Thymus negative selection

In 1959–1961, I was far from Burnet's influence studying the pathogenesis of virus-induced mouse leukaemia in London, UK. As a result of this work, I discovered the immunological function of the thymus.¹ How that came about has been recounted elsewhere.² Grafting thymus tissue reversed the immune defects of neonatally thymectomized mice but when thymus came from a strain of mice allogeneic to the thymectomized recipients, these were able to reject skin grafts from the allogeneic strains tested but not from the thymus donor strain (Figure 1). This led me to suggest, 'when one is inducing a state of immunological tolerance in a newly born animal, one is in effect performing a thymectomy, not a complete thymectomy but a partial, selective or immunological thymectomy. In accordance with selective theories of antibody formation, injected antigenic material might make contact with certain cell types differentiating in the thymus and in some way prevent these cells from maturing to a stage when they would be capable of reacting immunologically.³ In other words, the thymus could negatively select antigen-specific clones in vivo and might be the seat where self-tolerance was induced. It was, therefore, a great surprise to me when Burnet asked me to see him during his visit in London in early 1962 and I was immensely pleased to show him the thymus graft results that were in press in the Proceedings of the Royal Society.³ He was, at that time, one of the few immunologists convinced of the immune function of the thymus, as was evident in the lecture he later gave in London.⁴

Figure 1.

Skin graft survival in neonatally thymectomized (Ak T6) F1 mice (H-2k) grafted with 1-day-old Ak (H-2k) (green), C3H (H-2k) (blue) or C57BL (H-2b) (red) thymus. Data from Miller.³

Full figure and legend (78K)

The finding that thymus-deficient mice were much more prone than normal mice to develop tumours after treatment with certain carcinogenic chemical agents and oncogenic viruses^{5, 6} provided some experimental support for Burnet's concept of immunological surveillance.⁷ Although T cells can, in some experimental situations, act as killer cells against tumour cells, I believe that if tumour antigens do cross-react with self-antigens that are ectopically expressed in thymus medullary epithelial cells, one will not be able to prime the immune system to these antigens.²

Positive selection of antigen-specific T-cell clones

In 1966, I headed the Experimental Pathology Unit of the Walter and Eliza Hall Institute of Medical Research following the invitation of the then director, Gustav Nossal. With my first PhD student, Graham Mitchell, we gathered more evidence for in vivo selection of antigen-specific clones. As admirably recounted by Graham Mitchell in this symposium,⁸ we used genetically marked cells and adoptive transfer to established unequivocally and for the first time: (a) that thymus-derived cells could be activated specifically by antigen⁹ (b) that they were not the precursors of antibody-forming cells,^{10, 11} (c) that they were essential to help, via some type of collaboration, other lymphocytes derived from bone marrow to respond to antigen by producing antibody.¹¹ These results were initially greeted with surprise and scepticism (for example, ref¹²), a common criticism being that two rare, clonally individuated lymphocytes would never find each other, let alone meet. The discovery of T and B cell collaboration was a major milestone: it not only opened up the field of cell interactions, but literally changed the course of immunology; previously unexplained, immunological phenomena, such as the carrier effect, tolerance, memory, genetically determined unresponsive states, immunodeficiency and autoimmunity had to be re-assessed in terms of the role played by either T or B cells.

Another PhD student, Jonathan Sprent, joined my unit and became very proficient in the arduous task of collecting lymphocytes from the mouse thoracic duct. Thanks to his efforts, we were able to show in vivo positive selection of antigen-specific clones from the recirculating lymphocyte pool into areas where antigen had been deposited. We injected sheep erythrocytes into normal adult CBA mice that were then cannulated to obtain thoracic-duct lymphocytes (the majority of which were known to be T cells) at various times thereafter. These lymphocytes were then injected intravenously together with sheep and horse erythrocytes (known to be non-cross reactive) into groups of T-cell deficient adult thymectomized, irradiated and marrow protected CBA mice that served as a source of unstimulated B cells (Figure 2). Thoracic duct cells obtained 1–2 days after injection of sheep erythrocytes were deficient in adoptively transferring immune reactivity to sheep erythrocytes but were normal with respect to horse erythrocytes (Figure 3).¹³ This suggested that sheep erythrocyte antigen-specific clones had been recruited out of the recirculating pool into the spleen where the antigen had been deposited by the intravenous injection. Thoracic duct cells collected at 3 days had normal reactivity, but cells collected at 5 days allowed their T-cell deficient hosts to produce an enhanced response to sheep erythrocytes and a slightly depressed response to horse erythrocytes. Similar findings were made using histoincompatible cells as antigen.¹³

