| ObjectiveDendritic cells (DCs) are the most potent antigen - presenting cells in human immune system and play an important role in both natural and adaptive immune response. CD123~+ DC and CDllc~+ DC are the two main-subtypes of DCs in peripheral blood. In recent, it was found that DC might have the ability to inhibit HIV replication and to eliminate HIV. on the other hand, DC might promote the transmission of HIV in human body. It is not clear that the role and mechanism of DC in HIV propagation. DC - specific ICAM - 3 - grabbing non-integrin ( DC - SIGN) is a C - type integrin receptor with uniquely expression on DC and the capacity for specific - combination with intercellular adhesion molecule -3 (ICAM -3) on the surface of na ve T cells. The question is that whether or not DC - SIGN play an role in HIV transmission.The studies on the relation of DC and HIV from alien researchers are very few and the conclusions are evident controversory. Foreign researcher mainly focused on the population infected by sexual transmission. The pathway and im-munological features of Chinese subjects infected by HIV are different from those of foreigners. It is not reported that the function of DC in Chinese infected by HIV. This study is aimed at the changes of the number and function of DC in different stages of HIV infection and after HAART therapy as well as the role of DC in HIV transmission to discuss the relation of DC absolute counts to HIV infection and disease progression in Chinese HIV/AIDS patients as well as the function of different mature state of DC and the relation of DC to HIV transmission.Material and Methods1. HIV - infected subjects and blood samplesHIV - seropositive patients ( n = 62) enrolled in this study were not receiving antiretroviral therapy. All patients were from Liaoning, Jilin and Henan province in China. HIV serology was determined by ELISA ( Vironostika, Orga-non Tednika, The Netherlands) and confirmed by Western blot (Genelab Diagnostic, Singapore) in the HIV Confirmation Laboratory at University Hospital. Ethical approval and informed consent were obtained before blood donation. The following conditions, which can nonspecifically affect blood cell counts, were used as exclusion criteria-, previous cytotoxic chemotherapy, splenectomy, hy-persplensim, and blood transfusion within the past 4 weeks. HIV - infected subjects were classified into 3 clinial stages11"12. The first stage was long term non - progressors ( LTNPs ) and included subjects with persistent CD4 T - cell counts more than 414 cell/fxl, no antiretroviral therapy, and no clinical sign of disease for at least 10 years. The second stage was chronical HIV - infected subjects and included patients who had CD4+ T - cell counts less than 414 cell/uj, and no AIDS -defining condition without antiretroviral therapy. The third stage was AIDS and consisted of patients with an AIDS - defining condition according to the World Health Organization classification, including CD4 T — cells less than 200 cells/jxl, present or previous opportunistic infections or HIV - related neoplasms. The median age of the study population was 39 years (range: 30 -59), 37 males and 25 females, and all of the subjects were infected through blood transmission. Blood was collected in EDTA tubes (Becton Dickinson) and processed within 12 hours.2. Healthy controlsPeople from the First Affiliated Hospital Physical Examination Center were used as healthy control. They were randomly enrolled and then matched for age and gender with the subjects'population. Thirty - six seronegative subjects that never exposed to HIV -1 were selected as normal controls. They were 20 males and 16 females with median age of 40 years (25 -61).3. CD4 + T cell absolute count and HIV viral loadCD4+ T cell absolute counts were defined according to 1997 Revised Guidelines for Performing CD4 + T - cell Determinations in Persons Infected with Human Immunodeficiency Virus (HIV).Plasma HIV RNA level was measured by reverse transcriptase - polymerase chain reaction (RT - PCR; Roche Amplicor monitor kit, Roche Diagnostics, USA). Results are expressed as the number of viral RNA copies/ml. The lower detection limit of the assay was 400 copies/ml. When Statistical analysis was performed, values below 400 were assumed to be equivalent to 400 copies/ml.4. Enumeration of CD123 + DCsFor direct labeling, 100 fxl of whole blood were stained with the mixtures of different monoclonal antibodies in TruCount ( Becton Dickinson [BD] ; http:// www. bd. com) tubes containing internal bead standards. After incubation for 15 min at room temperature, samples were fixed with FACS Lyse buffer ( BD). Approximately 200,000 events were acquired within 12 hours of staining and analyzed on a fluorescence - activated cell - sorter scanner ( FACS; FACSCalibur, Becton Dickinson) using Cell Quest software. Fluorescent