Universidad Autónoma de Madrid Facultad de Ciencias
Departamento de Biología Molecular
Programa de Doctorado en Biociencias Moleculares
Regulation of pre-TCR and TCR function by the transmembrane domain of CD3ζ chain
during T cell development
Tesis Doctoral
Elena Rodríguez Bovolenta Madrid, 2018
Doctoral Thesis
Regulation of pre-TCR and TCR function by the transmembrane domain of CD3ζ chain during T cell development
This thesis is submitted by Elena Rodríguez Bovolenta in fulfilment of the requirements for the degree of Doctor in Molecular Biology.
Thesis directors:
Dr. Hisse Martien van Santen
Assistant professor of the Consejo Superior de Investigaciones Científicas Centro de Biología Molecular Severo Ochoa (CBMSO-‐‑CSIC)
Universidad Autónoma de Madrid
Dr. Balbino Alarcón Sánchez
Research Professor of the Consejo Superior de Investigaciones Científicas Centro de Biología Molecular Severo Ochoa (CBMSO-‐‑CSIC)
Universidad Autónoma de Madrid
This work has been performed at the Centro de Biología Molecular Severo Ochoa under the supervision of Drs. Hisse Martien van Santen and Balbino Alarcón Sánchez and the work has been funded by the MINECO project SAF2013-‐‑47075-‐‑R. Short term period in Dr. Wolfgang Schamel´s laboratory at the BIOSS Centre in Freiburg im Breisgau was supported by EMBO Short Term Fellowship program.
A MIS PADRES
CONTENTS
LIST OF THE FIGURES AND TABLES 13
ABBREVIATIONS 17
SUMMARY 21
RESUMEN 23
INTRODUCTION 25
A brief introduction to the adaptive immune system 25
T cell development: from the thymus to the periphery 26
Pre-‐‑TCR and TCR complexes 31
TCR and pre-‐‑TCR signalling 34
TCR triggering models: TCR clustering and conformational change 36 Role of the preTCR-‐‑CD3 and associated signalling molecules in early T cell development 41
MATERIALS AND METHODS 47
1. MATERIALS 47
Cells 47
Mice 49
Reagents 51
Antibodies and fluorescent probes 53
Vectors 55
Oligonucleotides 56
2. METHODS 59
Generation of CD3ζ-‐‑/-‐‑ / TgCD3ζWT-‐‑GFP and CD3ζ-‐‑/-‐‑ / TgCD3ζL19A-‐‑GFP mouse lines 59
Lentiviral production and cell line transduction 60
Generation of a CD3ζ-‐‑deficient pre-‐‑T cell lines 60
Generation of the SCBζWT and SCBζL19A cell lines 62
Obtention of cell suspension from mice lymphoid organs 62
FACS staining protocol 62
Annexin V staining 63
BrdU staining 63
Proliferation and functional assays 64
Mouse peripheral blood collection for phenotyping 64
FACS analysis 64
Mouse genotyping 64
Electron Microscopy (EM) 65
Western Blotting 66
Blue Native –PAGE (BN-‐‑PAGE) 67
Total Internal Reflection Fluorescen Microscopy (TIRFM) 68
Statistical analysis 69
OBJECTIVES 71
OBJETIVOS 73
RESULTS 77
ANALYSIS OF THE EFFECTS OF THE L19A MUTATION ON EARLY T CELL DEVELOPMENT
I. The L19A mutation alters thymus size and cellularity 77 II. L19A DN thymocytes fail in the upregulation of maturation markers during DN3 to DN4
transition 81
III. Proliferation and cell death are altered in L19A DN thymocytes 83 IV. L19A pre-‐‑TCR has a reduced proximal signalling capacity 86
V. Pre-‐‑TCR clustering 90
STUDY OF THE EFFECTS OF THE L19A MUTATION IN TRC-DEPENDENT THYMOCYTE DEVELOPMENT AND MATURE T LYMPHOCYTE PHENOTYPE AND ACTIVATION
I. Effect of L19A mutation in TCRαβ-‐‑expressing thymocytes 99
II. Generation of natural Treg cells in thymus 101
III. Phenotype of L19A immature thymocytes in a OT-‐‑I and OT-‐‑II TCR repertoire 102 IV. Phenotype and function of mature CD3ζ-‐‑GFP transgenic lymphocytes 106 V. L19A CD4 and L19A CD8 mature T cell function upon TCR stimulation 109
DISCUSSION 115
How is the CD3ζ transmembrane domain involved in signalling through the pre-‐‑TCR? 115
Pre-‐‑TCR in β-‐‑selection, the importance of the complex integrity 121
Negative and positive selection on a context of reduced TCR signalling 123
TCR signal strength, T cell commitment and peripheral T cell repertoire properties 126
CONCLUSIONS 133
CONCLUSIONES 135
BIBLIOGRAPHY 137
ACKNOWLEDGMENTS 151
LIST OF THE FIGURES AND TABLES
INTRODUCTION
Figure 1. Thymus structure and the dinamics of the thymocyte development. 28
Figure 2. Scheme of the αβ T cell development. 30
Figure 3. Scheme of the immature pre-‐‑TCR and mature TCR complexes. 32
Table 1. Comparison of pre-‐‑TCR and TCR characteristics. 33
Figure 4. Pre-‐‑TCR signalling. 36
Table 2. Summary of TCR characteristics 38
MATERIALS AND METHODS
• MATERIALS
Table 1. Material used for culture cells 48
Table 2. Reagents, source and application 51
Table 3. Buffers 52
Table 4. Primary Antibodies. 53
Table 5. Secondary antibodies 55
Table 6. Fluorescent probes 55
Table 7. Vectors 55
Table 8. Oligonucleotides used for pcr typing 56
Table 9. Oligonucleotides used in CRISPR/Cas9 System 56
• METHODS
Figure 1. Scheme of the generation of CD3ζ-‐‑/-‐‑ / TgCD3ζWT-‐‑GFP and CD3ζ-‐‑/-‐‑ / TgCD3ζL19A-‐‑
GFP mouse lines. 59
Figure 2. Scheme of the generation of the SCBζWTGFP and SCBζL19AGFP pre-‐‑TCR cell lines.60 Figure 3. Elution picks of the fragments amplified in CRISPR-‐‑STAT method of both clones
ζKO obtained. 61
RESULTS
ANALYSIS OF THE EFFECTS OF THE L19A MUTATION ON EARLY T CELL DEVELOPMENT
Figure 1. Thymus size and cellularity in CD3ζWT-‐‑GFP and CD3ζL19A-‐‑GFP transgenic mice. 77
Figure 2. Percentage and size of the thymocytes subsets in CD3ζWT-‐‑GFP and CD3ζL19A-‐‑GFP
transgenic mice. 78
Figure 3. DN thymocyte populations in CD3ζWT-‐‑GFP and CD3ζL19A-‐‑GFP transgenic mice. 79
