Immune Mechanisms of Drug Allergy

CHAPTER 4

Immune Mechanisms of Drug Allergy KATIE D. WHITE, MD, PHD  •  KATHERINE KONVINSE, MSC  •  RANNAKOE LEHLOENYA, MD  •  ALEC REDWOOD, PHD  •  ELIZABETH J. PHILLIPS, MD

KEY POINTS

• Many adverse drug reactions (ADRs) are not predictable based solely on the pharmacologic action of the drug. • We now know that these reactions stem from specific off-target drug activity that includes the interaction of drugs with immune receptors and pharmacologic drug effects such as those seen in non-IgE-mediated mast cell activation syndrome • Some reactions that display a clinical phenotype consistent with an immunologically mediated reaction, such as anaphylaxis, angioedema, urticaria, maculopapular exanthema, fever, and internal organ involvement (e.g., hepatitis), are related to an adaptive immune response and are associated with immunologic memory. • These reactions encompass a number of phenotypically distinct clinical diagnoses that comprise both B cell–mediated (antibody-mediated, Gell-Coombs Types I–III) and purely T cell–mediated reactions (Gell-Coombs Type IV).

INTRODUCTION Adverse drug reactions (ADRs) are major causes of iatrogenic, and potentially preventable, patient morbidity and mortality. These reactions are the source of approximately 3%–6% of inpatient admissions, are estimated to be the fourth most common cause of death, and comprise 5%–10% of inpatient costs that total over $4 billion annually in the United States alone.1–4 Many ADRs are not predictable based solely on the pharmacologic action of the drug, and we now know that these reactions stem from specific off-target drug activity that includes the interaction of drugs with immune receptors and pharmacologic drug effects such as those seen in nonIgE-mediated mast cell activation syndrome (Fig. 4.1). Some reactions that display a clinical phenotype consistent with an immunologically mediated reaction, such as anaphylaxis, angioedema, urticaria, maculopapular exanthema, fever, and internal organ involvement (e.g., hepatitis), are related to an adaptive immune response and are associated with immunologic memory. These encompass a number of phenotypically distinct clinical diagnoses that comprise both B cell–mediated (antibody-mediated, Gell-Coombs Types I–III) and purely T cell–mediated reactions (Gell-Coombs Type IV).

Gell-Coombs Classification of ImmuneMediated Adverse Drug Reactions According to the Gell and Coombs schema, developed in 1963, drug hypersensitivity reactions are classified

into four types based on the immune mediators of disease. This mechanism-based classification system is still widely used today. Type I, also known as immediate, hypersensitivity reactions, typically occurs within 30–60 min, but can occur within seconds, of drug exposure, depending on the mode of drug administration. On the first exposure to the implicated drug, drug-specific IgE antibodies are formed and bind to Fcε receptors on the surface of mast cells and basophils. Later exposure to the same drug results in cross-linking of cell-bound antibodies on the mast cells and basophils, leading to cell degranulation and the release of histamine, leukotriene, prostaglandin, and a variety of other immune mediators (Fig. 4.2). The principal effects of these products are vasodilation, increased vessel permeability, smooth muscle contraction, and leukocyte extravasation into tissues, resulting in clinical features ranging from irritating (pruritus) to life-threatening (anaphylaxis). Urticarial rash, flushing, angioedema, and gastrointestinal symptoms are also typical of Type I hypersensitivity reactions. Drugs commonly associated with Type I hypersensitivity reactions include antimicrobials, neuromuscular blocking agents, some NSAIDS, proton pump inhibitors, insulin, and chimeric monoclonal antibodies. These reactions are associated with immunologic memory; however, immune responses are lost over time. For instance, for patients with a history of IgE-mediated hypersensitivity to 27

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On-target ADRs

Off-target ADRs

Effect at site of pharmacological activity

Effect distant to site of pharmacological activity

Predictable based on drug action

Interaction with off-target receptor

Modifiers may include dose and PK/PD/pharmacogenomi cs (ADME genes, drug transporters, receptors)

Drug allergy (Immunological memory) Antibody mediated (Gell-Coombs Type I-III)

T-cell mediated (Gell-Coombs Type IV)

