In Situ Vaccination with a TLR9 Agonist and Anti-OX40 Antibody Leads to Tumor Regression and Induces Abscopal Responses in Murine Lymphoma

Abstract Background: Toll-like receptors (TLRs) are components of the innate immune system that recognize pathogen-associated molecular patterns on bacterial, fungal, or viral pathogens. Intratumoral (IT) injection of unmethylated CG-enriched oligodeoxynucleotide (CpG), a TLR9 agonist, results in local tumor eradication but on its own is not able to induce a systemic anti-tumor immune response. OX40 is a potent costimulatory receptor that can potentiate the action of conventional T cells leading to their proliferation, effector function and survival, but can also inhibit or kill T regulatory cells by ADCC. In previous preclinical studies systemic injection of OX40 agonists increased antitumor immunity and improved survival. Scientific question: Does local injection of both CpG and low doses of an anti-OX40 agonistic antibody trigger a systemic anti-tumor immune response? Results: We implanted the A20 lymphoma tumor bilaterally on opposite sides of the abdomen. After tumors were established we administered microgram quantities of CpG and anti-OX40 antibody into the tumor on one side and monitored both the injected and the uninjected tumor sites. This treatment resulted in both a local and an abscopal effect on the contralateral, untreated tumor. In addition, the animals were protected from a second challenge with A20 cells. This anti-tumor effect was T cell dependent, since depletion of either CD4+ or CD8+ T cells abrogated the therapeutic effect. There was no evidence of toxicity or autoimmunity in the treated animals. To examine the potential of this maneuver to treat spontaneous, non-transplanted cancers we chose the mouse mammary tumor model- FVB/N-Tg(MMTV-PyVT)634Mul/J. These animals all develop invasive breast cancer tumors in all of their mammary glands by 12 weeks of age. Injections of CpG and anti-OX40 antibody into the first arising tumor not only prevented its growth but significantly reduced the incidence and outgrowth of subsequent tumors at un-injected susceptible mammary glands and reduced the number of lung metastases. Significance: TLR9 agonists and anti-OX40 antibodies are currently under clinical development for cancer treatment. We show here that combining anti-OX40 antibody with a TLR9 agonist at a single established tumor is sufficient to trigger a systemic anti-tumor response able to eradicate tumor at distant sites in both transplantable and spontaneously occurring oncogene-driven murine tumors. This anti-tumor effect was long lasting, specific and required T cells. Impact: Our current results suggest that CpG and anti-OX40 are sufficient to induce fully protective and curative anti-tumor immune responses, even in spontaneously arising cancer. Anti-OX40 and CpG are both currently in phase-I clinical trials as single agents. Our results provide the rationale for testing these agents clinically in combination as described here, injected locally in low doses in order to induce therapeutic anti-lymphoma immunity. Disclosures Levy: Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

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768 Systemic delivery of a tumor-targeted TLR9 agonist conjugate transforms the tumor immune landscape and induces tumor regression in mice

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.768 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A803-A803

Author(s):

