scholarly journals CMTM6-Deficient Monocytes in ANCA-Associated Vasculitis Fail to Present the Immune Checkpoint PD-L1

2021 ◽  
Vol 12 ◽  
Author(s):  
Markus Zeisbrich ◽  
Nina Chevalier ◽  
Bettina Sehnert ◽  
Marta Rizzi ◽  
Nils Venhoff ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Surface Expression ◽  
Molecular Defect ◽  
Lysosomal Degradation ◽  
Lysosomal Function ◽  
Immune Checkpoint Molecules

ObjectivesANCA-associated vasculitides (AAV) affect small- and medium-sized blood vessels. In active disease, vessel wall infiltrates are mainly composed of monocytes and macrophages. Immune checkpoint molecules are crucial for the maintenance of self-tolerance and the prevention of autoimmune diseases. After checkpoint inhibitor therapy, the development of autoimmune vasculitis has been observed. However, defects of immune checkpoint molecules in AAV patients have not been identified yet.MethodsMonocytes and monocyte-derived macrophages from AAV patients and healthy age-matched controls were tested for surface expression of immunoinhibitory checkpoint programmed cell death ligand-1 (PD-L1). Using in vitro co-culture approaches, the effect of monocyte PD-L1 expression on CD4+ T cell activation and proliferation was tested.ResultsMonocytes from AAV patients displayed lower PD-L1 expression and a defective PD-L1 presentation upon activation, an effect that was correlated with disease activity. Lower PD-L1 expression was due to increased lysosomal degradation of PD-L1 in AAV monocytes. We identified a reduced expression of CMTM6, a protein protecting PD-L1 from lysosomal breakdown, as the underlying molecular defect. PD-L1low AAV monocytes showed increased stimulatory capacity and induced T cell activation and proliferation. Inhibiting lysosomal function corrected this phenotype by increasing PD-L1, thus normalizing the pro-stimulatory behavior of AAV monocytes.ConclusionsThis study identifies a defect of the immunoinhibitory checkpoint PD-L1 in monocytes from patients with AAV. Low expression of CMTM6 results in enhanced lysosomal degradation of PD-L1, thus providing insufficient negative signaling to T cells. Correcting this defect by targeting lysosomal function may represent a novel strategy to treat AAV.

scholarly journals Novel immune checkpoint targets: moving beyond PD-1 and CTLA-4

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Shuang Qin ◽  
Linping Xu ◽  
Ming Yi ◽  
Shengnan Yu ◽  
Kongming Wu ◽  
...  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Programmed Cell Death ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
De Novo ◽  
Checkpoint Inhibitors ◽  
Immune Checkpoints ◽  
Immune Checkpoint Molecules

Abstract The emergence of immune checkpoint inhibitors (ICIs), mainly including anti-programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) and anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) monoclonal antibodies (mAbs), has shaped therapeutic landscape of some type of cancers. Despite some ICIs have manifested compelling clinical effectiveness in certain tumor types, the majority of patients still showed de novo or adaptive resistance. At present, the overall efficiency of immune checkpoint therapy remains unsatisfactory. Exploring additional immune checkpoint molecules is a hot research topic. Recent studies have identified several new immune checkpoint targets, like lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin and mucin-domain containing-3 (TIM-3), T cell immunoglobulin and ITIM domain (TIGIT), V-domain Ig suppressor of T cell activation (VISTA), and so on. The investigations about these molecules have generated promising results in preclinical studies and/or clinical trials. In this review, we discussed the structure and expression of these newly-characterized immune checkpoints molecules, presented the current progress and understanding of them. Moreover, we summarized the clinical data pertinent to these recent immune checkpoint molecules as well as their application prospects.

