601 research outputs found
The differential regulation of Lck kinase phosphorylation sites by CD45 is critical for T cell receptor signaling responses
No full textThe molecular mechanisms whereby the CD45 tyrosine phosphatase (PTPase) regulates T cell receptor (TCR) signaling responses remain to be elucidated. To investigate this question, we have reconstituted CD45 (encoded by Ptprc)-deficient mice, which display severe defects in thymic development, with five different expression levels of transgenic CD45RO, or with mutant PTPase null or PTPase-low CD45R0. Whereas CD45 PTPase activity was absolutely required for the reconstitution of thymic development, only 3% of wild-type CD45 activity restored T cell numbers and normal cytotoxic T cell responses. Lowering the CD45 expression increased CD4 lineage commitment. Peripheral T cells with very low activity of CD45 phosphatase displayed reduced TCR signaling, whereas intermediate activity caused hyperactivation of CD4+ and CD8+ T cells. These results are explained by a rheostat mechanism whereby CD45 differentially regulates the negatively acting pTyr-505 and positively acting pTyr-394 p56(lck) tyrosine kinase phosphorylation sites. We propose that high wild-type CD45 expression is necessary to dephosphorylate p56(lck) pTyr-394, suppressing CD4 T+ cell lineage commitment and hyperactivity
Studies of suppression using monoclonal regulatory T cells and the importance of co-receptor Lck coupling ratios for negative selection
No full textNormal physiology is not given without immunological tolerance. Depending on the origin of induction, tolerance can be divided into central and peripheral tolerance. Central tolerance comprises depletion of autoreactive T cells in the thymus (i.e. negative selection), and with this prevents autoimmunity. Peripheral tolerance critically depends on regulatory T cells (Tregs) maintaining lymphocyte homeostasis. Hallmark features of CD4+ Tregs are the expression of high surface IL-2-Rα (CD25) and the transcription factor Forkhead box protein P3 (Foxp3). Expression of non-functional Foxp3 results in lethal multi-organ lymphocytic infiltrations and cytokine secretions. Numerous data agree on an indispensable role for Tregs enabling physiological immunity. Yet an understanding of how Tregs function at a cellular and molecular level has not been fully elucidated.
In the first part of the thesis we made use of a RagKO TCR transgenic mouse expressing a Foxp3 transgene. This mouse provides a source of monoclonal CD4+, Foxp3+ T cells with a defined specificity. We show that monoclonal B3K506 Tregs are fully functional in vitro and in vivo and clearly require cognate antigen to be suppressive. We further show that the strength of Treg stimulation determines the strength of Treg mediated suppression. Finally we analysed various suppressive mechanisms used by monoclonal Tregs and found that Treg-Tconv proximity is an important parameter, which correlates with effective suppression.
In the second part of the thesis we aimed to understand the molecular mechanism underlying the affinity threshold for negative selection. We quantified the amount of Lck coupled to CD8 or CD4 coreceptors. We found that CD4 co-receptors have higher Lck coupling ratios than do CD8 co-receptors. In addition we determined the absolute numbers of surface molecules (i.e. CD8α, CD4 and CD3ε) on double positive (DP) thymocytes in B6 and MHC- class I restricted, TCR Tg mouse strains. A model, explaining how the TCR measures antigen affinity to initiate a negative selection signal, was generated. Here we show that the affinity threshold for different co-receptors depends on the probability that a peptide-MHC- TCR complex will collide with a co-receptor carrying Lck during the time pMCH binds to the TCR (Stepanek, O. et al. Cell, 2014
Lipids Regulate Lck Protein Activity through Their Interactions with the Lck Src Homology 2 Domain.
