FOXP3 and helios expressing CD4+ T conventional cells are a regulatory-like subset that is induced after orca-T allogeneic immunotherapy
Bibliographic record
Abstract
Abstract 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.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.003 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".