Pharmacodynamics of Rituximab in Kidney Allotransplantation
2006; Elsevier BV; Volume: 6; Issue: 10 Linguagem: Inglês
10.1111/j.1600-6143.2006.01497.x
ISSN1600-6143
AutoresHelena Genberg, A. Hansson, Annika Wernerson, Lars Wennberg, Gunnar Tydén,
Tópico(s)T-cell and B-cell Immunology
ResumoAmerican Journal of TransplantationVolume 6, Issue 10 p. 2418-2428 Free Access Pharmacodynamics of Rituximab in Kidney Allotransplantation H. Genberg, Corresponding Author H. Genberg Division of Transplantation Surgery *Corresponding author: H. Genberg, helena.genberg@karolinska.seSearch for more papers by this authorA. Hansson, A. Hansson Department of Laboratory Medicine, Division of Pathology Karolinska Institute Karolinska University Hospital, Huddinge SwedenSearch for more papers by this authorA. Wernerson, A. Wernerson Department of Laboratory Medicine, Division of Pathology Karolinska Institute Karolinska University Hospital, Huddinge SwedenSearch for more papers by this authorL. Wennberg, L. Wennberg Division of Transplantation SurgerySearch for more papers by this authorG. Tydén, G. Tydén Division of Transplantation SurgerySearch for more papers by this author H. Genberg, Corresponding Author H. Genberg Division of Transplantation Surgery *Corresponding author: H. Genberg, helena.genberg@karolinska.seSearch for more papers by this authorA. Hansson, A. Hansson Department of Laboratory Medicine, Division of Pathology Karolinska Institute Karolinska University Hospital, Huddinge SwedenSearch for more papers by this authorA. Wernerson, A. Wernerson Department of Laboratory Medicine, Division of Pathology Karolinska Institute Karolinska University Hospital, Huddinge SwedenSearch for more papers by this authorL. Wennberg, L. Wennberg Division of Transplantation SurgerySearch for more papers by this authorG. Tydén, G. Tydén Division of Transplantation SurgerySearch for more papers by this author First published: 21 August 2006 https://doi.org/10.1111/j.1600-6143.2006.01497.xCitations: 137AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract The anti-CD20 antibody rituximab has recently gained interest as a B-cell depleting agent in renal transplantation. However, little is known about the pharmacodynamics of rituximab in renal transplant recipients. We have therefore studied the effect of single-dose rituximab in combination with conventional triple immunosuppressive therapy on the B-cell population in peripheral blood as well as in tissues. A total of 49 renal transplant recipients received single-dose rituximab, as induction therapy (n = 36) or as anti-rejection therapy (n = 13). We counted B cells in peripheral blood and performed immunohistochemical staining on lymph nodes and kidney transplant tissue samples to assess the prevalence of B cells. In all but 6 patients (88%) complete depletion of B cells in peripheral blood was achieved. In adults, 15 months after treatment the CD19+ and CD20+ cell counts were still below 5 cells/μL in the majority of patients (78%). The immunohistochemical staining showed a complete elimination of B cells in kidney tissue and a reduction of B cells in lymph nodes. In conclusion, single-dose rituximab in kidney transplant recipients evokes a long-term elimination of B cells in peripheral blood as well as within the kidney transplant. The effect seems to extend beyond the expected 3–12 months observed in lymphoma patients. Introduction Rituximab (MabThera®, Rituxan®, Roche, Basel, Switzerland) is a lytic chimeric mouse/human anti-CD20 antibody of the IgG1 subtype (1). CD20 is a transmembrane protein expressed on all mature B cells but not on plasma cells, hematopoietic stem cells or any other cell type. At present, rituximab is primarily used in the treatment of B-cell lymphoma. In addition, it has become a therapeutic alternative for various autoimmune diseases (2). Recently, rituximab has also gained interest as a B-cell depleting agent in renal transplantation. The target of most immunosuppressives currently used, is the T-cell population. For B-cell