524 Tim-4+ resident macrophages impair anti-tumor immunity in the serous body cavities By sequestering viable and cytotoxic CD8+ T cells expressing high levels of phosphatidylserine

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A560-A560
Author(s):  
Andrew Chow ◽  
Sara Schad ◽  
Michael Green ◽  
Matthew Hellmann ◽  
Nicholas Ceglia ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Peritoneal Carcinomatosis ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Pleural Effusions ◽  
Phosphatidylserine Receptor

BackgroundMalignant pleural effusions and peritoneal carcinomatosis are associated with poor outcomes in patients with cancer.1–3 Macrophages in these serous body cavities express the phosphatidylserine receptor Tim-4.4–8 Prior reports demonstrated that Tim-4 abrogation is associated with improved anti-tumor activity.9–11 Whether macrophages expressing Tim-4 contribute to immunosuppression in the serous body cavities has not been previously investigated.MethodsWe retrospectively annotated sites of metastases in 500 patients with lung cancer and assessed for clinical outcomes. Utilizing a combination of flow cytometry, immunohistochemistry, and antibody biodistribution assays, we surveyed for Tim-4 expression across various tissues and cell types. We performed flow cytometry on 55 consecutive pleural and peritoneal effusions from patients with lung cancer. We utilized murine models of peritoneal carcinomatosis to determine whether Tim-4 abrogation could enhance the anti-tumor efficacy of anti-PD-1 therapy. We characterized CD8+ T cells with high levels of phosphatidylserine (PShigh) with flow cytometry, cytotoxicity assays, and paired single cell RNA and TCR sequencing. Confocal microscopy was utilized to visualize interactions between Tim-4+ macrophages and PShigh CD8+ T cells.ResultsMetastatic disease involvement of the pleural or peritoneal cavity was associated with reduced response rate and progression-free and overall survival. We demonstrate that Tim-4 is highly expressed on pleural and peritoneal macrophages and other select resident macrophages, but not on monocytes, tumor-associated macrophages, or tumor cells in mice and humans. High levels of Tim-4 on macrophages from fluid biospecimens is associated with reduced levels CD39+ CD8+ T cells, which comprise the tumor-reactive portion of CD8+ T lymphocytes. In order to further elucidate the mechanism of Tim-4+ macrophage-mediated immunosuppression, we established a murine model of peritoneal carcinomatosis with MC38 and CT26 colon carcinoma. Genetic or pharmacologic abrogation of Tim-4 improved the efficacy of anti-PD-1 therapy and was associated with enhanced CD39+ CD8+ T cell numbers. In parallel, we observed in mice and humans that CD8+ T cell activation results in PS upregulation despite not undergoing cell death. PShigh CD8+ T cells expressed genes associated with cytotoxicity, activation/exhaustion, and proliferation, and mediated greater cytotoxicity. Mechanistic studies revealed that Tim-4 mediates sequestration of PShigh CD8+ T cells by macrophages which subsequently impedes CD8+ T cell cytotoxicity of tumor cells.Abstract 524 Figure 1After activation by antigen-presenting cells in the lymph nodes, viable CD8+ T cells express high levels of phosphatidylserine, which coincides with a highly proliferative and cytotoxic state. As they migrate towards tumors cells in the serous body cavities, they are sequestered by Tim-4+ resident macrophages which impede their anti-tumor cytotoxicity. Tim-4 abrogation can alleviate this sequestration and enhance anti-tumor immunityConclusionsWe demonstrate that Tim-4+ resident macrophages impair anti-tumor CD8+ T cell immunity in the serous body cavities and Tim-4 blockade represents on a novel therapeutic strategy to overcome resistance to immune checkpoint blockade (figure 1).Ethics ApprovalThe retrospective clinical analysis was approved by Memorial Sloan Kettering Cancer Center IRB #16-1566. The human biospecimen analyses were approved by Memorial Sloan Kettering Cancer Center IRB #06-107 and 14-091.ReferencesPorcel JM, et al., Clinical features and survival of lung cancer patients with pleural effusions. Respirology 2015;20:654–659.Donnenberg AD, Luketich JD, Dhupar R, Donnenberg VS. Treatment of malignant pleural effusions: the case for localized immunotherapy. J Immunother Cancer 2019;7:110.Morano WF, et al., Intraperitoneal immunotherapy: historical perspectives and modern therapy. Cancer Gene Ther 2016;23:373–381.Bain CC, et al., Long-lived self-renewing bone marrow-derived macrophages displace embryo-derived cells to inhabit adult serous cavities. Nat Commun 2016;7:ncomms11852.Wong K, et al., Phosphatidylserine receptor Tim-4 is essential for the maintenance of the homeostatic state of resident peritoneal macrophages. Proc Natl Acad Sci U S A 2010;107:8712–8717.Miyanishi M, et al., Identification of Tim4 as a phosphatidylserine receptor. Nature 2007;450:435–439.Rodriguez-Manzanet R, et al. T and B cell hyperactivity and autoimmunity associated with niche-specific defects in apoptotic body clearance in TIM-4-deficient mice. Proc Natl Acad Sci U S A 2010;107:8706–8711.Kobayashi N, et al. TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells. Immunity 2007;27:927–940.LD Cunha et al. LC3-Associated phagocytosis in myeloid cells promotes tumor immune tolerance. Cell 2018;175:429–441 e416.Baghdadi M, et al, TIM-4 glycoprotein-mediated degradation of dying tumor cells by autophagy leads to reduced antigen presentation and increased immune tolerance. Immunity 2013;39:1070–1081.Baghdadi M, et al. Combined blockade of TIM-3 and TIM-4 augments cancer vaccine efficacy against established melanomas. Cancer Immunol Immunother 2013;62:629–637.

