624 IFNγ secreted by tebentafusp (IMCgp100)-redirected T cells inhibits expression of melanin synthesis pathway genes in healthy melanocytes

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A660-A660
Author(s):  
Mariantonella Vardeu ◽  
David Depoil ◽  
Camille Britton-Rivet ◽  
Jane Houghton ◽  
Jane Harper ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adverse Events ◽  
Cancer Cells ◽  
Cell Lines ◽  
Uveal Melanoma ◽  
Melanin Synthesis ◽  
Activated T Cells ◽  
Melanoma Cancer ◽  
Melanoma Cell Lines

BackgroundTebentafusp (IMCgp100) is a bispecific T cell redirector comprised of an affinity-enhanced TCR recognising melanocyte lineage antigen gp100 and a T cell engaging anti-CD3 scFv domain. Tebentafusp has shown activity as monotherapy in advanced cutaneous and uveal melanoma (Middleton et al., ASCO 2019), and we have previously reported that over half of uveal melanoma patients treated with tebentafusp display melanocyte-related adverse events (MRAE). These include vitiligo/skin hypopigmentation, leukotrichia, and hyperpigmentation and, collectively, are associated with better overall survival in uveal patients receiving tebentafusp (Orloff et al, AACR 2020). In this study, we dissected the mechanisms by which tebentafusp may induce MRAE and highlight the potential clinical significance.MethodsIn vitro studies were conducted to assess the direct and indirect effects of tebentafusp on epidermal melanocytes from healthy donors. Expression of gp100 and the gp100:HLA*02:01 target complex by melanocytes were quantified at the mRNA level and on the cell surface by confocal microscopy, respectively. Melanocytes co-cultured with PBMC and increasing concentrations of tebentafusp were assessed for their susceptibility to lysis and/or ability to stimulate cytokine production. These readouts were compared to gp100-positive and negative melanoma cancer cell lines. Melanin production by melanocytes was quantified and the melanin synthesis pathway interrogated at the mRNA and protein level following exposure to secretomes from tebentafusp-redirected PBMC against melanoma cancer cells.ResultsHealthy melanocytes expressed 2 to 3-fold lower levels of gp100 peptide-HLA complexes on their surface compared to gp100-positive melanoma cell lines. In the presence of tebentafusp, this lower target expression translated into 3–6 fold lower levels of IFNγ and more than 100 fold lower granzyme B production by redirected T cells and these melanocytes were resistant to direct tebentafusp-induced killing (EC50 for melanocytes greater than 1nM vs EC50 melanoma cell lines of 23–50 pM). Supernatants from T cells activated in response to melanoma cancer cells by tebentafusp downregulated the melanin content of healthy melanocytes (20–30% reduction). Western blotting revealed 30–40% inhibition of two key components of the melanin synthesis pathway; the tyrosinase-related protein (TRP)-1 and TRP-2. This inhibition was reversed by blocking IFNγ in supernatants from activated T cells.ConclusionsMRAEs, especially vitiligo, associated with response to tebentafusp, may be explained, at least in part, by the downregulation of melanin biosynthesis pathway genes by IFNγ secreted by tebentafusp-activated T cells.Ethics ApprovalThe study was approved by the South Central - Oxford A Research Ethics Committee (UK), REC reference 13/SC/0226ReferencesMiddleton, et al., Relationship between clinical efficacy and AEs of IMCgp100, a novel bispecific TCR–anti-CD3, in patients with advanced melanoma. Journal of Clinical Oncology. 2019.Orloff, et al., Vitiligo and other clinical melanocyte-related adverse events following tebentafusp (IMCgp100) exposure in patients with uveal melanoma. AACR (American Association for Cancer Research), 2020.

scholarly journals T cell growth factor receptors. Quantitation, specificity, and biological relevance

1981 ◽  
Vol 154 (5) ◽  
pp. 1455-1474 ◽  
Author(s):  
RJ Robb ◽  
A Munck ◽  
KA Smith
Keyword(s):  
T Cells ◽  
T Cell ◽  
Growth Factor ◽  
Cell Lines ◽  
Binding Sites ◽  
Activated T Cells ◽  
Receptor Sites ◽  
Polypeptide Hormones ◽  
T Cell Lines ◽  
T Cell Growth Factor

