Phenotypic changes in CD38 + NK and CD38 + NKT Cells in CIA peripheral blood
We collected peripheral blood from 30 CIA rats and 24 normal rats. The proportions of CD4 + T cells, CD3-CD19 + B cells and CD3-CD161 + NK cells in total lymphocytes (CD45 + lymphocytes) in CIA group were significantly higher than those of the normal control group (p = 0.00467, 0.0415 and 0.0164, respectively), whereas the proportions of CD8 + T cells, CD3 + CD161 + NKT cells and CD4 + CD25 + FOXP3 + Treg cells were significantly lower than those of the normal control group (p = 0.0037, 0.0436 and 0.005, respectively). There was no significant difference in the proportion of CD3 + T cells between the two groups (p = 0.618). The results are shown in Fig. 1A.
Compared with the normal control group, the proportions of CD38 + CD3- CD19 + B (CD38 + B) cells and CD38 + CD3- CD161 + NK (CD38 + NK) cells in the peripheral blood of CIA rats were significantly increased (p = 0.0062 and 0.0016, respectively), the proportion of CD38 + CD3 + CD161 + NKT (CD38 + NKT) cells was significantly decreased (p = 0.0028), and there was no significant change in the proportion of CD38 + CD3+ (CD38 + T) cells (p = 0.3639) (Fig. 1B). The CD38 + NK/CD38 + NKT ratio values of the CIA rats ranged from 0.83 to 16.28, with an average of 5.16, which was significantly higher than that of the normal control group (range: 0.08–5.62, mean: 1.66, p = 0.0012). Meanwhile, the NK/NKT ratio values of the CIA rats ranged from 1.13 to 26.69, with an average of 8.34, which was significantly higher than that of the normal control group (range: 0.18–14.39, mean: 3.86, p = 0.007) (Fig. 1C). In addition, the ratio of CD38 + NK/CD38 + NKT in the CIA rats was positively correlated with the CIA inflammation index (r = 0.76, p < 0.0001), and the NK/NKT ratio was also positively correlated with the disease activity score (r = 0.69, p = 0.0002) (Fig. 1D).
Phenotypic changes in CD38 + NK and CD38 + NKT cells in RA peripheral blood and synovial fluid
We collected peripheral blood from 30 RA patients and 30 healthy volunteers. The proportions of CD45 + lymphocytes, CD4 + T, CD3- CD19 + B and CD3- CD16 + CD56 + NK cells in total lymphocytes (CD45 + lymphocytes) in the RA group were significantly higher than those in the control group (p = 0.0007, 0.0167 and < 0.0001, respectively), whereas the proportions of CD4 + CD25 + FOXP3 + Treg cells in RA group were significantly lower than those in the healthy controls (p = 0.0037 and 0.0029). There were no significant differences in the proportions of CD3 + T, CD8 + T and CD3 + CD16 + CD56 + NKT cells between the two groups (p > 0.05). The results are shown in Fig. 2A.
Compared with healthy volunteers, CD38 + CD3- CD19 + B (CD38 + B) and CD38 + CD3- CD16 + CD56 + NK (CD38 + NK) cells were significantly elevated in the peripheral blood of RA patients (p = 0.0001 and 0.0008, respectively), the proportion of CD38 + CD3 + CD16 + CD56 + NKT (CD38 + NKT) cells was significantly reduced (p = 0.0121), and there was no significant change in the proportion of CD38 + CD3 + cells (p = 0.3689) (Fig. 2B). The ratio of CD38 + NK/CD38 + NKT in RA patients ranged from 0.62 to 23, with an average of 4.545, which was significantly higher than that of the healthy control group (range: 0.31–11.26, mean: 1.153, p = 0.0004). There was no significant difference in NK/NKT ratio between the RA patients and healthy subjects (p = 0.9227) (Fig. 2C). The CD38 + NK/CD38 + NKT ratio was positively correlated with the RA DAS28 (r = 0.716, p < 0.0001), whereas the NK/NKT ratio was slightly correlated with DAS28 (r = 0.465, p = 0.0096) (Fig. 2D).
We collected synovial fluid samples from 30 RA patients and 30 OA patients. The proportions of CD4 + T, CD3- CD19 + B and CD3- CD16 + CD56 + NK cells in total lymphocytes (CD45 + lymphocytes) in RA group were significantly higher than those in OA group (p = 0.0007, 0.0124 and 0.0021, respectively), whereas the proportions of CD3 + CD16 + CD56 + NKT, Treg and CD8 + T cells were significantly lower than those in the control group (p = 0.0019, 0.0002 and 0.0094, respectively). There was no significant difference in the proportions of CD3 + T cells and CD45 + lymphocytes between the two groups (p > 0.05). The results are shown in Fig. 3A.
