Retargeting IL-2 Signaling to NKG2D-Expressing Tumor-Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy.

Ex vivo expansion followed by reinfusion of tumor-infiltrating leukocytes (TILs) has been used successfully for the treatment of multiple malignancies. Most protocols rely on the use of the cytokine IL-2 to expand TILs prior to reinfusion. In addition, TIL administration relies on systemic administration of IL-2 after reinfusion to support transferred cell survival. The use of IL-2, however, can be problematic because of its preferential expansion of regulatory T and myeloid cells as well as its systemic side effects. In this study, we describe the use of a novel IL-2 mutant retargeted to NKG2D rather than the high-affinity IL-2R for TIL-mediated immunotherapy in a murine model of malignant melanoma. We demonstrate that the NKG2D-retargeted IL-2 (called OMCPmutIL-2) preferentially expands TIL-resident CTLs, such as CD8+ T cells, NK cells, and γδT cells, whereas wild-type IL-2 provides a growth advantage for CD4+Foxp3+ T cells as well as myeloid cells. OMCPmutIL-2-expanded CTLs express higher levels of tumor-homing receptors, such as LFA-1, CD49a, and CXCR3, which correlate with TIL localization to the tumor bed after i.v. injection. Consistent with this, OMCPmutIL-2-expanded TILs provided superior tumor control compared with those expanded in wild-type IL-2. Our data demonstrate that adoptive transfer immunotherapy can be improved by rational retargeting of cytokine signaling to NKG2D-expressing CTLs rather than indiscriminate expansion of all TILs.

Ischemia reperfusion injury facilitates lung allograft acceptance through IL-33-mediated activation of donor-derived IL-5 producing group 2 innate lymphoid cells.

Pathways regulating lung alloimmune responses differ from most other solid organs and remain poorly explored. Based on our recent work identifying the unique role of eosinophils in downregulating lung alloimmunity, we sought to define pathways contributing to eosinophil migration and homeostasis. Using a murine lung transplant model, we have uncovered that immunosuppression increases eosinophil infiltration into the allograft in an IL-5-dependent manner. IL-5 production depends on immunosuppression-mediated preservation of donor-derived group 2 innate lymphoid cells (ILC2). We further describe that ischemia reperfusion injury upregulates the expression of IL-33, which functions as the dominant and nonredundant mediator of IL-5 production by graft-resident ILC2. Our work thus identifies unique cellular mechanisms that contribute to lung allograft acceptance. Notably, ischemia reperfusion injury, widely considered to be solely deleterious to allograft survival, can also downregulate alloimmune responses by initiating unique pathways that promote IL-33/IL-5/eosinophil-mediated tolerance.

Vendor-specific microbiome controls both acute and chronic murine lung allograft rejection by altering CD4+ Foxp3+ regulatory T cell levels.

Despite standardized postoperative care, some lung transplant patients suffer multiple episodes of acute and chronic rejection while others avoid graft problems for reasons that are poorly understood. Using an established model of C57BL/10 to C57BL/6 minor antigen mismatched single lung transplantation, we now demonstrate that the recipient microbiota contributes to variability in the alloimmune response. Specifically, mice from the Envigo facility in Frederick, Maryland contain nearly double the number of CD4+ Foxp3+ regulatory T cells (Tregs ) than mice from the Jackson facility in Bar Harbor, Maine or the Envigo facility in Indianapolis, Indiana (18 vs 9 vs 7%). Lung graft recipients from the Maryland facility thus do not develop acute or chronic rejection. Treatment with broad-spectrum antibiotics decreases Tregs and increases both acute and chronic graft rejection in otherwise tolerant strains of mice. Constitutive depletion of regulatory T cells, using Foxp3-driven expression of diphtheria toxin receptor, leads to the development of chronic rejection and supports the role of Tregs in both acute and chronic alloimmunity. Taken together, our data demonstrate that the microbiota of certain individuals may contribute to tolerance through Treg -dependent mechanisms and challenges the practice of indiscriminate broad-spectrum antibiotic use in the perioperative period.

