STUDIES ON THE RECONSTITUTION OF T CELL PRODUCTION IN ZAP-70 DEFICIENT MICE

The ZAP-70 (70 kDa Zeta-Chain Associated Protein) kinase plays a crucial role in the signal transduction by the antigen receptor during T cell activation. It is essential in T cell differentiation as well, in its absence, T cell maturation is blocked in the double positive (CD4+CD8+) stage in the thymus, leading to the complete lack of mature αβ T cells in the periphery which results in severe immunodeficiency in both human and mice. T cell maturation could be stably restored in ZAP-70 deficient mice by simple intraperitoneal (ip.) injection of thymocytes isolated from their wild type (ZAP-70 expressing) siblings. T cells appeared in the blood and lymphoid organs of the transferred mice and, importantly, the survival of the animals increased significantly, which clearly demonstrated the correction of the severe immunodeficiency. In our present work investigated the characteristics and kinetics of the T cell reconstitution by monitoring the changes in the morphology and cellular composition of the thymus and peripheral lymphoid organs. Following the thymocyte transfer of ZAP-70-/- mice the histology of the thymus and the appearance of T cells in the periphery were analyzed regularly. Flow cytometry revealed that 3 weeks after the ip. thymocyte transfer αβ T cells appeared in significant numbers in the blood, spleen and lymph nodes of the animals. This was preceded by the appearance of mature CD4+ or CD8+ cells in the thymus 2 weeks after the transfer. Using quantitative immunohistology we have found that the area of the medullary region increased after the transfer, which also indicated the normalisation of T cell maturation. In future experiments, we plan to test the in vivo effects of ZAP-70 point mutations using ZAP-70 deficient mice. We will reconstitute the ZAP-70 molecule under the control of T cell specific promoter(s) into T cell precursors of ZAP-70 deficient mice. Using various mutant forms of ZAP-70 or the normal molecule we can investigate the effects on T cell development and function. As an important preliminary step, we have cloned the T cell specific proximal- and distal Lck promoters and a CD4 minimal promoter construct. The T cell-specific activity of these promoter constructs was verified on cell lines using a lentiviral expression system. We have managed to correct the severe T cell deficiency using adoptive thymocyte transfer. According to our results the transferred T cell progenitors with normal ZAP-70 expression level can induce long-term normalisation of T cell maturation. Combined with the T cell-specific expression of ZAP-70 variants we will possess an excellent tool to study T cell differentiation and function. This work was funded by OTKA, K101493. and Bolyai Janos Reseach Scholarship to FB

A T sejt képzés helyreállításának kinetikai vizsgálata ZAP-70 deficiens egerekben.

A ZAP-70 kináz (70kDa zéta lánc aszociált kináz) központi szerepet tölt be a T sejtek antigén receptoron keresztüli aktivációjának jeltovábbításában. A ZAP-70 kináz a T sejt differenciáció során is kritikus szerepet tölt be, hiányában a T sejtek fejlődése leáll a thymusban a kettős pozitív (CD4+CD8+) stádiumban, aminek következtében a perifériás nyirokszervekben nincsenek érett αβ T sejtek, így súlyos immundeficiencia alakul ki. Előzetesen már kimutattuk, hogy ZAP-70 deficiens egerekben, vad típusú (azaz ZAP-70-et expresszáló) tetvéreikből izolált csontvelő vagy thymus sejtek adoptív transzferével stabilan helyreállítható a T sejt fejlődés. A transzferált állatok vérében ill. nyirokszerveiben T sejtek jelentek meg az egészségesekhez hasonló módon, továbbá az immundeficiencia megszűnésére utalt a szignifikánsan meghosszabodott élettartam is. Jelen vizsgálataink célja a T sejt képzés helyreállítás kinetikájának ill. korai eseményeinek pontosabb megismerése volt. Ehhez 15-20 ZAP-70-/- egér egyidejű transzferét követően rendszeresen ellenőriztük a thymus összetételét ill. a T sejtek megjelenését a periférián. Áramlási citometriás eredményeink szerint, intraperitonealis thymocyta transzfert követően 3 héttel jelentek meg szignifikáns mennyiségben az αβ T sejtek a vérben, a nyirokcsomókban illetve a lépben. Ezt valamivel megelőzve, már a transzfert követő második héten a thymusban is megjelentek CD4+ ill CD8+ érett sejtek. Kvantitatív immunhisztológia segítségével igazoltuk, hogy a thymusban megnövekedett a medulláris állomány, amely szintén a T sejt érés fokozódására utalt. Intrahepatikus csontvelő sejt transzfer után hasonlóképpen megnövekedett medulláris állomány volt detektálható. Transzfer kísérleteink segítségével sikerült a ZAP-70 deficiencia által okozott T sejt hiányos immundeficienciát korrigálnunk. Eredményeink szerint a bejuttatott normális ZAP-70 expressziójú T sejt progenitorok a recipiensben megtapadva képesek stabilan fokozni a T sejt képzést. Munkánkat az OTKA-K101493. sz pályázata támogatta. Boldizsár Ferenc MTA, Bolyai János Kutatói Ösztöndíjban részesül

