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Table of contents
Abstract ... 8
List of abbreviations ... 9
I. Introduction ...11
1. γδ T cells, the unconventional T lymphocytes ... 11
1.1. Discovery of γδ T cells and differences from αβ T cells ... 12
1.2. T cell development in the thymus ... 14
1.2.1. VDJ recombination ... 15
1.2.2. γδ T cell subsets ... 21
1.2.3. Development scheme in mouse and human ... 22
2. Vγ9Vδ2 T cells, activation and signalling ... 26
2.1. TCR activation by (phospho)antigens ... 26
2.1.1. Butyrophilins, sensor of phosphoantigens ... 28
2.2. Co-stimulation and signalling ... 31
3. Effector functions of the Vγ9Vδ2 T cells ... 34
3.1. Cytotoxicity – the granule exocytosis pathway... 34
3.2. Cytokine production ... 36
3.3. Regulation of the immune response ... 37
4. Vγ9Vδ2 T cells in cancer immunotherapy ... 40
4.1. Strategies for therapeutic manipulation of Vγ9Vδ2 T cells ... 41
4.2. Obstacles and how to overcome them ... 43
5. Immune response of Vγ9Vδ2 T cells to Mycobacterium ... 46
5.1. Mycobacterium tuberculosis... 46
5.2. Attenuated Mycobacterium bovis ... 49
6. Immune system in neonate and infant ... 51
6.1. Bridge between the tolerant foetal and vigorous adult immune system ... 51
6.2. Intervening in the neonatal host defence ... 53
II. Objectives ...55
III. T cell receptor sequencing reveals the distinct development of foetal and adult human Vγ9Vδ2 T cells ...56
Abstract ... 56
1. Introduction ... 57
2. Materials and Methods ... 59
2.1. Human cell material ... 59
2.2. Flow cytometry, sorting and cell cultures ... 59
2.3. TCRγ and TCRδ NGS ... 60
2.4. Statistical analysis ... 62
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3.1. Adult and foetal blood Vγ9Vδ2 T cells show a different CDR3 repertoire ... 63
3.2. Expansion of phosphoantigen-reactive foetal blood Vγ9Vδ2 T cells does not lead to an adult-type CDR3 repertoire ... 70
3.3. Foetal and post-natal Vγ9Vδ2 thymocytes express a different CDR3 repertoire . 72 3.4. Recombination of the germline-encoded public TRGV9-TRJP CDR3 sequence . 78 3.5. HMB-PP-expanded post-natal Vγ9Vδ2 thymocytes express an adult blood-type CDR3 repertoire ... 81
3.6. The generation of foetal-like or adult-like Vγ9Vδ2 T cells is HSPC-dependent .... 84
4. Discussion ... 86
IV. Foetal public Vγ9Vδ2 T cells expand and gain potent cytotoxic functions early after birth ...90
Abstract ... 90
1. Introduction ... 91
2. Materials and Methods ... 93
2.1. Study Population - Human cell material ... 93
2.2. Flow cytometry, sorting and cell cultures ... 94
2.3. TCRγ and TCRδ high-throughput sequencing ... 96
2.4. Statistical analysis ... 97
3. Results ... 98
3.1. Vγ9Vδ2 T cells expand early after birth ... 98
3.2. Only the 10-week-old Vγ9Vδ2 TRD repertoire is public and foetal-derived ... 100
3.3. Foetal-derived Vγ9Vδ2 T cells get activated and become highly cytotoxic rapidly after birth ... 108
3.4. Cytokine expression capacity by Vγ9Vδ2 T cells is mainly determined before birth ... 114
3.5. Phosphoantigen-reactivity remains stable early after birth ... 114
3.6. BCG vaccination at birth does not alter the TCR repertoire nor functional differentiation ... 117
4. Discussion ... 125
V. General discussion ... 128
1. Insight by CDR3 repertoire analysis ... 128
1.1. Gamma versus delta chain repertoire ... 128
1.2. Early life versus adult repertoire ... 129
1.3. Driving the generation of Vγ9Vδ2 T cells ... 130
1.4. Country of origin impact on repertoire ... 131
2. Expansion of foetal-derived Vγ9Vδ2 at 10 weeks after birth ... 131
2.1. Vγ9Vδ2 innateness versus nonVγ9Vδ2 γδ T cells ... 131
2.2. Function at 10 weeks after birth ... 132
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4. Effect of BCG immunisation on Vγ9Vδ2 T cells ... 133
4.1. BCG influence on repertoire and function ... 133
4.2. Speculations on absence of BCG effect on neonatal Vγ9Vδ2 T cells ... 134
VI. Conclusion ... 136
VII. Perspectives ... 138
VIII. References ... 141
