Adenosine Signaling Mediates Hypoxic Responses in the Chronic Lymphocytic Leukemia Microenvironment

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4145-4145
Author(s):  
Sara Serra ◽  
Davide Brusa ◽  
Tiziana Vaisitti ◽  
Roberta Buonincontri ◽  
Valentina Audrito ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Lymphocytic Leukemia ◽  
Nucleoside Transporters ◽  
Leukemic Cells ◽  
T Cell Proliferation ◽  
A2a Adenosine Receptor ◽  
Hypoxic Conditions ◽  
A2a Receptor

Abstract Extracellular adenosine generated from ATP/ADP through the concerted action of the ectoenzymes CD39 and CD73 elicits potent cytoprotective and immunosuppressive effects mediated by type-1 purinergic receptors. Chronic lymphocytic leukemia (CLL) cells expressing the ectoenzymes CD39 and CD73 can actively produce adenosine, activating an autocrine adenosinergic axis that supports engraftment of leukemic cells in a growth-favorable environment. These effects are mediated by the A2A adenosine receptor, which inhibits chemotaxis and limits spontaneous and drug-induced apoptosis of CLL cells. Following the reported cross-talk between hypoxia and adenosine, we tested the hypothesis of a functional interplay between the adenosinergic axis and hypoxic signals in the CLL microenvironment. Results indicate that culture of CLL cells under hypoxic conditions, such as those observed in lymph nodes from CLL patients, boosts adenosine production, mainly because of the significant increase in the mRNA and protein levels of CD73, the rate-limiting enzyme in adenosine synthesis. CLL also underwent a robust up-regulation of CD26, which functions as an adenosine-deaminase scaffold protein, in keeping with the hypothesis that extracellular nucleotides enter a scavenging pathway, with conversion to inosine and re-uptake by the leukemic cells. Confirmation was obtained using HPLC assays, which showed increased inosine generation under hypoxia. Consistently, expression of membrane nucleoside transporters was also significantly up-regulated. However, hypoxic CLL cells also expressed high levels of the A2A adenosine receptor, which delivered cytoprotective signals and which supported CLL proliferation in response to TLR signaling. Attention was then focused on the stromal and T cell compartments, which are critical to the formation and maintenance of the leukemic niche. Hypoxia enhanced differentiation of circulating monocytes into nurse-like cells, macrophages of the M2 type playing an essential role in nurturing leukemic cells. The enhancement of NLC differentiation under hypoxic conditions relied, at least in part, on the activation of A2A: its engagement by a pharmacological agonist favored NLC generation, with overexpression of indoleamine 2,3-dioxygenase (IDO) and of the M2 macrophage markers CD163 and CD206. Moreover, activation of A2A induced secretion of immunomodulatory cytokines, such as IL-6, IL-10 and CCL18, while pharmacological blockade of A2A under hypoxia prevented NLC differentiation, expansion, expression of immunosuppressive molecules and secretion of cytokines and chemokines. In the T cell compartment, hypoxic cultures were followed by the sharp up-regulation of A2A, without significantly affecting the enzymes that generate adenosine, which were anyway restricted to the regulatory T cell (Treg) compartment. Co-cultures of T lymphocytes and CLL cells under hypoxia resulted in a dramatic decrease of T cell proliferation, partially rescued by A2A receptor antagonists. Furthermore, hypoxic T cells underwent a metabolic switch, with increased expression of nucleoside transporters and enzymes involved in glucose metabolism, suggesting a Warburg effect. This was accompanied by the differentiation of a population of Tr1 cells, characterized by the expression of LAG3 and CD49b and by the secretion of high levels of IL-10 and VEGF. Expression of the PD-1 immuno-inhibitory receptor was enhanced in hypoxic T cells, suggesting that multiple inhibitory mechanisms are activated. We also observed expansion of classical Tregs, defined on the basis of a CD4+/CD25high/CD127low/foxp3+ phenotype. Blockade of the A2A receptor prevented this phenotype, partially restoring T cell proliferation and immune competence. Together, these findings indicate that the adenosinergic and hypoxic axes synergize in shaping the CLL niche, suggesting that pharmacological inhibition of the adenosinergic signals may counteract some of the effects mediated by an hypoxic environment, contributing to disrupt the leukemic niche and to restore the immune system. Disclosures Gaidano: Celgene: Research Funding; MorphoSys; Roche; Novartis; GlaxoSmithKline; Amgen; Janssen; Karyopharm: Honoraria, Other: Advisory boards.

scholarly journals Defective interleukin-2 production and responsiveness by T cells in patients with chronic lymphocytic leukemia of B cell variety

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 279-284 ◽  
Author(s):  
O Ayanlar-Batuman ◽  
E Ebert ◽  
SP Hauptman
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Proliferative Response ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Activated T Cells