To examine directly the hypothesis that T cell growth factor (TCGF) interacts with target cells in a fashion similar to polypeptide hormones, the binding of radiolabeled TCGF to various cell populations was investigated. The results indicate that TCGF interacts with activated T cells via a receptor through which it initiates the T cell proliferative response. Internally radiolabeled TCGF, prepared from a human T leukemia cell line and purified by gel filtration and isoelectric focusing, retained biological activity and was uniform with respect to size and charge. Binding of radiolabeled TCGF to TCGF-dependent cytolytic T cells occurred rapidly (within 15 rain at 37 degrees C) and was both saturable and largely reversible. In addition, at 37 degrees C, a receptor- and lysosome-dependent degradation of TCGF occurred. Radiolabeled TCGF binding was specific for activated, TCGF-responsive T cells. Whereas unstimulated lymphocytes of human or murine origin and lipopolysaccharide-activated B cell blasts expressed few if any detectable binding sites, lectin- or alloantigen-activated cells had easily detectable binding sites. Moreover, compared with lectin- or alloantigen-activated T cells, long-term TCGF-dependent cytolytic and helper T cell lines and TCGF-dependent neo-plastic T cell lines bound TCGF with a similar affinity (dissociation constant of 5-25 pM) and expressed a similar number of receptor sites per cell (5,000-15,000). In contrast, a number of TCGF-independent cell lines of T cell, B cell, or myeloid origin did not bind detectable quantities of radiolabeled TCGF. Binding of radiolabeled TCGF to TCGF-responsive cells was specific, in that among several growth factors and polypeptide hormones tested, only TCGF competed for binding. Finally, the relative magnitude of T cell proliferation induced by a given concentration of TCGF closely paralleled the fraction of occupied receptor sites. As the extent of T cell clonal expansion depends on TCGF and on the TCGF receptor, the dissection of the molecular events surrounding the interaction of TCGF and its receptor that these studies permit, should provide new insight into the hormonelike regulation of the immune response by this lymphokine.

scholarly journals GD2-specific Chimeric Antigen Receptor-modified T Cells for the treatment of Refractory and/or Recurrent Neuroblastoma in Pediatric Patients

2021 ◽  
Author(s):  
Lihua Yu ◽  
Lulu Huang ◽  
Danna Lin ◽  
Xiaorong Lai ◽  
Li Wu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adverse Events ◽  
Chimeric Antigen Receptor ◽  
Pediatric Patients ◽  
Antigen Receptor ◽  
Progression Free Survival ◽  
Fourth Generation ◽  
Activated T Cells ◽  
T Cell Therapy

Abstract PurposeThis study aimed to evaluate the safety and efficacy of the fourth-generation chimeric antigen receptor (CAR) disialoganglioside 2 (GD2)-specific (4SCAR-GD2) T cells for treatment of refractory and/or recurrent neuroblastoma (NB) in pediatric patients. Experimental DesignA phase I clinical study using 4SCAR-GD2 T cells for the treatment of NB in pediatric patients was conducted. This study was registered at www.clinicaltrials.gov (NCT02765243). A lentiviral CAR with the signaling domains of CD28/4-1BB /CD3ζ-iCasp9 was transduced into activated T-cells. The response to 4SCAR-GD2 T cell treatment, and 4SCARGD2 T cell expansion and persistence in patients were evaluated. Toxicities were determined based on the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v4.03. ResultsTwelve patients were enrolled and finally ten ptients were included in this clinical trial which occurred from January 1, 2016, to August 1, 2017. These patients had progressive disease (PD) before CAR T cell infusion. After 4SCAR-GD2 T cell treatment, 6 (6/10) had stable disease (SD) at 6 months, and 4(4/10) remained SD at 1 year and alive after 3-4 years of follow up. Six patients died due to disease progression by the end of July 1, 2020. The median overall survival (OS) time was 25 months (95% CI, 0.00 to 59.43), and the median progression-free survival (PFS) time was 8 months (95% CI, 0.25 to 15.75). Grade 3 or 4 hematological toxicities were the common adverse events frequently occurred after fludarabine and cyclophosphamide (Flu/cy) chemotherapy. Grade 1-2 toxicities like cytokine release syndrome and neuropathic pain were common, transient and mild. ConclusionsThe 4SCAR-GD2 T cell therapy demonstrated antitumor effect and manageable toxicities, indicating its potential to benefit children with refractory and/or recurrent NB.

