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

2. 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.

3. 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

4. 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 ? ? IL-7 @ Y449, STAT5 docking sites P MCB 2004, 24(14):6501-6513 J Exp Med. 2004,200(5):659-69 Immunol Rev. 2003,192:7-20

5. 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

6. 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 271281306267 252*28428655 *median fluorescen t intensity high low Stain with Abs to CD8, CD45RA, CCR7 and IL-7Rα, γC or isotype

7. 010 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 39.7 15.2 0.2744.7 010 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 19.7 73 1.45.62 010 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 47.7 18.8 0.8132.6 010 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 19.8 67.7 5.037.71 010 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 11.6 80.9 2.614.72 010 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 3.18 96.6 0.0430.21 EM CD45RA EM 010 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 12.3 1.16 0.685.9 010 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 28 9.93 1.0961 010 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 3.44 1.47 0.8694.2 010 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 12 30.3 3.6954 010 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 7.58 64.6 5.4922.5 0 10 2 3 4 5 : CD28 0 10 2 3 4 5 : CD27 7.8 89.2 0.42.61 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

8. 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+

9. 10 0 1 2 3 4 FL1-H: FITC annexin 10 0 1 2 3 4 FL3-H: 7 AAD 0.5155.5 24.519.5 10 0 1 2 3 4 FL1-H: FITC annexin 10 0 1 2 3 4 FL3-H: 7 AAD 0.2968.4 17.613.7 10 0 1 2 3 4 FL1-H: FITC annexin 10 0 1 2 3 4 FL3-H: 7 AAD 0.3210.6 9.3779.8 10 0 1 2 3 4 FL1-H: FITC annexin 10 0 1 2 3 4 FL3-H: 7 AAD 0.0110.1 12.677.2 10 0 1 2 3 4 FL1-H: FITC annexin 10 0 1 2 3 4 FL3-H: 7 AAD 0.144.48 5.5989.8 10 0 1 2 3 4 FL1-H: FITC annexin 10 0 1 2 3 4 FL3-H: 7 AAD 0.1128 13.558.4 10 0 1 2 3 4 FL1-H: FITC annexin 10 0 1 2 3 4 FL3-H: 7 AAD 0.0626.6 9.5463.8 10 0 1 2 3 4 FL1-H: FITC annexin 10 0 1 2 3 4 FL3-H: 7 AAD 0.0519.4 16.663.9 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

10. Elderly (n=5) IL-7R  GABP  GFI-1 0 10 20 30 40 50 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

11. 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

12. 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