Vaccination with Single Chain Antigen Receptors for Islet-Derived Peptides Presented on I-A^g7 Delays Diabetes in NOD Mice by Inducing Anergy in Self-Reactive T-Cells

<div><p>To develop a vaccination approach for prevention of type 1 diabetes (T1D) that selectively attenuates self-reactive T-cells targeting specific autoantigens, we selected phage-displayed single chain antigen receptor libraries for clones binding to a complex of the NOD classII MHC I-A^g7 and epitopes derived from the islet autoantigen RegII. Libraries were generated from B-cell receptor repertoires of classII-mismatched mice immunized with RegII-pulsed NOD antigen presenting cells or from T-cell receptor repertoires in pancreatic lymph nodes of NOD mice. Both approaches yielded clones recognizing a RegII-derived epitope in the context of I-A^g7, which activated autoreactive CD4⁺ T-cells. A receptor with different specificity was obtained by converting the BDC2.5 TCR into single chain form. B- but not T-cells from donors vaccinated with the clones transferred protection from diabetes to NOD-SCID recipients if the specificity of the diabetes inducer cell and the single chain receptor were matched. B-cells and antibodies from donors vaccinated with the BDC2.5 single chain receptor induced a state of profound anergy in T-cells of BDC2.5 TCR transgenic NOD recipients while B-cells from donors vaccinated with a single chain receptor specific for I-A^g7 RegII peptide complexes induced only partial non-responsiveness. Vaccination of normal NOD mice with receptors recognizing I-A^g7 RegII peptide complexes or with the BDC2.5 single chain receptor delayed onset of T1D. Thus anti-idiotypic vaccination can be successfully applied to T1D with vaccines either generated from self-reactive T-cell clones or derived from antigen receptor libraries.</p></div

Adoptive transfer of type 1 diabetes to NOD-SCID recipients by diabetogenic CD4⁺ T-cells (inducer cells) can be prevented by B- but not T-cells (test cells) from donors immunized with scFvs if the specificity of the scFv and the inducer cell are matched.

<p>Test cells were obtained from NOD mice immunized with the indicated scFvs at 4 and 7 weeks of age (experiment A and C) or at 8 and 11 weeks of age (experiment B). NtfrRII specific inducer cells were either obtained by depletion of B and CD8⁺ T-cells (A and C) or by negative isolation of CD4⁺ T-cells from NtfrRII-immunized mice (B). BDC2.5 inducer cells were obtained by negative isolation of CD4⁺ T-cells (B) or were non-separated spleen cells of BDC2.5 TCR tg mice (C). All mice in experimental groups with diabetes induced by NtfrRII specific T-cells had developed the disease by 82 days post transfer. Mice in groups free of diabetes were observed to 200 days post transfer. All mice in experimental groups with diabetes induced by BDC2.5 T-cells had developed the disease by 37 days post transfer. Mice in groups free of diabetes were observed to 100 days post transfer. B-cells were necessary to mediate prevention of disease and, for prevention to occur in this model, the specificity of the inducer cells has to match the specificity of the scFv used to vaccinate test cell donors.</p

Autoreactive CD4⁺ T-cells and BscFv D9 recognize the same I-A^g7 NtfrRII peptide complex.

