Improvement of herpetic stromal keratitis with fumaric acid derivate is associated with systemic induction of T helper 2 cytokines

Fumaric acid derivates have been shown to stimulate T helper-2-cytokines (interleukin (IL)-4, -5) without affecting the T-helper-1-cytokine (IL-2, interferon (IFN)-gamma)-response. Herein, the influence of systemic treatment with the fumaric acid derivate dimethylfumarate (DMF) on the secretion of T helper-cytokines and the development of HSV-1 stromal keratitis (HSK) was studied in mice. The corneas from BALB/c mice were infected with 10(5) PFU of HSV-1 (KOS strain). While one group of mice was treated intraperitoneally with PBS, another group of mice received DMF at 15 mg/kg of body weight. Expression of IL-2, -4, -10 and IFN-gamma was analysed in HSV-1 activated lymphocytes by ELISA. The severity of epithelial and stromal herpetic keratitis was investigated clinically. Corneas were studied for the inflammatory cell infiltration, and the CD3-, CD4- and CD8-positive cells were analysed by immunohistochemistry. The IL-2, -4, 10 and IFN-gamma content was measured in the corneas. Virus replication in the eyes was analysed by a plaque-assay. The DTH-response, the HSV-specific T cell proliferation and the serum neutralizing antibody-titres were investigated. DMF increased IL-4 and IL-10, but not IL-2 and IFN-gamma, secretion in activated lymphocytes from the spleen. Incidence and severity of stromal HSV-1 keratitis was reduced in the DMF group (P < 0.01). In the corneas from DMF-treated mice, the numbers of CD3+ and CD4+ cells were decreased and IL-4 was increased. Severity of epithelial disease and the virus-clearance from the eyes did not differ between the PBS and DMF group of mice. DTH, HSV-specific T cell proliferation and the neutralizing antibody-titres were not impaired. DMF increased the T helper-2-cytokine secretion in activated lymphocytes. After corneal HSV-1 infection, corneas from DMF treated mice had increased IL-4 content. This is associated with an improvement of herpetic stromal keratitis and reduced corneal T cell infiltration. DMF did not impair the systemic antiviral response.     

CD4(+) and CD8(+) cells are key participants in the development of recurrent herpetic stromal keratitis in mice

Ocular herpes simplex virus (HSV) infection results in an immune-mediated inflammation of the corneal stroma known as herpetic stromal keratitis (HSK). Recurrent HSK is a common cause of virus-induced corneal blindness in humans. The role of CD4(+) and CD8(+) T cell subsets in the disease pathogenesis is ill defined and varies with the virus strain and host genetic background. To examine the contribution of T cell subsets to corneal disease, we studied the development of recurrent HSK in CD4 or CD8 gene knockout (KO) mice ocularly infected with HSV-1 McKrae strain. Following UV-B induced viral reactivation, corneal opacity in latently infected BALB/c (HSV sensitive) CD4 and CD8 KO mice was reduced compared to infected BALB/c mice with normal genotype. In contrast, opacity in C57BL/6 (HSV resistant) CD4 and CD8 KO latent mice did not differ from genetically normal latent mice. Virus-induced corneal opacity was not demonstrable in C57BL/6 CD4/CD8 double KO mice. Increased viral shedding, measured by reactivation rate, days shedding or viral titers, occurred in CD4 KO mice of both strains. Our findings indicate that both CD4(+) and CD8(+) cells play a role in the immunopathogenesis of recurrent HSK, and their role is dependent upon the host genetic profile.     

IL-18, but not IL-12, is required for optimal cytokine production by influenza virus-specific CD8+ T cells

The potent innate cytokines IL-12 and IL-18 are considered to be important antigen-independent mediators of IFN-gamma production by NK cells and T lymphocytes. The present analysis addresses the physiological role of IL-12 and IL-18 in the generation of virus-specific CD8+ T cells. Both wt C57BL/6J (B6) mice and mice with disrupted IL-12p40 (IL-12p40(-/-)) or IL-18 (IL-18(-/-)) genes were infected with an influenza A virus and the characteristics of the resultant epitope-specific CD8+ T cell responses were compared. While IL-12 appeared to have no notable effect on either virus growth or on CD8+ T cell response profiles, the absence of IL-18 was associated with delayed virus clearance from the lung and, despite normal numbers, a significantly reduced production of IFN-gamma, TNF-alpha, and IL-2 by epitope-specific CD8+ T cells. While this cytokine phenotype was broadly maintained in IL-12p40/IL-18 double-knockout mice, no evidence was seen for any additive effect. Together, our results suggest that IL-18, but not IL-12, induces optimal, antigen-specific production of key cytokines by CD8+ T cells for the efficient clearance of influenza virus from the lungs of infected mice.     

