Transforming Growth Factor β1, in the Presence of Granulocyte/Macrophage Colony-stimulating Factor and Interleukin 4, Induces Differentiation of Human Peripheral Blood Monocytes into Dendritic Langerhans Cells

Langerhans cells (LCs) are dendritic cells (DCs) that are present in the epidermis, bronchi, and mucosae. Although LCs originate in bone marrow, little is known about their lineage of origin. In this study, we demonstrate that in vitro LCs may originate from monocytes. Adult peripheral blood CD14⁺ monocytes differentiate into LCs (CD1a⁺, E-cadherin⁺, cutaneous lymphocyte-associated antigen⁺, Birbeck granules⁺, Lag⁺) in the presence of granulocyte/macrophage colony-stimulating factor, interleukin 4, and transforming growth factor β1 (TGF-β1). This process occurs with virtually no cell proliferation and is not impaired by 30 Gy irradiation. Selection of monocyte subpopulations is ruled out since monocyte-derived DCs can further differentiate into LCs. Our data suggest that in vivo LC differentiation may be induced peripherally, from a nonproliferating myeloid precursor, i.e., the monocyte, in response to a TGF-β1–rich microenvironment, as found in the skin and epithelia. Therefore, the monocyte may represent a circulating precursor critical to the immune response in vivo.     

Tumor Necrosis Factor α and Lymphotoxin α Are Not Required for Induction of Acute Experimental Autoimmune Encephalomyelitis

Immunization of mice with myelin components results in experimental autoimmune encephalomyelitis (EAE), which is mediated by myelin-specific CD4⁺ T cells and anti-myelin antibodies. Tumor necrosis factor α (TNF-α) and lymphotoxin α (LT-α) are thought to be involved in the events leading to inflammatory demyelination in the central nervous system. To ascertain this hypothesis 129 × C57BL/6 mice with an inactivation of the tnf and lta genes (129 × C57BL/6^−/−) and SJL/J mice derived from backcrosses of the above mentioned mutant mice (SJL^−/−) were immunized with mouse spinal cord homogenate (MSCH) or proteolipid protein. Both 129 × C57BL/6^−/− mice and SJL^−/− mice developed EAE. In SJL^−/− mice immunized with MSCH, a very severe form of EAE with weight loss, paralysis of all four limbs, and lethal outcome was observed. The histologic hallmark was an intense perivascular and parenchymal infiltration with predominantly CD4⁺ T cells and some CD8⁺ T cells associated with demyelination in both brain and spinal cord. These results indicate that TNF-α and LT-α are not essential for the development of EAE.     

α6 Integrins Are Required for Langerhans Cell Migration from the Epidermis

Topical exposure of mice to chemical allergens results in the migration of epidermal Langerhans cells (LCs) from the skin and their accumulation as immunostimulatory dendritic cells (DCs) in draining lymph nodes. Epidermal cell–derived cytokines have been implicated in the maturation and migration of LCs, but the adhesion molecules that regulate LC migration have not been studied. We hypothesized that integrin-mediated interactions with extracellular matrix components of the skin and lymph node may regulate LC/DC migration. We found that α₆ integrins and α₄ integrins were differentially expressed by epidermal LCs and lymph node DCs. A majority of LCs (70%) expressed the α₆ integrin subunit, whereas DCs did not express α₆ integrins. In contrast, the α₄ integrin subunit was expressed at high levels on DCs but at much lower levels on LCs. The anti-α₆ integrin antibody, GoH3, which blocks binding to laminin, completely prevented the spontaneous migration of LCs from skin explants in vitro and the rapid migration of LCs from mouse ear skin induced after intradermal administration of TNF-α in vivo. GoH3 also reduced the accumulation of DCs in draining lymph nodes by a maximum of 70% after topical administration of the chemical allergen oxazolone. LCs remaining in the epidermis in the presence of GoH3 adopted a rounded morphology, rather than the interdigitating appearance typical of LCs in naive skin, suggesting that the cells had detached from neighboring keratinocytes and withdrawn cellular processes in preparation for migration, but were unable to leave the epidermis. The anti-α₄ integrin antibody PS/2, which blocks binding to fibronectin, had no effect on LC migration from the epidermis either in vitro or in vivo, or on the accumulation of DCs in draining lymph nodes after oxazolone application. RGD-containing peptides were also without effect on LC migration from skin explants.