SJS/TEN Fluoroquinolone non-IgE mediated mast cell activation

Penicillin anaphylaxis

DRESS/DIHS FIG. 4.1  Classification of Adverse Drug Reactions (ADRs).  ADRs may result from either on-target

or off-target interactions between the drug and cellular proteins. On-target adverse effects, traditionally referred to as Type A reactions, account for the majority of ADRs (≥80%) and are generally predictable based on the pharmacology of the drug. Variation in the cellular processes that modulate drug absorption, distribution, metabolism, and excretion (ADME); drug transporters; target receptor expression; and drug dosing or administration errors contribute to ADRs that are primarily mediated by pharmacologic mechanisms. Although off-target adverse effects account for a smaller proportion of total ADRs (≤20%), the cost, morbidity, and mortality associated with these reactions is significant. Off-target reactions may occur by both nonimmune-mediated and immune-mediated mechanisms. For example, the non-IgE-mediated mast cell activation syndrome, which phenotypically resembles anaphylaxis, has recently been shown in a murine model to result from off-target binding of drug to G protein–coupled receptors without the involvement of the adaptive immune system.8 The immune-mediated ADRs include both B cell/antibody and T cell–mediated reactions (Type I–IV reactions according to the Gell-Coombs schema). It should be noted that all ADRs are dose-dependent although the degree to which drug concentration contributes to phenotype varies for individual reactions. (Adapted from Peter JG, Lehloenyz R, Dlamini S, Risma K, White KD, Konvinse KC, Phillips EJ. Severe delayed cutaneous and systemic reactions to drugs: a global perspective on the Science and Art of Current Practice. J Allergy Clin Immunol Prac. 2017;5(3):547–563.)

β-lactam antibiotics, as determined by clinical history and skin test reactivity, approximately 10% of patients per year will lose skin test reactivity to penicillins and other β-lactams.5,6 Lack of reexposure to the implicated drug may explain some of this phenomenon, but it has also been observed that resensitization to the drug is uncommon even in the setting of repeat exposure.7 The term “pseudoallergic” or “anaphylactoid” is used

to describe reactions that appear clinically similar to Type I hypersensitivity reactions but are not mediated by cross-linking of IgE antibodies. These reactions may be triggered by peptidergic drugs such as ciprofloxacin signaling through a specific mast cell–related G protein–coupled receptor.8 Type II hypersensitivities are IgG or IgM (non-IgE) antibody–mediated reactions, which typically occur

CHAPTER 4  Immune Mechanisms of Drug Allergy

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Drug TH0 MHC II

TCR MHC II

IL-4

B cell IL-4/IL-13

Plasma cell

TH2

Dendritic cell TCR

Fcε receptor

Cell-bound IgE Vasoactive mediators

Vascular permeability Smooth muscle contraction Leukocyte extravasation

Drug-specific IgE

IgE sensitized mast cell

IgE crosslinking and degranulation FIG. 4.2  Pathogenesis of Type I Hypersensitivity Reactions.  In Type I hypersensitivity reactions, dendritic cells bind and internalize drug antigen for presentation to TH0 (naïve) CD4+ T cells. In the presence of IL-4, the naïve T cell develops into a drug-specific TH2 cell. Drug antigen is processed by the B cell and is presented to the drug-specific TH2 cells. TH2 cells produce cytokines (IL-4, IL-13) that induce B cell differentiation into a plasma cell that secretes drug-specific IgE. Soluble IgE then binds to Fcε receptors on the surface of mast cells and basophils. A second encounter with drug induces cross-linking of cell surface– bound IgE antibodies to induce mast cell degranulation with the release of soluble mediators that cause vascular permeability, smooth muscle contraction, and leukocyte extravasation into tissues.

minutes to several hours after drug exposure. These reactions occur when IgG or IgM antibodies bind cell surface–associated drug or drug metabolite. These antibody-coated cells are recognized and killed by innate immune cells, such as natural killer (NK) cells, monocytes, and macrophages, which bind to the target cells via the Fc region of the antibody. Clinically, Type II hypersensitivities may present as drug-induced hemolytic anemia (some β-lactam antibiotics, quinidine, α-methyldopa), thrombocytopenia (quinine, acetaminophen, vancomycin, and sulfonamide antibiotics), or granulocytopenia (some anticonvulsants, pyrazolone drugs, thiouracil, sulfonamides, and phenothiazines). Type III, also known as immune complex, hypersensitivity reactions occur when an antibody (IgG > IgM) binds to a soluble antigen (often a drug or a drug metabolite), forming a circulating immune complex, which can deposit into small vessels, joints, and renal

