Pembrolizumab in Combination with Antigen-Specific Cytotoxic T Lymphocytes Enhances Killing of Acute Myeloid Leukemia

Background Pembrolizumab, an antibody that blocks programmed cell death protein 1 (PD-1), has been FDA approved for several solid tumors and hematologic malignancies. Currently, pembrolizumab is under investigation for acute myeloid leukemia (AML) in combination with hypomethylating agents. It is established that AML is highly responsive to immunotherapy, as seen with the anti-leukemic effect of allogeneic hematopoietic stem cell transplantation (alloHSCT). However, response to cytotoxic T lymphocytes (CTLs) that target leukemia-associated antigens (LAAs) has been less reliable in eradicating disease. This insufficient response to LAA-specific CTLs is likely partially accounted for by the immune dysregulation seen in AML. Because of promising murine data that blockade of the PD1/PD-L1 pathway enhances the graft versus leukemia effect of alloHSCT, we investigated if adding pembrolizumab to CTLs that target the two LAAs CG1 and PR1 will enhance CTL antileukemia activities. CG1 and PR1are two HLA-A2 restricted nonameric peptides that we validated as promising AML targets derived from cathepsin G (i.e. CG1), and proteinase 3 (P3) and neutrophil elastase (NE) (i.e. PR1). We hypothesized that pembrolizumab added to CG1-CTLs and PR1-CTLs, will enhance their anti-leukemic effects with minimal off target toxicities. Methods Using a standard calcein AM in vitro cytotoxicity assay, we co-cultured AML targets, including U937 HLA-A2 + (U937-A2) AML cell line and primary patient HLA-A2 + AML samples, with CG1-CTL and PR1-CTL. AML cells were loaded with calcein AM and then incubated with CG1- and PR1-CTLs at increasing effector to target ratios. Pembrolizumab or isotype antibody were added to the cultures. After 4 hours, calcein AM was measured to determine cell viability. T2 cells pulsed with PR1 or CG1, were used as a positive control and non-pulsed T2 cells were used as negative control cells. For in vivo experiments, we used a human established AML xenograft mouse treatment model to determine the effect of pembrolizumab when added to CG1- or PR1- CTL in vivo. CG1-CTL and PR1-CTLs were expanded from the same donor and used as the effector cells. NOD/SCID gamma (NSG) mice (4-6-week-old females) were engrafted with AML samples by tail vein injection. After confirming engraftment, CG1- and/or PR1-CTL were administered to mice and pembrolizumab or isotype antibody [100ug/mouse] were given three times over two weeks. Mice were monitored for clinical GVHD and AML three times/week. Mice were sacrificed at approximately two weeks following treatment. To assess for GVHD, mouse tissues including spleen, liver, kidney, BM, intestine, brain, heart, and lung were harvested and fixed in 10% formalin. The fixed tissue samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin prior to histologic examination. Bone marrow (BM) was processed using standard methodology and analyzed for residual AML by flow cytometry. Results: In vitro our data demonstrate that U937-A2 and two primary patient HLA-A2 AML samples, have enhanced cell lysis when treated with pembrolizumab (CTL+ pembrolizumab) in comparison with isotype (Iso) + CTL, pembrolizumab only, iso only or CTL only groups (Figure 1). In vivo data show a decrease in U937-A2 disease burden and primary patient AML after treatment with CG1and/or PR1-CTL (CTL-treated) . This decrease was enhanced when pembrolizumab was added to the CTL-treated mice (CTL+ pembrolizumab) in comparison with mice treated with pembrolizumab only (pembrolizumab-treated), or CTL only (CTL-treated) (Figure 2). Pathology data to assess toxicity in mice treated with CTL +/- pembrolizumab showed an enhanced pulmonary (perivascular and parabronchial) lymphocytic infiltration in mice treated with the combination of CTL and pembrolizumab. The other organs investigated, showed no change when combination therapy was used. Conclusion: We have validated in vitro and in vivo the enhanced killing of AML (cell lines and primary patient samples) by CG1-CTL and/or PR1-CTL after addition of pembrolizumab. Toxicity data show mild enhancement of lymphocytic infiltrate in the lungs after addition of pembrolizumab to CTL. Our data suggest that the strategy of combining LAA-specific CTL with immune checkpoint blockade could prove beneficial in the setting of adoptive T cell therapy and allogeneic stem cell transplantation for AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Broadly Neutralizing Anti- HIV-1 Antibodies Do Not Inhibit HIV-1 Env-Mediated Cell-Cell Fusion

