Protective Extrafollicular Responses are Driven by Intrinsic and Extrinsic TLR-Mediated Signals
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Protective Extrafollicular Responses are Driven by Intrinsic and Extrinsic TLR-Mediated Signals


Affinity for antigen is not only crucial for antibodies’ protective capacity but is also considered the main inducer of plasma cells that secrete them. This has been demonstrated through use of model antigens where reductions in epitope valency or disruptive conformational alteration led to reductions in formation of antibody-secreting cells (ASCs). Surprisingly, the presence of abundant antigen does not always lead to high-affinity plasma cell generation such as during Salmonella and Ehrlichia infections. During these challenges, extrafollicular B cell responses (EFRs), foci of local, rapidly-formed, and short-lived ASCs known as plasmablasts, dominate the antibody response but fail to generate antigen-specific antibodies. This coincides with the kinetics of affinity maturation towards hapten antigen, where early antibody responses are of lower affinity than ones generated by highly matured germinal center (GC) reactions. Contradictory to this, antibody affinity towards vesicular stomatitis virus (VSV) antigen in mice does not increase over the course of infection, indicating a lack of overall affinity maturation within the anti-VSV repertoire. Additionally, mice either infected or inoculated intravenously (i.v.) with influenza generate high-affinity and neutralizing antibodies against hemagglutinin (HA) before formation of GCs, indicating that under certain physiological circumstances, EFRs likely generate early, protective antibodies. Given annual influenza outbreaks and the current COVID-19 pandemic, it is of the utmost interest to generate protective antibodies as quickly as possible, as vaccines generally elicit protective serum antibody levels only after multiple weeks or multiple boosts. Using influenza infection and immunization in mice as a model, we sought to characterize EFRs, determine the signals responsible for their generation, and ascertain the protective capacity of EFR-derived antibodies. Firstly, this work demonstrates EFRs generated after influenza infection produce fully protective antibodies, independent of GCs, while subcutaneous immunization with influenza virion in alum produced no EFRs. This indicated that infection-induced signals were required for EFRs generation and sustainment. Single knockout of a plethora of inflammatory and anti-viral signaling pathways had no impact on early EFRs formation. Only did complete loss of Toll-like receptor (TLR) signaling result in changes to EFR dynamics in both a B cell intrinsic and extrinsic manner, with complete deletion of TLR adaptors MyD88 and TRIF leading to both defective early EFR formation and loss of immune protection. Therefore, TLR-derived signaling is required for optimal generation of EFRs against influenza. Intrinsically, B cells required the presence of TLR components, from either MyD88/TRIF or unc93b and TLR2/4, for antigen-stimulated survival and expansion in vitro. This correlated with the inability of TLR-null B cells to upregulate IRF4, the master transcription factor of B cell differentiation. Additionally, TLR-null B cells had dysfunctional nuclear localization of c-Rel, an NF-kB factor that regulates IRF4 expression, and could not sustain upregulated c-Rel expression post-activation. Thus, B cells require TLR-mediated activation of c-Rel for survival and expansion after antigen-mediated stimulation, even in the absence of explicit TLR ligands, and likely need additional inflammatory stimuli to overcome abrogation of this signaling circuit. While subcutaneous immunization with alum-adjuvanted influenza virus particles produced no EFRs, boosting with a lipopolysaccharide (LPS), a TLR4 agonist that activates both TLR signaling adapters MyD88 and TRIF, led to robust, antigen-specific EFRs. Boosting with both influenza virus and LPS longer-term suppressed antigen-specific GC responses while enhancing antigen-specific EFRs, generating higher concentrations of antigen-specific serum antibodies which led to greater protection against lethal influenza infection than serum from mice boosted with virion alone. Thus, EFRs are an un-tapped source for kinetically advantageous, protective antibodies whose effectors form from a crucible of antigen and innate, inflammatory signals. This work not only elucidates crucial inputs of EFR generation, but also further clarifies the nature of B cell responses, which should be applied to other physiological and disease contexts.

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