No full textLymphocyte-specific protein-tyrosine kinase (Lck) plays an essential role in T cell receptor (TCR) signaling and T cell development, but its activation mechanism is not fully understood. To explore the possibility that plasma membrane (PM) lipids control TCR signaling activities of Lck, we measured the membrane binding properties of its regulatory Src homology 2 (SH2) and Src homology 3 domains. The Lck SH2 domain binds anionic PM lipids with high affinity but with low specificity. Electrostatic potential calculation, NMR analysis, and mutational studies identified the lipid-binding site of the Lck SH2 domain that includes surface-exposed basic, aromatic, and hydrophobic residues but not the phospho-Tyr binding pocket. Mutation of lipid binding residues greatly reduced the interaction of Lck with the chain in the activated TCR signaling complex and its overall TCR signaling activities. These results suggest that PM lipids, including phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, modulate interaction of Lck with its binding partners in the TCR signaling complex and its TCR signaling activities in a spatiotemporally specific manner via its SH2 domain.1175Ysciescopu
Lipids Regulate Lck Protein Activity through Their Interactions with the Lck Src Homology 2 Domain
No full textLymphocyte-specific protein-tyrosine kinase (Lck) plays an essential role in T cell receptor (TCR) signaling and T cell development, but its activation mechanism is not fully understood. To explore the possibility that plasma membrane (PM) lipids control TCR signaling activities of Lck, we measured the membrane binding properties of its regulatory Src homology 2 (SH2) and Src homology 3 domains. The Lck SH2 domain binds anionic PM lipids with high affinity but with low specificity. Electrostatic potential calculation, NMR analysis, and mutational studies identified the lipid-binding site of the Lck SH2 domain that includes surface-exposed basic, aromatic, and hydrophobic residues but not the phospho-Tyr binding pocket. Mutation of lipid binding residues greatly reduced the interaction of Lck with the chain in the activated TCR signaling complex and its overall TCR signaling activities. These results suggest that PM lipids, including phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, modulate interaction of Lck with its binding partners in the TCR signaling complex and its TCR signaling activities in a spatiotemporally specific manner via its SH2 domain.
Roles of the Src tyrosine kinases Lck and Fyn in regulating gammadeltaTCR signal strength.
No full textLck and Fyn, members of the Src family of tyrosine kinases, are key components of the alphabetaTCR-coupled signaling pathway. While it is generally accepted that both Lck and Fyn positively regulate signal transduction by the alphabetaTCR, recent studies have shown that Lck and Fyn have distinct functions in this signaling pathway, with Lck being a positive regulator and Fyn being a negative regulator of alphabetaTCR signal transduction. To determine whether Lck and Fyn also differentially regulate gammadeltaTCR signal transduction, we analyzed gammadelta T cell development and function in mice with reduced Lck or Fyn expression levels. We found that reducing Lck or Fyn levels altered the strength of the gammadeltaTCR signaling response, with low levels of Lck weakening gammadeltaTCR signal strength and low levels of Fyn augmenting gammadeltaTCR signal strength. These alterations in gammadeltaTCR signal strength had profound effects not only on alphabeta/gammadelta lineage choice, but also on gammadelta thymocyte maturation and gammadelta T cell effector function. These results indicate that the cellular levels of Lck and Fyn play a role in regulating the strength of the gammadeltaTCR signaling response at different stages in the life of the gammadelta T cell
SVM Model for Virtual Screening of Lck Inhibitors
