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Domenico Ribatti

of Immunologic

The Development of Immunologic Competence

Domenico Ribatti The Development of Immunologic Competence .

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.................................... 2 Human Hematopoietic Development ..................... 3........... 5...........................................1 The Hemangioblast and the Yolk Sac ............ 3...................................................................2 Studies of the Thymus in the Chick and in the Mouse ...................................................3 The Functional Anatomy of the Human Thymus......................................................................................... 3.................................................................................................. 4............. 5................... 25 25 26 28 34 Clinical Correlates ...................................................................... 4..........................................................................................4 Mammalian “Bursa-Equivalent” Organs and the Role of Liver and Bone Marrow in Lymphopoiesis ............................................................................. 5......4 The Effects of Neonatal Thymectomy ..... 10 2.......................... 13 13 18 19 The Thymus ........................... 7 2.3 The Fetal Liver and the Placenta................. 7 2... 4..................... 11 3 The Bursa of Fabricius ..4 The Bone Marrow ............3 Thymoma with Immunodeficiency ......................... 3...................6 Removal of Either the Thymus or Bursa of Fabricius ....... 8 2.1 Immunodeficiencies .....................1 The Discovery of the Thymus and Its Function ..................2 Bursal Regulation of Antibody Production ..........Contents 1 Introduction .............................................. 4................2 Di George Syndrome .........................1 The Discovery of the Bursa of Fabricius and Its Structure .............3 Regulation of the Synthesis of Antibodies .......................................... 4.............................................................................5 The Thymus Is Essential for Normal Development of the Immune System . 39 39 40 42 4 5 1 21 35 36 v ...................... 4...................................................2 The Aorta-Gonad-Mesonephros................................. Contents 5...4 Severe Combined Immunodeficiencies and Ataxia-Teleangectasia........................................ 43 5........................................ 47 Index ........................................................ 59 ............................................... 44 References ....................................5 The Role of Thymus and of Bursa Equivalent Organs in the Development of Tumors ................................

Abbreviations ALL Ang-1 AGM APECED AIRE BCG BL-CFC BMP BSA BALT BFU-E CFU-S FGF-1 FAE GVH GM-CSF GALT HSCs HLA-DR IFE IL LSF MHC MALT NK PDGF Runx1 SCID SRBC SCL TCR Acute lymphoblastic leukemia Angiopoietin-1 Aorta-gonad-mesonephros Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy Autoimmune regulator Bacillus Calmette-Guerin Blast colony-forming cell Bone morphogenetic protein Bovine serum albumin Bronchus-associated lymphoid tissue Burst-forming units-erythroid Colony-forming units-spleen Fibroblast growth factor-1 Follicle-associated epithelium Graft-versus-host Granulocyte-macrophage colony-stimulating factor Gut-associated lympho-epithelial tissues Hematopoietic stem cells Human leukocyte antigen-DR Interfollicular epithelium Interleukin Lymphocytosis-stimulating factor Major histocompatibility complex Mucosa-associated lymphoid tissues Natural killer Platelet-derived growth factor Runt domain factor x1 Severe combined immunodeficiency disease Sheep red blood cells Stem cell leukemia T-cell receptor vii .

viii TGF-β VE VEGF VEGFR-2 XLA Abbreviations Transforming growth factor beta Vascular endothelial Vascular endothelial growth factor Vascular endothelial growth factor receptor-2 X-linked agammaglobulinemia .

1007/978-3-319-24663-5_1 1 . The first faint trace seems to have appeared some 400 million years ago among primitive marine vertebrates and perhaps among some of the invertebrates as well as. Another major step forward among amphibian was the appearance of plasma cells in intestinal tract. Between the 12th © Springer International Publishing Switzerland 2015 D. pericardial tissue. Good (Fig. The lamprey possesses the most primitive known thymus. including amphibian. In the chicken embryo. A system involved in the synthesis of immunoglobulins arose about 250 million years ago in the higher sharks and paddle fish. the thymus is the first lymphoid organ to develop. Ribatti. in a meeting organized by Robert A. Gabrielsen was established that T and B cells are different and that in the immune system it is possible distinguish central and peripheral organs (Good and Gabrielsen 1964). and gonad. kidney. thymus-dependent and thymusindependent. in different animal species. The latest stages of this phylogenetic process are represented by the development of bursal like tissues. In 1962. In all of the placoderm-derived vertebrates the basic structure of immunoglobulins includes a composite polypeptide chain structure based on both high. The thymus-dependent system developed at a relatively early stage in the evolution of vertebrate forms. and true lymph nodes.Chapter 1 Introduction Keywords Immune system • Thymus • Bursa of Fabricius • Immunoglobulins • Plasma cells • T cells • B cells • Chick embryo • Bursectomy • Thymectomy • Irradiation • Clonal selection theory • Post-capillary venules • Recirculation The immune system is distinct in two components. The distinction in thymus and bursal systems in the chick was firstly suggested by Szenberg and Warner (1962). they also produce gamma globulins. The Development of Immunologic Competence. Ribatti 2006a) and Ann E. These species show the first well defined plasma cells in the spleen. and mammals. DOI 10. and that in bursectomized chickens the synthesis of antibodies is suppressed (Glick and Whatley 1967). birds.1. and a primitive spleen. and the thymus is a fully developed lymphoid organ by the 12th day of incubation. Bruce Glick and co-workers (Glick et al. 1956) demonstrated that the bursa is involved in the antibody production. The epithelial component is evident before the 9th day of incubation.and low molecular weight (heavy and light) polypeptide chains. not a single organ but a number of scattered foci of 5–20 lymphoid cells. 1.

although it was unclear at the time whether the thymus and the bursa lead synergistic or independent . 1965..1 A portrait of Robert A. Good and Max D. Parrott et al. budding of the epithelial fold of the bursa is observed. Cooper has pointed out that: “Chickens offered an animal model in which to test the possibility of alternative lymphocyte lineages. Cooper (Fig. Good et al. By contrast.3. and on the 14th day the lymphoid structures begin to develop by direct transformation of epithelial cells to lymphoid cells. Stutman et al. Metcalf 1960. even as late as two or three months after hatching. Cooper as postdoctoral fellow in the laboratory of Good. when the lymphoid tissue is forming and immunologic capacity is maturing (Miller 1962c. 1. 1964. the thymic cells had matured to a population with the largest number of lymphoid cells with small volume. 1966a. The crucial period during which thymus and bursa influence immunologic development occurs during early life. Good (1922– 2003) with two patients and the 14th day. 1.4).2. Good 1955. 2006) in Australia demonstrated that thymectomy was responsible of a reduction in the number of lymphocytes and that the earlier thymectomy produced the greater deficiency of lymphocytes in other lymphoid organs. Ribatti 2014) demonstrated that thymus was involved in both the development of cellular immunity and antibody production in chickens (Cooper et al. In 1964. 1. 1961). More recently. the bursal population. By the time of hatching. By the 18th day the bursa has a well-organized lymphoid structure.2 1 Introduction Fig. Francis Albert Pierre Miller (Fig. contained few such small cells but had a preponderance of cells with higher volume. 1966. 1969a). Three days before hatching. Ribatti et al. Observations on the changes in the lymphoid organs after bursectomy and thymectomy in chickens have indicated the possible existence of two almost completely separate lymphocytopoietic systems. 1. discovered the dual origin of lymphoid cells in the chicken demonstrating that earlier thymectomy and bursectomy are essential to explain their role in the development of immune system (Fig. Gowans et al. Peterson and Good (1965) found a different maturation of the lymphoid cells in the bursa and thymus of the chicken. In 1958. only the thymus and the bursa have identifiable lymphoid tissue (Papermaster and Good 1962).

2 A portrait of Francis Albert Pierre Miller (1931-) Fig. Cooper (1933-) roles and just how they might function.3 A portrait of Max D. probably because of the fairly mature status of the immune system in newly hatched chicks. Defining the respective roles of the thymus and the bursa would thus require either removing one or the other early in embryonic life or removing them after hatching in conjunction with . 1. 1.1 Introduction 3 Fig. It proved difficult to show that early thymectomy affected either cellular of humoral immunity.

when it was realized in the hatched chickens followed by sublethal X irradiation. In response to the stimulation of a specific antigen. Moore and Owen (1965. are mimicked by bursectomy or thymectomy alone or combined (Peterson et al.4 An original model of Max D. IgG Stream ? .Specific Antibodies Lymphoid Bursa Plasma Cell Bursal Hormone Intestinal Lumen BURSAL SYSTEM DEVELOPMENT Fig. Bursectomy within the egg blocked antibody production (Perey and Good 1968) while. Otherwise. 1968) The most important human immunodeficiencies. b) proposed that the lymphocyte precursors were blood-borne of extrinsic origin which colonized the thymic and bursal rudiments at a precise stage of their ontogeny. according to instructive mechanism of antibody production (Fig. 1. IgM.Homografi Rejection . IgA. 1.” (Cooper 2015). undergo antigen-independent proliferation and differentiate into immunocompetent T and B lymphocytes. allowed the development of T and B cell systems in the peripheral lymphoid organs (Van Alten et al. . is responsible for the synthesis of a specific antibody. a specific gene. and severe combined immunodeficiency disease (SCID).5. and the connective tissues of the body. Di George syndrome. T and B cells differentiate into cytotoxic T lymphocytes and plasma cells. including Bruton’s X-linked agammaglobulinemia.6. the spleen. These reenter the bloodstream and populate the lymph nodes. 1967a.Graft vs Host Reactivity Blood Lymphoid Immunoglobulins. The clonal selection theory formulated by Sir Frank Macfarlane Burnet (Fig. stem cells arising from the yolk sac in the embryo and from the bone marrow in the adult.4 1 Introduction THYMUS SYSTEM DEVELOPMENT Pharyngeal Pouches 4th 3rd Parathyroid Glands Mesenchymal Inducer Thymus Hormone Precursors Myeloid Epithelial Thymus Bone Marrow Stem Cell ? PERIPHERAL LYMPHOID TISSUES Lymphoid Thymus Erythroid Lymphatic Recirculation Megakaryocyte Cellular Immunity . Under the influence of thymus and bursa or bursa equivalent. 1965). Cooper concerning the different development of thymus and bursal systems (Reproduced from Cooper et al.) (1959) sustained that an antigen is able to induce the proliferation and differentiation in plasma cells producing antibodies of only a clone of lymphocytes carrying the genes for the corresponding antibody (Ribatti 2009). Pauling 1940). 1968) the destruction of cells that have developed earlier under their influence.Delayed Allergy . 1. as a component of the genome of each immune cells. respectively.

