We mechanistically explored the effect of increased hydrophobicity of the polycation on the efficacy and specificity of gene delivery in mice. N-Alkylated linear PEIs with varying alkyl chain lengths ...and extent of substitution were synthesized and characterized by biophysical methods. Their in vivo transfection efficiency, specificity, and biodistribution were investigated. N-Ethylation improves the in vivo efficacy of gene expression in the mouse lung 26-fold relative to the parent polycation and more than quadruples the ratio of expression in the lung to that in all other organs. N-Propyl-PEI was the best performer in the liver and heart (581- and 3.5-fold enhancements, resp.) while N-octyl-PEI improved expression in the kidneys over the parent polymer 221-fold. As these enhancements in gene expression occur without changing the plasmid biodistribution, alkylation does not alter the cellular uptake but rather enhances transfection subsequent to cellular uptake.
Significance
The recombinatorial process of V(D)J rearrangement generates a vast antibody repertoire from a limited number of genes. The joints generated in the course of V(D)J recombination are ...imprecise thus yielding greater diversity but also resulting in frequent generation of nonproductive VDJ rearrangements. We have previously shown that B cells with two nonproductive IgH rearrangements can be efficiently rescued by a form of secondary V(D)J recombination called V
H
replacement. We now demonstrate that V
H
replacement also contributes to the diversity of the immune repertoire by modifying productive IgH rearrangements. Results presented herein suggest that V
H
replacement occurs exclusively during early stages of B-cell development and therefore does not contribute to the editing of self-reactive antibodies.
The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. V
H
replacement represents a RAG-mediated secondary rearrangement in which an upstream V
H
element recombines with a rearranged V
H
D
H
J
H
joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original V
H
element and the conventional RSS of the invading V
H
gene, leaving behind a footprint of up to five base pairs (bps) of the original V
H
gene that is often further obscured by exonuclease activity and
N
-nucleotide addition. We have previously demonstrated that V
H
replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive V
H
D
H
J
H
allele. Using this mouse model, we characterized the role of V
H
replacement in the diversification of the primary Ig repertoire through the modification of productive V
H
D
H
J
H
rearrangements. Our results indicate that V
H
replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.
The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies ...on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. V... replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged ... joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original V... element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original V... gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive ... allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive ... rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies. (ProQuest: ... denotes formulae/symbols omitted.)
The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies ...on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VHreplacement represents a RAG-mediated secondary rearrangement in which an upstream VHelement recombines with a rearranged VHDHJHjoint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VHelement and the conventional RSS of the invading VHgene, leaving behind a footprint of up to five base pairs (bps) of the original VHgene that is often further obscured by exonuclease activity andN-nucleotide addition. We have previously demonstrated that VHreplacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJHallele. Using this mouse model, we characterized the role of VHreplacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJHrearrangements. Our results indicate that VHreplacement occurs before Ig light chain rearrangement and thus is not involved in the editing of selfreactive antibodies.
The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies ...on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. V... replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged ... joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original V... element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original V... gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive ... allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive ... rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies. (ProQuest: ... denotes formulae/symbols omitted.)
Immunoglobulin light chain (IgL) rearrangements occur more frequently at Igκ than at Igλ. Previous results suggested that the unrearranged Igκ locus negatively regulates Igλ transcription and/or ...rearrangement. Here, we demonstrate that expression of a VJλ1-joint inserted into its physiological position in the Igλ locus is independent of Igκ rearrangements. Expression of the inserted VJλ1 gene segment is developmentally controlled like that of a VJκ-joint inserted into the Igκ locus and furthermore coincides developmentally with the occurrence of Igκ rearrangements in wild-type mice. We conclude that developmentally controlled transcription of a gene rearrangement in the Igλ locus occurs in the presence of an unrearranged Igκ locus and is therefore not negatively regulated by the latter. Our data also indicate light chain editing in ∼30% of λ1 expressing B cell progenitors.
Effective clinical application of antiviral immunotherapies necessitates enhancing the functional state of natural killer (NK) and CD8 super(+) T cells. An important mechanism for the establishment ...of viral persistence in the liver is the activation of the PD-1/PD-L1 inhibitory pathway. To examine the role of hepatic myeloid PD-L1 expression during viral infection, we determined the magnitude and quality of antiviral immune responses by administering PD-L1 short-interfering RNA (siRNA) encapsulated in lipidoid nanoparticles (LNP) in mice. Our studies indicate that Kupffer cells (KC) preferentially engulfed PD-L1 LNP within a short period of time and silenced Pdl1 during adenovirus and MCMV infection leading to enhanced NK and CD8 super(+) T cell intrahepatic accumulation, effector function (interferon (IFN)- gamma and granzyme B (GrB) production), CD8 super(+) T cell-mediated viral clearance, and memory. Our results demonstrate that PD-L1 knockdown on KCs is central in determining the outcome of liver viral infections, and they represent a new class of gene therapy.Molecular Therapy - Nucleic Acids (2013) 2, e72; doi:10.1038/mtna.2012.63; published online 19 February 2013
Immunoglobulin light chain (IgL) rearrangements occur more frequently at Ig kappa than at Ig lambda. Previous results suggested that the unrearranged Ig kappa locus negatively regulates Ig lambda ...transcription and/or rearrangement. Here, we demonstrate that expression of a VJ lambda 1-joint inserted into its physiological position in the Ig lambda locus is independent of Ig kappa rearrangements. Expression of the inserted VJ lambda 1 gene segment is developmentally controlled like that of a VJ kappa-joint inserted into the Ig kappa locus and furthermore coincides developmentally with the occurrence of Ig kappa rearrangements in wild-type mice. We conclude that developmentally controlled transcription of a gene rearrangement in the Ig lambda locus occurs in the presence of an unrearranged Ig kappa locus and is therefore not negatively regulated by the latter. Our data also indicate light chain editing in approximately 30% of lambda 1 expressing B cell progenitors.
In mouse mutants incapable of expressing mu chains, VkappaJkappa joints are detected in the CD43(+) B cell progenitors. In agreement with these earlier results, we show by a molecular single cell ...analysis that 4-7% of CD43(+) B cell progenitors in wild-type mice rearrange immunoglobulin (Ig)kappa genes before the assembly of a productive VHDHJH joint. Thus, mu chain expression is not a prerequisite to Igkappa light chain gene rearrangements in normal development. Overall, approximately 15% of the total CD43(+) B cell progenitor population carry Igkappa gene rearrangements in wild-type mice. Together with the results obtained in the mouse mutants, these data fit a model in which CD43(+) progenitors rearrange IgH and Igkappa loci independently, with a seven times higher frequency in the former. In addition, we show that in B cell progenitors VkappaJkappa joining rapidly initiates kappa chain expression, irrespective of the presence of a mu chain.
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