The Development of Immunologic Competence

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Domenico Ribatti

The

Developmentof Immunologic

Competence


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Domenico Ribatti

The Development ofImmunologic Competence


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ISBN 978-3-319-24661-1 ISBN 978-3-319-24663-5 (eBook)DOI 10.1007/978-3-319-24663-5

Library of Congress Control Number: 2015956545

Springer Cham Heidelberg New York Dordrecht London

© Springer International Publishing Switzerland 2015This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part ofthe material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,broadcasting, reproduction on microfilms or in any other physical way, and transmission or informationstorage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodologynow known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publicationdoes not imply, even in the absence of a specific statement, that such names are exempt from the relevantprotective laws and regulations and therefore free for general use.The publisher, the authors and the editors are safe to assume that the advice and information in this bookare believed to be true and accurate at the date of publication. Neither the publisher nor the authors or theeditors give a warranty, express or implied, with respect to the material contained herein or for any errors

or omissions that may have been made.

Printed on acid-free paper

Springer International Publishing AG Switzerland is part of Springer Science+Business Media ( www.springer.com)

Domenico RibattiDepartment of Basic Medical Sciences,

Neurosciences, and Sensory OrgansUniversity of Bari Medical School

Bari, Italy

http://www.springer.com/http://www.springer.com/http://www.springer.com/http://www.springer.com/http://www.springer.com/


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Contents

1 Introduction ................................................................................................. 1

2 Human Hematopoietic Development ........................................................ 7

2.1 The Hemangioblast and the Yolk Sac .................................................. 7

2.2 The Aorta-Gonad-Mesonephros........................................................... 8

2.3 The Fetal Liver and the Placenta .......................................................... 10

2.4 The Bone Marrow ................................................................................ 11

3 The Bursa of Fabricius ............................................................................... 13

3.1 The Discovery of the Bursa of Fabricius and Its Structure .................. 133.2 Bursal Regulation of Antibody Production .......................................... 18

3.3 Regulation of the Synthesis of Antibodies ........................................... 19

3.4 Mammalian “Bursa-Equivalent” Organs

and the Role of Liver and Bone Marrow in Lymphopoiesis ................ 21

4 The Thymus ................................................................................................. 25

4.1 The Discovery of the Thymus and Its Function ................................... 25

4.2 Studies of the Thymus in the Chick and in the Mouse ........................ 26

4.3 The Functional Anatomy of the Human Thymus................................. 28

4.4 The Effects of Neonatal Thymectomy ................................................. 34

4.5 The Thymus Is Essential for Normal Development

of the Immune System ......................................................................... 35

4.6 Removal of Either the Thymus or Bursa of Fabricius ......................... 36

5 Clinical Correlates ...................................................................................... 39

5.1 Immunodeficiencies ............................................................................. 39

5.2 Di George Syndrome ........................................................................... 40

5.3 Thymoma with Immunodeficiency ...................................................... 42


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5.4 Severe Combined Immunodeficiencies

and Ataxia-Teleangectasia.................................................................... 43

5.5 The Role of Thymus and of Bursa Equivalent

Organs in the Development of Tumors ................................................ 44

References......................................................................................................... 47

Index................................................................................................................... 59

Contents


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ALL Acute lymphoblastic leukemia

Ang-1 Angiopoietin-1

AGM Aorta-gonad-mesonephros

APECED Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy

AIRE Autoimmune regulator

BCG Bacillus Calmette-Guerin

BL-CFC Blast colony-forming cell

BMP Bone morphogenetic protein

BSA Bovine serum albuminBALT Bronchus-associated lymphoid tissue

BFU-E Burst-forming units-erythroid

CFU-S Colony-forming units-spleen

FGF-1 Fibroblast growth factor-1

FAE Follicle-associated epithelium

GVH Graft-versus-host

GM-CSF Granulocyte-macrophage colony-stimulating factor

GALT Gut-associated lympho-epithelial tissues

HSCs Hematopoietic stem cellsHLA-DR Human leukocyte antigen-DR

IFE Interfollicular epithelium

IL Interleukin

LSF Lymphocytosis-stimulating factor

MHC Major histocompatibility complex

MALT Mucosa-associated lymphoid tissues

NK Natural killer

PDGF Platelet-derived growth factor

Runx1 Runt domain factor x1SCID Severe combined immunodeficiency disease

SRBC Sheep red blood cells

SCL Stem cell leukemia

TCR T-cell receptor

Abbreviations


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TGF-β Transforming growth factor beta

VE Vascular endothelial

VEGF Vascular endothelial growth factor

VEGFR-2 Vascular endothelial growth factor receptor-2

XLA X-linked agammaglobulinemia

Abbreviations


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1© Springer International Publishing Switzerland 2015

D. Ribatti, The Development of Immunologic Competence,

DOI 10.1007/978-3-319-24663-5_1

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, thymus-dependent and thymus-

independent, in different animal species, including amphibian, birds, and

mammals.

The thymus-dependent system developed at a relatively early stage in the evolu-

tion of vertebrate forms. The first faint trace seems to have appeared some 400 mil-

lion years ago among primitive marine vertebrates and perhaps among some of the

invertebrates as well as. The lamprey possesses the most primitive known thymus,

not a single organ but a number of scattered foci of 5–20 lymphoid cells, and a

primitive spleen.A system involved in the synthesis of immunoglobulins arose about 250 million

years ago in the higher sharks and paddle fish. These species show the first well

defined plasma cells in the spleen, pericardial tissue, kidney, and gonad; they also

produce gamma globulins. In all of the placoderm-derived vertebrates the basic

structure of immunoglobulins includes a composite polypeptide chain structure

based on both high- and low molecular weight (heavy and light) polypeptide chains.

Another major step forward among amphibian was the appearance of plasma

cells in intestinal tract. The latest stages of this phylogenetic process are represented

by the development of bursal like tissues, and true lymph nodes. The distinction inthymus and bursal systems in the chick was firstly suggested by Szenberg and

Warner (1962). Bruce Glick and co-workers (Glick et al. 1956) demonstrated that

the bursa is involved in the antibody production, and that in bursectomized chickens

the synthesis of antibodies is suppressed (Glick and Whatley 1967).

In 1962, in a meeting organized by Robert A. Good (Fig. 1.1 , Ribatti 2006a) and

Ann E. 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).

In the chicken embryo, the thymus is the first lymphoid organ to develop. Theepithelial component is evident before the 9th day of incubation, and the thymus is

a fully developed lymphoid organ by the 12th day of incubation. Between the 12th


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and the 14th day, budding of the epithelial fold of the bursa is observed, and on the

14th day the lymphoid structures begin to develop by direct transformation of epi-

thelial cells to lymphoid cells. By the 18th day the bursa has a well-organized lym-

phoid structure. Three days before hatching, only the thymus and the bursa have

identifiable lymphoid tissue (Papermaster and Good 1962).

The crucial period during which thymus and bursa influence immunologic devel-

opment occurs during early life, when the lymphoid tissue is forming and immuno-

logic capacity is maturing (Miller 1962c; Good 1955; Good et al. 1964; Gowans

et al. 1961).

Peterson and Good (1965) found a different maturation of the lymphoid cells in

the bursa and thymus of the chicken. By the time of hatching, the thymic cells had

matured to a population with the largest number of lymphoid cells with small vol-

ume. By contrast, the bursal population, even as late as two or three months after

hatching, contained few such small cells but had a preponderance of cells with

higher volume.

In 1958, Francis Albert Pierre Miller (Fig. 1.2 , Ribatti et al. 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.

Observations on the changes in the lymphoid organs after bursectomy and thy-

mectomy in chickens have indicated the possible existence of two almost com-

pletely separate lymphocytopoietic systems. Good and Max D. Cooper (Fig. 1.3 .,

Ribatti 2014) demonstrated that thymus was involved in both the development of

cellular immunity and antibody production in chickens (Cooper et al. 1965, 1966a;

Metcalf 1960; Parrott et al. 1966; Stutman et al. 1969a). In 1964, Cooper as post-

doctoral fellow in the laboratory of Good, 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. 1.4 ). More recently, Cooper has pointed out that: “Chickens offered an animal model

in which to test the possibility of alternative lymphocyte lineages, although it was

unclear at the time whether the thymus and the bursa lead synergistic or independent

Fig. 1.1 A portrait of

Robert A. Good (1922–

2003) with two patients

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roles and just how they might function. It proved difficult to show that early thymec-tomy affected either cellular of humoral immunity, probably because of the fairly

mature status of the immune system in newly hatched chicks. Defining the respec-

tive 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


Francis Albert Pierre

Miller (1931-)

Fig. 1.3 A portrait of MaxD. Cooper (1933-)

1 Introduction


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the destruction of cells that have developed earlier under their influence.” (Cooper

2015).

