bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced innate CD161+ T cell responses in mucosal tissues of nonhuman primates
Authors: Rout Namita , Woods Adrienne
Published: January 2023

Innate T cells recognize non-peptide antigens presented by non-classical MHC molecules, such as CD1 and MR1. The three major populations within the innate T cell group, namely, γδ T cells, MAIT cells, and NKT cells express high levels of the C-type lectin molecule CD161. To understand the significance of CD161 expressing innate T cells in mucosal immunity, we investigated the phenotype and functions of these cells in mucosal tissues, lymph nodes and blood of rhesus macaques. The frequency of CD161+ T cells was higher in the lungs and colonic mucosa (9.4%±3.8 and 6.2%±1.9) in comparison to peripheral blood (2.9%±1) and mesenteric lymph nodes (0.5%±0.3). Regardless of the tissue compartment, 30 to 80% of the CD161+ T cells comprised of γδ T cells and TCR Vα 7.2+ MAIT cells in approximately equal proportions. However, only 1 to 6% consisted of CD1d tetramer+ NKT cells. Moreover, the mucosal tissue CD161+ T cells displayed a highly activated phenotype based on CD69 and HLA-DR expression in comparison to blood and lymph node cells. HMBPP stimulated γδ T cells from lungs and colonic mucosa produced significantly higher amounts of IFN-γ, Perforin and IL-17 than those in blood. Similarly, E.coli stimulated MAIT cells from mucosal tissues produced significantly higher amounts of IFN-γ and IL-17 than those in blood. The enhanced Th1 and Th17 type effector functions of mucosal innate T cells along with their cytotoxic potential suggests that they can mount early immune responses to mucosal pathogens like HIV and Mtb prior to the development of adaptive immune responses and thereby play a role in the initial control of pathogen burden/dissemination.
Loss of intestinal epithelial autophagy leads to catastrophic susceptibility to acute T. gondii-mediated inflammation
Authors: Burger Elise , Lopez-Yglesias Americo H , Yarovinsky Felix
Published: January 2023

The protozoan parasite Toxoplasma gondii triggers severe small intestinal immunopathology characterized by inflammation, Paneth cell loss and bacterial dysbiosis. Paneth cells are the predominant secretory epithelial cells which reside at the base of the crypt and release antimicrobial peptides that maintain intestinal homeostasis. T. gondii-triggered IFN-γ is a major inducer of Paneth cell death. However, mechanisms of IFN-γ-mediated Paneth cell death are unknown. We recently observed that under steady state conditions Paneth cells undergo active autophagy, which is both microbiota- and IFN-γ-dependent. To study autophagy’s role in T. gondii–induced Paneth cell loss we used mice deficient in the critical autophagy gene Atg5 in the intestinal epithelium (Villin-Cre × ATGfl/fl mice, E-ATG5−/−). Infected E-ATG5−/− mice exhibited catastrophic inflammation, characterized by the complete destruction of the intestinal crypts, establishing ATG5’s protective role during intestinal inflammation. To determine ATG5’s protective mechanism we generated ATG5-deficient intestinal organoids and observed that lack of ATG5 increased sensitivity to the TNF-induced cell death. Organoid death was further exacerbated when TNF stimulation occurred in combination with IFN-γ. Reexamining our in vivo model showed significantly elevated levels of TNF receptor 2 in infected E-ATG5−/− mice, indicating these mice are prone to TNF-triggered small intestinal immunopathology. Our results reveal that ATG5 expression within Paneth cells plays a highly protective role against cytokine-mediated immunopathology in the intestine during acute T. gondii infection.
The proteolytic activity of Porphyromonas gingivalis is a critical virulence factor in the murine model of periodontitis
Authors: Choi Youngnim , Baek Keum Jin , Choi Yun Sik
Published: January 2023

Porphyromonas gingivalis, a periodontal pathogen, has an inter-strain variability in virulence. We recently reported that the cell invasion ability, but not cytokine proteolytic activities, of P. gingivalis has strong positive correlations with clinical parameters of subjects who harbored the isolates. To determine the contribution of the invasion ability and proteolytic activity to the virulence of P. gingivalis in the murine model of periodontitis, three clinical isolates KUMC-P1 (P1: low cell invasion ability and low proteolytic activity), KUMC-P4 (P4: low cell invasion ability but high proteolytic activity), and KUMC-P8 (P8: high cell invasion ability but low proteolytic activity) were orally inoculated in Balb/c mice. The net alveolar bone loss induced by three strains was P4 > P8 > P1, where the difference between P8 and P1 was not significant. The amounts of P. gingivalis within the gingival tissues examined by in-situ hybridization using a P. gingivalis-specific probe were increased by all three strains, which had a significant positive correlation with alveolar bone loss. Whereas P1 and P8 induced a significant IgA antibody response, P4 induced a slight but not significant IgG1 antibody response against P. gingivalis. In vitro, P4 had the greater effect than other strains on the epithelial barrier disruption evidenced from the reduction in the levels of tight junction proteins and transepithelial resistance. In conclusion, the proteolytic activity of P. gingivalis was a critical virulence factor in the murine model of periodontitis, which may be attributed to the paracellular pathway-dependent bacterial invasion of the gingival tissue in mice.
Fast and efficient enrichment of functional ILC2 from human whole blood
Authors: Valdez Yanet , Kyei Stephen K. , Poon Grace F.T. , Takei Fumio , Peters Carrie , Woodside Steve M. , Eaves Allen C. , Thomas Terry E.
Published: January 2023

