Cytokine-Mediated Signaling Is Suppressed in Myeloid Cells and Enhanced in Lymphatic Cells in Patients with Chronic Myeloid Leukemia (CML) — Partial Normalization with Tyrosine Kinase Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2180-2180
Author(s):  
Sari Jalkanen ◽  
Satu Mustjoki ◽  
Kimmo Porkka ◽  
Jukka Vakkila
Keyword(s):  
T Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Ex Vivo ◽  
Healthy Controls ◽  
Gm Csf ◽  
Single Cell Profiling ◽  
Treg Lymphocytes

Abstract Abstract 2180 Poster Board II-157 Introduction. Aberrant phosphorylation of the BCR-ABL1 tyrosine kinase (TK) is characteristic of chronic myeloid leukemia (CML). This oncoprotein interacts directly with intracellular signaling proteins, alters the responsiveness of cytokine receptors and regulates secretion of autocrine cytokines. Targeted inhibition of BCR-ABL1 with TK inhibitor (TKI) imatinib mesylate (IM) is the current standard treatment of CML. For overcoming IM resistance or intolerance, 2nd generation TKIs (nilotinib, dasatinib) with broader kinase inhibition profile have been approved for clinical use. Although in vitro results suggest that TKIs are immunosuppressive, no increases in opportunistic infections or secondary malignancies have been observed to date. In contrast, in some TKI-treated patients immunoactivation in the form of chronic lymphocytosis linked to excellent therapy responses has recently been shown. Dynamic monitoring of aberrant cytokine signaling pathways would aid in understanding and predicting the development of TKI-resistance or adverse/off-target effects. The aim of this study was to analyze the responsiveness of leukocytes to cytokine stimuli in CML patients at diagnosis and during TKI therapy using single-cell profiling of phosphoprotein networks by multiparameter flow cytometry. Patients and methods. The study consisted of 4 healthy controls, 6 CML patients at diagnosis, 6 IM patients and 5 dasatinib patients. Stimuli included GM-CSF, IL-2+IL-10+IFNα and IL-4+IL-6+IFNγ and they were added immeadately to freshly drawn whole blood ex vivo. The readout phosphoproteins were pERK1/2, pSTAT1, pSTAT3, pSTAT5a and pSTAT6 (with isotype controls), and were analyzed separately from granulocytes, monocytes, CD4+ CD25neg T helper cells (Th), CD4neg lymphocytes and CD4+CD25+ T cells including regulatory T-cells (Treg). Analysis was performed with heatmap function of Cytobank software (http://cytobank.stanford.edu/public/). Results. Unstimulated phosphoprotein levels reflecting the activation state of leukocytes in vivo did not differ between healthy controls and CML patients at diagnosis or during dasatinib therapy. Strikingly, in IM patients, baseline levels of pSTAT3 were relatively high indicating in vivo occurring activation of leukocytes in this patient group. We next studied ex vivo responsiveness of immune effector cells with cytokines and found clear differences between healthy controls and CML patients. At CML diagnosis. GM-CSF/pERK1+pSTAT5a, IFNa/pSTAT1,and IL-4/pSTAT6 (stimulus/readout) as well as pSTAT3 responses with all stimuli were suppressed in monocytes. In granulocytes, GM-CSF/pSTAT1 levels were diminished. In Th and Treg lymphocytes, IL-6/pSTAT3 responses were markedly pronounced, while IL-10/pSTAT3 responses were not affected when compared to healthy controls. Such difference was not observed in CD4neg lymphocytes. During TKI therapy. Most patients (9/11) were in cytogenetic remission at the time of analysis. The unresponsiveness of myeloid cells at diagnosis was restored by IM or dasatinib therapy in most, but not all patients. Similarly, in Th and Treg lymphocytes TKI-therapy normalized the enhanced IL-6/pSTAT3 responses that were evident at diagnosis. However, in Th and Treg cells pSTAT3 responses provoked by IL-10 were particularly prominent. Interestingly, one dasatinib patient with aberrant constant blood NK-lymphocytosis and monocytosis had uniquely strong IFNg/pSTAT1 and IL-4/pSTAT6 responses in monocytes. Furthermore, one patient who have stayed in persistent remission after IM discontinuation had exceptionally high pSTAT3 responses with all of stimuli used. Similar kind of signaling profile was unseen with the other patients and could reflect immunoactivation related to leukemia control. Conclusions. Dynamic single-cell profiling of signaling networks is feasible in CML patients and can be used to study mechanisms of aberrant immune reactivity in TKI-treated patients. The method could be particularly suitable for assessing candidate patients for TKI discontinuation. Although in vitro results suggest immunosuppressive effects of TKIs on lymphocytes, leukocytes ex vivo from patients were able to respond similarly to cytokine stimuli as in healthy controls. Disclosures: Mustjoki: BMS: Honoraria. Porkka:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