inhibition the therapeutic options have been few. In renal transplantation, rituximab is now used as induction therapy (3-5) as well as in the treatment of renal allograft rejection (6). Even so, little is known about the pharmacodynamics of rituximab in renal transplant recipients (7, 8). The objective of this study was, therefore, to evaluate the effect of single-dose rituximab on the B-cell population (9) not only in peripheral blood but also within the transplanted kidney and in the lymphoid compartment. Patients A total of 49 kidney allograft recipients at our center received treatment with single-dose rituximab, 375 mg/m2 body surface area (BSA) in combination with oral triple immunosuppression (tacrolimus, mycophenolate mofetil/azathioprine, and corticosteroids) (Table 1). Table 1. Immunosuppressive protocol used in the study (adult patients) LD/CD POD Tacrolimus Mycophenolate mofetil Azathioprine Prednisolone (mg) LD −2 0.1 mg/kg b.i.d 1 g b.i.d. 3 mg/kg 30 −1 LD/CD 0 2 g 3 mg/kg 5001 1 0.1 mg/kg b.i.d. 1 g b.i.d. 3 mg/kg 100 2 Adjusted to a concentration of 15 ng/mL 90 3 80 4 70 5 60 6 50 7 According to AUC: 75–150 mikromol/L.h alternatively 30–60 mg/L.h 40 8 30 9–30 20 31–60 Adjusted to a concentration of 10 ng/mL 15 61–90 Adjusted to a concentration of 5–10 ng/mL 2 mg/kg 12.5 >91 Adjusted to a concentration of 5 ng/mL 1mg/kg 10 1Methylprednisolone. LD = living donor; CD = deceased donor; POD = postoperative day; AUC = area under the curve. The patients either received mycophenolate mofetil or azathioprine. Initial immunosuppression included tacrolimus, mycophenolate and corticosteroids for all but the three patients on a steroid-free protocol, receiving five doses of daclizumab instead. Azathioprine was only used when mycophenolate mofetil had to be discontinued due to side effects. The indications for rituximab treatment are shown in Table 2. Table 2. Patients included in the study and the indication for rituximab treatment Indication for rituximab treatment Number of patients Adults Children Immunoadsorption1 (percentage of patients) Graft survival (percentage of patients) Induction therapy ABOi 15 10 5 15/15 (100%) 14/15 (93%) FACS XM+ 8 8 0 7/8 (88%) 7/8 (87.5%) ABOi+FACS XM+ 3 3 0 3/3 (100%) 3/3 (100%) Steroid-free protocol 3 3 0 0/3 (0%) 3/3 (100%) PRA > 40% 6 6 0 0/6 (0%) 6/6 (100%) Cancelled transplantation 1 1 0 1/1 (100%) N/A Rejection Rejection (rescue) 13 12 1 5/13 (36%) 11/13 (85%) 1Immunoadsorption consisted of either antigen-specific immunoadsorption, protein A immunoadsorption or plasmapheresis. ABOi = ABO-incompatible; FACS = flow cytometric cross-match; flow-PRA = flow cytometric panel reactive class I antibodies; N/A = not applicable. Materials and Methods To study the effect of rituximab on the B-cell population in peripheral blood, 194 venous blood samples were analyzed by flow cytometry, 171 from the adult patients and 23 from the younger patients. The blood samples were grouped according to the time elapsed after treatment. (Table 3—adult patients). Table 3. FACS analysis of B-cell depletion in peripheral blood: Adult recipients Time in relation to rituximab treatment Number of samples CD19+ cells/microL CD20+ cells/microL Median Interquartile range Median Interquartile range Healthy controls 4 127.7 27.1 127.7 27.1 Day 0 14 62.6 65.6 62.6 57.4 Day (1–3) 11 17.3 29.6 5.7 20.1 Week (1–2) 14 2.9 8.5 0.0 1.2 Week (3–6) 17 0.0 0.9 0.0 0.0 Week (6–10) 8 0.0 0.2 0.0 1.2 Month (3–5) 20 0.0 0.9 0.0 0.9 Month (6–7) 13 0.5 1.0 0.5 1.0 Month (8–10) 20 0.6 2.7 0.6 2.0 Month (11–13) 15 3.2 18.1 3.2 18.1 Month (14–16) 9 2.8 1.8 2.5 3.3 Month (17–20) 8 5.4 8.9 6.0 19.1 Month (22–26) 8 5.9 11.5 5.6 7.1 Month (28–32) 5 13.8 12.0 13.8 8.8 Month (34–43) 5 19.0 13.3 19.0 17.6 The healthy controls did not receive rituximab or any other immunosuppressive treatment. Day 0 = prior to treatment with rituximab. As the analysis was done retrospectively a full dataset could not be obtained. For the same reason there was an unequal number of samples a the various time points. To