Radiation Bone Marrow Chimeras Research Articles

Minimally invasive longitudinal intravital imaging of cellular dynamics in intact long bone.

Intravital two-photon microscopy enables deep-tissue imaging at high temporospatial resolution in live animals. However, the endosteal bone compartment and underlying bone marrow pose unique challenges to optical imaging as light is absorbed, scattered and dispersed by thick mineralized bone matrix and the adipose-rich bone marrow. Early bone intravital imaging methods exploited gaps in the cranial sutures to bypass the need to penetrate through cortical bone. More recently, investigators have developed invasive methods to thin the cortical bone or implant imaging windows to image cellular dynamics in weight-bearing long bones. Here, we provide a step-by-step procedure for the preparation of animals for minimally invasive, nondestructive, longitudinal intravital imaging of the murine tibia. This method involves the use of mixed bone marrow radiation chimeras to unambiguously double-label osteoclasts and osteomorphs. The tibia is exposed by a simple skin incision and an imaging chamber constructed using thermoconductive T-putty. Imaging sessions up to 12 h long can be repeated over multiple timepoints to provide a longitudinal time window into the endosteal and marrow niches. The approach can be used to investigate cellular dynamics in bone remodeling, cancer cell life cycle and hematopoiesis, as well as long-lived humoral and cellular immunity. The procedure requires an hour to complete and is suitable for users with minimal prior expertise in small animal surgery.

The IL-4Rα Q576R polymorphism is associated with increased severity of atopic dermatitis and exaggerates allergic skin inflammation in mice

Atopic dermatitis (AD) is characterized by TH2-dominated skin inflammation and systemic response to cutaneously encountered antigens. The TH2 cytokines IL-4 and IL-13 play a critical role in the pathogenesis of AD. The Q576->R576 polymorphism in the IL-4 receptor alpha (IL-4Rα) chain common to IL-4 and IL-13 receptors alters IL-4 signaling and is associated with asthma severity. We sought to investigate whether the IL-4Rα R576 polymorphism is associated with AD severity and exaggerates allergic skin inflammation in mice. Nighttime itching interfering with sleep, Rajka-Langeland, and Eczema Area and Severity Index scores were used to assess AD severity. Allergic skin inflammation following epicutaneous sensitization of mice 1 or 2 IL-4Rα R576 alleles (QR and RR) and IL-4Rα Q576 (QQ) controls was assessed by flow cytometric analysis of cells and quantitative RT-PCR analysis of cytokines in skin. The frequency of nighttime itching in 190 asthmatic inner-city children with AD, as well as Rajka-Langeland and Eczema Area and Severity Index scores in 1116 White patients with AD enrolled in the Atopic Dermatitis Research Network, was higher in subjects with the IL-4Rα R576 polymorphism compared with those without, with statistical significance for the Rajka-Langeland score. Following epicutaneous sensitization of mice with ovalbumin or house dust mite, skin infiltration by CD4+ cells and eosinophils, cutaneous expression of Il4 and Il13, transepidermal water loss, antigen-specific IgE antibody levels, and IL-13 secretion by antigen-stimulated splenocytes were significantly higher in RR and QR mice compared with QQ controls. Bone marrow radiation chimeras demonstrated that both hematopoietic cells and stromal cells contribute to the mutants' exaggerated allergic skin inflammation. The IL-4Rα R576 polymorphism predisposes to more severe AD and increases allergic skin inflammation in mice.

Inhibition of macrophage proliferation dominates plaque regression in response to cholesterol lowering

Statins induce plaque regression characterized by reduced macrophage content in humans, but the underlying mechanisms remain speculative. Studying the translational APOE*3-Leiden.CETP mouse model with a humanized lipoprotein metabolism, we find that systemic cholesterol lowering by oral atorvastatin or dietary restriction inhibits monocyte infiltration, and reverses macrophage accumulation in atherosclerotic plaques. Contrary to current believes, none of (1) reduced monocyte influx (studied by cell fate mapping in thorax-shielded irradiation bone marrow chimeras), (2) enhanced macrophage egress (studied by fluorescent bead labeling and transfer), or (3) atorvastatin accumulation in murine or human plaque (assessed by mass spectrometry) could adequately account for the observed loss in macrophage content in plaques that undergo phenotypic regression. Instead, suppression of local proliferation of macrophages dominates phenotypic plaque regression in response to cholesterol lowering: the lower the levels of serum LDL-cholesterol and lipid contents in murine aortic and human carotid artery plaques, the lower the rates of in situ macrophage proliferation. Our study identifies macrophage proliferation as the predominant turnover determinant and an attractive target for inducing plaque regression.

