The 1960s were revolutionary in many ways, including in immunology. Immunologists had just begun to appreciate the importance of recirculating lymphocytes as mediators of immunity from the seminal work of Gowans et al. (1, 2) in Oxford. The central role of the thymus in immune responses was being elucidated by Cooper, Good, and colleagues (3, 4) in the United States and by Miller (5) in Australia from clinical immunodeficiency diseases and animal models, respectively; further studies on the role of the bursa in chickens (6) helped to define differential functions of cells derived from these organs. By the late 1960s, these studies all came together with the pioneering experiments demonstrating collaboration between cells from the thymus (as a source of T cells) and bone marrow (as a source of B cells) in Ab formation. In these studies, Miller and Mitchell (7, 8) in Melbourne, Australia, and Claman et al. (9) in Denver, CO, demonstrated that both types of cells were necessary for Ab formation, but that cells derived from the thymus and those from the bone marrow (bursa) had different functions (1012). These studies built the paradigm that the former “help” the latter to mature into Ab-secreting cells. This was demonstrated by Miller and Mitchell (8), who used semiallogeneic transfers and complement-mediated cytotoxicity and chromosomal analyses to prove the bone marrow origin of Ab-secreting cells.

Prior to that point, lymphocytes were just … lymphocytes, thought to be quiescent “end cells” owing to their high nucleus/cytoplasm ratio. Immunity could be transferred with lymphocytes from the thoracic duct (2) or from spleen and lymph node tissues. The cell collaboration experiments proved that there were at least two different cell types that cooperated to promote Ab formation and were each critical links in a chain of events. This was all accomplished before there were CD markers and monoclonals, when cytokines were just “supes,” and there were no transgenics or knockouts, and certainly no PCR or Tregs! MHC restriction was yet to be discovered. Adoptive cell transfer experiments with depletion using alloantibodies plus complement were sufficient proof of the B cell origin of Ab-forming cells and were proof that the cells from the thymus provided critical “help.”

So what about tolerance? Could T and B cells both be rendered tolerant? This was the question that Chiller et al. (13, 14) asked at the end of the decade.

Immunologists had become aware that tolerance could be acquired either in utero, based on Ray Owen’s (15) observations of dizygotic cattle twins, or was inducible in neonates, based on the Nobel Prize–winning experiments of Billingham, Brent, and Medawar (16). In 1945, Owen (15) observed that fraternal offspring had shared blood elements via vascular anastomoses and later would neither form Abs against their sibling’s red cell Ags nor reject their skin grafts. Building on that observation, the Medawar group (16) injected foreign lymphoid tissues into neonatal mice and found that these mice acquired tolerance to grafts from donors of the same strain but would reject “third party” unrelated grafts. Thus, tolerance could be acquired and was specific.

Mitchison (17) and Dixon and Mauer (18) had demonstrated that tolerance in adults could be induced to protein Ags delivered systemically via various dosing regimens. Using mice and rabbits, respectively, they found that after administering high doses of serum proteins such as albumins or γ-globulins, the recipients were unresponsive (tolerant) to subsequent challenge with the same serum protein Ag in an immunogenic form (e.g., in adjuvant).

In the later 1960s, Weigle and his colleagues at the Scripps Clinic in La Jolla, CA, built a model system that used heterologous γ-globulins as Ags. They first demonstrated that ultracentrifuged human IgG (human γ-globulin, or HGG), called “soluble” or “deaggregated” monomeric 7S IgG (19), was nonimmunogenic. However, the pelleted or aggregated HGG was highly immunogenic. When mice were first treated with the deaggregated HGG and then “challenged” with the aggregated HGG, they were specifically unresponsive. This defined the basic tolerance model they used for most of the next decade. They then repeated the basic thymus plus bone marrow collaboration experiments described above to demonstrate that the response to aggregated HGG was “T cell dependent”: only the transfer of a combination of thymus cells with bone marrow cells from a normal donor into an irradiated mouse would restore immunocompetence and Ab formation to aggregated HGG. Next, they injected donor mice with deaggregated HGG to render them tolerant and then transferred thymus cells with bone marrow cells from those donors into irradiated recipients. As expected, the recipients were tolerant to challenge with aggregated HGG, just as Gowans and colleagues (20) and others had demonstrated for whole lymphocyte populations. Finally, they mixed thymus from tolerant donors with bone marrow from normal donors (or vice versa) and transferred those mixtures to irradiated recipients as summarized in Table I (extracted from Ref. 14). Therein, they demonstrated that neither tolerant thymus nor tolerant bone marrow cells in the presence of their normal bone marrow or thymus cells, respectively, could restore Ab competence in the recipients. Akin to breaking a link in a chain, both lymphocytic links had to be competent to transfer responsiveness. This formally demonstrated that both thymus (T) and bone marrow (B) cells could be rendered tolerant (14).

