Michael Overstreet

CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes.

The success of immunization with irradiated sporozoites is unparalleled among the current vaccination approaches against malaria, but its mechanistic underpinnings have yet to be fully elucidated. Using a model mimicking natural infection by Plasmodium yoelii, we delineated early events governing the development of protective CD8+ T-cell responses to the circumsporozoite protein. We demonstrate that dendritic cells in cutaneous lymph nodes prime the first cohort of CD8+ T cells after an infectious mosquito bite. Ablation of these lymphoid sites greatly impairs subsequent development of protective immunity. Activated CD8+ T cells then travel to systemic sites, including the liver, in a sphingosine-1-phosphate (S1P)-dependent fashion. These effector cells, however, no longer require bone marrow–derived antigen-presenting cells for protection; instead, they recognize antigen on parenchymal cells—presumably parasitized hepatocytes. Therefore, we report an unexpected dichotomy in the tissue restriction of host responses during the development and execution of protective immunity to Plasmodium.

Protective CD8 T cells against Plasmodium liver stages: immunobiology of an 'unnatural' immune response.

Summary: Immunization with high doses of irradiated sporozoites delivered by the bites of infected mosquitoes has been shown to induce protective responses against malaria, mediated in part by CD8+ T cells. In contrast, natural transmission involving low exposure to live sporozoite antigen fails to elicit strong immunity. In this review, we examine how irradiated sporozoite immunization breaks the natural host–parasite interaction and induces protective CD8+ T cells. Upon biting, the malaria‐infected mosquitoes deposit parasites in the skin, many of which eventually exit to the bloodstream and infect hepatocytes. However, certain antigens, including the circumsporozoite (CS) protein, remain in the skin and are presented in the draining lymph node. These antigens prime specific CD8+ T cells, which migrate to the liver where they eliminate parasitized hepatocytes. We discuss the relevance of the different tissue compartments involved in the induction and effector phases of this response, as well as the cellular requirements for priming and memory development of CD8+ T cells, which include a complete dependence on dendritic cells and a near absolute need for CD4+ T‐cell help. Finally, we discuss the impact of the immunodominant CS protein on this protection and the implications of these findings for vaccine design.

Prolonged Antigen Presentation Is Required for Optimal CD8+ T Cell Responses against Malaria Liver Stage Parasites

Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization—a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

17β-estradiol alters the activity of conventional and IFN-producing killer dendritic cells

Estrogens increase aspects of innate immunity and contribute to sex differences in the prevalence of autoimmune diseases and in response to infection. The goal of the present study was to assess whether exposure to 17β-estradiol (E2) affects the development and function of bone marrow-derived dendritic cells and to determine whether similar changes are observed in CD11c+ splenocytes exposed to E2 in vivo. E2 facilitated the differentiation of BM precursor cells into functional CD11c+CD11b+MHC class II+ dendritic cells (DCs) with increased expression of the costimulatory molecules CD40 and CD86. Exposure of bone marrow-derived dendritic cells to E2 also enhanced production of IL-12 in response to the TLR ligands, CpG and LPS. In contrast, CD11c+ cells isolated from the spleens of female C57BL/6 mice that were intact, ovariectomized, or ovariectomized with E2 replacement exhibited no differences in the number or activity of CD11c+CD11b+MHC class II+ DCs. The presence of E2 in vivo, however, increased the number of CD11c+CD49b+NK1.1low cells and reduced numbers of CD11c+CD49b+NK1.1high cells, a surface phenotype for IFN-producing killer DCs (IKDCs). Ultrastructural analysis demonstrated that CD11c+NK1.1+ populations were comprised of cells that had the appearance of both DCs and IKDCs. CD11c+ splenocytes isolated from animals with supplemental E2 produced more IFN-γ in response to IL-12 and IL-18. These data illustrate that E2 has differential effects on the development and function of DCs and IKDCs and provide evidence that E2 may strengthen innate immunity by enhancing IFN-γ production by CD11c+ cells.

Effector CD8+ T Lymphocytes against Liver Stages of Plasmodium yoelii Do Not Require Gamma Interferon for Antiparasite Activity

ABSTRACT The protective immune response against liver stages of the malaria parasite critically requires CD8+ T cells. Although the nature of the effector mechanism utilized by these cells to repress parasite development remains unclear, a critical role for gamma interferon (IFN-γ) has been widely assumed based on circumstantial evidence. However, the requirement for CD8+ T-cell-mediated IFN-γ production in protective immunity to this pathogen has not been directly tested. In this report, we use an adoptive transfer strategy with circumsporozoite (CS) protein-specific transgenic T cells to examine the role of CD8+ T-cell-derived IFN-γ production in Plasmodium yoelii-infected mice. We show that despite a marginal reduction in the expansion of naive IFN-γ-deficient CS-specific transgenic T cells, their antiparasite activity remains intact. Further, adoptively transferred IFN-γ-deficient CD8+ T cells were as efficient as their wild-type counterparts in limiting parasite growth in naive mice. Taken together, these studies demonstrate that IFN-γ secretion by CS-specific CD8+ T cells is not essential to protect mice against live sporozoite challenge.

