Fluorinated Adenosine A2A Receptor Antagonists Inspired by Preladenant as Potential Cancer Immunotherapeutics

Antagonism of the adenosine A2A receptor on T cells blocks the hypoxia-adenosinergic pathway to promote tumor rejection. Using an in vivo immunoassay based on the Concanavalin A mouse model, a series of A2A antagonists were studied and identified preladenant as a potent lead compound for development. Molecular modeling was employed to assist drug design and subsequent synthesis of analogs and those of tozadenant, including fluorinated polyethylene glycol PEGylated derivatives. The efficacy of the analogs was evaluated using two in vitro functional bioassays, and compound 29, a fluorinated triethylene glycol derivative of preladenant, was confirmed as a potential immunotherapeutic agent

Yoga and Mindfulness as Therapeutic Interventions for Stroke Rehabilitation: A Systematic Review

Aim. This paper reports a systematic review and critical appraisal of the evidence on the effectiveness of behavioral therapies such as yoga and mindfulness practices for stroke rehabilitation. Background. The experience of stroke can have a negative impact on both psychological and physical health and on quality of life. Yoga and relevant practices are promising therapies that have been used with patients with a variety of conditions. In order to draw conclusions on effectiveness for stroke patients, the evidence requires systematic assessment. Methods. A comprehensive search of major biomedical and complementary medicine databases was conducted. Relevant research was categorized by study type and appraised according to study design. Results. Five randomized controlled clinical trials and four single case studies were found. Additionally, one qualitative research study was identified. Studies reported positive results, including improvements in cognition, mood, and balance and reductions in stress. Modifications to different yoga practices make comparison between studies difficult, and a lack of controlled studies precludes any firm conclusions on efficacy. Conclusion. Yoga and mindfulness could be clinically valuable self-administered intervention options for stroke rehabilitation. Further research is needed to evaluate these specific practices and their suitability in stroke rehabilitation

Targeted Elimination of Immunodominant B Cells Drives the Germinal Center Reaction toward Subdominant Epitopes

Rapidly evolving pathogens such as HIV or influenza can quickly mutate their antigenic profiles, reducing the efficacy of conventional vaccines. Despite this challenge, functionally required epitopes are highly conserved among heterologous viral strains and represent a key vulnerability that could be targeted during vaccine development. As the antigenicity of these conserved epitopes is frequently subdominant, there is a critical need for innovative vaccination strategies designed to target these neutralizing epitopes. Here, we immunized mice with antigens containing discrete immunodominant and subdominant moieties and show that treatment with soluble heterologous antigen bearing only the immunodominant epitope selectively suppresses these germinal center (GC) B cells. By exploiting this intrinsic tolerance mechanism, we promote the expansion of subdominant B cells in the GC and the subsequent long-lived components of the humoral response. We propose that this strategy may be applied to elicit preferential expansion of subdominant B cells that recognize weakly immunogenic epitopes on microbial pathogens

Hypoxia and hypoxia-inducible factor (HIF) downregulate antigen-presenting MHC class I molecules limiting tumor cell recognition by T cells

<div><p>Human cancers are known to downregulate Major Histocompatibility Complex (MHC) class I expression thereby escaping recognition and rejection by anti-tumor T cells. Here we report that oxygen tension in the tumor microenvironment (TME) serves as an extrinsic cue that regulates antigen presentation by MHC class I molecules. In support of this view, hypoxia is shown to negatively regulate MHC expression in a HIF-dependent manner as evidenced by (i) lower MHC expression in the hypoxic TME <i>in vivo</i> and in hypoxic 3-dimensional (3D) but not 2-dimensional (2D) tumor cell cultures <i>in vitro</i>; (ii) decreased MHC in human renal cell carcinomas with constitutive expression of HIF due to genetic loss of von Hippel-Lindau (VHL) function as compared with isogenically paired cells with restored VHL function, and iii) increased MHC in tumor cells with siRNA-mediated knockdown of HIF. In addition, hypoxia downregulated antigen presenting proteins like TAP 1/2 and LMP7 that are known to have a dominant role in surface display of peptide-MHC complexes. Corroborating oxygen-dependent regulation of MHC antigen presentation, hyperoxia (60% oxygen) transcriptionally upregulated MHC expression and increased levels of TAP2, LMP2 and 7. In conclusion, this study reveals a novel mechanism by which intra-tumoral hypoxia and HIF can potentiate immune escape. It also suggests the use of hyperoxia to improve tumor cell-based cancer vaccines and for mining novel immune epitopes. Furthermore, this study highlights the advantage of 3D cell cultures in reproducing hypoxia-dependent changes observed in the TME.</p></div

