gd T cells are innate-like T lymphocytes that play critical roles in tumor surveillance, and have been demonstrated to both promote tumor growth/metastasis and efficiently kill tumor cells. Specifically, gdT1 subsets have been associated with anti-tumor immunity, while gdT17 subsets have been associated with tumor growth and metastasis. However, the factors that control the selective activation of gdT1 vs. gdT17 in the tumor microenvironment remain unclear, as are the mechanisms through which gdT17 promote tumor growth/metastasis. To explore these questions, we developed an inducible mouse model of Kras-driven lung adenocarcinoma with or without a lung club cell-specific deletion of the Stk11 tumor suppressor. Upon induction of tumorigenesis, KrasG12D/Stk11fl mice exhibited significantly greater mortality than KrasG12D mice. Interestingly, the increased mortality in the KrasG12D/Stk11fl mice was associated with a selective expansion of Vg4 and Vg6 gdT17 subsets in the lungs of these mice. Characterization of these gd T cells using scCITEseq, revealed the presence of significant transcriptional heterogeneity in the gdT17 subsets that was associated with a significant expansion of a novel Il2ra+Ctla4+Areg+ Vg6 gdT17 subset in KrasG12D/Stk11fl lungs. Together these data suggest that tumor-specific Stk11 loss leads to the expansion of a novel subset of gdT17 cells with pro-tumor and immunosuppressive potential. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

During thymic development, most γδ T cells acquire innate-like characteristics that are critical for their function in tumor surveillance, infectious disease, and tissue repair. The mechanisms, however, that regulate γδ T cell developmental programming remain unclear. Recently, we demonstrated that the SLAM/SAP signaling pathway regulates the development and function of multiple innate-like γδ T cell subsets. Here, we used a single-cell proteogenomics approach to identify SAP-dependent developmental checkpoints and to define the SAP-dependent γδ TCR repertoire in mice. SAP deficiency resulted in both a significant loss of an immature Gzma+Blk+Etv5+Tox2+ γδT17 precursor population and a significant increase in Cd4+Cd8+Rorc+Ptcra+Rag1+ thymic γδ T cells. SAP-dependent diversion of embryonic day 17 thymic γδ T cell clonotypes into the αβ T cell developmental pathway was associated with a decreased frequency of mature clonotypes in neonatal thymus, and an altered γδ TCR repertoire in the periphery. Finally, we identify TRGV4/TRAV13-4(DV7)-expressing T cells as a novel, SAP-dependent Vγ4 γδT1 subset. Together, the data support a model in which SAP-dependent γδ/αβ T cell lineage commitment regulates γδ T cell developmental programming and shapes the γδ TCR repertoire.

Developments in mRNA/lipid nanoparticle (LNP) technology have advanced the fields of vaccinology and gene therapy, raising questions about immunogenicity. While some mRNA/LNPs generate an adjuvant-like environment in muscle tissue, other mRNA/LNPs are distinct in their capacity for multiple rounds of therapeutic delivery. We evaluate the adjuvancy of components of mRNA/LNPs by phenotyping cellular infiltrate at injection sites, tracking uptake by immune cells, and assessing the inflammatory state. Delivery of 9 common, but chemically distinct, LNPs to muscle revealed two classes of inflammatory gene expression programs: inflammatory (Class A) and noninflammatory (Class B). We find that intramuscular injection with Class A, but not Class B, empty LNPs (eLNPs) induce robust neutrophil infiltration into muscle within 2 h and a diverse myeloid population within 24 h. Single-cell RNA sequencing revealed SM-102-mediated expression of inflammatory chemokines by myeloid infiltrates within muscle 1 day after injection. Surprisingly, we found direct transfection of muscle infiltrating myeloid cells and splenocytes 24 h after intramuscular mRNA/LNP administration. Transfected myeloid cells within the muscle exhibit an activated phenotype 24 h after injection. Similarly, directly transfected splenic lymphocytes and dendritic cells (DCs) are differentially activated by Class A or Class B containing mRNA/LNP. Within the splenic DC compartment, type II conventional DCs (cDC2s) are directly transfected and activated by Class A mRNA/LNP. Together, we show that mRNA and LNPs work synergistically to provide the necessary innate immune stimuli required for effective vaccination. Importantly, this work provides a design framework for vaccines and therapeutics alike.

