1 A New Strategy of Tumor Vaccination Based on Mammary Adenocarcinoma Cells Transduced with the MHC Class II Transactivator CIITA. L. Mortara 1 , P. Castellani 2 , R. Meazza 3 , G. Tosi 1 , A. De Lerma Barbaro 1 , F. A. Procopio 1 , L. Zardi 4 , S. Ferrini 3 , R. S. Accolla 1 1 Dept. Clinical and Biological Sciences, University of Insubria, Varese, Italy 2 Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 3 Laboratory of Immunopharmacology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 4 Advanced Biotechnology Center, Philogen, Genova, Italy Here we demonstrate that the murine mammary adenocarcinoma cell line TS/A, a highly malignant MHC-II-negative tumor, is rejected in vivo if genetically engineered to express MHC-II mole- cules by transfer of the MHC-II transactivator CIITA. TS/A- CIITA cells are fully rejected by 93% of the syngeneic recipients and have a significantly lower growth rate in the remaining 7% of animals. Rejection requires CD4+ and CD8+ cells, but not B cells or NK cells. in vivo depletion experiments showed that CD4+ T cells are fundamental in the priming phase, whereas CTLs are the major anti-tumor effectors. All tumor rejecting animals are protected against rechallenge with the parental TS/A tumor. CTLs specific for a peptide of the envelope gp70 of an endogenous ecotropic retrovirus were identified and explained the specificity of the newly acquired effector mechanism of rejection against the TS/ A. Immunohistochemical analysis showed a dramatic subversion of the tumor microenvironment in TS/A-CIITA-injected as com- pared to parental TS/A-injected mice. At day 5 post-inoculation a higher infiltrate of CD4+ T cells in mice bearing TS/A-CIITA was observed. Subsequently, from day 7 through day 10, TS/A-CIITA tumors showed higher number of both CD4+ and CD8+ cells, dendritic cells, and neutrophils together with massive necrosis. The frequency of IFN-g-secreting splenocytes early after inoculations was also assessed by an ex vivo ELISPOT assay. Only rejecting TS/A-CIITA animals showed a high frequency of IFN-g-secreting cells. Importantly, CD4 and CD8 depletion experiments revealed that at the time of tumor resolution the major cell population recognizing the TS/A-CIITA cells was of CD4 origin. This is the first example of successful tumor vaccination by genetic transfer of CIITA. These results open the way to a possible use of CIITA for increasing both the inducing and the effector phase of the anti-tumor response. 2 The Molecular Basis of the Inhibition of HTLV-2 Retroviral Replication by the MHC Class II Transactivator (CIITA) G. Tosi 1 , E. Pilotti 2 , A. De Lerma Barbaro 1 , L. Mortara 1 , C. Casoli 2 , R. S. Accolla 1 1 Dept. Clinical and Biological Sciences, University of Insubria, Varese, Italy 2 Dept. of Clinical Medicine, Nephrology, and Prevention, University of Parma, Parma, Italy The transcriptional activator CIITA is the master regulator of the expression of MHC class II genes. In addition to this major role, we found that CIITA exerts an important inhibitory effect on the HTLV-2 replication. This inhibition is mediated by the N-terminal 1–321 region where we identified a minimal fragment of 80 aminoa- cids that specifically blocks the activity of the viral transactivator Tax2. To unveil the biochemical basis of the CIITA-mediated inhi- bition of Tax2 we first focussed on the identification of the cellular cofactors used by Tax2 to transactivate the viral promoter. Preliminary data indicate that the transactivation activity of Tax2 and Tax1, the HTLV-1 homologous of Tax2, is differently influ- enced by the hystone acetyltransferases CBP, p300 and PCAF providing new informations on the biology of HTLV-2. Furthermore, none of these factors was able to reverse the inhibitory action of CIITA on Tax2 function. Interestingly, we found that the B and, to a lesser extent, the A subunits of the NFY complex inhibit Tax2 activity when iper-expressed in cells. On the basis of our results and of the reported physical interactions between NFY and both CIITA and Tax1, a possible mechanism for the CIITA-mediated inhibition of Tax2 activity would be the binding of the CIITA-NFY complex to Tax2. When expressed in cells, CIITA interacts with the NFY complex; this interaction changes the conformation of NFY increasing its binding affinity for Tax2. Following this model the inhibition of Tax2 by CIITA it is not due to the squelching of a transcriptional positive co-activator, but instead to the recruitment of a cellular factor, NFY, with a negative regulatory action on Tax2. On the whole these results confirm that CIITA may represent a physiologic tool for novel therapeutic strategies aimed at counter- acting HLTV-2 replication and spreading. 3 Phorbol Esther-induced Differentiation in Human Macrophages Reveals the Existence of Factors Destabilizing MHC Class II Transactivator (CIITA) mRNA. A. De Lerma Barbaro, F. A. Procopio, L. Mortara, G. Tosi, R. S. Accolla Dept. Clinical and Biological Sciences, University of Insubria, Varese, Italy The human promyelocytic U937 and THP1 cell lines express detectable levels of MHC class II (MHC-II) molecules. Treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA), inducing macrophage-like differentiation, produces a dramatic decrease of MHC-II expression as result of down-modulation of the AIR-1-encoded MHC-II transcriptional activator CIITA. This event is specific, as MHC-I remains unaffected. Similar results are observed with U937 cells expressing an exogenous full length CIITA. Molecular studies demonstrate that TPA Tissue Antigens ISSN 0001-2815 Tissue Antigens 66 (2005) 343–604 ß 2005 Blackwell Munksgaard 343