Bursectomy within the egg blocked antibody production (Perey and Good 1968)

while, when it was realized in the hatched chickens followed by sublethal X irradia-tion, allowed the development of T and B cell systems in the peripheral lymphoid

organs (Van Alten et al. 1968) The most important human immunodeficiencies,

including Bruton’s X-linked agammaglobulinemia, Di George syndrome, and

severe combined immunodeficiency disease (SCID), are mimicked by bursectomy

or thymectomy alone or combined (Peterson et al. 1965).

The clonal selection theory formulated by Sir Frank Macfarlane Burnet (Fig.

1.5 .) (1959) sustained that an antigen is able to induce the proliferation and differ-

entiation in plasma cells producing antibodies of only a clone of lymphocytes car-

rying the genes for the corresponding antibody (Ribatti 2009). Otherwise, accordingto instructive mechanism of antibody production (Fig. 1.6 , Pauling 1940), a specific

gene, as a component of the genome of each immune cells, is responsible for the

synthesis of a specific antibody.

Moore and Owen (1965, 1967a, b) proposed that the lymphocyte precursors

were blood-borne of extrinsic origin which colonized the thymic and bursal rudi-

ments at a precise stage of their ontogeny. Under the influence of thymus and bursa

or bursa equivalent, stem cells arising from the yolk sac in the embryo and from the

bone marrow in the adult, undergo antigen-independent proliferation and differenti-

ate into immunocompetent T and B lymphocytes, respectively. These reenter thebloodstream and populate the lymph nodes, the spleen, and the connective tissues of

the body. In response to the stimulation of a specific antigen, T and B cells differen-

tiate into cytotoxic T lymphocytes and plasma cells.

THYMUS SYSTEM DEVELOPMENT

BURSAL SYSTEM DEVELOPMENT

PERIPHERAL LYMPHOID TISSUES

Thymus Hormone

Cellular Immunity

Immunoglobulins, IgM, IgA, IgG

- Homografi Rejection

- Delayed Allergy- Graft vs Host Reactivity

- Specific Antibodies

Plasma

Cell

Bursal Hormone

Intestinal Lumen

Lymphoid Bursa

Stream

Blood

Lymphatic

Recirculation

Lymphoid Thymus

Mesenchymal

Inducer

Lymphoid

? ?

Megakaryocyte

Erythroid

Bone

Marrow

Stem

Cell

Precursors

Parathyroid

Glands

Pharyngeal

EpithelialThymus

Pouches

3rd 4th

Myeloid

Fig. 1.4 An original model of Max D. Cooper concerning the different development of thymus

and bursal systems (Reproduced from Cooper et al. 1968)

1 Introduction


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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. Superimposed upon this slow traffic is a second type of migratory phe-

nomenon, called recirculation and measured in hours, by which long-lived smalllymphocytes rapidly move from blood to peripheral lymphoid organs and tissues

and back into the blood, by interacting with specific receptors expressed on the

surface of endothelial cells of postcapillary venules (Fig. 1.7 ). The majority of T

and B cells have one type of glycoprotein on their surface that is required for them


Sir Frank Macfarlane

Burnet (1899–1885)


Linus Pauling (1901–1994)

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to recirculate through lymph nodes, while other homing receptors are involved in

their localization in specific areas of secondary lymphoid organs.

Recirculation has been demonstrated by experimental drainage of lymphocytes

from a chronic fistula of the thoracic duct, which collects most of the lymph of the

body and returns it to the bloodstream. Prolonged drainage of the thoracic duct

lymph causes pronounced lymphopenia and extreme depletion of the lymphocytes

of the spleen, lymph nodes, and gut-associated lymphoid tissue. If thoracic duct

lymphocytes are recovered, labeled radioactively in vitro , and injected intravenously

into a syngeneic recipient, it can be shown that they moved rapidly from the blood-stream to the peripheral lymphoid organs, but leave them again to reenter the blood,

but they do not enter either the thymus or the bone marrow. The vast majority of the

recirculating lymphocytes belongs to T variety, the remaining B lymphocytes.

Fig. 1.7 Lymphocyte recirculation is a fast migratory phenomenon of small B- and T-lymphocytes

from blood to tissues and lymphopoietic organs and back into blood. The purpose of recirculation

is constant patrolling of immunocompetent lymphocytes throughout organism and informing lym-

phopoietic organs about presence or absence of antigens in body

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DOI 10.1007/978-3-319-24663-5_2

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

2.1 The Hemangioblast and the Yolk Sac

The existence of the hemangioblast has been proposed for the first time by Sabin

and Murray (Sabin 1920; Murray 1932), The first site of hematopoiesis is the yolk

sac where mesodermal cells aggregate into clusters to form blood islands or heman-

gioblasts, consisting of an inner core of hematopoietic cells and an external layer ofendothelial cells (Moore and Owen 1965; Moore and Metcalf 1970; Vogeli et al.

2006; Marshall and Thrasher 2001). The removal of the central cells precludes

blood formation, but not vascular endothelium differentiation (Goss 1928).

Yolk sac progenitors consists predominantly of large nucleated primitive eryth-

rocytes and primitive macrophages (Moore and Metcalf 1970), and do not possess

the capacity for B- or T-cell potential, even when placed in culture conditions that

permit lymphoid differentiation from definitive hematopoietic stem cells (HSCs)

(Tavian et al. 2001). HSCs have been identified in the human yolk sac as early as

day 18 of embryonic life, when they are exclusively comprised of erythrocytes

expressing embryonic hemoglobin and to a lesser extent monocytes and macro-

phages (Oberlin et al. 2002; Wilt 1974).

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 , therefore identi-

fying these cells as a common precursor for both lineages (Nishikawa et al. 1998;

Yokomizo et al. 2001).

Blood islands arise in the mouse from proximal mesodermal cells in the visceral

yolk sac. Cells constituting the outer layer of the blood islands assume a spindle shape

and differentiate into endothelial cells (Shepard and Zon 2000). Definitive hematopoi-

esis depends on the action of the transcription factor Runt domain factor x1 (Runx1).

Runx1 mutant embryos undergo normal primitive yolk sac hematopoiesis, but die


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between E11 and E12 because of failure of definitive hematopoiesis (Okuda et al.

1996). Runx1 expression and function might differentiate the primitive hemangioblast

from the later definitive hemogenic endothelium (North et al. 2002).

In the early 1960s, it was demonstrated that mouse hematopoietic tissues con-

tained a class of cells, colony forming units-spleen (CFU-S) (Till and Mc Culloch

1961). The colonies originated from pluripotent cells and able to generate granulo-

cytes, megacaryocytes, and erythroid elements. The phenotypes CD34-positive,

CD38-negative and CD45RAlow and CD71low contain >75 % HSCs. Human leuko-

cyte antigen-DR (HLA-DR) is absent or expressed at low levels on HSCs, but is

present on fetal or neonatal HSCs (Moore et al. 1980).

2.2 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. 2.1 ) (Tavian et al. 2001; De Bruijn et al. 2000, 2002). The

AGM develops at day 27 of gestation in the human, when human HSCs are gener-

ated as clusters of two or three cells arising from the endothelium on the ventral

surface of the pre-umbilical region of the aorta. The HSCs of the AGM do not pro-

duce mature cells in situ ; instead they migrate and colonize the fetal liver, and

hematopoiesis disappears entirely in the AGM by day 40 (Oberlin et al. 2002). Immunohistochemical analysis revealed an extensive overlap in the expression

of hematopoietic and endothelial markers in the clusters. VE-cadherin has been

widely used as a marker for endothelium (Breier et al. 1996). CD31 and CD34 are

expressed on endothelial cells as well as on HSCs in the embryo and adult (Wood

et al. 1997; Drake and Fleming 2000). CD45 is a pan-hematopoietic marker that is

absent from endothelium (Ledbetter and Herzenberg 1979).