study the effect of rituximab on the presence of B cells within the transplanted kidneys, kidney biopsies and explanted kidney transplants, obtained both before and after rituximab treatment, were studied with immunohistochemical techniques (Tables 4 and 5). Kidney biopsies were obtained either intraoperatively after reperfusion (0-biopsies) or when clinically indicated due to an impairment of the kidney function. In addition, eight control biopsies were obtained from patients not receiving rituximab. (Table 4). In total we collected 24 tissue samples from patients on conventional immunosuppression and 26 after exposure to rituximab. Table 4. Immunohistochemical studies of B-cell depletion in kidney transplants before/without treatment with rituximab, using CD79α and CD20 as B-cell markers (adult and pediatric patients) Patient (biopsy no.) Postoperative day Tissue source Histologic diagnosis Immunohistochemistry CD79a+ cells CD20+ cells Tissue samples before treatment with rituximab 22(1) 0 Kidney biopsy 0-biopsy 1 1 37(1) 1 Kidney transplant Acute rejection grade 2B 3 3 40 3 Kidney biopsy Acute rejection grade 2B 0 0 22(2) 6 Kidney biopsy Ischemia 0 0 22(3) 7 Kidney biopsy Acute rejection grade 1A 1 1 5 10 Kidney biopsy Acute rejection grade 2A N/A 2 P2 (1) 12 Kidney biopsy Calcifications + focal inflammation 1 1 P2(2) 19 Kidney biopsy Acute rejection grade 1A 1 2 35 26 Kidney biopsy Acute rejection grade 2B 1 3 18(1) 27 Kidney biopsy Acute rejection grade 1B 2 2 37(2) 37 Kidney biopsy Suspect vascular rejection 0 1 18(2) 49 Kidney biopsy Acute rejection 1 1 18(3) 58 Kidney biopsy Acute tubular necrosis 1 1 P2(3) 581 Kidney biopsy Acute rejection grade 1B 0 1 P2(4) 1253 Kidney transplant Chronic rejection 1 2 P2(5) 1568 Kidney biopsy Acute rejection grade 2A 2 2 Tissue samples from controls (no treatment with rituximab) C12(1) 7 Kidney biopsy Focal inflammation 1 2 C43 13 Kidney biopsy Acute rejection grade 2A 2 2 C13(1) 16 Kidney biopsy Acute rejection grade 2A 0 1 C34 26 Kidney biopsy Acute rejection grade 2B 1 3 C13(2) 29 Kidney biopsy Acute rejection grade 2A 0 0 C6(1) 60 Kidney biopsy Chronic interstitial inflammation 1 2 C6(2) 113 Kidney biopsy Borderline acute rejection 3 3 C12(2) 210 Kidney biopsy Acute rejection grade 1B 3 3 Histopathologic diagnosis of acute rejection is based on the Banff criteria. Samples are listed chronologically according to the postoperative day. The tissue samples either consisted of kidney biopsies (n = 22) or explanted kidney transplants (n = 2). N/A = not available; P = pediatric patient (<18 years of age); C = control (no treatment with rituximab, neither prior to the biopsy nor after); 0 = 0% positive cells; 1 = 1–10% positive cells; 2 = >10–25% positive cells; 3 = >25–50% positive cells; 4 = >50%–75% positive cells; 5 = >75% positive cells. Table 5. Immunohistochemical studies of B-cell depletion in kidney transplants after treatment with rituximab, using CD79α and CD20 as B-cell markers (adult and pediatric patients) Patient (biopsy no.) Postoperative day Time in relation to rituximab treatment (days) Histopathologic diagnosis Immunohistochemistry CD79a+ cells CD20+ cells 38 0 0 0-biopsy 0 0 37(1) 4 2 Acute rejection grade 2B 1 0 P2(1) 608 5 Acute rejection grade1A 0 0 37(2) 9 7 Acute rejection grade 3–4 0 0 22(1) 18 11 Antibody-mediated rejection 0 0 8 11 11 Acute rejection grade 2A 0 0 3(1) 30 14 Antibody-mediated rejection N/A 0 38 15 15 Acute rejection grade 2A N/A 0 24(1) 16 16 Thrombotic microangiopathy 0 0 16 8 17 Arterial thrombosis 0 0 5 37 26 Borderline acute rejection N/A 0 1 7 34 Antibody-mediated rejection 0 0 22(2) 47 40 Severe antibody-mediated rejection N/A 0 36 33 42 Unspecific changes 1 0 31(1) 33 51 Acute tubular necrosis 0 0 14 22 59 Suspect acute rejection grade 1A, pyelonephritis N/A 0 31(2) 47 65 Acute tubular necrosis N/A 0 P2(2) 699 96 Acute rejection grade 1B 0 0 9(1) 99 113 Acute rejection grade1A 0 0 9(2) 108 121 Acute rejection grade1A 0 0 31(3) 107 125 Borderline acute rejection 0 0 24(2) 128 128 Acute