| AIDS, the abbreviation for acquired immunodeficiency syndrome is caused by human immunodeficiency virus (HIV). It is a chronic infectious disease threatening the health of all mankind, with main features of progressively damaging immune function and ultimately leading to opportunistic infections and malignant tumors. HIV mainly infects and attacks immune cells, especially the CD4~+T lymphocytes. The functional T lymphocytes (CD4~+CD28~+, CD8~+CD28~+), na?ve subsets (CD4~+CD45RA+CD62L+) and memory subsets (CD4~+CD45RO+) can be seen significantly decreased in AIDS patients. While CD38 and HLA-DR, the markers for immune activation, will be sharply increased. HIV infection itself and the aberrant immune status can ruin the function of T lymphocytes and prevent the elimination of HIV.HIV-1 specific CTL plays a key role in controlling HIV-1 replication and slowing disease progression. Previous results showed that there existed a strong and extensive HIV-1 specific CTL response and low levels of virus replication in the long term nonprogressors, LNTP). But it decreased markedly in the advanced patients and failed to eliminate HIV. The mechanism of dysfunction of specific CTL has not been clear so far, the probable mechanism might be as follows: the entire or partial lose of the function of T helper cells, reduction of specific antigens and immunosuppressive and regulatory activity of CD4~+CD25+Tregs (Regulatory T cells, Treg cells). CD4~+CD25+Tregs can suppress immune activation to avoid excessive immune damage. Additionally, they can suppress cellular immune response by suppressing proliferation and function of CD4~+T cells and CD8~+T cells, which is not good for the elimination of HIV. Fluorescence intensity of CD25 levels on human CD4~+T cells is divided into three grades: low, median and high. Only the CD4~+CD25hi population could represent characteristics of CD4~+CD25+Treg cells and exhibit strong and specific regulatory function, but it should not be considered as the specific marker for its high expression on activated or memory CD4~+T cells. It has been demonstrated that IL-7 receptor (CD127) is down-regulated on CD4~+T cells, and it is negatively correlated with FoxP3 expression. CD4~+CD25+CD127loT lymphocytes possess approximately same immune-regulatory and immunosuppressive activity as CD4~+CD25+FoxP3+T lymphocytes. So CD127 and CD25 can be used to quantitate Treg subsets.Highly active antiretroviral therapy (HAART) is the most effective way to control and treat AIDS. It can suppress HIV-1 replication, restore CD4~+T lymphocytes, enhance CD28 expression and increase na?ve and memory CD4~+T cells,and correct abnormal immune activation. In most cases, HAART allows the inadequacy quantitative and functional immune defects to be reversed, but it could not retune the damaged immunity to the normal levels. There are three different views about the influence of HAART therapy on HIV-1 specific CTL response: 1. HIV-1 specific CTL response may reduce rapidly and soon disappear as viral load reaches undetectable. 2. HIV-1 specific CTL response could still be detected even though sustained undetectable plasma viremia has been achieved. 3. Low plasma viremia might be helpful and required to stimulate HIV-1 specific CTL response.The reports about the effect of HAART on the frequency and function of CD4~+CD25+Tregs are inconsistent. The reasons for that might be that different methods to identify Tregs and different inclusion criteria for recruiting patients are used.Benefited from the policy of"Four Frees and One Care"since 2004, more and more patients can receive HAART regimes based mainly on free domestic drugs. There are few prospective studies on virological and immunological response in AIDS patients treated with domestic-drug–based HAART, especially very few data about specific CTL response and the change of CD4~+CD25hiCD127lo/-Treg in these patients. Study purposeTo explore virological and immunological response in AIDS patients treated with HAART regimes based mainly on free domestic drugs. Study subjects and methods1. Study subjects(1)A total of 40 AIDS patients were recruited from AIDS department of Guangzhou No.8 People's Hospital with regular follow-up. Of the them 24 were males and 16 were females,with the average age being 37 years, ranging from 20 to 63 years. 