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High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals.

Pittet MJ, Valmori D, Dunbar PR, Speiser DE, Liénard D, Lejeune F, Fleischhauer K, Cerundolo V, Cerottini JC, Romero P - J. Exp. Med. (1999)

Bottom Line: High frequencies (>/=1 in 2,500 CD8(+) T cells) of Melan-A-specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals.Thus, it appears that high frequencies of naive Melan-A-specific CD8(+) T cells can be found in a large proportion of HLA-A*0201(+) individuals.Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A-specific cells can occur in vivo.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, Centre Hospitalier Universitaire Vaudois, Switzerland.

ABSTRACT
Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A-specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A-specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (>/=1 in 2,500 CD8(+) T cells) of Melan-A-specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A-specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RA(hi)/RO(-) phenotype, whereas variable proportions of Ag-experienced CD45RA(lo)/RO(+) Melan-A-specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix-specific CTLs from all individuals exhibited a CD45RA(lo)/RO(+) memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A(+) cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon gamma ELISPOT assays independently confirmed that most of the Melan-A-specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A-specific CD8(+) T cells can be found in a large proportion of HLA-A*0201(+) individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A-specific cells can occur in vivo.

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Phenotypic analysis of ex vivo circulating A2/Melan-A+ and A2/Flu-MA+ cells in melanoma patients and healthy donors. CD8+ lymphocyte populations were highly purified (>98%) from PBMCs, as illustrated in Fig. 1. The lymphocyte preparations were then stained with A2/Melan-A, or A2/Flu-MA tetramers together with anti-CD45RACYC and either anti-CD45ROFITC or anti-CD28FITC mAbs, and immediately analyzed by flow cytometry. (A) Pattern of expression of CD45RA/RO (top) or CD45RA/CD28 (bottom) in total circulating CD8+ T cells (left) from healthy donors (shown is HD 329). They were CD28+CD45RAhi/RO− in A2/Melan-A+ gated cells (middle) but CD28+CD45RAlo/RO+ in A2/Flu-MA+ gated cells (right). (B) Circulating A2/Melan-A+CD8+ T cells detected in the majority (7 out of 10) of melanoma patients presented a CD28+CD45RAhi/RO− phenotype (left). In contrast, tetramer+ cells from 2 out of 10 patients displayed variable proportions of CD45RAhi/RO− and CD45RAlo/RO+tetramer+ cells (middle) and a CD28−CD45RAint phenotype for 1 out of 10 patients (right). (C) Summary of phenotyping data obtained for melanoma patients and healthy donors. Frequencies of CD45RAlo cells detected in gated A2/Melan-A+ and A2/Flu-MA+CD8+ T cells were calculated with CellQuest™ software.
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Figure 2: Phenotypic analysis of ex vivo circulating A2/Melan-A+ and A2/Flu-MA+ cells in melanoma patients and healthy donors. CD8+ lymphocyte populations were highly purified (>98%) from PBMCs, as illustrated in Fig. 1. The lymphocyte preparations were then stained with A2/Melan-A, or A2/Flu-MA tetramers together with anti-CD45RACYC and either anti-CD45ROFITC or anti-CD28FITC mAbs, and immediately analyzed by flow cytometry. (A) Pattern of expression of CD45RA/RO (top) or CD45RA/CD28 (bottom) in total circulating CD8+ T cells (left) from healthy donors (shown is HD 329). They were CD28+CD45RAhi/RO− in A2/Melan-A+ gated cells (middle) but CD28+CD45RAlo/RO+ in A2/Flu-MA+ gated cells (right). (B) Circulating A2/Melan-A+CD8+ T cells detected in the majority (7 out of 10) of melanoma patients presented a CD28+CD45RAhi/RO− phenotype (left). In contrast, tetramer+ cells from 2 out of 10 patients displayed variable proportions of CD45RAhi/RO− and CD45RAlo/RO+tetramer+ cells (middle) and a CD28−CD45RAint phenotype for 1 out of 10 patients (right). (C) Summary of phenotyping data obtained for melanoma patients and healthy donors. Frequencies of CD45RAlo cells detected in gated A2/Melan-A+ and A2/Flu-MA+CD8+ T cells were calculated with CellQuest™ software.

Mentions: Clearly, circulating A2/Melan-A+ and A2/Flu-MA+ CD8+ T cells from healthy donors displayed distinct phenotypes (as illustrated for one donor in Fig. 2 A and summarized in Fig. 2 C). Practically all of the A2/Melan-A+ cells were CD28+CD45RAhi/RO− (range: 84–95%), corresponding to a naive phenotype. In marked contrast, most of A2/Flu-MA+ cells were CD45RAlo/RO+ (range: 83–97%). Thus, the phenotype of A2/Flu-MA+ cells corresponded to Ag-experienced memory T cells, compatible with the notion that the Flu-MA58–66 peptide probably represents a recall Ag in these HLA-A2+ individuals. In addition, ∼20% of CD45RAlo/RO+A2/Flu-MA+ cells from HD 099 and HD 604 presented a CD28− phenotype (data not shown).


High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals.

