Critical role of 4-1BB:4-1BBL axis in humoral immune responses against Plasmodium

Introduction/Rationale: Several costimulatory and coinhibitory signaling axis have been reported to modulate anti-Plasmodium immune response, albeit the precise role of 4-1BB:4-1BBL axis has yet to be investigated.

Methods: We used 4-1BB KO and 4-1BBL KO mice to investigate the effects of each receptor during Plasmodium infection. To temporally modulate 4-1BBL signaling, we treated WT mice with a 4-1BBL blocking antibody (TKS-1) at different timepoints. Considering the scenario of 4-1BB binding to Galectin-9 (GAL9) as previously reported, certain experiments involve treating WT mice with GAL9 blocking antibody. To understand the functional significance of 4-1BB-4-1BBL axis, we also performed single cell RNA-, V(D)J-, and CITE-seq on sort-purified WT, 4-1BB and 4-1BBL KO T and B cells sourced from three-way bone marrow chimeric mice infected with a rodent-specific Plasmodium strain.

Results: Here, we show that complete loss of 4-1BB resulted in delayed parasite control, impaired GC responses and memory B cell recall. The absence of 4-1BBL, however, resulted in enhanced parasite control without influencing the GC response. Early blockade of 4-1BB:4-1BBL signaling by using TKS-1 recapitulated 4-1BB KO data, an effect that was absent when 4-1BBL was blocked at effector time points. While exploring the possibility of other 4-1BB binding partners such as GAL9, we observed that combined blockade of GAL9 and 4-1BBL resulted in loss of parasite control as well as loss of protection following lethal challenge, similar to 4-1BB KO mice.

Conclusion: While these data show a preferential binding of 4-1BB to 4-1BBL mediating an inhibitory effect, our data suggest that 4-1BB can bind to GAL9 in the absence of 4-1BBL and potentiate stimulatory signals. Our future experiments are directed towards understanding the precise and differential role of ligands of 4-1BB and their temporal effects in governing anti-Plasmodium humoral immunity.