Group 2 innate lymphoid cells (ILC2) are a functionally distinct subset of recently identified immune cells with important roles in type-2 immunopathologies such as allergies, asthma, helminth infections and other metabolic diseases. Studying these rare cells is challenging due to a lack of specific surface markers, and currently multicolor flow cytometric analysis and cell sorting are the only methods to characterize and isolate ILC2s. However, the scarcity of these cells makes flow cytometry time-consuming, expensive and often results in low purities and recoveries. Thus, better approaches for effective identification and isolation are essential to further understanding of ILC2 biology and function. We have developed a rapid and efficient method for enrichment of human ILC2 from whole blood. In brief, non-ILC2 cells in whole blood were crosslinked to red blood cells already present in the sample using RosetteSep™. The sample was then layered over Lymphoprep in a SepMate™ tube, spun at 1200 x g for 10 minutes (min), and the untouched, desired cells simply poured off. Cells were washed once and were then ready for subsequent analysis. Starting with only 0.01 – 0.07% in whole blood, ILC2 were enriched 350 ± 220 fold to 8.2 ± 6.8% in 35 min (means ± SD, n=17). Subsequent cell sorting from these pre-enriched samples was faster and yielded higher purity ILC2 than sorting from non-enriched controls (n=3, p<0.05 paired t test). Sorted ILC2, both pre-enriched and non-enriched controls, were cultured and stimulated, and secreted similar high levels of IL-13 as assessed by ELISA, indicating that these cells are functional. In summary, ILC2 pre-enrichment improves sorting efficiency, increases ILC2 purity, and maintains ILC2 functionality.
Immunomagnetic enrichment of regulatory T cells directly from whole blood without the need for density gradient centrifugation.
Authors: Richter Anne , Steinbrück Philipp , Schenk Tobias , König Sindy , Siemer Ramona , Bacher Petra , Hohnstein Thordis , Scheffold Alexander , Dose Christian
Published: January 2023

Regulatory T cells (Tregs) play a pivotal role in maintaining peripheral tolerance of unspecific or excessive immune responses. Therefore, the enrichment of these cells and their cultivation is of great interest for basic and translational research.

Accordingly, there is a considerable need for continually improved and reliable cell isolation protocols. We introduce here a rapid, easy, and convenient strategy for the isolation of regulatory T cells directly from anticoagulated whole blood without the need for density gradient centrifugation.

The new immunomagnetic separation protocol provides an easy and fast solution for the highly specific isolation of CD4+ CD25highCD127− regulatory T cells directly from whole blood within 30 minutes ready for downstream application such as flow cytometric phenotyping, gene expression studies, cytokine analysis, suppression assays, or cell expansion.

The procedure, leads to populations of CD4+ CD25highCD127− cells with high yields (2×104/mL whole blood), purities (>90%) and viability (>98%) as proven by flow cytometric analysis. In addition, enriched Tregs were capable for in vitro expansion for several weeks while remaining stable FoxP3 expression.
Generation of functional mini thymus-like units with self-assembling EAK16-II/EAKIIH6 hydrogel
Authors: Tajima Asako , Liu Wen , Pradhan Isha , Bertera Suzanne , Lakomy Robert , Rudert William A , Trucco Massimo , Meng Wilson S , Fan Yong
Published: January 2023

A functional thymus is crucial to produce mature, self-tolerant T cells. Crosstalk between the residing thymic stromal cells, especially the predominant population of thymic epithelial cells (TECs), and the developing thymocytes is essential for thymopoiesis. The survival and proliferation of TECs depend on a unique 3 dimensional (3-D) configuration of the thymus microenvironment. Disorganization of TECs results in a loss of gene expressions essential for TEC growth and survival, as well as inability to produce functional T cells. Recapitulating the property of thymus has been proven to be challenging.

In our study, we have incorporated EAK16-II, a low molecular weight peptide that self-assembles into beta-sheet fibrils, and its histidinylated analogue, to induce 3-D aggregation of TECs. When the adaptor complex comprised of anti-EpCAM IgG, anti-His and recombinant protein A/G molecules were mixed with TECs in the hydrogel, TECs were captured into small clusters. TECs cultured in this condition maintained their molecular properties and were viable up to 2 weeks. Furthermore, when the TEC clusters in the hydrogel were transplanted into athymic nude mice, T cell development was observed, and these newly generated T cells proliferated upon stimulation with allogenic cells. These results demonstrate that self-assembling EAK16-II/EAKIIH6 system with addition of tri-component adaptor complex may be a useful tool to organize TECs in a 3-D-like structure to support T cell development, suggesting a possibility to generate injectable mini thymus-like units to restore adaptive immune system.
A SMARTer approach to profiling the human T-cell receptor repertoire
Authors: Taylor Sarah , Yasuyama Nao , Farmer Andrew
Published: January 2023

Profiling T-cell receptor (TCR) repertoires involves characterizing the diversity of TCR nucleotide sequences in a sample, and is an increasingly popular approach for analyzing the composition of the adaptive immune system. While low-throughput approaches have yielded important insights concerning TCR repertoire dynamics, development of next-generation sequencing (NGS) technologies has dramatically expanded research prospects.

Using SMART® technology, we have developed an NGS library preparation kit for TCR profiling that employs a 5′ RACE-like approach to capture full-length variable regions of TCR-α and/or TCR-β subunits. With this approach, 10 ng–3 μg of input RNA or 50–10,000 purified T cells undergo reverse transcription using dT priming. Following first-strand cDNA synthesis, semi-nested PCR is used to amplify TCR-specific sequences and add Illumina® sequencing adapters.