Figure 4. Intracellular TCRβ levels in DN3 subpopulation. 80
Figure 5. Levels of pre-‐‑TCR expression on L19A and WT DN CD44neg thymocytes. 82
Figure 6. Protein expression levels of the CD27, CD98 and CD71 maturation markers on the
surface of the DN3 and DN4 thymocytes. 83
Figure 7. FCS levels in DN thymocytes. 84
Figure 8. Ki67 levels in DN thymocytes. 85
Figure 9. BrdU incorporation assay in DN thymocytes. 86
Figure 10. Annexin V staining in DN thymocytes. 87
Figure 11. TCR expression levels in SCB.29 and derived cell lines. 88
Figure 12. Steady state phosphorylation levels of CD3ε in SCIET.27, SCB.29, ζKO and
reconstituted cell lines. 89
Figure 13. Phosphorylation levels in SCBζGFPWT and SCBζGFPL19A cell lines after
stimulation with anti-‐‑CD3ε antibody. 89
Figure 14. Pre-‐‑TCR distribution on the cell surface of the SCB.29, the SCBζWT 21.1 and the
SCBζL19A 21.1 cells. 91
Figure 15. Analysis of the pre-‐‑TCR clustering distribution using BN-‐‑PAGE. 92
Figure 16. TIFRM analysis of the pre-‐‑TCR on CD44neg primary thymocytes. 94
STUDY OF THE EFFECTS OF THE L19A MUTATION IN TRC-DEPENDENT THYMOCYTE DEVELOPMENT AND MATURE T LYMPHOCYTE PHENOTYPE AND ACTIVATION.
Figure 17. Single positive thymocyte populations in WT and L19A thymus of transgenic mice.99
Figure 18. Single positive thymocytes populations in WT and L19A thymus of transgenic
mice. 100
Figure 19. CD69 and CD5 expression levels in DP thymocytes. 101
Figure 20. Treg cells in WT and L19A mice thymus. 102
Figure 21. Thymic populations in CD3ζ-‐‑GFP OT-‐‑I double transgenic mice. 103
Figure 22. Number of cells in CD3ζ-‐‑GFP OT-‐‑I double transgenic thymuses. 104
Figure 23. Thymic populations in CD3ζ-‐‑GFP OT-‐‑II double transgenic mice. 105
Figure 24. Number of cells in CD3ζ-‐‑GFP OT-‐‑II double transgenic thymuses. 106
Figure 25. Mature lymphocytes in LNs of WT and L19A mice. 107
Figure 26. TCR expression levels in peripheral CD4 T and CD8 T lymphocytes. 108
Figure 27. Treg cells in peripheral LNs of WT and L19A mice. 109
Figure 28. In vitro proliferation and functional assay in mature CD4 and CD8 T polyclonal
lymphocytes 112
Figure 29. In vitro stimulation and proliferation assay in mature OT-‐‑II CD4 T and OT-‐‑I CD8 T
lymphocytes. 112
DISCUSSION
Figure 1. CRAC and CARC cholesterol binding motifs in the transmembrane domain of the pTα, TCRα, TCRβ, CD3ε, CD3γ, CD3δ, CD3ζ chains of the pre-‐‑TCR and TCR
complexes. 118
Figure 2. Transmembrane domain sequence of the CD3ζ in different mamal species. 119
Figure 3: Proposed cell model based in the data obtained in this work. 130
ABBREVIATIONS
4SP Single positive CD4 8SP Single positive CD8
ACK Erythrocytes lysis buffer BCR B cell receptor BSA Bovine Serum Albumin B2 B cell subtype
BN-PAGE Blue Native Polyacriladide gel electroforesis bp base pairs
BRS Basic amino acid-‐‑Rich Sequence BSA Bovine Serum Albumin
CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CS Cholesterol Sulfate
DMEM Dulbecco'ʹs Modified Eagle Medium DN Double Negative
DNA Deoxyribonucleic acid DP Double positive
EM Electron-‐‑mycroscopy
ERK Extracellular signal–regulated kinase ETPs Early T cell progenitors
FACS Fluorescent activated cell sorting FBS Fetal Bovine Serum
FSC Forward Scatter
GFP Green Flourescent protein HRP Horseradish peroxidase I.p Intraperitoneal
IP Immunoprecipitation
ISP8 Immature Single Positive CD8
ITAM Immunoreceptortyrosine-‐‑Based activation motif ITIM Immunoreceptortyrosine-‐‑Based inactivation motif kD Kilodalton
KO Knock Out
L19A Mutation Leucine 19 to Alanine L9A Mutation Leucine 9 to Alanine LAT linker for activation of T cells MFI Mean Fluorescence Intensity
MHC Molecular Histocompatibility complex OVA Ovalbumin
PBS Phosphate buffered saline PFA Paraformaldehyde
pre-TCR pre T Cell Receptor PRS Proline Rich Sequence PTKs Protein Tyrosine Kinases
PTPases Protein Tyrosine Phosphatases RPM Revolutions Per Minute
RPMI Roswell Park Memorial Institute Medium S1P1 Sphingosine 1-‐‑phosphate receptor type 1 SD Standard Deviation
SDS-PAGE Sodium Dodecyl Sulfate-‐‑PAGE SEM Standard Error of Mean
T-ALL T-‐‑cell Acute Lymphoblastic Leukemia TCR T cell receptor
TIRFM Total Internal Reflection Fluorecent Microscopy TM Transmembrane domain
WB Western Blot WT Wild-‐‑Type
ZAP70 Zeta-‐‑Associated Protein of 70 KD
SUMMARY
Signalling through pre-‐‑T Cell Receptor (pre-‐‑TCR) and the αβ T cell Receptor (TCR) complexes are key events during thymic T cell development, which assure the correct generation of a functional and self-‐‑tolerant mature T cell repertoire. Previous work of the laboratory, using a transmembrane point mutant of the TCR-‐‑associated CD3ζ chain (L19A), has shown that ligand-‐‑independent pre-‐‑clustering of the TCR plays an important role in providing sensitivity to mature T cells, possibly by facilitating receptor-‐‑proximal cooperative signalling mechanisms. Dimerization of the pre-‐‑TCR has been proposed as necessary for the correct function of the pre-‐‑TCR during the earliest steps of T cell development in the thymus, although its effect on signalling has not been directly addressed. Given the relevance of T cell development in determining the protective and pathogenic capacities of the mature T cell repertoire, it is important to understand the molecular mechanisms of pre-‐‑TCR and TCR function and signalling underlying the formation of this repertoire.
