Th2 cells inhibit growth of colon and pancreas cancers by promoting anti-tumorigenic responses from macrophages and eosinophils

The study addresses whether type II immune responses, specifically Th2 cells and their cytokines, can reprogram the tumor microenvironment to suppress gastrointestinal cancer growth. Immune cell composition within the TME correlates with cancer progression and patient survival, yet many gastrointestinal tumors are resistant to current immunotherapies due to immunosuppressive TMEs. Th2 cells, classically associated with allergic responses, have uncertain and contradictory roles in cancer. The authors hypothesize that adoptive transfer of Th2 cells can inhibit colon and pancreatic tumor growth by recruiting and activating innate immune cells (eosinophils, macrophages) and promoting cytotoxic/apoptotic pathways, with IL-5 as a key mediator. The purpose is to define mechanistic roles for Th2 cells and IL-5 in anti-tumor immunity and explore their therapeutic potential in gastrointestinal cancers.

Prior work links TME immune infiltration with outcomes in multiple cancers. While checkpoint inhibitors benefit a subset of patients (e.g., mismatch repair–deficient pancreatic tumors), many gastrointestinal cancers remain refractory due to immunosuppressive TMEs. The role of Th2/type II responses in cancer is mixed and often descriptive. IL-5 has been implicated in anti-tumor responses, potentially via eosinophils, which can exert cytotoxicity through granule proteins such as granzyme B (GZMB) and major basic protein (MBP). Some studies show eosinophils support tumoricidal actions in colon cancer models, whereas others report context-dependent effects. Th2 cells can recruit and modulate innate cells, and T-cell plasticity suggests simultaneous contributions from Th1 and Th2 phenotypes. Overall, mechanistic understanding of Th2 cells and IL-5 in solid tumor immunity is limited, motivating the present mechanistic in vivo study.

- Animal models: Murine allograft models of gastrointestinal cancer using BRAF (BRAF^V600E) colorectal tumor–derived cells (from organoid-derived tumors) and PKS1940 pancreatic cancer sphere cells (Kras mutant). Tumors established by subcutaneous inoculation; many experiments conducted in Rag1−/− mice to control adaptive immunity context and assess adoptively transferred Th2 effects. - Th2 cell generation: Naive CD4+ T cells isolated from spleens of C57BL/6 mice using magnetic separation. Cells cultured in MaxEAMS medium with IL-2 and anti-IFN-γ, activated with anti-CD3/CD28–coupled beads, and polarized to Th2 over 6 days (conditions including IL-4, IL-2, anti–IFN-γ). Post-polarization, Th2 cells exhibited high GATA3 expression, elevated IL-5, lower IL-4/IL-13, and minimal IFN-γ. - Eosinophil isolation: From C57BL/6 mice using Anti–Siglec-F magnetic microbeads; cultured in RPMI with serum, antibiotics, and IL-4. - Treatments: Adoptive transfer of Th2 cells into tumor-bearing mice (single or weekly injections). Separate cohorts received recombinant IL-5 at biologically relevant doses approximating levels observed in Th2-treated tumors. - Histology and immunostaining: Tumors fixed (formalin/paraffin) or frozen in OCT; immunofluorescence/IHC using antibodies for eosinophils (SIGLEC-F), macrophages (F4/80), myeloid cells (CD11b), proliferation (Ki-67), GATA3, MBP, MPO, and NOS2. Imaging for marker localization and qualitative/quantitative assessment. - Gene expression: Tumor RNA extracted (TRIzol), reverse-transcribed; real-time PCR (TaqMan) for markers including Adgre1 (F4/80), Siglec1, Gr1, Mbp, Mpo, Nos2, GzmB, Prf1, Fas, FasL, and others; Ct-based quantification normalized to Actb; fold-change analysis. - Tumor-killing assays: Co-culture of BRAF or PKS1940 tumor cells with Th2 cells or eosinophils at 12:1 ratio for 12 h; cell death measured via Live/Dead Caspase-3/7 reagent and flow cytometry. Supernatants collected for secreted factor analysis. - Cytokine/chemokine multiplex: Tumor explants incubated; supernatants analyzed by ELISA/Luminex for cytokines including IL-4, IL-5, IL-13 (where detectable), GZMB, and soluble FAS. - Statistics: Data presented as mean ± SEM; non-parametric Mann–Whitney U-test and two-way ANOVA for multiple comparisons; significance at P < 0.05.