Compared with the OA control group, the proportions of CD38 + CD3- CD19+ (CD38 + B) and CD38 + CD3- CD16 + CD56 + NK (CD38 + NK) cells in the synovial fluid of RA patients were significantly increased (p = 0.0088 and 0.0002, respectively), the proportion of CD38 + CD3 + CD16 + CD56 + NKT (CD38 + NKT) cells was significantly reduced (p = 0.0189), and there was no significant change in the proportion of CD38 + CD3 + cells (p = 0.7905) (Fig. 3B). The ratio of CD38 + NK/CD38 + NKT cells in RA patients ranged from 0.942 to 14.45, with an average of 3.07, which was significantly higher than that of the OA group (range: 0.234–4.407, mean: 1.58, p < 0.0001). The NK/NKT ratios in RA patients ranged from 1.463 to 9.713, with an average of 2.82, which was significantly higher than that in OA group (range: 0.503–5.326, mean: 1.54, p = 0.0004) (Fig. 3C). The CD38 + NK/CD38 + NKT ratio in RA patients was positively correlated with DAS28 (r = 0.747, p < 0.0001), whereas the NKT/NK ratio was not associated with DAS28 (r=-0.20, p = 0.2875) (Fig. 3D).
Effects of CD38 + NK and CD38 + NKT cells on MNCs
We collected synovial fluid from 9 patients with RA, isolated MNCs from the samples, and then prepared CD38 + NK cells, CD38 + NKT cells and MNCs depleted of CD38 + cells. The purity of CD38 + NK was 99.28%, whereas that of CD38 + NKT was 98.9%. The result is shown in Supplementary Fig. 1A, B. The CD38 + NK and CD38 + NKT cells were pretreated with CD38 monoclonal antibody (mAb) or BSA as a control and then cocultured with RA synovial fluid MNCs that were depleted of CD16 + CD56 + CD38 + cells. The experimental design is shown in Fig. 4A. Compared with non-cocultured synovial MNCs, the proportion of CD4 + CD25 + FOXP3 + Treg cells in total lymphocytes (CD45 + lymphocytes) in the MNCs was significantly decreased (p = 0.033), and the proportion of CD4 + IL-17 + Th17 (Th17) cells in lymphocytes was significantly increased (p = 0.0055) in MNCs after coculturing with the CD38 + NK cells. Meanwhile, the proportion of CD4 + CD25 + FOXP3 + Treg cells in MNCs was significantly increased (p = 0.0021), and the proportion of CD4 + IL-17 + Th17 cells in lymphocytes was significantly decreased (p = 0.0152) in the MNC after coculturing with CD38 + NKT cells. Compared with MNCs cocultured with the CD38 + NK cells, the proportions of CD3 + T cells and CD4 + CD25 + FOXP3 + Treg cells in lymphocytes were significantly higher (p = 0.0252 and 0.0021, respectively), and the proportion of CD4 + IL-17 + Th17 cells in lymphocytes was significantly reduced (p = 0.0025) in MNCs coculturing with the CD38 + NKT cells. Compared with MNCs cocultured with BSA-pretreated CD38 + NK cells, the proportions of CD4 + T cells and CD4 + IL-17 + Th17 cells were significantly reduced (p = 0.0402 and 0.0137, respectively), and the proportion of CD4 + CD25 + FOXP3 + Treg cells was significantly elevated (p = 0.0479) in MNCs after coculturing with the antibody-pretreated CD38 + NK cells (CD38 + NK + CD38 mAb). Conversely, compared with MNCs cocultured with BSA-pretreated CD38 + NKT cells, the proportion of CD4 + CD25 + FOXP3 + Treg cells was significantly reduced (p = 0.0139), and the proportion of CD4 + IL-17 + Th17 cells was significantly increased (p = 0.0224) in MNCs after coculturing with the antibody-pretreated CD38 + NKT cells (CD38 + NKT + CD38 mAb). The above results are shown in Fig. 4B.
Compared with non-cocultured synovial MNCs, the IFN-γ level in the medium was significantly increased (p = 0.0114), and IL-10 was significantly decreased (p = 0.0377), when MNCs were cocultured with CD38 + NK cells. In contrast, the IL-6 level was significantly decreased (p = 0.0434) when MNCs were cocultured with CD38 + NKT. Compared with MNCs cocultured with CD38 + NK cells, the IFN-γ level in the culture medium was significantly decreased (p = 0.0193) when MNCs were cocultured with CD38 + NKT cells. Compared with MNCs cocultured with BSA-pretreated with CD38 + NK cells, the levels of IL-2 and IL-10 in the culture medium were significantly increased (p = 0.0492 and 0.0275, respectively) when MNCs were cocultured with CD38 antibody-pretreated CD38 + NK cells, and the levels of IL-6 and IFN-γ were significantly reduced (p = 0.0200 and 0.0119, respectively). Compared with MNCs cocultured with BSA-pretreated CD38 + NKT cells, the cytokine levels in the culture medium were not changed when MNCs were cocultured with CD38 antibody-pretreated CD38 + NKT cells. The above results are shown in Fig. 4C.
