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. 2013 Jul 24;8(7):e69464. doi: 10.1371/journal.pone.0069464

Figure 6. BDC2.5 TscFv, an idiotype structurally different from D9 and S9/P2.

Figure 6

The availability of NOD mice transgenic for the TCR of the islet-autoreactive T-cell clone BDC2.5 provides a means to investigate in vivo effects induced by vaccination and, by generating an idiotype different from D9 and S9/P2, the specificity of the vaccination may be addressed. BDC2.5 TCR was therefore converted into a TscFv. Like the actual BDC2.5 TCR, BDC2.5 TscFv recognized NOD APCs pulsed with chromogranin A epitope 29–42 (24-DTKVMKCVLEVISD-42) (A) and NOD APCs pulsed with ChgA epitope 358–371 (358-WSRMDQLAKELTAE-371 (B). In contrast, NOD APCs pulsed with RegII epitope 48–64 were not recognized (C), thereby confirming that this process retains the idiotype of the original TCR. Staining of unpulsed or ChgA 358–375 or RegII 48–64-pulsed NOD APCs with BDC2.5 TscFv was routinely performed for each newly produced batch. One of the batches was also tested on ChgA 29–42-pulsed NOD APCs (A). Additionally, S9/P2 TscFv was tested on NOD APCs pulsed with ChgA 29–42 and 358–371 and failed to recognize these, whereas APCs pulsed with RegII 48–64 stained positive with S9/P2 (Fig. S3, File S3). To determine if recombinant BDC2.5 TscFv could attenuate the stimulation of BDC2.5 TCR tg T-cells, spleen cells from BDC2.5 TCR tg mice were stimulated with mimotope RTRPLWVRME (3 µg/ml) in the presence of recombinant BDC2.5 or S9/P2 TscFv (10 µg/ml). TscFv BDC2.5, but not S9/P2 attenuated proliferation of BDC2.5 T-cells (D). Three experiments with different batches of BDC2.5 and S9/P2 were performed. The capacity of anti BDC2.5 antiserum to bind BDC2.5 TCR tg cells was assessed using PBMCs collected from BDC2.5 tg mice double-stained with anti CD4-PE and with serum from pre bleed or with anti BDC2.5 or S9/P2 TscFv immune serum. The protocol is depicted in (E) and the cells analyzed were in gate 1 (F). Antiserum from mice vaccinated with BDC2.5 TscFv (G), but not antiserum from mice vaccinated with S9/P2 TscFv (H) preferentially bound CD4+ T-cells from BDC2.5 TCR tg mice. PBMCs from three different BDC2.5 TCR transgenic mice were stained.