Background: Immune checkpoint inhibitors (ICIs) such as anti-CTLA4 and anti-PD1 have revolutionized cancer therapy but can cause severe immune-related adverse events, notably immune checkpoint inhibitor-associated myocarditis (ICIM). Although clinically rare (0.06–1.14%), ICIM carries a mortality of up to 50%, highlighting the need for mechanistic insight. While ICI-induced immune activation is well characterized, the molecular drivers of myocarditis remain unclear. In previous work, we identified Guanylate-binding protein 5 (GBP5) as an ICIM-specific gene upregulated in vitro, in vivo, and in patient biopsies. GBP5 promotes NLRP3 inflammasome assembly and subsequent IL-1β and IL-18 release, but its precise role in ICIM pathogenesis remains to be defined.
Methods: We generated an inducible, conditional, cardiomyocyte-specific knock-in mouse, expressing a dysfunctional GBP5 molecule (GBP5-cKI), lacking the c-terminus which is necessary for NLRP3 assembly. To explore its role in cardiac inflammation during ICI therapy, tumor models were used a preclinical melanoma model, followed by treatment with anti-CTLA4 and anti-PD1 checkpoint inhibitors. Treated animal Interferon gamma was also so as to boost the autoimmune
Results:
Mice carrying the dysfunctional GBP5 knock-in exhibited mild to no myocarditis, with a clearly reduced rate of CD8+ and CD45+ cell infiltrates in the GBP5-cKI-animals’ hearts compared to treated controls (cell count 2.40/0.51 GBP5-cKI vs. wildtype (WT) controls; p=0.15). Cardiac function was largely preserved in treated knock-in animals (FS 24.92%±0.66 vs. 20,63%±1.18 GBP5-cKI vs. WT controls; p=0.021; EF 49.42%±1.09 vs. 42.20%±2.11 GBP5-cKI vs. WT controls; p=0.022; measured echocardiographically in anaesthetic animals). GBP5 knock-in (GBP5-cKI) mice exhibited attenuated inflammatory signaling compared with wild-type (WT) controls following ICI challenge. NF-κB pathway analysis revealed markedly reduced activation of key pro-inflammatory transcripts, including Il1b, Il18, and Ccrl2, in GBP5-cKI animals. In line with these findings, cytokine profiling demonstrated distinct transcriptional and proteomic signatures between WT and GBP5-cKI mice. Serum analysis showed robust induction of pro-inflammatory mediators such as Il6, Il1a, and Il10 in WT animals, which was substantially attenuated in GBP5-cKI mice. Protein profiling further revealed selective suppression of cytokines associated with inflammasome activation and Th1/Th17 signaling in GBP5-cKI mice, including Il1b, Il21, and Ifnl2. In contrast, WT animals displayed strong upregulation of Il2, Il5, Il31, and Il33, indicating a broad activation of both innate and adaptive immune pathways that was largely mitigated by GBP5 knock-in.
Implications: These findings identify GBP5 as a critical regulator of inflammasome activation and immune-mediated cardiac inflammation during ICI therapy. The protective phenotype observed in GBP5-cKI mice demonstrates that disruption of the GBP5-NLRP3 axis can effectively mitigate myocarditis without impairing systemic immune checkpoint activity.