glomeruli, resulting in serum sickness reactions. These small immune complexes induce complement fixation and unlike larger immune complexes are not cleared from the circulation by macrophages. Clinically, these reactions are characterized by fever, lymphadenopathy, urticaria, joint pain, and proteinuria. Drugs known to cause Type III hypersensitivity reactions include penicillins, cephalosporins, sulfonamides, trimethoprim– sulfamethoxazole, ciprofloxacin, tetracycline, lincomycin, NSAIDS, and carbamazepine. Type IV, also known as delayed, hypersensitivities are T cell–mediated reactions that occur days to weeks after drug initiation. The clinically relevant T cell–mediated drug reactions are collectively referred to as the drug hypersensitivity syndromes (DHSs) and have been classified into delayed exanthema without systemic symptoms (maculopapular eruption or MPE), contact dermatitis, drug-induced hypersensitivity syndrome (DIHS)/drug

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THE IMMUNOPATHOGENESIS OF T CELL– MEDIATED DRUG HYPERSENSITIVITY SYNDROME: ESTABLISHED MODELS

reaction with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), acute generalized exanthematous pustulosis (AGEP), fixed drug eruption, and single organ involvement pathologies such as drug-induced liver disease (DILI) and pancreatitis.9,10 In general, these reactions result from drug interaction with immune receptors involved in T cell immune activation, leading to aberrant CD4+ or CD8+ T cell immune responses. The rest of this chapter will focus on the immunopathogenesis of Type IV delayed hypersensitivity drug reactions with particular reference to SJS/TEN, DRESS/DIHS, and a unique, systemic T cell–mediated DHS termed the abacavir hypersensitivity syndrome (AHS). 

The role of T cells as pathogenic mediators of many DHSs has been firmly established. As described in Chapter 5, many Type IV DHSs are strongly associated with variation in the class I and class II HLA genetic loci, which encode the restricting elements for CD8+ and CD4+ T cells, respectively. Three nonmutually exclusive models describe how a small-molecule pharmaceutical might interact with immune proteins to elicit T cell reactivity. These are the hapten/prohapten model, the pharmacologic interaction (p-i) model, and the altered peptide repertoire model (Fig. 4.3).

Hapten/prohapten model

P-I model

Altered peptide repertoire model

Effector T cell

Effector T cell

Effector T cell

TCR

TCR

TCR

α β

α β

α β

HLA class I or II

HLA class I or II

HLA class I or II

Antigen presenting cell

Antigen presenting cell

Antigen presenting cell

Covalent binding of drug to peptide

Non-covalent drug peptide/HLA interaction

Non-covalent drug peptide/HLA interaction

Drug binds to peptide either in endoplasmic reticulum or at cell surface

Drug binds to peptide/HLA at cell surface

Drug binds to peptide/HLA in endoplasmic reticulum (likely)

Antigen processing required if hapten binding in ER Not dependent on antigen processing if hapten binds at cell surface

Not dependent upon antigen processing

Antigen processing required

Neo-epitope formed by drug binding to peptide

Drug binding results in formation of immunogenic complex

Drug binding results in a change in HLA peptide binding motif and selection of novel endogenous peptides

FIG. 4.3  Models of T Cell Activation by Small Molecules.  Three models have been proposed to explain

T cell stimulation by small-molecule pharmaceuticals. The hapten/prohapten model postulates that the drug binds covalently to peptide (either in the intracellular environment before peptide processing and presentation or at the cell surface) to generate a neoantigen that stimulates a T cell response. The p-i model proposes that a small molecule may bind to HLA in a noncovalent manner to directly stimulate T cells. The altered peptide model postulates that a small molecule can bind noncovalently to the MHC binding cleft to alter the specificity of peptide binding. This results in the presentation of novel peptide ligands to elicit an immune response.

CHAPTER 4  Immune Mechanisms of Drug Allergy For DHSs that adhere to the hapten/prohapten model, the drug or drug metabolite binds covalently to an endogenous protein that then undergoes intracellular processing to generate a pool of chemically modified peptides. When presented in the context of HLA proteins, these modified peptides will be recognized as “foreign” by T cells and elicit an immune response.11,12 Examples of DHS that are associated with hapten modification of endogenous proteins include the binding of penicillin derivatives to serum albumin13 and protein modification by the nitroso-sulfamethoxazole.14 Under the p-i model, the offending drug is postulated to bind noncovalently to either the T cell receptor (TCR) or HLA protein in a peptide-independent manner to directly activate T cells. This model has been hypothesized to explain in vitro T cell reactivity that is labile (i.e., reactivity is abrogated by washing drug from the surface of antigen presenting cells [APCs]) and/or is observed within seconds of drug exposure, a time course too short to require intracellular antigen processing.15,16 In DHSs that adhere to the altered peptide repertoire model, the offending drug occupies a position in the peptide-binding groove of the HLA protein, thereby changing the chemistry of the binding cleft and the peptide specificity of HLA peptide presentation. It is proposed that peptides presented in this context are recognized as “foreign” by the immune system and therefore elicit a T cell response.17,18

Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis SJS and TEN are two of the most severe immune mediated ADRs (IM-ADRs) with an estimated patient mortality rate over 30% at 1 year following the disease onset.19 Cardinal features of SJS/TEN include widespread epidermal necrosis, resembling a severe burn injury and manifesting clinically with skin, mucous membrane, and eye involvement (Fig. 4.1). SJS/TEN is considered to be a disease with a cohesive immunopathogenesis and is defined by the percentage of body surface area (BSA) involvement (SJS: 10% BSA affected; SJS/TEN overlap: 10%–30% BSA affected; TEN: >30% BSA affected). Internal organ failure and secondary complications such as infection, thrombosis, and deconditioning are frequently associated with SJS/TEN. Furthermore, the long-term sequelae of this disease, including scarring, blindness, and psychiatric illness, are a source of significant disability for survivors. Multiple studies have demonstrated strong associations between carriage of class I HLA alleles and risk of drug-induced SJS/(for full descriptions

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see Chapter 5). The best characterized examples of these associations include carriage of HLA-B*15:02 and risk of carbamazepine-induced SJS/TEN in Southeast Asian populations and carriage of HLA-B*58:01 and risk of allopurinol-induced SJS/TEN in both European and Southeast Asian populations, among others.20–25 Cytotoxic CD8+ T cells, NK cells, and CD3+CD56+ NK T cells (NKT cells) are enriched in blister fluid samples obtained from patients with acute SJS/TEN, and these cells are the primary mediators of pathogenesis (Fig. 4.4).26–29 Granulysin, a cytotoxic peptide produced by CD8+ T cells, NK cells, and NKT cells, is an important mediator of epidermal cell death in SJS/TEN. Granulysin is present in high concentrations in blister fluid. Serum levels of granulysin associate with the severity of acute SJS/TEN and predict mortality.30,31 Immunohistochemical analyses of skin biopsies taken during acute SJS/TEN show that early lesions are characterized by the infiltration of CD14+CD16+CD11c+HLA-DR+ monocytes into the epidermis and dermoepidermal junction before the onset of epidermal damage. These cells express CD137 and the costimulatory molecules CD80 and CD86 and are characteristic of monocytes poised to facilitate the proliferation of cytotoxic T cells recruited to the site of pathology.32 More recent studies applying next-generation sequencing technologies demonstrate that in some cases of HLA-B*15:02-associated carbamazepine-SJS/ TEN, the repertoire of CD8+ T cells recruited to the blister fluid are enriched for T cells bearing a common CDR3 sequence and that this dominant clonotype is shared among multiple patients with carbamazepineSJS/TEN (unpublished data). Another study, however, focusing on allopurinol-SJS/TEN using similar methods to evaluate the TCR repertoire, demonstrated some clonotypic restriction of TCR usage within individual patients but no public TCR common to multiple patients.30 

Drug Reaction With Eosinophilia and Systemic Symptoms Drug reaction with eosinophilia and systemic symptoms (DRESS) presents as a widespread rash of varying severity without skin separation or blistering and is often accompanied by fever, internal organ involvement (usually hepatitis), and hematologic abnormalities (often atypical lymphocytes and/or eosinophilia) (Fig. 4.1). Diffuse lymphadenopathy, pneumonitis, encephalitis, cardiac failure (myocarditis), and nephritis are variable features of this syndrome, which may mimic a viral illness. The onset of symptoms typically occurs 2–8 weeks following initiation of the inciting

