Memory B lymphocytes determine repertoire oligoclonality early after haematopoietic stem cell transplantation

Abstract

The objective of this study was to investigate if oligoclonality of the Ig repertoire post-haematopoietic stem cell transplantation (HSCT) is restricted to memory B lymphocytes or if it is a general property among B lymphocytes. As a measure of B lymphocyte repertoire diversity, we have analysed size distribution of polymerase chain reaction (PCR) amplified Ig H complementarity determining region 3 (CDR3) in naive and memory B lymphocytes isolated from patients before HSCT and at 3, 6 and 12 months after HSCT as well as from healthy controls. We demonstrate a limited variation of the IgH CDR3 repertoire in the memory B lymphocyte population compared to the naive B cell population. This difference was significant at 3 and 6 months post-HSCT. Compared to healthy controls there is a significant restriction of the memory B lymphocyte repertoire at 3 months after HSCT, but not of the naive B lymphocyte repertoire. Twelve months after HSCT, the IgH CDR3 repertoire in both memory and naive B lymphocytes are as diverse as in healthy controls. Thus, our findings suggest a role for memory B cells in the restriction of the oligoclonal B cell repertoire observed early after HSCT, which may be of importance when considering reimmunization of transplanted patients.

Fig. 1

Enrichment of naive and memory B lymphocytes. B cell subsets separated by flow cytometry, according to CD19/CD20 and CD27 expression as described in Methods. (a) PBMC from a donor before enrichment with immunomagnetic beads; (b) CD19⁺ CD27^– (naive) B lymphocytes; and (c) CD19⁺ CD27⁺ (memory) B lymphocytes after enrichment with immunomagnetic beads.

Fig. 2

CDR3 spectratype repertoire in four representative patients compared to a normal control. Size distribution of CDR3 fragments, also called CDR3 spectratypes, in memory (upper panels) and naive (lower panels) B lymphocytes from patient 1 (A + B), patient 6 (C + D), patient 7 (E + F) and patient 8 (G + H) before HSCT and at 3, 6 and 12 months post-HSCT. The vertical line in (g) and (h) indicates 100 bp. The peaks between 75 bp and 140 bp were counted from the datasheet generated by the computerized GeneScan analysis.

Fig. 2

CDR3 spectratype repertoire in four representative patients compared to a normal control. Size distribution of CDR3 fragments, also called CDR3 spectratypes, in memory (upper panels) and naive (lower panels) B lymphocytes from patient 1 (A + B), patient 6 (C + D), patient 7 (E + F) and patient 8 (G + H) before HSCT and at 3, 6 and 12 months post-HSCT. The vertical line in (g) and (h) indicates 100 bp. The peaks between 75 bp and 140 bp were counted from the datasheet generated by the computerized GeneScan analysis.

Fig. 2

CDR3 spectratype repertoire in four representative patients compared to a normal control. Size distribution of CDR3 fragments, also called CDR3 spectratypes, in memory (upper panels) and naive (lower panels) B lymphocytes from patient 1 (A + B), patient 6 (C + D), patient 7 (E + F) and patient 8 (G + H) before HSCT and at 3, 6 and 12 months post-HSCT. The vertical line in (g) and (h) indicates 100 bp. The peaks between 75 bp and 140 bp were counted from the datasheet generated by the computerized GeneScan analysis.

Fig. 2

CDR3 spectratype repertoire in four representative patients compared to a normal control. Size distribution of CDR3 fragments, also called CDR3 spectratypes, in memory (upper panels) and naive (lower panels) B lymphocytes from patient 1 (A + B), patient 6 (C + D), patient 7 (E + F) and patient 8 (G + H) before HSCT and at 3, 6 and 12 months post-HSCT. The vertical line in (g) and (h) indicates 100 bp. The peaks between 75 bp and 140 bp were counted from the datasheet generated by the computerized GeneScan analysis.

Fig. 3

CDR3 spectratype repertoire in memory and naive B lymphocytes in patients and controls. The number of peaks in the CDR3 spectratype profile for total (a), naive (b) and memory (c) B lymphocytes for all samples are plotted (▪). The median for each group is represented by a horizontal bar and the medians are connected with a line. The number of individuals in each group is indicated under the plot. **P < 0·01, ***P < 0·001 versus healthy controls.