CSF1R inhibition by a small-molecule inhibitor is not microglia specific; affecting hematopoiesis and the function of macrophages.

Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a method for microglia depletion, with the assumption that it does not affect peripheral immune cells. Here, we show that CSF1R inhibition by PLX5622 indeed affects the myeloid and lymphoid compartments, causes long-term changes in bone marrow-derived macrophages by suppressing interleukin 1ß, CD68, and phagocytosis but not CD208, following exposure to endotoxin, and also reduces the population of resident and interstitial macrophages of peritoneum, lung, and liver but not spleen. Thus, small-molecule CSF1R inhibition is not restricted to microglia, causing strong effects on circulating and tissue macrophages that perdure long after cessation of the treatment. Given that peripheral monocytes repopulate the central nervous system after CSF1R inhibition, these changes have practical implications for relevant experimental data.

Glaucoma after Ocular Surgery or Trauma: The Role of Infiltrating Monocytes and Their Response to Cytokine Inhibitors.

Glaucoma is a frequent and devastating long-term complication following ocular trauma, including corneal surgery, open globe injury, chemical burn, and infection. Postevent inflammation and neuroglial remodeling play a key role in subsequent ganglion cell apoptosis and glaucoma. To this end, this study was designed to investigate the amplifying role of monocyte infiltration into the retina. By using three different ocular injury mouse models (corneal suture, penetrating keratoplasty, and globe injury) and monocyte fate mapping techniques, we show that ocular trauma or surgery can cause robust infiltration of bone marrow-derived monocytes into the retina and subsequent neuroinflammation by up-regulation of Tnf, Il1b, and Il6 mRNA within 24 hours. This is accompanied by ganglion cell apoptosis and neurodegeneration. Prompt inhibition of tumor necrosis factor-α or IL-1ß markedly suppresses monocyte infiltration and ganglion cell loss. Thus, acute ocular injury (surgical or trauma) can lead to rapid neuroretinal inflammation and subsequent ganglion cell loss, the hallmark of glaucoma. Infiltrating monocytes play a central role in this process, likely amplifying the inflammatory cascade, aiding in the activation of retinal microglia. Prompt administration of cytokine inhibitors after ocular injury prevents this infiltration and ameliorates the damage to the retina-suggesting that it may be used prophylactically for neuroprotection against post-traumatic glaucoma.

Microglia Regulate Neuroglia Remodeling in Various Ocular and Retinal Injuries.

Reactive microglia and infiltrating peripheral monocytes have been implicated in many neurodegenerative diseases of the retina and CNS. However, their specific contribution in retinal degeneration remains unclear. We recently showed that peripheral monocytes that infiltrate the retina after ocular injury in mice become permanently engrafted into the tissue, establishing a proinflammatory phenotype that promotes neurodegeneration. In this study, we show that microglia regulate the process of neuroglia remodeling during ocular injury, and their depletion results in marked upregulation of inflammatory markers, such as Il17f, Tnfsf11, Ccl4, Il1a, Ccr2, Il4, Il5, and Csf2 in the retina, and abnormal engraftment of peripheral CCR2+ CX3CR1+ monocytes into the retina, which is associated with increased retinal ganglion cell loss, retinal nerve fiber layer thinning, and pigmentation onto the retinal surface. Furthermore, we show that other types of ocular injuries, such as penetrating corneal trauma and ocular hypertension also cause similar changes. However, optic nerve crush injury-mediated retinal ganglion cell loss evokes neither peripheral monocyte response in the retina nor pigmentation, although peripheral CX3CR1+ and CCR2+ monocytes infiltrate the optic nerve injury site and remain present for months. Our study suggests that microglia are key regulators of peripheral monocyte infiltration and retinal pigment epithelium migration, and their depletion results in abnormal neuroglia remodeling that exacerbates neuroretinal tissue damage. This mechanism of retinal damage through neuroglia remodeling may be clinically important for the treatment of patients with ocular injuries, including surgical traumas.

Permanent neuroglial remodeling of the retina following infiltration of CSF1R inhibition-resistant peripheral monocytes.

