Lymphocyte-mediated regulation of β-endorphin in the myenteric plexus

2007 ◽  
Vol 292 (1) ◽  
pp. G344-G348 ◽  
Author(s):  
Monica Verma-Gandhu ◽  
Elena F. Verdu ◽  
Daniel Cohen-Lyons ◽  
Stephen M. Collins
Keyword(s):  
T Cells ◽  
Nervous System ◽  
T Cell ◽  
Enteric Nervous System ◽  
Myenteric Plexus ◽  
Pain Perception ◽  
Visceral Pain ◽  
Receptor Expression ◽  
Visceral Hyperalgesia

Lymphocytes are antinociceptive and can modulate visceral pain perception in mice. Previously, we have shown that adoptive transfer of CD4+ T cells to severe combined immune-deficient (SCID) mice normalized immunodeficiency-related visceral hyperalgesia. Pain attenuation was associated with an increase in β-endorphin release by T cells and an upregulation of β-endorphin in the enteric nervous system. In this study, we investigated the relationship between T cells and opioid expression in the myenteric plexus. We examined opioid peptide and receptor expression in the myenteric plexus in the presence and absence of mucosal T cells. We found a positive association between T cells and β-endorphin expression; this was accompanied by a downregulation of the μ-opioid receptor (MOR). In vitro, T helper (Th) type 1 and type 2 cytokine stimulation of CD4+ T cells or isolation of T cells from in vivo Th-polarized mice did not increase T cell release of β-endorphin or the induction of β-endorphin expression in the myenteric plexus. However, exogenous β-endorphin did upregulate β-endorphin expression, and both cycloheximide and naloxone methiodide inhibited peptide upregulation. Therefore, our results suggest that nonpolarized CD4+ T cells release β-endorphin, which, through an interaction with MOR, stimulates an upregulation of β-endorphin expression in the myenteric plexus. Thus, we propose that the mechanism underlying lymphocyte modulation of visceral pain involves T cell modulation of opioid expression in the enteric nervous system.

scholarly journals CD4 T-cell immune stimulation of HER2+ breast cancer cells alters response to trastuzumab in vitro

2020 ◽  
Author(s):  
Patrick Song ◽  
Amer Mansur ◽  
Kari J. Dugger ◽  
Tessa R. Davis ◽  
Grant Howard ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Receptor Expression ◽  
Immune Stimulation ◽  
Her2 Breast Cancer ◽  
Stimulation Of

Abstract Introduction: The HER2+ tumor immune microenvironment is composed of macrophages, natural killer cells, and tumor infiltrating lymphocytes, which produce pro-inflammatory cytokines. Determining the effect of T-cells on HER2+ cancer cells during therapy could guide immunogenic therapies that trigger antibody-dependent cellular cytotoxicity. This study utilized longitudinal in vitro time-resolved microscopy imaging to measure T-cell influence on trastuzumab in HER2+ breast cancer.Methods: Fluorescently-labeled breast cancer cells (BT474, SKBR3, MDA-MB-453, and MDA-MB-231) were co-cultured with CD4+ T-cells (Jurkat cell line) and longitudinally imaged to quantify cancer cell viability when treated with or without trastuzumab (10, 25, 50 and 100 mg/mL). The presence and timing of T-cell co-culturing was manipulated to determine immune stimulation of trastuzumab-treated HER2+ breast cancer. HER2 and TNF-a expression were evaluated with western blot and ELISA, respectively. Significance was calculated using a two-tailed parametric t-test. Results: The viability of HER2+ cancer cells significantly decreased when exposed to 25 mg/mL trastuzumab and T-cells, compared to cancer cells exposed to trastuzumab without T-cells (p = 0.01). The presence of T-cells significantly increased TNF-a expression in trastuzumab-treated cancer cells (p = 0.02). Conversely, cancer cells treated with TNF-a and trastuzumab had a similar decrease in viability as trastuzumab-treated cancer cells co-cultured with T-cells (p = 0.32).Conclusions: The presence of T-cells significantly increases the efficacy of targeted therapies and suggests trastuzumab may trigger immune mediated cytotoxicity. Increased TNF-a receptor expression suggest cytokines may interact with trastuzumab to create a state of enhanced response to therapy in HER2+ breast cancer, which has potential to reducing tumor burden.