In the absence of added vascular endothelial growth factor (VEGF), the VEGF

receptor-2 (VEGFR-2)-positive, but not the VEGFR-2-negative precursors differen-

tiated to hematopoietic cells of different lineages. In the presence of VEGF, endo-thelial cell differentiation of the VEGFR-2-positive precursors was induced

(Eichmann et al. 1997), A VEGFR-2-positive cell would either differentiate to an

endothelial cell or an hematopoietic cell, but not both, precluding a direct demon-

stration of the existence of a hemangioblast.

Jaffredo et al. (1998) analyzed the characteristics of the cells lining the aortic

lumen at the time of hematopoietic emergence, using double staining with antibodies

anti-CD45 and anti-VEGFR-2. Before cluster emergence, aortic endothelial cells

were VEGFR-2-positive, while when clusters differentiate, VEGFR-2 was down-reg-

ulated and CD45 up-regulated in the ventral endothelium. As hematopoietic cellsbulge in the aortic lumen, all cells became CD45-positive (Jaffredo et al. 1998).

Marshall et al. (2007) isolated and identified from murine AGM a population of

CD34-positive,c-kit high, CD45-positive cells, which are hematopoietic-progenitors,

and a population of CD34-negative, c-kit low, VEGFR-2-positive,CD45-negative

cells, which resemble haemangioblast colonies.

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Bone morphogenetic protein-4 (BMP-4) induces the expansion of the CD34-

positive, c-kit low cells (Marshall et al. 2007), suggesting that BMP-4 regulates

c-kit expression and differentiation potential in CD34-positive cells. BMP signaling

is crucial for hematopoietic and vascular development (Larsson and Karlsson 2005;

Miyazono et al. 2005; Moser and Patterson 2005). BMP-4 induces specific differen-

tiation of VEGFR-2-positive mesodermal cells (Park et al. 2004). Gata-2 is a direct

target of BMP-4 and Gata-2 expression upregulates BMP-4, VEGFR-2 and Scl(Lugus et al. 2007). Moreover, Gata-2 induction results in a sensitive increase in

hemangioblast and endothelial cell generation (Lugus et al. 2007).

Hematopoietic and endothelial lineages share expression of a number of differ-

ent markers such as MB-1/QH-1 in the quail (Pardanaud et al. 1987; Peault et al.

1983) and CD31, CD34, stem cell leukemia (SCL)/Tal-1 and VEGFR-2 in the

mouse (Gering et al. 1998; Kallianpur et al. 1994; Kabrun et al. 1997; Watt et al.

1995; Young et al. 1995). Some of these genes are essential for the development of

both lineages (Robb et al. 1995; Shalaby et al. 1995; Shivdasani et al. 1995).

VEGFR-2 and SCL/Tal-1 regulate cell fate decisions for the formation of endothelialand hematopoietic cells in early development (Chung et al. 2002; Ema et al. 2003).

The development of hematopoietic and endothelial cells within embryoid bodies

mimics in vivo events (Doetschman et al. 1985). Embryoid bodies contain the blast

colony-forming cells (BL-CFC), which form colonies in the presence of VEGF

Fig. 2.1 (a ) Schematic drawing of a mouse embryo at E11. Legend: Yolk sac (YS ), head, somites,

limb buds ( Lb ), heart ( H ), liver ( L ), umbilical (U ) and vitelline (V ) arteries, aorta-gonad-

mesonephros ( AGM ) region were dissected and cells isolated for further testing. (b ) AGM region.

Legend: aorta ( A ), mesenchyme ( M ), gonads and mesonephros (GM ) (Reproduced from Mendes

et al. 2005)

2.2 The Aorta-Gonad-Mesonephros


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(Choi et al. 1998). Cells within blast colonies express genes common to both hema-

topoietic and endothelial lineages, including SCL/Tal-1, CD34, and VEGFR-2

(Kennedy et al. 1997). Embryonic stem cell derived VEGFR-2-positive cells can

also give rise to smooth muscle cells in the presence of platelet derived growth fac-

tor (PDGF) (Yamashita et al. 2000).

In the developing embryo, brachyury-positive, VEGFR-2-positive cells dis-

played hemangioblast activity as demonstrated by their in vitro potential to form

blast colonies (Huber et al. 2004).

2.3 The Fetal Liver and the Placenta

Most progenitors disappear from the yolk sac and begin to appear in the fetal liverby 5 weeks. The first cells to appear in the liver are macrophages, followed by ery-

throid elements. Early development of erythrocytes occurs in hepatocyte niches,

while production of granulocytes and macrophages occurs in the vascular areas of

portal triads.

By day 30, 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. 1999a, b).

Hematopoiesis in the fetal liver disappears around 11 weeks of gestation (Tavian

and Peault 2005). The first B cells detectable in the human fetus are found in the

fetal liver at approximately week 8 of gestation (Hayward 1981), with the appear-ance of cytoplasmic IgM-positive pre-B cells; by 10–12 weeks, surface IgM-positive

B cells are detectable (Dorshkind and Montecino-Rodriguez 2007; Solvason and

Kearney 1992).

Epitheliocytes, resident macrophages, and several stromal cell populations of

mesenchymal origin, including hepatic stellate cells, fibroblasts, myofibroblasts,

vascular smooth muscle and endothelial cells, and mesenchymal stem cells, contrib-

ute to hematopoiesis in fetal liver. They produce cytokines, chemoattractants, extra-

cellular matrix components, and so forth and directly interact with hematopoietic

cells, thereby providing for the functioning of the liver as a hematopoietic organduring a considerable period of prenatal development.

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; Ottersbach and

Dzierak 2005).

2.4 The Bone Marrow

From the end of the second trimester throughout adult life, bone marrow is the

exclusive site of B-cell development (Gathings et al. 1977). Thymic colonization by

fetal liver-derived progenitors and lymphocyte production begins at approximately

week 9 (Hayward 1981).



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The final weave of hematopoietic development takes place in the fetal marrow,

starting around 11 weeks of gestation and immature cells start to appear by 15

weeks and by 22 weeks marrow hematopoiesis is very active. The pre-hematopoietic

stroma in the medullary spaces of bone consists of loose connective tissue attached

to bone trabeculae with large sinusoids, reticular cells, and CD34-positive cells,

which behave functionally as true HSCs, generating B, T, natural killer (NK), and

myeloid and erythroid lineages (Tavian and Peault 2005).

The bone marrow is the major hematopoietic organ in humans and support dif-

ferentiation of all blood cells (Weiss 1981). However, T lymphocytes and mono-

cytes reach their final stages of maturation in locations outside the bone marrow.

The bone marrow is divided in two histologically distinct compartments: an extra-

vascular compartment, which is the site of hematopoiesis, and a vascular

compartment.

The sinusoidal endothelial cells regulate the traffic of leukocytes, platelets anderythrocytes between marrow and circulating pool. Moreover, they are a source of a

variety of cytokines and chemokines that influence hematopoietic development and

function. An inverse relationship exists between the number of marrow adipocytes

and hematopoiesis. During period of decreased hematopoiesis, there is an increase

in marrow adipocytes and lipid content.

Bone marrow microenvironment is composed by HSCs (Krause 2002) and non-

hematopoietic cells. These latter include endothelial cells, endothelial progenitor

cells, pericytes, fibroblasts, osteoblasts, osteoclasts, mast cells, macrophages, and

mesenchymal stem cells (Kopp et al. 2005).Stem cell niches or bone marrow niches are specific sites where stem cells reside,

undergo self-renewal and differentiate (Li and Xie 2005; Scadden 2006). Osteoblast

or endosteal niche and “vascular niche (Fig. 2.2 ) are important for HSCs differentia-

tion (Wilson and Trumpp 2006).

Quiescent HSCs reside in the endosteal niche, where their interaction is medi-

ated by several factor, including N-cadherins, integrins, Jagged-1, Notch, BMPs,

transforming growth factor beta (TGF-β), angiopoietin-1 (Ang-1), Wnt, and fibro-

blast growth factor-1 (FGF-1) (Faloon et al. 2000; Calvi et al. 2003; Rizo et al.