rejection grade 1B 0 0 18 223 142 Acute rejection grade 1B 0 0 P32 199 215 Acute tubular necrosis 0 0 P2(3) 879 276 Acute rejection grade 2B-3 0 0 35 7430 436 End-stage renal disease 1 2 Histopathologic diagnosis of acute rejection based is based on the Banff criteria. Samples are listed chronologically according to time after rituximab treatment. The tissue samples either consisted of kidney biopsies (n = 22) or explanted kidney transplants (n = 4). P = pediatric patient (<18 years of age); N/A = not available; 0 = 0% positive cells; 1 = 1–10% positive cells; 2 = >10–25% positive cells; 3 = >25–50% positive cells; 4 = >50–75% positive cells; 5 = >75% positive cells. To study the effect of rituximab on the lymphoid compartment, inguinal lymph nodes were obtained at transplantation from 13 consecutive patients having received induction therapy (Table 6). In order to compare the effect of rituximab in combination with other immunosuppressives to the effect of the conventional immunosuppression alone, lymph nodes were also assessed from one non-transplanted uremic patient and from seven patients on conventional therapy (tacrolimus, mycophenolate mofetil/azathioprine, and corticosteroids) (Table 7). From 1 patient two lymph nodes were obtained. A total of nine lymph nodes were used as controls. Table 6. Flow cytometry and immunohistochemical studies of B-cell depletion in inguinal lymph nodes after treatment with rituximab (adult and pediatric patients) Patient Time from rituximab treatment to extirpation (days) Immunohistochemistry Flow cytometry CD79a+ cells (graded 0–5) CD20+ cells (graded 0–5) CD19+ cells (%) CD20+ cells (%) 10 0 3 2 1 13.6 33 0 3 3 N/A N/A 19 1 2 1 18 0 P30 8 2 2 8.7 0 21 9 3 3 15 0 31 18 1 1 N/A N/A 25 19 4 4 62 62 23 30 2 1 8.5 1 14 30 1 1 11.3 0 27 32 1 1 N/A N/A P45 42 N/A 1 29.4 0 11 44 3 2 12 0 28 44 2 2 1 1 Samples are listed chronologically according to time from rituximab treatment. Some of the lymph nodes were formalin-fixed at surgery. Flow cytometric analysis could therefore not be conducted. Patient 10 and 33 received rituximab on day 0 prior to the lymph node extirpation. P = pediatric patient (<18 years of age); N/A = not available; 0 = 0% positive cells; 1 = 1–10% positive cells; 2 = >10–25% positive cells; 3 = >25–50% positive cells; 4 = >50–75% positive cells; 5 = >75% positive cells. Table 7. Flow cytometry and immunohistochemical studies of B-cell depletion in lymph nodes from patients treated with conventional immunosuppressive therapy Patient Immunohistochemistry Flow cytometry CD79a+ cells (graded 0–5) CD20+ cells (graded 0–5) CD19+ cells (%) CD20+ cells (%) C41 3 4 N/A N/A C7 4 5 13.3 13.3 C15 4 4 5.1 5.1 C26 3 4 10.3 11.3 C29(1) 2 3 25.8 27.6 C29(2) 3 3 N/A N/A C42 3 4 N/A N/A CP39 3 3 20 20 C41 3 3 27 27 1No immunosuppressive treatment at the time of lymph node extirpation. Samples are listed chronologically according to the date of the lymph node extirpation. Some of the lymph nodes were formalin-fixed at surgery. Flow cytometric analysis could therefore not be conducted. P = pediatric patient (<18 years of age); C = control; N/A = not available; 0 = 0% positive cells; 1 = 1–10% positive cells; 2 = >10–25% positive cells; 3 = >25–50% positive cells; 4 = >50–75% positive cells; 5 = >75% positive cells. Moreover, the immunoglobulin levels in peripheral blood were assessed in the first 26 rituximab-treated patients >8 years of age (Table 8), not receiving plasmapheresis or protein A immunoadsorption (Immunosorba®, Fresenius HemoCare, Redmond, WA, USA). The AB0-incompatible patients receiving antigen-specific immunadsorption (Glycosorb AB0®, Glycorex, Lund Sweden), however, were included in the analysis of immunoglobulins. A total of 110 samples after treatment with rituximab were obtained from these 26 patients. From 11 patients samples were available before as well as after treatment with rituximab. In 15 patients, only samples retrieved after rituximab treatment were available. We have assumed that the immunoglobulin levels were normal prior