scholarly journals Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1172-1172
Author(s):  
Maryam Kazerani Pasikhani ◽  
Anetta Marcinek ◽  
Bettina Brauchle ◽  
Jonathan Jonas Taylor ◽  
Helena Domínguez Moreno ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cell Function ◽  
Target Cells ◽  
Cell Cytotoxicity ◽  
T Cell Cytotoxicity ◽  
Support Research

Abstract Novel immunotherapeutic strategies like BiTE ® (bispecific T cell engager) constructs aim to eradicate neoplastic cells by TCR-independent T-cell activation, and therefore rely on the function of autologous T cells. Currently, their efficacy is also evaluated in heavily pre-treated patients with relapsed/refractory acute myeloid leukemia (AML). Previous data demonstrated dysfunction in CD8 + T cells of AML patients (Knaus et al 2018). Thus, we aimed to characterize the progressive modulation of T-cell activity over the course of AML progression to improve the optimal application of T-cell based immunotherapeutic approaches. Bone marrow mononuclear cells (BMMCs) from AML patients at time of initial diagnosis (ID), complete remission (CR), relapse (RL), as well as of age-matched healthy donors (HD) were analyzed for T-cell subset distribution and expression of exhaustion markers by flow cytometry. Additionally, T-cell function was assessed after stimulation with 1) CD3/CD28 beads; 2) AMG 330, a CD33/CD3 specific BiTE ® construct, after incubation with OCI-AML3 target cells; or 3) AMG 330 in an autologous ex vivo long-term culture system after incubation with primary AML cells (pAML). After 6 days, T cell proliferation, expression of effector molecules and cytokines, and AMG 330-mediated T-cell cytotoxicity were assessed by flow cytometry. Lastly, we performed longitudinal bulk RNA-sequencing on 5000 sorted T cells from 7 matched ID-RL primary AML samples. Immunophenotypic analysis of BM T-cell subsets revealed a shift from T NAIVE toward central/effector memory subsets during AML progression. We observed lower percentages of T NAIVE in RL (n=3) compared to CR (n=3) CD8 + T cells(11.8 vs. 45.2%, p=0.07; RL vs. CR). Conversely, RL patients showed increased percentages of CD8 + memory T cells (T CM: 23.4 vs. 6.7%; T EM: 29.4 vs. 20.2%; T EMRA: 35.3 vs. 27.8%; RL vs. CR). Further characterization of exhaustion markers exhibited a significantly higher percentage of both CD4 + and CD8 + T cells expressing 2B4 (CD244) in ID (n=19) and RL (n=13) compared to HD (n=10, both p < 0.001). A higher percentage of PD-1 + CD8 + and TIM-3 + CD4 + T cells was detected in both ID and RL relative to HD (all p < 0.05). However, a significantly increased percentage of CD8 + T cells expressing TIM-3 and CD160 was detected in ID relative to HD (p < 0.05). Intriguingly, RL CD4 + T cells demonstrated a significantly higher level of LAG3 compared to ID (p < 0.01). In line with phenotypic exhaustion features, ID (n=4) and RL (n=5) CD8 + T cells showed reduced proliferation compared to HD (n=4) CD8 + T cells after CD3/CD28 bead stimulation (both p < 0.01). Correspondingly, we observed a marked reduction in the expression of Granzyme B (GZMB) by CD8 + T cells (both p < 0.05). Interestingly, when compared to ID, RL CD4 + T cells showed decreased TNF-α secretion (p < 0.05). In contrast to these findings, AMG 330-mediated T cell cytotoxicity against OCI-AML3 target cells was superior with RL T cells compared to ID T cells (p < 0.001). The percentage of GZMB + CD8 + T cells strikingly enhanced in RL relative to ID (p < 0.01). In an autologous setting with pAML samples, T cells from RL patients (n=6) showed higher AMG 330-mediated cytotoxicity compared to ID (n=9) T cells (67.7 vs. 35.2; RL vs. ID). In our longitudinal RNA-sequencing, differentially expressed genes analysis detected 61 up- and 30 downregulated genes (log2 FC > 1 or < -1; p < 0.01) in RL T cells compared to their matched ID counterparts. Among the significantly upregulated genes in RL, we identified genes associated with memory T cell function (TP53INP2, DUSP4) and exhaustion (NR4A1, TOX2). Moreover, Gene set enrichment analysis showed significant enrichment of gene signatures associated to memory and exhausted T cells (normalized enrichment score (NES)=1.2 and 1.3; p-value= 0.026 and 0.008, respectively), depletion of oxidative phosphorylation (NES=-2.05; p adj < 0.0001) and protein secretion (NES=-1.49; p adj < 0.05) gene signatures in RL vs. ID T cells. Taken together, our data show that patient T cells acquire an activated/exhausted phenotype upon AML progression. However, this is not reflected in the T-cell effector functions upon AMG 330 stimulation, in contrast to bead stimulation. These observations may highlight the significant role of the AML target cells in shaping a T-cell response. To this end, we will further analyze the longitudinal communication between T cells and their corresponding AML blasts. Disclosures Brauchle: Adivo: Current Employment. Kischel: Amgen GmbH Munich: Current Employment. Buecklein: BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Miltenyi: Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Roche: Research Funding; Miltenyi: Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Klinikum der Universität München: Current Employment.

