| ObjectiveAcquired Immune Deficiency Syndrome (AIDS) is a kind of serious infectious disease caused by the human immunodeficiency virus (HIV). Human immunodeficiency virus mainly infects CD4+ T cells of acquired immune system, resulting in decreased function of immune system, eventually causes death due to a variety of opportunistic infections or tumors. In the course of HIV infection, on one hand, innate immune system and acquired immune system were damaged, and the number and function of cells were changed. On the other hand, cells still play a role of anti-virus, and inhibit viral replication. Natural killer cell (NK) is an important effector cell of the innate immune system, and plays an important role in controlling tumors and microbial infection. Most of the current international reports on NKG2 receptor expression focused on single receptor expression, but the reports on combinating expression of NKG2A and NKG2C were rare. As the study of single receptor expression can reflect the function of NK cells one-sided, we should study the combinating expression of inhibitory receptor and activating receptor to reflect the changes and roles of the different types of NK cells after HIV infection. Moreover, genetic characteristics and HIV strains of Chinese are also different from Europeans and Americans. Therefore, study on combinating expression of NKG2A and NKG2C on NK cell surface is very important. In addition, HIV primary infection individuals are difficult to obtain, and there are no reports on combinating expression of NKG2A and NKG2C. NK cells form a part of the first line of defense mechanism against viral spreading and infection, and protect the body prior to the acquired immune response. Study on NK receptor expression and cell function of primary infection cohort has important scientific significance. In this study, we applicate multi-color flow cytometry to study the expression of activating receptor NKG2C and inhibitory receptor NKG2A on NK cells.Methods1. Study population.22 cases of primary HIV infection (primary HIV infection, PHI) individuals are from the First Affiliated Hospital of China Medical University, Red Ribbon clinic, male, age from 21 to 53 (33±10), inclusion criteria to meet one or more of the following conditions:(1) HIV-antibody becoming positive within 6 months; (2)HIV-antibody negative but HIV-RNA positive; (3)HIV-antibody is weakly positive, and can be repeated within two weeks, and OD values were higher than the previous; (4)having cliniical symptoms of early HIV infection, HIV antigen positive, having positive Western blot (<4 bands).23 cases of HIV-antibody negative normal controls (NC), male, age from 22 to 60 (31±8), meet the following criteria:HIV antibody negative, blood and liver function normal, hepatitis B surface antigen and hepatitis C antibody negative, non-immune system diseases. Fiebig stage is a staging system for early HIV infection. We estimate the infected time of primary HIV infection individuals including to Fiebig staging system.2. Determination of CD4+T cell countsCD4+T cell counts were measured by flow cytometry (FACS Calibur, Becton-Dickinson, USA). A single platform lyses, but no wash, procedure was performed for CD4+T cell counts with Trucount tubes and TriTEST CD4FITC/CD8PE/CD3PerCP (Becton-Dickinson, USA).3. Determination of expression of NK cell receptors by flow cytometerBlood was drawn from each individual by venipuncture and collected into EDTA Vacutainer tubes. The blood samples were stained and immediately analyzed by flow cytometry. Mixtures of five antibodies consisting of CD3-FITC, CD16-Percp-Cy5.5 and CD56-PE-CY7 (Becton-Dickinson, USA), NKG2A-PE and NKG2C-APC (R&D, USA) were used. The frequency of NK cells expressing NK cell receptor was analyzed using FACSDiva software (Becton-Dickinson, USA).4. HIV viral load measurementHIV RNA extracted from plasma samples was amplified by a standardized reverse transcription PCR assay according to the manufacturer's recommendations (COBAS Amplicor, HIV-1 Monitor Test Version 1.5, Roche Diagnostics, USA).5. Statistical analysisBecause several distributions were non-normal, Mann-Whitney U tests were used for comparisons between groups. All analyses were carried out using SPSS 11.5 software and P values< 0.05 were considered significant.Results1. The expression of NKG2A/NKG2C in PHI individualsWe selected PHI individuals who infected HIV less than 6 months. According to infected time, they were divided into two groups respectively. There are 16 patients (infected time less than 3 months) in 3 Months group, and 13 patients (infected time from 3 months to 6 months) in 6 Months group. The percentage of NK cell subsets and receptor expression was compared with HIV-negative normal controls (NC).