Exosome-mediated genetic reprogramming of tumor-associated macrophages by exoASO-STAT6 leads to potent monotherapy antitumor activity.

Effectiveness of checkpoint immunotherapy in cancer can be undermined by immunosuppressive tumor-associated macrophages (TAMs) with an M2 phenotype. Reprogramming TAMs toward a proinflammatory M1 phenotype is a novel approach to induce antitumor immunity. The M2 phenotype is controlled by key transcription factors such as signal transducer and activator of transcription 6 (STAT6), which have been "undruggable" selectively in TAMs. We describe an engineered exosome therapeutic candidate delivering an antisense oligonucleotide (ASO) targeting STAT6 (exoASO-STAT6), which selectively silences STAT6 expression in TAMs. In syngeneic models of colorectal cancer and hepatocellular carcinoma, exoASO-STAT6 monotherapy results in >90% tumor growth inhibition and 50 to 80% complete remissions. Administration of exoASO-STAT6 leads to induction of nitric oxide synthase 2 (NOS2), an M1 macrophage marker, resulting in remodeling of the tumor microenvironment and generation of a CD8 T cell-mediated adaptive immune response. Collectively, exoASO-STAT6 represents the first platform targeting transcription factors in TAMs in a highly selective manner.

A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties.

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.

Methods of Isolation and Analysis of TREG Immune Infiltrates from Injured and Dystrophic Skeletal Muscle.

The immune infiltrate present in acutely injured or dystrophic skeletal muscle has been shown to play an important role in the process of muscle regeneration. Our work has described, for the first time, muscle regulatory T cells (Tregs), a unique population in phenotype and function capable of promoting skeletal muscle repair. Herein, we describe the methods we have optimized to study muscle Tregs, including their isolation from injured muscle, immuno-labeling for analysis/separation by flow cytometry, and measurement of their proliferation status.

Poor Repair of Skeletal Muscle in Aging Mice Reflects a Defect in Local, Interleukin-33-Dependent Accumulation of Regulatory T Cells.

Normal repair of skeletal muscle requires local expansion of a special population of Foxp3(+)CD4(+) regulatory T (Treg) cells. Such cells failed to accumulate in acutely injured muscle of old mice, known to undergo ineffectual repair. This defect reflected reduced recruitment of Treg cells to injured muscle, as well as less proliferation and retention therein. Interleukin-33 (IL-33) regulated muscle Treg cell homeostasis in young mice, and its administration to old mice ameliorated their deficits in Treg cell accumulation and muscle regeneration. The major IL-33-expressing cells in skeletal muscle displayed a constellation of markers diagnostic of fibro/adipogenic progenitor cells and were often associated with neural structures, including nerve fibers, nerve bundles, and muscle spindles, which are stretch-sensitive mechanoreceptors important for proprioception. IL-33(+) cells were more frequent after muscle injury and were reduced in old mice. IL-33 is well situated to relay signals between the nervous and immune systems within the muscle context.

MUCOSAL IMMUNOLOGY. Individual intestinal symbionts induce a distinct population of RORγ⁺ regulatory T cells.

T regulatory cells that express the transcription factor Foxp3 (Foxp3(+) T(regs)) promote tissue homeostasis in several settings. We now report that symbiotic members of the human gut microbiota induce a distinct T(reg) population in the mouse colon, which constrains immuno-inflammatory responses. This induction­which we find to map to a broad, but specific, array of individual bacterial species­requires the transcription factor Rorγ, paradoxically, in that Rorγ is thought to antagonize FoxP3 and to promote T helper 17 (T(H)17) cell differentiation. Rorγ's transcriptional footprint differs in colonic T(regs) and T(H)17 cells and controls important effector molecules. Rorγ, and the T(regs) that express it, contribute substantially to regulating colonic T(H)1/T(H)17 inflammation. Thus, the marked context-specificity of Rorγ results in very different outcomes even in closely related cell types.

Denervation protects limbs from inflammatory arthritis via an impact on the microvasculature.

Two-way communication between the mammalian nervous and immune systems is increasingly recognized and appreciated. An intriguing example of such crosstalk comes from clinical observations dating from the 1930s: Patients who suffer a stroke and then develop rheumatoid arthritis atypically present with arthritis on only one side, the one not afflicted with paralysis. Here we successfully modeled hemiplegia-induced protection from arthritis using the K/BxN serum-transfer system, focused on the effector phase of inflammatory arthritis. Experiments entailing pharmacological inhibitors, genetically deficient mouse strains, and global transcriptome analyses failed to associate the protective effect with a single nerve quality (i.e., with the sympathetic, parasympathetic, or sensory nerves). Instead, there was clear evidence that denervation had a long-term effect on the limb microvasculature: The rapid and joint-localized vascular leak that typically accompanies and promotes serum-transferred arthritis was compromised in denervated limbs. This defect was reflected in the transcriptome of endothelial cells, the expression of several genes impacting vascular leakage or transendothelial cell transmigration being altered in denervated limbs. These findings highlight a previously unappreciated pathway to dissect and eventually target in inflammatory arthritis.