scholarly journals Immune suppressed tumor microenvironment by exosomes derived from gastric cancer cells via modulating immune functions

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Juan Liu ◽  
Shaoxian Wu ◽  
Xiao Zheng ◽  
Panpan Zheng ◽  
Yuanyuan Fu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gastric Cancer ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Cancer Cells ◽  
Cell Lines ◽  
Gastric Cancer Cell ◽  
Gastric Cancer Cells ◽  
Gastric Cancer Cell Lines

Abstract Gastric cancer is one of the leading causes of cancer-related death due to late diagnosis with high metastatic frequency. In this study, the impact of tumor secreted exosomes on immune function in the tumor environment was investigated using exosomes isolated from gastric cancer cell lines MKN-28, MKN-45, and SGC-7901. Results show that exosomes derived from all of these cell lines changed the gene expression and cytokine secretion levels of CD8+ T cells. They also block cell cycle progression, induced apoptosis in CD8+ T cells. Image analysis of fluorescent labeled exosomes derived from three cell lines injected systemically into C57BL/6 mice revealed these exosomes primarily localize to the lungs. We further showed exosomes were mainly taken up by natural killer cells and macrophages in the lung. After long-term exposure to inject exosomes from MKN-45 cells, mice developed an immunosuppressive tumor microenvironment in the lung with increased frequency of effector memory CD4+ T and MDSC, decreased CD8+ T cell and NK frequency. This immune suppressive environment promotes gastric cancer lung metastasis. Lung metastasis sites developed after mice were exposed to exosomes isolated from all three gastric cancer cell lines when the mice were injected with MFC cells. Results suggest that exosomes derived from gastric cancer cells (especially MKN-45 and MKN-28) changed CD8+ T cell gene expression and cytokine secretion patterns to create an immunosuppressive condition for metastatic niche formation in the lung. Overall, this study provides new insights into how gastric cancer derived exosomes modulate the immune response to promote lung tumor metastasis.

Aberrant Expression and Activation of Nuclear Factor of Activated T cells (NFAT1) in Human T-Cell Acute Lymphocytic Leukemia (T-ALL) May Contribute to Leukemogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4474-4474
Author(s):  
Peter Haviernik ◽  
Mathew L Lesniewski ◽  
Richard Patrick Weitzel ◽  
Mary J Laughlin
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Jurkat Cells ◽  
Nuclear Factor ◽  
Flow Cytometry Analysis ◽  
Wild Type ◽  
Activated T Cells ◽  
Aberrant Expression

