In vitro expansion of CD3/TCR- human thymocyte populations that selectively lack CD3 delta gene expression: a phenotypic and functional analysis.

Abstract

SummaryHighly 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 CD3E chain. After culture with low doses of PMA (0.5 ng/ml) and subsequent addition (at 24 h) of recombinant interleukin 2 (rIIr2; 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 y /6+ (BB3 -A13+) cells. Further removal of CD3 +TCR-y/8+ 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 a/ß+ cells could be detected, thus indicating that PMA did not agect 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 FcyR+ 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 a (TNF-a) and interferon y (IFN-y) (but not 11,2) production by CD3 -thymocytes. Cloning of fresh CD1 -3 -4 -8 -thymocytes in the presence of PMA and rIIr2 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 CD3r, e, and y transcripts, while CD3E was undetectable. Mature transcripts for both y and 6 TCR chains could be detected, whereas no TCR-a mRNA and only a truncated (1.0 kb) form of TCR ß 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-6 gene rearrangements revealed that the cultured CD3 -thymocytes retained a germline configuration.

Human CD8 T lymphocytes recognize Mycobacterium tuberculosis antigens presented by HLA‐E during active tuberculosis and express type 2 cytokines

“…NK-cell clones were tested for cytolytic activity in a 4-h 51 Crrelease assay as described [50] in the presence or absence of anti-CD94 mAb Y9 (IgM, 10μg/mL). Target cells used in these experiments were RMA-S/HLA-E (described above) and the human erythroleukemic cell line K562 transfected with HLA-E*0103 (K562/HLA-E, kindly provided by E. H. Weiss, Department of Biology, Anthropology and Human Genetics, Ludwig-MaximiliansUniversität, Munich, Germany) [51].…”

Activation of human NK cells by plasmacytoid dendritic cells and its modulation by CD4+ T helper cells and CD4+ CD25hi T regulatory cells

“…To evaluate cytolytic activity of NK cells, we used MFO-1, LCL721.221 and K562 cell lines as target cells as described [28]. Assays were performed in triplicate at the indicated effector/target ratios.…”

HLA-E-restricted recognition of cytomegalovirus-derived peptides by human CD8+cytolytic T lymphocytes

“…NK-CTLs derived from four different donors were tested for cytolytic activity in a 4-h 51 Cr-release assay as described (28) in the presence or in the absence of mAbs. The concentration of the mAbs used for the masking experiments was 10 g͞ml.…”