It has recently been demonstrated that memory B cells can reenter and reengage germinal center (GC) reactions, opening the possibility that multi-hit lymphomagenesis gradually occurs throughout life during successive immunological challenges. Here, we investigated this scenario in follicular lymphoma (FL), an indolent GC-derived malignancy. We developed a mouse model that recapitulates the FL hallmark t(14;18) translocation, which results in constitutive activation of antiapoptotic protein B cell lymphoma 2 (BCL2) in a subset of B cells, and applied a combination of molecular and immunofluorescence approaches to track normal and t(14;18)+ memory B cells in human and BCL2-overexpressing B cells in murine lymphoid tissues. BCL2-overexpressing B cells required multiple GC transits before acquiring FL-associated developmental arrest and presenting as GC B cells with constitutive activation-induced cytidine deaminase (AID) mutator activity. Moreover, multiple reentries into the GC were necessary for the progression to advanced precursor stages of FL. Together, our results demonstrate that protracted subversion of immune dynamics contributes to early dissemination and progression of t(14;18)+ precursors and shapes the systemic presentation of FL patients.
CD8 T-cells contribute to long-term protection against Listeria monocytogenes (Lm) infection by differentiating into memory T-cells. These rapidly respond to Ag or inflammation upon secondary infection. In this study we used CD8 T-cells from OT1 and CD4 T-cells from OT2 mice expressing a fluorescent chimeric granzyme (GZMB-Tom) protein to monitor the primary response to infection with ovalbumin-expressing Lm (Lm-OVA). We show that, unlike poorly responding CD4 T cells, CD8 T cells readily proliferated and expressed high levels of GZMB-Tom as early as two days after infection. FACS analysis showed GZMB-Tom expression in un-divided CD8 T-cells, with its level increasing over 1-4 divisions. OT1 T-cells were visualized in the T-cell zone by confocal microscopy. This showed GZMB-Tom-containing granules oriented towards MHCII positive cells. Twenty hours later, most OT1 T-cells had divided but their level of GZMB-Tom expression was reduced. Recently divided OT1 cells failed to express GZMB-Tom. Fourteen hours after secondary infection, GZMB-Tom was re-expressed in memory OT1 T-cells responding either to Lm-OVA or Lm. Differences in the activation phenotype and in the splenic distribution of OT1 T-cells were observed, depending on the challenge. Notably, OTI T-cells with polarized granules were only observed after challenge with cognate Ag. This work showed that the GZMB-Tom KI-mice in which GZMB-Tom faithfully reproduced GZMB expression provide useful tools to dissect mechanisms leading to development of anti-bacterial effector and memory CD8 T-cells and reactivation of the memory response to cognate Ag or inflammatory signals. This article is protected by copyright. All rights reserved.
The transcription factor NF-?B is central to inflammatory signaling and activation of innate and adaptive immune responses. Activation of the NF-?B pathway is tightly controlled by several negative feedback mechanisms, including A20, an ubiquitin-modifying enzyme encoded by the tnfaip3 gene. Mice with selective deletion of A20 in myeloid, dendritic, or B cells recapitulate some human inflammatory pathology. As we observed high expression of A20 transcripts in dysfunctional CD8 T cells in an autochthonous melanoma, we analyzed the role of A20 in regulation of CD8 T-cell functions, using mice in which A20 was selectively deleted in mature conventional T cells. These mice developed lymphadenopathy and some organ infiltration by T cells but no splenomegaly and no detectable pathology. A20-deleted CD8 T cells had increased sensitivity to antigen stimulation with production of large amounts of IL-2 and IFN?, correlated with sustained nuclear expression of NF-?B components reticuloendotheliosis oncogene c-Rel and p65. Overexpression of A20 by retroviral transduction of CD8 T cells dampened their intratumor accumulation and antitumor activity. In contrast, relief from the A20 brake in NF-?B activation in adoptively transferred antitumor CD8 T cells led to improved control of melanoma growth. Tumor-infiltrating A20-deleted CD8 T cells had enhanced production of IFN? and TNF? and reduced expression of the inhibitory receptor programmed cell death 1. As manipulation of A20 expression in CD8 T cells did not result in pathologic manifestations in the mice, we propose it as a candidate to be targeted to increase antitumor efficiency of adoptive T-cell immunotherapy.