We collected peripheral blood from nine patients with RA, isolated MNCs from the samples, and then prepared CD38 + NK cells, CD38 + NKT cells and MNCs depleted of CD38 + cells using flow cytometry. The CD38 + NK and CD38 + NKT cells were treated with BSA or CD38 mAb. They were then cocultured with MNCs depleted of CD16 + CD56 + CD38 + cells including CD38 + NK and CD38 + NKT cells. The experimental design is also shown in Fig. 4A. Compared with non-cocultured peripheral MNCs, the proportion of CD4 + CD25 + FOXP3 + Treg cells was significantly decreased (p = 0.0007), and the proportion of CD4 + IL-17 + Th17 cells in the lymphocytes was significantly increased (p = 0.0162) in the blood MNCs after coculturing with CD38 + NK cells. The proportion of CD4 + CD25 + FOXP3 + Treg cells was significantly increased (p = 0.0269), and the proportion of CD4 + IL-17 + Th17 cells in lymphocytes was significantly decreased (p = 0.0006) in MNCs after coculturing with CD38 + NKT cells. Compared with MNCs cocultured with CD38 + NK cells, the proportion of Treg cells was significantly increased (p = 0.0327) in MNCs after coculturing with CD38 + NKT cells, and the proportion of CD4 + IL-17 + Th17 cells in lymphocytes was significantly decreased (p = 0.0004). Compared with MNCs cocultured with BSA-pretreated CD38 + NK cells, the proportion of Treg cells was significantly increased (p = 0.0454) after coculturing with CD38 antibody-pretreated CD38 + NK cells, and the proportion of CD4 + IL-17 + Th17 cells in lymphocytes was significantly decreased (p = 0.0381). Compared with MNCs cocultured with BSA-pretreated CD38 + NKT cells, the proportion of Treg cells was significantly decreased (p = 0.008), and the proportion of CD4 + IL-17 + Th17 cells in lymphocytes was significantly increased (p = 0.0342) in MNCs after coculturing with CD38 antibody-treated with CD38 + NKT cells. The results are shown in Fig. 5A.
Compared with non-cocultured blood MNCs, the level of IFN-γ in the culture medium was significantly increased (p < 0.0001), and IL-10 was significantly decreased (p = 0.0164), when MNCs were cocultured with CD38 + NK cells. The IL-6 level in the culture medium was significantly decreased (p = 0.0434) when MNCs were cocultured with CD38 + NKT cells. Compared with MNCs cocultured with CD38 + NK cells, IL-6 and IFN-γ levels in the culture medium were significantly decreased (p = 0.0155 and p = 0.0006, respectively) when MNCs were cocultured with CD38 + NKT cells, whereas the IL-10 level was significantly increased (p = 0.0416). Compared with MNCs cocultured with BSA-pretreated with CD38 + NK cells, the levels of IL-2 and IL-10 were significantly increased (p = 0.0499 and p = 0.0013, respectively), and the IL-6 and IFN-γ levels in the culture medium were significantly decreased (p = 0.0051 and p = 0.0454, respectively) when MNCs were cocultured with CD38 antibody-pretreated CD38 + NK cells. Compared to MNCs cocultured with BSA-pretreated CD38 + NKT cells, IL-6 level in the culture medium was significantly increased (p = 0.0341) when MNCs were cocultured with CD38 antibody-pretreated CD38 + NKT cells, but the levels of other cytokines were not significantly changed. The above results are shown in Fig. 5B.