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SJS/TEN PATHOGENESIS

EPIDERMIS

Blister fluid

Necrotic keratinocytes

Apoptotic keratinocytes CTL

CD69

CD103

NK NKT

CD103

CTL

CD69

NK Inflammation

Granulysin

Afferent lymph

DERMIS

DRESS PATHOGENESIS

Detachment of epidermis

Perforin granzyme B NKT

CTL

DC

Lymphatic infiltrate

TNFα / IFNγ

NK CTL

Efferent lymph Drug

BLOOD

DC

Proliferation

Drugspecific T-cell

Efferent lymph

Lymph node

Afferent lymph

DC

Lymph node Eosinophils

TNFα / IFNγ

Drug CD8+

CTL

PTSD Fever

Granulysin / IFNγ

Viral reactivation

CD4+

CD4+ or CD8+ T-cells

Fever

Vision loss Peptide APC

Mucosal involvement

Drug

HLA

+

TCR

CD8 T-cell

Drug Eosinophilia

Sepsis

Keratinocyte

CD4+ or CD8+ T-cell

Peptide HLA

APC

TCR

Rash IFNγ / TNFα

Granulysin Epidermal detachment

Erosive hemorrhagic lesions

Hepatitis Viral reactivation

Pain weakness

Delayed autoimmunity

FIG. 4.4  Current Models of Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN)

and Drug Reaction With Eosinophilia and Systemic Symptoms (DRESS) Pathogenesis. Widespread epidermal necrosis is a hallmark of SJS/TEN. It is likely that this process is initiated by drug interaction with HLA present on the surface of epidermal keratinocytes in an allotype-specific manner. Drug-peptide–HLA interaction generates an immunogenic epitope that is recognized by CD8+ effector T cells, NK cells, and NKT cells to stimulate a cytotoxic response. Keratinocyte death is mediated by granulysin, a cytotoxic peptide release by drug-reactive effector cells, leading to epidermal detachment and the formation of fluid filled bullae and sloughing. The dermis is the primary skin compartment involved in DRESS pathogenesis. Both effector T cells (CD8+ and CD4+) and CD4+FoxP3+ regulatory T cells contribute to disease. DRESS is also associated with reactivation of human herpesviruses although the exact role of viral reactivation in DRESS pathogenesis is currently unclear. (Adapted from Peter JG, Lehloenyz R, Dlamini S, Risma K, White KD, Konvinse KC, Phillips EJ. Severe delayed cutaneous and systemic reactions to drugs: a global perspective on the Science and Art of Current Practice. J Allergy Clin Immunol Prac. 2017;5(3):547–563.)

CHAPTER 4  Immune Mechanisms of Drug Allergy drug, and symptoms can persist for weeks. Prolonged or recurrent symptoms, sometimes weeks following the cessation of the offending drug, as well as lateonset autoimmune diseases including thyroiditis, systemic lupus, and type I diabetes, are not uncommon following resolution of acute disease.33 Numerous drugs are associated with the development of DRESS, including the antiretrovirals (abacavir, nevirapine, fosamprenavir), allopurinol, antiepileptic medications (carbamazepine, phenytoin, phenobarbital and lamotrigine), β-lactam antibiotics, NSAIDs, and sulfa antimicrobials. DRESS is associated with expansion of circulating and dermal-infiltrating effector T cells as well as CD4+FoxP3+ regulatory T cells (Tregs).34,35 Skinhoming CD4+FoxP3+ T cells are postulated to limit the severity of acute disease by suppressing effector T cell responses.36 Reactivation of human herpesviruses (HHVs), in particular HHV-6, but also Epstein-Barr virus (EBV), HHV-7, and cytomegalovirus, is universally observed during acute and recovery phase disease (Fig. 4.4). HHV-6 and EBV reactivation has been observed as early as 2–3 weeks after the onset of rash, and antiviral CD8+ effector T cells are expanded during this phase of disease. Whether viral replication contributes to the events inciting DRESS or is the result of general immune dysfunction, such as breakdown of Treg suppressor function or the upregulation of the HHV-6 receptor, CD134, on CD4+ T cells, has not been defined.35–38 Nevertheless, viral replication and a virusspecific T cell response likely contribute to the clinical features of DRESS, including prolonged duration, multiorgan involvement, and relapsing disease following withdrawal of glucocorticoid steroids. 

