scholarly journals Defining the Immune Checkpoint Landscape in Patients (pts) with Acute Myeloid Leukemia (AML)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2900-2900 ◽  
Author(s):  
Naval Daver ◽  
Sreyashi Basu ◽  
Guillermo Garcia-Manero ◽  
Jorge E. Cortes ◽  
Farhad Ravandi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Treg Cells ◽  
Immune Checkpoints ◽  
Live Cells ◽  
Median Frequency ◽  
Healthy Controls ◽  
T Cell Subpopulations

Abstract Introduction: Immune checkpoint therapy has been successful in many solid tumors andHodgkins lymphoma.The expression and clinical implications of immune checkpoints in the tumor microenvironment in patients (pts) with AML remain poorly defined.Identification and targeting of immune checkpoints in AMLwill guide the rational selection of specific antibodies for clinical trials. Methods:Between March, 2015 and April 2016 we performed17-colormulti-parameter flow-cytometry (MFC) on bone marrows (BM) aspirate from 74 pts with AML (36 untreated AML, 38 relapsed AML). We evaluated expression of immune receptors (inhibitory receptors: PD1, CTLA4, LAG3, TIM3, and activating receptors GITR, OX40, 41BB, ICOS) on T-cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. AML blasts were assessed for both immune receptors (as above), and ligands (4-1BBL, B7-1, B7-2, ICOSL, PDL-1, PDL-2, OX40L).Eight healthy human BMs were used as control. These analyses were performed on freshly collected BM aspirate by the M. D. Anderson Cancer Center Immunotherapy Platform. Results: A total of 74 pts were evaluated. Our analyses show that T-cell populations are preserved in BM of untreated and relapsed AML patients. In untreated AML BM, median frequency of live CD3+, CD4+, and CD8+ T cells were 16.5%, 10.4%, and 5.1% respectively. In relapsed AML BM, median frequency of live CD3+, CD4+, and CD8+ T cells were 19.2%, 9.5%, and 4.5% respectively. The expression of the8 costimreceptors and 7 ligands varied significantly between individual AML pts. On analysis of all markers PD-1 and OX40 emerged as potential checkpoint receptors of importance in AML.The baseline expression of OX40 and PD-1 on the CD8, Teff, and Treg cells are shown in Figure 1. All T cell subpopulations had significantly higher PD1 expression in relapsed AML (P<0.006) and untreated AML (P<0.05) compared to healthy controls. There were no significant differences in PD1 expression between relapsed and untreated AML. OX40 expression was significantly higher on all T cell subpopulations in relapsed AML pts (P<0.006) and specifically on Teff cells in untreated AML (P<0.006) when compared to healthy controls. Moreover, Treg cells in relapsed AML pts expressed significantly higher OX40 compared to untreated AML.The expression of costimulatory receptors and ligands differed significantly between BM and PB for the same time-point in the same pt (PB data not shown here). We compared the T-cell repertoire and checkpoint receptor/ligand expression to baseline clinical characteristics (age, cytogenetics, therapy-related status) and outcomes. Pts with untreated AML with adverse karyotype had a higher expression of PDL2 on AML blasts (P=0.07) in the BM. Pts with untreated AML who responded to induction therapy had a trend to higher Teff live cells (P=0.2), and lower OX40 on CD8 cells (P=0.04) in the BM. In relapsed AML higher T-eff live cells (P=0.04) and higher OX40 expression on CD8 (P=0.009) in BM were associated with improved survival. Conclusions: Total T-cell and T-cell subset populations are preserved in the BM of pts with untreated and relapsed AML and may be manipulated by checkpoint antibodies. Clinically targetable checkpoint receptors, specifically PD1 and OX40 are overexpressed in the BM of pts with AML and may be manipulated to unleash T-cells. PD1 and OX40 expression in new (N=34) and relapsed AML (N=38), and healthy controls (N=8) PD1 and OX40 expression in new (N=34) and relapsed AML (N=38), and healthy controls (N=8) Disclosures Daver: BMS: Research Funding; Kiromic: Research Funding; Karyopharm: Honoraria, Research Funding; Pfizer: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Otsuka: Consultancy, Honoraria; Ariad: Research Funding. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

High Frequency of Circulating CD8+ Memory Stem T Cells in Acquired Aplastic Anemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3613-3613
Author(s):  
Kohei Hosokawa ◽  
Pawel Muranski ◽  
Xingmin Feng ◽  
Danielle M. Townsley ◽  
Baoying Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Autoimmune Diseases ◽  
Aplastic Anemia ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Memory T Cells ◽  
Healthy Controls ◽  
Hematopoietic Stem ◽  
Ifn Γ