Figure 2.

Experimental design to show recruitment of antigen-specific clones from the recirculating lymphocyte pool into areas where antigen has been deposited. TDL=thoracic duct lymphocytes; ATXXBM=adult thymectomized, irradiated, marrow protected; SRBC=sheep erythrocytes; HRBC=horse erythrocytes; AFC=antibody-forming cells.

Full figure and legend (81K)

Figure 3.

Antibody-forming cell response of thymectomized, irradiated, marrow protected CBA mice given sheep () and horse () erythrocytes and thoracic duct lymphocytes. Abbreviations as in Figure 2. Data from Sprent et al.¹³

Full figure and legend (74K)

Conclusion

Taken together, the results I have outlined are clearly supportive of Burnet's clonal selection theory.

References

1. Miller JFAP. Immunological function of the thymus. Lancet 1961; 2: 748–749. | Article | PubMed | ISI | ChemPort |
2. Miller JFAP. Vestigial no more. Nat Immunol 2006; 7: 3–5. | Article | PubMed | ChemPort |
3. Miller JFAP. Effect of neonatal thymectomy on the immunological responsiveness of the mouse. Proc Roy Soc 1962; 156B: 415–428.
4. Burnet FM. The role of the thymus and related organs in immunity. Br Med J 1962; 2: 807–810. | PubMed |
5. Miller JFAP, Grant GA, Roe FJC. Effect of thymectomy on the induction of skin tumours by 3,4-benzopyrene. Nature 1963; 199: 920–922. | Article | PubMed | ChemPort |
6. Miller JFAP, Law LW, Ting RC. Influence of thymectomy on tumor induction by polyoma virus in C57BL mice. Proc Soc Exp Biol Med 1964; 116: 323–327. | PubMed | ISI | ChemPort |
7. Burnet FM. The concept of immunological surveillance. Progr Exp Tumor Res 1970; 1: 13–18.
8. Mitchell GF. Selection, memory and selective memories: T cells, B cells and Sir Mac 1968. Immunol Cell Biol 2008; in press.
9. Mitchell GF, Miller JFAP. Immunological activity of thymus and thoracic duct lymphocytes. Proc Nat Acad Sci USA 1968; 59: 296–303. | Article | PubMed | ChemPort |
10. Miller JFAP, Mitchell GF. Cell to cell interaction in the immune response. I. Hemolysin-forming cells in neonatally thymectomized mice reconstituted with thymus or thoracic duct lymphocytes. J Exp Med 1968; 128: 801–820. | Article | PubMed | ISI | ChemPort |
11. Mitchell GF, Miller JFAP. Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes. J Exp Med 1968; 128: 821–837. | Article | PubMed | ISI | ChemPort |
12. Gowans JL. Discussion after Miller JFAP. Cell to cell interaction in the immune response of mice to sheep erythrocytes. In: Landy Braun W (ed). 'Immunological Tolerance: a Reassessment of Mechanisms of the Immune Response', Symp on Immunological Tolerance, Brook Lodge, Michigan. Academic. Press: New York, 1969, pp 169.
13. Sprent J, Miller JFAP, Mitchell GF. Antigen-induced selective recruitment of circulating lymphocytes. Cell Immunol 1971; 2: 171–181. | Article | PubMed | ISI | ChemPort |