bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic changes in immune populations at the fetomaternal interface during term and preterm birth
Authors: Lewis Emma L. , Barila Guillermo , Brown Amy G. , Porrett Paige M. , Elovitz Michal A.
Published: January 2023

The biologic mechanisms by which birth is initiated are unknown. Recent evidence suggests that the immune system may modulate gestational length. We previously established that maternal immune cells enter the placenta by mid gestation and change in composition near term. We hypothesize that preterm birth also involves immunologic alterations at the fetomaternal interface.

To test this hypothesis, we used an established model of inflammation-induced preterm birth. Pregnant mice received intrauterine injections (IUI) of lipopolysaccharide (LPS) (250μg/100μl) (N=8) or saline (100μl) (N=7) on embryonic day 15 (E15). Control mice, without IUI, were sacrificed on E15 (N=7) and E19 (N=7). (Term gestation is E19.5.) Maternal blood, spleen, uterine implantation sites, and placentae were collected, processed, and analyzed for immune cells by flow cytometry (CD45, Ly6G, CD11c, CD11b, CD103, F4/80, IA/IE, CD3, CD4, CD8, CD19, CD49b, TCR γ/δ, FoxP3, IL-17).

At E19, term gestation, the frequencies of multiple immune cell types decrease in the placenta: regulatory T cells (1.6 to 0.8%; p=0.0004), NK cells (2.6 to 1.5%; p=0.009), CD11c+ myeloid cells (8.6 to 5.5%; p=0.013), and CD103+ dendritic cells (0.43 to 0.25%; p=0.013). Only NK cells decrease near term in the uterus (9.5 to 1.6%; p=0.011). In contrast, during inflammation-induced preterm birth, macrophages decrease in the placenta (4.4 to 2.5%; p=0.042) and neutrophils increase in the uterus (26 to 52%; p=0.0006). These findings demonstrate that the immune composition of the fetomaternal interface shifts prior to birth. However, both the cell types and location of this shift differ between term and preterm birth, suggesting distinct immunologic mechanisms contribute to these processes.
Surveying the TCR landscape of intestinal intraepithelial lymphocytes
Authors: Schattgen Stefan A. , Crawford Jeremy C. , Thomas Paul G.
Published: January 2023

The mucosal epithelial surface of the gastrointestinal tract (GIT) provides an important barrier against microbes, toxins, and dietary matter, with disruption of epithelial integrity underlying the pathologies of a number of autoimmune conditions. Residing in the gut mucosa, intestinal intraepithelial lymphocytes (IELs) expressing rearranged αβ T cell receptors (TCRs) are able to rapidly respond to antigens and mediate repair of damaged epithelium. IELs are continuously surveying a stream of microbial, food, and self-antigens, and while antigen-recognition via the TCR of IELs is presumed the major mechanism governing their functions, little is known about the TCR repertoire of IELs or its specificity. Even less is known about how the IEL repertoire is formed under “normal” conditions and whether the IEL repertoire is dynamically edited in response to changes in the gut. Here, we perform a comprehensive survey of the IEL landscape of mice for the three distinct subpopulations of TCRαβ+IELs (DN-nIELs, CD8-iIELs, and CD4-iIELs) by leveraging single cell and bulk level TCR repertoire analyses. Our data show the IEL repertoire contains a striking amount of diversity, landing between oligoclonal and polyclonal on the continuum of TCR diversity. We have identified numerous public TCR chains with distinct restrictions and found the repertoires within each subpopulation are close in distance between individuals. Long-term antibiotic treatment and synthetic diets led to an increase in the number, TCR diversity, and activation of the IELs, suggesting the abundance of microbial and dietary antigens are important, though not the only, factors that shape the IEL repertoire.
Obesity and Intraepithelial Lymphocyte Dysfunction
Authors: Jameson Julie M. , Park Christa S. , Limon Natalie
Published: January 2023

Obesity has become prevalent in the United States with over 30% of adults and 17% of children having a BMI over 30. Complications associated with obesity include chronic nonhealing wounds in the skin and increased susceptibility to more severe colitis in the intestine suggesting that obesity leads to barrier dysfunction. Intraepithelial lymphocytes regulate epithelial homeostasis through the production of growth factors and cytokines. Our laboratory has shown that obese mice exhibit reduced numbers of intraepithelial lymphocytes in both the skin and the intestine. The remaining intraepithelial lymphocytes have reduced growth factor and cytokine production resulting in dysregulation of the barrier. To better understand the mechanism of T cell dysfunction, we placed mice on high-fat diet immediately post-weaning when intraepithelial lymphocytes are seeding the intestine. After 7 weeks, mice fed normal chow had normal numbers of intraepithelial lymphocytes seeding the intestine, however mice fed a high-fat diet had almost 50% fewer cells. CCR9 and CD103 were both reduced on the lymphocytes suggesting problems with homing and retention. In addition, mice administered a high-fat diet were more susceptible to DSS-induced colitis. To determine whether intestinal epithelial cells could be restored to normal numbers in obese mice, obese mice were administered normal chow for 7 weeks. Indeed, reducing fat intake was sufficient to restore intraepithelial lymphocytes to normal levels and rescue mice from more severe colitis suggesting that dieting is effective in improving intestinal barrier function in obesity.
Osteopontin provides a survival signal for intestinal intraepithelial lymphocytes in mice
Authors: Nazmi Ali , Olivares-Villagomez Danyvid , Piazuelo M. Blanca
Published: January 2023

Intraepithelial lymphocytes (IEL) are a diverse immune population residing in between intestinal epithelial cells, which possess important roles during mucosal immune responses. The IEL family comprises cells such as TCRαb+CD4+, TCRαb+CD8+, TCRgd+, TCRab+CD8aa+, iCD8a and iCD3+, among others. Osteopontin (OPN), a pleiotropic cytokine encoded by the Spp-1 gene, is well known for its function in tissue remodeling, modulation of Th1 responses development, maintenance of Th17 function, and homeostasis of NK cells. Herein, we investigated the impact of OPN in the IEL compartment.