scholarly journals Acute Myeloid Leukemia (AML) Blasts Influence the Gene Expression Signature and Co-Signaling Receptor Expression of CD8+ T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1700-1700
Author(s):  
Hanna A. Knaus ◽  
Sofia Berglund ◽  
Hubert Hackl ◽  
Raúl Montiel-Esparza ◽  
Mark J. Levis ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Induction Chemotherapy ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Receptor Expression ◽  
Inhibitory Receptor ◽  
Canonical Pathways

Abstract Background: T cell dysfunction in AML remains poorly understood. Our previous studies of AML-associated T cell dysfunction (Knaus, ASH 2015) have focused on expression of multiple inhibitory receptors by T cells in AML patients. Transcriptional signatures, however, remain relatively unexplored, as does the role of Blast/T cell interactions on T cell function. Deciphering those could be crucial for integration of future immunotherapies into clinical practice. Therefore, we aimed to characterize CD8+ T cell gene expression signatures in newly diagnosed AML patients before and after treatment, and to decipher the effects of AML blasts on the expression of co-signaling molecules by CD8+ T cells in co-culture experiments. Methods: Serial peripheral blood (PB) samples (at diagnosis and at the recovery after induction chemotherapy) were collected. To study transcriptional signatures, RNA isolated from FACS-purified PB CD8+ T cells from 6 patients [3 responders (R) and 3 non-responders (NR)] and 4 healthy controls (HC) was analyzed with the Human Prime View Gene Expression Array (Affymetrix). The data were normalized and log transformed. Expression fold change (FC), p values and false discovery rate were determined. Enrichment of canonical pathways was determined using Ingenuity Pathway Analysis (IPA, QIAGEN). To study AML blast-T cell interactions, we FACS-purified T cells and primary AML blasts at diagnosis (n=13) and T cells from HC (n=12). T cells were cultured in vitro for 3 days in the presence or absence of blasts (T cell:blast ratio 1:10) and analyzed by flow cytometry. Results: The transcriptional profile of CD8+ T cells at AML diagnosis significantly differed from that of HC. Genes were selected based on >2 FC between patient and HC, and p< 0.01. We identified a total of 453 dysregulated genes, of which 237 were up- and 216 down-regulated. Upregulated genes included immune inhibitory receptors LILRB1, 2B4, KLRG1, CD160, the transcription factors EOMES, TBET, TIGIT and cytokines (granzyme-A/B/K). In contrast, co-stimulatory receptor genes were downregulated, including CD40LG, CD28, CD30LG and CD28H. Canonical pathways analysis with IPA revealed that the NFAT pathway (involved in T cell differentiation and self-tolerance) was highly upregulated, while co-stimulatory CD28, ICOS and OX40 signaling pathways were downregulated in CD8+ T cells at AML diagnosis. Next, we compared R to NR after induction chemotherapy. There were a total of 351 dysregulated genes; 108/243 genes were up-/down-regulated, respectively. R patients upregulated immune stimulatory receptor genes like ICOS, whereas the top expressed genes for NR patients included the co-inhibitory receptor TIM3; several members of the inhibitory LIR receptor family; LST1 (involved in inhibition of lymphocyte proliferation); TWEAK-APRIL (associated with T cell apoptosis); and CD39 (terminally exhausted CD8+ T cells). In line with these findings, IPA showed that the co-stimulatory ICOS and OX40 signaling pathways were enriched in R patients. In contrast, the NFAT pathway, which had been highly upregulated at diagnosis, remained enriched in NR, but not in R patients. Results were confirmed by qPCR. The culture assay showed that the presence of primary AML blasts significantly reduced the viability of both AML and HC T cells (p <0.005 in both cases). The presence of AML blasts also significantly decreased the frequency of primary AML T cells expressing co-stimulatory receptors 41BB, ICOS and OX40, while it increased the frequency of HC T cells expressing co-inhibitory receptor 2B4 and the senescence/exhaustion marker CD57 compared to their counterparts cultured without blasts. Conclusions: Our study provides insight into the genomic CD8+ T cell signatures of AML patients at diagnosis and following chemotherapy. At diagnosis, T cells overexpressed genes that negatively regulate T cell immune responses, while genes that positively regulate immune responses were downregulated. Interestingly, after induction chemotherapy these changes persisted in NR only. Additionally, a pattern of decreased viability and co-stimulatory receptor expression was seen after in vitro co-culture of T cells with AML blasts, whereas immune inhibitory receptor expression was increased. Our data suggests that the blasts themselves influence the T cell phenotype and genotype in AML patients and that remission is associated with reversion to HC pattern. Disclosures Levis: Astellas: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Millennium: Consultancy, Research Funding.

