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CD28 provides an essential costimulatory signal for T cell activation, and its function is critical in antitumor immunity. However, the molecular mechanism of CD28 transmembrane signaling remains elusive. Here we show that the conformation and signaling of CD28 are regulated by two counteractive charged factors, acidic phospholipids and Ca ions. NMR spectroscopy analyses showed that acidic phospholipids can sequester CD28 signaling motifs within the membrane, thereby limiting CD28 basal signaling. T cell receptor (TCR) activation induced an increase in the local Ca concentration around CD28, and Ca directly disrupted CD28-lipid interaction, leading to opening and signaling of CD28. We observed that the TCR, Ca, and CD28 together form a dual-positive-feedback circuit that substantially amplifies T cell signaling and thus increases antigen sensitivity. This work unravels a new regulatory mechanism for CD28 signaling and thus contributes to the understanding of the dependence of costimulation signaling on TCR signaling and the high sensitivity of T cells.

B cells that express the isotype-switched IgG-B cell receptor (IgG-BCR) are one of the driving forces for antibody memory. To allow for a rapid memory IgG antibody response, IgG-BCR evolved into a highly effective signalling machine. Here, we report that the positively charged cytoplasmic domain of mIgG (mIgG-tail) specifically interacts with negatively charged acidic phospholipids. The key immunoglobulin tail tyrosine (ITT) in mIgG-tail is thus sequestered in the membrane hydrophobic core in quiescent B cells. Pre-disruption of such interaction leads to excessive recruitment of BCRs and inflated BCR signalling upon antigen stimulation, resulting in hyperproliferation of primary B cells. Physiologically, membrane-sequestered mIgG-tail can be released by antigen engagement or Ca2+ mobilization in the initiation of B cell activation. Our studies suggest a novel regulatory mechanism for how dynamic association of mIgG-tail with acidic phospholipids governs the enhanced activation of IgG-BCR.

T-cell receptor-CD3 complex (TCR) is a versatile signaling machine that can initiate antigen-specific immune responses based on various biochemical changes of CD3 cytoplasmic domains, but the underlying structural basis remains elusive. Here we developed biophysical approaches to study the conformational dynamics of CD3ε cytoplasmic domain (CD3ε CD ). At the single-molecule level, we found that CD3ε CD could have multiple conformational states with different openness of three functional motifs, i.e., ITAM, BRS and PRS. These conformations were generated because different regions of CD3ε CD had heterogeneous lipid-binding properties and therefore had heterogeneous dynamics. Live-cell imaging experiments demonstrated that different antigen stimulations could stabilize CD3ε CD at different conformations. Lipid-dependent conformational dynamics thus provide structural basis for the versatile signaling property of TCR.

Because invasion of erythrocytes by Plasmodium falciparum merozoites involves multiple receptor-ligand interactions, it may be necessary to develop a multivalent malaria vaccine that is comprised of distinct parasite ligands. PfAMA-1, PfMSP1, and PfEBA-175 are merozoite proteins that play important roles in invasion. We have constructed a PfCP-2.9 chimeric protein consisting of PfAMA-1 and PfMSP1 and tested it for immunogenicity in animal models and humans. The F2 subdomain of PfEBA-175 (PfEBA-175II F2) was identified as the binding domain for glycophorin A on erythrocytes. In this study, we used the codon frequencies of the yeast Pichia pastoris to redesign and synthesize a gene encoding the F2 domain. We found that the codon-optimized gene was expressed at a high level in P. pastoris as a soluble protein with a yield of about 300 mg/liter. The expressed protein was able to bind normal erythrocytes but not those treated with neuraminidase or trypsin. Moreover, the protein was recognized by the sera of malaria patients and was highly immunogenic in mice, rabbits, and rhesus monkeys. Immunoglobulin G isolated from both immunized rabbits and monkeys inhibited in vitro parasite growth. Immunization of animals with a combination of PfEBA-175II F2 and PfCP-2.9 did not result in antigen (Ag) competition in animals. Moreover, antibodies to both PfEBA-175II F2 and PfCP-2.9, isolated from rabbits immunized with both constructs, inhibited parasite growth in vitro. The combination of high yield, functional folding, antibody inhibition, and lack of Ag competition provides support for inclusion of these merozoite proteins in a combination vaccine against infection with blood-stage parasites.Plasmodium falciparum and Plasmodium vivax are the causative agents of the majority of malaria cases in the world today. Of the two, P. falciparum is responsible for the most virulent form of the disease, causing over 2 million deaths per year, usually in children under 5 years of age. Malaria infections have traditionally been treated by chemotherapy. Another approach has been to use insecticides against the Anopheles sp. mosquito vectors that transfer the parasites between hosts. Because of the emergence and rapid spread of drug-resistant parasites and insecticide-resistant mosquitoes, there is an urgent need for the development of new tools to control malaria. Vaccination is one such tool that may control and even eradicate the disease from the world. Based on the life cycle of the parasite, merozoite invasion of host erythrocytes is an optimal target for vaccines against infection with blood-stage parasites. However, merozoite invasion is a complex process involving several steps. The initial step requires species-specific interactions between erythrocyte receptors and parasite ligands. Disruption of these interactions would, in principle, prevent invasion and all of the clinical manifestations of infection. The invasion of human erythrocytes by P. vivax requires recognition of the Duffy blood group antigen (Ag) (11,22), while invasio...

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Attenuated Salmonella strains are an attractive live vector for delivery of a foreign antigen to the human immune system. However, the problem with this vector lies with plasmid segregation and the low level of expression of the foreign gene in vivo when constitutive expression is employed, leading to a diminished immune response. We have established inducible expressions of foreign genes in the Salmonella enterica serovar Typhi CVD908 vaccine strain using the tetracycline response regulatory promoter. To set up this system, a tetracycline repressor (tetR) was integrated into a defined ⌬aroC locus of the chromosome via suicide plasmid pJG12/tetR-neo. To remove the neo gene conferring kanamycin resistance from the locus, a cre expression vector under the control of the tetracycline response promoter was transformed into the clone; expression of the Cre recombinase excised the neo gene and generated the end strain CVD908-tetR. Expression of the luciferase reporter gene in this strain is dependent on the presence of tetracycline in the medium and can be regulated up to 4,773-fold. Moreover, the tightly controlled expression of major merozoite surface protein 1 (MSP1) and parts of Plasmodium falciparum was achieved, and the product yield was increased when the inducible expression system was employed. Inoculation of bacteria harboring plasmid pZE11/MSP1 42 in mice produced the protein in liver and spleen controlled by the inducer. The persistence of the plasmid-carrying bacteria in mice was determined. Peak colonization of both liver and spleen was detected on the third day postinoculation and was followed by a decline in growth curves. After 14 days postinfection, the majority of the bacteria (>90%) recovered from the liver and spleen of the mice retained the plasmid when expression was induced; this clearly indicated that stability of the expression vector in vivo was improved by inducible expression. Establishment of the regulatory system in the vaccine strain may broaden the range of its use by enhancing plasmid stability and expression levels in vivo. Moreover, the availability of the vaccine strain inducibly expressing the entire MSP1 provides possibilities for examining its immunogenicity, particularly the cellular response in animal models.