1. 1. The majority of T and B cells have one type of glycoprotein on their surface that is required for them . by interacting with specific receptors expressed on the surface of endothelial cells of postcapillary venules (Fig.6 A portrait of Linus Pauling (1901–1994) The movement of stem cells from bone marrow to thymus and bursa and the subsequent seeding of lymphocytes to the peripheral lymphoid organs are measured in weeks. 1.7).1 Introduction 5 Fig. by which long-lived small lymphocytes rapidly move from blood to peripheral lymphoid organs and tissues and back into the blood. Superimposed upon this slow traffic is a second type of migratory phenomenon. called recirculation and measured in hours.5 A portrait of Sir Frank Macfarlane Burnet (1899–1885) Fig.

but they do not enter either the thymus or the bone marrow.and T-lymphocytes from blood to tissues and lymphopoietic organs and back into blood. The vast majority of the recirculating lymphocytes belongs to T variety. the remaining B lymphocytes. The purpose of recirculation is constant patrolling of immunocompetent lymphocytes throughout organism and informing lymphopoietic organs about presence or absence of antigens in body to recirculate through lymph nodes. . it can be shown that they moved rapidly from the bloodstream to the peripheral lymphoid organs.6 1 Introduction Fig. If thoracic duct lymphocytes are recovered. labeled radioactively in vitro. which collects most of the lymph of the body and returns it to the bloodstream. Recirculation has been demonstrated by experimental drainage of lymphocytes from a chronic fistula of the thoracic duct. and gut-associated lymphoid tissue. Prolonged drainage of the thoracic duct lymph causes pronounced lymphopenia and extreme depletion of the lymphocytes of the spleen. but leave them again to reenter the blood. and injected intravenously into a syngeneic recipient. lymph nodes. 1. while other homing receptors are involved in their localization in specific areas of secondary lymphoid organs.7 Lymphocyte recirculation is a fast migratory phenomenon of small B.

and do not possess the capacity for B.Chapter 2 Human Hematopoietic Development Keywords Hemangioblast • Yolk sac • Endothelial cells • Blood islands • Hematopoiesis • Vascular endothelial growth factor • Bone morphogenetic protein • Embryoid bodies • Liver • Bone marrow • Endosteal niche • Vascular niche • Sinusoidal cells • Osteoblasts 2. Ribatti. when they are exclusively comprised of erythrocytes expressing embryonic hemoglobin and to a lesser extent monocytes and macrophages (Oberlin et al. even when placed in culture conditions that permit lymphoid differentiation from definitive hematopoietic stem cells (HSCs) (Tavian et al. Marshall and Thrasher 2001).1 The Hemangioblast and the Yolk Sac The existence of the hemangioblast has been proposed for the first time by Sabin and Murray (Sabin 1920. Blood islands arise in the mouse from proximal mesodermal cells in the visceral yolk sac. but not vascular endothelium differentiation (Goss 1928). HSCs have been identified in the human yolk sac as early as day 18 of embryonic life. but die © Springer International Publishing Switzerland 2015 D. Vascular endothelial (VE)-cadherin-positive or CD34-positive CD45-negative endothelial cells. sorted from yolk sac and/or PAS/aorta-gonad-mesonephros (AGM) generate both hematopoietic and endothelial cells in vitro.1007/978-3-319-24663-5_2 7 . 2002. Murray 1932). Yokomizo et al. Runx1 mutant embryos undergo normal primitive yolk sac hematopoiesis. Vogeli et al. 2001). 1998. Wilt 1974). The Development of Immunologic Competence. Definitive hematopoiesis depends on the action of the transcription factor Runt domain factor x1 (Runx1). DOI 10. 2001). Cells constituting the outer layer of the blood islands assume a spindle shape and differentiate into endothelial cells (Shepard and Zon 2000). The first site of hematopoiesis is the yolk sac where mesodermal cells aggregate into clusters to form blood islands or hemangioblasts. consisting of an inner core of hematopoietic cells and an external layer of endothelial cells (Moore and Owen 1965. The removal of the central cells precludes blood formation. Moore and Metcalf 1970. 2006. therefore identifying these cells as a common precursor for both lineages (Nishikawa et al.or T-cell potential. Yolk sac progenitors consists predominantly of large nucleated primitive erythrocytes and primitive macrophages (Moore and Metcalf 1970).

Human leukocyte antigen-DR (HLA-DR) is absent or expressed at low levels on HSCs. In the early 1960s. The phenotypes CD34-positive. endothelial cell differentiation of the VEGFR-2-positive precursors was induced (Eichmann et al. using double staining with antibodies anti-CD45 and anti-VEGFR-2. In the absence of added vascular endothelial growth factor (VEGF). which are hematopoietic-progenitors. CD45 is a pan-hematopoietic marker that is absent from endothelium (Ledbetter and Herzenberg 1979). 2002). As hematopoietic cells bulge in the aortic lumen. while when clusters differentiate. VE-cadherin has been widely used as a marker for endothelium (Breier et al. 2000. VEGFR-2 was down-regulated and CD45 up-regulated in the ventral endothelium. In the presence of VEGF. 2. Drake and Fleming 2000). CD45-positive cells. but not both. c-kit low. colony forming units-spleen (CFU-S) (Till and Mc Culloch 1961). . De Bruijn et al.1) (Tavian et al. 2002). 1997). A VEGFR-2-positive cell would either differentiate to an endothelial cell or an hematopoietic cell. Immunohistochemical analysis revealed an extensive overlap in the expression of hematopoietic and endothelial markers in the clusters. Jaffredo et al. it was demonstrated that mouse hematopoietic tissues contained a class of cells. 1996).2 The Aorta-Gonad-Mesonephros Definitive hematopoiesis develops in the AGM region where CD34-positive cells with the capacity for full lymphoid and myeloid differentiation are first found in the human embryo (Fig. precluding a direct demonstration of the existence of a hemangioblast. and hematopoiesis disappears entirely in the AGM by day 40 (Oberlin et al. and a population of CD34-negative. 1998). CD31 and CD34 are expressed on endothelial cells as well as on HSCs in the embryo and adult (Wood et al. when human HSCs are generated as clusters of two or three cells arising from the endothelium on the ventral surface of the pre-umbilical region of the aorta.CD45-negative cells. 1996). but is present on fetal or neonatal HSCs (Moore et al. all cells became CD45-positive (Jaffredo et al. the VEGF receptor-2 (VEGFR-2)-positive. Runx1 expression and function might differentiate the primitive hemangioblast from the later definitive hemogenic endothelium (North et al. CD38-negative and CD45RAlow and CD71low contain >75 % HSCs. and erythroid elements. The HSCs of the AGM do not produce mature cells in situ. 2001. 2. but not the VEGFR-2-negative precursors differentiated to hematopoietic cells of different lineages. 1997. 1980).c-kit high. aortic endothelial cells were VEGFR-2-positive. instead they migrate and colonize the fetal liver. VEGFR-2-positive. The colonies originated from pluripotent cells and able to generate granulocytes.8 2 Human Hematopoietic Development between E11 and E12 because of failure of definitive hematopoiesis (Okuda et al. 2002). which resemble haemangioblast colonies. (2007) isolated and identified from murine AGM a population of CD34-positive. megacaryocytes. The AGM develops at day 27 of gestation in the human. Before cluster emergence. (1998) analyzed the characteristics of the cells lining the aortic lumen at the time of hematopoietic emergence. Marshall et al.

heart (H). 2007). suggesting that BMP-4 regulates c-kit expression and differentiation potential in CD34-positive cells. gonads and mesonephros (GM) (Reproduced from Mendes et al. 1985). 1995. 1995). 1998. Peault et al. which form colonies in the presence of VEGF . Legend: Yolk sac (YS). Gata-2 induction results in a sensitive increase in hemangioblast and endothelial cell generation (Lugus et al. Moser and Patterson 2005). 1987. VEGFR-2 and SCL/Tal-1 regulate cell fate decisions for the formation of endothelial and hematopoietic cells in early development (Chung et al. (b) AGM region. 2003). 2007). head. Ema et al. limb buds (Lb). liver (L). 2004). Miyazono et al. Shalaby et al. The development of hematopoietic and endothelial cells within embryoid bodies mimics in vivo events (Doetschman et al. Hematopoietic and endothelial lineages share expression of a number of different markers such as MB-1/QH-1 in the quail (Pardanaud et al. Some of these genes are essential for the development of both lineages (Robb et al. Young et al. Watt et al. umbilical (U) and vitelline (V) arteries. c-kit low cells (Marshall et al. 2007).2. 2005. 1997. Shivdasani et al. BMP-4 induces specific differentiation of VEGFR-2-positive mesodermal cells (Park et al. aorta-gonadmesonephros (AGM) region were dissected and cells isolated for further testing. 1994. somites. CD34. Embryoid bodies contain the blast colony-forming cells (BL-CFC). 2002. Kabrun et al. 1995). 1983) and CD31. Kallianpur et al. 2. stem cell leukemia (SCL)/Tal-1 and VEGFR-2 in the mouse (Gering et al.1 (a) Schematic drawing of a mouse embryo at E11.2 The Aorta-Gonad-Mesonephros 9 Fig. Moreover. VEGFR-2 and Scl (Lugus et al. Gata-2 is a direct target of BMP-4 and Gata-2 expression upregulates BMP-4. 1995. 1995. BMP signaling is crucial for hematopoietic and vascular development (Larsson and Karlsson 2005. 2005) Bone morphogenetic protein-4 (BMP-4) induces the expansion of the CD34positive. mesenchyme (M). Legend: aorta (A).

surface IgM-positive B cells are detectable (Dorshkind and Montecino-Rodriguez 2007. and mesenchymal stem cells. . 2000). chemoattractants. by 10–12 weeks. extracellular matrix components. 1998). The first cells to appear in the liver are macrophages. Epitheliocytes. with the appearance of cytoplasmic IgM-positive pre-B cells. Pluripotential HSCs appear to be generated along with the endothelium of the placental blood vessels and these cells appear in numbers large enough to account for the population of cells later found in the liver (Melchers 1979. 2004). resident macrophages.10 2 Human Hematopoietic Development (Choi et al. and several stromal cell populations of mesenchymal origin.4 The Bone Marrow From the end of the second trimester throughout adult life. They produce cytokines. VEGFR-2-positive cells displayed hemangioblast activity as demonstrated by their in vitro potential to form blast colonies (Huber et al. vascular smooth muscle and endothelial cells. Thymic colonization by fetal liver-derived progenitors and lymphocyte production begins at approximately week 9 (Hayward 1981). thereby providing for the functioning of the liver as a hematopoietic organ during a considerable period of prenatal development. and so forth and directly interact with hematopoietic cells. myofibroblasts. Hematopoiesis in the fetal liver disappears around 11 weeks of gestation (Tavian and Peault 2005). Cells within blast colonies express genes common to both hematopoietic and endothelial lineages. Solvason and Kearney 1992). followed by erythroid elements. Embryonic stem cell derived VEGFR-2-positive cells can also give rise to smooth muscle cells in the presence of platelet derived growth factor (PDGF) (Yamashita et al. while production of granulocytes and macrophages occurs in the vascular areas of portal triads. In the developing embryo. By day 30. Early development of erythrocytes occurs in hepatocyte niches. CD34. brachyury-positive. CD34-positive cells appear in the fetal liver and by day 32 these cells are able to maintain long term hematopoiesis in vitro (Tavian et al. 2. 1999a. and VEGFR-2 (Kennedy et al. The first B cells detectable in the human fetus are found in the fetal liver at approximately week 8 of gestation (Hayward 1981). Ottersbach and Dzierak 2005). bone marrow is the exclusive site of B-cell development (Gathings et al. b).3 The Fetal Liver and the Placenta Most progenitors disappear from the yolk sac and begin to appear in the fetal liver by 5 weeks. fibroblasts. including hepatic stellate cells. including SCL/Tal-1. 1997). contribute to hematopoiesis in fetal liver. 1977). 2.