2006; Zhang et al. 2003). Hypoxic environment contributes to maintain HSCs in theendosteal niche in a quiescent state (Eliasson and Jonsson 2010)

In vascular niche, endothelial cells, pericytes, and smooth muscle cells create a

microenvironment that recruits endothelial precursor cells, mesenchyme stem cells

and HSCs, and is important for stem cell recruitment (Abkowitz et al. 2003; Kopp

et al. 2005; Yin and Li 2006) Osteoblasts and vascular niches are adjacent and inti-

mately related establishing several interactions between hematopoietic and

non-hematopoietic cells (Li and Neaves 2006; Moore and Lemischka 2006), through

modulation of expression of growth factors, cytokines and adhesion molecules

(Carlesso and Cardoso 2010; Perry and Li 2007; Raaijmakers 2011).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.

Identifiable lymphocytes are found singly or in small groups near the sinusoidal

2.4 The Bone Marrow


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walls, with some of them in transit through the wall of the sinus (Osmond 1986). B

lymphocytes acquire membrane Ig while are located extravascularly.

HSCs express c-kit/CD117 (Gunjii et al. 1993) and can modulate between CD34-

positive and CD34-negative states depending upon their level of activation, withlack of expression by deeply quiescent cells, and up-regulation of CD34 as cells

enter the proliferative pool (Sato et al. 1999).

Fig. 2.2 Interactions between HSCs and their endosteal and vascular niches (Reproduced from

Levesque et al. 2010)



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DOI 10.1007/978-3-319-24663-5_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

3.1 The Discovery of the Bursa of Fabricius and Its

Structure

Student and successor of Andreas Vesalius (Fig. 3.1 ) and Gabriel Fallopius (Fig.3.2 ), Girolamo Fabrici or Fabrizio (Fig. 3.3 ), was professor of Surgery at the

University of Padova, Italy, from 1565 to 1613, and practiced and taught Anatomy

(Smith et al. 2004). In 1594, he built the first permanent theatre ever designed for

public anatomical dissection (Fig. 3.4 ). Fabricius is best known for his description

of the bursa that bears his name. A manuscript entitled “ De Formatione Ovi et

Pulli ”, found among his lecture notes was published in 1621 (Fig. 3.5 ). 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 uterusalready mentioned presents itself to view”.

The bursa may be described as a dorsal epithelial diverticulum of the proctadael

region of the cloaca (Fig. 3.6 ). The first appearance of the bursal anlage occurs

approximately on day 5 of embryonic development (Hamilton 1952). The bursa

grows during development and changes its form from round to oval, and hypertro-

phy of the mesenchyme surrounding the epithelium originates longitudinal plicae

that project into its lumen (Romanoff 1960). The bursa at first only epithelial, is

invaded by stem cells of yolk sac or fetal liver origin, undergoing rapid prolifera-

tion. The bursa reaches its maximum size at 8–10 weeks of age and by 6–7 monthsinvolutes (Ciriaco et al. 2003).

The surface epithelium consists of interfollicular epithelium (IFE) and follicle

associated epithelium (FAE), that form about 90 % and 10 % of the surface,


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14

respectively (Bockman and Cooper 1973; Olah and Glick 1978). Epithelial cells lining

the plicae extend into the lamina propria as epithelial buds, while FAE covers the bursal

folds filled with follicles and provides a direct connection between the follicular


Andreas Vesalius

(1514–1564)


Gabriel Fallopius

(1523–1562)

3 The Bursa of Fabricius


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15

medulla and the bursal lumen (Fig. 3.7 ). Follicles are present during late embryonic

development, after 16 days (Frazier 1974). The bursa has 8,000 to 12,000 follicles

(each of which contains 1000 bursal cells) each composed of a cortex,


Hyeronimous Fabricius ad

Acquapendente (1537–

1619) that hangs in

Palazzo del Bo, University

of Padova

Fig. 3.4 The anatomical theatre in Padova, constructed in 1594 by Fabricius

3.1 The Discovery of the Bursa of Fabricius and Its Structure


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16

cortico-medullary border, and medulla (Bockman and Cooper 1973; Glick 1983).

Medullary anlage emerges on the 11–12 day of incubation, followed by the forma-

tion of the FAE on 14–15 day (Bockman and Cooper 1973); first cortical cells

appearing around hatching (Olah et al. 1986), and the cortex is fully developed by

Fig. 3.5 Chick embryos at

different stages of

development (Reproduced

from H. Fabricius, De

Formatione Ovi et Pulli ,

Padua, 1621)

Fig. 3.6 A drawing showing the anatomical position of the bursa of Fabricius



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17

two weeks after hatching. Medulla consists of epithelial cells and blood-borne

hematopoietic cells, including dendritic cells, lymphoid cells and macrophages with

a few plasma cells in the involuting bursa. Hematopoietic colonization of the folli-

cles occurs through the formation of dendro-epithelial tissue (Olah et al. 1986); and

colonization of dendro-epithelial tissue by pre-B cells (Le Douarin et al. 1975).

At hatching, 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. These cells are similar to the M-cells of mamma-

lian appendix or Peyer’s patch (Bockman and Cooper 1973), and explains the move-

ment of antigen from the lumen into the medulla, where immature B cells develop

(Sayesh et al. 2000). The other major change at hatching is the segregation of bursal

follicles into cortical and medullary regions.

At least, 98 % of the lymphocytes are B-cells. Lymphopoiesis is active in the

medulla of the bursal follicle (Ackerman and Knouff 1959; Ackerman 1962).

Medullary B cells express surface IgM, while the major histocompatibility complex

(MHC) class II antigen appears only on cortical B cells. The first surface IgM-

positive cells are detected from 12 incubation day and at hatching more than 90 %

of bursal cells are mature B cells. During embryonic development, the bursa is colo-nized by B cell precursors undergone Ig gene rearrangement in the para-aortic foci

and in the bone marrow (Ratcliffe and Jacobsen 1994).

The bursa provides a unique microenvironment essential for proliferation and

differentiation of B cells (Ratcliffe 2006), and is colonized by lymphoid precursors

that expand and mature in the bursa before migrate to the periphery. B cell progeni-

tors responsible for colonizing the bursa and forming the B cell lineage colonize the

bursa from 8 to 14 incubation day (Le Douarin et al. 1975). Bursal extracts induce

both B and T cell differentiation, however the effect on B cells is dominant (Brand

et al. 1976). Cells which fails to express surface antibodies are eliminated by apop-tosis; only B cell precursors that positively rearrange the immunoglobulin gene are

able to express cell surface immunoglobulin and expand in bursal follicles. The

rearranged variable region, undergoes somatic diversification, and result in an

Fig. 3.7 Microscopic organization of the bursa of Fabricius at low magnification

3.1 The Discovery of the Bursa of Fabricius and Its Structure


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18

immunoglobulin repertoire of at least 1011 distinct antibody molecules (Mc Cormack

et al. 1991). A differentiating hormone isolated from the bursa called bursin induces

phenotypic differentiation of B cell precursors (Audhya et al. 1986).

3.2 Bursal Regulation of Antibody Production

In 1954, Timothy S. Chang, a guaduate student of Bruce Glick (Fig. 3.8 , Ribatti

2006b) at the Ohio State University, 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. Several of the pullets

died subsequently the immunization, and none of the surviving produces antibody.

All of these pullets had been bursectomized, and Glick concluded that the absencebursa was responsible of these results (Glick 1955).

He performed two types of different experiments in which, the pullets were bur-

sectomized at 12 day of age and injected with Salmonella typhimurium O antigen.

At 7 weeks of age, 7/10 bursectomized birds and 2/10 controls failed to produce

antibody (Glick 1955). These data were reinforced by a second experiment employ-

ing larger numbers and two different breeds of chickens (Chang et al. 1955; Glick

et al. 1956). Bursectomy at 2 weeks was more effective in suppressing antibody

production than at 5 or 10 weeks of age (Chang et al. 1957).

The first experiments to evaluate the existence of a functional period for thebursa (Meyer et al. 1959) took advantage of the regressive influence of androgens

on the post hatched bursa (Kirkpatrick and Andrews 1944; Glick 1957).


