IN MULTIPLE MYELOMA THERE ARE TWO SUBSETS OF IMMUNOSUPPRESSIVE POLYMORPHONUCLEAR NEUTROPHILS WITH INCREASED LEVELS OF ARGINASE
(Abstract release date: 05/19/16)
EHA Library. ROMANO A. 06/09/16; 132782; E1233
Dr. ALESSANDRA ROMANO
Contributions
Contributions
Abstract
Abstract: E1233
Type: Eposter Presentation
Background
Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. Development of MM is associated with accumulation of Myeloid-Derived suppressor cells (MDSC), mostly represented by pathologically activated relatively immature polymorphonuclear neutrophils (G-MDSC). Our previous work showed that also mature polymorphonuclear neutrophils (PMN) are activated and immunosuppressive in MM.
Aims
We hypothesized that two populations of polymorphonuclear neutrophils are present in MM, with differential distribution after a Ficoll separation and differential immunosuppressive activity
Methods
Using oligonucleotide microarrays we first evaluated the gene expression profile (GEP) of PMN at the steady state in 10 MM, 5 MGUS and 8 healthy subjects matched for sex and age, identifying Arg-1 as the first gene differentially expressed in MM versus healthy PMN.Thus, we validated Arg-1 by RT-PCR, immunohistochemistry and circulating levels in serum by ELISA in a series of 60 newly-diagnosed MM patients, 30 MGUS and 30 healthy subjects in both G-MDSC and PMN.Finally, we tested the immunosuppressive properties of G-MDSC (isolated by CD66b+ immunomagnetic beads in the upper Ficoll layer) and PMN (sorted from the lower Ficoll layer) isolated from the same MGUS or MM patients, through functional assays, based on in vitro co-culture with T-lymphocytes from healthy subjects.
Results
MM-PMN exhibit an increased expression of ARG-1 compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity of ARG-1, positively correlated with advanced disease. In MM patients, increased levels of ARG-1 were positively associated with advanced bone disease and unfavourable cytogenetics.Circulating Arg-1 in serum was higher in MM than MGUS and healthy subjects (183.6 ± 21.9 versus 88.3 ± 15.6 versus 98.8 ± 11.4 ng/ml, p=0.0022). In MM patients there was a progressive increase from ISS stage I through III (p=0.003).Immunostaining showed that Arg-1 was increased in MM versus healthy subjects, but higher levels were evident in G-MDSC than PMN. Moreover, only in MM-G-MDSC Arg-1 was evident at the nuclear level, while in PMN Arg-1 had an exclusive cytoplasmic localization.This differential distribution of Arg-1 was functionally evident, since G-MDSC were more immunosuppressive than PMN.Indeed, after 72 hours of co-culture with T-cells obtained from healthy donors in presence of mitogen stimulation (PHA), MM-PMN were able to reduce T-cell proliferation at both tested 1:2 and 1:8 ratios (respectively 38.3 ± 2.6 % and 14.3 ± 0.6 %, p<0.0001), while MGUS-PMN induced a partial inhibition only at the 1:8 (25.4 ± 4.3%, p=0.002). This effect was partially reverted with the treatment of 200 μM nor-NOHA, an Arg-1 inhibitor, since within first 24 hours T-cell proliferation increased in presence of MM-PMN (14.3 ± 0.6 versus 24.5 ± 1.3%, p<0.0001) and MGUS-PMN (25.4 ± 4.3 versus 31.6 ± 2.3 %, p<0.0001).MM-G-MDSC were more immunosuppressive than their MM-PMN counterpart, since at 72 hours T-cell proliferation was 22.3 ± 1.6 % and 8.4 ± 0.5 % (p<0.0001) at 1:2 and 1.8 ratio respectively.
Conclusion
Polymorphonuclear neutrophils in MM are immunosuppressive, but distinguished in two main subpopulations, at different stages of maturation, based on the expression of Arg-1 and grains distribution, to target in order to improve the effects of the immunochemotherapy.
Session topic: E-poster
Keyword(s): Arginase, Myeloma, Myelosuppression
Type: Eposter Presentation
Background
Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. Development of MM is associated with accumulation of Myeloid-Derived suppressor cells (MDSC), mostly represented by pathologically activated relatively immature polymorphonuclear neutrophils (G-MDSC). Our previous work showed that also mature polymorphonuclear neutrophils (PMN) are activated and immunosuppressive in MM.
Aims
We hypothesized that two populations of polymorphonuclear neutrophils are present in MM, with differential distribution after a Ficoll separation and differential immunosuppressive activity
Methods
Using oligonucleotide microarrays we first evaluated the gene expression profile (GEP) of PMN at the steady state in 10 MM, 5 MGUS and 8 healthy subjects matched for sex and age, identifying Arg-1 as the first gene differentially expressed in MM versus healthy PMN.Thus, we validated Arg-1 by RT-PCR, immunohistochemistry and circulating levels in serum by ELISA in a series of 60 newly-diagnosed MM patients, 30 MGUS and 30 healthy subjects in both G-MDSC and PMN.Finally, we tested the immunosuppressive properties of G-MDSC (isolated by CD66b+ immunomagnetic beads in the upper Ficoll layer) and PMN (sorted from the lower Ficoll layer) isolated from the same MGUS or MM patients, through functional assays, based on in vitro co-culture with T-lymphocytes from healthy subjects.
