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The Journal of Immunology, 2002, 169: 6753-6759.
Copyright © 2002 by The American Association of Immunologists

Activation of CD8 T Cells by Antigen Expressed in the Pituitary Gland

James de Jersey*, Danielle Carmignac{dagger}, Thomas Barthlott*, Iain Robinson{dagger} and Brigitta Stockinger1,*

Divisions of * Molecular Immunology and {dagger} Molecular Neuroendocrinology, The National Institute for Medical Research, Mill Hill, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ag expressed exclusively in the anterior pituitary gland and secreted locally by pituitary somatotrophs can gain access to the MHC class I presentation pathway and activate CD8 T cells. Influenza nucleoprotein (NP) was expressed as a transgene under the control of the human growth hormone (GH) locus control region. Activation of monoclonal F5 CD8 T cells specific for NP resulted in spontaneous autoimmune pathology of the pituitary gland in mice transgenic for both NP and the F5 TCR. Destruction of somatotrophs resulted in drastically reduced GH levels in adult mice and a dwarf phenotype. Adoptive transfer of F5 T cells into NP-transgenic hosts resulted in full T cell activation, first demonstrable in regional lymph nodes, followed by their migration to the pituitary gland. Despite the presence of activated, IFN-{gamma}-producing CD8 T cells in the pituitary gland and a slight reduction in pituitary GH levels, no effect on growth was observed. Thus, CD8 T cells have access to the neuroendocrine system and get fully activated in the absence of CD4 help, but Ag recognition in this location causes autoimmune pathology only in the presence of excessive CD8 T cell numbers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recognition of (self) Ag expressed in peripheral tissues depends on uptake by dendritic cells (DC),2 their migration into lymphoid organs, and presentation to naive T cells. Studies using TCR transgenic mouse models to investigate the response of CD8 T cells to peripherally expressed Ag generally concluded that such T cells can be activated, undergo a limited round of division, but then are eliminated or anergized and rarely cause autoimmune disease (1, 2, 3, 4, 5, 6). Autoreactive CD8 T cell responses are thought to be largely dependent on CD4 T cell help, reflecting either the need for activation of Ag-presenting DC (7, 8, 9) or a CD4 T cell requirement to support continued survival and expansion (10, 11). DC have the unique capacity to cross-present Ags acquired from an exogenous source (e.g., necrotic or apoptotic tissue throughout the body) in the context of MHC class I molecules and render them recognizable by CD8 T cells (12). The efficiency with which exogenous Ags are cross-presented depends on Ag dose and whether they are secreted or acquired by DC via phagocytosis of cell debris (13, 14, 15). Constitutive trafficking of DC from peripheral organs in which Ags are picked up to lymphoid organs in which they are presented (16) could cause either T cell activation or tolerance induction, depending on the state of differentiation of the DC (17). Thus, it was postulated that self Ag picked up from cells that are undergoing steady state apoptosis (18) would fail to activate DC (19) and might instead make them tolerogenic (20, 21).

We were interested to analyze to what extent the immune system has access to Ag expressed in the neuroendocrine system and what the consequences of recognition might be. To test this, we made use of a construct containing the extensively characterized human growth hormone (hGH) promoter and locus control region (LCR) (22). The hGH LCR reliably directs position-independent, copy number-dependent transgene expression in the pituitary somatotrophs of transgenic mice. This system is therefore ideal for studies of bona fide tissue-specific Ag expression, avoiding the pitfalls with tissue-specific promoters lacking major regulatory control elements, such as transgenic promoters that frequently show ectopic expression in the thymus or position effect variegation. The hGH LCR has been shown to direct heterologous proteins efficiently to the regulated growth hormone (GH) secretory pathway using GH signal peptide and N-terminal sequences (22, 23). The pituitary gland is a highly vascularized peripheral organ outside the blood brain barrier, with both arterial and venous blood supplies. Releasing hormones, secreted from hypothalamic nerve terminals into capillaries in the external zone of the median eminence, are collected and delivered via the hypophysial portal veins to the anterior pituitary gland. The endothelial cells lining the pituitary sinusoids provide little barrier to the passage of secreted proteins from the endocrine cells to the bloodstream, which drain rapidly via the cavernous sinus to the jugular veins. However, very little is known of the lymphatic drainage of the pituitary.