Effect of CD38 + NK and CD38 + NKT cells on CD4 + T cells
To investigate the effects of CD38 + NK and CD38 + NKT cells on CD4 + T cell differentiation, we collected synovial fluid from 9 RA patients and sorted homogenous CD38 + NK cells, CD38 + NKT cells and CD4 + T cells. The CD38 + NK and CD38 + NKT cells were treated with BSA or anti-CD38 antibody and were then cocultured with CD4 + T cells. The experimental design is shown in Fig. 6A. Compared with the non-cocultured CD4 + T cells, the ratios of Th1/Th2 and Th17/Treg cells were increased significantly (p = 0.0086 and 0.0186, respectively) in the CD4 + T cells after coculturing with CD38 + NK cells, whereas the ratios of Th1/Th2 and Th17/Treg cells were significantly decreased (p = 0.0061 and 0.0074, respectively) in the CD4 + T cells after coculturing with CD38 + NKT cells. Compared with CD4 + T cells cocultured with BSA-pretreated CD38 + NK cells, the ratios of Th1/Th2 and Th17/Treg cells were significantly decreased (p = 0.0325 and 0.0259, respectively) in CD4 + T cells after coculturing with CD38 antibody-treated CD38 + NK + CD38 cells. Compared with CD4 + cells with BSA-pretreated CD38 + NKT cells, the ratios of Th1/Th2 and Th17/Treg cells were significantly increased (p = 0.0006 and 0.0002, respectively) in CD4 + T cells after coculturing with CD38 antibody-pretreated CD38 + NKT cells. The results are shown in Fig. 6B and C.
To determine if CD38 + NK or CD38 + NKT cells had direct effect on Treg cell proliferation, we cocultured CD38 + NK cells or CD38 + NKT cells and Treg cells in place of CD4 + T cells in a transwell apparatus. The experimental design is shown in Supplementary Fig. 2A. We examined the cell proliferation of Treg cells using a CCK-8 kit. Treg cells following coculture with CD38 + NK cells showed no significant change in cell proliferation, compared with the Treg cells without coculture or Treg cells following coculture with anti CD38 antibody-pretreated CD38 + NK cells. The effect of CD38 + NKT on Treg cell proliferation was similar to that of CD38 + NK. The results are shown in Supplementary Fig. 2B. These results indicated that CD38 + NK cells and CD38 + NKT cells as well as CD38 molecular on the cells had no significant effect on Treg cell proliferation.
To investigate the molecular mechanisms by which CD38 + NK and CD38 + NKT cells act on the differentiation of CD4 + T cells, we collected the cocultured CD4 + T cells and then examined changes in the mTOR signaling pathway using Western blot analysis. Compared with CD4 + T cells cocultured with BSA-pretreated CD38 + NK cells, CD4 + T cells showed a significant decrease in the expression levels of phospho-P70S6, phospho-mTOR and total mTOR protein (p = 0.0473, 0.0127 and 0.0094, respectively) after coculturing with CD38 antibody-pretreated CD38 + NK cells. Compared with CD4 + T cells cocultured with CD38 + NK cells, CD4 + T cells also showed a significant decrease in phospho-P70S6, phospho-mTOR and total mTOR protein expression levels (p = 0.0004, 0.0037 and 0.0012, respectively) after coculturing with CD38 + NKT cells. Compared with CD4 + T cells cocultured with BSA-pretreated with the CD38 + NKT cells, CD4 + T cells showed significant increases in phospho-P70S6, phospho-mTOR and total mTOR protein expression levels (p = 0.003, 0.0019 and 0.0059, respectively) after coculturing with CD38 antibody-pretreated CD38 + NKT cells. Compared with non-cocultured CD4 + T cells, CD4 + T cells showed significant increases in the phospho-P70S6, phospho-mTOR, and total mTOR protein expression levels (p = 0.0075, 0.0425 and 0.0216, respectively) after coculturing with CD38 + NK cells, while CD4 + T cells also showed significant increases in phospho-P70S6, phospho-mTOR and total mTOR protein levels (p = 0.0029, 0.0358 and 0.0116, respectively) after coculturing with CD38 antibody-pretreated CD38 + NKT cells. The above results are shown in Fig. 6D and E.
We also collected CD38 + NK and CD38 + NKT cells from the cocultures described above and used real-time PCR to detect the mRNA levels of CD3, CD28 and TGF-β. Compared with the CD38 + NK cells, the CD38 + NKT cells showed a significant increase in CD3 mRNA expression (p = 0.0002), while the CD38 + NKT cells with pretreatment of CD38 antibody showed decreased CD3 mRNA expression (p = 0.0079). Regardless of presence or absence of the CD38 antibody, the CD28 level in CD38 + NK and CD38 + NKT cells remained unchanged. Compared with BSA-pretreated CD38 + NK cells, CD38 + NK cells with pretreatment of CD38 antibody showed significantly higher levels of TGF-β expression (p = 0.0056), but the antibody pretreatment did not affect TGF-β expression in CD38 + NKT cells. The above results are shown in Fig. 6F. Additionally, flow cytometry analysis of cytokines detected increased TGF-β expression in the cultured medium of CD38 + NK cells with pretreatment of CD38 antibody. The above results are shown in Fig. 6G and H.