Contribution of non-T cells to GVL against CML-like leukemia. (A) Survival curve for leukemic allogeneic chimeras (B10.D2→Balb/c) treated with imatinib (100 mg/kg) alone or in combination with DLI consisting of unfractionated splenocytes (SPL; 3 × 10⁷ total splenocytes per treatment, 4 infusions per recipient), CD5-depleted splenocytes (2.1 × 10⁷ CD5-depleted splenocytes per treatment), or CD5⁺ splenocytes (0.9 × 10⁷ CD5⁺ splenocytes per treatment). The numbers of CD5⁺ cells were adjusted to equal the number of T cells in the unfractionated DLI arm. Both imatinib and DLI treatments were discontinued at day 35 (vertical dotted line). The addition of delayed DLI to imatinib resulted in superior survival compared with imatinib alone (P < .0001 by Mantel-Cox test), whereas the survival differences between cohorts receiving CD5⁻ splenocytes, imatinib only, and control were not statistically significant. (B) Flow cytometric analysis of allogeneic chimerism (β2-microglobulin allotype, y-axis) and leukemic burden (GFP⁺ cells, x-axis) in peripheral blood leukocytes from 5 representative mice each from the cohorts in panel A analyzed at day 43 after transplantation. The percentage of allotype-positive and GFP⁺ cells in each plot is indicated. Note the eradication of leukemia in the majority of recipients treated with total splenocytes + imatinib and CD5⁺ splenocytes + imatinib compared with persistent leukemia in other groups. (C) Survival curve of leukemic allogeneic chimeras (B10.D2→Balb/c) treated with imatinib and weekly infusions of allogeneic splenocytes from wild-type B10.D2 allogeneic donors (B10.D2-wt, 3 × 10⁷ cells per treatment for a total of 4 infusions) or Rag2-deficient B10.D2 donors (B10.D2-Rag2^−/−, 8-10 × 10⁶ cells per treatment). Both treatments were discontinued at day 35 (vertical dotted line). The survival of recipients treated with imatinib + B10.D2-wt DLI was prolonged relative to the imatinib-only cohort (P = .012 by Mantel-Cox test), but there was no significant survival advantage imparted by B10.D2-Rag2^−/− DLI. (D) Flow cytometric analysis of peripheral blood of 5 representative leukemic chimeras, analyzed at day 34 after transplantation (after 3 doses of DLI treatment). The percentage of allogeneic chimerism (y-axis) and GFP⁺ leukemic cells (x-axis) in indicated. Note the relative reduction of GFP⁺ cells and the high degree of allogeneic chimerism in some recipients of B10.D2 Rag2^−/− DLI. (E) Percentage of GFP⁺ peripheral blood cells of individual recipients in each cohort over time. Note the relatively low burden of leukemic cells in some individuals receiving B10.D2-Rag2^−/− DLI, with subsequent relapse after treatment cessation at day 35 (dotted lines).