RESUMO
The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4+ helper T (TH) cells and CD8+ cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of TH1 and TH2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent TH1 or TH2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed TH1/TH2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid TH1/TH2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.
Assuntos
Diferenciação Celular , Ativação Linfocitária/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Análise de Célula Única/métodos , Células Th1/citologia , Células Th2/citologia , Antígenos/metabolismo , Antígeno CTLA-4/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/metabolismo , Fenótipo , Proteômica , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Using grazing-incidence small-angle X-ray scattering in a special configuration (parallel SAXS, or parSAXS), we mapped the crystallization of DNA-capped nanoparticles across a sessile droplet, revealing the formation of crystalline Gibbs monolayers of DNA-capped nanoparticles at the air-liquid interface. We showed that the spatial crystallization can be regulated by adjusting both ionic strength and DNA sequence length and that a modified form of the Daoud-Cotton model could describe and predict the resulting changes in interparticle spacing. Gibbs monolayers at the air-liquid interface provide an ideal platform for the formation and study of equilibrium nanostructures and may afford exciting routes toward the design of programmable 2D plasmonic materials and metamaterials.