The Mechanism of Splenic Invariant NKT Cell Activation Dictates Localization In Vivo Irah L. King, Eyal Amiel, Mike Tighe, Katja Mohrs, Natacha Veerapen, Gurdyal Besra, Markus Mohrs and This information is current as Elizabeth A. Leadbetter of September 27, 2021. J Immunol 2013; 191:572-582; Prepublished online 19 June 2013; doi: 10.4049/jimmunol.1300299 http://www.jimmunol.org/content/191/2/572 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/06/17/jimmunol.130029 Material 9.DC1 http://www.jimmunol.org/ References This article cites 46 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/191/2/572.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 2021 • 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 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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Mechanism of Splenic Invariant NKT Cell Activation Dictates Localization In Vivo Irah L. King,*,1 Eyal Amiel,* Mike Tighe,* Katja Mohrs,* Natacha Veerapen,† Gurdyal Besra,† Markus Mohrs,*,2 and Elizabeth A. Leadbetter*,2 Invariant NKT (iNKT) cells are glycolipid-specific innate lymphocytes emerging as critical players in the immune response to diverse infections and disease. iNKT cells are activated through cognate interactions with lipid-loaded APCs, by Ag- independent cytokine-mediated signaling pathways, or a combination of both. Although each of these modes of iNKT cell activation plays an important role in directing the humoral and cell-mediated immune response, the spatio-temporal nature of these inter- actions and the cellular requirements for activation are largely undefined. Combining novel in situ confocal imaging of aGalactosylceramide-loaded CD1d tetramer labeling to localize the endogenous iNKT cell population with cytokine reporter mice, we reveal the choreography of early murine splenic iNKT cell activation across diverse settings of glycolipid immunization and Downloaded from systemic infection with Streptococcus pneumoniae. We find that iNKT cells consolidate in the marginal zone and require dendritic cells lining the splenic marginal zone for activation following administration of cognate glycolipids and during systemic infection but not following exogenous cytokine administration. Although further establishing the importance of cognate iNKT cell inter- actions with APCs, we also show that noncognate iNKT-dependent mechanisms are sufficient to mediate effector outcomes, such as STAT signaling and dendritic cell licensing throughout the splenic parenchyma. Collectively, these data provide new insight into how iNKT cells may serve as a natural adjuvant in facilitating adaptive immune responses, irrespective of their tissue local- http://www.jimmunol.org/ ization. The Journal of Immunology, 2013, 191: 572–582. nvariant NKT (iNKT) cells are a distinct lineage of ab TCR+ marginal zone (MZ) of the spleen, which lies just outside of lymphocytes that rapidly exert effector functions, such as the lymphocyte-residing white pulp, contains a wide vascular bed, I secretion of the cytokines IL-4 and/or IFN-g following en- termed the “marginal sinus,” where the arterial blood supply ter- counter with lipid Ags presented in the context of the MHC I–like minates. This site serves as the primary access point for both par- molecule CD1 (1), through cytokine-regulated mechanisms, or ticulate and cellular entry into the spleen (5). The MZ contains both (2). In addition, iNKT cells have a low threshold of activa- a number of innate leukocytes specialized for rapid detection of tion, thus providing a functional bridge between initial innate blood-borne Ags. For example, SIGNR-1+MARCO+ macrophages by guest on September 27, 2021 immune defense and subsequent adaptive responses. These prop- lining the marginal sinus are critical for recognition and degrada- erties, in combination with their emerging role in diverse settings, tion of systemic pathogens and complement the function of innate- including infection, cancer, and metabolic syndrome (3, 4), indi- like MZ B cells that are well-establishedtoprovideearlyAbpro- cate that iNKT cells serve a critical role in the early events of duction for control of systemic pathogens (5). Notably, subsets of immune activation, as well as provide a motivation for exploiting dendritic cells (DCs) also reside in the MZ and bridging channels this cell type clinically to act as a natural adjuvant for enhancing under steady-state conditions (6), yet their role at this site is not protective immunity and/or controlling immune homeostasis. To clear. Thus, the splenic MZ is a tightly organized network of cells fully realize the function of iNKT cells, as well as their potential working together