Figure 3

Semi-invariant T-cell receptor (TCR) Vβ8:Vα14-Jα18 is preferentially used by T cells in Irga6 liver cored patches. (A) TCR Vβ8 is used preferentially in Irga6 liver cored patches. TCR Vβ sequences were amplified by nested-real-time polymerase chain reaction (RT PCR) and sequenced from microdissected cored patches as described (Methods). Lymph node cells (LN) served as control. In total 68 and 132 Vβ clones for liver cored patch (CP) and lymph node (LN) T cells were identified, respectively. The histogram shows the distribution of sequenced clones among Vβ subfamilies as % of total (error bars indicate standard deviations). (B) T cells in Irga6 liver cored patches express TCR Vβ8. Consecutive cryo-sections of C57BL/6 adult mice liver were probed for Irga6 protein (1, green) and for TCR Vβ8 protein (2, blue), respectively; frames show enlarged images. Arrows indicate T cells expressing Vβ8 TCR. Microscope magnification is 200x. (C) TCR Vβ8 chains in liver cored patches have variable junctional regions, shown in alignment of TCR Vβ8.2 junctions from (A) above. Junction sequences for all Vβ8 clones from liver cored patches are given in Additional file 5: Table S3. (D) TCR Vα14 dominates Vα usage in Irga6 liver cored patches. TCR Vα usage was assessed by amplifying Vα14, 2, 8 and 17 individually from cDNA of liver cored patches or lymph node with nested-RT PCR (see Methods section: upper panel). GAPDH served as control. RT-PCR products of Vα8 and Vα14 were cloned and sequenced. One hundred per cent of Vα8 PCR products from LN T cell were Vα8, whereas 100% of the Vα8 products from liver CP were non-specific. For liver CP, 70 out of 72 clones from the Va14 PCR were Vα14; two clones were Vα11. For LN all 40 identified Vα14 clones were Vα11. The histograms (lower panel) show the corrected distribution of Vα8 and Vα14/11 usage. (E) Alignment of representative TCR Vα14/Ja18 junction regions found in liver cored patches. For full junction information on all Vα14 and Vα11 clones see Additional file 5: Table S3.