Cell Activation Dictates Localization in Vivo the Mechanism of Splenic Invariant

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

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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 interactions 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 interactions 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. Glycolipid-loaded CD1d-tetramer was in combination with cytokine exposure to exclusively cytokine- used for all FACS and confocal analyses, unless otherwise noted. Intra- dominated activation (11). The ramifications of these different cellular IFN-g (XMG1.2; BD Biosciences) was detected by following forms of activation for iNKT effector function and the localiza- instructions in the eBioscience Foxp3 staining kit. Phosphorylation of Downloaded from STAT6 at tyrosine 641 (J71-773.58.11) and STAT1 at tyrosine 701 (4a) was tion of splenic iNKT cells following stimulation by these different detected as described previously (19). All flow cytometric data were acquired activators have not been described. The focus of the current study on a FACSCanto II and analyzed with FlowJo software (TreeStar) was to compare the dynamics of, and cellular requirements for, iNKT cell activation in response to blood-borne cognate lipid Ags Generation of mixed bone marrow chimeras compared with systemic cytokine or complex pathogen-mediated Bone marrow from B cell–deficient mMT mice heterozygous for the KN2 2/2 activation. To this end, we combined various chemokine and cyto- allele was mixed with either (wild-type) WT or CD1d bone marrow at http://www.jimmunol.org/ kine reporter mice with a new method of mCD1d-specific tetramer a 3:1 ratio and injected into the tail veins of lethally irradiated (1000 rad split into two doses) WT C57BL/6 hosts to generate mice selectively lacking staining of fresh spleen tissue to track the localization and activation CD1d expression by B cells. To evaluate the role of CD1d on DCs, some of the endogenous iNKT cell population in situ. Using these tech- irradiated mice received a 50:50 mix of CD1d+/+ and CD1d2/2 bone marrow. niques, we find that iNKT cells are widely distributed throughout All chimeras were used for experiments 6–8 wk following donor cell transplantation. Specific deletion of CD1d on splenic B220+ cells or CD11c+ the spleen during steady-state conditions, but they rapidly consoli- + date in the MZ following immunization with synthetic glycolipids MHCII DCs from test chimeras was confirmed by flow cytometry. and during systemic S. pneumoniae infection, where they produce Diphtheria toxin and liposome administration cytokine in a highly compartmentalized fashion. In contrast, in vivo

