IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 15. Evolution of the Immune System

AMPHIBIANS AS A MODEL FOR STUDYING THE ONTOGENY OF IMMUNITY

In recent years, several isogenic and inbred clones of Xenopus have been developed for immunological research. Various Xenopus clones, either MHC-compatible or differing by one or two haplotypes, have proven to be exceptionally valuable for studying the ontogeny of The Immune System.

Development of the Thymus

Thymus development and thymectomy experiments. The African clawed frog X. laevis is ideally suited for investigating The Role of the thymus in immune system development, as free-living tadpoles can be thymectomized at very early developmental stages before the thymus has matured (Figs. 15.35 and 15.36). The paired thymus in Xenopus develops from the dorsal epithelium of the second pharyngeal pouch. Experiments show that lymphoid Cell precursors first invade the epithelial thymic rudiment on days 3–4 of development. By day 7, the T-Cell Differentiation antigen—the XNLA-I marker (120 kDa), recognized by the anti-thymus murine monoclonal antibody XT-I—begins to appear on thymic lymphoid Cells. The majority of thymocytes express T-cell surface Antigens by day 10 of development, coinciding with the first appearance of T-lymphocyte progenitor cells in the periphery. Metamorphosis is accompanied by a new wave of stem cell colonization, followed by thymic involution. Following metamorphosis, the thymocyte count increases again, reaching a peak by months 15–16 of development. Experiments involving early thymectomy in Xenopus (on days 4–5 of development) clearly demonstrate the existence of T-dependent (T_d) and T-independent (T_i) components of Immunity (Fig. 15.37). Following such early thymectomy, XT-I⁺ T cells (as well as CD4⁺ and CD8⁺ T cells) are no longer detectable in the Lymphoid Organs of tadpoles and adults, whereas B cells bearing surface IgM are abundantly present (Fig. 15.38). Therefore, in animals at this evolutionary level, the thymus appears to be absolutely essential for T-cell maturation. It should be noted, however, that early-thymectomized Xenopus sometimes undergo chronic rejection of MHC-incompatible Skin grafts. Following rejection, splenocytes from these animals, when tested in a mixed lymphocyte reaction, respond even to stimulator cells whose MHC molecules differ from those of the original donor, yet fail to respond to stimulation by the T-cell mitogen PHA. The Nature of these alloreactive cells remains unknown.

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Fig. 15.35. Thymus of 3- and 7-day-old Xenopus. 1. On day 3, the developing thymus (T) is still attached to the pharyngeal epithelium (PE) and consists primarily of epithelial cells. The branchial cleft (B) is also visible. Hematoxylin-eosin staining, × 100. 2. On day 7, the thymus contains fewer than a thousand cells of two main types: epithelial cells (E) with clearly visible nucleoli, dispersed Chromatin, and pale-staining Cytoplasm, as well as lymphoid cells (L) with abundant well-staining cytoplasm and numerous free Ribosomes and Mitochondria. By day 7 of development, the XT-1 marker appears on thymic lymphoid cells, and epithelial cells begin to express MHC class II Proteins. Electron micrograph, × 500.

Fig. 15.36. Thymus of a 38-day-old Xenopus. The pigmented paired thymus is located behind the eyes (top); its absence is clearly noticeable in a 7-day-thymectomized sibling (bottom).

Fig. 15.37. HUMORAL Immune Response, cell-mediated response to skin allografts, and in vitro mitogenic responses were examined in Xenopus thymectomized on days 4–6 of development. (LPS — lipopolysaccharide; SRBC — sheep red Blood Cells; PHA — phytohemagglutinin; T_i — T-independent; T_d — T-dependent.)

Fig. 15.38. Thymus and Spleen cells from a control Xenopus (4 months old) and spleen cells from a sibling thymectomized at 7 days of age were first stained with either murine anti-T-cell (XT-1) Monoclonal Antibodies (mAbs) or murine anti-B-cell (anti-IgM) mAbs. Fluorescein isothiocyanate-labeled anti-mouse immunoglobulin antibodies served as secondary antibodies. T AND B cells were then detected using a fluorescence-activated cell sorter. In the early-thymectomized tadpole, the XT-1⁺ T-cell population has disappeared, being replaced by a corresponding number of B cells. The number and percentage in each plot represent the fraction of positive cells, i.e., those located to the right of the marker threshold (gray bar) (excluding 98% of Background fluorescence).

