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Clinical and Translational Research

Phenotypic Evaluation of B-Cell Subsets After Rituximab for Treatment of Acute Renal Allograft Rejection in Pediatric Recipients

Zarkhin, Valeriya1; Lovelace, Patricia A.2; Li, Li1; Hsieh, Szu-Chuan1; Sarwal, Minnie M.1,3

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doi: 10.1097/TP.0b013e318213df29
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Abstract

B cells play important role in acute and chronic graft rejection. Historically, the primary focus of research on B cells in transplantation has been on plasma cells (PCs) and their contributory role in antibody-mediated rejection (1). Recent studies hypothesized that intragraft B cells may function as efficient antigen-presenting and costimulatory cells, regulate dendritic cell function and migration, and support robust T-cell-mediated rejection (2–4). Recent studies of acute rejection (AR) in pediatric (5–8) and adult (9, 10) renal transplant recipients showed that an incidence of 22% to 53% of AR episodes being associated with intragraft CD20+ B-cell clusters. The inability to target B cells with standard immunosuppressive agents may result in a recalcitrant rejection with poor graft outcome (5). Recent studies showed transient circulating/intragraft B-cells depletion and favorable clinical responses after Rituximab treatment (11).

Rituximab, a humanized monoclonal antibody that reacts with the pan B-cell CD20 marker, is frequently used to treat a variety of B-cell lymphomas (12), autoimmune diseases with B-cell involvement (13), has demonstrated efficacy in ABO incompatible transplantation (14–17) in steroid- resistant and humoral rejections in adult kidney (18–22), heart (23–27), and liver (28) transplant recipients, for induction therapy in sensitized patients (18) and for the planned reduction of preformed antibodies in sensitized patients (29–31). Rituximab has been used in pediatric patients to treat autoimmune diseases (32–36), lymphomas (37–39), nephrotic syndrome (40, 41), focal segmental glomerulosclerosis (42–44), and CD20+ posttransplant lymphoproliferative disorder (45–48), although there are limited published case reports on the use of Rituximab for treatment of recalcitrant rejection in pediatric heart (49, 50) and renal transplant recipients (21, 51, 52).

Clinical benefits of Rituximab depend on effective B-cell depletion (11, 53). However, the regeneration profile of peripheral B-cell subsets after Rituximab treatment requires additional investigation. Despite the widespread use of Rituximab, the phenotype of the B cells emerging after Rituximab treatment has been recently described only in adult patients with lymphoma (54), systemic lupus erythematosus (SLE) (55, 56), Sjogren's syndrome (57, 58), rheumatoid arthritis (59–61), and high panel reactive antibody dialysis awaiting transplantation (62, 63), but not in transplanted patients treated with Rituximab for AR, especially pediatric patients. Recent study evaluated B cells repopulation in sensitized adult kidney transplant candidates after a single dose of Rituximab before transplantation, reporting a delay in functional maturity of repopulating human leukocyte antigen (HLA)-specific B cells, particularly those specific for donor HLA (63). Transient depletion of circulating and intragraft B cells after four doses of Rituximab treatment has been reported in pediatric patients, with mean repopulation times of ∼12 months (11), compared with mean repopulation times of more than 15 months in adult studies (20).

The goal of this study was to evaluate the phenotype of B-cell subsets after treatment of renal AR with or without exposure to B-cell-depletion therapy with Rituximab.

RESULTS

Peripheral blood CD19+ B cells were examined for the expression of CD27 and immunoglobin D (IgD) markers to differentiate an IgD+CD27 fraction of naive B cells and CD27+ memory B cells, which can be further separated into isotype nonswitched IgD+ and isotype switched IgD subsets, and CD19+/CD27/IgD double negative (DN) B cells (55). Human transitional B cells, those cells in developmental transition between immature B cells in the bone marrow and mature B cells in the periphery that are believed to represent a key negative selection checkpoint for autoreactive B cells, have been defined based on the high expression of CD38 and CD24 markers (56, 64, 65), whereas plasmablasts/PCs have been detected as CD19low/CD38high/CD27+/IgD cells. We evaluated circulating B-cell subsets in percentages from total peripheral blood CD19+ B cells.