The characteristics of ctDNA reveal the high complexity in matching the corresponding tumor tissues.

BACKGROUND: Next-generation sequencing (NGS) is an efficient and sensitive method to detect mutations from ctDNA. Many features and clinical conditions could significantly affect the concordance between ctDNA and corresponding tumor tissues. Our goal was to systematically investigate the critical factors contributing to different concordance between ctDNA and corresponding tumor tissues. METHODS: We recruited two groups of IIIB or IV lung cancer patients: The standard group to evaluate the accuracy of our method and the concordance between ctDNA and tumor tissues, and the study group with various clinical conditions. We applied our unique identification (UID) indexed capturing-based sequencing (UC-Seq) to ctDNA samples, and confirm the results by Droplet digital PCR (ddPCR). RESULTS: Considering mutations detected from NGS of tumor tissues as golden standard, UC-Seq achieved overall 93.6% sensitivity for SNVs and Indels, and 0.8 Pearson correlation between tumor TMB and bTMB. Efficacious treatments, long sampling date (more than 2 weeks) between tumor tissues and ctDNA and low concentrations of cfDNA (less than 9 ng/ml) could significantly decrease the concordance between ctDNA and tumor tissues. About 84% mutations showed shorter mutant fragment length than that of wild-type fragments, and the AFs of mutations could be significantly enriched in small-size ctDNA. CONCLUSIONS: In late-stage lung cancer patients, ctDNA generally has high concordance with tumor tissues. However it could be significantly affected by three clinical conditions which could dynamically change the content of ctDNA. Moreover, the detection limit could be further extended by enriching small-size ctDNA in the preparation of samples.

Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) Regulates Osteoclast Differentiation via the Ca2+ /NFATc1 Axis.

DC-STAMP is a multi-pass transmembrane protein essential for cell-cell fusion between osteoclast precursors during osteoclast (OC) development. DC-STAMP-/- mice have mild osteopetrosis and form mononuclear cells with limited resorption capacity. The identification of an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) on the cytoplasmic tail of DC-STAMP suggested a potential signaling function. The absence of a known DC-STAMP ligand, however, has hindered the elucidation of downstream signaling pathways. To address this problem, we engineered a light-activatable DC-STAMP chimeric molecule in which light exposure mimics ligand engagement that can be traced by downstream Ca2+ signaling. Deletion of the cytoplasmic ITIM resulted in a significant elevation in the amplitude and duration of intracellular Ca2+ flux. Decreased NFATc1 expression in DC-STAMP-/- cells was restored by DC-STAMP over-expression. Multiple biological phenotypes including cell-cell fusion, bone erosion, cell mobility, DC-STAMP cell surface distribution, and NFATc1 nuclear translocation were altered by deletion of the ITIM and adjacent amino acids. In contrast, mutations on each of the tyrosine residues surrounding the ITIM showed no effect on DC-STAMP function. Collectively, our results suggest that the ITIM on DC-STAMP is a functional motif that regulates osteoclast differentiation through the NFATc1/Ca2+ axis. J. Cell. Physiol. 232: 2538-2549, 2017. © 2016 Wiley Periodicals, Inc.

Eosinophils restrain humoral alloimmunity after lung transplantation.

While the function of many leukocytes in transplant biology has been well defined, the role of eosinophils is controversial and remains poorly explored. Conflicting data exist regarding eosinophils' role in alloimmunity. Due to their prevalence in the lung, and their defined role in other pulmonary pathologies such as asthma, we set out to explore the role of eosinophils in the long-term maintenance of the lung allograft. We noted that depletion of eosinophils results in the generation of donor-specific antibodies. Eosinophil depletion increased memory B cell, plasma cell, and antibody-secreting cell differentiation and resulted in de novo generation of follicular germinal centers. Germinal center formation depended on the expansion of CD4+Foxp3-Bcl6+CXCR5+PD-1+ T follicular helper (Tfh) cells, which increase in number after eosinophil depletion. Mechanistically, we demonstrate that eosinophils prevent Tfh cell generation by acting as the dominant source of IFN-γ in an established lung allograft, thus facilitating Th1 rather than Tfh polarization of naive CD4+ T cells. Our data thus describe what we believe is a unique and previously unknown role for eosinophils in maintaining allograft tolerance and suggest that indiscriminate administration of eosinophil-lytic corticosteroids for treatment of acute cellular rejection may inadvertently promote humoral alloimmunity.