Caitlyn Miller Candidate ◽

Idit Sagiv-Barfi ◽

Patrick Neuhoefer ◽

Debra Czerwinski ◽

Steven Artandi ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

B Cells ◽

Tumor Targeting ◽

Tumor Regression ◽

Breast Tumors ◽

Live Cells ◽

Systemic Delivery ◽

Tlr9 Agonist

BackgroundTumor-localized delivery of Toll-like receptor (TLR) agonists is a promising strategy to promote immune activation within the tumor microenvironment (TME) to overcome tumor immunosuppression and induce anti-tumor immune responses. To enable localization to multiple tumor sites following systemic administration, we developed a fully-synthetic tumor-targeting TLR9 agonist and demonstrate its potential to transform the tumor immune microenvironment for effective tumor regression in mice.MethodsAn engineered synthetic peptide (PIP) that binds to multiple integrin receptors overexpressed in many solid tumors was chemically conjugated to a synthetic CpG oligonucleotide (TLR9 agonist), thereby generating a tumor-targeting immune-stimulant referred to as PIP-CpG. To facilitate clinical translation, PIP-CpG is cross-reactive between mouse, non-human primate, and human. Therapeutic studies were conducted in immune-competent mice bearing breast or pancreatic tumors to evaluate the efficacy of intravenously (IV)-injected PIP-CpG compared to IV-injected unmodified CpG or vehicle (PBS). We then performed mechanistic studies to evaluate the immune response elicited by PIP-CpG therapy.ResultsIntravenous dosing of PIP-CpG led to tumor regression and prolonged survival, and in some cases cures, relative to vehicle or unmodified CpG therapy in both murine breast (4T1) and pancreatic cancer (KPC-G2) models. This tumor regression was dependent on T cells as T cell depletion resulted in loss of therapeutic response. To study the effect of systemic therapy on the cellular landscape in the TME, we analyzed 4T1 breast tumors by flow cytometry. We found that vehicle and CpG IV-dosed mice had immunosuppressive TMEs comprised mostly of myeloid-derived suppressor cells (MDSCs; 43–68% of live cells) with minimal infiltrating T cells and B cells (5–16% of live cells). In contrast, the TME of PIP-CpG treated mice was transformed into a lymphocyte-rich “hot” tumor phenotype with massive infiltration by T cells and B cells (92–95% of live cells) and plummeting levels of MDSCs (down to ~1%). In addition, tumor-specific effector CD8+ T cells were generated in response to PIP-CpG therapy, but not CpG dosed IV, indicating that PIP-CpG therapy transforms the TME and elicits a T cell-mediated tumor-specific immune response. Furthermore, PIP-CpG was effective for treating MMTV-PyMT transgenic mice, which spontaneously develop multiple breast tumors. Murine toxicity studies indicated that effects of PIP-CpG were similar to CpG dosed IV or intratumorally, which have been well-tolerated in human clinical trials.ConclusionsTumor-directed systemic delivery of a TLR9 agonist transforms the TME via activated B and T cells and is promising for further development in patients with solid tumors.Ethics ApprovalAll mouse experiments were performed in accordance with protocols approved by the Stanford Administrative Panel on Laboratory Animal Care.

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Interleukin 1-induced, T cell-mediated regression of immunogenic murine tumors. Requirement for an adequate level of already acquired host concomitant immunity.

Journal of Experimental Medicine ◽

10.1084/jem.168.6.2031 ◽

1988 ◽

Vol 168 (6) ◽

pp. 2031-2043 ◽

Cited By ~ 44

Author(s):

R J North ◽

R H Neubauer ◽

J J Huang ◽

R C Newton ◽

S E Loveless

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Tumor Growth ◽

Tumor Regression ◽

Interleukin 1 ◽

Host Immunity ◽

Antitumor Action ◽

Complete Regression ◽

Subtherapeutic Level

Intraperitoneal injection of human rIL-1 in a dose of 0.5 microgram daily for 5 d, or 1 microgram daily for 3 d, was capable of causing complete regression of immunogenic SA1 sarcoma growing subcutaneously in syngeneic or semisyngeneic mice. Higher doses of IL-1 were not more therapeutic against the SA1 sarcoma, but needed to be given to cause complete regression of the immunogenic L5178Y lymphoma. On the other hand, the P815 mastocytoma was much less responsive to IL-1 therapy, in that it failed to undergo complete regression in response to doses of IL-1 capable of causing regression of the L5178Y lymphoma. IL-1 caused regression of the SA1 sarcoma when given on days 6-8 of tumor growth, but not when given on days 1-3. This refractoriness of a small tumor to IL-1 therapy suggests that the antitumor action of IL-1 is based on an underlying host-immune response that is not generated until after day 3 of tumor growth. Direct evidence for the participation of host immunity in IL-1-induced tumor regression was supplied by results showing that IL-1 was not therapeutic against the SA1 sarcoma growing in T cell-deficient (TXB) mice, unless these mice were first infused with Ly-2+ and L3T4+ T cells from donor mice bearing an established SA1 sarcoma. In contrast, normal T cells, or T cells from donor mice bearing a YAC-1 lymphoma, failed to provide TXB recipients with the ability to cause regression of their SA-1 sarcoma in response to IL-1 treatment. The results are in keeping with the interpretation that exogenous IL-1, by augmenting the production of tumor-sensitized T cells, converts a subtherapeutic level of host immunity to a therapeutic level. The results suggest, in addition, that IL-1 only stimulates the replication of T cells that are already engaged in the antitumor immune response.

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Phase I/II Study of IPH1101, γσ T Cell Agonist, Combined with Rituximab, in Low Grade Follicular Lymphoma Patients.

Blood ◽

10.1182/blood.v114.22.1649.1649 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1649-1649 ◽

Cited By ~ 4

Author(s):

Guy Laurent ◽

Sylvie Lafaye de Micheaux ◽

Philippe Solal-Celigny ◽

Pierre Soubeyran ◽

Vincent Delwail ◽

...