We compared the cell numbers of IEL, lamina propria (LP) lymphocytes and splenocytes from Spp-1−/− and wild-type (WT) mice. OPN deficiency reduced the number of TCRγδ+, TCRβ+CD4+, TCRβ+CD8α+ and TCRβ+CD4+CD8α+ IEL, but had no effect on LP and spleen lymphocytes. In in vitro studies, IEL from WT mice cultured in the presence of rOPN showed better survival rates than IEL incubated alone. The increased survival was blunted to control levels when CD44, a receptor for OPN, was blocked with anti-CD44 antibody.

When total T cells (including Tregs) from WT splenocytes were transferred into Spp-1−/−Rag-2−/− and Rag2−/− mice, we observed that although OPN deficiency did not affect donor-derived IEL reconstitution at day 7 post-transfer, it decreased the number of CD4+ and CD4+CD8α+ IEL at day 28 post-transfer. As expected, Rag-2−/− mice remained healthy throught the experiment; however, Spp-1−/−Rag-2−/− recipient mice presented a remarkably loss of body weight and colon inflammation.

Together, our results indicate that OPN is a critical survival signal for IEL via CD44, and that OPN-deficiency promotes development of colitis even in the presence of Tregs.
Reciprocal activation of B cells and group 2 innate lymphoid cells
Authors: Feldman Scott , Kasjanski Richard , Norton James E. , Kern Robert C. , Conley David B. , Welch Kevin , Tan Bruce K. , Peters Anju T. , Grammer Leslie C. , Harris Kathleen E. , Carter Roderick G. , Suh Lydia , Stevens Whitney W. , Kato Atsushi , Schleimer Robert P. , Hulse Kathryn E.
Published: January 2023

B cells can be activated at peripheral sites in chronic inflammatory disease, but the mechanisms that drive this, especially in humans, are unclear. We have reported elevated frequencies of Epstein-Barr virus-induced molecule 2 (EBI2) expressing B cells in tissues from patients with chronic airway inflammation. EBI2 is a marker of extrafollicular plasmablasts, which are activated antibody-secreting cells (ASC). We sought to determine the mechanisms of extrafollicular B cell activation during chronic airway inflammation. ELISpot was used to determine ASC frequency in cells from inflamed nasal tissue or control tonsil tissue. B cells, T cells, and group 2 innate lymphoid cells (ILC2) were isolated from peripheral blood, and B cells were co-cultured with group 2 innate lymphoid cells (ILC2) or T cells. EBI2 expression was assessed by flow cytometry, and gene expression changes were assessed by single cell RNA-seq. Inflamed nasal tissue had a higher frequency of ASC compared to tonsil (p<0.05). The majority of the ASC were contained in the EBI2+ B cell subset in nasal and tonsil tissues. Co-culture of B cells and ILC2 significantly increased the frequency of EBI2+ B cells (p<0.01). B cells co-cultured with ILC2 expressed significantly higher levels of CCL17, CCL22, and FceR2 (>5 fold), compared to freshly isolated B cells, or B cells co-cultured with T cells. ILC2 co-cultured with B cells had significantly increased expression of IL-5, IL-13, and IL-2Ra (>7 fold) compared to freshly isolated ILC2. Our data suggest that not only can ILC2 directly activate B cells, but B cells can also enhance ILC2 function. These findings provide new insights into mechanisms that B cells may play in chronic inflammatory disease.
Novel cell-based high-throughput hybridoma screening method using the Celigo image cytometer for antibody discovery
Authors: Chan Leo , Zhang Haohai , Rice William , Kassam Nasim , Longhi Maria Serena , Zhao Haitao , Robson Simon , Gao Wenda , Wu Yan
Published: January 2023

Hybridoma screening is a highly important process that can identify potential clones from hybridoma cultures against the desired target antigen. Traditional screening methods using ELISA and flow cytometry presented technical issues such as limited accuracy, reduced high-throughput capability, increased time and cost. Here, we demonstrate a novel cell-based high-throughput screening method using the Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on CHO cells. Both target CHO and CFSE-stained wild type were co-cultured to simultaneously detect target antibodies and nonspecific binding, which can eliminate the need to produce a separate control sample for each hybridoma supernatant. Next, the image cytometer was used to screen 672 hybridoma supernatants to develop the screening workflow. The Celigo was used to quickly image and analyze antibody binding using Hoechst, Alexa Fluor 594 and CFSE fluorescence to identify high, medium, and low binding hits. In addition, the Celigo was used to measure the binding affinities of the target antibodies to membrane-bound CD39. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5 ng/mL. We propose that this method will greatly improve screening technologies and facilitate more efficient antibody discovery.
Sample multiplexing of peripheral immune populations for high throughput single-cell RNA-sequencing
Authors: Bansal Nidhanjali , Chang Christina , Liang Yuqiong , Shum Eleen Y. , Martin Jody C. , Ghadiali James , Jensen Devon , Hu Jing , Rosenfeld David , Zheng Ye , Fan H. Christina
Published: January 2023

Diverse immune populations reside in non-lymphoid organs and contribute to immune defense and tissue homeostasis. However, these cells are often hard to study due to low cell abundance and high heterogeneity. Recent advancements in single-cell sequencing technology provides a powerful high parameter tool to study these peripheral immune populations. But current single cell experiments are costly and limited by sample throughput. To address these limitations, we have developed a novel sample multiplexing approach for high throughput single-cell sequencing.

