Photochemically-Mediated Inflammation and Cross-Presentation of Mycobacterium bovis BCG Proteins Stimulates Strong CD4 and CD8 T-Cell Responses in Mice.

Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as Mycobacterium tuberculosis. The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose, Mycobacterium bovis BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated in vitro for analysis of their activation of T cells in vitro or in vivo after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1ß in vivo. Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of M. bovis BCG.

Sensitivity and specificity of T-cell receptor PCR BIOMED-2 clonality analysis for the diagnosis of cutaneous T-cell lymphoma.

BACKGROUND: Early and precise diagnosis of cutaneous T-cell lymphomas (CTCL) is challenging. Currently, polymerase chain reaction (PCR)-based clonality assessment of the T-cell receptor (TCR) is a helpful tool for this diagnosis. OBJECTIVES: In this retrospective study, we aimed to assess the sensitivity and specificity of this method for the diagnosis of CTCL. MATERIALS AND METHODS: Monoclonal rearrangement of the TCR was investigated retrospectively by PCR-based clonality assessment based on 292 DNA samples from skin biopsies of patients with a suspicion of CTCL. Algorithms were based on different ratios (three or five-fold difference) between the dominant PCR peak and the third highest PCR peak. RESULTS: A PCR peak five-fold higher than the third highest PCR peak demonstrated significantly greater specificity (83.7% versus 76.4%) but lower sensitivity (59.8% versus 68.6%) compared to a cut-off of three-fold higher. CONCLUSION: Our results confirm the diagnostic value of TCR clonality analysis which may be used to define the ideal cut-off in order to optimize sensitivity and specificity.

Microcrystalline Tyrosine and Aluminum as Adjuvants in Allergen-Specific Immunotherapy Protect from IgE-Mediated Reactivity in Mouse Models and Act Independently of Inflammasome and TLR Signaling.

Allergen immunotherapy (AIT) is the only modality that can modify immune responses to allergen exposure, but therapeutic coverage is low. One strategy to improve AIT safety and efficacy is the use of new or improved adjuvants. This study investigates immune responses produced by microcrystalline tyrosine (MCT)-based vaccines as compared with conventional aluminum hydroxide (alum). Wild-type, immune-signaling-deficient, and TCR-transgenic mice were treated with different Ags (e.g., OVA and cat dander Fel d 1), plus MCT or alum as depot adjuvants. Specific Ab responses in serum were measured by ELISA, whereas cytokine secretion was measured both in culture supernatants by ELISA or by flow cytometry of spleen cells. Upon initiation of AIT in allergic mice, body temperature and further clinical signs were used as indicators for anaphylaxis. Overall, MCT and alum induced comparable B and T cell responses, which were independent of TLR signaling. Alum induced stronger IgE and IL-4 secretion than MCT. MCT and alum induced caspase-dependent IL-1ß secretion in human monocytes in vitro, but inflammasome activation had no functional effect on inflammatory and Ab responses measured in vivo. In sensitized mice, AIT with MCT-adjuvanted allergens caused fewer anaphylactic reactions compared with alum-adjuvanted allergens. As depot adjuvants, MCT and alum are comparably effective in strength and mechanism of Ag-specific IgG induction and induction of T cell responses. The biocompatible and biodegradable MCT seems therefore a suitable alternative adjuvant to alum-based vaccines and AIT.