scholarly journals Sodium activates human monocytes via the NADPH oxidase and isolevuglandin formation

2020 ◽  
Author(s):  
Natalia Ruggeri Barbaro ◽  
Justin Van Beusecum ◽  
Liang Xiao ◽  
Luciana do Carmo ◽  
Ashley Pitzer ◽  
...  
Keyword(s):  
Risk Factors ◽  
T Cells ◽  
Nadph Oxidase ◽  
Immune Cells ◽  
Adduct Formation ◽  
High Salt ◽  
Human Monocytes ◽  
Activation Markers

Abstract Aims Prior studies have focused on the role of the kidney and vasculature in salt-induced modulation of blood pressure; however, recent data indicate that sodium accumulates in tissues and can activate immune cells. We sought to examine mechanisms by which salt causes activation of human monocytes both in vivo and in vitro. Methods and results To study the effect of salt in human monocytes, monocytes were isolated from volunteers to perform several in vitro experiments. Exposure of human monocytes to elevated Na+ex vivo caused a co-ordinated response involving isolevuglandin (IsoLG)-adduct formation, acquisition of a dendritic cell (DC)-like morphology, expression of activation markers CD83 and CD16, and increased production of pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-6, and IL-1β. High salt also caused a marked change in monocyte gene expression as detected by RNA sequencing and enhanced monocyte migration to the chemokine CC motif chemokine ligand 5. NADPH-oxidase inhibition attenuated monocyte activation and IsoLG-adduct formation. The increase in IsoLG-adducts correlated with risk factors including body mass index, pulse pressure. Monocytes exposed to high salt stimulated IL-17A production from autologous CD4+ and CD8+ T cells. In addition, to evaluate the effect of salt in vivo, monocytes and T cells isolated from humans were adoptively transferred to immunodeficient NSG mice. Salt feeding of humanized mice caused monocyte-dependent activation of human T cells reflected by proliferation and accumulation of T cells in the bone marrow. Moreover, we performed a cross-sectional study in 70 prehypertensive subjects. Blood was collected for flow cytometric analysis and 23Na magnetic resonance imaging was performed for tissue sodium measurements. Monocytes from humans with high skin Na+ exhibited increased IsoLG-adduct accumulation and CD83 expression. Conclusion Human monocytes exhibit co-ordinated increases in parameters of activation, conversion to a DC-like phenotype and ability to activate T cells upon both in vitro and in vivo sodium exposure. The ability of monocytes to be activated by sodium is related to in vivo cardiovascular disease risk factors. We therefore propose that in addition to the kidney and vasculature, immune cells like monocytes convey salt-induced cardiovascular risk in humans.

scholarly journals TRPM2 Is Not Required for T-Cell Activation and Differentiation

2022 ◽  
Vol 12 ◽  
Author(s):  
Niels C. Lory ◽  
Mikolaj Nawrocki ◽  
Martina Corazza ◽  
Joanna Schmid ◽  
Valéa Schumacher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transient Receptor Potential ◽  
Cell Function ◽  
Intestinal Inflammation ◽  
Activation Markers

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+ signal that is crucial for T-cell function. The Ca2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro, Trpm2-/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2-/- CD8+ T cells and unimpaired differentiation of CD4+ T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo, Trpm2-/- and WT CD8+ T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2-/- mice and after transfer of WT and Trpm2-/- CD8+ T cells into infected recipients. CD4+ T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2-/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

scholarly journals 708 Novel ways to exploit IL-21 to augment adoptive T cell transfer therapy against tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A737-A737
Author(s):  
Anna Cole ◽  
Guillermo Rangel RIvera ◽  
Aubrey Smith ◽  
Megan Wyatt ◽  
Brandon Ware ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Striking Difference ◽  
T Cell Therapy ◽  
Cell Immunity ◽  
Emory University

