Andrew Sullivan

Are drug metabolites able to cause T-cell-mediated hypersensitivity reactions?

Introduction: Adverse drug reactions with an immune pathogenesis are a problem in the clinic and an impediment to drug development. T lymphocytes are believed to play a role in the pathogenesis; however, the nature of the drug interaction with immune receptors remains an area of debate. Areas covered: This article reviews recent advances in our understanding of drug hypersensitivity focusing specifically on the way in which drugs are displayed in MHC molecules. Most drugs associated with a high incidence of reactions have been shown to form protein-reactive metabolites. Hence, the relationship between drug metabolism and T-cell activation is discussed in detail. Expert opinion: The role of metabolism in pathogenesis of immunological drug reactions has only been studied with a small number of drugs where synthetic metabolites are available for functional studies. In each case, metabolite-responsive T cells have been detected. However, the field is skewed by the fact that most research is conducted using the parent compound in metabolically inert cell systems. We propose that research efforts are directed towards the synthesis of drug metabolites and/or drug–protein conjugates. Furthermore, analytical methods need to be developed to relate metabolite exposure to the T-cell response. For now, our understanding of the chemical basis of drug hypersensitivity is incomplete.

Activation of carbamazepine-responsive T-cell clones with metabolically inert halogenated derivatives

mimicking an autoinflammatory syndrome until the development of overt sepsis and septic arthritis in the context of more profound pharmacological immunosuppression. In summary, this is the first description of an almost complete loss of the long splicing variant of CD33, containing the ligandbinding region, on peripheral leukocytes of a young female patient. Despite a negative cellular feedback role of this molecule, the autoinflammatory phenotype of the disease was not caused by this deficiency but rather by an occult bacterial infection. It is, however, conceivable that the defect resulted in an overshooting inflammatory response to bacterial stimuli. Maria L. Balmer, MD, PhD Beat Tr€ ueb, PhD Lei Zhuang, PhD Emma Slack, PhD Helmut Beltraminelli, MD Peter Matthias Villiger, MD

β-Lactam hypersensitivity involves expansion of circulating and skin-resident TH22 cells

&NA; Figure. No caption available. Background: &bgr;‐Lactam hypersensitivity has been classified according to the phenotype and function of drug‐specific T cells. However, new T‐cell subsets have not been considered. Objective: The objective of this study was to use piperacillin as a model of &bgr;‐lactam hypersensitivity to study the nature of the drug‐specific T‐cell response induced in the blood and skin of hypersensitive patients and healthy volunteers. Methods: Drug‐specific T cells were cloned from blood and inflamed skin, and cellular phenotype and function were explored. Naive T cells from healthy volunteers were primed to piperacillin, cloned, and subjected to the similar analyses. Results: PBMC and T‐cell clones (n = 570, 84% CD4+) from blood of piperacillin‐hypersensitive patients proliferated and secreted TH1/TH2 cytokines alongside IL‐22 after drug stimulation. IL‐17A secretion was not detected. Drug‐specific clones from inflamed skin (n = 96, 83% CD4+) secreted a similar profile of cytokines but displayed greater cytolytic activity, secreting perforin, granzyme B, and Fas ligand when activated. Blood‐ and skin‐derived clones expressed high levels of skin‐homing chemokine receptors and migrated in the presence of the ligands CCL17 and CCL27. Piperacillin‐primed naive T cells from healthy volunteers also secreted IFN‐&ggr;, IL‐13, IL‐22, and cytolytic molecules. Aryl hydrocarbon receptor blockade prevented differentiation of the naive T cells into antigen‐specific IL‐22–secreting cells. Conclusion: Together, our results reveal that circulating and skin‐resident, antigen‐specific, IL‐22–secreting T cells are detectable in patients with &bgr;‐lactam hypersensitivity. Furthermore, differentiation of naive T cells into antigen‐specific TH22 cells is dependent on aryl hydrocarbon receptor signaling.

Detection of drug‐responsive B lymphocytes and antidrug IgG in patients with β‐lactam hypersensitivity

Delayed‐type β‐lactam hypersensitivity develops in subset of patients. The cellular immunological processes that underlie the drug‐specific response have been described; however, little is known about involvement of the humoral immune system. Thus, the aim of this study was to utilize piperacillin hypersensitivity as an exemplar to (i) develop cell culture methods for the detection of drug‐specific B‐cell responses, (ii) characterize drug‐specific IgG subtypes and (iii) assess reactivity of IgG antibodies against proteins modified to different levels with piperacillin haptens.