Drugs are needed to protect against the neutrophil-derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but may cause secondary, deleterious effects such as excessive bleeding. Here, we demonstrate that suramin—a widely available polyanionic drug—completely neutralizes the toxic effects of histones. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone-induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial-dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sub-lethal doses of histones in vivo. Suramin also prevented lung edema, intra-alveolar hemorrhage and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels. Significance Statement Pathologic levels of circulating histones cause acute endotheliopathy, characterized by widespread disruption of critical endothelial functions and thromboinflammation. We discovered that suramin binds histones and prevents histone-induced endothelial dysfunction, thrombin generation, lung injury, and death. Histone binding is a novel mechanism of action for suramin, considered among the safest and most effective drugs by the World Health Organization. These results support the use of suramin for protection of blood vessels in conditions exacerbated by circulating histones including trauma and sepsis.

γδ T cells have been demonstrated to both promote tumor growth/metastasis and to efficiently kill tumor cells. This dual role of γδ T cells has been attributed to their ability to produce either IL-17 (γδT17) or IFNγ (γδT1), the former being associated with tumor progression and the latter being associated with anti-tumor activity. γδ T cell function is influenced by multiple factors, including the recognition of stress-induced ligands, as well as cytokines and chemokines. However, the specific signals that lead to the selective activation and function of γδT17 or γδT1 subsets in different cancers remains unclear. Here, we explored these questions using a novel inducible mouse model of Kras-driven lung adenocarcinoma with or without a co-mutation in Stk11, a master regulator of metabolic pathways. Preliminary characterization of this model revealed that Kras G12D/Stk11 fl/flmice exhibited significantly greater morbidity than Kras G12Dmice. While lung leukocyte numbers did not differ significantly between Kras G12Dand Kras G12D/Stk11 fl/flmice, we noted a significantly increased frequency of γδT17, but not γδT1, subsets in Kras G12D/Stk11 fl/fllungs. Interestingly, the increased frequency of γδT17 was due to increased Vγ1 and Vγ6, but not Vγ4, γδT17 suggesting a selective expansion of these subsets. To examine a potential role for butyrophilins in these models, we assessed lung butyrophilin gene expression using qPCR. This analysis revealed increased expression of Btnl1, Btnl2, and Btnl9 in Kras G12Dlungs mice as compared to controls. Together, these results suggest that this model has the potential to shed new light on how the interaction of the tumor microenvironment with specific γδ T cell subsets affects disease pathogenesis.

Innate-like T cells are unusual T cells that are enriched in mucosal tissues, where they constitute a prominent source of pro-inflammatory cytokines like IL-17 and IFN-γ. During thymic development, mouse γδ T cells commit to either γδT1, γδT2, or γδT17 phenotypes through mechanisms that remain unclear. Recent observations from our laboratory have suggested a role for the SLAM/SAP signaling pathway in γδ T cell thymic developmental programming. Here, we investigated the influence of the SLAM/SAP signaling pathway on γδ TCR signal strength during thymic development. Using Nur77 GFPexpression as a readout of TCR signal strength, we observed significantly greater Nur77 GFPexpression in B6.Nur77 GFP.SAP −/−thymic γδ T cells compared their B6.Nur77 GFPcounterparts, suggesting that SAP inhibited TCR signal strength. Consistent with these findings, we observed significantly greater Erk phosphorylation in CD3-stimulated B6.SAP −/−thymic γδ T cells. In vitrostimulation of thymic γδ T cells with anti-TCRδ and anti-SLAM family receptors revealed that SLAMF6 co-stimulation resulted in decreased Erk phosphorylation, but increased Nur77 GFPexpression. Surprisingly, the effects of SLAMF6 co-stimulation on γδ TCR signaling were SAP-independent. Together, these data suggest an inhibitory role for SAP in γδ TCR signaling during thymic developmental programming.