Mice were injected i.p. with 100 ng/mouse diphtheria toxin from Cory- by guest on September 27, 2021 exposure of iNKT cells to systemic cytokine fails to organize them nebacterium diphtheriae (D0564; Sigma-Aldrich) 24 h prior to immuni- along the MZ, despite dramatic activation and production of IFN-g. zation with aGalCer. For liposome experiments, mice were injected i.v. Given this localization pattern, it is not surprising that MZ DCs are with 0.2 ml clodronate (CL)-loaded or PBS liposomes either 24 h or 3 wk a critical contributor to iNKT cell activation in response to both prior to immunization with aGalCer. Cl2MDP (or CL) was a gift of Roche glycolipids and infection but are dispensable for activation by sys- Diagnostics (Mannheim, Germany) and was encapsulated in liposomes, as previously described (20). temic cytokines. iNKT cell cytokine production, in turn, mediates global effects on the splenic microenvironment by inducing cyto- Confocal/fluorescent microscopy kine-specific STAT phosphorylation across the splenic parenchyma For detection of GFP, YFP, and PBS57-CD1d tetramer staining, spleens and DC mobilization to the T cell zone (TZ) in a noncognate were harvested and immediately placed in 5% agarose blocks for sectioning. manner. Collectively, our results demonstrate that iNKT localiza- Spleen tissue was cut into 200-mm-thick sections using a vibrating mi- tion and site of activation are consistent with a requirement for crotome. Tissue Ab staining was performed on ice for 60 min, with the DCs under all conditions, with the exception of exogenous addition of exception of PBS57-CD1d or unloaded tetramer staining, which was performed at room temperature for 30 min prior to staining with other Abs cytokines. Furthermore, these activation conditions share a com- on ice and analysis by confocal microscopy (Leica TCS SP5). Glycolipid- mon, global effector outcome, further highlighting the function of loaded CD1d-tetramer was used for all FACS and confocal analyses, un- iNKT cells as a natural adjuvant during immunity and infection. less otherwise noted. Surface Ab staining included B220, CD11c, huCD2, CD169 (3D6.112; AbD Serotec), and SIGN-R1 (22D1; eBioscience). For in situ evaluation of splenic macrophage and DC subsets, spleens were Materials and Methods immediately frozen in optimal cutting temperature embedding compound Animals over liquid nitrogen. Frozen spleens were cut into 6-mm sections on a cryostat (Leica) and fixed in a mixture of ice-cold 75% acetone/25% 4get, 4get/KN2, STAT6-deficient, and CD1d-deficient mice on a BALB/c ethanol for 5 min. Sections were blocked in PBS plus 2% BSA and 5% background and CD11c-DTR.eGFP.KN2, mMT.KN2, Great (Ifngtm3.1Lky), normal mouse serum for 60 min prior to specific Ab staining. To quantitate CD1d-deficient (Mark Exley, Beth Israel Deaconess Medical Center, localization, pixel intensity and square area were determined by ImageJ Boston, MA), batf32/2, and CXCR6-GFP+/2 mice (The Jackson Labora- software for three defined regions: TZ, B cell follicles/zone, and MZ. tory, Bar Harbor, ME) on a C57BL/6 background were bred and kept under Regions were defined based on labeling with CD4/8 (TZ), B220 (B cell specific pathogen–free conditions at the Trudeau Institute and were used at zone), and SignR1 (MZ). Unit density was determined to be the specified 8–12 wk of age. All experiments were performed under Trudeau Institute pixel intensity per unit area. Normalized unit density is an expression of Animal Care and Use Committee–approved protocols. unit density standardized to the TZ value (i.e., a ratio of specific regional unit density/TZ unit density). Immunizations and S. pneumoniae infection IL-12 ELISA Mice were immunized i.v. with 0.5 mg/mouse aGalactosylceramide (aGalCer)or40mg/mouse GSL-1 in PBS containing 0.1% BSA and For detection of serum IL-12, ELISA plates were coated with anti-mouse ,0.25% DMSO or PBS/BSA/DMSO alone. Alternatively, some mice re- IL-12p40 (C15.6; BD) and then blocked with 1% BSA/PBS. Serum sam- ceived i.v. injection of IL-12 (0.5 mg) and IL-18 (1.0 mg) or 1 3 106 CFU ples were diluted in 0.1% BSA/PBS. Purified murine IL-12p40 (BD) was used 574 VISUALIZATION OF SPLENIC iNKT ACTIVATION for a standard curve. Bound IL-12 was detected with rat anti-mouse IL-12p40/ gens contain known CD1d cognate ligands (11). To compare these p70-biotin (C17.8; BD), followed by streptavidin-alkaline phosphatase two other methods of iNKT cell activation to encounter of cognate (BD) using pNpp (Sigma-Aldrich) diluted to 1 mg/ml in alkaline buffer Ag-mediated activation, WT mice were administered rIL-12 and (Sigma-Aldrich). Plates were read on a Molecular Devices SpectraMax Plus384 ELISA reader at 405 nm. rIL-18 i.v. or were infected with S. pneumoniae. We determined that IFN-g production by iNKT cells peaked at 2 h after cytokine ad- Statistical analysis ministration and 8 h after S. pneumoniae infection (Supplemental Nonparametric t tests and one-way ANOVA tests were performed using Fig. 2A, 2B). As previously shown, IL-4 production was not de- GraphPad PRISM 5 software. tectable under either condition (data not shown) (11). Interestingly, iNKT cells also accumulated in the splenic MZ during infection (Fig. 1L). This localization pattern is the same as we detected after Results cognate glycolipid immunization and suggests that the requirement Splenic iNKT cell localization differs according to the stimulus for CD1d engagement dictates the localization of iNKT cells in the To determine the location of the endogenous iNKT cell population in spleen. In contrast, i.v. IL-12 and IL-18 administration robustly WT mice under steady-state and various activating conditions, we activates iNKT cells, but they remain widely distributed throughout developed an in situ technique by which we detected glycolipid- the spleen (Fig. 1M), reflecting the same distribution observed in loaded CD1d tetramer labeling of iNKT cells in fresh splenic tis- PBS vehicle–injected mice (Fig. 1N). sue by confocal microscopy (Fig. 1). Abs against B220, CD4, and SIGN-R1 (a marker of MZ macrophages) were used to delineate the Consolidation of iNKT cells yields compartmentalization of

