Transplant Immunology

CHAPTER 1

Tissues and organs of the immune system

Isam W. Nasr, Qiang Zeng, and Fadi G. Lakkis

Thomas E. Starzl Transplantation Institute, Departments of Surgery, Immunology, and Medicine, University of Pittsburgh, Pittsburgh, USA

CHAPTER OVERVIEW

Lymphoid organs or tissues are specialized anatomic compartments where lymphocytes develop, reside, and function.

Primary lymphoid tissues are the sites where lymphocytes undergo development, education, and maturation.

Secondary lymphoid tissues are the main sites where naïve lymphocytes engage foreign antigens to mount a primary immune response.

Tertiary lymphoid tissues are secondary lymphoid tissue-like structures that are induced at sites of chronic inflammation, and the function of such structures is not fully defined.

Memory immune responses can occur outside secondary lymphoid tissues. Memory T cells can also be maintained without secondary lymphoid tissues.

Primary and secondary lymphoid tissues are also necessary for tolerance induction and maintenance.

Introduction

It is the nature of scientists to be perpetually occupied with questions like where, how, and why things happen the way they do. Immunologists, in particular, are keen on answering the where question as it is central to understanding how immune cells are generated, what is required for their maturation, and whether they might mount productive responses against foreign antigens or not. The immune system is a bona fide organ system comprising primary and secondary lymphoid tissues (Figure 1.1). Primary lymphoid tissues (the bone marrow and thymus) specialize in generating immune cells from hematopoietic progenitors and transforming immature cells into mature lymphocytes with high specificity to foreign antigens (non-self) but not self antigens. Secondary lymphoid tissues, namely the spleen, lymph nodes, and mucosa-associated lymphoid tissues (MALTs) on the other hand are organized structures that are strategically located throughout the body to trap foreign antigens and ensure that they are best presented to T and B lymphocytes. The ability of an animal to mount a productive immune response is therefore critically dependent on the presence of the primary and secondary lymphoid tissues as well as the coordinated migration of immune cells into and out of these tissues.

Figure 1.1 Lymphoid tissues of the human body. The primary lymphoid tissues are the bone marrow and thymus. The secondary lymphoid tissues consist of lymph nodes, the spleen, and MALTs. Lymph nodes are arranged in strings along lymphatic vessels where they trap antigens and cells traveling in the lymph. The spleen intercepts antigens and cells circulating in the bloodstream. MALT includes the Peyer’s patches, adenoids, tonsils, and appendix. Cells traveling in the lymphatic system re-enter the blood circulation via the thoracic duct.

Source: Redrawn from Murphy (2011). Reproduced by permission of Garland Science/Taylor & Francis LLC.

This chapter will provide a comprehensive overview of the anatomy and function of primary and secondary lymphoid tissues and consider their roles in both transplant rejection and tolerance. Tertiary lymphoid tissues, which are secondary lymphoid tissue-like structures that are induced at sites of chronic inflammation, will also be discussed as they are thought to influence allograft outcomes. Controversies and unresolved questions will be highlighted where appropriate to encourage future investigations.

Primary lymphoid tissues

Primary lymphoid tissues are sites where T cells and B cells develop and mature, and mainly include the bone marrow and the thymus in mammals.

Bone marrow

The bone marrow is the site where both red and white blood cells are generated, by a process known as hematopoiesis. The adult human has two types of bone marrow: the red marrow, in which hematopoiesis is actively taking place, and the yellow marrow, consisting mainly of fat cells and lacking hematopoietic activity. At birth, all marrow is red but it is slowly replaced by yellow marrow over time. By adulthood, red marrow is restricted to flat bones (cranium, sternum, vertebrae, pelvis, and scapulae) and the epiphyseal ends of long bones (e.g., the femur and humerus), while the remaining marrow cavities are being occupied by fat cells. The bone marrow also provides a place where subsets of lymphocytes (both T cells and B cells), especially those with memory phenotypes reside.

Structure

Histologically, the red marrow consists of hematopoietic islands; such islands are mixed with fat cells, surrounded by vascular sinusoids, and interspersed throughout a meshwork of trabecular bone (Figure 1.2). The hematopoietic islands are organized into three-dimensional structures that provide optimal microenvironment for hematopoiesis. They contain blood cell precursors at different stages of maturation, stromal reticular cells, endothelial cells, macrophages, osteoblasts, osteoclasts, and the extracellular matrix. Both hematopoietic and nonhematopoietic cells in the islands orchestrate blood cell maturation through cell–cell contacts as well as production of growth factors, cytokines, and chemokines. Mature blood cells enter the circulation by migrating through the discontinuous basement membrane and between the endothelial cells of the vascular sinusoids.

Figure 1.2 Structure of the bone marrow. Example of red bone marrow (vertebra). Arrow points to a hematopoietic tissue island. Note fat cells (white globules) admixed with hematopoietic cells. Trabecular bone fills the space between islands.