γδ T cells are non-conventional T cells that are highly enriched in the mucosal tissues where they play critical roles in immunity. We recently demonstrated that the SLAM/SAP signaling pathway regulates the thymic development of innate-like γδT17 and γδTIFN subsets, in addition to γδNKT cells. Here, we utilized a single-cell proteogenomics approach coupled with γδ V(D)J profiling to define the transcriptional landscape and developmental checkpoints of SAP-dependent γδ T cells. This analysis not only confirmed our previous finding that SLAMF1 and SLAMF6 expression marks γδT17 and γδTIFN subsets, respectively, it also identified SLAMF7 as a novel marker of the SAP-dependent innate-like CD44+ CD45RB+ γδTIFN cells in both the thymus and periphery. Next, our data indicated that disruption of SAP-dependent signaling impaired γδT17 development at a very early (CD24high CD73−) stage, and was associated with the decreased expression of critical regulators of γδT17 development such as Blk and c-Maf. In contrast, while SAP also impaired γδTIFN development at an early (CD24high CD73+) stage, SAP-deficient γδTIFN cells exhibited increased expression of genes associated with TCR signaling and thymic export such as Prkch, Dgka, Klf2, and S1p1r. Finally, our analysis revealed significant alterations in the γδT17 TCR repertoire in both embryonic/neonatal thymus and the lung, as well as the presence of a SAP-dependent IFN-γ-producing lung Vγ4 population that preferentially utilized TRDV7. Altogether, these data suggest that SLAM/SAP signaling acts during the very early stages of γδ T cell development where it regulates critical pathways in both γδT17 and γδTIFN development, and influences the development of the innate-like γδ TCR repertoire. Supported by NIH (R03AI153902, P30GM118228), AAI Careers in Immunology Fellowship

Decellularized pig lungs recellularized with human lung cells offer a novel approach for organ transplantation. However, the potential immunogenicity of decellularized pig lungs following exposure to human tissues has not been assessed. We found that exposure of native lungs from wildtype and transgenic pigs lacking alpha (1,3)-galactosyltransferase (α-gal KO) to sera from normal healthy human volunteers demonstrated similar robust IgM and IgG immunoreactivity, comparably decreased in decellularized lungs. Similar results were observed with sera from patients who had previously undergone transcutaneous porcine aortic valve replacement (TAVR) or from patients with increased circulating anti-α-gal IgE antibodies (α-gal syndrome). Depleting anti-α-gal antibodies from the sera demonstrated both specificity of α-gal immunoreactivity and also residual immunoreactivity similar between wildtype and α-gal KO pig lungs. Exposure of human monocytes and macrophages to native wildtype lungs demonstrated greater induction of M2 phenotype than native α-gal KO pig lungs, which was less marked with decellularized lungs of either type. Overall, these results demonstrate that native wildtype and α-gal KO pig lungs provoke similar immune responses that are comparably decreased following decellularization. This provides a further platform for potential use of decellularized pig lungs in tissue engineering approaches and subsequent transplantation schemes but no obvious overall immunologic advantage of utilizing lungs obtained from α-gal KO pigs.

