Optimizing Assays to Find Rare Antigen-Specific T cells in Cryopreserved PBMCs

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Optimizing Assays to Find Rare Antigen-Specific T

cells in Cryopreserved PBMCs
Posted by Jemima Escamilla on Fri, May 31, 2013

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Immunomonitoring of T cell based immune responses spans a wide field of therapeutic applications such as
infectious and autoimmune diseases and is particularly important for vaccine research. Regardless of the
therapeutic application, immunomonitoring can be a daunting task due to the variability of methods and protocols
available. There are several commonly used functional assays for the enumeration of antigen specific CD8 + T cells
and there is great variability in the protocols that are used for these assays. Thus, making it increasing difficult to
thoroughly interpret data obtained from multi-center clinical trials and to compare results between laboratories. In
order to address some of the issues associated with immunomonitoring of clinical trials, the Association for
Immunotherapy of Cancer (CIMT) formed a CIMT monitoring panel tasked to standardize protocols for assaying T

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cell antigen immune responses. Thirteen centers from 6 different European countries participated in this study. They
were given the same samples and asked to determine the number of antigen specific T cells and assess their

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antigen specific function using tetramer staining and a functional assay of their choice. Common techniques used
for monitoring antigen induced immune responses included ELISPOT assays, HLA-multimer staining and
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intracellular cytokine staining (ICS).

Pre-tested samples of peripheral blood mononuclear cells (PBMC), synthetic peptides, and PE-conjugated
HLA-tetramers were distributed to each center. Using HLA-typed healthy volunteers, PBMCs were isolated by
Ficoll density gradient separation. Each sample was tested for T cell reactivity against CMV and influenza. All
centers received an HLA-A negative control as well as HLA-A positive samples consisting of a combination of CMV
and influenza reactive PBMCs. The study comprised of 2 phases; Phase I consisted of all centers performing the
assays with their commonly used protocols, and in Phase II each center received optimized protocols based on the
findings from Phase I.

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http://info.sanguinebio.com/pbmc-basics-blog/bid/289781/Optimizing-Assays-to-Find-Rare-Antigen-Specific-T-cells-in-Cryopreserved-PBMCs[6/21/2013 12:23:33 AM]

Optimizing Assays to Find Rare Antigen-Specific T cells in Cryopreserved PBMCs

Cryopreserved PBMCs

For Phase I's tetramer-staining assay, the laboratories could

choose to stain samples with antibodies (Ab) for CD8 + alone,
CD3 + CD8 + , or CD4 + CD8 + and use their preferred Ab clone,
fluorescent dye, and Ab concentration. For the functional
assays synthetic peptides were provided and each group could
choose either the INF- ELISPOT assay, FACS-based
intracellular INF- staining or both with their antigen
concentration of choice ranging from 1-10 g/ml. To reduce
variability in FACS analysis, sample plots were provided as well
as gate settings and quadrants. Tetramer-staining data reported
included; number of viable cells post-thawing, cytometer model,
number of lymphocytes and/or CD8 + cells analyzed. Data was presented as percent of tetramer-positive cells
among CD8 + , CD3 + CD8 + , or CD4 + lymphocytes depending on what antibody cocktail was chosen. For the
functional assays each center reported the type of ELISPOT plates used, reagents and conditions used, and
number cells tested.

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Tetramer results from the Phase I study showed the number CD8 + cells analyzed significantly affected the
sensitivity of tetramer staining. Antigen-specific T cell reactivity when less than 30,000 CD8 + T cells were
counted resulted in only 70% responsiveness detected. In contrast, when more than 30,000 CD8 + cells were
counted, an 89% response was observed.Although, when antigen-specific T cells were present at high frequencies
the number of counted cells did not matter. Interestingly, Ab clone variability, Ab concentration, or cytometer type
did not result in any significant differences. Thus, the main factors affecting antigen-specific T cell reactivity by
tetramer staining is the number of CD8 + cells used. For Phase II it was then recommended at least 1 x106 PBMCs
are used for this assay.