Source: Reprinted from Travlos (2006). Reproduced by permission of SAGE publications.

Function

Hematopoietic stem cells (HSCs) are a pluripotent self-renewing cell type in the bone marrow that give rise to progenitor cells. These progenitor cells in turn generate all cells of the megakaryocytic (platelet), erythroid (RBC), myeloid, and lymphoid lineages (Figure 1.3). Myeloid cells (monocytes, dendritic cells or DCs, neutrophils, basophils, and eosinophils), natural killer (NK) cells, and B lymphocytes develop in the bone marrow, whereas T cell progenitors (pre-thymocytes) migrate to the thymus where they undergo further maturation (see section Thymus). The bone marrow also contains mesenchymal stem cells that give rise to nonhematopoietic tissues such as adipocytes, chondrocytes, osteocytes, and myoblasts. Mesenchymal stem cells have attracted considerable interest among transplant immunologists because of their immunosuppressive properties and prolonged survival features when adoptively transferred in select models.

Figure 1.3 Ontogeny of immune cells. Cells of the immune system arise from pluripotent HSCs in the bone marrow. The common lymphoid progenitor gives rise to B cells, T cells, and NK cells. The common myeloid progenitor gives rise to dendritic cells (DCs), monocytes, neutrophils, eosinophils, and basophils.

The bone marrow is the site where most stages of B cell maturation occur in mammals. B cell development in the bone marrow proceeds in a stepwise fashion from pro-B cells to pre-B cells, and lastly to immature B cells. During maturation in the bone marrow, B cells rearrange their immunoglobulin genes and express cell-surface IgM (the B cell receptor for antigen). These steps require close interactions with bone marrow stromal cells, which provide critical adhesion molecules, growth factors, chemokines, and cytokines (e.g., Flt3 ligand, thrombopoietin, CXCL12, and IL-7). Finally, autoreactive immature B cells are weeded out in the bone marrow through either clonal deletion or receptor editing before they are allowed into the circulation and complete their maturation in secondary lymphoid tissues.

In addition to serving as a primary lymphoid organ, the bone marrow is also a reservoir for mature myeloid and lymphoid cells. The bone marrow contains large numbers of neutrophils and monocytes that are mobilized into the circulation when needed (e.g., after infection). It is also the homing site for mature plasma cells, which are maintained in the bone marrow through the action of IL-6. Plasma cells are the principal source of antibodies in sensitized transplant recipients; therefore, investigators addressing the pathogenesis of antibody (historically referred to as humoral) rejection are increasingly interested in these bone marrow-resident plasma cells. There is also strong evidence that memory T cells home to or reside in the bone marrow where they can be activated by antigens. Other experiments have suggested that activation of naïve T cells could occur in the bone marrow under certain circumstances, raising the possibility that the bone marrow may additionally serve as a secondary lymphoid tissue (see section Secondary lymphoid tissues).

Cell trafficking

Cell trafficking is a dynamic process underlying allorecognition and transplantation responses, and remains a potential target in therapeutic strategies. Mature myeloid cells and certain precursor lymphoid cells (pre-thymocytes, immature B cells) migrate out of the bone marrow and enter the circulation. Conversely, mature lymphocytes (e.g., plasma cells) migrate into the bone marrow. The HSCs are also known to exit and re-enter the bone marrow. These trafficking events are primarily regulated by adhesion molecules and guided by chemokines. The integrin VLA-4, for example, maintains the developing B cells in tight contact with stromal cells by binding to VCAM-1. The chemokine CXCL12, which is produced by stromal reticular cells and osteoblasts, is responsible for retaining HSCs as well as myeloid and lymphoid cells in the bone marrow by binding to its receptor CXCR4. Some individuals with gain of function mutations in CXCR4 (e.g., WHIM syndrome) have pan-leukopenia because of increased retention of leukocytes in the bone marrow. Conversely, CXCR4 antagonism with the drug plerixafor mobilizes HSCs and myeloid and lymphoid cells from the bone marrow into the circulation. In the mouse, there is abundant evidence that CCR2 and its ligand CCL2 are responsible for monocytes exiting from the bone marrow into circulation.

