Imaging effector functions of human cytotoxic CD4+ T cells specific for Plasmodium falciparum circumsporozoite protein☆
Introduction
After transmission by the bite of an infected mosquito, Plasmodium sporozoites travel from the skin to the liver, their first site of replication in the mammalian host (Vanderberg and Frevert, 2004, Amino et al., 2005). Once arrested within a hepatic sinusoid, the parasites leave the bloodstream by traversing Kupffer cells, migrate through the parenchyma of the liver, infect hepatocytes and grow to exoerythrocytic forms (EEF) (Ishino et al., 2004, Frevert et al., 2005, Baer et al., 2007b, Usynin et al., 2007). Eventually, each infected hepatocyte releases thousands of erythrocyte-infective merozoites, which begin the cycle of blood stage infection, thus initiating the clinical phase of the disease (Sturm et al., 2006, Baer et al., 2007a).
The circumsporozoite protein (CSP), the major surface antigen of the sporozoites, has been the focus of malaria vaccine development because of its immunodominance and presence in both the sporozoite and EEF stage of Plasmodium (Kumar et al., 2006). Extensive studies over the past 30 years demonstrated that both sporozoite neutralizing antibodies and cell-mediated immunity directed against infected hepatocytes, contribute to sporozoite-induced immunity (Nardin and Nussenzweig, 1993). Based on data from a variety of rodent models, CD8+ T cells are generally regarded as the principal cytotoxic effector cells, while CD4+ T cells provide T helper factors required for memory CD8+ T cell and optimal antibody responses (Romero et al., 1989, Molano et al., 2000, Carvalho et al., 2002, Doolan and Martinez-Alier, 2006). However, in the absence of CD8+ cells, CD4+ T cells can also provide direct protective immunity in both sporozoite and peptide-immunised animals (Tsuji et al., 1990, Rénia et al., 1991, Rénia et al., 1993, Wang et al., 1996, Charoenvit et al., 1999, Oliveira et al., 2008). One major protective mechanism is dependent on IFN-γ, a cytokine produced by both CD4+ and CD8+ cells, which induces nitric oxide (NO) production in infected hepatocytes thus killing the intracellular parasites (Ferreira et al., 1987, Schofield et al., 1987, Mellouk et al., 1991, Seguin et al., 1994).
CD8+ CTL are essential for protection in some murine malaria models (Doolan and Hoffman, 2000). In other infectious diseases, human CD4+ CTLs, as well as CD8+ CTLs, have been shown to develop during viral, bacterial and fungal infections and following immunisation (Miskovsky et al., 1994, Zaunders et al., 2004, Aslan et al., 2006, Casazza et al., 2006, Mitra-Kaushik et al., 2007, Bastian et al., 2008, Leen et al., 2008). There is currently little information available about the interaction between Plasmodium-specific human CTLs, either CD4+ or CD8+, and their target cells.
CTLs utilise a granule exocytosis pathway for target cell killing (Russell and Ley, 2002, Catalfamo and Henkart, 2003, Clark and Griffiths, 2003, Lieberman, 2003, Trapani and Sutton, 2003). Cytotoxic granules are secretory lysosomes that store many of the apoptosis-inducing molecules including the pore-forming proteins perforin and granulysin, various serine proteases named granzymes, and the death receptor ligand FasL in their distinctive dense core (Peters et al., 1991, Clark and Griffiths, 2003). Under resting conditions, CD8+ CTLs are polar cells with an organelle-free leading edge and a tapered uropod containing the cytotoxic granules (Yannelli et al., 1986, Ng et al., 2008). Within minutes of specific antigen recognition, primed CD8+ CTLs round up and form a tight junction or immunological synapse with the target cell (Kupfer and Singer, 1989, Faroudi et al., 2003, Depoil et al., 2005, Dustin, 2005, Wiedemann et al., 2006). Intracellular signalling causes the microtubule organizing centre (MTOC), Golgi complex and cytotoxic granules to migrate vectorially along the microtubule network towards the site of contact (Yannelli et al., 1986, Kuhn and Poenie, 2002, Trambas and Griffiths, 2003, Dustin, 2005, Dustin, 2006). Immediately upon completion of reorientation, one or more of the granules rapidly fuse with the cell membrane and discharge their lethal content into the narrow gap between CTL and target cell. The fatal hit is delivered either via secretion of perforin and granzymes, (Keefe et al., 2005, Pipkin and Lieberman, 2007), or via release of Fas ligand (FasL), which causes the apoptotic death of cells expressing Fas on their surface (Berke, 1995). The perforin/granzyme pathway, rather than the Fas/FasL pathway, is essential for the clearance of many intracellular pathogens (Russell and Ley, 2002).
