The T-Cell/CLL/Macrophage Triad Shapes a Supportive Tumor Microenvironment in CLL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1715-1715
Author(s):  
Martijn H.A. van Attekum ◽  
Sanne Terpstra ◽  
Emilie Reinen ◽  
Marieke Von Lindern ◽  
Erik Slinger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Expression ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cellular Interactions ◽  
Activated T Cells ◽  
Cd40 Activation

Abstract Survival of CLL cells critically depends on heterotypic communication with benign bystanders cells in micro-environmental niches such as lymph node (LN) tissue. Here, mesenchymal stromal cells and macrophages, in concert with CD40L expressing T cells, are thought to participate in the dialog with the neoplastic B cells, but the mechanisms of this intricate interplay remain largely unknown. Moreover, whether CLL cells actively participate in shaping their prosurvival niche is poorly understood. We aimed to study 1) whether CD40 stimulation initiates active recruitment of monocytes by CLL cells, 2) whether CLL cells are able to differentiate these monocytes towards a supporting phenotype and 3) by which mechanism macrophages induce CLL survival. We first studied the chemokinome of CLL cells after T cell stimulation using both microarray and Luminex techniques. Co-culture of autologous activated T cells with CLL cells resulted in induction of mRNA expression of CCL2,3,4,5,22 and IL10, which are known chemo-attractants for monocytes. These effects could be mimicked by CD40 activation of CLL cells. Protein screens of supernatants of CD40 activated CLL cells by Luminex assays confirmed increased protein expression of these chemo-attractants. Indeed, transwell assays showed enhanced migration of primary monocytes towards supernatants of CD40L stimulated CLL cells. Inhibitor experiments furthermore showed that the migratory effects of these chemokines was largely governed via the CCR2 and CCR3 receptors. We next examined and compared polarization patterns of monocytes after differentiation with serum derived from CLL patients (N=25) or pooled healthy donor serum and found that CLL serum was able to differentiate macrophages towards a tumor supporting M2 phenotype. This finding was confirmed ex vivo by IHC, as M2 marker CD206 co-localizes with CD68 cells in CLL LNs, while the majority of macrophages in non-CLL derived LNs are CD80+ (M1 type). Lastly, we examined how these macrophages exert their pro-survival effect on CLL. From a variety of Bcl-2 family proteins investigated, only Mcl-1 protein expression levels increased after interaction with macrophages. The relevance of Mcl-1 upregulation was verified by MCL-1 siRNA interference studies. The mechanism of induction of Mcl-1 was independent on NF-κB signaling, Mcl-1 mRNA transcription levels or protein stability, but rather unexpectedly appeared as a result of recruitment of polysomes to Mcl-1 mRNA, resulting in an increase in translation. This increase was accompanied by an increased phosphorylation of the rate-limiting translation initiation factor 4E-BP1 and ribosomal protein S6. The increase in Mcl-1 translation could be attributed to macrophage-induced Akt signaling. In conclusions, these studies shed light on reciprocal cellular interactions in the CLL LN that shape pro-tumor differentiation of supporting cells, that in turn cause survival by changing the apoptotic balance. These interactions can be targeted at different levels, creating new treatment venues for this still incurable disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals PD-L1 Checkpoint Blockade Augments Anti-Tumor Immune Response of GD2 or HER2-Bsab Ex Vivo Armed T-Cells (EVAT) Therapy in Osteosarcoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1959-1959
Author(s):  
Jeong A Park ◽  
Hong fen Guo ◽  
Hong Xu ◽  
Nai-Kong V. Cheung
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Bispecific Antibody ◽  
Ex Vivo ◽  
Activated T Cells ◽  
The Impact ◽  
Anti Tumor Activity

