Key interactions in the trimolecular complex consisting of the rheumatoid arthritis-associated DRB1*04:01 molecule, the major glycosylated collagen II peptide and the T-cell receptor

Objectives Rheumatoid arthritis (RA) is an autoimmune disease strongly associated with the major histocompatibility complex (MHC) class II allele DRB1*04:01, which encodes a protein that binds self-peptides for presentation to T cells. This study characterises the autoantigen-presenting function of DRB1*04:01 (HLA-DRA*01:01/HLA-DRB1*04:01) at a molecular level for prototypic T-cell determinants, focusing on a post-translationally modified collagen type II (Col2)-derived peptide. Methods The crystal structures of DRB1*04:01 molecules in complex with the peptides HSP70289-306, citrullinated CILP982-996 and galactosylated Col2259-273 were determined on cocrystallisation. T cells specific for Col2259-273 were investigated in peripheral blood mononuclear cells from patients with DRB1*04:01-positive RA by cytofluorometric detection of the activation marker CD154 on peptide stimulation and binding of fluorescent DRB1*0401/Col2259-273 tetramer complexes. The cDNAs encoding the T-cell receptor (TCR) α-chains and β-chains were cloned from single-cell sorted tetramer-positive T cells and transferred via a lentiviral vector into TCR-deficient Jurkat 76 cells. Results The crystal structures identified peptide binding to DRB1*04:01 and potential side chain exposure to T cells. The main TCR recognition sites in Col2259-273 were lysine residues that can be galactosylated. RA T-cell responses to DRB1*04:01-presented Col2259-273 were dependent on peptide galactosylation at lysine 264. Dynamic molecular modelling of a functionally characterised Col2259-273-specific TCR complexed with DRB1*04:01/Col2259-273 provided evidence for differential allosteric T-cell recognition of glycosylated lysine 264. Conclusions The MHC-peptide-TCR interactions elucidated in our study provide new molecular insights into recognition of a post-translationally modified RA T-cell determinant with a known dominant role in arthritogenic and tolerogenic responses in murine Col2-induced arthritis.


INTRODUCTION
Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease targeting diarthrodial cartilaginous joints. The disease is believed to be initiated by the development of autoantibodies to various altered self-antigens, predominantly modified by citrullination for yet unknown reasons, [1][2][3][4] followed years later by onset of joint inflammation and spreading of autoimmune responses to new structures, including cartilage proteins. [5][6][7] The strong association of RA with major histocompatibility complex class II (MHCII) suggests activation of T cells and a maturation of an autoreactive B cell response, capable of orchestrating the immune attack on the joints. 8 The origin of the T-cell activation is unknown but is likely to be dependent on presentation of antigenic peptides bound to the MHCII molecules. These peptides

