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

JAMES DE JERSEY,* DANIELLE CARMIGNAC,y PAUL LE TISSIER,y THOMAS BARTHLOTT,* IAIN ROBINSONy & BRIGITTA STOCKINGER* *Division of Molecular , 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 F5‡polyclonal ‡/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. de Jersey et al.

to aconcentrationof 1±5 ng/ mlin0Á5mM EDTA, 1 mM Tris± in 50 ml of phosphate-buffered saline (PBS). In some experi- HCl, pH 7Á5. Transgenic mice were generated by pronuclear ments, spleen and lymph node cells containing a total of microinjection of fertilized oocytes of superovulated Rag1À/À 5 Â 106 GFP-F5 CD8 T cells, were co-injected with virus H-2b mice, followed by oviductal transfer into pseudopregnant i.v., or transferred alone, into host mice, in a total volume of recipients. 0Á2 ml. To monitor in vivo proliferation, 10 Â 106 F5 T cells were labelled with CFSE, as described previously,25 and Genotyping of transgenic animals injected i.v. into recipient mice in atotalvolume of 0 Á2ml Genomic DNA from tail biopsies was analysed for transgene of air-buffered IMDM. Before injection, total spleen and lymph DNA by a standard PCR using NP-speci®c primers: forward, 50- node cells (107/ml) were incubated with 2Á5 mM CFSE in PBS at GCTCACCTAGCGGCAATGGC-30; and reverse, 50-AGGC- 378 for 10 min. Four days after transfer, T cells were recovered CCTCTGTTGATTAGTGTTTC-30. The PCR conditions were: from spleen and lymph nodes and analysed for CFSE by 948 for 45 seconds, 528 for 30 seconds, and 728 for 45 seconds, ¯uorescence-activated cell sorter (FACS) analysis. per cycle, for 35 cycles. FACS analysis Reverse transcription±polymerase chain reaction (RT±PCR) Single-cell suspensions of pituitaries, spleen or lymph nodes Total RNA was extracted from whole pituitaries using the were stained by sequential incubations on ice with antibodies TRIZOL1 reagent (Life Technologies, Rockville, MD). cDNA speci®c for cell-surface markers followed by streptavidin-¯uor- was generated with reverse transcriptase and random hexamers ochrome conjugates, when necessary. Non-speci®c staining via (GeneAmp1 RNA PCR Core Kit; Perkin Elmer, Shelton, NJ) and FcR binding was blocked by initial incubation with the anti-FcR used in a standard PCR with the NP-speci®c primers and con- clone, 2.4G2. Cell data were acquired on a FACScalibur (Bec- ditions speci®ed above. The semiquantitative RT±PCR was ton-Dickinson Co., San Jose, CA) using CELLQUEST software. carried out using age-matched pituitaries from male and female Antibodies and secondary reagents are listed as follows: CD8a- D.2-RagÀ/À and 48-RagÀ/À mice. cDNA was reverse transcribed APC (53-6.7); CD8a-phycoerythrin (PE) (53-6.7); T-cell from approximately 1 mgoftotalRNA,usingSuperscriptIII receptor (TCR)-b-APC (H57); CD44-biotin (IM7) and strepta- (Invitrogen, Carlsbad, CA) and an oligo-dT primer, according to vidin-Cy7PE (all Becton-Dickinson). NP-epitope (ASNENM- the manufacturer's instructions. Dilutions of the cDNAwere used DAM)-speci®c T cells were detected using recombinant soluble in aPCR with primers thatwere speci®c for GH gene sequences dimeric mouse H-2Db±immunoglobulin fusion proteins (DimerX