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FULLY FUNCTIONAL IMMUNE ORGAN GROWN IN MICE FROM LAB-CREATED CELLS Maleeha Akram 08-arid-1772 Ph.D. Scholar Department of Zoology 1 Contents Thymus ◦ Structure and Function ◦ Types ◦ Dysfunctions and Treatment 2 Thymic involution Generation of TECs Functional attributes of iTECs iTECs can form functional thymus Conclusion Reference Thymus The thymus is ◦ a soft ◦ roughly triangular in shape ◦ specialized organ of the immune system It is located ◦ in the mediastinum of the thoracic cavity between the lungs ◦ anterior and superior to the heart ◦ posterior to sternum 3 Structure of thymus The thymus is of a pinkish-gray color and lobulated on its surfaces. It has two distinct but identical lobes – each surrounded by a tough, fibrous capsule. 4 Within each lobe is a superficial region of tissue called the cortex and a histologically distinct deep region called the medulla. Epithelial tissues and lymphatic tissues containing dendritic cells and macrophages make up the majority of both regions of the thymus. Cortex 5 The cortical portion is mainly composed of thymocytes (developing T-lymphocytes) Supported by a network of finely-branched epithelial reticular cells, which is continuous with a similar network in the medullary portion. This network forms an adventitia to the blood vessels. Thymocytes Reticular cells Medulla 6 The medulla is continuous between adjacent lobules The medulla is paler staining, less densely cellular than the cortex and contains – More mature T-cells – Prominent epithelial cells – Hassalls corpuscles – Admixed macrophages – Dendritic cells – B lymphocytes Thymus function The thymus serves a vital role in the training and development of T-lymphocytes or T cells. ◦ T cells defend the body from potentially deadly pathogens such as bacteria, viruses and fungi. The function of the thymus is to receive immature T cells – that are produced by hematopoietic stem cells (HSC) in the red bone marrow – and train them into functional, mature T cells that attack only foreign cells. 7 It also secretes the hormone thymosin that stimulates the development of T-cells Immature T Lymphocytes Thymus Mature T-cells Thymosin Stimulates • Development of T-cells • Other immune cells 8 Types of T cells Helper T cells (TH cells) ◦ Assist other white blood cells in immunologic processes, maturation of B cells into plasma cells and memory B cells, activation of cytotoxic T cells and macrophages. ◦ Also known as CD4+ T cells because they express the CD4 glycoprotein on their surfaces. Cytotoxic T cells (TC cells) ◦ Destroy virus-infected cells and tumor cells, and are also implicated in transplant rejection. ◦ These cells are also known as CD8+ T cells since they express the CD8 glycoprotein at their surfaces. 9 Process of T-cell maturation T cells first reside within the cortex of the thymus – where they come in contact with epithelial cells presenting various antigens. The immature T cells that respond to the antigens corresponding to foreign cells are selected to survive, mature and migrate to the medulla The rest die via apoptosis and are cleaned up by macrophages. This process is known as positive selection. 10 Continued… Upon reaching the medulla – the surviving T cells continue to mature and are presented with the body’s own antigens. T cells that bind to the body’s own antigens test positively for autoimmunity. Autoimmune T cells are eliminated by apoptosis in a process known as negative selection – resulting in only around 2% of the immature T cells reaching maturity. 11 T-cells that leave the thymus (via the corticomedullarly junction) are singly positive, selfrestricted and self-tolerant. 12 Thymus dysfunctions People without a fully functioning thymus can't make enough T cells – as a result, they are very vulnerable to infections. This is a particular problem for bone marrow transplant patients – Because a functioning thymus is needed to rebuild the immune system once the transplant has been received. Some newborns also have malfunctioning or completely absent thymus – Due to conditions such as DiGeorge syndrome. 13 Treatment Thymus disorders can sometimes be treated with – Infusions of extra immune cells – Transplantation of a thymus organ soon after birth – But both are limited by a lack of donors and problems matching tissue to the recipient. 