Immunology 2004 111 254±261 doi: 10.1111/j.1365-2567.2004.01821.x Factors affecting the susceptibility of the mouse pituitary gland to CD8 T-cell-mediated autoimmunity JAMES DE JERSEY,* DANIELLE CARMIGNAC,y PAUL LE TISSIER,y THOMAS BARTHLOTT,* IAIN ROBINSONy & BRIGITTA STOCKINGER* *Division of Molecular Immunology, and yDivision of Molecular Neuroendocrinology, The National Institute for Medical Research, Mill Hill, London, UK SUMMARY We have previously shown, in a transgenic mouse model, that the pituitary gland is susceptible to CD8 T-cell-mediated autoimmunity, triggered by a cell-speci®c model autoantigen, resulting in pan-anterior pituitary hypophysitis and dwar®sm. In the present study, we now demonstrate that antigen dose, the T-cell precursor frequency, the degree of lymphopenia and the context of target antigen expression, are important parameters determining the time course and extent of the pathological consequences of CD8 T-cell-mediated autoimmunity. Furthermore, our data indicate that the pituitary gland is susceptible to CD8 autoimmunity following an in¯ammatory insult such as a viral infection. As lymphocytic hypophysitis may be manifest in other autoimmune conditions, and the pituitary gland may be susceptible to T- cell-mediated pathology after immunization with a virus expressing soluble pituitary antigen, it is important to consider that strategies based on vaccination against soluble pituitary gonadotrophins could have other unexpected endocrine consequences. presentation of soluble protein is relatively inef®cient,9 and INTRODUCTION proteins such as hormones, which occur in small quantities in Thymic clonal deletion is the major mechanism of T-cell the bloodstream, may not reach suf®ciently high concentrations tolerance to self-antigens. Recent studies have shown that even to be cross-presented to CD8 T cells. However, when soluble several `tissue-speci®c' and developmentally regulated proteins antigen is present in high quantities, CD8 T-cell tolerance may are expressed by rare medullary thymic epithelial cells occur by cross-presentation to T cells by professional antigen- (mTECs)1,2 and it has been proposed that T-cell engagement presenting cells (APCs)10 and, in some cases, non-professional of self-peptides, expressed by mTECs, may impose a regulatory APCs.11 Cell-associated (necrotic or apoptotic cells) antigen is T-cell phenotype. Despite this additional mechanism of thymic ef®ciently cross-presented to CD8 T cells9 and this is a well- regulation, CD8 T cells with self-antigen speci®city do leave the described mechanism of peripheral tolerance to tissue-restricted thymus and enter the periphery, with the potential to cause antigens.12 autoimmunity (reviewed in ref. 3). In most situations these T The role of CD8 T cells in autoimmune diseases is gradually cells will not become autoaggressive, but instead may engage becoming better understood. In mice, it has been shown that major histocompatibility complex (MHC) antigen yet remain CD8 T cells can mediate experimental autoimmune encepha- unresponsive,4 be activated and undergo proliferation before lomyelitis (EAE)13,14 and diabetes,15,16 caused by pancreatic b- dying of activation-induced cell death,5,6 or never encounter cell damage. Indeed, endocrine organs with a specialized their cognate antigen and remain ignorant.7,8 vasculature into which large quantities of secreted protein are Ignorance may occur because T cells do not migrate to the abruptly secreted, may be particularly susceptible to an auto- site of antigen expression, or the antigen may not be presented in immune pathology, readily detected by the obvious phenotypic the correct context for T-cell recognition. For example, cross- consequences of endocrine de®ciency.17 Although reportedly rarer than other autoimmune endocrinopathies, the pituitary gland of humans is susceptible to such attack.18 A putative Received 31 October 2003; revised 5 January 2004; accepted 5 autoantigen has recently been described,19 but the role of CD8 T January 2004. cells in lymphocytic hypophisitis is unknown. Correspondence: Dr B. Stockinger, Division of Molecular Immuno- We previously developed amodel of this condition in logy, The Ridgeway, Mill Hill, London NW7 1AA, UK. E-mail: transgenic mice and showed that the mouse pituitary gland is [email protected] susceptible to CD8 T-cell-mediated autoimmunity, triggered by 254 # 2004 Blackwell Publishing Ltd T-cell activation by secreted and intracellular protein 255 a cell-speci®c model autoantigen, resulting in pan-anterior pituitary hypophysitis, multiple hormone de®ciencies and dwar®sm.20 In the present study we now demonstrate that antigen dose, the context of expression of the target antigen, the T-cell precursor frequency