Efavirenz stimulates HIV-1 reverse transcriptase RNase H activity by a mechanism involving increased substrate binding and secondary cleavage activity.

Efavirenz is a non-nucleoside reverse transcriptase inhibitor used for treating HIV/AIDS. We found that polymerization activity of a reverse transcriptase (RT) with the E478Q mutation that inactivates the RNase H catalytic site is much more sensitive to efavirenz than wild-type RT, indicating that a functional RNase H attenuates the effectiveness of efavirenz. Moreover, efavirenz actually stimulated wild-type RNase H binding and catalytic functions, indicating another link between efavirenz action and RNase H function. During reverse transcription in vivo, the RT that is extending the DNA primer also periodically cleaves the genomic RNA. The RNase H makes primary template cuts ~18 nucleotides from the growing DNA 3'-end, and when the RT pauses synthesis, it shifts to make secondary cuts ~9 nucleotides from the DNA 3'-end. After synthesis, RTs return to bind the remaining template RNA segments at their 5'-ends and make primary and secondary cuts, 18 and 9 nucleotides in, respectively. We found that efavirenz stimulates both 3'- and 5'-directed RNase H activity. Use of specific substrates revealed a particular acceleration of secondary cuts. Efavirenz specifically promoted binding of the RT to RNase H substrates, suggesting that it stabilizes the shifting of RTs to make the secondary cuts. We further showed that efavirenz similarly stimulates the RNase H of an RT from a patient-derived virus that is highly resistant and grows more rapidly in the presence of low concentrations of efavirenz. We suggest that for efavirenz-resistant RTs, stimulated RNase H activity contributes to increased viral fitness.

Reverse transcriptase backbone can alter the polymerization and RNase activities of non-nucleoside reverse transcriptase mutants K101E+G190S.

Previous work by our group showed that human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing non-nucleoside RT inhibitor (NNRTI) drug resistance mutations has defects in RNase H activity as well as reduced amounts of RT protein in virions. These deficits correlate with replication fitness in the absence of NNRTIs. Viruses with the mutant combination K101E+G190S replicated better in the presence of NNRTIs than in the absence of drug. Stimulation of virus growth by NNRTIs occurred during the early steps of the virus life cycle and was modulated by the RT backbone sequence in which the resistance mutations arose. We wanted to determine what effects RT backbone sequence would have on RT content and polymerization and RNase H activities in the absence of NNRTIs. We compared a NL4-3 RT with K101E+G190S to a patient-isolate RT sequence D10 with K101E+G190S. We show here that, unlike the NL4-3 backbone, the D10 backbone sequence decreased the RNA-dependent DNA polymerization activity of purified recombinant RT compared to WT. In contrast, RTs with the D10 backbone had increased RNase H activity compared to WT and K101E+G190S in the NL4-3 backbone. D10 virions also had increased amounts of RT compared to K101E+G190S in the NL4-3 backbone. We conclude that the backbone sequence of RT can alter the activities of the NNRTI drug-resistant mutant K101E+G190S, and that identification of the amino acids responsible will aid in understanding the mechanism by which NNRTI drug-resistant mutants alter fitness and NNRTIs stimulate HIV-1 virus replication.

L74V increases the reverse transcriptase content of HIV-1 virions with non-nucleoside reverse transcriptase drug-resistant mutations L100I+K103N and K101E+G190S, which results in increased fitness.

