In the late 1960s and early 1970s, a shift occurred in the focus of immunological research from Ab-dominated studies to ones involving cellular components of immunity such as T cells, B cells, and APCs. The Ig nature of the B cell Ag receptor greatly facilitated the elucidation of how B cells recognized Ags. For T cells, significant progress was made with their identification, the establishment of the critical role of the thymus in their development, and their essential functional activities; however, how T cells recognized Ag was not fully understood until the mid-1980s. This month’s Pillars of Immunology article by Ziegler and Unanue made a seminal contribution to the understanding of T cell recognition of Ag by defining the macrophage Ag processing step required for the generation of the ligand recognized by the TCR (1).

Our current view of how T cells recognize Ag emerged from a series of findings that distinguished this recognition from that of B cells; many of these findings involved the now ignored research animal, the guinea pig. There was no evidence that T cells could directly bind Ag as B cells did, which raised the possibility that Ag was being modified prior to T cell recognition. There was some evidence, by Gell and Benacerraf, that delayed-type hypersensitivity in guinea pigs, a T cell function, could be stimulated by denatured Ag, whereas immediate-type hypersensitivity, an Ab-mediated function, could only be stimulated by native Ag (2). A conceptual breakthrough came from the use of in vitro T cell assays, mostly in the guinea pig system, which revealed that an accessory cell (macrophages in all of these early studies) was required for T cell recognition of Ag (3, 4). This requirement for a second cell type for T cell stimulation radically altered the conceptual landscape. In 1973, Shevach and Rosenthal made a key finding by demonstrating the requirement for MHC identity between the macrophages and T cells for an Ag-specific CD4+ T cell response (5), the phenomenon called MHC restriction. The finding of MHC restriction, coupled with the seminal work of Zinkernagel and Doherty involving CD8+ T cells (6), firmly established the critical role of MHC gene products in the recognition of Ag by T cells. However, the molecular nature of the Ag recognized remained a mystery and it was unclear whether MHC restriction and Ag recognition were two separate requirements in T cell activation or two aspects of the same recognition event. For a variety of reasons, the guinea pig has faded as the immunological animal model of choice, but it obviously played a very important role in these early studies. It seems so simple now that we know that a TCR recognizes a peptide bound to an MHC molecule, but at the time many different theories were proposed for the role of the MHC molecules and the form of Ag recognized, all of which lacked direct experimental proof.

From the early studies of Metchnikoff and continuing into the modern era, macrophages have been known to be involved in immunity against pathogens. They had been shown to readily digest bacterial and soluble Ags, and the zeitgeist in the 1970s was essentially that macrophages completely degraded Ags to their component amino acids. Thus, it was difficult to imagine how a macrophage could be involved in presenting Ag when they efficiently and completely degraded Ags. There were some studies with soluble model Ags that showed that macrophages could retain some “native” Ag, which could induce Ab responses in naive animals (7). The concept of Ag handling by the macrophage slowly developed, but precisely how they functioned as accessory cells and what was the relevant form of Ag was not known. Understanding of macrophage Ag degradation slowly increased, but it remained unclear whether this process was in any way relevant to its role in Ag presentation. Indeed, before the Ziegler and Unanue article, it seemed that Ag degradation was essentially antithetic to Ag processing.