PG9, PG16, PGT121, and PGT145 antibodies were identified from culture media of activated memory B-cells of an infected donor and shown to neutralize many HIV-1 strains. Since HIV-1 spreads via both free virions and cell-cell fusion, we examined the effect of the antibodies on HIV-1 Env-mediated cell-cell fusion. Clone69TRevEnv cells that express Env in the absence of tetracycline were labeled with Calcein-AM Green, and incubated with CD4+ SupT1 cells labeled with CellTrace™ Calcein Red-Orange, with or without antibodies. Monoclonal antibodies PG9, PG16, 2G12, PGT121, and PGT145 (at up to 50 µg/mL) had little or no inhibitory effect on fusion between HIV-Env and SupT1 cells. By contrast, Hippeastrum hybrid agglutinin completely inhibited fusion. Our results indicate that transmission of the virus or viral genetic material would not be inhibited by these broadly neutralizing antibodies. Thus, antibodies generated by HIV-1 vaccines should be screened for their inhibitory effect on Env-mediated cell-cell fusion.

ABCB1 Does Not Require the Side-Chain Hydrogen-Bond Donors Gln347, Gln725, Gln990 to Confer Cellular Resistance to the Anticancer Drug Taxol

The multidrug efflux transporter ABCB1 is clinically important for drug absorption and distribution and can be a determinant of chemotherapy failure. Recent structure data shows that three glutamines donate hydrogen bonds to coordinate taxol in the drug binding pocket. This is consistent with earlier drug structure-activity relationships that implicated the importance of hydrogen bonds in drug recognition by ABCB1. By replacing the glutamines with alanines we have tested whether any, or all, of Gln347, Gln725, and Gln990 are important for the transport of three different drug classes. Flow cytometric transport assays show that Q347A and Q990A act synergistically to reduce transport of Calcein-AM, BODIPY-verapamil, and OREGON GREEN-taxol bisacetate but the magnitude of the effect was dependent on the test drug and no combination of mutations completely abrogated function. Surprisingly, Q725A mutants generally improved transport of Calcein-AM and BODIPY-verapamil, suggesting that engagement of the wild-type Gln725 in a hydrogen bond is inhibitory for the transport mechanism. To test transport of unmodified taxol, stable expression of Q347/725A and the triple mutant was engineered and shown to confer equivalent resistance to the drug as the wild-type transporter, further indicating that none of these potential hydrogen bonds between transporter and transport substrate are critical for the function of ABCB1. The implications of the data for plasticity of the drug binding pocket are discussed.

Effects of Normal Synovial Fluid and Interferon Gamma on Chondrogenic Capability and Immunomodulatory Potential Respectively on Equine Mesenchymal Stem Cells

Synovial fluid contains cytokines, growth factors and resident mesenchymal stem cells (MSCs). The present study aimed to (1) determine the effects of autologous and allogeneic synovial fluid on viability, proliferation and chondrogenesis of equine bone marrow MSCs (BMMSCs) and (2) compare the immunomodulatory properties of equine synovial fluid MSCs (SFMSCs) and BMMSCs after stimulation with interferon gamma (INF-γ). To meet the first aim of the study, the proliferation and viability of MSCs were evaluated by MTS and calcein AM staining assays. To induce chondrogenesis, MSCs were cultured in a medium containing TGF-β1 or different concentrations of synovial fluid. To meet the second aim, SFMSCs and BMMSCs were stimulated with IFN-γ. The concentration of indoleamine-2,3-dioxygenase (IDO) and nitric oxide (NO) were examined. Our results show that MSCs cultured in autologous or allogeneic synovial fluid could maintain proliferation and viability activities. Synovial fluid affected chondrocyte differentiation significantly, as indicated by increased glycosaminoglycan contents, compared to the chondrogenic medium containing 5 ng/mL TGF-β1. After culturing with IFN-γ, the conditioned media of both BMMSCs and SFMSCs showed increased concentrations of IDO, but not NO. Stimulating MSCs with synovial fluid or IFN-γ could enhance chondrogenesis and anti-inflammatory activity, respectively, suggesting that the joint environment is suitable for chondrogenesis.

