Hepatitis durch Checkpunkt-Therapie – periphere Immunaktivierung als möglicher Biomarker

2021 ◽  
Vol 3 (2) ◽  
pp. 75-76
Author(s):  
Bertram Bengsch
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cytokine Production ◽  
Transcriptional Profiling ◽  
Granzyme B ◽  
Enhanced Production ◽  
Hla Dr ◽  
Cytotoxic Effector ◽  
Pro Inflammatory Cytokines

<b>Background &amp; aims:</b> Checkpoint inhibitor-related hepatitis (CPI-Hep) is an emerging clinical challenge. We aim to gain insights into the immunopathology of CPI-Hep by comprehensive characterisation of myeloid and lymphoid subsets. <b>Methods:</b> CPI-treated patients with or without related hepatitis (CPI-Hep; n = 22 and CPI-noHep; n = 7) were recruited. Phenotypic and transcriptional-profiling of peripheral immune subsets was performed and compared with 19 healthy controls (HC). In vitro monocyte-derived macrophages (MoMF) were assessed for activation and cytokine production. CD163, CCR2, CD68, CD3, CD8 and granzyme B expression was assessed using immunohistochemistry/immunofluorescence (n = 4). <b>Results:</b> A significant total monocyte depletion was observed in CPI-Hep compared with HC (p = 0.04), along with a proportionate increase in the classical monocyte population (p = 0.0002) and significant upregulation of CCR2, CD163 and downregulation of CCR7. Soluble CD163 levels were significantly elevated in CPI-Hep compared with HC (p &#x3c; 0.0001). In vitro MoMF from CPI-Hep showed enhanced production of pro-inflammatory cytokines. CD8+ T cells demonstrated increased perforin, granzyme B, ICOS and HLA-DR expression in CPI-Hep. Transcriptional profiling supported activated monocyte and enhanced effector CD8+ T cell populations in CPI-Hep. Immunohistochemistry demonstrated co-localisation of CD8+/granzyme B+ T cells with CD68+CCR2+/ CD68+CD163+ macrophages in CPI-Hep liver tissue. <b>Conclusions:</b> CPI-Hep is associated with an activation of peripheral monocytes and enhanced cytotoxic, effector phenotype of CD8+ T cells. These changes were reflected by liver inflammation composed of CD163+/CCR2+ macrophage and CD8+ T cells.

scholarly journals 663 Media based on the metabolic composition of tumor interstitial fluid reveals persistent T cell dysfunction induced through arginine deprivation and exposure to the oncometabolite phosphoethanolamine

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A691-A691
Author(s):  
Yupeng Wang ◽  
Chufan Cai ◽  
Dayana Rivadeneira ◽  
Alexander Muir ◽  
Greg Delgoffe
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Interstitial Fluid ◽  
Cytokine Production ◽  
Cell Dysfunction ◽  
Kennedy Pathway ◽  
T Cell Dysfunction ◽  
Tumor Interstitial Fluid