What does this study add?
► This study contributes new crystal structure information that reveals the DRB1*04:01 function in presenting post-translationally modified antigenic determinants to T cells in patients with RA. ► Through comparative analysis of RA T cells, T cell receptor (TCR) cloning and molecular modelling of a prototypic trimolecular complex, we gained insights into TCR recognition of unmodified and glycosylated Col2 in a DRB1*04:01 context.
How might this impact on clinical practice or future developments? ► Our findings contribute to a better understanding of the role of posttranslational Col2 modification for TCR recognition in CD4 + T cells and has potential implications for induction of tolerance and onset of pathogenic autoimmunity.
could have been derived from non-self-proteins, for example, from infectious agents giving help to autoreactive B cells, or could be modified self-peptides that have escaped tolerance selection.
The MHCII association with RA has been mapped to the DRB1 locus, 9 10 which encodes a DR β-chain (DRB) and forms a peptide-binding receptor together with an invariant α-chain (DRA). 11 According to a popular hypothesis, alleles of the highly polymorphic DRB1 locus associated with RA encode a shared peptide binding pocket for a selected set of self-peptides, thereby predisposing individuals to pathogenic T-cell activation. 12 13 Most of the RA-associated DRB1 molecules have positively charged amino acids at position 71, favouring interactions with peptides that contain a negatively charged amino acid at the P4 position. Although a favoured binding of peptides with citrulline at this position (in contrast to a positively charged arginine) has been proposed, 13 14 this hypothesis could not be confirmed in studies on larger sets of peptides. 15 16 A potentially relevant self-antigen in arthritis pathogenesis is type II collagen (Col2) due to its abundance in cartilage and proven role as an arthritogenic immunogen in experimental arthritis. 17 Moreover, antibodies to native and citrullinated Col2 [18][19][20][21][22] and Col2-specific T cells 18 are detectable in patients with RA. Interestingly, RA T-cell responses directed to the dominant Col2 259-273 peptide were restricted by the RA-associated DR alleles 0401 and 0101, which were demonstrated to confer susceptibility to collagen-induced arthritis on transgenic expression in mice. [23][24][25] However, it is crucial to consider that the major Col2 259-273 peptide can be both hydroxylated and galactosylated at lysine residues and that RA T cells predominantly recognise the galactosylated form. 18 Initial insight into the positioning of the galactosylated (gal) Col2 259-273 peptide (galCol2 259-273 ) in the binding pocket of the DRB1*04:01 molecule was provided by molecular modelling. 26 27 The same target peptide and its post-translational modification (PTM) is also recognised by arthritogenic T cells in mice, provided that they express the natural murine antigen MHCII allele or transgenic human DRB1*04:01 or DRB1*01:01 molecules. 18 23-25 Critical differences in central tolerance induction of T cells based on their specificity for unmodified (nCol2 259-273 ) or galactosylated Col2 259-273 (galCol2 259-273 ) 28 and in the tolerogenic potential of Col2 peptide vaccination against experimental arthritis have been observed in mice. 29 To further elucidate the role of PTM in Col2 recognition by human T-cell receptors (TCR), we performed a comparative study of Col2 259-273 recognition in either galactosylated or unmodified form using binding of tetramerised recombinant DRB1*04:01/Col2-peptide complexes and analysed Col2 peptide-induced T-cell activation in peripheral blood mononuclear cells (PBMCs) from patients with DRB1*04:01-positive RA. Moreover, we solved the X-ray crystallographic structure of DRB1*04:01(HLA-DRA1*01:01/HLA-DRB1*04:01) complexed with galCol2 259-273 to gain the first molecular insights into the structural basis of PTM-dependent differential Col2 recognition. Together with sequence information obtained from a cloned human galCol2-specific TCR, these crystal structures allowed us to perform molecular modelling on the trimolecular MHCII/peptide/TCR complex.

MATERIALS AND METHODS
See online supplemental material.

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The crystal structure of Col2 259-273 -bound DRB1*04:01 was determined at 1.9 Å resolution (online supplemental table S1). A weakly bound mutated human CLIP peptide (PVSKARMAT-GALAQA) occupying the DRB1*04:01 binding groove was exchanged with a synthetic glycosylated Col2 259-273 (mono-[β-D-galactopyranosyl]-moiety at a lysine [K264] side chain) prior to protein crystallisation. However, we did not observe electron density for the galactose moiety electron density in the structure, most likely because it is mobile at its solvent-exposed position on the protein surface. As expected, the peptide occupied the P1, P4, P6, P7 and P9 pockets with P1-Phe, P4-Glu, P6-Gly, P7-Pro and P9-Gly, respectively, whereas the potential TCR contact residues are P2-Lys, P5-Gln and P8-Lys. We also determined DRB1*04:01 in complex with covalently attached mutated Col2 259-273 (online supplemental table S1, figures 1 and 2, online supplemental figure S1 and figure S2). To investigate a possible influence on peptide binding by the two potential TCR contact residues P2-Lys and P8-Lys, we mutated them to Ala. We have previously found that the mutation of P2 from Lys to Ala slightly decreased the affinity of the peptide with DRB1*04:01, but also abolished T cell reactivity. 25 As can be seen from the structure, the mutated peptide mimics the conformation and location of the wildtype peptide (online supplemental figure S2), but its P4-Glu did not engage with Lys71β, thus explaining the reduced affinity.
As expected, the citCILP 982-996 peptide also binds in a linear, extended manner with P1-L, P4-I, P6-D and P9-S interacting with the DR molecules. Thus, the citrulline does not bind to the P4 pocket but instead is likely to face the TCR.
An overall comparison of the binding sites confirmed that the strongest DR binding site had a hydrophobic amino acid in the P1 position, whereas a considerable degree of flexibility was allowed at other positions. An important DR binding site is P4, in which an acidic side chain (glutamic acid) is favoured as it interacts with basic amino acids at position 71 in the beta chain, in line with the known association with RA. In contrast, the citrulline side chains of the CILP peptide did not bind within the P4 pocket. The TCR recognition sites for the Col2 peptide were the lysines at P2 and P10 as well as the glutamine at P5.

