Germinal centers in human lymph nodes contain reactivated memory B cells

Abstract

To reveal migration trails of antigen-responsive B cells in lymphoid tissue, we analyzed immunoglobulin (Ig)M-V(H) and IgG-V(H) transcripts of germinal center (GC) samples microdissected from three reactive human lymph nodes. Single B cell clones were found in multiple GCs, one clone even in as many as 19 GCs. In several GCs, IgM and IgG variants of the same clonal origin were identified. The offspring of individual hypermutated IgG memory clones were traced in multiple GCs, indicating repeated engagement of memory B cells in GC reactions. These findings imply that recurring somatic hypermutation progressively drives the Ig repertoire of memory B cells to higher affinities and infer that transforming genetic hits in non-Ig genes during lymphomagenesis do not have to arise during a single GC passage, but can be collected during successive recall responses.

Figure 1.

Genescanning analyses of the IgM-VH4 transcripts amplified out of four individual GC samples of LN1. Shown are four representative examples of genescanning analyses of IgM-VH4 transcripts present in microdissected tissue samples and amplified by RT-PCR. On the x axis are the lengths in basepairs as determined using internal length markers. The recurrent A-μ clone with an IgM-VH4 PCR product length of 481 bp is found in GC16, GC19, and in GC21, but not in GC27. These findings correlated well with the cloning experiments separately performed.

Figure 2.

Localization of recurrent IgM and IgG clones in LNs 1, 2, and 3. To visualize the proliferating B cells within GCs, the LN sections were immunohistochemically stained for Ki67 (red) and subsequently counterstained with hematoxylin (blue). LN1: The red-filled circles indicate the 19 GCs in which the IgM-VH4 A-μ clone was found. GCs highlighted by a yellow triangle (GC5 and GC25) and by a yellow box (GC11 and GC16) contained the IgM-VH4 H-μ and I-μ clones, respectively. GCs highlighted by a yellow star (GC5, 7, 16, 17, and 20) contained the recurrent IgG-VH4 C-γ clone. LN2: The red-filled GC4 and GC8 harbored the IgG-VH4 G-γ clone. LN3: The red-filled GCs harbored the four recurrent IgM-VH1 clones, i.e., the B-μ clone in GC1 and GC2, the C-μ clone in GC2 and GC3, and the D-μ and G-μ clones, both detected in GC11 and GC13. GCs marked by a yellow star (GC10, 11, and 13) contained the recurrent IgG-VH1 J-γ clone. Samples from FMs and TZs are indicated by m and t, respectively. Bars, 2 mm.

Figure 3.

Selection of the IgM-VH4 sequences and mutations therein of the recurrent A-μ clone in four GCs of LN1. Lollipop-shaped symbols indicate nucleotide differences as compared with the V4-30.4 germline IgV_H gene. Replacement and silent mutations are indicated by closed and open circles, respectively. Gray shaded bars at codons 37 and 56 indicate identical somatic mutations found in all A-μ clones. The gray shaded mutation at codon 93 in GC3 and GC5 represents an identical mutation that differed from the codon 93 mutation found in all A-μ clones of GC11. The total number of clones in which a particular IgV_H mutation pattern was found is indicated by (Nx).

Figure 4.

IgV_H sequences and mutations therein of related IgM and IgG clones found within individual GCs of LNs 1, 2, and 3. Lollipop-shaped symbols indicate nucleotide differences as compared with the respective germline IgV_H genes. Replacement and silent mutations are indicated by closed and open circles, respectively. Gray shaded bars covering mutations of IgM and IgG clones indicate identical mutations. The total number of clones in which a particular IgV_H mutation pattern was found is indicated by (Nx).

Figure 5.

Mean IgV_H mutation numbers of all clones analyzed ordered according to anatomical location in LNs 1, 2, and 3. FM, mantle zones; GC, samples from germinal centers; TZ, samples from T cell areas. Numbers underneath the base of the bars indicate the number of sequenced clones.

Figure 6.

Mean IgV_H mutation numbers of related IgM and IgG clones found within individual GCs. The mean IgV_H mutation numbers of the 11 clonal IgM/IgG sets of Fig. 4 are shown. M and G indicate related IgM and IgG clones, respectively.

Figure 7.

IgV_H sequences and mutations therein of the recurrent IgG clones found in LNs 1, 2, and 3. The C-γ clone found in five GCs of LN1 is shown in the top, the G-γ clone found in two GCs of LN2 is shown in the middle, and the J-γ clone found in three GCs of LN3 is shown in the bottom. Lollipop-shaped symbols indicate nucleotide differences as compared with the V4-4, V4-30.4, and the V1-69 germline IgV_H genes, respectively. Replacement and silent mutations are indicated by closed and open circles. Gray shaded bars indicate the identical somatic mutations in the different GCs. 2X, two mutations in the indicated codon. The total number of clones in which a particular IgV_H mutation pattern was found is indicated by Nx.