bluuebunny/crossref_metadata_2025 huggingface dataset:
CCL3 in the tumor microenvironment augments the antitumor immune response
Authors: Allen Frederick , Bobanga Iuliana , Barkauskas Deborah , Myers Jay , Huang Alex Y.
Published: January 2023

CCL3 is important in orchestrating cellular contacts in vaccinated lymph nodes (LNs) and enhancing memory T cell generation. Furthermore, dendritic cells (DCs) have been shown to mediate both tumor-tolerance and antitumor immunity in some tumor models. We hypothesize that incorporating CCL3 into tumor vaccines can enhance antitumor immunity. We injected into the footpad of naïve recipients, wild-type tumors (WTTUs) or CCL3-secreting (L3TUs) colon tumors (CT26). Immunocompetent mice injected with L3TUs resulted in a suppression of tumor growth compared to the WTTU group in a CD8+ T cell dependent manner. 1- and 5- days after injection with WTTUs or L3TUs, there was a superior accumulation of endogenous DCs and activated T cells in the draining lymph node (DLN) of the L3TU group. Concurrently bone marrow dendritic cells (BMDC)-tumor or -T cell co-culturing experiments were conducted to assess the effects the tumor and its microenvironment (+/− CCL3) has on DC function. In vitro experiments using BMDCs cultured with CCL3 enhanced their expression of MHC-I, CD86, and CCR7. BMDCs cultured with CCL3, then pulsed with SIINFEKL peptide, also showed an enhance capacity to proliferate OT-I (CD8+) T cells. In vivo analysis reveal that BMDCs injected directly into mice bearing L3TUs showed an enhanced ability to home to DLNs. Moreover, ElISpot and intracellular analysis of isolated T cells from LNs revealed an enhanced ability for T cells to upregulate IFNγ post-tumor injection with L3TUs. Together these data reveal that CCL3 in the tumor microenvironment leads to a reduction in tumor growth and improved T cell responses in the DLN, which may be a result of an enhanced capacity for DCs to home to the DLN and activate antitumor T cells.
Aβ vaccination in conjunction with bee venom derived phospholipase A2 (bvPLA2) ameliorates Alzheimer’s disease pathology through the induction of Aβ-specific Treg population in 3xTg-AD mice
Authors: Bae Hyunsu , Baek Hyunjung , Ye Minsook , Kang Geun-Hyung , Lee Chanju , Lee Gihyun , Choi Da Bin , Hwang Deok-Sang , Lee Junyoung
Published: January 2023

Alzheimer’s disease (AD) is the most common form of dementia and characterized by an imbalance between the production and clearance of amyloid-beta (Aβ) and tau proteins. Vaccination against Aβ peptide results in dramatic reduction of Aβ pathology in experimental mouse models. Our recent study demonstrated that bvPLA2, the major component of BV, causes immune tolerance by increasing the population of CD4+CD25+Foxp3+ Tregs in cisplatin-induced nephrotoxicity, an allergic asthma and Parkinson disease murine model. Here, we investigated that the effect of bvPLA2 to induce antigen-specific Tregs to ameliorate Alzheimer’s disease (AD) pathology through linked immunosuppression. First, we investigated whether bvPLA2 would improve the cognitive function and the pathological features of AD in Aβ-vaccinated-3xTg-AD mice. bvPLA2 treatment dramatically ameliorated learning and memory deficits in Aβ-vaccinated-AD mice. In addition, bvPLA2 significantly reduced Aβ deposits in the hippocampus and cortex region of AD mice, compared with the Aβ-vaccinated-AD mice group. Next, we systemically administered Aβ-specific Treg populations generated in the absence or presence of bvPLA2 into 3xTg-AD mice. Systemic transplantation of Aβ-specific Tregs into 3xTg-AD mice improved cognitive function and reduced deposition of Aβ plaques. Furthermore, adoptive transfer of Tregs generated in the presence of bvPLA2 showed reduced Aβ plaques and diminished learning and memory ability compared with Tregs generated in the absence of bvPLA2. This opens the possibility of new therapeutic strategy to target Tregs to tissue-specific antigens for the treatment of AD.
Direct activation of iNKT cells by CD1d bound glycolipid antigens leads to a different kinetics of iNKT activation and expansion
Authors: Kharkwal Shalu Sharma , Kharkwal Himanshu , Johndrow Christopher T , Carreno Leandro J , Jervis Peter , Zhang Lianjun , Hui Engyap , Fiser Andras , Zauderer Maurice , Besra Gurdyal S , Donda Alena , Porcelli Steven A
Published: January 2023