B cell follicles, T cell area, and MZ, respectively. Consistent with effector cytokine production Downloaded from a recent study that used confocal imaging in combination with in- As demonstrated in our in situ tetramer-labeling studies, some direct labeling techniques or transfer of purified iNKT cells into iNKT cells remained in the TZ following lipid administration. To congenic recipients to follow splenic iNKT cell migration (21), we delineate the exact location of iNKT cell activation and effector consistently found CD1d tetramer+ cells dispersed throughout the cytokine production, we used IL-4 dual reporter (4get/KN2) (24) and splenic white and red pulp during their endogenous resting state IFN-g reporter (Great) (25) mice. iNKT cells from 4get/KN2 mice + (Fig. 1A, 1B, data not shown). Tetramer labeling of spleens from are constitutively GFP as a result of the persistent expression of IL- http://www.jimmunol.org/ naive NKT-deficient CD1d2/2 mice demonstrated negligible back- 4 mRNA (26) and, upon activation, they rapidly express huCD2 ground binding (Fig. 1C, 1D), as did labeling with unloaded CD1d on their cell surface, indicating IL-4 protein production (24, 27). tetramer on WT spleen sections from uninjected mice (Supple- Following i.v. aGalCer administration, splenic CD1d-tetramer+ mental Fig. 1A, 1B). Kinetic experiments using upregulation of the iNKT cells began to express huCD2 at 2 h, with peak production high-affinity a-chain of IL-2R subunit (CD25) as an early marker of occurring at 4 h postimmunization (Fig. 2A, 2B), a kinetic similar to activation demonstrated that iNKT cells were activated within 4 h IFN-g production (Fig. 1E–G). Importantly, Fig. 2A confirms that after i.v. immunization with aGalCer (Fig. 1E). Similar activation production of IL-4 (i.e., huCD2 expression) was limited to CD1d2 kinetics were observed using intracellular IFN-g production as an tetramer+ iNKT cells following in vivo administration of aGalCer. outcome measure (Fig. 1F, 1G). We next determined the location of This observation allowed the use of huCD2 as a marker for local- by guest on September 27, 2021 CD1d-tetramer+ iNKT cells at this time of peak activation. Four izing the endogenous IL-4–producing iNKT cell population in the hours after aGalCer immunization, there was no significant change spleen of mice in situ. At 4 h postimmunization of 4get/KN2 mice, in the number of splenic CD1d-tetramer+ iNKT cells (Fig. 1H). we found IL-4–producing huCD2+ cells almost exclusively within However, confocal images and fluorescent quantification revealed the MZ and bridging channels of the spleen (Fig. 2C, left panel, Fig. that aGalCer stimulates iNKT cells to accumulate at the MZ and 2D). Mice homozygous for the 4get allele were used as a huCD2 bridging channels of the spleen (Fig. 1I) compared with vehicle- negative–staining control (Fig. 2C, middle panel). To corroborate injected controls (Fig. 1K). This is consistent with redistribution, but our flow cytometry data and confirm that huCD2-producing cells not enhanced recruitment, of this cell type from the peripheral cir- detected within the MZ were iNKT cells, we combined our in situ culation. To determine whether the iNKT cell response that we tetramer-labeling technique with huCD2 detection. Consistent with observed with a synthetic lipid agonist is similar to encounter with the results described above, CD1d-tetramer+ iNKT cells that cos- bacterial glycolipids, BALB/c mice were immunized with a glyco- tained with huCD2 were found outside of the white pulp clustered sphingolipid (GSL-1) expressed by the Sphingomonadaceae family around the bridging channels and MZ (Fig. 2C, right panel). To of bacteria (9). Intravenous administration of GSL-1 resulted in confirm that our characterization of effector cytokine-secreting rapid accumulation of iNKT cells within the MZ and bridging iNKT cells as consolidating in the MZ is not unique to IL-4–se- channels of the spleen at the same 4-h time point as for aGalCer creting iNKT cells, we evaluated the localization of iNKT cells administration compared with vehicle-injected controls (Fig. 1J, producing IFN-g using mice engineered to report IFN-g produc- 1K). The observations of iNKT cell location under steady-state and tion via YFP expression (Great) (25). aGalCer immunization glycolipid-mediated activation conditions were confirmed using showed that, at the time of iNKT consolidation in the MZ, the IFN- heterozygous CXCR6-eGFP knock-in mice in which iNKT cells g–producing cells were also consolidated in the MZ (Supplemental highly express eGFP (Supplemental Fig. 1C, 1D) (22). Collec- Fig. 2C) compared with the DMSO vehicle controls (Supplemental tively, these results indicate that iNKT cells rapidly localize to the Fig. 2D). At this time point following aGalCer administration, IFN- vascular-rich region of the spleen and cluster at the MZ and bridging g production is largely limited to iNKT cells (data not shown); channels following immunization with diverse cognate lipid Ags. therefore, this result is consistent with MZ consolidation of IFN-g– In addition to cognate lipid immunization, two other forms of secreting iNKT cells. We next determined that i.v. administration of activation for iNKT cells have been characterized: one driven ex- GSL-1 resulted in rapid production of IL-4 and huCD2 expression clusively by high levels of cytokines, such as IL-12 and IL-18 (23), by iNKT cells with a kinetic similar to aGalCer immunization (Fig. and the other induced by a combination of self-ligand–CD1d rec- 2E). Similar to our experiments using aGalCer, IL-4–producing ognition plus IL-12 costimulation (2). Indeed, we recently showed huCD2+ iNKT cells accumulated within the MZ and bridging that the cytokine-driven “indirect” form of iNKT cell activation channels after GSL-1 administration (Fig. 2F, 2G). In summary, dominates the immune response during infection, even if the patho- these results indicate that iNKT cells rapidly localize to the The Journal of Immunology 575 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Localization of iNKT cells under steady-state and various activating conditions. Confocal images of CD1d tetramer labeling of spleen sections from unimmunized WT mice (A, B) or CD1d-deficient BALB/c mice (C, D). (B) and (D) are the dashed insets in (A) and (C), respectively. (E) Graph depicting FACS measurement of CD25 expression by CD1d-tetramer+ splenocytes at 1 h (dotted line), 2 h (red line), and 4 h (solid line) following immunization. The filled graph represents the DMSO vehicle control at 4 h postimmunization. (F) Graph depicting intracellular IFN-g by tetramer+ cells, as described in (E). (G) Bar graph showing the number of IFN-g–producing CD1d-tetramer+ splenocytes detected by FACS analysis at various time points after DMSO vehicle or aGalCer immunization. (H) The total number of CD1d-tetramer+ iNKT cells/spleen detected by FACS following DMSO vehicle or aGalCer immunization over time. Confocal images and quantitation of CD1d tetramer labeling of spleen sections from BALB/c mice immunized 4 h prior with aGalCer (I) or GSL-1 (J), 8 h prior with S. pneumoniae (L), or 2 h prior with IL-12 + IL-18 (M). (K) and (N) show DMSO and PBS vehicle controls. The quantification of these confocal images was performed as described in Materials and Methods. All data shown are representative of three experiments with two mice/group. Scale bar, 100 mm(A, C, I, J, L, M) and 30 mm(B, D). *p , 0.05, **p , 0.001, ***p , 0.0001, ****p , 0.00001. BZ, B cell zone; ns, not significant. vascular-rich region of the spleen and produce the canonical cyto- unpublished observations). Instead, under those conditions, the lo- kines IL-4 and IFN-g in a highly compartmentalized fashion fol- calization pattern of iNKT cells, as determined by confocal imaging lowing immunization with cognate glycolipid Ags. (Fig. 1L, 1M), at the times of peak IFN-g production by iNKT cells These data clearly inform us as to the localization of specific IL- (Supplemental Fig. 2A, 2B) gives the clearest suggestion of local- 4– and IFN–g producing iNKT cells in response to cognate Ag. ization of cytokine-secreting iNKT cells. However, following recombinant cytokine administration or during S. pneumoniae infection, the production of IFN-g by other MZ DCs are critical for iNKT cell activation under diverse cells, including NK1.1+TCRb2 NK cells, precludes the ability to inflammatory conditions detect iNKT-specific IFN-g production in situ during those stim- The distinct localization patterns of iNKT cells following cognate ulating conditions (I.L. King, M. Mohrs, and E.A. Leadbetter, and, to a lesser extent, pathogen activation versus cytokine-driven 576 VISUALIZATION OF SPLENIC iNKT ACTIVATION