Keyword(s):

T Cells ◽

Follicular Lymphoma ◽

Research Funding ◽

Low Grade ◽

Low Doses ◽

Open Label ◽

Independent Panel ◽

Pharmacodynamic Profile ◽

Previously Treated Patients ◽

Previously Treated

Abstract Abstract 1649 Poster Board I-675 Non-conventional γσ T lymphocytes are innate immunity effectors bearing potent anti-tumoral activity, particularly against malignant B cells. IPH1101 is an agonist of γσ T cells, which in the presence of low doses of IL-2 potentiates their direct cytotoxic activity. ADCC is a major molecular mechanism underlying rituximab's efficacy. Increasing the number and the activation state of killer lymphocytes mediating ADCC is therefore believed to be beneficial for therapeutic potency. Since γσ T cells have been found to be capable of mediating ADCC, modulating γσ T cells in the context of rituximab is worth being tested in a clinical trial. The main purpose is to assess the clinical efficacy of IPH1101 with low doses of IL-2, combined with a standard rituximab treatment, in patients (pts) with follicular lymphoma. This is an open label, multinational study consisting of a dose escalation Phase (ph) I-like part followed by a ph II part. The ph I part has shown a good safety and immuno-biological efficacy profile for the highest dose of IL-2. Consequently, the pts of the phase II part were treated with the combination of rituximab (375 mg/m2) administered 4 times weekly, IPH1101 (750 mg/m2) administered i.v. 3 times (every 3 weeks) and IL-2 (8 MIU) administered daily s.c. for 5 days starting on the day of each IPH1101 administration. All pts presented low grade FL which had relapsed after 1 to 4 lines of previous therapy including at least one rituximab-containing line. Inclusion criteria set forth that pts should have no lesion > 7 cm. Results We report here the end of recruitment and updated data on 45 patients and more than 100 cycles of IPH1101. Overall safety was good, and most of the drug-related adverse events were, as expected, flu like symptoms of grade 1 or 2. The SAEs reported were 2 hypotensions, 1 bronchospasm, 1 allergic reaction (back pain), 1 glomerular filtration decrease, 1 ALAT elevation, 1 hypertension and 1 asthenia. The immuno-biological follow up demonstrated the very good pharmacodynamic profile of IPH1101 in these patients and these data are presented in details in another abstract from Lucas et al. To date, in the first set of evaluable patients (12 pts) and after at least 3 months post treatment, investigators reported about 75% of response and 50% of CR. Most of the responses are already confirmed by an independent panel. Conclusion These observations confirm the good safety and the biological rationale of this approach. Furthermore, the response rate in this first set of pts is encouraging in the context of previously treated patients. Updated results will be presented at the meeting. Disclosures Laurent: Innate pharma: Honoraria. Lafaye de Micheaux:Innate Pharma: Employment. Solal-Celigny:Innate Pharma: Research Funding. Soubeyran:Innate Pharma: Research Funding. Delwail:Innate Pharma: Honoraria. Ghesquières:innate pharma: Honoraria. Thieblemont:Innate Pharma: Honoraria. Jourdan:Innate Pharma: Honoraria. Beautier:Innate Pharma: Employment. Squiban:Innate pharma: Employment. Rossi:Innate Pharma: Honoraria.

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Immunomodulatory Effects and Adaptive Immune Response to Daratumumab in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.3037.3037 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3037-3037 ◽

Cited By ~ 8

Author(s):

