Summary Colonization with commensal flora in very early life may profoundly influence intestinal lymphoid development and bias later immune responses. We defined gut‐homing T cell phenotypes and the influence of flora on intestinal immune development in mice. Intestinal T cells were phenotyped and quantified in conventional (CV), germfree (GF) and conventionalized germfree (GF/CV) neonatal mice by immunohistochemistry. Mucosal adressin cell adhesion molecule 1 (MAdCAM‐1) was expressed by mucosal vessels at birth in CV and GF mice and was more prevalent in CV than GF small intestine, but was distributed similarly and did not change with age. Less MAdCAM‐1 was expressed in the colon; its distribution became restricted after weaning, with no difference between CV and GF mice. CD3+β7+ cells were present in similar numbers in CV and GF intestine at birth. They were CD62L– in CV mice and were accompanied by further CD3+β7+CD62L– T cells as development progressed, but in GF and GF/CV intestine they expressed CD62L and numbers did not change. IEL numbers increased at weaning in CV mice in both small and large intestine, but showed delayed development in GF intestine. Macrophages were present at high levels from birth in GF intestine, but dendritic cells did not develop until day 16. Thus, fetus‐derived T cells seed the intestinal lamina propria before birth via β–MadCAM interactions. Their activation status depends on the microbiological status of the dam, and without a commensal flora they remain naive. We propose that these cells regulate antigen responsiveness of the developing mucosal T cell pool.

Intestinal Ag exposure during neonatal life influences appropriate adult immune responses. To define the mechanisms shaping the T cell repertoire during this period, we examined T cell differentiation and receptor diversity in the intestine of human infants. Developmental phenotypes of intraepithelial and lamina propria intestinal T cells from infants aged 1 day to 2 years were assessed ex vivo by flow cytometry and in situ by triple-fluorescent immunohistochemistry. Gene recombination-specific enzymes were assessed by PCR. TCR β-chain V region gene diversity was determined by sequencing. Several different early lineage T cell populations were present neonatally: CD3+4−8− T cells were present at birth and numbers decreased during the neonatal period; CD3+4+8+ T cells were present in low numbers throughout infancy; and CD3+4+8− or CD3+4−8+ T cells increased with age. Very early lineage T cells, CD3−2−7+ and CD3−2+7+, were present neonatally, but were essentially absent at 1 year. Most lamina propria T cells differentiated rapidly after birth, but maturation of intraepithelial T cells took place over 1 year. Intestinal samples from infants less than 6 mo old contained transcripts of T early α and TdT, and 15 of 19 infant samples contained mRNA for RAG-1, some coexpressing RAG-2. TCR β-chain repertoires were polyclonal in infants. Immature T cells, pre-T cells, and genes involved in T cell recombination were found in the intestine during infancy. T cell differentiation occurs within the neonatal human intestine, and the TCR repertoire of these developing immature T cells is likely to be influenced by luminal Ags. Thus, mucosal T cell responsiveness to environmental Ag is shaped in situ during early life.

Using competitive binding experiments, it was found that native type XI collagen binds heparin, heparan sulfate, and dermatan sulfate. However, interactions were not evident with hyaluronic acid, keratan sulfate, or chondroitin sulfate chains over the concentration range studied. Chondrocyte-matrix interactions were investigated using cell attachment to solid phase type XI collagen. Pretreatment of chondrocytes with either heparin or heparinase significantly reduced attachment to type XI collagen. Incubation of denatured and cyanogen bromide-cleaved type XI collagen with radiolabeled heparin identified sites of interaction on the ␣1(XI) and ␣2(XI) chains. NH 2 -terminal sequence data confirmed that the predominant heparin-binding peptide contained the sequence GKPGPRGQRGPTGPRGSRGAR from the ␣1(XI) chain. Using rotary shadowing electron microscopy of native type XI collagen molecules and heparin-bovine serum albumin conjugate, an additional binding site was identified at one end of the triple helical region of the collagen molecule. This coincides with consensus heparin binding motifs present at the aminoterminal ends of both the ␣1(XI) and the ␣2(XI) chains. The contribution of glycosaminoglycan-type XI collagen interactions to cartilage matrix stabilization is discussed.Type XI collagen is a component of the heterotypic collagen fibrillar network found in cartilage that, along with proteoglycan, gives cartilage its unique structural and biomechanical properties. The type XI collagen molecule consists of three genetically distinct polypeptide chains, namely ␣1(XI), ␣2(XI), and overglycosylated ␣1(II) chains, and is typical of the fibrillar class of collagens having a 300-nm triple helical domain (1). Although type XI collagen is a relatively minor collagen in cartilage, it is believed to be important in the regulation of fibril diameter (2) and in maintaining tissue integrity and cohesion. Mice homozygous for the autosomal recessive chondrodysplasia (cho) mutation in the col11a1 gene (3) do not synthesize ␣1(XI) chains and have larger cartilage collagen fibrils, less cartilage matrix cohesion, and increased extractability of proteoglycans. However, neither the domains of type XI collagen responsible for these activities nor the molecules with which they interact are known.By immuno-electron microscopy, it was found that the triple helical domain of type XI collagen was buried within the heterotypic fibril (4). However, it has been detected without the use of chaotropic agents both pericellularly (5) and, also, more generally throughout the matrix (4, 6), suggesting that some type XI collagen molecules in cartilage are not buried and are therefore available for interaction. Type XI collagen has been shown to be associated with the surface of bovine articular chondrocytes in suspension culture (7). It is also known that the triple helical domain binds to heparin-agarose with greater affinity than other cartilage collagens, an activity that has been exploited as a tool for its purification (8). Therefore, the tripl...

Rationale: Laryngopharyngeal reflux (LPR) affects up to 20% of Western populations. Although individual morbidity is usually moderate, treatment costs are high and there are associations with other diseases, including laryngeal cancer. To date, there have been no studies of the mucosal immune response to this common inflammatory disease. Objectives: To determine the mucosal immune response to LPR. Methods: We performed a prospective immunologic study of laryngeal biopsies from patients with LPR and control subjects (n 5 12 and 11, respectively), and of primary laryngeal epithelial cells in vitro. Measurements and Main Results: Quantitative multiple-color immunofluorescence, using antibodies for lymphocytes (CD4, CD8, CD3, CD79, CD161), granulocytes (CD68, EMBP), monocytic cells (CD68, major histocompatibility complex [MHC] class II), and classical and nonclassical MHC (I, II, b 2 -microglobulin, CD1d). Univariate and multivariate analysis and colocalization measurements were applied. There was an increase in percentage area of mucosal CD8 1 cells in the epithelium (P , 0.005), whereas other leukocyte and granulocyte antigens were unchanged. Although epithelial MHC class I and II expression was unchanged by reflux, expression of the nonclassical MHC molecule CD1d increased (P , 0.05, luminal layers). In vitro, laryngeal epithelial cells constitutively expressed CD1d. CD1d and MHC I expression were inversely related in all subjects, in a pattern which appears to be unique to the upper airway. Colocalization of natural killer T (NKT) cells with CD1d increased in patients (P , 0.01). Conclusions: These data indicate a role for the CD1d-NKT cell axis in response to LPR in humans. This represents a useful target for novel diagnostics and treatments in this common condition.

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