Glucocorticoid-Induced TNFR-Related Protein (GITR) Ligand Mediates Tumor Immunoediting in Chronic Lymphocytic Leukemia and Impairs Direct and Rituximab-Induced NK Cell Anti-Leukemia Reactivity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4403-4403
Author(s):  
Corina Buechele ◽  
Tina Baessler ◽  
Benjamin J Schmiedel ◽  
Lothar Kanz ◽  
Helmut R Salih
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Nk Cells ◽  
Tumor Immunity ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
Cell Reactivity ◽  
Immune Effector ◽  
Ifn Γ

Abstract Abstract 4403 Members of the TNF/TNF receptor (TNFR) family of proteins govern differentiation, proliferation, activation, and death of both tumor and immune effector cells and thus play an important role in tumor immunoediting, the reciprocal interaction of tumor cells and anti-tumor immunity. Activation of the TNFR family member GITR has recently been shown to stimulate T cell-mediated anti-tumor immunity in mice. However, available data suggest that GITR mediates different effects in mice and men, and may impair anti-tumor immunity of human NK cells. Here we studied the expression and function of GITR ligand (GITRL) in patients with chronic lymphocytic leukemia (CLL) and the consequences of GITR-GITRL interaction for NK cell reactivity against CLL cells. Substantial GITRL expression was detected on primary B-CLL cells in 38 of 48 (79%) investigated patients. Upon interaction with its cognate receptor, GITRL induced the release of immunoregulatory cytokines like TNF by the leukemia cells, which demonstrated that CLL-expressed GITRL is functional and capable to transduce bidirectional signals. Moreover, disruption of GITR-GITRL interaction in cultures of allogenic NK cells with patient CLL cells by addition of blocking antibody caused a significant increase in NK cell granule mobilization, cytotoxicity and IFN-γ production. The inhibitory effect of tumor-expressed GITRL on the reactivity of human NK cells was also confirmed in cocultures of C1R lymphoma cells transfected to express GITRL with mock transfectants serving as control. In addition, blocking GITR-GITRL interaction also considerably augmented both antibody-dependent cellular cytotoxicity (ADCC) and antibody-induced IFN-γ production of NK cells in cultures with allogenic CLL cells upon Rituximab exposure. Of note, GITR blockade also significantly enhanced anti-leukemia reactivity of autologous NK cells among PBMC of B-CLL patients, and this reinforcement of NK cell effector functions was observed both regarding the direct and, more pronounced, Rituximab-induced anti-leukemia reactivity (both n=10, p<0.01, Student's T test). Thus, expression of functional GITRL by CLL cells potently influences tumor immunoediting and impairs anti-tumor immunity by diminishing both direct and Rituximab-dependent anti-leukemia reactivity of NK cells. Modulation of the GITR-GITRL system might therefore serve to enhance the efficacy of therapeutic approaches in CLL which, like Rituximab-induced ADCC or stem cell transplantation, rely on a sufficient NK cell anti-tumor response. Disclosures: No relevant conflicts of interest to declare.

The Role of CD137 and Its Ligand in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3130-3130
Author(s):  
Tina Baessler ◽  
Corina Buechele ◽  
Matthias Krusch ◽  
Benjamin J Schmiedel ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Nk Cells ◽  
Tumor Immunity ◽  
Nk Cell ◽  
Cell Subset ◽  
Surrogate Marker ◽  
Lymphocytic Leukemia ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Cell Reactivity

