CD5 Elimination Enhances CART Cell Proliferation through JAK-STAT Activation

Introduction: Chimeric antigen receptor T (CART) cell therapy targeting CD19 (CART19) or BCMA has demonstrated significant success in treating B-cell malignancies. Despite these advances, a substantial number of patients still experience relapse, highlighting the need for more effective CART cell approaches. Previously, we have shown that CD5 elimination (CD5KO) enhances CART cell efficacy, partly by increasing the phosphorylation of ERK1/2 and S6, suggesting the involvement of the MAPK and mTOR pathways (Patel et al., Sci Immunol, 2024). Here, we use bulk RNA sequencing and Luminex analyses to further explore the involvement of the IL-2 family, MAPK, and JAK-STAT signaling pathways in increasing cell proliferation to enhance the antitumor efficacy of CD5KO CART cells.

Methods and Results: We generated our CART cells through a 5-day rapid manufacturing process, in which CD5 wild-type (WT) and KO CART19 cells were activated by bead on day 0 and allowed to expand for five days in IL-2 – free culture media. At the end of day 5, these cells were collected and subjected to bulk RNA sequencing analysis. Our results showed distinct gene expression profiles at principle component analysis (PCA) between CD5 WT and KO cells, and confirmed strong gene knockout of CD5 in the KO group (p-value = 1.79e-46). Moreover, gene set enrichment analyses highlighted the enrichment of JAK-STAT and MAPK signaling pathways in CD5 KO CART cells as compared to WT, primarily through upregulation of cytokines and growth factors.

JAK-STAT pathway is a major regulator of immune cell homeostasis, contributing to a poised epigenetic and transcriptional state and helping to prepare these cells for rapid response to immune stimuli. We first noticed that several of the IL-2 cytokine family members (including IL-2, -4, -9, -21 and -15), which play crucial roles in proliferation, differentiation, etc., were significantly upregulated in CD5KO cells compared to WT cells. Further, unbiased Luminex analysis of CD5 WT and KO CART cells before and after CART activation identified increased levels of IL-13, IL-6, and IFN-γ in the supernatant of CD5KO CART19 cells. These cytokines are known to play roles in the JAK-STAT signaling pathway. We also found PDGFA growth factor, an activator of JAK-STAT signaling, to be significantly upregulated (p < 0.05). These cytokines and growth factors play major roles in the activation of JAK-STAT signaling. Moreover, several components of the AP-1 transcription factor (TF) were upregulated in CD5 KO CART cells as compared to WT. AP-1 TF can bind to NFAT promoter to trigger the production of IL-2 to activate PI3K/AKT, Ras-MAPK, and JAK-STAT pathways. Specifically, in the MAPK pathway, the RNA expression levels of MYC, growth factors CFS1 and FGF2, c-fos, and SRF were significantly upregulated in CD5KO cells (p-values < 0.05), potentially underlying their enhanced cell proliferation compared to their WT counterparts. MYC is a known master regulator of T-cell metabolism and plays an essential role in T-cell proliferation and growth. That observation aligns with our in vitro and in vivo studies, demonstrating outstanding proliferation and persistence of CD5KO CART cells.

Conclusions: In summary, this study demonstrates that CD5KO CART cells exhibit enhanced signaling through the JAK-STAT, MAPK, and mTOR pathways, mechanistically explaining the enhanced CART cell proliferation and a more sustained antitumor response. Our findings build on previously published work demonstrating the enhanced efficacy of CD5KO CART cells and provide deeper insights into the molecular mechanisms driving their superior efficacy. Therefore, CD5 deletion presents a promising strategy for enhancing CART therapies, with the potential to improve CART cell efficacy in hematological malignancies and solid tumors.