Starting with peripheral blood RNA, libraries containing TCR-α and TCR-β sequences were generated and sequenced. For each replicate >70% of reads were on-target, and the most highly represented clonotypes remained consistent across a range of input amounts. A separate sensitivity assay demonstrated that RNA corresponding to a single clonotype could be detected above background levels when spiked into input RNA at a relative concentration of 0.1 %.

Our approach enables the user to obtain sequencing-ready TCR libraries from RNA in ~2.5 hours of hands-on time. Whereas approaches that utilize genomic DNA as starting material require multiplexed PCR strategies, RNA based amplification of TCR sequences use single primer pairs for each subunit. In this way, our approach minimizes the likelihood of sample misrepresentation due to amplification biases.
Camelid nanobodies specific to human pancreatic glycoprotein 2
Authors: Hanack Katja , Schlör Anja , Holzlöhner Pamela , Listek Martin , Bauer Cindy , Butze Monique , Micheel Burkhard , Hentschel Christian , Sowa Mandy , Roggenbuck Dirk , Schierack Peter , Füner Jonas , Schliebs Erik , Goihl Alexander , Reinhold Dirk
Published: January 2023

Pancreatic secretory zymogen-granule membrane glycoprotein 2 (GP2) has been identified to be a major autoantigenic target in Crohn’s disease patients. It was discussed recently that a long and a short isoform of GP2 exists whereas the short isoform is often detected by GP2-specific autoantibodies. In the outcome of inflammatory bowel diseases, these GP2-specific autoantibodies are discussed as new serological markers for diagnosis and therapeutic monitoring. To investigate this further, camelid nanobodies were generated by phage display and selected against the short isoform of GP2 in order to isolate specific tools for the discrimination of both isoforms. Nanobodies are single domain antibodies derived from camelid heavy chain only antibodies and characterized by a high stability and solubility. The selected candidates were expressed, purified and validated regarding their binding properties in different enzyme-linked immunosorbent assays formats, immunofluorescence, immunohistochemistry and surface plasmon resonance spectroscopy. Four different nanobodies could be selected whereof three recognize the short isoform of GP2 very specifically and one nanobody showed a high binding capacity for both isoforms. The KD values measured for all nanobodies were between 1.3 nM and 2.3 pM indicating highly specific binders suitable for the application as diagnostic tool in inflammatory bowel disease.
Expression of Interferon-inducible Genes is Influenced by Sex Hormones in SLE
Authors: Singh Ram Pyare , Hahn Bevra , Hossain Awlad
Published: January 2023

SLE is a female-biased disease with female to male ratio of 9:1. Accumulating evidence suggests the existence of a nexus between inflammatory pathways and 17b-estradiol, resulting in increased interferon stimulated genes (ISGs), autoantibodies and dysregulation of immune cells in SLE. However, the mechanisms by which 17b-estradiol interacts with IFN and T cells, B cells and antigen presenting cells (APC) in SLE is poorly understood. Women may be more susceptible than men to SLE and other autoimmune diseases in part because many healthy women have higher interferon regulated genes. Similarly, female BWF1 lupus mice have increased expression of interferon genes compared to age and sex- matched males. We found that plasma estradiol levels positively correlated with levels of serum/plasma IL-6, and 21/IL-23 in SLE patients. Plasma 17b-estradiol levels are significantly increased in female SLE patients compared to healthy females (p<0.01). Furthermore, we found that estradiol increases proinflammatory cytokines, and level of estradiol positively correlated with expression of miR21, 25, and 186 in SLE patients. Expression of miR21, miR25 and miR186 are positively correlated with SLEDAI score in SLE. Understanding the role of 17b-estradiol in the regulation of pro-inflammatory pathways, ISGs, and microRNAs will further advance our understanding of SLE disease pathology and may identify additional novel therapeutic targets.
Modification of soluble common gamma chain function controls collagen-induced arthritis
Authors: Ko Eunhee , Goh Taesik , Lee Byunghyuk , Hong Changwan
Published: January 2023

IL-2Rγ (γc) is an essential subunit in γc cytokine signaling which is very important in development and homeostasis of immune cells. Recently, we showed that the soluble form of γc (sγc) is generated by alternative splicing and their production is enhanced in activated T cells. Moreover, we found that sγc inhibits γc cytokine signaling and enhances Th17 differentiation, consequently exacerbates an inflammation. Since Th17 cells are known to play pivotal roles in the pathogenesis of autoimmune rheumatoid arthritis (RA) disease, we have questioned whether sγc affects RA pathogenesis. Consistent with EAE model, we found that sγc overexpressing mice with collagen induced arthritis (CIA) displayed accelerated clinical score of arthritis compared to WT with CIA. In addition, serum level of sγc was significantly increased in CIA mice compared to WT, indicating that sγc may contribute to exacerbate the pathogenesis of RA. It is conceivable that a blockade of sγc function potentially improves autoimmune diseases. Although sγc dimerization is more functional than monomer, the dimerization mechanism of sγc is not fully understood. Using a mutant sγc form which cysteine (Cys) is replaced to alanine (Ala), we confirmed that the generation of sγc homodimer is elicited by disulfide bond. This result suggests that Cys is critical to form the dimer structure. To further study, we are designing sγc inhibitor to block sγc functions and then testing inhibitory effect in RA animal model.
Development of citrullinated-vimentin-specific CAR for targeting Tregs to treat autoimmune rheumatoid arthritis.
Authors: Raffin Caroline , Zhou Yu , Piccoli Luca , Lanzavecchia Antonio , Sadelain Michel , Bluestone Jeffrey A
Published: January 2023

