Interaction of Small Molecules with Specific Immune Receptors: The p-i Concept and its Consequences

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1 Send Orders for Reprints to Current Immunology Reviews, 2014, 10, Interaction of Small Molecules with Specific Immune Receptors: The p-i Concept and its Consequences Werner J. Pichler * and Stephen Watkins ADR-AC GmbH, Holligenstr. 91, 3008 Bern, Switzerland and Department of Rheumatology, Immunology and Allergology, University Hospital of Bern, 3010 Bern, Switzerland Abstract: Drugs may stimulate the immune system by forming stable new antigenic complexes consisting of the drug or drug metabolite which is covalently bound to a protein or peptide (hapten-carrier complex). Both, B- and T-cell immunity may arise, the latter directed to hapten modified peptides presented by HLA molecules. Beside this immunological stimulation, drugs can also stimulate the immune system through binding by non-covalent bonds to proteins like immune receptors. This so-called pharmacological interaction with immune receptors concept ( p-i concept ) may occur with HLA or TCR molecules themselves (p-i HLA or p-i TCR), and not the immunogenic peptide. It is a type of off-target activity of the drug on immune receptors, but more complex as various cell types, cell interactions and functionally different T cells are involved. In this review the conditions which lead to activation of T cells by p-i are discussed: important factors for a functional consequence of drug binding is the location of binding (p-i HLA or p-i TCR); the exact site within these immune receptors; the affinity of binding and the finding that p-i HLA can stimulate the immune system like an allo-allele. The p-i concept is able to solve some puzzles of drug hypersensitivity reactions and are a basis to better treat and potentially avoid drug hypersensitivity reactions. Moreover, the p-i concept shows that in contrast to previous beliefs small molecules do interact with immune receptors with functional consequence. But these interactions are not based on immune recognition, are at odds with some immunological concepts, but may nevertheless open new possibilities to understand and even treat immune reactions. Keywords: Alloreactivity, hapten, p-i concept, p-i HLA, p-i TCR. INTRODUCTION It is an old dogma in immunology and pharmacology that small sized chemicals like drugs are per se not immunogenic [1]. Only if they are able to bind covalently to a larger molecule like a protein and form a hapten-carrier complex they may stimulate the immune system [2, 3]. A drug may form this bond directly (hapten concept) or after a conversion to a reactive substance as a result of metabolism (pro-hapten concept). The chemical reactivity of a hapten may also lead to alteration of proteins involved in cell survival, cell activation, and this modification may lead to activation of cells of the innate immune system [4, 5, 6]. Together, the stimulation of innate immunity and the formation of neo-antigens leads to the development of a complete immune response with specificity against the hapten [7]. A typical hapten may thus elicit an immunoglobulin and a T-cell response, as the hapten-carrier complex offers antigenic sites for antibodies and for T-cell receptors (TCR) [7]. As the induction of a hapten-specific T- cell immune response relies on processing of haptenated proteins, followed by clonal expansion in lymph nodes and migration of reactive cells to the affected organ (often the skin), these immune reactions to drugs appear delayed, often days after drug exposure [7] (Fig. 1). In the last ~15-20 years it became clear that drugs may activate the immune system via alternative pathways as well. *Address correspondence to this author at the ADR-AC GmbH, Holligenstr. 91, 3008 Bern, Switzerland; Tel: ; Fax: ; werner.pichler@adr-ac.ch Based on the analysis of drug specific T cell clones (TCC) it was found that drugs can stimulate T cells also without the need to acquire antigenic features [8-11]. Drug specific T cell clones (TCC) reacted within seconds to the addition of the drug (immediate Ca++ influx), the presence of antigen presenting cells (APC) with HLA-peptide was needed, but fixing the APC by glutaraldehyde did not block the reactivity, which showed that drug uptake, processing and metabolism were not needed to elicit reactivity of T cells. Moreover, the drug binding to the cell surfaces was labile, as washing removed the drug and abolished the stimulatory potential (summary of p-i features in 10, 11). These findings resulted in the generation of the p-i concept, meaning that a pharmacological interaction of drugs with immune receptors was sufficient to stimulate T cells [10,11]. The conditions of stimulation were reminiscent of pharmacological interactions, and were in contrast to hapten binding via covalent bonds based on non-covalent interactions (van der Waals forces, hydrogen bonds, electrostatic interactions). How and to which immune receptor the drug was binding was initially not clarified. The drug stimulation by p-i was restricted to activation of a single T cell clone (TCC). Transfection of the relevant TCR into mouse hybridoma or Yurkat cell lines proved the involvement of certain T-cell receptors (TCR) and the need for HLA interaction [12-14] (See insert 1). Some initial data using CD4+ T cell clones specific for lidocain or sulfamethoxazole suggested that drug interaction may occur first with the TCR) [15-17]. However, further data pointed to a decisive role of a the presence of a certain HLAallele and appearance of drug hypersensitivity [18-22]. This drug-hla binding was further underscored by the X/14 $ Bentham Science Publishers

