Page 122 - Haematologica Vol. 109 - July 2024

ARTICLE - Cytotoxic reprogramming for BiTE immunotherapy M. Casey et al. AB
CD
Figure 3. IL-21 transcriptionally modulates the cytotoxic granule exocytosis pathway. (A) Isolated CD8 T cells were co-cultured with JJN-3 in the presence of T-cell-engaging bispecific antibody targeting B-cell maturation antigen (anti-BCMA T-BsAb, 0.1 μg/ mL) with or without recombinant interleukin-21 (rIL-21, 100 ng/mL), followed by sorting of CD8 T cells for RNA sequencing (RNA- seq). A schematic illustrates the sample preparation. (B) Principal component analysis (PCA) of log2-transformed normalized pro- tein-coding genes across all samples. Principal component (PC) 1 versus PC2 (left) and PC2 versus PC3 (right) of the scaled and centered protein-coding genes from 6 different donors colored by sample type. (C) Enriched Reactome pathways in differentially expressed genes (DEG) significantly upregulated in IL-21-primed CD8 T cells compared with control CD8 T cells. (D) Heatmap show- ing z-scored mRNA abundances of genes related to cytotoxic granules (GZMB, GZMH, NKG7, GNLY, and PRF1), pro-inflammatory cytokines (IFNG, TNF, and CSF2), and co-receptor (CD226, CD28, PCDC1, TIGIT, LAG3). DEG are indicated in bold text. Ctrl: control.
myeloma cell lines, JJN-3, RPMI8226, and KMS-11 (Figure 4B). Target myeloma cells expressed IL-21 receptor; how- ever, treatment with rIL-21 in these cells showed negligi- ble impacts on expression levels of BCMA and ligands for co-receptors in tumor cells (Online Supplementary Figure S2). While we observed that the expression level of CD226 was upregulated by rIL-21 (Figure 2D), anti-CD226 blocking monoclonal antibody (mAb) did not inhibit T-BsAb-mediated cytotoxicity (Figure 4C), suggesting that the enhanced in vitro cytotoxic activity was explained by other mechanisms. Importantly, T-BsAb-mediated tumor-killing activities were not ameliorated by anti-IFN-γ neutralizing mAb, regardless of IL-21 treatment (Figure 4C). By contrast, pretreatment with a perforin inhibitor, concanamycin A, largely abrogated cytotoxic activities in both control CD8 T cells and IL-21- primed CD8 T cells, indicating that the T-BsAb-mediated myeloma-killing activity was cytotoxic granule-dependent (Figure 4C). Additionally, IL-21-primed CD4 T cells showed upregulation of cytotoxic granules and enhanced myelo- ma-killing activities (Online Supplementary Figure S3). Thus,
IL-21 induces the cytotoxic program in both CD8 T cells and CD4 T cells.
The cytotoxic immunological synapse acts as a molecular platform, triggering dynamic cytoskeletal reorganization and delivery of cytotoxic granules via exocytosis.34 Given that granule exocytosis and signaling pathways related to cyto- skeletal reorganization were transcriptionally upregulated by IL-21 (Figure 3C), we next evaluated whether treatment with rIL-21 could promote immunological synapse formation and degranulation. Indeed, IL-21-primed CD8 T cells more rapidly expressed the degranulation marker CD107a, com- pared to control CD8 T cells (Figure 4D). The polarization of microtubule-organizing center (MTOC) toward the contact site is recognized as a crucial step for delivering cytotoxic granules.34 In IL-21-primed CD8 T cells, rapid MTOC polar- ization was observed after T-BsAb stimulation (Figure 4E, F). Together, IL-21 priming promotes the cytotoxic granule exocytosis pathway.
Haematologica | 109 July 2024
2136