A double-blind, placebo-controlled study of immunotherapy was conducted in 19 patients with grass-pollen hay fever to evaluate the efficacy and safety of a formalinized depot grass allergoid. The patients were assessed before and during IT by clinical (symptom-medication scores during the grass- pollen season, specific nasal and skin reactivity) and immunological (specific IgE, IgG, IgG1 and IgG4 antibodies) parameters. High doses of grass allergoid, corresponding to a cumulative pre-seasonal dosage of 46,050 PNU, were administered, with only one systemic reaction. The actively treated patients had significantly lower symptom-medication scores than placebo (p less than 0.01) during the month of May and showed a significant decrease in specific skin (p less than 0.01) and nasal (p less than 0.05) reactivity, and a significant early increase in specific IgE (p less than 0.01), IgG (p less than 0.0005), IgG1 (p less than 0.001) and IgG4 (p less than 0.05), with a subsequent decrease of IgE and IgG1. No differences were detected in any of these parameters in the placebo group. A correlation was found between high IgG4/IgG1 ratio and the specific skin reactivity decrease (r = 0.691, p less than 0.05), whereas a high IgG4/IgG1 ratio was associated with higher symptom-medication scores (r = 0.654, p less than 0.05). Possible explanations of these apparent discrepancies are proposed.

Initially thought as being non-immunogenic, recombinant AAVs have emerged as efficient vector candidates for treating monogenic diseases. It is now clear however that they induce potent immune responses against transgene products which can lead to destruction of transduced cells. Therefore, developing strategies to circumvent these immune responses and facilitate long-term expression of transgenic therapeutic proteins is a main challenge in gene therapy. We evaluated herein a strategy to inhibit the undesirable immune activation that follows muscle gene transfer by administration of CTLA-4/Ig to block the costimulatory signals required early during immune priming and by using gene transfer of PD-1 ligands to inhibit T cell functions at the tissue sites. We provide the proof of principle that this combination immunoregulatory therapy targeting two non-redundant checkpoints of the immune response, i.e., priming and effector functions, can improve persistence of transduced cells in experimental settings where cytotoxic T cells escape initial blockade. Therefore, CTLA-4/Ig plus PD-L1/2 combination therapy represents a candidate approach to circumvent the bottleneck of immune responses directed toward transgene products.

Objective. To investigate the influence of myoinjury on antigen presentation to T cells in draining lymph nodes (LNs).Methods. Muscle crush was performed in mice injected with exogenous ovalbumin (OVA) and in transgenic SM-OVA mice expressing OVA as a musclespecific self antigen. Antigen exposure and the resulting stimulation of T cell proliferation in draining LNs was assessed by transferring carboxyfluorescein succinimidyl ester (CFSE)-labeled OVA-specific CD8؉ and CD4؉ T cells from OT-I and OT-II mice and by measuring the dilution of CFSE, which directly reflects their proliferation. The role of monocyte-derived dendritic cells (DCs) in T cell priming was assessed using pharmacologic blockade of DC migration. Immunofluorescence was used to detect CD8؉ T cells, inflammatory monocyte-derived DCs, and type I major histocompatibility complex (MHC)-expressing myofibers in crushed muscle, and to assess expression of perforin, interferon-␥ (IFN␥), interleukin-2 (IL-2), IL-10, and transforming growth factor ␤1 (TGF␤1). Idiopathic inflammatory myopathies (IIMs) are autoimmune diseases with distinct histopathologic features that suggest either humorally mediated processes, primarily targeting the microcirculation (in dermatomyositis) and myofibers (in autoimmune necrotizing myopathies), or CD8ϩ T cell-mediated and class I major histocompatibility complex (MHC)-restricted autoimmune attack of myofibers (in polymyositis and inclusion body myositis) (1). Pathophysiologic studies have mainly explored how muscle cells can participate in immune cell interactions in polymyositis (2). In this setting, myofibers strongly express class I MHC molecules at their surface (1) and are invaded by autoinvasive T cells (3) expressing perforin (4). Clonal expansions of T cells are found in muscle and blood (5-8), and autoinvasive T cells exhibit selective gene rearrangement of their T cell receptor (TCR) with restricted

