1α,25(OH)2D3 Alleviates Exhaustion and Promotes Antitumor Function of Anti-CD19 Chimeric Antigen Receptor (CAR) T Cells in DLBCL
Authors: He Mengke , Zhang Fan , Shen Jie , Li Zeyi , Liu Xiaxin , Ye Chenjing , Yan Hua , Hu Jiong , Li Junmin , Jin Zhen , Xiang Rufang 
Published: December 2024

Background
Despite continuous improvements in the new target and construction of chimeric antigen receptor (CAR)-T, relapse remains a serious challenge following CAR-T cell therapy. It is previously reported that Vitamin D insufficiency is a potentially modifiable risk factor for poor outcomes in diffuse large B-cell lymphoma (DLBCL). In DLBCL recipients of anti-CD19 CAR-T administration, vitamin D insufficiency was associated with significantly lower cell viability of the infused CAR-T product, as well as inferior day 100 complete response and overall survival rates. However, the molecular mechanism by which vitamin D may augment CAR-T responses remains elusive.
Method
We first verified the proliferation inhibition of 1α,25(OH)2D3 in treating both ABC-DLBCL cell lines (Su-DHL2 and U2932) and GCB-DLBCL cell line (OCI-Ly1 and Su-DHL4). Under the maximum observed plasma concentration (75 nM) of 1α,25(OH)2D3, the growth inhibition in both cell line was minimal. We further examined the efficiency of 1α,25(OH)2D3 in anti-CD19 CAR-T cells. CD3+ T cells were isolated from healthy donors or DLBCL patients, expanded with CD3/CD28 beads, while infected with anti-CD19-CAR lentiviral vector. CAR expression was detected by flow cytometry. We found that 1α,25(OH)2D3 pretreatment was negatively correlated with expression of programmed cell death-1 (PD-1) and T-cell immunoglobulin and mucin-domaincontaining-3 (Tim-3) on anti-CD19 CAR-T cells, but positively correlated with cell activation (CD69), without effecting CAR expression, CAR-T cell expansion or differentiation. Since 1α,25(OH)2D3 could reverse the exhaustion phenotype of anti-CD19 CAR-T cells, we assumed that it might also regulate antitumor function. To explore this assumption, anti-CD19 CAR-T cells were pretreated with1α,25(OH)2D3 for 6 days in vitro and then co-culture with DLBCL cell lines, antitumor function were substantially boosted in the absence of 1α,25(OH)2D3. Therefore, we further explored the cytokine secretion of anti-CD19 CAR-T cells with or without 1α,25(OH)2D3. It showed that 1α,25(OH)2D3 promoted IFN-γ and TNF-α secretion. To further investigate the molecular mechanism, CD4+ or CD8+ anti-CD19 CAR-T cells treated with 1α,25(OH)2D3 or vehicle were sorted by flow cytometer and performed by RNA-seq. Unbiased principal component analysis (PCA) showed that CAR-T cells treated with 1α,25(OH)2D3 were transcriptionally distinct from counterparts. A total of 935 differentially expressed genes were identified, illustrating the high degree of transcriptomic reprogramming induced by 1α,25(OH)2D3. Consistent with the previous data, 1α,25(OH)2D3-treated cells displayed decreased expression of exhaustion-related genes, including PDCD1, HAVCR2, LAG3, BATF, CD40LG, NR4A1, NR4A2 and NR4A3. Gene set enrichment analysis (GSEA) of our data with T cell exhaustion genes identified in the lymphocytic choriomeningitis virus mouse model revealed significant enrichment of less-exhausted T cell populations in the treatment of 1α,25(OH)2D3.
Conclusion
Therefore, our findings illustrated that 1α,25(OH)2D3 alleviated exhaustion of anti-CD19 CAR-T cells, which might contribute to the superior antitumor function of anti-CD19 CAR-T cells. Further investigations are warranted to establish the in-vivo combination efficacy in the future. This potential enhancement of CAR-T cell therapy by 1α,25(OH)2D3 opens new avenues for improving treatment outcomes in patients with DLBCL.