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    PBMC Poster

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    米許
    Thawed PBMC displayed > 90% viability when tested for peptide or protein antigen-induced T cell recall responses in cytokine ELISPOT assays. Ready access to such PBMC should facilitate human immunological research and assay standardization within laboratories, and between different laboratories.

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    PBMC Poster

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    米許
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    Thawed PBMC displayed > 90% viability when tested for peptide or protein antigen-induced T cell recall responses in cytokine ELISPOT assays. Ready access to such PBMC should facilitate human immunological research and assay standardization within laboratories, and between different laboratories.

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    Thawed PBMC displayed > 90% viability when tested for peptide or protein antigen-induced T cell recall responses in cytokine ELISPOT assays. Ready access to such PBMC should facilitate human immunological research and assay standardization within laboratories, and between different laboratories.

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    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    70 views1 page

    PBMC Poster

    Uploaded by

    米許
    Thawed PBMC displayed > 90% viability when tested for peptide or protein antigen-induced T cell recall responses in cytokine ELISPOT assays. Ready access to such PBMC should facilitate human immunological research and assay standardization within laboratories, and between different laboratories.

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    HLA-Typed PBMC Samples with Established Antigen/Peptide Reactivity for Accelerating and Standardizing Human Immunological Research

    WJ Zhang, C Shive, N Sigmund, OS Targoni, PV Lehmann Cellular Technology Ltd, Cleveland, OH, USA
    Introduction
    We have developed a protocol to cryopreserve human peripheral blood mononuclear cells (PBMC) while maintaining full functionality. The thawed PBMC displayed > 90% viability, and when tested for peptide or protein antigen-induced T cell recall responses in cytokine ELISPOT assays, the frequencies and per-cell cytokine productivities of the thawed cells approximated 100% of the fresh PBMC. Since serum is a highly variable reagent that affects the results, we also developed serum free freezing and testing media (CTL-Cryo, CTL-Test) towards standardization. Based on these developments, we are building an immune-characterized PBMC library of HLA-typed healthy human donors intended as positive and negative controls for T cell monitoring in ELISPOT, ELISA, cytokine bead array, tetramer/pentamer, and cytokine capture assays. Ready access to such PBMC should facilitate human immunological research and assay standardization within laboratories, and between different laboratories.
    140 120 100

    CD4CD8-

    80 60 40 20 0

    Medium Medium

    FluFlu Mat1 58 58 Mat1

    HCMV pp65 HCMV pp65 495 495

    Candida Candida

    Mumps Mumps

    Fig 4. Defining recall antigen-induced responses as CD4 or CD8 cell mediated. PBMC were depleted of CD4 (blue) or CD8 (red) cells using magnetic beads, and the cell populations obtained for a donor were tested in an IFN- ELISPOT assay for the recall response to the antigens specified. The results show that CD8 cell depletion entirely abrogates the recall response to Flu Mat1 58 and the HCMV pp65 495 peptide but CD4 cell depletion does not affect the recall response identifying the cytokine-producing cells as CD8 cells. Candida and Mumps elicited recall responses by CD4 cells.

    # of spots

    Donor 1 Antigen Media HLA restriction A2, A3 B7,B44, Bw4, Bw6, C7 1 0 251 9.17 A1 A1 A2 A2 A2 A3 A3 A3 A11 A11 A24 AA68 B0702 B7 B8 B8 B8 B8 B27 B27 B35 B44 B44 1 1 1.3 0.58 62.7 9.07 21.7 7.21 0 0 9.7 2.08 4.3 2.08 2.3 1.15 7.7 4.16 0.3 0.58 1.7 1.15 36.7 5.51 3 1 48.3 14.64 2.3 2.31 9 13.86 0.7 1.15 1 1 0.7 0.58 1 1 2 1 62.7 12.58 1 0 280 7.55 66 14 33.3 19.66 46 11.53 30 13.11

    Donor 2 A2, B18, B51, Bw4, Bw6, C2 0.3 0.58 352.7 13.32 0.3 0.58 0.7 0.58 6 1.53 53 11.37 42 2.94 26 4.58 3.7 3.79 8.7 3.06 0.3 0.58 0.3 0.58 0.3 0.58 2 1 3.3 0.58 8.3 2.52 0.3 0.58 1.3 1.53 2 1 1 1 0.7 1.15 19.7 4.62 0 0 0.3 0.58 7 1 78 11.93 77.3 26.1 6.3 9.29 7.7 4.73 3 1.73

    Donor 3 A2 A11 B7,B35, Bw6, C4, C7 0 0 248.6 58.9 0.3 0.58 0.7 0.58 9.5 3.51 12.8 5.12 0.7 1.15 0.7 1.15 1.3 0.58 0 0 3 1 0.7 0.58 0.3 0.58 12.3 3.51 2.3 3.21 18 3.21 1.3 1.53 0.7 0.58 0.7 0.58 0.3 0.58 0 0 0.7 1.15 0 0 2.7 1.53 0.7 0.58 18.3 2.52 104.7 41.15 1.3 0.58 134.8 12.12 11.3 2.31