BackgroundWhile CD8 T cells are crucial for anti-tumor immunity, tumor infiltrating CD8 T cells encounter stressors which deviate their differentiation to a dysfunctional, exhausted phenotype. T cell functions are closely regulated by T cell metabolism, and the dysfunctional vasculature in tumor tissues and the deregulated metabolism of tumor cells lead to depletion of nutrients and accumulation of metabolic wastes in the tumor microenvironment (TME). Thus, the unbalanced levels of the nutrients and the metabolic wastes might skew the metabolism of T cells thus contributing to T cell dysfunction.MethodsOvalbumin-specific OT-I cells were activated with SIINFEKL/IL2 and cultured with IL2. The tumor interstitial fluid media (TIFM) was formulated based on the concentrations of the metabolites measured in the tumor interstitial fluid of pancreatic ductal adenocarcinoma.1 Purified arginine and phosphoethanolamine (PEtn) were used to change their levels in TIFM/RPMI1640 culture. Expression level of cytokines and PD-1 was measured by flow cytometry.ResultsWe sought to determine how T cells would differentiate, in vitro, if they were exposed only to the metabolites present in the TME. Using media formulated to model the metabolic composition of tumor interstitial fluid (TIFM),1 we show that CD8 T cells develop features of exhausted T cells in the TIFM culture: reduced proliferation, increased expression of PD-1 and decreased cytokine production. Using 'dropout' and 'add-back' approaches, we found arginine levels as a major contributor to the proliferation defect observed in TIFM-cultured T cells. Arginine was sufficient to restore proliferative capacity to T cells cultured in TIFM, but had no effect on the inhibited cytokine production. We then asked which metabolites were enriched in the TIFM, finding that PEtn, an intermediate in the ethanolamine branch of the Kennedy pathway and an oncometabolite enriched in the interstitial of many solid tumors, up-regulates PD-1 expression and compromises the cytokine production of the cells in culture. Depletion of Pcyt2, the metabolizing enzyme of PEtn and the rate limiting enzyme in the Kennedy pathway, makes CD8 T cells resistant to the effects of PEtn.ConclusionsOur data shows that the metabolic environment in the TME can be recapitulated in vitro and is sufficient to drive T cell dysfunction. Arginine depletion acts as a major inhibitor of T cell proliferation in the TME, but the oncometabolite PEtn drives a hypofunctional effector fate of T cells. Targeting PEtn metabolism via Pcyt2 depletion or inhibition is a potential target to reinvigorate T cells and enhance anti-tumor immunity.ReferenceSullivan MR, Danai LV, Lewis CA, Chan SH, Gui DY, Kunchok T, Dennstedt EA, Vander Heiden MG, Muir A. Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability. Elife 2019;;8:e44235. doi: 10.7554/eLife.44235. PMID: 30990168; PMCID: PMC6510537.

Study on the Pathways to Damage Hematopoiesis by CD8+ Effector T Cells of the Patients with Severe Aplastic Anemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4371-4371
Author(s):  
Zonghong Shao ◽  
Le Feng ◽  
Rong Fu ◽  
Jun Wang ◽  
Chunyan Liu ◽  
...  
Keyword(s):  
T Cells ◽  
Aplastic Anemia ◽  
Cd8 T Cells ◽  
Bone Marrow Failure ◽  
Granzyme B ◽  
Effector T Cells ◽  
Severe Aplastic Anemia ◽  
Control Group ◽  
Hla Dr ◽  
Normal Controls

Abstract Abstract 4371 Objective To investigate the quantity and their pathways to damage hematopoietic cells of CD8+CD25+ and CD8+HLA-DR+ effector T cells in peripheral blood (PB) of the patients with severe aplastic anemia(SAA) and explore the heterogeneous immunopathogenesis of SAA further. Methods The quantity of CD8+CD25+and CD8+HLA-DR+ cells in PB and the expressions of perforin, granzyme B, tumor necrosis factor-β(TNF -β) and FasL of 29 SAA (14 untreated and 15 recovered) patients and 12 normal controls were analyzed by flow cytometry. Results The ratio of CD8+CD25+T cells in CD8+ T cells was (3.67±2.58)% in untreated SAA patients, (5.19±4.29)% in recovered patients and (4.84±2.31)% in normal controls, and the ratios of CD8+CD25+T cells in CD3+ cells in three groups were (2.25±1.35)%, (2.98±1.35)% and (2.11±1.88)% respectively. There was no statistic difference among 3 groups(P>0.05). The ratio of CD8+HLA-DR+T cells in CD8+T cells was (39.30±8.13)% in untreated patients, which was significantly higher than that of recovered patients[(20.65±5.38%)] and controls [(18.34±6.68%)](P<0.001). There was no statistic difference between recovered patients and controls(P>0.05). CD8+HLA-DR+T cells in CD3+ cells was (27.81±7.10)% in untreated group, higher than that of recovered patients (12.02±3.03)% and controls(8.50±2.33)%(P<0.01). And the ratio in recovered group was higher than in control group(P<0.05). The expressions of perforin, granzyme B, TNF-β and FasL of CD8+HLA-DR+ T cells of untreated SAA patients were 8.51% A96.08% A72.11% and 94.25% respectively, higher than those of recovered patients(1.78% A85.20% A34.38% A51.20%)and controls(1.86% A82.09% A17.92% A32.91%). There was no statistic difference between recovered patients and controls(P>0.05). Conclusion There were elevated quantity of CD8+HLA-DR+ T cells and high expressions of perforin, granzyme B, TNF-β and FasL in SAA, which might contribute to the bone marrow failure of SAA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hypoxia Selectively Impairs CAR-T Cells In Vitro