Macrophage-specific IRF5 deficiency stabilizes atherosclerotic plaques in ApoE−/− mice

Abstract Background Interferon regulatory factor (IRF) 5 is a transcription factor promoting inflammatory macrophage polarization (M1 type). Given the central role of macrophages in atherosclerotic plaque development we hypothesized that macrophage specific deletion of IRF5 will protect from atherosclerosis. Purpose Investigate whether intrinsic blockade of M1 macrophage polarization ameliorates atherosclerosis Methods Female ApoE−/−LysMCre/wtIRF5flox/floxand ApoE−/−LysMwt/wtIRF5flox/floxmice were fed a high cholesterol diet for 3 months, and atherosclerotic plaque size and compositions as well as inflammatory gene expression were analyzed. Mechanistically, IRF5-dependend bone marrow derived macrophage cytokine profiles were tested under M1 and M2 polarizing conditions. Aortic macrophage chimerism in irradiated ApoE−/− mice reconstituted with a mixture of CD45.1+ ApoE−/− (WT) and CD45.2+ ApoE−/− LysMCre/WtIRF5flox/flox(KO) bone marrow was evaluated to distinguish systemic from intra-plaque effects on monocyte/macrophage kinetics. Results Macrophage-specific IRF5 deficiency blunted LPS/IFNg-induced IL-1β and TNFα gene expression in vitro. In ApoE−/− mice, macrophage-specific IRF5 deficiency did not alter lesion size in the aortic root but significantly reduced macrophage and lipid contents by about 25% while increasing collagen deposition by over 30%. This was accompanied by relative reductions in gene expressions of pro-inflammatory (IL-1β, IL-6, IL-12) and increases in anti-inflammatory (Mertk, TGFβ, CD206) markers in atherosclerotic aortas of ApoE−/−LysMCre/wtIRF5flox/floxmice. When competing with IRF5 deficient cells in mixed irradiation bone marrow chimeras, IRF5 competent macrophages showed an advantage in accumulating in atherosclerotic aortas as disease progressed independent of monocyte recruitment. Conclusion Transcription factor IRF5 promotes a pro-inflammatory response in macrophages leading to vulnerable plaque formation and plaque destabilization, providing genetic evidence for targeting macrophage polarization in atherosclerosis. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): DFG

Non-myeloablative busulfan chimeric mouse models are less pro-inflammatory than head-shielded irradiation for studying immune cell interactions in brain tumours

