What to Measure, How to Measure It
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1 Dale and Betty Bumpers Vaccine Research Center National Institute of Allergy and Infectious Diseases National Institutes of Health Monitoring Memory T-cells: What to Measure, How to Measure It Pratip K. Chattopadhyay, Ph.D. Research Fellow, Roederer Laboratory ImmunoTechnology Section Vaccine Research Center, NIAID, NIH
2 Vaccine Development Choices: antigen, formulation, regimen? How to recognize right choice? Reduced disease incidence/severity = long-term follow-up, or examine early correlates of immunity Efficacy Study Infection Protection Immune Response? Disease time weeks years / decades?
3 Early Correlates of T-cell Immunity What to Measure? Infection Efficacy Study Protection Immune Response? Disease Frequency of antigen-specific T-cells Phenotype Function Aidit Avidity
4 Early Correlates of T-cell Immunity How to Measure Frequency of Ag-specific T-cells? Classical methods: in vitro or ex vivo antigen stimulation, followed by: cell proliferation by tritiated thymidine, or measurement of IFN secretion by ELISA or ELISPOT Considerations: Proliferation assays miss terminal effectors, which do not divide but may still produce cytokines. ELISA and ELISPOT assays measure only IFN Not the only cytokine T-cells produce So, by limiting to IFN, are we missing Ag-specific cells?
5 Measuring the Frequency of Ag-specific T-cells Does IFN Mark All Ag-specific T-cells? 13-color Flow Cytometric Analysis, Acute/Early HIV, n = 471 patients TNFa IL CD IFNg CD CD45RO CD4+ CD8+ 7a CD CD Cells stimulated with overlapping peptides from HIV gagb IFNg CCR7
6 IFN Does Not Mark All Ag-specific T-cells IFN Expression among All Responding* CD8+ Cells 12 9 In this setting, 6% of Ag-specific cells would be missed by ELISpot. 6 IFN - IFN + 3 IFNg (Pies) ( ) + - * defined as a cell expressing at least defined as a cell expressing at least one of the functional markers measured, at levels above the unstimulated control.
7 Measuring the Frequency of Ag-specific T-cells Functional Profile of IFN - Cells 1% CD17a+ IL2- TNF - CD17a- IL2- TNF + Most IFN - cells are CD17a+. Measuring both IFN and CD17a marks 8% of Ag-specific T-cells. CD17a IL2 TNF
8 How to Measure Frequency of Ag-specific T-cells? Proliferation & IFN assays may not mark all Ag-specific cells. Other approaches: Intracellular cytokine staining (more later), including both IFN and CD17a. Tetramers
9 Frequency of Ag-specific T-cells by Tetramer Tetramers structural, not functional, identification Less likely to miss Ag-specific T-cells Antigen-Specific Cells Tetramer+ Cytokine+ Disadvantages: One specificity at a time, not easy for CD4+.
10 Tetramer Staining Considerations How do you know staining is real? 1. Rigorously exclude sources of non-specific binding. dead cells, monocytes, B-cells 2. Practice/optimize staining technique until signal/noise ratio is maximized. Trouble signs: Tetramer+ events in CD4+ population Events positive for more than one tetramer. Background events often naive. Requires combining phenotype + ag-specificity. Reviewed in Chattopadhyay, et al., Cytometry, 28.
11 Early Correlates of T-cell Immunity: Phenotype of Ag-specific T-cells Phenotype often suggests biology: CD57+ cells CD25 br cells CCR7+ cells = short telomeres, terminally differentiated = regulatory T-cells = lymph node homing But how do we choose which phenotypes to measure? Literature: Confusing multiple classification schemes for memory cell Confusing, multiple classification schemes for memory cell subsets are they equivalent?
12 T-cell Classification Schema Compared 13-color expt. Markers Stained: CD3 CD4 Subsets are not equivalent! CD8 CD14 Dead cells (PI) CD45RA CD62L CCR7 CD27 CD28 So how complex is the T-cell compartment? How many markers do we need to measure? CD7 (not shown) CD11a (not shown) CD57 (not shown) Gated on CD8+ T-cells.
13 Complexity of T-cell Compartment: 14-color Flow Di it A / ff t h t > 1% f ll! Diversity: Among memory/effector, no phenotype > 1% of cells! Over 9 phenotypically distinct populations using 6 markers.
14 Are Subsets Really Distinct? Though we find remarkable phenotypic diversity, perhaps some are functionally equivalent, and T-cell compartment not as diverse as it seems. Experiment: Sort various T-cell subsets Stimulate with Cytostim (pan T-cell stimulation) Measure secreted cytokines by Cytokine Bead darray.
