Journal of Immunology

Mice genetically deficient in IL-7R(alpha) are highly lymphopenic in the peripheral lymphoid organs. The functional competence of T cells that have developed in the absence of an IL-7R signal was investigated. Three important observations were made using several in vitro activation regimens. First, stimulation of T cells from IL-7R -/- mice at limiting dilution with immobilized Abs to CD3, CD4 or CD8, and CD18 revealed a six- to sevenfold reduction in the frequency of clonogenic T cells compared with T cells from IL-7R +/+ mice. IL-7R -/- T cells were also significantly less responsive to alloantigen as well as to receptor-independent stimuli such as PMA and ionomycin. Furthermore, the average clone size of single IL-7R -/- T cells was 50% smaller than that of IL-7R +/+ T cells. These data suggest that the reduced clonogenicity was predominantly due to intrinsic deficiencies in the ability of IL-7R -/- T cells to proliferate upon stimulation. Second, analysis of the kinetics of cell growth of IL-7R -/- T cells revealed that a significant proportion of T cells failed to proliferate within the first 72 h of in vitro stimulation, with the majority undergoing programmed cell death. Third, both clonogenic IL-7 -/- T cells and IL-7R +/+ T cells showed a similar proliferative response in the presence of IL-2 and similar survival kinetics, indicating that a subpopulation of IL-7R -/- T cells is functionally mature. We propose that an absence of IL-7R signaling not only affects T cell development in the thymus, but also results in the accumulation of functionally inactive T cells in the periphery.

Secretory phospholipases A2 (sPLA2s) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA sPLA2 has been detected in inflammatory fluids, and its plasma level is increased in inflammatory diseases. To investigate a potential mechanism of sPLA2-induced inflammation we studied the effect of group IA (from cobra venom) and group IIA (human synovial) sPLA2s on human macrophages. Both sPLA2s induced a concentration- and Ca2+-dependent, noncytotoxic release of β-glucuronidase (16.2 ± 2.4% and 13.1 ± 1.5% of the total content with groups IA and IIA, respectively). Both sPLA2s also increased the rate of secretion of IL-6 and enhanced the expression of IL-6 mRNA. Preincubation of macrophages with inhibitors of the hydrolytic activity of sPLA2 or cytosolic PLA2 did not influence the release of β-glucuronidase. Incubation of macrophages with p-aminophenyl-mannopyranoside-BSA (mp-BSA), a ligand of the mannose receptor, also resulted in β-glucuronidase release. However, while preincubation of macrophages with mp-BSA had no effect on β-glucuronidase release induced by group IIA sPLA2, it enhanced that induced by group IA sPLA2. A blocking Ab anti-mannose receptor inhibited both mp-BSA- and group IIA-induced β-glucuronidase release. Taken together, these data indicate that group IA and IIA sPLA2s activate macrophages with a mechanism independent from their enzymatic activities and probably related to the activation of the mannose receptor or sPLA2-specific receptors. The secretion of enzymes and cytokines induced by sPLA2s from human macrophages may play an important role in inflammation and tissue damage associated with the release of sPLA2s.

In vivo priming of CD8+ T lymphocytes against exogenously processed model Ags requires CD4+ T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4+ T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8+ T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8+ T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8+ T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8+ T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immunized with L. monocytogenes and OVA-loaded splenocytes, cross-priming of OVA-specific naive CD8+ T cells occurred. No significant decline in cross-priming against OVA was measured when either TNF or IFN-γ was neutralized in L. monocytogenes-infected animals, demonstrating that multiple signals exist to overcome CD40L blockade of CD8+ T cell cross-priming during bacterial infection. These data support a model in which DCs can be stimulated in vivo through signals other than CD40, becoming APC that can effectively stimulate CD8+ T cell responses against exogenous Ags during infection.

The contribution of B lymphocytes as APCs for CD4+ T cell priming remains controversial, based on findings that B cells cannot provide the requisite ligating and costimulatory signals for naive T cells to be activated. In the current study, we have examined Ag-specific T:B cell collaboration under circumstances in which B cells take up Ag through Ig receptors in vivo. This results in their activation and an ability to effectively stimulate naive CD4+ T cells both in vitro and in vivo. The aim of this work was to establish some of the key molecular interactions, as well as kinetics, between Ag-specific T and B cells that enable this priming to take place. Our approach was to amplify the starting pools of both Ag-specific T and B cell populations in vivo to track directly the events during initial T:B cell collaborations. We show that the induction of optimal levels of T cell priming to a protein Ag requires the involvement of Ag-specific B cells. The interaction that results between Ag-specific T and B cells prevents the down-modulation of B7 costimulatory molecules usually observed in the absence of appropriate T cells. Moreover, this prevention in down-modulation is independent of CD40:CD40 ligand contact. Finally, we present data suggesting that once Ag-specific T and B cells interact, there is a rapid (1–2-h) down-regulation of antigenic complexes on the surface of the B lymphocytes, possibly to prevent them from engaging other T cells in the vicinity and therefore focus the initial interaction.

