A minimal level of MHC class II expression is sufficient to abrogate autoreactivity

Eveline S. J. M. de Bont1,4, Christina R. Reilly3, David Lo3, Laurie H. Glimcher1,2 and Terri M. Laufer1,2,5

1 Department of Immunology and Infectious Diseases, Harvard School of Public Health, and
2 Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
3 Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA

Correspondence to: T. M. Laufer, Division of Rheumatology, University of Pennsylvania, BRB2, Rm 753, 421 Curie Boulevard, Philadelphia, PA 19104, USA


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The establishment of CD4+ T cell tolerance requires that self-reactive thymocytes are negatively selected during thymic development. A threshold of antigen concentration appears to exist for both MHC class I- and class II-mediated negative selection, below which clonal deletion of a self-reactive transgenic TCR does not occur. Similarly, both the specificity and thymic concentration of MHC molecules affect the efficiency with which autoreactive thymocytes are deleted. However, this threshold for MHC class II concentration has not been well established. Here, we show that this threshold must be extraordinarily low. We have used the human lysozyme promoter to re-express an Aßb cDNA on macrophages and other phagocytic myelomonocytic cells of class II-deficient Aßb –/– mice. Surface expression of I-Ab could be detected on mature peritoneal macrophages and, minimally, on thymic dendritic cells; however, this level of expression was not sufficient for antigen-specific T cell activation. Nevertheless, when backcrossed onto an autoreactive K14 background, this minimal level of class II was sufficient to induce negative selection of a polyclonal self-reactive population. We conclude that provision of extremely low levels of class II to thymic dendritic cells confers on them the ability to mediate clonal deletion of autoreactive T cells.

Keywords: dendritic cell, lysozyme promoter, macrophage, MHC class II, negative selection


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The normal development of T cells in the thymus requires that only those T cells that express TCR which recognize self-major MHC molecules complexed with foreign peptide will survive. The developing {alpha}ß TCR+ double-positive thymocyte must interact productively with self-MHC molecules to be positively selected (1,2). However, this selection process must also eliminate those thymocytes with potentially autoreactive receptors, a process termed negative selection (37). These two processes are rather stringent: only 5% of immature thymocytes develop to become mature T cells which are exported to the periphery (8).

Developing thymocytes traffic from the external thymic cortex towards the internal thymic medulla. This maturation involves contact with multiple different thymic stromal cells: in the cortex, thymocytes interact with thymic epithelial cells (the site of positive selection), and at the corticomedullary junction and medulla (the sites of negative selection) they encounter dendritic cells, macrophages, B cells and medullary epithelial cells. Mature single-positive T cells exit the thymus from the medulla (reviewed in 9).

Although it is well-established that positive selection of CD4+ T cells requires the selecting ligand to be present on thymic cortical epithelium (1,10,11), the cellular requirements for deletion of autoreactive cells are less clear. Both hematopoietic cells (12,13) and, to a lesser extent, medullary epithelium (14,15), can induce clonal deletion. For example, clonal deletion occurs virtually normally when the expression of the selecting MHC ligand is limited to thymic dendritic cells (16). Other work has suggested that—at least for endogenous superantigens—B cells may be able to induce negative selection (1719). Currently, there is no evidence that thymic macrophages mediate clonal deletion; rather, they primarily function as scavengers to remove apoptotic thymocytes (20). Complete negative selection, then, requires that the selecting ligand be present on appropriate thymic stroma and dendritic cells. However, the concentrations of peptide, MHC complexes and TCR are also crucial variables, as has been demonstrated for affinity/avidity models of class I-mediated negative selection (2124).

A threshold of antigen concentration also appears to exist for class II-mediated negative selection, below which clonal deletion of a transgenic TCR does not occur. Above this threshold, the amount of clonal deletion of a self-reactive transgenic TCR varies with the antigen concentration (25,26). Similarly, both the specificity and thymic concentration of MHC class II molecules affect the deletion of autoreactive class II-restricted TCR transgenic thymocytes. Thus, Spain and Berg demonstrated that double-positive thymocytes are deleted by a specific peptide in a dose-dependent manner, and that both the relative affinity and the gene dose of the particular MHC allele determined the necessary peptide concentration (27).

We have generated a new line of transgenic mice which allows us to follow the negative selection of a polyclonal population of autoreactive thymocytes, rather than a population expressing a single transgenic TCR. We previously described K14 mice in which MHC class II expression is limited to thymic cortical epithelium (28). In the absence of negative selection, both K14 thymocytes and peripheral single positive CD4 cells are activated by syngeneic antigen-presenting cells (APC). We have used the human lysozyme promoter (hLP) (29) to target Aßb to macrophages and other phagocytic myelomonocytic cells and have bred these transgenic mice onto the K14 background. Here, we show that the resultant level of MHC class II expression on peritoneal macrophages and dendritic cells, which is insufficient to elicit in vitro antigen-specific responses, is nonetheless sufficient to induce negative selection of the self-reactive K14 population.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
C57Bl/6 (B6) and BALB/c mice were purchased from Taconic (Germantown, NY). The Aßb –/– mice (30) and K14 mice (28) have been described previously. The hLP/Aßb –/– and K14/Aßb –/– mice were backcrossed to B6 mice 13 times. All mice were maintained pathogen-free in a barrier facility.

