imune system developement

ch08 Lymphocyte development

Neonatal Immunology

Introduction

Like many other systems in the body, the immune system is not fully functional at birth and therefore we are at an increased risk of infection. At the same time, the act of being born – moving from the sterile environment of the womb to the wider world, exposes us to a whole range of pathogens that we have never encountered and have no protection against. To supplement this period of immune immaturity and reduce the risk of infection, the mother transfers passive protection to the child, mainly in the form of antibody.

The immune response in early life is dampened compared to adults. This in part is caused by the immunosuppressive environment of the womb. Speculatively though it may also be an adaption to the exposure to larges amounts of new antigens in early life. The immune response is carefully regulated to ensure appropriate levels of immune response but avoiding inflammatory responses to benign or harmless antigens. Since there are more new antigens in early life, it may be that the response is skewed to suppression.

The reduction in immune response leads to increased susceptibility to pathogens and to reduced responses to vaccines that are effective in adults, necessitating research on infant-specific formulations. The altered function of the neonatal immune response may also influence the development of asthma and allergy in later life.

Importance

Five million infants die in the first year of life, 1.5 million of these deaths are due to infection. The most common causes are respiratory infection and diarrhoea. Sadly, current vaccines are not as effective in early life as they are in adulthood.

Development of the immune system

In many ways the immune system we are born with is the product of the immune environment during pregnancy. In order to maintain the foetus the mother needs to ignore the foetal alloantigens (half of the antigens being of paternal, and therefore of foreign origin). This leads to a scenario of immunosuppression/regulation during pregnancy and this carries over into early life. (For more about cellular development see: T-cell development in the thymus).

Transfer of protection from mother-to-child

The main component of immune protection transferred from mother to child is antibody. This is transferred across the placenta to the foetus using the FcRn (neonatal Fc receptor). Antibody is also transferred to the infant via breast milk. The main immunoglobulin class transferred is IgA, the transferred IgA works at mucosal surfaces, where it is able to prevent pathogen entry. However other important factors are transferred, including complement and commensal bacteria – which may provide protection against asthma and allergy in later life (see: Complement system).

Features of the neonatal immune system

Pattern recognition: Neonatal responses to pathogen associated molecular patterns (PAMPs) are reduced compared to adults. However pattern recognition receptor (PRR)-expression levels are similar. It appears that the molecules that transduce the signal (for example interferon response factor 3 – IRF3) have reduced function. This leads to reduced production of key inflammatory mediators, for example interleukin-12 (IL-12) and interferon-α (IFNα). PRR function increases over time, and the increase in capacity occurs in proportion to time since birth rather than ‘gestational’ age, suggesting that it is controlled by exposure to the environment and removal of maternal influence.

T-cell response: There is a well-documented skewing of the neonatal T-cell response towards T helper 2 (Th2). This is associated with the reduction in IL-12 and IFNα production by neonatal antigen-presenting cells (APC). This may have an effect on the immune response to antigens seen in early life – possibly inducing an allergic type response.

B-cell response: Antibody production in early life is reduced. In particular the antibody response to polysaccharide antigens is reduced. This is a particular problem with regards to bacterial infections, to which newborn children are highly susceptible. This failure to produce antibody is associated with several factors including reduced T cell help, fewer follicular dendritic cells and germinal centres and reduced signalling through the CD40 ligand family members.

Downstream effects of the neonatal immune response

The immaturity of the neonatal immune response has an effect on three important areas:

1. Increased susceptibility to infection. The recognition of infectious agents is reduced in early life and it is therefore easier for a pathogen to invade the host. Neonates are also less experienced so have no immune memory against infection.

2. Decreased vaccine efficacy. In a similar fashion to infection, reduced recognition of vaccine antigens as foreign means that induction of protective memory responses to vaccines are reduced. There is also an effect of maternally derived antibody which may mask key epitopes of the vaccine.

3. Development of asthma and allergy. The Th2 skewing of the T cell response is hypothesised to drive the development of allergic responses to antigen in early life.

Figure 1. Downstream effects of reduced antigen presenting cell function in early life.

The neonatal immune system is exposed to a large number of previously unseen antigens. The majority of these antigens are benign and therefore should be tolerised but some are dangerous and therefore should induce an immune response. The neonatal antigen presenting cells (APC) have reduced recognition of antigens regardless of source (self, benign, pathogenic, vaccine). This is mediated at the level of pattern recognition receptors (PRR) or their adaptor molecules. This leads to reduced immune responses to these antigens and has an impact on vaccine efficacy, disease susceptibility and, possibly by skewing responses to Th2, the development of asthma and allergy.

