Lenalidomide Treatment Enhances Immunological Synapse Formation of Cord Blood Natural Killer Cells with B Cells Derived From Chronic Lymphocytic Leukemia

Abstract Abstract 1794 Immune dysfunction is a hallmark of chronic lymphocytic leukemia (CLL) including suppressed humoral and cell-mediated immune responses. The immunomodulatory agent lenalidomide has shown effective clinical activity against CLL, but its mechanism of action is poorly understood. Previous work has demonstrated that the T cell immunological synapse and functional defects in CLL can be reversed following lenalidomide treatment (J Clin Invest. 2008; 118). Polymerization of F-actin at the NK cell immunological synapse with tumor cells is required for signaling molecules to assemble and regulate NK cell activation and effector function. Confocal microscopy was used to visualize and analyze F-actin polymerization at the immune synapse between NK cells and CLL cells. The impaired immune synapse defect identified in CLL could result from not only the defects of CLL B cells but also defects in the CLL NK cells or a combination of both factors. To investigate the contribution of each factor, we examined synapse formation in experiments using CLL B cells with autologous CLL NK cells or healthy allogeneic NK cells. Conjugates formed with healthy NK cells and CLL B cells exhibited a strong band of F-actin at the immune synapse. In contrast, significantly less actin polymerization at the synapse was observed in autologous CLL NK cells and CLL B cells (P < 0.01). These results indicate CLL B cells, together with CLL NK cells contributed to the immune dysfunction in CLL. As autologous NK cell function in CLL is suppressed, we investigated the utility of CB as a potential functional source of NK cells for CLL immunotherapy. We examined the effect of lenalidomide on NK cell immune synapse function with CLL B cells acting as APCs. We demonstrated that ex vivo treatment of CLL cells with lenalidomide (500 ng/ml) for 48 hours caused a significant increase in the ability of autologous CLL NK cells to form F-actin immune synapses with CLL B cells. The same treatment of CLL B cells also significantly increased the ability of CB-NK cells to form F-actin immunological synapses with these treated CLL B cells compared to untreated CLL B cells (33.6% to 67.3%, P < 0.01, n=6). Our results also show that lenalidomide treatment of autologous NK cells from CLL patients enhanced synapse formation with treated CLL cells compared to experiments using untreated NK cells, but with reduced function compared to CB NK cells. Of note, lenalidomide treatment was shown to increase the recruitment of the signaling molecule Lck to NK cell:CLL cell synapse site, that is known to regulate lytic synapse function. Importantly, lenalidomide treatment significantly increased CB-NK killing of CLL B cells compared to untreated CLL B cells (20.5% versus 48.2%, E:T ratio of 10:1, n = 6, p < 0.001). These results provide insight into the potential mechanism of action of lenalidomide's anti-leukemic function – priming CLL tumor cells for enhanced NK cell lytic synapse formation and effector function. In addition, the data suggests that immunotherapeutic strategies utilizing a combination of CB-NK cells and lenalidomide has an enhanced clinical efficacy in CLL. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

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Compared to Adult Peripheral Blood T Cells, Cord Blood T Cells Show Enhanced Immunological Recognition of Chronic Lymphocytic Leukemia Tumor Cells.

Blood ◽

10.1182/blood.v112.11.2333.2333 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2333-2333

Author(s):

Alan G. Ramsay ◽

Dong-Xia Xing ◽

William K. Decker ◽

Jared K. Burks ◽

William G. Wierda ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cord Blood ◽