Rheumatoid arthritis (RA) is one of the most common chronic autoimmune diseases with a frequency of 0.5–1.0% in the adult population of developed countries. RA is characterized by an aberrant inflammation of the synovial membrane that causes irreversible joint and bone damages. It has been previously described that regulatory T cells (Tregs) were defective in RA patients and that whereas Treg depletion increased the severity of the disease, Treg transfer efficiently reversed this effect in the collagen-induced arthritis (CIA) model, an animal model of RA. These studies strongly suggest that Treg therapy may be able to treat RA patients by inducing a long-term restoration of appropriate tolerance. Moreover, adding Ag-specificity to the expanded Tregs may promote their migration to the site of the abnormal inflammation and enhance their function. The aim of our study has been to engineer and expand chimeric antigen receptor (CAR)-expressing Tregs specifically targeting an antigen present in the joint of RA patients to induce a localized and effective immunosuppressive response. We developed a CAR directed against citrullinated vimentin (CV), a posttranslational modified intermediate filament protein known to be exclusively located in the extracellular matrix of the synovial tissue in 50% of the RA patients. We are currently working on transducing the CV-specific CAR into Tregs to then study the functional activity of these cells in vitro. We are also developing an in vivo protocol to assess the therapeutic efficacy of the CAR Treg transfer in CIA model. In conclusion, our project aiming to combine Treg therapy and CAR technology may lead to the development on a promising therapeutic tool to cure RA and potentially other autoimmune disorders.
Adenosine 2A Receptor Promotes Tolerance by Reducing Germinal Center Follicular Helper T cells and Pathogenic Class-Switched B cells
Authors: Schmiel Shirdi E , Yang Jessica , Kalekar Lokeshchandra , Jenkins Marc K , Mueller Daniel L
Published: January 2023

Enhanced production of high affinity class-switched autoantibodies contributes to pathological symptoms of autoimmune diseases. Full penetrance of autoantibody production appears to rely on help from germinal center follicular helper T cells (GC TFH cells). GC TFH cells promote the survival, differentiation, isotype class switch, and affinity maturation of B cells. Previous studies have reported that activation of Adenosine receptor 2a (A2aR) downstream signaling negatively regulates adaptive immune responses by limiting the function of effector T cells. Recent work from our lab has demonstrated that the A2aR-specific agonist blocks autoimmune arthritis that is mediated by GPI-specific KRN CD4 T cells. A2aR agonist caused a reduction of GC TFH cells that respond to autoantigen. Consistent with this, GPI-specific humoral immune responses were also reduced in A2aR agonist treated mice. To establish if the effect of A2aR agonist treatment on GC TFH differentiation was T cell intrinsic, CD4-Cre A2aR-floxed B6 mice were immunized with a protein complex containing a CD4 T cell antigen (2W1S peptide) and B cell antigen (PE). Polyclonal 2W1S/I-Ab-specific CD4 T cells and PE-specific B cells were then monitored in the presence or absence of T cell-specific A2a receptors. Our data show that in the absence of T cell-specific A2a receptors, agonist treatment fails to influence GC TFH cell differentiation. Therefore, A2aR downstream signaling can be stimulated within autoreactive CD4 T cells during their encounter with self antigen, and can serve to limit the differentiation of dangerous GC TFH cell effectors. Ongoing experiments are working to identify the T cell-intrinsic mechanism by which A2aR signaling inhibits GC TFH cell generation.
Targeting the phosphatidylinositol 3-kinase signaling pathway to inhibit pathogenic B cells in systemic lupus erythematosus (SLE)
Authors: Yin Catherine C , Trigunaite Abhishek , Dinh Dominic , Yiu Ying , Lannutti Brian J , Stein Paul L
Published: January 2023

Systemic Lupus Erythematosus (SLE) is an autoimmune disease with high levels of autoreactive B cells and circulating anti-nuclear autoantibodies. Circulating antibody-antigen immune complexes deposit in tissues, including the kidney, causing chronic inflammation and irreparable tissue damage. The hematopoietic system expresses a unique isoform of the phosphatidylinositol 3 kinase (PI3K) catalytic subunit, termed delta (PI3Kδ). It is critical for B cell survival and function, and its inhibition results in impaired antibody production. We hypothesized that targeting PI3Kδ will decrease levels of circulating auto-antibodies in a mouse model of SLE and reduce disease pathology. This study shows that a clinically utilized small molecule inhibitor of PI3Kδ (GS-9820) significantly inhibits naïve B cell differentiation into antibody secreting plasma cells as well as B cell proliferation in vitro. Furthermore, in the (NZBxNZW)F1 mouse model of SLE, treatment with the PI3Kδ inhibitor reduced autoantibody levels to background and increased overall survival. Within 2 weeks of treatment animals showed a marked reduction of germinal center B cells and T follicular helper cells (Tfh). The decrease in autoantibody levels ultimately prevented kidney damage by reducing immune complex deposition and complement fixation. Current treatments for lupus predominantly utilize systemic immunosuppressive drugs which have severe side effects. Since PI3Kδ plays a predominant role in B cells, our data suggests a clinical PI3Kδ inhibitor can be a therapeutic for lupus, potentially reducing autoantibody levels and associated pathologies. This therapy can result in better disease management and quality of life for lupus patients.
The role of IL-10 in B-cell chronic lymphocytic leukemia cell survival
Authors: Alhakeem Sara Samir , McKenna Mary Kathryn , Gachuki Beth W , Rangnekar Vivek R , Byrd John C , Muthusamy Natarajan , Bondada Subbarao
Published: January 2023