Abstract Tumor immunosurveillance is dependent on the reciprocal interaction between tumor cells and anti-tumor immunity mediated e.g. by NK cells. This has led to the concept of tumor immunoediting, which incorporates the multitude of mechanisms underlying this dual tumor- and immune-sculpting interaction. Various members of the TNF/TNFR family modulate differentiation, proliferation, activation, and death of both tumor and immune effector cells. Very recently the TNFR family member CD137 has been shown to be induced on NK cells by Fc receptor triggering indicating that not only the Fab region but also the Fc part of a given antibody may be responsible for effects attributed to CD137 modulation (Lin et al., Blood2008, 112: 699). Thus we here studied the role of human CD137 and its cognate counterpart, the CD137 ligand (CD137L) in the interaction of CLL with NK cells. High levels of CD137L expression were detected on B-CLL cells in all investigated patients (n=40). Incubation of CLL cells in the presence of an immobilized CD137-Ig fusionprotein significantly induced the release of the immunoregulatory cytokine TNF demonstrating that CLL-expressed CD137L was capable to transduce bidirectional signals. Furthermore, we found that NK cells of CLL patients displayed substantial CD137 expression. While being absent on resting NK cells, CD137 expression was upregulated on the CD56dimCD16+ but not the CD56brightCD16− NK cell subset of healthy donors upon activation e.g. with IL-2 or IL-15. In addition, CD137 was also induced on NK cells after incubation in supernatants of PBMC of CLL patients. Surprisingly, disruption of CD137-CD137L interaction in cocultures of allogenic NK cells with patient CLL cells by blocking CD137 antibody caused a significant increase in NK cell cytotoxicity. The observed inhibitory effect of CD137L on NK cell reactivity was confirmed in cytotoxicity assays using CD137L-transfectants with mock-transfectants as control. Furthermore, blocking CD137-CD137L interaction also substantially enhanced Rituximab-induced antibody dependent cellular cytotoxicity in an allogenic setting. Importantly, CD137 blockade also substantially enhanced CD107a expression as a surrogate marker for granule mobilization on autologous NK cells within PBMC of B-CLL patients, and this effect was observed both in the absence and more pronounced in the presence of Rituximab. Thus, expression of functional CD137L by CLL cells impairs anti-tumor immunity by diminishing both direct and antibody-dependent cellular cytotoxicity of allogenic and autologous NK cells. Modulation of the CD137-CD137L system might therefore be a suitable therapeutic approach in strategies like antibody therapy which rely on a sufficient NK cell anti-tumor response.

4-1BB Ligand Mediates a “Vicious Cycle” of Immune Evasion Upon Reciprocal Interaction of Chronic Lymphocytic Leukemia and NK Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2416-2416
Author(s):  
Corina Buechele ◽  
Tina Baessler ◽  
Benjamin J Schmiedel ◽  
Lothar Kanz ◽  
Helmut R. Salih
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Nk Cells ◽  
Immune Evasion ◽  
Nk Cell ◽  
Lymphocytic Leukemia ◽  
Antibody Treatment ◽  
Cell Reactivity ◽  
Allogenic Stem Cell Transplantation ◽  
Immunoregulatory Cytokines ◽  
Healthy Donors

Abstract Abstract 2416 NK cells play an important role in tumor immunosurveillance. Their reactivity is governed by various immunoregulatory molecules, which influence both direct anti-tumor immunity and NK responses induced by therapeutic antibodies like Rituximab. Various members of the TNF/TNFR family modulate differentiation, proliferation, activation, and death of both tumor and immune effector cells including NK cells. Recently we reported that the TNFR family member 4-1BB/CD137 is expressed on human NK cells following activation. In contrast to the stimulatory role of its murine counterpart, we found that human 4-1BB impairs NK anti-tumor reactivity upon interaction with its ligand 4-1BBL expressed on blasts of a substantial proportion of acute myeloid leukemia patients (Blood 115: 3058-69; 2010). In addition, we found that expression of 4-1BBL is general feature of leukemic cells of chronic lymphocytic leukemia (CLL) patients causing impaired direct and Rituximab-induced NK cell reactivity (Blood 114: 279; 2009). Here we report that reverse signaling via 4-1BBL into CLL cells following interaction with 4-1BB, which is absent on NK cells of healthy donors, but expressed at substantial levels on NK cells of CLL patients, induced pronounced production of immunoregulatory cytokines like TNF, IL-6 and IL-8 by the CLL cells. Moreover, we found that sera of CLL patients contained elevated levels of these immunoregulatory cytokines as compared to healthy controls. When PBMC of healthy donors were exposed to supernatants of in vitro cultured CLL cells or sera from CLL patients, this resulted in pronounced 4-1BB expression on the NK cells. This effect could be prevented by addition of the TNF blocker Infliximab to patient sera. The 4-1BB expression induced by CLL sera resulted in impaired NK reactivity specifically against 4-1BBL-expressing targets as revealed by functional analyses with 4-1BBL transfectants and the respective mock-controls. Moreover, the induced 4-1BB expression also impaired NK cell reactivity against primary CLL cells constitutively expressing 4-1BBL, thus closing a cycle of immune evasion. Taken together, our data demonstrate that 4-1BBL enables CLL cells to evade NK anti-tumor reactivity, and disruption of the “vicious 4-1BB-4-1BBL cycle” in CLL - NK interaction e.g. by TNF- or 4-1BB-blockade may serve well to enhance NK reactivity in therapeutic strategies like antibody treatment or allogenic stem cell transplantation in CLL, which rely on sufficient NK cell function. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Glucocorticoid-induced TNFR-related protein (GITR) ligand modulates cytokine release and NK cell reactivity in chronic lymphocytic leukemia (CLL)