Invariant Natural Killer T cells (iNKT) are innate like T lymphocytes that recognize glycolipid antigens bound to CD1d molecule expressed by antigen presenting cells (APCs). iNKT cells modulate many aspects of immune responses and hence are targeted in many immunotherapeutic strategies. However, the prototypical iNKT agonist α-galactosylceramide (α-GC) and its synthetic analogues have limitations that include but are not limited to toxicity and iNKT anergy. Here, we report the development of an iNKT activating tumor targeted tertiary complex composed of a CD1d-antitumor ScFv antibody fusion protein covalently coupled to novel benzophenone linked α-GC analogs (BPGCs). Covalent complexes have dramatically improved stability and higher avidity to iNKT TCR and hence up to ten times lower effective dosage of the complex can stimulate strong immune response. Notably, these complexes directly activate iNKT cells bypassing the need for APCs. Hallmarks of covalent complex mediated iNKT activation include strong and sustained IFN-y secretion with no extensive iNKT proliferation or upregulation of co-inhibitory markers such as PD-1, even after multiple treatments as opposed to free α-GC and non-covalent complexes. In vitro, anti Her2 ScFv.CD1d.BPGC complexes lead to iNKT mediated targeted killing of MC38 cells overexpressing human Her2. Preliminary antitumor immunotherapy experiments in mice have shown that one third of the covalent complex treated mice show substantial reduction in tumor growth, including apparent eradication of tumors in some cases. Altogether, our approach enables a strong iNKT-based immune activation without the need for APCs and provides long lived and localized NKT activation in the tumor microenvironment.
Fucoidan prevents LPS-induced production of nitric oxide and prostaglandin E2 by inhibiting MAPK-mediated signal pathways in RAW 264.7 macrophage cells
Authors: Jang Yong-Suk , Park Jisang , Hwang Seung-Mi , Ko Eun-Sil , Choi Kyung-Min , Cha Jeong Dan
Published: January 2023

Fucoidan is a sulfated polysaccharide that is primarily extracted from brown seaweeds which has been broadly studied in recent years due to its numerous biological properties, including anticoagulant, antithrombotic, antitumor, and antiviral activities. In this study, we investigated whether fucoidan has the ability to attenuate the expression of pro-inflammatory mediators such as NO, prostaglandin E2 (PGE2), TNF-α, and IL-1β in LPS-stimulated RAW 264.7 macrophage cells. Fucoidan inhibited the expression of LPS-induced pro-inflammatory mediators including NO, PGE2, TNF-α, and IL-1β. In addition, gene expression of iNOS, cyclooxygenase-2 (COX-2), TNF-α, and IL-1β was inhibited both at mRNA and protein levels by fucoidan treatment, without any cytotoxic effect. Moreover, fucoidan significantly inhibited LPS-induced NF-κB activity NF-κB translocation into the nucleus by preventing the degradation of iκB-α. Taken together, these results indicate that fucoidan downregulates the expression of pro-inflammatory genes involved in the synthesis of NO, PGE2, TNF-α, and IL-1β in LPS-stimulated RAW 264.7 macrophage cells by suppressing NF-κB activity.
Alternating electric fields (TTFields) induce immunogenic cell death resulting in enhanced antitumor efficacy when combined with anti-PD-1 therapy
Authors: Giladi Moshe , Voloshin Tali , Shteingauz Anna , Munster Mijal , Blat Roni , Porat Yaara , Schneiderman Rosa S , Cahal Shay , Itzhaki Aviran , Kirson Eilon , Weinberg Uri , Palti Yoram
Published: January 2023

Tumor Treating Fields (TTFields) are an effective anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. This therapy is approved for the treatment of patients with glioblastoma. Previous investigations have shown that TTFields disrupt microtubules and septin filaments, both of which govern key roles in mitosis. Some of the outcomes of mitosis under TTFields application include abnormal chromosome segregation and ER stress which trigger different forms of cell death. In this study, we demonstrate that application of TTFields leads to the exposure of calreticulin on the surface of different cancer cells in vitro. In vivo, the combined treatment of lung tumor-bearing mice with TTFields and anti-PD-1 led to a significant decrease in tumor volume compared to control group and to animals treated with anti-PD-1 alone. An increase in CD45+ tumor infiltrating cells was observed in both anti-PD-1 and TTFields+anti-PD-1 groups although statistical significance was reached only in the combination treatment group. Interestingly, CD45+ cells from the combination treatment group also demonstrated a significant upregulation of PD-L1 expression on the cell surface. Specifically, this upregulation in the PD-L1 expression was observed in both F4/80+CD11+ cells and CD11c+ cells whereas no significant effect on the infiltration pattern of these immune cell populations was noted. Taken together, our results demonstrate that TTFields application potentiates immunogenic cell death in cancer cells and that combining TTFields with specific immunotherapies such as anti-PD-1 might achieve tumor control by further enhancing antitumor immunity.
Pharmacological activation of the p53 pathway within the tumor microenvironment induces immunogenic cell death of tumors and enhances antitumor immunity
Authors: Cui Yan , Yu Miao , Xiao Wei , Guo Gang
Published: January 2023

The tumor suppressor p53 is the most frequently mutated genes in human tumors. Thus, targeting the p53 pathway is important. Small molecules that activate wild type p53 or revert p53 mutants to wild type conformation have been developed. Despite effective tumor killing, these molecules as mono-therapy fail to reach all tumor cells. Interestingly, recent studies from our laboratory and others showed that p53 inactivation/dysfunction in the TME skews the immunological landscape towards pro-tumor/inflammation. We, thus, propose that activating (re-activating) p53 reverses tumor-induced immunosuppression and enhances antitumor immunity. Here, we examined whether pharmacological activation of the p53 pathway in the TME lead to immunogenic cell death (ICD) of tumors. As expected, tumors maintaining functional p53, such as EL4 and B16, initiated ICD upon treatment with a p53 activator, nutlin-3a, shown as the release of HMGB1 and ATP to extracellular space. Intratumoral injection of nutlin lead to mobilization and activation of immune cells and antitumor immunity. This resulted in tumor regression not only on the side of injection, but also those contralateral to the injection. Moreover, mice experienced completely tumor regression due to nutlin-3a injection rejected the same tumor in subsequent re-challenges. This tumor rejection was mostly mediated by CD8 T cells because it was alleviated by CD8, but not CD4, depletion. Together, our results suggest that p53 activation within the TME serves as a dual agent, direct killing of tumors and subsequent activation of antitumor immunity. Thus, pharmacological activation of p53 can be used in combinational therapies for promoting more durable, specific, and systemic antitumor immunity.
Artificial antigen presenting cells with simultaneously releasing IL-2 and anti-CTLA-4 showed enhanced capability in antigen specific T-Cells activation and tumor inhibition
Authors: Zhang Lei , Zhang Yi-Ming , Shahzad Khawar Ali , Shen Chuan-Lai
Published: January 2023

