bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytotoxic polyfunctionality maturation of cytomegalovirus-pp65-specific CD4+ and CD8+ T-cell responses in the elderly positively correlates with response size
Authors: Chiu Yen-Ling , Sung Bo-Yi , Schneck Jonathan P , Lin Chung-Hao , Chuang Yi-Fang , Kern Florian , Pawelec Graham P , Wang George C
Published: January 2023

Cytomegalovirus (CMV) infection is one of the most common persistent viral infections in humans worldwide and is epidemiologically associated with many adverse health consequences during aging. Previous studies yielded conflicting results regarding whether large, CMV-specific T-cell expansions maintain their function during human aging. In the current study, we examined the in vitro CMV-pp65-reactive T-cell response by comprehensively studying five effector functions (i.e., interleukin-2, tumor necrosis factor-α, interferon-γ, perforin, and CD107a expression) in 76 seropositive individuals aged 70 years or older. Two data-driven, polyfunctionality panels (IL-2-associated and cytotoxicity-associated) derived from effector function co-expression patterns were used to analyze the results. We found that, CMV-pp65-reactive CD8+ and CD4+ T cells contained similar polyfunctional subsets, and the level of polyfunctionality was related to the size of antigen-specific response. In both CD8+ and CD4+ cells, polyfunctional cells with high cytotoxic potential accounted for a larger proportion of the total response as the total response size increased. Notably, a higher serum CMV-IgG level was positively associated with a larger T-cell response size and a higher level of cytotoxic polyfunctionality. These findings indicate that CMV-pp65-specific CD4+ and CD8+ T cell undergo simultaneous cytotoxic polyfunctionality maturation during aging.
Open chromatin landscapes of human CD8 T cell subsets reveal transcription factors associated with differentiation and aging
Authors: Zhang David
Published: January 2023

The epigenomic changes in differentiation and aging in T cells have broad impact on the efficacy of the adaptive immune system. Using the assay for transposase-accessible chromatin (ATAC-seq), we generated genome-wide chromatin accessibility maps in CD8 T cell subsets from age-segregated human donors. We find that distinct transcription factor families maintain the naïve and memory T cell states, including ETS and bZIP. In aging, we observe that differentiation-associated factors become more prominent. Additionally, a general shift in chromatin openness from promoters to enhancers is evident, with promoter-binding factors like NRF1 implicated as the drivers. Our results describe immune aging in T cells as the erosion of accessibility at promoters and predisposition toward a differentiated state.
FcγRIIIa (CD16a) drives the differentiation of peripheral human naive CD4+ T-cells
Authors: Chauhan Anil K , Moore Terry L , Chen Chen , Bi Ye
Published: January 2023

Activated human naïve CD4+ T-cells express FcγRIIIa receptors, which may serve as a checkpoint for immune homeostasis. A positive co-stimulation from FcγRIIIa (CD16a) will override the negative co-stimulation to drive immune expansion in autoimmunity. In an autoimmune background, FcγRIIIa via pSyk signal generated IFN-γhigh, TH17 and TFH populations. FcγRIIIa-pSyk signal up-regulated more than 80-fold toll-like receptor (TLR)3 and TLR8 proteins and enzymes that drive epigenetic modifications. FcγRIIIa-pSyk was observed both upon in vitro activation of naïve CD4+ T-cells and in vivo in CD4+ gated cells in SLE patients. Activated CD4+ T-cells expressing CD25, CD69 and CD98 also show FcγRIIIa-pSyk signaling. Cells with FcγRIIIa-pSyk signaling show ICOS and Bcl6 expression, markers of TFH. Adaptor and TLR interacting proteins BTK, HMGB1, HRAS, and MyD88 were upregulated. Labeled ICs that bound to FcγRIIIa also co-localize with HMGB1 and MyD88 in activated CD4+ T-cells. Western blots of IPs generated with anti-FcγRIIIa antibody show association of HMGB1 and MyD88 with FcγRIIIa. Three enzymes from epigenetic array ATF2, HAT6, and KAT6A showed several folds increase from FcγRIIIa engagement by ICs vs. CD28 co-signal.