scholarly journals Irradiation Suppresses IFNγ-Mediated PD-L1 and MCL1 Expression in EGFR-Positive Lung Cancer to Augment CD8+ T Cells Cytotoxicity

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2515
Author(s):  
Chun-I. Wang ◽  
Yi-Fang Chang ◽  
Zong-Lin Sie ◽  
Ai-Sheng Ho ◽  
Jung-Shan Chang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lung Cancer ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Irradiation Effect ◽  
Western Blots ◽  
Stat3 Phosphorylation

Tumor cells express immune checkpoints to exhaust CD8+ T cells. Irradiation damages tumor cells and augments tumor immunotherapy in clinical applications. However, the radiotherapy-mediated molecular mechanism affecting CD8+ T cell activity remains elusive. We aimed to uncover the mechanism of radiotherapy augmenting cytotoxic CD8+ T cells in non-small-cell lung cancer (NSCLC). EGFR-positive NSCLC cell lines were co-cultured with CD8+ T cells from healthy volunteers. Tumor cell viability and apoptosis were consequently measured. IFNγ was identified secreted by CD8+ T cells and PBMCs. Therefore, RNAseq was used to screen the IFNγ-mediated gene expression in A549 cells. The irradiation effect to IFNγ-mediated gene expression was investigated using qPCR and western blots. We found that the co-culture of tumor cells stimulated the increase of granzyme B and IFNγ in CD8+ T, but A549 exhibited resistance against CD8+ T cytotoxicity compared to HCC827. Irradiation inhibited A549 proliferation and enhanced apoptosis, augmenting PBMCs-mediated cytotoxicity against A549. We found that IFNγ simultaneously increased phosphorylation on STAT1 and STAT3 in EGFR-positive lung cancer, resulting in overexpression of PD-L1 (p < 0.05). In RNAseq analysis, MCL1 was identified and increased by the IFNγ-STAT3 axis (p < 0.05). We demonstrated that irradiation specifically inhibited phosphorylation on STAT1 and STAT3 in IFNγ-treated A549, resulting in reductions of PD-L1 and MCL1 (both p < 0.05). Moreover, knockdowns of STAT3 and MCL1 increased the PBMCs-mediated anti-A549 effect. This study demonstrated that A549 expressed MCL1 to resist CD8+ T cell-mediated tumor apoptosis. In addition, we found that irradiation suppressed IFNγ-mediated STAT3 phosphorylation and PD-L1 and MCL1 expression, revealing a potential mechanism of radiotherapy augmenting immune surveillance.

Generation of CD8 T Cell-Mediated Protective Immunity against Tumor Escapees

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2623-2623 ◽  
Author(s):  
Bindu Varghese ◽  
Behnaz Taidi ◽  
Adam Widman ◽  
James Do ◽  
R. Levy
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
B Cell Lymphoma ◽  
Cd8 T Cell

