Dynamic establishment and maintenance of the human intestinal B cell population and repertoire following transplantation in a pediatric-dominated cohort

Abstract

Introduction: It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of recipient gut lymphocyte populations in immunosuppressed conditions.

Methods: Using polychromatic flow cytometry that includes HLA allele group-specific antibodies distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients.

Results: We confirm the early presence of naïve donor B cells in the circulation (donor age range: 1-14 years, median: 3 years) and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa (recipient age range at the time of transplant: 1-44 years, median: 3 years). Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection (recipient age range at the time of transplant: 1-9 years, median: 2 years) revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in deceased adult donors. In available pan-scope biopsies from pediatric recipients, we observed higher percentages of naïve recipient B cells in colon allograft compared to small bowel allograft and increased BCR overlap between native colon vs colon allograft compared to that between native colon vs ileum allograft in most cases, suggesting differential clonal distribution in large intestine vs small intestine.

Discussion: Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of stabilization of the mucosal B cell repertoire in pediatric ITx patients.

Keywords: B cell repertoire sequencing; B cell subpopulations; human longitudinal studies; intestinal transplantation; resident memory B cells.

Figure 1

B cell chimerism in PBMCs and intestinal allografts post ITx and correlation with transplant type and donor age. (A) Chimerism of donor CD19⁺ B cells in recipient PBMC and recipient CD19⁺ B cells in ileum allograft post-Tx. Samples containing at least 40 cells in the parent gate (CD19⁺ B cells) are shown. MVTx patients are represented by circles and iITx represented by triangles. Symbols with black outline in recipient B cell chimerism in allograft plots indicate samples collected whose histology demonstrated mild to severe ACR, or concurrance of DSA⁺ AMR. Area under the curve (AUC) values normalized by days of measurement (POD_last – POD_first) of donor B cell chimerism in PBMC plots (B) and recipient B cell chimerism in ileal allograft plots (C) during early (POD0–90), mid (POD91–365) and late (POD366–1000) post-Tx periods were subgrouped by MVTx vs iITx (upper panels) and donor age <1 vs ≥1 year old (lower panels). For (B, C) dashed lines from the bottom to the top within each condition represent the first quartile, median, and third quartile of data distribution, respectively. The Mann-Whitney U test was performed to determine statistical significance (**p<0.01).

Figure 2

Naive and memory B cell chimerism in the PBMC and intestinal allograft over time. (A) Percentages of donor and recipient naive (CD27^-IgD⁺) and memory B cells (CD27⁺ IgD^+/-) in PBMC and ileal allograft during quiescence in post-Tx time brackets defined as in Figure 1 . The median percentage among samples for each patient in a given period (represented by the median of the timepoints sampled) are shown for (B, C). (B) Median donor naïve and memory B cell percentages among total donor B cells in PBMC and ileal allograft from early to late post-Tx periods were subgrouped by donor age. (C) Median recipient naïve and memory B cell percentages of total donor B cells in PBMC and ileal allograft during early, mid and late post-Tx periods were subgrouped by donor age. For (B, C) dashed lines from the bottom to the top within each condition represent the first quartile, median, and third quartile of data distribution, respectively. The Mann-Whitney U test was performed to determine statistical significance (*p<0.05, **p<0.01).

Figure 3

Memory subsets in donor and recipient B cells. Median percentages of donor (“D”: pink) and recipient (“R”: blue) BRM cells (CD69⁺CD45RB⁺), IgA⁺, and IgG⁺ B cells from the CD24⁺ memory gate ( Supplementary Figure 1 ) within total recipient B cells for each patient in the PBMC and ileal allograft during early, mid and late post-Tx periods. In all cases, only quiescent samples exceeding a 40 cell threshold in the preceding parent gate are shown. Low sample numbers in the late time bracket for donor cells reflect near-complete replacement by recipient B cells at this stage. Dashed lines from the bottom to the top within each condition represent the first quartile, median, and third quartile of data distribution, respectively. The Mann-Whitney U test was performed to determine statistical significance (*p<0.05).

Figure 4

Mutation levels of BCR clones in pre- and post-Tx samples of pediatric ITx patients and adult deceased donor controls. (A) Fraction of mutated clones (clones with an average v gene mutation frequency >2%) by tissue for pediatric Tx patients and adult controls (11). Each individual is represented by a unique marker. The color of the marker represents the tissue, and the pink marker outline indicates that the tissue is an allograft. Tissues from the pediatric cohort are post-Tx unless specifically labeled as pre-Tx. Ages are shown in years. The Wilcoxon two-sided paired test was used to test for significance when the categories being compared were from the same cohort. An asterisk (*) denotes p-values <0.05. (B) Fraction of clones with an average v gene mutation frequency >2% by POD bracket for blood and ileum allograft (or native ileum for the “Pre” time bracket). Each POD is grouped into either pre, early, mid, or late time brackets (POD0, POD1–90, POD91–365 and POD>365, respectively). For each individual, the median among samples at a given POD bracket is shown. The horizontal black line represents the median among the individuals. POD samples were filtered for having >5 clones. Tissues are colored as in Figure 4A . (A, B) Pre-Tx samples represent native recipient blood/tissue and were taken during transplantation. Comparisons performed here were based on previous knowledge obtained in the adult control cohort and to determine: 1) whether tissue differs from blood in B cell mutation levels; and 2) whether mutation levels of circulating and ileal allograft B cells increase post-transplant. Our significance level is α = 0.05.

Figure 5

Diversity of B cell clones in peripheral blood and ileal allograft over time. (A) Evenness of clone distribution across time brackets. Evenness is calculated by dividing diversity (order 1) by richness (see Methods). The black horizontal line represents the median. Individuals are marked as in Figure 4 . Tissues are colored as in Figure 4A (red for blood, light blue for ileum). The limited but novel and unprecedented data presented here exhibit a trend but are not sufficient to permit statistical analysis, as only two individuals span all 4 time points for ileum and those two individuals show differing trends. We therefore present the observed overall trends without attempting to generalize beyond the small population of individuals studied. (B) The number of clones that increase (red) or decrease (gray) in size (by number of unique instances) across two time brackets. The sign test was performed to determine significant difference in the direction of clonal growth between time brackets (*p <0.05). (A, B) Each POD is grouped into either pre (P), early (E), mid (M), or late (L) time brackets, as in Figure 4B .

Figure 6

Pediatric ITx patients exhibit increased clumpiness between the blood and ileum allograft compared to deceased adult donors. Median clone clumpiness per individual for a given pair of tissues is shown. Only clones with 3 or more unique sequences that were sampled in both tissues were included. Medians were filtered for having greater than 5 clones. The Mann-Whitney U test was performed to determine statistical significance (*p<0.05). Individuals are marked as in Figure 4 .

Figure 7

Phenotypic and repertoire analysis of recipient B cells in pan-scope biopsies of pediatric ITx recipients. (A) Percentages of recipient naive (CD27^-IgD⁺) and memory B cells (CD27⁺ IgD^+/-) in native colon, colon allograft and small bowel (ileum or duodenum) allograft during quiescence at late post-Tx time points (POD 400–1600) from two pediatric ITx patients. (B) Cosine similarities of BCR repertoires between native colon and colon allograft, and between native colon and ileum allograft for three pediatric ITx patients are shown at mid and/or late post-Tx time brackets (defined as in Figure 1 ). Due to the limited number of patients and variable number of time points available between patients for this analysis, we did not assess statistical significance but instead describe biologically informative trends.