Follicular lymphoma (FL) is a B-cell neoplasm resulting from the transformation of germinal center (GC) B cells. Although t(14;18) and ectopic B-cell lymphoma 2 (BCL2) expression constitute the genetic hallmark of FL, t(14;18)(pos) B cells bearing genotypic and phenotypic features of FL cells can be found in the blood of most healthy individuals. Nevertheless, the localization of these FL-like cells (FLLCs) in nonmalignant GC-rich tissues and the functional consequences of BCL2 overexpression have not been evaluated thus far. Among 85 reactive lymph node (RLN) samples, 14% were found to contain high levels of t(14;18) by quantitative polymerase chain reaction. In t(14;18)(hi) RLNs, CD20(pos)BCL2(pos)CD10(pos) FLLCs consistently accumulated within the GC, essentially as nonproliferative CXCR4(neg) centrocytes. Moreover, they displayed a reduced response to proliferative stimuli in vitro. Altogether, our findings provide new insights into in situ FLLC functional properties and suggest that these cells have not acquired the ultimate genetic events leading to FL transformation.
The membrane-bound Vanin-1 pantetheinase regulates tissue adaptation to stress. We investigated Vnn1 expression and its regulation in liver. Vnn1 is expressed by centrolobular hepatocytes. Using novel tools, we identify a soluble form of Vnn1 in mouse and human serum and show the contribution of a cysteine to its catalytic activity. We show that liver contributes to Vanin-1 secretion in serum and that PPARalpha is a limiting factor in serum Vnn1 production. Functional PPRE sites are identified in the Vnn1 promoter. These results indicate that serum Vnn1 might be a reliable reporter of PPARalpha activity in liver.
Galectin-1 (GAL1) is an S-type lectin with multiple functions, including the control of B cell homeostasis. GAL1 expression was reported to be under the control of the plasma cell master regulator BLIMP-1. GAL1 was detected at the protein level in LPS-stimulated B cells and was shown to promote Ig secretion in vitro. However, the pattern of GAL1 expression and function of GAL1 in B cells in vivo are still unclear. In this study, we show that, among B cells, GAL1 is only expressed by differentiating plasma cells following T-dependent or T-independent immunization. Using GAL1-deficient mice we demonstrate that GAL1 expression is required for the maintenance of Ag-specific Ig titers and Ab-secreting cell numbers. Using an in vitro differentiation assay we find that GAL1-deficient plasmablasts can develop normally but die rapidly, through caspase 8 activation, under serum starvation-induced death conditions. TUNEL assays show that in vivo-generated GAL1-deficient plasma cells exhibit an increased sensitivity to apoptosis. Taken together, our data indicate that endogenous GAL1 supports plasma cell survival and participates in the regulation of the humoral immune response.
MYC is a potent oncogene involved in ?70% of human cancers, inducing tumorigenesis with high penetrance and short latency in experimental transgenic models. Accordingly, MYC is recognized as a major driver of T-cell acute lymphoblastic leukemia (T-ALL) in human and zebrafish/mouse models, and uncovering the context by which MYC-mediated malignant transformation initiates and develops remains a considerable challenge. Because MYC is a very complex oncogene, highly dependent on the microenvironment and cell-intrinsic context, we generated transgenic mice (tgMyc(spo)) in which ectopic Myc activation occurs sporadically (<10(-6) thymocytes) within otherwise normal thymic environment, thereby mimicking the unicellular context in which oncogenic alterations initiate human tumors. We show that while Myc(+) clones in tgMyc(spo) mice develop and initially proliferate in thymus and the periphery, no tumor or clonal expansion progress in aging mice (n?=?130), suggesting an unexpectedly low ability of Myc to initiate efficient tumorigenesis. Furthermore, to determine the relevance of this observation in human pathogenesis we analyzed a human T-ALL case at diagnosis and relapse using the molecular stigmata of V(D)J recombination as markers of malignant progression; we similarly demonstrate that despite the occurrence of TAL1 and MYC translocations in early thymocyte ontogeny, subsequent oncogenic alterations were required to drive oncogenesis. Altogether, our data suggest that although central to T-ALL, MYC overexpression per se is inefficient in triggering the cascade of events leading to malignant transformation.
