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The Thymus and T-Cell Specificity

J. Sprent, Y. Adachi, I. Hwang, H. Kishimoto, S. Sun, D.F. Tough

T cells arise in the thymus by positive and negative selection directed to self-MHC molecules and then migrate into the peripheral lymphoid tissues to form a pool of long-lived mature T cells. We are interested in the selective mechanisms that shape the T-cell repertoire and the inductive processes that control primary and secondary immune responses.

FAS AND NEGATIVE SELECTION OF THYMOCYTES IN VIVO

Fas is a cell-surface molecule found on various cell types, including T cells, that conveys signals leading to cell death via apoptosis. To gain information on the role of Fas in negative selection of thymocytes, we examined subsets of thymocytes from normal neonatal mice and from neonatal Fas-deficient lpr/lpr mice; both groups of animals were injected with graded doses of antigen. In normal mice, injection of 1--100 µg of staphylococcal enterotoxin B induced clonal elimination of Vß8⁺ cells reactive with the enterotoxin at the level of the semimature population of HSA^hi CD4⁺ CD8^-- cells found in the thymic medulla; deletion of fully mature HSA^lo CD4⁺ CD8⁺ thymocytes was minimal. Injection of the enterotoxin also caused marked elimination of Vß8⁺ HSA^hi CD4⁺ CD8^-- thymocytes in lpr/lpr mice. Paradoxically, however, elimination of these cells in lpr/lpr mice was induced by low-to-moderate doses of enterotoxin (¾1 µg) but not by high doses (100 µg). Injection of specific peptide into mice transgenic for the T-cell receptor (TCR) resulted in similar findings.

These data suggest that clonal elimination of semimature medullary T cells is Fas independent at low doses of antigen but Fas dependent at high doses. Previous reports that negative selection is not obviously impaired in lpr/lpr mice could thus reflect that the antigens studied were expressed at only a low level.

LIFE SPAN OF / T CELLS

T cells expressing / TCR molecules form a small subset of T cells of largely unknown function. The turnover and life span of murine / cells was examined by administering the DNA precursor bromodeoxyuridine in the drinking water of mice and then determining incorporation of the precursor by lymphoid cells obtained from the mice. For / (V2) TCR transgenic mice, nearly all / thymocytes showed incorporation of bromodeoxyuridine within 2 days of treatment and were released rapidly into the peripheral lymphoid tissues. These recent thymic emigrants underwent phenotypic maturation in the periphery for several days, but most of the cells died within 4 weeks. In adult thymectomized transgenic mice, only a small proportion of / cells survived as long-lived cells; most of these cells had a slow turnover and retained a naive phenotype. As in transgenic mice, the majority of recent thymic emigrants generated in normal mice (C57BL/6) appeared to have a restricted life span as naive cells. However, in marked contrast to the findings in TCR transgenic mice, most of the / cells that survived in adult thymectomized normal mice had a rapid turnover and displayed an activated or memory phenotype, implying a long-term response to environmental antigens. Hence, in normal mice, many / recent thymic emigrants did not die but became memory cells.

DNA AS AN ADJUVANT

Responses of mature T cells to soluble antigens in vivo are generally low or undetectable unless the antigens are accompanied by an adjuvant. How strong adjuvants such as complete Freund's adjuvant promote T-cell priming, however, is still unclear. Because the nonmethylated CpG motifs in DNA of bacteria and other nonvertebrates are stimulatory for B cells and antigen-presenting cells, the strong adjuvanticity of complete Freund's adjuvant could be attributed, at least in part, to the presence of dead bacteria (i.e., a source of stimulatory DNA). In support of this possibility, evidence has been obtained that insect DNA in mineral oil has even stronger adjuvant activity than does complete Freund's adjuvant. Synthetic oligodeoxynucleotides containing nonmethylated CpG motifs mimic the effects of insect DNA, and, even in soluble form, oligodeoxynucleotides markedly potentiate clonal expansion of TCR transgenic T cells responding to specific peptides.

STIMULATION OF MEMORY PHENOTYPE CD8⁺ T CELLS IN VIVO BY IL-15

The intense proliferation of memory-phenotype (CD44^hi) CD8⁺ cells induced by infectious agents can be mimicked by injecting type I interferon (IFN I) and by IFN I--inducing agents such as lipopolysaccharide and polyinosinic-polycytidylic acid; such proliferation does not affect naive T cells and appears to be TCR independent. Because IFN I inhibits proliferation in vitro, IFN I--induced proliferation of CD8⁺ cells in vivo presumably occurs indirectly through production of secondary cytokines, such as IL-2 or IL-15. In support of this notion, we found that unlike IL-2, IL-15 closely mimics the effects of IFN I, causing strong and selective stimulation of memory-phenotype CD44^hi CD8⁺ (but not CD4⁺) cells in vivo; purified T cells in vitro have a similar specificity for IL-15, which correlates with much higher expression of IL-2Rß on CD8⁺ cells than on CD4⁺ cells. Hence, the marked bystander stimulation of CD44^hi CD8⁺ cells that occurs during infection may reflect IFN I--induced production of IL-15.

PUBLICATIONS

Kishimoto, H., Surh, C.C., Sprent, J. A role for Fas in negative selection of thymocytes in vivo. J. Exp. Med., in press.

Korngold, R., Sprent, J. Murine models for graft-versus-host disease. In: Hematopoietic Cell Transplantation, Vol. II. Forman, S.J., Blume, K.G., Thomas, E.D. (Eds.). Blackwell Science, Cambridge, England, in press.

Sprent, J., Cai, Z., Brunmark, A., Jackson, M.R., Peterson, P.A. Constructing artificial antigen-presenting cells from Drosophila cells. Adv. Exp. Med. Biol. 417:249, 1997.

Sprent, J., Kishimoto, H. T cell tolerance and the thymus. Ann. N.Y. Acad. Sci. 841:236, 1998.

Sun, S., Beard, C., Jaenisch, R., Jones, P., Sprent, J. Mitogenicity of DNA from different organisms for murine B cells. J. Immunol. 159:3119, 1997.

Sun, S., Kishimoto, H., Sprent, J. DNA as an adjuvant: Capacity of insect DNA and synthetic oligodeoxynucleotides to augment T cell responses to specific antigen. J. Exp. Med. 187:1145, 1998.

Tough, D.F., Sprent, J. Bystander stimulation of T cells in vivo by cytokines. Vet. Immunol. Immunopathol. 63:123, 1998.

Tough, D.F., Sprent, J. Lifespan of / T cells. J. Exp. Med. 187:357, 1998.