there is an increase in marrow adipocytes and lipid content. Labeling techniques have indicated that new lymphocytes are formed at the periphery of the bone marrow and move toward the center in a centripetal fashion. undergo self-renewal and differentiate (Li and Xie 2005. Notch. The bone marrow is divided in two histologically distinct compartments: an extravascular compartment. The bone marrow is the major hematopoietic organ in humans and support differentiation of all blood cells (Weiss 1981). BMPs.4 The Bone Marrow 11 The final weave of hematopoietic development takes place in the fetal marrow. T lymphocytes and monocytes reach their final stages of maturation in locations outside the bone marrow. osteoclasts. Wnt. generating B. Yin and Li 2006) Osteoblasts and vascular niches are adjacent and intimately related establishing several interactions between hematopoietic and non-hematopoietic cells (Li and Neaves 2006. Quiescent HSCs reside in the endosteal niche. angiopoietin-1 (Ang-1). 2005). Hypoxic environment contributes to maintain HSCs in the endosteal niche in a quiescent state (Eliasson and Jonsson 2010) In vascular niche. Kopp et al. An inverse relationship exists between the number of marrow adipocytes and hematopoiesis. Rizo et al. Stem cell niches or bone marrow niches are specific sites where stem cells reside. and mesenchymal stem cells (Kopp et al. which is the site of hematopoiesis. However. 2003. cytokines and adhesion molecules (Carlesso and Cardoso 2010. 2. starting around 11 weeks of gestation and immature cells start to appear by 15 weeks and by 22 weeks marrow hematopoiesis is very active. and a vascular compartment. transforming growth factor beta (TGF-β). natural killer (NK). Bone marrow microenvironment is composed by HSCs (Krause 2002) and nonhematopoietic cells. platelets and erythrocytes between marrow and circulating pool. and smooth muscle cells create a microenvironment that recruits endothelial precursor cells. Calvi et al. and CD34-positive cells. These latter include endothelial cells. 2006. Osteoblast or endosteal niche and “vascular niche (Fig. 2000. and myeloid and erythroid lineages (Tavian and Peault 2005). T. 2003). macrophages. Identifiable lymphocytes are found singly or in small groups near the sinusoidal . 2003. mesenchyme stem cells and HSCs. through modulation of expression of growth factors. Zhang et al. pericytes. including N-cadherins. During period of decreased hematopoiesis.2. endothelial progenitor cells. 2005. Scadden 2006). Perry and Li 2007. The pre-hematopoietic stroma in the medullary spaces of bone consists of loose connective tissue attached to bone trabeculae with large sinusoids. they are a source of a variety of cytokines and chemokines that influence hematopoietic development and function. which behave functionally as true HSCs. and is important for stem cell recruitment (Abkowitz et al. endothelial cells. fibroblasts. reticular cells. Raaijmakers 2011).2) are important for HSCs differentiation (Wilson and Trumpp 2006). osteoblasts. and fibroblast growth factor-1 (FGF-1) (Faloon et al. where their interaction is mediated by several factor. pericytes. The sinusoidal endothelial cells regulate the traffic of leukocytes. Jagged-1. mast cells. Moreover. Moore and Lemischka 2006). integrins.

2.2 Interactions between HSCs and their endosteal and vascular niches (Reproduced from Levesque et al. 1993) and can modulate between CD34positive and CD34-negative states depending upon their level of activation. with lack of expression by deeply quiescent cells. 1999).12 2 Human Hematopoietic Development Fig. HSCs express c-kit/CD117 (Gunjii et al. with some of them in transit through the wall of the sinus (Osmond 1986). and up-regulation of CD34 as cells enter the proliferative pool (Sato et al. B lymphocytes acquire membrane Ig while are located extravascularly. 2010) walls. .

© Springer International Publishing Switzerland 2015 D.1007/978-3-319-24663-5_3 13 . 3.Chapter 3 The Bursa of Fabricius Keywords Bursa of Fabricius • Bursectomy • Irradiation • Thymus • Thymectomy • Adaptive immunity • T cells • Lymphocytes • Immunological tolerance • Microenvironment • Pharyngeal pouch • Mediastinum • Post-capillary venules • Hassall’s corpuscles • Thymus involution • Epithelial-reticular cells • Thymocytes • Dendritic cells • Autoimmune regulator gene • Lymphocytosis-stimulating-factor (LSF) • Thymosin 3. and practiced and taught Anatomy (Smith et al.2). 3. was professor of Surgery at the University of Padova. The bursa grows during development and changes its form from round to oval. The first appearance of the bursal anlage occurs approximately on day 5 of embryonic development (Hamilton 1952). The bursa reaches its maximum size at 8–10 weeks of age and by 6–7 months involutes (Ciriaco et al. Girolamo Fabrici or Fabrizio (Fig. 3. 2004). found among his lecture notes was published in 1621 (Fig. 3. and hypertrophy of the mesenchyme surrounding the epithelium originates longitudinal plicae that project into its lumen (Romanoff 1960). he built the first permanent theatre ever designed for public anatomical dissection (Fig. undergoing rapid proliferation.6). It contains the first description of the bursa (Adelman 1967) : “The third thing which should be noted in the podex is the double sac (bursa) which in its lower portion projects toward the pubic bone and appears visible to the observer as soon as the uterus already mentioned presents itself to view”. that form about 90 % and 10 % of the surface. from 1565 to 1613. 3.5). Ribatti. is invaded by stem cells of yolk sac or fetal liver origin. The bursa at first only epithelial. The surface epithelium consists of interfollicular epithelium (IFE) and follicle associated epithelium (FAE). In 1594. A manuscript entitled “De Formatione Ovi et Pulli”. Italy. The Development of Immunologic Competence. 2003).1) and Gabriel Fallopius (Fig. 3. DOI 10. The bursa may be described as a dorsal epithelial diverticulum of the proctadael region of the cloaca (Fig.1 The Discovery of the Bursa of Fabricius and Its Structure Student and successor of Andreas Vesalius (Fig.3). Fabricius is best known for his description of the bursa that bears his name.4).

3.1 A portrait of Andreas Vesalius (1514–1564) Fig. Olah and Glick 1978).2 A portrait of Gabriel Fallopius (1523–1562) respectively (Bockman and Cooper 1973. while FAE covers the bursal folds filled with follicles and provides a direct connection between the follicular . Epithelial cells lining the plicae extend into the lamina propria as epithelial buds.14 3 The Bursa of Fabricius Fig. 3.

3.000 to 12. Follicles are present during late embryonic development.3 A portrait of Hyeronimous Fabricius ad Acquapendente (1537– 1619) that hangs in Palazzo del Bo.000 follicles (each of which contains 1000 bursal cells) each composed of a cortex. after 16 days (Frazier 1974). . 3.4 The anatomical theatre in Padova. University of Padova Fig. 3.1 The Discovery of the Bursa of Fabricius and Its Structure 15 Fig. constructed in 1594 by Fabricius medulla and the bursal lumen (Fig. 3.7). The bursa has 8.

and medulla (Bockman and Cooper 1973. Fabricius. and the cortex is fully developed by . first cortical cells appearing around hatching (Olah et al. followed by the formation of the FAE on 14–15 day (Bockman and Cooper 1973). 3.16 3 The Bursa of Fabricius Fig.6 A drawing showing the anatomical position of the bursa of Fabricius cortico-medullary border.5 Chick embryos at different stages of development (Reproduced from H. 3. 1986). Medullary anlage emerges on the 11–12 day of incubation. 1621) Fig. De Formatione Ovi et Pulli. Glick 1983). Padua.

and is colonized by lymphoid precursors that expand and mature in the bursa before migrate to the periphery. the bursal epithelium overlying each follicle generates epithelial tufts (Ackerman and Knouff 1959) which transport the content of the bursal lumen into the lymphoid compartment. however the effect on B cells is dominant (Brand et al. lymphoid cells and macrophages with a few plasma cells in the involuting bursa. The other major change at hatching is the segregation of bursal follicles into cortical and medullary regions. At least. These cells are similar to the M-cells of mammalian appendix or Peyer’s patch (Bockman and Cooper 1973). Lymphopoiesis is active in the medulla of the bursal follicle (Ackerman and Knouff 1959. including dendritic cells. the bursa is colonized by B cell precursors undergone Ig gene rearrangement in the para-aortic foci and in the bone marrow (Ratcliffe and Jacobsen 1994). Medulla consists of epithelial cells and blood-borne hematopoietic cells. Ackerman 1962). 1975). 1976). Medullary B cells express surface IgM. 1986). Bursal extracts induce both B and T cell differentiation. The rearranged variable region. 2000). The bursa provides a unique microenvironment essential for proliferation and differentiation of B cells (Ratcliffe 2006). The first surface IgMpositive cells are detected from 12 incubation day and at hatching more than 90 % of bursal cells are mature B cells. B cell progenitors responsible for colonizing the bursa and forming the B cell lineage colonize the bursa from 8 to 14 incubation day (Le Douarin et al. only B cell precursors that positively rearrange the immunoglobulin gene are able to express cell surface immunoglobulin and expand in bursal follicles. At hatching.7 Microscopic organization of the bursa of Fabricius at low magnification two weeks after hatching. where immature B cells develop (Sayesh et al. 98 % of the lymphocytes are B-cells. while the major histocompatibility complex (MHC) class II antigen appears only on cortical B cells. Cells which fails to express surface antibodies are eliminated by apoptosis. 3. and result in an . undergoes somatic diversification. Hematopoietic colonization of the follicles occurs through the formation of dendro-epithelial tissue (Olah et al.3.1 The Discovery of the Bursa of Fabricius and Its Structure 17 Fig. and explains the movement of antigen from the lumen into the medulla. During embryonic development. and colonization of dendro-epithelial tissue by pre-B cells (Le Douarin et al. 1975).

2 Bursal Regulation of Antibody Production In 1954. These data were reinforced by a second experiment employing larger numbers and two different breeds of chickens (Chang et al. Timothy S. A differentiating hormone isolated from the bursa called bursin induces phenotypic differentiation of B cell precursors (Audhya et al. 1959) took advantage of the regressive influence of androgens on the post hatched bursa (Kirkpatrick and Andrews 1944. and none of the surviving produces antibody. Fig. 1991).18 3 The Bursa of Fabricius immunoglobulin repertoire of at least 1011 distinct antibody molecules (Mc Cormack et al. Glick 1957).8. the pullets were bursectomized at 12 day of age and injected with Salmonella typhimurium O antigen. All of these pullets had been bursectomized. 1986). Chang. At 7 weeks of age. 3. 3. Glick et al. The first experiments to evaluate the existence of a functional period for the bursa (Meyer et al. obtained several 6-month-old pullets from Glick for the purpose of injecting them with Salmonella-type O antigen to obtain serum with a high antibody titer for a class demonstration. 1955. a guaduate student of Bruce Glick (Fig.8 A portrait of Bruce Glick . 7/10 bursectomized birds and 2/10 controls failed to produce antibody (Glick 1955). and Glick concluded that the absence bursa was responsible of these results (Glick 1955). 1957). He performed two types of different experiments in which. 3. Several of the pullets died subsequently the immunization. 1956). Ribatti 2006b) at the Ohio State University. Bursectomy at 2 weeks was more effective in suppressing antibody production than at 5 or 10 weeks of age (Chang et al.

