CTLA‐4 and HLA‐DQ are key molecules in the regulation of mDC‐mediated cellular immunity by Tregs in severe aplastic anemia

Abstract Background Regulatory T cells (Tregs) inhibit the activation of cluster of differentiation (CD) 4+, CD8+T cells and the antigen‐presenting process of antigen‐presenting cells, and may play an important role in acquired severe aplastic anemia (SAA). Methods Flow cytometry was used to measure CD4+CD25+CD127dim Tregs, cytotoxic T lymphocyte antigen 4 (CTLA‐4) expression on Tregs, and human leukocyte antigen (HLA)‐DQ expression on myeloid dendritic cells (mDCs). The correlations of CTLA‐4 and HLA‐DQ with immune status and clinical indicators and the changes in these indicators after immunosuppressive therapy (IST) were analyzed. Results In SAA patients, the number of Tregs and their CTLA‐4 expression were low but recovered after IST; the HLA‐DQ expression on mDCs was high but decreased after IST. The CTLA‐4 expression on Tregs and the HLA‐DQ expression on mDCs showed a negative correlation. The CTLA‐4 on Tregs was positively but HLA‐DQ on mDCs negatively correlated with the number of Tregs, natural killer (NK) cell number, and CD4+T/CD8+T ratio. CTLA‐4 was positively but HLA‐DQ negatively correlated with the percentage of granulocytoid and erythroid cells in bone marrow, white blood cell count in PB, absolute neutrophil count in PB, and the percentage of reticulocytes in PB. Conclusions CTLA‐4/HLA‐DQ may be key in the regulation of Tregs on mDCs in SAA patients. Our findings should be helpful for further investigation of the mechanism of immune pathogenesis in SAA patients. Studies on the regulators of Treg and CTLA‐4 activity will be valuable for SAA therapeutic target research and disease monitoring.


| INTRODUC TI ON
Acquired severe aplastic anemia (SAA) is a bone marrow failure disease caused by abnormal cellular immunity. Its immune pathogenesis is that under the stimulation of unknown antigens, myeloid dendritic cells (mDCs) become hyperfunctional, the ratio of T-helper 1 (Th1) and Th2 cells is out of balance, and the abnormally activated cluster of differentiation (CD)8 + T cells become hyperfunctional, attacking the bone marrow hematopoietic cells, which causes severe pancytopenia. Disorders of autoimmune tolerance also play an important role in the pathogenesis of SAA. 1,2 Regulatory T cells (Tregs) were first proposed to exist by Sakaguchi in 1995. Tregs inhibit the activation of CD4 + and CD8 + T cells, inhibit the antigen-presenting process of antigen-presenting cells (APCs), are a kind of T-cell subset with immunomodulatory function, and play an important role in maintaining autoimmune tolerance. Tregs express many functional molecules, such as CD25 (interleukin [IL]-2Ra chain), forkhead box protein 3 (FoxP3), and cytotoxic T lymphocyte antigen (CTLA-4, CD152). FoxP3 is an important protein necessary for Tregs to exert their inhibitory function. 3 CD4 + T cells expressing FoxP3 + with a high level of CD25 and low level of CD127 are referred to as having the phenotype FoxP3 + CD25 high CD127 low CD4 + . 4 The CD127 expression is closely related to the regulatory function of Tregs and is negatively correlated with the FoxP3 expression and the inhibitory activity of Tregs. 5 As our understanding of Tregs has grown, an increasing number of researchers have used CD4 + CD25 + CD127 dim as the molecular marker of Tregs. Treg dysfunction is associated with several common autoimmune diseases, including SAA. 6,7 In this study, we performed correlation analysis on the expression levels of CTLA-4 and human leukocyte antigen (HLA)-DQ on the surfaces of Tregs and mDCs, respectively, in the peripheral blood (PB) of SAA patients and explored the possible mechanism by which Tregs regulate mDCs in the SAA immune pathogenesis.