Cancers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 602 ◽  
Author(s):  
Robert Berahovich ◽  
Xianghong Liu ◽  
Hua Zhou ◽  
Elias Tsadik ◽  
Shirley Xu ◽  
...  
Keyword(s):  
T Cells ◽  
Solid Tumor ◽  
Cytokine Production ◽  
Granzyme B ◽  
Cytolytic Activity ◽  
Car T Cells ◽  
Hypoxic Conditions ◽  
Car T ◽  
Differentiation Status

Hypoxia is a major characteristic of the solid tumor microenvironment. To understand how chimeric antigen receptor-T cells (CAR-T cells) function in hypoxic conditions, we characterized CD19-specific and BCMA-specific human CAR-T cells generated in atmospheric (18% oxygen) and hypoxic (1% oxygen) culture for expansion, differentiation status, and CD4:CD8 ratio. CAR-T cells expanded to a much lower extent in 1% oxygen than in 18% oxygen. Hypoxic CAR-T cells also had a less differentiated phenotype and a higher CD4:CD8 ratio than atmospheric CAR-T cells. CAR-T cells were then added to antigen-positive and antigen-negative tumor cell lines at the same or lower oxygen level and characterized for cytotoxicity, cytokine and granzyme B secretion, and PD-1 upregulation. Atmospheric and hypoxic CAR-T cells exhibited comparable cytolytic activity and PD-1 upregulation; however, cytokine production and granzyme B release were greatly decreased in 1% oxygen, even when the CAR-T cells were generated in atmospheric culture. Together, these data show that at solid tumor oxygen levels, CAR-T cells are impaired in expansion, differentiation and cytokine production. These effects may contribute to the inability of CAR-T cells to eradicate solid tumors seen in many patients.

Activation and Effector Phenotype of CD8+ T Cells Crossprimed by a Minor Histocompatibility Antigen Expressed on Red Blood Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2571-2571
Author(s):  
Maxime Desmarets ◽  
James C. Zimring
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Whole Blood ◽  
Granzyme B ◽  
Solid Organ ◽  
Minor Histocompatibility Antigens ◽  
Viable Solution ◽  
Cfse Dilution ◽  
A Minor

Abstract Background: Transfusion immunology research has traditionally focused on humoral immunity to RBC antigens, with study of CD4+ T cell and B cell responses. However, minor histocompatibility antigens (mHAs) on transfused RBCs in theory can also be cross-presented by recipient APCs to CD8+ T cells. This has clinical relevance in the context of transfusion patients who undergo transplantation, as several lines of evidence suggest that immunization to mHAs can lead to rejection of bone marrow and solid organ transplants. To test the hypothesis that mHAs on RBCs are capable of inducing immune responses through cross priming of CD8+ T cells, we created a novel transgenic mouse (HOD mouse), which expresses Ovalbumin (OVA) on RBCs but neither on leukocytes nor platelets. Methods: 1×107 OT-I splenocytes (TCR transgenic for the OVA derived peptide SIINFEKL presented by H-2Kb) were adoptively transferred into B6.Thy1.1 mice. 100μl of packed HOD RBCs from whole or leukoreduced blood were then transfused intravenously. Control mice were transfused with FVB whole blood (HOD mice are on a FVB background). Three to 4 days post transfusion the OT-I T cells were visualized in the spleen by staining with anti-CD8 antibody and the Kb/SIINFEKL tetramer. Proliferation was evaluated by CFSE dilution. The activation phenotype was studied by staining with antibodies against CD44 and CD62L. Expression of effector molecules was studied by staining with antibodies against CD107a, Granzyme B, IFNγ, and IL-2. Results: Compared to background analysis of OT-I T cells in control mice that received FVB blood, OT-I T cells in HOD recipients had a maximal expansion 24 fold higher in mice transfused with HOD leukoreduced blood (p=.0001), and 26 fold in mice transfused with HOD whole blood (p=.0672). Compared to naïve OT-I T cells, cross-primed OT-I T cells were CD44high, CD62Llow, CD107ahigh, suggesting activation and degranulation. Expression of IFNγ was also increased. However, cross-primed OT-I T cells were Granzyme Blow and did not have increased IL-2 expression. Lack of increased Granzyme B and IL-2 was not an artifact of the assay not working, as strong expression of both Granzyme B and IL-2 were detected in OT-I T cells stimulated with OVA in vitro. Discussion: Overall these results demonstrate that RBC antigens can be cross-primed into the MHC class I pathway by recipient APCs. However, activation of CD8+ T cells specific for the RBC antigen demonstrates differentiation into an incomplete effector phenotype. The effect is unlikely due to contaminating donor leukocytes in the transfused unit since OVA is not expressed by WBCs. Moreover, we did not see any differences in proliferation or activation phenotype between the mice transfused with whole blood and leukoreduced blood. Activation was not due to direct presentation by residual cells, as the HOD donor mice are on an FVB (H-2q) background and cannot directly present mHAs to the H-2b restricted OT-I T cells. The reason for an incomplete effector profile is unclear, but may be because transfused RBCs are a sterile introduction of foreign antigen. These results also suggest better leukoreduction is not a viable solution to the problem of transfusion-induced immunization to mHAs, as the antigen is coming from the RBCs themselves. Matching for relevant mHAs or use of immunomodulatory treatments may be required.

