Intratumoral Injection of TLR4 Agonist (G100) Leads to Tumor Regression of A20 Lymphoma and Induces Abscopal Responses

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 820-820 ◽  
Author(s):  
Idit Sagiv-Barfi ◽  
Hailing Lu ◽  
Jessica Hewitt ◽  
Frank J Hsu ◽  
Jan ter Meulen ◽  
...  
Keyword(s):  
T Cell ◽  
Cancer Cells ◽  
Research Funding ◽  
Tumor Regression ◽  
Cd8 T Cell ◽  
Significant Loss ◽  
Dependent Manner ◽  
Th1 Cell ◽  
Water Emulsion ◽  
Tlr4 Agonist

Abstract Background: Toll-like receptors (TLRs) are components of the innate immune system that recognize pathogen-associated molecular patterns present on bacterial, fungal, or viral pathogens. Glucopyranosyl lipid A (GLA) is a synthetic TLR4 agonist that activates dendritic cells (DC) and enhances antigen-specific Th1 cell responses. Scientific question: Can an oil-in-water emulsion of GLA (G100) induce an anti-tumor effect due to modulation of the tumor microenvironment? Results: In our A20 murine lymphoma tumor model, intratumoral (i.t) administration of G100 resulted in tumor regression in approximately 50% of the mice in a dose dependent manner with an optimal dose of 10-20 μg/mouse. Surviving mice remained tumor-free at three months post G100 treatment and were resistant to a secondary tumor challenge with A20 cells, but not with CT26 colon cancer cells or 4T1 breast cancer cells. The effect was CD8+ T cell mediated as CD8+ depletion resulted in a significant loss of the effect. To examine the potential systemic effect of G100 i.t. treatment, Balb/c mice were implanted bilaterally with A20 tumors. Injection of G100 into the ipsilateral tumor three times a week resulted in an abscopal effect on the contralaterally implanted, untreated tumor, as evidenced by reduced tumor growth. Furthermore, the potential synergy between G100 and immunomodulatory antibodies was investigated: mice with bilateral A20 tumors received intratumoral G100 alone, systemic anti-PD-1 mAb alone, or a combination of both agents. Mice receiving combination therapy showed best overall survival, as measured by regression or reduced growth of both treated and untreated tumors. In addition, injection of low doses of anti-CTLA4 and anti-OX40 mAbs together with G100, directly into the tumor, was able to cure mice with established disease. Significance: Immunomodulatory antibodies are currently under clinical development for cancer treatment. We show here that combining these antibodies with a TLR4 agonist is sufficient to trigger a systemic anti-tumor response able to eradicate tumor at a distant site. This anti-tumor immune response was long lasting, specific and required CD8+ T cells as the effect was lost in CD8+ T cell depleted mice. Impact: We recently published positive results of intra-tumoral CpG (TLR9 agonist) in combination with immunomodulatory antibodies (Marabelle, Levy, et al. JCI, 2013). Both anti-CTLA4 and anti-PD1 antibodies have been FDA approved, and anti-PD-L1 and anti-OX40 antibodies are currently in advanced clinical trials. Our results suggest that G100 alone or in combination with immunomodulatory mAbs may be a promising new treatment for patients with injectable sites of lymphoma. Clinical studies investigating intratumoral G100 are underway. Disclosures Off Label Use: G100- a TLR4 agonist. Lu:Immune Design: Employment. Hewitt:Immune Design: Employment. Hsu:Immune Design: Employment. Meulen:Immune Design: Employment. Levy:Immune Design: Research Funding; Dynavax: Research Funding.

scholarly journals SLAT Regulates CD8+ T Cell Clonal Expansion in a Cdc42- and NFAT1-Dependent Manner

2012 ◽  
Vol 190 (1) ◽  
pp. 174-183 ◽  
Author(s):  
Sonia Feau ◽  
Stephen P. Schoenberger ◽  
Amnon Altman ◽  
Stéphane Bécart
Keyword(s):  
T Cell ◽  
Clonal Expansion ◽  
Cd8 T Cell ◽  
Dependent Manner ◽  
Cell Clonal Expansion

scholarly journals 584 Powerful synergistic effects of a STING agonist and the IL-2 superkine, H9, in eliciting NK and T cell responses against MHC I- and MHC I+ tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A614-A614
Author(s):  
Natalie Wolf ◽  
Cristina Blaj ◽  
Lora Picton ◽  
Gail Snyder ◽  
Li Zhang ◽  
...  
Keyword(s):  
T Cell ◽  
Combination Therapy ◽  
Nk Cells ◽  
Nk Cell ◽  
Tumor Regression ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Mhc I ◽  
Therapy Regimen ◽  
Cell Responses