FIGURE 2. Early IL-4 production by iNKT cells occurs at the splenic MZ. (A) 4get/KN2 mice were immunized with either DMSO vehicle or aGalCer, and live B2202 splenocytes were assessed for expression of huCD2 (IL-4 production) by ﬂow cytometry at the time points indicated. Numbers represent the frequency of huCD2+ cells of the CD1d-tetramer+ population. DMSO vehicle–treated mice were assessed at 4 h postimmunization. (B) The total number of huCD2+ cells/ spleen at various time points following DMSO vehicle or aGalCer immuni-

zation. (C) Spleens were harvested Downloaded from from aGalCer-immunized 4get/KN2 or 4get/4get mice and assessed for localization of huCD2 expression, as described in Materials and Methods. (D) Quantitation of huCD2 labeling intensity in distinct splenic regions following aGalCer or DMSO http://www.jimmunol.org/ vehicle immunization, as described in Fig. 1. (E) 4get/KN2 mice were immunized with either DMSO vehicle or GSL-1, and live B2202 splenocytes were assessed for huCD2 expression at 4 h postimmunization. Numbers represent the frequency of huCD2+ cells of the CD1d-tetramer+ population. (F) Spleens harvested from by guest on September 27, 2021 GSL-1–immunized 4get/KN2 mice were assessed for location of huCD2 expression. SIGN-R1 delineates the MZ. (G) Quantitation of huCD2-labeling intensity in distinct splenic regions following GSL-1 immunization. All data shown are representative of at least three individual experiments, with three or four mice/ group. Scale bar, 100 mm. Error bars represent SD. ****p , 0.00001. BZ, B cell zone; ns, not signiﬁcant.

