Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for many hematologic malignancies. The primary non-relapse complication preventing the widespread use of allo-HSCT is graft-versus-host disease (GVHD). The use of T regulatory cells (Tregs) to prevent GVHD has emerged as a promising allogeneic T cell immunotherapy in the form of Orca-T. Orca-T consists of the sequential infusion of CD34+ hematopoietic stem cells and high-purity Tregs followed by conventional T cells. However, the precise differences in immune states which may influence clinical outcomes after Orca-T compared with unmanipulated peripheral blood stem cell (PBSC) grafts remains unexplored. Using peripheral blood specimens longitudinally collected between 3 weeks and 1 year after leukemia treatment, we report single-cell mRNA sequencing (scRNA-seq) and flow cytometric analysis of 51 HLA-matched patients receiving either Orca-T or unmanipulated PBSC grafts at Stanford University.

To identify potentially important T cell populations that drive clinical differences between Orca-T and unmanipulated PBSC grafts, sorted T cell subsets or whole PBMC from 16 total patients were captured for scRNA-seq analysis 3 weeks post-treatment. Targeted transcriptomic and V(D)J analysis identified increased expression of FOXP3 and Helios amongst CD4+CD25- T conventional cells (Tcons) in Orca-T treated patients (7.8% Orca-T vs. 3.0% PBSC, p=0.035). Moreover, in Orca-T recipients, there was a trend towards a higher proportion of expanding clones (≥2 identical clones) which belonged to the CD4+CD25-FOXP3+Helios+ cluster than in PBSC graft recipients (14.7% vs. 2.3%, p=0.095). Separate single-cell whole transcriptome analysis also found increased expression of FOXP3 and Helios amongst activated CD4+ Tcons from Orca-T recipients compared to PBSC graft recipients. Flow cytometric analysis of 20 additional patients corroborated the increased frequency of CD4+FOXP3+Helios+ Tcons - but not Tregs – 3 weeks post-treatment in patients receiving Orca-T (11.6% Orca-T vs. 4.4% PBSC, p=0.016). Further, we discovered that this T cell subset correlated significantly with frequencies of HLA-DR+ and effector memory CD4+ T cells (p=0.002, p=0.032) and effector memory CD8+ T cells (p=0.014) 3 months post-treatment, regardless of which therapy patients received. These data suggest that Orca-T immunotherapy induces a FOXP3+Helios+ Tcon population which may play a role in long-term immune reconstitution after allo-HSCT.

Next, we confirmed the increased frequency of CD4+CD25-FOXP3+Helios+ T cells early after Orca-T immunotherapy in a second cohort of 19 cryopreserved patient samples collected 2 weeks after treatment, including 12 which were transplanted at other centers (11.7% Orca-T vs. 4.3% PBSC, p=0.029). In contrast, a comparative examination of 14 mobilized donor PBMC samples found that only 1% of donor Tcons expressed FOXP3 and Helios (p=<0.0001 vs. Orca-T, p=0.23 vs. PBSC). In week +2 recipient samples that were matched to mobilized donor PBMC, we found an average 10.3-fold increase (p=0.011) in CD4+CD25-FOXP3+Helios+ cell frequency in Orca-T recipients compared to their donor. In contrast, PBSC recipients exhibited a 5.9-fold increase (p=0.023), suggesting that Orca-T immunotherapy greatly promotes the in vivo expansion or differentiation of these cells compared to standard PBSC grafts.

We also identified that CD4+CD25-FOXP3+Helios+ T cells were phenotypically similar in donors, Orca-T recipients, and PBSC recipients, indicating a common expression profile regardless of overall immune state. Compared to other CD4+ Tcons and Tregs, CD4+CD25-FOXP3+Helios+ T cells represented a unique T cell subset with intermediate expression of immunomodulatory proteins including CD39, CTLA-4, HLA-DR, TIGIT, and the distribution of naïve and effector memory cells (ANOVA p=<0.0001 for each) along with distinct expression of CD73 (ANOVA p=0.007). We therefore propose that CD4+CD25-FOXP3+Helios+ cells are a regulatory-like subset of T cells which are induced by Orca-T immunotherapy and that this population contributes to GVHD suppression in concert with other regulatory populations after leukemia treatment. Overall, we hypothesize that the sequential addition of high-purity Tregs directs the immune reconstitution of CD4+CD25- Tcons towards a non-redundant, immunomodulatory, FOXP3+Helios+ phenotype which may be predictive of long-term immune activation after T cell infusion.

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2025
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