and the degree of lymphopenia, are important parameters in determining the time course, extent and pathological consequences of CD8 T-cell-mediated auto- immunity. This model also shows that the pituitary gland may be highly susceptible to CD8 autoimmunity following an in¯ammatory insult, such as a viral infection. MATERIALS AND METHODS Mice Rag1À/À H-2b (RagÀ/À), F5 Rag1À/À H-2b (RagÀ/À F5) mice21 and F5 Rag1À/À H-2b mice expressing green ¯uorescent protein (GFP) under control of the CD2 promoter in all T cells (GFP- F5),22 were bred in the animal facilities of the National Institute for Medical Research (London, UK), in accordance with estab- Figure 1. Expression of the in¯uenzanucleoprotein A/NT/60/68 (NP) À/À À/À lished guidelines. 48-Rag1À/À H-2b mice, transgenic for the in the pituitaries of D.2-Rag and 48-Rag mice. (a) Schematic in¯uenzanucleoprotein A/NT/60/68 (NP), andthe double- representation of the NP transgenic constructs used to generate the D.2- RagÀ/À and 48-RagÀ/À lines. The NP gene replaced the sequence transgenic F1 intercross with F5, were as described previously20 À/À À/À between exons 2 and 5 of the human growth hormone (hGH) gene. and were termed 48-Rag and 48-Rag F5 throughout this Note the deletion of the growth hormone (GH) signal sequence (SS) in study. These mice expressed the NP epitope fused to the human the D.2-RagÀ/À construct. Arrows indicate the binding sites of primers growth hormone (hGH) signal peptide, which was driven by a used for genotyping and non-quantitative reverse transcription±polymer- 40-kb hGH locus control region (LCR) promoter that restricts ase chain reaction (RT±PCR). (b) Non-quantitative RT±PCR of pituitary expression to the regulated secretory vesicle pathway in RNA and PCR of genomic DNA from adult D.2-RagÀ/À and 48-RagÀ/À pituitary GH cells (somatotrophs).20 In this study, additional mice. mice were created that had a truncated variant of this construct (see below); these D.2-Rag1À/À H-2b mice, and the double- AGCTGTGGCTTCTAGCTGCCCGGGTG-30 and 50-GCCTG- transgenic F1 intercross with F5, are termed D.2-RagÀ/À GGGAGAAACCAGAGGGCAAC-30 (50 phosphorylated). The and D.2-RagÀ/À F5, respectively. Finally, mice transgenic for deletion left the splice acceptor region between exon 1 and exon NP on a polyclonal C57BL/10 background are called 48-B10 and 2 intact and generated a PvuII site at the junction between exon D.2-B10. Table 1 summarizes the genotypes of the strains used. 2 and the remainder of exon 5. The NP insert was derived from in¯uenza A/NT/60/68 nucleoprotein (GenBank accession no. Construction of the D.2-GH-NP cosmid for generating J02137). An NP fragment was generated by PCR and was transgenic animals identical to the NP expressed in the previously described Polymerase chain reaction (PCR) mutagenesis was used to `48' line,20 except for changes to the 50 end to allow in-frame delete the hGH-coding sequence between the ®rst codon of fusion with the mutated hGH exon 2 (see above). The primers hGH exon 2 and the last four codons of exon 5 (Fig. 1a). The used for the PCR were: sense, 50-CAGCTGCGCGCTGGTG- deletion removed virtually all of the hGH signal peptide and was TGGATGG-30; antisense, 50-CAGCTGCCAGCGTAGTCTGG- created using the ExSiteTM PCR site-directed mutagenesis kit GACGTCGTATGGGTAATTGTCGTACTCCTCTGC-30. The (Stratagene, Cedar Creek, TX) and the following primers: 50- NP fragment was ¯anked by PvuII sites and encoded both a truncated NP protein (amino acids 2, 327±498) and the in¯u- Table 1. Genotypes of the mouse strains used in this study enza haemagglutinin (HA) epitope, YPYDVPDYA. NP was subcloned into the neo PvuII site in vector pN3/M-DGH-NP Mouse strain TCR Rag-1 MHC 48 D.2 GFP that contained the mutated hGH sequence ¯anked by MluI sites. RagÀ/À ± À/À b ÀÀ À The mutated, transgenic sequence was subcloned into the B2K 23 F5-RagÀ/À F5 À/À b ÀÀ À cosmid, containing a 40-kb insert including the hGH LCR, to 48-RagÀ/À ± À/À b À ± generate the construct for microinjection. The ®nal product 48-RagÀ/À F5 F5 À/À b À À would be predicted to express an HA-tagged NP fusion protein, 48-B10 polyclonal /À b À À but with only the ®rst three residues of the hGH signal peptide, 48-B10-F5 F5polyclonal /À b À À and would thus not be expected to enter the regulated secretory À/À D.2-Rag ± À/À b À À pathway. D.2-RagÀ/À F5 F5 À/À b À À D.2-B10 polyclonal /À b À À Generation of transgenic mice GFP-F5 F5 À/À b ÀÀ Cosmid cosDGH-NP DNA was linearized by digestion with GFP, green fluorescent protein; MHC, major histocompatibility complex; NotI. The fragment was puri®ed by gel extraction and column TCR, T-cell receptor. elution (Schleicher and Schuell, Dassel, Germany) and adjusted # 2004 Blackwell Publishing Ltd, Immunology, 111, 254±261 256 J.