and ampli®ed both endogenous and transgenic cDNA: forward, I; Becton-Dickinson). Dimers were reconstituted overnight at 50-AACCACTCAGGGTCCTGTGGACAG-30;andreverse,378 in PBS at a concentration of 10 mg/ml in a160- M excess of 50-ATGCAACTTAATTTTATTAGGACAA-30. The PCR condi- peptide. Cells were stained with the H-2Db±immunoglobulin tions were: 948 for 30 seconds, 588 for 30 seconds, 728 for 1 min, reagent (2Á5 mg/ml) for 1 hr on ice followed by incubation with per cycle, for 29 cycles (these conditions were previously tested PE-conjugated anti-mouse immunoglobulin G (IgG)1 monoclo- to show that at the dilution range used this number of cycles is nal antibody (mAb) A85-1 (Becton-Dickinson). Intracellular still in the linear range of ampli®cation). interferon-g (IFN-g) was detected in T cells following a 4-hr stimulation at 378 in complete IMDM containing phorbol-12,13 In vitro antigen-presentation assay dibutyrate (PdBu) (50 ng/ml), ionomycin (50 ng/ml) and brefel- The relative quantity of transgenic NP in D.2-RagÀ/À, din A (10 mg/ml). Cells were washed, stained for cell-surface 48-RagÀ/À and RagÀ/À (negative control) pituitaries was esti- markers (as described above) and then ®xed in 1% paraformal- mated in an in vitro cross-presentation assay. Pituitaries were dehyde for 15 min on ice and permeabilized in 1% Nonidet P-40 homogenized in complete Iscove's modi®ed Dulbecco's med- (NP-40) for 3 min on ice. Cells were incubated with rat anti- ium (IMDM) supplemented with 5% fetal calf serum (FCS). F5 interferon-g (IFN-g) or rat IgG1-PE antibody isotype control lymph node T cells were incubated at 378 for 10 min at a cell (Becton-Dickinson). Analysis of FACS data was performed with density of 107/ml with 1 mM carboxy ¯uorescein diacetate either CELLQUEST (Becton-Dickinson) or FLOWJO software (Tree succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) Star Inc., San Carlos, CA). in phosphate-buffered saline (PBS) and washed thoroughly. Bone-marrow-derived H-2b DCs were generated in vitro by Radioimmunoassays 6 days of culture in granulocyte±macrophage colony-stimulat- Mouse GH in pituitary extracts was assayed by radioimmuno- ing factor (GM-CSF)-containing medium, as previously des- assay (RIA),26 using reagents kindly provided by NIDDK cribed.24 Pituitary extracts were titrated and incubated with (Bethesda, MD). Unless otherwise stated, data are shown as 1 Â 104 dendritic cells (DCs) and 5 Â 104 CFSE-labelled F5 T mean Æ standard deviation (SD). A two-tailed Student's t-test cells in complete IMDM, containing 5% FCS, for 5 days at 378. was used to estimate P-values, and a P-value of <0Á05 was NP peptide (ASNENMDAM) (1 nM) was used as a positive considered signi®cant. control. Proliferation of F5 T cells was assessed by pooling cells from triplicate wells and analysis by ¯ow cytometry, as described below. RESULTS Relative expression of NP by pituitary somatotrophs Virus infection and cell transfers For virus infections, atotalof 10 6 plaque-forming units (PFU) To study the role of the context of antigen expression in vivo,we of in¯uenza virus A/NT/60/68 was injected intravenously (i.v.) used two distinct lines of transgenic mice that expressed an NP

