Regulatory T cells control type I food allergy to Beta-lactoglobulin in mice.

BACKGROUND: Regulatory T cells contribute to peripheral immune tolerance, yet their ability to control immediate-type hypersensitivity (ITH) reactions involved in IgE-mediated food allergy is still poorly documented. OBJECTIVES: We investigated in mice whether CD4+CD25+ regulatory T cells could control ITH to ß-lactoglobulin (BLG), a major allergen in cow's milk. METHODS: C3H/HeOuJ mice were sensitized by repeated oral gavage with BLG plus cholera toxin as adjuvant and orally challenged with BLG alone to elicit allergic symptoms. Mice were treated with the anti-CD25 mAb (PC61) before sensitization. Oral sensitization (afferent phase of ITH) was assessed by production of BLG-specific serum antibodies and Th1/Th2-type cytokines by specific CD4+ T cells in mesenteric lymph nodes. ITH was elicited by oral BLG challenge (efferent phase of ITH) and we monitored symptom scores, numbers and function of intestinal mast cells and serum level of the mucosal mast cell protease mMCP-1. RESULTS: Upon oral BLG challenge, orally sensitized mice developed only mild clinical signs. Anti-CD25 mAb-treated mice exhibited enhancement of both BLG-specific CD4+ T cell priming with IL-4, IL-5, IL-13 and IFN-γ production and total IgE, and BLG-specific IgE, IgG1 and IgG2a in serum. Anti-CD25 mAb treatment caused more severe symptoms upon BLG challenge, which correlated with enhanced serum levels of the mucosal mast cell protease mMCP-1. CONCLUSIONS: These data document that constitutive CD4+CD25+ regulatory T cells alleviate clinical signs of ITH to dietary BLG by modulating the priming of BLG-specific T and B cell responses during oral sensitization and enhancing mast cell degranulation.

Reversal of tumor-induced dendritic cell paralysis by CpG immunostimulatory oligonucleotide and anti-interleukin 10 receptor antibody.

Progressing tumors in man and mouse are often infiltrated by dendritic cells (DCs). Deficient antitumor immunity could be related to a lack of tumor-associated antigen (TAA) presentation by tumor-infiltrating DCs (TIDCs) or to a functional defect of TIDCs. Here we investigated the phenotype and function of TIDCs in transplantable and transgenic mouse tumor models. Although TIDCs could encompass various known DC subsets, most had an immature phenotype. We observed that TIDCs were able to present TAA in the context of major histocompatibility complex class I but that they were refractory to stimulation with the combination of lipopolysaccharide, interferon gamma, and anti-CD40 antibody. We could revert TIDC paralysis, however, by in vitro or in vivo stimulation with the combination of a CpG immunostimulatory sequence and an anti-interleukin 10 receptor (IL-10R) antibody. CpG or anti-IL-10R alone were inactive in TIDCs, whereas CpG triggered activation in normal DCs. In particular, CpG plus anti-IL-10R enhanced the TAA-specific immune response and triggered de novo IL-12 production. Subsequently, CpG plus anti-IL-10R treatment showed robust antitumor therapeutic activity exceeding by far that of CpG alone, and elicited antitumor immune memory.

Distinct and overlapping roles of interleukin-10 and CD25+ regulatory T cells in the inhibition of antitumor CD8 T-cell responses.

Lack of antitumor immunity is often related to impaired CD8 T-cell responses that could result from a poor priming capacity by tumor-infiltrating dendritic cells (TIDC) and/or further inhibition by regulatory T cells (T(reg)). Interleukin-10 (IL-10) has been implicated in the inhibition of TIDC as well as in the generation and functions of T(reg). Here, we address some of the respective and possibly overlapping roles of IL-10 and CD25+ T(reg) in CD8 antitumor immunity. Whereas tumor antigen-specific CD8 T cells proliferated in vivo in the presence of IL-10 or T(reg), optimal effector functions were observed in mice lacking both IL-10 and T(reg). Indeed, tumors grown in normal but not in IL-10-deficient or CD25-depleted mice induced tumor antigen-specific CD8 suppressor T cells. Suppression involved transforming growth factor-beta. Similarly, both IL-10 and T(reg) were responsible for impaired CD8 T cell priming by TIDCs, but IL-12 production by TIDCs was prevented only by T(reg)-independent IL-10. Subsequently, IL-10 defect and T(reg) depletion were required to achieve optimal induction of CD8 T-cell effectors by TIDC following CpG activation. Our results point out major redundant and nonredundant roles for IL-10 and T(reg) in the inhibition of TIDC-mediated generation of antitumor CD8 T-cell response.

