Single-cell transcriptomic profiles analysis has proposed new insights for understanding the behavior of human gastric cancer (GC). GC offers a unique model of intratumoral heterogeneity. However, the specific classes of cells involved in carcinogenetic passage, and the tumor microenvironment of stromal cells was poorly understood. We characterized the heterogeneous cell population of precancerous lesions and gastric cancer at the single-cell resolution by RNA sequencing. We identified 10 gastric cell subtypes and showed the intestinal and diffuse-type cancer were characterized by different cell population. We found that the intestinal and diffuse-type cancer cells have the differential metaplastic cell lineages: intestinal-type cancer cells differentiated along the intestinal metaplasia lineage while diffuse-type cancer cells resemble de novo pathway. We observed an enriched CCND1 mutation in premalignant disease state and discovered cancer-associated fibroblast cells harboring pro-stemness properties. In particular, tumor cells could be categorized into previously proposed molecular subtypes and harbored specific subtype of malignant cell with high expression level of epithelial-myofibroblast transition which was correlated with poor clinical prognosis. In addition to intratumoral heterogeneity, the analysis revealed different cellular lineages were responsible for potential carcinogenetic pathways. Single-cell transcriptomes analysis of gastric pre-cancerous lesions and cancer may provide insights for understanding GC cell behavior, suggesting potential targets for the diagnosis and treatment of GC.

Anti-4-1BB-mediated anticancer effects were potentiated by depletion of CD4 + cells in B16F10 melanoma-bearing C57BL/6 mice. Anti-4-1BB induced the expansion and differentiation of polyclonal tumor-specific CD8 + T cells into IFN-;-producing CD11c + CD8 + T cells. The CD4 + cell depletion was responsible for facilitating immune cell infiltration into tumor tissues and removing some regulatory barriers such as T regulatory and indoleamine-2,3-dioxygenase (IDO) + dendritic cells. Both monoclonal antibodies (mAb) contributed to the efficient induction of MHC class I molecules on the tumor cells in vivo. The effectors that mediated the anti-4-1BB effect were NKG2D + KLRG1 + CD11c + CD8 + T cells that accumulated preferentially in the tumor tissues. Blocking NKG2D reduced the therapeutic effect by 20% to 26%, which may indicate that NKG2D contributes partially to tumor killing by the differentiated CD8 + T cells. Our results indicate that the combination of the two mAbs, agonistic anti-4-1BB and depleting anti-CD4, results in enhanced production of efficient tumor-killing CTLs, facilitation of their infiltration, and production of a susceptible tumor microenvironment. [Cancer Res 2007;67(18):8891-9]

Anti-4-1BB and cisplatin showed synergistic anticancer effects in the CT-26 colon carcinoma model, producing complete regression in >60% of mice with either preventive or therapeutic treatment. The tumor-free mice formed longlasting CD8 + T cell-dependent tumor-specific memory. Anti-4-1BB induced rapid repopulation of T and B cells from cisplatin-mediated lymphopenia and differentiation and expansion of IFN-; + CD11c + CD8 + T cells. Cisplatin facilitated expansion of naïve, effector, and memory CD8 + T cells; combination therapy produced almost twice as many lymphoid cells as anti-4-1BB alone. Cisplatin increased 4-1BB on antigen-primed T cells and induced 4-1BB de novo on kidney tubular epithelium. Cross-linking of 4-1BB protected the T cells and kidney epithelium from cisplatin-mediated apoptosis by increasing expression of antiapoptotic molecules. Thus, cisplatin-induced 4-1BB provided a mechanism for amelioration of the lymphopenia and nephrotoxicity inherent in cisplatin treatment. We concluded that chemoimmunotherapy with anti-4-1BB and cisplatin is synergistic in tumor killing and prevention of organ-specific toxicity. [Cancer Res 2008;68(18):7264-9]

Agonistic anti–4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8+ T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8+ T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB–triggered suppression of autoimmune responses using IFN-γ- or IFN-γR–deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3+CD4+ regulatory T (Treg) cells, particularly among CD4+ T cells. This was not due to a direct effect of 4-1BB signaling on CD4+ T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8+ T cells, but also did so in IFN-γ–deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.

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