REDUCED FREQUENCY OF CIRCULATING CD4+CD25+CD127LOWFOXP3+ REGULATORY T CELLS IN PRIMARY MYELOFIBROSIS
(Abstract release date: 05/19/16)
EHA Library. Barosi G. 06/09/16; 132887; E1338
Prof. Giovanni Barosi
Contributions
Contributions
Abstract
Abstract: E1338
Type: Eposter Presentation
Background
A systemic dysregulation of immune system, represented by increased plasma levels of several inflammatory cytokines, and clinical and laboratory findings of autoimmunity, has been documented in primary myelofibrosis (PMF). Regulatory T cells (Tregs), currently identified as CD4+T cells with high expression of CD25 (interleukin-2 receptor), low expression of CD127 (IL-7 receptor), and high expression of FOXP3, are known to play a crucial role in the maintenance of T cell homeostasis and immunologic tolerance. Moreover, Treg cell defects have been associated with different autoimmune diseases, and a Treg cell deficit due to a mutation in the FoxP3 gene has been shown to cause aggressive autoimmunity and early death. Although a clinical study has indicated decreased number of Treg cells in the active status of PMF, conflicting results have been reported up to now.
Aims
In this study we investigated the frequency of Tregs, identified as CD4+CD25+CD127lowFoxP3+ cells, to clarify whether or not Treg cell number is reduced in PMF, and to investigate the associations between Tregs frequency and disease phenotype
Methods
Tregs frequency was assessed by flow cytometry in 202 patients with PMF, all out of therapy at time of sampling, and in 24 healthy subjects (HS), comparable for sex and age. Spleen-derived Tregs were evaluated in 17 different patients undergoing splenectomy for symptomatic splenomegaly and in 8 HS splenectomized for abdominal trauma. At study entrance, all participants gave their informed consent, and study protocol was approved by the Ethics Committee of our Hospital before implementation. A single cell suspension was obtained from splenic samples by means of mechanical dissociation followed by density gradient centrifugation. The frequency of CD4+CD25+CD127lowFoxP3+ cells was calculated as a percentage of positive cells in the total CD4 gate. For the Treg suppression assay circulating CD4+CD25+ T cells were immunoselected and cultured in triplicate with soluble anti-CD3/anti-CD28 and autologous irradiated cells. At day 4, 3H thymidine (2Ci/well) was added to cultures for 16 hours and proliferation measured by the amount of incorporated 3H.
Results
Compared with HS who had a median Treg frequency of 1.99% (range, 0.48% to 6.51%), patients with PMF had a median frequency of Tregs of 0.80% (range 0% to 10.2%; P<0.001). A similar trend was observed in the spleen were HS had a median Treg frequency of 1.20% (range 0.36% to 3.97%) and patients with PMF a median of 0.35% (range 0.04% to 1.26%; P<0.001). This result secures that the reduced frequency of circulating Tregs was not due to cell recruitment to sites of active malignancy, like spleen. To support that blood and spleen Tregs were independently regulated among total CD3+CD4+ cells, we measured the percentages of CD3+CD4+ in blood and in total spleen lymphocytes. They resulted non different from healthy individuals (data not reported). The percentage of suppression of Tregs on the proliferation of effector T-cells was comparable in patients with PMF (38.7%) and HS (36.5%) indicating that Tregs isolated from patients were functionally active. In patients bearing JAK2V617F mutation (N=126) we observed a higher frequency of circulating Tregs than those non mutated (N= 74; median, 1.00 vs 0.70; P= 0.002), and patients with high JAK2V617F allele burden (≥50%) had higher frequency of circulating Tregs than those with low allele burden (P= 0.07). In CALR mutants, the reduction of circulating Tregs was statistically associated with older age, longer disease duration, and higher IPSS/DIPSS prognostic score, and a strict direct correlation between Treg frequency and haemoglobin was documented.
Conclusion
Our findings showed that a large fraction of PMF patients had reduced frequency of Tregs, a non clonal component of bone marrow and spleen microenvironment. Blood Treg cell defect was particularly evident in patients lacking JAK2V617F mutation: in CALR mutated patients the defective Treg percentage was associated with an advanced and prognostically unfavourable disease, and strongly correlated with severity of anemia. These results open new research perspectives and support new therapeutic strategies aimed at increasing the number of Tregs in PMF.