In this thesis we have addressed whether and how the L19A mutation affects T cell differentiation and activation, focussing on the early pre-‐‑TCR-‐‑dependent differentiation steps, TCR-‐‑dependent positive and negative selection as well as activation of mature T cells and we have also investigated to what extent this is due to alterations in the organization of these receptors at the cell surface. To address these questions, we generated transgenic mice expressing recombinant CD3ζWT or CD3ζL19A chains coupled to GFP in an endogenous CD3ζ-‐‑deficient mouse background. Our data demonstrate that the L19A mutation partially arrests T cell development at the Double Negative (DN) stage, more specifically at the DN3 to DN4 transition. Data obtained with pre-‐‑T cell lines expressing the WT or L19A pre-‐‑TCR support the notion that this impairment in the DN3-‐‑DN4 transition is most likely due to an alteration in the transmission of the outside-‐‑in signal mediated by the pre-‐‑TCR.
L19A DN thymocytes present a more immature phenotype, proliferate less and are more prone to die compared to WT DN thymocytes. This defect is translated into a mild but significant peripheral lymphopenia, probably aggravated by a poorer efficiency of thymic selections and/or lineage commitment at the Double Positive (DP) stage. Notably, CD4 T lymphocytes are more affected than CD8 T lymphocytes. From a molecular perspective, TIRFM analysis provides evidence that the mutant pre-‐‑TCR on the plasma membrane of CD44-‐‑negative DN primary thymocytes are more mobile and less prone to clustering that the WT pre-‐‑TCR, suggesting a role of the Leu19 in the pre-‐‑
TCR cluster formation.
Together these data indicate that pre-‐‑TCR clustering optimizes the receptor signalling capacities and that the transmembrane region of the CD3ζ chain has an important role in pre-‐‑TCR clustering formation.
RESUMEN
RESUMEN
La correcta generación de linfocitos T maduros, funcionales y tolerantes a péptidos propios requiere una señalización adecuada a través de la forma inmadura y madura del Receptor para Células T (pre-‐‑TCR y TCR respectivamente) durante su desarrollo en el timo. Investigaciones previas en nuestro laboratorio con una cadena CD3ζ del TCR mutada puntualmente en su región transmembrana (L19A), mostraron que los clústeres de TCR independientes de ligando juegan un papel importante en la sensibilidad de las células T maduras por el antígeno, posiblemente debido a que estos clústeres favorecen mecanismos cooperativos durante la señalización temprana. A pesar de la falta de evidencias directas, actualmente se cree que la dimerización del pre-‐‑TCR es necesaria para su correcta función durante las primeras fases del desarrollo de los linfocitos T. Ya que las capacidades protectoras y patogénicas del repertorio periférico de células T maduras se establecen durante su desarrollo en procesos dependientes de los complejos pre-‐‑TCR y TCR, es esencial conocer el mecanismo molecular de la señalización y función de éstos complejos.
En esta tesis hemos abordado la caracterización del impacto de la mutación L19A durante el desarrollo de los linfocitos T. Nos hemos centrado en las etapas tempranas dependientes de pre-‐‑TCR, en las selecciones negativa y positiva y en el fenotipo y activación de los linfocitos maduros, además de investigar hasta que punto estas alteraciones son debidas a la organización de éstos receptores en la superficie celular.
Para ello, hemos generado ratones transgénicos donde las variantes CD3ζWT-‐‑GFP y CD3ζL19A-‐‑GFP de la cadena CD3ζ del TCR fueron reintroducidas en ratones deficientes para CD3ζ. Nuestros datos demuestran que la mutación L19A bloquea parcialmente el desarrollo de linfocitos T en el estadio Doble Negativo (DN). Más específicamente, en la transición entre los estadios DN3 y DN4. Los datos obtenidos en líneas celulares pre-‐‑TCR sugieren que este bloqueo se debe a una alteración en la transmisión de la señal a través de éste receptor. Los timocitos DN mutantes presentan un fenotipo más inmaduro, proliferan menos y son más sensibles a sufrir apoptosis que los timocitos DN silvestres. Éste efecto se traduce posteriormente en una ligera pero significativa linfopenia en los órganos linfoides periféricos, posiblemente agravada por la pobre eficiencia de las selecciones tímicas y/o un defecto en el establecimiento de linaje en el estadio Doble Positivo (DP). Es interesante destacar que los linfocitos CD4 están más afectados que los linfocitos CD8. Desde un punto de vista molecular, los análisis mediante TIRFM sugieren que el pre-‐‑TCR mutado es más móvil y menos propenso a la oligomerización en la membrana celular de timocitos primarios que el pre-‐‑TCR silvestre, indicando un papel de la Leu19 en la formación de clústeres de pre-‐‑TCR. En conjunto, estos datos indican que el la presencia de clústeres del pre-‐‑
TCR optimiza la capacidad de señalización del receptor y que la región transmembrana de la cadena CD3 ζ tiene un papel importante en este proceso.
