Absolute CD4+CD25+CD127low/-

T Cells (Treg ) in Peoples with HIV/AIDS

Sakchai DettrairatHIV Laboratory

Division of Clinical ImmunologyDepartment of Medical Technology

Faculty of Associated Medical SciencesChiang Mai University

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HIV Infection/AIDS

2http://emedicine.medscape.com/article/211316-media

Treg Activity in HIV infection

3http://journals.lww.com/aidsonline/Fulltext/2008/03300

Red: Treg Activity Green: Treg Activity

Treg Activity in HIV infection

• Treg เพิ่��มขึ้�น:– Treg ถู�กทำ��ล�ยน�อย เน��องจ�กม�ก�รติ�ดเชื้�อ HIV ใน Treg น�อย– gp120 เพิ่��มก�รแสดงออกขึ้อง Foxp3 และย!บย!งก�รเก�ด apoptosis

ขึ้อง Treg

– HIV Tat เพิ่��มก�รแสดงออกขึ้อง CTLA-4 และ suppressive activity

– Immune activation & imflammation เหน��ยวน��ให� Treg แบ%งติ!ว– Treg circulation between HIV-infected site and lymphiod

tissue

• Treg ลดลง:– ART จะลดจ��นวนไวร!สลง ทำ��ให� effect ขึ้อง gp120 และ HIV Tat

ลดลง4

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.

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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.

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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

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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

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MethodSection B: Positive selection

EasySep® Positive Selection Cocktail Tetrameric Antibody complex (TAC) - Anti-

CD25+ cell & Anti-Dextran

EasySep® Magnetic Nanoparticles Nanoparticle coated with dextran

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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

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 11

Cell staining

1. add Anti-CD25-FITC 5 µl + Anti-CD4-PE 5 µl + Anti-CD127-PerCP/Cy5.5 2.5 µl to cell suspension

2. incubate in dark, RT, 30 min.3. wash with PBS+2%FBS 300 µl 4 min4. add PBS+2%FBS 1 ml5. add Flow-CountTM fluorosphere (984 bead/µl ) 10 µl6. Analysis with Flow cytometer

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Flow cytometric analysis

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Flow-CountTM fluorosphere 8,000 beads

Flow cytometric analysis

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CD4 positive

CD4+CD25+CD127low/- T Cells

= 684 events

Calculation of Absolute Treg cells

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A: event no. of CD4+CD25+CD127low/- T cellsB: Flow-CountTM fluorosphere conccentration (9,840 beads)C: Flow-CountTM fluorosphere in bead gate (8,000 beads)

684 x 98408,000 =

= 841 cells/ml WB

Absolute Treg =A X B

C

ResultsAbsolute Treg cell counts of Nornal and

HIV/AIDSNornal %CD4 Abs Treg

cells/ml WB HIV/AIDS %CD4 Abs Tregcells/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 5 3

N8 42 748 P8 27 151

N9 41 433 P9 24 474

N10 38 595 P10 3 2 6

Mean±SD 37±5 635±171 Mean±SD 15±8 232±22216

Box plot of absolute Treg cell counts

17Normal HIV/AIDSp = 0.0007

Correlation of Treg and CD4

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r = -0.4604 r = 0.4877

Normal HIV/AIDS

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).

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Thank you for your attention

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