bluuebunny/crossref_metadata_2025 huggingface dataset:
Neoantigens derived from differential mutations of invasive ductal carcinomas with axillary lymph node metastasis
Authors: Bagriacik Emin Umit , Ermis Ilker , Ekinci Ozgur , Girgin Cansu , Oruklu Nihan , Bayram Orhan
Published: January 2023

Invasive ductal carcinomas (IDCs) are the most common breast cancer type. It was reported that almost 80% of breast cancers are IDCs. IDCs can be treated by immunotherapy using neoantigens. Therefore, common neoantigens that are specific for ductal carcinomas should be determined. We aimed to determine differential expression of neoantigens for IDCs in the presence or absence of axillary lymph node metastasis. DNA was isolated from paraffin-embedded (FFPE) tissue specimens by using GeneRead DNA FFPE Kit. QIAseq Targeted DNA panel for human breast cancer was used for sequencing of isolated tumor DNA. Next generation sequencing was performed using a Illumina MiSeq. Total exons of 93 genes were studied. A bioinformatic analysis was conducted by a platform-specific pipeline software (Qiagen Clinical Insight-AnalyseTM and Clinical Insight-InterpretTM). We found significant variations in several genes for individuals with or without axillary lymph node metastasis. In particular, variations in CDKN2A and PIK3R1genes were quite interesting to determine individual neoantigens of ductal carcinomas as potential immunotherapy targets. Currently, these findings are still under further investigations. Based on these findings, we concluded that NGS analysis is a very powerful technology in determination of specific neoantigens of individuals with ductal carcinomas. Also NGS can be used to discover new genes related to axillary lymph node metastasis for ductal carcinomas. Supported by Gazi University BAP.
Immune profiling of the tumor microenvironment (TME) using multiplexed ion beam imaging (MIBI)
Authors: Ptacek Jason , Johnson Robert W. , Palma Joann P , Tarolli Jay G. , Sigal Yari , Finck Rachel , Aksoy Murat , Zhang Yi , Finn Jessica
Published: January 2023

Understanding the cell types present in the tumor microenvironment and their activation state is at the forefront of immunotherapy research. To address this, MIBI has been developed to simultaneously image more than 40 markers at single cell resolution. Staining of 10 NSCLC formalin-fixed paraffin embedded tissue sections was performed with a panel of 20 metal labeled antibodies stained together. The tissue was imaged at subcellular resolution using an ion beam and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Masses of detected species were assigned to target biomolecules given the unique label of each antibody and multi-step processing and segmentation were performed to create images of the TME. Each tumor sample was imaged across 10 regions of interest (ROIs) to assess heterogeneity of the TME. The resulting single cell segmentation enabled quantitative analyses of both marker expression and the spatial relationships between cells of different types. At the highest level, cells were classified as immune (CD45) and tumor cells (keratin) based on measured intensities of marker expression. Co-expression of markers were used to classify B cells, macrophages, and T cells subsets. Markers associated with immune suppression such as PD-L1 were also quantified. Cell types and their frequency were compared within the 10 ROIs collected per sample as well as between samples. Finally, distances between tumor and the closest immune cell were measured as a means for describing the spatial organization of the TME, which has been linked to patient survival. In conclusion, MIBI offers high-parameter capability, at a sensitivity and resolution uniquely suited to understanding the complex tumor immune landscape.
A novel genetic screen to identify genes that enhance T cell trafficking into solid tumors
Authors: Rogers Laura M , Dupuy Adam , Weiner George
Published: January 2023

T cell mediated cancer immunotherapies, including anti-PD-1 and CAR-T, are becoming standard of care for a variety of cancer types. However, patients with tumors that lack T cell infiltration generally do not respond well to immunotherapy. One potential strategy to overcome immunotherapy resistance is to enhance the ability of T cells to traffic into the tumor. The objective of this study was to develop an in vivo genetic screen in endogenous T cells using the Sleeping Beauty (SB) mutagenesis system to identify candidate genes that drive intratumoral T cell accumulation.

Mutations in one candidate gene, Aak1, were identified in intratumoral T cells of multiple tumor models, including those treated with anti-PD-1. Insertions were highly clustered, suggesting that a gain-of-function Aak1 mutation may be favorable for driving T cells into the tumor. This gain-of-function is likely dominant-negative, as inhibition of Aak1 kinase activity enhanced intratumoral T cell infiltration. Surprisingly, inhibition of Aak1 kinase activity also abolished the therapeutic effect of anti-PD-1 in mouse models of lymphoma and melanoma.

Our data demonstrates that our SB mutagenesis approach can be used to identify target genes that could be therapeutically modified to enhance T cell infiltration into tumors. Our innovative method can be adapted to other immune cell types. At least one candidate gene we identified (Aak1) deserves further evaluation as a potential therapeutic target, though a better understanding of the antagonistic relationship between anti-PD-1 action and Aak1 kinase activity is needed. Immediate next steps also include assessing the impact of candidate gene modification in CAR-T cells on their ability to traffic into solid tumors.
High parameter immune cell quantification in a melanoma-derived tumor model by Chipcytometry
Authors: Gamber Kevin , Detmers Jan , Schupp Jonathan , Tuttenberg Andrea , Christians Arne
Published: January 2023

Immune evasion is a hallmark of cancer and the presence and interaction of immune cells within a tumor and its microenvironment can have a great impact on disease progression and prognosis. Unravelling the complex interplay of neoplastic and immune cells requires both phenotypic and spatial analysis of each involved cell population in the tissue.

