RAPID EXTRACELLULAR DEGRADATION OF SYNTHETIC CLASS-I PEPTIDES BY HUMAN DENDRITIC CELLS

Dendritic cells (DCs) effectively process exogenous and endogenous Ag and present peptide in the context of both class I and class II molecu les. We have demonstrated that peripheral blood DCs efficiently degrad e synthetic class I peptides at their cell surface within minutes as d etermined by analyzing DC supernatants by HPLC. Fragments were verifie d as bona fide cleavage products by direct sequencing using collision- induced dissociation tandem mass spectrometry. The predominant degrada tive activities were 1) not secreted but associated with activity at t he plasma membrane, 2) ecto-orientated, 3) mot induced by peptide-spec ific interactions, and 4) not associated with nonspecific uptake. Sequ ence analysis indicated that both N- and C-terminal as well as endopro teolytic events were occurring at the cell surface. The primary exopro teolytic event was identified as CD13 or CD13-like activity through in hibition studies and could be inhibited by ubiquitin and metal-chelati ng agents. Endoproteolytic events could be inhibited in the presence o f DTT, but the precise nature of this enzyme is still undetermined. Co mpared with the starting monocyte population, DCs cultured in the pres ence of granulocyte-macrophage CSF/IL-4 exhibited the highest degradat ive rate (4.3 nmol/min), followed by cultured monocytes (2.9 nmol/min) and freshly isolated monocytes (1.0 nmol/min), In addition to increas ed enzymatic activity, a change in substrate specificity was noted. Re sults are discussed with respect to APC loading, and alternatives are offered for circumventing such degradation.