A Dendritic Cell Based Vaccination Strategy Geared towards Eradication of Leukemic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2046-2046
Author(s):  
Hetty J Bontkes ◽  
Jurjen Ruben ◽  
Willemijn van den Ancker ◽  
Theresia M Westers ◽  
G. Ossenkoppele ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Tumor Antigens ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Vaccination Strategy ◽  
Leukemic Stem Cells ◽  
Cell Immunity

Abstract Abstract 2046 Poster Board II-23 Introduction: In the majority of cases, initial remission of acute myeloid leukemia (AML) is reached but unfortunately relapse rates remain high and therefore novel treatments are needed. It is thought that recurrent AML originates from chemotherapy resistant quiescent leukemic stem cells (LSC). The application of immunotherapeutic approaches to eradicate LSC remaining after first line chemotherapy may contribute to improved disease outcome. Vaccination strategies have often used dendritic cells (DC) ex vivo pulsed with tumor-derived whole lysates or peptides as modalities to present a broad range of tumor antigens to T cells to stimulate effective anti-tumor T-cell immunity in vivo. It is likely that certain proteins expressed by LSC have a distinct antigenicity as compared to more mature AML blasts and thus provide targets for specific T-cells. Even without identification of specific antigens, LSC can be a useful source of tumor antigens in DC vaccination-based immunotherapy. CD34+CD38- LSC can be identified using malignant stem cell associated cell surface markers including CLL-1 and lineage markers such as CD7, CD19 and CD56. However, the low frequency of these cells precludes the use of LSC derived apoptotic cells or lysates for DC loading. Alternatively, mRNA isolated from LSC can be amplified and subsequently transfected into DC. Materials and Methods: We have made use of the CD38- AML derived cell line MUTZ-3 which contains a subpopulation of CD34+CLL1+ cells which resembles the phenotype of a putative LSC. CLL1+CD34+ and CLL1-CD34- cells were isolated by FACS sorting and total RNA was isolated. mRNA was converted to cDNA and amplified by PCR using the SMART system. Subsequently, mRNA was in vitro transcribed from the amplified cDNA. Mature monocyte derived DC (MoDC) were generated from healthy donor blood and transfected with amplified CLL1+CD34+ derived mRNA and used to stimulate autologous CD8β+ T-cells. After three weekly re-stimulations with CLL1+CD34+ mRNA transfected DC, specificity of the T-cells was analyzed by intracellular IFNγ staining upon 5 hour stimulation with autologous immature MoDC transfected with GFP mRNA, mRNA amplified from unsorted, CLL1+CD34+ or CLL1-CD34- MUTZ-3 subpopulations. Results: Amplification of CLL1 and survivin (also expressed by MUTZ-3) transcripts was confirmed by RT-PCR. After 3 weekly re-stimulations with CLL1+CD34+ amplified RNA transfected DC, 0.04% (range 0.01-0.12%) of the T-cells were positive for IFNγ upon a 5 hr re-stimulation with GFP transfected DC. 0.44% (range 0.04-0.69%) of the T-cells responded to DC transfected with unsorted MUTZ-3 amplified mRNA (p<0.00005 versus GFP control, 2-sided student's T-test), 0.51% (range 0.24-1.35%) responded to DC transfected with CLL1+CD34+ amplified mRNA (p<0.005 versus GFP control) and 0.46% (range 0.24-0.94%) responded to DC transfected with CLL1-CD34- amplified mRNA (p<0.0001 versus GFP control). Conclusion: We show that MoDC transfected with RNA amplified from one MUTZ-3 sub-population resembling the phenotype of LCS cells are capable of inducing T-cells which recognize both cells transfected with mRNA from the LSC resembling MUTZ-3 subset as well as the CLL1-CD34- subset. We are currently testing the efficacy and feasibility of this approach in an autologous setting in vitro. CD8β+ T-cells are stimulated with autologous MoDC from AML patients transfected with amplified mRNA isolated from their own LSC enriched populations. The capacity of these T-cells to kill autologous AML blasts and LSC is subsequently analysed in a 6-colour FACS based cytotoxicity assay. Disclosures: No relevant conflicts of interest to declare.

