Immunology and Reproduction

Introduction

The relationship between a living organism and its environment is based on a tightly regulated balance between symbiosis and competition. Survival is dependent upon appropriate resource acquisition, permissive physicochemical environments, and competition for limiting resources and ecological niches exerted by other living organisms. The complexity of an organism is directly correlated to the sheer volume of challenges presented to its continued fertility (the ultimate currency of evolution). Generally, competition can be extra-organismal (acquisition of the same resource by competing organisms), interorganismal (direct predation), or intraorganismal (parasitism). The evolution of higher organisms required the development of advanced defense mechanisms to ensure survival in an overwhelmingly hostile biological environment. The systems in higher organisms that protect against biological insults are collectively referred to as the immune system. The immune system may act as friend or foe. Immune systems of higher vertebrates can be roughly subdivided into two major components: a phylogenetically older innate immune system and the more recently evolved adaptive immune system . Innate immunity is a genetically fixed, direct evolutionary relative of the ancient mechanisms of niche competition found in both plants and animals. The diversity of environments that humans occupy is staggering; thus, the human body has remarkably elaborate mechanisms to protect the individual from foreign pathogens.

During development, the innate and adaptive arms of the immune system are programmed and learn to distinguish self from nonself. The dangerous side effect of the variability/adaptability generation is that a substantial number of potentially autoaggressive cells reside in healthy individuals. Consequently, mechanisms of immune regulation that prevent autoimmune pathology operate as checkpoints at multiple levels of immune development and function. Despite the safeguards of the immune system, some self-reacting lymphocytes are not inactivated or removed and can result in one of several illnesses known as autoimmune diseases. Immunologists are well aware that if they understood all the mechanisms for tolerance induction, they might be able to prevent autoimmune diseases and, conversely, focus the potent toolkit of immunity against foreign pathogens and the “altered self” of cancer.

The immune system is capable of recognizing foreign proteins with chemistry slightly different from its own molecules using three critical processes:

specific methods for recognition of foreign antigens involving antigen-specific receptors on B lymphocytes and T lymphocytes;
control over responses produced by cellular “licensing” at T-cell and B-cell levels and via innate immune “alarm” signals;
processing and presentation of self-antigens, particularly as they relate to the induction of tolerance during ontogeny, are critical to the regulation of autoimmunity.

A major focus of attention is on the two- (three)-party interaction of antigen presented by major histocompatibility complex molecule to the T-cell receptor.

The failure of tolerance-induction mechanisms to prevent the recognition of “self” antigens as foreign by the immune system may result in an autoimmune response. Thus, autoimmunity refers to an immune reaction of the body against substances that are normally present in the body. To understand autoimmunity, it is important to first understand tolerance. Tolerance can best be defined as a state of antigen-induced immunologic unresponsiveness. (Autoimmunity can be considered a failure of tolerance.) Tolerance is not synonymous with generalized immunosuppression; it is antigen-specific and causes no impairment in the immune response to antigens other than those used to induce tolerance.

Because tolerance is antigen-specific, it must involve the T- and/or B-lymphocyte clone(s) specific to the antigen in question. At the clonal level, tolerance can only result from mechanisms that induce clonal deletion or clonal anergy. Clonal deletion mechanisms eliminate T-cell clones reactive to self-antigens in the thymus during development. Clonal anergy mechanisms allow regulatory self-reactive clones to exist but they are in a long-term state of unresponsiveness. Natural (induced in the thymus during development) and induced (result of effector T-cell differentiation) regulatory T cells (Tregs) are pivotal regulators of the remaining pool of peripheral autoreactive T cells. Indeed, Tregs are necessary for survival of allogeneic pregnancy.

Implantation is one of the central aspects of pregnancy targeted by immunologic studies. Implantation represents a critical developmental process in that it requires the interaction of immunologically and genetically distinct tissues. The immune system may influence pregnancy success or failure during any of the critical steps of implantation. Embryo attachment occurs only during the “implantation window,” the time period when the endometrial epithelium is receptive and the embryo is hatching and competent for attachment. Failure of this synchronization precludes success, as demonstrated in human studies of implantation.