Migration of dendritic cells (DC) from the tumor environment to the T cell cortex in tumor-draining lymph nodes (TDLN) is essential for priming naïve T lymphocytes (TL) to tumor antigen (Ag). We used a mouse model of induced melanoma in which similar oncogenic events generate two phenotypically distinct melanomas to study the influence of tumor-associated inflammation on secondary lymphoid organ (SLO) organization. One tumor promotes inflammatory cytokines, leading to mobilization of immature myeloid cells (iMC) to the tumor and SLO; the other does not. We report that inflammatory tumors induced alterations of the stromal cell network of SLO, profoundly altering the distribution of TL and the capacity of skin-derived DC and TL to migrate or home to TDLN. These defects, which did not require tumor invasion, correlated with loss of fibroblastic reticular cells in T cell zones and in impaired production of CCL21. Infiltrating iMC accumulated in the TDLN medulla and the splenic red pulp. We propose that impaired function of the stromal cell network during chronic inflammation induced by some tumors renders spleens non-receptive to TL and TDLN non-receptive to TL and migratory DC, while the entry of iMC into these perturbed SLO is enhanced. This could constitute a mechanism by which inflammatory tumors escape immune control. If our results apply to inflammatory tumors in general, the demonstration that SLO are poorly receptive to CCR7-dependent migration of skin-derived DC and naïve TL may constitute an obstacle for proposed vaccination or adoptive TL therapies of their hosts.
In the bone marrow (BM), stromal cells constitute a supportive tissue indispensable for the generation of pro-B/pre-BI, pre-BII, and immature B lymphocytes. IL-7-producing stromal cells constitute a cellular niche for pro-B/pre-BI cells, but no specific stromal cell microenvironment was identified for pre-BII cells expressing a functional pre-B cell receptor (pre-BCR). However expression of the pre-BCR represents a crucial checkpoint during B-cell development. We recently demonstrated that the stromal cell derived-galectin1 (GAL1) is a ligand for the pre-BCR, involved in the proliferation and differentiation of normal mouse pre-BII cells. Here we show that nonhematopoietic osteoblasts and reticular cells in the BM express GAL1. We observed that pre-BII cells, unlike the other B-cell subsets, were specifically localized in close contact with GAL1(+) reticular cells. We also determined that IL-7(+) and GAL1(+) cells represent 2 distinct mesenchymal populations with different BM localization. These results demonstrate the existence of a pre-BII specific stromal cell niche and indicate that early B cells move from IL-7(+) to GAL1(+) supportive BM niches during their development.
The oxidative burst produced by the NADPH oxidase (Phox) is an essential weapon used by host cells to eradicate engulfed pathogens. In Salmonella typhimurium, oxidative stress resistance has been previously proposed to be mediated by the pathogenicity island 2 type III secretion system (T3SS-2), periplasmic superoxide dismutases and cytoplasmic catalases/peroxidases. Here, we fused an OxyR-dependent promoter to the gfp to build the ahpC-gfp transcriptional fusion. This reporter was used to monitor hydrogen peroxide levels as sensed by Salmonella during the course of an infection. We showed that the expression of this fusion was under the exclusive control of reactive oxygen species produced by the host. The ahpC-gfp expression was noticeably modified in the absence of bacterial periplasmic superoxide dismutases or cytoplasmic catalases/peroxidases. Surprisingly, inactivation of the T3SS-2 had no effect on the ahpC-gfp expression. All together, these results led to a model in which Salmonella resistance relies on its arsenal of detoxifying enzymes to cope with Phox-mediated oxidative stress.
Cellular AF is usually considered a hindrance to flow cytometric analysis. Here, we incorporate AF into analysis of complex mixtures of leukocytes. Using a mouse model, we examined cellular AF at multiple excitation and emission wavelengths, and populations with discrete patterns were gated and examined for surface marker expression. In the spleen, all major myeloid populations were identified. In particular, the approach allowed simultaneous characterization of RPM and resident monocytes. When monocytes and RPM were compared, RPM exhibited a phenotype that was consistent with involvement in physiological processes, including expression of genes involved in lipid and iron metabolism. The presence of large amounts of stored ferric iron within RPM enabled purification of these cells using a magnetic-based approach. When adapted for use on leukocytes isolated from a range of other organs, incorporation of AF into analysis allowed identification and isolation of biologically important myeloid populations, including subsets that were not readily identifiable by conventional cytometric analysis.
We studied the effect of the immune system on two differentially aggressive melanomas developing in mice on conditional deletion of the INK4A/ARF tumor suppressor gene, with concomitant expression of oncogene H-Ras(G12V) and a natural cancer-germline tumor antigen (TA). "Slow progressor" melanomas contained no activated T lymphocytes (TL). In contrast, "aggressive" melanomas were infiltrated by activated TLs lacking effector molecules and expressing high levels of PD-1, indicating an exhausted phenotype. Aggressive melanomas were also infiltrated by immature myeloid cells (IMC). Infiltration was associated with local inflammation and systemic Th2/Th17-oriented chronic inflammation that seemed to impair further activation of TLs, as tumor-specific T cells adoptively transferred into mice bearing aggressive melanomas were poorly activated and failed to infiltrate the melanoma. This immunosuppression also led to the incapacity of these mice to reject inoculated TA-positive tumors, in contrast to slow-progressing melanoma-bearing mice, which were responsive. To test the role of adaptive immunity in tumor progression, we induced melanomas in immunodeficient RagKO compound mice. These mice developed aggressive but not slow-progressing melanomas at a higher frequency and with a shorter latency than immunocompetent mice. Immunodeficient mice also developed abnormal inflammation and infiltration of IMCs in a manner similar to immunocompetent mice, indicating that this phenotype was not dependent on adaptive immunity. Therefore, tumor-intrinsic factors distinguishing the two melanoma types control the initiation of inflammation, which was independent of adaptive immunity. The latter delayed development of aggressive melanomas but was overridden by inflammation.