    Results
    Fresh PBMC Donor A Test antigen Media Mumps EBV BMLF1 259 Flu Mat1 58 B Media Flu Mat1 58 EBV EBNA3c 281 HCMV pp65 C Media Mumps EBV BMLF1 259 Flu NP 91 Mean 1.00 66.00 73.30 14.00 0.30 19.70 77.70 7.00 0.90 127.00 20.30 18.70 SD 0.00 14.00 9.07 7.21 0.50 10.02 12.58 1.00 0.78 11.15 3.51 3.51 Frozen PBMC Mean 0.30 66.70 62.70 18.30 0.00 16.50 62.70 6.50 0.70 127.30 15.00 18.50 SD 0.50 14.99 11.59 3.51 0.00 9.40 12.01 2.52 0.58 9.00 2.16 4.80 P value 0.07 0.96 0.28 0.41 0.36 0.71 0.21 0.77 0.74 0.97 0.09 0.96

    CEF pool Flu PB 591

    Table1. No statistically significant difference between the IFN- recall response of fresh and cryopreserved PBMC. Freshly isolated PBMC were tested in 24h IFN- ELISPOT assays at 200,000 cells per well, directly ex vivo (Fresh PBMC). The test antigens were 23 individual peptides from a library of 23 MHC-Class I restricted CEF peptides, in addition to the protein antigens Candida, Tetanus, Mumps, Dust Mite, and PPD. After the fresh PBMC had been plated for these assays, the remaining PBMC were cryopreserved in CTL-Cryo serum free freezing medium according to CTL protocols (Frozen PBMC). These cells were thawed, and were retested under identical conditions as the fresh PBMC, using the serum free testing medium CTLTest. Representative data are shown as the mean number of IFN- spots induced by the specified antigens for the fresh and the frozen PBMC of 3 donors. The P values on the right show that there is no statistical significant difference between the performance of fresh and frozen PBMC.

    Flu NP 44 EBV BMLF1 259 Flu Mat1 58 HCMV pp65 495 Flu NP 265 EBV BRLF1 148 EBV EBNA3a 603 EBV EBNA3b 416 EBV BRLF1 134 EBV BRLF1 28 Flu NP 91 HCMV pp65 417 EBV EBNA3a 379 EBV EBNA3a 158 EBV EBNA3a 325 EBV BZLF1 190 FLU NP 380 EBV EBNA3c 258 FLU NP 383 EBV EBNA3a 458 EBV EBNA3c 281 HCMV pp65 Candida

    Panel A
    Subject ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 % Viable 95.3 91.4 91.3 91.5 95.3 95.0 93.2 91.3 95.0 93.3 94.6 87.0 89.8 86.7 92.1 92.6 95.4 95.6 % Recovery 80.8 80.6 87.1 92.7 98.3 82.6 94.8 80.5 99.6 94.7 96.2 97.2

    Panel B Viable Dead

    (95.3%) (4.7%)

    Mumps Dust Mite Mix PPD Tetanus

    Fig 5. Immune characterized cryopreserved PBMC library. The PBMC of each donor have been characterized for reactivity to a panel of 23 individual peptides (common viral Class I-restricted determinants) and 5 protein recall antigens, recognized by CD8 and CD4 cells, respectively. Up to 1,500 vials of each of the characterized samples have been cryopreserved. To date, samples from 21 donors have been frozen and characterized, continuing at the rate of 2 new donors per week.

    Panel C

    100.0 80.0

    Percent

    99.4 80.1 84.9 92.7 93.9 99.5

    Implications:
    - Ready access to PBMC: no need for IRB. - Experiments can be designed online based on established HLA-types and antigen reactivity. - Sizable donor pool facilitates screening for new reactivities and markers. - Unlimited cell numbers available for experimentation. - Continuous access to PBMC of the same donor and of the same bleed permit rapid progress with controlled experimentation. - Experiments can be readily repeated and extended, e.g. if reviewers ask for additional experiments. - Ability to share the same PBMC with colleagues anywhere helps collaborative work. - Assay comparisons and validations are facilitated by access to precharacterized reference samples. - Availability of positive and negative control samples, e.g., for clinical trials.

    60.0 40.0 20.0 0.0

    Viability

    Recovery

    Fig 2. Viability and recovery rate of cryopreserved PBMC. Cryopreserved PBMC of 18 donors were thawed according to CTL protocols. (A) The viability of the cells (number of viable cells/total number of cells x 100) was established by viable-dead cell staining using the Guava platform. Also shown are the recovery rates (cell number after thawing /number of cells frozen x 100). (B) A representative plot is shown. (C) The mean and standard deviation of viability and recovery rates for all 18 donors are shown.

    50 45 40 35 30 25 20 15 10 5 0 -2.80

    Panel A

    80 70 60
    Fresh Frozen

    Panel B

    50 40 30 20 10 0

    -2.60

    -2.40

    -2.20

    -2.00

    -1.80

    -1.60

    -1.40

    -1.20

    -1.00

    -3.00

    -2.80

    -2.60

    -2.40

    -2.20

    -2.00

    -1.80

    -1.60

    -1.40

    Fig 3. Cryopreservation does not impair the cytokine productivity of individual CD4 or CD8 T cells. In cytokine ELISPOT assays, the spot size reflects the cytokine productivity of individual cells. We used an ImmunoSpot Series 3B Analyzer for the morphometric analysis of IFN- spots induced by antigens that trigger CD4 (Panel A) cells or CD8 (Panel B) cells. The spot size distribution for each category is shown for fresh (green) and frozen (blue) cells.

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