Abstract The present studies were designed to investigate the mechanism(s) of the defective T cell proliferative response to various stimuli in patients with B cell chronic lymphocytic leukemia B-CLL. In 14 patients with advanced B-CLL (stage III or IV) we found the T cell response in the autologous (auto) and allogeneic (allo) mixed lymphocyte reaction (MLR) to be 35.7% and 30% of the controls, respectively. Proliferation in the MLR depends upon the production of and response to interleukin 2 (IL 2), a T cell growth factor. IL 2 production in eight B-CLL patients was 22% of the control. The response to IL 2 was measured by the increase in the T cell proliferation in the MLR with the addition of IL 2. T cell proliferation in both the auto and allo MLR of CLL patients was significantly lower than in the controls after the addition of IL 2. The proliferative response of normal T cells to stimulation by CLL B cells was 50% of the control. This latter response was increased to control levels when cultures were supplemented with exogenous IL 2, suggesting that CLL B cells could stimulate IL 2 receptor generation in normal T cells in an allo MLR, but not IL 2 production. The presence of IL 2 receptors on activated T cells was directly determined using anti- Tac, a monoclonal antibody with specificity for the IL 2 receptor. Of the mitogen- or MLR-activated T cells in CLL patients, 6% and 10%, respectively, expressed Tac antigen, whereas identically stimulated control T cells were 60% and 47% Tac+, respectively. Our findings suggest that T cells in B-CLL are defective in their recognition of self or foreign major histocompatibility antigens as demonstrated by their impaired responsiveness in the MLR. Thus, these cells are unable to produce IL 2 or generate IL 2 receptors.

Chronic Lymphocytic Leukemia Cells Acquire a Regulatory B-Cell Phenotype Modified By PD1 Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3319-3319 ◽  
Author(s):  
Shimrit Ringelstein-Harlev ◽  
Irit Avivi ◽  
Shoham Shivtiel-Arad ◽  
Tami Katz
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Immune Responses ◽  
B Cell ◽  
Immune Modulation ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Cell Contact

Abstract Introduction: Chronic lymphocytic leukemia (CLL) cells utilize several mechanisms of survival, some propagating proliferation and preventing apoptosis through intrinsic cell cycle signals, and others suppressing anti-tumor immune responses. Patients often present with a predominant population of regulatory T-cells (Tregs), and general features of T-cell exhaustion. Given the unique phenotype of CLL cells and the observed T-cell abnormalities we hypothesized that these cells function as regulatory B-cells (Bregs). Bregs, mostly explored in the autoimmune disease setting, produce interleukin-10 (IL10), which mediates attenuation of effector T-cell responses and enhances regulatory activity. These features have also been suggested to be responsible for weakening of anti-tumor immune responses. Breg activation requires stimulation of various combinations of Toll-like receptors (TLRs), the B-cell receptor (BCR) and CD40. Our previous studies have demonstrated that TLR9-stimulated CLL cells "acquire" Breg markers as well as PD1 and PDL1, which, while not being classic Breg discriminators, are established players in immune modulation. Moreover, such stimulation resulted in inhibition of proliferation of autologous T-cells. The current study aimed to further explore the regulatory characteristics of CLL cells focusing on additional suppressive mechanisms that may have a role in CLL immune evasion, particularly, the PD1/PDL1 axis. Methods: B-cells were isolated from peripheral blood mononuclear cells (PBMCs) of untreated CLL patients (Rai stages 0-IV). These B-CLL cells were stimulated with TLR-9 agonist (ODN) or CD40 ligand (CD40L) followed by their co-culture with isolated autologous CD4+ T cells. The regulatory features of B-CLL cells were studied by testing their effect on T cells. Their proliferation was evaluated using the CFSE method following stimulation with anti-CD3/CD28 antibodies and IL2; induction of Tregs (CD4+CD25highFoxp3+ population) was assessed by FACS analysis. The involvement of the PD1/PDL1 axis was examined by incubating B-cells with antiPD1 neutralizing antibodies prior to co-culture. Cell contact dependence was evaluated by plating B-cells in hanging cell culture inserts denying B and T cell contact while allowing flow of small soluble molecules. Results: CLL cells stimulated with ODN or CD40L, induced a significant increase in Tregs: 1.35±0.1-fold (p=0.03, N=12) for ODN and 1.7±0.2-fold (p=0.008, N=14) for CD40L, occurring in 68% and 80% of patients, respectively, while co-culture with unstimulated B-CLL cells did not result in the expansion of the Treg population. Treg induction was observed only under contact conditions (N=5), suggesting that this regulatory function requires cell-to-cell contact and cannot be carried out solely by secreted factors like IL10. Neutralization of PD1 on CLL B-cells affects both Treg induction and T-cell proliferation. Following CD40L stimulation, a 1.3-fold reduction in Treg percentage was observed when PD1 signaling was blunted (N=10). In contrast, PD1 blockage of ODN-stimulated CLL cells did not reduce Treg induction; however, it did adversely affect inhibition of T-cell proliferation (10%-decrease in inhibited T-cells; N=6). Conclusions: CLL cells "acquire" a Breg phenotype and function, inhibiting T-cell proliferation and inducing Tregs. These properties, while working together to promote immune regulation and cancer evasion, are elicited by different ligands in the cell environment and are likely to be mediated via separate pathways. The involvement of B-cell-associated PD1 in the induction of Tregs and inhibition of T-cell proliferation suggests a biologic role of PD1 signaling in CLL cells, strengthening the Breg phenotype. The current study has shown that CLL cells recruit several mechanisms operating cooperatively to support immune modulation and promote their survival. Disclosures No relevant conflicts of interest to declare.

