Myeloid-lymphoid ontogeny in the rhesus monkey (Macaca mulatta)

Abstract

Establishment of a functional immune system has important implications for health and disease, yet questions remain regarding the mechanism, location, and timing of development of myeloid and lymphoid cell compartments. The goal of this study was to characterize the ontogeny of the myeloid-lymphoid system in rhesus monkeys to enhance current knowledge of the developmental sequence of B-cell (CD20, CD79), T-cell (CD3, CD4, CD8, FoxP3), dendritic cell (CD205), and macrophage (CD68) lineages in the fetus and infant. Immunohistochemical assessments addressed the temporal and spatial expression of select phenotypic markers in the developing liver, thymus, spleen, lymph nodes, gut-associated lymphoid tissue (GALT), and bone marrow with antibodies known to cross-react with rhesus cells. CD3 was the earliest lymphoid marker identified in the first trimester thymus and, to a lesser extent, in the spleen. T-cell markers were also expressed midgestation on cells of the liver, spleen, thymus, and in Peyer's patches of the small and large intestine, and where CCR5 expression was noted. A myeloid marker, CD68, was found on hepatic cells near blood islands in the late first trimester. B-cell markers were observed mid-second trimester in the liver, spleen, thymus, lymph nodes, bone marrow spaces, and occasionally in GALT. By the late third trimester and postnatally, secondary follicles with germinal centers were present in the thymus, spleen, and lymph nodes. These results suggest that immune ontogeny in monkeys is similar in temporal and anatomical sequence when compared to humans, providing important insights for translational studies.

Keywords: fetus; immune system; infant; monkey; ontogeny.

Figure 1. Rhesus monkey liver, spleen, and thymus

A. CD68+ cells were only found within blood islands (examples circled) in the liver in the first trimester, but were also observed within hepatocytes (H) in the second trimester. Near term, blood islands were no longer present and infrequent CD68+ cells with morphology typical of Kupffer cells (K) were noted. In postnatal liver, CD68+ cells were most frequently found near venules (V). B. CD3+ cells were noted occasionally in the splenic mesenchyme in the first trimester. By the second trimester, organization of early follicles (dotted circle) with white pulp (WP) and red pulp (RP) regions were evident. Central arterioles (CA) were surrounded by CD3+ cells in developing periarteriolar lymphatic sheaths (PALS). CD3+ cells were concentrated in the PALS in postnatal follicles with a few positive cells noted in germinal centers (GC) and marginal zones (MZ). C. CD20+ cells were not observed until the second trimester in the spleen and were usually associated with white pulp follicles (dotted circle) by the third trimester. CD20+ cells were concentrated in the germinal center (GC) and marginal zone of the white pulp at 3 months of age. D. Developing lobules of the thymus were composed of CD3+ cells in the first trimester. Cortical (C) and medullary (M) regions were evident from the second trimester onward with nearly all cortical cells expressing CD3. The medulla contained CD3+ cells and unstained thymic epithelial cells. Small clusters of epithelial cells in Hassall’s corpuscles (HC) were noted from the early second trimester and increased in frequency with advancing gestation. E. FoxP3 expression was similar to CD3 in the first trimester with strong positive staining of nearly all thymic lobular cells. Expression of FoxP3 was restricted to a few cells in the medulla from the second trimester and onward. All images 20x except first trimester CD68 (40x), third trimester CD3 (10x), and postnatal CD20 (10x). Calibration bar = 100 μm.

Figure 2. Rhesus monkey axillary and mesenteric lymph nodes

A. In the second trimester, lymph nodes were surrounded by subcapsular sinuses. CD3 was strongly expressed in cells surrounding venules (solid ovals). As lymph nodes developed, CD3 expression was noted on nearly all cells of the paracortex (PC) except trabeculae (arrow), and less frequently in the follicle (F). B. CD4 was expressed near the subcapsular sinus (SS), and with follicle maturation was noted most frequently in the paracortex (PC) and near the medullary sinuses (M). Follicles (dotted circles) were present in the third trimester, with progression to secondary follicles containing germinal centers (GC) surrounded by coronal regions (Co) by 3 months postnatal age. C. Cells expressing FoxP3 were found as individual or small clusters of cells (arrows) in the cortical parenchyma (C), and were also noted with greater frequency in the paracortex (PC). With follicular maturation, frequency of CD68+ cells decreased and individual positive cells were noted in germinal centers (GC) and the surrounding corona (Co) of the follicles after birth. D. Expression of CD20 was concentrated near subcapsular sinuses (SS) opposite the hilum (H) of the developing lymph nodes. Individual CD20+ cells were also found throughout the developing parenchyma. Beginning with the third trimester most CD20+ cells were localized in developing follicles (F) (dotted circles). After birth, positive cells were also noted infrequently in the paracortex (PC). E. CD68+ cells were found along the border of the developing subcapsular sinus (SS), and localized to the cortical margins (C) of maturing follicles and medullary regions as development progressed. After birth, positive cells were less frequent and, when observed, were located in the medulla or the germinal centers (GC) of cortical (Co) follicles (dotted circle). All images 20x except second trimester CD20 and CD68 (10x), and postnatal CD3 (10x). Calibration bar = 100 μm.