THE ROLE OF ZAP-70 KINASE IN THE FINE-TUNING OF TCR SIGNALLING : IMPLICATIONS FOR IMMUNOPATHOLOGY AND –THERAPY

ZAP-70 (zeta-chain associated 70 kDa) kinase is a key regulator of T cell receptor signaling. After ligand binding of the T cell receptor (TcR), Lck kinase phosphorylates tyrosine (Y) residues of the CD3 ζ chains and ZAP-70, which, in turn, phosphorylates a number of downstream target proteins (eg. LAT, SLP-76, PLCγ, Cbl). ZAP-70 itself contains a number of Y residues, which can be phosphorylated. Using an array of mutant cell lines where targeted Y-Phenylalanine (F) mutations were introduced into ZAP-70, we were able to characterize the fine details of TcR signaling. Our data confirmed the function of earlier described activator (Y315, Y493) and inhibitory (Y292, Y492) residues; moreover, we described the regulatory role of previously less-known (Y069, Y126, Y178) positions. Glucocorticoid treatment is widely used for suppressing the immune response, primarily through the inhibition of T cell functions. Our earlier work demonstrated, that ZAP-70 is also involved in non-genomic (rapid) GC signaling mechanisms. Using our Y-F mutant ZAP-70 expressing cell line array, we identified that Y315 and Y492 were phosphorylated upon short-term high dose GC analogue treatment. These results confirmed that ZAP-70 represents an important link between the non-genomic GC and TcR/CD3 signaling pathways. Moreover, potential role of ZAP-70 kinase was implicated in chronic lymphoid leukemia (CLL) and autoimmune arthritis. It has been shown in a subgroup of patients with CLL that the malignant B-lymphocytes express ZAP-70 kinase, which was associated with inferior clinical outcome and prognosis. Using two ZAP-70 specific antibodies recognizing different epitopes in the kinase, we performed intracellular staining of malignant B cells from CLL patients. Based on our preliminary experiments, it seems possible that the ZAP-70 molecule expressed in the tumorous B-cells is structurally different from that found in normal T-cells, as some patients showed positivity with either one or the other antibody, while the normal T-cells were positive with both antibodies, just as expected. A spontaneous single point mutation at 163 from Triptophane (W) to Cysteine (C) in the SH2 domain of ZAP-70 caused altered thymic selection and leads to the development of autoimmune arthritis in SKG mice. Another study has shown that targeted simultaneous mutation at positions Y315 and Y319 to Alanine led to similar defects in T cell development than in SKG mice, interestingly, however, these mice did not develop autoimmune arthritis despite the presence of rheuma factor in the sera, increased IL-17 production and impaired Treg development. These data clearly show, how our understanding about ZAP-70 kinase has emerged from being exclusively a T cell specific signaling molecule to an important therapeutic target and potential regulator of pathologies like CLL or autoimmune arthritis

