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The Journal of Immunology, 2004, 172: 155-161.
Copyright © 2004 by The American Association of Immunologists

Fully Functional HLA B27-Restricted CD4+ as well as CD8+ T Cell Responses in TCR Transgenic Mice

Matthew Roddis*, Robert W. Carter*, Mei-Yi Sun{dagger}, Thomas Weissensteiner*, Andrew J. McMichael{dagger}, Paul Bowness{dagger} and Helen C. Bodmer1,*

* Edward Jenner Institute for Vaccine Research, Compton, Newbury, United Kingdom; and {dagger} Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The strong association of HLA B27 with spondyloarthropathies contrasts strikingly with most autoimmune diseases, which are HLA class II associated and thought to be mediated by CD4+ T lymphocytes. By introducing a human-derived HLA B27-restricted TCR into HLA B27 transgenic mice, we have obtained a functional TCR transgenic model, GRb, dependent on HLA B27 for response. Surprisingly, HLA B27 supported CD4+ as well as CD8+ T cell responses in vivo and in vitro. Further, HLA B27-restriced CD4+ T cells were capable of differentiation into a range of Th1 and Th2 T cell subsets with normal patterns of cytokine expression. The transgenic T cells were also able to enhance clearance of recombinant vaccinia virus containing influenza nucleoprotein in vivo. This is the first description of a human HLA class I-restricted TCR transgenic line. The existence of CD4+ MHC class I-restricted T cells has significant implications for immune regulation in autoimmunity and, in particular, in HLA B27-associated arthritis. We believe that this model provides a novel system for the study of unusual T cell behavior in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A current paradigm for the pathogenesis of many autoimmune diseases involves recognition by CD4+ T cells of peptide autoantigens presented by MHC class II molecules. This is consistent with the observation that the vast majority of autoimmune diseases have HLA class II associations. Key exceptions are the association of HLA B27 with the spondyloarthropathies (1, 2) and of HLA A29 with birdshot retinopathy (3).

The association of the seronegative spondyloarthropathies with the HLA class I molecule, HLA B27, is one of the strongest associations of any autoimmune disease (odds ratio, 171:1) (1). There is a variety of hypotheses proposed for this strong association, including those related to Ag presentation properties (4), interaction with NK cells (5), biochemical properties such as homodimer formation (6), and effects on responses to intracellular bacterial infection (7, 8, 9, 10, 11). One of the fundamental principals in T cell immunology is that CD4+ T cells recognize antigenic peptides in association with MHC class II molecules, whereas CD8+ T cells recognize peptide/MHC class I complexes. Increasing evidence supports the idea of CD4+ T cells as the controllers of an established immune response through a balance of differentiation into immune regulatory cell subtypes secreting different combinations of cytokines. Tight regulation of expression of MHC class II molecules together with other regulatory mechanisms on class II-positive APC are thought to be important elements in the overall induction and control of CD4+ T cell responses. By contrast, although regulatory functions for CD8+ T cells have been described, these cells are one of the major effector arms of the adaptive immune response, for instance in the lysis of infected cells. MHC class I is constitutively expressed on virtually all cell types in the body, with only a few exceptions where expression is low, unless in the presence of inflammation.

That it is largely the CD4+ T cells that control immune responses is consistent with the observation that in the vast majority of autoimmune diseases that have HLA associations, these associations are with MHC class II molecules. In a recent report, Gaston and colleagues (12) have described the existence of HLA B27-specific or -restricted T cells with a CD4+ phenotype. In addition, CD4+ T cells have been implicated in the inflammatory disease seen in susceptible HLA B27 transgenic rats, where transfer of CD4+ T cells is more efficient in the induction of disease than that of CD8+ T cells (13).

To study the potential roles and behavior of an HLA B27-restricted T cell in vivo, we have created a model system in which variable portions of a TCR (denoted GRb) originally derived from an HLA B27-positive volunteer are expressed as a transgene in an HLA B2705, human {beta}2-microglobulin (h{beta}2m)2 transgenic (TG) mouse (14) using a mouse genomic cassette vector (15). We demonstrate that the receptor is expressed and produces fully functional T cells in the context of HLA B27. In addition, we show that CD4+ T cells expressing the TG TCR receptor can function as HLA B27-restricted T cells and are capable of differentiation to secrete cytokines indicative of either Th1 or Th2 subsets of T cells.