Hypoxia downregulates MHC class I expression via HIF transcription factors.

<p><b>(A-C):</b> siRNA mediated knockdown of HIF-1α reversed hypoxic downregulation of MHC class I expression as compared with the scrambled, non-targeting (NT) siRNA control. MCA205 tumor cells were reverse transfected with scrambled siRNA (NT; red histogram) or with HIF-1α specific siRNA (blue histogram) and cultured as 3D spheroids under 1% <b>(A)</b> or 21% <b>(B)</b> oxygen for 48h. Levels of MHC class I surface expression was determined using flow cytometry. Efficacy of gene knockdown was assessed using Western blot <b>(C)</b>. β-Actin was used as the loading control. Representative data of 3 independent experiments shown. <b>(D-F):</b> Flow cytometry assessment of surface expression of HLA-ABC on paired isogenic renal cell carcinoma cell lines RCC4 <b>(D)</b>, UMRC2 <b>(E)</b> and CAKI2 <b>(F)</b>. Each pair had the parental cell line that lacked endogenous wild-type VHL (VHL null, transfected with empty vector) and one with vector stably expressing functional VHL (VHL restored). Restoring VHL function and thereby reducing HIF expression, significantly increased HLA-ABC expression on the cells. Representative histograms of 4 independent experiments are shown. Grey filled: unstained control; red: VHL null genotype; blue: VHL restored genotype. <b>(D1-F1):</b> Inactivation of HIF-1α by restoring VHL expression was verified by Western blotting for RCC4 <b>(D1)</b>, UMRC2 <b>(E1)</b> and CAKI2 <b>(F1)</b> cells. β-Actin was used as the loading control.</p

Hypoxia downregulates and hyperoxia upregulates expression levels of TAPs and LMPs.

<p><b>(A, C)</b> MCA205 tumor cells were cultured <i>in vitro</i> as 3D spheroids for 48h under 1%, 21% or 60% oxygen. <b>(C)</b> Relative band intensity, normalized to the loading control and 21% oxygen samples is shown. <b>(B, D)</b> For <i>in vivo</i> experiments, tumor nodules (MCA205 pulmonary tumors) were harvested from mice exposed to respiratory hypoxia (10% oxygen), normoxia (21% oxygen) or hyperoxia (60% oxygen) for 48h. <b>(D)</b> Relative band intensity, normalized to the loading control and 21% oxygen samples is shown. Protein levels were determined by Western blot. β-actin was used as loading control. Representative blots with samples from 2 independent experiments are shown.</p

Hypoxia downregulates MHC class I expression <i>in vitro</i> in 3D but not in 2D culture systems and requires the deeper hypoxia achieved in the 3D system.