scholarly journals Mutated GM-CSF-Based CAR T-Cells Targeting CD116/CD131 Complexes Exhibit Enhanced Anti-Tumor Effects Against Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-37
Author(s):  
Shoji Saito ◽  
Aiko Hasegawa ◽  
Mika Nagai ◽  
Yoichi Inada ◽  
Hirokazu Morokawa ◽  
...  
Keyword(s):  
T Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Antigen Recognition ◽  
Gm Csf ◽  
Car T Cells ◽  
Car T ◽  
Myelomonocytic Leukemia

Background: The prognosis of relapsed/refractory (R/R) acute myeloid leukemia (AML) remains poor; therefore, novel treatment strategies are required urgently. Meanwhile, recent clinical trials have demonstrated that CAR-T cells for AML have been less successful than those targeting CD19 for B cell malignancies. Recently, we developed piggyBac-modified ligand-based CAR-T cells that target CD116, also called granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) α chain, for treating juvenile myelomonocytic leukemia (Nakazawa, et al. J Hematol Oncol. 2016). Since CD116 is overexpressed in 60%-80% of AML cases, the present study aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. Methods: CD116 expression in AML cell lines or primary leukemia cells were examined using flow cytometry. The original piggyBac transposon plasmid for GMR CAR comprises GM-CSF as an antigen recognition site, IgG1 CH2CH3 hinge region, CD28 costimulatory domain, and CD3ζ chain. To improve the in vivo persistency and anti-tumor effects, two types of spacer (∆CH2H3 and G4S) that lack CH2CH3 lesion were newly constructed. In order to modulate the antigen recognition ability, mutated ligand-based GMR CAR vectors were constructed with a mutation at residue 21 of GM-CSF that is reported to play a critical role in its biological activity (Lopez, et al. Embo j. 1992). All the GMR CAR-T cells were generated with piggyBac gene modification. To investigate the in vitro anti-tumor activity, GMR CAR-T cells were co-cultured with AML cell lines. In order to evaluate the in vivo anti-tumor effects, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were intravenously injected with THP-1, THP1-ffLuc, or MV4-11 and then treated with GMR CAR-T cells. To characterize the safety profile of GMR CAR-T cells, peripheral blood mononuclear cells or polymorphonuclear cells were co-cultured with GMR CAR-T cells at an effector:target ratio of 1:1 for 3 days. Thereafter, B cells, NK cells, neutrophils, and monocytes were quantified using flow cytometry using counting beads. Results: Approximately 80% of the AML cells predominant in myelomonocytic leukemia expressed CD116. PiggyBac-modified GMR CAR-T cells displayed a favorable CD45RA+CCR7+-dominant phenotype, consistent with our previous findings. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. GMR CAR-T cells incorporating a G4S spacer significantly improved the long-term in vitro and in vivo anti-tumor effects as compared to those incorporating a ∆CH2CH3 spacer. Furthermore, by employing a mutated GM-CSF at residue 21 (E21K and E21R) as an antigen recognition site, the in vivo anti-tumor effects were also substantially improved along with prolonged survival (Figure 1) over controls (PBS or CD19.CAR-T cells) (all, p < 0.01) as well as over GMR CAR-T cells with a wild-type GM-CSF ligand (E21R: p < 0.01; E21K: p = 0.02), with 4 out of 5 mice surviving for > 150 days. Safety tests revealed that the toxicity of GMR CAR-T cells was restricted to normal monocytes. It is noteworthy that the cytotoxic effects of GMR CAR-T cells on normal neutrophils, T cells, B cells, and NK cells were minimal. Conclusions: GMR CAR-T cell therapy appears to be a potentially useful strategy for CD116+ R/R AML. Based on the promising results, we plan to perform the first-in-human clinical trial of GMR CAR-T cells. Disclosures Saito: Toshiba Corporation: Research Funding. Hasegawa:Toshiba Corporation: Research Funding. Inada:Kissei Pharmaceuticals: Ended employment in the past 24 months. Nakashima:Toshiba Corporation: Research Funding. Yagyu:Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.