3. In a few birds.and IgG-producing cells. Romppanen and Sorvari 1980). and allowed allogenic spleen cells to are more effectively in antibody synthesis (Papermaster et al. Cooper et al. but foreign skin was rejected. In most of these chickens. and probably IgM. when specific goat antiserum against mu chains was tagged with fluorescin isothiocianate and specific antiserum against gamma chains was tagged with rhodamine. it was clear that the bursa was the first site to develop both . Finally. As Miller pointed out: “They inoculated chickens in ovo with testosterone to impair bursa development. 1962a. Chickens irradiated at hatching and subjected to bursectomy were unable to form circulating antibodies. While injection of bovine serum albumin (BSA) into chicks hatched from eggs injected with testosterone on day 5 of incubation determined complete antibody elimination.3 Regulation of the Synthesis of Antibodies 19 Different experimental conditions. developed normal peripheral small lymphocytes. 1966). 1960. 1964. (1966b). 1969. b. b. c).” (Miller 2002). followed by IgG on the 20th and than IgA (Cooper et al. 1959. cyclophosphamide administration (Lerman and Weidanz 1970. 1962). Both antibody production and delayed-type hypersensitivity were impaired. Kincade and Cooper 1971). The bursa is the first site where cells produce mu chains. Moreover. including testosterone and colchicines treatment (Meyer et al. are able to prevent antibody production and lymphoid development. Injection of an antibody anti-mu chain into the chick embryo at the moment of appearance of IgMstaining cells in the bursa prevents the development of both IgM. Mixture of cells from bone marrow and thymus together which antigen in irradiated mice produced far more antibody than when given antigen with either cell source alone (Claman et al. 1962a. the thymus and spleen developed normally but not the bursa. bursa was absent in 19-day embryos that had received testosterone prior to the 8 day of incubation (Warner and Burnet 1961). b). Eskola and Toivanen 1974). hormonal bursectomy enhanced graft versus host activity of injected homologous cells. Glick 1957. Warner and Burnet 1961. as demonstrated by means of immunofluorescence with antisera specific for mu and gamma chains (Cooper et al. in which they demonstrated that the precursors of the hemolysin-forming cells were derived not from either thymus or thoracic duct lymphocytes but from the bone marrow.3 Regulation of the Synthesis of Antibodies The B cell differentiates in the bursa and produces IgM on the 14th day of embryo development. rejected skin syngeneic grafts and showed normal graft versus host reactions. These birds failed to reject homografts. were sick and rarely survived more that a few weeks. Glick and Sadler 1961. 1966a. chicks from eggs injected on day 12 or 13 possessed reduced levels of antibody (Mueller et al. 3. Moreover. lymphoid atrophy had also involved the thymus. This evidence was confirmed by Miller and Mitchell (1967) in a study on the role of various cell types in reconstituting immune functions in immune-incompetent mice. Papermaster et al.

1972. CD24. Kincade and Cooper 1973). plasma cell generation and antibodies production (Cooper et al. indicating that while all B cells express IgM initially. or IgM Memory B cell: CD19. CD24.9). CD40 Pre-B cell: CD19+. We showed that bursectomy of chickens at different times during development interrupted this progression. many immunologists had shown that IgM antibodies are produced before IgG antibodies in antigeninduced responses and during ontogeny. CD40 IgG. More recently. 3. CD40 µ chain in cytoplasm Immature B cell: CD19+. Cooper has pointed out that: “By the late 1960s. These results could . CD40 IgM & IgD on surface Plasma cell: secrete IgG. The infusion of autologous bursal lymphocytes in these animals restored germinal center development.and IgG-producing lymphocytes. CD24+. CD40 IgM on surface Mature B cell: CD19. CD24+. and chicken bursectomized and irradiated at hatching fail to develop either IgM or IgG and cannot make antibodies. 1966a). they can switch to the production of other isotypes (Fig.9 B cell differentiation during the antibody response IgM. IgA.20 3 The Bursa of Fabricius Stem Cell: CD34+ Pro-B cell: CD24+. 3. IgA. IgE or IgM on surface Fig. The anti-mu inhibition of IgM B cells inhibited development of the IgG and IgA B cells (Lawton et al. Bursectomy at the end of embryonic development prevented development of a population of IgG-producing plasma cells. IgE.

and include Meckel’s diverticulum. b)’. Archer et al. Presumably. In favour of the possibility that a single lienage of B cells switches from IgM to IgG production. including gut-associated lympho-epithelial tissues (GALT) tissues (Cooper et al. 1962). our efforts proved largely futile. 3. However. Ablation of this organ in neonatal life resulted in a lifelong immunodeficiency (Perey and Good 1968. including GALT and bronchus-associated lymphoid tissue (BALT).” (Cooper 2015). 1966b) and the bone marrow in primates.3. although the class-switch mechanism was not elucidated until the recombinant DNA technology in the 1980s. but only when antibody administration was initiated at birth and not a week later. however. Burnet raised ‘the rather urgent question of whether there is a functional equivalent of the bursa in the mammal (Burnet 1962a. Different structures have been identified as bursa equivalents in mammals.and IgA-producing cells in mice.10). respiratory. embryonic treatment with IgMspecific antibodies prevented the development of IgG-producing cells.4 Mammalian “Bursa-Equivalent” Organs and the Role of Liver and Bone Marrow in Lymphopoiesis As Miller pointed out: “As early as 1962. These findings suggested that IgM-positive B cells give rise to B cells that produce other immunoglobulins classes. or by the capacity of a single lineage of B cells to switch from IgM to IgG production. My work had. 1962). As Good pointed out: “The bursa of Fabricius is present in all orders of birds. shown that neonatal thymectomy in the mouse was associated not only with defective cellular immunity but also with impaired antibody-producing capacity to certain antigens. develop within follicular out-pouching of the lower gut (Archer et al. reproductive and urinary tracts are collectively termed mucosa-associated lymphoid tissues (MALT). The GALT comprises lymphoid cells residing in epithelial lining and distributed in the underlying lamina propria as well as specialized lymphoid structures located at strategic sites along the gut. In our quest to find bursal-equivalents in burslaess vertebrates.4 Mammalian “Bursa-Equivalent” Organs and the Role of… 21 be explained either by there being separate lineages of B cells committed to making either IgM or IgG antibodies.” (Miller 2002). which is to feed into the body the cells whose descendants will produce antibody (Burnet 1962b)’. including humans. in the rabbit we recognized very early in the course of our . Peyers’s patches and coecal tonsils. 3. whereas the inhibitory effects of antibodies against IgG were class-specific. which later were known as thymusdependent antigens. Treatment with IgM-specific antibodies also inhibited the development of IgG. this work led Burnet to the view that in ‘mammals it is highly probable that the thymus also carries out the function performed by the bursa of Fabricius in the chicken. and mediate influences similar to those of the bursa on the humoral system (Knight and Crane 1994). The lymphoid tissues in the walls of the alimentary. The rabbit appendix and sacculus rotundus (intestinal tonsil) located at the ileocoecal valve (Fig.

Although neonatal thymectomy reduced the circulating lymphocyte count. When both the thymus and appendix (Sutherland and co-workers 1964) were removed in the neonatal period.11) may be special sites where antigen-driven proliferation can lead to great expansion of a B-cell population and to a switching of capacity to produce one kind of immunoglobulin. these animals recovered to near normal structure and function between 9 and 16 weeks after birth. they showed that the appendix-sacculus rotundus tissue of the rabbit functions quite similarly to the bursa of Fabricius of chickens in generating a normal and diverse antibody repertoire.” (Good 2002). we could produce an impressive immunodeficiency of antibody production that lasted through the lifetime of the rabbit (Archer et al. IgM or IgG. My original morphologic analysis of similarities of the development of the bursa of Fabricius of chicks and appendix-sacculus rotundus of rabbit also attracted the attention of Katherine Knight of Loyola University. 1962). 1962). to capacity to produce IgA immunoglobulin. DLT diffuse lymphoid tissue) studies that the origins and development of the appendix-sacculus rotundus. depleted lymphatic structure in spleen and lymph nodes. as in the bursa (Archer et al. . depletion of lymphocyte count and organized lymphoid structure was more profound. We also showed that if we took to extirpate all of the appendixsacculus rotundus immediately in the newborn rabbit. 1965). With several of her students. Knight was able to demonstrate that the bursa of chickens and the appendix-sacculus rotundus of rabbits mediate very similar influence on humoral immune system. 3. 1966a). Chicago (Knight and Crane 1994). and interfered with development of immunologic capacity in rabbits. and the deficiency thus induced persisted far longer (Archer et al. immunologic capacity was depressed more regularly and more completely then when either organ was removed alone. Peyer’s patches (Fig. Using molecular genetic approaches. Neonatal appendectomy followed by Peyer’s patch removal in combination with whole body irradiation to destroy pre-existing lymphocytes in rabbits induced immunological defects comparable to those observed in older chickens subjected to bursectomy and irradiation (Cooper et al. 3.10 Histological picture of the sacculus rotundus of rabbits (GC germinal center. develop within follicular outpouching of the lower intestinal tract.22 3 The Bursa of Fabricius Fig.

and like T cells. which may protrude into the lumen and also extend into the submucosa Immunoglobulin-bearing cells first appear in the liver during mouse embryogenesis and after their colonization with HSCs. B cells differentiate in the bone marrow. It is now clear that in mammals. 3. 1977).4 Mammalian “Bursa-Equivalent” Organs and the Role of… 23 Fig. . with the addition of conspicuous patches of lymphoid tissue called Peyers patches. 1976. also fetal long bones can also generate B cell ex vivo.3. 1974. B cells circulate and re-circulate.11 The mucosa of the ileum is typical of the small intestine. (Owen et al.