Circulating B-Cell Phenotypes, Recipient Age, and Time Posttransplantation

To exclude the impact of patient age and time posttransplantation on the phenotype of circulating B cells, we studied correlations of these parameters with percentages of naive, memory, DN, PC, and transitional B cells in transplanted pediatric patients with stable graft function (n=18), defined as patients with stable serum creatinine and absence of rejection on protocol surveillance biopsies (performed in our program at 3, 6, 12, and 24 months posttransplantation). There was no impact of time posttransplantation (5–25 months) on different examined populations of B-cell subsets. To evaluate the impact of young age on B-cell subsets, a subanalysis of B-cell subsets was performed on children older than and younger than 5 years. Pediatric patients with stable graft function (STA) younger than 5 years (n=4) had nonsignificant trend toward less memory B cells (9.8%±6.4% from CD19+ cells) and more naive B cells (86.8%±8.1%) compared with children older than 5 years (n=14, 16.2%±9.4% and 77.9%±12%, respectively, both P=0.2). There was no impact of recipient age on the frequency of circulating PC and DN B cells: patients younger than 5 years had similar frequency of PC (1.8%±1.4%, P=0.3) and DN cells (3.3%±1.8%, P=0.12) compared with patients older than 5 years (3.2%±4.2% and 5.8%±4.04%, respectively). Younger patients (those younger than 5 years) showed a reduction in naive (r=−0.97) and an elevation in memory (r=0.97) B cells with longer time posttransplantation.

Infections Complications Increase the Percentage of Circulating Memory B Cells

We next studied B-cell subsets frequency in the peripheral blood of patients with stable graft function without (hSTA; n=8) and with concomitant viremia with cytomegalovirus, Epstein-Barr virus, or polyoma virus infection (iSTA; n=10). There was a significant difference in naive/memory B-cells ratio between the hSTA and iSTA groups. We observed decrease in naive CD19+/CD27/IgD+ (delta=−12.9%, P=0.01) and elevation in total memory B cells (delta=9.1%, P=0.02; Fig. 1) alone with elevation in DN B cells (delta=3.8%, P=0.02) in iSTA compared with hSTA patients. There was no discernable impact of viremia on the frequency of early (delta=0.3%, P=0.8), late (delta=−3.9%, P=0.1), or overall total transitional B cells (delta=−3.5%, P=0.3) or PC (delta=0.5%, P=0.8).

FIGURE 1.
FIGURE 1.:
Effect of active infection complications on the frequency of circulating B-cell subsets in pediatric and young adult patients with stable renal allograft function. Frequency of circulating naive (CD19+/IgD+/CD27−), memory (CD19+/CD27+), double-negative (DN, CD19+/IgD−/CD27−), plasmablasts and PCs (CD19low/CD38high/CD27+/IgD−), and transitional (CD19+/CD24high/CD38high) cells were studied by multicolor flow cytometry analysis in 18 pediatric patients with stable renal allograft function (STA) and correlated with age, time posttransplantation, and infection complications. iSTA, STA patients with interval infection; hSTA, healthy STA patients.

Acute Renal Rejection Decreases the Ratio of Naive/Memory B Cells

Peripheral blood B cells were examined at the time of biopsy-proven diagnosis of AR (Banff criteria (66)), before steroid immunosuppression intensification. As patients in the rejection group had no reported active bacterial or viral infections at the time of the study, stable patients without interval infection were chosen as the control group for further analyses (hSTA group, n=8). There was a significant decrease in naive/memory B-cell ratio at AR (delta=8.8%, P=0.01), mostly due to a significant elevation of memory B cells (12.4% increase, P=0.02) as compared with hSTA, with no changes in the frequency of early, late, or total transitional (P=0.4), DN (P=0.4), or PC (P=0.6) cells (Fig. 2A and B, Table 1). This increase in memory B cells at AR was significantly greater than increase in memory B cells observed with viral infection (7.9% increase, P=0.01).

