T Cell Exhaustion and Downregulation of Cytotoxic NK Cells – an Immune Escape Mechanism in Adult Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3781-3781
Author(s):  
Eolia Brissot ◽  
Sawa Ito ◽  
Kit Lu ◽  
Carly Cantilena ◽  
B. Douglas Smith ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Research Funding ◽  
Immune Escape ◽  
Nk Cell ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Healthy Donors

Abstract Adult acute lymphoblastic leukemia (ALL) remains a therapeutic challenge with less than 40% long term survival. There is growing evidence that malignant diseases exert an “immune editing” effect which blocks antitumor immunity and permits tumor growth through immune evasion. Such tumor escape represents an obstacle for anticancer immunotherapy. In ALL such immune escape mechanisms are not well characterized. We therefore profiled cellular immunity in ALL, by characterizing the subsets of T cells, regulatory T cells (Treg), natural killers (NK) cells and γd T cells, using various functional markers including T cell exhaustion and NK cell activating or inhibitory molecules. Forty ALL patients were included in the study. The median age was 39 y (range, 18-75). Thirty-six presented with B-lineage ALL and 4 with T-lineage ALL. Mononuclear cells were isolated from blood (n=19) or bone marrow (n=21) at the onset of leukemia or at relapse. The median infiltration of blasts was 85% (range 24-96%). Healthy donor peripheral blood (n=12) and bone marrow (n=9), from age and gender matched population, were simultaneously analyzed as controls. Extra-and intra cellular staining were performed using using antibodies directed against CD3, CD4, CD8, CD45, CD45, CD45RA, CD45RO, CCR7, CD95, CD27, CD19, CD14, CD127, CD25, Foxp3, Helios, αβTCR, HLA-DR, CD117, CD20, CD10, CD22, CD34, LAG3, PD1, PDL1, CD56, NKG2A, NKG2C, NKG2D, KIR2DL1, KIR2DL3, CD57, CD33, CD11b, CD15, CD38 and CD24. Data were acquired on a BD LSRFORTESSA flow cytometer. The expression of programmed cell death 1 (PD-1, CD279) receptor on CD8+T cells was significantly increased in blood and bone marrow of ALL patients compared to healthy donors (p<0.0001 and p=0.004, respectively) (Fig. 1). Focusing on the different subsets, CD8+ effector memory T cells significantly over-expressed PD-1 in blood and bone marrow of ALL patients compared to healthy donors (p=0.008 and p=0.04, respectively). Moreover, there was a significant positive correlation between PD-1 expression on CD8+ effector memory T cells and blast infiltration (R2=0.23, 95%CI 0.026-0.76, p=0.04). Expression of the co-inhibitory receptor lymphocyte-activation gene 3 (LAG-3, CD223) was similar in ALL patients compared to healthy donors. A significantly higher frequency of T regulators (CD25+, CD127 low, Foxp3+) was found in bone marrow microenvironment in ALL patients (4.3% versus 1.6%, p=0.02). Concerning γd T cells, frequency was similar in blood and bone marrow of ALL patients compared with healthy donors. There was a significantly lower frequency of CD56dimNKG2A+KIR-CD57- (p=0.02) in the bone marrow of ALL patients indicating a maturation arrest. Interestingly, expression of the activating receptor NKG2D which plays an important role in triggering the NK cell–mediated tumor cell lysis was significantly reduced in NK cells of ALL patients while no difference in NK cell expression of NKG2C was found(Fig. 2). Adult patients with ALL show evidence of immune-editing of T cells and NK cells. This global immunosuppressive mechanism may contribute to the eventual escape of ALL from immune control. PD-1, overexpression, described in acute myeloid leukemia and chronic myeloid leukemia has been implicated in T-cell exhaustion and subsequent tumor immune evasion. Our data suggests similar immune escape mechanisms pertain in ALL. Effective antileukemia immunotherapy will require targeting one or more of these immunosuppressive pathways to achieve optimum results. Disclosures Fathi: Seattle Genetics, Inc.: Consultancy, Research Funding; Takeda pharmaceuticals International Co.: Research Funding; Exelixis: Research Funding; Ariad: Consultancy.

scholarly journals Single-Cell Multi-Omic Analysis Uncovers Comprised Immune Function and Primary Resistance Mechanism in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 378-378
Author(s):  
Jianbiao Zhou ◽  
Jonathan Adam Scolnick ◽  
Stacy Xu ◽  
Melissa Ooi ◽  
Priscella Shirley Chia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Single Cell ◽  
Research Funding ◽  
Immune Escape ◽  
Nk Cell ◽  
Primary Resistance ◽  
Bm Niche