Peripheral Blood ◽

Actin Polymerization ◽

Synapse Formation ◽

Immunological Synapse ◽

Lymphocytic Leukemia ◽

Adult Peripheral Blood

Abstract Following allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion (DLI) from adult peripheral blood (APB), chronic lymphocytic leukemia (CLL) cells are good targets of a graft-versus-leukemia effect. However, some patients eligible for this treatment do not have a suitable allogeneic donor and CLL B cells have been shown to be dysfunctional antigen-presenting cells (APCs) for allogeneic APB T cells. As a result, allogeneic APB T cells show suppressed immunological synapse formation with CLL cells. Umbilical cord blood (CB) is a promising source of hematopoietic cells for allogeneic transplantation and can be obtained from matched unrelated donors with greater tolerance for incompletely HLA-matched recipients. Moreover, we have successfully expanded CB T cells ex vivo (anti-CD3/CD28 beads and rIL-2) using a protocol that retains a naïve and diverse immune population including central memory cells. In this present study we used confocal microscopy to visualize F-actin polymerization to assess immunological synapse formation of CB T cells compared to APB T cells with CLL B cells with and without superantigen as APCs. Our results identify the ability of unexpanded and expanded CB CD4 and CD8 T cells to form F-actin immune synapses with CLL B cells and of note, CB was more effective than unexpanded or expanded APB T cells (p<0.05). Of interest, the expansion protocol maintained immune synapse formation with a trend towards increased F-actin polymerization. As control, we examined the ability of unexpanded and expanded T cells to form F-actin synapses with allogeneic healthy B cells with or without superantigen as APCs and found no significant difference between CB and APB as a source of T cells. Our results demonstrate that CB T cells have an enhanced ability to recognize CLL B cells as allogeneic APCs compared to APB T cells and provide important and exciting pre-clinical data for the potential use of expanded CB T cells in the setting of CB transplantation in CLL.

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Lenalidomide Alone and in Combination with Rituximab Enhances NK Cell Immune Synapse Formation in Chronic Lymphocytic Leukemia (CLL) Cells in Vitro through Activation of Rho and Rac1 GTPases.

Blood ◽

10.1182/blood.v114.22.3441.3441 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3441-3441 ◽

Cited By ~ 5

Author(s):

Svetlana Gaidarova ◽

JianWu Li ◽

Laura G Corral ◽

Emilia Glezer ◽

Peter H Schafer ◽

...

Keyword(s):

B Cells ◽

Nk Cells ◽

Synapse Formation ◽

Nk Cell ◽

Employment Equity ◽

Immune Synapse ◽

Immunological Synapses ◽

Immune Synapses ◽

Cd20 Expression ◽

Equity Ownership

Abstract Abstract 3441 Poster Board III-329 Background CLL is characterized by the progressive accumulation of monoclonal B lymphocytes. One theory to explain how CLL cells avoid elimination through immune surveillance mechanisms is through a defect in the ability of T-cells to form immunological synapses with antigen-presenting tumor B-cells (Ramsay et al JCI 2008). Lenalidomide is an immunomodulatory agent with clinical activity in the treatment of B-cell malignancies. Recent laboratory studies showed that lenalidomide not only stimulates T- and natural killer (NK)-cell-mediated ADCC, it also restores the T-cell-mediated ability to form immunological synapses with CLL tumor cells. Since NK cells also exert cytotoxicity through immune synapse formation, here we explore how lenalidomide affects NK-cell-mediated cytotoxicity mechanisms and whether this activity is altered in the presence of rituximab since published studies showed that lenalidomide-pretreated B-cells have a down-regulated surface CD20 expression. Further, we investigated the molecular events associated with immune synapse formation and the effect of lenalidomide. Methods Immune synapse formation was assessed in NK cells (from healthy donors PBMCs) co-cultured with either B-CLL cells derived from pts or with K562 cells (positive control). Cells were fixed and the ability to form synapses was assessed via immunohistochemisty co-staining for either F-actin and CD2, or F-actin and perforin (a cytolytic protein found in NK cells). Synapse formation was visualized by microscopy and measured via relative mean fluorescent intensity. Activity of RhoA, Rac1, Cdc42 were measured using Rho GTPases assay kits. Inhibition of lenalidomide-mediated immune synapse activity was assayed using the cell permeable Rho inhibitor C3 (0.5 mM). Flow cytometry was used to measure changes in surface CD20 and CD54 (ICAM-1) expression in B-CLL samples from 3 pts after treatment with lenalidomide. Results Lenalidomide induced the formation of immunological synapses between NK cells and primary B-CLL cells (p<.01) or the K562 cell line. Lenalidomide activated NK cells regardless of the presence of target cells, as measured by F-actin and perforin staining. RhoA and Rac1 were activated at the immunological synapse in the presence of lenalidomide. Inhibition of RhoA by the C3 inhibitor blocked F-actin localization, as well as perforin accumulation induced by lenalidomide at cell-cell contact sites, indicating inhibition of immune synapses and the associated cytolytic activity. This was also observed with Rac1 inhibition, but to a lesser degree than with RhoA inhibition. Functionality of formed synapses was confirmed by co-localization of F-actin and perforin at the synapse sites. 3 CLL pt samples treated ex vivo with lenalidomide demonstrated variable changes in CD20 expression: a 20-30% decrease in CD20 expression was observed in 2 B-CLL pt samples, whereas CD20 levels remained unchanged in the third. In the presence of rituximab, lenalidomide-induced synapse formation between NK cells and B-cells from CLL patients was further enhanced. This was accompanied by upregulation of costimulatory and adhesion molecule CD54 on B-CLL cells suggesting increased antigen presentation, which might contribute to the increased synapse formation. Conclusion Lenalidomide can directly activate NK-cell-mediated anti-tumor activity through enhanced formation of immune synapses via the regulation of Rho and Rac1 GTPases and the cytoskeleton. Despite some down-modulation of CD20 expression in lenalidomide-pretreated B-CLL cells, the immune synapse activity increases when lenalidomide is combined with rituximab suggesting that combining lenalidomide and anti-CD20 antibodies warrants exploration in the CLL clinical setting. Disclosures Gaidarova: Celgene: Employment, Equity Ownership. Li:Celgene: Employment. Corral:Celgene: Employment. Glezer:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment. Xie:Celgene: Employment. Lopez-Girona:Celgene: Employment.