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

All mice were bred in the specific pathogen-free facility at the Institute for Animal Health (Compton, U.K.). B27{beta}2m (BALB/c) mice, expressing HLA B2705 and human {beta}2m were originally obtained from E. Weiss (14) and were bred as a homozygous inbred line. The GRb TCR TG line, expressing both the {alpha}- and {beta}-chains of the GRb TCR (as below), was maintained as a heterozygous line backcrossed to B27{beta}2m (BALB/c), except where stated. To obtain mice lacking MHC class II, GRb+ B27{beta}2m mice were crossed to A{beta} knockout mice (16). F1 mice that were GRb positive were interbred to produce offspring for analysis.

For in vivo infection with influenza A virus A/X31, mice were lightly anesthetized with isoflurathane, and 20 hemagglutinin units of virus in 30 µl of PBS was administered intranasally.

All animal experiments were performed under a Home Office project license in compliance with relevant laws and local guidelines and were approved by the Institute for Animal Health ethical committee.

Creation of the GRb TG mouse line

Genomic DNA was isolated from a well-characterized CD8+ T cell clone, GRb, which was HLA B27-restricted and specific for influenza A virus nucleoprotein (NP383–391) peptide (17), V{alpha}14.1 J{alpha}9.3, and V{beta}7.1 J{beta}2.3. The sequence, previously obtained from cDNA from the clone, was used to deduce the full genomic sequences of the V and J elements to design primers upstream of the V leader and in the intron following the specific J regions for insertion into cassette vectors with unique cloning sites as previously described (15). Genomic DNA from the T cell clone was prepared and used as a template for PCR amplification of the TCR{alpha} and TCR{beta} variable elements. For TCR{alpha} cloning, the forward primer, 5'-CACCCTGCACCCGGGACCTG-3', sequence was derived from the published V{alpha}14.1 (hADV38S2; accession no. AE000661; bases 33975–33994) with a G to C change at 33,984 and an A to C change at 33,986 to introduce an XmaI site. The reverse primer, 5'-TCTTTGTACCGCGGAGTTCTAATCCCTC-3', sequence was derived from the published J{alpha}9.3 (J 54) genomic sequence (accession no. M94081) with the SacII site 141 bases downstream of the J 54 coding sequence, bases 23,443–23,470, but introducing a SacII site at bases 23,457–23,462. Fragments were digested with XmaI and SacII and cloned into a shuttle vector for sequencing, and a correct clone was selected for insertion into the pT{alpha}cass (15). Similarly, for the TCR {beta}-chain cloning the forward primer, 5'-CTCAGACTCGAGGCTAGCATGG-3', sequence was derived from the published V{beta}7.1 (TCRBV7S1A1N2T; accession no. U66059, bases 105,756–105,777) with a C to T change at 105,763 to introduce an XhoI site. The reverse primer, 5'-CGGCCGCGGCTTACCCAG-3', sequence was derived from the published J{beta}2.3 (TCRBJ2S3; accession no. U66061, bases 198,776–198,793) with a SacII site introduced 158 bases downstream of coding sequence at bases 198,786–198,791. PCR fragments were digested with XhoI and SacII and cloned into the pT{beta}cass after verifying the correct sequence in a shuttle vector. SalI and KpnI fragments from the {alpha} and {beta} cassettes, respectively, were purified mixed in a 1:1 ratio and microinjected into CB6F1 x B27{beta}2m (BALB/c). A founder expressing both {alpha}- and {beta}-chains was identified and maintained as described above. All mice used in these experiments were homozygote for the H-2d haplotype, except when backcrossed to A{beta} knockout mice.

In vitro stimulation of T cell bulk cultures

Bulk cultures of splenocytes from either naive or in vivo primed mice were set up as follows. For peptide-stimulated cultures, stimulator splenocytes from either the same mouse or a naive B27{beta}2m (BALB/c) mouse were pulsed with 1 µM of the appropriate peptide for 1 h and washed. Stimulator cells (3 x 106) were cultured with 1.5 x 107 responder spleen cells in complete medium containing 10% FCS, with the addition of 5 U/ml recombinant mouse IL-2 (R&D Systems, Minneapolis, MN) in a 15-ml volume in a 25-cm2 flask. Cultures were incubated for 5–8 days at 37°C in 5% CO2. If influenza A virus was used as a stimulus, then stimulator cells were first infected with 100 hemagglutinin units of A/X31 virus/107 cells for 60–90 min in serum-free medium before addition of 3 x 106 flu-infected cells to 1.5 x 107 responder cells, as described above, except that no exogenous IL-2 was added to the cultures.

