Evolution of T Cell Repertoire Diversity After Reduced-Intensity Conditioning and Double Umbilical Cord Blood Transplantation with or without Exposure to FT1050 (16,16-dimethyl Prostaglandin E2)

In double umbilical cord blood (UCB) transplantation (dUCBT), ex vivo modulation of a single UCB unit with 16, 16 dimethyl prostaglandin E₂ (dmPGE₂) accelerates neutrophil engraftment and leads to preferential hematopoietic dominance with complete T cell chimerism as early as 13 days from dUCBT. We sought to determine whether dmPGE₂ affected the recovery of T cell diversity after dUCBT.  Methods: dUCBT recipients received fludarabine, melphalan, and ATG conditioning with Tacrolimus and Sirolimus GvHD prophylaxis. TCRb CDR3 regions were amplified and sequenced using the ImmunoSeq assay (Adaptive Biotechnologies) from a median of 171 ng of T cell gDNA (range 0.8 - 400.0 ng) at 3 and 12 months from dUCBT. We analyzed 5 paired samples with dmPGE₂-dominance (PD); 3 paired and 7 single 12 month untreated (U) samples; and 5 paired and 1 single 12 month dmPGE₂ treated sample with untreated-dominance (UD). The U+UD groups were combined as one control cohort.  Results: PD CD3⁺ cells were lower at 3 months versus the U+UD group (p = 0.057, Table) but this trend diminished by 12 months (p = 0.11). Lower CD3⁺ counts were reflected in lower total TCR alpha diversity in PD samples at both time points (3 month median 585 vs. 3905 unique TCR sequences, p = 0.079; 12 month median 2729 vs. 12062.5 unique TCR sequences, p = 0.22). Median lymphocyte counts did not increase between 3 and 12 months in the PD group as in the U+UD group, but the proportion of CD3⁺ cells within lymphocytes increased in both groups. PD T cell repertoires were less clonal than U+UD samples at 3 months (1-normalized entropy: 0.14 vs. 0.25, p = 0.09) and 12 months (1-normalized entropy: 0.11 vs. 0.23, p = 0.95), with more even frequency distribution of TCRs. PD T cell repertoire was more dynamic than the U+UD controls with a significantly decreased T cell clonal persistence between 3 and 12 months (TCR sequence overlap: 8.73% vs. 38.84%, p = 0.027).  Conclusions:  Ex vivo dmPGE₂ modulation with dmPGE₂ dominance results in delayed T cell and lymphocyte recovery, but less clonality despite a T cell repertoire restricted by low CD3⁺ counts. The reduced oligoclonality and rapid T cell turnover may indicate enhanced thymopoiesis. Correlation with TRECs and clinical infectious outcomes is ongoing.

	3 Months after dUCBT				12 Months after dUCBT
	dmPGE2-dominant	Untreated / Untreated-dominant	P value  - T test	P value - U test	dmPGE2-dominant	Untreated / Untreated-dominant	P value -  T test	P value - U test
WBC	5900.0	4850.0	0.377	1	4300.0	6500.0	0.049	0.035
CD3+ count	10.6	50.0	0.097	0.26	159.4	444.2	0.016	0.014
% CD3+ of lymphocytes	2.55%	11.00%	0.066	0.057	12.30%	31.85%	0.161	0.11
Richness Alpha Diversity	585	3905		0.079	2729	12062.5		0.22
Clonality 1-Normalized Entropy	0.1381	0.2454	0.090	0.19	0.1105	0.2326	0.945	0.54
Total Repertoire Overlap Index					0.0873	0.3884	0.027	0.057
Persistence of Top 10 Clones					2	10		0.040