Jakub Krejcik ◽

Tineke Casneuf ◽

Inger Nijhof ◽

Bie Verbist ◽

Jaime Bald ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Immune Cell ◽

Adaptive Immune Response ◽

Advisory Committees ◽

Adaptive Immune ◽

T Cell Clonality ◽

Cell Clonality

Abstract Introduction: Daratumumab (DARA) is a novel human monoclonal antibody that targets CD38, a protein that is highly expressed on multiple myeloma (MM) cells. DARA acts through multiple immune effector-mediated mechanisms, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis. In two clinical studies (NCT00574288 [GEN501] and NCT01985126 [Sirius]) of DARA monotherapy in patients with relapsed and refractory MM, overall response rates were 36% and 29%, respectively. CD38 is highly expressed in myeloma cells but also expressed in lymphocytes and other immune cell populations. Therefore, the effects of DARA on immune cell populations and adaptive immune response pathways were investigated. Methods: The patient population investigated included treated subjects with MM that were relapsed after or were refractory to ≥2 prior therapies (GEN501) or had received ≥3 prior therapies, including a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD), or were refractory to both a PI and an IMiD (Sirius). Patients assessed in this analysis were treated with 16 mg/kg DARA. When both studies were combined, median age (range) was 64 (31-84) years and median time from diagnosis was 5.12 (0.77-23.77) years. Seventy-six percent of patients had received >3 prior therapies and 91% were refractory to their last treatment. Clinical response was evaluated using IMWG consensus recommendations. Peripheral blood (PB) samples and bone marrow (BM) biopsies/aspirates were taken at prespecified time points and immunophenotyped by flow cytometry to enumerate various T-cell sub-types. T-cell clonality was measured by TCR sequencing. Antiviral T-cell response and regulatory T-cell (Treg) activity were analysed by functional in vitro assays. T-cell subpopulation counts were modelled over time with linear mixed modelling. Two group comparisons were performed using non-parametric Wilcoxon rank sum tests. Results: Data from 148 patients receiving 16 mg/kg DARA in GEN501 (n = 42) and Sirius (n = 106) were analyzed for changes in immune response. In PB, robust mean increases in CD3+ (44%), CD4+ (32%) and CD8+ (62%) T-cell counts per 100 days were seen with DARA treatment. However, responding evaluable patients (n = 45) showed significantly greater increases from baseline than nonresponders (n = 93) in CD3+ (P = 0.00012), CD4+ (P = 0.00031), and CD8+ (P = 0.00018) T cells. In BM aspirates the number of CD3+, CD4+, and CD8+ T-cells increased during treatment compared to baseline (the median percent increases were 19.95%, 5.66%, and 26.99% [n = 58]). Additionally, CD8+: CD4+ T-cell ratios significantly increased compared to baseline in both PB (P = 0.00017), and BM (P = 0.00016). T cell clonality, assessed by TCR sequencing, increased after DARA treatment compared with pretreatment (P = 0.049), with greater sums of absolute expansion in the repertoire (P = 0.037), as well as greater maximum expansion of a single clone (P = 0.048) in responders compared to nonresponders. Increased antiviral T-cell responses were observed post-DARA treatment, particularly in responders. Interestingly, a novel subpopulation of regulatory T cells was identified that expressed high levels of CD38. These cells comprised ~10% of all Tregs and were depleted by one DARA infusion. In ex vivo analyses, CD38+ Tregs appeared to be highly immune suppressive compared to CD38-Tregs. Conclusions: Robust T cell increases, increased CD8+: CD4+ ratios, increased antiviral responses, and increased T cell clonality were all observed after DARA treatment in a heavily pretreated, relapsed, and refractory patient population not expected to have strong immune responses. Improved clinical responses were associated with changes in these parameters. In addition, a sub-population of regulatory T cells expressing high CD38 levels was determined to be extremely immune suppressive and sensitive to DARA treatment. These data suggest a previously unknown immune modulatory role of DARA that may contribute to its efficacy, and a potential role for CD38 immune targeted therapies. We postulate that there are several distinct and complementary mechanisms that contribute to DARA's efficacy including increased antigen presentation through phagocytosis, targeting of immune suppressive Tregs, and increased adaptive immune responses. JK and TC contributed equally to this work. Disclosures Casneuf: Janssen: Employment. Verbist:Janssen: Employment. Bald:Janssen: Employment. Plesner:Genmab: Membership on an entity's Board of Directors or advisory committees; Roche and Novartis: Research Funding; Janssen and Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Liu:Janssen: Employment. van de Donk:Janssen Pharmaceuticals: Research Funding; Amgen: Research Funding; Celgene: Research Funding. Weiss:Janssen and Onclave: Research Funding; Janssen and Millennium: Consultancy. Ahmadi:Janssen: Employment. Lokhorst:Genmab: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria. Mutis:Janssen: Research Funding; Genmab: Research Funding.

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Preoperative treatment to modify the immune microenvironnement of liver colorectal metastases.

Journal of Clinical Oncology ◽

10.1200/jco.2015.33.3_suppl.602 ◽

2015 ◽

Vol 33 (3_suppl) ◽

pp. 602-602 ◽

Cited By ~ 1

Author(s):

