Abstract 4482: Monitoring T cell reactivity to allogeneic placental cell products using multiple lymphocyte reaction and TCR sequencing approaches
Authors: Stout Bhavani , Mahlakoiv Tanel , Hariri Robert , Zhang Xiaokui , van der Touw William 
Published: September 2020

Introduction: Allogeneic stem cell-derived therapies offer patients a readily accessible source of off-the-shelf product. However, allogeneic cells may be recognized by the recipient's immune system thus negatively affecting persistence and efficacy. Among people, allelic differences in human leukocyte antigen (HLA) complex genes are the major source of alloantigen which can elicit immunity. While there are established assays for measuring alloantibodies, monitoring T cell alloreactivity is more challenging. Unique clonal populations vary between individuals and functional assays are difficult to scale and standardize if alloreactive populations are rare/infrequent. Here, we evaluate how alloreactive T cells from patients may be identified using multiple lymphocyte reaction (MLR). Next generation sequencing of the TCR locus permits the subsequent identification and monitoring of allogeneic T cell clones in a patient over time.

Methods: Placental leukocytes depleted of CD34 cells or peripheral blood mononuclear cells (PBMC) were enriched for monocytes using magnetic-bead isolation followed by dendritic cell (DC) maturation in the presence of GM-CSF and IL-4 for 5 days. DC were activated with lipopolysaccharide for 16 hours then cultured in the presence of CD3+ T cells isolated from healthy 3rd party PBMC for 5 days in the presence of IL-2. Proliferating alloantigen-specific T cells were observed by flow cytometry. TCR locus sequencing was performed on DNA libraries generated from unstimulated and post-MLR cultures using the Illumina platform.

Results: In contrast to monocyte derived DCs from PBMC, monocyte derived DCs from placenta required higher levels of cytokine and lipopolysaccharide stimulation to achieve similar level of maturation. Co-culture of placental-derived DCs with unrelated T cells over 5 days resulted in the outgrowth of T cell colonies visible by brightfield microscopy. Flow cytometry confirmed clonal expansion of CD4 and CD8 T cell populations based upon the expression of Ki-67, CD25, and CD69 on cells with a higher forward scatter. To assess feasibility of performing TCR sequencing in unstimulated and post-MLR T cells, one T cell responder was evaluated in response to 3 different DC donors. For each DC donor, a single T cell clone representing 0.35% of the starting repertoire expanded to become the dominant clone at 44%, 61%, and 66% following MLR with the next most frequent clones represented 4%, 3%, and 2% among total T cells, respectively. Our data demonstrate the feasibility of identifying alloreactive T cell clones for translational research monitoring in patients. This approach could inform correlations with cell persistence and help optimize allogeneic cell delivery and dosage strategies in the context of disease state and lymphodepletion.

Citation Format: Bhavani Stout, Tanel Mahlakoiv, Robert Hariri, Xiaokui Zhang, William van der Touw. Monitoring T cell reactivity to allogeneic placental cell products using multiple lymphocyte reaction and TCR sequencing approaches [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4482.