Leukemia ◽  
2011 ◽  
Vol 26 (5) ◽  
pp. 991-1000 ◽  
Author(s):  
C Buechele ◽  
T Baessler ◽  
S Wirths ◽  
J U Schmohl ◽  
B J Schmiedel ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Nk Cell ◽  
Lymphocytic Leukemia ◽  
Cytokine Release ◽  
Related Protein ◽  
Cell Reactivity

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

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.

Role of CD137/4-1BB and Its Ligand in the NK Cell Mediated Immune Surveillance of Acute Myeloid Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 880-880
Author(s):  
Tina Baessler ◽  
Matthias Krusch ◽  
Katrin M. Baltz ◽  
Benjamin J. Schmiedel ◽  
Helga M. Schmetzer ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Immune Surveillance ◽  
Cell Reactivity ◽  
Ifn Γ ◽  
Tnfr Superfamily ◽  
Acute Myeloid

Abstract NK cells play an important role in the reciprocal interaction of tumor cells with the immune system and participate in the surveillance and eradication of hematological malignancies including acute myeloid leukemia (AML). NK cell reactivity is governed by a balance of activating and inhibitory receptors including various members of the TNF receptor (TNFR) superfamily. The TNFR superfamily member CD137/4-1BB has been shown to stimulate proliferation and IFN-γ production, but not cytotoxicity of NK cells in mice. Surprisingly, yet nothing is known regarding the consequences of CD137-CD137 ligand (CD137L) interaction for NK cell reactivity in humans. In this study we demonstrate that CD56dimCD16+ but not CD56brightCD16− NK cells express CD137 upon stimulation with the activating cytokines IL-2 and IL-15 with peak expression between 48 and 60h. Furthermore, we found that 5 of 7 investigated AML cell lines and 16 of 51 (33%) primary AML cells of patients expressed substantial CD137L levels, while no CD137L expression was detected on CD34+ cells of healthy donors (n=5). CD137L expression was not restricted to a specific French-American-British (FAB) subtype, but was significantly (p<0.05, one-way ANOVA) associated with monocytic (FAB M4, M5) differentiation. In addition, no association with a particular cytogenetic abnormality or with expression of MHC class I was observed. Reverse signaling via CD137L into AML cells (n=10) significantly induced the release of the immunoregulatory cytokines IL-10 and TNF (both p<0.05, Mann-Whitney U-test). Surprisingly and in contrast to available data regarding the function of murine CD137, we found that in humans blocking CD137-CD137L interaction caused a significant increase in NK cell cytotoxicity and IFN-γ production about 50% (both p<0.05, Mann-Whitney U-test) in coculture assays with CD137L-expressing patient AML cells and AML cell lines. The inhibitory effect of CD137 on NK cell reactivity was further confirmed in cocultures of NK cells with CD137L-transfectants and by triggering CD137 with an agonistic monoclonal antibody. This indicates that CD137 mediates opposite effects in murine compared to human NK cells. Furthermore we conclude that CD137L expression substantially influences tumor immunoediting by AML cells and diminishes NK cell reactivity against AML.

scholarly journals Intrinsic Resistance of Chronic Lymphocytic Leukemia Cells to NK Cell-Mediated Lysis Can Be Overcome In Vitro by Pharmacological Inhibition of Cdc42-Induced Actin Cytoskeleton Remodeling

2021 ◽  
Vol 12 ◽  
Author(s):  
Hannah Wurzer ◽  
Liza Filali ◽  
Céline Hoffmann ◽  
Max Krecke ◽  
Andrea Michela Biolato ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Actin Cytoskeleton ◽  
Nk Cells ◽  
Nk Cell ◽  
Granzyme B ◽  
Lymphocytic Leukemia ◽  
Cell Cytotoxicity ◽  
Cytoskeleton Remodeling ◽  
Nk Cell Cytotoxicity

