bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorescence-optimized 2-photon lifetime and kinetic imaging reveals reanimation of tumor immune surveillance by hyper-oxygenation
Authors: Rytelewski Mateusz , Harutyunyan Karine , Wspanialy Patrick , Nwajei Felix , Zal Malgorzata Anna , Vinogradov Sergei , Konopleva Marina , Zal Tomasz
Published: January 2023

Lymphocytes encounter varying oxygen levels as they traverse through healthy and diseased tissue. Hypoxia is a hallmark of the tumor microenvironment and can affect the anti-tumor immune response. However, despite the importance of T cell adaptation to differing oxygen concentrations in the tumor niche, there has been no method to study T cell spatiotemporal dynamics in the context of oxygen in vivo. To this end, we developed phosphorescence-optimized 2-photon lifetime and kinetic (2pOLAK) microscopy which enables co-imaging of phosphorescence lifetimes and cellular dynamics in highly fluorescent biological reporter systems. In conjunction with the PtP-C343 oxygen probe, 2pOLAK microscopy revealed the tissue oxygen landscapes and individual oxygen “experiences” of T cells as they moved through tissues in syngeneic models of metastatic lung cancer and acute leukemia. We found that T cells experienced hypoxia in leukemic bone marrow, and that the motility of these cells was significantly decreased relative to non-hypoxic T-cells in healthy bone marrow. Inhibition of oxidative phosphorylation slowed non-hypoxic T cell motility to a level comparable with that of hypoxic T cells. T cell motility was also decreased in hypoxic lung tumor cores, and it was significantly lower than in the tumor margin, where T cells experienced higher oxygen. Supplemental oxygenation increased the oxygen experienced by T cells in the tumor core and reanimated T cell motility. These studies describe a novel method for co-imaging tissue oxygen and cellular behavior, shed light on the role that oxygen availability plays in T cell dynamics in vivo, and suggest that counteracting hypoxia can improve tumor immune surveillance by restarting T cell motility.
Interaction between Kynurenine and the AhR is an effector mechanism of tumor immunosuppression and represents a potential immunotherapy target
Authors: Campesato Luis Felipe , Budhu Sadna , Tchaicha Jeremy , Pourpe Stephane , Liu Cailian , Manfredi Mark G. , McGovern Karen , Wolchok Jedd D. , Merghoub Taha
Published: January 2023

Although with notable impact on cancer treatment, immune-checkpoint blockade (ICB) have multiple mechanisms of resistance. The catabolism of Tryptophan into metabolites known as Kynurenines (Kyn) by enzymes such as IDO or TDO plays a major suppressive role in different tumor types. Recently it was shown that Kyn acts as an endogenous agonist of the Aryl-hydrocarbon Receptor (AhR). In order to gain a better understanding of this pathway, we sought to characterize the mechanisms of immunosuppression associated with AhR and evaluate its potential as therapeutic target. Gene-expression analysis of IDO-overexpressing melanomas (B16-IDO vs B16-WT) present reduced expression levels of Type-1 inflammatory genes, including IFNy, TNFa, Granzyme B and CD40. In addition, B16-IDO presents higher infiltration of TAMs, which up-regulate classic AhR-regulated genes (Cyp1a1 and Cyp1b1) and are differentially skewed towards an immunosuppressive M2-phenotype. Tumor-antigen specific CD8+T cells present reduced expression of activation markers (GzmB and CD44) and proliferation rate when primed by Kyn-treated BMDMs. Also, B16-IDO TAMs suppressed activation of CD8+T cells in vitro and their depletion abrogated tumor growth. Implantation of B16-IDOs in FoxP3-depleted mice prevents TAMs accumulation. Treatment of B16-IDO tumors with an AhR-specific antagonist (CH-223191) up-regulates MHC II in APCs, activation markers in CD8s and reduced frequency of T-regs. AhR inhibition slows down tumor growth and prolongs survival, which is improved in combination with PD-1 blockade. In summary, our findings demonstrate that targeting the Kyn pathway through AhR-inhibition represents a promising approach in cancer patients who are resistant to ICB.
Rapamycin prevents surgery-induced T cell exhaustion in patients with bladder cancer
Authors: Svatek Robert S. , Ji Niannian , Mukherjee Neelam , de Leon Essel , Kabra Aashish , Hurez Vincent , Nicolas Marlo , Michalek Joel E. , Javors Martin , Livi Carolina , Sharp Z. Dave , Curiel Tyler J.
Published: January 2023

The mechanistic target of rapamycin (mTOR) integrates environmental inputs to regulate cellular growth and metabolism in tumors. However, mTOR also regulates T cell differentiation and activation, rendering applications of mTOR inhibitors towards treating cancer complex. Recent preclinical data supports distinct biphasic effects of rapamycin with higher doses directly suppressing tumor cell growth and lower doses enhancing T cell immunity. We conducted a randomized controlled pre-surgical clinical trial to study the tumor and immune effects of low dose mTOR complex 1 inhibition with rapamycin for patients with invasive, non-metastatic bladder cancer. At 3 mg daily for four weeks, rapamycin significantly inhibited tumor mTORC1 (ribosomal protein S6 phosphorylation), and achieved high bladder tissue levels. Rapamycin also reduced surgery-mediated T cell exhaustion, evidenced by a significant decrease in the prevalence of dysfunctional programmed death-1 (PD-1) expressing circulating T cells without decreasing T cell proliferation or cytokine production. In mice challenged with MB49 orthotopic bladder tumors, a 2 cm surgical laparotomy reduces the efficacy of α-PD-L1 immunotherapy but efficacy can be partly restored with presurgical administration of rapamycin. These findings support favorable delivery, pharmacodynamic activity, and immune modulatory profile of low dose rapamycin in bladder cancer and identifies a novel role for mTOR inhibition in modulating surgery-induced immune dysfunction.
Crosstalk of sphingolipids and inflammasomes modulates tumor microenvironment and promotes cancer progression
Authors: Guo Beichu , Kantono Melvin , Sun Wei , Ogretmen Besim
Published: January 2023