Role in rejection and tolerance

The role of the bone marrow in organ transplantation has been studied in the context of tolerance induction. Investigators hoping to achieve solid organ allograft acceptance without immunosuppression have used simultaneous bone marrow and solid organ transplantation from the same donor. In this regimen, recipients receive partial myeloablative conditioning, followed by infusion of donor HSCs, with the goal being to induce mixed hematopoietic chimerism (both donor and host cells co-exist). Stable mixed chimerism can be attained in small experimental animals. It is more difficult in nonhuman primates (NHPs) and humans, but does result in long-lasting tolerance to allografts as re-emerging donor-specific B cells and T cells in transplant recipients are deleted or become anergic upon encountering donor antigens in the bone marrow and thymus, respectively. One obstacle to the widespread clinical use of the mixed chimerism is the need for toxic myeloablation conditioning prior to HSC infusion, as donor HSC engraftment is dependent on the presence of unoccupied hematopoietic niches or space in the recipient’s bone marrow. A very small proportion (0.1–1%) of niches are unoccupied under homeostatic conditions, severely limiting the number of exogenous HSCs that could engraft, even if infused in very large numbers. While irradiation and cytotoxic drugs (e.g., cyclophosphamide) have been the mainstay of freeing up niches in the recipient, less toxic therapies that target chemokines and chemokine receptors in the bone marrow are currently being investigated. Finally, the bone marrow alone is insufficient for sustaining primary immune responses as seen in mice that lack secondary lymphoid tissues but have an intact bone marrow are severely compromised in their ability to reject a transplanted organ.

Thymus

The thymus is the primary lymphoid organ where mature T cells are generated from bone marrow-derived progenitors (pre-thymocytes). The emergence of the thymus in evolution coincides with the emergence of adaptive immunity in jawed fish. In mammals, it is located in the upper anterior thorax above the heart. It owes its name to its lobular shape, which in the eyes of the Greek physician Galen resembled the thyme leaf. The thymus was considered to be a nonimmune organ for a long time until seminal work in mice by Jacques F. A. P. Miller and others in the 1960s demonstrated its central role in T lymphocyte development. It was found that removal of the thymus at birth leads to severe immune defects, including the abrogation of skin allograft rejection. Thymectomizing mice that had already reached puberty, on the other hand, had no significant effects on the immune response, indicating that the mature T cell repertoire had completely formed by then. The critical role of the thymus in T cell development in humans was confirmed in individuals with the congenital absence or severe hypoplasia of the thymus (DiGeorge syndrome). These individuals have very few T cells but normal B cell counts. Unlike in mice, removal of the thymus in infants or children does not lead to any obvious immune abnormalities, as T cell development in humans is largely completed prior to birth. Although the human thymus involutes significantly in size after puberty, thymic function persists in adults, especially in those who become lymphopenic secondary to either infection (e.g., HIV) or lymphodepletion (e.g., induction therapy at the time of transplantation).

Structure

Histologically, thymic lobes are made up of two clearly distinguishable areas: the cortex and the medulla and are separated by a highly vascularized corticomedullary border (Figure 1.4). The stroma in both regions consists of a three-dimensional network of thymic epithelial cells (TECs) surrounded by T cells. TECs are known by the acronyms cTECs and mTECs depending on whether they are located in the cortex or medulla, respectively. The cortex is densely populated with immature T cells in various stages of development, while the medulla harbors less tightly packed mature T cells. In addition to T cells, the thymus also contains DCs, macrophages, and B cells. As will be highlighted in the next section, cTECs, mTECs, DCs, and B cells play critical roles in T cell development, selection, and education. The thymic medulla in humans also contains distinct structures known as Hassall’s corpuscles that consist of concentric layers of keratinizing epithelium. These structures are a prominent site of T cell apoptosis and of thymic stromal lymphopoeitin (TSLP) production. TSLP is an IL-7-like cytokine believed to activate thymic DC (see section Function).

Figure 1.4 Structure of the thymus. The thymus consists of outer (cortical) and central (medullary) regions. Thymocyte maturation and T cell selection occur in both the cortex and medulla, with the outer cortical layer containing mainly proliferating, immature thymocytes and the deeper cortical and medullary areas containing immature T cells undergoing selection. Cortical and medullary epithelial cells, as well as bone-marrow derived DCs and macrophages, participate in the selection process.

Source: Redrawn from Murphy (2011). Reproduced by permission of Garland Science/Taylor & Francis LLC.

Function

The thymus is the primary site where T cell maturation and selection occur. The end result of these processes is the generation of a mature T cell repertoire that recognizes myriad foreign peptides in the context of self-MHC (self-restricted) and yet has been successfully purged of autoreactive T cells. Bone marrow-derived T cell progenitors enter the thymus via venules near the corticomedullary junction and begin their maturation in the thymic cortex where they proliferate extensively, acquire classical T cell markers (e.g., the CD4 and CD8 co-receptors), and undergo random rearrangement of T cell receptor (TCR) genes to form mature TCRs. Thymocytes that express functional TCRs then undergo positive and negative selection. Most (>95%) die by negative selection because they either fail to sufficiently bind self-MHC and self-peptide complexes, and thus be destined to be poor antigen recognizing cells (leading to immunodeficiency) or bind these complexes too strongly (destined to become potentially autoreactive cells). Only those with proper TCR affinity for self-MHC and peptide complexes are selected to undergo further maturation (positive