Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry.

Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.

Keratinocytes produce IL-17c to protect peripheral nervous systems during human HSV-2 reactivation.

Despite frequent herpes simplex virus (HSV) reactivation, peripheral nerve destruction and sensory anesthesia are rare. We discovered that skin biopsies obtained during asymptomatic human HSV-2 reactivation exhibit a higher density of nerve fibers relative to biopsies during virological and clinical quiescence. We evaluated the effects of HSV infection on keratinocytes, the initial target of HSV replication, to better understand this observation. Keratinocytes produced IL-17c during HSV-2 reactivation, and IL-17RE, an IL-17c-specific receptor, was expressed on nerve fibers in human skin and sensory neurons in dorsal root ganglia. In ex vivo experiments, exogenous human IL-17c provided directional guidance and promoted neurite growth and branching in microfluidic devices. Exogenous murine IL-17c pretreatment reduced apoptosis in HSV-2-infected primary neurons. These results suggest that IL-17c is a neurotrophic cytokine that protects peripheral nerve systems during HSV reactivation. This mechanism could explain the lack of nerve damage from recurrent HSV infection and may provide insight to understanding and treating sensory peripheral neuropathies.

Genetic deletion of toll-like receptor 4 on platelets attenuates experimental pulmonary hypertension.

RATIONALE: Recent studies demonstrate a role for toll-like receptor 4 (TLR4) in the pathogenesis of pulmonary hypertension (PH); however, the cell types involved in mediating the effects of TLR4 remain unknown. OBJECTIVES: The objective of this study was to determine the contribution of TLR4 expressed on nonparenchymal cells to the pathogenesis of PH. METHODS AND RESULTS: TLR4 bone marrow chimeric mice revealed an equal contribution of TLR4 on nonparenchymal and parenchymal cells in the pathogenesis of PH as determined by measuring right ventricular (RV) systolic pressure and RV hypertrophy. However, the deletion of TLR4 from myeloid lineage cells had no effect on the development of PH because we found no difference in RV systolic pressure or RV hypertrophy in wild-type versus LysM-TLR4(-/-) mice. To explore the potential role of platelet TLR4 in the pathogenesis of PH, platelet-specific TLR4(-/-) mice were generated (PF4-TLR4(-/-) mice). TLR4(-/-) platelets from either global TLR4(-/-) or PF4-TLR4(-/-) mice were functional but failed to respond to lipopolysaccharide, demonstrating a lack of TLR4. PF4-TLR4(-/-) mice demonstrated significant protection from hypoxia-induced PH, including attenuated increases in RV systolic pressure and RV hypertrophy, decreased platelet activation, and less pulmonary vascular remodeling. The deletion of TLR4 from platelets attenuated serotonin release after chronic hypoxia, and lipopolysaccharide-stimulated platelets released serotonin and promoted pulmonary artery smooth muscle cell proliferation in a serotonin-dependent manner. CONCLUSIONS: Our data demonstrate that TLR4 on platelets contributes to the pathogenesis of PH and further highlights the role of platelets in PH.

Inducible nitric oxide synthase contributes to immune dysfunction following trauma.

Trauma results in a persistent depression in adaptive immunity, which contributes to patient morbidity and mortality. This state of immune paralysis following trauma is characterized by a change in cell-mediated immunity, specifically a depression in T-cell function and a shift toward TH2 T-cell phenotype. Upregulation of inducible nitric oxide synthase (iNOS) is well recognized after injury and contributes to the inflammatory response and organ damage early after trauma. However, it is unknown whether iNOS plays a role in adaptive immune dysfunction after trauma. This study utilized a murine model of severe peripheral tissue injury to show that iNOS is rapidly upregulated in macrophages and a (Gr-1-CD11b) myeloid-derived suppressor cell subpopulation in the spleen. Through the use of iNOS knockout mice, a specific iNOS inhibitor, and a nitric oxide (NO) scavenger, this study demonstrates that iNOS-derived NO is required for the depression in T-lymphocyte proliferation, interferon γ, and interleukin 2 production within the spleen at 48 h after trauma. These findings support the hypothesis that iNOS regulates immune suppression following trauma and suggest that targeting the sustained production of NO by iNOS may attenuate posttraumatic immune depression.

A role for cGMP in inducible nitric-oxide synthase (iNOS)-induced tumor necrosis factor (TNF) α-converting enzyme (TACE/ADAM17) activation, translocation, and TNF receptor 1 (TNFR1) shedding in hepatocytes.

