Anchored polymerase chain reaction based analysis of the Vβ repertoire in the non-obese diabetic (NOD) mouse

We have performed extensive analyses of T cell receptor Vβ usage in the thymus, the spleen and the infiltrated islets of preclinical non-obese diabetic (NOD) mice. A semiquantitative anchored polymerase chain reaction (An-PCR) protocol has been developed for this purpose. The validity of the method has been first assessed by antibody staining with a panel of anti-Vβ monoclonal antibodies (mAb). The results obtained by An-PCR are accurate, reproducible, and in good agreement with cell surface protein staining. A strict comparison between thymus and spleen repertoires reveals no major Vβ-specific deletion except the already reported Vβ3 deletion due to Mtv-3. Certain Vβ such as Vβ15, 18, 20 are found with a low frequency in the spleen, but the fact that they are also scarce in the thymus probably reflects a poor availability of these genetic elements during β chain rearrangement rather than negative selection. Other Vβ, such as Vβ2, Vβ12 and Vβ14 are significantly more abundant in the spleen than in the thymus. This finding was confirmed by mAb staining for Vβ2 and Vβ14. The expansion asymetrically affects the CD4⁺ subset and can be traced back to the mature, single-positive thymocyte subset, suggesting an intrathymic positive selection event. Vβ repertoires in infiltrated islets of 13- and 18-week-old, non-diabetic mice are polymorphic. Practically all the Vβ found in the peripheral lymphoid tissues are present in the islets, in similar proportions. The major exception is Vβ12, one of the Vβ which is subject to expansion during intrathymic differentiation and which is further augmented in the islets, both at 13 and 18 weeks. This increase probably reflects further peripheral amplification of the Vβ12-bearing subset due to encounter with the same ligand as in the thymus or with a cross-reactive motif. Finally, the nucleotide sequencing of all the Vβ segments in usage in the NOD strain confirms the absence of allelic polymorphism of Vβ-coding regions.