Sphingosine-1-phosphate (S1P), a bioactive lipid, plays important roles in tumor development through its ability to promote tumor cell growth, migration, and invasion. S1P, synthesized by sphingosine kinase 1 and 2 (SK1 and SK2), can initiate multiple signal events through intracellular and extracellular mechanisms. In human cancer patients, increased levels of S1P and SK1 are correlated with poor prognosis and high incidence of diseases recurrence. How sphingolipid signaling regulates the function of immune cells and inflammation during tumor development remains poorly understood. Our results demonstrate that sphingolipid-induced inflammasome activation and IL-1β production promotes tumorigenesis and metastasis. Interestingly, our data indicate that SK-1/S1P signaling induces inflammasome activation and IL-1 production. We found that in MMTV-PyT transgenic mouse mammary gland tumor model, genetic loss of SphK1 significantly inhibited breast cancer progression and metastasis, accompanied by reduced levels of IL-1β at primary tumors and metastatic sites. Our results further show that SK1 and S1P modulate tumor microenvironments through induction of inflammasome activation and recruitment of myeloid cells. These findings indicate that the cross-talk between the SK1/S1P and inflammasome/IL-1 pathways promotes tumor growth and metastasis.
The efficacy of PD-L1 blockade on PD-L1 negative medulloblastoma is dependent on timing and the tumor microenvironment
Authors: Allen Frederick , Dorand Rodney Dixon , Rauhe Peter , Petrosiute Agne , Huang Alex Y.
Published: January 2023

PD-L1 blockade has shown great success in the rejection of specific PD-L1+ tumor-types within various tissues. Blocking PD-L1 interactions with PD-1 molecules expressed on effector T cells allow them to engage their targets unimpeded and enhances tumor rejections. However, most pediatric tumors and some adult tumors express low to no surface PD-L1. Interestingly, some reports have demonstrated the rejection of PD-L1 negative tumors after treatment with anti-PD-L1 antibodies (Abs). When we administered anti-PD-L1 Abs 7 days after injection of CRISPR-negative PD-L1 (crPD-L1neg) medulloblastoma (MM1) tumors, all tumors were rejected. However, treating with anti-PD-L1 Abs at the time of tumor inoculation (day 0), resulted in a greatly diminished response to further treatment and a loss of protection. In addition, analysis of day 7 treated tumors in comparison to isotype and day 0 treated tumors, revealed an ~4- and ~2-fold increase in PD-1+ CD8+ T cells by day 21, respectively. These data suggest that the efficacy of anti-PD-L1 Abs on PD-L1 negative tumors is dependent on the timing and the microenvironment in which the T cell priming occurs. Our lab is investigating this phenomenon by interrogating the interaction of DC-T cell priming in the tumor draining lymph nodes (TDLNs) during the early stages of tumor growth. These studies may lead to improved efficacy of immune checkpoint blockade in PD-L1 negative tumors.
The intrinsic pro-tumorigenic role of IRF1
Authors: Shao Lulu , Hou Weizhou , Scharping Nicole , Delgoffe Greg M. , Sarkar Saumendra N.
Published: January 2023

Interferon regulatory factor 1 (IRF1) is a nuclear transcription factor which appears to have anti-tumor activity in human and mice. IRF1 can be induced by interferon-gamma (IFNγ). Although recent studies indicated that the IFNγ-PD-L1 axis played an important role during T cell exhaustion, and promoted tumorigenesis, the cell intrinsic role of IRF1 is not completely clear in this context. In this study, we investigated the intrinsic role of IRF1 during tumor progression. We generated several IRF1-deficient (IRF1-KO) murine tumor cell lines (CT26, MC38 and B16-F10) via CRISPR/Cas9-mediated genome editing, and compared their growth rates with wild type (WT) cells both in vitro and in vivo using syngeneic mouse tumor models. We found that the loss of IRF1 did not affect cell growth rates in vitro. However, the IRF1-KO cells showed significantly slower tumor growth than WT cells in mice models. Depletion of CD8+ T cell using antibody rescued the ability of IRF1-KO cells to grow tumors. Furthermore, analysis of tumor infiltrating T cells showed more activated CD8+ T cells and less exhausted T cells in IRF1-KO induced tumors than those induced by WT cells. To confirm whether these results were caused by IFNγ-IRF1-PD-L1 axis, we evaluated IFNγ-induced inhibitory ligands on tumor cells in both WT and IRF1 KO cell lines. We found that the expression levels of MHC class I were not affected by loss of IRF1, whereas, the induction of PD-L1 by IFNγ was abolished in IRF1 KO cells. In conclusion, intrinsic loss of IRF1 in tumor cells significantly reduces tumorigenesis in solid tumors. This phenomenon depends on the regulation of PD-L1 expression on tumor cells via IFNγ-IRF1-PD-L1 axis, and eventually affecting host anti-tumor immune responses, such as CD8+ T cells.
ST8Sia6 overexpression in cancer and its effect on the tumor microenvironment
Authors: Friedman David J. , Shapiro Michael J. , Shapiro Virginia Smith
Published: January 2023