15 Phenotypic Diversity Reflects Functional Diversity IFN IL1 IFN IL2 TNF IL2 TNF IFN IL2 TNF Total Cytokine Response Sorted Subset Three CD28+ subsets compared. Some are IL1+ Others do not make IL1. Pheno and function don t always correlate. Two central memory-like populations compared. CD62L+ CCR7+ CD27+ CD28+, IL2 predominates. However, in CD62L+ CD28+ cells TNF predominates. Precise phenotypic definitions important.
16 Phenotypic Correlates of T-cell Immunity: Summary Memory T-cell compartment is diverse. measure more markers : find more distinct subsets Subsets distinct, both in terms of quality and quantity of cytokines. We don t know which to measure, so perhaps better to measure as many as possible?
17 More Colors Help Detect Correlates of Vaccine Efficacy % RA A- X X X X Total CD8+ 1 marker 2 markers 3 markers 4 markers A+B+C+D+ A+B+C+D A+B A B C D A % RA % RA- 57+ X X X X X X X X A = cells expressing a certain combination of proteins (e.g., CD45RA- CD57+ CD27+ CD28+). Bulk measurements include irrelevant populations and reduce our power to detect the cells of interest. RA % X X X X Disease or Vax Efficacy
18 More Colors = More Power the rationale underlying multicolor flow cytometry.
19 More Colors = More Problems? 18-color flow cytometry y described in: Perfetto, Chattopadhyay, et al. Nature Reviews Immunology (24) Chattopadhyay, et al. Nature Medicine (26) Standardization issues: Fluorochrome choice Reagent manufacture Panel design Quality control Data analysis Statistical issues <B515-A>: <G78> <G78-A>: TNFa TNFA 1 4 IFN-G <R71> <R71-A>: CCR7 <R66> <R66-A>: IL2 IL <G56-A>: CD154 Multicolor technology becoming more and more accessible. QuickTime and a decompressor are needed to see this picture.
20 Multicolor Leads to Multiplexing: Qdots Help Organic fluorochromes: 13-color maximum. Broad emission spectra. Overlap = poor staining. Quantum Dots: Inorganic nanocrystals Leap to 18-colors. Narrow emission spectra. Less overlap = sensitive staining Series of Quantum Dots illuminated by violet (45nm) laser.
21 Phenotype + Tetramers : An Example of Multiplexing 17-color experiment: Nature Medicine, August 26 Measured 9 T-cell differentiation markers in four Ag-specific T-cell populations. Qdot-conjugated antibodies to CD45RA and CD57. Qdot pmhci multimers (tetramers) for CMV, EBV, HIV gag, and HIV nef. Data from single tube, one patient sample. Maximum data from minimum sample.
22 Tools to Measure Avidity of Ag-specific T-cells Tetramers : TCR binding stabilized by CD8. Tetramers with mutations in CD8 binding region bind TCR with: less affinity it than wild-type (Choi, et al. JI, 23) = high avidity T-cells, sold to reduce background. more affinity than wild-type (Melenhorst, et al. JIM, 28) = low avidity T-cells May be important in cancer immunology Tumor Ags = self Ags? High affinity tetramers pick up Ag-specific cells that are missed by wild-type tetramers. (Blood, in revision)
23 Early Correlates of T-cell Immunity: Functionality of Ag-Specific T-cells Tetramers: one specificity at a time. difficult for Ag-specific CD4+ T-cells Functional assays: multiple l specificities iti (whole protein, overlapping peptides) can assay CD4 and CD8 responses Recent literature relates simultaneous expression of multiple functions (polyfunctionality) y) to immunity. polyfunctional
24 Summary: Monitoring Memory T-cells may require polychromatic (8+ color) flow cytometry: to enhance the power to detect phenotypes of interest, to improve accuracy, to identify polyfunctional cells. However, T-cell monitoring: doesn t always have to be done in one tube, may only require live purification of Ag-specific cells, simplified by markers that correlate with complex function.
25 Simple Purification of Live Ag-specific T-cells Live = can t use ICS, fix/perm is lethal CD8+ Tetramers or CD17a (6-7% of Ag-specific T-cells) CD4+ CD154 upon stimulation, use to isolate Ag-specific cells? Problem: transient expression, rapidly internalized. Solution: CD154 in culture during stim + monensin (Chattopadhyay, et al. Nature Medicine, 25.)
