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PubMed2005

Secretion of type III effectors into host cells in real time.

Type III secretion (T3S) systems are key features of many gram-negative bacteria that translocate T3S effector proteins directly into eukaryotic cells. There, T3S effectors exert many effects, such as cellular invasion or modulation of host immune responses. Studying spatiotemporal orchestrated secretion of various effectors has been difficult without disrupting their functions. Here we developed a new approach using Shigella flexneri T3S as a model to investigate bacterial translocation of individual effectors via multidimensional time-lapse microscopy. We demonstrate that direct fluorescent labeling of tetracysteine motif-tagged effectors IpaB and IpaC is possible in situ without loss of function. Studying the T3S kinetics of IpaB and IpaC ejection from individual bacteria, we found that the entire pools of IpaB and IpaC were released concurrently upon host cell contact, and that 50% of each effector was secreted in 240 s. This method allows an unprecedented analysis of the spatiotemporal events during T3S.

Antigens, Bacterial↗
PubMed2006

The discovery of SycO highlights a new function for type III secretion effector chaperones.

Bacterial injectisomes deliver effector proteins straight into the cytosol of eukaryotic cells (type III secretion, T3S). Many effectors are associated with a specific chaperone that remains inside the bacterium when the effector is delivered. The structure of such chaperones and the way they interact with their substrate is well characterized but their main function remains elusive. Here, we describe and characterize SycO, a new chaperone for the Yersinia effector kinase YopO. The chaperone-binding domain (CBD) within YopO coincides with the membrane localization domain (MLD) targeting YopO to the host cell membrane. The CBD/MLD causes intrabacterial YopO insolubility and the binding of SycO prevents this insolubility but not folding and activity of the kinase. Similarly, SycE masks the MLD of YopE and SycT covers an aggregation-prone domain of YopT, presumably corresponding to its MLD. Thus, SycO, SycE and most likely SycT mask, inside the bacterium, a domain needed for proper localization of their cognate effector in the host cell. We propose that covering an MLD might be an essential function of T3S effector chaperones.

Bacterial Proteins↗
PubMed1989

Characteristics necessary for an interconvertible enzyme cascade to generate a highly sensitive response to an effector.

A monocyclic interconvertible enzyme cascade, in which active and inactive states of an enzyme are interconverted by two opposing enzyme-catalysed reactions, does not necessarily produce a greater degree of sensitivity to an effector than one could expect from direct interaction between effector and target reaction. On the contrary, a cascade in which an effector acts on one of the enzymes catalysing the interconversion reactions by altering the apparent value of its specificity constant will always generate a less sensitive response than direct interaction would give. Nonetheless, even if both interconversion reactions obey Michaelis-Menten kinetics with the ordinary types of inhibition and activation, one can easily generate an enormous sensitivity in which a 0.5% change in concentration can increase the proportion of target enzyme in the active state from 10% to 90%: this corresponds approximately to a Hill coefficient of 800. To maximize the sensitivity, the following conditions must be satisfied: (1) both modifier enzymes must act under conditions of near saturation; (2) the effector must act on both of them in opposite directions; (3) it must alter the apparent values of their catalytic constants; (4) the enzyme subject to inhibition by the effector must respond at much lower effector concentrations than the enzyme subject to activation. As the last of these conditions appears to be counter-intuitive, it suggests that feeble activation of modifier enzymes in real systems may have passed unnoticed, or been dismissed as physiologically insignificant, although in reality crucial to the effective response of the system.

Animals↗
PubMed1997

Effector molecules of the host defence mechanism against Mycobacterium avium complex: the evidence showing that reactive oxygen intermediates, reactive nitrogen intermediates, and free fatty acids each alone are not decisive in expression of macrophage antimicrobial activity against the parasites.