Reagents
The following antibodies for flow cytometry were purchased from PharMingen (San Diego, CA): I-Ab (AF6-120.1), CD11b/Mac 1(M1/70), CD11c (HL3), CD4 (RM4-5) and CD8 (Ly-2). Anti-DEC205 (NLDC 145) (31) was used as a culture supernatant and developed with goat anti-rat–FITC (PharMingen). Streptavidin–TriColor was purchased from Gibco/BRL (Gaithersburg, MD). Propidium iodide was purchased from Sigma (St Louis, MO).

Hybridomas producing the mAb anti-CD4 (GK1.5), anti-CD8 (2.43), anti-I-Ab,d (M5/114) and anti-Thy1.2 (AT83A) were obtained from ATCC (Manassas, VA). They were used as culture supernatants for cell depletions.

Transgene construction and injection
An 850 bp Aßb cDNA was subcloned into the EcoRI site of pBSKSII(–) vector (Stratagene, La Jolla, CA). A 2.5 kb BamHI–HindIII fragment containing the coding sequence of the human growth hormone gene was cloned into the BamHI and XbaI sites in pBSKSII 3' of the Aßb cDNA, destroying the HindIII and XbaI sites. The 3.5 kB KpnI–HincII fragment containing the proximal promoter of the human lysozyme gene (29) was excised from pUC19.hLP using KpnI and HindIII and subcloned 5' of the Aßb cDNA utilizing the KpnI and HindIII sites of pBSKSII. The hLP/Aßb transgene was removed from plasmid sequences with KpnI and NotI, and the construct was microinjected into fertilized B6 eggs.

Transgene-positive founders were identified by Southern blot analysis using a 0.8 kB EcoRI Aßb cDNA fragment. Founder animals were backcrossed to Aßb –/– mice or K14/Aßb –/– mice. Subsequent generations were assayed for transgene expression by PCR analysis using hLP promoter-specific primers (sense, 5'-CCA ATT CTT CCA GAG CCA CTA C-3'; antisense, 5'-GTC AGA GTG CTA GGC TGA CCA G-3').

RNA preparation and analysis
RNA from whole spleen, whole thymus and thioglycollate-elicited peritoneal cells was extracted by homogenization of tissue samples in Trizol (Gibco/BRL). First-strand cDNA was synthesized with SuperScript2 (Gibco/BRL) according to the manufacturer's instructions. PCR analysis of the cDNA was performed using primers specific for the hLP Aßb transgene (Aßb, 5'-CCG GAA TGG CCA GGA GGA GAC G-3'; hGH, 5'-AAG GGA ATG GTT GGG AAG GCA CTG-3'). Mouse primers specific for the ß-actin transcript were used for RNA from all tissues as an internal control to verify the efficiency of cDNA synthesis.

Flow cytometry
Stained single-cell suspensions were analyzed on a Becton Dickinson FACSCalibur using CellQuest software.

Immunohistochemistry
Cryostat sections (10 µm) of thymus were fixed in cold acetone, then incubated with primary antibody (M5/114) against murine MHC class II I-A and the lectin UEA-1 to mark thymic cortical epithelium (32). Detection was done using mouse anti-rat IgG (Jackson ImmunoResearch, West Grove, PA). Signal amplification was performed using the TSA tyramide kit (DuPont/NEN, Wilmington, DE) and AEC as the chromagen. Sections were counterstained with hematoxylin.

Cell preparation and tissue culture
DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 1% non-essential amino acids and 50 µM mercaptoethanol was used for all stimulations. Cells were maintained at 37°C in a 5% CO2 environment.

The IH3.1 Y-Ae-restricted T–T hybrid (33) was a gift of Dr A. Rudensky (HHMI, Seattle, WA). The cognate E{alpha} 52–68 peptide (ASFEAQEALANIAVDKA) was purchased from Quality Controlled Biochemicals (Hopkinton, MA).

Peritoneal exudate cells were obtained by saline lavage from the peritoneal cavity of either untreated mice or mice pretreated with i.p. injection of 1.5 ml 3% thioglycollate (Sigma) 3–4 days prior to harvest.