INTRODUCTION
A brief introduction to the adaptive immune system
Each individual has an intrinsic capacity to defend itself against environmental pathogens. In vertebrates, this capacity is known as the immune system and it consists of an organization of cells and molecules with specialized roles that will trigger an immune response against potential pathogens, such as bacteria and viruses, or in order to prevent the uncontrolled growth of cells that may form a tumour.
Two interacting defences form the immune system: the innate and the adaptive immunity. The innate immunity provides an earliest line of defence. It is activated by pattern recognition receptors that identify unique, pathogen-‐‑derived molecules (i.e. not shared with eukaryotic cells), which are well conserved among different pathogens. The principal components of this defence system are physical and chemical barriers, phagocytic cells (neutrophils, macrophages), dendritic cells, natural killer cells and blood proteins, such as the molecules of the complement. All of them are in place even before infection and are poised to response rapidly. In contrast, activation of the adaptive immune system enables antigen-‐‑specific recognition of pathogens and tumoral cells resulting in long-‐‑term immunity, including immunological memory; however, the adaptive immune response requires time to arise and, hence, it is not as effective as the innate immune response at the onset of a new antigenic challenge. The adaptive immune system is further differentiated into a humoral component, which is mediated by antibody-‐‑producing B lymphocytes, and a cellular response, which is mediated by T lymphocytes (Fayard et al., 2010).
• B CELLS: Express on their membrane the B Cell Receptor (BCR) that is able to bind intact antigens. They have to principal roles: to produce specific antibodies/immunoglobulins, and to function as antigen-‐‑presenting cells (APCs).
For this second role, the bound antigen molecules are engulfed by the B cells where it is then digested into fragments and displayed at the cell surface nested inside a class II major histocompatibility complex molecule (MHC-‐‑II). Helper T
cells bind the B cell through the T Cell Receptor (TCR) and secrete cytokines that stimulate the B cell to become and effector-‐‑antibody-‐‑producer B cell (Cantrell, 2015).
• T CELLS: Express on their surface the T Cell Receptor (TCR). According to their TCR, it is possible to distinguish gamma/delta (γδ) and alpha/beta (αβ) T cells.
The vast majority of αβ T cells recognize antigen peptides presented by
“classical” class I or class II major histocompatibility complex molecules (MHC-‐‑I and MHC-‐‑II respectively), and are referred to as “conventional” T cells.
Conventional T cells are distributed into two subsets that differ by their function and their expression of CD4 or CD8, two surface glycoproteins that contribute to recognition of MHC molecules and signalling:
> CD8+ T cells, also known as cytotoxic T lymphocytes (CTLs). These cells recognize epitopes presented by the MHC-‐‑I molecules and are involved in the immune defence against intracellular pathogens.
> CD4+ T cells, that bind epitopes presented by the MHC-‐‑II and are essential for both cell-‐‑mediated and antibody-‐‑mediated branches of the immune system. They are also known as helper T cells because they produce regulatory cytokines and chemokines that contribute in the activity and function of other immune cells.
Establishment of a functional and competent CD4 T and CD8 T cell repertoire endowed with the capacity to respond to pathogens and tolerate self-‐‑antigens, happens during the lymphocyte development. This process take place most efficiently in the thymus, which provides a complexity of discrete cellular microenvironments specialized in supporting each of the steps of this developmental process (Cantrell, 2015).
T cell development: from the thymus to the periphery
The formation of a functional T cell population with a broad TCR repertoire depends on a precisely controlled developmental process that takes place in the thymus, in which epithelial, mesenchymal and hematopoietic stromal cells provide a unique
microenvironment and the signals necessary for proper thymocyte differentiation. There are several observations supporting the importance of this organ in the generation of the T cell repertoire; in the absence of the thymus, a reduction of peripheral T cells is observed, resulting in severe immunodeficiency (Bosma et al., 1983; Miller, 2002) . Also, contrary to the B cells that can be easily differentiated in vitro from bone marrow precursors, the in vitro generation of T cells require complex thymic organ cultures for their successful differentiation (Jenkinson and Owen, 1990). T cell development and differentiation is also a dynamic process; developing thymocytes moves through defined thymic regions, with lymphoid progenitors entering the thymus at the cortico–
medullary junction, then migrating to the outer cortex, and finally returning to the medulla (Figure 1) (Lind et al., 2001). These coordinated movements are directed by chemokines, and mediated by interactions between integrins expressed on T cell progenitors and their ligands on stromal cells. In summary, T cell development is a complex, thymic-‐‑dependent process that involves multiple and precisely coordinated differentiation and proliferation events. During this process, hematopoietic precursors give rise to T cells able to respond to antigen stimulation and ready for effector differentiation (Fayard et al., 2010; Zuniga-‐‑Pflucker, 2004).
The development of T cells can be separated in three broad steps: Thymic colonization by bone marrow precursors followed by the divergence of αβ and γδ lineages. Finally, αβ and γδ lineage cells complete their differentiation and acquire immunological properties (Carpenter and Bosselut, 2010). The γδ T cell is a very heterogeneous population with a not yet fully understood developmental process that takes place in waves (Prinz et al., 2013). The work presented in this thesis has been performed with αβ T cells and their precursors therefore from here on I will exclusively focus on this T cell type.