(1) NK cell subset distributionThe proportion of CD56dimCD16+NK cells in 3 Months and 6 Months group was significantly lower than HIV-negative normal controls (P=0.014, P=0.040). The proportion of CD56-CD16+NK cells in 3 Months group and 6 Months group was significantly higher than HIV-negative normal controls (P=0.001, P=0.001). The proportion of CD56briCD16+/-NK cells in 6 Months group was significantly lower than HIV-negative normal controls (P=0.017).(2) Expression of NKG2A/NKG2C on total NK cellsNo significant changes were found in the percentage of NK cells expressing NKG2A between groups. The percentage of NK cells expressing NKG2C in 3 Months group and 6 Months group was significantly higher than HIV-negative normal controls (P=0.010,P=0.005).The percentage of NK cells expressing NKG2A+NKG2C- in 6 Months group was significantly lower than HIV-negative normal controls (P=0.022). The percentage of NK cells expressing NKG2C+NKG2A- in 3 Months group and in 6 Months group was significantly higher than HIV-negative normal controls (P=0.002, P=0.002). No significant changes were found in the percentage of NK cells expressing NKG2A+NKG2C+ between groups. (3) Expression of NKG2A/NKG2C on CD56dimCD16+NK cellsNo significant changes were found in the percentage of NK cells expressing NKG2A between groups. The percentage of NK cells expressing NKG2C in 3 Months group and in 6 Months group was significantly higher than HIV-negative normal controls (P=0.003, P=0.001).The percentage of NK cells expressing NKG2A+NKG2C- was significantly decreased in 6 Months group compared with HIV-negative normal controls (P=0.023). The percentage of NK cells expressing NKG2C+NKG2A- was significantly increased in 3 Months group and in 6 Months group compared with HIV-negative normal controls(P=0.002, P=0.003). No significant changes were found in the percentage of NK cells expressing NKG2A+NKG2C+ between groups.(4) Expression of NKG2A/NKG2C onCD56-CD16+NK cellsNo significant changes were found in the percentage of NK cells expressing NKG2A between groups. The percentage of NK cells expressing NKG2C was significantly increased in 3 Months group and 6 Months group compared with HIV-negative normal controls (P=0.001, P=0.001).No significant changes were found in the percentage of NK cells expressing NKG2A+NKG2C- between groups. The percentage of NK cells expressing NKG2C+NKG2A- in 3 Months group and 6 Months group was significantly higher than HIV-negative normal controls (P=0.001, P=0.001). The percentage of NK cells expressing NKG2A+NKG2C+ was significantly increased in 6 Months group compared with HIV-negative normal controls (P=0.028).(5) Expression of NKG2A/NKG2C on CD56briCD16+/-NK cellsThe percentage of NK cells expressing NKG2A in 3 Months group and in 6 Months group was significantly lower than HIV-negative normal controls (P=0.043, P=0.043). The percentage of NK cells expressing NKG2C in 3 Months group and in 6 Months group was significantly higher than HIV-negative normal controls (P=0.009, P=0.002).The percentage of NK cells expressing NKG2A+NKG2C- was significantly decreased in 6 Months group compared with HIV-negative normal controls (P=0.009). The percentage of NK cells expressing NKG2C+NKG2A- was significantly increased in 3 Months group and in 6 Months group compared with HIV-negative normal controls (P=0.013, P=0.013). No significant changes were found in the percentage of NK cells expressing NKG2A+NKG2C+ between groups.2. Trends of NKG2A/NKG2C expression on NK cells in Primary HIV infected individuals(1) Trends of single expression of NKG2A/NKG2C on NK cellsThe percentage of all NK cells expressing NKG2A was not significantly changed. The percentage of all NK cells expressing NKG2C was increased with the infected time.