Inhibitory Effects of Lactic Acid on Human Antigen-Specific CD8+ T-Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3844-3844
Author(s):  
Marina Kreutz ◽  
Karin Fischer ◽  
Petra Hoffmann ◽  
Simon Volkl ◽  
Matthias Edinger ◽  
...  
Keyword(s):  
T Cells ◽  
Lactic Acid ◽  
T Cell ◽  
Cd8 T Cells ◽  
Acid Production ◽  
Cell Function ◽  
Immune Escape ◽  
Lactic Acid Production ◽  
Target Cells ◽  
Thymidine Uptake

Abstract A characteristic feature of inflammatory lesions or tumor sites is local acidosis, which is attributed to the local increase in lactic acid production. We studied the effect of such an acidic environment on the immune functions of antigen-specific CD8+ T-cells by incubating the cells in the presence of various concentrations of lactic acid for up to 48h. CD8+ T-cells were isolated from healthy donors and expanded by weekly stimulation with autologous dendritic cells pulsed with a mutated HLA-A2-binding Melan-A (ELAGIGILTV) peptide. The obtained T cell population consisted of at least 90% CD8+ and about 60% Melan-A specific T cells, as determined by Melan-A multimer staining. Incubation of CD8+ T cells with up to 20mM lactic acid for 24h did not cause T-cell apoptosis or cell death as determined by combined annexin/propidium iodide staining. However, the proliferative capacity of CD8+ T cells, as determined by 3H-thymidine uptake, was strongly inhibited. Similar results were obtained when we determined cytokine production and cytotoxic activity of the cells after a 24h culture period in 5-20 mM lactic acid. Production of both, IL-2 and IFN-gamma was strongly diminished in comparison to untreated cells, as determined by intracellular staining after stimulation with PMA/ionomycin for 5h in the presence of monensin. Analysis of the antigen-specific cytolytic capacity revealed that CD8+ T cells pre-cultured with lactic acid were less effective in killing antigen-loaded T2 target cells as compared to untreated CD8+ T cells. In parallel, the intracellular contents of the cytotoxic effector molecules granzyme-B and perforin was diminished. Re-adjusting the pH of the medium to a physiological value of pH7.4 could partially revert the effect of lactic acid. Treatment of CD8+ T cells with sodium lactate instead of lactic acid had no inhibitory effect. We conclude, that lactic acid is an important modulator of CD8+ T-cell function and may contribute, together with other factors, to immune escape mechanisms in the tumor environment.

WT1-Specific T Cells Exhibiting a Non-Exhausted, Functional Phenotype Can Be Generated From The Natural Repertoire Of Healthy Donors For Clinical Use

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4504-4504 ◽  
Author(s):  
Sabine Schmied ◽  
Anne Richter ◽  
Mario Assenmacher ◽  
Juergen Schmitz
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Leukemic Cells ◽  
Target Cells ◽  
Clinical Use ◽  
Healthy Donors ◽  
Effector Cytokines

Background The Wilms tumor antigen 1 (WT1) is a self-antigen expressed at high levels in leukemic cells, but not in healthy tissue. As WT1 expression in leukemic cells drives leukemogenesis, it is a favorable target antigen for immunotherapy, e.g. adoptive transfer of allogeneic T cells, to prevent or treat leukemic relapse after stem cell transplantation (Cheever et al., Clin Cancer Res 2009;15(17)). WT1-specific CD8+ T cells have been detected in healthy individuals at low frequencies (Rezvani et al., Blood 2003;102). However, a comprehensive characterization of CD4+ and CD8+WT1-specific T cells is missing and the efficient expansion of a polyclonal WT1-reactive T cell population for clinical use has remained a major challenge. In this study we aim to directly ex vivo characterize WT1-specific T cells present in the blood of healthy donors at high-resolution and to develop a rapid method for the generation of functionally potent, polyclonal CD4+ and CD8+WT1-specific T cells for clinical use. Methods For direct ex vivo analysis of CD4+ WT1-specific T cells peripheral blood mononuclear cells (PBMC) of healthy blood donors were in vitro stimulated with a pool of overlapping peptides spanning the WT1 protein for 7 hours. Subsequently CD154 (CD40L)-expressing cells were magnetically enriched and flow cytometrically examined for expression of effector cytokines and their differentiation status. Presence and phenotype of CD8+ WT1-specific T cells have been studied after stimulation of presorted naïve and memory T cell populations with WT-1 peptide pool for 30 hours, magnetic enrichment of CD137+ (4-1BB) cells and subsequent staining using pMHCI-Tetramers. For the generation of polyclonal WT1-specific CD4+ and CD8+ T cells PBMC were in vitro activated with WT-1 peptide pool for 30 hours. CD137+cells were magnetically selected and expanded for 9 days in the presence of the cytokines IL-7, IL-15 and IL-21 at low doses. Expanded T cells were analyzed for their phenotype, the expression of co-stimulatory and exhaustion markers and were tested for their functionality and cytotoxicity by restimulation experiments with antigen-loaded target cells. Results Ex vivo frequencies of WT1-specific T cells are low, 1 to 10 WT1-specific CD154+ CD4+ T cells can be detected within 1x106 CD4+ T cells. In about 80% of healthy donors (n=15) a CD4+ memory response, accompanied by production of effector cytokines like IFNγ, TNFα and IL-2, against WT1 peptides is present. Additionally, in all donors naïve WT1-specific CD4+ T cells can be detected. In contrast, detected CD137+CD8+ WT1-reactive T cells exhibit a naïve phenotype (CD45RA+CCR7+) in all donors (n=5), no WT1-reactive CD8+T cells could be enriched from presorted memory T cells. To evaluate the usefulness of our improved short-term expansion protocol to generate potent WT1-specific T cell cultures for clinical use, we characterized CD137 enriched and expanded T cells. Notably, a high frequency of CD4+ and CD8+ T cells show specific reactivity against WT1-presenting autologous cells as detected by production of effector cytokines like IFNγ, TNFα and IL-2 after antigen-specific restimulation. Cytotoxic activity against antigen-loaded target cells could be shown by direct flow-cytometry-based cytotoxicity assays and antigen-specific upregulation of the degranulation marker CD107a. Stainings using multiple WT1-MHCI-tetramers furthermore confirmed antigen-specificity and suggested polyclonality within the CD8+T cell population. In contrast to previous expansion protocols our polyclonally expanded T cells exhibit a favourable, unexhausted memory phenotype, express co-stimulatory markers CD27 and CD28 and the IL7R-a chain (CD127) which has been shown to mark cells with stem T cell like properties. Furthermore exhaustion markers like CD279 (PD-1), CD178 (FasL) and CD57 are scarcely expressed. Conclusions Functional, polyclonal, CD4+ and CD8+ WT1-specific, reactive T cells can be efficiently enriched directly ex vivo from the natural repertoire by magnetic separation of T cells after antigen-specific stimulation. Phenotypic and functional characterization revealed a non-exhausted phenotype of expanded WT1-specific T cells, thereby suggesting good persistence and functionality of the obtained T cell product in vivo. Thus, our approach holds great potential for the GMP-compliant generation of WT1-specific T cells for future clinical use. Disclosures: Schmied: Miltenyi Biotec GmbH: Employment. Richter:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec: Employment.

