Pharmacologic Inhibition with Duvelisib (IPI-145) and Genetic Inhibition of PI3K p110δ Antagonizes Intrinsic and Extrinsic Survival Signals in Chronic Lymphocytic Leukemia (CLL)

Abstract Background: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen, and bone marrow. CLL cells display up-regulated B-cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton’s tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. Both the PI3K p110δ inhibitor, idelalisib, and the BTK inhibitor, ibrutinib, have received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies. Methods/Results: Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, duvelisib (IPI-145), which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of duvelisib in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM duvelisib for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to duvelisib, displaying about 20% decrease in viability at concentrations greater than 0.5μM; however, the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM duvelisib for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, duvelisib is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in the leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose-dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, duvelisib. Disclosures Dubovsky: Principia Inc.: Research Funding. Kutok:Infinity Pharmaceuticals, Inc.: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

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Expression of the Tigit/CD226/CD155 Receptors/Ligand System in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2019-128308 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5454-5454 ◽

Cited By ~ 2

Author(s):

Francesca Arruga ◽

Giulia Guerra ◽

Denis Baev ◽

Catherine Hoofd ◽

Marta Coscia ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

B Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Advisory Committees

Introduction: T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a surface receptor mainly expressed by CD8+, regulatory T lymphocytes and natural killer (NK) cells, but not by normal B cells. It performs as an inhibitory immune checkpoint, activated through binding of CD155. TIGIT competes with CD226 for CD155 binding, resulting in opposite outcomes: while CD226 enhances cytotoxicity of T lymphocytes and NK cells, TIGIT exerts immunosuppressive effects. Whether TIGIT engagement triggers an alternative signaling cascade, or whether it simply prevents CD226 activation, remains an open point. Tumor-infiltrating T lymphocytes generally express high levels of the molecule, together with the other checkpoint inhibitor PD-1. On this basis, antagonist antibodies targeting TIGIT are under evaluation to restore immunity and treat cancer patients, alone or in various combinations. Chronic lymphocytic leukemia (CLL), the most common adult leukemia, is characterized by a highly heterogeneous clinical outcome. Several molecular markers can help in stratifying patients, including the presence or absence of somatic mutations in B cell receptor, cytogenetic aberrations and single gene mutations. Interestingly, CLL cells express several T cell specific antigens, including CD5. A previous report indicates that, in CLL, TIGIT is expressed by circulating CD4+T cells, increasing during disease progression, while nothing is known about its expression on CLL cells. Aim:This work was undertaken with the aim of studying expression of the TIGIT/CD226/CD155 axis in CLL. Methods:We assembled a cohort of 101 primary CLL samples (40% females, mean age of 61). All patients were either untreated or had not received treatment in the 6 months prior to analysis. PBMC samples were tested for expression of TIGIT, CD155 and CD226 in both T and B subsets. A multiparametric flow cytometry strategy was designed, combining anti-TIGIT, anti-CD155 and anti-CD226 antibodies with a panel of B- (anti-CD19, anti-CD5, anti-CD38, anti-CD49d and anti-CD73) and T-mono/NK specific (anti-CD3, anti-CD8, anti-CD4, anti-CD14 and anti-CD56) markers. The number of TIGIT molecules on leukemic cells was estimated by interpolating values of mean fluorescence intensity (MFI) of each sample with that of PE-Quantibrite beads. Results:CLL cells heterogeneously express surface TIGIT, ranging from 0.2 to 81% (mean value 20%, median 10%, SEM ±2.145). The estimated number of molecules per cell was in the range of 32.5-3571 (mean 1140, median 841.1, SEM ±83.6). Expression of TIGIT was independent of gender or age at diagnosis and there was no correlation between TIGIT levels and lymphocyte counts in peripheral blood. In contrast, in this cohort of untreated patients, we observed a significantly lower TIGIT expression in samples with advanced disease (RAI III-IV) compared to early stages (RAI 0-I). Accordingly, low TIGIT associated with unmutated (UM) IGHVgenes and with an unfavorable FISH profile (trisomy 12, deletion 17 and deletion 11 vs. deletion 13 or normal karyotype). Lower, although not significant, TIGIT levels were observed in NOTCH1-mutated CLL samples (n=11) compared to counterpart (n=89). Looking at the T cell population, we observed overall higher TIGIT levels in the CD8+vs CD4+subset (mean %TIGIT+cells in CD8+56.7±1.8 vs 27.2±1.3 in CD4+). In line with reported observations, we found a modest but significant increase of TIGIT+T cells in advanced stage CLLs, at variance with what observed on the leukemic B cell side. Accordingly, we observed higher percentages of TIGIT+/CD4+cells in CLL samples carrying UM IGHVgenes. CD226 and CD155 were more homogeneously expressed in all subsets without significant differences, both in CLL and T cell components. Conclusions: This work shows that CLL cells express the immunomodulatory molecule TIGIT, particularly in the early stages of the disease in untreated patients. While further studies are needed to characterize its functional implications as well as treatment effect on TIGIT expression, it is tempting to speculate that TIGIT expression by CLL cells may serve to trigger an immunosuppressive behavior in these cells, which is no longer needed when the disease becomes advanced. This observation represents a starting point for future studies investigating the role of TIGIT in CLL and hints to a possible use of anti-TIGIT antibodies to target different cellular components of the disease. Disclosures Hoofd: iTeos Therapeutics: Employment. Coscia:Abbvie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Research Funding. Gaidano:Sunesys: Consultancy, Honoraria; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra-Zeneca: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Furman:Acerta Pharma: Consultancy; Beigene: Consultancy; Incyte: Consultancy; Janssen: Consultancy; Oncotracker: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Genentech: Consultancy; Abbvie: Consultancy; AstraZeneca: Consultancy. Deaglio:VelosBio Inc.: Research Funding; Verastem Inc: Research Funding; iTeos Therapeutics: Research Funding.