<p><b>(A-C):</b> MCA205 tumor cells were cultured as 2D monolayers <b>(A)</b> or as 3D spheroids <b>(B,C)</b> and cultured under 21% O₂ or 1% O₂ for 48h. Levels of MHC class I expression was determined using flow cytometry. Representative histograms <b>(B)</b> and associated quantification and statistics <b>(C)</b> of 4 independent experiments are shown. The significance of differences was analyzed by the Student’s t-test (two-sided); p = 0.013 (C). Grey filled: Unstained control; Red: Hypoxia; Blue: Normoxia. MFI: mean fluorescence intensity. Inset: 40X magnification of MCA205 grown at 1% O₂ in 2D culture <b>(A)</b> or in 3D culture <b>(B)</b>. Error bars indicate SD. <b>(D)</b> Representative flow cytometry histograms of Hypoxyprobe-1 (HP) indicating significantly increased levels of hypoxia in MCA205 cells grown under 1% oxygen for 48h as 3D spheroids (Red histogram) as compared with 2D monolayers (Blue histogram). n = 4. <b>(E)</b> Contour plots representing intensity of hypoxia within MCA205 cultures grown as 2D monolayers show 98% of the population was intermediately hypoxic. <b>(F)</b> MCA205 cells grown as 3D spheroids show two distinct populations of intermediately hypoxic (56%; HP MFI = 309) and severely hypoxic (39.5%; HP MFI: 1412) regions. <b>(G, H)</b> Gating on the 2 distinct hypoxic populations in the spheroid revealed inverse correlation between MHC class I and hypoxia levels. Less hypoxic cells had significantly higher percentage of MHC class I positive cells <b>(G)</b> and more hypoxic regions had lower percentage of MHC class I positive cells <b>(H)</b>. <b>(D-H)</b> Representative data of 4 independent experiments.</p

Hypoxia-mediated downregulation of MHC class I expression impairs recognition and killing of tumor cells by CTLs.

<p>(<b>A</b>) Hypoxic-grown MCA205-OVA cells with downregulated MHC class I expression were poorly recognized and killed by effector OT.1 T cells compared to normoxic controls. OVA expressing MCA205 tumor cells were grown in 3D cultures for 48h in 21% O₂ conditions. A subset of these cells were then moved to hypoxic (1% O2) conditions for an additional 24h. The hypoxic and normoxic spheroids were subsequently co-cultured with activated OT-I T cells. Tumor cells were identified by CellTracker staining (stained prior to co-culture) and cytotoxicity was assessed based on percent propidium idodide positive tumor cells. Each data point represents a replicate. Data is representative of 3 independent experiments. (<b>B</b>) Flow cytometry assessment of surface expression of MHC-SIINFEKL on OVA transfected MCA-205 cells. The tumor cells were grown as 3D spheroids for 48h in either 1% or 21% O₂ conditions. Each data point represents an independent experiment. n = 4. Error bars indicate SD. The significance of differences was analyzed by the Student’s t-test (two-sided); *p = 0.02, ** p = 0.003 (A), p = 0.005 (B).</p

Molecular oxygen regulates MHC class I expression transcriptionally.

<p><b>(A)</b> Mice bearing MCA205 pulmonary tumors were exposed to either respiratory hypoxia (10% O₂), normoxia (21% O₂), or respiratory hyperoxia (60% O₂) for 48h. <b>(B)</b> MCA205 tumors grown <i>in vitro</i> in 3D spheroids under hypoxia (1% O₂), normoxia (21% O₂), or hyperoxia (60% O₂) for 48h. Hypoxia significantly downregulated whereas hyperoxia significantly upregulated MHC class I transcripts as compared normoxic controls both <i>in vivo</i> and <i>in vitro</i>. RT-qPCR was used to analyze MHC class I (H-2Kb) transcript levels. Ribosomal protein L32 was used as internal control. Y- axis represents transcript levels relative to normoxic controls. n = 4. The significance of differences was analyzed by the Student’s t-test (two-sided); p values are as indicated in the figure. Error bars indicate SD.</p

Hyperoxia upregulates MHC class I expression equally in 2D and 3D cultures.

<p><b>(A-D):</b> MCA205 tumors were grown as 2D monolayers <b>(A, C)</b> or as 3D spheroids <b>(B, D)</b> at 21% O₂ or 60% O₂ for 48h. MHC class I levels were determined by flow cytometry. The magnitude of MHC class I upregulation was similar in 2D and 3D cultures. Representative histograms (<b>A, B</b>) and associated quantification and statistics (<b>C, D</b>) of 4 independent experiments shown. The significance of differences was analyzed by the Student’s t-test (two-sided); p = 0.002 (C), p = 0.001 (D). Grey filled: Unstained control; Blue: Normoxia (21% O₂); Green: Hyperoxia (60% O₂). Error bars indicate SD.</p