Induction of bystander tolerance and immune deviation after Fel d 1 peptide immunotherapy.

BACKGROUND: Treatment of patients with cat allergy with peptides derived from Fel d 1 (the major cat allergen) ameliorated symptoms of cat allergy in phase 2 clinical trials. OBJECTIVE: We sought to demonstrate that the tolerance induced by Fel d 1 peptide immunotherapy can be exploited to reduce allergic responses to a second allergen, ovalbumin (OVA), in mice sensitized dually to OVA and Fel d 1. METHODS: Induction of tolerance to OVA was achieved through simultaneous exposure to both allergens after peptide treatment. Functional tolerance to each allergen was assessed in a model of allergic airways disease in which treated mice were protected from eosinophilia, goblet cell hyperplasia, and TH2 cell infiltration. RESULTS: Suppression of allergic responses to cat allergen challenge was associated with significant increases in numbers of CD4+CD25+Foxp3+ T cells, IL-10+ cells, and CD19+IL-10+ B cells, whereas the response to OVA was associated with a marked reduction in numbers of TH2 cytokine-secreting T cells and less prominent changes in outcomes associated with immune regulation. CONCLUSIONS: These observations suggest that immune tolerance induced by peptide immunotherapy can be used experimentally to treat an allergic response to another allergen and that the molecular mechanisms underlying induction of tolerance to a treatment-specific allergen and a bystander allergen might be different.

Modulation of CRTh2 expression on allergen-specific T cells following peptide immunotherapy.

BACKGROUND: Allergen immunotherapy using synthetic peptide T-cell epitopes (Cat-PAD) from the major cat allergen Fel d 1 has been shown, in allergen exposure studies, to significantly reduce symptoms of allergic rhinoconjunctivitis in cat-allergic subjects. However, the immunological mechanisms underlying clinical benefit remain only partially understood. Since previous studies of whole allergen immunotherapy demonstrated a reduction in the frequency of allergen-specific (MHC II tetramer+ ) CD4+ T cells expressing the chemokine receptor CRTh2, we assessed the impact of Cat-PAD on the frequency and functional phenotype of Fel d 1-specific CD4+ T cells. METHODS: Using before and after treatment samples from subjects enrolled in a randomized, double-blind, placebo-controlled trial of Cat-PAD, we employed Fel d 1 MHC II tetramers and flow cytometry to analyze the expression of chemokine receptors CCR3, CCR4, CCR5, CXCR3, and CRTh2, together with markers of memory phenotype (CD27 and CCR7) on Fel d 1-specific CD4+ T cells. RESULTS: No statistically significant change in the frequency of Fel d 1-specific CD4+ T cells, nor in their expression of chemokine receptors or memory phenotype, was observed. However, a significant reduction in cell surface expression of CRTh2 was observed between the placebo and active groups (P = 0.047). CONCLUSIONS: Peptide immunotherapy with Cat-PAD does not significantly alter the frequency or phenotype of Fel d 1-CD4+ T cells, but may decrease their expression of CRTh2.

The number of responding CD4 T cells and the dose of antigen conjointly determine the TH1/TH2 phenotype by modulating B7/CD28 interactions.

Our previous in vivo studies show that both the amount of Ag and the number of available naive CD4 T cells affect the Th1/Th2 phenotype of the effector CD4 T cells generated. We examined how the number of OVA-specific CD4 TCR transgenic T cells affects the Th1/Th2 phenotype of anti-SRBC CD4 T cells generated in vivo upon immunization with different amounts of OVA-SRBC. Our observations show that a greater number of Ag-dependent CD4 T cell interactions are required to generate Th2 than Th1 cells. We established an in vitro system that recapitulates our main in vivo findings to more readily analyze the underlying mechanism. The in vitro generation of Th2 cells depends, as in vivo, upon both the number of responding CD4 T cells and the amount of Ag. We demonstrate, using agonostic/antagonistic Abs to various costimulatory molecules or their receptors, that the greater number of CD4 T cell interactions, required to generate Th2 over Th1 cells, does not involve CD40, OX40, or ICOS costimulation, but does involve B7/CD28 interactions. A comparison of the level of expression of B7 molecules by APC and CD4 T cells, under different conditions resulting in the substantial generation of Th1 and Th2 cells, leads us to propose that the critical CD28/B7 interactions, required to generate Th2 cells, may directly occur between CD4 T cells engaged with the same B cell acting as an APC.

Direct demonstration of CD4 T cell cooperation in the primary in vivo generation of CD4 effector T cells.

Many observations bear upon the cellular and molecular requirements for CD4 T cell activation. The interaction of CD4 T cells with dendritic cells (DC), central to the induction of most immune responses, is the most studied. However, leukocytes other than DC can dramatically affect the induction and differentiation of CD4 T cells into effector cells. We recently provided indirect evidence that in vivo CD4 T cooperation facilitates the activation of CD4 T cells. Here, we demonstrate that the activation of CD4 T cells, specific for the hen egg lysozyme (HEL)(105) (-120) peptide, is optimally achieved when BALB/c mice are immunized with additional MHC class II-binding HEL peptides in incomplete Freund's adjuvant. This cooperation cannot be mimicked by the coadministration of LPS or of an agonistic antibody to CD40, at the time of immunization. In contrast, OX40-OX40L interactions are necessary for CD4 T cell cooperation in that an OX40 agonistic antibody can replace, and an OX40L-blocking antibody can abrogate, CD4 T cell cooperation in situations where such cooperation would otherwise enhance the activation of CD4 T cells.

