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The Journal of Immunology, 2001, 167: 3250-3256.
Copyright © 2001 by The American Association of Immunologists

T Cell Selection and Differential Activation on Structurally Related HLA-DR4 Ligands1

John A. Gebe*, Erik J. Novak*,{dagger}, William W. Kwok*, Andy G. Farr*,{ddagger}, Gerald T. Nepom*,{ddagger} and Jane H. Buckner2,*,§

* Benaroya Research Institute, Virginia Mason Research Center, Seattle, WA 98101; and {dagger} R. H. Williams Laboratory and Molecular and Cellular Biology Program, and Departments of {ddagger} Immunology and § Rheumatology, University of Washington School of Medicine, Seattle, WA 98101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Plasticity of TCR interactions during CD4+ T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4+ T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307–319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307–319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The specificity of CD4+ T cells in the immune response results from TCR interactions with MHC class II-peptide complexes. Allelic variation in MHC class II molecules contributes to the specificity of this interaction in two ways: first, by selecting peptides that bind within the class II binding groove itself, thus determining which peptides are available for MHC-peptide recognition of the TCR, and second, by influencing MHC-peptide-TCR interaction, which involves TCR contact with amino acid residues from the MHC molecule itself (1, 2). Thus, the MHC class II molecules play a role in T cell activation via two interfaces: binding of specific peptides and direct contact with the TCR.

The plasticity of TCR recognition of MHC-peptide ligands accommodates some structural variation, as seen in the setting of one MHC molecule presenting related peptides (3, 4, 5), a single peptide presented in the context of different MHC molecules (6, 7), or even in the recognition of unrelated peptide-MHC ligands (8, 9). In the setting of altered peptide ligands, amino acid substitutions in a peptide Ag have been shown to alter the character of the T cell response, leading to changes in the proliferation, cytotoxicity, and cytokine profiles induced upon TCR-peptide-MHC recognition. In some cases altered peptide ligands act through an alteration in the avidity of the TCR-peptide-MHC interaction, which translates into a change in the signal transmitted via the TCR and the subsequent response to activation (5, 10, 11). These findings and the observation that the TCR interacts directly with residues of the MHC (1, 2) suggest that TCR recognition of a peptide in the context of an alternative MHC molecule other than the cognate restriction element of the T cells may lead to an alteration in the T cell activation and response profile.

We now address this issue through the analysis of two systems: 1) a system in which immature thymocytes have undergone positive selection on one MHC class II molecule and their subsequent activation on multiple class II molecules, and 2) a system in which we examine a human T cell clone where TCR recognition of a peptide occurs in the context of two class II molecules, DRB1*0401 and DRB1*0404. A setting in which a T cell response to an altered MHC ligand (same peptide, different MHC) that is intriguing is that of individuals who are heterozygous for these two closely related class II alleles. Both DRB1*0401 and DRB1*0404 are associated with an increased risk of development of rheumatoid arthritis (RA)3; however, heterozygous individuals with this genotype have an amplification of that risk and increased severity of disease (12, 13, 14). These two HLA-DR4 molecules have very similar peptide-binding motifs and differ from each other only in two amino acid polymorphisms in the {beta}1 domain, including a disease-associated sequence known as the shared epitope (SE) (15, 16).

In this study we provide evidence that T cells that respond to altered MHC ligands containing the SE can arise in a thymic reaggregation culture (RC) system. In this system thymocytes positively selected on DRB1*0401 can be activated by the structurally similar SE-containing DRB1*0404, but not the non-SE-containing DRB1*0402. We also demonstrate that cross-reactive T cells can be isolated from mature PBMC populations using MHC class II tetramers. We then show that the cytokine response of a hemagglutinin (HA) 307–319-specific T cell clone from a DRB1*0401 homozygous individual can be skewed in response to an altered MHC ligand (DRB1*0404) containing the same peptide.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reaggregation culture