scholarly journals Defective interleukin-2 production and responsiveness by T cells in patients with chronic lymphocytic leukemia of B cell variety

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 279-284 ◽  
Author(s):  
O Ayanlar-Batuman ◽  
E Ebert ◽  
SP Hauptman
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Proliferative Response ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Activated T Cells

The present studies were designed to investigate the mechanism(s) of the defective T cell proliferative response to various stimuli in patients with B cell chronic lymphocytic leukemia B-CLL. In 14 patients with advanced B-CLL (stage III or IV) we found the T cell response in the autologous (auto) and allogeneic (allo) mixed lymphocyte reaction (MLR) to be 35.7% and 30% of the controls, respectively. Proliferation in the MLR depends upon the production of and response to interleukin 2 (IL 2), a T cell growth factor. IL 2 production in eight B-CLL patients was 22% of the control. The response to IL 2 was measured by the increase in the T cell proliferation in the MLR with the addition of IL 2. T cell proliferation in both the auto and allo MLR of CLL patients was significantly lower than in the controls after the addition of IL 2. The proliferative response of normal T cells to stimulation by CLL B cells was 50% of the control. This latter response was increased to control levels when cultures were supplemented with exogenous IL 2, suggesting that CLL B cells could stimulate IL 2 receptor generation in normal T cells in an allo MLR, but not IL 2 production. The presence of IL 2 receptors on activated T cells was directly determined using anti- Tac, a monoclonal antibody with specificity for the IL 2 receptor. Of the mitogen- or MLR-activated T cells in CLL patients, 6% and 10%, respectively, expressed Tac antigen, whereas identically stimulated control T cells were 60% and 47% Tac+, respectively. Our findings suggest that T cells in B-CLL are defective in their recognition of self or foreign major histocompatibility antigens as demonstrated by their impaired responsiveness in the MLR. Thus, these cells are unable to produce IL 2 or generate IL 2 receptors.

GITR Ligation with GITR Ligand On Leukemic Cells Suppresses Induction of Leukemia-Associated Antigen Specific T Cells by Increasing Indoleamine 2, 3-Dioxygenase (IDO) Activity Leading to Kynurenine Secretion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3557-3557
Author(s):  
Yukio Kondo ◽  
Luis Espinoza ◽  
Takamasa Katagiri ◽  
Zhirong Qi ◽  
Shinji Nakao
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Culture Supernatant ◽  
Cd8 T Cell ◽  
Cd4 T Cell ◽  
Leukemic Cells ◽  
T Cell Proliferation ◽  
Dependent Manner ◽  
Specific Ctls