Single-cell suspensions were prepared from either whole thymus or whole spleen by treatment with collagenase (CLS4) 1.6 mg/ml (Worthington Biochemical, Freehold, NY) and DNase I 1 mg/ml (Boehringer Mannheim, Indianapolis, IN) for 1–2 h at 37°C. The suspensions were placed into tissue culture-treated dishes for 3 h at 37°C and non-adherent cells were removed by vigorous washing. For preparation of semi-adherent or adherent splenocytes, the remaining cells were cultured for 48 h in either 1 ng/ml IL-4 (Genzyme, Cambridge, MA) and 50 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF; PharMingen) or 100 U/ml IFN-{gamma} (Genzyme) or 50 mg/ml lipopolysaccharide (LPS). On day 2, the non-adherent (semi-adherent) cells were used as a dendritic cell-enriched population; the adherent cells were removed by treatment with 0.6 mM EDTA at 37°C and used as a macrophage-enriched population.

For preparation of thymic NLDC145+/CD11c+ cells, a single-cell suspension of collagenase/DNase-treated thymus was resuspended in Percoll (density 1.092) and layered over Percoll (density 1.109). These two layers were topped with layers of 1.079 and 1.060, and centrifuged at 800 g for 20 min. The top layer was washed in media and plated at 37°C. After 3 h, non-adherent cells were removed by vigorous washing. The remaining cells were cultured overnight. In the morning, the non-adherent population was removed and used as a dendritic cell-enriched population. Where indicated, this population was further purified by staining with NLDC 145 and CD11c, and flow cytometric sorting.

Splenic APC were prepared from single-cell suspensions. When indicated, the cell suspension was depleted of T cells by incubation with 1:3 dilutions of anti-CD4, anti-CD8 and anti-Thy1.2 supernatants for 30 min on ice followed by treatment with rabbit complement (Cedarlane, Hornby, Ontario, Canada) at 37°C for 45 min. Dead cells were removed by centrifugation over Lympholyte-M (Cedarlane). All APC were irradiated 2000 rad with a cesium source prior to use.

CD4+ T cells were prepared from spleen and lymph node. Single-cell suspensions were plated on Petri dishes coated with 25 µg/ml goat anti-mouse IgG (Cappel, Organon Teknica, Durham, NC). Non-adherent cells were stained with anti-CD4 antibody and purified on paramagnetic MiniMACS columns according to the manufacturer's instructions (Miltenyi Biotec, Auburn, CA). The final population was always >95% pure as assessed by flow cytometry.

Mixed lymphocyte reactions (MLR) and hybrid stimulation
MLR were performed by culturing either 1x105 CD4+ responders or 4x105 thymic responders with graded numbers of irradiated APC in 200 µl in round-bottom 96-well plates for 4 days. Proliferation was assessed by [3H]thymidine (Dupont/NEN) incorporation (1 µCi/well) for the final 18 h.

IH3.1 cells (1x105) were incubated with varying numbers of APC (as described subsequently) and increasing concentrations of E{alpha} peptide in 200 µl for 24 h. After 24 h, 100 µl of culture supernatant was removed and assayed for IL-2 production using the indicator cell line, HT-2 (34).

Precursor frequency analysis
Purified CD4+ T cells (2x106) were cultured with 5x106 T cell-depleted stimulators in one well of a 24-well plate for 2–3 days in the presence of 1 µM colchicine (Sigma) and 5 µg/ml BrdU (Sigma) essentially as described by Tough and Sprent (35).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Re-expression of MHC class II molecules in transgenic hLP Aßb –/– mice
Lysozyme is expressed by mature phagocytic cells of the myelomonocytic lineage such as monocytes and neutrophils (36). Gordon and colleagues have shown that 3.5 kB of the human lysozyme gene (hLP) proximal promoter can appropriately direct heterologous expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in vivo (29). They detected enzymatic CAT activity in myeloid-rich tissues such as bone marrow, spleen, lung and the thymus of neonatal transgenic mice. Transgene activity was also apparent in thioglycollate-elicited peritoneal macrophages and Mycobacterium bovis (BCG strain)-induced hepatic macrophages.

We utilized the hLP to selectively re-express I-A molecules in Aßb –/– mice using the construct shown in Fig. 1Go(a). Transgene-positive founders were backcrossed two generations to the Aßb –/– mice (30) and analyzed for restoration of class II expression. Three different founder lines transmitted the transgene and had detectable levels of I-Ab protein. Two of these lines, 3483 and 3485, displayed the same serologic and functional phenotype, and will be described interchangeably. Appropriate expression of transgene mRNA could be detected by reverse transcription and subsequent PCR using transgene-specific primers with RNA prepared from resident peritoneal cells, thymus and spleen as shown in Fig. 1Go(B).




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Fig. 1. Construction and expression of the hLP/Aßb transgene. (A) Schematic representation of the hLP/Aßb transgene. (B) The hLP/Aßb transgene is expressed in the thymus, spleen and peritoneal cells of transgenic animals. RT-PCR with primers specific for the transgene identifies transcripts in the lymphoid organs of transgenic animals.