B cell activation and the germinal CENTER response

B cell activation

B cells are activated when their B cell receptor (BCR) binds to either soluble or membrane bound antigen. This activates the BCR to form microclusters and trigger downstream signalling cascades. The microcluster eventually undergoes a contraction phase and forms an immunological synapse, this allows for a stable interaction between B and T cells to provide bidirectional activation signals.

Once activated B cells may undergo class switch recombination. In their inactivated state B cells express IgM/IgD but once activated they may express IgA, IgE, IgG or retain IgM expression. They do this by excision of the unwanted isotypes (Figure 2). Cytokines produced by T cells and other cells are important in determining what isotype the B cells express.

Figure 2: Class switch recombination. After VDJ recombination class switch recombination may occur. In this process unwanted Immunoglobulin (Ig) genes are excised so that the desired gene can be expressed. In this depiction excision occurs and IgE is expressed. There are five isotypes which can be found in difference circumstances. For example, IgE is common in allergic responses such as asthma.

Figure 3: The migration of B cells in an immune response. When B cells (B) first encounter antigen (★) they migrate to the T-B border to receive survival signals from T cells (T). If they receive survival signals they will begin to proliferate and either become plasmablasts (Bl) or form a germinal centre (Blue). B cells can migrate between the light zone and dark zone of the germinal centre to undergo somatic hypermutation and class switch recombination. Eventually they may leave the GC as high-affinity memory cells (M) or plasma cells (P).

The germinal centre

B cells have two main types of immune responses. In a T-Independent immune response B cells can respond directly to the antigen. In a T-dependent immune response the B cells need assistance from T cells in order to respond.

In this situation activated B cells move to the border of the T cell zone to interact with T cells (Figure 3). CD40 ligand is found on these T helper cells and interacts with CD40 on the B cells to form a stable attraction. Cytokines secreted by T cells encourage proliferation and isotype switching and maintain germinal centre size and longevity. Without these signals the germinal centre response will quickly collapse.

B cells that have encountered antigen and begun proliferating may exit the follicle and differentiate into short-lived plasma cells called plasmablasts (Figure 3). They secrete antibody as an early attempt to neutralize the foreign antigen. They do not survive more than three days but the antibody produced can provide important assistance to stop fast-dividing pathogens such as viruses.

The germinal centre has a light zone and a dark zone. The germinal centre response begins in the dark zone where the B cells rapidly proliferate and undergo somatic hypermutation. During somatic hypermutation, random mutations are generated in the variable domains of the BCR by the enzyme activation-induced cytidine deaminase (AID). B cells then enter the light zone and compete with each other for antigen. If the mutation resulted in a BCR with an improved affinity to the antigen the B cell clone can out-compete other clones and survive. The light zone is also thought to be where B cells undergo class switch recombination, although a germinal centre is not crucial for this process. The B cells may migrate between both zones to undergo several rounds of somatic hypermutation and class switch recombination. The ultimate goal of the germinal centre is to produce B cells with a BCR which has high affinity for the initial antigen.

Plasma and memory cells

B cells leave the germinal centre response as high-affinity plasma cells and memory B cells (Figure 4). Plasma cells secrete antigen-binding antibodies for weeks after activation. They migrate to the bone marrow soon after formation where they can reside indefinitely, ready to encounter the antigen again and respond. Memory B cells circulate throughout the body on the lookout for antigen with a high-affinity for their BCR and then quickly respond to the antigen, stopping infection. This is how vaccination works. As your body has been previously exposed to the antigen the immune cells can quickly respond to remove the antigen if it is encountered again, stopping you getting sick.

Figure 4: B cell differentiation after activation. When a mature B cell encounters antigen that binds to its B cell receptor it becomes activated. It then proliferates and becomes a blasting B cell. These B cells form germinal centres. The germinal centre B cells undergo somatic hypermutation and class switch recombination. Plasma cells and memory B cells with a high-affinity for the original antigen stimuli are produced. These cells are long lived and plasma cells may secrete antibody for weeks after the initial infection.