FACS staining

Cells from in vitro culture or disrupted lymph nodes, spleen, or thymus from naive or primed mice were stained by standard methods with anti-CD4 and anti-CD8 directly conjugated Abs (BD Biosciences, Oxford, U.K.) plus a third Ab or HLA B27/h{beta}2m/NP383–391 (B27/NP383) tetramer (18) as indicated. Where tetramer was used, cells were first incubated at 37°C for 10 min and put on ice before addition of Abs.

For intracellular cytokine assays, 3 x 105–106 cells were incubated directly with peptide or with peptide-pulsed stimulator cells as indicated in 1 ml at 37°C in 5% CO2. After 3 h, brefeldin A was added to a final concentration of 10 µg/ml, and cells were incubated for an additional 4 h. For staining, cells were washed in facswash (1% FCS and 0.1% azide in PBS) and surface-stained with anti-CD4-PE and anti-CD8 CyChrome (BD Biosciences). Cells were then fixed in 2% paraformaldehyde, plus 5µg/ml brefeldin A vortexed vigorously, and incubated at room temperature for 20 min. Cell were washed twice in facswash and resuspended in 0.5% saponin in facswash for 10 min. Cells were then incubated with 50 µl of anti-IFN-{gamma}-FITC and anti-TNF-{alpha}-allophycocyanin where indicated or with isotype control Abs (1/50 dilution made up in 0.5% saponin) for 30 min at room temperature. Cells were resuspended in 1% paraformaldehyde before analysis on a FACSCalibur (BD Biosciences).

Vaccinia virus (VV) clearance assay

Female B27+/h{beta}2m+ mice that were either positive or negative for the GRb TCR were infected i.p. with 107 PFU VV recombinant for flu NP (VV-NP). Ovaries were removed 5 days after infection, and a VV plaque assay was performed. Ovaries were homogenized in 1 ml of PBS. Serial dilutions of homogenate were added to confluent layers of 143 TK cells in six-well plates and incubated for 1 h at 37°C in 5% CO2. Cultures were then further incubated for 3 days under a layer of 0.5% agarose in medium. Cells were fixed by adding 2 ml of a 25% solution of formalin in PBS, agarose was stripped off the wells, and the cell monolayer was stained with crystal violet to reveal plaques for counting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of the GRb TCR in GRb TCR TG mice

To date, there are no published reports of human MHC class I-restricted TCR TG mice. We chose to use TCR {alpha} and {beta} cassette vectors (15), which would allow expression of the variable parts of the human receptor in conjunction with the respective mouse constant regions. This is an approach that has been successful for the expression of HLA class II-restricted TCRs (19). Mice TG for both {alpha}- and {beta}-chains of the receptor in the presence of HLA B27 and human {beta}2m expressed slightly reduced number of T cells, with both CD4+ and CD8+ single-positive T cells present in lymph nodes (Fig. 1a), spleen, and thymus (data not shown). In addition, >90% of all CD4+ and CD8+ cells in the lymph nodes expressed the transgene-derived human V{beta}7 molecule. Mouse V{beta}8, normally one of the most common variable genes used in BALB/c mice, was virtually undetectable in lymph nodes of GRb+ HLA B27+/h{beta}2m+ mice (Fig. 1a), implying allelic exclusion of the mouse TCR {beta}-chains by the TG human TCR {beta}-chain. In contrast, murine V{alpha}2 was expressed on comparable numbers of T cells in TCR TG and non-TG mice, indicating that this {alpha}-chain was not affected by allelic exclusion (Fig. 1a). No Ab was available for the transgene-derived human V{alpha}14 chain, but expression of the complete GRb receptor was detected using an HLA B2705/h{beta}2m tetramer presenting the specific peptide NP383–391 (B27/NP383). Fig. 1b (top row) shows representative staining of CD4+ and CD8+ T cells from a GRb+ HLA B27+/h{beta}2m+ mouse. To determine the relevance of HLA B27 to the pattern of expression of the GRb receptor, some mice were backcrossed to BALB/c to eliminate the expression of HLA B27 (with or without h{beta}2m). Although effects were variable, the absence of HLA B27 frequently resulted in a lower percentage of B27/NP383 tetramer-positive cells in both the CD8 and CD4 compartments (Fig. 1b, lower rows), which we have not seen in any of the HLA B27+ mice we have used in the various studies. This was matched by higher numbers of human V{beta}7-negative cells and an increase in the mouse V{beta}8.1 populations (data not shown).



