Transnuclear mice reveal Peyer's patch iNKT cells that regulate B‐cell class switching to IgG1
2019; Springer Nature; Volume: 38; Issue: 14 Linguagem: Inglês
10.15252/embj.2018101260
ISSN1460-2075
AutoresEleanor Clancy‐Thompson, Gui Zhen Chen, Nelson M. LaMarche, Lestat R. Ali, Hee‐Jin Jeong, Stephanie J. Crowley, Kelly Boelaars, Michael B. Brenner, Lydia Lynch, Stephanie K. Dougan,
Tópico(s)T-cell and B-cell Immunology
ResumoArticle31 May 2019Open Access Transparent process Transnuclear mice reveal Peyer's patch iNKT cells that regulate B-cell class switching to IgG1 Eleanor Clancy-Thompson Eleanor Clancy-Thompson Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Gui Zhen Chen Gui Zhen Chen Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Nelson M LaMarche Nelson M LaMarche Department of Rheumatology, Brigham and Women's Hospital, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Lestat R Ali Lestat R Ali orcid.org/0000-0003-2673-1592 Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Hee-Jin Jeong Hee-Jin Jeong Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Stephanie J Crowley Stephanie J Crowley Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Kelly Boelaars Kelly Boelaars Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA VU University Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Michael B Brenner Michael B Brenner Department of Rheumatology, Brigham and Women's Hospital, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Lydia Lynch Lydia Lynch Department of Rheumatology, Brigham and Women's Hospital, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Stephanie K Dougan Corresponding Author Stephanie K Dougan [email protected] orcid.org/0000-0002-2263-363X Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Eleanor Clancy-Thompson Eleanor Clancy-Thompson Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Gui Zhen Chen Gui Zhen Chen Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Nelson M LaMarche Nelson M LaMarche Department of Rheumatology, Brigham and Women's Hospital, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Lestat R Ali Lestat R Ali orcid.org/0000-0003-2673-1592 Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Hee-Jin Jeong Hee-Jin Jeong Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Stephanie J Crowley Stephanie J Crowley Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Kelly Boelaars Kelly Boelaars Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA VU University Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Michael B Brenner Michael B Brenner Department of Rheumatology, Brigham and Women's Hospital, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Lydia Lynch Lydia Lynch Department of Rheumatology, Brigham and Women's Hospital, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Stephanie K Dougan Corresponding Author Stephanie K Dougan [email protected] orcid.org/0000-0002-2263-363X Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Eleanor Clancy-Thompson1,‡, Gui Zhen Chen1,‡, Nelson M LaMarche2,3, Lestat R Ali1, Hee-Jin Jeong1,5, Stephanie J Crowley1, Kelly Boelaars1,4, Michael B Brenner2,3, Lydia Lynch2,3 and Stephanie K Dougan *,1,3 1Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA 2Department of Rheumatology, Brigham and Women's Hospital, Boston, MA, USA 3Program in Immunology, Harvard Medical School, Boston, MA, USA 4VU University Amsterdam, Amsterdam, The Netherlands 5Present address: Hongik University, Seoul, Korea ‡These authors contributed equally to this work *Corresponding author. Tel: +1 617 582 9609; Fax: +1 617 582 9610; E-mail: [email protected] The EMBO Journal (2019)38:e101260https://doi.org/10.15252/embj.2018101260 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens. Synopsis We used iNKT transnuclear mice to detect iNKT cells from tissues where they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches that produces IL-4 and helps B-cell class switching to IgG1. iNKT transnuclear mice faithfully model tissue-resident iNKT cell populations. iNKT cells are found in Peyer's patches of both wild type and transnuclear mice and can be used as cellular adjuvants for oral vaccination. PP-NKT cells produce the majority of the IL-4 in Peyer's patches, and provide indirect help for B-cell class switching to IgG1. Introduction iNKT cells are T cells with semi-invariant TCRs that recognize lipid antigens presented on CD1d. They exist as a pre-expanded pool and can rapidly respond by producing