It does not cease to exist in later life no more than would the Anglo-Saxon race disappear were the British Isles to sink beneath the waves. and grafted with an unmarked thymus have shown that the original lymphocyte population of the grafted thymus is replaced by a new population of cells bearing the bone marrow karyotype (Feldman and Globerson 1964).1). or from the fancied resemblance of the thymus to the leaf of the plant Thymus vulgaris (Fig. and has there set up colonies for itself and for its increase. for increase. Ribatti.Chapter 4 The Thymus Keywords Bursa of Fabricius • Bursectomy • Irradiation • Thymus • Thymectomy • Adaptive immunity • T cells • Lymphocytes • Immunological tolerance • Microenvironment • Pharyngeal pouch • Mediastinum • Post-capillary venules • Hassall’s corpuscles • Thymus involution • Epithelial-reticular cells • Thymocytes • Dendritic cells • Autoimmune regulator gene • Lymphocytosis-stimulating-factor (LSF) • Thymosin 4. so the original leukocytes. Galen of Pergamum (130–200 AD) first described the morphology of the gland and noted that the thymus was largest during infancy. 1963). colonize the spleen and lymph nodes and constitute the immunologically competent cells of the lymphoid system (Auerbach 1961. This provides further evidence that the lymphocyte population of the thymus arises from immigration and differentiation of blood-borne © Springer International Publishing Switzerland 2015 D. starting from their birth place in the thymus. lymphocytes differentiate from the epithelial component of the thymus anlage.1 The Discovery of the Thymus and Its Function The word thymus is derived from a Greek word meaning the heart or soul. Experiments in animals which were thymectomized. irradiated. DOI 10. and have there created new centres for growth. John Beard suggested that: “the thymus must be regarded as the parent source of all the lymphoid structures of the body. 4.1007/978-3-319-24663-5_4 25 .” (Beard 1990). The Development of Immunologic Competence. and they migrate out. have penetrated into almost every part of the body. At the beginning of the twentieth century. “reconstituted” with bone marrow cells bearing a chromosomal marker. The immunological competence of the thymus was demonstrated by Billingham et al. For just as the Anglo-Saxon stock has made its way from its original home into all parts of the world. (1956) and by Gowans et al. and for useful work for themselves and for the body. (1962). During embryonic life.

and subsequently the thymic epithelial cells were surrounded by mesenchymal cells derived from the grafted neural crest (Le Douarin 1973a. In the quail-chick chimaeras the third and fourth endodermal pouches. possible under local inductive influences. b). arising in this instance from the bone marrow. Moreover. they differentiate into thymic lymphocytes. Once stem cells have migrated into the thymus. 4.2 Studies of the Thymus in the Chick and in the Mouse In birds. the potentiality of the endoderm to give rise to lymphocytes . if ventro-lateral part of the pharyngeal endoderm of the third and fourth branchial pouches is associated with an appropriate mesenchyme. thymic histogenesis proceeds. demonstrating that no intervention of ectoderm is required for thymus differentiation (Le Douarin 1967). 4.1 Thymus vulgaris is a species of flowering plant in the mint family Lamiaceae stem cell precursors.26 4 The Thymus Fig.

suggesting that an inflow of blood-borne stem cells is responsible for lymphoid differentiation in the chick thymus. 1957). TCR 1 and TCR2. developed a normal immune system. Implants of thymus tissue depleted of lymphocytes by irradiation stimulated lymphopoiesis. 1963). only low levels of chimaerism were found. Miller further investigated the effect of injecting lymphoid cells into neonatally thymectomized mice and found that: (i) Syngeneic thymus cells from 1-day-old mice injected intravenously to newborn mice immediately after thymectomy did not prevent immunological failure (Miller 1962b).and 30-somite quail embryo into the somatopleure of a chick (Le Douarin and Jotereau 1973). b) used a sex-chromosome marker system in paired chick embryos joined by vascular anastomoses of chorioallantoic or yolk-sac blood vessels.2 Studies of the Thymus in the Chick and in the Mouse 27 was tested by transplanting the third and fourth pharyngeal pouches endoderm of a 15. while TCR3 appears to be unique to birds. (iii) Allogeneic lymphoid cells from 2-month-old mice caused a severe graft-versus-host (GVH) reaction when injected intravenously into newborn mice immediately after thymectomy (Miller 1962a). Good investigated the possibility that the thymus was involved in adaptive immunity. 1963). Lymphocytes restored immune capabilities. 1964). whereas lymph node and muscle implants had no such effect (Grégoire and Duchateau 1956). Non-thymectomized mice recovered normal lymphoid functions. and they were given bone marrow cells.4. Mature T cells express either CT4 or CT8. . They demonstrated that chromosome analysis following yolk sac anastomosis at 4–5 days of incubation revealed high levels of chimaerism in the thymus. Different mammalian homologues of T cell surface markers have been identified in chickens. These results led the authors to formulate the “haematogenous theory of blood forming organ histogenesis”. As in mammals. while thymectomized mice did not (Miller 1962a. correspond to their mammalian counterparts. (ii) Syngeneic lymphoid cells from 8-week-old mice pre-sensitized against Ak skin conferred adoptive immunity. in chickens CT4 cells have helper functions and the cells expressing CT8 cells have cytotoxic activity. When the anastomosis was established later in the development. Neonatal thymectomized mice. The Ak skin was rejected within 12 days and the mice showed evidence of immunity to a second-set graft (Miller et al. Three sublineages of the CT3 positive cells have been recognized designated as TCR1. they were specifically tolerant of thymus-donor type skin only (Miller 1962b. Antigen receptors on chicken T cells appear as CT3/T cell receptor (TCR) complex. According to Moore and Owen. but only if the donor was syngeneic (Miller 1964). 1956. TCR2 and TCR3. total body irradiated. When grafted with foreign thymus tissue. and performed thymectomies on 4–5 week old rabbits. the source of the blood-borne stem cells that invade the primary lymphoid organs is located in the yolk sac and it would be attractive to consider the hypothesis of a single cell precursor of all blood cells of both erythroid and lymphocytic series. In this context. Miller predicted that recovery of immune functions following irradiation would be thymus-dependent. Adult mice were thymectomized . implanted with syngeneic thymus tissue soon after birth. without no demonstrable effects on the antibody response (Maclean et al. Moore and Owen (1967a.

The thymus and the parathyroid glands develop from epithelial anlagen of the third and fourth pharyngeal pouches (Fig. It develops from an ectodermal-endodermal juncture and its epithelial components contain derivative of both ectodermal and endodermal germ layers. invade the epithelial anlage and they move toward the subcapsular region and acquire T lineage commitment. The thymic mass gradually increases with colonization of blood-borne HSCs (Le Douarin and Jotereau 1975) and the rapid increase. spleen. During its development the thymus undergoes a descensus which brings it to lie in the anterior mediastinum.3 The Functional Anatomy of the Human Thymus Human thymus receives stem cells from the bone marrow and provides the microenvironment for them to develop in T cells. therefore. 4. a few days before hatching. Accordingly. the local cardiac neural crest mesenchyme controls the pattern and development of the gland. adhere posteriorly to the carotid sheath and extend into the retropharyngeal space (Ahsan et al. peripheral lymph nodes. as I believe. and gut. Ectopic thymus is usually located anteriorly and deep to the middle third of the sternocleidomastoid muscle. Ectopic thymus in both humans and mice reflect a failed migration of thymic tissue from third pharyngeal pouch endoderm during organogenesis.2). HSCs enter through postcapillary venules at the cortical-medullary junction. 4. in close connection with the pericardium and the great veins at the base of the heart. The lower border of the thymus reaches the level of the fourth costal cartilage. while superiorly. The thymus is the first lymphoid organ to develop followed in turn by the central lymph nodes. results in the appearance of the medulla. As the thymus proliferate and descends. . Macfarlane Burnet in a lecture in June 1962 at the University of London stated: “If. 2010). 4. which are released to begin a long life circulating and recirculating through blood and lymph. able to circulate in blood and lymph for many months in rodents and years in man (Miller and Osoba 1967).28 4 The Thymus This finding led Miller to postulate that “when one is inducing a state of immunological tolerance in a newly born animal. we must also endow it with another function. the thymus is the site where proliferation and differentiation of lymphocytes into clones with definable immunological functions occurs. Uncommitted hematopoietic progenitors. the elimination or inhibition of self-reactive clones” (Burnet 1962a).3). slowly moving through T-cell zones in peripheral lymphatic tissue (Fig. Defective development of cardiac neural crest also results in thymic deficiency as seen in the Di George syndrome. extensions into the neck are common reflecting the embryonic origin of the thymus. one is an effect performing a selective or immunological thymectomy” (Miller 1962b). where the endodermal epithelial masses fuse in the midline in the 12th week of embryonic life. Experiments combining the techniques of thymectomy and injection of marked thymus cells led to the conclusion that thymus-derived cells were small lymphocytes.

Vascularized mesenchyme transforms into connective septa that invade epithelial strands up to the medulla. Export ‘Promiscuous’ gene expression by medullary TECs SP mature Apoptosis by negative selection SP (CD4+ or CD8+) immature Cortical TEC DC Cyst Medullary TEC Macrophage Myoid cell Thymocyte Hassal’s body Neuroendocrine-like cell TRENDS in Immunology Fig. subdividing the cortical zone into lobules.J. Legend: thymic epithelial cells (TEC).M. SP single-positive thymocytes (Reproduced from Crivellato et al.4. 4. DP doublepositive thymocyte. surrounded by a basal lamina and a vascularized mesenchyme. During . T-lineage commitment Expansion.2 Structural and functional architecture of the thymus. loss of B and NK potential DN2 Apoptosis by neglect Positive selection by cortical TECs Expansion DN1 Entry Medulla C. dendritic cells (DC). Later.3 The Functional Anatomy of the Human Thymus 29 DN3 DP TCRα recombination Expansion TCRβ Cortex recombination. cortical epithelial cells begin to separate while cells of the medulla remain densely packed. 4.3 An overview of thymus development (Reproduced from Gordon and Manley 2011) At the beginning of development. T-cell receptor (TCR). DN double-negative thymocyte. but not completely subdividing the medullary zone. 2004) Fig. the thymus is a solid epithelial strand composed of densely packed epithelial cells.

T trabecula. This barrier is formed by the continuous blood capillaries in the thymic cortex. Only a small proportion of T cells is carried out from the thymus by the efferent lymphatic vessels. Legend: C cortical region. This increases to about 30–40 g. 4. Some continue through the cortex to join larger veins running in the capsule and so leave the thymus.5) (Lind et al. creating a specific microenvironment in which T cells develop into mature T cells (Kato and Schoefl 1989). At birth the thymus weighs 12–15 g.30 4 The Thymus Fig. LT thymic lobule further development. After this it begins to involute. 2001). The large medullary vessels are highly permeable to substances in the plasma and lymphocytes traverse the walls of the post-capillary venules of the corticomedullary junction and those of the medulla (Fig. the thymus is largest during embryonic life and in childhood up to the period of puberty. 1997) after which it begins to decrease in weight. In contrast to the cuboidal endothelium of post-capillary venules of the appendix. and lymph nodes. a process which proceeds gradually and continuously throughout life under normal condition. protecting T cells against foreign antigens. Most cortical capillaries loop around at different depths in the cortex and join venous vessels at the cortico-medullary junction. thymic lobules with their well-delimited cortex and medulla become packed tightly together (Fig. the endothelium of thymic post-capillary venules is flattened. Peyer’s patches. completely isolates the thymus cortex. and the epithelial cells. 4. capillary basal lamina. as a consequence of the blood-thymus barrier. 4. In relation to body weight. M medullary region.4 Microscopic organization of the fetal human thymus at a low magnification. The rate of thymic . at puberty (Hasselbalch et al. The blood-thymus barriers separates cortical T cells from the blood of cortical vessels. There is very little movement of macromolecules from blood to thymic parenchyma across the capillary walls in the cortex. so that at 60 years it weighs only 10–15 g (Linton and Dorshkind 2004).4). tonsils. basal lamina of epithelial cells.