Abstract Introduction: Anti-idiotype antibodies against B cell lymphoma have shown remarkable success in causing tumor regression in the clinic. In addition to their known ability to mediate ADCC, anti-idiotype antibodies have also been shown to directly inhibit the proliferation of tumor cells by sending negative growth signals via the target idiotype. However, further studies to investigate this mechanism have been hindered by the failure of patient tumor cells to grow ex vivo. Methods and Results: In order to study this phenomenon further, we developed an antibody against the idiotype on an A20 mouse B lymphoma cell line. A radioactive thymidine incorporation assay showed decreased A20 cell proliferation in the presence of the anti-id antibody ex vivo. In vivo, when mice were treated intraperitoneally (i.p.) with 100 μg of antibody 3 hours post-tumor inoculation (1×106 A20 subcutaneously (s.c.)), tumor growth was delayed for greater than 40 days after which the tumor began to grow once again. Further analysis of these escaping tumor cells by flow cytometry showed that that the tumor cells escaped the antibody-mediated immune response by down-regulating expression of idiotype and IgG on their surfaces although the cells retained idiotype expression intracellularly. This down-regulation of surface idiotype rendered the tumor cells resistant to both ADCC and signaling-induced cell death. The addition of an immunostimulatory bacterial mimic (CpG-DNA; 100 μg × 5 intratumoral (i.t.) injections; Days 2, 3 4, 6 & 8) to antibody therapy (Day 0; 100 μg i.p.) cured large established tumors (Day 0 = 1 cm2) and prevented the occurrence of tumor escapees (p&lt;0.0001). Antibody plus CpG combination therapy in tumor-bearing mice deficient for CD8+ T cells demonstrated the critical role of CD8+ T cells in A20 tumor eradication (p&lt;0.005). Depletion of CD4+ T cells was found to have no significant impact on the therapy. We also found that when mice were inoculated with two tumors and treated with anti-idiotype antibody (i.p.) followed by intratumoral CpG in just one tumor (Day 0=1 cm2; anti-idiotype antibody 100 μg Day 0; 100 μg CpG Days 2, 3, 4, 6 & 8), untreated tumors regressed just as well as CpG-treated tumors indicating a systemic anti-tumor immune response was generated. Conclusion: Anti-idiotype therapy, although effective in delaying tumor growth, frequently generates antigen-loss variants. However, we found that when anti-idiotype antibodies were combined with CpG, even large established tumors were cured due to systemic CD8+ T cell-dependent tumor immunity. Rather than simply mediating ADCC against a single tumor antigen, which requires the constant infusion of antibody to hamper tumor growth, we hypothesize a cytotoxic T-cell response against many tumor antigens was also generated. Such a diverse T-cell repertoire can prevent the emergence of tumor escapees and collectively provide long-lasting tumor protection. These pre-clinical results suggest that anti-tumor antibodies combined with CpG warrant further study in patients with B cell lymphoma.

scholarly journals Regulating immune memory and reversing tumor thermotolerance through a step-by-step starving-photothermal therapy

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Lihua Luo ◽  
Bing Qin ◽  
Mengshi Jiang ◽  
Lin Xie ◽  
Zhenyu Luo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Photothermal Therapy ◽  
Cd8 T Cell ◽  
Immune Memory ◽  
In Situ Vaccine

Abstract Background Photothermal therapy (PTT) is a highly effective treatment for solid tumors and can induce long-term immune memory worked like an in situ vaccine. Nevertheless, PTT inevitably encounters photothermal resistance of tumor cells, which hinders therapeutic effect or even leads to tumor recurrence. Naïve CD8+ T cells are mainly metabolized by oxidative phosphorylation (OXPHOS), followed by aerobic glycolysis after activation. And the differentiate of effector CD8+ T cell (CD8+ Teff) into central memory CD8+ T cell (CD8+ TCM) depends on fatty acid oxidation (FAO) to meet their metabolic requirements, which is regulated by adenosine monophosphate activated protein kinase (AMPK). In addition, the tumor microenvironment (TME) is severely immunosuppressive, conferring additional protection against the host immune response mediated by PTT. Methods Metformin (Met) down-regulates NADH/NADPH, promotes the FAO of CD8+ T cells by activating AMPK, increases the number of CD8+ TCM, which boosts the long-term immune memory of tumor-bearing mice treated with PTT. Here, a kind of PLGA microspheres co-encapsulated hollow gold nanoshells and Met (HAuNS-Met@MS) was constructed to inhibit the tumor progress. 2-Deoxyglucose (2DG), a glycolysis inhibitor for cancer starving therapy, can cause energy loss of tumor cells, reduce the heat stress response of tumor cell, and reverse its photothermal resistance. Moreover, 2DG prevents N-glycosylation of proteins that cause endoplasmic reticulum stress (ERS), further synergistically enhance PTT-induced tumor immunogenic cell death (ICD), and improve the effect of immunotherapy. So 2DG was also introduced and optimized here to solve the metabolic competition among tumor cells and immune cells in the TME. Results We utilized mild PTT effect of HAuNS to propose an in situ vaccine strategy based on the tumor itself. By targeting the metabolism of TME with different administration strategy of 2DG and perdurable action of Met, the thermotolerance of tumor cells was reversed, more CD8+ TCMs were produced and more effective anti-tumor was presented in this study. Conclusion The Step-by-Step starving-photothermal therapy could not only reverse the tumor thermotolerance, but also enhance the ICD and produce more CD8+ TCM during the treatment. Graphical Abstract