Previous studies have demonstrated that ocular injury can lead to prompt infiltration of bone-marrow-derived peripheral monocytes into the retina. However, the ability of these cells to integrate into the tissue and become microglia has not been investigated. Here we show that such peripheral monocytes that infiltrate into the retina after ocular injury engraft permanently, migrate to the three distinct microglia strata, and adopt a microglia-like morphology. In the absence of ocular injury, peripheral monocytes that repopulate the retina after depletion with colony-stimulating factor 1 receptor (CSF1R) inhibitor remain sensitive to CSF1R inhibition and can be redepleted. Strikingly, consequent to ocular injury, the engrafted peripheral monocytes are resistant to depletion by CSF1R inhibitor and likely express low CSF1R. Moreover, these engrafted monocytes remain proinflammatory, expressing high levels of MHC-II, IL-1ß, and TNF-α over the long term. The observed permanent neuroglia remodeling after injury constitutes a major immunological change that may contribute to progressive retinal degeneration. These findings may also be relevant to other degenerative conditions of the retina and the central nervous system.

The Role of Microglia and Peripheral Monocytes in Retinal Damage after Corneal Chemical Injury.

Eyes that have experienced alkali burn to the surface are excessively susceptible to subsequent severe glaucoma and retinal ganglion cell loss, despite maximal efforts to prevent or slow down the disease. Recently, we have shown, in mice and rabbits, that such retinal damage is neither mediated by the alkali itself reaching the retina nor by intraocular pressure elevation. Rather, it is caused by the up-regulation of tumor necrosis factor-α (TNF-α), which rapidly diffuses posteriorly, causing retinal ganglion cell apoptosis and CD45+ cell activation. Herein, we investigated the involvement of peripheral blood monocytes and microglia in retinal damage. Using CX3CR1+/EGFP::CCR2+/RFP reporter mice and bone marrow chimeras, we show that peripheral CX3CR1+CD45hiCD11b+MHC-II+ monocytes infiltrate into the retina from the optic nerve at 24 hours after the burn and release further TNF-α. A secondary source of peripheral monocyte response originates from a rare population of patrolling myeloid CCR2+ cells of the retina that differentiate into CX3CR1+ macrophages within hours after the injury. As a result, CX3CR1+CD45loCD11b+ microglia become reactive at 7 days, causing further TNF-α release. Prompt TNF-α inhibition after corneal burn suppresses monocyte infiltration and microglia activation, and protects the retina. This study may prove relevant to other injuries of the central nervous system.

Mechanisms of Retinal Damage after Ocular Alkali Burns.

Alkali burns to the eye constitute a leading cause of worldwide blindness. In recent case series, corneal transplantation revealed unexpected damage to the retina and optic nerve in chemically burned eyes. We investigated the physical, biochemical, and immunological components of retinal injury after alkali burn and explored a novel neuroprotective regimen suitable for prompt administration in emergency departments. Thus, in vivo pH, oxygen, and oxidation reduction measurements were performed in the anterior and posterior segment of mouse and rabbit eyes using implantable microsensors. Tissue inflammation was assessed by immunohistochemistry and flow cytometry. The experiments confirmed that the retinal damage is not mediated by direct effect of the alkali, which is effectively buffered by the anterior segment. Rather, pH, oxygen, and oxidation reduction changes were restricted to the cornea and the anterior chamber, where they caused profound uveal inflammation and release of proinflammatory cytokines. The latter rapidly diffuse to the posterior segment, triggering retinal damage. Tumor necrosis factor-α was identified as a key proinflammatory mediator of retinal ganglion cell death. Blockade, by either monoclonal antibody or tumor necrosis factor receptor gene knockout, reduced inflammation and retinal ganglion cell loss. Intraocular pressure elevation was not observed in experimental alkali burns. These findings illuminate the mechanism by which alkali burns cause retinal damage and may have importance in designing therapies for retinal protection.

Laboratory Evaluation of Hemolysis and Systemic Inflammatory Response in Neonatal Nonpulsatile and Pulsatile Extracorporeal Life Support Systems.