PA317 cells were infected from virus-secreting PE501 cells transfected with a pLNCX vector (Clontech, Palo Alto, CA) containing a bicistronic DNA element encoding the DRA1*0101 and DRB1*04 cDNAs separated by an internal ribosome entry site (sequence from polio virus). All cDNAs encoding the DRA1*0101, DRB1*0401, *0402, and *0404 MHC molecules were sequenced in the pLNCX vector. PA317 cells expressing the MHC genes were cloned and used to infect actively dividing ANV41.2 thymic epithelial cells and also mouse A20 cells (ATCC TIB208; American Type Culture Collection, Manassas, VA). Retrovirally infected cells were selected with 1 mg/ml G-418 (Life Technologies, Gaithersburg, MD), and surface expression was checked using FITC-labeled anti-DR L243 Ab. RCs were performed using a modified technique developed by Jenkinson and Owens (17, 18). I-A{beta}o/o {beta}2mo/o mice were obtained from Taconic Farms (Germantown, NY). Thymi from 1- to 3-wk-old mice were dissociated into single-cell suspension with Blendzyme 3 (Life Technologies) following the manufacturer’s instructions. CD4+CD8+TCRlow thymocytes were mixed with MHC expressing ANV41.2 cells at a ratio of 25:1 (25 x 106 thymocytes/RC). The cell mixture was spun down in microfuge tubes at 300 x g for 6 min, followed by medium aspiration. The resulting cell slurry was pipetted onto a 0.65-µm pore size filter (DVPP 01300; Millipore, Bedford, MA) lying atop gelfoam sponges (Pharmacia-Upjohn, Kalamazoo, MI) in six-well tissue culture plates containing 6 ml of RPMI 10 medium (Life Technologies; RPMI 1640, no HEPES), 10% serum (50% FBS/BCS), penicillin (100 U/ml), streptomycin (100 µg/ml), 1x nonessential amino acids (Life Technologies), and 50 µM 2-ME. Plates were incubated for 7 days in a 5% CO2 incubator at 37°C. On days 2 and 4 of the culture, 200 µl of murine IL-7 (Endogen, Woburn, MA) at 10 ng/ml was added. After 7 days RC were gently dissociated using frosted slides, which left most of the stromal tissue intact and facilitated separating stromal cells from the thymocytes. One-fourth of the thymocyte suspension from each reaggregate was incubated with 105 HLA-transfected mouse A20 cells in round-bottom wells of a 96-well plate (150 µl of complete RPMI 10, total volume). Following overnight culture (18 h) cells were stained with anti-CD4, -CD8, and -CD69 Abs and run on a FACSort or FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Live cells were gated on using forward and side scatter. We found that >99% of this population did not stain with the viability indicator 7-amino actinomycin D (Sigma, St. Louis, MO).

CD4-FITC (clone RM4-5), CD8a-CyChrome (clone 53-6.7), CD69-PE (clone H1.2F3), DR-FITC (clone G46-6, equivalent to L243), and TCR-CyChrome (clone H57-597) were obtained from BD PharMingen (San Diego, CA).

Tetramer identification of Ag-specific T cells

Soluble MHC molecules were loaded with peptides and tetramerized as described previously (19). Briefly, cDNAs encoding the extracellular portions of MHC {alpha}- and {beta}-chains were ligated to opposing leucine zipper regions. At the end of the {beta}-chain cDNA is a biotinylation sequence that allows site-specific biotinylation using the Bir A enzyme (20). Soluble {alpha}{beta} MHC molecules were produced in insect cells and affinity purified using an anti-MHC matrix. Following biotinylation, {alpha}{beta} MHC molecules were incubated with peptide for 3 days at 37°C. Multimeric units of MHC molecules were prepared by 24-h room temperature incubation of the peptide-loaded MHC complexes with PE-labeled streptavidin (BioSource International, Camarillo, CA).