- Th2 cell therapy suppresses tumor growth: In both BRAF^V600E colorectal and PKS1940 pancreatic allograft models, adoptive transfer of Th2 cells markedly reduced tumor growth versus controls. In PKS1940 tumors, a single Th2 injection reduced growth by approximately 50%, while weekly injections had stronger effects; Th2 cells (GATA3+) were detected within tumors at endpoint, indicating persistence. - Innate immune cell recruitment: Th2 treatment increased tumor mRNA of Siglec1 (reported as eosinophil marker) by ~2-fold in PKS1940 and ~3-fold in BRAF tumors, and Adgre1 (F4/80, macrophage marker) up to ~3-fold in both models; Gr1 (neutrophils/other granulocytes) was not significantly changed. IHC confirmed increased SIGLEC-F+ eosinophils and F4/80+ macrophages. - Activation of cytotoxic/apoptotic programs: Th2-treated tumors showed increased expression of Mbp, Mpo, and Nos2, and elevated cytotoxic/apoptotic genes GzmB, Prf1, Fas, and FasL. Tumor supernatants had increased soluble GZMB and FAS. IHC confirmed increased MBP, MPO, and NOS2. - Direct tumor killing: In co-culture assays, both Th2 cells and eosinophils increased the percentage of caspase 3/7–positive PKS1940 tumor cells, indicating direct cytotoxicity. Supernatants from Th2/tumor or eosinophil/tumor co-cultures contained elevated GZMB. - IL-5 as key mediator: Th2 treatment increased IL-5 in tumor supernatants; IL-4 showed no significant increase and IL-13 was largely undetectable. Recombinant IL-5 administration to tumor-bearing mice reduced tumor volumes by approximately 25-fold in both models, increased eosinophil influx (SIGLEC-F+), and elevated tumor GZMB and FAS (protein and 2–3-fold mRNA increases), though generally to a lesser extent than full Th2 cell therapy. - Statistical significance: Reported differences generally reached statistical significance (e.g., P < 0.05 or P < 0.001 as indicated in figure legends).

The findings directly support the hypothesis that Th2 cells can reprogram the gastrointestinal tumor microenvironment to an anti-tumor state. Adoptive Th2 transfer enhanced recruitment and activation of eosinophils and macrophages, key effectors that contributed to tumor cytotoxicity via MBP, GZMB, MPO, and NOS2, and upregulated apoptotic pathways (FAS/FASL, perforin/granzyme). Elevated IL-5 in Th2-treated tumors and the independent anti-tumor effect of recombinant IL-5 implicate IL-5 as a mechanistic link between Th2 cells and eosinophil-mediated tumor killing. While prior literature offered mixed, largely correlative data regarding Th2 responses in cancer, these mechanistic in vivo results demonstrate that type II immunity can be protective in colon and pancreatic tumor models by recruiting and activating innate immune effectors and inducing apoptotic signaling in tumor cells. The results highlight the therapeutic potential of Th2-based adoptive cell therapy and IL-5–mediated strategies to overcome immunosuppression in the TME, potentially complementing existing immunotherapies.

Type II immune responses, driven by Th2 cells, inhibit colon and pancreatic tumor growth by reprogramming the TME to recruit and activate eosinophils and macrophages, enhancing cytotoxic and apoptotic mechanisms. IL-5 is a key mediator linking Th2 activity to eosinophil-driven anti-tumor effects, and recombinant IL-5 alone confers substantial tumor suppression. These findings position Th2 cells, eosinophils, and IL-5 as promising therapeutic targets for gastrointestinal cancers. Future research should: (1) define the precise phenotypes and polarization states of recruited macrophages and eosinophils; (2) dissect signaling pathways downstream of IL-5 and Th2-derived factors in the TME; (3) evaluate synergy of Th2/IL-5–based approaches with checkpoint blockade or chemotherapy; (4) assess safety, dosing, and durability in additional preclinical models and ultimately in clinical studies; and (5) explore contributions of other type II–associated cells (e.g., mast cells) in these anti-tumor responses.

- Preclinical murine allograft models may not fully recapitulate human gastrointestinal cancer biology and TME complexity. - Many experiments used Rag1−/− mice, limiting adaptive immunity to transferred Th2 cells and potentially altering TME dynamics versus immunocompetent hosts. - Some cytokines (e.g., IL-13) were not detectable, and protein-level validation was limited to select markers and supernatant analyses; comprehensive phenotyping of macrophage polarization states was not performed. - The relative contributions of Th2 cells versus eosinophils and macrophages to tumor killing were inferred from association and co-culture assays; definitive lineage-specific depletion studies were not reported. - Potential roles of other type II–related cells (e.g., mast cells) and off-target or pro-tumor effects of IL-5 were not examined.