# 2004 Blackwell Publishing Ltd, Immunology, 111, 254±261 T-cell activation by secreted and intracellular protein 257 epitope exclusively in the somatotrophs of the anterior pituitary gland. In `48' strain mice (described above and previously20), pituitary somatotrophs produce NP with a signal peptide that directs its packaging to secretory vesicles and secretion in a manner similar to that of GH. The new line reported here as `D.2', expresses the same NP epitope, but from a truncated product that lacks a signal peptide, so that the protein is retained in the cytoplasm of somatotrophs and does not reach the regulated secretory pathway. Each of these transgenic lines was bred with either RagÀ/À F5 mice, which express amono- clonal population of CD8 T cells speci®c for in¯uenza A/NT/ 60/68 NP epitope 366±374, RagÀ/À mice or C57BL/10 (B10) mice. Expression of NP mRNA in both the 48-RagÀ/À and D.2- RagÀ/À transgenic strains was demonstrated by RT±PCR (Fig. 1b), con®rming that mRNA transcripts of the correct size were present in transgenic pituitaries. NP protein was detected by Western blotting and immunocy- tochemistry in the 48-RagÀ/À strain only and not in the D.2- RagÀ/À strain. This indicated that the D.2-RagÀ/À strain expressed lower amounts of NP protein. Alternatively, faster degradation of Figure 2. (a) In vitro proliferation of carboxy ¯uorescein diacetate NP protein in the cytoplasmic compartment in the D.2-RagÀ/À succinimidyl ester (CFSE)-labelled F5 T cells in response to cross- strain might compromise its detection with HA antibody. presentation of D.2-RagÀ/À, 48-RagÀ/À and RagÀ/À pituitary extracts by To determine relative expression levels in a semiquantitative bone marrow-derived H-2b dendritic cells (DCs). (b) In vitro prolifera- manner, two assays were performed. First, a novel T-cell-based tion of CFSE-labelled F5 T cells incubated with DCs and 1 nM bioassay was devised to directly measure the amount of protein ASNENMDAM peptide. (c) Semi-quantitative PCR analysis of in¯u- enza nucleoprotein A/NT/60/68 (NP) transgene expression. RNA was in pituitaries. Pituitary extracts from age- and gender-matched À/À À/À À/À À/À extracted from the pituitaries of 48-Rag and D.2-Rag mice and 48-Rag and D.2-Rag mice were titrated in culture with reverse transcribed, as described. Dilutions of cDNA were ampli®ed b CFSE-labelled F5 T cells and H-2 DCs. Both extracts were with primers speci®c to NP and growth hormone (GH). The GH processed by DCs and caused a comparable low level of semiquantitative PCR was used to control for the concentration of proliferation by the F5 T cells (Fig. 2a). The level of prolifera- cDNA in each sample. tion of the F5 T cells should be related to the quantity of pituitary-derived NP peptide processed and presented by DCs. animals with more slowly developing GH de®ciency can achieve More considerable proliferation of T cells was observed when normal size in adulthood.27,28 The difference in growth pheno- DCs were coated with 1 nM NP peptide (Fig. 2b). Pituitary types between 48-RagÀ/À F5 mice and D.2-RagÀ/À F5 mice extract from control RagÀ/À mice did not cause any F5 T-cell could be explained by the lower level of antigen expression proliferation (Fig. 2a). combined with its intracellular context in the D.2-RagÀ/À F5 Second, a semiquantitative RT±PCR was used to measure mice. Both parameters might result in delayed T-cell recognition NP mRNA levels in the pituitaries of the 48-RagÀ/Àand D.2- and activation, resulting in a delayed onset and severity of GH RagÀ/À mice. While the bioassay suggested that pituitaries from de®ciency. the two strains contained similar quantities of NP protein, the Non-secreted NP appears to take longer to reach peripheral T semiquantitative RT±PCR demonstrated considerably lower cells when compared with secreted NP (Fig. 3d), as shown by levels of NP mRNA in the D.2-RagÀ/À strain (Fig. 2c). the fact that CFSE-labelled F5 T cells transferred into T-cell- replete D.2-B10 hosts of different ages only divided in the lymph nodes of mice more than 2 weeks old (Fig. 3d). This D.2-RagÀ/À F5 double-transgenic mice have a normal delay in activation of F5 T cells may explain the normal growth growth phenotype of D.2-RagÀ/À F5 mice. The gradual accumulation of T cells in We previously reported that 48-RagÀ/À mice are normal, but that the pituitaries of these mice eventually overcomes the ability of 48-RagÀ/À F5 mice, which are double transgenic for the secreted the somatotroph population to proliferate in response to somato- form of NP and the F5 TCR, show an early massive destruction of troph damage, so that GH de®ciency becomes manifest in GH-producing cells and develop a dwarf phenotype.20 We now adulthood. In contrast, the pituitaries of 48-RagÀ/À F5 mice show that the growth phenotype of both D.2-RagÀ/À and D.2- expressing the secreted form of the NP antigen are in®ltrated by RagÀ/À F5 mice was normal (Fig. 3a). However, T-cell in®ltra- T cells as early as 2 weeks after birth (J. de Jersey, unpublished tion was seen in the pituitary of some adult D.2-RagÀ/À F5 mice observation). Transferred CFSE-labelled F5 T cells divided in (Fig. 3c), and when GH levels were measured in 16-week-old the lymph nodes of 2-week-old 48-B10 hosts (Fig. 3d). D.2-RagÀ/À F5 mice, they were signi®cantly (P ˆ 0Á003) lower than in age-matched RagÀ/À F5 control mice (Fig. 3b). In Pituitary pathology is less severe on a Rag‡ background rodents, growth becomes GH dependent at  3 weeks of age. Thus, even with severe GH de®ciency from birth, growth defects In our previous study, the marked and early-onset pituitary only become apparent during the third postnatal week, and damage occurred in Rag-negative mice (`48-RagÀ/À F5') that