Modulating T-cell activity has been the primary focus in cancer immunotherapy. However, cancer cells can promote differentiation of macrophages into tolerogenic “M2” macrophages, which support tumor growth. Macrophage differentiation by cancer cells is influenced by many receptors including Siglec-E. Siglec-E recognizes α2,8-linked disialic acids on O-linked glycoproteins, and functions to inhibit activation of innate immune cells including macrophages. One enzyme that generates α2,8-linked disialic acids is ST8Sia6. COSMIC database analysis revealed that 389 tumors overexpressed, while none under-expressed, ST8Sia6, indicating a potential selective advantage for increased ST8Sia6 expression on tumors. Additionally, on a variety of cancers many patients with high expression of ST8Sia6 exhibit reduced survival as compared to patients with low expression. Therefore, we hypothesized that ST8Sia6 overexpressing tumors will engage Siglec-E, causing differentiation and expansion of M2 macrophages, and accelerating tumor growth. We demonstrated that ST8Sia6 increases M2 macrophage polarization in cell lines expressing ST8Sia6 compared to parental lines in vitro and in vivo. In vivo murine models also demonstrate increased tumor burden and reduced survival in mice injected with ST8Sia6 overexpressing MC38 tumors. Therefore, ST8Sia6 expression in tumors may suppress the immune response and promote tumor growth. ST8Sia6 may prove to be a “druggable” target for future cancer immunotherapies.
Single cell sequencing reveals regulatory role for T cell expressed microRNA-155 within the tumor microenvironment.
Authors: Huffaker Thomas , O’Connell Ryan
Published: January 2023

MicroRNA-155 (miR-155) is a pro-inflammatory miRNA that has been shown to regulate antitumor immunity. We recently demonstrated, utilizing miR-155 T cell conditional knockout mice (TCKO), that miR-155 is required by T cells to inhibit the growth of transplanted B16f10 melanoma tumors. To further understand the role of miR-155 expression within tumor infiltrating T cells and their impact upon the tumor microenvironment, we performed 10× single cell RNA-sequencing on tumor infiltrating immune cells from miR-155 TCKO mice and controls. At 12 days post administration of B16f10 OVA syngeneic tumors, CD45+ cells from the tumors of miR-155 TCKO mice and control mice were sorted via flow cytometry and single cell sequencing was performed. Results indicate that the immune cell landscape within these tumors consists of at least 11 distinct populations. This included an expanded population of Arg1 and Ym1 expressing cells of the myeloid lineage in tumors from miR-155 TCKO mice. Tumors from these mice were also deficient in a specific subset of CD8+ T cells that highly express interferon gamma, granzyme B, and perforin. miR-155 host gene was expressed by this population of effector CD8+ T cells in Wt control mice. These results demonstrate the utility of using single cell RNA-Seq to comprehensively profile immune cells that are responding to solid tumors. Further, the data clearly indicate that miR-155 expression by T cells is necessary for their recruitment and production of antitumor molecules, while at the same time preventing the accumulation of myeloid populations with known roles in suppressing tumor immunity. These data have implications for manipulating miR-155 in the clinic as a means to bolster tumor immunotherapy.
DcR3 acts as a tumor marker of metastatic cancers
Authors: Wan Xiaochun , Li Junxin
Published: January 2023

Decoy receptor 3 (DcR3) is a soluble protein that competitively binds Fas ligand and has an anti-apoptotic role. Abnormally elevated DcR3 in malignant tumors may help cancer cells metastasize by suppressing immune responses and establishing metastatic lesions. However, whether DcR3 is valuable in differentiating cancer metastasis from non-metastasis remains largely unclear. In this study, we produced anti-DcR3 monoclonal antibodies and established a sensitive ELISA (11–12000 pg/ml, R2= 0.9941) to measure serum levels of DcR3 in cancer patients. DcR3 was significantly elevated in gastric cancer (2.04 ± 1.01, P = 0.0061), lymphoma (1.62 ± 0.75, P = 0.041) and breast cancer (1.53 ± 0.51, P = 0.023). ROC (Receiver Operating Characteristic) analysis suggested DcR3 was a suitable biomarker for the diagnosis of gastric cancer (Sensitivity = 85.7%, Specificity = 90.0%, AUC = 82.3%). Importantly, serum DcR3 was positively correlated with platelet distribution width (PDW) (P = 2.45×10−6, R = 0.63) in metastatic cancers while negatively correlated with hemoglobin (HGB) (P = 0.002, R = −0.59) and hematocrit (HCT) (P = 0.001, R = −0.62) in non-metastatic cancers. DcR3×PDW÷(HGB×HCT), the novel indicator could be used to differentiate cancer metastasis from non-metastasis (Sensitivity = 75.0%, Specificity = 80.9%, AUC = 79.0%). These results reveal that DcR3 has a predictive value for cancer metastasis in combination with hematological traits.
Tumor necrosis factor receptor II (TNFR2) promotes the growth of mouse CT26 colon cancer
Authors: Chen Xin , Li Ping , Yang Xinyu , Miao Xiaosu , Luo Hua
Published: January 2023

TNFR2 is expressed by several tumor cells including colon cancer, Hodgkin lymphoma, myeloma, renal carcinoma and ovarian cancer. Recently, TNFR2 is becoming appreciated as an oncogene. However, the exact role of TNFR2 on the tumor development remains to be fully understood. In this study, we examined the tumorigenesis and growth of mouse CT26 colon cancer cell lines with genetically modified TNFR2 expression in syngeneic Balb/c mice. We used CRISPR/Cas9- induced loss-of-function approaches to obtain stable TNFR2 KO CT26 cell line and gain-of-function techniques to obtain TNFR2 overexpressing CT26 cell line. WT, TNFR2 KO and TNFR2 overexpressing CT26 cells were inoculated s.c. into the mice. Although TNFR2 overexpressing CT26 tumor grew markedly faster than TNFR2 KO CT26 cells, there was no difference in the initial tumorigenesis, Intriguingly, all TNFR2 KO CT26 tumors completely regressed 2 weeks after inoculation. The proportion of Foxp3+ Tregs, as well as their TNFR2 expression, in the draining LNs was markedly higher in mice bearing TNFR2 overexpressing CT26 tumor. The levels of soluble TNFR2 in the peripheral blood of CT26 tumor bearing mice were ~3 folds higher than that in tumor free mice, which is comparable with the elevated levels of soluble TNFR2 in human tumor patients. In mice bearing TNFR2 overexpressing CT26 tumors, the levels of soluble TNFR2 was >4 folds higher than that in mice bearing WT CT26 tumors. In vitro, exogenous TNF was able to up-regulate TNFR2 surface expression on CT26 tumor cells. Taken together, our data suggest that TNF-TNFR2 signaling pathway may play a major role in tumor growth and tumor immune evasion, and thus may represent an important therapeutic target.
Cheating death: a Fas-41BB immunomodulatory fusion protein obviates a death signal to enhance T cell function and adoptive therapy targeting leukemia and solid tumors
Authors: Oda Shannon K. , Anderson Kristin G. , Garcia Nicolas , Ravikumar Pranali , Daman Andrew , Bonson Patrick , Greenberg Philip D.
Published: January 2023