Abstract Nuclear Factor of Activated T-cells 1 (NFAT1) is a member of the NFAT family of transcription factors (NFAT1-NFAT5) that has been shown to play an important role in regulating genes related to T-cell expansion, differentiation, and apoptosis. As murine NFAT1 knockout mice exhibit lymphoproliferative disease, we hypothesize that aberrant expression of NFAT1 may impact cell cycle dysregulation underlying T-ALL pathogenesis. Four T-ALL cell lines – including 3 mature T-cell lines: CCRF-CEM (no clear chromosomal abnormalities), Jurkat (karyotype 46, XY, -2, -18, del(2) (p21p23), del(18) (p11.2)), Loucy (translocation t(16;20)(p12;q13), and p53 overexpression), and one immature T-cell line MOLT-4 (hypertetraploid chromosomes and 6q-, t(7;7)) (ATCC Manassas, VA), established from peripheral blood of T-ALL patients were analyzed and compared to normal resting adult CD4+ T-cells. Flow cytometry analysis was performed as previously described including CD34, CD38, HLA-DR, CD3, CD4, CD8, CD2, and CD7 to determine maturation stage; MOLT-4 being the most primitive and Jurkat the most mature. Methods: Growth curves were determined and proliferation potential was determined using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. The cell cycle phase distribution was assessed by flow cytometry analysis of Hoechst 33342-stained cells. mRNA expression was examined by quantitative rtPCR using TaqMan probes (ABI) on cDNA derived from TRIzol purified total RNA. For stimulation, we used 2 μM ionomycin treatment for 3 hours. Proteins were prepared as whole cell extract from radioimmunoprecipitation assay (RIPA) buffer lysed cells. Protein expression was evaluated by Western blotting analysis of 20 μg of proteins using anti-NFAT1 antibody (BD Biosciences) and anti-γ-tubulin antibody (Sigma) as a loading control. Transient transfection of GFP vector or plasmid containing the wild-type or constitutively active NFAT1 gene along with the GFP gene was performed by either electroporation (Amaxa) or Lipofectamine 2000 (Invitrogen). GFP positive cells were sorted by FACSAria sorter. Results: Despite their different maturation states, all cell lines (except Loucy) have similar high growth rates. However, the cell cycle distribution analysis of Hoechst-stained cells revealed a lower proportion of Loucy cells in the G0/G1 phase (35% vs. 48–51% for the other 3 cell lines) and a higher proportion in the G2/M phase (37% vs. 19–24% for the other 3 cell lines). In addition, Loucy cells have a higher rate of apoptosis as measured by Annexin V staining. Analysis of NFAT1 expression demonstrated decreased levels of NFAT1 mRNA (30-70-fold) and protein (2-10-fold) in these cell lines compared to resting adult peripheral blood CD4+ T lymphocytes. Figure Figure Moreover, ionomycin stimulation of the calcium-calcineurin pathway in these cells revealed aberrant activation of NFAT1. There was no dephosphorylated or activated NFAT1 in MOLT-4 and Jurkat cells; in contrast, dephosphorylated or activated NFAT1 was degraded in CCRF-CEM and Loucy. As NFAT1 is implicated in the regulation of cell cycle and apoptosis, the expression of cell cycle and apoptosis genes was measured by qrtPCR. Consistent with the negative regulatory role of NFAT1 in cell cycle, T-ALL cells expressing low level of NFAT1 showed upregulation of cyclin A2 (20-80-fold), cyclin E2 (5-8-fold) and CDK4 (3-7-fold) as well as downregulation of p21Cip1 (20-470-fold) and p27Kip1 (20-180-fold). In addition, these cells also demonstrated downregulation of the expression of the pro-apoptotic gene Nur77 (2.5-10-fold). Introduction of exogenous NFAT1 gene into Jurkat cells resulted in decreased proliferation rate to 64% and 42%, for wild-type and constitutively active form of NFAT1 gene, respectively, compare to the cells transfected with the empty GFP vector. Conclusion: Aberrant expression of NFAT1 contributes to leukemogenesis via dysregulation of proliferation and apoptosis. Targeted NFAT1 expression may be an effective therapeutic strategy in T-ALL.

scholarly journals A bispecific T cell engager targeting Glypican-1 redirects T cell cytolytic activity to kill prostate cancer cells

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Maria E. Lund ◽  
Christopher B. Howard ◽  
Kristofer J. Thurecht ◽  
Douglas H. Campbell ◽  
Stephen M. Mahler ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Cell Lines ◽  
Inflammatory Cytokines ◽  
Cytolytic Activity ◽  
Activated T Cells ◽  
Bispecific T Cell Engager

