Antibody repertoire development in fetal and newborn piglets, III. Colonization of the gastrointestinal tract selectively diversifies the preimmune repertoire in mucosal lymphoid tissues

Abstract

SUMMARYChanges in the V H -region repertoire of isolator piglets reared for 6 weeks under germ-free (GF) conditions and those colonized (COL) with a de®ned exclusion¯ora on the 1st day of life were compared. Although serum immunoglobulin levels were 20±100-fold higher in COL piglets than GF piglets, an analysis of peripheral blood B cells (PBBs) indicated that: GF and COL piglets used the same four V H genes and two D H segments during the 6-week period; proportional usage of V H genes and D H segments was the same as in fetal animals; and V H and D H usage did not differ between COL and GF animals. This pattern differed from the PBBs from 6-week-old conventional (CONV) piglets. When the sequences of 73 splenic CDR3 segments were analysed, D H usage and mutation frequency were the same in sequences from both 6-week-old GF and COL piglets; mutations were infrequent and occurred with the same frequency as in 110-day fetal spleen. However, the median CDR3 length in COL piglets was shifted upward due to 3k D H N-nucleotide additions. Neither COL nor GF animals made speci®c serum antibodies to phosphoryl choline given parenterally on a T-cell dependent carrier. In contrast to the near absence of a colonization effect in PBBs and splenic DNA, rearranged variable heavy-chain gene segments (VDJs) recovered from the DNA of mucosal lymphoid tissues of COL piglets showed pronounced differences from those recovered from GF animals in usage of D H A-, D H B-and V H B-and in the frequency of point mutation. The mucosal VDJ transcripts and those from the spleen were similarly affected by colonization. This effect on mucosal lymphoid tissue was consistent with the ®ve-fold selective increase in serum immunoglobulin A (IgA) levels relative to IgM and IgG. Comparison of IgM and IgA transcripts from mucosal tissues suggested that IgA and IgM clones diversify in parallel. Our ®ndings are the ®rst to show that colonization of the gastrointestinal tract of offspring separated from their mothers, differs from`conventionalized' GF animals in that colonization preferentially in¯uences diversi®cation and expansion of the preimmune IgM and IgA repertoire in mucosal lymphoid tissues but not in PBBs and seldom/modestly in VDJs from splenic DNA.

Antibody Repertoire Development in Fetal and Neonatal Piglets. II. Characterization of Heavy Chain Complementarity-Determining Region 3 Diversity in the Developing Fetus

“…In fact, the mean CDR3 length at 30 days (Fig. 4B) is only one codon shorter than reported for adult humans (40) and is the same length as we have observed in both colonized isolator piglets and adults (21). Consistent with studies in mice (39) and humans (16,36,40), piglets also first use their shortest D H segment (D H B; Fig.…”

“…On the other hand, reported differences in CDR3s between fetal and adult mice or humans is quite likely another matter, and some differences may be the result of extrinsic factors acting on the fetal immune system, since these cannot or have not been controlled in rodent and primate studies. In the piglet we have already shown that there are no differences in V H usage, D H usage, or the characteristics of CDR3 in piglets reared germfree for 6 wk in an isolator (21). On the other hand, both colonized isolator piglets and adults use D H A two or three times more frequently than D H B, suggesting that pronounced D H A usage is a marker for diversification of the VDJ repertoire in swine as is increased CDR3 length due to N region additions 3Ј of D H (21).…”

“…In the piglet we have already shown that there are no differences in V H usage, D H usage, or the characteristics of CDR3 in piglets reared germfree for 6 wk in an isolator (21). On the other hand, both colonized isolator piglets and adults use D H A two or three times more frequently than D H B, suggesting that pronounced D H A usage is a marker for diversification of the VDJ repertoire in swine as is increased CDR3 length due to N region additions 3Ј of D H (21). In fetal piglets we observed a trend to greater D H A usage during development, but unlike colonized or conventional animals, no increase in N region additions was seen (Fig.…”

“…CDR3 sequences were compared as previously described (21) in terms of total length, P and N nucleotide additions both 5Ј and 3Ј of D H , D H usage, D H length, and D H reading frame usage ( Fig. 3 and Table I).…”

Antibody Repertoire Development in Fetal and Neonatal Piglets. XX. B Cell Lymphogenesis Is Absent in the Ileal Peyer’s Patches, Their Repertoire Development Is Antigen Dependent, and They Are Not Required for B Cell Maintenance

“…IgM levels were higher on day 7 after surgery in all surgically manipulated animals but thereafter levels paralleled those in colonized controls. Ig levels in GF piglets remained within the range for newborn levels throughout the study (58).…”