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Defective T cell-mediated, isotype-specific immunoglobulin regulation in B cell chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v71.4.1012.bloodjournal7141012 ◽

1988 ◽

Vol 71 (4) ◽

pp. 1012-1020 ◽

Cited By ~ 1

Author(s):

JS Moore ◽

MB Prystowsky ◽

RG Hoover ◽

EC Besa ◽

PC Nowell

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Activity ◽

Lymphocytic Leukemia ◽

Specific Immunoglobulin ◽

Cell Chronic Lymphocytic Leukemia

The consistent occurrence of T cell abnormalities in patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that the non- neoplastic host T cells may be involved in the pathogenesis of this B cell neoplasm. Because potential defects of immunoglobulin regulation are evident in B-CLL patients, we investigated one aspect of this by studying the T cell-mediated immunoglobulin isotype-specific immunoregulatory circuit in B-CLL. The existence of class-specific immunoglobulin regulatory mechanisms mediated by Fc receptor-bearing T cells (FcR + T) through soluble immunoglobulin binding factors (IgBFs) has been well established in many experimental systems. IgBFs can both suppress and enhance B cell activity in an isotype-specific manner. We investigated the apparently abnormal IgA regulation in a B-CLL patient (CLL249) whose B cells secrete primarily IgA in vitro. Enumeration of FcR + T cells showed a disproportionate increase in IgA FcR + T cells in the peripheral blood of this patient. Our studies showed that the neoplastic B cells were not intrinsically unresponsive to the suppressing component of IgABF produced from normal T cells, but rather the IgABF produced by the CLL249 host T cells was defective. CLL249 IgABF was unable to suppress IgA secretion by host or normal B cells and enhanced the in vitro proliferation of the host B cells. Size fractionation of both normal and CLL249 IgABF by gel-filtration high- performance liquid chromatography (HPLC) demonstrated differences in the ultraviolet-absorbing components of IgABF obtained from normal T cells v that from our patient with defective IgA regulation. Such T cell dysfunction may not be restricted to IgA regulation, since we have found similar expansion of isotype-specific FcR + T cells associated with expansion of the corresponding B cell clone in other patients with B-CLL. These data suggest that this T cell-mediated regulatory circuit could be significantly involved in the pathogenesis of B-CLL.