2 8 Current Immunology Reviews, 2014, Vol. 10, No. 1 Pichler and Watkins Fig. (1). Hapten concept: A drug/chemical able to covalently interact may form a stable, covalent bond between an AA and the drug. This hapten-protein complex is taken up and processed. The hapten-peptide bond persisits the processing and the modified peptide can be presented by HLA molecules. Concomitantly, the hapten may also form hapten-protein conjugates inside the cell and thereby activate the dendritic cell. This is an important costimulatory signal facilitating an immune response to the hapten-peptide complex. crystallographic analysis of HLA-drug-peptide complexes [21, 22]. Thus, in some hypersensitivity reactions a particular HLAallele might be the initial protein to which a drug binds. In this review recent findings on the p-i concept, in particular functional consequences of drug binding to TCR or HLA proteins are highlighted. The data show that in contrast to previous beliefs that small molecules do not interact with the specific immune system, there is now clear evidence that a non-immunological ( pharmacological ) stimulation of the specific immune system by drugs exist [23]. The drug interactions involve TCR and HLA molecules, are actually very complex and result in many different clinical outcomes, some of them mimicking allo- or autoimmune stimulations [7]. Thus, the interaction of small chemicals with immune receptors is a highly relevant topic in our societies where chemicals are not only widely used as drugs but are also a common part of our environment. BOX I: P-I CONCEPT The p-i Concept: A Particular Off Target Activity of a Drug The p-i concept is a general model to understand drug stimulations of immune receptors without postulating antigen-like features of the drug (e.g. by forming haptencarrier complexes): It is simply applying pharmacological concepts to immune receptors: The drug binds to the immune-receptor protein, and not to the antigen (antigenic peptide itself). It is thus not an immune reaction to the antigen, but a drug (pharmacological) stimulation of immune receptor proteins by drug binding. The interactions are reversible and the strengths of binding depend on the amount and type of van der Waals forces, hydrogen and electrostatic bonds between the drug and the particular immune receptor. It is also influenced by the size of the drug, chirality, the confirmation of the protein sequence to which the drug binds etc. [24, 25]. It may occur with inert drugs which are not haptens [8, 9], or with drugs, which act as haptens as well [26]. Actually, a drug which binds covalently to a certain amino-acid within a protein may first bind by non-covalent bonds to this site within the protein [27, 28]. In addition, also a drug-metabolite like oxypurinol can elicit p-i reactions itself, independent of an ability of this metabolite to bind covalently [29]. The features of the p-i concept do actually correspond to an off target activity of a drug. Such an off target activity of drugs directed to proteins of the immune system may actually occur rather frequently, as the antigen-specific immune receptors are very polymorphic and thus offer

3 Interaction of Small Molecules with Specific Immune Receptors Current Immunology Reviews, 2014, Vol. 10, No. 1 9 Box 1. The p-i concept Pharmacological Interaction with Immune Receptors A chemically inert drug, unable to covalently bind to proteins, happens to bind by non-covalent bonds to some of the many immune receptors. This drug-receptor interaction can occur with the phla complex or with the TCR. It can result in activation and expansion of T cells, which has some bizarre immunological and clinical features. abundant possibilities that a drug may bind at least to some of them. One estimates that there are about >10 11 different T cell receptors (TCR) per individual and over > 9900 different HLA-class I and > 3000 class II alleles in the human population ( Thus the chances of a drug to find a suitable binding site is probably higher with these polymorphic proteins than with monomorphic receptor structures. The majority of such drugimmune receptor interactions may be without functional consequences, but a few bindings may occur at a relevant position and be affine enough to cause a functional consequence. While the idea of the p-i concept as off-target activity of a drug is intriguing and helpful to emphasize the pharmacological nature of the reaction, the complexity of the immune system make the drug interaction with immune receptors still unique and more complicated than a simple off target activity of a drug. Therefore it makes sense to keep the term p-i concept, which is quite broad and includes different drug bindings and target structures. Some peculiarities of the off-target activity of a drug on immune receptors is summarized below. In p-i, Always Two Cells are Involved The start of T-cell stimulations is normally a consequence of cell interactions, namely of the T-cell with a professional antigen presenting cell (APC, e.g. a dendritic cell) or, if cells are already activated, with target cells in the tissue. In p-i (both, p-i HLA and p-i TCR) this intercellular interaction is also necessary: The need of TCR-drug-peptide HLA interaction for full T cell activation (measured by immediate Ca 2+ influx in specific T-cells, cytokine synthesis, proliferation and/or cytotoxicity) was proven by TCR transfections (sulfamethoxazole [SMX], chinolone, abacavir or radio contrast media [RCM] specific TCR) [12-14]. The TCR-transduced hybridoma cells did not develop a full response (IL-2 secretion) to addition of the drug. Only if APC with human (fitting) MHC were provided, they reacted. If the drug protein modification occurred on/in the APC, a stimulation of a second reactive cell, the T cell, takes place. Thus not the cell expressing the drugmodified protein (the APC) but the reactive cytokine secreting or cytotoxic T cell is causing clinical symptoms. This is quite different from other off-target reactions. Moreover, even if the drug would bind to the TCR, the T cell activation still depends on the interaction with HLA-peptides presented by another cell type [24, 25]. Complexity of the Immune System Types of immune responses following p-i