Failure of Rituximab in Immune Thrombocytopenia Is Associated with the Activation of Splenic CD8 T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 623-623
Author(s):  
Sylvain Audia ◽  
Maxime Samson ◽  
Malika Trad ◽  
Nona Janikashvili ◽  
Denis Caillot ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Immune Thrombocytopenia ◽  
Granzyme B ◽  
Humoral Response ◽  
Interferon Γ ◽  
Off Label Use ◽  
Platelet Destruction ◽  
Off Label ◽  
Hla Dr

Abstract Abstract 623 Background: Different mechanisms are involved in the pathogenesis of immune thrombocytopenia (ITP). Platelet production is impaired as result of an immune response directed against megakaryocytes and an inappropriate level of thrombopoietin. Furthermore, platelets targeted by autoantibodies against their membrane glycoproteins are cleared by the reticuloendothelial system, mainly in the spleen. Thus, treatments targeting the autoimmune humoral response, notably rituximab (off-label use), have been proposed. However, the one-year response rate after rituximab (RTX) is not greater than 40% and the mechanisms involved in its failure remain to be defined. CD8 T cells which have been demonstrated to be involved in platelet destruction both in a mouse ITP model and in humans may play a part in the inefficiency of RTX. Purpose: To compare the splenic CD8 T cell response of ITP patients who have been refractory to RTX with untreated ITP patients Methods: The spleens of 23 primary ITP patients were assessed: 12 patients previously treated with RTX without improvement were examined as RTX refractory patients and 11 patients who have not received RTX were referred as untreated patients. Splenectomy was performed at a median of 7.1 months after RTX administration. Nine post-traumatic spleens were used as controls. Flow cytometry analysis was performed on CD8+ splenocytes to determine the expression of activation marker (HLA-DR), memory T cell markers (CD27, CD28), chemokine receptor (CCR7), adhesion molecule (CD62L) and intracellular cytotoxic proteins (granzyme B, perforin). Lineage commitment of T cells was determined after stimulation for 4 hours with PMA and ionomycin in presence of brefeldin-A, by intracellular staining of interferon-γ (Tc1, Th1), IL-4 (Tc2, Th2) and IL-17 (Tc17, Th17). Results are expressed by median and [interquartile range]. Statistical analysis was performed using non parametric tests (Kruskal-Wallis and Mann-Whitney) to compare the different groups. Results: After RTX infusion, B cells represented only 1.4% [0.3–3.4] in total splenic lymphocytes compared to about 35% in controls and untreated patients. No alteration in the CD8/CD4 ratio was observed after RTX. Nevertheless, expression of HLA-DR and granzyme B by splenic CD8 T cells was increased in RTX refractory patients when compared to untreated patients: 60% [50.6–68.9] vs. 41.8% [33.7–54.2] (p=0.02) and 50.3% [32.2–72.3] vs. 23.9% [14.4–27.7] (p=0.006), respectively. Upon stimulation, interferon-γ expression was significantly higher in CD3+CD8+ (68% [59.1–81.6]) but also in CD3+CD8− (32.5% [24.8–44]) in RTX refractory patients compared to untreated patients. The percentage of CCR7−CD62L− and CD27−CD28− among CD8+ T cells was increased in RTX refractory patients compared to untreated patients, respectively 85.4%[77.1–93.2] vs. 66.5%[56.2–73.2] (p=0.001) and 33.2% [23.3–67.1] vs. 13.4%[11.6–24.9] (p=0.04). Conclusions: Our results show that splenic CD8 T cells from RTX refractory patients express more HLA-DR, granzyme B and interferon-γ, whereas CCR7, CD62L, CD27 and CD28 are lower in comparison to untreated patients. This phenotype is consistent with effector T cells localizing in the red pulp as they lack CCR7 expression, and presumably playing a main role in splenic platelet destruction. Our results strongly support that platelet destruction is preferentially mediated by CD8 T cells rather than by the humoral response in some ITP patients, which may explain their unresponsiveness to rituximab. Disclosures: Off Label Use: Rituximab used during Immune Thrombocytopenia.

