Rituximab Mediated Depletion of B Cells Following Transplant of Human CD20 Transgenic Mouse Bone Marrow Results in Prolonged B Cell Depletion and Augmented Anti-Tumor Immune Response.

We previously demonstrated rejection of murine tumors due to enhanced anti-tumor cytolytic and Th1 cytokine responses in mice lacking B cells (BCDM) compared to immunocompetent mice. We wished to compare immune and anti-tumor responses in BCDM to mice functionally depleted of B cells using an anti-human CD20 antibody (Rituximab). We evaluated Rituximab effects on the outgrowth of human CD20 transgenic mouse (huCD20 Tg) B cells following the transplant of huCD20Tg CD45.2+ bone marrow (BM) into sublethally irradiated congenic CD45.1+ mice. CD45.1 mice were irradiated with 9.5Gy at day -1, followed by infusion of 3x10⁶ huCD20Tg BM cells on day 0, and by treatment with or without Rituximab at 400ug/mouse given once weekly i.v. for 4 weeks. Without Rituximab treatment, B cell percentages in the peripheral blood (PB) were approximately 46.4%±1.3; spleen, 77.8%±1.5; and peripheral lymph nodes (pLNs), 43%±1.5 at day 47. With Rituximab treatment, CD19+ cells in the PB, spleen and pLNs were 1.8%±0.4, 4.3%±1.3, and 0.8%±0.2 respectively at 47days post transplant. Higher percentages of CD3+ T cells in PB, spleen and pLNs (66.1%±0.5, 51.7%±1.1 and 43.0%±3.8) were noted in the Rituximab treated group compared to controls (19%±0.9, 10.3%±1.0 and 32.3%±1.7 respectively). The percentage of remaining B cells in PB declined progressively from 5.6% at day 13 to 1.8% at day 47 post transplant indicating that Rituximab depletion of huCD20+ transgenic B cells was progressive, durable and very effective. In contrast, CD3+ cells in PB increased from 11.2% (day 13) to 66.1 % (day 47) following Rituximab treatment. Interestingly reconstitution with endogenous murine B cells in huCD20 transplanted mice was minimal. The huCD20 Tg donor-derived T cells in the Rituximab treated group maintained a CD44^low naive phenotype.

The growth of MC38 tumors implanted 15 days following transplant was significantly slowed in the Rituximab treated group. Increased IFN-γ production and higher expression of the cytolytic marker CD107a by T cells was seen in the Rituximab treated group compared to controls. The combination of myeloablative irradiation, huCD20 transgenic bone marrow transfer and Rituximab depletion, was able to generate mice devoid of CD20+ B cells. Rituximab treated huCD20+ reconstituted B cell depleted mice have substantially higher percentages of T cells than control non-Rituximab treated mice, show sustained depression of B cell numbers and manifest an increased anti-tumor Th1 response. The huCD20 transplant model will be useful in studying effects of post-transplant B cell depletion on immune reconstitution and in the design of future clinical transplant and immunization strategies.