Limits...
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.

Show MeSH

Related in: MedlinePlus

Rapid cloning of gp100154–162 specific CD8+ T cells from peripheral blood. (A.) On Day 0, PBMC from four HLA-A2+ melanoma patients underwent staining with gp100154–162 peptide/HLA-A*0201 tetramers and anti-CD8 APC to determine natural precursor frequency. None of the patients demonstrated a significant population of tetramer positive CD8+ T cells by FACS. PBMC from each patient were plated in replicate microwells (n = 96) containing ~300,000 cells and sensitized for 10 days with 1 μM of gp100154–162 peptide in the presence of IL-2 (90 IU/ml). (B.) On day 10, a sample from every microculture was screened using the qPCR assay for specific recognition of the gp100154–162 peptide versus the HIVpol peptide. The wells with the highest SI reactivity (denoted by **) were selected for limiting dilution cloning. After approximately 2 weeks, growth positive wells were functional screened for their ability to lyse peptide pulsed T2 cells. Selected T cell clones were expanded and underwent FACS analysis (C.) between days 25 and 34 to reveal highly enriched (99%) populations of gp100154–162 tetramer positive CD8+ T cells. Values in FACS dot plots correspond to the percentage of total CD8 + T cells that are tetramer-positive. (O) represents the SI for each microwell. Shaded area represents range of non-specific reactivity (SI = 0.5–2.0).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2577625&req=5

Figure 5: Rapid cloning of gp100154–162 specific CD8+ T cells from peripheral blood. (A.) On Day 0, PBMC from four HLA-A2+ melanoma patients underwent staining with gp100154–162 peptide/HLA-A*0201 tetramers and anti-CD8 APC to determine natural precursor frequency. None of the patients demonstrated a significant population of tetramer positive CD8+ T cells by FACS. PBMC from each patient were plated in replicate microwells (n = 96) containing ~300,000 cells and sensitized for 10 days with 1 μM of gp100154–162 peptide in the presence of IL-2 (90 IU/ml). (B.) On day 10, a sample from every microculture was screened using the qPCR assay for specific recognition of the gp100154–162 peptide versus the HIVpol peptide. The wells with the highest SI reactivity (denoted by **) were selected for limiting dilution cloning. After approximately 2 weeks, growth positive wells were functional screened for their ability to lyse peptide pulsed T2 cells. Selected T cell clones were expanded and underwent FACS analysis (C.) between days 25 and 34 to reveal highly enriched (99%) populations of gp100154–162 tetramer positive CD8+ T cells. Values in FACS dot plots correspond to the percentage of total CD8 + T cells that are tetramer-positive. (O) represents the SI for each microwell. Shaded area represents range of non-specific reactivity (SI = 0.5–2.0).