scholarly journals Interferon-Gamma Modulation of the Local T Cell Response to Alphavirus Encephalomyelitis

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 113 ◽  
Author(s):  
Victoria K. Baxter ◽  
Diane E. Griffin
Keyword(s):  
T Cells ◽  
T Cell ◽  
Interferon Gamma ◽  
Cd8 T Cells ◽  
Local Development ◽  
Granzyme B ◽  
T Cell Response ◽  
Viral Rna ◽  
Ifn Γ

Infection of mice with Sindbis virus (SINV) provides a model for examining the role of the immune response to alphavirus infection of the central nervous system (CNS). Interferon-gamma (IFN-γ) is an important component of this response, and we show that SINV-infected differentiated neurons respond to IFN-γ in vitro by induction of antiviral genes and suppression of virus replication. To determine the in vivo effects of IFN-γ on SINV clearance and T cell responses, C57BL/6 mice lacking IFN-γ or IFN-γ receptor-1 were compared to wild-type (WT) mice after intracranial SINV infection. In WT mice, IFN-γ was first produced in the CNS by natural killer cells and then by CD4+ and CD8+ T cells. Mice with impaired IFN-γ signaling initiated clearance of viral RNA earlier than WT mice associated with CNS entry of more granzyme B-producing CD8+ T cells. However, these mice established fewer CD8+ tissue-resident memory T (TRM) cells and were more likely to experience reactivation of viral RNA synthesis late after infection. Therefore, IFN-γ suppresses the local development of granzyme B-expressing CD8+ T cells and slows viral RNA clearance but promotes CD8+ TRM cell establishment.

The Mutated Region of Cytoplasmatic Nucleophosmine 1 (NPM1) Elicits Both CD4+ and CD8+ T Cell Responses

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2569-2569
Author(s):  
Jochen Greiner ◽  
Yoko Ono ◽  
Susanne Hofmann ◽  
Vanessa Schneider ◽  
Anita Schmitt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Healthy Volunteers ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Granzyme B ◽  
T Cell Responses ◽  
Interferon Γ ◽  
Cell Responses ◽  
Hla Dr