Past literature from over three decades have suggested that CD4+ T-cells do not express Fc-receptors. Although, this may be true for quiescent normal CD4+ T-cells, activated CD4+ T-cells not only express FcγRIIIa but signal via Syk. FcγRIIIa mediated signal efficiently replaces CD28 co-signal and rescue naïve cells activated with sub-optimal CD3 ligation. FcγRIIIa will act as vehicle for DNA delivery to endosomes in T-cells. We describe a new role for FcγRIIIa in adaptive immunity mediated by CD4+ T-cell responses.
T cell up-regulation of CD127 is associated with reductions in the homeostatic set point of the peripheral T cell pool during malnourishment.
Authors: Gubbels Bupp Melanie R , Murphy Sarah , Patrick Kristin , Thoner Timothy , Edwards R. Whitney , Litvin Seth
Published: January 2023

The following study was undertaken to better understand the impact of short-term malnutrition on T cell homeostasis, with particular attention focused on the CD127/IL-7 signaling dynamic of peripheral T cells in mice. We report that the total number of peripheral naïve and memory CD4+ and CD8+ T cells notably declined after one week of mild malnourishment, a time period too short to be entirely due to malnutrition-induced thymic involution. Peripheral malnourished T cells expressed higher levels of the IL-7 receptor component, CD127, and were less sensitive to death-by-neglect as compared to control T cells. Adoptive transfer studies revealed that CD127 expression did not correlate with increased survival in vivo and that all naïve CD8+ T cells upregulated CD127, regardless of initial expression levels. Corticosterone levels were elevated in malnourished mice and this correlated in time with peripheral T cell up-regulation of CD127 and the diminishment of the peripheral T cell pool. Overall, these data suggest a model in which CD127 levels are up-regulated quickly during malnourishment, thereby increasing the scavenge rate of IL-7, and providing a mechanism to quickly adjust the total number of T cells during malnutrition.
Regulation of Regulatory B Cells by Th2 Cytokines
Authors: Taitano Sophina Horne , Lundy Steven K
Published: January 2023

Regulatory B cells (Breg) have emerged as important members of the immune regulatory network, particularly in the context of autoimmunity and tissue transplant. Previously we found that IL-5 supported the growth and functions of Breg cells, including production of IL-10 and FasL-mediated T cell killing. In contrast, IL-4 did not support regulatory B cells growth or function and instead antagonized the effects of IL-5. Signal transducer and activator of transcription-6 (STAT6) is the primary STAT activated by IL-4. We hypothesized IL-4 effects on B cells were mediated by STAT6, and that STAT6−/− mice would have more abundant and effective regulatory B cells. Flow cytometric analysis of naïve B cells from STAT6−/− and BALB/c wild-type mice indicated differences in some Breg phenotypes. Culture with CD40L, IL-5, and/or IL-4 stimulation were used to assess the IL-10 production, FasL mediated killing, and activated phenotypes. As anticipated, STAT6−/− B cells did not lose regulatory function when cultured with CD40L, IL-5, and IL-4. Interestingly, when BALB/c B cells were cultured with CD40L and IL-5 to expand regulatory B cells and then re-cultured in the presence of IL-4, regulatory functions were not affected. These results suggested the inhibitory effects of IL-4 on Breg cells is likely to be due to competition of other B cells in the culture rather than direct inhibition. To confirm these findings in vivo, STAT6−/− and control mice were immunized with OVA emulsified in IFA and their B cells were compared phenotypically and functionally. Our data suggest that regulatory B cell growth, IL-10 production, and FasL mediated killing may be supported by IL-5 and CD40L stimulation, and are not directly affected by IL-4 as previously thought.
Antigen presentation via intravascular extensions of renal DC is necessary for antigen-specific T Cell migration into the kidney
Authors: Yatim Karim , Gosto Minja , Humar Rishab , Williams Amanda L. , Oberbarnscheidt Martin H.
Published: January 2023

We have previously shown that CX3CR1+ renal DC sample antigen via intravascular processes in the context of systemic infection and immune complex disease. Here, we investigated the role of intravascular DC processes in antigen presentation and effector T cell migration to the kidney.

To investigate T cell migration, CFP-OVA E. coli and CD8+ DsRed+ OT-I effector T cells, pretreated with Pertussis toxin (PTX), were i.v. injected into CX3CR1gfp/+ mice 24h before 2P intravital microscopy (2PIM) of the kidney was performed.

OT-I T cells migrated into the kidney, with 2PIM showing prolonged (30 min) DC-T cell interactions. OT-I migration was significantly higher (2-fold) when OVA was present (CFP-OVA E. coli vs WT E.coli), demonstrating that migration of effector T cells was antigen-specific. To test if antigen presentation by DC was necessary to mediate migration of T cells, we used CD11c-YFP Kb−/− bone marrow chimeric recipients (hematopoietic cells lack H-2k(b), parenchymal cells express H-2k(b)). CFP-OVA E. coli injection into a CD11c YFP Kb−/− bone marrow chimeric mouse abrogated effector OT-I T cell migration into the renal tissue.

