T follicular helper (T) cells are a heterogeneous subset of immunocompetent T helper (T) cells capable of augmenting B cell responses in lymphoid tissues. In transplantation, exposure to allogeneic tissue activates T cells increasing the risk of the emergence of de novo donor-specific HLA-antibodies (dnDSA). These can cause antibody-mediated rejection (AMR) and allograft loss. Follicular regulatory T (T) cells counteract T cell activity. Here, we investigated the implications of T and T cells on dnDSA formation after renal transplantation (RTX). Considering T cells to be CXCR5 and IL-21, we found by flow cytometry that patients with dnDSA produced IL-21 more abundantly compared to healthy volunteers. In in vitro alloreactivity assays, patients with dnDSA featured an enhanced alloreactive T cell pool in response to donor-specific HLA antigens. Besides, longitudinal investigations suggested enhanced alloreactivity shortly after transplantation increasing the risk of dnDSA development. Taken together, in spite of continuous immunosuppression we report a strong IL-21 response in T cells and an expanded reservoir of donor-specific memory T cells in patients with dnDSA. This warrants further investigations if aberrant T cell activation may precede the formation of dnDSA and their inherent risk of promoting AMR.Copyright © 2021. Published by Elsevier Inc.