Toshiya Sakai

Hypogammaglobulinemia with a selective delayed recovery in memory B cells and an impaired isotype expression after rituximab administration as an adjuvant to autologous stem cell transplantation for non-Hodgkin lymphoma.

Abstract:  Objectives: Some studies have indicated patients who received rituximab as adjuvant to stem cell transplantation had an increased risk of developing severe hypogammaglobulinemia. The mechanism of this hypogammaglobulinemia is unknown, although investigators have hypothesized a further delay in the B‐cell recovery as one potential etiology. The aim of this study is to clarify the mechanism(s) of this hypogammaglobulinemia. Methods: A total of 14 patients with high‐risk CD20+ lymphoma underwent an autologous peripheral blood stem cell transplantation (APBSCT). After a hematological recovery, rituximab was given weekly for up to four doses as an adjuvant therapy. Results: After a median follow up of 33.5 months, we found six patients (group A) who had hypogammaglobulinemia, while the eight other patients (group B) had normal serum immunoglobulin levels. A phenotypical analysis revealed that group A patients had already achieved B‐cell recovery. However, we found a severe delay in the recovery of CD27+ memory B cells, especially in the IgD−/CD27+ switched populations in group A, but CD27 negative naive B‐cells reverted to a normal range in both groups. Consistent with this, reverse transcriptase‐polymerase chain reaction studies with peripheral blood mononuclear cells revealed that most patients in group A lacked more than two classes of isotype transcripts. Conclusions: Abnormal repertoires and impaired isotype expression are seen in patients with common variable immunodeficiency, these data suggested that rituximab after APBSCT can affect not only the B‐cell quantities, but also the recovery of the B‐cell repertoires.

Tumor necrosis factor-α inhibits generation of glycophorin A+ cells by CD34+ cells

Abstract Objective The inhibitory effects of tumor necrosis factor-α (TNF-α) on cytokine-induced proliferation and differentiation of normal human erythroid progenitors have been characterized extensively, yet little is known about the maturation level of erythroid progenitors that are sensitive to TNF-α or of the expression of TNF receptors (TNFRs) in erythroid lineage. The aim of this study was to determine the extent to which human erythroid progenitor cells are sensitive to TNF-α, and to relate this to the expression of TNFRs in the erythroid lineage. Materials and Methods Highly purified human CD34 + cells underwent erythroid differentiation, with or without TNF-α. We used colony assay as well as a method by which colony-forming unit-erythroid (CFU-E) and glycophorin A (GPA; a specific marker for erythroid lineage) positive cells can be generated in liquid phase from purified human CD34 + cells in the presence of multiple cytokines, including stem cell factor (SCF), interleukin-3 (IL-3), and erythropoietin (EPO). During erythroid differentiation of CD34 + cells, TNFRs expression were monitored. Results TNF-α inhibited the generation of GPA + cells by CD34 + cells as well as the proliferative capacity of GPA + cells supported by EPO, IL-3, and SCF. Erythroid progenitors became resistant to the inhibitory effect of TNF-α as they matured. The detectable expression of TNFR-I was transient in the early phase of erythroid differentiation, whereas TNFR-II was expressed through the entire course of erythroid differentiation of CD34 + cells. Conclusions TNF-α suppresses erythropoiesis by inhibiting the generation of GPA + cells derived from CD34 + cells as well as by inhibiting the proliferative capacity of GPA + cells. Although the presence of TNFRs does not directly indicate that the receptor(s) mediates death signaling, altered expression of TNFRs depending on the level of maturation may imply altered sensitivities to TNF-α in various stage of erythroid progenitors.

