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The immune response to the protozoan pathogens, Leishmania spp., Trypanosoma spp. and Plasmodium spp., has been studied extensively with particular focus on regulation of the immune response by immunological mechanisms. More specifically, in diseases caused by parasites, immunosuppression frequently prevents immunopathology that can injure the host. However, this allows a small number of parasites to evade the immune response and remain in the host after a clinical cure. The consequences can be chronic infections, which establish a zoonotic or anthroponotic reservoir. This review will highlight some of the identified regulatory mechanisms of the immune system that govern immune responses to parasitic diseases, in particular leishmaniasis, trypanosomiasis and malaria, and discuss implications for the development of efficient vaccines against these diseases.

The atypical chemokine receptor CCX-CKR regulates bioavailability of CCL19, CCL21, and CCL25, homeostatic chemokines that play crucial roles in thymic lymphopoiesis. Deletion of CCX-CKR results in accelerated experimental autoimmunity induced by immunization. Here we show that CCX-CKR deletion also increases incidence of a spontaneous Sjögren's syndrome-like pathology, characterized by lymphocytic infiltrates in salivary glands and liver of CCX-CKR−/− mice, suggestive of a defect in self-tolerance when CCX-CKR is deleted. This prompted detailed examination of the thymus in CCX-CKR−/− mice. Negatively selected mature SP cells were less abundant in CCX-CKR−/− thymi, yet expansion of both DP and immature SP cells was apparent. Deletion of CCX-CKR also profoundly reduced proportions of DN3 thymocyte precursors and caused DN2 cells to accumulate within the medulla. These effects are likely driven by alterations in thymic stroma as CCX-CKR−/− mice have fewer cTECs per thymocyte, and cTECs express the highest level of CCX-CKR in the thymus. A profound decrease in CCL25 within the thymic cortex was observed in CCX-CKR−/− thymi, likely accounting for their defects in thymocyte distribution and frequency. These findings identify a novel role for CCX-CKR in regulating cTEC biology, which promotes optimal thymocyte development and selection important for self-tolerant adaptive immunity.

In the absence of TNF, the normally resistant C57BL/6 (B6.WT) strain develops a fatal, progressive form of leishmaniasis after infection with Leishmania major. It is not yet understood which TNF activity or the lack thereof is responsible for the dramatic progression of leishmaniasis in TNF-negative (B6.TNF−/−) mice. To elucidate the underlying mechanisms resulting in the fatal outcome of L. major infection in this gene-deficient mouse strain, we analyzed the monocytic component of the inflammatory infiltrate in the draining popliteal lymph node and the site of the infection using multicolor flow cytometry. The leukocytic infiltrate within the draining lymph node and footpad of B6.TNF−/− mice resembled that of B6.WT mice over the first 2 wk of cutaneous L. major infection. Thereafter, the B6.TNF−/− mice showed an increase of CD11c+Ly-6C+CCR2+ monocytic dendritic cells within the popliteal lymph node in comparison with B6.WT mice. This increase of inflammatory dendritic cells was paired with the accumulation of a novel CD11b+Ly-6ClowCCR2low population that was not present in B6.WT mice. This B6.TNF−/−- and B6.TNFR1−/−-specific cell population was CD115+Ly-6G−iNOS−, not apoptotic, and harbored large numbers of parasites.

Background Tumour necrosis factor - alpha (TNF-α) is a pro-inflammatory cytokine that combines a plethora of activities in the early stages of an immune response. TNF-α has gained increasing importance given TNF-α upregulation in multiple brain pathologies like neuropsychiatric conditions such as depression, schizophrenia, as well as neuroinflammatory disorder like multiple sclerosis (MS).