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  		FIGURE 1. TCR expression in lymph nodes of GRb+ and GRb- mice. a, Comparison of representative GRb+ and GRb- lymph node cells in the presence of HLA B27 and h{beta}2m. Density plot, CD4 vs CD8; top histograms, expression of human V{beta}7 on CD4+ or CD8+ lymph node T cells; middle histograms, expression of mouse V{alpha}2 on CD4+ or CD8+ T cells; bottom histograms, expression of mouse V{beta}8.1 on CD4+ or CD8+ T cells. b, B27/NP383 tetramer on CD4+ or CD8+ T cells from GRb+ females in the presence or the absence of HLA B27 and/or h{beta}2m transgenes as indicated. The top row shows a representative HLA B27-positive mouse, and the middle rows are representative of the range of staining in GRb mice lacking HLA B27. c, Percentages of CD4-positive cells in PBL of HLA B27+ h{beta}2m+ or HLA B27- mice in mice expressing MHC class II (top panel) or in mice lacking MHC class II (bottom panel).

 
To examine the role of HLA B27 in the selection or maintenance of the CD4 T cell population, GRb mice were crossed to mice lacking MHC class II. Fig. 1c shows analysis of the percentages of CD4+ T cells in PBL of F2 mice (all GRb+) in either the presence (top panel) or absence (bottom panel) of MHC class II. There was a significant decrease in the percentages of CD4+ T cells in B27-negative vs B27-positive mice only in the presence of MHC class II.

GRb mice make efficient responses to NP383–391, but poor responses to third-party Ags

To assess the function of the TCR transgene, GRb+ or GRb-, HLA B27+/h{beta}2m+ mice were infected with influenza A virus A/X31. Two weeks later, spleen cells were isolated, and bulk cultures were set up to analyze responses to the H-2 Kd (NP147–155) or HLA B27 (NP383–391)-restricted epitopes in influenza A virus NP. Fig. 2a shows FACS staining of the various cultures compared with unstimulated spleen. Stimulation with the NP383–391 peptide resulted in a considerable expansion of the CD8+, B27/NP383 tetramer-positive cells. In contrast, there was only a small expansion of this population in GRb- mice. Culturing the GRb+ spleen with NP147–155, however, failed to stimulate the cultures, with few viable cells resulting. In a standard 51Cr release cytotoxicity assay, cultures from the TCR TG mice were able to lyse HLA B27-transfected L cells pulsed with NP383–391 peptide (Fig. 2b). Some non-TCR TG mice were also able to respond to this peptide, although the response was lower and more variable (Fig. 2b) (14). GRb+ mice failed to make a significant cytotoxic response to the Kd epitope NP147–155. In addition, they made poor responses to other third-party Ags, such as hen egg lysozyme (data not shown). Despite this, after flu infection, GRb+ mice remained healthy, with at most a mild transient systemic illness seen in non-TG littermates.



