Longitudinal studies of a B cell-derived signature of tolerance in renal transplant recipients

Abstract

Biomarkers of transplant tolerance would enhance the safety and feasibility of clinical tolerance trials and potentially facilitate management of patients receiving immunosuppression. To this end, we examined blood from spontaneously tolerant renal transplant recipients and patients enrolled in two interventional tolerance trials using flow cytometry and gene expression profiling. Using a previously reported tolerant cohort as well as newly identified tolerant patients, we confirmed our previous finding that tolerance was associated with increased expression of B cell-associated genes relative to immunosuppressed patients. This was not accounted for merely by an increase in total B cell numbers, but was associated with the increased frequencies of transitional and naïve B cells. Moreover, serial measurements of gene expression demonstrated that this pattern persisted over several years, although patients receiving immunosuppression also displayed an increase in the two most dominant tolerance-related B cell genes, IGKV1D-13 and IGLL-1, over time. Importantly, patients rendered tolerant via induction of transient mixed chimerism, and those weaned to minimal immunosuppression, showed similar increases in IGKV1D-13 as did spontaneously tolerant individuals. Collectively, these findings support the notion that alterations in B cells may be a common theme for tolerant kidney transplant recipients, and that it is a useful monitoring tool in prospective trials.

Figure 1

B cell subset definitions by flow cytometry. Switched memory (SM) IgD⁻CD27⁺; Unswitched memory (UM) IgD⁺CD27⁺; Naïve + Transitional (N+T) IgD⁺CD27⁻ and Double Negative memory (DN) IgD⁻CD27⁻. The Naïve + Transitional cells were further subset into T1 + T2, T3 and naïve subsets based on MitoTracker Green (MTG) extrusion and CD24/CD38 expression as follows: Naïve IgD⁺CD27⁻MTG⁻ ; T3: IgD⁺CD27⁻MTG⁺CD24^+/−CD38^+/− ; T1 + T2: IgD⁺CD27⁻MTG⁺CD24⁺⁺CD38⁺⁺. The bimodal distribution of MTG staining from total B cells (inset) allowed positioning of the MTG cutoff for analysis of the IgD⁺CD27⁻ B cells. https://www.itntrialshare.org/FACTOR_fig1.url

Figure 2

Distributions of B cell populations for each study group. Each plot summarizes distribution of frequencies, where: B Cell = CD19+ lymphocytes; SM = switched memory; USM = unswitched memory; DN = double negative memory; T1 + T2 = transitional type 1 and type 2; T3 = transitional type 3; and N = naïve. n = 35 for HC, n = 26 for SI, and n = 27 for TOL. Boxes reflect 25^th through 75^th percentile of data, black line indicates the median, circles are outliers and whiskers indicate range of non-outlier data. Outliers are data that are 1.5 times the interquartile range beyond the 25^th or 75^th percentiles. A one-way ANOVA was performed for each subset. Pairwise differences are indicated by bars above the plots as a result of a multiple comparisons test performed after the ANOVA; color reflects the p-value for the ANOVA: purple, p<0.001; blue, p<0.005; green, p<0.05. https://www.itntrialshare.org/FACTOR_fig2.url

Figure 3

Panel A. Clustered, stacked bar plot of memory and transitional B cell subset frequencies comprising a 6-part composition. Each column of data is derived from a single sample from one of four sample groups (indicated by a symbol above each column). Each color represents the proportion of a particular cell subset in the sample (of CD19⁺ cells). The percentages of CD19+ (of Lymphocytes) are shown beneath the stacked bar plot. 88 B cell profiles are hierarchically clustered (see Methods) resulting in three clusters and a singleton. Panel B. Same as A except samples are ordered by the percentages of CD19⁺ cells in the lymphocyte gate. https://www.itntrialshare.org/FACTOR_fig3.url

Figure 4

B cell numbers and BAFF levels. Absolute number of CD19⁺ B cells (left panel) and BAFF levels (right panel), on a log2 scale, for tolerant (TOL) patients and subjects on standard immunosuppression (SI). https://www.itntrialshare.org/FACTOR_fig4.url

Figure 5

IGKV1D-13 expression normalized by CD19⁺ cells (left panel) and naïve plus transitional B cells (right panel). IGKV1D-13 levels as measured by Sequenom analysis in 2011 (see Figure S1) in tolerant patients (TOL) and subjects on standard immunosuppression (SI). Left panel shows expression levels normalized by total number of CD19⁺ cells, and right panel shows levels normalized by numbers of naïve plus transitional B cells (CD19⁺IgD⁺CD27⁻). https://www.itntrialshare.org/FACTOR_fig5.url

Figure 6

Longitudinal IGKV1D-13 and IGLL-1 expression. A. Scatter plots for normalized IGKV1D-13 and IGLL-1 expression in all available samples. The time points indicate the point at which the sample was obtained relative to the start of the study, i.e., year 0 is the first blood sample we obtained, year 1 is one year later, etc. Mean values for each group at each time point are represented by * and connected by dashed lines. P-values represent comparisons between the groups at each time point. B. Longitudinal IGKV1D-13 expression as a function of months post-transplant. Tolerant patients (TOL) are shown in blue, patients on standard immunosuppressive therapy (SI) are shown in red. Points joined by a line depict sequential samples from the same participant. Arrows indicate four patients that were operationally tolerant when first studied, but later went back on immunosuppressive therapy. https://www.itntrialshare.org/FACTOR_fig6A.url https://www.itntrialshare.org/FACTOR_fig6B.url

Figure 6

Longitudinal IGKV1D-13 and IGLL-1 expression. A. Scatter plots for normalized IGKV1D-13 and IGLL-1 expression in all available samples. The time points indicate the point at which the sample was obtained relative to the start of the study, i.e., year 0 is the first blood sample we obtained, year 1 is one year later, etc. Mean values for each group at each time point are represented by * and connected by dashed lines. P-values represent comparisons between the groups at each time point. B. Longitudinal IGKV1D-13 expression as a function of months post-transplant. Tolerant patients (TOL) are shown in blue, patients on standard immunosuppressive therapy (SI) are shown in red. Points joined by a line depict sequential samples from the same participant. Arrows indicate four patients that were operationally tolerant when first studied, but later went back on immunosuppressive therapy. https://www.itntrialshare.org/FACTOR_fig6A.url https://www.itntrialshare.org/FACTOR_fig6B.url

Figure 7

Cross-sectional IGKV1D-13 expression in renal transplant recipients enrolled in multiple ITN studies. ITN507 is the tolerant renal transplant registry (TOL n=19; New-TOL n=5; SI n=25); ITN010 and 036 are the mixed chimerism trials with combined bone marrow and renal transplants. Samples from 7 operationally tolerant patients (TOL) were collected 0.33 – 7 years after discontinuation of immunosuppression (no differences in IGKV1D-13 expression were seen in relation to time, not shown). The three patients that eventually had to restart immunosuppression are indicated by light blue circles. One patient who was returned to SI (Return to SI) had their sample obtained one year after transplantation (which was 2 months after the patient rejected and was returned to immunosuppression). ITN013 is the Campath-1H followed by tacrolimus plus sirolimus trial where samples were obtained 4 years after transplantation. In ITN013, 7 patients were weaned and stable on sirolimus monotherapy (Sirolimus Mono), while 2 patients required re-institution of tacrolimus plus mycophenolate (SI Multiagent). https://www.itntrialshare.org/FACTOR_fig7.url