Figure 3. Ontogeny of T lymphocytes in rhesus monkey jejunum, ileum, and colon

A. CD8 was expressed in the lamina propria (LP) of elongated villi and in the submucosa (SM) in first trimester jejunum. CD8+ cells were also located within the surface epithelium in the ileum. Muscularis mucosa (MM). B. In the second trimester, CD3+ cells were located in the lamina propria (LP), developing Peyer’s patches (PP), and submucosa (SM). C. Mature follicles (F) were evident within Peyer’s patches in the third trimester. CD4+ cells were found with greater frequency in the interfollicular regions than within the follicle center. Submucosa (SM). D. FoxP3+ cells were found as individual cells within Peyer’s patches (arrows), submucosa (SM), and lamina propria (LP). All images 20x except third trimester jejunum and colon (10x). Calibration bar = 100 μm.

Figure 4. Ontogeny of B lymphocytes, macrophages, and dendritic cells in rhesus monkey jejunum, ileum, and colon

A. Cells expressing B cell markers CD20 or CD79 (not shown) were not found in the submucosa (SM), lamina propria (LP), or surface epithelium in the late first trimester. B. Cells expressing the dendritic marker, CD205 (small arrows), were noted within the pseudostratified surface epithelium between the villi and within Peyer’s patches (PP) in the second trimester. Submucosa (SM). C. Cells expressing CD68 (macrophage marker) (arrows) were more abundant in the third trimester jejunum and ileum, were located in the tips of the lamina propria (LP), and as individual cells in Peyer’s patches (PP). Submucosa (SM). D. Postnatal Peyer’s patches contained distinct follicles (F) with a majority of cells CD20+ (not shown) or CD79+ (arrow). Dense CD20+ cells were found in the coronal region (Co) between the germinal center of the follicles and the surface epithelium. All images 20x except first trimester colon (10x), second trimester ileum (40x), and postnatal jejunum, ileum, and colon (10x). Calibration bar = 100 μm.

Figure 5. Expression of CD4 and CCR5 in the developing colon of rhesus monkeys

A. Cells expressing CD4 were found in developing Peyer’s patches, submucosa (SM), and lamina propria surrounding the crypts (Cr). CD4+ cells were not observed in the muscularis (MM). B. Cells expressing CCR5 were observed in the epithelium, Peyer’s patches, submucosa (SM), and lamina propria at mid-gestation and at later gestational ages. In sequential sections, CCR5+ cells were noted in locations where CD4+ cells were found (representative regions highlighted with dotted circles). All images 20x. Calibration bar = 100 μm.

Figure 6. Lymphoid and myeloid ontogeny in rhesus monkey bone marrow

A. Developing long bones were composed largely of cartilage (C) and epiphyseal plates (EP) in the first trimester. B. Medullary cavities in the second trimester contained individual cells positive for CD20 (arrows) or CD68 (arrows), but not CD3. C. CD68 was noted with greater frequency than CD3 or CD20 in the third trimester marrow cavities. Arrows denote representative positive cells within CD3 or CD20 panels. D. Postnatal long bones at 3 months contained adipose (A) and reticular cells (R) with modest expression of CD3 and CD68 and frequent expression of CD20. All images 20x except third trimester CD20 (40x). Calibration bar = 100 μm.

Figure 7. Temporal sequence of immune ontogeny in human, monkey, and mouse

The liver is the major site of hematopoiesis in human and nonhuman primates from the mid-first trimester until bone marrow hematopoiesis is established in the mid-second trimester. Thymic and splenic demarcation is completed by mid-gestation followed by the presence of B and T cells within Peyer’s patches prior to the third trimester. Lymph node morphogenesis including formation of germinal centers is completed prior to birth in both species. In contrast, the mouse immune system is immature at birth with developmental events beginning in mid-gestation and with little or no development of Peyer’s patches and lymph nodes until after birth.