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Ex Vivo IL-2 Expansion of CB-NK Cells Promotes Synergistic LFA-1 and CD2 Engagement at the NK Cell Lytic Immune Synapse; Implications for Adoptive CB-NK Cell Therapy in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.3029.3029 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3029-3029

Author(s):

Dongxia Xing ◽

Alan G. Ramsay ◽

William Decker ◽

Sufang Li ◽

Simon Robinson ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Synapse Formation ◽

Immunological Synapse ◽

Nk Cell ◽

Ex Vivo ◽

Immune Therapy ◽

Cytolytic Activity ◽

Lymphocyte Function ◽

Immune Synapse

Abstract Abstract 3029 Poster Board II-1005 Donor peripheral blood (PB) natural killer (NK) cell have shown clinical promise in cancer immunotherapy. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. Umbilical cord blood (CB) represents an important alternative source of NK cells for adoptive immune therapy. We first demonstrated that cord blood (CB) derived NK cells have poor cytolytic activity and deficiency in the formation of the F-actin immunological synapse with HLA class I deficient target K562 cells and primary AML blasts compared to PB-NK cells. In this study, we explored the cellular mechanism of these dysfunctions. We hypothesized that adhesion and signaling molecules may be defective in unmanipulated CB NK cells. Activating receptor Both CD2 and the integrin lymphocyte function-associated antigen (LFA-1) play important roles in both T lymphocyte and NK cell immune synapse formation and their trafficking to the immune synapse regulates both T and NK cell function. We now show that unmanipulated CB NK cells exhibit reduced LFA-1 mediated adhesion to mobilized ICAM-1 compared to IL-2 expanded CB NK cells (CB NK 29.7+/- 3.2 %, vs expanded CB NK 78.5+/- 6.1%, n=6). Moreover, unmanipulated CB-NK cells demonstrated reduced surface expression of CD2, and high affintyLFA-1 detected by the specific antibody (MHM24). There was decreased recruitment of CD2 and LFA-1 to the NK cell immune synapse site as quantified by confocal microscope analysis (RRI CD2 CB NK 2.02 vs PB NK 4.98, n=3). Furthermore, defective LFA-1 trafficking lead to a decrease in downstream cytotoxic granules that traffic to the immunological synapse as demonstrated by decreased perforin trafficking to the CB-NK synapse site (> 60% reduction).We next wanted to confirm that CD2 or LFA-1 play a role in restoring the immune synapseformation for IL-2 expanded CB NK cells. We incubated expanded CB NK cells with blocking antibodies specific for LFA-1 or CD2 prior to conjugation to the K562 target cells. After CD2 or LFA-1 blocking there was decreased synapse formation, with a resultant decrease in cytotoxic function. When monoclonal antibodies against both CD2 and LFA-1 were used there was significant blockade of the formation of the immune synapse, and a marked reduction of CB NK cell cytolytic activity (Mean specific lysis of K562 targets at E:T ratio 20:1 was 81% IgG control vs 22% with anti-CD2; and 29% with anti-LFA-1, n=6, P<0.001). This data shows that CD2 and LFA-1 are defective in unmanipulated CB NK cells resulting in impaired immune synapse formation. In contrast, ex vivo IL-2 expansion of CB-NK cells enhanced lytic synapse formation with the synergistic repair of CD2 and LFA-1 localization and activity. We believe our results provide important mechanistic insights for the potential use of IL-2 expanded CB-derived NK cells for adoptive immune therapy in leukemia. Disclosures No relevant conflicts of interest to declare.

