RESUMEN
CCR5 is not only a coreceptor for HIV-1 infection in CD4+ T cells, but also contributes to their functional fitness. Here, we show that by limiting transcription of specific ceramide synthases, CCR5 signaling reduces ceramide levels and thereby increases T-cell antigen receptor (TCR) nanoclustering in antigen-experienced mouse and human CD4+ T cells. This activity is CCR5-specific and independent of CCR5 co-stimulatory activity. CCR5-deficient mice showed reduced production of high-affinity class-switched antibodies, but only after antigen rechallenge, which implies an impaired memory CD4+ T-cell response. This study identifies a CCR5 function in the generation of CD4+ T-cell memory responses and establishes an antigen-independent mechanism that regulates TCR nanoclustering by altering specific lipid species.
Asunto(s)
Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Ceramidas/inmunología , Memoria Inmunológica , Receptores CCR5/deficiencia , Animales , Antígenos/genética , Linfocitos T CD4-Positivos/citología , Ceramidas/genética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Receptores CCR5/inmunologíaRESUMEN
The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.
Asunto(s)
Antígenos/metabolismo , Fitocromo B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Humanos , Células Jurkat , Cinética , Luz , Modelos Teóricos , Optogenética/métodos , Unión ProteicaRESUMEN
AIM: Although prostate cancer is one of the most commonly diagnosed malignancies in men, there is no effective curative therapy for the advanced disease. Therefore, the aim of the present study was to generate prostate-specific membrane antigen (PSMA)×CD3 diabodies as a novel treatment option for this tumor. METHODS: A PSMA×CD3 diabody and a covalently linked single-chain diabody were constructed from the anti-PSMA single-chain Fv fragment D7 and an anti-CD3 single-chain Fv fragment. The fusion proteins were periplasmatically expressed in Escherichia coli. The binding properties were tested on PSMA-expressing C4-2 prostate cancer cells and CD3(+) Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability assay was used. T-cell activation was determined by flow cytometry. In vivo activity of the diabody was tested in SCID mice reconstituted with human peripheral blood lymphocytes bearing C4-2 tumor xenografts. RESULTS: Bacterial expression levels were significantly higher for the diabody (1-1.5 mg/l culture) compared with the single-chain diabody (0.2-0.4 mg/l culture). Specific binding on CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown with both diabody formats. In vitro, both diabodies proved to be potent agents for retargeting human CD4(+) and CD8(+) lymphocytes to lyse C4-2 prostate cancer cells. The formation of conjugates between T cells and target cells with clustering of the diabody at sites of interaction could be shown. SCID mice reconstituted with human peripheral blood lymphocytes bearing C4-2 tumor xenografts with the diabody showed an efficient inhibition of tumor growth. CONCLUSION: Both diabody formats showed a highly efficient and specific T cell-mediated killing of prostate cancer cells and are encouraging for further development in preclinical and clinical studies.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Complejo CD3/uso terapéutico , Antígeno Prostático Específico/uso terapéutico , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/terapia , Animales , Anticuerpos Biespecíficos/inmunología , Complejo CD3/administración & dosificación , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Humanos , Células Jurkat , Activación de Linfocitos , Masculino , Ratones , Ratones SCID , Antígeno Prostático Específico/administración & dosificación , Antígeno Prostático Específico/inmunología , Anticuerpos de Cadena Única , Resultado del TratamientoRESUMEN
BACKGROUND: Studying the stoichiometry and intracellular trafficking of the T cell antigen receptor (TCR) is pivotal in understanding its mechanisms of activation. The alphabetaTCR includes the antigen-binding TCRalphabeta heterodimer as well as the signal transducing CD3epsilongamma, CD3epsilondelta and zeta2 subunits. Although the TCR-interacting molecule (TRIM) is also part of the alphabetaTCR complex, it has not been included in most reports so far. RESULTS: We used the native antibody-based mobility shift (NAMOS) assay in a first dimension (1D) blue native (BN)-PAGE and a 2D BN-/BN-PAGE to demonstrate that the stoichiometry of the digitonin-solublized TRIM-containing alphabetaTCR is TCRalphabetaCD3epsilon2gammadeltazeta2TRIM2. Smaller alphabetaTCR complexes possess a TCRalphabeta CD3epsilon2gammadeltazeta2 stoichiometry. Complexes of these sizes were detected in T cell lines as well as in primary human and mouse T cells. Stimulating the alphabetaTCR with anti-CD3 antibodies, we demonstrate by confocal laser scanning microscopy that CD3epsilon colocalizes with zeta and both are degraded upon prolonged stimulation, possibly within the lysosomal compartment. In contrast, a substantial fraction of TRIM does not colocalize with zeta. Furthermore, TRIM neither moves to lysosomes nor is degraded. Immunoprecipitation studies and BN-PAGE indicate that TRIM also associates with the gammadeltaTCR. CONCLUSIONS: Small alphabetaTCR complexes have a TCRalphabeta CD3epsilon2gammadeltazeta2 stoichiometry; whereas those associated with one TRIM dimer are TCRalphabeta CD3epsilon2gammadeltazeta2TRIM2. TRIM is differentially processed compared to CD3 and zeta subunits after T cell activation and is not degraded. The gammadeltaTCR also associates with TRIM.