a range of different cytokines (Brennan et al, 2013). iNKT cell functional subsets have been described that parallel the CD4 T-cell subsets: NKT1 cells express T-bet and are poised to secrete IFNγ; NKT2 cells express high levels of PLZF and are poised to secrete IL-4, while NKT17 cells are RORγt+ and poised to secrete IL-17 (Kim et al, 2015; Wang & Hogquist, 2018). Each of these subsets can be found in the thymus and appear at different ratios in spleen and liver, which are the most abundant sources of iNKT cells in the mouse (Engel et al, 2016; Tuttle & Gapin, 2018). iNKT cells are also found in disparate tissues such as lung, adipose tissue, and intestinal lamina propria (Crosby & Kronenberg, 2018). In lung, iNKT cell production of GM-CSF helps control Mycobacterium tuberculosis infection (Rothchild et al, 2017). iNKT cells in the gut interact with CD1d on epithelial cells to cause feedback production of IL-10 under homeostatic conditions (Olszak et al, 2014), but can be activated by oxazolone-induced inflammation to trigger colitis (Heller et al, 2002; Iyer et al, 2018). Gut iNKT cells are also induced by microbial ligands early in life (Olszak et al, 2012; An et al, 2014) and help shape the nascent microbiome (Selvanantham et al, 2016; Saez de Guinoa et al, 2018). In adipose tissue, iNKT cell interactions with macrophages set the metabolic tone of the whole animal and affect insulin sensitivity and propensity toward obesity (Lynch et al, 2012, 2015; Exley et al, 2014). In addition to the well-described NKT1/2/17 subsets, iNKT cells can also have follicular helper function (Chang et al, 2011; King et al, 2011; Dellabona et al, 2014; Doherty et al, 2018) and regulatory function (Monteiro et al, 2010; Sag et al, 2014), or produce primarily IL-9 (Kim & Chung, 2013; Monteiro et al, 2015). The role of iNKT cell subsets in distinct tissue environments has been often difficult to elucidate due to poor cell recovery and a paucity of iNKT cells at baseline. iNKT cells can provide B-cell help in two fashions: either by cognate interactions between CD1d-expressing B cells and CD40L-expressing iNKT cells (Galli et al, 2007; Barral et al, 2008; Leadbetter et al, 2008) or by non-cognate interactions whereby iNKT cells license dendritic cells to prime CD4 Tfh cells (Tonti et al, 2009; Vomhof-DeKrey et al, 2014). Cognate interactions generate short-term bursts of Ig production, but do not sustain long-term B-cell memory or generate long-lived plasma cells (King et al, 2011; Tonti et al, 2012; Vomhof-DeKrey et al, 2015). Non-cognate interactions do generate long-term memory, and several studies have shown differences between help provided by splenic iNKT Tfh versus CD4 Tfh, despite the fact that both cell types produce IL-21 and express CD40L (King et al, 2011; Tonti et al, 2012). In addition to cognate and non-cognate help, early production of IL-4 by iNKT cells in the lung was demonstrated to be critical for B-cell survival and entry into germinal centers upon infection with viral pathogens (Gaya et al, 2018). These studies defined iNKT cell provision of IL-4 as a third mechanism by which iNKT cells offer B-cell help (Gaya et al, 2018). NKT cell functional differentiation can begin as early as thymic development (Lee et al, 2013). Signal strength through the TCR during positive selection can skew function, with higher affinity or more TCR signaling leading to NKT2 cells and lower TCR signaling required for NKT1 cell development (Matulis et al, 2010; Cruz Tleugabulova et al, 2016; Tuttle et al, 2018; Zhao et al, 2018). We previously reported a panel of iNKT cell transnuclear mice, cloned by somatic cell nuclear transfer from the nuclei of individual iNKT cells, which express monoclonal Vβ7 or Vβ8.2 TCRs (Clancy-Thompson et al, 2017). Tissue-specific factors have been implicated in iNKT cell subset specification, and our study unequivocally showed that monoclonal iNKT cells with different ligand specificities differentiate in vivo into all iNKT subsets at relatively normal frequencies, with a slight skewing of particular TCRs toward or away from NKT17 profiles. TCR specificity does not measurably affect localization of iNKT cells, their accumulation in tissues, or the expression of CD4 and has only a modest impact on transcription factor expression and cytokine production (Clancy-Thompson et al, 2017). Instead, tissue of origin plays a more dominant role in determining iNKT cell function, with iNKT cells