scholarly journals Cannabinoid CB2 receptors in the enteric nervous system modulate gastrointestinal contractility in lipopolysaccharide-treated rats

2008 ◽  
Vol 295 (1) ◽  
pp. G78-G87 ◽  
Author(s):  
Marnie Duncan ◽  
Abdeslam Mouihate ◽  
Ken Mackie ◽  
Catherine M. Keenan ◽  
Nancy E. Buckley ◽  
...  
Keyword(s):  
Nervous System ◽  
Enteric Nervous System ◽  
Myenteric Plexus ◽  
Electrical Field Stimulation ◽  
Receptor Expression ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Receptor Agonists ◽  
Lps Treatment

Enhanced intestinal transit due to lipopolysaccharide (LPS) is reversed by cannabinoid (CB)2 receptor agonists in vivo, but the site and mechanism of action are unknown. We have tested the hypothesis that CB2 receptors are expressed in the enteric nervous system and are activated in pathophysiological conditions. Tissues from either saline- or LPS-treated (2 h; 65 μg/kg ip) rats were processed for RT-PCR, Western blotting, and immunohistochemistry or were mounted in organ baths where electrical field stimulation was applied in the presence or absence of CB receptor agonists. Whereas the CB2 receptor agonist JWH133 did not affect the electrically evoked twitch response of the ileum under basal conditions, in the LPS-treated tissues JWH133 was able to reduce the enhanced contractile response in a concentration-dependent manner. Rat ileum expressed CB2 receptor mRNA and protein under physiological conditions, and this expression was not affected by LPS treatment. In the myenteric plexus, CB2 receptors were expressed on the majority of neurons, although not on those expressing nitric oxide synthase. LPS did not alter the distribution of CB2 receptor expression in the myenteric plexus. In vivo LPS treatment significantly increased Fos expression in both enteric glia and neurons. This enhanced expression was significantly attenuated by JWH133, whose action was reversed by the CB2 receptor antagonist AM630. Taking these facts together, we conclude that activation of CB2 receptors in the enteric nervous system of the gastrointestinal tract dampens endotoxin-induced enhanced intestinal contractility.

scholarly journals In Vitro Cellular and Molecular Interplay between Human Foreskin-Derived Mesenchymal Stromal/Stem Cells and the Th17 Cell Pathway

Pharmaceutics ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1736
Author(s):  
Mehdi Najar ◽  
Makram Merimi ◽  
Wissam H. Faour ◽  
Catherine A. Lombard ◽  
Douâa Moussa Agha ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Th17 Cell ◽  
T Cell Response ◽  
Receptor Expression ◽  
Human Model ◽  
Cell Based Therapy ◽  
Stromal Stem Cells

