COMPARTMENTALIZATION OF DEFINED EPITOPES EXPRESSED IN ESCHERICHIA-COLI HAS ONLY A MINOR INFLUENCE ON EFFICIENCY OF PHAGOCYTIC PROCESSING FOR PRESENTATION BY CLASS-I AND CLASS-II MAJOR HISTOCOMPATIBILITY COMPLEX-MOLECULES TO T-CELLS

The effect of abundance and compartmentalization of antigenic epitopes expressed in Escherichia coli on phagocytic processing was studied by expressing fusion proteins containing the epitope from positions 52 t o 61 of hen egg white lysozyme [HEL(52-61)], which binds the I-A(k) mu rine major histocompatibility complex class II (MHC-II) molecule or th e epitope from positions 257 to 264 of chicken egg ovalbumin [OVA(257- 264)], which binds the K(b) murine MHC-1 molecule. Epitopes expressed as fusion proteins in the outer membrane protein LamB allowed exposure of the epitopes either at the bacterial surface, in the periplasmic s pace, or in the cytoplasm. Regardless of epitope compartmentalization within the bacterium, MHC-H-restricted or MHC-1-restricted presentatio n to T hybridoma cells occurred after macrophages phagocytosed bacteri a producing the HEL(52-61) epitope or the OVA(257-264) epitope, respec tively. Increased epitope abundance within a given microbial compartme nt resulted in increased processing and presentation to epitope-specif ic T hybridoma cells. Minor differences in the efficiency of epitope p rocessing between the constructs was observed, and the HEL or OVA epit ope exposed in the periplasmic space was processed most efficiently co mpared with the surface- or cytoplasm-localized epitopes. These differ ences could be overcome by increasing the amount of epitope per bacter ium as little as two to five times. The minor differences in processin g efficiency may be due to differing protein contexts of the epitope a s well as differing epitope compartmentalizations within the bacteria. Thus, production of abundant epitope is the important parameter influ encing processing of epitopes expressed in E. coli to induce T-cell re sponses rather than targeting of an epitope to a specific bacterial co mpartment.