and capsule (Fig. 4. the thymus remains a functional organ. Mast cells may be present in large numbers in aged thymuses. . 2005) growth in the child and involution in the adult is extremely variable.6). where they are largely confined to the inner medulla.3 The Functional Anatomy of the Human Thymus 31 Fig. 4.5 A post-capillary venule (V) at the cortico-medullary junction in human thymus Fig.6 An electron microscopic picture showing a mast cell surrounded by red blood cells and thymocytes in the thymic medulla (Reproduced for Crivellato et al. septae.4. Although there is a considerable age involution. and so it is difficult to determine weight appropriate for age (Levine and Rosal 1978). 4.

the cortex appears dark blue to purple because of the predominance of lymphocytes (80–85 %). Ultrastructural studies of these cells reveal evidence of their epithelial nature such as desmosomes. containing scattered islands of parenchyma consisting mainly of enlarged reticular cells. IL-6. and many other organelles found in epithelial cells. they swell. Types 2 and 3 create microenvironment niches in the outer cortex. cytoplasmic tonofilament. Six types of epithelial cells can be identified. Keratohyalin granules and numerous tonofilaments appear in central cells. degenerate. due to massive death of cortical small lymphocytes and their destruction by macrophages. Thymic epithelial-reticular cells are present in the cortex and in the medulla (Anderson and Jenkinson 2001). As the innermost cells gradually become distant from blood capillaries. increasing in number and size with age. the thymus rapidly diminishes in size. with distinct cortical and medullary compartments. IL-3. swelling of its nucleus. the thymus is transformed into a mass of adipose tissue (corpus adiposum thymi). and granulocyte-macrophage colony stimulating factor (GM-CSF)]. whereas the medulla appears clear (eosinophilic) because of the predominance of the epithelial cells. 4. This cell becomes surrounded by one or more other epithelial cells which are organized circumferentially and connected closely to one another by numerous desmosomes. Hassall’s corpuscles frequently measure 100 μm in diameter. called thymic nurse cell complexes (Brelinska and Warchol 1997.7). 1995). Under these conditions. Formation of Hassall’s corpuscles begins with degeneration of an epithelial cell. IL-2. The medulla contains also mature thymocytes and the thymic or Hassall’s corpuscles (Fig. In hematoxylin-eosin-stained sections. The gland displays a lobuled pattern. 4. chemokines . cytoplasm. The changes affect both the cortex and the medulla but are most pronounced in the cortex. Reike et al. and mitochondria. Thymic epithelial cells secrete cytokines [interleukin (IL)-1.7 An Hassall’s corpuscle (CH) in the medullary region of human thymus In adults.32 4 The Thymus Fig. Injections of glucocorticoids eliminates as much as 75 % of thymocytes within 2–3 days. and transform into keratinized and/or necrotic material which often calcifies.

2008). They have well developed processes spreading in the adjacent areas surrounding T cells. and small amounts of B cells. Maturation of T cells is accompanied by the sequential acquisition of the various T cell markers. 2003). In the cortical region are localized macrophages with flat shape and scanty cytoplasm. Most of these cells are rapidly dividing cortical thymocytes that are actively rearranging TCR genes. Thymic cortical dendritic macrophages have been described (Wakimoto et al. Dendritic cells are involved in shaping and maturating T cells by deleting self-reactive thymocytes to established central tolerance (Varas et al. characterized by loss of self-tolerance to multiple organs and abnormal structure of the thymic medulla (Ramsey et al. and localized in the medulla. adult and old humans (Varas et al. perivascular space and in the medulla.3 The Functional Anatomy of the Human Thymus 33 (Savino et al. containing apoptotic thymocytes. The existence of the Thy-1 antigen on T cells (Reif and Allen 1964) and the high density of surface Ig on B cells (Raff 1971) allowed to distinguish and separate T from B cells. but not by anti-F4/80 antibodies (Liu et al. The double negative cells give rise to the doublepositive cells localized in the cortex. Keratin-negative cells include fibroblasts. anti-F4/80 and anti-Mac-2 (Liu et al. 2002). Fibroblasts are found in the capsule. while only 3–5 % become fully competent T cells. It has been suggested that their contraction might aid the movement of lymphoid cells across or out of the thymus. but are infrequent in the cortex. and both cortex and medulla provide selective signals leading to cell survival or death (Sprent and Kishimoto 2002). Type 1. These cells become the CD4-positive or CD-8 positive cells. 2. Mutations in the AIRE gene are responsible for an autoimmune syndrome called APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy). 2003). 2013).4. The proportion of human thymic dendritic cells remain constant between postnatal. while all six types are localize in the medulla (Rezzani et al. non fibroblastic mesenchymal cells and endothelial cells. an actin binding protein. as well as some dendritic cell associated molecules. except in the involuted thymus. About 96 % of thymocytes become apoptotic. The correct expression of the product of the autoimmune regulator (AIRE) gene correlates with a normal organization of the medullary stroma (Zuklys et al. 2000). including fascin. Myoid cells are situated mainly in the medulla and at the cortico-medullary junction. under the guidance of contact and paracrine signals from the epithelium (Petrie 2002). Electron microscopic detection of Barbera granules in the cytoplasm of dendritic cells indicate that they express a Langherans cells like phenotype during human ontogeny (Valledeau et al. do not express CD4 or CD8 (double negative). 2000). They are stained by anti-Mac-2. including interdigitating dendritic cells. 3 and 4 thymic epithelial cells are localized in the cortex. 2002). indistinguishable from peripheral T cells and expressing high levels of TCR. express CD8 and MHC II molecules. 2008). are placed in all thymus regions and are positive for two antibodies. A massive rate of cell death (“apoptosis . Terminal deoxynucleotidyl transferase is found in pro-thymocytes and immature thymocytes but is absent in mature T cells (Hale 2004). 2013). The second thymic population is composed of thymocytes plus a variety of antigen presenting cells. and thymic hormones and neuropeptides (Mentlein and Kendall 2000). macrophages particularly at the cortico-medullary junction. The most immature cells in the thymic cortex.

beginning at 6 incubation day. 1962b. Dalmasso et al. that they had independently shown that neonatally thymectomized mice were somewhat immunodeficient but that. Good and co-workers in rabbits (Archer and Pierce 1961. E-cadherin is strongly expressed on epithelia cells as well as on the double negative thymocytes in mice. they admitted that the difference between our results on skin graft rejection may have been due to the fact that they had not completely thymectomized their mice. Klein and Kyewski 2000) affects the majority of the double-positive cells (McPhee et al. Later on. in contrast to my findings. publish their independent finding in later 1962. in February 1962. without providing data. Their mice rejected skin from H-2-incompatible strains. In the mouse. Cells deriving from the thymus are both short-lived and long-lived. It was disappointing and indeed surprising that this group. again emphasizing the ability of their thymic-deficient mice to reject H-2 incompatible grafts. They did. Three different experimental approach showed that neonatal thymectomy has a significant effect on immunologic reactivity: (i) The studies of Fichtelius et al. 1964). Papermaster et al. suggested that the thymus might participate in the control of antibody formation. who claimed ‘at long last’ to have ‘established the essential function of the thymus’ prior to the April 1961 meeting of the American Association of Immunologists.34 4 The Thymus by neglect”. (iii) The studies of Miller (Miller 1961a. 1962. Martinez et al. However. 1962). 1962a). b. E-cadherin is expressed only on epithelial cells (Kutlesa et al. 1962. a paper that was not on the thymus and in which the word thymus did not appear. emphasizing that mice thymectomized at birth failed to reject skin both from totally unrelated strains (H-2-incompatible) and from other species as rats. In the discussion that followed. Martinez from Good’s group claimed. Long-lived cells in man have a life-span upward of 5 years and perhaps over 10 years. in human thymus. There I gave my data in great detail. gave at this New York Meeting. Archer et al. 1962) and mice (Good et al. suggesting its participation in the interactions between these two cell types.” (Miller 2002). 1979). deficient transplantation immunity and delayed-type hypersensitivity (Waksman et al. however. In the mouse their life-span is more than 80 days (Everett et al. (1961) in young guinea pigs. 1963). until 18 incubation day (Coltey et al. immunologic depression is profound after thymectomy in neonatal animals. As Miller has remembered: “ The February 1962 New York Academy of Sciences Fifth Tissue Homotransplantation Conference was a unique opportunity to present results on the inability of neonatally thymectomized mice to reject foreign skin grafts. Good et al. prolonged skin graft survival occurred only in mice identical at the H-2 histocompatibility locus but differing at other weaker histocompatibility genes. 1962a. T cell progenitors colonize the epithelial thymus in three successive waves. resulting in considerable depression of antibody production. (ii) The experiments of Archer. 1989). […] It still seems likely that some . 2002). Good wrote that: “The simultaneous occurrence of acquired agammaglobulinemia and benign thymoma in a human being.4 The Effects of Neonatal Thymectomy When the thymus was removed from 3 to 7 incubation day quail or chick embryos and grafted into the somatopleure of the other species. 4.

and the fact that neonatal thymectomized mouse retained an immunologic reactivity suggests that the thymus influence on immunologic development may already have been excised before birth. After thymectomy in the newborn. A second case of acquired agammaglobulinemia with thymoma present itself and strengthens the conviction that the two phenomena are related in some essential manner” (Good 2002). In neonatal rodents. Moreover. Miller sustained that: “During embryogenesis the thymus would produce the originators of immunologically competent cells many of which would have migrated to other sites at about the time of birth. few germinal centres with low cellularity. The lymph nodes were smaller and displayed inactive follicles and poor cellularity. Neonatal thymectomy is responsible of gross deficiencies in the distribution of T lymphocytes.5 The Thymus Is Essential for Normal Development of the Immune System Thymectomy (or congenital athymia) results in severe immunological defects due to a deficiency of T cells. b). Once the periphery has become populated with T lymphocytes. Cells of spleen and lymph nodes extert a very low immunologic activity (Dalmasso et al. 1962b). 1963). and few mitoses (Miller 1961a. Moreover. These data indicate that in the mouse the thymus is the primary central lymphoid organ. thymectomized mice failed to reject skin from foreign mouse strains (Miller 1962b). The immune defects observed after neonatal thymectomy were confirmed by Arnason et al. Lymphoid tissues showed minimal development and circulating lymphocyte were greatly reduced. Neonatal thymectomized mice showed a marked deficiency of lymphocytes in the circulation and in the lymphoid tissues. (1962a. but the peripheral population of thymus-dependent lymphocytes has not yet been established. homografts may persist indefinitely instead of being rejected within a week or two. This would suggest that lymphocytes leaving the thymus are specially selected cells” (Miller 1962b). thymectomy is not longer followed by a dramatic deficiency of cell-mediated immune responses. At 6 weeks. Thymectomy in adults causes no such changes because the extrathymic lymphatic tissues and circulation are already stocked with T cells. Mice were vulnerable to homologous disease when injected with parent strain lymphoid cells (Parrott 1962). the thymus has completed its development.5 The Thymus Is Essential for Normal Development of the Immune System 35 essential relationship exist between the thymic tumor and the acquisition of an acquired agammaglobulinemia. 4. but rejected allogeneic grafts (Miller 1961b). if thymectomy is performed in the neonatal period before the thymus seeds peripheral lymphatic organs with T cells. the spleen was greatly reduced in size (Miller 1962a) and displayed inactive follicles.4. When mice were thymectomized after . Peyer’s patches were also smaller (Miller 1961a. antibody production against antigens that require cooperation of T cells and B cells is also impaired. (1962) and Martinez et al. 1962b).