 
To assess levels of surface class II I-Ab, we performed FACS analysis of CD11b (Mac-1)+ macrophages from different lymphoid organs, keeping in mind that surface MHC class II expression is only expected where transgenic Aßb expression overlaps with endogenous A{alpha}b expression. Similarly, in vitro and in vivo activation of macrophages will result in an increase in expression of class II only when those stimuli increase both endogenous class II A{alpha}b and the activity of the lysozyme promoter. Two-color flow cytometric analysis of resident peritoneal cells (Fig. 2Go) revealed a low level of expression of I-Ab on CD11b (Mac 1)+ macrophages. This result is in agreement with the work of Dighe et al. (37) who found that an hLP-targeted mutant IFN-{gamma} receptor transgene was only expressed in mature F4/80+ peritoneal macrophages. However, in our experiments, there was significant mouse-to-mouse variation in constitutive peritoneal macrophage class II expression which was independent of the founder line observed. Thus, approximately one-third of the animals examined had no detectable surface I-Ab on peritoneal macrophages (data not shown). As expected, neither potato starch nor thioglycolate-elicited peritoneal CD11b+ cells expressed I-Ab since these stimuli typically do not induce class II expression on macrophages (38). Immature, unactivated transgenic splenic macrophages had no detectable surface class II expression.



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Fig. 2. Surface expression of I-Ab can be detected on the peritoneal and thymic dendritic cells of transgenic animals. Peritoneal cells, splenocytes or thymic dendritic cells were purified from Aßb–/– (faint line), Aßb +/– (dashed line) or hLP/Aßb (bold line) mice, stained with anti-I-Ab and either CD11b (Mac 1) (A–C) or NLDC145 and CD11c (D), and analyzed on a FACScan. I-Ab expression by CD11b+ (A–C) or NLDC145+/CD11c+ (D) cells is shown. In (D), the mean fluorescent intensities of the dendritic cells from each animal are: Aßb –/–, 21; Aßb +/–, 168; and hLP/Aßb, 26.

 
Thymic dendritic cells express low levels of transgene-driven class II
We next determined the cell source of transgene expression detected in the thymus by RT-PCR. The thymi of wild-type, Aßb –/– and hLP Aßb –/– mice were analyzed by immunohistochemistry for I-Ab expression, and the results are shown in Fig. 3Go. Occasional punctate regions of staining were apparent in the hLP Aßb –/– thymi and were limited to the medulla. However, this staining was not uniform throughout the medulla and was weak enough that it could only be detected with additional amplification of the signal (cf. Fig. 3CGo with E and F). As seen in Fig. 3Go(E and F), only a subpopulation of medullary cells was I-Ab-positive. The absence of anti-I-Ab staining in the cortex suggested that the class II+ cells were not macrophages since macrophages are evenly distributed between thymic cortex and medulla (39). Further, the cells were UEA-1 and had the stellate appearance of dendritic cells. To confirm the identity of the thymic transgene-expressing cells, dendritic cells were purified from DNase/collagenase-dispersed thymi through adherence and density gradient centrifugation. Flow cytometric analysis revealed low but reproducible levels of I-Ab staining on NLDC145+/CD11c+ cells, consistent with their classification as dendritic cells (Fig. 2CGo). In contrast, I-Ab-staining could not be detected on CD11b+ cells obtained in parallel (data not shown). Thus, hLP-driven expression of Aßb restores low-level MHC class II expression to mature peritoneal macrophages and a subpopulation of dendritic cells within the thymic medulla.



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Fig. 3. Immunohistochemical analysis of transgene expression in the thymus. The figure shows staining for MHC class II I-Ab (red) and the medullary epithelial cell marker UEA-1 (blue) (A, C, D and E) in Aßb –/– (A and D), Aßb +/– (B) and hLP/Aßb (C, E and F) thymic sections. In (D and F), the anti-I-Ab staining is enhanced with tyramide amplification. The I-Ab-positive cells in the hLP transgenic sections (E and F) are indicated by arrows. Note that the transgenic I-Ab-positive cells are UEA. Photographs were taken at x200 original magnification.

 
Antigen presentation function of transgenic APC in vitro
Two different in vitro systems were used to assess the antigen presentation ability of the transgenic class II+ APC. We asked whether the transgenic APC could present an E{alpha} peptide to the IH3.1 Y-Ae-restricted T hybrid (33) and whether such APC could stimulate autoreactive K14 I-Ab-restricted CD4 cells. Neither freshly isolated peritoneal cells, adherent splenocytes nor semi-adherent thymic APC could present E{alpha} peptide to a Y-Ae-specific hybrid over a broad range of peptide concentrations, despite flow cytometry demonstrating that the APC were, indeed, class II+ (Fig. 4A and BGo, and data not shown). However, spleen cells treated with IL-4 and GM-CSF minimally, but reproducibly, induced IL-2 production by the I-Ab-restricted hybrid (Fig. 4CGo, inset), despite such cells having undetectable surface levels of class II.