The aim of this study is to compare 3 methods of biomarker analysis in tissues, flow cytometry, immunohistochemistry (IHC) and Chipcytometry, in their ability to detect immune cells in a tumor model.

NOD-Scid tg HLA-A2.1 mice were implanted with human Ma-Mel-19 melanoma cells. After 21 days, the mice were injected with human PBMC at the tumor site. Local treatment with Tasquinimod was performed three times once a week. Tumors, including the neighbouring PBMC injection site were excised on day 48 and immune cell content was analysed by Chipcytometry, flow cytometry and IHC. All three methods were successful in detecting human immune cells in the samples consisting mostly of T-cells (CD45+CD3+). However, flow cytometry and IHC were unable to provide either accurate localization or phenotyping data, respectively. With Chipcytometry we were able to specifically quantify tumor-infiltrating immune cells. In treated samples immune cell count (110/mm^2) and composition (72% T-cells) was significantly higher than in the untreated samples (54/mm^2, 49% T-cells). We show that Chipcytometry is able to produce “flow-like” quantitative data, while also retaining spatial information. This makes Chipcytometry a powerful tool for the study of immunological processes in tumors.
High-throughput screening for rare antigen-reactive TCRs using natively-paired TCRαβ expression libraries generated from millions diverse primary T cells
Authors: Spindler Matthew J , Nelson Ayla L , Heather James M , Wagner Ellen K , Adler Adam S , Johnson David S
Published: January 2023

A major goal of T cell research is to profile the repertoire of antigen-reactive TCRs targeting specific peptide:MHCs (pMHC). Single cell sorting of pMHC-binding T cells using multimers and ex vivo antigen expansion are the current gold standards for identifying antigen-reactive primary T cells. However, functional validation of the TCRs identified by these approaches requires resource intensive cloning of each individual TCRαβ pair. Additionally, primary T cells, especially tumor infiltrating lymphocytes (TILs), are a limited resource, which restricts the number of antigens that can be screened.

To address this, we developed a microfluidic approach to capture and functionally express natively-paired TCRαβ libraries from millions of single T cells. Unlike DNA barcoding approaches that mark single cells by adding a sequence tag, we physically link the TCRα-TCRβ chains to generate sequencing and full-length expression libraries which we introduce into Jurkat cells for functional testing.

Using these methods, we captured over 2.9 million TCRαβ clonotypes from six healthy PBMC donors and over 0.5 million from expanded melanoma TIL samples. We applied pMHC binding and cellular activation screens to identify and validate 14 TCRs reactive to common viral and tumor associated antigens with starting library frequencies of <0.0001 – 0.3% and functional avidities ranging from 0.3 – 592nM. We found that including the cellular activation screen reduced our false-positive TCR discovery rate from 65% for pMHC binding alone to 18%. We are further developing these cellular activation enrichment approaches and applying them to broadly profile anti-tumor TCR reactivity from TILs.
Rational targeting of Cdc42 GTPase destabilizes Treg to promote anti-tumor T cell immunity
Authors: Nguyen Thi Thuy Phuong , Kalim Khalid , Zheng Yi , Guo Fukun
Published: January 2023

Targeting of regulatory T cells (Treg) is a long-sought approach to overcome tumor immune evasion. However, systemic depletion of Treg likely causes autoimmune diseases. Indeed, we have recently reported that Treg-specific homozygous deletion of Cdc42 GTPase caused a drastic reduction in Treg numbers and systemic inflammatory diseases. Here we show that heterozygous deletion of Cdc42 (Cdc42+/−) did not affect Treg homeostasis but destabilized them, leading to effector T cell reprogramming in these Treg and impaired function in suppressing effector T cells. Accordingly, Cdc42+/− mice had increased effector T cells. Intriguingly, Cdc42+/− mice showed a remarkable suppression of growth of mouse colon and pancreatic tumors, with no noticeable autoimmunity. Mechanistically, Cdc42+/− Treg exhibited a significantly increased expression of carbonic anhydrase 1 (CA1), reversal of which by CA1 shRNA or a CA inhibitor restored Treg stability and tumor growth. Pharmacological inactivation of Cdc42 by a chemical inhibitor, CASIN, destabilized wild type (WT) Treg in mice and triggered anti-tumor T cell immunity, with no detectable autoimmune responses. CASIN also destabilized WT Treg in vitro and adoptive transfer of CASIN-treated Treg with congenic effector T cells into Rag1−/− mice suppressed tumor growth, demonstrating that CASIN-induced Treg instability caused tumor suppression. Combined CASIN and Anti-PD-1 antibody led to an additive suppression of mouse tumors as well as human tumors in human immune cell-reconstituted NSG-SGM3 mice. Collectively, our data suggest that rational targeting of Cdc42-regulated Treg stability holds promise in counteracting tumor immune evasion.
Interferon gamma production by regulatory T cells is required for response to cancer immunotherapy
Authors: Gocher Angela Marie , Handu Sanah , Workman Creg J , Vignali Dario A. A.
Published: January 2023

