Effective clinical application of antiviral immunotherapies necessitates enhancing the functional state of natural killer (NK) and CD8+ 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+ T cell intrahepatic accumulation, effector function (interferon (IFN)-γ and granzyme B (GrB) production), CD8+ 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.

In mouse mutants incapable of expressing μ chains, VκJκ 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)κ genes before the assembly of a productive VHDHJH joint. Thus, μ chain expression is not a prerequisite to Igκ light chain gene rearrangements in normal development. Overall, ∼15% of the total CD43+ B cell progenitor population carry Igκ 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 Igκ loci independently, with a seven times higher frequency in the former. In addition, we show that in B cell progenitors VκJκ joining rapidly initiates κ chain expression, irrespective of the presence of a μ chain.

Inflammatory monocytes -- but not the non-inflammatory subset -- depend on the chemokine receptor CCR2 for distribution to injured tissue and stimulate disease progression. Precise therapeutic targeting of this inflammatory monocyte subset could spare innate immunity's essential functions for maintenance of homeostasis and thus limit unwanted effects. Here we developed siRNA nanoparticles targeting CCR2 expression in inflammatory monocytes. We identified an optimized lipid nanoparticle and silencing siRNA sequence that when administered systemically, had rapid blood clearance, accumulated in spleen and bone marrow and showed high cellular localization of fluorescently tagged siRNA inside monocytes. Efficient degradation of CCR2 mRNA in monocytes prevented their accumulation in sites of inflammation. Specifically, the treatment attenuated their number in atherosclerotic plaques, reduced infarct size following coronary artery occlusion, prolonged normoglycemia in diabetic mice after pancreatic islet transplantation and resulted in reduced tumor volumes and lower numbers of tumor-associated macrophages. Taken together, siRNA nanoparticle-mediated CCR2 gene silencing in leukocytes selectively modulates functions of innate immune cell subtypes and may allow for the development of specific anti-inflammatory therapy.

Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.

Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.

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.

Analysis of mononuclear cells in the adult mouse liver revealed that B cells represent as much as half of the intrahepatic lymphocyte population. Intrahepatic B cells (IHB cells) are phenotypically similar to splenic B2 cells but express lower levels of CD23 and CD21 and higher levels of CD5. IHB cells proliferate as well as splenic B cells in response to anti-IgM and LPS stimulation in vitro. VDJ gene rearrangements in IHB cells contain insertions of N,P region nucleotides characteristic of B cells maturing in the adult bone marrow rather than in the fetal liver. To evaluate whether B cells can have an impact on liver pathology, we compared CCl4-induced fibrosis development in B cell–deficient and wild-type mice. CCl4 caused similar acute liver injury in mutant and wild-type mice. However, following 6 weeks of CCl4 treatment, histochemical analyses showed markedly reduced collagen deposition in B cell–deficient as compared with wild-type mice. By analyzing mice that have normal numbers of B cells but lack either T cells or immunoglobulin in the serum, we established that B cells have an impact on fibrosis in an antibody- and T cell–independent manner.

V(D)J recombination at the immunoglobulin heavy chain (IgH) locus follows the 12/23 rule to ensure the correct assembly of the variable region gene segments. Here, we report characterization of an in vivo model that allowed us to study recombination violating the 12/23 rule, namely a mouse strain lacking canonical D elements in its IgH locus. We demonstrate that VH to JH joining can support the generation of all B cell subsets. However, the process is inefficient in that B cells and antibodies derived from the DH-less allele are not detectable if the latter is combined with a wild-type IgH allele. There is no preferential usage of any particular VH gene family or JH element in VHJH junctions, indicating that 23/23-guided recombination is possible, but is a low frequency event at the IgH locus in vivo.

V(D)J recombination at the immunoglobulin heavy chain (IgH) locus follows the 12/23 rule to ensure the correct assembly of the variable region gene segments. Here, we report characterization of an in vivo model that allowed us to study recombination violating the 12/23 rule, namely a mouse strain lacking canonical D elements in its IgH locus. We demonstrate that VH to JH joining can support the generation of all B cell subsets. However, the process is inefficient in that B cells and antibodies derived from the DH-less allele are not detectable if the latter is combined with a wild-type IgH allele. There is no preferential usage of any particular VH gene family or JH element in VHJH junctions, indicating that 23/23-guided recombination is possible, but is a low frequency event at the IgH locus in vivo.

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.

Mantle cell lymphoma is characterized by a genetic translocation results in aberrant overexpression of the CCND1 gene, which encodes cyclin D1. This protein functions as a regulator of the cell cycle progression, hence is considered to play an important role in the pathogenesis of the disease. In this study, we used RNA interference strategies to examine whether cyclin D1 might serve as a therapeutic target for mantle cell lymphoma. Knocking down cyclin D1 resulted in significant growth retardation, cell cycle arrest, and most importantly, induction of apoptosis. These results mark cyclin D1 as a target for mantle cell lymphoma and emphasize the therapeutic potential hidden in its silencing.