Our data demonstrate that migration of effector T cells in the setting of systemic infection is antigen-specific. In addition, antigen presentation by hematopoietic cells, most likely DC, is necessary to mediate antigen-specific effector T cell migration. H-2k(b) expression on parenchymal and endothelial cells does not mediate antigen-specific effector T cell migration. Our findings show that renal DC have two roles: immune surveillance by sampling and presenting antigen via intravascular processes, and guiding effector T cells to the site of antigen.
H. pylori infection promotes intestinal inflammation and colitis-associated colon cancer in mice with a lineage-restricted deletion of the Smad4 gene in T cells.
Authors: Kim Byung-Gyu , Choi Sung Hee Hee , Bagheri Nayer , Nedrud John , Letterio John
Published: January 2023

Recent data suggest a protective effect of Helicobacter pylori (H. pylori) infection in inflammatory bowel disease (IBD), but the mechanisms mediating this protective effect are poorly understood. We previously reported that selective deletion in T cells of the TGF-β signaling intermediate in mice, Smad4 (Smad4TKO mice) leads to spontaneous gastrointestinal inflammation which progresses to mucosal epithelia cancers. Thus we infected Smad4TKO mice with H. pylori SS1 and PMSS1. Smad4TKO mice developed thickened intestinal mucosa, epithelial hyperplasia, crypt abscesses, and abundant inflammatory cell infiltrates between 2 and 3 months post infection. Some mice also developed colonic adenocarcinoma while wild type (WT) infected mice exhibited no gross or histologic signs of colitis. Smad4TKO mice infected with H. pylori had a increase in the number of mucosal effector CD4+ T cells producing IFN-g and IL-17 and a decrease in the mucosal Foxp3+CD4+ T cells (Tregs) in vivo, compared to WT mice infected with H. pylori. We also observed enhanced epithelia expressing PD-L1 in colon of Smad4TKO mice with H. pylori infection. The colon epithelia of Smad4TKO mice with H. pylori infection exhibited an oncogenic signature with an increase in the expression of iNOS and p-STAT1. Mutations in Smad4 are found in large percentage of human Juvenile Polyposis Syndrome (JPS) and Hereditary Hemorrhagic Telangiectasia (HTT) patients who are at risk of developing both adolescent and adult colon cancer. Our results raise the intriguing possibility that H. pylori infection could be a co-factor in the development of colon cancer by JPS/HTT patients.
MAdCAM as a potential biomarker of HIV-induced inflammation
Authors: Andrews Omozusi E , Van Ryk Donald , Ansari Aftab , Cicala Claudia , Fauci Anthony , Arthos James
Published: January 2023

HIV causes a slowly progressive immunodeficiency disease via depletion of helper T cells and other immune cells, eventually resulting in AIDS. Currently, no effective vaccine exists for HIV. Acute HIV infection is associated with viral replication in, and of destruction of gut tissues. A key subset of gut-homing immune cells, CD4+ T cells expressing the integrin receptor α4β7, are highly susceptible to HIV infection. α4β7+/CD4+T cells gain entry to gut tissues by binding to MAdCAM, a receptor that is expressed on the surface of endothelial cells that line gut-associated lymphoid tissues (GALT). In a recent study of rhesus macaques infected with SIV we observed dramatic increases in levels of soluble MAdCAM in the serum shortly after infection. The presence of soluble MAdCAM in serum, correlated with viral replication in GALT, and suggested that MAdCAM was shed from the surface of endothelial cells in response to immune activation and inflammation of gut tissues. To investigate the regulation and functionality of MAdCAM, we generated distinct recombinant Rhesus macaque MAdCAM proteins including both soluble and membrane bound forms. Plate bound assays demonstrated that soluble rhesus MAdCAM can function as a T cell co-stimulatory signal. Human CD4+ T cells substantially proliferated in the presence of soluble rhesus MAdCAM proteins and proliferation diminished in the presence of the anti-α4β7 (ACT1) antibody. Importantly, since HIV infects GALT, which contains the majority of immune cells, the study of MAdCAM-α4β7 signaling and mechanisms of MAdCAM shedding may provide insight on potential mucosal-based therapies for treating HIV and serve as a biomarker of HIV-induced gut inflammation, respectively.
Intestinal inflammation drives differentiation of tissue-resident memory CD8+ T cell populations with enhanced migratory ability
Authors: Bergsbaken Tessa , Fink Pamela J
Published: January 2023