Abstract Background. Aplastic anemia (AA), the prototypical bone marrow (BM) failure syndrome, is caused by immune-mediated destruction of hematopoietic stem/progenitor cells (HSPCs). CD8+ cytotoxic T cells with restricted TCR diversity (oligoclonal T cells) are expanded in AA, leading to production of proinflammatory cytokines, such as IFN-γ, which induce apoptosis of HSPCs. Recent studies have identified a new subset of memory T cells with stem cell-like properties, TSCM, which are the least differentiated cells of all distinct memory populations. Functionally, TSCM possess an enhanced capacity for self-renewal and can generate multiple memory T cell populations, and they likely have an important role in controlling immunity. In autoimmune diseases, there is abnormal CD4+ and CD8+ T cell activation. We evaluated TSCM frequency in AA and its association with severity, treatment response, relapse, and changes after immunosuppressive therapy (IST). Further, to evaluate the TSCM in other autoimmune diseases, we examined CD4+ and CD8+ TSCM frequencies in uveitis, systemic lupus erythematosus (SLE), and sickle cell disease (SCD), as compared with healthy controls. Method. We retrospectively analyzed CD4+ and CD8+ TSCM populations by flow cytometry. PB specimens were collected from 55 AA samples and 41 age-matched healthy donor samples. Among 55 AA samples, 21 samples were analyzed at diagnosis and 34 after IST. For comparison, blood samples were obtained from 34 uveitis patients (27 inactive or 7 active cases), 43 SLE patients who met the American College of rheumatology (ACR) criteria for the disease [19 inactive SLE (SLE disease activity index-2K (SLEDAI-2K) score < 3; and 24 active SLE (SLEDAI-2K score > 3)], and 5 SCD patients who were receiving frequent transfusions. TSCM was defined as CD3+ CD4 (CD8)+ CD45RO- CD45RA+ CCR7+ CD27+ CD95+ population. Results and Discussion. In healthy controls, TSCM represented a relatively small percentage of circulating CD4+ or CD8+ T cells (median 2.4% CD4+ TSCM and 2.1% CD8+ TSCM, Fig. 1A). A significantly higher CD8+ TSCM frequency was detected in AA patients (4.2% vs. 2.1%, p < 0.05) while there was no difference in the CD4+ TSCM frequency (p > 0.05), compared to controls (Fig. 1B-C). In AA, high CD8+ TSCM frequency at diagnosis correlated with complete (CR) or partial response (PR) to IST [5.0 % in CR and PR vs 2.8 % in non-responders (NR), p < 0.05). In AA patients prior to IST (n=21), CD8+ TSCM frequency was not correlated with age, sex, absolute neutrophil count, platelet count, time from diagnosis to therapy, and serum ferritin levels. CD8+ TSCM were significantly increased in the two AA cohorts (with or without IST), relative to controls (p < 0.05, respectively). Higher CD8+ TSCM frequency after IST associated with treatment-failure (3.5 % in responders vs 5.5 % in NR or relapse, p < 0.05). Stimulation with anti-CD3/CD28 beads successfully induced cytokine production in CD4+ and CD8+ T cells from AA and healthy controls. Elevated IFN- γ and IL-2 levels were seen in CD4+ and CD8+ TSCM in AA compared to healthy controls. We next compared CD4+ or CD8+ TSCM frequency between each patient group (AA, uveitis, SLE, or SCD) and a healthy control group. Among the four patient groups, the uveitis group alone displayed a reduction in CD4+ TSCM frequency (1.8%) relative to the healthy controls (2.4 %; p < 0.05). An elevated CD8+ TSCM frequency was observed in AA (4.2 %), uveitis (3.6 %), and SCD (4.3 %), but not in SLE, compared to controls (2.1%; p < 0.05) (Fig. 2A). Positive correlation between CD4+ and CD8+ TSCM frequencies was found in AA, autoimmune uveitis, and SLE (Fig. 2B). Evaluation of PD-1 expression revealed that TSCM were the least exhausted T cell compartment, as compared to other types of memory T cells. Immune therapies appeared to have negative effects on the TSCM population both in uveitis and SLE patient cohorts, as well as in AA. Conclusion. We provide evidence for increased circulating CD8+ TSCM in AA, underscoring the importance of this novel subset in regulation of immune responses and pathogenesis of autoimmunity. Our work described previously unknown potential roles of TSCM in AA, such as cytokine secretion correlated with effector functions. Understanding the CD8+ TSCM population may offer new therapeutic strategies and novel mechanistic insight into the various autoimmune diseases. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding. Dumitriu:Novartis: Research Funding; GSK: Research Funding. Young:Novartis: Research Funding; GSK: Research Funding.

scholarly journals Tyrosine Kinase Inhibitor Imatinib Enhances Tumor Immunity By Depleting Functionally Mature Regulatory T Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2219-2219 ◽  
Author(s):  
Shinsuke Noguchi ◽  
Hiroyoshi Nishikawa ◽  
Hirobumi Saitoh ◽  
Atsushi Ohshima ◽  
Kenichi Sawada ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Board Of Directors ◽  
Tumor Immunity ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Treg Cells ◽  
Advisory Committees