Adenoviruses of subgroup C can establish persistent infections in human beings. The exact site of persistence has not been established, but lymphoid tissues are certainly one reservoir. Experimental evidence suggests that early transcription unit 3 (E3) of the virus is involved in this phenomenon. In particular, the most abundant protein of this region, the E3/19K protein, seems to fulfill an important role in viral escape from the immune response. We previously demonstrated that in nonlymphoid cells E3/19K interferes with the antigen presentation function of class I major histocompatibility complex (MHC) antigens by inhibiting their transport to the cell surface. However, the function of the E3 products in lymphoid cells was not investigated. To examine this, the T-lymphoma cell line Jurkat was transfected with a DNA fragment comprising the entire E3 region of adenovirus type 2. We show here that E3/19K is expressed in the absence of the viral transactivator E1A with a rate of biosynthesis similar to that in nonlymphoid 293 cells. Furthermore, inhibition of transport and down-regulation of MHC antigens was comparable in both cell lines. In contrast, various T-cell molecules containing immunoglobulin-like domains showed a normal expression pattern in the transfected cells. A detailed analysis of the interaction between E3/19K and the MHC class I antigens of Jurkat (HLA-A3 and HLA-B35) revealed a differential sensitivity for down-regulation by E3/19K. The data demonstrate that E3/19K exerts its function also in lymphoid cells without affecting other lymphoid cell surface molecules. The implications for persistence of adenovirus in lymphoid cells in vivo are discussed.

Leishmania (L.) major is a protozoan parasite that infects mammalian hosts and causes a spectrum of disease manifestations that is strongly associated with the genetic background of the host. Interleukin (IL)-6 is an acute phase proinflammatory cytokine, known in vitro to be involved in the inhibition of the generation of regulatory T cells. IL-6-deficient mice were infected with L. major, and T cell and monocyte subsets were analyzed with flow cytometry. Our data show that at the site of infection in the footpad and in the draining popliteal lymph node, numbers of regulatory T cells remain unchanged between WT and IL-6-deficient mice. However, the spleens of IL-6(-/-) mice contained fewer regulatory T cells after infection with L. major. The development of cutaneous lesions is similar between WT and IL-6-deficient mice, while parasite burden in IL-6(-/-) mice is reduced compared to WT. The development of IFN-γ or IL-10 producing T cells is similar in IL-6(-/-) mice. Despite a comparable adaptive T cell response, IL-6-deficient mice develop an earlier peak of some inflammatory cytokines than WT mice. This data indicate that the role of IL-6 in the differentiation of regulatory T cells is complex in vivo, and the effect of an absence of this cytokine can be counter-intuitive.

The accumulation, uptake mechanism, cytotoxicity, cellular localisation of-and mode of cell death induced by-dinuclear ruthenium(II) complexes ΔΔ/ΛΛ-[{Ru(phen)(2) }(2) {μ-bb(n) }](4+) (Rubb(n)), where phen is 1,10-phenanthroline, bb(n) is bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n=2, 5, 7, 10, 12 or 16), and the corresponding mononuclear complexes containing the bb(n) ligands, were studied in L1210 murine leukaemia cells. Cytotoxicity increased with linker chain length, and the ΔΔ-Rubb(16) complex displayed the highest cytotoxicity of the series, with an IC(50) value of 5 μM, similar to that of carboplatin in the L1210 murine leukaemia cell line. Confocal microscopy and flow cytometry studies indicated that the complexes accumulate in the mitochondria of L1210 cells, with the magnitude of cellular uptake and accumulation increasing with linking chain length in the bb(n) bridge of the metal complex. ΔΔ-Rubb(16) entered the L1210 cells by passive diffusion (with a minor contribution from protein-mediated active transport), inducing cell death via apoptosis. Additionally, metal-complex uptake in leukaemia cells was approximately 16-times that observed in healthy B cells highlighting that the bb(n) series of complexes may have potential as selective anticancer drugs.

Our previous in vitro studies led to proposals that the atypical chemokine receptor CCX-CKR is a scavenger of CCR7 ligand homeostatic chemokines. In the present study, we generated CCX-CKR−/− mice and confirm this scavenger function in vivo. Compared with wild-type mice, CCX-CKR−/− have a 5-fold increase in the level of CCL21 protein in blood, and 2- to 3-fold increases in CCL19 and CCL21 in peripheral lymph nodes. The effect of these protein increases on immunity was investigated after immunization with MOG35-55 peptide emulsified in complete Freund adjuvant (CFA). The subsequent characteristic paralysis develops with enhanced kinetics and severity in CCX-CKR−/− versus wild-type mice. Despite this effect, antigen-specific immune responses in the draining lymph nodes are diminished in CCX-CKR−/− mice. Instead, the earlier onset of disease is associated with enhanced T-cell priming in the CCX-CKR−/− spleen and a skewing of CD4+ T-cell responses toward Th17 rather than Th1. This observation correlates with increased expression of IL-23 in the CCX-CKR−/− spleen and increased CCL21 levels in the central nervous system postimmunization. The early onset of disease in CCX-CKR−/− mice is reversed by systemic administration of neutralizing anti-CCL21 antibodies. Thus, by regulating homeostatic chemokine bioavailability, CCX-CKR influences the localization, kinetics, and nature of adaptive immune responses in vivo.