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Lymphocyte aggregation in response to adrenergic stimulation

Blood ◽

10.1182/blood.v71.5.1470.1470 ◽

1988 ◽

Vol 71 (5) ◽

pp. 1470-1474 ◽

Cited By ~ 5

Author(s):

DE Hammerschmidt ◽

C Jeanneret ◽

M Husak ◽

M Lobell ◽

HS Jacob

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Lymphocytic Leukemia ◽

Binding Studies ◽

Beta Adrenergic ◽

Cell Counts ◽

Significant Difference ◽

Induced Aggregation

Abstract A nonanemic chronic lymphocytic leukemia patient with nearly 500,000 lymphocytes/microL underwent leukapheresis when she presented with CNS symptoms and retinal vascular engorgement. Respiratory distress developed during the cell separator run, which led us to ask whether the procedure could have changed the adhesive properties of her cells. C5a desarginine, N-f-Met-Leu-Phe, adenosine diphosphate, and collagen all failed to aggregate her lymphocytes in vitro, but arachidonic acid, excess free calcium, and 4 mumol/L epinephrine did aggregate the cells. Arachidonate-induced aggregation appeared to be a toxic phenomenon: the ED50 for aggregation was statistically indistinguishable from that for cytotoxicity, and aspirin only mildly blunted the response. In contrast, epinephrine-induced aggregation was not associated with lactic dehydrogenase release or the loss of trypan blue exclusion and was blunted by propranolol; radiopindolol-binding studies confirmed the presence of a beta-adrenergic receptor. There were approximately 3,000 receptors/cell, with no statistically significant difference between normal and chronic lymphocytic leukemia B cells or between B cells and T cells (separated by rosetting techniques). The Kd for the B cells' receptor, however, was less than that for T cells by a factor of ten (P less than .01). We conclude that B cells may aggregate when stimulated and that they--like T cells--have beta-adrenergic receptors. Adrenergically mediated changes in B cell adhesiveness may play a role in regulating lymphocyte traffic; in the rare patient with truly enormous B cell counts, we postulate that they may be an occasional cause of morbidity.

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ZAP-70 Is Not Phosphorylated on the Activating Tyrosine Residue (Tyr319) in Chronic Lymphocytic Leukemia and B-Cell Lymphoma Cells.

Blood ◽

10.1182/blood.v106.11.178.178 ◽

2005 ◽

Vol 106 (11) ◽

pp. 178-178

Author(s):