Abstract Introduction Naturally occurring regulatory (Treg) cells expressing the transcription factor FoxP3 are actively engaged in the maintenance of immunological self-tolerance and homeostasis. On the other hand, they are hindering effective anti-tumor immune responses in cancer patients, indicating possible augmentation of tumor immunity by depletion of FoxP3+ Treg cells. Here we show that this can be achieved by imatinib, a tyrosine kinase inhibitor of the oncogenic BCR-ABL fusion protein specifically expressed in chronic myelogenous leukemia (CML) cells. Methods Peripheral blood samples were obtained from healthy individuals and CML patients. PBMCs were directly subjected to ex vivo staining for the analyses of FoxP3+CD4+ T cells and activation status of CD8+ T cells. Cytokine production by CD8+ T cells was detected by intracellular cytokine staining after stimulation with PMA/Ionomycin using BD Cytofix/Cytoperm Fixation/Permeabilization Kit. To detect the effect of TKI on T cells, CFSE-labelled CD8+ T cells, CD45RA+CD25lo nTreg cells, CD45RA-CD25hi eTreg cells and CD25- T cells were stimulated for 5 days with Dynabeads Human T-Activator CD3/CD28 (Life technologies) in the presence/absence of imatinib, nilotinib or dasatinib, and then assessed for proliferation and induction of apoptosis. Flow cytometric analyses were performed with an LSR Fortessa (BD Bioscience) and analysed with FlowJo Version 9.6.2 software. For investigation of imatinib target(s), expression levels of mRNA, and total and phosphorylated protein by sorted CD8+ T cells, CD45RA+CD25lo nTreg cells, CD45RA-CD25hi eTreg cells and CD25- T cells were analysed by standard quantitative real-time PCR and western blotting. Results Long-term imatinib-treated CML patients in complete molecular remission with no detection of BCR-ABL mRNA in the blood cells showed selective depletion of terminally differentiated and highly suppressive FoxP3+ effector Treg cells whereas those not in molecular remission did not (Figure1). The former concurrently exhibited a general increase in the number of effector- or memory-type CD8+ T cells producing multiple cytokines. In vitro, imatinib induced apoptosis predominantly in effector Treg cells, augmenting CD8+ T-cell responses against various tumor antigens in healthy individuals and cancer patients (Figure 2). It inhibited tyrosine phosphorylation of LCK (lymphocyte-specific protein tyrosine kinase), a T cell-specific signaling molecule, as an off-target effect. In Treg cells, FoxP3-dependent gene repression maintained at low levels the expression of LCK and its down-stream signaling molecule zeta-chain-associated protein kinase 70 (ZAP-70) (Figure 3). Conclusion Imatinib is, therefore, able to attenuate T-cell receptor (TCR) signaling intensity more profoundly in Treg cells than in other T cells, rendering the former more susceptible to signal-deprived apoptotic cell death. Thus, small molecules that can selectively deplete Treg cells via exploiting FoxP3-dependent Treg-cell functions are instrumental in evoking and augmenting anti-tumor immunity against various cancers. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Takahashi: Celgene: Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sysmex: Research Funding, Speakers Bureau; Masis: Consultancy; Otsuka: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Speakers Bureau.

scholarly journals Somatic Mutations in CD8+ T Cells in Patients with Chronic Immune Thrombocytopenia Are Associated with Increased Clonality and Cytotoxic Phenotype of CD8+ T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 131-131
Author(s):  
Hanna Rajala ◽  
Paula Savola ◽  
Sofie Lundgren ◽  
Samuli Eldfors ◽  
Tiina Kelkka ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Deep Sequencing ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Somatic Mutations ◽  
Healthy Controls ◽  
Variant Allele Frequency

Abstract Background The role of self-antigen-targeting T cells is well established in multiple autoimmune disorders. In immune thrombocytopenia (ITP), CD8+ cytotoxic T cells target both peripheral blood platelets and bone marrow megakaryocytes. In addition, CD4+ helper T cell imbalance and cytokine secretion leads to the release of autoantibodies by B cells favoring the destruction of platelets. In order to understand if somatic mutations play a role and drive the aberrant immune responses in ITP, we analyzed sorted CD4+ and CD8+ T cells with deep sequencing panel. Methods The study population consisted of 13 adult patients diagnosed with chronic ITP (median age 47 years) at least one year before the first sampling. The median number of lines of therapy was 4, (range 0-8). In addition, 11 healthy control samples were collected for T cell phenotyping and TCR analysis (median age 63 years). After separation of peripheral blood mononuclear cells, immunophenotyping by flow cytometry was performed for CD4+ and CD8+ T cells and both fractions were sorted using magnetic beads. The CD4+ and CD8+ clonality was analyzed by deep sequencing of TCRB CDR3 using Adaptive platform. Custom-made deep sequencing gene panel covering 2433 genes related to immunity and cancer was used to analyze somatic mutations from 11 ITP patients. Mean target coverage was 567X (range 405-757X). Somatic variants with over 2% variant allele frequency (VAF) were called using Varscan2-based bioinformatics tool and CD4+ and CD8+ fractions were used as each other's germline control. Candidate somatic variants were visually validated with Integrative Genomics Viewer. Results ITP patients harbored clonal TCR rearrangements both in CD8+ and CD4+ fractions. The median percentage of the largest T cell clone of all TCR rearrangements in an individual sample was 9.5% (range 1.8%-31.9%) in CD8+ cells and 7.1% (range 0.9-13.6%) in CD4+. The median clone size or clonality index did not differ between ITP patients and healthy controls. Age correlated with the largest rearrangement and clonality index both in CD4+ and CD8+ fractions in ITP patients (r=0.70, p=0.015 and r=0.60, p=0.043 for CD4+ and r=0.63, p=0.033 and r=0.77, p=0.0050 for CD8+), but no correlation was observed in healthy controls. The size of the maximum CD8+ TCR rearrangement and clonality index correlated positively with the percentage of highly cytotoxic CD8+CD57+ (r=0.80, p=0.0029 and r=0.90, p=0.0002) and terminally differentiated CD8+ effector memory with CD45RA (TEMRA) T cells (r=0.66, p=0.022 and r=0.78, p=0.0038). In healthy controls positive correlation was observed only between CD8+ clonality index and CD8+CD57+ T cells, but not with TEMRAs. Somatic mutations in CD8+ T cells were detected in 7/11 (64%) of ITP patients. Somatic variants were distinct between individuals and their mean variant allele frequency (VAF) was 4.5% (range 2.0-13.6%). The mutated genes included those related to IFNg-signaling pathway (STAT1, NLRP4, CARD6, and EBF1), apoptosis (CASP6), and inflammation and cancer (ATM, EGR1, NOD1). ITP patients with CD8+ mutations had larger immunodominant CD8+ TCR clone (Figure 1A) and higher CD8+ clonality index (Figure 1B). The patients with CD8+ mutations had more cytotoxic CD8+CD57+ T cells than mutation-negative cases, but CD8+TEMRAs did not differ between the two groups (Figure 1C). The mutation profile of the CD4+ samples was different: 3/11 (27%) of the ITP patients had mutations in the genes connected to clonal hematopoiesis of indeterminate potential (CHIP). One patient with a maximum of 8% clone in CD4+ T cells harbored mutations both in TET2 (VAF 13.5%) and PPM1D (VAF 12.8%): the VAFs suggest that the mutation was not restricted to the largest CD4+ clone. The second patient had a different mutation in PPM1D gene (VAF 2.8%) and the third one had a PPM1B-mutation both in CD4+ (VAF 18.0%) and CD8+ (VAF 11.1%) T cells. Conclusions To our knowledge, this is the first report of somatic variants in T cells in patients with ITP. Discovered somatic mutations in CD8+ T cells were associated with increased clonality and highly cytotoxic phenotype of CD8+ T cells. Interestingly, three patients harbored mutations in CHIP-associated genes in CD4+ and also in CD8+ fraction, with recurrent PPM1D-mutations. Further studies and screening of a larger ITP patient cohort is warranted to understand the meaning and functional consequence of somatic mutations in the pathogenesis of chronic ITP. Disclosures Ebeling: Boehringer Ingelheim: Consultancy; Celgene: Speakers Bureau; Otsuka Pharma Scandinavia AB: Consultancy. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Research Funding; Celgene: Honoraria; Pfizer: Honoraria, Research Funding.