Artificial antigen presenting cells (aAPCs) are thought to be a powerful tool to induce antigen specific T cells responses. Thus far, aAPCs were synthesized by coupling antigen specific signals (signal 1) and the co-stimulatory molecules (signal 2) onto microparticles surface. To fully activate and proliferate antigen specific T cells, the optimized aAPCs should be consider signal 3 in the form of various cytokines secretion.

IL-2 and anti-CTLA-4 are both effective immunotherapeutic agents for tumors. Therefore, encapsulating these two important molecules into aAPCs might increase their stimulating function through modulation of two oppositely cell signaling pathways.

Here, we prepared aAPCs with simultaneously releasing IL-2 and anti-CTLA4 (aAPCIL-2+ anti-CTLA4) based on PLGA microparticles. A high density of functional molecular ligands for recognition (H-2Kb/TRP2) and co-stimulation (anti-CD28) of T cells were attached to the particles surface.

Our results showed that a suitable aAPCIL-2+ anti-CTLA4 were fabricated successfully, IL-2 and anti-CTLA-4 were released in a sustained manner. Our modified aAPCIL-2+ anti-CTLA4 showed an enhanced capability to generate and expand antigen specific T cells. Furthermore, the aAPCIL-2+anti-CTLA4 generated T cells displayed more effective specific killing abilities. In addition, in vivo studies also demonstrated an enhanced tumor inhibition capability over traditional aAPCs.

Thus, the novel aAPC system aAPCIL-2+ anti-CTLA4 we fabricated here showed a unique potential to enhance the application of the aAPC technology for future immunotherapy approaches.
The TLR4 agonist vaccine adjuvant, GLA-SE, requires canonical and atypical mechanisms of action for TH1 induction
Authors: Cauwelaert Natasha Dubois , Desbien Anthony L , Hudson Thomas E , Pine Samuel O , Reed Steven G , Coler Rhea N. , Orr Mark T
Published: January 2023

The Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant formulated in a stable emulsion (GLA-SE) promotes strong TH1 and balanced IgG1/IgG2 responses to protein vaccine antigens. This enhanced immunity is sufficient to provide protection against many diseases including tuberculosis and leishmaniasis. To better characterize the adjuvant action it is important to understand how the different cytokines and transcription factors contribute to the initiation of immunity. In the present study using T-bet−/− and IL-12−/− mice and a blocking anti-IFNαR1 monoclonal antibody, we define mechanisms of adjuvant activity of GLA-SE. In accordance with previous studies of TLR4 agonist based adjuvants, we found that TH1 induction via GLA-SE was completely dependent upon T-bet, a key transcription factor for IFNγ production and TH1 differentiation. Consistent with this, deficiency of IL-12, a cytokine canonical to TH1 induction, ablated TH1 induction via GLA-SE. Finally we demonstrate that the innate immune response to GLA-SE, including rapid IFNγ production by memory CD8+ T cells and NK cells, was contingent on type I interferon, a cytokine group whose association with TH1 induction is contextual, and that they contributed to the adjuvant activity of GLA-SE.
CD16-IL15-CD33 Trispecific Killer Engager (TriKE) induces NK cell expansion, persistence, and myeloid blast antigen specific killing.
Authors: Felices Martin , Vallera Daniel A. , McElmurry Ron , Lenvik Alexander J.L. , Chu Sami , Todhunter Deborah , Taras Elizabeth P. , Tolar Jakub , Blazar Bruce R , Miller Jeffrey S
Published: January 2023

NK cell infusions induce complete remissions in 30–50% of patients with refractory AML when combined with lymphodepleting chemotherapy and IL-2. NK cell therapy is limited by lack of antigen specificity and by IL-2 mediated induction of Tregs. To overcome these limitations we developed a 161533 trispecific killer engager (TriKE) molecule containing an anti-CD16 scFv, to engage NK cells, an anti-CD33 scFv, to engage AML targets, and an IL-15 molecule. We have shown previously that a 1633 bispecific killer engager (BiKE) is capable of creating an immunologic synapse between NK cells and AML targets leading to target killing. These molecules generate higher affinity interactions than natural CD16 binding to Fc portions of therapeutic antibodies, resulting in increased NK cell functionality. Addition of IL-15 in the TriKE molecule is meant to enhance NK cell priming, proliferation, and maintenance without inducing Tregs. Cytotoxicity assays and cytokine secretion assessment of NK cells incubated with CD33+ HL-60 target cells indicated that the TriKE induced greater NK cell activation than the BiKE or no molecule control. The TriKE was able to induce potent NK cell proliferation and survival when compared to the BiKE. Interestingly, when compared to recombinant IL-15 the TriKE induced less proliferation of T cells while inducing similar proliferation of NK cells from healthy donors, indicating specificity. The TriKE was capable of rescuing NK cell function of post-transplant patients. In vivo functionality was tested in an NSG xenogeneic model engrafted with HL-60-luc targets and NK cells. TriKE treatment resulted in reduced tumor burden and NK cell maintenance at day 21, displaying the immunotherapeutic potential of the molecule.
A small-molecule RIG-I agonist functions to enhance vaccine protection against influenza A virus infection.
Authors: Hemann Emily A , Grigg John B , Wilkins Courtney R , Knoll Megan , Iadonato Shawn P , Bedard Kristin , Probst Peter , Loo Yueh-Ming , Gale Michael
Published: January 2023