These results identify an important role for α₆ integrins in the migration of LC from the epidermis to the draining lymph node by regulating access across the epidermal basement membrane. In contrast, α₄ integrins, or other integrin-dependent interactions with fibronectin that are mediated by the RGD recognition sequence, did not influence LC migration from the epidermis. In addition, α₄ integrins did not affect the accumulation of LCs as DCs in draining lymph nodes.

     

In Vitro and In Vivo Evidence that Thrombospondin-1 (TSP-1) Contributes to Stirring- and Shear-Dependent Activation of Platelet-Derived TGF-β1

Thrombospondin 1 (TSP-1), which is contained in platelet α-granules and released with activation, has been shown to activate latent TGF-β1 in vitro, but its in vivo role is unclear as TSP-1-null (Thbs1^−/−) mice have a much less severe phenotype than TGF-β1-null (Tgfb1^−/−) mice. We recently demonstrated that stirring and/or shear could activate latent TGF-β1 released from platelets and have now studied these methods of TGF-β1 activation in samples from Thbs1^−/− mice, which have higher platelet counts and higher levels of total TGF-β1 in their serum than wild type mice. After either two hours of stirring or shear, Thbs1^−/− samples demonstrated less TGF-β1 activation (31% and 54% lower levels of active TGF-β1 in serum and platelet releasates, respectively). TGF-β1 activation in Thbs1^−/− mice samples was normalized by adding recombinant human TSP-1 (rhTSP-1). Exposure of platelet releasates to shear for one hour led to near depletion of TSP-1, but this could be prevented by preincubating samples with thiol-reactive agents. Moreover, replenishing rhTSP-1 to human platelet releasates after one hour of stirring enhanced TGF-β1 activation. In vivo TGF-β1 activation in carotid artery thrombi was also partially impaired in Thbs1^−/− mice. These data indicate that TSP-1 contributes to shear-dependent TGF-β1 activation, thus providing a potential explanation for the inconsistent in vitro data previously reported as well as for the differences in phenotypes of Thbs1^−/− and Tgfb1^−/− mice.     

Transforming Growth Factor β Production Is Inversely Correlated with Severity of Murine Malaria Infection

We have examined the role of the immunomodulatory cytokine transforming growth factor (TGF)-β in the resolution and pathology of malaria in BALB/c mice. Circulating levels of TGF-β, and production of bioactive TGF-β by splenocytes, were found to be low in lethal infections with Plasmodium berghei. In contrast, resolving infections with P. chabaudi chabaudi or P. yoelii were accompanied by significant TGF-β production. A causal association between the failure to produce TGF-β and the severity of malaria infection was demonstrated by treatment of infected mice with neutralizing antibody to TGF-β, which exacerbated the virulence of P. berghei and transformed a resolving P. chabaudi chabaudi infection into a lethal infection, but had little effect on the course of P. yoelii infection. Parasitemia increased more rapidly in anti–TGF-β–treated mice but this did not seem to be the explanation for the increased pathology of infection as peak parasitemias were unchanged. Treatment of P. berghei–infected mice with recombinant TGF-β (rTGF-β) slowed the rate of parasite proliferation and prolonged their survival from 15 to up to 35 d. rTGF-β treatment was accompanied by a significant decrease in serum tumor necrosis factor α and an increase in interleukin 10. Finally, we present evidence that differences in TGF-β responses in different malaria infections are due to intrinsic differences between species of malaria parasites in their ability to induce production of TGF-β. Thus, TGF-β seems to induce protective immune responses, leading to slower parasite growth, early in infection, and, subsequently, appears to downregulate pathogenic responses late in infection. This duality of effect makes TGF-β a prime candidate for a major immunomodulatory cytokine associated with successful control of malaria infection.     