# 2004 Blackwell Publishing Ltd, Immunology, 111, 254±261 258 J. de Jersey et al.

(a) 35 (b) 60 g) µ 25 40

15 Weight (g) 20 Pituitary GH ( 5 0 46810121416 ∆.2-Rag-/--F5 Age (weeks) Rag-/--F5

(c)

CD8 Figure 4. Characteristics of F5 T-cell receptor (TCR) transgenic mice CD44 on a Rag-positive background. (a) Absolute numbers of F5 T cells in spleens of age- and gender-matched B10-F5 (Rag‡, n ˆ 5) and RagÀ/À (d) B10 48-B10 ∆.2-B10 F5(n ˆ 5) mice. (b) Lymph node cells from B10-F5 (Rag‡, n ˆ 5) and 20 RagÀ/À F5 (n ˆ 5) mice were stained with anti-CD8 (y-axis) and the 15 major histocompatibility complex (MHC)±peptide dimer reagent b 10 [D ::NP68(366±374)]. Plots are representative examples from each group of mice and percentages are mean values. Error bars are standard 0/5 8/8 0/7 3/5 7/10 % T cells divided 5 deviations.

0 various 13 14 23 >35 Age (days) quanti®ed with a dimeric major histocompatibility complex (MHC) reagent, the Rag‡ B10-F5 mice contained a greater Figure 3. Characteristics of double-transgenic D.2-RagÀ/À F5 mice. (a) number of F5 T cells than age- and gender-matched RagÀ/À F5 Growth of male D.2-RagÀ/À F5 (*)micecomparedwithF5(*) and 48- counterparts (Fig. 4a). A high degree of allelic exclusion was À/À À/À Rag F5 (Â) mice. Growth data for the 48-Rag F5 mice have been observed in B10-F5 mice, with 96% of lymph node CD8 T 20 previously published and are included here for comparison only. (b) cells bearing the transgenic F5 TCR (Fig. 4b). Therefore, a Pituitary GH levels in individual 16-week-old male D.2-RagÀ/À F5 (*; reduced frequency of F5 T cells was not likely to be the reason n ˆ 4) and RagÀ/À F5 (*; n ˆ 6) mice. The mean value for each group is indicated by a horizontal line. (c) High CD44 expression in for the less severe phenotype observed in 48-B10 mice. Other pituitary-in®ltrating CD8‡ T-cell receptor (TCR)‡ cells isolated from a probable factors include the lack of lymphopenia in 48-B10-F5 12-week-old D.2-RagÀ/À F5 mouse. (d) In vivo proliferation of trans- mice that had normal-sized spleens. Thus, activated F5 T cells ferred, CFSE-labelled F5 T cells in the cervical lymph nodes of B10, had less `space' to ®ll in these mice. Additionally, it is con- 48-B10 and D.2-B10 mice of various ages. B10 mice (n ˆ 5), 48-B10 ceivable that some of the F5 TCR-bearing CD8 T cells had a (13 days old; n ˆ 8) and D.2-B10 mice (14 days old, n ˆ 7; 23 days old, second TCR, possibly reducing the functional capacity of these n ˆ 5; and >35 days old, n ˆ 10) were injected intravenously (i.v.) with dual-receptor cells.29,30 The mean ¯uorescence intensity of F5 6 10  10 T cells. The percentage of transferred T cells that had under- TCR staining in B10-F5 mice (397 Æ 22) was slightly reduced gone one or more divisions was determined by ¯uorescence-activated cell compared to that of RagÀ/À F5 mice (433 Æ 51). The normal sorter (FACS) analysis, 4 days after cell transfer. Numbers indicate the pituitary phenotype in 48-B10 mice suggests that their NP- proportion of mice in each group in which the F5 T-cell proliferation was observed. speci®c T-cell precursor frequencies are too low to allow for functional recognition of pituitary-derived NP, or that if such activation occurs, it is either controlled or is insuf®cient to contained a high number of monoclonal transgenic T cells.20 overcome the normal mechanisms that maintain an adequate Here, we address the effect of T-cell-precursor frequency and number of GH-producing somatotrophs.31 lymphopenia on the degree of pituitary pathology. 48-RagÀ/À F5 mice were bred with B10 mice to generate two CD8 T cells cause pituitary pathology after virus infection different strains: 48-B10-F5 and 48-B10. 48-B10-F5 mice were Rag‡/± and heterozygous for the transgenic F5 TCR. 48-B10 In the absence of immunization, 48-B10 mice do not suffer any mice no longer had a transgenic TCR and were also Rag- pituitary damage. Their pituitaries are free of T cells until an old suf®cient. We previously reported that the pituitaries of 16- age and they have a normal growth phenotype and normal GH week-old 48-B10-F5 mice were GH-de®cient (3% of normal levels (Fig. 5). When 48-B10 mice were infected with in¯uenza GH levels), but the de®ciency was far less severe than that seen virus A/NT/60/68, NP-speci®c T cells were detectable in the in age-matched 48-RagÀ/À F5 mice (undetectable levels).20 48- peripheral lymph nodes and spleen at day 7 after infection. This B10 mice, without any transgenic T cells, had normal levels of population of endogenous ASNENMDAM (NP 366±374) epi- pituitary GH (Fig. 4). tope-speci®c CD8 T cells was undetectable in uninfected mice. This result suggested that the degree of autoimmune pathol- At day 7 after infection, endogenous NP 366±374-speci®c T ogy was related to T-cell precursor frequency and/or lympho- cells comprised 2Á2 Æ 0Á7% (n ˆ 3) of the total CD8 T-cell penic status. When the absolute number of F5 T cells was population in the spleens of 48-B10 mice. Six weeks after virus