T-cell development in thymus

T cells are derived from haematopoietic stem cells that are found in the bone marrow. The progenitors of these cells migrate to and colonise the thymus. The developing progenitors within the thymus, also known as thymocytes, undergo a series of maturation steps that can be identified based on the expression of different cell surface markers. The majority of cells in the thymus give rise to αβ T cells, however approximately 5% bear the γδ T cell receptor (TCR). Developing thymocytes interact with the thymus stromal (non-haematopoietic) cells, and undergo the process described below in distinct regions of the thymus. The thymus is made up of an outer cortex and an inner medulla region.

The earliest developing thymocytes lack the expression of the co-receptors CD4 and CD8 and are termed double negative (DN) cells. The DN population can be further sub-divided by the expression of CD44 (an adhesion molecule) and CD25 (Interleukin-2 receptor α chain), Figure 5 shows the ordered expression of these markers. Cells that lack expression of CD44, but express CD25 (DN3) undergo a process termed beta-selection. This process selects for cells that have successfully rearranged their TCR-β chain locus. The β chain then pairs with the surrogate chain, pre-Tα, and produces a pre-TCR, which forms a complex with CD3 molecules. This complex leads to the survival, proliferation, arrest in further β chain loci rearrangement, and further differentiation by up-regulation and expression of CD4 and CD8, these cells are termed double positive (DP) cells. Cells that do not undergo beta-selection die by apoptosis.

Figure 5. αβ T cell development, showing the different cell surface markers expressed at the different stages of T cell development in the mouse.

DP cells rearrange their TCR-α chain loci, to produce an αβ-TCR. These cells then undergo positive selection, in the cortex. DP cells interact with self-antigens in the context of major histocompatabilty complex (MHC) class I or class II molecules. Those cells that engage antigen/MHC with an appropriate affinity survive, whereas those cells that interact with a weaker affinity die by apoptosis. Thymocytes then migrate into the medulla to undergo negative selection. They are presented self-antigens on antigen presenting cells (APCs), such as dendritic cells and macrophages. Thymocytes that interact too strongly with antigen undergo apoptosis. The majority of developing thymocytes die during this process. Following selection, down-regulation of either co-receptor produces either naïve CD4 or CD8 single positive cells that exit the thymus and circulate the periphery.

Bone Marrow

Bone marrow is found in the medullary cavities – the centres of bones. The bone marrow is where circulating blood cells are produced – a process known as haematopoiesis. Early on in a human’s life, this takes place in many bones, but during development haematopoiesis increasingly centres on flat bones so that by puberty, blood production takes place predominantly in the sternum, vertebrae, iliac bones and ribs. Bone marrow undergoing haematopoiesis is coloured red due to the presence of red blood cells, whereas bone marrow that is not undergoing haematopoiesis is yellow.The red marrow consists of long trabeculae (beam-like structures) within a sponge-like reticular framework. Spaces around this framework are filled with fat cells, stromal fibroblasts and blood cell precursors. A healthy bone marrow biopsy is shown in Figure 6.

Figure 6. Haematoxylin-Eosin stain of healthy bone marrow (from http://bonemarrowbiopsy.wordpress.com/normal-results/) The haematoxylin has stained the cells’ nuclei purple and the eosin has stained the cells’ cytoplasm pink. The white patches are fat stored in fat cells.

Figure 7. Bone Marrow Morphology. Adapted from Nagasawa Nat Rev Immunol. 2006 Feb;6(2):107-16.

During the development of the blood cells, these haematopoietic precursors migrate from the subendosteal region (the inner bone surface) towards a central region (Figure 7). The matured blood cells exit through a dense network of vascular sinuses.

During haematopoiesis the haematopoietic stem cells (HSC) divide, and one daughter cell remains in the bone marrow to continue renewing the HSC pool. The other daughter cell will pass through several stages of development (see Figure 9) to become a mature blood cell and leave the bone marrow to enter the circulation.

Figure 8. Stain of CLL patient bone marrow showing heavy infiltration of leukaemic cells (from http://bonemarrowbiopsy.wordpress.com)

Figure 9. A model for blood and tissue production in Bone Marrow. HSCs (haematopoeitic stem cells) are self-renewing stem cells that can differentiate into any blood cell type. MPPs (multipotent progenitors) still have the potential differentiate into any cell type, but cannot divide continuously so must be renewed by the differentiation of HSCs. CLPs (common lymphoid progenitors) differentiate into lymphoid cell types whereas CMPs (common myeloid progenitors) differentiate into myeloid cells types via the GMP (granulocyte-macrophage progenitor) or MKEP (megakaryocyte-erythrocyte progenitor). Adapted from Yin, Li J Clin Invest. 2006 May;116(5):1195-201

Mesenchymal stem cells (MSC) are found in the bone marrow cavity and differentiate into a number of stromal lineages such as chondrocytes (cartilage generation), osteoblasts (bone formation), adipocytes (adipose), myocytes (muscle), endothelial cells and fibroblasts.