The effective maturation of dendritic cells (DC) is complex and highly regulated and requires the presence of a variety of signals. Tumor necrosis factor (TNF) and its receptors or innate pattern recognition receptors such as the toll-like receptors have been shown to contribute to this process. DC derived from bone marrow cells in the presence of granulocyte/macrophage colony-stimulating factor can be used as a model to ascertain the contribution of different signals to DC maturation. Analysis of DC activated by addition of the mycobacterial vaccine strain Bacillus Calmette-Guérin showed that of the effector molecules studied only interleukin-10 expression was significantly reduced in TNF-negative (B6.TNF(-/-)) DC. Another effector molecule produced by DC, inducible nitric oxide synthase, was largely unchanged.

In many chronic neurodegenerative diseases including Frontotemporal Dementia and Alzheimer's disease (AD), microglial activation is suggested to be involved in pathogenesis or disease progression. Activated microglia secrete a variety of cytokines, including interleukin-1beta, interleukin-6, and tumor necrosis factor as well as reactive oxygen and nitrogen species (ROS/RNS). ROS and RNS contribute to alterations in neuronal glucose uptake, inhibition of mitochondrial enzymes, a decrease in mitochondrial membrane potential, impaired axonal transport, and synaptic signaling. In addition, ROS act as signaling molecules in pro-inflammatory redox-active signal transduction pathways. To establish a high throughput screening system for anti-inflammatory and neuroprotective compounds, we have constructed an "Enhanced Green Fluorescent protein" (EGFP) expressing neuronal cell line and set up a murine microglia/neuron co-culture system with these EGFP expressing neuronal cells. We show that microglia activation leads to neuronal cell death, which can be conveniently measured by loss of neuronal EGFP fluorescence. Moreover, we used this system to test selected polyphenolic compounds for their ability to downregulate inflammatory markers and to protect neurons against microglial insult. We suggest that this system might allow accelerated drug discovery for the treatment of inflammation-mediated neurodegenerative diseases.

Memory B cells (MBCs) are rapidly activated upon Ag re-exposure in vivo, but the precise requirements for this process are still elusive. To address these requirements, T cell-independent reactivation of MBCs against virus-like particles was analyzed. As few as 25 MBCs are sufficient for a measurable Ab response after adoptive transfer. We found that MBCs were reactivated upon antigenic challenge to normal levels after depletion of macrophages, CD11c+ dendritic cells, and matured follicular dendritic cells. Furthermore, MBC responses were possible in TNF/lymphotoxin α double-deficient mice after partial normalization of lymphoid architecture by means of long-term reconstitution with wild-type bone marrow. Activation did not occur when chimeric mice, which still lack all lymph nodes and Peyer’s patches, were splenectomized prior to MBC transfer. Together with our finding that MBC responses are weak when Ag was administered within minutes after adoptive MBC transfer, these results strongly suggest that MBCs have to occupy specific niches within secondary lymphoid tissue to become fully Ag-responsive. We provide clear evidence that MBCs are not preferentially resident within the splenic marginal zones and show that impaired homing to lymphoid follicles resulted in significantly diminished activation, suggesting that reactivation of MBCs occurred inside lymphoid follicles. Furthermore, comparison of virus-specific MBC T cell-independent reactivation versus primary T cell-independent type II B cell activation revealed unique requirements of MBC activation.

