The Impact of Aging on CD4 T Cell Function

1. The Impact of Aging on CD4 T Cell Function • Define the impact of the defects in aged naïve CD4 T cells in memory development? • When in CD4 T cell development do the defects develop? • How and why do they develop?

2. In Vivo Effector Generation from Aged Naïve CD4 T Cells Isolate:Young or Aged Naïve CD4 T Cells Inject into Young host Harvest Spleen Days 1-4 Determine: 1) Division 2) Expansion 3) Cytokine CFSE Label Young or Aged TcR Tg mouse: Homogeneous naïve CD4 T cells Prime with peptide Ag and Alum IP Compare relative response of young and aged naïve CD4 T cellswith all other components young.

3. Aged Naïve CD4 T Cell Defect In Vitro and In Vivo • Aged naïve CD4 T cells make less IL-2 following stimulation. • Expansion of responding aged CD4 cells is much reduced. • Effectors which develop are not fully differentiated, and are not well-polarized to make effector cytokines or help B cells. • IL-2 restores effector generation. • Proinflammatory cytokines (TNF, IL-1/6) enhance response. Thus defects in CD4 T cells may be largely responsible for the inability of the aged to be well vaccinated Linton, Haynes, Klinman and Swain. 1996. J. Exp. Med. Haynes, Linton, Eaton, Tonkonogy and Swain. 1999. J. Exp. Med. Haynes, Eaton and Swain. 2002. J. Immunol.

4. Memory from Aged Naïve:Does restoration of primary effector formation overcome aging defects in memory? Inject Effectors Tg Naïve CD4 From Young or Aged Ag/APC + IL-2 4 days In vitro effectors Wait >6 wks Generate Th1 or Th2 Effectors in vitro (with IL-2 and polarizing cytokine) Re-isolate donor memory cells Test function in vitro

5. Defect in Memory from Aged Naïve CD4 CD4 memory T cells derived from “rescued” effectors re-express defects in cytokine production (Haynes et. al. PNAS, 2003)

6. Ex Vivo Expansion of Memory Memory cell recovery equivalent, but memory from aged effectors expand little. Primary in vitro Transfer to host and restimulate

7. Function of Th2 Memory Cells Memory cells from aged effectors are defective in division and cytokine production and are not enhanced by IL-2.

8. Aged naïve CD4 T cells make defective memory • Even though addition of IL-2 restores effector cell generation, the memory derived from those effectors is defective. Rescue is transient. • Poor cytokine production following ex vivo restimulation • Poor expansion following ex vivo restimulation • Poor help for B cells (not shown) • Response no longer rescued by IL-2 Apparently, when effectors revert to resting memory, they remember their defects. Suggests an epigenetic, age-associated event that was present in aged naïve CD4 T cells.

9. Effect of Aging on Memory Cells Prepare Effectors Inject into Young host (ATXBM) Isolate: Naïve CD4 T Cells 4 mo recent Young AND TcR Tg (6-8 wk) Harvest Memory 3-6 wk Vs 12 mo. Compare 12 mo old Recover memory and restimulate ex vivo. Compare response (cytokines, expansion) and effector function of 4 and 12 month old memory cells

10. Memory Derived from Young Naïve CD4 T Cells Retains Function with Aging Older memory cells from young naïve CD4 T cells expand and make IL-4, like younger ones (Haynes et. al. 2004, PNAS). Memory cells are resistant to the development of aging defects.

11. Memory cells from Young Naïve CD4 T Cells Retain Function with Aging Older Memory cells divide as quickly as younger ones (or more quickly) in response to restimulation and they express a comparable memory effector phenotype.

12. Aging and Memory • Memory cells that were developed from young effectors seem resistant to the effects of aging. • Several other researchers have findings supporting this concept including Ahmed (Kapasi et. al Eur. J. Immunol., 2002). If confirmed in additional studies in mice and in humans, this would imply that vaccine programs would best be directed at the young and middle aged people.

13. When Does the Aging Defect Develop? New Naïve CD4 T Cell Old Naïve CD4 T Cell Thymocyte Effector Cell 1. Arebone marrow stem cells in aged mice defective? Are freshly generated naïve CD4 T cells in aged mice defective. Does increasing the chronologic age of a cohort of naïve CD4 T cells lead to development of the defect ? Stages of CD4 T cell: BM precursor Memory Cell Defect ?

