Dynamic establishment and maintenance of the human intestinal B cell population and repertoire following transplantation

Abstract

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 gut lymphocyte populations. Using polychromatic flow cytometry that includes HLA allele group-specific mAbs 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. We confirm the early presence of naïve donor B cells in the circulation and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa. 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 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 healthy control adults. Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of establishment of a stable mucosal B cell repertoire.

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 peroids were subgrouped by MVTx vs iITx (upper panels) and donor age <1 vs ≥1 year old (lower panels). 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 and 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 peroids were subgrouped by donor age. The Mann-Whitney U test was performed to determine statistical significance (*p<0.05, **p<0.01).

Figure 3.. Memory subsets in recipient B cells.

Median percentages of recipient BRM cells (CD69⁺CD45RB⁺), IgA⁺, and IgG⁺ B cells from the CD24⁺ memory gate (Figure S1) within total recipient B cells for each patient in the PBMC and ileal allograft during early, mid and late post-Tx peroids. In all cases, only quiescent samples exceeding a 40 cell threshold in the preceding parent gate are shown. 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 clones with an average V gene mutation frequency >2% by tissue. Each individual is represented by a unique marker (filled markers for pediatrics, unfilled markers for adults). The color of the marker represents the tissue label at the top of the graph, and the pink marker outline represents the tissue is an allograft. Pre-Tx, pretransplant; Post-Tx, post-transplant. Age ranges in years are shown. The Wilcoxon two-sided paired test was used to test for significance when the categories being compared were from the same cohort. The Mann-Whitney U test was used for significance testing between children and adults. 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 the PODs within a POD bracket is shown. For each POD, the samples within the POD were combined, and PODs that did not have >5 clones were omitted from the median calculation of a given POD bracket. The horizontal black line represents the median among the individuals. Tissues are colored as in Figure 4A (red for blood, light blue for ileum). (A and B) Pretransplant samples represent native recipient blood/tissue and were taken during transplantation.

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). (B) The number of clones that increase (green) or decrease (red) 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 and 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 adult deceased donors.

Median clumpiness per individual for a given pair of tissues is shown. Clones were filtered for having 3 or more sequence nodes in their lineages (i.e., unique sequences) and being sampled in both tissues that were compared. Only medians of individuals with more than 5 clones were included. The Mann-Whitney U test was performed to determine statistical significance (*p<0.05). Individuals are marked as in Figure 4.