Background Ex Vivo Armed T-cells (EVAT) carrying zeptomoles (10-21M) of T-cell engaging GD2-bispecific antibody (GD2-EVAT) or HER2-bispecific antibodies (HER2-EVAT) have potent anti-tumor activity against GD2(+) and/or HER2(+) solid tumors. Strategies to further optimize this approach are highly relevant. PD-1 is a key immune checkpoint receptor expressed mainly by activated T-cells and mediates immune suppression by binding to its ligands PD-L1 or PD-L2. Upregulation of PD-L1 has been found in many cancers including osteosarcoma and associated with aggressive disease and poor outcome. While the use of immune checkpoint inhibitors (ICIs) seems logical, the ideal timing when combined with T-cell engaging bispecific antibody (T-BsAb) or EVAT has yet to be defined. Here, we described the effects of anti-PD-1 or anti-PD-L1 antibodies on GD2-EVAT or HER2-EVAT therapy and explored the impact of its timing in the treatment of osteosarcoma which is GD2(+), HER2(+) and PD-L1(+). Methods GD2-BsAb and HER-BsAb were built using the IgG(L)-scFv format (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from healthy volunteer donors were isolated, and cultured ex vivo in the presence of CD3/CD28 beads plus 30 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T-cells (ATCs) were harvested and armed for 20 minutes at room temperature with GD2-BsAb or HER2-BsAb. In vivo anti-tumor activity against GD2(+), HER2(+), and PD-L1(+) osteosarcoma cell line xenografts was tested in BALB-Rag2-/-IL-2R-γc-KO mice. Anti-human PD-1 antibody (pembrolizumab, anti-PD-1) or anti-human PD-L1 antibody (atezolizumab, anti-PD-L1) were tested for synergy with GD2-EVAT or HER2-EVAT therapy. Results The PD-1 expression increased among T-cells that circulated in the blood, that infiltrated the spleen or the tumor after EVAT therapy. While anti-PD-L1 combination therapy with GD2-EVAT or HER2-EVAT improved anti-tumor response against osteosarcoma (P=0.0123 and P=0.0004), anti-PD-1 did not (all P>0.05). The addition of anti-PD-L1 significantly increased T-cell survival in blood and T-cell infiltration of tumor when compared to GD2-EVAT or HER2-EVAT alone (all P<0.0001). Treatment of GD2-EVAT or anti-PD-L1 plus GD2-EVAT downregulated GD2 expression on tumors, but anti-PD-1 plus GD2-EVAT did not. For the next step we tested the impact of different combination schedules of ICIs on GD2-EVAT therapy. Concurrent anti-PD-1 (6 doses along with GD2-EVAT therapy) interfered with GD2-EVAT, while sequential anti-PD-1 (6 doses after GD2-EVAT) did not make a significant effect (P>0.05). On the other hand, while the concurrent use of anti-PD-L1 did not show benefit on GD2-EVAT, sequentially administered anti-PD-L1 produced a significant improvement in tumor control when compared to anti-PD-L1 or GD2-EVAT alone (P=0.002 and P=0.018). When anti-PD-L1 treatment was extended (12 doses after GD2-EVAT), the anti-tumor effect was most pronounced compared to GD2-EVAT alone (P <0.0001), which translated into improved survival (P=0.0057). These in vivo anti-tumor responses were associated with increased CD8(+) tumor infiltrating lymphocytes (TILs) of tumor. Conclusion In the arming platform, large numbers of target-specific T-cells can be generated, and this EVAT therapy is a highly effective cellular treatment with high potency in preclinical models. In addition, the advantage of ex vivo cytokine release following T-cell arming and activation could reduce or avoid life threatening cytokine storm if such activation was to proceed in vivo. Adoptive T-cell therapy induced immune response upregulates the inhibitory immune checkpoint PD-1/PD-L1 pathway, and combination treatment with anti-PD-L1 antibody, especially when combined as sequential therapy and continuously treated, significantly improved anti-tumor effect of EVAT, partly through increase in CD8(+) TILs infiltration. Disclosures Xu: MSK: Other: co-inventors in patents on GD2 bispecific antibody and HER2 bispecific antibody. Cheung:Ymabs: Patents & Royalties, Research Funding.

The effect of adoptive transfer of ex vivo activated T cells on the efficacy and tumor penetrance of intravenously-administered CD3-engaging bispecific antibody.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 30-30
Author(s):  
Patrick C. Gedeon ◽  
Carter M. Suryadevara ◽  
Bryan D. Choi ◽  
John H. Sampson
Keyword(s):  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
Adoptive Transfer ◽  
Bispecific Antibody ◽  
Ex Vivo ◽  
The Body ◽  
Whole Body ◽  
Activated T Cells ◽  
T Cell Migration

