B cell-mediated tumor antigen presentation is required for therapeutic efficacy of a combined immune-stimulatory/conditional cytotoxic gene therapeutic approach for glioblastoma

MARIANELA CANDOLFI; JAMES F CURTIN; KADER YAGIZ; HIKMAT ASSI; KURT M KROEGER; MATTHEW TESARFREUND; ANDREW ALDEN; CHUNYAN LIU; ROBERT DUNN; PEDRO R LOWENSTEIN; MARIA G CASTRO

San Diego, CA, EEUU

Congreso; American Society of Gene Therapy 12 Annual Meeting; 2009

Although B cells are well known by producing Ag specific IgGs, they can also act as efficient antigen presenting cells (APCs). Clonal expression of BcR and high specific avidity for Ag allow B cells to concentrate low density extracellular Ag for presentation to T cells, lowering the threshold of Ag density required for triggering adaptive immune responses. The role of B cells as APCs mediating brain tumor regression in response to immunotherapy is not clearly understood. We developed adenoviral vectors (Ads) expressing HSV1-TK (Ad-TK; a conditional cytotoxic molecule) and Flt3L (Ad-Flt3L; an immune-stimulatory cytokine), which induce CD4+ and CD8+ T cell-mediated tumor regression, long term survival and immunological memory. Here, we investigated whether B cells play a role in Ad-TK+Ad-Flt3Lmediated GBM regression. While Ad-TK+Ad-Flt3L induced tumor regression and long term survival in 60% of the wild type (WT) mice, the treatment completely failed in B-cell deficient Igh6-/- mice. Similarly, in tumor-bearing WT mice depleted of total B cells (using anti-mouse CD20 antibodies), or marginal zone B cells (MZB, using antibodies against CD11a and CD49d), treatment with Ad-TK+Ad-Flt3L failed. Examination of the spleen indicated that CD19+ B cells do not expand, while the number of MZB cells increases at day 7 post-treatment. The number of B cells within the tumor mass as well as in the draining lymph nodes did not change after the treatment; we could not detect circulating antibodies against tumor cells in the treated mice. Since B cells can concentrate antigen via specific B cell receptor interactions and upregulate the expression of co-stimulatory molecules, we studied whether B cells could present tumor antigen during the onset of the anti-tumoral immune response induced by Ad-TK+Ad-Flt3L. We found that B cells purified from the draining lymph nodes of Ad-TK+Ad-Flt3L-treated, but not from control tumor bearing C57/B6 mice significantly increased the proliferation of T cells purified from spleen of na ve Balb/c mice. Also, tumor-specific T cell clonal expansion was studied in an ELISPOT using splenocytes from tumor-bearing WT or Igh6-/- mice treated with saline or Ad- TK+Ad-Flt3L, and stimulated with Trp2180-188, an MHCI associated GL26 tumor antigen. Trp2- specific T cell precursors were detected only in Ad-TK+Ad-Flt3L-treated WT mice, but we did not observe any increase in the frequency of tumor antigen specific T cells in Igh6-/- mice. Together, our results indicate that B cells play an important role in facilitating clonal expansion of tumor antigen specific T cells and subsequent elimination of brain tumor antigen, most likely by acting as APCs, rather than antibody-producing cells.