Is Toll-like Receptor 4 Required for Antibody Production?: Using Genetically Defined Mouse Models to Settle the Dispute 2260073

Abstract

Abstract Introduction Toll-like receptors (TLRs) play an integral role in activating immune pathways in response to the presence of a pathogen. Although mechanisms of action in mediating innate immune responses are well-researched, it is still unclear whether TLR expression regulates adaptive immunity. Data from current literature lead to conflicting conclusions. Some studies show that TLRs, specifically TLR4, are critical to initiating adaptive immune responses, and therefore the production of antibodies, while others indicate that TLR signaling is not required. Methods This study uses a two week prime-boost schedule of intramuscular vaccination using the model antigen ovalbumin (Ova) in wild-type (WT) and TLR4-deficient (TLR4-/-) mice. Serum ELISAs indicate that TLR4-/- mice (strain B6(Cg)-Tlr4tm1.2Karp/J) have significantly less antibody production than WT mice (strain C57BL/6) after vaccination with Ova. To investigate whether the lack of TLR4 could be overcome by immunoadjuvants, TLR4-/- mice (strain B6(Cg)-Tlr4tm1.2Karp/J) were injected with adjuvanted Ova on days 1 and 14. The adjuvants CpG DNA (TLR9 ligand), PHAD (TLR4 ligand), and Alum (alhydrogel) were used. Serum was collected and antibody production was measured on days 14 and 28. Results Serum ELISAs indicate extremely low levels of antibody. To confirm the lack of antibody production is due to the TLR4 deficiency, the adjuvant study was repeated in a different strain of WT (C3HeB/FeJ) and TLR4-/- mice (C3H/HeJ). Serum ELISA data is inconclusive due to the lack of antibody production exhibited in the WT mice. Conclusion Further studies must be performed to determine whether insufficient antibody production is a phenotype exhibited by all TLR4-/- mice. Identifying pathways that are critical in stimulating adaptive immunity will potentially lead to the development of more effective vaccines, especially in immunosuppressed individuals. Funding Source Towson University Office of Undergraduate Research and Creative Inquiry 2025 Summer Project Grant and 2024 Fisher Endowed Chair of Biology Grant. Topic Categories Vaccines and Immunotherapy (VAC)