We and others have previously shown that the inducible nitric-oxide synthase (iNOS) and nitric oxide (NO) are hepatoprotective in a number of circumstances, including endotoxemia. In vitro, hepatocytes are protected from tumor necrosis factor (TNF) α-induced apoptosis via cGMP-dependent and cGMP-independent mechanisms. We have shown that the cGMP-dependent protective mechanisms involve the inhibition of death-inducing signaling complex formation. We show here that LPS-induced iNOS expression leads to rapid TNF receptor shedding from the surface of hepatocytes via NO/cGMP/protein kinase G-dependent activation and surface translocation of TNFα-converting enzyme (TACE/ADAM17). The activation of TACE is associated with the up-regulation of iRhom2 as well as the interaction and phosphorylation of TACE and iRhom2, which are also NO/cGMP/protein kinase G-dependent. These findings suggest that one mechanism of iNOS/NO-mediated protection of hepatocytes involves the rapid shedding of TNF receptor 1 to limit TNFα signaling.

Nonhemopoietic cell TLR4 signaling is critical in causing early lipopolysaccharide-induced ileus.

Endotoxin-mediated ileus is poorly understood. Our objective was to mechanistically investigate the role of cell-specific TLR4 expression/signaling in causing gastrointestinal dysmotility. TLR4 chimeras and CSF-1-dependent macrophage-deficient mice were subjected to i.p. ultrapure (UP)-LPS (5 mg/kg). At 6 h, gastric emptying and gastrointestinal transit assessed in vivo motility, and jejunal circular muscle contractility was measured in vitro. Muscularis infiltration of neutrophils and monocytes were counted, and intestinal muscularis inflammatory mediators were quantified by quantitative PCR. Demonstrating TLR4 dependency, UP-LPS-induced gastric stasis and ileus of TLR4(WT) mice were absent in mutant TLR4(LPS-d) mice. Unexpectedly, engraftment of TLR4-mutant bone marrow into TLR4-competent mice (bmTLR4(LPS-d)/TLR4(WT)) exhibited a significant transit delay to UP-LPS similar to bmTLR4(WT)/TLR4(WT) mice. CSF-1(-/-) mice were not protected from ileus. Contrary, UP-LPS-treated bmTLR4(WT)/TLR4(LPS-d) and bmTLR4(LPS-d)/TLR4(LPS-d) mice had normal transit. No leukocytic infiltration was detected at 6 h. Spontaneous jejunal contractions were markedly suppressed in UP-LPS-treated TLR4-competent mice, but bethanechol-stimulated contractions were not altered by UP-LPS in any group. UP-LPS-induced inflammatory mRNAs in a TLR4-dependent manner, but TLR4 mRNA itself was not significantly altered. In chimera mice, UP-LPS induction of IL-1beta and IL-10 were hemopoietic dependent, and GM-CSF was nonhemopoietic dependent, whereas IL-6 and inducible NO synthase were derived from both cell types. Hemopoietic and nonhemopoietic cells contribute to TLR4-sensitive muscularis inflammatory signaling, but nonhemopoietic TLR4 signaling plays an exclusive primary role in causing functional UP-LPS-induced gastric stasis and ileus. Direct LPS suppression of spontaneous contractility participates in mediating early TLR4-transduced dysmotility.

Proinflammatory role of leukocyte-derived Egr-1 in the development of murine postoperative ileus.

BACKGROUND & AIMS: Early growth response gene-1 (Egr-1) is an important inflammatory transcription factor. We hypothesize that leukocyte-derived Egr-1 plays a key inflammatory role in causing postoperative ileus. METHODS: Wild-type, Egr-1 knockout, and chimera mice (constructed by irradiation followed by injection with Egr-1(+/+) or Egr-1(-/-) bone marrow) were subjected to surgical manipulation of the gastrointestinal tract to induce ileus. Reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry quantified and localized Egr-1. Lumenal transit of nonabsorbable fluorescein isothiocyanate-labeled dextran and in vitro organ bath techniques measured functional gastrointestinal motility. Inflammatory mediator expressions were measured by Griess reaction, enzyme-linked immunosorbent assay, and multiplex Luminex assay. RESULTS: Intestinal manipulation rapidly and significantly induced Egr-1 messenger RNA and protein within the inflamed muscularis externa. Egr-1 was colocalized early to smooth muscle and enteric neurons and later in extravasated monocytes after surgery when postoperative ileus was functionally prominent. The functional severity of postoperative ileus was significantly ameliorated in mice deficient in Egr-1(-/-) and chimera wild-type mice transplanted with Egr-1(-/-) bone marrow, whereas knockout mice with Egr-1(+/+) bone marrow again displayed significant ileus. Motility was mechanistically associated in Egr-1(-/-) gene deficiency with a down-regulation in the release of nitric oxide, prostanoids, monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, interleukin-6, interleukin-1, and granulocyte colony-stimulating factor, as well as a decrease in the recruitment of leukocytes into the manipulated muscle wall of the intestine compared with wild-type mice. CONCLUSIONS: Leukocyte-derived Egr-1 plays an early critical inflammatory role in the initiation of the postoperative inflammatory response, which leads to a prolonged decreased in gastrointestinal motility after intestinal surgery.