Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving

The nature of certain clinical samples (tissue biopsies, fluids) or the subjects themselves (pediatric subjects, neonates) often constrain the number of cells available to evaluate the breadth of functional T-cell responses to infections or therapeutic interventions. The methods most commonly used to assess this functional diversity ex vivo and to recover specific cells to expand in vitro usually require more than 106 cells. Here we present a process to identify antigen-specific responses efficiently ex vivo from 104–105 single cells from blood or mucosal tissues using dense arrays of subnanoliter wells. The approach combines on-chip imaging cytometry with a technique for capturing secreted proteins—called “microengraving”—to enumerate antigenspecific responses by single T cells in a manner comparable to conventional assays such as ELISpot and intracellular cytokine staining. Unlike those assays, however, the individual cells identified can be recovered readily by micromanipulation for further characterization in vitro. Applying this method to assess HIV-specific T cell responses demonstrates that it is possible to establish clonal CD8+ T-cell lines that represent the most abundant specificities present in circulation using 100- to 1,000-fold fewer cells than traditional approaches require and without extensive genotypic analysis a priori. This rapid (<24 h), efficient, and inexpensive process should improve the comparative study of human T-cell immunology across ages and anatomic compartments

Rapid and Comprehensive Bacterial Identification and Antimicrobial Resistance Determination Using Microfluidic Enrichment and Whole-Genome Sequencing

Sepsis and other severe bacterial infections affect over 750,000 Americans annually, resulting in a 28% mortality rate and $16 billion in health care costs due to delays in proper diagnosis. The current gold-standard method for determining the species identification (ID) and antimicrobial resistance (AMR) profile of a clinical pathogen relies on the century-old method of growing bacteria in culture media, which takes 2-5 days, and frequently fails to cultivate the causative pathogen. In the absence of microbiologic data, physicians are forced to treat suspected infections with broad-spectrum empiric antibiotics that can have significant toxicity to the patient, are not always effective due to the rise of drug resistant pathogens, and can be expensive. Here we describe a process to perform bacterial ID and AMR determination directly from patient samples with a novel enrichment process combined with downstream agnostic DNA sequencing and real-time analysis with a MinION sequencer. By combining a microfluidic cell sorter and immunomagnetic depletion, we achieve at least 2.5x10^4-fold depletion of leukocytes, 10^2-fold depletion of erythrocytes, and 10^2-fold depletion of platelets, while retaining an average of 75% of bacterial cells. With the MinION sequencer, we can achieve ten-fold genome coverage of a bacterial isolate in 30 minutes of sequencing time, even with sample multiplexing. Overall, we show that this approach is feasible when combined with additional human cell and DNA depletion steps, and has the potential to improve clinical care and antibiotic stewardship

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information

Epithelial adhesion molecules can inhibit HIV-1–specific CD8+ T-cell functions

Under persistent antigenic stimulation, virus-specific CD8+ T cells become increasingly dysfunctional and up-regulate several inhibitory molecules such as killer lectin-like receptor G1 (KLRG1). Here, we demonstrate that HIV-1 antigen-specific T cells from subjects with chronic-progressive HIV-1 infection have significantly elevated KLRG1 expression (P < .001); show abnormal distribution of E-cadherin, the natural ligand of KLRG1, in the intestinal mucosa; and have elevated levels of systemic soluble E-cadherin (sE-cadherin) that significantly correlate with HIV-1 viral load (R = 0.7, P = .004). We furthermore demonstrate that in the presence of sE-cadherin, KLRG1hi HIV-1–specific CD8+ T cells are impaired in their ability to respond by cytokine secretion on antigenic stimulation (P = .002) and to inhibit viral replication (P = .03) in vitro. Thus, these data suggest a critical mechanism by which the disruption of the intestinal epithelium associated with HIV-1 leads to increased systemic levels of sE-cadherin, which inhibits the effector functions of KLRG1hi-expressing HIV-1–specific CD8+ T cells systemically