Viral sensing by RIG-I and downstream activation of antiviral defenses along with the induction of innate immune cytokines is essential from protection against influenza A virus (IAV) infection. We have identified a novel, small-molecule RIG-I agonist, KIN1148, which binds and activates RIG-I to signal the activation of IRF3 and the innate immune response. We are developing this molecule as an adjuvant to enhance vaccination against pandemic H1N1 (pH1N1) IAV. Ex vivo treatment of dendritic cells with KIN1148 leads to their activation and maturation. We determined the ability of KIN1148 to enhance suboptimal IAV vaccine responses in vivo. Administration of KIN1148 leads enhanced protection during high dose pH1N1 infection following a single, intramuscular administration of KIN1148 with IAV vaccine. This increase in protection is accompanied by a significant reduction in virus titers, as well as lung pathology. Analysis of the immune response induced following vaccination with KIN1148 as well as challenge demonstrates an increase in chemoattractant cytokines, germinal center B cells, IAV-specific antibodies, and IAV-specific CD4 and CD8 T cells compared to vaccination alone, indicating the induction of a broad anti-IAV immune response. Together these results demonstrate that prophylactic drug targeting of the RIG-I pathway with a small molecule enhances vaccine protection and highlight the potential of KIN1148 to enhancing vaccines against RNA virus infection.
A trivalent alphavirus virus-like replicon particle vaccine induces cell-mediated responses against conserved glycoprotein regions
Authors: Cohen Courtney A , Bakken Russ , Glass Pamela J
Published: January 2023

Venezuelan (VEEV), eastern (EEEV) and western (WEEV) equine encephalitis viruses are highly infectious and neuroinvasive when aerosolized, making them threats for weaponization. In contrast to subcutaneous infection, studies suggest that protection from aerosol challenge is partially cell-mediated, albeit poorly understood. A trivalent (VEEV/EEEV/WEEV) virus-like replicon particle (triVRP) vaccine is protective against aerosol challenge in mice, yet the correlates of protection are unclear. To investigate, mice were vaccinated (day 0 and 28; PBS or triVRP) and spleens were harvested for analysis. Using an ex vivo splenocyte expansion assay with peptides from VEEV, EEEV and WEEV glycoprotein libraries, IFNγ-producing memory CD8+ T cells were identified. T cell-specific epitopes were mapped, and the most immunodominant were found within regions of sequence homology between strains. Thus, ongoing studies evaluate potential monovalent vaccine cross-protection, which would greatly reduce manufacturing costs. Understanding vaccine-induced responses will inform rational design and clinical evaluation.
Rapamycin adjuvant therapy did not improve the outcome of severe influenza virus infection in an experimental mouse model system
Authors: Huang Ching-Tai , Dutta Avijit , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Kao Kuo-Chin , Hung Chen-Yiu , Chang Chia-Shiang , He Yueh-Chia , Huang Yu-Lin
Published: January 2023

The host immune system responds to influenza virus infection for eradication of the virus but the immune response associated inflammation causes tissue injury and damage to the host as well. Severe influenza with morbidity and mortality is commonly believed to be the result of overwhelming immune response. In addition to antiviral agent, adjuvant therapy with immune modulating agent is a reasonable approach for severe influenza and has been reported to be effective in clinical series. As the immunity is also critical for virus eradication and recovery, we investigated to try to define the appropriate window for immune modulation in our murine animal model of influenza virus infection. Rapamycin treatment in a wide dose range did not alter the clinical course of severe influenza. In combination with antiviral agent Oseltamivir, rapamycin failed to offer additional benefit beyond treatment with Oseltamivir alone. Oseltamivir barely rescued the mice from severe influenza if the treatment was delayed until 48 hours after infection. Rapamycin did not provide salvage effect in this scenario. In fact, rapamycin suppressed influenza antigen specific CD4+ T cell response and impaired the virus eradication capacity. The virus burden in the lung increased with intense neutrophil infiltration and hyaline deposition in the alveoli. In conclusion, rapamycin cannot be relied upon as a salvage measure in severe influenza with progressive illness under treatment with Oseltamivir. T cell immunity is suppressed but other arms of immunity are recruited with undesirable effect on the disease outcome.
Identification of a unique lipid from Francisella tularensis as an effective therapeutic for viral mediated inflammation
Authors: Bosio Catharine M , Nardone Glenn , Broeckling Corey , Chiramel Abilash , Best Sonja , Ireland Robin
Published: January 2023

Francisella tularensis (FT) is a highly pathogenic bacterium which can cause acute, lethal disease following exposure to fewer than 20 bacteria. FT is a successful pathogen due to its ability to potently and rapidly suppress inflammatory responses in host cells. We previously established that crude lipid preparations from virulent FT dampen inflammation in vitro and in vivo, but did not characterize the active specie(s). Utilizing a variety of biochemical techniques, we identified one of the active lipids found in FT as a unique phosphatidylethanolamine (PE) consisting of tetracosanoyl and decanoyl acyl chains (PE2410). Similar to crude lipid, enriched PE2410 restricted production of cytokines in human and mouse cells stimulated with well-defined microbial ligands. PE2410 was also effective at dampening inflammation provoked by infection with West Nile Virus. Activity of PE2410 was dependent on presence of the host phosphatase SHP-1. Importantly, a synthetic version of the PE2410 mimicked the ability of natural PE2410 to inhibit inflammation triggered by a variety of sources. Together our data support use of PE2410 as a novel therapeutic for pathogenic inflammation triggered by viral, or other, infection.
Suppression of dendritic cell necroptosis by pandemic influenza virus
Authors: Hartmann Boris M. , Albrecht Randy A , Marjanovic Nada , Sealfon Stuart C
Published: January 2023

