scholarly journals PD-1 expression on NK cells can be related to cytokine stimulation and tissue residency

2021 ◽  
Author(s):  
Arnika K Wagner ◽  
Nadir Kadri ◽  
Chris Tibbitt ◽  
Koen van de Ven ◽  
Sunitha Bagawath-Singh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Mhc Class I ◽  
Sequencing Analysis ◽  
Single Cell Rna Sequencing ◽  
Cytokine Stimulation ◽  
Deficient Mice

ABSTRACTAlthough PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells which in part could be attributed to a decrease in tumor-infiltrating NK cells in PD-1-deficient mice. NK cells from PD-1-deficient mice exhibited a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Finally, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wildtype mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells.

scholarly journals Decreased tumor surveillance in perforin-deficient mice.

1996 ◽  
Vol 184 (5) ◽  
pp. 1781-1790 ◽  
Author(s):  
M E van den Broek ◽  
D Kägi ◽  
F Ossendorp ◽  
R Toes ◽  
S Vamvakas ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Mhc Class I ◽  
Cell Lines ◽  
Tumor Cell Lines ◽  
Tumor Surveillance ◽  
Induced Tumors ◽  
Deficient Mice

Immune surveillance against tumors usually depends on T cell recognition of tumor antigens presented by major histocompatibility complex (MHC) molecules, whereas MHC class I- tumors may be controlled by natural killer (NK) cells. Perforin-dependent cytotoxicity is a major effector function of CD8+ MHC class I-restricted T cells and of NK cells. Here, we used perforin-deficient C57BL/6 (PKO) mice to study involvement of perforin and Fas ligand in tumor surveillance in vivo. We induced tumors in PKO and normal C57BL/6 mice by (a) injection of different syngeneic tumor cell lines of different tissue origin in naive and primed mice; (b) administration of the chemical carcinogens methylcholanthrene (MCA) or 12-O-tetradecanoylphorbol-13-acetate (TPA) plus 7,12-dimethylbenzanthracene (DMBA), or (c) by injection of acutely oncogenic Moloney sarcoma virus. The first set of models analyzes the defense against a tumor load given at once, whereas the last two sets give information on immune defense against tumors at the very moment of their generation. Most of the tumor cell lines tested were eliminated 10-100-fold better by C57BL/6 mice in an unprimed situation; after priming, the differences were more pronounced. Lymphoma cells transfected with Fas were controlled 10-fold better by PKO and C57BL/6 mice when compared to untransfected control cells, indicating some role for FasL in tumor control. MCA-induced tumors arose more rapidly and with a higher incidence in PKO mice compared to C57BL/6 or CD8-deficient mice. DMBA+TPA-induced skin papillomas arose with similar high incidence and comparable kinetics in both mouse strains. C57BL/6 and PKO mice have a similar incidence of Moloney murine sarcoma and leukemia virus-induced sarcomas, but tumors are larger and regression is retarded in PKO mice. Thus, perforin-dependent cytotoxicity is not only a crucial mechanism of both cytotoxic T lymphocyte- and NK-dependent resistance to injected tumor cell lines, but also operates during viral and chemical carcinogenesis in vivo. Experiments addressing the role of Fas-dependent cytotoxicity by studying resistance to tumor cell lines that were stably transfected with Fas neither provided evidence for a major role of Fas nor excluded a minor contribution of Fas in tumor surveillance.

Role of Il-2, Il-7, and IL-15 in T Cell Mediated Graft-vs-Leukemia Against Human Acute Lymphoblastic Leukemia in a NOD/scid Chimeric Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5256-5256
Author(s):  
Doug Cipkala ◽  
Kelly McQuown ◽  
Lindsay Hendey ◽  
Michael Boyer
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Mouse Model ◽  
Scid Mouse ◽  
In Vitro Cytotoxicity ◽  
Scid Mouse Model