Tissue-resident memory (TRM) CD8+ T cells represent a distinct population of memory T cells that are maintained independently of the circulation and are positioned to respond rapidly to reinfection of the tissue. Using intestinal infection with the bacterial pathogen Yersinia pseudotuberculosis, we determined that CXCR3-mediated recruitment to areas of inflammation during bacterial colonization drives differentiation of a CD103− subset of lamina propria (LP) CD8+ TRM cells. However, the inflammatory cytokines that regulate the development of this subset remain unidentified. Ex vivo cytokine stimulation of Yersinia-primed CD8+ effector T cells identified several cytokines capable of preventing CD103 upregulation by TGF-beta, including IFN-beta and IL-12. Yersinia infection resulted in induction of IL-12 and IFN-beta in infected macrophages both in vitro and in proinflammatory microenvironments in the intestinal tissue. Eliminating IFN-beta signaling in T cells resulted in the preferential development of CD103+ T cells during infection, indicating type I interferon plays a role in the differentiation of the CD103− LP TRM population. Ex vivo migration assays indicated CD103− LP TRM cells are able to migrate more readily than CD103+ cells; these populations also display distinct chemokine receptor profiles, supporting differential migration of LP TRM populations in response to secondary challenge. These studies identify inflammatory cytokines encountered in the intestinal tissue that can dictate the phenotype and function of CD8+ TRM cells.
Colonic intraepithelial lymphocytes produce IL-6 in response to resident bacteria to modulate epithelial barrier function
Authors: Kuhn Kristi , Regner Emilie H. , Mehta Gaurav , Hendrickson Jason D. , Whitney Alyssa K. , Campbell Eric L. , Ohri Neha , Colgan Sean P.
Published: January 2023

Interactions between the microbiota and distal gut are important for the maintenance of a healthy immune system. Dysbiosis of colon bacteria has emerged as a likely contributor to diseases that arise at the level of the mucosa. Intraepithelial lymphocytes (IELs) are positioned within the epithelial barrier, and in the small intestine, function to maintain epithelial homeostasis. We hypothesized that IELs of the colon modulate epithelial barrier function through the liberation of cytokines stimulated by interactions with resident bacteria. Our data demonstrate that IL-6 is a major cytokine secreted by colonic IELs in a microbe-dependent fashion. We identify Alistipes species of the phylum Bacteroidetes as candidates to recruit IELs and stimulate their IL-6 secretion. IEL-derived IL-6 is functionally important in the maintenance of the epithelial barrier as IL-6−/− and antibiotic-treated mice were noted to have increased paracellular permeability and closer interaction with luminal bacteria. IL-6 was found to signal in colonic epithelial cells and resulted in increased epithelial barrier integrity and claudin1 expression in model epithelia. Therefore, we conclude that the host microbiota provides a homeostatic role for epithelial barrier function through regulation of IEL derived IL-6.
Vitamin D deficiency results in fewer innate lymphoid cells and severe infection with Citrobacter rodentium.
Authors: LIN YANG-DING , Cantorna Margherita T
Published: January 2023

Vitamin D deficiency results in severe experimental inflammatory bowel disease. We investigated the role of vitamin D following an infectious model of inflammation in the gut following Citrobacter rodentium infection. Vitamin D sufficient (D+) and vitamin D deficient (D−) wild-type (WT) and Cyp27B1 knock-out (CypKO) mice, which lack the enzyme to produce the high affinity ligand, 1,25(OH)2D (1,25D), were infected. In the absence of 1,25D, the low affinity vitamin D ligand (25(OH)D) accumulates in CypKO mice and can replace 1,25D. Therefore it was not surprising that D+ WT and D+ CypKO cleared C. rodentium with identical kinetics. D− WT mice cleared C. rodentium slower than D+ mice and D− CypKO mice developed a lethal infection. Infected D− CypKO and D− WT mice had increased colonic inflammation, systemic bacterial dissemination, but only D− CypKO mice died following infection. Increased colonic IL-17 mRNA in D− versus D+ mice at d14 post-infection was perhaps too late to protect the D− mice from C. rodentium. Innate lymphoid cells (ILCs) are critical sources of early IL-17 and IL-22 that are essential for clearance of C. rodentium. Uninfected D− mice had reduced ILCs in the gut and decreased ILC-derived IL-17 and IL-22 in the small intestine, which is the site of early colonization of C. rodentium. Vitamin D deficiency resulted in delayed clearance of C. rodenitum that was associated with reduced ILC produced early IL-17 and IL-22, more inflammation and IL-17 at the peak of infection. The data suggests that vitamin D may be an important regulator of early cytokine production by ILC in the gut and therefore protection from enteric infection.
T-bet-independent TH1 immunity to Toxoplasma gondii drives intestinal immunopathological response
Authors: Lopez-Yglesias Americo H , Burger Elise , Yarovinsky Felix
Published: January 2023