beads were simultaneously acquired with cellular events. CD123 + DCs were identified by labeling for HLA - DR - PerCP, CD123 - PE and absence of staining with the Linl - FITC - conjugated antibody mixture.5. Flow - cytometryAcquisition threshold was set on FL - 3, excluding most cells negative for HLA - DR - PerCP (Fig. 5B) . For DCs analysis, an Rl region was drawn, including lymphocytes and monocytes but excluding granulocytes and beads (Fig. 5A). DCs were further defined by tight gating on HLA - DR - PerCP - expressing cells in FL -3 (R2 in Fig. 5B) , and finally by limiting to only those cells (R3 in Fig. 5C) that stained negative/dim for a cocktail of FITC - conjugated monocyte/B - cell/T - cell/NK - cell lineage ( Lin) markers ( CD14, CD19/ 20, CD3, CD16/56). CD123+DCs were identified within the HLA -DRV Lin" cells ( compound gated Rl R2 R3) as those cells that stained with CD123 + - PE above that of isotype control (Fig. 5D).Absolute counts of CD 123 + DCs were calculated by determining the ratio ofbeads to the cell population of interest and then multiplying this ratio with the number of beads placed in the tube17. The following formula was used. Dr ( 11 / n _ CD123 * DCs cells Internal standard beads acquiredacquired 1006. TCIDjo assay of virus strains; 2 Primary M - tropic and 2 Primary T -tropic - HIV strains were isolated by our laboratory . Abstracted PBMC from healthy donors and stimulated with PHA for 3 days. Suspended by RPMI - 1640 with 100U IL -2 and moderated concentration to 4 x lOVml before cultured in 31X. Selected 12 wells in 96 wells plates, added 180ul RPMI - 1640 and PBMC 50jul1 in each wells. And added 200|uJ virus , serial dilution by 10 - fold. Changed half supernatant and detected p24 after 7 days. Calculated TCID50.7. The isolation of CD4+T cells and CD14+ monocytes: PBMC was isolated from 5 healthy donors by density gradient centrifugation . Isolated cells using MACS immunomagnetic beads ( Miltenyi , Germany). Added 20ul anti - CD4 and anti - CD14 immunomagnetic beads to perl x 10 cells. Centrifuged lOmin at 300g after Cultured 15min at 10^1. Suspended cells with 500ul buffer solutions and positive isolation by LS column . Added 20ui CD4 - FITCN20fxLCD8- Pe and 20|jd CD3 - PerCP in few cells and assayed cell purity by FCM with CELLQUEST software.8. Cell surface staining; Examined 100,000 cells by FCM, and analyzed surface marker expressed on DCs.9. DC culture in vitro; CD14+ monocytes were cultured by RPMI - 1640 with 10% FCS, GM -CSF(800U/ml)and IL -4(800U/ml). Immature dendritic cells (iDC) were cells cultured after 7 days, then matured after 3 - day stimulation with TNF - a (10|xg/ml). Examined CD4, CCR5, CXCR4, CD14, HLA -DR^CD80 and CD86 expressions on CD14+ monocytes by FCM.10. Analyses of HIV - 1 transmissibility: M - tropic and T - tropic HIV strains were isolated from HIV/AIDS patients. 5 x 104 imDCs and mDCs were cocultured with 100 -^OOOTCID^M - tropic and T - tropic HIV strains respectively for 2 hours. Wash twice. Added GM - CSF(800U/ml)and IL -4(800U/ ml)and cultured 10 - 12 days. Changed half supernatants per 3 days. Collectedsupematants and detected p24 antigen to analyze HIV -1 replication.2. 5 x 104imDCs and CD4 + T cells cocultured 20 minutes with 20jxg/ml anti- DC - SIGN respectively. Stimulated with 400TCID.5O M - tropic . strains 2 hours, and cocultured with active 5 x 105CD4 +T cells for 10 days. Collected supematants per 3 days and detected p24 antigen on the l8t, 4th, 7th and 10th day. Compared with negative controls to investigate effects of anti - DC - SIGN to anti-HIV.11. p24 detection; p24 antigen was detected by Diomerieux kit according to instruction.12. Statistical analysisSPSS 11.0 software package was used for the statistical analysis. Geometric means were determined for log - distributed variables. The significance o￡ the differences in CD123 +DCs true count in patients and controls was evaluated with one - way ANOVA. A P value of 0. 05 or less was considered to represent significance. Correlations between CD123+DCs and CD4 counts and percentage, viral load were performed by the Spearman's rank test. The group variable was HIV clinical stage. The P values are based on comparisons of group mean throughout the study.Results1. Blood CD123 + DCs severely reduced in Chinese HIV/AIDS patients The CD 123 + DCs true counts in chronical HIV - infected individuals ( median: 5. 36 cells/jjlI; range; 2. 26 ~9. 80 ) were significantly lower than that of healthy controls(median :7.92 cells/ui; range: 3. 30 ~ 14. 25 ; P <0.05). The CD4+ - cell count in chronical HIV - infected individuals (median; 326 cells/ jxl; range; 233 -400) were significantly lower than that of healthy controls(PIn AIDS patients, the CD123 + DCs count was found markedly decreased, with a median of 2. 43 cells/jxl (range; 0.06 ~5. 