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Objective:To investigate the therapeutic effect of humanized TRAB domain-containing protein 2A (TRABD2A) monoclonal blocking antibody to HIV-1 reservoir cells, and to explore novel methods for measuring the sizes/capacities of HIV-1 infected reservoirs in HIV-1 infected individuals on receiving combined antiretroviral therapy (cART).Methods:A total number of 51 subjects were collected from the First Affiliated Hospital of China Medical University from May 2021 to December 2021. Among them, there were 2 healthy persons, 41 HIV-1 infected persons receiving cART (cART group) and 8 HIV-1 infected persons not receiving cART (no cART group). Humanized TRABD2A monoclonal antibody was constructed based on the phage display technology, the PBMCs and CD4+T cells separated from the peripheral blood mononuclear cells (PBMCs) and CD4+T cells of HIV-1 infected patients treated with receiving cART, or the HIV-1 infected patients without cART treatment and healthy controls were treated with TRABD2A monoclonal antibodies. The luciferase reporter system, single molecule immune array detection technology and other methods were used to detect the virus content in the supernatant of cell culture. At the same time, flow cytometry and fluorescence real-time quantitative polymerase chain reaction were used to detect the activation of the treated cells and the expression of virus genes. The statistical differences between different treatment the amount of virus release and the level of surface activation markers CD25, CD69, human leukocyte antigen DR (HLA-DR) of different groups in the amount of virus release and the expression of surface activation markers CD25, CD69, HLA-DR were compared.Results:The PBMCs of HIV-1 infected persons receiving cART were tested for HIV-1 production after being treated with humanized TRABD2A monoclonal antibody. The amount of virus released by the untreated group was 0 (0, 440), and the amount of virus released by the use of negative antibody was 0 (0, 390). There was no significant difference between the two ( P>0.05). The amount of virus released by the use of positive antibody was 1 259 (0, 4 269), 3 142 (1 292, 5 060), compared with the amount of virus released by the use of negative antibody, The difference was statistically significant ( P<0.05). The healthy control PBMC was used to conduct multiple dilutions to the infected PBMC. After positive antibody treatment, the amount of virus release decreased in equal proportions [the HIV-1 production corresponding to 5, 25, 125, 625 times of undiluted, diluted PBMC was 4 670 (3 339, 7 697), 1 860 (1 509, 4 615), 1 550 (1 150, 2 680), 602 (255, 1 441), 2 (0, 37), respectively].In addition, there was no significant difference in the resting state of cells treated with TRABD2A antibodies compared with the untreated group (The percentage of CD25 positive cells in the untreated group and positive antibody 1 treated group were 3.89±1.31 and 4.60±1.74, the percentage of CD69 positive cells were 2.50±1.27 and 2.18±0.51, and the percentage of HLA-DR positive cells were 7.66±3.78 and 8.79±3.42, respectively, P>0.05). The viral gag expression levels of untreated and positive antibody 1 were 1 and 0.82±0.55, respectively, with no significant difference. Conclusions:The humanized TRABD2A monoclonal antibody can effectively block the protein activity of TRABD2A, and can significantly promote the release of progeny viruses from viral reservoir in the peripheral blood of HIV-1 infected persons without changing the cell resting state and the whole genome transcription level. The amount of virus released in this way is positively related to the number of reservoir cells.
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Objective@#To explore whether human adipose-derived stem cells (ADSCs) express PD-L1 and Gal-9 and its potential influence factors.@*Methods@#ADSCs isolated from 28 healthy female donors who underwent liposuction of the abdomen or breast tissue were cultured and characterized. The expression of PD-L1 and Gal-9 were detected using flow cytometry. The impact of donor age, body mass index (BMI), donor site and interferon-γ (IFN-γ) on the expression of PD-L1 and Gal-9 was analyzed using multivariate linear regression analysis.@*Results@#The cultured cells fulfilled the criteria for defining MSCs according to the international standards and expressed PD-L1 and Gal-9. Breast-derived ADSCs had higher expression levels of PD-L1 (37.24%±8.20%) and Gal-9 ( 4.41%±2.65%) than those in abdomen-derived ADSCs (28.80%±8.59% and 2.51%±1.39%, respectively) (P=0.018, P=0.039). Multivariate linear regression analysis showed that donor age and site were independent risk factors affecting PD-L1 expression (P=0.009, P=0.006). The expression of PD-L1 decreased by 0.385% for every one year of age increase, and it was 8.58% higher in the breast-derived ADSCs than in the abdomen-derived ADSCs. Donor site was an independent risk factor for Gal-9 expression (P=0.041). Gal-9 positive expression rate in the breast-derived ADSCs was 1.898% higher than that in the abdomen-derived ADSCs. BMI was not a risk factor affecting PD-L1 or Gal-9 expression on ADSCs. The expression of PD-L1 and Gal-9 on ADSCs were significantly upregulated after 48 hours stimulation with 20 ng/ml IFN-γ in vitro.@*Conclusions@#Human ADSCs expressed PD-L1 and Gal-9. Donor age, site and IFN-γ treatment were independent risk factors affecting the expression of PD-L1 and Gal-9 on ADSCs.