Implantation is the most important limiting factor in human reproduction. Only 25% of all fertilized ova will generate a live birth and 50% appear to fail at time of implantation. In specific situations, such as early recurrent pregnancy loss (RPL), karyotype analysis on products of conception has shown that chromosomal abnormalities are not the only cause of pregnancy losses, particularly in women under the age of 36 years. ^– Human preimplantation embryos express major histocompatibility antigens theoretically capable of inducing an immune response, but the role of these antigens in pregnancy has not been delineated. It is possible that maternal immune responses play a role in the failure of implantation.

Basic Immune Principles

Responses of the innate immune system are generally rapid, do not exhibit antigen specificity, and lack memory; those of the adaptive/acquired immune system are antigen-specific and include primary responses and secondary memory-driven responses .
Immune specificity relies on a lymphocyte education process that distinguishes self from nonself. T-lymphocyte education is dependent on recognition of self-major histocompatibility complex (MHC) restriction; B-lymphocyte education is not .
As a part of the mucosal immune system, which is the first line of defense against many pathogens, the immunologic interactions in the female reproductive tract are biased toward innate immunity .

Innate and Acquired/Adaptive Immune Defense Systems

The importance of effective defenses against foreign invaders has led to evolution of innate (or natural) and subsequently adaptive (acquired or specific) immune systems. While these immune systems often cooperate in defense against foreign invaders, their responses differ in intensity, timing, and specificity ( Table 16.1 . Section II.A).

Table 16.1

Features of Innate and Acquired Immunity

Characteristic	Innate Immunity	Acquired Immunity
Specificity	Nonspecific	Antigen-specific
Physicochemical barriers	Skin, mucous membranes	Peripheral and mucosal immune systems
Effector cells	ILCs, NK cells, macrophages	B and T lymphocytes
Circulating molecules	Complement	Antibodies
Soluble mediators	Macrophage-derived cytokines (IFN-α, IFN-β, TNF-α)	Lymphocyte-derived cytokines (IFN-γ)
Response time	Rapid	Primary and secondary
Memory	None	Long-lived

IFN-α, Interferon-alpha; IFN-β, Interferon-beta; IFN-γ, Interferon-gamma; TNF-α, Tumor necrosis factor-alpha

Innate Immunity

The innate or natural defenses against pathogens are fixed, genetically encoded, and consist of tissue barriers, phagocytic and cytotoxic cells, and a variety of effector molecules. These defenses at the skin and mucous membranes may be perfectly adequate to clear many potentially harmful environmental antigens. The initial entry of foreign organisms or cells is met by an initial inflammatory response, which may be amplified by the activation of serum complement components resulting in the generation of enzymes and the deposition of their products on the surface of foreign antigens. The innate system can recognize a harmful invader such as bacteria or viruses by promoting an offensive environment. Other invaders, such as sperm or blastocyst, result in an inflammatory response that is accommodating to the guest. However, this first exposure to an antigen, regardless of the intensity of the response elicited, will not alter the type and magnitude of the innate immune response upon reexposure. In this sense, innate response to reexposure to same or unrelated antigen(s) will not exhibit “memory.”

Acquired/Adaptive Immunity

The acquired immune response is characterized by an adaptation to the first exposure to the foreign antigen, known as the primary response . A subsequent exposure to the same antigen results in a secondary response that is quantitatively and qualitatively different than the primary response. The acquired immune response consists of antigen-specific cells and molecules that often interface with components of the innate immune defense systems. During opsonization , antibodies bind to bacterial surface antigens facilitating their phagocytosis by macrophages. Conversely, macrophages may process and present protein antigens to specific T cells during a primary immune response. Thus, innate immunity facilitates activation of adaptive immunity and adaptive immunity facilitates innate responses in the context of reexposure (memory).