14. Bone Marrow Transfer to Create New CD4 T from Old Bone Marrow Lethal Irradiation Young AND TcR Tg Young BALB/c Recipients Aged AND TcR Tg Recover BM-derived, CD4 T cells and evaluate function ex vivo. Is it defective?

15. No Defect in Bone Marrow Precursors Haynes et. al. , J. Exp. Med. 2005

16. Even aged bone marrow in aged mice gives rise to functional naïve CD4 T cells….confirm in a second model. Generation of New CD4 by anti-CD4 Depletion Young Tg Mouse Isotype (Young cells/Young mouse) Anti-CD4=Only new emigrants (Younger cells/Young mouse) Aged Tg Mouse Isotype (Aged Cells/Aged Mouse) Anti-CD4: only new emigrants (Young Cells/Aged Mouse) 69 Days : Isolate and Test Naïve CD4 T Cells

17. "Young" CD4 T cells generated in Aged Host 10 weeks after Ab treatment Conclude: No defect in "young" CD4 T cells developed in aged host after CD4 depletion..

18. Isotype CD4-depleted 15 10 # of NP+ B Cells x 106 5 0 Young Aged Restoration of Helper Function “New” CD4 T cells from aged mice have enhanced helper function

19. Generation of new CD4 T Cells Overcomes Aging Defect Bone marrow of aged mice, gives rise to a population of functional naïve CD4 T cells in young or aged mice. 2. “New” T cells arising after anti-CD4 treatment of aged mice are not defective. In aged mice reconstitution is slower, but the resultant naïve CD4 T cells are none-the-less functional. (Haynes et. al. J. Exp. Med., 2005) Bone marrow stem cells in aged mice are able to give rise to naïve CD4 T cells which do not appear defective. Suggests “age” of cell not environment is key.

20. Shift in CD4 Population with Age Peripheral CD4 Numbers Frequency of CD44hi CD4 Cells Hypothesis:Increased lifespan and Homeostatic division act to maintain CD4 numbers into old age, and are responsible for the aging defect. Naïve CD4 T Cells (increased lifespan with aging) Thymic CD4 Output Age in Years

21. Making Older Cells by Thymectomy In a TX mouse the naïve CD4 cohort ages more rapidly

22. Effect of Cellular Age Early onset of aging defect after thymectomy: (Haynes et al. J. Exp. Med. 2005)

23. Does homeostatic division lead to an aging like defect? Class II dependent HD, leads to a loss of IL-2 production. Could this be what happens as naïve CD4 T cells age in situ? (Karen Clise-Dwyer, unpublished)

24. Defective Ca++ Mobilization in HD Cells Undivided Divided (ATXBM) Agonist: TCR Tg Donor Cells aCD3 + 20Ab Relative Intracellular [Ca++] i CFSE Ionomycin Time (5 min)

25. ATXBM ATxBM Class II KO Intact B6 Effect of Homeostatic Division (HD) on Naïve CD4 T Cell Function HD • Sort Donor Cells into HD and Undivided Populations • Culture in vitro with Ag+APC • Monitor Proliferation and Cytokine Production 106 CFSE+ Naïve CD4 +++ --- +/- TCR Tg Donor

26. Defects in Homeostatically Divided Cells Reduced Proliferative Response to Ag MHC Class II KO ATxBM CFSE hi Relative cpm ATxBM CFSE lo C57BL/6 NA MHC Class II KO Day Post-Transfer ATxBM CFSE hi Reduced IL-2 Production Isotype ATxBM CFSE lo IL-2

27. Cells which have undergone HD appear less functional • Lower Ca++ Flux • Less IL-2 Production • Lower Proliferative Response to Ag • Aged naïve CD4 T cells actually undergo more HD than young ones (not shown). We suggest that post thymic “age” and homeostatic division play roles in the development of the aged defects.

28. Collaborators in Aging Studies Eve Burns Laura Haynes Sheri Eaton Karen Clise-Dwyer