Targeting Truncated O-Glycan Elicits Dual T Cell Immunity and Overcomes MHC-I Heterogeneity to Design Effective Cancer Vaccine

Aberrant O-glycosylation is a hallmark of cancer, but its contribution to immune evasion is unclear. Using CRISPR–Cas9 to delete GalNAc-transferases, particularly Galnt7, in tumor cells, we reduced mucin-type O-glycan truncation. This remodeling activated dendritic cell– and T cell–dependent immunity, eradicating established tumors, preventing metastasis, and generating durable memory. It enhanced clearance of MHC class I–positive tumors via coordinated CD8⁺ and CD4⁺ T cell responses, and uniquely enabled CD4⁺ T cell–mediated elimination of MHC class I–deficient tumors, which evade cytotoxic T lymphocyte surveillance. Tumor cells with reduced O-glycan truncation served as potent whole-cell vaccines, with efficacy further improved by radiotherapy. These findings establish glycosylation as a central regulator of tumor immune evasion and define a broadly applicable vaccine platform that bypasses the limitations of neoantigen-based approaches for low-immunogenicity cancers. RNA-seq profiling was performed on: 1) Colorectal tumor tissues from the AOM/DSS-induced tumorigenesis model at day 70 (n = 4). 2) The MC38 cell line (n = 3). 3) Tumor tissues collected on day 8 from wild-type C57BL/6J mice subcutaneously injected with Ctrl or Galnt7⁻/⁻ MC38 cells (n = 3). 4) CD8⁺ T cells isolated on day 14 from the spleens of C57BL/6J mice inoculated with MC38-Ctrl or MC38-Galnt7⁻/⁻ cells (n = 3). TCR-seq profiling was performed on CD8⁺ T cells isolated on day 14 from the spleens of C57BL/6J mice inoculated with MC38-Ctrl or MC38-Galnt7⁻/⁻ cells.

异常O-糖基化是癌症的标志性特征之一，但其在肿瘤免疫逃逸中的作用机制尚未明确。本研究借助CRISPR–Cas9技术敲除肿瘤细胞中的GalNAc-转移酶（GalNAc-transferase），尤其是Galnt7，从而减少黏蛋白型O-聚糖的截短修饰。该糖基化重塑可激活树突状细胞与T细胞依赖的免疫应答，根除已形成的实体瘤、抑制肿瘤转移，并诱导持久的免疫记忆。其通过协同CD8⁺与CD4⁺ T细胞应答，增强了对MHC I类分子（MHC class I）阳性肿瘤的清除效果；且可特异性介导CD4⁺ T细胞清除MHC I类分子缺陷的肿瘤——这类肿瘤可逃避细胞毒性T淋巴细胞的监视。O-聚糖截短程度降低的肿瘤细胞可作为高效的全细胞疫苗，联合放射治疗可进一步提升其疫苗效力。本研究证实糖基化是肿瘤免疫逃逸的核心调控因子，并构建了一套普适性的疫苗平台，可规避基于新抗原的疗法在低免疫原性癌症中的应用局限。

本研究对以下样本进行了RNA测序（RNA-seq）分析：
1）AOM/DSS诱导的结直肠癌发生模型小鼠在造模第70天的结直肠肿瘤组织（n=4）；
2）MC38细胞系（n=3）；
3）野生型C57BL/6J小鼠皮下接种Ctrl或Galnt7基因敲除（Galnt7⁻/⁻）的MC38细胞后第8天采集的肿瘤组织（n=3）；
4）接种MC38-Ctrl或MC38-Galnt7⁻/⁻细胞的C57BL/6J小鼠在第14天分离的脾脏CD8⁺ T细胞（n=3）。

本研究对接种MC38-Ctrl或MC38-Galnt7⁻/⁻细胞的C57BL/6J小鼠在第14天分离的脾脏CD8⁺ T细胞进行了T细胞受体测序（TCR-seq）分析。