Abstract Background: Approximately 20% of AML patients do not respond to induction chemotherapy (primary resistance) and 40-60% of patients develop secondary resistance, eventually leading to relapse followed by refractory disease (RR-AML). Diversified molecular mechanisms have been proposed for drug resistance and RR phenotype. However, we still cannot predict when relapse will occur, nor which patients will become resistant to therapy. Single-cell multi-omic (ScMo) profiling may provide new insights into our understanding of hematopoietic stem cell (HSC) differentiation trajectories, tumor heterogeneity and clonal evolution. Here we applied ScMo to profile bone marrow (BM) from AML patients and healthy controls. Methods: AML samples were collected at diagnosis with institutional IRB approval. Cells were stained with a panel of 62 DNA barcoded antibodies and 10x Genomics Single Cell 3' Library Kit v3 was used to generate ScMo data. After normalization, clusters were identified using Uniform Manifold Approximation and Projection (UMAP) and annotated using MapCell (Koh and Hoon, 2019). We analyzed 23,933 cells from 4 adult AML BM samples, and 39,522 cells from 2 healthy adults and 3 sorted CD34+ normal BM samples. Gene set enrichment analysis (GSEA) and Enrichr program were used to examine underlying pathways among differentially expressed genes between healthy and AML samples. Results: We identified 16 cell types between the AML and normal samples (Fig 1a) amongst 45 clusters in the UMAP projection (Fig 1b). Comparative analysis of the T cell clusters in AML samples with healthy BM cells identified an "AML T-cell signature" with over-expression of genes such as granzymes, NK/T cell markers, chemokine and cytokine, proteinase and proteinase inhibitor (Fig 2a). Among them, IL32 is known to be involved in activation-induced cell death in T cells and has immunosuppressive role, while CD8+ GZMB+ and CD8+ GZMK+ cells are considered as dysfunctional or pre-dysfunctional T cells. Indeed, Enrichr analysis showed the top rank of phenotype term - "decreased cytotoxic T cell cytolysis". We next examined whether NK cells, are similarly dysfunctional in the AML ecosystem. The "AML NK cell signature" includes Fc Fragment family, IFN-stimulated genes (ISGs), the effector protein-encoding genes and other genes when compared to normal NK cells (Fig 2b). GSEA analysis revealed "PD-1 signalling" among the top 5 ranked pathways in AML-NK cells, though no increase in PD-1 protein nor PDCD1 gene were identified in these cells. Inhibitory receptor CD160 was expressed higher in AML samples along with exhaustion (dysfunction) associated genes TIGIT, PRF1 and GZMB (Fig 2c). Enrichr analysis uncovered enrichment of "abnormal NK cell physiology and "impaired natural killer cell mediated cytotoxicity". Similarly, the "AML monocyte signature" was significantly enriched with genes in "Tumor Infiltrating Macrophages in Cancer Progression and Immune Escape" and "Myeloid Derived Suppressor Cells in Cancer Immune Escape". We also analyzed HSPC component in one pair of cytogenetically matched, untreated complete remission (CR) /RR AML pair (Fig 2d). Notably, half of the 10 genes overexpressed in RR-AML, CXCR4, LGALS1, S100A8, S100A9, SRGN (Serglycin), regulate cell-matrix interaction and play pivotal roles in leukemic cells homing bone marrow niche. The first 4 of these genes have been demonstrated as prognostic indicators of poor survival and associated with chemo-resistance and anti-apoptotic function. Furthermore, single-cell trajectory analysis of this CR/RR pair illustrated a change in differentiation pattern of HSPCs in CR-AML to monocytes in RR-AML. We are currently analyzing more AML samples to validate these findings. Conclusions: Our ScMo analysis demonstrates that the immune cells are systematically reprogrammed and functionally comprised in the AML ecosystem. Upregulation of BM niche factors could be the underlying mechanism for RR-AML. Thus, reversing the inhibited immune system is an important strategy for AML therapy and targeting leukemic cell-BM niche interaction should be considered for cases with high expression of these molecules on AML HSPCs. Note: J.Z. and J.A.S. share co-first authorship. Figure 1 Figure 1. Disclosures Scolnick: Proteona Pte Ltd: Current holder of individual stocks in a privately-held company. Xu: Proteona Pte Ltd: Current Employment. Ooi: Jansen: Honoraria; Teva Pharmaceuticals: Honoraria; GSK: Honoraria; Abbvie: Honoraria; Amgen: Honoraria. Lovci: Proteona Pte Ltd: Current Employment. Chng: Aslan: Research Funding; Takeda: Honoraria; Johnson & Johnson: Honoraria, Research Funding; BMS/Celgene: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Antengene: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria.