We introduced influenza nucleoprotein (NP), containing an epitope recognized by H-2Db-restricted CD8 T cells from the F5 TCR transgenic strain (24), into a cosmid construct containing the full hGH LCR, promoter, and signal sequences, and generated transgenic mice. With this construct, NP should be stored in secretory vesicles of somatotrophs and released in a pulsatile manner, generating high local Ag concentrations in the anterior pituitary sinusoids that are rapidly diluted in the systemic blood volume.

Tissue-specific expression of NP in somatotrophs resulted in activation of monoclonal F5 CD8 T cells in the absence of CD4 T cell help. In double-transgenic mice with both influenza NP-specific T cells and cognate Ag (NP), T cell activation led to rapid autoimmune destruction of GH-producing cells and a dwarf phenotype. We found that NP secreted locally in the pituitary gland was cross-presented by DC and gained access to CD8 T cells in peripheral lymphoid organs draining the pituitary gland. Adoptively transferred F5 T cells first proliferated in lymph nodes and subsequently migrated to the pituitary gland.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Rag1-/- H-2b (Rag-/-), F5 Rag1-/- H-2b (F5) mice (24), and F5 Rag1-/- H-2b mice expressing green fluorescent protein (GFP) under control of the CD2 promoter in all T cells (GFP-F5) were bred in the animal facilities of the National Institute for Medical Research in accordance with established guidelines. The 48-Rag1-/- H-2b mice and the double-transgenic F1 intercross with F5 are termed 48-Rag-/- and 48-Rag-/--F5 throughout this work. Mice transgenic for NP ± the F5 TCR on a polyclonal C57BL/10 background are called 48-B10 and 48-B10-F5.

Construction of the 48GH-NP cosmid for generating transgenic animals

A PCR fragment encoding influenza A/NT/60/68 NP (GenBank Accession J02137) was generated. This NP gene fragment was flanked by PvuII restriction enzyme sites (5'-CAGCTG-3') and contained the sequence encoding a truncated NP protein lacking the ATG translation start codon (aa 2, 327–498), and the influenza hemagglutinin (HA) epitope YPYDVPDYA. The sense and antisense primers had the following sequences, respectively: 5'-CAGCTGGCGCTGGTGTGGATGG-3' and 5'-CAGCTGCCAGCGTAGTCTGGGACGTCGTATGGGTAATTGTCGTACTCCTCTGC-3'. The antisense primer contained the sequence encoding the last 6 C-terminal aa of NP, the HA epitope, and a PvuII site. A plasmid containing the truncated NP gene (MI3) was used as the PCR template (25). The PCR product was subcloned into pGEMT-Easy (Promega, Madison, WI) for sequencing, and pRSET (Invitrogen, Carlsbad, CA) for protein expression. The NP PCR product had the correct sequence and encoded a protein of the predicted size that could be detected on a Western blot with an anti-HA Ab (data not shown). The chimeric 48GH-NP sequence was generated when the NP PCR fragment was ligated into the PvuII sites of a modified hGH genomic clone containing an MluI linker (Fig. 1GoA). The 48GH-NP insert contains a genomic sequence encoding the first 48 aa of the hGH gene product (signal peptide and N-terminal 22 residues of hGH) fused in frame with the NP cDNA. The hGH gene sequences of a 40-kb cosmid (B2K) (23) were excised as a single MluI fragment and replaced with the MluI-linked 48GH-NP sequence to give the cosmid cos48GH-NP. Thus, the final cosmid contained a ~40-kb insert containing the hGH LCR, 5' and 3' untranslated sequences of the hGH gene driving expression of the 48GH-NP fusion protein.