Stefania Gobessi ◽

Aleksandar Petlickovski ◽

Luca Laurenti ◽

Dimitar G. Efremov

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Progressive Disease ◽

Cell Receptor ◽

Kinase Domain ◽

Lymphocytic Leukemia ◽

Bcr Signaling ◽

Cell Receptor Signaling

Abstract The protein tyrosine kinase ZAP-70 is expressed at high levels in leukemic B-cells from chronic lymphocytic leukemia (CLL) patients with progressive disease and short survival. ZAP-70 is a key component of the proximal T-cell receptor signaling pathway and is highly homologous to Syk, an important B-cell receptor signaling (BCR) molecule. Recent studies indicate that ZAP-70 may participate in BCR signaling as well, but the mechanism of action is still not well understood. In T-cells, upon TCR stimulation ZAP-70 becomes phosphorylated on Tyr319 by the Src-like kinase Lck, which results in the release of the ZAP-70 kinase domain from an autoinhibited state to a fully active conformation. The Tyr319 site in ZAP-70 corresponds to the Tyr352 site in Syk, which is phosphorylated in B-cells following BCR stimulation. We therefore investigated the activation status of ZAP-70 and Syk in BCR stimulated CLL B-cells, using phosphorylation of Tyr319 and Tyr352 as markers of their activation. Analysis of 10 ZAP-70-positive CLL samples by immunoblotting with the phospho-ZAP70Tyr319/SykTyr352 antibody revealed that ZAP-70 is not phosphorylated at this site either before or after BCR stimulation, although in control experiments with Jurkat T-cells ZAP-70 became phosphorylated on Tyr319 upon TCR stimulation. Moreover, the Tyr352 site in Syk was phosphorylated following BCR stimulation in 6 of the 10 CLL B-cell samples. To further investigate the reasons for the unexpected lack of ZAP-70 activation in CLL B-cells, we produced stable transfectants of the BJAB lymphoma B-cell line that expressed ZAP-70 at levels similar to those found in CLL cases with progressive disease. In agreement with the CLL B-cell experiments, the Tyr319 site in ZAP-70 was not phosphorylated either before or after BCR stimulation. Since phosphorylation of Tyr319 is Lck-dependent in T-cells, and this kinase is expressed also in CLL B-cells, we ectopically expressed Lck in the ZAP-70-positive BJAB clones. Again, the Tyr319 site was not phosphorylated, indicating that ZAP-70 does not undergo activation of the kinase domain also in this cellular system. In contrast, BCR crosslinking in BJAB cells induced significant phosphorylation of Tyr352 in Syk, which was further enhanced in the clones that coexpressed ZAP-70. Furthermore, analysis of downstream signaling pathways following BCR stimulation showed stronger and prolonged activation of ERK and to a lesser extent Akt in the ZAP-70 positive clones, whereas no difference was observed in terms of activation of PLC-γ 2, JNK and degradation of the NF-kB inhibitor IkB. These data indicate that ZAP-70 does not undergo full activation in B-cells, but can still enhance activation of certain downstream BCR signaling pathways, possibly by affecting the activity of the related PTK Syk.

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Different Expression of FcgammaRIIb in Chronic Lymphocytic Leukemia and Human Normal B Lymphocytes

Blood ◽

10.1182/blood.v112.11.3134.3134 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3134-3134

Author(s):

Carol Moreno ◽

Rajendra Damle ◽

Sonia Jansa ◽

Gerardo Ferrer ◽

Pau Abrisqueta ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

B Lymphocytes ◽

Surface Membrane ◽

Memory B Cells ◽

Lymphocytic Leukemia ◽

Cd38 Expression ◽

B Cell Subsets

Abstract The Fcgamma receptors (FcγRs) are a family of molecules that modulate immune responses. FcγRIIb is an inhibitory FcγR that bears immunoreceptor tyrosine-based inhibitory motifs which transduce inhibitory signals on coligation with the surface membrane Ig of the B-cell antigen receptor (BCR). The role of FcγRIIb in controlling B cell activation through inhibition of BCR signaling has been extensively studied in animal models. Nevertheless, data on FcγRIIb are scant in human normal and neoplastic B cells, this being due to the lack of a specific antibody for human FcγRIIb. Consequently, there is little information on this receptor in chronic lymphocytic leukemia (CLL). Considering the activated nature of CLL cells and the central role of the BCR in the biology of the disease, studies of FcγRs are warranted. We used a novel specific mAb directly conjugated with Alexa 488 fluorophore that solely reacts with the human FcγRIIb (MacroGenics, Inc.) to investigate the receptors expression on CLL and normal human B cells. The study population included 84 patients with CLL and 24 age- and sex-matched controls. FcγRIIb expression was assessed as the mean fluorescence intensity (MFI) of surface membrane staining. In CLL cells, FcγRIIb was measured on CD19+CD5+ cells in combination with CD38, CD49d or CD69. Normal B cells were immunostained for CD19, CD5, IgD and CD38 expression and B cell subsets: naïve (IgD+CD38−), activated (IgD+CD38+) and memory B cells (IgD−CD38−) were studied for their relative expression of FcγRIIb. FcγRIIb expression was found significantly higher in naïve B cells compared to activated and memory B cells [median MFI: 17420 (11960–21180) vs. 11.140 (7899–16970) and 11.830 (6984–17100); p<0.001]. Significant differences were also observed between CD5− and CD5+ normal B cells. In contrast, FcγRIIb expression was lower in CLL cells than in CD5+ and CD5− normal B lymphocytes [median MFI: 6901(1034–42600), 10180 (5856–14820) and 12120 (7776–16040); p<0.05)]. Interestingly, FcγRIIb expression was variable within individual CLL clones, this being higher in CD38+ and CD49d+ cells than in CD38− and CD49d− cells (p<0.05). Furthermore, the highest density of FcγRIIb was observed on those cells which coexpressed CD38 and CD49d. In contrast, no significant differences were observed between FcγRIIb and the expression of the activation antigen CD69. Although CD69 and CD38 expression was significantly higher on unmutated IGHV cases, no correlation was found between FcγRIIb levels and IGHV mutational status. Similarly, there was no correlation between FcγRIIb and other poor prognostic variables such as ZAP-70 (≥20%), CD38 (≥ 30%) or high risk cytogenetics. Nevertheless, cases with ≥ 30% CD49d+ cells had higher FcγRIIb expression than those with <30% CD49d+ cells (p=0.006). The findings presented in this study suggest a hierarchy of FcγRIIb expression in normal B-cells, CLL cells and their subpopulations: circulating normal CD5− B cells > circulating normal CD5+ B cells > circulating CD5+ CLL B cells. In addition, although FcγRIIb is present on all normal B cell subsets its expression is higher in naïve B cells. Furthermore, in CLL FcγRIIb density is greater in CD38+ and CD49d+ cells within the clone. Although CD49d and FcγRIIb on CLL clones is linked in a direct manner, there is no relationship with FcγRIIb density and IGHV mutations, ZAP-70, CD38 and unfavorable cytogenetic markers. Finally, the relationship between FcγRIIb expression on CLL cells and functional responses to BCR and other receptor-mediated signals deserve further investigation.