CTIM-11. PHASE 2 STUDY OF SL-701, A NOVEL IMMUNOTHERAPY, IN ADULTS WITH RECURRENT GBM: A HIGH PARAMETER FLOW CYTOMETRY ANALYSIS OF CD8+ T CELLS AND POTENTIAL IMPLICATIONS FOR PATIENT ENRICHMENT STRATEGIES

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi51-vi51
Author(s):  
David Peereboom ◽  
Ross Lindsay ◽  
Michael Badruddoja ◽  
L Burt Nabors ◽  
Priya Kumthekar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Clinical Outcome ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Phase 2 ◽  
Duration Of Response ◽  
Recurrent Gbm

Abstract Treatment of glioblastoma (GBM) remains a critical challenge and unmet medical need due to limited treatment options. SL-701 is a novel immunotherapy comprised of synthetic peptides designed to elicit a target-specific anti-tumor immune response against the GBM antigens IL-13Rα2, ephrinA2, and survivin. A multicenter, 2-stage, phase 2 clinical trial (NCT02078648) that evaluated the safety and efficacy of SL-701 in 74 adults with recurrent GBM was previously reported. This report describes preliminary data to suggest a correlation of immunocompetence to clinical outcome. In stage 2 (SL-701 + bevacizumab + poly-ICLC) the overall survival at 12 months was 50%. Two of 28 patients enrolled in stage 2 achieved CR (duration of response: 7.8 and 8.8 months) and 2 achieved PR (duration of response: 7.9 and 8.8 months). In a preliminary analysis to assess CD8+ T-cell responses, long-term survivors were comprised largely of subjects with an SL-701-induced target-specific CD8+ T-cell response, indicating a potential correlation of immunocompetence to clinical outcome. By week 24, SL-701-induced target-specific CD8+ T cells expressing IFNg were detected in 8 of 27 patients (30%) who had sufficient samples, with co-expression of PD-1, TIM3, and LAG3 detected in 4 patients. To further understand the T-cell response to SL-701, deep sequencing of target-specific CD8+ T cells using whole transcriptome-based molecular cytometry and high parameter (25+ color) flow cytometry is currently underway and updated data will be reported.

Combining Early Heat Shock Protein Vaccination with Directed IL-2 Leads to Effective Anti-Tumor Immunity in Autologous Hematopoietic Cell Transplantation Recipients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 998-998
Author(s):  
Robert G. Newman ◽  
Eckhard R. Podack ◽  
Robert B. Levy
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Hematopoietic Cell Transplantation ◽  
Cd8 T Cell ◽  
Tumor Bearing ◽  
Donor T Cells ◽  
Vaccine Site ◽  
Autologous Hematopoietic Cell Transplantation

