Adrenergic nerves regulate B cell responses to large antigens through modulation of lymph node permeability

Technical Summary: Adrenergic Nerves Regulate B Cell Responses to Large Antigens via Lymph Node Permeability

Problem Statement
Adrenergic nerves innervate all secondary lymphoid organs, yet their specific role in adaptive immune responses remains unclear. While systemic depletion of these nerves is known to compromise germinal center (GC) formation after infection or vaccination, the local mechanisms by which they influence B cell responses are not defined. A critical gap in understanding exists regarding how the lymph node (LN) conduit system—a reticulin fiber network typically restricted to molecules smaller than 70 kDa—handles large antigens. It is unknown whether this system gains permeability to high molecular weight (HMW) draining antigens during an immune response and if adrenergic signaling drives this process. This is clinically relevant as patients with peripheral nerve injuries exhibit susceptibility to recurrent infections, suggesting a defect in immune memory.

Methodology
The study employed a combination of local and systemic nerve depletion models in C57Bl/6 mice to isolate the effects of adrenergic nerves on LN physiology.

• Nerve Depletion: Two approaches were used: (1) Systemic chemical sympathectomy using 6-hydroxydopamine (6OHDA) and (2) Local depletion using a TH-Cre iDTR mouse model treated with diphtheria toxin (DT) in a specific footpad. This local method allowed for the ablation of tyrosine hydroxylase (TH)+ adrenergic fibers in the draining popliteal LN while sparing non-draining nodes and systemic physiology.
• Antigen Models: Mice were vaccinated with small antigens (NP-Ovalbumin, ~45 kDa) or large antigens (NP-Keyhole Limpet Hemocyanin, ~450 kDa; Staphylococcus aureus ~100 gigadaltons).
• Permeability Assays: To assess conduit system permeability, mice were injected with fluorescent dextrans of varying sizes (2 kDa FITC and 70 kDa TRITC) into the footpad 7 days post-vaccination or infection.
• Structural Analysis: Serial sections of LNs were stained with a modified Gomori's reticulum stain to visualize reticulin fibers and trabeculae. Fiber diameter and network conformation were quantified.
• Immunological Readouts: Germinal center formation was assessed via flow cytometry (GL-7, Bcl-6, CXCR5, PD-1) and immunofluorescence. Antibody titers (anti-NP IgG) were measured by ELISA, and plasma cell generation was quantified via ELISPOT. Antigen accumulation in GCs was visualized using FITC-Ova and Crimson bead-conjugated KLH (Red-KLH).

Key Contributions and Results

1. Vaccination-Induced Remodeling of the Conduit System:
   The study demonstrated that vaccination or infection induces a structural remodeling of the LN reticulin fiber network. In nerve-intact LNs, this remodeling creates a "loose, net-like" conformation of trabeculae with an average diameter of 2.2–2.6 µm (compared to ~1.4 µm in PBS controls). This expansion allows HMW molecules (70 kDa dextran, NP-KLH) to penetrate from the subcapsular sinus (SCS) into the B cell follicles and the conduit system. In contrast, low molecular weight molecules (2 kDa dextran, NP-Ova) access the conduit system regardless of this remodeling.

2. Adrenergic Nerves are Required for Remodeling and HMW Permeability:
   Local depletion of adrenergic nerves prevented the expansion of trabeculae. In nerve-depleted LNs, the reticulin network remained in a compact, "closed" conformation (~1.5 µm diameter). Consequently, 70 kDa dextran and large antigens were restricted to the SCS and could not access the cortex or B cell follicles, whereas 2 kDa dextran access remained unaffected. This indicates that local adrenergic signaling is necessary to render the conduit system permeable to large draining antigens.

3. Differential Impact on B Cell Responses:
   The inability of large antigens to access the LN cortex in the absence of nerves led to specific defects in B cell immunity:

   • Large Antigens (NP-KLH): Local nerve depletion significantly impaired germinal center formation, reduced T follicular helper (Tfh) cells, and diminished the accumulation of large antigens within developing GCs. While primary antibody titers were comparable, the secondary (recall) antibody response was blunted in nerve-depleted mice.
   • Small Antigens (NP-Ova): The germinal center response, Tfh cell numbers, and antibody titers for small antigens were largely unaffected by nerve depletion, as these antigens do not require conduit remodeling for access.
   • Rescue by Antigen Dose: Increasing the dose of a large antigen (Big NP-Ova conjugated to 2 µm beads) from 10 µg to 100 µg rescued the germinal center formation and recall antibody response in nerve-depleted mice, suggesting that the defect is one of antigen availability/access rather than a fundamental inability to respond.

4. Antigen Accumulation in Germinal Centers:
   In nerve-intact mice, both small (FITC-Ova) and large (Red-KLH) antigens accumulated in GCs. In nerve-depleted mice, while small antigens accumulated normally, the accumulation of large antigens (Red-KLH) in GCs was substantially reduced, correlating with the impaired GC response.

Significance and Claims
The paper claims that adrenergic nerves play a critical, local role in remodeling the lymph node's reticulin fiber network to permit the entry of large antigens into the B cell follicles. This permeability is essential for optimal B cell responses to large antigens, including the formation of germinal centers and the generation of robust memory responses.

The authors posit that these findings provide a mechanistic explanation for why patients with peripheral nerve injuries or dysfunction are more susceptible to recurrent infections: the failure of nerve-mediated remodeling prevents large antigens (and potentially pathogens) from accessing the conduit system and germinal centers, thereby impairing the priming of adequate memory B cell responses. The study concludes that the physical expansion of the trabecular network is a conserved attribute of responding LNs driven by adrenergic signaling, distinct from the systemic effects of stress or blood pressure changes.