Detection of T lymphocytes specific for Col2 259-273
DRB1*04:01 allele carriers (patients with RA and healthy donors) were investigated for the presence of Col2 259-273 peptide (nCol2 or gal 264 Col2)-specific CD4 + T cells in peripheral blood by flow cytometric analysis of tetramer-stained PBMCs. A representative result in figure 3A shows the detection of Col2 epitopespecific cells that stain double-positive for two identical but differently fluorescence-labelled DRB1*04:01/Col2 tetramer complexes. The frequency of double-positive cells was 0.030%

Figure 3
Detection of human antigen-specific T cells in the peripheral blood of HLA-DRB1*04:01 carriers by flow cytometry using # DR4 (HLADRB1*04:01)-tetramers with specificity for the gal 264 Col2 259-273 or nCol2 259-273 peptide. (A) Representative dot blots: the CD4 + enriched T cells from PBMCs were stained with a dead/live marker and DRB1*04:01/Col2 peptide tetramers conjugated with two different fluorophores (PE and APC). Subsequent flowcytometric analysis reveals the Col2-specific cells in the live double positive stained subpopulation. Representative dot blots of specific DR4/gal 264 Col2 and DR4/nCol2 tetramer binding to T helper cells in samples from a patient with RA and a HD are shown. Biotin-streptavidin complexes without a specific peptide conjugate served as a negative control. (B) Specific tetramer binding of T helper cells from patients with RA (n=55) and HD (n=20) using # DR4 (DRB1*04:01)-tetramers with different peptide specificity (gal 264 Col2 vs nCol2). In PBMCs of patients with RA, the frequencies of DR4/nCol2 and DR4/gal 264 Col2 staining CD4+T cells do not differ significantly (n.s.). Depicted values represent processed data in which for each tetramer staining datapoint the respective biotin background has already been subtracted from the raw value. (C) Detection of antigenspecific T cells in PBMCs of patients with RA (HLA-DRB1*04:01) on in vitro stimulation by synthetic Col2 peptides using flow cytometry. PBMCs from patients with RA were stimulated with a Col2 peptide (gal 264 Col2, nCol2) and anti-CD40 for 7 hours. Subsequently, peptide-induced upregulation of the activation marker CD154 on the surface of the live CD4+T cell population was detected by flow cytometry. The frequency of CD154-positive CD4positive T helper cells in PBMCs was significantly elevated in response to vitro challenge by the galCol2 peptide (n=41) compared with unmodified nCol2 (n=35, p=0.0262). Statistical significance was determined using the Mann-Whitney test. APC, allophycocyanin; HD, healthy donors; PBMC, peripheral blood mononuclear cells; PE, phycoerythrin; RA, rheumatoid arthritis; TCR, T-cell receptor.
for the gal 264 Col2 259-273 -peptide and 0.025% for the nCol2 259-273peptide containing tetramers in the total CD4 + T-lymphocyte population. In a concomitantly analysed blood sample from a healthy donor, no DRB1*04:01/Col2-specific T cells could be identified.
The studies on our entire sample size using DRB1*04:01 tetramers containing either unmodified nCol2 or gal 264 Col2 259-273 peptide confirmed detectability of antigen-specific T cells in PBMCs from patients with RA at the expected low precursor frequencies. The frequencies of DRB1*04:01/nCol2-staining and DRB1*04:01/gal 264 Col2-staining CD4 + T cells staining CD4 + T cells were identical: 27.27%. Staining positivity was defined by a value exceeding the threshold set by the mean of negative biotin control +3 SD (see Methods section). Interestingly, CD4 + T cells staining with the DRB1*04:01/nCol2tetramers as well the DRB1*04:01/gal 264 Col2-tetramers were also detectable at a percentage of 5% and 30%, respectively, in the small cohort of healthy DRB1*04:01 carriers (figure 3B). No significant differences were detectable between the RA and healthy donor groups (figure 3B). In this respect, the small sample size constitutes a certain constraint of our study mainly due to limited access to biomaterial from HLA-typed healthy blood donors.
Interestingly, the DRB1*04:01/Col2-tetramer-positive T-cell population detectable in the peripheral blood of patients with RA exhibited a difference in responsiveness to in vitro stimulation of PBMCs with synthetic Col2 peptides. Stimulation with the gal 264 Col2 peptide resulted in an elevated frequency of antigen-activated CD4 + T lymphocytes compared with nCol2 as determined by peptide-induced upregulation of the activation marker CD154 detected by flow cytometry ( figure 3C). Taken together, the results demonstrate a functional impact of the Col2 peptide structure presented in the context of a DRB1*04:01encoded MHCII molecule on autoimmune recognition by CD4 + T cells in the peripheral blood of patients with RA.