Mentions: We next sought to incorporate the qPCR functional assay into a novel strategy for high throughput isolation of low frequency antigen specific CD8+ T cell clones from bulk PBMC for use in adoptive immunotherapy trials. We initially aimed to isolate CD8+ T cell clones reactive against the gp100154–162 epitope given their extremely low natural frequency in peripheral blood, the difficulty in prospectively isolating these T cells, and the limited published reports of T cell clones reactive against this epitope [7]. The basic isolation strategy is summarized in Figure 4. PBMC from an HLA-A2+ patient with metastatic melanoma are used to establish 96 independent microcultures which are sensitized for 10 days with 1 uM of gp100154–162 in the presence of IL-2 (90 IU/ml). On day 10, a sample from every microculture is screened using the qPCR assay for specific recognition of the gp100154–162 peptide versus the HIVpol peptide. The SI reactivities for the 96 wells are stratified by their magnitude and the most reactive microcultures are selected for the next step, limiting dilution cloning. After approximately 2 weeks, growth positive wells are screened for their ability to lyse peptide pulsed T2 cells and melanoma tumor lines. Selected T cell clones are then expanded in the final step for further analysis and potential use in adoptive immunotherapy trials. This strategy was applied to PBMC from four melanoma patients (patients 2, 5, 6, and 7) (Figure 5). A sample of the bulk PBMC from each patient, prior to any in vitro manipulation underwent staining with the gp100154–162 tetramer to determine natural precursor frequency. None of the patients demonstrated a significant population of tetramer positive CD8+ T cells by FACS on day 0 (Figure 5A). After 10 days of sensitization, the 96 independent microcultures for each patient were screened for peptide reactivity using the qPCR assay (Figure 5B). The stratified results for Patients 2, 5, and 6 demonstrated that only 7%, 12%, and 8% of the wells had a SI ≥ 2, respectively; 1%, 3%, and 1% of the wells had a SI ≥ 10, respectively; and the remaining wells had no detectable peptide reactivity (SI < 2). In contrast, for Patient 7, 92% of the wells had a SI ≥ 2 and 60% of the wells had a SI ≥ 10. The highest reactive microcultures from patients 2, 5, 6, and 7 (qPCR SI = 45, 635, 23, and 78, respectively) were selected for limiting dilution cloning. The frequencies of growth positive clones with lytic ability against peptide pulsed targets were 0.2%, 28%, 0.1%, and 2.3% for the respective patients, which directly correlated with the qPCR SI (r2 = 0.99, p < 0.0001). These selected clones were expanded and underwent FACS analysis between days 25 and 34 to reveal highly enriched populations (99%) of gp100154–162 tetramer positive CD8+ T cells (Figure 5C). Further, the derived populations were confirmed to be clonal by the sequencing of a single T cell receptor Vα and Vβ chain for each patient (Table 1). The functional avidity of these isolated T cell clones was high, as measured by their ability to recognize 10-10 to 10-11 M of gp100154–162 peptide pulsed onto T2 cells and HLA A2+/gp100+ melanoma tumor lines in vitro (Table 2). The phenotype of these cells was assessed by cell surface FACS for CD27, CD28, CD45RO, CD45RA, CD62L, and CD25 (Figure 6). The gp100154–162 tetramer positive cells from patients 2, 5, and 7 all were uniformly CD45RO+ and CD62L-, consistent with an effector memory phenotype. However, unlike typical antigen experienced T cells, there was persistent variable expression of CD45RA (19–96%). In addition all of the isolated clones continued to have significant expression of the costimulatory molecule CD27 (90–99%). This phenotype differed from the terminally differentiated TIL derived MART27–35 specific clone, JKF6, which had no significant expression of CD27. Since initiating these isolation studies we have successfully cloned gp100154–162 CD8+ T cells in 6 of 8 patients (75%). In pilot clinical scale expansions, these clones demonstrated between 850–1000 fold expansions in cell numbers over the initial 14 days after a single rapid expansion in flasks. A second serial expansion of these clones resulted in an additional 400–600 fold expansion over the ensuing week. Thus, we found that with two consecutive rapid expansions ~1010 cells could be generated for potential clinical adoptive transfer from each starting clone isolated.


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)

Rapid cloning of gp100154–162 specific CD8+ T cells from peripheral blood. (A.) On Day 0, PBMC from four HLA-A2+ melanoma patients underwent staining with gp100154–162 peptide/HLA-A*0201 tetramers and anti-CD8 APC to determine natural precursor frequency. None of the patients demonstrated a significant population of tetramer positive CD8+ T cells by FACS. PBMC from each patient were plated in replicate microwells (n = 96) containing ~300,000 cells and sensitized for 10 days with 1 μM of gp100154–162 peptide in the presence of IL-2 (90 IU/ml). (B.) On day 10, a sample from every microculture was screened using the qPCR assay for specific recognition of the gp100154–162 peptide versus the HIVpol peptide. The wells with the highest SI reactivity (denoted by **) were selected for limiting dilution cloning. After approximately 2 weeks, growth positive wells were functional screened for their ability to lyse peptide pulsed T2 cells. Selected T cell clones were expanded and underwent FACS analysis (C.) between days 25 and 34 to reveal highly enriched (99%) populations of gp100154–162 tetramer positive CD8+ T cells. Values in FACS dot plots correspond to the percentage of total CD8 + T cells that are tetramer-positive. (O) represents the SI for each microwell. Shaded area represents range of non-specific reactivity (SI = 0.5–2.0).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577625&req=5

