Yersinia species use a variety of type III effector proteins to target eukaryotic signaling systems. The effector YopJ inhibits mitogen-activated protein kinase (MAPK) and the nuclear factor kappaB (NFkappaB) signaling pathways used in innate immune response by preventing activation of the family of MAPK kinases (MAPKK). We show that YopJ acted as an acetyltransferase, using acetyl-coenzyme A (CoA) to modify the critical serine and threonine residues in the activation loop of MAPKK6 and thereby blocking phosphorylation. The acetylation on MAPKK6 directly competed with phosphorylation, preventing activation of the modified protein. This covalent modification may be used as a general regulatory mechanism in biological signaling.

Summary Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBC). Recent studies of MBC development and function after protein immunization have uncovered significant MBC heterogeneity. To clarify functional roles for distinct MBC subsets during malaria infection, we generated tetramers that identify Plasmodium-specific MBCs in both humans and mice. Long-lived murine Plasmodium-specific MBCs consisted of three populations: somatically hypermutated IgM+ and IgG+ MBC subsets and an unmutated IgD+ MBC population. Rechallenge experiments revealed that high affinity, somatically hypermutated Plasmodium-specific IgM+ MBCs proliferated and gave rise to antibody secreting cells that dominated the early secondary response to parasite rechallenge. IgM+ MBCs also gave rise to T cell-dependent IgM+ and IgG+B220+CD138+ plasmablasts or T cell-independent B220−CD138+ IgM+ plasma cells. Thus, even in competition with IgG+ MBCs, IgM+ MBCs are rapid, plastic early responders to a secondary Plasmodium rechallenge and should be targeted by vaccine strategies.

T cells are involved in the early identification and clearance of viral infections and also support the development of antibodies by B cells. This central role for T cells makes them a desirable target for assessing the immune response to SARS-CoV-2 infection. Here, we combined two high-throughput immune profiling methods to create a quantitative picture of the T-cell response to SARS-CoV-2. First, at the individual level, we deeply characterized 3 acutely infected and 58 recovered COVID-19 subjects by experimentally mapping their CD8 T-cell response through antigen stimulation to 545 Human Leukocyte Antigen (HLA) class I presented viral peptides (class II data in a forthcoming study). Then, at the population level, we performed T-cell repertoire sequencing on 1,015 samples (from 827 COVID-19 subjects) as well as 3,500 controls to identify shared "public" T-cell receptors (TCRs) associated with SARS-CoV-2 infection from both CD8 and CD4 T cells. Collectively, our data reveal that CD8 T-cell responses are often driven by a few immunodominant, HLA-restricted epitopes. As expected, the T-cell response to SARS-CoV-2 peaks about one to two weeks after infection and is detectable for several months after recovery. As an application of these data, we trained a classifier to diagnose SARS-CoV-2 infection based solely on TCR sequencing from blood samples, and observed, at 99.8% specificity, high early sensitivity soon after diagnosis (Day 3-7 = 83.8% [95% CI = 77.6-89.4]; Day 8-14 = 92.4% [87.6-96.6]) as well as lasting sensitivity after recovery (Day 29+/convalescent = 96.7% [93.0-99.2]). These results demonstrate an approach to reliably assess the adaptive immune response both soon after viral antigenic exposure (before antibodies are typically detectable) as well as at later time points. This blood-based molecular approach to characterizing the cellular immune response has applications in vaccine development as well as clinical diagnostics and monitoring.

Exposure to inhaled allergens generates T helper 2 (Th2) CD4+ T cells that contribute to episodes of inflammation associated with asthma. Little is known about allergen-specific Th2 memory cells and their contribution to airway inflammation. We generated reagents to understand how endogenous CD4+ T cells specific for a house dust mite (HDM) allergen form and function. After allergen exposure, HDM-specific memory cells persisted as central memory cells in the lymphoid organs and tissue resident memory (Trm) cells in the lung. Experimental blockade of lymphocyte migration demonstrated that lung resident cells were sufficient to induce airway hyper-responsiveness, which depended upon CD4+ T cells. Investigation into the differentiation of pathogenic Trm cells revealed that interleukin-2 (IL-2) signaling was required for residency and directed a program of tissue homing migrational cues. These studies thus identify IL-2-dependent resident Th2 memory cells as drivers of lung allergic responses.

