Effective therapies for treating patients with steroid-refractory acute graft-versus-host-disease (SR-aGVHD) are urgently needed. aGvHD affects mostly the skin and gut, and is a leading cause of morbidity and mortality. We conducted a phase I/II trial in 20 patients with SR-aGVHD, to examine the safety and efficacy of a mixture of anti-CD3 and anti-CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-IT). On day 28 after the start of CD3/CD7-IT therapy, the overall response rate was 60%, with 50% complete remissions. The 6-month overall survival rate was 60%. Using flow cytometric analysis and T cell receptor (TCR) sequencing, we show CD3/CD7-IT caused profound but transient depletion of T cells and NK cells, followed by rapid recovery of the immune system with a diverse TCR Vβ repertoire, and preservation of Epstein-Barr virus- and cytomegalovirus-specific T cell clones. Different characteristics of responders (Rs) versus non-responders (NR) were noticed. Rs had a more swift recovery of T cells in the blood after treatment, and CD4 and CD8 subsets reconstitute differently between groups. Before treatment, Rs had significantly higher amounts of regulatory T cells (Tregs), CD4:CD8 ratios and higher clonal T cell diversity. While T cells reconstitution is not associated with disease severity, there seems to be an inverse relationship between disease severity and response rate. We further noticed a correlation between the conditioning regimen and amount Tregs, CD4:CD8 ratio and T cell diversity. These results suggest CD3/CD7-IT is safe and well tolerated, with a relatively low prevalence of reversible adverse events. It can be effective in all aGvHD grades and in patients with different conditioning regimens. Determining the Treg counts, CD4:CD8 ratio and T cell repertoire diversity before CD3/CD7-IT may predict response and select patients that benefit from CD3/CD7-IT therapy.