Immune reconstitution following memory T-cell add back and prophylactic blinatumomab post TCRαβ/CD19-depleted haploidentical transplantation in pediatric patients with high-risk acute leukemia

Abstract

Abstract TCRαβ depletion of haploidentical donor (haplo) grafts enables low rates of graft-versus-host disease (GVHD) but delays immune reconstitution in hematopoietic cell transplant (HCT) recipients. In our recent trial (NCT03849651), we combined CD45RA-depleted donor lymphocyte infusion (memory DLI; memDLI) following TCRαβ/CD19-depleted haploHCT to enhance immune reconstitution without increasing risk of GHVD for children with high-risk acute leukemia. The clinical outcomes of this trial were previously presented (Naik S, Blood Vol 144 (Supplement 1);144:383). Here, we report detailed immune reconstitution data from the trial. Patients received a preparative regimen consisting of fludarabine, melphalan, cyclophosphamide, thiotepa, and anti-thymocyte globulin. Patients with B-cell ALL received one 4-week cycle of prophylactic blinatumomab (blina). No GVHD prophylaxis was used. Patients were enrolled to receive memDLI in 2 cohorts: a dose escalation cohort (1×105, 1×106, or 1×107 cells/kg) of 29 patients and a dose expansion cohort (n=40) at the maximal effective memDLI dose (1×107cells/kg). Peripheral blood samples were collected on days 30, 60, 100, 180, and 365 post-HCT for flow cytometric immunophenotyping of lymphocyte subsets. T-cell receptor Vβ spectratyping for T-cell diversity, T-cell receptor excision circles (TRECs) for naïve T-cell production, and Enzyme-Linked ImmunoSpot (Elispot) for viral-specific T-cell responses were performed at day 100, 180, and 365 post-HCT. From 2019 to 2023, 69 patients received haploHCT at a median age of 8.8 years (range 0.5-21.6). At a median of 29 days (range 22-57), 67 patients received memDLI. At 30, 60, 100, 180, and 365 days post-HCT median CD3+ cell counts were 130, 280, 382.5, 835, and 1,280 cells/μL, CD4+ cell counts 27.5, 70, 105, 270, and 530 cells/μL, and CD8+ cell counts 40, 100, 125, 410, and 620 cells/μL, respectively. TCRgd T cells dominated early post-HCT (day +30: median 92.4% [74.8-97.3%]), and by day +60 TCRαβ T cells had become the dominant T cell subset (61.1% [33.4-75.4%]). The early TCRαβ T-cell recovery was driven by memory (CD45RO+) T cells as naïve (CD45RA+) T cell counts (cells/μL), were consistently lower at day +60 (14.9 vs. 102.5), +100 (35.2 vs. 163.5), and +180 (143.6 vs. 277.9) until day +365 (428.4 vs. 309.5). This phenotypic analysis was corroborated by TREC analysis, and only at 1-year post-HCT normal TREC levels were reached (4.5 vs. 4.6 (HCT donors) log/mL; P=0.62). The emergence of memory T cells was directly linked to memDLI as demonstrated by comparing T cell counts (cells/mL) pre and 4 weeks post memDLI (CD3: 106 vs. 361, P<0.0001; CD4+: 7 vs. 80, P<0.0001; CD8+: 36 vs. 198, P=0.001, CD45RO+ T cells: 48.3 vs. 236, P=0.003), CD45RA+ T cells: 18.3 vs. 20.5, P=0.64). These memory T cells were functional as measured by Elispot assays with significant expansion of CMV-specific T-cells 4 weeks post-memDLI (pre: 0, post: 121.9 spot-forming cells; P=0.008) and contributed to broad diversity as assessed by TCRVβ diversity analysis (post-HCT day + 100 : 135.3; day +180: 168.6 vs. 178.4 (HCT donors); P=0.13) Median B-cell recovery at 30, 60, 100, 180, and 365 days post-HCT was 60, 196.5, 162.5, 230, and 316 cells/mL, respectively. Patients received blina (n=26) at a median of 63 days (range 44-135) post-HCT and developed B-cell aplasia at a median of 6.5 days post-blina infusion, with a median duration of B-cell aplasia of 65.5 days. Patients who received blina had significantly lower B-cell counts at day +100 compared to those who did not receive blina (91.11 vs. 232.7 cells/mL; P=0.003); this difference had resolved by day +180 (196.4 vs. 232.5 cells/mL; P=0.51) and +365 (202.6 vs. 339.4 cells/mL; P=0.07) post-HCT. Immunoglobulin (Ig)G levels were maintained with IVIG until evidence of antibody production through rising IgM, before day +180 post-HCT (median IgM: 75 mg/dL, IgA: 46mg/dL) with no difference amongst patients who did and did not receive blina. In conclusion, the use of memDLI at doses up to 1 x107/kg was associated with robust phenotypic and functional immune reconstitution, while the use of blina early post-HCT did not impact long-term B-cell reconstitution. Thus, our approach presents a promising strategy to enhance immune reconstitution following TCRαβ-depleted haplo-HCT.