Klara Martinsson

In silico analysis of HLA associations with drug-induced liver injury: use of a HLA-genotyped DNA archive from healthy volunteers

BackgroundDrug-induced liver injury (DILI) is one of the most common adverse reactions leading to product withdrawal post-marketing. Recently, genome-wide association studies have identified a number of human leukocyte antigen (HLA) alleles associated with DILI; however, the cellular and chemical mechanisms are not fully understood.MethodsTo study these mechanisms, we established an HLA-typed cell archive from 400 healthy volunteers. In addition, we utilized HLA genotype data from more than four million individuals from publicly accessible repositories such as the Allele Frequency Net Database, Major Histocompatibility Complex Database and Immune Epitope Database to study the HLA alleles associated with DILI. We utilized novel in silico strategies to examine HLA haplotype relationships among the alleles associated with DILI by using bioinformatics tools such as NetMHCpan, PyPop, GraphViz, PHYLIP and TreeView.ResultsWe demonstrated that many of the alleles that have been associated with liver injury induced by structurally diverse drugs (flucloxacillin, co-amoxiclav, ximelagatran, lapatinib, lumiracoxib) reside on common HLA haplotypes, which were present in populations of diverse ethnicity.ConclusionsOur bioinformatic analysis indicates that there may be a connection between the different HLA alleles associated with DILI caused by therapeutically and structurally different drugs, possibly through peptide binding of one of the HLA alleles that defines the causal haplotype. Further functional work, together with next-generation sequencing techniques, will be needed to define the causal alleles associated with DILI.

The Development of In Vitro Culture Methods to Characterize Primary T-Cell Responses to Drugs

Adverse drug reactions represent a major stumbling block to drug development and those with an immune etiology are the most difficult to predict. We have developed an in vitro T-cell priming culture method using peripheral blood from healthy volunteers to assess the allergenic potential of drugs. The drug metabolite nitroso sulfamethoxazole (SMX-NO) was used as a model drug allergen to establish optimum assay conditions. Naive T cells were cocultured with monocyte-derived dendritic cells at a ratio of 25:1 in the presence of the drug for a period of 8 days, to expand the number of drug-responsive T cells. The T cells were then incubated with fresh dendritic cells, and drug and their antigen responsiveness analyzed using readouts for proliferation, cytokine secretion, and cell phenotype. All five volunteers showed dose-dependent proliferation as measured by 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester content and by (3)H-thymidine uptake. CD4 T cells that had divided in the presence of SMX-NO had changed from a naive phenotype (CD45RA+) to a memory phenotype (CD45RO+). These memory T cells expressed the chemokine receptors CCR2, CCR4, and CXCR3 suggesting a mixture of T(H)1 and T(H)2 cells in the responding population, with a propensity for homing to the skin. Drug stimulation was also associated with the secretion of a mixture of T(H)1 cytokines (interferon γ) and T(H)2 cytokines (interleukin [IL]-5 and IL-13) as detected by ELISpot. We are currently developing this approach to investigate the allergenic potential of other drugs, including those where an association between specific human leucocyte antigen alleles and susceptibility to an immunological reaction has been established.

T-cells from HLA-B*57:01+ human subjects are activated with abacavir through two independent pathways and induce cell death by multiple mechanisms.

Susceptibility to abacavir hypersensitivity has been attributed to possession of the specific human leukocyte antigen allele HLA-B*57:01. HLA-B*57:01-restricted activation of CD8+ T-cells provides a link between the genetic association and the iatrogenic disease. The objectives of this study were to characterize the functionality of drug-responsive CD8+ T-cell clones generated from HLA-B*57:01+ drug-naive subjects and to explore the relationship between abacavir accumulation in antigen presenting cells and the T-cell response. Seventy-four CD8+ clones expressing different Vβ receptors were shown to proliferate and kill target cells via different mechanisms when exposed to abacavir. Certain clones were activated with abacavir in the absence of antigen presenting cells. Analysis of the remaining clones revealed two pathways of drug-dependent T-cell activation. Overnight incubation of antigen presenting cells with abacavir, followed by repeated washing to remove soluble drug, activated approximately 50% of the clones, and the response was blocked by glutaraldehyde fixation. In contrast, a 1 h antigen presenting cell pulse did not activate any of the clones. Accumulation of abacavir in antigen presenting cells was rapid (less than 1 h), and the intracellular concentrations were maintained for 16 h. However, intracellular abacavir was not detectable by mass spectrometry after pulsing. These data suggest that T-cells can be activated by abacavir through a direct interaction with surface and intracellular major histocompatibility complex (MHC) molecules. With the former, abacavir seemingly participates in the MHC T-cell receptor binding interaction. In contrast, the latter pathway likely involves MHC binding peptides displayed as a consequence of abacavir exposure, but not abacavir itself.