scholarly journals Identifying Immune Resistance Mechanisms to CD33/CD3 BiTE® Antibody Construct (AMG 330) Mediated Cytotoxicity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3677-3677
Author(s):  
Christina Krupka ◽  
Thomas Köhnke ◽  
Peter Kufer ◽  
Roman Kischel ◽  
Felix S. Lichtenegger ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Constitutive Expression ◽  
Resistance Mechanisms ◽  
Adaptive Resistance ◽  
Immune Checkpoint Molecules ◽  
Immune Resistance

Abstract In our previous work, we showed elimination of primary acute myeloid leukemia (AML) cells by CD33/CD3 BiTE® antibody construct (AMG 330) mediated cytotoxicity (Krupka et al, Blood 2014). The goal of the present study was to identify innate and adaptive resistance mechanisms to AMG 330 mediated lysis of AML cells. Immune checkpoint molecules have been shown to be a highly relevant escape mechanism of malignant cells to evade innate and adaptive immunity. Previously, it was shown that AML cells upregulate the expression of inhibitory ligands in response to proinflammatory cytokines (Krönig et al, 2014). As AMG 330 mediated T-cell activation induces high levels of the proinflammatory cytokines IFNγ and TNFα, we assessed the constitutive and inducible expression profile of different immune checkpoint molecules on AML cell lines and primary AML cells, including PD-L1, HVEM, ILT3 and SLAMF7 by flow cytometry. No constitutive expression was observed for PD-L1 at time of primary diagnosis in 83.7% of the cases (103/123). In contrast, constitutive expression of HVEM and ILT3 was detected in 73.7% (42/57) and 91.9% (68/74) of patient samples, respectively. Adaptive resistance was evaluated by incubating AML cell lines and primary AML samples with IFNγ and TNFα. We observed an upregulation of PD-L1 and SLAMF7 on AML cell lines and on primary AML patient samples whereas HVEM and ILT3 did not show a significant change in expression level. To test the functional relevance of the immune checkpoint molecules upon AMG 330 mediated lysis, we used an ex vivo long term culture system that enabled us to analyse the dynamic process of receptor-ligand interaction over time. Blockade of the PD-1/PD-L1 interaction resulted in a significantly increase in AMG 330 mediated lysis of primary AML cells (n=9, p=0.03). Currently, blockade of the inducible molecule SLAMF7 in AMG 330 mediated cytotoxicity is being tested. Blocking of HVEM or ILT3 did not result in a significant increase in T cell activation and concomitant lysis of AML cells suggesting a less relevant role of HVEM and ILT3 in resistance to AMG 330 mediated cytotoxicity. The latter might also be influenced by the cytokine microenvironment which favours immune resistance of AML cells. Using a bead based multiplex assay we screened the bone marrow (BM) plasma from 16 AML patients and 3 healthy donors (HD) for the presence of 33 cytokines. The cytokine profile differed between AML patients and healthy donors (HDs). The plasma levels of IL-8, IP-10 and CXCL-16 were higher in the AML samples compared to those of HDs (p=0.0041, 0.0248 and 0.0289, respectively). In contrast, EGF, FLT3-ligand, RANTES and IL-4 were significantly lower in AML samples compared to HDs (p=0.0227, 0.0145, 0.0041 and 0.0041, respectively). However, we did observe a high inter-patient variability of cytokine composition in AML. To explore the functional relevance of the BM plasma on AMG 330 mediated cytotoxicity, cocultures of AML cell lines and HD T cells were set up using different sources of plasma including fetal calf serum (FCS) and patient derived BM plasma. Interestingly, AMG 330 mediated cytotoxicity was significantly reduced using patient derived BM plasma (n=5) compared to cultures containing FCS (n=4) (mean % lysis FCS 97.4 vs PT 70.6). This was accompanied by a considerable impairment in T-cell proliferation (mean % proliferation FKS 44.7% vs PT 26.6%). Currently, we are investigating which soluble factors are responsible for the immunosuppressive effects and if we can increase lysis efficacy and T-cell proliferation through specific blocking of them. In summary we have identified possible resistance mechanisms of AML cells to AMG 330 mediated cytotoxicity. Dynamic receptor-ligand interactions between target and effector cells as well as soluble factors contribute to AMG 330 mediated lysis of primary AML cells. We hypothesize that AMG 330 mediated cytotoxicity can be augmented through combinatorial approaches including PD-1 blockade. The significance of our findings will first be validated in an in vivo mouse model and prospectively translated into human studies. Disclosures Krupka: AMGEN Research (Munich): Research Funding. Kufer:AMGEN Research (Munich): Employment, Equity Ownership. Kischel:AMGEN Research (Munich): Employment, Equity Ownership. Subklewe:AMGEN Research (Munich): Research Funding.