The activation, by antigen, of naïve TCR transgenic CD4 T cells cultured at physiological, rather than artificially high, frequencies more accurately reflects the in vivo activation of normal numbers of naïve CD4(+) T cells.

The majority of in vitro studies investigating the activation of naïve TCR transgenic T cells routinely employ an artificially high frequency of such cells. To assess whether employing high frequencies of TCR transgenic cells in vitro accurately reflects the in vivo activation of a normal number of T cells, we cultured between 300 and 3×10(6) Rag2(-/-) DO11.10 T cells per well under otherwise identical conditions. We find that those T cells cultured at low frequencies proliferate more and are more potently activated, as assessed by the expression of CD44 and CD62L, each giving rise to a much larger number of cytokine producing cells, comparable to the number generated in vivo when a normal number of CD4(+) T cells are activated. The effect of T cell frequency on the level of their activation was not due to differences in MHCII or CD80/86 expression by B cells, the major APC population present, nor to increased death of B cells in high frequency cultures. Taken together, our observations illustrate the necessity of culturing naïve TCR transgenic CD4(+) T cells at a physiological frequency if one is to more accurately recapitulate the in vivo activation of naïve CD4(+) T cells.

Prospective Analysis of the Effects of Maternal Immune Activation on Rat Cytokines during Pregnancy and Behavior of the Male Offspring Relevant to Schizophrenia.

Influenza during pregnancy is associated with the development of psychopathology in the offspring. We sought to determine whether maternal cytokines produced following administration of viral mimetic polyinosinic-polycytidylic acid (polyI:C) to pregnant rats were predictive of behavioral abnormalities in the adult offspring. Timed-pregnant Sprague Dawley rats received a single intravenous injection of 4-mg/kg polyI:C or saline on gestational day (GD)15. Blood was collected 3 h later for serum analysis of cytokine levels with ELISA. Male offspring were tested in a battery of behavioral tests during adulthood and behavior was correlated with maternal cytokine levels. Maternal serum levels of CXCL1 and interleukin (IL)-6, but not tumor necrosis factor (TNF)-α or CXCL2, were elevated in polyI:C-treated dams. PolyI:C-treated dams experienced post-treatment weight loss and polyI:C pups were smaller than controls at postnatal day (PND)1. Various behavior alterations were seen in the polyI:C-treated offspring. Male polyI:C offspring had enhanced MK-801-induced locomotion, and reduced sociability. PolyI:C offspring failed to display crossmodal and visual memory, and oddity preference was also impaired. Set-shifting, assessed with a lever-based operant conditioning task, was facilitated while touchscreen-based reversal learning was impaired. Correlations were found between maternal serum concentrations of CXCL1, acute maternal temperature and body weight changes, neonatal pup mass, and odd object discrimination and social behavior. Overall, while the offspring of polyI:C-treated rats displayed behavior abnormalities, maternal serum cytokines were not related to the long-term behavior changes in the offspring. Maternal sickness effects and neonatal pup size may be better indicators of later effects of maternal inflammation in the offspring.

An Update on Lymphocyte Subtypes in Asthma and Airway Disease.

Inflammation is a hallmark of many airway diseases. Improved understanding of the cellular and molecular mechanisms of airway disease will facilitate the transition in our understanding from phenotypes to endotypes, thereby improving our ability to target treatments based on pathophysiologic characteristics. For example, allergic asthma has long been considered to be driven by an allergen-specific T helper 2 response. However, clinical and mechanistic studies have begun to shed light on the role of other cell subsets in the pathogenesis and regulation of lung inflammation. In this review, we discuss the importance of different lymphocyte subsets to asthma and other airway diseases, while highlighting the growing evidence that asthma is a syndrome that incorporates many immune phenotypes.

Antigen presenting B cells facilitate CD4 T cell cooperation resulting in enhanced generation of effector and memory CD4 T cells.

We show that the in vivo generation of cytokine-producing CD4 T cells specific for a given major histocompatibility class-II (MHCII)-binding peptide of hen egg lysozyme (HEL) is facilitated when mice are immunized with splenic antigen presenting cells (APC) pulsed with this HEL peptide and another peptide that binds a different MHCII molecule. This enhanced generation of peptide-specific effector CD4 T cells requires that the same splenic APC be pulsed with both peptides. Pulsed B cells, but not pulsed dendritic cells (DCs), can mediate CD4 T cell cooperation, which can be blocked by disrupting OX40-OX40L (CD134-CD252) interactions. In addition, the generation of HEL peptide-specific CD4 T cell memory is greater when mice are primed with B cells pulsed with the two peptides than with B cells pulsed with the HEL- peptide alone. Based on our findings, we suggest CD4 T cell cooperation is important for vaccine design, underlies the phenomenon of "epitope-spreading" seen in autoimmunity, and that the efficacy of B cell-depletion in the treatment of human cell-mediated autoimmune disease is due to the abrogation of the interactions between autoimmune CD4 T cells that facilitates their activation.