Analysis, Isolation, and Activation of Antigen-Specific CD4 + and CD8+ T Cells by Soluble MHC-Peptide Complexes

T cells constitute the core of adaptive cellular immunity and protect higher organisms against pathogen infections and cancer. Monitoring of disease progression as well as prophylactic or therapeutic vaccines and immunotherapies call for conclusive detection, analysis, and sorting of antigen-specific T cells. This is possible by means of soluble recombinant ligands for T cells, i.e., MHC class I-peptide (pMHC I) complexes for CD8(+) T cells and MHC class II-peptide (pMHC II) complexes for CD4(+) T cells and flow cytometry. Here we review major developments in the development of pMHC staining reagents and their diverse applications and discuss perspectives of their use for basic and clinical investigations

Analysis of CD4+ and CD8+ T cells by defined MHC-peptide complexes

MHC class II-peptide multimers are important tools for the detection, enumeration and isolation of antigen-specific CD4+ Τ cells. However, their erratic and often poor performance impeded their broad application and thus in-depth analysis of key aspects of antigen-specific CD4+ Τ cell responses. In the first part of this thesis we demonstrate that a major cause for poor MHC class II tetramer staining performance is incomplete peptide loading on MHC molecules. We observed that peptide binding affinity for "empty" MHC class II molecules poorly correlates with peptide loading efficacy. Addition of a His-tag or desthiobiotin (DTB) at the peptide N-terminus allowed us to isolate "immunopure" MHC class II-peptide monomers by affinity chromatography; this significantly, often dramatically, improved tetramer staining of antigen-specific CD4+ Τ cells. Insertion of a photosensitive amino acid between the tag and the peptide, permitted removal of the tag from "immunopure" MHC class II-peptide complex by UV irradiation, and hence elimination of its potential interference with TCR and/or MHC binding. Moreover, to improve loading of self and tumor antigen- derived peptides onto "empty" MHC II molecules, we first loaded these with a photocleavable variant of the influenza A hemagglutinin peptide HA306-318 and subsequently exchanged it with a poorly loading peptide (e.g. NY-ESO-1119-143) upon photolysis of the conditional ligand. Finally, we established a novel type of MHC class II multimers built on reversible chelate formation between 2xHis-tagged MHC molecules and a fluorescent nitrilotriacetic acid (NTA)-containing scaffold. Staining of antigen-specific CD4+ Τ cells with "NTAmers" is fully reversible and allows gentle cell sorting. In the second part of the thesis we investigated the role of the CD8α transmembrane domain (TMD) for CD8 coreceptor function. The sequence of the CD8α TMD, but not the CD8β TMD, is highly conserved and homodimerizes efficiently. We replaced the CD8α TMD with the one of the interleukin-2 receptor a chain (CD8αTac) and thus ablated CD8α TMD interactions. We observed that ΤΙ Τ cell hybridomas expressing CD8αTacβ exhibited severely impaired intracellular calcium flux, IL-2 responses and Kd/PbCS(ABA) P255A tetramer binding. By means of fluorescence resonance energy transfer experiments (FRET) we established that CD8αTacβ associated with TCR:CD3 considerably less efficiently than CD8αβ, both in the presence and the absence of Kd/PbCS(ABA) complexes. Moreover, we observed that CD8αTacβ partitioned substantially less in lipid rafts, and related to this, associated less efficiently with p56Lck (Lck), a Src kinase that plays key roles in TCR proximal signaling. Our results support the view that the CD8α TMD promotes the formation of CD8αβP-CD8αβ dimers on cell surfaces. Because these contain two CD8β chains and that CD8β, unlike CD8α, mediates association of CD8 with TCR:CD3 as well as with lipid rafts and hence with Lck, we propose that the CD8αTMD plays an important and hitherto unrecognized role for CD8 coreceptor function, namely by promoting CD8αβ dimer formation. We discuss what implications this might have on TCR oligomerization and TCR signaling. - Les