Elena G. Kamburova

A single dose of rituximab does not deplete B cells in secondary lymphoid organs but alters phenotype and function

A single dose of the anti‐CD20 monoclonal antibody rituximab induces a nearly complete B cell depletion in peripheral blood, but not in secondary lymphoid organs. Modulation of this remaining B cell population due to rituximab treatment may contribute to the therapeutic effects of rituximab. To assess the in vivo effects of rituximab we used lymph nodes (LNs) collected during renal transplant surgery in patients who had received rituximab 4 weeks earlier in preparation for an ABO‐incompatible transplantation. Rituximab treatment resulted in a lower percentage of naïve (IgD+CD27−) and a higher percentage of switched memory (IgD−CD27+) B cells. Remarkably, transitional (CD24++CD38++) B cells were virtually lacking in the LNs of rituximab‐treated patients. Moreover, LN‐derived B cells from rituximab‐treated patients produced different amounts of various Ig‐subclasses after anti‐CD40/IL‐21 stimulation ex vivo. Finally, after stimulation of allogeneic T cells with LN‐derived B cells from rituximab‐treated patients, the proliferated T cells showed a decreased production of IL‐17. In conclusion, after treatment with rituximab there remains a B cell population with different functional capacities. Consequently, the effect of rituximab on the immune response will not only be determined by the extent of B cell depletion, but also by the functional properties of the remaining B cells.

Rituximab as induction therapy after renal transplantation: a randomized, double-blind, placebo-controlled study of efficacy and safety

We evaluated the efficacy and safety of rituximab as induction therapy in renal transplant patients. In a double‐blind, placebo‐controlled study, 280 adult renal transplant patients were randomized between a single dose of rituximab (375 mg/m2) or placebo during transplant surgery. Patients were stratified according to panel‐reactive antibody (PRA) value and rank number of transplantation. Maintenance immunosuppression consisted of tacrolimus, mycophenolate mofetil and steroids. The primary endpoint was the incidence of biopsy proven acute rejection (BPAR) within 6 months after transplantation. The incidence of BPAR was comparable between rituximab‐treated (23/138, 16.7%) and placebo‐treated patients (30/142, 21.2%, p = 0.25). Immunologically high‐risk patients (PRA >6% or re‐transplant) not receiving rituximab had a significantly higher incidence of rejection (13/34, 38.2%) compared to other treatment groups (rituximab‐treated immunologically high‐risk patients, and rituximab‐ or placebo‐treated immunologically low‐risk (PRA ≤ 6% or first transplant) patients (17.9%, 16.4% and 15.7%, p = 0.004). Neutropenia (<1.5 × 109/L) occurred more frequently in rituximab‐treated patients (24.3% vs. 2.2%, p < 0.001). After 24 months, the cumulative incidence of infections and malignancies was comparable. A single dose of rituximab as induction therapy did not reduce the overall incidence of BPAR, but might be beneficial in immunologically high‐risk patients. Treatment with rituximab was safe.

Longitudinal Analysis of T and B Cell Phenotype and Function in Renal Transplant Recipients with or without Rituximab Induction Therapy

Background Prevention of rejection after renal transplantation requires treatment with immunosuppressive drugs. Data on their in vivo effects on T- and B-cell phenotype and function are limited. Methods In a randomized double-blind placebo-controlled study to prevent renal allograft rejection, patients were treated with tacrolimus, mycophenolate mofetil (MMF), steroids, and a single dose of rituximab or placebo during transplant surgery. In a subset of patients, we analyzed the number and phenotype of peripheral T and B cells by multiparameter flow cytometry before transplantation, and at 3, 6, 12, and 24 months after transplantation. Results In patients treated with tacrolimus/MMF/steroids the proportion of central memory CD4+ and CD8+ T cells was higher at 3 months post-transplant compared to pre-transplant levels. In addition, the ratio between the percentage of central memory CD4+ and CD4+ regulatory T cells was significantly higher up to 24 months post-transplant compared to pre-transplant levels. Interestingly, treatment with tacrolimus/MMF/steroids resulted in a shift toward a more memory-like B-cell phenotype post-transplant. Addition of a single dose of rituximab resulted in a long-lasting B-cell depletion. At 12 months post-transplant, the small fraction of repopulated B cells consisted of a high percentage of transitional B cells. Rituximab treatment had no effect on the T-cell phenotype and function post-transplant. Conclusions Renal transplant recipients treated with tacrolimus/MMF/steroids show an altered memory T and B-cell compartment post-transplant. Additional B-cell depletion by rituximab leads to a relative increase of transitional and memory-like B cells, without affecting T-cell phenotype and function. Trial Registration ClinicalTrials.gov NCT00565331

Anti-B cell therapy with rituximab as induction therapy in renal transplantation.

Traditionally, antirejection therapy in organ transplantation has mainly been directed at T cells. During recent years, the role of B cells in acute rejection has attracted more attention. In the Radboud University Medical Center (Nijmegen, The Netherlands) we performed a randomized, placebo controlled study to assess the efficacy and safety of rituximab as induction therapy after renal transplantation. In parallel we investigated the effects of rituximab on the numbers and function of B and T cells. An overview of the results, which have largely been published in peer reviewed papers, is presented below.

Cytokine Release After Treatment With Rituximab in Renal Transplant Recipients

Background Treatment with rituximab may be accompanied by a systemic cytokine release. We studied the effects of a single dose of rituximab on cytokine levels in transplant patients and examined the underlying mechanism. Methods Twenty renal transplant recipients (10 rituximab-treated, 10 placebo-treated) were recruited from a randomized clinical trial. Rituximab or placebo was infused during surgery, and blood samples were taken before, during, and after surgery and analyzed for interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12, IL-17, interferon-&ggr;, macrophage inflammatory protein (MIP)-1&bgr;, transforming growth factor-&bgr;, and tumor necrosis factor-&agr;. in vitro, healthy donor peripheral blood mononuclear cells, purified B cells, monocytes, natural killer (NK) cells, or combinations thereof were incubated with rituximab, rituximab-F(ab′)2, or medium and MIP-1&bgr;, IL-10, interferon-&ggr;, and tumor necrosis factor-&agr; levels were measured in the supernatant. Results Rituximab-treated patients had higher serum levels of IL-10 (101 ± 35 pg/mL vs 41 ± 9 pg/mL; P < 0.01) and MIP-1&bgr; (950 ± 418 pg/mL vs 125 ± 32 pg/mL; P < 0.001) compared to placebo-treated patients at 2 hours after start of infusion. There was no difference in the level of other cytokines. In vitro, the addition of rituximab, but not rituximab-F(ab′)2 fragments, only led to significantly increased levels of MIP-1&bgr; in co-cultures of B and NK cells. Levels of MIP-1&bgr; were higher in patients with a high affinity Fc-receptor compared to those with a lower affinity Fc&ggr;RIIIa (1356 ± 184 pg/mL vs 679 ± 273 pg/mL; P < 0.01). Conclusions In addition to B-cell depletion, rituximab can modulate the immune response by inducing cytokine secretion, especially IL-10 and MIP-1&bgr;. Rituximab-induced MIP-1&bgr; secretion depends on the combined presence of B cells and FcR-bearing cells, especially NK cells.

Effects of the Anti-CD20 Antibody Rituximab on B Cells in Human Secondary Lymphoid Organs: 929

Introduction: A single dose of the anti-CD20 monoclonal antibody rituximab (RTX) induces a nearly complete B-cell depletion in peripheral blood. However, there remains a residual B-cell population in secondary lymphoid organs, such as spleen and lymph nodes. An intriguing question that remains to be answered is whether these remaining B cells are modified due to binding by RTX. Methods: To mimic the in vivo situation, where B cells are exposed to RTX but only partially depleted, spleen cells obtained from organ donors were incubated in vitro with RTX and a low concentration of complement. The distribution of B-cell subsets was analyzed after 3 days of culture in the presence or absence of CpG-B. Results: Incomplete B-cell depletion followed by culture in the absence of CpG-B resulted in a IgD+CD27(naïve) : IgD-CD27+ (switched memory) B-cell ratio that was comparable to the non-depleted condition. However, after culture in the presence of CpG-B, the B cells that survived the depletion procedure showed a reduced naïve : switched memory ratio. In contrast to the total splenic B-cell population, the depletion surviving B cells did not contain IL-10 producing cells after culture with CpG-B. Moreover, B cells were also characterized ex vivo in peripheral blood and lymph nodes obtained during renal transplant surgery from patients (n=4) who had received RTX 4 weeks before transplantation. As described in literature, a complete depletion of B cells in the peripheral blood was obtained after a single dose of RTX, however in the lymph nodes the frequency of B cells was comparable to that of untreated control patients. Remarkably, the remaining lymph node B cells from RTX-treated patients showed a lower percentage of IgD+CD27and a higher percentage of IgD-CD27+ B cells compared to lymph node B cells of untreated patients. Conclusion: Exposure of both spleen and lymph node B cells to RTX results in a shift of B cells from a naïve to a switched memory phenotype. 1389

IN VITRO EFFECTS OF RITUXIMAB ON PROLIFERATION, ACTIVATION, AND DIFFERENTIATION OF CD4+ T CELLS: 1595

Introduction: Rituximab is a chimeric anti-CD20 monoclonal antibody used in various autoimmune diseases and recently also in transplantation to reduce the level of autoor alloreactive antibodies. However, not all therapeutic effects can be ascribed to this antibody depletion. It is conceivable that the absence or modulation of antigen-presenting B cells can influence T cell activation, which might contribute to the therapeutic effects. Objective: To investigate the in vitro effects of rituximab on proliferation, activation and differentiation of CD4+CD25conventional T cells (Tconv) and CD4+CD25+ regulatory T cells (Treg). Methods: Tconv and Treg isolated from peripheral blood of healthy donors were stimulated with allogeneic B cells, monocytes, PBMC or anti-CD3/CD28 beads. T cell proliferation was assessed by means of a CFSE-based proliferation assay as well as 3H incorporation. Expression of cell surface markers was assessed by flow cytometry. Cytokine production was evaluated by intracellular flow cytometry, ELISA and quantitative PCR. Results: Rituximab inhibited the proliferation of Tconv following stimulation with allogeneic B cells. IL-2 enhanced proliferation of Treg was not affected in this experimental setting. Rituximab did not affect T cell stimulation by allogeneic monocytes, PBMC, or anti-CD3/CD28 beads. When Tconv were stimulated with allogeneic B cells, rituximab treatment resulted in a reduced production of the effector cytokines IL-2, IL-21, IFNγ, TNFα and TGFβ, and an increased production of IL-10. Addition of rituximab did not alter the expression of CD25, CD27, CD62L, CD70 and FOXP3 of Tconv and Treg, and did not induce regulatory capacity in Tconv. Conclusion: Rituximab inhibits the proliferation of Tconv, but not of Treg, when stimulated by B cells. In addition, our data suggest that rituximab treatment induces IL-10 production by Tconv in vitro, which might be due to altered B cell differentiation as a consequence of rituximab treatment.