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Fig. 4. hLP/Aßb transgenic APC only minimally present E{alpha} peptide to the 1H3.1 T-T hybrid. Either 5x104 peritoneal cells (A), untreated semi-adherent splenic APC (B) or semi-adherent APC cultured in IL-4 and GM-CSF (C) were cultured with 1x105 1H3.1 in the presence of increasing concentrations of exogenous E{alpha} 52-68 peptide. The inset in (C) shows the data from the Aßb –/– and hLP/Aßb –/– experiment. IL-2 production by the T cell hybrid was assessed by [3H]thymidine uptake of the indicator cell line, HT-2.

 
Given the minimal responses elicited in a peptide-restricted T hybrid by the transgenic APC, we turned to an ostensibly more sensitive assay of antigen presentation—proliferation of autoreactive I-Ab-restricted primary CD4+ T cells. We previously described K14 mice, in which Aßb expression is restricted to the thymic cortical epithelium, resulting in positive selection of CD4+ T cells in the absence of negative selection. These peripheral T cells proliferated in vitro to I-Ab-positive APC (28). We thus asked if hLP/Aßb –/– APC could stimulate the proliferation in vitro of primary K14 CD4+ T cells. APC were obtained from hLP/Aßb –/–, Aßb –/– or Aßb +/– animals as above and co-cultured with K14 CD4 cells. [3H]Thymidine incorporation was measured on day 4. Despite the consistent presence of surface class II, freshly isolated peritoneal cells only minimally presented antigen to K14 CD4+ T cells (Fig. 5Go). Splenic APC enriched by brief adherence to plastic could induce proliferation of K14 CD4 cells, although to only one-third the level induced by adherent wild-type cells. However, treatment of these semi-adherent splenic APC with IL-4 and GM-CSF improved the antigen presentation ability of both wild-type and hLP APC ~3-fold. Treatment with either IFN-{gamma} alone or IFN-{gamma} in combination with LPS did not improve the antigen presentation function of either the semi-adherent or macrophage-like adherent cells (data not shown). The response of semi-adherent APC to IL-4 and GM-CSF, rather than IFN-{gamma} or LPS, suggests that the population of APC which express the transgene and are competent to present antigen are almost certainly dendritic cells. Of note, CD11c+/NLDC 145+ thymic dendritic cells—which we previously showed were class II+—did not stimulate proliferation by K14 CD4+ T cells (data not shown).



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Fig. 5. hLP/Aßb transgenic APC stimulate self-reactive K14 CD4 cells to proliferate in vitro. Increasing numbers of peritoneal cells (A), untreated semi-adherent splenic APC (B) or semi-adherent APC cultured in IL-4 and GM-CSF (C) were irradiated at 2000 rad and cultured with 1x105 purified self-reactive K14 CD4+ T cells for 4 days. [3H]Thymidine was added for the last 18 h.

 
In vivo function of transgenic APC
Given that peripheral hLP/Aßb transgenic APC could weakly stimulate autoreactive K14 CD4+ T cells in vitro, we were interested in determining the effect of introducing the hLP transgene on the self-reactivity and immune function of K14 animals. Therefore, hLP/Aßb –/– animals were bred to K14 Aßb –/– mice and double transgenic offspring were analyzed.

The T cell populations of the thymus and spleen of double transgenic hLP/K14 Aßb –/–, single transgenic, and Aßb –/– and +/– control mice were analyzed by flow cytometry, and the data are summarized in Table 1Go. Single hLP/Aßb –/– transgenic mice, like Aßb –/– mice, have very few thymic and peripheral CD4 cells, consistent with previous reports that neither hematopoietic cells nor dendritic cells can mediate the positive selection of CD4+ T cells (16,40). As we noted previously, ~8–10% of peripheral T cells in the K14 Aßb –/– mouse are CD4+ (28). Introduction of the hLP transgene caused a significant decrease in the number of SP CD4+ T cells present in both the thymus (to 2–4%) and periphery (to 4%) of double transgenic hLP/K14 Aßb –/– mice as compared with single transgenic K14 mice. This reduction in CD4+ T cells could be secondary to the effect of the transgene in either the thymus—allowing the negative selection of autoreactive cells—or in the periphery—by allowing the induction of peripheral tolerance. In either case, the reduction of the CD4 T cell population in the double transgenic animals clearly demonstrates that the transgenic APC are functional in vivo.