Results
MM-PMN exhibit an increased expression of ARG-1 compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity of ARG-1, positively correlated with advanced disease. In MM patients, increased levels of ARG-1 were positively associated with advanced bone disease and unfavourable cytogenetics.Circulating Arg-1 in serum was higher in MM than MGUS and healthy subjects (183.6 ± 21.9 versus 88.3 ± 15.6 versus 98.8 ± 11.4 ng/ml, p=0.0022). In MM patients there was a progressive increase from ISS stage I through III (p=0.003).Immunostaining showed that Arg-1 was increased in MM versus healthy subjects, but higher levels were evident in G-MDSC than PMN. Moreover, only in MM-G-MDSC Arg-1 was evident at the nuclear level, while in PMN Arg-1 had an exclusive cytoplasmic localization.This differential distribution of Arg-1 was functionally evident, since G-MDSC were more immunosuppressive than PMN.Indeed, after 72 hours of co-culture with T-cells obtained from healthy donors in presence of mitogen stimulation (PHA), MM-PMN were able to reduce T-cell proliferation at both tested 1:2 and 1:8 ratios (respectively 38.3 ± 2.6 % and 14.3 ± 0.6 %, p<0.0001), while MGUS-PMN induced a partial inhibition only at the 1:8 (25.4 ± 4.3%, p=0.002). This effect was partially reverted with the treatment of 200 μM nor-NOHA, an Arg-1 inhibitor, since within first 24 hours T-cell proliferation increased in presence of MM-PMN (14.3 ± 0.6 versus 24.5 ± 1.3%, p<0.0001) and MGUS-PMN (25.4 ± 4.3 versus 31.6 ± 2.3 %, p<0.0001).MM-G-MDSC were more immunosuppressive than their MM-PMN counterpart, since at 72 hours T-cell proliferation was 22.3 ± 1.6 % and 8.4 ± 0.5 % (p<0.0001) at 1:2 and 1.8 ratio respectively.
Conclusion
Polymorphonuclear neutrophils in MM are immunosuppressive, but distinguished in two main subpopulations, at different stages of maturation, based on the expression of Arg-1 and grains distribution, to target in order to improve the effects of the immunochemotherapy.
Session topic: E-poster
Keyword(s): Arginase, Myeloma, Myelosuppression
Abstract: E1233
Type: Eposter Presentation
Background
Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. Development of MM is associated with accumulation of Myeloid-Derived suppressor cells (MDSC), mostly represented by pathologically activated relatively immature polymorphonuclear neutrophils (G-MDSC). Our previous work showed that also mature polymorphonuclear neutrophils (PMN) are activated and immunosuppressive in MM.
Aims
We hypothesized that two populations of polymorphonuclear neutrophils are present in MM, with differential distribution after a Ficoll separation and differential immunosuppressive activity
Methods
Using oligonucleotide microarrays we first evaluated the gene expression profile (GEP) of PMN at the steady state in 10 MM, 5 MGUS and 8 healthy subjects matched for sex and age, identifying Arg-1 as the first gene differentially expressed in MM versus healthy PMN.Thus, we validated Arg-1 by RT-PCR, immunohistochemistry and circulating levels in serum by ELISA in a series of 60 newly-diagnosed MM patients, 30 MGUS and 30 healthy subjects in both G-MDSC and PMN.Finally, we tested the immunosuppressive properties of G-MDSC (isolated by CD66b+ immunomagnetic beads in the upper Ficoll layer) and PMN (sorted from the lower Ficoll layer) isolated from the same MGUS or MM patients, through functional assays, based on in vitro co-culture with T-lymphocytes from healthy subjects.
