CD40 Is Required for Protective Immunity against Liver Stage Plasmodium Infection Sara A. Murray, Isaac Mohar, Jessica L. Miller, Katherine J. Brempelis, Ashley M. Vaughan, Stefan H. I. Kappe and Ian This information is current as N. Crispe of October 2, 2021. J Immunol published online 2 February 2015 http://www.jimmunol.org/content/early/2015/01/31/jimmun ol.1401724 Downloaded from Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision http://www.jimmunol.org/ • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription by guest on October 2, 2021 Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 2, 2015, doi:10.4049/jimmunol.1401724 The Journal of Immunology CD40 Is Required for Protective Immunity against Liver Stage Plasmodium Infection Sara A. Murray,* Isaac Mohar,† Jessica L. Miller,‡ Katherine J. Brempelis,* Ashley M. Vaughan,‡ Stefan H. I. Kappe,*,‡ and Ian N. Crispe† The costimulatory molecule CD40 enhances immunity through several distinct roles in T cell activation and T cell interaction with other immune cells. In a mouse model of immunity to liver stage Plasmodium infection, CD40 was critical for the full maturation of liver dendritic cells, accumulation of CD8+ T cells in the liver, and protective immunity induced by immunization with the Plasmodium yoelii fabb/f2 genetically attenuated parasite. Using mixed adoptive transfers of polyclonal wild-type and CD40- deficient CD8+ T cells into wild-type and CD40-deficient hosts, we evaluated the contributions to CD8+ T cell immunity of CD40 expressed on host tissues including APC, compared with CD40 expressed on the CD8+ T cells themselves. Most of the effects of CD40 could be accounted for by expression in the T cells’ environment, including the accumulation of large numbers of CD8+ Downloaded from T cells in the livers of immunized mice. Thus, protective immunity generated during immunization with fabb/f2 was largely dependent on effective APC licensing via CD40 signaling. The Journal of Immunology, 2015, 194: 000–000. espite the recent success of public health measures, dicate that, during immunization with RAS, CD4+ T cells are malaria remains widespread in Sub-Saharan Africa, needed to generate optimal numbers of CD8+ T cells, although Southeast Asia, and South America and continues to they appear not to shape the quality of effector function or D http://www.jimmunol.org/ cause morbidity and mortality and impede socioeconomic progress. memory response (11). Climate change threatens to extend the range of Plasmodium- There are several routes by which CD4+ T cells provide help to infected Anopheles mosquitoes and the complexity of vector CD8+ T cells including licensing APC to better prime CD8+ control and swift spread of drug resistance make development of T cells and signaling CD8+ T cells directly via cytokines or sur- an effective vaccine imperative (1). Live, attenuated Plasmodium face molecules. Interaction between CD40, a costimulatory mol- vaccines against liver stage infection have shown great promise in ecule expressed on APC and CD8+ T cells, and CD40L expressed the mouse model and are now being optimized in preliminary on CD4+ T cells is a core mechanism of CD4+ T cell help (12, 13). human clinical trials (1–3). These attenuated strains are invaluable Frequently used to improve responses in antipathogen or antitu- as a model of effective sterilizing immunity and can be used to mor vaccine studies (14, 15), CD40 stimulation induces APC to by guest on October 2, 2021 determine the mechanisms that must be triggered during immu- secrete inflammatory Th1 cytokines such as IL-12 and IFN-g and nization to generate a protective and long-lasting response that can to upregulate Ag presentation and costimulatory molecules, en- prevent symptomatic blood stage infection. hancing the cells’ ability to recruit and prime T cells (16). IL-12, Radiation-attenuated sporozoites (RAS) and genetically atten- IFN-g, and Th1 responses have been strongly implicated in pro- uated parasites (GAP) elicit strong CD8+ T cell responses that tection against liver stage Plasmodium infection and other protect immunized mice from infectious challenge (4–6). It is intracellular parasites (17–20). IL-4–secreting CD4+ T cells, unclear by which cells CD8+ T cells are primed; hepatocytes, liver a hallmark of Th2 responses, may also be required for protective dendritic cells (DC), and APC in the skin-draining lymph node immunity conferred by RAS, throwing into question whether Th2 have all been implicated (7–9). Although they are not involved in or Th1 responses aid immunity against liver stage infection (21, the effector response, CD4+ T cells are required during immuni- 22). zation to induce protective immunity (6, 10). Recent