Characterization and allogeneic transplantation of a novel transgenic cone-rich donor mouse line.

OBJECTIVES: Cone photoreceptor transplantation is a potential treatment for macular diseases. The optimal conditions for cone transplantation are poorly understood, partly because of the scarcity of cones in donor mice. To facilitate allogeneic cone photoreceptor transplantation studies in mice, we aimed to create and characterize a donor mouse model containing a cone-rich retina with a cone-specific enhanced green fluorescent protein (EGFP) reporter. METHODS: We generated OPN1LW-EGFP/NRL-/- mice by crossing NRL-/- and OPN1LW-EGFP mice. We characterized the anatomical phenotype of OPN1LW-EGFP/NRL-/- mice using multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, immunohistology, and transmission electron microscopy. We evaluated retinal function using electroretinography (ERG), including 465 and 525 nm chromatic stimuli. Retinal sheets and cell suspensions from OPN1LW-EGFP/NRL-/- mice were transplanted subretinally into immunodeficient Rd1 mice. RESULTS: OPN1LW-EGFP/NRL-/- retinas were enriched with OPN1LW-EGFP+ and S-opsin+ cone photoreceptors in a dorsal-ventral distribution gradient. Cone photoreceptors co-expressing OPNL1W-EGFP and S-opsin significantly increased in OPN1LW-EGFP/NRL-/- compared to OPN1LW-EGFP mice. Temporal dynamics of rosette formation in the OPN1LW-EGFP/NRL-/- were similar as the NRL-/- with peak formation at P15. Rosettes formed preferentially in the ventral retina. The outer retina in P35 OPN1LW-EGFP/NRL-/- was thinner than NRL-/- controls. The OPN1LW-EGFP/NRL-/- ERG response amplitudes to 465 nm stimulation were similar to, but to 535 nm stimulation were lower than, NRL-/- controls. Three months after transplantation, the suspension grafts showed greater macroscopic degradation than sheet grafts. Retinal sheet grafts from OPN1LW-EGFP/NRL-/- mice showed greater S-opsin + cone survival than suspension grafts from the same strain. CONCLUSIONS: OPN1LW-EGFP/NRL-/- retinae were enriched with S-opsin+ photoreceptors. Sustained expression of EGFP facilitated the longitudinal tracking of transplanted donor cells. Transplantation of cone-rich retinal grafts harvested prior to peak rosette formation survived and differentiated into cone photoreceptor subtypes. Photoreceptor sheet transplantation may promote greater macroscopic graft integrity and S-opsin+ cone survival than cell suspension transplantation, although the mechanism underlying this observation is unclear at present. This novel cone-rich reporter mouse strain may be useful to study the influence of graft structure on cone survival.

Quantifiable In Vivo Imaging Biomarkers of Retinal Regeneration by Photoreceptor Cell Transplantation.

Purpose: Short-term improvements in retinal anatomy are known to occur in preclinical models of photoreceptor transplantation. However, correlative changes over the long term are poorly understood. We aimed to develop a quantifiable imaging biomarker grading scheme, using noninvasive multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, to enable serial evaluation of photoreceptor transplantation over the long term. Methods: Photoreceptor cell suspensions or sheets from rhodopsin-green fluorescent protein mice were transplanted subretinally, into either NOD.CB17-Prkdcscid/J or C3H/HeJ-Pde6brd1 mice. Multimodal cSLO imaging was performed serially for up to three months after transplantation. Imaging biomarkers were scored, and a grade was defined for each eye by integrating the scores. Image grades were correlated with immunohistochemistry (IHC) data. Results: Multimodal imaging enabled the extraction of quantitative imaging biomarkers including graft size, GFP intensity, graft length, on-target graft placement, intra-graft lamination, hemorrhage, retinal atrophy, and periretinal proliferation. Migration of transplanted material was observed. Changes in biomarker scores and grades were detected in 14/16 and 7/16 eyes, respectively. A high correlation was found between image grades and IHC parameters. Conclusions: Serial evaluation of multiple imaging biomarkers, when integrated into a per-eye grading scheme, enabled comprehensive tracking of longitudinal changes in photoreceptor cell grafts over time. The application of systematic multimodal in vivo imaging could be useful in increasing the efficiency of preclinical retinal cell transplantation studies in rodents and other animal models. Translational Relevance: By allowing longitudinal evaluation of the same animal over time, and providing quantifiable biomarkers, non-invasive multimodal imaging improves the efficiency of retinal transplantation studies in animal models. Such assays will facilitate the development of cell therapy for retinal diseases.