Human adenovirus (Ad) can cause persistent infections in humans. Early region 3 (E3) of the virus appears to be implicated in this phenomenon. This transcription unit encodes proteins that interfere in various ways with host cell functions, including (i) cell-surface expression of histocompatibility class I antigens (HLA), (ii) cell-surface expression of the epidermal growth factor receptor (EGF-R), and (iii) the biological activity of tumor necrosis factor alpha (TNF-alpha). We transfected the human cell line 293 with the entire E3 region of Ad2 and investigated the influence of the cytokines TNF-alpha and interferon gamma (IFN-gamma) on cell-surface expression of HLA class I and the EGF-R. Whereas IFN-gamma treatment induced expression of HLA to some extent but not that of the EGF-R, TNF-alpha treatment augmented the reduction of these cell-surface molecules. Subsequent studies on the mechanism of this effect showed a TNF-alpha-dependent upregulation of E3 protein (E3/19K) and mRNA. The significance of this phenomenon was confirmed in infection experiments. A dramatic increase in the amount of E3/19K, even after short induction with low doses of TNF-alpha could be demonstrated. The study provides evidence for an interaction between the immune system and Ad in which the virus takes advantage of an immune mediator to escape immunosurveillance of the host.

Chemokines are essential for homeostasis and activation of the immune system. The chemokine ligand/receptor pairing CCL20/CCR6 is interesting because these molecules display characteristics of both homeostatic and activation functions. These dual characteristics suggest a role for CCR6 in the priming and effector phases of the immune response. However, while CCR6 has been implicated in the effector phase in several models, a role in the priming phase is less clear. Herein we analyze the role of CCR6 in these two important arms of the immune response during experimental autoimmune encephalomyelitis (EAE). Both CCR6 and its chemokine ligand CCL20 were up-regulated in the draining lymph nodes and spinal cord during EAE, and CCR6 was up-regulated on CD4(+) T cells that had divided following induction of EAE. The functional role of this expression was demonstrated by impaired development of EAE in gene-targeted CCR6-deficient mice and in mice treated either with a neutralizing anti-CCR6 Ab or with a novel receptor antagonist. Inhibition Of EAE was due to reduced priming Of autoreactive CD4(+) T cells probably as a result of impaired late-stage influx of dendritic cells into draining lymph nodes. This was accompanied by reduced egress of activated lymphocytes from the lymph nodes. These results demonstrate a novel role for CCR6 in the mechanism of autoreactive lymphocyte priming and emigration to the efferent lymphatics. The Journal of Immunology, 2009, 182: 3121-3130. ispartof: Journal of Immunology vol:182 issue:5 pages:3121-3130 ispartof: location:United States status: published

The chemokine receptor CCR6 is expressed on naïve B cells, dendritic cell and T-cell subpopulations and is involved in cell navigation during organogenesis and recruitment in response to inflammatory stimuli. Gene-deficient C57BL/6 CCR6(-/-) mice infected with the protozoan parasite Leishmania (L.) major were able to mount a protective immune response and survived the infection. Whereas macrophage production of nitric oxide (NO), the key leishmanicidal effector molecule during the immune response to L. major, did not require CCR6, the migration of CD4(+) T cells to the site of infection was reduced in CCR6(-/-) mice. Furthermore, the induction of a T-cell-dependent delayed-type-hypersensitivity (DTH) reaction was defective in CCR6(-/-) mice, whereas resistance to re-infection was maintained in the absence of CCR6. We conclude that CCR6 contributes to the recruitment of T cells to the site of infection, but is largely dispensable for the control of L. major parasites during primary or secondary infection.

Intensive world wide development work in high-speed bipolar RF technologies leads to a rapid increase in transistor performance. This paper describes a manufacturable leading-edge 0.35 /spl mu/m bipolar SiGe:C technology offering balanced transistor parameter sets with more than 150 GHz f/sub T/ and 180 GHz f/sub max/ as well as further integrated active and passive devices to serve a broad spectrum of applications.

Activation of resting T cells usually leads to their proliferation and differentiation into effector cells and a subsequent decline following elimination of the antigen. A situation of excessive antigen density may result in T cell receptor (TCR)-induced deletion of T effector cells, a process termed antigen-induced cell death (AgICD). Previous studies indicate that AgICD of cytotoxic T cells may be induced by either of the two key cytotoxic processes, granule exocytosis, including perforin and granzymes, or the Fas ligand (FasL)/Fas pathway. By using in vitro-polyclonally activated or ex vivo-derived virus-induced T cell populations from mice with mutations or targeted gene defects in one or more components of the two key cytolytic pathways we now show that TCR-induced apoptosis is only impaired in the absence of FasL and/or Fas, but not in the absence of perforin and/or granzymes. Furthermore, antibody-blockage of FasL alone is sufficient to inhibit early T cell death. Inhibition of both, FasL and tumor necrosis factor (TNF-alpha) is required to abrogate late apoptosis by AgICD. The fact that antibodies to IFN-gamma also inhibit AgICD suggests that the perforin plus granzyme-independent and FaSL and/or TNF-alpha facilitated process of AgICD of T effector cells is tightly regulated by endogenous IFN-gamma.

