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mizing 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 PBMCsPosted by Jemima Escamillaon Fri, May 31, 2013

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Immunomonitoringof 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 , theAssociation for

Immunotherapy o f Cancer (CIMT)formed a CIMT monitoring panel tasked to standardize protocols for assaying T

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 theirantigen 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

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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2/4



Cryopreserved PBMCs

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knocks out Tregs!

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cytokine levels in serum or pla

Using Application Settings to

Standardize Flow Cytometry

Results Across Experiments a

Instruments

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monitor lymphocyte proliferatio

Identification of Type I InnateLymphoid Cells that functiona

resemble TH1 and NK cells

Going Serum-Free in

Cryopreserving PBMCs: Bette

Immunoassay Performance?

Antigen Cross-Presentation by

Human Dendritic Cell Subsets

Expansion of NK cells from

Human PBMC

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INF ELISPOT (1)

For Phase I'stetramer-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

assayssynthetic 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.

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 frequenciesthe 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 x106PBMCs

are used for this assay.

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

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

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

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

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

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

Student t test and triplicates were sufficient to reach statistical significance. Addition of allogenic-APCs for bingingand presentation of the synthetic peptides was found to have a negative effect on detection response (28% of all

responses vs. 58%). When looking at the number of cells seeded per well, those with more than 4 x105PBMC

detected 71% positive samples and those with less than 4 x10 5only detected 43%. Granted, when antigen specific

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

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

be performed for each test antigen (2)avoid using allogenic-APCs (3)include a resting phase (4)use over 4 x 105

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

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

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

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

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

the assay.

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

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

are particularly important when assaying antigen-specific T cell populations present at low frequencies.
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innate lymphoid cells (1)

jemima escamilla (3)

MFI (1)

Multiple sclerosis (1)

NK cell (2)

oncology (2)

PBMC (28)

personalized medicine (1)

protocols (1)

regulatory T cells (1)

research (1)

Rheumatoid Arthritis (1)

standardization (1)

Reference:

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

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

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

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

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

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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