Abstract The use of cytotoxic T-lymphocytes (CTL) has been attempted experimentally with various tumors to achieve disease control. Factors that may influence GVT include CTL cytotoxicity, ability to home to disease sites, and survival of T cells in the host. The objective of our study is to evaluate the GVL effects of human alloreactive CTL against ALL in a chimeric NOD/scid mouse model. CTL were generated from random blood donor PBMCs stimulated with the 697 human ALL cell line and supplemented with IL-2, -7, or -15. CTL were analyzed for in vitro cytotoxicity against 697 cells, phenotype, and in vitro migration on day 14. NOD/scid mice were injected with 107 697 ALL cells followed by 5x106 CTL. Mice were sacrificed seven days following CTL injection and residual leukemia was measured in the bone marrow and spleen via flow cytometry. The ratios of CD8/CD4 positive T cells at the time of injection were 46/21% for IL-2, 52/31% for IL-7, and 45/14% for IL-15 cultured CTL (n=13). Control mice not receiving CTL had a baseline leukemia burden of 2.01% and 0.15% in the bone marrow and spleen, respectively (n=15). Mice treated with IL-15 cultured CTL had a reduction in tumor burden to 0.2% (n=13, p=0.01) and 0.05% (n=13, p=0.01) in bone marrow and spleen, respectively. Those treated with IL-2 or IL-7 cultured CTL showed no significant difference in leukemia burden in either the bone marrow (IL-2 1.28%, Il-7 5.97%) or spleen (IL-2 0.4%, IL-7 0.33%). No residual CTL could be identified in the bone marrow or spleen at the time of sacrifice in any CTL group. CTL grown in each cytokine resulted in similar in vitro cytotoxicity at an effector:target ratio of 10:1 (IL-2 41.3%, IL-7 37.7%, IL-15 45.3%, n=12–15, p>0.05 for all groups) and had statistically similar intracellular perforin and granzyme-B expression. In vitro CTL migration to a human mesenchymal stem cell line was greatest with IL-15 CTL (30.5%, n=4), followed by IL-7 CTL (18.9%, n=4), and least in IL-2 CTL (17.9%, n=4), though the differences were not significant. In vitro CTL migration was analyzed to an SDF-1α gradient as CXCR4/SDF-1α interactions are necessary for hematopoietic progenitor cell homing to the bone marrow. IL-15 cultured CTL showed the highest migration (48.8%, n=8) as compared to IL-2 (21.7%, n=6, p=0.048) or IL-7 CTL (35.9%, n=8, p>0.05). However, surface expression of CXCR4 measured by flow cytometry was significantly higher in IL-7 CTL (89.4%, n=9) compared to IL-2 CTL (52.2%, n=9, p<0.001) and IL-15 CTL (65.4%, n=10, p=0.002). Experiments are currently underway to further evaluate the role of CXCR4/SDF-1α in GVL. Preliminary in vivo experiments do not suggest any significant differences in CTL engraftment when evaluated at 24 hours post injection. Expression of the anti-apoptotic bcl-2 protein was greatest on IL-7 (MFI=5295, n=13) and IL-15 (MFI=4865, n=14) when compared to IL-2 CTL (MFI=3530, n=13, p=0.02 vs. IL-7, p=0.05 vs. IL-15), suggesting an increased in vivo survival ability. We hypothesize that IL-15 cultured CTL have greater GVL effects due to either higher in vivo survival, greater bone marrow homing efficiency, or both. Future experiments are planned to evaluate in vivo administration of IL-2 to enhance CTL survival in the host. In conclusion, IL-15 cultured CTL had significantly greater in vivo GVL effects compared to IL-2 and IL-7 CTL in the NOD/scid mouse model. This model can be utilized to evaluate the mechanism of T cell mediated GVL against ALL and potentially other human malignancies.