BackgroundChimeric mouse models generated via adoptive bone marrow transfer are the foundation for immune cell tracking in neuroinflammation. Chimeras that exhibit low chimerism levels, blood-brain barrier disruption and pro-inflammatory effects prior to the progression of the pathological phenotype, make it difficult to distinguish the role of immune cells in neuroinflammatory conditions. Head-shielded irradiation overcomes many of the issues described and replaces the recipient bone marrow system with donor haematopoietic cells expressing a reporter gene or different pan-leukocyte antigen, whilst leaving the blood-brain barrier intact. However, our previous work with full body irradiation suggests that this may generate a pro-inflammatory peripheral environment which could impact on the brain’s immune microenvironment. Our aim was to compare non-myeloablative busulfan conditioning against head-shielded irradiation bone marrow chimeras prior to implantation of glioblastoma, a malignant brain tumour with a pro-inflammatory phenotype.MethodsRecipient wild-type/CD45.1 mice received non-myeloablative busulfan conditioning (25 mg/kg), full intensity head-shielded irradiation, full intensity busulfan conditioning (125 mg/kg) prior to transplant with whole bone marrow from CD45.2 donors and were compared against untransplanted controls. Half the mice from each group were orthotopically implanted with syngeneic GL-261 glioblastoma cells. We assessed peripheral blood, bone marrow and spleen chimerism, multi-organ pro-inflammatory cytokine profiles at 12 weeks and brain chimerism and immune cell infiltration by whole brain flow cytometry before and after implantation of glioblastoma at 12 and 14 weeks respectively.ResultsBoth non-myeloablative conditioning and head-shielded irradiation achieve equivalent blood and spleen chimerism of approximately 80%, although bone marrow engraftment is higher in the head-shielded irradiation group and highest in the fully conditioned group. Head-shielded irradiation stimulated pro-inflammatory cytokines in the blood and spleen but not in the brain, suggesting a systemic response to irradiation, whilst non-myeloablative conditioning showed no cytokine elevation. Non-myeloablative conditioning achieved higher donor chimerism in the brain after glioblastoma implantation than head-shielded irradiation with an altered immune cell profile.ConclusionOur data suggest that non-myeloablative conditioning generates a more homeostatic peripheral inflammatory environment than head-shielded irradiation to allow a more consistent evaluation of immune cells in glioblastoma and can be used to investigate the roles of peripheral immune cells and bone marrow-derived subsets in other neurological diseases.

Trefoil Factor 2 Promotes Type 2 Immunity and Lung Repair through Intrinsic Roles in Hematopoietic and Nonhematopoietic Cells

Trefoil factors (TFFs) are small secreted proteins that regulate tissue integrity and repair at mucosal surfaces, particularly in the gastrointestinal tract. However, their relative contribution(s) to controlling baseline lung function or the extent of infection-induced lung injury are unknown issues. With the use of irradiation bone marrow chimeras, we found that TFF2 produced from both hematopoietic- and nonhematopoietic-derived cells is essential for host protection, proliferation of alveolar type 2 cells, and restoration of pulmonary gas exchange after infection with the hookworm parasite Nippostrongylus brasiliensis. In the absence of TFF2, lung epithelia were unable to proliferate and expressed reduced lung mRNA transcript levels for type 2 response-inducing IL-25 and IL-33 after infectious injury. Strikingly, even in the absence of infection or irradiation, TFF2 deficiency compromised lung structure and function, as characterized by distended alveoli and reduced blood oxygen levels relative to wild-type control mice. Taken together, we show a previously unappreciated role for TFF2, produced by either hematopoietic or nonhematopoietic sources, as a pro-proliferative factor for lung epithelial cells under steady-state and infectious injury conditions.

Peroxisome Proliferator-Activated Receptor Delta (PPARδ) activity is required for normal T cell development

Abstract PPARδ is a nuclear receptor that functions as a transcriptional regulator of various processes including metabolic homeostasis, cell proliferation and differentiation; however, whether PPARδ regulates T cell development is not known. We observed that PPARδ-deficient (PPARδ−/−) mice have smaller spleens and thymi compared to wild-type (WT) mice. This reduction in thymus cellularity was apparent across all stages of thymocyte development. To further narrow in on the cellular basis of this effect, radiation bone marrow chimeras were generated where WT or PPARδ−/− mice were reconstituted with WT or PPARδ−/− bone marrow. These experiments clearly revealed that the defects in thymus and spleen mapped to the radio-resistant compartment. This was furthered confirmed by the finding that mice with T cell-restricted deficiency of PPARδ did not exhibit defective thymocyte homeostasis. Given that thymic epithelial cells (TECs) are the major radio-resistant cell population in the thymus, we next generated mice with a TEC-restricted deficiency of PPARδ (PPARδflox/floxFoxN1-Cre+). We observed that PPARδflox/floxFoxN1-Cre+ mice exhibit a trend for reduced thymus and spleen cellularity compared to PPARδflox/flox mice; however, this phenotype was not as striking as seen in PPARδ−/− mice. These results suggest that, in addition to acting within TECs, PPARδ may regulate the production of a circulating factor that in turn regulates thymocyte development. Large-scale serum profiling studies are underway to identify this factor. Altogether, this work highlights a role for PPARδ in T cell development.