Leukemic Dendritic Cells Expand Central Memory T Lymphocytes From HCT Donors Able to React against the Original Leukemia in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4090-4090
Author(s):  
Monica Casucci ◽  
Serena Kimi Perna ◽  
Attilio Bondanza ◽  
Zulma Magnani ◽  
Massimo Bernardi ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Dendritic Cells ◽  
T Lymphocytes ◽  
Central Memory ◽  
Ionophore A23187 ◽  
Leukemic Stem Cells ◽  
Memory T Lymphocytes

Abstract Abstract 4090 Poster Board III-1025 Allogeneic hematopoietic transplantation (allo-HCT) is the only curative option for patients affected by high-risk acute myeloid leukemia (AML). This is largely due to the ability of allogeneic immune system to eradicate leukemic stem cells (LSC). However, the fact that some patients still relapse after allo-HCT, suggests that strategies to increase LSC targeting by donor T cells are needed. For this purpose, we exploited the unique ability of myeloid blasts to differentiate into leukemic dendritic cells (LDC). We observed that a short (48h) exposure to calcium ionophore A23187 and IL-4 is able to induce LDC differentiation in 14/16 (86%) of AML that we studied, both de novo and secondary. Importantly, despite phenotypic and functional changes indicative of differentiation into DC-like cells, the process was accompanied by the maintenance of disease markers such as CD34 and CD117. Moreover, LDC maintained the expression of the oncogenic protein WT1, which is a putative LSC antigen. Thanks to these favourable characteristics, LDC proved to be superior to the original blasts in expanding leukemia-reactive T lymphocytes both in the autologous and allogeneic HCT setting (on average, 5-fold expansion of blasts-stimulated T cells vs 95-fold expansion of LDC-stimulated T cells, SEM=2,7 and 67,7 respectively, p=0,01). We observed that the level of T-cell expansion directly correlate with the percentage of LDC obtained upon treatment with A23187 and IL-4. Most importantly, LDC proved to be more potent than blasts in expanding central memory T lymphocytes (TCM), which are known to confer superior anti-tumor immunity (on average, 29% of TCM upon stimulation with blasts vs 53% TCM upon stimulation with LDC, SEM=7,2 and 5,7 respectively, p=0,01). LDC-expanded T lymphocytes were able to efficiently recognize and kill leukemic blasts in vitro (on average, 953 specific spots of IFN-g/50'000 effectors at E:T ratio of 10:1 -SEM=120- and 29% of specific killing at E:T ratio of 50:1 -SEM=7,4-). Importantly, analysis of different HLA-settings and different targets of patient origin, suggests that LDC can expand T lymphocytes with specificities against multiple antigens expressed by the original leukemia. In particular, we observed the expansion of WT-1 specific T cells upon LDC stimulation. Finally, when infused in NOD/Scid mice transplanted with the original leukaemia, LDC-stimulated T lymphocytes were able to induce long-term complete remissions (>16 weeks) in all mice analyzed, suggesting that this approach may be active against leukemic stem cells. These results show for the first time that LDC-stimulated human T cells could exert a strong GvL activity in vivo. Disclosures: Bordignon: Molmed Spa: Employment.

Strong CD28 Costimulation Suppresses Induction of Regulatory T Cells From Naïve Precursors through Lck Signaling.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3728-3728
Author(s):  
Kenrick Semple ◽  
Antony Nguyen ◽  
Yu Yu ◽  
Claudio Anasetti ◽  
Xue-Zhong Yu
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Conflicts Of Interest ◽  
Graft Versus Host ◽  
Cell Immunity ◽  
Cd28 Costimulation ◽  
Insight Into

Abstract Abstract 3728 CD28 costimulation is required for the generation of naturally-derived regulatory T cells (nTregs) in the thymus through Lck-signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naïve CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25+Foxp3+) from naïve CD4 T cells (CD25−Foxp3−) by TCR-stimulation with additional TGFβ in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. By using a series of transgenic mice on CD28-deficient background that bears WT CD28 or mutated CD28 in its cytosolic tail incapable of binding to Lck, PI3K or Itk, we found that CD28-mediated Lck-signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide the rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28-signaling. Disclosures: No relevant conflicts of interest to declare.