After leaving the bone marrow and undergoing further development, activated antigen-experienced B cells differentiate into plasma cells which return to, and colonise the bone marrow cavity.

The framework of the bone marrow and all the cells present within it, along with their secreted cytokines and cell surface receptors make up a complex microenvironment. Maintenance of this microenvironment is important to promote haematopoiesis, cell development and prevent haematological disorders.

Bone Marrow Disorders

Leukaemias are malignant diseases of the bone marrow and occur during haematopoietic development of either lymphoid lineages in acute or chronic lymphoblastic leukaemia (ALL/CLL) (Figure 8); or myeloid lineages in acute or chronic myeloid leukaemia (AML/CML).

Myeloproliferative disorders are related to leukaemias in that they are characterised by the overproduction of one type of blood cell and in some cases develop into leukaemias. There are three main forms: essential thrombocythaemia, polycythaemia vera and myelofibrosis in which the overproduced cell types are platelets, red blood cells and fibroblasts, respectively.

Myelodysplastic syndromes (MDS) are a spectrum of disorders resulting from overproduction of one or more type of blood cell. Some cases transform into AML. Multiple myeloma is a malignancy of plasma cells which leads to excessive production of a single paraprotein. Aplastic anaemia is characterised by reduced blood production as a result of loss of haematopoietic stem cells and replacement by fat cells.

Lymph Node

A number of specialised tissues are important for the proper functioning of the immune system. Among these are the lymph nodes, which provide an ideal environment for communication between immune cells. This environment is necessary for proper activation of the T and B cells (or lymphocytes) that are required for defence against many pathogens. A number of features of lymph nodes help them to perform their functions.

Lymph node location

The lymph nodes are strategically located at anatomical locations where they are most able to receive immunological signals from around the body. The total number of nodes is not known, but there are likely to be hundreds. Each node is well-supplied by both lymphatic and blood vessels, which allow lymphocytes to enter and exit. They are contained within a tough capsule, and surrounded by specialised fatty deposits, which may give some physical protection.

Lymph node structure

Each human lymph node is up to 20mm in diameter, and is divided into compartments, with important functions in enabling communication between lymphocytes. The outer layer (Cortex) contains the B-cell areas, or follicles. The middle layer (Paracortex) is mostly populated by T cells and dendritic cells (Figure 10). The paracortex also contains specialised blood vessels (high endothelial venules) through which many B and T cells enter the node. The lymph vessels enter the nodes at the outer edge, between the capsule and the cortex, and also penetrate deep within the nodes, via conduits. T and B cells leave the node via “efferent” lymphatic vessels, found in the central “medullary” region. All these structures are maintained by a network of non-lymphoid cells that also actively influence immune responses.

Figure 10. A slice through a lymph node, showing B and T cell areas where lymphocytes are tightly packed (purple)

Co-ordinated movement of cells in lymph nodes

T cells enter the lymph nodes through high endothelial venules, and move around within the T-cell area, transiently interacting with large numbers of dendritic cells. They finally leave the node via the efferent lymphatic vessels. B cells enter by the same route, and migrate through the T-cell area to the follicles, before finally leaving the node and re-entering the circulation. These migratory patterns give dendritic cells, T cells, and B cells many opportunities to interact. The cells may travel on a network of microscopic fibres, further increasing their chances of interacting.

Responding to new infections

Lymph nodes are extremely important in responses to pathogens, especially those that an individual has not previously encountered. Proteins from the pathogen will reach dendritic cells in the lymph node, or will be carried to the lymph node by migrating dendritic cells, and protein fragments will be “presented” to T cells. The continual interactions between dendritic cells and T cell ensure that a T cell will soon be found that recognises the pathogenic protein fragment. This T cell will then divide and coordinate the immune response against the pathogen. Crucially, some of the dividing T cells’ daughters will travel to the B cell follicle and promote B-cell division and maturation, enabling the production of the antibodies that are essential for fighting many infections.

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