Abstract Abstract 998 Tumor relapse is still the major cause of morbidity and mortality in patients with hematologic cancers that undergo aggressive chemo-radiotherapy followed by autologous hematopoietic cell transplantation (auto-HCT). Hence, there is a critical need for new anti-tumor therapies. Heat shock protein (HSP) based vaccines elicit innate and adaptive immune responses in murine studies and have shown promise in clinical trials. The pre-clinical studies here investigated the efficacy of vaccination with tumor cells secreting the HSP fusion gp96-Ig together with directed IL-2 in tumor bearing auto-HCT recipients. To mimic clinical T cell replete auto-HCT, transplanted donor T cells were obtained from congenic tumor bearing mice (C57BL/6 CD45.2+ CD90.1+) that had been previously inoculated intraperitoneally (ip) with 4×106 OVA expressing lymphoma cells (E.G7). Some of these donor mice received 0.5×106 CD8 T cells specific for OVA257–264 (OT-I) to allow for tumor antigen specific T cell monitoring. Three weeks later, T cells were harvested from these animals bearing progressively growing tumor for use in T cell replete auto-HCT. Recipient mice (C57BL/6 CD45.2+ CD90.2+) received 9.5 Gy TBI with subsequent infusion of 5×106 congenic T cell depleted bone marrow cells (C57BL/6 CD45.1+ CD90.2+) supplemented with 2×106 enriched T cells from the tumor bearing donors. The following day, recipients were inoculated ip with 1×105 viable E.G7 lymphoma cells. Based on our prior findings, a multiple vaccination protocol was employed utilizing 1×107 irradiated E.G7 cells transfected to secrete the HSP fusion gp96-Ig (E.G7-gp96-Ig). Some recipients were administered IL-2 via specific antibody-cytokine complexes comprised of IL-2 and αIL-2 mAb clone S4B6 (IL-2/αIL-2CD122). This specific IL-2 complex has been shown to interact with cells expressing the β chain (CD122) of the IL-2 receptor, such as memory CD8 T cells and NK cells, but not with cells expressing the α chain (CD25). Compared to recipients of T cell replete auto-HCT vaccinated with parental E.G7 tumor cells who exhibited virtually no increase in antigen-specific CD8 T cells, marked expansion was detected in the blood after 2 vaccinations with E.G7-gp96-Ig, i.e. within 1 week of auto-HCT. This response reached a plateau after 3 vaccinations, and persisted throughout the 5 vaccine protocol. To quantitate this vaccine induced CD8 T cell expansion, analysis of the vaccine site, splenic and lymph node compartments was performed following 3 vaccinations, i.e. 2 weeks post-HCT. In contrast to the modest 25× increase observed after vaccination with parental E.G7 cells, a 175× expansion was detected following E.G7-gp96-Ig vaccination (6.8×106 vs. 3.8×104 input). Moreover, 75% of these gp96-Ig expanded CD8 T cells at the vaccine site were bifunctional, expressing IFN-γ and TNF-α following antigen specific stimulation ex vivo. Strikingly, combined treatment with vaccine cells secreting gp96-Ig together with IL-2/αIL-2CD122 complex resulted in a 1000× enhancement of antigen specific CD8 T cell numbers in all compartments analyzed. Tumor bearing auto-HCT recipients exhibited a median survival time (MST) of 1 month if not vaccinated or if vaccinated with parental E.G7 cells (Figure). However, vaccination with E.G7-gp96-Ig extended the MST by more than 2 weeks and ∼20% of recipients survived long term (>100 days). This effect was dependent on T cells since gp96-Ig vaccination alone without donor T cells resulted in no MST extension. Combination therapy with tumor cells secreting gp96-Ig and IL-2/αIL-2CD122 complex markedly elevated total CD8 T cells as well as NK cells at the vaccine site and in secondary lymphoid tissues, two populations that have been shown to facilitate HSP based vaccines. Notably, this strategy resulted in a MST >100 days with ∼60% of mice surviving indefinitely. We propose that 3 components are required together with auto-HCT to avoid relapse related mortality: (1) transplanted autologous T cells, (2) a pan-antigen vaccination approach that induces potent antigen presentation and activation of multiple antigen specific T cells, i.e. tumor cells secreting gp96-Ig, and (3) an adjuvant that potentiates this vaccine induced response, i.e. IL-2 delivered in the form of an antibody-cytokine complex. In total, this combinatorial protocol represents a promising regimen that could be translated into the clinic for patients with hematologic cancers. Disclosures: Podack: Heat Biologics, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals 303 Non-functional T cell responses in non-small cell lung cancer are induced during tumor antigen-specific T cell priming in the mediastinal lymph node

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A326-A326
Author(s):  
Brendan Horton ◽  
Duncan Morgan ◽  
Elen Torres-Mejia ◽  
Maria Zagorulya ◽  
Vidit Bhandarkar ◽  
...  
Keyword(s):  
Lung Cancer ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Lung Tumor ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Lung Tumors ◽  
Tumor Control ◽  
Cell Responses