HLA-A2.1–restricted Education and Cytolytic Activity of CD8+ T Lymphocytes from β2 Microglobulin (β2m) HLA-A2.1 Monochain Transgenic H-2Db β2m Double Knockout Mice

Three different HLA-A2.1 monochains were engineered in which either the human or mouse β2-microglobulin (β2m) is covalently linked to the NH₂ terminus of the heavy chain by a 15– amino acid long peptide: HHH, entirely human, HHD, with the mouse H-2D^b α3, transmembrane, and cytoplasmic domains, and MHD, homologous to HHD but linked to the mouse β2m^b. The cell surface expression and immunological capacities of the three monochains were compared with transfected cells, and the selected HHD construct was introduced by transgenesis in H-2D^b−/− β2m^−/− double knockout mice. Expression of this monochain restores a sizable peripheral CD8⁺ T cell repertoire essentially educated on the transgenic human molecule. Consequently, infected HHD, H-2D^b−/− β2m^−/− mice generate only HLA-A2.1–restricted CD8⁺ CTL responses against influenza A and vaccinia viruses. Interestingly, the CTL response to influenza A virus is mostly, if not exclusively, directed to the 58-66 matrix peptide which is the HLA-A2.1–restricted immunodominant epitope in humans. Such mice might constitute a versatile animal model for the study of HLA-A2.1–restricted CTL responses of vaccine interest.     

Suppressive Role of B Cells in Chronic Colitis of T Cell Receptor α Mutant Mice

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-α^−/− mice was explored by creating double mutant mice (TCR-α^−/− × immunoglobulin (Ig)μ^−/−), which lack B cells. TCR-α^−/− × Igμ^−/− mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-α^−/− mice. Colitis was induced in recombination-activating gene-1 (RAG-1^−/−) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-α^−/− × Igμ^−/− mice. When purified B cells from TCR-α^−/− mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1^−/− mice. Administration of the purified Ig from TCR-α^−/− mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-α^−/− × Igμ^−/− mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-α^−/− × Igμ^−/− mice as compared to Igμ^−/− mice and TCR-α^−/− mice. Administration of the purified Ig from TCR-α^−/− mice into TCR-α^−/− × Igμ^−/− mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.     

T Cell Development in Mice Lacking All T Cell Receptor ζ Family Members (ζ, η, and FcεRIγ)

The ζ family includes ζ, η, and FcεRIγ (Fcγ). Dimers of the ζ family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking ζ/η chains, T cell development is impaired, yet low numbers of CD4⁺ and CD8⁺ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a ζ family dimer can promote T cell maturation, or that in the absence of ζ/η, Fcγ serves as a subunit in TCR complexes. To elucidate the role of ζ family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only ζ/η or Fcγ. The data reveal that surface complexes that are expressed in the absence of ζ family dimers are capable of transducing signals required for α/β–T cell development. Strikingly, T cells generated in both ζ/η^−/− and ζ/η^−/−–Fcγ^−/− mice exhibit a memory phenotype and elaborate interferon γ. Finally, examination of different T cell populations reveals that ζ/η and Fcγ have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of ζ family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations.     

α/β–T Cell Receptor (TCR)+CD4−CD8− (NKT) Thymocytes Prevent Insulin-dependent Diabetes Mellitus in Nonobese Diabetic (NOD)/Lt Mice by the Influence of Interleukin (IL)-4 and/or IL-10

We have previously shown that nonobese diabetic (NOD) mice are selectively deficient in α/β-T cell receptor (TCR)⁺CD4⁻CD8⁻ NKT cells, a defect that may contribute to their susceptibility to the spontaneous development of insulin-dependent diabetes mellitus (IDDM). The role of NKT cells in protection from IDDM in NOD mice was studied by the infusion of thymocyte subsets into young female NOD mice. A single intravenous injection of 10⁶ CD4^−/lowCD8⁻ or CD4⁻CD8⁻ thymocytes from female (BALB/c × NOD)F1 donors protected intact NOD mice from the spontaneous onset of clinical IDDM. Insulitis was still present in some recipient mice, although the cell infiltrates were principally periductal and periislet, rather than the intraislet pattern characteristic of insulitis in unmanipulated NOD mice. Protection was not associated with the induction of “allogenic tolerance” or systemic autoimmunity. Accelerated IDDM occurs after injection of splenocytes from NOD donors into irradiated adult NOD recipients. When α/β-TCR⁺ and α/β-TCR⁻ subsets of CD4⁻CD8⁻ thymocytes were transferred with diabetogenic splenocytes and compared for their ability to prevent the development of IDDM in irradiated adult recipients, only the α/β-TCR⁺ population was protective, confirming that NKT cells were responsible for this activity. The protective effect in the induced model of IDDM was neutralized by anti–IL-4 and anti–IL-10 monoclonal antibodies in vivo, indicating a role for at least one of these cytokines in NKT cell-mediated protection. These results have significant implications for the pathogenesis and potential prevention of IDDM in humans.     