Fas/FasL signaling plays a significant role in the generation and persistence of tumors. We and others have detected FasL expression in the tumor microenvironment (TME) of human ovarian, pancreatic, and other cancers, where it can function to protect tumor cells from tumor-infiltrating lymphocytes. Adoptive immunotherapy, a promising treatment option, uses genetically modified T cells to eliminate tumors. However, efficacy of T cell immunotherapy is dampened by limited costimulation as well as increased inhibitory and death signals in the TME. We previously showed enhanced therapeutic efficacy by engineering T cells to express an immunomodulatory fusion protein (IFP) to convert the inhibitory CD200R signal to a costimulatory CD28 signal and questioned if IFPs could use a 4-1BB signal to enhance in vivo persistence and memory formation. 4-1BB forms a trimer when binding its ligand, that is requisite for an effective costimulatory signal. We and others engineered T cells to express 4-1BB with an ectodomain that dimerizes, however this IFP did not improve T cell function. We hypothesized a Fas IFP with a 4-1BB signal would effectively replace Fas signaling and provide costimulation, as Fas also trimerizes when engaged by ligand. T cells transduced with the Fas-4-1BB IFP exhibited enhanced accumulation and function in vitro. In a model of leukemia, Fas-4-1BB-transduced T cells eradicated otherwise lethal disease and exhibited increased persistence. Preliminary data in the autochthonous KPC pancreatic and ID8 ovarian cancer models support increased efficacy. We describe for the first time that an IFP can generate an effective 4-1BB signal to overcome obstacles in the TME and enhance T cell immunotherapy of solid and liquid malignancies.
Adoptive cell therapy using allergen-specific CD4-CD8-T regulatory cells to treat airway allergic disease in mice
Authors: Tian Dan , Zhang Chunpan , Jin Hua , Tian Yue , Sun Guangyong , Liu Kai , Shi Wen , Zhang Dong
Published: January 2023

Allergic asthma is a chronic inflammatory disorder of the airways. Traditional therapeutic strategies have been unsatisfactory in controlling the underlying pathology. New approaches that specific overcome the detrimental effects of immune dysregulation in allergic asthma are required. We have identified a new differentiation pathway for CD4−CD8− (double negative, DN) T cells that exhibits the potent immunosuppressive function in vitro and in vivo.

In this study, we adoptive transferred OVA323–339 primed DNT cells to a mouse model of ovalbumin (OVA)-induced allergic asthma intravenously. The adoptive transferred DN T cells mainly migrated to mediastinal lymph node and lung, exerted strong suppression on CD11b+ dendritic cells, T follicular helper (Tfh) cell proliferation and IL-21 secretion, which resulted in significantly ameliorated airway hyperresponsiveness, lung inflammation and granulocyte accumulation, markedly reduced mucus formation and OVA-IgG/IgE production.

To understand the antigen-specific mechanism of DN T cell, which is lack of CD4 molecule, we investigated the role of Lag3 in allergen recognition of DN T cell. We demonstrated that Lag3 deficiency impaired the generation of OVA-specific DN T cells. DN T cells acquired MHC-II of dendritic cells to obtain antigen specificity with the assistance of Lag3 molecule mainly through a trogocytosis process.

The effectiveness of ex vivo generated OVA-specific DN T cell to alleviate airway inflammation support the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the treatment of allergic asthma.
Differential induction of antibodies against immunogenic V1V2 and V3 regions of HIV-1 envelope by immune complex vaccines
Authors: Hioe Catarina E. , Kumar Rajnish , Upadhyay Chitra , Fox Alicia , Itri Vincenza , Peachman Kristina , Rao Mangala , Jiang Xungqing , Kong Xiangpeng , Zolla-Pazner Susan
Published: January 2023

Antibodies (Abs) are critical elements of immune defense against HIV-1. Abs induced during infection or after immunization with HIV-1 envelope (Env)-based vaccines often target immunogenic sites on the V1V2 and V3 regions of HIV-1 Env. Most of these Abs have poor neutralizing activity but display Fc functions, such as Ab-dependent cellular cytotoxicity and phagocytosis, that also can contribute to protection against HIV-1. Nonetheless, eliciting high-titer and durable Ab responses to V1V2 and V3 by active vaccination remains a challenge.