Marc Van Den Eynde ◽

Bernhard Mlecnik ◽

Jean-Pascal H. Machiels ◽

Daphne Debetancourt ◽

Gabriela Bindea ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

B Cells ◽

Tumor Regression ◽

Colorectal Metastases ◽

Tumor Regression Grade ◽

Preoperative Treatment ◽

Prognostic Impact ◽

Histological Response ◽

Primary Colorectal Tumor

602 Background: We previously reported that an adaptive Th1 immune response (CD3/CD8/CD45RO T-cells) observed in resected primary colorectal tumor and liver colorectal metastases (LCM) is an important prognostic factor. B and FoxP3 regulatory lymphocytes participate to the modulation of this response. We aimed to investigate whether the preoperative treatments influenced the quality and the density of the immune infiltrates previously reported in the LCM. Methods: We used a cohort of metastatic colorectal patients (n=107) engaged for curative liver surgery with available FFPE blocks for all resected LCM to confirm the prognostic impact of the immune response. Among this cohort of 338 LCMs, 46 were completely resected after chemotherapy (CT) alone, 130 after CT + anti-VEGF, 118 after CT + anti-EGFR and 44 after surgery alone. LCMs were analyzed for histological response according the Tumor Regression Grade (TRG) and regrouped as Response (R, TRG1-3) or No Response (NR, TRG4-5). The density of CD3+ (T-cells), CD8+ (cytotoxic), CD45RO+ (memory), CD20+ (B-cells) and FoxP3+ (regulatory) in the core (CT) and invasive margin (IM) of all LCM was quantified on immunostained slides. The mean density value (CT/IM) was calculated for each marker with a dedicated image analysis software on whole-slide imaging. Comparisons were made using the Wilcoxon-Mann-Whitney test. Results: LCMs showing R (compared to NR and untreated LCM) were more frequently associated with a high immune infiltrate for CD3+ (CT: p<0.005; IM: p<0.05), CD8+ (CT: p<0.005; IM: p<0.005) and CD20+ (CT: p<0.05). Conversely, high FoxP3+ density in the CT and IM was related to NR and untreated LCMs (p<0.01). LCMs treated with an anti-EGFR therapy showed higher densities of CD3+ (CT: p<0.005; IM: p<0.01), CD8+ (CT: p<0.005), CD45RO+ (CT: p<0.005), CD20+ (CT: p<0.005) and FoxP3+ (CT: p<0.05; IM: p<0.005) compared to other treatments and untreated LCMs. Conclusions: Preoperative treatment modifies the LCM immune microenvironnement. LCMs with a histological response show a cytotoxic immune response (CD3+/CD8+) with associated B-cells (CD20+) and downregulated Tregs (FoxP3+). The use of an anti-EGFR therapy significantly increases immune infiltration in the CT.

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Human squamous cell carcinomas evade the immune response by down-regulation of vascular E-selectin and recruitment of regulatory T cells

Journal of Experimental Medicine ◽

10.1084/jem.20071190 ◽

2008 ◽

Vol 205 (10) ◽

pp. 2221-2234 ◽

Cited By ~ 161

Author(s):

Rachael A. Clark ◽

Susan J. Huang ◽

George F. Murphy ◽

Ilse G. Mollet ◽

Dirkjan Hijnen ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Squamous Cell ◽

Transforming Growth Factor ◽

Tumor Regression ◽

Squamous Cell Carcinomas ◽

Skin Cancers ◽

Immune Defects

Squamous cell carcinomas (SCCs) of the skin are sun-induced skin cancers that are particularly numerous in patients on T cell immunosuppression. We found that blood vessels in SCCs did not express E-selectin, and tumors contained few cutaneous lymphocyte antigen (CLA)+ T cells, the cell type thought to provide cutaneous immunosurveillance. Tumors treated with the Toll-like receptor (TLR)7 agonist imiquimod before excision showed induction of E-selectin on tumor vessels, recruitment of CLA+ CD8+ T cells, and histological evidence of tumor regression. SCCs treated in vitro with imiquimod also expressed vascular E-selectin. Approximately 50% of the T cells infiltrating untreated SCCs were FOXP3+ regulatory T (T reg) cells. Imiquimod-treated tumors contained a decreased percentage of T reg cells, and these cells produced less FOXP3, interleukin (IL)-10, and transforming growth factor (TGF)-β. Treatment of T reg cells in vitro with imiquimod inhibited their suppressive activity and reduced FOXP3, CD39, CD73, IL-10, and TGF-β by indirect mechanisms. In vivo and in vitro treatment with imiquimod also induced IL-6 production by effector T cells. In summary, we find that SCCs evade the immune response at least in part by down-regulating vascular E-selectin and recruiting T reg cells. TLR7 agonists neutralized both of these strategies, supporting their use in SCCs and other tumors with similar immune defects.

