McCann, C. D. | Search bioRxiv

bioRxiv2025

REFLEX, a Novel Immune Profiling Assay, Combining TCR Repertoire and Multiome at Massively Scalable Single-cell Resolution to Catapult Exploration of T-cell Derived Immunity

Single-cell profiling of T cell state with immune repertoire is critical for understanding heterogenous T cell phenotypes and responses to antigen, however, existing technologies struggle to generate this information at sufficient throughput to match biological complexity. We present "REFLEX", a novel single-cell method enabling highly scalable, cost-efficient, multiomic profiling with paired-chain TCR sequencing. REFLEX utilizes in-situ reverse transcription with integrated sample multiplexing barcodes in a way that merges seamlessly with the commonly used 10x FLEX platform to allow capture of TCR sequences at unprecedented scale and depth. We profile >2 million cells from CMV-peptide-pulsed T cell expansions, capturing TCR sequences and rich multiomic information from 1.4M T cells, identifying many putative novel CMV reactive clonotypes and illustrating the scale and transformative impact on our understanding of T cell mediated adaptive immunity achievable with REFLEX.

immunology↗

bioRxiv2020

Deep Sequencing Reveals Compartmentalized HIV-1 in the Semen of Men with and without STI-associated Urethritis

Concurrent sexually transmitted infections (STI) can increase the probability of HIV-1 transmission primarily by increasing the viral load present in semen. In this study, we explored the relationship of HIV-1 in blood and seminal plasma in the presence and absence of urethritis and after treatment of the concurrent STI. Primer ID deep sequencing of the V1/V3 region of the HIV-1 env gene was done for paired blood and semen samples from ART-naive men living in Malawi with (n = 19) and without (n = 5) STI-associated urethritis; for a subset of samples full length env genes were generated for sequence analysis and to test entry phenotype. Cytokine concentrations in the blood and semen were also measured, and a reduction in the levels of pro-inflammatory cytokines was observed following STI treatment. We observed no difference in the prevalence of diverse compartmentalized semen-derived lineages in men with or without STI-associated urethritis, and these viral populations were largely stable during STI treatment. Clonal amplification of one or a few viral sequences accounted for nearly 50% of the viral population indicating a recent bottleneck followed by limited viral replication. We documented a case of superinfection where the new strain was restricted to the genital tract. We conclude that the male genital tract is a site where virus can be brought in from the blood, where localized sustained replication can occur, where a superinfecting strain can persist, and where specific genotypes can be amplified perhaps initially by cellular proliferation but further by limited viral replication. ImportanceHIV-1 is a sexually transmitted infection that co-exists with other STIs. Here we examine the impact of a concurrent STI resulting in urethritis on the HIV-1 population within the male genital tract. We found that viral populations remain largely stable even with treatment of the STI. These results show that viral populations within the male genital tract are defined by factors beyond transient inflammation associated with a concurrent STI.

microbiology↗