TCR ITAM multiplicity is required for the generation of follicular helper T-cells.

The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3ζ subunits. We generated 'knock-in' mice that express non-signalling CD3ζ chains in lieu of wild-type CD3ζ. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR-ligand interactions, but is not essential for 'general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire.

Reduced TCR signaling potential impairs negative selection but does not result in autoimmune disease.

Negative selection and regulatory T (T reg) cell development are two thymus-dependent processes necessary for the enforcement of self-tolerance, and both require high-affinity interactions between the T cell receptor (TCR) and self-ligands. However, it remains unclear if they are similarly impacted by alterations in TCR signaling potential. We generated a knock-in allele (6F) of the TCR ζ chain gene encoding a mutant protein lacking signaling capability whose expression is controlled by endogenous ζ regulatory sequences. Although negative selection was defective in 6F/6F mice, leading to the survival of autoreactive T cells, 6F/6F mice did not develop autoimmune disease. We found that 6F/6F mice generated increased numbers of thymus-derived T reg cells. We show that attenuation of TCR signaling potential selectively impacts downstream signaling responses and that this differential effect favors Foxp3 expression and T reg cell lineage commitment. These results identify a potential compensatory pathway for the enforcement of immune tolerance in response to defective negative selection caused by reduced TCR signaling capability.

Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice.

The human epithelial mucin MUC1 is a heavily glycosylated transmembrane protein that is overexpressed and aberrantly glycosylated on over 90% of human breast cancers. The altered glycosylation of MUC1 reveals an immunodominant peptide along its tandem repeat (TR) that has been used as a target for tumour immunotherapy. In this study, we used the MUC1 TR peptide as a test antigen to determine whether a plant-expressed human tumour-associated antigen can be successfully expressed in a plant system and whether it will be able to break self-antigen tolerance in a MUC1-tolerant mouse model. We report the expression of MUC1 TR peptide fused to the mucosal-targeting Escherichia coli enterotoxin B subunit (LTB-MUC1) in a plant host. Utilizing a rapid viral replicon transient expression system, we obtained high yields of LTB-MUC1. Importantly, the LTB-MUC1 fusion protein displayed post-translational modifications that affected its antigenicity. Glycan analysis revealed that LTB-MUC1 was glycosylated and a MUC1-specific monoclonal antibody detected only the glycosylated forms. A thorough saccharide analysis revealed that the glycans are tri-arabinans linked to hydroxyprolines within the MUC1 tandem repeat sequence. We immunized MUC1-tolerant mice (MUC1.Tg) with transiently expressed LTB-MUC1, and observed production of anti-MUC1 serum antibodies, indicating breach of tolerance. The results indicate that a plant-derived human tumour-associated antigen is equivalent to the human antigen in the context of immune recognition.

Analysis of a cholera toxin B subunit (CTB) and human mucin 1 (MUC1) conjugate protein in a MUC1-tolerant mouse model.

Since epithelial mucin 1 (MUC1) is associated with several adenocarcinomas at the mucosal sites, it is pertinent to test the efficacy of a mucosally targeted vaccine formulation. The B subunit of the Vibrio cholerae cholera toxin (CTB) has great potential to act as a mucosal carrier for subunit vaccines. In the present study we evaluated whether a MUC1 tandem repeat (TR) peptide chemically linked to CTB would break self-antigen tolerance in the transgenic MUC1-tolerant mouse model (MUC1.Tg) through oral or parenteral immunizations. We report that oral immunization with the CTB-MUC1 conjugate along with mucosal adjuvant, unmethylated CpG oligodeoxynucleotide (ODN) and interleukin-12 (IL-12) did not break self-antigen tolerance in MUC1.Tg mice, but induced a strong humoral response in wild-type C57BL/6 mice. However, self-antigen tolerance in the MUC1.Tg mouse model was broken after parenteral immunizations with different doses of the CTB-MUC1 conjugate protein and with the adjuvant CpG ODN co-delivered with CTB-MUC1. Importantly, mice immunized systemically with CpG ODN alone and with CTB-MUC1 exhibited decreased tumor burden when challenged with a mammary gland tumor cell line that expresses human MUC1.

Themis, a T cell-specific protein important for late thymocyte development.

During positive selection, thymocytes transition through a stage during which T cell antigen receptor (TCR) signaling controls CD4-versus-CD8 lineage 'choice' and subsequent maturation. Here we describe a previously unknown T cell-specific protein, Themis, that serves a distinct function during this stage. In Themis(-/-) mice, thymocyte selection was impaired and the number of transitional CD4(+)CD8(int) thymocytes as well as CD4(+) or CD8(+) single-positive thymocytes was lower. Notably, although we detected no overt TCR-proximal signaling deficiencies, Themis(-/-) CD4(+)CD8(int) thymocytes showed developmental defects consistent with attenuated signaling that were reversible by TCR stimulation. Our results identify Themis as a critical component of the T cell developmental program and suggest that Themis functions to sustain and/or integrate signals required for proper lineage commitment and maturation.

Plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice.

Yersinia pestis, the causative agent of plague, is an extremely virulent bacterium but there are no approved vaccines for protection against it. Our goal was to produce a vaccine that would address: ease of delivery, mucosal efficacy, safety, rapid scalability, and cost. We developed a novel production and delivery system for a plague vaccine of a Y. pestis F1-V antigen fusion protein expressed in tomato. Immunogenicity of the F1-V transgenic tomatoes was confirmed in mice that were primed subcutaneously with bacterially-produced F1-V and boosted orally with transgenic tomato fruit. Expression of the plague antigens in fruit allowed producing an oral vaccine candidate without protein purification and with minimal processing technology.

Application of Quillaja saponaria extracts as oral adjuvants for plant-made vaccines.

Extracts from the Quillaja saponaria tree are known to provide immune potentiating responses and, hence, can be useful as adjuvants. Partial purification from the crude (food-grade) extract results in Quil A, which is contained in several veterinary vaccines. Further purification can provide concentrated saponin fractions such as QS-21, which is currently under investigation as a potential adjuvant for use in humans. Purified saponins have proven safe and effective when injected and have significantly enhanced the efficacy of some oral vaccines under clinical investigation. Toxicity of the food-grade extract from Quillaja saponaria has limited its use as a parenteral adjuvant; however, this toxicity seems to be abated when delivered orally. It is commonly used within the food and beverage industries and has no documented toxicity in humans at the present levels of consumption. Use of transgenic plants has been proposed as an alternative system for oral vaccine production and administration, and it is likely that an oral adjuvant will be required in most cases. Food-grade saponins have significant advantages for use with plant-made vaccines and are likely to provide a broad adjuvant effect due to the multiple saponin components. A review of the origin, production, biological activity, toxicity and use in the food industry is provided for Quillaja saponaria extract. Previous evaluation of this adjuvant in preclinical studies with plant made vaccines is discussed and a proposed level of experimental use in humans is provided.