Abstract Innate-like T cells are unusual T cells that are enriched in mucosal tissues, and which constitute a prominent source of pro-inflammatory cytokines like IL-17 and IFN-γ. While it is known that SLAM/SAP signaling is required for the development of innate-like iNKT and MAIT αβ T cells, far less is known about the role of this pathway in the development and function of γδ T cells. Here, we utilized a single-cell proteogenomics approach coupled with γδ V(D)J profiling to define the transcriptional landscape and developmental checkpoints of SAP-dependent γδ T cells. We found that SAP-dependent γδNKT TCRs utilized TRGV1 paired with TRAV15N-1 or TRAV15-1/DV6-1, and we identified two distinct developmental γδNKT stages in the neonatal thymus that were distinguished by SLAMF6 and SLAMF7 expression. Moreover, we found that a significant fraction of SLAMf1+innate-like γδT17 cells utilized TRGV4 and TRGV6, γ-chains paired with TRDV2 or TRDV5 δ-chains of limited diversity. Examination of SAP-deficient neonatal thymus revealed decreased numbers of mature SLAMF1+ γδT17 cells, which was associated with lower numbers of an invariant germline-encoded TRGV4/TRDV5 clonotype. Accordingly, we observed significant alterations in the SLAMF1+ γδT17 TCR repertoire in adult lung. We found that lung γδTIFN were CD44+CD45RB+CD27+ cells that co-expressed SLAMF6and SLAMF7, and that these cells predominantly utilized a diverse TRDV7 paired with TRGV4. Interestingly, we observed a specific decrease of these TRDV7+ γδTIFN cells in SAP-deficient mice, which we confirmed using qPCR. Altogether, these data indicate a crucial link between SLAM/SAP signaling and the development of functionally distinct innate-like γδ TCR clonotypes.

γδ T cells are unusual T cells that are highly enriched in mucosal tissues where they constitute a prominent source of pro-inflammatory cytokines like IL-17 and IFN-γ. The TCR usage of tissue-specific γδ T cells is often associated with specific effector functions. We have recently found that IL-17- and IFN-γ-producing Vγ4 γδ T cells in the mouse lung are marked by the expression of SLAMF1 and SLAMF6 receptors, respectively. The objective of this study was to investigate a possible link between SLAM-associated effector function and γδ TCR usage. We first identified the major Vγ4 γδ TCR clonotypes in the mouse lung (n=13 mice). Vγ4 γδ T cells were single cell-sorted and paired TCR clonotypes were identified using next-gen sequencing of TCR amplicon libraries. The data revealed that TRDV5, TRDV2, and TRDV7 chains accounted for 51.18%, 29.61%, and 14.96% of the productive TCRδ chain rearrangements, respectively. While TRDV5 and TRDV2 CDR3 sequences were limited in diversity, TRDV7 CDR3 sequences were highly diverse. A significant fraction (30.46%) of the TRDV5 sequences were characterized by an invariant germline-encoded Vδ5Dδ2Jδ1 sequence. A comparison between sorted SLAMF1+ and SLAMF6+lung Vγ4 γδ T cells revealed that TRDV5 and TRDV2 chains were predominantly associated with SLAMF1+IL-17+ γδ T cells while the TRDV7 chain was predominantly associated with SLAMF6+IFN-γ+ γδ T cells. Moreover, the invariant germline-encoded TRDV5 sequence was primarily associated with SLAMF1+IL-17+ cells. These data indicate that the lung Vγ4 γδ TCR repertoire is limited in diversity and that specific lung Vγ4 γδ TCR clonotypes segregate with the expression of discrete SLAM family receptors.

