Abstract: Our study delves into the intricate interplay between Src homology region 2 domain-containing phosphatases 1 and 2 (SHP-1 and SHP-2) in orchestrating the functionality of CD8+ T cells during both acute and chronic lymphocytic choriomeningitis virus (LCMV) infection. Through the utilization of gene knockout mouse models, we elucidate the distinct yet complementary roles of SHP-1 and SHP-2 in regulating effector function and mitigating T cell exhaustion, both in vitro and in vivo. These findings provide valuable insights into potential therapeutic avenues for chronic viral infections and tumor immunotherapy.Introduction: The modulation of CD8+ T cell responses in immunological contexts, particularly in the realm of viral infections and tumor immunology, demands a deeper understanding of the underlying molecular pathways. While SHP-1 has been recognized as a negative regulator of T cells and SHP-2 primarily associated with PD-1 signaling, recent discoveries suggest a non-redundant functionality for these phosphatases, particularly in the realm of T cell exhaustion.
Study Design: Led by G.F. and B.H., our research team adopted a comprehensive approach to delineate the contributions of SHP-1 and SHP-2 in CD8+ T cell regulation during LCMV infection. Leveraging gene knockout technologies and immunological assays, we meticulously dissected the stage-specific functions of these phosphatases.
Mice models including SHP-1 conditional knockout (SHP-1 cKO), SHP-2 conditional knockout (SHP-2 cKO), and double knockouts for both SHP-1 and SHP-2 (DKO) were generated and bred at Xiamen University. The LCMV model was employed to mimic acute and chronic viral infections, with mice subjected to thorough behavioral, immunopathological, and viral-specific T cell response assessments.
All experimental procedures adhered to the guidelines provided by the Institutional Animal Care and Use Committee of Xiamen University, ensuring ethical treatment of the animals involved. Quantitative RT-PCR was employed to analyze virus titers, and histology images were evaluated to assess pathology.
This study provides a comprehensive elucidation of the roles of SHP-1 and SHP-2 in CD8+ T cells, particularly in the context of T cell exhaustion, with implications for chronic infection management and antitumor immune responses. Contributions from a diverse array of researchers and guidance from H.-R.W., B.X., and N.R.J.G. were instrumental in shaping the manuscript, with no influence from funders on the study design, data collection, analysis, or publication decisions.
Materials and Methods: We utilized conditional knockout (cKO) and double knockout (DKO) mouse models to systematically explore the roles of SHP-1 and SHP-2 in CD8+ T cell responses during LCMV infection. Our experimental design aimed to decipher the impact of these phosphatases on both effector function and exhaustion of T cells. Ethical approval for animal experimentation was secured from the relevant Institutional Review Board.
Results: Our comprehensive investigation into the roles of SHP-1 and SHP-2 in CD8+ T cell regulation during lymphocytic choriomeningitis virus (LCMV) infection revealed multifaceted dynamics underlying T cell functionality across different infection stages.
During acute LCMV infection, SHP-1 emerged as a critical negative regulator of CD8+ T cell effector function. Mice lacking SHP-1 displayed heightened effector responses characterized by increased cytokine production and cytotoxic activity. Conversely, SHP-2 deficiency did not significantly impact effector function during this early phase of infection. The distinct roles of SHP-1 and SHP-2 were further underscored by the observation that double knockout (DKO) mice, deficient in both phosphatases, exhibited an intermediate phenotype, suggesting partial compensation or functional redundancy between SHP-1 and SHP-2 during acute infection.
As the infection progressed to the chronic phase, the regulatory landscape shifted. While SHP-1 continued to exert a modulatory influence on CD8+ T cell responses, its significance diminished compared to the acute phase. Notably, SHP-2 deficiency now revealed its distinct impact, leading to enhanced T cell exhaustion characterized by elevated expression of exhaustion markers such as PD-1, Tim-3, and Lag-3. The DKO mice, lacking both SHP-1 and SHP-2, displayed a unique phenotype characterized by prolonged survival without immunopathology, indicating a complex interplay between these phosphatases in shaping the course of chronic viral infection.
Further analysis of T cell functionality in DKO mice revealed an unexpected responsiveness to PD-1 blockade therapy. Despite the absence of SHP-1 and SHP-2, exhausted CD8+ T cells retained their ability to respond to immunomodulatory interventions, suggesting the existence of alternative regulatory mechanisms or compensatory pathways that enable therapeutic intervention even in the absence of canonical negative regulators.
Histological examination of tissue samples provided additional insights into the pathological consequences of SHP-1 and SHP-2 deficiency. While acute infection in SHP-1 knockout mice was associated with minimal immunopathology, chronic infection in SHP-2-deficient mice exhibited signs of exacerbated tissue damage, indicating distinct roles for SHP-1 and SHP-2 in modulating immunopathological outcomes.
Discussion: This study offers significant insights into the differential contributions of SHP-1 and SHP-2 throughout the course of CD8+ T cell-mediated antiviral responses. While SHP-1 emerges as a prominent negative regulator during acute infection, its involvement becomes more stage-specific in chronic infection settings. The survival observed in the double deficiency scenario suggests a complex, non-redundant interplay between these phosphatases, challenging conventional views on the PD-1/PD-L1 pathway and T cell exhaustion and potentially influencing the design of future immunotherapeutic strategies.
Conclusion: Our research advances the understanding of the molecular regulation underlying CD8+ T cell dynamics, highlighting the stage-specific roles of SHP-1 and SHP-2 in both effector function and exhaustion. These findings hold implications for the treatment of chronic infections and cancer immunotherapy, offering novel insights into therapeutic targeting strategies.
The collaborative effort in this study was led by Guo Fu and Bing Xu, both of whom are affiliated with Xiamen University in China. Guo Fu, affiliated with the State Key Laboratory of Cellular Stress Biology at the School of Medicine, played a crucial role in designing the study and is the corresponding author. Bing Xu, the first author, holds positions at various institutions within Xiamen University, including the Department of Hematology at The First Affiliated Hospital and Institute of Hematology, as well as the State Key Laboratory of Cellular Stress Biology. Additionally, Bing Xu contributed to the Immunology Translational Research Programme at the National University of Singapore. Nicholas R. J. Gascoigne, also a corresponding author, is associated with the Immunology Translational Research Programme at the National University of Singapore and provided assistance in manuscript writing. This collaboration underscores the interdisciplinary nature of the research, bridging institutions in China and Singapore. Contact information for Guo Fu and Nicholas R. J. Gascoigne is available for further inquiries.
