The role of the liver in immunity to blood-stage murine malaria.

Mice vaccinated with fixed parasitized red blood cells and Bordetella pertussis can clear an otherwise lethal Plasmodium yoelii infection in 7 days; this protection is abolished by splenectomy before vaccination. Most mice splenectomized following vaccination were able to clear their infections, although their recovery was delayed. When labelled parasitized red cells were injected into mice during an infection, splenic uptake fell from day 3 onwards while uptake by the liver increased. Lymphocytes (mainly T cells) formed the majority of the live cells extracted from livers 7 days after infection, although blasts and myeloid cells were also present. Infected livers from vaccinated mice contained most cells. Less marked increases were observed 7 days after P. berghei infection of vaccinated mice. Examination of liver tissue showed that the sinusoids contained increased numbers of cells and suggested that activation of Kupffer cells was occurring, particularly in vaccinated mice infected with P. yoelii. Homing experiments confirmed the increased trapping of various cells in livers of vaccinated mice infected with P. yoelii. These results suggest an important role for the liver in recovery from blood-stage malaria infection.     

T cells and protective immunity to Plasmodium berghei in rats.

Experiments were carried out in which unfractionated spleen cells, and T lymphocyte subpopulations characterized by certain experimental criteria, were isolated at various times from rats infected with Plasmodium berghei. By adoptive transfer it was shown that unfractionated spleen cells, and T cells alone, could transfer protection to syngenic recipients as early as 11 days after infection of the cell donors. The protection conferred by T cells increased with the duration of the infection in the donors, at least up to 100 days. The additional presence of B cells in transferred lymphocyte populations enhanced their protective capacity over that shown by T cells alone. The role of T cells in protective immunity to malaria is discussed.     

A hybrid multistage protein vaccine induces protective immunity against murine malaria

We have previously reported the design and expression of chimeric recombinant proteins as an effective platform to deliver malaria vaccines. The erythrocytic and exoerythrocytic protein chimeras described included autologous T helper epitopes genetically linked to defined B cell epitopes. Proof-of-principle studies using vaccine constructs based on the Plasmodium yoelii circumsporozoite protein (CSP) and P. yoelii merozoite surface protein-1 (MSP-1) showed encouraging results when tested individually in this mouse malaria model. To evaluate the potential synergistic or additive effect of combining these chimeric antigens, we constructed a synthetic gene encoding a hybrid protein that combined both polypeptides in a single immunogen. The multistage vaccine was expressed in soluble form in Escherichia coli at high yield. Here we report that the multistage protein induced robust immune responses to individual components, with no evidence of vaccine interference. Passive immunization using purified IgG from rabbits immunized with the hybrid protein conferred more robust protection against the experimental challenge with P. yoelii sporozoites than passive immunization with purified IgG from rabbits immunized with the individual proteins. High antibody titers and high frequencies of CD4(+)- and CD8(+)-specific cytokine-secreting T cells were elicited by vaccination. T cells were multifunctional and able to simultaneously produce interleukin-2 (IL-2), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α). The mechanism of vaccine-induced protection involved neutralizing antibodies and effector CD4(+) T cells and resulted in the control of hyperparasitemia and protection against malarial anemia. These data support our strategy of using an array of autologous T helper epitopes to maximize the response to multistage malaria vaccines.     

A role for natural regulatory T cells in the pathogenesis of experimental cerebral malaria

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection that is responsible for a significant number of deaths in children and nonimmune adults. A failure to control blood parasitemia and subsequent sequestration of parasites to brain microvasculature are thought to be key events in many CM cases. Here, we show for the first time, to our knowledge, that CD4(+)CD25(+)Foxp3(+) natural regulatory T (Treg) cells contribute to pathogenesis by modulating immune responses in P. berghei ANKA (PbA)-infected mice. Depletion of Treg cells with anti-CD25 monoclonal antibody protected mice from experimental CM. The accumulation of parasites in the vasculature and brain was reduced in these animals, resulting in significantly lower parasite burdens compared with control animals. Mice lacking Treg cells had increased numbers of activated CD4(+) and CD8(+) T cells in the spleen and lymph nodes, but CD8(+) T-cell recruitment to the brain was selectively reduced in these mice. Importantly, a non-Treg-cell source of interleukin-10 was critical in preventing experimental CM. Finally, we show that therapeutic administration of anti-CD25 monoclonal antibody, even when blood parasitemia is established, can prevent disease, confirming a critical and paradoxical role for Treg cells in experimental CM pathogenesis.     

