A novel cytolysis assay using fluorescent labeling and quantitative fluorescent scanning technology

Roden, Maureen; Lee, Kang-Hun; Panelli, Monica C.; Marincola, Francesco M.

doi:10.1016/s0022-1759(99)00039-3

Cited by 77 publications

(66 citation statements)

References 14 publications

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“…Beside serious biosafety questions raised by viral lysates, reliability is also critical, because APC function can differ between patients. In our previous work, we found that peptide libraries comprising 15-aa peptides successfully stimulated CD4 + and CD8 + T lymphocytes [7,11]. In this report, we integrated peptide stimulation in a T cell generation process for antigen-specific CD4 + and CD8 + lymphocytes.…”

Section: Discussionmentioning

confidence: 92%

“…Of our donors, 3/8 were HLA-A02 positive, but specific binding of NLVP A02 tetramers was found in only one of them -a well-known phenomenon [7,11]. Next, we generated a new T cell line from this donor by stimulating with our pp65 peptide library and NLVP peptides according to protocol I.…”

Section: Multiple Epitope Specificity Of Generated T Cell Linesmentioning

confidence: 99%

“…For cytotoxicity testing, a modified Calcein-AM release assay was used [11]. Target cells were labeled with Calcein-AM (15 mM) according to the manufacturer's instructions (Molecular Probes, Eugene, OR).…”

Section: Cytotoxicity Assay and Imaging Experimentsmentioning

confidence: 99%

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HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

Hammer

Meyer²,

Brestrich³

et al. 2005

Eur. J. Immunol.

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Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4 + and CD8 + T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-c-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4 + and CD8 + lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.

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Section: Discussionmentioning

confidence: 92%

Section: Multiple Epitope Specificity Of Generated T Cell Linesmentioning

confidence: 99%

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HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

Hammer

Meyer²,

Brestrich³

et al. 2005

Eur. J. Immunol.

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“…In fact, Tax 301-309 was found to be the immunodominant epitope for anti-Tax CTL generated in ATL patients with HLA-A*2402 who underwent allogeneic hemopoietic stem cell transplantation (20). Nine other epitopes, Tax [12][13][14][15][16][17][18][19][20] , Tax 178 -186 , Tax [187][188][189][190][191][192][193][194][195] , Tax 289 -297 , Tax 309 -315 , Tax 311-319 , Env [11][12][13][14][15][16][17][18][19] , Env [21][22][23][24][25][26][27][28][29] , and Env 153-161 , were likely to be minor epitopes for generation of anti-HTLV-1 CD8 ϩ T cells. It is possible that even minor epitopes have significant immune function in vivo, although we have no direct evidence.…”

Section: Discussionmentioning

confidence: 99%

Reduced Frequency, Diversity, and Function of Human T Cell Leukemia Virus Type 1-Specific CD8+ T Cell in Adult T Cell Leukemia Patients

Kozako

Arima

Toji

et al. 2006

The Journal of Immunology

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Human T cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-γ, perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53 vs 90%; p = 0.001), whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11–19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301–309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax11–19 with HLA-A*0201 and Tax301–309 with HLA-A*2402 at frequencies of 30 and 55%. There were also significant differences in percentage of cells binding Tax11–19/HLA-A*0201 and Tax301–309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-γ in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.

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“…This assay was performed using the nonradioactive fluorescence-based assay we described elsewhere. 56 Briefly, target cells were resuspended in complete medium at a final concentration of 10 6 /ml and incubated with 15 mM calcein-AM (Molecular Probes, Eugene, OR, USA) for 60 min at 371C with occasional shaking. After two washes in CM, cells were adjusted to 10 5 /ml.…”

Section: Cytotoxicity Assaymentioning

confidence: 99%

IL-10 stimulatory effects on human NK cells explored by gene profile analysis

et al. 2004

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The molecular mechanisms underlying the increase of natural killer (NK) cell anticancer activity mediated by interleukin (IL)-10 have not been elucidated. The aim of this study was to identify potential molecular mediators of IL-10 stimulatory effects by exploring the NK cell gene display induced by this cytokine. Gene profile was determined by high-throughput cDNA microarray and quantitative real-time PCR. In vitro, NK cells resting or conditioned with IL-10 were tested for cytotoxicity, migration and proliferation. IL-10 enhanced mRNA levels of cell activation/cytotoxicity-related genes (eg secretogranin, TIA-1, HMG-1, interferon-inducible genes) not upregulated by IL-2. In line with these findings, IL-10 increased NK cell in vitro cytotoxicity against Daudi cells. Unlike IL-2, IL-10 did not show any significant effect on NK cell in vitro proliferation and migration. However, gene profile analysis showed that IL-10 increased the expression of cell migration-related genes (eg L-selectin, vascular endothelium growth factor receptor-1, plasminogen activator, tissue; formyl peptide receptor, lipoxin A4 receptor), which might support a stimulatory effect not evident with the in vitro functional assay. Overall, gene profiling allowed us to formulate new hypotheses regarding the molecular pathways underlying the stimulatory effects of IL-10 on NK cells, supporting further investigation aimed at defining its role in cancer immune rejection.

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A novel cytolysis assay using fluorescent labeling and quantitative fluorescent scanning technology

Cited by 77 publications

References 14 publications

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

Reduced Frequency, Diversity, and Function of Human T Cell Leukemia Virus Type 1-Specific CD8+ T Cell in Adult T Cell Leukemia Patients

IL-10 stimulatory effects on human NK cells explored by gene profile analysis

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