FIGURE 2.
FIGURE 2.:
Phenotyping B-cell subsets after B cell reconstitution. (A) Expansion of memory B cells in acute rejection. We studied the distribution of circulating B cells in AR. CD19+-gated B cells were examined for the expression of CD27 and IgD markers to delineate naive (CD19+/IgD+/CD27−), immunoglobulin isotype nonswitched (CD19+/IgD+/CD27+), and immunoglobulin isotype switched (CD19+/IgD−/CD27+) B-cell subsets. Presented flow cytometry dot plots show studied B-cell subsets in different representative patients. In patient with stable graft function (a) who has never had an AR episode, the majority of B cells (87%) were naive and 10.5% were memory B cells. Within days after AR (b), we detected increase in both isotype nonswitched and isotype-switched memory B cells (23% of total memory B cells). One week after treatment of AR with pulse steroids (S-AR) (c), the percentage of memory B cells was still high (29%). Rituximab treatment (R-AR) resolved in depletion of 99.9% of circulating CD19+ B cells within a week (e) (this plot was obtained from one of the R-AR patients at 7 days post-Rituximab to monitor B-cell depletion and was included in this study at 27.5 months post-Rituximab). One year post-S-AR treatment (d), we observed persistence of memory B cells (37%). However, B cells repopulating at 1 year post-R-AR were mostly naive, and percentages of circulating B-cell subsets were similar to those in stable patients (f). (B) Persistent decrease in memory and increase in naive B cells after Rituximab. Regarding Flow cytometry data, naive/total memory B-cells ratio was calculated for all patients. Naive/memory B-cell ratio decreased two times within couple of days after acute rejection (AR) in the S-AR patients, compared with stable patients without infection complications (hSTA group, P=0.01). S-AR treatment resulted in persistence of low naive/memory B-cell ratio for at least 17.3±26.04 months post-AR. Rituximab treatment resulted in long-term persistent (23.5±10.9 months post-Rituximab) recovery of naive/memory B-cell ratio that was significantly higher than at AR (P=0.004) and after S-AR treatment given for AR (P=0.001). Adult healthy volunteers had naive/memory B-cell ratio (1.35±0.15) similar to AR (P=0.3) and S-AR-treated rejectors (P=0.21), but significantly lower compared with hSTA (P=0.007) and Rituximab-treated rejectors (P=0.0005).
TABLE 1
TABLE 1:
Frequency of circulating B-cell subsets in the patients in the study

Rituximab Therapy Alters the Distribution of B-Cell Subsets

As Rituximab results in complete peripheral CD19+ B-cell depletion for 6 to 9 months with B-cell repopulation by 1 year (11), here we examined the proportions of different B-cell subsets 1 year after intent to treat the AR episode with pulse steroids alone (S-AR group), or a combination of pulse steroids with Rituximab therapy (R-AR group). After treatment of AR with pulse methylprednisolone, nevertheless the total percentage of circulating CD19+ B cell from total peripheral blood mononuclear cell (PBMC) count was similar to that in hSTA patients (3.8%±3.9% in S-AR vs. 6.1%±3.4% in hSTA, P=0.3, Table 1), increase in memory B cells persisted even 17.3±26.04 months post-AR treatment. Thus, a year after AR treatment, we identified a reduction in the naive B cells in the peripheral blood of S-AR rejectors compared with hSTA patients (63.8%±8.5% vs. 87.14%±5.5%, respectively, P=0.002) along with expansion of total memory B cells (28.4%±6.7% vs. 9.74%±4.2%, P=0.0003), mostly due to isotype switched B cells (delta=10.96, P=0.02; Fig. 2A and B, Table 1). Interestingly, the percentage of DN B cells at 1 year after S-AR treatment was significantly higher compared with hSTA (7.9%±2.9% vs. 3.1%±1.8%, respectively, P=0.0001; Table 1). There was no significant difference in PC cell count between S-AR and hSTA patients (P=0.7).

One year after treatment of AR with Rituximab, the total percentage of circulating CD19+ B cell from total PBMC count had reverted to normal (6.4%±5.1% in R-AR vs. 6.1%±3.4% in hSTA, P=0.9; Table 1). Rituximab-treated patients had a significant increase in naive/memory B-cell ratio over S-AR group (9.9%±5.6% vs. 2.14%±0.76%, respectively, P=0.001; Fig. 2B) at the expense of a significant reduction in memory B cells (16.6% reduction compared with S-AR, P=0.0006), mostly due to reduction in isotype switched memory B cells (7.3% reduction, P=0.02), as well as an increase in repopulated naive B cells (17.9% increase, P=0.004; Table 1). The repopulated naive/memory B-cell ratio in R-AR group was similar to hSTA group (9.9±5.6 vs. 11.7±7.9, respectively, P=0.6). The percentage of circulating DN B cells in R-AR group was lower compared with S-AR treated rejectors (P=0.04) and similar to that in hSTA patients (P=0.13; Table 1). There were no significant differences in the number of PC (2.37%±1.2% in R-AR vs. 4.1%±4.3% in S-AR, P=0.38) and transitional B cells (6.5%±9.4% in R-AR vs. 4.8%±4.7% in S-AR, P=0.6) between R-AR and S-AR groups (Table 1). To summarize, 1-year post-Rituximab treatment, the pattern of circulating B cells in R-AR patients was similar to that in hSTA patients without significant differences in the frequency of naive (P=0.1), memory (P=0.5), DN (P=0.13), transitional (P=0.4), and PC (P=0.7) B cells (Fig. 2B, Table 1).