T-cell immunity in murine malaria: adoptive transfer of resistance to Plasmodium chabaudi adami in nude mice with splenic T cells.

Acute infections caused by the murine malarial parasite Plasmodium chabaudi adami are resolved by antibody-independent mechanisms of immunity. The fact that athymic nude mice developed high-grade unrelenting malaria and died when infected with this parasite suggested a significant role for T lymphocytes. Using adoptive transfer techniques, we demonstrated that spleen cells from either nonimmune or immune donor BALB/c mice eventually suppressed P. chabaudi adami infections in histocompatible recipient nude mice in a dose-dependent manner. Infections in recipients of "immune" spleen cells were less severe, demonstrating a depressed peak parasitemia and a shortened duration of patent infection, than was observed in recipients of normal spleen cells. Also, when sufficient numbers of immune spleen cells were transferred, the second wave of parasitemia (characteristic of this infection in nonimmune mice) failed to occur. T lymphocytes mediated protection in recipient mice, since T-cell-enriched, but not B-cell-enriched, spleen cell fractions suppressed P. chabaudi adami infections in nude mice. Protection was best achieved with T cells that bore the L3T4 phenotype. Patent parasitemias developed in all recipient mice, suggesting that the grafted cells did not limit parasite growth directly but achieved this end by activating other as yet unidentified inhibiting cell systems.     

The parasitology of malaria and the study of protective immunity

Phenotypic antigenic variation within parasite strains and antigenic diversity between strains are important parameters in understanding the development of immunity to malaria. With the Plasmodium chabaudi chabaudi model in inbred mice, a combination of serological tests and DNA technology can be used to study the specificity of inducer and effector mechanisms in an infection with characteristics similar in many ways to those of P. falciparum in man.     

The regulation and role of T follicular helper cells in immunity

It is well established that the generation of a high-affinity long-lived antibody response requires the presence of T cells, specifically CD4+ T cells. These CD4+ T cells support the generation of a germinal centre (GC) response where somatic hypermutation and affinity maturation take place leading to the generation of memory B cells and plasma cells, which provide long-lasting protection. Greater insight into the nature of the CD4+ T cells involved in this process was provided by two studies in 2000 that described CD4+ T cells residing in the B cell follicle that expressed CXCR5. As a result these cells were named follicular B helper T cells, now more commonly known as T follicular helper (Tfh) cells. Since then there has been enormous growth in our understanding of these cells, now considered a distinct T helper (Th) cell lineage that can arise from naive CD4+ T cells following activation. This review summarizes some of the most recent work that has characterized Tfh cells and the pathways that lead to their generation.     

Tumor immunity to murine plasma cell tumors. II. Essential role of T lymphocytes in immune response

Spleen cells, thoracic duct lymphocytes and adherent peritoneal exudate cells from mice immunized to syngeneic plasma cell tumors were capable of transferring specific protective immunity to these tumors. Pre-treatment of these cells with anti-Θ serum or anti-lymphocyte serum, but not with anti-ΘK serum, effectively abolished the ability of lymphoid cells to transfer this immune response. These studies demonstrate that T cells are essential in the adoptive transfer of immunity. Furthermore, immunization of athymic (nude) mice to plasma cell tumors was not achieved. Attempts to activate normal macrophages in vitro with sensitized lymphoid cells were unsuccessful. Admixture of immune lymphoid cells and target tumor cells to unrelated tumor cells did not inhibit the growth of the unrelated tumor cells. It is proposed that the immune response to syngeneic tumor associated antigens of plasma cell tumors not only involves essential sensitization of T lymphocytes, but that activated cytotoxic T lymphocytes are directly responsible for inhibiting target tumor growth.     