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Impaired Actin Polymerization Results in Defective Immunological Synapse Formation in T Cells in Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v110.11.338.338 ◽

2007 ◽

Vol 110 (11) ◽

pp. 338-338 ◽

Cited By ~ 1

Author(s):

Alan G. Ramsay ◽

Abigail M. Lee ◽

John G. Gribben

Keyword(s):

Immune Deficiency ◽

T Cells ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Molecular Basis ◽

Actin Polymerization ◽

Immunological Synapse ◽

Lymphocytic Leukemia ◽

Donor T Cells

Abstract Cancer is associated with immune deficiency, but the molecular basis for this is poorly defined. We have previously demonstrated that multiple gene expression abnormalities are induced in patients with chronic lymphocytic leukemia (CLL) including defects within the actin cytoskeleton formation pathways. Based on this data, we hypothesized that failure of actin polymerization would result in defects in the formation of the immunological synapse (IS) which is critical for T cell activation and effector function. To assess this, actin polymerization at the IS in T cells in response to superantigen-pulsed B cells (APCs) was visualized using confocal microscopy. We observed significantly reduced ability to polymerize actin at the IS (> 50% reduction) in autologous CD4 and CD8 T cells from previously untreated CLL patients compared to age-matched healthy donors (p<0.05). Since reduced IS formation could result from defects in T cells, APCs or both, we examined IS formation in mixing experiments using T cells or APCs from leukemic patients with healthy allogeneic cells. These experiments demonstrated impaired IS formation using T cells from patients with CLL (p<0.01) or CLL cells as APCs (p<0.01), in keeping with defects in both T cells and APC function of CLL cells. We further postulated that interaction of CLL cells with healthy T cells would induce similar changes. Healthy allogeneic T cells were co-cultured for 48 hours with either allogeneic CLL cells or healthy B cells. Co-culture with CLL cells resulted in subsequent significant impairment in IS formation of the T cells with healthy superantigen pulsed APCs (p<0.01). Blocking experiments using anti-LFA-1 and anti-ICAM1 monoclonal antibodies with CLL B cells prevented subsequent actin remodelling impairment at the IS in the healthy allogeneic donor T cells. Further evidence that direct cell contact with CLL cells and not soluble factors is required to induce this T cell immune defect was provided by the finding that there was no impairment on IS formation when the T cells were co-cultured with CLL cells in transwell culture assays. The finding that direct contact of CLL cells with allogeneic T cells induces impairment in IS formation is relevant for the use of donor lymphocyte infusions in the setting of bulk disease. Co-localization experiments assessed by confocal microscopy suggest that the molecular basis for the defective T cells function stems from inability in T cells from CLL patients to recruit key proteins to the IS efficiently compared to healthy donor T cells. Greater than 50% reduction in co-localization at the IS was seen for dynamin 2, filamin A and LFA-1 integrin (p<0.05). These assays provide a rapid and simple method to assess T cell impairment in cancer and can be used to determine if steps to attempt to improve defective T cell function in cancer are successful. The finding of impaired IS formation as a key T cell defect in these cancer bearing patients has implications for both autologous and allogeneic immunotherapy approaches and identify both IS formation and the molecules regulating its organisation as potential functional markers and targets for the reversal of immune deficiency in cancer.