stimulation prompted us to investigate which splenic APC(s) are early iNKT activation in vivo by depleting CD11c+ cells from mice required for elaboration of the iNKT cell effector program under expressing the simian diphtheria toxin receptor under control of the these conditions. Given that iNKT cells accumulate and produce CD11c promoter (CD11c-DTR mice) prior to aGalCer adminis- IL-4 and IFN-g at the splenic MZ in response to cognate lipid Ag tration. Crossing CD11c-DTR mice to the KN2 IL-4 reporter strain, administration, we first determined the cell type resident in the we obtained similar results using IL-4 (huCD2 expression) as an MZ niche responsible for cognate activation of iNKT cells fol- outcome measure (Fig. 3B). However, a recent report indicated that lowing systemic glycolipid administration, an event dependent diphtheria toxin treatment to these mice depletes DCs, as well as on CD1d-restricted Ag presentation (3). Although the splenic CD169+ and SIGNR-1+ macrophage subsets lining the MZ (31). To MZ contains distinct subsets of macrophages and DCs, MZ investigate the potential contribution of macrophages in this system, B cells express particularly high levels of CD1d (28) and are we next depleted both macrophage subsets by i.v. administration of potent activators of iNKT cells in vitro (13, 29). However, analysis liposome-encapsulated CL to WT mice, a well-established method of mixed bone marrow chimeras, in which B cells were selectively of splenic macrophage depletion (Fig. 3C) (32). In contrast to pre- deficient in CD1d, did not result in any loss of iNKT cell IL-4 vious observations (30), however, CL treatment also abrogated production following aGalCer administration in vivo (Fig. 3A). A splenic iNKT cell production of IL-4 (Fig. 3D), despite having no study by Schmieg et al. (30) found that DCs were indispensable in impact on the frequency of total splenic iNKT cells (Fig. 3E). Thus, The Journal of Immunology 577 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021 FIGURE 3. Depletion of MZ phagocytes compromises iNKT cell activation. (A) Frequency of IL-4–producing huCD2+dump2 (B2202CD11b2) splenocytes following aGalCer or DMSO vehicle immunization of bone marrow chimeras containing either WT or CD1d2/2 B cells, as described in Materials and Methods.(B) Frequency of huCD2+dump2 splenocytes following aGalCer or DMSO vehicle immunization of B6.KN2 (WT) or CD11c- DTR.KN2 (DTR) mice 24 h after diphtheria toxin or PBS treatment. (C) Confocal images of spleen sections depicting the effects of CL treatment on the SIGNR-1+ MZ and CD169+ metallophilic macrophage subsets. (D) Frequency of huCD2+dump2 splenocytes following aGalCer or DMSO vehicle immunization of 4get/KN2 mice treated 24 h prior with either CL-loaded or PBS liposomes. (E) Frequency of splenic CD1d2tetramer+ cells under the conditions described in (D). (F and G) Absolute number of total CD11c+MHCII+ DCs or CD8+ and CD82 DC subsets 24 h after PBS or CL treatment. (H) Spleen sections showing the localization of total CD11c+ splenocytes following PBS or CL injection 24 h earlier. Data shown are representative of two (A– C) or three (D–F) individual experiments with three or more mice/group. Scale bar, 100 mm. Error bars represent SD. *p , 0.05, **p , 0.001. ns, Not significant. further investigation into the relevant APC(s) for iNKT cell acti- immunized animals 24 h after CL injection, a time point at which vation was required. both MZ DCs and macrophages were depleted (Fig. 4A, 4B). In In the course of our studies, we consistently observed an ∼50% contrast, iNKT cell production of IL-4 in response to aGalCer im- reduction in total splenic CD11c+MHCII+ DCs24hafterCL munization was completely restored in mice 3 wk after CL treat- treatment (Fig. 3F), with a more profound loss in the CD8+ subset ment when macrophages were still absent but MZ DCs had been than in the CD82 subset (Fig. 3G). When we examined the spleens restored. The numbers of IFN-g–producing iNKT cells generated in of CL-treated mice histologically, we found selective depletion of response to aGalCer was also restored (Fig. 4F) but not quite to the CD11c+ cells lining the MZ, whereas the CD11c+ cells in the TZ extent achieved in intact mice, which suggests that the DCs may not appeared to be relatively unaffected (Fig. 3H), a result consistent be completely reconstituted yet or there could be different cellular with a previous report (33). To more closely test a role for MZ DCs requirements for iNKT production of IFN-g than for IL-4. Collec- versus macrophages in early iNKT cell activation, we took advan- tively, these results indicate that MZ DCs are a critical APC for tage of the distinct reconstitution kinetics of splenic macrophages iNKT cell activation following cognate lipid immunization. compared with DCs (32). Consistent with previous observations, To further investigate the APC requirements for iNKT cell acti- both SIGN-R1+ MZ and CD169+ metallophilic splenic macrophage vation during systemic infection, we repeated these CL-depletion populations remained depleted for $3 wk after a single injection of studies with S. pneumoniae–infected mice. As expected, iNKT CL, whereas DC numbers were restored in frequency and location cells failed to become activated during S. pneumoniae infection in within 1 wk (Fig. 4A–C) (31, 32). Using this strategy, we immu- mice treated with CL 24 h prior to infection (Fig. 5A). Given our nized mice with aGalCer at either 24 h or 3 wk post-CL treatment previous work describing the importance of both CD1d and IL-12 to assess iNKT cell canonical cytokine production. iNKT cell IL-4 from APCs in mediating iNKT activation during infection (2), these (Fig. 4D, 4E) and IFN-g (Fig. 4F) production was lost in aGalCer- data suggested that acute treatment of mice with CL prior to 578 VISUALIZATION OF SPLENIC iNKT ACTIVATION

