Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens.

Abstract

The a/fl T-cell receptor recognies a complex ligand formed by the association ofantigenic peptides with molecules ofthe major histocompatibility complex (MHC). The inherent limitations of the conventional T-cell activation assays used to detect these peptide/MHC ligands have, until now, hampered the development of expression cloning systems for T-cell antigens. To overcome these limitations, we have recently introduced a method for detecting ligand-induced activation of individual T cells. This assay, which makes use of a lacZ reporter construct, differs from conventional ligadinduced activation assays in that it allows the detection of single, activated T cells in large pools of resting cells. We applied the IacZ assay to the problem of screening expression libraries, which requires the ability to detect ligand-bearing antigen-presenting cells when they are present at very low frequency. We show here that ligand-expressing antigenpresenting cells can be detected at frequencies of 1:103-104, a level of sensitivity compatible with the screening of cDNA libraries. Furthermore, by using as antigen-presenting cells COS-7 cells stably transfected with the murine Kb class I MHC molecule, we demonstrate that transiently expressed ovalbumin is efficiently processed and presented to an ovalbumin/Kb_ specific T-cell hybridoma. IacZ expression is induced in a detectable number of cocultured T cells, even when the ovalbumin cDNA consists of only 1:104 of the total DNA used to transfect the COS cells. These results suggest that unknown T-cell antigens may be identified by screening cDNA libraries in MHC-expressing COS cells using lacZ-inducible T cells as indicators of peptide antigen expression.The a/X3 T-cell receptor (TCR) recognizes small antigenic peptides bound to major histocompatibility complex (MHC)

Targeting dendritic cells with antigen via dendritic cell-associated promoters

“…Upon engagement of its TCR, which recognizes the OVA peptide (257 --264) in the context of H-2k b IL-2 is produced along with b-Galactosidase. 26 As a negative control, the same constructs were also transfected into murine embryonic fibroblasts expressing H-2k b The pCMV/OVAtransfected murine embryonic fibroblast cells were able to induce IL-2 production in the B3Z cells (Figure 3a). However, none of the OVA constructs were capable of activating IL-2 production in this non-DC cell line.…”

“…The DC cell line, D1 (kindly provided by Dr Paola Ricciardi-Castagnoli) was maintained in culture as previously described. 25 The B3Z T-cell hybridoma 26 and the OVA-transfected, murine melanoma cell line, B16OVA, 27 (derived from B16F10, clone MO5) was kindly provided by Dr Kenneth Rock. The medium used for the in vitro T-cell stimulation was RPMI 1640 supplemented with 5% FCS, glutamine, 50 mM b-Mercaptoethanol and antibiotics/antimycotics.…”

Alternative processing for MHC class I presentation by immature and CpG‐activated dendritic cells

“…pathway for processing of OVA DC were tested for the processing of OVA and presentation of the SIINFEKL peptide on H2-K b using the specific T cell hybridoma B3Z, which secretes IL-2 in response to TCR activation [18]. DC from different days of tissue culture were incubated with OVA, with or without the Golgi inhibitor BFA, for 4 h and then fixed and tested for T hybridoma activation.…”

“…The proteasomal inhibitor lactacystin had an effect similar to BFA in the present system, with limited inhibition of OVA processing and presentation in iDC (mean 11.4% in three separate experiments) compared to imDC (mean 95.3%, data not shown). The presence of an alternative MHC class I processing pathway was further established with iDC from TAP -/-mice, which were less active in triggering the T cell OVA processing in DC is reflected by IL-2 secretion of the specific B3Z T cell hybridoma [18] with background subtracted. For details regarding the OVA processing assay see the Sect.…”

“…Antigen processing was terminated by the fixation of the cells with 1% paraformaldehyde at room temperature for 15 min and the addition of 0.1 M glycine for another 15 min, followed by two washes. The T cell hybridoma B3Z (1×10 5 cells/well) [18], which recognizes the processed SIINFEKL peptide bound to H2-K b , was added for an additional 24 h before the collection of supernatants. IL-2 secreted by activated B3Z cells was measured using a mouse ELISA kit (PharMingen) according to the manufacturer's instructions.…”