Abstract LB037:89ZED88082A PET imaging to visualize CD8+T cells in patients with cancer treated with immune checkpoint inhibitor

BackgroundNearly half of all U.S. oncology patients meet FDA eligibility criteria to receive treatment with an immune checkpoint inhibitor (ICI). With increasing use of ICIs, preventing, diagnosing and treating immune-related adverse events (irAEs) are urgent clinical challenges. Myocarditis is an uncommon irAE, affecting < 1% of ICI-treated patients, but is highly fatal, with a mortality rate of nearly 50%. Genetically altered Pdcd1-/-Ctla4± mice die prematurely and specifically due to myocarditis. This model recapitulates the clinical and pathological features of ICI-myocarditis, including abundant cardiac infiltrating CD8+ T cells. The potential autoantigen(s) involved in ICI-myocarditis are unknown for both human disease and our murine model.MethodsWe used Pdcd1-/-Ctla4± mice on the C57BL6 background as a model of ICI-myocarditis. Single cell RNA and T cell receptor (TCR) sequencing was performed on sorted CD45+ cardiac immune cells from four affected Pdcd1-/-Ctla4± mice compared to six healthy wild type mice. The most three clonal TCRs (TCR-A, B, C), derived from two independent Pdcd1-/-Ctla4± mice, were reconstructed using stiTChR and transduced into reporter T cell lines for antigen discovery. Alpha-myosin was selected as a candidate autoantigen due to lack of presentation in the thymus. Reporter TCR-A, B, and C cells were screened using a library of overlapping 20 amino acid peptides derived from alpha-myosin in co-culture with bone marrow derived dendritic cells.ResultsTreatment with anti-CD8, but not anti-CD4, depleting antibodies rescues survival of Pdcd1-/-Ctla4± mice. Furthermore, adoptive transfer of splenocytes from Pdcd1-/-Ctla4± mice with myocarditis to Rag1-/- recipient mice was sufficient to induce fatal myocarditis. Single cell RNA/TCR sequencing on the cardiac immune infiltrate of Pdcd1-/-Ctla4± mice identified highly activated, clonal CD8+ T cells as the dominant cell population. The TCR-A cell line, the most clonal TCR identified in single cell TCR sequencing, activates NFAT, NFkB, and AP-1 reporters in response to the alpha-myosin epitope VIQYFASI. The TCR-B and TCR-C cell lines activate their reporters in response to the alpha myosin peptide DALLVIQWNIRAFMGVKNWP, indicating that alpha-myosin is an autoantigen in this mouse model of ICI-myocarditis.ConclusionsClonal, activated CD8+ T cells are critical for the development of ICI-myocarditis. Alpha-myosin is an autoantigen recognized by the most clonal cardiac CD8+ T cells. Efforts are currently underway to determine whether human TCRs derived from ICI-myocarditis samples recognize similar antigens. These studies are the first to identify a candidate autoantigen in ICI-myocarditis and may yield new insights into irAE pathogenesis.Ethics ApprovalAll animal experiments were in accordance with the VUMC Institutional Animal Care and Use Committee (IACUC), protocol # M2000067

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Abstract 1044: Frequency of circulating CX3CR1+ CD8+ T cells to predict response to immune checkpoint inhibitor therapy

10.1158/1538-7445.am2020-1044 ◽

2020 ◽

Author(s):

Takayoshi Yamauchi ◽

Toshifumi Hoki ◽

Takaaki Oba ◽

Kristopher Attwood ◽

Sebastiano Battaglia ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Immune Checkpoint ◽

Immune Checkpoint Inhibitor ◽

Checkpoint Inhibitor ◽

Inhibitor Therapy ◽

Checkpoint Inhibitor Therapy

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Granzyme B PET Imaging of Immune Checkpoint Inhibitor Combinations in Colon Cancer Phenotypes

Molecular Imaging and Biology ◽

10.1007/s11307-020-01519-3 ◽

2020 ◽

Vol 22 (5) ◽

pp. 1392-1402

Author(s):

J. L. Goggi ◽

Y. X. Tan ◽

S. V. Hartimath ◽

B. Jieu ◽

Y. Y. Hwang ◽

...

