Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza’s Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology.
CD4 T cell activation and differentiation mechanisms constitute a complex and intricate signaling network involving several regulatory proteins. IRF2BP2 is a transcriptional repressor that is involved in gene-expression regulation in very diverse biologic contexts. Information regarding the IRF2BP2 regulatory function in CD4 T lymphocytes is very limited and suggests a role for this protein in repressing the expression of different cytokine genes. Here, we showed that Irf2bp2 gene expression was decreased in CD4 T cells upon activation. To investigate the possible regulatory roles for IRF2BP2 in CD4 T cell functions, this protein was ectopically expressed in murine primary-activated CD4 T lymphocytes through retroviral transduction. Interestingly, ectopic expression of IRF2BP2 led to a reduction in CD25 expression and STAT5 phosphorylation, along with an impaired proliferative capacity. The CD69 expression was also diminished in IRF2BP2-overexpressing cells, whereas CD44 and CD62L levels were not altered. In vivo, transferred, IRF2BP2-overexpressing, transduced cells displayed an impaired expansion capacity compared with controls. Furthermore, overexpression of IRF2BP2 in differentiated Th cells resulted in slightly reduced IL-4 and pro-TGF-β production in Th2 and iT but had no effect on IFN-γ or IL-17 expression in Th1 and Th17 cells, respectively. Taken together, our data suggest a role for IRF2BP2 in regulating CD4 T cell activation by repressing proliferation and the expression of CD25 and CD69 induced by TCR stimuli.
These authors contributed equally to this work.Chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of cancer is now an approved treatment for B cell malignancies. However, the use of viral vectors to provide long-term CAR expression is associated with high production costs and cumbersome quality controls, impacting the final cost of CAR T cell therapies. Nonviral integrative vectors, such as Sleeping Beauty (SB) transposons, provide an alternative to modify primary T cells. Therefore, we developed a protocol to expand SB-transfected 19BBf CAR T cells using a lymphoblastoid cell line, and evaluated T cell phenotype as well as function along the T cell expansion. Electroporation of PBMCs with transposon plasmid decreased cell viability on day 1 but had a minor impact on the frequency of memory subpopulations when compared to mock condition. CAR+ lymphocytes showed increased proliferation compared to mock control and high cytotoxic activity towards CD19+ cells without significant differences in exhaustion markers expression. Moreover, CAR+ lymphocytes showed an increased frequency by the end of the stimulation cycle compared with day 1, suggesting that CAR expression confers a selective proliferation advantage. Immunodeficient NOD scid gamma chain knockout (NSG) mice engrafted with the human pre-B leukemic cell line RS4;11 and treated with 19BBf CAR T cells showed improved overall survival when compared to mock T cells treated animals. The results showed that electroporation using the SB system is a simple and affordable method for inducing long-term CAR expression in T lymphocytes. Expansion of gene-modified T cells with the lymphoblastoid cell line provided up to 2 cycles of stimulations, generating effective T cells against leukemia in vitro and in vivo.
Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza's Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology.
Peripheral blood sample was donated by a 61years old female patient diagnosed with acute myeloid leukemia secondary to a primary myelofibrosis harboring the 52-bp deletion in the CALR gene (c.1092_1143del, p.L367fs*46) and the R693X mutation in the ASXL1 gene (c.2077C>T, p.R693X). CD34+ cells were isolated from the sample and subjected to the reprogramming procedure by using the Sendai virus carrying the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc. iPS colonies generated retained the original mutations and displayed all the features of bona fide iPS cells.
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