BackgroundIL-21 enhances the anti-tumor capacity of adoptively transferred CD8+ T cells, while IL-2 and IL-15 impair T cell immunity by driving their expansion to a more differentiated status. Yet, these cytokines can act on many different immune cells. Given the potency of IL-21, we tested if this cytokine directly augments T cells or rather if it enhances other immune cells in the culture that indirectly improves T cell therapy.MethodsTo test this question, splenocytes from pmel-1 transgenic mice were used, as all CD8+ T cells express a transgenic TCR specific for tumor-antigen gp10025–33 overexpressed on melanoma. We then peptide activated naïve CD8+ T cells enriched or not from the spleen of pmel-1 mice and expanded them in the presence of IL-21 or IL-2 (10 ng/mL) for four days. Expanded pmel-1 from these various cultures were then restimulated with irradiated splenocytes pulsed with gp10025–33 and grown an additional seven days with IL-2 (10 ng/mL), irrespective of their initial cytokine condition. The in vitro memory phenotype, exhaustion profile, and cytokine secretion of these cultures were then assayed. Furthermore, mice bearing B16KVP melanoma tumors were infused with pmel-1 T cells expanded via these various approaches and compared for their relative capacity to engraft, persist, and regress tumor in vivo.ResultsInterestingly, we discovered that IL-21-treated T cells generated from bulk splenocytes are phenotypically and functionally distinct from IL-21-treated isolated T cells. Upon restimulation, IL-21-treated T cells from bulk splenocytes exhibited an exhausted phenotype that was like anergic IL-2-treated T cells. Moreover, few cells expressed CD62L but expressed heightened markers of suppression, including TIM3, PD-1, and EOMES. Moreover, they produced more effector molecules, including granzyme B and IFN-gamma. In vivo IL-21-treated T cells expanded from bulk splenocytes engrafted and persisted poorly, in turn mediating suboptimal regression of melanoma. Conversely, IL-21 dramatically bolstered the engraftment and antitumor activity of T cells only if they were first isolated from the spleen prior to their expansion and infusion into the animal.ConclusionsCollectively, our data shows that IL-21 may improve ACT therapy best when used directly on antitumor CD8+ T cells. Further studies will illuminate the mechanism behind this striking difference and determine whether other cell subsets reactive to IL-21 cause T cell dysfunction and/or reduced bioavailability. These findings are important for defining the best culture conditions in which to use IL-21 for ACT.AcknowledgementsWe would like to acknowledge Emory University, The Winship Cancer Institute, and the Pediatrics/Winship Flow Cytometry Core.Ethics ApprovalAll animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University, protocol number 201900225.

scholarly journals In Vivo Suppression of Autophagy via Lentiviral shRNA Targeting Atg5 Improves Lupus-Like Syndrome

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Chi-Jui Liu ◽  
Shye-Jye Tang ◽  
Chun-Che Chou ◽  
Guang-Huan Sun ◽  
Kuang-Hui Sun
Keyword(s):  
T Cells ◽  
Immune Cells ◽  
Plasma Cells ◽  
B Cell Activating Factor ◽  
Dsdna Antibody ◽  
Lymph Node Cells ◽  
Complex Deposition ◽  
Novel Strategy

In both mouse models and clinical patients with lupus, autophagy levels were significantly elevated and correlated with disease activity. Furthermore, autophagy can promote the survival of B and T cells, plasma cell differentiation, and antibody production. These results suggest that autophagy may promote the progression of lupus by regulating the survival of autoreactive immune cells. Therefore, we aimed at studying whether suppressing autophagy can modulate lupus progression in vivo. First, we found that the autophagy levels in splenocytes and lymphocytes of peripheral blood (PB) were elevated and positively correlated with disease severity in lupus-prone mice. The shAtg5-lentivirus, which effectively inhibits autophagy in vitro, was then injected into the lupus-prone mice. Autophagy levels in lymph node cells and PB lymphocytes were reduced following Atg5 suppression. We also found that lymphadenopathy and the numbers of plasma cells, CD4-CD8-, and CD4+ T cells decreased in mice treated with the shAtg5-lentivirus. The mice treated with shAtg5-lentivirus exhibited lower levels of proteinuria, serum anti-dsDNA antibody, B-cell activating factor (BAFF), and glomerular immune complex deposition. Therefore, targeting autophagy to moderate overactivated autophagy in immune cells seems to be a novel strategy for combination therapy of lupus.