In this study, we evaluated the roles of reactive oxygen intermediates (ROI), reactive nitrogen intermediates (RNI), and free fatty acids (FFA) as effectors of the macrophage-mediated host defence mechanism against Mycobacterium avium complex (MAC). First, M. avium (three strains) and M. intracellulare (two strains) were treated with the H2O2-Fe2+-mediated halogenation system, acidified NaNO2-derived RNI, or FFA (linolenic acid) in sodium acetate buffer pH 5.5, and then counted for the number of residual colony-forming units (CFU) of organisms. Although these effectors exerted strong bactericidal activity against the MAC, the susceptibility of test organisms markedly varied from strain to strain. There was no significant relationship between the degree of resistance of a given MAC strain to these effectors and its virulence in mice, indicating that ROI, RNI, and FFA each alone are not decisive as the effector components of the host defence mechanism against the MAC. Second, the increase in ROI-producing ability in murine peritoneal macrophages due to tumour necrosis factor-alpha (TNF-alpha) treatment was not accompanied by parallel potentiation of anti-MAC activity of the same macrophage population. This excludes the possibility that ROI play a central role in macrophage-mediated killing and inhibition of MAC organisms. Third, anti-MAC activity of BAM3 macrophage cell line was not significantly attenuated by N(G)-monomethyl-L-arginine (NO synthase-inhibitor causing reduction of RNI production) or by quinacrine (phospholipase A2-inhibitor causing reduction of FFA release), indicating that RNI and FFA each alone do not play crucial roles in the expression of macrophage antimicrobial activity against the MAC. The present findings suggest important roles of collaborating actions of various antimicrobial effectors and/or the participation of other kinds of effectors in macrophage-mediated killing and inhibition of MAC organisms.

Animals↗
PubMed1998

YopT, a new Yersinia Yop effector protein, affects the cytoskeleton of host cells.

Extracellular Yersinia disarm the immune system of their host by injecting effector Yop proteins into the cytosol of target cells. Five effectors have been described: YopE, YopH, YpkA/YopO, YopP and YopM. Delivery of these effectors by Yersinia adhering at the cell surface requires other Yops (translocators) including YopB. Effector and translocator Yops are secreted by the type III Ysc secretion apparatus, and some Yops also need a specific cytosolic chaperone, called Syc. In this paper, we describe a new Yop, which we have called YopT (35.5kDa). Its secretion required an intact Ysc apparatus and SycT (15.0kDa, pl4.4), a new chaperone resembling SycE. Infection of macrophages with a Yersinia, producing a hybrid YopT-adenylate cyclase, led to the accumulation of intracellular cAMP, indicating that YopT is delivered into the cytosol of eukaryotic cells. Infection of HeLa cells with a mutant strain devoid of the five known Yop effectors (deltaHOPEM strain) but producing YopT resulted in the alteration of the cell cytoskeleton and the disruption of the actin filament structure. This cytotoxic effect was caused by YopT and dependent on YopB. YopT is thus a new effector Yop and a new bacterial toxin affecting the cytoskeleton of eukaryotic cells.

Amino Acid Sequence↗
PubMed2003

Synergistic roles for the Map and Tir effector molecules in mediating uptake of enteropathogenic Escherichia coli (EPEC) into non-phagocytic cells.

Enteropathogenic Escherichia coli (EPEC) are a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. Enteropathogenic Escherichia coli encode a type III secretion system (TTSS) to transfer effector proteins into host cells, a process which is essential for virulence. In addition to generation of A/E lesions, the TTSS is also implicated in the ability of EPEC to invade cultured cells but the effector proteins responsible for promoting invasion have not been identified. In this paper we confirm the requirement of TTSS in EPEC invasion and demonstrate important roles for the Map and Tir effector molecules. Whereas in trans expression of Tir in the tir mutant restored invasion to wild-type levels, similar complementation of the map mutation by in trans expression of Map results in a hyperinvasive phenotype. The Map effector protein has two distinct functions within host cells, mediating Cdc42-dependent filopodia formation and targeting mitochondria to elicit dysfunction. The former function appears to be related to Map's ability to promote invasion as this was inhibited by interference with Cdc42 signalling. Conversely, Map targeting to mitochondria is not necessary for invasion. Promotion of EPEC invasion by Tir appears to involve interaction with intimin but is independent of pedestal formation, and intimin-Tir interaction is neither necessary nor sufficient for invasion. Comparison of the invasiveness of strains lacking Tir and/or Map with wild-type or mutant strains expressing the effectors in trans provides evidence that Map and Tir stimulate invasion by synergistic mechanisms. This synergism, which is in stark contrast to the antagonistic actions of Map and Tir in regulating filopodia and pedestal formation, further illustrates the complex interplay between EPEC effectors.

Adhesins, Bacterial↗
PubMed2002

Frequencies of HCV-specific effector CD4+ T cells by flow cytometry: correlation with clinical disease stages.