multimères de complexes MHC classe II-peptide sont des outils importants pour la détection, le dénombrement et l'isolation des cellules Τ CD4+ spécifiques pour un antigène d'intérêt. Cependant, leur performance erratique et souvent inadéquate a empêché leur utilisation généralisée, limitant ainsi l'analyse des aspects clés des réponses des lymphocytes Τ CD4+. Dans la première partie de cette thèse, nous montrons que la cause principale de la faible efficacité des multimères de complexes MHC classe II-peptide est le chargement incomplet des molécules MHC par des peptides. Nous montrons également que l'affinité du peptide pour la molécule MHC classe II "vide" n'est pas nécessairement liée au degré du chargement. Grâce à l'introduction d'une étiquette d'histidines (His-tag) ou d'une molécule de desthiobiotine à l'extrémité N-terminale du peptide, des monomères MHC classe II- peptide dits "immunopures" ont pu être isolés par chromatographic d'affinité. Ceci a permis d'améliorer significativement et souvent de façon spectaculaire, le marquage des cellules Τ CD4+ spécifiques pour un antigène d'intérêt. L'insertion d'un acide aminé photosensible entre l'étiquette et le peptide a permis la suppression de l'étiquette du complexe MHC classe- Il peptide "immunopure" par irradiation aux UV, éliminant ainsi de potentielles interférences de liaison au TCR et/ou au MHC. De plus, afin d'améliorer le chargement des molécules MHC classe II "vides" avec des peptides dérivés d'auto-antigènes ou d'antigènes tumoraux, nous avons tout d'abord chargé les molécules MHC "vides" avec un analogue peptidique photoclivable issu du peptide HA306-318 de l'hémagglutinine de la grippe de type A, puis, sous condition de photolyse, nous l'avons échangé avec de peptides à chargement faible (p.ex. NY-ESO-1119-143). Finalement, nous avons construit un nouveau type de multimère réversible, appelé "NTAmère", basé sur la formation chélatante reversible entre les molécules MHC-peptide étiquettés par 2xHis et un support fluorescent contenant des acides nitrilotriacetiques (NTA). Le marquage des cellules Τ CD4+ spécifiques pour un antigène d'intérêt avec les "NTAmères" est pleinement réversible et permet également un tri cellulaire plus doux. Dans la deuxième partie de cette thèse nous avons étudié le rôle du domaine transmembranaire (TMD) du CD8α pour la fonction coréceptrice du CD8. La séquence du TMD du CD8α, mais pas celle du TMD du CD8β, est hautement conservée et permet une homodimérisation efficace. Nous avons remplacé le TMD du CD8α avec celui de la chaîne α du récepteur à l'IL-2 (CD8αTac), éliminant ainsi les interactions du TMD du CD8α. Nous avons montré que les cellules des hybridomes Τ T1 exprimant le CD8αTacβ présentaient une atteinte sévère du flux du calcium intracellulaire, des réponses d'IL-2 et de la liaison des tétramères Kd/PbCS(ABA) P255A. Grâce aux expériences de transfert d'énergie entre molécules fluorescentes (FRET), nous avons montré que l'association du CD8αTacβ avec le TCR:CD3 est considérablement moins efficace qu'avec le CD8αβ, et ceci aussi bien en présence qu'en absence de complexes Kd/PbCS(ABA). De plus, nous avons observé que le CD8αTacβ se distribuait beaucoup moins bien dans les radeaux lipidiques, engendrant ainsi, une association moins efficace avec p56Lck (Lck), une kinase de la famille Src qui joue un rôle clé dans la signalisation proximale du TCR. Nos résultats soutiennent l'hypothèse que le TMD du CD8αβ favorise la formation des dimères de CD8αβ à la surface des cellules. Parce que ces derniers contiennent deux chaînes CD8β et que CD8β, contrairement à CD8α, favorise l'association du CD8 au TCR:CD3 aussi bien qu'aux radeaux lipidiques et par conséquent à Lck, nous proposons que le TMD du CD8α joue un rôle important, jusqu'alors inconnu, pour la fonction coreceptrice du CD8, en encourageant la formation des dimères CD8αβ. Nous discutons des implications possibles sur l'oligomerisation du TCR et la signalisation du TCR

Analiza koncentracije toksičnih i esencijalnih elementa (as, cd, cu, cr, hg, ni, pb, sr, zn) u zooplanktonu sa šaranskog ribnjaka

