RESUMO
Phosphoantigens (PAgs)-like HMBPP ((E)-4-hydroxy-3-methyl-but-2-enyl diphosphate) and butyrophilin 3 (BTN3A, CD277)-specific monoclonal antibody 20.1 induce TCR-mediated activation of Vγ9Vδ2 T cells. Here, we compared murine reporter cells transduced with Vγ9Vδ2 TCRs G115, D1C55, and MOP for the activation in culture with human RAJI cells and PAgs or mAb 20.1 and its single-chain (sc) derivative. All transductants responded readily to PAg but only TCR MOP γ-chain-expressing cells responded to mAb/sc 20.1. Furthermore, both antagonist and agonist mAb and sc of the agonist mAb inhibited the PAg response of TCR-transduced murine reporter cells. These findings suggest that, in contrast to stimulation by physiological stimulators (PAg), the responsiveness to mAb 20.1 depends strongly on CDR3 sequences of the TCR, and that mAb 20.1 can interfere with the PAg-response. Mouse or human origin of reporter cells might affect the mAb 20.1 response since all three TCR-mediated mAb 20.1-induced activation of TCR-transduced Jurkat cells. The pronounced differences between PAg and mAb 20.1-induced activation observed here help to understand the often contradictory published data. This study provides novel perspectives on the physiological mechanism of Vγ9Vδ2 T-cell activation, and highlights the complex mode of action of BTN3A-specific antibodies as agents in cancer immunotherapy.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Butirofilinas/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Células HEK293 , Humanos , CamundongosRESUMO
We have shown previously that acute heat shock protein (Hsp) 70 induction in a human melanoma cell line containing a doxycycline-inducible Hsp70 expression construct increases lysability of these tumor cells by cytotoxic T lymphocyte (CTL) without interfering with MHC class I expression and antigen presentation. The same parental melanoma cell line has now been transduced retrovirally to overexpress Hsp70 permanently. Here we demonstrate that MHC class I cell surface expression is again not altered and that these cells, in contrast with acutely Hsp70 overexpressing cells, do not show augmentation of CTL-mediated apoptosis. Also, long-term induction of Hsp70 in cells with the doxycycline-inducible Hsp70 construct leads to abrogation of increased lysability. Because, furthermore, after heat shock the same permanently Hsp70 overexpressing cells show Hsp70 induction and increased lysability, it is hypothesized that acutely available Hsp70 is able to chaperone proteins that are involved in CTL-mediated apoptosis of target cells and to thereby improve their lysability. We also observed that permanent but not acute Hsp70 overexpression resulted in decreased levels of Hsc70, the constitutively expressed member of the Hsp70 family. Down-regulation of Hsc70 occurs at the post-transcriptional level and can be observed also after long-term induction of Hsp70 in cells containing the doxycycline-inducible expression system. Hsc70 down-regulation might reflect a functional integration of the overexpressed Hsp70 on the basis of a chaperone network so that only acute induction will provide Hsp70 that can improve tumor cell lysability. The implications of the differential effect of acute versus permanent Hsp70 overexpression for tumor therapy are discussed.
Assuntos
Proteínas de Choque Térmico HSP70/imunologia , Melanoma/imunologia , Linfócitos T Citotóxicos/imunologia , Linhagem Celular Tumoral , Clatrina/metabolismo , Regulação para Baixo , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Transdução GenéticaRESUMO
The MHC class II molecule RT1Bl covalently linked with gpMBP-71-90 was expressed in P80 cells (mouse mastocytoma P815 expressing rat-CD80) and i.v. injection ameleriorated active and adoptive transfer (AT) experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Spinal cord of animals with AT-EAE showed significant increase of apoptotic T-cells at maximum of disease after injection of P80-RT1Bl-MBP-71-90 but not of P80RT1Bl or P80 cells. The data demonstrate a possible therapeutic effect on EAE by provision of T-cell receptor (TCR) and costimulatory signals by genetically engineered antigen presenting cells (APC) and suggest induction of T-cell apoptosis as important mechanism of action.
Assuntos
Transferência Adotiva , Encefalomielite Autoimune Experimental/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Medula Espinal/imunologia , Sequência de Aminoácidos , Animais , Apoptose/imunologia , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Proteína Básica da Mielina/química , Proteína Básica da Mielina/imunologia , Peptídeos/imunologia , Ratos , Medula Espinal/patologia , Linfócitos T/imunologiaRESUMO
In experimental autoimmune encephalomyelitis (EAE) of LEW rats, BV8S2(+) (V(beta)8.2) T cells dominate the RT1B(l)-restricted response to guinea pig myelin basic protein (gpMBP), and respond to the superantigens (SAg) Staphylococcus enterotoxin C1 (SEC1), Mycoplasma arthritidis SAg (MAS) and Yersinia pseudotuberculosis mitogen (YPM). T cells expressing the closely related BV8S4 differ from BV8S2 T cells in their response to gpMBP, and the SAg SEC1 and MAS, but not in their response to YPM. The functional differences between BV8S2 and BV8S4, which vary in complementarity-determining/hypervariable region 4 (CDR4/HV4) and CDR2, were analyzed by cloning and mutating a TCR with features typical for gpMBP-specific BV8S2(+) TCR. The wild-type BV8S2 receptor and the BV8S4-like CDR2 + 4beta double mutant of BV8S2 showed the same differences in ligand specificity as polyclonal BV8S2(+) and BV8S4(+) lymphocyte populations. The CDR2beta mutant lost its reactivity for SEC1 and gpMBP(68-88), but the CDR4/HV4beta mutation abolished only activation by SEC1. Thus, CDR2 and HV4 contribute not only differently to recognition of peptide antigens, but also to recognition of different types of bacterial SAg.