Imaging flow cytometry challenges the usefulness of classically used EV labelling dyes and qualifies that of a novel dye, named Exoria for the labelling of MSC-EV preparations

Extracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is central for mediating the EVs role in intercellular communication processes, labelling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of the EVs target cells and tissues. However, the accuracy and specificity of commonly utilized labelling dyes has not been sufficiently analyzed. By combining recent advancements in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant MSC-EV preparations, we explored the EV labelling efficacy of various fluorescent dyes, specifically of CFDA-SE, Calcein AM, PKH67, BODIPY-TR-Ceramide and a novel lipid dye named Exoria. Our analyses qualified Exoria as the only dye which specifically labels EVs within our MSC-EV preparations. Furthermore, we demonstrate Exoria labelling does not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labelled EVs were differentially taken-up by different immune cell types. Overall, our results qualify Exoria as an appropriate dye for the labelling of EVs derived from our MSC-EV preparations, this study also demonstrates the need for the development of next generation EV characterization tools which are able to localize and confirm specificity of EV labelling.

BROADLY NEUTRALIZING ANTI- HIV-1 ANTIBODIES DO NOT INHIBIT HIV-1 ENV-MEDIATED CELL-CELL FUSION

PG9, PG16, PGT121, and PGT145 antibodies were identified from culture media of activated memory B-cells of an infected donor and shown to neutralize many HIV-1 strains. Since HIV-1 spreads via both free virions and cell-cell fusion, we examined the effect of the antibodies on HIV-1 Env-mediated cell-cell fusion. Clone69TRevEnv cells that express Env in the absence of tetracycline were labeled with Calcein-AM Green, and incubated with CD4+ SupT1 cells labeled with CellTrace Calcein Red-Orange, with or without antibodies. Monoclonal antibodies PG9, PG16, 2G12, PGT121, and PGT145 (at up to 50 μg/mL) had little or no inhibitory effect on fusion between HIV-Env and SupT1 cells. By contrast, Hippeastrum hybrid agglutinin completely inhibited fusion. Our results indicate that transmission of the virus or viral genetic material would not be inhibited by these broadly neutralizing antibodies. Thus, antibodies generated by HIV-1 vaccines should be screened for their inhibitory effect on Env-mediated cell-cell fusion.

ABCB1 Does Not Require the Side-Chain Hydrogen-Bond Donors Q347, Q725, Q990 to Confer Cellular Resistance to the Anticancer Drug Taxol

The multidrug efflux transporter ABCB1 is clinically important for drug absorption and distribution and can be a determinant of chemotherapy failure. Recent structure data shows that three glutamines donate hydrogen bonds to co-ordinate taxol in the drug binding pocket. This is consistent with earlier drug structure-activity relationships that implicated the importance of hydrogen bonds in drug recognition by ABCB1. By replacing the glutamines with alanines we have tested whether any or all of Q347, Q725 and Q990 are important for the transport of three different drug classes. Flow cytometric transport assays show that Q347A and Q990A act synergistically to reduce transport of Calcein-AM, BODIPY-verapamil and OREGON GREEN-taxol bisacetate but the magnitude of the effect was dependent on the test drug and no combination of mutations completely abrogated function. Surprisingly, Q725A mutants generally improved transport of Calcein-AM and BODIPY-verapamil, suggesting that engagement of the wild-type Q725 in a hydrogen bond is inhibitory for the transport mechanism. To test transport of unmodified taxol, stable expression of Q347/725A and the triple mutant was engineered and shown to confer equivalent resistance to the drug as the wild-type transporter, further indicating that none of these potential hydrogen bonds between transporter and transport substrate are critical for function of ABCB1. The implications of the data for plasticity of the drug binding pocket are discussed.

Viability of preloaded Descemet membrane endothelial keratoplasty grafts with 96-hour shipment

ObjectiveTo assess feasibility and compare the effects of 96-hour shipment of Descemet membrane endothelial keratoplasty (DMEK) grafts as a scroll or a tri-fold on cell viability.Methods and analysisDMEK grafts were prepared at the Rocky Mountain Lions Eye Bank. Twenty pre-stripped DMEK grafts, paired from 10 donors, were either tri-folded in an endothelium-in configuration using microforceps and loaded into a plastic Treyetech cartridge, or suctioned in a scrolled endothelium-out configuration into a modified Jones Tube. Grafts were shipped via FedEx to a secondary location and back for 48 hours each way, resulting in a total shipping time of 96 hours. After shipping, grafts were removed from inserters onto glass slides and unfolded using viscoelastic with endothelium facing upwards. Calcein-AM stained grafts were imaged with a fluorescent microscope and endothelial cell loss (ECL) was measured using trainable segmentation in Fiji by a masked grader.ResultsA total of 20 grafts were shipped for 96 hours, split between preloaded tri-folded (n=10) and preloaded scrolled (n=10) tissues. No significant difference in ECL was observed across groups after prolonged shipping (14.8% vs 13.7% ECL respectively, p=0.68).ConclusionFor preloaded DMEK after 96 hours, both scrolled and tri-folded tissue demonstrated clinically acceptable levels of ECL. The data suggest a wider window of time for endothelial cell viability and is promising for the prospect of international shipment of preloaded grafts.

