Immune Suppression By High-Density Neutrophils Predicts Short Progression-Free Survival in Multiple Myeloma
Authors: Romano Alessandra , Manteiga Jose , Simeon Vittorio , Parrinello Nunziatina L , Giallongo Cesarina , La Cava Piera , Conticello Concetta , Tibullo Daniele , Fontana Francesca , Ciceri Fabio , Cremasco Floriana , Fabbri Monica , Musto Pellegrino , Cenci Simone , Di Raimondo Francesco 
Published: October 2019

Abstract
Background and Objective :
Our previous work showed that neutrophils sedimenting on top of red cells after density gradients, defined as high-density neutrophils (HDN) are emerging as pro-tumoral players with immunesuppressive properties, by depleting the surrounding microenvironment from arginine (arg) and tryptophan (trp) through increased expression of arginase (Arg-1) and 2-3 indoleamine deoxygenase (IDO), but little is known about their clinical relevance in Multiple Myeloma (MM).
Experimental design : We integrated two distinct unbiased approaches. First, we assessed gene expression profiles (GEP) and functional evaluation of HDN sorted from 60 newly diagnosed MM patients, 30 patients with MGUS, and 30 healthy subjects.. Second, we adopted ultra-high performance liquid and gas chromatography followed by mass spectrometry (UHPLC/GC-MS) on an independent series of 167 samples of bone marrow (BM) and peripheral plasma collected ad hocfrom 125 individuals, comprising newly diagnosed MM, MGUS, smoldering MM, and age-matched healthy volunteers (n=29). Progression-free survival in newly diagnosed MM patients was correlated respectively to HDN functional and expression features or metabolic profiles in the two series.
Results :
GEP analyses disclosed that, compared to MGUS and HD, MM-HDN showed increased expression of Arg-1, IDO-1 and the angiogenic factor PROK-2/BV8 , confirmed by qRT-PCR and immunofluorescence. Indeed, MM-HDN (but not MGUS- and HD-HDN) revealed immunosuppressive activity in co-cultures with allogeneic T-cells, reverted by the selective Arg-1 inhibitor nor-NOHA.
In addition, circulating Arg-1 in serum was higher in MM (169.5 ± 17.6) than MGUS (93.2 ± 10.4, p=0.0028) and healthy subjects (70.1 ± 16.5 ng/ml, p=0.0017). In MM patients there was a progressive increase from ISS stage I through III (p=0.014). High Arg-1 in MM patients' sera at diagnosis predicted clinical outcome after first line therapy, being associated to shorter progression-free survival (respectively 22.4 months versus unreached median, p=0.01).
Metabolic profiling independently identified the same immunosuppressive Arg-1-urea-arginine (arg) and tryptophan (trp)-kinurenine-IDO pathways, with arg and trp significantly reduced along MM evolution. Lower levels of arg were associated to shorter PFS (37.2 versus 17.6 months, p=0.023).
We then tested if aminoacid depletion (arg and trp) favors PCs growth. When exposed to trp-free or arg-free medium, human MM cell lines expressed more CHOP, p62, m-TOR and Blimp-1 (markers respectively of autophagy induction and immunoglobulin production); conversely, p62 silencing reduced Blimp-1, confirming the important role of p62-dependent mechanism in supporting PCs fitness.
Conclusion : In MM, HDN are immunosuppressive due to increased Arg-1 and IDO expression. We propose the arginine/Arg-1 axis as a novel potential biomarker for MM prognosis.

Disclosures
Ciceri: MolMed SpA: Consultancy.