Immune-Mobilizing Monoclonal T Cell Receptors Mediate Specific and Rapid Elimination of Hepatitis B-Infected Cells.

BACKGROUND AND AIMS: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV-specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity-enhanced T Cell receptor with an anti-CD3 T Cell-activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus-derived peptides presented by human leukocyte antigen (HLA). APPROACH AND RESULTS: ImmTAV molecules specific for HLA-A*02:01-restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV-Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging-based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV-Env can redirect T cells from healthy and HBV-infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV-Env redirection of T cells induced cytolysis of antigen-positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA. CONCLUSIONS: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non-HBV-specific T cells, bypassing exhausted HBV-specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials.

Immunomodulatory glycan lacto-N-fucopentaose III requires clathrin-mediated endocytosis to induce alternative activation of antigen-presenting cells.

The mechanism of alternative activation of antigen-presenting cells (APCs) is largely unknown. Lacto-N-fucopentaose III (LNFPIII) is a biologically conserved pentasaccharide that contains the Lewis(x) trisaccharide. LNFPIII conjugates and schistosome egg antigens, which contain the Lewis(x) trisaccharide, drive alternative activation of APCs and induce anti-inflammatory responses in vivo, preventing inflammation-based diseases, including psoriasis, transplant organ rejection, and metabolic disease. In this study, we show that LNFPIII conjugates and schistosome egg antigens interact with APCs via a receptor-mediated process, requiring internalization of these molecules through a clathrin/dynamin-dependent but caveolus-independent endocytic pathway. Using inhibitors/small interfering RNA (siRNA) against dynamin and clathrin, we show for the first time that endocytosis of Lewis(x)-containing glycans is required to drive alternative maturation of antigen-presenting cells and Th2 immune responses. We identified mouse SIGNR-1 as a cell surface receptor for LNFPIII conjugates. Elimination of SIGNR-1 showed no effect on uptake of LNFPIII conjugates, suggesting that other receptors bind to and facilitate uptake of LNFPIII conjugates. We demonstrate that disruption of actin filaments partially prevented the entry of LNFPIII conjugates into APCs and that LNFPIII colocalizes with both early and late endosomal markers and follows the classical endosomal pathway leading to lysosome maturation. The results of this study show that the ability of LNFPIII to induce alternative activation utilizes a receptor-mediated process that requires a dynamin-dependent endocytosis. Thus, key steps have been defined in the previously unknown mechanism of alternative activation that ultimately leads to induction of anti-inflammatory responses.

A randomised controlled pilot feasibility study of the physical and psychological effects of an integrated support programme in breast cancer.

A pilot study was conducted to assess recruitment and effectiveness of an integrated support programme in women with breast cancer. Twelve participants were randomised to receive medical care with or without the support programme. Psychosocial questionnaires and immune/hormonal assays were completed at baseline, three and six months. Recruitment was problematic. In the intervention group, mental fatigue was significantly improved (p = 0.016) compared to controls; increased NK cell activity suggested an improvement in immune function. Total stress (p = 0.009), anxiety (p = 0.032) and endocrine-specific (p = 0.032) symptoms were significantly improved in the controls. A large-scale randomisation trial appears warranted, dependent upon effective recruitment.

Metabolic characterization of Leishmania major infection in activated and nonactivated macrophages.

Infection with Leishmania spp. can lead to a range of symptoms in the affected individual, depending on underlying immune-metabolic processes. The macrophage activation state hereby plays a key role. Whereas the l-arginine pathway has been described in detail as the main biochemical process responsible for either nitric oxide mediated parasite killing (classical activation) or amplification of parasite replication (alternative activation), we were interested in a wider characterization of metabolic events in vitro. We therefore assessed cell growth medium, parasite extract, and intra- and extracellular metabolome of activated and nonactivated macrophages, in presence and absence of Leishmania major. A metabolic profiling approach was applied combining 1H NMR spectroscopy with multi- and univariate data treatment. Metabolic changes were observed along both conditional axes, that is, infection state and macrophage activation, whereby significantly higher levels of potential parasite end products were found in parasite exposed samples including succinate, acetate, and alanine, compared to uninfected macrophages. The different macrophage activation states were mainly discriminated by varying glucose consumption. The presented profiling approach allowed us to obtain a metabolic snapshot of the individual biological compartments in the assessed macrophage culture experiments and represents a valuable read out system for further multiple compartment in vitro studies.

Proteophosophoglycans regurgitated by Leishmania-infected sand flies target the L-arginine metabolism of host macrophages to promote parasite survival.

All natural Leishmania infections start in the skin; however, little is known of the contribution made by the sand fly vector to the earliest events in mammalian infection, especially in inflamed skin that can rapidly kill invading parasites. During transmission sand flies regurgitate a proteophosphoglycan gel synthesized by the parasites inside the fly midgut, termed promastigote secretory gel (PSG). Regurgitated PSG can exacerbate cutaneous leishmaniasis. Here, we show that the amount of Leishmania mexicana PSG regurgitated by Lutzomyia longipalpis sand flies is proportional to the size of its original midgut infection and the number of parasites transmitted. Furthermore, PSG could exacerbate cutaneous L. mexicana infection for a wide range of doses (10-10,000 parasites) and enhance infection by as early as 48 hours in inflamed dermal air pouches. This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours. Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth. The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo. Thus, PSG is an essential component of the infectious sand fly bite for the early establishment of Leishmania in skin, which should be considered when designing and screening therapies against leishmaniasis.

