Background: Oncogenic alteration in RET, representing 1-2% of NSCLC, is an important target. Pralsetinib is a selective RET inhibitor targeting RET fusions. We present a case series on opportunistic infections and the mechanism of immunosuppression. Methods: From October 2021 to March 2022, we administered pralsetinib to a total of 15 patients with NSCLC harboring RET fusion. We retrospectively analyzed the clinical efficacy and adverse events related to pralsetinib. To investigate pralsetinib’s impact on T-cells, we examined cytokine release using ELISA from Jurkat T (JT) cells after pralsetinib treatment. To test if Stat5 binds to the IL-2 promoter, we conducted Chromatin Immunoprecipitation (ChIP). Results: Out of 18 patients with measurable disease, 14 (93%) achieved a partial response. With a median follow-up duration of 8.7 months, four cases of opportunistic infections occurred. Notably, three patients (16.6%) experienced invasive pulmonary aspergillosis, and one patient (5.5%) experienced cytomegalovirus pneumonia. To investigate mechanisms behind these observations, we aimed to replicate conditions similar to those of activated T cells. Thus, JT cells were treated with PMA and ionomycin to induce IL-2 release through NFAT, AP-1, and NF-κB binding to the IL-2 promoter. After ∼2 weeks of pralsetinib treatment, Jak3 signaling was inhibited, leading to decreased IL-2 release by downregulating transcription factors associated with IL-2 production, such as JunB/c-Jun and Stat5, which are downstream components of Jak3 signaling. Additionally, to investigate the direct correlation between Jak3 and IL-2 release, JT cells were treated with ritlecitinib, a Jak3-selective inhibitor, for ∼2 weeks. Like pralsetinib, a reduction in IL-2 release was observed. Furthermore, based on the evidence that Stat5 inhibition leads to reduced IL-2 release, we showed that Stat5, a direct downstream component of Jak3, binds to the IL-2 promoter during activation of Jurkat T cells. Conclusion: Pralsetinib, not Selpercatinib, inhibits additional IL-2 production by blocking Jak3/Stat5 activation triggered by IL-2 released during early T cell activation. Consequently, Jak3 inhibition by pralsetinib suppresses IL-2 release by inhibiting the activation of transcription factors for IL-2, such as JunB/c-Jun and Stat5, thereby inducing opportunistic infections, including invasive pulmonary aspergillosis (IPA), cytomegalovirus (CMV) pneumonia, CMV viremia, and pneumocystis pneumonia. Citation Format: Shinkyo Yoon, Hyun-Min Ryu, Eunjin Lee, Yunkyung Sung, Ho-Su Lee, Ji Eun Park, Chang Hoon Lee, Seyoung Seo, Sang-We Kim, Kang-Seo Park, Dae Ho Lee. Pralsetinib induces opportunistic infection in RET fusion-positive NSCLC patients via inhibition of IL-2 production by blocking Jak3/Stat5 activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3112.