Harnessing the potential of the NALT and BALT as targets for immunomodulation using engineering strategies to enhance mucosal uptake

Abstract

Mucosal barrier tissues and their mucosal associated lymphoid tissues (MALT) are attractive targets for vaccines and immunotherapies due to their roles in both priming and regulating adaptive immune responses. The upper and lower respiratory mucosae, in particular, possess unique properties: a vast surface area responsible for frontline protection against inhaled pathogens but also simultaneous tight regulation of homeostasis against a continuous backdrop of non-pathogenic antigen exposure. Within the upper and lower respiratory tract, the nasal and bronchial associated lymphoid tissues (NALT and BALT, respectively) are key sites where antigen-specific immune responses are orchestrated against inhaled antigens, serving as critical training grounds for adaptive immunity. Many infectious diseases are transmitted via respiratory mucosal sites, highlighting the need for vaccines that can activate resident frontline immune protection in these tissues to block infection. While traditional parenteral vaccines that are injected tend to elicit weak immunity in mucosal tissues, mucosal vaccines (i.e., that are administered intranasally) are capable of eliciting both systemic and mucosal immunity in tandem by initiating immune responses in the MALT. In contrast, administering antigen to mucosal tissues in the absence of adjuvant or costimulatory signals can instead induce antigen-specific tolerance by exploiting regulatory mechanisms inherent to MALT, holding potential for mucosal immunotherapies to treat autoimmunity. Yet despite being well motivated by mucosal biology, development of both mucosal subunit vaccines and immunotherapies has historically been plagued by poor drug delivery across mucosal barriers, resulting in weak efficacy, short-lived responses, and to-date a lack of clinical translation. Development of engineering strategies that can overcome barriers to mucosal delivery are thus critical for translation of mucosal subunit vaccines and immunotherapies. This review covers engineering strategies to enhance mucosal uptake via active targeting and passive transport mechanisms, with a parallel focus on mechanisms of immune activation and regulation in the respiratory mucosa. By combining engineering strategies for enhanced mucosal delivery with a better understanding of immune mechanisms in the NALT and BALT, we hope to illustrate the potential of these mucosal sites as targets for immunomodulation.

Keywords: adaptive immunity; antigen specific immunotherapy (ASIT); bronchus associated lymphoid tissue (BALT); drug delivery; germinal center (GC); mucosal vaccine; nasal associated lymphoid tissue (NALT); secretory IgA (SIgA).

Figure 1

UPPER RESPIRATORY TRACT. (A) Nasal anatomy and location of NALT: Murine nasal anatomy consists of the naso-, ethmo-, and maxilo- turbinates (NT, ET, MT, respectively). The nasal-associated lymphoid tissue (NALT) is found medially in the hard palate, positioned proximal to the ethmoturbinates. (B) Germinal center formation and immune priming in NALT (1). Antigen uptake across follicle-associated epithelium (FAE) results in (2) activated B cell class switch recombination (CSR) in the subepithelial dome (SED) signaled by DCs or by T cells. Class switched B cells preferentially form IgA through TGF-b signaling, (and (3) seed germinal centers (GCs) in the B cell zone of the NALT where somatic hypermutation (SHM) occurs (4). B cells within the GC compete for T follicular helper (Tfh) cell signaling which results either in the return to GCs or (5) B cell differentiation towards memory B cells, plasma cells, or apoptosis (6). Differentiated B cells migrate through high endothelial venules (HEVs) and lymphatics to turbinates where they reside as IgA-secreting cells. (Created with BioRender.com).

Figure 2

LOWER RESPIRATORY TRACT. (A) BALT follicular anatomy in lungs: Lymphocytes home to the bronchus subepithelial space via lymphatic vessels and HEVs where they form an ectopic lymphoid structure known as the bronchus associated lymphoid tissue (BALT). This tissue is characterized by densely packed B cell follicles with distinct germinal center (GC) behavior. (B) Outcomes of BALT induction: The BALT plays a positive protective role against respiratory pathogens via antibody (IgA, IgG) clearance. However, during prolonged chronic inflammation, BALT is associated with harmful conditions such as autoimmunity and cancer. (C) Timeline of BALT formation and degradation in humans: Infant BALT often forms in fetal lungs, reaches maximum size by year two to three, and fully degrades by year four or five. As an adult, BALT can be transiently induced by respiratory challenge but will degrade following clearance and resolution. However, chronically induced BALT that persists over time is a marker for harmful chronic immune activation and inflammation in the bronchials. (Created with BioRender.com).