Our previous studies have shown Env/mAb immune complexes (ICs) to be superior vaccine immunogens vs Env alone for eliciting anti-V3 Abs. Our current study examined parameters that enhance IC immunogenicity by comparing IC vaccines made with different HIV Env (gp120-JRFL clade B, gp140-CN54 clade C, gp120-A244 clade E) and mAbs of different specificities (V3, V1V2, CD4 binding site [CD4bs]) in BALB/c mice. The data showed that greater titers of V3-binding and virus neutralizing Abs were induced by ICs made of clade B gp120 and mAbs to V1V2 or CD4bs vs uncomplexed gp120. IC made of clade B gp120 and mAb to V1V2 also induced greater V1V2-specific Ab-dependent phagocytosis. On the other hand, the gp120/V3 mAb IC elicited lower Ab titers to V3, V1V2, and gp120. Immunization with IC made with clade C gp140 and CD4bs mAb also induced higher titer of anti-V3 Abs, although it was not as potent as the clade B IC. In contrast, ICs made with clade E gp120 did not have the same effect. These results demonstrate the capacity of IC vaccines made of HIV-1 Env and selected anti-Env mAbs to modulate induction of Ab responses to V1V2 and V3, and this activity depends on the Env proteins used to form ICs.
Monoclonal antibody prophylaxis protects against lethal Ebola virus infection
Authors: James Rebekah , Stonier Spencer W. , Bakken Russell , Bornholdt Zachary A. , Dye John M.
Published: January 2023

Pan-Ebolavirus neutralizing antibodies MBP087 and MBP047, derived from a human EBOV survivor, are protective in rodent models when administered therapeutically two days after infection. Mutations in the Fc region of hIgG, known as YTE and XTND, prolong the half-life of hIgG so we incorporated these mutations into the framework of MBP087 an MBP047 to determine if the longer half-lives could provide protection with an extended pretreatment prior to infection. Wildtype (wt) (C57Bl/6) and mFcRn−/− hFcRn tg+/+ mice were given a single dose of Nicotiana (N)-derived MBP087 or MBP047 bearing a wt antibody framework or with the YTE or XTND mutations. MBP087-N or MBP087-YTE-N protected 80–100% of hFcRn tg mice against EBOV when administered 30 or 60 days prior to infection. Surprisingly, both MBP087-N and MBP087-XTND-N were also highly protective (80%) in wt mice over the same 30 or 60d window, showing surprising longevity and potency. Interestingly, wt MBP047-N protected 80% of wt mice when administered 30d prior to infection but provided little protection over 60d, regardless of YTE or XTND mutations. These data indicate that the utility of pan-Ebolavirus neutralizing antibodies can be expanded from a therapeutic use to a prophylactic use 30–60d prior to exposure. Expanding the utility of antibodies to prophylaxis may be advantageous in cases where vaccines are lacking or contraindicated due to patient restrictions.
Novel MVA-based vaccine expressing influenza NP and M1 activates cross-reactive T cell responses in human nasopharynx-associated lymphoid tissue (NALT)
Authors: Puksuriwong Suttida , Ahmed Muhammad Shamsher , Sharma Ravi , Krishnan Madhan , Sood Salil , McCormick Max , Leong Sam , Lambe Teresa , McNamara Paul , Gilbert Sarah C. , Zhang Qibo
Published: January 2023

Influenza virus infection continues to cause widespread morbidity and mortality, resulting in major challenges for healthcare systems. Current influenza vaccines are of limited efficacy against mismatched or emerging new virus strains. Recent efforts are focused on the development of novel vaccines capable of conferring broad protection against various influenza virus subtypes. We have studied the potential of Modified Vaccinia Ankara (MVA)-vectored vaccines expressing nucleoprotein and matrix protein 1 (MVA-NP+M1) to induce cross-reactive mucosal immunity using human adenotonsillar mononuclear cell (MNC) culture system with flow cytometry and ELISPOT analysis.

Influenza NP and M1 antigens were abundantly expressed following vaccine stimulation in MNC particularly in B cells and dendritic cells. M1-specific T cell response was analyzed in detail, and MVA-NP+M1 was shown to activate IFN-γ-producing CD8+ T cells against conserved M1 epitopes. M158–66-specific CD8+ T cells in HLA-matched individuals were markedly increased in an age-dependent manner. These M1-specific T cells coexpressed cytotoxic molecules such as perforin and granzyme A and B. Upon the recognition of M158–66 peptide, these cells displayed marked increase in surface CD107a expression (implicating degranulation), together with the production of pro-inflammatory cytokines including IFN-γ and TNF-α, that correlated to their ability to kill the peptide-pulsed target cells.

In summary, we showed MVA-NP+M1 activates conserved influenza antigen-specific cytotoxic T cell response in human NALT. The results suggest MVA-NP+M1 vaccine has the potential to induce cross-reactive mucosal T cell immunity against different types of influenza viruses.
Age-specific TLR7/8 adjuvant formulation overcomes hyporesponsiveness to neonatal acellular pertussis vaccination in a mouse model
Authors: Dowling David J. , Smith Alyson J. , Borriello Francesco , Brightman Spencer , Schüller Simone , Bazin Hélène , Burkhart David J. , Levy Ofer , Evans Jay T.
Published: January 2023

Infection is the most common cause of mortality early in life, in large measure due to suboptimal early life vaccination strategies as compared to older age groups. New adjuvants are absolutely cardinal to further optimize current immunization approaches. However, only a few classes of adjuvants are presently incorporated in vaccines approved for human use. Recent advances in the discovery and delivery of Toll-like receptor (TLR) adjuvants have provided a new toolbox for vaccinologists. Prominent among these candidate adjuvants are synthetic small molecule TLR7/8 agonists. The development of an effective infant Bordetella pertussis vaccine is now urgent because of the resurgence of pertussis in many countries, contemporaneous and likely related to the switch from whole cell to acellular vaccines. In this context, age-specific adjuvant strategies may be key to enhance early life immunogenicity by creating a vaccine formulation that induces both robust and persistent immunity (i.e., overcome waning immunity) to B. pertussis. In the present study, we firstly optimized the a) formulation delivery system, b) stability, and c) immunologic activity of novel small molecule imidazoquinoline TLR7/8 adjuvants towards human infant leukocytes. Next, in both adult and neonatal mouse models, we demonstrate that this TLR7/8 adjuvant can overcome neonatal hyporesponsiveness to acellular pertussis vaccination by driving Th1 favoring responses to a licensed acellular vaccine (DTaP). This potent immunization strategy may be of fundamental importance in vaccine development and could represent a new paradigm for effective pertussis immunization in early life.
Innate immunity limits protective adaptive immune responses against pre-erythrocytic malaria infection.
Authors: Minkah Nana K. , Sack Brandon K. , Sheikh Amina A. , Vaughan Ashley M. , Kappe Stefan H.I
Published: January 2023

