ACE configurator for ELISpot: optimizing combinatorial design of pooled ELISpot assays with an epitope similarity model.

The enzyme-linked immunosorbent spot (ELISpot) assay is a powerful in vitro immunoassay that enables cost-effective quantification of antigen-specific T-cell reactivity. It is used widely in the context of cancer and infectious diseases to validate the immunogenicity of predicted epitopes. While technological advances have kept pace with the demand for increased throughput, efforts to increase scale are bottlenecked by current assay design and deconvolution methods, which have remained largely unchanged. Current methods for designing pooled ELISpot experiments offer limited flexibility of assay parameters, lack support for high-throughput scenarios and do not consider peptide identity during pool assignment. We introduce the ACE Configurator for ELISpot (ACE) to address these gaps. ACE generates optimized peptide-pool assignments from highly customizable user inputs and handles the deconvolution of positive peptides using assay readouts. In this study, we present a novel sequence-aware pooling strategy, powered by a fine-tuned ESM-2 model that groups immunologically similar peptides, reducing the number of false positives and subsequent confirmatory assays compared to existing combinatorial approaches. To validate ACE's performance on real-world datasets, we conducted a comprehensive benchmark study, contextualizing design choices with their impact on prediction quality. Our results demonstrate ACE's capacity to further increase precision of identified immunogenic peptides, directly optimizing experimental efficiency. ACE is freely available as an executable with a graphical user interface and command-line interfaces at https://github.com/pirl-unc/ace.

Mycobacterium tuberculosis precursor rRNA as a measure of treatment-shortening activity of drugs and regimens.

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.

Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature.

Lymphocytes in barrier tissues play critical roles in host defense and homeostasis. These cells take up residence in tissues during defined developmental windows, when they may demonstrate distinct phenotypes and functions. Here, we utilized mass and flow cytometry to elucidate early features of human skin immunity. Although most conventional αß T (Tconv) cells in fetal skin have a naive, proliferative phenotype, a subset of CD4+ Tconv and CD8+ cells demonstrate memory-like features and a propensity for interferon (IFN)γ production. Skin regulatory T cells dynamically accumulate over the second trimester in temporal and regional association with hair follicle development. These fetal skin regulatory T cells (Tregs) demonstrate an effector memory phenotype while differing from their adult counterparts in expression of key effector molecules. Thus, we identify features of prenatal skin lymphocytes that may have key implications for understanding antigen and allergen encounters in utero and in infancy.