Immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical SCT, but infection and leukaemic relapse remain problematic. To develop a rational approach to refining allodepletion, we characterized the expression of surface markers and cytokines on proliferating alloreactive T-cells flow cytometrically. CD25 was expressed on 83 % of CFSE-dim alloreactive T-cells, confirming this as an excellent target for allodepletion. 70 % of the alloreactive CD25-ve population expressed CD71, identifying this as a novel marker to target alloreactive T-cells that persist after CD25 depletion. We compared residual alloreactivity to host or 3rd party after CD25 vs combined CD25/71 immunomagnetic depletion in 8 HLA-mismatched donor-recipient pairs. In 1o MLRs, residual responses to host were undetectable after CD25/71 depletion. In 2o MLRs, CD25/71 depletion resulted in significantly lower residual proliferative response to host than CD25 depletion (median 4.8% of the response of unmanipulated PBMC vs 9.9%, p < 0.01). Likewise, the median residual reactivity to host in IFN-γ ELISPOT assays was significantly lower after combined CD25/71 than CD25 allodepletion (14.1 % vs 54.6%, p < 0.05). Third party responses after CD25/71 allodepletion were equivalent to unmanipulated PBMCs in both assays. In pentamer and IFN-γ ELISPOT assays, anti-viral responses to CMV, EBV and adenovirus were preserved after combined CD25/71 allodepletion. Finally, we showed that CD25/71 allodepleted T-cells can be redirected to recognize and secrete IFN-γ and granzyme B in response to CD19 cell lines and primary ALL blasts through lentiviral transfer of a chimeric αCD19ζ TCR. This strategy may facilitate immunotherapy with larger doses of allodepleted T-cells after haplo-SCT, enhancing graft versus leukaemia and anti-viral effects.