<p>Vaccine adjuvants typically contain pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) that activate innate immune cells. The FDA’s approval of multiple PAMP-DAMP combinations for clinical use has generated increased momentum in this field in recent years. Here, we report the use of DAMP-inducing peptide nanofibers (PNFs) and CL429 (PAMP) combinations as subunit boosters for Bacille Calmette-Guérin (BCG). We demonstrate that pulmonary boosting with PNFs and CL429 enhances the lung-resident memory phenotype, effector cytokine profiles, and transcription factor bias of antigen-specific CD4⁺ T cell populations compared to PNFs alone. Importantly, the combination significantly improved the frequency of lung-specific memory T cells, which have been shown to provide superior protection compared to circulating memory T cells. Interestingly, the T helper (Th) subset profile was driven in part by the vaccination route, resulting in a Th17 bias via the mucosal route or a Th1 bias when delivered intravenously. We show that, following pulmonary administration, lung-resident antigen-presenting cells (APCs) efficiently internalize PNFs and upregulate crucial co-stimulatory markers that promote T cell priming and activation. Our findings suggest that heterologous booster vaccines composed of DAMP-inducing PNFs and PAMP combinations can engage both innate and adaptive immunity to generate memory T cells that protect against tuberculosis (TB) and potentially other respiratory diseases.</p> Graphical Abstract <p></p>