Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses

Abstract

CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9–guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.

Several cell-intrinsic effectors drive type I interferon-mediated restriction of HIV-1 in primary CD4+T cells

“…Notably, editing of up to three ISGs in one cell pool did not impact cell viability ( Figure S5A ). We did however observe a 34% reduction in the number of cells in the five-ISG KO condition, similar to a prior report that tested a six-gene KO in CD4 + T cells 54 . To assess whether multi-gene editing influences HIV infection or IFN restriction non-specifically, we compared the increase in HIV Levels with IFN for HM13 single KO cells to that of cells edited for both HM13 and the negative control B cell marker CD19 ( Figure S5B ).…”

Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells

“…To validate if these receptors are indeed transferred from macrophages to T cells, the genes of two top transfer candidates, HLA-DRA and DC-SIGN ( Figures 1 C and 1D), were knocked out by CRISPR-Cas9 in M2 (knockout [KO] efficiency over 90%, Figure S3 A). Co-culture with M2 KOs abolished HLA-DR or DC-SIGN ( Figures 1 C, 1D, and S3 A) surface exposure on co-cultured CD4 T cells, respectively, whereas KO of HLA-DR in CD4 T cells 20 did not impact on their HLA-DR surface exposure following co-culture with HLA-DR + wild-type (WT) M2 ( Figure 1 H). These results strongly suggested the myeloid cells as the source of these surface-exposed receptors on co-cultured autologous CD4 T cells ( Figures 1 A and S1 A).…”

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression

“…We are aware that the model of stimulated CD4+ primary cells does not recapitulate the actual state of the reservoir, which is mainly comprise of resting CD4+ T cells that do not support HIV infection. As this is a limitation of the current study, we are trying to adopt a recently developed gene editing approach to lncRNAs to deplete CYTOR in this unique cell population and monitor the effects of latency kinetics without altering its activation [ 56 ]. Mechanistically, our observations show that CYTOR directly binds to the HIV promoter and enhances the phosphorylation of the Ser2 CTD of RNAPII through association with P-TEFb to activate viral gene expression (Figs 4 and 5 ).…”