FIGURE 4. MZ DCs mediate cyto- Downloaded from kine production by iNKT cells. (A and B) Immunoﬂuorescent images of spleen sections from 4get/KN2 mice 24 h or 3 wk after CL treatment. Mice treated with PBS liposomes are shown for comparison. Scale bar, 200 mm. (C) Contour plots showing the frequency http://www.jimmunol.org/ of total splenic CD11c+MHCII+ DCs at each time point described in (A). (D) Percentages of CD1d-tetramer+ splenocytes producing IL-4 (huCD2+) from representative individuals. A summary of the percentages of IL-4–producing (huCD2+) (E)andIFN-g–producing (F) iNKT cells after DMSO vehicle or

aGalCer immunization from all 4get/ by guest on September 27, 2021 KN2 mice treated with PBS or CL. Results shown are representative of two individual experiments with three to ﬁve mice/group. **p , 0.001, ***p , 0.0001.

pneumococcal infection eliminated self-lipid–presenting CD1d, the SIGN-R1+ macrophages, were allowed to repopulate the splenic source of IL-12 critical for iNKT cell activation, or both. Indeed, the MZ. To corroborate our depletion studies and to conﬁrm the im- increase in serum IL-12p40 detected 8 h after systemic S. pneu- portance of DC-derived IL-12 in combination with CD1d to me- moniae infection was not observed in infected, CL-treated animals diate iNKT cell activation in vivo during infection, we assessed (Fig. 5B). Importantly, both iNKT cell activation and serum IL- iNKT cell activation following pneumococcal infection of batf32/2 12p40 levels were completely restored in mice infected 3 wk after mice, which have a speciﬁc deletion of splenic CD8+ DCs (34). CL treatment (Fig. 5A, 5B), a time point at which MZ DCs, but not Importantly, this subset was shown to reside in the MZ during The Journal of Immunology 579

conﬁrmed that cytokines are sufﬁcient to activate iNKT cells, and MZ DCs are no longer required if the cytokine is extrinsically provided. Collectively, these results indicate that MZ-resident DCs are the critical APC responsible for presenting foreign or self-Ags and providing cytokines for iNKT cell activation during both immunization and infection. iNKT cells exert their effector program beyond their cognate partners Given the restricted localization of iNKT cells under cognate- activating conditions, we next considered whether iNKT cells direct their effector functions to their local site of activation. In the course of our studies, we observed that CD11c+MHCII+ DCs dramatically upregulated CCR7 expression (Fig. 6A) and relocated from the MZ and bridging channels to the TZ following aGalCer administration (Fig. 6B), a response previously observed following activation with TLR agonists (38, 39) or CD40–CD40L interactions (39). Importantly, DC relocation occurred in a CD1d-dependent

manner (Fig. 6B). To consider whether this effector outcome re- Downloaded from quired cognate interactions, we generated mixed bone marrow chimeras, in which 50% of DCs lacked CD1d expression but 50% of DCs expressed normal levels of CD1d (Fig. 6C, 6D). Unexpectedly, we found that cognate iNKT–DC interactions were dispensable for the upregulation of CCR7 by splenic DCs, because both CD1d+ 2

and CD1d DCs in these chimeras upregulated CCR7 expression http://www.jimmunol.org/ equally after aGalCer immunization (Fig. 6E). We also demonstrated previously that, during infection, despite localized production, cytokine signaling can saturate the entire immune compartment of lymphoid organs (19). Given that iNKT cell activation can take place, depending on the stimulus, at distinct loci within the splenic parenchyma, we further postulated that the effector range of their cytokines extends beyond their immediate microenvironment. To investigate this question, we measured the levels of phospho-STAT6

