Monocytes as effector cells: Activated Ly-6Chigh mouse monocytes migrate to the lymph nodes through the lymph and cross-present antigens to CD8+T cells

The anti-tumour immune response is often not potent enough to prevent or eradicate disease. Dendritic cells (DCs) are professional antigen-presenting cells that are critical for the initiation of immune responses. While DCs frequently infiltrate tumours, lack of activation together with immuno-suppressive factors from the tumour can hamper an effective anti-tumour immune response. In this thesis, the ability of microbial stimuli and danger signals to overcome suppression and re-programme DCs and macrophages to an immuno-stimulatory phenotype was investigated. Whole live Mycobacterium smegmatis and BCG were used to provide multiple pathogen-associated molecular patterns. The intracellularly-recognised toll-like-receptor (TLR) ligands CpG and Poly IC, as well as the extracelullarly recognised TLR ligand LPS, and the danger signal monosodium-urate crystals (MSU) were also included. Bone-marrow derived DCs were found to respond to all adjuvants in vitro and DCs in tumour cell suspensions could be activated ex vivo. To assess the ability of adjuvants to enhance anti-tumour responses in vivo, immune-competent mice bearing established subcutaneous B16F1 melanomas were injected peri-tumorally with the different adjuvants. In line with previous reports, CpG treatment was effective in delaying tumour growth and increasing survival. A similar effect was found with Poly IC, but not with LPS, M. smegmatis, BCG or MSU alone. Combination of M. smegmatis + MSU, however, significantly delayed tumour growth and prolonged survival, while combinations of MSU + BCG or LPS were ineffective. Similar results were obtained using the B16.OVA melanoma and E.G7-OVA thymoma subcutaneous tumour models. In addition, Poly IC and MSU + M. smegmatis reduced primary tumour growth as well as lung metastases in the orthotopic 4T1 breast carcinoma model. Both Poly IC and MSU + M. smegmatis elicited an anti-tumour immune response that required CD8 T cells as well as NK cells. These treatments also resulted in increased proliferation of CD8 T cells and NK cells in tumour-draining lymph nodes, augmented infiltration of effector cells into the tumour, as well as enhanced production of in ammatory cytokines by effector cells and DCs in tumours. In addition, MSU + M. smegmatis also stimulated CD4 T cell proliferation, tumour-infiltrationand activation, while at the same time decreasing the frequency of regulatory T cells in tumours. Activation of a successful immune response to tumours was associated with early induction of IL-12 and IFNʸ, as well as moderate levels of pro-inflammatory cytokines at the tumour site and systemically. Furthermore, anti-tumour activity correlated with the induction of inflammatory monocyte-derived DCs in tumour-draining lymph nodes. These DCs were also observed in adjuvant treated tumours and their appearance was preceded by accumulation of inflammatory monocytes at the tumour site. These findings suggest that specific natural adjuvants can successfully modify the tumour environment and enhance the innate and adaptive anti-tumour immune response to delay tumour progression and increase survival.

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Using Natural Adjuvants to Stimulate Anti-Tumour Immune Responses

10.26686/wgtn.17011490 ◽

2021 ◽

Author(s):

◽

Sabine Kuhn

Keyword(s):