The fitness of non-nucleoside reverse transcriptase inhibitor (NNRTI) drug-resistant reverse transcriptase (RT) mutants of HIV-1 correlates with the amount of RT in the virions and the RNase H activity of the RT. We wanted to understand the mechanism by which secondary NNRTI-resistance mutations, L100I and K101E, and the nucleoside resistance mutation, L74V, alter the fitness of K103N and G190S viruses. We measured the amount of RT in virions and the polymerization and RNase H activities of mutant RTs compared to wild-type, K103N and G190S. We found that L100I, K101E and L74V did not change the polymerization or RNase H activities of K103N or G190S RTs. However, L100I and K101E reduced the amount of RT in the virions and subsequent addition of L74V restored RT levels back to those of G190S or K103N alone. We conclude that fitness changes caused by L100I, K101E and L74V derive from their effects on RT content.

Circ8199 encodes a protein that inhibits the activity of OGT by JAK2-STAT3 pathway in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is an invasive malignant tumor with a high incidence rate and mortality. It is imperative to study its tumorigenesis and development for better treatment. CircRNA has been proven to play an important role in various cancers. Our previous studies found that the circ8199 gene is associated with tumor prognosis. To further clarify the role of circ8199 in ESCC, we performed functional experiments and found that overexpression of circ8199 significantly inhibited the proliferation of ESCC cells and the activity of O-linked N-acetylglucosamine transferase (OGT) simultaneously. Further experiments demonstrated that circ8199 could interact with OGT, leading to a decrease in OGT's activity. The reduction of circ8199 expression stimulated the binding activity between OGT and its downstream gene JAK2, promoting the O-GlcNAc glycosylation modification of JAK2 and activating the JAK2-STAT3 pathway. Our study indicated that circ8199 regulates the JAK2-STAT3 pathway through OGT, providing a candidate mechanism for drug discovery and development.

Nonnucleoside reverse transcriptase inhibitor-resistant HIV is stimulated by efavirenz during early stages of infection.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent and commonly prescribed antiviral agents used in combination therapy (CART) of human immunodeficiency virus type 1 (HIV-1) infection. The development of drug resistance is a major limitation of CART. Reverse transcriptase (RT) genotypes with the NNRTI resistance mutations K101E+G190S are highly resistant to efavirenz (EFV) and can develop during failure of EFV-containing regimens in patients. We have previously shown that virus with K101E+G190S mutations can replicate more efficiently in the presence of EFV than in its absence. In this study, we evaluated the underlying mechanism for drug-dependent stimulation, using a single-cycle cell culture assay in which EFV was added either during the infection or the virus production step. We determined that EFV stimulates K101E+G190S virus during early infection and does not affect late steps of virus replication, such as increasing the amount of active RT incorporated into virions. Additionally, we showed that another NNRTI, nevirapine (NVP), stimulated K101E+G190S virus replication during the early steps of infection similar to EFV, but that the newest NNRTI, etravirine (ETR), did not. We also showed that EFV stimulates K101E+Y188L and K101E+V106I virus, but not K101E+L100I, K101E+K103N, K101E+Y181C, or K101E+G190A virus, suggesting that the stimulation is mutation specific. Real-time PCR of reverse transcription intermediates showed that although the drug did not stimulate minus-strand transfer, it did stimulate minus-strand strong-stop DNA synthesis. Our results indicate that stimulation most likely occurs through a mechanism whereby NNRTIs stimulate priming or elongation of the tRNA.

Loss of stromal cell Thy-1 plays a critical role in lipopolysaccharide induced chronic lung allograft dysfunction.

