Relationship of Frequency of CD4+CD25+Foxp3+ Regulatory T Cells with. Disease Progression in Antiretroviral-Naïve HIV-1 Infected Chinese. Zining Zhang ...
Jpn. J. Infect. Dis., 61, 391-392, 2008
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Relationship of Frequency of CD4+CD25+Foxp3+ Regulatory T Cells with Disease Progression in Antiretroviral-Naïve HIV-1 Infected Chinese Zining Zhang, Yongjun Jiang, Min Zhang, Wanying Shi, Jing Liu, Xiaoxu Han, Yanan Wang, Xin Jin and Hong Shang* Key Laboratory of Immunology of AIDS, Ministry of Health, the First Affiliated Hospital, China Medical University, Shenyang, P. R. China (Received January 9, 2008. Accepted June 16, 2008) SUMMARY: Forty-five antiretroviral-naïve HIV-1 infected patients and 14 healthy controls in North China were enrolled in this study. The frequency of CD4+CD25+Foxp3+ regulatory T cells (Tregs) and levels of expression of CD95, HLA-DR and CD38 in T cells were detected by flow cytometry. We found that the frequency of Tregs was higher in AIDS patients than in asymptomatic HIV-1 infected patients (P = 0.004). The frequency of Tregs was significantly correlated with absolute CD4 count, viral load, CD4+CD95+ T cells and CD8+CD95+ T cells (P < 0.05). The relationship between the frequency of Tregs and immune activation was not found in HIVinfected patients. We concluded that the frequency of Tregs in HIV-infected Chinese patients was significantly correlated with disease progression. manufacturer’s protocol (eBioscience, San Diego, Calif., USA). Cells were analyzed on a FACSAria (BD Biosciences) with FACSDiva software. Tregs were identified as CD25positive and Foxp3-positive cells among CD3+CD4+ T lymphocytes, and absolute Treg numbers were determined by multiplying the proportion of CD4+CD25+Foxp3+ by the total CD4 cell count. For the detection of activation and CD95 expression of T cells, fresh whole blood samples were incubated with the following monoclonal antibody combinations and appropriate isotypes: anti-CD8-FITC/anti-CD95-PE/antiCD3-PerCP/anti-CD4-APC-Cy7, anti-HLA-FITC/anti-CD38PE/anti-CD8-APC/anti-CD4-APC-Cy7. After the cells were lysed and washed, the proportions of CD4+ and CD8+ T cells expressing HLA-DR, CD38, and CD95 were analyzed with FACSDiva software. We found the frequency of CD4+CD25+Foxp3+ T cells in AIDS patients was significantly higher than that of asymptomatic HIV-infected patients (P = 0.004) (Table 1). In contrast, no significant differences in the frequency of CD4+CD25+Foxp3+ Tregs were observed between healthy controls and HIV/AIDS patients (Table 1). The absolute numbers of Tregs in AIDS patients and asymptomatic HIVinfected patients were lower than in healthy controls (P < 0.05), which shared the same tendency of the variety of CD4+ T cell counts as in these groups (P < 0.05) (Table 1). The frequency of CD4+CD25+Foxp3+ Tregs was significantly related to viral load (r = 0.520, P < 0.001) and inversely related to CD4 count (r = –0.406, P = 0.001) in HIV-infected patients. As shown in Table 1, a significant increase in the level of activation of T cells was observed in AIDS patients and asymptomatic HIV-infected patients when compared with healthy controls. The expression of CD95 in both CD4+ and CD8+ T cells in AIDS patients was significantly increased in comparison with asymptomatic HIV-infected patients and normal controls (Table 1). The frequency of CD4+CD25+Foxp3+ Tregs in HIV-infected patients was significantly correlated with the level of CD95 expression in T cells (CD95/CD4: r = 0.3, P = 0.024; CD95/CD8: r = 0.284, P = 0.032). No relationship between the frequency of Tregs and immune activation was seen in HIV-infected patients (P
Studies on the role of CD4+CD25+ regulatory T cells (Tregs) in HIV-induced immune dysfunction have shown that Treg-mediated immunosuppression may play a role in the degradation of HIV-specific CD4+ and CD8+ T cell responses (1). Human Treg activity is primarily confined to CD4+CD25bright cells (1). The medical research community has greeted with excitement the identification of phenotypic markers that purportedly allow a better discrimination of Tregs, such as CTLA-4, GITR, CD62L, CD103 and LAG-3 (1), and transcription factor Foxp3 is generally accepted as the best available marker for CD4+CD25+ Tregs (1). Several reports have investigated the expression of Foxp3+ Tregs (2,3), but the relationship between the