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Table 1. Quantitation of thymic and splenic subpopulations
 
We next examined the functional consequences of introducing the hLP transgene into the K14 mice by determining the response of the CD4 cells to stimulation. Similar to what we observed in vitro using the YAe-restricted hybrid, the class II+ hLP/Aßb APC were not competent to initiate an antigen-specific response in vivo as double transgenic animals failed to mount either antibody or T cell responses to s.c. or i.p. immunization with a protein antigen (data not shown). The proliferative response of total thymocytes or peripheral CD4+ T cells to syngeneic and allogeneic stimulator cells in MLR was therefore next examined. As we previously reported, single transgenic K14 Aßb –/– thymocytes or CD4+ splenocytes proliferate in response to syngeneic B6 stimulators, in agreement with our interpretation that these cells are self-reactive due to expression of class II limited to cortical epithelium. Under the conditions employed in these assays, an allogeneic proliferative response by wild-type cells is not detected. However, as seen in Fig. 6Go(B), CD4+ T cells purified from double transgenic hLP/K14 Aßb –/–, single transgenic K14 and +/– control mice are all functional in that they proliferate to allogeneic BALB/c stimulators. The double transgenic hLP/K14 thymocytes and peripheral CD4+ T cells did not display the striking anti-B6 self-response seen in the K14 animals despite maintaining a peripheral allo-response. Thus, introduction of the hLP Aßb transgene is sufficient to prevent the autoreactivity of K14 CD4 cells. Further, the loss of the syngeneic response by double transgenic thymocytes suggests that this is a thymic, rather than a peripheral, event.



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Fig. 6. Introduction of the hLP transgene abolishes almost all anti-B6 reactivity from K14 mice. Either 4x105 thymocytes (A) or 1x105 CD4+ T cells (B) from the indicated mice were cultured with titrations of irradiated spleen APC from syngeneic C57Bl/6 or allogeneic BALB/c mice for 4 days. [3H]Thymidine was added for the last 18 h.

 
A careful examination of the hLP/K14 anti-B6 MLR reveals that the autoreactive response has not been completely ablated. There is a minimal, but reproducible, amount of persistent proliferation to syngeneic B6 stimulators present in the double transgenic hLP/K14 Aßb –/– responder CD4 cells. In three different experiments (using both founder lines), hLP/K14 Aßb –/– CD4+ T cells activated with a large number (8x105) of B6 stimulators proliferated ~3-fold over baseline and a dose response to increasing numbers of stimulators was present. In the setting of this very small proliferative response, we were, however, unable to detect the production of the cytokines, IL-2, IL-4 or IFN-{gamma} (data not shown).

The hLP Aßb transgene eliminates 75% of autoreactive K14 CD4 cells
To determine the percentage of autoreactive CD4+ T cells remaining in the periphery of hLP/K14—as compared with K14—animals, we made an indirect measurement of the precursor frequency of self-reactive CD4+ T cells. Previous analyses have suggested that the number of cells which incorporate BrdU—a measure of DNA synthesis—in the presence of a particular stimulus accurately reflects the precursor frequency of cells present in vivo (41). By limiting dilution analysis, we previously estimated that ~5% (one of 21) of splenic K14 CD4+ T cells was self-reactive. As seen in Fig. 7, Go14.5% (ranging between 14 and 15 %) of K14 CD4+ T cells incorporate BrdU on day 2 in response to syngeneic B6 stimulators. The difference between our previously published figure of 5% and this figure probably reflects plating inefficiency in the precursor frequency analysis. A relative increase in the number of T cells incorporating BrdU secondary to the death of those cells which are not activated is also possible, although all cells irrespective of their forward and side scatter profiles (both dead and live cells) were included in the analysis.



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Fig. 7. Precursor frequency of hLP/K14 CD4+ T cells reactive to B6 APC in vitro. CD4+ T cells (2x106) from the mice indicated (A–C) were cultured with T-depleted irradiated B6 APC (5x106) in 24-well plates for 3 days in the presence of 1 µM colchicine and 5 µg/ml BrdU. Cells were harvested, stained with anti-CD4 and anti-BrdU antibodies, and analyzed by flow cytometry. Histograms are gated on CD4+ cells. Background staining of the K14 anti-B6 response in the absence of BrdU (D) was ~1%. A representative experiment of three is shown.