The tetravalent live attenuated dengue vaccine candidate TV003 induces neutralizing antibodies against all four dengue virus serotypes (DENV1-4), and protects against experimental DENV2 challenge in humans. Protection occurred with or without a secondary post-challenge rise in DENV2-specific neutralizing antibodies. Here we combined high resolution temporal sampling and analysis of antigen-specific responses after vaccination and challenge to determine the cellular underpinnings of the B cell response in relation to vaccine viremia and serum antibodies. TV003 vaccine-related viremia was associated with an acute plasmablast response that correlated with the development of serum neutralizing antibodies. At six-months following immunization, subjects had developed DENV2-specific memory B cells including serotype-specific and multi-valent responders. DENV2 challenge of vaccinees did not induce a post-challenge plasmablast response, though stronger and earlier post-vaccine plasmablast responses were associated with sterile humoral protection from DENV2 challenge (i.e. a lack of DENV2 antibody boosting). Our findings demonstrate that TV003 vaccine triggers a durable B cell response containing plasmablasts and memory B cells which functionally link early vaccine viremia and the serum antibody responses.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease. The current lack of an effective vaccine to simultaneously protect against the four serotypes of DENV in seronegative individuals is a major unmet medical need. Further, the immunological basis for protective immunity in the setting of DENV infection or vaccination is not fully understood. Our team has developed a live attenuated tetravalent dengue virus vaccine that provides complete protection in a human model of dengue virus challenge. The goal of this study was to define, in the context of protective human vaccination, the quality of vaccine-induced DENV-specific CD8(+) and CD4(+) T cells and the temporal dynamics associated with their formation and maintenance. Multifunctional, DENV-specific CD8(+) and CD4(+) T cells developed 8-14 days after vaccination and were maintained for at least 6 months. Virus-specific CD8 T+ cells were a mixture of effector memory T cells (T-EM) and effector memory T cells re-expressing CD45RA (T-EMRA), with T-EM cells predominating until day 21 post-vaccination and T-EMRA cells thereafter. The majority of virus-specific CD4(+) T cells were T-EM with a small fraction being T-EMRA. The frequency of virus-specific CD8(+) and CD4(+) T cells were further skewed to the T-EMRA phenotype following either a second dose of the tetravalent vaccine or challenge with a single serotype of DENV. Collectively, our study has defined the phenotypic profile of antiviral CD8(+) and CD4(+) T cells associated with protective immunity to DENV infection and the kinetics of their formation and maintenance.

During thymic development, mouse γδ T cells commit to either an IFN-γ- or an IL-17-producing phenotype through mechanisms that remain unclear. In this study, we investigated the extent to which the SLAM/SAP signaling pathway regulates the functional programming of γδ T cells. Characterization of SLAM family receptor expression revealed that thymic γδ T cell subsets were each marked by distinct coexpression profiles of SLAMF1, SLAMF4, and SLAMF6. In the thymus, Vγ1 and Vγ4 T cells that exhibited an SLAMF1+SLAMF6+ double positive phenotype were largely contained within immature CD24+CD73- and CD24+CD73+ subsets, whereas SLAMF1 single positive, SLAMF6 single positive, or SLAMF1SLAMF6 double negative cells were found within mature CD24-CD73+ and CD24-CD73- subsets. In the periphery, SLAMF1 and SLAMF6 expression distinguished IL-17- and IFN-γ-producing γδ T cells, respectively. Disruption of SLAM family receptor signaling through deletion of SAP resulted in impaired thymic Vγ1 and Vγ4 T cell maturation at the CD24+CD73-SLAMF1+SLAMF6+ double positive stage that was associated with a decreased frequency of CD44+RORγt+ γδ T cells. Impaired development was in turn associated with decreased γδ T cell IL-17 and IFN-γ production in the thymus as well as in peripheral tissues. The role for SAP was subset-specific, as Vγ1Vδ6.3, Vγ4, Vγ5, but not Vγ6 subsets were SAP-dependent. Together, these data suggest that the SLAM/SAP signaling pathway plays a larger role in γδ T cell development than previously appreciated and represents a critical checkpoint in the functional programming of both IL-17- and IFN-γ-producing γδ T cell subsets.