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Notch1-Activated B Cells Have an Immunomodulatory Function Enhancing Th2 and Treg Immune Response Via IL-33-ST2 Pathway

Blood ◽

10.1182/blood.v128.22.130.130 ◽

2016 ◽

Vol 128 (22) ◽

pp. 130-130

Author(s):

Hiroshi Arima ◽

Momoko Nishikori ◽

Yasuyuki Otsuka ◽

Kiyotaka Izumi ◽

Wataru Kishimoto ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cell Fate ◽

Research Funding ◽

Pharmaceutical Research ◽

Fate Decision ◽

Activated B Cells

Abstract Notch1 signaling pathway is involved in T-cell fate decision and development, but it is also known to be activated in B cells upon anti-IgM or LPS stimulation. In addition to its physiological upregulation in B cells, Notch1 signaling is often aberrantly activated in several lymphoid malignancies of B-cell origin, such as classical Hodgkin lymphoma, mantle cell lymphoma and chronic lymphocytic leukemia. However, functional roles of Notch1 in B cells have not been well elucidated to date. Here we report a novel immunomodulatory role of Notch1-activated B cells that alters T-cell immune response in an IL-33-dependent manner. Functional analysis of Notch1 in mature B cells had been hampered by its substitutability for Notch2, which is involved in early B-cell fate decision towards marginal zone B cells (Zhang et. al. J Immunol 2013). To eliminate such irrelevant effect of Notch1 on early B-cell differentiation, we generated a mouse model in which Notch1 intracellular domain (NICD), a constitutively active form of Notch1, began to be expressed in mature B cells after AICDA promoter-dependent Cre expression in germinal centers (StopFloxed-NICD Tg mice×Aicda-Cre mice, hereby designated as NICD Tg mice). In this mouse model, NICD transgene was expressed in about 5% of total splenic B cells, with normal B cell maturation and differentiation. Alternatively, subsets of splenic CD4+ T cells were significantly altered, with increase in Th2 and Treg cells and decrease in Th1 and Th17 cells. IFN-γ production by CD8+ T cells was also significantly reduced. Consequently, NICD Tg mice were susceptible to fungal infections, and more importantly, they began to die of spontaneous malignant neoplasms such as sarcoma and lymphoma at 9 months of age. The tumor development was further increased when TP53 gene was heterozygously deleted in NICD Tg mice. None of the tumors having developed in NICD Tg mice expressed the NICD transgene, suggesting that these tumors did not develop as a result of direct oncogenic effect of NICD. As serum levels of IFN-γ and TNF-α were significantly lower in NICD Tg mice than in control mice, it was rather suggested that these tumors had developed under a condition of suppressed anti-tumor immunity. To elucidate the mechanism of immunomodulatory activity of Notch1-activated B cells, we performed a comparative gene expression analysis using B cells from NICD Tg and control mice. Among several candidate genes whose expression levels were increased in Notch1-activated B cells, we focused on elevated IL-33 as a potential cause for the immunomodulation. Upregulation of IL-33 protein in Notch1-activated B cells was validated by intracellular cytokine flow cytometry. IL-33 is a cytokine that is expressed in nuclei of broad types of cells in their resting state. However, we found that it was also present in the cytoplasm of Notch1-activated B cells, suggesting that IL-33 is actively produced in these cells. To confirm whether extracellular release of IL-33 from B cells was enhanced through Notch1, we cultured splenic B cells from wild-type mice with LPS stimulation in the presence of L cells with or without Notch1 ligand Delta-like 1 (Dll1) expression. We found that IL-33 secretion from B cells was increased twofold in the presence of Dll1-positive compared to Dll1-negative L cells. As expected, the Dll1-mediated increase in IL-33 levels was successfully blocked by DAPT, a Notch signaling inhibitor. To determine whether the IL-33 secreted from Notch1-activated B cells was responsible for the functional modulation of T cells, we cultured wild-type CD4+ T cells with B cells from NICD Tg or control mice, and measured cytokine levels produced by T cells. As a result, IL-4, IL-13 and IL-10 secretion was markedly increased when T cells were cocultured with Notch1-activated B cells. Strikingly, the increase in these Th2- and Treg-associated cytokine levels was completely canceled by addition of a blocking antibody against the IL-33 receptor ST2. In summary, we have shown that Notch1-activated B cells have a novel immunomodulatory function to alter T-cell immunity towards Th2 and Treg immune response via IL-33 secretion, thereby suppressing cellular immunity. This immunomodulatory mechanism may potentially be utilized by Notch1-activated B-cell neoplasms to escape anti-tumor immunity, and we propose that the Notch1-IL-33-ST2 axis can be a promising target for immunotherapy of lymphoid malignancies. Disclosures Nishikori: Kyowa Kirin: Honoraria; Eisai: Honoraria, Research Funding; Janssen Pharmaceutical: Honoraria. Takaori-Kondo:Alexion Pharmaceuticals: Research Funding; Mochida Pharmaceutical: Research Funding; Shionogi: Research Funding; Eisai: Research Funding; Takeda Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Toyama Chemical: Research Funding; Cognano: Research Funding.