Abstract Abstract 3557 Poster Board III-494 Immunization of allogeneic stem cell transplant (SCT) recipients with leukemia-associated antigens (LAAs) is an attractive approach to the augmentation of graft-versus-leukemia (GVL) effect. However, the induction of CTLs specific to LAAs is hampered by various inhibitory molecules expressed on leukemic cells that restrain the T cell function in connection with their receptors on T cells. Even if the cellular immunity is rebuilt after SCT by T cells of the donor origin, overcoming such an escape mechanism is required to effectively induce the CTLs specific to TAAs by vaccination after allogeneic SCT. Glucocorticoid-induced TNFR-related protein (GITR) belongs to the TNF receptor superfamily and is expressed on NK cells, CD25+ regulatory T cells and activated T cells. The binding of GITR ligand (GITRL) on leukemic cells to GITR on NK cells restrains NK cell activity but the influence on T cells of the GITR/GITRL binding has not been clarified. Myeloid dendritic cells derived from myeloid leukemic cells express GITRL which inhibits induction of LAA-specific CTLs (Blood 2008; 112:817a). The mechanisms of the negative effect on the induction of LAA-specific T cells through the GITR/GITRL interaction was investigated to improve the efficiency of the CTL induction. The expression of GITRL was observed on leukemic cells from 9 of 16 patients with myeloid leukemia and a monocytic leukemia cell line THP-1, and soluble GITRL (sGITRL) was detectable in the serum from 3 of 5 patients as well as in the culture supernatant of THP-1 cells. CFSE-labeled pan T cell, CD4+ T cell and CD8+ T cell proliferation in response to microbeads coated with anti-CD3 and anti-CD28 monoclonal antibodies (CD3/CD28 microbeads) was suppressed to 55.0%, 63.6%, 65.8% of the controls in a culture supernatant of THP-1 cells, and was restored to 86.9%, 65.1% and 76.8% respectively by addition of sGITR to block the binding of sGITRL in the supernatant and GITR on T cells. Flow cytometry detected GITRL in exosomes, which express HLA class II, purified from the culture supernatant of THP-1 with anti-HLA class II antibody-coated microbeads, and CFSE-labeled pan T cell, CD4+ T cell and CD8+ T cell proliferation was restrained as well by the addition of GITRL+ exosomes in a dose dependent manner (27.6%, 54.1%, 27.9% reduction of proliferation with 10 μl exosome, respectively). Indoleamine 2, 3-dioxygenase (IDO) activity in plasmacytoid DC (pDC) is negatively correlated with the activity of CD4+ T cells induced by their interaction with the pDC through the GITR/GITRL interaction in a mouse model. Kynurenine (Kyn), a metabolite of tryptophan in leukemic cells that is broken down by IDO, suppressed CFSE-labeled pan T cell, CD4+ T cell and CD8+ T cell proliferation in response to CD3/CD28 microbeads in a dose dependent manner (24.5%, 12.3%, 18.3% reduction in the proliferation at 100 μM, respectively). Significantly higher concentrations of Kyn were detected in the supernatant of THP-1 cells after incubation in the presence of sGITR than a control, and the production of Kyn was suppressed by the addition of an IDO inhibitor, 1-Methyl Tryptophan (1MT) (Fig). Moreover, the addition of sGITR to leukemic cells from five patients with AML induced Kyn (Fig). These findings indicate that GITRL on leukemic cells and sGITRL secreted by leukemic cells as an exosome protein suppress the induction of LAA-specific CTLs by directly binding GITR on LAA-specific CTLs, increasing the IDO activity in leukemic cells and inducing Kyn secretion from leukemic cells. The administration of anti-IDO agents or anti-GITRL blocking Abs combined with LAA vaccination may therefore effectively induce LAA-specific T cells in SCT recipients. Fig GITR/GITRL binding induces kyn secretion from THP-1 cell and primary AML cells. Fig. GITR/GITRL binding induces kyn secretion from THP-1 cell and primary AML cells. Disclosures: No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia Cells Exhibit Inhibitory Properties of Regulatory B Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3874-3874
Author(s):  
Shimrit Ringelstein-Harlev ◽  
Irit Avivi ◽  
Lina Bisharat ◽  
Tamar Katz
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Fold Increase ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Cell Contact ◽  
Stimulation Of

Abstract Abstract 3874 Background: Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy, characterized by distinct immune suppression rendering both tumor cells and invading pathogens invisible to the immune system. However, CLL cells also display profound immune sensitivity as proven by long-term remissions achieved with allogeneic bone marrow transplantation. Many phenotypic properties of B-CLL cells resemble a subset of B-cells, studied mostly in autoimmunity and termed regulatory B cells (Bregs). Bregs are thought to suppress CD4+ T-cell mediated immune responses, directly through cell contact and indirectly through inhibitory cytokines. This study aims to define whether malignant B-CLL cells exhibit Breg suppressive properties, contributing to immune dysfunction in this disease. Methods: B-cells were isolated from peripheral blood mononuclear cells (PBMCs) of untreated CLL patients (Rai stages 0-IV) using immunomagnetic separation (STEMCELL technologies). Naïve cells and those stimulated with B-cell activators TLR-9 agonist or CD40Ligand (CD40L) were analyzed by FACS for Breg phenotypic markers and intracellular IL-10. Additionally, B-CLL cell effects on autologous CD4+ T cells (isolated by immunomagnetic beads; Miltenyi Biotec) were studied. T-cells were stimulated with anti-CD3/CD28 antibodies and IL-2, and exposed to B-cells either directly or through hanging cell culture inserts (Millipore) preventing physical cell-cell contact. T-cell proliferation was assessed using the carboxyfluorescein diacetate succinimidyl ester (CFSE) method and phenotype was analyzed by FACS. Results: B-cell phenotype was studied in 11 patients. Breg markers (CD5, CD38, CD25 and intracellular IL-10) as well as inhibitory molecules PD-1 and PDL-1 were expressed at high levels on B-CLL cells (62%, 37%, 50%, 52%, 29%, 61%, respectively), although not every patient expressed all markers. These expression levels were higher than those reported for normal peripheral blood B-cells. TLR-9 stimulation of B-CLL cells resulted in a 5.7-fold increase in expression of CD25 in 77% of patients. Increments were also observed in IL-10 (1.9-fold; 62% of patients), PDL-1 (1.96-fold; 83% of patients) and PD-1 (2.19-fold; 57% of patients). Of 13 patients whose T-cell proliferation potential was evaluated after exposure to B-CLL cells, proliferation was induced in only 69%; in the other 31% (4 patients) no proliferation was observed; moreover, inhibition was demonstrated in one of them. Among the former group only 33% of patients expressed CD25 on their B-cells, whereas within the latter group, 75% of patients' B-cells were CD25-positive. Stimulation of B-CLL cells with TLR-9 markedly increased their inhibitory capacity (72% of 11 patients tested), while CD40L stimulation caused a weaker effect (50% of 6 patients tested). T-cell proliferation remained unchanged when evaluated using a Transwell system versus a contact system, as demonstrated in 3 of 4 experiments. T-cells exposed to B-CLL cells altered the ratio of CD25high vs. CD25low T-cells in favor of CD25 high cells (2.44-fold increase for stimulation with naïve B-CLL cells, 4.94-fold increase with TLR-9 stimulated cells; in all the 5 tested patients). Conclusions: Previously identified Breg markers as well as PD-1 and PDL-1 were highly expressed in B-CLL cells, supporting the role of these cells in shaping an immune tolerant environment, enabling tumor growth. Stimulation of B-CLL cells with TLR-9 agonist enhanced this phenotype and resulted in consistent inhibition of T-cell proliferation, likely to be independent of cell-to-cell contact. These findings demonstrate the presence of Breg features within the CLL clone. The observed alterations in CD4+CD25+ T-cell populations after exposure to B-CLL cells suggest induction of T-regulatory cells, another mechanism supposedly used by Bregs for immune suppression. The enhancement of Breg properties in B-CLL cells following B-cell activation can serve as a platform for further studies of the innate regulatory mechanisms utilized by tumor cells. Disclosures: No relevant conflicts of interest to declare.

