum titers against AChR more than against the cytokines (31,32) and so may again favor cytokine-specific plasma cells.
Resting but PWM-responsive B cells specific for IFN- or IL-12 were detected in some peripheral blood lymphocyte samples, as noted previously for AChR in cases with high serum titers (unpublished). However, we have never seen significant spontaneous antibody production by peripheral blood lymphocytes, consistently with the general rarity of circulating plasma cells (48). Interestingly, one of the four thymomas with memory B cell responses to AChR (patient 8; corticosteroid pre-treated) also gave the only clear response to titin (22%, again with PWM, not shown); the same sample had previously also shown a very striking Th response to tuberculin (49). That indicates immigration of memory cells from the periphery, possibly into the perivascular spaces so characteristic of thymomas in MG (4,5), in which B cell clusters have also been observed (16). If they were very receptive to activated cells, that could explain their anti-AChR (Fig. 1a) and anti-striational muscle antigen responses (9,10).
In conclusion, our results suggest active autoimmunization against IFN- and/or IL-12 in a dangerous thymoma microenvironment, and offer novel clues to the autoimmunizing cell type(s), which may be relevant in other MG subsets or other diseases.
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Acknowledgements |
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Abbreviations |
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AChacetylcholine
AChRacetylcholine receptor
APCantigen-presenting cell(s)
DCdendritic cell
EOMGearly-onset myasthenia gravis
MGmyasthenia gravis
PWMpokeweed mitogen
RIAradioimmunoassay
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References |
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