The objective of this study was to compare the systemic inflammatory response and hemolytic characteristics of a conventional roller pump (HL20-NP) and an alternative diagonal pump with nonpulsatile (DP3-NP) and pulsatile mode (DP3-P) in simulated neonatal extracorporeal life support (ECLS) systems. The experimental neonatal ECLS circuits consist of a conventional Jostra HL20 roller pump or an alternative Medos DP3 diagonal pump, and Medos Hilite 800 LT hollow-fiber oxygenator with diffusion membrane. Eighteen sterile circuits were primed with freshly donated whole blood and divided into three groups: conventional HL20 with nonpulsatile flow (HL20-NP), DP3 with nonpulsatile flow (DP3-NP), and DP3 with pulsatile flow (DP3-P). All trials were conducted for durations of 12 h at a flow rate of 500 mL/min at 36°C. Simultaneous blood flow and pressure waveforms were recorded. Blood samples were collected to measure plasma-free hemoglobin (PFH), human tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-8, in addition to the routine blood gas, lactate dehydrogenase, and lactic acid levels. HL20-NP group had the highest PFH levels (mean ± standard error of the mean) after a 12-h ECLS run, but the difference among groups did not reach statistical significance (HL20-NP group: 907.6 ± 253.1 mg/L, DP3-NP group: 343.7 ± 163.2 mg/L, and DP3-P group: 407.6 ± 156.6 mg/L, P = 0.06). Although there were similar trends but no statistical differences for the levels of proinflammatory cytokines among the three groups, the HL20-NP group had much greater levels than the other groups (P > 0.05). Pulsatile flow generated higher total hemodynamic energy and surplus hemodynamic energy levels at pre-oxygenator and pre-clamp sites (P < 0.01). Our study demonstrated that the alternative diagonal pump ECLS circuits appeared to have less systemic inflammatory response and hemolysis compared with the conventional roller pump ECLS circuit in simulated neonatal ECLS systems. Pulsatile flow delivered more hemodynamic energy to the pseudo-patient without increased odds of hemolysis compared with the conventional, nonpulsatile roller pump group.

Apolipoprotein E levels in pediatric patients undergoing cardiopulmonary bypass.

Apolipoprotein E (apoE) may play a critical role in modulating the response to neurological injury after cardiopulmonary bypass (CPB) in children. Plasma samples were collected from 38 pediatric patients. Half of the patients received nonpulsatile flow and the other half underwent pulsatile flow during CPB. Plasma samples were collected at three time points: at baseline prior to incision (T1), 1 h after CPB (T2), and 24 h after CPB (T3). The study included 38 pediatric patients undergoing heart surgery (mean age 2.5 ± 2.1 years). Baseline apoE levels were low (<30 µg/mL) in 21 patients (55%). ApoE levels were significantly decreased at 1 h after CPB compared with baseline (22 ± 14 vs. 34 ± 18 µg/mL, P = 0.001). At 24 h after CPB, apoE levels were significantly increased compared with baseline (47 ± 25 vs. 34 ± 18 µg/mL, P = 0.002). Pulsatile mode was associated with lower apoE levels at 24 h after CPB compared with nonpulsatile mode (38 ± 14 vs. 57 ± 29 µg/mL, P = 0.018). ApoE levels correlated negatively with pump time (r = -0.525, P = 0.021) and cross-clamp time (r = -0.464, P = 0.045) at 24 h following CPB for the nonpulsatile group but not for the pulsatile group. In this cohort of young children with congenital heart disease, baseline apoE levels were low in the majority of patients prior to surgery. ApoE levels decreased further at 1 h after CPB, and then significantly increased by 24 h. The mode of perfusion and the duration of pump time and clamp time influence the apoE levels after CPB. An improved understanding of these mechanisms may translate into the development of new techniques to improve the clinical outcomes after pediatric CPB.

Transgenic expression of survivin compensates for OX40-deficiency in driving Th2 development and allergic inflammation.

Survivin, an inhibitor of apoptosis family molecule, has been proposed as a crucial intermediate in the signaling pathways leading to T-cell development, proliferation, and expansion. However, the importance of survivin to T-cell-driven inflammatory responses has not been demonstrated. Here, we show that survivin transgenic mice exhibit an increased antigen-driven Th2 lung inflammation and that constitutive expression of survivin reversed the defective lung inflammation even in the absence of OX40 costimulation. We found that OX40-deficient mice were compromised in generating Th2 cells, airway eosinophilia, and IgE responses. In contrast, OX40-deficient/survivin transgenic mice generated normal Th2 responses and exhibited strong lung inflammation. These results suggest that OX40 costimulation crucially engages survivin during antigen-mediated Th2 responses. These findings also promote the notion that OX40 costimulation regulates allergic responses or lung inflammation by targeting survivin thereby enhancing T-cell proliferation and resulting in more differentiated Th2 cells in the allergic inflammatory response.