from liver, skin-draining lymph nodes, spleen, and thymus having distinct cytokine and transcription factor profiles (Clancy-Thompson et al, 2017). Given the importance of tissue-resident iNKT cells, we further investigated whether our panel of transnuclear mice could be used as an abundant source of tissue-resident iNKT cells. We here show that iNKT cells from mesenteric lymph node, skin-draining lymph node, adipose tissue, lung, liver, and spleen coordinate distinct cytokine profiles. These cytokine profiles are similar among polyclonal and each of our monoclonal lines, suggesting that TCR specificity plays a minor role in the differentiation of tissue-resident iNKT cells. Our transnuclear iNKT cells faithfully recapitulate the skewing of NKT1/2/17 ratios seen in disparate tissues from C57BL/6 mice, and transnuclear iNKT cells from adipose tissue are similar to those reported from C57BL/6 mice as well. Furthermore, we uncovered a novel population of iNKT cells residing in Peyer's patches and show that PP-iNKT cells are critical for B-cell class switching to IgG1+ B cells in both steady state and upon oral vaccination. Results Tissue-resident iNKT cells are greatly enriched in iNKT transnuclear mice We used somatic cell nuclear transfer to generate three independent lines of transnuclear (TN) mice, all of which use the identical Vα14Jα18 TCRα chain, but with three distinct TCRβ rearrangements (Clancy-Thompson et al, 2017, 2018). When crossed to C57BL/6 mice, the TN TCR alleles segregate independently, which allowed us to establish a line of Vα14 TN mice that inherited only the rearranged TCRα locus and therefore develop polyclonal iNKT cells. These mice contain many-fold more iNKT cells in peripheral tissues than wild-type B6 mice (Fig 1A and B). The fold increase is especially pronounced in tissues where iNKT cells are rare, such as skin-draining and mesenteric lymph nodes, spleen, and lung. Figure 1. iNKT TN mice have increased numbers of tissue-resident iNKT cells Diagram representing the placement of various tissues analyzed for iNKT cells. mLN = mesenteric lymph node; sdLN = skin-draining lymph node. Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL/6 mouse lines. Tissues were isolated from indicated C57BL/6 or iNKT TN mouse lines and stained with anti-CD3 and CD1d-(PBS57)-tetramer. Spleen, mLN, sdLN, liver, adipose, and lung lymphocytes from Jα18−/− or Vα14 mice were stimulated in vitro with RAW-CD1d cells and 1 μg α-GalCer. An additional sample of Vα14 lymphocytes from each organ was plated with RAW-CD1d cells but no α-GalCer. Supernatants were collected after 24 h and cytokine concentration determined by cytokine bead array. Error bars are SD of mean values from three different mice per group. Results shown are representative of two independent experiments where n = 3 biological replicates. Download figure Download PowerPoint To better profile cytokine and chemokine production by iNKT cells from different tissues, lymphocytes were harvested from spleen, mLN, sdLN, liver, adipose tissue, and lung of Vα14 and Jα18−/− mice (lacking iNKT cells), and cocultured with RAWd cells with or without α-GalCer for 24 h. Culture supernatants were then analyzed by cytokine bead array. Unfractionated lymphocyte populations were used; thus, the cytokines analyzed were not necessarily secreted by iNKT cells directly. To determine which cytokines and chemokines were produced in an iNKT cell-dependent manner, lymphocytes from Jα18−/− mice stimulated with α-GalCer were included as a negative control. As a second negative control, Vα14 lymphocytes were cultured in the absence of added antigen to determine the production of iNKT-dependent cytokines in response to endogenous ligands. Of the 31 analytes examined, 15 cytokines and chemokines showed iNKT cell-dependent production as defined by increased production in Vα14 cultures compared to Jα18−/− cultures across most tissues (Fig 1C). Mesenteric lymph node iNKT cells produced IL-4 and IL-13, as well as LIF and IL-2, suggesting that a large fraction of these cells are NKT2 (Fig 1C and Lee et al, 2015). Liver iNKT cells adopted more of an NKT1-like profile and produced CXCL9, CXCL10, and IFNγ. Liver iNKT cells also produced some IL-4, consistent with a previous report of IL-4 secretion by iNKT cells during sterile liver injury (Liew et al, 2017). Both inguinal LN and lung iNKT cells produced IL-17, although lung iNKT also produced IL-10 (Fig 1C). Adipose