PBMC from a DRA1*0101/B1*0401 homozygous individual previously vaccinated for influenza virus were separated from heparinized venous blood by gradient centrifugation (Lymphoprep; Nycomed Pharma, Oslo, Norway.). Cells were cultured in RPMI 1640 (Life Technologies) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin (100 U/ml), streptomycin (100 µg/ml), and 15% (v/v) pooled human serum. Adherent cells were prepared by plating PBMC at 5 x 106 cells/well in 24-well plates for 1 h. Nonadherent cells were removed using a transfer pipette. Adherent cells were incubated for 3 h with 10 µg/ml influenza HA307–319. The nonadherent fraction was passed through a nylon wool column, added back to the adherent cells at a density of 2.5 x 106 cells/well, and cultured for 7 days.

DRA1*0101/B1*0401 and DRA1*0101/B1*0404 PE-labeled tetramers were constructed and loaded with HA307–319 as previously described (19). On day 7 of culture, 5 x 105 cells were incubated with 0.5 µg PE-labeled tetramer in 50 µl of culture medium at 37°C for 1–2 h and then stained with anti-CD4-FITC (BD PharMingen) for 15 min on ice. Cells were washed and analyzed using a BD Bioscience FACSVantage flow cytometer. Cells that were positive for a particular tetramer were single-cell sorted into 96-well U-bottom plates. Sorted cells were expanded with 1.5 x 105 unmatched, irradiated (5000 rad) PBMC/well as feeders, with 2.5 µg/ml PHA and 10 U/ml IL-2 added 24 h later.

The specificity and DRB1*0401/DRB1*0404 cross-reactivity of T cell clones were examined by staining with DRA1*0101/B1*0401 and DRA1*0101/B1*0404 tetramers loaded with either the specific peptide, HA307–319 (herein called DR0401-HA and DR0404-HA, respectively) or an irrelevant peptide, VP16465–484 (VP16 is a tegument protein of HSV-2 virus).

T cell assays

PBMC (105) from DRB1*0401- or DRB1*0404-positive donors were incubated for 2 h with peptide, irradiated with 5000 rad, and then coincubated with 2 x 104 T cell clones in 96-well round-bottom plates. [3H]thymidine was added to the plates for the last 16 h, and plates were harvested at 72 h. For assays using plate-bound HLA class II monomers, 96-well flat-bottom, high binding plates (Costar, Cambridge, MA) were coated with class II monomers preloaded with the peptide of interest, incubated at 4°C overnight, washed with PBS, and blocked with medium containing 15% serum for 1 h. T cell clones (2 x 104) were then added to each well in 200 µl of medium containing 15% pooled human serum. Proliferation was measured at 48 or 72 h by addition of [3H]thymidine 16 h before cell harvesting. Media removed from wells at 24 or 48 h were used for examination of cytokine production.

Human IL-5 and IFN-{gamma} cytokine production were detected by use of a standard sandwich ELISA method. Fifty microliters of supernatant was added to wells precoated with a capture Ab (Endogen), and cytokine was detected using a biotinylated anti-cytokine (IL-5 or IFN-{gamma}) mAb (Endogen), followed by avidin-peroxidase and o-phenylenediamine dihydrochloride. OD490 was read using a microplate reader (Bio-Tek Instruments, Winooski, VT). A standard calibration curve, in the range of 0–2 ng/ml, was also performed for each assay using rIL-5 or rIFN-{gamma} (Endogen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
T cells positively selected on DRB1*0401 can be stimulated by DRB1*0404 APC