P-Selectin and Platelet Clearance

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4446-4452 ◽  
Author(s):  
Gaëtan Berger ◽  
Daqing W. Hartwell ◽  
Denisa D. Wagner
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Platelet Activation ◽  
Soluble Form ◽  
Wild Type ◽  
Adhesion Receptor ◽  
Activated Platelets ◽  
Deficient Mice

P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin–deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin–deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4°C caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.

scholarly journals Constitutive versus Activation-dependent Cross-Presentation of Immune Complexes by CD8+ and CD8− Dendritic Cells In Vivo

2002 ◽  
Vol 196 (6) ◽  
pp. 817-827 ◽  
Author(s):  
Joke M.M. den Haan ◽  
Michael J. Bevan
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Immune Complexes ◽  
Class I ◽  
Cross Presentation ◽  
Deficient Mice ◽  
Mhc Class I Molecules

Murine splenic dendritic cells (DCs) can be divided into two subsets based on CD8α expression, but the specific role of each subset in stimulation of T cells is largely unknown. An important function of DCs is the ability to take up exogenous antigens and cross-present them in the context of major histocompatibility complex (MHC) class I molecules to CD8+ T cells. We previously demonstrated that, when cell-associated ovalbumin (OVA) is injected into mice, only the CD8+ DC subset cross-presents OVA in the context of MHC class I. In contrast to this selectivity with cell-associated antigen, we show here that both DC subsets isolated from mice injected with OVA/anti-OVA immune complexes (OVA-IC) cross-present OVA to CD8+ T cells. The use of immunoglobulin G Fc receptor (FcγR) common γ-chain–deficient mice revealed that the cross-presentation by CD8− DCs depended on the expression of γ-chain–containing activating FcγRs, whereas cross-presentation by CD8+ DCs was not reduced in γ-chain–deficient mice. These results suggest that although CD8+ DCs constitutively cross-present exogenous antigens in the context of MHC class I molecules, CD8− DCs only do so after activation, such as via ligation of FcγRs. Cross-presentation of immune complexes may play an important role in autoimmune diseases and the therapeutic effect of antitumor antibodies.

scholarly journals Essential role of CCL21 in establishment of central self-tolerance in T cells

2017 ◽  
Vol 214 (7) ◽  
pp. 1925-1935 ◽  
Author(s):  
Mina Kozai ◽  
Yuki Kubo ◽  
Tomoya Katakai ◽  
Hiroyuki Kondo ◽  
Hiroshi Kiyonari ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Essential Role ◽  
Functional Inequality ◽  
Cell Accumulation ◽  
Self Tolerance ◽  
Thymic Medulla ◽  
Deficient Mice

The chemokine receptor CCR7 directs T cell relocation into and within lymphoid organs, including the migration of developing thymocytes into the thymic medulla. However, how three functional CCR7 ligands in mouse, CCL19, CCL21Ser, and CCL21Leu, divide their roles in immune organs is unclear. By producing mice specifically deficient in CCL21Ser, we show that CCL21Ser is essential for the accumulation of positively selected thymocytes in the thymic medulla. CCL21Ser-deficient mice were impaired in the medullary deletion of self-reactive thymocytes and developed autoimmune dacryoadenitis. T cell accumulation in the lymph nodes was also defective. These results indicate a nonredundant role of CCL21Ser in the establishment of self-tolerance in T cells in the thymic medulla, and reveal a functional inequality among CCR7 ligands in vivo.

scholarly journals TIM-1 Serves as a Nonredundant Receptor for Ebola Virus, Enhancing Viremia and Pathogenesis

2018 ◽  
Author(s):  
Bethany Brunton ◽  
Kai Rogers ◽  
Elisabeth K. Phillips ◽  
Rachel B. Brouillette ◽  
Ruayda Bouls ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ebola Virus ◽  
Apoptotic Bodies ◽  
Virus Load ◽  
Ebov Infection ◽  
Model Virus ◽  
Deficient Mice