Session topic: E-poster
Keyword(s): Myelofibrosis, Peripheral blood, Regulatory T cell, Spleen
Type: Eposter Presentation
Background
A systemic dysregulation of immune system, represented by increased plasma levels of several inflammatory cytokines, and clinical and laboratory findings of autoimmunity, has been documented in primary myelofibrosis (PMF). Regulatory T cells (Tregs), currently identified as CD4+T cells with high expression of CD25 (interleukin-2 receptor), low expression of CD127 (IL-7 receptor), and high expression of FOXP3, are known to play a crucial role in the maintenance of T cell homeostasis and immunologic tolerance. Moreover, Treg cell defects have been associated with different autoimmune diseases, and a Treg cell deficit due to a mutation in the FoxP3 gene has been shown to cause aggressive autoimmunity and early death. Although a clinical study has indicated decreased number of Treg cells in the active status of PMF, conflicting results have been reported up to now.
Aims
In this study we investigated the frequency of Tregs, identified as CD4+CD25+CD127lowFoxP3+ cells, to clarify whether or not Treg cell number is reduced in PMF, and to investigate the associations between Tregs frequency and disease phenotype
Methods
Tregs frequency was assessed by flow cytometry in 202 patients with PMF, all out of therapy at time of sampling, and in 24 healthy subjects (HS), comparable for sex and age. Spleen-derived Tregs were evaluated in 17 different patients undergoing splenectomy for symptomatic splenomegaly and in 8 HS splenectomized for abdominal trauma. At study entrance, all participants gave their informed consent, and study protocol was approved by the Ethics Committee of our Hospital before implementation. A single cell suspension was obtained from splenic samples by means of mechanical dissociation followed by density gradient centrifugation. The frequency of CD4+CD25+CD127lowFoxP3+ cells was calculated as a percentage of positive cells in the total CD4 gate. For the Treg suppression assay circulating CD4+CD25+ T cells were immunoselected and cultured in triplicate with soluble anti-CD3/anti-CD28 and autologous irradiated cells. At day 4, 3H thymidine (2Ci/well) was added to cultures for 16 hours and proliferation measured by the amount of incorporated 3H.
Results
Compared with HS who had a median Treg frequency of 1.99% (range, 0.48% to 6.51%), patients with PMF had a median frequency of Tregs of 0.80% (range 0% to 10.2%; P<0.001). A similar trend was observed in the spleen were HS had a median Treg frequency of 1.20% (range 0.36% to 3.97%) and patients with PMF a median of 0.35% (range 0.04% to 1.26%; P<0.001). This result secures that the reduced frequency of circulating Tregs was not due to cell recruitment to sites of active malignancy, like spleen. To support that blood and spleen Tregs were independently regulated among total CD3+CD4+ cells, we measured the percentages of CD3+CD4+ in blood and in total spleen lymphocytes. They resulted non different from healthy individuals (data not reported). The percentage of suppression of Tregs on the proliferation of effector T-cells was comparable in patients with PMF (38.7%) and HS (36.5%) indicating that Tregs isolated from patients were functionally active. In patients bearing JAK2V617F mutation (N=126) we observed a higher frequency of circulating Tregs than those non mutated (N= 74; median, 1.00 vs 0.70; P= 0.002), and patients with high JAK2V617F allele burden (≥50%) had higher frequency of circulating Tregs than those with low allele burden (P= 0.07). In CALR mutants, the reduction of circulating Tregs was statistically associated with older age, longer disease duration, and higher IPSS/DIPSS prognostic score, and a strict direct correlation between Treg frequency and haemoglobin was documented.
Conclusion
Our findings showed that a large fraction of PMF patients had reduced frequency of Tregs, a non clonal component of bone marrow and spleen microenvironment. Blood Treg cell defect was particularly evident in patients lacking JAK2V617F mutation: in CALR mutated patients the defective Treg percentage was associated with an advanced and prognostically unfavourable disease, and strongly correlated with severity of anemia. These results open new research perspectives and support new therapeutic strategies aimed at increasing the number of Tregs in PMF.