RCs using the class II null thymic epithelial cell line (ANV41.2) transfected with an MHC restriction element have been shown to allow TCR transgenic thymocytes to differentiate into a mature phenotype capable of Ag-specific proliferation (21, 22). To examine how positive selection influences T cell cross-recognition of DR4 MHC molecules, we have extended the use of the ANV41.2 cell line to examine maturation of nontransgenic thymocytes into phenotypically mature T cells (Fig. 1Go). Thymocytes from I-A{beta}o/o {beta}2mo/o mice lack murine class I and II molecules and have arrested thymocyte maturation at the CD4+/CD8+/TCRlow stage. These thymocytes were coincubated with the ANV41.2 cell line expressing one of the RA-associated human MHC class II molecules, DRA1*0101/B1*0401 or DRA1*0101/B1*0404. As shown in Fig. 2Go, A and B, immature thymocytes from I-A{beta}o/o {beta}2mo/o mice mature from a CD4+/CD8+/CD69low to CD4+/CD8+/CD69high to a CD4+/CD8-/CD69low phenotype over a 7-day period, with day 7 thymocytes having a mature phenotype. This positive selection of CD4+/CD8- T cells on day 7 is seen in 16.5 and 14.9% of the 7-day RC T cell population using DRB1*0401- and DRB1*0404-transfected ANV41.2 cells, respectively, but in only 2.6% of the vector-only-transfected ANV41.2 control cultures (Fig. 2GoC). An increase in TCR expression was also observed between day 0 thymocytes and day 7 CD4+/CD8- cells (data not shown).



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  		FIGURE 1. Schematic representation of the protocol used in thymocyte RC experiments. Dissociated thymus from I-A{beta}o/o{beta}2mo/o mice are deficient in MHC class I and class II molecules. These cells are coincubated with ANV41.2 cells containing transfected DRA1*0101/B1*0401, DRA1*0101/B1*0404, or vector-only plasmids. After 7 days cells are taken from the RC and assayed for positive selection and activation specificity.

 


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  		FIGURE 2. CD4, CD8, and CD69 expression on thymocytes from RCs. A, CD4 and CD8 expression on days 0, 4, and 7 of thymocyte RC with ANV41.2-DRB1*0401. B, CD69 expression on thymocytes shown in A. C, CD4 and CD8 expression on day 7 of RC with ANV41.2 cells transfected with control-pLNCX, DRB1*0401, or DRB1*0404. CD4+SP denote cells with a CD4+/CD8- phenotype, and DP denote cells with a CD4+/CD8+ phenotype. The percentages of cells that are CD4+/CD8- are shown below the dot-plots.

 
To analyze the potential for cross-recognition of the TCR for other DR4 class II molecules with the SE during thymic selection, we examined the ability of CD4 single-positive cells generated in this system to be activated by other DR4 molecules. Thymocytes from the RCs were stimulated on day 7 with mouse A20 B cells expressing DRB1*0401, *0402, or *0404. The T cells were coincubated for 18 h with HLA expressing A20 cells and analyzed for CD69 expression on gated CD4+/CD8- cells. Fig. 3GoA shows that these CD4+/CD8- cells could be activated (as measured by CD69 up-regulation) by APC expressing the selecting HLA class II molecule, consistent with the positive selection and maturation of the thymocyte cells in the RC based on the transfected human MHC introduced in the ANV41.2 thymic epithelial cell. When examined for cross-recognition of HLA class II molecules, cells selected on either DRB1*0404 or DRB1*0401 were stimulated by APC expressing other class II molecules containing the SE (Fig. 3GoA). These cells were not activated (as measured by CD69 up-regulation) above background levels by the closely related HLA class II DRB1*0402, which does not contain the SE. In RCs containing plasmid-only (pLNCX)-transfected ANV41.2 cells there was no difference between CD69 levels after overnight culture with APCs expressing DRB1*0401, DRB1*0402, or DRB1*0404 (Fig. 3GoA). These findings suggest that the residues of the SE are involved in positive selection and that the potential for cross-recognition of other SE-containing DR4 molecules is present after positive thymic selection. Fig. 3GoB shows the percent increase in the number of CD69+ cells after 18-h A20 APC culture with thymocytes from DRB1*0401 and DRB1*0404 RC relative to that after 18-h A20 APC culture with thymocytes from pLNCX control RC. The statistical analysis in Fig. 3GoB is on CD4+/CD8- gated cells from two independent RC experiments.