A ZAP-70 kináz részleges hiányának vizsgálata rheumatoid arthritis egér modellben

A rekombináns humán G1 (rhG1) domén indukált arthritis (GIA) egér modell sok tekintetben hasonlít a humán rheumatoid arthritishez (RA) főbb klinikai tüneteit és immunológiai paramétereit tekintve. Az autoimmun arthritis BALB/c egerekben humán proteoglikán aggrekán rekombináns G1 doménjével történő immunizálással váltható ki. Ismert, hogy a T-sejtek fontos szerepet töltenek be az arthritis indukciójában; aktivációjuk szabályozásában a T-sejt receptor jelátvitel egyik különösen fontos molekulája a ZAP-70 tirozin kináz is szerepet játszik. Munkánk során azt vizsgáltuk, hogy a ZAP-70 részleges hiánya milyen hatással van az autoimmun arthritis kialakulására a GIA modellben. Vad típusú BALB/c (WT) és ZAP-70 heterozigóta knockout (ZAP-70+/-) egereket párhuzamosan háromhetente immunizáltunk rhG1-el, összesen három alkalommal. A betegség klinikai tüneteit egy 4-es pontrendszer segítségével értékeltük, a gyulladást in vivo lumineszcens képalkotó vizsgálattal jelemeztük, a végtagfunkció mérésére pedig kapaszkodási tesztet végeztünk. A T sejt közvetített immunválasz jellemzésére in vitro lépsejt kultúrákban proliferációs tesztet végeztünk és citokin termelést mértünk. A részleges ZAP-70 hiány érdekes módon nem gátolta a GIA kialakulását, sőt várakozásainkkal ellentétben a pontrendszer és a kapaszkodási idők alapján ZAP-70+/- egerekben WT egerekhez képest kissé súlyosabb arthritis alakult ki. Egyértelmű összefüggést találtunk a pontszám és a funkcionális teszt eredményei között, valamint megfigyeltük, hogy a fizikai teljesítményben jelentkező változások (pl. csökkent kapaszkodási idő) megelőzték az izületi gyulladás megjelenését. A lumineszcens képalkotó vizsgálatok szintén megnövekedett gyulladásos aktivitást mutattak ki a ZAP-70+/- egerek érintett végtagjaiban a WT állatokéhoz képest. A ZAP-70+/- egerek in vitro tesztekben csökkent T sejt proliferációt és IL-4,-6 termelést mutattak, szignifikáns IL-17, IFNγ és TNFα termeléssel. Előzetes várakozásainkkal ellentétben a T-sejt jelátvitelben kiemelkedő fontosságú ZAP-70 molekula részleges hiánya nem csökkentette az autoimmun arthritis súlyosságát GIA egérmodellben. Ebben feltételezhetően a megváltozott T sejt aktiváció, differenciáció és apoptózis játszhat szerepet. Munkánkat az OTKA-K101493. sz pályázata támogatta. Boldizsár Ferenc MTA, Bolyai János Kutatói Ösztöndíjban részesül