Keyword(s):

Colon Cancer ◽

T Cells ◽

Pet Imaging ◽

Immune Checkpoint ◽

Immune Checkpoint Inhibitor ◽

Checkpoint Inhibitor ◽

Granzyme B ◽

Cell Populations ◽

Combination Therapies ◽

Cancer Phenotypes

Abstract Purpose Immune checkpoint inhibitor (ICI) monotherapy and combination regimens are being actively pursued as strategies to improve durable response rates in cancer patients. However, the biology surrounding combination therapies is not well understood and may increase the likelihood of immune-mediated adverse events. Accurate stratification of ICI response by non-invasive PET imaging may help ensure safe therapy management across a wide number of cancer phenotypes. Procedures We have assessed the ability of a fluorine-labelled peptide, [18F]AlF-mNOTA-GZP, targeting granzyme B, to stratify ICI response in two syngeneic models of colon cancer, CT26 and MC38. In vivo tumour uptake of [18F]AlF-mNOTA-GZP following ICI monotherapy, or in combination with PD-1 was characterised and correlated with changes in tumour-associated immune cell populations. Results [18F]AlF-mNOTA-GZP showed good predictive ability and correlated well with changes in tumour-associated T cells, especially CD8+ T cells; however, overall uptake and response to monotherapy or combination therapies was very different in the CT26 and MC38 tumours, likely due to the immunostimulatory environment imbued by the MSI-high phenotype in MC38 tumours. Conclusions [18F]AlF-mNOTA-GZP uptake correlates well with changes in CD8+ T cell populations and is able to stratify tumour response to a range of ICIs administered as monotherapies or in combination. However, tracer uptake can be significantly affected by preexisting phenotypic abnormalities potentially confusing data interpretation.

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2416PD1-blocking immune checkpoint inhibitor therapy for malignant melanoma induces left ventricular dysfunction

European Heart Journal ◽

10.1093/eurheartj/ehz748.0164 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

L Michel ◽

U B Hendgen-Cotta ◽

I Helfrich ◽

D Schadendorf ◽

T Rassaf ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Immune Checkpoint ◽

Immune Checkpoint Inhibitor ◽

Checkpoint Inhibitor ◽

Underlying Mechanism ◽

Left Ventricular ◽

Inhibitor Therapy ◽

Cardiotoxic Effect ◽

Checkpoint Inhibitor Therapy

Abstract Background Immune checkpoint inhibitor therapy has significantly improved treatment of advanced malignant diseases. However, patients receiving immune checkpoint inhibitor therapy with programmed death 1 (PD1) blocking agents are at risk for cardiotoxicity with high mortality. The underlying pathomechanisms have not yet been elucidated. Purpose This study aims to evaluate the cardiotoxic effect of PD1-blocking agents and its underlying mechanism with focus on myocardial inflammation and metabolism. Methods A transplantable melanoma mouse model was used to study PD1 blocking therapy in a preclinical setting. In brief, mice were subcutaneously transplanted with a melanoma cell line and treated with anti-PD1 antibodies or non-specific immunoglobulin control for 14 days. Murine transthoracic echocardiography including strain analysis was conducted to assess left ventricular (LV) function. Pressure/volume analysis was performed using a micro-tip catheter introduced into the LV via the right commune carotid artery. Inotropic stress was induced by dobutamine. Myocardial immune cell infiltration and expression of PD1/PD-L1 was assessed using flow cytometry. A combined approach for mass spectrometry-guided profiling of proteome, lipids and metabolites was applied to evaluate changes in cardiomyocyte function and metabolism. Results Reduced tumor size in anti-PD1-treated animals confirmed response to treatment (n=7; p=0.018). Echocardiographic examination revealed reduced LV ejection fraction (EF) (n=7–8; p=0.001) and reduced global radial strain in anti-PD1-treated mice compared to control littermates (n=3–4; p=0.004). Remarkably, pressure/volume catheterization indicated reduced EF, stroke volume and stroke work under dobutamine stress in anti-PD1-treated mice (p=0.013; n=3–4). Anti-PD1 treatment was associated with a 2-fold elevated level of CD4+ and CD8+ T-cells in murine hearts (n=8; p=0.009 and p=0.049). CD44 expression was upregulated in CD8+ T-cells of anti-PD1-treated animals (n=8; p=0.024). Proteomics revealed downregulation of proteins critical for cardiomyocyte contraction, e.g. ryanodine receptor 2 and L-type calcium channel beta 2 (n=4; p<0.05). Analysis of metabolites and lipids indicated dysfunctional energy metabolism. To identify a potential underlying mechanism, expression of PD1 and its ligand PD-L1 on cardiac cell populations was examined. PD-L1 was mainly expressed on cardiac endothelial cells while PD1 was expressed on 10–20% of murine cardiomyocytes (n=12; p<0.001 and p=0.004). Conclusion The obtained results point towards a cardiotoxic effect of PD1 blocking therapy with severely disturbed cardiac function and disrupted cardiomyocyte functional integrity. Myocardial expression of the PD1 receptor could mediate the observed effect. This could potentially promote the development of PD1 immune checkpoint inhibitor-associated myocarditis in patients. Acknowledgement/Funding IFORES research grant of the Medical Faculty, University Duisburg-Essen, Essen, Germany