Dietary Vitamin D3 Suppresses Pulmonary Immunopathology Associated with Late-Stage Tuberculosis in C3HeB/FeJ Mice.

Tuberculosis (TB) is a significant human disease caused by inhalation of Mycobacterium tuberculosis. Left untreated, TB mortality is associated with a failure to resolve pulmonary immunopathology. There is currently widespread interest in using vitamin D3 (VitD3) as an adjunct therapy for TB because numerous in vitro studies have shown that VitD3 has direct and indirect mycobactericidal activities. However, to date, there have been no in vivo studies addressing whether VitD3 affects experimental TB outcome. In this study, we used C3HeB/FeJ mice to determine whether dietary VitD3 influences the outcome of experimental TB. We observed that although M. tuberculosis burdens did not differ between mice on a VitD3-replete diet (VitD(HI) mice) and mice on a VitD3-deficient diet (VitD(LO) mice), the inflammatory response in VitD(HI) mice was significantly attenuated relative to VitD(LO) controls. Specifically, the expression of multiple inflammatory pathways was reduced in the lungs at later disease stages as were splenocyte IL12/23p40 and IFN-γ levels following ex vivo restimulation. Dietary VitD3 also suppressed the accumulation of T cells in the mediastinal lymph nodes and lung granulomatous regions while concomitantly accelerating the accumulation of F4/80(+) and Ly6C/Ly6G(+) lineages. The altered inflammatory profile of VitD(HI) mice also associated with reductions in pulmonary immunopathology. VitD receptor-deficient (vdr(-/-)) radiation bone marrow chimeras demonstrate that reductions in pulmonary TB immunopathology are dependent on hematopoietic VitD responsiveness. Collectively, our data support a model wherein the in vivo role of VitD3 during TB is not to promote M. tuberculosis killing but rather to function through hematopoietic cells to reduce M. tuberculosis-elicited immunopathology.

Bone Marrow and Fetal Liver Radiation Chimeras.

Radiation chimeras are prepared by subjecting recipient mice to sublethal or lethal dose of irradiation and injecting them with hematopoietic stem cells (HSC) from untreated donor mice. HSC can be obtained from bone marrow or fetal liver. This technique is a powerful tool when coupled with gene targeting strategies to investigate function of HSCs, thymocyte development, and T cell function. This protocol describes how to produce bone marrow or fetal liver chimeras.

Peroxisome proliferator-activated receptor delta (PPARδ) limits Th1 inflammation through effects on interferon (IFN)-γ transcription and lymphocyte homeostasis (IRM7P.480)

Abstract PPARδ is a nuclear receptor that functions as a transcriptional regulator of various processes including metabolic homeostasis, cell proliferation and differentiation. We previously reported that mice deficient in PPARδ (KO) exhibit more severe experimental autoimmune encephalomyelitis than wild type (WT) mice. This more severe inflammation correlated with an increased expansion of myelin-reactive IFNγ-producing cells in the spinal cord, suggesting that PPARδ may act to limit Th1 inflammatory responses. However, the mechanism of how PPARδ accomplishes this is not clear. Characterization of the spleen and thymus compartments revealed that KO mice exhibit reduced spleen and thymus cellularity as compared to WT mice. The resultant lymphopenia in KO mice associated with a higher frequency of memory CD4+ T cells in the periphery. Radiation bone marrow chimeras were generated and the defect in thymocyte development was found to map to the radio-resistant compartment, suggesting that PPARδ may operate in stromal cells to regulate thymocyte development. This was furthered confirmed by the finding that mice with T cell-restricted deficiency of PPARδ did not exhibit defective thymocyte homeostasis. Furthermore, we observed that naïve CD4+ T cell from both KO and T cell-restricted PPARδ-deficient mice had a higher propensity to produce IFNγ upon stimulation. Thus PPARδ appears to limit Th1 inflammation both through the control of lymphocyte homeostasis and IFNγ production.