Antigen-specific lymphocyte responses are characterized by their activation and proliferation of previously antigen-naïve lymphocytes, followed by functional differentiation toward production of soluble mediators including antibodies and cytokines and the development of antigen-specific memory. A typical primary response to antigen has a lag phase of about 5 days, a slow rate of increase to a low plateau, and a low sensitivity to the antigen. The low affinity antibody elicited in a primary response is predominantly pentameric IgM. Over the course of approximately 4 to 8 weeks, B cells specific for antigen proliferate and undergo affinity maturation (selection for B cells that produce higher-affinity antibodies) and class switching (from IgM to IgG, IgA, or IgE). The secondary response (and all subsequent memory responses ) to the same antigen are quantitatively and qualitatively different from the primary response. The secondary response occurs in 1 to 2 days, rises rapidly to a high plateau, and has a high degree of sensitivity to the antigen. The antibody affinity is high in a secondary response and may be predominated by IgG, IgA, or IgE, depending on the location in which the immune response takes place.

The major features of the specific immune response are consistent with Burnett’s clonal selection theory. This hypothesis states that: (1) the antigen specificity of a single lymphocyte is established in the absence of antigen during its initial maturation, (2) each lymphocyte has a unique antigen specificity manifested by multiple copies of the same receptor, and (3) the proliferation and differentiation of the lymphocyte is induced by antigen binding to the specific receptors. The interaction of antigens and lymphocyte receptors is governed by the relative affinity of the receptor for the antigen. Clonal selection explains subsequent responses to the same antigen that are more rapid, robust, and long lasting. The proliferation of specific lymphocytes in response to antigen results in the generation of antigen-responsive clones of cells.

As a consequence of antigen-induced proliferation, a small proportion of the proliferating lymphocytes commits to a long-term memory cell fate that persists for many years. ^, Activated B cells may undergo isotype switching by changing from the production of IgM to IgG, IgE, or IgA, thus allowing several different physiologic and biologic outcomes. By these mechanisms, a maturing immune response maintains antigen specificity but establishes memory and functional diversity. The development of adaptive immunity based on the clonal selection of lymphocytes bearing specific receptors is critical in allowing all pathogens to be recognized (no matter how novel) and in allowing the development of immunologic memory.

Basic Effectors of an Immune Responses

Cellular Effectors

Leukocytes are the cellular effectors of immunity and include lymphocytes, monocytes, macrophage, dendritic cells, neutrophils, basophils, and eosinophils ( Table 16.2 . Section II.B.1). Lymphocytes can be further divided into subclasses based on function and upon cell surface markers, called “cluster of differentiation” or CD markers. Lymphocyte subclasses include T cells, B cells, and innate lymphoid cells (ILCs) – including natural killer (NK) cells. Both B and T lymphocytes originate in the bone marrow and participate in antigen-specific immune responses.

Table 16.2

Characteristics of T, B, and NK Lymphocytes

Characteristic	T Cytotoxic T Regulatory	T Helper T Inducer	B Cells	NK Cells
Symbol	Tc/reg	Th	B	NK
Surface antigen	CD8+	CD4+	CD19+	CD56+
MHC restricted	Class I	Class II	Class I and II	None
Target cells	Tumors, virally infected cells, allografts	B cells, Tc cells and precursors, macrophages		Tumor cells, virally infected autologous cells
Function	Kill foreign cells, downregulate cells	Interleukin secretion	Immunoglobulin production	Immune surveillance, cytotoxic
Differentiation	Thymus	Thymus	Bone marrow	Bone marrow
Specificity	Antigen peptides	Antigen peptides	Native antigen epitopes	Unknown
Antigen receptor	T-cell receptor	T-cell receptor	Cell surface immunoglobulin	Fc portion of immunoglobulin

