Monitoring Antigen-Specific T-Cells by Multiparameter Flow Cytometry

1
Monitoring Antigen-Specific T-Cells by
Multiparameter Flow Cytometry

• Holden T. Maecker

2
CMV as a model of a successful cellular immune
response

• Chronic, latent infection
• No pathology in immunocompetent hosts
• Usually no measurable viral titer in blood
• Highly expanded populations of memory T cells

3
What can flow cytometry tell us about
antigen-specific T cells?

• T cell subsetting
• CD4, CD8
• Cytokine profiles
• IFNg, IL-2, TNFa, MIP-1b
• Differentiation status
• CD27, CD28, CD45RA, CD45RO, CCR7, CD127, CD57
• Degranulation capacity
• CD107, perforin, granzyme B

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Production of IL-2 and IFNg by antigen-specific T
cells
Antigen-Experienced
Naïve
IL-2 IFNg-
IL-2 IFNg
IL-2- IFNg
Increasing cytotoxic potential Decreasing
proliferative potential
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Reported IL-2 defects in progressive HIV disease

• Various authors Lack of IL-2 production by
  HIV-specific CD4 and/or CD8 T cells
• Sekaly et al. HIV-specific CD4 T cells lack
  ability to make IL-2 or proliferate in
  progressive disease
• Pantaleo et al. HIV-specific CD4 T cells of
  progressors make IFNg but not IL-2, while
  non-progressors make IFNg and IL-2.

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Simplified CD8 T cell differentiation
Antigen-Experienced
Naïve
CD45RA CD27 CD28
CD45RA- CD27 CD28
CD45RA CD27- CD28-
effector
Increasing cytotoxic potential Decreasing
proliferative potential
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Reported differentiation defects in progressive
HIV disease

• Incomplete effector differentiation of CD4 or
  CD8 T cells
• Impaired cytolytic function of CD8 T cells
• Low perforin content in HIV-specific CD8 T cells

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Hypothesis

• IL-2 drives the differentiation of effector T
  cells
• Lack of IL-2 production by HIV-specific T cells
  results in impaired effector cell differentiation

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HIV progressors and healthy controls

• 19 HIV subjects with CD4 count decline to lt500
  cells/ml, persistent viremia gt2000 copies/ml
• 15 off therapy, 4 on therapy
• All CMV
• 20 healthy HIV- healthy subjects
• All CMV

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8-color Antigen-Specific Immunophenotyping
Ab Conjugate Laser

CD28 PerCP-Cy5.5 488
CD45RA PE-Cy7 488
CD27 APC 633 Surface
CD8 APC-Cy7 633 staining
CD3 Pacific Blue 405
CD4 AmCyan 405

Anti-IFNg FITC 488 Intracellular
Anti-IL-2 PE 488 staining

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All events
CD3 gate
B
CD8 APC-Cy7
Side Light Scatter
C
A
CD3 Pacific Blue
CD4 AmCyan
CD4 CD8- gate
CD4- CD8 gate
IFNg FITC
IFNg FITC
IL-2 PE
IL-2 PE
CD8 IFNg
CD4 IFNg
CD8 IFNg IL-2
CD4 IFNg IL-2
Side Light Scatter
Side Light Scatter
Side Light Scatter
Side Light Scatter
CD27 APC
CD27 APC
CD27 APC
CD27 APC
CD45RA PE-Cy7
CD45RA PE-Cy7
CD45RA PE-Cy7
CD45RA PE-Cy7
CD45RA PE-Cy7
CD45RA PE-Cy7
CD45RA PE-Cy7
CD45RA PE-Cy7
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
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Assay standardization for clinical trials

• Target channels for PMT voltages using mid-range
  calibration particles
• Pre-configured lyophilized reagent plates
• Stimulation
• Staining
• Gating
• Centralized analysis (single template)
• Dynamic gates

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Transfer of data
Statistics files
Custom Excel spread- sheet
Web database
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Stability of phenotypic markers
A.
B.
0 h stimulation
6 h stimulation
1.8
1.8
CMV tetramer PE
CMV tetramer PE
IFNg FITC
IFNg FITC
66
34
66
34
Side Light Scatter
Side Light Scatter
CD27 APC
CD27 APC
90
1.7
92
2.3
75
5.0
65
6.3
CD45RA PE-Cy7
CD45RA PE-Cy7
CD45RA PE-Cy7
CD45RA PE-Cy7
8.3
0.2
5.7
0.2
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5.3
20
8.9
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
CD28 PerCP-Cy5.5
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Results CMV response