Allelic Exclusion in pTα-deficient Mice: No Evidence for Cell Surface Expression of Two T Cell Receptor (TCR)-β Chains, but Less Efficient Inhibition of Endogeneous Vβ→ (D)Jβ Rearrangements in the Presence of a Functional TCR-β Transgene

Although individual T lymphocytes have the potential to generate two distinct T cell receptor (TCR)-β chains, they usually express only one allele, a phenomenon termed allelic exclusion. Expression of a functional TCR-β chain during early T cell development leads to the formation of a pre-T cell receptor (pre-TCR) complex and, at the same developmental stage, arrest of further TCR-β rearrangements, suggesting a role of the pre-TCR in mediating allelic exclusion. To investigate the potential link between pre-TCR formation and inhibition of further TCR-β rearrangements, we have studied the efficiency of allelic exclusion in mice lacking the pre-TCR-α (pTα) chain, a core component of the pre-TCR. Staining of CD3⁺ thymocytes and lymph node cells with antibodies specific for Vβ6 or Vβ8 and a pool of antibodies specific for most other Vβ elements, did not reveal any violation of allelic exclusion at the level of cell surface expression. This was also true for pTα-deficient mice expressing a functionally rearranged TCR-β transgene. Interestingly, although the transgenic TCR-β chain significantly influenced thymocyte development even in the absence of pTα, it was not able to inhibit fully endogeneous TCR-β rearrangements either in total thymocytes or in sorted CD25⁺ pre-T cells of pTα^−/− mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.     

The Common Cytokine Receptor γ Chain Controls Survival of γ/δ T Cells

We have investigated the role of common γ chain (γ_c)-signaling pathways for the development of T cell receptor for antigen (TCR)-γ/δ T cells. TCR-γ/δ–bearing cells were absent from the adult thymus, spleen, and skin of γ_c-deficient (γ_c⁻) mice, whereas small numbers of thymocytes expressing low levels of TCR-γ/δ were detected during fetal life. Recent reports have suggested that signaling via interleukin (IL)-7 plays a major role in facilitating TCR-γ/δ development through induction of V-J (variable-joining) rearrangements at the TCR-γ locus. In contrast, we detected clearly TCR-γ rearrangements in fetal thymi from γ_c⁻ mice (which fail to signal in response to IL-7) and reduced TCR-γ rearrangements in adult γ_c thymi. No gross defects in TCR-δ or TCR-β rearrangements were observed in γ_c⁻ mice of any age. Introduction of productively rearranged TCR Vγ1 or TCR Vγ1/Vδ6 transgenes onto mice bearing the γ_c mutation did not restore TCR-γ/δ development to normal levels suggesting that γ_c-dependent pathways provide additional signals to developing γ/δ T cells other than for the recombination process. Bcl-2 levels in transgenic thymocytes from γ_c⁻ mice were dramatically reduced compared to γ_c⁺ transgenic littermates. We favor the concept that γ_c-dependent receptors are required for the maintenance of TCR-γ/δ cells and contribute to the completion of TCR-γ rearrangements primarily by promoting survival of cells committed to the TCR-γ/δ lineage.     

Thermal stability and oxidation characteristics of α-pinene, β-pinene and α-pinene/β-pinene mixture

Turpentine is a renewable resource, has good combustion performance, and is considered to be a fuel or promising additive to diesel fuel. This is very important for the investigation of thermal stability and energy oxidation characteristics, because evaluation of energy or fuel quality assurance and use safety are necessary. The main components of turpentine are α-pinene and β-pinene, which have unsaturated double bonds and high chemical activity. By investigating their thermal stability and oxidation reaction characteristics, we know the chemical thermal properties and thermal explosion hazard of turpentine. In this present study, the thermal stability and oxidation characteristics of α-pinene, β-pinene and α-pinene/β-pinene mixture were investigated using a high sensitivity accelerating rate calorimeter (ARC) and C80 calorimeter. The important parameters of oxidation reaction and thermal stability were obtained from the temperature, pressure and exothermic behavior in chemical reaction. The results show that α-pinene and β-pinene are thermally stable without chemical reaction under a nitrogen atmosphere even when the temperature reaches 473 K. The initial exothermic temperature of the two pinenes and their mixture is 333–338 K, and the heat release (−ΔH) of their oxidation is 2745–2973 J g⁻¹. The oxidation activation energy (E_a) of α-pinene, β-pinene and α-pinene/β-pinene mixture is 116.25 kJ mol⁻¹, 121.85 kJ mol⁻¹, and 115.95 kJ mol⁻¹, respectively. There are three steps in the oxidation of pinenes: the first is the induction period of the oxidation reaction; the second is the main oxidation stage, and the pressure is reduced; the third is thermal decomposition to produce gas.

