0031-3998/90/2703-0239$02.00/0 PEDIATRIC RESEARCH Vol. 27, No.3, 1990 Copyright © 1990 International Pediatric Research Foundation, Inc. Primed in U. SA. The Distribution of T and B Lymphocyte Populations and MHC Class II Expression in Human Fetal and Postnatal Intestine GARY J. RUSSELL, ATUL K. BHAN, AND HARLAND S. WINTER Combined Program in Pediatric Gastroenterology and Nutrition, The Children 's Hospital and the Department of Pathology, M assachusells General Hospital; Harvard Medical School, Boston, M assachusells 02115 ABSTRACT. The development of the intestinal mucosal MATERIALS AND METHODS immune barrier is an important protective adaptation for Tissue samples. Fetal tissues were obtained within 2 h after postnatal life. The distribution and phenotype of T and B prostaglandin/saline-induced abortions. The estimated fertiliza­ lymphocytes in human fetal intestine and lymphoid tissues tion age of the fetuses was determined by crown-rump length, have been characterized and compared to the distribution foot length, and fetal wt during postmortem examination ( 10, and phenotype of lymphocytes in postnatal intestine. The II). The following tissues were harvested from six fetuses, 17- characterization of lymphocyte phenotype and MHC class 21 wk estimated fertilization age: proximal jejunum, distal ileum, II antigen distribution was done using MAb and an avidin­ cecum, colon, spleen, thymus, and liver. Procurement of human biotin complex immunohistochemical staining technique. tissues and the consent forms were approved by the Committee Intraepithelial lymphocytes were occasionally present in for the Protection of Human Subjects from Research Risks at fetal intestine and were primarily CD3+, ens+. T lympho­ the Brigham and Women's Hospital and The Children's Hospi­ cytes were readily identified in the lamina propria of fetal tal, in accordance with the Declaration of Helsinki. intestine, but most were clustered in lymphoid aggregates. Histologically normal intestinal tissue from children ranging Cells identified by anti-IgA and anti-lgA2 were the most 1 in age from I d to 3 y, 10 mo was obtained from surgical numerous cells of B cell lineage in the lamina propria of specimens submitted to the Pathology Department (Table 1). postnatal intestine, whereas lgM+ and lgD+ lymphocytes The tissue samples were embedded in OCT Compound (Ames predominated in fetal tissues. However, lgA-bearing cells Co., Elkhart, IN), snap frozen in liquid nitrogen or isopentane were identified in lymphoid aggregates of the intestine or (-70oC) and stored at -70oC. spleen of some fetuses. This finding suggests that B lym­ MAb. The MAb used in this study include B I (CD20), B2 phocytes can undergo Ig switching in utero. Additionally, (CD21), (Coulter Corp., Hialeah, FL), anti-IgA2, anti­ fetal intestinal epithelial cells did not express MHC class IgG, anti-IgM, anti-IgD, Leu 2a (CDS), Leu 3a (CD4), Leu 4 II antigens, unlike some postnatal intestinal tissues. It is (CD3), Leu 6 (CD!), anti-HLA-DR, anti-HLA-DQ, anti-HLA­ possible that postnatal events such as antigen exposure DP (Becton Dickenson, Mountain View, CA), anti-HLA-A,B,C may be important for the induction of these class II anti­ gens on intestinal epithelial cells. (Pediatr Res 27:239- (Bioproducts for Science Inc., Indianapolis, IN) and OKTS (CDS) (Ortho Pharmaceutical Corp., Raritan, NJ). This panel of 244,1990) antibodies was chosen so that aspects ofB cell, T cell, and antigen presenting cell development could be evaluated. Immunoperoxidase staining. Frozen tissue sections 4- to 6-JLm thick were fixed in acetone and stained by the previously de­ scribed avidin-biotin complex method (12). Briefly, the tissue The postnatal development and maturation of the intestinal sections were incubated with MAb at an optimal dilution for 1 mucosal immune barrier is an important host defense mecha­ h followed by incubations with 1: 240 dilution of biotinylated nism to prevent the penetration and uptake of bacteria, toxins, horse anti-mouse antibody and a I: 120 dilution of avidin-biotin­ and potentially damaging antigens ( 1). Previous reports on the ylated peroxidase complex (Vector Laboratories, Inc., Burlin­ phenotypic characterization and tissue distribution of human game, CA). Negative control slides were incubated with PBS, pH fetal lymphocytes have focused primarily on lymphocytes found 7.3 instead of a primary antibody. A positive control known to in thymus, spleen, liver, lymph nodes, and bone marrow (2-6). express the antigen of interest was also processed in parallel. Limited information is currently available on the development Each incubation was followed by three washes in PBS. Endoge­ of the human gut-associated lymphoid tissue. Immunohisto­ nous peroxidase activity was blocked by a 20-min incubation in chemical examination of human fetal terminal ileum has dem­ 0.3% hydrogen peroxide in PBS. Tissue sections of fetal liver onstrated that by 19-22 wk gestation, the gut epithelium and lamina propria contain many isolated T cells and the lamina propria contains aggregates ofT and