T (Thymus Derived) Cells

In humans, T cells circulate through the thymus, where they gain specific CD markers, antigen specificity, and tolerance to self (see below). T lymphocytes maturing in the thymus express both CD4 and CD8 cell surface receptors during early development, but those exiting the thymus express only one of these cell surface markers. CD4-positive T cells typically develop into helper T cells when they reach peripheral lymphoid tissues. Helper T cell effector functions include the secretion of soluble mediators of immune responses such as cytokines. These cytokines, in turn, modulate B-cell, T-cell, and macrophage responses. A subset of CD4 T cells differentiates into regulatory T cells (CD4+CD25+Foxp3+). These latter cells are key suppressors of autoimmunity. CD8-positive T cells leaving the thymus are destined to mature into cytolytic T cells (CTLs), which function to lyse infected or otherwise altered target cells. ^,

B (Bone Marrow Derived) Cells

B cells appear to be educated in the bone marrow prior to exit into the peripheral immune system. Like T lymphocytes, B lymphocytes are antigen-specific. Unlike T lymphocytes, B cells function to secrete antibodies, which characterize humoral immune responses (see below).

ILC (Innate Lymphoid) Cells

ILCs are the third general class of lymphocytes, sharing transcriptional and cytokine effector profiles with adaptive T cells but lacking specific antigen receptors. Natural killer (NK) cells were previously thought to be the only innate lymphocytes but are now recognized to comprise only a subset of a much more diverse and complex system collectively known as ILCs. A classification system for ILCs has been recently agreed upon and categorizes ILCs into three groups: (1) Group 1: ILC1s and NK cells in intraepithelial (intestinal and other epithelial, inflamed mucosal, and tonsillar) locations; (2) Group 2-ILC2s in human lung and intestinal tissue (also in spleen and liver in mice); and (3) Group 3- ILC3s, which are predominantly gut-associated mucosal and lymphoid tissues.

NK cells have characteristic cell surface markers and receptors and their activities are typically not antigen-specific, although a form of surface receptor repertoire tuning is reflected in their long-term cellular identity. NK cells function in the recognition of cells lacking MHC class I products (see below) and in first-line defense against virally infected or oncogenically transformed target cells. NK cells also display cell surface receptors allowing recognition of antibody-coated target cells. Through these receptors, NK cells function as the major effector of antibody-dependent cellular cytotoxicity (ADCC).

Monocytes, Macrophage, and Dendritic Cells

Like other leukocytes, monocytes are derived from bone marrow stem cells. Monocytes circulate in the peripheral blood and populate nearly every tissue. Within specific tissues, monocytes mature into macrophages. In some tissues, these macrophages have been given specific names (e.g., alveolar macrophages in the lung and Kupffer cells in the liver). Macrophages are phagocytic cells that participate in development of innate as well as antigen-specific immune responses. They can signal T and B cells via secretion of cytokines and via delivery of antigen in forms recognized by these cells (antigen presentation). They may also stimulate direct lysis of altered target cells. Dendritic cells are closely related to macrophages and most derive from a similar cellular lineage in the bone marrow. Like macrophages, dendritic cells are phagocytic. Unlike macrophages, dendritic cells are distinguished by their pivotal role in antigen presentation, a process necessary for naïve T cells priming (initiation of primary immune responses) and subsequent development of antigen-specific B cell responses. ^,

Other Effector Cells

Neutrophils, eosinophils, and basophils are effector cells with specific importance in innate defense against pathogens. Each has also been associated with specific immune-mediated disease states. Eosinophils, for instance, are classically associated with defense against parasites and have pathophysiologic importance in asthma.