cultures produced IL-10, as previously reported (Lynch et al, 2012, 2015; Sag et al, 2014), as well as GM-CSF and eotaxin (Fig 1C). Spleen appeared to contain the most diverse iNKT population, capable of making nearly all cytokines and chemokines examined, although this likely reflects a mixture of several different functional subsets. Therefore, iNKT cells coordinate a signature cytokine profile dependent on the tissue of origin. The impact of tissue of origin on cytokine profiles was apparent in cultures containing polyclonal iNKT as well as monoclonal iNKT cells from Vβ7A, Vβ7C, and Vβ8.2 TN mice, although subtle differences in the relative magnitude of cytokine production from iNKT cells bearing different TCRs may exist (Fig EV1). Click here to expand this figure. Figure EV1. Cytokine production is influenced by tissue microenvironment, not by TCR specificitySpleen, mLN, sdLN, liver, and adipose iNKT cells from Vα14, Vβ7A, Vβ7C, and Vβ8.2 mice (n = 3 mice per group) were stimulated in vitro with RAW-CD1d cells and 1 μg α-GalCer. Supernatants were collected after 24 h and cytokine concentrations determined by cytokine bead array. Error bars show SD of mean values. Results shown are representative of three independent experiments where n = 3 biological replicates per experiment. Download figure Download PowerPoint Tissue-specific imprinting of TN iNKT cells recapitulates that seen in wild-type iNKT cells To ensure that tissue-resident iNKT cells obtained from our TN mice faithfully recapitulate the phenotype of tissue-resident iNKT cells from wild-type mice, we examined expression of lineage-specific transcription factors in iNKT cells across multiple tissues in Vα14 and C57BL/6 mice (Fig 2A–F). IFNγ-poised NKT1 cells are characterized by expression of T-bet, while NKT2 cells are PLZFhigh, and NKT17 cells express RORγt (Kim et al, 2015). Thymic differentiation is altered in iNKT TN mice, with an increase in the NKT2:NKT1 ratio in the thymus, consistent with our previous report (Clancy-Thompson et al, 2017). However, across all peripheral tissues, Vα14 TN iNKT cells showed similar frequencies of NKT1/2/17 cells when compared to iNKT cells in those same tissues from C57BL/6 mice, with the exception of slightly higher frequencies of NKT2 cells in the lungs of Vα14 TN mice (Fig 2G). Inguinal LN iNKT cells from both Vα14 and C57BL/6 mice had an increased frequency of RORγt+ NKT17 cells—a population that was notably absent from mesenteric LN iNKT cells in both groups of mice. Liver iNKT cells were more strongly T-bet+, indicating an increased frequency of NKT1 cells in the liver (Fig 2G). Adipose iNKT cells are among the more distinct iNKT cell lineages and express the transcription factor E4BP4 rather than PLZF. We analyzed adipose iNKT cells from Vα14 TN mice and found low levels of PLZF and high expression of E4BP4, similar to C57BL/6 mice and previous reports (Fig EV2A–D and Lynch et al, 2015). Figure 2. iNKT cells from TN and C57BL/6 mice show similar influence of tissue microenvironment on NKT1, NKT2, and NKT17 subsets A–F. Lymphocytes from the indicated tissues of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d-(PBS57)-tetramer, before they were fixed, permeabilized, and stained with antibodies to T-bet, RORγt, and PLZF. Results shown are gated on CD3+CD1d-tetramer+ cells. G. The percentage of CD3+ CD1d-tetramer+ iNKT cells in each organ that stained positively for PLZF, T-bet, and RORγt are shown. **P < 0.01, Mann–Whitney test. Error bars are SD. Data information: Results shown are representative of three independent experiments where n = 3 biological replicates. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Adipose iNKT cells from Vα14 TN mice are indistinguishable from C57BL/6-derived adipose iNKT cells Flow cytometry analysis of iNKT cell abundance in white adipose tissue from a Vα14 TN mouse. Spleen cells and stromal vascular fractions of white adipose tissue from Vα14 TN or C57BL/6 mice were stained intracellularly with anti-PLZF and analyzed by flow cytometry. Histograms shown are gated on CD1d-(PBS57)-tetramer+ CD3+ cells. Thymus, spleen, and adipose tissue were harvested from C57BL/6 mice and Vα14 TN mice. Cell suspensions were stained with antibodies to CD3, Nur77, E4BP4, and CD1d-(PBS57)-tetramer and analyzed by flow cytometry. Mean fluorescence intensity of Nur77 and E4BP4 staining after gating on iNKT cells is shown. N = 3 per group. Error bars are SEM. Representative