Nlrp3 activation in the intestinal epithelium protects against a mucosal pathogen.

Polymorphisms in the intracellular pattern recognition receptor gene NLRP3 have been associated with susceptibility to Crohn’s disease, a type of inflammatory bowel disease (IBD). Following tissue damage or infection, NLRP3 triggers the formation of inflammasomes, containing NLRP3, ASC and caspase-1, which mediate secretion of IL-1β and IL-18. However, the precise role of NLRP3 inflammasomes in mucosal inflammation and barrier protection remains unclear. Here we show that upon infection with the attaching/effacing (A/E) intestinal pathogen Citrobacter rodentium, Nlrp3−/− and Asc−/− mice displayed increased bacterial colonization and dispersion, more severe weight loss and exacerbated intestinal inflammation. Analyses of irradiation bone marrow chimeras revealed that protection from disease was mediated through Nlrp3 activation in non-hematopoietic cells and was initiated very early after infection. Thus, early activation of Nlrp3 in intestinal epithelial cells limits pathogen colonization and prevents subsequent pathology, potentially providing a functional link between NLRP3 polymorphisms and susceptibility to IBD.

MHC Class II–Alpha Chain Knockout Mice Support Increased Viral Replication That Is Independent of Their Lack of MHC Class II Cell Surface Expression and Associated Immune Function Deficiencies

MHCII molecules are heterodimeric cell surface proteins composed of an α and β chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα-/- (I-Aα-/-), MHC-IIAβ-/- (I-β-/-) and the double knockout (I-Aαxβ-/-). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα-/-, but not in I-β-/- or I-Aαxβ-/-, mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα-/- mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα-/- bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα-/- mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα-/- compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα-/- mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.

IL12B expression is sustained by a heterogenous population of myeloid lineages during tuberculosis

IL12B is required for resistance to Mycobacterium tuberculosis (Mtb) infection, promoting the initiation and maintenance of Mtb-specific effector responses. While this makes the IL12-pathway an attractive target for experimental tuberculosis (TB) therapies, data regarding what lineages express IL12B after infection is established are limited. This is not obvious in the lung, an organ in which both hematopoietic and non-hematopoietic lineages produce IL12p40 upon pathogen encounter. Here, we use radiation bone marrow chimeras and Yet40 reporter mice to determine what lineages produce IL12p40 during experimental TB. We observed that hematopoietic IL12p40-production was sufficient to control Mtb, with no contribution by non-hematopoietic lineages. Furthermore, rather than being produced by a single subset, IL12p40 was produced by cells that were heterogenous in their size, granularity, autofluorescence and expression of CD11c, CD11b and CD8α. While depending on the timepoint and tissue examined, the surface phenotype of IL12p40-producers most closely resembled macrophages based on previous surveys of lung myeloid lineages. Importantly, depletion of CD11c(hi) cells during infection had no affect on lung IL12p40-concentrations. Collectively, our data demonstrate that IL12p40 production is sustained by a heterogenous population of myeloid lineages during experimental TB, and that redundant mechanisms of IL12p40-production exist when CD11c(hi) lineages are absent.

Akt and mTOR Pathways Differentially Regulate the Development of Natural and Inducible IL-17-Producing CD4+ T Cells