Table I.

Transfer of tolerance with either thymus or bone marrow cellsa

Thymus CellsBone Marrow CellsResponse to Aggregated HGG
Normal Normal ++++ 
Tolerantb Normal — 
Normal Tolerantb — 
Tolerant Tolerantb — 
Thymus CellsBone Marrow CellsResponse to Aggregated HGG
Normal Normal ++++ 
Tolerantb Normal — 
Normal Tolerantb — 
Tolerant Tolerantb — 
a

Adapted from Chiller et al. (Table 3 in Ref. 14).

b

Donor treated with soluble HGG.

In this Pillars article, Weigle and coworkers (13) examined the kinetics of tolerance induction in T and B cells. Using the HGG tolerance model, they injected mice with a tolerogenic dose of deaggregated HGG and then removed thymus or bone marrow populations at different times after injection of tolerogen. The thymocytes from tolerized mice were mixed with normal bone marrow, and tolerant bone marrow cells were mixed with normal thymus cells in the transfer model described above (14). Mice in each of these groups were then challenged with aggregated HGG, as was a separate group of donor mice. If tolerance had been achieved in either thymus or bone marrow cells by the time of transfer, then those cells would fail to collaborate with their normal cooperating population. As shown in figure 1 of Chiller et al. (13), thymus cells became tolerant rapidly, within a day after the host received the deaggregated HGG. This tolerant state lasted 49 d according to this report but was later found to be stable for >150 d, exactly matching the tolerant state of the donor mice.

Interestingly, cells from the bone marrow required more than a week to achieve immune tolerance, and tolerance began to wane by day 35. Bone marrow transferred at day 49 was normal in terms of being able to cooperate with thymus cells. Importantly, even though the bone marrow was “normal” at day 49, the host remained tolerant. One link in the chain of immunocompetence was clearly lost. The difference in kinetics between T and B cell tolerance was postulated to either reflect the necessity of an active process in bone marrow cell tolerance or a quantitative dose-dependent difference. The latter hypothesis appeared to be more likely, as they found that tolerance in the bone marrow population required a higher dose of deaggregated HGG, although this may also reflect an active process or maturation step for bone marrow B cell precursors. The different kinetics for tolerance in T and B cells were reproduced virtually identically in rats in a hapten-carrier model described by Sanfilippo and Scott (21).

The waning of tolerance in both lymphocyte populations most likely reflected the generation of new T and B cell precursors, combined with the catabolism of available deaggregated HGG to tolerize both mature and newly emerging precursors. This was evidenced by the fact that waning was prevented or prolonged by continued exposure to low doses of deaggregated HGG or adult thymectomy (22, 23). Thus, Ag was shown to drive both immunity and tolerance, and newly generated T cells were necessary for responsiveness.

In the last 40 y, we have learned details of tolerance mechanisms in T and B cells from the use of TCR and BCR transgenics and cloned lines. Nonetheless, these classic cell transfer experiments by Weigle and colleagues continue to teach us the value of elegantly designed but simple cell transfer experiments.

This article is dedicated to the memory of Byron H. Waksman, who taught me more than tolerance.

Abbreviation used in this article:

HGG

human γ-globulin.

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The author has no financial conflicts of interest.