14 Being able to create a complete transplantable thymus from cells in a lab would be a huge step forward in treating such conditions. Thymic involution Unlike most organs that grow until the age of maturity – the thymus enlarges throughout childhood – but slowly shrinks from the onset of puberty and throughout adulthood. 15 As the thymus shrinks, its tissues are replaced by adipose tissue. This process is called thymic involution The shrinking may be due to the reduced role of the thyroid in adulthood or increased secretion of sex steroids The immune system produces most of its T cells during childhood and requires very few new T cells after puberty. 16 17 Need for research Thymus transplantations soon after birth can increase adaptive immunity in patients who are congenitally athymic But these transplantations are limited by donor tissue supply and histocompatibility These limitations would be overcome if functional thymic epithelial cells (TECs) could be generated or expanded in vitro, i.e. they are able to 18 ◦ produce mature T-cells ◦ produce well-formed organ with the same structure as a healthy thymus History of research Several investigators have reported derivation of TEC-like cells from pluripotent cells 1. Lai and Jin, (2009) and Lai et al., (2011) used mouse embryonic stem cells to produce the cells that had phenotype of thymic epithelial cells using FGF-7, FGF10 and BMP-4. 2. Inami et al., (2011) produced thymic epithelial progenitor cells (TEPCs) by phenotype – by culturing induced pluripotent stem cells (iPSCs) with collagen IV coated dishes in the presence of activin A and lithium chloride (LiCl) in mice 3. Parent et al., (2013) used human pluripotent stem cells to generate thymic epithelial cells – by regulating TGFβ, BMP4, RA,Wnt, Shh and FGF signaling 19 Continued… Sun et al., (2013) produced TECs after transplantation of the pluripotent cell-derived TECs using a transcription factor autoimmune regulator (AIRE) But in the experiment, scientists were unable to produce cortical and medullary compartments of thymus. 20 In all these experiments ◦ Unable to produce TEC-like cells of an organized thymus containing all TEC subtypes ◦ TEC-like cells had no capacity to support T-cell development in vitro Generation of TECs Bredenkamp et al., 2014 from University of Edinburgh carried out their study using cells called fibroblasts taken from mouse embryos (MEFs). The transcription factor forkhead box N1 (FOXN1) is critically required for development of thymic epithelial cells (TECs), during embryonic development By increasing levels of the protein FOXN1, scientists observed that the morphology of MEFs had changed from fibroblasts’ cell shape to epithelial cells shape. 21 The experiment Rosa26CreERt2/+ mice were taken as controls Transgenic mouse line was developed in which ◦ Foxn1 cDNA controlled by CAG promoter was knocked into ROSA26 locus with a LoxP-flanked transcriptional stop cassette was inserted between CAG promoter and Foxn1 cDNA 22 Continued… These mice were then crossed with Rosa26CreERt2 generating Rosa26CreERt2/CAG-STOP-Foxn1-IRES-GFP embryos. From these embryos, primary mouse embryonic fibroblast (MEFs) were generated These MEFs were then treated with tamoxifen to remove STOP cassette generating Rosa26CreERt2/CAG-Foxn1-IRES-GFP iFoxn1 MEFs After 10 days of iFoxn1 expression initiation, the morphology of cells changed from elongated, bipolar shape characteristic of fibroblast cells to broader, polygonal shape, characteristic of epithelial cells 23 Bright-field (left) and immunofluorescence images (right) showing morphology and K8 staining, 10 days after 4OHT treatment. 24 Checking the identity of cells The identity of these cells was further confirmed by using ◦ Epithelial-specific markers keratin 8 (K8) and ◦ Epithelial cell adhesion molecule (EpCAM) These are expressed by all TECs during early thymus development. Most of iFoxn1 MEFs, but no control MEFs, showed K8 expression Approximately 15% iFoxn1 MEFs expressed EpCAM This suggested that FOXN1 induction had converted the fibroblasts to an epithelial-like state. 