Programming of regulatory T cells from pluripotent stem cells and prevention of autoimmunity.

Regulatory T (Treg) cells are being used to treat autoimmunity and prevent organ rejection; however, Treg cell-based therapies have been hampered by the technical limitation in obtaining a high number of functional Treg cells. In this study, we show how to generate functional Treg cells from induced pluripotent stem (iPS) cells and to determine the potential role of such cells for Treg cell-based immunotherapy against autoimmunity in a therapeutic setting. Ligation of a Notch ligand and transduction of the gene Foxp3 induce iPS cells to differentiate into Treg cells. Expression of Foxp3 and coculture on Notch ligand-expressing stromal cells augment expression of CD3, TCR, CD4, CD25, and CTLA-4 on iPS cell-differentiated Treg cells, which are able to secrete TGF-ß and IL-10 both in vivo and in vitro. Importantly, adoptive transfer of iPS cell-derived Treg cells expressing large amounts of Foxp3 and Bcl-x(L) significantly suppresses host immune responses and reduces arthritis development within murine models. These data suggest that Notch signaling and Foxp3 regulate the development and function of Treg cells derived from iPS cells. Our results provide a novel approach for generating potentially therapeutic Treg cells for the treatment of autoimmune diseases.

Comparative effects of pulsatile and nonpulsatile flow on plasma fibrinolytic balance in pediatric patients undergoing cardiopulmonary bypass.

In the brain, the components of the fibrinolytic system, tissue plasminogen activator (tPA) and its endogenous inhibitor plasminogen activator inhibitor-1 (PAI-1), regulate various neurophysiological and pathological responses. Fibrinolytic balance depends on PAI-1 and tPA concentrations. The objective of this study is to compare the effects of pulsatile and nonpulsatile perfusion on fibrinolytic balance in children undergoing pediatric cardiopulmonary bypass (CPB). Plasma PAI-1 antigen and tPA antigen were measured in 40 children (n = 20 pulsatile and n = 20 nonpulsatile group). Plasma samples (1.5 mL) were collected (i) prior to incision, (ii) 1 h after CPB, and (iii) 24 h after CPB. PAI-1 and tPA levels were measured at each time point. PAI-1 and tPA levels were significantly increased at 1 h after CPB, followed by a decrease at 24 h. Nonpulsatile but not pulsatile CPB lowered PAI-1 : tPA ratio significantly at 24 h (median PAI-1 : tPA ratio 4.63 ± 0.83:1.98 ± 0.48, P = 0.03, for the nonpulsatile group and 4.50 ± 0.92:3.56 ± 1.28, P = 0.2, for the pulsatile group). These results suggest that pulsatile flow maintains endogenous fibrinolytic balance after pediatric cardiopulmonary bypass. Further studies are needed to define the clinical significance of these differences.

The prophylactic value of TNF-α inhibitors against retinal cell apoptosis and optic nerve axon loss after corneal surgery or trauma.