The public health risk of a new pandemic influenza virus (IAV) approaching the scope and mortality of the devastating 1918 strain makes it important to identify virus-host mechanisms unique to pandemic strains. Human dendritic cell infection by two different H1N1 seasonal IAV strains (1991 and 1999) induces widespread RNA degradation and programmed cell death beginning at 4 h after infection. Characteristic of necroptosis, the cell death showed nuclear condensation and fragmentation that was caspase independent and RIP3 kinase dependent. Notably, infection with the pandemic 1918 and 2009 H1N1 IAV strains did not induce dendritic cell RNA degradation and necroptosis. Furthermore, necroptosis induced in dendritic cells by poly I:C exposure was suppressed by concomitant infection with the pandemic 2009 IAV. These results indicate that infection with the pandemic viruses not only fails to induce the dendritic cell necroptosis seen with the seasonal IAV strains, but that it actively suppresses necroptosis. Virus chimera studies implicate the HA virus segment as mediating the necroptosis inhibition by the pandemic IAV strains. Necroptosis leads to the release of intracellular components which serve as danger associated molecular patterns (DAMP), making this a highly immunogenic cell death mechanism. Consistent with this view, we find that the suppression of virus-induced necroptosis by the pandemic IAV reduces T cell activation. These studies show that pandemic viruses are unique in suppressing a key immunological danger signal. The suppression of the generation of DAMPs from infected immune cells may contribute to the pathogenicity of pandemic H1N1 IAV viruses.
Influenza exposure history dictates cellular dynamics in CD8 T cell-mediated lung protection
Authors: Van Braeckel-Budimir Natalija , Martin Matthew D , Hartwig Stacey , Badovinac Vladimir P , Harty John T
Published: January 2023

Influenza-specific memory (M) CD8 T cells directed against conserved viral antigens can mediate protection against heterosubtypic influenza infections. In the light of flu seasonality, it is important to understand how multiple influenza exposures shape specific M CD8 T cells and their capacity to provide protection. To address this major knowledge gap we studied primary (1M) and secondary (2M) influenza-specific endogenous M CD8 T cell responses in mice. We show that secondary influenza infection generates large, long lasting, Ag-specific CD8 T cell responses, which exceed 1M responses by 5–20× in all tissues analyzed. To evaluate CD8 T cell-mediated protection, without the contribution of other immune mediators induced by multiple influenza exposures, we used intranasal infection with vaccinia virus expressing influenza Ag. Interestingly, despite the large difference in numbers of Ag-specific 1M and 2M CD8 T cells, both groups of mice showed similar control of infection in the lung. Importantly, initial numerical differences between 1M and 2M CD8 T cells in the lung disappear with the course of infection (days 0–3), due to superior accumulation of 1M compared to 2M CD8 T cells, as shown by Pertussis toxin-induced migration block. Thus, while protection mediated by 2M CD8 T cells relies mainly on a large number of lung-residing 2M CD8 T cells, 1M CD8 T cells display superior recruitment to the target tissue and exhibit higher proliferative capacity, allowing them to compensate for fewer M numbers at the time of re-infection. Therefore, repetitive influenza exposures have the capacity to change numerical and dynamic properties of protective M CD8 T cell responses, which should inform the design of more efficient influenza vaccines.
Antibody idiotype plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity (ADCC) against influenza A virus
Authors: Miller Matthew S , He Wenqian , Tan Gene S , Mullarkey Caitlin E , Krammer Florian , Palese Peter
Published: January 2023

The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. Recently, elicitation of broadly-neutralizing antibodies which target the conserved hemagglutinin stalk domain have emerged as a promising “universal” influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions, such as antibody-dependent cell-mediated cytotoxicity, has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza-binding antibody idiotypes in vivo has never been defined. Here, we demonstrate that complex interactions among viral glycoprotein-binding antibody idiotypes regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We propose that antibody idiotype serves as a critical determinant in the activation of ADCC. This is the first demonstration that cross-talk among antibody idiotypes is important for the regulation of Fc-dependent effector functions. This phenomenon will have major implications for not only the development of universal influenza virus vaccines, but also in any scenario wherein Fc-dependent effector functions are engaged in the context of polyclonal responses containing multiple antibody idiotypes.
ADAMTS4 modulates lung tissue repair following lethal influenza A infection in mice
Authors: Boyd David F , Sanders Catherine J , Bajracharya Resha , Diercks Alan H , Thomas Paul G
Published: January 2023