scholarly journals CD4+ T Cell Fate in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4494-4494
Author(s):  
Rachel Elizabeth Cooke ◽  
Jessica Chung ◽  
Sarah Gabriel ◽  
Hang Quach ◽  
Simon J. Harrison ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Flow Cytometric Analysis ◽  
Advisory Committees ◽  
Mitochondrial Mass ◽  
Flow Cytometric

Abstract The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells. We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex. We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up. We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND. To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways. PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist. ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. Disclosures Quach: Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Immunomodulatory Effects of Pevonedistat, a NEDD8-Activating Enzyme (NAE) Inhibitor, in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2946-2946
Author(s):  
Scott R Best ◽  
Adam Kittai ◽  
Taylor Rowland ◽  
Nur Bruss ◽  
Stephen E Spurgeon ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cell Function ◽  
Cell Subset ◽  
Continuous Treatment ◽  
Cd4 T Cell ◽  
Cytotoxic Function

Abstract Introduction: T cells from patients with CLL and lymphoma show highly impaired immune synapse formation, cytotoxic function, and adhesion and migration capabilities. Recent advances in immunooncology led to the emergence of therapeutic agents that permit reversal of T-cell exhaustion in cancer. However, rational development of novel combination approaches in immunotherapy requires detailed understanding of how targeted therapies influence T-cell function. We have shown that pevonedistat (TAK-924), an investigational NAE inhibitor, abrogates NFκB activation in CLL cells. Pevonedistat forms a covalent adduct with NEDD8, a ubiquitin-like modifier, thereby disrupting its interaction with NAE. This leads to reduced activity of Cullin-RING ligases (CRLs), a group of ubiquitin ligases that require modification by NEDD8 for their function. Ultimately, a decrease in CRL activity leads to reduced ubiquitination and proteasomal degradation of CRL substrates, extending the half-life of these proteins, including inhibitor of NFκB (IκB). Moreover, NFκB is critical in T-cell function. However, limited data exist on the effects of targeting neddylation on T-cell response. Here, we demonstrate that targeting neddylation in vitro preserves T-cell functionality and may lead to favorable T-cell population shifts in CLL. Methods: Peripheral blood mononuclear cells were isolated from patients with CLL (n=50), and T cells were purified using Dynabeads. Pevonedistat was obtained from Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA). Results: In vitro T-cell receptor (TCR; CD3/CD28) stimulation induced T-cell activation and proliferation. Continuous treatment of T cells with pevonedistat led to rapid (2 hour) disruption of cullin neddylation, followed by a significant reduction in activity of NFκB and NFAT as assessed by immunoblotting and immunofluorescence. Despite this reduction, CD4 and CD8 T cells continued to respond to TCR stimulation, with relative abundance of early markers of activation (CD40L, CD69). However, we observed reduced expression of CD25 and PD-1 at 72 hours. Continuous treatment with pevonedistat led to a dose-dependent decrease in IL-2 secretion and reduced proliferation of the CD4 T-cell subset (CFSE, Ki-67) but did not induce apoptosis. Unlike CLL cells, CD4 T cells did not undergo DNA re-replication and G2/M arrest in response to pevonedistat. We further analyzed T-cell subsets following TCR stimulation. Concurrent treatment with pevonedistat led to an increase in IFNγ-secreting CD4 T cells, whereas IL-4 production decreased, suggesting a shift toward the Th1 phenotype. Furthermore, we observed a robust decrease of the iTreg population, accompanied by downregulation of FoxP3 mRNA and protein within the CD4 T-cell subset, indicating that targeting neddylation may help to reverse the immunosuppressive phenotype in CLL. To mimic the in vivo pharmacokinetics of pevonedistat, we performed drug washouts where CLL-derived T cells were exposed to 2-hour pulse treatment with 1 µM pevonedistat prior to TCR stimulation. Under these conditions, cullin neddylation and NFκB activity began to recover by 8 hours, with near complete recovery by 24 hours. Moreover, pevonedistat did not disrupt allogeneic (OCI-LY19 cells) or autologous (CD40L-stimulated CLL cells) T-cell cytotoxicity. Meanwhile, CD8 T cells continued to produce perforin and granzyme B. Conclusions: Our data suggest that pharmacologic targeting of NAE preserves T-cell cytotoxic function and may enhance anti-tumor immunity in CLL. Combined with our earlier reports that targeting NAE kills CLL cells under lymph node-mimicking conditions, these data provide a strong rationale for continued investigation of pevonedistat in CLL and lymphoid malignancies. Disclosures Spurgeon: Bristol Myers Squibb: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Oncternal: Research Funding; Acerta: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Research Funding; MEI Pharma: Consultancy. Berger:Takeda Pharmaceuticals International Co.: Employment. Danilov:Gilead Sciences: Consultancy, Research Funding; Astra Zeneca: Consultancy; Verastem: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Aptose Biosciences: Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy; Bayer Oncology: Consultancy, Research Funding.