No full textLymphocyte-specific protein tyrosine kinase (Lck) inhibitors have treatment potential for autoimmune diseases and transplant rejection. A support vector machine (SVM) model trained with 820 positive compounds (Lck inhibitors) and 70 negative compounds (Lck noninhibitors) combined with 65 142 generated putative negatives was developed for predicting compounds with a Lck inhibitory activity of IC50 ≤ 10 μM. The SVM model, with an estimated sensitivity of greater than 83% and specificity of greater than 99%, was used to screen 168 014 compounds in the MDDR and was found to have a yield of 45.8% and a false positive rate of 0.52%. The model was also able to identify novel Lck inhibitors and distinguish inhibitors from structurally similar noninhibitors at a false positive rate of 0.27%. To the best of our knowledge, the SVM model developed in this work is the first model with a broad applicability domain and low false positive rate, which makes it very suitable for the virtual screening of chemical libraries for Lck inhibitors
Μελέτη της αναστολής της τυροσινικής κινάσης Lck στα λεμφοκύτταρα
No full textThe Lymphocyte-specific protein tyrosine kinase Lck, a member of the evolutionarily conserved family of Src kinases (SFKs), is predominantly expressed in T cell and is responsible for the differentiation, proliferation, and activation of T cells, because of its vital role in the initiation of T cell receptor (TCR) signaling following engagement with an antigen. Based on its essential role in these cells, Lck is implicated in aberrant T-cell responses such as autoimmunity, leukemias, allograft rejections and Graft Versus Host Disease as well as the occurrence of toxicity accompanying immunotherapy using Chimeric Antigen Receptor (CAR)- T cells. As a result, Lck has become a highly attractive target for the development of small molecule inhibitors. However, designing these inhibitors has been exceptionally challenging, as currently available compounds exhibit very low target specificity. The primary obstacle is the remarkable homology shared among SFK members within their catalytic centers, which makes the development of selective inhibitors exceedingly difficult. Recent studies from our laboratory and others have identified an N-terminal regulatory domain (LcKSH4) located outside the catalytic center, as mandatory for Lck anchoring on plasma membrane and its activity. Importantly, LckSH4 displays negligible homology with other members of the SFK family or any other kinases, making it a perfect target for selective Lck inhibition. The goal of the current study is the efficient and highly selective inhibition of Lck by blocking the LckSH4 domain, which in turn will prohibit anchoring of the kinase at the plasma membrane and decrease its enzymatic activity. The LckSH4 blocking will be accomplished using nanobodies (anti-LckSH4 Nbs). Nbs are single-domain antibody fragments derived from heavy chain-only antibodies (HcAbs) which are naturally found in camelids. Nbs consist of one single variable heavy chain (VHH) region, which is the site of epitope binding. In recent years Nbs have been characterized as the “rising stars” of the antibody world, occupying a central position in cutting-edge applied medical research, particularly in the field of diagnosis and treatment of diseases. Due to their unique structural features and advantageous properties, Nbs are expected to quickly replace conventional monoclonal antibodies as therapeutic and diagnostic tools. 30 different anti-LckSH4 Nbs were tested for their ability to bind Lck intracellularly. Initial data revealed that the Nbs predominantly localized to intracellular compartments different from those of Lck, preventing effective binding. To address this, we introduced various N-terminal modifications to the Nbs with the aim to enforce their colocalization with Lck, thereby increasing the likelihood of successful Lck binding. To select the top Nb Lck-binder, we assessed whether the N-terminal modifications successfully directed the Nbs to the same intracellular compartments as Lck, as well as their ability to bind Lck in different cell lines (HEK293 and Jurkat). Additionally, we evaluated the capacity of these Nbs to interfere with Lck activation and block Lck-mediated TCR signaling. While the majority of N-terminal modifications successfully directed the Nbs to the same compartments as Lck, only one modification—the addition of the LckSH4 domain to NbGT, termed Lck-NbGT—resulted in significant Lck binding. The other modifications exhibited minimal or