Abstract 838Interleukin-17 (IL-17) producing CD4+ T helper (Th) cells, Th17 cells, are essential for immune responses against extracellular pathogens. Dysregulation of Th17 cells, meanwhile, leads to the pathogenesis of many inflammatory and autoimmune disorders, and the role of IL-17 and Th17 cells in cancer is the focus of extensive investigation. Recent studies have revealed that two distinct populations of Th17 cells exist. Inducible Th17 (iTh17) cells differentiate from naïve CD4+T cells in response to antigenic stimulation in the presence of an appropriate cytokine environment in the periphery, most notably the intestine, while natural Th17 (nTh17) cells acquire the capability of producing IL-17 during development in the thymus without a required differentiation step in the periphery. While iTh17 and nTh17 cells share many features, the signaling pathways essential for their development and function are not yet well understood. Using both genetic and pharmacological modulation of Akt activity, we show that Akt regulates the development of both nTh17 and iTh17 cells. Upon investigating the mechanism by which Akt controls the generation of both Th17 cell populations, we found that selective deficiency of mTORC1 activity did not affect nTh17 cells in contrast to the defective iTh17 cell generation in these mice (Rheb-deficient mice). The absence of mTORC2 activity, by deleting Rictor, an mTORC2-specific subunit, led to a severe defect in nTh17 cell development, while iTh17 cells were preserved in these mice. In line with the role of mTORC2 in nTh17 cells, mice deficient in both FoxO1 and FoxO3a, inhibitory proteins whose function is blocked by Akt and mTORC2, showed greatly enhanced nTh17 cell development. Mice receiving chronic rapamycin treatment, which has been demonstrated to inhibit not only mTORC1 but also mTORC2 activity, had greatly decreased nTh17 cells (Control: 29080±2426 nTh17s vs. Chronic Rapa: 1515±393.0 nTh17s, P=0.0004), while single dose of rapamycin, which interferes selectively with mTORC1 function, had no affect on these cells (Control: 29080±2426 nTh17s vs. Single Dose Rapa: 25310±2770 nTh17s, P=0.3631). In contrast, iTh17 cells were greatly diminished under both chronic and single dose of rapamycin treatment, supporting the distinct roles of mTORC1 and mTORC2 in controlling iTh17 versus nTh17 cell development. Finally, Akt isoform-specific activity also differentially contributes to nTh17 and iTh17 cell development. Selective deletion of Akt2, but not Akt1, resulted in defective iTh17 cell differentiation both in vitro and in vivo but preservation of nTh17 cells. Using mixed radiation bone marrow chimeras, we found that the aberrant iTh17 phenotype in Akt2-deficient mice is cell-intrinsic. Collectively, these data reveal novel mechanisms regulating nTh17 and iTh17 cell development and critical roles of Akt isoforms and the two distinct mTOR complexes in controlling the development of the Th17 cell subsets. Given the increasing interest in modulating Akt and mTOR pathways in various malignancies and the usage of mTOR inhibitors as immunosuppressants following tissue transplantation, our findings suggest that the effect on this aspect of immune system development should be taken into consideration when targeting these signaling pathways. Disclosures:No relevant conflicts of interest to declare.

Resolution of Experimental Lung Injury by Monocyte-Derived Inducible Nitric Oxide Synthase

Although early events in the pathogenesis of acute lung injury (ALI) have been defined, little is known about the mechanisms mediating resolution. To search for determinants of resolution, we exposed wild type (WT) mice to intratracheal LPS and assessed the response at intervals to day 10, when injury had resolved. Inducible NO synthase (iNOS) was significantly upregulated in the lung at day 4 after LPS. When iNOS-/- mice were exposed to intratracheal LPS, early lung injury was attenuated; however, recovery was markedly impaired compared with WT mice. iNOS-/- mice had increased mortality and sustained increases in markers of lung injury. Adoptive transfer of WT (iNOS+/+) bone marrow-derived monocytes or direct adenoviral gene delivery of iNOS into injured iNOS-/- mice restored resolution of ALI. Irradiated bone marrow chimeras confirmed the protective effects of myeloid-derived iNOS but not of epithelial iNOS. Alveolar macrophages exhibited sustained expression of cosignaling molecule CD86 in iNOS-/- mice compared with WT mice. Ab-mediated blockade of CD86 in iNOS-/- mice improved survival and enhanced resolution of lung inflammation. Our findings show that monocyte-derived iNOS plays a pivotal role in mediating resolution of ALI by modulating lung immune responses, thus facilitating clearance of alveolar inflammation and promoting lung repair.

