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