Generation

Like CD4+ TR cells, CD8+CD25- Ts cells can be generated in vitro aftermultiple rounds of stimulation of human PBMCs with either allogeneic or xenogeneic donor APCs. Similary, CD8+CD25= Ts can be generated in vitro by rpiming PBMCs with self APCs pulsed with nomianl antigens such as MHC antigens or tetanus toxin.

Phenotype

Ts are characterized by their CD8+CD28- phenotype. Ts cells are MHC class I-restricted and suppress antigen-specific CD4+ Th cells responses, inhibiting their capacity to rpoduce IL_2 and preventing upregulation of CD40 ligand (CD40L).

Mechanism of suppression

Inhibition of CD4+ Th cell proliferation is not cause by killing either APCs or CD4+ Th cells. Neither is the suppressor effect mediated by the production of cytokines; instead it requires direct interactions between Ts cells and the APCs used for priming. In this system, the APCs act as a bridge between Ts cells, which recognize peptide-MHC class I complexes on their cell surfaces- and CD4+ Th cells, which recognize peptid-MHC class II complexes on their cell surfaces.

Inhibition of Costimulatory Molecules

Ts cells inhibit CD40 mediated upregulation of costimulatory molecules such as CD80 and CED86 on APCs that represent the peptide MHC class I complexes to which the CD8+CD28-Ts cells have been previously primed. The supressed APCs are rendered unable to induce and sustain the full program of CD4+Th cell activation due, at least in part, to the inhbiition of NF-kB activation and transcription of costimulatory molecules in APCs.

Induction of ILT3/ILT4 on APCs

Ts cells induce the up regulation of ILT3 and ILT4 on monocytes and DCs, rednering these APC capabile of anergizing CD4+Th cells. The inhbitiory receptors ILT3/ILT4, which are expresed by monocytes and DCs, belong to a family of Ig-like inhibitory receptors that are structurally and functionally related to killer cell inhibitory receptors (KIRs). They display a long cytoplasmic tail containing immunoreceptor tyrosine based inhibitory motifs (ITIMs). These receptors mediate inhibition of cell activation by recruiting tyrosine phosphatase SHP-1. Co-ligation of ILTs in monocytes inhibits Ca2+ mobilization and tyrosin phosphoyrlation triggered by antibody ligation of FcyRII (CD32), HLA-DR and FcyRI (CD64). Although the ligand for ILT3 is unknown, ILT4 binds HLA-A, HLA-B, HLA-c and HLA-G.

upregulation of ILT3 and ILT4 renders DCs tolerogenic. The finding that overexpression of ILT3 is associated with inhibition of NFkB activation shows that, in the presence of Ts cells, APCs have a reduced capacity to transcribe NFkB dependent costimulatory molecules. Although it is not clear how ILT3 and ILT4 overexpression interferes wtih CD40 signaling, it is beleived that these receptors act through SHP posphatases to modulate IkB phosphorylation and degradation, thus affecting NFkB activation. This would inhibit transcription of NFkB dependent genes that encodes costimulatory molecules in DCs, thus promoting their capacity to induce CD4+ Th cell anergy.