It is our goal to delineate therapies that do not suppress immune systems and could be used in with vaccines or other immune therapies to boost responses to tumors.Pancreas cancer patients have significant levels of tumor specific immune activity despite the aggressive nature of their disease.Clinical trials of vaccines in pancreas cancer patients have confirmed activation of immune responses;however significant effects on tumor growth and overall survival have not yet been achieved.The failure of vaccines to control tumor growth may be due to a number of factors including a hostile immunosuppressive tumor microenvironment.We studied immune systems of pancreas cancer patients to determine the effects of B7-H3 expression in pancreatic tumors and whether pemetrexed preceding gemcitabine could be combined with immunotherapy or if it was immunosuppressive.Baseline levels of innate and adaptive immune cell functions demonstrated a strong positive correlation between pre-therapy absolute numbers of NK cells and overall survival (p=0.039).Elevated levels of memory (CD45RO+) T-cells however correlated negatively with survival (p=0.011) but were decreased by pemetrexed therapy.Greater B7-H3 expression in tumor tissue correlated with increased memory T-cells (p =0.019), and decreased OX40+ activated total T-cells and helper T-cell subset in peripheral blood (p =0.02 and p =0.039, respectively).B7-H3 expression in tumors thus is significantly detrimental and patients whose tumor tissue have high levels of B7-H3 expression may not benefit from immunotherapy.Pemetrexed caused an increase in NK cells producing IFNγ detected 7 days after therapy which correlated negatively with survival (p=0.038).Addition of gemcitabine to pemetrexed decreased NK cells producing IFNγ to baseline levels, resulting in increased levels of NK cells producing IL-2 which in turn led to a positive correlation with survival (p=0.041).A potentially deleterious effect of pemetrexed thus was alleviated by gemcitabine.Both innate NK-cell immunity and FoxP3+,CD8+ T-cells were found to be important in defense against pancreatic cancer.Despite the negative effect pemetrexed had on inducing increased activation of a subset of NK cells to produce IFNγ, its reversal following addition of gemcitabine led to progression-free intervals and survival that were significantly longer when compared to results of a clinical phase Ⅲ in which gemcitabine preceded pemetrexed.These data suggest that a larger clinical trial of pemetrexed preceding gemcitabine is merited.Furthermore, information gained from this study can be used to generate future immunotherapeutic clinical trials in pancreatic cancer.