BackgroundIn non-small cell lung cancer (NSCLC), response to checkpoint blockade therapy (CBT) is associated with tumor-infiltrating CD8+ T cells, but not all T cell-infiltrated tumors respond to CBT. The subgroup of T cell-infiltrated but CBT-resistant tumors has been clinically described as containing ”non-functional” T cell responses. Mechanisms governing the generation of non-functional T cell responses remain poorly understood, and treatment options for this subgroup are limited.MethodsWe utilized a transplantable, syngeneic murine NSCLC cell line derived from an autochthonous NSCLC driven by KrasG12D expression and p53 deletion (KP cell line) to model non-functional T cell responses. To study antigen-specific responses, we engineered KP cells to express the model CD8+ T cell antigen SIY for certain experiments. CBT consisted of combined anti-CTLA-4 and anti-PD-L1 therapy.ResultsOrthotopic KP lung tumors failed to respond to CBT, but KP flank tumors were controlled by CBT. Lung and flank tumors contained activated CD8+ T cells, providing a platform to compare functional and non-functional CD8+ T cell responses in NSCLC. Single-cell RNA sequencing revealed that lung tumor-infiltrating CD8+ T cells lacked effector and exhaustion molecules despite clonal expansion. Analysis of antigen-specific CD8+ T cells revealed that this lung cancer-specific T cell dysfunction was established during priming in lung-draining mediastinal lymph nodes (mLN) despite robust T cell proliferation. RNA sequencing and flow cytometry of antigen-specific CD8+ T cells found that T cells primed in the mLN underwent blunted effector differentiation characterized by a lack of effector molecules CD25, Granzyme B, and TIM-3, but retention of TCF-1. This phenotype persisted in lung tumors, consistent with our initial observations of absent effector and exhaustion molecule expression. Many CD8+ T cells from NSCLC patients expressed an analogous gene expression program distinct from T cell exhaustion. TCF-1+ CD8+ T cells in lung tumors did not mediate tumor control and failed to differentiate into effector cells after CBT. To investigate alternative therapeutic strategies of reinvigorating lung tumor-reactive T cells, we focused on IL-2 and IL-12, as expression of their receptors was reduced in mLN-primed T cells. Administering recombinant IL-2 and IL-12 was sufficient to restore effector T cell differentiation, induce lung tumor control, and significantly extend survival of lung tumor-bearing mice.ConclusionsOur results suggest that non-functional CD8+ T cell responses in NSCLC arise from failed effector T cell differentiation during priming. Transient combination therapy with IL-2 and IL-12 overcomes this dysfunctional state to induce protective T cell responses in CBT-resistant tumors.Ethics ApprovalAll mouse experiments were approved by MIT’s Committee on Animal Care (CAC) - DHHS Animal Welfare Assurance # D16-00078

scholarly journals Irradiation Mediates IFNα and CXCL9 Expression in Non-Small Cell Lung Cancer to Stimulate CD8+ T Cells Activity and Migration toward Tumors

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1349
Author(s):  
Chun-Chia Cheng ◽  
Yi-Fang Chang ◽  
Ai-Sheng Ho ◽  
Zong-Lin Sie ◽  
Jung-Shan Chang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Nuclear Imaging ◽  
Cd8 T Cell ◽  
Small Cell Lung ◽  
T Cell Migration

Irradiation-broken DNA fragments increase type I interferon and chemokines secretion in tumor cells. Since radiotherapy may augment tumor immunotherapy, we hypothesize that the chemokines increased by irradiation could recruit CD8+ T cells to suppress tumor proliferation. This study intended to unveil the secreted factors activating and recruiting CD8+ T cells in non-small-cell lung cancer (NSCLC). EGFR-positive A549 was selected and treated by X-irradiation (IR) to identify the overexpression of chemokines associated to CD8+ T cell cytotoxicity and recruitment. A transwell assay with Alexa 488-labeled CD8+ T cells was used to evaluate CD8+ T cell motility in vitro. A nuclear imaging platform by In111-labeled nivolumab was used to track CD8+ T cells homing to tumors in vivo. The activation markers GZMB, PRF-1, and IFNγ, migration marker CD183 (CXCR3), and inhibitory marker CD274 (PD-1), were measured and compared in CD8+ T cells with A549 co-cultured, chemokines treated, and patients with late-stage lung cancer. We found that IR not only suppressed A549 proliferation but also induced IFNα and CXCL9 expression (p < 0.05). IFNα majorly increased IFNγ levels in CD8+ T cells (p < 0.05) and synergistically with CXCL9 enhanced CD8+ T cell migration in vitro (p < 0.05). We found that CXCR3 and PD-1 were down-regulated and up-regulated, respectively, in the peripheral blood CD8+ T cells in patients with lung cancer (n = 4 vs. healthy n = 3, both p < 0.05), which exhibited reduction of cell motility (p < 0.05). The in vivo nuclear imaging data indicated highly CD8+ T cells migrated to A549-induced tumors. In addition, we demonstrated that healthy PBMCs significantly suppressed the parallel tumor growth (p < 0.05) and the radioresistant tumor growth in the tumor xenograft mice (p < 0.05), but PBMCs from patients with lung cancer had lost the anti-tumor capacity. We demonstrated that IR induced IFNα and CXCL9 expression in A549 cells, leading to CD8+ T cell migration. This study unveiled a potential mechanism for radiotherapy to activate and recruit CD8+ T cells to suppress lung tumors.

scholarly journals Regulating Immune Memory and Reversing Tumor Thermotolerance through a Step-by-Step Starving-Photothermal therapy

2021 ◽  
Author(s):  
Lihua Luo ◽  
Bing Qin ◽  
Mengshi Jiang ◽  
Lin Xie ◽  
Zhenyu Luo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Photothermal Therapy ◽  
Aerobic Glycolysis ◽  
Cd8 T Cell ◽  
Immune Memory