In this study, we performed single-cell sequencing analysis of thousands of immune cells isolated from peripheral tissues in mice, and utilized a DNA barcoded universal antibody to sample multiplex up to 12 samples in a single experiment. This allowed us to combine samples from different mice and tissue types into a single pooled sample, significantly reducing experimental scale and cost, while eliminating potential batch effects. The sample pool was captured on the BD Rhapsody™ system and a targeted assay was performed to measure gene expression of ~400 genes. We were able to de-multiplex the pooled samples with high specificity after sequencing. The targeted gene panel provided robust clustering of the major immune cell types, enabling us to perform immuno-profiling and compare gene expression of major cell populations across different tissues. We observed distinct tissue-specific expression profiles of major immune populations, and further investigation of the differentially expressed genes may provide better understanding of the interactions between these tissue immune populations and their local environment.
Tumor infiltrating T cells: complete workflows allow faster and improved analysis
Authors: Evaristo Cesar , Siemer Ramona , Agorku David , Brauner Janina , Hardt Olaf , Dose Christian , Richter Anne
Published: January 2023

T cells can eliminate tumors, but tumor infiltrating (TI) T cells can become intrinsically anergic or extrinsically suppressed. Identifying the mechanisms of T cell dysfunction is necessary to improve immunotherapy efficacy. Mouse tumor models are useful tools in tumor T cell research, but analysis of TI T cells is technically challenging, time consuming and labor intensive. Their frequency can be very low and small populations may escape analysis as they get lost in the background. Importantly, TI T cells are in an antigen-rich and highly immunomodulatory cell environment, such that cell intrinsic functional analysis is hindered. It is fundamental to use new tools to streamline the workflow and generate reliable data.

We established workflows combining tissue dissociation, T cell isolation and phenotyping. Tumor dissociation was automated using the gentle MACS™ and optimized for epitope preservation. Enzymatic dissociation was essential for analysis of PD1hiTim3+Lag3+CD39+ CD8 T cells in tumors. Using new T cell-specific microbeads for magnetic cell sorting we increased TI T cell purity by up to 500-fold, while maintaining activation status and phenotype. Importantly T cell isolation from the tumor’s highly immunomodulatory cellular environment was essential to assess cell-intrinsic functional properties. Finally, our data show the use of recombinant RE Afinity antibodies eliminated non-specific labeling of cells in the tumor microenvironment.

We conclude that standardized processing of tumor samples and magnetic isolation of tumor infiltrating T cells greatly reduces time and cost of total workflow and downstream analysis while significantly increasing reproducibility and the quality of data obtained from TIL analysis.
Dissection of single-cell gene regulation by simultaneous digital mRNA and protein quantification
Authors: Lam Regina , Shum Eleen , Chang Christina Christina , Lam Gretchen , Bansal Nidhanjali , Shah Hemi , Jensen Devon , Hu Jing , Ghadiali James , Martin Jody , Rosenfeld David , Fan H Christina
Published: January 2023

The immune system consists of complex gene regulatory networks that allow a rapid transition of different cellular states during an immune response. Cell-surface marker analysis using flow cytometry or single cell RNA-seq has allowed characterization of immune subpopulations and a greater understanding of the complexity of immune cells. However, restrictions on protein-only or RNA-only analysis can greatly limit the understanding of how genes are regulated in cells. For example, many cell surface markers – such as CD4 in T cells – have thousands of protein copies per cell, yet is fueled by a small number of mRNA transcripts.

To bridge the understanding of protein and mRNA expression differences, we used BD™ AbSeq on the Rhapsody™ platform to provide digital quantification of both protein and mRNA expression level in single cells. An oligo-conjugated antibody panel against immune-relevant cell-surface markers was created and used for this multi-omic gene expression profiling effort. This approach is coupled with mRNA analysis using the BD Rhapsody Immune Response Panel, a targeted method of RNA-seq that allows a higher sensitivity of immune markers than conventional whole transcriptome assays.

We studied the dynamics of early T cell activation in vitro to understand this response on transcriptional, post-transcriptional, and translational levels. Different time points following anti-CD3 and anti-CD28 treatment were collected and multiplexed on to BD Rhapsody cartridge for single cell capture and analysis. Using AbSeq on BD Rhapsody, we were able to detect the difference in mRNA and protein levels of crucial markers, allowing us to dissect the intricate gene regulatory pathways during an immune response in a single cell level.
Modular, defined assembly of antibody-based constructs using click chemistry
Authors: Langsdorf Chris , Aggeler Robert , Chen Aimei , Dolman Nicholas , Hu Yi-Zhen , Kamen Lynn , Mandavilli Bhaskar , Ordonia Benjamin , Wickett Marcy
Published: January 2023

Immunoglobulin antibodies contribute to a broad range of research assays and also provide the foundation for a diverse variety of biopharmaceutical agents. Here we describe the use of bio-orthogonal click chemistry reactions to synthesize several common antibody-based structures and demonstrate their use in cell-based applications. First, defined antibody-fluorophore conjugates were synthesized, characterized and functionally tested in receptor occupancy and antibody internalization assays. Next, a double conjugation strategy was employed to synthesize antibody drug conjugates which become brightly fluorescent upon endocytosis. ADC uptake and targeted cell killing were demonstrated using cell line and spheroid models. Then bispecific antibodies were constructed using a click chemistry reaction to create tetravalent heterodimeric antibodies. Structure and function of these bispecific constructs are demonstrated in cell-free and flow cytometry assays.
Validation of a Protein A-based ELISA for quantifying Immunoglobulin G in a non-traditional wildlife species, the Steller sea lion (Eumatopias jubatus)
Authors: Kennedy Stephanie N. , Wilhite Brittany , Castellini J. Maggie , Rea Lorrie D. , Kuhn Tom , O’Hara Todd
Published: January 2023