Abstract Abstract 2569 Introduction In AML, mutations in the nucleophosmin (NPM1) gene are one of the most frequent molecular alterations and predominantly occur in AML with normal cytogenetics. Patients with NPM1 mutation without FLT3-ITD mutation show a favourable prognosis of their disease. The functional role of mutated NPM1 for the improved clinical outcome is under evaluation. Immune responses might be involved in the clinical outcome of the disease. In this work, we demonstrate both CD4+ and CD8+ T cell responses against the mutated region of NPM1. Methods The entire amino acid sequences of the NPM1 wild type protein as well as of the mutated cytoplasmic NPM1 types A, B, C and D were screened for HLA-A*0201 binding T cell epitopes using the algorithms of the SYFPEITHI, the Rankpep and the HLA-Bind software programs. Ten peptides with most favourable characteristics were subjected to ELISpot analysis for interferon-γ and granzyme B in 22 healthy volunteers and 27 AML patients to test specific T cell responses of CD8+ T cells. Tetramer assays against the two most interesting epitopes have been performed and chromium release assays have been used to show the cytotoxicity of peptide-specific T cells to lyse T2 cells and leukemic blasts. Moreover, HLA-DR binding epitopes were screened in algorithmic analysis and HLA-DR*0701 binding peptides were exploited to stimulate CD4+ T cells. In the presence of overlapping peptide stimulated CD4+ T cells, NPM1-A specific CD8+ T cells revealed augmented interferon-γ and granzyme B secretion and up-regulation of intracellular interferon-γ. CD4+, CD4-CD8+, CD4-CD8- cell fractions were separated from PBMCs of HLA-A2+DR*0701+ healthy volunteers using a combination of CD4 and CD8 MicroBeads. Results Two epitopes (P3 and P9) derived from the NPM1-mutated protein showed specific T cell responses in healthy volunteers and AML patients. In NPM1-mutated AML patients 33% showed immune responses of CD8+ T cells against peptide P3 and 42% against peptide P9. Specific lysis was detected in chromium release assays NPM1 peptide-primed effector T cells generated from NPM1-mutated AML patients. Tetramer assays showed peptide-specific T cells. To obtain a robust and effective immune response against tumor cells, the activation of CD4 + helper T cells is crucial. Thus NPM1-peptide-A overlapping MHC class II epitopes were searched by primary structure analysis program. Based on plenary search, eight favourable overlapping peptides OL 1–8 were synthesized and exploited for CD4+ T cell stimulation. In granzyme B ELISPOT assay, OL8 co-pulsed NPM1-A CD8+ T cells indicated notable S.I., in contrast other OL1-7 disabled to increase granzyme B secretion. To ensure that Th1 cytokine secretion, under the condition of CD8+ and CD4+ T cells mixed culture, was resulted from NPM1-A CD8+ T cells but not HLA-DR epitope stimulated CD4+ T cells activation, HLA-A2 blocking effect was confirmed in ELISPOT assay. NPM1-A CD8+ T cells co-pulsed with OL6, 7 and 8 showed lesser interferon-γ secretion after HLA-A2 blocking antibody exposure as 73, 35 and 57%. Of note, 83–94% of granzyme B secretion levels were reduced by HLA-A2 blockade administration, and by which NPM1-A CD8+ T cells seemed to be the most probable IFN-gamma and granzyme B producers and CD4+ T cells to interfere with CD8+ T cells. Conclusion Taken together, mutated NPM1 is a promising target structure for specific immunotherapies in AML patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role ofIn VitroStimulation with Lipopolysaccharide on T-Cell Activation in HIV-Infected Antiretroviral-Treated Patients

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Camilla Tincati ◽  
Giusi M. Bellistrì ◽  
Giuseppe Ancona ◽  
Esther Merlini ◽  
Antonella d’Arminio Monforte ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Hiv Positive ◽  
Cd38 Expression ◽  
Hla Dr ◽  
Measured Flow

We investigated the effect of LPSin vitrostimulation on T-cell activation in HIV-infected patients with different CD4+ recovery on HAART. PBMCs from 30 HIV-positive, HAART-treated, aviremic individuals with different CD4+ reconstitution (Low Responders: CD4+ < 350/μL; Intermediate Responders: CD4+ 350–599/μL; High Responders: CD4+ ≥ 600/μL) were cultured with LPS and the proportion of HLA-DR/CD38- and Ki67-expressing CD4+/CD8+ T-cells was measured (flow cytometry). Upon LPS stimulation, significantly higher CD4+ and CD8+HLA-DR+ cells were shown in LR and IR versus HIV-negative controls. While no differences in the proportion of LPS-stimulated CD4+CD38+ cells were recorded amongst HIV-positive subgroups, CD8+CD38+ cells were more elevated in patients with lower CD4+ recovery on HAART (i.e., LR and IR). Uponin vitroLPS stimulation, HLA-DR and CD38 expression on T-cells are differentially regulated. While HLA-DR induction reflects impaired CD4+ reconstitution on HAART, cell-surface CD38 expression is increased only on CD8+ T-cells, allowing to speculate that the sole induction of CD38 on CD4+ cells may not be sufficient to depict LPS-driven immune activation in HIV.