# 2004 Blackwell Publishing Ltd, Immunology, 111, 254±261 T-cell activation by secreted and intracellular protein 259

antigen density in lymph nodes, in the form of DCs presenting 75±18 100

g) the antigen, which, if exceeded, results in T-cell activation. The µ 80 delay in T-cell activation in the D.2-RagÀ/À F5 mice suggests 60 35±8 * 27±12 ** that it took longer to reach the critical antigen density in these 40 mice. This may have been because of a lower level of antigen 20 expression in the D.2-RagÀ/À F5 strain and/or the difference in Pituitary GH ( 0 antigen context. The difference in phenotype in this model has a Nil Virus Virus + F5 simple endocrine explanation. To affect growth, pituitary damage must be signi®cant and uncompensated from 3 weeks Figure 5. Immunization with in¯uenza virus causes a reduction in onwards. The D.2 strain achieves a normal growth curve pituitary growth hormone (GH) levels in 48-B10 mice. Pituitary GH was measured in unimmunized 48-B10 mice (n ˆ 6), in mice immu- because T cells did not in®ltrate the pituitary and cause suf®- nized with in¯uenzavirus alone( n ˆ 4) and in mice that received a cient GH de®ciency in time to affect growth. In the pituitary, combination of virus and 5  106 naõÈve green ¯uorescent protein DCs can only be loaded with NP as the result of the steady-state (GFP)-F5 T cells (n ˆ 6). Pituitary GH was measured 14 weeks after turnover (apoptosis/necrosis) of NP-transgenic somatotrophs, immunization. Unimmunized mice were 16 weeks of age. Mean Æ which is not likely to exceed 2% per day, based on previous standard deviation (SD) values are indicated, and mean values are work in rats.35,36 In contrast, pituitary DCs in the 48-strain could represented by a horizontal line. Mean GH values of the treatment be loaded with soluble NP from somatotrophs that secrete groups were signi®cantly different from the mean GH of the control actively from birth and generate high local concentrations of group: *P ˆ 0Á004, **P ˆ 0Á0004. antigen. Interestingly, whilst the onset of pituitary pathology is infection, the circulating pool of NP-speci®c CD8 T cells had delayed in D.2-RagÀ/À F5 mice, the autoimmune consequences contracted and was no longer detectable by FACS analysis with are similar to those seen in the 48-RagÀ/À F5 mice. Thus, in this the dimeric MHC reagent. However, 3Á5 months after immu- case, the context of antigen expression had little in¯uence on the nization, a signi®cant reduction in pituitary GH was observed in predisposition to autoimmunity and CD8 T cells could be 48-B10 mice (P ˆ 0Á004, Fig. 5). When the precursor fre- primed, albeit with different kinetics, independently of antigen quency of NP-speci®c T cells was raised, by transferring context. The non-secreted form of NP must somehow access GFP-F5 T cells at the time of virus infection, the reduction DCs that activate T cells in the lymph nodes. in GH was more severe than observed after virus infection alone Once T cells have encountered antigen, they will expand (Fig. 5). In parallel experiments, the pituitaries of B10 control only to the extent that their surroundings will allow. The rapidity mice were unaffected by any of the infections and T-cell and extent to which T cells can expand is affected by the `space' transfers (results not shown). available to ®ll. In RagÀ/À mice there is abundant space to ®ll and T-cell expansion is unhindered. However, in a Rag-suf®- cient mouse, expansion may be inhibited by the presence of T DISCUSSION and B cells in the lymph nodes and spleen. The presence of We have previously shown that in mice which express a CD4‡ CD25‡ cells may also actively inhibit CD8 T-cell pro- transgene-encoded antigen in the pituitary in a secretory form, liferation.37±39 This could explain why 48-B10-F5 mice have a T cells are activated in the periphery and a fulminant auto- milder phenotype despite having a higher number of F5 TCR- immune hypophysitis ensues. In the present study, we compared bearing T cells. Whilst in this model, and in other models of this strain with another mouse strain in which the transgene spontaneous CD8-mediated autoimmunity,39 Rag insuf®ciency antigen is expressed in the same cells, but in a non-secreted either allows the disease to proceed or accelerates disease onset, form. Semiquantitative RT±PCR suggested that the amount of other models have shown the opposite effect.16 T-cell activation/ NP mRNA was much lower in the D.2-RagÀ/À F5 strain. In expansion in 48-B10-F5 mice could also have been restrained contrast, an in vitro functional assay, measuring T-cell prolif- because of a reduced functionality of F5 T cells that may eration in response to pituitary-derived NP cross-presented by express two T-cell receptors, resulting in lower levels of surface DCs, indicated that pituitary NP levels were equivalent. Despite F5 TCR and thus reduced sensitivity to activation by NP.29,30 the apparent discrepancy in antigen-expression levels, cross- Nevertheless, 48-B10-F5 mice are clearly autoimmune, but presentation of antigen in vivo occurred in the lymph nodes, their GH de®ciency is delayed and less severe. presumably by migratory DCs of pituitary origin, and resulted Neither the 48-B10 nor D.2-B10 mice (i.e. expressing NP but in T-cell activation in both lines, but differences in the timing of in the absence of transgenic F5 T cells) exhibit autoimmune cross-presentation eventually produced two disparate pheno- pathology under normal circumstances. Presumably, antigen is types. being processed and presented by DCs in the lymph nodes of The different phenotypes observed in the F5 TCR transgenic these mice, but the number of NP-speci®c T cells is vastly models re¯ect the initial differences in the processing and reduced compared with the F5 TCR transgenic strains. When presentation of NP to T cells. It is unlikely that the difference the NP-speci®c CD8 T-cell pool was increased after infection between the two strains is a result of the fact that secreted with virus, the number of speci®c T cells expanded to a level protein reaches lymph nodes directly, as discussed previously.20 suf®cient to cause GH de®ciency. At the height of the immune The amount of soluble antigen outside the pituitary gland would response evoked by virus, F5 T cells constituted 2% of the be far below the threshold required for cross-presentation.32±34 total CD8 pool. This expansion, combined with the in¯amma- The data suggest that there must be a threshold of critical tion caused by viral infection, and, of course, the possibility that

