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?

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In Vivo Effector Generation from Aged Naïve CD4 T
Cells
IsolateYoung 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 cells with all other components
young.
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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.
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Memory from Aged NaïveDoes 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 gt6 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)
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Ex Vivo Expansion of Memory
Memory cell recovery equivalent, but memory from
aged effectors expand little.
Primary in vitro
Transfer to host and restimulate
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Function of Th2 Memory Cells
Memory cells from aged effectors are defective
in division and cytokine production and are not
enhanced by IL-2.
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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.
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Effect of Aging on Memory Cells
Inject into Young host (ATXBM)
Prepare Effectors
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
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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.
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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.
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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.

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When Does the Aging Defect Develop?

• 1. Are bone 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 ?
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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?
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No Defect in Bone Marrow Precursors
Haynes et. al. , J. Exp. Med. 2005
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Generation of New CD4 by anti-CD4 Depletion
Even aged bone marrow in aged mice gives rise to
functional naïve CD4 T cells.confirm in a
second model.
Young Tg Mouse
Isotype (Young cells/Young mouse)
Anti-CD4Only new emigrants (Younger cells/Young
mouse)
Isotype (Aged Cells/Aged Mouse)
Aged Tg Mouse
Anti-CD4 only new emigrants (Young Cells/Aged
Mouse)
69 Days
Isolate and Test Naïve CD4 T Cells
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"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..
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Restoration of Helper Function
New CD4 T cells from aged mice have enhanced
helper function
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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.
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Shift in CD4 Population with Age
Peripheral CD4 Numbers
Frequency of CD44hi CD4 Cells
Naïve CD4 T Cells (increased lifespan with aging)
Thymic CD4 Output
Age in Years

• HypothesisIncreased lifespan and Homeostatic
  division act to maintain CD4 numbers into old
  age, and are responsible for the aging defect.

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)
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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)
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Effect of Homeostatic Division (HD) on Naïve CD4
T Cell Function
HD

• Sort Donor Cells into HD and Undivided
  Populations
• Culture in vitro with AgAPC
• Monitor Proliferation and Cytokine Production

106 CFSE Naïve CD4
--- /-
TCR Tg Donor
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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
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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.
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Collaborators in Aging Studies
Eve Burns
Laura Haynes
Sheri Eaton
Karen Clise-Dwyer