Treg Selection from G-CSF Stimulated Donors Improves Outcomes of Immune-Suppression Free HLA-Haploidentical Hematopoietic Cell Transplantation with T Cell Adoptive Immunotherapy

Background

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for patients with high-risk acute leukemias. We have recently employed an immune-suppression free HSCT approach with regulatory and conventional T cell immunotherapy (Treg-Tcon HSCT) for acute myeloid leukemia (AML) patients that allowed for a potent antileukemic activity. Indeed, leukemia relapse was very low (4%). Major complication of such approach was acute Graft-versus-Host Disease (aGVHD, 33%) that, despite being manageable in most patients, impacted on HSCT morbidity (Pierini et al. Blood Adv 2021).

In this study, Tregs have been isolated through two steps magnetic separation (first step: CD19 and CD8 negative selection; second step: CD25 positive selection) from a dedicated unstimulated donor leukapheresis. The infused final product contained a median of 71% (range 62.5-79.5%) FOXP3+CD127- Tregs. We hypothesized that contaminating cells might account for the relevant incidence of aGVHD. To improve purity of the final Treg product without increasing the complexity of the procedure, we selected Tregs from G-CSF stimulated donor apheresis. In fact, G-CSF treatment allowed for a more efficient Treg selection procedure that resulted in increased Treg purity.

Here, we report preclinical and early clinical data that support the use of G-CSF stimulated Tregs (Stim-Tregs) to possibly reduce incidence of aGVHD in HLA-haploidentical Treg/Tcon HSCT.

Methods

We tested Tregs products from leukapheresis of unstimulated (Unstim-Tregs) or Stim-Tregs healthy donors for purity by FACS analysis and function by cell cultures and mouse models of GVHD. We also analyzed outcomes of acute leukemia patients that received Stim-Tregs in HLA-haploidentical Treg/Tcon HSCT.

Results

CD25 expression was similar between Stim- and Unstim-Tregs. Final purity was higher in Stim-Tregs as CD127 expression was lower in Stim-Tregs (11.6%±7.3% vs 27%±7.6%., p<0.01), while FOXP3 expression tended to be higher (94.01%±5.63%). Also, human Stim-Tregs conserved good ability to suppress HLA-matched (same donor) Tcons proliferation in vitro and protected immune-deficient NSG mice from Tcon-induced lethal aGVHD.

We translated such results in a pilot HLA-haploidentical Treg/Tcon HSCT study where 24 patients (median age 49.6, range: 22-68; 13 male and 11 female) with high-risk acute leukemia and myelodysplastic syndrome (19 AML, 2 MDS, 2 B-ALL, 1 T-ALL). Twenty acute leukemia patients were in complete remission. Eight (33%) were minimal residual disease (MRD) negative, 12 (50%) MRD positive, and 2 with active disease at transplant.

Patients received an irradiation-based (total body irradiation, TBI, or total marrow lymphoid irradiation, TMLI, in old or unfit for TBI) myeloablative conditioning regimen. Donor Stim-Tregs (2x10⁶/kg) were given at day 0 together with a mega-dose of purified donor CD34+ cells (11x10⁶/kg±2x10⁶) followed by the infusion of previously frozen unstimulated donor Tcons (1x10⁶/kg) at day +4. One patient received TBI and 23 patients received TMLI. Toxicity was mild as oral mucositis was grade III in 2 patients, grade II in 17 patients, and grade I in 5 patients. All but one patient (successfully rescued with a second transplant) reached neutrophil engraftment after a median of 13.3 days (range 10-18). Only 3 patients developed grade ≥2 aGvHD. All of them are alive and off-therapy. Such outcome compares favorably (12.5% vs 33%, p=0.05) with historical 66 patients who received unstim-Tregs in HLA-haploidentical Treg/Tcon HSCT (Pierini A., et al, Blood advances 2021, updated). No patient developed moderate/severe chronic GvHD (cGvHD). Despite the high-risk diseases, only two patients relapsed after a median follow up of 14 months (range 3-25). Thus, moderate/severe cGvHD-relapse free survival was 82% and GvHD-Relapse free survival was 75%.

Conclusions

Our study shows that Treg selection from G-CSF stimulated donors allows for higher purity of Treg final product. Infusion of stim-Tregs together with previously collected unstimulated donor Tcons in the absence of post-transplant immune suppression reduced rates of aGvHD and maintained a potent antileukemic activity of Treg-Tcon HSCT approach. While larger and randomized studies are needed to confirm such findings, the use of Tregs derived from G-CSF stimulated donors improved outcomes of HLA-haploidentical Treg/Tcon HSCT.