Abstract Background Glypican-1 is a heparan sulfate proteoglycan that is overexpressed in prostate cancer (PCa), and a variety of solid tumors. Importantly, expression is restricted in normal tissue, making it an ideal tumor targeting antigen. Since there is clinical and preclinical evidence of the efficacy of Bispecific T cell Engager (BiTE) therapy in PCa, we sought to produce and test the efficacy of a GPC-1 targeted BiTE construct based on the Miltuximab® sequence. Miltuximab® is a clinical stage anti-GPC-1 antibody that has proven safe in first in human trials. Methods The single chain variable fragment (scFv) of Miltuximab® and the CD3 binding sequence of Blinatumomab were combined in a standard BiTE format. Binding of the construct to immobilised recombinant CD3 and GPC-1 antigens was assessed by ELISA and BiaCore, and binding to cell surface-expressed antigens was measured by flow cytometry. The ability of MIL-38-CD3 to activate T cells was assessed using in vitro co-culture assays with tumour cell lines of varying GPC-1 expression by measurement of CD69 and CD25 expression, before cytolytic activity was assessed in a similar co-culture. The release of inflammatory cytokines from T cells was measured by ELISA and expression of PD-1 on the T cell surface was measured by flow cytometry. Results Binding activity of MIL-38-CD3 to both cell surface-expressed and immobilised recombinant GPC-1 and CD3 was retained. MIL-38-CD3 was able to mediate the activation of peripheral blood T cells from healthy individuals, resulting in the release of inflammatory cytokines TNF and IFN-g. Activation was reliant on GPC-1 expression as MIL-38-CD3 mediated only low level T cell activation in the presence of C3 cells (constitutively low GPC-1 expression). Activated T cells were redirected to lyse PCa cell lines PC3 and DU-145 (GPC-1 moderate or high expression, respectively) but could not kill GPC-1 negative Raji cells. The expression of PD-1 was up-regulated on the surface of MIL-38-CD3 activated T cells, suggesting potential for synergy with checkpoint inhibition. Conclusions This study reports preclinical findings into the efficacy of targeting GPC-1 in PCa with BiTE construct MIL-38-CD3. We show the specificity and efficacy of the construct, supporting its further preclinical development.

scholarly journals The human OX40/gp34 system directly mediates adhesion of activated T cells to vascular endothelial cells.

1996 ◽  
Vol 183 (5) ◽  
pp. 2185-2195 ◽  
Author(s):  
A Imura ◽  
T Hori ◽  
K Imada ◽  
T Ishikawa ◽  
Y Tanaka ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Cell Leukemia ◽  
Activated T Cells ◽  
T Cell Lines

Fresh leukemic cells from patients with adult T cell leukemia (ATL) and some ATL-derived T cell lines show adhesion to human umbilical vein endothelial cells (HUVECs) mainly through E-selectin, but a proportion of this binding remains unaffected by the addition of combinations of antibodies against known adhesion molecules. By immunizing mice with one of such cell lines, we established monoclonal antibodies (mAbs), termed 131 and 315, that recognize a single cell surface antigen (Ag) and inhibit the remaining pathway of the adhesion. These mAbs did not react with normal resting peripheral blood mononuclear cells (PBMC) or most of the cell lines tested except for two other human T cell leukemia virus type I (HTLV-I)-infected T cell lines. After stimulation with phytohemagglutinin (PHA), PBMC expressed Ag 131/315 transiently, indicating that these mAbs define a T cell activation Ag. Western blotting and immunoprecipitation revealed that Ag 131/315 has an apparent molecular mass of 50 kD. Expression cloning was done by transient expression in COS-7 cells and immunological selection to isolate a cDNA clone encoding Ag 131/315. Sequence analysis of the cDNA indicated that it is identical to human OX40, a member of the tumor necrosis factor/nerve growth factor receptor family. We then found that gp34, the ligand of OX40, was expressed on HUVECs and other types of vascular endothelial cells. Furthermore, it was shown that the adhesion of CD4+ cells of PHA-stimulated PBMC to unstimulated HUVECs was considerably inhibited by either 131 or 315. Finally, OX40 transfectants of Kit 225, a human interleukin 2-dependent T cell line, were bound specifically to gp34 transfectants of MMCE, a mouse epithelial cell line, and this binding was blocked by either 315 or 5A8, an anti-gp34 mAb. These results indicate that the OX40/gp34 system directly mediates adhesion of activated T cells or OX40+-transformed T cells to vascular endothelial cells.