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  		FIGURE 1. A, Construction of the cos48GH-NP transgenic cosmid. The hgh-N gene sequence, between the PvuII (P) restriction enzyme sites of exons 2 and 5, was replaced with influenza NP and HA epitope cDNA sequences (see Materials and Methods). Cos48GH-NP was generated by replacement of the MluI (M)-flanked hgh-N sequence in cosmid B2K with the 48GH-NP sequence. Arrows indicate DNase I-hypersensitive regions of the hGH LCR sequence. B, NP and GH colocalization in the pituitary. Conventional fluorescence microscope images of an area of the anterior pituitary of a 4-wk-old 48-Rag-/- mouse, stained with anti-HA (green for NP) and anti-GH (red) Abs and their overlay. NP appears to be contained within secretory granules, as visualized by confocal microscopy (see bottom right image).

 
Generation of transgenic animals

Cosmid cos48GH-NP DNA was linearized by NotI digestion. The fragment was purified by gel extraction and column elution (Schleicher & Schuell, Dassel, Germany) and adjusted to a concentration of 1–5 ng/µl in 0.5 mM EDTA, 1 mM Tris-HCl, pH 7.5. Transgenic mice were generated by pronuclear microinjection of fertilized oocytes of superovulated Rag-/- H-2b mice, followed by oviductal transfer into pseudopregnant recipients.

Genotyping of transgenic animals

Genomic DNA from tail biopsies was analyzed for transgene DNA by a standard PCR using NP-specific primers: 5'-GCTCACCTAGCGGCAATGGC-3' and 5'-AGGCCCTCTGTTGATTAGTGTTTC-3'. Thirty-five cycles of amplification were performed under the following conditions: 94°C for 45 s, 52°C for 30 s, and 72°C for 45 s per cycle.

Immunocytochemistry

Mouse pituitaries were embedded in OCT compound (BDH, Poole, U.K.) and frozen. Tissue sections (6 µm) were collected on Superfrost Plus slides (BDH), air dried, and fixed in acetone for 5 min before immunostaining. Sections were incubated with blocking solution (20% normal mouse serum in PBS) and then with primary Abs against either cell surface markers, GH or HA, for 1 h at room temperature. Sections were washed in PBS and incubated with secondary and tertiary reagents (when necessary). Reagents and Abs were: rat anti-HA FITC (3F10; Roche, Basel, Switzerland); monkey anti-rat GH (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD); rat anti-mouse CD3 (KT3); hamster anti-mouse CD11c biotin (N418); donkey anti-rat IgG FITC (Jackson ImmunoResearch, West Grove, PA); biotinylated goat anti-human Ab (Jackson ImmunoResearch); streptavidin FITC (BD Biosciences, San Jose, CA); and streptavidin Texas Red (BD Biosciences). Stained sections were mounted in Fluoromount (BDH) and viewed with either a conventional or confocal fluorescence microscope.

Histology

Pituitaries were fixed in phosphate-buffered 4% paraformaldehyde, pH 7.2, and embedded in paraffin. Sagittal sections (10 µm) were cut, dewaxed, and stained with H&E.

FACS analysis

Single cell suspensions of pituitaries, spleen, or lymph nodes were stained by sequential incubations on ice with Abs specific for cell surface markers, followed by streptavidin-fluorochrome conjugates when necessary. Nonspecific staining via FcR binding was blocked by initial incubation with anti-FcR Ab clone 2.4G2. Cell data were acquired on a FACSCalibur machine (BD Biosciences) with CellQuest software. Abs and secondary reagents are as follows: CD8 PE; TCR{beta} APC (clone H57); CD44 biotin; CD4 FITC; streptavidin Cy7PE (all BD Biosciences). Intracellular IFN-{gamma} was detected in T cells following a 4-h stimulation with phorbol dibutyrate (50 ng/ml), ionomycin (50 ng/ml), and brefeldin A (10 µg/ml) in complete IMDM at 37°C. Cells were washed, stained for cell surface markers (as above), and then fixed in 1% paraformaldehyde for 15 min on ice and permeabilized in 1% Nonidet P-40 for 3 min on ice. Cells were incubated with rat anti-IFN-{gamma} PE or rat IgG1 PE Ab isotype control (BD Biosciences).

Adoptive transfer

A total of 2–5 x 106 GFP-F5 CD8 T cells were injected i.v. into Rag-/-, 48-Rag-/-, B10, 48-B10, or double-transgenic 48-Rag-/--F5 hosts. To monitor cell proliferation, F5 T cells were labeled with CFSE (Molecular Probes, Eugene, OR), as previously described (26). Briefly, pooled spleen and lymph node cells were incubated at 37°C for 10 min at a cell density of 107/ml in 2 µM CFSE and washed thoroughly before i.v. injection.