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Natural Killer Cell Dysfunction in Chronic Lymphocytic Leukemia Is Associated with Loss of the Mature KIR3DL1+ Subset

Blood ◽

10.1182/blood.v124.21.3318.3318 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3318-3318 ◽

Cited By ~ 1

Author(s):

Alexander W. MacFarlane ◽

Mowafaq Jillab ◽

Mitchell R Smith ◽

R. Katherine Alpaugh ◽

Marion E. Cole ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Nk Cells ◽

Peripheral Blood ◽

Cell Function ◽

Nk Cell ◽

Tumor Burden ◽

Lymphocytic Leukemia ◽

Healthy Controls

Abstract Background: B-cell chronic lymphocytic leukemia (CLL) is a common blood cancer characterized by high prevalence of malignant B cells in peripheral blood. Small lymphocytic lymphoma (SLL) is considered to be a different presentation of the same disease, with the malignant B cells primarily localized in lymph nodes. Natural killer (NK) cells are innate immune effectors that can spontaneously identify and kill malignant cells, especially hematopoietic cancers. In peripheral blood of CLL patients, NK cells are chronically exposed to significant tumor burden, which is predicted to influence their phenotype and function. Effective NK cell function may be particularly beneficial in CLL patients, since commonly-used monoclonal antibody therapies (e.g. rituximab, alemtuzumab) rely at least partially on ADCC-mediated by NK cells. Methods: We performed a prospective analysis of biomarkers on fresh peripheral blood lymphocytes from 25 untreated CLL patients, 10 untreated SLL and 17 age-matched healthy controls by 10-color flow cytometry. All subjects signed IRB approved informed consent forms. Our study analyzed 180 distinct biomarker parameters, with a particular focus on NK and T cells. Differences in biomarker expression between patients with SLL, CLL, and healthy controls were compared by Wilcoxon rank-sum test. Results: Absolute numbers of NK and T cells per µl of blood were significantly higher in CLL patients, and this correlated with increased B cell numbers. As indicators of immune suppression, the frequency of regulatory T cells was significantly increased in CLL samples, as were levels of PD-1 expression on T cells and CD56dim NK cells. NK cells in CLL expressed higher levels of CD27, which is characteristic of a less mature phenotype, and CD56dim cells expressed lower levels of NKG2D. Compared to healthy controls, CLL samples displayed a marked reduction in degranulation by CD56dim NK cells in response to transformed 721.221 B cells, either with or without rituximab. CD56dim NK cells from CLL patients were also less viable under resting conditions or when challenged with target cells, especially in ADCC responses. We further observed a striking reduction in the frequency and viability of KIR3DL1+ NK cells, which progressed over time in most CLL patients. Surprisingly, CLL patients with the highest levels of PD-1 expression on NK cells possessed genes for both KIR3DL1 and its ligand, HLA-Bw4. Our findings were also clearly evident in a CLL patient compared to her healthy monozygotic twin, thereby providing compelling support for the results in the full patient cohort. The altered expression levels of nearly all of the NK cell biomarkers and degranulation were less pronounced in blood samples from SLL patients, presumably due to low tumor burden in peripheral blood. Conclusions: CLL patients have increased numbers of NK cells in peripheral blood, but these NK cells are less mature, are significantly depleted of the KIR3DL1+ subset, and have deficits in degranulation response, reduced expression of NKG2D activating receptor, increased expression of inhibitory PD-1, and enhanced susceptibility to activation-induced death when challenged with tumor targets and rituxumab. Our findings support the hypothesis that immune dysfunction in CLL may be due in part to a selective loss of mature KIR3DL1+ NK cells, possibly upon encountering overwhelming tumor burden in peripheral blood, and CLL patients may benefit from therapeutic strategies that augment NK cell function. Disclosures No relevant conflicts of interest to declare.

