The Human Immune Monitoring Center
Protocols
The HIMC is pleased to offer a comprehensive package of standardized immunological assays. These include:
- Quantitation of 50 human cytokines in serum using a Luminex system;
- Enumeration of leukocyte subsets by 8-color immunophenotyping using a custom BD LSRII flow cytometer;
- Functional analysis of leukocyte responses using phosphoepitope flow cytometry, intracellular cytokine staining, and/or proliferation assays;
- RNA extraction with or without stimulation, for RNA microarray analysis (done in collaboration with the Stanford Functional Genomics Center).
In addition, access to other complementary technologies is available through the HIMC, including:
1) Analysis of phosphorylated proteins in cell lysates using the CellBioSciences
Firefly system;
2) Analysis of biomolecular interactions using a ForteBio Octet instrument;
3) Readout of user-processed ELISPOT plates using a CTL reader.
Finally, experimental technologies being tested in the HIMC include:
1) Flow cytometry by time of flight mass spectroscopy (CyTof);
2) Multiplexed tetramer analysis by flow cytometry;
3) Electrochemiluminescence analysis of cytokines using a MesoScaleDiscovery
instrument.
In order to insure sample integrity and cell functionality, the HIMC requires
adherence to strict blood-handling protocols. We prefer to work with the Stanford
General Clinical Research Center (GCRC) for blood processing, for samples
originating at Stanford. With the exception of limited absolute counting assays
done on fresh whole blood, the HIMC will perform its assays on cryopreserved
PBMC, serum frozen at –80C, or stabilized whole blood designed for RNA
extraction (Paxgene tubes).
One of the objectives of the HIMC is to create HIPPA compliant database of immune system information across a wide spectrum of clinical indications. One year after the results of these assays are released to the investigator (or earlier if requested), the data will be released for all registered users of the facility to access. We also plan to analyze large numbers of healthy individuals using these assays and to make this data available to HIMC users.
1. Multiplex cytokine assay (Luminex 200 IS platform)
Secreted cytokines are characteristic of many types of immune response and inflammatory conditions. To measure large numbers of cytokines simultaneously in clinical isolates, we use a Luminex 200 IS System, which is a very flexible analysis platform based on flow cytometry. Our current kits allow multiplexing of 50 human cytokines in a single microplate well, using very small (100 microliter) sample volumes.
Analytes contained in the current human 50-plex assay, with sensitivity in pg/ml
in parentheses, include:
| IFN-beta (30) | FGF-basic (55) | VEGF (56) |
TNFalpha (45) |
IL-2 (19) | RANTES (42) |
| IL-1beta (18) | IL-4 (20) | NGF (38) |
| IL-5 (21) | IL12p40 (33) | IL-18 (57) |
| IL-6 (25) | TGF-beta (29) | IFN-alpha (48) |
| IL-7 (26) | TNF-beta (32) | IL15 (65) |
| IL-8 (27) | LEPTIN (6) | HGF (79) |
| IL-10 (28) | IP-10 (22) | PAI-1 (14) |
| IL12p70 (34) | MIP-1alpha (12) | Resistin (86) |
| IL-13 (35) | ENA-78 (17) | sVCAM-1 (88) |
| IL-17A (36) | MIG (11) | sICAM-1 (73) |
| IFN-gamma (43) | MCP-3 (13) | TGFa (23) |
| GM-CSF (44) | IL-1alpha (62) | sFas Ligand (16) |
| G-CSF (46) | PDGF-BB (37) | M-CSF (90) |
| MIP-1beta (47) | GRO-alpha (61) | LIF (49) |
| MCP-1 (51) | IL-1RA (63) | SCF (7) |
| EOTAXIN (52) | IL-17F (39) |
2. Leukocyte phenotyping by flow cytometry (BD LSR II)
Human blood contains a variety of immunologically important cell types, including conventional B and T lymphocytes, regulatory T cells, natural killer (NK) and NKT
cells, monocytes, and dendritic cells. Within these cell types, subsets can be
identified using additional markers that might be associated with specific effector
functions, proliferative capacity, functional exhaustion, etc. An ideal platform for
analyzing such subsets is flow cytometry, which uses fluorescently labeled
antibodies to specifically identify multiple proteins on individual cells. We have
designed a set of 8-color immunofluorescence assays that are run on a custom
BD LSRII flow cytometer. The current analytes are designed to measure subsets
of specific cell types, as follows:
T cells: CD3, CD4, CD8, CD27, CD45RA, CD161, PD-1
Activated T cells: CD3, CD4, CD8, CD38, Bcl-2, HLA-DR, Ki-67
NK/NKT cells: CD3, CD8, CD16, CD56, CD94, CD314 (NKG2D), HLA-DR
B cells: CD3, CD20, CD24, CD27, CD38, IgD, PD-L1
Regulatory T cells: CD3, CD4, CD8, CD25, CD127, CD161
In addition, we have the ability to measure absolute counts of basic cell subsets
and dendritic cell subsets using the following markers:
T/B/NK/monocytes: CD45, CD3, CD4, CD8, CD14, CD19, CD16, CD56
Dendritic cells: CD45, lineage cocktail, HLA-DR, CD11c, CD123, CD86,
CD16, CD56
3a. Phosphoepitope analysis by flow cytometry (BD LSRII)
Signaling by cytokines or other cell stimulators is mediated by cascades that
involve phosphorylation of specific signaling proteins. Recently, antibodies have
been developed that recognize many of these specifically phosphorylated
proteins, allowing their analysis by flow cytometry. Defects in signaling
cascades, or abnormally amplified signaling, may be associated with specific
disease states, making this an ideal method for interrogating cellular responses.