and phospho-STAT1 in the total splenic B cell compartment as by guest on September 27, 2021 readouts of IL-4 and IFN-g signaling, respectively, by intracellular FIGURE 5. CD8+ MZ DCs play a critical role in iNKT cell cytokine flow cytometry (19). Although we found iNKT-derived IL-4 and production following pneumococcal infection. (A) The frequency of IFN- IFN-g to be produced in a tightly restricted manner in proximity to g–producing iNKT cells or serum IL-12p40 levels (B)inS. pneumoniae– the MZ of the spleen (Figs. 1, 2). FACS analysis detected CD1d- infected animals under conditions shown in Fig. 4A. Frequency of IFN-g– dependent induction of high levels of both phospho-STAT6 and C D producing iNKT cells ( ) or serum IL-12p40 levels ( ) in naive or S. phospho-STAT1 in the entire splenic B220+ B cell population within pneumoniae–infected WT or batf32/2 animals. (E) Frequency of IFN-g– 4 h after aGalCer administration (Fig. 6F–H). Because all splenic producing iNKT cells following PBS, aGalCer, or IL-12/IL-18 administration 24 h after CL-mediated phagocyte depletion. Data are representa- B cells show evidence of having received IL-4 signals, these results tive of three (E) and two (A–D) experiments. Error bars represent SD of at suggest that, irrespective of the location in which iNKT cells are least three mice/group. *p , 0.02, **p , 0.001, ***p , 0.0001. ns, Not activated, they contain the potential to condition the entire lymphoid significant. organ environment to either regulate or potentiate the immune response. homeostasis (6) and to be the dominant source of IL-12 in a number Discussion of adjuvant- and infection-based models of immunity (35–37). In- The inability to localize and track the endogenous iNKT population deed, iNKT cells from batf32/2 mice produced significantly less as it initiates the effector program in vivo has been a long-standing IFN-g in response to S. pneumoniae infection than did WT controls roadblock to advancing our understanding of this innate T lym- (Fig. 5C), which correlated with reduced serum levels of IL-12p40 phocyte subset. Other groups (40, 41) tried unsuccessfully to identify from the same animals (Fig. 5D). The residual IFN-g production in endogenous individual splenic NKT cells by tetramer in WT mice S. pneumoniae–infected batf32/2 mice could be a result of activa- and instead used TCRb NK1.1 labeling of endogenous cells, adop- tion through cytokines and/or CD1d on other DC subsets or partial tive transfer of sorted iNKT cells, or fluorescent-diffusion strategies contribution of an entirely different APC (2). (21) to probe the localization of iNKT cells in a posttransfer state. One concern with the above studies was that acute treatment of These approaches found the majority of resting iNKT cells to be CL may have off-target effects on iNKT cells and inhibit their localized in the TZ, or periarteriolar lymphoid sheath (PALS) (41), activation, irrespective of phagocyte depletion. To address this or in the MZ, PALS, and red pulp (21). However, using a novel concern, we again treated animals with CL and, 24 h later, ad- method of in situ mCD1d tetramer staining, in combination with ministered rIL-12 and rIL-18, as described above. As shown in Fig. relevant cytokine reporter mice, we can now unambiguously iden- 5E, cytokine-driven iNKT activation was similar in both PBS- and tify a restricted localization and cytokine-production profile for CL-treated immunized mice, indicating that iNKT cells are not endogenous splenic iNKT cells. In agreement with previously intrinsically defective under these conditions. These results also published reports, we find that endogenous iNKT cells are widely 580 VISUALIZATION OF SPLENIC iNKT ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. Noncognate effector functions of iNKT cells in vivo. (A) Graphs showing CCR7 expression of CD11b+CD82 (left panel) or CD11b2CD8+ (right panel) DCs from WT or CD1d-deficient mice 12 h after immunization with aGalCer or DMSO vehicle. (B) Confocal microscopy analysis of spleen sections from WT or CD1d-deficient mice 12 h after immunization with aGalCer or DMSO vehicle. Scale bar, 200 mm. (C) Frequency of CD45.1+ WT or CD45.2+ CD1d2/2 DCs from unimmunized mixed chimeric mice, as described in Materials and Methods.(D) CD1d expression by WT or CD1d2/2 CD8a+ DCs from mixed chimeras. (E) CCR7 geometric mean fluorescence intensity (MFI) of WT or CD1d2/2 CD8a+ DCs in mixed chimera from DMSO vehicle– or aGalCer-immunized mice at the 12-h time point. (F) Phosphorylation of STAT6 in the total B220+ splenocyte population was assessed by flow cytometry in WT, CD1d2/2, and STAT62/2 mice 4 h after aGalCer immunization. Geometric mean fluorescence intensity (MFI) of pSTAT6 (G) and pSTAT1 (H) after DMSO vehicle or aGalCer immunization. All data shown are representative of at least two individual experiments with three or four mice/group. **p , 0.001. The Journal of Immunology 581 and evenly distributed throughout the parenchyma of the spleen, suggests that the conditions that produce MZ localization of iNKT including B and T cell follicles in the PALS, the MZ, and red pulp cells are relevant for iNKT responses to both highly pure glycolipid (data not shown). Importantly, we find that this distribution only Ags and systemic infections. Furthermore, early production of changes to a restricted MZ localization following cognate activation IFN-g by this population of iNKT cells throughout the spleen condi- by model and bacterially derived glycolipids or S. pneumoniae in- tions the entire environment for a rapid coordinated immune de- fection, presumably as a result of arrest in proximity to Ag-loaded fense, including enhancing activation of innate responders like NK APCs. Barral et al. (42) showed that iNKT cells arrest near lymph cells, macrophages, DCs, and neutrophils, as well as driving rele- node subcapsular sinus macrophages in response to particulate vant activation of adaptive defenders, such as CD4 T cells and glycolipid Ag and similarly described how iNKT cells arrest in the B cells. Perhaps as one example of this, we also noted iNKT- MZ in close proximity to Ag-rich regions within 2 h of adminis- dependent upregulation of CCR7 and relocation by MZ DCs from tration of particulate aGalCer (21). This is consistent with live- the MZ and bridging channels into the TZ in the spleens of mice imaging studies from the Kubes laboratory (43) characterizing immunized with aGalCer. Furthermore, we demonstrate that these iNKT arrest in the liver following Kupffer cell capture and pre- changes, evidence of DC licensing, occur independently of cognate sentation of Borrelia burgdorferi Ags. Michael Dustin and col- interactions with iNKT cells (i.e., the DCs themselves do not need to leagues (22, 44) provided the earliest description of iNKT cell arrest specifically engage an iNKT cell via CD1d to receive this licensing in the liver in response to aGalCer and GSL-1. Interestingly, the signal), suggesting that iNKT cells mediate this aspect of DC li- Dustin group (44) also found that iNKT cells arrest in the liver in censing via a soluble mediator. The specific soluble factor(s) me- response to systemic IL-12 plus IL-18. This contrasts with our data diating DC relocation to the TZ requires further investigation. showing that iNKT cells remain widely and evenly distributed In summary, our data suggest that iNKT cells use diverse methods Downloaded from throughout the spleen following activation by systemic cytokines. to exert their effector functions, the results of which can have a The difference between our findings and those of the Dustin group profound effect on the entire lymphoid tissue landscape, further ex- could be explained by the different subsets of iNKT cells or APCs emplifying their critical role as a bridge between innate and adap- that populate the spleen versus the liver, or it could be a consequence tive immune responses. In the future, combining tetramer labeling of different integrin- or chemokine-trafficking requirements or of fresh tissue sections for confocal imaging with genetically engi-