stimulations are heterogeneous [7]: the same drug may in one individual elicit a pustular, in another individual a bullous skin reaction. This heterogeneity is based three factors: First, the drug stimulations affect different T cell subsets, which differ in specificity and function, namely cytokine production and cytotoxicity [7]. Second, the drug reactive T cells stem from a repertoire of >10 11 different T cells with distinct antigenspecificities. The real peptide specificity of T cells stimulated by drugs via p-i is unknown with the exception of some allo-reactivity described [25, 30]. Third, while the majority of drug induced reactive T cells do not carry an identical TCR or TCR-variable region (CDR3), there might be drug binding to the public part of the TCR [25]. Drug binding to this part of the TCR may be insufficient for stimulation. Only if T cells are pre-activated, e.g. by cytokines secreted during generalized virus infections or a previous drug allergy, the T cell may react [23, 31]. Thus, functional differences, antigen specificity and level of T-cell activation may all affect reactivity to p-i. T Cell Stimulations Outside the Rules In p-i, drugs bind to HLA or TCR receptors and not the antigen (like an immunogenic peptide presented by HLA and recognized by a TCR). These two immune receptors are quite special molecules as the TCR is connected to a very efficient signal transduction component: a T cell can sense minimal changes, e.g. a single hydrogen bond change, of the peptide-hla complex and react to it [32, 33]. It is feasible that binding of a drug at the right position together with HLA interaction may be sufficient to activate the signal machinery at least if the drug binds to a pre-activated, memory T cell [25]. Second, the HLA-molecule is the signature for self, and changing this signature is an abnormal and dangerous process, which in the evolution of the immune system has probably not been considered : it can lead to an abnormal T cell stimulation, which may cause symptoms similar to a gvh disease [34, 35]. Analysis of drug specific T cell clones revealed that the functional consequence of p-i stimulation of a single T cell appears to be similar (at least not inferior) to its stimulation by a classical peptide antigen: The drug reactive T cells synthesize cytokines, exert cytotoxicity by various means, proliferate, and affected patients have more drug specific T

4 10 Current Immunology Reviews, 2014, Vol. 10, No. 1 Pichler and Watkins cells in the circulation. It looks like a normal sensitization with a memory response, which results in faster responses upon reexposure and e.g. positive skin test reactions. However, more work is needed to understand whether the bizarre clinical picture of some severe drug hypersensitivity reactions (Stevens-Johnson Syndrome / toxic epidermal necrolysis [SJS/TEN]; Drug rash with eosinophilia and systemic symptoms [DRESS]) is due to an abnormal (increased) stimulation of many single cells by p-i stimulation with e.g. synthesis of many cytokines simultaneously, some of which are also exerting cytotoxicity, or whether the bizarre clinic is due to massive stimulation of many different cells, each of which reacts in a normal way, but together are resulting in a bizarre clinic. Subdivision and Functional Consequence of p-i: The partners involved in the p-i concept are the drug, the TCR and the peptide-hla complex (pepthla). It is always the combination of these three components which lead to functional consequences. These are limited to T-cell activations, as stimulation of B cells or NK cells has not been shown or have not yet been investigated. All data regarding p-i TCR and p-i HLA refer to αβtcr, and only one drug (lidocain) specific γδtcr within many 1000 TCC with different specificities have been described, but the reactive TCC is lost and not available for further analysis [15]. The drug may directly interact with a certain region of the TCR (p-i TCR), or it may bind to the HLA-protein on antigen presenting cells (p-i HLA) [23]. In p-i TCR, stimulation depends on an interaction of the drug affected TCR with pepthla, in p-i HLA the T cells are reactive to drug modified pepthla {drug-pepthla} complex. At present, the majority of data support the concept that in both, p-i TCR and in p-i HLA, the TCR is peptide-reactive (and only exceptionally recognizing the drug per se (see Fig. 5) [21, 22]: The drug binding may occur on the polymorphic part of the HLA-peptide complex like the peptide binding groove, which is hidden and not accessible for TCR [21, 22]; or it may bind to the TCR outside the peptide interaction site, exerting an allosteric effect on the configuration of the TCR and its ability to bind to pept/hla [25]. The functional consequence of this p-i stimulation is always T cell activation like cytokine secretion, proliferation, cytotoxicity and homing to different organs. Drug binding to HLA or TCR activates T cells, which have already a normal peptide specificity. This original peptide specificity is normally not known and the functional and clinical consequences of T cell stimulation by an altered pepthla complex or of an allosteric effect of a drug-binding on TCR are difficult to judge if the specificity is not clear. Since some drugs tend to induce often a rather similar clinical phenotype (e.g. kidney damage with allopurinol, lung damage with abacavir) [36] it is possible that at least in some HLA allele restricted reactions - a certain drug activates consistently a set of T cells with similar peptide specificities and homing to a certain tissue, where it is harmful. Since alterations of pepthla occur more commonly with HLA-class I molecules (e.g. F pocket in B*57:01 for abacavir and in B*58:01 for oxypurinol), p-i directed to HLA-class I tends to cause more HLA-class I restricted, CD8+ T cell reactions. These are often strongly cytotoxic and thus more cell damaging than CD4+ T cell reactions. p-i TCR The data on p-i TCR are still rather