We routinely use the following individual stimuli for phophoepitope flow
cytometry:
IFNalpha
IFNgamma
IL-6
IL-7
IL-10
IL-21
IL-2
B cell receptor
CD3+CD28
Depending upon the stimulus, we read out the following phosphorylated analytes
and cell types:
On T and B cells: pErk1/2, p38 MAP kinase, Lck
On B cells and monocytes: pStat1, pStat3, pStat5
T/B/NK/monocytes: CD45, CD3, CD4, CD8, CD14, CD19, CD16, CD56
Dendritic cells: CD45, lineage cocktail, HLA-DR, CD11c, CD123, CD86,
CD16, CD56
3b. Intracellular cytokine staining for flow cytometry (BD LSRII)
Another short-term consequence of immune cell activation is the production of
cytokines by responsive cells. The frequency of responsive cells and the types
of cytokines they produce can be useful biomarkers for monitoring immune
responses to vaccines or specific antigens, or to monitor immune function as
related to disease progression. We have developed 8-color flow cytometry
panels for enumerating multiple cytokines on important immune cell subsets,
using the following stimuli:
IFNalpha
Lipopolysaccharide (LPS)
CD3+CD28
Cytomegalovirus (CMV) peptides + cell lysate
Using subsets of these stimulators, we read out the following cytokines and cell
types:
On T cells: IL-2, IFNgamma, TNF, IL-4, IL-10, IL-17
On NK cells: IFNgamma, TNF
On monocytes: IL-1alpha, IL-1beta, IL-12, TNFalpha
3c. Proliferation (BD LSRII)
The ability of T cells to proliferate in response to generalized stimulation is a
good measure of cellular immune function. Using CD3+CD28 to stimulate T cells
through the T cell receptor complex, we measure proliferation through dilution of
the cytophilic dye, CFSE. This is measured by flow cytometry in combination
with markers of cell activation (CD38, HLA-DR) and exhaustion (PD-1).
4. RNA microarray analysis (Agilent or Ilumina platform)
The ability to interrogate genome-wide RNA expression in parallel with protein-based
immunological analyses provides a powerful platform for both additional
biomarker discovery and for confirmation of findings at the protein level. In
collaboration with the Stanford Functional Genomics Center, we prepare RNA for
running RNA expression microarrays on either Agilent or Illumina platforms. We
extract RNA either from stabilized whole blood (using the Paxgene system), or
from isolated PBMC, which can be either stimulated or not. RNA samples are
checked by bioanalyzer and nanodrop to ensure their quality prior to microarray
analysis.
Sample handling
Detailed protocols for sample handling are available when samples are not being routed through the Stanford GCRC for processing of serum and cells.
Data entry
Data entry begins with the assigning of a barcode at blood draw. The barcode will be the only way that the sample is linked to assay results and additional information. The data will be entered to the database using a web based interface which is designed to collect de-identified patient data that is relevant to the data set. The barcode is scanned into the system when the blood arrives and each tube or 96 well plate location is assigned that barcode as the sampes are processed. Authorized personnell will be able to log into the system and track sample progress and also view assay results in a simplified html format. This software will also be able to show statistics of the number of samples collected and processed as well as the amounts and location in the archives.
Sample Results
We are migrating reporting of results to an electronic database format. Existing
Stanford databases such as CytoBank (for flow cytometry) and Stanford
Microarray Database (SMD, for microarray data) allow the archiving of raw and
analyzed data, and communicating that data to investigators in a collaborative
fashion. We are establishing a similar database for reporting of Luminex results.
These systems will eventually be integrated using visualization and mining tools,
which will allow us to mine data across platforms and across studies.
For more information contact:
Holden T. Maecker, PhD
Director, Human Immune Monitoring Center (HIMC)
maecker at stanford dot edu [maecker]
phone: +1.650.723.1671
Stanford School of Medicine
CCSR Building 0125A
269 Campus Drive
Stanford, CA 94305-5166
fax: +1.650.498.6345
ITI Internal