Bruce Glick



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Different experimental conditions, including testosterone and colchicines treat-

ment (Meyer et al. 1959; Glick 1957, 1964; Glick and Sadler 1961; Warner and

Burnet 1961; Papermaster et al. 1962a, b; Romppanen and Sorvari 1980), cyclo-

phosphamide administration (Lerman and Weidanz 1970; Eskola and Toivanen

1974), are able to prevent antibody production and lymphoid development. While

injection of bovine serum albumin (BSA) into chicks hatched from eggs injected

with testosterone on day 5 of incubation determined complete antibody elimination,

chicks from eggs injected on day 12 or 13 possessed reduced levels of antibody

(Mueller et al. 1960, 1962). Moreover, bursa was absent in 19-day embryos that had

received testosterone prior to the 8 day of incubation (Warner and Burnet 1961).

Finally, hormonal bursectomy enhanced graft versus host activity of injected

homologous cells, and allowed allogenic spleen cells to are more effectively in anti-

body synthesis (Papermaster et al. 1962a, b).

As Miller pointed out: “They inoculated chickens in ovo with testosterone toimpair bursa development. In most of these chickens, the thymus and spleen devel-

oped normally but not the bursa. Both antibody production and delayed-type hyper-

sensitivity were impaired, but foreign skin was rejected. In a few birds, lymphoid

atrophy had also involved the thymus. These birds failed to reject homografts, were

sick and rarely survived more that a few weeks.” (Miller 2002).

Chickens irradiated at hatching and subjected to bursectomy were unable to form

circulating antibodies, developed normal peripheral small lymphocytes, rejected

skin syngeneic grafts and showed normal graft versus host reactions. Cooper et al.

(1966b).Mixture of cells from bone marrow and thymus together which antigen in irradi-

ated mice produced far more antibody than when given antigen with either cell

source alone (Claman et al. 1966). 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, in which they demonstrated that the pre-

cursors of the hemolysin-forming cells were derived not from either thymus or tho-

racic duct lymphocytes but from the bone marrow.

3.3 Regulation of the Synthesis of Antibodies

The B cell differentiates in the bursa and produces IgM on the 14th day of embryo

development, followed by IgG on the 20th and than IgA (Cooper et al. 1969;

Kincade and Cooper 1971). The bursa is the first site where cells produce mu chains,

and probably IgM, as demonstrated by means of immunofluorescence with antisera

specific for mu and gamma chains (Cooper et al. 1966a, b, c). Injection of an anti-

body anti-mu chain into the chick embryo at the moment of appearance of IgM-staining cells in the bursa prevents the development of both IgM- and IgG-producing

cells. Moreover, 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

3.3 Regulation of the Synthesis of Antibodies


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IgM- and IgG-producing lymphocytes. Bursectomy at the end of embryonic devel-opment prevented development of a population of IgG-producing plasma cells, and

chicken bursectomized and irradiated at hatching fail to develop either IgM or IgG

and cannot make antibodies. The infusion of autologous bursal lymphocytes in

these animals restored germinal center development, plasma cell generation and

antibodies production (Cooper et al. 1966a).

The anti-mu inhibition of IgM B cells inhibited development of the IgG and IgA

B cells (Lawton et al. 1972; Kincade and Cooper 1973), indicating that while all B

cells express IgM initially, they can switch to the production of other isotypes

(Fig. 3.9 ). More recently, Cooper has pointed out that: “By the late 1960s, many immunolo-

gists had shown that IgM antibodies are produced before IgG antibodies in antigen-

induced responses and during ontogeny. We showed that bursectomy of chickens at

different times during development interrupted this progression. These results could

Stem Cell:CD34+

Pro-B cell:CD24+, CD40

Pre-B cell:CD19+, CD24+, CD40

µ chain in cytoplasm

Immature B cell:CD19+, CD24+, CD40

IgM on surface

Mature B cell:CD19, CD24, CD40

IgM & IgD on surfacePlasma cell:secrete IgG, IgA, IgE,

or IgM

Memory B cell:CD19, CD24, CD40

IgG, IgA, IgE or IgM

on surface

Fig. 3.9 B cell differentiation during the antibody response



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be explained either by there being separate lineages of B cells committed to making

either IgM or IgG antibodies, or by the capacity of a single lineage of B cells to

switch from IgM to IgG production. In favour of the possibility that a single lienage

of B cells switches from IgM to IgG production, embryonic treatment with IgM-

specific antibodies prevented the development of IgG-producing cells, whereas the

inhibitory effects of antibodies against IgG were class-specific. Treatment with

IgM-specific antibodies also inhibited the development of IgG- and IgA-producing

cells in mice, but only when antibody administration was initiated at birth and not a

week later. These findings suggested that IgM-positive B cells give rise to B cells

that produce other immunoglobulins classes, although the class-switch mechanism

was not elucidated until the recombinant DNA technology in the 1980s.” (Cooper

2015).

3.4 Mammalian “Bursa-Equivalent” Organs and the Role

of Liver and Bone Marrow in Lymphopoiesis

As Miller pointed out: “As early as 1962, Burnet raised ‘the rather urgent question

of whether there is a functional equivalent of the bursa in the mammal (Burnet

1962a, b)’. My work had, however, 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, which later were known as thymus-dependent antigens. Presumably, 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, which is to feed into the body the cells whose

descendants will produce antibody (Burnet 1962b)’.” (Miller 2002).

Different structures have been identified as bursa equivalents in mammals,

including gut-associated lympho-epithelial tissues (GALT) tissues (Cooper et al.

1966b) and the bone marrow in primates, including humans. The lymphoid tissues

in the walls of the alimentary, respiratory, reproductive and urinary tracts are col-

lectively termed mucosa-associated lymphoid tissues (MALT), including GALTand bronchus-associated lymphoid tissue (BALT).

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, and include Meckel’s diverticulum, Peyers’s patches

and coecal tonsils.

The rabbit appendix and sacculus rotundus (intestinal tonsil) located at the ileo-

coecal valve (Fig. 3.10 ), develop within follicular out-pouching of the lower gut

(Archer et al. 1962), and mediate influences similar to those of the bursa on the

humoral system (Knight and Crane 1994). Ablation of this organ in neonatal liferesulted in a lifelong immunodeficiency (Perey and Good 1968; Archer et al. 1962).

As Good pointed out: “The bursa of Fabricius is present in all orders of birds. In

our quest to find bursal-equivalents in burslaess vertebrates, our efforts proved

largely futile. However, in the rabbit we recognized very early in the course of our

3.4 Mammalian “Bursa-Equivalent” Organs and the Role of…


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studies that the origins and development of the appendix-sacculus rotundus , as in

the bursa (Archer et al. 1962), develop within follicular outpouching of the lowerintestinal tract. We also showed that if we took to extirpate all of the appendix-

sacculus rotundus immediately in the newborn rabbit, we could produce an impres-

sive immunodeficiency of antibody production that lasted through the lifetime of

the rabbit (Archer et al. 1962). 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,

Chicago (Knight and Crane 1994). With several of her students, Knight was able to

demonstrate that the bursa of chickens and the appendix-sacculus rotundus of rab-

bits mediate very similar influence on humoral immune system. Using moleculargenetic approaches, 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).

Although neonatal thymectomy reduced the circulating lymphocyte count,

depleted lymphatic structure in spleen and lymph nodes, and interfered with devel-

opment of immunologic capacity in rabbits, these animals recovered to near normal

structure and function between 9 and 16 weeks after birth. When both the thymus

and appendix (Sutherland and co-workers 1964) were removed in the neonatal

period, immunologic capacity was depressed more regularly and more completelythen when either organ was removed alone, depletion of lymphocyte count and

organized lymphoid structure was more profound, and the deficiency thus induced

persisted far longer (Archer et al. 1965).

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. 1966a). Peyer’s patches (Fig. 3.11 ) 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 immuno-

globulin, IgM or IgG, to capacity to produce IgA immunoglobulin.

Fig. 3.10 Histological

picture of the sacculus

rotundus of rabbits

(GC germinal center,

DLT diffuse lymphoid

tissue)



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Immunoglobulin-bearing cells first appear in the liver during mouse embryogen-esis and after their colonization with HSCs, also fetal long bones can also generate

B cell ex vivo . (Owen et al. 1974, 1976, 1977). It is now clear that in mammals, B

cells differentiate in the bone marrow, and like T cells, B cells circulate and

re-circulate.

