We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

Adjuvants can affect APCs function and boost adaptive immune responses post-vaccination. However, whether they modulate the specificity of immune responses, particularly immunodominant epitope responses, and the mechanisms of regulating antigen processing and presentation remain poorly defined. Here, using overlapping synthetic peptides, we screened the dominant epitopes of Th1 responses in mice post-vaccination with different adjuvants and found that the adjuvants altered the antigen-specific CD4⁺ T-cell immunodominant epitope hierarchy. MHC-II immunopeptidomes demonstrated that the peptide repertoires presented by APCs were significantly altered by the adjuvants. Unexpectedly, no novel peptide presentation was detected after adjuvant treatment, whereas peptides with high binding stability for MHC-II presented in the control group were missing after adjuvant stimulation, particularly in the MPLA- and CpG-stimulated groups. The low-stability peptide present in the adjuvant groups effectively elicited robust T-cell responses and formed immune memory. Collectively, our results suggest that adjuvants (MPLA and CpG) inhibit high-stability peptide presentation instead of revealing cryptic epitopes, which may alter the specificity of CD4⁺ T-cell-dominant epitope responses. The capacity of adjuvants to modify peptide–MHC (pMHC) stability and antigen-specific T-cell immunodominant epitope responses has fundamental implications for the selection of suitable adjuvants in the vaccine design process and epitope vaccine development.