B cells (7-9). Our study Table I. Tissue samples ofpo stnatal intestine evaluates the distribution ofT and B lymphoid populations and MHC class II antigen expression in human fetal intestine in Diagnosis Age comparison with normal postnatal intestine. Duodenal atresia ( 1) I d Received June 29, 1989; accepted November 2, 1989. Biliary atresia ( 1) 4 mo Correspondence Gary J. Russell, M.D., Combined Program in Pediatric Gastro­ Intestinal stricture (I) 8 mo enterology and Nutrition Unit, Hunnewell Ground, The Children's Hospital, 300 Hirschsprung's disease ( 1) 14 mo Longwood Ave., Boston, MA 02115. Intussusception (I) 16 mo Supported by a grant from The Hood Foundation to G.J.R., NIH Grant Al- 27747 to H.S.W., and NIH Grant DR-33506 to A.K.B. Choledochal cyst (2) 3 y 10 mo, 6 mo 239 240 RUSSELL ET AL. HUMAN FETAL INTESTINAL LYMPHOCYTES 241 were also incubated with avidin and then biotin (Vector Labo­ Small intestine. The lymphoid cells in the fetal intestinal ratories) before the incubation with biotinylated horse anti­ mucosa were distributed as scattered cells or in small aggregates mouse antibody to block endogenous biotin. The sections were (primary follicles) . Sparsely scattered lymphocytes stained with stained by incubating in a solution of 3-amino-9-ethylcarbazole antibody against CD20, IgM, lgD, and occasionally CD21 and (Aldrich Chemical Co., Inc., Milwaukee, WI), postfixed in 4% were considered to be B lymphocytes. No IgA,+, IgA2+, or IgG+ formaldehyde, counter stained with hematoxylin and mounted cells were present in the lamina propria of fetal intestine or the in Glycergel (Dako Corp., Santa Barbara, CA). proximal jejunum of the 1-d-old infant; however, as expected, IgA, + and IgA2+ cells represented the predominant B cell popu­ RESULTS lation in the lamina propria of postnatal small intestine (Fig. 1b). Lymphocyte staining for IgM, IgD, CD20, and occasionally Thymus. The staining observed in the fetal thymus with anti­ C02! was present in the small intestinal lamina propria of bodies against T cells was consistent with previous report> on postnatal specimens. In order of predominance, CD4+, CD3+, fetal thymocyte suspensions (2, 13) and immunohistochemical and cos+ cells were readily identified in the lamina propria of staining of postnatal thymic tissue ( 14 ). Cortical thymocytes all fetal intestine as well as all specimens obtained postnatally. demonstrated peripheral staining with antibodies against CD1, Many of the CD4+ cells had the morphologic appearance of CD3, CD4, and CDS. The majority of the medullary thymocytes macrophages or dendritic cells. was CD4+ but many were CDS+. IgM+ and IgD+ cells were Most B cells in fetal intestinal tissue were clustered in lymphoid present in the medulla, but no IgG+, IgA, +, or IgA2 + cells were aggregates, characteristic of primary follicles staining with anti­ identified. Many CD20+, and to a lesser extent CD21 +,cells were bodies against IgM, IgD, CD20, and faintly CD21. Many primary found in the medulla. These cells were larger than medium size follicles were present in fetal cecum and appendix. In two spec­ lymphocytes and demonstrated membrane staining. Prominent imens some lgA 1+, lgA2+, and IgG+ cells were prominently diffuse (cytoplasmic) and some membrane staining was present stained in lymphoid aggregates (Fig. I a). in the medulla with the antibodies against MHC class II antigens. Both primary and secondary lymphoid follicles were found in Patchy dendritic staining occurred in the cortex with anti-HLA­ the postnatal specimens except for the 1-d-old infant. Germinal DR and anti-HLA-DQ, but there was minimal cortical staining centers stained for IgM, IgG, and CD20; whereas, the mantle with anti-HLA-DP. This staining pattern was most consistent zones demonstrated the staining characteristics of a primary with the staining of epithelial cells; however, staining of some follicle. Interfollicular cells stained with antibodies against CD4, thymocytes cannot be excluded. CD3, and CDS with CD4+ cells predominating. These results are consistent with the protein that immature Occasional intraepithelial lymphocytes (0-1 cells per high­ thymocytes reside in the cortex and mature thymocytes are powered field) stained with CD3 and CDS antibodies were pres­ present in the medulla; however, the expression of CD3 and ent in all six fetuses and the 1-d-old infant (Fig. 2a). CD4+ CDS antigens by the majority of cortical thymocytes raises the intraepithelial lymphocytes were absent in four fetuses and the possibility that the maturation ofthymocytes starts in the cortex. 1-d-old infant and were observed only rarely in two fetuses. Spleen. B lymphocytes identified with antibodies against IgM, Similarly, intraepithelial lymphocytes that were slightly more IgD, C020, and to a lesser extent CD21, were found mainly in numerous (one to two cells per high-powered field) in the intes­ primary follicles of the white pulp with some cells scattered tine of older infants and children were predominately CD3+ and throughout the red pulp. A few IgA,+ and IgA2+ cells were cos+ (Fig.