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 subsequent text). 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, key suppressors of autoimmunity. CD8 positive T cells leaving the thymus are destined to mature into either cytolytic T cells (CTLs), which function to lyse infected or otherwise altered target cells, or into suppressor T cells, which are currently called regulatory T (Treg) cells.

B (Bone Marrow Derived) Cells

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

Innate Lymphoid Cells

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. 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 in 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

Similar to 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 the 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. Similar to macrophages, dendritic cells are phagocytic. Unlike macrophages, dendritic cells are distinguished by their pivotal role in antigen presentation, a process necessary for naïve T-cell priming 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.

Immunoglobulins and Humoral Immunity

Ig molecules are composed of dimerized subunits of heavy and light chains ( Fig. 13.1 ). The N-terminal portions of each of the heavy and light chains are highly polymorphic and are termed variable regions . The variable region of one heavy chain combines with the variable region of one light chain to give the Ig its antigen specificity. The C-terminal segments of the Ig heavy and light chains are called constant segments and have minimal polymorphism. The constant portion of the Ig heavy chain interacts with other immune response components. Since these interactions are governed by heavy chain constant region isotypes, the Ig effector functions are also governed by the isotypes. Ig molecules 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. 13.2 ).

A typical immunoglobulin (Ig) molecule.

The constant region (carboxy [COOH] terminal) isotype of the heavy chain determines function. The combined variable regions (amino [NH ₂ ] terminal) of the heavy and light chains determine antigen specificity. IgG, IgE, and IgD isotypes are typically monomers of this basic structure.

Immunoglobulin (Ig) A dimers and IgM pentamers.

Each structure is connected by a joining chain (J-chain) .

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 components. 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 (DTH) 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 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 and soluble cytokines), allowing this B cell to proliferate and produce antibody molecules—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 secretion than IgG 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 to 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 Ig 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, promoting a process called ADCC. Finally, IgG molecules can activate the complement cascade.

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. 13.3 ). 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. Similar to 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.

Activation pathways for complement-mediated cytolysis.

The classical pathway of complement activation is initiated by the binding of antigen-antibody complexes to complement factor Cl. The binding of C3b to activating surfaces such as a bacterial cell wall activates the alternative pathway. Activation of either pathway results in a cascade of subsequent enzymatic activations. These pathways converge, forming the membrane attack complex (MAC) . The MAC allows osmotic shifts that lyse the affected target cell. Complement-mediated activities can also include promotion of inflammatory responses, direct opsonization of pathogenic substances, and clearance of unwanted immune complexes.

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. 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. 13.4 ). Certain cytokines appear to be of particular importance in the polarization of T helper cell responses. If antigen is recognized by Th0 cells in an environment dominated by the cytokines interleukin 12 (IL-12), IL-18, and interferon gamma (IFN-γ), the resulting T helper effector phenotype is characterized by the secretion of inflammatory-type cytokines, including IFN-γ, interleukin 2 (IL-2), 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 IL-4, the predominant T helper response is of the Th2 type and is characterized by secretion of IL-4, IL-5, IL-9, and IL-13. These Th2 cytokines, in turn, stimulate allergic-type responses, including mast cell and eosinophil activation and antibody production. Th1 responses enhance Th1 responses while inhibiting Th2 responses. Th2 responses initiate a positive feedback loop, which promotes further Th2 activity while inhibiting Th1 effects. Several other T helper responses have also been distinguished. Th3 and Treg1 responses are characterized by the secretion of transforming growth factor beta (TGF-β); Treg responses are defined by the secretion of IL-10, and Th17 cells are defined by the secretion of IL-17.

The Th1/Th2 polarization paradigm.

The cytokine microenvironment in which antigen is presented by antigen-presenting cells to immature (Th0) T helper cells promotes development into Th1, Th2, Th17, and Treg phenotypes. It appears that each T helper phenotype supports further development of itself while opposing developments of its counterparts. FoxP3 , Forkhead box P3; IFN-γ , interferon gamma; IL , interleukin; TGF-β , transforming growth factor beta.

Altered T helper cell polarization may lead to 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 towards 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 (RPL) may be the result of an insufficient Th2 polarization of maternal lymphocytes in the presence of placental antigens.

Immunoglobulins in Genital Tract Secretions

Ig concentrations in the genital tract are dependent on hormonal and local factors such as the presence of inflammation. Several studies concerning the origin of antigen specific IgG in the female genital tract secretions suggest that locally produced, as well as plasma-derived, IgG contribute to the IgG pool in genital tract secretions. Regardless of the source, the route of passage of IgG into the lumen of the genital tract—passive, paracellular diffusion or active, receptor-mediated transport—has not been elucidated.