BACKGROUND: Long-term survival of lung transplants lags behind other solid organs due to early onset of a fibrotic form of chronic rejection known as chronic lung allograft dysfunction (CLAD). Preventing CLAD is difficult as multiple immunologic and physiologic insults contribute to its development. Targeting fibroblast activation, which is the final common pathway leading to CLAD, offers the opportunity to ameliorate fibrosis irrespective of the initiating insult. Thy-1 is a surface glycoprotein that controls fibroblast differentiation and activation. METHODS: To study the role of Thy-1 in CLAD, we utilized the minor antigen mismatched C57BL/6 (B6wild-type) or B6Thy-1-/-→C57BL/10 (B10) model of murine orthotopic lung transplantation with postoperative bacterial infection modeled by intratracheal lipopolysaccharide (LPS) administration. The effects of LPS on Thy-1 expression, proliferation, and gene expression were assessed in fibroblasts in vitro and the therapeutic potential of Thy-1 replacement was assessed in vivo. RESULTS: More severe CLAD was evident in B6Thy-1-/- →B10 grafts compared to B6wild-type →B10 grafts. LPS further accentuated fibrosis in B6wild-type →B10 grafts with some, but limited, effects on B6Thy-1-/- →B10 grafts. LPS contributed to Thy-1 loss from Thy-1(+) fibroblasts in vitro due to a decrease in mRNA expression. In addition, LPS promoted proliferation and upregulation of multiple inflammatory pathways in Thy-1(-) fibroblasts by gene expression analysis. Most importantly, replacement of Thy-1 through exogenous administration ameliorated the fibrotic phenotype post-LPS mediated modeling of infection. CONCLUSIONS: Our findings suggest that the loss of Thy-1 on fibroblasts is a previously unrecognized cause of CLAD and its replacement may offer therapeutic applications for amelioration of this disease post-transplantation in the setting of infectious stress responses.

A reengineered common chain cytokine augments CD8+ T cell-dependent immunotherapy.

Cytokine therapy is limited by undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here, we describe the signaling properties of a potentially unique cytokine by design, where T cell surface binding and signaling are separated between 2 different families of receptors. This fusion protein cytokine, called OMCPmutIL-2, bound with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signaled through the common γ chain cytokine receptor. In addition to precise activation of cytotoxic T cells due to redirected binding, OMCPmutIL-2 resulted in superior activation of both human and murine CD8+ T cells by improving their survival and memory cell generation and decreasing exhaustion. This functional improvement was the direct result of altered signal transduction based on the reorganization of surface membrane lipid rafts that led to Janus kinase-3-mediated phosphorylation of the T cell receptor rather than STAT/AKT signaling intermediates. This potentially novel signaling pathway increased CD8+ T cell response to low-affinity antigens, activated nuclear factor of activated T cells transcription factors, and promoted mitochondrial biogenesis. OMCPmutIL-2 thus outperformed other common γ chain cytokines as a catalyst for in vitro CD8+ T cell expansion and in vivo CD8+ T cell-based immunotherapy.

Eosinophils downregulate lung alloimmunity by decreasing TCR signal transduction.

Despite the accepted notion that granulocytes play a universally destructive role in organ and tissue grafts, it has been recently described that eosinophils can facilitate immunosuppression-mediated acceptance of murine lung allografts. The mechanism of eosinophil-mediated tolerance, or their role in regulating alloimmune responses in the absence of immunosuppression, remains unknown. Using lung transplants in a fully MHC-mismatched BALB/c (H2d) to C57BL/6 (H2b) strain combination, we demonstrate that eosinophils downregulate T cell-mediated immune responses and play a tolerogenic role even in the absence of immunosuppression. We further show that such downregulation depends on PD-L1/PD-1-mediated synapse formation between eosinophils and T cells. We also demonstrate that eosinophils suppress T lymphocyte responses through the inhibition of T cell receptor/CD3 (TCR/CD3) subunit association and signal transduction in an inducible NOS-dependent manner. Increasing local eosinophil concentration, through administration of intratracheal eotaxin and IL-5, can ameliorate alloimmune responses in the lung allograft. Thus, our data indicate that eosinophil mobilization may be utilized as a novel means of lung allograft-specific immunosuppression.

Prenatal exposure to fluoxetine induces fetal pulmonary hypertension in the rat.

