FOXP3/Helios Expression in CD4+ Tcons Correlates With T-Cell Activation After Orca-T in Hematologic Malignancies

Treatment with the allogeneic immunotherapy Orca-T led to more frequent increases in CD4-, FOXP3-, and Helios-positive conventional T cells (Tcons) compared with conventional unmanipulated peripheral blood stem cell (PBSC) grafts in patients with hematologic malignancies, according to findings from a study presented at the 2025 Transplantation and Cellular Therapy Meetings.

Furthermore, the activation of CD4-, FOXP3-, and Helios-positive Tcons at 3 weeks post-treatment correlated with activated T-cell phenotypes 3 months following treatment.

Single-cell mRNA sequencing was used to evaluate FOXP3 and Helios expression on CD4-positive T cells, and findings from this method also revealed a diverse population of CD4-positive Tcons and regulatory T cells (Tregs).

Using flow cytometry, investigators confirmed the increase of CD34-, FOXP3-, and Helios-positive Tcons in patients treated with Orca-T compared with those who underwent a PBSC graft.

“Overall, this study identifies an increase in T-cell activation early after Orca-T immunotherapy, and we propose that the sequential addition of high-purity Tregs directs the immune reconstitution of CD4[-positive] T cells toward a more activated, but also potentially immunomodulatory, FOXP3- and Helios-expressing phenotype, which contributes to immune protection without graft-vs-host disease [GVHD],” lead study author Cameron S. Bader, PhD, a postdoctoral scholar at Stanford Medicine, said in a presentation of the data.

Orca-T Background and Study Design

Orca-T is an allogeneic immunotherapy that is administered to patients over multiple days. On day 0, patients receive CD34-positive hematopoietic stem and progenitor cells, plus Tregs. On day 2/3, patients then receive Tcons. Conversely, patients undergoing unmanipulated PBSC grafts receive mixed mobilized cells on day 0.

“Allogeneic hematopoietic cell transplantation [allo-HCT] is a potentially curative therapy for many malignant and non-malignant diseases. Donor T cells are transplanted, along with the stem cell graft, to harvest beneficial graft-vs-tumor effects and to provide protective immunity,” Bader said. “However, donor T-cell recognition of recipient alloantigen in healthy tissues can result in GVHD, which remains the major non-relapse complication limiting the use of allo-HCT.”

Bader and fellow study investigators hypothesized that the transcriptional and protein signatures of recipient immune cells could indicate potential mechanisms for GVHD suppression with Orca-T compared with conventional allo-HCT.

The study included patients with hematologic malignancies who received Orca-T (n = 23) or unmanipulated PBSC grafts (n = 28) at day 0. At week 3, patients were evaluated via single-cell mRNA sequencing and flow cytometry (n = 16) or flow cytometry alone (n = 31). Flow cytometry assessments were also conducted at month 3 (n = 33) and month 6 (n = 30).

The median age was 48 years (range, 21-65) in the Orca-T arm vs 51 years (range, 27-63) in the PBSC graft arm. Sixty-one percent of patients in the Orca-T group were female vs half of patients in the PBSC graft group. Most patients in the Orca-T (96%) and PBSC graft (79%) arms received busulfan, fludarabine, and thiotepa as a conditioning regimen; the remainder of patients underwent fractionated total body irradiation and cyclophosphamide.

Disease histologies included acute myeloid leukemia (Orca-T, 61%; PBSC graft, 68%), myelodysplastic syndrome (30%; 7%), acute lymphoblastic leukemia (9%; 18%), and other acute leukemia (0%; 7%). All patients in the Orca-T arm received tacrolimus as GVHD prophylaxis; all patients in the PBSC graft arm were given tacrolimus plus methotrexate.Donor types included related HLA-matched (65%; 46%) and unrelated HLA-matched (35%; 50%); notably, 1 patient (4%) in the PBSC graft arm had a related permissible-mismatch donor.

Cytomegalovirus status at baseline comprised donor negative, recipient negative (Orca-T, 22%; PBSC graft, 14%), donor positive, recipient negative (4%; 4%), donor negative, recipient positive (30%; 36%), and donor positive, recipient positive (43%; 46%).

The median time to neutrophil engraftment was 14 days (range, 10-18) in the Orca-T arm vs 13 days (range, 9-22) in the PBSC graft arm. The median times to platelet engraftment were 16 days (range, 10-19) and 16 days (range, 12-23), respectively.

HLA-DR Expression and GVHD

During the study, investigators also examined HLA-DR expression at 90 days post-transplant, and they found that lower levels of this expression could correlate with the incidence of GVHD.

Among patients between both arms with high CD4-positive HLA-DR expression at day 90 (n = 12), the 100-day cumulative incidence of GVHD was 8.3% (95% CI, 0.4%-32.3%) compared with 29.2% (95% CI, 12.6%-48.0%) in patients with low or medium expression (n = 24; P = .15).

“[Although] this preliminary univariate analysis, which does not take into account the many GVHD- and treatment-related confounders, was not a goal of our original study...it does indicate that there may be a potential link between low levels of [HLA-DR expression] of activated CD4 T cells and negative [GVHD] outcomes that is worth studying further across multiple transplantation strategies,” Bader concluded.

Disclosures: Bader reported receiving research funding from Orca Bio.

Reference

Bader CS, Killian S, Le CT, et al. FOXP3 and Helios expressing CD4+ T conventional cells correlate with T cell activation after Orca-T allogeneic T cell immunotherapy. Presented at: 2025 Transplantation and Cellular Therapy Meetings; February 12-15, 2025; Honolulu, HI. Abstract 87.