1. Absolute CD4+CD25+CD127low/- 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

3. Treg Activity in HIV infection
Red: Treg Activity
Green: Treg Activity

4. Treg Activity in HIV infection
Treg ถูกทำลายน้อย เนื่องจากมีการติดเชื้อ HIV ใน Treg น้อย
gp120 เพิ่มการแสดงออกของ Foxp3 และยับยั้งการเกิด apoptosis ของ Treg
HIV Tat เพิ่มการแสดงออกของ CTLA-4 และ suppressive activity
Immune activation & imflammation เหนี่ยวนำให้ Treg แบ่งตัว
Treg circulation between HIV-infected site and lymphiod tissue
Treg ลดลง:
ART จะลดจำนวนไวรัสลง ทำให้ effect ของ gp120 และ HIV Tat ลดลง

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 CD4+CD25+CD127low/- 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 CD4+CD127lowCD49d-CD25+ regulatory T cells (StemCell Technologies)
Anti-CD4:PE, Anti-CD25:FITC, Anti-CD127:PerCP/Cy5.5 (BioLegend)
PBS+2%FBS
Ficoll-Hypaque solution
EasySep® Magnet
BC FC500 Flow cytometer

8. Method
Cell Isolation
COMPLETE KIT FOR CD4+CD127lowCD49d-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® CD4+CD127lowCD49d- T Cell Pre-Enrichment Cocktail (50 µl) to whole blood (1 ml)
mix, incubate 20 min. at RT
dilute sample with PBS+2%FBS 1 ml
over-layer diluted sample on Ficoll-Hypaque 1.5 ml
centrifuge at 400g for 30 min
collect enriched cells, wash 2x with PBS+2% FBS at 200g
resuspend cells in PBS+2%FBS+ 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 PBS+2% BS+1 mM EDTA to cell suspension (total volume 2.5 ml)
place tube in magnet for 5 min, pour off supernatant
add PBS+2% FBS+1 mM EDTA 2.5 ml and place back tube in magnet for 3 additional rounds of separation
add PBS+2%FBS+1 mM EDTA 100 µl to resuspend cells

12. Cell staining
add Anti-CD25-FITC 5 µl + Anti-CD4-PE 5 µl + Anti-CD127-PerCP/Cy µl to cell suspension
incubate in dark, RT, 30 min.
wash with PBS+2%FBS 300 µl 4 min
add PBS+2%FBS 1 ml
add Flow-CountTM fluorosphere (984 bead/µl) 10 µl
Analysis with Flow cytometer

13. Flow cytometric analysis
Flow-CountTM fluorosphere
8,000 beads

14. Flow cytometric analysis
CD4 positive
CD4+CD25+CD127low/- T Cells
= 684 events

15. Calculation of Absolute Treg cells
684 x 9840
8,000 =
841
cells/ml WB
Absolute Treg
A X B C
A: event no. of CD4+CD25+CD127low/- T cells
B: Flow-CountTM fluorosphere conccentration (9,840 beads)
C: Flow-CountTM fluorosphere in bead gate (8,000 beads)

16. Results Absolute Treg cell counts of Nornal and HIV/AIDS
%CD4
Abs Treg
cells/ml WB
HIV/AIDS 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 N7 27
754 P7 13 53 N8
748 P8
151 N9 41
433 P9 24
474
N10 38
595
P10 3 26
Mean±SD
37±5
635±171
15±8
232±222

17. Box plot of absolute Treg cell counts
Normal
HIV/AIDS

18. Correlation of Treg and CD4
Normal
HIV/AIDS

19. Conclusions
Absolute Treg numbers in normal is significantly higher than that of PHAs
635 ± 171 cells/ml vs. 232 ± 222 cells/ml
p =
Absolute Treg numbers in normal inversely correlate with %CD4+ T cell (r = ).
Absolute Treg numbers in PHAs positively correlate with %CD4+ T cell (r = ).

20. Thank you for your attention