Immunization with whole pre-erythrocytic stage malaria parasites that infect hepatocytes but are unable to cause blood stage infection constitutes one of the most promising strategies to protect against malaria infection. In rodent malaria models, whole parasite vaccines that exhibit substantial replication within hepatocytes engender superior, long-lasting strain- and stage-transcending immunity as compared to replication-deficient whole parasite vaccines. Liver stage infection with replication-competent Plasmodium parasites induces an innate immune response that is dependent on type I Interferon (IFN-1) signaling. However, it remains unknown how this IFN-1 response contributes to vaccine-engendered adaptive immunity. Using a replication-competent genetically attenuated parasite vaccine in an immunization regimen that confers partial protection in wildtype (WT) mice, we examined vaccine efficacy in mice lacking IRF3 or IFNAR. Surprisingly, IRF3−/− and IFNAR−/− mice showed superior protection against an infectious sporozoite challenge when compared to WT mice. Better protection correlated with a greater frequency of liver-resident memory CD8 T cells in immunized IFNAR−/− mice. Furthermore, IFNAR−/− mice exhibited lower levels of the T cell co-inhibitory receptors, PD-1 and LAG-3, a greater percentage of IFNγ-producing CD8 T cells, and eliminated liver stages more effectively. Taken together, we show that the Plasmodium engendered IFN-1 response impairs the generation of an optimal memory T cell response. To our knowledge, this study is the first description of a detrimental role for IFN-1 signaling on malaria vaccine efficacy and as such may necessitate a paradigm shift in rational malaria vaccine design.
A unique adjuvant combination modulates immune responses preventing vaccine-enhanced pulmonary histopathology after vaccination with fusion protein and challenge with respiratory syncytial virus
Authors: Lee Youri , Ko Eun-Ju , Lee Young-Tae , Kim Ki-Hye , Kwon Young-Man , Kang Sang-Moo
Published: January 2023

Vaccination of young children with alum-adjuvanted formalin-inactivated respiratory syncytial virus (FI-RSV) induced vaccine-enhanced disease (VED) after natural infection during epidemic season, resulting in hospitalizations and two deaths. There is no licensed RSV vaccine. VED was also observed in previous reports with fusion (F) protein RSV vaccines and adjuvants including oil-in-water emulsion, delta-inulin, and natural killer T cell agonist α-GalCer. In this study to search for adjuvants preventing RSV VED, we investigated a novel combination of monophosphoryl lipid A (MPL TLR4 agonist) and oligodeoxynucleotide CpG (CpG TLR9 agonist) in the RSV F protein vaccination. RSV specific IgG antibodies and neutralizing titers, and controlling RSV lung viral loads after challenge were similarly observed in the alum and MPL+CpG adjuvant groups of mice that received a single dose prime immunization with RSV F protein vaccine. Analysis of IgG isotypes and cytokines suggested the induction of T helper type 1 (Th1) immune responses by inclusion of MPL+CpG adjuvant in contrast to alum adjuvant biasing Th2 type immune responses. Most importantly, lung histopathology and infiltration of inflammatory innate immune cells (eosinophils, neutrophils) were not observed after F protein vaccination with MPL+CpG adjuvant whereas severe lung histopathology was induced after alum adjuvanted F protein vaccination and RSV challenge. The results in this study suggest a novel adjuvant approach to improve efficacy and safety of RSV subunit vaccines.
Reversing T cell Exhaustion Through Discovery of Small Molecules
Authors: Marro Brett S. , Zavareh Reza Beheshti , Teijaro John R. , Lairson Luke L. , Oldstone Michael B.A.
Published: January 2023

Inhibitory receptors (IR) function as critical regulators of immunity by tempering T cell responses to microbial infections. In humans, persistent viruses such as HIV, HCV and HBV as well as several types of cancer have adapted to exploit IR signaling by upregulating IR ligands, resulting in the suppression of T cell function (exhaustion) and escape from immunity. T cell exhaustion and T cell recovery, by blocking IR signaling, was first defined in C57BL/6 mice following infection with the Clone 13 variant of LCMV that results in varying degrees of suboptimal CD4 and CD8 T cell polyfunctionality, ranging from reduced to absent cytotoxic capacity of CD8 T cells to decreased production of anti-viral effector molecules such as IFN-γ and TNF-α. Chronicling the resurrection of anti-viral T cell effector function using antibodies that target IRs suggests that an in vitro phenotypic assay using the LCMV system could be an effective screening platform to identify small molecule(s) that disrupt pathways regulating T cell dysfunction. To this end, we developed a screening strategy that utilizes transgenic mice (IFN-γ-YFP) combined with high-throughput (HTS) flow cytometry to examine compounds that potentiate IFN-γ expression in exhausted T cells isolated from LCMV infected animals. Using this approach, we have screened a library of 10,000 investigational new drugs (IND). We have identified several unique hits among the INDs that encompass a broad range of known targets. Thus, this novel screening strategy offers an unprecedented opportunity to identify potent small molecules that influence T cell function in the context of persistent infection and cancer.
Hematopoietic stem/progenitor cells engineered with T cell receptors for immunotherapy for HIV infection
Authors: Joglekar Alok , Sandoval Salemiz , Jeppson John , Liu Zhe , Leonard Michael Troy , Swift Margaret , Baltimore David
Published: January 2023