Immunoglobulins are key proteins that function to preserve immune homeostasis and are quantified to infer changes to the acquired humoral immune response in mammals. Measuring immunoglobulins in non-traditional wildlife for immune surveillance often requires ingenuity, and rigorous standardization of methodologies to provide consistent and reliable results especially for species lacking species-specific reagents. We modified and optimized existing ELISA methodology that utilizes the binding properties of Staphylococcus derived Protein A (Prt A) with immunoglobulin G (IgG). We validated the assay for accurately quantifying IgG in Steller sea lion (SSL) serum using critical measures of validation including parallelism, spike and percent recoveries, and internal controls. Of the modifications made, heat inactivation of SSL serum proved pivotal to enhance accuracy and precision of IgG detection by improving linearity and percent recovery in parallelism dilutions and serum spikes. Purified canine IgG standard was not affected by the heat inactivation protocol. These results support that confounding serum proteins likely interfere with the binding of Prt A with IgG, and demonstrate the need for heat inactivation of serum to ensure optimal IgG quantification using the PrtA-ELISA. Further, incorporation of spike and recovery are essential validation measures to ensure proper standardization of this assay. Consequently, the improved and validated PrtA-ELISA guarantees a species-independent IgG detection with validation criteria to enhance accuracy, and precision for addressing future immunological questions concerning SSLs and other non-traditional wildlife in a clinical, ecological, and conservation context.
NCR−ILC3 as the initial responders to liver inflammatory environment promote HCC development through IL-23/IL-17 axis
Authors: Liu Yonghao , Song Yuan , Lei Lei , Huang Yuhui , Liu Haiyan
Published: January 2023

IL-17 is crucial in tumor-promoting inflammation and the development of hepatocellular carcinoma (HCC). However, the role of IL-23, an important IL-17-inducing inflammatory cytokine, as well as the immune cells responsible for initiating IL-17 production in the tumor microenvironment are not clear. In the current study, we assessed the development of HCC and the function of the adaptive anti-tumor immune response, as well as the kinetics of the development of IL-17-producing cells in an IL-23-rich tumor microenvironment. Our results demonstrated that IL-23 promoted HCC tumor growth and inhibited anti-tumor adaptive immune response by enhancing IL-17A production in the tumor microenvironment. Furthermore, our kinetic analysis showed that NCR−ILC3 were the first responders to IL-23, producing IL-17 during the early phase of tumor development. Their proliferation in vivo was facilitated by IL-23, and adoptive transfer of NCR−ILC3 cells promoted HCC development. NCR−ILC3 could suppress IFN-γ production of both CD4+ and CD8+ T cells, and inhibit proliferation and survival of CD8+ T cells in vitro. Moreover, the percentage of IFN-γ-producing ILC1 were significantly reduced by IL-23 in the liver during tumor development, and they were shown to be able to differentiate into ILC3 in the presence of IL-1β and/or IL-23 in vitro. The adoptive transfer experiment further demonstrated that ILC1 could gain RORγt expression in an IL-23-expressing HCC model. Therefore, our findings demonstrated that IL-23 could promote HCC development through enhancing IL-17 production, and NCR−ILC3 as the initial IL-17-producing cells in response to IL-23 in the liver could be the initiator of the IL-17-rich immune-suppressive tumor microenvironment.
Phosphatidylserine targeting antibody in combination with tumor radiation and immune checkpoint blockade promotes anti-tumor activity in mouse B16 melanoma
Authors: Budha Sadnu , Giese Rachel , Gupta Aditi , De Henau Olivier , Zappasodi Roberta , Campesato Luis Felipe , Barker Christopher , Shan Joseph , Wolchok Jedd D , Merghoub Taha
Published: January 2023

Phosphatidylserine (PS) is a phospholipid that is exposed on surface of apoptotic cells, some tumor cells and tumor endothelium. It has been shown to promote immunosuppressive signals in the tumor microenvironment. Radiation therapy (RT) is an effective focal treatment of primary solid tumors, but is less effective in treating metastatic solid tumors as a monotherapy. There is evidence that RT induces immunogenic tumor cell death and enhances tumor-specific T cell infiltration in treated tumors. In this study, we show that irradiation of B16 melanoma causes an increase in PS expression on the surface of viable tumor and immune infiltrates. We subsequently examined the effects of combining RT with an antibody that targets PS (mch1N11) and immune checkpoint blockade (anti-PD-1) in B16 melanoma. We found that treatment with mch1N11 synergizes with RT to improve anti-tumor activity and overall survival in tumor bearing mice. In addition, the triple combination of mch1N11, RT and anti-PD-1 treatment displayed even greater anti-tumor and survival benefit. Analysis of immune responses in the tumors of treated animals revealed a shift towards a pro-inflammatory M1 phenotype after treatment with RT and mch1N11. In addition, analysis of the adaptive immune response revealed an increase in antigen-specific CD8 T cell infiltration in the tumors as well as increased activation, effector function and differentiation in the triple combination therapy. This finding highlights the potential of combining these agents to improve outcome in patients with advanced-stage cancer and may inform the design of future clinical trials.
Blocking colony stimulating factor 1 receptor (CSF-1R) and tropomyosin receptor kinase A (TrkA) improves the antitumor efficacy of immune checkpoint blockade
Authors: Duffy Colm R. , Mok Stephen , Allison James P.
Published: January 2023

Established tumors can escape immune responses by secreting the cytokine colony stimulating factor 1 (CSF-1), stimulating the proliferation and recruitment of immunosuppressive myeloid cells to the tumor microenvironment by binding to colony stimulating factor-1 receptor (CSF-1R). Additionally, the neurotrophin nerve growth factor (NGF) is a ligand for tropomyosin receptor kinase A (TrkA), which is over-expressed on multiple tumor types. Signaling through TrkA via the Akt and MAPK pathways regulates survival, proliferation and invasion of tumor cells. This invasion can occur around nerves, and perineural invasion (PNI) has emerged as a critical pathological feature of multiple malignancies, resulting in poor prognosis.