TLR9-Regulated IL10 Mediates Immune Inhibitory Activity of Chronic Lymphocytic Leukemia B-Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4377-4377 ◽  
Author(s):  
Shimrit Ringelstein-Harlev ◽  
Mona Fanadka ◽  
Netanel A. Horowitz ◽  
Irit Avivi ◽  
Tami Katz
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cd4 T Cells ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Cytokine Array ◽  
Cell Inhibition ◽  
Tlr9 Activation

Abstract Introduction: Chronic lymphocytic leukemia (CLL) cells exhibit inhibitory features of regulatory B-cells (Bregs), which are suspected to modulate cancer immune evasion on the one hand, and to promote immunosuppression leading to opportunistic infections on the other. One of the major mechanisms through which Bregs restrain the immune system is production of the anti-inflammatory cytokine interleukin 10 (IL10). Our previous study has demonstrated that CLL cells generate IL10, inhibit autologous CD4+ T-cell proliferation and induce regulatory T-cells (Tregs) from autologous CD4+ T-cells. All these abilities were found to be triggered by activation of toll like receptor 9 (TLR9) with the agonist CpG-ODN (ODN). Of note, Treg induction was also observed following CD40 activation with CD40 ligand (CD40L), while intracellular IL10 levels and proliferation inhibition were unaffected by this stimulant. Given the magnitude of the regulatory effects induced by ODN, the current study aimed to explore the contribution of TLR9 activation to the control of IL10 generation by CLL cells and to assess the role of this inhibitory cytokine in mediating the above Breg abilities of these cells. Methods: B-cells were isolated from peripheral blood mononuclear cells (PBMCs) of untreated CLL patients (Rai stages 0-IV). B-CLL cells were stimulated with ODN or CD40L and either examined directly, or co-cultured with isolated autologous CD4+ T-cells. A cytokine array was used to detect and estimate the concentration of a variety of cytokines (n = 42) secreted to the medium. FACS analysis was employed to examine intracellular IL10 levels in CLL cells, either untreated or treated with an IRAK1/4 inhibitor, obstructing upstream TLR signaling, at doses of 2µM or 5µM. For co-culture experiments, CLL cells were transfected with IL10 siRNA or scramble RNA (control), using the nucleofector technology by Amaxa. T-cell proliferation was evaluated using carboxyfluorescein succinimidyl ester (CFSE) staining for cell tracking after stimulation with anti-CD3/CD28 antibodies and interleukin 2 (IL2). Percentage of cells retained in G0 (non-proliferating pool) was assessed for each condition. Results: The cytokine array showed IL10 to be the only cytokine secreted by CLL cells to the medium under baseline conditions. The secretion was significantly enhanced following ODN stimulation [fold induction >1.65 (N=3)], but not after CD40L activation. Since in our studies, ODN emerged as the sole activator of IL10 generation, levels of this cytokine were assessed following IRAK1/4 inhibition. The results showed a dose-dependent attenuation of intracellular IL10 levels, with percentage of IL10 expression being 5.88 (±2.01) at baseline, 5.18 (±2.01) for low-dose and 4.83 (±1.93) for high-dose IRAK1/4 inhibition (N=5, p<0.05 for 2µM and p<0.01 for 5µM). To define the contribution of IL10 to Breg functions of CLL cells, the impact of IL10 RNA interference was examined. CLL cell transfection with IL10 siRNA diminished the inhibitory effect of these cells on proliferation of co-cultured autologous T-cells, demonstrating a 21% reduction in T-cell inhibition. In the control co-culture, 12.4 (±2.3)% of stimulated CD4+ T-cells remained in G0, whereas in the presence of IL10-depleted CLL cells, only 9.8 (±1.87)% of CD4+ T-cells remained in the non-proliferating pool (N=8, p<0.05). Conclusions: In the current study, IL10 emerges as the only inhibitory cytokine secreted at biologically relevant levels by CLL cells, and the only one whose quantity significantly increases following ODN-activation. The TLR9 signaling pathway directly controls IL10 generation, which in turn contributes to the inhibition of T-cell proliferation. While normal B-cells are known to respond to TLR9 activation by secreting a variety of cytokines, the exclusive generation of IL10 by activated CLL cells, resulting in T-cell inhibition, may represent a unique clonal quality promoting disease progression. Disclosures Avivi: Tel Aviv Sourasky Medical center: Consultancy, Other: consultancy to :BMS Roche.