scholarly journals Pembrolizumab Enhances the Anti-Leukemia Activity of Antigen Specific Cytotoxic T Lymphocytes

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4542-4542
Author(s):  
Mao Zhang ◽  
Pariya Sukhumalchandra ◽  
Anne V. Philips ◽  
Na Qiao ◽  
Celine Kerros ◽  
...  
Keyword(s):  
T Cells ◽  
Cytotoxic T Lymphocytes ◽  
Immune Checkpoint ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Immune Checkpoint Blockade ◽  
Cellular Immunotherapy ◽  
Calcein Am

Abstract Many aggressive hematologic malignancies are exquisitely responsive to immunotherapy. While allogeneic (allo) hematopoietic stem cell transplantation (SCT) is the prime example of anti-leukemia immunotherapy, it is highly toxic, limiting its application to patients with aggressive disease and a good performance status. Although targeting leukemia-associated antigens (LAA) with antigen specific cytotoxic T lymphocytes (CTL) and chimeric antigen receptor (CAR) T cells has proven to be a successful immunotherapeutic option, the immune response generated with these approaches often fails to fully eradicate the underlying malignancy. The expression of immune inhibitory molecules and their ligands by myeloid leukemia cells and leukemia-specific T cells could contribute to the failure of cellular immunotherapy approaches for acute myeloid leukemia (AML). Specifically, blocking the PD1/PD-L1 pathway using antibodies was shown to enhance the graft versus leukemia response in murine leukemia transplant models, and promising results with immune checkpoint blockade have been demonstrated in clinical trials for patients with AML. We therefore investigated the role of blocking the PD1/PD-L1 pathway in combination with adoptive cellular immunotherapy in the setting of AML. We used pembrolizumab in combination with CTLs that target the HLA-A2 restricted myeloid leukemia antigens CG1, derived from cathepsin G, and PR1, derived from neutrophil elastase and proteinase 3. Using a standard calcein AM in vitro cytotoxicity assay, we co-cultured AML targets, including U937 HLA-A2+ (U937-A2) AML cell line and primary patient AML, with CG1-CTL and PR1-CTL. AML cells were loaded with calcein AM and then incubated with CTL at increasing effector to target ratios. Pembrolizumab or isotype antibody was added to the cultures. After 4 hours, calcein AM was measured to determine cell viability. Our results demonstrated that U937-A2 cells and patient primary AML samples can be effectively lysed by CG1/PR1 CTL. In vitro lysis of AML by CTL was enhanced after the addition of pembrolizumab in comparison with the AML cells that were co-cultured only with CTL. Killing was dose-dependent and decreased at the lower effector:target ratios. We then tested whether anti-leukemia activity can be enhanced in vivo. For these experiments, NOD/SCID gamma (NSG) mice were engrafted with U937-A2 cells or patient AML intravenously (IV) via tail vein at a dose of 1 x 106 to 1 x 107 cells. After confirming engraftment (1-5% PB HLA-A2+/human (h) CD45+ cells), CG1-CTL and PR1-CTL (0.5 x 106) were administered to mice IV via tail vein and pembrolizumab or isotype antibody [100ug/mouse] was given intraperitoneally 4 times over 2 weeks. Mice were monitored for clinical graft versus host disease (GVHD) and AML 3 times/week. Mice were sacrificed at approximately 2 weeks following treatment and bone marrow (BM) was processed and analyzed for residual AML (Human[h] CD45+/mouse[m] CD45- cells) by flow cytometry. Our data demonstrate a decrease in U937-A2 (Figure) and primary AML after treatment with CG1/PR1-CTL. This decrease was enhanced when pembrolizumab was administered to the CTL-treated mice in comparison with mice treated with pembrolizumab only, CTL only, or CTL+ isotype. Furthermore, there was no increased toxicity or GVHD after the addition of pembrolizumab. Together these data highlight the potential for combination immune checkpoint blockade and antigen specific CTL to eradicate AML in vitro and in vivo. Figure. Figure. Disclosures Daver: Incyte: Consultancy; BMS: Research Funding; ImmunoGen: Consultancy; ARIAD: Research Funding; Sunesis: Consultancy; Alexion: Consultancy; Novartis: Research Funding; Daiichi-Sankyo: Research Funding; Sunesis: Research Funding; Incyte: Research Funding; Karyopharm: Research Funding; Otsuka: Consultancy; Novartis: Consultancy; Kiromic: Research Funding; Karyopharm: Consultancy; Pfizer: Consultancy; Pfizer: Research Funding.