Lymphotoxin (LT)alpha in combination with LTbeta forms membrane-bound heterotrimeric complexes with a crucial function in lymph node (LN) organogenesis and correct morphogenesis of secondary lymphoid tissue. To study the role of membrane LT (mLT) in lymphoid tissue organogenesis we generated an LTbeta-deficient mouse strain on a pure genetic C57BL/6 background (B6.LTbeta-/-) and compared it to a unique series of LTalpha-, TNF- and TNF/LTalpha-gene-targeted mice on an identical genetic background (B6.LTalpha-/-, B6.TNF-/- and B6 TNF/LTalpha-/-). B6.LTbeta-/- mice lacked peripheral LN with the exception of mesenteric LN, and displayed a disturbed micro-architecture of the spleen, although less profoundly than LTalpha- or TNF/LTalpha-deficient mice. Radiation bone marrow chimeras (B6.WT--B6.LTbeta-/- developed Peyer's patch (PP)-like lymphoid aggregates in the intestinal wall indicating a possible role for soluble LTalpha(3) in the formation of the PP anlage. After infection with Leishmania major, B6.LTbeta-/- mice developed a fatal disseminating leishmaniasis resulting in death after 8 to 14 weeks, despite the natural resistance of the C57BL/6 genetic background (B6.WT) mice to the parasite. Both, the cellular and the humoral anti-L. major immune responses were delayed and ineffective. However, the expression pattern of the key cytokines IFN-gamma and IL-12 were comparable in B6.WT and B6.LTbeta-/- mice. Infection of radiation bone marrow chimeras showed that it is the LTbeta-dependent presence of lymphoid tissue and not the expression of mLT itself that renders mice resistant to leishmaniasis.

Tumor necrosis factor (TNF)-mediated activities are potentially numerous, although in TNF-deficient ( −/− ) mice, altered spatial orientation of leukocytes within tissues is the predominant phenotype. Here, we propose that TNF plays a fundamental role in the control of leukocyte movement by virtue of a pivotal role in regulation of chemokine expression.

Tumor necrosis factor (TNF) and Fas ligand (FasL) play major roles in the homeostasis of the peripheral immune system. This becomes dramatically obvious in the absence of a functional FasL. Mice with such a deficiency develop a profound lymphadenopathy, splenomegaly, hypergammaglobulinemia, and strain-dependent systemic autoimmune disease, and succumb to premature death. It is consequently termed generalized lymphoproliferative disorder (gld). By contrast, TNF deficiency alone does not result in a striking phenotype. Thus, we sought to determine what role TNF might play in contributing to the gld phenotype by creating C57BL/6.gld.TNF−/− mice. Contrary to the expected outcome, mice deficient for both FasL and TNF had a substantially milder gld phenotype with regard to mortality, lymphoaccumulation, germinal center formation, and hypergammaglobulinemia. To confirm these data in a strain highly permissive for the phenotype, C3H/HeJ.gld and C3H.HeJ.lpr mice were treated with a TNF-specific monoclonal antibody. This transient neutralization of TNF also resulted in a significantly attenuated lymphoproliferative phenotype. We conclude that TNF is necessary for the full manifestation of the lymphoproliferative disorder, in particular playing a critical role in lymphoaccumulation. Most importantly, absence of TNF protects gld mice against premature death.