BackgroundMost current cancer immunotherapies are based on mobilizing CD8 T cell responses. However, many types of tumors evade CD8 T cell recognition by displaying few or no antigens, or losing expression of MHC I. These considerations underlie the need for complementary therapies that mobilize other antitumor effector cells, such as NK cells, which preferentially kill MHC I-deficient cells. Cyclic dinucleotides (CDNs) activate the cGAS-STING pathway of the innate immune system and are candidates as immunotherapy agents. Intratumoral CDN injections induce type I IFNs and other mediators that amplify the CD8 T cell response and induce tumor regression [1]. CDN therapy also induces long-term tumor regressions in some MHC I-deficient tumor models, mediated primarily by NK cells [2].MethodsTo extend the efficacy of CDN therapy, we combined the IL-2 superkine, H9, or half-life extended H9, with CDNs to target and activate NK cells in the tumor microenvironment and prevent or delay the onset of NK cell desensitization [3,4]. In these studies, we utilized B16-F10 and MC38 tumor cells lacking B2m to examine effects of the combination therapy on MHC I-deficient tumor growth as well as to examine the activation of NK cells by flow cytometry and cytotoxicity assays. We also utilized B16-F10 WT and the spontaneous tumor model, MCA, to assess the effect of the combination therapy on MHC I+ tumors.ResultsHere we show that H9 synergized with CDN therapy to mobilize much more powerful antitumor responses against MHC I-deficient tumors than CDN alone. The responses were mediated by NK cells and in some cases CD4 T cells, and were accompanied by increased recruitment to and sustained activation of NK cells in the tumor. This combination therapy regimen activated NK cells systemically, as shown by antitumor effects distant from the site of CDN injection and enhanced cytolytic activity of splenic NK cells against tumor cell targets ex vivo. Finally, the same combination therapy regimen synergistically mobilized powerful CD8 T cell responses in the case of MHC I+ tumor cells, suggesting the generality of the approach. The approach was effective against primary sarcomas, as well, especially when combined with checkpoint therapy, leading to tumor regressions and long-term survival of many mice with MCA-induced sarcoma.ConclusionsOverall, our work demonstrates the impact of a novel combination therapy in mobilizing powerful NK and T cell-mediated antitumor activity, providing important justification for evaluating this approach for treating cancers that are refractory to available treatment options.ReferencesCorrales, L., Glickman, L.H., McWhirter, S.M., Kanne, D.B., Sivick, K.E., Katibah, G.E., Woo, S.R., Lemmens, E., Banda, T., Leong, J.J., et al. (2015). Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. Cell Rep 11, 1018–1030.Nicolai, C.J., Wolf, N., Chang, I.C., Kirn, G., Marcus, A., Ndubaku, C.O., McWhirter, S.M., and Raulet, D.H. (2020). NK cells mediate clearance of CD8(+) T cell-resistant tumors in response to STING agonists. Science immunology 5, eaaz2738.Levin, A.M., Bates, D.L., Ring, A.M., Krieg, C., Lin, J.T., Su, L., Moraga, I., Raeber, M.E., Bowman, G.R., Novick, P., et al. (2012). Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’. Nature 484, 529–533.Ardolino, M., Azimi, C.S., Iannello, A., Trevino, T.N., Horan, L., Zhang, L., Deng, W., Ring, A.M., Fischer, S., Garcia, K.C., and Raulet, D.H. (2014). Cytokine therapy reverses NK cell anergy in MHC-deficient tumors. J Clin Invest 124, 4781–4794.

Overcoming resistance to anti-PD-1 with tumor agnostic NBTXR3: From bench to bed side.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 2591-2591
Author(s):  
Tanguy Y. Seiwert ◽  
Colette Shen ◽  
Jessica M. Frakes ◽  
Jiaxin Niu ◽  
Jared Weiss ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cell ◽  
Tumor Regression ◽  
Single Agent ◽  
Locally Advanced ◽  
Cd8 T Cell ◽  
Intratumoral Injection ◽  
Post Treatment ◽  
Primary Tumors ◽  
Systemic Control