Drug-Specific Models: The Aromatic Amine Anticonvulsants Carbamazepine is an aromatic amine anticonvulsant used for the treatment of epilepsy, bipolar disorder, and trigeminal neuralgia. Multiple DHS are associated with carbamazepine, including SJS/TEN, MPE, and DRESS/ DIHS. Carriage of the HLA-B*15:02 allele was first associated with carbamazepine-induced SJS/TEN in Han Chinese patients (negative predictive value [NPV] approaches 100%) and later to others of Southeast Asian ethnicity.20,39 Mutagenesis studies demonstrate that carbamazepine binding to HLA-B*15:02 maps to the B pocket of the peptide-binding groove with a likely primary contact at the Arg62 residue, a conserved amino acid among HLA B75 serotypes. Additional contacts at the Asn63, Ile95, and Leu156 residues also likely participate in carbamazepine–B*15:02 interactions, as

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alteration of these residues results in reduced affinity of carbamazepine binding.40 The observation that neither drug nor antigen processing is required for T cell activation supports the p-i model of drug–immune receptor interaction.40 Recent experiments aimed at defining the CD8+ T cell repertoire in carbamazepine-SJS/TEN have employed next-generation sequencing of circulating and blister fluid–derived T cells from multiple Taiwanese patients with HLA-B*15:02-associated carbamazepine-SJS/ TEN. These studies have identified a shared CD8+ T cell clonotype, bearing a common CDR3 sequence, that is present in blood and blister fluid among patients with carbamazepine-SJS/TEN but not in the peripheral blood of drug-tolerant controls or in blister fluid from patients with SJS/TEN secondary to another causative drug (Hung and Chung, unpublished data and Chung et al.30) This is significant as it suggests for the first time the concomitant involvement of both a specific HLA allotype and a specific TCR clonotype in the pathogenesis of a serious DHS and in this case SJS/TEN. 

Drug-Specific Models: Allopurinol Allopurinol is a xanthine oxidase inhibitor used to treat hyperuricemia/gout and is associated with DHS of primarily cutaneous phenotype in approximately 2% of patients who initiate therapy. The HLA-B*58:01 genotype is associated with allopurinol-induced SJS/ TEN and DRESS/DIHS in persons of Han Chinese ancestry (100% NPV, 3% positive predictive value [PPV]), an association that has since been identified in other Asian populations.24,41,42 Oxypurinol, the active metabolite of allopurinol, is the primary agent in the pathogenesis in allopurinol-DHS. In vivo, allopurinol is rapidly metabolized to oxypurinol (T1/2 = 1–2 h for allopurinol; oxypurinol is metabolized within 15 h in the setting of normal renal function). In keeping with the necessity of noncovalent and dose-dependent interactions between oxypurinol immune receptors (HLA and/or TCR) to activate T cells, in the setting of renal insufficiency, oxypurinol accumulates and serum concentrations of oxypurinol remain elevated for considerably longer periods. As such, impaired renal function and increased plasma concentrations of oxypurinol correlate with disease severity and mortality in allopurinol-SJS/TEN/DRESS.31,43 Cell culture experiments have shown that T cells isolated from patients with allopurinol-DHS expand and acquire effector functions following stimulation with allopurinol or oxypurinol but that high concentrations of oxypurinol (up to 100 μg/mL, 10× that expected with a therapeutic dose) are the primary drivers of T

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cell reactivity.30,44 These findings are consistent with observations that allopurinol is rapidly converted to oxypurinol in vivo and that elevated serum concentrations of oxypurinol in the setting of delayed renal clearance contribute to risk of allopurinol-DHS. In vitro generation of allopurinol- and oxypurinol-reactive T cell lines was shown to be independent of intracellular metabolism or antigen processing, consistent with a p-i mechanism of drug-HLA-peptide–TCR interaction.45,46 Mutagenesis studies suggest that oxypurinol binds in the HLA-B*58:01 peptide-binding groove and that binding is likely dependent on the presence of Arg97 because mutation of this residue to valine substantially decreases T cell proliferation in the presence of oxypurinol relative to wild-type HLA-B*58:01.45 In silico modeling experiments predict that oxypurinol docks within the F-pocket of HLA-B*58:01 and that stable association requires the formation of a hydrogen bond with Arg97, consistent with site-directed mutagenesis data. These studies also suggest that allopurinol binding to HLA-B*58:01 occurs at reduced affinity than that predicted for oxypurinol, which supports the in vivo and in vitro data, showing that reactivity is mediated by oxypurinol in a dose-dependent manner.46 The mechanisms by which oxypurinol stimulates TCRs are currently under investigation. One study that examined the CD8+ T cell repertoire among blister fluid and peripheral blood cells obtained from multiple patients with allopurinol-associated SJS/TEN by bulk next-generation TCR sequencing demonstrated some degree of clonotypic restriction for some, but not all, patients and no evidence of a shared clonotype as seen for carbamazepine-associated SJS/TEN.30 Which, if any, of these clonotypes mediates pathogenesis is not known. Granulysin is a key mediator of this disease and levels of granulysin correlate with disease severity and mortality in allopurinol-SJS/TEN and DRESS.31 