Coordinated production of IFN-γ by innate and adaptive immune cells is central for host defense, but can also trigger immunopathology. The investigation of lymphoid cell specific contribution to the IFN-γ-mediated intestinal pathology during Toxoplasma gondii (T. gondii) infection identified that CD4+ T cells but not group 1 innate lymphoid cells (ILC1s) are responsible for IFN-γ-dependent Paneth cell death, inflammation, and bacterial dysbiosis. We identified that lack of T-bet, the lineage-defining transcription factor for ILC1s and a maturation factor for intraepithelial lymphocytes had no detectable effect on T. gondii induced intestinal inflammation, ruling out contribution of these cells in the parasite-induced immunopathology. Instead, we show that T-bet deficient CD4+ T helper 1 (TH1) cells are necessary and fully sufficient for the T. gondii triggered acute ileitis. These results revealed that T-bet-independent TH1 effector cells are functional mediators of type I immunopathological response during acute gastrointestinal infection.
Batf3-dependent dendritic cells impact T-cell responses differently between adult and neonatal mice in an intestinal viral infection.
Authors: Sun Tian , Philpott Dana J , Gommerman Jennifer L
Published: January 2023

The role of dendritic cells (DCs) in shaping the immunity against bacterial infections in the colon (e.g. Citrobacter rodentium) has been well investigated; however, what subset modulates the immune response against viral infections in the small bowel is less known. Rotavirus (RV) almost exclusively infects and replicates in the small intestinal villi resulting in both cellular and humoral adaptive immunity, and RV infection serves as a model to study the DC-elicited antiviral responses in adult and neonatal mice. Batf3−/− adult and neonatal mice lack CD103+CD11b− DCs in the small intestinal lamina propria (SILP) and displayed a delay in viral clearance. CD8+ T cells have a role in the resolution of primary RV infection, and indeed, we found that while no antigen-specific CD8+ T cell response was detected in the Batf3−/− neonatal mice, a residual CD8+ T cell response was observed in the Batf3−/− adult mice, suggesting compensatory antigen-presentation from other adult DC subsets in the SILP (such as CD103+CD11b+ DCs). In terms of the CD4+ T cell response, no Th1 response was found in adult Batf3-deficient or –sufficient mice, however, a robust Th1 response was present in neonatal mice which was dependent on Batf3, implying a requirement of CD103+CD11b−DCs for priming/maintaining a Th1 response only in the neonatal period. Both the local and systemic anti-RV IgA response was normal in adult Batf3−/− mice, however, neonatal Batf3−/− mice displayed a stronger systemic IgA response than littermate controls. Our results demonstrate a requirement for Batf3-dependent DCs in antiviral responses which differs between neonates and adults, and these results may shed light on vaccine design.
VDJML – tools for capturing the results of inferring immune receptor rearrangements
Authors: Toby Inimary , Breden Felix , Buntzman Adam , Christley Scott , Corrie Brian , Fonner John , Gupta Namita , Hershberg Uri , Jordan Chris , Kim Min S , Kleinstein Steven , Marthandan Nishanth , Mock Stephen , Monson Nancy , Rounds William H , Rojas Manual , Rosenfeld Aaron , Rubelt Florian , Scarborough Walter , Scheuermann Richard H , Scott Jamie , Uduman Mohamed , Heiden Jason Vander , Cowell Lindsay G
Published: January 2023

Despite the widespread use of immune repertoire profiling, there is currently no standardized format for output files from VDJ analysis. Researchers utilize software such as IgBlast and IMGT/High V-Quest to perform VDJ analysis and infer germline rearrangements. Each of these software tools produces results in a different file format, and can identify the same result using different labels. These differences make it challenging for users to perform additional analysis using the output file from one software to the next. We have addressed this problem by developing a standardized file format for representing results. The purpose of VDJML is to provide a common standardized format for different VDJ analysis applications and to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format is accompanied by a suite of analysis tools, which are accessible via command line and written in C++ and python. The VDJML suite will allow users to streamline their VDJ analysis and facilitate the sharing of scientific knowledge within the community. The VDJML suite and documentation are available from https://www.vdjserver.org/software. We welcome participation from others in developing the file format standard, as well as code contributions.
Characterization of monoclonal antibodies generated by in vitro immunization
Authors: Maier Natalia , Holzloehner Pamela , Hoenow Anja , Scheunemann Astrid , Weschke Daniel , Hanack Katja
Published: January 2023

