A Study on the Dual Role of IFN-γ Protein in CAR-T Therapy

I. Research Background and Scientific Questions

CAR-T therapy has demonstrated promising efficacy in patients with hematological malignancies, but it is often accompanied by clinical toxicities such as cytokine release syndrome (CRS), which not only affects patient health but also increases the medical burden. Currently, clinically used cytokine antagonists primarily target downstream inflammatory factors, making it difficult to fully alleviate these side effects. IFN-γ protein, as one of the biomarkers of CRS, is considered a critical activator of innate immune cells. Based on the strong clinical correlation between IFN-γ and CRS, researchers have identified this signaling pathway as a potential intervention target. This strategy directly targets CAR-T cells themselves rather than downstream macrophage functions, offering the potential to control clinical toxicity before it occurs.

II. Impact of IFN-γ Blockade on CAR-T Antitumor Function

For a long time, IFN-γ protein has been closely associated with T cell effector functions and regarded as an important indicator for evaluating CAR-T functionality. However, this study found that in hematological tumor models, blocking or knocking out IFN-γ does not affect the antitumor activity of CAR-T cells. In vitro experiments, after antibody blockade of IFN-γ, CAR-T cells exposed to CD19-positive tumor cell lines showed no difference in Granzyme B expression, and tumor cell lysis efficiency remained unaffected. In vivo studies demonstrated that mice treated with IFN-γ-blocked or IFN-γ-knockout CAR-T cells exhibited reduced serum IFN-γ levels but similar tumor clearance effects, CAR-T cell engraftment levels, and overall survival rates compared to the control group. In more aggressive leukemia models, the persistence and efficacy of CAR-T cells were not compromised, and mice receiving IFN-γ blockade showed improved survival. Similar results were validated in BCMA-targeted CAR-T cells, indicating the target-agnostic nature of this phenomenon.

III. Effect of IFN-γ Knockout on CAR-T Cell Phenotype

IFN-γ-knockout CAR-T cells constructed using CRISPR/Cas9 technology exhibited similar cell subset composition to control products, with comparable CD4 and CD8 ratios. Most cells displayed a naive phenotype, and no significant differences were observed in memory subsets or chemokine receptor expression. In co-culture systems with macrophages, IFN-γ-knockout CAR-T cells demonstrated comparable proliferation capacity but lower expression levels of inhibitory molecules such as LAG-3, PD-1, and TIM-3, particularly in the CD4-positive subset. For CAR-T cells based on the 28ζ structure, IFN-γ knockout enhanced their expansion capacity while reducing inhibitory molecule expression. These results suggest that IFN-γ protein may, to some extent, limit CAR-T cell expansion and promote upregulation of inhibitory molecules.

IV. Mechanism of IFN-γ in Macrophage Activation

Macrophages, as innate immune cells, can amplify IFN-γ signals and contribute to CRS development. Studies have shown that serum levels of IFN-γ, IL-6, and IL-10 are significantly elevated in CAR-T-treated patients. Adding macrophages to CAR-T and tumor cell co-culture systems or exposing them to the supernatant of such systems can activate macrophages and induce the release of multiple CRS-related cytokines. When IFN-γ-knockout CAR-T cells or IFN-γ-blocking antibodies were applied, macrophage activation was attenuated, activation marker expression decreased, downstream IFN-γ receptor signaling weakened, and PD-L1 expression declined. An in vitro hybrid model further confirmed that macrophages exposed to serum from IFN-γ-knockout CAR-T-treated mice produced fewer cytokines and exhibited weaker activation.

V. Comparison of Targeting IFN-γ with Existing Intervention Strategies

Researchers compared targeting IFN-γ with current clinical intervention strategies. In vitro models treated with IFN-γ antibodies, IL-6 receptor antibodies, or IL-1 receptor antagonists showed that IFN-γ antibody treatment was more effective in reducing cytokine levels. This result was validated across multiple macrophage subsets and undifferentiated monocytes. Transcriptional profiling of macrophages revealed that IFN-γ antibody treatment upregulated costimulatory gene expression and downregulated genes encoding PD-L2 and TIM-3, molecular features potentially associated with improved CAR-T efficacy.

VI. Differences Between Solid and Hematological Tumors and Future Research Directions

Notably, in solid tumor models, IFN-γ deficiency led to moderate treatment resistance, and IFN-γ antibody blockade reduced CAR-T cytotoxicity against glioblastoma. This difference suggests that, compared to hematological malignancies, IFN-γ protein may play a more critical role in CAR-T efficacy against solid tumors. These findings challenge the traditional view that IFN-γ is indispensable for T cell therapies, demonstrating that CAR-T efficacy and toxicity can be decoupled under certain conditions. For clinical translation, direct knockout of IFN-γ in CAR-T cells may be more feasible than using neutralizing antibodies, which could affect other immune cell functions. Further investigation into the mechanisms underlying differences between solid and hematological tumors will enhance understanding of the complex role of IFN-γ in CAR-T therapy.

VII. Which Companies Provide IFN-γ Protein?

Nanjing UA-Bio Technology Co., Ltd. (UA-Bio) has independently developed "IFN-γ Protein, Mouse," a high-quality recombinant protein reagent specifically designed for mouse Th1 immune responses, macrophage activation, and preclinical efficacy studies. This mouse-derived interferon-gamma (IFN-γ) belongs to the type II interferon family and efficiently activates the JAK-STAT signaling pathway, inducing immune cell activation, enhancing antigen presentation, and inhibiting viral replication. It provides a stable and reliable standardized tool for research in anti-infection immunity, tumor immunotherapy, and autoimmune diseases in mouse models.

Nanjing UA-Bio Technology Co., Ltd. remains committed to providing cutting-edge, high-quality core reagents and tools for immunology, cell therapy, and innovative drug development. For detailed technical parameters, validation data, or specific application inquiries regarding "IFN-γ Protein, Mouse" (Catalog No.: UA040065), please feel free to contact us.