Holden T. Maecker, PhD Stanford University Mass Cytometry Assays for Antigen-specific T cells using...
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Holden T. Maecker, PhDStanford University
Mass Cytometry Assays for Antigen-specific T cells using CyTOF
Immunity to chronic infections: HIV, CMV
Both require cellular immunity for protectionVirus-infected cells are targets for CTL killing
Both result in chronic antigen persistenceBut CMV is usually at undetectable levels
CMV does not cause pathology in immunocompetent hosts
What is unique about the CMV signature?
Early & Late Functions of Cellular Immunity
IL-4IL-2
TNFαIFNγ
TCR engagement
Cytokineexpression
Proliferation/Death
Cyto-toxicity
Why multicolor flow cytometry
The concept of a T cell response signature:CD4 + CD8 + Multiple cytokines + memory/effector
markers
Based on the idea that quality as well as quantity of responding T cells is important:
Polyfunctional T cells associated with LTNP (Betts et al.)Central memory T cells assocated with protection from SIV
challenge (Letvin et al.)
Principle of ICS Assays
Antigenic stimulus + brefeldin A
and/or monensin
Incubate 6-10 h
• Fix cells• Permeabilize• Stain
PBMC orwhole blood
Gate on cellsof interest
Magnitude of CMV & HIV responses
CD4+ IFNγ+
CMV HIV
105
104
103
102
101
0
Abso
lute
cells
/ml
CD8+ IFNγ+
CMV HIV
105
104
103
102
101
0
Nomura et al., BMC Immunol 2006
Function of CMV & HIV responses
0.0005
CMV HIV
105
104
103
102
101
0
0.0005
0.0
0.1
0.2
0.3
0.4
CMV HIV
Absolute counts of IL-2+ cells: Ratio IL-2+/IFNγ+ cells:
Nomura et al., BMC Immunol 2006
Phenotype of CMV & HIV responses
CMV
HIV
CD27: + + - - + + - - + + - - + + - -
CD28: + + + + - - - - + + + + - - - -
CD45RA: + - - + - + - + + - - + - + - +
Cyto
kine+
cells
/ml blo
od
0
20002000
9000
0
2000
4000
CD4+ CD8+
HIV-responsive CD8+ T cells lack IL-2 production regardless of phenotype
+ - - + + - -
+ + + - - - -
- - + - + - +
HIVresponse
0.0
0.1
0.2
0.3
Rati
o o
f IL
-2+
to IFN
γ+ c
ells
CD27:+ - - + + - -
CD28:+ + + - - - -
CD45RA: - - + - + - +
0.0
0.1
0.2
0.3 CMVresponse
Hypothesis
IL-2 producing CD8+ T cells may be required to drive terminal effector differentiation
The signature of HIV responses
Magnitude: similar to CMV
Functions: Lack of IFNγ+IL-2+ CD8+ T cells
Phenotype: High fraction of intermediately differentiated CD8+ T cells (CD27+CD28-CD45RA-)
But what about other functions and phenotypes?
T cell markers of interest
Lineage markers: CCR7 CD3 CD4 CD8 CD14 CD16 CD19 CD20 CD25 CD27 CD28 CD33 CD38 CD45RA CD49d CD56 CD57 CD85j CD94 CD127 HLA-DR IgD TCR /g d
Activation markers: CD154 IL-2 IL-4 IL-10 IL-17 IFNg GM-CSF TNFa Granzyme B Perforin CD38 CD69 CD107a PD-1
Mass Cytometry Rationale
Fluorescence cytometry Mass cytometryY Y
• Many more labels (antibodies)• Little or no spillover
CyTOF Principle
Element-LabeledAntibodies
tim
e
atomic mass
Initial gating of CyTOF data
Intact cell gate:
Singlet gate:
Live/dead discrimination:
Lymph vs mono gate:
CyTOF vs. fluorescence
IgD CD4 CD56C
D16
CD
8
CD
27
B cells: T cells: NK cells:
CyT
OF
LSR
II
So what’s wrong with mass cytometry?