scholarly journals Role of Recipient CD8+ T Cell Exhaustion in the Rejection of Adoptively Transferred Haploidentical NK Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 503-503
Author(s):  
Robin Williams ◽  
Sarah Cooley ◽  
Veronika Bachanova ◽  
Thomas A Waldmann ◽  
Bruce R. Blazar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
Cd8 T Cell ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Refractory Aml

Natural killer (NK) cells are cytotoxic innate lymphoid cells, which play a major role in tumor surveillance. We have tested the safety and efficacy of allogeneic NK cell adoptive transfer from heathy haploidentical donors and demonstrate that in vivo expansion and persistence of the adoptively transferred NK cells at Day 14 after infusion correlates with 30-50% remission in patients with refractory AML. However, the factors that influence successful persistence of donor-derived NK cells are unclear. We hypothesized that recipient T cells play a role in the rejection of allogeneic NK cells and a correlation could exist between persistence of donor-derived NK cells and exhaustion in recipient T cells. T cell exhaustion, a well-established state of T-cell dysfunction occurring in response to chronic and continuous antigen stimulation, is well-documented in human cancer, and characterized by progressive and hierarchical loss of effector functions including sustained up-regulation and co-expression of multiple inhibitory receptors such as PD-1 and Tim-3 and altered expression of key transcription factors including the gain of Eomes and T-bet. We used samples from a phase I/II trial of CD3/CD19 depleted, IL-15-activated, haploidentical donor NK cells delivered following conditioning with cyclophosphamide (50mg/kg) and fludarabine (35 mg/m2 x 3days) in adults with chemotherapy refractory AML. Patients received donor NK cells on Day 0 followed by 10 doses of recombinant human (rh) IL-15 (2 mcg/kg/day) manufactured by the NCI and delivered SQ on Days 1-5 and 8-12. A significant proportion of patients experienced donor NK cell expansion at Day 14 (expanders), but there were some that did not (non-expanders). Therapeutic benefit has only been noted among the expanders. We examined samples from a total of 10 patients with refractory AML, 5 expanders and 5 non-expanders, along with their 10 respective donors. Cryopreserved patient PBMCs were thawed and rested overnight in RPMI-1640 with 2% FBS. The cells were stained for viability, for surface markers using antibodies against CD3, CD8, CD56, PD-1, and Tim-3, intracellularly stained for Eomes and T-bet. We evaluated CD8+ T cell expression of PD-1 and Tim-3, in expanders and non-expanders, prior to chemotherapy and at Day 14. Paired donor T cells from the non-mobilized apheresis products served as controls. Prior to chemotherapy, both patient groups had equivalently elevated expression of both PD1 and Tim-3 on CD8+ T cells. However, at Day 14, the expanders had persistence of PD-1 and Tim-3 while expression on non-expander CD8+ T cells fell to donor level (Figure 1A). Furthermore, expanders had a significantly higher proportion of CD8+ T cells that either co-expressed PD-1 and Tim-3 (p=0.017) or had a PD-1high phenotype (p=0.032) at Day 14, both of which are suggestive of an exhausted state, as opposed to an activated one (Figure 1B,C). Next, we examined Eomes and T-bet expression in recipient T cells. While generally low among healthy T cell populations, as T cells become exhausted, they gain expression of these transcription factors. We looked specifically at the expression of these transcription factors in the recipient CD8+ T cell populations with the highest likelihood of being exhausted, i.e. those co-expressing PD-1 and Tim-3 or those with the PD-1high phenotype. Eomes expression in recipient PD-1high CD8+ T cells and in PD-1+Tim-3+ CD8+ T cells at Day 14 was significantly higher (p=0.01 and p=0.04, respectively) among expanders compared to non-expanders (Figure 2A,B). Likewise, T-bet expression was greater (p=0.004) among expanders in the PD-1high population (Figure 2A). There was no difference in the T-bet expression in PD-1+Tim-3+ CD8+ T cells between groups (Figure 2B). While all patients with refractory AML receiving NK cell adoptive transfer had an elevated percentage of CD8+ T cells with an exhausted phenotype prior to therapy, only patients with donor-derived NK cell expansion had persistence of the exhausted T cell phenotype at Day 14. Thus, T cell mediated rejection is a major obstacle to overcome for successful adoptive NK cell transfer which could in part be aided by a link between recipient T cell exhaustion and expansion of NK cells. This might further suggest that IL-15 reverses T cell exhaustion among those who failed to achieve donor-derived NK cell expansion. Disclosures Miller: Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

scholarly journals Natural Killer Cell–Mediated Eradication of Neuroblastoma Metastases to Bone Marrow by Targeted Interleukin-2 Therapy