Blood-‐‑borne T cell precursors continuously access the vascularized microenvironment of the thymus at its cortico-‐‑medullary junction. Once situated within the thymus, these precursors begin to proliferate and give rise to cells designated as early T lineage progenitors (ETPs). The immediate progeny of ETPs is phenotypically characterized by the lack of CD4 and CD8 proteins at the cell surface, known as Double-‐‑Negative (DN)
Figure 1. Thymus structure and the dinamics of the thymocyte development.
Figure is a modification of (Germain, 2002).
thymocytes. In mouse, four successive DN subpopulations have been distinguished based on the expression of the surface molecules CD25 and CD44 (inset in Figure 2):
DN1 (CD44posCD25neg), DN2 (CD44posCD25pos), DN3 (CD44negCD25pos) and DN4 (CD44negCD25neg) (Godfrey et al., 1993). It is normally at the DN2 stage where the thymocytes become firmly committed to the αβ T lineage fate and begin to rearrange their Tcrβ locus. Rearrangement of Tcrβ is a mechanism of genetic recombination that randomly selects and assembles V, D and J segments of the Tcrβ locus, resulting in the generation of a diverse repertoire of TCRβ chains (Mallick et al., 1993). DN3 cells can be further subdivided into DN3a and DN3b: DN3a cells that have productively rearranged the Tcrβ locus become DN3b cells that express at the cell surface a functional TCRβ chain together with the invariant pre-‐‑TCRα chain (pTα) and the CD3 components. Whereas thymocytes that fail to form the pre-‐‑TCR die through apoptosis, signalling through this receptor rescues DN3 cells from programmed cell death, inhibits further rearrangement of the Tcrβ locus (resulting in allelic exclusion, a process by which only one allele of a gene is expressed while the other allele is silenced), initiates cell proliferation and enables the developmental progression to the DN4 phenotype (Hoffman et al., 1996).
These events, from the successful TCRβ rearrangement, are referred to as β-‐‑selection (Hoffman et al., 1996) and this process is also companied by changes in molecular expression of various membrane receptors such as CD98, CD71, CD25, CD27 and CD28 (Kelly et al., 2007; Teague et al., 2010). At the DN4 stage, thymocytes may also begin to express CD8 on their cell surface, becoming cells known as immature single CD8 positive (ISP8). DN4-‐‑ISP8 cells progress to a developmental stage characterized by the concomitant presence of CD4 and CD8 and hence called Doube-‐‑Positive (DP) population (Figure 2). Rearrangement of the Tcrα locus occurs in DP thymoyctes and eventually enables the expression of a mature αβTCR-‐‑CD3 complex where the pTα is replaced by a TCRα chain. The replacement of the pTα chain is important to ensure the further development of thymocytes beyond the DN4 stage. It has been seen that overexpression of pTα accelerates the transition from DN3 to DN4 but reduces the rate of assembly of αβTCR, increasing the apoptosis of DP thymocytes (Lacorazza et al., 2001). At the DP stage, thymocytes are subjected to a strictly TCR-‐‑dependent selection process in the thymic cortex and medulla that is aimed to test the specificity of their TCR (Figure 1).
Thymocytes with a TCR able to bind with sufficient affinity to self-‐‑peptide-‐‑MHC present on epithelial cells of the cortex, leads to positive selection and continue their intrathymic maturation. In contrast, DP thymocytes that have a non-‐‑functional TCR fail to receive any further survival signal and are thus eliminated in a process referred as “death by neglect”. In a subsequent stage, which typically but not exclusively takes place in the medulla, positively selected thymocytes are submitted yet to another selection step known as negative selection, which it is the result of strong TCR ligation of self-‐‑peptide-‐‑
MHC. Here, thymocytes with a TCR that binds to self-‐‑peptide MHC complexes with an affinity above a critical threshold are removed through induction of programmed cell death. This selection process thus eliminates potentially self-‐‑reactive thymocytes that otherwise may elicit a harmful autoimmune response as peripheral T cells. The affinity window for positive versus negative selection is narrow and, remarkably, small changes in the affinity of the TCR signal re-‐‑direct the fate of immature thymocytes. For instance, impairment of TCR signal strength results in a shift from negative to positive and from positive selection to neglect (Hogquist, 2001; Moran and Hogquist, 2011).
Figure 2. Scheme of the αβ T cell development.
Characteristic protein markers for each cell at the specific stage of development are shown. The development of the DN subpopulations is presented in more detail in the inset. ETP: Early T cell Progenitors, DN; Double Negative, ISP: Intermediate Single Positive, DP: Double Positive, 4SP:
Single Positive CD4, 8SP: Single Positive CD8.
Finally, selected thymocytes that have a TCR restricted to MHC class I molecules will develop into single positive CD4negCD8pos phenotype (8SP), whereas cells with an MHC class II-‐‑restricted TCR will accomplish a single positive CD4posCD8neg phenotype (4SP).
SP cells exit the thymus to the periphery as naïve T cells through a mechanism that
involves signalling by the sphingosine 1-‐‑phosphate receptor type 1 (S1P1) (Allende et al., 2004; Fayard et al., 2010).
Pre-TCR and TCR complexes
The TCR consists of six different types of transmembrane (TM) proteins that assemble in dimers: the ligand-‐‑binding TCRαβ heterodimer and the signal-‐‑transducing dimers of CD3εγ, CD3εδ, and CD3ζζ (Figure 3, right). The variable immunoglobulin (Ig) domains of the TCRα and TCRβ heterodimer form the binding surface for its ligand, the MHC-‐‑
peptide, while the constant Ig and TM regions couple to the CD3 dimers. The CD3 subunits contain an extracellular Ig domain (except for CD3ζ), a TM region and a cytoplasmic tail that contains one or more copies of a conserved sequence motif, known as the Immunoreceptor Tyrosine-‐‑based Activation Motif (ITAM).