30 Background: Activated T cells are known to traffic throughout the body including past the blood-brain barrier where they perform routine immune surveillance. Whether activated T cells can be used to enhance the efficacy and delivery of intravenously-administered, immunotherapeutic antibodies has yet to be explored. Methods: To examine efficacy, T cell migration and antibody delivery in vivo, the invasive murine glioma, CT-2A-EGFRvIII, was implanted orthotopically in human CD3 transgenic mice. Cohorts of mice were given vehicle or 1x107 non-specifically activated, syngeneic T cells intravenously. Beginning the subsequent day, groups were treated with daily intravenous infusions of human-CD3-binding, tumor-lysis-inducing bispecific antibody (hEGFRvIII-CD3 bi-scFv) or control bispecific antibody. To block T cell extravasation, cohorts received natalizumab or isotype control via intraperitoneal injection every other day beginning on the day of adoptive cell transfer. T cell migration was assessed using whole body bioluminescence imaging of activated T cells transduced to express firefly luciferase. Bispecific antibody biodistribution was assessed using PET-CT imaging of iodine-124 labeled antibody. Results: Following intravenous administration, ex vivo activated T cells tracked to invasive, syngeneic, orthotopic glioma, reaching maximal levels on average four days following adoptive transfer. Administration of ex vivo activated T cells enhanced bispecific antibody efficacy causing a statistically significant increase in survival (p = 0.007) with 80% long-term survivors. Treatment with the T cell extravasation blocking molecule natalizumab abrogated the increase in efficacy to levels observed in cohorts that did not receive adoptive transfer of activated T cells (p = 0.922). Pre-administration with ex vivo activated T cells produced a statistically significant increase in tumor penetrance of radiolabeled bispecific antibody (p = 0.023). Conclusions: Adoptive transfer of non-specifically activated T cells enhances the efficacy and tumor penetrance of intravenously-administered CD3-binding bispecific antibody.

Ex vivo–expanded DCs induce donor-specific central and peripheral tolerance and prolong the acceptance of donor skin grafts

Blood ◽  
2011 ◽  
Vol 117 (9) ◽  
pp. 2640-2648 ◽  
Author(s):  
Tomoyoshi Yamano ◽  
Sho Watanabe ◽  
Hiroyuki Hasegawa ◽  
Toshihiro Suzuki ◽  
Ryo Abe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Peripheral Tolerance ◽  
Skin Grafts ◽  
Major Histocompatibility ◽  
Clonal Deletion ◽  
Activated T Cells ◽  
Donor Skin ◽  
Major Histocompatibility Complexes

Abstract Dendritic cells (DCs) are known to regulate immune responses by inducing both central and peripheral tolerance. DCs play a vital role in negative selection of developing thymocytes by deleting T cells with high-affinity for self-peptide–major histocompatibility complexes. In the periphery, DCs mediate peripheral tolerance by promoting regulatory T-cell development, induction of T-cell unresponsiveness, and deletion of activated T cells. We studied whether allogeneic DCs, obtained from bone marrow cultured with either Flt3L (FLDCs) or granulocyte-macrophage colony-stimulating factor (GMDCs), could induce allospecific central and peripheral tolerance after IV injection; B cells were used as a control. The results showed that only FLDCs reached the thymus after injection and that these cells induced both central and peripheral tolerance to donor major histocompatibility complexes. For central tolerance, injection of FLDCs induced antigen-specific clonal deletion of both CD8 and CD4 single-positive thymocytes. For peripheral tolerance, injection of FLDCs induced donor-specific T-cell unresponsiveness and prolonged survival of donor-derived skin grafts. Tolerance induction by adoptive transfer of FLDCs could be a useful approach for promoting graft acceptance after organ transplantation.

scholarly journals CNS-localized myeloid cells capture living invading T cells during neuroinflammation

2020 ◽  
Vol 217 (6) ◽  
Author(s):  
Beatrice Wasser ◽  
Dirk Luchtman ◽  
Julian Löffel ◽  
Kerstin Robohm ◽  
Katharina Birkner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Myeloid Cells ◽  
Initial Response ◽  
Cellular Interactions ◽  
T Cell Infiltration ◽  
Th17 Lymphocytes ◽  
The Central Nervous System