Results
MM-PMN exhibit an increased expression of ARG-1 compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity of ARG-1, positively correlated with advanced disease. In MM patients, increased levels of ARG-1 were positively associated with advanced bone disease and unfavourable cytogenetics.Circulating Arg-1 in serum was higher in MM than MGUS and healthy subjects (183.6 ± 21.9 versus 88.3 ± 15.6 versus 98.8 ± 11.4 ng/ml, p=0.0022). In MM patients there was a progressive increase from ISS stage I through III (p=0.003).Immunostaining showed that Arg-1 was increased in MM versus healthy subjects, but higher levels were evident in G-MDSC than PMN. Moreover, only in MM-G-MDSC Arg-1 was evident at the nuclear level, while in PMN Arg-1 had an exclusive cytoplasmic localization.This differential distribution of Arg-1 was functionally evident, since G-MDSC were more immunosuppressive than PMN.Indeed, after 72 hours of co-culture with T-cells obtained from healthy donors in presence of mitogen stimulation (PHA), MM-PMN were able to reduce T-cell proliferation at both tested 1:2 and 1:8 ratios (respectively 38.3 ± 2.6 % and 14.3 ± 0.6 %, p<0.0001), while MGUS-PMN induced a partial inhibition only at the 1:8 (25.4 ± 4.3%, p=0.002). This effect was partially reverted with the treatment of 200 μM nor-NOHA, an Arg-1 inhibitor, since within first 24 hours T-cell proliferation increased in presence of MM-PMN (14.3 ± 0.6 versus 24.5 ± 1.3%, p<0.0001) and MGUS-PMN (25.4 ± 4.3 versus 31.6 ± 2.3 %, p<0.0001).MM-G-MDSC were more immunosuppressive than their MM-PMN counterpart, since at 72 hours T-cell proliferation was 22.3 ± 1.6 % and 8.4 ± 0.5 % (p<0.0001) at 1:2 and 1.8 ratio respectively.
Conclusion
Polymorphonuclear neutrophils in MM are immunosuppressive, but distinguished in two main subpopulations, at different stages of maturation, based on the expression of Arg-1 and grains distribution, to target in order to improve the effects of the immunochemotherapy.
Session topic: E-poster
Keyword(s): Arginase, Myeloma, Myelosuppression
Type: Eposter Presentation
Background
Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. Development of MM is associated with accumulation of Myeloid-Derived suppressor cells (MDSC), mostly represented by pathologically activated relatively immature polymorphonuclear neutrophils (G-MDSC). Our previous work showed that also mature polymorphonuclear neutrophils (PMN) are activated and immunosuppressive in MM.
Aims
We hypothesized that two populations of polymorphonuclear neutrophils are present in MM, with differential distribution after a Ficoll separation and differential immunosuppressive activity
Methods
Using oligonucleotide microarrays we first evaluated the gene expression profile (GEP) of PMN at the steady state in 10 MM, 5 MGUS and 8 healthy subjects matched for sex and age, identifying Arg-1 as the first gene differentially expressed in MM versus healthy PMN.Thus, we validated Arg-1 by RT-PCR, immunohistochemistry and circulating levels in serum by ELISA in a series of 60 newly-diagnosed MM patients, 30 MGUS and 30 healthy subjects in both G-MDSC and PMN.Finally, we tested the immunosuppressive properties of G-MDSC (isolated by CD66b+ immunomagnetic beads in the upper Ficoll layer) and PMN (sorted from the lower Ficoll layer) isolated from the same MGUS or MM patients, through functional assays, based on in vitro co-culture with T-lymphocytes from healthy subjects.
Results
MM-PMN exhibit an increased expression of ARG-1 compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity of ARG-1, positively correlated with advanced disease. In MM patients, increased levels of ARG-1 were positively associated with advanced bone disease and unfavourable cytogenetics.Circulating Arg-1 in serum was higher in MM than MGUS and healthy subjects (183.6 ± 21.9 versus 88.3 ± 15.6 versus 98.8 ± 11.4 ng/ml, p=0.0022). In MM patients there was a progressive increase from ISS stage I through III (p=0.003).Immunostaining showed that Arg-1 was increased in MM versus healthy subjects, but higher levels were evident in G-MDSC than PMN. Moreover, only in MM-G-MDSC Arg-1 was evident at the nuclear level, while in PMN Arg-1 had an exclusive cytoplasmic localization.This differential distribution of Arg-1 was functionally evident, since G-MDSC were more immunosuppressive than PMN.Indeed, after 72 hours of co-culture with T-cells obtained from healthy donors in presence of mitogen stimulation (PHA), MM-PMN were able to reduce T-cell proliferation at both tested 1:2 and 1:8 ratios (respectively 38.3 ± 2.6 % and 14.3 ± 0.6 %, p<0.0001), while MGUS-PMN induced a partial inhibition only at the 1:8 (25.4 ± 4.3%, p=0.002). This effect was partially reverted with the treatment of 200 μM nor-NOHA, an Arg-1 inhibitor, since within first 24 hours T-cell proliferation increased in presence of MM-PMN (14.3 ± 0.6 versus 24.5 ± 1.3%, p<0.0001) and MGUS-PMN (25.4 ± 4.3 versus 31.6 ± 2.3 %, p<0.0001).MM-G-MDSC were more immunosuppressive than their MM-PMN counterpart, since at 72 hours T-cell proliferation was 22.3 ± 1.6 % and 8.4 ± 0.5 % (p<0.0001) at 1:2 and 1.8 ratio respectively.
Conclusion
Polymorphonuclear neutrophils in MM are immunosuppressive, but distinguished in two main subpopulations, at different stages of maturation, based on the expression of Arg-1 and grains distribution, to target in order to improve the effects of the immunochemotherapy.
Session topic: E-poster
Keyword(s): Arginase, Myeloma, Myelosuppression
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