Investigating Human T cells Using Multicolor Flow Cytometry Insoo Kang Section of Rheumatology.

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Investigating Human T cells Using Multicolor Flow Cytometry Insoo Kang Section of Rheumatology

Aging and IL-7-mediated CD8+ T cell survival IL-7, largely produced from thymus, is critical for the development and survival (homeostasis) of CD8+ T cells. Decreased naïve CD8+ T cells and impaired memory CD8+ T cell responses occur with aging. Decreased plasma levels of IL-7 have been observed with aging. IL-7 has been used to rejuvenate T cell immunity in aged mice and non-human primates, without significant success. It is critical to determine whether aging also affects other steps involved in IL-7-mediated CD8+ T cell survival pathway.

Objective: Investigating IL-7 receptor expression on CD8+ T cell subsets and their signaling and survival responses to IL-7 in healthy young (≤ 40 years) and elderly (≥ 65 years) people. Exclusion criteria: Individuals who were taking immunosuppressive drugs or who had a disease potentially affecting the immune system including infection, cancer, asthma, autoimmunity and diabetes were excluded from the study

IL-7 receptor (R) signal transduction pathways STAT5 X  cc Jak1 Jak3 PI3 kinase P PTEN Akt P BAD inactivation FOXO inactivation GSK inhibition Survival Proliferation Glucose use P + Survival (major) Retention of Bax Box1 region P BCL-2 ? ? Y449, STAT5 docking sites P MCB 2004, 24(14): J Exp Med. 2004,200(5): Immunol Rev. 2003,192:7-20

Peripheral blood mononuclear cells (PBMCs) from human subjects Measure IL-7Rα and γC expression by different CD8+ T cell subsets as well as their cellular characteristics using flow cytometry (FACSCalibur® and LSRII® ). Measure cell signaling using flow cytometry (FACSCalibur®) Stimulate cells with IL-7 or PBS. Stain cells with Abs Using FACSAria® Sort cells into different cell subsets Flow cytometry is the key step for experiments Conduct functional studies

CD8+ T cells CD45RA CCR7 CMNaive EM CD45RA+ EM Naive CM EM EM CD45RA+ IL-7R  Elderly Young PBMCs IL-7R  expression by subsets of CD8+ T cells Isotype anti-IL-7R  Ab * *median fluorescen t intensity high low Stain with Abs to CD8, CD45RA, CCR7 and IL-7Rα, γC or isotype

: CD : CD : CD : CD : CD : CD : CD : CD : CD : CD : CD : CD EM CD45RA EM : CD : CD : CD : CD : CD : CD : CD : CD : CD : CD : CD : CD NaiveCM IL-7R  high IL-7R  low Young NaiveCM IL-7R  high IL-7R  low Elderly CD28 CD27 Late Early Int More than 4 markers can be simultaneously measured using LSRII® Markers used CD8, CD45RA, CCR7, IL-7R  CD27 and CD28

P-STAT5 in subsets of CD8+ T cells in response to IL-7* can be measured using flow cytometry IL-7R  expression Phospho-STAT5 IgG  -IL-7R  PBS IL-7 *10 min at 10 ng/ml Elderly PBMCs Measure expression of P-STAT5 by CD8+ T cell subsets using flow cytometry (FACSCalibur®) Stimulate cells with IL- 7 or PBS. Permeabilize and stain cells with Abs to P-STAT5 or isotype. Stain cells with CD8,CD45RA and CCR7 Advantage of flow cytometry: Cell signaling can be measured in a small number of cells (<100,000) and quantitative analysis is possible. Naive CM EM EM CD45RA+

FL1-H: FITC annexin FL3-H: 7 AAD FL1-H: FITC annexin FL3-H: 7 AAD FL1-H: FITC annexin FL3-H: 7 AAD FL1-H: FITC annexin FL3-H: 7 AAD FL1-H: FITC annexin FL3-H: 7 AAD FL1-H: FITC annexin FL3-H: 7 AAD FL1-H: FITC annexin FL3-H: 7 AAD FL1-H: FITC annexin FL3-H: 7 AAD NaiveEM PBS IL-7 EM CD45RA+ IL-7R  high EM CD45RA+ IL-7R  low IL-7R  low cells have decreased survival in response to IL-7 PBMCsSorted into subsets Using FACSAria® Incubate cells with IL-7 Measure live cells using flow cytometry

Elderly (n=5) IL-7R  GABP  GFI Naive EM CD45RA+ IL-7R  high EM CD45RA+ IL-7R  low FACS sorting can be used to measure gene expression in a small number (100,000) of specific cell subsets P > 0.05 P < 0.05

Conclusions Our findings suggest that aging affects IL-7R  expression by CD8+ T cells leading to impaired signaling and survival responses to IL-7. Flow cytometry is valuable in investing the phenotypes and functions of human immune cells. Kim et al, Blood 06

Acknowledgements Yale Flow Cytometry Facility Hartford Foundation Arthritis Foundation NIH/NIAMS Lupus Foundation AFAR Yale Pepper Center Hang-Rae Kim Myung Sun Hong Kyung-A Hwang Ping Zhu Chris Bailey Barbara Foster Lynne Iannone