Fig. 3.11 The mucosa of the ileum is typical of the small intestine, with the addition of conspicu-

ous patches of lymphoid tissue called Peyers patches, which may protrude into the lumen and also

extend into the submucosa

3.4 Mammalian “Bursa-Equivalent” Organs and the Role of…


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DOI 10.1007/978-3-319-24663-5_4

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

4.1 The Discovery of the Thymus and Its Function

The word thymus is derived from a Greek word meaning the heart or soul, or from the

fancied resemblance of the thymus to the leaf of the plant Thymus vulgaris (Fig. 4.1 ).Galen of Pergamum (130–200 AD) first described the morphology of the gland and

noted that the thymus was largest during infancy. At the beginning of the twentieth cen-

tury, John Beard suggested that: “the thymus must be regarded as the parent source of

all the lymphoid structures of the body. 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. For just as the Anglo-Saxon stock has made its way from its original home into

all parts of the world, and has there set up colonies for itself and for its increase, so the

original leukocytes, starting from their birth place in the thymus, have penetrated into

almost every part of the body, and have there created new centres for growth, for

increase, and for useful work for themselves and for the body.” (Beard 1990).

The immunological competence of the thymus was demonstrated by Billingham

et al. (1956) and by Gowans et al. (1962). During embryonic life, lymphocytes dif-

ferentiate from the epithelial component of the thymus anlage, and they migrate out,

colonize the spleen and lymph nodes and constitute the immunologically competent

cells of the lymphoid system (Auerbach 1961, 1963).

Experiments in animals which were thymectomized, irradiated, “reconstituted”

with bone marrow cells bearing a chromosomal marker, 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). This provides further evidence that the lymphocyte

population of the thymus arises from immigration and differentiation of blood-borne


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stem cell precursors, arising in this instance from the bone marrow. Once stem cells

have migrated into the thymus, they differentiate into thymic lymphocytes, possibleunder local inductive influences.

4.2 Studies of the Thymus in the Chick and in the Mouse

In birds, if ventro-lateral part of the pharyngeal endoderm of the third and fourth

branchial pouches is associated with an appropriate mesenchyme, thymic histogen-

esis proceeds, demonstrating that no intervention of ectoderm is required for thy-mus differentiation (Le Douarin 1967). In the quail-chick chimaeras the third and

fourth endodermal pouches, and subsequently the thymic epithelial cells were sur-

rounded by mesenchymal cells derived from the grafted neural crest (Le Douarin

1973a, b). Moreover, the potentiality of the endoderm to give rise to lymphocytes

Fig. 4.1 Thymus vulgaris is a species of flowering plant in the mint family Lamiaceae

4 The Thymus


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was tested by transplanting the third and fourth pharyngeal pouches endoderm of a

15- and 30-somite quail embryo into the somatopleure of a chick (Le Douarin and

Jotereau 1973).

Moore and Owen (1967a, b) used a sex-chromosome marker system in paired

chick embryos joined by vascular anastomoses of chorioallantoic or yolk-sac blood

vessels. They demonstrated that chromosome analysis following yolk sac anasto-

mosis at 4–5 days of incubation revealed high levels of chimaerism in the thymus.

When the anastomosis was established later in the development, only low levels of

chimaerism were found, suggesting that an inflow of blood-borne stem cells is

responsible for lymphoid differentiation in the chick thymus. These results led the

authors to formulate the “haematogenous theory of blood forming organ histogen-

esis”. According to Moore and Owen, the source of the blood-borne stem cells that

invade the primary lymphoid organs is located in the yolk sac and it would be attrac-

tive to consider the hypothesis of a single cell precursor of all blood cells of botherythroid and lymphocytic series.

Different mammalian homologues of T cell surface markers have been identified

in chickens. As in mammals, in chickens CT4 cells have helper functions and the

cells expressing CT8 cells have cytotoxic activity. Mature T cells express either

CT4 or CT8. Antigen receptors on chicken T cells appear as CT3/T cell receptor

(TCR) complex. Three sublineages of the CT3 positive cells have been recognized

designated as TCR1, TCR2 and TCR3. TCR 1 and TCR2, correspond to their mam-

malian counterparts, while TCR3 appears to be unique to birds.

Good investigated the possibility that the thymus was involved in adaptive immu-nity, and performed thymectomies on 4–5 week old rabbits, without no demonstra-

ble effects on the antibody response (Maclean et al. 1956, 1957). Implants of thymus

tissue depleted of lymphocytes by irradiation stimulated lymphopoiesis, whereas

lymph node and muscle implants had no such effect (Grégoire and Duchateau

1956). In this context, Miller predicted that recovery of immune functions following

irradiation would be thymus-dependent. Adult mice were thymectomized , total

body irradiated, and they were given bone marrow cells. Non-thymectomized mice

recovered normal lymphoid functions, while thymectomized mice did not (Miller

1962a, 1963). Miller further investigated the effect of injecting lymphoid cells intoneonatally thymectomized mice and found that: (i) Syngeneic thymus cells from

1-day-old mice injected intravenously to newborn mice immediately after thymec-

tomy did not prevent immunological failure (Miller 1962b); (ii) Syngeneic lym-

phoid cells from 8-week-old mice pre-sensitized against Ak skin conferred adoptive

immunity. The Ak skin was rejected within 12 days and the mice showed evidence

of immunity to a second-set graft (Miller et al. 1964); (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).Lymphocytes restored immune capabilities, but only if the donor was syngeneic

(Miller 1964). Neonatal thymectomized mice, implanted with syngeneic thymus

tissue soon after birth, developed a normal immune system. When grafted with

foreign thymus tissue, they were specifically tolerant of thymus-donor type skin

only (Miller 1962b, 1963).

4.2 Studies of the Thymus in the Chick and in the Mouse


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This finding led Miller to postulate that “when one is inducing a state of immu-

nological tolerance in a newly born animal, one is an effect performing a selective

or immunological thymectomy” (Miller 1962b). Accordingly, Macfarlane Burnet in

a lecture in June 1962 at the University of London stated: “If, as I believe, the thy-

mus is the site where proliferation and differentiation of lymphocytes into clones

with definable immunological functions occurs, we must also endow it with another

function, the elimination or inhibition of self-reactive clones” (Burnet 1962a).

Experiments combining the techniques of thymectomy and injection of marked

thymus cells led to the conclusion that thymus-derived cells were small lympho-

cytes, able to circulate in blood and lymph for many months in rodents and years in

man (Miller and Osoba 1967).

4.3 The Functional Anatomy of the Human Thymus

Human thymus receives stem cells from the bone marrow and provides the micro-

environment for them to develop in T cells, which are released to begin a long life

circulating and recirculating through blood and lymph, slowly moving through

T-cell zones in peripheral lymphatic tissue (Fig. 4.2 ). The thymus is the first lym-

phoid organ to develop followed in turn by the central lymph nodes, spleen, periph-

eral lymph nodes, and gut. The thymus and the parathyroid glands develop from

epithelial anlagen of the third and fourth pharyngeal pouches (Fig. 4.3 ). It developsfrom an ectodermal-endodermal juncture and its epithelial components contain

derivative of both ectodermal and endodermal germ layers. The thymic mass gradu-

ally increases with colonization of blood-borne HSCs (Le Douarin and Jotereau

1975) and the rapid increase, a few days before hatching, results in the appearance

of the medulla. Uncommitted hematopoietic progenitors, therefore, HSCs enter

through postcapillary venules at the cortical-medullary junction, invade the epithe-

lial anlage and they move toward the subcapsular region and acquire T lineage

commitment.

During its development the thymus undergoes a descensus which brings it to liein the anterior mediastinum, in close connection with the pericardium and the great

veins at the base of the heart, where the endodermal epithelial masses fuse in the

midline in the 12th week of embryonic life. The lower border of the thymus reaches

the level of the fourth costal cartilage, while superiorly, extensions into the neck are

common reflecting the embryonic origin of the thymus.

Ectopic thymus in both humans and mice reflect a failed migration of thymic

tissue from third pharyngeal pouch endoderm during organogenesis. Ectopic thy-

mus is usually located anteriorly and deep to the middle third of the sternocleido-

mastoid muscle, adhere posteriorly to the carotid sheath and extend into theretropharyngeal space (Ahsan et al. 2010).