PLX7486 is a novel orally bioavailable small molecule Trk and CSF-1R dual-inhibitor that is now being studied in Phase I clinical trials. We hypothesized that PLX7486 would synergize with immune checkpoint blockade to result in a greater antitumor effect, by targeting Trk signaling directly on cancer cells and by inhibiting the recruitment of immunosuppressive myeloid cells through CSF-1R, thus enabling an improved antitumor T cell response.

We confirmed the expression of TrkA receptor on multiple murine cancer cell lines in vitro and exposed them to PLX7486, showing a direct cytotoxic effect with an IC50 of 5–8μM on most of the cell lines and an inhibition of AKT pathway signaling in MC38 cells. PLX7486 also had a direct cytotoxic effect on bone marrow-derived macrophages and the murine macrophage cell line RAW264.7 with an IC50 <1μM, while it had no effect on activated T cells in vitro. Combining PLX7486 and anti-CTLA-4 or anti-PD-1 in vivo in MC38 and B16F10 tumor models resulted in increased antitumor effects.
The Curative Effect of Cryptotanshinone and anti-PD-L1 on Mouse Hepatocellular Carcinoma
Authors: Han Zhen , Liu Shuo , Lin Hongsheng , Trivett Anna , Yang De , Oppenheim Joost J.
Published: January 2023

Cryptotanshinone (CT), isolated, purified and crystalized from the dried roots of Salvia militorrhiza, has been reported to have cytotoxic antitumor effects on a human liver cancer cell line in vitro. However, we have shown that CT also has immunoadjuvant effects and augments Th1 antitumor immunity. CT inhibited the proliferation of mouse hepatoma (Hepa1-6) cells by inducing Hepa1-6 cells apoptosis by dose dependent inhibition of the STAT3 signaling pathway. Furthermore, CT could also induce RAW264.7 cells polarize toward a M1 phenotype presumably with cytotoxic anti-tumor effects. CT was effective in the treatment of established Hepa1-6 hepatoma tumors in mice in combination with anti-PD-L1, and can cure Hepa1-6-bearing mice with the induction of subsequent anti-Hepa1-6 specific long-term immunity. Our data indicate that CT and anti-PD-L1 result in an effective immunotherapeutic regimen for the treatment of human hepatocellular carcinoma (HCC) based both on the cytotoxic effects and induction of immunity to the tumor.
Improvement of agonist cancer immunotherapy by CD73 blockade
Authors: Chen Siqi , Fan Jie , zhang minghui , Qin Lei , Dominguez Donye , Long Alan , Zhang Bin
Published: January 2023

Agonist antibodies (Ab) can directly target costimulatory molecule on the surface of antigen-primed T-lymphocytes. Among those, tumor necrosis factor receptor superfamily (TNFRSF) agonists are attractive and some of them are currently in various stages of pre-clinical and clinical trials, including anti-4-1BB, anti-GITR and anti-OX40, albeit with limited therapeutic benefit as single agents. Here, we examined the inhibitory role of ecto-enzyme CD73 (generating adenosine) on the antitumor efficacy of agonistic anti-4-1BB/CD137 Ab therapy, as anti-4-1BB treatment preferentially drove CD73− effector T cell response within TGF-β-low tumor microenvironment. The anti-CD73/anti-4-1BB combination therapy induced tumor regression associated with enhanced antitumor CD8+ T cell responses and reduced CD4+Foxp3+ regulatory T cell (Treg) accumulation. However, the TGF-β-rich tumor milieu confered resistance to anti-4-1BB therapy with sustained CD73 expression on both infiltrating CD4+ and CD8+ T cells across several tumor models. Our study establishes a novel mechanistic link between the CD73 axis and engagement of 4-1BB and possibly other costimulatory molecules. Thus, CD73 blockade has great potential for more effective clinical targeting of TNFRSF agonists.
Immunological Insights Into Liver Metastasis Associated Resistance To Checkpoint Blockade Cancer Immunotherapy
Authors: Lee James , Mehdizadeh Sadaf , Tsai Katy , Algazi Alain , Rosenblum Michael , Daud Adil , Bluestone Jeffrey A.
Published: January 2023

Despite advancements in cancer immunotherapy, we have previously reported that stage IV melanoma and lung cancer patients with liver metastasis have decreased response rate to PD-1 blockade and shorter survival. A uniquely tolerogenic organ, the liver is the most common site of metastases of the majority of cancers, and cancer patients with liver metastasis have much worse survival overall. Thus, we hypothesized that cancer cells that metastasize to the liver may utilize liver-mediated tolerance mechanisms to decrease systemic anti-tumor immunity and render immunotherapies less effective.

