Microenvironmental factors contribute to the immune dysfunction characterizing acute myeloid leukemia (AML). Indoleamine 2,3-dioxygenase 1 (IDO1) is an interferon (IFN)-γ-inducible enzyme that degrades tryptophan into kynurenine, which, in turn, inhibits effector T cells and promotes regulatory T-cell (Treg) differentiation. It is presently unknown whether childhood AML cells express IDO1 and whether IDO1 activity correlates with patient outcome.

Purpose: The α₆β₄ integrin, a laminin receptor, has been implicated from many studies in tumor progression and invasion. We showed that the β₄ integrin subunit associates with the ErbB-2 tyrosine kinase in human mammary carcinoma cell lines and that its overexpression in NIH3T3/ErbB-2–transformed cells causes a constitutive activation of phosphatidylinositol 3-kinase (PI3K), inducing a strong increase of their invasive capacity. In this study, we investigated the biological consequences of interference with the endogenous β₄ integrin subunit expression. Experimental Design:In vitro and in vivo tumor growth and the biochemical consequences of β₄ integrin inactivation were studied in mammary tumor cells by using short hairpin RNA approach. Results: Our data show that tumor growth of mammary tumor cells strictly depends on β₄ expression, confirming the relevance of β₄ protein in these cells. Moreover, interference with β₄ expression significantly reduces endogenous PI3K activity and AKT and mammalian target of rapamycin phosphorylation. Accordingly, with these results and considering that PI3K activity in mammary tumor plays a relevant role in hormone resistance, we asked whether β₄ expression might be relevant for hormone responsiveness in these cells. Data reported indicate that the interference with endogenous β₄ expression, upon hormone deprivation, induces caspase-9 and cytochrome c–mediated apoptosis, which is enhanced upon tamoxifen treatment. On the other hand, the expression of myr-AKT in MCF7 β₄–short hairpin RNA cells rescues the cells from apoptosis in the absence of hormones and upon tamoxifen treatment. Conclusions: Overall, these results confirm the relevance of β₄ expression in mammary tumors and indicate this integrin as a relevant target for tumor therapy.

Background: Multiple myeloma (MM) is a plasma cell malignancy with a multifaceted immune dysfunction. Indoleamine 2,3-dioxygenase 1 (IDO1) degrades tryptophan into kynurenine (KYN), which inhibits effector T cells and promote regulatory T-cell (Treg) differentiation. It is presently unknown whether MM cells express IDO1 and whether IDO1 activity correlates with immune system impairment. Methods: We investigated IDO1 expression in 25 consecutive patients with symptomatic MM and in 7 patients with either monoclonal gammopathy of unknown significance (MGUS; n=3) or smoldering MM (SMM; n=4). IDO1-driven tryptophan breakdown was correlated with the release of hepatocyte growth factor (HGF) and with the frequency of Treg cells and NY-ESO-1-specific CD8⁺ T cells. Results: KYN was increased in 75% of patients with symptomatic MM and correlated with the expansion of CD4⁺CD25⁺FoxP3⁺ Treg cells and the contraction of NY-ESO-1-specific CD8⁺ T cells. In vitro, primary MM cells promoted the differentiation of allogeneic CD4⁺ T cells into bona fide CD4⁺CD25^hiFoxP3^hi Treg cells and suppressed IFN-γ/IL-2 secretion, while preserving IL-4 and IL-10 production. Both Treg expansion and inhibition of Th1 differentiation by MM cells were reverted, at least in part, by d,l-1-methyl-tryptophan, a chemical inhibitor of IDO. Notably, HGF levels were higher within the BM microenvironment of patients with IDO⁺ myeloma disease compared with patients having IDO^- MM. Mechanistically, the antagonism of MET receptor for HGF with SU11274, a MET inhibitor, prevented HGF-induced AKT phosphorylation in MM cells and translated into reduced IDO protein levels and functional activity. Conclusions: These data suggest that IDO1 expression may contribute to immune suppression in patients with MM and possibly other HGF-producing cancers.

NK cells expressing TIM-3 show a marked increase in IFNγ production in response to acute myeloid leukemia (AML) blast cells that endogenously express Gal-9. Herein, we demonstrate that NK cell-mediated production of IFNγ, induced by TIM-3/Gal-9 interaction and released in bone marrow microenvironment, is responsible for IDO1 expression in AML blasts. IDO1-expressing AML blasts consequently down-regulate NK cell degranulation activity, by sustaining leukemia immune escape. Furthermore, the blocking of TIM-3/Gal-9 interaction strongly down-regulates IFNγ-dependent IDO1 activity. Thus, the inhibition of TIM-3/Gal-9 immune check point, which affects NK cell-dependent IFNγ production and the consequent IDO1 activation, could usefully integrate current chemotherapeutic approaches.

Techniques for medical tissue regeneration require an abundant source of human adult stem cells. There is increasing evidence that adipose stem cells contribute to restoration of tissue vascularization and organ function. The object of our study was to isolate and characterize adult adipose-derived stem cells from patients undergoing on lipoaspirate transplant with the aim to improve tissue regeneration. Adipose-derived stem cells were isolated and purified from the lipoaspirate of 15 patients and characterized for CD markers and the ability to differentiate toward the adipogenic lineage. We found that purified adipose stem cells express high level of CD49d, CD44, CD90, CD105, CD13, and CD71 and these markers of staminality were maintained at high level for at least 3 months and seven passages of in vitro culture. As expected, these cells resulted negative for the endothelial and hematopoietic-specific markers CD31, CD106, CD34, and CD45. Differentiation towards adipogenic lineage demonstrated that purified adipose-derived stem cells are still able to become adipocytes at least 3 months after in vitro culture. The analysis of Akt and MAPK phosphorylation confirmed a modulation of their activity during differentiation. Interestingly, we established for the first time that, among the p53 family members, a strong upregulation of p63 expression occurs in adipocytic differentiation, indicating a role for this transcription factor in adipocytic differentiation. Taken together, these data indicate that purified lipoaspirate-derived stem cells maintain their characteristic of staminality for a long period of in vitro culture, suggesting that they could be applied for cell-based therapy to improve autologous lipoaspirate transplant.