Single-Cell Profiling of Aberrant Cytokine Signaling in Patients with Chronic Myeloid Leukemia (CML) at Diagnosis and during Dasatinib Therapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4214-4214
Author(s):  
Sari Hernesniemi ◽  
Jukka Vakkila ◽  
Kimmo Porkka ◽  
Satu Mustjoki
Keyword(s):  
Growth Factor ◽  
Adverse Effects ◽  
Ex Vivo ◽  
Healthy Controls ◽  
Gm Csf ◽  
Tki Resistance ◽  
Single Cell Profiling ◽  
Vitro Data ◽  
In Vitro Data

Abstract Aberrant cytokine and growth factor signaling is the hallmark of CML and results from constitutive oligomerization of the oncogenic BCR-ABL tyrosine kinase (TK). Inhibition of BCR-ABL by imatinib mesylate is the current standard of care of CML and results in durable responses in majority of patients. However, a proportion of patients shows primary or secondary resistance to imatinib, which can be attributed either to selection of clones harboring mutations in the kinase domain of BCR-ABL or activation of a BCR-ABL independent pathway. Dasatinib, a potent multikinase inhibitor, can rescue some imatinib-resistant patients, but carries an increased risk of adverse effects due to inhibition of off-target wild-type kinases, particularly in immune effector cells. In concord, recent in vitro data indicate a profound immunosuppressive effect of dasatinib. The aim of this study was to analyze and predict TK inhibitor (TKI) resistance and off-target effects using single-cell profiling of aberrant phosphoprotein networks upon cytokine stimulus by multiparameter flow cytometry. The study cohort consisted of 5 healthy controls, 4 non-treated CML patients at diagnosis and 5 CML patients on dasatinib therapy and in cytogenetic remission. Stimuli included GM-CSF, IL-4+IL-6+IFNγ and IL-2+IL-10+IFNα and they were added to freshly drawn whole blood or bone marrow. The readout phosphoproteins were pERK1/2, pSTAT1, pSTAT3, pSTAT5a and pSTAT6 (with isotype controls), and were analyzed separately from granulocytes, monocytes, CD3+, CD4+ and CD8+ lymphocytes and regulatory T-cells. In unstimulated blood samples from healthy controls the phosphoproteins were essentially unphosphorylated. The responses to cytokines were consistent among individuals resulting in phosphorylation of ERK1/2, STAT3 and STAT5a on GM-CSF stimulus, STAT-1, STAT-3 and STAT-5a on IL-2+IL10+IFNα and STAT-1, STAT-3 and STAT-6 on IL4+IL6+IFN-γ. Compared to healthy controls, increased baseline phosphorylation of STAT-1, STAT-3 and STAT5a, but not ERK1/2 was seen in CML patients at diagnosis, especially in myeloid cell lineages (neutrophils/monocytes), but also in lymphocyte subgroups. The responses to cytokine stimulation were modest overall, in particular the ERK1/2 responses to GM-CSF were absent. This indicated the inactivation of the Ras/MEK/MAPK pathway and saturation of other BCR-ABL downstream pathways. Already at diagnosis, the phosphorylation pattern of a TKI primary resistant patient differed profoundly from the responding patients. Marked activation of STAT-1 and STAT-3 was seen in granulocytes and monocytes stimulated either by GM-CSF or by combination of IL2+IL10+IFN-α, suggesting activation by a pathway circumventing BCR-ABL. In dasatinib treated patients, the baseline activation status was similar in granulocytes and monocytes and slightly diminished in lymphocytes when compared to healthy controls. Similarly, the responses to cytokines resembled those seen in healthy controls, in contrast to published in vitro data. Remarkably, in some of the dasatinib treated patients, STAT1 and STAT3 responses were even more pronounced than in healthy controls. This underlines the importance of studying the in vivo/ex vivo effects of TKIs on off-target kinases, in particular of drugs with a short half-life such as dasatinib. In conclusion, inter-individual differences in TKI response and immunomodulatory effects of pan-TKI dasatinib can readily be discerned by analyzing key intracellular phosphoprotein responses to cytokine and growth factor stimuli ex vivo. The method allows profiling of aberrant signaling pathways in different subsets of leukocytes in CML patients and can be used to predict TKI resistance and spectrum of potential adverse effects due to inhibition of wildtype targets. Similar analyses of signaling pathways at the stem cell level are ongoing and may aid in understanding TKI resistance of CML stem cells.