no binding. Importantly, Lck-NbGT was specific for Lck, as it failed to bind chimeric forms of Lck in which the LckSH4 domain had been replaced by those of other SFK members, and it did not interfere with signaling in B cell lines, in which another SFK member-Lyn is expressed. Although, our initial working hypothesis that Nb-mediated masking of LckSH4 will abolish Lck enzymatic activity, could not been proved, we identified a specific and efficient Lck binder, which we then employed for the selective knock down of the kinase. Specifically, we repurposed the usage of Lck-NbGT as a mediator for Lck degradation using the proteasome system. Lck-NbGT was fused with the ubiquitin E3 ligase RNF4, with the purpose to bind and lead Lck to ubiquitin proteasome-mediated destruction. This fusion showed promising results since endogenous Lck expression levels were significantly reduced. Additionally, collective data from confocal microscopy and co-immunoprecipitation experiments revealed that NbGT can be used as a non-invasive, specific and high resolution Lck probe, able to pinpoint native Lck with high accuracy. Based on these data, we propose the broader usage of Nbs as a new biochemical tool to determine the intracellular localisation of target molecules with superior resolution than the one provided by imaging techniques.Η ειδική λεμφοκυτταρική τυροσινική κινάση-Lck είναι μέλος της εξελικτικά συντηρημένης οικογένειας των Src κινασών (SFK) και εκφράζεται κυρίως στα Τ λεμφοκύτταρα. Η Lck είναι υπεύθυνη για την διαφοροποίηση, πολλαπλασιασμό και ενεργοποίηση των Τ λεμφοκυττάρων, λόγω του καίριου ρόλου της στην επαγωγή της Τ κυτταρικής σηματοδότησης μετά την πρόσδεση ενός αντιγόνου από τον Τ κυτταρικό υποδοχέα που έχουν στην επιφάνεια τους. Βασιζόμενη στο ουσιαστικό ρόλο που έχει στα κύτταρα αυτά, η Lck εμπλέκεται σε ποικίλες Τ παρεκτρεπόμενες αποκρίσεις, όπως είναι η αυτοανοσία, οι λευχαιμίες, η νόσος μοσχεύματος έναντι του ξενιστή και στην εμφάνιση τοξικότητας από ανοσοθεραπείες που χρησιμοποιούν CAR-T λεμφοκύτταρα. Λόγω της σημασίας που έχει η Lck στα Τ λεμφοκύτταρα, η κινάση αυτή έχει αποτελέσει ελκυστικό στόχο για την ανάπτυξη μικρών μορίων αναστολέων. Ωστόσο ο σχεδιασμός τέτοιων αναστολέων είναι εξαιρετικά δύσκολος, καθώς οι μέχρι τώρα διαθέσιμοι αναστολείς έχουν ιδιαίτερα χαμηλή ειδικότητα. Το κύριο εμπόδιο είναι η αξιοσημείωτη ομολογία που παρουσιάζουν οι Src κινάσες στα καταλυτικά τους κέντρα, κάνοντας την δημιουργία ειδικών Lck αναστολέων εξαιρετικά απαιτητική. Πρόσφατες έρευνες από το δικό μας και άλλα εργαστήρια έχουν εντοπίσει ότι η SH4 περιοχή της Lck (LckSH4), η οποία βρίσκεται εκτός του καταλυτικού κέντρου της κινάσης, είναι υπεύθυνη για την πρόσδεση της Lck στην πλασματική μεμβράνη των Τ λεμφοκυττάρων, πρόσδεση η οποία είναι απαραίτητη για την ενεργοποίηση της κινάσης. Αξιοσημείωτο είναι επίσης το γεγονός ότι η LckSH4 περιοχή παρουσιάζει ελάχιστη ομολογία με αντίστοιχες περιοχές άλλων Src κινασών, κάνοντας την ιδανικό στόχο για την επιλεκτική αναστολή της Lck. Στόχος της παρούσας μελέτης είναι η αποτελεσματική και επιλεκτική αναστολή της Lck, στοχεύοντας την LckSH4 περιοχή με σκοπό την παρεμπόδιση της αγκυροβόλησης της Lck στην πλασματική μεμβράνη των κυττάρων και κατά συνέπειά την μείωση της ενζυμικής ενεργότητας της κινάσης. Η στόχευση αυτή θα επιτευχθεί με την χρήση νανοαντισωμάτων έναντι της LckSH4 περιοχής (anti-LckSH4 Nbs). Τα νανοαντισώματα (Nbs) είναι μια ειδική κατηγορία μικρών, γενετικά τροποποιημένων αντισωμάτων που απαντώνται στα καμηλοειδή και αποτελούνται αποκλειστικά από μία μεταβλητή περιοχή βαριάς αλύσου (VHH), περιοχή η οποία είναι υπεύθυνη για την πρόσδεση σε ένα επίτοπο. Τα τελευταία χρόνια τα Nbs έχουν χαρακτηριστεί ως «ανερχόμενα αστέρια» στο κόσμο των αντισωμάτων, κατέχοντας κεντρική θέση στην καινοτόμα εφαρμοσμένη βιοϊατρική έρευνα και ιδιαίτερα στο τομέα της διάγνωσης και της θεραπείας ασθενειών. Λόγω των μοναδικών δομικών χαρακτηριστικών και ιδιοτήτων τους, τα Nbs αναμένονται να αντικαταστήσουν γρήγορα τα συμβατικά μονοκλωνικά αντισώματα ως θεραπευτικά και διαγνωστικά εργαλεία. Στην παρούσα μελέτη 30 διαφορετικά anti-LckSH4 Nbs δοκιμάστηκαν για την ικανότητα τους να προσδέσουν την Lck ενδοκυτταρικά. Τα αρχικά αποτελέσματα έδειξαν ότι τα Nbs εντοπίζονταν