studies in- CD40 signaling also promotes CD8+ T cell activation and proliferation and can influence the memory program and prevent T cell exhaustion (23, 24). In noninflammatory model systems, + *Department of Global Health, University of Washington, Seattle, WA 98195; CD40 expressed on the CD8 T cell is critical for the development †Department of Pathology, University of Washington, Seattle, WA 98195; and ‡ of an effective memory response, whereas in viral and bacterial Seattle Biomedical Research Institute, Seattle, WA 98109 infections, it is not required, and CD40 on the APC drives the Received for publication July 8, 2014. Accepted for publication January 2, 2015. CD8+ T cell response (25–27). Whether immunity to an intra- This work was supported by the Seattle Biomedical Research Institute, the University cellular eukaryotic parasite such as the liver stage of Plasmodium of Washington Department of Pathology, and the University of Washington Pathobi- + ology Program “Diseases of Public Health Importance” National Institutes of Health relies on CD40 as a route of CD4 T cell help is unclear. In this Training Grant T32AI007509-13. study, we explore the role of CD40 in generating a protective Address correspondence and reprint requests to Dr. Ian N. Crispe, Department of immune response during primary immunization with the late- Pathology, University of Washington, 1595 Northeast Pacific Street, Seattle, WA arresting attenuated strain Plasmodium yoelii fabb/f2 (28). 98195. E-mail address: [email protected] 2 2 Rather than using a single T cell specificity to investigate the Abbreviations used in this article: CD40 / , CD40-deficient; CD62L, L-selectin; CS, 2 circumsporozoite; DC, dendritic cell; GAP, genetically attenuated parasite; HCV, response to fabb/f immunization, we chose to examine the total + hepatitis C virus; KC, Kupffer cell; LSEC, liver sinusoidal endothelial cell; MPEC, CD8 T cell response to be able to draw conclusions that would memory precursor effector cell; RAS, radiation-attenuated sporozoite; WT, wild- apply to the full range of polyclonal responses to the parasite’s type. many Ags. An alternative approach to using Ag-specific T cell Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 clones or tetramers would be to examine activated Ag-specific www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401724 2 CD40 SUPPORTS CD8+ T CELLS IN MALARIA LIVER STAGE IMMUNITY CD8+ T cells that are CD11ahiCD8alo (29); however, the high remove hepatocytes. The supernatant was applied to a 20% iodixanol frequency of activated T cells present in both the resting and the (Optiprep) gradient and spun at 2500 rpm for 25 min to isolate non- immunized liver makes this method difficult to apply to cells parenchymal cells for flow cytometric and gene expression analysis. Several lymph nodes (the inguinal, celiac, and portal vein lymph nodes) collected from the liver (30, 31). We find that without CD40, mice 2 and the spleen were collected and made into separate lymph node and normally protected by P. yoelii fabb/f immunizations are not able spleen single-cell suspensions for flow cytometric analysis. to withstand infectious challenge. Moreover, CD40 signaling is a key requirement for multiple components of the response in- Cell staining for flow cytometry and cell sorting 2 duced by fabb/f immunization, and CD40 expressed on the CD8+ Lymphocytes from the liver, spleen, and lymph nodes were stained and T cell has a distinct function from that of CD40 expressed on the interrogated using panels that included the following Abs from BioLegend: APC. anti-CD3 (145-2C11), anti-CD8 (53-6.7), anti-CD4 (RM4-5), anti-CD90.2 (53-2.1), anti–L-selectin (CD62L) (MEL-14), anti-CD44 (IM7), anti- CD11b (ICRF44) anti-CD122 (5H4), anti-CD86 (GL-1), anti-CD80 (16- Materials and Methods 10A1), and anti-IA/IE (M5/115.15.2); from BD Biosciences: anti-CD45.1 Ethics statement (A20), anti–IFN-g (XMG1.2), anti-CD25 (7D4), and anti-CD11c (N418); and from eBioscience: anti-KLRG1 (2F1). Intracellular anti–IFN-g stain- The animal experiments described in this study were performed according ing was performed using a Cytofix/Cytoperm kit (BD Biosciences) after to the regulations of the Institutional Animal Care and Use Committee and cells were stimulated with ionomycin and PMA for 4 h and treated with the Office of Laboratory Animal Welfare, under the protocol NC-06 ap- brefeldin A to inhibit protein transport. proved by the Seattle BioMed Animal Care and Use Committee. Liver To conduct surface marker expression analysis and to sort liver CD8+ and perfusions were performed under terminal anesthetization with Avertin CD4+ T cells and DC into separate populations for gene expression (tribromoethanol).