RESUMEN
The blood-brain barrier acts as a major barrier for the entrance of most therapeutics into the brain, impeding treatment for neurological disorders. Intracerebroventricular (ICV) injection of T cells is a useful tool for cell therapy of neurological disorders including neurodegenerative and neuropsychiatric diseases and brain tumors. Here, we present an optimized ICV injection of T cells with improved injection efficiency at pathological sites within the brain parenchyma. We describe details of the surgical procedure and verification of injection via immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Fisher et al. (2014); Strominger et al., (2018); Mittal et al. (2019); Eremenko et al. (2019).
Asunto(s)
Barrera Hematoencefálica/metabolismo , Inyecciones Intraventriculares/métodos , Inyecciones/métodos , Animales , Barrera Hematoencefálica/inmunología , Encéfalo/metabolismo , Linfocitos T CD4-Positivos/inmunología , Inmunohistoquímica/métodos , Infusiones Intraventriculares , Sistema Linfático/inmunología , Recuento de Linfocitos/métodos , Ratones , Tejido Parenquimatoso , Linfocitos T/inmunologíaRESUMEN
Transduction of primary T cells has become prominent with the introduction of chimeric antigen receptor T-cell therapy. Although there are many protocols for the transduction of human T cells, it remains a challenge to transduce murine T cells. We present an optimized protocol for the retroviral transduction of murine CD4 T cells, which overcomes major challenges including large-scale production and long-term culturing of transduced cells. The optimized protocol combines high transduction efficiency with a low rate of cell death. For complete details on the use and execution of this protocol, please refer to Eremenko et al., 2019.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Transducción Genética/métodos , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citometría de Flujo/métodos , Vectores Genéticos/genética , Ratones , Retroviridae/genéticaRESUMEN
IL-2 is the master-regulator cytokine for T cell dependent responses and is crucial for proliferation and survival of T cells. However, IL-2-based treatments remained marginal, in part due to short half-life. Thus, we aimed to extend IL-2 half-life by flanking the IL-2 core with sequences derived from the extensively glycosylated hinge region of the NCR2 receptor. We termed this modified IL-2: "S2A". Importantly, S2A blood half-life was extended 14-fold compared to the clinical grade IL-2, Proleukin. Low doses inoculation of S2A significantly enhanced induction of Tregs (CD4+ Regulatory T cells) in vivo, as compared to Proleukin, while both S2A and Proleukin induced low levels of CD8+ T cells. In a B16 metastatic melanoma model, S2A treatment was unable to reduce the metastatic capacity of B16 melanoma, while enhancing induction and recruitment of Tregs, compared to Proleukin. Conversely, in two autoimmune models, rheumatoid arthritis and DSS-induced colitis, S2A treatment significantly reduced the progression of disease compared to Proleukin. Our results suggest new avenues for generating long-acting IL-2 for long-standing treatment and a new technique for manipulating short-life proteins for clinical and research uses.