scholarly journals Mass Cytometry Reveals Heterogeneity in the Exhaustion Profile of T-Cells in Chronic Lymphocytic Leukemia and the CD4:CD8 Ratio May be a Reliable Predictor of CD8+ T Cell Compartment "Fitness"

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 353-353 ◽  
Author(s):  
Muharrem Muftuoglu ◽  
Li Li ◽  
Han Chen ◽  
Duncan Mak ◽  
Elif Gokdemir ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Healthy Controls ◽  
Inhibitory Receptors ◽  
Cell Exhaustion ◽  
Healthy Donors ◽  
Checkpoint Receptors ◽  
Inhibitory Molecules

Abstract T cell exhaustion is characterized by coordinated expression of a series of negative checkpoint receptors such as programmed death-1 (PD-1), 2B4, CD160 and TIGIT, resulting in T cell dysfunction and immune evasion. Under physiological states, these inhibitory molecules maintain self-tolerance and prevent autoimmunity by applying a break on cytotoxic T cells. In cancer, T-cells exhibit features of T-cell exhaustion including increased expression of PD-1, 2B4 and CD160, coupled with reduced T cell proliferation, altered synapse formation and impaired cytotoxicity. Although the role of the PD-1/PD-L1 axis in mediating T cell defects in chronic lymphocytic leukemia (CLL) is well-studied, the contribution of other checkpoint molecules such as 2B4, CD160 and TIGIT in mediating tumor-induced immune dysfunction remains to be determined. Checkpoint inhibitors have provided a paradigm-shifting approach to cancer treatment. We hypothesized that the expression levels of checkpoint receptors on T-cells, as well as the "fitness" of the T cell compartment may provide a prognostic stratification system to predict response to checkpoint inhibitors in CLL. To determine if the number of inhibitory receptors per cell and their expression level may identify patient-to-patient differences that may not be easily deciphered using conventional research tools, we performed a detailed single-cell analysis of the T-cell repertoire, using 40-parameter mass cytometry (CyTOF) in 12 untreated CLL and 12 healthy controls. Consistent with previous reports, we found that expression of 2B4 (43.7% vs 30.8%), PD1 (28.8% vs 21%) and CD160 (17% vs. 9.7%) was significantly higher on CLL CD8+ T cells compared to healthy controls. In addition, CD8+T cells in CLL expressed higher levels of TIGIT (48.2% vs 25.2%), CD57 (43.9% vs 17.9%) and KLRG1 (49.5% vs. 29.7%). We clearly distinguished 2 patterns of exhaustion marker distribution in CLL. In one group of patients, the expression of checkpoint receptors was similar to that seen in healthy controls, whereas in the second group, CD8+ T-cells expressed higher levels of PD1, 2B4, TIGIT, CD160 as well as markers of terminal differentiation such as CD57 and KLRG1. Compared to healthy donors, CLL was characterized by an inversion in the CD4:CD8 ratio. Interestingly, CD8+ T cells in patients with a low CD4:CD8 ratio (defined as <2.5) expressed significantly higher levels of 2B4 (56.6% vs 31.25%), TIGIT (62.9% vs 37%), CD160 (22.8% vs 12.6%), CD57 (57% vs 28.7), PD-1 (34.6% vs 24.5%) and KLRG1 (62.3% vs 36.3%). In contrast, the expression levels of PD-1, 2B4 and CD160 in CLL patients with a CD4:CD8 ratio of ≥2.5 were similar to that seen in healthy controls, suggesting that the CD4:CD8 ratio may be a valuable marker of T cell exhaustion in CLL. Next, we compared the number of checkpoint molecules expressed per CD8+ T-cells in CLL patients versus healthy donors. Whereas a similar proportion of CD8+ T-cells in CLL (mean 19.56%, range 18.34-31.73%) and healthy donors (mean 22.13%, 14.17-41.19%) expressed one inhibitory receptor, a significantly higher proportion of CLL patients expressed 2 and more inhibitory receptors (mean 28.4, range 10.52-48.78%) compared to healthy controls (mean 15.38%, range 9.67-21.94%). PD-1 was mostly co-expressed with TIGIT, although TIGIT+PD-1+CD4+ and CD8+ T-cells were higher in CLL compared to healthy controls (12.9% vs 7.1%). Interestingly the predominant population of PD-1+CD8+ T cells in CLL was also positive for 2B4 and TIGIT, whereas expression of TIGIT was more diverse and was seen in association with PD-1, 2B4, KLRG1 or CD57. Taken together, our findings indicate a remarkable heterogeneity in the expression patterns of inhibitory molecules on CD8+ and CD4+ T-cells in CLL. While CLL patients with a normal CD4:CD8 ratio expressed comparable levels of inhibitory molecules to that seen in healthy controls, a low CD4:CD8 ratio was indicative of higher expression of checkpoint molecules. On a per cell basis, CLL CD8+ T cells expressed more inhibitory receptors compared to healthy controls, suggesting that certain patients may benefit from combinational use of checkpoint molecules. A more detailed data and analysis, including transcription and functional profile of exhausted CLL T cells, will be presented in the meeting. Disclosures Wierda: Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding; Gilead: Research Funding; Genentech: Research Funding. Jain:Incyte: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Abbvie: Research Funding; Infinity: Research Funding; BMS: Research Funding; Genentech: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Seattle Genetics: Research Funding; Celgene: Research Funding; Servier: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria.