(2) Trends of combinating expression of NKG2A and NKG2C on NK cellsThe percentage of all NK cells expressing NKG2A+NKG2C- was slightly reduced. The percentage of all NK cells expressing NKG2C+NKG2A- and NKG2A+NKG2C+ was increased slowly with the infected.3. The expression of NKG2A/NKG2C in PHI individuals who have different viral set pointWe selected PHI individuals who had different viral set point and infected HIV less than 120 days. According to set point, they were divided into two groups respectively. Low set point group has 8 patients (log (v1)<4) and High set point group has 8 patients (log (v1)>4). The percentage of NK cell subsets and receptor expression were compared with each other.(1) Expression of NKG2A/NKG2C on total NK cellsThe percentage of NK cells expressing NKG2A and NKG2A+NKG2C- in Low set point group was significantly lower than High set point group (P=0.021, P=0.036).The percentage of NK cells expressing NKG2C+NKG2A- in Low set point group was significantly higher than High set point group (P=0.021).(2) Expression of NKG2A/NKG2C on CD56dimCD16+NK cellsThe percentage of NK cells expressing NKG2A in Low set point group was significantly lower than High set point group (P=0.027).The percentage of NK cells expressing NKG2C+NKG2A- in Low set point group was significantly higher than High set point group (P=0.021).4. Correlations of Immune and virus indicators in Primary HIV infected individuals We selected PHI individuals who infected HIV less than 120 days, and analysis the correlations of immune and viral indicators.(1) Correlations between the expression of NKG2A/NKG2C and viral load on total NK cellsThe percentage of NK cells expressing NKG2A and NKG2A+NKG2C- positively correlated with viral load (R=0.650, P=0.002; R=0.457, P=0.043).The percentage of NK cells expressing NKG2C+NKG2A-negatively correlated with viral load (R=-0.567, P=0.009).(2) Correlations between the expression of NKG2A/NKG2C and viral load on CD56dimCD16+NK cellsThe percentage of NK cells expressing NKG2A and NKG2A+NKG2C-positively correlated with viral load (R=0.697, P=0.001; R=0.499, P=0.025).The percentage of NK cells expressing NKG2C+NKG2A- negatively correlated with viral load (R=-0.595, P=0.006).(3) Correlations between the expression of NKG2A/NKG2C on NK cell subsetsThe percentage of total NK cells expressing NKG2A negatively correlated with expressing NKG2C (R=-0.676, P=0.000). The percentage of total NK cells expressing NKG2A+NKG2C- negatively correlated with expressing NKG2C+NKG2A-(R=-0.829, P=0.000).The correlation of the expression of NKG2A/NKG2C on CD56dimCD16+NK cells which is the same as on total NK cells.Conclusion1. When separately analyzing the expression of NKG2A on total NK cells, there is no significant changes were found between groups. When simultaneously analyzing the expression of NKG2A and NKG2C, the percentage of NK cells expressing NKG2C+NKG2A- decreases significantly on total NK cells and other NK subset cells. This indicates that simultaneous detection of NKG2A/NKG2C, and analyzing different NK subsets, can truly reflects the changes of NK cells after HIV infection.2. The percentage of NK cells expressing NKG2A+NKG2C- significantly decreases in primary HIV infected individuals, while expressing NKG2C and NKG2C+NKG2A- significantly increases in primary HIV infected individuals. These results indicate that activating signal increases, and the cytotoxicity of NK cell is enhanced to play a role of killing the infected cells. 3. In primary HIV infected individuals, the percentage of NK cells expressing NKG2A and NKG2A+NKG2C- is higher, and the percentage of NK cells expressing NKG2C+NKG2A- is lower in Low set point group. That hints that the decreased NKG2A expression and the increased NKG2C expression enhance the activity of NK cells. Therefore, NK cells can repress the duplication of viral in early stage of infection, and affected the virus set point as well as the progression of disease.4. The expression of inhibitory receptor NKG2A positively correlates with viral load, and negatively correlates with the expression of activatory receptor NKG2C. The expression of NKG2C+NKG2A- negatively correlates with viral load, and negatively correlates with the expression of NKG2A+NKG2C-. The expression of NKG2A and NKG2C on NK cell surface is an indicator of disease progression.
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