Drug-Specific Models: Abacavir Hypersensitivity Syndrome Abacavir is a guanosine analog that is used as part of combination antiretroviral therapy for the treatment of HIV-1 infection. In early studies, hypersensitivity type reactions were reported in approximately 5%–8% of patients within the first 6 weeks following the initiation of abacavir. These reactions were named the abacavir hypersensitivity syndrome and were characterized by fever, malaise, gastrointestinal and respiratory symptoms, and/or generalized rash. In 2002, a strong association between carriage of the HLA class I allele HLA-B*57:01 and AHS was reported.47 Key clinical studies that confirmed the

immunologic basis of this syndrome included the application of abacavir in varying concentrations in a patch test on the skin that demonstrated consistent and visibly positive results indicative of in vivo T cell responses to abacavir in HLA-B*57:01 positive AHS patients.48 These observations were followed by the PREDICT-1 and SHAPE trials, which showed that screening for and exclusion of HLA-B*57:01 carriers from abacavir drug exposure could completely eliminate the incidence of immunologically confirmed (patch test positive) AHS with a 100% NPV and a 55% PPV.49,50 The functional basis for AHS was defined in 2012 with the simultaneous publication of two crystal structures of HLA-B*57:01 in complex with abacavir and peptide.17,18 These studies supported an altered peptide repertoire model for abacavir-specific activation of T cells. According to this model abacavir binds to the HLA-B*57:01 peptide-binding groove underneath the C-terminus of the bound peptide, and this induces a change in the binding properties of HLA-B*57:01 such that peptides with a small aliphatic C-terminal residue (Ile, Leu, Val) are preferentially selected, a repertoire that is distinct from that presented by HLA-B*57:01 in the absence of drug binding. Approximately 20%–45% of the peptides eluted from abacavir-treated HLAB*57:01 APCs were distinct from those recovered from untreated cells, illustrating the dramatic shift in the repertoire of HLA-B*57:01 bound peptide in the presence of abacavir. This work defined the altered peptide repertoire model of DHS and predicts that in the context of drug, self-peptides that are normally not “seen” by the immune system are presented to T cells and recognized as “foreign” to elicit an immune response. 

Unexplained Features of T Cell–Mediated Adverse Drug Reactions There remain many unanswered questions regarding the immunopathogenesis of T cell–mediated DHS. First, all of the T cell–mediated DHS described in this chapter are generally characterized by a high NPV for HLA association (approaching 100% in many cases) but a low PPV meaning that only a small proportion of those carrying an HLA risk allele will develop DHS. For instance, why is it that 55% of HLA-B*57:01 carriers will develop a hypersensitivity reaction in response to abacavir exposure but only 3% of HLA-B*15:02 carriers exposed to carbamazepine will develop SJS/TEN? What immunologic factors differ among those individuals who develop DHS and those who carry the risk HLA allele but tolerate the drug? Second, what might explain the variable clinical phenotypes of T cell–mediated

CHAPTER 4  Immune Mechanisms of Drug Allergy DHS? AHS is characterized predominantly by systemic symptoms and internal organ involvement and is less commonly associated with rash. In contrast, carbamazepine-associated DHS most commonly manifest as severe cutaneous and systemic reactions such as SJS/ TEN and DRESS. Carbamazepine causes the full spectrum of DHS (SJS/TEN, DRESS, AGEP, FDE, MPE, DILI) and specific phenotypes have been associated with carriage of different HLA alleles. What explains the phenotypic variation seen among these HLA-restricted DHS? Third, there exists wide variability in the timing of clinical onset of many DHS. For instance, immunologically mediated abacavir hypersensitivity, defined as patch test positive HLA-B*57:01 positive abacavir reactivity, occurs in a narrow time window of <2 days to 3 weeks from the first exposure to drug. SJS/TEN has a shorter latency period than DRESS and typically occurs 4–28  days following the first drug exposure. Many carbamazepine-associated DHS have been observed to occur over a broad and longer timeframe with the onset of symptoms often occurring later, 2–8 weeks after drug therapy. Factors playing into the variable timing of these syndromes are currently unclear. Finally, in some cases, immunologically mediated drug-specific recall reactions have been demonstrated years after drug exposure and withdrawal. For example, abacavirspecific in vivo (skin patch test) and ex vivo (ELISpot) responses remain positive years after clinical abacavir hypersensitivity in the absence of subsequent reexposure to abacavir. Similarly, long-lived T cell responses have been observed in patients with history of carbamazepine-SJS/TEN years after clinical DHS. Factors that lead to the maintenance of this long-lasting memory T cell response and the specific antigen leading to the maintenance of this response are similarly unknown. Ongoing work to define the pathogenesis of immune-mediated ADRs might shed light on these outstanding questions. One focus of current research seeks to evaluate the role of preexisting memory T cell responses in the pathogenesis of DHS. It has been shown that T cells isolated from abacavir-naïve, HLAB*57:01 positive individuals proliferate and become activated in response to abacavir exposure in 14-day cell culture systems and that abacavir-reactive T cells derive from both memory and naïve T cell populations.51 These findings raise the possibility that a proportion of drug-reactive T cells may stem from memory populations that were generated before drug exposure against an unrelated antigen, such as an infectious pathogen. If this hypothesis is correct, then the requirement for preexisting memory T cells may, at least in part,