Immune Response ◽

T Cells ◽

Immune Responses ◽

Lymph Nodes ◽

Nk Cells ◽

Cd8 T Cells ◽

Tumour Growth ◽

Tumour Site ◽

Effector Cells ◽

Draining Lymph Nodes

The anti-tumour immune response is often not potent enough to prevent or eradicate disease. Dendritic cells (DCs) are professional antigen-presenting cells that are critical for the initiation of immune responses. While DCs frequently infiltrate tumours, lack of activation together with immuno-suppressive factors from the tumour can hamper an effective anti-tumour immune response. In this thesis, the ability of microbial stimuli and danger signals to overcome suppression and re-programme DCs and macrophages to an immuno-stimulatory phenotype was investigated. Whole live Mycobacterium smegmatis and BCG were used to provide multiple pathogen-associated molecular patterns. The intracellularly-recognised toll-like-receptor (TLR) ligands CpG and Poly IC, as well as the extracelullarly recognised TLR ligand LPS, and the danger signal monosodium-urate crystals (MSU) were also included. Bone-marrow derived DCs were found to respond to all adjuvants in vitro and DCs in tumour cell suspensions could be activated ex vivo. To assess the ability of adjuvants to enhance anti-tumour responses in vivo, immune-competent mice bearing established subcutaneous B16F1 melanomas were injected peri-tumorally with the different adjuvants. In line with previous reports, CpG treatment was effective in delaying tumour growth and increasing survival. A similar effect was found with Poly IC, but not with LPS, M. smegmatis, BCG or MSU alone. Combination of M. smegmatis + MSU, however, significantly delayed tumour growth and prolonged survival, while combinations of MSU + BCG or LPS were ineffective. Similar results were obtained using the B16.OVA melanoma and E.G7-OVA thymoma subcutaneous tumour models. In addition, Poly IC and MSU + M. smegmatis reduced primary tumour growth as well as lung metastases in the orthotopic 4T1 breast carcinoma model. Both Poly IC and MSU + M. smegmatis elicited an anti-tumour immune response that required CD8 T cells as well as NK cells. These treatments also resulted in increased proliferation of CD8 T cells and NK cells in tumour-draining lymph nodes, augmented infiltration of effector cells into the tumour, as well as enhanced production of in ammatory cytokines by effector cells and DCs in tumours. In addition, MSU + M. smegmatis also stimulated CD4 T cell proliferation, tumour-infiltrationand activation, while at the same time decreasing the frequency of regulatory T cells in tumours. Activation of a successful immune response to tumours was associated with early induction of IL-12 and IFNʸ, as well as moderate levels of pro-inflammatory cytokines at the tumour site and systemically. Furthermore, anti-tumour activity correlated with the induction of inflammatory monocyte-derived DCs in tumour-draining lymph nodes. These DCs were also observed in adjuvant treated tumours and their appearance was preceded by accumulation of inflammatory monocytes at the tumour site. These findings suggest that specific natural adjuvants can successfully modify the tumour environment and enhance the innate and adaptive anti-tumour immune response to delay tumour progression and increase survival.

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EXTH-14. A NOVEL LONG-ACTING INTERLEUKIN-7 AGONIST, NT-I7, INCREASES CYTOTOXIC CD8 CELLS AND ENHANCES SURVIVAL IN MOUSE GLIOMA MODELS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.368 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii89-ii89

Author(s):

Subhajit Ghosh ◽

Ran Yan ◽

Sukrutha Thotala ◽

Arijita Jash ◽

Anita Mahadevan ◽

...

Keyword(s):

T Cells ◽

Lymph Nodes ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cervical Lymph Nodes ◽

Cd8 Cells ◽

Interleukin 7 ◽

Long Acting ◽

Spleen And Lymph Nodes ◽

Mouse Glioma

Abstract BACKGROUND Patients with glioblastoma (GBM) are treated with radiation (RT) and temozolomide (TMZ). These treatments can cause prolonged severe lymphopenia, which is associated with shorter survival. NT-I7 (efineptakin alfa) is a long-acting recombinant human IL-7 that supports the proliferation and survival CD4+ and CD8+ cells in both human and mice. We tested whether NT-I7 would protect T cells from treatment-induced lymphopenia and improve survival. METHODS C57BL/6 mice bearing intracranial tumors (GL261 or CT2A) were treated with RT (1.8 Gy/day x 5 days), TMZ (33 mg/kg/day x 5 days) and/or NT-17 (10 mg/kg on the final day of RT completion). We followed for survival and profiled CD3, CD8, CD4, FOXP3 in peripheral blood over time. In parallel, we assessed cervical lymph nodes, bone marrow, thymus, spleen, and the tumor 6 days after NT-I7 treatment. RESULTS Median survival in mice treated with NT-I7 combined with RT was significantly better than RT alone (GL261: 40d vs 34d, p< 0.0021; CT2A: 90d vs 40d, p< 0.0499) or NT-I7 alone (GL261: 40d vs 24d, p< 0.008; CT2A: 90d vs 32d, p< 0.0154). NT-17 with RT was just as effective as NT-I7 combined with RT and TMZ in both GL261 (40d vs 47d) and CT2A (90d vs 90d). NT-I7 treatment significantly increased the amount of CD8+ cells in the peripheral blood and tumor. NT- I7 rescued CD8+ T cells from RT induced lymphopenia in peripheral blood, spleen, and lymph nodes. NT-I7 alone or NT-I7 in combination with RT increased the CD8+ T cells in peripheral blood and tumor while reducing the FOXP3+ T-reg cells in the tumor microenvironment. CONCLUSIONS NT-I7 protects T-cells from RT induced lymphopenia, improves cytotoxic CD8+ T lymphocytes systemically and in the tumor, and improves survival. Presently, a phase I/II trial to evaluate NT-I7 in patients with high-grade gliomas is ongoing (NCT03687957).