scholarly journals Ex vivo modelling of PD-1/PD-L1 immune checkpoint blockade under acute, chronic, and exhaustion-like conditions of T-cell stimulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander Roberts ◽  
Lindsay Bentley ◽  
Tina Tang ◽  
Fay Stewart ◽  
Chiara Pallini ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Inflammatory Cytokine ◽  
Cell Activation ◽  
Ex Vivo ◽  
In Vitro Assays ◽  
The Impact

AbstractBlockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1—revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1—to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

scholarly journals Effect of B7-2 and CD40 Signals from Activated Antigen-Presenting Cells on the Ability of Zwitterionic Polysaccharides To Induce T-Cell Stimulation

2005 ◽  
Vol 73 (4) ◽  
pp. 2184-2189 ◽  
Author(s):  
Tom Li Stephen ◽  
Marcus Niemeyer ◽  
Arthur O. Tzianabos ◽  
Martin Kroenke ◽  
Dennis L. Kasper ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Surface Expression ◽  
Costimulatory Molecules ◽  
T Cell Proliferation ◽  
Hla Dr ◽  
Antigen Presenting

ABSTRACT Carbohydrates have been thought to stimulate immune responses independently of T cells; however, zwitterionic polysaccharides (ZPSs) from the capsules of some bacteria elicit potent CD4+-T-cell responses in vivo and in vitro. We demonstrated that HLA-DR on professional antigen-presenting cells (APCs) is required for ZPS-induced T-cell proliferation in vitro (15). Recently, it was shown that ZPSs are processed to low-molecular-weight carbohydrates by a nitric oxide-mediated mechanism in endosomes and locate in the major histocompatibility complex class II pathway (5, 15). The effect of the ZPS-mediated expression of HLA-DR and costimulatory molecules on the APC and T-cell engagement and subsequent T-cell activation has not been elucidated. Herein, we report that ZPS-mediated induction of HLA-DR-surface expression and T-cell proliferation are maximally enhanced after incubation of APCs for 8 h with ZPS. Treatment of APCs with bafilomycin A inhibits the up-regulation of ZPS-mediated HLA-DR surface expression and leads to inhibition of T-cell proliferation. Monoclonal antibodies (MAbs) to the costimulatory molecules B7-2 and CD40L specifically block ZPS-mediated T-cell activation, while a MAb to B7-1 does not. Surface expression of B7-2 and B7-1 but not of CD40 is maximally enhanced at 8 to 16 h of treatment of APCs with ZPS. The results demonstrate that the cellular immune response to ZPS depends on the translocation of HLA-DR to the cell surface and requires costimulation via B7-2 and CD40 on activated APCs. The implication is that activation of ZPS-specific T cells requires an orchestrated arrangement of both presenting and costimulatory molecules to form an immunological synapse.

scholarly journals L5 RIG-I activation enhances melanoma immunogenicity and improves anti-tumor T cell responses in combination with anti-PD-1 immune checkpoint blocking antibodies

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A3.1-A3
Author(s):  
B Thier ◽  
L Such ◽  
M Schwamborn ◽  
A Sucker ◽  
C Coch ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
T Cell Responses ◽  
Surface Expression ◽  
Melanoma Cells ◽  
Facs Analysis ◽  
Cell Responses