To study the role of myeloid cells in the central nervous system (CNS) in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), we used intravital microscopy, assessing local cellular interactions in vivo in EAE animals and ex vivo in organotypic hippocampal slice cultures. We discovered that myeloid cells actively engulf invading living Th17 lymphocytes, a process mediated by expression of activation-dependent lectin and its T cell–binding partner, N-acetyl-D-glucosamine (GlcNAc). Stable engulfment resulted in the death of the engulfed cells, and, remarkably, enhancement of GlcNAc exposure on T cells in the CNS ameliorated clinical EAE symptoms. These findings demonstrate the ability of myeloid cells to directly react to pathogenic T cell infiltration by engulfing living T cells. Amelioration of EAE via GlcNAc treatment suggests a novel first-defense pathway of myeloid cells as an initial response to CNS invasion and demonstrates that T cell engulfment by myeloid cells can be therapeutically exploited in vivo.

Effect of Ivig On Human T Cells: Interference with Activation Rather Than Suppression of Activated Cell Functions.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4738-4738
Author(s):  
Lauriane Padet ◽  
Isabelle St-Amour ◽  
Eric Aubin ◽  
Real Lemieux ◽  
Renee Bazin
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
In Vitro Assays ◽  
Activated T Cells ◽  
Cell Functions ◽  
Human T Cells

Abstract Abstract 4738 Introduction IVIg is known to have immunosuppressive effects in a variety of inflammatory and autoimmune diseases, which may be caused by modulations of T cell functions in treated patients. The mechanisms responsible for these modulations have been mostly investigated using in vitro stimulated T cells. These studies revealed that IVIg inhibited the proliferation of activated T cells possibly by interfering with the secretion of cytokines important for T cell proliferation, such as IL-2. In the present study, we sought to determine the precise mechanism by which IVIg inhibited cytokine secretion by stimulated T cells. Methods Human PBMC and Jurkat T cells were stimulated with PHA. Human T cells purified from PBMC were stimulated with CD3/CD28 T cell expander beads. Cells were cultured in presence or not of 10 mg/ml of IVIg for 24 hours. IL-2 secretion was measured in the culture supernatants by ELISA. IVIg was depleted of PHA-reactive IgG by passage on a PHA-Sepharose column. The extent of depletion was evaluated by ELISA using PHA as capture antigen. The role of F(ab')2 fragments in the inhibitory effect of IVIg on IL-2 secretion was determined using pepsin-generated fragments. Results IVIg inhibited IL-2 secretion by PHA-stimulated T cells, as previously reported. However, the use of increasing concentrations of PHA for T cell stimulation led to a decreased ability of IVIg to inhibit IL-2 production, suggesting that IVIg acted by neutralizing PHA or by competition for receptor occupancy on the cell surface. Pre-incubation of T cells with IVIg followed by washing and addition of PHA did not result in inhibition of IL-2 secretion, indicating that competition for receptor was not involved in this IVIg-mediated inhibition. In contrast, inhibition of IL-2 production by IVIg was completely abrogated using IVIg depleted from PHA-reactive IgG, indicating that IVIg-mediated inhibition of IL-2 secretion was the consequence PHA neutralization. Testing of F(ab')2 fragments of IVIg showed that these fragments bound to PHA and inhibited IL-2 secretion as efficiently as IVIg. Using another activation strategy, we showed that IVIg could also decrease IL-2 secretion by purified human T cells following anti-CD3/CD28 stimulation. Preliminary data using light microscopy indicated that IVIg interfered with the binding of CD3/CD28 beads on T cells, therefore reducing cell activation as evaluated by IL-2 secretion. Conclusion Altogether, our results suggest that the inhibition of T cell responses by IVIg occurs during the cell activation step, by preventing the binding of mitogens or antibody-coated beads on the cell surface. These observations emphasize the importance of ruling out the possible interactions of IVIg with culture medium additives, mitogens or activating agents before deriving strong conclusions on the mechanisms of action of IVIg based on their apparent immunomodulatory effects observed in vitro assays. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Zoledronic Acid Boosts γδ T-Cell Activity in Children Receiving αβ+ T and CD19+ CELL-Depleted Grafts from a Haplo-Identical DONOR

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5771-5771
Author(s):  
Giulia Barbarito ◽  
Irma Airoldi ◽  
Alessia Zorzoli ◽  
Alice Bertaina ◽  
Andrea Petretto ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Zoledronic Acid ◽  
Conflicts Of Interest ◽  
High Resolution Mass Spectrometry ◽  
Ex Vivo ◽  
Cell Activity ◽  
Γδ T Cells ◽  
Leukemia Cells ◽  
Γδ T Cell

