Development of regulatory T cells in the human thymus

...........................................................................................................................................7 TIIVISTELMÄ.......................................................................................................................................9 ABBREVIATIONS ..............................................................................................................................11 ORIGINAL PUBLICATIONS ............................................................................................................14 REVIEW OF THE LITERATURE ....................................................................................................15 1 T CELL SUBSETS .........................................................................................................................15 1.1 CD8 T cells + ............................................................................................................................15 1.2 CD4 T cells + ............................................................................................................................16 1.2.1 Th1 Cells .........................................................................................................................................16 1.2.2 Th2 cells..........................................................................................................................................16 1.2.3 Th17 cells........................................................................................................................................17 1.2.4 Natural Treg cells............................................................................................................................17 1.3 Other regulatory T cells ..........................................................................................................17 2 ANTIGEN RECOGNITION AND T CELL ACTIVATION ..........................................................18 2.1 T cell receptor complex...........................................................................................................18 2.2 Antigen recognition by T cells................................................................................................19 2.3 Co-receptors............................................................................................................................20 2.4 T cell activation.......................................................................................................................21 2.4.1 First signal.......................................................................................................................................21 2.4.2 Second signal ..................................................................................................................................21 2.4.3 Third signal .....................................................................................................................................21 2.4.4 Consequences of activation.............................................................................................................21 3 T CELL DEVELOPMENT AND CENTRAL TOLERANCE ........................................................22 3.1 Thymus structure ....................................................................................................................22 3.2 Entry into thymus and early stages of T cell development .....................................................24 3.3 -rearrangements ....................................................................................................................24 3.4 -rearrangements ....................................................................................................................25 3.5 Allelic exclusion .....................................................................................................................27 3.6 Dual-specific T cells ...............................................................................................................27 3.7 Positive selection ....................................................................................................................28 3.8 Negative selection...................................................................................................................29 3.9 AIRE and central tolerance .....................................................................................................29 4 DEVELOPMENT OF NATURAL REGULATORY T CELLS .....................................................30 4.1 Identification of natural regulatory T cells..............................................................................30 4.2 FOXP3 gene............................................................................................................................31 4.2.1 Significance of FOXP3 to Treg cells...............................................................................................31 4.2.2 Control of FOXP3 expression .........................................................................................................31 4.2.3 Functions of transcription factor FOXP3 ........................................................................................32 4.3 Surface markers of Treg cells .................................................................................................33 4.4 Commitment to Treg cell lineage............................................................................................33 4.4.1 FoxP3 thymocyte subsets + ...............................................................................................................33 4.4.2 Evidence for commitment in SP stage.............................................................................................34 4.4.3 Evidence for commitment in DP stage ............................................................................................34 4.4.4 Evidence for commitment in DN stage ...........................................................................................35 4.5 Selection and TCR specificities of Treg cells .........................................................................35 4.6 Cytokines and additional signals.............................................................................................36 5 PERIPHERAL TOLERANCE........................................................................................................37 5.1 Natural Treg cell function .......................................................................................................37 5.1.1 Cellular contacts..............................................................................................................................38 5.1.2 Cytokines ........................................................................................................................................38 5.1.3 Suppressive role of Treg cells in immune responses.......................................................................39 5.1.4 Activating role of Treg cells in immune responses .........................................................................39 5.2 Peripheral conversion of Treg cells.........................................................................................39 5.2.1 Peripheral induction of FOXP3 Treg cells + .....................................................................................39 5.2.2 Nonregulatory induction of FOXP3 ................................................................................................40 5.3 Other mechanisms of peripheral