AbstractBackground.T cell immunoglobulin mucin domain-1 (TIM-1) is a phosphatidylserine (PS) receptor, mediating filovirus entry into cells through interactions with PS on virions. TIM-1 expression has been implicated in Ebola virus (EBOV) pathogenesis; however, it remains unclear whether this is due to TIM-1 serving as a filovirus receptor in vivo or, as others have suggested, TIM-1 induces a cytokine storm elicited by T cell/virion interactions. Here, we use a BSL2 model virus that expresses EBOV glycoprotein and demonstrate the importance of TIM-1 as a virus receptor late during in vivo infection.Methodology/Principal findings.We used an infectious, recombinant vesicular stomatitis virus expressing EBOV glycoprotein (EBOV GP/rVSV) to assess the role of TIM-1 during in vivo infection. TIM-1-sufficient or TIM-1-deficient BALB/c interferon α/β receptor-/-mice were challenged with EBOV GP/rVSV-GFP or G/rVSV-GFP. While G/rVSV caused profound morbidity and mortality in both mouse strains, TIM-1-deficient mice had significantly better survival than TIM-1-expressing mice following EBOV GP/rVSV challenge. EBOV GP/rVSV load in spleen was high and unaffected by expression of TIM-1. However, infectious virus in serum, liver, kidney and adrenal gland was reduced late in infection in the TIM-1-deficient mice, suggesting that virus entry via this receptor contributes to virus load. Consistent with higher virus loads, proinflammatory chemokines trended higher in organs from infected TIM-1-sufficient mice compared to the TIM-1-deficient mice, but proinflammatory cytokines were more modestly affected. To assess the role of T cells in EBOV GP/rVSV pathogenesis, T cells were depleted in TIM-1-sufficient and -deficient mice and the mice were challenged with virus. Depletion of T cells did not alter the pathogenic consequences of virus infection.Conclusions.Our studies provide evidence that at late times during EBOV GP/rVSV infection, TIM-1 increased virus load and associated mortality, consistent with an important role of this receptor in virus entry. This work suggests that inhibitors which block TIM-1/virus interaction may serve as effective antivirals, reducing virus load at late times during EBOV infection.Author summaryT cell immunoglobulin mucin domain-1 (TIM-1) is one of a number of phosphatidylserine (PS) receptors that mediate clearance of apoptotic bodies by binding PS on the surface of dead or dying cells. Enveloped viruses mimic apoptotic bodies by exposing PS on the outer leaflet of the viral membrane. While TIM-1 has been shown to serve as an adherence factor/receptor for filoviruses in tissue culture, limited studies have investigated the role of TIM-1 as a receptor in vivo. Here, we sought to determine if TIM-1 was critical for Ebola virus glycoprotein-mediated infection using a BSL2 model virus. We demonstrate that loss of TIM-1 expression results in decreased virus load late during infection and significantly reduced virus-elicited mortality. These findings provide evidence that TIM-1 serves as an important receptor for Ebola virus in vivo. Blocking TIM-1/EBOV interactions may be effective antiviral strategy to reduce viral load and pathogenicity at late times of EBOV infection.

scholarly journals P025 Identification and validation of predictors for tofacitinib through supervised and unbiased approaches in Inflammatory Bowel Disease

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S141-S141
Author(s):  
B Liu ◽  
M Spalinger ◽  
L G Perez ◽  
A Machicote ◽  
N Gagliani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Signaling Pathway ◽  
Cd4 T Cells ◽  
Cytokine Production ◽  
Stat Signaling ◽  
Deficient Mice