Abstract 003: Direct and Indirect Effects of Prostaglandin E 2 and Its EP1/EP3 Receptors on Dendritic Cell Activation in Mice with L-NAME/High Salt-induced Hypertension

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Liang Xiao ◽  
Hana A Itani ◽  
Maria P Kraemer ◽  
Richard M Breyer ◽  
David G Harrison
Keyword(s):  
Cell Activation ◽  
Systolic Pressure ◽  
Adduct Formation ◽  
High Salt ◽  
Effector Memory ◽  
Prostaglandin E ◽  
Dendritic Cell Activation ◽  
Ep3 Receptor

We recently identified a pathway underlying immune activation in hypertension. Proteins oxidatively modified by reactive γ-ketoaldehydes (isoketals) accumulate in dendritic cells (DCs). These are immunogenic and lead to subsequent T lymphocytes activation. The local signals that stimulate DCs to accumulate isoketal adducts remain undefined. Prostaglandin E 2 (PGE 2 ) has been implicated in the inflammation associated with hypertension. We hypothesized that PGE 2 via its EP3 receptor contributes to DC activation in hypertension. EP3 -/- mice and wild type (WT) littermates were exposed to sequential hypertensive stimuli involving an initial 2-week exposure to the NOS inhibitor L-NAME (LN) in drinking water, a 2 week washout period, and a subsequent 4% high salt diet (HS) for 3 weeks. In WT mice, this protocol increased systolic pressure from 123±2 to 148±8 mmHg (p<0.05), and renal CD4 + and CD8 + effector memory T cells by 2 to 3 fold. This was associated with a striking accumulation of isoketal protein adducts in splenic DCs. However, the increases in blood pressure, renal T cell infiltration and DC isoketal formation were completely prevented in EP3 -/- mice. We further hypothesized that EP3 receptors contribute to oxidative stress production in the kidney. As measured by dihydroethidium with confocal microscopy, the LNHS protocol induced marked increases in superoxide production in WT mice, but not in EP3 -/- mice. To examine the direct effects of PGE 2 , splenic DCs were incubated with PGE 2 in vitro for 24 hours. PGE 2 dose-dependently increased isoketal-adduct formation in DCs (vehicle: 8.8±5.1% vs. 50 nM PGE 2 : 41.4±11.7%, p<0.05). Interestingly, this effect was not blocked by the EP3 receptor antagonist DG-041 (30 nM), but was completely prevented by the EP1 receptor blocker SC-51322 (20 μM). These data indicate both direct and indirect roles of PGE 2 in DC activation in hypertension. In vivo, PGE 2 has a predominant effect on EP3 receptors to enhance renal vascular ROS production, which likely leads to isoketal-adduct formation and accumulation in DCs. PGE 2 also acts directly on DCs via its EP1 receptors to stimulate intracellular isoketal formation. Together, these findings provide additional information as to how PGE 2 modulates inflammation in hypertension.

Mesenchymal Stromal Cells Suppress T-and B-Cells via Galectin-9 in a Donor Dependent Manner

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1248-1248
Author(s):  
Christopher Ungerer ◽  
Patricia Quade-Lyssy ◽  
Reinhard Henschler ◽  
Erhard Seifried ◽  
Heinfried Radeke ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Immune Suppression ◽  
Immune Cells ◽  
Stromal Cells ◽  
Coagulation Factor ◽  
T And B Cells ◽  
Ifn Γ