The IgA of cervical mucus is primarily secretory-IgA, with a small component of monomeric IgA. The IgA subclass distribution resembles that of the lower intestinal tract with IgA1 and IgA2 present in roughly equal proportions. The increased proportions of IgA2 may provide a functional advantage to certain specific antibodies, because they are resistant to the IgA1 proteases of many pathogenic bacteria.

Cytokines in Genital Tract Secretions

Unlike other mucosal tissues, the reproductive tract is characterized by progressive tissue growth and remodeling during each menstrual cycle. Cell proliferation, apoptosis, and cell migration are essential for reproductive tract renewal to occur. Growth in preparation for fertilization, implantation, and successful pregnancy is mediated by a changing pattern of cytokine and adhesion molecule expression that is regulated by the sex steroid hormones. Multifunctional cytokines such as Il-1β, IL-6, and IL-10 that are produced throughout the reproductive tract have important roles in the maturation of B lymphocytes to Ig-producing plasma cells. Estrogen increases the expression of polymeric IgA receptor, thereby increasing the transport of IgA into the lumen, when cultured human endometrial cells are incubated with IL-4 and IFN-γ. Regulatory cytokines, such as IL-10, IL-1β, and IL-6 are present in human cervical mucus, and their levels correlate with the cyclic reproductive hormones estradiol and progesterone. Cytokines and chemokines are differentially secreted from immortalized human endocervix–derived epithelial cell lines depending on the hormone milieu in which the cells are exposed.

Cells of the Female Reproductive Tract

Estradiol and progesterone secreted during the menstrual cycle act both directly and indirectly on epithelial cells and other immune cells to modify immune function in a way that is unique to specific sites throughout the female reproductive tract. The female reproductive tract contains a full spectrum of leukocytes with the potential to function in ways similar to those present in other mucosal tissues and in the peripheral blood. Macrophages, distributed primarily between the epithelial and myometrial layer, are found both scattered throughout the fallopian tubes, cervix, vagina, and in small aggregates in the uterus. A tissue-specific population of uterine/decidual CD56 ^bright CD16 ⁻ natural killer (uNK) cells, distinct from peripheral blood CD56 ^low CD16 ⁺ NK cells, are present in uterine endometrium and increase in number during the secretory phase of the menstrual cycle and during the first trimester of pregnancy. They are present at the maternal-fetal interface and have been implicated in cytotrophoblast (cytoT) guidance into the decidua and vascular remodeling necessary for proper decidualization. Similar to other mucosal tissues, it is also now apparent that uNK cells are merely a part of a more complex milieu of ILCs newly discovered in the uterine tissue. Immune cells make up 6 to 20% of the total nonmyometrial cells in the fallopian tube, uterus, endocervix, ectocervix, and vagina and are not significantly influenced by the stage of the menstrual cycle. Approximately 50% of the leukocytes in the reproductive tract are CD3+T cells, with the remainder consisting of neutrophils (CD66b+), B cells (CD19+), and monocytes (CD14+). In particular, NK cells and CD3+ T lymphocytes, including the CD4+ T helper/regulatory cells and CD8+ T cytotoxic cells, are the two major leukocyte subsets present of the endometrial leukocytes. Studies have demonstrated that cervical class I MHC-restricted CD8+ and class II MHC-restricted CD4+ cytotoxic T cells can lyse autologous target cells made to express HIV-1 and other viral proteins.

Discrete lymphoid aggregate (follicles) in the endometrium are composed primarily of a B-cell core (CD19+) surrounded by T cells (CD3+, CD8+, CD4−) and an outer halo of macrophages (CD14+). Antigen-presenting cells in the reproductive tract, which are responsible for initiating the immune response by presenting antigens to T lymphocytes, are derived proportionally from reproductive tract cells and peripheral blood mononuclear cells.

Immune Cell Subtypes

As mentioned previously, the immune cell repertoire of the human endometrium differs dramatically from that of the peripheral blood and is characterized by very few B lymphocytes, a larger complement of T cells, macrophages, ILCs/uNKs, and a number of more specialized immune effector cell subtypes. The relative proportions of these resident cells demonstrate remarkable menstrual cyclicity. A few of these cellular populations deserve specific description, as their presence in both the female reproductive tract and in the periimplantation decidua implicate a particularly important role for innate immune interactions during implantation and early pregnancy.