scholarly journals A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes

1998 ◽  
Vol 188 (9) ◽  
pp. 1593-1602 ◽  
Author(s):  
George R. Ehring ◽  
Hubert H. Kerschbaum ◽  
Claudia Eder ◽  
Amber L. Neben ◽  
Christopher M. Fanger ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Lines ◽  
K Channel ◽  
Na Channel ◽  
Activated T Cells ◽  
K Channels ◽  
Voltage Gated

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin–resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl− channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression.

scholarly journals 760 In vivo demethylation-mediated reversal of tumor cell-intrinsic cGAS silencing improves the efficacy of STING agonist therapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A795-A795
Author(s):  
Rana Falahat ◽  
Patricio Perez-Villarroel ◽  
Anders Berglund ◽  
Shari Pilon-Thomas ◽  
Glen Barber ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
Fold Increase ◽  
Cell Recognition ◽  
Agonist Therapy ◽  
T Cell Recognition ◽  
Tumor Bearing ◽  
Melanoma Cell Lines

BackgroundWhile STING-activating agents have shown limited efficacy in early phase clinical trials, multiple lines of evidence suggest the importance of the so far unappreciated tumor cell-intrinsic STING function in antitumor immune responses. Accordingly, we have shown that although there is a widespread impairment of STING signaling among human melanomas, its restoration through epigenetic reprogramming can augment antigenicity and T cell recognition of melanoma cells.1 2 In this study, we determined if rescue of tumor cell-intrinsic STING signaling using a DNA methyltransferase inhibitor can improve the therapeutic efficacy of a STING agonist in mouse models of melanoma.MethodsWe subjected three distinct murine melanoma cell lines (B16-F10, B16-ISG and Yumm1.7) to treatment with 5-aza-2'-deoxycytidine (5AZADC) and evaluated their activation of STING following stimulation with the STING agonist ADU-S100. Using a B16-F10 subcutaneous model, we assessed the effect of 5AZADC treatment on the efficacy of intratumorally administered ADU-S100 in STINGgt/gt mice. Additionally, we performed mechanistic studies using T-cell depletion experiments as well as phenotypic and gene expression profiling.ResultsWe observed reconstitution of cGAS in all three 5AZADC-pretreated cell lines as well as up to a 46-fold increase in induction of IFN-beta (p < 0.001) and a 4.5-fold increase in MHC class I surface expression (p < 0.01) compared to untreated controls following stimulation with ADU-S100. In B16-F10 tumor-bearing mice, while treatment with a combination of 5AZADC plus ADU-S100 resulted in a marked increase in Ifnb1 transcripts within tumors (p < 0.001), it significantly delayed tumor growth compared to treatment with ADU-S100 alone (p = 0.0244 on day 22). Antibody-mediated depletion studies in mice receiving the combination therapy further indicated that this antitumor activity depends on the generation of functional tumor antigen-specific CD8+ T cells (p = 0.0111 on day 22); however, tumor growth remained unaltered by the depletion of CD4+ T cells.ConclusionsWe show that reversal of methylation silencing of cGAS in murine melanoma cell lines using a clinically available DNA methylation inhibitor can improve agonist-induced STING activation and type I IFN induction, which in tumor-bearing mice is capable of inducing tumor regression through a CD8+ T cell-dependent immune response. These findings not only provide mechanistic insight into how STING signaling dysfunction in tumor cells can contribute to impaired responses to STING agonist therapy, but also suggest, depending on tumor cell-intrinsic STING signaling status, its pharmacologic restoration should be considered for improving therapeutic efficacy of STING agonists in future clinical studies.AcknowledgementsFunding: NCI P50 CA168536, Cindy and Jon Gruden Fund, Chris Sullivan Fund, V Foundation, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.ReferencesFalahat R, Perez-Villarroel P, Mailloux AW, Zhu G, Pilon-Thomas S, Barber GN, Mulé JJ. STING signaling in melanoma cells shapes antigenicity and can promote antitumor T-cell activity. Cancer Immunol Res 2019;7(11):1837–48.Falahat R, Berglund A, Putney RM, Perez-Villarroel P, Aoyama S, Pilon-Thomas S, Barber GN, Mulé JJ. Epigenetic reprogramming of tumor cell–intrinsic STING function sculpts antigenicity and T cell recognition of melanoma. PNAS 2021;118(15).

