Recombinant food allergens

Allergenic (glyco)proteins are the elicitors of food allergies and can cause acute severe hypersensitivity reactions. Recombinant food allergens are available in standardised quantity and constant quality. Therefore, they offer new perspectives to overcome current difficulties in the diagnosis, treatment and investigation of food allergies. This review summarises the expression strategies and characteristics of more than 40 recombinant food allergens that have been produced until today. Their IgE-binding properties are compared to those of their natural counterparts, in addition their application as diagnostic tools, the generation of hypoallergenic recombinant isoforms and mutants for therapeutic purposes, the determination of epitopes and cross-reactive structures are described.     

Recombinant allergens in the US

Recombinant allergens may increase the safety and efficacy of allergen immunodiagnostics and immunotherapy, and may prove to be excellent tools for the standardization of existing, natural allergens. However, there are several biological and regulatory issues that need to be addressed as these products are moved from bench to bedside. This article discusses some of these issues.     

Antigen delivery systems for analysing host immune responses and for vaccine development.

Overall, the meeting was timely and worthwhile. It is evident that there is a need for comparative studies to establish a firm foundation for future work. This is particularly important since it appears that the responses observed are influenced by the particular strain used for attenuation, the means of attenuation, the antigen-presentation system employed and the animal most immunized. In this last regard, one must recognize that most work to date has been with inbred strains of mice which differ from the genetic diversity of most hosts that are the intended beneficiaries of the immunization strategies being developed.     

Major T-cell responses in multiple sclerosis

Evidence is emerging that the major T- and B-cell response in multiple sclerosis (MS) is directed to a region of myelin basic protein (MBP) between residues 84 and 103. In rodent models of MS, immunization to this component of MBP evokes experimental autoimmune encephalomyelitis (EAE). T cells found in EAE lesions show similarities in the VJ and VDJ regions of their alpha and beta chains with T cells in MS lesions, and with T cells that are specific for MBPp84-103 isolated from patients with MS. If this region of MBP is critical in the pathogenesis of MS, then therapy aimed at controlling the immune response to this immunodominant region of MBP may be beneficial in treating MS.     

Consequences of suboptimal priming are apparent for low-avidity T-cell responses

The emergence of the novel reassortant A(H1N1)-2009 influenza virus highlighted the threat to the global population posed by an influenza pandemic. Pre-existing CD8(+) T-cell immunity targeting conserved epitopes provides immune protection against newly emerging strains of influenza virus, when minimal antibody immunity exists. However, the occurrence of mutations within T-cell antigenic peptides that enable the virus to evade T-cell recognition constitutes a substantial issue for virus control and vaccine design. Recent evidence suggests that it might be feasible to elicit CD8(+) T-cell memory pools to common virus mutants by pre-emptive vaccination. However, there is a need for a greater understanding of CD8(+) T-cell immunity towards commonly emerging mutants. The present analysis focuses on novel and immunodominant, although of low pMHC-I avidity, CD8(+) T-cell responses directed at the mutant influenza D(b)NP(366) epitope, D(b)NPM6A, following different routes of infection. We used a C57BL/6J model of influenza to dissect the effectiveness of the natural intranasal (i.n.) versus intraperitoneal (i.p.) priming for generating functional CD8(+) T cells towards the D(b)NPM6A epitope. In contrast to comparable CD8(+) T-cell responses directed at the wild-type epitopes, D(b)NP(366) and D(b)PA(224), we found that the priming route greatly affected the numbers, cytokine profiles and TCR repertoire of the responding CD8(+) T cells directed at the D(b)NPM6A viral mutant. As the magnitude, polyfunctionality, and T-cell repertoire diversity are potential determinants of the protective efficacy of CD8(+) T-cell responses, our data have implications for the development of vaccines to combat virus mutants.     

Mouse ephrinB3 augments T-cell signaling and responses to T-cell receptor ligation

Ephrins (EFN) are cell-surface ligands of Ephs, the largest family of cell-surface receptor tyrosine kinases. The function of EFNs in the immune system has not been well studied, although some EFNs and Ephs are expressed at high levels on certain leukocytes. We report here that EFNB3 and its receptors (collectively called EFNB3Rs, as EFNB3 binds to multiple EphBs) were expressed in peripheral T cells and monocytes/macrophages, with T cells being the dominant EFNB3+ and EFNB3R+ cell type. Solid-phase EFNB3-Fc in the presence of suboptimal anti-CD3 crosslinking enhanced T-cell responses in terms of proliferation, activation marker expression, interferon-gamma but not interleukin-2 production, and cytotoxic T-cell activity. EFNB3R costimulation in the presence of phorbol 12-myristate 13- acetate was insensitive to cyclosporin A, similar to CD28 costimulation, suggesting they might share a part of the signaling pathway. After crosslinking, T-cell receptor and EFNB3R congregated into aggregated rafts, and this provided a morphological basis for signaling pathways of T-cell receptor and EFNB3R to interact. Solid-phase EFNB3-Fc augmented p38 and p44/42 MAPK activation further downstream of the signaling pathway. These data suggest that EFNB3 is important in T-cell/T-cell and T-cell/antigen-presenting cell collaboration to enhance T-cell activation and function.     