| Study subjects
Twenty-one SAA patients admitted to the Tianjin Medical University General Hospital between January 2018 and December 2019 were enrolled (SAA group), including 13 males and 8 females, with a median age of 38 (9-77) years. Twenty-four recovering SAA patients were enrolled (RSAA group), including 11 males and 12 females, with a median age of 34  years. Twenty-four healthy controls (HC group) were also enrolled, including 12 males and 12 females, with a median age of 33    were used. Then, 2 mL of red blood cell lysis buffer was added, and the tube was incubated for 10 minutes in the dark to remove red blood cells.

| Experimental methods
After centrifugation at 1300 rpm for 5 minutes, the supernatant was discarded. After washing with phosphate-buffered saline, the cells were resuspended in 300 μL for detection, and at least 50 000 cells/ tube were recorded. In this study, CD4 + CD125 + CD127 dim defined Tregs, HLA-DR + CD11c + Lin -CD123defined mDCs, and the mouse IgG1 was used as the isotype control to analyze the expression levels of CTLA-4 and HLA-DQ. SPSS 19.0 software (IBM) was used for statistical analysis. The

| Statistical analysis
Kolmogorov-Smirnov test was used to determine whether the data were normally distributed. When the data were normally distributed, the independent-sample t test was used to compare values between groups. When data were not normally distributed, Kruskal-Wallis one-way analysis of variance (ANOVA) was used to compare values between groups. Correlation analysis between variables was performed using the Spearman rank correlation test. When a significant result was found, the correlation coefficient (r) is reported. A paired-sample t test was used to analyze the changes from before to after the IST. Quantitative data are expressed as the mean ± SD, and P < .05 was considered statistically significant.

| The number of Tregs and CTLA-4 expression in SAA patients and HCs
The ratio of CD4 + T cells among the PBLs (CD4 + T/PBL) in the SAA, RSAA, and HC groups was 27.28 ± 11.43%, 30.57 ± 17.12%, and 36.47 ± 7.13%, respectively ( Figure 1A,B). The CD4 + T/PBL in SAA group was significantly lower than in the HC group (P < .01), and there was no significant difference between the RSAA and HC groups ( Figure 1C). In the SAA group, the percentage of Tregs in CD4 + T cells (Treg/CD4 + T) was 3.01 ± 0.73%, which was significantly lower than that (3.97 ± 1.38%) in the RSAA group (P < .01) and that (5.39 ± 1.18%) in HC group (P < .001) ( Figure 1A,B,D), and the Treg/CD4 + T ratio in the RSAA group was significantly lower than that in the HC group (P < .001, Figure 1D). The product of Treg/CD4 + T and CD4 + T/PBL equals the ratio of Tregs among PBLs (Treg/PBL). The Treg/PBL ratio in the SAA group was 0.83 ± 0.43%, significantly lower than that (1.19 ± 0.71%) in the RSAA group (P < .05) and that (1.94 ± 0.43%) in HC group (P < .001), and the Treg/PBL ratio in the RSAA group was significantly lower than that in the HC group (P < .001, Figure 1E). The expression percentage of CTLA-4 on Tregs in the PB in the SAA group (CTLA-4 + Treg/ Treg) was 9.55 ± 2.28%, which was significantly lower than that (11.88 ± 3.96%) in the RSAA group (P < .05) and that (13.71 ± 4.48%) in the HC group (P < .05). There was no statistically significant difference in CTLA-4 + Treg/Treg between the SAA and HC groups (P < .001) (Figure 2A-C).

| HLA-DQ expression on mDCs in SAA patients and HCs
The expression percentage of HLA-DQ on mDCs (HLA-DQ + mDC/ mDC) from PB in the SAA and RSAA groups were 18.44 ± 8.53% and 14.88 ± 13.33%, respectively, significantly higher than that (8.59 ± 6.14%) in the HC group (P < .001, P < .05). The HLA-DQ + mDC/ mDC in the SAA group was not significantly different from that in the RSAA group ( Figure 3).