BACKGROUND AND PURPOSE: Late secondary glaucoma is an often-severe complication after acute events like anterior segment surgery, trauma and infection. TNF-α is a major mediator that is rapidly upregulated, diffusing also to the retina and causes apoptosis of the ganglion cells and degeneration of their optic nerve axons (mediating steps to glaucomatous damage). Anti-TNF-α antibodies are in animals very effective in protecting the retinal cells and the optic nerve-and might therefore be useful prophylactically against secondary glaucoma in future such patients. Here we evaluate (1) toxicity and (2) efficacy of two TNF-α inhibitors (adalimumab and infliximab), in rabbits by subconjunctival administration. METHODS: For drug toxicity, animals with normal, unburned corneas were injected with adalimumab (0.4, 4, or 40 mg), or infliximab (1, 10, or 100 mg). For drug efficacy, other animals were subjected to alkali burn before such injection, or steroids (for control). The rabbits were evaluated clinically with slit lamp and photography, electroretinography, optical coherence tomography, and intraocular pressure manometry. A sub-set of eyes were stained ex vivo after 3 days for retinal cell apoptosis (TUNEL). In other experiments the optic nerves were evaluated by paraphenylenediamine staining after 50 or 90 days. Loss of retinal cells and optic nerve degeneration were quantified. RESULTS: Subconjunctival administration of 0.4 mg or 4.0 mg adalimumab were well tolerated, whereas 40.0 mg was toxic to the retina. 1, 10, or 100 mg infliximab were also well tolerated. Analysis of the optic nerve axons after 50 days confirmed the safety of 4.0 mg adalimumab and of 100 mg infliximab. For efficacy, 4.0 mg adalimumab subconjunctivally in 0.08 mL provided practically full protection against retinal cell apoptosis 3 days following alkali burn, and infliximab 100 mg only slightly less. At 90 days following burn injury, control optic nerves showed about 50% axon loss as compared to 8% in the adalimumab treatment group. CONCLUSIONS: Subconjunctival injection of 4.0 mg adalimumab in rabbits shows no eye toxicity and provides excellent neuroprotection, both short (3 days) and long-term (90 days). Our total. accumulated data from several of our studies, combined with the present paper, suggest that corneal injuries, including surgery, might benefit from routine administration of anti-TNF-α biologics to reduce inflammation and future secondary glaucoma.

Disrupted mitochondrial transcription factor A expression promotes mitochondrial dysfunction and enhances ocular surface inflammation by activating the absent in melanoma 2 inflammasome.

PURPOSE: Severe dry eye disease causes ocular surface damage, which is highly associated with mitochondrial dysfunction. Mitochondrial transcription factor A (TFAM) is essential for packaging mitochondrial DNA (mtDNA) and is crucial for maintaining mitochondrial function. Herein, we aimed to explore the effect of a decreased TFAM expression on ocular surface damage. METHODS: Female C57BL/6 mice were induced ocular surface injury by topical administrating benzalkonium chloride (BAC). Immortalized human corneal epithelial cells (HCECs) were stimulated by tert-butyl hydroperoxide (t-BHP) to create oxidative stress damage. HCECs with TFAM knockdown were established. RNA sequencing was employed to analyze the whole-genome expression. Mitochondrial changes were measured by transmission electron microscopy, Seahorse metabolic flux analysis, mitochondrial membrane potential, and mtDNA copy number. TFAM expression and inflammatory cytokines were determined using RT-qPCR, immunohistochemistry, immunofluorescence, and immunoblotting. RESULTS: In both the corneas of BAC-treated mice and t-BHP-induced HCECs, we observed impaired TFAM expression, accompanied by mitochondrial structure and function defects. TFAM downregulation in HCECs suppressed mitochondrial respiratory capacity, reduced mtDNA content, induced mtDNA leakage into the cytoplasm, and led to inflammation. RNA sequencing revealed the absent in melanoma 2 (AIM2) inflammasome was activated in the corneas of BAC-treated mice. The AIM2 inflammasome activation was confirmed in TFAM knockdown HCECs. TFAM knockdown in t-BHP-stimulated HCECs aggravated mitochondrial dysfunction and the AIM2 inflammasome activation, thereby further triggering the secretion of inflammatory factors such as interleukin (IL) -1ß and IL-18. CONCLUSIONS: TFAM reduction impaired mitochondrial function, activated AIM2 inflammasome and promoted ocular surface inflammation, revealing an underlying molecular mechanism for ocular surface disorders.

CSF1R inhibition is not specific to innate immune cells but also affects T-helper cell differentiation independently of microglia depletion.

Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a specific method for microglia depletion. However, recent work revealed that in addition to microglia, CSF1R inhibition also affects other innate immune cells, such as peripheral monocytes and tissue-resident macrophages of the lung, liver, spleen, and peritoneum. Here, we show that this effect is not restricted to innate immune cells only but extends to the adaptive immune compartment. CSF1R inhibition alters the transcriptional profile of bone marrow cells that control T helper cell activation. In vivo or ex vivo inhibition of CSF1R profoundly changes the transcriptional profile of CD4+ cells and suppresses Th1 and Th2 differentiation in directionally stimulated and unstimulated cells and independently of microglia depletion. Given that T cells also contribute in CNS pathology, these effects may have practical implications in the interpretation of relevant experimental data.