Abstract Abstract 1248 Therapeutic approaches using multipotent mesenchymal stromal cells (MSCs) are advancing in regenerative medicine, transplantation and autoimmune diseases. Until now the way of action for MSC-mediated immune suppression is still controversial and relies most probably on a multifactorial mechanism. MSCs have been demonstrated to produce the suppressive molecules hepatocyte growth factor (HGF), tumor growth factor-β (TGF-β), prostaglandin E2 (PGE2) and indoleamine 2,3-dioxygenase (IDO). Furthermore, it has been described that immunosuppression by MSCs is enhanced via stimulation with interferon-γ (IFN-γ). Recently, galectin-1, a β-galactoside binding lectin with immune modulatory properties, has been added to the group of immune modulatory molecules that are responsible for MSC-mediated immune suppression. Here, we identified galectin-9 (Gal-9) as a new molecule involved in MSC-mediated immune modulation. First, we isolated MSCs from bone marrow of randomly selected donors and performed several in vitro experiments regarding their immune modulatory potential (e.g proliferation and IgG production). Interestingly, Gal-9 was the only investigated protein, which was strongly upregulated in MSCs upon activation with IFN-γ. We moreover demonstrate that Gal-9 is a major mediator of the anti-proliferative effect of MSCs on T-cells. Although a B-cell suppressive function of Gal-9 has previously not been reported, we were surprised to detect the same inhibitory effect on isolated B-cells. Proliferation of immune cells was triggered upon either stimulation with either PHA and LPS, or CD40L and PHA. Activation of MSCs with IFN-γ resulted in a major decrease of proliferation of both T-cells and B-cells. In addition, Gal-9 and activated MSCs contribute to the suppression of VZV triggered immunoglobulin release as well. Again activation of MSCs with IFN-γ decreased the IgG release, whereas blocking Gal-9 with lactose, a well characterized inhibitor of Gal-9 function, reversed the effect almost completely. Further, we determined that Gal-9 expression levels (mRNA and protein) distinguish between MSC cultures from different donors after activation. Among donors, we could differentiate between individuals with high Gal-9 levels and higher immune modulatory potential and such with low Gal-9 expression and lower immune modulatory potential. Compared to untreated MSCs we demonstrated a three- to fifty-fold rise in Gal-9 levels after prior activation with IFN-γ. In addition, we demonstrated the upregulation of Gal-9 in MSCs by cell-cell contacts with either T-or B-cells. The upregulation was additionally at least two fold increased by previeously activating MSCs with IFN-γ. Because our group is interested in the therapy of hemophilia A and because of the unxpected suppressive effect of Gal-9 on B-cells and B-cell function, we next tested the effect of MSCs and Gal-9 on the induction of inhibitory antibodies to coagulation factor VIII (FVIII). Mice were immunized with human coagulation factor VIII (FVIII) in the presence or absence of either human MSCs, anti-murine Gal-9 or human Gal-9. As predicted, MSCs suppressed and anti-Gal-9 antibodies anhanced antibody formation. However in contrary to the expected, human Gal-9 co-treatment enhanced the anti-FVIII antibody response. A set of additional experiments revealed, that human Gal-9 suppresses murine regulatory T-cells in vivo. Further, in contrast to human immune cells, murine-derived T- and B-cells did not respond to human recombinant Gal-9 in vitro, but human IFN-γ activated MSCs were able to suppress proliferation of murine immune cells. Because of only 60% homology of murine and human Gal-9 we assume that the murine model cannot predict the function of human Gal-9 and that MSC-mediated immune modulatory functions are exerted via alternative pathways in this setting. Experiments with murine Gal-9 to demonstrate the in vivo function of Gal-9 are ongoing. In conclusion, Gal-9 is novel mediator of MSC immunomodulatory functions and affectsmultiple immune cell types including B-cells. Gal-9 is differentially expressed in MSCs from different donors and may therefore serve as a predictive indicator for clinical MSC functionality. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Eradication of disseminated murine leukemia by chemoimmunotherapy with cyclophosphamide and adoptively transferred immune syngeneic Lyt-1+2- lymphocytes.