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Deep immune profiling reveals targetable mechanisms of immune evasion in immune checkpoint inhibitor-refractory glioblastoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-002181 ◽

2021 ◽

Vol 9 (6) ◽

pp. e002181

Author(s):

Erin F Simonds ◽

Edbert D Lu ◽

Oscar Badillo ◽

Shokoufeh Karimi ◽

Eric V Liu ◽

...

Keyword(s):

T Cells ◽

Tumor Cells ◽

Immune Evasion ◽

Immune Checkpoint ◽

Immune Checkpoint Inhibitor ◽

Checkpoint Inhibitor ◽

Prolonged Survival ◽

Tumor Associated Macrophages ◽

The Brain ◽

Immune Profiling

BackgroundGlioblastoma (GBM) is refractory to immune checkpoint inhibitor (ICI) therapy. We sought to determine to what extent this immune evasion is due to intrinsic properties of the tumor cells versus the specialized immune context of the brain, and if it can be reversed.MethodsWe used CyTOF mass cytometry to compare the tumor immune microenvironments (TIME) of human tumors that are generally ICI-refractory (GBM and sarcoma) or ICI-responsive (renal cell carcinoma), as well as mouse models of GBM that are ICI-responsive (GL261) or ICI-refractory (SB28). We further compared SB28 tumors grown intracerebrally versus subcutaneously to determine how tumor site affects TIME and responsiveness to dual CTLA-4/PD-1 blockade. Informed by these data, we explored rational immunotherapeutic combinations.ResultsICI-sensitivity in human and mouse tumors was associated with increased T cells and dendritic cells (DCs), and fewer myeloid cells, in particular PD-L1+ tumor-associated macrophages. The SB28 mouse model of GBM responded to ICI when grown subcutaneously but not intracerebrally, providing a system to explore mechanisms underlying ICI resistance in GBM. The response to ICI in the subcutaneous SB28 model required CD4 T cells and NK cells, but not CD8 T cells. Recombinant FLT3L expanded DCs, improved antigen-specific T cell priming, and prolonged survival of mice with intracerebral SB28 tumors, but at the cost of increased Tregs. Targeting PD-L1 also prolonged survival, especially when combined with stereotactic radiation.ConclusionsOur data suggest that a major obstacle for effective immunotherapy of GBM is poor antigen presentation in the brain, rather than intrinsic immunosuppressive properties of GBM tumor cells. Deep immune profiling identified DCs and PD-L1+ tumor-associated macrophages as promising targetable cell populations, which was confirmed using therapeutic interventions in vivo.

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