An Essential Role for STAT1 Signaling in Regulation of Donor CD4+ T Cell Dependent Acute Graft Versus Host Disease (GVHD) Following Allogeneic Bone Marrow Transplantation (BMT).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3551-3551
Author(s):  
Huihui Ma ◽  
Caisheng Lu ◽  
Judith Ziegler ◽  
Suzanne Lentzsch ◽  
Markus Y. Mapara
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Allogeneic Bone Marrow Transplantation ◽  
Treg Cells ◽  
Allogeneic Bone ◽  
Target Tissue

Abstract Abstract 3551 Poster Board III-488 We have previously demonstrated that activation of STAT1 and STAT3 in target tissue and secondary lymphoid organs belong to the earliest events during induction of GVHD. Using STAT1-gene-deficient (STAT1KO) mice we tested the role of donor STAT1 in fully MHC-mismatched (129Sv[H2b] to BALB/c [H2d]) and MHC-matched minor histocompatibility antigen (mHA)-mismatched strain combinations (129Sv[H2b] to B6[H2b]). GVHD was induced lethal irradiation and transplantation of allogeneic donor bone marrow cells and whole spleen cells. GVHD in the MHC-mismatched model is primarily CD4 dependent. Induction of GVHD was associated with activation of STAT1 and significant expansion of activated STAT1 expressing CD4+ and CD8+ T cells as assessed by analysis of STAT1 Tyr701 phosphorylation using phosphoflow staining. Using STAT1KO whole splenocytes we were able to show that lack of STAT1 significantly inhibited development of GVHD in both major and mHA mismatched recipients with significantly extended median survival times (MST) and lower GVHD morbidity. Protection against GVHD in recipients of STAT1KO splenocytes was associated with significant contraction of CD8+ T cells, but expansion of CD4 T cells on days +3 and +6 post-BMT in the MHC-mismatched setting. Most importantly, we observed a significant expansion of CD4+CD25+ FOXP3+ Treg cells in recipients of STAT1KO splenocytes. Lack of STAT1 in donor splenocytes resulted in a significantly attenuated and skewed systemic inflammatory response on day +6 post-BMT as demonstrated by significantly reduced IFN-g levels 508pg/ml vs 84.pg/ml (p<0.05), but significantly increased IL-4 (p=0.003), IL-5 (p=0.007) and IL-17 (p=0.03) levels. IL-6 levels were also increased with a trend towards statistical significance (p=0.08). In vitro studies demonstrated that STAT1KO CD8+ T cells produced much less IFN-g upon combined engagement of TCR and costimulation, but that this decrease in IFN-g secretion could be rescued if cells were simultaneously cultured under Th1 conditions (ie in the presence of IL-12 and anti-IL4 antibody). In contrast, lack of STAT1 completely inhibited the differentiation of naïve CD4+ T cells to IFN-g -producing cells upon TCR commitment and this capacity was also severely impaired under Th1 conditions. Furthermore, we observed a significantly reduced number of CXCR3−expressing CD4+ T cells in recipients of STAT1 KO splenocytes. In parallel to the afore-mentioned observations, tissue samples from BMT mice on day +3 and day +6 showed significantly less inflammation in liver and gut in recipients of STAT1 KO splenocytes compared to wild type cells. These data indicate that donor STAT1 is important for the induction of acute GVHD and that attenuation of GVHD in the absence of STAT1 involves expansion of Treg cells, perturbation of T cell polarization and subsequent reduced expression of the chemokine receptor CXCR3 on donor T cells leading to impaired target organ infiltration. Disclosures: Lentzsch: Celgene: Consultancy, Research Funding; cephalon: Consultancy, Research Funding. Mapara:Genzyme: Membership on an entity's Board of Directors or advisory committees; Resolvyx: Consultancy, Honoraria, Research Funding; Gentium: Stock Ownership.