Abstract Background: Photothermal therapy (PTT) is a highly effective treatment for solid tumorsand can induce long-term immune memory worked like an in situ vaccine. Nevertheless, PTT inevitably encounters photothermal resistance of tumor cells, which hinders therapeutic effect or even leads to tumor recurrence. Naïve CD8+T cells are mainly metabolized by oxidative phosphorylation (OXPHOS), followed by aerobic glycolysis after activation. And the differentiate of effector CD8+ T cell (CD8+Teff) into central memory CD8+ T cell (CD8+TCM) depends on fatty acid oxidation (FAO) to meet their metabolic requirements, which is regulated by adenosine monophosphate activated protein kinase (AMPK). In addition, the tumor microenvironment (TME) is severely immunosuppressive, confering additional protection against the host immune response mediated by PTT.Methods: Metformin (Met) down-regulates NADH/NADPH, promotes the FAO of CD8+T cells by activating AMPK, increases the number of CD8+TCM, which boosts the long-term immune memory of tumor-bearing mice treated with PTT. Here, a kind of PLGA microspheres co-encapsulated hollow gold nanoshells and Met (HAuNS-Met@MS) was constructed to inhibit the tumor progress. 2-Deoxyglucose (2DG), a glycolysis inhibitor for cancer starving therapy, can cause energy loss of tumor cells, reduce the heat stress response of tumor cell, and reverse its photothermal resistance. Moreover, 2DG prevents N-glycosylation of proteins that cause endoplasmic reticulum stress (ERS), further synergistically enhance PTT-induced tumor immunogenic cell death (ICD), and improve the effect of immunotherapy. So 2DG was also introduced and optimized here to solve the metabolic competition among tumor cells and immune cells in the TME.Results: We utilized mild PTT effect of HAuNS to propose an in situ vaccine strategy based on the tumor itself. By targeting the metabolism of TME with different administration strategy of 2DG and perdurable action of Met, the thermotolerance of tumor cells was reversed, more CD8+TCMs were produced and more effective anti-tumor was presented in this study.Conclusion: The Step-by-Step starving-photothermal therapy could not only reverse thetumor thermotolerance, but also enhance the ICD and produce more CD8+TCM during the treatment.

Blocking VIP Signaling Abrogates Upregulation Of PD1 and Increases Proliferation Of Allo-Reactive T-Cells In a Mixed Lymphocyte Reaction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1041-1041
Author(s):  
Emily R Summerbell ◽  
Cynthia R. Giver ◽  
Sravanti Rangaraju ◽  
Katarzyna Anna Darlak ◽  
Edmund K. Waller
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Immunity ◽  
T Cell Proliferation ◽  
Cell Immunity

Abstract Introduction Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that suppresses Th1 immunity and inhibits antiviral immunity. Decreased Th1 immunity is problematic for allogeneic bone marrow transplant (allo-BMT) patients requiring T-cell immunity against blood cancers (Graft-versus-Tumor) and against secondary infections such as CMV. VIPhyb, a modified VIP peptide, is a VIP receptor antagonist that decreases VIP signaling. VIP-knockout mice and mice treated with VIPhyb after allo-BMT are known to have better antiviral immunity and survival after CMV infection without increasing GvHD (Li et al. PLoS One. 2013 May 27;8(5):e63381) (Li et al. Blood. 2013 Mar 21;121(12):2347-51.), thus making VIPhyb of interest for pharmacological use in humans to improve the efficacy of allo-BMT The effects of VIPhyb on T-cell immunity are not yet fully profiled. This study aimed to analyze the effects of VIPhyb on CD4+ and CD8+ T-cell proliferation and activation in order to better understand the mechanistic implications of VIP inhibition on T-cell adaptive immunity. This study also aimed to show that mixed lymphocyte reactions (MLRs), an in vitro allo-BMT model, could be used to provide rapid and reliable results that are consistent with in vivo data. It was hypothesized that VIPhyb would increase T-cell immunity as profiled by: increased T-cell proliferation, CD69 and PD1 co-upregulation in early T-cell activation, and PD1 downregulation in T-cells after initial activation. Methods Splenocytes from two histoincompatible mice were cultured together at 37°C in a 1:1 ratio in a one-way MLR. BALB/c splenocytes (stimulators) were irradiated at 20Gy, and Pepboy splenocytes (responders) were labeled with CFSE to trace proliferation. VIPhyb was added daily to the cell cultures in doses of 0.1μM, 0.3μM, 1μM, or 3μM. Treatment groups were compared to a PBS control. Proliferation, CD69, and PD1 were assessed by flow cytometry on the BD FACSAria. All results are shown as mean ± SEM (n=3). One-way ANOVA tests with Dunnett post-tests were calculated using Prism software. *p < 0.05; **p < 0.01; ***p < 0.001 Results VIPhyb increased CD4+ and CD8+ T-cell proliferation: 3, 5, and 7 days after initiating a one-way MLR, CFSE expression of Pepboy responder T-cells was assessed using flow cytometry (Figure 1). As the VIPhyb dose increased, the percentage of initial splenocytes that underwent proliferation increased in both CD4+ and CD8+ T-cells. VIPhyb increased early T-cell CD69 expression and abrogated later PD1 upregulation in CD8+ T-cells: 3, 5, and 7 days after initiating a one-way MLR, expression levels of CD69 and PD1 on Pepboy responder T-cells were assessed by flow cytometry. Significant upregulation of CD69 on CD4+ and CD8+ T-cells on day 3 occurred with increasing VIPhyb doses (Figures 2A and 2B). PD1 was co-upregulated with CD69 during early activation, and VIPhyb significantly decreased PD1 expression on CD8+ T-cells on days 5 and 7 (Figures 2C and 2D). Conclusions VIPhyb increased T-cell proliferation; CD8+ T-cells were affected more significantly. VIPhyb increased early co-upregulation of CD69 and PD1 in all T-cells and significantly decreased later CD8+ T-cell PD1 expression, indicating that VIPhyb increases T-cell activation. We hypothesize that the decreased PD1 expression will be critical for understanding the pathways involved in VIP inhibition. Importantly, since it has been shown in vivo that VIPhyb does not increase GvHD, then it can be assumed that the VIPhyb-induced T-cell proliferation and activation will increase GvL and adaptive immunity without increasing alloreactivity. Notably, these results are consistent with published in vivo data, which demonstrates that the MLR can be used as a faster method of analyzing pharmacological compounds than in vivo experiments. Given these results, VIPhyb is still of interest as a potential therapy for allo-BMT patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals P06.14 Characterization of tumor-infiltrating T cells by highly multiplexed immunofluorescence imaging