Permanent ectoplasmic structures in deep-sea Cibicides/oides taxa – long-term observations at in situ pressure

Deep-sea Cibicidoides pachyderma (forma mundulus) and related Cibicidoides spp. were cultured at in situ pressure for 1-2 days, or 6 weeks to 3 months. During that period, fluorescence analyses following BCECF-AM (2’,7’-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester) or Calcein AM (4,5-Bis((N,N-bis(carboxymethy)amino)methyl)fluorescein acetoxymethylester) labelling, revealed a persisting cytoplasmic sheet or envelope surrounding the Cibicidoides tests. Thus, the Cibicidoides shell can be considered rather as an internal than an external cell structure. A couple of days to a week after being transferred into high-pressure aquaria and adjusted to a pressure of 115 bar, the foraminifera changed from a mobile to a more or less sessile living mode. During this quasi sessile way of life, a series of comparably thick static ectoplasmic structures developed that were not resorbed or remodelled but, except for occasional further growth, remained unchanged throughout the experiments. Three different types of these ‘permanent structures’ were observed: A) Ectoplasmic ‘roots’ were common in adult C. pachyderma, C. lobatulus and C. wuellerstorfi specimens. In our experiments single ectoplasmic ‘roots’ grew to maximum 700 times the individuals shell diameter and were presumably used to anchor the specimen in an environment with strong currents. B) Ectoplasmic ‘trees’ describe rigid ectoplasmic structures directed into the aquarium’s water body and were used by the foraminifera to climb up and down these ectoplasmic structures. Ectoplasmic ‘trees’ were so far only observed in C. pachyderma and enabled the ‘tree’-forming foraminifera to elevate itself above ground. C) Ectoplasmic ‘twigs’ were used to guide and hold the more delicate pseudopodial network when distributed into prevailing currents, and were, in our experiments, also only developed in C. pachyderma specimens. Relocation of a specimen usually required to tear apart and leave behind the rigid ectoplasmic structures, eventually also the envelope surrounding the test. Apparently, these rigid structures could not be resorbed or reused.

Влияние цитофлавина на жизнеспособность лейкоцитов периферической крови пациентов с ишемической болезнью сердца

Для определения характера влияния цитофлавина в терапевтической концентрации на жизнеспособность лейкоцитов проведено исследование образцов крови 30 пациентов с ишемической болезнью сердца. Лейкоциты из цельной крови выделяли с помощью микропипетки, смешивали с питательной средой RPMI, в опытные лунки добавляли цитофлавин в концентрации, близкой к терапевтической и пробы инкубировали в течение 3 ч в инкубаторе с 5 % содержанием СО2 при температуре 37 °C, затем пробы окрашивали флуоресцентными красителями (Mito TrackerTM Red CMXRos, Calcein AM, Ethidium bromide) и снова инкубировали при тех же условиях ещё в течение 30 мин. Результаты детектировали методом флуоресцентной микроскопии при помощи инвертированного флуоресцентного микроскопа EclipseTi-U (Nikon, Япония) и анализировали с помощью специализированного программного обеспечения EZ-C1 Free Viewer Ver 3.90. Оценивали интенсивность флуоресценции митохондрий и вычисляли индекс жизнеспособности лейкоцитов по разработанной методике. После введения цитофлавина в пробу с лейкоцитарной взвесью наблюдалось два варианта изменения жизнеспособности клеток: у большинства (80 %) больных отмечено увеличение жизнеспособности лейкоцитов, в среднем, на 60 % (p < 0,001) и у 20 % — тенденция к уменьшению данного показателя, в среднем, на 47 % (p < 0,1). Величина флуоресценции митохондрий изменялась в обоих случаях в сторону увеличения, но без достижения достоверности различий. Достоверной корреляции между показателями жизнеспособности клеток и интенсивностью флуоресценции митохондрий обнаружено не было (коэффициент корреляции составил 0,28, p > 0,05). Цитофлавин у 80 % пациентов с ишемической болезнью сердца способен повышать жизнеспособность клеток (лейкоцитов крови), в среднем, на 60 % и таким образом проявлять цитопротекторный эффект.