Mice deficient in the cytokines tumor necrosis factor (TNF) or lymphotoxin (LT) alpha/beta lack polarized B cell follicles in the spleen. Deficiency in CXC chemokine receptor 5 (CXCR5), a receptor for B lymphocyte chemoattractant (BLC), also causes loss of splenic follicles. Here we report that BLC expression by follicular stromal cells is defective in TNF-, TNF receptor 1 (TNFR1)-, LTalpha- and LTbeta-deficient mice. Treatment of adult mice with antagonists of LTalpha1beta2 also leads to decreased BLC expression. These findings indicate that LTalpha1beta2 and TNF have a role upstream of BLC/CXCR5 in the process of follicle formation. In addition to disrupted follicles, LT-deficient animals have disorganized T zones. Expression of the T cell attractant, secondary lymphoid tissue chemokine (SLC), by T zone stromal cells is found to be markedly depressed in LTalpha-, and LTbeta-deficient mice. Expression of the SLC-related chemokine, Epstein Barr virus-induced molecule 1 ligand chemokine (ELC), is also reduced. Exploring the basis for the reduced SLC expression led to identification of further disruptions in T zone stromal cells. Together these findings indicate that LTalpha1beta2 and TNF are required for the development and function of B and T zone stromal cells that make chemokines necessary for lymphocyte compartmentalization in the spleen.

Natural killer (NK) cells are thought to provide the first line of defence against tumors, particularly major histocompatibility complex (MHC) class I− variants. We have confirmed in C57BL/6 (B6) mice lacking perforin that peritoneal growth of MHC class I− RMA-S tumor cells in unprimed mice is controlled by perforin-dependent cytotoxicity mediated by CD3− NK1.1+ cells. Furthermore, we demonstrate that B6 mice lacking tumor necrosis factor (TNF) are also significantly defective in their rejection of RMA-S, despite the fact that RMA-S is insensitive to TNF in vitro and that spleen NK cells from B6 and TNF-deficient mice are equally lytic towards RMA-S. NK cell recruitment into the peritoneum was abrogated in TNF-deficient mice challenged with RMA-S or RM-1, a B6 MHC class I− prostate carcinoma, compared with B6 or perforin-deficient mice. The reduced NK cell migration to the peritoneum of TNF-deficient mice correlated with the defective NK cell response to tumor in these mice. By contrast, a lack of TNF did not affect peptide-specific cytotoxic T lymphocyte–mediated rejection of tumor from the peritoneum of preimmunized mice. Overall, these data show that NK cells delivering perforin are the major effectors of class I− tumor rejection in the peritoneum, and that TNF is specifically critical for their recruitment to the peritoneum.

There are significant advantages to the production of gene-knockout mice directly in mouse strains other than 129. The availability now of ES cells derived from the C57BL/6 mouse strain presents workers with a valuable alternative. A major difficulty, however, is the requirement for BALB/c blastocysts as recipients for ES cell injection. Using standard procedures, few BALB/c blastocysts can be obtained. This limitation has now been resolved by harvesting BALB/c embryos at the early morula stage and maturing these to blastocysts by in vitro culture. Of early morulae harvested and cultured, over 70% were recovered as fully expanded and injectable blastocysts. C57BL/6 ES cell injection of these blastocysts has enabled the production of a number of gene-knockout mice with a success rate similar to that reported for ES cells derived from the 129 mouse strains.

Recent studies demonstrated that administration of a p55-tumor necrosis factor (TNF) receptor IgG-fusion protein (TNFR-IgG) prevented the clinical onset of experimental autoimmune encephalomyelitis but did not alter the number or tissue distribution of autoantigen-specific CD4+ effector T cells which trafficked into the central nervous system. To determine whether specific target tissues of autoimmune damage remain intact after TNFR-IgG treatment despite the presence of inflammatory cells within the tissues, we examined rats with experimental autoimmune uveoretinitis (EAU), as in this model, the main target of autoreactive CD4+ T cells, the retinal rod outer segments (ROS), can be examined readily by light microscopy. As judged by direct ophthalmoscopy, the onset of inflammation in the anterior chamber of the eye in EAU following administration of TNFR-IgG was delayed by 6 days compared to untreated controls, but the magnitude of the response was only slightly less than controls. Histological examination of the retinae and direct assessment of retinal inflammation revealed a disproportionate sparing of ROS in the TNFR-IgG-treated animals despite a level of retinal inflammation not substantially less than controls in which ROS damage was marked. Analysis of retinal leukocytes by immunofluorescence microscopy and flow cytometry indicated that approximately equal numbers of CD4+ alpha beta TCR+ lymphocytes were present in treated and control retinae, more than 30% of CD4+ cells in both experimental groups expressed the CD25 or MRC OX40 activation markers and most cells, which would include the CD4+ T lymphocytes, were activated as evidenced by MHC class II expression. Fewer activated macrophages and granulocytes were present in the treated retinae, possibly reflecting the lower level of tissue damage and subsequent accumulation of these inflammatory cells. The results demonstrate directly that a tissue specifically targeted for autoimmune destruction can be protected despite the influx of fully activated CD4+ T cells.