TCR and DNAM Dependent Lysis of Acute Monocytic Leukemia (AMoL) by Vg9Vd2 T Lymphocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1041-1041
Author(s):  
Julie Gertner-Dardenne ◽  
Eloise Perrot ◽  
Thomas Prebet ◽  
Aude Charbonnier ◽  
Helene Sicard ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
T Lymphocytes ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Acute Myeloid

Abstract Abstract 1041 Poster Board I-63 BACKGROUNd: Compelling evidences have demonstrated the role of the immune system in the control of acute myeloid leukemia (AML). So far, T cells and natural killer (NK) cells are the major immune effectors shown to be involved in AML control. The graft-versus-leukemia (GVL) effect following allogenic stem cell transplantation as well as donor lymphocyte infusions indicate that T lymphocytes can control and eliminate AML cells. Leukemia-specific antigenic peptides have been characterized (proteinase-3 and Wilms tumor 1 protein) and serve as targets for peptide-based vaccine trials in AML. Allogenic NK cells have anti-leukemic activity as shown by killer cell inhibitory receptor (KIR)-mismatched haplo-identical stem cell transplantation. Less is known regarding the role of gd T cells in the control of AML. Recently the reconstitution of Vd1 T lymphocytes post transplantation has been shown to correlate with a better prognosis. In the present study, we have analyzed gd T cells in patients with AML and in a mouse model of human AML and focused on (Vg9) Vd2 T cells, the main subset of circulating gd T cells with anti-neoplastic activity. Human Vg9Vd2 T lymphocytes can be activated by nonpeptidic antigens such as the mevalonate pathway-derived isopentenyl pyrophosphate or synthetic phosphoantigen such as bromohydrin pyrophosphate (BrHPP). This population may be suitable for the adoptive immunotherapy of acute myeloid leukemia (AML). However little is known about the frequency, the function and the mechanisms underlying Vg9Vd2 T-cell recognition of AML. We have focused this study on AMoL which are targets of NK and ab T cells. OBJECTIVE OF THE STUDY to describe Vg9Vd2 T cells in patients with AML and investigate their ability to induce an effective cytotoxic response against autologous AML blast in vitro and in vivo. EXPERIMENTAL PROCEDURe: We compared the phenotype and the absolute circulating Vg9Vd2 T cell levels in the blood and the bone marrow (BM) in 12 patients with AMoL (FAB AML-M4 and -M5) and 12 healthy volunteers (HV) using multi parametric flow cytometry. All patients and volunteers gave written informed consent. Vg9Vd2 T cells of AML patient were expanded ex vivo using BrHPP or Zoledronic acid plus IL2. The functions of expanded Vg9Vd2 T cells were assessed in vitro by their cytotoxicity against leukemic blasts (CD107a staining, 51Cr assay) and in vivo in immunodeficient mice transplanted with human AML cell line (U937). In these experiments, the ability of adoptively transferred Vg9Vd2 T cells to migrate into BM and improve mice survival was assessed after i.v. infusion of U937 cells into healthy female NOD-SCID, common _-chain knockout mice (NOG mice). Mice were then treated twice i.v. with 40.106 Vg9Vd2 T cells. RESULTs: Vg9Vd2 T lymphocytes are present in the blood as well as BM of AMoL patients at a lower frequency as compared to HV (median 2.07/μl vs 34/μL respectively P<0.001). Vg9Vd2 T lymphocytes from AML patients are endowed with in vitro proliferation in response to BrHPP or Zoledronic acid plus IL2 but lower than HV (fold increase median 33 versus 69, P=0.051). Expanded Vg9Vd2 express activation markers (CD69 and CCR5) and exhibit an effector/memory phenotype (CD45RA- CD27-). Their lytic potential toward autologous AML blast was equivalent to those of HV by 51Cr experiments and CD107a staining and involves the perforin-granzyme pathway. Their activity depends on both TCRVd2 and DNAX accessory molecule-1 (DNAM-1) as demonstrated by antibody blockade. In vivo data show that, upon sacrifice, Vg9Vd2 were detected in BM, spleen and blood of mice. Preliminary Kaplan-Meier analysis of pooled cohorts of Vg9Vd2-treated and untreated mice reveals that mice receiving Vg9Vd2 T cells displayed superior survival compared with untreated controls (P=0.0047). CONCLUSIOn: Altogether, our data indicate that Vg9Vd2 T cells are decreased in AML patients and have a more limited expansion potential. However, they are able to kill autologous AML blast upon stimulation in a TCRVd2 as well as the DNAM-1 receptor dependent manner. These results provide a rationale for the clinical evaluation of adoptive transfer of ex vivo expanded allogenic Vg9Vd2T cells or direct activation of Vg9Vd2T cells with IL2 + phosphoantigens in patients with AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Novel CD34-Specific T-Cell Engager Efficiently Depletes Stem Cells and Acute Myeloid Leukemia Cells in Vitro and In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2861-2861
Author(s):  
Lucas C M Arruda ◽  
Liqing Jin ◽  
Melanie Lambert ◽  
Laura Sanchez Rivera ◽  
Renato Alvez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
T Cell Activation ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Cell Activation ◽  
Privately Held