Turpentine is a renewable resource, has good combustion performance, and is considered to be a fuel or promising additive to diesel fuel.     

Positive Selection of γδ CTL by TL Antigen Expressed in the Thymus

To elucidate the function of the mouse TL antigen in the thymus, we have derived two TL transgenic mouse strains by introducing Tla^a-3 of A strain origin with its own promoter onto a C3H background with no expression of TL in the thymus. These transgenic mouse strains, both of which express high levels of Tla^a-3-TL antigen in their thymus, were analyzed for their T cell function with emphasis on cytotoxic T lymphocyte (CTL) generation. A T cell response against TL was induced in Tg.Tla^a-3-1, Tg.Tla^a-3-2, and control C3H mice by skin grafts from H-2K^b/T3^b transgenic mice, Tg.Con.3-1, expressing T3^b-TL ubiquitously. Spleen cells from mice that had rejected the T3^b-TL positive skin grafts were restimulated in vitro with Tg.Con.3-1 irradiated spleen cells. In mixed lymphocyte cultures (MLC), approximately 20% and 15% of Thy-1⁺ T cells derived from Tg.Tla^a-3-1 and Tg.Tla^a-3-2, respectively, expressed TCRγδ, whereas almost all those from C3H expressed TCRαβ. The MLC from Tg.Tla^a-3-2 and C3H demonstrated high CTL activity against TL, while those from Tg.Tla^a-3-1 had little or none. The generation of γδ CTL recognizing TL in Tg.Tla^a-3-2, but not C3H mice, was confirmed by the establishment of CTL clones. A total of 14 γδ CTL clones were established from Tg.Tla^a-3-2, whereas none were obtained from C3H. Of the 14 γδ CTL clones, 8 were CD8⁺ and 6 were CD4⁻CD8⁻ double negative. The CTL activity of all these clones was TL specific and inhibited by anti-TL, but not by anti-H-2 antibodies, demonstrating that they recognize TL directly without antigen presentation by H-2. The CTL activity was blocked by antibodies to TCRγδ and CD3, and also by antibodies to CD8α and CD8β in CD8⁺ clones, showing that the activity was mediated by TCRγδ and coreceptors. The thymic origin of these γδ CTL clones was indicated by the expression of Thy-1 and Ly-1 (CD5), and also CD8αβ heterodimers in CD8⁺ clones on their surfaces and by the usage of TCR Vγ4 chains in 12 of the 14 clones. Taken together, these results suggest that Tla^a-3-TL antigen expressed in the thymus engages in positive selection of a sizable population of γδ T cells.     

NF-κB RelA-deficient Lymphocytes: Normal Development of T Cells and B Cells, Impaired Production of IgA and IgG1 and Reduced Proliferative Responses

To investigate the function of NF-κB RelA (p65), we generated mice deficient in this NF-κB family member by homologous recombination. Mice lacking RelA showed liver degeneration and died around embryonic day 14.5. To elucidate the role of RelA in lymphocyte development and function, we transplanted fetal liver cells of 13.5-day embryos from heterozygote matings into irradiated SCID mice. Within 4 weeks, both T and B cells had developed in the SCID mice receiving relA−/− fetal liver transplants, similar to the relA+/+ and +/− cases. T cells were found to mature to Thy-1⁺/TCRαβ⁺/CD3⁺/CD4⁺ or CD8⁺, while B cells had the ability to differentiate to IgM⁺/B220⁺ and to secrete immunoglobulins. However, the secretion of IgG1 and IgA was reduced in RelA-deficient B cells. Furthermore, both T and B cells lacking RelA showed marked reduction in proliferative responses to stimulation with Con A, anti-CD3, anti-CD3+anti-CD28, LPS, anti-IgM, and PMA+calcium ionophore. The results indicate that RelA plays a critical role in production of specific Ig isotypes and also in signal transduction pathways for lymphocyte proliferation.     