Antiviral memory T-cell responses in the lung

Recent studies have identified distinct populations of memory T cells that persist in the lungs following respiratory virus infections, and contribute to the control of secondary virus infections. Here we discuss the establishment, maintenance and recall of memory T cells in the lung.     

Suppression of T-cell responses by tumor metabolites

Tumor cells have developed multiple mechanisms to escape T-cell-mediated immune recognition. Recent work has revealed that the altered tumor metabolism depletes essential nutrients or leads to the accumulation of immunosuppressive metabolites in the tumor microenvironment. In this review, we discuss the suppressive activity of some metabolic key players, which are upregulated in human tumor cells, including indolamine-2,3-dioxygenase (IDO), arginase, inducible nitric oxide synthetase (iNOS), and lactate dehydrogenase (LDH)-A, on the adaptive immune system. A better understanding of the impact of metabolic alterations of tumor cells on effector T-cell functions could lead to new therapeutic strategies to improve the efficacy of cancer immunotherapy.     

A defined basal medium for study of in vitro T-cell responses

A basal serum-free medium has been devised which supports short-term murine T-cell proliferative and functional assays in the absence of added lipids or serum albumin. Immune responses obtained with this medium are totally dependent on cell-cell interactions known to generate the lymphokines and monokines essential for replication and differentiation. Background activity is minimal, allowing better control of the specificity of response than is possible with serum-containing media. This medium is also suitable for the identification of added agents which modulate immune responses, and may facilitate the purification of secreted factors.     

Hexadecylphosphocholine-mediated enhancement of T-cell responses to interleukin 2

The effect of low-dose hexadecylphosphocholine (He-PC) on normal peripheral mononuclear cells (PMNC) was studied. Interferon-gamma (IFN-g) production, interleukin 2 (IL-2) receptor, and HLA-DR antigen expression were investigated, representing typical T-cell activation parameters. In PMNC cultures, He-PC dose-dependently enhanced the production of IFN-g, provided IL-2 had been added exogenously. Without IL-2 He-PC was ineffective. In some cultures, at a concentration of 8 micrograms/ml He-PC stimulated the secretion of IFN-g more than 20-fold compared to untreated controls. Although He-PC by itself lacked mitogenic activity, this compound also stimulated IFN-g production in the presence of suboptimal doses of phytohemagglutinin (PHA). Immunofluorescence studies demonstrated that He-PC also increased IL-2 receptor and HLA-DR antigen expression under these experimental conditions. Taken together, these results indicate that He-PC may possess immunomodulatory activity also in vivo, acting as a costimulator for the IL-2-mediated T-cell activation process.     

T-cell responses at the host: tumour interface

The evidence considered here reinforces the conclusion that T-cell responses to tumours involve complex cellular interactions. An attempt to summarize some of these interactions is shown. This emphasizes that not only are the interactions between the effector cell populations complicated, but that the target cell surface is also subject to variation and modification as a result of the immune response. A feature that also emerges from these studies is that most cells apparently responding to or infiltrating a tumour do not necessarily participate in its destruction, and it is in this area that experimental tumour systems have particular value. This also perhaps explains the preoccupation of experimentalists with the identification of 'the' effector cell crucial to tumour rejection. However, there is heterogeneity between systems in terms of the type of rejection response induced, but a logical basis for this heterogeneity is not established. If experimental studies could define the nature of the immune response generated by a tumour in the context of the biological features of the tumour itself, this could lead to the prediction of the immunogenicity and potential for induction of a rejection response for that tumor. Clearly, experimental tumour systems do not provide an exact reflection of the situation with human tumours. However, they may provide systems that illuminate particular aspects of the human response, and give precedents to guide the interpretation of data derived from human systems. This form of assessment is still at an early stage, but developments in the experimental field should provide a framework for the development and exploitation of T-cell responses to tumours.     

Surfactant Protein-A inhibits Aspergillus fumigatus-induced allergic T-cell responses

Background

Recent studies suggest that HCV infection is associated with progressive declines in pulmonary function in patients with underlying pulmonary diseases such as asthma and chronic obstructive pulmonary disease. Few molecular studies have addressed the inflammatory aspects of HCV-associated pulmonary disease. Because IL-8 plays a fundamental role in reactive airway diseases, we examined IL-8 signaling in normal human lung fibroblasts (NHLF) in response to the HCV nucleocapsid core protein, a viral antigen shown to modulate intracellular signaling pathways involved in cell proliferation, apoptosis and inflammation.

Methods

NHLF were treated with HCV core protein and assayed for IL-8 expression, phosphorylation of the p38 MAPK pathway, and for the effect of p38 inhibition.