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Increased Expression of PD-1 on CD4+ T Cells from Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v112.11.4154.4154 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4154-4154

Author(s):

Mary M Sartor ◽

David J Gottlieb

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Nk Cells ◽

Cd4 T Cells ◽

Mononuclear Cells ◽

Lymphocytic Leukemia ◽

Effector Memory ◽

Cd4 Cells ◽

Normal Peripheral Blood

Abstract Although the predominant finding in patients with chronic lymphocytic leukemia (CLL) is an expansion of monoclonal B lymphocytes, a polyclonal expansion of T cells co-exists in CLL patients. Allogenic stem cell transplants for CLL suggest that a significant graft versus leukaemia effect mediated through recognition of minor MHC or leukaemia specific antigens can be achieved. Since it appears that the immune system and probably T cells recognise CLL cells, it is possible that one or more T cell defects might contribute to the initiation or maintenance of a clone of CLL lymphocytes. PD-1 is a coinhibitory molecule that is expressed on T cells in patients with chronic viral infections. It has been suggested that PD-1 expression might be a marker of cell exhaustion due to antigenic overstimulation. We examined the expression of PD-1 and its naturally occurring ligands PD-L1 and PD-L2 on both B and T cells in patients with CLL and compared this with expression on normal peripheral blood mononuclear cells. We found that PD-1 was expressed on over 10% of CD4+ T cells in 7 of 9 cases of CLL (mean 22±16%) but not on CD4+ T cells in any of 9 normal donors (mean 0±0%), p=0.0009. There was no difference in PD-1 expression on CD8+ or CD14+ PBMCs from CLL patients and normal donors (for CD8+ 24±21% and 19±16% for CLL and normals; for CD14+ 58±16% and 71±31% for CLL and normals). More than 10% of CD5+/19+ CLL cells expressed PD-1 in 7 of 10 cases (mean 18±18%) while more than 10% of normal B cells from 6 of 7 donors also expressed PD-1 (mean 49±30%). We examined the expression of PD-1 on naïve, central memory, effector memory and terminally differentiated subsets of CD4+ cells (CD62L+CD45RA+, CD62L+CD45RA−, CD62L−CD45RA− and CD62L−CD45RA+ respectively) from CLL patients and normal donors. The expression of PD-1 was higher on CD4+ cells from CLL patients in all subsets. The effect was most prominent in the effector memory subset (mean 54±4% for CLL patients versus 26±17% for normal donors, p=0.02). We looked for expression of PD-L1 and PD-L2 on T cells, B cells, monocytes and NK cells from CLL patients and normal donors. PD-L1 was only expressed on monocytes (mean 30±23%) and NK cells (mean 14±19%) from CLL patients and on monocytes from normal donors (mean 35±26%). There was no expression of PD-L2 on any cell type in either CLL patients or normal donors. We conclude that there is increased expression of the co-inhibitory molecule PD-1 on CD4+ T cells in patients with CLL. Ligation of PD-1 by PD-L1 expressed on monocytes or NK cells could inhibit immune responses to tumor and infectious antigens leading to persistence of clonally expanded cells and predisposition to opportunistic pathogens.