Session topic: E-poster
Keyword(s): Myelofibrosis, Peripheral blood, Regulatory T cell, Spleen
Abstract: E1338
Type: Eposter Presentation
Background
A systemic dysregulation of immune system, represented by increased plasma levels of several inflammatory cytokines, and clinical and laboratory findings of autoimmunity, has been documented in primary myelofibrosis (PMF). Regulatory T cells (Tregs), currently identified as CD4+T cells with high expression of CD25 (interleukin-2 receptor), low expression of CD127 (IL-7 receptor), and high expression of FOXP3, are known to play a crucial role in the maintenance of T cell homeostasis and immunologic tolerance. Moreover, Treg cell defects have been associated with different autoimmune diseases, and a Treg cell deficit due to a mutation in the FoxP3 gene has been shown to cause aggressive autoimmunity and early death. Although a clinical study has indicated decreased number of Treg cells in the active status of PMF, conflicting results have been reported up to now.
Aims
In this study we investigated the frequency of Tregs, identified as CD4+CD25+CD127lowFoxP3+ cells, to clarify whether or not Treg cell number is reduced in PMF, and to investigate the associations between Tregs frequency and disease phenotype
Methods
Tregs frequency was assessed by flow cytometry in 202 patients with PMF, all out of therapy at time of sampling, and in 24 healthy subjects (HS), comparable for sex and age. Spleen-derived Tregs were evaluated in 17 different patients undergoing splenectomy for symptomatic splenomegaly and in 8 HS splenectomized for abdominal trauma. At study entrance, all participants gave their informed consent, and study protocol was approved by the Ethics Committee of our Hospital before implementation. A single cell suspension was obtained from splenic samples by means of mechanical dissociation followed by density gradient centrifugation. The frequency of CD4+CD25+CD127lowFoxP3+ cells was calculated as a percentage of positive cells in the total CD4 gate. For the Treg suppression assay circulating CD4+CD25+ T cells were immunoselected and cultured in triplicate with soluble anti-CD3/anti-CD28 and autologous irradiated cells. At day 4, 3H thymidine (2Ci/well) was added to cultures for 16 hours and proliferation measured by the amount of incorporated 3H.
Results
Compared with HS who had a median Treg frequency of 1.99% (range, 0.48% to 6.51%), patients with PMF had a median frequency of Tregs of 0.80% (range 0% to 10.2%; P<0.001). A similar trend was observed in the spleen were HS had a median Treg frequency of 1.20% (range 0.36% to 3.97%) and patients with PMF a median of 0.35% (range 0.04% to 1.26%; P<0.001). This result secures that the reduced frequency of circulating Tregs was not due to cell recruitment to sites of active malignancy, like spleen. To support that blood and spleen Tregs were independently regulated among total CD3+CD4+ cells, we measured the percentages of CD3+CD4+ in blood and in total spleen lymphocytes. They resulted non different from healthy individuals (data not reported). The percentage of suppression of Tregs on the proliferation of effector T-cells was comparable in patients with PMF (38.7%) and HS (36.5%) indicating that Tregs isolated from patients were functionally active. In patients bearing JAK2V617F mutation (N=126) we observed a higher frequency of circulating Tregs than those non mutated (N= 74; median, 1.00 vs 0.70; P= 0.002), and patients with high JAK2V617F allele burden (≥50%) had higher frequency of circulating Tregs than those with low allele burden (P= 0.07). In CALR mutants, the reduction of circulating Tregs was statistically associated with older age, longer disease duration, and higher IPSS/DIPSS prognostic score, and a strict direct correlation between Treg frequency and haemoglobin was documented.
Conclusion
Our findings showed that a large fraction of PMF patients had reduced frequency of Tregs, a non clonal component of bone marrow and spleen microenvironment. Blood Treg cell defect was particularly evident in patients lacking JAK2V617F mutation: in CALR mutated patients the defective Treg percentage was associated with an advanced and prognostically unfavourable disease, and strongly correlated with severity of anemia. These results open new research perspectives and support new therapeutic strategies aimed at increasing the number of Tregs in PMF.
