Cognate T-cell labeling by conventional and unconventional antigen-presenting cells in vivo using a modular SrtA-based toolkit

While different antigen-presenting cell (APC) populations can drive divergent T-cell fates even when presenting identical antigens (Ags), our ability to trace these specific in vivo interaction histories remains constrained by a lack of compatible molecular tools. Here, we describe an enhanced Labeling Immune Partnerships by SorTagging Intercellular Contacts (LIPSTIC) platform utilizing the immunologically inert human CD58/CD2 receptor-ligand pair (SrtA-hCD58/G5-hCD2). This system provides a modular alternative to previous LIPSTIC animal models and enables the detection and recovery of T cells following Ag-dependent interactions with distinct APC populations in vitro and in vivo. We applied this approach to interrogate the role of type 3 innate lymphoid cells (ILC3s), an unconventional ROR{gamma}t Ag-presenting population whose role in shaping Ag-specific T cells in vivo remain poorly defined. Modified LIPSTIC successfully recorded cognate interactions between ILC3s and naive CD4 T cells in the spleen and enabled direct comparison with CD4+ T cells activated by conventional dendritic cells (cDCs). Although interactions with ILC3s resulted in lower frequency of in vivo-labeled T cells than cDC2s, CD4+ T cells showed comparable cell division and expression of activation markers irrespectively of an APC type. Together, these findings establish SrtA-hCD58/G5-hCD2 as a versatile LIPSTIC receptor-ligand pair for tracking cell-cell interactions in vivo and provide a robust tool for studying how APC identity influences Ag-specific T-cell responses.