High-frequency clonotypes in mice with GVHD-associated autoimmune colitis. (A) Experimental approach used to examine the development of GVHD-associated autoimmunity. Lethally irradiated (900 cGy) Balb/c mice were transplanted with C57BL/6 (B6) BM (10 × 10⁶) and 0.4 × 10⁶ spleen cells to induce moderate GVHD. Animals were then euthanized 19-21 days after transplantation and pooled spleen cell suspensions from fully donor-engrafted mice adjusted to yield a T-cell dose of 0.5 × 10⁶ were injected intravenously into nonirradiated B6 Rag animals. Mice were typically analyzed 60-70 days after transfer. (B) Serial weight curve after transfer of spleen cells from primary GVHD mice (n = 7). (C) Pathologic damage in the colon using a semiquantitative scoring system as detailed in the “Methods” section. The maximal pathologic score was 12. Histology of colon from a representative animal that received adoptive transfer of primary GVHD spleen cells showing extensive inflammation in the lamina propria, goblet cell depletion, and crypt cell destruction. Magnification is 20× for the upper panel and 100× for the lower panel. (D) Pathologic score in the colons of B6 Rag mice (n = 6-7 group) 60-70 days after transplantation that received spleen cells from primary B6 → Balb/c GVHD mice alone or together with purified CD4⁺ CD25⁺ Tregs from normal B6 animals. Data are presented as the means ± SEM. **P < .01. (E) Colons from replicate B6 Rag mice (n = 4) that received the same pooled spleen cells from GVHD animals were harvested and RNA was isolated for spectratype analysis. A representative spectratype showing dominant skewed peaks within the Vβ4 family is depicted. Arbitrary units reflecting peak intensity are on the y-axis, and CDR3 region nucleotide sequence length are on the x-axis. (F) Nonirradiated B6 Rag mice were adoptively transferred with pooled spleen cells from primary GVHD B6 → Balb/c chimeras that had undergone transplantation 19-21 days previously. Animals were euthanized 60-70 days after transfer and TCRβ spectratype and clonotype analysis was performed on processed colon tissue. The name and number of CDR3 region amino acid sequences derived from dominant peaks in specified Vβ families is depicted. Colors in bar graphs represent individual mice.