histograms of E4BP4 staining are shown. Plots are gated on total CD3+ cells. Download figure Download PowerPoint iNKT cells are found in Peyer's patches of both wild-type and Vα14 TN mice and correlate with increased IgG1+ B cells iNKT cells with follicular helper-like function have been previously defined; immunization with α-GalCer induces the formation of NKTfh in the spleen (Chang et al, 2011). However, NKTfh have not previously been shown in Peyer's patches, an important site for germinal center formation that is continuously exposed to gut antigens. When we examined Peyer's patches from Vα14 TN mice, we found CD1d-tetramer+ iNKT cells at greatly increased frequency compared to C57BL/6 mice (Fig 3A and B). Importantly, we could detect a small population of iNKT cells in C57BL/6 mice as compared to Jα18−/− mice, indicating that iNKT cells are also present in Peyer's patches in wild-type mice (Fig 3A and B). Figure 3. iNKT TN mice show increased IgG1 production and IgG1+ B cells in the mLN and Peyer's patches A. Lymphocytes from Peyer's patches of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d-(PBS57)-tetramer. B. Percentage of lymphocytes that were CD3+CD1d-tetramer+ iNKT cells among Peyer's patches of C57BL/6, Vα14, and Jα18−/− mice are shown. Mann–Whitney test. Error bars are SD. C57BL/6 n = 22; Vα14 n = 16; Jα18−/− n = 8. C–E. Mice were analyzed for total fecal IgA (C), total fecal IgG1 (D), or total serum IgG1 (E) by ELISA. Mann–Whitney test. Error bars are SD. C57BL/6 n = 14; Vα14 n = 11; Jα18−/− n = 13. F. Percentages of total B cells that were IgG1+ in the spleen, mLN, and Peyer's patches of C57BL/6, Vα14, and Jα18−/− mice are shown. Mann–Whitney test. Error bars are SD. C57BL/6 n = 15; Vα14 n = 15; Jα18−/− n = 5. Download figure Download PowerPoint Peyer's patches are important sites of germinal center activity to produce antigen-specific antibodies (Reboldi and Cyster, 2016). We first measured fecal IgA titers and found only modest differences in total IgA among mice with low, high, and zero levels of PP-NKT cells (Fig 3C). IgA can be produced in both T-cell-dependent and T-cell-independent fashion and correlates with the amount of TGF-β present in Peyer's patches. Although IgA is the predominant isotype in the gut lumen, IgG is also secreted into and recycled from the gut lumen through binding to FcRn (Rath et al, 2013). IgG sampling of gut luminal contents is an important source of antigen acquisition and has a protective role against some enteric pathogens (Bry & Brenner, 2004; Maaser et al, 2004). To investigate IgG antibody production, we measured IgG1 titers by ELISA and IgG1+ B cells by flow cytometry (Fig 3D–F). IgG1+ B-cell frequencies were similarly low (< 1%) in spleens of C57BL/6, Vα14 TN, and Jα18−/− mice; total serum IgG1 was also not different among the groups. However, C57BL/6 and Vα14 mice had significantly increased numbers of IgG1+ B cells in both mLN and Peyer's patches and compared to Jα18−/− mice (Fig 3F), and increased fecal IgG1 titers (Fig 3D). Jα18−/− mice have somewhat limited TCR repertoire diversity (Bedel et al, 2012); thus, we also examined fecal IgG1 in CD1d−/− mice and age- and sex-matched C57BL/6 control mice. Both fecal IgG1 and the frequencies of IgG1+ B cells in mLN and Peyer's patches were reduced in CD1d−/− mice (Fig EV3A and B). Thus, PP-NKT cells are critical for homeostatic levels of IgG1+ B cells in the gut, but do not appear to be dose-limiting, as even the low frequencies of iNKT cells found in wild-type mice are sufficient to allow for class switching to IgG1. This requirement of iNKT cells for IgG1+ B cells in Peyer's patches was observed across two different mouse facilities (Fig EV3C). Click here to expand this figure. Figure EV3. Magnitude of regulation of IgG1+ B cells by NKT cells varies by mouse facility, but is dependent on CD1d Stool collected from C57BL/6 and CD1d−/− mice was analyzed by ELISA for IgG1 as described in Fig 3. Mice were age- and sex-matched and housed in the Longwood Center facility. C57BL/6 n = 18; CD1d−/− n = 5. Mann–Whitney test. Error bars are SD. Spleen, mLN, and PP cells were harvested from wild-type or CD1d−/− mice, stained with antibodies to B220 and IgG1, and analyzed by flow cytometry. Mice were age- and sex-matched and housed in the Longwood Center facility. C57BL/6 n = 5; CD1d−/− n = 3. Mann–Whitney test. Error bars are SD. Analysis was performed identically to that shown in Fig 