Title: Absolute CD4 CD25 CD127low/- T Cells (Treg) in Peoples with HIV/AIDS
1Absolute CD4CD25CD127low/- T Cells (Treg)
in Peoples with HIV/AIDS
- Sakchai Dettrairat
- HIV Laboratory
- Division of Clinical Immunology
- Department of Medical Technology
- Faculty of Associated Medical Sciences
- Chiang Mai University
2HIV Infection/AIDS
http//emedicine.medscape.com/article/211316-media
3Treg Activity in HIV infection
http//journals.lww.com/aidsonline/Fulltext/2008/0
3300
4Treg Activity in HIV infection
- Treg ?????????
- Treg ???????????? ?????????????????????? HIV ??
Treg ???? - gp120 ?????????????????? Foxp3 ?????????????????
apoptosis ??? Treg - HIV Tat ?????????????????? CTLA-4 ??? suppressive
activity - Immune activation imflammation ????????????
Treg ??????? - Treg circulation between HIV-infected site and
lymphiod tissue - Treg ????
- ART ???????????????? ????? effect ??? gp120 ???
HIV Tat ????
5Regulatory T cells
- Suppressor T cells or Treg cells
- Molecules that used to charaterize Treg cells
- CD4
- CD25 (IL-2 receptor)
- Foxp3
- CD127 (IL-7 receptor) negative/low expression
- etc.
6Objective
- Enumeration of absolute CD4CD25CD127low/- T
Cells (Treg) numbers in people with HIV/AIDS
receiving ART by using pre-enrichment technique
and flow cytometry.
7Samples Materials
- Blood samples
- 10 EDTA blood samples of ARV-treated PHAs
- 10 EDTA blood samples of normal subjects
- Complete kit for CD4CD127lowCD49d-CD25
regulatory T cells (StemCell Technologies) - Anti-CD4PE, Anti-CD25FITC, Anti-CD127PerCP/Cy5.
5 (BioLegend) - PBS2FBS
- Ficoll-Hypaque solution
- EasySep Magnet
- BC FC500 Flow cytometer
8Method
- Cell Isolation
- COMPLETE KIT FOR CD4CD127lowCD49d-CD25
REGULATORY T CELLS - Section A Negative Selection
- RosetteSep CD4 CD127lowCD49d- T Cell
Pre-Enrichment Cocktail - Tetrameric Antibody complex (TAC) - Anti-Unwanted
cell Anti-glycophorin A -
9Method
- Section B Positive selection
- EasySep Positive Selection Cocktail
- Tetrameric Antibody complex (TAC) - Anti-CD25
cell Anti-Dextran - EasySep Magnetic Nanoparticles
- Nanoparticle coated with dextran
-
10- Section A
- Add RosetteSep CD4CD127lowCD49d- T Cell
Pre-Enrichment Cocktail (50 µl) to whole blood
(1 ml) - mix, incubate 20 min. at RT
- dilute sample with PBS2FBS 1 ml
- over-layer diluted sample on Ficoll-Hypaque 1.5
ml - centrifuge at 400g for 30 min
- collect enriched cells, wash 2x with PBS2 FBS
at 200g - resuspend cells in PBS2FBS 1 mM EDTA 500 µl
- transfer enriched cells to polystyrene tube
11- Section B
- add EasySep Positive Selection Cocktail 25 µl to
Enriched cell in polystyrene tube - incubate 15 min. at RT
- add EasySep Magnetic Nanoparticles 25 µl
- mix, incubate 10 min. at RT
- add PBS2 BS1 mM EDTA to cell suspension (total
volume 2.5 ml) - place tube in magnet for 5 min, pour off
supernatant - add PBS2 FBS1 mM EDTA 2.5 ml and place back
tube in magnet for 3 additional rounds of
separation - add PBS2FBS1 mM EDTA 100 µl to resuspend cells
12Cell staining
- add Anti-CD25-FITC 5 µl Anti-CD4-PE 5 µl
Anti-CD127-PerCP/Cy5.5 2.5 µl to cell suspension - incubate in dark, RT, 30 min.
- wash with PBS2FBS 300 µl 4 min
- add PBS2FBS 1 ml
- add Flow-CountTM fluorosphere (984 bead/µl) 10 µl
- Analysis with Flow cytometer
-
13Flow cytometric analysis
14Flow cytometric analysis
15Calculation of Absolute Treg cells
A event no. of CD4CD25CD127low/- T cells B
Flow-CountTM fluorosphere conccentration (9,840
beads) C Flow-CountTM fluorosphere in bead gate
(8,000 beads)
16ResultsAbsolute Treg cell counts of Nornal and
HIV/AIDS
Nornal CD4 Abs Treg cells/ml WB HIV/AIDS CD4 Abs Treg cells/ml WB
N1 40 841 P1 23 736
N2 34 421 P2 11 91
N3 30 913 P3 9 284
N4 33 699 P4 14 374
N5 42 499 P5 22 66
N6 43 450 P6 6 66
N7 27 754 P7 13 53
N8 42 748 P8 27 151
N9 41 433 P9 24 474
N10 38 595 P10 3 26
MeanSD 375 635171 MeanSD 158 232222
17Box plot of absolute Treg cell counts
p 0.0007
Normal
HIV/AIDS
18Correlation of Treg and CD4
19Conclusions
- Absolute Treg numbers in normal is significantly
higher than that of PHAs - 635 171 cells/ml vs. 232 222 cells/ml
- p 0.0007
- Absolute Treg numbers in normal inversely
correlate with CD4 T cell (r -0.4604). - Absolute Treg numbers in PHAs positively
correlate with CD4 T cell (r 0.4877).
20Thank you for your attention