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

Abstract

Changes in the VH-region repertoire of isolator piglets reared for 6 weeks under germ-free (GF) conditions and those colonized (COL) with a defined exclusion flora 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 VH genes and two DH segments during the 6-week period; proportional usage of VH genes and DH segments was the same as in fetal animals; and VH and DH 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, DH 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 3' DH N-nucleotide additions. Neither COL nor GF animals made specific 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 DHA-, DHB-and VHB- 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 five-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 findings are the first to show that colonization of the gastrointestinal tract of offspring separated from their mothers, differs from 'conventionalized' GF animals in that colonization preferentially influences diversification 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.

Figure 1

Serum concentration of IgM, IgG and IgA in colonized (COL) and germ-free (GF) piglets (a–c). Note differences in the scale of the y-axis. All differences between COL and GF piglets are highly significant. The error bars represent the standard deviation of at least nine values in the titration range of the standard curve in the assays used as described for ELISANALYSIS. (d) Relative proportion of serum immunoglobulins in COL and GF animals compared to adult animals.

Figure 5

DPPH values for D_H and V_H usage in DNA and transcripts from three different lymphoid tissues of 6-week-old GF and COL piglets. (a, b) D_HA and D_HB, respectively; (c) combined fetal V_H gene usage; (d) V_HB usage. Animals 4-1 and 4-2 remained GF while 3-1 and 3-2 were COL piglets. In the case of D_H values, all data were normalized to values obtained for the IPP of animals 4-1 and this was assigned a value of 100. Mean values for cDNA and DNA were compared by t-test to those for animal 4-1; **Significant at 0·01 level; *significant at 0·05 level. In the case of DPPH values for V_H genes, proportional usage is actual and mean values were compared to those for GF animal 4-1.

Figure 7

DPPH analysis of V_HB and D_HB usage by IgM and IgA transcripts from the parotid gland (Par) and ileal Peyers patches (IPP) of COL and GF isolator piglets. Animals 4-1 and 4-2 remained GF, while 3-1 and 3-2 were COL piglets.

Figure 2

V_H usage in DNA from peripheral blood B cells (PBBs). (a) The combined usage of four fetal V_H genes (V_HA, V_HB, V_HC and V_HE) as a proportion of total V_H usage in four isolator and four conventional piglets (6 weeks CONV). Usage of fetal V_H genes in the 6-week-old CONV animals is shown as the righthand bar in each histogram cluster. (b–e) The proportional usage of V_HA, V_HB, V_HC and V_HE on weeks 2–6 in GF piglets and COL piglets compared to DPPH usage values from 6-week-old CONV piglets. All data are expressed as mean± SD of four independent measurements. Note differences in the scale of the y-axis. Statistically significant at **P < 0·01 or *P < 0·05 when compared to 6-week-old GF or COL piglets.

Figure 3

The relative usage of D_HA and D_HB in the DNA of PBBs from GF and COL piglets. Data are presented for samples collected on weeks 2, 4 and 6 after birth. All values for D_HA were normalized to animal 3-1 on week 2 and for D_HB, to animal 4-2 on week 2. These were assigned a value of 100. Data are expresses as the mean ± SD of four independent determinations. Mean DPPH values were significantly lower on weeks 4 and 6 than values for week 2 at the 0·01 (**) or 0·05 (*) level for animals 4-1 and 4-2.

Figure 6

Comparative analysis of random cDNA transcripts from COL and GF isolator piglets. (a) Proportional V_H and D_H usage and comparison of CDR3 lengths; (b) frequency of mutations in CDR1, -2 and -3 in transcripts from GF piglets; (c) frequency of CDR mutation among transcripts from colonized piglets. All sequences submitted to GenBank and presented under Accession numbers.

Figure 4

Summary of sequence analysis of CDR3 segments cloned from 73 splenic DNA clones (randomly selected) from 6-week-old COL and GF piglets and from 17 110-day fetal splenic clones. (a) D_H and V_H usage in randomly selected splenic clones; (b) 3′ N-nucleotide additions; (c) frequency of point mutation. Comparative data are provided for 30 previously published adult transcripts.