As the thymus proliferate and descends, the local cardiac neural crest mesen-

chyme controls the pattern and development of the gland. Defective development of

cardiac neural crest also results in thymic deficiency as seen in the Di George

syndrome.

4 The Thymus


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At the beginning of development, the thymus is a solid epithelial strand com-posed of densely packed epithelial cells, surrounded by a basal lamina and a vascu-

larized mesenchyme. Later, cortical epithelial cells begin to separate while cells of

the medulla remain densely packed. Vascularized mesenchyme transforms into con-

nective septa that invade epithelial strands up to the medulla, subdividing the corti-

cal zone into lobules, but not completely subdividing the medullary zone. During

DN3

DN2

DN1

Export

Entry

C o r t e x

M e d u l l a

C.M.J.

Cortical TEC

Medullary TEC

Thymocyte

DC

Macrophage

Cyst

Myoid cell

Neuroendocrine-like cell

TRENDS in Immunology

Hassal’s body

SPmature

Expansion

Expansion

TCRβ

recombination,T-lineagecommitment

Expansion,loss of B andNK potential

Apoptosisby neglect

Apoptosisbynegativeselection

Positiveselectionby corticalTECs

‘Promiscuous’gene expressionby medullaryTECs SP (CD4+ or

CD8+) immature

DP TCRαrecombination

Fig. 4.2 Structural and functional architecture of the thymus. Legend: thymic epithelial cells

(TEC ), dendritic cells ( DC ), T-cell receptor (TCR ), DN double-negative thymocyte, DP double-

positive thymocyte, SP single-positive thymocytes (Reproduced from Crivellato et al. 2004)

Fig. 4.3 An overview of thymus development (Reproduced from Gordon and Manley 2011)



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30

further development, thymic lobules with their well-delimited cortex and medulla

become packed tightly together (Fig. 4.4 ).

Most cortical capillaries loop around at different depths in the cortex and join

venous vessels at the cortico-medullary junction. Some continue through the cortex to

join larger veins running in the capsule and so leave the thymus. There is very little

movement of macromolecules from blood to thymic parenchyma across the capillary

walls in the cortex, as a consequence of the blood-thymus barrier. This barrier is

formed by the continuous blood capillaries in the thymic cortex, capillary basal lam-

ina, basal lamina of epithelial cells, and the epithelial cells. The blood-thymus barriers

separates cortical T cells from the blood of cortical vessels, protecting T cells against

foreign antigens, completely isolates the thymus cortex, creating a specific microenvi-

ronment in which T cells develop into mature T cells (Kato and Schoefl 1989).The large medullary vessels are highly permeable to substances in the plasma

and lymphocytes traverse the walls of the post-capillary venules of the cortico-

medullary junction and those of the medulla (Fig. 4.5 ) (Lind et al. 2001). Only a

small proportion of T cells is carried out from the thymus by the efferent lymphatic

vessels. In contrast to the cuboidal endothelium of post-capillary venules of the

appendix, Peyer’s patches, tonsils, and lymph nodes, the endothelium of thymic

post-capillary venules is flattened.

In relation to body weight, the thymus is largest during embryonic life and in

childhood up to the period of puberty. After this it begins to involute, a processwhich proceeds gradually and continuously throughout life under normal condition.

At birth the thymus weighs 12–15 g. This increases to about 30–40 g, at puberty

(Hasselbalch et al. 1997) after which it begins to decrease in weight, so that at 60

years it weighs only 10–15 g (Linton and Dorshkind 2004). The rate of thymic

Fig. 4.4 Microscopic organization of the fetal human thymus at a low magnification. Legend:

C cortical region, M medullary region, T trabecula, LT thymic lobule

4 The Thymus


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growth in the child and involution in the adult is extremely variable, and so it is dif-ficult to determine weight appropriate for age (Levine and Rosal 1978). Mast cells

may be present in large numbers in aged thymuses, where they are largely confined

to the inner medulla, septae, and capsule (Fig. 4.6 ). Although there is a considerable

age involution, the thymus remains a functional organ.

Fig. 4.5 A post-capillary venule (V) at the cortico-medullary junction in human thymus

Fig. 4.6 An electron

microscopic picture

showing a mast cell

surrounded by red blood

cells and thymocytes in thethymic medulla

(Reproduced for Crivellato

et al. 2005)



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In adults, the thymus is transformed into a mass of adipose tissue (corpus adipo-

sum thymi ), containing scattered islands of parenchyma consisting mainly of

enlarged reticular cells. Injections of glucocorticoids eliminates as much as 75 % of

thymocytes within 2–3 days. The changes affect both the cortex and the medulla but

are most pronounced in the cortex. Under these conditions, the thymus rapidlydiminishes in size, due to massive death of cortical small lymphocytes and their

destruction by macrophages.

The gland displays a lobuled pattern, with distinct cortical and medullary compart-

ments. In hematoxylin-eosin-stained sections, the cortex appears dark blue to purple

because of the predominance of lymphocytes (80–85 %), whereas the medulla appears

clear (eosinophilic) because of the predominance of the epithelial cells. The medulla

contains also mature thymocytes and the thymic or Hassall’s corpuscles (Fig. 4.7 ).

Formation of Hassall’s corpuscles begins with degeneration of an epithelial cell,

swelling of its nucleus, cytoplasm, and mitochondria. This cell becomes surroundedby one or more other epithelial cells which are organized circumferentially and con-

nected closely to one another by numerous desmosomes. Keratohyalin granules and

numerous tonofilaments appear in central cells. As the innermost cells gradually

become distant from blood capillaries, they swell, degenerate, and transform into

keratinized and/or necrotic material which often calcifies. Hassall’s corpuscles fre-

quently measure 100 μm in diameter, increasing in number and size with age.

Thymic epithelial-reticular cells are present in the cortex and in the medulla

(Anderson and Jenkinson 2001). Ultrastructural studies of these cells reveal evi-

dence of their epithelial nature such as desmosomes, cytoplasmic tonofilament, andmany other organelles found in epithelial cells. Six types of epithelial cells can be

identified. Types 2 and 3 create microenvironment niches in the outer cortex, called

thymic nurse cell complexes (Brelinska and Warchol 1997; Reike et al. 1995).

Thymic epithelial cells secrete cytokines [interleukin (IL)-1, IL-2, IL-3, IL-6, and

granulocyte-macrophage colony stimulating factor (GM-CSF)], chemokines

Fig. 4.7 An Hassall’s corpuscle (CH) in the medullary region of human thymus

4 The Thymus


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(Savino et al. 2002), and thymic hormones and neuropeptides (Mentlein and Kendall

2000). Type 1, 2, 3 and 4 thymic epithelial cells are localized in the cortex, while all

six types are localize in the medulla (Rezzani et al. 2008).

Keratin-negative cells include fibroblasts, non fibroblastic mesenchymal cells

and endothelial cells. Fibroblasts are found in the capsule, perivascular space and in

the medulla, but are infrequent in the cortex, except in the involuted thymus. Myoid

cells are situated mainly in the medulla and at the cortico-medullary junction. It has

been suggested that their contraction might aid the movement of lymphoid cells

across or out of the thymus.

The second thymic population is composed of thymocytes plus a variety of anti-

gen presenting cells, including interdigitating dendritic cells, macrophages particu-

larly at the cortico-medullary junction, and small amounts of B cells. Dendritic cells

are involved in shaping and maturating T cells by deleting self-reactive thymocytes

to established central tolerance (Varas et al. 2003). Electron microscopic detectionof Barbera granules in the cytoplasm of dendritic cells indicate that they express a

Langherans cells like phenotype during human ontogeny (Valledeau et al. 2000).

Thymic cortical dendritic macrophages have been described (Wakimoto et al. 2008),

containing apoptotic thymocytes, express CD8 and MHC II molecules, as well as

some dendritic cell associated molecules, including fascin, an actin binding protein.

They have well developed processes spreading in the adjacent areas surrounding T

cells, are placed in all thymus regions and are positive for two antibodies, anti-F4/80

and anti-Mac-2 (Liu et al. 2013). The proportion of human thymic dendritic cells

remain constant between postnatal, adult and old humans (Varas et al. 2003). In thecortical region are localized macrophages with flat shape and scanty cytoplasm.