Direct and Continuous Inhibition of ADAM17 Using a Novel Selective Inhibitor Restores Functional Platelet Yield From Human Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2323-2323
Author(s):  
Shinji Hirata ◽  
Ryoko Jono-Ohnishi ◽  
Satoshi Nishimura ◽  
Naoya Takayama ◽  
Sou Nakamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Imaging System ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
P38 Map Kinase

Abstract Abstract 2323 Platelet transfusion is therapeutically important for patients with thrombocytopenia and/or bleeding disorders. Problems associated with a lack of donors and unknown infections in the blood have not been fully resolved, however. In that context, human induced pluripotent stem cells (hiPSCs) are a potentially abundant source of infection-free platelets. The pluripotent state of hiPSCs and their differentiation depend upon appropriate culture conditions defined in part by oxygen and temperature. We therefore initially examined whether temperatures at or below 24°C, which are required for preservation of platelet concentrates ex vivo, allow hiPSC differentiation to generate platelets. We found that only at 37°C were platelets generated. But at 37°C in vitro, platelets are subject to degradation exemplified by the shedding of GPIbα, a receptor for von Willebrand factor (vWF), which is caused by a disintegrin and metalloprotease (ADAM) 17. We therefore developed KP-457, a novel ADAM17 inhibitor that has a reverse hydroxamic acid structure and has been found safe in rats and dogs. Although inhibition of p38 MAP kinase, putatively upstream of ADAM17, reportedly inhibits GPIbα shedding in stored human platelets, even at 37°C, administration of the p38 inhibitor SB203580 induces cytotoxicity during differentiation, leading to a loss of platelet yield from hiPSCs. By contrast, KP-457 significantly protected GPIbα expression in platelets from hiPSCs and in aged human platelets in culture at 37°C. Moreover, iPSC-derived platelets generated in the presence of KP-457 displayed improved hemostatic function when studied using an imaging system that enables characterization of single-platelet kinetics during thrombus formation after laser-induced injury in vivo. We propose this new drug could markedly improve the maintenance of functional platelets generated in culture, particularly those derived from hiPSCs. Disclosures: No relevant conflicts of interest to declare.

GPR84, a Proinflammatory Receptor, Sustains Wnt/β-Catenin Signaling In Leukemic Stem Cells For Maintenance Of MLL-AF9-Induced Leukemogenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3781-3781
Author(s):  
Philipp A Dietrich ◽  
Murray D Norris ◽  
Jenny Yingzi Wang
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Active Form ◽  
Leukemic Stem Cells ◽  
Poor Survival ◽  
Functional Link ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Inappropriate activation of Wnt/β-catenin signaling confers hematopoietic progenitors the property of self-renewal that promotes malignant transformation in MLL-rearranged acute myeloid leukemia (AML). However, it has been noted that activation of β-catenin is observed in tumors without clear mutations in the major components of the pathway or increase in Wnt signaling. This suggests that other developmental signaling pathways may be capable of inducing activation or downstream signaling of β-catenin. Recently, a number of G protein-coupled receptors (GPCRs) have been shown to activate β-catenin signaling to recruit the key downstream components of the canonical Wnt pathway in distinct cell types, including stem cells. GPCRs, the largest family of cell-surface molecules involved in signal transmission, have emerged as crucial players in tumor growth and metastasis, and represent one of the most important drug targets in pharmaceutical development. Given the close functional link with activation of β-catenin signaling, a GPCR signaling pathway may act as the upstream regulator of β-catenin signaling in the establishment of leukemic stem cells (LSC). In this study, our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified GPR84, a proinflammatory GPCR, as a potential LSC-specific candidate target. An analysis of the comprehensive patient outcome database (Oncogenomics – maintained by the National Cancer Institute) showed that high levels of GPR84 were significantly associated with poor survival in patients with leukemia (P=0.0048), implying its potential clinical relevance in predicting disease prognosis. Western blot and flow cytometric analyses confirmed the microarray results and revealed a positive correlation between GPR84 and β-catenin expression. We previously demonstrated that β-catenin was highly expressed in HSC transformed by MLL-AF9 (HSC-MLLAF9) and had lower expression in HSC transduced with leukemic oncogenes Hoxa9/Meis1 (HSC-Hoxa9/Meis1), while increased β-catenin expression was correlated with a poor survival rate in vivo. Herein, our results showed that forced expression of GPR84 induced a robust upregulation of β-catenin in HSC-Hoxa9/Meis1. Conversely, shRNA-mediated ablation of GPR84 in HSC-MLLAF9 led to highly significant downregulation of both GPR84 (P=0.0003) and β-catenin (P=0.0008). Further in vitro functional studies showed that GPR84 knockdown significantly reduced HSC-MLL-AF9 colony forming units (P=0.0006), and induced a marked reduction of cells in S-phase (P=0.0017). This deficient phenotype could be rescued by expression of a constitutively active form of β-catenin. Importantly, subsequent in vivo survival studies using leukemia transplantation mouse models showed that GPR84 knockdown significantly reduced LSC frequency and severely impaired maintenance (P<0.0001; 11 mice per cohort) of HSC-MLL-AF9 induced leukemia, a highly aggressive and drug-resistant subtype of AML. The defect in disease phenotype resulted from inhibited expression of both GPR84 and β-catenin. Furthermore, forced overexpression of GPR84 alone was not sufficient for leukemic transformation of HSC but conferred a growth advantage in vivo to HSC-Hoxa9/Meis1 cells and significantly accelerated the onset of Hoxa9/Meis1-induced AML (P=0.0039), establishing a completely malignant phenotype similar to HSC-MLL-AF9 in vivo (P=0.9986). These data support an oncogenic role of GPR84 in MLL-AF9-induced leukemogenesis. In conclusion, our studies have identified a novel β-catenin regulator that contributes to leukemia maintenance by sustaining aberrant activation of a stem cell self-renewal pathway in LSC, and drugs targeting GPR84 may represent a novel and promising strategy for improving the therapy and outcome of AML patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Use of CD70 Targeted Chimeric Antigen Receptor (CAR) T Cells for the Treatment of Acute Myeloid Leukemia (AML)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4443-4443 ◽  
Author(s):  
Mark Leick ◽  
Irene Scarfò ◽  
Bryan D. Choi ◽  
Rebecca Larson ◽  
Amanda A Bouffard ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Synergistic Activity ◽  
Leukemic Stem Cells ◽  
Cytotoxicity Assays ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