2591 Background: Despite recent advances, resistance to immune checkpoint inhibitors (ICI), observed in over 80% of treated patients, is currently the main challenge in immuno-oncology. Intense efforts are being made to identify combination therapies that could improve ICI response rates. NBTXR3, a novel radioenhancer administered by direct intratumoral injection (ITI), is designed at the nanoscale to increase radiation therapy (XRT) dose deposit within tumor cells and subsequent tumor cell killing, without increasing toxicity to surrounding healthy tissue. Here we present evidence that NBTXR3 activated by XRT primes the immune system, producing an anti-tumor response, including activation of the cGAS-STING pathway, that overcomes anti-PD-1 resistance both in mice models and patients. Methods: Abscopal assays were conducted in immunocompetent mice. Anti-PD-1 sensitive or resistant tumor cell lines, were injected in both flanks of mice. Intratumoral injection of NBTXR3 (or vehicle) followed by XRT was performed in right flank (primary) tumors only. Some mice also received anti-PD-1 injections. Tumor growth was monitored, and tumor immune cell infiltrates analyzed by immunohistochemistry (IHC). Separately, in the phase II/III randomized Act.in.Sarc [NCT02379845] trial patients with locally advanced soft tissue sarcoma (STS) received either NBTXR3+XRT or XRT alone followed by tumor resection. Pre- and post-treatment tumor samples from patients in both groups were analyzed by IHC and Digital Pathology for immune biomarkers. The safety and efficacy (RECIST 1.1/iRECIST) of NBTXR3 plus stereotactic body radiotherapy (SBRT) in combination with anti-PD-1 is being evaluated in three cohorts of patients with advanced cancers [NCT03589339]. Results: Pre-clinical studies demonstrated that NBTXR3+XRT induces an immune response not observed with XRT alone and enhances systemic control. IHC showed significant increase of CD8+ T-cell infiltrates in both NBTXR3+XRT treated and untreated tumors compared to XRT alone. Similarly, increased CD8+ T-cell and decreased FOXP3+ Treg density (pre- vs post-treatment) was observed in tumor tissues from STS patients treated with NBTXR3+XRT. Furthermore, NBTXR3+XRT in combination with anti-PD-1 improved local and systemic control in mice bearing anti-PD-1 resistant lung tumors, as well as reduced the number of spontaneous lung metastases. Preliminary efficacy data from the first in human trial of NBTXR3+XRT in combination with anti-PD-1 showed tumor regression in the majority of patients (8/9). Of note, tumor regression was observed in 6/7 pts who had progressed on prior anti-PD-1. Conclusions: The clinical efficacy of NBTXR3+XRT has been demonstrated as a single agent. We now demonstrate that it potentiates anti-PD-1 treatment to overcome resistance mechanisms. These results highlight the potential of NBTXR3+XRT to positively impact the immuno-oncology field. Clinical trial information: NCT03589339.

Effect of intratumoral (IT) injection of the toll-like receptor 4 (TLR4) agonist G100 on a clinical response and CD4 T-cell response locally and systemically.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 71-71
Author(s):  
Yongwoo David Seo ◽  
Jing Zhou ◽  
Kevin Morse ◽  
Jeremy Patino ◽  
Sean Mackay ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Local Control ◽  
Soft Tissue Sarcomas ◽  
T Cell Response ◽  
Cd4 T Cell ◽  
Tumor Control ◽  
Water Emulsion ◽  
Tlr4 Agonist

71 Background: Soft tissue sarcomas (STS) are heterogeneous tumors which are morbid and lethal. G100 is under investigation in multiple clinical trials and contains a potent TLR4 agonist (oil-in-water emulsion of glucopyranosyl lipid A) that has been tested as vaccine adjuvant. We hypothesized IT G100 would induce robust local and potentially systemic anti-tumor immune response, leading to improved outcomes. Methods: 15 metastatic STS patients with superficial lesions were treated with weekly IT G100 for 8-12 wks; 12 patients received radiation (RT) for 2 wks to start, while 3 received IT G100 for 6 wks prior to RT. Biopsies and PBMC were collected pre and post treatment, and flow cytometry was performed on biopsies. TIL and PBMC were analyzed with TCR deep sequencing. PBMC were analyzed by single cell multiplex cytokine profiling. Results: No grade ≥ 3 toxicity was observed, and local tumor control was achieved in all evaluable tumors (14/14). Treated tumors tracked post-trial (mean 156 days) had persistent local control with 1 CR (7%), 1 PR (7%), and 11 SD (79%). In 3 patients with long term follow up, treated lesions remained controlled vs index lesions (-53% vs +31% at mean 235 days, p = 0.002). In all tumors after G100 alone, T cell infiltration increased. In P14, CD3 live cells in tumor rose from < 1% to 62%. PBMC clonality increased in 8/14 tested including P06, who had 4× increase in clonality and CR in the injected lesion; PBMC and TIL TCR overlap increased from 13.4% to 21.5%. P13 had a 2.3× rise in TIL clonality; the top clone (a CD4 T cell) expanded from 0.1% to 38% and expressed more TNFα than the rest (p < 0.0001). Single cell cytokine analysis of PBMC showed 7/13 (54%) increased in polyfunctionality (producing > 2 cytokines) in CD4 T cells; no consistent increase was seen in CD8 T cells. TNFα levels in pre-treatment monocytes correlated with PFS (R2= 0.5, p = 0.02). Conclusions: IT G100 is a viable agent for local control of metastatic STS lesions. With or without RT, G100 appears to cause CD4 T cell mediated local and systemic response. Combination of G100 with other immunomodulators could induce clinically significant systemic responses, as seen in follicular NHL treated with G100. Clinical trial information: NCT02180698.