Toll-like receptors (TLRs) are evolutionary conserved molecules that recognize various microbial components and host-derived agonists from damaged cells and play a central role in innate and adaptive immunity. It has been reported that MyD88, the adaptor molecule downstream of all TLRs, except TLR3, is essential for initiation of experimental autoimmune myocarditis (EAM). To determine the role of the intracellular TLRs in EAM, TLR3(-/-), TLR7(-/-), and TLR9(-/-) mice were immunized with cardiac alpha-myosin heavy chain peptide (MyHC-alpha) in Complete Freunds Adjuvant (CFA) and their EAM scores and associated immunological responses were compared to wild-type (WT) and MyD88(-/-) mice. MyD88(-/-) mice were completely resistant to EAM and had a profound defect in all the parameters we tested. Myocardial cellular infiltration and in vitro proliferation of MyHC-alpha-restimulated splenocytes were markedly reduced in TLR7(-/-) mice, while TLR3(-/-) and TLR9(-/-) mice showed similar inflammatory cell infiltration in the heart-like WT mice. Thus, the resistance of MyD88(-/-) mice to EAM can be attributed to a certain degree to TLR7 signaling. Moreover, upon murine cytomegalovirus-induced myocarditis, we found that the severity of myocardial inflammation was higher in TLR9(-/-) and MyD88(-/-) mice compared with WT, TLR3(-/-), or TLR7(-/-) mice and paralleled the ability of the mice to fight the viral infection.
Macrophages are central players in both lipid metabolism and innate immunity. Their determinant role in the pathogenesis of atherosclerosis is under the control of the ATP-binding cassette transporter (ABCA1), which by minimizing cellular lipid content, limits development of pro-inflammatory foam cells. Considering the differential contribution of monocyte subsets to the generation of vascular lesions we analyzed the immunophenotype of ABCA1-expressing cells in the myeloid lineage, by the combined use of flow cytometry and real-time quantitative RT-PCR. ABCA1 expression is limited to "non-inflammatory" Ly6C(lo) circulating monocytes and tissue-resident macrophages expressing markers of alternative activation. In ABCA1(-/-) peritoneal macrophages the transcriptional programs induced by LPS/IFN-gamma or IL-4 cytokines are altered and deviated phosphorylation patterns of STAT transcriptional regulators in response to stimuli are observed.
The INK4/ARF locus encodes three tumor suppressor genes (p15(Ink4b), Arf and p16(Ink4a)) and is frequently inactivated in a large number of human cancers. Mechanisms regulating INK4/ARF expression are not fully characterized.
NKp46+CD3- natural killer lymphocytes isolated from blood, lymphoid organs, lung, liver and uterus can produce granule-dependent cytotoxicity and interferon-gamma. Here we identify in dermis, gut lamina propria and cryptopatches distinct populations of NKp46+CD3- cells with a diminished capacity to degranulate and produce interferon-gamma. In the gut, expression of the transcription factor RORgammat, which is involved in the development of lymphoid tissue-inducer cells, defined a previously unknown subset of NKp46+CD3- lymphocytes. Unlike RORgammat- lamina propria and dermis natural killer cells, gut RORgammat+NKp46+ cells produced interleukin 22. Our data show that lymphoid tissue-inducer cells and natural killer cells shared unanticipated similarities and emphasize the heterogeneity of NKp46+CD3- cells in innate immunity, lymphoid organization and local tissue repair.