Muscle potentially represents the most abundant source of autoantigens of the body and can be targeted by a variety of severe autoimmune diseases. Yet, the mechanisms of immunological tolerance toward muscle autoantigens remain mostly unknown. We investigated this issue in transgenic SM-Ova mice that express an ovalbumin (Ova) neo-autoantigen specifically in skeletal muscle. We previously reported that antigen specific CD4+ T cell are immunologically ignorant to endogenous Ova in this model but can be stimulated upon immunization. In contrast, Ova-specific CD8+ T cells were suspected to be either unresponsive to Ova challenge or functionally defective. We now extend our investigations on the mechanisms governing CD8+ tolerance in SM-Ova mice. We show herein that Ova-specific CD8+ T cells are not detected upon challenge with strongly immunogenic Ova vaccines even after depletion of regulatory T cells. Ova-specific CD8+ T cells from OT-I mice adoptively transferred to SM-Ova mice started to proliferate in vivo, acquired CD69 and PD-1 but subsequently down-regulated Bcl-2 and disappeared from the periphery, suggesting a mechanism of peripheral deletion. Peripheral deletion of endogenous Ova-specific cells was formally demonstrated in chimeric SM-Ova mice engrafted with bone marrow cells containing T cell precursors from OT-I TCR-transgenic mice. Thus, the present findings demonstrate that immunological tolerance to muscle autoantigens involves peripheral deletion of autoreactive CD8+ T cells.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous B cell neoplasm ranging from indolent to rapidly progressive disease. Leukemic cell subsets with regulatory properties evade immune clearance; however, the contribution of such subsets during CLL progression is not completely elucidated. Here, we report that CLL B cells crosstalk with their immune counterparts, notably by promoting the regulatory T (Treg) cell compartment and shaping several helper T (Th) subsets. Among various constitutively- and BCR/CD40-mediated factors secreted, tumour subsets co-express two important immunoregulatory cytokines, IL10 and TGFβ1, both associated with a memory B cell phenotype. Neutralizing secreted IL10 or inhibiting the TGFβ signalling pathway demonstrated that these cytokines are mainly involved in Th- and Treg differentiation/maintenance. In line with the regulatory subsets, we also demonstrated that a CLL B cell population expresses FOXP3, a marker of regulatory T cells. Analysis of IL10, TGFβ1 and FOXP3 positive subpopulations frequencies in CLL samples discriminated 2 clusters of untreated CLL patients that were significantly different in Tregs frequency and time-to-treatment. Since this distinction was pertinent to disease progression, the regulatory profiling provides a new rationale for patient stratification and sheds light on immune dysfunction in CLL.

Zur Untersuchung der Siliziumversorgung und des Einflusses Silizium‐haltiger Dünger auf die Mineralstoffzusammensetzung von Kulturpflanzen wurden in Schleswig‐Holstein Felderhebungsuntersuchungen und Feldversuche zu Getreide und Raps durchgeführt. Getreidepflanzen wiesen im Gegensatz zu Rapspflanzen eine deutliche Differenzierung der Silizium‐Gehalte nach den Naturräumen des Landes auf. Zu Schoßbeginn wurden auf den Podsolen der Geest in Getreidepflanzen durchschnittlich 2,5 mg Si/g TS, auf den Braunerden des östlichen Hügellandes 5,9 mg Si/g TS und auf den Marschböden 11,5 mg Si/g in der Trockensubstanz festgestellt. Die Silizium‐Gehalte von Wintergetreide lagen dabei infolge des bei gleichem Entwicklungsstadium höheren Alters der Pflanzen um 50% höher als von Sommergetreide. Die Ursachen der regionalen Differenzierung scheinen im wesentlichen in den niedrigen verfügbaren Silizium‐Gehalten der Podsole einerseits und in der vom mobilen Eisen abhängigen Kieselsäureadsorption auf Braunerden und Marschen andererseits zu liegen. Eine direkte kausale Beziehung zwischen pH‐Wert der Böden und Silizium‐Versorgung der Pflanzen war demgegenüber nicht festzustellen. Silizium‐haltige Dünger vermochten nur bei Anwendung über den Böden, nicht jedoch bei Blattapplikation die Silizium‐Gehalte der Pflanzen zu erhöhen. Reine amorphe Kieselsäure erwies sich dabei als effektiver als basische Silikate des Hüttenkalkes. Unabhängig davon, ob reine Kieselsäure oder Hüttenkalk verwendet wurden, konnten keine Effekte der Düngung auf die Nährstoffgehalte der Pflanzen und den Kornertrag festgestellt werden.

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