The Effect of Acute Myeloid Leukemia Cells Expressing PD-L2 Co-Stimulatory Molecule on Helper T Cell Activation

The binding of PD-1 to its ligands (PD-L1 and PD-L2) inhibits and/or regulates T cell responses. Myeloid leukemia cells can express both co-activatory and co-inhibitory molecules simultaneously. The function of PD-L1 in regulation of antitumor responses is well-known. Although under physiological conditions, PD-L2 is expressed on mature antigen presenting cells (APCs), it can also be highly expressed on monocytic leukemia that is composed of immature blasts. In this study, THP-1 was used as monocytic leukemia cell line and it resembles to primary monocytes. Monocytes were cultured in vitro to differentiate, while THP-1 cells were differentiated into monocyte/macrophage-like cells with protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) and these cells were called “pTHP-1 cells”. Then, the expression of PD-1 ligands and B7 molecules were modulated and their effect on T helper responses was evaluated. Primary monocytes used as controls were maturated in suspension in vitro. By this way, the expressions of B7 molecules (especially PD-L2) were modulated and morphologically, macrophage-like cell type was obtained. However, because PD-L1 could not have been upregulated on pTHP-1 cells and cultured monocytes, THP-1 and pTHP-1 were stimulated with IFN-γ as a critical cytokine in anti-tumor responses. These cells were co-cultured with Th cells or peripheral blood mononuclear cells (PBMCs). In the co-cultures, the first signal was provided by anti-CD3 mAb and the second signal was provided by co-stimulatory molecules expressed on monocyte/macrophage-like cells. Although THP-1 cells and monocytes resemble to each other, they behaved differentially. It was also found that the effect of THP-1 cells on Th cells was generally maintained through co-stimulatory mechanisms. Upon IFN-γ treatment, monocytes gained less stimulatory character. When PD-L2 molecule was blocked on IFN-γ-treated and control monocytic cells, it led to increase in the proliferation of Th cells and promote Th1 differentiation. Upon PD-L2 blockade, especially in the co-cultures with control monocytes, the production of Th2-related cytokines were also increased. To confirm the results, HEK293T cells were transfected with PD-L1, PD-L2 and CD86 genes. No Treg differentiation or T cell exhaustion was observed. On the other hand, blockade of PD-L2 molecule promoted immune activation. Consequently, in this study, the immune modulatory effects of PD-L2 on primary monocytes cells and on monocytic leukemia cells were compared and novel findings were obtained.Hacettepe Üniversitesi Bilimsel Araştırma Birimi (Proje No: TSA-2017-12739).PD-1 reseptörü ve ligandları (PD-L1 ve PD-L2), T lenfosit yanıtını inhibe eder ve/veya düzenler. Miyeloid lösemi hücreleri hem ko-aktivatör hem de koinhibitör molekülleri eş zamanlı olarak hücre yüzeyinde ifade etme kapasitesine sahiptir. PD-L1’in kanserde immün düzenlenme üzerine etkisi daha net olarak bilinmektedir. Fizyolojik koşullar altında, PD-L2 matür antijen sunan hücrelerin üzerinde bulunmasına rağmen, immatürite ile karakterize olan monositik lösemilerde de yüksek düzeyde bulunmaktadır. Bu çalışmada, monositlere olan benzerliğiyle bilinen monositik lösemi hücre hattı THP-1 kullanılmıştır. Monositler in vitro kültür ve THP-1 hücreleri de protein kinaz C aktivatörü, forbol 12-miristat 13-acetat (PMA) etkisi altında farklılaştırılarak (pTHP-1 hücreleri) alternatif monositik/makrofajbenzeri hücre türevleri yaratılmıştır. Bu hücrelerin karakterizasyonunun ardından, PD-1 ligandlarının ve diğer B7 ailesi kostimülatör moleküllerinin ekspresyonu modüle edilmiş ve yardımcı T hücre yanıtlarına etkileri değerlendirilmiştir. Kontrol olarak kullanılan primer monositler süspansiyon kültürde olgunlaşma basamağına itildi. Bu şekilde B7 ailesi kostimulatör moleküllerinin (özellikle PD-L2’nin) modülasyonu sağlandı ve morfolojik olarak makrofaj-benzeri bir hücre tipi elde edildi. Ancak, hem pTHP-1’de hem de kültürde bekletilen monositlerde PD-L1 yüksek düzeyde upregüle edilemedi. Bu yüzden, THP-1, pTHP-1 ve primer kültürdeki monositler her iki PD-1 ligandını artıran ve anti-tümör yanıtlarda da önemli rol oynayan IFN-γ ile muamele edildi. Bu hücreler yardımcı T lenfositler ve/veya periferik kan mononükleer hücreleri ile bir arada tutuldu. Gerçekleştirilen bu ko-kültürlerde ilk sinyal anti-CD3 antikoru aracılığıyla verilerek, ikinci sinyalin monositik/makrofaj-benzeri hücrelerdeki kostimülatör moleküller aracılığıyla verilmesi sağlandı. THP-1 ve monositlerin benzerliklerinin yanı sıra oldukça farklı davranabildiği görüldü. THP-1’in yardımcı T hücreler üzerindeki etkisinin büyük ölçüde kostimülasyon mekanizmaları üzerinden gerçekleştiğine dair bulgular elde edildi. IFN-γ ile muamele edilmiş monositik hücrelerin daha az uyarıcı olduğu gözlendi. Gerek kontrol gerekse IFN-γ ile muamele edilen monositik hücreler varlığnda PD-L2 molekülü bloklanarak yardımcı T hücre proliferasyonunun ve Th1 dönüşümünün arttığı gözlemlendi. Th1-ilişkili sitokinlerin yanısıra özellikle kontrol monositlerde PD-L2’nin blokajı ile Th2-ilişkili sitokin artışları da izlendi. Proliferasyon bulguları PD-L1, PD-L2 ve CD86 genleri aktarılan HEK293T hücreleri ile de doğrulandı. Treg dönüşümü veya T hücre yorulması izlenmedi. Diğer taraftan, PD-L2’nin tek başına inhibe edilmesi de önemli ölçüde immün aktivasyon sağladı. Sonuç olarak, normal monositlerde ve monositik lösemi hücrelerinde bulunan PD-L2’nin immün yanıtları düzenleme yeteneği karşılaştırılmış ve önemli bilgiler elde edilmiştir

