Absolute CD4 CD25 CD127low/- T Cells (Treg) in Peoples with HIV/AIDS - PowerPoint PPT Presentation

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Absolute CD4 CD25 CD127low/- T Cells (Treg) in Peoples with HIV/AIDS

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Title: Absolute CD4 CD25 CD127low/- T Cells (Treg) in Peoples with HIV/AIDS


1
Absolute 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

2
HIV Infection/AIDS
http//emedicine.medscape.com/article/211316-media
3
Treg Activity in HIV infection
http//journals.lww.com/aidsonline/Fulltext/2008/0
3300
4
Treg 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 ????

5
Regulatory 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.

6
Objective
  • Enumeration of absolute CD4CD25CD127low/- T
    Cells (Treg) numbers in people with HIV/AIDS
    receiving ART by using pre-enrichment technique
    and flow cytometry.

7
Samples 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

8
Method
  • 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

9
Method
  • 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

12
Cell 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

13
Flow cytometric analysis
14
Flow cytometric analysis
15
Calculation 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)
16
ResultsAbsolute 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
17
Box plot of absolute Treg cell counts
p 0.0007
Normal
HIV/AIDS
18
Correlation of Treg and CD4
19
Conclusions
  • 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).

20
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