Public and private human T cell clones respond differentially to HCMV antigen when boosted by CD3 co-potentiation

Human cytomegalovirus (HCMV) induces long-lasting T cell immune responses that control but do not clear infection. Typical responses involve private T cell clones, expressing T cell antigen receptors (TCR) unique to a person, and also public T cell clones with identical TCRs active in different people. Here, we report the development of a pre-therapeutic immunostimulation modality against HCMV for human T cells, CD3 co-potentiation , and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 co-potentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was co-engaged with antigen. When applied in recall assays, CD3 co-potentiation enhanced the expansion of both public and private T cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLV peptide from HCMV pp65 protein. Interestingly, public versus private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmaco-immunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 co-potentiation of T cell clones from HLA-A2(-) donors and one HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T cell copotentiation can increase the expansion of different classes of T cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV. Key Points ### Competing Interest Statement AGS and DG declare a patent filed on the use of anti-CD3 Mono-Fabs for immunotherapy. The remaining authors declare no competing financial interests.