Eomesodermin Regulates The Early Activation Of Alloreactive CD4 T Cells and Is Critical For Both Gvh and GVL Responses

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 133-133 ◽  
Author(s):  
Barbara Du Rocher ◽  
Odette M Smith ◽  
Andrew M. Intlekofer ◽  
Jarrod A Dudakov ◽  
Emily Levy ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Function ◽  
T Cell Differentiation ◽  
Early Activation ◽  
Donor T Cells ◽  
Cd4 Help

Abstract Despite increasing insights into its immunobiology, graft vs host disease (GVHD) remains a major obstacle for successful allogeneic hematopoietic stem/progenitor cell transplantation (allo-HCT). Separation of GVHD from graft vs. leukemia/lymphoma (GVL) responses also remains an elusive goal for allo-HSCT. Efforts to delineate the transcriptional networks regulating T cell differentiation post-HCT have suggested that multiple transcription factors may be involved in the regulation of alloreactive helper T (Th) cells and GVHD. However, conflicting data have emerged regarding the role of Th1 and Th17 pathways, and it remains unclear which transcription factors mediate the early activation of alloreactive T cells necessary for subsequent GVHD development. The T-box transcription factor eomesodermin (Eomes) cooperates with T-bet to regulate CD8 T cell cytotoxic function, IFNy production, and memory cell formation. Recently, a role for Eomes in CD4 Th cell polarization has been described as well. In order to evaluate the role of Eomes in T cell function in the context of allo-HCT, we used a MHC-disparate mouse model (C57BL/6 into BALB/c) with T cell depleted donor bone marrow (TCD-BM) and wild-type (WT) or Eomes knock out (KO) donor T cells. Recipients were conditioned with lethal total body irradiation. Eomes deficiency in donor T cells led to a significant reduction in GVHD mortality (Fig 1, p<.001), morbidity (p<.001), and intestinal pathology (p<.05, colon). Notably, Eomes KO T cells exerted significantly less GVHD mortality than T-bet KO T cells (Fig 1, p<.001). Given the reduced gastrointestinal (GI) GVHD observed with Eomes KO T cells, we next analyzed the expression of homing molecules important for T cell migration to the GI tract. Consistent with reduced GI GVHD, we detected reduced expression of α4β7 integrin on Eomes KO donor CD8 T cells one week post-HCT. We also observed an increase in the proportion and absolute numbers of Foxp3+ regulatory T cells, as well as a decrease in expression of T-bet in mesenteric lymph nodes (MLNs). Moreover, we found decreased production of IFNy by Eomes KO donor CD4 T cells two weeks (spleen and MLN, p<.001) and three weeks (spleen, p<.01) post-HCT without a comcomitant increase in IL-17. We also found increased IL-4 production by Eomes KO CD4 T cells two weeks post-HCT (MLN, p<.05), indicating a shift from Th1 to Th2 polarization in the absence of Eomes. Strikingly, one of the greatest differences we observed between WT and Eomes KO donor T cells was impaired early activation of CD4 T cells; Eomes deficiency was associated with reduced proliferation (p<.001), reduced expression of CD25 (p<.001, spleen; p<.001, MLN), and increased expression of CD62L (p<.01, spleen; p<.001, MLN) in CD4 T cells within the first 72 hours post-HCT (Fig 2). In order to determine if Eomes was important for T cell-mediated GVL responses, we performed allo-HCT in the presence of A20 lymphoma cells. Despite the reduction in GVHD mortality as described above, A20 tumor challenge led to increased mortality in recipients of Eomes KO T cells, indicating that Eomes was also critical for effective GVL function. Given the importance of Eomes in early alloactivation of CD4 T cells, we evaluated if the impaired GVL function was due to an intrinsic CD8 defect or lack of CD4 help. B6 TCD-BM was transplanted into BALB/c recipients along with either WT or Eomes KO CD4 or CD8 T cells. Eomes deficiency in both CD4 and CD8 T cells again led to significant mortality, but HCT with Eomes KO CD4 T cells and WT CD8 T cells led to the greatest survival due to less GVHD and intact GVL (Fig 3), suggesting that Eomes is essential for intrinsic CD8 function during GVL, but not for CD4 help. In summary, we identified distinct requirements for Eomes in CD4 versus CD8 T cells in the context of allo-HCT. Eomes regulated multiple aspects of CD4 T cell function following allo-HCT, including early activation, cytokine production, and gut trafficking. The multifacted functions of Eomes in CD4 T cells likely explain its requirement for GVHD. In contrast, Eomes deficiency in CD8 T cells led to impaired GVL, consistent with its established importance for cytotoxic CD8 T cell differentiation. To our knowledge, this is one of the first descriptions of a transcription factor necessary for effective GVL capacity. Our results suggest that selective manipulation of Eomes function in T cell subsets may be useful for both limiting GVHD and enhancing GVL. Disclosures: No relevant conflicts of interest to declare.