Peripheral immune cells have been described that have characteristics of both NK cells and T cells. These NKT cells and their ligands (e.g., CD1) have been detected in the endometrium and decidua of humans. NKT cell interactions with the MHC-like antigen presenting molecule, CD1d, can result in proinflammatory or in tolerogenic effector functions, suggesting that these interactions may be important in maintaining the delicate immune balance at the maternal-fetal interface. Other NK-like cells also populate the late luteal phase endometrium and decidua of women. These unusual cells have been variably called decidual granular lymphocytes, large granular lymphocytes (LGLs), and decidual natural killer cells (dNKs), and they exhibit phenotypic differences in their cell surface CD expression from typical NK cells found in the periphery. Still, most investigators consider them to be NK cell variants. The proportion of uNK cells in the decidua of early pregnancy rises to nearly 70% to 90% of the total endometrial lymphocyte population. Recent studies reveal that this lymphocyte mix is even more complex than previously understood as all three ILC groups, in addition to uNKs, were recently discovered at this site. While NK cells can display activating and inhibitory receptors on their cell surfaces, the predominant interactions of those in the decidua involve killer inhibitory receptor (KIR) ligation with trophoblast MHC class I products (see previous text) and the outcome of these interactions is unlikely to be cytotoxic. Rather, the cytokine and chemokine products of the decidual NK cell tend to be tolerogenic and to support trophoblast invasion and decidual spiral artery remodeling. Very elegant investigations have shown that decidual NK cells are derived from a small population of endometrial NK cells that await pregnancy for robust expansion. These cells do not appear to be recruited from the peripheral blood, as previously hypothesized. Thus the measurement of peripheral NK cell number and/or activity as a predictor of pregnancy outcome is unlikely to reflect aberrancy in the function of decidual NK cells in pregnancy maintenance.

Animal and human data have also suggested roles for “suppressor” macrophages and TCR γδ+ T lymphocytes in the immunology of early pregnancy. Mimicking the subcategorization of Th cells, macrophages have been classified based on their cytokine secretion patterns and the abundant macrophages of the uterine decidua typically exhibit an immunosuppressive phenotype or M2 polarization. The M1 phenotype is proinflammatory. In contrast, M2 cells exert robust IL-10 secretion and indoleamine 2,3-dioxygenase (IDO) activity. As mentioned earlier, TCRγδ+ T cells have presumed importance in innate immunity. Their presence within the human endometrium increases dramatically in early pregnancy, they preferentially secrete IL-10 and TGF-β, and they support trophoblast invasion and inhibit trophoblast apoptosis. All should promote pregnancy maintenance.

Soluble Immune Effectors

The human endometrium and decidua are replete with immune and inflammatory cells capable of cytokine and chemokine secretion, but the role of individual factors in pregnancy maintenance may be fairly minimal. Most cytokine genes have been individually deleted in animal models and few, if any, of these factors appear to be absolutely required in pregnancy maintenance. Still, two are frequently discussed as particularly integral. Leukemia inhibitory factor (LIF), and IL-11, both IL-6 family cytokines, have been demonstrated to be required for blastocyst implantation, but not for subsequent embryogenesis in mice, indicating that the defect induced by their deletion is in the maternal compartment. Further delineation of the mechanisms for these implantation abnormalities indicate that LIF functions at the blastocyst attachment/adhesion phase and IL-11 in the response to the implanted blastocyst. The fact that essential roles for individual cytokines in pregnancy maintenance are so limited speaks to the likely redundancy in this system and spurs future studies addressing the integrated effects of multiple cytokines.

Application of the Th1/Th2 paradigm to pregnancy maintenance has led to the hypothesis that the intrauterine environment surrounding normal human pregnancies is Th2-dominant and in vitro data have shown that peripheral blood cells from women with normal pregnancy histories secrete Th2 type cytokines when exposed to placental antigens; some women with histories of RPLs have Th1 responses in the same in vitro assay systems. Inflammatory cytokines characteristic of Th1 responses (e.g., IFN-γ) have been shown to be harmful to the implanting embryo. While the Th1/Th2 paradigm has been useful as a stepping stone in our understanding of the immunology of pregnancy maintenance, rapid expansion in descriptions of cytokine/chemokine activities and in T helper cell subpopulations suggests that the Th1/Th2 paradigm must also be expanded to include Th17, Th3, Treg, and possibly even more cytokine-secreting immune cells at the maternal-fetal interface. Furthermore, the antigen recognized at the site of implantation that might cause dysregulation of the local T helper response phenotype has yet to be identified in vivo. In vitro data have been based on the supposition that the antigen(s) is placental in origin, which is a reasonable, but unverified hypothesis. Cytokine profiles in the microenvironment of an implanting fetus are also difficult to document, and dysregulation in the presence of a failing pregnancy will always be obfuscated by the question of cause versus effect. The possibility that dysregulation of T helper response causes pregnancy loss remains intriguing but unproven.