limited although early data on the p-i concept actually favored a p-i TCR over p-i HLA concept [8, 9, 15]. It was shown already in 2002 using SMX and lidocain specific T cell clones (TCC) that some drug specific T cells react if the peptide or even the HLA-molecule was exchanged [37-39]. Newer data support two versions of T cell stimulations via the p-i TCR concept for SMX stimulations [24, 25]. Furthermore, a recent example of p-i TCR is the finding that a certain TCR clonotype is involved in carbamazepine (CBZ) hypersensitivity in HLA-B*15:02 positive individuals [40]: Elution of peptides from CBZ pulsed APC was not presenting hapten-modified peptides [41]. The T-cells of B*15:02+ patients with CBZ related SJS/TEN used mainly the TCR Vβ-11-ISGSY clonotype [40]. This clonotype was present in 16 of 19 patients and absent in all 17 carbamazepin tolerant patients [40]. These data showed that in addition to B*15:02 [19, 42], also the availability of TCR carrying a certain sequence may be crucial for disease manifestations. Moreover, in PBMCs of healthy subjects, who are carriers of HLA-B*15:02 and of Vβ-11-ISGSY, CBZ-specific cytotoxicity could also be primed in vitro. Thus, the data suggest that only if both, a certain HLA and a certain TCR-sequence are present, a strong and disease causing T cell stimulation develops [40-42]. The early data with SMX specific TCC [8, 16, 17] were recently further extended by an in depths analysis of two sulfamethoxazole (SMX) specific CD4+ TCC and their TCR ( H13 and 1.3 ) (Fig. 2). These TCC stem from the same individual and are restricted to HLA-DR-B1*10:01: The study combined in vitro stimulations of the two TCC by SMX and 11 structurally similar sulfanilamides (SA), the blocking effect of some sulfanilamides on SMX stimulation and docking of SMX and 11 sulfanilamides to the two TCR [24]. Moreover, an extensive molecular modelling analysis of the TCR H13 was performed [25]. The data show that SMX bound to two different sites on the two TCR with rather distinct functional consequences. An allosteric effect was seen on the TCR H13, where SMX (and five of the 11 other sulfanilamides) bind outside the peptide interacting site on the TCR-CDR2Vβ. Molecular modeling of SMX binding to the TCR revealed that SMX binding to TCRVβ20-1 on TCR-CDR2β alters the configuration of TCR H13, which, after drug interaction, binds with higher affinity (Gibbs forces between the TCR and pepthla 7fold higher with compared to without SMX) to typical HLA-DR-B1*10:01 presented peptides (exemplified by a laminin derived peptide). Of note, this binding site is not unique as Vβ 20.1 is found on ~0,8-2,4% of normal circulating T cells. Thus, drug binding to TCR can result in an allosteric effect and alteration which enhances the reactivity with presented (self) peptides [25]. The elucidation of SMX binding to the second TCR gave a completely different picture: SMX binds directly to the unique CDR3α of TCR 1.3, and stimulate the TCR similar to

5 Interaction of Small Molecules with Specific Immune Receptors Current Immunology Reviews, 2014, Vol. 10, No a peptide or a hapten-modified peptide presented by HLA- DR [24]. Interestingly, this SMX stimulation could be blocked by any of the 11 other sulfanilamides analysed in a dose dependent way. This blocking effect could be explained by docking studies, which revealed that SMX and the 11 other sulfanilamides (SA) bound to the same binding site on CDR3α. What is the difference between binding of SA which stimulate (SMX) and binding which do not stimulate (11 SA)? A closer look suggested that all 11 non stimulatory SA bound only in one orientation into the CDR3α pocket, namely via their NH2 ending directed to the TCR (Fig. 3). But SMX could also bind in another orientation to the CDR3α, namely with the NH2 group pointing to the peptide binding groove. This orientation would be similar to the orientation of the hapten SMX-NO, if it would be bound to a peptide presented by HLA-DR*10:01. Indeed, we and others have already described TCC which react on one hand with SMX (via p-i), and on the other side with the hapten SMX-NO [17, 43]. Thus our explanation of SMX binding to TCR-CDR3α supports and explains the existence of such hapten and p-i cross-reactive TCC. It also shows that under these stimulatory conditions the drug itself (and not a peptide, see TCC H13 and discussion on Stimulating interactions peptide Blocking interactions Fig. (2). 3D model of 2 SMX-specific TCR (A: 1.3, B: H13). The TCR were screened for SMX docking using a methodical approach with Autodock and Autodock Vina software. A) TCC 1.3: a classically restricted T cell CDR3α hypervariable region against a sulfamethoxazole. Eleven other, structurally related sulfanilamides bound to the same site, but were not stimulatory. They even blocked SMX reactivity [24]. B) TCC H13: a non-classical interaction of sulfamethoxazole with the TCR H13 - CDR2β, an conserved region of T cell receptor variable β20-1. The TCC H13 was stimulated by SMX and 5 additional sulfanilamides, while 6 other sulfaniamides could not be docked to this site. Note that this SMX binding site is free of interaction with the HLA or peptide in any way. Binding of SMX induces a conformational change with increased affinity of TCR with peptide (allosteric effect) [24, 25]. The small molecule binding to the respective sites is colored in pink. Fig. (3). Orientation of SMX is decisive for stimulatory effect: The TCC 1.3 bound SMX and 11 SA at the CDR3α loop. However, only SMX was stimulatory: It was the only sulfanilamide which could bind to the TCR in two opposing directions. The confirmation with NH2 pointing to the HLA-peptide complex may be the stimulatory conformation; this conformation of SMX would also occur if SMX-NO is presented as hapten bound to HLA-presented peptide. The other conformations where a contact with Tyr 31 in TCR-CDR2 loop takes place is not stimulatory and actually blocking the SMX induced activation [24].

