Adverse drug reactions (ADRs) are a major obstacle to drug development, and some of these, including hypersensitivity reactions to the HIV reverse transcriptase inhibitor abacavir (ABC), are associated with HLA alleles, particularly HLA-B*57:01. However, not all HLA-B*57:01+ patients develop ADRs, suggesting that in addition to the HLA genetic risk, other factors may influence the outcome of the response to the drug. To study HLA-linked ADRs in vivo, we generated HLA-B*57:01-Tg mice and show that, although ABC activated Tg mouse CD8+ T cells in vitro in a HLA-B*57:01-dependent manner, the drug was tolerated in vivo. In immunocompetent Tg animals, ABC induced CD8+ T cells with an anergy-like phenotype that did not lead to ADRs. In contrast, in vivo depletion of CD4+ T cells prior to ABC administration enhanced DC maturation to induce systemic ABC-reactive CD8+ T cells with an effector-like and skin-homing phenotype along with CD8+ infiltration and inflammation in drug-sensitized skin. B7 costimulatory molecule blockade prevented CD8+ T cell activation. These Tg mice provide a model for ABC tolerance and for the generation of HLA-B*57:01-restricted, ABC-reactive CD8+ T cells dependent on both HLA genetic risk and immunoregulatory host factors.

Glutamine is the preferred AA used by polymorphonuclear leukocytes (PMN) during the inflammatory response. However, the effect of other AA on bovine PMN response during inflammation and how this is altered by stage of lactation has not been fully elucidated. The objective of this study was to determine the effect of additional AA supplementation (pool of AA excluding Gln) on AA profiles, gene expression, and inflammatory function of PMN from dairy cows in early and mid lactation in vitro. We used 18 Holstein cows for this study. Polymorphonuclear leukocytes were isolated. Working solutions of AA (0 or 4 mM) and LPS (0 or 50μg/mL) were added to cell populations suspended in RPMI and incubated for 2h at 37°C. We used a subset of samples for gene and protein expression. Concentrations of AA in medium were determined using gas chromatography-mass spectrometry with norleucine as an internal standard. Apparent AA and glucose utilization were calculated by subtracting the concentration after from that of before incubation. Data were analyzed as a randomized block design. Challenge with LPS increased the expression of proinflammatory genes and AA supplementation decreased both the expression of some proinflammatory genes and the media concentrations of tumor necrosis factor-α. Neither stage of lactation, LPS challenge, nor AA supplementation altered the chemotactic or phagocytic abilities of PMN in vitro. Polymorphonuclear leukocytes supplemented with AA had greater concentrations and apparent utilization of most of the supplemented AA, whereas the unsupplemented group had greater apparent utilization of glucose. Alanine was not provided in the media but was present in spent media, and Ile, Gly, and Pro were greater in spent media than in media before incubation indicating synthesis of these AA. Regarding expression of genes involved in nutrient metabolism, the expression of G6PD, coding for the enzyme glucose 6-phosphate dehydrogenase, was increased and that of PDHA1, coding for the enzyme pyruvate dehydrogenase α 1, tended to increase with AA supplementation. Due to the lower concentration of tumor necrosis factor-α in media coupled with a downregulation of several proinflammatory genes, we concluded that AA, rather than Gln, alter the inflammatory response of bovine blood PMN. Independent from Gln, blood PMN from cows in early lactation may use certain AA as their primary carbon source for energy than cows in later lactation. Evaluating cows during the early postpartum period will provide additional information on the effect of stage of lactation and nutrient supplementation on PMN function.

Adverse drug reactions (ADR) are a major obstacle to drug development. Genome-wide association studies identified several human leukocyte antigens (HLA)-class I alleles as risk factors for ADR. The HLA-B*57:01 allele has been found to be associated with the development of abacavir (ABC) hypersensitivity syndrome (AHS) and with the induction of liver injury by the β-lactam antibiotic flucloxacillin. The nucleoside analog ABC, an inhibitor of the HIV reverse transcriptase, can induce severe multi-organ toxicity in >50% of HLA-B*57:01+ patients with HIV infection. In a previous study we showed that ABC induced binding to the HLA-B*57:01 of altered self-peptides containing predominantly isoleucine or leucine residues at the carboxyl terminus. Recognition of these self-peptides drives in vitro activation of cytotoxic CD8+ T cells. However, the early immune events/danger signals required to overcome the immune tolerance that otherwise suppress ADR are still unknown. In order to study HLA-linked drug hypersensitivity in vivo and to understand better drug immune tolerance, HLA-B transgenic mice were generated. Here, we show that ABC activates HLA-B*57:01 transgenic CD8+ T cells in vitro via CD8 and HLA. A systemic, but partial, early response to the drug is also observed in vivo, however, ABC fails to promote skin hypersensitivity in immunocompetent transgenic mice. Instead, depletion of CD4+ T cells breaks immune tolerance to ABC and promotes ADR in HLA-B*57:01 transgenic mice. Supported by the Intramural Research Program of the NIAID, NIH, and CDER, FDA.