Chronic Lymphocytic Leukemia Patients and Eµ-TCL1 Mice Share a Phenotype of Functional Granulocyte-like and Dysfunctional Monocyte-like Myeloid Derived Suppressor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 614-614
Author(s):  
Gerardo Ferrer ◽  
Xiao-Jie Yan ◽  
Brendan Franca ◽  
Jacqueline C. Barrientos ◽  
Jonathan E. Kolitz ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Suppressor Cells ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
T Cell Proliferation ◽  
Myeloid Derived Suppressor Cells ◽  
Cell Responses

Abstract Immune imbalance is a common characteristic of patients with chronic lymphocytic leukemia (CLL). This feature is shared with Eμ-TCL1 transgenic mice that, like CLL patients, exhibit an expansion of CD5+ B cells with associated non-B-cell defects. In patients and in mice, T-cell responses are often ineffective. This alteration is generally considered due to the direct effects of the leukemic cells. The expansion of myeloid derived suppressor cells (MDSCs), which play a major role in helping tumor cells escape immune surveillance by inhibiting T-cell responses, is promoted by many cancers. MDSCs are a heterogeneous population of cells that are subdivided into monocyte-like (m-MDSC) and granulocyte-like (g-MDSC) subsets, both in humans and mice. There we have investigated the extent that patients with CLL have expansions of MDSCs, what are their types and functions, and how these correlate with the Eμ-TCL1 mice model. Using flow cytometry on cryopreserved PBMCs, we found that the absolute numbers of MDSCs (HLA-DRlo/CD11b+/CD33+) in 49 untreated CLL patients were significantly higher than 15 healthy controls (HCs) (966 446 vs. 163 578 cells/ml, P<0.001). Moreover, we observed that the absolute numbers of MDSCs significantly correlated with CLL B-cell counts in the blood (P=0.005, Spearman r=0.423). Of note, the distribution between m-MDSCs (CD14+) and g-MDSCs (CD15+) was dramatically different, with CLL patients exhibiting significantly higher numbers and percentages of g-MDSCs than HCs (702 296 vs. 26 818 cells/ml, P<0.001; 50.89 vs. 16.98%, P<0.001).In line with these results, when we explored the MDSC populations (CD11b+/GR1+) in Eμ-TCL1 mice of 5-16 months of age with leukemia cell blood counts ranging from 0.1 to 100 x 106 cell/ml. This analysis indicated a positive correlation between MDSCs and leukemic CD19+ CD5+ cells (P=0.003; Spearman r=0.328). Furthermore, the dot-plot analysis of GR1 and CD11b showed three well defined cell populations: one monocytic (Ly6-C+) and two granulocytic (Ly6-G+ CD11blo and Ly6-G+ CD11bhi). As in patients, the g-MDSC population was larger than the m-MDSC population (884 100 vs. 454 700,P=0.016). However in this case, the m-MDSCs correlated with the numbers of circulating leukemic cells (P<0.001; Spearman r=0.463) and the g-MDSCs did not. The latter was the case even when they were subdivided into both CD11blo and CD11bhi subgroups. A similar pattern was observed when analyzing single cell suspensions from murine spleens. When we evaluated the ability of MDSCs to inhibit autologous T-cell proliferation in CLL patients (n=7), we observed a consistent reduction of proliferation only when co-culturing with g-MDSCs(P=0.034). In contrast, the effects of m-MDSCs on T-cell expansion were varied and insignificant statistically. In 5 CLL samples, we induced m-MDSCs (im-MDSCs) from purified CD33+ cells in vitro with GM-CSF, IL10, and IL6; the im-MDSCs effectively suppressed T-cell proliferation in 4 of 5 cases at an average inhibition of 33% (range: 10-79%). Thus, dysfunctional m-MDSC suppression was not inherent and functional suppression could be achieved by stimulation of CLL precursor cells. Similarly in 3 independent experiments performed with MDSCs from Eμ-TCL1 mice (12-14 months of age), we observed a reduction of in vitro proliferation with g-MDSCs (P=0.049) and not with m-MDSCs. In addition, for those Eμ-TCL1 animals for which sufficient sample was available, we subdivided the g-MDSC population into the two subpopulations based on CD11b density; the CD11blo subset present less nuclear segmentation and higher suppressive activity. In summary, absolute numbers of MDSCs in the blood of CLL patients and Eμ-TCL1 mice are elevated and correlate with the levels of expansion of the leukemia. The major subtype in both situations was g-MDSCs.These g-MDSCs were functionally competent suppressors, whereas m-MDSCs were impaired in this function. In CLL patients, this m-MDSC suppressor defect could be corrected by in vitro stimulation with growth factors that support monocyte differentiation. The high similarity between CLL patients and Eμ-TCL1 mice in relation to MDSC number and function suggest that an imbalance in g-MDC vs. m-MDSC function may affect CLL development and expansion, altering interactions with members of the microenvironment such as T cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals BET Inhibition As a Targeted Epigenetic Approach to Reverse T Cell Dysfunction in Chronic Lymphocytic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3715-3715
Author(s):  
Audrey L Smith ◽  
Alexandria P Eiken ◽  
Sydney A Skupa ◽  
Dalia Y Moore ◽  
Avyakta Kallam ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Cell Dysfunction ◽  
T Cell Dysfunction