Dasatinib-Induced Reduction of Tumor Growth Is Accompanied By the Changes in the Immune Profile in a Melanoma B16.OVA Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1408-1408
Author(s):  
Mette Matilda Ilander ◽  
Can Hekim ◽  
Markus Vähä-Koskela ◽  
Paula Savola ◽  
Siri Tähtinen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Research Funding ◽  
K562 Cells ◽  
Cell Cytotoxicity ◽  
Cd8 Cells

Abstract Background: Dasatinib is a 2nd generation tyrosine kinase inhibitor (TKI) used in the treatment of chronic myeloid leukemia (CML). Its kinase inhibition profile is broad and includes several kinases important in the immune cell function such as SRC kinases. Furthermore, it is known that dasatinib has immunomodulatory effects in vivo. Recently, we observed that dasatinib induces a rapid and marked mobilization of lymphocytes, which closely follows the drug plasma concentration. The phenomenon is accompanied by an increase of NK-cell cytotoxicity. In addition, we have shown that dasatinib alters T-cell responses long-term favoring Th1 type of responses. Interestingly, the dasatinib induced immune effects have been associated with better treatment responses. We now aimed to characterize the dasatinib-induced antitumor immune responses in a syngeneic murine melanoma model to address whether dasatinib-induced immunoactivation affects tumor growth. Methods: Direct cytotoxic effect of dasatinib on B16.OVA melanoma cells in vitro was assessed with an MTS cell viability assay. T-cell cytotoxicity was assessed by preincubating splenocytes isolated from naïve and OT-I mouse spleen with 100 nM dasatinib and measured their cytotoxic capacity against B16.OVA cells. To further evaluate the dasatinib induced antitumor immune effects in vivo, B16.OVA cells were implanted subcutaneously in C57BL/6J mice. The mice (n=6/group) were treated daily i.g. either with 30 mg/kg dasatinib or vehicle only. Blood was collected before tumor transplantation, before treatment, and on treatment days 4, 7 and 11. Tumor volumes were measured manually and specific growth rate was calculated based on the first and the last day of the treatment. In addition to white blood cell differential counts, immunophenotyping of blood and tumor homogenate was performed by flow cytometry using antibodies against CD45.1, CD3, CD4, CD8b, NK1.1, CTLA4, PD-1 and CD107. Immunohistochemical staining of CD8+ T-cells was performed from the paraffin embedded tumor samples. Results: In vitro incubation of B16.OVA cells with dasatinib showed only a moderate unspecific cytotoxicity with the two highest concentrations of dasatinib (1- and 10 µM), whereas in K562 cells (a CML blast crisis cell line) almost complete killing was observed already with the 100nM concentration. The cell viability of B16.OVA cells was 90% with at 100 nM of dasatinib concentration (as compared to 21% of K562 cells) suggesting that there was no direct dasatinib sensitive target oncokinase in this cell line. In contrast, a significant enhancement in the cytotoxic capacity of splenocytes was observed when they were pretreated with 100nM dasatinib (60% of target cells were alive when incubated with dasatinib pretreated naïve splenocytes compared to 100% with control treated splenocytes, p=0.004). The in vivo tumor experiments demonstrated that the tumor volumes were smaller in dasatinib group, and there was a significant decrease in the specific tumor growth rate (0.06 vs. 0.18, p=0.01) on the 11th day of treatment. Interestingly, dasatinib treated mice had increased proportion of CD8+cells in the circulation (17.9% vs. 14.4%, p=0.005) and the CD4/CD8 ratio was significantly decreased (1.39 vs. 1.52, p= 0.04). During the tumor growth the mean CTLA-4 expression on CD8+ cells in PB increased from 1.2% to 9% in the control group, whereas, in dasatinib group the increase was more modest (1.2% to 5.7%). When the tumor content was analyzed, dasatinib treated mice had significantly more tumor infiltrated CD8+ T-cells (median 17 vs. 4/counted fields, p=0.03). In dasatinib group 80% of the tumor infiltrating CD8+ cells expressed PD-1 antigen compared to <5% of PD1 positive CD8+ cells in the peripheral blood suggesting either tumor induced CD8 T-cell exhaustion or the presence of tumor-reactive effector cells. Lastly, when CD4 and CD8 cells were depleted before tumor inoculation, dasatinib was no longer able to slow down the tumor growth. Conclusions: Dasatinib treatment slowed the tumor growth in a B16.OVA mouse model. The growth retardation was due to immunomodulatory properties of dasatinib as the drug was not directly cytotoxic and depletion of T-cells abolished the effect. Dasatinib may be a therapeutically useful immunomodulatory agent for targeting tumor-associated anergy, particularly in combination with novel checkpoint inhibitors and tumor-targeting drugs. Disclosures Hemminki: Oncos Therapeutics Ltd: Shareholder Other; TILT BioTherapeutics Ltd: Employment, Shareholder, Shareholder Other. Porkka:BMS and Novartis: Honoraria, Research Funding; Pfizer: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

scholarly journals 692 AMV564, a clinically active T cell engager, induces a target-dependent adaptive immune response

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A733-A734
Author(s):  
Sterling Eckard ◽  
Aurelien Sarde ◽  
Li Mei ◽  
Curtis Ruegg ◽  
Patrick Chun ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Ovarian Cancer Patient ◽  
Target Cells ◽  
Cell Cytotoxicity