IL‐7 is essential for lymphopenia‐driven turnover of colitogenic CD4+ memory T cells in chronic colitis

We previously demonstrated that IL‐7 is essential for the persistence of T‐cell‐mediated colitis, by showing that adoptive transfer of CD4⁺CD45RB^high T cells into IL‐7^?/?×RAG‐1^?/? mice did not induce colitis; and that intestinal IL‐7 is not essential for this colitis model, by showing that IL‐7^?/?×RAG‐1^?/? mice parabiosed with colitic CD4⁺CD45RB^high T‐cell‐transferred RAG‐1^?/? mice developed colitis. Here, we investigated the role of IL‐7 in the maintenance of colitogenic CD4⁺ T cells by surgically separating these parabionts. Surprisingly, the separated IL‐7^?/?×RAG‐1^?/? mice were consistently diseased after separation, although no IL‐7 mRNA was detected in the tissues of separated IL‐7^?/?×RAG‐1^?/? partners. CD4⁺ T cells isolated from the separated RAG‐1^?/? or IL‐7^?/?×RAG‐1^?/? mice were then transferred into new RAG‐1^?/? or IL‐7^?/?×RAG‐1^?/? mice. Regardless of the source of donor cells, RAG‐1^?/? recipients developed colitis, whereas IL‐7^?/?×RAG‐1^?/? recipients did not. Collectively, these results demonstrate that IL‐7 is essential for lymphopenia‐driven turnover of colitogenic CD4⁺ T cells rather than the maintenance of those cells in established colitic mice. They also provide a basis for the timing of IL‐7/IL‐7R blockade for the treatment of inflammatory bowel diseases.     

Accumulation and activation of epidermal γδ T cells in a mouse model of chronic dermatitis is not required for the inflammatory phenotype

Chronic skin inflammation resulting from a defective epidermal barrier is a hallmark of atopic dermatitis (AD). We previously demonstrated that mice lacking FGF receptors 1 and 2 in keratinocytes (K5‐R1/R2 mice) develop an AD‐like chronic dermatitis as a result of an impaired epidermal barrier. Here, we show that γδ T cells, which rapidly respond to various insults, accumulate in the epidermis of K5‐R1/R2 mice before the development of histological abnormalities. Their number and activation further increase as the phenotype progresses, most likely as a consequence of increased expression of Il‐2 and Il‐7 and the stress‐induced proteins Rae‐1, H60c, Mult1, PlexinB2, and Skint1. To determine the role of γδ T cells in the skin phenotype, we generated quadruple mutant K5‐R1/‐R2 mice lacking γδ T cells. Surprisingly, loss of γδ T cells did not or only marginally affect keratinocyte proliferation, epidermal thickness, epidermal barrier function, and accumulation and activation of different immune cells in the skin of K5‐R1/R2 mice, possibly due to partial compensation by αβ T cells. These results demonstrate that γδ T cells do not contribute to the development or maintenance of chronic inflammation in response to a defect in the epidermal barrier.     

Targeting the EGFR, VEGFR, and PDGFR on colon cancer cells and stromal cells is required for therapy

Immunohistochemical analysis of human colon cancers growing in the cecal walls of nude mice revealed that epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) were expressed by different tumor cells and tumor-associated endothelial cells, whereas platelet-derived growth factor receptor (PDGFR)beta was expressed by tumor-associated endothelial cells and pericytes. We hypothesized that treatment of nude mice with AEE788 (an inhibitor of EGFR and VEGFR phosphorylation) and STI571 (an inhibitor of PDGFRbeta phosphorylation) combined with irinotecan would overcome the intratumoral heterogeneity of these growth factors and efficiently inhibit colon cancer growth and metastasis. We implanted HT29 and KM12SM cells into the cecal walls of nude mice. Two weeks later, the mice were treated with oral vehicle solution; oral AEE788, oral STI571, or intraperitoneal injection of irinotecan as single agents; or the various combinations of these agents. We then assessed the mice for tumor growth and metastasis. Immunohistochemical analyses revealed that oral AEE788 suppressed proliferation and increased apoptosis of tumor cells and tumor-associated endothelial cells. Oral STI571 increased apoptosis of tumor-associated endothelial cells and pericytes. The combination of AEE788, STI571, and irinotecan produced the greatest inhibition of primary tumor growth and metastasis. Collectively, these data demonstrate that only targeting multiple tyrosine kinase receptors on colon cancer cells and tumor-associated stromal cells can overcome the effects of biologic heterogeneity for resistance to treatment and has the potential to improve therapeutic outcome for patients with this disease.     