25 Continued… Further, EpCAM+ cells were isolated and analyzed for expression of specific FOXN1-regulated genes ◦ ◦ ◦ ◦ TEC- (Dll4, Ccl25 and Kitl) Cutaneous epithelium- (Fgf2 and Krt1) Epithelial V-like antigen (Eva or Mpzl2) non-Foxn1 target TEC-associated genes (Pax9 and Trp63) The iFoxn1 MEFs but not control MEFs expressed all genes except cutaneous epithelium genes. All these expressions suggested FOXN1-mediated conversion of MEFs into TEC-like cells 26 27 Functional attributes of iTECs To test whether induced thymic epithelial cells (iTECs) were functional or not, scientists cultured a monolayer of iTECs with early T lineage progenitors (ETPs) Lin-CD25-C-Kit+ Analysis after 12 days of co-culture revealed the presence of CD4+CD8+ double positive, CD4+ and CD8+ single positive cells resembling that of adult mouse thymus 28 Continued… These T cells expressed both CD3 (role in antigen recognition) and T-cell antigen receptor beta While, ETPs seeded onto control MEFs did not enter thymopoiesis Interestingly, the capacity of iTECs to support thymocyte development was dependent on their plating density ◦ high density (>500 cellsmm-2) producing >3 times more CD4+ CD8+ T cells within 12 days than a lower density (<250 cellsmm-2) 29 iTECs can form functional thymus 30 iTEC were then grafted under the kidney capsule of genetically identical adult mice Fetal thymic mesenchyme was included to ensure that growth factors essential for expansion of the thymus, including FGF10 and IGF, were available within the graft After four weeks, the cells had produced macroscopic, well-formed organs with the same structure as a healthy thymus, with clearly defined regions (known as the cortex and medulla). All EpCAM+ cells in the iTEC grafts expressed GFP, reporting the transgenic iFoxn1-IRES-GFP messenger RNA, confirming they were derived from the input iTECs 31 Hematoxylin and eosin staining reveals cortical and medullary portions of thymus 32 Pan-cytokeratin staining reveals reticular network of epithelial cells throughout the organs Advantages iTECs are a new and readily available source of TECs, that may provide the basis for thymus transplantation therapies aimed at boosting adaptive immune system function in immuno-compromised patients. The technique may also offer a way of making patientmatched T cells in the laboratory that could be used in cell therapies. Such treatments could benefit bone marrow transplant patients, by helping speed up the rate at which they rebuild their immune system after transplant. The discovery offers hope to babies born with genetic conditions that prevent the thymus from developing properly. Older people could also be helped as the thymus is the first organ to deteriorate with age. 33 Future enhancements This is an exciting study but much more work will be needed before this process can be reproduced in a safe and tightly controlled way suitable for use in humans With further refinements, the researchers hope that their lab-grown cells could form the basis of a thymus transplant treatment for people with a weakened immune system. 34 Conclusion Enforced expression of FOXN1 is sufficient to convert fibroblasts into iTECs ◦ an in vitro generated cell type that exhibits phenotypic and functional properties of in vivo TECs. iTECs are able to promote full T-cell development in vitro iTECs generate a properly patterned, functional organ on transplantation in vivo, composed of cortex and medulla of thymus 35 Reference 36 Bredenkamp, N., S. Ulyanchenko, K. E. O'Neill, N. R. Manley, H. J. Vaidya and C. C. Blackburn. 2014. An organized and functional thymus generated from FOXN1reprogrammed fibroblasts. Nat. Cell Biol., 1-15. Abbreviations used 37 HSC = Hematopoietic stem cells MHC = Major histocompatibility complex FGF = Fibroblast growth factor BMP = Bone morphogenetic protein TECs = Thymic epithelial cells MEFs = Mouse embryonic fibroblasts TGFβ = Transforming growth factor β RA = Retinoic acid Wnt = Wingless type Shh = Sonic hedgehog IRES = Internal ribosome entry site GFP = Green fluorescent protein K8 = Keratin 8 EpCAM = Epithelial cell adhesion molecule Continued… 38 4OHT = 4-hydroxy tamoxifen Dll4 = Delta like ligand 4 Ccl25 = Chemokine ligand 25 Kitl = Kit ligand Krt1= Keratin 1 Mpzl2 = Myelin protein zero-like 2 Pax9 = Paired box gene 9 Trp63 = Transformation related protein 63 CD3 = Cluster of differentiation 3 IGF = Insulin like growth factor iTECs = Induced thymic epithelial cells