Tadpole thymectomy experiments indicate that the appearance of functionally diverse peripheral T cells requires the presence of the thymus at various developmental stages (Fig. 15.39). Studies on intact animals have shown that responses to allogeneic Tissues (in vivo and in vitro), together with the capacity of splenocytes to respond to T-cell mitogens, appear early in tadpole development, whereas a robust IgY response is achieved only in the adult, concurrent with The Emergence of T-cell helper function.

Fig. 15.39. Ontogeny of immunoreactivity in Xenopus and The Effect of thymectomy at various developmental stages. Allograft rejection, mixed lymphocyte reaction, and T-cell mitogenic responses are observed as early as the initial Selection/3.html">Stages of development. T_h cells, particularly those mediating the primary "IgY" response, appear significantly later. (Circles indicate the age after which thymectomy no longer impairs the respective function.)

Thymic "education" in Xenopus involves both positive and limited negative selection. A foreign thymus grafted onto a frog following early thymectomy Supports the differentiation of recipient precursor cells along the T-cell Lineage (Fig. 15.40). By joining the anterior part of a 24-hour embryo containing the epithelial thymic rudiment with the posterior part of an MHC-incompatible embryo that generates hematopoietic stem cells, including lymphocytes, it is straightforward to produce in vivo thymuses containing epithelial and lymphoid components that express distinct MHC markers (Fig. 15.41).

Fig. 15.40. Thymus implantation in a thymectomized Xenopus. 1. An African clawed frog (X. laevis) was thymectomized on day 7 of development and subcutaneously implanted with a thymus from a late-stage donor tadpole (X. borealis). Following metamorphosis, excellent growth of the implant (I) adjacent to the left eye was observed. 2. Section of the implanted thymus viewed under a fluorescence Microscope. Donor cells (D) can be distinguished from recipient cells because X. borealis nuclei fluoresce as bright dots, whereas X. laevis cells exhibit a uniform green color. The thymus is populated by recipient lymphocytes, while many stromal cells remain "patched" (i.e., of donor origin). Quinacrine staining, × 300.

Fig. 15.41. Chimeric frogs. Xenopus chimeras were generated by cross-transplanting the anterior and posterior parts of two embryos 24 hours post-Fertilization. At this stage, the thymic rudiment (i.e., its epithelium) is located in the anterior part, while all lymphocyte precursors reside in the posterior part of the embryo. One embryo was an albino variant (with white skin and red eyes), and the other was a normal Xenopus. These chimeras were used to study cellular "education" within the thymus. (Photographs kindly provided by Dr. M. Flajnik and Dr. L. DuPasquier.)

Both of these experimental models have been utilized to investigate the role of thymic stromal cells in "education" processes, which include negative selection (induction of T-cell tolerance to autoantigens) and positive selection (restriction to specific MHC antigens predominantly recognized by helper and effector T-cell populations). Results from such experiments in Xenopus indicate that foreign thymic epithelium participates in positive selection and induces tolerance to skin grafts bearing the MHC Specificity of the thymus, although, surprisingly, this tolerance does not prevent mixed lymphocyte reactions directed against the donor thymic cells. Similar findings have been obtained in avian and mammalian embryos. However, in mammals, negative selection (the deletion of T cells with high affinity for self-MHC molecules) is believed to be mediated primarily not by thymic epithelial cells, but by immigrant interdigitating (dendritic) Cells of the thymic stroma.