We have shown that murine regulatory T cells (Tregs) produce the cytokine interferon gamma (IFNγ) during anti-PD1 immunotherapy and that IFNγ-driven Treg dysfunction is necessary for tumor clearance by anti-PD1. However, it is unknown how Treg production of IFNγ dictates response to cancer immunotherapy. It has been shown that IL12-induced production of IFNγ is required for response to anti-PD1 therapy. However, it has yet to be determined whether Tregs are a key responder to IL12. Thus, elucidating the interplay of IL12, IFNγ and Tregs in the tumor microenvironment (TME) is essential for understanding the mechanistic events required for response to immunotherapy. Our lab has generated two novel murine models that allow for Treg-restricted deletion of Ifng (IfngL/LFoxp3Cre-YFP) or Il12rb2 (Il12rb2L/LFoxp3Cre-YFP). These murine models were used to assess the contribution of Treg-derived IFNγ and response to IL12 on the growth of various syngeneic tumor models and response to checkpoint blockade, vaccination and tumor-specific antibodies. Surprisingly, mice with Ifng-deficient Tregs were completely resistant to anti-PD1 immunotherapy. Preliminary experiments suggest that IFNγ production by Tregs is an early requirement to produce an effector-like Treg state in order to mediate an effective anti-tumor response. Mice with Il12rb2-deficient Tregs had diminished therapeutic response to anti-PD1. These data suggest that IFNγ producing Tregs are required to shift the balance from an immunosuppressive TME to one that favors the reinvigoration of the anti-tumor response generated by cancer immunotherapies. Future studies will determine the mechanisms of resistance and whether IFNγ+ Tregs predict response to immunotherapy.
Influenza vaccines using liposomal formulations of toll-like receptor (TLR) 7/8 and 4 agonists as adjuvants
Authors: Cole Shelby L , Cybulski Van A , Whitacre Margaret , Lathrop Stephanie K , Bazin-Lee Helene , Burkhart David , Evans Jay T
Published: January 2023

Influenza virus infection is a global health concern and causes severe illness and thousands of deaths each year. While there are currently licensed vaccines available for both seasonal and pandemic influenza viruses there are several issues to consider; 1) current vaccines do not provide supra-seasonal or universal protection against influenza subtypes, 2) antigenic mismatch between the vaccine and circulating influenza strains impact vaccine efficacy, 3) and the efficacy of influenza seasonal vaccines could be improved in the elderly and immune compromised populations. Therefore, vaccine adjuvants capable of inducing more robust and broadly cross-protective immune responses to various subtypes of influenza virus is a critical unmet need. To address this challenge, we developed a synthetic dual TLR adjuvant combination system (TRAC-478) to improve influenza vaccine immunity. Preliminary data in human peripheral blood mononuclear cells demonstrate that combining our synthetic TLR7/8 and TLR4 agonists results in a synergistic innate immune responses. Influenza vaccine immunogenicity studies in both murine and pig models demonstrate the safety and efficacy of this new dual TLR adjuvant system. Using a co-encapsulated TRAC-478 liposome delivery system with a split-flu vaccine resulted in strong anti-influenza humoral, Th1 cell mediated immunity, and protection from a heterologous influenza challenge in mice. We hypothesize that using the TRAC-478 adjuvant system in combination with current influenza vaccines will induce a more robust and broadly cross-protective immune responses to circulating or pandemic influenza virus.
Influenza-specific ex vivo immune responses in ferrets commensurate with number of vaccinations despite decreased protection for repeat vaccines
Authors: Hofstetter Amelia R , Music Nedzad , Kamal Ram P. , York Ian A.
Published: January 2023

There is controversy in the literature whether consecutive year influenza vaccinations lead to decreased vaccine efficacy. Evidence for this effect is strongest in the context of repeated H3N2 vaccinations. As recently reported (NPJ Vaccines 4:28 (2019)), we recreated this phenomenon in the ferret model of influenza. Ferrets exposed to consecutive year vaccinations had significantly increased weight loss, viral shedding and fever after A/Hong Kong/4801/2014 (HK4801) virus challenge than ferrets only exposed to the 2017–2018 vaccine. One possible explanation for this phenomenon is that the repeated vaccination induces non-protective antibodies against influenza virus, which might include low avidity antibodies, non-neutralizing antibodies, and antibodies that otherwise do not elicit a protective immune response. Ferrets that received repeated vaccination had higher avidity antibodies to HK4801 HA at Day 2 and 14 post-infection than did the other two groups. Preliminary data do not support increased levels of HA stem-binding antibodies in repeat vaccinees’ serum. NA and NP are two additional viral proteins expressed on the cell surface, with greater or lesser evidence for their role in protective antibody responses, respectively. Serum reactivity to both proteins correlated with the number of vaccinations. Together, these results do not support a model of increased non-protective antibodies after repeat vaccination. Furthermore, the breadth of the T cell response against H3N2 viruses from different clades was similar between both vaccinated groups. Ongoing studies continue to explore the mechanism for the decreased protection observed in the repeat vaccination group compared with the single year vaccination group.
Understanding memory B cell responses to a chimeric-based universal influenza virus vaccine candidate
Authors: Lan Linda (Yu-Ling) , Li Lei , Huang Min , Henry Carole , Zheng Nai-Ying , Chen Yao-Qing , McNair Mahalia , Meade Philip , Guthmiller Jenna , Dugan Haley , Tepora Micah , Palm Anna-Karin , Shaw Dustin , Stamper Christopher , Khan Aly Azeem , Nachbagauer Raffael , García-Sastre Adolfo , Palese Peter , Krammer Florian , Wilson Patrick C
Published: January 2023

