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Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy.

Kammula US, Serrano OK - J Transl Med (2008)

Bottom Line: In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients.In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells.This screening was combined with early limiting dilution cloning to rapidly isolate gp100154-162 reactive CD8+ T cell clones.

View Article: PubMed Central - HTML - PubMed

Affiliation: Surgery Branch, National Cancer Institute, Bethesda, MD, USA. udai_kammula@nih.gov

ABSTRACT

Background: The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients.

Methods: We developed a novel in vitro T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay) as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients.

Results: In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100154-162 reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (approximately 1010 cells).

Conclusion: This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.

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Related in: MedlinePlus

The magnitude of qPCR reactivity prospectively identifies selected wells enriched for functionally active antigen specific T cells. Day 6 gp100209–217 sensitized microcultures (n = 24) from patients 1 (A.) and 3 (B.) were evaluated in parallel using qPCR and ELISA assays. Significant well reactivity was detected with the qPCR assay, but not with ELISA. Microcultures with the highest and lowest qPCR SIs were selected (see arrow) for rapid expansion. On day 14, the phenotype of the expanded cultures was assessed by staining with gp100209–217 peptide/HLA-A*0201 tetramers and anti-CD8 antibody and analysis by flow cytometric analysis. Dot plots are shown for propidium iodide–negative gated cells. Values in FACS dot plots correspond to the percentage of total CD8+ T cells that are tetramer-positive calculated as the number of CD8+ tetramer+ cells divided by the total number of CD8+ T cells minus the CD8- tetramer+ background × 100. Functional reactivity of the expanded cultures was assessed by co-culture with peptide pulsed T2 cells and supernatant analysis by standard ELISA for IFN-γ protein at 24 hrs. ELISA data represents the average of replicate co-culture wells. (O) represents the SI for each microwell. Shaded area represents range of non-specific reactivity (SI = 0.5–2.0). (*), not detectable.
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Figure 3: The magnitude of qPCR reactivity prospectively identifies selected wells enriched for functionally active antigen specific T cells. Day 6 gp100209–217 sensitized microcultures (n = 24) from patients 1 (A.) and 3 (B.) were evaluated in parallel using qPCR and ELISA assays. Significant well reactivity was detected with the qPCR assay, but not with ELISA. Microcultures with the highest and lowest qPCR SIs were selected (see arrow) for rapid expansion. On day 14, the phenotype of the expanded cultures was assessed by staining with gp100209–217 peptide/HLA-A*0201 tetramers and anti-CD8 antibody and analysis by flow cytometric analysis. Dot plots are shown for propidium iodide–negative gated cells. Values in FACS dot plots correspond to the percentage of total CD8+ T cells that are tetramer-positive calculated as the number of CD8+ tetramer+ cells divided by the total number of CD8+ T cells minus the CD8- tetramer+ background × 100. Functional reactivity of the expanded cultures was assessed by co-culture with peptide pulsed T2 cells and supernatant analysis by standard ELISA for IFN-γ protein at 24 hrs. ELISA data represents the average of replicate co-culture wells. (O) represents the SI for each microwell. Shaded area represents range of non-specific reactivity (SI = 0.5–2.0). (*), not detectable.

Mentions: To determine whether the immune reactivity identified at day 6 by the qPCR assay could also be detected by ELISA, gp100209–217 sensitized microcultures from patients 1 and 3 were evaluated using both assays with an equivalent number of sampled PBMC (~100,000 cells) from each of the replicate wells (Figure 3A and 3B). ELISA evaluation did not identify any wells from either patient with reactivity above background. In contrast the qPCR assay performed on the same wells demonstrated multiple cultures with detectable peptide reactivity. To confirm that the qPCR reactivity in these early cultures independently correlated with the presence of gp100209–217 specific T cells, the microcultures with the highest and lowest SIs were rapidly expanded with anti-CD3, allogenic feeder cells, and IL-2 over 1 week and evaluated for the presence and activity of gp100209–217 reactive CD8+ T cells (Figure 3A and 3B). By day 14, the expanded cultures from the wells with the high SI (Patient 1, SI = 11.1 and 12.4; Patient 3, SI = 3.3) demonstrated a distinct population of antigen specific CD8+ T cells when stained with the gp100209–217 tetramer (3–5% of CD8+ cells). When samples of these expanded cultures were tested for functional recognition of T2 cells pulsed with the gp100209–217 peptide, they released significant amounts of interferon-γ protein that could now easily be detected by ELISA. In contrast, the expanded cultures from the low SI wells (Patient 1, SI = 1.1; Patient 3, SI = 0.8) had neither discernable tetramer positive cells nor functional activity against peptide pulsed targets. We concluded that the qPCR assay could be used at an early time point to stratify the epitope reactivity of short term sensitized PBMC microcultures to prospectively identify selected wells enriched for functionally active antigen specific T cells and to identify wells with no evidence of reactivity.


Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy.

Kammula US, Serrano OK - J Transl Med (2008)

The magnitude of qPCR reactivity prospectively identifies selected wells enriched for functionally active antigen specific T cells. Day 6 gp100209–217 sensitized microcultures (n = 24) from patients 1 (A.) and 3 (B.) were evaluated in parallel using qPCR and ELISA assays. Significant well reactivity was detected with the qPCR assay, but not with ELISA. Microcultures with the highest and lowest qPCR SIs were selected (see arrow) for rapid expansion. On day 14, the phenotype of the expanded cultures was assessed by staining with gp100209–217 peptide/HLA-A*0201 tetramers and anti-CD8 antibody and analysis by flow cytometric analysis. Dot plots are shown for propidium iodide–negative gated cells. Values in FACS dot plots correspond to the percentage of total CD8+ T cells that are tetramer-positive calculated as the number of CD8+ tetramer+ cells divided by the total number of CD8+ T cells minus the CD8- tetramer+ background × 100. Functional reactivity of the expanded cultures was assessed by co-culture with peptide pulsed T2 cells and supernatant analysis by standard ELISA for IFN-γ protein at 24 hrs. ELISA data represents the average of replicate co-culture wells. (O) represents the SI for each microwell. Shaded area represents range of non-specific reactivity (SI = 0.5–2.0). (*), not detectable.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: The magnitude of qPCR reactivity prospectively identifies selected wells enriched for functionally active antigen specific T cells. Day 6 gp100209–217 sensitized microcultures (n = 24) from patients 1 (A.) and 3 (B.) were evaluated in parallel using qPCR and ELISA assays. Significant well reactivity was detected with the qPCR assay, but not with ELISA. Microcultures with the highest and lowest qPCR SIs were selected (see arrow) for rapid expansion. On day 14, the phenotype of the expanded cultures was assessed by staining with gp100209–217 peptide/HLA-A*0201 tetramers and anti-CD8 antibody and analysis by flow cytometric analysis. Dot plots are shown for propidium iodide–negative gated cells. Values in FACS dot plots correspond to the percentage of total CD8+ T cells that are tetramer-positive calculated as the number of CD8+ tetramer+ cells divided by the total number of CD8+ T cells minus the CD8- tetramer+ background × 100. Functional reactivity of the expanded cultures was assessed by co-culture with peptide pulsed T2 cells and supernatant analysis by standard ELISA for IFN-γ protein at 24 hrs. ELISA data represents the average of replicate co-culture wells. (O) represents the SI for each microwell. Shaded area represents range of non-specific reactivity (SI = 0.5–2.0). (*), not detectable.
Mentions: To determine whether the immune reactivity identified at day 6 by the qPCR assay could also be detected by ELISA, gp100209–217 sensitized microcultures from patients 1 and 3 were evaluated using both assays with an equivalent number of sampled PBMC (~100,000 cells) from each of the replicate wells (Figure 3A and 3B). ELISA evaluation did not identify any wells from either patient with reactivity above background. In contrast the qPCR assay performed on the same wells demonstrated multiple cultures with detectable peptide reactivity. To confirm that the qPCR reactivity in these early cultures independently correlated with the presence of gp100209–217 specific T cells, the microcultures with the highest and lowest SIs were rapidly expanded with anti-CD3, allogenic feeder cells, and IL-2 over 1 week and evaluated for the presence and activity of gp100209–217 reactive CD8+ T cells (Figure 3A and 3B). By day 14, the expanded cultures from the wells with the high SI (Patient 1, SI = 11.1 and 12.4; Patient 3, SI = 3.3) demonstrated a distinct population of antigen specific CD8+ T cells when stained with the gp100209–217 tetramer (3–5% of CD8+ cells). When samples of these expanded cultures were tested for functional recognition of T2 cells pulsed with the gp100209–217 peptide, they released significant amounts of interferon-γ protein that could now easily be detected by ELISA. In contrast, the expanded cultures from the low SI wells (Patient 1, SI = 1.1; Patient 3, SI = 0.8) had neither discernable tetramer positive cells nor functional activity against peptide pulsed targets. We concluded that the qPCR assay could be used at an early time point to stratify the epitope reactivity of short term sensitized PBMC microcultures to prospectively identify selected wells enriched for functionally active antigen specific T cells and to identify wells with no evidence of reactivity.

Bottom Line: In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients.In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells.This screening was combined with early limiting dilution cloning to rapidly isolate gp100154-162 reactive CD8+ T cell clones.

View Article: PubMed Central - HTML - PubMed

Affiliation: Surgery Branch, National Cancer Institute, Bethesda, MD, USA. udai_kammula@nih.gov

ABSTRACT

Background: The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients.

Methods: We developed a novel in vitro T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay) as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients.

Results: In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100154-162 reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (approximately 1010 cells).

Conclusion: This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.

Show MeSH
Related in: MedlinePlus