Background: CAR-T cells have led to a revolution in the treatment of advanced hematologic malignancies. Since these cells target antigens that are expressed on the cellular surface, it is imperative that there is near ubiquitous tumor expression with minimal expression vital human tissues. Finding targets with these characteristics in myeloid malignancies has been challenging. Typical markers expressed on the surface of AML are also expressed on essential innate immune effector cells (e.g. neutrophils) which, if targeted, could lead to prolonged absence of this immune arm, which is not survivable or replaceable. Current approaches rely on the use of CAR-T cells against common myeloid targets (e.g. CD123, CD33) as an ablative strategy with a planned allogeneic stem cell transplant rescue to eradicate the CAR-T cells afterwards. These solutions have resulted in significant toxicity with several deaths resulting from CD123-targeted CAR-T cells. Another approach has involved gene editing donor progenitor cells to delete CD33, repopulation of the marrow with these CD33 negative cells, and then treatment with CD33-targeted CAR-T cells. (Kim, Cell 2018). However, this approach is challenging, costly, and genomic editing of stem cells remains a concern. CD70 is an immune checkpoint found on antigen presenting cells and activated T cells. Multiple studies have shown a strong degree of expression on AML blasts and leukemic stem cells, with minimal normal tissue expression (Perna, Cell 2017, Riether J Exp Med 2017). A Phase 1 study of a CD70 targeted antibody drug conjugate in combination with azacitidine (which has been shown to increase CD70 expression on leukemic stem cells) for untreated AML patients has shown impressive results (Blood 2018 132:2680, Blood 2017 130:2652). Based on these findings, we explored CD70-targeting CARs for the treatment of AML. Methods: Based on our success with a trimeric ligand-based CAR of another TNFα family member, APRIL, for multiple myeloma (Schmidt Blood 2018 132:2059), we generated monomeric and trimeric second-generation ligand-based CAR constructs to target CD70 on AML. In vitro effector function was compared by cytotoxic potency and cytokine production. In vivo anti-tumor efficiency was assessed in a xenograft mouse model of AML. Effect of surface CD70 expression on AML cell lines after co-culture with azacitidine was assessed. Results: CAR T cell manufacturing of both constructs was accomplished successfully (transduction efficiency 70-93%) from three different healthy donors with no apparent fratricide. CD70 CARs were efficacious in in vitro cytotoxicity assays targeting an AML cell line Molm13. Unexpectedly, monomeric CD70 targeted CAR-T cells were superior to trimeric in cytotoxicity assays and, thus, were carried forward for in vivo assays. Next, we treated NSG mice that had been engrafted with Molm13 and demonstrated a substantial dose-dependent therapeutic effect with prolonged survival of CAR treated mice compared to those treated with untransduced T-cells (UTD). Treated mice demonstrated a CAR-T robust expansion in the peripheral blood assessed by flow cytometry that was commensurate with individual animal treatment responses. Bone marrow from these mice revealed substantially reduced CD70 in all groups. Preliminary in vitro co-culture of AML cells with azacitidine showed increased CD70 expression. Conclusion: CD70 based CAR-T targeting of AML is effective in vitro and in vivo. Combination treatment with azacitidine may increase target antigen expression and lead to synergistic activity and represents a viable therapeutic strategy that warrants further investigation. Treatment of AML engrafted NSG mice with CD70 CAR-T cells in conjunction with azacitidine is ongoing. Disclosures Frigault: Xenetic: Consultancy; Novartis: Consultancy; Juno/Celgene: Consultancy; Foundation Medicine: Consultancy; Incyte: Consultancy; Nkarta: Consultancy; Kite/Gilead: Honoraria. Maus:INFO PENDING: Other: INFO PENDING.

