TCR-transduced CD8⁺ T cells mediate strong GVL effects with decreased GVHD rates when administered early after HCT. A total of 40 × 10⁶ TCR gene-transduced allogeneic (B10.A) or syngeneic (B6) T cells were adoptively transferred early (21 days after HCT) into established allogeneic (B10.A → B6) or syngeneic recipients (B6 → B6). (A) On days 1, 3, and 7 after AT, peripheral blood was monitored for GFP expressing allogeneic (●) or syngeneic (○) TCR-transduced T cells. ***P < .0001 for day 1. **P < .005 for day 3. P = not significant (n.s.) for day 7. n = 14 to 17 per group. (B) Mice received early AT with either allogeneic (■) or syngeneic (□) TCR-transduced T cells. Cohorts treated with naive DLI (▴) or PBS (*) were used as controls. Seven days after AT, mice were challenged with 1.2 × 10⁶ C1498-OVA cells intravenously. n = 8 to 10 per group. P < .05 between cohorts that received DLI or allogeneic TCR-transduced T cells. P = not significant (n.s.) between cohorts that received syngeneic or allogeneic TCR-transduced T cells. (C) For a sensitive GVHD setup, mixed chimeras were established by reconstituting lethally irradiated B6 recipients with a mixture of 15 × 10⁶ TCD B10.A BM plus 5 × 10⁶ TCD B6 BM. A total of 40 × 10⁶ TCR-transduced (■), nontransduced (♦) B10.A T cells, or DLI (▴) were adoptively transferred on day 21 after HCT. Clinical GVHD scores were performed weekly for 8 weeks after AT. The highest GVHD score/observed individual is graphed. n = 8 to 10. ***P < .0001 between cohorts that received TCR-transduced T cells or DLI. **P < .005 between cohorts that received TCR-transduced or nontransduced T cells. P = not significant (n.s.) for cohorts that received nontransduced T cells or DLI. (D) Representative examples of CDR3-size spectratype analysis of TCR-transduced T cells are shown. A total of 40 × 10⁶ TCR gene-transduced B10.A T cells were adoptively transferred early (21 days after HCT) into established allogeneic (B10.A → B6) or syngeneic recipients (B10A → B10A). Forty days after AT, GFP⁺CD8⁺ T cells were sorted from recipient spleens, and CDR3-size spectratype analysis was performed as described in “TCR spectratype analysis and assessment of proliferation.” Naive CD8⁺ B10.A T cells (used for transduction) and TCR-transduced T cells (used for AT) were used as controls. Spectratype histograms show an example for a skewed (Vβ 16) and a nonskewed (Vβ 8.1) Vβ-family.