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, affecting approximately 2% of the world's population. The immune mechanisms responsible for the highly variable natural history in a given individual are unknown. We used a multiparameter flow cytometric technique to functionally and phenotypically characterize HCV-specific effector T cells in the peripheral blood of 32 individuals with different stages of hepatitis C disease (resolved, mild chronic, advanced chronic) and normal controls. We found the highest frequencies of virus-specific effector cells with an activated memory phenotype (CD45RO+CD69+) in subjects who had resolved HCV infection, either spontaneously or with antiviral therapy. Effector cells from patients with resolved infection produced Th1 type cytokines following stimulation with nonstructural antigens (NS3 and NS4), whereas effector cells from chronically infected patients produced Th1 type cytokines predominantly following stimulation with the HCV core antigen. Stimulation with superantigen staphylococcal enterotoxin (SEB) induced the same levels of cytokine production in the different patient groups. Among the HCV-seropositive patients, viral load inversely correlated with the Th1 effector cell response to NS3. Interleukin (IL)-4 was produced only in response to the control antigens, but not in response to the HCV recombinant proteins. Taken together, these findings suggest that a vigorous HCV-specific CD4+ Th1 response, particularly against the nonstructural proteins of the virus, may be associated with viral clearance and protection from disease progression. Prospective studies using this new flow cytometric assay will be required to determine whether antiviral therapy modifies the frequency, specificity, and function of these virus-specific effector cells.

Adult↗
PubMed2006

RAR1, a central player in plant immunity, is targeted by Pseudomonas syringae effector AvrB.

Pathogenic bacterial effectors suppress pathogen-associated molecular pattern (PAMP)-triggered host immunity, thereby promoting parasitism. In the presence of cognate resistance genes, it is proposed that plants detect the virulence activity of bacterial effectors and trigger a defense response, referred to here as effector-triggered immunity (ETI). However, the link between effector virulence and ETI at the molecular level is unknown. Here, we show that the Pseudomonas syringae effector AvrB suppresses PAMP-triggered immunity (PTI) through RAR1, a co-chaperone of HSP90 required for ETI. AvrB expressed in plants lacking the cognate resistance gene RPM1 suppresses cell wall defense induced by the flagellar peptide flg22, a well known PAMP, and promotes the growth of nonpathogenic bacteria in a RAR1-dependent manner. rar1 mutants display enhanced cell wall defense in response to flg22, indicating that RAR1 negatively regulates PTI. Furthermore, coimmunoprecipitation experiments indicated that RAR1 and AvrB interact in the plant. The results demonstrate that RAR1 molecularly links PTI, effector virulence, and ETI. The study supports that both pathogen virulence and plant disease resistance have evolved around PTI.

Arabidopsis↗
PubMed2001

Dynamic properties of the Ras switch I region and its importance for binding to effectors.

We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)(.)GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. (31)P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. (31)P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wild-type and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.

Amino Acid Substitution↗
PubMed2003

S-adenosyl-L-methionine is an effector in the posttranscriptional autoregulation of the cystathionine gamma-synthase gene in Arabidopsis.

Cystathionine gamma-synthase, the first committed enzyme of methionine biosynthesis in higher plants, is encoded by the CGS1 gene in Arabidopsis thaliana. We have shown previously that the stability of the CGS1 mRNA is negatively regulated in response to methionine application [Chiba, Y., Ishikawa, M., Kijima, F., Tyson, R. H., Kim, J., Yamamoto, A., Nambara, E., Leustek, T., Wallsgrove, R. M. & Naito, S. (1999) Science 286, 1371-1374]. To determine whether methionine itself is the effector of the CGS1 exon 1-mediated posttranscriptional regulation, we carried out transfection experiments. The results suggested that, rather than methionine, S-adenosyl-L-methionine (AdoMet), or one of its metabolites, acts as the effector of this regulation. To further identify the actual effector, we exploited the wheat germ in vitro translation system. The effects of various metabolites and analogs of AdoMet were tested by using RNA carrying a CGS1 exon 1-reporter fusion. These tests identified AdoMet as the effector of this regulation. S-adenosyl-L-ethionine, an analog of AdoMet, also had effector activity. A. thaliana mto1 mutants, which are deficient in this regulation, showed a much reduced response to AdoMet in vitro, with a leaky allele showing a less reduced response. RNA translated in vitro in the presence of AdoMet contained a 5'-truncated RNA species, similar to the one that we previously suggested was an in vivo degradation intermediate of CGS1 mRNA. Together, the results show that the basic reactions of CGS1 exon 1-mediated posttranscriptional regulation occur in the wheat germ in vitro translation system, and that AdoMet acts as the effector.

Amino Acid Sequence↗
PubMed1997

Genghis Khan (Gek) as a putative effector for Drosophila Cdc42 and regulator of actin polymerization.