Abstract A new method of graft manipulation based on physical removal of αβ+ T cells and CD19+ B cells, leaving mature NK cells and γδ T cells in the graft, has been recently developed for HLA-haploidentical HSCT. We demonstrated that γδ T cells collected from transplanted patients are endowed with capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we hypothesized that infusion of ZOL in patients receiving this type of graft, may boost γδ T cell cytotoxic activity against leukemia cells. Thirty-three patients were treated with ZOL every 28 days at least twice. γδ T cells before and after ZOL treatments were studied till at least 7 months after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. Proteomic analysis of γd T cells purified from patients showed that, starting from the first infusion, ZOL caused up-regulation of proteins involved in activation processes and immune response, paralleled by down-regulation of proteins involved in proliferation. These findings are consistent with an induction of Vδ2 cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts. Furthermore, a proteomic signature was identified for each individual ZOL treatment. Patients given 3 or more ZOL infusions had a better probability of survival in comparison to those given 1 or 2 treatments. In conclusion,ZOL influences Vδ2 cell activity, determines a specific proteomic signature and enhances anti-leukemia activity, this potentially resulting into an increased anti-tumor effect. Disclosures No relevant conflicts of interest to declare.

scholarly journals Adverse Events Following Infusion of T Cells for Adoptive Immunotherapy: A 10 Year Experience.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3212-3212
Author(s):  
Conrad Russell Y. Cruz ◽  
Patrick Hanley ◽  
Hao Liu ◽  
Vicky Torrano ◽  
Yu-Feng Lin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adverse Events ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Safe Procedure ◽  
Cell Product ◽  
Increased Risk ◽  
Infusion Reactions ◽  
Severe Adverse Events

Abstract Abstract 3212 Poster Board III-149 Following administration of ex-vivo expanded T cells, the FDA currently recommends at least 4 hours of recipient monitoring to detect early infusion reactions. Recent catastrophic reactions to “first in man” biological agents have emphasized the importance of this rule for initial studies of new products, but its value for longer established agents is less evident. We have therefore reviewed the incidence and nature of infusion-related adverse events (AEs) associated with administration of ex-vivo expanded T cell products (antigen specific CTLs, allodepleted T cells, and genetically modified T cells) on Investigational New Drug (IND) studies in our center over the last decade. From 1998 to 2008, we infused 381 T cell products to 180 recipients who were enrolled on 18 such studies, receiving T cells targeting malignancies or post-transplant viral infections. The age of these recipients ranged from 9 months to 80 years. Cell doses were protocol specific and ranged from 104/kg up to 3 ×108/m2. Patients were premedicated with diphenhydramine (0.5-1mg/kg) and acetaminophen (10mg/kg up to a maximum of 625mg) prior to infusion. All cellular products were cryopreserved and administered intravenously over 1-15 minutes immediately after thawing. There were no Grade 3-4 infusion reactions during initial monitoring or 24 hour follow-up. Twenty four grade 1-2, non-severe adverse events (AEs) occurred in 21 infusions either during or immediately following infusion (up to 6 hours). The most common AEs were nausea and vomiting (10/24; 41.6%), most likely due to DMSO used in cryopreservation of T cell products, and hypotension (20.8%), attributable to diphenhydramine pre-medication. An additional 22 non-severe events within 24 hours of infusion were reported, the most common of which were fever (with negative blood cultures)/chills/constitutional symptoms (6/22; 27.3%) and nausea/vomiting (4/22; 18.2%) Overall, a total of 46 non-severe adverse events were noted within 24 hours of T cell product infusion (12.56%). A Fisher's exact analysis of all T cell product infusions grouped by patient age, patient ethnicity, or cell source revealed no association with increased risk. T cells from both allogeneic and autologous sources produced similar adverse events in terms of type, frequency, and severity, and allogeneic cells that were mismatched at > 2 HLA antigens had the same AEs as donor T cells matched at 5/6 or 6/6 HLA loci. We further analyzed the data using Poisson regression and the general estimating equation (GEE) model for correlated counts, to seek associations that may have been missed because AEs within a subject may not be statistically independent. By this analysis, we found decreased risks of adverse events in older patients (IRR 0.98; 95% CI 0.96-1.00; p=0.05), and increased risks of immediate (defined as occurring during the monitoring period) infusion-related events in patients reporting allergies (IRR 2.72; 95% CI 1.00-7.40; p=0.05). We thus conclude that infusion of the ex-vivo expanded T cell products used in these studies is a safe procedure associated with no severe reactions, that it is safe in the outpatient setting and that monitoring can be limited to an hour after infusion. As many of the AEs observed were due to diphenhydramine premedication, a lower dose (0.25mg/kg) of this agent may be preferred. Disclosures No relevant conflicts of interest to declare.