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  		FIGURE 2. GRb+ response to virus infection. GRb+ or GRb- HLA B27+/h{beta}2m+ mice were infected with influenza A virus. Two weeks later mice were sacrificed, and splenocytes were restimulated in vitro with the Kd-restricted epitope peptide NP147–155 (TYQRTRALV) or the HLA B27-restricted epitope peptide NP383–391 (SRYWAITRTR; 1 µmol) in the presence of 5 U/ml IL-2. a, CD4-FITC, CD8-CyChrome and B27/NP383 tetramer. Top row, Spleen cells before stimulation of one representative GRb+ and one GRb- mouse. Middle rows, After stimulation with NP383–391 peptide, two representative GRb+ and two negative mice are shown. Bottom row, One representative of each after NP147–155 stimulation. b, GRb+-derived bulk cultures are cytotoxic against HLA B27-positive cells pulsed with the cognate peptide. Top row, Standard 51Cr release assay with the NP383–391-stimulated cultures from two GRb+ or two GRb- individuals. Targets cells are L cells transfected with HLA B2705 and h{beta}2m (40 ) pulsed with 1 µm NP383–391 (•) or unpulsed ({circ}). Bottom row, Standard 51Cr release assay with the NP147–155-stimulated cultures from two GRb+ or two GRb- individuals. Target cells are P815 cells pulsed with 1 µM NP147–155 ({blacktriangleup}) or unpulsed ({triangleup}).

 
We next tested the in vivo function of GRb+ T cells. Female HLA B27+/h{beta}2m+ TG mice, which were either positive or negative for the GRb transgenes, were infected with VV-NP. Five days later mice were sacrificed, and ovaries were removed to assess the virus titer (20). Fig. 3 shows that the virus titers of four GRb-positive mice were reduced by >1000-fold compared with their GRb-negative littermates. Thus, GRb T cells are functional in vivo and are able to clear viral infections. In addition, as VV-NP is recombinant for the whole NP protein, this also demonstrates that the HLA B27 epitope NP383–391 is naturally processed and presented in vivo.



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  		FIGURE 3. GRb+ mice clear primary VV-NP infection more rapidly than negative littermates. Female B27+/h{beta}2m+ mice that were either positive or negative for GRb TCR were infected i.p. with 107 PFU VV-NP. Ovaries were removed 5 days after infection, and a VV plaque assay was performed. Results are expressed as log virus titer per ovary and are representative of two experiments.

 
CD8 depletion does not completely abrogate the IFN-{gamma} response to NP383–391

Bulk cultures from the GRb+ animals stimulated with NP383–391 showed moderately higher CD4+ CD8+ T cell ratios than non-TG littermates (Fig. 2a). This was not due solely to IL-2 in the medium, as it did not occur with the NP147–155 peptide. We therefore determined whether these mice were capable of making an HLA B27-restricted CD4+ T cell response. Fig. 4 shows that NP383–391 (but not NP147–155)-specific IFN-{gamma} ELISPOT responses could still be detected after depletion of CD8+ T cells from splenocytes of flu-infected mice. The residual response represents about one-third of the total, even though the depleted population comprised <0.3% CD8+ T cells (data not shown). This strongly implies that CD8-negative T cells are able to respond specifically to NP383–391 peptide.



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  		FIGURE 4. Depletion of CD8 T cells does not abrogate the IFN-{gamma} ELISPOT response to NP383–391 in GRb+ mice. Ex vivo IFN-{gamma} ELISPOTs on whole spleen cells or spleen cells depleted of CD8+ T cells 2 wk after influenza A virus A/X31 infection. Responder splenocytes were either untreated or were depleted of CD8+ T cells using CD8-conjugated Dynabeads. Serial 1/3 dilutions of responder cells are shown from 5 x 105/well for each group. Results are representative of more than three experiments.

 
Cytokine production in GRb CD4+ and CD8+ T cells

To confirm that GRb+CD4+ T cells were able to produce IFN-{gamma} specifically in response to NP383–391 peptide, spleen cells from the VV-NP clearance mice (from Fig. 3) were assayed ex vivo for their ability to secrete IFN-{gamma} by intracellular cytokine staining. Fig. 5a shows examples from two GRb+ mice of spleen cells stimulated with either NP383–391 (top row) or a control peptide, NP366–374 (bottom row). More than one-third of the CD8+ T cells produce IFN-{gamma} specifically in response to the B27-restricted peptide, but not the control peptide. There was also a small, but specific, response from CD4+ T cells. In the same experiment, GRb-negative mice did not show significant responses to any peptide (data not shown).