Session topic: E-poster
Keyword(s): Myelofibrosis, Peripheral blood, Regulatory T cell, Spleen
Type: Eposter Presentation
Background
A systemic dysregulation of immune system, represented by increased plasma levels of several inflammatory cytokines, and clinical and laboratory findings of autoimmunity, has been documented in primary myelofibrosis (PMF). Regulatory T cells (Tregs), currently identified as CD4+T cells with high expression of CD25 (interleukin-2 receptor), low expression of CD127 (IL-7 receptor), and high expression of FOXP3, are known to play a crucial role in the maintenance of T cell homeostasis and immunologic tolerance. Moreover, Treg cell defects have been associated with different autoimmune diseases, and a Treg cell deficit due to a mutation in the FoxP3 gene has been shown to cause aggressive autoimmunity and early death. Although a clinical study has indicated decreased number of Treg cells in the active status of PMF, conflicting results have been reported up to now.
Aims
In this study we investigated the frequency of Tregs, identified as CD4+CD25+CD127lowFoxP3+ cells, to clarify whether or not Treg cell number is reduced in PMF, and to investigate the associations between Tregs frequency and disease phenotype
Methods
Tregs frequency was assessed by flow cytometry in 202 patients with PMF, all out of therapy at time of sampling, and in 24 healthy subjects (HS), comparable for sex and age. Spleen-derived Tregs were evaluated in 17 different patients undergoing splenectomy for symptomatic splenomegaly and in 8 HS splenectomized for abdominal trauma. At study entrance, all participants gave their informed consent, and study protocol was approved by the Ethics Committee of our Hospital before implementation. A single cell suspension was obtained from splenic samples by means of mechanical dissociation followed by density gradient centrifugation. The frequency of CD4+CD25+CD127lowFoxP3+ cells was calculated as a percentage of positive cells in the total CD4 gate. For the Treg suppression assay circulating CD4+CD25+ T cells were immunoselected and cultured in triplicate with soluble anti-CD3/anti-CD28 and autologous irradiated cells. At day 4, 3H thymidine (2Ci/well) was added to cultures for 16 hours and proliferation measured by the amount of incorporated 3H.
Results
Compared with HS who had a median Treg frequency of 1.99% (range, 0.48% to 6.51%), patients with PMF had a median frequency of Tregs of 0.80% (range 0% to 10.2%; P<0.001). A similar trend was observed in the spleen were HS had a median Treg frequency of 1.20% (range 0.36% to 3.97%) and patients with PMF a median of 0.35% (range 0.04% to 1.26%; P<0.001). This result secures that the reduced frequency of circulating Tregs was not due to cell recruitment to sites of active malignancy, like spleen. To support that blood and spleen Tregs were independently regulated among total CD3+CD4+ cells, we measured the percentages of CD3+CD4+ in blood and in total spleen lymphocytes. They resulted non different from healthy individuals (data not reported). The percentage of suppression of Tregs on the proliferation of effector T-cells was comparable in patients with PMF (38.7%) and HS (36.5%) indicating that Tregs isolated from patients were functionally active. In patients bearing JAK2V617F mutation (N=126) we observed a higher frequency of circulating Tregs than those non mutated (N= 74; median, 1.00 vs 0.70; P= 0.002), and patients with high JAK2V617F allele burden (≥50%) had higher frequency of circulating Tregs than those with low allele burden (P= 0.07). In CALR mutants, the reduction of circulating Tregs was statistically associated with older age, longer disease duration, and higher IPSS/DIPSS prognostic score, and a strict direct correlation between Treg frequency and haemoglobin was documented.
Conclusion
Our findings showed that a large fraction of PMF patients had reduced frequency of Tregs, a non clonal component of bone marrow and spleen microenvironment. Blood Treg cell defect was particularly evident in patients lacking JAK2V617F mutation: in CALR mutated patients the defective Treg percentage was associated with an advanced and prognostically unfavourable disease, and strongly correlated with severity of anemia. These results open new research perspectives and support new therapeutic strategies aimed at increasing the number of Tregs in PMF.
Session topic: E-poster
Keyword(s): Myelofibrosis, Peripheral blood, Regulatory T cell, Spleen
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