1981 ◽  
Vol 154 (3) ◽  
pp. 952-963 ◽  
Author(s):  
P D Greenberg ◽  
M A Cheever ◽  
A Fefer
Keyword(s):  
T Cells ◽  
Immune Cells ◽  
Positive Contribution ◽  
Friend Virus ◽  
Effector Cells ◽  
Cytotoxic Responses ◽  
Murine Leukemia ◽  
T Cell Growth Factor

The phenotype of T cells therapeutically effective in immunotherapy of advanced Friend virus-induced (FBL) leukemia in vivo and cytotoxic to FBL in vitro was determined. Mice bearing disseminated FBL leukemia were successfully treated by a combination of cyclophosphamide and adoptive transfer of syngeneic immune lymphocytes. Therapeutic efficacy was largely dependent on the presence of Lyt-1+2- T cells in the transferred cells, whereas cells cytotoxic to FBL tumor in vitro were derived from the Lyt-1+2+ and Lyt-1-2+ subsets. Thus, the predominate cell required to eradicate tumor in adoptive chemoimmunotherapy was not cytolytic to tumor in vitro. Potentially, the Lyt-1+2- cell may operate in vivo as an amplifier cell rather than by a direct anti-tumor effect. Elimination of the Lyt-1+ population with alpha-Lyt-1 and complement prevented the generation of significant cytotoxic responses during both primary in vitro sensitization to alloantigens and in vitro sensitization of tumour-primed cells. The capacity of Lyt-1+ cell-depleted population to generate cytotoxic responses was partially reconstituted by addition, at the initiation of culture, of interluekin 2, a T cell growth factor derived from Lyt-1+2- cells, which contain the CTL and CTL precursors, were nearly as effective in vitro as unseparated immune cells. If the remaining effector cells (i.e., Lyt-1+2- T cells) function in vivo predominantly as amplifier cells, than the tumour-bearing host must be capable of making a positive contribution to the outcome of therapy.

scholarly journals In Vitro-Generated Antigen-Specific CD4+ CD25+ Foxp3+ Regulatory T Cells Control the Severity of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions

2008 ◽  
Vol 82 (14) ◽  
pp. 6838-6851 ◽  
Author(s):  
Sharvan Sehrawat ◽  
Susmit Suvas ◽  
Pranita P. Sarangi ◽  
Amol Suryawanshi ◽  
Barry T. Rouse
Keyword(s):  
T Cells ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Regulatory T Cells ◽  
Inflammatory Disease ◽  
Activation Markers ◽  
Control Virus ◽  
Simplex Virus

ABSTRACT Generating and using regulatory T cells (Tregs) to modulate inflammatory disease represents a valuable approach to therapy but has not yet been applied as a means to control virus-induced immunopathological reactions. In this report, we developed a simplified technique that used unfractionated splenocytes as a precursor population and showed that stimulation under optimized conditions for 5 days with solid-phase anti-CD3 monoclonal antibody in the presence of transforming growth factor β (TGF-β) and interleukin-2 could induce up to 90% of CD4+ T cells to become Foxp3+ and able to mediate suppression in vitro. CD11c+ dendritic cells were intricately involved in the conversion process and, once modified in the presence of TGF-β, could convert Foxp3− CD4+ cells into Foxp3+ CD4+cells by producing TGF-β. The converted cells had undergone cell division, and the majority of them expressed activation markers along with surface molecules that would facilitate their migration into tissue sites. The primary reason for our study was to determine if such in vitro-converted Tregs could be used in vivo to influence the outcome of a virus-induced immunoinflammatory lesion in the eye caused by herpes simplex virus infection. We could show in three separate models of herpetic stromal keratitis that adoptive transfers of in vitro-converted Tregs effectively diminished lesion severity, especially when given in the initial phases of infection. The suppression effect in vivo appeared to be polyspecific. The protocol we have developed could provide a useful additional approach to control virus-induced inflammatory disease.