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1706-1715 ◽  
Author(s):  
Holger N. Lode ◽  
Rong Xiang ◽  
Torsten Dreier ◽  
Nissi M. Varki ◽  
Stephen D. Gillies ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
Nk Cells ◽  
Natural Killer ◽  
Therapeutic Effect ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Interleukin 2

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody–IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell–dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell–deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell–stimulating agents, such as poly I:C or recombinant mouse interferon-γ. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.

scholarly journals Treatment-Free Intervals Mitigate T-Cell Exhaustion Induced By Continuous CD19xCD3-BiTE® Construct Stimulation in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 44-45
Author(s):  
Nora Zieger ◽  
Alyssa Nicholls ◽  
Jan Wulf ◽  
Gerulf Hänel ◽  
Maryam Kazerani Pasikhani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Function ◽  
Granzyme B ◽  
T Cell Exhaustion ◽  
Specific Lysis ◽  
Cell Exhaustion ◽  
T Cell Function

The bispecific T-cell engager (BiTE®) blinatumomab is approved for treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and applied as continuous infusion over 28 days. The overall response rate to blinatumomab reported in clinical trials was 43 % and correlated to T-cell expansion (Zugmaier et al. 2015). In chronic viral infections, continuous antigen stimulation induces T-cell exhaustion, defined by phenotypic changes and functional impairment (Wherry 2011). Thus, we hypothesized that continuous BiTE® construct stimulation leads to T-cell exhaustion and that a treatment-free interval (TFI) reverses progressive T-cell dysfunction. To simulate continuous application of a BiTE® construct in vitro, T-cell long-term co-cultures were set up. Healthy donor T cells were stimulated in the presence of CD19+ OCI-Ly1 cells for 28 days with AMG 562, a half-life extended CD19 and CD3 specific BiTE® construct. T cells were harvested from the co-culture every 3-4 days between day 7 and 28 and assessed for markers of T-cell exhaustion: (1) AMG 562-mediated cytotoxicity of T cells was evaluated as specific lysis of CD19+ Ba/F3 target cells after 3 days, (2) T-cell expansion during the cytotoxicity assay was calculated as fold change (FC) of CD2+ counts, (3) Cytokine secretion of AMG 562-stimulated T cells was evaluated in co-culture supernatants by cytometric bead array (CBA) or after PMA/Ionomycine stimulation via intracellular cytokine staining (ICCS), (4) T-cell metabolic fitness was determined by Mito- and Glycolytic Stress Test using a Seahorse Analyzer, and (5) expression of the exhaustion-related transcription factor TOX was assessed by multiparameter flow cytometry. In order to assess the effect of a TFI on T-cell function, we cultured T cells and CD19+ OCI-Ly1 cells in the absence of AMG 562 from day 7-14 and 21-28 and compared their activity to T cells stimulated continuously with AMG 562. On day 7 of continuous (CONT) AMG 562 stimulation, we observed high cytotoxic and proliferative potential (% specific lysis=93±0.2, FC=2.9±0.2) as well as high IFN-g and TNF-a secretion analyzed by ICCS (% CD8+IFN-g+TNF-a+=23±6.7). However, cytotoxicity and proliferation decreased gradually until day 28 (% specific lysis=28±8.9; FC=0.6±0.1). CBA analysis confirmed decreasing secretion of IFN-g (day 3: 61113±12482, day 24: 3085±1351 pg/ml) and TNF-a (day 3: 1160±567, day 24: 43±7.6 pg/ml) as well as decreased IL-2 and granzyme B levels in culture supernatants. We furthermore observed highest mitochondrial fitness and basal glycolysis in T cells on day 7 of stimulation (basal OCR=2.2±0.6, maximal OCR=3.7±1.0, SRC=1.5±1.1 pmol/min/1000 cells, basal ECAR=2.0±0.4 mpH/min/1000 cells) which decreased until day 28 (basal OCR=0.4±0.2, maximal OCR=1.5±0.5, SRC=1.0±0.2 pmol/min/1000 cells, basal ECAR=0.5±0.2 mpH/min/1000 cells). In concordance, TOX increased during continuous stimulation (MFI ratio CD8+ day 7=6±0.8 to 12±0.8 on day 28). Strikingly, implementation of a TFI of 7 days led to superior cytotoxicity in T cells compared to continuously stimulated T cells (% specific lysis on day 14 CONT=34±4.2, TFI=99±2.2) and granzyme B production (CD8+; MFI ratio on day 14 CONT=124±11, TFI=303±34). Furthermore, increased proliferation during the cytotoxicity assay was observed in previously rested T cells (FC CONT=0.2±0.0, TFI=1.6±0.6). Although T cell function also decreased over time in TFI T cells, they maintained a strikingly higher cytotoxic potential (CONT=6±4.4, TFI=52±9.9) as well as higher granzyme B production (CONT=25±2, TFI=170±11) on day 28 compared to continuously stimulated T cells. In addition, TFI T cells showed increased IFN-g and TNF-a secretion after PMA/Ionomycine stimulation on day 28 (% CD8+IFN-g+TNF-a+ CONT=21±3.8, TFI=38±11.6). Our in vitro results demonstrate that continuous AMG 562 exposure negatively impacts T-cell function. Comprehensive analysis of T-cell activity in an array of functional assays suggests that continuous BiTE® construct exposure leads to T-cell exhaustion which can be mitigated through TFI. Currently, T cells from patients receiving blinatumomab are being analyzed to confirm the clinical relevance of our findings. Furthermore, RNA-Seq of continuously vs. intermittently AMG 562-exposed T cells will help us to understand underlying transcriptional mechanisms of BiTE® construct induced T-cell exhaustion. Disclosures Zieger: AMGEN Research Munich: Research Funding. Buecklein:Pfizer: Consultancy; Novartis: Research Funding; Celgene: Research Funding; Amgen: Consultancy; Gilead: Consultancy, Research Funding. Brauchle:AMGEN Inc.: Research Funding. Marcinek:AMGEN Research Munich: Research Funding. Kischel:AMGEN: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe:Gilead Sciences: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