The Checkpoint for Agonist Selection Precedes Conventional Selection in Human Thymus

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 860-860
Author(s):  
Greet Verstichel ◽  
David Vermijlen ◽  
Liesbet Martens ◽  
Glenn Goetgeluk ◽  
Yvan Saeys ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Double Positive ◽  
Human Thymus ◽  
Tcr Signals

Abstract The thymus plays a central role in self-tolerance by preventing strongly self-reactive thymocytes from accumulating as naïve T cell receptor (TCR) αβ+ T cells in the periphery. The elimination of auto-reactive T cells from the naïve pool is in part mediated by deletion during conventional negative selection. Alternatively, self-reactive thymocytes can also be positively selected in response to strong TCR signals during agonist selection and functionally differentiate to innate TCRαβ + T cells such as the CD8αα+ double negative (DN) T cells. How thymocytes discriminate between these opposite outcomes remains unclear. We identified a novel agonist-selected PD-1+ CD8αα+ subset of mature CD8+ T cells in human thymus. Using the same markers a similar population was also identified in cord blood at about the same frequency as TCRγδ+ cells. This population expresses high levels of Helios, indicative of strong TCR engagement, and displays an effector phenotype associated with agonist selection. Indeed, PD-1+CD8αα+ T cells exhibit innate production of IFN-γ and an elevated T-bet to Eomes ratio typical of effector CD8 T cells. These cells are CD62L-, CXCR3+ and Hobit high suggesting that these cells leave the thymus and home to the tissues. Interestingly, in vitro CD3/TCR stimulation of sorted early post-β-selection thymocyte blasts uniquely gives rise to this innate subset, whereas small CD4+CD8+ double positive precursors fail to survive strong TCR signals. The generation of the innate subset seems to arise also in vivo from early post-β-selection thymocyte blasts as these two populations have an identical TCRα repertoire: ex vivo isolated PD-1+CD8αα+ thymocytes are skewed for early 3' TRAV and 5' TRAJ rearrangements compared to conventional CD8 T cells. A similar skewing was found in early post-β-selection thymocyte blasts. As TCRα rearrangements are terminated by TCR engagement of agonist selection, this is strong evidence for a precursor progeny relationship. Together, we conclude that human CD8αα+ T cells are preferentially selected by strong TCR engagement on a subset of progenitors that express a full TCRαβ early on, leading to the generation of a post-selection T cell population with innate functional capacity and a markedly distinct TCR repertoire. These findings uncover the heterogeneity among DP precursors in their potential to survive strong selection signals and suggests that the decision making in the thymus to divert immature thymocytes to the agonist selection pathway occurs early before conventional selection of DP cells. We propose that progression through the immature thymic developmental program influences the outcome of TCR engagement with early post-β-selection thymocytes triggered by strong TCR signals preferentially giving rise to innate CD8αα+ T cells in humans. Disclosures No relevant conflicts of interest to declare.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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.

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