Lung tissue damage is a major contributor to morbidity and mortality following infection with influenza A virus. Tissue repair, including remodeling of the extracellular matrix (ECM), is a critical process in recovery from infection. A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family proteins are secreted zinc metalloendopeptidases and important mediators of ECM remodeling. The role of ADAMTS proteins in lung tissue repair following influenza A infection remains unclear. To investigate the regulation of ADAMTS genes in the context of influenza A infection, we performed a microarray analysis of whole lung homogenates harvested at 24 hours post-infection from B6 mice that were uninfected or infected with mouse-adapted influenza A PR8. In the context of infection, several ADAMTS genes, including ADAMTS4, −6, and −9 exhibited a ≥ 3-fold increase in gene expression compared to uninfected lungs. ADAMTS-4 exhibited the most dramatic increase in gene expression (>100-fold) at this early timepoint. We further investigated the role of ADAMTS4 in survival and recovery from influenza A infection by infecting wild-type (WT) and ADAMTS4 −/− knockout B6 mice with a lethal dose of influenza A PR8. Interestingly, ADAMTS4 −/− mice exhibited significantly improved survival compared to WT littermates (log-rank test, p= 0.04) with 75% vs. 41% survival at 15 days post-infection, despite no observable difference in weight loss. Consistent with a role in tissue repair and recovery, differences in survival were observed after 9 days post-infection. These results raise the possibility that increased expression of ADAMTS4 following influenza A infection modulates the tissue repair process and contributes to lung pathology.
SP-R210 (Myo18A) enhances influenza A virus pathogenicity by alveolar macrophages and dendritic cells in vivo
Authors: Fino Kristin K , Yang Linlin , Hu Sanmei , Halstead Scott E , DiAngelo Susan L , Silveyra Patricia , Chroneos Zissis C
Published: January 2023

Alveolar macrophages (AMs) are critical for the host response to severe influenza A virus (IAV) infection; IAV is known to cause abortive infection in AMs that is thought to initiate the lung’s immune response to the virus. Our recent studies indicate that the surfactant protein A receptor SP-R210 mediates IAV entry in AMs. Based on these findings, we asked whether SP-R210 modulates the host response to IAV infection.

To address this question, we used SP-R210 floxed mice to delete SP-R210 in CD11c-Cre (dendritic cells and AMs) or Clec9A-Cre (CD103+ dendritic cells) expressing cells. Mice were infected intranasally with a sublethal dose of 1,000 FFU IAV PR8 strain and morbidity was monitored daily by body weight. We found that disruption of SP-R210 significantly reduced susceptibility to infection in both CD11c-CRE/SPR210+/− and Clec9A-Cre/SP-R210+/− as indicated by accelerated body weight recovery 10 days after infection compared to control littermates. The increased recovery of SP-R210-deficient mice was accompanied by a faster decrease in viral titer 7 to 14 days post infection (dpi) as well as increased numbers of CD4+ and CD8+ T cells 14 dpi. Furthermore, flow cytometry analysis revealed significantly increased number of CD11b+Ly6C++Ly6G- and CD11b+SiglecF+ macrophages and AMs in SP-R210-deficient mice, respectively. Consistent with these results, real time qPCR showed persistent suppression of the AM marker SiglecF in control littermates whereas inhibition of SiglecF expression in SP-R210-deficient mice was transient returning to near normal levels by 14 dpi. Taken together, these findings support the model that IAV causes depletion of AMs through SP-R210 disrupting restoration of immune homeostasis after IAV infection.
Decreased polyfunctionality and PD-1 elevation of CD8+ T cells in sepsis patients with human cytomegalovirus reactivation
Authors: Choi Young Joon , Kim Sun Bean , Kim Jong Hoon , Park Su-Hyung , Park Moo Suk , Kim June Myung , Han Sang Hoon , Shin Eui-Cheol
Published: January 2023

Human cytomegalovirus (HCMV) establishes a lifelong chronic latent infection and often reactivates in immunocompromised patients. Additionally, HCMV reactivates in patients with sepsis or other critical illnesses, particularly in patients with poor prognoses. However, the immunological characteristics of sepsis patients with HCMV reactivation have not been elucidated. In the present study, we examined T cell responses in severe sepsis patients with and without HCMV reactivation. First, we found that PD-1 expression was significantly increased in the total CD8+ T cell population in sepsis patients with HCMV reactivation, indicating CD8+ T cell exhaustion in these patients. Next, we studied HCMV pp65-specific T cell functions by intracellular cytokine staining for IFN-γ, TNF-α, and MIP-1β and CD107a staining. When we analyzed the data for each single function, we found no difference between the patient groups. However, the relative frequency of polyfunctional CD8+ T cells was significantly decreased in sepsis patients with HCMV reactivation and inversely correlated with the relative frequency of PD-1+ cells in the CD8+ T cell population. Herein, we demonstrate that HCMV reactivation in severe sepsis patients is associated with CD8+ T cell exhaustion and impaired polyfunctionality of CD8+ T cells.
The Herpes Simplex Virus LAT Gene is Associated with a Broader Repertoire of Virus-Specific Exhausted CD8+ T Cells Retained Within The Trigeminal Ganglia of Latently Infected HLA Transgenic Rabbits
Authors: Benmohamed Lbachir , Srivastava Ruchi , Khan Arif A
Published: January 2023