6 12 Current Immunology Reviews, 2014, Vol. 10, No. 1 Pichler and Watkins p-i HLA) is recognized. It also raises the question whether SMX-NO as rather immunogenic hapten is initiating this reactivity to SMX binding via p-i. But as this cross-reactivity is rare, we consider it as an unlikely explanation for the majority of SMX reactive T cells. In conclusion, the detailed analysis of SMX stimulation revealed that drugs may bind to TCR. It may or may not result in T cell activation [24, 25]. Compounds may bind but remain non-stimulatory. They can even block the activation by an activating drug like SMX. Binding may occur on different sites (CDR3α or CDR2β), and even binding outside the HLA-peptide interactions site might be stimulatory, as it can elicit an allosteric effect. Small alteration in the drug structure may lead to inability to bind, or inability to bind in the correct (stimulatory) orientation [24]. In both, the TCR 1.3 and the TCR H13, an interaction with the HLA-peptide complex is necessary for full stimulation. p-i HLA An decisive step forward in understanding severe, T-cell mediated drug hypersensitivity reactions was the description of HLA-allele associations [18-22] for severe drug hypersensitivity reactions elicited by certain drugs: These associations were very high and highly specific for one drug, with npv of close to 100%, while the ppv was mostly low (<3%). A notable exception is abacavir, where 47% of HLA- B*57:01 carriers developed a hypersensitivity reaction upon exposure [44]. The frequency of the involved allele in the population was important for linkage associations [45]: the HLA-B*15:02 association for carbamazepine induced SJS/TEN in Han Chinese could not be confirmed in Europeans, where HLA-B*15:02 is rare. Carbamazepine hypersensitivity in Europeans could be linked to HLA- A*31:01, in particular to DRESS, less so to SJS/TEN [46, 47]. Approximately 60% of patients with hypersensitivity carried this allele. In follow up studies many more associations were described but with a substantially lower risk. The majority of high risk alleles were HLA-class I, but some less stringent associations were also found for HLAclass II alleles [48, 49]. Fig. (4). Schematic representation of TCR-peptide HLA complex and where, according to crystallographic [21, 22] and docking data, abacavir is binding: It is a non-covalent interaction of abacavir (yellow) within the peptide groove (F pocket) of HLA B*57:01. Note that the drug is not accessible to the TCR and that the TCR stimulation occurs by conformational change of {abacavirpeptide/hla} complex. (See color image online) The strong HLA-association raised a lot of questions, which could partly be answered in follow up studies: Elution of the peptide from HLA-class I alleles showed that in carbamazepine or abacavir hypersensitivity reactions the immunogenic peptides were not modified by covalent drug binding. This was in agreement with the p-i concept implying that the immune receptor protein and not the antigen, namely the immunogenic peptide, is modified [21, 22, 41]. It was also in agreement with common concepts of immunology. The peptides presented by structurally related HLA-alleles are often identical, and thus more than a single allele would be expected to be associated with drug hypersensitivity. As this is not the case, and as the linkage is mostly exclusive for a single allele, p-i-hla is better explained by direct drug binding to the HLA-protein itself [47]. This direct drug binding to the immune receptor was finally proven by the crystallographic structure of abacavir binding to HLA-B*57:01 [21, 22] (Fig. 4). In allopurinol reactivity, a detailed functional and docking analysis of allopurinol or oxypurinol specific TCC and HLA-restriction revealed that the described linkage is actually mainly due to oxypurinol binding to the F-pocket of B*58:01 [28, 50, 51], and that allopurinol is less stringently associated with B*58:01. The crystallographic structure of abacavir bound to B*57:01 showed that the drug is hidden below the peptide [21, 22], and the docking of allopurinol and oxypurinol also located the drug below the peptide presented by the HLAallele [51] (Fig. 3). On the other hand, if TCC were incubated with APC and the drug (abacavir, flucloxacillin, allopurinol and oxypurinol) an immediate (seconds to <2min) recognition of T cells could be observed in Ca++ assays. This raises two questions: a) where are the drugs loaded onto the HLA-allele? and b) what is actually stimulating the drug reactive T cells? Drug Loading Can Occur on the Surface or Inside the APC If specific TCC were tested, an immediate reactivity (within seconds, mostly < 2min) could be observed with allopurinol, oxypurinol, flucloxacillin. Ca. 40% of abacavir reactive TCC reacted also immediately [52]. Incubation of the cell mixture containing APC and drug specific TCC for 30min to 14hrs with flucloxacillin, allopurinol or oxypurinol, followed by a washing step, did not lead to stimulation [26, 51]: This suggest that uptake and intracellular drug binding to HLA-protein, followed by a subsequent presentation of the drug-pepthla complex is exceptional. Only an instable association between allopurinol, oxypurinol and flucloxacillin and the relevant HLA allele (B*57:01 or B*58:01) on the surface seems to take place and may be sufficient to stimulate the T cell. This is in contrast to abacavir binding: washing of APC after 15 min did not abrogate reactivity to abacavir, and after a few hours of incubation the drug pulsed APC were again stimulatory. In 60% of abacavir specific TCC the reaction occurred only to pulsed APC, but did not occur immediately. Thus abacavir binds to HLA-B*57:01 intracellularly and reaches as {abacavir-pepthla} complex the surface [52, 53]. The stimulatory potential of {abacavir-pepthla} complex could be linked to the density of these complexes on the cell surface: increasing the density of {abacvir-pepthla}