Isolation and characterization of protective cytolytic T cells in a rodent malaria model system.

Protective immunity against malaria is induced by immunization with irradiation-attenuated sporozoites. Here we report the isolation of cytolytic T-cell (CTL) clones from BALB/c (H-2d) mice immunized with either Plasmodium berghei or Plasmodium yoelii sporozoites. The epitopes recognized by these CTL can be mimicked by synthetic peptides corresponding to a homologous region in the CS proteins of both rodent malaria species. Both peptides are recognized by the CTL in the context of the same MHC class I molecule, H-2 Kd. In vivo adoptive transfer of the CTL clones into non-immune syngeneic mice protected them from a lethal challenge of infectious sporozoites.     

The role of CD4+ T cells in BKV-specific T cell immunity

Reactivation of polyomavirus BK (BKV) infection represents a severe complication in kidney transplant (KTX) patients. We previously reported an association between a declining BK viral load and the reconstitution of CD4⁺ T cell BKV-specific immunity in patients following kidney transplantation. However, the specific contribution of CD4⁺ T cells in the regulation of BKV-replication is unknown. Nevertheless, in vitro enrichment of BKV-specific T cells and subsequent adoptive T cell transfer may improve the restoration of immune competence in KTX patients with BKV infection. To date, strategies to capture human BKV-specific T cells with the ensuing expansion to clinically useful numbers are lacking. Here, we demonstrated a comprehensive flow cytometric analysis of the BKV-specific T cell response that permits access to the majority of T cells specific for immunodominant BKV antigens. A full-spectrum evaluation of the BKV-specific T cell response was performed by stimulating peripheral blood mononuclear cells (PBMC) with a mixture of BKV immunodominant peptide pools at varying concentrations and measuring activation marker expression and cytokine secretion. We also examined the effects of co-stimulation and PBMC resting time prior to activation. We defined the narrow range of stimulation conditions that permit the capture and expansion of functional BKV-specific T cell lines. The generated BKV-specific T cell lines showed the highest specificity and functionality when the T cells were captured according to IFNγ-secretion. This study highlights the multifunctional and cytolytic BKV-specific CD4⁺ T cells as a dominant population within the generated T cell product. This method offers a novel approach for the generation of BKV-specific T cell lines for adoptive immunotherapy and underscores the critical role of CD4⁺ T cells in the clearance of BKV.     

Antigenic variation and protective immunity in Plasmodium knowlesi malaria

Rhesus monkeys were immunized with defined strains and variants of Plasmodium knowlesi and their immunity on challenge was correlated with serum levels of schizont agglutinins and specific inhibitory antibody assayed by in vitro parasite culture. The results indicate that the inhibitory antibody provides a consistent index of immune status and probably represents the protective antibody which initiates specific immune reactions in vivo. The relationship between inhibitory and schizont agglutinating antibodies is discussed.

Inhibitory antibody is predominantly specific for those variants which have produced patent infections. However, antibody active against other variants is also present at lower titre and is associated with clinical immunity on challenge with such variants. The presence of this antibody could explain why P. knowlesi parasites, which arise by antigenic variation during the course of a chronic infection, produce mild parasitaemia in the host and yet are fully virulent in normal monkeys.

The occurrence of cross-immunization between variants and between some strains of P. knowlesi is encouraging from the point of view of malaria vaccine production. A preparation containing common antigens may induce a degree of clinical immunity comparable with that appearing during the course of chronic infection.

     

The role of interleukin-5 in protective immunity to Strongyloides venezuelensis infection in mice.