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Leukemia-associated monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2002-03-0746 ◽

2003 ◽

Vol 101 (3) ◽

pp. 1063-1070 ◽

Cited By ~ 50

Author(s):

Mohammad-Reza Rezvany ◽

Mahmood Jeddi-Tehrani ◽

Hans Wigzell ◽

Anders Österborg ◽

Håkan Mellstedt

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cd8 T Cells ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Cell Chronic Lymphocytic Leukemia

Abstract T-cell receptor–B-variable (TCR-BV) gene usage and the CDR3 size distribution pattern were analyzed by reverse transcription–polymerase chain reaction (RT-PCR) in patients with B-cell chronic lymphocytic leukemia (B-CLL) to assess the T-cell repertoire. The use of TCR-BV families in CD4 and CD8 T cells stimulated with autologous activated leukemic cells was compared with that of freshly obtained blood T cells. Overexpression of individual TCR-BV families was found in freshly isolated CD4 and CD8 T cells. Polyclonal, oligoclonal, and monoclonal TCR-CDR3 patterns were seen within such overexpressed native CD4 and CD8 TCR-BV families. In nonoverexpressed TCR-BV families, monoclonal and oligoclonal populations were noted only within the CD8 subset. After in vitro stimulation of T cells with autologous leukemic B cells, analyses of the CDR3 length patterns showed that in expanded TCR-BV populations, polyclonal patterns frequently shifted toward a monoclonal/oligoclonal profile, whereas largely monoclonal patterns in native overexpressed TCR-BV subsets remained monoclonal. Seventy-five percent of CD8 expansions found in freshly obtained CD8 T cells further expanded on in vitro stimulation with autologous leukemic B cells. This suggests a memory status of such cells. In contrast, the unusually high frequency of CD4 T-cell expansions found in freshly isolated peripheral blood cells did not correlate positively to in vitro stimulation as only 1 of 9 expansions continued to expand. Our data suggest that leukemia cell–specific memory CD4 and CD8 T cells are present in vivo of patients with CLL and that several leukemia cell–associated antigens/epitopes are recognized by the patients' immune system, indicating that whole leukemia cells might be of preference for vaccine development.

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Suppression of Anti-Tumor Immunity in Chronic Lymphocytic Leukemia Via Interleukin-10 Production

Blood ◽

10.1182/blood.v128.22.3215.3215 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3215-3215

Author(s):

Sara S Alhakeem ◽

Mary K McKenna ◽

Sunil K Nooti ◽

Karine Z Oben ◽

Vivek M Rangnekar ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Tumor Immunity ◽

