In pancreatic cancer (PC) patients the immune response is not robust enough to counteract cell growth and metastases. Tumor cells may neutralize the immune system by evading detection and/or by inhibiting immune cells function. To test this latter hypothesis we verified in vitro whether PC cells conditioned media modify CD4+ T cell proliferation, migration and activation. CD4+ T lymphocytes were purified by negative selection using a RosetteSep kit (StemCell Technologies). CD4+ cells were cultured for 4 days in control medium or CAPAN1 (a PC cell line) fresh conditioned medium. Migration was assessed using a transwell system in the presence or absence of the chemoattractant hSDFα; the number of migrating cells was FACS counted. Proliferation was FACS analysed in control and conditioned CD4+ cells, labelled with carboxyfluorescein succinimidyl ester, after 72 hrs of co-culture with allogenic PBMC. Control and conditioned CD4+ cells were co-cultured for 24 hrs with unpulsed or staphylococcal enterotoxins (0.25, 0.5 and 1.0 μg/mL) pulsed EBV-B cells. In the supernatants interferon-γ (IFNγ) and IL4 were assayed. The number of control and conditioned migrating CD4+ T cells did not differ in the absence of hSDFα (t=1.0, p:ns). In the presence of hSDFα, migrating CAPAN1 conditioned lymphocytes were less (3337±390, mean±SEM) than control lymphocytes (6413±660) (t=7.55, p<0.001). CAPAN1 conditioned medium stimulated CD4+ T cell proliferation both in the presence (t=2.27, p<0.05) or absence (t=3.87, p<0.001) of allogenic PBMC. IL4 concentration did not significantly vary between conditioned or non conditioned APC stimulated CD4+ cells. Stimulation of CD4+ T cells by pulsed or unpulsed EBV-B induced higher IFNγ concentrations (pg/mL) in conditioned than in control cells (table 1). Control and conditioned media were ultrafiltered (mw cut-offs=30,000 and 10,000 Da). The effects of CAPAN1 conditioned medium on CD4+ T cell migration and IFNγ production were recovered in the mw fraction of >30.000 Da. In
Conclusion: we demonstrated that PC cells release soluble mediator/s of more than 30.000 Da which inhibit CD4+ T cell migration, activate proliferation and Th1 differentiation, supporting the hypothesis that tumor cells alter immune cells function.
Table 1

	CD4+T + unpulsed EBV-B Mean±SEM	CD4+T + 0.25ug/mL pulsed EBV-B Mean±SEM	CD4+T + 0.5ug/mL pulsed EBV-B Mean±SEM	CD4+T + 1.0ug/mL pulsed EBV-B Mean±SEM
Control	10.5±1.7	178.1±39.5	261.4±56.7	499.1±111.6
Conditioned	69.7±24.7	293.4±66.1	478.5±94.7	998.0±236.6
Student’st test for paired data	t=2.56, p<0.05	t=2.92, p<0.05	t=3.30, p<0.05	t=2.82, p<0.05