κυρίως σε ενδοκυττάρια διαμερίσματα διαφορετικά από αυτά που βρίσκεται η Lck, αποτρέποντας την αποτελεσματική πρόσδεση της Lck από τα Nbs. Προκειμένου να αντιμετωπίσουμε αυτό το πρόβλημα, εισαγάγαμε διάφορες τροποποιήσεις στο αμινοτελικό άκρο των Nbs, με στόχο να αυξηθεί ο συνεντοπισμός μεταξύ της κινάσης και των Nbs, αυξάνοντας με την σειρά του τις πιθανότητες επιτυχούς δέσμευσης της Lck. Η επιλογή του καλύτερου Nb ως δεσμευτή της Lck, έγινε κατόπιν ελέγχου της ικανότητας των αμινοτελικών τροποποιήσεων να κατευθύνουν τα Nbs στα ίδια ενδοκυττάρια διαμερίσματα με την Lck καθώς και της ικανότητας των Nbs να προσδένουν την Lck. Επιπρόσθετα, αξιολογήθηκε η ικανότητα των Nbs να μειώνουν την ενεργότητα της κινάσης και να αναστέλλουν την Lck-επαγόμενη Τ κυτταρική σηματοδότηση. Παρά το γεγονός ότι η πλειοψηφία των αμινοτελικών τροποποιήσεων κατηύθυνε τα Nbs στα ίδια ενδοκυττάρια διαμερίσματα στα οποία βρίσκεται η Lck, μόνο μία τροποποίηση, η προσθήκη της SH4 περιοχής της Lck στο NbGT (Lck-NbGT) οδήγησε στην αποτελεσματική πρόσδεση της Lck. Σε αντίθεση, οι άλλες τροποποιήσεις στα Nbs παρουσίασαν ελάχιστη έως μηδαμινή πρόσδεση στην κινάση. Παράλληλα, το Lck-NbGT είναι ιδιαίτερα επιλεκτικό έναντι της Lck, καθώς δεν είχε την ικανότητα πρόσδεσης χιμαιρικών μορφών της Lck, στις οποίες η LckSH4 αντικαταστάθηκε από τις αντίστοιχες άλλων Src κινασών, ούτε παρεμπόδισε την επαγωγή της ενδοκυττάριας σηματοδότησης στα Β λεμφοκύτταρα, τα οποία εκφράζουν ενδογενώς μια άλλη Src κινάση, την Lyn και όχι την Lck. Παρόλο που η αρχική υπόθεση εργασίας, δηλαδή η στόχευση της LckSH4 περιοχής από τα Nbs θα απωλέσει την ενζυμική ενεργότητα της κινάσης, δεν μπόρεσε να επαληθευτεί, έγινε δυνατή η έρευση ενός αποτελεσματικού και ειδικού προσδέτη της LCK, ο οποίος στην συνέχεια χρησιμοποιήθηκε για τη επιλεκτική αποικοδόμηση της κινάσης. Πιο συγκεκριμένα, επαναπροσδιορίσαμε την χρήση του Lck-NbGT ως επαγωγέα αποικοδόμησης της Lck, χρησιμοποιώντας το σύστημα του πρωτεασώματος. Πιο αναλυτικά, το Lck-NbGT ενώθηκε με τη Ε3 λιγάση ουβικουιτίνης RNF4, με σκοπό την πρόσδεση της Lck από το Lck-NbGT και την αποικοδόμηση της από το πρωτεάσωμα. Αυτό το καινούργιο είδος anti-LckSH4 Nb έδειξε ενθαρρυντικά αποτελέσματα, καθώς η μείωση των επιπέδων της Lck ήταν ιδιαίτερα σημαντική. Τέλος, συγκεντρωτικά δεδομένα από συνεστιακή μικροσκοπία και πειράματα ανοσοκατακρίμνισης κατέδειξαν ότι το NbGT μπορεί να χρησιμοποιηθεί ως ειδικός, μη επεμβατικός και υψηλής ανάλυσης ανιχνευτής της Lck, ικανός να εντοπίζει την φυσική τοποθεσία της κινάσης με υψηλή ακρίβεια. Με βάση αυτά τα δεδομένα, προτείνουμε την ευρύτερη χρήση των Nbs ως ένα νέο βιοχημικό εργαλείο για τον προσδιορισμό του ενδοκυτταρικού εντοπισμού των μορίων-στόχων με ανώτερη ανάλυση από αυτή που παρέχεται από τις συμβατικές τεχνικές απεικόνισης
Primary T-cell immunodeficiency with immunodysregulation caused by autosomal recessive LCK deficiency
No full textInternational audienceSignals emanating from the antigen T-cell receptor (TCR) are required for T-cell development and function. The T lymphocyte-specific protein tyrosine kinase (Lck) is a key component of the TCR signaling machinery. On the basis of its function, we considered LCK a candidate gene in patients with combined immunodeficiency. Objective: We identify and describe a child with a T-cell immunodeficiency caused by a homozygous missense mutation of the LCK gene (c.1022T>C) resulting from uniparental disomy. Methods: Genetic, molecular, and functional analyses were performed to characterize the Lck deficiency, and the associated clinical and immunologic phenotypes are reported. Results: The mutant LCK protein (p.L341P) was weakly expressed with no kinase activity and failed to reconstitute TCR signaling in LCK-deficient T cells. The patient presented with recurrent respiratory tract infections together with predominant early-onset inflammatory and autoimmune manifestations. The patient displayed CD41 T-cell lymphopenia and low levels of CD4 and CD8 expression on the T-cell surface. The residual T lymphocytes had an oligoclonal T-cell repertoire and exhibited a profound TCR signaling defect, with only weak tyrosine phosphorylation signals and no Ca21 mobilization in response to TCR stimulation. Conclusion: We report a new form of T-cell immunodeficiency caused by a LCK gene defect, highlighting the essential role of Lck in human T-cell development and responses. Our results also point out that defects in the TCR signaling cascade often result in abnormal T-cell differentiation and functions, leading to an important risk factor for inflammation and autoimmunit
