Ligation of CD24 on T cells and B cells modulates the cytokine production profile of activated tumor-draining lymph node cells

4627 CD24 is a heat-stable antigen (HSA) expressed on the surface of a variety of cell types. AlthoughCD24 expression onT cells is required for optimal homeostatic T cell proliferation, the role of CD24 on tumor-primed T cells and B cellsis largely unknown. In this study, we evaluated the regulation of CD24 expression on activated tumor-draining lymph node (TDLN) cells and examined the cytokine production by these activated TDLN cells after CD24 ligation using an anti-CD24 mAb. Freshly harvested TDLNs from MCA205 tumor-bearing mice are comprised of 50-60% T cells and 20-30% B cells. By FACS analysis, approximately 5% of the TDLN cells are T cells expressing CD24 (CD3+CD24+), whereas almost all of the B cells express CD24 (CD19+CD24+). TDLN cells activated in vitro with anti-CD24 alone did not result in proliferation. Activation of TDLN cells with anti-CD3 or anti-CD3/anti-CD28 increased the percentage of CD3+CD24+ cells to approximately 15% and 25% respectively, while all the B cells remained CD24 positive. Culture supernatants of antibody-activated TDLN cells, with or without CD24 engagement, were collected for cytokine production assay. While CD3/CD28 co-stimulation significantly increased IFNγ and GM-CSF production compared with CD3 activation alone, CD3/CD24 co-stimulation failed to do so. However, CD24 ligation in addition to CD3/CD28 co-stimulation resulted in significantly enhanced IFNγ and GM-CSF production compared with CD3/CD28 activation. In these experiments, the IL-10 levels were not drastically changed. Furthermore, CD24 engagement with simultaneous T cell activation with anti-CD3/anti-CD28 and B cell activation with LPS plus anti-CD40 further significantly enhanced IFNγ and GM-CSF production. However, under these culture conditions, IL-10 secretion was also markedly up-regulated. These results indicated that CD24 expression on TDLN T cells can be up-regulated by CD3 or CD3/CD28 activation. While CD24 was unable to provide a co-stimulation signal to T cells activated via the primary TCR pathway, ligation of CD24 was found to act as a third signal to boost CD3/CD28-activated T cells. In addition, simultaneous ligation of CD24 on anti-CD3/anti-CD28-activated T cells and LPS/anti-CD40-activated B cells could further modulate the cytokine profile of these TDLN cells. Our results suggest that the in vitro ligation of CD24 on tumor-primed T and B cells may have utility in generating tumor reactive effector cells for cancer immunotherapy.