Ontogeny of alloimmunoreactivity/tolerance and antibody production. Immune responses to alloantigens (MHC) and The production of specific antibodies are "switched on" in tadpoles concurrently with the appearance of the necessary T- and B-cell populations in the periphery (Fig. 15.42); this can occur when the lymphoid system contains fewer than a million lymphocytes. Immunological memory persists through metamorphosis, although whether this relies on The transfer of memory cells or the persistence of antigen remains unknown. Nevertheless, immunocompetent tadpoles (though not adults) can readily be rendered tolerant to allogeneic skin; such "allotolerance" is particularly easily induced during metamorphosis (Fig. 15.43). Graft size and the degree of histoincompatibility are critical factors in this process. In Xenopus, tadpoles and metamorphosing individuals are invariably tolerant to grafts whose MHC molecules differ only slightly from those of the host. Tolerance induced in tadpoles by transplantation of foreign skin or lymphoid tissue is rarely "complete," as certain "antidonor" reactions (e.g., the MLR) persist both before and after metamorphosis. The underlying mechanism of this tolerance appears to be suppression or anergy.

Fig. 15.42. Embryonic tissue transplantation in Rana: the ontogeny of "alloimmunity". 1. A small piece of the neural fold, removed from an embryo at the tail-bud stage, was grafted into the middle of the ventral surface of another embryo (the host). Elements of the neural crest, which are precursors of various cell types including pigment cells, are closely associated with the neural folds. Distinctive pigment cells provide an excellent marker for tracking The Fate of the embryonic graft. In the host tadpole, pigment cells derived from the graft form a clearly visible mass. 2. A histological section (15 days post-transplantation) reveals differentiated graft elements, large ganglionic cells (G) with distinct nucleoli, other neural tissue (N), and melanin (M). Despite the short time interval, lymphocytes and granulocytes have already infiltrated the graft (L — leukocyte). Hematoxylin-eosin staining, x 100. (Photographs kindly provided by Dr. E. Volpe)

Fig. 15.43. Skin graft tolerance in Xenopus. A recipient at the tadpole or metamorphic stage can become tolerant to allogeneic skin grafts, even from an MHC-mismatched donor. Subsequent skin grafts (here, a piece of white belly skin) from the same donor successfully engraft in the adult frog. However, skin from a different donor is rejected within 3 weeks (at 25 °C).

Model systems for studying the origins of lymphoid cells

Experiments involving the transplantation of cytogenetically distinct gill primordia in Rana pipiens, Xenopus laevis, and the newt Pleurodeles waltlii have confirmed that, as in homeothermic animals, thymic lymphocytes develop from precursor cells that colonize the thymus from outside. In Xenopus, lymphoid precursor destined to enter the thymus originate from both the ventrolateral (ventral blood islands) and dorsolateral mesodermal plates of the embryo. In young Xenopus, stem cells colonize the thymus in two distinct waves: the first occurs during early tadpole development, and the second during metamorphosis, which presumably ensures the "education" of mature T cells in an environment where adult-specific antigens are expressed.

It has been established that in Xenopus, cells of certain hematopoietic differentiation lineages express the pan-leukocyte antigen CD45, which exhibits Tyrosine phosphatase activity.

Metamorphosis imposes new demands on the immune system

It is extremely interesting to understand how amphibians avoid death from autoimmune diseases during metamorphosis, given that mature cell-specific markers appear for the first time during this period. The role of class I MHC molecules, which are newly expressed during metamorphosis and very likely perform suppressive Functions, also remains to be elucidated. At the same time, metamorphosis is accompanied by high plasma corticosteroid levels and increased expression of corticosteroid receptors on lymphocytes. Such hormonal shifts can lead to the direct suppression of cell-mediated immunity, possibly by inhibiting IL-2 production. Amphibian metamorphosis provides a highly convenient model for studying neuroendocrine-immune interactions, and not only from a phylogenetic perspective.

Questions for Thought

■ What cellular and humoral immune mechanisms have been discovered in invertebrates?

■ Discuss the main differences between the invertebrate and vertebrate immune systems. What evolutionary factors might have driven The Development of the vertebrate immune system?

■ How is antibody diversity generated in various vertebrates?

■ What is currently known about the Evolution of the Major Histocompatibility Complex and the associated phylogeny of T cells?

■ What are the advantages of the amphibian Xenopus laevis as a model Organism for studying the Ontogeny of the immune system?

■ What are the major gaps in our current understanding of the evolution of immunity, and what approaches could be proposed to address these questions?

Introduction/47.html">Further Reading

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