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Hyperthermia by Near Infrared Radiation Induced Immune Cells Activation and Infiltration in Breast Tumor

10.21203/rs.3.rs-125646/v1 ◽

2021 ◽

Author(s):

Wan Fatin Amira Wan Mohd Zawawi ◽

Merilyn Hibma ◽

Maheza Irna Salim ◽

Khairunadwa Jemon

Keyword(s):

Breast Cancer ◽

Immune Response ◽

T Cells ◽

Side Effects ◽

Infrared Radiation ◽

Near Infrared ◽

Tumor Regression ◽

Immunohistochemical Analysis ◽

Therapeutic Effects ◽

Near Infrared Radiation

Abstract Breast cancer is the most common cancer that causes death in women. Conventional therapies, including surgery and chemotherapy, have different therapeutic effects and are commonly associated with risks and side effects. Near infrared radiation is a technique with few side effects that is used for local hyperthermia, typically as an adjuvant to other cancer therapies. The understanding of the use of near NIR as a monotherapy, and its effects on the anti-tumour immune response, is limited. In this study we investigate the effects of HT treatment using NIR on tumor regression and on the anti-tumor immune response in breast tumors. Results demonstrated that local HT by NIR at 43oC reduced tumor progression and prolonged the survival of tumor-bearing mice. Immunohistochemical analysis revealed a significant reduction in proliferation in treated tumor, which was accompanied by an abundance of heat shock protein 70 (Hsp70). Increased numbers of activated dendritic cells were observed in the draining lymph nodes of the mice, along with infiltration of T cells, NK cells and B cells into the tumor. In contrast, tumor-infiltrated regulatory T cells were largely diminished from the tumor. In addition, higher IFN-γ and lower IL-10 secretion was observed in blood of treated mice. Overall, this present study extends the understanding of using local HT by NIR to stimulate a favourable anti-tumor immune response against breast cancer.

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Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-000996 ◽

2020 ◽

Vol 8 (2) ◽

pp. e000996

Author(s):

Marion v Guerin ◽

Fabienne Regnier ◽

Maxime Thoreau ◽

Lene Vimeux ◽

Matthieu Benard ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Tumor Regression ◽

Time Frame ◽

Complete Regression ◽

Systemic Injection ◽

Tumor Relapse ◽

Tumor Outgrowth ◽

High Cytotoxicity

BackgroundTumor relapse constitutes a major challenge for anti-tumoral treatments, including immunotherapies. Indeed, most cancer-related deaths occur during the tumor relapse phase.MethodsWe designed a mouse model of tumor relapse in which mice transplanted with E7+ TC1 tumor cells received a single therapeutic vaccination of STxB-E7+IFNα. Unlike the complete regression observed after two vaccinations, such a treatment induced a transient shrinkage of the tumor mass, followed by a rapid tumor outgrowth. To prevent this relapse, we tested the efficacy of a local administration of IFNα together with a systemic therapy with anti-PD1 Ab. The immune response was analyzed during both the tumor regression and relapse phases.ResultsWe show that, during the regression phase, tumors of mice treated with a single vaccination of STxB-E7 + IFNα harbor fewer activated CD8 T cells and monocytes than tumors doomed to fully regress after two vaccinations. In contrast, the systemic injection of an anti-PD1 Ab combined with the peri-tumoral injection of IFNα in this time frame promotes infiltration of activated CD8 T cells and myeloid cells, which, together, exert a high cytotoxicity in vitro against TC1 cells. Moreover, the IFNα and anti-PD1 Ab combination was found to be more efficient than IFNα or anti-PD1 used alone in preventing tumor relapse and was better able to prolong mice survival.ConclusionsTogether, these results indicate that the local increase of IFNα in combination with an anti-PD1 therapy is an effective way to promote efficient and durable innate and adaptive immune responses preventing tumor relapse.

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Effect of Bruton Tyrosine Kinase Inhibitor on Serologic and Cellular Immune Responses to Recombinant Zoster Vaccine

Blood ◽

10.1182/blood-2021-148539 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1556-1556

Author(s):

Christopher Pleyer ◽

Kerry J Laing ◽

Mir Ali ◽

Christopher L McClurkan ◽

Susan Soto ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Immune Responses ◽

