Qualitative, rather than quantitative, differences between HLA-DQ alleles affect HLA-DQ immunogenicity in organ transplantation.

Prolonging the lifespan of transplanted organs is critical to combat the shortage of this life-saving resource. Chronic rejection, with irreversible demise of the allograft, is often caused by the development of donor-specific HLA antibodies. Currently, enumerating molecular (amino acid) mismatches between recipient and donor is promoted to identify patients at higher risk of developing HLA antibodies, for use in organ allocation, and immunosuppression-minimization strategies. We have counseled against the incorporation of such approaches into clinical use and hypothesized that not all molecular mismatches equally contribute to generation of donor-specific immune responses. Herein, we document statistical shortcomings in previous study design: for example, use of individuals who lack the ability to generate donor-specific-antibodies (HLA identical) as part of the negative cohort. We provide experimental evidence, using CRISPR-Cas9-edited cells, to rebut the claim that the HLAMatchmaker eplets represent "functional epitopes." We further used unique sub-cohorts of patients, those receiving an allograft with two HLA-DQ mismatches yet developing antibodies only to one mismatch (2MM1DSA), to interrogate differential immunogenicity. Our results demonstrate that mismatches of DQα05-heterodimers exhibit the highest immunogenicity. Additionally, we demonstrate that the DQα chain critically contributes to the overall qualities of DQ molecules. Lastly, our data proposes that an augmented risk to develop donor-specific HLA-DQ antibodies is dependent on qualitative (evolutionary and functional) divergence between recipient and donor, rather than the mere number of molecular mismatches. Overall, we propose an immunological mechanistic rationale to explain differential HLA-DQ immunogenicity, with potential ramifications for other pathological processes such as autoimmunity and infections.

Estimating alloantibody levels in highly sensitized renal allograft candidates: Using serial dilutions to demonstrate a treatment effect in clinical trials.

Small reductions in calculated panel-reactive antibody (cPRA) are associated with increased kidney transplantation in 100% cPRA patients. However, the high level of antibody in these patients is such that desensitization may reduce antibody but not cPRA, thus the cPRA change on undiluted serum with desensitization is an insensitive measure of effectiveness. We evaluated cPRA reduction, calculated per antibody titer, as a desensitization trial endpoint. To accomplish this, two serum samples from 20 kidney transplant candidates with cPRA ≥99.9% (100%) were obtained and serially diluted in triplicate to determine the titer of individual human leukocyte antigen (HLA) antibody specificities. CPRA was computed per dilution to identify the titer at which cPRA drops below 98%. Inter- and intra-assay variability and changes overtime were determined. The dilution needed to reach a cPRA <98% was within 1 titer for replicates from the same sample, with 90% (36/40) concordance. This indicates that only changes >2 titers can be deemed clinically meaningful. The median (IQR) titer difference was 0 (0-1) from baseline to follow-up within 12 months. The cPRA per titer also risk-stratified candidates for trial inclusion. In conclusion, determining the cPRA per titer is a reliable approach to simplify complex antibody data and an ideal endpoint for desensitization trials.

Measuring human leukocyte antigen alloantibodies: beyond a binary decision.

PURPOSE OF REVIEW: Accurate measurement of human leukocyte antigen antibodies is critical for making clinical decisions treating patients awaiting transplantation or monitoring them post transplantation. Single antigen bead assay results are given as Mean Fluorescence Intensity, falling short of providing the required quantitative measure. RECENT FINDINGS: Titration studies were shown to circumvent the limitation of target-saturation that affect interpretation of single antigen bead assays especially in highly sensitized patients with strong antibodies. In fact, titration information can serve to measure efficacy of antibody removal during pretransplant desensitization using plasmapheresis/intravenous immunoglobulin (PP/IVIg) approaches. Moreover, recent studies indicate that knowing the donor-specific antibody titer has prognostic value that can guide PP/IVIg desensitization treatments. Newer data demonstrates an additional layer of information obtained by titration studies allowing to stratify patients with very high cPRA (>99%) based on the strength of the antibodies present, rather than the breadth. This data can thereby identify patients that are more likely to benefit from desensitization approaches on the transplant wait-list. SUMMARY: Titration studies have a prognostic value with regards to quantifying antibody strength. Obtaining this information does not require performing the complete set of dilutions. In fact, performing two to three specific dilutions can provide relevant information while maintaining practical cost.

Allergens stimulate store-operated calcium entry and cytokine production in airway epithelial cells.

Aberrant immune responses to environmental allergens including insect allergens from house dust mites and cockroaches contribute to allergic inflammatory diseases such as asthma in susceptible individuals. Airway epithelial cells (AECs) play a critical role in this process by sensing the proteolytic activity of allergens via protease-activated receptors (PAR2) to initiate inflammatory and immune responses in the airway. Elevation of cytosolic Ca(2+) is an important signaling event in this process, yet the fundamental mechanism by which allergens induce Ca(2+) elevations in AECs remains poorly understood. Here we find that extracts from dust mite and cockroach induce sustained Ca(2+) elevations in AECs through the activation of Ca(2+) release-activated Ca(2+) (CRAC) channels encoded by Orai1 and STIM1. CRAC channel activation occurs, at least in part, through allergen mediated stimulation of PAR2 receptors. The ensuing Ca(2+) entry then activates NFAT/calcineurin signaling to induce transcriptional production of the proinflammatory cytokines IL-6 and IL-8. These findings highlight a key role for CRAC channels as regulators of allergen induced inflammatory responses in the airway.

Dead or alive? Viability of chytrid zoospores shed from live amphibian hosts.

Pathogens vary in virulence and rates of transmission because of many differences in the host, the pathogen, and their environment. The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), affects amphibian hosts differently, causing extinction and population declines in some species but having limited effects on others. Phenotypic differences in zoospore production rates among Bd lineages likely contribute to some of the variation observed among host responses, although no studies have quantified the viability of zoospores shed from live animals. We compared host survivorship, infection intensity, shedding rates, and zoospore viability between 2 species of endangered tropical frogs, Hylomantis lemur and Atelopus zeteki, when exposed to a highly virulent lineage of Bd (JEL 423). We applied a dye to zoospores 30 to 60 min following animal soaks, to estimate shedding rate and proportion of live zoospores shed by different species. The average infection intensity for A. zeteki was nearly 17 times higher (31,455 ± 10,103 zoospore genomic equivalents [ZGEs]) than that of H. lemur (1832 ± 1086 ZGEs), and A. zeteki died earlier than H. lemur. The proportion of viable zoospores was ~80% in both species throughout the experiment, although A. zeteki produced many more zoospores, suggesting it may play a disproportionate role in spreading disease in communities where it occurs, because the large number of viable zoospores they produce might increase infection in other species where they are reintroduced.