DCs are the most capable APCs, express all the necessary machinery and have a high capacity of efficient presentation. We have established that the peptidome from unpulsed professional APC can be studied without having to resort to cell lines, using small numbers of monocyte-derived DCs that tend to select very high-affinity peptides forming nested sets as a norm. However, they also presented unconventional peptides. 20% of the peptides presented by all alleles were N-terminal or C-terminal peptides. Most C-terminal peptides were located at the very extreme of the protein, pertained to cytosolic proteins and did not cluster in nested sets. They also show preferent cleavage residues, compared to internal peptides._x000D_ We have used MoDCs and an in vitro digestion model set up as a cell-free system (CFS) to analyze the processing and presentation of an AITD autoantigen, thyroglobulin. Native or denatured purified thyroglobulin captured by MoDC generated many peptides, with dominant nested sets and no peptide derived from the N- or C-terminus of the protein. Very similar pattern was obtained wen MoDC were pulsed with colloid-enriched thyroid tissue extracts. Yet, if thyroglobulin was digested by the colloid cathepsins (B, L and S) at pH 7.4 prior to pulsing, MoDCs presentation of thyroglobulin peptides was almost completely abrogated. The large amount of intact thyroglobulin in the tissue extract could be accountable for these data and that the pre-digestion must have destroyed any thyroglobulin epitope that could be presented. However, when we did the same experiment using the CFS, predigested thyroglobulin was as efficiently presented as purified protein. Therefore, pre-digestion did not destroy epitopes, the fragments may have been degraded before reaching the MIIC in MoDCs. Thus, the state of the antigen is extremely relevant for its presentation by MoDCs but, the CFS method may help identifying steps of the processing events that may be lost when analyzing DC-presented peptides._x000D_ Two abundant and high affinity dominant nested sets were identified from thyroglobulin. One, associated to HLA-DR3 with the VVVDPSIRH core and one associated to HLA-DR15 with the core IMQYFSHFI. The VVVDPSIRH set contains peptide Tg2098, defined as immunodominant in an in vivo model of thyroiditis in HLA-DR3 transgenic mice. The same peptide was identified in the HLA-DR3+ peptidome from GD patients' thyroids. HLA-DR15 is not negatively associated to AITD but in a similar model, HLA-DR15 transgenic mice did not_x000D_ develop the disease using the same conditions. Interestingly, in HLA-DR15/DR3 MoDCs, most thyroglobulin peptides were presented by HLA-DR15 but peptides with this core were not identified in HLA-DR15+ thyroid samples affected by GD. A second nested set associated to HLA-DR3 was also found, independent of the source of antigen or the processing method. This second HLA-DR3 nested set, around the VIFDANAPV core, was not as abundant as the VVVDPSIRH and contained a peptide (Tg1574), known not to generate T cell responses in the same EAT model as the Tg2098 peptide._x000D_ The functional difference between these two peptides correlated with two characteristics that are important in the definition of immunodominance, i.e. sensitivity to cathepsins and to HLA-DM. Tg1574 was cathepsin-resistant whereas Tg2098 was partially sensitive. Upon digestion with several combinations of cathepsins, Tg1574 did not generate any intermediate variants and between 45 and 100% remained intact. In contrast, Tg2098 was trimmed at the peptide ends generating a number of variants, its core was maintained resistant to cleavage and only between 6 and 26% remained intact. In addition, Tg1574 was much more sensitive to HLA-DM than Tg2098. Interpreting these data after identifying what of the two peptides is preferentially presented in thymus will be necessary to fully demonstrate our hypothesis.
Autoantigen processing. How immunodominant thyroglobulin peptides are generated and presented by HLA-DR molecules
Ciudad García, M. T. (Author). 27 Nov 2015
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis