Immune reconstitution following rituximab-based immunochemotherapy in pediatric patients with B-cell non-Hodgkin lymphomas

Rituximab is a therapeutic monoclonal antibody (mAb) directed against the CD20 molecule, which is highly expressed on normal and malignant B-cells. For over 20 years, this mAb has been applied in the treatment of B-cell non-Hodgkin lymphomas (B-NHLs) in adults [1]. Rituximab has proven to be effective in low-grade and aggressive B-NHLs [1]. The survival rates of adults with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma/leukemia (BL) have improved remarkably after rituximab was added to the baseline chemotherapy regimens [2]. This positive experience has prompted the use of the same approach in pediatric patients. There is growing evidence for the efficacy and safety of rituximab for children and adolescents with B-NHLs [3]. During B-cell development, CD20 first appears at the pre-B stage and then becomes downregulated at the plasma cell stage [1]. Rituximab causes depletion of CD20-expressing B-cell subsets but does not affect stem cells. It usually takes 6 to 9months for B-cells to reappear after therapy completion and a few more months or even years for them to reach the pretreatment levels [4]. The understanding of post-rituximab B-cell kinetics comes mostly from adults treated for autoimmune disorders or lymphomas [5]. In contrast, data regarding immune reconstitution in children are rare. Additionally, since B-cells participate in the regulation of T-cell immune responses, more information about T-cells under B-cell depletion is needed [6]. We have previously conducted two major trials of rituximab combined with chemotherapy in pediatric patients with aggressive B-NHLs [7,8]. Here we report characteristics of Band T-cell subsets in the peripheral blood of patients included in either of these studies. The majority of observations were made at very distant posttreatment time points. However, a small group of patients was examined serially at early time points to provide insight into the dynamics of post-rituximab immune recovery. We examined 253 peripheral blood samples from 154 patients (male/female ratio 4:1; median age 12 years; range 3–28 years) enrolled in one of the two trials of combined immunochemotherapy for pediatric patients with aggressive B-NHLs in Russia [7,8]. At the time of enrollment, all patients were under 18 years of age and had a newly diagnosed CD20-positive B-NHL: BL (n1⁄4 125, 81.2%), DLBCL (n1⁄4 23, 14.9%) or primary mediastinal Bcell lymphoma (PMBCL, n1⁄4 6, 3.9%). In both trials, modified Berlin-Frankfurt-M€ unster protocols were coupled with the use of rituximab (MabThera, F. Hoffmann-La Roche Ltd., Switzerland). Rituximab was infused at a standard dose (375mg/m) biweekly 4 times as described previously [7,8]. The majority of patients (n1⁄4 127, 82.5%) were examined only once after therapy completion (Group 1). They were in complete remission at the time of sampling. The median follow-up was 3 years (range 4months 12.5 years). The rest of the patients (Group 2, n1⁄4 27, 17.5%) had multiple follow-up points during the early post-rituximab phase (median follow-up 6months, range 1–30months). The pretreatment levels of Band T-cell subsets were available for 13 patients. Band T-cells were analyzed by multicolor flow cytometry. Samples were stained with fluorochrome-conjugated mAbs (Table S1) according to a standard procedure [9]. B-cells (CD19þ) were classified as follows: CD27IgDþ IgMþ (naïve B-cells), CD27þ IgD-IgM(switched memory B-cells), CD27þ IgD-IgMþ (IgM-only memory B-cells), CD27þ IgDþ IgMþ (natural effector B-cells), and CD5þ (B1a cells). The following T-cell subsets were assessed: CD3þCD4þ (T-helpers), CD3þCD8þ (cytotoxic T-cells), CD3þCD4þCD45RAþCD45RO(naïve T-helpers), CD3þ CD4þ D45RA-CD45ROþ (memory T-helpers),