T-Lymphocyte Suppression By ULK2 High Expression of CD14+ Monocytes in Patients with Multiple Myeloma

Background: Multiple myeloma (MM), a plasma cell malignancy, is incurable and highly prone to relapse. The immunosuppressive cells in the bone marrow environment inhibit endogenous T-lymphocyte activity and reduce the efficacy of T-cell immunotherapies. Bone marrow monocytes in patients with MM correlate with poor survival. This study explored the mechanism of T-lymphocyte suppression by bone marrow CD14⁺ monocytes in MM.

Methods: CD14⁺ monocytes from the bone marrow of patients with newly-diagnosed MM (NDMM) were analyzed, and RNA sequencing was performed. We investigated the regulation effects on T lymphocytes by studying the interaction between CD14⁺ monocytes and T lymphocytes, as well as the downstream signaling mechanism through in vitro and in vivo experiments.

Results: By analyzing single-cell RNA sequence data (GSE124310), we discovered that the incoming and outgoing signal strength of CD14⁺ monocytes from MM was increased compared to healthy controls (HCs). The change in MHC Ⅱ gene signaling of outgoing interaction in CD14⁺ monocytes was decreased in MM. In our experiment, the proportion of intermediate monocytes was higher in NDMM than in HCs, mean fluorescence intensity (MFI) of surface CD86, CD80, and HLA-DR in intermediate and classical monocytes was decreased. Abnormal numbers, defective antigen presentation, and downregulated surface co-stimulatory molecules of bone marrow CD14⁺ monocytes in patients with NDMM were verified. We collected bone marrow samples from 10 NDMM and 5 HCs, and sorted CD14⁺ monocytes for RNA sequencing. The Unc-51 like autophagy activating kinase 2 (ULK2) gene was significantly upregulated NDMM. The overexpressing ULK2 (oeULK2) gene THP1 cell line was constructed. The oeULK2 and vector THP1 cells underwent high-throughput RNA sequencing. The antigen processing and presentation pathway, involved in T lymphocyte activation, was validated. Compared with the vector THP1, mRNA levels of MHC Ⅰ, TAP1, PSME1/2, HLA-DQB, HLA-DMA, and CTSB were downregulated in the oeULK2 THP1 cells. Reduced levels of MHC Ⅰ, TAP1, PSME1, MHC Ⅱ, CTSB, and CTSL proteins in oeULK2 THP1 were confirmed by WB. The MFI of CD86 of oeULK2 THP1 was found to be decreased. The protein levels of MHC Ⅰ, TAP1, PSME1, MHC Ⅱ, CTSB, CTSL, and the MFI of CD86 in oeULK2 THP1 was upregulated accompanying with various concentrations of ULK1/ULK2 inhibitor (MRT68921) co-culture for 48h. Additionally, the MFI of CD86 and HLA-DR in primary CD14⁺ monocytes from patients with NDMM was found to be upregulated after 0.5 μM MRT68921 co-culture for 48h. Bone marrow CD14⁺ monocytes in NDMM suppressed T-lymphocyte activation, indicated by a decrease in the MFI of CD69, GZMB, and TNFα. Administration of MRT68921 to CD14⁺ monocytes alleviated T-lymphocyte suppression. In vivo experiments were conducted using a mouse xenograft model implanted with RPMI-8226 cells and reconstituted with human CD3⁺ T lymphocytes and ULK2^high or ULK2^low/- CD14⁺ monocytes. Tumor growth was significantly accelerated by infusing ULK2^high monocytes. The MFI of CD69, GZMB, and TNFαin CD3⁺ T lymphocytes from the tumor was significantly decreased after infusion of ULK2^high monocytes compared to that in the ULK2^low/- monocytes group. The level of serum IFN γwas also decreased in the ULK2^high monocytes group.

Conclusions: We demonstrated that ULK2^high CD14⁺ monocytes in MM hinder the delivery of antigenic peptides through the antigen processing and presentation pathway. This disruption affects T-lymphocyte activation and attenuates their ability to kill and secrete cytokines. This study provides a theoretical basis for understanding the formation of the myeloma immunosuppressive microenvironment, enhancing the efficacy of T lymphocyte-based immunotherapy, and developing new targeted therapies ULK2.