Within the R-AR group, the percentage of repopulated naive B cells was significantly higher in patients with a sustained response without relapse of rejection (n=8; 85.8%±6%) compared with the patients who had rejection relapse within 12 months of initial therapy due to immunosuppression nonadherence (n=3; 70.5%±3.4%, P=0.004). Kaplan-Meier survival analysis (Fig. 3) shows that patients with a higher ratio of reconstituted naive/memory B cells had trend toward better graft survival compared with other patients in R-AR group (P=0.06).

FIGURE 3.
FIGURE 3.:
Graft loss in Rituximab-treated rejectors is associated with decrease in naive and increase in memory B-cells. Kaplan-Meier survival analysis shows that patients with high ratio of naive/memory cells (12.4±5.3, 8 patients) had trend to longer graft survival compared with patients with low ratio (4.9±3.6, 3 patients, P=0.06).

There was a trend toward lower numbers of transitional B cells (3.6%±3.2%) and higher numbers of DN B cells (10.2%±6.4%) in the relapsing group compared to sustained responders (8.3%±12.2%, P=0.4 and 4.04%±2.2%, P=0.2, respectively) but likely due to the small number of relapsing patients in the R-AR group these differences are not significant. Because plasmablasts/PCs are not directly targeted by Rituximab, we evaluated circulated PC as percentages from CD19+ B cells after Rituximab therapy. There was higher percentage of PC in relapsers (3.6%±0.9% vs. 1.9±1.1 in sustained responders, P=0.06).

Serum BAFF Level and Surface BAFF-R Expression

We studied whether differences in repopulating B-cell subsets could be related to differences in circulating B-cell activating factor (BAFF) or the expression of the BAFF-R in the hSTA and the rejection patient groups. Although circulating BAFF levels by enzyme-linked immunosorbent assay trended higher in the R-AR group (2.6±3.4 ng/mL) than in hSTA patient (0.96±0.2 ng/mL) or S-AR group (1.04±0.4 ng/mL), the results did not reach statistical significance (P=0.3). When CD19+ cells were studied by flow cytometry for the surface BAFF-R expression, we detected no significant difference in between hSTA patients (93.9%±7.4% of total CD19+ cells), or in patients after treatment of graft rejection (92.6%±7.3% in S-AR and 96.2%±2.2% in R-AR).

DISCUSSION

Despite a growing awareness of the role of intragraft B cells in acute cellular and humoral renal rejections (1, 5, 6, 8, 9, 67), little is known about the pattern of circulating B cells in stable transplant patients, at graft rejection and after different approaches to rejection treatment. Transient B-cell depletion using Rituximab has been shown to be clinically efficacious in the treatment of AR in pediatric renal transplant patients (11). There is a paucity of information on circulating B-cells repopulation profiles in patients after Rituximab therapy for AR, particularly in pediatric patients. Thus, here we characterized, for the first time, frequency of different B-cell subsets in pediatric patients after renal transplantation.

This study maps differences in naive and memory B-cell subsets impacted by young recipient age (younger than 5 years) and interval viral replication in stable pediatric kidney transplant recipients. The percentages of memory (P=0.02) and DN B cells (P=0.02) were significantly increased and naive B cells significantly decreased (P=0.01) in iSTA patients with viremia, compared with hSTA patients without interval infection. Time posttransplantation was not a confounder for the frequency of B-cell subsets, except in the youngest patients younger than 5 years who showed an expansion of memory B cells over time.

This study also confirms that there is heterogeneity in the B-cells reconstitution process after different modalities of treatment. We have tested the effect of Rituximab (CD20+ B-cell depleting antibody) therapy with steroid pulsing to steroid therapy alone on circulating B cells patterns. During initial ontogeny, memory B cells accumulate at significant rate and typically represent 10% to 20% of all peripheral blood B cells by 2 years of age and reach adult levels (30%) by 5 years of age (68). The results of this analysis reveal that the frequency of memory B cells can increase almost 2.5-fold in patients with biopsy-proven AR over hSTA patients without rejection (P=0.004). Treatment of AR with methylprednisolone pulsing results in long-term persistence of these increased circulating memory and DN B cells.