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  		FIGURE 3. CD69 expression on CD4+/CD8- (RC) thymocytes cultured with HLA-transfected mouse A20 cells. A, Thymocytes from 7-day RC were cultured for 18 h with A20 cells transfected with DRA1*0101/B1*0402 ({blacksquare}), DRA1*0101/B1*0401 (), or DRA1*0101/B1*0404 () HLA genes. The percentage of CD69+ cells after 18-h culture for one RC experiment is shown. B, The percent increase in the number of CD69+ cells (on CD4+/CD8--gated thymocytes) for a particular A20-HLA 18-h culture was expressed as the [(number of CD69+ cells from ANV41.2-w RC) - (number of CD69+ cells from ANV41.2-pLNCX RC)]/(number of CD69+ cells from ANV41.2-w RC), where w is the HLA molecule in the RC (x-axis on graph). Data are shown for two experiments. In experiments using vector-only-transfected A20 cells instead of DRA1*0101/B1*0402-transfected A20 cells as control cells, the results were equivalent (data not shown).

 
DRA1*0101/B1*0401-HA 307–319 restricted T cells can be detected by DR0404-HA tetramers

In vivo, thymic selection establishes the peripheral T cell repertoire, and the in vitro evidence presented above suggested that circulating T cells from HLA DRB1*0401 individuals may demonstrate a similar cross-recognition of HLA DRB1*0404. To study this we examined the T cell response to the influenza peptide HA307–319 in a DRA1*0101/B1*0401 homozygous individual. HA307–319 is bound by both DRB1*0401 and DRB1*0404 (6, 23, 24) and is therefore a suitable Ag with which to evaluate recognition in the context of DRB1*0401 and DRB1*0404. PE-labeled HLA tetramers were constructed for both DRB1*0401 and DRB1*0404 molecules. PBMC from a homozygous DRA1*0101/DRB1*0401 individual previously vaccinated for influenza virus were stained with CFSE before stimulation with HA307–319 peptide to allow identification of cells proliferating in response to specific Ag stimulation. On day 7 of culture, cells were harvested and stained with PE-labeled DR0401-HA or DR0404-HA tetramers. As shown in Fig. 4GoA, 3.7% of the cells presented as tetramer positive and CFSElow upon staining with the cognate DR0401-HA tetramer, consistent with Ag-specific expansion of the HA307–319-responsive cells during the culture period. Interestingly, staining with the DR0404-HA tetramer also identified a small, but detectable, fraction of proliferating cells (~0.1%), as shown in Fig. 4GoB. This suggests that a portion of the HA307–319-specific T cells from this DRB1*0401 individual were capable of allelic promiscuity.



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  		FIGURE 4. MHC tetramer staining on human T cells stimulated for 7 days with HA307–319 peptide. Donor T cells were obtained from a DRA1*0101/B1*0401 homozygous individual previously vaccinated against influenza virus. Before stimulation cells were stained with CSFE. On day 7 cells were harvested and stained with PE-labeled DR0401-HA (A) or DR0404-HA (B) tetramers loaded with HA307–319 peptide. Percentages above the dot-plots are the percentage of cells in the gate that are CFSE-divided cells and are tetramer positive. Previous experiments performed using this donor’s PBMC demonstrated no background staining with 0401 or 0404 tetramers containing an irrelevant peptide.

 
To test the Ag specificity of the T cells identified by DR0401-HA and DR0404-HA staining, tetramer-positive cells were single-cell sorted, and 15 clones sorted from the DR0401-HA tetramer and 25 clones sorted from the DR0404-HA tetramer were expanded for further characterization. All clones isolated responded to Ag-specific stimulation on DRB1*0401 and showed positive staining with the DR0401-HA tetramer. Staining of clones with the DR0401-HA tetramer was generally quite strong, as shown for the representative clone B16 in Fig. 5GoA. Eleven of the 15 clones initially sorted using DR0401-HA tetramer showed no DR0404-HA tetramer binding, as shown for clone B16 in Fig. 5GoA. The remaining four clones showed variable levels of staining with DR0404-HA, ranging from 3.4 to 15.5%. In contrast, those clones initially sorted using DR0404-HA tetramer all showed heterogeneous staining when restained with DR0404-HA, as shown in Fig. 5GoB for clone B2. The heterogeneity in staining seen in these clones probably represents the upper end of a spectrum of TCR expression and cell activation. The percentage of cells classified as DR0404-HA tetramer positive ranged from 1.4 to 31.2% for the 25 (DR0404-HA sorted) clones analyzed (data not shown). The selective binding of DRB1*0404 tetramer to these clones indicates that some T cells that are selected in vivo on DRB1*0401 are able to cross-react on DRB1*0404.