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the progressive accumulation of abnormal lymphocytes in the periphery. Typically the disease progression is slow, but as the number of leukemic cells increases, they interfere with the production of other important blood cells, causing the patients to be in an immunosuppressive state. To study B-CLL we use cells from the transgenic Eμ-Tcl1 mouse, which spontaneously develop B-CLL. Previously we showed that Eμ-Tcl1 CLL cells produce an anti-inflammatory cytokine, IL-10, constitutively. Here we studied the role of IL-10 in CLL cell survival in vitro and the development of CLL in vivo. We found that neutralization of IL-I0 using anti-IL-10 antibodies or blocking the IL-10 receptor (IL-10R) using anti-IL-10R antibodies did not effect the survival of CLL cells in vitro. When we adoptively transferred Eμ-Tcl1 cells into IL-10R KO mice, we found that cells grew at a slower rate in IL-10R KO mice vs. wild type mice. We also found that there was a significant reduction in CLL cell engraftment in the spleen, bone marrow, peritoneal cavity and liver of the IL-10R KO mice. Further studies revealed that IL-10 could be playing a role in the tumor microenvironment possibly by affecting anti-tumor immunity. This was seen by a reduction in the activation of CD8+ T cells as well as a significantly lower production of IFN-γ by CD4+ T cells purified from CLL-injected WT mice in compare to the IL-10R KO injected mice. In current in vivo studies, CLL growth will be monitored in WT mice after IL-10 neutralization using anti-IL-10 monoclonal antibody to further elucidate the role of IL-10 in CLL development. These studies demonstrate that CLL cells suppress host anti-tumor immunity via IL-10 production.
Caspase-1 from MDSC promote MyD88 dependent carcinogenesis that is T-cell independent
Authors: Zeng Qi , Qualliotine Jesse R , Blosser Lee , pardoll Drew , Kim Young
Published: January 2023

Myeloid-derived-suppression cells (MDSC) are functionally defined to suppress T-cells in their primary means of immune evasion mechanism in cancer. However, we have discovered that MDSC can have direct, T-cell independent pro-carcinogenic effect on tumor cells. Sorted monocytic CD14+/CD11b+/HLA-DRlowMDSCs from head and neck squamous cell carcinoma (HNSCC) patients were found to increase the proliferation index of HNSCC cells. This induction of tumor proliferation was not dependent on MDSC-tumor cell contact. Exome analysis showed that these human MDSC from cancer patients expressed high levels of inflammasome complex, and supernatant from the MDSCs were found to secrete IL-1b and IL-18. When we probed the functional significance of these inflammasome complex, we found that MDSC’s promotion of tumor proliferative index of the tumor was found to be caspase-1 dependent. To test this in vivo, T-cell depleted caspase-1 null mice showed significant decrease in tumor growth rate. To confirm the importance of myeloid inflammasome signaling in carcinogenesis, we suppressed MyD88 gene in tumor cell line Cal27 and found that the ability of MDSC to promoting tumor proliferation is diminished. Taken together, our findings demonstrate that tumor infiltrating MDSCs may play a prominent role in chronic inflammation associated carcinogenesis.
Positive Feedback Loop between B cells/plasma cells and Macrophages Promote M2 Macrophage-Elicited Hepatoma Progression
Authors: Kuang Dong-Ming
Published: January 2023

Tumor progression has been recognized as the product of an evolving crosstalk between different cell types within tumor and its stroma. However, the crosstalk between humoral immunity and innate immunity within human cancer microenvironments is poorly defined. We show that infiltration of plasma cells orchestrates the differentiation and function of protumorigenic macrophages in human hepatocellular carcinoma (HCC): specialized polarized immunoglobulin G (IgG)-secreting plasma cells in HCCs trigger the differentiation of protumorigenic M2b polarized macrophages. Reciprocally, autocrine interleukin 6-elicited-interferon-inducible protein 10 (IP-10) and membrane-bound intercellular cell adhesion molecule-1/vascular cell adhesion molecule (ICAM-1/VCAM) expressed by macrophages selectively induces long-lived IgG-secreting plasma cells, forming a positive feedback loop, in coculture systems and mice. Plasma cell infiltration determines macrophage-associated clinical outcome of HCC patients. These findings suggest that bidirectional communications between macrophages and B cells in cancer microenvironments support humoral immunity-elicited myeloid cell suppression and disease progression.
Time-course analysis of SHIP-1 modulation and expression in a pancreatic cancer model
Authors: McGill Andrew Robert , Ghansah Tomar , Ortiz-Rivera Ivannie , Iclozan Cristina
Published: January 2023