# 2004 Blackwell Publishing Ltd, Immunology, 111, 254±261 260 J. de Jersey et al.

T cells with different NP-epitope speci®cities were also acti- 14 Huseby ES, Liggitt D, Brabb T, Schnabel B, Ohlen C, Goverman J. vated, would be suf®cient to cause pituitary autoimmunity. It is A pathogenic role for myelin-speci®c CD8(‡) T cells in amodel for well known that lymphocytic hypophysitis may be manifest in multiple sclerosis. J Exp Med 2001; 194:669. other autoimmune conditions, including those affecting other 15 Graser RT, DiLorenzo TP, Wang F, Christianson GJ, Chapman HD, Roopenian DC, Nathenson SG, Serreze DV. Identi®cation of a CD8 endocrine systems,40,41 and we showed previously that the that can independently mediate autoimmune diabetes devel- autoimmune reaction triggered by a somatotroph-speci®c epi- opment in the complete absence of CD4 T cell helper functions. J 20 tope extends to other pituitary cell types. Our present results Immunol 2000; 164:3913. now suggest that the pituitary gland could be susceptible to 16 Verdaguer J, Schmidt D, Amrani A, Anderson B, Averill N, Santa- T-cell-mediated pathology after infection with a virus expres- maria P. Spontaneous autoimmune diabetes in monoclonal T cell sing a soluble pituitary antigen. If so, strategies for immuno- nonobese diabetic mice. J Exp Med 1997; 186:1663. contraceptive immunity based on vaccination against soluble 17 Anderson MS. Autoimmune endocrine disease. Curr Opin Immunol pituitary gonadotrophins could have other unexpected endo- 2002; 14:760. crine consequences. 18 Tanaka S, Tatsumi KI, Kimura M et al. Detection of autoantibodies against the pituitary-speci®c proteins in patients with lymphocytic hypophysitis. Eur J Endocrinol 2002; 147:767. ACKNOWLEDGMENTS 19 O'Dwyer DT, Smith AI, Matthew ML, Andronicos NM, Ranson M, Robinson PJ, Crock PA. Identi®cation of the 49-kDa autoantigen We are grateful to Nancy Cooke for providing us with the hGH LCR associated with lymphocytic hypophysitis as alpha-enolase. J Clin cosmid, K2B. We thank T. Norton, R. Simmonds, L. Allan and K. Endocrinol Metab 2002; 87:752. Williams for the breeding and welfare of transgenic mice. 20 de Jersey J, Carmignac D, Barthlott T, Robinson I, Stockinger B. Activation of CD8 T cells by antigen expressed in the pituitary REFERENCES gland. J Immunol 2002; 169:6753. 21 Mamalaki C, Elliott J, Norton T, Yannoutsos N, Townsend AR, 1 Derbinski J, Schulte A, Kyewski B, Klein L. Promiscuous gene Chandler P, Simpson E, Kioussis D. Positive and negative selection expression in medullary thymic epithelial cells mirrors the periph- in transgenic mice expressing a T-cell receptor speci®c for in¯uenza eral self. Nat Immunol 2001; 2:1032. nucleoprotein and endogenous superantigen. Dev Immunol 1993; 2 Kyewski B, Derbinski J, Gotter J, Klein L. Promiscuous gene 3:159. expression and central T-cell tolerance: more than meets the eye. 22 De Boer J, Williams A, Skavdis G et al. Transgenic mice with Trends Immunol 2002; 23:364. hematopoietic and lymphoid speci®c expression of Cre. Eur J 3 Liblau RS, Wong FS, Mars LT, Santamaria P. Autoreactive CD8 T Immunol 2003; 33:314. cells in organ-speci®c autoimmunity: emerging targets for thera- 23 Magoulas C, McGuinness L, Balthasar N et al. A secreted ¯uor- peutic intervention. Immunity 2002; 17:1. escent reporter targeted to pituitary growth hormone cells in trans- 4 Schwartz RH. T cell anergy. Annu Rev Immunol 2003; 21:305. genic mice. Endocrinology 2000; 141:4681. 