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ABSTRACT Vaccine development requires innovative approaches to improve immune responses while reducing the number of immunizations. In this study, we explore the impact of controlled antigen release on immune activation and regulation using programmable infusion pumps and biodegradable biomaterials in OT‐II and wild‐type mice to understand the adaptive immune response through controlled antigen delivery in the absence of adjuvant. Ovalbumin (OVA) was delivered via an exponentially decreasing profile, mimicking clearance of infection, and an exponentially increasing profile, mimicking induction of infection. Exponentially decreasing OVA delivery through infusion pumps promoted regulatory T‐cell (Treg) activation in secondary lymphoid organs and suppressed pro‐inflammatory T‐helper type 17 (Th17) responses in blood. An exponentially increasing OVA profile enhanced central memory T‐cell (TCM) populations in submandibular blood and humoral immune responses in cardiac blood serum, demonstrating distinct immune modulation based on release kinetics. OVA was also delivered using a biodegradable PLGA‐PEG‐PLGA (PPP) depot, which provided controlled OVA release in an exponentially decreasing pattern. PPP‐OVA treatment significantly reduced the frequency of pro‐inflammatory T‐helper type 1 (Th1) cells while increasing CD25⁺FOXP3⁺Treg cells in the spleen. Moreover, to identify T‐cell populations that most accurately characterize the divergence in Treg and T‐helper response to OVA kinetics, a Sequential Feature Selection (SFS) algorithm with Machine Learning (ML) models was used. ML algorithms identified gMFI of RORγt⁺as a key feature in submandibular blood and the ratio of gMFI of FOXP3⁺to GATA3⁺as the marker that was significantly changed by treatments in inguinal lymph nodes (iLN) when infusion pumps were used to deliver OVA. In addition, ML‐based SFS identified CD25⁺FOXP3⁺regulatory T cells as the most important feature, influencing the expression of other cell types in both inguinal lymph nodes (iLN) and spleen when PPP was used to deliver OVA. This finding suggests that the exponentially decreasing profile may generate anti‐inflammatory responses. Overall, these findings suggest that controlled antigen delivery enhances immune regulation and memory T cells, providing new insights into immune responses mediated by the release kinetics. 

Abstract Cells employ multiple systems to maintain cellular integrity, including mechanosensitive ion channels and the cell wall integrity (CWI) pathway. Here, we use pollen as a model system to ask how these different mechanisms are interconnected at the cellular level. MscS-Like 8 (MSL8) is a mechanosensitive channel required to protect Arabidopsis thaliana pollen from osmotic challenges during in vitro rehydration, germination, and tube growth. New CRISPR/Cas9 and artificial miRNA-generated msl8 alleles produced unexpected pollen phenotypes, including the ability to germinate a tube after bursting, dramatic defects in cell wall structure, and disorganized callose deposition at the germination site. We document complex genetic interactions between MSL8 and two previously established components of the CWI pathway, MARIS and ANXUR1/2. Overexpression of MARISR240C-FP suppressed the bursting, germination, and callose deposition phenotypes of msl8 mutant pollen. Null msl8 alleles suppressed the internalized callose structures observed in MARISR240C-FP lines. Similarly, MSL8-YFP overexpression suppressed bursting in the anxur1/2 mutant background, while anxur1/2 alleles reduced the strong rings of callose around ungerminated pollen grains in MSL8-YFP overexpressors. These data show that mechanosensitive ion channels modulate callose deposition in pollen and provide evidence that cell wall and membrane surveillance systems coordinate in a complex manner to maintain cell integrity.