They are stained by anti-Mac-2, but not by anti-F4/80 antibodies (Liu et al. 2013).

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).

Mutations in the AIRE gene are responsible for an autoimmune syndrome called

APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy),

characterized by loss of self-tolerance to multiple organs and abnormal structure of

the thymic medulla (Ramsey et al. 2002).

About 96 % of thymocytes become apoptotic, while only 3–5 % become fullycompetent T cells, and both cortex and medulla provide selective signals leading to

cell survival or death (Sprent and Kishimoto 2002). 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. Maturation of

T cells is accompanied by the sequential acquisition of the various T cell markers.

Terminal deoxynucleotidyl transferase is found in pro-thymocytes and immature

thymocytes but is absent in mature T cells (Hale 2004).

The most immature cells in the thymic cortex, do not express CD4 or CD8 (dou-

ble negative). Most of these cells are rapidly dividing cortical thymocytes that areactively rearranging TCR genes. The double negative cells give rise to the double-

positive cells localized in the cortex. These cells become the CD4-positive or CD-8

positive cells, under the guidance of contact and paracrine signals from the epithe-

lium (Petrie 2002), and localized in the medulla, indistinguishable from peripheral

T cells and expressing high levels of TCR. A massive rate of cell death (“apoptosis



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by neglect”, Klein and Kyewski 2000) affects the majority of the double-positive

cells (McPhee et al. 1979). E-cadherin is strongly expressed on epithelia cells as

well as on the double negative thymocytes in mice, suggesting its participation in

the interactions between these two cell types. However, in human thymus, E-cadherin

is expressed only on epithelial cells (Kutlesa et al. 2002).

Cells deriving from the thymus are both short-lived and long-lived. Long-lived

cells in man have a life-span upward of 5 years and perhaps over 10 years. In the

mouse their life-span is more than 80 days (Everett et al. 1964).

4.4 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, T cell progenitors colonize theepithelial thymus in three successive waves, beginning at 6 incubation day, until 18

incubation day (Coltey et al. 1989). In the mouse, immunologic depression is pro-

found after thymectomy in neonatal animals, resulting in considerable depression of

antibody production, deficient transplantation immunity and delayed-type hypersen-

sitivity (Waksman et al. 1962). Three different experimental approach showed that

neonatal thymectomy has a significant effect on immunologic reactivity: (i) The stud-

ies of Fichtelius et al. (1961) in young guinea pigs; (ii) The experiments of Archer,

Good and co-workers in rabbits (Archer and Pierce 1961; Archer et al. 1962; Good

et al. 1962) and mice (Good et al. 1962; Martinez et al. 1962a, b; Dalmasso et al.1963); (iii) The studies of Miller (Miller 1961a, 1962b; Papermaster et al. 1962a).

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.

There I gave my data in great detail, 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, without providing data, that they had independently shown that neonatally

thymectomized mice were somewhat immunodeficient but that, in contrast to my find-ings, prolonged skin graft survival occurred only in mice identical at the H-2 histo-

compatibility locus but differing at other weaker histocompatibility genes. Their mice

rejected skin from H-2-incompatible strains. It was disappointing and indeed surpris-

ing that this group, who claimed ‘at long last’ to have ‘established the essential func-

tion of the thymus’ prior to the April 1961 meeting of the American Association of

Immunologists, gave at this New York Meeting, in February 1962, a paper that was

not on the thymus and in which the word thymus did not appear. They did, however,

publish their independent finding in later 1962, again emphasizing the ability of their

thymic-deficient mice to reject H-2 incompatible grafts. Later on, they admitted thatthe difference between our results on skin graft rejection may have been due to the fact

that they had not completely thymectomized their mice.” (Miller 2002).

Good wrote that: “The simultaneous occurrence of acquired agammaglobulin-

emia and benign thymoma in a human being, suggested that the thymus might par-

ticipate in the control of antibody formation. […] It still seems likely that some

4 The Thymus


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35

essential relationship exist between the thymic tumor and the acquisition of an

acquired agammaglobulinemia. 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).

These data indicate that in the mouse the thymus is the primary central lymphoid

organ, 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. Mice were vulnerable to homologous dis-

ease when injected with parent strain lymphoid cells (Parrott 1962). Lymphoid tis-

sues showed minimal development and circulating lymphocyte were greatly

reduced. Cells of spleen and lymph nodes extert a very low immunologic activity

(Dalmasso et al. 1963).

In neonatal rodents, the thymus has completed its development, but the peripheral

population of thymus-dependent lymphocytes has not yet been established. Neonatalthymectomy is responsible of gross deficiencies in the distribution of T lymphocytes.

Once the periphery has become populated with T lymphocytes, thymectomy is not

longer followed by a dramatic deficiency of cell-mediated immune responses.

4.5 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, if thymectomy is performed in the neonatal period before

the thymus seeds peripheral lymphatic organs with T cells. After thymectomy in the

newborn, homografts may persist indefinitely instead of being rejected within a

week or two. Moreover, antibody production against antigens that require

cooperation of T cells and B cells is also impaired. Thymectomy in adults causes no

such changes because the extrathymic lymphatic tissues and circulation are already

stocked with T cells.

Neonatal thymectomized mice showed a marked deficiency of lymphocytes inthe circulation and in the lymphoid tissues. At 6 weeks, the spleen was greatly

reduced in size (Miller 1962a) and displayed inactive follicles, few germinal centres

with low cellularity, and few mitoses (Miller 1961a, 1962b). The lymph nodes were

smaller and displayed inactive follicles and poor cellularity. Peyer’s patches were

also smaller (Miller 1961a, 1962b). Moreover, thymectomized mice failed to reject

skin from foreign mouse strains (Miller 1962b), but rejected allogeneic grafts

(Miller 1961b). 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. This would suggest that lym-phocytes leaving the thymus are specially selected cells” (Miller 1962b). The

immune defects observed after neonatal thymectomy were confirmed by Arnason

et al. (1962) and Martinez et al. (1962a, b). When mice were thymectomized after

4.5 The Thymus Is Essential for Normal Development of the Immune System


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birth, i.e. when their lymphoid system and the immune mechanisms had developed

to a certain extent, only negligible effects were observed.

A specific lymphocytosis-stimulating-factor (LSF) heat-labile and filterable, but

non-dialyzable was demonstrated in the thymus (Metcalf 1956). If a thymus

wrapped in a cell-tight filter was inserted into the peritoneal cavity of mouse thy-

mectomized at birth, the animal showed partial restoration of T cells and no immu-

nological deficiencies. Factors appear to diffuse through the wrapping and largely

substitute for the thymus. The best characterized of thymic humoral factors is thy-

mosin, which restores T-cell deficiencies in thymectomized mice. Thymosin has

been demonstrated in thymic epithelial cells by immunohistochemistry and is

secreted by them (Schulof et al. 1987). These factors, when added to culture of

thymic cells, can induce the appearance of T-cell differentiation markers, activate

cyclic GMP or AMP, and induce mature T-cell functions.

4.6 Removal of Either the Thymus or Bursa of Fabricius

In a series of experiments, Cooper removed either the thymus or the bursa from

some newly hatched chicks, and both from others, and to destroy all peripheral

lymphoid components, he subjected the chicks to intense x-irradiation, and waited

several weeks until the animals recovered from the irradiation effects. In the mind

of Cooper: “The plan was to compare the immunological status of the differentexperimental groups, after they recovered from the effects of surgery and irradia-

tion.” (Cooper 2010). He specified that: “I devised an alternative strategy that would

combine post-hatching thymectomy or bursectomy together with whole body irra-

diation 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. In

these experiments I removed either the thymus or the bursa, 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.” (Cooper 2002).

Bursectomized and irradiated birds were completely devoid of germinal centers,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. 1965, 1966a, b). 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, but they still produced germinal centers, plasma cells and circulating immu-

noglobulins (Cooper et al. 1965, 1966a, b; Miller et al. 1963). Birds subjected to

combined thymectomy, bursectomy and irradiation had severe cellular and humoral

immune system deficit (Cooper et al. 1965, 1966a, b).More recently, Cooper pointed out that: “we decided to combine whole-body

irradiation wi

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The Development of Immunologic Competence