Pseudomonas aeruginosa is a major human opportunistic pathogen associated with a high incidence of multi-drug resistance. The antibody-based blockade of P. aeruginosa virulence factors represents a promising alternative strategy to mitigate its infectivity. In this study, we employed single B cell sorting from cystic fibrosis patients to isolate human monoclonal antibodies (mAbs) targeting proteins from the P. aeruginosa Type 3 Secretion System (T3SS) and characterized a panel of mAbs directed at PscF and PcrV. Among those, two mAbs, P5B3 and P3D6, that bind to the injectisome tip protein PcrV, exhibited T3SS blocking activity. We solved the crystal structure of the P3D6 Fab-PcrV complex, which revealed that the Ab binds to the C-terminal region of PcrV. In addition, we compared the T3SS-blocking activity of three PcrV-targeting mAbs, including two from previous independent studies, using two distinct assays to evaluate pore formation and toxin injection. We conducted a mechanistic and structural analysis of their modes of action through modeling based on the known structure of a functional homolog, SipD from Salmonella typhimurium. The analysis suggests that anti-PcrV mAbs may act through different mechanisms, ranging from preventing PcrV oligomerization to disrupting PcrV's scaffolding function, thereby inhibiting the assembly and function of the translocon pore. Our findings provide additional evidence that T3SS-targeting Abs, some capable of inhibiting virulence, are elicited in P. aeruginosa-infected patients. The results offer deeper insights into PcrV recognition by mAbs and their associated mechanisms of action, helping to identify which Abs are more likely to be therapeutically useful based on their mode of action and potency. This paves the way for the development of effective alternatives to traditional antibiotics in the fight against this resilient pathogen.
© 2025, Desveaux, Faudry et al.

Immunoglobulin A (IgA) is the most abundantly produced antibody isotype and mediates protection and homeostatic regulation at mucosal surfaces. Steady-state IgA production is supported by multiple pathways, including chronic germinal centers in gut inductive lymphoid tissues. However, we lack a detailed understanding of how IgA responses are temporally integrated across inductive and effector sites. Here, we dissect homeostatic IgA responses from the perspective of clonal repertoires in inductive compartments and the gut lamina propria as the main effector compartment. We show that unique clonal patterns dominate across gut inductive sites and that plasma cell (PC) clones in gut lamina propria entail progressive stages of differentiation. We demonstrate that ongoing diversification of recurrent clones continuously seeds the gut PC population. These observations suggest that clonal rather than cellular longevity shapes IgA responses and that dynamic modulation of recurrent clones may balance stability and flexibility of the gut PC repertoire.
Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.

Syndecans (SDCs) are glycosaminoglycan-containing cell surface proteins with diverse functions in the immune system with SDC1 (CD138) and SDC4 expressed in B-lineage cells. Here, we show that stem cells lacking either molecule generate fewer B-cell progenitors but give rise to mature B cells in vivo. Deletion of the plasma cell "marker" CD138 has no effect on homeostatic or antigen-induced plasma cell formation. Naive B cells express high SDC4 and encounter with cognate antigen results in transient CD138 upregulation and SDC4 loss, both further modulated by IL-4, IL-21, and CD40 ligation. SDC4 is downregulated on germinal center B cells and absent on most memory B cells. Glycosaminoglycans such as those attached to SDCs, and heparin, a commonly used therapeutic, regulate survival and activation of naive B cells by limiting responsiveness to cognate antigen. Conversely, ablation of SDC4 results in increased baseline and antigen-induced B-cell activation. Collectively, our data reveal B-cell activation- and subset-dependent SDC expression and show that SDC4 and GAGs can limit antigen-induced activation to promote B-cell survival and expansion.
© 2025. The Author(s).

Long-lived humoral memory is key to durable immunity against pathogens yet remains challenging to define due to heterogeneity among antigen-reactive B cells. We addressed this gap through longitudinal sampling over the course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccinations with or without breakthrough infection. High-dimensional phenotypic profiling performed on ∼72 million B cells showed that receptor-binding domain (RBD) reactivity was associated with five distinct immunoglobulin G (IgG) B cell populations. Two expressed the activation marker CD71, both correlated with neutralizing antibodies, yet the one lacking the memory marker CD27 was induced by vaccination and blunted by infection. Two were resting memory populations; one lacking CD73 arose early and contributed to cross-reactivity; the other, expressing CD73, arose later and correlated with neutralizing antibodies. The fifth, a rare germinal center-like population, contributed to recall responses and was highly cross reactive. Overall, robust and distinct responses to booster vaccination overcame the superiority of hybrid immunity provided by breakthrough infection.
Published by Elsevier Inc.

Immune memory is influenced by the frequency and type of antigenic challenges. Here, we performed a cross-sectional comparison of immune parameters following a BA.1 breakthrough infection in individuals with prior hybrid immunity (conferred by infection and vaccination) versus those solely vaccinated in a cohort of health care workers in Lyon, France. The results showed higher levels of serum anti-receptor binding domain (RBD) antibodies and neutralizing antibodies against BA.1 post-infection in the vaccine-only group. Individuals in this group also showed a decrease in memory B cells against the ancestral strain but an increase in those specific and cross-reactive to BA.1, suggesting a more limited immune imprinting. Conversely, hybrid immunity prevents the decrease in antibody dependent cellular cytotoxicity (ADCC) response, possibly by limiting IgG4 class-switching and enhanced anti-N responses post-infection. This highlights that BA.1 breakthrough infection induces different immune responses depending on prior history of vaccination and infection, which should be considered for further vaccination guidelines.
© 2025 The Authors.