Vpliv različnih fluorokromov na učinkovitost znotrajceličnega označevanja transkripcijskega dejavnika FoxP3 in aplikacija v in vitro testu indukcije celic Treg

Kramar, Eva (author), Švajger, Urban (mentor)

Regulatorne limfocite T (Treg) uvrščamo v skupino CD4 pozitivnih limfocitov T. Sodelujejo pri uravnavanju imunskega sistema, in sicer tako, da preko različnih mehanizmov uravnavajo ravnotežje med imunskim odzivom in imunsko toleranco. V osnovi jih delimo na naravne Treg, ki nastanejo med limfopoezo v priželjcu, in na Treg, ki se inducirajo na periferiji. Prepoznamo jih lahko preko molekul, ki jih izražajo na svoji površini; le-te za namen laboratorijskih raziskav označimo s protitelesi konjugiranimi z različnimi barvili. Ena od molekul, ki nam omogoča prepoznavo Treg, je tudi znotrajcelični transkripcijski dejavnik FoxP3. FoxP3 ima ključno vlogo v razvoju in delovanju Treg in posledično igra pomembno vlogo v regulaciji tolerance do lastnega ter sodeluje pri preprečevanju avtoimunskih bolezni. V magistrski nalogi smo preučevali vpliv treh v pretočni citometriji najpogosteje uporabljenih fluorokromov (Alexa Fluor 488, fikoeritrin, alofikocianin) – opazovali smo zmožnost njihovega prehajanja preko permeabilizirane celične membrane in primernost za označevanje FoxP3. Ugotovili smo, da je za označevanje FoxP3 najprimernejše barvilo Alexa Fluor 488, saj nam daje največji odstotek FoxP3+ dogodkov. Izbrani fluorokrom smo v nadaljevanju uporabili za preučevanje indukcije FoxP3+ Treg v testu in vitro. Hkrati smo preučevali tudi pomen signalizacijske osi PD-1/PD-L1 v tem procesu. Za indukcijo FoxP3 Treg iz naivnih CD4 pozitivnih limfocitov T smo uporabili dendritične celice (DC), ki smo jih pridobili iz osamljenih monocitov in nato aktivirali na različne načine. Za aktivacijo DC smo uporabili lipopolisaharid in interferon-γ (IFN-γ) oziroma IFN-γ v različnih koncentracijah. Uspeli smo pokazati, da DC, ki jih aktiviramo s 1000 IU/mL IFN-γ inducirajo največji odstotek FoxP3+ limfocitov T. Prav tako smo pokazali, da dodatek nevtralizacijskega protitelesa, usmerjenega proti PD-L1, občutno zniža sposobnost z IFN-γ-tretiranih DC, da inducirajo FoxP3+ Treg. S tem smo pokazali povečano sposobnost z IFN-γ-tretiranih DC za indukcijo FoxP3+ Treg in pomembnost signalizacije PD-1/PD-L1 za njihov nastanek. Fenotip DC, tretiranih s 1000 IU/mL IFN-γ, je prikazoval največje izražanje inhibitorne molekule PD-L1. S tem se nakazuje tolerogenost tako pripravljenih DC, ki jo povezujemo z visokim izražanjem inhibitornih molekul in istočasno nizkim izražanjem kostimulacijskih molekul.

• Language: Slovenian
• Keywords: Regulatorni limfociti T, transkripcijski dejavnik FoxP3, molekula PD-L1.
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-125999
• Publication date in RUL: 11.04.2021

Secondary language

• Language: English
• Title: The influence of various fluorochromes on intracellular staining efficiency of the transcription factor FoxP3 and application in in vitro assay of Treg cell induction

Abstract

Regulatory T cells (Tregs) are a subset of CD4 positive T lymphocytes. They are involved in the regulation of the immune system. Through different mechanisms, they regulate the balance between immune activation and immune tolerance. Tregs are divided into two groups – those originating from the thymus and peripheral Tregs. We can identify them by the different molecules on their surface. For analysis purpose, we label those molecules with antibodies conjugated with different fluorochromes. One of the molecules that enable their recognition is the FoxP3 transcription factor. FoxP3 has a key role in Treg development and function. Therefore, it plays an important role in the maintenance of self-tolerance and participates in the prevention of autoimmune diseases. We were observing the three most frequently used fluorochromes in flow cytometry (Alexa Fluor 488, phycoerythrin, allofycocyanin) – we checked their ability to penetrate the cell membrane and their ability to label FoxP3 transcription factor. We found out that the greatest percentage of FoxP3+ cells is reached when we use antibodies conjugated with Alexa Fluor 488. We subsequently used this fluorochrome to measure the induction of FoxP3+ Tregs in an in vitro assay. At the same time, we were investigating the importance of the PD-1/PD-L1 signal pathway in that process. For the induction of FoxP3+ Tregs, we were using dendritic cells (DC) differentiated from isolated monocytes and later activated in a different manner. For activation of DC, we used lipopolysaccharide in combination with interferon-γ (IFN-γ) or IFN-γ alone. We managed to prove that DCs activated with 1000 IU/mL IFN-γ induce the highest percentage of FoxP3+ T lymphocytes. We also showed that adding of neutralizing antibody against the PD-L1 molecule significantly lowers the ability of DC treated with IFN-γ to induce FoxP3+ Tregs. Thus, we demonstrated a higher ability of DC treated with IFN-γ to induce FoxP3+ Tregs and the importance of PD-1/PD-L1 signal pathway for their generation. The phenotype of DCs treated with 1000 IU/mL IFN-γ showed the highest expression of PD-L1 molecule. Thus we indicate tolerability of DC prepared with 1000 IU/mL IFN-γ related with high expression of inhibitory molecules and low expression of co-stimulatory molecules at the same time.

• Keywords: Regulatory T cells, transcription factor FoxP3, PD-L1 molecule.