Injection Site ◽

Cellular Immune Response ◽

Research Funding ◽

Vaccine Response ◽

Cellular Immune Responses ◽

Serologic Response ◽

Cellular Immune

Abstract Introduction The recombinant zoster vaccine (RZV) is effective in preventing herpes zoster reactivation in the general population. We previously showed that patients with chronic lymphocytic leukemia (CLL), particularly those receiving Bruton tyrosine kinase inhibitors (BTKis), have decreased humoral immune responses following vaccination. The impact of vaccination on cellular immune responses in CLL patients is not well characterized. Understanding the effect of humoral and cellular immunity in CLL patients who are treatment naïve or receiving BTKis can inform vaccination strategies in this immunosuppressed patient population. Methods In this phase II open-label study (NCT03702231), patients with CLL who were either treatment naïve (TN) or receiving a BTKi (ibrutinib or acalabrutinib) received 2 doses of RZV via intramuscular injection at baseline and 3 months. Subjects were followed for 6 months and assessed for serologic response at 3 and 6 months. Serologic response was defined as a ≥ four-fold rise in anti-glycoprotein E (anti-gE) IgG serum titer at the 6 month timepoint. Cellular immune response was assessed by intracellular cytokine staining and flow cytometric analysis of gE-specific CD4+ T cells expressing upregulation of ≥2 effector molecules (interferon-γ, interleukin-2, tumor necrosis factor-α, and/or CD40 ligand). Cellular response was defined as ≥ two-fold rise over baseline and ≥320 net gE-specific CD4(2+) cells per million CD4+ T cells. Descriptive statistics were used to report vaccine response rates. Mann-Whitney test and Fisher's exact test were used to compare titers and response rates between different groups. Spearman r was used to measure the correlation between vaccine responses and clinical characteristics. All subjects completed an adverse event (AE) diary documenting any local (injection site) or systemic AE that started within 7 days after receiving the first and second vaccine dose. Results 106 subjects had serologic response assessment at 6 months. Baseline characteristics are shown in Table 1. The serologic response rate to RZV was significantly higher in the TN cohort (76.8%, 95% CI, 64.2-85.9; n = 56) compared to patients receiving a BTKi (40.0%, 95% CI,27.6-53.8; n = 50; P = .0002). Cellular vaccine response was assessed in 94 subjects at 6 months. Similarly, the rate of cellular immunity was significantly higher in the TN cohort (69.4%, 95% CI,55.5-80.5; n = 49) compared to patients treated with a BTKi (40.0%, 95% CI,27.0-54.5; n = 45, P = .0067). Paired serologic and cellular responses were available in 93 subjects. 68.5% (95% CI,55.3-79.3; n = 54) of subjects with a serologic response also had a positive cellular immune response, whereas 35.9% (95% CI,22.7-51.6; n = 39) of subjects attained a cellular immune response in absence of a serologic response (P = .0029) (Figure 1). Among subjects with a negative serologic response and a positive cellular immune response, 42.9% were TN (n = 6) and 57.1% (n = 8) received a BTKi. There was no difference in serologic or cellular responses between patients treated with ibrutinib and acalabrutinib (P > 0.05). Serologic antibody titers and T cell responder frequencies were weakly positively correlated (r = 0.26; 95%CI .05-.44; P = .0127). Serologic titers and T cell responses were not correlated with age, beta-2 microglobulin, absolute lymphocyte count, absolute peripheral blood CD19+, CD3+, CD4+ or CD8+ counts or serum immunoglobulin levels (IgA, IgG, IgM) (all P > 0.05). The most frequent local and systemic AEs were injection site pain (98.3%), injection site reaction (97.4%), headache (51.7%), and generalized myalgias (51.7%). Most AEs were grade 1-2 and all AEs resolved or returned to baseline within 7 days of vaccine administration. Conclusions RZV is safe in CLL patients and can induce both humoral and cellular immune responses. BTKi treatment was associated with impaired serologic and cellular vaccine responses compared to TN patients. Although BTKi therapy may inherently decrease vaccine immunogenicity, TN CLL patients could be more immunocompetent because of less advanced disease, thereby permitting more effective immune responses. The majority of patients with a positive antibody response also developed virus-specific T cells following vaccination. Approximately one third of patients without a positive serologic response developed virus reactive T cells. Figure 1 Figure 1. Disclosures Laing: Curevo Vaccine: Consultancy; MaxHealth LLC: Consultancy. Wiestner: Acerta Pharma: Research Funding; Pharmacyclics LLC: Research Funding; Merck: Research Funding; Nurix: Research Funding; Verastem: Research Funding; Genmab: Research Funding. Koelle: Merck: Research Funding; Curevo Vaccine: Other: Scientific Advisory Board ; MaxHealth LLC: Other: Scientific Advisory Board ; Oxford Immunotec: Research Funding; Sensei Biotherapeutics: Research Funding; Sanofi Pasteur: Research Funding. Sun: Genmab: Research Funding.

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