The Tn antigen (GalNAc-α-1-O-Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche ...of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp2-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.
The first examples of amino acid (Ser/Thr)-sp(2)-iminosugar glycomimetic conjugates featuring an α-O-linked pseudoanomeric linkage are reported. The key synthetic step involves the completely ...diastereoselective α-glycosylation of Ser/Thr due to strong stereoelectronic and conformational bias imposed by the bicyclic sp(2)-iminosugar scaffold. Mucin-related glycopeptides incorporating these motifs were recognized by the monoclonal antibody (mAb) scFv-SM3, with activities depending on both the hydroxylation pattern (Glc/Gal/GlcNAc/GalNAc) of the sp(2)-iminosugar and the peptide aglycone structure (Ser/Thr).
The first examples of amino acid (Ser/Thr)–sp 2-iminosugar glycomimetic conjugates featuring an α-O-linked pseudoanomeric linkage are reported. The key synthetic step involves the completely ...diastereoselective α-glycosylation of Ser/Thr due to strong stereoelectronic and conformational bias imposed by the bicyclic sp 2-iminosugar scaffold. Mucin-related glycopeptides incorporating these motifs were recognized by the monoclonal antibody (mAb) scFv-SM3, with activities depending on both the hydroxylation pattern (Glc/Gal/GlcNAc/GalNAc) of the sp 2-iminosugar and the peptide aglycone structure (Ser/Thr).
T cells recognize antigens via their cell surface TCR and are classified as either αβ or γδ depending on the variable chains in their TCR, α and β or γ and δ, respectively. Both αβ and γδ TCRs also ...contain several invariant chains, including CD3δ, which support surface TCR expression and transduce the TCR signal. Mutations in variable chains would be expected to affect a single T cell lineage, while mutations in the invariant chains would affect all T cells. Consistent with this, all CD3δ-deficient patients described to date showed a complete block in T cell development. However, CD3δ-KO mice have an αβ T cell-specific defect. Here, we report 2 unrelated cases of SCID with a selective block in αβ but not in γδ T cell development, associated with a new splicing mutation in the CD3D gene. The patients' T cells showed reduced CD3D transcripts, CD3δ proteins, surface TCR, and early TCR signaling. Their lymph nodes showed severe T cell depletion, recent thymus emigrants in peripheral blood were strongly decreased, and the scant αβ T cells were oligoclonal. T cell-dependent B cell functions were also impaired, despite the presence of normal B cell numbers. Strikingly, despite the specific loss of αβ T cells, surface TCR expression was more reduced in γδ than in αβ T cells. Analysis of individuals with this CD3D mutation thus demonstrates the contrasting CD3δ requirements for αβ versus γδ T cell development and TCR expression in humans and highlights the diagnostic and clinical relevance of studying both TCR isotypes when a T cell defect is suspected.
The Tn antigen (GalNAc-α-1- O -Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising ...niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp 2 -iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.
► We analyze signaling events in human CD3γ−/− primary and HVS-transformed T cells. ► CD3γ is dispensable for certain early signaling pathways and late iNKT cell functions. ► Other signaling pathways ...are affected, such as TCRζ phosphorylation. ► TCRVβ11bright iNKT cells are preferentially selected in response to α-GalCer. ► CD3γ chain contributes to improve TCR signaling efficiency and survival.
Antigen recognition by T-lymphocytes through the T-cell antigen receptor, TCR–CD3, is a central event in the initiation of an immune response. CD3 proteins may have redundant as well as specific contributions to the intracellular propagation of TCR-mediated signals. However, to date, the relative role that each CD3 chain plays in signaling is controversial. In order to examine the roles of CD3γ chain in TCR signaling, we analyzed proximal and distal signaling events in human CD3γ−/− primary and Herpesvirus saimiri (HVS)-transformed T cells. Following TCR–CD3 engagement, certain early TCR signaling pathways (ZAP-70, ERK, p38 and mTORC2 phosphorylation, and actin polymerization) were comparable with control HVS-transformed T cells. However, other signaling pathways were affected, such TCRζ phosphorylation, indicating that the CD3γ chain contributes to improve TCR signaling efficiency and survival. On the other hand, CD3γ−/− primary invariant NKT cells (iNKT cells) showed a normal expansion in response to alpha-galactosylceramide (α-GalCer) and TCRVβ11bright iNKT cells were preferentially selected in this in vitro culture system, perhaps as a consequence of selective events in the thymus. Our results collectively indicate that a TCR lacking CD3γ can propagate a number of signals through the remaining invariant chains, likely the homologous CD3δ chain, which replaces it at the mutant TCR.
T cells recognize antigens via their cell surface TCR and are classified as either alphabeta or gammadelta depending on the variable chains in their TCR, alpha and betaor gamma and delta, ...respectively. Both alphabeta and gammadelta TCRs also contain several invariant chains, including CD3delta, which support surface TCR expression and transduce the TCR signal. Mutations in variable chains would be expected to affect a single T cell lineage, while mutations in the invariant chains would affect all T cells. Consistent with this, all CD3delta-deficient patients described to date showed a complete block in T cell development. However, CD3delta-KO mice have an alphabeta T cell-specific defect. Here, we report 2 unrelated cases of SCID with a selective block in alphabeta but not in gammadelta Tcell development, associated with a new splicing mutation in the CD3D gene. The patients' T cells showed reduced CD3D transcripts, CD3delta proteins, surface TCR, and early TCR signaling. Their lymph nodes showed severe T cell depletion, recent thymus emigrants in peripheral blood were strongly decreased, and the scant alphabeta T cells were oligoclonal. T cell-dependent B cell functions were also impaired, despite the presence of normal B cell numbers. Strikingly, despite the specific loss of alphabeta T cells, surface TCR expression was more reduced in gammadelta than in alphabeta T cells. Analysis of individuals with this CD3D mutation thus demonstrates the contrasting CD3delta requirements for alphabeta versus gammadelta Tcell development and TCR expression in humans and highlights the diagnostic and clinical relevance of studying both TCR isotypes when a T cell defect is suspected.
T cells recognize antigens via their cell surface TCR and are classified as either αbeta or γδ depending on the variable chains in their TCR, α and beta or γ and δ, respectively. Both αbeta and γδ ...TCRs also contain several invariant chains, including CD3δ, which support surface TCR expression and transduce the TCR signal. Mutations in variable chains would be expected to affect a single T cell lineage, while mutations in the invariant chains would affect all T cells. Consistent with this, all CD3δ-deficient patients described to date showed a complete block in T cell development. However, CD3δ-KO mice have an αbeta T cell-specific defect. Here, we report 2 unrelated cases of SCID with a selective block in αbeta but not in γδ T cell development, associated with a new splicing mutation in the CD3D gene. The patients' T cells showed reduced CD3D transcripts, CD3δ proteins, surface TCR, and early TCR signaling. Their lymph nodes showed severe T cell depletion, recent thymus emigrants in peripheral blood were strongly decreased, and the scant αbeta T cells were oligoclonal. T cell-dependent B cell functions were also impaired, despite the presence of normal B cell numbers. Strikingly, despite the specific loss of αbeta T cells, surface TCR expression was more reduced in γδ than in αbeta T cells. Analysis of individuals with this CD3D mutation thus demonstrates the contrasting CD3δ requirements for αbeta versus γδ T cell development and TCR expression in humans and highlights the diagnostic and clinical relevance of studying both TCR isotypes when a T cell defect is suspected.
The Tn antigen (GalNAc-α-1-
O
-Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising ...niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp
2
-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments
in vivo
have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.
An anti-cancer vaccine based on an unnatural antigen with an sp
2
-iminosugar fragment.