T Cells from Chronic Lymphocytic Leukemia (CLL) Patients Display Dysregulated Expression of Immune Checkpoint Molecules and Activation Markers Mainly Restricted to CD4+ Cells and Correlated with Disease Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4132-4132
Author(s):  
Marzia Palma ◽  
Giusy Gentilcore ◽  
Fariba Mozaffari ◽  
Kia Heimersson ◽  
Barbro Näsman-Glaser ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Immune Checkpoints ◽  
Activated T Cells ◽  
Activation Markers ◽  
Cd8 Cells

Abstract Background CLL patients (pts) have impaired humoral and cellular immune functions, which is largely due to profound defects of T-cells. Regulation and activation of T lymphocytes depend not only on T cell receptor signaling but also on co-signaling receptors delivering either inhibitory or stimulatory signals, known as immune checkpoints. CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) is transiently expressed on activated T cells, binding the same ligands as CD28, inhibiting T-cell activation. Similarly, programmed cell death protein 1 (PD-1) is expressed on activated CD4+ and CD8+ T cells inhibiting T-cell functions upon binding to the ligands B7-H1 (PD-L1, CD274) and B7-DC (PD-L2, CD273). CD137 is an inducible costimulatory receptor expressed by activated T cells. Dysregulated expression of immune checkpoint receptors on T cells of CLL pts may have an impact on T-cell responsiveness and might be a mechanism for the immune deficiency in the disease. Aim To evaluate the expression of the immune checkpoint molecules CTLA-4, PD-1 and CD137 as well as of the cell proliferation marker Ki67, the activation marker CD69 and of CD103, a marker expressed on regulatory T cells, in T cells from CLL pts in different disease phases. Methods Peripheral blood samples were obtained from 69 CLL pts and 13 healthy control donors (HD). Pts were sub-grouped according to disease phase: indolent vs progressive (i.e. fulfilling criteria for active disease). The expression of CTLA-4, PD-1, PD-L1, CD69, CD103, CD137 and Ki-67 was assessed by flow-cytometry on CD4+ and CD8+ T cells. We also analysed the change in expression of these markers on T cells after 72 hours of PHA stimulation. Results CLL pts (n=17) had a significanty higher percentage of proliferating (Ki67+) CD3+ cells compared to HD (n=7) (median 3.7% in progressive vs 1.7% in indolent CLL vs 0.9% in HD, p=0.004 and p=0.04, respectively) (Fig.1). Progressive CLL pts had a significantly higher percentage Ki67+ CD4+ compared to indolent pts as well as HD (p=0.007 and p=0.001, respectively). Both indolent and progressive pts had higher percentage of Ki67+ CD8+ T cells compared to HD (p=0.01 and p=0.03, respectively). The percentage of CTLA-4+ CD4+ and CTLA-4+ CD8+ cells was low in CLL pts as well as in HD. However, the percentage of PD-1+ CD4+ T cells was significantly higher in progressive (n=32) as compared to indolent (n=35) CLL pts (median 40.3% vs 23.3%, p<0.0001) and HD (n=13) (median 21.5%, p<0.0001) (Fig.2) and correlated positively to the white blood cell counts (WBC) at the time of testing (r=0.29, p=0.03), while no difference was found with regard to the percentage of PD-1+ CD8+ T cells. No difference was observed between CLL pts and HD regarding the expression of PD-L1 on T cells. Both the percentage of CD69+ CD4+ and CD137+ CD4+ T cells were significantly higher in progressive as compared to indolent disease and correlated positively to WBC while no difference was found seen in CD8+ T cells. The percentage of CD103+ T cells was significantly lower in progressive compared to and HD within both the CD4+ (p=0.02) and the CD8+ subpopulations (p=0.02). After 72-hrs of PHA stimulation, PD-1 and CTLA-4 expression increased in CD4+ and CD8+ cells to a similar extent in CLL pts and HD, while PD-L1 increased in HD but not in progressive CLL pts (p=0.03 and p=0.007 for CD4+ and CD8+ cells, respectively). CD69 expression increased to a similar extent in CLL pts and HD, while CD137 expression increased more in T cells from progressive pts compared to HD (p=0.03 and 0.01 for the CD4+ and CD8+ cells, respectively). No increase in CD103 on CD8+ T-cells was observed in CLL pts compared to HD (p=0.04 and p=0.01 for the indolent and progressive pts, respectively). Conclusions Progressive CLL pts have more proliferating (Ki67+) T cells in both the CD4+ and CD8+ compartments compared to HD. CD4+ T-cells in progressive CLL pts display an activated phenotype (CD69+) and express the immune co-stimulatory molecule CD137 at a significantly higher level compared to indolent pts and HD. Nevertheless, the expression of the inhibitory immune checkpoint molecule PD-1 is so high that it is reasonable to assume that these cells are heavily impaired in their immune functions. The differences observed in the expression of immune checkpoints and activation markers between CLL pts in different phases of the disease suggest that major changes occur in the CD4+ T-cell compartment during disease progression. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Hansson: Jansse Cilag: Research Funding. Österborg:Janssen, Pharmacyclics, Gilead: Consultancy, Research Funding; Novartis: Research Funding.

scholarly journals Decreased numbers and sex-based differences of circulating regulatory T cells in patients with seropositive undifferentiated arthritis

2021 ◽  
Vol 12 ◽  
pp. 204062232098672
Author(s):  
Hong-Qing Niu ◽  
Chenrui Yuan ◽  
Chenglan Yan ◽  
Na Li ◽  
Yuan-Sheng Lei ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Flow Cytometric Analysis ◽  
Treg Cells ◽  
T Cell Subsets ◽  
Joint Disease ◽  
Healthy Controls ◽  
Undifferentiated Arthritis ◽  
Cell Subpopulations ◽  
T Cell Subpopulations