A special population of regulatory T cells potentiates muscle repair.

Long recognized to be potent suppressors of immune responses, Foxp3(+)CD4(+) regulatory T (Treg) cells are being rediscovered as regulators of nonimmunological processes. We describe a phenotypically and functionally distinct population of Treg cells that rapidly accumulated in the acutely injured skeletal muscle of mice, just as invading myeloid-lineage cells switched from a proinflammatory to a proregenerative state. A Treg population of similar phenotype accumulated in muscles of genetically dystrophic mice. Punctual depletion of Treg cells during the repair process prolonged the proinflammatory infiltrate and impaired muscle repair, while treatments that increased or decreased Treg activities diminished or enhanced (respectively) muscle damage in a dystrophy model. Muscle Treg cells expressed the growth factor Amphiregulin, which acted directly on muscle satellite cells in vitro and improved muscle repair in vivo. Thus, Treg cells and their products may provide new therapeutic opportunities for wound repair and muscular dystrophies.

Regulatory T cells in nonlymphoid tissues.

Both Foxp3(+)CD4(+) regulatory T cells (Treg cells) and local immune responses in nonlymphoid tissues have long been recognized as important elements of a well-orchestrated immune system, but only recently have these two fields of study begun to intersect. There is growing evidence that Treg cells are present in various nonlymphoid tissues in health and disease, that they have a unique phenotype and that their functions go beyond the classical modulation of immune responses. Thus, tissue Treg cells might add yet another level to classification of the Treg cell compartment into functional and/or phenotypic subtypes. In this Review, we summarize recent findings in this new field, discussing knowns and unknowns about the origin, phenotype, function and memory of nonlymphoid tissue-resident Treg cells.

Increases in IgA(+) B cells in Peyer's patches during milk-borne mouse mammary tumor virus infection are influenced by Toll-like receptor 4 and are completely dependent on the superantigen response.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Although mammary glands are the final target of infection, Peyer's patches (PP) are the entry site of the virus. Herein, we show that the infection induces increases in the number of PP IgA(+) B cells and higher expression of the α circular transcript, which is a specific marker of the switch to IgA. In addition, IgA(+) B-cell increases correlated with higher levels of cytokines related to IgA class switching, such as interleukin (IL)-5 and IL-6. Of interest, the increases in IgA(+) B cells were lower in Toll-like receptor 4-deficient mice and were completely dependent on the presence of superantigen-reactive T cells. Our results point to a novel mechanism involved in MMTV infection and suggest that IgA(+) B cells may play an important role in carrying the virus to the mammary glands.

Early increases in superantigen-specific Foxp3+ regulatory T cells during mouse mammary tumor virus infection.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Here, we show in a natural model of MMTV infection that the virus causes early and progressive increases in superantigen (SAg)-specific Foxp3(+) regulatory T cells (T(reg)) in Peyer's patches (PP). These increases were shown to be dependent on the presence of dendritic cells. CD4(+) CD25(+) T cells from the PP of infected mice preferentially suppress the proliferative response of T cells to SAg-expressing antigen-presenting cells ex vivo. We investigated the influence of the depletion of CD25(+) cells at different stages of the infection. When CD25(+) cells were depleted before MMTV infection, an increase in the number of PP SAg-cognate Foxp3(-) T cells was found at day 6 of infection. Since the SAg response is associated with viral amplification, the possibility exists that T(reg) cells attenuate the increase in viral load at the beginning of the infection. In contrast, depletion of CD25(+) cells once the initial SAg response has developed caused a lower viral load, suggesting that at later stages T(reg) cells may favor viral persistence. Thus, our results indicated that T(reg) cells play an important and complex role during MMTV infection.

Critical role of dendritic cells in mouse mammary tumor virus in vivo infection.

Mouse mammary tumor virus (MMTV) is a milk-transmitted betaretrovirus that causes mammary tumors in mice. Although mammary epithelial cells are the ultimate targets of MMTV, the virus utilizes components of the host immune system to establish infection. Previous studies indicated that dendritic cells play a role in MMTV infection. Here we show that dendritic cells are the first cells to be infected by MMTV in vivo and that they are capable of producing infectious virus that can be transmitted to other cell types. Moreover, upon contact with the virus, dendritic cells became more mature and migrated in response to the chemokine macrophage inflammatory protein 3beta. Finally, we demonstrate that targeted ablation of dendritic cells in vivo dramatically attenuated MMTV infection. These data indicate that MMTV infection of dendritic cells is critical to initial propagation of the virus in vivo.

Binding of natural variants of staphylococcal superantigens SEG and SEI to TCR and MHC class II molecule.