Expression of LAG-3 and efficacy of combination treatment with anti-LAG-3 and anti-PD-1 monoclonal antibodies in glioblastoma.

Like in many tumor types, immunotherapy is currently under investigation to assess its potential efficacy in glioblastoma patients. Trials are under way to assess the efficacy of new immune checkpoint inhibitors including anti-PD-1 or CTLA4. We here investigate the expression and efficacy of a novel immune-checkpoint inhibitor, called LAG-3. We show that LAG-3 is expressed in human glioblastoma samples and in a mouse glioblastoma model we show that knock out or LAG-3 inhibition with a blocking antibody is efficacious against glioblastoma and can be used in combination with other immune checkpoint inhibitors toward complete eradication of the model glioblastoma tumors. From a mechanistic standpoint we show that LAG-3 expression is an early marker of T cell exhaustion and therefore early treatment with LAG-3 blocking antibody is more efficacious than later treatment. These data provide insight and support the design of trials that incorporate LAG-3 in the treatment of glioblastoma.

Absence of host NF-κB p50 induces murine glioblastoma tumor regression, increases survival, and decreases T-cell induction of tumor-associated macrophage M2 polarization.

High-grade gliomas harbor abundant myeloid cells that suppress anti-tumor immunity and support tumor growth. Targeting transcription factors, such as NF-κB p50, that mediate suppressive myeloid M2 polarization may prove therapeutic. GL261-Luc glioblastoma cells were inoculated into wild-type and p50-/- mice, followed by analysis of tumor growth, survival, tumor myeloid cells, and T cells. The absence of host p50 slows tumor growth and enables regression in 30% of recipients, leading to prolonged survival. Tumors developing in p50-/- mice possess a greater concentration of tumor-infiltrating myeloid cells (TIMs) than those in wild-type mice. TIMs are predominantly F4/80hi macrophages which, along with tumor-associated microglia, express increased pro-inflammatory M1 and reduced immune-suppressive M2 markers. In p50-/- mice, total tumor CD4 T cells are threefold more abundant, whereas CD8 T-cell numbers are unchanged, and both produce increased IFNγ and Granzyme B. Naïve splenic p50-/- CD8 T cells manifest increased activation, whereas naïve p50-/- and WT CD4 T cells show similar Th1, Th2, and Th17 polarization. Antibody targeting CD4, but not CD8, fully obviates the p50-/- survival advantage. Combined CD4 and CD8 T-cell depletion reverses myeloid M2 polarization in wild-type hosts, without affecting myeloid M1 polarization in p50-/- hosts. Finally, gliomas grow similarly in p50(f/f) and p50(f/f);Lysozyme-Cre mice, the latter having reduced p50 specifically in myeloid cells and tumor microglia. Thus, high-grade glioma T cells play a key role in directing M2 polarization of tumor myeloid cells, and reducing NF-κB p50 in both tumor myeloid cells and T cells may contribute to glioma therapy.

PD-L1 expression in medulloblastoma: an evaluation by subgroup.

BACKGROUND: This study evaluated the expression of PD-L1 and markers of immune mediated resistance in human medulloblastoma (MB), the most common malignant pediatric brain tumor. RESULTS: Overall levels of PD-L1 in human MB were low; however, some cases demonstrated robust focal expression associated with increased immune infiltrates. The case with highest PD-L1 expression was a sonic hedgehog (SHH) MB. In cell lines, SHH MB, which are low-MYC expressing, demonstrated both constitutive and inducible expression of PD-L1 while those in Group 3/4 that expressed high levels of MYC had only inducible expression. In vitro, IFN-γ robustly stimulated the expression of PD-L1 in all cell lines while radiation induced variable expression. Forced high MYC expression did not significantly alter PD-L1. METHODS: Human MB tumor samples were evaluated for expression of PD-L1 and immune cell markers in relation to molecular subgroup assignment. PD-L1 expression was functionally analyzed under conditions of interferon gamma (IFN-γ), radiation, and MYC overexpression. CONCLUSIONS: MB expresses low levels of PD-L1 facilitating immune escape. Importantly, TH1 cytokine stimulation appears to be the most potent inducer of PD-L1 expression in vitro suggesting that an inflamed tumor microenvironment is necessary for PD-1 pathway activation in this tumor.