Soluble Components of Immune Responses

Immunoglobulins and Humoral Immunity

Immunoglobulin (Ig) molecules are comprised of dimerized subunits of heavy and light chains ( Fig. 16.1 . Section II.B.2.a.1). The N-terminal portions of each of the heavy and light chains are highly polymorphic and are termed their variable regions. The variable region of one heavy chain combines with the variable region of one light chain to give the immunoglobulin its antigen specificity. The C-terminal segments of the immunoglobulin heavy and light chains are called their constant segments and have minimal polymorphism. The constant portion of the immunoglobulin heavy chain interacts with other immune response components. Since these interactions are governed by heavy chain constant region isotype, so too is immunoglobulin effector function. Ig molecule isotypes include IgA, IgD, IgE, IgG, and IgM. IgG, IgE, and IgD molecules are typically present as single Ig molecules (monomers). IgA molecules usually circulate as dimers and IgM molecules as pentamers ( Fig. 16.2 . Section II.B.2.a.2).

Fig. 16.1, A typical immunoglobulin molecule.

Fig. 16.2, IgA dimers and IgM pentamers.

Each Ig isotype has distinctive functions. IgA dimers, connected by a joining or J chain, have been best described in the mucosa of the intestine and the female reproductive tract. Here, they are actively transported into the mucosal lumen via interaction of the J chain with mucosally derived secretory component. This allows exposure of IgA to antigens at the mucosal surface. IgE molecules interact with eosinophils in parasitic infections allowing eosinophil-mediated target lysis. IgE molecules are also implicated in delayed-type hypersensitivity via mast cell interactions. Membrane-bound IgD and IgM monomers are components of the antigen-recognizing B-cell receptor on naïve B cells. Pentavalent IgM molecules activate the complement cascade. Secreted IgM multimers are also characteristic of the early antigen-specific responses of naïve B cells.

Primary and Secondary Immune Responses

Collaboration between helper T cells and B cells is essential during the generation of primary immune responses. B cells expressing an antibody surface receptor bind to and internalize an antigenic particle (e.g., bacterium, virus). The particle is digested within B cells and peptide fragments are presented to circulating helper T cells by B cell-expressed MHC molecules. If a circulating, activated helper T cell recognizes the peptide antigen on the B cell, it will in turn provide an activating signal (through direct cell-cell receptor/ligand pairing and soluble cytokines) allowing the B cell to proliferate and produce antibody molecules in a process termed “licensing.” Continued helper T-cell activation in local lymph nodes is necessary to trigger affinity maturation, antibody class switching appropriate to tissue locale, and the formation of immunologic memory. Without strong and sustained T-cell help, B cells will produce mostly IgM and will not form immunological memory. As described earlier, the initial or primary immune response requires fairly significant antigenic stimulus and typically peaks approximately 5 to 10 days after antigen exposure. Primary responses usually involve more IgM than IgG secretion and the magnitude of the response is often lower than that after a second exposure to peptide antigen. Upon reexposure to the same antigen, a secondary response ensues. Secondary responses require less antigenic exposure than primary responses and they peak more rapidly (2–5 days postexposure). Secondary responses are typically more robust than primary responses and often do not require T-cell help. As a result of isotype switching, IgG rather than IgM molecules represent the predominant immunoglobulin subtype in secondary immune responses.

These IgG molecules have multiple immune effector functions. They can cross the placenta, allowing immune transfer from mother to fetus. IgG can directly bind antigen via its variable region. This allows the free constant portion of the IgG heavy chain (Fc portion) to be recognized and internalized by phagocytic cells in a process termed opsonization. Similarly, IgG bound to cell-associated antigen can signal lytic attack by cytotoxic T cells, NK cells, or NKT cells via a process called antibody-dependent cellular cytotoxicity (ADCC). Finally, IgG molecules can activate the complement cascade, resulting in cellular lysis.