Early Control of Mycobacterium tuberculosis Infection Requiresil12rb1Expression byrag1-Dependent Lineages

IL12RB1 is essential for human resistance to Mycobacterium tuberculosis infection. In the absence of a functional IL12RB1 allele, individuals exhibit susceptibility to disseminated, recurrent mycobacterial infections that are associated with defects in both RAG1-dependent and RAG1-independent hematopoietic lineages. Despite this well-established association, a causal relationship between M. tuberculosis susceptibility and IL12RB1 deficiency in either RAG1-dependent or RAG1-independent lineages has never been formally tested. Here, we use the low-dose aerosol model of experimental tuberculosis (TB) to both establish that infected il12rb1(-/-) mice recapitulate important aspects of TB in IL12RB1 null individuals and, more importantly, use radiation bone marrow chimeras to demonstrate that restriction of il12rb1 deficiency solely to rag1-dependent lineages (i.e., T and B cells) allows for the full transfer of the il12rb1(-/-) phenotype. We further demonstrate that the protection afforded by adaptive lymphocyte il12rb1 expression is mediated partially through ifng and that, within the same infection, il12rb1-sufficient T cells exhibit dominance over il12rb1-deficient T cells by enhancing ifng expression in the latter population. Collectively, our data establish a basic framework in which to understand how IL12RB1 promotes control of this significant human disease.

Naïve CD8 T cell activation by liver bone marrow-derived cells leads to a “neglected” IL-2low Bimhigh phenotype, poor CTL function and cell death

The occurrence of primary CD8 T cell activation within the liver, unique among the non-lymphoid organs, is now well accepted. However, the outcome of intrahepatic T cell activation remains controversial. We have previously reported that activation initiated by hepatocytes results in a tolerogenic phenotype characterized by low expression of CD25 and IL-2, poor cytotoxic T lymphocyte (CTL) function, and excessive expression of the pro-apoptotic protein Bim. To investigate whether this phenotype was due to activation in the absence of co-stimulation, we generated bone marrow (bm) radiation chimeras in which adoptively transferred naïve transgenic CD8 T cells were activated in the presence of co-stimulation by liver bm-derived cells. Despite expressing pro-inflammatory cytokines, high levels of CD25 and CD54, donor T cells activated by liver bm-derived cells did not produce detectable IL-2 and displayed poor CTL function, suggesting incomplete acquisition of effector function. Simultaneously, these cells expressed high levels of Bim and died by neglect. Transfer of Bim-deficient T cells resulted in increased T cell numbers. These results imply that expression of CD25 and CD54 is co-stimulation dependent and distinguishes T cell activated by hepatocytes and liver bm-derived cells. In contrast, low expression of IL-2, poor CTL function and excess Bim production represent a more universal phenotype defining T cells undergoing primary activation by both types of hepatic antigen presenting cells (APC). These results have important implications for transplantation, in which all liver antigen presenting cells contribute to activation of T cells specific for the allograft.

A T Cell Receptor Signaling Mutant Reveals Two Distinct Populations of IL-17 Producing CD4+ T Cells