The first evidence for human CD4+ T cells with cytotoxic properties directed against a protozoan parasite came from class II-restricted T cells isolated from a sporozoite-immunised volunteer, who was protected against challenge with Plasmodium falciparum sporozoites for at least 1 year (Moreno et al., 1991). Herein we demonstrate that additional clones from sporozoite-immunised volunteers are able to lyse autologous B cells pulsed with the same epitope, the T∗ peptide (EYLNKIQNSLSTEWSPCSVT), in the context of HLA-DR 4, 7 and 9. In addition, cytotoxic CD4+ T cells that can specifically recognise autologous target cells pulsed with T∗ peptide were also isolated from volunteers immunised with a (T1BT∗) peptide vaccine containing the T∗ epitope. Upon activation, these cytotoxic subsets of both sporozoite and peptide-induced CD4+ T cell clones, which have the phenotype of memory T cells (Moreno et al., 1993b, Calvo-Calle et al., 2005), were shown to express perforin and release serine esterases. These CD4+ CTLs form typical immunological synapses with peptide-primed target cells and induce apoptosis. Together, our findings suggest that in addition to their indirect role as helper cells in the humoral immune response against sporozoites (Molano et al., 2000) multifunctional CD4+ T cells have the potential to eliminate EEFs from the infected liver through direct effector mechanisms, similar to CD8+ T cells. This work provides the basis for more detailed spatial and temporal analysis of the interaction between cytotoxic T cells and the malaria-infected liver.
Section snippets
Human CD4+ T cell lines and clones
The CD4+ T cell clones have been described in detail previously and were established either from protected volunteers immunised with irradiated P. falciparum sporozoites (Moreno et al., 1991, Moreno et al., 1993a) or volunteers immunised with (T1BT∗)4-P3C malaria peptide vaccine containing immunodominant T and B cell epitopes of the P. falciparum CSP (Moreno et al., 1991, Moreno et al., 1993a, Nardin et al., 2001, Calvo-Calle et al., 2005). Sporozoite-induced clones were isolated from
Sporozoite-induced CD4+ CTL clones
A series of CD4+ T cell clones were derived from three sporozoite-immunised volunteers by stimulation of their immune PBMC with peptide representing the T∗ epitope of P. falciparum CSP (NF54 isolate; aa 326-345). These CD4+ T cell clones proliferated when stimulated with T∗ peptide in the context of multiple class II molecules, DR 1, 4, 7 or 9 (Moreno et al., 1993a). Consistent with previous observations using a DR7 restricted CTL clone derived from one of the sporozoite-immunised volunteers (
Discussion
These studies demonstrate that Plasmodium-specific cytotoxic CD4+ T cells can be elicited in volunteers by immunisation with irradiated sporozoites as well as by immunisation with a synthetic peptide vaccine containing a universal T cell epitope of P. falciparum CSP. The high-avidity CD4+ CTLs induce apoptosis in autologous target cells primed with cognate peptide. These human CD4+ CTLs are multifunctional as, in addition to cytolytic activity, they secrete a broad range of cytokines when
Acknowledgements
We acknowledge the expert technical assistance provided by Rita Altszuler. Human rIL2 was obtained through the AIDs Research and Reference Reagent Program, NIAID, NIH: donated by Dr. Maurice Gately, Hoffman-La Roche Inc. The work was supported by NIH Grants RO1 AI 25085 and RO1 AI45138 to EN and RO1 AI51656 and S10 RR019288 to U.F. C.K. received a postdoctoral fellowship from the American Liver Foundation.
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Note: Supplementary data associated with this article.
- 1
Emory Vaccine Center at Yerkes National Primate Research Center and Division of Infectious Diseases, Department of Medicine, Emory University, Atlanta, GA, USA.
- 2
Department of Pathology, University of Massachusetts Medical School, Worcester, MA, USA.
- 3
Department of Molecular Parasitology, Humboldt-University Berlin, Germany.