In cancer treatment, immunotherapy has offered new hope to patients. However, the effectiveness of this approach is often limited by the complex effects of the tumor microenvironment (TME) on immune cell function. Cytotoxic CD8(+) T lymphocytes (CTLs) are crucial for tumor immune defense, but they can gradually become exhausted and lose their ability to combat tumors due to the influence of the TME.

Wei Jian, a researcher from the School of Basic Medical Sciences at Shandong University, recently published new findings in the journal *Cancer Immunology Research*. The study, titled “IL3-Driven T Cell–Basophil Crosstalk Enhances Antitumor Immunity,” offers a comprehensive analysis of how IL3 mediates interactions between cytotoxic T lymphocytes and basophils. This research highlights IL3’s crucial role in modulating the body’s anti-tumor immune response. Additionally, the findings suggest that targeting IL-3 or basophils could be a promising approach to improve and enhance the effectiveness of cancer immunotherapy, potentially guiding future developments in the field.

The author used the B16 melanoma model to study T cell exhaustion during tumor progression. As the tumor grows, there’s an increase in the expression of PD1 and TIM-3, while T-bet and key effector cytokines (IFNγ, TNFα, and IL2) decrease. It suggests that T cells become progressively exhausted as the tumor develops. Additionally, the study observed that in vitro activated CD8(+) and CD4(+) T cells initially show high levels of IL3. Early tumors also feature CD8(+) and CD4(+) T cells producing a lot of IL3, but IL3 levels drop as the tumor progresses. Further experiments revealed that culturing CD8(+) T cells under low glucose and continuous activation conditions, which mimic the tumor microenvironment (TME), results in reduced IL3 expression and secretion. This indicated that glucose deprivation in the TME is a key factor contributing to the decline of IL3 in T cells under ongoing antigen stimulation (see Figure 1).

The author further investigated whether supplementing IL-3 could inhibit tumor growth. Intraperitoneal injections of IL-3 into tumor-bearing mice, or genetically modifying tumor cells to overexpress IL-3, both significantly suppressed tumor growth. Even when only 1% of the tumor cells overexpressed IL-3, it still markedly reduced tumor growth. In a metastatic melanoma model, IL-3 supplementation also decreased the formation of liver metastases. Additionally, transferring IL-3-overexpressing OT1 cells into tumor-bearing mice also inhibited tumor growth and extended the host’s survival. These findings indicated that both direct IL-3 supplementation and its combination with T cell activity can effectively control tumor growth. (Figure 2)

Although IL-3 supplementation effectively inhibits tumor growth, the underlying mechanism is not yet fully understood. The study revealed that the anti-tumor activity mediated by IL-3 relies on the function of lymphocytes. This inhibitory effect was not observed in Rag1 KO mice, which lack mature T and B cells. Additionally, depleting CTLs in the mouse models eliminated the tumor growth suppression and survival benefits associated with IL-3. These findings indicated that CTLs play a crucial role in IL-3-mediated tumor control.

The study further explored the effects of IL-3 supplementation on CTLs. It was observed that IL-3 increased the expression of CD44, T-bet, the proliferation marker Ki67, IFNγ, and TNFα in CTLs, while decreasing PD1 expression and slightly reducing TIM3 expression. However, IL-3 had no impact on granzyme B. Additionally, CD8(+) T cells treated with IL-3 displayed similar metabolic profiles, effector functions, vitality, and in vitro anti-tumor capabilities compared to the control group. This suggests that IL-3 does not directly enhance the anti-tumor immune activity of CD8(+) T cells but rather exerts its effects through indirect mechanisms. (Figure 3)

The research team discovered that the enhanced anti-tumor activity of CTLs mediated by IL-3 might involve intermediary cell populations. Compared to purified CD8(+) T cells (pCD8), CD8(+) T cells co-cultured with splenocytes (bCD8) exhibited a glycolytic metabolic profile, increased IFNγ production, and enhanced survival. Moreover, bCD8 OT1 cells showed stronger anti-tumor immune activity when transferred into tumor-bearing mice. Transwell assays revealed that soluble factors released by other cell populations mediated the enhanced effector functions of CD8(+) T cells. These soluble effectors produced by T cells act on specific non-T/B cell subpopulations, thereby improving CTLs’ effector functions. IL-3 plays a crucial role in mediating the interactions between T cells and non-B/T cells (Figure 4).

(The authors used the RWD Nano Bead Cell Seperation System to isolate and purify CD8+ T cells.)

To identify the cellular targets of IL3, the author isolated different cell populations from the spleens of Rag1 KO mice for evaluation. He found that only the conditioned media from IL3-treated CD45(+)Lin(–) cells enhanced IFNγ production in CD8(+) T cells. Similarly, conditioned media from IL3-treated tumor-infiltrating CD45(+)Lin(–) cells increased IFNγ production and vitality in CTLs.

Among these, eosinophils, a subset of CD45(+)Lin(–) leukocytes, express high levels of IL3 receptors. IL3 treatment of eosinophils enhances IFNγ production and vitality in CD8(+) T cells. Additionally, IL3 supplementation significantly increases the number of eosinophils in both the spleens and tumors of mice. Depleting eosinophils reduces the anti-tumor efficacy of IL3 supplementation. Eosinophils isolated from the spleens of B16-IL3 melanoma-bearing Rag1 KO mice were able to limit tumor growth. These results suggest that IL3-activated eosinophils play a crucial role in enhancing anti-tumor immunity. (Figure 5)

The study further investigated through RNA-seq and pathway analysis, discovering that the enhanced efficacy of T cells by the conditioned media from IL3-treated eosinophils is likely due to the activation of IL4 signaling pathways. IL3 treatment significantly increased the expression of the Il4 gene in eosinophils. Blocking IL4 with neutralizing antibodies notably reduced the effects of the IL3-treated eosinophil-conditioned media on CD8(+) T cells. Injection of IL3 into Il4 gene knockout mouse models had no impact on tumor growth. Conversely, IL4 treatment remodeled CD8(+) T cell metabolism, boosting IFNγ production and survival, and adoptive transfer of IL4-treated OT1 cells significantly reduced tumor volume. These findings suggest that IL3-treated eosinophils produce more IL4, thereby enhancing CTL-mediated anti-tumor immunity. (Figure 6)

To better address cancer treatment in clinical practice, the author also assessed the impact of chemotherapy drugs on IL3 expression and its effector functions, as well as the role of IL3-mediated immune cell interactions in the prognosis of melanoma patients. The data suggest that targeting the IL-3-mediated interaction between cytotoxic T lymphocytes and basophils may be an effective approach to enhance anti-tumor immunity and improve patient outcomes. (Figure 7)

Research Method Highlights

This study provided an in-depth exploration of the IL-3-mediated crosstalk between cytotoxic T lymphocytes and basophils. It employs a range of experimental models and techniques, including cell culture, animal studies, genetic engineering, cell sorting, and molecular biology assays. The research thoroughly uncovers the complex role of IL-3 in tumor immunity, offering new insights into the mechanisms of tumor immune suppression.

In this study, the researchers utilized RWD Nano Beads Cell Separation System. Additionally, RWD provides comprehensive solutions for primary cell extraction, cell purification, and other preclinical research techniques related to cancer treatment. To date, RWD’s products and services are available in over 100 countries and regions worldwide, serving more than 2,500 hospitals, 1,000 research institutions, and 6,000 higher education institutions. RWD has supported global researchers in publishing over 14,500 SCI articles and has earned widespread recognition in the industry.