Gene targeting by homologous recombination is a powerful technique, generating mouse strains with defined mutations in their genome. These genetically modified, 'designer' animals allow us for the first time to ask simple questions about elaborate and complex biological systems. Dissecting the function of individual components of the immune system is a perfect application of this technology. Although the techniques involved in the generation of gene knock-out mice are increasingly well defined, to many immunologists the language and concepts are confusing. This review presents the essentials of the technology in a form digestible by the non-expert.

TNF is a key cytokine in the effector phase of both graft-versus-host disease (GVHD) and the graft-versus leukemia (GVL) effect after bone marrow transplantation (BMT). TNF neutralizing antibodies are now established as effective therapeutic adjuncts for the treatment of severe acute GVHD. TNF signals through the p55 and p75 TNF receptors (TNFR), which are also receptors for the soluble lymphotoxin homotrimer (LTα3). The membrane-bound lymphotoxin heterotrimeric molecule (LTα1β2) signals through the LTβ receptor. The function of these molecules in GVHD remains unknown. Pharmacological agents are available which block either TNF alone, or both TNF and LTα3 and elucidating the roles of these molecules in GVHD is essential for the design of rational therapeutic strategies. We have employed multiple well established preclinical models of GVHD to investigate the roles of these molecules. We first used a functional bioassay to confirm that a human TNFR:Fc construct was capable of preventing physiological activity of both recombinant murine TNF and LTα3. Irradiated B6D2F1 recipients were then transplanted with allogeneic B6.TNF−/− bone marrow and T cells, then treated with control Ig or the TNFR:Fc. Suprisingly, the TNFR:Fc provided significant protection from GVHD mortality in the absence of donor derived TNF (median survival 25.5 vs 35 days, P 3 logs more LTα than LTβ). We subsequently developed the first reported ELISA to assess murine LTα3 protein and were able to clearly demonstrate LTα3 production by wild-type (WT) CD4 T cells seven days after BMT. To further examine this molecule, we performed experiments using WT, LTα−/− or LTβ−/− donor grafts in two BMT systems (B6 → B6D2F1 and B6 → BALB/c). Recipients of LTα−/− grafts were protected from GVHD (P

Lymphotoxin (LT)alpha in combination with LTbeta forms membrane-bound heterotrimeric complexes with a crucial function in lymph node (LN) organogenesis and correct morphogenesis of secondary lymphoid tissue. To study the role of membrane LT (mLT) in lymphoid tissue organogenesis we generated an LTbeta-deficient mouse strain on a pure genetic C57BL/6 background (B6.LTbeta-/-) and compared it to a unique series of LTalpha-, TNF- and TNF/LTalpha-gene-targeted mice on an identical genetic background (B6.LTalpha-/-, B6.TNF-/- and B6 TNF/LTalpha-/-). B6.LTbeta-/- mice lacked peripheral LN with the exception of mesenteric LN, and displayed a disturbed micro-architecture of the spleen, although less profoundly than LTalpha- or TNF/LTalpha-deficient mice. Radiation bone marrow chimeras (B6.WT-->B6.LTbeta-/- developed Peyer's patch (PP)-like lymphoid aggregates in the intestinal wall indicating a possible role for soluble LTalpha(3) in the formation of the PP anlage. After infection with Leishmania major, B6.LTbeta-/- mice developed a fatal disseminating leishmaniasis resulting in death after 8 to 14 weeks, despite the natural resistance of the C57BL/6 genetic background (B6.WT) mice to the parasite. Both, the cellular and the humoral anti-L. major immune responses were delayed and ineffective. However, the expression pattern of the key cytokines IFN-gamma and IL-12 were comparable in B6.WT and B6.LTbeta-/- mice. Infection of radiation bone marrow chimeras showed that it is the LTbeta-dependent presence of lymphoid tissue and not the expression of mLT itself that renders mice resistant to leishmaniasis.