Local suppression of T cell responses by arginase-induced L-arginine depletion in nonhealing leishmaniasis.

The balance between T helper (Th) 1 and Th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized Th1 or Th2 responses are not sufficient to account for healing or nonhealing. Here we show that high arginase activity, a hallmark of nonhealing disease, is primarily expressed locally at the site of pathology. The high arginase activity causes local depletion of L-arginine, which impairs the capacity of T cells in the lesion to proliferate and to produce interferon-gamma, while T cells in the local draining lymph nodes respond normally. Healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. Moreover, competitive inhibition of arginase as well as supplementation with L-arginine restored T cell effector functions and reduced pathology and parasite growth at the site of lesions. These results demonstrate that in nonhealing leishmaniasis, arginase-induced L-arginine depletion results in impaired T cell responses. Our results identify a novel mechanism in leishmaniasis that contributes to the failure to heal persistent lesions and suggest new approaches to therapy.

L-arginine deprivation impairs Leishmania major-specific T-cell responses.

The amino acid L-arginine plays a crucial role in the regulation of immune responses. We have recently shown that uncontrolled replication of Leishmania parasites at the site of pathology correlates with high levels of arginase activity in nonhealing leishmaniasis and that this elevated arginase activity causes local depletion of L-arginine. To further our understanding of the impact of L-arginine deprivation in experimental leishmaniasis, here we characterize in detail the effects of L-arginine deprivation on antigen-specific T cells and MPhi. The results of our study show that decrease of L-arginine levels in the extracellular milieu affects the biological activities of Leishmania major-specific T cells, both at the level of the magnitude and the quality of their responses. L. major-specific CD4(+) T cells rendered hyporesponsive by L-arginine deprivation can be partially rescued by addition of exogenous L-arginine to produce IL-4 and IL-10, but not to produce IFN-gamma. Furthermore, our results show that L-arginine deprivation also greatly impacts parasite growth in activated macrophages. In summary, our results suggest that L-arginine levels affect both Th cell responses and parasite replication.

Age-related alteration of arginase activity impacts on severity of leishmaniasis.

BACKGROUND: The leishmaniases are a group of vector-borne parasitic diseases that represent a major international public health problem; they belong to the most neglected tropical diseases and have one of the highest rates of morbidity and mortality. The clinical outcome of infection with Leishmania parasites depends on a variety of factors such as parasite species, vector-derived products, genetics, behaviour, and nutrition. The age of the infected individuals also appears to be critical, as a significant proportion of clinical cases occur in children; this age-related higher prevalence of disease is most remarkable in visceral leishmaniasis. The mechanisms resulting in this higher incidence of clinical disease in children are poorly understood. We have recently revealed that sustained arginase activity promotes uncontrolled parasite growth and pathology in vivo. Here, we tested the hypothesis that arginase-mediated L-arginine metabolism differs with age. METHODOLOGY: The age distribution of patients with visceral or cutaneous leishmaniasis was determined in cohorts of patients in our clinics in endemic areas in Ethiopia. To exclude factors that are difficult to control in patients, we assessed the impact of ageing on the manifestations of experimental leishmaniasis. We determined parasite burden, T cell responses, and macrophage effector functions in young and aged mice during the course of infection. RESULTS: Our results show that younger mice develop exacerbated lesion pathology and higher parasite burdens than aged mice. This aggravated disease development in younger individuals does not correlate with a change in T helper cytokine profile. To address the underlying mechanisms responsible for the more severe infections in younger mice, we investigated macrophage effector functions. Our results show that macrophages from younger mice do not have an impaired capacity to kill parasites; however, they express significantly higher levels of arginase 1 than aged mice and promote parasite growth more efficiently. Thus, our results demonstrate that ageing differentially impacts on L-arginine metabolism and subsequent effector functions of physiologically distinct macrophage subsets. CONCLUSIONS: Here, we show that arginase-mediated L-arginine metabolism is modulated with age and affects the capacity of macrophages to express arginase; the increased capacity to upregulate this enzyme in younger individuals results in a more permissive environment for parasite growth, increased disease severity and pathology. These results suggest that the difference in arginase-mediated L-arginine catabolism is likely to be an important factor contributing to the increased incidence of clinical cases in children. Thus, targeting L-arginine metabolism might be a promising therapeutic strategy against leishmaniasis, especially in children and young adults.

Arginase activity mediates reversible T cell hyporesponsiveness in human pregnancy.

Complex regulation of T cell functions during pregnancy is required to ensure materno-fetal tolerance. Here we reveal a novel pathway for the temporary suppression of maternal T cell responses in uncomplicated human pregnancies. Our results show that arginase activity is significantly increased in the peripheral blood of pregnant women and remarkably high arginase activities are expressed in term placentae. High enzymatic activity results in high turnover of its substrate L-arginine and concomitant reduction of this amino acid in the microenvironment. Amino acid deprivation is emerging as a regulatory pathway of lymphocyte responses and we assessed the consequences of this enhanced arginase activity on T cell responses. Arginase-mediated L-arginine depletion induces down-regulation of CD3 zeta, the main signalling chain of the TCR, and functional T cell hyporesponsiveness. Importantly, this arginase-mediated T cell suppression was reversible, as inhibition of arginase activity or addition of exogenous L-arginine restored CD3 zeta chain expression and T cell proliferation. Thus, L-arginine metabolism constitutes a novel physiological mechanism contributing to the temporary suppression of the maternal immune response during human pregnancy.