Immunophenotyping of T cells Combined with Vβ antibodies identifies long lasting CMV related T cell Expansions with a consistent TIGIT and PD-1 phenotype

Simultaneous analysis of T cell receptor repertoire and T cell phenotype is of high relevance to gain a deeper understanding on the T cell response in terms of differentiation, kinetics and persistence. Also it is currently unknown how the repertoire of immune checkpoints on T cells evolves over time. To fully capture the heterogenous response, TCR repertoire and T cell phenotype analysis is preferably performed at the single-cell level, but this is costly and analytically challenging. Therefore, we developed a flow cytometry based method that allows for simultaneous characterization of the T cell TCR Vβ and the phenotype. We generated a 9-tube 24-color panel with antibodies against maturation and activation markers, and 24 TCR Vβ chains. Peripheral blood mononuclear cells from 5 healthy controls were analyzed, revealing the presence of oligoclonal expansions. Within the CD27+CD28+ memory T cell population, oligoclonal expansions were characterized by a T regulatory, or KLRG1+CCR7-CD27low phenotype. Within the late CD27- and/or CD28-memory T cells, the populations with the lowest TCR Vβ diversity expressed CX3CR1. As a proof of principle, we demonstrate the dynamics of circulating oligoclonal T cell expansions that emerged following hematopoietic stem cell transplantation in the presence of CMV in 2 patients. We provide evidence that oligoclonal T cells transferred from donor to recipient can be traced up to at least 2.5 years while maintaining its phenotype. Mostly TIGIT and PD-1 were critical in defining T cell expansions, while CD45RA, CD57, CD56 and NKG2A were variably expressed. Overall, we developed a phenotype based method to trace T cell populations, that is widely applicable to different settings in which the T cell response needs to be monitored.