Radioimmunoassays

Mouse GH in pituitary extracts was assayed by RIA, as previously described for the rat (27), using mouse reagents kindly provided by National Institute of Diabetes and Digestive and Kidney Diseases. Unless otherwise stated, data are shown as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tissue-specific expression of influenza NP

To direct expression of influenza NP to pituitary somatotrophs, we used a construct containing the GH promoter and the well-characterized hGH LCR. The 48GH-NP transgenic line was generated by injecting a 40-kb cosmid clone containing a genomic sequence encoding the signal peptide and initial N-terminal sequences of the hGH gene fused to a truncated influenza NP (aa 2, 327–498; Fig. 1GoA) with a C-terminal HA tag. Fertilized eggs from Rag-/- H-2b mice were injected, and one founder female was bred to Rag-/- H-2b to generate the 48-Rag-/- line. Transgene expression was visualized by staining pituitary sections for the HA tag and colocalized with GH expression in somatotrophs. NP appeared to be packaged into secretory granules (Fig. 1GoB). Mice carrying the 48GH-NP transgene showed normal weight gain; pituitary GH levels were comparable to those of nontransgenic Rag-/- mice; and 48-Rag-/- mice exhibited no phenotypic changes in their pituitary glands (Fig. 2Go). Thus, transgenic expression of NP does not interfere with normal somatotroph physiology.



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  		FIGURE 2. Characteristics of 48-Rag-/- mice. A, Average body weight of sex-matched (female) 48-Rag-/- mice ({blacksquare}, n = 4) and nontransgenic Rag-/- littermates ({square}, n = 2). B, Pituitary GH content in 48-Rag-/- and Rag-/- mice (from A) at 58 days of age. C and D, H&E-stained sections of anterior pituitaries of a 48-Rag-/- mouse (C) and a nontransgenic F5 mouse (D).

 
GH-producing cells are destroyed by F5 CD8 T cells

We crossed 48-Rag-/- mice with the F5 strain that expresses a monoclonal population of CD8 T cells specific for influenza NP epitope 366–374, to investigate whether NP-specific T cells would have access to Ag expressed in the pituitary gland. Analysis of the thymus from double-transgenic mice revealed that absolute numbers of T cells, composition of thymocyte subpopulations, and levels of TCR expression were similar to those found in normal F5 littermates (data not shown). This confirms the absence of ectopic expression, as has been consistently observed with the hGH LCR by others. Although GH expression was reported in the subcapsular cortex of human thymus (28), it appears that expression in such locations has no influence on T cell development, because this would have resulted in at least a degree of negative selection in developing F5 thymocytes. Tissue-specific expression of NP in the pituitary gland of mice transgenic for both NP and the F5 TCR was manifested by a striking dwarf phenotype (Fig. 3GoB, inset). In normal mice, GH-dependent weight increase is evident from 4 wk after birth, but the 48-Rag-/--F5 double-transgenic mice failed to gain weight from about this time onward (Fig. 3GoA). A dramatic reduction of GH levels in the pituitaries of double-transgenic mice was already evident at 4 wk of age. GH decreased to almost undetectable levels (<0.1% of normal) by 8 wk of age (Fig. 3GoB). Double-transgenic mice on a Rag-/- background also showed reduced levels of other hormones produced in the anterior pituitary gland, including prolactin (Fig. 3GoC) and thyroid-stimulating hormone (Fig. 3GoD). Severe reductions in GH levels were also evident in 48-B10-F5 mice on a Rag+ background (Fig. 3GoE), although not as severe as that seen in 48-Rag-/--F5 mice; this was reflected in a less dramatic effect on growth (data not shown). These differences were presumably because of a lower frequency of F5 T cells in the Rag+/- mice, probably resulting in a delayed onset of somatotroph destruction.