Lymphocytic Leukemia ◽

Clonal Variation ◽

Bcr Signaling

Abstract The most common human leukemia is B-cell chronic lymphocytic leukemia (B-CLL), which is characterized by a progressive accumulation of abnormal B-lymphocytes in blood, bone marrow and secondary lymphoid organs. Typically disease progression is slow, but as the number of leukemic cells increases, they interfere with the production of other important blood cells, causing the patients to be in an immunosuppressive state. To study the basis of this immunoregulation, we used cells from the transgenic Eμ-Tcl1 mouse, which spontaneously develop B-CLL due to a B-cell specific expression of the oncogene, Tcl1. Previously we showed that Eμ-Tcl1 CLL cells constitutively produce an anti-inflammatory cytokine, IL-10. Here we studied the role of IL-10 in CLL cell survival in vitro and the development of CLL in vivo. We found that neutralization of IL-I0 using anti-IL-10 antibodies or blocking the IL-10 receptor (IL-10R) using anti-IL-10R antibodies did not affect the survival of CLL cells in vitro. On the other hand, adoptively transferred Eμ-Tcl1 cells grew at a slower rate in IL-10R KO mice vs. wild type (WT) mice. There was a significant reduction in CLL cell engraftment in the spleen, bone marrow, peritoneal cavity and liver of the IL-10R KO compared to WT mice. Further studies revealed that IL-10 could be playing a role in the tumor microenvironment possibly by affecting anti-tumor immunity. This was seen by a reduction in the activation of CD8+ T cells as well as a significantly lower production of IFN-γ by CD4+ T cells purified from CLL-injected WT mice compared to those purified from CLL-injected IL-10R KO mice. These studies demonstrate that CLL cells suppress host anti-tumor immunity via IL-10 production. This led us to investigate possible mechanisms by which IL-10 is produced. We found a novel role of B-cell receptor (BCR) signaling pathway in constitutive IL-10 secretion. Inhibition of Src or Syk family kinases reduces the constitutive IL-10 production by Eμ-Tcl1 cells in a dose dependent manner. In addition, we found that Eμ-Tcl1 CLL cells exhibit clonal variation in their IL-10 production in response to BCR cross-linking. Further studies are being performed to understand the mechanisms by which BCR signaling affects IL-10 production. Disclosures No relevant conflicts of interest to declare.

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Indoleamine 2,3-Dioxygenase Mediates Survival of Chronic Lymphocytic Leukemia B Cells through Aryl Hydrocarbon Receptor By Inducing Mcl1

Blood ◽

10.1182/blood-2020-142345 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 19-19

Author(s):

Claudio Giacinto Atene ◽

Rossana Maffei ◽

Stefania Fiorcari ◽

Silvia Martinelli ◽

Patrizia Zucchini ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Aryl Hydrocarbon Receptor ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Strong Increase ◽

Indoleamine 2,3 Dioxygenase ◽

Aryl Hydrocarbon

Introduction: Chronic lymphocytic leukemia (CLL) is a dynamic disease in which monoclonal B cells proliferate within the pseudo-follicular centers in lymphoid organs and then they accumulate due to an intrinsic defect of apoptosis. Leukemic cells are considered as "addicted to the host" since extrinsic signals from the microenvironment strongly influence the establishment of a progressive immunosuppression for malignant cell growth and survival. The cytoplasmic enzyme indoleamine 2,3-dioxygenase (IDO) mediates the conversion of the essential amino acid tryptophan (Trp) into metabolic byproducts such as kynurenine (Kyn). Kyn and other secondary metabolites are endogenous activators of the aryl hydrocarbon receptor (AHR), a ligand-controlled transcription factor that mediates cellular responses to toxins or endogenous ligands. The IDO-Kyn-AHR axis plays important roles in carcinogenesis and cancer progression. The mechanisms that promote inflammation around tumor tissues and determine immune tolerance consist in Trp depletion, which induces T cell apoptosis, and in Kyn-mediated AHR activation that inhibits effector T cells and promotes regulatory T cells differentiation. IDO protein is expressed in human hematologic malignancies and its level is correlated with a poor prognosis and chemoresistance. The IDO activity, measured as the Kyn/Trp ratio, was reported to be increased in CLL cases comparing to normal controls. Aim: We wondered to characterize the expression of IDO and AHR in CLL patients and to dissect the biological function of the IDO-Kyn-AHR axis. Methods: Gene transcription and protein expression were evaluated by real time PCR and western blot. Enzymatic activity was assessed through ELISA. Survival was measured with PI/annexin V assay. Overexpression and silencing of target genes was obtained by nucleofection. Results: Firstly, we observed that CLL cells expressed both IDO and AHR at variable levels. Moreover, we found that several microenvironmental signals such as IFNγ, LPS, anti-IgM, CpG oligo DNA, CD40L and TNFα were able to up-regulate IDO and AHR mRNA and protein. To characterize the pathways able to mediate IDO expression, we stimulated CLL cells with IFNγ and CD40L. Using ruxolitinib, an inhibitor of JAK-STAT pathway, we found that IFNγ induced IDO through STAT1 signaling. Again, CD40L stimulation determined IDO overexpression through the non-canonical NF-kB pathway, as assessed by treating cells with NF-κB inducing kinase inhibitor, NIK SMI1. We also confirmed that IFNγ-treated CLL cells were able to produce a functional IDO enzyme by measuring Kyn production and Trp consumption by ELISA. The strong increase in the Kyn/Trp ratio induced by IFNγ was significantly reduced by ruxolitinib treatment. To verify if Kyn produced by CLL cells could act through an autocrine loop on AHR, leukemic cells were treated with Kyn. We observed that Kyn mediated AHR translocation from the cytoplasm to the nuclei, inducing its activation as assessed by up-regulation of CYP1A1, a known AHR target gene. Of interest, we found that Kyn treatment improved CLL cells survival. Analyzing the anti-apoptotic proteins of the Bcl2 family after Kyn treatment, we found the induction of Mcl1, that was affected by adding CH-223191, an antagonist of AHR. Moreover, we transfected CLL cells with an IDO vector. The up-regulation of IDO increased CLL cells survival through the induction of Mcl1. Accordingly, when CLL cells were silenced for AHR, we observed a reduction of their survival. Conclusion: Our data demonstrate the constitutive expression of IDO and AHR in CLL cells. Furthermore, the tumor microenvironment promotes the induction of IDO and AHR through a complex signaling crosstalk with leukemic cells. Our findings underline that IDO-Kyn-AHR axis is active in CLL cells and promotes Mcl1 expression, sustaining the survival of CLL cells. Disclosures Luppi: Gilead Sci: Consultancy, Speakers Bureau; MSD: Consultancy; Sanofi: Consultancy; Abbvie: Consultancy; Daiichi-Sankyo: Consultancy; Novartis: Consultancy, Speakers Bureau. Marasca:Gilead Sci: Honoraria, Research Funding; Roche: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Janssen: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria.