to treatment with rituximab for these 15 patients, as they were normal for the other 11 patients. Table 8. Immunoglobulin levels before treatment with rituximab and at nadir after treatment rituximab Immunoglobulins (g/L) Before rituximab treatment After rituximab treatment Time in days after rituximab treatment IgM (ref. 0.27–2.10) Median 0.9 0.4 86.0 Interquartile range 0.3 0.5 199.5 Number of samples 8 26 IgA (ref. 0.70–3.65) Median 1.5 1.0 136.0 Interquartile range 1.0 0.8 261.5 Number of samples 7 24 IgG (ref. 6.9–15.7) Median 9.6 6.9 73.5 Interquartile range 6.5 3.5 266.3 Number of samples 11 26 Patients >8 years of age; ref = reference range at our laboratory. Flow cytometry Samples of venous blood in EDTA and unfixed lymph nodes were used for immunophenotyping by flow cytometry. The details of the flow cytometry were as follows: Ammonium chloride lysing solution was added to samples of venous blood to eliminate erythrocytes and serum immunoglobulins. Antibody panels for detection of cell surface markers were dispersed into polystyrene vials (Table 9). Then, 200 μL of the leukocyte cell suspension was mixed with the antibody reagents and incubated for 15 min. Subsequently, 2 mL of buffered sodium chloride pH 7.0 (PBS) was added and the samples centrifuged. The cell suspension was diluted in 200 μL PBS to achieve a cell rate of 1000–1200 cells/s. Flow cytometry was performed in a FACSCalibur flowcytometer™ (BD Biosciences, San Jose, CA, USA) using CELLQuest software (BD Biosciences) for statistical analyses. Table 9. Antibody panels used in the flow cytometry Vial Antigen Conjugate Clone Source Vial 1 CD14 FITC M0P9 Dako CD19 APC SJ25C1 BD Biosciences CD20 PE L27 BD Biosciences CD45 PerCpCy5.5 2D1 BD Biosciences CD235a (Glycophorin A) FITC JC159 Dako Vial 2 Lambda light chain FITC Polyclonal rabbit anti-human Dako Kappa light chain PE Polyclonal rabbit anti-human Dako CD19 PerCpCy5.5 SJ25C1 BD Biosciences CD20 APC L27 BD Biosciences Vial 3 CD4 FITC MT310 Dako CD8 PE DK25 Dako CD3 PerCpCy5.5 SK7 BD Biosciences CD56 APC NCAM 16.2 BD Biosciences Lymph nodes were extirpated from the inguinal area during kidney transplantation (n = 20) or other surgical procedures (n = 2). The lymph nodes were cut in a standardized manner to allow for histologic and flow cytometric analyses from adjacent areas of the tissue. Pieces of tissue of approximately 8 mm3, were inserted into polyethylene chambers, known as Medicons, (BD Biosciences) together with 1 mL of PBS. The Medicons were placed in a Medimachine™ (BD Biosciences) and the tissue disaggregated for 15–20 s in order to obtain a cell suspension. Ammonium chloride lysing buffer was added to the cell suspension and flow cytometry was performed using the technique describe above for peripheral blood. CD19 and CD20, expressed on all normal mature B cells, were used as specific B-cell markers (10). Histopathology Kidney tissue samples were fixed in 4% buffered formalin and embedded in paraffin. And 1–2 μm thick sections were cut on a rotation microtome. Routine stainings, including hematoxylin & eosin, periodic acid Schiff (PAS) and Ladewig were done and the tissue samples assessed by a transplant pathologist. Diagnosis of acute cellular and antibody-mediated rejection was based on the Banff criteria (11, 12) Immunohistochemistry Immunohistochemical stainings were performed on formalin fixed, paraffin embedded 1–2 μm thick sections, using a TechMate™500 Plus machine (Dako, Glostrup, Denmark). CD20 and CD79α were used as B-cell markers. There is no anti-CD19 antibody suitable for immunohistochemistry on formalin fixed, paraffin embedded samples. CD79α, expressed on all normal B cells (9, 10) was used instead. CD3 and CD5 were used as T-cell markers. Monoclonal anti-CD20 clone L26, anti-CD79α clone JCB117 and polyclonal anti-CD3 (Dako) were used together with anti-CD5 (Novocastra Laboratories Ltd, Newcastle upon Tyne, UK). The sections were evaluated in a blinded fashion and the inflammatory infiltrate was semi-quantitatively graded on a scale ranging from 0 to 5 as outlined below. For each antibody, one section was randomly chosen and evaluated. Only tissue samples with inflammation were included, as tissue samples without a lymphocytic infiltrate were assumed to be devoid of B cells regardless of immunosuppressive treatment. Grading used for the semi-quantitative analyses in the immunohistochemical studies: The numbers refer to the percentage of cells staining positive in relation to the entire population of inflammatory cells within the tissue. 