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  		FIGURE 5. Binding of T cell clones B16 (A) and B2 (B) with DR0401-HA and DR0404-HA MHC tetramers. CD4 staining is shown on the x-axis, and tetramer staining is shown on the y-axis.

 
We further analyzed cytokine and proliferation profiles in response to specific Ag for six of the DR0404-HA-sorted clones and identified one clone, B2, that had an altered response when stimulated on DRB1*0404 compared with stimulation on the cognate restriction DRB1*0401.

DRB1*0404 can stimulate Ag-specific DRB1*0401-restricted T cells and alter the cytokine response

The two clones shown in Fig. 5Go were further examined for their response to activation with HA307–319 in the context of DRB1*0401 and DRB1*0404. T cell clones B2 (cloned using DR0404-HA tetramers) and B16 (cloned using DR0401-HA tetramers) were isolated from the same DRB1*0401 homozygous individual, and both clones bound DR0401-HA tetramer and demonstrated a proliferative response to activation by peptide in the context of DRB1*0401 PBMC (Fig. 6Go, A and B). Clone B16 lacked staining with the DR0404-HA tetramer (Fig. 5GoA) and demonstrated a peptide-specific, DRB1*0401-restricted response with significant production of IFN-{gamma} and IL-5 upon activation (Fig. 6Go, B, D, and F). Relatively minimal proliferation was elicited by DRB1*0404-restricted Ag presentation, and only at the highest peptide concentration was some IFN-{gamma} production noted. In contrast, the B2 clone was activated by HA in the context of either DRB1*0401 or DRB1*0404, with the proliferative response and production of IFN-{gamma} similar with both class II molecules (Fig. 6Go, A and C). Moreover, the B2 clone produced IL-5 when activated in the context of DRB1*0404, but not with DRB1*0401 (Fig. 6GoE).



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  		FIGURE 6. HA peptide dose response of CD4 T cell clones B2 and B16. B2 (A, C, and E,) and B16 (B, D, and F) were stimulated with DRB1*0401 (•) or DRB1*0404 ({circ}) APC. Cells were assayed for proliferation, (A and B), IFN-{gamma} production (C and D), and IL-5 production (E and F). Proliferation was measured at 72 h, and cytokines were assayed at 48 h.

 
Alterations in cytokine production in this setting could be secondary to differences in the costimulatory signals produced by the APC. To remove this variable, we used plate-bound recombinant MHC monomers containing HA peptide to stimulate clone B2. Fig. 7Go demonstrates that plate-bound monomer, alone or with the addition of anti-CD28, induced proliferation of the B2 T cell in a concentration-dependent manner. Similarly, cytokine profiles examined after stimulation with DR0401-HA or DR0404-HA monomer were consistent with the profiles seen when PBMC APC were used (Fig. 7Go, B and C). Specifically, IL-5 was only produced when activation of the clone was triggered by DR0404-HA. The addition of soluble anti-CD28 in this system decreased the concentration of monomer required for stimulation, but did not alter the pattern of cytokine production (Fig. 7Go). No activation was seen when empty class II monomer was used or an irrelevant peptide was bound in the class II monomer (data not shown).



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  		FIGURE 7. Response of CD4 T cell clone B2 to plate-bound HLA monomers. Clone B2 was stimulated with plate-bound DR0401-HA (•) or DR0404-HA ({circ}) MHC monomers alone (A, C, and E) or with soluble anti-CD28 (B, D, and F). Cells was assayed for proliferation (A and B), IFN-{gamma} production (C and D), and IL-5 production (E and F). Proliferation was measured at 48 h, and cytokines were assayed at 24 h.