Multi-Omic Based Antigen Discovery for the Immunotherapy of Pediatric Acute T Cell Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Nikhil Hebbar ◽  
Chunxu Qu ◽  
Hong Wang ◽  
Ying Shao ◽  
Phuong Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
T Cell ◽  
Cell Surface ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Differentially Expressed ◽  
Activated T Cells ◽  
Car T Cells ◽  
Car T

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk disease due to treatment related complications and poor prognosis of patients with relapsed disease. Immunotherapy with monoclonal antibodies (MAbs) and/or chimeric antigen receptor (CAR) T-cells for T-ALL is limited by identification of tumor specific target antigens. Differential expression is necessary to prevent on-target/off-tumor toxicities and fratricide of activated T-cells. Targeting multiple antigens can bypass immune escape and result in improved T-cell effector function, since antigen density correlates with T-cell activation. Here we designed a pipeline (Figure 1) to identify unique surface antigens expressed in T-ALL using proteomic and transcriptomic analyses followed by flow cytometry validation, and functional studies with CAR T cells targeting the identified antigens. We generated an Illumina total stranded RNAseq library from healthy donor myeloid and lymphoid cells of bone marrow, peripheral blood and cord blood (N= 116). We compared data to 265 St. Jude pediatric T-ALL samples and against 53 normal tissue expression data from the GTEx (Genotype-Tissue Expression) project. To analyze the T-cell surface proteome, we isolated plasma membrane fractions from 11 samples including healthy T-cells and T-ALL cell lines using a differential centrifugation-based method. The purity of the plasma membrane fraction was confirmed by western blot. Na+/K+ ATPase and GAPDH were used as controls for the plasma membrane and cytosolic fractions respectively. Following plasma membrane enrichment, the membrane proteins were applied for proteomic analysis using an advanced TMT-L/LC-MS/MS pipeline, and the acquired proteomic data were further processed via the JUMP software suite. 997 unique proteins were quantified from the membrane fractions. Integrated analysis the transcriptomic and proteomic datasets showed significant correlation and yielded a list of candidate genes, which were validated by flow cytometry on a panel of T-ALL cell lines (CCRF, RPMI8402, and MOLT3) and resting and activated T-cells from healthy donors. We identified GRP78 as one of the differentially expressed cell surface antigens and further confirmed its expression on additional T-ALL cell lines (KE37, PF382, PEER, CEMC7) and 3 PDX samples. Finally, we generated GRP78-CAR T cells and demonstrate that GRP78-CAR T cells recognize and kill GRP78+ T-ALL cells and have potent antitumor activity in xenograft and PDX models. We have established an unbiased pipeline to identify differentially expressed antigens on the cell surface of T-ALL blasts and created a healthy tissue RNAseq library. The results from our analyses are encouraging and interrogation of our pipeline has yielded differentially expressed immunotherapy targets for the treatment of relapsed refractory T-ALL. Our results highlight the importance of integrated surface proteomics and transcriptomics analysis. Figure 1: Outline of strategy for target selection: Figure Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Gottschalk:Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; TESSA Therapeutics: Other: research collaboration; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Merck and ViraCyte: Consultancy. Mullighan:AbbVie, Inc.: Research Funding; Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties.