bursectomy and irradiation had severe cellular and humoral immune system deficit (Cooper et al. the animal showed partial restoration of T cells and no immunological deficiencies. activate cyclic GMP or AMP. Factors appear to diffuse through the wrapping and largely substitute for the thymus. In the mind of Cooper: “The plan was to compare the immunological status of the different experimental groups. but non-dialyzable was demonstrated in the thymus (Metcalf 1956). which restores T-cell deficiencies in thymectomized mice.36 4 The Thymus birth. In these experiments I removed either the thymus or the bursa. plasma cells and the capacity to make antibodies yet they had perfectly normal development of thymocytes and lymphocytes elsewhere in the body that mediated cellular immune reactions (Cooper et al. when added to culture of thymic cells. When examined as young adults.” (Cooper 2002). b. Cooper removed either the thymus or the bursa from some newly hatched chicks. and induce mature T-cell functions. can induce the appearance of T-cell differentiation markers. and both from others. and waited several weeks until the animals recovered from the irradiation effects. after they recovered from the effects of surgery and irradiation. then subjected the newly hatched chicks to near lethal irradiation and waited several weeks until they and their irradiated controls recovered from the irradiated effects. 1963). but they still produced germinal centers. More recently. Thymosin has been demonstrated in thymic epithelial cells by immunohistochemistry and is secreted by them (Schulof et al. 1965. i. 1966a. 1966a. The best characterized of thymic humoral factors is thymosin. Thymectomized and irradiated animals were deficient in lymphocytes that mediated cellular immunity as assessed by skin graft rejection. delayed-type hypersensitivity and graft versus host assays.” (Cooper 2010).e. 4. 1965. He specified that: “I devised an alternative strategy that would combine post-hatching thymectomy or bursectomy together with whole body irradiation to destroy cells that might have seeded earlier from the thymus and bursa or that could have been influenced by postulated thymic and bursal humoral factors. 1965. b). If a thymus wrapped in a cell-tight filter was inserted into the peritoneal cavity of mouse thymectomized at birth. when their lymphoid system and the immune mechanisms had developed to a certain extent. A specific lymphocytosis-stimulating-factor (LSF) heat-labile and filterable. the birds that were irradiated and . 1966a. b). only negligible effects were observed. These factors. Cooper pointed out that: “we decided to combine whole-body irradiation with removal of the thymus and/or the bursa (or neither) immediately after hatching. plasma cells and circulating immunoglobulins (Cooper et al. Miller et al. and to destroy all peripheral lymphoid components.6 Removal of Either the Thymus or Bursa of Fabricius In a series of experiments. Bursectomized and irradiated birds were completely devoid of germinal centers. 1987). he subjected the chicks to intense x-irradiation. Birds subjected to combined thymectomy.

Conversely. Irradiated. These composite findings 50 years ago offered a clear view of separate thymus-dependent and bursa-dependent lineages of lymphocytes that mediate cellular and humoral immunity. .4. these experimental data indicate that at some point differentiation along two distinctly different pathways occurs within the lymphoid system and that the critical point seems to focus about two separate central lymphoid organs. birds that were irradiated and bursectomized at hatching resembled boys with X-linked agammaglobulinemia. bursectomized and thymectomized birds resembled infection-prone infants who had congenital agammaglobulinemia no lymphocytes and deficient cell-mediated and humoral immune responses. respectively. Overall. as indicated by deficiencies in delayed-type hypersensitivity. despite having an abundance of lymphocytes and normal –mediated immune responses.” (Cooper 2015). graft-versus-host reactivity and allograft rejection capability. germinal centre and plasma cell development could be restored in irradiated and bursectomized birds by reinfusion of their bursal cells. They were lymphopenic and had severely impaired cell-mediated immunity. in that they had no germinal centres or plasma cells and made no antibodies. Notably.6 Removal of Either the Thymus or Bursa of Fabricius 37 thymectomized at hatching resembled neonatally thymectomized mice. antibody responses to some antigens were also reduced even though immunoglobulin production and the development of germinal centres and of plasma cells seemed to be normal.

X linked agammaglobulinemia (XLA) there is a complete absence of plasma cells and blood B lymphocytes (Janeway et al. The most common organisms responsible for infection are Streptococcus pneumoniae and Haemophilus influenzae. 1956. deep cortical areas. Cooper et al. These patients have intact cell-mediated immunity. 1971. © Springer International Publishing Switzerland 2015 D. between 6 months and 2 years of life.Chapter 5 Clinical Correlates Keywords Immunodeficiencies • B cells • T cells • Bruton’s congenital agammaglobulinemia • X linked agammaglobulinemia • Immunoglobulins • Antibody • Di George syndrome • Hypoparathyroidism • Thymoma • Myasthenia gravis • Autoimmune diseases • Severe combined immunodeficiency • Ataxia-teleangestasia 5. The Development of Immunologic Competence.1007/978-3-319-24663-5_5 39 . In XLA. Initial symptoms consists of recurrent bacterial otitis media. and reject skin allografts. cytotoxic drugs. respond normally to viral infections. Ribatti. which are genetically determined. and B-cell lymphoproliferative disorders. Gitlin et al.1 Immunodeficiencies Immunodeficiencies include primary disorders. Odgen Bruton described a male child with hypogammaglobulinemia and this is recognized as the first clinical description of an immunodeficiency disorder (Bruton 1952. DOI 10. In Bruton’s congenital agammaglobulinemia the production of immune globulins is grossly depressed and there are few lymphoid follicles or plasma cells in lymph node biopsies. malnutrition. Good 1955. thymus is mormal. Patients with XLA remain asymptomatic until 9 months of age. X-rays. show abundant cellularity in normals and agammaglobulinemic patients following antigenic stimulation. and dermatitis. pneumonia. associated with viral infections. By contrast. Good and Zak 1956. and they are noted because of the susceptibility of infants to recurrent infections. In another primary immunodeficiency. The diagnosis of XLA is based in on the demonstration of absence or marked deficiency of all immunoglobulin classes. meningitis. 1952). and secondary forms. 1953. while in lymph nodes. paracotrex. bronchitis. Dent and Good 1965). Bruton et al. corticosteroids. Good and Varco 1955. deficiency of cells in cortical areas and absence of germinal centers are recognizable. Primary immunodeficiencies manifest themselves in infancy. In 1952. when passively transferred maternal immunoglobulins.

they often died from overwhelming infection with viruses. The thymus. fungal or protozoal agents.40 5 Clinical Correlates As Good pointed out: “Bruton’s initial reports were published in 1952. Morphology and the thymus-dependent lymphocytes were also normal in XLA patients and their functions appeared to be quite normal. 1979). Thus. often with congenital cardiac defects. We and others found that the patients lacked all of the subsequently described immunoglobulins. these patients exhibited striking absence of identifiable thymus. this organ was found almost normal. hypoparathyroidism. and these children lacked thymus-dependent lymphocytes in the blood . which proved to be genetically X-linked”. He described a patient for whom life was one severe life-threatening bacterial infection after another. Immediately after Bruton’s description of the association of immunodeficiency with agammaglobulinemia. However. antibody and Ig deficiency represent a rare disease described by Bruton. Parathyroid glands were also absent. the 22q11 deletion (Conley et al. congenital heart disease. at cardiac surgery. These patients’ lymph nodes showed characteristic morphology. Later on. We encountered a group with XLA who showed an extreme deficiency of gammaglobulins. but not in peripheral . fungi.2 Di George Syndrome The Di George syndrome described for the first time by Angelo Di George (Fig. 5. Patients who survive to neonatal period may then develop recurrent or chronic infections with various viral. when studies were made of the thymus in the XLA patients. Although these children frequently presented with hypocalcemic tetany. was quite normal in size and cellularity as in Bruton’s XLA. or atypical acid-fast bacteria. we found to be strikingly under populated. They lacked intact cell-mediated immunity and lacked normal lymphoid cell populations within the deep cortical regions. by contrast. These patients who could not make antibodies were found not to have plasma cells or germinal centers in their hematopoietic and lymphoid tissues. bacterial. As Good pointed out: “Di George and his colleague had seen a number of children born without a thymus or parathyroid glands. where germinal centers usually appear in abundance. The far-cortical areas. with consequent aplasia of the parathyroid and thymus glands. and cellular immunodeficiency. The most frequent clinical sign is hypocalcemia in the first 24 h of life. The syndrome may be associated with a genetic mutation. The complete syndrome consists of the following features: abnormal facies.1) is the result of interference with normal embryologic development of the third and fourth pharyngeal arches. 5. Janeways’s group in Boston and our group in Minneapolis launched studies on series of patients with X-linked agammaglobulinemia (XLA). (Good 2002). while in Di George patients. and have a high incidence of autoimmune diseases. Most of these agammaglobulinemic patients exhibited normal delayed-type hypersensitivity. they possessed circulating lymphocytes in normal numbers. and rejected primary as well as secondary skin allografts.

They possess germinal centers and plasma cells. These are the areas in which immunologically normal person develop B-cell germinal centers. In partial Di George syndrome. offered further support for the two-component concept of the immune system that we had constructed. of the lymph nodes. . 1970. This can result in permanent reconstitution of T cell immunity. which suggests that lymphocytic recognition mechanisms in these infants are intact.1 Angelo Di George (right) and Bob Shprintzen at the “Deletion 22q11” Meeting in Rome in 2002 cortical regions. these corrections have become apparent as early as 48 h after the transplants. The subcortical thymusdependent region exhibits moderate or severe depletion of lymphocytes and the lymphoid sheats of the spleen are also depleted of lymphocytes. 1972). Furthermore. but in the far cortical areas a lymphocyte population is more abundant. but had plasma cells. but fail to produce cellular immunity.2 Di George Syndrome 41 Fig. named Di George syndrome. Although they had normal immunoglobulins. fetal thymus transplant should be given as soon as possible following diagnosis (Cleveland et al. The thymus is almost completely lacking and grossly deficient.” (Good 1991). Patients with Di George syndrome develop all immunoglobulins well and form antibody to many antigens very well. Peyer’s patches and tonsils. patients may show impressive corrective immunological development. The paracortical or deep cortical regions of lymph nodes of patients with Di Gorge syndrome show very few lymphocytes.5. 5. 1968. Biggar et al. they could not make antibodies well and did not form germinal centers. The disease. but germinal centers are not completely developed in patients with di George syndrome. In the treatment of Di George syndrome. August et al.

some patients have deficient T cell immunity. 1957. stomatitis. Ramos 1956. 5. The weakness is often noted in the ocular muscles and manifested as diplopia or ptosis. resulting in dysphagia. and urinary infection. Pharyngeal and facial muscle weakness. 1956. 1956. In no instance the removal of the thymoma resulted in improvement of immunodeficiency. Myasthenia gravis is a chronic autoimmune disease of adults and presents as a diminution in power of repetitive contraction in certain voluntary muscles. 5. chronic diarrhea. About 10 % of individuals with myasthenia gravis have a thymoma and 50 % have medullar follicular hyperplasia. septicemia. Marked hypogammaglobulinemia is present and the antibody response following immunization may be abnormal. 1968). Martin et al. 5.3 Thymoma with Immunodeficiency Recurrent infection may be sign if thymoma (Fig. Many patients affected by thymoma have myasthenia gravis (the most common) and other autoimmune diseases. The overall prognosis is poor.42 5 Clinical Correlates Good recognized overlapping between Bruton’s agammaglobulinemia and Glick’s bursectomy and between Di George syndrome and Miller’s thymectomy. Lambie et al. Moreover. and death secondary to infection is common. Infection takes the form of pulmonary infection. As he has emphasized: “Our experiments with irradiated bursectomized chicken combined with our similarly extensive histopathologic studies of patients with primary immunodeficiency diseases provided a clear perspective for understanding several of these immunodeficiency diseases and also the entire lymphoid system in both chickens and humans” (Cooper et al. dysarthria. dermatitis.2) is associated with immunodeficiency (Maclean et al. and Fig.2 CT scan of a mediastinic thymoma . 1960). Gafni et al.