Abstract ASH Abstract. Intro Acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS) are poor prognosis hematological malignancies characterized by abnormal hematopoiesis and dysfunctions of the hematopoietic stem cell system. Chemotherapy remains the standard of care but is associated with side effects and often high rates of relapse. Today, less than a third of patients diagnosed with AML are cured. Bispecific T-cell engagers (BiTEs) are promising immunotherapeutic agents intended for cancer treatment. BiTEs are small molecules constructed of two single chain variable fragments (scFv) connected in tandem by a flexible linker that acts by retargeting T-cells against tumor cells. One scFv binds to CD3, while the second scFv binds to a tumor-associated antigen. This structure and specificity allow a BiTE construct to physically link a T-cell to a tumor cell, stimulating effector cell activation ultimately leading to cytokine production and tumor killing. Material BiTEs against CD34/CD3 and relevant controls were constructed by recombinant DNA technology and purified from the supernatants of transfected CHO cells following standard procedures. The scFv domain binding to CD34 is positioned N-terminally, and the scFv binding to CD3e C-terminally followed by a hexa-histidine sequence. Results By co-culturing T-cells and target AML cells for 48 h in the presence of increasing concentrations of BiTE or controls, we observed that CD34-BiTE efficiently triggered T-cell-mediated depletion of the CD34 hi and CD34 low cell lines, while negative controls killed none of the target cell lines. Next, we examined the T-cell activation and proliferation. We observed that both CD4+ and CD8+ T-cells presented high levels of CD25/CD69 expressions when the CD34+ cell lines were co-cultured with T-cells in the presence of the CD34/CD3 BiTE. No unspecific activation was found when CD34- cell line was used as target cell. Since CD34 is constitutively expressed by HSCs, the CD34-specific BiTE may deplete not only CD34 +AML blasts but also healthy HSCs. To test this, T-cells and HSCs were purified from PBSC grafts and co-cultured in the presence of either CD34/CD3 BiTE or controls. After co-culture, a significant depletion of CD34 + HSCs was observed for the CD34/CD3 BiTE. To address the potential of the anti-CD34 BiTE in vivo, we next established a human CD34 + cell line in NSG mice per intravenous injection and randomized into three different groups and started treatment the day after. Two groups of mice received two consecutive cycles of one intraperitoneal injection of freshly isolated human T-cells followed by daily intravenous injections of either BiTE or control. The mice were euthanatized at day 21 by which the AML burden was measured, and T-cells quantified. No side effects of the treatment, including after BiTE administration, was observed. There were statistically significant reductions of leukemia burden in both bone marrow and spleen in mice receiving T-cells and BiTE compared to T-cells only and control. Conclusions We show that the CD34/CD3 BiTE is able to promote T-cell activation and killing of CD34-expressing target cells with high efficacy in vitro and in vivo, supporting the translation of this drug into clinical trials. In this scenario, the treatment with CD34-targeting BiTE prior to HSCT would trigger the patient's T-cells to deplete CD34 + leukemic blasts and HSCs. As consequence, this adjuvant treatment would decrease the use of cytotoxic and cytostatic conditioning drugs before HSCT, reducing life-threatening complications such as GvHD and infections. Disclosures Arruda: Anocca: Current Employment, Research Funding. Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mattsson: MattssonAB medical: Current Employment, Current holder of individual stocks in a privately-held company. Onfelt: Desumo: Current Employment, Current holder of individual stocks in a privately-held company. Uhlin: XNK therapeutics: Current Employment, Current holder of stock options in a privately-held company.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals IMMU-31. QUANTITATIVE EVALUATION OF ROUTES OF ADMINISTRATION OF IMMUNOTHERAPEUTIC T CELLS TO ORTHOTOPIC GLIOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii111-ii111
Author(s):  
Lan Hoang-Minh ◽  
Angelie Rivera-Rodriguez ◽  
Fernanda Pohl-Guimarães ◽  
Seth Currlin ◽  
Christina Von Roemeling ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Ex Vivo ◽  
Adoptive T Cell Therapy ◽  
Whole Body ◽  
T Cell Therapy ◽  
Clinical Responses