Foreskin, considered a biological waste material, has been shown to be a reservoir of therapeutic cells. The immunomodulatory properties of mesenchymal stromal/stem cells (MSCs) from the foreskin (FSK-MSCs) are being evaluated in cell-based therapy for degenerative, inflammatory and autoimmune disorders. Within the injured/inflamed tissue, proinflammatory lymphocytes such as IL-17-producing T helper cells (Th17) may interact with the stromal microenvironment, including MSCs. In this context, MSCs may encounter different levels of T cells as well as specific inflammatory signals. Uncovering the cellular and molecular changes during this interplay is central for developing an efficient and safe immunotherapeutic tool. To this end, an in vitro human model of cocultures of FSK-MSCs and T cells was established. These cocultures were performed at different cell ratios in the presence of an inflammatory setting. After confirming that FSK-MSCs respond to ISCT criteria by showing a typical phenotype and multilineage potential, we evaluated by flow cytometry the expression of Th17 cell markers IL-17A, IL23 receptor and RORγt within the lymphocyte population. We also measured 15 human Th17 pathway-related cytokines. Regardless of the T cell/MSC ratio, we observed a significant increase in IL-17A expression associated with an increase in IL-23 receptor expression. Furthermore, we observed substantial modulation of IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, INF-γ, sCD40, and TNF-α secretion. These findings suggest that FSK-MSCs are receptive to their environment and modulate the T cell response accordingly. The changes within the secretome of the stromal and immune environment are likely relevant for the therapeutic effect of MSCs. FSK-MSCs represent a valuable cellular product for immunotherapeutic purposes that needs to be further clarified and developed.

scholarly journals CD4 T-cell immune stimulation of HER2+ breast cancer cells alters response to trastuzumab in vitro

2020 ◽  
Author(s):  
Patrick Song ◽  
Amer Mansur ◽  
Kari J. Dugger ◽  
Tessa R. Davis ◽  
Grant Howard ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Receptor Expression ◽  
Immune Stimulation ◽  
Her2 Breast Cancer ◽  
Stimulation Of

Abstract Introduction: The HER2+ tumor immune microenvironment is composed of macrophages, natural killer cells, and tumor infiltrating lymphocytes, which produce pro-inflammatory cytokines. Determining the effect of T-cells on HER2+ cancer cells during therapy could guide immunogenic therapies that trigger antibody-dependent cellular cytotoxicity. This study utilized longitudinal in vitro time-resolved microscopy to measure T-cell influence on trastuzumab in HER2+ breast cancer.Methods: Fluorescently-labeled breast cancer cells (BT474, SKBR3, MDA-MB-453, and MDA-MB-231) were co-cultured with CD4+ T-cells (Jurkat cell line) and longitudinally imaged to quantify cancer cell viability when treated with or without trastuzumab (10, 25, 50 and 100 mg/mL). The presence and timing of T-cell co-culturing was manipulated to determine immune stimulation of trastuzumab-treated HER2+ breast cancer. HER2 and TNF-a expression were evaluated with western blot and ELISA, respectively. Significance was calculated using a two-tailed parametric t-test.Results: The viability of HER2+ cancer cells significantly decreased when exposed to 25 mg/mL trastuzumab and T-cells, compared to cancer cells exposed to trastuzumab without T-cells (p = 0.01). The presence of T-cells significantly increased TNF-a expression in trastuzumab-treated cancer cells (p = 0.02). Conversely, cancer cells treated with TNF-a and trastuzumab had a similar decrease in viability as trastuzumab-treated cancer cells co-cultured with T-cells (p = 0.32).Conclusions: The presence of T-cells significantly increases the efficacy of targeted therapies and suggests trastuzumab may trigger immune mediated cytotoxicity. Increased TNF-a receptor expression suggest cytokines may interact with trastuzumab to create a state of enhanced response to therapy in HER2+ breast cancer, which has potential to reducing tumor burden.

scholarly journals In Situ Tolerance within the Central Nervous System as a Mechanism for Preventing Autoimmunity