In vivo manipulation of dendritic cell migration and activation to elicit antitumour immunity.

Two approaches have been pursued to elicit antitumour immunity: (i) induce recruitment of immature dendritic cells or their precursors at a site of antigen delivery, and (ii) induce activation of tumour-infiltrating dendritic cells (DCs). The recruitment of selected DC subtype conditions the class of the immune response. Each immature DC population displays a unique spectrum of chemokine responsiveness. For examples, Langerhans cells (LCs) migrate selectively in response to CCL20/MIP-3alpha (through CCR6), blood CD11c+ DC to MCP chemokines (through CCR2). All these chemokines are inducible in response to inflammatory stimuli. CCL20/MIP-3alpha in particular is only detected within inflamed epithelium, at the site of antigen entry, which is infiltrated by immature DCs. Furthermore, to reach the site of injury, sequential responsiveness might operate, blood DC precursors are recruited by a set of chemokines (MIP, MCP) while within the tissue other chemokines will direct their navigation (CCL20/MIP-3alpha). Of interest, when injected in vivo together with antigen, MCP-4/CCL13, but not CCL20/MIP-3alpha, recruits blood monocytes or blood DC precursors that promptly differentiate into typical DCs and that improve antitumour immune responses. After antigen uptake, DCs acquire, upon maturation, responsiveness to CCR7 ligands (CCL21/SLC/6Ckine, CCL19/ELC/MIP-3beta) due to receptor up-regulation. In particular, in the periphery, CCL21/SLC/6Ckine expressed by lymphatic vessels may direct into the lymph stream, antigen-loaded maturing DCs leaving the site of infection; while within lymph-node, CCL21/SLC/6Ckine plays a critical role in the entry of naïve T cells from the blood through HEV. In regard to its central role, we decided to investigate whether the expression of CCL21/SLC/6Ckine in tumour may lead to antitumour immune responses. C26 colon carcinoma tumour cell line transduced with CCL21/SLC/6Ckine showed reduced tumorigenicity when injected in vivo into immunocompetent mice. The protection was CD8 dependent and associated with an important intratumoral infiltration of DCs. Most tumour infiltrating DCs (TIDCs) had an immature phenotype, were able to present TAA in the context of MHC class I, but were refractory to stimulation with the combination of LPS, IFNgamma and anti-CD40 antibody. TIDC paralysis could be reverted, however, by in vitro or in vivo stimulation with the combination of a CpG immunostimulatory sequence and an anti-interleukin 10 receptor (IL10R) antibody. CpG or anti-IL10R alone were inactive in TIDC, while CpG triggered activation in normal DC. In particular, CpG plus anti-IL10R enhanced the TAA-specific immune response and triggered de novo IL-12 production. Subsequently, CpG plus anti-IL10R treatment showed robust antitumour therapeutic activity exceeding by far that of CpG alone, and elicited antitumour immune memory.

Depletion of human NK and CD8 cells prior to in vitro H1N1 flu vaccine stimulation increases the number of gamma interferon-secreting cells compared to the initial undepleted population in an ELISPOT assay.

In order to study the respective roles of CD4, CD8, and CD56 (NK) cells in gamma interferon (IFN-gamma) production after in vitro stimulation with flu vaccine in a healthy adult human population, we depleted these cellular subtypes before stimulation with antigen (inactivated split vaccine, A/Texas H1N1, or A/Sydney H3N2). We observed that while CD4 cells were required for IFN-gamma secretion in both conditions in vitro, CD56 (NK) cells and, to a lesser extent, CD8 cells had a negative effect on such synthesis upon H1N1 stimulation, as judged by an increased number of spots compared to the initial undepleted population. This regulation of IFN-gamma secretion was associated with an increase in ICAM-1 expression, in particular on T and B cells. This study points out the importance of evaluating in vitro immune responses on a whole-cell population in addition to isolated subtypes if one needs to address potential cellular interactions occurring in vivo in some situations (H1N1 stimulation in the present case). Such cross-regulations occur even in vitro during the antigenic stimulation step.