Natural killer (NK) cells are innate effector lymphocytes with strong antitumor effects against hematologic malignancies such as chronic lymphocytic leukemia (CLL). However, NK cells fail to control CLL progression on the long term. For effective lysis of their targets, NK cells use a specific cell-cell interface, known as the immunological synapse (IS), whose assembly and effector function critically rely on dynamic cytoskeletal changes in NK cells. Here we explored the role of CLL cell actin cytoskeleton during NK cell attack. We found that CLL cells can undergo fast actin cytoskeleton remodeling which is characterized by a NK cell contact-induced accumulation of actin filaments at the IS. Such polarization of the actin cytoskeleton was strongly associated with resistance against NK cell-mediated cytotoxicity and reduced amounts of the cell-death inducing molecule granzyme B in target CLL cells. Selective pharmacological targeting of the key actin regulator Cdc42 abrogated the capacity of CLL cells to reorganize their actin cytoskeleton during NK cell attack, increased levels of transferred granzyme B and restored CLL cell susceptibility to NK cell cytotoxicity. This resistance mechanism was confirmed in primary CLL cells from patients. In addition, pharmacological inhibition of actin dynamics in combination with blocking antibodies increased conjugation frequency and improved CLL cell elimination by NK cells. Together our results highlight the critical role of CLL cell actin cytoskeleton in driving resistance against NK cell cytotoxicity and provide new potential therapeutic point of intervention to target CLL immune escape.

The FLT3-Inhibitors Midostaurin, Sunitinib, Sorafenib, and TKI258 Differentially Affect NK Cell-Mediated Immunesurveillance of Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3785-3785
Author(s):  
Julia Salih ◽  
Lothar Kanz ◽  
Helmut R Salih ◽  
Matthias Krusch
Keyword(s):  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Immune Surveillance ◽  
Leukemia Cells ◽  
Target Cells ◽  
Cell Reactivity ◽  
Flt3 Inhibitors ◽  
Ifn Γ

Abstract Abstract 3785 Poster Board III-721 FLT3 is a receptor tyrosine kinase with an important role in hematopoietic progenitor cell survival and proliferation. The discovery of internal tandem duplication mutations (ITD) in FLT3 was a major breakthrough in understanding the role of abnormally activated FLT3 in myeloid transformation. Between 15% and 34% of AML patients show FLT3-ITD mutations, and thus the inhibition of FLT3 in combination with chemotherapeutic agents may be a promising stragety in the treatment of Acute Myeloid Leukemia (AML). Several protein kinase inhibitors (PKI) targeting FLT3 like e.g. Midostaurin, Sunitinib, Sorafenib, and TKI258 are currently under preclinical and/or clinical evaluation (http://clinicaltrials.gov/ct2/results?term=AML+and+FLT3). Since those PKI, besides targeting their eponymous enzyme FLT3, also inhibit signaling via other molecules they may impair the effector function of various components of anti-tumor immunity. NK cells as part of the innate immune system play an important role in the immune surveillance of tumors due to their ability to directly kill target cells and to shape adaptive immune responses by secreting cytokines like IFN-γ. Clinical evidence for the particularly important role of NK cells in leukemia has recently been provided by studies of haploidentical stem cell transplantation (Ruggeri et al., Science 2002). We report here that CD107a expression as a surrogate marker for degranulation of NK cells within PBMC is inhibited by pharmacological concentrations of Sorafenib (10μg/ml) and Midostaurin (2μg/ml), but not by Sunitinib (200ng/ml) and TKI258 (125ng/ml). In line, pharmacological concentrations of Sunitinib and TKI258 did not affect NK cell cytotoxicity and IFN-γ production in cocultures with leukemia cells. Sorafenib and Midostaurin caused a clear concentration-dependent inhibition of NK cell cytokine production in response to target cells both in resting and in IL-2 activated state (92% and 66%, respectively at plasma peak levels). Furthermore, pharmacological concentrations of Sorafenib and Midostaurin also reduced lysis of leukemia cells by NK cells (54% and 58%, respectively, E:T ratio 10:1) and thus generally compromised NK cell reactivity. Analysis of NK cell signaling revealed that Sorafenib, but not Midostaurin decreased phosphorylation of PI3K and ERK which are important regulators of NK cell reactivity. Thus, Midostaurin inhibits yet undefined signaling events which are crucial for NK effector functions, but are independent of the “classical” PI3K – Rac – PAK – MEK – ERK pathway and are presently under study. Moreover, in light of the important role of NK cells in the immune surveillance of leukemia and the differential influence of clinically used FLT3-inhibitors on NK cell functions our data indicate that the choice and dosing of the most suitable compound in the treatment of AML requires further characterization and careful consideration. Disclosures: No relevant conflicts of interest to declare.