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  		FIGURE 5. Both CD4+ and CD8+ cells respond to NP383–391 peptide on intracellular cytokine staining. a, Ex vivo intracellular IFN-{gamma} staining showing specific release on stimulation with NP383–391 (top row), but not control peptide NP366–374 (ASNENMETM; bottom row). Spleen cells from mice that had been infected with VV-NP 5 days previously were stimulated in vitro with 1 µmol peptide for a total of 6 h, with addition of brefeldin A (10 µg/ml) for the final 3 h. Results from two representative GRb+ mice are shown. b, Ex vivo intracellular IFN-{gamma} and TNF-{alpha} staining showing specific release on stimulation with NP383–391 (top row), but not control peptide NP366–374 (bottom row). Spleen cells from naive GRb+ mice were treated as described above with the addition of staining for TNF-{alpha} in a four-color analysis. c, Intracellular IFN-{gamma} and TNF-{alpha} release from GRb+ T cells after an 8-day in vitro stimulation with influenza A virus (A/X31)-infected stimulator spleen cells. Results are representative of at least three experiments.

 
Fig. 5b shows the ex vivo TNF-{alpha} and IFN-{gamma} responses from naive GRb+ spleen cells when they were stimulated directly ex vivo with NP383–391 or control peptide in the intracellular cytokine assay. Less than 1% of the CD4+ cells produced IFN-{gamma}, whereas there was a significant population of CD4+ cells secreting TNF-{alpha}. Approximately 3% of naive CD8+ T cells produced IFN-{gamma} with or without TNF-{alpha} ex vivo. Cells from these same mice were then cultured in vitro with A/X31 flu-infected stimulator cells for 7 days. After this primary in vitro stimulation, there was a very vigorous response from the CD8+ T cells, with ~36% producing IFN-{gamma}, of which about half also made TNF-{alpha} specifically in response to NP383–391 (Fig. 5c, top row), but not the control peptide (bottom row). In the same cultures, NP383–391 stimulated 35% of the CD4+ T cells to produce TNF-{alpha}. The pattern of response in CD4+ T cells was very different from that in CD8+ cells, as only 7% of the cells produced IFN-{gamma} (Fig. 5c). Despite the low levels of IFN-{gamma} secretion, virtually all responding cells in a similar assay were positive for IL-18R, a marker of Th1 cells (21), and were negative for ST2L, a marker of Th2 cells (22) (data not shown). These results show the existence of a significant NP383–391-specific Th1 CD4+ T cell population.

The response of CD4+ T cells, like that of CD8+ T cells, is dependent on presentation of the peptide by HLA B27, because when cultures were stimulated with BALB/c-derived stimulators pulsed with NP383–391 peptide there was no response from either the CD4+ or CD8+ T cells (data not shown).

CD4+ and CD8+ T cells respond with similar sensitivity to peptide

To date we have shown that CD4+ cells are induced either by natural infection of the host or by in vitro stimulation with virus-infected cells. This indicates that the CD4+ T cells are detecting naturally processed peptide and are able to respond to physiological levels of peptide presented on an APC. Fig. 6 shows a direct and simultaneous comparison of the peptide sensitivity of CD4+ and CD8+ T cells. Naive, GRb+, HLA B27+ h{beta}2m+ spleen cells were first stimulated in culture for 5 days with influenza A virus, A/X31-infected HLA B27+ h{beta}2m+ splenocytes. An intracellular cytokine assay was then performed in the presence of titration of NP383–391 peptide in culture. Cells were stained for CD4, CD8, IFN-{gamma}, and TNF-{alpha} in a four-color FACS analysis. The density plots show examples of staining at three concentrations of peptide. The graphs below show total specifically responding cells from two individual mice. As shown in Fig. 6, although the patterns of cytokine response differ, the shape of the curve for titration is similar for CD4+ and CD8+ T cells.



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  		FIGURE 6. GRb+ CD4 and CD8 T cells respond to NP383–391 peptide with similar sensitivity. Spleen cells from naive GRb+ HLA B27+ h{beta}2m+ animals were first stimulated in vitro with 20% the cell number of A/X31-infected stimulator HLA B27+ h{beta}2m+ spleen cells for 5 days. Responder cells (5 x 105 in 1 ml) were stimulated with decreasing concentrations of NP383–391 peptide (or 1 µM control peptide NP366–374), and an intracellular cytokine assay was performed as described in Fig. 5. Density plots show representative staining gated on CD8+ (top row) or CD4+ (bottom row) T cells at 2, 0.2, and 0.002 µM peptide. Graphs show comparisons of percent total responding cells (less percentages of control peptide, >0.4%) for two individual mice. Results are representative of two experiments and five mice.