Sustained Inhibition of VEGF and TNF-α Achieves Multi-Ocular Protection and Prevents Formation of Blood Vessels after Severe Ocular Trauma.

PURPOSE: This study aimed to develop a clinically feasible and practical therapy for multi-ocular protection following ocular injury by using a thermosensitive drug delivery system (DDS) for sustained delivery of TNF-α and VEGF inhibitors to the eye. METHODS: A thermosensitive, biodegradable hydrogel DDS (PLGA-PEG-PLGA triblock polymer) loaded with 0.7 mg of adalimumab and 1.4 mg of aflibercept was injected subconjunctivally into Dutch-belted pigmented rabbits after corneal alkali injury. Control rabbits received 2 mg of IgG-loaded DDS or 1.4 mg of aflibercept-loaded DDS. Animals were followed for 3 months and assessed for tolerability and prevention of corneal neovascularization (NV), improvement of corneal re-epithelialization, inhibition of retinal ganglion cell (RGC) and optic nerve axon loss, and inhibition of immune cell infiltration into the cornea. Drug-release kinetics was assessed in vivo using an aqueous humor protein analysis. RESULTS: A single subconjunctival administration of dual anti-TNF-α/anti-VEGF DDS achieved a sustained 3-month delivery of antibodies to the anterior chamber, iris, ciliary body, and retina. Administration after corneal alkali burn suppressed CD45+ immune cell infiltration into the cornea, completely inhibited cornea NV for 3 months, accelerated corneal re-epithelialization and wound healing, and prevented RGC and optic nerve axon loss at 3 months. In contrast, anti-VEGF alone or IgG DDS treatment led to persistent corneal epithelial defect (combined: <1%; anti-VEGF: 15%; IgG: 10%, of cornea area), increased infiltration of CD45+ immune cells into the cornea (combined: 28 ± 20; anti-VEGF: 730 ± 178; anti-IgG: 360 ± 186, cells/section), and significant loss of RGCs (combined: 2.7%; anti-VEGF: 63%; IgG: 45%) and optic nerve axons at 3 months. The aqueous humor protein analysis showed first-order release kinetics without adverse effects at the injection site. CONCLUSIONS: Concomitant inhibition of TNF-α and VEGF prevents corneal neovascularization and ameliorates subsequent irreversible damage to the retina and optic nerve after severe ocular injury. A single subconjunctival administration of this therapy, using a biodegradable, slow-release thermosensitive DDS, achieved the sustained elution of therapeutic levels of antibodies to all ocular tissues for 3 months. This therapeutic approach has the potential to dramatically improve the outcomes of severe ocular injuries in patients and improve the therapeutic outcomes in patients with retinal vascular diseases.

Opposing Roles of Blood-Borne Monocytes and Tissue-Resident Macrophages in Limbal Stem Cell Damage after Ocular Injury.

Limbal stem cell (LSC) deficiency is a frequent and severe complication after chemical injury to the eye. Previous studies have assumed this is mediated directly by the caustic agent. Here we show that LSC damage occurs through immune cell mediators, even without direct injury to LSCs. In particular, pH elevation in the anterior chamber (AC) causes acute uveal stress, the release of inflammatory cytokines at the basal limbal tissue, and subsequent LSC damage and death. Peripheral C-C chemokine receptor type 2 positive/CX3C motif chemokine receptor 1 negative (CCR2+ CX3CR1-) monocytes are the key mediators of LSC damage through the upregulation of tumor necrosis factor-alpha (TNF-α) at the limbus. In contrast to peripherally derived monocytes, CX3CR1+ CCR2- tissue-resident macrophages have a protective role, and their depletion prior to injury exacerbates LSC loss and increases LSC vulnerability to TNF-α-mediated apoptosis independently of CCR2+ cell infiltration into the tissue. Consistently, repopulation of the tissue by new resident macrophages not only restores the protective M2-like phenotype of macrophages but also suppresses LSC loss after exposure to inflammatory signals. These findings may have clinical implications in patients with LSC loss after chemical burns or due to other inflammatory conditions.

