Cellular Immune Reconstitution Varies Across GVHD Prophylaxis Strategies in HSCT

Although graft-versus-host disease (GVHD) prophylaxis strategies are designed to improve outcomes following hematopoietic stem cell transplantation (HSCT), differences in long-term survival and immune recovery remain poorly understood. At the 2026 Joint American Society of Transplantation and Cellular Therapy + European Society for Blood and Marrow Transplantation Basic and Translational Scientific Meeting in Philadelphia, Pennsylvania, Rachel Lorenc, MD, PhD student, and Maxim Maron, MD, PhD, both of Memorial Sloan Kettering Cancer Center, presented new translational findings examining how distinct GVHD prophylaxis approaches shape immune reconstitution at a cellular level following transplant.¹

Leveraging Data from the Phase 3 BMT CTN 1301 Clinical Trial

Lorenc opens the presentation by providing background on the landmark case trial, BMT CTN 1301 (NCT02345850)², a 3-arm, randomized multicenter phase 3 trial comparing 2 calcineurin inhibitor (CNI)-free strategies for graft-versus-host disease (GVHD) prophylaxis—CD34-selected peripheral blood stem cell or in vivo post-transplant cyclophosphamide (PTCy) after a bone marrow (BM) graft—to standard tacrolimus and methotrexate (Tac/Mtx) in patients with hematologic malignancies undergoing myeloablative conditioning hematopoietic stem cell transplantation (HSCT). The trial’s primary outcome was to compare chronic GVHD/relapse-free survival (CRFS) as a time-to-event end point after HSCT between each of the CNI-free interventions and a Tac/Mtx control. Secondary objectives were to compare rates of grades 2 through 4 as well as grades 3 and 4 acute GVHD, immunosuppression-free survival at the 1-year period, incidence of infections, immune reconstitution, quality of life, and overall survival (OS), among other outcomes.²

Among the 346 patients enrolled, 327 received HSCT, 300 per protocol. Intent-to-treat rates of 2-year CRFS were approximately 50.6% for CD34 selection (HR, 0.80 [95% CI, 0.56-1.15]; P = .24), 48.1% for PTCy (HR, 0.86 [95% CI, 0.61-1.23]; P = .41), and 41.0% for control. Corresponding rates of OS were approximately 60.1% (HR, 1.74 [95% CI,1.09-2.80]; P = .02), 76.2% (HR, 1.02 [95% CI, 0.60-1.72]; P = .95), and 76.1%. CD34 selection was found to be associated with lower moderate to severe cGVHD (HR, 0.25 [95% CI, 0.12-0.52]; P = .02) but higher transplant-related mortality (HR, 2.76 [95% CI, 1.26-6.06]; P = .01). Additionally, PTCy was associated with comparable cGVHD and survival outcomes to control and a trend toward lower disease relapse (HR, 0.52 [95% CI, 0.28-0.96]; P = .037).^1,3

Lorenc explained that these findings demonstrated that CNI-free interventions as performed did not result in superior CRFS compared with Tac/Mtx with BM, and lower rates of moderate and severe cGVHD did not translate into improved survival.^1,3

“These differential clinical outcomes suggest that GVHD prophylaxis strategy is not an important consideration when thinking about patient outcomes,” Lorenc said. “Which led us to ask the question, ‘How did each of these prophylaxis strategies shape the reconstitution of the immune system at a cellular level?’”¹

How Do These Prophylaxis Strategies Shape Immune System Reconstitution?