Heme oxygenase-1 is not required for mouse regulatory T cell development and function

CD4 regulatory T cells (Treg) ensure peripheral tolerance to self-antigens and limit the deleterious effects associated with inflammatory and immune responses by mechanisms that remain to be fully understood. The enzyme heme oxygenase-1 (HO-1), through its known anti-inflammatory activity, is a candidate for a functional role in Treg activity. We compared wild-type and heme oxygenase-1-deficient (hmox-1(-/-)) mice in order to assess the role of HO-1 in mouse Treg development and function under physiologic conditions. The frequency of CD25+ and Foxp3+ Treg was similar in hmox-1(-/-) and hmox-1(+/+) mice. More importantly, CD4+ CD25+ Treg purified from either hmox-1(-/-) or hmox-1(+/+) mice were equally efficient in controlling the proliferation in vitro and the expansion in vivo of CD4+ CD25- T cells, whether or not these responder cells expressed HO-1. In addition, induction of expression of HO-1 in vivo did not affect Treg suppressor function. As shown before, expression of HO-1 was higher in Treg than in naive T cells; however, naturally activated Foxp3- T cells displayed equal amount of HO-1 mRNA as Treg. Finally, we conclude that under physiological conditions in mice, Treg development, maintenance and function are independent of HO-1 activity.     

Notch1 expression on T cells is not required for CD4+ T helper differentiation

Notch1 proteins are involved in binary cell fate decisions. To determine the role of Notch1 in the differentiation of CD4(+) Th1 versus Th2 cells, we have compared T helper polarization in vitro in naive CD4(+) T cells isolated from mice in which the N1 gene is specifically inactivated in all mature T cells. Following activation, Notch1-deficient CD4(+) T cells transcribed and secreted IFN-gamma under Th1 conditions and IL-4 under Th2 conditions at levels similar to that of control CD4(+) T cells. These results show that Notch1 is dispensable for the development of Th1 and Th2 phenotypes in vitro. The requirement for Notch1 in Th1 differentiation in vivo was analyzed following inoculation of Leishmania major in mice with a T cell-specific inactivation of the Notch1 gene. Following infection, these mice controlled parasite growth at the site of infection and healed their lesions. The mice developed a protective Th1 immune response characterized by high levels of IFN-gamma mRNA and protein and low levels of IL-4 mRNA with no IL-4 protein in their lymph node cells. Taken together, these results indicate that Notch1 is not critically involved in CD4(+) T helper 1 differentiation and in resolution of lesions following infection with L. major.     

IRAK-4 kinase activity is required for IRAK-4-dependent innate and adaptive immune responses

Interleukin-1 receptor-associated kinase (IRAK)-4 is a serine-threonine kinase that plays an important role in innate and adaptive immune responses. While the requirement of IRAK-4 kinase activity has been studied in the context of IL-1R signaling, it is not clear whether IRAK-4 requires its kinase function for all of its roles in the immune system. IRAK-4 kinase-dead knock-in (IRAK-4KD/KD) mice were generated to further elucidate whether IRAK-4 kinase activity is required for IRAK-4 to induce cytokine production. IRAK-4KD/KD mice were impaired in their ability to produce cytokines in response to in vivo challenge with lipopolysaccharide (LPS), a potent TLR4 ligand. Cytokine production was also reduced in macrophages and dendritic cells from IRAK-4KD/KD mice in response to LPS and other TLR ligands. In addition, adaptive immune responses were impaired in IRAK-4KD/KD mice. Although in vitro T cell proliferation in response to TCR activation was unaffected in IRAK-4-deficient mice, in vivo T cell responses to lymphocytic choriomeningitits virus infection were significantly impaired in IRAK-4-knockout mice or mice expressing the kinase-dead mutant of IRAK-4. Collectively, these results indicate that IRAK-4 kinase activity is required for IRAK-4-dependent signaling in innate and adaptive immunity.     