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explain the low PPV for HLA allele carriage as a sole predictor of DHS risk, the rapid onset of symptoms following drug exposure in some DHS, the variable phenotypes seen among different HLA- and drug-specific DHS, and the long-lasting immunologic memory after drug withdrawal. Other avenues of research that may define components of the immunopathogenesis of T cell–mediated DHS include investigations into how variation in genes encoding proteins that are involved in drug metabolism and/or intracellular peptide processing and presentation, such as the cytochrome P450 (CYP) enzymes and/or endoplasmic reticulum aminopeptidase variants, might contribute to DHS risk in the setting of HLA risk allele carriage.52–54 

FUTURE RESEARCH AND IMPLICATIONS FOR CLINICAL PRACTICE Identification of patients who are at risk for an ADR via pharmacogenomics screening before drug administration is desirable given the substantial cost, morbidity, and mortality associated with these reactions. Methods to incorporate such screening strategies into clinical practice have been successfully implemented for a small number of well-characterized T cell–mediated DHS. For example, it is now a standard-of-care practice to test patients for carriage of the HLA-B*57:01 gene before the initiation of abacavir and to exclude any patient who carries this allele from receiving abacavir. This practice has eliminated AHS in populations where screening is routinely performed. From a drug safety standpoint, the 100% NPV for lack of reaction in the absence of allele carriage in the target population, the low number needed to test to prevent one case, and the paucity of safe and efficacious therapeutic alternatives are critical for incorporation of these screening strategies into clinical care. However, given the low PPVs for the HLA-associated ADRs defined to date, screening strategies that focus solely on HLA genotyping will inevitably result in the denial of therapy to a large number of carriers of HLA risk alleles who would ultimately tolerate the drug in question without complication and benefit from its use. Further delineation of the factors that contribute to DHS risk, in addition to HLA genotype, will be critical to defining disease pathogenesis and potentially allow us to refine our screening protocols to more precisely identify those patients who are truly at risk for ADR. This will, in turn, improve drug safety, improve the efficiency of drug design, and reduce the cost of drug development.

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ABBREVIATIONS ADR  Adverse drug reaction AGEP  Acute generalized exanthematous pustulosis AHS  Abacavir hypersensitivity syndrome APC  Antigen presenting cell BSA  Body surface area CDR  Complementarity-determining regions DHS  Drug hypersensitivity syndrome DIHS  Drug-induced hypersensitivity syndrome DILI  Drug-induced liver injury DRESS  Drug reaction with eosinophilia and systemic symptoms EBV  Epstein-Barr virus HLA  Human leukocyte antigen HSV  Herpes simplex virus MPE  Maculopapular eruption NPV  Negative predictive value PK/PD  Pharmacokinetic/pharmacodynamic NSAID  Nonsteroidal anti-inflammatory drug PPV  Positive predictive value SJS  Stevens-Johnson syndrome TCR  T cell receptor TEN  Toxic epidermal necrolysis

ACKNOWLEDGMENTS National Institute of Health:1P50GM115305-01 (EJP, KK, KDK), 1R01AI103348-01 (EJP), 1P30AI11052701A1 (EJP), 5T32AI007474-20 (EJP), 1 R13AR7126701(EJP), National Health & Medical Research Council of Australia (EJP,AR), Australian Centre for HIV and Hepatitis Virology Research (EJP).

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