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The majority of groups chose the INF- ELISPOTas their functional assay. Results showed a

Adam Best (1)

large amount of heterogeneity between the centers. Some centers included a resting phase

Andrea Miyahira (25)

after thawing the cells, of 2-20 hours, resulting in 73% positive reactivity (number of spot

antibody (3)

forming cells per seeded PBMC). In contrast, not allowing a resting phase resulted in only

Arijit Bhowmick (2)

detecting 30% of the positive cells. Additionally, intra-center replicate reproducibility was

arthritis (1)

significantly affected by the number of replicates used, where duplicates often failed the

Autoantibodies (1)

Student t test and triplicates were sufficient to reach statistical significance. Addition of allogenic-APCs for binging

Autoimmunity (3)

and presentation of the synthetic peptides was found to have a negative effect on detection response (28% of all

biobank (1)

responses vs. 58%). When looking at the number of cells seeded per well, those with more than 4 x105 PBMC

biomarker research (1)

blood (2)

detected 71% positive samples and those with less than 4 x105 only detected 43%. Granted, when antigen specific

Cancer (2)

T cells were available at high frequencies the number of counted cells did not affect the response rates.

cancer research (3)

Consequently, Phase IIs minimum requirements for the INF- ELISPOT protocol included: (1) triplicates should

CD14+ Monocytes (5)

be performed for each test antigen (2) avoid using allogenic-APCs (3) include a resting phase (4) use over 4 x 10 5
PBMCs per well.
Another interesting finding from this study was that lab experience in performing these assays had no effect on the
performance of the assays compared to labs that had just adopted the techniques. Further highlighting the

CD19+ B Cell (4)

CD4+ T Cell (13)
CD56+ NK cells (2)
CD8+ cell (9)
Cell Isolation (2)
Cell-based therapies (1)

importance of developing standardized protocols for immunomonitoring assays. This study did not however,

Chemotherapy (1)

address specific detection limits for the ELISPOT assays, the variability between ELISPOT plate readers, nor

clinical research (1)

serum source effects on background and specificity. In addition, it was not reported whether live/dead cell stains

Colt Egelston (4)

where included in the tetramer assays and how combinations of these may have had an effect on the sensitivity of

Dendritic cell (5)

the assay.

Flow Cytometry (21)

Gerhard Wingender (3)

Overall, this study identified several factors that should be generally implemented when performing tetramer

IgM (1)

staining and INF- ELISPOT assays with cryopreserved PBMC samples. Furthermore, these protocol modifications

immune therapy (1)

are particularly important when assaying antigen-specific T cell populations present at low frequencies.

Immunology (4)
Immunology Research Methods
(7)
immunotherapy (1)
INF ELISPOT (1)

http://info.sanguinebio.com/pbmc-basics-blog/bid/289781/Optimizing-Assays-to-Find-Rare-Antigen-Specific-T-cells-in-Cryopreserved-PBMCs[6/21/2013 12:23:33 AM]

Optimizing Assays to Find Rare Antigen-Specific T cells in Cryopreserved PBMCs

innate lymphoid cells (1)
jemima escamilla (3)

Reference:

MFI (1)

The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T

Multiple sclerosis (1)

lymphocytes by structural and functional assays. Britten CM, Gouttefangeas C, Welters MJ, Pawelec G, Koch S,

NK cell (2)

Ottensmeier C, Mander A, Walter S, Paschen A, Mller-Berghaus J, Haas I, Mackensen A, Kllgaard T, thor

oncology (2)

Straten P, Schmitt M, Giannopoulos K, Maier R, Veelken H, Bertinetti C, Konur A, Huber C, Stevanovi S, Wlfel T,

PBMC (28)

van der Burg SH. Cancer Immunol Immunother. 2008 Mar;57(3):289-302. Epub 2007 Aug 25.

personalized medicine (1)

protocols (1)
regulatory T cells (1)
research (1)
Rheumatoid Arthritis (1)
standardization (1)

Jemima Escamilla is a senior Ph.D candidate in Molecular and Medical Pharmacology at the UCLA
David Geffen School of Medicine. Her research focus integrates hormonally regulated cancer
progression and cancer immunology.

Tags:CD8+ cell, Flow Cytometry, jemima escamilla, immunotherapy, Cell-based therapies, INF ELISPOT

Comments
Thank you for sharing this information. This is very useful.
Posted @ Saturday, June 01, 2013 11:49 AM by Jacky Woo

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Optimizing Assays to Find Rare Antigen-Specific T cells in Cryopreserved PBMCs

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