Expression of class II,but not class I,major histocompatibility complex molecules is required for granuloma formation in infection with Schistosoma mansoni

Previous studies have suggested that granulomatous inflammation in schistosomiasis is mediated by CD4⁺ T helper lymphocytes sensitized to parasite egg antigens. However, CD8⁺ T cells have also frequently been associated with the immune response to schistosome eggs. To examine more precisely the role of CD4⁺ and CD8⁺ T cells in the pathology of the schistosomal infection, we used mice with targeted mutations in major histocompatibility complex (MHC) class II or class I molecules. These mutations lead, respectively, to the virtual absence of CD4⁺ and CD8⁺ T cells. The results clearly show that schistosome-infected MHC class II mutant mice failed to form granulomas around parasite eggs. In contrast, infected MHC class I mutant mice displayed characteristic granulomatous lesions that were comparable to those in wild-type control mice. Moreover, lymphoid cells from MHC class II mutant mice were unable to react to egg antigens with either proliferative or cytokine [interferon-gamma, interleukin (IL)-4, IL-10] responses; nor were they able to present egg antigens to specifically sensitized CD4⁺ T helper cells from infected syngeneic control mice. By comparison, cells from MHC class I mutant mice exercised all these functions in a manner comparable with those from wild-type controls. These observations clearly demonstrate that schistosomal egg granulomas are mediated by MHC class II-restricted CD4⁺ T helper cells. They also suggest that CD8⁺ T cells do not become sensitized to egg antigens and play little role, if any, in the pathogenesis of schistosomiasis.     

IL‐4 promotes stromal cell expansion and is critical for development of a type‐2, but not a type 1 immune response

IL‐4 is critical for differentiation of Th2 cells and antibody isotype switching, but our work demonstrated that it is produced in the peripheral LN under both Type 2, and Type 1 conditions, raising the possibility of other functions. We found that IL‐4 is vital for proper positioning of hematopoietic and stromal cells in steady state, and the lack of IL‐4 or IL‐4Rα correlates with disarrangement of both follicular dendritic cells and CD31⁺ endothelial cells. We observed a marked disorganization of B cells in these mice, suggesting that the lymphocyte‐stromal cell axis is maintained by the IL‐4 signaling pathway. This study showed that absence of IL‐4 correlates with significant downregulation of Lymphotoxin alpha (LTα) and Lymphotoxin beta (LTβ), critical lymphokines for the development and maintenance of lymphoid organs. Moreover, immunization of IL‐4 deficient mice with Type 2 antigens failed to induce lymphotoxin production, LN reorganization, or germinal center formation, while this process is IL‐4 independent following Type 1 immunization. Additionally, we found that Type 1 antigen mediated LN reorganization is dependent on IFN‐γ in the absence of IL‐4. Our findings reveal a role of IL‐4 in the maintenance of peripheral lymphoid organ microenvironments during homeostasis and antigenic challenge.     

PD‐1 signalling in CD4+ T cells restrains their clonal expansion to an immunogenic stimulus,but is not critically required for peptide‐induced tolerance

The ultimate outcome of T‐cell recognition of peptide–major histocompatibility complex (MHC) complexes is determined by the molecular context in which antigen presentation is provided. The paradigm is that, after exposure to peptides presented by steady‐state dendritic cells (DCs), inhibitory signals dominate, leading to the deletion and/or functional inactivation of antigen‐reactive T cells. This has been utilized in a variety of models providing peptide antigen in soluble form in the absence of adjuvant. A co‐inhibitory molecule of considerable current interest is PD‐1. Here we show that there is the opportunity for the PD‐1/PD‐L1 interaction to function in inhibiting the T‐cell response during tolerance induction. Using traceable CD4⁺ T‐cell receptor (TCR) transgenic cells, together with a blocking antibody to disrupt PD‐1 signalling, we explored the roles of PD‐1 in the induction of tolerance versus a productive immune response. Intact PD‐1 signalling played a role in limiting the extent of CD4⁺ T‐cell accumulation in response to an immunogenic stimulus. However, PD‐1 signalling was not required for either the induction, or the maintenance, of peptide‐induced tolerance; a conclusion underlined by successful tolerance induction in TCR transgenic cells genetically deficient for PD‐1. These observations contrast with the reported requirement for PD‐1 signals in CD8⁺ T‐cell tolerance.     