a combination of both. The MZ is often invoked as the likely point neered reporter- or gene-deficient mice will provide a unique ap- http://www.jimmunol.org/ of interaction for iNKT cells and activating APCs bearing pathogens proach to dissecting the migration requirements for splenic iNKT and pathogen-derived Ags collected from the blood flowing into the cells under various conditions and to answer many future ques- spleen through the marginal sinus (13). Our data showing consoli- tions about the kinetics and localization of iNKT cells in other dation by activated iNKT cells and the requirement for MZ DCs primary and secondary lymphoid organs. after glycolipid immunization and during systemic infection are also consistent with a key activation mechanism of iNKT cells being via Disclosures engagement of CD1d-presented foreign or self-Ag in the MZ (11). The authors have no financial conflicts of interest. iNKT cells can be activated by a combination of TCR-mediated recognition of CD1d and TCR-independent activation by cytokines by guest on September 27, 2021 on a sliding scale of relative ratios (45). Specifically, iNKT cells are References activated to produce IFN-g and IL-4 by TCR recognition of the 1. Cohen, N. R., S. Garg, and M. B. Brenner. 2009. Antigen Presentation by CD1 Lipids, T Cells, and NKT Cells in Microbial Immunity. Adv. Immunol. 102: 1–94. glycolipids tested in this study in the context of CD1d. They can also 2. Brigl, M., L. Bry, S. C. Kent, J. E. Gumperz, and M. B. Brenner. 2003. Mech- produce IFN-g following TCR engagement of CD1d-presenting anism of CD1d-restricted natural killer T cell activation during microbial infection. Nat. Immunol. 4: 1230–1237. self-glycolipids when receiving costimulation from IL-12. Finally, 3. Van Kaer, L., V. V. Parekh, and L. Wu. 2011. 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