Hydrophilic and organophilic pervaporation of industrially important α,β and α,ω-diols

The distillation-based purification of α,β and α,ω-diols is energy and resource intensive, as well as time consuming. Pervaporation separation is considered to be a remarkable energy efficient membrane technology for purification of diols. Thus, as a core pervaporation process, hydrophilic polyvinyl alcohol (PVA) membranes for the removal of water from 1,2-hexanediol (1,2-HDO) and organophilic polydimethylsiloxane–polysulfone (PDMS–PSF) membranes for the removal of isopropanol from 1,5 pentanediol (1,5-PDO) were employed. For 1,2-HDO/water separation using a feed having a 1 : 4 weight ratio of 1,2-HDO/water, the membrane prepared using 4 vol% glutaraldehyde (GA4) showed the best performance, yielding a flux of 0.59 kg m⁻² h⁻¹ and a separation factor of 175 at 40 °C. In the organophilic pervaporation separation of the 1,5-PDO/IPA feed having a 9 : 1 weight ratio of components, the PDMS membrane prepared with a molar ratio of TEOS alkoxy groups to PDMS hydroxyl groups of 70 yielded a flux of 0.12 kg m⁻² h⁻¹ and separation factor of 17 638 at 40 °C. Long term stability analysis found that both hydrophilic (PVA) and organophilic (PDMS) membranes retained excellent pervaporation output over 18 days'' continuous exposure to the feed. Both the hydrophilic and organophilic membranes exhibited promising separation performance at elevated operating conditions, showing their great potential for purification of α,β and α,ω-diols.

The distillation-based purification of α,β and α,ω-diols is energy and resource intensive, as well as time consuming.     

GSK-3β Is Required for Memory Reconsolidation in Adult Brain

Activation of GSK-3β is presumed to be involved in various neurodegenerative diseases, including Alzheimer's disease (AD), which is characterized by memory disturbances during early stages of the disease. The normal function of GSK-3β in adult brain is not well understood. Here, we analyzed the ability of heterozygote GSK-3β knockout (GSK+/−) mice to form memories. In the Morris water maze (MWM), learning and memory performance of GSK+/− mice was no different from that of wild-type (WT) mice for the first 3 days of training. With continued learning on subsequent days, however, retrograde amnesia was induced in GSK+/− mice, suggesting that GSK+/− mice might be impaired in their ability to form long-term memories. In contextual fear conditioning (CFC), context memory was normally consolidated in GSK+/− mice, but once the original memory was reactivated, they showed reduced freezing, suggesting that GSK+/− mice had impaired memory reconsolidation. Biochemical analysis showed that GSK-3β was activated after memory reactivation in WT mice. Intraperitoneal injection of a GSK-3 inhibitor before memory reactivation impaired memory reconsolidation in WT mice. These results suggest that memory reconsolidation requires activation of GSK-3β in the adult brain.     

Transferable Anergy: Superantigen Treatment Induces CD4+ T Cell Tolerance That Is Reversible and Requires CD4−CD8− Cells and Interferon γ

Bacterial superantigens induce peripheral unresponsiveness in CD4⁺ T cell populations that express appropriate Vβ chains. We have used Vβ3/Vα11 T cell receptor transgenic (Tg) mice and the Vβ3-specific superantigen staphylococcal enterotoxin A (SEA) to further investigate the mechanisms that contribute to such unresponsiveness. As in other models, in vivo exposure to SEA rendered the Tg CD4⁺ cells unresponsive to subsequent restimulation in vitro with antigen or mitogens. However, when the SEA-treated CD4⁺ cells were completely purified away from all other contaminating cells, they regained the ability to proliferate and secrete cytokines. Moreover, enriched CD4⁻CD8⁻ cells from the SEA-treated mice suppressed the responses of fresh control CD4⁺ cells in mixed cultures indicating that the apparent “anergy” was both transferable and reversible. Further analysis demonstrated that interferon γ, but not the Fas receptor, played a critical role in the suppression.     