SEG and SEI are staphylococcal superantigens (SAgs) identified recently that belong to the egc operon and whose genes are in tandem orientation. Only a few allelic variants of SEG and SEI have been reported. Here we analyzed four Staphylococcus aureus strains with genotypic variation in both SAgs. However, both SAgs retain key residues in their putative TCR and MHC binding sites and, accordingly, their superantigenic properties. Thus, SEI significantly stimulates mouse T-cells bearing Vbeta3, 5 and 13, while SEG stimulates Vbeta7 and 9 in the draining node when inoculated in the footpad. As another member of the SEB subfamily, SEG also stimulates mouse Vbeta8.1+2. However, the increase in Vbeta8.1+2 T-cells observed at day 2 after inoculation reverts to normal values at day 4, whereas it remains high at day 4 following inoculation with SEC3 or SSA. T-cell stimulation assays in the mouse and analysis of the putative Vbeta8.2 binding site on SEG, which includes three non-conserved residues, suggest a possibly unique interaction between Vbeta8.2 and SEG. We also analyzed biochemical and biophysical characteristics of SEI and SEG binding to their cognate human beta chains by surface plasmon resonance, and binding to the HLA-DR1 MHC class II molecule by gel filtration. SEI binds human Vbeta5.2 and Vbeta1 with apparent K(D)'s of 23 and 118 microM, respectively; SEG binds Vbeta13.6 with a K(D) of 5 microM. As suggested by sequence homology, SEI requires Zn2+ for strong binding to DR1, which goes undetected in the presence of EDTA. SEG and SEI have characteristics such as co-expression, different interaction with MHC class II and stimulation of completely different subsets of human and mouse T-cells, which indicate complementary superantigenic activity and suggest an important advantage to staphylococcal strains in producing them both.

Cathepsin-L influences the expression of extracellular matrix in lymphoid organs and plays a role in the regulation of thymic output and of peripheral T cell number.

Nackt mice, which are deficient in cathepsin-L (CTSL), show an early impairment during positive selection in the context of class II MHC molecules and as a consequence, the percentage and absolute number of CD4(+) thymocytes are significantly decreased. In this study, we show that lymph nodes from nackt mice are hypertrophied, showing normal absolute numbers of CD4(+) T cells and marked increases in the number of CD8(+) T lymphocytes. Basal proliferative levels are increased in the CD4(+) but not in the CD8(+) population. Lymph node T cells show increases in the expression of alpha(5), alpha(6), and beta(1) integrin chains. These alterations correlate with increases in the expression of extracellular matrix (ECM) components in lymph nodes. Interestingly, laminin, fibronectin, and collagen I and IV are markedly decreased in nackt thymus which shows an augmented output of CD8(+) cells. These results demonstrate that a mutation in the Ctsl gene influences the levels of ECM components in lymphoid organs, the thymic output, and the number of T cells in the periphery. They further raise the possibility that, by regulating the level of expression of ECM components in lymphoid organs, CTSL is able to broadly affect the immune system.

Toll-like receptor 4-dependent activation of dendritic cells by a retrovirus.

Mouse mammary tumor virus (MMTV) is a milk-borne retrovirus that exploits the adaptive immune system. It has recently been shown that MMTV activates B cells via Toll-like receptor 4 (TLR4), a molecule involved in innate immune responses. Here, we show that direct virus binding to TLR4 induced maturation of bone marrow-derived dendritic cells and up-regulated expression of the MMTV entry receptor (CD71) on these cells. In vivo, MMTV increased the number of dendritic cells in neonatal Peyer's patches and their expression of CD71; both these effects were dependent on TLR4. Thus, retroviral signaling through TLRs plays a critical role in dendritic-cell participation during infection.

Decrease in cAMP levels modulates adhesion to fibronectin and immunostimulatory ability of human dendritic cells.

The aim of the present study was to analyze the early events elicited by tumor necrosis factor alpha (TNF-alpha) on monocyte-derived dendritic cells (moDC) adhesion to fibronectin (FN) and the involvement of cAMP in the signal transduction mechanism. The intracellular concentration of cAMP and moDC adhesion to FN decreased after TNF-alpha treatment. An inverted dose-dependency for TNF-alpha effect was observed for adhesion and cAMP levels. The presence of a phosphodiesterase (PDE) inhibitor (IBMX) and cAMP analogs (8Br-cAMP, Db-cAMP) reversed the observed TNF-alpha effects. The role of cAMP was analyzed further by examining the cAMP levels in nonadhered and adhered, TNF-alpha-treated moDC. Nonadhered moDC showed lower cAMP levels compared with adhered moDC. Furthermore, nonadhered moDC showed higher IL-12 content and allostimulatory ability compared with adhered moDC. The higher allostimulatory capacity was abolished in the presence of cAMP analogs and a PDE inhibitor. These results suggest that cAMP levels correlate with TNF-alpha-induced changes of moDC adhesion and allostimulatory capacity.