An increased expression of focal adhesion kinase (FAK) in a variety of cancers is associated with a poor disease prognosis. To study the role of FAK in breast tumor growth and metastasis formation, we used conditional doxycycline-regulated expression of a dominant-negative acting splice variant of FAK, FAK-related non-kinase (FRNK), in MTLn3 mammary adenocarcinoma cells in a syngeneic Fischer 344 rat tumor and metastasis model. In cell culture, doxycycline-mediated expression of FRNK inhibited MTLn3 cell spreading and migration in association with reduced formation of focal adhesions and phosphorylation of FAK on Tyr 397 , but FRNK did not cause apoptosis. Continuous expression of FRNK decreased the primary tumor growth in the mammary fat pad by 60%, which was not due to induction of apoptosis. Lung metastasis formation was almost completely prevented when FRNK was already expressed 1 day before tumor cell injection, whereas expression of FRNK 11 days after injection did not affect lung metastasis formation. FRNK expression during the first 5 days was sufficient to block metastasis formation, excluding the possibility of FRNK-induced dormancy of tumor cells. Together, these data fit with a model wherein FAK is required for breast tumor cell invasion/migration processes that take place in the early phase of metastasis formation. Our findings suggest that FAK is a good candidate for therapeutic intervention of metastasis formation. (Cancer Res 2005; 65(11): 4698-706)

Epstein-Barr virus (EBV) is associated with several human malignancies that each show different viral gene expressionprofiles. In malignancies such as Hodgkin's disease and nasopharyngeal carcinoma only Epstein-Barr nuclear antigen 1 (EBNA1) and varying levels of latent membrane proteins 1 and 2 (LMP1 and -2) are expressed. Since endogenously expressed EBNA1 is protected from CTL recognition, LMP1 and LMP2 are the most likely target antigens for anti-tumor immunotherapy. Therefore, we sought to identify in a systematic way CD8 ؉ T-cell responses directed against eptitopes derived from LMP1 and LMP2. Using IFN␥-ELISPOT assays of interferon-␥ release, peripheral blood mononuclear cells (PBMC) of healthy donors were screened with peptide panels (15 mer overlapping by 10) spanning the LMP1 and LMP2 sequences of the prototype EBV strain B95.8. When positive responses were found, CD4 ؉ or CD8 ؉ T cells were depleted from donor PBMC to determine the origin of the responder population. We detected CD8 ؉ T-cell responses to LMP1 in 9/50(18%) donors and to LMP2 in 15/28 (54%) donors. In addition to the already described epitopes, 3 new LMP1-and 5 new LMP2-derived CD8 ؉ epitopes were identified. In most donors LMP1-and LMP2-specific CD8 ؉ precursor frequencies were low compared with precursors against immunodominant EBV epitopes from latent (EBNA3A, -3B and -3C) and lytic cycle antigens. These results demonstrate that CD8 ؉ memory T cell responses to LMP1 and especially to LMP2 do exist in Caucasians, albeit at low levels and could potentially be exploited for therapeutic use. © 2002 Wiley-Liss, Inc. Key words: CTL; LMP1; LMP2; Epstein-Barr virusEpstein-Barr virus (EBV) can induce fatal B lymphoproliferative lesions in immunocompromised patients and is etiologically linked to several malignancies arising in the immunocompetent host. Examples of the latter include all cases of endemic Burkitt's lymphoma (BL), all undifferentiated nasopharyngeal carcinomas (NPC), 10% of gastric carcinomas, certain T-/NK-cell lymphomas and 40 -90% of Hodgkin's Disease (HD) cases. 1 EBV is transcriptionally active in the tumor cells but expresses only a restricted set of EBV-encoded proteins; expression patterns range from the Epstein-Barr nuclear antigen 1 (EBNA1) only in BL through to EBNA1 plus high levels of both latent membrane proteins 1 and 2 (LMP1, -2) in HD. 2-4 EBV-associated carcinomas are intermediate in that EBNA1 and LMP2 appear to be expressed, with or without LMP1. 5 Interestingly many EBV-positive carcinomas also show detectable transcription of a gene, BARF1, which is otherwise thought to be associated only with the virus lytic cycle. 6,7 Given the presence of at least some viral antigens in tumor cells, EBV-positive malignancies are potential candidates for immunotherapy. Because endogenously expressed EBNA1 is protected from proteasomal processing and therefore escapes cytotoxic T lymphocyte (CTL) recognition, 8 the prime targets for therapeutic responses in the context of HD and NPC are LMP1 and LMP2. Both are multiple mem...

IL22 is an important cytokine involved in the intestinal defense mechanisms against microbiome. By using ileum-derived organoids, we show that the expression of anti-microbial peptides (AMPs) and anti-viral peptides (AVPs) can be induced by IL22. In addition, we identified a bacterial and a viral route, both leading to IL22 production by T cells, but via different pathways. Bacterial products, such as LPS, induce enterocyte-secreted SAA1, which triggers the secretion of IL6 in fibroblasts, and subsequently IL22 in T cells. This IL22 induction can then be enhanced by macrophage-derived TNFα in two ways: by enhancing the responsiveness of T cells to IL6 and by increasing the expression of IL6 by fibroblasts. Viral infections of intestinal cells induce IFNβ1 and subsequently IL7. IFNβ1 can induce the expression of IL6 in fibroblasts and the combined activity of IL6 and IL7 can then induce IL22 expression in T cells. We also show that IL22 reduces the expression of viral entry receptors (e.g. ACE2, TMPRSS2, DPP4, CD46 and TNFRSF14), increases the expression of anti-viral proteins (e.g. RSAD2, AOS, ISG20 and Mx1) and, consequently, reduces the viral infection of neighboring cells. Overall, our data indicates that IL22 contributes to the innate responses against both bacteria and viruses.

To study the tumor cell autonomous processes of metastasis, an in vivo tumor metastasis model is required that excludes the involvement of the innate immune system. For this purpose we used the established syngeneic