Monoclonal antibodies are highly valuable tools in biomedicine but the generation by hybridoma technology is very time-consuming and elaborate. In order to circumvent the consisting drawbacks an in vitro immunization approach was established by which murine as well as human monoclonal antibodies against a viral coat protein could be developed. The in vitro immunization process was performed by isolation of murine hematopoietic stem cells or human monocytes and an in vitro differentiation into immature dendritic cells. After antigen loading the cells were co-cultivated with naive T and B lymphocytes for three days in order to obtain antigen-specific B lymphocytes in culture, followed by fusion with murine myeloma cells or human/murine heteromyeloma cells. Antigen-specific hybridomas were selected and the generated antibodies were purified and characterized in this study by ELISA, western blot, gene sequencing, affinity measurements. Further the characteristics were compared to a monoclonal antibody against the same target generated by conventional hybridoma technology. Isotype detection revealed a murine IgM and a human IgG4 antibody in comparison to an IgG1 for the conventionally generated antibody. The antibodies derived from in vitro immunization showed indeed a lower affinity for the antigen as compared to the conventionally generated one, which is probably based on the significantly shorter B cell maturation (3 days) during the immunization process. Nevertheless, they were suitable for building up a sandwich based detection system. Therefore, the in vitro immunization approach seems to be a good and particularly fast alternative to conventional hybridoma technology.
Immunomagnetic enrichment of human ILC2 cells from peripheral blood leukopak
Authors: Poon Grace FT , Kyei Stephen K , Valdez Yanet , Takei Fumio , Peters Carrie , Woodside Steven M , Eaves Allen C , Thomas Terry E
Published: January 2023

Group 2 innate lymphoid cells (ILC2s) are essential for airway tissue repair, protection against helminth infection, metabolic homeostasis, and the development of allergic airway diseases. They are widely distributed throughout the human body in lung, intestine, palatine tonsils, skin and peripheral blood, but are extremely rare within a given tissue. In healthy individuals, ILC2s comprise 0.01–0.04% of leukocytes in the peripheral blood. Currently, flow cytometric cell (FACS) sorting is the only method to purify ILC2s, which is time-consuming due to their low frequency. We have adapted EasySep™ to pre-enrich ILC2s from peripheral blood leukopaks, and thereby shorten subsequent FACS analysis and sorting times. Non-ILC2 cells in an unprocessed leukopak were immunomagnetically labeled using a cocktail of antibody complexes and magnetic particles. The sample was then placed in a magnet for 10 minutes, the labeled cells were retained, and the unlabeled cells, containing the ILC2 population, were recovered. ILC2s were enriched from 5 x108unprocessed cells in 30 minutes. Enriched cells were counted and evaluated by FACS. ILC2s, defined as lineage negative (CD1a− CD3− CD11c− CD14− CD19− CD34− CD123− TCRαβ− TCRγδ− BDCA2− FcɛR1−), CRTH2+ CD161+CD127+, were enriched 268±167 fold, with 74± 34% recovery (means ± SD, n=7). EasySep™ pre-enrichment of ILC2s reduced subsequent FACS analysis and sorting time compared to non-enriched samples. Importantly, ILC2s enriched with EasySep™ were functional, as shown by production of the type 2 cytokine IL-13 upon stimulation with IL-33 and IL-2. Overall, EasySep™ is a simple and rapid method to pre-enrich ILC2s from leukopak for subsequent FACS analysis or sorting and functional analyses.
MojoSort™ kit isolated cells maintain physical function in magnetic cell separator and column
Authors: Zhang Hong , Tam Miguel , Gohel Dhanesh , Koury Jeffrey , Shen Crystal , Chen David , Yang Xifeng , Lay Gene
Published: January 2023