scholarly journals Differential Regulation of Antigen-Specific CD8+ T Cell Responses by IL-12p40 in a Dose-Dependent Manner

2008 ◽  
Vol 180 (11) ◽  
pp. 7167-7174 ◽  
Author(s):  
Doo-Jin Kim ◽  
Je-In Youn ◽  
Sang-Hwan Seo ◽  
Hyun-Tak Jin ◽  
Young-Chul Sung
Keyword(s):  
T Cell ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Differential Regulation ◽  
Dependent Manner ◽  
Cell Responses ◽  
Dose Dependent

scholarly journals Mammalian Target of Rapamycin Complex 2 Controls CD8 T Cell Memory Differentiation in a Foxo1-Dependent Manner

Cell Reports ◽  
2016 ◽  
Vol 14 (5) ◽  
pp. 1206-1217 ◽  
Author(s):  
Lianjun Zhang ◽  
Benjamin O. Tschumi ◽  
Isabel C. Lopez-Mejia ◽  
Susanne G. Oberle ◽  
Marten Meyer ◽  
...  
Keyword(s):  
T Cell ◽  
Mammalian Target Of Rapamycin ◽  
Cd8 T Cell ◽  
Target Of Rapamycin ◽  
T Cell Memory ◽  
Dependent Manner ◽  
Cell Memory ◽  
Memory Differentiation ◽  
Cd8 T Cell Memory

scholarly journals IL-4 sensitivity shapes the peripheral CD8+ T cell pool and response to infection

2016 ◽  
Vol 213 (7) ◽  
pp. 1319-1329 ◽  
Author(s):  
Kristin R. Renkema ◽  
June-Yong Lee ◽  
You Jeong Lee ◽  
Sara E. Hamilton ◽  
Kristin A. Hogquist ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Antigen Specificity ◽  
Th1 Cell ◽  
Memory Cd8 T Cells ◽  
Expression Levels ◽  
Cell Pool

Previous studies have revealed that a population of innate memory CD8+ T cells is generated in response to IL-4, first appearing in the thymus and bearing high expression levels of Eomesodermin (Eomes) but not T-bet. However, the antigen specificity and functional properties of these cells is poorly defined. In this study, we show that IL-4 regulates not only the frequency and function of innate memory CD8+ T cells, but also regulates Eomes expression levels and functional reactivity of naive CD8+ T cells. Lack of IL-4 responsiveness attenuates the capacity of CD8+ T cells to mount a robust response to lymphocytic choriomeningitis virus infection, with both quantitative and qualitative effects on effector and memory antigen-specific CD8+ T cells. Unexpectedly, we found that, although numerically rare, memory phenotype CD8+ T cells in IL-4Rα–deficient mice exhibited enhanced reactivity after in vitro and in vivo stimulation. Importantly, our data revealed that these effects of IL-4 exposure occur before, not during, infection. Together, these data show that IL-4 influences the entire peripheral CD8+ T cell pool, influencing expression of T-box transcription factors, functional reactivity, and the capacity to respond to infection. These findings indicate that IL-4, a canonical Th2 cell cytokine, can sometimes promote rather than impair Th1 cell–type immune responses.

scholarly journals The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3202-3202
Author(s):  
Cameron S. Bader ◽  
Henry Barreras ◽  
Casey O. Lightbourn ◽  
Sabrina N. Copsel ◽  
Dietlinde Wolf ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
Advisory Committees ◽  
Hematopoietic Stem

