Why and How to Measure Proliferative Capacity

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Why and How to Measure Proliferative Capacity

• Vernon (Skip) Maino
• BD Biosciences

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OUTLINE

• Flow methods for characterizing proliferating
  cells
• Memory T cell proliferative capacity in vitro
  analysis
• Memory T cell proliferative capacity in vivo
  analysis in Rhesus Macquaque

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T Cell Activation Effector function,
Proliferation, Death
IL-4
IL-2
TNFa
IFNg
APC-T cell interactions
Cytokine expression
Proliferation/ Death
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Proliferation Assays

• In vivo proliferation BrdU injection, Ki-67
  staining
• In vitro proliferation (proliferative capacity)
• 3H-thymidine incorporation (MTT, XTT)
• Cellular markers (Ki67, CD71)
• BrdU labeling
• CFSE labeling

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Importance of Proliferative CapacityHints from
HIV

• Rosenberg et al., Science (1997)
  CD4 T cell proliferation correlates with
  control of viremia
• Migueles et al, Nature Immunology (2002) CD8 T
  cell proliferative capacity differentiates
  non-progressors from progressors
• Lieberman, Shearer, etc
  Importance of IL-2, Anergy,
  APC defects

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Memory T Cell Proliferative Capacity

• Relevance to Immune Protection

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HypothesisDetection of a given frequency of
antigen-specific T cells may be a necessary but
not sufficient condition for protection from
disease progression.Other potential factors -
proliferative capacity - effector function -
Ratio of anergic or regulatory cells
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Flow Cytometric Techniques for Characterizing
Proliferating T Cells

• CFSE
• BrdU
• Ki-67
• Multi-color Combinations

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CFSE Labeling

• Requires pre-labeling of targets with precise
  level of CFSE
• Allows multiparameter analysis of other
  phenotypic markers, including cytokines
• Semi-quantitative, allows calculation of initial
  burst size as well as number of cell divisions
• Does not account for frequency of dead cells

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CFSE Example - anti-CD3 Stimulation
Day 0
Day 4 - IL-4 cells
No CD81
CD81
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BrdU Incorporation

• Simple, requires no pre-labeling of targets
• Allows multiparameter analysis of other
  phenotypic markers, including cytokines
• Provides cumulative frequency of cells which have
  undergone DNA synthesis but does not calculate
  divisions
• Does not account for frequency of apoptotic cells

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BrdU Incorporation as a Measure of Proliferation

• BromoDeoxyUridine is a thymidine analog which is
  incorporated into DNA of the replicating cells
• Detected by staining the cells with fluorescence
  labeled anti-BrdU mAb in presence of DNAase

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or
PBMC Ag (peptides or proteins)
48 96h
1X FACSLYSE
Wash 1X FACSPERM
10 min
CD71 PE
Wash Stain ?-BrdU DNAase
BrdU-FITC
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Combined CFSE and BrdU Labeling
unstimulated
SEB
CFSE
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Equivalence of 3H-thymidine and BrdU
Mean cpm X 10-3
T cells incorporating BrdU
CD4
CD8
3H-TdR
0
1
2
0.5
1.5
SEB concentration (mg/ml)
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Equivalence of 3H-thymidine and BrdU
? cpm
? cpm
? CD4BrdU
? CD4BrdU cells
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SEB Activation 6h Pulse vs. Continuous BrdU
Labeling
80
60
40
CD4IFNg cells incorporating BrdU
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pulse (6 h)
continuous (72 h)
0
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CMV-specific Proliferation in CMV seropositive
Individual Comparison of Ki67 vs CD71 as
Additional Marker
BrdU FITC
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Proliferation Cytokine Expression in
CMV-Activated PBMC 48 h Culture / CD4 gated
unstimulated
CMV lysate
anti-IFNg FITC
3.6
anti-BrdU FITC
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Use of BrdU Staining to Identify Antigen-Specific
Proliferating CD4 Cells
48h culture/CD4 gated
p55 gag
HIV-REMUNE
Unstimulated
0.61
2.3
2.5
CD71PE
Anti-BrdU FITC
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(No Transcript)
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Relationship of HIV Proliferative Responses and
HAART Therapy
50000
p 0.07
45000
Absolute Number of Proliferating CD4 T cells /
ml Blood
20000
15000
mean
10000
7042
mean
5000
2484
0
no therapy
HAART therapy
(n 14)
(n 12)
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Lack of Correlation between CD4 Counts and Number
of Proliferating CD4 T Cells or Stimulation
Index (Treated HIV-infected Individuals)
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Proliferative T Cell Response as a Measure of
Dendritic Cell Function
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Conclusions

• Proliferative capacity of antigen-specific T
  cells is a potentially important predictor of
  disease protection.
• CFSE and BrdU incorporation are both valuable,
  multiparametric methods to assess proliferative
  capacity of specific cell subsets.