Metastatic melanoma patients with liver metastasis or other organ metastasis were biopsied pre and post PD-1 treatment and their distant primary site’s tumor immune infiltrates (TILs) were analyzed by FACS. Patients with liver metastasis had significantly lower CD8/Foxp3+ Treg ratio and decreased percentage of activated PD-1+/CTLA-4+ CD8 cells from their biopsy samples, suggesting a systemic influence on tumor immunity. To investigate the mechanism, C57BL/6 mice were implanted with “primary” subcutaneous (SQ) tumors and experimental “metastatic” tumors in the liver or control organ (lungs) and given systemic PD-1 blockade. Mice with liver metastasis had shortened survival and lower response rate to PD-1 blockade at the SQ site. TILs analysis within the SQ tumor of animals with liver metastasis shows lower CD8/Foxp3+ Treg ratio, lower expression of post-activation makers PD-1, CTLA-4, ICOS, and Ki67, and decreased tumor antigen tetramer-positive CD8 cells in a Treg dependent manner. These results indicate liver metastasis may induce systemic immune suppression to the detriment of stage IV cancer patients undergoing PD-1 blockade therapy.
Commensal dysbiosis modulates the tumor microenvironment in breast cancer and diminishes efficacy of PD-L1 inhibition
Authors: Rutkowski Melanie R. , Rosean Claire M. , Bostic Raegan , Feng Tzu-Yu
Published: January 2023

For patients with HR+ breast cancer, non-responsiveness to PD-1/PD-L1 blockade cannot be attributed to poor immunogenicity, as immune-stimulatory gene signatures are associated with greater long-term survival and enhanced responsiveness to therapy. Targeting myeloid-driven immune suppression has also been shown to enhance efficacy of PD-L1 blockade in non-responsive tumors. However, we find that in HR+ breast cancer, synergy with PD-L1 blockade is negatively influenced by a pre-established loss of commensal homeostasis, or commensal dysbiosis. Using a clinically relevant non-responsive HR+ breast tumor model, establishing commensal dysbiosis prior to breast tumor initiation drives significant and irreversible T cell dysfunction within primary tumors. Additionally, commensal dysbiosis alters the cellular composition within the tumor microenvironment, culminating in the recruitment and polarization of M2-like macrophages with distinct tumor-suppressive attributes. Systemically, tumor-bearing animals with established commensal dysbiosis have significantly higher levels of prostaglandin E2 with the serum, which is independent of tumor volume. However, blockade of COX-2 only synergizes with PD-L1 blockade in animals without commensal dysbiosis. Unexpectedly, we found that commensal dysbiosis enhances COX-1/PGE2 signaling within the mammary epithelium during early tumor progression. Inhibition of COX-1-mediated PGE2 synthesis during early stages of tumor progression resulted in delayed tumor progression only in dysbiotic tumor-bearing mice, suggesting that dysbiosis-induced COX-1 mediated tissue inflammation is sufficient to drive irreversible immune suppression during advanced breast cancer.
IL-17C inhibits the development of hepatocellular carcinoma through the modulation of gut microbiota
Authors: Gong Huanle , Ma Shoubao , Dong Chen , Liu Haiyan
Published: January 2023

IL-17C has been recently reported to play an important role in infections and inflammatory diseases. However, its function in anti-tumor immune response is not clear. Here, we investigated the role of IL-17C in murine hepatocellular carcinoma (HCC) models and found that genetic depletion of IL-17C significantly promoted HCC growth, whilst overexpression of IL-17C inhibited HCC development. Flow cytometry analysis further demonstrated that the percentage of CD8+ T cells was reduced in IL-17C−/− mice. Moreover, the activation of T cells was also significantly reduced when IL-17C was deficient. Interestingly, depletion of gut microbiota with antibiotics treatment diminished the difference in tumor burdens between WT and IL-17C−/− groups, indicating microbiota might be involved in the process of IL-17C regulating HCC development. In addition, 16sRNA sequencing results revealed that IL-17C deficiency significantly reduced the Lactobacillus acidophilus colonization. Collectively, we found that IL-17C suppressed HCC growth in a gut microbiota-dependent manner. Further studies are needed to confirm the contribution of L.acidophilus to HCC development and investigate the detailed mechanisms of IL-17C regulating anti-tumor immune response.
Tim-3 and TIGIT mark Natural Killer cells susceptible to effector dysfunction in human bladder cancer
Authors: Farkas Adam M. , Audenet Francois , Anastos Harry , Galsky Matthew , Sfakianos John , Bhardwaj Nina
Published: January 2023

Natural Killer cell (NK) effector functions are diminished during cancer but the phenotype and mediators of this process are unclear. In contrast, dysfunctional T cells express markers of “exhaustion” (e.g. CTLA-4 and PD-1), and inhibition of signaling through these molecules has improved clinical outcome in multiple tumors. Our data from 100+ patients with bladder cancer (BC) suggests that NK undergo an analogous process of exhaustion marked by up-regulation of Tim-3 and TIGIT in both the periphery and tumor. Tim-3 and TIGIT are induced on both immature and mature NK subsets suggesting implications for normal NK maturation. The magnitude of Tim-3 expression in both tissues is tuned to the invasiveness of the primary tumor, while TIGIT is not, making Tim-3 function as a rheostat for primary tumor invasiveness. Both molecules are expressed at equivalent frequencies in both tissues independent of the magnitude of overall expression but define NK with different effector functions. Peripheral NK from BC patients are functionally comparable to NK from healthy donors in their ability to produce IFNγ and degranulate in response to target cells, while tumor NK are refractory to both stimuli. NK from tumors are not terminally exhausted as they make IFNγ, TNFa, and degranulate after “resting” ex vivo prior to stimulation. Blockade of Tim-3, but not TIGIT, enhances effector function in peripheral NK from BC patients, but is ineffective for NK in tumor tissue implicating tumor-specific factors in mediating NK dysfunction. Tim-3 blockade was most efficient in NK from BC patients after activation with IL-15 suggesting specific inflammatory cues affect responsiveness to checkpoint blockade. Ongoing work will develop strategies to reverse NK exhaustion.
Investigation of novel TLR7/8 ligands in combination with TLR4 ligands as adjuvants to drive cell mediated anti-influenza immunity
Authors: Miller Shannon M. , Cybulski Van , Walsh Lois , Livesay Mark , Bess Laura , Burkhart David J. , Bazin-Lee Helene , Evans Jay T.
Published: January 2023