561 DuoBody®-PD-L1×4–1BB (GEN1046) induces superior immune-cell activation, cytokine production and cytotoxicity by combining PD-L1 blockade with conditional 4–1BB co-stimulation

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A595-A595
Author(s):  
Alexander Muik ◽  
Isil Altintas ◽  
Rachelle Kosoff ◽  
Friederike Gieseke ◽  
Kristina Schödel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cytokine Production ◽  
Immune Cell ◽  
Anti Tumor Activity

BackgroundCheckpoint inhibitors targeting the PD-1/PD-L1 axis (CPI) have changed the treatment paradigm and prognosis for patients with advanced solid tumors; however, many patients experience limited benefit due to treatment resistance. 4-1BB co-stimulation can activate cytotoxic T-cell- and NK-cell-mediated anti-tumor immunity and has been shown to synergize with CPI in preclinical models. DuoBody-PD­L1×4-1BB is a first-in-class, Fc-silenced, bispecific next-generation checkpoint immunotherapy that activates T cells through PD-L1 blockade and simultaneous PD-L1-dependent 4-1BB co-stimulation. Here we present preclinical evidence for the mechanism of action of DuoBody-PD-L1×4-1BB, and proof-of-concept using mouse-reactive mbsAb-PD-L1×4-1BB in vivo.MethodsRNA sequencing analyses was performed on primary human CD8+ T cells that were co-cultured with PD-L1+ monocytes in the presence of anti-CD3/anti-CD28 and test compounds. T-cell proliferation and cytokine production were analyzed in primary human T-cell and mixed lymphocyte reaction (MLR) assays in vitro, and using patient-derived tumor-infiltrating lymphocytes (TILs). Cytotoxic activity was assessed in co-cultures of CLDN6+PD-L1+ MDA-MB-231 tumor cells and CLDN6-TCR+CD8+ T cells. Anti-tumor activity of mbsAb-PD-L1×4-1BB was tested in vivo using the CT26 mouse tumor model. Immunophenotyping of the tumor microenvironment (TME), tumor-draining lymph nodes (tdLNs) and peripheral blood was performed by flow cytometry.ResultsDuoBody-PD-L1×4-1BB significantly induced expression of genes associated with immune cell proliferation, migration and cytokine production in activated CD8+ T cells, which were not altered by CPI. DuoBody-PD-L1×4-1BB dose-dependently enhanced expansion of human TILs ex vivo. DuoBody-PD-L1×4-1BB dose-dependently enhanced T-cell proliferation and pro-inflammatory cytokine production in vitro (e.g. IFNγ and TNFα; in polyclonal and antigen-specific T-cell proliferation assays and MLR), which was dependent on crosslinking to PD-L1+ cells and superior to CPI or the combination of Fc-silenced PD-L1- and 4-1BB-specific antibodies. DuoBody-PD-L1x4-1BB induced upregulation of degranulation marker CD107a and granzyme B in CD8+ T cells, resulting in antigen-specific T-cell-mediated cytotoxicity of MDA-MB-231 tumor cells in vitro, superior to CPI. In mice bearing subcutaneous CT26 tumors, a model that was insensitive to PD-L1 blockade, mbsAb-PD-L1×4-1BB elicited tumor rejection in the majority of the mice at active dose levels and significantly improved survival. Dose-dependent anti-tumor activity was associated with expansion of tumor antigen-specific T cells in the blood and enhanced immune-cell activation in tdLNs and TME.ConclusionsCombining PD-L1 blockade with conditional 4-1BB co-stimulation using bispecific antibodies induced T-cell activation, expansion, and cytotoxic activity in vitro and potent anti-tumor activity in vivo superior to CPI. DuoBody-PD-L1×4-1BB is currently being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT03917381).Ethics ApprovalAll mice studies were performed by BioNTech SE at its research facilities in Germany, and the mice were housed in accordance with German federal and state policies on animal research. All experiments were approved by the regulatory authorities for animal welfare in Germany. The use of tumor tissue resections was approved by BioNTech SE‘s Ethics Board, approval number 837.309.12 (8410-F).

