This month’s Pillars of Immunology papers by Av Mitchison (1, 2) from 1971 provided extensive evidence for an emerging consensus on the role of cellular cooperation in the Ab response. The papers are concerned with a phenomenon called the carrier effect, defined below, that was intensely studied at that time for clues to the nature of Ag recognition and the mechanisms of immunological memory and tolerance. Specifically, these papers mapped the carrier effect onto the new finding of cellular cooperation between T lymphocytes and B lymphocytes in the Ab response.

In a paper in 1957 followed by a monograph in 1959, Macfarlane Burnet (3) proposed the clonal selection theory of Ab formation and launched the modern age of cellular immunology. In the 1950s and early 1960s, Peter Medawar, James Gowans, Jacques Miller, Robert Good, Baruj Benacerraf, and their colleagues, along with others, showed that lymphocytes are the agents of specific, adaptive immunity, and split immunity into two effector arms, cellular and humoral (Ab-mediated). Transfer experiments from immunized to naive animals demonstrated that cell-mediated immunity was responsible for graft rejection and delayed type hypersensitivity. Later in the 1960s, Jacques Miller (4, 5) and Henry Claman (6) and their colleagues showed that Ag-primed thymus-derived lymphocytes (now called T cells) cooperated with bone marrow–derived lymphocytes (now called B cells) to enable Ab formation against sheep erythrocytes. B cells were shown to be the source of the specific Abs, but the role of Ag-specific T cells in the Ab response remained mysterious. This history is nicely reviewed by Mitchison (7).

In 1967, I was a second year graduate student at the University of California, Berkeley, and my mentor, Leon Wofsy, kindly brought me along to the 1967 Cold Spring Harbor Symposium on Quantitative Biology. It was a very exciting time in immunology, and many of the leaders in Ab structure and genetics as well as the new field of cellular immunology were there. Many of the tools and concepts we take for granted had not yet been conceived: we did not have mAbs, flow cytometers, transgenic or knockout animals, or nucleic acid clones or sequences. The basic structure of Ab molecules had just been solved, and laborious amino acid sequencing of homogenous Igs from myeloma patients gave us a first look at the constant and variable regions of the component H and L chains. The enormous potential variability of the N-terminal regions of the Ig polypeptides provided a structural basis for Ab specificity, but also posed the molecular genetic puzzle of how the body could generate polypeptides that were identical at one end and variable at the other. On the cellular side, one cell to one Ab had been experimentally established in several diverse systems, and clonal selection had become the dominant paradigm, although I heard at that meeting the last gasp of instructional theories of Ab formation from Felix Haurowitz, who elegantly reviewed the history of those theories and proposed that an Ag fragment might sit on the ribosome in an Ab-forming cell, and together with the mRNA, direct the sequence of amino acids in the growing Ab chains.

Av Mitchison and Klaus Rajewsky were also present at that meeting, talking about what they had found by investigating the carrier effect. Immunologists at that time were very familiar with the hapten-carrier systems that had been used earlier in the century by Karl Landsteiner and others to precisely define the specificity of antisera. A hapten is a small, chemically defined molecule that by itself is unable to elicit an Ab response. To make Abs to the hapten, the hapten must be coupled to an immunogenic protein, a carrier (or “schlepper”), and injected with adjuvant into an animal. Some of the Abs in the resulting antiserum are specific for the hapten and will bind it directly as a small molecule or when it is coupled to an unrelated carrier protein. The carrier effect is the finding that a secondary response to the hapten requires challenging the primed animal with the homologous hapten-carrier conjugate, the same carrier that was used in the priming injection. When the primed animal is injected with the hapten coupled to an unrelated protein (a heterologous carrier), a much smaller Ab response is obtained, similar in size to the response of an unprimed (or naive) animal.

There were two contending theories to explain the carrier effect (Fig. 1) (8). One was known as the “local environment hypothesis,” the idea that the Ag receptors responsible for triggering the secondary response recognized the hapten together with adjacent structures on the carrier. To investigate the specificity of these Ag receptors, measurements were made on the resulting Abs in anti-hapten antisera, because, by the clonal selection theory, the secreted Abs are identical in specificity to the Ag receptors on the precursors of the Ab-forming cells. Some Abs in the antisera could be demonstrated to react better with the hapten on the homologous carrier, supporting the local environment hypothesis. However, other anti-hapten Abs in such antisera bound the hapten without a measureable contribution from the carrier, which could not easily be explained by the local environment hypothesis. The competing theory was that two receptors cooperatively recognized Ag: one receptor specific for the hapten that was later secreted as an Ab, and a more mysterious receptor for the carrier.

FIGURE 1.

Alternative hypotheses to account for the carrier effect. Reprinted from Mitchison (8) (with permission from Cold Spring Harbor Laboratory Press).