To investigate how these strategies influenced immune reconstitution at a cellular level, Lorenc and Maron first identified several important clinical paradoxes. Although the CD34 selection group was shown to demonstrate a significantly lower incidence of chronic GVHD, this benefit came with a trade-off in OS due to infection-related complications. Notably, relapse rates did not significantly differ across the 3 treatment arms. These findings prompted the investigators to ask how each prophylaxis strategy uniquely shaped the reconstitution of the immune system.^1,3

To address their question, Lorenc and Maron selected a specific patient cohort consisting of 5 patients from each treatment arm. They intentionally chose individuals who experienced “successful” immune reconstitution, which was defined as having no relapse and minimal GVHD 1 year following transplant. All patients in the cohort received sex-mismatched grafts from their donors, enabling researchers to track donor versus recipient chimerism using single-cell sequencing.¹

Using this carefully selected cohort, they designed flow cytometry panels to monitor the development of B cells, dendritic cells, and NK cells. They also applied advanced multiomics approaches, including single-cell RNA sequencing and TCR sequencing, to evaluate the phenotypes of the reconstituting immune system. This generated a high-resolution view of more than 300,000 cells, allowing for a detailed comparison of immune profiles across the different treatment strategies.¹

Lorenc and Maron found that, although different GVHD prophylaxis strategies resulted in similar relapse rates, they ultimately produced different immune environments that help explain variations in GVHD incidence and OS. For example, patients who received Tac/Mtx demonstrated a rapid return to a normal immune process; however, they experienced severe B-cell depletion through approximately day 100. Most cell populations in this group were donor-derived, with the exception of some CD4 and naïve cell populations.

Conversely, they observed that patients receiving PTCy exhibited an immune system that was dominated by the CD8 compartment in the early posttransplant period. Large clones were found to dominate the CD8 effector compartment within these samples, and naïve T cells remained effectively absent until 1 year following transplant. Similarly, the CD34-selected graft group displayed an immune profile persistently dominated by NK cells through the 1-year mark. Like the PTCy arm, naive T cells were effectively absent until a year post-transplant. Notably, many CD4 and CD8 cells in the CD34-selected group remained recipient-derived, despite the use of myeloablative conditioning.¹

Additional transcriptional and cellular analyses revealed important differences between treatment groups. Both the PTCy and CD34 selection arms demonstrated a more cytotoxic transcriptional signature in CD8 effector memory cells compared with the control group. Gamma delta T cells in the CD34 selection arm also exhibited a heightened cytotoxic signature. When Lorenc and Maron compared reconstituted cells to a reference map of 40 million healthy cells, most aligned closely with expected healthy immune labels. There was only a minimal statistical difference observed in the memory CD8 compartment, where Tac/Mtx samples appeared slightly further from the “normal” baseline.¹

Based on these findings, Lorenc and Maron concluded that distinct clinical outcomes—such as the trade-off between reduced GVHD and increased infection-related complications observed with CD34 selection—are driven by the proportional kinetics of immune reconstitution. Specifically, the depletion of naïve T cells—which serve as mediators of GVHD but are also essential for infection control—along with the rate at which immune cell subsets recover, appears to be the primary driver of these clinical differences.¹

“[Although] the overall conscriptional programs across the 3 prophylaxis [methods] are relatively similar, we predict that it's actually the rate at which each subset recovers that drives the distinct clinical outcomes we observed,” Lorenc concluded.¹

REFERENCES

1. Lorenc R, Maron M. Myeloid Cells in Immunotherapy Outcomes: Rebuilding the Immune System: A Single Cell Map of Immune Reconstitution Across GvHD Prophylaxis Strategies from BMT CTN 1301. Presented at: Joint American Society of Transplantation and Cellular Therapy + European Society for Blood and Marrow Transplantation Basic and Translational Scientific Meeting; Philadelphia, Pennsylvania. May 28–29, 2026.

2. Calcineurin Inhibitor-Free Interventions BMT CTN 1301 for Prevention of Graft-versus-Host Disease (BMT CTN 1301). ClincialTrials.gov identifier: NCT02345850. Updated March 7, 2023. Accessed May 29, 2026. https://clinicaltrials.gov/study/NCT02345850

3. Randomized Phase III BMT CTN Trial of Calcineurin Inhibitor–Free Chronic Graft-Versus-Host Disease Interventions in Myeloablative Hematopoietic Cell Transplantation for Hematologic Malignancies. J Clin Oncol. 2021;40(4):356-368. doi:10.1200/JCO.21.02293