Magnetic cell sorting has been widely used to isolate different cell types as it is a fast and reliable method to obtain discrete populations with high purity and yield. However, to maintain cell functionality with this approach has been concerned for different studies. To address this, the cells were isolated with our recently developed MojoSort™ isolation kit using commercially available magnetic cell separation column, and compared that with other commercial magnetic column related products. The stickiness of the magnetic particles of those isolated cells were then imaged with electron microscope, and the cellular functionality was tested by activation of MAPK/ERK pathway and cell proliferation assay. Our data show that MojoSort™ negative isolated cells with MojoSort™ separator or magnetic column do not display beads on cell surface, phosphorylation of ERK1/2 level did not increase as detected by western blot, and the isolated cells showed fully functional. In addition, both positively isolated cells with either MojoSort™ kit or magnetic separation column related kit preserved functionality. Furthermore, MojoSort™ isolated cells only have <10% of particles covered on the cell surface. Finally, when analyzing positively selected cells using MojoSort™ kit with MojoSort™ magnet, we observed a higher number of beads on the cell surface as compared to cells isolated using separation columns, but the cells still maintain their functionality. Thus, our data demonstrates that cells isolated with MojoSort™ reagents are viable and functional, independently of the magnetic separator used, either column-based or handheld magnet.
Systems Biology Approach to Uncover Signaling Defects in Primary Immunodeficiency Diseases
Authors: Choi Jeff , Fernandez Rosemary , Maecker Holden T , Butte Manish J
Published: January 2023

To improve diagnoses and further our understanding of functional defects in subjects with primary immunodeficiency (PI) diseases, we developed a new screen to broadly study the signaling of defective human immune responses to infections. Using time-of-flight mass cytometry (CyTOF) and phospho-specific antibodies, we simultaneously examined the major signaling pathways of all circulating innate and adaptive immune cell types in response to cytokines and TLR agonists to identify signaling responses. We studied healthy controls and developed software tools to generate a systematic map of signaling responses. CyTOF successfully recapitulated established signaling responses, and further, elucidated new pathways that would not have been appreciated with traditional studies. Signaling map of controls was compared to maps generated by analyzing PI subjects with gain-of-function STAT1 and autosomal dominant STAT3 deficiency diseases. CyTOF allowed us to perform unbiased studies to uncover many ex-vivo signaling defects simultaneously to better understand how known genetic etiologies manifest functionally, and exactly which cell subtypes are affected. We identified more glaring defective signaling responses in these subjects aside from the canonical defects, elucidating the true breadth of cell subtypes and pathways affected. Broad, unbiased, functional analysis can complement whole exome sequencing to facilitate identification of novel or rare immune diseases. Comprehensive view of signaling across all immune cell subsets facilitates learning more about abnormal signaling patterns that confer susceptibility to infection or autoimmunity, improving our understanding of immune responses and can lead to new therapies.
Controlling role of Ly6Chigh Monocytes in breast cancer and C26 colon carcinoma
Authors: Zare Fatemeh
Published: January 2023

Tumor microenvironment encompasses a wide variety of immune cells, which macrophages comprise the most dominant portion of them and thus are the major players in the connection between inflammation and cancer. These tumor-associated macrophages (TAMs) are derived from blood monocyte precursors and subsequently acquire distinct characteristics as a result of tumor micro-environmental cues. Monocytes are a heterogeneous population in the blood with an enormous plasticity whose fate and functions are dictated by the microenvironment. Therefore, the roles of specific Monocytes subsets in tumor progression and the molecular mechanisms for their impacts need to be elucidated. Ly6Chigh and Ly6Clow are two main different types of murine monocytes subsets that have been defined by distinct phenotypes and immunoregulatory functions. Recent data demonstrates that Ly6Chigh monocytes can recruit to inflammation loci while Ly6Clow monocytes are patrolling cells. We have developed a method to produce large number of Ly6Chigh monocytes in vitro. In our study we observed that, injection of these cells affects tumor progression in breast cancer and C26 colon carcinoma animal models. Activation of Ly6Clow monocytes by pro- or anti-inflammatory cytokines, results in displaying a genetic expression profile, corresponding to pro- or anti-inflammatory genes, respectively. Injection of pre- activated Ly6Clow monocytes toward pro- or anti-inflammatory polarization in C26 colon carcinoma showed that anti-inflammatory activated monocytes are more beneficial in delaying cancer cachexia. Increased knowledge of monocytes improves the chances to find therapies against a broad spectrum of diseases including cancer.
Foxp3 controls oxidative phosphorylation in regulatory T cells
Authors: Beier Ulf H , Angelin Alessia , Jiao Jing , Xiao Haiyan , Wang Liqing , Dahiya Satinder , Wells Andrew D , Wallace Douglas C , Hancock Wayne W
Published: January 2023