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells, leading to damage of particular host tissues characteristic of GVHD. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to pro-inflammatory cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) aHSCT models. Here we show that STING rapidly promotes donor CD8+ T cell activation and recipient APC death early after aHSCT. To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of MUD C3H.SW bone marrow (BM) + T cells into irradiated B6 wildtype (WT) or STING-/- recipients. Colon tissue from STING-/- recipients had >2x reduction in IFNβ, TNFα and IL-6 mRNA vs WT. MUD STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 wks post-HSCT vs WT. Double chimerism studies showed that the absence of STING in non-hematopoietic cells was responsible for GVHD amelioration. Conversely, a single dose of the highly specific STING agonist DMXAA given in vivo increased IFNβ, TNFα and IL-6 mRNA expression in WT, but not STING-/-, colon tissue 48 hrs after transplant and increased GVHD scores and lethality post-HSCT. Post-transplant cytoxan treatment abolished the ability of DMXAA to augment GVHD, supporting the notion that STING signaling increases donor T cell activation during aHSCT. To evaluate the potential impact of STING in the clinical setting, we transplanted C3H.SW BM + T cells into mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STINGHAQ/HAQ) and found that these mice also exhibited diminished GVHD. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD aHSCT contrasts to reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) aHSCT (Fischer J, et al, Sci. Transl. Med. 2017). Since CD4+ and CD8+ T cells are central in MMUD and MUD GVHD, respectively, we hypothesized that STING's effect on the predominant T cell subset in each model may explain these seemingly paradoxical results in STING-/- vs WT recipients. Therefore, we transplanted MMUD BALB/c BM + CD8+ T cells into B6-WT and STING-/- mice and found that - in contrast to MMUD recipients of combined CD4+ and CD8+ T cells - STING-/- recipients developed lower GVHD clinical scores, reduced skin pathology and had lower frequencies of activated T cells 8 wks post-HSCT vs WT, supporting a role for STING in the promotion of CD8+ T cell-mediated GVHD. Next, we investigated if recipient APCs played a role in STING's enhancement of CD8+ T cell-mediatedGVHD. We found that STING-/- mice had greater frequencies and numbers of recipient splenic CD11b+CD11c+ APCs 1 day after MMUD B6 into BALB/c aHSCT (Fig. A). BALB/c-STING-/- APCs also expressed reduced MHC class I protein levels (Fig. B). Moreover, STING-/- recipient spleens contained lower numbers of donor CD8+ T cells producing IFNγ and TNFα (Fig. C). These data support the hypothesis that STING contributes to early activation of donor CD8+ T cells and elimination of recipient APCs. Next, to identify if the loss of host MHC II+ APCs affected subsequent donor CD4+ T cell activation, B6-Nur77GFP transgenic donor T cells were used to explicitly monitor T cell receptor signaling. Consistent with increased numbers of host MHC II+ APCs in the spleens of STING-/- recipients 1 day post-aHSCT, we found greater frequencies and numbers of donor Nur77GFP CD4+ T cells expressing GFP, CD69 and IFNγ in STING-/- spleens 6 days after transplant (Fig. D). In summary, our studies demonstrate that STING plays an important role in regulating aHSCT and provide one potential mechanism by which STING could promote CD8+ T cell-mediated GVHD yet diminish CD4+-mediated GVHD. Overall, our studies suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. Disclosures Blazar: BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding. Levy:Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

scholarly journals Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5

2020 ◽  
Author(s):  
Angel K. Kongsomboonvech ◽  
Felipe Rodriguez ◽  
Anh L. Diep ◽  
Brandon M. Justice ◽  
Brayan E. Castallanos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Toxoplasma Gondii ◽  
Cd8 T Cells ◽  
Immune Cell ◽  
Cd8 T Cell ◽  
Inflammasome Activation ◽  
Phenotypic Variance ◽  
Dependent Manner ◽  
Presentation Pathway

ABSTRACTHost resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite’s protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including ‘avirulent’ ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.AUTHOR SUMMARYParasites are excellent “students” of our immune system as they can deflect, antagonize and confuse the immune response making it difficult to vaccinate against these pathogens. In this report, we analyzed how a widespread parasite of mammals, Toxoplasma gondii, manipulates an immune cell needed for immunity to many intracellular pathogens, the CD8 T cell. Host pathways that govern CD8 T cell production of the immune protective cytokine, IFNγ, were also explored. We hypothesized the secreted Toxoplasma virulence factor, ROP5, work to inhibit the MHC 1 antigen presentation pathway therefore making it difficult for CD8 T cells to see T. gondii antigens sequestered inside a parasitophorous vacuole. However, manipulation through T. gondii ROP5 does not fully explain how CD8 T cells commit to making IFNγ in response to infection. Importantly, CD8 T cell IFNγ responses to T. gondii require the pathogen sensor NLRP3 to be expressed in the infected cell. Other proteins associated with NLRP3 activation, including members of the conventional inflammasome activation cascade pathway, are only partially involved. Our results identify a novel pathway by which NLRP3 regulates T cell function and underscore the need for inflammasome-activating adjuvants in vaccines aimed at inducing CD8 T cell IFNγ responses to parasites.

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