We depleted or neutralized interleukin-5 (IL-5) and IL-5 receptor of C57BL/6 mice, using rat anti-murine IL-5 monoclonal antibody (NC17) and anti-murine IL-5 receptor monoclonal antibody (H7). Mice treated with these monoclonal antibodies were infected with Strongyloides venezuelensis larvae. The time-course of faecal egg output and peripheral eosinophilia were monitored. In a primary infection, anti-IL-5 treatment did not affect faecal egg output, although the eosinophil count in peripheral blood was markedly reduced. There was no difference in intestinal worm burden or faecal egg output between anti-IL-5 treated and non-treated mice. In a secondary infection, worms were expelled from the small intestine of anti-IL-5-treated mice as well as from non-treated mice. Worm recovery from the lungs of mice treated with either anti-IL-5 or anti-IL-5 receptor monoclonal antibody was the same as that of normal controls. However, a marked reduction in worm recovery was observed in re-infected mice that had not been treated with monoclonal antibodies. Treatment with anti-IL-5 or anti-IL-5 receptor monoclonal antibody suppressed blood and tissue eosinophilia. Thus the results suggested that the host's protective immunity against tissue-migrating larvae was IL-5-dependent but intestinal immunity was not.     

Mesenchymal stem cells play an important role in host protective immune responses against malaria by modulating regulatory T cells

Plasmodium spp. parasites, the causative agents of malaria, survive and replicate in human hosts by modulating host protective immune responses. In a rodent model, malaria manifests as a severe splenomegaly, with infiltration of cells and lympho‐proliferation as major contributing factors of the immunopathology. However, the cellular contents and the functions of these cells have not been well studied. Here, we report that Plasmodium berghei infection of mice leads to massive recruitment of mesen‐chymal stem cells (MSCs) in secondary lymphoid organs. Infusion of these cells into naïve mice was able to confer host resistance against malaria. Furthermore, MSCs augmented interleukin (IL)‐12 production but suppressed IL‐10 production in recipient animals. In addition, we observed dramatic reductions of regulatory T (Treg) cells in animals that received MSCs. Taken together, our findings have identified recruitment of MSCs as a novel host protective mechanism adopted by the host to combat malaria by modulating Treg‐cell responses.     

Differential involvement of non-specific suppressor T cells in two lethal murine malaria infections.

The suppression of the contact sensitivity of oxazolone in murine malaria is shown to be mediated by non-specific T suppressor cells, but to a different extent in infection caused by two different species of parasite. Depletion of T suppressor cells in vivo and/or anti-Thy 1.2 treatment in vitro indicated that in mice infected with P. berghei the suppressor effect was largely mediated by T cells. By contrast, in mice infected with a lethal strain of P. yoelii it was only partly due to T cells; B suppressor cells and/or macrophages may also be involved. However, depletion of T suppressor cells in vivo had no effect on the course of the parasitaemia or on the survival time. Therefore, we postulate that this kind of non-specific immunosuppression cannot be regarded as a major cause of lethality.     

The role of gamma/delta T cells in immunity to infection and regulation of inflammation

The role of gamma/delta T cells in immunity to bacterial infection and control of inflammation is discussed. Special emphasis is placed on the use of murine models in which various aspects of immune function can be monitored in the absence of gamma/delta T cells. Issues discussed include the response to and control of cytokine production by gamma/delta T cells. Especially relevant is the apparent paradox that gamma/delta T cells play both pro-inflammatory and antiinflammatory roles in response to infection.     

The role of γ/δ T cells in immunity to infection and regulation of inflammation

The role of γ/δ T cells in immunity to bacterial infection and control of inflammation is discussed. Special emphasis is placed on the use of murine models in which various aspects of immune function can be monitored in the absence of γ/δ T cells. Issues discussed include the response to and control of cytokine production by γ/δ T cells. Especially relevant is the apparent paradox that γ/δ T cells play both pro-inflammatory and antiinflammatory roles in response to infection.     

Macrophages as effector cells in immunity to malaria

Experiments with malaria in mice suggest that protective immunity depends not only on antibody but also on activation of macrophages. Activated macrophages may cause intra-erythrocytic death of parasites by releasing reactive oxygen intermediates and/or tumour necrosis factor. Macrophage activation for both types of product correlates well with the timing of recovery in a range of different malaria infections and in mice protected by vaccination.