γδ T cells are innate-like T cells that are one of the first responders in the defense against pathogens through their rapid production of pro-inflammatory cytokines like IL-17 and IFN-γ. Currently, the mechanisms that direct γδ T cells to exert these distinct effector functions are unknown. The limited TCR diversity of tissue-specific γδ T cells is often associated with specific effector functions. We have recently found that IL-17- and IFN-γ-producing Vγ4 γδ T cells in the lung are marked by SLAM1 and SLAM6 cell surface receptors, respectively. Our long-term goal is to investigate the link between γδ T cell effector function, specific SLAM family receptor expression, and TCR clonotype usage. The objective of this study was to identify the major Vγ4 γδ TCR clonotype(s) in the mouse lung. Vγ4 γδ T cells were single cell-sorted and paired TCR clonotypes were identified using next-generation sequencing of TCR amplicon libraries. Out of 789 sorted lung C57BL/6 Vγ4 T cells, we obtained paired TCR γ and δ chain sequences from 396 cells. These data revealed that Vδ5 and Vδ2 chains accounted for 56.20% and 35.77% of the productive TCRδ chain rearrangements, respectively. A significant fraction of the Vδ5 sequences were characterized by invariant germline-encoded (28.57%) or highly restricted (9.10%) Vδ5Dδ2Jδ1 sequences. Likewise, 55.10% of the Vδ2 sequences were characterized by a highly restricted Vδ2Dδ2Jδ1 sequence. These data reveal that the lung Vγ4 γδ T cell population is characterized by extremely limited delta chain usage. Since lung Vγ4 T γδ cells are programmed to be SLAM1+IL-17+ or SLAM6+IFN-γ+, future studies will determine whether there is a link between this limited TCRδ chain usage, cytokine production, and SLAM receptor expression.

Tissue-resident γδ T cells constitute a prominent source of IL-17 and IFN-γ in mucosal tissues where they provide an important first line of defense against pathogens. Unlike conventional αβ T cells, most γδ T cells are programmed during thymic development to produce either IFN-γ or IL-17. The mechanisms, however, that direct γδ T cell functional programming in the thymus remain unclear. Slam receptors are a family of nine (SLAMf1 to SLAMf9) cell surface receptors that are expressed only on hematopoietic cells and which play a central role in the host immune response. Most Slam receptors transduce signals via the small adapter protein SAP, and mice deficient in SAP exhibit numerous immunological defects. Relatively little is known, however, of the function of this signaling pathway in γδ T cells. Here, we report that distinct Slam receptor expression profiles mark functional γδ T cell subsets. Whereas SLAMf1 expression was associated with IL-17-producing CD27− RORγt+ γδ T cells, SLAMf6 expression was associated with IFNγ-producing CD27+ cells. We found that Slam receptor expression profiles were established on discrete thymic γδ T cell subsets as early as embryonic day 17 (E.17), as was the association of SLAMf1 with RORγt expression. Disruption of Slam receptor signaling by deletion of SAP resulted in a significant loss of E.17 thymic CD44hiRORγt+ γδ T cells, and a significant shift in the ratio of IFN-γ/IL-17-producing γδ T cells in both thymus and peripheral tissues. These results demonstrate for the first time that the Slam/SAP signaling pathway plays a significant role in the functional programming that regulates the γδ T cell IFN-γ/IL-17 axis during thymic development.

Invariant NKT (iNKT) cells are tissue-resident innate-like T cells critical to the host immune response. We previously identified a 6.6 Mbp region on chromosome 1 as a major regulator of iNKT cell number and function in C57BL/6 and 129X1/SvJ mice. Here, we fine-mapped this locus by assessing the iNKT cell response to alpha-galactosylceramide (αGalCer) in a series of B6.129 congenic lines. This analysis revealed the presence of at least two genetic elements that regulate iNKT cell cytokine production in response to αGalCer. While one of these genetic elements mapped to the B6.129c6 interval containing Slam genes, the dominant regulator in this region mapped to the 0.14 Mbp B6.129c3 interval. In addition, we found that numbers of thymic iNKT cells and DP thymocytes were significantly lower in B6.129c3 mice, indicating that this interval also regulates iNKT cell development. Candidate gene analysis revealed a fivefold increase in Fcgr3 expression in B6.129c3 iNKT cells, and we observed increased expression of FcγR3 protein on B6.129c3 iNKT cells, NK cells, and neutrophils. These data identify the B6.129c3 interval as a novel locus regulating the response of iNKT cells to glycosphingolipid, revealing a link between this phenotype and a polymorphism that regulates Fcgr3 expression.