Persistent pathogens, such as herpes simplex virus type 1 (HSV-1), have evolved a variety of immune evasion strategies to avoid being detected and destroyed by the host’s immune system. A dynamic cross talk appears to occur between the HSV-1 Latency-Associated Transcript (LAT), the only viral gene that is abundantly transcribed during latency, and the CD8+ T cells that reside in HSV-1 latently infected human and rabbit trigeminal ganglia (TG). The reactivation phenotype of TG that are latently infected with wild type HSV-1 (i.e. LAT(+) TG) is significantly higher than TG latently infected with LAT null mutant (i.e. LAT(−) TG). Whether LAT promotes virus reactivation by selectively shaping a unique repertoire of HSV-specific CD8+ T cells retained in LAT(+) TG is unknown. In the present study, we assessed the frequency, function, and exhaustion status of TG-resident CD8+ T cells specific to 40 different epitopes derived from HSV-1 gB, gD, VP11/12 and VP13/14 proteins, in Human Leukocyte Antigen (HLA-A*0201) transgenic rabbits infected ocularly with LAT(+) vs. LAT(−) virus. Compared to CD8+ T cells from LAT(−) TG, CD8+ T cells from LAT(+) TG: (i) recognized a broader selection of non-overlapping HSV-1 epitopes; (ii) expressed higher levels of PD-1, TIM-3 and CTLA-4 markers of exhaustion; and (iii) produced less TNF-a, IFN-g, and GzmB. These results suggest a novel immune evasion mechanism by which the HSV-1 LAT may contribute to the shaping of a broader repertoire of exhausted HSV-specific CD8+ T cells in latently infected TG, thus, allowing for increased viral reactivation.
An essential role of ITK in T cell migration to the gut during mouse γ-herpesvirus 68 infection
Authors: Cho Hyoung-Soo , Shin Hyun Mu , Berg Leslie J.
Published: January 2023

IL-2-inducible T cell kinase (ITK) is the predominant Tec family kinase in T cells. ITK functions to regulate the magnitude of T cell receptor signaling. Deficiencies in ITK in humans leads to a fatal lymphoproilferative syndrome due to uncontrolled Epstein-Barr virus (EBV) infection. Yet, Itk−/− mice mount protective immune responses against several acute viral infections. To address this dichotomy, we have studied the role of ITK in anti-viral T cell responses to latent γ-herpesvirus infection using mouse γ-herpesvirus 68 (MHV68). Despite a delayed response at D7 post-infection, Itk−/− mice controlled MHV68 replication in the spleen at D14, and WT and Itk−/− mice showed no differences in their viral titers in the spleen during latency. However, MHV68-infected Itk−/− mice (n=11/24) spontaneously developed a severe gastrointestinal pathology after D100 post-infection, whereas WT mice (n=24/24) showed no disease symptoms. Following LPS-induced virus reactivation, Itk−/− mice also showed this pathology in the gut accompanied by a high viral titer in the intestine at various time points during viral latency, while WT mice remained healthy. Consistently, the number of gut-resident T cells were substantially less in Itk−/− mice than WT with or without infection. We also found that P-selectin binding on activated Itk−/− T cells or small molecule inhibitor of ITK (PRN694)-treated WT T cells is greatly impaired. Overall, these data suggest that ITK plays an important role in T cell migration to γ-herpesvirus-infected gut tissue following reactivation from latency.
Heterologous protective immunity can lead to impaired activation, memory differentiation and inflation of new naive responses
Authors: Agarwal Priti , Kraft Anke , Watkin Levi B , Kenney Laurie Lea , Daniels Keith , Chen Alex , Brehm Michael A , Selin Liisa K
Published: January 2023

Activation of crossreactive memory CD8 T cells leads to heterologous protective immunity. Here we question whether early activation of crossreactive memory CD8 T cells alters the activation of new naïve CD8 responses to the second virus. Pichinde virus (PICV) and lymphocytic choriomeningitis virus (LCMV) contain a crossreactive usually subdominant epitope, NP205, which differs in 6 of 8 amino acids. We find that during PICV infection of LCMV-immune mice the NP205 memory response becomes immunodominant and mediates protective immunity. PICV is cleared early and does not persist. The usually dominant non-crossreactive new naïve PICV NP38 CD8 T cell response appears to be subdominant day 7-post infection. Surprisingly over the next 100 days post infection the NP38 response continues to gradually expand and then stabilizes. These NP38-specific CD8 T cells appear to arrest in an intermediate effector T cell phenotype (KLRG1low, CD62Llow IL-7Rhi), with altered functionality (decreased TNFalpha production). They present with altered expression of apoptotic genes on gene array. This NP38 inflationary response did not occur when mice were immunized with an LCMV mutant that did not present the crossreactive NP205 epitope. Our data shows that the strong crossreactive response kills DC’s expressing NP205 early (day 2–3) in PICV infection. This may result in an early termination of antigen-presentation leading to inadequate activation of the naïve NP38 response and inflation. (NIH U19 AI109858)
IL-2 and IL-15 on CD8 T cells during chronic viral infection: The exhaustion program awakens
Authors: Beltra Jean-Christophe , Bourbonnais Sara , Bédard Nathalie , Charpentier Tania , Boulangé Moana , Michaud Eva , Boufaied Ines , Bruneau Julie , Shoukry Naglaa H , Lamarre Alain , Decaluwe Helene
Published: January 2023

CD8 T cell exhaustion is an immunosuppressive mechanism by which chronic viruses escape adaptive immune response. Yet, the direct implication of cytokines as well as the precise candidates involved in this process have remained elusive. Here we demonstrate the direct contribution of IL-2 and IL-15 on several aspects of CD8 T cell exhaustion. Using a mouse model of chronic viral infection (LCMV clone-13), we show that IL2Rβ (CD122; the receptor chain common to IL-2 and IL-15) is maintained on CD8 T cells during chronicity. Its expression correlates with deeper exhaustion and selectively marks terminally exhausted CD8 T cells in mice and humans. Adoptive transfer of IL2Rβ−/− P14 CD8 T cells (unresponsive to combined IL-2 and IL-15-dependent signals) revealed that IL-2 and IL-15 directly control the expression of a specific pattern of inhibitory receptors and are mandatory for the development of PD-1hi effectors. Further, IL-2 and IL-15 instigate the arrested development of central memory cells seen during antigen persistence and preclude responsiveness to IL-7-dependent signals. Together, we ascribe a novel role to IL-2 and IL-15 as instigators of CD8 T cell exhaustion during chronic viral infection.
TCR modifications that increase proper chain pairing also enhance reactivity against naturally occurring HCV mutant peptides
Authors: Foley Kendra C , Spear Timothy , Nagato Kaoru , Murray David , Garrett-Mayer Elizabeth , Nishimura Michael I.
Published: January 2023