Implications of HLA-allele associations for the study of type IV drug hypersensitivity reactions

ABSTRACT Introduction: Type IV drug hypersensitivity remains an important clinical problem and an obstacle to the development of new drugs. Several forms of drug hypersensitivity are associated with expression of specific HLA alleles. Furthermore, drug-specific T-lymphocytes have been isolated from patients with reactions. Despite this, controversy remains as to how drugs interact with immune receptors to stimulate a T-cell response. Areas covered: This article reviews the pathways of T-cell activation by drugs and how the ever increasing number of associations between expression of HLA alleles and susceptibility to hypersensitivity is impacting on our research effort to understanding this form of iatrogenic disease. Expert opinion: For a drug to activate a T-cell, a complex is formed between HLA molecules, an HLA binding peptide, the drug and the T-cell receptor. T-cell responses can involve drugs and stable or reactive metabolites bound covalently or non-covalently to any component of this complex. Recent research has linked the HLA associations to the disease through the characterization of drug-specific T-cell responses restricted to specific alleles. However, there is now a need to identify the additional genetic or environment factors that determine susceptibility and use our increased knowledge to develop predictive immunogenicity tests that offer benefit to Pharma developing new drugs.

Negative regulation by Programmed Death Ligand-1 during drug-specific priming of T-cells and the influence of Programmed Death-1 on effector T-cell function

PD-1 (Programmed Death-1) has been classified as a marker of T-cell exhaustion, however, several recent studies suggest that most PD-1 high T-cells are highly proliferative effector memory cells that maintain effector function during chronic infection. Furthermore, activation of PD-1 on T-cells is thought to inhibit antigen-specific T-cell priming and regulate T-cell differentiation. Thus, we sought to measure the drug-specific activation of naive T-cells after perturbation of PD-L1/2/PD-1 binding and investigate whether PD-1 signalling influences the differentiation of T-cells. Naive T-cells were cocultured with monocyte-derived dendritic cells in the presence of a drug (flucloxacillin, nitroso sulfamethoxazole) for a period of 8 days (±PD-L1/2 block), to expand the number of drug-responsive T-cells. The T-cells were then incubated with fresh dendritic cells and the drug. The antigen responsiveness was analyzed using readouts for proliferation, cytokine secretion, and cell phenotype. Cell phenotype was characterised by flow cytometry. T-cell clones were generated following priming and from drug hypersensitive patients to characterize the cytokine signature(s) of antigen specific T-cells and to study whether PD-1 expression/signalling governs the differentiation of T-cells into effector/helper subsets. Priming of naive CD4+ and CD8+ T-cells against drug antigens was found to be more effective when PD-L1 signaling was blocked. Upon restimulation, T-cells proliferated more vigorously and secreted increased levels of IFN-γ, IL-13 and IL-22, but not IL-17. Naive T-cells expressed low levels of PD-1; however, CFSE analysis revealed a transient increase during priming. Drug-specific T-cell clones generated through priming and from hypersensitive patients were found to secrete IFN-γ, IL-5 and IL-13. More detailed analysis revealed two different cytokine signatures. Clones secreted either FasL/IL-22 or granzyme B. The FasL/IL-22 secreting clones expressed the skin homing receptors CCR4, CCR10 and CLA and migrated in response to CCL17/CCL27. PD-1 was stably expressed at different levels on clones; however, PD-1 expression did not correlate with the strength of the antigen-specific proliferative response or the secretion of cytokines/cytolytic molecules. This study shows that PD-L1/PD-1 binding negatively regulates the priming of drug-specific T-cells. ELIspot analysis uncovered an antigen-specific FasL/IL-22 secreting T-cell subset with skin homing properties.