| Correlation analysis of CTLA-4 expression on Tregs and HLA-DQ expression on mDCs in SAA patients
There was a negative correlation between CTLA-4 expression on Tregs and HLA-DQ expression on mDCs in SAA patients (r = −.500, P < .05, Figure 4).

| Changes in the number of Tregs, CTLA-4 expression on Tregs, HLA-DQ expression on mDCs, NK cell number, CD8 + T-cell number, and CD4 + T/ CD8 + T ratio in SAA patients after IST
To study the changes in the number of Tregs, CTLA-4 expression on Tregs, HLA-DQ expression on mDCs, NK cell number, CD8 + T-cell number, and CD4 + T/CD8 + T ratio in SAA patients from before to after enhanced IST, 10 patients in the SAA group, whose IST with rATG and CsA was effective, were observed before and at 3 and 6 months after IST.

| D ISCUSS I ON
Severe aplastic anemia is a severe hematologic disease characterized by a severe reduction in whole blood cells and bone marrow failure.
Its pathogenesis is directly related to the damage done to autologous FoxP3 gene mutations in human and mouse can cause severe autoimmune diseases in multiple organs. In the CD4 + CD25 + FoxP3cells of AA patients transfected with NFAT1, the expression of NFAT1 is increased, and the expression of FoxP3 is also increased. In contrast, when NFAT1 is knocked out from Tregs, Foxp3, and NFAT1 expression is decreased, suggesting that the cells that induce immune tolerance are seriously insufficient in SAA patients.
During the immune response, DCs recognize, take in, and process antigens; synthesize MHC-antigen (peptide) complexes and costimulatory molecules on the surface; and present antigens to naïve T cells. This process plays an important role in the immune response.
Our previous results suggested that the number of mDCs in SAA was increased, the mDC/pDC ratio was increased, and the mDCs were hyperfunctional. 13 The abnormal antigen-presenting process of DCs. 16 Tregs can also inhibit the functions of the above immune cells through direct contact. 17 DCs are the main regulatory target of Tregs, and this regulatory process relies on antigenic stimulation. 18 This study analyzed the regulatory mechanism of Tregs on mDCs.
The experimental results suggested that the number of Tregs in the PB of SAA patients was decreased significantly and gradually recov- pathways and adverse reactions of inflammatory bowel disease. 26,27 We also examined the expression of MHC class II molecule HLA-DQ the NK cell number at 3 months after IST were not different from those before IST, but they were significantly higher 6 months after IST than before IST; the CTLA-4 expression on Tregs and CD8 + T-cell number at 3 months after IST was significantly higher than before IST, and those at 3 months after IST were not significantly different from those at 6 months after IST; the CD4 + T/CD8 + T ratio was significantly higher at 3 months after IST than before IST and continued to on SAA and other autoimmune diseases. 29 The CD8 + T-cell number and CD4 + T/CD8 + T ratio were significantly corrected at 3 months after IST and continued to recover over time. The NK cell number started to recover at 6 months after IST, which was similar to the recovery of the number of Tregs, suggesting that the inhibitory factors in the SAA immunization cascade recover less readily. MHC class II HLA-DQ on mDCs also showed this pattern after IST, which suggested that it may be a target in Tregs regulating mDCs, providing an idea for research on the poor efficacy of IST and on the recurrence monitoring of diseases.
In summary, the number of Tregs and their functional molecule CTLA-4 were significantly downregulated in SAA, and the expression of the MHC class II molecule HLA-DQ on mDCs was significantly upregulated. These changes might be key to the loss of regulation by Tregs of mDCs in SAA patients. These findings are conducive to further exploring the mechanism of SAA immune pathogenesis. Research on the key regulatory mechanisms of Tregs and CTLA-4 has great potential to help find therapeutic targets of SAA and to monitor this disease. and Natural Science Foundation of Tianjin City (16JCZDJC35300, 17JCQNJC11500, 18JCYBJC91700, 18ZXDBSY00140).

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.