Figure 3

Barriers and uptake mechanisms in the respiratory epithelium: The transport of antigens across respiratory epithelial cells (ECs) is controlled through dense, negatively charged cilia that repel negatively charged antigens to prevent adhesion. (A) ECs express pattern recognition receptors that signal to dendritic cells (DCs) the presence of infection and (B) triggers them to extend transepithelial dendrites (TEDs) to sample the lumenal space. (C) Antigen uptake also occurs by microfold (M) cells. Class switch recombination in B cells can be induced by (D) ECs or (E) DCs, followed by (F) potential DC migration to MALT or draining cervical or mediastinal lymph nodes (dLNs). (Created with BioRender.com)| .

Figure 4

Mucosal vaccine approaches: All mucosal vaccines that are clinically approved are based on live attenuated or inactivated pathogens; however, ongoing clinical trials are investigating other strategies to overcome challenges associated with pathogenic vaccines. (A) Live attenuated virus infects the host cell, undergoes replication, and uses cellular machinery to produce viral antigens. (B) Inactivated virus is phagocytosed by APCs, broken down into viral antigens, then presented on the cell surface. (C) Vaccine genome is encapsulated into an unrelated viral vector that infects the host cell then uses cellular machinery to produce viral antigens. (D) Genetic content from the vaccine (either DNA or RNA) is encapsulated in lipid nanoparticles or cationic polymers for cellular uptake followed by translation of viral proteins. (E) Subunit vaccines can utilize two delivery mechanisms: Particulate vaccine containing virus-like particles is phagocytosed by APCs and digested into viral antigen fragments followed by antigen presentation on the cell surface. Or, recombinant and/or particulate subunit vaccines can directly bind to the B cell receptor (BCR) on the surface of B cells followed by receptor-mediated endocytosis and antigen presentation on the cell surface. (Created with BioRender.com).

Figure 5

Engineering approaches to enhance active or passive uptake across the respiratory mucosa: (A) Protein/peptide antigens conjugated to an amphiphile tail that ‘hitchhike’ on albumin and (B) Fc-fusion proteins consisting of protein antigen fused directly to IgG Fc fragment are transcytosed across respiratory mucosa by binding the neonatal Fc receptor (FcRn). (C) Protein antigens tethered to secretory IgA (sIgA) are transcytosed across respiratory mucosa by binding to Dectin-1 on the surface of M cells. (D) Protein antigens fused to C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) are transcytosed across respiratory mucosa by binding the tight junction protein Claudin-4 on the surface of M cells. (E) Polyethyleneimine (PEI) reversibly opens tight junctions and increases uptake of cyclodextrin-conjugated PEI mRNA polyplexes across the respiratory mucosa. (F) Inclusion of polyethylene glycol (PEG) into PACE-mRNA polyplexes increases mucus transport and therefore increases passive uptake across the respiratory mucosa. (Created with BioRender.com).

Figure 6

Protective and pathogenic mechanisms of IgA in autoimmunity. Protective – (A) Immune exclusion: IgA binds viral or bacterial pathogens in the mucosa, forming immune complexes to prevent pathogenic entry into or across the mucosal epithelium. (B) Pathogenic clearance: IgA clears pathogens that cross or infect the mucosal epithelium. (C) Induction of tolerogenic DCs: sIgA induces tolerogenic DCs via ICAM3 signaling. Pathogenic – (D) Compromised mucosal barrier: sIgA deficiency in combination with breaks in the mucosal barrier can allow for pathogen entry and infection. (E) Inflammation: IgA immune complexes in the lamina propria and serum can lead to crosslinking activation of CD89/FcαRi on monocytes and subsequent inflammation, contributing to autoimmune pathogenesis. (F) Anti-IgA autoantibodies: often IgG, can cause IgA deficiency. ITAMi and ITAM Signaling: Monomeric engagement of CD89 leads to ITAMi signaling and cell inhibition. Multivalent crosslinking of CD89 by IgA immune complexes with moderate avidity leads to ITAM signaling and cell activation. IgA affinity for CD89 varies based on isoform. (Created with BioRender.com).