tolerance..............................................................................40 6 FAILURE OF TOLERANCE: APECED AS A MODEL................................................................41 6.1 Human disease ........................................................................................................................41 6.2 Aire knock-out mouse and ectopic transcription.....................................................................42 6.3 Other functions of AIRE.........................................................................................................43 AIMS OF THE STUDY .......................................................................................................................44 SUMMARY OF MATERIALS AND METHODS ............................................................................45 1 SAMPLES..........................................................................................................................................45 1.1 Healthy donors (I-IV) .............................................................................................................45 1.2 APECED patients (IV)............................................................................................................45 1.3 Mice (IV) ................................................................................................................................45 2 CELL SEPARATION AND FLOW CYTOMETRY (I-IV) ...........................................................................46 2.1 Immunomagnetic sorting ........................................................................................................46 2.2 Flow cytometry .......................................................................................................................46 2.3 Control staining.......................................................................................................................46 3 CELL CULTURE (III, IV) ...................................................................................................................46 4 PCR.................................................................................................................................................47 4.1 RNA/DNA isolation and cDNA synthesis (I-IV) ...................................................................47 4.2 Quantitative PCR (I-IV)..........................................................................................................47 4.3 TCR repertoire analysis (IIIV)..............................................................................................47 5 IMMUNOHISTOCHEMISTRY (IV) .......................................................................................................48 6 STATISTICAL ANALYSES ..................................................................................................................48 7 ETHICAL CONSIDERATIONS..............................................................................................................48 RESULTS..............................................................................................................................................49 1 UP-REGULATION OF FOXP3 IN THYMUS (I) .....................................................................................49 1.1 FOXP3 expression in DN thymocytes ....................................................................................49 1.2 TCR expression in FOXP3 thymocytes + .................................................................................49 1.3 FOXP3 DN thymocytes have characteristics of Treg cells + ....................................................51 2 FOXP3 EXPRESSION IN DP CELLS (II)...............................................................................................51 2.1 CD25 DP thymocytes express as much FOXP3 as CD25 CD4 SP thymocytes + + + .................51 2.2 Markers of positive selection on FOXP3 DP thymocytes + .....................................................52 2.3 FOXP3 DP thymocytes have features of immature cells + .......................................................53 2.4 TCR repertoires of CD25 DP and CD4 CD25 SP cells + + + .......................................................53 2.5 FOXP3 expression in CD8 SP thymocytes + ............................................................................54 3 DUAL-SPECIFICITY OF TREG CELLS (III) ..........................................................................................54 3.1 Increased TCR C mRNA levels in CD4 CD25 SP thymocytes + + ..........................................54 3.2 Expression of two different TCR V genes in CD4 CD25 thymocytes + + ...............................54 3.3 Dual-specific cells express more FOXP3................................................................................55 3.4 V2 and V12 are comparable with the whole repertoire......................................................56 3.5 Most human Treg cells express two functional TCR  chains ...............................................57 4 TREG CELLS IN APECED (IV) .........................................................................................................57 4.1 Impaired function of Treg cells...............................................................................................57 4.2 Activation profile of Treg cells ...............................................................................................58 4.3 FOXP3 expression is diminished ............................................................................................58 4.4 TCR repertoire of Treg cells ...................................................................................................60 4.5 AIRE localization in human thymus .......................................................................................60 DISCUSSION........................................................................................................................................61 1 TREG CELLS AND TRANSCRIPTION FACTOR FOXP3 .........................................................................61 1.1 FOXP3 as a marker for Treg cells...........................................................................................61 1.2 Factors affecting the up-regulation of FOXP3........................................................................61 2 THYMIC DEVELOPMENT OF NATURAL REGULATORY T CELLS ..........................................................62 2.1 Relationships of FOXP3 precursors + ......................................................................................62 2.2 CD8 T cells expressing FOXP3 + .............................................................................................64 2.3 Significance of TCR in Treg cell development.......................................................................64 2.4 Negative selection of Treg cells..............................................................................................65 2.5 Treg cells express two different TCRs....................................................................................65 2.6 Consequences of dual-specificity on Treg cells ......................................................................66 2.7 AIRE and Treg cells................................................................................................................67 CONCLUDING REMARKS...............................................................................................................70 ACKNOWLEDGEMENTS .................................................................................................................71 REFERENCES 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