Collection speed: ~500 events per second maximum to avoid too many doublets
Cell efficiency: only ~1/3 of injected cells are collected
Sensitivity: no channel as bright as PE in fluorescence cytometryBut multiple markers for each cell subset can partially
overcome this
Destructive: can’t sort cells of interest for downstream applicationsProbably not that important
Overcoming CyTOF speed limitations
Enrichment of activated cells (Axel Schultz, HIMC):
% IFNg+
Pre-enrichment Negative fraction Positive fraction
0.40 % 0.08 % 35 %
Enrichment - - 86x
Loss - ≈ 20 % -
IFNg
CD
40L
Cell-surface barcoding(Henrik Mei, HIMC)
• Small sample loading time savings
• Large savings of staining Abs
• Improved staining consistency
CD
45
Pd
10
5
CD
45
Pd
10
4
CD
45
Pd
11
0
CD
45
Pd
10
6
CD
45
Pr1
41
CD
45
Pd
10
8
Composite sample
CD45 Pr141 CD45 Pd110 CD45 Pd108 CD45 Pd106 CD45 Pd105 CD45 Pd104
CyTOF ICS: CMV pp65 stimulation(Sheena Gupta, HIMC)
Dead CD14 CD3 CD4
CCR7 IFNg TNF GM-CSF
CCR7 IFNg TNF GM-CSF
CD
33
CD
8
CD
45
RO
IL-2
MIP
-1b
IL-1
7
CD
45
RO
IL-2
MIP
-1b
IL-1
7
Basicgates
CD4+
CD8+
Alternative visualization/analysis approaches
SPADE (Qiu et al., 2011):Clustering of events in N dimensionsDisplay of clusters by relatedness in a 2-dimensional “tree”:
CD4 naïve/CMCD4 EM CD8 naïve/CM
CD8 EM/effector
CD45RA
SPADE – CMV-specific cytokine expression
IFNγ TNFα
CD4 CM
CD4 EM
CD8 EM
CD8 CM
CD4 CM
CD4 EM
CD8 EM
CD8 CM
SPADE – CMV-specific cytokine expression
IL-2 GM-CSF
CD4 CM
CD4 EM
CD8 EM
CD8 CM
CD4 CM
CD4 EM
CD8 EM
CD8 CM
Alternative visualization/analysis approaches
Principal components analysis (PCA):No clusteringAll events are shown in a 2- or 3-dimentional space, with
axes that are composite vectors of the actual markers usedIdea is to maximize separation of events
PCA analysis of CMV responseto pp65 vs. IE-1
Alternative visualization/analysis approaches
Sparse clustering (Tyson Holmes):K-means type clustering in N
dimensions, after filtering for relevant dimensions
All events are shown in a 2-dimensional projection, with vectors that show the individual dimensions that were useful in cluster definition
Can also test for significant differences between groups for each cluster
EBV – Solid organ transplant study(Olivia Martinez, Dongxia Lin)
Organ transplant recipients are at risk for EBV disease, including post-transplant lymphoproliferative disorder (PTLD)
Anti-viral prophylaxis is expensive, has side effects, and is not always effective for EBV
Following viral load and adjusting immunosuppression is not idealToo little, too lateEarlier prognosis of risk is needed: T cell response?
unstimulated Lytic proteins mix Latent proteins mix
Example of CD8+ T cell response to EBV peptide mixes
Sparse clustering of EBV-responsive CD8+ T cells
Latent Lytic
Hea
lthy
Tra
nspl
ant
Unstimulated
Sparse clustering of EBV-responsive CD8+ T cells
Conclusions (1)
CMV- and EBV-specific T cell responses can be measured by mass cytometry, and analyzed by:SPADEPrincipal Components Analysis (PCA)Sparse clustering analysis
EBV-specific T cell responses in organ transplant patients may lack polyfunctional cells responding to lytic antigens
The argument for measuring immunocompetence in cancer patients
Tumors are treated with surgery, radiation, and chemotherapy, all of which are immunosuppressive
There is growing interest and success in combining these with immunotherapyBlockade of inhibitory pathways (CTLA-4, PD-1)Augmentation of costimulatory pathways (CD137)Direct vaccination for inducing tumor-specific immunity
Yet we don’t usually check the immunocompetence of patients to respond to immunotherapyCould be prognostic for response generally, or to specific
agents
IL-2 expression (PMA+ionomycin)
PatientsControls
