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To start addressing these issues, we have in this study determined the ex-vivo frequencies, phenotype and functional properties of HLA-A*0201 HLA-A*0201s speCD8ic for different peptides of Mtb proteinspecificlt subjects with LTBI and adult TB patients with active disease, both before andifferentng four months of anti-mycobacterial therapeptides
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In the present study, we investigated the ex-vivo frequencies, multifunctional cytokine production and memory phenotype of circulating CD8 T-cells specific for different peptide-nonamers of Mtb proteins in adult HLA-A*0201 HLA-A*0201ith LTBI and in TB patientsubjects (T0) and after four months of anti-mycobacterial therapy (T4).
Binders specific for the C-terminal domain were blocked to different degrees by anti-sera specific for three different peptides derived from this domain.
However, we consider this unlikely given that the same cell culture protocol was previously used by us to isolate T cells specific for different GAD peptides (p206 and p221) as well as for an unrelated peptide (a mimetic epitope, p79, that is highly active in stimulating BDC2.5 T cells).
This motivates the new models that allow for different variances for different peptide groups.
We successfully sorted and short-term expanded T-cell populationspecificic for five different peptide sequences from gp100 (2), tyrosinase (1) and TRP-2 (2).
Some of the peptide residues are buried in the MHC groove and act as anchor residues, which define binding motifs specific for different MHC alleles.
In C57BL/6 (B6) mice acutely infected with murine cytomegalovirus (MCMV), CD8+ T cells specific for up to 26 different peptides from 19 viral proteins can be detected directly ex vivo by intracellular cytokine stimulation [6].
To enrich target bacterial cells, an immunocapture approach was performed using a mixture of rabbit-derived polyclonal antibodies(GenScript Corp , NJ USA) directed against 'Ca. L. solanacearum' OMP-A (Ab-OMP-A) and 'Ca. L. solanacearum' OMP-B (Ab-OMP-B) which were specific for two different synthesized peptides (OMP-A "GKDKKDSYGGKEQLC" and OMP-B "VIRRELGFSEGDPIC").
This is because the ionization efficiency might be different for the different peptides.
Our results suggested the established NR286 T cell line was p286-specific, as cells were positively stained by tetAg7/p286, but not by a control tetramer (tetAg7/p206) specific for a different GAD peptide, p206; additionally, they produced IL-2 in response to p286, but not to p206 (Fig. 1A and 1B).
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