The TCRβ chain of the pre-‐‑TCR is bound to an invariant pre-‐‑T cell receptor α (pTα) chain instead of to the rearranged TCRα chain (Groettrup et al., 1993) (Figure 3, left). The pre-‐‑TCR complex can be viewed as a variant of the mature αβTCR, but the pTα chain provides the pre-‐‑TCR with a unique function due to its different capabilities compared with the TCRα chain as has been demonstrated over the years (Table 1). The pTα chain possesses a single immunoglobulin-‐‑like domain that is more structurally similar to the constant domain of an antibody light chain than to the constant domain of the TCRα.
Even so, there is some homology between the Ig-‐‑like domain of the pTα and the two Ig-‐‑
like constant domain of the TCRα. The mode of association between pre-‐‑Tα and TCRβ resembles that mediated by the constant domains of the heterodimer TCRαTCRβ, although a cystein residue in pTα, responsible for interchain disulphide bond formation, is in a different position from that in the TCRα chain and allows less efficient heterodimerization with the TCRβ chain as well as only weak association with the CD3ζζ homodimer (Boehmer and Fehling, 1997). In addition, both pTα and TCRα transmembrane domains present polar residues in the same position and orientation, allowing the association with the signal-‐‑transducing CD3 molecules.
Figure 3. Scheme of the immature pre-‐‑TCR and mature TCR complexes.
CD3 chain names are displayed bellow each chain while the TCR chain names are shown above them.
The requirement of the engagement of mature TCRs by agonist pMHC for signal transduction is very well established. It is also well described that after TCR ligation, there is an accumulation of TCRs in the area of contact where an immune synapse structure is formed (Dustin and Shaw, 1999; Monks et al., 1998). In addition, there are several studies demonstrating that at least part of the TCRs in resting T cells, i.e. before interaction with pMHC agonist pMHC ligands, are organized in clusters of nanoscale size known as nanoclusters (Lillemeier et al., 2010; Schamel et al., 2005; Sherman et al., 2011; Zhong et al., 2009). Unlike for the mature αβTCR and despite its structural similarities, it has been suggested that the pre-‐‑TCR signals in a ligand-‐‑independent manner, even though the molecular mechanism underlying the autonomous signalling is still unclear. Some publications proposed that this ligand-‐‑independent signalling is not attributable to the pre-‐‑TCR per se; instead, it is a property of the DN cell in which pre-‐‑
TCR signalling occurs. This was illustrated by studies in which retroviral transduction of the TCRα chain into pTα-‐‑deficient thymocytes restored T cells development in fetal
Table 1. Comparison of pre-‐‑TCR and TCR characteristics.
thymus organ culture (Haks et al., 2003). However, in vivo experiments showed that the replacement of the pTα coding sequence by a rearranged TCRα chain using a knock-‐‑in approach, impaired the efficiency of thymocyte development form DN to DP meaning that pTα has an advantage over the TCRα in β-‐‑selection (Borowski et al., 2004) but in DP the advantage is inverted. This highlights that preTα and TCRα are not interchangeable and execute different genetic programs. Other evidence supporting the autonomous signalling of the pre-‐‑TCR was obtained using transgenic mice expressing a mutant pTα and TCRβ lacking extracellular immunoglobulin (Ig)-‐‑like domains thereby impeding any potential extracellular receptor-‐‑ligand interaction. Thymocytes expressing these truncated chains could still bypass the β-‐‑selection, suggesting that no extracellular ligand binding was necessary (Irving et al., 1998). On other hand, (Yamasaki et al., 2006) observed that charged amino acids in the extracellular domain of pTα are essential for pTα self-‐‑oligomerization and critical for optimal traversal of the β-‐‑selection checkpoint, suggesting that the extracellular domain is necessary for oligomerization although not
pre-‐‑TCR TCR
TCRβ bound to pTα invariant chain. TCRβ bound to a rearranged TCRα chain.
pTα: weak association with TCRβ and CD3ζζ
homodimer (Boehmer and Fehling, 1997) TCRα: high efficiency of dimerization with TCRβ.
Low levels of expression at the plasma membrane.
Peripheral and SP cells present high levels of expression on cell surface while in DP is lower but still more than the pre-‐‑TCR.
No ligand binding to promote signalling (Irving et al., 1998; Saint-‐‑Ruf et al., 2003;
Yamasaki et al., 2006) Ligand binding needed to promote signalling.
At least forming dimers (Yamasaki et al., 2006), but BN unpublished data suggest that is highly clustered (Thesis of Dr. Gina Fiala on Prof. Wolfgang Schamel’s Laboratory)
TCR in clusters of diverse size (Schamel et al., 2005).
Constitutively active. Ligand-‐‑dependent activation.
Degradation of TCR due to the continuous signalling. Similar phenotype as activated mature T cells (Carrasco et al., 2003; Panigada et al., 2002; Valitutti et al., 1997)
In resting T cells, TCR is recycled back to the membrane and very few TCR are degraded.
TCR in activated T cells is internalized and degraded (Carrasco et al., 2003; Panigada et al., 2002; Valitutti et al., 1997).
Localized in raft domains (Saint-‐‑Ruf et al., 2003).