Abstract SIGNIFICANCE Adoptive T cell therapy (ACT) has emerged as the most effective treatment against advanced malignant melanoma, eliciting remarkable objective clinical responses in up to 75% of patients with refractory metastatic disease, including within the central nervous system. Immunologic surrogate endpoints correlating with treatment outcome have been identified in these patients, with clinical responses being dependent on the migration of transferred T cells to sites of tumor growth. OBJECTIVE We investigated the biodistribution of intravenously or intraventricularly administered T cells in a murine model of glioblastoma at whole body, organ, and cellular levels. METHODS gp100-specific T cells were isolated from the spleens of pmel DsRed transgenic C57BL/6 mice and injected intravenously or intraventricularly, after in vitro expansion and activation, in murine KR158B-Luc-gp100 glioma-bearing mice. To determine transferred T cell spatial distribution, the brain, lymph nodes, heart, lungs, spleen, liver, and kidneys of mice were processed for 3D imaging using light-sheet and multiphoton imaging. ACT T cell quantification in various organs was performed ex vivo using flow cytometry, 2D optical imaging (IVIS), and magnetic particle imaging (MPI) after ferucarbotran nanoparticle transfection of T cells. T cell biodistribution was also assessed in vivo using MPI. RESULTS Following T cell intravenous injection, the spleen, liver, and lungs accounted for more than 90% of transferred T cells; the proportion of DsRed T cells in the brains was found to be very low, hovering below 1%. In contrast, most ACT T cells persisted in the tumor-bearing brains following intraventricular injections. ACT T cells mostly concentrated at the periphery of tumor masses and in proximity to blood vessels. CONCLUSIONS The success of ACT immunotherapy for brain tumors requires optimization of delivery route, dosing regimen, and enhancement of tumor-specific lymphocyte trafficking and effector functions to achieve maximal penetration and persistence at sites of invasive tumor growth.

scholarly journals In Vivo Role of Dendritic Cells in a Murine Model of Pulmonary Cryptococcosis

2006 ◽  
Vol 74 (7) ◽  
pp. 3817-3824 ◽  
Author(s):  
Karen L. Wozniak ◽  
Jatin M. Vyas ◽  
Stuart M. Levitz
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Mouse Lung

ABSTRACT Dendritic cells (DC) have been shown to phagocytose and kill Cryptococcus neoformans in vitro and are believed to be important for inducing protective immunity against this organism. Exposure to C. neoformans occurs mainly by inhalation, and in this study we examined the in vivo interactions of C. neoformans with DC in the lung. Fluorescently labeled live C. neoformans and heat-killed C. neoformans were administered intranasally to C57BL/6 mice. At specific times postinoculation, mice were sacrificed, and lungs were removed. Single-cell suspensions of lung cells were prepared, stained, and analyzed by microscopy and flow cytometry. Within 2 h postinoculation, fluorescently labeled C. neoformans had been internalized by DC, macrophages, and neutrophils in the mouse lung. Additionally, lung DC from mice infected for 7 days showed increased expression of the maturation markers CD80, CD86, and major histocompatibility complex class II. Finally, ex vivo incubation of lung DC from infected mice with Cryptococcus-specific T cells resulted in increased interleukin-2 production compared to the production by DC from naïve mice, suggesting that there was antigen-specific T-cell activation. This study demonstrated that DC in the lung are capable of phagocytosing Cryptococcus in vivo and presenting antigen to C. neoformans-specific T cells ex vivo, suggesting that these cells have roles in innate and adaptive pulmonary defenses against cryptococcosis.