to optimally mediate rapid immunity for host as a therapeutic target, it is critical to identify the earliest acti- protection. vation events of this cell type in situ. Given the accelerated effector responses of iNKT cells and their The spleen is the primary site of systemic immune surveillance relative abundance in the spleen compared with other lymphoid and activation in response to blood-borne Ags. In particular, the organs (7), it stands to reason that these innate leukocytes may also communicate with resident MZ cell types to immediately and dramatically impact the outcome of immune responses directed *Trudeau Institute, Saranac Lake, NY 12983; and †School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom toward systemic infections or other foreign Ags. Specifically, 1Current address: McGill University, Montreal, Quebec, Canada. iNKT cells recognize numerous bacterial glycolipids in the con- 2 text of CD1d (8, 9), including one recently identified in Strepto- M.M. and E.A.L. contributed equally to this work. coccus pneumoniae (10), and they are robustly activated during S. Received for publication January 30, 2013. Accepted for publication May 15, 2013. pneumoniae infection by DC-derived IL-12 plus self-glycolipid This work was supported by the Trudeau Institute and the National Institutes of Health (Grant R01 AI0764). containing CD1d (11). We (12, 13) and other investigators (14) recently showed that iNKT cells can provide cognate help to B Address correspondence and reprint requests to Dr. Elizabeth Leadbetter, Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: eleadbetter@ cells, resulting in extrafollicular plasmablast formation and IgM trudeauinstitute.org and IgG3 production reminiscent of MZ B cell activation. MZ The online version of this article contains supplemental material. B cell Ab production is a key component of immune defense Abbreviations used in this article: CL, clodronate; DC, dendritic cell; aGalCer, against S. pneumoniae (15), so B cell help from iNKT cells during aGalactosylceramide; iNKT, invariant NKT; MZ, marginal zone; PALS, periarter- this infection might be highly relevant but remains largely un- iolar lymphoid sheath; TZ, T cell zone; WT, wild-type. studied. Furthermore, iNKT cells have been identified by us (11) Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 and other investigators (16) as critical for survival following in- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300299 The Journal of Immunology 573 fection with URF918, a serotype 3 clinical isolate of S. pneumo- S. pneumoniae (strain URF918; provided by K. Kawakami, University of niae. Systemic S. pneumoniae infection, described by the World the Ryukyus, Nishihara, Okinawa, Japan). Health Organization as a significant public health threat, is re- Flow cytometry sponsible for the deaths of more than half a million children a year, despite the introduction of multiple vaccines in the last 10 years. S. iNKT cell analysis was performed by dissociating spleens through a 0.7-mm cell strainer with the end of a 5-ml syringe plunger, to generate single- pneumoniae causes pulmonary pneumonia, otitis media, meningi- cell suspensions, followed by RBC lysis. For DC analysis, spleens were tis, and invasive disease or septicemia. S. pneumoniae of the se- injected with a solution of 0.1 mg/ml Liberase TM and 20 mg/ml DNase I rotype 3 is also one of a select group of serotypes found to be (Roche) in RPMI 1640 (Invitrogen) and shaken for 20 min at 37˚C. A final associated with increased risk for death during invasive disease in concentration of 5 mM EDTA diluted in FCS was added to each sample for humans (17), with the rate of in-hospital death for patients sys- an additional 5 min at 37˚C. Splenocytes were filtered through a 100-mm cell strainer, red cell lysed, and maintained on ice for Ab staining and flow temically infected with serotype 3 reaching 50% (18). As such, it cytometric analysis. Splenocytes were stained with Abs against TCR-b (H57- is important to dissect the role of splenic iNKT cells during this 597), CD11b (M1/70) from BioLegend, and huCD2 (RPA.2-10), CD11c systemic infection relevant to public health. (HL3), MHC II (M5/114.15.2), CD8a (53-6.7), and B220 (RA3-6B2) from BD Furthermore, iNKT cells are susceptible to activation across a Biosciences. CD1d tetramers loaded with PBS57 (an aGalCer homolog) or left unloaded were obtained from the National Institutes of Health Tetramer spectrum of stimuli, ranging from selectively TCR–CD1d–glyco- Core Facility and incubated with splenocytes at room temperature for 20 min lipid–mediated activation to TCR-CD1d–self glycolipid stimulation prior to surface Ab staining on ice.