A universal influenza virus vaccine candidate trial was recently conducted that utilized novel influenza viruses expressing chimeric hemagglutinin (HA) proteins. The chimeric HAs present in the vaccines are composed of a typical stalk domain (from an H1 HA) paired with an exotic head from a strain to which humans are naive (avian H5 or H8). Given the conserved nature of the HA-stalk region, broadly neutralizing antibodies that target this region would confer protection against a wide range of influenza virus strains. The major objective of the trial is to determine whether the human immune response to influenza virus can be re-programmed to target universally conserved stalk epitopes. In this study, we collected antigen-specific memory B cells at day 28 post-vaccination at prime, boost and 1-year time points, and subsequently characterized both the transcriptome by single cell RNA-seq and antibody specificity by generating monoclonal antibodies (mAbs) from the sequenced immunoglobulin genes of the same B cells. Our research has suggested HA stalk-reactive B cells were selected after the boost time point. In addition, we have identified clonal expansions within an individual (referred to as private clones) and across multiple individuals (referred to as public clones) that have neutralizing capability and are able to bind divergent influenza strains. Furthermore, we have identified different memory B cell subsets induced by universal influenza virus vaccination at the single-cell level.
Simultaneous mRNA, protein, and immune repertoire profiling of antigen-specific T cells by single cell sequencing
Authors: Shi Xiaoshan , Siddique Imteaz , Nakamoto Margaret , Mortimer Stefanie
Published: January 2023

High-throughput single cell RNA-seq (scRNA-seq) has transformed our understanding of complex and heterogenous immune populations. New advances in scRNA-seq are expanding the molecules that can be profiled at the single cell level, such as oligo-conjugated antibody technologies that enable protein expression profiling alongside mRNA. Although the ability to sequence the immune repertoire can provide crucial insights into understanding the complexities of the adaptive immune system and advancing discoveries in immuno-oncology, the ability to extract this information from single cells requires new technology to profile regions of the mRNA that are missed by conventional 3′ scRNA-seq. In this study we utilized an ex vivo antigen stimulation system to measure antigen-specific T-cell activation and clonal amplification. Stimulated T cells from two donors were loaded onto the BD Rhapsody™ Single-Cell Analysis System to extract immune repertoire information in addition to gene and protein expression information from the same cells. In a single workflow, we profiled a panel of 400 mRNA targets, 20 BD® AbSeq protein markers associated with different status of T-cell activation, and the hypervariable region of T-cell receptors at the single cell resolution. The study is a proof of concept of combining mRNA, protein and immune repertoire analysis at the single cell level to deconvolute the heterogeneity and different activation of stimulated T cells.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.
CAR T cells generated in situ using gene therapy in naturally activated lymph node T cells
Authors: Pease Larry R , Guttormson Matthew C , Parks Christopher A , Felts Sara J , Van Keulen Virginia P , Castro Sarah A , Evgin Laura , Vile Richard G , Barry Michael A
Published: January 2023

T cells expressing chimeric antigen receptors (CAR T cells) are manufactured outside the body using non-physiological activation and in vitro culture protocols. The procedure takes 3 weeks and costs approximately $450,000 to administer. We have devised a scheme using mouse models for manufacturing CAR T cells in just 3 days employing a natural, polyclonal anti-viral immune response inside a lymph node (LN) using off the shelf reagents that can be applied widely in populations. CAR T cells emerge from the LN within a week, invade solid tumor, and eliminate the targeted antigen-positive tumor cells. Polyclonal activation of T cells is achieved by modifying the antigen presenting properties of lymph node resident professional antigen presenting cells (APC) through viral expression of allogeneic major histocompatibility antigens. The ensuing polyclonal activation occurs in the context of virus activated APC within the normal aarchitecture of the LN and confers robust T cell activation signals directing the development of cytolytic T cells. During the course of T cell activation, the T cells are transduced in situ with retroviruses encoding selected CARs. This approach could be applied to any number of different CARs to target a variety of tumors. T cells activated in the lymph node are functionally distinct from those activated using standard CAR T manufacturing protocols. We anticipate that CAR T cells generated in situ to natural stimuli will exhibit more normal immune regulatory properties that will impact their therapeutic utility. Because our approach uses off the shelf reagents and is administered using standard clinical procedures, cost to patients should be lowered and access to therapy increased.
A single administration of Fc-fused recombinant human IL-7 induces expansions of T lymphocytes in healthy adults and cancer patients
Authors: Kim Sojeong , Heo Minkyu , Jeong Sohee , Woo Jungwon , Sung Young Chul , Shin Eui-Cheol , Park Su-Hyung
Published: January 2023

Interleukin-7 (IL-7) is a cytokine that plays important roles in T cell development and homeostasis. IL-7 is a potent agent for promoting T cell reconstruction in lymphopenia patients. In this study, we investigated the effects of GX-I7 (human IL-7 fused to hyFcTM for half-life extension and improved stability) in healthy volunteers (20~60 μg/kg, n=24) and advanced solid cancer patients who had no available effective treatments (60~1,200 μg/kg, n=33).

We observed significant increases in the number of circulating CD8+ and CD4+ T cells in healthy volunteers, the peak of which was seen on days 14-21. Importantly, naïve, TEM, and TCM subsets of CD8+ and CD4+ T cells increased, coupled with upregulation of Ki-67. TCR diversity of the naïve T cells tends to be increased in healthy adults by single administration of GX-I7. In addition, cytokine secretion assay using PBMCs revealed that Ag–specific (CMV-specific) T cell responses were maintained after GX-I7 administration in healthy volunteers. Moreover, in advanced solid cancer patients, GX-I7 administration significantly increased the number of all subsets (naïve, TEM, TCM and TEMRA) of both CD8+ and CD4+ T cells in a dose-dependent manner. More importantly, single GX-I7 administration increased the absolute lymphocyte count above the normal range in all patients with lymphopenic conditions (ALC < 1,000/mm3).