FIGURE 1.

Alternative hypotheses to account for the carrier effect. Reprinted from Mitchison (8) (with permission from Cold Spring Harbor Laboratory Press).

Close modal

Mitchison did the experiments reported in these papers during several years at the National Institute of Medical Research at Mill Hill in London. The initial findings were reported at meetings and published in summary form in various conference proceedings in the late 1960s. When Mitchison helped Otto Westphal found the European Journal of Immunology, these two papers were part of a backlog of work that was now ready for formal publication. These are the first two of six papers on the carrier effect in the first volume of that new journal by Mitchison and his colleagues at the National Institute of Medical Research.

In these papers, Mitchison used the cell transfer systems in inbred mice pioneered by Medawar and applied to cellular cooperation in the Ab response by Miller and Claman. These Pillars of Immunology papers are distinguished by extensive, sensitive, quantitative measurements of Ab levels in serum from animals challenged with a broad range of Ag concentrations—over 4 logs in some experiments. This enabled Mitchison to account for the intrinsic potency of his various carrier proteins and accurately compare the anti-hapten Ab responses to the homologous and heterologous carriers in terms of the displacement of the dose response curves (for example, see Fig. 2 in Ref. 1). In the first of these two papers, Mitchison introduced his quantitative methods to measure the carrier effect in cell transfer systems and presented some strong evidence against the local environment hypothesis (1). In the second paper, he showed in transfer experiments that different cell populations recognized hapten and carrier (2). Carrier-specific helper cells were primed in one animal and hapten-specific cells in another. The primed cells were then transferred into an irradiated recipient, where they produced a secondary anti-hapten response to the hapten-carrier conjugate, with appropriate reciprocal specificity controls. Moreover, Mitchison showed that the carrier-specific cells were the thymus-derived lymphocytes of cellular immunity (the T cells), because spleen cells from lethally irradiated, thymocyte-repopulated, carrier-immunized donors provided help, as did cells from the thymus-derived fraction of thymus/marrow chimeras. Thus, the hapten-carrier cooperative response was mapped onto the well-established synergy of thymus and bone marrow in the response to foreign erythrocytes (2).

In 1971, the nature of T cell help for the Ab response remained obscure. Having shown that the T cell could help the B cell only when both antigenic determinants were on the same Ag particle, Mitchison and Rajewsky et al. (9) conceived of T cell/B cell cooperation as an “antigen bridge” between a rare Ag-specific T cell and a rare Ag-specific B cell. Mitchison thought the parallel slopes of the dose–response curves in his paper indicated that the role of the T cell was to concentrate and present Ag to the B cell. It took more than another decade of work to reveal that it was the other way around: the B cell presents Ag to the T cell. I spent a couple of years as a postdoctoral fellow in Mitchison’s laboratory at University College London, much of that time unsuccessfully trying, with help from Marc Feldmann, to purify and identify “IgT,” a hypothetical Ag-specific, Ab-like molecule secreted by Th cells that was thought to bind to macrophages and allow them to present Ag in a multivalent immunogenic form to B cells.

The local environment hypothesis and the question of one versus two receptors was recapitulated in the next decade in the debate over how MHC molecules controlled Ag recognition by T cells. When I was in Mitchison’s laboratory, Gordon Ada came through and told us of Zinkernagel and Doherty’s unpublished results on MHC restriction of cytolytic T cells: they needed to recognize both the Ag and a self allele of an MHC molecule to kill target cells (10). About the same time, Rosenthal and Shevach (11) showed that a similar phenomenon governed Ag recognition on macrophages by CD4+ T cells. It was hotly debated for several years whether T cells used a single receptor or two receptors to recognize both Ag and MHC. The riddle was solved by the identification of the TCR by several laboratories in 1983, the functional studies of Ag processing and antigenic peptide presentation (1214), and finally the solution of the crystal structure of an MHC molecule with a peptide-binding groove (15), proving that T cells see Ag only as peptide fragments bound to an MHC molecule on an APC.

The analysis of the carrier effect established the role of Th cells in the Ab response, but a more complete understanding of the mechanism of T cell help had to wait until the nature of T cell recognition of Ag was discovered as described above. Now we know that B cells are Ag-specific APCs, and cooperating B cells and T cells recognize Ag sequentially and in different forms. First, the Ag binds directly to the Ag receptors on the B cell, delivering an activating signal when the Ag is multivalent. Then, the receptor-bound Ag is efficiently internalized, processed, and presented to the Th cell as a peptide fragment bound to an MHC class II molecule (16, 17, reviewed in Ref. 18). When the T cell sees Ag presented by the B cell, it delivers CD40 ligand by cell contact to enable the B cell to proliferate and produce a clone of Ab-forming cells (19, 20).

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The author has no financial conflicts of interest.