Dynamics of B Cell Recovery In Kidney/Bone Marrow Transplant Recipients

Abstract

Background: Previous studies identified B cell gene signatures and predominance of specific B cell subsets as a marker of operational tolerance after kidney transplantation. These findings suggested a role for B cells in the establishment or maintenance of tolerance. Here we analyzed B cell recovery in 4 subjects, 3 of whom achieved tolerance after combined kidney/bone marrow transplantation.

Methods: Peripheral B cell subsets were examined longitudinally by flow cytometry. Immunoglobulin heavy chain repertoire analysis was performed using next-generation sequencing. Lastly, the patients' serum reactivity to HLA was assessed by Luminex.

Results: B cell counts recovered approximately 1 year posttransplant except for 1 subject who experienced delayed reconstitution. This subject resumed immunosuppression for acute rejection at 10 months posttransplant and underwent preemptive retransplantation at 3 years for chronic rejection. B cell recovery was accompanied by a high frequency of CD20 + CD24CD38 transitional B cells and a diversified clonal repertoire. However, all 4 subjects showed prevalence of CD20 + CD27+ memory B cells around 6 months posttransplant when B cell counts were still low and the clonal B cell repertoire very limited. The predominance of memory B cells was also associated with high levels of somatically mutated immunoglobulin heavy chain variable sequences and transient serum reactivity to HLA.

Conclusions: Our observations reveal the presence of memory B cells early posttransplant that likely escaped the preparative regimen at a time consistent with the establishment of tolerance. Further studies are warranted to characterize the functional properties of these persisting memory cells and evaluate their potential contribution to tolerance induction.

Figure 1. Phenotypic assessment of B cell reconstitution

Peripheral CD19+ B cell absolute number (A), percentages of CD19+CD24^highCD38^hightransitional cells (B) and CD19+CD27+ memory B cells (C) were determined by flow cytometry on serial samples collected from the 4 subjects at various times after transplantation.

Figure 2. Subject 6 longitudinal peripheral blood IGHV repertoire analysis

Pie chart representation of the frequency of clonotypic CDR3 sequences for each the 6 IGHV families (VH1~VH6) in the peripheral blood of subject 6. Each pie chart section represents a distinct clonotypic CDR3. The size of each section corresponds to the frequency of the clonotypic sequences among all analyzed sequences. The frequency of each clonotypic sequence is also indicated by a color code (right box). The total number of sequences analyzed for each IGHV family is indicated below each pie. IGHV, immunoglobulin heavy chain variable gene.

Figure 3. Shannon Diversity Index of the IGHV sequence pools

All sequences including identical CDR3 amino acid segments were considered originating from the same clone. Shannon Diversity Index was calculated to show diversity of sequences generated from 4 subjects’ specimens at various time points. Shannon Index was calculated using the formula: $H^{\prime }=-{\displaystyle \sum _{i=1}^R}{p}_{i}ln{p}_i$.

Figure 4. Somatic hypermutation rate of IGHV sequences

Somatic mutations (SHM) within IGHV sequences were identified using IgAT software. SHM rates were calculated for all time points. The total number of sequences analyzed for each sample is indicated below the time point markers. Somatic mutation rate = (Number of mutated sequences/Number of total sequences) ×100%.

Figure 5. Longitudinal assessment of serum reactivity to HLA molecules

The reactivity of subject 6, 7, 9 and 10, serum specimens collected at various times to HLA Class I and HLA Class II was assessed using beads coated with mixed HLA molecules. Results are reported as mean fluorescence intensity (MFI).

Figure 6. Subject 6 serum reactivity to HLA class I

The reactivity of subject 6 serum at Day 182 posttransplant to HLA class I antigens was tested by Luminex using single antigen beads. Each bar represents reactivity to 1 corresponding class I molecule labeled below. Only the 50 antigens towards which the serum reacted the most are depicted. Antigens are sorted based on reactivity. Donor and recipient HLA are listed below the x-axis.

Figure 7. Subject 7 serum reactivity to HLA class I

The reactivity of subject 7 serum at Day 20 posttransplant to HLA class I antigens was tested by Luminex using single antigen beads. Each bar represents reactivity to 1 corresponding class I molecule labeled below. Only the 50 antigens towards which the serum reacted the most are depicted. Antigens are sorted based on reactivity. Donor and recipient HLA are listed below the x-axis.

Figure 8. Subject 9 serum reactivity to HLA class I and class II

The reactivity of subject 9 serum at Day 121 posttransplant to HLA class I and class II antigens was tested by Luminex using single antigen beads. Each bar represents reactivity to 1 corresponding class I or class II molecule labeled below. Only the 50 antigens towards which the serum reacted the most are depicted. Antigens are sorted based on reactivity. Donor and recipient HLA are listed below the x-axis.