Ateşle emeğin birleştiği yer : Paşabahçe

Ankara : İhsan Doğramacı Bilkent Üniversitesi İktisadi, İdari ve Sosyal Bilimler Fakültesi, Tarih Bölümü, 2013.This work is a student project of the The Department of History, Faculty of Economics, Administrative and Social Sciences, İhsan Doğramacı Bilkent University.by Berna Kamay.Kamay, Berna. HIST 200-20KAMAY HIST 200-20/2 2012-1

Effects of Particle Geometry for PLGA-Based Nanoparticles: Preparation and In Vitro/In Vivo Evaluation

The physicochemical properties (size, shape, zeta potential, porosity, elasticity, etc.) of nanocarriers influence their biological behavior directly, which may result in alterations of the therapeutic outcome. Understanding the effect of shape on the cellular interaction and biodistribution of intravenously injected particles could have fundamental importance for the rational design of drug delivery systems. In the present study, spherical, rod and elliptical disk-shaped PLGA nanoparticles were developed for examining systematically their behavior in vitro and in vivo. An important finding is that the release of the encapsulated human serum albumin (HSA) was significantly higher in spherical particles compared to rod and elliptical disks, indicating that the shape can make a difference. Safety studies showed that the toxicity of PLGA nanoparticles is not shape dependent in the studied concentration range. This study has pioneering findings on comparing spherical, rod and elliptical disk-shaped PLGA nanoparticles in terms of particle size, particle size distribution, colloidal stability, morphology, drug encapsulation, drug release, safety of nanoparticles, cellular uptake and biodistribution. Nude mice bearing non-small cell lung cancer were treated with 3 differently shaped nanoparticles, and the accumulation of nanoparticles in tumor tissue and other organs was not statistically different (p > 0.05). It was found that PLGA nanoparticles with 1.00, 4.0 ± 0.5, 7.5 ± 0.5 aspect ratios did not differ on total tumor accumulation in non-small cell lung cancer