scholarly journals Peripheral Natural Killer cells from chronic hepatitis B patients display molecular hallmarks of T cell exhaustion

2020 ◽  
Author(s):  
M. Marotel ◽  
M. Villard ◽  
I. Tout ◽  
L. Besson ◽  
O. Allatif ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chronic Hepatitis ◽  
T Cell ◽  
Hepatitis B ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Cell Dysfunction

AbstractA significant proportion of individuals infected by HBV develops chronic infection. Antiviral effectors such as Natural Killer (NK) cells have impaired functions in these patients, but the molecular mechanism responsible for this dysfunction remains poorly characterized. Here, we show that peripheral NK cells from chronic hepatitis B (CHB) patients have a defective capacity to produce IFN-γ, MIP1-β and TNF-α but retain an intact killing capacity. This functional phenotype was associated with a decrease in the expression of NKp30 and CD16, combined with defects in IL-15 stimulation of the mTOR pathway. Transcriptome analysis of NK cells in CHB patients further revealed a strong enrichment for transcripts typically expressed in exhausted T cells suggesting that NK cell dysfunction and T cell exhaustion rely on common molecular mechanisms. In particular, the transcription factor thymocyte selection-associated HMG box protein (TOX) and several of its targets, including immune checkpoints, were over-expressed in NK cells of CHB patients. This T cell exhaustion signature was predicted to be dependent on the calcium (Ca2+)-associated transcription factor NFAT. In line with this, when stimulating the Ca2+-dependent pathway in isolation, we recapitulated the dysfunctional phenotype. Thus, deregulated Ca2+ signalling could be a central event in both T cell exhaustion and NK cell dysfunction that occur during chronic infections.

Hemophagocytic lymphohistiocytosis in syntaxin-11–deficient mice: T-cell exhaustion limits fatal disease

Blood ◽  
2013 ◽  
Vol 121 (4) ◽  
pp. 604-613 ◽  
Author(s):  
Tamara Kögl ◽  
Jürgen Müller ◽  
Birthe Jessen ◽  
Annette Schmitt-Graeff ◽  
Gritta Janka ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Hemophagocytic Lymphohistiocytosis ◽  
Cytolytic Activity ◽  
T Cell Exhaustion ◽  
Inhibitory Receptors ◽  
Cell Exhaustion ◽  
Syntaxin 11 ◽  
Deficient Mice