scholarly journals 720 CUE-102 selectively activates and expands WT1-specific T cells for the treatment of patients with WT1+ malignancies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A749-A749
Author(s):  
Christie Zhang ◽  
Natasha Girgis ◽  
Zohra Merazga ◽  
Steven Hatfield ◽  
Alex Histed ◽  
...  
Keyword(s):  
T Cells ◽  
Transgenic Mice ◽  
Cd8 T Cells ◽  
Wilms Tumor ◽  
Interleukin 2 ◽  
Target Cells ◽  
Tolerability Profile ◽  
Activation Markers

BackgroundWilms' Tumor 1 (WT1) was ranked as the highest priority antigen for therapeutic targeting in an effort by the National Cancer Institute. Development of novel modalities targeting WT1 provide a significant opportunity to address high unmet medical need in WT1-positive malignancies, including AML, ovarian, endometrial, breast, lung, colorectal and pancreatic cancer. Leveraging the Immuno-STAT platform of targeted IL-2 therapies, and the ongoing development of CUE-101, CUE-102 is being developed as a novel therapeutic fusion protein to selectively activate tumor antigen-specific T cells to treat WT1-expressing cancers. CUE-102 consists of two human leukocyte antigen (HLA) molecules presenting a WT1 peptide, four affinity-attenuated human interleukin-2 (IL-2) molecules, and an effector attenuated human immunoglobulin G (IgG1) Fc domain.MethodsHuman PBMCs were tested to demonstrate cellular activity and specificity of CUE-102, while in vivo activity of CUE-102 was assessed in HLA-A2 transgenic mice. HLA-A2/WT1-specific TCRs were validated and expressed in primary human CD8 T cells. Tetramer staining and flow cytometry identified cell populations and activation markers.ResultsMultiple in vitro assessments demonstrate that CUE-102 selectively activates and expands WT1-specific CD8+ T cells from PBMC of healthy and cancer bearing donors. These CUE-102-expanded CD8+ T cells exhibit polyfunctional and cytotoxic responses upon challenge with WT1-presenting target cells. In addition, significant functional attenuation of the IL-2 components of CUE-102 was shown, similar to preclinical results obtained with CUE-101. In vivo studies in HLA-A2 transgenic mice confirm that CUE-102 elicits and expands polyfunctional WT1-specific CD8+ T cells from naïve and previously immunized mice without significantly altering the frequencies of other immune lineages. The WT1-specific CD8+ T cells expanded in vivo exhibit polyfunctionality in response to peptide-loaded target cells, and selectively kill WT1-presenting target cells in vivo.ConclusionsCUE-102 elicits selective expansion of a WT1-specific population of cytotoxic CD8+ T cells both in vitro and in vivo. These results, together with its similarity to CUE-101, support its anticipated tolerability profile and potential for clinical efficacy in a Phase 1 trial planned to initiate in 2022.Ethics ApprovalAll animal studies followed guidance from the SmartLabs Institutional Animal Care and Use Committee protocol MIL-100 and were performed in compliance with federal guidelines.

scholarly journals High Salt Promotes Human Monocytes Activation In Vitro and In Vivo

2018 ◽  
Vol 32 (S1) ◽  
Author(s):  
Natalia Ruggeri Barbaro ◽  
Jason D. Foss ◽  
Aseel Alsouqi ◽  
Roxana Loperena ◽  
Justin Van Beusecum ◽  
...  
Keyword(s):  
High Salt ◽  
Human Monocytes

scholarly journals Pre-Clinical Development of Gene Modification of Hematopoietic Stem Cells with Chimeric Antigen Receptors for Cancer Immunotherapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4430-4430
Author(s):  
Laurel Christine Truscott ◽  
Sarah Larson ◽  
Amie Patel ◽  
Roy L. Kao ◽  
Satiro N. De Oliveira
Keyword(s):  
Stem Cells ◽  
Clinical Trials ◽  
T Cells ◽  
Immune Cells ◽  
Suicide Gene ◽  
Antigen Specificity ◽  
Hematopoietic Stem ◽  
Cancer Activity