Studies comparing endogenous and recombinant serum amyloid A (SAA) have generated conflicting data on the proinflammatory function of these proteins. In exploring this discrepancy, we found that in contrast to commercially sourced recombinant human SAA1 (hSAA1) proteins produced in Escherichia coli, hSAA1 produced from eukaryotic cells did not promote proinflammatory cytokine production from human or mouse cells, induce Th17 differentiation, or stimulate TLR2. Proteomic analysis of E. coli–derived hSAA1 revealed the presence of numerous bacterial proteins, with several being reported or probable lipoproteins. Treatment of hSAA1 with lipoprotein lipase or addition of a lipopeptide to eukaryotic cell–derived hSAA1 inhibited or induced the production of TNF-α from macrophages, respectively. Our results suggest that a function of SAA is in the binding of TLR2-stimulating bacterial proteins, including lipoproteins, and demand that future studies of SAA employ a recombinant protein derived from eukaryotic cells.

Serum amyloid A (SAA) proteins are a family of acute phase apolipoproteins implicated to directly modulate innate and adaptive immune responses. However, new studies comparing endogenous SAAs and recombinant forms of these proteins have questioned the function of SAA in inflammation and immunity. We generated SAA3 knockout mice to evaluate the contribution of SAA3 to lung development and immune-mediated lung disease. While SAA3 deficiency does not affect the generation of house dust mite-induced allergic asthma, mice lacking SAA3 develop adult-onset obesity, intrinsic airway hyperresponsiveness, increased inflammatory and fibrotic gene expression in the lung, and elevated levels of lung citrullinated proteins. Polyclonally stimulated CD4+ T cells from SAA3−/− mice exhibit impaired glycolytic activity, decreased TH2 and TH1 cytokine secretion, and elevated IL-17A production compared to wild type cells. Polyclonally stimulated CD8+ T cells from SAA3−/− mice also exhibit impaired glycolytic activity as well as a diminished capacity to produce IL-2 and IFNγ. Finally, SAA3−/− mice demonstrate increased mortality in response to H1N1 influenza infection, along with higher copy number of viral RNAs in the lung, a lack of CD8+ T cell IFNγ secretion, and decreased flu-specific antibodies. Our findings indicate that endogenous SAA3 regulates lung development and homeostasis, and is required for protection against H1N1 influenza infection.

NKT cells play a critical role in tissue-specific immune responses by rapidly producing cytokines in response to glycolipid stimulation. We previously reported that host genetic background played a substantial role in the NKT cell response to the agonist glycolipid α-galactosylceramide (αGalCer). A 6.6 Mbp region on chromosome 1 was identified as a major regulator of the NKT cell cytokine response between C57BL/6 (B6) and 129X1/SvJ mice. To fine-map this locus, we generated several B6.129 congenic lines with overlapping 129X1/SvJ intervals from 0.1 to 1.1 Mbp, and assessed the response of NKT cells to αGalCer. An analysis of serum and intracellular cytokine production after αGalCer challenge revealed significantly reduced levels of all cytokines tested in the B6.129c3 (c3) congenic strain that possessed the minimal 129 interval containing 5 genes. In vitro analysis of sorted splenic NKT cells indicated that the reduced cytokine production was NKT cell-intrinsic. In addition, we observed significantly fewer thymic NKT cells in the B6.129c3 congenic mice suggesting that the c3 interval may also regulate NKT cell development. Analysis of candidate genes within the c3 interval revealed a 5-fold increase in Fcgr3 gene expression on c3 NKT cells, which was associated with increased expression of FcγR3 protein on B6.129c3 NKT, NK and neutrophils. Taken together, these data identify the c3 interval as a novel locus regulating the response of NKT cells to glycosphingolipid, and reveal a link between this phenotype and an expression polymorphism that regulates Fcgr3 expression.