frequency of CD4+CD25+Foxp3+ Tregs and disease progression has not been described fully, especially among HIV-infected Chinese. In the present paper, we studied the correlation of the alternation of CD4+CD25+Foxp3+ Tregs and disease progression in HIV-infected Chinese. A total of 45 treatment-naïve HIV-infected patients from Liaoning, Jilin and Henan provinces in North China were enrolled (24 males, 21 females; median age 40 years, range 30 - 68 years). Phylogenetic analysis of the PR-RT sequences revealed that 39 (86.7%) of the sequences were subtype B’, the main subtype found in former blood donors in China. The patients were clinically classified according to the 1993 revised criteria from the Centers for Disease Control and Prevention and comprised 25 asymptomatic HIV-1 infected patients (CD4 ≥200/μL, A1, A2) and 20 AIDS patients (CD4 0.05) (data not shown). Methodological discrepancies make it difficult to conclude with absolute certainty as to the variety of the frequency of Tregs in HIV infection. In the present study, we characterized the Tregs of HIV-infected Chinese as the percentage of CD3+CD4+CD25+Foxp3+ T cells, and found that the frequency of Tregs in AIDS patients was significantly higher than that of asymptomatic HIV-infected patients. This result is consistent with findings of several published studies, which showed a marked increase of the level of Tregs in individuals with very low CD4 cell counts (3,4), or reported that HIV can selectively promote Treg survival (6). We also observed that the up-regulation of the frequency of Tregs was significantly correlated with an increased plasma viral load, suggesting that the increase of Tregs may be due to the continuous replication of HIV over the course of disease progression. We also quantified the count of Tregs and found a decreased level of Treg numbers in HIV/AIDS patients when compared with that of healthy controls. These results are consistent with the findings of Montes et al. (3) and Kinter et al. (5), but quite different from those of Epple et al. (2), which did not find any differences in absolute numbers of Foxp3+ Tregs between untreated HIV-infected patients and healthy controls. The reason for the different results between our study and that of Epple et al. (2) may be related to methodological differences and sample size. We used CD25+ while Epple et al. used CD25high as one of the markers of Tregs: this different identification may have affected the final results. The total sample size enrolled in our study (45 HIV-infected patients) was larger than that in Epple’s (13 treatment-naïve patients), which may improve the statistical significance of our findings. We found that the decreased absolute count of Tregs was consistent with the alternation of CD4+ T cells as the disease progresses, indicating that the greater dynamic range of CD4 cell numbers may overshadow the increases in the proportion of cells positive for Foxp3 in the advanced disease (3). Recent studies have found that the control of inflammatory or autoimmune T cell responses by Tregs can be accomplished via induction of apoptosis (7). Although the expression of CD95 on the cell surface of lymphocytes does not always indicate that the cells are dying, high levels of CD95 expression on T cells may reflect the potential for apoptosis. Our
study showed that the frequency of Tregs was significantly correlated to the level of CD95 expression of CD4+ and CD8+ T cells, indicating the possibility that Tregs may also exert its function via induction of effector cell apoptosis in HIV infection. A strong association between immune activation and Treg number has been reported (1), but we did not observe a correlation between the frequency of Tregs and activation. One possible explanation for this may be that we measured the activation markers on T cells instead of on memory cells, which may correlate better with disease progression. Because Tregs play an important role in the interplay of the human immune system and HIV, further study may provide new targets for therapy or the design of a vaccine for HIV prevention. ACKNOWLEDGMENTS This study was supported by the fund of National Natural Science of P. R. China (30600532), Japanese Foundation for AIDS Prevention and 973 programme about the development of national significant elementary research (2006CB504206).
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