 
CD4+ GRb T cells can be differentiated into both Th1 and Th2 subtypes

We have seen that as well as the expected CD8+ T cell response in GRb+ mice, we are easily able to generate CD4+ T cells that specifically respond to the HLA B27-binding peptide, NP383–391, but not to the control peptide. To establish whether HLA B27-restricted NP383–391-specific CD4+ T cell responses in these mice are capable of differentiation into different subsets of Th cells, culture conditions were set up to favor the development of either Th1- or Th2-polarized cells (23). Enriched populations of CD4+ T cells from splenocytes were cultured with NP383–391 peptide in either Th1-driving (IL-12 and anti-IL-4 Ab) or Th2-driving (IL-4, anti-IFN-{gamma}, and anti-IL-12) conditions. After two successive in vitro stimulations, CD4+ T cells were tested for the production of intracellular cytokines in a 6-h stimulation assay with either NP383–391 or NP366–374 peptides. Peptides were added directly to the T cells without additional APCs, and as mouse T cells never express MHC class II, the numbers of class II-positive APC should be very low. Fig. 7 shows that all CD4+ cells in both Th1 and Th2 cultures were B27/NP383 tetramer-positive. Stimulation of Th1-driven cells with NP383–391, but not with control peptide, led to >80% of the cell population producing IFN-{gamma}, with no IL-4-producing cells detected. In contrast, Th2-driven cells were specifically stimulated to produce IL-4, with only 2% of the total producing any IFN-{gamma}. Thus, GRb+ CD4+ T cells are capable of responding to the HLA B27-restricted peptide, NP383–391 and are capable of differentiating into both the Th1 and Th2 subsets of T cells seen with classical MHC class II-restricted CD4+ T cell responses.



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  		FIGURE 7. Both Th1 and Th2 CD4+ HLA B27-restricted, NP383–391-specific T cells can be derived from GRb+ animals. Purified CD4+ T cells from a naive GRb+ spleen underwent two rounds of stimulation in either Th1- or Th2-driving conditions. All cells were uniformly positive for B27/NP383 tetramer (top row). Cells were stimulated with control peptide, NP366–374 (top density plots) or NP383–391 (bottom density plots) by addition of 1 µmol peptide directly to the T cells without addition of further stimulators in a 6-h assay. Brefeldin A was added for the final 3 h, and cells were fixed and stained intracellularly with anti-IFN-{gamma} and anti-IL-4 or isotype control Abs. Results are representative of at least three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results clearly show that GRb+ TCR transgenic mice can mount a CD4+ T cell response that is restricted by the spondyloarthritis-associated class I allele HLA B27. Further, these CD4+ T cells exhibit apparently physiological characteristics of CD4+ T cells, such as the potential for different patterns of cytokine production. Our data provide evidence that in this system, HLA B27 expression may influence the positive selection of CD4+ T cells, in that there is an increased percentage of B27/NP383 tetramer-positive CD4+ T cells in HLA B27+, as opposed to B27-, mice (Fig. 1b). However, in the absence of MHC class II, very few CD4+ T cells are selected, and HLA B27 has no apparent influence on the numbers present (Fig. 1c). This indicates that HLA B27 is unable directly to positively select CD4+ GRb+ T cells. The absence of TCR {alpha}-chain allelic exclusion is known to permit the maturation of T cells with dual receptor specificity, expressing the transgenic {beta}-chain paired with both the transgenic {alpha}-chain and also an endogenous mouse {alpha}-chain (24). Thus, even in GRb+ mice, in which virtually all cells are B27/NP383 tetramer-positive, we found that ~10% of cells expressed an additional mouse TCR V{alpha}-chain (Fig. 1a, see histograms for V{alpha}2). The latter specificity could lead to positive selection of T cells on murine MHC class I or class II. The presence of GRb+ T cells in HLA B27-negative mice (Fig. 1b) suggests that that this process is of physiological importance. T cells with dual specificity have been described previously in TCR TG mice (25), and this mechanism has been shown to enable the expression of self-specific T cells (26). Thus, in our system we propose that HLA B27 alone does not select CD4+ GRb+ T cells, but that these cells are selected on mouse MHC class II, presumably through dual receptor expression of the GRb {beta}-chain pairing with a mouse endogenous {alpha}-chain. However, once these cells reach the periphery, they are maintained in statistically greater relative numbers in the presence of HLA B27 than in its absence (Fig. 1c). A mouse line that expresses only the {beta}-chain of the GRb TCR, to the virtually complete exclusion of mouse TCR {beta}, has normal numbers of CD4- and CD8-positive T cells, indicating that mouse endogenous {alpha}-chains can pair with the GRb {beta}-chain, allowing T cell selection in the mouse thymus. These cells are B27/NP383 tetramer negative (our unpublished observations).