Multivirus-Specific T Cell Immunotherapy to Prevent or Treat Infections of Stem Cell Transplant Recipients.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2207-2207
Author(s):  
Ulrike Gerdemann ◽  
Anne Christin ◽  
Carlos A. Ramos ◽  
Yuriko Fujita ◽  
Juan F. Vera ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cell Lines ◽  
Stem Cell Transplant ◽  
Acute Infection ◽  
Functional Characterization ◽  
Cell Transplant ◽  
Activated T Cells ◽  
Hematopoietic Stem

Abstract Viral infections caused by community viruses frequently cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. Antiviral drugs are costly, often have severe adverse effects, and are frequently ineffective. Treatment of the underlying problem, namely lack of antigen-specific T cells, should offer effective and longterm protection. Our group has produced trivirus-reactive T cells targeting EBV, CMV, and adenoviruses (Adv) using monocytes and EBV-transformed lymphoblastoid cell lines (EBV-LCL) expressing pp65 from an adenoviral vector as antigen-presenting cells to present CMV, Adv and EBV antigens. As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs post-infusion into HSCT recipients and appeared to protect the recipients against all three viruses. Despite these encouraging clinical results, the broader implementation of the approach is limited by (i) the infectious virus material (EBV/Adv) required for CTL generation and (ii) the prolonged culture required to produce the EBV-LCL, increase viral specificity and reduce alloreactivity (3 months) which means T cells must be produced “speculatively” for all patients. Finally, (iii) “antigenic competition” between multiple viruses limits the extension of the approach to additional problematic pathogens. To overcome these limitations we have developed an approach to rapidly produce multivirus-specific CTL with broad spectrum specificity without using adenoviral vectors or EBV-LCL. Using the Amaxa system to nucleofect monocyte-derived DCs we consistently detected GFP transgene expression in 39% (median; range 30–58%) of cells 24hrs posttransfection. Viability was ~70% and the DC maturation state, as measured by CD80, 83, 86, and HLA-DR expression, was unaffected by the transfection. To show that nucleofected DCs reactivated virus-specific T cells in vitro, we cocultured p-Shuttle-pp65-GFP-transfected DCs from CMV seropositive donors with PBMCs at a responder:stimulator ratio of 20:1. After nine days, phenotypic and functional characterization of the responder T cell lines showed higher or comparable frequencies of pp65-specific T cells in IFN-g ELIspot and minimal alloreactivity when compared to pp65-specific T cells lines generated from the same donors using our standard protocol with Ad5f35pp65-transduced DCs as APCs. Pentamer analysis of pShuttle-pp65-generated CTL lines also showed a higher frequency of pp65 pentamer-directed T cells than the Ad5f35pp65-transduced counterparts (median 2.05 fold higher frequency of HLA-A2 NLV-directed T cells; range 1.34–3.35 fold) (n=4 donors). Importantly, this protocol could also be used to reactivate T cells against multiple viruses for which high (EBV), intermediate (BK), and low (Adv) frequencies of reactive memory T cells circulate. Using a panel of p-Shuttle plasmids encoding LMP2 and BZLF1 (EBV), Large T (BK), and Hexon and Penton (Adv), we amplified CTLs from seropositive donors, using as stimulators DCs transfected with each construct. This modification overcomes the need for EBV-LCL generation. Furthermore, we demonstrated that by pooling transfected DCs prior to coculture with PBMC, we could reproducibly generate multivirus-specific CTL lines with specificity for all the stimulating antigens, irrespective of the circulating memory T cell frequency. To further shorten the CTL production process, we established that virus-activated T cells could be specifically selected by IFN-g capture 24 hours after DC stimulation and that the selected cells were highly specific for the stimulating antigens as measured by IFN-g ELIspot, proliferation and cytotoxicity assay. In summary, we have established a GMP-applicable protocol for the rapid generation (&lt;10 days) of two different CTL products without using infectious viral material. In 10 days we can generate virus-specific CTLs with broad specificity which can be administered prophylactically to high risk SCT recipients. However by combining DC transfection with IFN-g selection we can also rapidly generate mono- or multivirus-specific CTL products for treatment of acute infection. We demonstrate the feasibility of generating CTL lines targeting 6 different antigens from 4 common viruses without using infectious viral material. Future studies will extend our approach to additional viral, fungal, and bacterial antigens.

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