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565 A novel long-acting interleukin-7 agonist, NT-I7, increases cytotoxic CD8+ T cells and enhances survival in mouse glioma models

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0565 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A599-A599

Author(s):

Subhajit Ghosh ◽

Ran Yan ◽

Sukrutha Thotala ◽

Arijita Jash ◽

Anita Mahadevan ◽

...

Keyword(s):

T Cells ◽

Lymph Nodes ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Standard Of Care ◽

Control Group ◽

Cervical Lymph Nodes ◽

Cd8 Cells ◽

Interleukin 7 ◽

Long Acting

BackgroundRadiation (RT) and temozolomide (TMZ), which are standard of care for patients with glioblastoma (GBM), can cause prolonged severe lymphopenia. Lymphopenia, in turn, is an independent risk factor for shorter survival. Interleukin-7 (IL-7) is a cytokine that is required for T cell homeostasis and proliferation. IL-7 levels are inappropriately low in GBM patients with lymphopenia. NT-I7 (efineptakin alfa) is a long-acting recombinant human IL-7 that supports the proliferation and survival CD4+ and CD8+ cells in both human and mice. We tested whether NT-I7 rescues treatment-induced lymphopenia and improves survival.MethodsImmunocompetent C57BL/6 mice bearing two intracranial glioma models (GL261 and CT2A) were treated with RT (1.8 Gy/day x 5 days), TMZ (33 mg/kg/day x 5 days) and/or NT-I7 (10 mg/kg on the final day of RT completion). We profiled the CD3, CD8, CD4, FOXP3 cells in peripheral blood over time. We also immunoprofiled cervical lymph nodes, bone marrow, thymus, spleen, and the tumor 6 days after NT-I7 treatment. Survival was monitored daily.ResultsMedian survival in mice treated with NT-I7 combined with RT was significantly longer than RT alone (GL261: 40d vs 34d, p<0.0021; CT2A: 90d vs 40d, p<0.0499) or NT-I7 alone (GL261: 40d vs 24d, p<0.008; CT2A: 90d vs 32d, p<0.0154). NT-I7 with RT was just as effective as NT-I7 combined with RT and TMZ in both GL261(40d vs 47d) and CT2A (90d vs 90d). Cytotoxic CD8+ T cells were increased in both peripheral blood (0.66 x 105 to 3.34 x 105; P≤0.0001) and tumor (0.53 x 103 to 1.83 x 103; P≤0.0001) in mice treated with NT-I7 when compared to control. Similarly, NT-I7 in combination with RT increased the CD8+ T cells in peripheral blood (0.658 x 105 to 1.839 x 105 P≤0.0001) when compared to RT alone. There were decreases in tumor infiltrating FOXP3+ T-reg cells in mice treated with NT-I7 (1.9 x 104 to 0.75 x 104 P≤0.0001) and NT-I7 + RT (1.9 x 104 to 0.59 x 104 P≤0.0001) when compared to the control group without NT-I7. In addition, NT- I7 treatment increased CD8+ T cells in thymus, spleen, and lymph nodes.ConclusionsNT-I7 enhances cytotoxic CD8+ T lymphocytes systemically and in the tumor microenvironment, and improves survival. A phase I/II trial to evaluate NT-I7 in patients with high-grade gliomas is ongoing (NCT03687957).