26 Kinetics of CD154 Expression Time point = 5 1 Time point = 1 hours 5 1 Time point = 2 5 Time point = 3 hours 1 2 hours 3 hours d4 <Cblue-A>: c d4 <Cblue-A>: c d4 <Cblue-A>: c d4 <Cblue-A>: c % %.3% 2.5% <PE-A>: cd154 keepers Event 1Count: Time point = 4 hours 4 hours <PE-A>: cd154 keepers 1 Event 1Count: Time point = 5 hours <PE-A>: cd154 keepers 2 Event 1Count: Time point = 6 hours <PE-A>: cd154 keepers 3 Event 1Count: Time point = 12 hours 5 hours 6 hours 8 hours <Cblue-A>: cd d <Cblue-A>: cd d <Cblue-A>: cd d <Cblue-A>: cd d % 6.3% 14% 16% 21% <PE-A>: cd154 keepers 4 Event Count: Time point = 18 hours <Cblue-A>: cd4 CD4 12 hours <PE-A>: cd154 keepers 6 Event Count: hours Time point = 24 hours keepers 18 Event Count: <PE-A>: cd154 CD <Cblue-A>: cd % 23% keepers 24 Event Count: <PE-A>: cd <PE-A>: cd154 keepers 8 Event Count: <PE-A>: cd154 keepers 12 Event Count: Hours
27 Cytokine Expressing Cells Are CD <FIT TC-A>: IFN FNG <PE E-A>: CD % % % 91.6% <Ax68-A>: IL2 IL <Cy7PE-A>: TNF TNFA % of IL2+ or IFN + cells express CD % of these cells also express TNF. Most cytokine+ cells express CD154.
28 Not All CD154+ Cells Are IFN IL2, or TNFa+ TNF Negative TNF and IL2 and IFN and negative CD4 T IL2 Negative and 5.69 IFN Negative Boolean Gating CD154 Assay identifies responding cells that make IL4, IL5, and IL1 thirty hours later.
29 Isolation of Live, Highly Cytotoxic Cells CD17a = not specific for cytotoxic activity. Any stimulated CD8+ cell will degranulate, regardless of endosomal content. Cytotoxic enzymes (CEs): granzyme A, granzyme B, perforin CE combination matters: knockouts of each more susceptible to infection than wildtype. Immunity may require cooperative action. Can we identify cells with all three CEs, without fix/perm?
30 Isolation of Live, Highly Cytotoxic Cells CD57 bright marks grza+ grzb+ perforin high cells. Means to identify these cells without fix/perm, and only one marker. CD57 delineates function in a way common classification schemes don t. Chattopadhyay, et al. Journal Leukocyte Biology, in press.
31 Summary Many ways to monitor human memory T-cells, each with special considerations. To quantify Ag-specific cells: IFN alone is not enough! Tetramers least likely to miss responses but limited specificities, not easy for CD4 Functional assays biologically relevant, assay multiple epitopes which function? polychromatic flow cytometry Live cell assays for Ag-specific cells CD4+ : CD154 assay CD8+ : CD17a assay
32 Summary Is quantity important, or does cell quality matter? Phenotypic analysis can be combined with tetramer or functional assays, and checks quality of tetramer staining. Note that memory cell compartment very diverse, cells identified with one marker set not equivalent to others. Functional analysis polyfunctional cells imp. in many successful immune responses. live cell purification: highly cytotoxic cells expressing CD57. Avidity another potential way of assessing quality tools emerging, relevance not fully known yet.
33 Acknowledgements David Price Joseph Melenhorst, AJ Barrett Mario Roederer Steve Perfetto Joanne Yu Tess Brodie Yolanda Mahnke Carl-Magnus Hogerkorp Diane Bolton Enrico Lugli Kaimei Song Steve De Rosa Mike Betts Rick Koup Danny Douek Bob Seder
34
35 Highly Cytotoxic Cells Found in All Subsets N CM CM Naïve Central Mem Memory Effector E CM E CM M A- B- P- A+ B- P- A+ B+ P- A+ B+ P+ grza+ grzb+ perforin high found in many memory subsets. CD57 delineates function in a way common classification schemes don t. With only one marker! Not seen before polychromatic flow.
36 CD154 : Marker for CD4+ T-cell Responses? Only expressed upon stimulation, restricted to CD Fresh PBMC Cblue-A> >: CD4 1 4 CD4 < % <PE-A>: CD154 CD hours SEB stimulation Surface staining CD3, CD4, CD8, CD154 Gated on CD3+ lymphocytes. H i ll l l CD4+ ll d IFN However, in parallel sample, more CD4+ cells expressed IFN So, surface staining cannot identify all Ag-responsive CD4+ s.
37 Improving CD154 Measurements Problem: Transient expression. Surface CD154 6 hours Solution: Stain with CD154 PE during stimulation. Detect CD154 recycled from cell surface. Use monensin. Once internalized, complex may degrade in acidic endosome. Monensin prevents endosomal acidification. So, it s added d to prevent PE degradation d during long staining. i
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