Critical media attributes in E-beam sterilization of corneal tissue.

When ionizing irradiation interacts with a media, it can form reactive species that can react with the constituents of the system, leading to eradication of bioburden and sterilization of the tissue. Understanding the media's properties such as polarity is important to control and direct those reactive species to perform desired reactions. Using ethanol as a polarity modifier of water, we herein generated a series of media with varying relative polarities for electron beam (E-beam) irradiation of cornea at 25 kGy and studied how the irradiation media's polarity impacts properties of the cornea. After irradiation of corneal tissues, mechanical (tensile strength and modulus, elongation at break, and compression modulus), chemical, optical, structural, degradation, and biological properties of the corneal tissues were evaluated. Our study showed that irradiation in lower relative polarity media improved structural properties of the tissues yet reduced optical transmission; higher relative polarity reduced structural and optical properties of the cornea; and intermediate relative polarity (ethanol concentrations = 20-30% (v/v)) improved the structural properties, without compromising optical characteristics. Regardless of media polarity, irradiation did not negatively impact the biocompatibility of the corneal tissue. Our data shows that the absorbed ethanol can be flushed from the irradiated cornea to levels that are nontoxic to corneal and retinal cells. These findings suggest that the relative polarity of the irradiation media can be tuned to generate sterilized tissues, including corneal grafts, with engineered properties that are required for specific biomedical applications. STATEMENT OF SIGNIFICANCE: Extending the shelf-life of corneal tissue can improve general accessibility of cornea grafts for transplantation. Irradiation of donor corneas with E-beam is an emerging technology to sterilize the corneal tissues and enable their long-term storage at room temperature. Despite recent applications in clinical medicine, little is known about the effect of irradiation and preservation media's characteristics, such as polarity on the properties of irradiated corneas. Here, we have showed that the polarity of the media can be a valuable tool to change and control the properties of the irradiated tissue for transplantation.

Cooperation between molecular targets of costimulation in promoting T cell persistence and tumor regression.

Costimulation regulates multiple cellular processes of T cells inducing proliferation, expansion, and survival. The molecular targets of costimulation might then be useful to augment T cell activities. Two defined targets of costimulatory signals in primary T cells are the anti-apoptotic bcl-2 family molecule Bcl-x(L), and survivin, an inhibitor of apoptosis family member that might regulate both cell division and survival. However, the relative importance of, and relationship between, these molecules in primary T cells is not clear. To understand whether they have overlapping or cooperative functions, we used retrovirus-mediated transduction to introduce Bcl-x(L) and survivin separately, or together linked by a 2A picornavirus self-cleaving peptide, into Ag-responding CD8(+) T cells. We found that CD8(+) effector T cells expressing both Bcl-x(L) and survivin strongly expanded at an early stage and had a long-term survival advantage over cells transduced with either molecule alone. In vivo, with response to tumor-expressed Ag following adoptive T cell transfer, Ag-reactive CD8(+) T cells expressing both Bcl-x(L) and survivin displayed greatly enhanced tumor protective activity compared with CD8(+) T cells expressing either molecule introduced separately. These results indicate that Bcl-x(L) and survivin can critically contribute in a cooperative, nonredundant manner to augment the accumulation and persistence of CD8(+) T cells following encounter with Ag. The data provide new insights into why costimulatory signals might need to be sustained over time and suggest a potential novel approach to augment cellular immunotherapy for cancer.

An optimized protocol for the generation of HBV viral antigen-specific T lymphocytes from pluripotent stem cells.

In T cell-based cancer immunotherapy, tumor antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) can specifically target tumor Ags on malignant cells. This promising approach drove us to adopt this strategy of T cell transfer (ACT)-based immunotherapy for chronic viral infections. Here, we describe the generation of hepatitis B virus (HBV) Ag-specific CTLs from induced pluripotent stem cells (iPSCs), i.e., iPSC-CTLs. Ag-specific iPSC-CTLs can target HBV Ag+ cells and infiltrate into the liver to suppress HBV replication in a murine model. For complete details on the use and execution of this protocol, please refer to Haque et al. (2020).