Abstract Introduction : Chronic Lymphocytic Leukemia (CLL) is characterized by the clonal expansion of mature CD19+/CD5+ lymphocytes in the peripheral blood and secondary lymphoid organs. The accumulation of B-CLL cells yields profound immune defects in the CLL tumor microenvironment (TME), promoting evasion of immune surveillance that contributes to tumor persistence and thus relapsed/refractory disease. The bromodomain and extra-terminal domain (BET) family of proteins are epigenetic readers that bind acetylated histone residues to regulate transcription of numerous genes involved in critical CLL protumor pathways. Of the BET family proteins, BRD4 is overexpressed in CLL and highly enriched at super-enhancers of genes that regulate CLL-TME interactions such as B cell receptor pathway components, chemokine/cytokine receptors, and immune checkpoint molecules. Pan BET inhibitors (BET-i), such as PLX51107 (Plexxikon Inc.) significantly improve survival in aggressive CLL murine models. Here we demonstrate that blocking BRD4 function with PLX51107 (PLX5) can alleviate the inherent immune defects observed in CLL, hence reducing B-CLL induced T cell dysfunction and allowing for robust B-CLL cell elimination. This therapeutic strategy may be vital in overcoming frequent drug resistance and/or bolstering the anti-tumor effect of current CLL therapies. Methods : Primary leukemic B cells were isolated from the peripheral blood of CLL patients and co-cultured with healthy donor T cells to evaluate the effect of PLX5 (0.1-0.5μM) on CLL-induced T cell immunosuppression ex vivo via an array of flow cytometry assays. T cell proliferation was assessed using CFSE after 96 h co-culture with α-CD3/α-CD28 stimulation. Effector cytokine production was evaluated after 48 h co-culture in the presence of PMA/ionomycin (final 6 h) and brefeldin A (final 5 h). Immune inhibitory molecule surface expression was measured following 48 h co-culture with α-CD3/α-CD28 stimulation. To further validate our ex vivo findings, the E μ-TCL1 adoptive transfer model was used. Once disease onset was confirmed in recipient WT B6 mice (&gt;10% CD45+/CD19+/CD5+ peripheral blood lymphocytes), mice were randomized to receive either PLX5 (20 mg/kg) or vehicle (VEH) equivalent daily by oral gavage for 4 weeks. Following treatment, mouse spleens were processed to evaluate exhaustion marker expression, T cell proliferation (CellTrace™ Violet, 72 h a-CD3/α-CD28 stimulation ex-vivo), and T-cell effector function (ex-vivo mitogenic stimulation, 6 h). Results : T cell proliferation indices were reduced following ex vivo co-culture with primary B-CLL cells (mean ± SEM for T cells vs. co-culture, 2.0 ± 0.13 vs. 1.57 ± 0.05; P&lt;0.01). This suppression was significantly alleviated in 0.5μM PLX5-treated co-cultures (1.84 ± 0.08; P&lt;0.01). In a similar fashion, the percentage of polyfunctional TNF-α+/IFN-γ+ CD4+ T cells markedly increased in PLX5-treated co-cultures (VEH vs. 0.5μM PLX5, 10.0% ± 0.76% vs. 15.2% ± 0.92%; P&lt;0.01). Notably, BET inhibition with PLX5 also bolstered T cell inflammatory function (%TNF-α+/IFN-γ+) in the absence of B-CLL cells (VEH vs. PLX5, 12.9% ± 1.0% vs. 15.3% ± 0.69%; P&lt;0.05). Remarkably, the expression of numerous immune inhibitory molecules (e.g., PDL1, PD1, CTLA4, LAG3) was consistently reduced between 1.8- and 3-fold in PLX5-treated co-cultures (0.1μM). In the adoptive transfer E μ-TCL1 model, mice receiving PLX5 displayed reduced expansion of B-CLL cells and increased T cell infiltration in the spleen (Fig. 1A). Splenic CD4+ T cells from PLX5-treated mice had significantly greater proliferative capacity (Fig. 1B) and pro-inflammatory functionality (Fig. 1C). Finally, PLX5 treatment markedly reduced the surface expression of immune inhibitory molecules (e.g., PDL1, LAG3, VISTA) on CD4+ and CD8+ T cells in the spleen (Fig. 1D). Studies to evaluate the effects of PLX5 on malignant B-CLL and T cells within the bone marrow niche and soluble factors in the plasma are ongoing. Collectively, our data indicate that the novel BET-i, PLX5, exerts beneficial immunomodulatory effects on T cells within the CLL TME. Conclusion : Epigenetic-targeted therapies such as BET-i have the potential to alleviate CLL-induced T cell dysfunction while eliminating B-CLL cells and preventing tumor expansion. Future profiling studies are pending to further illuminate how BET proteins regulate immune function in CLL. Figure 1 Figure 1. Disclosures Lunning: AstraZeneca: Consultancy; Legend: Consultancy; Acrotech: Consultancy; ADC Therapeutics: Consultancy; Kyowa Kirin: Consultancy; Myeloid Therapeutics: Consultancy; Beigene: Consultancy; Celgene, a Bristol Myers Squibb Co.: Consultancy; Verastem: Consultancy; Janssen: Consultancy; Daiichi-Sankyo: Consultancy; Morphosys: Consultancy; TG Therapeutics: Consultancy; Novartis: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Spectrum: Consultancy; Kite, a Gilead Company: Consultancy. Vose: Kite, a Gilead Company: Honoraria, Research Funding. Powell: Plexxikon Inc.: Current Employment.