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  		FIGURE 3. Characteristics of double-transgenic 48-Rag-/--F5 and 48-B10-F5 mice. A, Body weights of male NP-transgenic, 48-Rag-/--F5 mice (•) compared with male nontransgenic F5 mice ({circ}). B, Pituitary GH content in 48-Rag-/--F5 mice (•) compared with F5 mice ({circ}). Each data point represents mean µg of GH (+SD) per pituitary of three to five pituitaries. Inset photograph, 4-mo-old male littermates F5 on the left, and 48-Rag-/--F5 on the right. C, Pituitary prolactin content in 4-mo-old male 48-Rag-/--F5 mice compared with F5 nontransgenic controls (n = 3). Mean values are in micrograms. D, Pituitary thyroid-stimulating hormone (TSH) content in 4-mo-old 48-Rag-/--F5 mice (n = 2) compared with F5 nontransgenic controls (n = 4). Mean values are in nanograms. E, Pituitary GH content in 4-mo-old 48-B10-F5 mice (n = 4) compared with F5 nontransgenic controls (n = 6). Mean values are in micrograms. All error bars are SDs.

 
H&E-stained sections of pituitary glands from double-transgenic mice showed extensive apoptosis and cellular infiltrates restricted to the anterior lobe of the pituitary gland (Fig. 4GoA). Immunostaining of cryosections revealed substantial infiltration of the anterior pituitary by CD8 T cells in double-transgenic mice (Fig. 4GoB) evident as early as 15 days after birth (not shown). Pituitary glands of all 48-Rag-/--F5 mice contained activated F5 CD8 T cells expressing IFN-{gamma} (Fig. 4GoC).



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  		FIGURE 4. Histology and T cells in the pituitary of 48-Rag-/--F5 mice. A, H&E-stained sections of pituitaries from a 33-day-old 48-Rag-/--F5 mouse showing dense apoptotic bodies. B, Pituitary section from a male, 34-day-old 48-Rag-/--F5 mouse stained for CD3 (T cells; green) and CD11c (DC; red). Arrows indicate the location of DC. T cells do not appear in the posterior (P) pituitary, but exclusively in the anterior (A) pituitary. C, Intracellular IFN-{gamma} staining of pituitary-infiltrating T cells (gated on TCR+/CD8+ cells). Values represent the percentages of IFN-{gamma} (29%) IFN-{gamma}-negative (71%) events.

 
Activation of adoptively transferred naive F5 T cells in NP-transgenic mice

To follow the sequence of events leading to destruction of GH-producing somatotrophs, we performed adoptive transfers of naive T cells from GFP-F5 mice. Transfers were done into transgenic (48-Rag-/-, 48-Rag-/--F5, or 48-B10) or nontransgenic (Rag-/-, F5, or B10) hosts. Seven days after transfer into 48-Rag-/- (Fig. 5GoA) or double-transgenic 48-Rag-/--F5 (Fig. 5GoB) mice, a proportion of the GFP-positive T cells found in cervical lymph nodes had up-regulated the activation marker CD44. GFP-F5 T cells transferred into nontransgenic Rag-/- adoptive hosts did not up-regulate CD44 at this early time point, because homeostatic expansion of F5 T cells in lymphopenic hosts does not result in acquisition of activation markers early after transfer (29) (Fig. 5GoC). A similar picture was found in superficial cervical, deep cervical, axillary, brachial, inguinal, and mesenteric lymph nodes (not shown). Seven days after transfer, no cells were found in the pituitary glands and very few in the spleen of 48-Rag-/- hosts (not shown), indicating an early time point of activation in the regional lymph nodes. Activated T cells were first detected in the pituitary gland of 48-Rag-/- mice (and not Rag-/- controls) 21 days after transfer (Fig. 5GoI), whereas double-transgenic hosts showed an earlier influx of GFP-F5 T cells by 7 days after transfer (Fig. 5GoJ), presumably because of the already severe inflammatory conditions prevalent in these hosts from an early age (see also the large population of GFP-negative, host-derived CD8+ CD44+ T cells in this location). Therefore, despite the dramatic destruction of somatotrophs in 48-Rag-/--F5 mice, there is clearly sufficient ongoing Ag expression for activation of further T cells.