BackgroundAMV564 is a potent bispecific T cell engager that binds CD3 and CD33. Due to its bivalent structure, AMV564 is selective for MDSCs via clustered CD33 expressed on the cell surface both in vitro and in patients. MDSCs are responsible for local and systemic suppression of the immune response to both circulating and solid cancers. Targeting MDSC suppression allows T cell priming to be restored in both the lymph nodes and tumor microenvironment, and expands previously activated tumor-specific T cells. Here we report clinical observations and results of our ex vivo assay development.MethodsCell lines, primary human cells, and patient samples were analyzed using flow cytometry with appropriate marker panels including AMV564 directly labeled (phycoerythrin) or detected with labeled anti-AMV564 antibodies. T cell cytotoxicity assays were conducted using primary human T cells and leukemic blast or other target cells (3:1 ratio) for 48 or 72 hours. Patient peripheral blood was sequenced for TcRbeta CDR3 variable chain on the hsTCRBv4b.ResultsAMV564 is currently under investigation in a Phase 1 clinical trial (NCT04128423). There have been no dose-limiting toxicities and clinical activity has been observed (RECIST complete response in an ovarian cancer patient) when dosed once daily as a subcutaneous injection. In patients, T cell redistribution is consistent with activation and depletion of both monocytic and granulocytic MDSCs. Immune profile changes consistent with CD8 and Th1 cell activation are observed (figure 1). Furthermore, TCR sequencing data indicate that one cycle of treatment is sufficient to expand and generate de novo clones (figure 2). We developed a primary cell cytotoxicity assay and observe that cytotoxic potency is target dependent. Target cell killing and T cell activation/proliferation depend on CD33 clustering, and both CD4 and CD8 T cells can engage and kill target cells. This is illustrated in assays with KG-1 (M2, clustered) and KG-1a (M0, not clustered) cell lines, in which the KG-1 cells have an EC50 15–20 fold lower than the M0 cell line (figure 3). In addition, there is little to no detectable binding or killing of monocytes or neutrophils, which is consistent with the absence of neutropenia in patients enrolled in the trial to date.Abstract 692 Figure 1Peripheral blood of a solid tumor patient shows robust activation of CD8 T cells over 5 cycles of AMV564 therapy. Significant increases in effector CD8 for patients treated with 15 or 50 mcg AMV564 as monotherapy (n = 8, *** p < 0.001)Abstract 692 Figure 2TcRb CDR3 sequencing of an ovarian cancer patient shows extensive clonal expansion upon treatment. Scatter plots represent clonal abundance in the periphery between Baseline (C1D1) and C1D12 using the differential Abundance tool (Adaptive Biotechnology)Abstract 692 Figure 3Cytotoxicity assay using fresh primary T cells demonstrates target selectivity on two cell lines with equivalent CD33 surface expression. T cell activation by AMV564 at clinically relevant doses is equivalent to CD3/CD28 stimulationConclusionsAMV564 is a potent conditional T cell agonist which is clinically active. We demonstrate that the combination of T cell activation, increased T cell diversity, and target specificity allow AMV564 to deplete MDSCs and restore a native immune response to cancer.Ethics ApprovalThis study was approved by the Institutional Review Board (IRB) or Independent Ethics Committee (IEC) at each participating institution.

scholarly journals DSP107, a Novel Bi-Functional Fusion Protein That Combines Inhibition of CD47 with Targeted Activation of 4-1BB to Trigger Innate and Adaptive Anticancer Immune Responses

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 19-20
Author(s):  
Ewa Cendrowicz ◽  
Lisa Jacob ◽  
Shirley Greenwald ◽  
Ami Tamir ◽  
Yosi Gozlan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cancer Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Innate Immune ◽  
Cell Cytotoxicity ◽  
Murine Models ◽  
T Cell Cytotoxicity

The mainstay of treatment for Diffuse Large B cell Lymphoma (DLBCL) is conventional chemotherapy combined with anti-CD20 monoclonal antibody rituximab (RTX). However, a subset of patients is refractory to treatment and between 20 to 50% of patients will, after experiencing an initial complete response (CR), develop resistance to treatment and relapse with poor prognosis. Therefore, additional therapeutic options are urgently needed. In this respect, combination of RTX treatment with CD47 monoclonal antibodies has yielded high objective response rates in patients with relapsed/refractory DLBCL in recent phase I trials. Interestingly, although CD47-targeting specifically activates the innate immune system, treatment with CD47 antibodies augments antigen-presentation in the context of MHC by macrophages and dendritic cells, thereby, triggering cross-priming of T cells in murine models. This T cell activation was pivotal in vivo efficacy in these murine models. Thus, a clear rationale exists for the development of novel therapeutics that exploit CD47 checkpoint inhibition while simultaneously stimulating anticancer T cell immunity. Here, we report on such an immunotherapeutic, termed Dual Signaling Protein 107 (DSP107), comprising a computationally-designed fusion of human soluble SIRPα and 4-1BBL. DSP107 was designed to bind to CD47 on cancer cells and block the CD47/SIRPα inhibitory signal delivered to phagocytes. Further, DSP107 was designed to bind to 4-1BB, a costimulatory receptor upregulated upon TCR/MHC interaction and a validated surrogate marker for the tumor-reactive subset of T cells in tumor tissue. Since 4-1BB activation by soluble 4-1BBL requires cross-linking, DSP107 will trigger 4-1BB signaling only after binding to CD47. This CD47-mediated surface immobilization of DSP107 enables delivery of the 4-1BBL-4-1BB costimulatory signal to tumor localized T cells. This dual immunomodulatory effect of DSP107 is designed to unleash both innate and adaptive immune responses targeted to the tumor site (Figure 1). Treatment with DSP107 alone or in combination with RTX triggered significant phagocytosis of a panel of DLBCL cancer cell lines as well as primary patient-derived DLBCL cells by macrophages and neutrophils within 3 hours. Further, after longer term incubation of 24h an ~85% reduction in remaining tumor cells was detected upon combined DSP107 and RTX treatment compared to medium control, whereas an increase in apoptosis was detected in the remaining cells. The pro-phagocytic activity of DSP107 was equal to both CD47 antibody as well as SIRPα:Fc. Simultaneously, binding of DSP107 to CD47 enabled 4-1BB costimulatory signaling by reporter cell line HT1080.4-1BB only on CD47-coated plates. Further, in co-cultures of HT1080.4-1BB with CHO.wt and CHO cells ectopically expressing human CD47, 41BB activation was only observed after binding of DSP107 to human CD47. This activation of 4-1BB costimulatory signaling triggered prominent T cell proliferation in mixed cultures of isolated peripheral blood T cells with cancer cells and augmented T cell cytotoxicity in vitro in a concentration and Effector to Target ratio dependent manner. Finally, injection of peripheral blood mononuclear cells (PBMCs) in mice with established SUDHL6 xenografts and simultaneous treatment with DSP107 triggered a strong reduction in tumor size compared to treatment with PBMCs alone. In conclusion, DSP107 clearly inhibits the CD47/SIRPα inhibitory axis and augments phagocytic removal of cancer cells by innate immune cells. Moreover, binding of DSP107 to CD47 enables the 4-1BBL-mediated costimulation of antitumor T cell cytotoxicity. Thus, DSP107 activates both innate and adaptive anticancer immunity and may be of use for the treatment of DLBCL alone or in combination with RTX. Disclosures Cendrowicz: Kahr Medical: Research Funding. Jacob:Kahr Medical: Current Employment. Greenwald:Kahr Medical: Current Employment. Tamir:Kahr Medical: Current Employment. Huls:Kahr Medical: Research Funding. Foley-Comer:Kahr Medical: Current Employment. Pereg:Kahr Medical: Current Employment. Chajut:Kahr Medical: Current Employment. Peled:Kahr Medical: Consultancy. Bremer:Kahr Medical: Consultancy, Research Funding.