In contrast to euthymic adult Fischer rats, immunocompromised Nu/Nu animals develop a lethal infection when inoculated with the RH strain of the protozoan Toxoplasma gondii. However, a significant period of survival is obtained when Nu/Nu rats are passively transferred with sera from 28-day infected Fischer +/+ (euthymic) animals. Specific IgE are involved since IgE-depleted sera are unable to afford such a protection. Only excreted/secreted Ag or living tachyzoites are able to induce a significant protective IgE response in intact animals. In addition, platelets or, to a lesser extent, eosinophil-rich populations from Toxoplasma infected or excreted-secreted Ag-immunized euthymic animals bear surface IgE and are cytotoxic for the parasite in vitro. Also, adoptive transfer of immune platelets confers a significant degree of protection to Toxoplasma-infected Nu/Nu animals. Our results clearly show the key role of Ag present in both living parasites and excreted-secreted Ag to induce, in this model, a protective IgE response. In addition, as in other parasitic infections, platelets and probably eosinophils are the effector cells involved in controlling parasitic dissemination during Toxoplasma infection in immunocompromised rats.

Attenuation of signaling is a key step in controlling the cytotoxic potential of leukocyte responses to chemotactic factors. Antipeptide antibodies, directed against the N-formyl chemotactic peptide receptor (FPR) and the activation peptide from the fifth component of C (C5a) anaphylatoxin receptor (C5aR) of human neutrophils, were used to analyze the ability of these receptors to be phosphorylated. Our data show that, in granulocyte-like differentiated HL-60 cells, both FPR and C5aR undergo an agonist dose-dependent phosphorylation that reaches completion in less than 2 to 3 min, consistent with the rate and the dose-dependent attenuation of signaling in phagocytes. Therefore, phosphorylation might be one of the possible mechanisms involved in the desensitization process of FPR and C5aR. Addition of either C5a or the protein kinase C activator (PMA) did not appear to induce the phosphorylation of FPR in the absence of FMLP or to modulate the phosphorylation of the latter at low concentrations of agonist. In contrast, although FMLP at a saturating concentration barely stimulated the phosphorylation of unoccupied C5aR, it markedly potentiated C5aR phosphorylation in cells exposed to low concentrations of C5a. Moreover, PMA was able to induce C5aR phosphorylation in the absence of agonist, indicating that protein kinase C or protein kinase C-activated kinase(s) could be involved in the phosphorylation of C5aR. Pretreatment of cells with staurosporine, a potent but nonspecific inhibitor of protein kinase C, resulted in the partial inhibition of both FPR and C5aR phosphorylation induced by saturating concentrations of agonist, suggesting that a kinase different from protein kinase C might be mainly responsible for the phosphorylation of these chemotactic receptors.

The developmental stages of malaria parasites that infect E are responsible for the morbidity and mortality associated with this disease. One of the leading candidates for a blood-stage vaccine against malaria is a surface protein of merozoites, the infectious stages for E, designated merozoite surface protein-1 (MSP-1). The rodent malarial parasite Plasmodium yoelii yoelii (Py) has provided a model system for the study of this Ag, and previous studies from our laboratory had demonstrated that the carboxyl-terminal, cysteine-rich region of MSP-1, when expressed in a native configuration, could immunize mice against a normally lethal challenge infection with Py. We have now prepared a new fusion construct with the glutathione-S-transferase gene of Schistosoma japonicum joined to the carboxyl-terminal 11 kDa of Py MSP-1. This includes only the two epidermal growth factor-like domains of the MSP-1 protein. When expressed in recombinant Escherichia coli, the fusion protein induces a strong protective response in BALB/c mice as judged by the resistance of immunized animals to a virulent challenge infection. Moreover, we demonstrate that this resistance can be transferred passively by immune serum or by purified Ig, establishing a significant role for humoral immunity in protection. No role for CD4+ or CD8+ T cells could be identified in the first 12 days after challenge infection in immune mice selectively depleted of these cells; however, after this time, parasitemias gradually increased in mice depleted of CD4+ T cells, suggesting an active host response is necessary to completely eliminate the infection.

The 195-kDa major merozoite surface antigen of Plasmodium falciparum (Pf PMMSA) is a potential candidate for the development of a blood-stage malarial vaccine. We have focused on an analogous 230-kDa Ag of the rodent malarial parasite, Plasmodium yoelii, in an effort to study this protein in an experimental model system. Previously we reported the cloning and sequencing of a 2.1-kb portion of the gene encoding the carboxyl-terminal 77 kDa of the Py PMMSA. This region contained the B cell epitope recognized by mAb 302, a mAb shown to protect mice passively against P. yoelii challenge infection. To localize this B cell epitope, we have inserted various restriction fragments of the cloned Py PMMSA sequence into the bacterial expression vector pMG27NSTerm. Recombinant peptides of 74, 40, 34, 17, and 10 kDa have been produced which bear the epitope recognized by mAb 302. The results demonstrate that this B cell epitope is located within the most carboxyl-region of the Py PMMSA which contains a series of ten cysteine residues, also found in the PMMSA of P. falciparum. Further analysis showed that the reduction of disulfide bonds as well as the deletion of CYS-607 of the cloned sequence, resulted in the loss of the expression of this epitope. It is of interest that this epitope does not appear to be a dominant B cell determinant of the Py PMMSA molecule during infection. Inasmuch as this cysteine-rich domain of Py PMMSA displays considerable homology with that of Pf PMMSA, our data suggest that this region of the Pf PMMSA should be considered for inclusion in the development of a blood-stage vaccine.