Abstract Syntaxin-11 (Stx11), an atypical member of the SNARE protein family, is part of the cytolytic machinery of T and NK cells and involved in the fusion of lytic granules with the plasmamembrane. Functional loss of syntaxin-11 in humans causes defective degranulation and impaired cytolytic activity of T and NK cells. Furthermore, patients with STX11 deficiency develop familial hemophagocytic lymphohistiocytosis type 4 (FHL4), a life-threatening disease of severe hyperinflammation. We established Stx11-deficient mice as an animal model for FHL4. Stx11-deficient mice exhibited severely reduced degranulation and cytolytic activity of CTL and NK cells and developed all clinical symptoms of hemophagocytic lymphohistiocytosis (HLH) after infection with lymphocytic choriomeningitis virus (LCMV). The HLH phenotype was further characterized by hyperactive CD8 T cells and continuous IFN-γ production. However, in contrast to perforin-deficient mice, which represent a model for FHL2, progression of HLH was not fatal. Survival of Stx11-deficient mice was determined by exhaustion of antigen-specific T cells, characterized by expression of inhibitory receptors, sequential loss of effector functions, and finally T-cell deletion. Blockade of inhibitory receptors on T cells in Stx11-deficient mice converted nonfatal disease course into fatal HLH, identifying T-cell exhaustion as an important factor for determination of disease severity in HLH.

NK Cells Induce Alloreactive Donor T Cell Apoptosis and Decrease Proliferation in GVHD Induction.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2162-2162
Author(s):  
Janelle A. Olson ◽  
Dennis B. Leveson-Gower ◽  
Andreas Beilhack ◽  
Robert S. Negrin
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Trafficking ◽  
Peripheral Lymph ◽  
Donor T Cells

Abstract Natural Killer (NK) cells have the ability to suppress graft-versus-host disease (GVHD) while inducing a graft-versus-tumor response (GVT) during allogeneic bone marrow transplantation (BMT). Previous studies in allogeneic BMT models have shown NK cell trafficking to and proliferation in lymphoid organs and GVHD target organs, which are also sites of donor T cell trafficking. This study aims to investigate the impact of NK cells on alloreactive, GVHD-inducing donor T cells. Interleukin-2 activated allogeneic NK cells isolated from C57Bl6 (H–2b) or FVB (H–2q) animals were transplanted along with T cell-depleted bone marrow into lethally irradiated BALB/c (H–2d) mice, followed 2 days later by luciferase-expressing CD4+ and CD8+ conventional T cells from the same donor strain (NK+Tcon group). Control mice received lethal irradiation and T cell-depleted bone marrow on day 0, and luciferase-expressing T cells on day 2 after transplant (Tcon group). Bioluminescence imaging of NK+Tcon mice revealed a significantly lower T cell bioluminescent signal (p=0.03 for FVB into BALB/c on day 6) than from Tcon mice. CFSE proliferation analysis of alloreactive T cells on day 3 after transplant showed no significant change in the percent of donor T cells that have divided in the spleen, and only a slight decrease in the percent of T cells that have divided in the lymph nodes when NK cells are present. However, at this timepoint 82% of the proliferating cells have divided past the third generation, in contrast to 64% in the NK+Tcon mice. Donor T cells in both groups become equally activated in vivo, expressing similar levels of the early-activation marker CD69. T cells re-isolated from NK+Tcon animals at day 5 stained 2 to 10-fold higher for the TUNEL apoptosis marker than those from Tcon mice in the mesenteric and peripheral lymph nodes, respectively (p&lt;0.0001). Additionally, decreased numbers of T cells were re-isolated from the peripheral lymph nodes in the NK+Tcon group as compared to the Tcon group. This increase in TUNEL staining was not seen when the transplanted NK cells were isolated from a perforin-deficient donor. This indicates that NK cells in lymph nodes use a perforin-dependent mechanism to increase apoptosis in proliferating, alloreactive donor T-cells, which are syngeneic to the transplanted NK cells. Donor T cells re-isolated from the lymph nodes of transplanted mice up-regulate the NKG2D ligand Rae1γ as compared to naïve T cells, as shown by FACS. This suggests that NK cells may cause direct lysis of alloreactive donor T cells in vivo during GVHD induction, mediated by the NK cell activating receptor NKG2D. This study provides crucial mechanistic information regarding the function of NK cells in suppressing GVHD.