 
Whereas 14–15% of the K14 CD4+ T cells incorporate BrdU, 3–4% of double transgenic hLP/K14 CD4+ T cells were activated by syngeneic B6 stimulators. Thus, ~75% of the self-reactive CD4+ T cells were eliminated from the periphery of K14 mice upon introduction of the hLP transgene.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have utilized the human lysozyme promoter to re-express the MHC class II polypeptide Aßb in an Aßb –/– environment. Introduction of the hLP/Aßb transgene restored low-level I-Ab surface expression to mature peritoneal macrophages and thymic dendritic cells. We found that a population of transgenic, semi-adherent splenic APC—which responded to IL-4 and GM-CSF—functioned weakly as APC to both a peptide-specific T cell hybrid and to primary autoreactive K14 CD4+ T cells in vitro. In contrast, transgenic class II+ peritoneal macrophages and thymic dendritic cells failed to present antigen to these populations. Nevertheless, the minimal levels of surface class II expressed by transgenic thymic APC, while not sufficient for T cell activation, were sufficient to ablate the autoreactive response of K14 CD4 T cells. From these data we draw two conclusions. First, as has been previously shown, the level of class II expression on APC is a critical, but not the only, factor that determines their capacity to activate T cells. Second, since negative selection fails to occur in the K14 animals, we conclude that provision of extremely low levels of class II to thymic APC confers on them the ability to mediate clonal deletion of autoreactive T cells.

The distribution of transgene expression in the hLP Aßb –/– mice is similar to that described by other groups utilizing the same promoter. Dighe et al. found that an hLP-driven mutant IFN-{gamma} receptor was expressed in mature peritoneal and peripheral blood macrophages and ascribed functional activity of the transgene to these cells (37). Using a more sensitive assay (bacterial CAT activity), Clarke et al. measured hLP-driven gene expression in extracts derived from liver, spleen, bone marrow and thymus, but did not fractionate these tissues to identify the cell type responsible for the transgene expression (29). In contrast with these earlier studies, we were unable to detect I-Ab expression in vivo on peritoneal cells after treatment of the animals with either thioglycollate or M. bovis (BCG). Similarly, in vitro treatment of semi-adherent peritoneal cells, splenic APC or thymic APC, with IFN-{gamma}, LPS or the combination of the two had no effect on surface class II levels. While it is possible that different 3' splicing cassettes used in the transgene constructs (human growth hormone versus SV40) account for this, it is more likely that the discrepant results reflect the requirement, in our system, for expression of both the hLP-driven transgene and the endogenous A{alpha}b gene. In the hLP Aßb –/– animal, I-Ab complexes will only be detected at those sites in which transcription of the lysozyme-driven Aßb chain overlaps with transcription of the endogenous A{alpha}b gene. Lysozyme is an antibacterial protein produced by macrophages treated with inflammatory stimuli (36). Its production is induced by bacterial products, such as LPS and BCG, as well as, phagocytic stimuli such as latex particles. Expression of the lysozyme gene does not parallel the differentiation of macrophages into effective antigen presenting cells with enhanced co-stimulatory activity and increased MHC class II expression. Thus, treatments such as IFN-{gamma} or tumor necrosis factor (TNF)-{alpha} which up-regulate endogenous class II transcription will have the opposite effect on transcription of the lysozyme/Aßb gene.

What cell type then is the effective APC in the transgenic animal? The antigen presentation function of splenic APC was enhanced by treatment of semi-adherent splenocytes with IL-4 and GM-CSF, cytokines which induce the maturation of myeloid dendritic cells. There are no published descriptions of expression of the endogenous lysozyme gene by dendritic cells. However, there is evidence in the human system that monocytes can differentiate either into macrophages through treatment with macrophage colony stimulating factor or into immature dendritic cells through treatment with IL-4 and GM-CSF, the stimuli we utilized to enhance the antigen presentation function of splenic transgenic APC (42). Maturation into fully differentiated dendritic cells requires treatment with anti-CD40, LPS and TNF-{alpha}, and is an irreversible event. We therefore believe that the functional peripheral APC in the hLP transgenic mouse is an immature dendritic cell of the myeloid (rather than lymphoid) lineage.

The identity of the functional thymic APC, as defined by its ability to eliminate autoreactive T cells, is clearer. The I-Ab-positive transgenic cells detected by immunohistochemistry had a stellate pattern and were limited to the medulla, characteristics of thymic dendritic cells. In two different founder lines, these class II+ cells expressed the surface markers N418+ and NLDC145+, markers which are specific for dendritic cells. Further, these cells are unlikely to be macrophages since there is no evidence that fully differentiated macrophages can mediate negative selection in the thymus (20), a function generally ascribed to dendritic cells and thymic medullary epithelium. Taken together, these data suggest that the functional class II+ transgenic APC in the thymus is also a dendritic cell. Interestingly, in contrast to the splenic dendritic cell, these thymic dendritic cells could not act as APC as measured by K14 T cell proliferation. This difference may reflect phenotypic differences (i.e. co-stimulatory potential or cytokine secretion) between thymic and peripheral dendritic cells. Alternatively, a weaker affinity of thymocyte–APC interactions may be required for the negative selection of self-reactive thymocytes as compared with the activation of peripheral cells.

