CD312 Promotes Paediatric Acute Lymphoblastic Leukaemia Through GNA15-Mediated Non-Classical GPCR Signalling Pathway

Abstract

The bone marrow-infiltrated immune microenvironment plays a crucial role in blood system diseases, such as leukaemia. In this study, we aimed to investigate the critical role of the immune microenvironment in the onset and progression of childhood acute lymphoblastic leukaemia (ALL). Through high-throughput detection and screening of the GPCR database in the childhood ALL immune microenvironment, we identified CD312 as a candidate target. CD312 is associated with the distribution of Treg and CTL cells within the bone marrow immune microenvironment of ALL children. After CD312 knockdown, the proportion of the Treg subgroup in immune cells was significantly reduced, whereas the proportion of CTL subgroup cells was increased. CD312 exhibited good affinity with GNA15 in the transmembrane intracellular segment, and it could interact with GNA15. The BrdU staining assay revealed that the proliferation of leukaemia cells was enhanced in the CD312-overexpressed CD3+ T cells group via the phosphorylation of ERK, JNK and p38, whereas it was decreased by GNA15 knockdown in the co-culture system. In conclusion, our study suggests that CD312 fosters a suppressive immune microenvironment in the onset and progression of paediatric ALL through a GNA15-mediated non-classical GPCR signalling pathway.

Keywords: CD312; GPCR; acute lymphoblastic leukaemia; immune microenvironment.

FIGURE 1

The expression of GPCRs in ALL children. (A) Experimental design. Three bone marrow samples from ALL children selected as patients group samples, three bone marrow samples from children without non‐tumour diseases were used as controls. (B) Flow cytometry was used to observe the distribution of Treg cell subpopulations and CTL cell subpopulations in the bone marrow. n = 5. (C, D) Heatmap of differentially genes in CD8+ T cells (C) and CD4+ T cells(C) by the high‐throughput screening. (E) 6 GPCRs exhibited differential expression in both CD8+ and CD4+ cells. *p < 0.05.

FIGURE 2

CD312 expression was related to immunosuppressive state. (A) The expression of CD312 in both CD8⁺ T cells was detected by RT‐PCR (n = 40). (B) The expression of IFN‐ γ in CD8⁺ T cells was detected by RT‐PCR (n = 40). (C) Pearson correlation analysis indicated that CD312 expression was a negative correlation with IFN‐ γ. (D) The expression of CD312 in both CD4⁺ T cells was detected by RT‐PCR (n = 40). (E) The expression of IFN‐ γ in CD4⁺ T cells was detected by RT‐PCR (n = 40). (F) Pearson correlation analysis indicated that CD312 expression was a negative correlation with Foxp3. *p < 0.05, **p < 0.01, *p < 0.001.

FIGURE 3

CD312 was related to the distribution of Treg and CTL cells. (A, B) The proportion of Treg cells was detected by flow cytometry in the normal control group, CD312 high expression group (CD312 high) and CD312 low expression group (CD312 low). n = 40. (C, D) The proportion of CTL cells was detected by flow cytometry in the normal control group, CD312 high expression group (CD312 high) and CD312 low expression group (CD312 low). n = 40. **p < 0.01.

FIGURE 4

CD312 knockdown regulated Treg and CTL cells distribution. (A, B) The RT‐PCR and western blot assays were used to detect the expression of the CD312 level in the CD312 shRNA group. n = 3. (C, D) The distribution of Treg and CTL in the cell subpopulations was detected by flow cytometry after CD312 knockdown. n = 3. *p < 0.05, **p < 0.01.

FIGURE 5

CD312 in CD3 + T cells promoted the proliferation of leukaemia cells. (A) The design of the co‐culture system. (B, C) The CCK8 assay and BrdU staining were conducted to evaluate the proliferation of leukaemia cells after CD312 knockdown in CD3 + T cells. n = 3. (D, E) The flow cytometry and TUNEL staining were conducted to evaluate the apoptosis of leukaemia cells after CD312 knockdown in CD3 + T cells. n = 3. **p < 0.01.

FIGURE 6

CD312 interacts with GNA15. (A) Prediction of potential binding sites between CD312 and GNA15. (B, C) Immunofluorescence assay and coimmunoprecipitation assay were performed to indicate the interaction between CD312 and GNA15. Scale bar = 20 μm. (D) PLA assay was used to detect the interaction between CD312 and GNA15. Scale bar = 20 μm. (E, F) The proportion of the CTL subgroup and Treg subgroup in immune cells was detected by flow cytometry upon CD312 overexpression and GNA15 knockdown or not. n = 3. **p < 0.01.

FIGURE 7

CD312 promoted the proliferation in leukaemia cells through binding to GNA15. (A) The BrdU staining assay was used to detect the proliferation of leukaemia cells, which was increased in the CD312 overexpressed CD3 + T cells group, while it was downregulated by GNA15 knockdown. n = 3. (B) The TUNEL staining assay was used to detect the apoptosis of leukaemia cells, which was increased in the CD312 overexpressed CD3 + T cells group, while it was downregulated by GNA15 knockdown. n = 3. (C) The phosphorylation of ERK, JNK and p38 was detected by western blot in CD312 overexpressed CD3 + T cells and GNA15 knockdown or not. n = 3. **p < 0.01.