0 = 0% positive cells 1 = 1–10% positive cells 2 = >10–25% positive cells 3 = >25–50% positive cells 4 = >50–75% positive cells 5 = >75% positive cells Statistics As there was a relatively small number of samples and sometimes a positively skewed distribution, averages were calculated using the median and the interquartile range. Wilcoxon's rank sum test was used for non-parametric statistical comparisons. Results Safety and patient survival Patient survival was 100%. One patient developed an infusion-related allergic reaction resulting in hypotension and syncope. The patient responded immediately to therapy. Three months after rituximab treatment one patient developed hypogammaglobulinemia, still persisting at the 18-month follow-up, and despite a normal B-cell count. The patient receives immunoglobulin infusions subcutaneously twice monthly. There were no other serious adverse events associated with the administration of single-dose rituximab. Efficacy Overall graft survival for patients receiving induction therapy was 94.3% (33/35). One patient was not transplanted and is therefore excluded in the analysis. For patients receiving rituximab as anti-rejection therapy graft survival was 85% (11/13) (Table 2). As the study was uncontrolled, it did not allow for any evaluation of the therapeutic effect, as in preventing acute rejection or improving graft survival. In the group of patients treated with rituximab in combination with immunoadsorption, all but one responded to treatment by an elimination of the donor-specific antibodies (ABO-antibodies or HLA-antibodies). At transplantation, the ABO-incompatible patients had anti-ABO IgM and IgG titers equal to or below 1: 4. In the patients with a positive flow cytometric cross-match prior to treatment, 91% (10/11) received rituximab in combination with immunoadsorption. All these 10 patients responded to treatment by seroconversion to a negative flow cytometric cross-match at transplantation. Flow cytometry of B-cell depletion in peripheral blood The effect of single-dose rituximab on the B-cell population in peripheral blood in adults is shown in Table 3 and Figure 1. Prior to rituximab administration (day 0) these patients had already a depression in the number of B cells: 62 cells μL (interquartile range 57.8) versus the normal reference value of 50–440 cells/μL in peripheral blood obtained previously from healthy adults at our laboratory. Complete B-cell depletion was defined as < 5 CD19+ cells/μL and was achieved in 91% of the adult patients (39/43). In 9.3% of the adult patients (4/43) complete B-cell depletion could not be demonstrated. Two of these patients lost their grafts in the early postoperative period. At day one to three after rituximab treatment there was already a reduction in the levels of B cells (median 17.3; interquartile range 29.6 cells) compared to the day 0 count. At 3–6 weeks the levels of B cells ranged from 0 cells/μL to10.9 cells/μL, 94% (16/17) of adult patients having a complete elimination of B cells. The effect of rituximab was also long term; 77.7% (7/9) of adult patients remained at <5 CD19+ cells/μL, (p < 0.001 compared to the pre-rituximab level) for as long as 14–16 months. At 2 years the B cells reappeared but remained suppressed compared to normal values (50–440 cells/μL). Only two of the adult patients with an initial complete elimination of B cells reached a B-cell count in peripheral blood within the normal range during the study period. In the children (n = 6) complete B-cell depletion