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  		FIGURE 5. Adoptive transfer of naive F5 T cells into NP-transgenic hosts. CD44-positive (activated) GFP-F5 T cells appear in the cervical lymph nodes of 48-Rag-/- (A), 48-Rag-/--F5 (B), and 48-B10 (F) mice, but not Rag-/- (C), F5 (not shown), or B10 (E) mice at 7 days posttransfer. The numbers within each GFP vs CD44 plot refer to the percentage of GFP+ cells that are CD44+ and are contained within the small box. When 10 x 106 CFSE-labeled F5 T cells were transferred into B10 (G) and 48-B10 (H) hosts, a higher rate of cell division was observed in the 48-B10 mice, in lymph nodes at 3 days posttransfer. Dot plots of TCR vs CFSE (G and H) show CD8+/TCR+-gated events only, and the numbers shown refer to the percentage of transferred CFSE-labeled cells that remained undivided. Transferred GFP-F5 T cells appear and accumulate in the pituitaries of 48-Rag-/- (I, day 21 shown), 48-Rag-/--F5 (J, day 7 shown), and 48-B10 mice (K, day 62 shown), but not Rag-/-, F5, or B10 mice (not shown). Note that the pituitaries of 48-Rag-/--F5 and 48-B10 mice also contain endogenous GFP-negative T cells (J and K). All dot plots of GFP vs CD44 show CD8+/TCR+-gated events only, and are true representations of results obtained from several animals at each time point. Pituitary-infiltrating GFP-F5 T cells in 48-Rag-/- (not shown) and 48-B10 (K and L) mice were positive for IFN-{gamma}. IFN-{gamma}-producing T cells were predominantly CD44high (L). Recipient mice were aged between 4 and 6 wk upon adoptive transfer. Additional groups of young 48-Rag-/- (n = 3) and Rag-/- (n = 3) mice received 3 x 106 naive F5 T cells i.p. at 21 days of age. Pituitary GH was measured 3 mo posttransfer (D). Values refer to mean GH content per pituitary in micrograms, and error bars are SDs.

 
To verify that activation and migration to the pituitary gland are not just observed in artificially lymphopenic mice, we transferred either GFP-F5 (Fig. 5Go, E and F) or CFSE-labeled F5 (Fig. 5Go, G and H) into polyclonal B10 mice or polyclonal transgenic 48-B10 mice. Up-regulation of CD44 was again observed in a proportion of GFP-F5 T cells 7 days after transfer, and 48-B10 pituitary glands contained a substantial number of activated GFP-F5 T cells that also produced IFN-{gamma} (Fig. 5Go, K and L). CFSE-labeled F5 T cells transferred into 48-B10 mice underwent a number of divisions within a 3-day period, whereas no division was seen in F5 cells transferred into nontransgenic polyclonal controls.

Despite the continuous and long-term presence of transferred Ag-specific T cells in peripheral lymphoid organs and the pituitary gland, no fulminant autoimmune pathology was observed, and GH levels were only slightly lower than in control animals (Fig. 5GoD).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show in this study that an autoantigen expressed and secreted in a peripheral organ, the pituitary gland, can gain access to CD8 T cells and cause a CD8 T cell-mediated autoimmune response. The involvement of CD8 T cells in a number of organ-specific autoimmune diseases has received increased attention in recent years, and it has become clear that these cells contribute significantly to damage in autoimmune disorders (reviewed in Ref. 30).

Although it has been shown previously that systemic administration of soluble Ag in very high doses can lead to cross-presentation by DC and activation of CD8 T cells (13, 31, 32), little is known about the recognition of endogenously synthesized and locally secreted Ag by CD8 T cells. In fact, given the extraordinarily large doses of soluble Ag necessary to achieve activation of CD8 T cells, we had anticipated that F5 CD8 T cells would remain ignorant of soluble NP expressed in the pituitary gland, unless they were specifically activated by Ag in an optimal immunogenic form, e.g., following infection with influenza virus. However, the local concentration of NP, mimicking that of GH, as anticipated by its localization in secretory granules, would be orders of magnitude higher than levels found in peripheral blood. In contrast, NP stores in the anterior pituitary will be somewhat lower than those of GH, because GH has specific sequences mediating zinc-dependent aggregation and packing to form a dense granule protein core (33), resulting in a higher GH concentration than that of a heterologous protein targeted to the same granule (23).