The role of Fc-receptors in murine mercury-induced systemic autoimmunity

Inorganic mercury (Hg) in genetically susceptible mouse strains induces a T cell‐dependent, systemic autoimmune condition (HgIA) characterized by immunostimulation, anti‐nuclear antibodies (ANA) and systemic immune‐complex (IC) deposits. The exact phenotypic expression of HgIA in different strains depends on H‐2 and non‐H‐2 genes. Fc receptors (FcRs) are important in the development of many autoimmune diseases. In this study, the effect of targeted mutations for activating and inhibiting FcRs in the BALB/c model of HgIA was examined. Hg‐treated BALB/c mice without mutation (wild‐type, wt) showed heavy IC deposits in the renal glomerular mesangium, as well as in renal and splenic vessel walls. The renal mesangial IC deposits were severely reduced in Hg‐treated BALB/c mice without the γ‐chain (lack of the activating receptors FcγRI, FcγRIII and Fc∈RI), but unchanged in mice lacking the inhibitory FcγRIIB. The Hg‐induced vessel wall IC deposits present in wt mice were abolished and reduced in the FcRγ and FcγRIIB strains, respectively. Hg‐treated BALB/c wt mice and mice without the γ‐chain showed an increase in serum IgE, while the increase in IgG1 was attenuated in the latter strain. In contrast, absence of the inhibiting FcγRIIB augmented the Hg‐induced increase of both serum IgG1 and IgE. In conclusion, FcRs are important mainly for the induction of systmeic IC deposits in the HgIA model, but also affects serum IgG1 and IgE levels.

The effect of activating and inhibiting Fc-receptors on murine mercury-induced autoimmunity

Fc-receptors for IgG (FcgammaR) link cellular and humoral immune responses, controlling the balance between activating and inhibitory immune responses, and are involved in autoimmune diseases. Mercury (Hg) induces an autoimmune condition in genetically (H-2(s,q,f)) susceptible mice characterized by lymphoproliferation, hypergammaglobulinemia and IgG antinucleolar antibodies (ANoA). Here we investigate the role of activating (FcgammaRI, FcgammaRIII) and inhibitory (FcgammaRIIb) Fc-receptors on mercury-induced autoimmunity (HgIA) using DBA/1 mice (H-2(q)) with targeted FcgammaR mutations and wild type (wt) mice. Mice deficient for the FcRgamma-chain or FcgammaRIII and treated with 15 mg/L HgCl(2) showed a delayed and attenuated IgG1, IgG2a and IgG2b ANoA response compared to wt mice. Female Hg-treated FcgammaRIIB(-/-) mice showed a significant increased of IgG2b ANoA development compared to wt mice. The total serum IgG1 response due to Hg was attenuated in FcRgamma(-/-) and FcgammaRIII(-/-) mice compared to wt mice. Hg-treated FcgammaRIIB(-/-) mice showed an increase of both serum IgG1 and IgE compared to wt mice. We conclude that FcgammaRIII is of importance for the rapidity and final strength of the ANoA response and the increase in serum IgG1 in HgIA, while lack of FcgammaRIIb increases the IgG2b ANoA response and the serum IgG1 and IgE response.

Deficiency of Activating Fcγ-Receptors Reduces Hepatic Clearance and Deposition of IC and Increases CIC Levels in Mercury-Induced Autoimmunity

Background Inorganic mercury (Hg) induces a T-cell dependent, systemic autoimmune condition (HgIA) where activating Fcγ-receptors (FcγRs) are important for the induction. In this study we examined the influence of activating FcγRs on circulating levels and organ localization of immune complexes (IC) in HgIA. Methods and Principal Findings Mercury treated BALB/c wt mice showed a significant but modest increase of circulating IC (CIC) from day 12 until day 18 and day 35 for IgG2a- and IgG1- CIC, respectively. Mercury-treated mice lacking the trans-membrane γ-chain of activating FcγRs (FcRγ−/−) had significantly higher CIC levels of both IgG1-CIC and IgG2a-CIC than wt mice during the treatment course. The hepatic uptake of preformed CIC was significantly more efficient in wt mice compared to FcγR−/− mice, but also development of extrahepatic tissue IC deposits was delayed in FcRγ−/− mice. After 35 days of Hg treatment the proportion of immune deposits, as well as the amounts was significantly reduced in vessel FcRγ−/− mice compared to wt mice. Conclusions We conclude that mice lacking functional activating FcγRs respond to Hg with increased levels and altered quality of CIC compared with wt mice. Lack of functional activating FcγRs delayed the elimination of CIC, but also significantly reduced extrahepatic tissue localization of CIC.