Pittet MJ, Valmori D, Dunbar PR, Speiser DE, Liénard D, Lejeune F, Fleischhauer K, Cerundolo V, Cerottini JC, Romero P - J. Exp. Med. (1999)

Phenotypic analysis of ex vivo circulating A2/Melan-A+ and A2/Flu-MA+ cells in melanoma patients and healthy donors. CD8+ lymphocyte populations were highly purified (>98%) from PBMCs, as illustrated in Fig. 1. The lymphocyte preparations were then stained with A2/Melan-A, or A2/Flu-MA tetramers together with anti-CD45RACYC and either anti-CD45ROFITC or anti-CD28FITC mAbs, and immediately analyzed by flow cytometry. (A) Pattern of expression of CD45RA/RO (top) or CD45RA/CD28 (bottom) in total circulating CD8+ T cells (left) from healthy donors (shown is HD 329). They were CD28+CD45RAhi/RO− in A2/Melan-A+ gated cells (middle) but CD28+CD45RAlo/RO+ in A2/Flu-MA+ gated cells (right). (B) Circulating A2/Melan-A+CD8+ T cells detected in the majority (7 out of 10) of melanoma patients presented a CD28+CD45RAhi/RO− phenotype (left). In contrast, tetramer+ cells from 2 out of 10 patients displayed variable proportions of CD45RAhi/RO− and CD45RAlo/RO+tetramer+ cells (middle) and a CD28−CD45RAint phenotype for 1 out of 10 patients (right). (C) Summary of phenotyping data obtained for melanoma patients and healthy donors. Frequencies of CD45RAlo cells detected in gated A2/Melan-A+ and A2/Flu-MA+CD8+ T cells were calculated with CellQuest™ software.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195613&req=5

Figure 2: Phenotypic analysis of ex vivo circulating A2/Melan-A+ and A2/Flu-MA+ cells in melanoma patients and healthy donors. CD8+ lymphocyte populations were highly purified (>98%) from PBMCs, as illustrated in Fig. 1. The lymphocyte preparations were then stained with A2/Melan-A, or A2/Flu-MA tetramers together with anti-CD45RACYC and either anti-CD45ROFITC or anti-CD28FITC mAbs, and immediately analyzed by flow cytometry. (A) Pattern of expression of CD45RA/RO (top) or CD45RA/CD28 (bottom) in total circulating CD8+ T cells (left) from healthy donors (shown is HD 329). They were CD28+CD45RAhi/RO− in A2/Melan-A+ gated cells (middle) but CD28+CD45RAlo/RO+ in A2/Flu-MA+ gated cells (right). (B) Circulating A2/Melan-A+CD8+ T cells detected in the majority (7 out of 10) of melanoma patients presented a CD28+CD45RAhi/RO− phenotype (left). In contrast, tetramer+ cells from 2 out of 10 patients displayed variable proportions of CD45RAhi/RO− and CD45RAlo/RO+tetramer+ cells (middle) and a CD28−CD45RAint phenotype for 1 out of 10 patients (right). (C) Summary of phenotyping data obtained for melanoma patients and healthy donors. Frequencies of CD45RAlo cells detected in gated A2/Melan-A+ and A2/Flu-MA+CD8+ T cells were calculated with CellQuest™ software.
Mentions: Clearly, circulating A2/Melan-A+ and A2/Flu-MA+ CD8+ T cells from healthy donors displayed distinct phenotypes (as illustrated for one donor in Fig. 2 A and summarized in Fig. 2 C). Practically all of the A2/Melan-A+ cells were CD28+CD45RAhi/RO− (range: 84–95%), corresponding to a naive phenotype. In marked contrast, most of A2/Flu-MA+ cells were CD45RAlo/RO+ (range: 83–97%). Thus, the phenotype of A2/Flu-MA+ cells corresponded to Ag-experienced memory T cells, compatible with the notion that the Flu-MA58–66 peptide probably represents a recall Ag in these HLA-A2+ individuals. In addition, ∼20% of CD45RAlo/RO+A2/Flu-MA+ cells from HD 099 and HD 604 presented a CD28− phenotype (data not shown).

Bottom Line: High frequencies (>/=1 in 2,500 CD8(+) T cells) of Melan-A-specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals.Thus, it appears that high frequencies of naive Melan-A-specific CD8(+) T cells can be found in a large proportion of HLA-A*0201(+) individuals.Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A-specific cells can occur in vivo.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, Centre Hospitalier Universitaire Vaudois, Switzerland.

ABSTRACT
Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A-specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A-specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (>/=1 in 2,500 CD8(+) T cells) of Melan-A-specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A-specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RA(hi)/RO(-) phenotype, whereas variable proportions of Ag-experienced CD45RA(lo)/RO(+) Melan-A-specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix-specific CTLs from all individuals exhibited a CD45RA(lo)/RO(+) memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A(+) cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon gamma ELISPOT assays independently confirmed that most of the Melan-A-specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A-specific CD8(+) T cells can be found in a large proportion of HLA-A*0201(+) individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A-specific cells can occur in vivo.

Show MeSH
Related in: MedlinePlus