Although Rituximab treatment results in rapid depletion of 99.9% of circulating CD19+ B cells within a week (11), total B-cell counts return to normal a year later. Unlike the S-AR patients, the R-AR group had prolonged reduction in memory B cells (P=0.0006) with predominance of naive B cells (P=0.002) after complete B-cell repopulation. These results are consistent with similar data reporting delay in reconstitution of peripheral blood CD27+ memory B cells after B-cell depletion therapy in adult patients with rheumatoid arthritis (59–61), SLE (55, 56), Sjögren's syndrome (57, 58), and in desensitization prior transplantation (62, 63). Recent studies have also reported a delay in functional maturity of repopulating HLA-specific B cells, in particular those specific for donor HLA (63) after Rituximab usage for desensitization in sensitized adult renal transplant candidates. In R-AR group, the repopulating B cells were predominantly naive in sustained responders, whereas in relapsers these were mostly memory B cells. Additional corroborative studies are required to confirm whether these different B-cell populations confer differential risks to graft survival. This observation suggests process after Rituximab treatment of renal AR in pediatric/young adult patients, which impacts clinical outcomes, possibly because of different sensitivity of the B-cell clones to the Rituximab or conversely a differential impact presence of survival factors. Serum BAFF levels are known to be positively associated with the peripheral blood CD19+ B-cell count (r=0.8) in Rituximab-treated patients (69). Despite our investigation into the B-cell survival pathways, we could not demonstrate any differences in circulating BAFF levels or expression of the BAFF-R in the repopulated CD19+ B cells at 1 year post-AR treatment.

Because CD20 PC cells are not directly targeted by Rituximab, we were interested in their frequency in R-AR group. Interestingly, we observed 1.9 times higher percentage of PC within relapsers compared with sustained responders (P=0.06). Unlike previous studies with Rituximab therapy in adults with autoimmune diseases and lymphoma reporting reconstitution of the peripheral blood with immature transitional B cells similar to what has been described during the original ontogeny of the immune system and after bone marrow transplantation (53), we did not detect any statistically significant differences in the percentage of repopulating total transitional B cells.

Recently, Anolik et al.(55, 70) reported that some patients with SLE display an expansion of circulating CD19+/IgD/CD27 (DN) B cells, whereas in healthy subjects, DN cells are always lower than 10% of all CD19+ B cells. Further detailed analysis of DN cells in patients with SLE characterized them as memory B cells and, most important, increased frequency of CD19+/CD27/IgD memory B cells was significantly associated with higher disease activity index, a history of nephritis, and disease-specific autoantibodies (70). In this study, we detected significantly higher level of DN cells in the peripheral blood of iSTA patients with infectious complications (P=0.02) compared with hSTA patients without complications, and 2.5-fold increase in DN cells in Rituximab-treated relapsers compared with sustained responders. The observation of negative association between the percentages of DN cells and CrCl (r=−0.8) in sustained responders may suggests that patients repopulating with higher percentage of DN cells after Rituximab treatment have worse graft function. However, further detailed phenotypical and functional analysis of this B-cell subset in pediatric patients with kidney transplant is needed to understand the role of CD19+/CD27/IgD B cells in graft function and AR treatment responsiveness.

CONCLUSIONS

Increases in circulating memory B cells in pediatric/young adult transplant patients are associated with active viral replication and AR and their persistence in higher numbers after treatment of graft rejection may adversely impact graft outcomes. Transplant protocols that can harness the immunologic advantage of young recipient age should be explored as the memory B-cell pool is evolutionarily lower early in life and may support better long-term graft survival, as previously noted (71). Significant differences in peripheral B-cell phenotypes are observed in patients receiving pulse steroids therapy alone, where the expansion of memory B cells at the time of rejection is seen to persist even 1 year later; versus Rituximab therapy, where the repopulating B-cell pool returns as preferentially naive. Therapies that can skew the population of naive/memory B cells to increase may result in improved and sustained resolution of rejection. Further studies on the correlations of different B-cell subsets with transplant outcomes are warranted.