Detection of interleukin-22 secreting T-cells in piperacillin hypersensitive patients with cystic fibrosis

Delayed-type cutaneous drug hypersensitivity is a T-cell mediated disease; the spectrum of conditions has been classified according to the phenotype and function of drug antigen-specific T-cells sub-sets. Importantly, this classification does not take into account tissue-specific cytokines such as interleukin-17 and interleukin-22. Both of these cytokines have been shown to play a role in numerous physiological and pathological processes. For example, elevated levels have been detected in patients with psoriasis and allergic contact dermatitis. To investigate this, we have focused on piperacillin, a β-lactam antibiotic used for the treatment of pulmonary exacerbations in patients with cystic fibrosis. Unfortunately hypersensitivity reactions (mean onset = 9.1 days; symptoms include maculopapular rash, fever and/or flu-like symptoms) to this drug develop in 26-50% of treated patients. Piperacillin-specific T-cell clones were generated from 3 lymphocyte transformation test positive hypersensitive patients and 4 healthy donors, following priming of naive T-cells against piperacillin-exposed dendritic cells, to characterize T-cell phenotype (CD and chemokine receptor expression) and function (proliferation and secretion of cytokines/cytolytic molecules [IFNγ, 13, 17, 22 by ELIspot]). Seventy six CD4+ and CD8+ clones were isolated from the hypersensitive patients and shown to proliferate in the presence of piperacillin. Drug stimulation was associated with the secretion of IFN-γ and IL13 from all clones. IL-22 secretion was detected from 64% and 75% of the CD4+ and CD8+ clones, respectively. In contrast, IL17 was not detected. Naive T-cells co-cultured with piperacillin and autologous dendritic cells showed concentration-dependent proliferation and cytokine (IFNγ, IL13 and IL22) secretion. CD4+ and CD8+ clones generated from the primed T-cells also secreted IFNγ, IL13 and IL22 (CD4+ 50%, CD8+ 67%) following piperacillin stimulation. CCR1, CCR4 and CCR10 were expressed on all clones. Other chemokine receptors expressed on a limited number of clones included CXCR3, CXCR6, CCR2 and CCR9. In conclusion, these data show the involvement of IL22 secreting T-cells in the pathogenesis of piperacillin hypersensitivity reactions in patients with cystic fibrosis.

Characterization of anti-drug antibodies and drug-responsive B-lymphocytes in piperacillin hypersensitive patients with cystic fibrosis

Cystic fibrosis is the most common autosomal recessive condition in Caucasians and recurrent infections lead to a plethora of complications. Repeated courses of high dose intravenous β-lactam antibiotics, such as piperacillin, are employed for the treatment of respiratory exacerbations. Unfortunately, delayed-type hypersensitivity reactions develop in between 26-50% of treated patients. We have recently described the cellular immunological processes that underlie drug-specific response in hypersensitive patients; however, the involvement of the humoral immune system has not been studied. The aim of this study was to quantify piperacillin-specific antibodies in plasma of hypersensitive and tolerant patients and investigate whether B-cells can be stimulated to secrete antibodies in vitro following drug stimulation. Drug-specific antibodies were quantified by ELISA using piperacillin-modified BSA as an antigen. Adducts generated using different drug-protein ratios were used to measure the degree of conjugation that elicits an antibody response. BSA was modified with different β-lactam antibiotics to define structural specificity. Specificity for the piperacillin BSA adduct was confirmed by hapten inhibition. Mass spectrometry was used to characterize the Lys residues modified with piperacillin. B-cells isolated from PBMC were cultured with piperacillin for 5 days and IgG secretion and B-cell activation was measured using ELISpot and flow cytometry (CD19, CD27), respectively. A significantly higher level of anti-piperacillin IgG antibody was detected in plasma of hypersensitive patients when hypersensitive and tolerant patients were compared. Hapten inhibition ELISA confirmed specificity for the piperacillin antigenic determinant. Antibody binding was detected with adducts generated at piperacillin:BSA ratios between 1:1 and 100:1. In contrast, antibody binding was not detectable with penicillin G, amoxicillin or aztreonam BSA conjugates. IgG antibody secretion was also detected from purified B-cells from hypersensitive patients cultured with soluble piperacillin. Drug treatment was associated with increased expression of the B-cell activation marker CD27. These data begin to describe the drug-specific humoral immune response in piperacillin hypersensitive patients with cystic fibrosis. Further work is needed to define the role different antibody subtypes play in the disease pathogenesis.