A Triple-Transgenic Immunotolerant Mouse Model

Avoiding unwanted immunogenicity is of key importance in the development of therapeutic drug proteins. Animal models are of less predictive value because most of the drug proteins are recognized as foreign proteins. However, different methods have been developed to obtain immunotolerant animal models. So far, the immunotolerant animal models have been developed to assess one protein at a time and are not suitable for the assessment of combination products. Our aim was to develop an animal model for evaluating the impact of manufacturing and formulation changes on immunogenicity, suitable for both single protein and combination products. We constructed two lines of transgenic mice expressing the three human coagulation factors, II, VII, and X, by inserting a single vector containing the three coagulation factors encoding sequences separated by insulator sequences derived from the chicken beta-globin locus into the mouse genome. Immunization of transgenic mice from the two lines and their wild-type littermates showed that transgenic mice from both lines were immunotolerant to the expressed human coagulation factors. We conclude that transgenic mice immunotolerant to multiple proteins can be obtained, and that these mice are potentially useful as animal models in the assessment of immunogenicity in response to manufacturing changes.

HLA-DR7 and HLA-DQ2: Transgenic mouse strains tested as a model system for ximelagatran hepatotoxicity

The oral thrombin inhibitor ximelagatran was withdrawn in the late clinical trial phase because it adversely affected the liver. In approximately 8% of treated patients, drug-induced liver injury (DILI) was expressed as transient alanine transaminase (ALT) elevations. No evidence of DILI had been revealed in the pre-clinical in vivo studies. A whole genome scan study performed on the clinical study material identified a strong genetic association between the major histocompatibility complex alleles for human leucocyte antigens (HLA) (HLA-DR7 and HLA-DQ2) and elevated ALT levels in treated patients. An immune-mediated pathogenesis was suggested. Here, we evaluated whether HLA transgenic mice models could be used to investigate whether the expression of relevant HLA molecules was enough to reproduce the DILI effects in humans. In silico modelling performed in this study revealed association of both ximelagatran (pro-drug) and melagatran (active drug) to the antigen-presenting groove of the homology modelled HLA-DR7 molecule suggesting “altered repertoire” as a key initiating event driving development of DILI in humans. Transgenic mouse strains (tgms) expressing HLA of serotype HLA-DR7 (HLA-DRB1*0701, -DRA*0102), and HLA-DQ2 (HLA-DQB1*0202,–DQA1*0201) were created. These two lines were crossed with a human (h)CD4 transgenic line, generating the two tgms DR7xhCD4 and DQ2xhCD4. To investigate whether the DILI effects observed in humans could be reproduced in tgms, the mice were treated for 28 days with ximelagatran. Results revealed no signs of DILI when biomarkers for liver toxicity were measured and histopathology was evaluated. In the ximelagatran case, presence of relevant HLA-expression in a pre-clinical model did not fulfil the prerequisite for reproducing DILI observed in patients. Nonetheless, for the first time an HLA-transgenic mouse model has been investigated for use in HLA-associated DILI induced by a low molecular weight compound. This study shows that mimicking of genetic susceptibility, expressed as DILI-associated HLA-types in mice, is not sufficient for reproducing the complex pathogenesis leading to DILI in man.

Immunoglobulin (Ig)G1 and IgG4 anti-cyclic citrullinated peptide (CCP) associate with shared epitope, whereas IgG2 anti-CCP associates with smoking in patients with recent-onset rheumatoid arthritis (the Swedish TIRA project).

Given the possible importance of anti‐citrullinated peptide/protein antibodies (ACPA) for initiation and progression of rheumatoid arthritis (RA), extended knowledge about the different isotypes and subclasses is important. In the present study, we analysed the immunoglobulin (Ig)G subclasses regarding reactivity against cyclic citrullinated peptides (anti‐CCP) among 504 clinically well‐characterized patients with recent‐onset RA in relation to smoking habits, shared epitope (SE) status and IgA and pan‐IgG anti‐CCP antibodies. All patients, regardless of pan‐IgG anti‐CCP status, were analysed for IgG1–4 CCP reactivity. Sixty‐nine per cent were positive in any IgG anti‐CCP subclass, and of these 67% tested positive regarding IgG1, 35% IgG2, 32% IgG3, and 59% IgG4 anti‐CCP. Among ever‐smokers the percentages of IgG2 anti‐CCP (P = 0·01) and IgA anti‐CCP (P = 0·002)‐positive cases were significantly higher compared to never‐smokers. A positive IgG anti‐CCP subclass ‐negative cases. Combining SE and smoking data revealed that IgG1 and IgG4 anti‐CCP were the IgG anti‐CCP isotypes associated with expression of SE, although the lower number of patients positive for IgG2 or IgG3 anti‐CCP could, however, have influenced the results. High levels of IgG2 anti‐CCP were shown to correlate with expression of the ‘non‐SE’ allele human leucocyte antigen (HLA)‐DRB1*15. In conclusion, in this study we describe different risk factor characteristics across the IgG anti‐CCP subclasses, where IgG2 appears similar to IgA anti‐CCP regarding the predominant association with smoking, while IgG1 and IgG4 related more distinctly to the carriage of SE genes.