The Impact of Aging on CD4 T Cell Function 1.Define the impact of the defects in aged naïve CD4 T cells in memory development? 2.When in CD4 T cell development do the defects develop? 3.How and why do they develop?

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

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. Linton, Haynes, Klinman and Swain J. Exp. Med. Haynes, Linton, Eaton, Tonkonogy and Swain J. Exp. Med. Haynes, Eaton and Swain J. Immunol. Thus defects in CD4 T cells may be largely responsible for the inability of the aged to be well vaccinated

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

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)

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

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

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.

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

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.

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.

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.

When Does the Aging Defect Develop? 1. Are bone marrow stem cells in aged mice defective? 2.Are freshly generated naïve CD4 T cells in aged mice defective. 3.Does increasing the chronologic age of a cohort of naïve CD4 T cells lead to development of the defect ? BM precursor Memory Cell Defect ? Thymocyte New Naïve CD4 T Cell Old Naïve CD4 T Cell Effector Cell Stages of CD4 T cell:

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

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

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

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

Restoration of Helper Function YoungAged # of NP+ B Cells x IsotypeCD4-depleted “New” CD4 T cells from aged mice have enhanced helper function

Generation of new CD4 T Cells Overcomes Aging Defect 1.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. 3.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.

Shift in CD4 Population with Age Thymic CD4 Output Peripheral CD4 Numbers Frequency of CD44 hi CD4 Cells Age in Years 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)

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

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

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)

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

Effect of Homeostatic Division (HD) on Naïve CD4 T Cell Function 10 6 CFSE+ Naïve CD4 TCR Tg Donor Sort Donor Cells into HD and Undivided Populations Culture in vitro with Ag+APC Monitor Proliferation and Cytokine Production ATxBM ATXBM Class II KO Intact B6 HD /-

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

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.

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