7 Interaction of Small Molecules with Specific Immune Receptors Current Immunology Reviews, 2014, Vol. 10, No complexes increased the reactivity of the TCC raised against abacavir [52]. Some TCC required only few {abacavirpepthla} complexes (high avidity TCR/TCC), others needed more (low avidity TCR/TCC). Importantly, all TCC reacting to abacavir immediately (surface loading) did also react after pulsing (internal loading)! This shows that the reactive T cells recognize abacavir-modified pepthla independent of the location of peptide loading. Addition of B*57:01-binding peptides of various origin to abacavir pulsing hardly affected reactivity of the abacavir induced TCC. It suggests that abacavir modified pepthla are stimulatory rather independent of the type of peptide or/and that at least different sets of peptides are recognized in the context of (abacavir-hla) [52, 56]. Internal loading has also been suggested in radio contrast media (RCM) hypersensitivity [14]: Various TCC from different individuals with delayed hypersensitivity reactions to RCM were analysed. The TCC were mainly CD4+ and HLA-class II restricted, but some TCC were CD8 and class I restricted; an iomeron specific TCR from a CD8+ TCC was also transfected into a mouse hybridoma cell line, maintaining reactivity. Some TCCs and the transfectant reacted to RCM independent of uptake by APCs because proliferation/il-2 secretion occurred in the presence of glutaraldehyde-fixed APCs, and intracellular calcium within reactive T cells increased within seconds after drug addition. Other TCCs required functional APCs, compatible with uptake and presentation of RCM on HLA-class II molecules: this was demonstrated by using glutaraldehyde fixed APCs, which resulted in loss of presentation; Additionally, RCM could not be washed away from RCM-pre-incubated APCs; and the optimal pulsing time for APC was 10 20h. The data suggested that RCM may be stimulatory for T cells either by direct binding to the surface of (pept)hla (including HLAclass II), but that also RCM representation after uptake and processing by APCs is possible [14]. The data on loading suggest the following scenario: Internal loading: Some drugs (like abacavir) are taken up, reach the endoplasmatic reticulum and are loaded onto the still empty HLA-B*57:01 very effectively [21, 22, 53]. Actually very low concentrations, namely at 1-10ng/ml are sufficient to cause a strong stimulation of abacavir specific TCC [52]. The abacavir-hla complex is further loaded with peptides: the majority (>80%) represent normal B*57:01 binding peptides, a smaller fraction are altered peptides (e.g. valin instead of tryptophan at the F-pocket binding position of the immunogenic peptide), as the F-pocket is already occupied by abacavir [21, 22, 53]. In RCM hypersensitivity, which is probably not linked to a single HLA-allele, higher concentrations (100μg/ml) are required to bind to the HLAmolecule [14]. External, surface loading: It has been well demonstrated that drugs can also associate with the pepthla complex on the cell surface. This is documented by the rapidity of the reaction and that even fixed APC may allow stimulation of TCC [52]. Somehow a labile complex consisting of {drug and pepthla} must be formed, which is stimulatory enough to cause an Ca++ influx in reactive TCC, followed by cytokine secretion and/or cytotoxicity. Yun et al. proposed that the flexibility of HLA-binding peptides still allowed binding of drugs to the HLA-pockets in spite of peptide presence [51]. This flexibility of HLA-class I bound peptides has been shown by molecular modelling [54], demonstrating that normal HLA peptide complexes are not fixed but flexible and may allow access of the drug to the F-pocket. The flexibility would allow partial detachment of the peptide and exposure to the binding site of the drug; conditions for binding are that the drug must have sufficient affinity or concentration to allow the binding to take place; and that the peptide must be able to bind in a new conformation in the presence of drug-hla complexes, as the {drug-pepthla} complex is recognized by T cells as foreign namely as drug altered pepthla complex. What is Stimulatory for the Reactive T Cell? In principle three possibilities exist: 1) recognition of the drug on pepthla: In p-i HLA the drug itself may be presented and recognized by the reactive T-cell together with the pepthla-complex; this seems to be unlikely considering the location of oxypurinol and abacavir in the F-pocket, actually covered by the peptide and not accessible to the TCR. On the other hand, the location of carbamazepine in the middle of the peptide binding groove may make the drug still accessible to the TCR and the hypervariable CDR3 region on TCR, which is linked to carbamazepine induced SJS-disease [21, 42]. In analysing T cell reactions to the RCM, our group has developed iomeprol specific TCCs and a TCR transfected hybridoma cell line, which react immediately and also after pulsing to iomeprol presented by the HLA-B*51:01. Preliminary docking data suggested that iomeprol, which is rather large with a MW of 777D, spans the peptide binding groove of HLA and may be presented even without a peptide [St. Watkins, doctoral thesis Univ. of Bern]. 2) Altered peptide model: this model has been established with abacavir: It links presentation of an altered peptide to immune stimulation [21, 22, 53]: it is well documented regarding exchange of peptide in the HLA-B*57:01 allele where abacavir binding may alter peptide presentation or peptide repertoire. But the documentation that T cells are indeed reactive to the altered peptide is still rather limited: - the ELISPOT data and only moderate IFNγ stimulations by presumably peptide specific T cell lines is very not strong [21, 22], - TCC specific for the altered peptide could not be induced in abacavir driven cultures - the concentration of abacavir used to achieve peptide exchange were high (100μg/ml) [22, 53]. As shown, specific TCC was already stimulated using concentration of 1ng and 10ng/ml abacavir for APC pulsing, - the capacity of altered peptides to stimulate an immune response without CD4 cell help is assumed to be limited to high affinity interactions, and - last but not least, the altered peptide model does not explain the immediate reactivity of Ca. 40% of

