CD8+ T cells and Cross-Presentation Pathways in Leishmaniasis Sylvie Bertholet,1 Alain Debrabant,2Michel Desjardins,3 and David Sacks1 1) Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. 2) Division of Emerging and Transfusion Transmitted Diseases, OBRR, CBER, Food and Drug Administration, Bethesda, MD 20892, USA. 3) Departement de pathologie et biologie cellulaire, Universite de Montreal, Montreal, Quebec Canada H3T 1J4. Specialized APC such as macrophages, B cells and especially DC have the capacity to capture, process and present in a MHC class-I restricted manner various exogenous cell associated antigens, including minor histocompatibility antigens, tumor antigens, or antigens derived from apoptotic, necrotic and/or virus-infected cells (1-4), a process referred to as cross-presentation. Cross-presentation can be extended to a wide range of exogenous antigens derived from pathogens residing transiently or permanently in the phagosome of the host cell, as indicated by the strong CD8+ T cell response induced by infection with Leishmania, Toxoplasma gondii, Trypanosoma cruzi, mycobacteria, Salmonella typhimurium, Brucella abortus, and Listeria monocytogenes (5-9). The cross-presentation of antigens derived from intracellular pathogens might occur as described for other exogenous cell associated antigens, via the uptake of infected apoptotic or necrotic cells by DC, or by direct infection of the DC. Dependence on TAP is taken as evidence that translocation of antigens to the cytosol is required, whereas TAP-independent cross-presentation indicates that peptide ligands are generated and loaded onto MHC class I directly in the phagosome or on the cell surface following peptide regurgitation. Recently, the observation of ER-mediated phagocytosis of antigen-coated latex beads (10) has led to the description of a novel phagosome-associated cross-presentation pathway. We (11) and others (12, 13) demonstrated that OVA coated on latex beads is retrotranslocated to the cytosol, and targeted to a phagosome-associated proteasome for degradation. The peptides are then transported by TAP back to the phagosome, and onto the MHC class I/peptide loading complex. As Leishmania parasites were also shown to enter macrophages by ERmediated phagocytosis (10) and reside in a similar phagolysosomal compartment as latex beads, we were interested to know whether Leishmania antigens can be processed for cross-presentation using the same phagosome-associated pathway. In the case of Leishmania, CD8+ T cells play an essential role in primary immunity to infection with Leishmania major, as cutaneous lesions fail to heal in C57BL/6 CD8/- mice, or in anti–CD8 treated mice (14). They are also required for resistance to reinfection (15). In clinical studies of cutaneous leishmaniasis, efficient priming of CD8+ T cells and their presence within healing lesions has been a consistent finding (15, 16). We have addressed the mechanisms underlying cross-presentation of L. major parasite antigens by infected DC. L. major expressing a chimeric OVA protein was used to specifically follow the processing of the SIINFEKL epitope and its presentation to OT-I TCR transgenic CD8+ T cells. The participation of TAP was evaluated in vitro and in vivo for the presentation of the SIINFEKL epitope to OT-I CD8+ T lymphocytes. The study was further extended to the processing and presentation of wild-type L. major antigens to a primed polyclonal CD8+ T cell population. The presentation of the SIINFEKL peptide by L. major NT-OVA-infected DC in vitro or by APCs involved in cross-priming in vivo, was not affected by the absence of TAP1, suggesting that the conventional MHC class I processing pathway is not used for this particular epitope. When we extended these studies to the crosspresentation of wild-type L. major antigens recognized by in vivo primed CD8+ T cells, a dispensable role for TAP1 was still observed. Infection of TAP1 KO mice with L. major indicated a normal ability to control infection, despite the fact that acquired resistance in these mice still required the contribution of CD8+ T cells. Thus in the case of L. major, cross-presentation does not appear to follow a phagosome-to-cytosol pathway as has been reported for antigen–coated latex beads (11-13) and a number of intracellular microorganisms, including Mycobacterium tuberculosis, Listeria monocytogenes , E. coli , or Streptoccocus gordonii (18-21). Analysis of the processing of OVA originating within model latex bead phagosomes indicated that the requirement for TAP could be bypassed with higher concentrations of antigen, suggesting that TAP-dependent cross-presentation is more efficient than the TAP-independent pathway. In addition, higher concentrations of OVA were also required for cross-presentation by DC following uptake of coated beads specifically complexed with L. major promastigotes. In the presence of L. major, DC required 8 fold higher concentration of OVA on the beads to activate the OT-I cells. Thus avoidance or inhibition of the more efficient, ER-based, TAP dependent cross-presentation machinery by antigens sequestered within the Leishmania phagosome may represent a strategy whereby the parasite can delay the onset of CD8+ T cell priming, as has been clearly observed in experimental mouse models (14). This data suggests that remodeling of the phagosome by Leishmania effects the ability of exogenous antigen to be targeted through a properly constituted, ER-based processing pathway. The absence of a requirement for TAP also suggests that the crosspresentation of Leishmania antigens relies on the generation of peptide ligands and post-Golgi loading of MHC class I molecules within the phagosome. The requirement for endocytic proteases to generate TAP-independent ligands was demonstrated by the inhibition of Leishmania OVA cross-presentation by leupeptin. The requirement for phagosome maturation/ endocytic proteases may explain why DC infected with T. gondii expressing a chimeric protein, P30-OVA, residing within vacuoles that do not fuse with endosomes/lysosomes, were unable to use a TAP-independent pathway for OVA cross-presentation, even at high multiplicities of infection. DC appear capable of multiple phagosomal processing pathways, perhaps reflecting their encounter with intracellular pathogens such as Leishmania that have co-evolved to inhibit or avoid the efficient processing machinery associated with ERmediated phagocytosis. Understanding these alternative MHC class I processing and presentation pathways may prove useful for appropriate targeting of exogenous vaccine antigens for efficient priming of protectiveCD8+ T cell responses against tumors, viruses, and intracellular parasites and bacteria. References 1. Bevan, M.J. 1976. 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