In conclusion, following GX-I7 administration, dose-dependent increase of ALC and T cell subsets were observed. Our study suggests that GX-I7 as a T cell amplifier provides a unique opportunity for immuno-oncology combination strategies by reconstituting persistent T cell immunity, where anti-tumor effects are mediated by T cells.
Bispecific CAR T cells for multiple myeloma: natural ligand compared to tandem scFv design
Authors: Larson Rebecca , Castano Ana , Bouffard Amanda , Scarfo Irene , Kann Michael , Schmidts Andrea , Gallagher Kathleen , Maus Marcela Valderrama
Published: January 2023

One of the most promising candidates for Chimeric Antigen Receptor (CAR) T cell therapy beyond CAR19 is treatment of multiple myeloma with CAR T cells targeting B cell maturation antigen (BCMA). However, current reports of BCMA CAR in the clinic have a median progression free survival of 11.8 months, suggesting that targeting BCMA alone may not be sufficient. Evidence suggests patients treated with BCMA-targeted therapies may be thwarted by BCMA negative relapse. We have developed a novel second generation CAR T cell against another multiple myeloma target: transmembrane activator and CAML interactor (TACI). Mice were immunized against TACI and we designed a new scFv targeting TACI based on the resulting antibodies. Anti-TACI CAR T cells are cytotoxic in vitro and in vivo against multiple myeloma. To overcome single antigen loss, we designed bispecific tandem scFv CAR T cells targeting BCMA and TACI. These two antigens share a natural ligand, A Proliferation-Inducing Ligand (APRIL), which has also been used to design a dual-targeting CAR called TriPRIL. We show that these dual targeting CARs, based on tandem scFv or natural ligand design, have similar efficacy against wild type multiple myeloma models. However, this changes in the context of single antigen loss. We have characterized the proliferative (population doublings) and activation capability (CD69) of these CARs, as well as their memory (CCR7, CD45RA) and exhaustion phenotype (PD-1, TIM-3, LAG-3), with long term exposure to single antigen. Our studies show that sensitivity to antigen density differs between the tandem bispecifics and the natural ligand CAR. These data provide insight into how structural differences between dual-targeting CAR T cells affects their function.
The IL-15 receptor agonist N-803 combined with the anti-CD20 monoclonal antibody rituximab expands NK and CD8 T cells and alters single cell immune transcriptomes in a phase 1 clinical trial in lymphoma
Authors: Foltz-Stringfellow Jennifer Ann , Bartlett Nancy L. , McClain Ethan , Becker-Hapak Michelle , Hess Brian T. , Bachanova Veronika , Berrien-Elliott Melissa M. , Foster Mark , Schappe Timothy , Kahl Brad , Mehta-Shah Neha , Cashen Amanda F. , McDaniels Kristin , Moreno Chaz , Gao Feng , Griffith Obi , Griffith Malachi , Wagner Julia A. , Rock Amy D. , Soon-Shiong Patrick , Lee John , Petti Allegra A. , Fehniger Todd A
Published: January 2023

IL-15 is a cytokine that is crucial for the development, survival, and activation of NK cells, and pre-clinical studies demonstrated that IL-15 augments antibody-dependent cellular cytotoxicity. We hypothesized that IL-15 would augment anti-CD20 mAb (rituximab)-directed responses in patients with indolent non-Hodgkin’s lymphoma. To address this, we performed a first-in-human combination clinical trial of the IL-15 super agonist N-803 with rituximab, and investigated the impact of this novel immune combination on NK, CD8 T cells, and monocytes. The combination was safe and demonstrated clinical activity, including in patients refractory to rituximab. To understand the in vivo impact on the patients’ immune systems, we performed mass cytometry and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), which measures single-cell RNA-seq and cell surface proteins simultaneously. N-803 induced expansion of NK (12.5-fold) and CD8 T cells (2-fold) in serial blood samples and upregulated CD38, chemokines, and MHC class II family member expression while maintaining CD16 expression on NK. On CD56dim NK, N-803 increased activating receptors, decreased CD57, and modulated transcription factor expression (AP-1, Eomes, CEBPB/D). CD56bright NK cells were primed, with increases in granzyme A, B, and K. Therapy also markedly altered the monocyte compartment, significantly upregulating type 1 interferon and interferon gamma pathways in CD14+ monocytes, and decreased the percent of CD16+ monocytes, suggestive of potential trafficking to the tumor. Collectively, our data demonstrates that N-803 plus rituximab induces complex immune activation, and supports N-803 combination with other mAbs and immunotherapies.
IFN-lambda 3 directly induces antiviral responses in human B and T cells leading to inhibition of HIV-1 infection of CD4+ T cells
Authors: Santer Deanna M. , Minty Gillian ES , Golec Dominic , Lu Julia , May Julia , Namdar Afshin , Shah Juhi , Elahi Shokrollah , Proud David , Joyce Michael , Tyrrell D. Lorne , Houghton Michael
Published: January 2023