Influenza is a highly communicative viral pathogen that infects 9–35 million individuals and results in 12,000–56,000 deaths in the US annually. For the most commonly used seasonal influenza vaccine, historical efficacy rates range from 10–60% thus improvements to the current vaccine are needed. We identified a novel series of synthetic TLR7/8 ligands that improve influenza vaccine efficacy when used alone or in combination with our synthetic TLR4 ligand. In human PBMCs, TLR7/8 ligands elicited secretion of the inflammatory cytokines TNFα, IL-6, and IL-1β as well as the Th1-polarizing cytokine IL12-p70. These responses can be altered by modification of novel structural features of the TLR7/8 ligands. The addition of a synthetic TLR4 ligand modified the cytokine expression profile, indicating that the structure of a TLR7/8 ligand and/or addition of a TLR4 ligand can drive substantive changes in the innate immune response. To evaluate the adjuventicity of these TLR ligands, Balb/c mice were vaccinated with split-flu vaccine containing TLR7/8 ligands alone or in combination with our TLR4 ligand. TLR7/8 ligands increased influenza-specific total IgG and IgG2a, but not IgG1, over antigen alone. Addition of a TLR4 ligand further increased influenza-specific total IgG and IgG2a. These data demonstrate that these ligands act as adjuvants to promote a Th1 skewed humoral response. The combination of TLR7/8 and TLR4 ligands elicited superior cell mediated immunity compared to either TLR7/8 or TLR4 ligand alone. These data demonstrate that heightened innate and adaptive immune responses can be elicited through a combination adjuvant using novel synthetic TLR7/8 and TLR4 ligands.
Poly-gamma glutamic acid/alum enhances the efficacy of vaccine antigen by modulating antigen transport to lymph nodes and the adaptive immune responses
Authors: Nguyen Quyen Thi , Yang Jihyun , Kim Jaemoo , Kwak Chaewon , Poo Haryoung
Published: January 2023

Vaccination is the most effective strategy to protect against infectious diseases and adjuvants are proven to be the key component in vaccines capable of improving antigen-specific immune responses. Conventional vaccine adjuvants, however, still have several limitations such as safety concerns and insufficient induction of vaccine efficacy. Here, we developed a novel adjuvant by combining poly-gamma glutamic acid (γ-PGA) with alum that is already approved for human use worldwide. The physiochemical properties, efficacy, and underlying mechanism of the PGA/Alum as an adjuvant were investigated using ovalbumin (OVA) as a model antigen in vivo and in vitro. PGA/Alum-OVA administered mice showed the robust increase of antigen-specific humoral and cellular immune responses compared to mice administered with γ-PGA-OVA or alum-OVA. Importantly, the PGA/Alum-OVA-induced antibodies had significantly higher antibody-dependent cellular cytotoxicity (ADCC) activity. The enhanced activation and antigen processing of dendritic cells (DCs) and the facilitated migration of antigen-loaded DCs from the injection sites to draining lymph nodes were observed in PGA/Alum-OVA administered mice. Finally, the efficacy of the PGA/Alum as a vaccine adjuvant was evaluated using pandemic H1N1 influenza vaccine antigen. The PGA/Alum drastically enhanced protective efficacy of the pandemic H1N1 vaccine against influenza virus infection. Taken together, PGA/Alum may be a promising adjuvant to improve the influenza vaccine efficacy.
CD8+ T cells control HSV-1 replication in the nervous system but not the cornea in HSV-1 0ΔNLS-vaccinated mice lacking virus-neutralizing antibody
Authors: Carr Daniel J. J. , Montgomery Micaela Lee , Royer Derek
Published: January 2023

HSV-1 is a successful human pathogen that can elicit benign pathology including cold sores to more severe to extreme life-threatening manifestations like blinding necrotizing stromal keratitis and encephalitis. To date, there is no commercially available vaccine for HSV-1 despite many failed experimental prototypes. We have evaluated the use of a live-attenuated HSV-1 strain (HSV-1 0ΔNLS) as a vaccine in a mouse model of ocular infection. This vaccine has been shown to prevent virus replication, spread, and establishment of latency while functionally preserving the visual axis and corneal integrity. The correlate of protection elicited by the vaccine is antibody, and optimal efficacy requires expression of the neonatal Fc receptor in the corneal epithelium. The present study was undertaken to determine if T cell receptor transgenic mice expressing CD8 T cells specific for glycoprotein B of HSV-1 (gBT-I.1) benefit from vaccination. gBT-I.1 mice were vaccinated with HSV-1 0ΔNLS or vehicle and subsequently ocularly challenged with HSV-1. The results showed HSV-1 0ΔNLS vaccinated gBT-I.1 mice had no measureable neutralizing antibody titer but did show enhanced survival compared to vehicle-vaccinated gBT-I.1 mice. The efficacy correlated with less infectious virus in the nervous system of HSV-1 0ΔNLS-vaccinated mice during acute infection (p < 0.05). However, viral titers and pathology were similar in the corneas of both groups. Collectively, the results suggest in the absence of antibody, vaccine-primed HSV-specific CD8+ T cells enhance control of HSV-1 replication and spread in the nervous system but are not effective in the cornea.
A Bovine Adenovirus (BAdv) Expressing Mycobacterial Secreted Ag85B and a TLR-2 Stimulating Adjuvant Peptide Induces Robust Antigen Presentation In vitro and Improved Protection against Tuberculosis in Mice
Authors: Khan Arshad , Mani Arunmani , Hwang Shen-An , Mann Lovepreet , Papanna Ramesha , Won Jong Hak , Canaday David , Hunter Robert L. , Wang Jin , Mittal Suresh , Jagannath Chinnaswamy
Published: January 2023