Correction to: a novel biallelic lck variant resulting in profound t-cell ımmune deficiency and review of the literature
No full textLymphocyte-specific protein tyrosine kinase (LCK) is an SRC-family kinase critical for initiation and propagation of T-cell antigen receptor (TCR) signaling through phosphorylation of TCR-associated CD3 chains and recruited downstream molecules. Until now, only one case of profound T-cell immune deficiency with complete LCK deficiency [1] caused by a biallelic missense mutation (c.1022T>C, p.L341P) and three cases of incomplete LCK deficiency [2] caused by a biallelic splice site mutation (c.188-2A>G) have been described. Additionally, deregulated LCK expression has been associated with genetically undefined immune deficiencies and hematological malignancies. Here, we describe the second case of complete LCK deficiency in a 6-month-old girl born to consanguineous parents presenting with profound T-cell immune deficiency. Whole exome sequencing (WES) revealed a novel pathogenic biallelic missense mutation in LCK (c.1393T>C, p.C465R), which led to the absence of LCK protein expression and phosphorylation, and a consecutive decrease in proximal TCR signaling. Loss of conventional CD4+ and CD8+ αβT-cells and homeostatic T-cell expansion was accompanied by increased γδT-cell and Treg percentages. Surface CD4 and CD8 co-receptor expression was reduced in the patient T-cells, while the heterozygous mother had impaired CD4 and CD8 surface expression to a lesser extent. We conclude that complete LCK deficiency is characterized by profound T-cell immune deficiency, reduced CD4 and CD8 surface expression, and a characteristic TCR signaling disorder. CD4 and CD8 surface expression may be of value for early detection of mono- and/or biallelic LCK deficiency. © 2023, The Author(s)
Influence of Lck abundance on thymic selection, peripheral T cell activation and the formation of T cell memory
No full textSelection of the T cell repertoire in the thymus is governed by the need to
create a repertoire of peripheral T cells that can respond to any foreign
antigen in the context of self-major histocompatibility complex (MHC), while
enforcing central tolerance to self-antigens. Perturbations in signalling
molecules, that reduce the affinity of thymic selection, can lead to the
production of a peripheral repertoire with increased autoimmunity, as has
been shown for mutations in the Zap-70 kinase. Upstream of Zap-70 is Lck,
the most proximal tyrosine kinase required for T cell receptor (TCR)
triggering upon TCR engagement by peptide:MHC.
In order to study how Lck influences T cell activation, a transgenic mouse
model (LckVA), in which Lck is expressed constitutively from a T cell specific
transgene and mice have very low expression of Lck (~5% of WT) in both the
thymus and periphery, was used.
It has been shown that Lck is critical for successful T cell development, yet
the results of this thesis show that even 5% of WT levels of Lck are sufficient
for selection of thymic T cells on both polyclonal and F5 TCR transgenic
backgrounds.
Previous studies utilising mice expressing an inducible Lck transgene, which
also had reduced Lck expression in the periphery, showed Lck to be critical
in determining the activation threshold of T cells. In contrast, peripheral T
cells in LckVA mice had similar activation thresholds to wild type T cells, as
measured by in vitro upregulation of early activation markers. Further
analysis of LckVA peripheral T cells revealed differential influences of low
expression of Lck on downstream signalling pathways upon TCR
engagement. For example, ERK signalling was impaired, while calcium flux
and proliferation were enhanced in LckVA T cells. Finally, LckVA T cells were
altered in their ability to differentiate, showing enhanced production of
cytokines and retaining the capacity to form memory cells
- …