Analysis of partial ZAP-70 deficiency in a murine model of rheumatoid arthritis

Recombinant human G1 (rhG1) induced arthritis (GIA) model resembles human rheumatoid arthritis (RA) both in immunological characteristics and clinical parameters. Immunization of BALB/c mice with rhG1 domain of human proteoglycan aggrecan induces arthritis. T cells are involved in the pathogenesis of arthritis; their activation is regulated by ZAP-70, a key molecule in T cell receptor signaling. The aim of our study was to assess the effect of partial ZAP-70 deficiency on autoimmune arthritis in the GIA model. Wild-type BALB/c (WT) and ZAP-70 heterozygous knockout (ZAP-70+/-) mice were immunized with rhG1 side-by-side. Surprisingly, partial ZAP-70 deficiency did not inhibit the development of GIA; moreover, the arthritis was more severe in ZAP-70+/- mice than in WT as assessed by the physical scoring system. Luminescence imaging confirmed the increased inflammatory activity in affected limbs of ZAP-70+/- mice compared to WT animals. There was a clear correlation between the results of the functional test (hanging time measurements) and the clinical scores. Alterations in the physical performance preceded the clinical onset of arthritis. Investigation of the T cell mediated immune response indicated decreased T cell proliferation and IL-4,-6 production accompanied by significant IL-17, IFNγ and TNFα production measured from in vitro splenocyte cultures. Contrary to our expectations partial deficiency of ZAP-70 did not ameliorate the severity of arthritis in GIA model, which may be due to alterations in T cell activation and apoptosis. This work was funded by OTKA, K101493. and Bolyai Janos Reseach Scholarship of the Hungarian Academy of Sciences to FB

Screening and monitoring of the BTK C481S mutation in a real-world cohort of patients with relapsed/refractory chronic lymphocytic leukaemia during ibrutinib therapy

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has revolutionised the therapeutic landscape of chronic lymphocytic leukaemia (CLL). Acquired mutations emerging at position C481 in the BTK tyrosine kinase domain are the predominant genetic alterations associated with secondary ibrutinib resistance. To assess the correlation between disease progression, and the emergence and temporal dynamics of the most common resistance mutation BTKC481S , sensitive (10-4 ) time-resolved screening was performed in 83 relapsed/refractory CLL patients during single-agent ibrutinib treatment. With a median follow-up time of 40 months, BTKC481S was detected in 48·2% (40/83) of the patients, with 80·0% (32/40) of them showing disease progression during the examined period. In these 32 cases, representing 72·7% (32/44) of all patients experiencing relapse, emergence of the BTKC481S mutation preceded the symptoms of clinical relapse with a median of nine months. Subsequent Bcl-2 inhibition therapy applied in 28/32 patients harbouring BTKC481S and progressing on ibrutinib conferred clinical and molecular remission across the patients. Our study demonstrates the clinical value of sensitive BTKC481S monitoring with the largest longitudinally analysed real-world patient cohort reported to date and validates the feasibility of an early prediction of relapse in the majority of ibrutinib-treated relapsed/refractory CLL patients experiencing disease progression

How do metaphors shape our world

We tend to think of metaphors as poetic language, but we actually use them all the time in our everyday speech. But how do metaphors in different languages work? And can the metaphors we use affect our thinking? In this episode of LinguaMania, we explore how we use metaphors across the world, looking at the different ways of representing abstract concepts, such as emotion and time, through idioms and metaphors

ZAP-70 Regulates Autoimmune Arthritis via Alterations in T Cell Activation and Apoptosis

T cells play an essential role in the pathogenesis of both human rheumatoid arthritis (RA) and its murine models. A key molecule in T cell activation is ZAP-70, therefore we aimed to investigate the effects of partial ZAP-70 deficiency on the pathogenesis of recombinant human G1(rhG1)-induced arthritis (GIA), a well-established mouse model of RA. Arthritis was induced in BALB/c and ZAP-70+/− heterozygous mice. Disease progression was monitored using a scoring system and in vivo imaging, antigen-specific proliferation, cytokine and autoantibody production was measured and T cell apoptotic pathways were analyzed. ZAP-70+/− mice developed a less severe arthritis, as shown by both clinical picture and in vitro parameters (decreased T cell proliferation, cytokine and autoantibody production). The amount of cleaved Caspase-3 increased in arthritic ZAP-70+/− T cells, with no significant changes in cleaved Caspase-8 and -9 levels; although expression of Bim, Bcl-2 and Cytochrome C showed alterations. Tyrosine phosphorylation was less pronounced in arthritic ZAP-70+/− T cells and the amount of Cbl-b—a negative regulator of T cell activation—decreased as well. We hypothesize that the less severe disease seen in the partial absence of ZAP-70 might be caused by the decreased T cell activation accompanied by increased apoptosis