Gastrointestinal cancers pose a significant challenge for immunotherapy. While adoptive transfer of immune cells shows promise, the role of T helper 2 (Th2) cells in this context remains poorly understood. Existing literature on type II immune responses in cancer is contradictory, with some studies suggesting a protective role but lacking mechanistic detail. The impact of Th2 cells and associated cytokines, particularly interleukin-5 (IL-5), on cancer progression requires further investigation. IL-5 is known to promote eosinophil function, and eosinophils are hypothesized to exert cytotoxic effects on cancer cells, potentially through factors like granzyme B (GZMB) and major basic protein (MBP). This study aims to evaluate the therapeutic potential of Th2 cells in gastrointestinal cancers by exploring their effects on tumor growth and the composition of the tumor microenvironment (TME). The researchers hypothesize that Th2 cells are crucial regulators of colon and pancreatic cancer progression, impacting both immune cell responses and the overall TME. This study will utilize murine allograft models of colon and pancreatic cancers to examine the effects of Th2 cell adoptive transfer on tumor burden, focusing on the interplay between Th2 cells, eosinophils, macrophages, and IL-5.

The bidirectional communication between cancer cells and immune cells significantly influences cancer development and progression. Immune cell infiltration and composition within the TME correlate with cancer progression and patient survival. Immunotherapy holds promise, but its effectiveness varies across cancer types, with only a subset of gastrointestinal tumors responding well. Studies have shown that patients with specific characteristics, like those with defective mismatch repair in pancreatic cancer, are better candidates for immunotherapy based on immune checkpoint inhibition. Adoptive immunotherapy using stimulated cytotoxic T cells combined with gemcitabine has demonstrated some success in preventing metastasis in pancreatic ductal adenocarcinoma, although challenges remain due to the immunosuppressive TME. The existing literature on Th2 cells in cancer is limited and largely descriptive, with conflicting findings regarding their impact. While some studies suggest a protective role of type II immune responses in certain cancers, the mechanisms involved remain unclear. The role of IL-5 in antitumorigenic responses, potentially through eosinophils, requires further exploration. Previous research has shown that IL-5 promotes eosinophil function and eosinophils may have cytotoxic effects on cancer cells, potentially through secreted cytotoxic factors such as GZMB and MBP. However, the available data on the involvement of IL-5 in tumor immunity is sparse and requires further investigation. The study postulates that Th2 cells are crucial regulators of colon and pancreatic cancer progression by influencing immune cell responses within the TME.

The study employed in vivo experiments using murine allograft models of gastrointestinal cancers. **Isolation of immune cells:** Eosinophils were isolated from C57BL/6 wild-type mice using magnetic separation with Anti-Siglec-F MicroBeads. Naive CD4+ T cells were isolated from mouse spleens using a Naive CD4+ T Cell Isolation Kit. Naive CD4+ T cells were then differentiated into Th2 cells in vitro using activation beads coupled with anti-CD3 and anti-CD28 antibodies and supplemented with IL-2 and anti-IFN-γ antibody. **Murine allograft models:** BRAF (BRAF^V600E)-derived cells (from mouse tumor organoids) and PKS1940 cells (Kras mutation) were used to induce tumors in mice. **Treatments:** Mice received adoptive transfer of Th2 cells or recombinant IL-5 injections. **Immunohistochemistry analysis:** Tumor samples were processed for paraffin-embedding and immunofluorescence staining to assess immune cell infiltration (F4/80 for macrophages, Siglec-F for eosinophils, and GATA3 for Th2 cells). **Tumor-killing assay:** In vitro co-cultures of tumor cells (BRAF and PKS1940) with Th2 cells or eosinophils were used to assess tumor cell killing using Live/Dead® Caspase-3® Green Detection Reagent and flow cytometry. Supernatants were collected for multiplex analysis. **RNA extraction, reverse transcription, and real-time PCR:** RNA was extracted from tumor tissues and used for real-time PCR to quantify the expression of various immune cell markers and cytotoxic/apoptotic factors. **Multiplex analysis:** Tumor supernatants were analyzed by ELISA and Luminex to measure cytokine and chemokine levels. **Statistical analysis:** Data were analyzed using GraphPad Prism 5.0, employing the Mann–Whitney U-test and two-way ANOVA for statistical comparisons.

Adoptive transfer of Th2 cells significantly suppressed the growth of both BRAF and PKS1940 cell-derived allograft tumors in mice. Th2 cell administration led to a substantial increase in eosinophil and macrophage influx into the tumors, as indicated by real-time PCR and immunofluorescence analysis showing increased expression of Siglec1 (eosinophil marker) and Adgre1 (F4/80, macrophage marker). Th2 cells and eosinophils induced the expression of cytotoxic/apoptotic factors such as GZMB, Prf1, Fas, and FasL, as demonstrated by real-time PCR. In vitro tumor-killing assays showed that both Th2 cells and eosinophils directly contributed to cancer cell death. Furthermore, IL-5 was significantly increased in tumors from Th2 cell-treated mice. Administration of recombinant IL-5 also inhibited tumor growth, accompanied by increased eosinophil influx and elevated levels of GZMB and Fas. These findings demonstrate that Th2 cells, eosinophils, and IL-5 play crucial roles in inhibiting gastrointestinal tumor growth through reprogramming of the tumor microenvironment and the induction of cytotoxic/apoptotic responses.

This study provides strong evidence for a protective role of Th2 cells in gastrointestinal cancers, challenging the traditional view of Th2 cells solely in allergic responses. The data demonstrate a novel mechanism of Th2 cell-mediated anti-tumorigenic immunity involving the recruitment and activation of macrophages and eosinophils, leading to the production of cytotoxic factors such as MBP, MPO, and NOS2. The significant increase in IL-5 and its independent effect on tumor growth highlight its importance in this process. The observed direct cytotoxic activity of Th2 cells against cancer cells is a significant finding that warrants further exploration. While some studies have shown that IL-4 promotes tumor clearance, the present study observed only low levels of IL-4 production, emphasizing the prominent role of IL-5 in the observed anti-tumor effects. The results are consistent with previous studies showing an association between high levels of tumor-infiltrating eosinophils and improved prognosis in various cancers. The findings suggest that reprogramming the tumor microenvironment towards a type II immune response might be a valuable therapeutic strategy for gastrointestinal cancers.

This study demonstrates that Th2 cells inhibit the growth of colon and pancreas cancers by promoting anti-tumorigenic responses from macrophages and eosinophils. The data highlight the importance of IL-5 in this process and suggest that targeting Th2 cells and IL-5 could be a promising therapeutic strategy for gastrointestinal cancers. Future research should focus on further elucidating the complex interplay among Th2 cells, eosinophils, macrophages, and IL-5 in the tumor microenvironment and exploring the potential of Th2 cell-based immunotherapies.

The study utilized murine models, which may not perfectly recapitulate the complexities of human cancer. Further research is needed to translate these findings to human clinical settings. The study focused on specific cancer cell lines, and the findings may not be generalizable to all types of gastrointestinal cancers. The mechanisms underlying Th2 cell-mediated cytotoxicity require more detailed investigation.