was observed in four patients. For the other two children data were missing. The children experienced a faster recovery, the B cells reappearing at a median of 9 months after rituximab treatment. At the 15-month follow-up (n = 4) the B-cell count was within the normal range in all of the children. Figure 1Open in figure viewerPowerPoint Change (median) in CD19+ and CD20+ cell count (cells/μL peripheral blood) after treatment with rituximab. Immunophenotyping by flow cytometry. Day 0 = prior to treatment. Healthy controls = no treatment with rituximab or any other immunosuppressive. Histopathology The results of the histopathologic assessments of the kidney biopsies and explanted kidney transplants are shown in Tables 4 and 5. Histopathology of lymph nodes is not shown as the morphology was normal. Immunohistochemical studies of B-cell depletion in kidney transplants The results from the immunohistochemical staining of kidney biopsies and explanted kidney transplants were grouped according to the immunosuppressive regimen. (Tables 4 and 5). In patients treated with conventional immunosuppression only, 24 samples were stained for CD20. In 87.5% (21/24) of the analyzed samples CD20+ cells were detected. Twenty-three tissue samples were stained for CD79α+ cells and 73.9% (17/23) of these had a positive staining. Twenty-six samples collected after treatment with rituximab were stained, 96% (25/26) being negative for CD20. CD79α+ cells were only detected in 15% (3/20) of the tissue samples stained (Figures 2 and 3). Comparing CD20+ cells to CD79α+ cells prior to treatment with rituximab, did not demonstrate any statistically significant difference. The same observation was done after treatment with rituximab. However, comparing CD20+ cells within the kidney tissue in patients treated with rituximab to those on conventional immunosuppression only, the CD20+ cells were significantly reduced (p < 0.0001). The same result was obtained for the CD79α+ cells (p < 0.001). Figure 2Open in figure viewerPowerPoint Immunohistochemistry of kidney transplant biopsies and explanted kidney transplants, comparing triple immunosuppression (tacrolimus, mycophenolate mofetil/azathioprine and corticosteroids) with rituximab in combination with oral immunosuppression (tacrolimus, mycophenolate mofetil/azathioprine and corticosteroids). Columns representing the percentage of tissue samples with detectable CD20+ and CD79α+ cells. RIT = rituximab + oral immunosuppression. IS = conventional immunosuppression. Figure 3Open in figure viewerPowerPoint Immunohistochemistry of kidney biopsies before and after rituximab treatment. 1: Biopsy obtained postoperative day 10 prior to treatment with rituximab and diagnosed as acute rejection grade 2A (Table 4, patient 5). 2: Biopsy obtained postoperative day 37 after treatment with rituximab and diagnosed as a borderline acute rejection (Table 5 patient 5). The condition resolved spontaneously. No anti-rejection therapy was given. H&E = hematoxylin and eosin staining. CD20 = immunohistochemstry using anti-CD20 MAb L26 (BD Bioscience) and CD79a = immunohistochemstry using anti-CD79α MAb JCB117 (Dako). Original magnification 20×. Flow cytometry and immunohistochemical studies of B-cell depletion in lymph nodes In the immunohistochemical stainings of lymph nodes obtained at transplantation from patients on conventional therapy, the CD20+ cell infiltrate corresponded to 25–100% of all inflammatory cells, (median > 50–75%) (Table 7; Figures 4 and 5). In the lymph nodes from patients treated with rituximab prior to transplantation, the B-cell infiltrate corresponded to 1–75% (median > 10–25%) (Table 6; Figures 4 and 5). When comparing the two groups, the difference in CD20+ cells was statistically significant (p = 0.001). A reduction in CD79α+ cells was also observed although it was less pronounced (p = 0.01). Figure 4Open in figure viewerPowerPoint Immunohistochemical stainings of lymph nodes. 1: Normal lymph node from a ur
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