Induction of NK Cell Reactivity Against AML Cells by Fc-Engineered RANK-Ig Fusion Proteins.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2625-2625
Author(s):  
Tina Nuebling ◽  
Benjamin J Schmiedel ◽  
Miyuki Azuma ◽  
Pascal Schneider ◽  
Ludger Grosse-Hovest ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Nk Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Target Antigen ◽  
Dependent Manner ◽  
Cell Reactivity ◽  
Allogenic Stem Cell Transplantation

Abstract Abstract 2625 NK cells are cytotoxic lymphocytes that play an important role in anti-tumor immunity. A clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of anti-tumor antibodies. In acute myeloid leukemia (AML) NK cells largely contribute to the therapeutic efficacy allogenic stem cell transplantation (SCT). Recently we demonstrated that AML cells functionally express the TNF family member RANK ligand (RANKL) which impairs NK cell anti-leukemia reactivity (Schmiedel et al., ASH annual meeting 2011). Here we developed a strategy to combine blocking of the NK inhibitory effects of RANKL with targeting of AML cells for NK cell ADCC. To this end we generated fusion proteins consisting of the extracellular domain of RANK and a human IgG1 Fc part that was modified by amino acid exchange. Compared to wild type RANK-Fc fusion protein (RANK-Fc-WT), our mutant RANK-Fc-ADCC (S239D/I332E) displayed highly enhanced affinity to FcγRIIIa (CD16) on NK cells. Primary AML cells expressed substantial levels of RANKL in 53 of 78 (68%) investigated patient cases, and our RANK-Ig fusion proteins bound to AML cells in a target antigen-specific manner. Treatment with both RANK-Fc-WT and RANK-Fc-ADCC clearly reduced the release of RANKL-induced immunomodulatory factors like TNF, IL-6, IL-8 and IL-10 by AML cells. When the effects of the fusion proteins on NK cell ADCC were studied we found that treatment with RANK-Fc-WT only slightly enhanced NK cell reactivity against RANKL-positive patient AML cells. However, RANK-Fc-ADCC potently induced NK cell ADCC and cytokine production in response to AML targets in a target antigen-dependent manner due to the functional properties of its engineered Fc moiety. Taken together, our Fc-engineered RANK-Fc-ADCC fusion protein may serve to modulate the cytokine milieu involved in AML pathophysiology and target RANKL-expressing leukemia cells for NK anti-tumor reactivity. Thus, RANK-Fc-ADCC constitutes an attractive immunotherapeutic means for the treatment of AML, e.g. for elimination of minimal residual disease after conventional therapy including SCT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Involvement of NFAT Transcription Factors in NK Cell Reactivity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1344-1344 ◽  
Author(s):  
Kathrin Rothfelder ◽  
Melanie Märklin ◽  
Julia Wild ◽  
Daniela Dörfel ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nk Cells ◽  
Nk Cell ◽  
Inhibitory Effect ◽  
Target Cells ◽  
Cell Reactivity ◽  
Tumor Immunosurveillance ◽  
Metastatic Burden ◽  
And Function

Abstract NK cells are lymphoid components of innate immunity and play an important role in tumor immunosurveillance. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), as highlighted by its important role in T and B cell development and function. With regard to NK cells, available data indicate that NFAT is dispensable for development. However, several lines of evidence including the observation that the immunosuppressive drugs cyclosporin A and tacrolimus, which mediate their effects through inhibition of calcineurin and consecutively NFAT, influence NK reactivity implicate a role of this family of transcription factors in NK cell reactivity and function. Here we employed different genetic mouse models on the C57BL/6 background to directly study the functional role of NFAT in NK cells. We found that except for NFAT3 mRNA and protein of all family members (NFAT 1, 2, 4 and 5) was expressed in resting NK cells of wild type (WT) mice with NFAT1, 2 and 4 being most abundantly detectable. When we employed NK cells with knockout (KO) of NFAT 1, 2, and 4 in comparative in vitro analyses, we surprisingly found that lack of NFAT resulted in enhanced NK cell activation, degranulation and release of immunomodulatory cytokines like IFN-γ after co-culture with YAC-1 target cells as well as increased production of granzyme B and perforin after cytokine activation. The inhibitory effect of NFAT on NK cell effector function was further confirmed in vivo by employing WT and germ line NFAT KO animals in the syngeneic B16 melanoma model, which revealed a significantly reduced metastatic burden in NFAT KO mice. Depletion of NK cells in this model system in turn resulted in increased metastasis, however, with WT animals displaying significantly higher metastatic burden compared to NFAT KO mice. As this pointed to the fact that NFAT influences metastasis via both NK-dependent and independent mechanisms, we further generated mice with a NK cell-specific (conditional Ncr1-Cre dependent) NFAT2 KO. When these animals were employed again in analyses of B16 lung metastasis, comparative analyses with WT animals confirmed the inhibitory effect of NFAT on NK tumor immunosurveillance. Taken together, these results provide the first direct evidence for the functional involvement of NFAT in NK cell antitumor reactivity and, in contrast to T and B cells, identify NFAT as a negative regulator of NK cell function. Disclosures No relevant conflicts of interest to declare.