 
Persistence of the 0404-HA signal

In a heterozygous individual the ability of one MHC class II molecule to modify the response of a T cell could potentially be dependent on the relative quantity of class II peptide complex available on the APC. In turn, this would be determined by the level of class II expression; the ability of the Ag to be processed, bound as peptide, and presented by the class II molecule at the cell surface; and the relative predominance of one signal over the other. To address this issue when both altered MHC and cognate MHC-peptide complexes are present, we used the MHC monomeric stimulation assay system. In this system class II monomers were preloaded with HA peptide, diluted, and then added to a 96-well plate at varying concentrations and combinations. In this assay the presence of DR0404-HA, even at low concentration, leads to the production of IL-5 by clone B2 (Fig. 8Go), consistent with a specific bias toward IL-5 secretion elicited by altered MHC recognition. Changes in the DR0401-HA concentration had no consistent effect on IL-5 levels. Proliferation and IFN-{gamma} production were present at low monomer concentrations, but decreased with higher concentrations in this assay. This may be due to accelerated proliferation and cytokine secretion followed by activation-induced cell death at these high concentrations of MHC-monomer. We have observed this behavior in other MHC-monomer-stimulated systems (our unpublished observations). This finding suggests that the T cell response to the altered MHC ligand (DRB1*0404-HA) persists when more than one type of MHC molecule is present. The presence of both genotypes in a heterozygous individual could lead to a fundamental alteration in the T cell response to an Ag compared with that of an individual who expresses only DRB1*0401.



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  		FIGURE 8. The relative influences of DRB1*0401-HA and DRB1*0404-HA complexes on the cytokine response of clone B2. Ninety-six-well plates were coated with either monomer alone or in combination. A, Proliferation at 48 h; B, production of IFN-{gamma} at 24 h; C, production of IL-5 at 24 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
T cell recognition of a peptide in the context of multiple MHC molecules has been termed promiscuous recognition and includes examples of peptide-specific recognition with restriction by both DR molecules of a heterozygous individual (25, 26). In this paper we investigate the potential for generation and activation of such cells in individuals carrying the RA-associated alleles DRB1*0401 and DRB1*0404. We demonstrate using thymocyte RCs that at the level of positive selection, thymocytes selected in the context of SE-containing DRB1*0401 can respond to activation signals from SE-containing DRB1*0404, but not from a non-SE-containing DRB1*0402 APC. Thymocytes selected on DRB1*0404 also demonstrate a restriction to activating signals, where DRB1*0401(SE HLA) APC is capable of up-regulating CD69, but not DRB1*0402 APC (non-SE HLA). The ability of thymocytes selected on SE-containing molecules to receive and respond to activation signals only from SE-containing MHC suggests that at the level of positive selection TCR-MHC interactions do play a significant major role in thymic selection in addition to TCR-peptide interactions. The number of CD4+/CD8- cells in our RC capable of being recognized by DRB1*0401 and DRB1*0404 is ~5–10%. However, because the ANV41.2 cell line does not express B7 molecules, and thus lacks the ability to negatively select, our data represent the results of positive selection only. As such, our estimate of the number of cross-reactive T cells in the RC probably represents the high end of what one might find in the peripheral T cell pool. However, such cross-reactive T cells have been identified in the periphery by ourselves and others. In this paper we use HLA tetramers to identify such cross-reactive T cells and have used this technology to identify a T cell clone that is differentially activated by its cognate and altered self ligands.

The human T cell clone B2 responds to HA307–319 in the context of DRB1*0401 with a Th1 phenotype characterized by proliferation and production of IFN-{gamma}. However, when activated by HA307–319 in the context of the closely related DRB1*0404, IL-5 is produced as well. This pattern of cytokine production is consistent over a large range of peptide concentrations and time points (data not shown) and occurs with APC alone or class II monomers alone, with or without CD28 costimulation.