22 volunteers severed ashealthy controls including 8 males, 14 females, with a average age of 26 years, ranging from 18 to 37 years.(2)The diagnoses of HIV infection for all cases were confirmed by Western Blot by Guangdong provincial CDC or Guangzhou municipal CDC.(3)The average of absolute CD4~+T cell count is 201cells/uL (range 10-340cells/uL) before HAART.(4)HAART regimes: D4T+3TC+NVP (n=21), AZT+3TC+NVP (n=19). D4T, NVP and AZT were bought from Disainuo Biological Pharmaceutical Company(Shanghai), Mike Pharmaceutical Company(Xiamen) and Northeast Pharmaceutical Factory(Shenyang) respectively; 3TC were bought from Glaxosmithkline Company Limited.(5)All cases received standard HAART and were followed-up regularly during treatment. 2. Study methods(1) Sample collection Patients'blood samples were collected before therapy and at every visit during treatment on schedule. Plasma and peripheral blood mononuclear cells (PBMC) were isolated and reasonably preserved.(2)Virological evaluation The quantification of HIV-1 RNA in the plasma was performed by using COBAS TaqMan 48 with a lower limit of detection of 40copies/mL.(3)Detection of absolute CD4~+T cell count Absolute CD4~+T cell count was determined in EDTA-treated whole blood by FACSCalibur flow cytometry Multiset software.(4)Detection of CD4~+CD28~+ and CD8~+CD28~+ Percentage of CD4~+CD28~+ and CD8~+CD28~+ in PBMC was detected by Three-color flow cytometry by using monoclonal antibodies.(5)Detection of absolute CD4~+CD45RA+ and CD4~+CD45RO+ numbers First we detected absolute numbers of leukocytes in peripheral blood and lymphocytes percentage, and then calculated the absolute numbers of lymphocytes. Double positive CD4 and CD45RA or CD45RO in PBMC was detected by Three-color flow cytometry. Finally we obtained absolute numbers of na?ve and memory cells by multiplying absolute numbers of lymphocytes and double positive cells in the percentage of lymphocytes.(6)Detection of percentage of CD8~+CD38~+and CD8~+HLA-DR+ Percentage of CD8~+CD38~+ and CD8~+HLA-DR+ in PBMC was measured by Three-color flow cytometry.(7)Detection of frequency and magnitude of HIV-1 specific CTL response Frequency of Interferon-γ(IFN-γ) secreting cells in PBMC in 40 patients pre-HAART and post-HAART were assessed by stimulation with a peptide pool containing 12 overlapping peptides in HIV-1 P24, by using assay of enzymed-linked immunospot (ELISPOT)( IFN-γkit BD America).( 8 ) Detection of frequency and absolute CD4~+CD25hiCD127lo/-Treg numbersFirst we detected absolute numbers of leukocytes in peripheral blood and percentage of lymphocytes, then and then calculated the absolute numbers of lymphocytes. Frequency of CD4~+CD25hiCD127lo/-T lymphocytes was determined by Three-color flow cytometry using monoclonal antibodies. We gained absolute numbers of CD4~+CD25hiCD127lo/-T lymphocytes by multiplying absolute numbers of lymphocytes and frequency of CD4~+CD25hiCD127lo/-T lymphocytes.Results1. Virological responseThe average HIV viral load (HIVRNA) level in 40 patients before treatment was 4.54 log10copies/mL (range 2.36-5.98 log10 copies/mL). It dramatically dropped to 2.28 log10 copies/mL (range 1.70-3.07 log10 copies/mL), 1.87 log10 copies/mL (range 0.00-2.61 log10 copies/mL), 1.60 log10 copies/mL (range 0.00-3.56 log10 copies/mL), 0.00 log10 copies/mL (range 0.00-3.10 log10 copies/mL), and 0.00 log10 copies/mL (range 0.00-4.80 log10 copies/mL) respectively 4 ,12, 24 ,36 and 48 weeks after treatment.The percentage of patients with HIVRNA below 40 copies/mL 4, 12, 24, 36 and 48 weeks'after therapy was 0%, 30%, 70%, 80% and 92.5% respectively. HIV RNA level at each following-up time point was significantly lower than that of pre-HAART (all P<0.05).2. Immunological response2.1 Absolute CD4~+T cell countThe average absolute CD4~+T cell count before treatment in 40 patients was 201.80±91.84 cells/uL. It ascended to 306.75±146.4, 356.23±149.09, 379.35±169.20, 401.30±190.11 and 348.23±149.75 cells/uL at the end of week 4, 12, 24, 36 and 48 of treatment, respectively. Following an initial rapidest augmentation of CD4~+T cell count in the first 4 weeks of treatment, CD4~+T-cell count at each following-up time point was significantly higher than that of pre- treatment (all P<0.05).2.2 Percentage of CD4~+CD28~+ and CD8~+CD28~+ The percentage of of CD4~+CD28~+ before treatment (n=22) was 78.94±13.80%. It was significantly lower than that in the healthy controls 98.36±1.62% (P=0.000). It increased to 88.78±7.25%, 91.35±6.08% and 92.90±3.39% at the end of week 24, 36 qnd 48 weeks of treatment respectively. The percentage of CD4~+CD28~+ at these following-up time points was significantly higher than that of pretreatment (all P<0.05).The percentage of CD8~+CD28~+ before treatment was 16.19±7.01%. It was significantly lower than that in the healthy controls 50.17±18.31% (P=0.000). It increased to 20.63±8.55%, 20.28±9.13% and 18.42±11.98% at the end of week 24, 36 and 48 of treatment respectively. The percentage of CD8~+CD28~+ at these following-up time points was higher than that of pretreatment, but the differences did not reach statistical significance (all P>0.05).2.3 Absolute CD4~+CD45RA+ and CD4~+CD45RO+ numbersThe absolute CD4~+CD45RA+ number before treatment (n=25) was 42±29 cells/uL. It was 37±24, 59±32, 64±40, 72±47 and 74±36 cells/uL respectively at the end of week 4, 12, 24, 36 and 48 of treatment. There was a significant increase in CD4~+CD45RA+ from week 12 compared with that of pre-treatment (all P<0.05).The absolute CD4~+CD45RO+ number before treatment was 55±24 cells/uL. It was 69±30, 92±33, 107±43, 129±42 and 143±48 cells/uL respectively at the end of week 4, 12, 24, 36 and 48 of treatment. It was significantly higher at each follow-up time point than that of pretreatment (all P<0.05).2.4 Percentage of CD8~+CD38~+ and CD8~+HLA-DR+The percentage of CD8~+CD38~+ (n=14) before treatment was 42.70±17.10%. It was significantly higher than that in the healthy controls 22.21±6.80%(P=0.001). After therapy, there was a continuous decrease in the percentage of CD8~+CD38~+. It reached 38.2±22.78%, 34.78±17.04%, 28.02±11.72%, 18.29±7.72% and 21.59±11.77% at the end of week 4, 12, 24, 36 and 48 of treatment respectively. Except for week 4, the percentage of CD8~+CD38~+ at other time points were all significantly lower than that of pretreatment (all P<0.05). The percentage of CD8+CD38+ at week 36, 48 was close to that in healthy controls (P=0.25).The percentage of CD8+HLA-DR+ before treatment was 40.49±13.15% .It was significantly higher than that in healthy controls 22.21±6.80% (P=0.001). After 4-weeks of therapy, the percentage of CD8+HLA-DR+ slightly increased to 47.33±31.06% with no statistical difference compared with that of pre-HAART (P>0.05). Then it decreased to 27.70±19.76%, 24.45±12.46%, 17.81±7.07% and 14.30±10.27% at the end of week 12, 24, 36 and 48 of treatment respectively. The percentage of CD8+HLA-DR+ at these time points was significantly lower than that of pretreatment (all P<0.05).2.5 The frequency and magnitude of HIV-1 specific CTL2.5.1 The relationship between HIV-1 specific CTL response and HAARTThe positive frequency of HIV-1 specific CTL response before teatment in 40 patients was 15%. It increased to 17.5%, 35%, 50%, 50% and 40% at the end of week 4, 12, 24, 36 and 48 of treatment respectively. There was a fast increase in the frequency from week 12 and then keep at a relative high level in the remaining course of HAART. The positive frequency at the end of week 12, 24, 38 and 48 was significantly higher than that of pretreatment (all P<0.05).The magnitude of HIV-1 specific CTL response before treatment was 0 SFC/106PBMC (range0-987 SFC/106PBMC). It was 0 SFC/106PBMC (range0-2796 SFC/106PBMC), 26 SFC/106PBMC (range 0-1780 SFC/106PBMC), 53.5 SFC/106PBMC (range0-2110 SFC/106PBMC), 46.5 SFC/106PBMC (range0-1830 SFC/106PBMC) and 11.5 SFC/106PBMC (range 0-1295 SFC/106PBMC) at the end of week 4, 12, 24, 36 and 48 of treatment respectively. We observed an obvious increase in specific CTL response magnitude from week 12 and it remained at a relative high level. Specific CTL response magnitude in week12, 24, 36, 48 was much higher than that of pretreatment (all P<0.05).2.5.2 The relationship between HIV-1 specific CTL response and HIVRNAAccording to the level of HIVRNA at different time points, plasma viremia was divided into two groups: HIVRNA≤40copies/mL group (n=109) and HIVRNA >40 copies/mL group (n=91). The positive response rate of HIV-1 specific CTL in the group with HIV RNA≤40 copies/mL (45.87%, n=109) was significantly higher than