We compared gene expression signatures of aggressive amelanotic (Amela) melanomas with those of slowly growing pigmented melanomas (Mela), identifying pathways potentially responsible for the aggressive Amela phenotype. Both tumors develop in mice upon conditional deletion in melanocytes of Ink4a/Arf tumor suppressor genes with concomitant expression of oncogene H-Ras(G12V) and a known tumor antigen. We previously showed that only the aggressive Amela tumors were highly infiltrated by leukocytes concomitant with local and systemic inflammation. We report that Amela tumors present a pattern of de-differentiation with reduced expression of genes involved in pigmentation. This correlates with reduced and enhanced expression, respectively, of microphthalmia-associated (Mitf) and Pou3f2/Brn-2 transcription factors. The reduced expression of Mitf-controlled melanocyte differentiation antigens also observed in some human cutaneous melanoma has important implications for immunotherapy protocols that generally target such antigens. Induced Amela tumors also express Epithelial-Mesenchymal-Transition (EMT)-like and TGF?-pathway signatures. These are correlated with constitutive Smad3 signaling in Amela tumors and melanoma cell lines. Signatures of infiltrating leukocytes and some chemokines such as chemotactic cytokine ligand 2 (Ccl2) that contribute to leukocyte recruitment further characterize Amela tumors. Inhibition of the mitogen-activated protein kinase (MAPK) activation pathway in Amela tumor lines leads to reduced expression of EMT hallmark genes and inhibits both proinflammatory cytokine Ccl2 gene expression and Ccl2 production by the melanoma cells. These results indicate a link between EMT-like processes and alterations of immune functions, both being controlled by the MAPK pathway. They further suggest that targeting the MAPK pathway within tumor cells will impact tumor-intrinsic oncogenic properties as well as the nature of the tumor microenvironment.
Toll-like receptor (TLR)-dependent pathways control the activation of various immune cells and the production of cytokines and chemokines that are important in innate immune control of viruses, including mouse cytomegalovirus (MCMV). Here we report that upon MCMV infection wild-type and TLR7(-/-) male mice were more resistant than their female counterparts, while TLR9(-/-) male and female mice showed similar susceptibility. Interestingly, 36 h upon MCMV infection TLR9 mRNA expression was higher in male than in female mouse spleens. MCMV infection led to stronger reduction of marginal zone (MZ) B cells, and higher infiltration of plasmacytoid dendritic cells and neutrophils in wild-type male than female mice, while no such sex differences were observed in TLR9(-/-) mice. In accordance, the serum levels of KC and MIP-2, major neutrophil chemoattractants, were higher in wild-type, but not in TLR9(-/-), male versus female mice. Wild-type MCMV-infected female mice showed more severe liver inflammation, necrosis and steatosis compared to infected male mice. Our data demonstrate sex differences in susceptibility to MCMV infection, accompanied by a lower activation of the innate immune system in female mice, and can be attributed, at least in a certain degree, to the lower expression of TLR9 in female than male mice.
Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition due to the association of an infectious agent with lymphocyte cytotoxicity defects, either of congenital genetic origin in children or presumably acquired in adults. In HLH patients, an excess of lymphocyte or macrophage cytokines, such as IFN-? and TNF? is present in serum. In animal models of the disease, IFN-? and TNF-? have been shown to play a central pathogenic role. In humans, unusually high concentrations of IL-18, an inducer of IFN-?, and TNF-? have been reported, and are associated with an imbalance between IL-18 and its natural inhibitor IL-18 binding protein (IL-18BP) resulting in an excess of free IL-18. Here we studied whether IL-18BP could reduce disease severity in an animal model of HLH. Mouse cytomegalovirus infection in perforin-1 knock-out mice induced a lethal condition similar to human HLH characterized by cytopenia with marked inflammatory lesions in the liver and spleen as well as the presence of hemophagocytosis in bone marrow. IL-18BP treatment decreased hemophagocytosis and reversed liver as well as spleen damage. IL-18BP treatment also reduced both IFN-? and TNF-? production by CD8(+) T and NK cells, as well as Fas ligand expression on NK cell surface. These data suggest that IL-18BP is beneficial in an animal model of HLH and in combination with anti-infectious therapy may be a promising strategy to treat HLH patients.
In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation that exhibits specific mechanisms to control the immune response. Here we show that in response to polyriboinosinic:polyribocytidylic acid (pI:C), DCs mount a specific integrated stress response during which the transcription factor ATF4 and the growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), a phosphatase 1 (PP1) cofactor, are expressed. In agreement with increased GADD34 levels, an extensive dephosphorylation of the translation initiation factor eIF2? was observed during DC activation. Unexpectedly, although DCs display an unusual resistance to protein synthesis inhibition induced in response to cytosolic dsRNA, GADD34 expression did not have a major impact on protein synthesis. GADD34, however, was shown to be required for normal cytokine production both in vitro and in vivo. These observations have important implications in linking further pathogen detection with the integrated stress response pathways.
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