The ability of altered MHC class II monomers to activate the T cell clone and modify the cytokine profile is reminiscent of the interactions of the TCR-peptide-MHC complex that determines the outcome of cytokine production, as has been described with altered peptide ligands. Altered peptide ligands have been shown to function by modifying the TCR signaling pathway (5, 27, 28, 29), in part by changing the avidity of the TCR-peptide-MHC interaction due to alterations in binding affinity of the TCR-peptide-MHC contacts (30, 31, 32, 33, 34). Our finding of reduced tetramer staining with the DR0404-HA tetramer supports the concept that a common underlying mechanism for altered T cell function is altered TCR avidity to the peptide-MHC complex (35). DRB1*0401 and DRB1*0404 differ in only two amino acids: an arginine to lysine at position 71, and a glycine to valine at position 86. Position 86 lies at the end of the {beta}-chain {alpha} helix and forms part of the position P1 peptide binding pocket. This alteration leads to lower affinity of the HA peptide to DRB1*0404 relative to DRB1*0401 (24) and most likely alters the stability of the trimolecular complex. Position 71 is located approximately in the middle of the {alpha} helix on the {beta}-chain and interacts with both the bound peptide and the CDR3 regions of the TCR (36). It has been suggested that the lysine to arginine difference in {beta}71 could directly alter the TCR affinity for the complex by the addition of a key hydrogen bond interaction between TCR and MHC (37). The persistence of IL-5 production in clone B2 over a range of peptide concentrations suggests that the altered cytokine response is not solely due to the reduced affinity of the peptide to the MHC. We suggest that the altered cytokine response is due to the reduced avidity of the TCR to the peptide-MHC resulting from affinity changes between TCR and MHC contacts.

There are several potential immunologic consequences of altered self recognition by T cells in heterozygous individuals. Activation of the B2 clone with the combination of altered MHC and cognate MHC leads to persistence of the lower TCR-peptide-MHC affinity response. This is conceptually analogous to the phenomenon of T cell antagonism elicited by altered peptide ligands. In vivo, one could envision a T cell selected on one MHC molecule that, due to alterations in the APC, Ag concentration, or cytokine milieu, could become more favorable for activation in the context of the alternative class II molecule (altered MHC), leading to a deviant immune response. Although our studies were performed with HLA-DR4 alleles, presumably this may also occur with other structurally related class II molecules. However, this paradigm may be particularly apropos in individuals who are heterozygous for DRB1*0401/DRB1*0404, in that the susceptibility to RA is synergistically increased for those individuals carrying both DRB1*0401 and DRB1*0404. Moreover, RA patients carrying both these alleles exhibit a more severe and erosive form of RA, with a substantially poorer long-term prognosis (12, 13, 14, 38). Our findings raise the possibility that in these individuals the pathogenesis and progression of disease may be influenced by the altered MHC-peptide recognition and provide a basis for further studies to evaluate whether such a mechanism plays a role in these patients with RA.


   Acknowledgments
 
We thank Sharon Kochik, Megan Van Landeghen, Nicole Pratt, and Laura Tsaknaridis for their excellent technical assistance; Janice Abbas for help with preparation of the manuscript; and Helena Reijonen for critical review of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AR37296 (to G.T.N.) and AI24137 (to A.G.F.). J.H.B. was supported by grants from the Arthritis Foundation, Paul G. Allen Foundation Clinical Scholars Program. J.A.G. was supported in part by National Institutes of Health Postdoctoral Training Grant T32AR07108-24. E.J.N. was supported by Achievement Rewards for College Scientists and Poncin predoctoral fellowships. This work was also supported by the M. J. Murdock Charitable Trust, the William Randolph Hearst Foundation, and the Elmer and Mary Louise Rasmuson Center for Rheumatic Disease. Back

2 Address correspondence and reprint requests to Dr. Jane Hoyt Buckner, Virginia Mason Research Center, 1201 Ninth Avenue, Seattle, WA 98101. E-mail address: jbuckner{at}vmresearch.org Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; SE, shared epitope; HA, hemagglutinin; RC, reaggregation culture. Back

Received for publication March 19, 2001. Accepted for publication July 17, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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