<p>Four groups of NOD mice were immunized s.c. with the four peptides of set L in alum, one peptide for each group of mice. Set L covers NtfrII and by applying the procedure shown for peptide 1 (A) to the remaining peptides in set L the ability of each peptide to activate NtfrRII specific CD4⁺ islet self-reactive T-cells could be assessed. Set L Peptide 1 (P1) 22-GQVAEEDFPLAEKDLPSAKINC-43 Peptide 2 (P2) 34-KDLPSAKINCPEGANAYGSY-53 Peptide 3 (P3) 44-PEGANAYGSYCYYLIEDRLT-63 Peptide 4 (P4) 59-EDRLTWGEADLFCQNMN-75 This was accomplished by transferring CD4⁺ T-cells from the vaccinated NOD donor mice to NOD-SCID recipients and then - four weeks after transfer - studying the islet histology in the NOD-SCID recipients. Few infiltrated islets existed in recipients of CD4⁺ T-cells derived from P1- and P2-vaccinated donors. In contrast, as shown in B, extensive infiltration with islet destruction was observed in recipients of CD4⁺ T-cells from P3- and P4-vaccinated donors (scale bar: 50 µm). For each peptide a total of 60–80 islets from pancreata of three recipients were assessed for islet inflammation (C) (white bars: no infiltration; vertically striped bars: peri islet infiltration; horizontally striped bars: intra islet infiltration; black bars: islet destruction). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069464#pone-0069464-g003" target="_blank">Figure 3D</a> demonstrates that the region of NtfrRII covered by peptide pool 7 of set S (RegII 48–64, as indicated in red), which was preferentially bound by BscFv D9 when presented in the context of I-A^g7, overlaps to a large extent with peptide 3 of set L (RegII 44–63). Since peptide 3 specific T-cells infiltrate and destroy islets it is highly likely that RegII specific autoaggressive CD4⁺ T-cells exist in the NOD mouse that recognize the same I-A^g7 RegII peptide complex as does BscFv D9. To test this possibility further we examined if recombinant D9 could attenuate the proliferation of CD4⁺ T-cells from peptide 3 set L-vaccinated NOD mice. For this purpose recombinant clone D9 (10 µg/ml) was added to a proliferation assay of spleen cells depleted of CD8⁺ T-cells from peptide 3 set L-vaccinated NOD mice (E). A second BscFv clone, termed C8 that suppressed proliferation of NtfrRII specific CD4⁺ T-cells was tested with a T-cell assay similar to that used to test D9. However, in this case donors were vaccinated with the full-length NtfrRII given that the epitope recognized by C8 in the context of I-A^g7 had not been mapped (F). To determine whether the attenuation of proliferation was antigen specific we used a previously established CD4⁺ T-cell line, which recognizes peptide 35 of the autoantigen glutamic acid decarboxylase (GAD) in the context of I-A^g7. GAD peptide 35 (524-SRLSKVAPVIKARMMEYGTT-543) is one of several epitopes identified in this protein that can activate NOD self-reactive CD4⁺ T-cells. GAD peptide 35 presented in the context of I-A^g7 should not be recognized by either C8 or D9, which are idiotypes binding to RegII (NtfrRII)-derived epitopes presented in the context of I-A^g7. Neither C8 nor D9 attenuated proliferation of the GAD peptide 35 specific T-cell line (G). Statistical evaluation was performed by t-test. Representative graphs are shown in E, F and G; three independent experiments were performed. For T-cell proliferation assays in this report triplicates were counted.</p

TscFv S9/P2, an idiotype structurally similar to that of BscFv D9.

<p>Given that D9 (a BscFv) is an idiotype with known specificity the aim was to isolate another idiotype structurally similar to D9, but different in its primary sequence. To achieve this aim a TscFv library was generated and selected by applying two different selection methods: 1) precipitation of the TscFv library with anti D9 antiserum that had been adsorbed with D9mut to reduce the presence of antibodies recognizing linear epitopes and 2) selection of the TscFv library on NtfrRII-pulsed NOD APCs. The criterion to screen the two resulting selected libraries P and S (same clone isolated by both selection methods) was chosen to yield an idiotype that recognized the same pMHC complex as D9. Spleen cells of NOD mice were pulsed with NtfrRII and stained with TscFv S9/P2 (A) or pulsed with NtfrRII peptide pools of set S (B). As with D9, spleen cells pulsed with peptide pools outside the region 44–68 of RegII did not stain with S9/P2. Given that BscFv D9 and TscFv S9/P2 represent two idiotypes with different primary sequences, but recognizing the same pMHC complex, we assessed their structural relatedness. A direct comparison of the CDR3 loops of D9 (green) with S9/P2 (blue) revealed structural overlap despite significant differences on the primary sequence level (Table S10 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069464#pone.0069464.s004" target="_blank">File S4</a>). The structural overlap was most notable for the β-chain of TscFv S9/P2 and the heavy chain of BscFv D9 (C). In contrast, a comparison of the CDR3 loops of TscFv S9/P2 (blue) and TscFv BDC2.5 (orange), which recognize different epitopes in the context of I-A^g7, showed marked differences (D). The structural homology between BscFv D9 and TscFv S9/P2 extended beyond the CDR3 regions to encompass the entire molecule (idiotype) as shown in (E) and as evidenced by the template modeling (TM) scores for this alignment, which are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069464#pone-0069464-t001" target="_blank">Table 1</a>. In keeping with this observation, anti D9 serum was able to precipitate both BscFv D9 and TscFv S9/P2, anti D9mut serum precipitated D9, but only marginally TscFv S9/P2 (F). Lysates of bacteria expressing D9 or S9/P2 were precipitated with sera from anti D9 or anti D9mut-immunized mice. Precipitates were captured by immobilized protein A/G, were run in adjacent lanes of an SDS-PAGE gel and blotted to nitrocellulose. Bands were detected with an anti c-myc, HRP-labeled antibody. The bands corresponding to the scFvs run at an apparent molecular weight of ∼ 29 kD. The experiment was repeated once yielding the same result.</p

Template modeling scores [TM] and root mean square deviation scores (RMSD) for the comparison of structural similarities between scFvs.

<p>The higher the template modeling score [TM] and the lower the root mean square deviation score (RMSD) the more closely two structures are related. Comparison of two identical structures (D9 and S9/P2 each with itself) yields 1 for the TM score and 0 for the RMSD score. The TM score weighting algorithm is less sensitive to distant than to close matches and is therefore a more sensitive measure for structural similarity than the RMSD score <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069464#pone.0069464-Zhang3" target="_blank">[46]</a>.</p