First Clinical Evidence of In Vivo Natural Killer (NK) Cell Modulation in Chronic Lymphocytic Leukemia (CLL) Patients (pts) Treated with Lenalidomide (L).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2109-2109 ◽  
Author(s):  
Swaminathan Padmanabhan ◽  
Noreen Ersing ◽  
Paul K. Wallace ◽  
Kena C. Miller ◽  
Laurie Musiel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Nk Cells ◽  
Phase Ii ◽  
Nk Cell ◽  
Cytokine Profile ◽  
Lymphocytic Leukemia ◽  
Appreciable Change

Abstract Introduction: Pts with Chronic Lymphocytic Leukemia (CLL) are reported to have quantitative and qualitative T and NK cell dysfunction. While NK cells act through non-specific killing, T-cells are more specific. The 2 types of T-lymphocytes, CD4+ (Th; helper) and CD8+ (Ts; cytotolytic/suppressor) are subcategorized based on cytokine secretion profile upon activation. Release of different cytokines from these immune cells modulates the host response. T1 cells (Th1, Ts1) secrete IL-2 and interferon-g which initiate the Th1 response- mainly CD4+ activation along with B and T cells, leading to proliferation and differentiation of these cells. T2 (Th2, Ts2) cells initiate the Th2 response (release of TNF-a, IL-10) resulting in direct lysis of the target cell by production of cytokines such as IL-4, IL-5 and IL-10. Hypothesis: To decipher this antitumor mechanism of L in CLL pts we investigated its effect on the efferent arm of immune response by evaluating the T cell population and the afferent response by change in expression of co-stimulatory molecules on B-CLL cells and cytokine profile in these pts treated on a phase II clinical study. Methods: CLL pts treated with L were evaluated for absolute number of T (CD4+, CD8+) and NK (CD56+) cells by flow cytometry on day before (day0) start and on Day 8 of treatment with L. Peripheral blood was collected and ficolled to obtain enriched mononuclear cells. The serum was used to study the cytokines. Activation status was determined by co-expression of CD45+. Serum cytokine profile was measured by Flow cytometry using the Luminex system. B-CLL surface co-stimulatory molecules were detected by flow cytometry and analyzed by FACS. These responses were correlated with the tumor flare (TF) reaction that the patients developed during the first week of treatment with L. Results: Eighteen out of 45 pts have so far been evaluated for immunomodulatory activity of L. There were 2 complete responders (CRs) and 6 partial responders (PRs); while 4 had stable disease (SD), 4 were clinically unevaluable and 2 were too early for response in this group. Mean baseline (bl) NK cell count pretreatment was 251 (range 31–1510) vs. post treatment was 193 (range 6–13,482). Six out of 18 patients showed an increase, ranging from 20 −199% in the absolute NK (CD16+/CD56+/CD45+). While there was no appreciable change in CD4+ numbers there was a general trend in increase of CD8+ cells. No change in monocyte population was noted. Concurrent increase in the expression of co-stimulatory molecules such as CD95 and CD80 was noted. This response in co-stimulation was confirmed by in vitro experiments done on isolated B-CLL cells (n=4)treated with L. An increase in Th-2 cytokines such as IL-4, IL-5, IL-6 and IL-10 was noted in all eight responders, while VEGF levels were decreased in 6/18 patients. 99% of patients had a TF and the grade of TF correlated with the changes in T cells and cytokine profile. Conclusion: It appears that in vivo L is able to orchestrate an anti-tumor response in CLL by modulating the NK cells, changing the cytokine profile and up-regulating co-stimulatory molecules. This change in the immune effector cell repertoire and the Th2 skewing may explain the initial flare reaction noted in these L treated pts. Data from these correlative studies is being evaluated in the context of the phase II clinical trial to be reported at the 48th ASH annual meeting.

Sign in / Sign up

Export Citation Format

Share Document