5.4 Severe Combined Immunodeficiencies and Ataxia-Teleangectasia


difficulty in chewing, commonly occur. Patients with thymoma lacked all the
immunoglobulins (Good 1954; Janeway et al. 1953; Gitlin et al. 1956; Bridges and
Good 1960).
Beneficial effects from thymectomy are seen in the majority of cases of patients
with myasthenia gravis. The response to corticosteroids and immunosuppressive
agents, such as cyclophosphamide, are encouraging.


Severe Combined Immunodeficiencies
and Ataxia-Teleangectasia

In 1950, Glanzmann and Riniker described for the first time the severe combined
immunodeficiency (SCID) named “essential lymphocytophthisis” in two infants
with lymphopenia, which died during the second year as a consequence of a long
series of infections (Peterson et al. 1965). In SCID, serum concentrations of immune
globulins are very low and the IgG may exhibit restricted heterogeneity. No antibody synthesis can be detected. Electron microscopy reveals that blood lymphocytes are mostly very immature forms resembling lymphoblasts, and that abnormal
granulation of eospinophils are recognizable. Transplants of thymus in SCID
patients improved T cell functions (Hong et al. 1976). In CID, a residual function of
T cell is still recognizable, while the clinical features are similar to those of SCID.
Ataxia-teleangestasia is a disease of children, caused by mutations in the ATM
gene, characterized by progressive neurologic impairment (progressive cerebellar
ataxia and choreoathetoid movements), oculo-cutaneous teleangectasia, accompanied by unusual susceptibility to bacterial and viral infections of the sinuses and
lungs in most of the patients. The disorder is progressive with both neurological
abnormalities and immunologic deficiencies, in both T and B cells, becoming more
severe with time (Peterson et al. 1964).
Another immunologic disease is the so-called Swiss type of lymphopenic agammaglobulinemia. Infants affected with this condition fail to develop any primordial
lymphoid tissue and have neither a thymus-dependent nor an immunoglobulinproducing system. The thymus in these babies is a small vestigial organ, plasma
cells are lacking, and there is no delayed hypersensitivity.
Defects of immune functions have long been regarded as factors in the etiology
of such autoimmune diseases as rheumatoid arthritis and systemic lupus erythmatous. Rheumatoid arthritis is often associated with agammaglobulinemia; it’s
incidence is about 30 times higher among patients with Bruton’s syndrome than
among general population, and it commonly occurs in primary acquired agammaglobulinemia, which most often develops in adult life. IgA deficiency, is associated
with a variety of autoimmune conditions, including rheumatoid arthritis, systemic
lupus erythematosus, pernicious anemia.




Clinical Correlates

The Role of Thymus and of Bursa Equivalent Organs
in the Development of Tumors

Different tumors, including carcinomas, Kaposi sarcoma, carcinoid tumors,
Hodgkin’s and non Hodgkin’s lymphomas, chronic myeloid leukemia, and multiple
myeloma, are associated with thymoma (Souadjian et al. 1974; Lindstrom et al.
1968; Skinnider et al. 1982; Knowles 1976; Nemoto et al. 1987; Gross 1951;
Stutman et al. 1967, 1968, 1969b; Vogler et al. 1978; Mc Endy et al. 1944; Miller
1960). Thymectomy in mice injected at birth with leukemic extracts prevented the
development of leukemia (Miller 1959a, 1961a), while implantation of syngeneic
thymus restored leukemia development (Miller 1959b).
As Miller has remembered: “In 1958, having recently graduated in Medicine
from the University of Sydney, I was awarded a Gaggin Fellowship which enabled
me to go to the Chester Beatty Research Institute in London to study for a Ph.
D. Degree. As space was at a premium, even in those days, I was sent to one of the
satellites of the Institute at a place called Pollards Wood in Chalfont St Giles, about
one hour by train from Greater London, and I was given a shack in which to work
and a little mouse space. For the Ph. D. Degree I had decided to study the pathogenesis of mouse lymphocytic leukemia.” (Miller 2002). “I was attracted by the work
of Dr. J.C. Harris who was investigating tumours produced by the Rous sarcoma
virus in chickens. (…) Harris suggested that I might study the pathogenesis of
mouse leukaemia, as a leukaemogenic virus had recently been discovered by
Ludwik Gross. I was delighted to take up this challenge. As adult thymectomy had
been shown by several investigators to prevent spontaneous mouse leukaemia developing in high leukaemic strains and leukaemia induced in low-leukaemic strains by
ionizing radiation and chemical carcinogens, my immediate plan was to determine
whether it would also prevent the disease in virus-inoculated mice.(…) I followed
Gross’s procedure, which was to inoculate mice of a low leukaemic strain with filtered extracts of leukaemic tissues obtained from high-leukaemic strain mice. The
extracts had to be given to newborn mice, for otherwise leukaemia would not
develop. I thymectomized these mice a few weeks after weaning and none developed leukaemia. Thymus implantation six months after adult thymectomy restored
the potential for leukaemogenesis in neonatally inoculated mice. Hence the virus
must have remained latent and I went on to show that it was extractable from the
nonleukaemic tissues of thymectomized mice. Could it multiply outside thymus
tissue? Since, however, it had to be given at birth, this could be determined only by
thymectomizing the mice before the virus was inoculated and therefore immediately after birth. After many attempts, I finally worked out the technique of thymectomizing newborn mice. Immediate surgical mortality was low but the rate of
cannibalism by the mothers was high and I had to thymectomize large numbers of
baby mice.(…) The survivors fared well until sometime after weaning when many
died prematurely, whether inoculated with virus or not. As adult thymectomy had
never been associated with any untoward effect, I formulated the bold conclusion
‘that the thymus at birth may be essential to life’. Further investigations showed

5.5 The Role of Thymus and of Bursa Equivalent Organs…


clearly that mice thymectomized at one day of age, but not later than a few days,
were highly susceptible to infections, had a marked deficiency of lymphocytes in
the circulation and in the lymphoid tissues and failed to reject foreign skin grafts.
Since circulating lymphocytes were known to be immunologically competent and
skin graft rejection had been shown by Medawar and colleagues to be the result of
a lymphocyte-mediated immune response, it seemed to me logical to postulate that
‘during embryogenesis the thymus would produce the originators of immunologically competent cells, many of which would have migrated to other sites at about the
time of birth. This would suggest that lymphocytes leaving the thymus are specially
selected cells’. In this very first publication on the immunological function of the
thymus, which appeared in 1961 in The Lancet, I therefore held the unorthodox
conclusion that the thymus was the site responsible for the development of immunologically competent cells.” (Miller 1994).

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7. 28. 20. 21–23. 4. 21–23. 27. 27. 36–37 Bursectomy. 17. 21. 33–37. 43 Blood islands. 33 Di George syndrome. 43 Autoimmune regulator (AIRE) gene. 11 Hypoparathyroidism. 22. 29. 25. 5. 16. 13 Follicles. 11. 43. 32 Hemangioblasts. 11 Endothelial cell. 27. 35–37. 4. 17. 41 Immunodeficiencies. 36. 32 © Springer International Publishing Switzerland 2015 D. 8. 17. 7–10 Hematopoiesis. 27. 10. 4–6. 21. DOI 10. 11 Gut-associated lympho-epithelial tissue. 23. 42. 13–18. 21 Bruton’s congenital agammaglobulinemia. 30. 36. 36. 4. 9 Endosteal niche. 18–20. 19. 30. 11. 19. 28 Irradiation. 17. 10. 45 Lymphocytosis-stimulating-factor (LSF). 4–6. 17. 27 M Mediastinum. 17. 1. 41 Immunological tolerance. 8–10. 27. 34 Clonal selection theory. 19. 21 H Hassall’s corpuscles. 39–40. 12.Index A Adaptive immunity. 7 Bone marrow. 40. 4. 21. 12. 7–11. 2. 28 Bone morphogenetic protein. 17. 22. 1–3. 40–42 E Embryoid bodies. 4. 42–43 Immunoglobulins. 9 Bronchus-associated lymphoid tissue. 19–21. 27 Antibodies. 22. 10. 5. 36 L Liver. 8. 25–28. 1. 2. 32 F Follicle associated epithelium. 39 Bursa of Fabricius. The Development of Immunologic Competence. 33 Epithelial-reticular cell. 42 C Chick embryo. 43 Autoimmune diseases. 13. 39–41. 39 G Growth factor. 40 I Immune system. 33. 22. 4. 40–43 Ataxia-teleangestasia. 11. 8. 33 B B cells. 40. 28 Microenvironment. 13–15. 1. Ribatti. 35 Lymphopoiesis.1007/978-3-319-24663-5 59 . 35. 2. 4 D Dendritic cell. 35. 23. 21–23 Lymphocytes. 22. 28. 17–22.

4. 29. 6 X X linked agammaglobulinemia. 2–4. 27. 7. 23. 22 Severe combined immunodeficiency. 34–36. 12 Y Yolk sac. 17. 20. 13. 34. 30. 12 R Recirculation. 1. 10. 30–32 V Vascular endothelial. 2. 43–45 Thymus involution. 39 S Sacculus rotundus. 21. 9. 41–43 Thymectomies. 22. 39–41. 17. 21 Myasthenia gravis. 4–6. 19. 27. 22. 27 . 11. 36. 42. 28 Plasma cells. 30. 4. 31–33. 39–41. 23 Pharyngeal pouches. 28. 33–36. 43 Post-capillary venules. 5. 11. 43 O Osteoblasts. 1. 36 Thymoma. 7. 31 Index T T cells. 8 Vascular niches.60 Mucosa-associated lymphoid tissue. 4. 27. 43 Sinusoidal cells. 21. 25–37. 36 Thymus. 42–44 Thymosin. 11 P Peyers patches. 42–44 Thymocytes.