Antibody repertoire development in fetal and neonatal piglets. XI. The thymic B-cell repertoire develops independently from that in blood and mesenteric lymph nodes

Abstract

The origin and function of thymic B cells is currently unresolved. In the present study we compared V(H) gene repertoire diversification in >3500 cloned VDJs (from 11 animals at three time-points, using three to five animals per time-point) that were expressed with immunoglobulin (Ig)M, IgD, IgG, IgA and IgE in thymus, mesenteric lymph nodes (MLN) and peripheral blood B cells (PBB) of newborn piglets and 5-week-old isolator piglets maintained germfree (GF) or colonized with Escherichia coli. The results showed that the repertoire expressed with IgM, IgD, IgG and IgA in PBB and MLN remained polyclonal, undiversified and unselected in piglets maintained GF for 5 weeks, that age and colonization resulted in significant repertoire diversification of IgG and IgA in the MLN and of IgG in blood, that the thymic B-cell repertoire was polyclonal, unaffected by colonization and showed no clonal selection in any isotype, and that the thymic IgA and IgE repertoires were more diverse at birth than the repertoire of any isotype in MLN or PBB. IgD was seldom recovered from the PBB of newborn piglets or at any time-point in thymus, but was recovered in the MLN of all 11 animals examined. The IgD and IgM repertoires in all tissues remained polyclonal and unselected, although V(H) usage by IgD transcripts did not always parallel that of IgM in the same tissue. Therefore, isotype-switched B cells in the thymic medulla cannot be accounted for by immigration of B cells diversified by colonization of the gut, and thymic B cells undergo switch recombination and repertoire diversification before birth without clonal selection.

Figure 1

Characterization of V_H gene usage by hybridization using V_H-specific probes. Top panel: hybridization, using a V_HC-specific probe, of cloned VDJs expressed with immunoglobulin M (IgM) from the thymus of three different piglets. Bottom panel: hybridization of the same clones with an FR2, pan-specific V_H probe. Data from the example provided show that ≈25% of all VDJs expressed with IgM contain non-mutated V_HC and that animal differences are minimal. The specificity of the CDR1 and CDR2 probes employed for different V_H genes has been previously published in greater detail.,

Figure 2

Basis of repertoire diversification index (RDI) (a) and analysis of V_H gene usage in blood by immunoglobulin M (IgM) transcripts (b) and by immunoglobulin A (IgA) transcripts from mesenteric lymph nodes (MLN) (c) and thymus (d). Data are expressed as mean ± standard deviation (SD) for three to five animals at each time/treatment point. The number of clones tested is shown in parentheses at the top of each panel. Data in (a) are based on heavy chain variable regions from 251 IgM transcripts from the preimmune repertoire of newborn piglets and on 295 IgG and IgA transcripts from antigen-stimulated isolator piglets. The difference between these two sources was highly significant (P = 0·000001) and forms the basis of the RDI. χ²-Test analyses indicate that differences among treatment groups in V_H usage by IgA transcripts in the MLN (c) is highly significant for all three treatment groups, whereas those for IgM transcripts in blood (b) or for IgA transcripts in thymus (d) are not. Arrows indicate the direction of significant changes in V_H usage; horizontal arrows indicate that changes were not significant. The legends for (b), (c) and (d) are the same as for (a).

Figure 3

Isotype-associated V_H gene usage in the mesenteric lymph node (MLN) of individual newborn, 5-week germfree and 5-week colonized isolator piglets. The key to the different isotypes is shown in the upper left panel. Data are presented for two representative animals in each category. Data are based on 28–31 clones for each isotype. The numbers and letters simply identify the particular animal. The category ‘other’ includes mutated variants V_HA, V_HB, V_HC and V_HE and VDJs that use other germline V_H genes.

Figure 4

Isotype-associated V_H gene usage in the peripheral blood B cells (PBB) of individual newborn, 5-week germfree and 5-week colonized isolator piglets. The key to the different isotypes is shown in the upper left panel. Data are presented for two representative animals in each category. Data are based on 27–31 clones for each isotype. The numbers and letters identify the specific animals. The category ‘other’ includes mutated variants V_HA, V_HB, V_HC and V_HE and VDJs that use other germline V_H genes.

Figure 5

Isotype-associated V_H gene usage in the thymus of individual newborn, 5-week germfree and 5-week colonized isolator piglets. The key to the different isotypes is shown in the upper left panel. Data are presented for two representative animals in each category. Data are based on 27–31 clones for each isotype. The numbers and letters identify the specific animals. The category ‘other’ includes both mutated variants V_HA, V_HB, V_HC and V_HE and VDJs that use other germline V_H genes.

Figure 6

Spectratype analyses of B cells. Isotypes are indicated by their corresponding capital letter, e.g. A = IgA. S = CDR3 length standards. (a) B cells expressing IgA, IgD, IgG and IgM from newborn germfree (GF) and colonized (COL) mesenteric lymph node (MLN). The spectratypes on the right (FOL 1 and 2) illustrate extreme oligoclonal patterns and are from B cells recovered from follicles of the ideal Peyer's patches. (b) Spectratype of transcripts of IgA, IgD, IgG and IgM from peripheral blood B cells (PBB) of 5-week old GF and colonized piglets. NB = IgM spectratype from a newborn piglet. (c) Spectratype of transcripts from 5-week-old germfree (GF) MLN and thymus. (d) Spectratype of transcripts from the MLN of 5-week-old colonized piglet and from a colonized, 5-week-old thymus.