Pancreatic cancer (PC) is an aggressive disease with minimally effective therapies due to immunosuppression. Src Homology Inositol Phosphatase (SHIP-1) expression is essential in regulating myeloid derived suppressor cell (MDSC) and T regulatory cell (Treg) homeostasis and function, which can positively or negatively impact antitumor immunity. Inflammatory factors play an important role in the regulation of SHIP-1 expression. We demonstrated in our pancreatic cancer tumor bearing mice (TB) that SHIP-1 gene and protein expression is diminished in lymphoid tissues, correlating with an expansion of MDSC and Treg percentages compared to control mice (CTRL). These TB mice are generated via subcutaneous injection with Panc02 cells. Furthermore, we showed that inflammatory factors were elevated in TB mice compared to CTRL mice. In this study we sought to pinpoint the exact time when SHIP-1 expression was reduced, in correlation with the expansion of MDSC and Treg, along with the increase in production of inflammatory factors in TB mice. To evaluate the loss of SHIP-1, weekly time-course experiments were performed via collection of peripheral blood (PB) and lymphoid tissues for a total of 4 weeks. Preliminary data indicates a reduction of SHIP-1 expression two weeks post tumor inoculation, which correlated with the expansion of MDSC and Treg percentages and an increase in tumor and spleen weights in TB mice compared to CTRL mice. Also, PB from TB mice showed a weekly increase in the production of inflammatory factors compared to CTRL mice. The results from this study may further establish SHIP-1 as a potential therapeutic target that may help combat PC in humans.
CXCL9, CXCL10 and IFNγ favor the accumulation of infused T cells in tumors following IL-12 plus doxorubicin treatment
Authors: Hu Jiemiao , Bernatchez Chantale , Xia Xueqing , Xu Zihang , Hwu Patrick , Li Shulin
Published: January 2023

The infiltration of cytotoxic CD8+T cells suggests a positive prognosis in a number of established tumors. T cell therapy is rapidly growing in the past decade, and achieved big success in treating hematological tumors. Despite autologous TIL infusion made remarkable breakthroughs in treating refractory melanoma, the highest respond rates are only around 50%. Therefore, approaches which guide TILs to reach tumor sites more efficiently are to be developed. We employed the approach of IL-12 plus doxorubicin prior to T cell infusion in a murine lung cancer model and a human xenograft model. IL-12 plus doxorubicin were administered when tumors reached 5–7 mm in diameter. One day after, immune competent T cells were transferred intravenously into tumor bearing NSG mice. We also used the same strategy to infuse autologous TILs to human melanoma xenograft tumors. Not only did our approach increase the numbers of infiltrated CD8+T cells in tumors, and more significantly, it raised the ratios of immune stimulatory and immune inhibitory markers. As a result, large number of tumor cells undergoes apoptosis, along with delayed tumor development and extended survival time. The mechanism study revealed that T cell-attracting chemokines CXCL9 and CXCL10 induced by doxorubicin accounted for the recruitment of T cells to tumors, and the tumor-specific upregulation of IFNγ following IL-12 signaling initiate the positive feedback to stimulate T cell proliferation. Here, we showed the approach of IL-12 plus doxorubicin followed by murine T cell and human TIL infusion yielded striking response in murine and human xenograft solid tumors. In light of our findings, our approach might broaden the application of T cell therapy in a wider variety of solid tumors.
In vivo antigen-specific activation of CD8 T cells following radiation therapy can be monitored using Nur77-GFP transgenic mice
Authors: Uhde Lauren K , Moran Amy E , Crittenden Marka R , Gough Michael J
Published: January 2023

Current studies monitoring T cell infiltration within tumors using surface markers (i.e. CD69, CD25) are limited by their inability to distinguish activation through the T cell receptor (TCR) or through other inflammatory pathways. Here we use a reporter mouse expressing green fluorescent protein (GFP) with the immediate early gene Nr4a1 (Nur77), in which the level of GFP expression corresponds only to the strength of TCR stimulation. Research indicates that radiation therapy (RT) modifies tumors for improved lymphocyte infiltration and T cell killing. We investigated whether the proportion of antigen-specific infiltrating CD8 T cells increased following irradiation, hypothesizing that RT increases this number in irradiated tumors.

We bred C57BL/6J-Tg(Nur77-GFP) mice with FVB/N-Tg(MMTV-PyMT) spontaneous mammary tumor mice to yield GFP-expressing F1 mice with and without the MMTV-PyMT oncogene. Age-matched tumor-bearing mice were left untreated or treated with 10 Gy CT-guided RT to the mammary tumors. Alternatively, untreated tumors were harvested, transferred into Nur77-GFP+ MMTV-PyMT- littermates, and then treated. Tumors were harvested one week later.

Antigen-specific (Nur77-GFP+) CD8 T cells could be detected in both spontaneous and transplanted tumors, indicating an endogenous adaptive immune response to tumor-associated antigens. Our hypothesis proved false as no difference was found in the proportion of GFP+ T cells following RT in either the parental (p=0.727) or the transferred (p=0.803) tumors compared to untreated controls. These data suggest that improvements in T cell mediated tumor killing following RT are due to changes in quality, not quantity, of antigen-specific T cells.
sCYLD expression leads to CLL in mice and men
Authors: Hövelmeyer Nadine , Hahn Matthias , Reissig Sonja , Müller Claude , Wunderlich Thomas , Waisman Ari
Published: January 2023