5 Webb S, Morris C, Sprent J. Extrathymic tolerance of mature T cells: 24 Stockinger B, Hausmann B. Functional recognition of in vivo clonal elimination as a consequence of immunity. Cell 1990; processed self antigen. Int Immunol 1994; 6:247. 63:1249. 25 Lyons AB, Parish CR. Determination of lymphocyte division by 6 Moskophidis D, Lechner F, Pircher H, Zinkernagel RM. Virus ¯ow cytometry. J Immunol Methods 1994; 171:131. persistence in acutely infected immunocompetent mice by exhaus- 26 Carmignac DF, Robinson IC. Growth hormone (GH) secretion in the tion of antiviral cytotoxic effector T cells. Nature 1993; 362:758. dwarf rat: release, clearance and responsiveness to GH-releasing 7 Heath WR, Karamalis F, Donoghue J, Miller JF. Autoimmunity factor and somatostatin. J Endocrinol 1990; 127:69. caused by ignorant CD8‡ T cells is transient and depends on avidity. 27 McGuinness L, Magoulas C, Sesay AK et al. Autosomal dominant J Immunol 1995; 155:2339. growth hormone de®ciency disrupts secretory vesicles in vitro and in 8 Ohashi PS, Oehen S, Buerki K et al. Ablation of `tolerance' and vivo in transgenic mice. Endocrinology 2003; 144:720. induction of diabetes by virus infection in viral antigen transgenic 28 Eicher EM, Beamer WG. Inherited ateliotic dwar®sm in mice: mice. Cell 1991; 65:305. characteristics of the mutation, little, on chromosome 6. J Hered 9 Li M, Davey GM, Sutherland RM, Kurts C, Lew AM, Hirst C, 1976; 67:87. Carbone FR, Heath WR. Cell-associated ovalbumin is cross-pre- 29 Fossati G, Cooke A, Papa®o RQ, Haskins K, Stockinger B. Trigger- sented much more ef®ciently than soluble ovalbumin in vivo.J ing a second T cell receptor on diabetogenic T cells can prevent Immunol 2001; 166:6099. induction of diabetes. J Exp Med 1999; 190:577. 10 Kurts C, Miller JF, Subramaniam RM, Carbone FR, Heath WR. 30 Heath WR, Carbone FR, Bertolino P, Kelly J, Cose S, Miller Major histocompatibility complex class I-restricted cross-presenta- JF. Expression of two T cell receptor alpha chains on the tion is biased towards high dose antigens and those released during surface of normal murine T cells. Eur J Immunol 1995; 25: cellular destruction. J Exp Med 1998; 188:409. 1617. 11 Limmer A, Ohl J, Kurts C et al. Ef®cient presentation of exogenous 31 Mayo KE, Miller T, DeAlmeida V, Godfrey P, Zheng J, Cunha SR. antigen by liver endothelial cells to CD8‡ T cells results in antigen- Regulation of the pituitary somatotroph cell by GHRH and its speci®c T-cell tolerance. Nat Med 2000; 6:1348. receptor. Recent Prog Horm Res 2000; 55:237. 12 Kurts C, Kosaka H, Carbone FR, Miller JF, Heath WR. Class I- 32 Merkenschlager M, Power MO, Pircher H, Fisher AG. Intrathymic restricted cross-presentation of exogenous self-antigens leads to deletion of MHC class I-restricted cytotoxic T cell precursors by deletion of autoreactive CD8(‡) T cells. J Exp Med 1997; 186:239. constitutive cross-presentation of exogenous antigen. Eur J Immunol 13 Sun D, Whitaker JN, Huang Z, Liu D, Coleclough C, Wekerle H, 1999; 29:1477. Raine CS. Myelin antigen-speci®c CD8‡ T cells are encephalito- 33 Kurts C, Sutherland RM, Davey G et al. CD8 T cell ignorance or genic and produce severe disease in C57BL/6 mice. J Immunol tolerance to islet antigens depends on antigen dose. Proc Natl Acad 2001; 166:7579. Sci USA 1999; 96:12703.