MATERIALS AND METHODS

Rituximab Drug Dosage

Rituximab, a genetically engineered, chimeric, murine/human monoclonal antibody directed against the CD20 antigen, was provided by Genentech (San Francisco, CA) and Biogen Idec (San Diego, CA). For the treatment of CD20+ renal AR in pediatric patients, Rituximab was administered by intravenous infusion at a standard dose of 375 mg/m2 weekly for 4 consecutive weeks. The details of Rituximab administration have been described previously in our clinical trial (11).

Study Patients

Thirty-five pediatric/young adult transplant patients (1.3–22.3 years at study) were included in this study and all AR were biopsy proven. Three patient groups were studied for B-cells phenotyping: 17 transplant patients with CD20+ AR who received Rituximab therapy with steroid pulsing (R-AR; n=11) or steroid pulsing alone (S-AR; methylprednisolone at 10/mg/kg/dose for 3 consecutive doses; n=6), 18 stable transplant patients on maintenance immunosuppression with (iSTA; n=10) or without interval infection (hSTA; n=8), and three normal healthy young adult controls. iSTA patients had concomitant viremia with cytomegalovirus, Epstein-Barr virus (3000–5000 whole blood polymerase chain reaction), or polyoma virus (30,000 blood polymerase chain reaction).

Rejections were classified as CD20+ dense if they had one or more B-cell-infiltrating clusters with absolute count of at least 100 CD20+ cells/hpf as reported in our clinical trial (11) and previous studies of CD20+ rejections (8). Rejection episodes in this study were captured at a mean time of 33 months posttransplantation. As mean repopulation times have been previously evaluated at ∼12 months from our previous study (11), B-cell subsets were analyzed after 1 year in the groups.

Relevant demographic and clinical data were collected on all patients (Table 2). The study was approved by the institutional review board of Stanford University (Institutional Review Board# 13688, IND# BB-IND 11788, www.clinicaltrial.gov identifier is NCT00697996).

TABLE 2
TABLE 2:
Demographics of the patients in the study

Flow Cytometry Analysis

PBMCs were prepared by Ficoll-Paque PLUS separation (GE Healthcare, Munich, Germany) and B-cell subsets were immunophenotyped by five colors flow cytometry according to the standard protocols. Subsets of naive, memory, transitional, DN, and PC B cells were classified regarding previously described protocols (Figs. 2A and 3A) (54, 55) using following antibodies: phycoerythrin-Cy7-labeled CD19, allophycocyanin-labeled CD27, peridinin chlorophyll A protein–Cy5.5–labeled CD38, phycoerythrin- labeled IgD, fluorescein isothiocyanate–labeled anti-human CD24, and fluorescein isothiocyanate–labeled anti-human BAFF-R (Becton Dickinson, San Jose, CA). Data acquisition was performed on a BD LSRII Flow Cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) with BD FACSDiva Software followed by data analysis using FlowJo software. Cell viability was evaluated using Vi-Cell XR cell viability analyzer (Beckman Coulter, Fullerton, CA) and samples with cell viability more than 90% were stained for Flow Cytometry analysis. Ten thousand events were collected for each analysis.

Serum BAFF Measurements

Red-top whole blood tubes were collected from all patients at the time of PBMC collection for the B-cell subsets analysis. Serum samples were obtained by centrifugation of red-top tubes at 2000g for 10 min. Serum BAFF levels were measured by enzyme-linked immunosorbent assay (Cat. No. DBLYS0; R&D Systems, Inc. Minneapolis, MN) according to the manufacturer's instructions.

Statistical Analysis

The t test, chi-square test, and Pearson correlation coefficients were calculated for parametric clinical parameters, and Spearman correlation coefficients and Wilcoxon signed-rank test were calculated for nonparametric clinical parameters. Results have been presented as mean±SD, and P values equal to 0.05 or less is considered significant. All statistical analyses were performed using SAS 9.1.2 (SAS Institute Inc., Cary, NC). Kaplan-Meier survival analysis was performed to compare graft loss accidence in patients with high versus low ratio of naive/memory cells.

ACKNOWLEDGMENTS

The authors thank Amery Chen for her help with clinical samples collection and David L. Hirschberg for his purchase of flow cytometry antibodies and help in creation of six-color flow cytometry panel. They also thank the Beta Sigma Phi support for the Sarwal Lab for funding part of this work.

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Keywords:

Rituximab; B cells; Acute rejection; Pediatric transplantation

© 2011 Lippincott Williams & Wilkins, Inc.