Results

Our studies demonstrate that soluble HCV core protein induces significant increases in both IL-8 mRNA and protein expression in a dose- and time-dependent manner. Treatment with HCV core led to phosphorylation of p38 MAPK, and expression of IL-8 was dependent upon p38 activation. Using TNFα as a co-stimulant, we observed additive increases in IL-8 expression. HCV core-mediated expression of IL-8 was inhibited by blocking gC1qR, a known receptor for soluble HCV core linked to MAPK signaling.

Conclusion

These studies suggest that HCV core protein can lead to enhanced p38- and gC1qR-dependent IL-8 expression. Such a pro-inflammatory role may contribute to the progressive deterioration in pulmonary function recently recognized in individuals chronically infected with HCV.     

Antigen-driven T-cell repertoire selection during adaptive immune responses

Protective immunity against a variety of infections depends on the amplification and differentiation of rare na?ve antigen-specific CD4 and CD8 T cells. Recent evidence indicates that the clonotypic composition of the responding T-cell compartment has a critical role in the immune defense against pathogens. The present review compares and contrasts how naive CD4 and CD8 T cells recognize their cognate antigen, and discusses the factors that regulate the genesis and maintenance of the CD4 and CD8 T-cell receptor repertoire diversity.     

Virus-specific proliferative T-cell responses: Parameters and specificity

The genetic requirements for inducing virus-specific T-cell proliferation were investigated by taking spleen cells from animals primed with vaccinia virus in vivo, then culturing the cells in vitro with vaccinia virus-infected syngeneic peritoneal macrophages, and finally restimulating these cells a second time in vitro with vaccinia virus-infected macrophages from several strains of mice. Under these conditions, T cells proliferated in the tertiary response to virus-specific stimulation, whereas background proliferation caused by allogeneic differences between stimulator and responder cells was minimal. Compatibility between T cells and infected stimulator cells at the K or I regions alone or at I-A or I-A + I-B regions of the major histocompatibility complex (MHC) produced strong proliferative responses, whereas compatibility at D alone often resulted in somewhat weaker responses. However, these responses were rarely as great as in combinations of completely syngeneic stimulator and responder cells. Homology between responding and virus-infected stimulating cells in more than one of the H-2K, D, or I regions resulted in an additive, but not potentiating, effect. Genes coded outside the H-2 region did not seem to play a role in this system. In some rare cases, a weak response occurred across allogeneic barriers, but in general, virus-specific T-cell proliferation was strongly H-2 restricted.     

Bone marrow as a priming site for T-cell responses to blood-borne antigen

Although bone marrow is known as a primary lymphoid organ, its potential to serve as a secondary immune organ has hardly been explored. Here we demonstrate that naive, antigen-specific T cells home to bone marrow, where they can be primed. Antigen presentation to T cells in bone marrow is mediated via resident CD11c+ dendritic cells. They are highly efficient in taking up exogenous blood-borne antigen and processing it via major histocompatibility complex class I and class II pathways. T-cell activation correlates with dendritic cell-T cell clustering in bone marrow stroma. Primary CD4+ and CD8+ T-cell responses generated in bone marrow occur in the absence of secondary lymphoid organs. The responses are not tolerogenic and result in generation of cytotoxic T cells, protective anti-tumor immunity and immunological memory. These findings highlight the uniqueness of bone marrow as an organ important for hemato- and lymphopoiesis and for systemic T cell-mediated immunity.     

Human T-cell responses to vaccinia virus envelope proteins

One approach for a safer smallpox vaccine is to utilize recombinant subunits rather than live vaccinia virus (VACV). The products of the VACV envelope genes A27L, L1R, B5R, and A33R induce protective antibodies in animal models. We propose that proteins that elicit T-cell responses, as well as neutralizing antibodies, will be important to include in a molecular vaccine. To evaluate VACV-specific memory T-cell responses, peripheral blood mononuclear cells (PBMC) from four VACV vaccinees were tested against whole VACV and the individual envelope proteins A27, B5, L1, and A33, using gamma interferon enzyme-linked immunospot and cytokine flow cytometry assays. PBMC were stimulated with autologous dendritic cells infected with VACV or electroporated with individual VACV protein mRNAs. T-cell lines from all donors, vaccinated from 1 month to over 20 years ago, recognized all four VACV envelope proteins. Both CD4(+) and CD8(+) T-cell responses to each protein were detected. Further analysis focused on representative proteins B5 and A27. PBMC from a recent vaccinee exhibited high frequencies of CD4(+) and CD8(+) T-cell precursors to both B5 (19.8 and 20%, respectively) and A27 (6.8 and 3.7%). In comparison, B5- and A27-specific T-cell frequencies ranged from 0.4 to 1.3% in a donor vaccinated 3 years ago. Multiple CD4(+) and CD8(+) T-cell epitopes were identified from both A27 and B5, using overlapping 15-mer peptides. These data suggest that all four VACV envelope proteins may contribute to protective immunity, not only by inducing antibody responses, but also by eliciting T-cell responses.