58cells/jxl). It was significantly lower than in healthy controls, chronical HIV - infected individuals, and LTNPs (P<0. 01 for all pairwise comparions) . As expected, the AIDS groupalso had the lowest CD4+ T -cell count (median; 145 cells/jxl; range; 17 ~ 286)2. The number of CD 123 + DCs in Chinese LTNPs was significantly higher than that of normal controls.Blood CD123 + DCs in asymptomatic LTNPs group were significantly higher (median; 12.74 cells/jjlI; range; 4.95-22.43) as compared to healthy controls (median:7. 92 cells/jxl; range; 3. 30 ~ 14. 25) chronical HIV - infected individuals and AIDS patients. In contrast to the CD123 + DCs number, similar CD4+ T lymphocytes counts were observed in LTNPs (median; 736cells/ui; range; 414 -1945 cells/jxl) and healthy controls(median: 823cells/|xl; range; 429~1632ceUs/fJLl). .Blood CD123 + DCs true count of Chinese HIV/AIDS patients correlated significantly to disease progression. Blood CD123 + DCs true count correlated positively with CD4+ T count and percentage. Within the total 62 HIV - infected subjects, a strong positive correlation was found between the CD123 +DCs number and the CD4 + T - cell true count and percentage( r = 0. 660, P < 0. 01;r = 0. 445,P<0.01).3. Blood CD 123 + DCs true count correlated negatively with the HIV viral loadAs shown by the quantification of CD123 + DCs in different groups of HIV -infected subjects, CD123 + DCs were increased in the LTNPs group, which has the lower HIV viral load levels (median; 3230 copies/ml; range; < 400 ~ 9840 copies/ml) , and are decreased in AIDS, where the higher viral loads were observed (median; 24886 copies/ml; range; <400 -760000 copies/ml; P <0. 01). We therefore determined the relationship between CD 123 + DCs counts and HIV viral load in the 56 HIV - infected subjects ( Figure 4). A statistically significant negative correlation was found between those 2 paramerers ( r = - 0. 484,P<0.05).4. The relation of maturation of DC to different trophism of HIV: After T -trophic HIV - pulsed, the level of p24 antigen in culture supernatant of immature DC and mature DC didn' t increase with the duration of culture. After M -trophic HIV - pulsed, the level of p24 antigen in culture supernatant of imma-ture DC became increased with the duration of culture but the level of p24 antigen in culture supernatant of mature DC didnt increase with the duration of culture.5. The transmission of M - tropic HIV by immature and mature DC After coculture of M - tropic HIV - pulsed immature DC with CD4+ T cells,the p24 level in supernatant increased with the duration of time and significantly higher than that of control in the absence of CD4 + T cells. After coculture of M- tropic HIV - pulsed mature DC with CD4+ T cells, the p24 level in supernatant increased with the duration of time and significantly higher than that of control in the absence of CD4 + T cells.6. The transmission of T - tropic HIV by immature and mature DCAfter coculture of T - tropic HIV - pulsed immature DC with CD4 + T cells, the p24 level in supernatant increased with the duration of time and significantly higher than that of control in the absence of CD4 +T cells. After coculture of T -tropic HIV - pulsed mature DC with CD4+ T cells, the p24 level in supernatant increased with the duration of time and significantly higher than that of control in the absence of CD4 + T cells.7. The inhibition of HIV - 1 infection by anti - DC - SIGN antibody After coculture of immature or mature DC pulsed by low concentration of M- tropic HIV with CD4+ T cells, the high levels of p24 in culture supernatant were detected. The levels of p24 in culture supernatant of the control group didn t change.The levels of p24 in culture supernatant of immature and mature DC significantly deceased after addition of 20|xg/ml of DC -SIGN antibody, approaching to the level of control group.Conclusion1. The counts of CD123 + DC in blood of Chinese HIV/AIDS patients was depleted and decreased continuously with disease progression. CD123 + DCs in peripheral blood played an important role in control HIV infection and correlated with HIV disease progression.2. The numbers of CD 123 + DCs in blood of Chinese LTNP were significantly higher than that of chomical HIV - infected individual and AIDS patients, even higher than the normal control. It indicated that CD 123 + DC possessed the protective function in the progression of LTNP.3. Chinese T - tropic HIV didnt replicate in the Chinese immature and mature DC. M - tropic HIV replicated in immature DC but not in mature DC, indicating that the replication of HIV in DC was dependent on the HIV trophism and the maturation of DC.4. The infection of activated CD4+ T cells by HIV was inhibited by the antibody of DC - SIGN. It indicated that DC - SIGN might play an important role in the delivery of HIV from DCs to T cells.
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