In vitro expansion of CD3/TCR- human thymocyte populations that selectively lack CD3δ gene expression

A phenotypic and functional analysis

Alessandro Poggi, Roberto Biassoni, Nicoletta Pella, Francesca Paolieri, Rosanna Bellomo, Alberto Bertolini, Lorenzo Moretta, Maria Cristina Mingari

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Highly purified CD1-3-4-8- human thymocytes were obtained by panning techniques combined with cell depletion with antibody-coated magnetic beads. Most of these cells expressed cytoplasmic CD3 antigen, as assessed by mAbs known to react with the CD3∈ chain. After culture with low doses of PMA (0.5 ng/ml) and subsequent addition (at 24 h) of recombinant interleukin 2 (rIL-2; 100 U/ml) cells underwent extensive proliferation (40-60-fold of the initial cell input after 2 wk). The majority of the proliferating cells were CD3-TCR-. The remaining cells (5-40%) were represented by CD3+TCR γ/δ+ (BB3-A13+) cells. Further removal of CD3+TCR-γ/δ+ cells resulted in highly purified CD3- populations that further proliferated in culture with no substantial phenotypic changes. When CD3+ thymocytes were cultured under the same experimental conditions, only CD3+ TCR-α/β+ cells could be detected, thus indicating that PMA did not affect the surface expression of the CD3/TCR complex, but rather induced preferential growth of CD3- thymocytes. Surface marker analysis of cultured CD3- thymocytes showed that they were homogeneously CD7+, whereas low proportions of cells expressed CD2 and CD8 antigens. Among the natural killer (NK) cell markers, CD56 was highly expressed by all cells, whereas CD16, CD57, CD11b, NKH2, and GL183 were absent. Importantly, these cells were different from peripheral NK cells, as 80-95% of them expressed cytoplasmic CD3 antigen. Functional analysis revealed a strong cytolytic activity against both NK-sensitive (K562) and NK-resistant (M14, Daudi) human target cells. In a redirected killing assay against the FcγR+ P815 cells, mAbs specific for triggering molecules including CD3, CD2, and CD16 failed to augment target cell lysis, while a strong cytolytic effect was induced by PHA. In addition, PHA alone or in combination with PMA induced tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) (but not IL-2) production by CD3- thymocytes. Cloning of fresh CD1-3-4-8- thymocytes in the presence of PMA and rIL-2 resulted in CD3-CD56+ clones that displayed a pattern of cytolytic activity and lymphokine production similar to that of the polyclonal populations. Northern blot analysis of transcripts coding for CD3/TCR molecules revealed the presence of CD3ζ, ε, and γ transcripts, while CD3δ was undetectable. Mature transcripts for both γ and δ TCR chains could be detected, whereas no TCR-α mRNA and only a truncated (1.0 kb) form of TCR-β mRNA were revealed. These data suggest that CD3- cultured thymocytes may be, at least in part, representative of a T cell maturation stage that precedes the surface expression of the CD3/TCR molecular complex. Southern blot analysis of the TCR-δ gene rearrangements revealed that the cultured CD3- thymocytes retained a germline configuration.

Original languageEnglish
Pages (from-to)1409-1418
Number of pages10
JournalJournal of Experimental Medicine
Volume172
Issue number5
Publication statusPublished - Nov 1 1990

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Thymocytes
Gene Expression
Population
T-Cell Antigen Receptor-CD3 Complex
CD3 Antigens
In Vitro Techniques
Natural Killer Cells
Interleukin-2
CD2 Antigens
CD8 Antigens
Messenger RNA
Gene Rearrangement
Lymphokines
Southern Blotting
Northern Blotting
Interferons
Organism Cloning

ASJC Scopus subject areas

  • Immunology

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In vitro expansion of CD3/TCR- human thymocyte populations that selectively lack CD3δ gene expression : A phenotypic and functional analysis. / Poggi, Alessandro; Biassoni, Roberto; Pella, Nicoletta; Paolieri, Francesca; Bellomo, Rosanna; Bertolini, Alberto; Moretta, Lorenzo; Mingari, Maria Cristina.

In: Journal of Experimental Medicine, Vol. 172, No. 5, 01.11.1990, p. 1409-1418.

Research output: Contribution to journalArticle

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abstract = "Highly purified CD1-3-4-8- human thymocytes were obtained by panning techniques combined with cell depletion with antibody-coated magnetic beads. Most of these cells expressed cytoplasmic CD3 antigen, as assessed by mAbs known to react with the CD3∈ chain. After culture with low doses of PMA (0.5 ng/ml) and subsequent addition (at 24 h) of recombinant interleukin 2 (rIL-2; 100 U/ml) cells underwent extensive proliferation (40-60-fold of the initial cell input after 2 wk). The majority of the proliferating cells were CD3-TCR-. The remaining cells (5-40{\%}) were represented by CD3+TCR γ/δ+ (BB3-A13+) cells. Further removal of CD3+TCR-γ/δ+ cells resulted in highly purified CD3- populations that further proliferated in culture with no substantial phenotypic changes. When CD3+ thymocytes were cultured under the same experimental conditions, only CD3+ TCR-α/β+ cells could be detected, thus indicating that PMA did not affect the surface expression of the CD3/TCR complex, but rather induced preferential growth of CD3- thymocytes. Surface marker analysis of cultured CD3- thymocytes showed that they were homogeneously CD7+, whereas low proportions of cells expressed CD2 and CD8 antigens. Among the natural killer (NK) cell markers, CD56 was highly expressed by all cells, whereas CD16, CD57, CD11b, NKH2, and GL183 were absent. Importantly, these cells were different from peripheral NK cells, as 80-95{\%} of them expressed cytoplasmic CD3 antigen. Functional analysis revealed a strong cytolytic activity against both NK-sensitive (K562) and NK-resistant (M14, Daudi) human target cells. In a redirected killing assay against the FcγR+ P815 cells, mAbs specific for triggering molecules including CD3, CD2, and CD16 failed to augment target cell lysis, while a strong cytolytic effect was induced by PHA. In addition, PHA alone or in combination with PMA induced tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) (but not IL-2) production by CD3- thymocytes. Cloning of fresh CD1-3-4-8- thymocytes in the presence of PMA and rIL-2 resulted in CD3-CD56+ clones that displayed a pattern of cytolytic activity and lymphokine production similar to that of the polyclonal populations. Northern blot analysis of transcripts coding for CD3/TCR molecules revealed the presence of CD3ζ, ε, and γ transcripts, while CD3δ was undetectable. Mature transcripts for both γ and δ TCR chains could be detected, whereas no TCR-α mRNA and only a truncated (1.0 kb) form of TCR-β mRNA were revealed. These data suggest that CD3- cultured thymocytes may be, at least in part, representative of a T cell maturation stage that precedes the surface expression of the CD3/TCR molecular complex. Southern blot analysis of the TCR-δ gene rearrangements revealed that the cultured CD3- thymocytes retained a germline configuration.",
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AU - Poggi, Alessandro

AU - Biassoni, Roberto

AU - Pella, Nicoletta

AU - Paolieri, Francesca

AU - Bellomo, Rosanna

AU - Bertolini, Alberto

AU - Moretta, Lorenzo

AU - Mingari, Maria Cristina

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