Abstract Background: Patients with refractory or recurrent B-lineage hematological malignancies have less than 50% of chance of cure, despite intensive therapy. Innovative approaches are needed to decrease their morbidity and mortality. Chimeric Antigen Receptors (CAR) successfully engineer antigen specificity in immune cells, with clinical trials currently being conducted using ex vivo expanded gene-modified mature T cells. Results from preclinical studies and clinical trials show that effector cells usually have transient in vivo persistence that could significantly limit clinical efficacy and allow tumor recurrence. Building upon the standard of care and seeking an approach that could foster persistence of the CAR-modified cells, we have published studies using anti-CD19 CAR-modified human hematopoietic stem cells (HSC) engrafted in NSG immunodeficient mice. We hypothesize that modification of HSC with CD19-specific CAR will generate persistent multi-lineage anti-tumor activity for immunotherapy of CD19+ hematological malignancies. To increase the safety of the modification of HSC, a suicide gene can be inserted into the vector to eradicate the modified cells in the setting of toxicity. Thorough evaluation of this approach in relevant study models is required for advancement to clinical trials. Significance: This approach is untested in clinical translation to this date, and implies harnessing a patient's own HSC to create a whole self-renewing immune system directed to destroy cancer, a concept that can be applied to different cancers just by adjusting the target specificity. The prospect of modifying autologous cells to enhance graft-versus-cancer activity bears the possibility of decreased morbidity and mortality, being desirable for specifically vulnerable populations, as children and elderly patients, and offering alternative therapy for those without cell sources available for allogeneic HSC transplantation, benefiting patients from ethnic minorities. This approach can be easily adopted in the clinical setting for patients planned to receive autologous HSC transplant as their standard therapy, enhancing graft-versus-cancer activity with anti-CD19 specificity. Methods: High-titer third-generation lentiviral constructs were produced carrying second-generation CD19-specific CAR co-stimulated by CD28. These vectors also co-delivered HSV-sr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Human HSC isolated from umbilical cord blood and G-CSF-mobilized apheresed peripheral blood stem cells (PBSC) were transduced with such lentiviral vectors and injected into NSG pups after irradiation for in vivo evaluation of engraftment, function and suicide gene activation. Results: We have consistently achieved engraftment of human cells in about 95% of study mice, with engraftment of CAR+ cells in about 80% of the animals. Human HSC were successfully transduced with lentiviral vectors carrying anti-CD19 CAR with no impairment of differentiation or proliferation in vitro and in vivo. Immune cells differentiated in vivo from CAR+ HSC had antigen-specific cytotoxicity directed by CAR. CAR+ human cells were detected in BM, spleen, blood and thymus of injected mice. CAR+ T cells were stably detected in the blood of engrafted mice up to 40 weeks post-injection, demonstrating lymphopoiesis of CAR+ T cells successfully escaping thymic deletion and persisting throughout murine lifetime. As a surrogate of the antigen specificity and efficacy, CD19+ cells were significantly decreased in all mice engrafted with anti-CD19 CAR demonstrating that CAR+ immune cells were not inactivated or developed tolerance. Mice humanized with at least 4% of CAR+ cells in blood had significant protection against challenge with CD19+ tumor cell line, with inhibition or elimination of tumor development and consequent survival advantage. Activation of HSV-sr39TK suicide gene by ganciclovir treatment successfully led to ablation of gene-modified cells in vitro and in vivo. Conclusions: Our results demonstrate feasibility of CAR modification of human HSC for cancer immunotherapy. It could be easily employed in the context of HSC transplantation to augment the anti-cancer activity, with CAR-expressing myeloid and NK cells to ensure tumor-specific immunity until de novo production of T cells from CAR-modified HSC. Disclosures Larson: BMS: Consultancy.

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