that in the group with HIVRNA >40 copies/mL (29.67%, n=96) (χ2=5.498, P=0.019).HIV-1 specific CTL response magnitude in the group with HIVRNA≤40 copies/mL [40 SFC/106PBMC (range0-1830 SFC/106PBMC)] was significantly higher than that in the group with HIVRNA >40 copies/mL [20 SFC/106PBMC (range 0-2976 SFC/106PBMC)] (Z= -2.500, P=0.012).2.5.3The relationship between HIV-1 specific CTL response and absolute CD4~+T cell countAccording to CD4~+T cell count at different time points, it was divided into two groups: CD4~+≤350 cells/uL group (n=96) and CD4~+>350cells/uL (n=104) group. The frequency of HIV-1 specific CTL response in the group with CD4~+>350 cells/uL (42.31%, n=104) was higher than that in the group with CD4~+≤350cells/uL (34.38%, n=96), but the difference did not reach statistical significance (χ2=1.327, P=0.249). HIV-1 specific CTL response magnitude in the group with CD4~+≤350 cells/uL and group with CD4~+>350cells/uL were [25 SFC/106PBMC (range0-1295 SFC/106PBMC)] and [29 SFC/106PBMC (0-2976 SFC/106PBMC)] respectively. No statistical significance was find between these two groups (Z= -0.786, P=0.432).2.6 The frequency of CD4~+CD25hiCD127lo/-Treg cells2.6.1 The frequency of CD4~+CD25hiCD127lo/-Treg cells in AIDS patients and its relationship with absolute CD4~+T cell countThe frequency of CD4~+CD25hiCD127lo/-T reg cells before treatment was 4.55±2.83%. It was significantly higher than that in healthy controls 3.29±1.47%(P<0.05).It was significantly higher in patients with CD4~+T cell count≤200cells/uL (6.02±3.65%, n=15) than that in patients with CD4~+T cell>200cells/uL (3.71±1.85%, n=25) (P=0.008).Taken all CD4~+T cell count at all time points together, it was divided into two groups: CD4~+T cell≤200cells/uL group (n=54) and CD4~+T cell >200cells/uL group (n=186). The frequency of CD4~+CD25hiCD127lo/-Treg cells in the group with CD4~+T cell count≤200cells/uL (5.04±2.61%, n=54) was significantly higher that in the group with CD4~+T cell>200cells/uL (3.86±2.07%, n=186) (P=0.007). 2.6.2 The relationship of the frequency and absolute count of CD4~+CD25hiCD127lo/-Treg cells with HAARTThe frequency of CD4~+CD25hiCD127lo/-T reg cells before treatment was 4.55±2.83%. It was 3.65±1.65%, 4.51±2.10%, 4.31±2.48% and 3.78±1.96% at the end of week 12, 24, 36 and 48 of treatment respectively. The frequency did not change significantly in the course of HAART (P=0.244).The absolute CD4~+CD25hiCD127lo/-Treg cells count before treatment was 6.68±4.48 cells/uL. It was 6.51±3.49, 7.32±3.31, 8.41±3.62, 6.95±4.22 cells/uL at the end of week 12, 24, 36 and 48 of treatment respectively. There was a slight increase from week 24 but the difference did not reach statistical significance (all P >0.05). 2.6.3 The correlation of frequency of CD4~+CD25hiCD127lo/-Treg cells with magnitude of HIV-1 specific CTL response, level of CD8~+CD38~+ and CD8~+HLA-DR+According to CTL frequency detection at different time points, it was divided into two groups: the CTL response group (n=54) and non CTL response group (n=46).The frequency of CD4~+CD25hiCD127lo/-Treg cells was lower in the group with specific CTL response (3.98±1.72%, n=54) than that in the group without CTL response (4.28±2.12%, n=46), but the difference was not statistically significant (F=0.606, P=0.438).There was a negative correlation between the frequency of CD4~+CD25hiCD127lo/-Treg cells and magnitude of HIV-1 specific CTL response (n=100 r=-0.267, P=0.05).There was no correlation between the frequency of CD4~+CD25hiCD127lo/-Treg cells and the level of CD8~+CD38~+ and CD8~+HLA-DR+.Conclusions1. HAART regimes based mainly on free domestic drugs can effectively inhibit HIV replication, restore CD4~+, na?ve and memory CD4~+ T lymphocytes, decrease immune activation and partiallly reconstitute the immunity. 2. HAART regimes based mainly on free domestic drugs can enhance HIV-1 specific CTL response.3. The frequency of CD4~+CD25hiCD127lo/-Treg cells is associated with disease progression. HAART has no impact on the frequency and absolute count of CD4~+CD25hiCD127lo/-Treg cells.4. The frequency of CD4~+CD25hiCD127lo/-Treg cells was negatively correlated with the magnitude of HIV-1 specific CTL response, but did not correlate with degree of immune activation. |
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