We also provide evidence that HLA class I-restricted CD4+ T cells are functional in vivo and in vitro. Despite the requirement for MHC class II expression for the development of a substantial CD4+ population, CD4+ GRb T cell responses can be generated in the absence of mouse MHC class II (data not shown). There are few reports of class I-restricted CD4+ T cells in TCR TG mice, and in most cases a certain amount of manipulation is required for these to be expressed (27, 28). There are some reports of CD4+ HLA class I-restricted T cells that have been isolated in humans. Bagot et al. (29) isolated CD4+ HLA class I-restricted, tumor-infiltrating lymphocytes from an HLA class II-negative lymphoma. Several groups have described HLA A2-restricted CD4+ T cells from a melanoma patient (30), derived after H-Y immunization (31) or alloreactive cells (32). More importantly, in relation to HLA B27, CD4+ HLA B27-reactive T cell clones have been isolated in ankylosing spondylitis patients (12).

In addition, our data formally demonstrate that a human HLA class I-restricted TCR can support a functional CTL response in the absence of the human CD8 coreceptor, even though mouse CD8 may interact suboptimally with HLA B27 (33). However, despite the absence of human CD8 expression, HLA B27-restricted T cell responses can be elicited in HLA B27 TG mice (Fig. 2) (14, 34). Interestingly whereas the original GRb CTL clone could be inhibited by Abs to CD8 (P. Bowness, unpublished observations), this TCR has been shown to be capable of specific recognition and signaling when transfected into a reporter cell line in the absence of CD8 (18). HLA A2-restricted responses are relatively poor in mice that do not express human CD8 (35, 36). However, in the case of GRb mice, the receptor favors CD8+ T cell responses despite the absence of human CD8. Ab to mouse CD8 is able partially to inhibit the generation of CD8+ T cell responses in GRb mice, whereas Ab to mouse CD4 had little effect on the induction of CD4+ T cell responses (data not shown). Overall, there appears to be considerable variability in the ease with which human HLA class I-restricted responses can be induced in HLA TG mice in the absence of human CD8, and it is possible that HLA B27-restricted responses are less CD8 dependent than other HLA class I transgenics.

Our findings taken together with the recent demonstration of human HLA class I-restricted CD4+ T cell lines and clones (12) have significant implications for immune regulation in autoimmune disease. The expression of MHC class II molecules and hence their ability to stimulate CD4+ T cell responses are tightly regulated and mainly restricted to specialized APCs and, in the presence of inflammation, some epithelial cells. In contrast, HLA class I molecules are expressed on most nucleated cells. The tight regulation of MHC class II expression is a powerful mechanism for immunoregulatory control of CD4+ T cell activation (37, 38), which may be broken if HLA class I can present to CD4+ T cells. By introducing the NP383–391 target Ag into different tissues, including joints (39), the model described in this study will enable us to study such a pathogenic mechanism in the pathogenesis of autoimmune disease and to determine the importance of CD4+ T cell stimulation at ectopic sites.


   Acknowledgments
 
We thank C. Benoist and D. Mathis for provision of the TCR cassette vectors, and J. Taurog for provision of the L cells transfected with HLA B2705 and h{beta}2m.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Helen C. Bodmer, Edward Jenner Institute for Vaccine Research, Compton, Nr. Newbury, Berkshire, U.K. RG20 7NN. E-mail address: helen.bodmer{at}jenner.ac.uk Back

2 Abbreviations used in this paper: h{beta}2m, human {beta}2-microglobulin; NP, influenza A virus nucleoprotein; TG, transgenic; VV, vaccinia virus. Back

Received for publication July 9, 2003. Accepted for publication October 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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