Exhausted T Cells Characterized By Upregulation of Specific Transcription Factors Are Increased in a Mouse Model of De Novo Mature B Cell Neoplasms

Introduction: Recent studies demonstrated that exhausted T cells expressing immune checkpoint molecules such as PD-1, Tim-3, and TIGIT are increased at tumor sites in diffuse large B cell lymphoma (DLBCL) and multiple myeloma (MM) patients. However, the pathogenetic significance of exhausted T cells remains controversial and needs thorough investigation.

Method: In this study, we used a mouse model with human MM mutations of conditional Utx loss and Braf^V600E expression only in mature B cells, which developed mature B cell neoplasms de novo with time. Therefore, we expected this mouse model to show immune escape similar to human patients, rather than the currently available transplanted mouse models. To examine the level of T cell exhaustion, we analyzed the expression of PD-1 and Tim-3 on CD8⁺ T cells. To understand the characteristics of exhausted T cells, we performed flow cytometry (FCM) and RNA-seq analyses.

Result: In mice with mature B cell neoplasms, PD-1⁺ Tim-3⁺ and PD-1⁺ Tim-3^- T cells were significantly increased in BM, spleen, and lymph nodes, compared with control mice (p<0.0001). The expression level of TIGIT, which was reported to be expressed on CD8⁺ T cells of MM patients more frequently than other inhibitory receptors, was significantly higher in PD-1⁺ Tim-3⁺ T cells. These data suggest that the increase in PD-1⁺ Tim-3⁺ and PD-1⁺ Tim-3^- T cells at the tumor site may be related to the tumor pathogenesis in our mouse model. We then investigated the functional and transcriptional features of these exhausted T cells. The FCM analyses revealed that PD-1⁺ Tim-3⁺ T cells had the highest production capacity of granzyme B and expressed the highest level of Ki-67 and annexin V, compared with PD-1⁺ Tim-3^- and PD-1^- Tim-3^- T cells. These characteristics are consistent with those of terminally exhausted T cells previously reported. RNA-seq analyses also revealed the similarities between PD-1⁺ Tim-3⁺ T cells in this mouse model and terminally exhausted T cells. The transcription factors Tox and Nr4a2, which reportedly contribute to the progression of T cell exhaustion, were up-regulated in PD-1⁺ Tim-3⁺ T cells. On the other hand, PD-1⁺ Tim-3^- T cells showed high production capacity of IFN-γ and TNF-α and expressed moderate levels of Ki-67 with less apoptotic features. RNA-seq demonstrated the enrichment of gene sets related to immune system process. Tox and Nr4a2 were also up-regulated in PD-1⁺ Tim-3^- T cells but their levels were lower than in PD-1⁺ Tim-3⁺ T cells. Furthermore, PD-1⁺ Tim-3^- T cells retained the expression of Tcf7, encoding TCF-1 which plays a significant role in maintaining stem-like features. These results indicate that PD-1⁺ Tim-3⁺ T cells have direct cytotoxicity and strong proliferative capacity, however most of these cells are severely apoptotic. Therefore, PD-1⁺ Tim-3⁺ T cells are possibly unable to keep long-term anti-tumor functions. In contrast, PD-1⁺ Tim-3^- T cells show immunological responsiveness and progenitor-like transcriptional features, suggesting that these cells may maintain effective immune functions for a long time. When we stimulated CD8⁺ T cells in vitro in the presence or absence of lenalidomide or pomalidomide, these IMiDs down-regulated the expression of Tox and Nr4a2, suggesting that they might prevent T cell exhaustion.

Conclusion: The present study using our mouse model revealed the heterogeneity and the increase in exhausted T cells during the development of de novo mature B cell neoplasms. PD-1⁺ Tim-3⁺ T cells exhibit terminally exhausted features, whereas PD-1⁺ Tim-3^- T cells retain stem-like transcriptional features. IMiDs might prevent excessive T cell exhaustion and contribute to the immune response with long-term effects.