Human Adenovirus (HAdv) vectors expressing Mycobacterium tuberculosis (Mtb) derived antigens, have been tested as candidate boosters for BCG vaccine in animal models and Phase I human studies but failed to provide significant additional protection. Presumably, pre-existing immunity to HAdv among humans has been suggested to limit their efficacy as vectors. We earlier reported that novel bovine adenovirus (BAdv)-vectors induced robust T cell mediated immunity in mice and carried less risk of interference by human innate immunity. Thus, we evaluated BAdv vectors expressing Mtb derived antigen 85B and a novel TLR-2 activating peptide as booster vaccines for BCG in vitro and in mouse model for tuberculosis. Both mouse and human APCs (macrophages and dendritic cells) infected with BAdv-85B showed robust presentation of p25 epitope to T cells within 18 hrs of infection. BAdv-Ag85B-TLR2 was better than BAdv-85B in inducing antigen presentation. Antigen presentation by both BAdv-Ag85B and BAdv-Ag85B-TLR2 peptide were significantly decreased when DCs were derived from atg7 knockout mice or when 3-methyladenine was used for blockade, indicating that autophagy played a role during adenovirus-induced antigen presentation. Consistent with in vitro data, BAdv-Ag85B-TLR-2 and BAdv-Ag85B vaccinated mice exhibited reduced lung CFUs of Mtb by 1.5 log and 1.7 log respectively whereas BCG vaccinated mice could reduce CFUs by 0.95 log as compared to unvaccinated mice. In addition, the BAdv vaccines also protected spleens better than BCG suggesting improved protection against disseminated tuberculosis. Since BAdv can be given through mucosal route, we propose that they offer a novel mean to enhance lung and systemic immunity against tuberculosis.
Polarization of Acellular Pertussis Vaccine-primed Immune Response to the TH17 Response
Authors: Kapil Parul , Merkel Tod J.
Published: January 2023

The resurgence of pertussis in the United States has stimulated interest in the development of improved pertussis vaccines. Studies in the baboon model demonstrated that responses induced by infection were dominated by TH1 and TH17 and resulted in near sterilizing immunity. In contrast, the response to vaccination with licensed acellular pertussis (aP) vaccine was dominated by TH2 responses that prevented clinical disease following challenge with pertussis, but did not prevent colonization or transmission. These results suggest that B. pertussis will circulate more freely in an aP-vaccinated population.

A conservative approach for addressing the resurgence of pertussis would be replacement of the aP booster vaccines with an acellular vaccine that generates a balanced TH1/TH2/TH17 response. Before pursuing this strategy, it will be important to understand the degree to which a booster vaccine can polarize an aP-primed TH2 immune response. In this study, aP-primed and unvaccinated baboons were challenged with wild-type B. pertussis. Six months following the first challenge, all baboons and additional unvaccinated, unexposed (naïve) baboons were challenged with wild-type B. pertussis. Our results demonstrated that natural infection of the aP-primed animals resulted in polarization of the TH2-dominated immune response towards a TH17 but not a TH1 response. In contrast to the convalescent animals that were not detectably colonized following the second challenge, the aP-primed, convalescent animals were transiently infected. This data indicates that the aP-primed immune response can be reprogrammed towards a more protective TH17 response albeit not to the same degree as induced by natural infection of unvaccinated animals.
Validation of a fluorescent microsphere multiplex serology assay for differential diagnosis of exposure to Zika virus and other closely related arboviruses
Authors: Venkateswaran Neeraja , Sarwar Jawad , Parameswaran Nishanth , Krishnan Krish VV. , Nelson William M.
Published: January 2023

Arboviruses cause a serious public health problem in tropical and subtropical countries and are emerging as a threat in western hemisphere also. Rapid and accurate detection of these infections can help in improving patient care and disease control. The cross-reactivity of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to ZIKV with dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV) and some other related flaviviruses poses a significant challenge for an accurate detection of ZIKV infection in serological assay such as IgM capture ELISA. To overcome these challenges we developed a multiplex serological assay that will simultaneously detect antibody response to ZIKV, other related flaviviruses and Chikungunya virus (CHIKV). Various recombinant arboviral antigens were coupled to optically encoded microspheres from Luminex Corporation. This fluorescent microsphere immunoassay was used to differentiate between recent, past, and/or co-infections that may occur in endemic regions. We also developed an easy detection algorithm for quick multiplex data analysis to differentiate ZIKV IgM response from other arboviruses. This rapid assay can be performed from sample to answer in less than 2 hours. This assay was validated with more than 200 samples procured from commercial sources; ZIKV, DENV, CHIKV, and WNV positive samples for sensitivity; EBV, CMV, flu positive samples, and normal samples for specificity. We obtained good concordance with the results provided by the vendor. Results indicated that serological response to ZIKV NS1 antigen is more specific than ZIKV envelope antigen. A significant IgM antibodies to ZIKV envelope antigen was detected in samples that were positive for other flaviviruses.