HIV infection causes progressive depletion of CD4+ T cells and leads to severe immunodeficiency if left untreated. Current antiretroviral therapy can control infection, but does not cure it. The goal of this study was to develop a “functional cure” for HIV infection by providing a long-term, self-renewing source of antiviral immunity. To that end, we hypothesized that immunotherapy using engineered hematopoietic stem/progenitor cells (HSPCs) to express HIV-specific T cell receptors (TCRs) will be effective at controlling HIV. We first isolated six TCRs specific to the KK10 epitope from HLA-B27+ individuals and compared their function in vitro. Two TCRs, EC27 and EC5.5, were chosen for immunotherapy studies based on their superior function. To test HSPC-based immunotherapy, we transduced mobilized peripheral blood CD34+ HSPCs from HLA-B27+ healthy donors to express these TCRs and engrafted them in immunocompromised NOD/SCID/IL2Rγc−/− (NSG) mice. Transduced HSPCs were able to engraft mice and differentiate into T cells expressing the TCRs in peripheral blood and lymphoid tissues. Furthermore, engineered T cells isolated from engrafted mice showed KK10-driven expansion, cytotoxicity, and cytokine secretion. Expanded T cells were able to inhibit HIV-infection in vitro. Moreover, the functional activity of engineered T cells isolated from mice was comparable to modified primary T cells, highlighting their therapeutic potential. We are currently testing whether mice engrafted with transduced HSPCs can suppress HIV infection in vivo. If successful, these studies would demonstrate a “functional cure” of HIV infection, warranting further testing in clinical trials.
Co-delivery of mucosa-associated epithelial chemokine (MEC/CCL28) enhances anti-HIV-1 mucosal responses through CCR10 in the context of DNA vaccination
Authors: Gary Ebony Nicole , Kathuria Noshin , Bernui Mariana Estrella , Humeau Laurent , Weiner David B. , Kutzler Michele A.
Published: January 2023

Mucosal antibody plays a vital role in protection against HIV transmission. Mucosa-associated epithelial chemokine (CCL28) is secreted by mucosal epithelial cells following commensal colonization, and binds CC-chemokine receptor 10 (CCR10), expressed on IgA+ plasma cells. Deletion of CCR10 impairs mucosal IgA production and memory responses. Thus, the CCL28/CCR10 axis is critical for regulating mucosal IgA production and memory immunity. Mice were vaccinated twice with consensus HIV-1 gp160 DNA alone (antigen-only), or antigen and plasmid-encoded CCL28 (pCCL28) via intramuscular injection and in vivo electroporation (EP). pCCL28 increased antigen-specific IgA in fecal extracts (average 36.7ng/ml IgA in the antigen only group compared with 90.9ng/ml in the pCCL28 group). The intestinal compartment from animals co-immunized with pCCL28 harbored a higher frequency of HIV-specific, IgA+ B cells compared to the antigen-alone group (13.1% compared to 5.7%, respectively), and these cells expressed significantly-higher levels of CCR10 than those from mice in the non-adjuvanted group (MFI 413 compared to 181, respectively). Colonization with commensals induces CCL28 secretion by intestinal epithelial cells. Indeed, In vitro experiments with human colonic epithelial cell lines indicate that these organisms increased expression of CCL28 at the transcript and protein level. These studies suggest that electroporated delivery of the mucosal chemokine CCL28, can enhance antigen-specific anti-HIV immunity in the mucosa. Ongoing studies will determine if supplementation of commensal flora during vaccination can enhance CCL28 secretion in vivo and promote retention of vaccine-induced IgA+ B cells at mucosal surfaces.
Overcoming obesity-induced immunotherapeutic failure
Authors: Boi Shannon Kathryn , Orlandella Rachael , Gibson Justin Tyler , Starenki Dmytro , Makkouk Amani , Joshi Vijaya , Gross Brett , Salem Aliasger , Weiner George , Norian Lyse A.
Published: January 2023

Obesity is a leading public health concern that affects >37% of US adults and increases the risk of developing 13 types of cancer, including renal cancer. Renal cancer is treated by immunotherapy, but the impact of obesity on immunotherapy efficacy remains unclear. We addressed this issue by evaluating treatment efficacy in lean or diet-induced obese (DIO) mice with orthotopic renal tumors. Endogenous T cell priming with adenovirus-encoded TRAIL + CpG (AdTR/CpG) significantly reduced renal tumors in lean mice, but failed in ~80% of DIO mice. We then asked: 1) if failure were specific to this model and 2) if it could be reversed by reinvigoration of T cells with checkpoint blockade. Here, we report that obesity-induced impairment occurs across multiple immunotherapies and tumor types, and is independent of diet alone. Combining AdTR/CpG with checkpoint blockade (either anti-PD-1 or anti-CTLA-4) improved response rates in DIO mice with renal tumors to 44% and 67%, respectively. nanoString immunogenetic profiling of responder versus non-responder tumors revealed increases in T cell-related genes with concomitant decreases in myeloid/dendritic cell genes in responder mice (coined a ‘T cell-myeloid-inversion (TMI) signature’). These results were supported by flow cytometric data, which revealed increased intratumoral CD8+ T cell : Treg ratios and decreased myeloid-derived suppressor cells and tolerogenic dendritic cells in responders. Our results identify obesity as a confounder of immunotherapeutic efficacy that can be partially overcome by combining upstream T cell priming agents with checkpoint blockade. Further investigations will be aimed at targeting immunosuppressive populations to further boost therapeutic efficacy.
Characterization of a Single Nucleotide Variant of Ccdc85c as a Tumor Rejection-Mediating Neoepitope of Meth A Fibrosarcoma
Authors: Ebrahimi-Nik Hakimeh , Corwin William L. , Mandoiu Ion I. , Srivastava Pramod K.
Published: January 2023