Type III interferons (IFN-lambdas(λ)) are the most recently discovered interferon cytokine family that inhibit viruses by signaling through a unique IFN-λR1/IL-10RB heterodimeric receptor. Until now, IFN-λs were thought to primarily act on anatomical barrier epithelial cells, neutrophils and a subset of dendritic cells, although the majority of studies have been performed in mice. Here, we examine regulation of IFN-λR1 expression and downstream effects of IFN-λ3 stimulation of primary human blood immune cells and compare them to lung or liver epithelial cells. IFN-λ3 directly bound and upregulated IFN-stimulated gene (ISG) expression in freshly purified human B cells and CD8+ T cells, but not monocytes, neutrophils, natural killer cells and CD4+ T cells. Despite similar IFNLR1 transcript levels in B cells and lung epithelial cells, lung epithelial cells bound more IFN-λ3, which resulted in a 50-fold greater ISG induction when compared to B cells. The reduced response of B cells could be explained by the higher expression of the soluble splice variant of IFN-λR1, which inhibited ISG induction when added with IFN-λ3 to peripheral blood mononuclear cells. Human CD4+ T cells gained responsiveness to IFN-λ3 upon T-cell receptor stimulation since activation signals potently, and specifically upregulated membrane-bound IFN-λR1 expression. Importantly, IFN-λ3 treatment of activated CD4+ T cells caused a significant decrease in human immunodeficiency virus-1 (HIV-1) infection. Collectively, our data demonstrate that IFN-λ3 directly interacts with the human adaptive immune system, and thus can promote antiviral immunity at mucosal and peripheral sites, unlike what has been previously shown in published mouse models.
HIV-1 envelope glycan composition is a key determinant of efficient virus transmission via DC-SIGN and resistance to antibodies and inhibitory lectins
Authors: Hioe Catarina E , Jan Muzafar , Upadhyay Chitra
Published: January 2023

HIV-1 envelope (Env) undergoes extensive N-linked glycosylation that cloaks its surface with an assortment of oligomannose-, hybrid-, and complex-type glycans and enables virus interaction with carbohydrate-recognizing lectins. This study examined the importance of Env glycan heterogeneity for HIV-1 transmission via DC-SIGN, a mannose-binding lectin expressed on macrophages and immature dendritic cells. A diversity of glycan content was observed among HIV-1 strains and associated with varying degrees of trans-infection via DC-SIGN. When Env glycans were modified to display only oligomannose-type, DC-SIGN-mediated virus capture was enhanced. However, virus trans-infection was diminished due to increased degradation, which was alleviated by incorporation of hybrid-type glycans. HIV-1 strains also displayed varying sensitivity to trans-infection blockage by antiviral lectins GNA and GRFT, which correlated with sensitivity to neutralizing antibodies. Tier 1 viruses sensitive to neutralizing antibodies were more readily inhibited by GNA and GRFT than tier 2 and tier 3 viruses. Correspondingly, tier 1 Envs showed a greater reactivity with GNA and GRFT, which recognizes high-mannose-type N-glycans, suggesting enrichment of high-mannose type on tier 1 Envs. Of note, HIV-1 Env glycan content is influenced by amino-acid variations in the Env signal peptide (SP) and single-point mutations in the Env SP were sufficient to affect Env glycan content, leading to alterations in DC-SIGN-dependent trans-infection and virus sensitivity to antiviral lectins. Hence, SP variation and glycosylation that confer varied types of oligosaccharides to HIV-1 Env are critical determinants for virus fitness and phenotypic diversity.
Zika virus suppresses maturation of human dendritic cells
Authors: Wang Ying-Ting , Branche Emilie , Viramontes Karla M. , Cuevas Joan Valls , Carlin Aaron F. , Shresta Sujan
Published: January 2023

Zika virus (ZIKV) belongs to the genus Flavivirus, which includes several medically relevant viruses, including dengue, yellow fever and Japanese encephalitis viruses. ZIKV causes congenital Zika syndrome in neonates and Guillain-Barré syndrome in adults, and has emerged as a major public health problem worldwide. Dendritic cells (DCs) serve as the major cellular hosts of ZIKV, and T cell responses to flaviviruses may play a dual role in protection and pathogenesis. To decipher the precise interactions between DCs and ZIKV, we profiled the transcriptomic features of ZIKV-infected DCs versus uninfected bystander DCs in primary human blood monocyte-derived DC cultures. RNA sequencing data revealed that the expression of genes related to antigen processing/presentation pathways was reduced in ZIKV-infected DCs relative to uninfected bystander DCs that were exposed to the same environmental stimuli as ZIKV-infected DCs. Flow cytometric analysis of ZIKV-infected DCs for the expression of key DC maturation markers confirmed the RNA sequencing data. Thus, ZIKV hinders DC maturation, suggesting that ZIKV may modulate T cell responses by directly manipulating DC functions. Studies are in progress to determine how ZIKV inhibits DC maturation and influences T cell responses. Results of these studies will provide key insights towards the development of safe and effective vaccines against ZIKV and related flaviviruses.
Zika virus infection of mast cells is not associated with a degranulation response
Authors: Coish Jeremia M , Crozier Robert W. E. , Schieffelin John S. , Coorssen Jens R. , Hunter Fiona F. , MacNeil Adam J.
Published: January 2023

Dengue virus (DENV) infection is augmented by a process called antibody-dependent enhancement (ADE), in which pre-existing DENV immunity can render an individual more susceptible to a subsequent DENV exposure. Based on Zika virus (ZIKV) and DENV structural similarities, emerging evidence suggests anti-DENV antibodies can cross-react with ZIKV at non-neutralizing levels characteristic of ADE. Mast cells (MC), sentinel white blood cells integral in coordinating early immune defences, have been identified as a principle contributor to DENV vascular leakage. This clinical manifestation is attributed to significant histamine release caused by MC degranulation, a characteristic MC activation response. However, to our knowledge ZIKV-MC interactions remain unexplored. MC responses to virus can be modelled in vitro, including via use of the well-characterized KU812 cell line which express Fcγ receptors. Here, we sought to determine if the KU812 MC is susceptible to (1) direct ZIKV infection; and (2) ADE in the presence of anti-DENV antibodies that cross-react with ZIKV. A significant increase in viral titre (104 PFU/mL) was detected, by plaque assay, in MC directly infected with ZIKV compared to MC infected with UV-inactivated ZIKV (0 PFU/mL). Furthermore, a significant viral titre (106 PFU/mL) was detected in MC infected with ZIKV pre-incubated with anti-DENV antibodies when compared to MC infected with ZIKV pre-incubated with isotype control antibodies (104 PFU/mL). Furthermore, unlike DENV induced degranulation of MC, ZIKV did not activate a degranulation response in this model or in a primary murine model. Therefore, MC may be a contributor in ZIKV pathogenesis without the classical degranulation response seen in DENV infected MC.
Murine Ifit3 restricts West Nile virus infection and pathogenesis
Authors: Case James Brett , VanBlargan Laura A. , White James P. , Diamond Michael S.
Published: January 2023