Resting TCR localized in non-‐‑raft domains (Beck-‐‑García et al., 2015); can move to rafts upon stimulation (Burack et al., 2002).
necessarily for interaction with a ligand. However, a more recent publication suggests a self-‐‑peptide MHC interaction with the pre-‐‑TCR. The authors of this work propose that in addition to the autonomous signalling mechanism, there is also an interaction of the pre-‐‑
TCR with the pMHC or other ligands, that tunes selection of β-‐‑chains to be used by αβTCR-‐‑expressing DP thymocytes (Mallis et al., 2015). In agreement with a continuous signalling mode of the pre-‐‑TCR, it has been shown that the receptor is internalized and routed constitutively to lysosomes in absence of deliberate stimulation, similar to ligand-‐‑
induced internalization of αβTCRs on mature T cells (Carrasco et al., 2003; Panigada et al., 2002; Valitutti et al., 1997). This observation can also explain the low surface expression of pre-‐‑TCRs in DN thymocytes. Finally, there are others mechanisms proposed for the ligand-‐‑independent signalling such a low threshold signalling of the DN thymocytes compared with DP thymocytes (Haks et al., 2003) or their constitutive localization in raft domains (Saint-‐‑Ruf et al., 2003). Lipid rafts are membrane microdomains, which are enriched in sphingolipids, cholesterol and numerous signalling molecules, including Lck or LAT. It is suggested that the lipid raft content is elevated in DN thymocytes, which could facilitate pre-‐‑TCR signalling through increasing the frequency or duration of the interaction of the pre-‐‑TCR complex with signalling molecules (Yamasaki and Saito, 2006). As has been exposed above, many hypotheses have been proposed but, unfortunately, the complete structural basis for the oligomerization of pre-‐‑TCRs and the mechanisms allowing continuous signalling are still unclear and further studies are required. One of the main objectives in the work presented in this thesis is the better understanding of the pre-‐‑TCR clustering and its role in pre-‐‑TCR function.
Altogether, correct temporal regulation, stoichiometry and assembly of the pre-‐‑TCR and TCR are processes required to generate functional pre-‐‑TCR and TCR complexes that are essential in the T cell development and T cell function.
TCR and pre-TCR signalling
The biochemical events activated downstream of the TCR after its engagement were initially identified in mature T cells and involve not only the regulation of a number of protein tyrosine kinases (PTKs) and the phosphorylation of their substrates, but also the
activation of several protein tyrosine phosphatases (PTPases) (Fayard et al., 2010).
The earliest step in intracellular signalling following TCR ligation is the activation of Src (Lck and Fyn) PTKs, leading to phosphorylation of the CD3 ITAMs. The CD45 receptor tyrosine phosphatase modulates the phosphorylation and activation of Lck and other Src kinases that in turn promote the recruitment of the Zeta-‐‑chain associated protein kinase (Zap-‐‑70) to the TCR/CD3 complex where it becomes active and phosphorylates the adaptor proteins LAT and SLP-‐‑76. These two adapters form the backbone of the complex that organizes effector molecules in the correct spatiotemporal manner to allow for the activation of multiple signalling pathways (Figure 4).
Phosphorylation of SLP-‐‑76 promotes recruitment of Vav (a guanine nucleotide exchange factor), the adaptor proteins NCK and GADS, and an inducible T cell kinase (Itk).
Phosphorylation of phospholipase Cγ1 (PLCγ1) by Itk results in the hydrolysis of phosphatidylinositol 4,5-‐‑bisphosphate (PIP2) to produce the second messengers, diacylglycerol (DAG) and inositol trisphosphate (IP3). These two messengers are essential for T cell function. The production of DAG results in the activation of two major pathways: the PKCθ and the MAPK/Erk pathways, both promoting transcription factor NF-‐‑κB activation. NF-‐‑κB is translocated into the nucleus, where it activates genes involved in the function, survival, and homeostasis of T cells. On the other hand, IP3 triggers the release of Ca2+ from the endoplasmic reticulum (ER), which promotes entry of extracellular Ca2+ into cells through calcium release-‐‑activated Ca2+ (CRAC) channels.
Calcium-‐‑bound calmodulin (Ca2+/CaM) activates the phosphatase calcineurin, which in turn promotes the activation of the nuclear factor of activated T cells (NFAT). Feedback regulation at several points within these pathways allows for different outcomes, depending on the cell type and environment. The incorporation of signals from additional cell surface receptors (such as CD28 or LFA-‐‑1) further regulates cellular response (Malissen and Bongrand, 2015; Smith-‐‑Garvin et al., 2009).
The pre-‐‑TCR signalling in immature T cells occurs through pathways that are similar to signalling after ligation of the αβTCR in mature T cells (Michie and Zuniga-‐‑Pflucker, 2002). However, despite activation of the same transcription factors, the consequences
are different and this probably reflects the differential accessibility of specific genes in lymphocytes that are at different stages of maturation.
Figure 4. Pre-‐‑TCR signalling.
Only signalling via the pre-‐‑TCR complex has been represented, but signalling through the mature TCR is similar to pre-‐‑TCR signalling as discussed in the main text. Asterisk (*) close to protein names, indicates that in the absence of that particular molecule, T cell development is partially or totally arrested
TCR triggering models: TCR clustering and conformational change
TCRs on the membrane of T cells are able to bind to pMHC on the surface of the antigen presenting cells (APC). The T cells are able to identify a small numbers of foreign antigenic peptides in a context of thousands of self-‐‑pMHC in a rapid, highly sensitive and selective way. Moreover, each TCR is cross-‐‑reactive; i.e, it is capable to interact with a small spectrum of pMHC ligands with different affinities in the 1-‐‑200 µμM range. A given TCR is capable of triggering a diverse range of biological responses according to the affinity for the pMHC ligand that it encounters (Hogquist and Jameson, 2014). This is at the basis of self-‐‑nonself discrimination in both the thymus and the periphery.
In the periphery, naive T cells are continuously subjected to weak chronic TCR interactions with endogenous self pMHC that maintain the T cells in a state of heightened antigenic reactivity, whereas stronger interactions with foreign agonist pMHCs induce the T cells full-‐‑fledged activation (Malissen and Bongrand, 2015). A T cell also needs to discriminate between foreign pMHCs and self pMHC molecules even though the affinity and the kinetics of binding to self and foreign pMHCs and also the differences in the molecular sequence of the peptides are not large (Chakraborty and Weiss, 2014).