2000 ◽  
Vol 192 (6) ◽  
pp. 871-880 ◽  
Author(s):  
Thea Brabb ◽  
Peter von Dassow ◽  
Nadia Ordonez ◽  
Bryan Schnabel ◽  
Blythe Duke ◽  
...  
Keyword(s):  
Central Nervous System ◽  
T Cells ◽  
Nervous System ◽  
T Cell ◽  
Autoimmune Disease ◽  
Mononuclear Cells ◽  
Antigenic Stimulation ◽  
The Central Nervous System

Multiple sclerosis (MS) is believed to be an autoimmune disease in which autoreactive T cells infiltrate the central nervous system (CNS). Animal models of MS have shown that CNS-specific T cells are present in the peripheral T cell repertoire of healthy mice and cause autoimmune disease only when they are activated by immunization. T cell entry into the CNS is thought to require some form of peripheral activation because the blood–brain barrier prohibits trafficking of this tissue by naive cells. We report here that naive T cells can traffic to the CNS without prior activation. Comparable numbers of T cells are found in the CNS of both healthy recombinase activating gene (Rag)−/− T cell receptor (TCR) transgenic mice and nontransgenic mice even when the transgenic TCR is specific for a CNS antigen. Transgenic T cells isolated from the CNS that are specific for non-CNS antigens are phenotypically naive and proliferate robustly to antigenic stimulation in vitro. Strikingly, transgenic T cells isolated from the CNS that are specific for myelin basic protein (MBP) are also primarily phenotypically naive but are unresponsive to antigenic stimulation in vitro. Mononuclear cells from the CNS of MBP TCR transgenic but not nontransgenic mice can suppress the response of peripheral MBP-specific T cells in vitro. These results indicate that naive MBP-specific T cells can traffic to the CNS but do not trigger autoimmunity because they undergo tolerance induction in situ.

scholarly journals The Enteric Nervous System in Inflammation and Pain: The Role of Proteinase-Activated Receptors

2003 ◽  
Vol 17 (10) ◽  
pp. 589-592 ◽  
Author(s):  
Nathalie Vergnolle
Keyword(s):  
Nervous System ◽  
Animal Models ◽  
Enteric Nervous System ◽  
Neurogenic Inflammation ◽  
Pain Perception ◽  
Visceral Pain ◽  
Signalling Molecules ◽  
Proteinase Activated Receptors ◽  
Intestinal Hypersensitivity

The enteric nervous system (ENS) plays a pivotal role in inflammatory and nociceptive processes. Drugs that interact with the ENS have recently raised considerable interest because of their capacity to regulate numerous aspects of the gut physiology and pathophysiology. The present article summarizes recent research on proteinases and proteinase-activated receptors (PARs) as signalling molecules in the ENS. In particular, experiments in animal models suggest that PAR2is important to neurogenic inflammation in the intestine. Moreover, PAR2agonists seem to induce intestinal hypersensitivity and hyperalgesic states, suggesting a role for this receptor in visceral pain perception. Thus, PARs, together with the proteinases that activate them, represent exciting new targets for therapeutic intervention on the ENS.

scholarly journals Methods to Study the Myenteric Plexus of Rat Small Intestine

2021 ◽  
Author(s):  
Ines Hecking ◽  
Lennart Norman Stegemann ◽  
Sarah Stahlke ◽  
Verena Theis ◽  
Matthias Vorgerd ◽  
...  
Keyword(s):  
Nervous System ◽  
Enteric Nervous System ◽  
Myenteric Plexus ◽  
Laser Microdissection ◽  
In Vitro Studies ◽  
Sample Collection ◽  
Tissue Quality ◽  
Specific Tissue

