RESUMO
The B cell receptors (BCRs) for antigen express variable (V) regions that are enormously diverse, thus serving as markers on individual B cells. V region-derived idiotypic (Id) peptides can be displayed as pId:MHCII complexes on B cells for recognition by CD4+ T cells. It is not known if naive B cells spontaneously display pId:MHCII in vivo or if BCR ligation is required for expression, thereby enabling collaboration between Id+ B cells and Id-specific T cells. Here, using a mouse model, we show that naive B cells do not express readily detectable levels of pId:MHCII. However, BCR ligation by Ag dramatically increases physical display of pId:MHCII, leading to activation of Id-specific CD4+ T cells, extrafollicular T-B cell collaboration and some germinal center formation, and production of Id+ IgG. Besides having implications for immune regulation, the results may explain how persistent activation of self-reactive B cells induces the development of autoimmune diseases and B cell lymphomas.
Assuntos
Antígenos de Histocompatibilidade Classe II/metabolismo , Neuropeptídeos/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos T/imunologia , Animais , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/imunologia , Doenças Autoimunes/metabolismo , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/metabolismo , Modelos Animais de Doenças , Imunoglobulina G , Camundongos , Camundongos Endogâmicos BALB CRESUMO
T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2-inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239-274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239-256 and aa 257-274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242-268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Células HEK293 , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/genética , Proto-Oncogene Mas , Domínios de Homologia de srcRESUMO
BACKGROUND: Multiple myeloma (MM) cancers originate from plasma cells that have passed through the germinal center reaction where somatic hypermutation of Ig V regions takes place. Myeloma protein V regions often express many mutations and are thus a rich source of neoantigens (traditionally called idiotopes (Id)). Therefore, these are highly tumor-specific and excellent targets for immunotherapy. METHODS: We have developed a DNA Id vaccine which as translated protein targets conventional dendritic cells (cDC) for CCL3-mediated delivery of myeloma protein V regions in a single-chain fragment variable (scFv) format. Vaccine efficacy was studied in the mouse MM model, mineral oil-induced plasmacytoma 315.BM. RESULTS: The Id vaccine protected mice against a challenge with MM cells. Moreover, the vaccine had a therapeutic effect. However, in some of the vaccinated mice, MM cells not producing H chains escaped rejection, resulting in free light chain (FLC) MM. Depletion of CD8+ T cells abrogated vaccine efficacy, and protection was observed to be dependent on cDC1s, using Batf3-/- mice. Modifications of scFv in the vaccine demonstrated that CD8+ T cells were specific for two mutated VH sequences. CONCLUSIONS: VH neoantigen-specific CD8+ T cells elicited by CCL3-containing Id vaccines had a therapeutic effect against MM in a mouse model. MM cells could escape rejection by losing expression of the H chain, thus giving rise to FLC MM.
Assuntos
Mieloma Múltiplo , Vacinas de DNA , Animais , Camundongos , Mieloma Múltiplo/terapia , Linfócitos T CD8-Positivos , Imunoterapia , Células DendríticasRESUMO
Antibodies are important for vaccine efficacy. Targeting antigens to antigen-presenting cells (APCs) increases antibody levels. Here, we explore the role of antigen valency in MHC class II (MHCII)-targeted vaccines delivered as DNA. We design heterodimeric proteins that carry either two identical (bivalent vaccines), or two different antigens (monovalent vaccines). Bivalent vaccines with two identical influenza hemagglutinins (HA) elicit higher amounts of anti-HA antibodies in mice than monovalent versions with two different HAs. Bivalent vaccines increase the levels of germinal center (GC) B cells and long-lived plasma cells. Only HA-bivalent vaccines completely protect mice against challenge with homologous influenza virus. Similar results are obtained with other antigens by targeting CD11c and Xcr1 on dendritic cells (DCs) or when administering the vaccine as protein with adjuvant. Bivalency probably increases B cell responses by cross-linking BCRs in readily observable DC-B cell synapses. These results are important for generating potent APC-targeted vaccines.
Assuntos
Vacinas Anticâncer , Vacinas contra Influenza , Vacinas de DNA , Animais , Anticorpos Antivirais , Células Apresentadoras de Antígenos , Hemaglutininas , Camundongos , Vacinas Combinadas , Vacinas de DNA/genéticaRESUMO
The substrate specificity of Src family kinases (SFKs) is partly determined by their Src homology 2 (SH2) domains. Thus, transient alterations in the SH2 domain of SFKs might change their binding partners and affect intracellular signaling pathways. Lck is an SFK that is central to the initiation of T cell activation in response to ligand binding to the T cell receptor (TCR) and is also critical for later signaling processes. The kinase activity of Lck requires both the phosphorylation of an activating tyrosine residue and the dephosphorylation of an inhibitory tyrosine residue. We found that a third conserved tyrosine phosphorylation site (Tyr(192)) within the SH2 domain of Lck was required for proper T cell activation and formation of cell-cell conjugates between T cells and antigen-presenting cells. Through phosphopeptide arrays and biochemical assays, we identified several regulators of the actin cytoskeleton that preferentially bound to Lck phosphorylated at Tyr(192) compared to Lck that was not phosphorylated at this site. Two of these phosphorylation-dependent binding partners, the kinase Itk (interleukin-2-inducible Tec kinase) and the adaptor protein TSAd (T cell-specific adaptor), promoted the TCR-dependent phosphorylation of Lck at Tyr(192). Our data suggest that phosphorylation transiently alters SH2 domain specificity and provide a potential mechanism whereby SFKs may be rewired from one signaling program to another to enable appropriate cell activation.