An 18-mer peptide (named Neo1) containing a single amino acid variation (deduced from comparison of Meth A transcriptome with normal mouse genome) is capable of eliciting complete protection of BALB/c mice against a lethal challenge with Meth A fibrosarcoma cells. A similar peptide containing an un-mutated sequence does not elicit tumor rejection. In an attempt to identify the exact MHC-binding epitope within Neo1, nineteen smaller and distinct peptide constructs derived from Neo1 were tested in a prophylactic tumor rejection assay. The exact epitope was observed to be a 9-mer. This 9-mer neoepitope was not predicted to bind MHC I by NetMHC; NetMHC did predict four other peptides within Neo1 to bind Kd and Ld. None of these four predicted peptides elicited tumor rejection. Of note, even seemingly minor changes in the flanking regions of the precise neoepitope on either side of the peptide had a dramatic influence on anti-tumor immunogenicity of the peptide.
Roles of nitric oxide in hematopoietic differentiation induced by the vaccine adjuvant
Authors: Yang Ya-Wun , Liu Yen-Chi , Gupta Amit
Published: January 2023

N(ω)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), is involved in the pathogenesis of inflammatory disorders. The roles of nitric oxide (NO) in myeloid differentiation induced by the cancer vaccine adjuvant is unknown. The objective this study was to examine the effect of NOS inhibitor in the hematopoietic differentiation induced by the L121-adjuvant, an emulsion vaccine adjuvant containing Pluronic L121 previously shown to exhibit potent immune responses when combined with soluble protein antigens. C57BL/6J mice were vaccinated by subcutaneous injection on days 0 and 7 with 100 μl L121-adjuvant, containing 50 μg ovalbumin (OVA), followed by daily treatment with L-NAME (10 mg/kg). The animals were sacrificed on day 10, and bone marrow cells and splenocytes were isolated, lysed with the ammonium-chloride-potassium (ACK) lysing buffer, followed by flow cytometric analysis. Our results showed that vaccination of mice with the L121-adjuvant stimulated a considerable generation of CD11b+Gr-1+ myeloid populations in the bone marrow and spleens. Treatment of animals with L-NAME significantly upregulated the lineage−Sca-1+c-kit+ (LSK) frequency, but down-regulated the lineage− Sca-1− c-kit+CD16/CD32+ granulocytic/monocytic progenitor (GMP) populations in the bone marrow. These data demonstrated the involvement of NO in the modulation of granulocytic/monocytic differentiation and generation of inflammatory response in cancer vaccine adjuvant-mediate hematopoietic differentiation.
Immunologic synergy between intermittent NSAID administration and antigen specific vaccination in inhibiting the development of intestinal polyps in the APC(Min/+)
Authors: Gad Ekram , Corulli Lauren , Cowan Paul , Sei Shizuko , Grubbs Clinton , Disis Mary L.
Published: January 2023

Introduction

Studies from our group have shown that administration of NSAIDs in mouse models of Familial Adenomatous Polyposis results in a decrease in polyp formation and a significant increase in CD8 tumor infiltrating lymphocytes with downregulation of PD-L1 expression in tumors. We questioned whether a short course of NSAID could be used in combination with vaccination to even further reduce the development of intestinal polyps.

Methods

At 4–6 weeks of age, APC(Min/+) male/female mice were given CFA/IFA adjuvant with or without a multi-antigen (EGFR, COX2, CDC25B) peptide vaccine. Upon start of vaccinations, normal food or 400ppm naproxen mixed with food was provided either continuously (CON; 18w on) or intermittently (INT; 3w on + 3w off for 3 cycles). Animals were euthanized at 22±1 weeks and tumors quantified. Systemic and tissue specific immunity was measured by ELISPOT, FACS, and direct tumor staining.

Results

Tumor development was significantly inhibited both by vaccine alone and naproxen alone (p<0.0001). Combining naproxen (INT or CON) with vaccine was additive compared to vaccine alone (p<0.0001) and naproxen alone (INT+Vaccine p<0.0007; CON+Vaccine p<0.0003). INT naproxen was as effective as CON, with or without vaccine (p>0.999). Mice with greater tumor inhibition demonstrated higher magnitude antigen specific T-cell responses (p=0.0441, r=−0.423) and CD8+ T-cell infiltration (p<0.0001, r=−0.506) than those with less protection.

Conclusions

Data demonstrates that short-term administration of NSAIDs may be as effective as the more toxic long-duration alternative. Further, a short course of NSAID significantly synergizes with antigen specific vaccination to inhibit the development of polyps.
Role of glucose metabolism in herpes simplex encephalitis
Authors: Rajasagi Naveen K. , Rouse Barry T.
Published: January 2023

Herpes simplex virus-1 (HSV-1) is an important human pathogen without an effective vaccine. Following ocular infection the virus replicates in the epithelial tissues and is transported to the trigeminal ganglia (TG) where it establishes latency. Upon periodic reactivation, the virus multiplies in the TG and the replicating virus can reenter the cornea which could result in clinical consequences in many cases. Frequent recurrences can result in chronic inflammatory reactions in the corneal stroma which impairs vision and such recurrences can ultimately result in blindness. Although rare, the virus can spread to the brain and can cause serious complications such as encephalitis especially in immunocompromised individuals. The host immune responses, especially CD8 T cells play a crucial role in controlling virus replication in the TG and prevent spread to the brain. In mouse model of ocular infection, we found that blocking glucose metabolism using 2-deoxy glucose (2DG) resulted in increased virus spread to the brain. The majority of animals treated with 2DG succumbed to herpes simplex encephalitis (HSE). Our results demonstrated that 2DG treatment impacted on both the magnitude and function of the CD8 T cells in the draining lymph nodes and TG. These data show that glucose uptake is essential for HSV specific CD8 T cell responses to be appropriately induced and function and suggest that manipulation of glucose metabolism may serve to enhance anti-viral CD8 T cell responses acting to more effectively control infection.