CD4⁺ T cells are necessary for the induction of GVHD-associated autoimmunity. Lethally irradiated (900 cGy) Balb/c mice received transplants of TCD B6 BM plus 3 × 10⁵ B6 spleen cells. At 19 to 21 days after BMT, animals were killed and cohorts of nonirradiated B6 Rag mice received transplants of either purified CD4⁺ (n = 18), CD8⁺ (n = 8), or the combination of CD4⁺ and CD8⁺ T cells (n = 24) from pooled spleen cell suspensions using the MACS isolation system. B6 Rag animals were then killed 35 to 74 days after transfer. (A) Overall pathological score, as defined in “Histologic analysis,” of mice that received transplants of either CD4⁺ (□), CD8⁺ (▨), or CD4⁺ and CD8⁺ T cells (■). The CD4⁺ T-cell dose ranged from 0.3 to 0.5 × 10⁶ (mean, 0.43 × 10⁶), while the CD8⁺ T-cell dose ranged from 0.35 to 0.5 × 10⁶ (mean, 0.43 × 10⁶). Animals that received transplants of the combination of CD4⁺ and CD8⁺ T cells received 0.5 × 10⁶ cells (mean, 3 × 10⁵ CD4⁺ and 2 × 10⁵ CD8⁺ T cells). Results are derived from 3 to 5 experiments per T-cell group. Data are presented as the mean plus or minus SEM. (B) Absolute number of CD4⁺ (□) or CD8⁺ (■) T cells in the spleens of mice that received transplants of purified CD4⁺, CD8⁺, or CD4⁺ and CD8⁺ T cells from B6→ Balb chimeras. Data are presented as the mean plus or minus SEM. (C-F) Histology of colon (C,E) and liver (D,F) from representative B6 Rag recipients that received either purified CD4⁺ T cells (C,D) or both CD4⁺ and CD8⁺ T cells (E,F) from primary (B6→ Balb) GVHD animals. Colons show marked infiltration of mononuclear and granulocytic cells into the lamina propria along with extensive goblet cell depletion. Livers reveal portal (dashed arrows) and lobular (solid arrows) inflammation with mononuclear and granulocytic infiltrates. (G) Immunofluorescence staining from the colon of a representative B6 Rag mouse that received purified CD4⁺ and CD8⁺ T cells from primary GVHD animals showing infiltration of the lamina propria with both CD4⁺ (blue) and CD8⁺ (red) T cells. Statistics: *P ≤ .05; **P < .01.