Delayed Redistribution of CD27, CD40 and CD80 Positive B Cells and the Impaired In Vitro Immunoglobulin G Production in Patients with Non-Hodgkin Lymphoma Treated with Rituximab.

Rituximab (RT) has been proven to be very effective in depleting normal and malignant B lymphocytes in vivo and it is widely used for the treatment of B cell malignancies, particularly B cell non-Hodgkin’s lymphoma (NHL). RT alone does not appear to cause severe hypogammaglobulinemia according to initial clinical trials. However, recent studies revealed that patients who received RT as an adjuvant to stem cell transplantation (SCT) demonstrated an increased risk of developing severe hypogammaglobulinemia. We have found such hypogammaglobulinemia to be due to the delayed recovery of CD27 positive memory B cells and an impaired isotype expression. (Nishio et al. Eur J Haematol, 2006). This finding suggests that RT can influence not only the quantity, but also the quality of B-cell redistribution. Nevertheless, to our knowledge, precisely how the B-cell repertoire regenerates after anti-CD20-mediated transient B-cell depletion in patients with NHL remains to be elucidated. To clarify this, we performed a phenotypical analysis of B cells.

A total of 22 patients with NHL who received RT combined with autologous SCT (n=17) or CHOP (n=5) were evaluated to identify their immunophenotype. The median period after the last administration of RT was 33.5 months (range from 12 to 56 months). We investigated the expression of various markers, including CD27, CD38, CD40, CD80, CD86 and CD95 on B cells by immunofluorescence staining with a flowcytometry analysis. A statistically significant difference was noted in three of the six surface antigens when the expressions of those antigens were compared with those in the healthy control populations (N=14). The most striking differences we found was the expression levels of CD27. The healthy control group had a much higher expression of CD27 in comparison to those of the patients treated with RT (28.1±14.1% vs 8.2±6.1%, p<0.001). In addition, significant differences in the expression of CD40 and CD80 were also noted. While the positive rates of CD80 and CD40 on B cells from healthy controls were 21.5±10.8% and 80.5±16.7%, those of patients treated with RT were 9.9±6.9% and 49.7±33.5%, respectively (p<0.01 and p<0.05).

Since CD40-CD40L and CD80-CD28 pathways between B and T cells are necessary for the development of CD27 positive polyclonal B-cell activation and immunoglobulin production, we hypothesized that the B cells from patients treated with RT thus had a reduced ability to differentiate into plasma cells and immunoglobulin production in vitro. To test this hypothesis, we purified the B cells from ten patients with NHL treated with RT and then cultured them upon the engagement of immunoglobulin receptor and CD40 in the presence of IL-2 and IL-10. After eight days of stimulation, the supernatants of the culture were harvested and the concentrations of immunoglobulin were measured by ELISA. As a result, the IgG production was found to be significantly impaired in patients with NHL in comparison to those from the healthy controls.

The observation of a delayed recovery of the memory B cells with an abnormal cell marker expression and function demonstrates that naive B cells may therefore be responsible for their failure to differentiate into plasma cells after RT therapy.