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Role of NKG2D signaling in the cytotoxicity of activated and expanded CD8+ T cells

Blood ◽

10.1182/blood-2003-06-2125 ◽

2004 ◽

Vol 103 (8) ◽

pp. 3065-3072 ◽

Cited By ~ 254

Author(s):

Michael R. Verneris ◽

Mobin Karami ◽

Jeanette Baker ◽

Anishka Jayaswal ◽

Robert S. Negrin

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Interleukin 2 ◽

Cell Receptor ◽

Malignant Cell ◽

Leukemic Cells ◽

Target Cells ◽

High Dose ◽

Effector Cells ◽

Nkg2d Expression

Abstract Activating and expanding T cells using T-cell receptor (TCR) cross-linking antibodies and interleukin 2 (IL-2) results in potent cytotoxic effector cells capable of recognizing a broad range of malignant cell targets, including autologous leukemic cells. The mechanism of target cell recognition has previously been unknown. Recent studies show that ligation of NKG2D on natural killer (NK) cells directly induces cytotoxicity, whereas on T cells it costimulates TCR signaling. Here we demonstrate that NKG2D expression is up-regulated upon activation and expansion of human CD8+ T cells. Antibody blocking, redirected cytolysis, and small interfering RNA (siRNA) studies using purified CD8+ T cells demonstrate that cytotoxicity against malignant target cells occurs through NKG2D-mediated recognition and signaling and not through the TCR. Activated and expanded CD8+ T cells develop cytotoxicity after 10 to 14 days of culture, coincident with the expression of the adapter protein DAP10. T cells activated and expanded in low (30 U/mL) and high (300 U/mL) concentrations of IL-2 both up-regulated NKG2D expression equally, but only cells cultured in high-dose IL-2 expressed DAP10 and were cytotoxic. Collectively these results establish that NKG2D triggering accounts for the majority of major histocompatibility complex (MHC)–unrestricted cytotoxicity of activated and expanded CD8+ T cells, likely through DAP10-mediated signaling. (Blood. 2004;103: 3065-3072)

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CXCR5 + CD8 + T cells infiltrate the colorectal tumors and nearby lymph nodes, and are associated with enhanced IgG response in B cells

Experimental Cell Research ◽

10.1016/j.yexcr.2017.04.014 ◽

2017 ◽

Cited By ~ 9

Author(s):

Junjie Xing ◽

Chenxin Zhang ◽

Xiaohong Yang ◽

Shaoxuan Wang ◽

Zhongchuan Wang ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Lymph Nodes ◽

Cd8 T Cells ◽

Colorectal Tumors

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Phase 2 Study of Combination of Cytarabine, Idarubicin, and Nivolumab for Initial Therapy of Patients with Newly Diagnosed Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.815.815 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 815-815

Author(s):

Farhad Ravandi ◽

Naval Daver ◽

Guillermo Garcia-Manero ◽

Christopher B Benton ◽

Philip A Thompson ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Myeloid Leukemia ◽