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A47.2-A48
Author(s):  
E Criado-Moronati ◽  
A Gosselink ◽  
J Kollet ◽  
A Dzionek ◽  
B Heemskerk
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
T Cell Subsets ◽  
Imaging Technology ◽  
Part Time

BackgroundThe adoptive cell transfer (ACT) of tumor-infiltrating T lymphocytes (TILs) has shown remarkable results in patients with different cancer types. The antitumor effect of this therapy is mainly attributed to a small fraction of tumor-reactive T lymphocytes (TRLs) that recognize mutated peptides as well as overexpressed self-antigens. Therefore, the enrichment and expansion of TRLs constitutes a promising immunotherapy approach. However, the specific targeting of individual mutated antigens represents a daunting challenge for widespread therapeutic application. Alternatively, we hypothesize that TRLs could be identified and enriched by a surface marker (or combination thereof) in an antigen-independent manner as a result of the chronic antigen exposure and other factors present in the tumor microenvironment (TME).Materials and MethodsWe screened T cell activation and exhaustion markers, among others, on different tumor tissues using the MACSima™ Imaging Platform, an instrument for the highly multiplexed immunofluorescence imaging technology MICS (Multiparameter Imaging Cell Screen), enabling investigation of hundreds of markers on a single section. Moreover, flow cytometry and single-cell RNA sequencing analyses of T cells from tumor digests were performed to complement the characterization of TILs.ResultsThe MICS results highlighted the complexity of the TME, mainly composed of tumor cells, fibroblasts and endothelial vessels. In some cases, an extensive immune infiltrate consisted of T cells, plasma cells, some B cells and distinct myeloid cells was observed. Particularly, CD8 T cells from different tumor areas exhibited a tissue-resident memory phenotype with the expression of CD69, CD45RO or CD103. Activated/exhausted CD8 T cells were homogenously found across the imaged tumor areas. However, there was a tendency to find them in close proximity to tumor cells, especially for CD8 subsets expressing CD39 and other relevant markers, which may suggest the identification of tumor-reactive CD8 T cell populations. Flow cytometry data revealed the presence of similar T cell phenotypes in the patient´s TILs from tumor digests.ConclusionsThis imaging technology offers the possibility to study multiple parameters—including the localization—of relevant cells in the TME such as T cells. The phenotypic and functional characterization of different T cell subsets will allow the further investigation of their anti-tumor reactivity. Ultimately, the enrichment and expansion of the identified tumor-reactive T cell population hold great promises to improve the efficiency of T cell therapy against cancer.Disclosure InformationE. Criado-Moronati: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. A. Gosselink: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. J. Kollet: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. A. Dzionek: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. B. Heemskerk: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG.

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