Kitt Paraiso

TCR- and BCR-sequencing (TCR/BCR-seq) are two important technologies in studying the immune repertoire of samples such as PBMCs or tumors. In their most common form, these assays combine multiplex PCR of the repertoire using primers targeting regions of the V(D)J and the constant region with next-generation sequencing (NGS). The data produced by this assay provide a slew of information regarding immune repertoire(s) including the presence critical clonotypes, repertoire diversity, variable (V) gene usage, analysis of public clonotypes, etc. One issue that can arise during generation of the TCR/BCR-seq data is sequence bias during the PCR or NGS steps. To combat this, unique molecular identifiers (UMIs) have been used to identify and eliminate sequence bias. However, UMI fragments can be long and very diverse, resulting in the UMI sequences interfering with any of the multitude of primers during multiplex PCR. Here, we introduce Validator Barcodes (VBCs), a set of eight short barcodes (6-9 nucleotides in length). This compact set of barcodes improves PCR efficiency and facilitates PCR primer designs. Also, like UMIs, the VBCs may be used to estimate the number of template molecules (RNA or DNA). Using VBC-labeled primers for TCR and BCR repertoire profiling from PBMCs produces highly comparable results and similarly template values to those obtained through UMI-based assay counts. Overall, VBCs are a useful and simpler alternative to UMIs in assaying TCR and BCR repertoires.

How changes in the quality of anti-viral antibody (Ab) responses due to pre-existing or acquired CD4 T cell insufficiency affect virus evolution during persistent infection are unknown. Using mouse polyomavirus (MuPyV), we found that CD4 T cell depletion before infection results in short-lived plasma cells secreting low-avidity antiviral IgG with limited BCR diversity and weak virus-neutralizing ability. CD4 T cell deficiency during persistent infection incurs a shift from a T-dependent (TD) to T-independent (TI) Ab response, resembling the pre-existing TI Ab response. CD4 T cell loss before infection or during persistent infection is conducive for emergence of Ab-escape variants. Cryo-EM reconstruction of complexes of MuPyV virions with polyclonal IgG directly from infected mice with pre-existing or acquired CD4 T cell deficiency enabled visualization of shortfalls in TI IgG binding. By debilitating the antiviral IgG response, CD4 T cell deficiency sets the stage for outgrowth of variant viruses resistant to neutralization. ONE SENTENCE SUMMARY Pre-existing and acquired CD4 T cell deficiency facilitates outgrowth of Ab-escape viral variants during persistent infection.

In Finland the frequency of isolated cleft palate (CP) is higher than that of isolated cleft lip (CL) or cleft lip with cleft palate (CLP). This trend contrasts to that in other countries but its genetic underpinnings are unknown. We performed a genome-wide association study for orofacial cleft, which includes CL, CLP, and CP in the Finnish population. We identified rs570516915, a single nucleotide polymorphism that is virtually specific to Finns and Estonians, as being strongly associated with orofacial cleft, and predominantly with CP (p = 5.25 x 10^-34, OR = 8.65, 95% CI 6.11-12.25). The risk allele frequency for rs570516915 parallels the regional variation of CP prevalence in Finland, and the association was replicated in an independent sample of CP cases from Estonia (p < 1.3 x 10^-11). The rs570516915 variant lies in an IRF6 (Interferon Regulatory Factor 6) enhancer active in ectodermal and oral epithelial cells. Other variants in the IRF6 locus, including those in the same enhancer, are associated with orofacial cleft, but predominantly with CL or CLP, suggesting shared mechanisms in the distinct processes of lip and palate development. By luciferase and transgenic mouse reporter assays we found that the risk allele of rs570516915 diminishes the activity of the enhancer. CRISPR-Cas9 edited oral epithelial cells demonstrate that rs570516915 disrupts an IRF6 binding site and decreases the expression of IRF6, suggesting impaired IRF6 autoregulation as the molecular mechanism underlying risk for CP.

Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving more than 1000 nuclear-encoded proteins. Transcriptional networks controlling the nuclear-encoded mitochondrial genes remain to be fully elucidated. Here, we show that histone demethylase LSD1 KO from adult mouse liver (LSD1-LKO) reduces the expression of one-third of all nuclear-encoded mitochondrial genes and decreases mitochondrial biogenesis and function. LSD1-modulated histone methylation epigenetically regulates nuclear-encoded mitochondrial genes. Furthermore, LSD1 regulates gene expression and protein methylation of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), which controls the final step of NAD ⁺ synthesis and limits NAD ⁺ availability in the nucleus. Lsd1 KO reduces NAD ⁺ -dependent SIRT1 and SIRT7 deacetylase activity, leading to hyperacetylation and hypofunctioning of GABPβ and PGC-1α, the major transcriptional factor/cofactor for nuclear-encoded mitochondrial genes. Despite the reduced mitochondrial function in the liver, LSD1-LKO mice are protected from diet-induced hepatic steatosis and glucose intolerance, partially due to induction of hepatokine FGF21. Thus, LSD1 orchestrates a core regulatory network involving epigenetic modifications and NAD ⁺ synthesis to control mitochondrial function and hepatokine production.