Single-cell NAD(H) levels predict clonal lymphocyte expansion dynamics.

Adaptive immunity requires the expansion of high-affinity lymphocytes from a heterogeneous pool. Whereas current models explain this through signal transduction, we hypothesized that antigen affinity tunes discrete metabolic pathways to license clonal lymphocyte dynamics. Here, we identify nicotinamide adenine dinucleotide (NAD) biosynthesis as a biochemical hub for the T cell receptor affinity-dependent metabolome. Through this central anabolic role, we found that NAD biosynthesis governs a quiescence exit checkpoint, thereby pacing proliferation. Normalizing cellular NAD(H) likewise normalizes proliferation across affinities, and enhancing NAD biosynthesis permits the expansion of lower affinity clones. Furthermore, single-cell differences in NAD(H) could predict division potential for both T and B cells, before the first division, unmixing proliferative heterogeneity. We believe that this supports a broader paradigm in which complex signaling networks converge on metabolic pathways to control single-cell behavior.

Persistent Zika Virus Clinical Susceptibility despite Reduced Viral Burden in Mice with Expanded Virus-Specific CD8+ T Cells Primed by Recombinant Listeria monocytogenes.

Vaccines against Zika virus (ZIKV) infection that target CD8+ T cells are of considerable interest because Abs may enhance infection susceptibility. However, whether CD8+ T cells are protective or promote susceptibility to clinical infection symptoms remains uncertain. To more precisely investigate ZIKV-specific CD8+ T cells in isolation, we engineered a Listeria monocytogenes-based vector to express a single MHC class I-restricted immune dominant peptide, E294-302, from ZIKV envelope protein. We show accumulation of activated ZIKV-specific CD8+ T cells primed by recombinant L. monocytogenes is associated with reductions in circulating virus levels after ZIKV challenge in type I IFN receptor-deficient mice and wildtype mice administered neutralizing Abs against type I IFN receptor. Interestingly, susceptibility to ZIKV clinical infection including weight loss and mortality each persists and is neither significantly improved nor worsened compared with isogenic L. monocytogenes-primed control mice. These data demonstrating persistent ZIKV clinical susceptibility despite reduced viral burden in mice with expanded virus-specific CD8+ T cells highlights the need for targeting other adaptive immune components in developing vaccines against ZIKV infection.

CD8+ T Cell Functional Exhaustion Overrides Pregnancy-Induced Fetal Antigen Alloimmunization.

Pregnancy necessitates physiological exposure, and often re-exposure, to foreign fetal alloantigens. The consequences after pregnancy are highly varied, with evidence of both alloimmunization and expanded tolerance phenotypes. We show that pregnancy primes the accumulation of fetal-specific maternal CD8+ T cells and their persistence as an activated memory pool after parturition. Cytolysis and the potential for robust secondary expansion occurs with antigen re-encounter in non-reproductive contexts. Comparatively, CD8+ T cell functional exhaustion associated with increased PD-1 and LAG-3 expression occurs with fetal antigen re-stimulation during subsequent pregnancy. PD-L1/LAG-3 neutralization unleashes the activation of fetal-specific CD8+ T cells, causing fetal wastage selectively during secondary but not primary pregnancy. Thus, CD8+ T cells with fetal alloantigen specificity persist in mothers after pregnancy, and protection against fetal wastage in subsequent pregnancies is maintained by their unique susceptibility to functional exhaustion. Together, distinct mechanisms whereby fetal tolerance is maintained during primary compared with subsequent pregnancies are demonstrated.

Preconceptual Zika virus asymptomatic infection protects against secondary prenatal infection.

Pregnant women, and their fetal offspring, are uniquely susceptible to Zika virus and other microbial pathogens capable of congenital fetal infection. Unavoidable exposure to Zika virus in endemic areas underscores the need for identifying at-risk individuals, and protecting expecting mothers and their fetal offspring against prenatal infection. Here we show that primary Zika virus asymptomatic infection in mice confers protection against re-infection, and that these protective benefits are maintained during pregnancy. Zika virus recovery was sharply reduced in maternal tissues and amongst fetal concepti after prenatal challenge in mothers with resolved subclinical infection prior to pregnancy compared with mice undergoing primary prenatal infection. These benefits coincide with expanded accumulation of viral-specific antibodies in maternal serum and fetal tissues that protect against infection by the identical or heterologous Zika virus genotype strains. Thus, preconceptual infection primes Zika virus-specific antibodies that confer cross-genotype protection against re-infection during pregnancy.

Commensal Fungi Recapitulate the Protective Benefits of Intestinal Bacteria.

Commensal intestinal microbes are collectively beneficial in preventing local tissue injury and augmenting systemic antimicrobial immunity. However, given the near-exclusive focus on bacterial species in establishing these protective benefits, the contributions of other types of commensal microbes remain poorly defined. Here, we show that commensal fungi can functionally replace intestinal bacteria by conferring protection against injury to mucosal tissues and positively calibrating the responsiveness of circulating immune cells. Susceptibility to colitis and influenza A virus infection occurring upon commensal bacteria eradication is efficiently overturned by mono-colonization with either Candida albicans or Saccharomyces cerevisiae. The protective benefits of commensal fungi are mediated by mannans, a highly conserved component of fungal cell walls, since intestinal stimulation with this moiety alone overrides disease susceptibility in mice depleted of commensal bacteria. Thus, commensal enteric fungi safeguard local and systemic immunity by providing tonic microbial stimulation that can functionally replace intestinal bacteria.

Biochemical, mechanistic, and spectroscopic characterization of metallo-ß-lactamase VIM-2.

This study examines metal binding to metallo-ß-lactamase VIM-2, demonstrating the first successful preparation of a Co(II)-substituted VIM-2 analogue. Spectroscopic studies of the half- and fully metal loaded enzymes show that both Zn(II) and Co(II) bind cooperatively, where the major species present, regardless of stoichiometry, are apo- and di-Zn (or di-Co) enzymes. We determined the di-Zn VIM-2 structure to a resolution of 1.55 Å, and this structure supports results from spectroscopic studies. Kinetics, both steady-state and pre-steady-state, show that VIM-2 utilizes a mechanism that proceeds through a very short-lived anionic intermediate when chromacef is used as the substrate. Comparison with other B1 enzymes shows that those that bind Zn(II) cooperatively are better poised to protonate the intermediate on its formation, compared to those that bind Zn(II) non-cooperatively, which uniformly build up substantial amounts of the intermediate.