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Contributors

• Smita Ghanekar
• Bela Mehta
• Sonny Bhatia
• Holden Maecker
• Jay Levy

BD Biosciences San Jose, CA
UCSF
Louis Picker U. Oregon Health Sci
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Study of T Cell Dynamics in Rhesus Macaque

• RM present the most readily available human-like
  animal model
• Model will allow quantitative analysis of whole
  immune system
• Increasing availability of sophisticated analytic
  tools
• Direct application to central issues of HIV/SIV
  pathogenesis

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Investigation of T Cell Turnover in RM

• Mechanisms controlling peripheral T cell
  turnover (proliferation/death) are critical for
  the maintenance of naive and memory T cell
  populations and immune homeostasis but few
  avenues are available for in vivo exploration of
  these issues in the human.
• Basic questions include
• turnover characteristics of naïve, total memory,
  and effector/central memory populations, their
  homeostatic relationship to each other, and the
  regulatory mechanisms governing this homeostasis.
• contribution of homeostatic mechanisms to
  pathobiology of viral infection (particularly
  HIV/SIV).

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Tools For In Vivo Analysis of T Cell Homeostasis
in RM

• Ki-67 expression
• nuclear Ag expressed by cycling (non-G0) cells
• issues non-discrete staining significance of in
  vivo expression poorly characterized
• BrdU incorporation
• thymidine analogue metabolized by salvage
  pathway incorporated into proliferating cells
  during S-phase
• very discrete staining (reactivity indicates
  S-phase during the time of BrdU administration)
• useful for both proliferation analysis and
  tracking
• issues potential toxicity metabolic
  heterogeneity among different cell types

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Ki-67 Expression Correlation With BrdU Labeling
Time
BrdU pulse
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Ki-67 vs. BrdU During Washout Phase(CD4 T cells)
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In vivo Meaning of Ki-67 Expression

• 3-4 days BrdU labeling achieves near equivalence
  between Ki-67high and BrdU cells however,
  complete loss of Ki-67 expression among cells
  leaving cell cycle takes another 7-10 days.
• Thus,total Ki-67 expression reflects an
  integrated average of cells undergoing S-phase in
  the previous 7-10 days with higher levels of
  Ki-67 expression highly weighted towards the
  previous 3-4 days.

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Simultaneous BrdU/Ki-67 evaluation A poor
mans double label?

• Co-analysis of BrdU and Ki-67 in the immediate
  BrdU labeling period delineates overall
  proliferation, and efficiency of BrdU
  incorporation.
• Co-analysis in the first 1-2 weeks following BrdU
  pulsing, delineates cell cycle progression,
  length of the proliferative burst.
• Co-analysis at later time points delineates
  re-entry into the proliferating pool.
• Decay of BrdU cells occurs with either cell loss
  or proliferative dilution (gt 5 divisions), but
  relative death rates can be inferred by
  comparison of populations with equivalent Ki-67
  reactivity.

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Ki-67 Expression Naïve vs. Memory Subsets
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Ki-67 expression by CMV-specific CD4 memory T
cells before and after CMV challenge of
CMV-immune RM
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Analysis of In Vivo Turnover CD28-Defined and
CMV-Specific Memory Subsets ( 15449)
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Summary T cell proliferation/turnover in healthy
macaques

• Steady state proliferation of memory subset 10
  fold greater than that of naïve subset.
• CD4 and CD8 proliferation highly correlated.
• Decay of BrdU memory T cells is biphasic, with
  half of initial label cells lost in first month
  and the next half over the following 5 months.
• Decay of BrdU naïve T cells is not discernable
  over 6 months.
• Proliferation/turnover of CD28 vs. - memory T
  cell subsets are not significantly different.
• Ag provides strong proliferative stimulus in vivo.

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Conclusions

• Proliferative capacity of antigen-specific T
  cells is a potentially important predictor of
  disease protection.
• CFSE and BrdU incorporation are both valuable,
  multiparameter methods to assess proliferative
  capacity of specific cell subsets.