Delayed redistribution of CD27, CD40 and CD80 positive B cells and the impaired in vitro immunoglobulin production in patients with non-Hodgkin lymphoma after rituximab treatment as an adjuvant to autologous stem cell transplantation

Recent studies have indicated that patients who received rituximab as an adjuvant to stem cell transplantation (SCT) demonstrated an increased risk of developing severe hypogammaglobulinaemia, which was found to be a result of delayed recovery of CD27 positive memory B cells and impaired isotype expression. It appears that rituximab influences both the quantity and quality of B‐cell redistribution. Precisely how the B‐cell repertoire regenerates after anti‐CD20‐mediated transient B‐cell depletion in patients with non‐Hodgkin lymphoma (NHL) remains to be elucidated. This study performed a phenotypical analysis of B cells in 17 NHL patients who received rituximab as an adjuvant to autologous SCT. The median period after final administration of rituximab was 36 months (range, 12–43 months). Surface antigen expression of CD27, CD40 and CD80 in NHL patients was statistically significantly different from healthy controls (n = 14). Moreover, B cells from NHL patients showed significantly impaired IgG and IgA production upon engagement of surface immunoglobulin receptors in the presence of interleukin (IL)‐2, IL‐10 and CD40 ligand in comparison with samples from healthy controls. The delayed recovery of memory B cells with an abnormal cell marker expression and function demonstrates that naive B cells may fail to differentiate into plasma cells, resulting in hypogammaglobulinaemia after autologous SCT and rituximab therapy.

Persistent panhypogammaglobulinemia with selected loss of memory B cells and impaired isotype expression after rituximab therapy for post-transplant EBV-associated autoimmune hemolytic anemia

To the Editor: Miles and McGratten (1) reported persistent panhypogammaglobulinemia after CHOP-rituximab for HIV-related lymphoma. Prolonged hypogammaglobulinemiawith rituximab has also been seen in patients with post-transplant Epstein–Barr virus (EBV)-associated lymphoproliferative disorder (2, 3), as well B-cell lymphoma when rituximab is used as maintenance after stem cell transplantation (4, 5). We report a case of severe persistent panhypogammaglobulinemia after treatment of a posttransplant EBV-associated autoimmune hemolytic anemia (AIHA) with rituximab. The patient is a 32-yr-old Japanese woman with severe aplastic anemia who underwent non-myeloablative allogeneic peripheral blood stem cell transplantation from her HLA-matched sister in September 2000 (6). On days 90 and 150, chronic graft versus host disease occurred, but was alleviated with tacrolimus and predonisolone. In April 2002, 19 months after transplantation, the serum immunoglobulin M (IgM) level increased and immuno-electrophresis analysis revealed monoclonality of IgM. A remarkable proliferation of EBV-DNA also occurred. Tacrolimus and predonisolone were rapidly tapered off and the IgM level and EBV-DNA decreased. At that time, the IgG level was 757 mg/ dL (normal range 870–1700); low but stable after tapering of these immunosuppressive agents. In July 2002, severe AIHA occurred (7). Due to the existence of predictive factors of post-transplant lymphoproliferative disease (PTLD), conventional immunosuppressive therapy for AIHA was not considered. We gave rituximab (Chugai Pharmaceutical, Tokyo, Japan), 375 mg/m once a week for a total of four doses. The clinical and laboratory signs of hemolysis rapidly improved and the hemoglobin level began to improve 2 wk after the start of rituximab treatment. B cells in her peripheral blood rapidly disappeared, as expected. But all serum immunoglobulin levels kept on decreasing and remained extremely low (IgG < 200 mg/dL, IgA < 10 mg/dL and IgM < 10 mg/dL) unless intravenous immunoglobulin (IVIG) was administered for more than 2 yr. She developed repeated bacterial infections, such as pneumonia and sepsis, and was treated with IVIG and antibiotics. In February 2005, 30 months after last administration of rituximab, we found her blood B-cell number reverted to a normal range (8% of total lymphocytes, 180/lL), despite the still severe hypogammaglobulinemia. Flow cytometry analysis revealed that her B cells were composed with only CD27 negative naı̈ve B cells. CD27 positive memory B cells, both IgD-positive non-switched and IgDnegative switched populations, were hardly seen (Fig. 1A). In addition, we evaluated immunoglobulin isotype production by analyzing immunoglobulin transcripts with reverse transcriptase-polymerase chain reaction as described (8). Her peripheral blood mononuclear cells (PBMC) only expressed the transcriptions of IgM, IgG1, IgA1 and IgA2, but no IgG2, IgG3 and IgG4 (Fig. 1B). Rituximab is known to induce B-cell depletion for up to 6 months post-treatment (9). Moreover, in patients treated with high dose chemotherapy and maintenance rituximab, it takes longer (18– 24 months) for B-cell recovery (4). This delay of B-cell recovery is likely to contribute to hypogammaglobulinemia, especially when rituximab is used for treatment of EBV-PTLD (2, 3, 10). But this does not seem to be the case in our patient who Eur J Haematol 2005: 75: 527–529 doi:10.1111/j.0902-4441.2005.t01-1-EJH2327.x All rights reserved Copyright Blackwell Munksgaard 2005