U poslednjih 20 godina zagađivanje slatkovodnih ekosistema toksičnim elementima je u porastu širom sveta. Zagađenjem su pogođeni pre svega izvori za vodosnabdevanje stanovništva i životno okruženje, ali i industrija kao i privreda uopšte. Međutim zbog perzistentnosti i transfera kroz lance ishrane i potencijalnog akumuliranja u ribama i drugim vodenim organizmima koji se koriste u ishrani, toksični elementi predstavljaju stalnu pretnju ljudskom zdravlju. Zagađivanje naših reka teškim metalima nameće pitanje ne samo zdravstvene ispravnosti riba iz reka već i riba iz ribnjaka obzirom da se većina šaranskih ribnjaka napaja vodom iz sistema kanala DTD. Cilj ove studije je bio da se analizira koncentracija 9 elemenata u zooplanktonu koji predstavlja značajnu prirodnu hranu šarana u poluintenzivnom sistemu gajenja. Istraživanje je obavljeno na 4 ribnjačka objekta, tokom dva ciklusa gajenja šarana, od juna do oktobra, na ribnjaku „Despotovo“. Uzorci zooplanktona za analizu elemenata su uzimani sa tri tačke u svakom jezeru pomoću planktonske mrežice veličine 250 µm jednom mesečno. Na ovaj način su sakupljene samo krupnije veličinske klase zooplanktona (Cladocera i Copepoda), koje šaranska mlađ najviše konzumira. Sa svakog jezera je uziman po još jedan uzorak zooplanktona za kvantitativnu i taksonomsku analizu. Koncentracija elemenata je analizirana induktivno spregnutom plazma masenom (ICP-MS) i optičkom emisionom spektrometrijom (ICP-OES). Rezultati su obrađeni jednofaktorijalnom analizom varijanse (ANOVA) u statističkom programu PAST 3.06. Značajnost razlika testirana je primenom Tukey’s post hoc testa. Podaci su klasifikovani na prolećni, letnji i jesenji aspekt tokom jednog proizvodnog ciklusa. Cladocera su dominirale u populaciji zooplanktona, osim u junu kada su Copepoda bile zastupljenije. Iako nije bilo značajnih razlika u koncentraciji elemenata između godina, osim za Cu i Sr, uočen je karakterističan sezonski obrazac kretanja koncentracija elemenata tokom celog istraživanja. Prolećni i jesenji aspekti u 2012 su bili veći nego u 2013, dok je letnji aspekt u 2013 bio viši nego u 2012. godini. Izuzetak je bila koncentracija Zn u zooplanktonu gde je situacija bila obrnuta. Povišene vrednosti većine toksičnih metala u zooplanktonu na ribnjaku Despotovo se mogu objasniti relativno velikim afinitetom egzoskeleta Cladocera za većinu dvovalentih jona. Nakon adsorbcije elementi na površini ljuštura ovih životinja, tokom vremena bivaju absorbovani kroz telesni zid u unutrašnje organe. Neke studije čak navode da površinski akumulirani kontaminanti na plenu mogu biti dostupniji predatorima od onih akumuliranih u tkivima, zbog niskog pH i visokog nivoa jonske kompleksacije koji vladaju na mestu abstorpcije, u digestivnom traktu većine životinja, Ovi rezultati nameću zaključak da je u budućim istraživanjima kontaminacije vodenih ekosistema i riba poželjno uključiti analize ne samo vode kao izvora toksičnih elemenata, već i odgovarajućih izvora hrane (plena) koji, kako je pokazano, sadrži potencijal ne samo za značajnu akumulaciju elemenata već i njihovu potencijalnu veću biodostupnost konzumentima

MAGE-A3 and MAGE-A4 specific CD4+ T cells in head and neck cancer patients: detection of naturally acquired responses and identification of new epitopes

Frequent expression of cancer testis antigens (CTA) has been consistently observed in head and neck squamous cell carcinomas (HNSCC). For instance, in 52 HNSCC patients, MAGE-A3 and -A4 CTA were expressed in over 75% of tumors, regardless of the sites of primary tumors such as oral cavity or hypopharynx. Yet, T-cell responses against these CTA in tumor-bearing patients have not been investigated in detail. In this study, we assessed the naturally acquired T-cell response against MAGE-A3 and -A4 in nonvaccinated HNSCC patients. Autologous antigen-presenting cells pulsed with overlapping peptide pools were used to detect and isolate MAGE-A3 and MAGE-A4 specific CD4+ T cells from healthy donors and seven head and neck cancer patients. CD4+ T-cell clones were characterized by cytokine secretion. We could detect and isolate MAGE-A3 and MAGE-A4 specific CD4+ T cells from 7/7 cancer patients analyzed. Moreover, we identified six previously described and three new epitopes for MAGE-A3. Among them, the MAGE-A3111-125 and MAGE-A3161-175 epitopes were shown to be naturally processed and presented by DC in association with HLA-DP and DR, respectively. All of the detected MAGE-A4 responses were specific for new helper epitopes. These data suggest that naturally acquired CD4+ T-cell responses against CT antigens often occur in vivo in HNSCC cancer patients and provide a rationale for the development of active immunotherapeutic approaches in this type of tumo