• IL-2 production
• Differentiation status

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A small fraction of IFNg cells make IL-2
CD4 IFNg
CD8 IFNg
105
104
103
CD8 IL-2 cells/ml
CD8 IL-2 cells/ml
102
101
0
IFNg
IFNg IL-2
HIV- subjects pp65IE-1 response
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CMV responses are effector-dominated
CD4 IFNg
CD8 IFNg
4000
HIV- subjects pp65IE-1 response
2000
Cytokine cells/ml blood
0
CD27 - - - - - -
- - CD28 - - - -
- - - - CD45RA - - - -
- - - -
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Terminal Effector Cell ResponseCorrelates with
Spontaneous IFNg
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IL-2 production is correlated with phenotype
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Results HIV response

• IL-2 production
• Differentiation status

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HIV responses lack CD8 IL-2 cells
Ratio IL-2/IFNg cells
Absolute counts of IL-2 cells
0.0005
105
0.0005
0.5
104
0.4
103
0.3
Ratio of IL-2 to IFNg cells
CD8 IFNg IL-2 cells/ml
102
0.2
101
0.1
0
0.0
HIV- pp65IE-1
HIV pp65IE-1
HIV gagenv
HIV- pp65IE-1
HIV pp65IE-1
HIV gagenv
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CD4 IFNg
CD8 IFNg
HIV- subjects pp65IE-1
HIV subjects pp65IE-1
Cytokine cells/ml blood
HIV subjects gagenv
CD27 - - - - - -
- - CD28 - - - -
- - - - CD45RA - - - -
- - - -
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Phenotypes of total CD4 and CD8 T cell pools
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Relationship of IL-2 and differentiation

• Two major differences between CMV and HIV
  responses
• Ability of CD8 T cells to make IL-2
• Differentiation state of CD8 (and CD4) T cells
• Are these two defects related?
• IL-2 producing CD8 T cell phenotypes might be
  lost in HIV responses
• IL-2 might drive differentiation of effector CD8
  T cells

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Reduced IL-2 from all phenotypes of HIV-specific
CD8 T cells
0.3
HIV- subjects pp65IE-1
0.2
0.1
0.0
0.3
HIV subjects pp65IE-1
0.2
Ratio of IL-2 to IFNg cells
0.1
0.0
0.3
HIV subjects gagenv
0.2
0.1
0.0
CD27 - - - - CD28 - - -
- CD45RA - - - -
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Correlation of IL-2 production and presence of
effector cells (HIV)
CD8 T cells only
All T cells
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Correlation of CD8 IL-2 production and presence
of effector cells (CMV)
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Correlation of CD8 IL-2 production and presence
of effector cells
CD8 T cells only
All T cells
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Addition of IL-2 can increase the proportion of
effector cells during in vitro culture (CMV)
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Model of IL-2 effect on differentiation
Antigen-Experienced
Naïve
CD45RA CD27 CD28
CD45RA CD27- CD28-
CD45RA- CD27 CD28
CD45RA- CD27 CD28-
CMV IL-2
HIV IL-2-

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Additional questions

• Can IL-2 therapy in vivo alter the
  differentiation state of antigen-specific T
  cells?
• To what extent do other cytokines contribute to
  effector T cell differentiation in vivo?

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Other factors that may lead to differentiation
changes in HIV

• Availability of IL-7, IL-15 or other T cell
  survival factors
• Expression of CD70 (CD27L) on HIV-infected T
  cells
• Production of IFNa by dendritic cells

CpG-stimulated
Gated on Lineage-negative, HLA-DR-bright
leukocytes
CD123 PerCP-Cy5.5
Anti-IFNa PE
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Summary

• There are significantly more CD8 IL-2 cells
  responding to CMV compared to HIV.
• CD8 CMV responses are dominated by effector
  cells (CD27-CD28-CD45RA), while CD8 HIV
  responses are dominated by cells of intermediate
  differentiation (CD27CD28-CD45RA-).
• IL-2 production is mostly from non-effector cells
  (CD28).
• The proportion of CD8 IL-2 cells is correlated
  with the presence of CD8 effector cells.
• IL-2 may drive effector cell differentiation in
  vitro.

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Implications

• Lack of IL-2 production by CD8 T cells
  responding to HIV likely results in incomplete
  effector cell generation.
• Immune monitoring of HIV should include
  determination of HIV-specific CD8 IL-2 T cells.

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4-color CD4/CD8/IFNg/IL-2 staining
0.1
1.3
0.7
1.6
12.3
3.8
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Acknowledgements

• Laurel Nomura
• Smita Ghanekar
• Maria Jaimes
• Eugene Veteska
• Perry Haaland

• Brinda Emu
• Rebecca Hoh
• Steve Deeks
• Jeff Martin
• Mike McCune
• Doug Nixon

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