Type D SRV‐2 virus‐specific CD8 + and CD4 ‐ CD8 ‐ T cells that regulate virus‐induced T cell proliferation in Celebes macaques

1993 ◽  
Vol 22 (2-3) ◽  
pp. 80-85
Author(s):  
A. Malley ◽  
N. Pangares ◽  
S.K. Mayo ◽  
M. Zeleny‐Pooley ◽  
J.V. Torres ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Proliferation ◽  
Type D

scholarly journals Costimulation by B7 Modulates Specificity of Cytotoxic T Lymphocytes: A Missing Link That Explains Some Bystander T Cell Activation

1997 ◽  
Vol 186 (10) ◽  
pp. 1787-1791 ◽  
Author(s):  
Pan Zheng ◽  
Yang Liu
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Influenza Virus ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Target Cells ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation

It has been proposed that some bystander T cell activation may in fact be due to T cell antigen receptor (TCR) cross-reactivity that is too low to be detected by the effector cytotoxic T lymphocyte (CTL). However, this hypothesis is not supported by direct evidence since no TCR ligand is known to induce T cell proliferation and differentiation without being recognized by the effector CTL. Here we report that transgenic T cells expressing a T cell receptor to influenza virus A/NT/68 nucleoprotein (NP) 366-374:Db complexes clonally expand and become effector CTLs in response to homologous peptides from either A/PR8/34 (H1N1), A/AA/60 (H2N2), or A/NT/68 (H3N2). However, the effector T cells induced by each of the three peptides kill target cells pulsed with NP peptides from the H3N2 and H2N2 viruses, but not from the H1N1 virus. Thus, NP366–374 from influenza virus H1N1 is the first TCR ligand that can induce T cell proliferation and differentiation without being recognized by CTLs. Since induction of T cell proliferation was mediated by antigen-presenting cells that express costimulatory molecules such as B7, we investigated if cytolysis of H1N1 NP peptide–pulsed targets can be restored by expressing B7-1 on the target cells. Our results revealed that this is the case. These data demonstrated that costimulatory molecule B7 modulates antigen specificity of CTLs, and provides a missing link that explains some of the bystander T cell activation.

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