The anterior pituitary gland contains numerous DC that have been ascribed a role in regulation of endocrine cells (34). They are identified by their morphology, high expression of MHC class II, and the capacity to stimulate T cells in a MLR. The presence of DC in the vicinity of somatotrophs secreting NP makes uptake of soluble NP and processing in the MHC class I pathway feasible. We designed the adoptive transfer experiments shown in Fig. 5Go with the aim to determine where T cell activation takes place and whether this can cause autoimmune pathology. The kinetics of T cell activation following their transfer into 48-Rag-/- mice suggests that migratory DC carry NP internalized and cross-processed with MHC class I from the pituitary gland into lymphoid organs for activation of T cells. This is in agreement with earlier data showing cross-priming of OVA-specific CD8 T cells in lymph nodes draining the pancreas that expressed OVA as a transgene (35). Whether the migratory DC present Ag to T cells directly or are themselves phagocytosed by resident DC (36) is unclear. It is unlikely that Ag gains access to lymphoid organs in soluble form. First, its concentration in blood, like that of GH, is far below the amount required for cross-presentation by DC (13). Second, despite the much higher blood supply for the spleen (37), activated T cells were first detected in lymph nodes and were not apparent in the spleen until the later stages after they had already entered the pituitary gland. Third, there is no evidence of Ag access to the thymus via the blood judged by unimpaired development of F5 thymocytes.

The current view is that cross-presentation of Ag in the absence of danger signals, resembling pathogen-associated molecules, fails to give maturation signals to T cells (19, 38, 39) and favors tolerance induction rather than activation of CD8 T cells (20, 40). The expression of NP in somatotrophs of 48-Rag-/- transgenic mice has no discernible adverse effect on these cells, as shown by their normal morphology, GH concentrations, and unaffected growth rates. However, the type and concentration of Ag, as well as the affinity and frequency of responding CD4 or CD8 T cells will influence whether activation and autoimmunity or tolerance are the outcome of peripheral Ag expression (1, 2, 4, 41, 42, 43). It is likely that the high frequency of Ag-specific T cells in double-transgenic 48-Rag-/--F5 mice can also overcome the requirement for T cell help (44, 45) that was previously shown to prevent T cell deletion and favor autoimmunity (10). Infiltration of the pituitary gland was also observed following transfer of small numbers of T cells. Despite being fully activated to IFN-{gamma} secretion, they caused only a marginal reduction in GH levels that did not result in a dwarf phenotype (Fig. 5Go, D and L). This suggests that the reduced influx of Ag-specific activated T cells, as would more closely resemble physiological conditions in a polyclonal setting, does not cause the fulminant pathology seen in the double-transgenic mice. Preliminary data at a later time point, however, indicate that eventually significant damage to the pituitary does occur. Autoimmune reactions against the anterior pituitary gland arising either spontaneously in patients (46) or following injections of pituitary homogenate in rats (47) have been described, but were only correlated with Abs to pituitary constituents. Our data indicate that neuroendocrine self Ag secreted in a hormone-like fashion is indeed visible for CD8 T cells and under certain conditions can cause autoimmune activation.


   Acknowledgments
 
We are grateful to Nancy Cooke for providing us with the hGH LCR cosmid K2B. We thank D. Kioussis for critical review of the manuscript, T. Norton and K. Williams for the breeding and welfare of transgenic mice, A. Cooke and J. Phillips (Department of Pathology, University of Cambridge, Cambridge, U.K.) for help and advice with immunochemistry, and E. Grigorieva for histology.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Brigitta Stockinger, Division of Molecular Immunology, The Ridgeway, Mill Hill, London NW7 1AA, U.K. E-mail address: bstocki{at}nimr.mrc.ac.uk Back

2 Abbreviations used in this paper: DC, dendritic cell; GFP, green fluorescent protein; GH, growth hormone; HA, hemagglutinin; hGH, human GH; LCR, locus control region; NP, nucleoprotein. Back

Received for publication August 21, 2002. Accepted for publication October 17, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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