The host interferon (IFN) response is the first line of defense for mammalian cells against invading pathogens. Among the most upregulated IFN-stimulated genes are members of the IFN-induced protein with tetratricopeptide repeats (IFIT) family of proteins. IFITs play a critical role in the host antiviral response by distinguishing between ‘self’ and ‘non-self’ RNA. Recent findings demonstrate that human IFIT3 binding to IFIT1 alters the viral RNA binding specificity of IFIT1. Uniquely, mice encode two copies of the Ifit3 gene, Ifit3a and Ifit3b, that differ by only five amino acids. To determine the function of mouse Ifit3 and to gain further understanding of the role of IFIT3 in antiviral immunity, we used CRISPR/Cas9 gene editing to generate Ifit3a/b DKO mice. Our results demonstrate that Ifit3 is important for survival of infection with West Nile virus (WNV, strain NY99). Whereas approximately 65% of WT C5BL/6J mice survive infection with WNV-NY99, Ifit3a/b DKO mice were more susceptible with only 20% of animals surviving infection. Virological analysis of lymphoid and CNS tissues revealed that Ifit3a/b DKO mice have increased viral loads in the brain, spinal cord, and liver compared to WT animals, and gene expression analysis of WT animals corroborated that Ifit3a and Ifit3b are highly induced in these tissues during infection. Detailed immunological analysis of Ifit3a/b DKO animals did not show a loss in the ability to generate WNV-specific antibodies, but did reveal a significant decrease in the number of CD8+ T cells in the spleen and a delayed CD8+ T cell response in the spinal cord. Altogether, our data suggest that Ifit3 restricts WNV infection and pathogenesis in distinct anatomic locations, most notably in specific regions of the CNS.
Integrated single-cell analysis of multicellular immune dynamics during hyper-acute HIV infection
Authors: Kazer Samuel W , Aicher Toby P , Muema Daniel M , Miao Vincent N , Ordovas-Montanes Jose , Ziegler Carly G K , Moodley Amber , Dong Krista L , Ndhlovu Zaza M , Ndung’u Thumbi , Walker Bruce D , Shalek Alex K
Published: January 2023

Development of effective vaccines and therapeutics is facilitated by understanding the earliest immune responses to infection. Here, we apply single-cell RNA-sequencing (scRNA-seq) and a novel gene module discovery-based framework to longitudinally profile peripheral multicellular immune responses in untreated hyper-acute HIV infection.

We profiled >59,000 single cells from four individuals across multiple acute infection timepoints (including pre-infection), utilizing scRNA-seq data to identify transient immune responses with cell subset resolution. Onset of viremia induced interferon stimulated gene (ISG) responses integrated across multiple cell types, wherein monocytes notably contributed to the cytokine milieu. Otherwise obscured in bulk analyses, we describe a second set of responses following ISG upregulation that align in time: pro-inflammatory T cell differentiation, prolonged monocyte MHC-II upregulation, and persistent NK cytolytic killing. Leveraging our temporal, cell-type resolved data, we nominate putative cell-cell signaling networks and their key cytokines during infection. Two participants who later develop viremic control associated with elevated frequencies of proliferating cytotoxic cells, inclusive of a previously unappreciated NK cell subset, immediately following HIV detection.

Our study reveals both cooperative and cell subset specific immune responses during untreated hyper-acute HIV infection with temporal resolution, nominating monocytes, NK cells, and pro-inflammatory signaling as perturbation targets for future vaccine studies. Moreover, our gene module discovery framework can be readily applied to other longitudinal studies in humans and other model organisms.
Plasmacytoid dendritic cells impair HIV-specific CD8 T cells to maintain HIV-1 reservoir persistence during chronic HIV infection under cART
Authors: Li Guangming , Ma Jianping , Yu Haisheng , Cheng Liang , Su Lishan
Published: January 2023

Plasmacytoid dendritic cells (pDC) are the major type I interferon (IFN-I) producing cells and play important roles in antiviral immune responses during acute virus infection. However, sustained pDC activation and IFN-I induction has been correlated with disease progression in chronic virus infection. We have recently functionally defined the HIV-pDC-IFN axis in HIV-1 immuno-pathogenesis, and studied the mechanisms of pDC/IFN-induced immune suppression, and its role in HIV reservoir persistence. We have discovered that depleting pDC during HIV infection reverses HIV-related diseases in lymphoid organs even in the presence of higher levels of HIV replication. We further show that low levels of pDC/IFN-I signaling contribute to the immune dysfunction and foster HIV-1 persistence in cART-treated hosts. In this study, we demonstrate that pDCs-depletion during suppressive cART reversed T cell exhaustion and reduced HIV-1 reservoir cells. This control of HIV-1 reservoir induced by pDC depletion is dependent on CD8 T cells, as depletion of both pDCs and CD8 T cells failed to reduce HIV-1 reservoir cells during cART in humanized mice. Our study demonstrates a key role of pDC-dependent CD8 T cell tolerance in maintaining HIV-1 reservoir persistence during cART in vivo. We are currently characterizing the CD8 changes induced by pDC depletion from HIV-infected lymphoid organs by genomic, epigenomic and metabolomic analyses.
SIVmac239 infection dysregulates anti-mycobacterial immunity at the granuloma level and contributes to severe tuberculosis in cynomolgus macaques
Authors: Mattila Joshua T , Talukdar Priyanka , Jespersen Alyssa , Fallert Junecko Beth A , Moriarty Ryan , O’Connor Shelby , Scanga Chuck A
Published: January 2023