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
TNFa expression (PMA+ionomycin)
PatientsControls
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
IFNg expression (PMA+ionomycin)
PatientsControls
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
IL-17 expression (PMA+ionomycin)
PatientsControls
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
PD-1 expression (unstimulated)
PatientsControls
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
monocytesCD4+ T cells
CD8+ T cells NK cells
B cells
CD27+CD27-
CD45RO+CD45RO-CD49d+CD49d-CD57+CD57-
CD85j+CD85j-CD94+CD94-
CD137+CD137-
CD137L+CD137L-
GranzymeB+GranzymeB-
IFNg+IFNg-IL-2+IL-2-
IL-17+IL-17-IL10+IL10-
MIP1b+MIP1b-
Perforin+Perforin-
Q1: CD69–, CD107a+Q2: CD69+, CD107a+Q3: CD69+, CD107a–Q4: CD69–, CD107a–
Q5: IL-2–, GMCSF+Q6: IL-2+, GMCSF+Q7: IL-2+, GMCSF–Q8: IL-2–, GMCSF–
Q9: GranzymeB–, Perforin+Q10: GranzymeB+,
Perforin+Q11: GranzymeB+, Perforin–Q12: GranzymeB–, Perforin–
Q13: IFNg–, IL10+Q14: IFNg+, IL10+Q15: IFNg+, IL10–Q16: IFNg–, IL10–
Q17: TNF–, IL-17+Q18: TNF+, IL-17+Q19: TNF+, IL-17–Q20: TNF–, IL-17–
Q21: MIP1b–, PD1+Q22: MIP1b+, PD1+Q23: MIP1b+, PD1–Q24: MIP1b–, PD1–
Q25: CD25–, CD127+Q26: CD25+, CD127+Q27: CD25+, CD127–Q28: CD25–, CD127–
Q29: CD45RA–, CCR7+Q30: CD45RA+, CCR7+Q31: CD45RA+, CCR7–Q32: CD45RA–, CCR7–
Q33: CD45RO–, CD45RA+Q34: CD45RO+, CD45RA+Q35: CD45RO+, CD45RA–Q36: CD45RO–, CD45RA–
Q37: CD127–, CCR7+Q38: CD127+, CCR7+Q39: CD127+, CCR7–Q40: CD127–, CCR7–Q41: CD27–, CCR7+Q42: CD27+, CCR7+Q43: CD27+, CCR7–Q44: CD27–, CCR7–
TNF+TNF-
P1 P2 P3 P4 P5 P6 P7 C1 C2 C3
Summary of patients and controls:CD4+ T cells, PMA+ionomycin
patients ctrls
P1 P2 P3 P4 P5 P6 P7 C1 C2 C3
CD8+ T cells, PMA+ionomycin
CD27+CD27-
CD45RO+CD45RO-CD49d+CD49d-CD57+CD57-
CD85j+CD85j-CD94+CD94-
CD137+CD137-
CD137L+CD137L-GMCSF+GMCSF-
GranzymeB+GranzymeB-
IFNg+IFNg-IL-2+IL-2-
IL-17+IL-17-IL10+IL10-
MIP1b+MIP1b-PD1+PD1-
Perforin+Perforin-
Q1: CD69–, CD107a+Q2: CD69+, CD107a+Q3: CD69+, CD107a–Q4: CD69–, CD107a–
Q5: IL-2–, GMCSF+Q6: IL-2+, GMCSF+Q7: IL-2+, GMCSF–Q8: IL-2–, GMCSF–
Q9: GranzymeB–, Perforin+Q10: GranzymeB+, Perforin+Q11: GranzymeB+, Perforin–Q12: GranzymeB–, Perforin–
Q13: IFNg–, IL10+Q14: IFNg+, IL10+Q15: IFNg+, IL10–Q16: IFNg–, IL10–
Q17: TNF–, IL-17+Q18: TNF+, IL-17+Q19: TNF+, IL-17–Q20: TNF–, IL-17–
Q21: MIP1b–, PD1+Q22: MIP1b+, PD1+Q23: MIP1b+, PD1–Q24: MIP1b–, PD1–
Q25: CD25–, CD127+Q26: CD25+, CD127+Q27: CD25+, CD127–Q28: CD25–, CD127–
Q29: CD45RA–, CCR7+Q30: CD45RA+, CCR7+Q31: CD45RA+, CCR7–Q32: CD45RA–, CCR7–
Q33: CD45RO–, CD45RA+Q34: CD45RO+, CD45RA+Q35: CD45RO+, CD45RA–Q36: CD45RO–, CD45RA–
Q37: CD127–, CCR7+Q38: CD127+, CCR7+Q39: CD127+, CCR7–Q40: CD127–, CCR7–Q41: CD27–, CCR7+
Q42: CD27+, CCR7+Q43: CD27+, CCR7–Q44: CD27–, CCR7–
TNF+TNF-
patients ctrls
Conclusions (2)
Mass cytometry (CyTOF) is a powerful platform for both broad immune profiling and detailed tracking of rare (antigen-specific) populations
Cancer patients appear to have more heterogenous immune profiles than healthy controls
Specific immune profiles may be prognostic for responses to immunotherapy
HIMC:• Mike Leipold
• Serena Chang
• Sheena Gupta
• Meena Malipatlolla
• Rosemary Fernandez
• Dongxia Lin
• Xuahai Ji
• Igor Goncharov
• Iris Herschmann
• Ajay Fernandez
• Sanchita Bhattacharya
• Nicole Dalal
• Henrik Mei
• Axel Schultz
• Rohit Gupta
• Janine Sung
• Alaina Puleo
• Blanca Calvillo
• Glenn Dawes
• Yael Rosenberg-Hasson
• Other Stanford:
• Holbrook Kohrt
• Olivia Martinez
• Evan Newell
• Mark Davis
• Sean Bendall
• Garry Nolan
• Tyson Holmes
• BD Biosciences:
• Laurel Nomura
• Maria Jaimes
• Skip Maino
• UCSF:
• Mike McCune
• Doug Nixon
• Steve Deeks
AcknowledgementsThe Human Immune Monitoring Centerat Stanford
Immunology for the People!