8 14 Current Immunology Reviews, 2014, Vol. 10, No. 1 Pichler and Watkins abacavir reactive TCC and that the same TCC do also react to APC pulsed by abacavir [52, 55], nor does it explain the immediate reactivity to oxypurinol, allopurinol or flucloxacillin specific TCC [26, 51], which occurs within seconds. Thus, the altered peptide model has only limited applicability [55]. BOX 2: (THYMUS MATURATION/ALLOSTIMULATION) 3) drug altered pepthla complex: this concept implies that the pepthla complex is modified by drug binding and that this {drug-pepthla} complex is stimulatory for drug specific (or better drug induced) T-cells [56]: One has to be aware that in p-i HLA, not the peptide, but the immune receptor (HLA protein) is modified, mostly in the peptide binding groove [21, 22, 51, 53]. Our T cell system is trained to react with self-hla plus self or foreign-peptide. It is not trained to react with an alloallele instead of a self-allele as such an allo-allele is not present in the thymus, where the T cell learns how to react. If a drug binds to the self-hla protein, it can make an auto-pepthla looking like an allo-pepthla! This is an unusual and highly stimulatory situation [34, 35, 56], where 5-15% of matured T cells react directly with such allo-hla-peptide complexes (direct allorecognition, a main factor for early transplant rejection). Thereby, even a tolerogenic self-peptide if presented by an altered HLA (e.g. a drug altered HLA) - may become suddenly part of an highly stimulatory (allo/altered)-allele-peptide complex. An exchange of peptide is not (!) a prerequisite to cause such an immune stimulation. The T-cells activated by the {drug-pepthla} complex are rather unusual: they are induced by an {drug-pepthla} complex and are reacting like allo-specific T cells. Such an allo-stimulation is rather strong, and is stimulating naïve or memory T cells [34, 35, 56]. These T cells react directly with the preformed {drug-pepthla} complex, similar to the direct stimulation by an allo-allele [56]. Thus, no involvement of prior dendritic cell activation or of antigen processing is needed to present the stimulatory HLAcomplex. The p-i stimulation bypasses the activation of innate immunity [56]. The activated T cells have another particular feature: allo-reactive T cells are known to be polyspecific [34, 35, 57]: they react not only with one specific peptide, but with many, also structurally unrelated peptides. This may also explain the strengths of allo-allele and {drug-pepthla} complex related T-cell activations which often exceeds the stimulation by a single peptide [52]. Thus, in the context of an (drug-) altered HLA, the selective peptide-specificity of the T cell is lost and quite a few peptides presented by the altered HLA are stimulatory. The cause for this polyspecificity is not yet known in detail. Importantly, these features of allo- or {drug-pepthla} induced reactivity can explain, why so many T cells react to a drug like abacavir, as not a single peptide-abacavir HLA complex but many different peptide-abacavir-hla complexes are stimulatory. And polyspecificity of drug induced T cells explains, why those T cells which react to abacavir immediately (to {abacavir-pepthla}, where abacavir is loaded on the cell surface) do also react to abacavir pulsed APC, where abacavir is loaded onto HLA-B*57:01 in the endoplasmatic reticulum [52, 55]. And polyspecificity explains why the {abacavir-pepthla} complex is stimulatory independent on the type of APC used, which could be EBV transformed B- LCL, dendritic cells, and B*57:01 transduced hybridoma cells [56]: the peptides involved in these different conditions Thymus selection, allo-reactivity & polyspecificity: The fine specificity of TCR is the result of thymus selection: the majority of thymocytes die during the thymus selection process: During positive selection T cells capable of recognizing peptide-mhc complexes with appropriate affinity (not too strongly or too weakly) will receive a vital "survival signal, while the great majority of cells die by neglect. Then the negative selection in the medulla follows, which removes thymocytes that interact too strongly with the self-antigen presented. They receive an apoptotic signal that leads to cell death. It is important to realize that this selection process of T cells occurs only on own HLAalleles and thus the fine specificity of TCR for peptides is limited to the recognition of peptides in the context of own HLA-alleles. 5-15% of the matured T cells keep the ability that their TCR is stimulated by other peptide-hla-alleles as well (allo-reactive T cells). T cells react differently, if they are stimulated by peptide and allo-allele than by peptide and self allele: allo-stimulated T cells appear to be polyspecific: more than one peptide is stimulatory. If presented by an allo-allele (or drug altered allele, which look like an allo-allele, see text) even own peptides to which tolerance on own HLA has been generated - are stimulatory. Box. 2.

9 Interaction of Small Molecules with Specific Immune Receptors Current Immunology Reviews, 2014, Vol. 10, No Hapten p-i TCR SMX-NO 3 TCR HLA SMX-NO SMX SMX p-i HLA TCR HLA CBZ ABC ABC, ALP, OXY FLUX,... Fig. (5). Scheme of p-i stimulations discussed in this review: Upper part (1 ) hapten und p-i TCR ( ). In 1, hapten concept: the drug binds to a protein, which is processed to peptide, and the immunogenic hapten-peptide complex is presented. In he drug binds to the TCR, but stimulates like a hapten bound to an hapten modified immunogenic peptide presented by HLA (like 1 ). In, SMX is binding outside the peptide interaction site, acutally in the example analysed, to Vβ20-1. Binding induces alteration of TCR-conformation with increased affinity for an endogenous peptide. Lower part: p-i HLA: : The drug may be placed beside the peptide and be accessible to the TCR (discussed for CBZ in B*15:02). : the drug may be bound to the F-pocket of certain HLA allele (model abacavir-b*57:01). This binding may occur inside cells (endoplasmic reticulum), and be rather stable. It may even lead to altered peptide presentation (altered peptide model [21, 22, 53]). The {drug-pepthla} complex is altered and induces an auto- or allo-immune response. : some drugs bind from outside to surface HLA: the flexibility of the bound peptides may allow access of drugs to the HLA site (51). This complex {drug-pepthla} is strongly stimulatory for T cells, as it looks like and stimulates like an allo-allele. In 3,4and5 a peptide is stimulatory; recognized. The TCR in 4 (?), 5 and 6 may be stimulated like by an allo-like stimulation; detail see text. (immediate presentation or after pulsing, different APC) are most likely not the same, but they all are still stimulatory together with {abacavir-hla}: thus, the {abacavirpepthla} appears to be stimulatory for the same TCC, even if different peptides are involved! CONCLUSION AND OUTLOOK Drug hypersensitivity has been an enigmatic topic for many years and contradicted many concept of classical immunology [58]. This did not contribute to a better image of this difficult area. But the p-i concept with the detailed analyses of the direct, non-covalent (p-i) interactions of drugs with HLA or TCR now explain a lot of these puzzling findings, as the immune stimulations are not due to a classical immunological process. There is Light at the End of the Tunnel: Drug Hypersensitivity as Result of an Abnormal, Allo-Like T- Cell Stimulation Interactions of small molecules like drugs with immune receptors are probably frequent, but only a minor part of these interactions may also be functionally and clinically relevant. Such pharmacological interactions with immune receptors (p-i) are a kind of off-target activity of a drug on immune receptors. However, the p-i mediated stimulations are more complex than normal off target activities, which often concern a single receptor molecule and single cell type,