In their 1981 study, reported in this featured Pillars of Immunology article, Ziegler and Unanue made a major advance in our understanding of the role of macrophages by establishing that the macrophage performed an essential intracellular Ag processing step, after which time all of the necessary signals for stimulating a T cell were present on the surface of the macrophage. They used a mouse model of the bacterial pathogen Listeria monocytogenes, which Mackaness had established as the paradigm for cell-mediated immunity (8). The use of a bacterial Ag greatly facilitated the authors’ ability to track the fate of the T cell Ag as it entered the macrophage. They first established the kinetics of the binding, ingestion, and catabolism of Listeria by macrophages and related these events to the macrophage’s ability to stimulate Listeria-specific T cells. The binding and ingestion of Listeria were rapid events, with subsequent catabolism detectable after 15 min. Ziegler and Unanue then used multiple assays to analyze the T cell-macrophage interaction, including T cell binding, cytokine production, and proliferation. The use of the T cell binding assay was key to their ability to establish a direct link between macrophage Ag processing and later recognition by the T cell. T cell binding to macrophages occurred within minutes of Listeria phagocytosis, which was the same time frame as the processing events they were investigating; in contrast, T cell proliferation took several days, making it too distal an event for measuring events occurring within the macrophage. They found that there was a lag period of 20 min after the macrophages had ingested Listeria before they were able to bind T cells. The T cell binding activity reached maximal levels by 60 min. Thus, for the first time these findings established that Ag processing occurred 20–60 min after macrophage contact with Ag. Ag processing required metabolically active macrophages but not protein synthesis. A subsequent study by these same authors reported that Ag processing required acidic intracellular vesicles, thereby establishing that the endosomal/lysosomal pathway was involved (9). Fixation of the macrophages with paraformaldehyde at various points after exposure to Listeria allowed a dramatic demonstration of the kinetics of Ag processing. If they were fixed immediately after uptake of the Listeria, the macrophages could not stimulate T cells; however, if the macrophages were allowed a 60-min processing period and then fixed they were fully able to stimulate the T cells. Thus, after this Ag processing period all of the necessary signals for presentation to a CD4+ T cell existed on the surface of these fixed APCs, and no further macrophage metabolic steps were required.

This conclusion from the Ziegler and Unanue article resulted in a major shift in thinking about macrophage Ag presentation within the immunologic community. It firmly established that, after internalization by a macrophage, a form of the Ag was rescued from total degradation. Initially, the concept of Ag processing was met with skepticism by some investigators (10), but the evidence for Ag processing and presentation became overwhelming, even for the most the hardened skeptic (11). This, importantly, brought the immunological phenomenon of Ag processing into a cell biological context that greatly facilitated the delineation of the class II Ag processing pathway. The role of the MHC molecule in Ag processing and presentation was not established in these studies; this occurred in 1985, when it was shown that a processed peptide could bind to an MHC molecule directly, as has been highlighted in a previous Pillars of Immunology article (12). Thus, these findings by Ziegler and Unanue were paradigm shifting by establishing and defining the obligatory intracellular processing step by an APC required for an Ag to be presentable to T cells.

Ziegler, K., E. R. Unanue.
. Identification of a macrophage antigen-processing event required for I-region-restricted antigen presentation to T lymphocytes.
J. Immunol.
Gell, P. G., B. Benacerraf.
. Studies on hypersensitivity. II. Delayed hypersensitivity to denatured proteins in guinea pigs.
Hersh, E. M., J. E. Harris.
. Macrophage-lymphocyte interaction in the antigen-induced blastogenic response of human peripheral blood leukocytes.
J. Immunol.
Waldron, J. A., Jr, R. G. Horn, A. S. Rosenthal.
. Antigen-induced proliferation of guinea pig lymphocytes in vitro: obligatory role of macrophages in the recognition of antigen by immune T-lymphocytes.
J. Immunol.
Rosenthal, A. S., E. M. Shevach.
. Function of macrophages in antigen recognition by guinea pig T lymphocytes. I. Requirement for histocompatible macrophages and lymphocytes.
J. Exp. Med.
Zinkernagel, R. M., P. C. Doherty.
. Immunological surveillance against altered self components by sensitised T lymphocytes in lymphocytic choriomeningitis.
Unanue, E. R., B. A. Askonas.
. Persistence of immunogenicity of antigen after uptake by macrophages.
J. Exp. Med.
Mackaness, G. B..
. The immunological basis of acquired cellular resistance.
J. Exp. Med.
Ziegler, H. K., E. R. Unanue.
. Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells.
Proc. Natl. Acad. Sci. USA
Walden, P., Z. A. Nagy, J. Klein.
. Induction of regulatory T-lymphocyte responses by liposomes carrying major histocompatibility complex molecules and foreign antigen.
Klein, J..
. Alone on the heart of the earth: an immunogeneticist’s journey into the past.
Adv. Cancer Res.
Babbitt, B. P., P. M. Allen, G. Matsueda, E. Haber, E. R. Unanue.
. Binding of immunogenic peptides to Ia histocompatibility molecules.