CHARACTERIZATION OF TUMOR RESPONSIVE CD4⁺ AND CD8⁺ T CELLS IN THE PRESENCE OF INTERLEUKIN-12.

Parul Singh, Scott Cutro, Jessica Hu, Nicholas M. Ponzio

    B cell lymphomas (aka RCS) that develop spontaneously in 90% of aging SJL/J mice exhibit Reverse Immune Surveillance, wherein responding host T helper-2 (Th2) cells produce cytokines that promote tumor growth. Addition of IL-12 to co-cultures of spleen + irradiated (g-) RCS cells caused a switch to a Th1 response with the appearance of tumor-specific Cytotoxic T Lymphocytes (CTL). Our objective was to characterize the T cell responses to RCS using dilution of florescent dye PKH26 to measure the proliferation index, number of cell divisions, and precursor frequency of Th and CTL precursor cells. Co-cultures of PKH26-labeled spleen or lymph node cells and g-RCS cells +/- IL12, were examined by FACS; PKH26 levels were analyzed with modfit software; CTL function and mRNA expression for granzyme and perforin were also determined. PKH26 analysis showed that CD4⁺ and CD8⁺ T cells proliferated to tumor, but no cytotoxicity was observed without IL-12. Precursor frequency (PF) values for CD8⁺ splenic T cells from cultures without IL-12 (1.1 x 10^-2) was lower than the PF for cultures with IL-12 (2.7 x10^-2). In lymph nodes, the PF of tumor-specific CD8⁺ T cells was 5-fold higher than in spleen. A majority of the cytotoxicity against tumor targets was deleted after treatment of effector cells with anti-CD8 mAb + C, and this correlated very well with mRNA expression of granzyme and perforin. Enhanced granzyme B expression was seen in cells from cultures containing IL-12, but was reduced in populations depleted of CD8⁺ cells. These results indicate that CD8⁺ T cells respond to RCS antigens to a similar degree as CD4⁺ T cells, but despite their proliferation, CTL precursors do not develop cytotoxic effector function without IL-12. We previously showed in this model that IL-12 caused Th1 differentiation of naïve tumor-responsive CD4⁺ T cells (J Immunol, 174:1405-1415, 2005). Therefore, lack of CTL development against RCS cells in vitro and in vivo is likely due to a defect in production of requisite cytokines rather than failure of naïve precursor CD8⁺ T cells to undergo clonal expansion.