Analysis of a cloned TCR derived from a single sorted Col2reactive T lymphocyte
We next aimed to characterise TCRs with DRB1*04:01restricted recognition of Subsequent functional studies using lentiviral gene transfer from a single sorted gal 264 Col2 259-273 -specific T-cell of a patient with HLA-DRB1*04:01-positive RA demonstrated the selective capability of recombinant monomeric DRB1*04:01/ Col2-peptide complexes to induce IL-2 production in TCRreconstituted Jurkat 76 cells ( figure 4B). The challenge with monomeric DRB1*04:01/gal 264 Col2 259-273 induced the strongest IL-2 response. Stimulation with DRB1*04:01 complexes containing the nCol2 peptide resulted in a considerably reduced IL-2 release that nevertheless clearly exceeded the levels induced by DRB1*04:01/HA306-318 or A q /gal 264 Col2 259-273 control complexes ( figure 4B). To confirm this result, we tested antigenpresenting cells (APCs) homozygously expressing DR*0401 obtained from DRB1*04:01 knock-in mice as well as APCs from the peripheral blood of DR*0401individuals (online supplemental figure S6). The presentation of gal 264 Col2 259-273 in a DRB1*04:01 context on the surface of either murine or human fixed APC after preloading with peptides was specifically recognised by TCR#16 mRNA transfected nuclear factor of activated T cells (NFAT) luciferase Jurkat reporter cells and associated with stronger NFAT activation compared with the stimulatory effect of the nCOL2 peptide under identical conditions. The CLIP control peptide did not lead to any activation of the TCR mRNA transfected Jurkat reporter cells. Accordingly, these results are in agreement with the studies on the specificity of tetramer-induced IL-2 responses via the recombinantly expressed TCR #16 in lentivirally transduced Jurkat 76 cells lacking an endogenous TCR.

Modelling of molecular interactions in the trimolecular complex of the DRB1*04:01, Col2 259-273 peptide and TCR
Based on the identified sequence of the human Col2-specific TCR#16 and the solved crystal structure of the DRB1*04:01/ Col2 259-273 complex, molecular modelling was performed using the template of a published TCR cocrystallised with an influenza peptide-containing DRB1*04:01 molecule (HA1.7). 32 The overview of the entire modelled 3D structure of the multicomponent system consisting of the DRB1*04:01/Col2 peptide/TCR complex is shown in online supplemental figure S7. More detailed insights into critical amino acid residues involved in interactions between the unmodified or gal 264 Col2 peptides and TCR #16 variable regions in the DRB1*04:01 complex are provided in figure 5, which depicts the superimposition of the minimised starting geometries for the two trimolecular complexes. Molecular interaction of the Col2 peptide with the TCR occurs via three side chain bonds irrespective of Col2 259-273 galactosylation. Two interactions involve the CDR3 region of the TCRα-chain