scholarly journals P01.02 TLR-mediated suppression of the CCL22-CCR4 axis as a new target for tumor immunotherapy

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A3.2-A4
Author(s):  
J Grün ◽  
I Piseddu ◽  
C Perleberg ◽  
N Röhrle ◽  
S Endres ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
Regulatory T Cells ◽  
Type I ◽  
List Type ◽  
Gm Csf

BackgroundUnmethylated CpG-DNA is a potent ligand for the endosomal Toll-like-receptor-9, important for the immune activation to pathogen-associated molecules.1 CpG and other TLR-ligands show effective immunotherapeutic capacities in cancer treatment by inducing an antitumorigenic immunity.2 They are able to reduce tumor progression by reduction of intratumoral secretion of the immunoregulating chemokine CCL223 and subsequent recruitment of immunosuppressive regulatory T cells (Treg), which express CCR4 the only so far known receptor for CCL22.4 Our recent work has shown that CCL22 secretion by dendritic cells (DC) in the lymph node, mediates tolerance by inducing DC-Treg contacts.5 Indeed, in the absence of CCL22, immune responses to vaccination were stronger and resulted in tumor rejection.6 Therefore, we are aiming to investigate the effects of TLR-ligands on systemic CCL22 levels, elucidating all involved mechanisms to identify new targets for cancer immunotherapy.Materials and MethodsT, B and CD11c+ DCs of wildtype (wt) and RAG1-/- mice were isolated from splenocytes by magnetic-activated cell sorting for in vitro assays. Different co-cultures were incubated with CpG and GM-CSF, known as an CCL22 inducer.5 For in vivo experiments, wt mice were treated with CpG, R484 or poly(I:C) alone and in combination with GM-CSF. CCL22-levels in a number of organs were analyzed.ResultsAnalyzing the different immune cell compartments in vitro, we found that DCs in whole splenocytes secrete CCL22 during culture while DC cultured alone showed no CCL22 secretion. When treated with CpG, CCL22-levels were reduced in splenocytes, while it was induced in DC culture alone. The same results were seen when RAG splenocytes, that lack functional B and T cells, were cultured with CpG. CpG treated B cells were able to suppress CCL22 secretion by DC unlike T cells alone. Co-cultures of T and B cells treated with CpG, however, induced the strongest CCL22 suppression in DC. In vivo, we could show that all TLR ligands tested reduced CCL22 in a number of organs significantly. Furthermore, CpG showed the strongest suppression of CCL22 even in the presence of the CCL22 inducer GM-CSF.5ConclusionsWe could show that B cells with T cells mediate CCL22 suppression by TLR ligands. The fact that CpG was able to reduce CCL22 levels even in the presence of the inducer GM-CSF demonstrates the potent CCL22 suppressive capacity of TLR ligands.ReferencesO’Neill LA, et al. The history of toll-like receptors – redefining innate immunity. Nat Rev Immunol 2013;13(6):453–60.Rothenfusser S, et al. Recent advances in immunostimulatory CpG oligonucleotides. Curr Opin Mol Ther 2003;5(2):98–106.Wang S, et al. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A 2016;113(46): E7240–E7249.Rapp M, et al. CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes. J Exp Med 2019;216(5):1170–1181.Piseddu I, et al. Constitutive expression of CCL22 is mediated by T cell-derived GM-CSF. J Immunol 2020;205(8):2056–2065.Anz D, et al. Suppression of intratumoral CCL22 by type i interferon inhibits migration of regulatory T cells and blocks cancer progression. Cancer Res 2015;75(21):4483–93.Disclosure InformationJ. Grün: None. I. Piseddu: None. C. Perleberg: None. N. Röhrle: None. S. Endres: None. D. Anz: None.

Sign in / Sign up

Export Citation Format

Share Document