AbstractThe close interaction between the enteric nervous system, microbiome, and brain in vertebrates is an emerging topic of recent studies. Different species such as rat, mouse, and human are currently being used for this purpose, among others. The transferability of protocols for tissue isolation and sample collection is not always straightforward. Thus, the present work presents a new protocol for isolation and sample collection of rat myenteric plexus cells for in vivo as well as in vitro studies. With the methods and chemicals described in detail, a wide variety of investigations can be performed with regard to normal physiological as well as pathological processes in the postnatal developing enteric nervous system. The fast and efficient preparation of the intestine as the first step is particularly important. We have developed and described a LIENS chamber to obtain optimal tissue quality during intestinal freezing. Cryosections of the flat, snap-frozen intestine can then be prepared for histological examination of the various wall layers of the intestine, e.g. by immunohistochemistry. In addition, these cryosections are suitable for the preparation of defined regions, as shown here using the ganglia of the mesenteric plexus. This specific tissue was obtained by laser microdissection, making the presented methodology also suitable for subsequent analyses that require high quality (specificity) of the samples. Furthermore, we present here a fully modernized protocol for the cultivation of myenteric neurons from the rat intestine, which is suitable for a variety of in vitro studies.

scholarly journals 161 Development of a CD8 co-receptor independent T cell receptor specific for tumor-associated antigen MAGE-A4 for next generation T cell-based immunotherapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A175-A175
Author(s):  
Kathrin Davari ◽  
Tristan Holland ◽  
Laura Prassmayer ◽  
Giulia Longinotti ◽  
Kenneth Ganley ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Receptor Expression ◽  
T Cell Subsets ◽  
High Avidity

BackgroundThe cancer-testis antigen MAGE-A4 is an attractive target for T cell-based immunotherapy, especially for indications with unmet clinical need like non-small-cell lung carcinoma or triple-negative breast cancer. Overcoming high tumor burden using adoptive transfer of T cells modified to express a transgenic T cell receptor (TCR) demands optimal recognition of the corresponding target on tumor cells by the TCR-modified T cells (TCR-Ts). Here we describe the isolation and pre-clinical characterization of high avidity TCR-Ts expressing a human leucocyte antigen (HLA)-A*02:01-restricted MAGE-A4-specific TCR that is fully functional in T cells irrespective of CD4 or CD8 co-receptor expression.MethodsAn unbiased CD137-based sorting approach was first used to identify an immunogenic MAGE-A4-derived candidate epitope that was properly processed and presented on HLA-A2 molecules encoded by the HLA-A*02:01 allele. To isolate high avidity T cells via subsequent multimer sorting, an in vitro priming approach using HLA-A2-negative donors (allogeneic-HLA-restricted priming approach) was conducted to bypass central tolerance to this self-antigen. Pre-clinical parameters of safety and activity were assessed in a comprehensive set of in vitro and in vivo studies of the lead TCR candidate derived from a selected T cell clone.ResultsA TCR recognizing the MAGE-A4-derived decapeptide GVYDGREHTV was isolated from primed T cells of a non-tolerant HLA-A2-negative donor. The respective TCR-T cell product bbT485, expressing the lead TCR in T cells from healthy donors, was demonstrated pre-clinically to have a favorable safety profile and superior in vivo potency compared to TCR-Ts made using a TCR derived from an HLA-A2-positive donor bearing a tolerized T cell repertoire to self-antigens. The natural high avidity allogeneic (allo)-derived TCR was found to be CD8 co-receptor-independent, allowing effector functions to be elicited in transgenic CD4+ T helper cells. These CD4+ TCR-T cells not only supported an anti-tumor response by direct killing of MAGE-A4-positive tumor cells, but also upregulated hallmarks associated with helper function, such as CD154 expression and release of key cytokines upon tumor-specific stimulation.ConclusionsThe extensive pre-clinical assessment of safety and in vivo potency of this non-mutated high avidity, CD8 co-receptor-independent, MAGE-A4-specific HLA-A2 restricted TCR provide the basis for its use in clinical TCR-T immunotherapy studies. The ability of this co-receptor-independent TCR to activate all transduced T cells (irrespective of CD4 or CD8 expression) could potentially provide enhanced cellular responses in the clinical setting through the induction of functionally diverse T cell subsets that goes beyond what is currently tested in the clinic.