Abstract 186Interleukin-17 (IL-17) producing CD4+ T cells (Th17 cells) are essential for immune responses in mucosal and epithelial sites which are the first line of host defense. Th17 cells play a critical role in the pathogenesis of many inflammatory and autoimmune diseases, and the role of IL-17 and Th17 cells in cancer has recently become the focus of extensive investigation. Most studies to date have focused on elucidating the cell extrinsic requirements for differentiation of Th17 cells from naïve CD4+ T cells in peripheral effector sites. Here we report an unconventional population of Th17 cells, “natural Th17 cells” (nTh17), that acquire effector function during development in the thymus, thereby distinguishing them from conventional Th17 cells which require antigen encounter and differentiation in the periphery. We show that these nTh17 cells are present and indeed develop in the thymus using fetal thymic organ culture. nTh17 cells express surface markers consistent with an innate and/or activated phenotype and their development is dependent on selection by MHC class II in the thymus. Yet unlike conventional CD4+ T cells, MHC class II expression on thymic cortical epithelium is not sufficient for their development, rather expression on medullary epithelium is necessary. In addition, T cell receptor (TCR) repertoire analysis of nTh17 cells revealed unique characteristics in TCR gene usage compared to conventional Th17 cells. A mouse model with a mutation in the TCR signaling protein SLP76 (SLP76 Y145F mice) further highlights the difference between the two distinct Th17 populations. SLP76 Y145F mice have increased numbers of nTh17 cells in the thymus compared to wild-type mice. However, peripheral naïve CD4+ T cells from these mice showed severely defective IL-17 production when cultured in vitro under conditions promoting Th17 cell differentiation. This defect was reflected in vivo as CD4+ T cells in the small intestinal lamina propria of SLP76 Y145F mice fail to produce IL-17. Using mixed radiation bone marrow chimeras, we found that the aberrant Th17 phenotype in the thymus and periphery of SLP76 Y145F mice is cell-intrinsic. Finally, adoptive transfer of purified nTh17 cells into RAG-deficient host mice revealed preferential homing of nTh17 cells to thymus and lung compared to other comparison/competitive populations that were co-transferred. Taken together, our data suggest a distinct population of Th17 cells that have characteristics of innate lymphocytes that function at the interface between innate and adaptive immunity. Understanding the biology of nTh17 cells will provide insight into the recently identified Th17 cells in human thymi and umbilical cord blood. Disclosures:No relevant conflicts of interest to declare.

IL-15–High-Responder Developing NK Cells Bearing Ly49 Receptors in IL-15−/− Mice

In mice lacking IL-15, NK cell development is arrested at immature stages, providing an opportunity to investigate the earliest developing NK cells that would respond to IL-15. We show in this study that immature NK cells were present in the spleen as well as bone marrow (BM) and contained IL-15-high-responder cells. Thus, mature NK cells were generated more efficiently from IL-15(-/-) than from control donor cells in radiation BM chimeras, and the rate of IL-15-induced cell division in vitro was higher in NK cells in the spleen and BM from IL-15(-/-) mice than in those from wild-type mice. Phenotypically, NK cells developed in IL-15(-/-) mice up to the minor but discrete CD11b(-)CD27(+)DX5(hi)CD51(dull)CD127(dull)CD122(hi) stage, which contained the majority of Ly49G2(+) and D(+) NK cells both in the spleen and BM. Even among wild-type splenic NK cells, IL-15-induced proliferation was most prominent in CD11b(-)DX5(hi) cells. Notably, IL-15-mediated preferential expansion (but not conversion from Ly49(-) cells) of Ly49(+) NK cells was observed in vitro only for NK cells in the spleen. These observations indicated the uneven distribution of NK cells of different developing stages with variable IL-15 responsiveness in these lymphoid organs. Immature NK cells in the spleen may contribute, as auxiliaries to those in BM, to the mature NK cell compartment through IL-15-driven extramarrow expansion under steady-state or inflammatory conditions.

Monocytes control natural killer cell differentiation to effector phenotypes

AbstractNatural killer (NK) cells play a major role in immunologic surveillance of cancer. Whether NK-cell subsets have specific roles during antitumor responses and what the signals are that drive their terminal maturation remain unclear. Using an in vivo model of tumor immunity, we show here that CD11bhiCD27low NK cells migrate to the tumor site to reject major histocompatibility complex class I negative tumors, a response that is severely impaired in Txb21−/− mice. The phenotypical analysis of Txb21-deficient mice shows that, in the absence of Txb21, NK-cell differentiation is arrested specifically at the CD11bhiCD27hi stage, resulting in the complete absence of terminally differentiated CD11bhiCD27low NK cells. Adoptive transfer experiments and radiation bone marrow chimera reveal that a Txb21+/+ environment rescues the CD11bhiCD27hi to CD11bhiCD27low transition of Txb21−/− NK cells. Furthermore, in vivo depletion of myeloid cells and in vitro coculture experiments demonstrate that spleen monocytes mediate the terminal differentiation of peripheral NK cells in a Txb21- and IL-15Rα–dependent manner. Together, these data reveal a novel, unrecognized role for Txb21 expression in monocytes in promoting NK-cell development and help appreciate how various NK-cell subsets are generated and participate in antitumor immunity.