Myeloid Derived Suppressor Cells (MDSCs) Regulate Tumor Growth, Immune Response and Regulatory T Cell (Treg) Development in the Multiple Myeloma Bone Marrow Microenvironment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 565-565
Author(s):  
Gullu Topal Gorgun ◽  
Gregory Whitehill ◽  
Jennifer Lindsey Anderson ◽  
Teru Hideshima ◽  
Jacob P. Laubach ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Tumor Development ◽  
Suppressor Cells ◽  
Advisory Committees ◽  
Healthy Donors

Abstract Abstract 565 Background: The interaction of myeloma (MM) cells with bone marrow accessory cells induces genomic, epigenomic and functional changes which promote tumor development, progression, cell adhesion mediated-drug resistance (CAM-DR), and immune suppression. As in other cancers, bidirectional interaction between MM cells and surrounding cells regulates tumor development on the one hand, while transforming the BM microenvironment into a tumor promoting and immune suppressive milieu on the other. Recent developments in targeted therapies have indicated that generation of the most effective therapeutic strategies requires not only targeting tumor or stroma cells, but also methods to overcome blockade of anti-tumor immune responses. In addition to lymphoid immune suppressor cells such as regulatory T cells (Tregs), distinct populations of myeloid cells such as myeloid derived suppressor cells (MDSCs) can effectively block anti-tumor immune responses, thereby representing an important obstacle for immunotherapy. While MDSCs are rare or absent in healthy individuals, increased numbers of MDSCs have been identified in tumor sites and peripheral circulation. Recent studies have in particular focused on MDSCs in the context of tumor promoting, immune suppressing, stroma in solid tumors. However, their presence and role in the tumor promoting, immune suppressive microenvironment in MM remains unclear. Methods: Here we assessed the presence, frequency, and functional characteristics of MDSCs in patients with newly diagnosed or relapsed MM compared to MM patients with response and healthy donors. We first identified a distinct MDSC population (CD11b+CD14−HLA-DR-/lowCD33+CD15+) with tumor promoting and immune suppressive activity in both PB and BM of MM patients. Moreover, we determined the immunomodulatory effects of lenalidomide and bortezomib on induction of MDSCs by MM cells, as well as on MDSC function. Results: MDSCs were significantly increased in both PB and BM of patients with active MM compared to healthy donors and MM in response (p<0.01). To determine whether the CD11b+CD14−HLA-DR-/lowCD33+CD15+ myeloid cell population represents functional MDSCs, we first assessed tumor promoting role of MDSCs in the MM microenvironment by culturing MM cell lines with MM patient bone marrow stroma cells (BMSC), with or without depletion of MDSCs. Importantly, BMSC-mediated MM growth decreased to baseline levels of MM cells alone when MDSCs were removed from the BMSC microenvironment. Moreover, MDSCs isolated from MM-BM using magnetic-Ab and/or FACS sorting cell separation, directly induced MM cell growth and survival, evidenced by 3H-thymidine incorporation and MTT assays. Since the interaction between tumor and stromal accessory cells is bidirectional, we next analysed the impact of MM cells on MDSC development. Importantly, MM cell lines cultured with PBMCs from healthy donors induced a 7 fold increase in MDSCs. We also examined the immune suppressive functions of MDSCs in cultures of autologous T cells with T cell stimulators, in the presence and absence of MDSCs from MM-PB or MM-BM. Freshly isolated MDSCs from both MM-PB and MM-BM induced significant inhibition of autologous T cell proliferation. Moreover, MDSC-associated immune inhibitory molecules arginase-1 (ARG-1) and reactive oxygen species (ROS), as well as inhibitory cytokines IL-6 and IL-10, were significantly increased in BM MDSCs, evidenced by intracellular flow cytometry analysis. In addition, MM BM MDSCs induced development of Treg from autologous naïve CD4+T cells. Finally, we analysed whether MDSCs impacted response to bortezomib and lenalidomide. Culture of MDSCs with MM cell lines, with or without bortezomib (5nM) and lenalidomide (1uM), demonstrated that less MM cell cytotoxicity in the presence of MDSCs. Conclusions: Our data show that MDSCs are increased in the MM microenvironment and mediate tumor growth and drug resistance, as well as immune suppression. Therefore targeting MDSCs represents a promising novel immune-based therapeutic strategy to both inhibit tumor cell growth and restore host immune function in MM. Disclosures: Raje: Onyx: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding. Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

Examining the Heterogeneity of Follicular Lymphoma By Multi-Parameter Flow Cyotmetry in Previously Untreated Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2947-2947
Author(s):  
Debra K Czerwinski ◽  
Steven R Long ◽  
Michael Khodadoust ◽  
Matthew J. Frank ◽  
Adel Kardosh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Tumor Cells ◽  
Immune Cells ◽  
Research Funding ◽  
T Cell Exhaustion ◽  
Cell Exhaustion