scholarly journals Timely Controlled T Cell Receptor Expression of Genetically Engineered Precursor T Cells Requires Early Transgene Induction for Leukemia Control after Hematopoietic Stem Cell Transplantation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 657-657
Author(s):  
Sayed Shahabuddin Hoseini ◽  
Martin Hapke ◽  
Jessica Herbst ◽  
Dirk Wedekind ◽  
Rolf Baumann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Negative Selection ◽  
T Cell Development ◽  
Leukemia Cell ◽  
Cell Development ◽  
Receptor Expression ◽  
Hematopoietic Stem

Abstract BACKGROUND: The co-transplantation of hematopoietic stem cells (HS) with those that have been engineered to express tumor-reactive T cell receptors (TCRs) and differentiated into precursor T cells (preTs) may optimize tumor reduction. Since expression of potentially self-(tumor-) reactive TCRs will lead to negative selection upon thymic maturation, we investigated whether preTs forced to express a leukemia-reactive TCR under the control of a tetracycline-inducible promoter would allow timely controlled TCR expression thereby avoiding thymic negative selection. METHODS: Using lentiviral vectors, murine LSK cells were engineered to express a Tetracycline-inducible TCR directed against a surrogate leukemia antigen. TCR-transduced LSK cells were co-cultured on T cell development-supporting OP9-DL1 cells to produce preTs. Lethally-irradiated B6/NCrl recipients received syngeneic T cell-depleted bone marrow and 8 × 106 syngeneic or allogeneic (B10.A) TCR-engineered preTs. An otherwise lethal leukemia cell (C1498) challenge was given 28 days later. RESULTS: After in vivo maturation and gene induction up to 70% leukemia free survival was achieved in recipients of syngeneic TCR-transduced preTs (p<0.001) as shown in figure 1A. Importantly, transfer of allogeneic gene-manipulated preTs increased the survival of recipients (p<0.05) without inducing graft versus host disease (GVHD). Non-transduced preTs provided significantly lower leukemia protection being not significantly superior to the PBS controls. The progenies of engineered preTs gave rise to effector and central memory cells providing protection even after repeated leukemia challenge (Figure 1B and 1C). In vitro transduction and consecutive expansion of mature T cells required at least 40 × 106 cells/recipient to mediate similar anti-leukemia efficacy, risking the development of severe GVHD if of mismatched origin, and providing no long-term protection. Importantly, while transgene induction starting immediately after transplant forced CD8+ T cell development and was required to obtain a mature T cell subset of targeted specificity, late induction favored CD4 differentiation and failed to produce a leukemia-reactive population due to missing thymic positive selection. CONCLUSION: Co-transplanting TCR gene-engineered preTs is of high clinical relevance since small numbers of even mismatched HS can be transduced at a reasonable cost, expanded in vitro, stored if needed, and provide potent and long lasting leukemia protection. Figure 1 Figure 1. Co-transplantation of engineered preTs provides potent long-lasting anti-leukemia effects upon TCR-induction in vivo. (A) Lethally-irradiated B6 mice received syngeneic TCDBM cells and either non-transduced or TCR gene-transduced preTs. Doxycycline was given starting the day of transplantation. One month later, 1.2 x 106 C1498-OVA leukemia cells were injected via tail vein. Controls did not receive preTs. n = 10 to 15 per group. (B) Surviving mice of the co-transplantation experiments were re-challenged with C1498-OVA leukemia three months after the first challenge. Age matched non-transplanted mice were used as controls. Pooled data of two independent transplantations (n = 10) are shown. (C) 95 days after the second challenge, spleens of surviving animals were harvested (n = 4) and analyzed for the expression of T cell memory markers on the progenies of co-transplanted preTs. Disclosures No relevant conflicts of interest to declare.

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