Tuberculosis (TB) is a global pandemic caused by Mycobacterium tuberculosis (Mtb) infection that is characterized by formation of lung lesions called granulomas. TB has a synergistic relationship with HIV and TB often occurs in HIV-infected individuals with near normal CD4+ T cell counts. Despite the importance of this co-infection to public health, interactions between HIV and lesion-level immunity remain poorly understood. To address this issue, we used SIVmac239-infected or SIV-negative Mauritian cynomolgus macaques with active TB to identify which cell types are infected in granulomas and how SIV modulates local immunity. Using RNAscope and immunohistochemistry, we found that CD11c+ macrophages were the most-commonly infected cell type in granulomas. To identify how viral infection changes the immune landscape in granulomas, we performed a transcriptional analysis on RNA isolated from lung granulomas. We found that SIV infection downregulated large numbers of macrophage-activating cytokines and receptors, toll-like receptors, and T cell activation markers. In contrast, anti-viral responses including type 1 interferons and receptors expressed by NK cells were upregulated. IL-10 was among the genes that were not differentially regulated. Pathway analysis indicated that many pathways were downregulated, including pathways associated with Th1-, Th2-, and NFkB-mediated responses. These data suggest that SIV infection deactivates protective responses in granulomas but activates antiviral responses that are counterproductive in TB while leaving detrimental IL-10 expression intact. We hypothesize that this disequilibrium leads to an inability to kill bacteria and restrict dissemination that exacerbates TB.
Functional MAIT cells are associated with reduced simian-human immunodeficiency virus infection
Authors: Velu Vijayakumar , Murugesan Amudhan , Ibegbu Chris , Turner Tiffany , Jones Andrew , Shanmugasundaram Uma , Reddy Pradeep , Rahman Sadia Jannath , Saha PIu , Matam Vijay-kumar , Muthu Shankar Esaki , Amara Rama Rao
Published: January 2023

Mucosa-associated invariant T-cells (MAITs) are recently characterized novel subset of innate-like T-cells that recognize microbial metabolites presented by MHC-1b-related protein MR1. The significance of MAIT cells in anti-bacterial defense is well understood but not clearly in viral infections such as SIV/HIV infection. Here, we studied the phenotype, distribution, function of MAITs and their association with viral levels during chronic SHIV infection in rhesus macaques (RM). Two groups of healthy and chronic SHIV-infected RM were characterized for MAITs in blood and tissues. Similar to human, we found a significant fraction of RM CD8+ T cells express markers CD161, TCRVα□7.2 with high levels of IL-18R, CCR6, CD28 and CD95 that correlates with MR1 tetramer. During chronic SHIV, the frequency of MAITs are enriched in blood but unaltered in rectum, which displayed higher proliferative and cytotoxic capacity. Blood and rectal MAITs correlated inversely with plasma viral RNA and correlated directly with CD4 T cells. They respond to LPS and correlates with serum flagellin levels. Tissue analysis of MAITs during chronic SHIV showed significant enrichment in non-lymphoid tissues (lung, rectum and liver) with higher levels of tissue resident markers CD69 and CD103 unlike spleen/LN. In vitro treatment with cytokines during SHIV infection revealed that IL-7 is key for their proliferation and IL-12, IL-18 are important for their cytotoxicity. Thus our results demonstrates that functional MAITs are enriched in blood and rectum during chronic SHIV infection that inversely correlates with plasma viral levels. Therapies such combined cytokines could be beneficial for enhancing functional MAIT cells during chronic HIV infection in vivo.
Studies of incomplete control of Epstein-Barr virus infection in association with aging by using cynomolgus macaque colony
Authors: Urano Emiko , Murakata Yoshiko , Tsujimura Yusuke , Yasutomi Yasuhiro
Published: January 2023

The analysis of age-related immune senescent is extremely meaningful to understand the vital homeostasis in individuals among the aging society. Epstein-Barr virus (EBV) establishes latent infection in >90% of adults worldwide. In general, chronically infected EBV is controlled well for a lifelong in healthy adults. Since, EBV is reactivated by the failure of the host immune system, it can be an index to understand the hypofunction. A non-human-primate, controlled experimental animal, cynomolgus macaque (CM) has a long life span and can be used for longevity studies using aged animals that are more than 20 years old, which correspond to ≥60 years of age in humans. In this study, the state of EBV infection in the colony of CM was explored by detecting EBV in saliva as EBV reactivation, and the immunological functions were examined using PBMC from CMs concerned with the control of EBV. Age-related alteration of T cell and B cell subsets was observed in CM colony comparison with young (4–6 years) and elderly (24–26 years) group. T cell distribution was similar among young and elderly groups respectively; however, CD3 and CD28 stimulated cells from elderly EBV (+) group showed the lowest proliferation than the others. EBV-specific immune responses are under consideration. The CM colony will be a useful model of aging and the extensive study will help to elucidate the mechanisms of hypofunction through a long-life span.