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(figure 6A). By contrast, the presence of a galactose residue at position 264 allowed formation of a hydrogen bond to the TCRα-CDR3 backbone, also resulting in the stabilisation of both side chain bonds likely due to limitation in lysine 264 mobility ( figure 6B). The stabilising allosteric effect is depicted in figure 7, which shows a comparative overview of all intermolecular bonds formed in the trimolecular complexes consisting of TCR#16 and DRB1*04:01 associated with either nCol2 or gal 264 Col2. The graphic illustrates that the galactose is the only moiety bonded to all three molecules (the Col2 peptide, the DRB1*04:01 β-chain and the TCR). The molecular dynamics simulations for a 1000 ns period exhibited a rather low degree of molecular fluctuation in the TCR V-regions contacting the DRB1*04:01/Col2 peptide complex ( figure 8). An exception was a peak of molecular mobility detectable in a solventexposed loop with reduced protein contacts carboxyterminal of TCRβ-CDR2 in the complex with nCol2-bound DRB1*04:01 (figure 8). Even more remarkable was the increase in molecular fluctuations in the constant region of TCR#16 affecting a Cβ domain just proximal to the so-called FG-loop and a Cα region that included the AB-loop. 33 The molecular flexibility of these functional domains, which localise near ectodomains of the signal-transducing CD3 membrane complex, 33 was clearly more pronounced in the trimolecular complex containing gal 264 Col2 (figure 8).

DISCUSSION
Our studies characterised molecular details of antigen presentation by the DRB1*04:01 molecule, an allelic variant strongly associated with RA in Caucasian patients, by structure determinations following co-crystallisation with several peptides known to trigger autoreactive RA T cells. A crucial residue for DR molecules associated with RA is position 71 in the β-chain. This residue, which is a lysine in DRB1*04:01, critically interacts with glutamic acid in position 266 of the Col2 peptide. However, there is some freedom in this interaction as other peptides derived from HSP and CILP contain different amino acids in position 266. The residues of valine 11 (V11b) and histidine 13 (H13b) of the β chain in HLA-DRB1*04:01 have been shown previously to be associated with susceptibility of seropositive RA in genome-wide association studies. 11 Our crystal structure analysis reveals that both residues likely contribute to the stability of the MHCII molecule as they are located where α and β chains pair in the beta-plated sheet. Moreover, a likely contribution to antigen presentation of the CILP 982-996 peptide is provided by the residue H13β that forms a hydrogen bond with the carboxyl group of the aspartic acid residue in the P6 pocket (P6D) of the CILP peptide. This hydrogen bond is missing in the other three complexed MHCII/peptide crystals, as the P6A (HSP70 289-306 ) and P6G (Col 259-273 ) lack the corresponding carboxyl group. In addition, the side chains of A74β orientate toward the P4 pocket, likely influencing the binding specificity of the P4 residue.
Similar to its interaction with murine A q , 34 the DRB1*04:01 bound Col2 259-273 peptide exposes two lysine residues that are physiologically modified by hydroxylation and glycosylation, and these modified variants can be recognised by T cells. 18 35 Whereas in the mouse Col2 immunisation activates Col2 259-273 -specific T cells, thereby inducing a severe form of erosive arthritis, it is not clear to what extent MHCII-restricted Col2specific T cells play a regulatory role in humans. In this context, there has been a long-standing question concerning why these T cells are not deleted from the repertoire by central tolerance. A possible explanation could be that TCR affinity for PTM variants of Col2 peptides promotes escape from thymic selection; this hypothesis is supported by our previous finding that nonmodified Col2, but not glycosylated Col2-determinants, could be expressed by mouse or human thymic epithelium. 29 Notably, earlier studies have detected glycopeptide-reactive T cells in patients with DRB1*04:01-positive and DRB1*01:01-positive RA, 18 36 and the present investigation provides new evidence that peripheral T cells of healthy individuals also express TCRs with binding affinity for DRB1*01:01/ gal 264 Col2 complexes in respective MHCII allele carriers.
The present investigation of human PBMCs from RA and healthy DRB1*04:01 carriers, which used tetramer staining as well as parallel peptide-induced T cell activation assays, provides new insight into the impact of PTM on Col2 T-cell recognition. In contrast to a comparable prevalence of CD4 + T cells that recognise nCol2 or gal 264 Col2 in DRB1*04:01 tetramer complexes, T-cell responsiveness was increased on peptide challenge with the galactosylated variant. The mechanism for this glycosylation-dependent impact on T-cell activation is not entirely clear and might be multifactorial, but one attractive hypothesis is that an altered TCR interaction with the DRB1*04:01-bound galactosylated Col2-peptide affects TCR signal transmission. Accordingly, our functional and structural characterisation of a prototypic human TCR and its interaction with nCol2 or gal 264 Col2 in the context of DRB1*04:01 presentation provide first experimental support for this hypothesis. The comparative dynamic modelling of the respective trimolecular complexes containing either the galactosylated or unmodified