scholarly journals Antigen Presenting Cells from Tumor and Colon of Colorectal Cancer Patients Are Distinct in Activation and Functional Status, but Comparably Responsive to Activated T Cells

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5247
Author(s):  
Frank Liang ◽  
Azar Rezapour ◽  
Louis Szeponik ◽  
Samuel Alsén ◽  
Yvonne Wettergren ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Functional Status ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Activated T Cells ◽  
Ifn Γ ◽  
Colorectal Cancer Patients

Although mouse models of CRC treatments have demonstrated robust immune activation, it remains unclear to what extent CRC patients’ APCs and TILs interact to fuel or quench treatment-induced immune responses. Our ex vivo characterization of tumor and adjacent colon cell suspensions suggest that contrasting environments in these tissues promoted inversed expression of T cell co-stimulatory CD80, and co-inhibitory programmed death (PD)-ligand1 (PD-L1) on intratumoral vs. colonic APCs. While putative tumor-specific CD103+CD39+CD8+ TILs expressed lower CD69 (early activation marker) and higher PD-1 (extended activation/exhaustion marker) than colonic counterparts, the latter had instead higher CD69 and lower PD-1 levels. Functional comparisons showed that intratumoral APCs were inferior to colonic APCs regarding protein uptake and upregulation of CD80 and PD-L1 after protein degradation. Our attempt to model CRC treatment-induced T cell activation in vitro showed less interferon (IFN)-γ production by TILs than colonic T cells. In this model, we also measured APCs’ CD80 and PD-L1 expression in response to activated co-residing T cells. These markers were comparable in the two tissues, despite higher IFN- γ exposure for colonic APCs. Thus, APCs within distinct intratumoral and colonic milieus showed different activation and functional status, but were similarly responsive to signals from induced T cell activation.

scholarly journals LB-ARHGDIB-1R As Novel Minor Histocompatibility Antigen For Therapeutic Application

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4465-4465
Author(s):  
Margot J Pont ◽  
Willemijn Hobo ◽  
M. Willy Honders ◽  
Simone A.P. van Luxemburg-Heijs ◽  
Michel G.D. Kester ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Hematological Malignancies ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Specific Lysis ◽  
Hematopoietic Stem ◽  
Therapeutic Relevance