Distinct Roles for Signals Relayed through the Common Cytokine Receptor γ Chain and Interleukin 7 Receptor α Chain in Natural T Cell Development

The commitment, differentiation, and expansion of mainstream α/β T cells during ontogeny depend on the highly controlled interplay of signals relayed by cytokines through their receptors on progenitor cells. The role of cytokines in the development of natural killer (NK)1⁺ natural T cells is less clearly understood. In an approach to define the role of cytokines in the commitment, differentiation, and expansion of NK1⁺ T cells, their development was studied in common cytokine receptor γ chain (γc) and interleukin (IL)-7 receptor α (IL-7Rα)–deficient mice. These mutations block mainstream α/β T cell ontogeny at an early prethymocyte stage. Natural T cells do not develop in γc-deficient mice; they are absent in the thymus and peripheral lymphoid organs such as the liver and the spleen. In contrast, NK1⁺ T cells develop in IL-7Rα–deficient mice in the thymus, and they are present in the liver and in the spleen. However, the absolute number of NK1⁺ T cells in the thymus of IL-7Rα–deficient mice is reduced to ~10%, compared to natural T cell number in the wild-type thymus. Additional data revealed that NK1⁺ T cell ontogeny is not impaired in IL-2– or IL-4–deficient mice, suggesting that neither IL-2, IL-4, nor IL-7 are required for their development. From these data, we conclude that commitment and/or differentiation to the NK1⁺ natural T cell lineage requires signal transduction through the γc, and once committed, their expansion requires signals relayed through the IL-7Rα.     

Proliferation Kinetics Associated with T Cell Receptor-β Chain Selection of Fetal Murine Thymocytes

After productive rearrangement of a TCRβ chain gene, CD4⁻8⁻ double negative (DN) thymocytes express TCRβ polypeptide chains on the cell surface together with pre-Tα and the CD3 complex forming the pre-TCR. Signals transmitted through the pre-TCR select TCRβ⁺ DN thymocytes for further maturation to the CD4⁺8⁺ double positive stage, whereas DN cells that fail to generate a productive TCRβ gene rearrangement do not continue in development. This process is termed TCRβ chain selection. Although it is likely that differences between proliferation dynamics of TCRβ⁺ and TCRβ⁻ cells may play a role, the exact mechanisms of TCRβ chain selection have not been elucidated. We therefore studied the proliferation dynamics of TCRβ⁺ and TCRβ⁻ thymocytes during fetal development, i.e., when TCRβ chain selection takes place for the first time. We analyzed in situ accumulation of TCRβ⁺ thymocytes by confocal microscopy, and determined cell cycle and division parameters of TCRβ⁺ and TCRβ⁻ populations by flow cytometry. About 600 TCRβ⁺ cells/thymic lobe are generated by independent induction events between days of gestation (dg) 13.5. and 15.5. As of dg 14.5, most TCRβ⁺ cells have entered S/G2 phase of cell cycle, followed by seven to eight rapid cell divisions in fetal thymic organ culture, suggesting a corresponding burst of nine cell divisions within 4 d in vivo. By dg 18.5, the division rate of TCRβ⁺ cells has slowed down to less than 1/d. About three quarters of TCRβ⁻ cells divide at a slow rate of 1/d on dg 14.5, the proportion of nondividing cells increasing to 50% within the following four d. From dg 16.5 onwards, TCRβ⁻ cells, but not TCRβ⁺ cells, contain a significant proportion of apoptotic cells. The results suggest that failure to become selected results in shutdown of proliferation and eventual programmed cell death of fetal TCRβ⁻ cells. Positive selection of fetal TCRβ⁺ cells is achieved by an increased rate of cell divisions lasting for approximately 4 d.     

Activin A Induces Langerhans Cell Differentiation In Vitro and in Human Skin Explants

Langerhans cells (LC) represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFβ. Ιn vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFβ. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFβ family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFβ. Activin A-induced LC are Langerin⁺, Birbeck granules⁺, E-cadherin⁺, CLA⁺ and CCR6⁺ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a⁺ and Langerin⁺ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a⁺ and Langerin⁺ cells. This study reports that Activin A induces the differentiation of circulating CD14⁺ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFβ, responsible for LC differentiation during inflammatory/autoimmune conditions.