Effective in vivo purging with rituximab and autologous peripheral blood stem cell transplantation in a woman with CD5 positive primary cutaneous diffuse large B-cell lymphoma

Abstract:  Generalized subcutaneous tumors developed without any other sites of the disease in a Japanese woman. Skin biopsy revealed CD5+ and CD20+ atypical diffuse large cells infiltrating subcutaneous tissues. The diagnosis was CD5+ primary cutaneous diffuse large B‐cell lymphoma. Tumor‐specific PCR showed the existence of malignant cells in the peripheral blood and bone marrow. After three cycles of chemotherapy, she was remained in partial remission. Peripheral blood stem cells (PBSC) were harvested after the fourth cycles of chemotherapy combined with rituximab for in vivo purging. The contamination of tumor cells in PBSC was negative with PCR. She then underwent autologous peripheral blood stem cell transplantation using purged PBSC and has remained in complete remission for the past 24 month.

A preliminary analysis of the balance between Th1 and Th2 cells after CD34+ cell-selected autologous PBSC transplantation

BACKGROUND CD34+ cell-selected autologous PBSC transplantation (CD34+ APBSCT) is a procedure used for the treatment of patients with malignant disease that is intended to eliminate residual tumor cells from autologous grafts. However, frequent infectious complications after CD34+ APBSCT can occur. A delay of recovery of the absolute number of CD4+ T cells after transplantation was reported to be one disadvantageous factor. As data on T-cell function after CD34+ APBSCT are scanty, we analyzed changes in T-helper cell 1 (Th1) and T-helper cell 2 (Th2) after CD34+ APBSCT to evaluate immune reconstitution. METHODS Twelve patients underwent APBSCT (CD34+APBSCT group, n=4, and unselected APBSCT, n=8). Peripheral blood (PB) samples were obtained at 2, 4, 8, 12 and 16 weeks after the transplantation. The dynamics of the Th1 and Th2 were analyzed at a single-cell level, using flow cytometry. RESULTS In the CD34+ APBSCT group, not only the absolute count of CD4+ T cells but also the proportion of Th1 cells in CD4+ T cells and the ratio of Th1 to Th2 after transplantation were significantly decreased at 2 and 4 weeks after transplantation compared with findings in the unselected APBSCT group. DISCUSSION We suggest that higher rates of infectious complications after CD34+ APBSCT may be due to the inability of residual T cells from the CD34+ cell selection to generate mature T cells that function adequately against infection. Although further study would be required, our preliminary data provide some information on the immune reconstitution after CD34+ APBSCT and differentiation of T lymphocytes into Th1 and Th2 in vivo.

Granular lymphocyte proliferative disorder after autologous peripheral blood stem cell transplantation for multiple myeloma

Abstract: A 57‐yr‐old woman with multiple myeloma underwent tandem autologous peripheral blood stem cell transplantation (APBSCT). Fever, anemia, and thrombocytopenia, followed by the proliferation of granular lymphocytes in the peripheral blood occurred, after a second APBSCT. Clonal rearrangement of the T‐cell receptor was detected using Southern blot analysis of peripheral blood samples. Granular lymphocyte proliferative disorders (GLPD) were diagnosed. After steroid therapy, the symptoms resolved. Lymphocytosis did not recur after the discontinuation of steroids. There have been a few reports of GLPD after solid organ and allogeneic hematopoietic stem cell transplantations. We report a first case of GLPD after APBSCT.