scholarly journals The Immune Micro-Environment in Diffuse Large B Cell Lymphoma Is Characterised By an Immunosuppressive Shift in T Cell Subsets

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5240-5240
Author(s):  
Edward Truelove ◽  
Frances Seymour ◽  
Joseph G Taylor ◽  
Mariarita Calaminici ◽  
Andrew James Clear ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cytokine Production ◽  
B Cell Lymphoma ◽  
T Cell Subsets ◽  
Large B Cell Lymphoma ◽  
Car T

Diffuse large B-cell Lymphoma (DLBCL) is the most frequent non-Hodgkin's lymphoma with 3 molecularly distinct subtypes based on cell of origin. Genetic alterations in DLBCL, expression of checkpoint molecules and an immunosuppressive microenvironment (ME) all contribute to escape from host anti-lymphoma immunity. The clinical success of monoclonal antibodies that engage the immune system and CAR-T cellular therapy have further highlighted the importance and therapeutic potential of the immune ME in DLBCL. Here we present data from comprehensive phenotyping of cell suspensions from diagnostic DLBCL and reactive lymph node / tonsil (RLNT) biopsies by cytometry by time of flight (CyTOF), with a focus on the T-cell compartment. Cryopreserved samples from 6 DLBCL (5 LN, 1 spleen) at diagnosis and 5 RLNT (3 LN, 2 tonsil) were stained with a panel of metal-tagged antibodies and analysed by CyTOF2. Samples were acquired in 2 batches with the same RLNT (LN) sample with each to ensure staining consistency. Data were normalised, uploaded to Cytobank, gated to CD45+ CD3+ live single cells and exported for further analysis with Cytofkit in R. CD3+ events were gated further into CD4+ and CD8+ subsets, which demonstrated that CD4+ T cells were the predominant phenotype in all samples. However, there was a marked skewing of the CD4:CD8 ratio, with CD4+ T cells lower as a percentage of CD3+ T cells in the DLBCL samples (55.84 v 78.18, p=0.0173*). CD8+ T cells were higher as a percentage in DLBCL (36.22 v 16.75, p=0.03*) with no difference seen in double negative (DN) T cells. CD3+ T cells were then clustered with FlowSOM and visualised according to the tSNE algorithm. A heatmap of median marker expression intensity was generated to facilitate cluster identification. This revealed a number of differences in cluster abundance between the groups, with a significant shift in differentiation away from naïve and towards an effector memory (EM) phenotype in DLBCL. There were fewer cells in the CD27+ CD28+ CCR7+ CD45RA+ CD4+ naïve cluster in the DLBCL samples than the RLNT (p=0.0173*). Although the DLBCL samples showed an overall reduction in CD4+ T cells, the clusters of regulatory T cells (Treg: CD4+ CD25+ FOXP3+ and CD127-/low) consisted of more cells from these cases than the RLNT (p=0.0043**). Within the Treg population, the DLBCL patients had more Th1 polarised (T-bet+) Tregs and more PD-1 expressing Tregs. The Th1 Tregs predominantly secreted the suppressive cytokines IL-2, IL-10 and TGF-β on stimulation and may play a role in inhibiting Th1 responses. Conventional Th1 were not increased in DLBCL resulting in a higher Th1 Treg to Th1 ratio than in RLNT. There was a trend for RLNT samples to contribute more cells to the PD-1 high follicular helper T cell (TFH) cluster and DLBCL to the PD-1+ TIM-3+ DN cluster. The DLBCL ME had relatively more CD8+ T cells and contributed more to the CCR7- CD45RA- CD8+ EM clusters (p=0.0173*) but the CD8+ T cells in the RNLT samples tended to a naïve CCR7+ CD45RA+ PD-1- phenotype (p=0.0519). The CD8+ EM cells enriched in the DLBCL ME expressed the cytotoxic markers granzyme and perforin and responded to stimulation with degranulation (CD107a) and cytokine production (IFNγ, TNFα, TGFβ and IL-10), not suggestive of exhaustion. It is also notable that a cluster of PD-1+ TIM-3+ CD8+ EM with reduced markers of cytotoxicity, low CD107a expression and poor cytokine production after stimulation was predominantly made up of cells from DLBCL suspensions (p=0.002**). CyTOF analysis of the DLBCL ME has demonstrated a shift in the balance of T cell subsets and CD4:CD8 ratio with a relative abundance of immunosuppressive Tregs despite an overall reduction in the CD4+ population and a skew towards differentiation in CD4+ and CD8+ populations. The cytotoxic T cells in DLBCL tended to have an EM phenotype and express immune checkpoint molecules but remained capable of cytokine production. However, the production of IFNγ by these effector T cells may play a role in the development of inhibitory Tregs with a Th1 phenotype, which were enriched in these patients. A cluster of CD8+ EM cells expressing checkpoint molecules and displaying characteristics of exhaustion following stimulation was also seen in these DLBCL patients. These data provide new insights into the immunosuppressive nature of the DLBCL ME and provide a rationale for targeting the ME alongside existing therapeutic approaches, including CAR-T cells to improve outcomes. Disclosures Gribben: Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