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Targeting the Tumor Evasion Interaction of NKG2A and Its Ligand HLA-E Increases Natural-Killer Cell Activity in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.1289.1289 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1289-1289 ◽

Cited By ~ 1

Author(s):

Emily McWilliams ◽

Jennifer M Mele ◽

Faraz Fiazuddin ◽

Carolyn Cheney ◽

Natarajan Muthusamy ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Nk Cells ◽

Natural Killer ◽

Cell Activation ◽

Nk Cell ◽

Lymphocytic Leukemia ◽

Target Cells ◽

Cell Dysfunction ◽

Direct Cytotoxicity

Abstract Chronic Lymphocytic Leukemia (CLL) represents the most frequent adult leukemia, and remains incurable with current standard therapies. Natural Killer (NK) cell count is predictive of CLL disease progression and their dysfunction in mediating cytokine release and direct or antibody dependent cellular cytotoxicity (ADCC) against CLL B-cells is well documented. Detailed mechanistic insight into the etiology of NK-cell dysfunction in CLL patients is currently lacking. CLL B-cells overexpress HLA-E, the natural ligand for heterodimer CD94/NKG2A receptor complex that is expressed on the surface of NK cells, and this interaction suppresses NK cell activation. While NKG2A/CD94/HLA-E interaction is known to assist NK cells in recognizing "self", tumor cells utilize this mechanism to evade effector cell killing. Utilizing a novel anti-NKG2A monoclonal blocking antibody (mab) we explored the in vitro preclinical activity of targeting the NKG2A receptor, and the NKG2A/HLA-E interaction as a mechanism of tumor evasion in patients with CLL. We hypothesized that limiting the interaction of HLA-E/NKG2A will reverse NK cell anergy and result in increased direct cytotoxicity of CLL cells. Our results confirm the over expression of HLA-E on CLL B-cells and demonstrate NKG2A expression on CD16+ NK cells from CLL patients. Next, we examined the effect of anti-NKG2A mab on NK cell direct cytotoxicity. Treatment of NK cells, from both healthy donor and CLL patients, with anti-NKG2A mab increased direct cytotoxicity over isotype control on targets at various effector to target ratios of 25:1 (54% vs. 46%, p< 0.05, n= 12), 12:1 (43% vs. 35%, p<0.05, n=14), and 6:1 (31% vs. 23%, p<0.05, n= 12, for anti-NKG2A mediated cytotoxicity vs isotype mediated cytotoxicity respectively). These results were also validated with HLA-E over and underexpressing target cells. Fc-gamma receptor blocking experiments were also performed to confirm the specificity of the interaction. Further studies are being performed to confirm the specific activity of the antibody including its ability to modulate NK cell activation, enhance ADCC, and the impact of anti-NKG2A therapy for reversing ibrutinib mediated NK-cell dysfunction. This work has laid the foundation for the clinical utility of this reagent in patients with relapsed CLL in combination with ibrutinib. Disclosures No relevant conflicts of interest to declare.

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Battle of Thermopylae: 300 Spartans (natural killer cells plus obinutuzumab) versus the immortal warriors (chronic lymphocytic leukemia cells) of Xerxes’ army

Future Science OA ◽

10.2144/fsoa-2019-0064 ◽

2019 ◽

Vol 5 (10) ◽

pp. FSO425

Author(s):

Ricardo García-Muñoz ◽

María-Josefa Nájera ◽

Jesús Feliu ◽

Judith Antón-Remírez ◽

Enrique Ramalle-Gómara ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Nk Cell ◽

Killer Cell ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Lymphokine Activated Killer Cells ◽

Killer Cells

Aim: To analyze the effects of subcutaneous or intravenous rituximab + lymphokine-activated killer cells, obinutuzumab or ibrutinib on natural killer (NK) cell levels in chronic lymphocytic leukemia and follicular lymphoma patients. Patients & methods: The distribution of peripheral blood NK cells of 31 patients was analyzed by flow cytometry. Results: We detected a decrease of NK cells in peripheral blood below normal range after obinutuzumab treatment. During maintenance treatment with subcutaneous rituximab, an NK cell reduction was less pronounced than after intravenous rituximab treatment, despite lymphokine-activated killer cell infusions. Conclusion: After one dose of obinutuzumab, each NK cell in peripheral blood destroys 25 leukemic cells.