BackgroundClinical efficacy of immune checkpoint blocking (ICB) therapy critically relies on the killing of melanoma cells by CD8+ T cells, becoming activated upon recognition of tumor antigens presented by HLA class I (HLA-I) surface molecules. Patient-derived melanoma cells can escape from cytotoxic T cell effector functions by loss of HLA-I surface expression due to the silencing of HLA-I antigen processing and presentation machinery (APM) genes.Material and MethodsSeeking for a strategy to restore HLA-I expression, we transfected melanoma cells obtained from distinct patient metastasis with synthetic short double stranded RNA (3pRNA), an activating ligand of the cytosolic innate pattern recognition receptor RIG-I. 3pRNA-transfected melanoma cells were analyzed for HLA-I surface expression by FACS analysis and gene expression of HLA-I APM components by qPCR. In vivo 3pRNA-transfected tumors were analyzed for HLA-I expression by immunohistochemistry staining. Furthermore, T cell activation after coincubation with 3pRNA-transfected melanoma cells was determined by IFNγ-ELISpot assay. The effect of combined 3pRNA and blocking anti-PD-1 antibody treatment on T cell activation was measured by intracellular cytokine staining and FACS analysis.ResultsActivation of RIG-I by 3pRNA increased the expression of HLA-I APM components and strongly enhanced recognition of melanoma cells by autologous CD8+ T cells. Based on these findings, we asked whether the combination of 3pRNA and blocking anti-PD-1 antibodies could improve anti-melanoma T cell responses in an anti-PD-1 non-responder patient model. Indeed, T cell activation by 3pRNA-transfected melanoma cells was significantly increased in the presence of anti-PD-1 antibodies. In line with the enhancement of anti-tumor T cell responses, we found an association of elevated RIG-I mRNA levels with prolonged patient survival in TCGA melanoma samples.ConclusionsIn summary, this study demonstrates a beneficial effect of RIG-I activation on antigen presentation and T cell recognition of melanoma cells. Improved T cell responses by combined 3pRNA and anti-PD-1 treatment suggests that combinational therapy could be a strategy to overcome T cell resistance in melanoma.Disclosure InformationB. Thier: None. L. Such: None. M. Schwamborn: None. A. Sucker: None. C. Coch: None. D. Schadendorf: None. K. Griewank: None. M. Trilling: None. F. Zhao: None. A. Paschen: None.

scholarly journals B cells sustain inflammation and predict response to immune checkpoint blockade in human melanoma

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Johannes Griss ◽  
Wolfgang Bauer ◽  
Christine Wagner ◽  
Martin Simon ◽  
Minyi Chen ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Immune Checkpoint Blockade ◽  
Human Melanoma ◽  
Checkpoint Blockade ◽  
Inflammatory Factors

Abstract Tumor associated inflammation predicts response to immune checkpoint blockade in human melanoma. Current theories on regulation of inflammation center on anti-tumor T cell responses. Here we show that tumor associated B cells are vital to melanoma associated inflammation. Human B cells express pro- and anti-inflammatory factors and differentiate into plasmablast-like cells when exposed to autologous melanoma secretomes in vitro. This plasmablast-like phenotype can be reconciled in human melanomas where plasmablast-like cells also express T cell-recruiting chemokines CCL3, CCL4, CCL5. Depletion of B cells in melanoma patients by anti-CD20 immunotherapy decreases tumor associated inflammation and CD8+ T cell numbers. Plasmablast-like cells also increase PD-1+ T cell activation through anti-PD-1 blockade in vitro and their frequency in pretherapy melanomas predicts response and survival to immune checkpoint blockade. Tumor associated B cells therefore orchestrate and sustain melanoma inflammation and may represent a predictor for survival and response to immune checkpoint blockade therapy.

scholarly journals MARCH1-mediated MHCII ubiquitination promotes dendritic cell selection of natural regulatory T cells