Aims: CD4+ T cells play crucial roles as both mediators and regulators of the pathogenesis of rheumatoid arthritis (RA). However, the characteristics of CD4+ T cell subpopulations in the earliest stage of RA development remain unclear. Hence, we determined the proportions and absolute counts of circulating CD4+ T cell subsets in patients with seropositive undifferentiated arthritis (SUA), the early and preclinical stage of RA. Methods: Peripheral blood samples and clinical information were collected from 177 patients with SUA, 104 patients with RA, and 120 healthy controls. All patients were newly diagnosed and untreated. Proportions and absolute counts of CD4+ T cell subpopulations were determined by flow cytometric analysis. Results: In patients with SUA, percentages and absolute counts of circulating regulatory T (Treg) cells were decreased significantly and Th17/Treg cell ratios were abnormally increased, whereas Th17 cell numbers were similar to those in healthy controls. In addition, sex-based differences in circulating Treg cells were observed, with female SUA patients having lower proportions and absolute counts of Treg cells than those in males. Moreover, female patients with SUA had higher erythrocyte sedimentation rates and 28-joint Disease Activity Scores than those in males. Conclusion: Immune tolerance deficiency resulting from an abnormal reduction in circulating Treg cells might be the most crucial immunological event in the earliest stage of RA. The sex-specific disparity in Treg cells should also be considered for immunoregulatory and preventive strategies targeting early RA.

scholarly journals Alterations in regulatory T cells and immune checkpoint molecules in pancreatic cancer patients receiving FOLFIRINOX or gemcitabine plus nab-paclitaxel

2021 ◽  
Author(s):  
L. Sams ◽  
S. Kruger ◽  
V. Heinemann ◽  
D. Bararia ◽  
S. Haebe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Progression Free Survival ◽  
Ductal Adenocarcinoma ◽  
Immune Checkpoints ◽  
Prognostic Information ◽  
Peripheral Blood Mononuclear

Abstract Purpose This pilot study aimed on generating insight on alterations in circulating immune cells during the use of FOLFIRINOX and gemcitabine/nab-paclitaxel in pancreatic ductal adenocarcinoma (PDAC). Patients and methods Peripheral blood mononuclear cells were isolated before and 30 days after initiation of chemotherapy from 20 patients with advanced PDAC. Regulatory T cells (FoxP3+) and immune checkpoints (PD-1 and TIM-3) were analyzed by flow cytometry and immunological changes were correlated with clinical outcome. Results Heterogeneous changes during chemotherapy were observed in circulating T-cell subpopulations with a pronounced effect on PD-1+ CD4+/CD8+ T cells. An increase in FoxP3+ or PD-1+ T cells had no significant effect on survival. An increase in TIM3+/CD8+ (but not TIM3+/CD4+) T cells was associated with a significant inferior outcome: median progression-free survival in the subgroup with an increase of TIM-3+/CD8+ T cells was 6.0 compared to 14.0 months in patients with a decrease/no change (p = 0.026); corresponding median overall survival was 13.0 and 20.0 months (p = 0.011), respectively. Conclusions Chemotherapy with FOLFIRNOX or gemcitabine/nab-paclitaxel induces variable changes in circulating T-cell populations that may provide prognostic information in PDAC.

scholarly journals 754 TIGIT-PVR is a key immune checkpoint and therapeutic target in HPV-positive head and neck squamous cell carcinomas

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A788-A788
Author(s):  
Xiuning Le ◽  
Minghao Dang ◽  
Venkatesh Hegde ◽  
Bo Jiang ◽  
Ravaen Slay ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Murine Model ◽  
Immune Checkpoint ◽  
Therapeutic Target ◽  
Therapeutic Efficacy ◽  
Cd8 T Cell ◽  
Immune Checkpoints ◽  
Rna Seq

BackgroundHuman papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HPV+ HNSCC) is a disease that has moderate response to anti-PD-1/L1 immune checkpoint blockade, with the response rates less than 20% and median progression-free survival less than 3 months. A greater understanding of tumor intrinsic and extrinsic factors that restrict anti-tumor immunity in the tumor immune microenvironment (TIME) is needed to identify other immune checkpoints to enhance therapeutic efficacy.MethodsTwo cohorts (TCGA n=72 and a separate cohort n=84) of surgically resected, treatment-naïve HPV+ HNSCC with RNA-seq were analyzed to understand the immune features. In addition, single-cell RNA-seq and TCR-seq were performed on 18 cases to further delineate the immune molecules' interactions. An immune-competent murine HPV+ HNSCC model was used to preliminarily evaluate the therapeutic efficacy.ResultsIn two bulk-sequenced HPV+ HNSCC cohorts, TIGIT ligands PVR and NECTIN2 were found to associate with an epithelial-to-mesenchymal gene expression signature, suppression of IFNα and IFNγ signaling, a stromal-enriched or immune-excluded TIME, and poor survival. Single-cell RNA-seq of over 72,000 cells of HPV+ HNSCC revealed that the PVR/NECTIN ligand TIGIT was highly prevalent in T-cells (34%), significantly higher than PD1- (20%, p<0.01). There is an enrichment of cell-cell interactions mediated by TIGIT-PVR/NECTIN2 in the TIME of HPV+HNSCC versus normal tonsil. TIGIT was the most differentially upregulated immune checkpoint on clonally expanded CD8+T-cells and was abundant on antigen-experienced, tissue-resident memory CD8+T-cell and T-regulatory subsets. TIGIT ligands PVR, NECTIN1, and NECTIN2 were abundant on mature regulatory dendritic cells (DCs), immunosuppressive plasmacytoid (p)DCs, and macrophages, respectively. TIGIT and PD-1 co-blockade in the mEER syngeneic murine model significantly reduced tumor growth, improved survival, restored effector function of HPV16E7-specific CD8+T cells, natural killer cells, and DCs, and conferred tumor re-challenge protection.ConclusionsTIGIT-PVR/NECTIN receptors/ligands are more abundant than PD-1/L1 in the TIME of HPV+ HNSCC. Co-blockade of TIGIT and PD-1 immune checkpoints enhanced anti-tumor efficacy in a CD8+ T-cell-dependent manner and conferred long-term immune protection in a murine model. Our study nominates TIGIT as a therapeutic target for HPV+ HNSCC.