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors. One of these effectors, Vibrio outer protein A (VopA), inhibits MAPK signaling via a novel mechanism, distinct from those described for other bacterial toxins, that disrupts this signaling pathway. VopA is an acetyltransferase that potently inhibits MAPK signaling pathways not only by preventing the activation of MAPK kinases (MKKs) but also by inhibiting the activity of activated MKKs. VopA acetylates a conserved lysine found in the catalytic loop of all kinases and blocks the binding of ATP, but not ADP, on the MKKs, resulting in an inactive phosphorylated kinase. Acetylation of this conserved lysine inhibits kinase activity by a new mechanism of regulation that has not been observed previously. Identifying the target of VopA reveals a way that the reversible post-translational modification of lysine acetylation can be used to regulate the activity of an enzyme.Vibrio parahemeolyticus is a marine bacterium and causative agent of gastroenteritis associated with the consumption of contaminated seafood. It is endemic to Southeast Asia and is the leading cause of gastroenteritis in Japan (1). Sequencing of the V. parahemeolyticus genome revealed the presence of two type III secretion systems each encoded in separate pathogenicity islands (2). Type III secretion systems are used by bacterial pathogens to inject effector proteins into the cytoplasm of the host cells (3). Upon delivery into the host cytosol, the effectors manipulate host signaling to gain an advantage for the infecting pathogen. The bacterial effectors are proposed to usurp or mimic eukaryotic activity that is then used to target and deregulate normal functions in the host cell by either up-or downregulating signaling pathways (4). However, the bacterial effector proteins must initially be kept in a quiescent state within the bacterium. This is accomplished by a variety of methods, including their association with a chaperone in the bacterial cytosol, their specificity for a substrate, and/or their requirement for an activator or modification that is only provided by a eukaryotic source (4, 5).VopA (Vibrio outer protein A) is an effector found within one of the V. parahemeolyticus pathogenicity islands, and is a member of a family of type III effectors called YopJ-like proteins that are found in a wide variety of pathogens including other animal pathogens, plant pathogens, and the plant symbiont Rhizobium (5-7). The founding member of the family, YopJ from Yersinia spp., inhibits the mitogen-activated protein kinase (MAPK) 3 and NFB signaling cascades within the host cell, thereby inhibiting the host's innate immune response (7). All YopJ-like proteins contain a catalytic triad, and it was recently shown that YopJ functions as an acetyltransferase (8 -10). It has been speculated that other members of the family of YopJ-like proteins might also possess this activity (8). YopJ was demonstrated to inhibit the MAPK and NFB...

Summary Many current malaria vaccines target the pre-erythrocytic stage of infection in the liver. However, in malaria endemic regions, increased blood stage exposure is associated with decreased vaccine efficacy, thereby challenging current vaccine efforts. We hypothesized that pre-erythrocytic humoral immunity is directly disrupted by blood stage infection. To investigate this possibility, we used Plasmodium-antigen tetramers to analyze B cells after infection with either late-liver stage arresting parasites or wild type parasites that progress to the blood stage. Our data demonstrate that IgG antibodies against the pre-erythrocytic antigen, circumsporozoite protein (CSP), are generated only in response to the attenuated, but not the wild-type infection. Further analyses revealed that blood stage malaria inhibits CSP-specific germinal center B cell differentiation and modulates chemokine expression. This results in aberrant memory formation and the loss of a rapid secondary B cell response. These data highlight how immunization with attenuated parasites may drive optimal immunity to malaria.

Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum–specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines.

No abstract