Combination Therapy with Anti-PD-1, Anti-TIM-3, and Focal Radiation Results in Regression of Murine Gliomas.

PURPOSE: Checkpoint molecules like programmed death-1 (PD-1) and T-cell immunoglobulin mucin-3 (TIM-3) are negative immune regulators that may be upregulated in the setting of glioblastoma multiforme. Combined PD-1 blockade and stereotactic radiosurgery (SRS) have been shown to improve antitumor immunity and produce long-term survivors in a murine glioma model. However, tumor-infiltrating lymphocytes (TIL) can express multiple checkpoints, and expression of ≥2 checkpoints corresponds to a more exhausted T-cell phenotype. We investigate TIM-3 expression in a glioma model and the antitumor efficacy of TIM-3 blockade alone and in combination with anti-PD-1 and SRS. EXPERIMENTAL DESIGN: C57BL/6 mice were implanted with murine glioma cell line GL261-luc2 and randomized into 8 treatment arms: (i) control, (ii) SRS, (iii) anti-PD-1 antibody, (iv) anti-TIM-3 antibody, (v) anti-PD-1 + SRS, (vi) anti-TIM-3 + SRS, (vii) anti-PD-1 + anti-TIM-3, and (viii) anti-PD-1 + anti-TIM-3 + SRS. Survival and immune activation were assessed. RESULTS: Dual therapy with anti-TIM-3 antibody + SRS or anti-TIM-3 + anti-PD-1 improved survival compared with anti-TIM-3 antibody alone. Triple therapy resulted in 100% overall survival (P < 0.05), a significant improvement compared with other arms. Long-term survivors demonstrated increased immune cell infiltration and activity and immune memory. Finally, positive staining for TIM-3 was detected in 7 of 8 human GBM samples. CONCLUSIONS: This is the first preclinical investigation on the effects of dual PD-1 and TIM-3 blockade with radiation. We also demonstrate the presence of TIM-3 in human glioblastoma multiforme and provide preclinical evidence for a novel treatment combination that can potentially result in long-term glioma survival and constitutes a novel immunotherapeutic strategy for the treatment of glioblastoma multiforme. Clin Cancer Res; 23(1); 124-36. ©2016 AACR.

Erratum to: Agonist anti-GITR monoclonal antibody and stereotactic radiation induce immune-mediated survival advantage in murine intracranial glioma.

[This corrects the article DOI: 10.1186/s40425-016-0132-2.].

Agonist anti-GITR monoclonal antibody and stereotactic radiation induce immune-mediated survival advantage in murine intracranial glioma.

BACKGROUND: Glioblastoma (GBM) is a poorly immunogenic neoplasm treated with focused radiation. Immunotherapy has demonstrated synergistic survival effects with stereotactic radiosurgery (SRS) in murine GBM. GITR is a co-stimulatory molecule expressed constitutively on regulatory T-cells and by effector T-cells upon activation. We tested the hypothesis that anti-GITR monoclonal antibody (mAb) and SRS together would confer an immune-mediated survival benefit in glioma using the orthotopic GL261 glioma model. METHODS: Mice received SRS and anti-GITR 10 days after implantation. The anti-GITR mAbs tested were formatted as mouse IgG1 D265A (anti-GITR (1)) and IgG2a (anti-GITR (2a)) isotypes. Mice were randomized to four treatment groups: (1) control; (2) SRS; (3) anti-GITR; (4) anti-GITR/SRS. SRS was delivered to the tumor in one fraction, and mice were treated with mAb thrice. Mice were euthanized on day 21 to analyze the immunologic profile of tumor, spleen, and tumor draining lymph nodes. RESULTS: Anti-GITR (1)/SRS significantly improved survival over either treatment alone (p < .0001) with a cure rate of 24 % versus 0 % in a T-lymphocyte-dependent manner. There was elevated intratumoral CD4+ effector cell infiltration relative to Treg infiltration in mice treated with anti-GITR (1)/SRS, as well as significantly elevated IFNγ and IL-2 production by CD4+ T-cells and elevated IFNγ and TNFα production by CD8+ T-cells. There was increased mRNA expression of M1 markers and decreased expression of M2 markers in tumor infiltrating mononuclear cells. The anti-GITR (2a)/SRS combination did not improve survival, induce tumor regression, or result in Treg depletion. CONCLUSIONS: These findings provide preclinical evidence for the use of anti-GITR (1) non-depleting antibodies in combination with SRS in GBM.