scholarly journals Phase 2 Study of Combination of Cytarabine, Idarubicin, and Nivolumab for Initial Therapy of Patients with Newly Diagnosed Acute Myeloid Leukemia

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 815-815
Author(s):  
Farhad Ravandi ◽  
Naval Daver ◽  
Guillermo Garcia-Manero ◽  
Christopher B Benton ◽  
Philip A Thompson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
T Cell Subsets ◽  
Newly Diagnosed ◽  
Effector Cells ◽  
T Effector Cells ◽  
Grade 3

Abstract Background: Blocking PD-1/PD-L1 pathways enhances anti-leukemia responses by enabling T-cells in murine models of AML (Zhang et al, Blood 2009). PD-1 positive CD8 T-cells are increased in bone marrow (BM) of pts with AML (Daver et al, AACR 2016). PD1 inhibition has shown activity in AML (Berger et al, Clin Cancer Res 2008). We hypothesized that addition of nivolumab to an induction regimen of ara-C and idarubicin may prolong relapse-free survival (RFS) and overall survival (OS); this study was designed to determine the feasibility of this combination. Methods: Pts with newly diagnosed acute myeloid leukemia (by WHO criteria; ≥20% blasts) and high risk MDS (≥10% blasts) were eligible to participate if they were 18-65 yrs of age and had adequate performance status (ECOG ≤3) and organ function (LVEF ≥ 50%; creatinine ≤ 1.5 g mg/dL, bilirubin ≤ 1.5 mg/dL and transaminases ≤ 2.5 times upper limit of normal). Treatment included 1 or 2 induction cycles of ara-C 1.5 g/m2 over 24 hours (days 1-4) and Idarubicin 12 mg/m2 (days 1-3). Nivolumab 3 mg/kg was started on day 24 ± 2 days and was continued every 2 weeks for up to a year. For pts achieving complete response (CR) or CR with incomplete count recovery (CRi) up to 5 consolidation cycles of attenuated dose ara-C and idarubicin was administered at approximately monthly intervals. Eligible pts received an allogeneic stem cell transplant (alloSCT) at any time during the consolidation or thereafter. Results: 3 pts with relapsed AML were treated at a run-in phase with a dose of nivolumab 1 mg/kg without specific drug-related toxicity. Subsequently, 32 pts (median age 53 yrs; range, 26-65) were treated as above including 30 with AML (24 de novo AML, 2 therapy-related AML, 3 secondary AML and 1 therapy-related secondary AML) and 2 high risk MDS. Pre-treatment genetic risk by ELN criteria was 11 adverse, 16 intermediate, and 5 favorable, including 2 FLT3 -ITD mutated, 5 NPM1 mutated, and 7 TP53 mutated. All 32 pts were evaluable for response and 23 (72%) achieved CR/CRi (19 CR, 4 CRi). The 4-week and 8 week mortality was 6% and 6%. The median number of doses of nivolumab received was 6 (range, 0-13); one pt did not receive nivolumab due to insurance issues. 9 pts underwent an alloSCT. After a median follow-up of 8.3 mths (range, 1.5-17.0) the median RFS among the responding pts has not been reached (range, 0.1 - 15.8 mths) and the median OS has not been reached (range 0.5-17.0 mths). Grade 3/4 immune mediated toxicities have been observed in 5 pts and include rash, pancreatitis, and colitis. Other grade 3/4 toxicities thought to be potentially related to nivolumab include cholecystitis in one pt. 9 pts proceeded to an alloSCT. Donor source was matched related in 2, matched unrelated in 6 and haplo-identical in 1 pt. Conditioning regimen was Fludarabine plus busulfan-based in 8, and fludarabine plus melphalan in 1 pt. 4 pts developed graft versus host disease (GVHD)(grade I/II in 3, grade III/IV in 1), which responded to treatment in 3. Multicolor flow-cytometry studies are conducted by the Immunotherapy Platform on baseline (prior to first dose of nivolumab) and on-treatment BM aspirate and peripheral blood to assess the T-cell repertoire and expression of co-stimulatory receptors and ligands on T-cell subsets and leukemic blasts, respectively. The baseline BM was evaluated on 23 of the 32 evaluable pts, including 18 responders and 5 non-responders. Pts who achieved a CR/CRi had a trend of higher frequency of live CD3+ total T cell infiltrate as compared to non-responders in the baseline BM aspirates (Fig 1A). We evaluated expression of immune markers on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. At baseline, BM of non-responders had significantly higher percentage of CD4 T effector cells co-expressing the inhibitory markers PD1 and TIM3 (p&lt;0.05) and a trend towards higher percentage of CD4 T effector cells co-expressing PD1 and LAG3 compared to responders (Fig 1B). Co-expression of TIM3 or LAG3 on PD1+ T cells have been shown to be associated with an exhausted immune phenotype in AML (Zhou et al., Blood 2011). Conclusion: Addition of nivolumab to ara-C and anthracycline induction chemotherapy is feasible and safe in younger pts with AML. Among the pts proceeding to alloSCT the risk of GVHD is not significantly increased. Figure 1 Figure 1. Disclosures Daver: Pfizer Inc.: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Kiromic: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Incyte Corporation: Honoraria, Research Funding. Thompson: Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour: Bristol-Myers Squibb: Consultancy. Takahashi: Symbio Pharmaceuticals: Consultancy. DiNardo: Novartis: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Sharma: Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Astellas: Consultancy; EMD Serono: Consultancy; Amgen: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy; Consetellation: Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Kite Pharma: Consultancy, Other: stock; BMS: Consultancy. Cortes: BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; ImmunoGen: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding. Kantarjian: Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding.

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