IL‐4 induces a wide‐spectrum intracellular signaling cascade in CD8+ T cells

IL‐4 has distinct effects on the differentiation and functional properties of CD8+ T cells. In vivo studies have shown that it is critical for the development of protective memory responses against tumors and infections by Leishmania and Plasmodium parasites. The intracellular signaling events mediated by IL‐4/IL‐4 receptor (IL‐4R) interactions on CD4+ T cells have been studied extensively; however, the nature of IL‐4‐induced signaling on CD8+ T cells has not been characterized. Using naïve, activated, as well as differentiated CD8+ T cells, we show that IL‐4 has a strong in vivo and in vitro antiapoptotic effect on activated and resting CD8+ T cells. We demonstrate that IL‐4 induces the phosphorylation of the IL‐4R, which is followed by the activation of at least two distinct intracellular signaling cascades: the Jak1/STAT6 and the insulin receptor substrate/PI‐3K/protein kinase B pathways. We also found that IL‐4 induces the Jak3‐mediated phosphorylation and nuclear migration of STAT1, STAT3, and STAT5 in naïve, activated, as well as differentiated, IFN‐γ‐producing CD8+ T cells. The induction of this broad signaling activity in CD8+ T cells coincides with a transcriptional activity of suppressors of cytokine signaling genes, which are decreased significantly in comparison with CD4+ T cells. To our knowledge, this report constitutes the first comprehensive analysis of the signaling events that shape CD8+ T cell responses to IL‐4.

Memory CD8+ T Cell Responses Expand When Antigen Presentation Overcomes T Cell Self-Regulation

Antimicrobial memory CD8+ T cell responses are not readily expanded by either repeated infections or immunizations. This is a major obstacle to the development of T cell vaccines. Prime-boost immunization with heterologous microbes sharing the same CD8+ epitope can induce a large expansion of the CD8+ response; however, different vectors vary greatly in their ability to boost for reasons that are poorly understood. To investigate how efficient memory T cell expansion can occur, we evaluated immune regulatory events and Ag presentation after secondary immunization with strong and weak boosting vectors. We found that dendritic cells were essential for T cell boosting and that Ag presentation by these cells was regulated by cognate memory CD8+ T cells. When weak boosting vectors were used for secondary immunization, pre-established CD8+ T cells were able to effectively curtail Ag presentation, resulting in limited CD8+ T cell expansion. In contrast, a strong boosting vector, vaccinia virus, induced highly efficient Ag presentation that overcame regulation by cognate T cells and induced large numbers of memory CD8+ T cells to expand. Thus, efficient targeting of Ag to dendritic cells in the face of cognate immunity is an important requirement for T cell expansion.

CD4+ T Cells Modulate Expansion and Survival but Not Functional Properties of Effector and Memory CD8+ T Cells Induced by Malaria Sporozoites

CD4+ helper T cells are critical orchestrators of immune responses to infection and vaccination. During primary responses, naïve CD8+ T cells may need “CD4 help” for optimal development of memory populations. The immunological factors attributed to CD4 help depend on the context of immunization and vary depending on the priming system. In response to immunization with radiation-attenuated Plasmodium yoelii sporozoites, CD8+ T cells in BALB/c mice fail to generate large numbers of effector cells without help from CD4+ T cells – a defect not observed in most systems. Given this unique early dependence on CD4 help, we evaluated the effects of CD4+ cells on the development of functional properties of CD8+ T cells and on their ability to abolish infection. First, we determined that this effect was not mediated by CD4+ non-T cells and did not involve CD1d-restricted NKT cells. We found that CD8+ T cells induced by sporozoites without CD4 help formed memory populations severely reduced in magnitude that could not limit parasite development in the liver. The inability of these “helpless” memory T cells to protect is not a result of defects in effector function, as their capacity to produce cytokines and undergo cytotoxic degranulation was indistinguishable from control memory T cells. These data indicate that CD4+ T help may not be necessary to develop the functional attributes of CD8+ T cells; however they are crucial to ensure the survival of effector and memory cells induced in primary responses.

COLLAGEN MICROCARRIER SPINNER CULTURE PROMOTES OSTEOBLAST PROLIFERATION AND SYNTHESIS OF MATRIX PROTEINS

SummaryIn vitro propagation of osteoblasts in three-dimensional culture has been explored as a means of cell line expansion and tissue engineering purposes. Studies investigating optimal culture conditions are being conducted to produced bone-like material. This study demonstrates the use of collagen microcarrier beads as a substrate for three-dimensional cell culture. We have earlier reported that microcarriers consisting of cross-linked type I collagen support chondrocyte proliferation and synthesis of extracellular matrix. In this study, we investigated the use of collagen microcarriers to propagate human trabecular bone-derived osteoblasts. Aggregation of cell-seeded microcarriers and production of extracellular matrix-like material were observed after 5 d in culture. Expression of extracellular matrix proteins osteocalcin, osteopontin, and type I collagen was confirmed by messenger ribonucleic acid analysis, radioimmunoassay, and Western blot analysis. The efficient recovery of viable cells was achieved by collagenase digestion of the cell-seeded microcarriers. The collagen microcarrier spinner culture system provides an efficient method to amplify large numbers of healthy functional cells that can be subsequently used for further in vitro or transplantation studies.

Adenovirus particles that display the Plasmodium falciparum circumsporozoite protein NANP repeat induce sporozoite-neutralizing antibodies in mice

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world.