At the beginning of the 1970s, researchers thought that macrophages might help initiate the immune response, but the underlying mechanisms were obscure. Macrophages might process antigens, according to various theories, by producing immunogenic RNA (Askonas and Rhodes, 1965), acting as reservoirs that exocytosed antigen (Cruchaud and Unanue, 1971), altering antigens extracellularly (Shortman and Palmer, 1971), or, perhaps most popular, retaining whole protein antigen on their surface (Unanue et al., 1969).

Ralph Steinman remembers the puzzle surrounding the macrophage's role and how it might process antigens. He and Zanvil Cohn at the Rockefeller University in New York decided that the horseradish peroxidase (HRP) enzyme—newly adapted as part of an assay system for EM (Graham and Karnovsky, 1966)—would serve as both a model antigen and a clear and sensitive endocytic tracer. They reasoned that HRP's journey into the macrophage as an active enzyme would be easy to quantify and follow at the subcellular level. This represented one of the earliest forays of immunologists into the world of cell biology.

Although other studies had concluded that a small amount of intact antigen remained on the cell surface of macrophages (Unanue et al., 1969; Unanue and Cerottini, 1970; Schmidtke and Unanue, 1971), Steinman and Cohn could find no HRP bound extracellularly (Steinman and Cohn, 1972). Instead they saw HRP internalized, incorporated into secondary lysosomes, and presumably digested as activity fell off. In addition, radioactively labeled HRP appeared to be hydrolyzed down to single amino acids, which argued against macrophages retaining any antigenic information.

“At that time, the biological function of the major histocompatibility complex [MHC] had not been defined beyond its role in encoding antigens for transplant rejection,” says Steinman. MHC function was defined in 1975 (Doherty and Zinkernagel, 1975; Zinkernagel and Doherty, 1975) and eventually led to the discovery of the “fascinating [antigen] processing pathway.” Emile Unanue's group eventually showed that macrophage MHC could bind and retain antigen peptide fragments, which were recognized as peptide–MHC complexes by T cells (Allen et al., 1984).

But in 1973 the concept of antigens being presented as peptide fragments “was not envisioned at all,” says Steinman. Antigens were seen as a stable entity, so disappearance of HRP enzymatic activity was equated with disappearance of antigen. Steinman and Cohn used trypsin digestion to enhance release of any surface-bound HRP, but the tiny quantity of radioactive HRP peptide left on MHC would probably not have been detected by their methods—MHC is resistant to release by proteases, and many antigenic peptides may have lacked the radioactive tags from the original proteins.

Ingested HRP (black) appeared to be digested rather than processed.

STEINMAN

Steinman and Cohn's data had led them to have doubts about antigen processing by macrophages. Later it became clear that macrophages did present antigen as peptide–MHC, but meanwhile the work had several other, more positive consequences. The group used the HRP technique to track fluid and membrane movement, thus demonstrating that extensive membrane recycling must be happening at the plasma membrane. And the concept of presenting whole antigens was borne out in lymphoid areas rich in B cells, where intact, extracellular antigen is held on the surfaces of another cell type to stimulate B cells (Chen et al., 1978).

But for T cells, the action was elsewhere. “Our focus was the initiation of the immune response,” says Steinman. “When we couldn't find retained antigen in macrophages, we thought maybe we were looking at the wrong cell and that took us to the spleen where we found dendritic cells.” This related cell type turned out to be the “professional” antigen-presenting cell (Steinman and Cohn, 1973), and one of the key components of the immune system. It is that work—not the macrophage detour—that marks the highlight of Steinman's career. KP

Allen, P.M., et al.
1984
.
Proc. Natl. Acad. Sci. USA.
81
:
2489
–2493.

Askonas, B.A., and J.M. Rhodes.
1965
.
Nature.
205
:
470
–474.

Chen, L.L., et al.
1978
.
J. Cell Biol.
79
:
184
–199.

Cruchaud, A., and E.R. Unanue.
1971
.
J. Immunol.
107
:
1329
–1340.

Doherty, P.C., and R.M. Zinkernagel.
1975
.
Lancet.
305
:
1406
–1409.

Graham, R.C., Jr., and M.J. Karnovsky.
1966
.
J. Histochem. Cytochem.
14
:
291
–302.

Schmidtke, J.R., and E.R. Unanue.
1971
.
J. Immunol.
107
:
331
–338.

Shortman, K., and J. Palmer.
1971
.
Cell. Immunol.
2
:
399
–410.

Steinman, R.M., and Z.A. Cohn.
1972
.
J. Cell Biol.
55
:
186
–204.

Steinman, R.M., and Z.A. Cohn.
1973
.
J. Exp. Med.
137
:
1142
–1162.

Unanue, E.R., et al.
1969
.
Nature.
222
:
1192
–1195.

Unanue, E.R., and J.-C. Cerottini.
1970
.
J. Exp. Med.
131
:
711
–725.

Zinkernagel, R.M., and P.C. Doherty.
1975
.
J. Exp. Med.
141
:
1427
–1436.