Complement

The complement cascade is a vital part of innate immunity. It may be useful to compare the complement cascade to a more familiar clinical entity, the coagulation cascade. Both have components that circulate in inactive forms. Activation of each pathway can occur via two mechanisms. Reminiscent of the intrinsic and extrinsic coagulation pathways, the complement cascade can be activated via classical and alternative pathways ( Fig. 16.3 . Section II.B.2.c). The classical pathway is activated when a complement component C1 binds to antigen-antibody complexes (IgG or IgM). The alternative pathway is activated when complement component C3b binds to activating surfaces such as the cell wall of a bacterial pathogen. Each pathway sets off a series of activating enzymatic digestions of subsequent complement cascade components. Like the coagulation cascade, both of the activation pathways in the complement cascade intersect, joining as a final common effector pathway. Late components of the complement cascade can interact with specific complement receptors, resulting in pathogen phagocytosis and/or activation of the humoral immune system. Alternatively, activation of the complement cascade can result in the formation of a membrane attack complex (MAC) that results in indirect osmotic lysis of a target cell via the formation of an ion permeable transmembrane pore in the target cell.

Cytokines

The rapidly enlarging and pleiomorphic family of cytokines joins the immunoglobulins and the components of the complement cascade to complete a list of important soluble mediators of immune responses. Cytokines are a family of secreted proteins, all of which are generated by immune cells. They include the interleukins, the interferons, tumor necrosis factors, transforming growth factors, and the chemokines. The actions of cytokines may be autocrine, paracrine, or endocrine, and effects are mediated by specific cytokine receptors. Most cytokines have short half-lives, so direct actions are typically of short duration. However, cytokines often stimulate additional immune cell activity with cascading cytokine secretion and effects by target cells. Members of this large family of soluble immune mediators often have complementary and/or redundant activities.

In addition to myriad other roles in immunobiology, cytokines help to direct T helper cell differentiation. CD4+ T helper cells appear to circulate from the thymus to peripheral tissues in a functionally immature state (Th0). The T helper cell phenotype/polarization model holds that Th0 cells differentiate into their mature phenotypes based largely upon the cytokine microenvironment in which they reside at the time when they first recognize antigen and on the cell surface coreceptors expressed on the Th0 cell ( Fig. 16.4 . Section II.B.2.d). Certain cytokines appear to be of particular importance in the polarization of primary T helper cell responses. If antigen is recognized by naïve Th0 cells in an environment dominated by the cytokines interleukin 12 (IL12), IL18 and interferon gamma (IFNγ), the resulting T helper effector phenotype is characterized by the secretion of inflammatory-type cytokines, including interferon gamma (IFNγ), interleukin 2 (IL2), and tumor necrosis factor alpha (TNFα). This type of response is called a T helper 1 (Th1) response. Alternatively, if Th0 cells recognize antigen in a microenvironment dominated by IL4, the predominant T helper response is of the Th2 type and is characterized by secretion of IL4, IL5, IL9, and IL13. These Th2 cytokines, in turn, stimulate antibody production and allergic-type responses, including mast cell and eosinophil activation. Th1 responses enhance Th1 responses while inhibiting Th2 responses. Th2 responses initiate a positive feedback loop that promotes further Th2 activity while inhibiting Th1 effects. Several other T helper responses have also been distinguished. Th3 and Tr1 responses, ^, are characterized by the secretion of transforming growth factor beta (TGFβ); T regulatory (Treg) responses are defined by the secretion of IL10, and Th17 responses by the secretion of IL-17.

Fig. 16.4, The TH1/TH2 polarization paradigm.

Altered T helper cell polarization may lead to development or exacerbation of immune-mediated diseases. Although the etiologies are often incompletely described, these disorders are characterized by a dysregulation of cytokine secretion. For instance, patients with some forms of asthma appear to have inappropriate polarization of immune responses in their lungs toward the Th2 and Th17 phenotypes. ^, Those with Crohn disease polarize intestinal mucosal immune responses toward the Th1 phenotype. It has been hypothesized that some cases of spontaneous isolated and/or recurrent pregnancy loss may be the result of an insufficient Th2 polarization of maternal lymphocytes in the presence of placental antigens.

Basis of Immune Specificity and Immune Cell Education

While many of the salient attributes of the immune system can be addressed in a description of the cellular and soluble components of immune responses, there are two additional defining features of immune responses that require specific discussion: the specificity of immunologic antigen recognition and T lymphocyte education.

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