Mice without secreted TNF but with functional, normally regulated and expressed membrane-bound TNF (memTNF(Delta/Delta) mice) were created by knocking-in the uncleavable Delta 1-9,K11E TNF allele. In contrast to TNF-deficient mice (TNF(-/-)), memTNF supported many features of lymphoid organ structure, except generation of primary B cell follicles. Splenic chemokine expression was near normal. MemTNF-induced apoptosis was mediated through both TNF-R1 and TNF-R2. That memTNF is suboptimal for development of inflammation was revealed in experimental autoimmune encephalomyelitis. Disease severity was reduced in memTNF(Delta/Delta) mice relative to wild-type mice, and the nature of spinal cord infiltrates resembled that in TNF(-/-) mice. We conclude that memTNF supports many processes underlying lymphoid tissue structure, but secreted TNF is needed for optimal inflammatory lesion development.

SummaryAccumulation of T follicular helper (Tfh) cells and proinflammatory cytokines drive autoantibody-mediated diseases. The RNA-binding protein Roquin-1 (Rc3h1) represses the inducible costimulator ICOS and interferon-γ (IFN-γ) in T cells to prevent Tfh cell accumulation. Unlike Rc3h1san mice with a mutation in the ROQ domain of Roquin-1, mice lacking the protein, paradoxically do not display increased Tfh cells. Here we have analyzed mice with mutations that eliminate the RING domain from Roquin-1 or its paralog, Roquin-2 (Rc3h2). RING or ROQ mutations both disrupted Icos mRNA regulation by Roquin-1, but, unlike the ROQ mutant that still occupied mRNA-regulating stress granules, RING-deficient Roquin-1 failed to localize to stress granules and allowed Roquin-2 to compensate in the repression of ICOS and Tfh cells. These paralogs also targeted tumor necrosis factor (TNF) in nonlymphoid cells, ameliorating autoantibody-induced arthritis. The Roquin family emerges as a posttranscriptional brake in the adaptive and innate phases of antibody responses.

SummaryNeutralization or deletion of tumor necrosis factor (TNF) causes loss of control of intracellular pathogens in mice and humans, but the underlying mechanisms are incompletely understood. Here, we found that TNF antagonized alternative activation of macrophages and dendritic cells by IL-4. TNF inhibited IL-4-induced arginase 1 (Arg1) expression by decreasing histone acetylation, without affecting STAT6 phosphorylation and nuclear translocation. In Leishmania major-infected C57BL/6 wild-type mice, type 2 nitric oxide (NO) synthase (NOS2) was detected in inflammatory dendritic cells or macrophages, some of which co-expressed Arg1. In TNF-deficient mice, Arg1 was hyperexpressed, causing an impaired production of NO in situ. A similar phenotype was seen in L. major-infected BALB/c mice. Arg1 deletion in hematopoietic cells protected these mice from an otherwise lethal disease, although their disease-mediating T cell response (Th2, Treg) was maintained. Thus, deletion or TNF-mediated restriction of Arg1 unleashes the production of NO by NOS2, which is critical for pathogen control.

Although studies indicate LIGHT (lymphotoxin (LT)-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes) enhances inflammation and T cell-mediated immunity, the mechanisms involved in this process remain obscure. In this study, we assessed the role of LIGHT in IL-12 production and development of CD4+ Th cells type one (Th1) in vivo. Bone marrow-derived dendritic cells from LIGHT−/− mice were severely impaired in IL-12p40 production following IFN-γ and LPS stimulation in vitro. Furthermore, blockade of LIGHT in vitro and in vivo with HVEM-Ig and LT β receptor (LTβR)-Ig leads to impaired IL-12 production and defective polyclonal and Ag-specific IFN-γ production in vivo. In an infection model, injection of HVEM-Ig or LTβR-Ig into the usually resistant C57BL/6 mice results in defective IL-12 and IFN-γ production and severe susceptibility to Leishmania major that was reversed by rIL-12 treatment. This striking susceptibility to L. major in mice injected with HVEM-Ig or LTβR-Ig was also reproduced in LIGHT−/− → RAG1−/− chimeric mice. In contrast, L. major-infected LTβ−/− mice do not develop acute disease, suggesting that the effect of LTβR-Ig is not due to blockade of membrane LT (LTα1β2) signaling. Collectively, our data show that LIGHT plays a critical role for optimal IL-12 production by DC and the development of IFN-γ-producing CD4+ Th1 cells and its blockade results in severe susceptibility to Leishmania major.