scholarly journals P862 Clinical benefit potentially evident with immunopharmacodynamic responses in prior-checkpoint failed metastatic melanoma patients treated with imprime PGG and pembrolizumab

2020 ◽  
Vol 8 (Suppl 1) ◽  
pp. A10.1-A10
Author(s):  
Anissa Chan ◽  
Nandita Bose ◽  
Nandita Bose ◽  
Nadine Ottoson ◽  
Xiaohong Qiu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Disease Control ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Clinical Benefit ◽  
Secondary Resistance ◽  
Enhanced Production ◽  
Hla Dr ◽  
Melanoma Patients

BackgroundCheckpoint inhibitor (CPI) monotherapy has revolutionized the treatment of melanoma, yet most patients are primary nonresponders or develop secondary resistance. Lack of antigen-specific T cell priming and/or immunosuppressive mechanisms leading to T cell exhaustion are critical cancer-extrinsic factors contributing to CPI resistance mechanisms. Immunotherapeutic agents capable of sparking de novo anti-tumor T cell responses or reinvigorating pre-existing exhausted T cell immunity could help reinstate the activity of CPI.MethodsOur Phase 2, multi-center, open label study, NCT02981303 in collaboration with Merck & Co., Inc., is evaluating Imprime PGG (Imprime), a novel yeast derived, Dectin-1 agonist, β-glucan PAMP in combination with pembrolizumab (KEYTRUDA®, pembro) in heavily CPI pre-treated melanoma patients (20 patients; 65% had >2 prior CPI regimens with 17/20 having previously progressed on pembro). Patients received Imprime (4 mg/kg) + pembro (200 mg) intravenously in a 3-week cycle. Here, we present the immunopharmacodynamic (IPD) responses elicited by Imprime and pembro in the peripheral blood of 19 patients.ResultsIn the intent-to-treat population (ITT; N=20), the disease control rate was 45% (1 CR and 8 SD), 6-month and 12-month OS rates were 65% and 45% respectively, and median OS (mOS) was 8.8 months. In the patients showing disease control, a significant increase in CH50, the classical pathway complement function (~0.7-2.6-fold), HLA-DR expression on classical monocytes (~0.61-1.94-fold) and reduction of frequency of PD-1+Tbet-EOMES+ exhausted CD8 T cells (~0.9-4-fold) was observed. Stimulation of peripheral blood mononuclear cells from a subset of patients by CD3/CD28 beads showed enhanced production of IL-2 and IFN-gamma in the CD8 T cells. Some of these IPD responses were also associated with 6-month landmark OS analyses. Additionally, whole blood gene expression analyses showed >2-fold upregulation of several myeloid and T cell activation genes including IFNg, CD83, IP-10, and IL-2RA. Enhanced OS was observed in patients with >1.3 fold increase in CH50 (8/19; HR 0.385; p=0.1) or >1.5-fold reduction in the frequency of exhausted CD8 T cells (8/19; HR 0.102; p=0.001). The IPD responses observed in the ITT population included formation of circulating immune complexes (peak levels ranging from ~4.5-16.1-fold) and production of complement activation protein SC5b9 (~3.4-25.6-fold), and increase in the frequency of HLA-DR+ myeloid cells (~0.43-3.71-fold).ConclusionsOverall, these data, albeit in a small population, demonstrate that Imprime/pembro combination can drive the innate/adaptive IPD responses that are critical for providing clinical benefit to the patients who have progressed through prior CPI treatments.Ethics ApprovalThe study was approved by central and local ethics committees depending on site requirements. The central IRB for the study is Western Institutional Review Board (WIRB), approval number 20162506; all sites received IRB approval before opening the study at the respective sites.

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