Abstract Background Inflammatory Bowel Disease (IBD) is characterized by an overwhelming gut inflammation, where CD4+ effector T cells are main mediators of the inflammatory response. Tofacitinib, a small molecular drug recently used in IBD patients, blocks the JAK/STAT signaling pathway necessary for CD4+ effector T-cell activation. However, clinical data show that a percentage of patients do not respond to the treatment. Our main goal is to identify biomarkers predicting the response of patients to tofacitinib. Methods Tofacitinib efficacy was studied in vivo in wild type (WT) and T-cell-specific PTPN2 deficient mice (CD4-Cre;Ptpn2 floxed) in which the JAK/STAT signaling pathway is over activated. WT and PTPN2 deficient mice were gavaged with tofacitinib (50mg/kg, twice daily) or vehicle. Acute DSS-colitis was induced. Colitis development was evaluated by weight loss, colonoscopy and histology. CD4+ T cells were isolated from the colon and analyzed by flow cytometry. To study the effect of tofacitinib on T-cell differentiation, we isolated naïve T cells from mouse spleen and polarized them in vitro to different T-cell subsets with or without tofacitinib. CD4+ T cells differentiation and cytokine production were analyzed by flow cytometry. To evaluate the influence of tofacitinib on human CD4+ T cells, human peripheral blood mononuclear cells (PBMCs) from healthy donors and IBD patients were stimulated in presence of tofacitinib, and analyzed by flow cytometry. Results While no protective effect was found after tofacitinib treatment in WT mice, PTPN2 deficient mice were protected from colitis based on less weight loss, lower endoscopic and histological scores. The expression of pro-inflammatory cytokines such as IL-17 and IFN-γ by colonic CD4+ T cells was also decreased by tofacitinib. Consistent with the in vivo observations, in vitro experiments revealed a strong impact of tofacitinib on CD4+ T-cells cytokine production. In PBMCs from IBD patients, IFN-γ and TNF-α expression was strongly impacted. In contrast, in healthy donors, IL-10 was the most impacted cytokine. Finally, tofacitinib decreased the in vitro differentiation of Th1, Th2, Th17, Th22, Treg and Tr1. Conclusion In the T-cell-specific PTPN2 deficient mice, tofacitinib exerts a protective effect after DSS-induced colitis. In line with the in vivo findings, in vitro experiments show that tofacitinib has a strong impact on pro-inflammatory cytokine production, especially in the IBD patients. Taken together, these data suggest that tofacitinib might be suitable primarily for IBD patients where the JAK/STAT signaling pathway is over activated.

698 Targeting HLA-G-mediated immunosuppression with a first-in-class antagonist antibody

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A740-A740
Author(s):  
Douglas Hodges ◽  
Christina Kochel ◽  
Michael Totagrande ◽  
Jeffrey Jones ◽  
Megan Welch ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Solid Tumors ◽  
Immune Cells ◽  
Target Cells ◽  
Human Antibody ◽  
Advanced Solid Tumors ◽  
Tumor Types

BackgroundHuman leukocyte antigen-G (HLA-G) is an immune checkpoint molecule that belongs to the non-classical HLA-class I family of receptors. HLA-G restrains immune cell activation and effector function by engaging with inhibitory receptors ILT2 and ILT4. While expression of HLA-G is highly restricted under normal healthy conditions, we have demonstrated that its expression in cancer is aberrantly upregulated and broadly detected across a variety of tumor types. Tizona Therapeutics has generated a novel, fully human antibody that specifically targets HLA-G and reverses HLA-G-mediated immunosuppression. Here we present in vitro and in vivo data demonstrating the functional impact of HLA-G blockade on immune cells and evidence to support the use of TTX-080 in the clinic to treat patients with advanced solid tumors.MethodsEvaluation of HLA-G expression in cancer was performed using immunohistochemistry, flow cytometry, and gene profiling. Expression of ILT2 and ILT4 was assessed on tumor infiltrating leukocytes by flow cytometry. To demonstrate the suppressive function of HLA-G, primary human NK cells, T cells, and monocyte-derived macrophages were cultured with target cells expressing HLA-G. TTX-080 was then evaluated for its ability to reverse this suppression. In addition, TTX-080 was investigated in vivo using a disseminated xenograft tumor model.ResultsExpression of HLA-G was detected on tumor cells and tumor infiltrating leukocytes across a variety of solid tumor types. TTX-080 blocked interaction of HLA-G with both ILT2 and ILT4 and restored cytotoxicity in multiple assays using either primary NK cells or NKL cell lines. Monocyte-derived macrophages expressing ILT2 and ILT4 exhibited decreased phagocytosis of HLA-G+ target cells; this inhibition was reversed with an antigen-binding fragment of TTX-080. TTX-080 was also able to reverse HLA-G-mediated suppression of ILT2+ CD8+ T cells as assessed by degranulation and proinflammatory cytokine secretion. Notably, mice with disseminated tumors had extended median survival when treated with a single dose of TTX-080.ConclusionsTTX-080 reverses HLA-G-mediated suppression of ILT2+ and ILT4+ immune cells that are found within the tumor microenvironment. Blockade of HLA-G using TTX-080 therefore has the potential to reverse broad immune suppression in patients with advanced solid tumors by reinvigorating CD8+ T cells, enhancing NK cytolytic activity, and increasing macrophage phagocytosis.