Figure 5: Rapid cloning of gp100154–162 specific CD8+ T cells from peripheral blood. (A.) On Day 0, PBMC from four HLA-A2+ melanoma patients underwent staining with gp100154–162 peptide/HLA-A*0201 tetramers and anti-CD8 APC to determine natural precursor frequency. None of the patients demonstrated a significant population of tetramer positive CD8+ T cells by FACS. PBMC from each patient were plated in replicate microwells (n = 96) containing ~300,000 cells and sensitized for 10 days with 1 μM of gp100154–162 peptide in the presence of IL-2 (90 IU/ml). (B.) On day 10, a sample from every microculture was screened using the qPCR assay for specific recognition of the gp100154–162 peptide versus the HIVpol peptide. The wells with the highest SI reactivity (denoted by **) were selected for limiting dilution cloning. After approximately 2 weeks, growth positive wells were functional screened for their ability to lyse peptide pulsed T2 cells. Selected T cell clones were expanded and underwent FACS analysis (C.) between days 25 and 34 to reveal highly enriched (99%) populations of gp100154–162 tetramer positive CD8+ T cells. Values in FACS dot plots correspond to the percentage of total CD8 + T cells that are tetramer-positive. (O) represents the SI for each microwell. Shaded area represents range of non-specific reactivity (SI = 0.5–2.0).
Mentions: We next sought to incorporate the qPCR functional assay into a novel strategy for high throughput isolation of low frequency antigen specific CD8+ T cell clones from bulk PBMC for use in adoptive immunotherapy trials. We initially aimed to isolate CD8+ T cell clones reactive against the gp100154–162 epitope given their extremely low natural frequency in peripheral blood, the difficulty in prospectively isolating these T cells, and the limited published reports of T cell clones reactive against this epitope [7]. The basic isolation strategy is summarized in Figure 4. PBMC from an HLA-A2+ patient with metastatic melanoma are used to establish 96 independent microcultures which are sensitized for 10 days with 1 uM of gp100154–162 in the presence of IL-2 (90 IU/ml). On day 10, a sample from every microculture is screened using the qPCR assay for specific recognition of the gp100154–162 peptide versus the HIVpol peptide. The SI reactivities for the 96 wells are stratified by their magnitude and the most reactive microcultures are selected for the next step, limiting dilution cloning. After approximately 2 weeks, growth positive wells are screened for their ability to lyse peptide pulsed T2 cells and melanoma tumor lines. Selected T cell clones are then expanded in the final step for further analysis and potential use in adoptive immunotherapy trials. This strategy was applied to PBMC from four melanoma patients (patients 2, 5, 6, and 7) (Figure 5). A sample of the bulk PBMC from each patient, prior to any in vitro manipulation underwent staining with the gp100154–162 tetramer to determine natural precursor frequency. None of the patients demonstrated a significant population of tetramer positive CD8+ T cells by FACS on day 0 (Figure 5A). After 10 days of sensitization, the 96 independent microcultures for each patient were screened for peptide reactivity using the qPCR assay (Figure 5B). The stratified results for Patients 2, 5, and 6 demonstrated that only 7%, 12%, and 8% of the wells had a SI ≥ 2, respectively; 1%, 3%, and 1% of the wells had a SI ≥ 10, respectively; and the remaining wells had no detectable peptide reactivity (SI < 2). In contrast, for Patient 7, 92% of the wells had a SI ≥ 2 and 60% of the wells had a SI ≥ 10. The highest reactive microcultures from patients 2, 5, 6, and 7 (qPCR SI = 45, 635, 23, and 78, respectively) were selected for limiting dilution cloning. The frequencies of growth positive clones with lytic ability against peptide pulsed targets were 0.2%, 28%, 0.1%, and 2.3% for the respective patients, which directly correlated with the qPCR SI (r2 = 0.99, p < 0.0001). These selected clones were expanded and underwent FACS analysis between days 25 and 34 to reveal highly enriched populations (99%) of gp100154–162 tetramer positive CD8+ T cells (Figure 5C). Further, the derived populations were confirmed to be clonal by the sequencing of a single T cell receptor Vα and Vβ chain for each patient (Table 1). The functional avidity of these isolated T cell clones was high, as measured by their ability to recognize 10-10 to 10-11 M of gp100154–162 peptide pulsed onto T2 cells and HLA A2+/gp100+ melanoma tumor lines in vitro (Table 2). The phenotype of these cells was assessed by cell surface FACS for CD27, CD28, CD45RO, CD45RA, CD62L, and CD25 (Figure 6). The gp100154–162 tetramer positive cells from patients 2, 5, and 7 all were uniformly CD45RO+ and CD62L-, consistent with an effector memory phenotype. However, unlike typical antigen experienced T cells, there was persistent variable expression of CD45RA (19–96%). In addition all of the isolated clones continued to have significant expression of the costimulatory molecule CD27 (90–99%). This phenotype differed from the terminally differentiated TIL derived MART27–35 specific clone, JKF6, which had no significant expression of CD27. Since initiating these isolation studies we have successfully cloned gp100154–162 CD8+ T cells in 6 of 8 patients (75%). In pilot clinical scale expansions, these clones demonstrated between 850–1000 fold expansions in cell numbers over the initial 14 days after a single rapid expansion in flasks. A second serial expansion of these clones resulted in an additional 400–600 fold expansion over the ensuing week. Thus, we found that with two consecutive rapid expansions ~1010 cells could be generated for potential clinical adoptive transfer from each starting clone isolated.

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