2013 ◽  
Vol 210 (6) ◽  
pp. 1069-1077 ◽  
Author(s):  
Jaehak Oh ◽  
Nan Wu ◽  
Günther Baravalle ◽  
Benjamin Cohn ◽  
Jessica Ma ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Substantial Reduction ◽  
Surface Expression ◽  
Selection Of

Membrane-associated RING-CH1 (MARCH1) is an E3 ubiquitin ligase that mediates ubiquitination of MHCII in dendritic cells (DCs). MARCH1-mediated MHCII ubiquitination in DCs is known to regulate MHCII surface expression, thereby controlling DC-mediated T cell activation in vitro. However, its role at steady state or in vivo is not clearly understood. Here, we show that MARCH1 deficiency resulted in a substantial reduction in the number of thymus-derived regulatory T cells (T reg cells) in mice. A specific ablation of MHCII ubiquitination also significantly reduced the number of thymic T reg cells. Indeed, DCs deficient in MARCH1 or MHCII ubiquitination both failed to generate antigen-specific T reg cells in vivo and in vitro, although both exhibited an increased capacity for antigen presentation in parallel with the increased surface MHCII. Thus, MARCH1-mediated MHCII ubiquitination in DCs is required for proper production of naturally occurring T reg cells, suggesting a role in balancing immunogenic and regulatory T cell development.

scholarly journals 781 Validation of the combinatorial effect of blinatumomab and nivolumab in cancer therapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A831-A831
Author(s):  
Tienan Wang ◽  
Qing Lin ◽  
Jie Zhang
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Culture System ◽  
Cell Activation ◽  
Checkpoint Inhibitors

BackgroundCancer immunotherapies, including immune checkpoint inhibitors, CAR-T, cancer vaccines and bispecific antibodies, have been brought to spot light in recent years as several therapeutic strategies targeting the immune system have produced exciting clinical results. Bispecific antibody typically play dual roles in blocking the immune checkpoint and redirecting/re-boosting the function of the immune effector cells. Blinatumomab belongs to CD3 bispecific T cell engager (CD3 BiTE), which was engineered to harbor two arms binding with CD3 and CD19 simultaneously and direct CD8+ T cells to specifically recognize CD19 positive lymphoma cells to execute cytotoxicity. Approval of Blinatumomab for patients with relapse/refractory B cell acute lymphoblastic leukemia (ALL) has driven remarkable increase in combination studies of Blinatumomab with other immunotherapies such as checkpoint inhibitors.MethodsIn this study, we developed CD8+ T cytotoxic system targeting different B lymphoma cell line and fully validated the function of Blinatumomab in promoting target tumor cell lysis by primary CD8+ T cells (figure 1). In addition, we established a mixed lymphocyte and tumor system to mimic physiological TME to dissect the combinational role of Nivolumab and Blinatumomab (figure 2).ResultsThe result suggest that combinatory therapy is highly depend on the dosage of Blinatumomab and also T cell number in the TME, which might give an instruction for ongoing clinical trial design. Finally, we have employed humanized mouse models bearing Raji or Daudi tumor cells to further validate this combination treatment in vivo. Both In-vivo and In-vitro data support that Blinatumomab is dominant in activing T cell and Nivolumab can only exhibit synergistic effect under suboptimal dosage of Blinatumomab.Abstract 781 Figure 1Establishment of In vitro co-culture system for CD3 BiTEestablish in vitro human PBMC based system to validate CD3 BiTE functionAbstract 781 Figure 2Opdivo and CD3 BiTE CombinationOpdivo could further promote T cell activation under the treatment of CD3 BiTEConclusionsSuccessfully establish in vitro system to evaluate the function of CD3 BiTE and also take advantage of MLR/tumor co-culture system to demonstrate PD1 antibody could further promote T cell activation under appropriate dosage of CD3 BiTE.

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