The small GTPases Cdc42 and Rac regulate a variety of biological processes, including actin polymerization, cell proliferation, and JNK/mitogen-activated protein kinase activation, conceivably via distinct effectors. Whereas the effector for mitogen-activated protein kinase activation appears to be p65PAK, the identity of effector(s) for actin polymerization remains unclear. We have found a putative effector for Drosophila Cdc42, Genghis Khan (Gek), which binds to Dcdc42 in a GTP-dependent and effector domain-dependent manner. Gek contains a predicted serine/threonine kinase catalytic domain that is 63% identical to human myotonic dystrophy protein kinase and has protein kinase activities. It also possesses a large coiled-coil domain, a putative phorbol ester binding domain, a pleckstrin homology domain, and a Cdc42 binding consensus sequence that is required for its binding to Dcdc42. To study the in vivo function of gek, we generated mutations in the Drosophila gek locus. Egg chambers homozygous for gek mutations exhibit abnormal accumulation of F-actin and are defective in producing fertilized eggs. These phenotypes can be rescued by a wild-type gek transgene. Our results suggest that this multidomain protein kinase is an effector for the regulation of actin polymerization by Cdc42.

Actins↗
PubMed2000

Pseudomonas syringae Hrp type III secretion system and effector proteins.

Pseudomonas syringae is a member of an important group of Gram-negative bacterial pathogens of plants and animals that depend on a type III secretion system to inject virulence effector proteins into host cells. In P. syringae, hrp/hrc genes encode the Hrp (type III secretion) system, and avirulence (avr) and Hrp-dependent outer protein (hop) genes encode effector proteins. The hrp/hrc genes of P. syringae pv syringae 61, P. syringae pv syringae B728a, and P. syringae pv tomato DC3000 are flanked by an exchangeable effector locus and a conserved effector locus in a tripartite mosaic Hrp pathogenicity island (Pai) that is linked to a tRNA(Leu) gene found also in Pseudomonas aeruginosa but without linkage to Hrp system genes. Cosmid pHIR11 carries a portion of the strain 61 Hrp pathogenicity island that is sufficient to direct Escherichia coli and Pseudomonas fluorescens to inject HopPsyA into tobacco cells, thereby eliciting a hypersensitive response normally triggered only by plant pathogens. Large deletions in strain DC3000 revealed that the conserved effector locus is essential for pathogenicity but the exchangeable effector locus has only a minor role in growth in tomato. P. syringae secretes HopPsyA and AvrPto in culture in a Hrp-dependent manner at pH and temperature conditions associated with pathogenesis. AvrPto is also secreted by Yersinia enterocolitica. The secretion of AvrPto depends on the first 15 codons, which are also sufficient to direct the secretion of an Npt reporter from Y. enterocolitica, indicating that a universal targeting signal is recognized by the type III secretion systems of both plant and animal pathogens.

Bacterial Proteins↗
PubMed1997

The coiled-coil region of the G protein beta subunit. Mutational analysis of Ggamma and effector interactions.

The beta and gamma subunits of the heterotrimeric G proteins remain tightly associated throughout the signaling cycle as the betagamma dimer interacts with Galpha, receptors, and effectors. A coiled-coil structure involving alpha-helical segments at the N termini of the beta and gamma subunits contributes to the dimerization interface and has been implicated in effector signaling in yeast. Scanning mutagenesis of the coiled-coil region of the mammalian beta1 subunit was performed to examine the effect of point mutations on betagamma assembly and effector signaling in COS cell cotransfection assays. In addition to the E10K mutation described previously, mutations A11E, L14E, and I18E in beta1 were found to block betagamma association, as evidenced by the failure of the Gbeta mutants to undergo cytosolic translocation with cotransfected nonisoprenylated Ggamma. Although none of 14 beta1 point mutations prevented the betagamma-dependent activation of the c-Jun N-terminal kinase (JNK) effector pathway, the D20K point mutation enhanced JNK but not phospholipase C-beta2 activation. These findings implicate the coiled-coil region of Gbeta in JNK signaling, provide further evidence that the structural features of the betagamma complex mediating effector regulation may differ among effectors, and identify single codons in the mammalian beta subunit where mutation might yield a phenotype of defective signal transduction.

Amino Acid Sequence↗
PubMed2002

Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions.

A peptide corresponding to the basic (+13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic residues, Lys-13 and Arg-13, bind to PIP(2)-containing vesicles with the same high affinity as the effector domain peptide. Second, removing basic residues from the effector domain peptide reduces the binding energy by an amount that correlates with the number of charges removed. Third, peptides corresponding to a basic region in GAP43 and MARCKS effector domain-like regions in other proteins (e.g. MacMARCKS, adducin, Drosophila A kinase anchor protein 200, and N-methyl-d-aspartate receptor) also bind with an energy that correlates with the number of basic residues. Kinetic measurements suggest the effector domain binds to several PIP(2). Theoretical calculations show the effector domain produces a local positive potential, even when bound to a bilayer with 33% monovalent acidic lipids, and should thus sequester PIP(2) laterally. This electrostatic sequestration was observed experimentally using a phospholipase C assay. Our results are consistent with the hypothesis that MARCKS could reversibly sequester much of the PIP(2) in the plasma membrane.