Patients with hematological malignancies can be successfully treated with allogeneic hematopoietic stem cell transplantation (alloSCT). Beneficial Graft-versus-Leukemia (GvL) reactivity, however, is often accompanied with undesired Graft-versus-Host Disease (GvHD). In HLA-matched alloSCT, donor T-cells can mediate GvL reactivity by recognition of minor histocompatibility antigens (MiHA) on malignant cells, but also GvHD when MiHA are recognized on non-hematopoietic tissues. To decrease the incidence and severity of GvHD, T-cells can be (partially) depleted from the graft and re-administered later as donor lymphocyte infusion (DLI). MiHA are polymorphic peptides that are presented in HLA-molecules and can be recognized as non-self by donor-derived T-cells. Only a minority of MiHA have therapeutic relevance based on hematopoietic-restricted expression and only 25% and 40% of recipients transplanted with sibling and unrelated donors, respectively, are eligible for T-cell therapies in which one of the known hematopoietic-restricted MiHA is targeted. Therefore, to increase the efficacy and limit the toxicity of T-cell therapy, more therapeutic MiHA are needed. Recently, we identified a MiHA encoded by ARHGDIB, a gene that has been described to be highly expressed on hematopoietic cells, and we here studied the therapeutic relevance of LB-ARHGDIB-1R in detail. First, we confirmed hematopoietic-restricted expression of ARHGDIB by microarray gene expression analysis and demonstrated >10-fold overexpression in the majority of malignant and healthy hematopoietic versus non-hematopoietic cells. In line with its hematopoietic-restricted gene expression profile, LB-ARHGDIB-1R in the context of HLA-B* 07:02 was specifically recognized on different hematological malignancies, but not on non-hematopoietic fibroblasts and keratinocytes cultured in the absence or presence of IFN-γ, which was added to mimick the pro-inflammatory cytokine milieu of the early post-transplantation period. Next, we investigated the cytolytic capacity of LB-ARHGDIB-1R specific T-cells, and demonstrated specific lysis of patient, but not donor, EBV-B cells and specific lysis of an ALL sample in a 10 hrs 51Cr-release assay. Specific lysis of additional ALL and AML samples could be measured after 48 hrs of co-incubation in a flowcytometry-based cytotoxicity assay. To determine the in vivo immunogenicity of LB-ARHGDIB-1R, 11 MiHA-disparate HLA-B* 07:02 positive patient-donor pairs were screened for specific CD8+ T-cells by dual color tetramer analysis. All patients received partial T-cell depleted alloSCT and sampling was done at different time points after alloSCT (and DLI). In 4 out of 11 patients, LB-ARHGDIB-1R-specific T-cells (>0.01%) could be detected directly ex vivo and in 4 additional patients after 7 days of in vitro peptide stimulation, indicating that LB-ARHGDIB-1R is highly immunogenic. High frequencies of LB-ARHGDIB-1R specific T-cells were measured ex vivo in a patient whose hematological relapse was successfully treated with DLI, without development of GvHD, further supporting the therapeutic relevance of LB-ARHGDIB-1R. In summary, we confirmed hematopoietic-restricted expression of LB-ARHGDIB-1R and demonstrated T-cell mediated lysis of primary leukemic cells in long-term co-incubation assays. Furthermore, we showed that LB-ARHGDIB-1R is highly immunogenic and that specific T-cells could be detected in a patient who responded to DLI in the absence of GvHD. Altogether, our data support the clinical relevance of LB-ARHGDIB-1R as therapeutic MiHA with the potential to shift the delicate balance between GvL and GvHD in favor of a desired anti-tumor effect. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Selective Killing of Activated T Cells by 5-Aminolevulinic Acid Mediated Photodynamic Effect: Potential Improvement of Extracorporeal Photopheresis

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 377
Author(s):  
Sagar Darvekar ◽  
Petras Juzenas ◽  
Morten Oksvold ◽  
Andrius Kleinauskas ◽  
Toril Holien ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Blue Light ◽  
T Cell Activation ◽  
Cell Activation ◽  
Aminolevulinic Acid ◽  
Ex Vivo ◽  
Light Exposure ◽  
Extracorporeal Photopheresis ◽  
Activated T Cells

Extracorporeal photopheresis (ECP), a modality that exposes isolated leukocytes to the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light, is used to treat conditions such as cutaneous T-cell lymphoma and graft-versus-host disease. However, the current procedure of ECP has limited selectivity and efficiency; and produces only partial response in the majority of treated patients. Additionally, the treatment is expensive and time-consuming, so the improvement for this modality is needed. In this study, we used the concept of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA), a precursor of an endogenously synthesized photosensitizer protoporphyrin IX (PpIX) in combination with blue light to explore the possibility of targeting activated human blood T cells ex vivo. With various T-cell activation protocols, a high ALA-induced PpIX production took place in activated CD3+, CD4+CD25+, and CD8+ T cell populations with their subsequent killing after blue light exposure. By contrast, resting T cells were much less damaged by the treatment. The selective and effective killing effect on the activated cells was also seen after co-cultivating activated and resting T cells. Under our clinically relevant experimental conditions, ALA-PDT killed activated T cells more selectively and efficiently than 8-MOP/UV-A. Monocyte-derived dendritic cells (DCs) were not affected by the treatment. Incubation of ALA-PDT damaged T cells with autologous DCs induced a downregulation of the co-stimulatory molecules CD80/CD86 and also upregulation of interleukin 10 (IL-10) and indoleamine 2,3-dioxygenase expression, two immunosuppressive factors that may account for the generation of tolerogenic DCs. Overall, the data support the potential use of ALA-PDT strategy for improving ECP by selective and effective killing of activated T cells and induction of immune tolerance.

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