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Lymphocyte aggregation in response to adrenergic stimulation

Blood ◽

10.1182/blood.v71.5.1470.bloodjournal7151470 ◽

1988 ◽

Vol 71 (5) ◽

pp. 1470-1474

Author(s):

DE Hammerschmidt ◽

C Jeanneret ◽

M Husak ◽

M Lobell ◽

HS Jacob

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Lymphocytic Leukemia ◽

Binding Studies ◽

Beta Adrenergic ◽

Cell Counts ◽

Significant Difference ◽

Induced Aggregation

A nonanemic chronic lymphocytic leukemia patient with nearly 500,000 lymphocytes/microL underwent leukapheresis when she presented with CNS symptoms and retinal vascular engorgement. Respiratory distress developed during the cell separator run, which led us to ask whether the procedure could have changed the adhesive properties of her cells. C5a desarginine, N-f-Met-Leu-Phe, adenosine diphosphate, and collagen all failed to aggregate her lymphocytes in vitro, but arachidonic acid, excess free calcium, and 4 mumol/L epinephrine did aggregate the cells. Arachidonate-induced aggregation appeared to be a toxic phenomenon: the ED50 for aggregation was statistically indistinguishable from that for cytotoxicity, and aspirin only mildly blunted the response. In contrast, epinephrine-induced aggregation was not associated with lactic dehydrogenase release or the loss of trypan blue exclusion and was blunted by propranolol; radiopindolol-binding studies confirmed the presence of a beta-adrenergic receptor. There were approximately 3,000 receptors/cell, with no statistically significant difference between normal and chronic lymphocytic leukemia B cells or between B cells and T cells (separated by rosetting techniques). The Kd for the B cells' receptor, however, was less than that for T cells by a factor of ten (P less than .01). We conclude that B cells may aggregate when stimulated and that they--like T cells--have beta-adrenergic receptors. Adrenergically mediated changes in B cell adhesiveness may play a role in regulating lymphocyte traffic; in the rare patient with truly enormous B cell counts, we postulate that they may be an occasional cause of morbidity.

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