RATIONALE: Fluoxetine is a selective serotonin reuptake inhibitor antidepressant widely used by pregnant women. Epidemiological data suggest that fluoxetine exposure prenatally increases the prevalence of persistent pulmonary hypertension syndrome of the newborn. The mechanism responsible for this effect is unclear and paradoxical, considering the current evidence of a pulmonary hypertension protective fluoxetine effect in adult rodents. OBJECTIVES: To evaluate the fluoxetine effect on fetal rat pulmonary vascular smooth muscle mechanical properties and cell proliferation rate. METHODS: Pregnant rats were treated with fluoxetine (10 mg/kg) from Day 11 through Day 21 of gestation. MEASUREMENTS AND MAIN RESULTS: Fetuses were delivered by cesarean section. As compared with controls, fluoxetine exposure resulted in fetal pulmonary hypertension as evidenced by an increase in the weight ratio of the right ventricle to the left ventricle plus septum (P = 0.02) and by an increase in pulmonary arterial medial thickness (P < 0.01). Postnatal mortality was increased among experimental animals, and arterial oxygen saturation was 96 +/- 1% in 1-day-old control animals and significantly lower (P < 0.01) in fluoxetine-exposed pups (79 +/- 2%). In vitro, fluoxetine induced pulmonary arterial muscle contraction in fetal, but not adult, animals (P < 0.01) and reduced serotonin-induced contraction at both ages (P < 0.01). After in utero exposure to a low fluoxetine concentration the pulmonary arterial smooth muscle cell proliferation rate was significantly increased in fetal, but not adult, cells (P < 0.01). CONCLUSIONS: In contrast to the adult, fluoxetine exposure in utero induces pulmonary hypertension in the fetal rat as a result of a developmentally regulated increase in pulmonary vascular smooth muscle proliferation.

Identification and functional analysis of the novel ORF6 protein of porcine circovirus type 2 in vitro.

In the present study, the function of a novel ORF6 gene in the PCV2 genome was determined and functionally analyzed in vitro. ORF6 expression was demonstrated by indirect immunofluorescence in PCV2-infected cells. The antibody against ORF6 was detected in PCV2-infected pigs. The start codon of ORF6 was mutated and an infectious clone was used to create an ORF6-deficient mutant virus. Viral DNA replication curves and immunofluorescence analysis indicated that ORF6 is unnecessary for viral replication and ORF6 deletion reduces viral DNA replication in PK-15 cells. The activities of caspases 3 and 8 in ORF6-deficient virus-infected cells were significantly different from those in wild-type virus-infected cells. The ORF6 protein can increase the expression of IFN-ß, TNF-α, IL-1b, IL-10, and IL-12p40. These results demonstrated that the newly discovered ORF6 protein may be involved in caspases regulation and the expression of multiple cytokines in PCV2-infected cells. The functions of this gene in viral pathogenesis remain to be further elucidated.

Airway epithelial-derived factor relaxes pulmonary vascular smooth muscle.

The factors controlling the pulmonary vascular resistance under physiological conditions are poorly understood. We have previously reported on an apparent cross talk between the airway and adjacent pulmonary arterial bed where a factor likely derived from the bronchial epithelial cells reduced the magnitude of agonist-stimulated force in the vascular smooth muscle. The main purpose of this investigation was to evaluate whether bronchial epithelial cells release a pulmonary arterial smooth muscle relaxant factor. Conditioned media from SPOC-1 or BEAS-2B, a rat- and a human-derived bronchial epithelial cell line, respectively, were utilized. This media significantly relaxed precontracted adult but not fetal pulmonary arterial muscle in an oxygen tension-dependent manner. This response was mediated via soluble guanylate cyclase, involving AKT/PI3-kinase and neuronal nitric oxide synthase. Airway epithelial cell-conditioned media increased AKT phosphorylation in pulmonary smooth muscle cells (SMC) and reduced intracellular calcium change following ATP stimulation to a significantly greater extent than observed for bronchial SMC. The present data strongly support the evidence for bronchial epithelial cells releasing a stable and soluble factor capable of inducing pulmonary arterial SMC relaxation. We speculate that under physiological conditions, the maintenance of a low pulmonary vascular resistance, postnatally, is in part modulated by the airway epithelium.