TCR gene modified T cells in adoptive cell transfer have been shown to have clinical success in treating melanoma and other malignancies. One of the challenges in using TCR gene modified T cells include proper expression of the introduced TCR and function. Mispairing between endogenous and introduced TCR chains allows for the potential of unanticipated off-target reactivity, autoimmunity, and impaired therapeutic efficacy against targeted antigens. One approach to improve TCR expression, pairing, and T cell function involves modifying the introduced TCR genes to promote proper pairing by introducing a disulfide bridge in the alpha/beta constant regions, murine constant regions, codon optimization, TCR chain leucine zipper fusions, or a single chain TCR. To compare these modifications, we used a TCR we previously cloned from a HLA-A2 restricted, HCV NS3:1406-1415-reactive T cell clone. Results revealed that the murine Cβ2 and the leucine zipper TCR have the highest levels of expression on transduced T cells when compared to the wild type TCR and on a transduced cell by cell basis, these cells have the highest percentages of bi-functionality. Our data showed proper chain pairing can have an impact on the T cell’s ability to be reactive against naturally occurring HCV NS3 mutant peptides and lead to loss of CD8 dependence. Although this could be therapeutically beneficial for viral infections with unstable genomes, cross reactivity against nonspecific antigens due to high TCR density on the cell surface could be detrimental. These studies have given us a better understanding of how TCR modifications can impact expression and function that may allow for optimization of adoptive T cell transfer to treat viral infections and malignancies.
Aminobisphosphonates reveal a selective anti-viral potential of Vγ9Vδ2 T cells
Authors: Champagne Eric , Barragué Hugo , Daguzan Charline , Abravanel Florence , Bellvert Floriant , Peyrottes Suzanne , Izopet Jacques
Published: January 2023

Vγ9Vδ2 cells are activated by stressed cells producing endogenous phosphoantigens (PAgs) following tumoral transformation or treatment with aminobisphosphonates (ABP) such as zoledronate (Zol). Alternatively they respond to exogenous PAgs produced by bacteria. Their potential interest in viral infections is not well established. We analyzed in vitro their activation and activity against HCMV-infected cells. Although they were not activated by HCMV-infected fibroblasts, following treatment of targets with ABPs they were preferentially and more potently activated by infected cells, resulting in a stronger IFNγ production and induction of TNF. In conditions where uninfected cells were not affected, they limited viral replication through an IFNγ and TNF-dependent mechanism, whereas cytotoxicity did not play a significant role. This could be explained by an increased uptake of a fluorescent Zol derivative by infected cells. In addition, HCMV infection stimulated the transcription of mevalonate pathway enzymes HMG-CoA reductase and synthase evaluated by RT PCR. As a result, the production of endogenous PAgs IPP and ApppI following low-dose treatment with Zol was also boosted. Cytokines such as IL18, putatively produced by infected cells did not play a significant role in the synergistic effect of HCMV and Zol. In order to examine whether this was true for other viruses, we infected hepatocytoma cells with the hepatitis E virus and treated them with Zol. In this case, viral infection only moderately increased Zol-induced IFNγ production, but not TNF, by Vγ9Vδ2 cells. Our results suggest that ABPs could be used to target the anti-viral potential of Vγ9Vδ2 T cells against selected viruses responsible for chronic infections.
Deep-sequencing of longitudinal samples from RSV-infected adults and infants reveals heterogeneous dynamics of within-host viral diversification
Authors: Kim-Hoehamer Young-In , Lau Jessica , Murphy Ryan , Newman Ruchi , Yang Xiao , Zody Michael , Grad Yonatan H , DeVincenzo John
Published: January 2023

With high mutation rates, large population sizes, and rapid turnover, RNA viruses can evolve over the course of individual infections. While the dynamics of viral evolution under selective pressures from host immunity has been described for chronic infections, such as those from HIV and HCV, the extent and nature of viral diversification in acute viral infections remains uncharacterized. Here, we investigate the within-host diversification of human respiratory syncytial virus (RSV), a highly prevalent cause of respiratory infections, in healthy adults who were experimentally infected with identical virus and in infants hospitalized with severe manifestations of naturally acquired infections. In aggregate, viral diversification in the adults peaked at day 3, with statistically significant overrepresentation of diversity in the matrix protein 2 (M2) and non-structural protein 2 (NS2) genes. A later peak in diversity at day 10 was attributable to a single study subject, with diversity predominantly in known and predicted B and T cell epitopes. In the infant infections, much less diversity was observed, possibly related to sampling starting at the time of diagnosis and hospitalization, and trended towards overrepresentation in the G surface protein. Our findings raise the possibility of multiple mechanisms for control of RSV infection that vary over the course of an infection and by host, and establish a framework for further investigation into the basis of heterogeneous host response to infection. Moreover, the results have broad implications for investigations into immune response and the use of deep sequencing in viral pathogenicity and the molecular epidemiology of pathogen spread.