Despite the minimal in vitro antigen presentation and absent in vivo antigen presentation of transgenic APC, introduction of the hLP/Aßb transgene was sufficient to abrogate almost all of the autoreactivity of K14 CD4+ T cells. We were able to show that IL-4/GM-CSF-treated splenic APC could activate primary K14 CD4 cells in vitro, whereas the I-Ab-positive NLDC145+/N418+ population of thymic APC did not have this function. Thus, it is worth considering whether the induction of tolerance in the double transgenic animals occurs centrally or peripherally. The phenotype observed could arise from either thymic (central) deletion of autoreactive cells or from the introduction of peripheral tolerance into a system in which the CD4 cells are dysregulated secondary to the absence of peripheral class II+ APC. Our data support the introduction of thymic deletion for two reasons. First, flow cytometric analysis showed that double transgenic hLP/K14 Aßb –/– mice had approximately half the number of thymic single-positive CD4 cells that single-positive K14 Aßb –/– mice have, consistent with a direct effect on the thymic development of CD4+ T cells. We do not believe that this reflects an effect of the hLP transgene on the positive selection of CD4 cells in the K14 thymus as single transgenic hLP Aßb –/– mice have the same number of peripheral CD4 cells as Aßb –/– mice in agreement with previous observations that neither total bone marrow (40) nor dendritic cells (16) are capable of inducing the positive selection of CD4 cells. Second, introduction of the hLP transgene eliminated the anti-self MLR present in both the periphery and thymus of K14 mice. These data are suggestive of a central effect. Ultimately, this question can best be addressed by mating the double transgenic hLP/K14 and single transgenic K14 mice to an I-Ab-restricted autoreactive TCR transgenic and monitoring the deletion of TCR transgenic thymocytes in the presence of the hLP transgene, and this experiment is currently underway.

Although negative selection is believed to be a highly efficient process, the quantitative limits of the process continue to be defined. The variation in clonal deletion associated with alterations in thymic peptide concentration has been established by examining the thymic selection of MHC class I-restricted transgenic TCRs in fetal thymic organ culture (FTOC) and quantitating the frequency of peripheral cytotoxic T lymphocytes restricted to self-peptides appearing at low peptide concentrations. In a study designed to examine the requirements for positive selection, Merkenschlager et al. followed the clonal deletion of HY-restricted TCR transgenic thymocytes in fetal thymic re-aggregation chimeras with increasing ratios of male and female thymic stromal cells. They found that deletion of H-Y-specific cells could be detected at the smallest number of input male cells examined (~4%) and increased proportionately with the percentage of male cells added (43). Spain and Berg examined the role played by MHC class II surface density in the efficiency of negative selection and showed that 10-fold higher concentrations of cognate peptide were required in MHC class II-heterozygous FTOC to induce the same amount of clonal deletion as in homozygous thymi (27). This study clearly demonstrated that the level of presenting class II molecules was as important in determining the efficiency of clonal deletion as the concentration of deleting peptide. However, there is a threshold of peptide concentration below which negative selection does not occur (44). Similarly, a threshold of MHC class II surface density below which negative selection does not occur should also be present. In the current study, we have shown that the threshold for both class II level and effective cell number must be extraordinarily low. In hLP/K14 double transgenic mice, a small subpopulation of dendritic cells whose mean fluorescent intensity of surface class II was barely greater than matched class II cells efficiently deleted 75% of a polyclonal population of autoreactive cells revealing the extraordinary efficiency of the negative selection process. Although we have not yet determined the specificity of those few autoreactive CD4+ cells which persist in the hLP/K14 thymus, we would predict that they are cells whose TCR either have very low affinity for I-Ab–peptide complexes or whose cognate ligand occurs at very reduced concentration (if at all) on the surface of dendritic cells.


    Acknowledgments
 
The authors would like to thank Lian Fan for outstanding technical assistance. This work was funded by grants from the NIH to T. M. L. (AR01940), D. L. (AI38375) and L. H. G. (AI21569), and a grant to L. H. G. from the G. Harold and Leila Y. Mathers Foundation. E. S. J. M. de B. was supported by a grant from the Koningin Wilhelmina Fonds/Nederlandse Kankerbestrijding and the Foundation for Pediatric Oncology Research Groningen.


    Abbreviations
 
APCantigen-presenting cell
CATchloramphenicol acetyltransferase
FTOCfetal thymic organ culture
GM-CSFgranulocyte macrophage colony stimulating factor
hLPhuman lysozyme promoter
LPSlipopolysaccharide
MLRmixed lymphocyte reaction
TNFtumor necrosis factor

    Notes
 
Transmitting editor: A. Singer

4 Present address: Department of Pediatric Oncology, University Hospital Groningen, 9700 RB Groningen, The Netherlands Back

5 Present address: Division of Rheumatology, University of Pennsylvania, Philadelphia, PA 19104, USA Back

Received 29 October 1998, accepted 28 April 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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