A tightly regulated NF-κB pathway is very important for B cell homeostasis. Aberrant activation of NF-κB is involved in the development of chronic lymphocytic leukemia (CLL). The deubiquitinating enzyme CYLD, acts as a negative regulator of NF-κB by removing K63-linked ubiquitin chains from its target proteins. Here, we show that the B cell specific deletion of full-length CYLD and expression of the shorter splice form (sCYLD) lacking the binding sites for TRAF2 and NEMO, leads to the dramatic expansion of CD5 positive B cells, a hallmark of CLL. These cells accumulate in the blood and lymphatic organs with age, and show characteristic up-regulation of typical CLL-markers such as ZAP-70 and Bcl-2. A20, another negative regulator of NF-κB targets some of the same proteins as CYLD, thereby potentially compensating for the lack of FL-CYLD. We crossed sCYLD mice to a B cell specific A20 knockout. In these mice, the CLL phenotype progresses more rapidly probably due to an increase of NF-κB activity, up to the point where 100% of the B cells are CD5+. These cells show oligoclonal expansion, infiltrate the liver and the lungs, and accumulate in all lymphatic organs. Interestingly, we could show that some human CLL patients also express sCYLD, whereas none of the healthy controls do. These findings indicate that sCYLD is a new risk factor for CLL and implies that disrupted alternative splicing of key NF-κB regulators lead to the development of CLL.
Regulation of peripheral node addressin in tumor-associated CD31+ vascular endothelial cells.
Authors: Rodriguez Anthony B , Peske J David , Woods Amber N. , Engelhard Victor H
Published: January 2023

Naïve T cells use peripheral node addressin (PNAd), normally found on lymph node (LN) high endothelial venules (HEV), to traffic into lymphoid organs. We previously reported that the vasculature of murine tumors expressed PNAd and CCL21, which supports infiltration and intratumoral activation of naïve T cells, in turn delaying tumor growth. PNAd expression on tumor vasculature depends on effector CD8+ T cells that secrete LTα3, which signals through TNFR on tumor-associated CD31+ endothelial cells. PNAd expression is low on tumor vasculature, suggesting that it might be regulated differently than PNAd on HEV. Using RT-qPCR, we found that most of the enzymes responsible for PNAd synthesis are expressed comparably in CD31+ cells from tumors and LN. However, the primary sulfotransferase responsible for the generation of the 6-sulfo sialyl Lewis X structure on PNAd HEV, GlcNAc6ST-2, is minimally expressed in tumors. Tumor CD31+ cells expressed equivalent levels of GlcNAc6ST-1, suggesting that it is responsible for low-level PNAd expression. Of the scaffolding proteins that make up PNAd, GlyCAM-1 was minimally expressed in tumor CD31+ cells, whereas all others were expressed comparably. To identify the components of PNAd biosynthesis in tumor CD31+ cells that are controlled by LTα3-TNFR signaling, we evaluated tumors from WT and TNFR1/2KO mice. GlyCAM-1 was the only assessed PNAd component that was ablated in TNFR1/2KO tumors. However, based on transcript levels, GlyCAM-1 accounts for only about 10% of the PNAd scaffolding proteins in tumor CD31+ cells. This suggests that TNFR signaling regulates additional PNAd biosynthetic components yet to be examined, or that it regulates one or more components at the level of protein translation.
Spleen actively recruits, maintains and expands hematopoietic stem and progenitor cells in tumor-bearing hosts
Authors: Zheng Limin , Wu Chong , Ning Huiheng
Published: January 2023

We recently found that circulating hematopoietic stem and progenitor cells (HSPC) from various cancer patients exhibit a generalized myeloid bias with a skew toward granulocytic differentiation. These circulating myeloid precursors are highly enriched in tumors and serve as an important link between dysregulated hematopoiesis and accumulated myeloid-derived suppressor cells (MDSC) in cancer patients. Here, we showed that, in various murine tumor models, spleen is significantly superior to bone-marrow in inducing expansion of both hematopoietic stem cells and discrete intermediary progenitors, with a skew toward myelopoiesis. The bone-marrow derived HSPC are actively recruited and maintained in the spleen of tumor-bearing mice, a process that is regulated by CCL2/CCR2 axis, but not CXCL12. Splenic HSPC from tumor-bearing mice exhibits significantly higher proliferative activity and gives rise to CD11b+Gr-1intLy6G− Ly6Chi multipotent monocytic cells, the committed progenitors and immediate precursors of MDSC. Accordingly, abrogation of splenic hematopoiesis synergistically enhanced the therapeutic efficacy of anti-PD-L1 treatment. Moreover, we found that hematopoietic precursors and myeloid cells are accumulated in the spleen of patients with various solid tumors. These data suggest that the spleen is an important extramedullary reservoir and source of myeloid progenitors. Identifying unique splenic signals that instruct myeloid cells and their precursors might provide a novel strategy for anticancer therapy.
Infiltrate of effector regulatory T cells predicts outcome in a cohort of New Zealand colorectal cancer patients – a pilot study
Authors: Kemp Roslyn , Hartstonge Kirsten A Ward , McCulloch Tim , Kamps Ann-Kristin , Cretney Erika , McCall John
Published: January 2023

Staging of patients with colorectal cancer is currently based on tumour morphology and does not take into account the complexity of the anti-tumour immune response. Early-stage colorectal patients are usually treated with surgery alone and are not recommended additional adjuvant therapy. Up to twenty-five percent of these patients have relapse of disease, indicating that the current staging does not accurately predict disease-free survival. The Immunoscore has been proposed to incorporate the T cell infiltrate in the tumour into current staging protocols to improve estimates of disease-free survival. However, T cell subsets are complex and therefore may have different effects on patient outcome. Immunofluorescence was used to analyse immune cell infiltrates in early stage (II) colorectal cancer patients and to compare those with recurrent and non-recurrent disease (n=33). CD3 and CD8 were used for the Immunoscore. FoxP3, Blimp-1 and CD3 were used to identify effector regulatory T cells. Patients with a high Immunoscore (high T cell infiltrate) had increased disease-free survival than patients with a low Immunoscore (low T cell infiltrate, Log-rank test p>0.001). The ability to predict patient outcome was improved by measuring the infiltrate of CD4+FoxP3+Blimp-1+ cells (effector regulatory T cells, Log-rank test p>0.001). Patients with a low Immunoscore but high infiltrate of CD4+FoxP3+Blimp-1+ cells at the centre of the tumour had increased disease-free survival than those with a low Immunoscore and a low infiltrate of CD4+FoxP3+Blimp-1+ cells. These results show that incorporating the complexity of the local immune response into current practice may improve prediction of patient outcome in colorectal cancer.
Functional reprograming of chemokine microenvironment in colorectal cancer to promote intratumoral accumulation of immune effector cells
Authors: Kalinski Pawel , Zureikat Amer H. , Muthuswamy Ravikumar , Obermajer Natasa , Urban Julie , Butterfield Lisa H. , Gooding William , Zeh Herbert J. , Bartlett David L.
Published: January 2023