PD‐1 is not required for natural or peripherally induced regulatory T cells: Severe autoimmunity despite normal production of regulatory T cells

The expression of the coinhibitor PD‐1 on T cells is important for the establishment of immune homeostasis. We previously found that PD‐1 is particularly critical for the control of self‐tolerance during lymphopenia‐induced proliferation of recent thymic emigrants (RTEs). Previous studies suggested that PD‐1 modulates the generation of Treg cells, particularly peripherally induced Treg (pTreg) cells, and controls Th17 cells. However, these conclusions were derived indirectly from studies on the ligand PD‐L1, and not PD‐1 itself. Herein we directly tested whether T‐cell PD‐1 expression was needed for Treg cell generation and examined if a paucity of Treg cells or enhanced Th17 cells could explain the severe lymphopenia‐potentiated autoimmunity caused by PD‐1 KO RTEs. Employing the murine FoxP3^EGFP reporter system to simultaneously monitor conversion of WT and PD‐1 KO T cells to pTreg cells in the same animal, we found that PD‐1 deficiency did not inhibit pTreg cell generation or lead to Th17‐cell‐mediated autoimmunity. Surprisingly, pTreg cell numbers were increased in PD‐1 KO versus WT cell populations. Furthermore, we noted an increased conversion to pTreg cells by RTEs. Our data suggest that the primary role for PD‐1 is to restrain T‐cell activation/proliferation to self‐Ags rather than promote generation of Treg cells.     

A Ca2+ concentration of 1.5 mM,as present in IMDM but not in RPMI,is critical for maximal response of Th cells to PMA/ionomycin

Restimulation of memory–effector T helper lymphocytes with PMA/ionomycin is an established technique to determine their imprinting for the expression of cytokine genes, that is their functional potential. Here, we show that for the maximal reexpression of cytokine genes, the Ca²⁺ concentration of the medium has to be greater than 1.5 mM. For RPMI (Roswell Park Memorial Institute 1640), the most commonly used medium, the Ca²⁺ concentration has to be increased from the regular 0.42 to 1.5 mM.     

Dendritic cells and macrophages are required for Th1 development of CD4+ T cells from {alpha}{beta} TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFN-{gamma} production is IFN-{gamma}-dependent

We have examined the antigen presenting cell (APC) requirementsfor primary T cell activation and T helper (Th) cell phenotypedifferentiation using naive CD4⁺ T cells from ß TCRtransgenic mice. Purified dendritic cells were the principalcell required for induction of primary ovalbumln peptide specificT cell activation and clonal expansion. However, dendritic cellsdid not induce differentiation of T cells toward Th1 or Th2phenotype. Addition of IL-4 during primary dendritic cell stimulationsof T cells resulted in the development of a Th2 phenotype whichproduced high levels of IL-4 during secondary and tertiary stimulation.In contrast, development of Th1 cells producing high levelsof IFN- could not be induced with dendritic cells alone butrequired the addition of appropriately activated macrophages.Addition of splenic or peritoneal B cells did not induce Th1development. Activated splenic macrophages induced Th1 developmentvia a non-MHC restricted mechanism. Thus, requirements for inductionof proliferation of naive CD4⁺ T cells are distinct from thosedirecting Th1 phenotype development. IL-12 could replace therequirement for macrophages to induce Th1 development when Tcells were activated with dendritic cells. Furthermore, thisIL-12 mediated development of Th1 cells producing high levelsof IFN- was dependent on IFN-.     

IL-4 synthesis byin vivo-primed memory CD4+ T Cells: II. Presence of IL-4 is not required for IL-4 synthesis in primed CD4+ T Cells

Previous studies have shown that the presence of IL-4 is required for the development of IL-4 synthesis in naive CD4⁺ T cells. The purpose of our current studies was to investigate the role of IL-4 in the development of IL-4 synthesis in primed memory T cells. We therefore examined CD4⁺ T cells taken from lymph nodes of BALB/c mice immunized with keyhole limpet hemocyanin (KLH) and restimulatedin vitro with KLH. Our results with such primed resting CD4⁺ T cells programmed to produce IL-4 indicated that the production of IL-4 did not require the presence of IL-4 (although the presence of IL-2 was absolutely necessary), and was only slightly limited by the presence of anti-IL-4 MAb. These results with resting memory T cells were not biased by the presence of activated T cells already producing substantial quantities of IL-4, since we demonstrated that high-density memory T cells could produce IL-4 in the absence of IL-4, and because T cells that actively produce IL-4 do not persistin vivo very long after antigen exposure. These results indicate that IL-4 synthesis in T cells committed to IL-4 production can indeed occur in the absence of IL-4 when culture conditions have been optimized and suggest that therapies with anti-IL-4 MAb or with soluble IL-4 receptors designed to control the development of IL-4 synthesis in memory T cells from individuals exhibiting excessive IL-4 synthesis will be unsuccessful. Therefore, other therapies, for example, utilizing IL-12, will be required to modulate the relatively fixed programs in memory T cells that direct the development of cytokine synthesis.