3F, except that mice were housed in the Smith Building at Dana-Farber Cancer Institute prior to moving to the Longwood Center at Dana-Farber Cancer Institute. Results in Fig 3 are entirely from mice housed in the Longwood Center facility. C57BL/6 n = 10; Vα14−/− n = 12; Jα18−/− n = 3. Mann–Whitney test. Error bars are SD. Culture supernatants from Fig 5D and E were measured by ELISA for IgM. np = not performed. Mann–Whitney test. Error bars are SD. Download figure Download PowerPoint PP-NKT cells provide indirect help to B cells through production of IL-4 and are important in oral vaccination To determine how PP-NKT cells might regulate B-cell class switching, we sorted PP-NKT cells from Vα14 TN, as well as iNKT cells from spleen and CD4 T cells from Peyer's patches. Cell yields were adequate such that transcriptional profiling could be performed on bulk populations of cells isolated from 3 individual mice (n = 3 biological replicates). Cluster analysis revealed that PP-NKT cells were more similar to spleen iNKT cells than CD4 T cells, thereby confirming their identity as bona fide iNKT cells (Fig 4A). Genes associated with Tfh cell identity or required for their function were highly expressed in Peyer's patch CD4 T cells, but absent from PP-NKT (Fig 4B). Notably, PP-NKT expressed undetectable levels of CD40L and CXCR5, making it unlikely that PP-NKT cells make direct cell–cell contact with germinal center B cells. Figure 4. PP-NKT cells produce IL-4 in vitro and in vivo A. CD1d-(PBS57)-tetramer+ CD3+ cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4+CD3+CD1d-tetramer− cells from PP (PP CD4). RNAseq was performed. B. Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample. C, D. Spleen, mLN, and PP lymphocytes from Vα14 and C57BL/6 mice were stimulated with PMA and ionomycin. Lymphocytes were stained with anti-CD3, CD1d-(PBS57)-tetramer, anti-IL-4, and anti-IFNγ. Percentages of iNKT cells and non-iNKT T cells within the population of CD3+IL-4+ cells and CD3+IFNγ+ cells, n = 5 mice for C57BL/6 group and n = 4 mice for Vα14 group. Error bars are SD. E. Vα14 TN mice were administered α-GalCer either 2 μg intravenously or 5 μg by oral gavage. Mice were given brefeldin A intraperitoneally after 30 min, and tissues were harvested 3 h later. Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with antibodies to IL-4, IFNγ, and IL-17. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells. F. Quantification of data from (E), n = 2 mice per group. Mann–Whitney test. Error bars are SEM. Download figure Download PowerPoint IL-4 is important for regulating B-cell class switching to IgG1, and early production of IL-4 by iNKT cells in the lung was previously reported to be critical for supporting B cells en route to germinal centers (Gaya et al, 2018). We therefore examined IL-4 and IFNγ production from cells cultured from the spleen, mLN, or Peyer's patches (Fig 4C and D). Following stimulation, we analyzed the relative proportions of iNKT cells and non-iNKT T cells producing IL-4 or IFNγ and found that the majority of the IL-4 was derived from iNKT cells. In contrast, although iNKT cells produced IFNγ, the majority of the IFNγ was derived from other T cells in the cultures. We therefore conclude that PP-NKT cells could be an important local source of IL-4, which supports B-cell class switching and production of IgG1. To determine the relevant function of PP-NKT cells in vivo, we challenged mice with α-GalCer either intravenously or by oral gavage. Mice were treated with brefeldin A to prevent cytokine secretion, and then, cells from the indicated tissues were analyzed by intracellular cytokine staining (Fig 4E and F). NKT cells from spleen produced both IFNγ and IL-4 upon intravenous α-GalCer, but oral gavage failed to induce activation of spleen iNKT (Fig 4E and F). Peyer's patch iNKT cells produced primarily IL-4 upon oral administration of α-GalCer. Vα14 TN iNKT cells pooled from spleen and LNs, and cocultured with naïve B cells and α-GalCer yielded no detectable IgG1, consistent with the lack of detectable CD40L expression by iNKT cells (Figs 4B and 5A). Provision of agonistic anti-CD40 induced robust B-cell activation as evidenced by IgM secretion. IgM levels were not augmented by the presence of iNKT cells (Fig 5B). In contrast, IgG1 production was dependent on the presence of iNKT cells, was increased by α-GalCer, and was diminished by the
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