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Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2007-05-089409 ◽

2007 ◽

Vol 110 (9) ◽

pp. 3316-3325 ◽

Cited By ~ 130

Author(s):

Andrea Bürkle ◽

Matthias Niedermeier ◽

Annette Schmitt-Gräff ◽

William G. Wierda ◽

Michael J. Keating ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Actin Polymerization ◽

Serum Levels ◽

Lymphocytic Leukemia ◽

Lymphoid Tissues ◽

Accessory Cells ◽

Mitogen Activated Protein ◽

B1 Cells

Abstract CXCL13 is a homeostatic chemokine for lymphocyte homing and positioning within follicles of secondary lymphoid tissues, acting through its cognate receptor, CXCR5. Moreover, the CXCR5-CXCL13 axis plays a unique role in trafficking and homing of B1 cells. Here, we report that chronic lymphocytic leukemia (CLL) B cells express high levels of functional CXCR5. CXCR5 expression levels were similar on CLL B cells and normal CD5+ B cells, and higher compared with normal CD5− B cells, follicular B-helper T cells (TFH cells), or neoplastic B cells from other B-cell neoplasias. Stimulation of CLL cells with CXCL13 induces actin polymerization, CXCR5 endocytosis, chemotaxis, and prolonged activation of p44/42 mitogen-activated protein kinases. Anti-CXCR5 antibodies, pertussis toxin, and wortmannin inhibited chemotaxis to CXCL13, demonstrating the importance of Gi proteins and PI3 kinases for CXCR5 signaling. Moreover, CLL patients had significantly higher CXCL13 serum levels than volunteers, and CXCL13 levels correlated with β2 microglobulin. We detected CXCL13 mRNA expression by nurselike cells, and high levels of CXCL13 protein in supernatants of CLL nurselike cell cultures. By immunohistochemistry, we detected CXCL13+ expression by CD68+ macrophages in situ within CLL lymph nodes. These data suggest that CXCR5 plays a role in CLL cell positioning and cognate interactions between CLL and CXCL13-secreting CD68+ accessory cells in lymphoid tissues.

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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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Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells

Blood ◽

10.1182/blood.v94.11.3658.423k11_3658_3667 ◽

1999 ◽

Vol 94 (11) ◽

pp. 3658-3667 ◽

Cited By ~ 6

Author(s):

Jan A. Burger ◽

Meike Burger ◽

Thomas J. Kipps

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Pertussis Toxin ◽

Stromal Cells ◽

Stromal Cell ◽

Actin Polymerization ◽

Kinase Inhibitor ◽

Critical Role ◽

Lymphocytic Leukemia ◽

Marrow Stromal Cells

Chemokines play a central role for lymphocyte trafficking and homing. The mechanisms that direct the tissue localization of B cells from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We found that CLL B cells express functional CXCR4 receptors for the chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by receptor endocytosis, calcium mobilization, and actin polymerization assays. Moreover, CLL B cells displayed chemotaxis to this chemokine that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4, pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase inhibitor. That this chemotaxis may be involved in the homing of CLL cells is argued by studies in which CLL B cells were cocultured with a murine marrow stromal cell line that secretes SDF-1. Within 2 hours, CLL B cells spontaneously migrated beneath such stromal cells in vitro (pseudoemperipolesis). This migration could be inhibited by pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1, or pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4 on CLL B cells that migrated into the stromal cell layer. These findings demonstrate that the chemokine receptor CXCR4 on CLL B cells plays a critical role for heterotypic adherence to marrow stromal cells and provide a new mechanism to account for the marrow infiltration by neoplastic B cells.

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