Abstract BACKGROUND: Follicular lymphoma (FL) is an indolent form of Non-Hodgkin B cell lymphoma that remains incurable with present therapies. Derived from germinal center B cells, FL B cells experience ongoing hypermutation of the immunoglobulin variable region gene. In addition, Michael Green, et al (PNAS; 2015), reported the presence of numerous somatic mutations to include those of the chromatin-modifying genes. These mutations accumulate over the course of the disease and play an important role in regulating gene transcription, B cell development and immune interactions. Furthermore, FL tumors maintain a resemblance to primary lymphoid follicles, and as such, present with a number of infiltrating immune cells, especially T cells, the numbers of which vary from patient to patient. The close association and interaction of these immune cells with the tumor B cells play an important part in determining the disease biology (Dave SS, et al. N Engl J Med; 2004). For instance, tumor B cells, through cell-cell contact with these immune cells and/or through secretion of inhibitory cytokines such as TGF-b and IL-10, induce T cell exhaustion and apoptosis as well as suppressive T cell phenotypes (FoxP3+ T Regulatory cells) thus evading immune eradication (Yang Z-Z, et al. Blood 2007 and Ai WZ, et al. IntJ Cancer; 2009). They also promote their own survival and proliferation through their interaction with resident T follicular helper cells via CD40L/CD40 interactions (Ame'-Thomas P, et al. Blood; 2005). As a corollary to an ongoing clinical trial, we received fine needle aspirates (FNAs) of easily accessible tumors from 14 patients with FL prior to any treatment. 6 of these patients had samples taken from a second site simultaneously. All samples were processed within 24 hours into a single-cell suspension; red blood cells were lysed. Cells were then stained with antibodies to delineate T, B, NK, dendritic, and myeloid cells, as well as their subsets. Antibodies against activation antigens, T cell exhaustion, inhibition and function were also used to characterize these cells. Finally, the cells were run on a 17-parameter LSRII (Becton Dickinson) and data analyzed via Cytobank, a web-based data storage and analysis tool. PURPOSE: To better understand the biology of FL as represented by protein expression by the tumor cells and the immune cells that make up the microenvironment. We will especially look to evaluate the heterogeneity inherent in FL by flow cytometry across patients as well as within any one individual. RESULTS: Each sample is stained with 4 panels of antibodies, 13 antibodies each, allowing us to measure over 100 cell subsets. A quick preview of all data shows that there is a high variability between patients in the percentage of T cells within the microenvironment (37.7% + 16.6% of all cells collected from all samples). This variability is represented by the differences in the CD4 T cell compartment (27.6 + 12.9%) and to a lesser degree in the CD8 compartment (7.7 + 3.7%). To note, this variability in T cells does not correlate with time from diagnosis to sample collection which ranged from 3.4 years to approximately 5 months. Also, this is in contrast to the similar percentage of CD4 and CD8 T cells expressing PD-1 (55.5 + 8.8% and 46.0 + 8.9%, respectively) across patients. Notably, there is much less variability from site to site within each patient then between patients as demonstrated by Figure 1 where Site A and Site B are 2 separate lesions within each patient listed, sampled at the same time. Since FL presumably begins in a single site in the body and then becomes disseminated, the fact that a characteristic relationship exists between tumor cells and immune cells wherever the disease is found implies a mutual interdependence of the tumor cells in each case and their immune host component. CONCLUSION: Follicular lymphoma is a very heterogeneous disease as would be expected by the diversity of mutations seen at the genomic level. This heterogeneity is also apparent in the microenvironment from one patient to another. Conversely, different tumor sites within each patient have a characteristic and fixed relationship to their immune microenvironment. The emergence of novel therapies for FL, including checkpoint antibodies such as anti-PD-1 and anti-PD-L1 and small molecules such as Ibrutinib, will be informed by understanding the differences as well as the similarities in each case of FL. Disclosures Levy: Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

scholarly journals Natural Killer Cell–Mediated Eradication of Neuroblastoma Metastases to Bone Marrow by Targeted Interleukin-2 Therapy

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1706-1715 ◽  
Author(s):  
Holger N. Lode ◽  
Rong Xiang ◽  
Torsten Dreier ◽  
Nissi M. Varki ◽  
Stephen D. Gillies ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
Nk Cells ◽  
Natural Killer ◽  
Therapeutic Effect ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Interleukin 2

Abstract Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody–IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell–dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell–deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell–stimulating agents, such as poly I:C or recombinant mouse interferon-γ. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.

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