scholarly journals Modulation of T-Cell Function in the Microenvironment of Emu-TCL1 CLL Bearing Mice By Btki Appears Independent of ITK

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3139-3139 ◽  
Author(s):  
Mark-Alexander Schwarzbich ◽  
Arantxa Romero-Toledo ◽  
Melanie Frigault ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Synapse Formation ◽  
Cell Function ◽  
Cytotoxic T Cells ◽  
Tumour Microenvironment ◽  
T Cell Function ◽  
Cell Synapse

Abstract Background: Chronic lymphocytic leukemia (CLL) development is associated with global immunodeficiency including T-cell exhaustion. We hypothesise that repairing T cell functions would improve outcome and decrease infectious complications which cause significant morbidity in CLL patients. Chronic B-cell receptor (BCR) activation as well as close interactions with the tumour microenvironment promote survival of malignant CLL B-cells, supporting their ability to induce immune suppression. To date, the most clinically successful approach to BCR-signalling inhibition is by the use of BTK inhibitors (BTKi). It has been suggested that the BTKi Ibrutinib has the ability to modulate T-helper cell polarity from Th2 to Th1 and thus would be a step towards repairing CLL associated T-cell defects (1). We were therefore interested to determine whether the second generation BTKi Acalabrutinib which has no reported inhibitory capacity towards ITK would have similar effects than Ibrutinib in modulating T cell responses. Materials and Methods: To address this question in vivo C57/Bl6 animals 2.5 months of age were injected with 40x10e6 purified CLL B-cells pooled from Eµ-TCL1 mice with CLL. When peripheral blood CLL load reached >10% animals were randomized (mean day 14) to either vehicle treatment (2% HPBD) or Acalabrutinib treatment (0.15 mg/l in 2% HPBC) for 21 days. 17 animals were group were analysed. Splenic cells were isolated, the cellular component characterized by CyTOF and T cell function assessed by multi-parameter flow cytometry and T-cell synapse formation assay. Results: Treatment with Acalabrutinib resulted in increased expression of IL2 (p<0.0001) in CD4+ T cells and decreased expression of IL4 among both CD4+ T cells (p=0.0016) but not CD8+ T-cells. There was a reduction in Interferon gamma production in both CD4 T-cells (p=0.0463) and CD8+ T-cells (p=0.0064) with Acalabrutinib treatment. In addition, treatment resulted in an increase in CD107a+/CD107a- ratio among both CD44+ and CD44- CD8+ cytotoxic T-cells. This effect was pronounced in the antigen experienced CD44+ cytotoxic T-cells (p<0.0001) but only moderate (p=0.0056) in the CD44- cytotoxic T-cells. Lastly, we find a statistically significant increase in T-cell synapse area (p<0.0001) with Acalabrutinib treatment (Figure 1). Conclusion: We find that treatment with both Ibrutinib and Acalabrutinib result in a similar shift of T cell function with cytokine secretion with increased IL2 and decreased IL4. T-cells in CLL have increased Interferon gamma production (2) and the observed decrease seen with Acalabrutinib is in keeping with a normalization of T cell function. Moreover, overall CD8+ T-cell function is increased with Acalabrutinib treatment as evidenced by an increase in cytotoxic T-cell function and immune synapse formation. We speculate that inhibition of ITK is not the leading cause for this phenomenon as Acalabrutinib does not have inhibitory capacity toward this kinase. These changes suggest that BTKi modulate T cell mediated immune responses indirectly via either their effects in the CLL B-cell or myeloid cells in the tumour microenvironment. References Dubovsky JA, Beckwith KA, Natarajan G, Woyach JA, Jaglowski S, Zhong Y, et al. Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes. Blood. 2013;122(15):2539-49. Riches JC, Davies JK, McClanahan F, Fatah R, Iqbal S, Agrawal S, et al. T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production. Blood. 2013;121(9):1612-21. Disclosures Frigault: Acerta Pharma: Employment. Gribben:Abbvie: Honoraria; Acerta Pharma: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Unum: Equity Ownership; Roche: Honoraria; TG Therapeutics: Honoraria; NIH: Research Funding; Medical Research Council: Research Funding; Cancer Research UK: Research Funding; Kite: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria.

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