Research Funding ◽

T Cell Subsets ◽

Newly Diagnosed ◽

Effector Cells ◽

T Effector Cells ◽

Grade 3

Abstract Background: Blocking PD-1/PD-L1 pathways enhances anti-leukemia responses by enabling T-cells in murine models of AML (Zhang et al, Blood 2009). PD-1 positive CD8 T-cells are increased in bone marrow (BM) of pts with AML (Daver et al, AACR 2016). PD1 inhibition has shown activity in AML (Berger et al, Clin Cancer Res 2008). We hypothesized that addition of nivolumab to an induction regimen of ara-C and idarubicin may prolong relapse-free survival (RFS) and overall survival (OS); this study was designed to determine the feasibility of this combination. Methods: Pts with newly diagnosed acute myeloid leukemia (by WHO criteria; ≥20% blasts) and high risk MDS (≥10% blasts) were eligible to participate if they were 18-65 yrs of age and had adequate performance status (ECOG ≤3) and organ function (LVEF ≥ 50%; creatinine ≤ 1.5 g mg/dL, bilirubin ≤ 1.5 mg/dL and transaminases ≤ 2.5 times upper limit of normal). Treatment included 1 or 2 induction cycles of ara-C 1.5 g/m2 over 24 hours (days 1-4) and Idarubicin 12 mg/m2 (days 1-3). Nivolumab 3 mg/kg was started on day 24 ± 2 days and was continued every 2 weeks for up to a year. For pts achieving complete response (CR) or CR with incomplete count recovery (CRi) up to 5 consolidation cycles of attenuated dose ara-C and idarubicin was administered at approximately monthly intervals. Eligible pts received an allogeneic stem cell transplant (alloSCT) at any time during the consolidation or thereafter. Results: 3 pts with relapsed AML were treated at a run-in phase with a dose of nivolumab 1 mg/kg without specific drug-related toxicity. Subsequently, 32 pts (median age 53 yrs; range, 26-65) were treated as above including 30 with AML (24 de novo AML, 2 therapy-related AML, 3 secondary AML and 1 therapy-related secondary AML) and 2 high risk MDS. Pre-treatment genetic risk by ELN criteria was 11 adverse, 16 intermediate, and 5 favorable, including 2 FLT3 -ITD mutated, 5 NPM1 mutated, and 7 TP53 mutated. All 32 pts were evaluable for response and 23 (72%) achieved CR/CRi (19 CR, 4 CRi). The 4-week and 8 week mortality was 6% and 6%. The median number of doses of nivolumab received was 6 (range, 0-13); one pt did not receive nivolumab due to insurance issues. 9 pts underwent an alloSCT. After a median follow-up of 8.3 mths (range, 1.5-17.0) the median RFS among the responding pts has not been reached (range, 0.1 - 15.8 mths) and the median OS has not been reached (range 0.5-17.0 mths). Grade 3/4 immune mediated toxicities have been observed in 5 pts and include rash, pancreatitis, and colitis. Other grade 3/4 toxicities thought to be potentially related to nivolumab include cholecystitis in one pt. 9 pts proceeded to an alloSCT. Donor source was matched related in 2, matched unrelated in 6 and haplo-identical in 1 pt. Conditioning regimen was Fludarabine plus busulfan-based in 8, and fludarabine plus melphalan in 1 pt. 4 pts developed graft versus host disease (GVHD)(grade I/II in 3, grade III/IV in 1), which responded to treatment in 3. Multicolor flow-cytometry studies are conducted by the Immunotherapy Platform on baseline (prior to first dose of nivolumab) and on-treatment BM aspirate and peripheral blood to assess the T-cell repertoire and expression of co-stimulatory receptors and ligands on T-cell subsets and leukemic blasts, respectively. The baseline BM was evaluated on 23 of the 32 evaluable pts, including 18 responders and 5 non-responders. Pts who achieved a CR/CRi had a trend of higher frequency of live CD3+ total T cell infiltrate as compared to non-responders in the baseline BM aspirates (Fig 1A). We evaluated expression of immune markers on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. At baseline, BM of non-responders had significantly higher percentage of CD4 T effector cells co-expressing the inhibitory markers PD1 and TIM3 (p<0.05) and a trend towards higher percentage of CD4 T effector cells co-expressing PD1 and LAG3 compared to responders (Fig 1B). Co-expression of TIM3 or LAG3 on PD1+ T cells have been shown to be associated with an exhausted immune phenotype in AML (Zhou et al., Blood 2011). Conclusion: Addition of nivolumab to ara-C and anthracycline induction chemotherapy is feasible and safe in younger pts with AML. Among the pts proceeding to alloSCT the risk of GVHD is not significantly increased. Figure 1 Figure 1. Disclosures Daver: Pfizer Inc.: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Kiromic: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Incyte Corporation: Honoraria, Research Funding. Thompson: Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour: Bristol-Myers Squibb: Consultancy. Takahashi: Symbio Pharmaceuticals: Consultancy. DiNardo: Novartis: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Sharma: Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Astellas: Consultancy; EMD Serono: Consultancy; Amgen: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy; Consetellation: Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Kite Pharma: Consultancy, Other: stock; BMS: Consultancy. Cortes: BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; ImmunoGen: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding. Kantarjian: Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding.

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