10 16 Current Immunology Reviews, 2014, Vol. 10, No. 1 Pichler and Watkins and not a whole cascade of reacting cells, as it is typical in immune stimulations. Moreover, drug binding can occur on HLA or on TCR, and even if the same molecule (HLA or TCR) is involved, the drug binding to different sites can result in quite distinct functional consequences. There are still discussions how the cells are stimulated by drugs: The possibilities on drug interactions with immune receptors may be enormous. Some of them have an experimental basis and are discussed and schematically shown in Fig. (5): Drugs stimulating via p-i TCR may imitate a hapten like recognition of the drug. The drug is located in the interface of pepthla and variable region of CDR3/TCR; It interacts with the TCR as if it would be presented as hapten bound to an immunogenic peptide presented by HLA. Such a situation allows recognition of the drug itself. A second possibility is an allosteric effect of the drug on the TCR. The data are based on SMX stimulations and need to be substantiated in other drug/tcr/hla combinations to generalize these two quite distinct concepts. It is well possible that other pathways of p- i TCR stimulations exist as well. In p-i HLA, the distinctions of the different concepts are less clear: The drug may be partly accessible or may cause presentation of an altered peptide (Fig. 5). Most relevant seems to be the concept that p-i HLA may lead to an allolike stimulation, as this model does not only explain drug altered pepthla as stimulatory complex, but also implies an abnormal T cell stimulation, namely that T cells are stimulated outside the rules (no involvement of innate immunity) [56]. It has been shown for abacavir and needs to be shown for other drugs as well: The allo-model for drug hypersensitivity would explain the often described but not understood puzzles of T cell reactions to drugs [58, 59, 60]: that drug induced T cells show an increased frequency of allo-reactivity, that T cells can even be stimulated after exchange of the peptide from the presenting HLA structure [30, 37, 39]. It explains the finding of HLA-class I reactive CD4 and class II reactive CD8+ T cells as well as the frequent occurrence of double CD4+/CD8+ drug reactive T cells. Direct allo - or {drug-pepthla} stimulation may not need costimulation by CD4 or CD8 coreceptors [8, 9, 15, 16, 59]. And it would also explain the well-known clinical similarity between allo-immune reactions (gvh-disease) and some forms of drug allergy [61] as well as that some models of SJS are based on an gvhd reaction [62]. A Consequent Application of the p-i Concept Opens New Possibilities in and Beyond Drug Hypersensitivity Risk assessment for drug hypersensitivity: The detailed structure and immune sequence of most immune receptors (TCR, HLA) are well known. It is feasible to screen for drug binding to these proteins using docking and molecular modelling. This might be followed by in vitro assays and, if possible, analysis of patients with drug hypersensitivity to verify the relevance. Such an approach may help to better identify drugs which carry a risk for immune mediated side effects already early in development and open the possibility to use the drug outside the risk group, which makes it safer. Use of p-i HLA or p-i TCR to stimulate T cells with a defined peptide specificity: The drug induced stimulations become apparent as severe side effects. These reactions are often fulminant, partly deadly and illustrate that such stimulations are extremely powerful: The p-i concept postulates and it has been shown by competitive binding experiments - that immune receptors are amenable for pharmacological intervention [24]: One might block or stimulate the TCR namely induce a drug induced T cell reaction versus a certain cell or e.g. tumor antigen. Alternatively, one could attempt to stimulate or eliminate certain T cells with certain TCR by selective drug binding. However, one has to be aware that a drug which blocks the TCR A carrying e.g. TCR Vβ20.1 may activate another T cell/tcr B via binding at another location, e.g. on the variable CDR3 site of TCR [24]. Is the p-i concept and its consequences also relevant for other small molecules and potentially involved in some auto-inflammatory diseases? The p-i concept postulates that drugs/chemicals can bind to immune receptors and activate them: This does not exclude small molecules/chemicals, even if synthesized by the body, like e.g. xanthin or hypoxanthin or nucleotides, which may bind to some of the immune receptors as well. Such interactions may rarely be functionally relevant, but exceptions might exist: Hypoxanthin specific T cells were described, which were cytotoxic for APC upon addition of hypoxanthin [51]. Similarly, nucleotides with binding affinity for TCR have been described [63]. The in vivo relevance is still unclear. It is a challenging idea that some of these molecules are stimulatory and cause (auto-inflammatory) diseases under certain circumstances opening a completely new area of research. E.g., the single amino acid replacement Asp116His distinguishes the two subtypes HLA-B*2705 and HLA-B*2709 which are, respectively, associated and non-associated with Ankylosing Spondylitis, a chronic autoinflammatory disease [64]. It might be worthwhile to pursue not only the search for immunogenic peptides in this disease which is in contrast to other autoimmune diseases HLA-classI linked and investigate whether a small molecular compound is related to the pathogenesis and particular disease association of these HLA-B*27 subtypes. Indeed, the extreme HLA-subtype specificity has been observed for binding of drug or chemicals to a certain HLA-allele (abacavir for B*57:01 but not B57:03, B*58:01, etc), while the peptide-binding is normally broader. In conclusion, it is possible that drug hypersensitivity reactions are not only a nuisance for patients and treating physician, but may actually teach us a lot about the etiology of other diseases and the insight gained from drug hypersensitivity research may even help to treat some diseases better. LIST OF ABBREVIATIONS APC = APC antigen presenting cell p-i = Pharmacological interaction with immune receptors