Infiltration of tumor lesions with effector CD8+ T cells (CTLs) has been shown to predict improved clinical outcomes in multiple cancer types and predict patients’ responsiveness to checkpoint blockers. In contrast, local prevalence of regulatory T(reg) cells has been shown to be a negative prognostic factor.

To correct the balance between intratumoral CTLs and Tregs in patients with advanced cancer, we developed a combinatorial approach to modify the balance between the CTL- and Treg-attracting chemokines in tumor tissues. Our preclinical studies in ex vivo tissue explant culture- and mouse tumor models, showed that TLR3 ligands combined with IFNα and PGE2 antagonists can reprogram tumor microenvironments to selectively promote the accumulation of CTLs and suppress Treg attraction.

Based on these preclinical data, we developed a phase I/II clinical trial (UPCI 10-131/NCT01545141), to determine the safety and local effectiveness of the intravenous infusion of Rintatolimod (selective TLR3 ligand) combined with Intron A and oral celecoxib, in patients with resectable recurrent colorectal cancer. In the completed phase I of this trial, we observed very good safety profile of this combination and, in accordance with our expectations, selective reduction in the markers of CTLs, TH1- and NK cells in circulation, lasting 24–48 hours after Rintatolimod and Intron A infusion. Comparison of the resected tumors demonstrated an enhanced ratio of CXCL10 (CTL-attractant) to CCL22 (Treg-attractant) and an increased ratio of CD8α (CTL marker) to FoxP3 (Treg marker), in the chemokine modulation cohort, compared to patients receiving standard care at our center (non-randomized control). The randomized phase II portion of the trial is ongoing.
The epigenetic cross-talk between tumor cells and immune microenvironment in colorectal cancer
Authors: Kryczek Ilona , Nagarsheth Nisha B , Peng Dongjun , Lin Yanwei , Wei Shuang , Zhao Ende , Huang Emina , Zhao Lili , Vatan Linda , Szeliga Wojciech , Zou Weiping
Published: January 2023

Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation and influence the local anti-tumor immune response. In this study, we examined the relationship between the H3K27me3 and effector T-cell trafficking. We showed that H3K27me3 repress the tumor production of T helper 1 (TH1)-type chemokines CXCL9 and CXCL10, and subsequently determine effector T-cell trafficking to the tumor microenvironment. On the other hand, local trafficking in response to CCR6/CCL20 ligation is associated with increased Th22 phenotype. Th22 derived IL-22 activates DOT1L complex which turned on stem cell gene expression, increased cancer stemness, and promoted tumorigenic potential. The knowledge about the alterations in the epigenetic landscape of colorectal cancer may lead to understand colon cancer immunology and design effective treatments.
Immunosuppression limits CD8+ T cell entry into subcutaneous B16 melanoma
Authors: Wilson Ashley Lauren , Woods Amber N. , Engelhard Victor H.
Published: January 2023

Interactions between homing receptors (HR) and ligands on the vasculature facilitate effector CD8 T cell entry into tissues. HR ligand expression on normal inflamed tissue vasculature is induced by the local release of pro-inflammatory cytokines. In tumors, however, it is likely that expression of HR ligands on tumor vasculature is suboptimal due to immunosuppression. We hypothesized that disruption of immunosuppression would increase HR ligand expression on tumor vasculature, and consequently, enhance CD8 T cell entry. B16-F1 melanoma tumor-bearing mice were treated with blocking antibodies against CTLA-4 or PD-1. HR ligand expression on tumor vasculature and CD8 T cell representation were assessed. We found that CTLA-4 or PD-1 blockade significantly increased intravascular CXCL9 expression in subcutaneous B16-F1 tumors. Unlike PD-1 blockade, CTLA-4 blockade was associated with increased E-selectin expression on tumor vasculature, which we have shown to be important for CD8 T cell entry into subcutaneous tumors. CTLA-4 blockade also enhanced CD8 T cell representation and reduced tumor size. We observed a very low percentage of PD-1+ CD8 T cells in B16-F1 tumors, which may account for the limited efficacy of PD-1 blockade. Interestingly, the percentage of intratumoral PD-1+ CD8 T cells increased post-treatment with anti-CTLA-4. Our data suggest that CTLA-4 blockade may act to enhance T cell priming in the lymph node to increase the number of CD8 T cell effectors available to infiltrate tumors. It is also possible that CTLA-4 blockade may act on CTLA-4+ intratumoral Tregs, thereby promoting reactivation of tumor-resident CD8 T cell effectors that secrete pro-inflammatory cytokines that upregulate HR ligands on tumor vasculature.