Amino Acid Sequence↗
PubMed2004

Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase.

Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related. With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs). We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric alpha(2) beta(2) structure. The component alpha(2)called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the beta(2) component called R2 harbors the tyrosyl radical necessary for catalysis. Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2. Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2-3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments. The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction. Based on the experimental setup, we propose that the inhibition of enzyme activity in the E. coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2. Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold.

Allosteric Regulation↗
PubMed2000

Interaction between actin and the effector peptide of MARCKS-related protein. Identification of functional amino acid segments.

It is widely assumed that the members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with actin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. To clarify the mechanisms by which this interaction takes place, we have examined the effect of a peptide corresponding to the effector domain of MRP, the so-called effector peptide, on both the dynamic and the structural properties of actin. We show that in the absence of cations the effector peptide polymerizes monomeric actin and causes the alignment of the formed filaments into bundle-like structures. Moreover, we document that binding of calmodulin or phosphorylation by protein kinase C both inhibit the actin polymerizing activity of the MRP effector peptide. Finally, several effector peptides were synthesized in which positively charged or hydrophobic segments were deleted or replaced by alanines. Our data suggest that a group of six positively charged amino acid residues at the N-terminus of the peptide is crucial for its interaction with actin. While its actin polymerizing activity critically depends on the presence of all three positively charged segments of the peptide, hydrophobic amino acid residues rather modulate the polymerization velocity.

ATP-Binding Cassette Transporters↗
PubMed1978

H-2-restricted cytotoxic effectors generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins.

Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 mug of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.

Animals↗
PubMed1991

Characterization of antigen-specific CD4+ effector T cells in vivo: immunization results in a transient population of MEL-14-, CD45RB- helper cells that secretes interleukin 2 (IL-2), IL-3, IL-4, and interferon gamma.

In previous studies we demonstrated that, following activation by mitogens or alloantigens, helper T cell precursors proliferate and differentiate in vitro to produce a population of effector cells that secrete high titers of lymphokines upon restimulation. In this report, we demonstrate that a similar effector population develops in vivo following primary antigen stimulation. When restimulated with specific antigen in vitro, CD4+ T cells from mice primed 5 to 7 days previously by subcutaneous administration of keyhole limpet hemocyanin (KLH) in adjuvant, produced high levels of interleukin 2 (IL-2), IL-4, and IL-3, and little or no interferon gamma (IFN-gamma) or IL-5. The effector T cells provided excellent helper activity for in vitro antibody responses of 4-hydroxy-5-iodo-nitrophenyl acetic acid-primed B cells with the production principally of the immunoglobulin G1 (IgG1) and IgM isotypes, small quantities of IgG3, and no detectable IgG2a, or IgG2b. Antigen-specific secretion of IL-2, IL-3, and IL-4 by in vivo effectors was detectable by 12 hours following in vitro restimulation. IFN-gamma and IL-5 were not detected until 48 and 72 hours of culture, respectively, and low levels of these lymphokines were produced. Lymphokine production by primed CD4+ T cells could be induced as early as 3 days following immunization, peaked on day 5, and declined thereafter. The kinetics of in vivo appearance of effector CD4+ T cells that produce lymphokines upon restimulation in vitro were similar for each of the lymphokines examined. Mice depleted of precursor CD4+ T cells by adult thymectomy exhibited limited capacity to generate lymphokine secreting CD4+ T cells in response to primary immunization with KLH, suggesting that the majority of lymphokine producing T cells arise from short-lived and/or precursor cells. Separation of CD4+ T cells from KLH-primed mice on the basis of expression of the lymph node-specific homing receptor, MEL-14, revealed that antigen-specific production of IL-2, IL-3, IL-4, and IFN-gamma was exclusively associated with the MEL-14- subset of CD4+ T cells. Separation on the basis of CD45RB expression, demonstrated that antigen-specific lymphokine production was primarily associated with the minor CD45RB- population, which has been previously associated with memory activity. Our results indicate that primary in vivo immunization leads to the development of a transient population of helper-effectors with a unique phenotype that can produce large quantities of lymphokines and mediate excellent helper activity for B cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