Ispitivanje odabranih fizičko–hemijskih osobina i sadržaja makro- i mikroelemenata u vodi jezera Gruža

Water is one of the most precious natural resources. The downtrend in the quality of lake water and pollution with heavy metals cause serious threats to the environment in last decades. Particularly vulnerable are the lakes that are located near towns and villages. The aim of this study was to investigate the physicochemical parameters and levels of macro– and microelements in order to determine the water quality of Lake Gruža in the summer period. The results showed that the studied parameters were within the permissible levels, except of concentrations of iron, cobalt and vanadium.Voda predstavlja jedan od najdragocenijih prirodnih resursa. Opadanje kvaliteta jezerskih voda i zagađenje teškim metalima, predstavljaju ozbiljne pretnje za životnu sredinu u poslednjih nekoliko decenija. Posebno su ugrožena jezera koja se nalaze u blizini gradova i naselja. Cilj rada bio je ispitivanje osnovnih fizičko–hemijskih parametara i sadržaja makro– i mikroelemenata kako bi se utvrdio kvalitet vode jezera Gruža u letnjem periodu. Rezultati su pokazali da su ispitivani parametri u vodi bili u okviru dozvoljenih vrednosti, osim sadržaja gvožđa, kobalta i vanadijuma

Cell cycle, apoptosis, cellular uptake and whole-transcriptome microarray gene expression analysis of HeLa cells treated with a ruthenium(II)-arene complex with an isoquinoline-3-carboxylic acid ligand

Ruthenium(II)-arene complexes are promising drug candidates for the therapy of solid tumors. In previous work, seven new compounds of the general formula [Ru(η6-p-cymene)(L1–7)Cl] were synthesized and characterized, of which the complex with L = isoquinoline-3-carboxylic acid (RuT7) was two times as active on HeLa cells compared to normal cell line MRC-5, as indicated by IC50 values determined after 48 h of incubation (45.4 ± 3.0 vs. 84.2 ± 5.7 μM, respectively). In the present study, cell cycle analysis of HeLa cells treated with RuT7 showed S phase arrest and an increase in sub-G1 population. The apoptotic potential of the title compound was confirmed with the Annexin V-FITC/PI assay together with a morphological evaluation of cells using fluorescent microscopy. Analysis of the intracellular accumulation of ruthenium showed 8.9 ng Ru/106 cells after 6 h of incubation. To gain further insight in the molecular mechanism of action of RuT7 on HeLa cells, a whole-transcriptome microarray gene expression analysis was performed. Analysis of functional categories and signaling and biochemical pathways associated with the response of HeLa cells to treatment with RuT7 showed that it leads the cells through the intrinsic (mitochondrial) apoptotic pathway, via indirect DNA damage due to the action of reactive oxygen species, and through direct DNA binding of RuT7. Statistical analysis for enrichment of gene sets associated with known drug-induced toxicities identified fewer associated toxicity profiles in RuT7-treated cells compared to cisplatin treatment. Altogether these results provide the basis for further development of RuT7 in animal and pre-clinical studies as a potential drug candidate

Spectroscopy and spectropolarimetry of AGN: from observations to modelling

Active galactic nuclei (AGN) are one of the most luminous objects in the Universe, emitting powerful continuum and line emission across all wavelength bands. They represent an important link in the investigations of the galaxy evolution and cosmology. The resolving of the AGN inner structure is still a difficult task with current instruments, therefore the spectroscopy and spectropolarimetry are crucial tools to investigate these objects and their components, such as the properties of the supermassive black hole, the broad line region, and the dusty torus. In this review, we present the results of the project "Astrophysical spectroscopy of extragalactic objects", from the observations, data processing and analysis, to the modelling of different regions in AGN.Comment: Proceedings of the Serbian Astronomical Conference 201