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.

CAL-101, a Selective Inhibitor of the p110δ Isoform of Phosphatidylinositol 3-Kinase, Effectively Induces Apoptosis in Primary Chronic Lymphocytic Leukemia Cells Providing a Novel Therapeutic Strategy for the Treatment of This Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3165-3165 ◽  
Author(s):  
Sarah E. May ◽  
Adam Kashishian ◽  
Thomas S. Lin ◽  
Jeffrey A. Jones ◽  
Joseph M. Flynn ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Phase 1 ◽  
Phosphatidylinositol 3 Kinase ◽  
Phase 1 Clinical Trial

Abstract The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a pivotal role in cell proliferation and survival that underlies the biology of many cancers including CLL. Of the eight distinct mammalian isoforms of PI3K, it is the class IA PI3Ks (p110α, p110β and p110δ) that are responsible for Akt activation and cellular transformation. The p110α and p110β isoforms both have a ubiquitous tissue distribution in adults, whereas p110δ expression is restricted to cells of hematopoietic origin. Recent studies have established a dominant role of p110δ isoform in B-cell responses. Deletion or inactivation of p110δ ablates B-cell antigen receptor (BCR)-induced phosphorylation of Akt and impairs cell cycle progression. Furthermore, CD40-ligand (CD40L) dependent survival is compromised in the absence of p110δ activity. Considering the role of p110δ-mediated BCR and CD40L-CD40 signaling to the enhanced survival in normal B cells, we hypothesized that inhibition of this kinase will induce cytotoxicity in B-CLL cells. We first examined the protein expression of p110δ in primary tumor CD19+ B cells from CLL patients and show that 24/24 CLL consistently overexpressed p110δ. The expression levels of p110α and p110β however, varied more widely, and were often undetectable. Treatment of primary tumor cells with CAL-101, a novel selective p110δ inhibitor, at concentrations of 0.1–10μM resulted in significant cell killing (linear mixed model; p=0.0004). As an example, 5μM CAL-101 resulted in a median of 59.6% viable cells (n=18 CLL patient samples). CAL-101 induced cytotoxicity was accompanied by PARP and caspase 3 cleavage. Previous published studies have demonstrated that CD40L-CD40 signaling promotes activation of CLL cells (as measured by up-regulation of CD40 and CD86) and also protection from spontaneous apoptosis ex vivo. Treatment of CLL cells in the presence of CAL-101 diminished the activation markers CD40 and CD86 induced by CD40L. In addition, an increase in CLL cell viability induced by CD40L was reversed by CAL-101 treatment. Contrasting with this, diminishment of apoptosis with IL-4 was not observed. Given the common finding of innate and cellular immune effects induced by therapies utilized in CLL, we next assessed the effect of CAL-101 on normal NK cells and T cells. Treatment of NK cells and T cells in vitro from healthy volunteers had no effect on cell viability. The lack of cytotoxic effect on normal NK cells and T cells was also assessed in vivo from a completed phase I trial of healthy volunteers that serves as a forerunner to the phase 1 clinical trial in CLL and related lymphoproliferative diseases currently ongoing. Here, treatment of normal human volunteers with CAL-101 for seven days achieved peak plasma concentrations up to 5μM without changes in general hematology or subpopulations of NK cells and T cells. Overall, our results identify the p110δ isoform as a potential therapeutic target in CLL where selective cytotoxicity is observed as compared to normal immune effector cells and the very important CD40-CD40L survival pathway is disrupted. Together, these in vitro and in vivo data provide sound validation for the ongoing Phase 1 clinical trial for the treatment of patients with CLL and related lymphoid malignancies.

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