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Col2 variant revealed a major difference in the propagation of TCR dynamics from the V-regions to key allosteric sites in the Cα and Cβ region pertaining particularly to the so-called CαAB loop. 33 This domain was previously described for its role in the allosteric regulation of TCR signalling as evidenced by fluorescence-based conformational changes on MHCII/peptide perception and signalling impairment by mutational analysis. 37 Another region exhibiting reinforced molecular fluctuations on TCR recognition of the DRB1*04:01/gal 264 Col2 in our studies is in immediate proximity to the CβFG loop that has been critically incriminated in T-cell activation and thymic selection. 38 39 Thus, our dynamic modelling studies provide evidence for allosteric changes initiated by CDR3α-region interaction with a single galactose residue in the DRB1*04:01-bound gal 264 Col2 peptide, which propagates to result in increased conformational flexibility at distant sites in the constant TCR regions contacting the CD3 signalling complex. Whereas it remains enigmatic how these allosteric changes are transmitted across the cell membrane, our complementary functional T-cell studies provide experimental support for translation into a reinforced TCR signal in response to challenge by gal 264 Col2-bound versus nCol2-bound DRB1*04:01complexes. Moreover, the data obtained by analysis of RA T cells and the prototypic TCR #16 might reflect mechanisms of central tolerance by thymic medullary epithelial cells that do not express galactosylated Col2 and accordingly execute clonal deletion exclusively via presentation of unmodified Col2 determinants. 29 Thus, T cells rescued from thymic selection due to weak TCR recognition of DRB1*04:01/ nCol2 complexes and the resulting low TCR signal intensities could subsequently become activated on MHCII presentation of physiologically galactosylated Col2 in peripheral tissues 39 via reinforced signalling dynamics initiated by the recognition of posttranslational Col2 peptide modifications. However, the outcome of the activation, which can result in either arthritogenic or tolerogenic T cell responses, would remain context dependent. An obvious objection to our findings on the lack of a contribution of citrulline residues to MHCII binding in the two crystallised control complexes containing citrullinated epitopes of RA T cell responses and our focus on TCR recognition of Col2 peptides is how our proposed concept could be linked to citrulline-specific immunity and its strong association with DRB1*04:01 in RA. However, it has been shown that Col2 can be citrullinated in vivo, both in mice and in humans with RA. 22 Moreover, the autoantibody response to citrullinated Col2 is prominent in B cell recognition of Col2. Accordingly, it is easily conceivable that B cells specific for citrullinated Col2 can present the non-citrullinated 259-273 peptide to T cells and vice versa. 40 Such T cells could help to activate B cells specific for citrullinated epitopes, potentially breaking tolerance and allowing pathogenic epitope spreading of the anticitrullinated protein antibody response target, joint cartilage. 40 Although we have provided indirect evidence for the proposed scenario, further studies are clearly needed to provide additional experimental support and to answer the question of whether this hypothesis might also apply to citrullinated antigens, such as the CILP-derived peptide cocrystallised with DRB1*04:01 in this study.  In complex with the unmodified Col2 peptide, a peak of molecular mobility is detectable carboxyterminal of the TCRβ-CDR2 domain and to a minor degree in TCRα-CDR2 in complex with the gal264Col2 peptide. Most notable is the increase of molecular fluctuations in the constant region of TCR#16 (arrows), especially in the α-chain, also affecting the CαAB loop and clearly much more pronounced in complex with gal264 compared with the unmodified Col2 peptide. CDR, complementarity-determining region; RMS, root mean square; TCR, T-cell receptor.