# 2004 Blackwell Publishing Ltd, Immunology, 111, 254±261 T-cell activation by secreted and intracellular protein 261

34 Morgan DJ, Kreuwel HT, Sherman LA. Antigen concentration and 38 Kursar M, Bonhagen K, Fensterle J, Kohler A, Hurwitz R, Kamradt precursor frequency determine the rate of CD8‡ T cell tolerance to T, Kaufmann SH, Mittrucker HW. Regulatory CD4‡ CD25‡ T cells peripherally expressed antigens. J Immunol 1999; 163:723. restrict memory CD8‡ T cell responses. J Exp Med 2002; 196:1585. 35 Nolan LA, Kavanagh E, Lightman SL, Levy A. Anterior pituitary 39 Van de Keere F, Tonegawa S. CD4(‡) T cells prevent spontaneous cell population control: basal cell turnover and the effects of experimental autoimmune encephalomyelitis in anti-myelin basic adrenalectomy and dexamethasone treatment. J Neuroendocrinol protein T cell receptor transgenic mice. J Exp Med 1998; 188:1875. 1998; 10:207. 40 Collin P, Kaukinen K, Valimaki M, Salmi J. Endocrinological 36 Levy A. Physiological implications of pituitary trophic activity. disorders and celiac disease. Endocr Rev 2002; 23:464. J Endocrinol 2002; 174:147. 41 Cemeroglu AP, Bober E, Dundar B, Buyukgebiz A. Autoimmune 37 Murakami M, Sakamoto A, Bender J, Kappler J, Marrack P. CD25‡ polyglandular endocrinopathy and anterior hypophysitis in a 14 CD4‡ T cells contribute to the control of memory CD8‡ T cells. year-old girl presenting with delayed puberty. J Pediatr Endocrinol Proc Natl Acad Sci USA 2002; 99:8832. Metab 2001; 14:909.

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