The mechanisms by which information is passed from the TCR antigen binding site through the plane of the membrane and results in the earliest events of T cell activation is termed TCR triggering (Malissen and Bongrand, 2015). These earliest events must reflect the remarkable and above-‐‑described features of the TCR:
high sensitivity and selectivity for agonist pMHC (summarised in Table 2). A number of competing models for receptor triggering has been proposed, driven in part by persisting uncertainties about the stoichiometry of the complex and the arrangement of its components. From our point of view, there are, so far, two main mechanisms that are able to enclose the specific characteristics of the TCR. These triggering models are the conformational change and the TCR clustering and both are at the basis of the kinetic segregation model, in which T cell activation is made possible by the segregation of proteins with large extracellular domain, such as CD45, from areas of the membrane where the TCR and other proteins with shorter extracellular domains are accumulating, and the mechanoreceptor model in which a force tangential to the cell surface is applied to the ligand binding site of the TCR promoting a rotation of the complex that in turns allows the exposure of the ITAMs of the CD3ε ectodomains in a piston-‐‑like manner.
CONFORMATIONAL CHANGE TCR CHARACTERISTICS
Very sensitive. TCR can recognize 1–10 agonist pMHC in a sea of 103-‐‑104 times more of self pMHC
High molecular specific
Wide dynamic range in their response to antigen (1-‐‑200uM range)
Low affinity for MHC (1-‐‑10µμM) Short half-‐‑life of the TCR-‐‑pMHC interaction (t1/2≈15-‐‑0.1s)
Table 2. Summary of TCR characteristics.
The conformational change model is based on the allosteric control, in which binding of a ligand at one site of a protein affects a distant functional site through a conformational change. Studies focused on the understanding of the triggering mechanism of the TCR have documented that conformational changes could be implicated in the outside-‐‑in transmission of the TCR signal. Three conformational changes have been proposed as mechanisms for TCR-‐‑inducible ITAM phosphorylation: Two separate but not mutually exclusive conformational changes within the CD3 cytoplasmic tails and a third involving the TCRαβ heterodimer.
The first mechanism stems from the observation that the cytoplasmic tails of CD3ε and CD3ζ fold or interact with the lipid of the inner leaflet of the plasma membrane preventing the phosphorylation of the ITAMs. The cytoplasmic tail of the CD3ζ chain might convert from a lipid-‐‑bound helical structure to an unfolded structure upon TCR triggering (Aivazian and Stern, 2000; Sigalov et al., 2006; Xu et al., 2008; Zhang et al., 2011). Therefore, upon assembly of the TCR-‐‑CD3 complex, the CD3ζ juxtamembrane regions are forced apart adopting an inactive conformation. TCR engagement then triggers a conformational change, where the CD3ζ cytosolic juxtamebrane regions move together acquiring an active conformation (Gagnon et al., 2012). The group of Michael S.
Kuhns (Lee et al., 2015) suggested that the structural features most likely to facilitate these changes are the transmembrane domain charge interactions between CD3ζζ and TCRαβ and that this region could serve as a pivot point around which the interacting subunits could move without risk of separation
A second mechanism involves the CD3ε chain. Upon TCR ligation a proline rich sequence (PRS) in CD3ε is exposed and is available to recruit the adaptor protein Nck via one of its SH3 domains (Gil et al., 2002). This so-‐‑called active conformation can also be detected by a monoclonal antibody (Apa1/1) specific for the intracellular domain of CD3ε (Salmerón et al., 1991). In relation with this data, some amino acids residues were identified in the membrane-‐‑proximal stalk region of the CD3ε, in close proximity to or part of an evolutionary conserved CXXC motif, that were critical for the formation of the rigid stalk conformation (Wang et al., 2009). Single mutation of two amino acid residues (Lys76 and Cys80) where seen to change particularly their conformation in the bound vs
unbound status. Mutation in both residues inhibited or abrogated the adoption of the active conformation by the PRS of CD3ε and prevented T cell activation upon antibody or pMHC triggering (Martinez-‐‑Martin et al., 2009) thus suggesting that these residues are important in the transmission of outside-‐‑in information.
Finally, the crystal structure of the αβLC13 TCR showed a shift in the AB loop of the Cα domain induced by the pMHC ligand binding. These data lead to propose that the transmission of the outside-‐‑in signalling may rely on conformational changes that reoriented αβTCR in relation to the CD3 components (Beddoe et al., 2009; Kjer-‐‑Nielsen et al., 2003). Importantly, all these conformational changes occur prior to CD3ζ and CD3ε ITAM phosphorylation and the maintenance of the conformational change depends on continued ligand binding (Minguet et al., 2007).
During T cell development, only high affinity-‐‑negative selection inducing interactions gave rise to a conformational change (Risueño et al., 2006). This discriminating capacity is developmentally regulated as in preselection thymocytes both low and high affinity pMHC ligands induced the conformational change, whereas in positive selected thymocytes and mature T cells only high affinity ligands induced the conformational change (Gil et al., 2008; 2005). In this regard, the work of (Blanco et al., 2014) make clear that the conformational change is also a necessary step during αβ T cell development. A mutation in the Cys80 (C80G) of the cytoplasmic tail of the CD3ε abrogates the T cell development at the DN3 level. These data reflect the absolute dependence of pre-‐‑TCR signalling on adopting the active conformation.
TCR CLUSTERING
Independent groups, using biochemical, high resolution light microscopy and electron microscopy techniques, have reported that TCR clusters are not only induced upon ligand binding but that they are already present before antigen encounter (Lillemeier et al., 2010; Schamel et al., 2005; Sherman et al., 2011; Zhong et al., 2009). These oligomers may reach a size of up to 20 units and will be referred to as TCR nanoclusters. A functional explanation for the existence of these nanoclusters is that the TCR oligomerization allows the propagation of the activating signalling from ligand bound