Background: An increased understanding of the requirements for antigen presentation has encouraged development of cell-based cancer vaccines. Trials using dendritic cells (DC) as antigen presenting cells (APC) for immunotherapy of several malignancies have shown considerable success. However, the difficulty in generating large numbers of DC required for these immunizations has led to the search for alternative APC. One such candidate is the CD40 ligand (CD40L)-activated B cell, populations of which can readily be expanded in vitro. To be an effective vehicle for antigen presentation to T cells, CD40L-activated B cells must be capable of migrating to secondary lymphoid organs. Therefore, CD40L-activated B cell migration following subcutaneous or intravenous injection was evaluated.

Methods: Splenic B cells from GFP transgenic mice were activated with CD40L + IL-4 and expanded in vitro prior to i.v. or s.c. injection of 3–4 x 107 into C57BL/6 mice. Recipient mice were sacrificed 2 hrs or 1–14 days thereafter and the percentage of GFP+/B220+ B cells quantified in spleens and lymph nodes by flow cytometry. Localization of these cells within lymphoid organs was determined by immunohistochemistry. In some experiments, activated C57BL/6 B cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) to evaluate cell growth in vivo.

Results: Murine B cell populations were readily expanded by culture on CD40L-transfected L cells in the presence of IL-4. CD40L-activated B cells expressed high levels of CD80, CD86, and LFA-1 but decreased levels of L-selectin relative to naive cells. Following i.v. injection, activated B cells were detected in spleens and lymph nodes within 1 day. Peak concentrations of activated B cells were noted in spleens and lymph nodes on days 7 (4.8% of injected cells) and 10 (1.25% of injected cells) respectively, suggesting expansion of the activated B cell population in vivo. Naive B cells injected i.v. were detected within 1 day but their number declined precipitously thereafter. Following s.c. injection, peak levels of CD40L-activated B cells were noted on day 5 (spleens) and day 7 (lymph nodes). As determined by immunohistochemistry, both CD40L-activated and naïve B cells injected i.v. appeared in B cell regions of spleens and lymph nodes. While the kinetics of accumulation of CD40L-activated B cells injected s.c. or i.v. were similar, s.c. injected CD40L-activated B cells homed to the T cell regions of spleens and lymph nodes. CFSE experiments indicated that these activated B cells continue to grow in vivo. In contrast, naïve B cells injected s.c. only appeared in B cell regions.


  1. CD40L-activated B cell populations can readily be expanded in vitro,

  2. CD40L-activated B cells migrate to secondary lymphoid organs even when injected s.c.,

  3. activated B cell populations expand in vivo, and

  4. s.c. injected, CD40L-activated B cells preferentially home to T cell regions of secondary lymphoid organs.

These results suggest that this effective APC may serve as an important vehicle for delivery and presentation of exogenous (e.g. tumor) antigens to T cells in vivo.

Disclosure: No relevant conflicts of interest to declare.

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