the impact of aging on cd4 t cell function 1.define the impact of the defects in aged naïve cd4 t...
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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 AgedTcR Tg mouse:Homogeneous naïve CD4T cells
Isolate:Young or AgedNaïve CD4 T Cells
Inject into Young host
Prime with peptide Agand Alum IP
CFSE Label
Harvest Spleen Days 1-4Determine:1) Division2) Expansion3) 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. 1996. J. Exp. Med. Haynes, Linton, Eaton, Tonkonogy and Swain. 1999. J. Exp. Med.Haynes, Eaton and Swain. 2002. 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 CD4From Young or Aged
Generate Th1 or Th2Effectors in vitro (with IL-2 and polarizing cytokine)
Ag/APC + IL-2 4 days
Wait>6 wks
Re-isolate donor memory cellsTest function in vitro
Inject Effectors
In vitroeffectors
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)
IL-2 IFNγ -4IL -5IL0
1000
2000
10000
0
200001Th 2Th
/cytokine ml
Primary effectors
Young
Aged
1 Th Memory
0
400
800
0
500
1000
1500
-2IL IFNγ* 0
2000
4000
-5IL
*
0
10000
20000
-4IL
*
2 Th Memory
Cytokine Production from Effectors and Memory
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 CellsDAY 1 DAY 2 DAY 3 DAY 4
Th2Memory
Th2Memory+IL-2
YOUNGAGED
CFSE
Memory cells from aged effectors are defective in divisionand cytokine production andare 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 morecent
12 mo old
Memory Derived from Young Naïve CD4 T Cells Retains Function with Aging
12 Months
01
2
34
5
6
Foldexpansion
1 Month
0
3000
6000
9000
IL-4 (pg/mlL)
CD4 Memory Response to RestimulationExpansion IL-4 Production
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
Division and Phenotype of Th2 memory effectors CFSE CD44 CD62L CD25
1 Month 12 Months
Fluorescence
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
MemoryCell
Defect ?
Thymocyte New NaïveCD4 T Cell
Old NaïveCD4 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 cellsdeveloped in agedhost after CD4 depletion..
CFSE
YOUNGANTI-CD4
YOUNGISOTYPE
AGED ANTI-CD4
AGEDISOTYPE
Donor of Naive CD4 T Cells
0
500
1000
1500
2000
2500
IL-2 (U/ML)
Fold Expansion
ISOTYPE
*
Young Aged
CD4 Depleted
0
5
10
15*
Young Aged
10 weeks after Ab treatment
Restoration of Helper Function
Young Aged
# of
NP
+ B
Cel
ls x
106
0
5
15
10
Isotype CD4-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 youngor 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 CD44hi 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
Cellular Age-Months
8 Week Mouse
1 2 3 4
# Cells
# Cells
12 Month Mouse
8 Month Mouse TX at 3 weeks
1 2 3 4
# Cells
5 86 7
1 2 3 4 5 86 7
Hypothetical Age of Naive CD4 T Cells in Different Mice
In a TX mousethe naïve CD4 cohortages 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?
Recover,Sort
Donor Cells, Day 0
Class II KO HostsDay 7
ATXBM HostsDay 7 (HDD+)
ATXBM HostsDay 7
Class II KO HostsDay 7
IL-2 Production
RestimulateEx-Vivo
IL-2
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
CD3 + 20Ab
Ionomycin
UndividedDivided (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
106 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
Rel
ativ
e cp
m
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
0.0
0.5
1.0
1.5
2 6 10 14
ABOATxBM HiATxBM LoC57BL/6
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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.
Collaborators in Aging Studies
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Laura HaynesSheri Eaton Karen Clise-Dwyer
Eve Burns