Many T cell subsets have distinct metabolic requirements. Activated CD4 helper and cytotoxic CD8 T cells require glycolysis for optimal function, whereas Foxp3+ T-regulatory (Treg) cells are thought to be dependent on oxidative phosphorylation. We questioned if Foxp3 is involved with re-programming T cell metabolism. We retrovirally transduced murine T cells with Foxp3 or empty vector (EV), and also cultured CD4+Foxp3− T cells from Foxp3YFPcre mice under polarizing conditions with TGFβ to form induced Tregs, using YFP to track Foxp3 expression. Our analysis of FACS-sorted YFP+ iTreg and YFP− non-iTreg showed that both iTreg and Foxp3-transduced T cells had significantly higher basal oxygen consumption rates than non-iTreg or EV transduced T cell controls (44.1±49% and 76.1±76.5%, respectively, p<0.05, n=4), despite equal mitochondrial mass. Using Foxp3 ChIP-seq and microarray studies, we identified multiple binding sites of Foxp3 and Foxp3-dependent transcripts among key genes involved in control of oxidative phosphorylation as well as nuclear encoded electron transport chain genes. FACS-sorted YFP+ iTreg showed increased protein expression of electron transport chain complexes, especially complex I, compared to YFP− non-iTreg. We found that mice deficient in ND6, a mitochondrial encoded gene of complex I, have deficient Treg function. Furthermore, 50 nM rotenone, a complex I inhibitor, could selectively inhibit Treg suppressive function without affecting Tconv cell proliferation. The effects of Foxp3 on Treg metabolism may be physiological important to enable Tregs operating in low glucose environments such as the tumor microenvironment, and could offer an opportunity for therapeutic immune modulation.
Staphylococcal enterotoxin G and I: augmenting the T cell response to murine melanoma.
Authors: Knopick Peter Leo , Benedict Katherine C , Alvine Travis D , Nilles Matthew L , Terman David S , Bradley David S
Published: January 2023

Staphylococcal enterotoxins (SEs) have been shown to be effectual proteins which demonstrate anti-tumor activity. SEG and SEI, produced from the enterotoxin gene cluster (egc) of Staphylococcal aureus, activate T cells with Vβ specificity to elicit robust T cell proliferation, T helper cell 1 (TH1) and TH2 cytokine secretion, and nitric oxide dependent tumor lysis. Our data highlight the effects of SE stimulated T cells and the subsequent consequence on aggressive, metastatic cancer progression using B16-F10 murine melanoma model. Specifically, we explore the differences between major histocompatibility complex (MHC) and human leukocyte antigen (HLA) SE presentation to the T cells via the T cell receptor (TCR). Our data demonstrates increased survival of B16-F10 tumor bearing mice, C57BL/6 and DQ8 (HLA-DQA1*0301, HLA-DQB1*0302) transgenic mice, after irradiated B16-F10 vaccination and SE stimulation. Interestingly, SEG and SEI induce strong T cell proliferation yet do not induce neutralizing antibodies to the extent of classical SEs, SEA and SEB, nor induce autoimmune pathology. Our findings demonstrate that superantigen administration enhances lymphocytic tumor killing and suggest a potential role for SEG and SEI as potent immunotherapeutics for human cancer.
Inhibition of Tumor-Derived Semaphorin7A Decreases Tumor Progression in a Murine Model of Metastatic by modulating production angiogenic chemokines and immune infiltration
Authors: Garcia-Areas Ramon Antonio , Libreros Stephania , Simoes Michael , Gazaniga Nathalia , Schilling Kathy , Iragavarapu-Charyulu Vijaya
Published: January 2023

It is estimated that one in eight women will be diagnosed with breast cancer. Developing an understanding of the tumor microenvironment is critical for developing treatments for breast cancer patients. Poorly differentiated blood vessels can fashion an oxygen-deprived microenvironment that triggers the expression of transcription factor Hypoxia Inducible Factor alpha (HIF-1alpha) that in turn can up regulate genes mediating a pro-tumor effect. Our laboratory has discovered that mammary tumors express Semaphorin7a (SEMA7A), a HIF-1alpha inducible protein. Tumor-derived SEMA7A induces monocytes to secrete proangiogenic chemokines to enhance tumor growth. Decreasing tumor-derived SEMA7A limits tumor growth and the production of angiogenic CXCL-2/MIP-2 and VEGF-A by monocytes. Immune cells also express elevated levels of SEMA7A in tumors, however the role of stromal-derived SEMA7A in tumorigenesis is largely unknown. We hypothesized that SEMA7A may be a hypoxia-inducible factor and that inhibition may deter the effects of hypoxia on tumor and immune cells. Following hypoxic stimuli, WT monocytes increased production of CXCL2, VEGF-A and Chitinase-3-like protein1. However, SEMA7A null monocytes failed to respond to hypoxia, which may be attributed to an impaired MAPK signaling response. Monocytes from SEMA KO tumor bearing mice showed a decreased production of angiogenic molecules. More importantly, we found that SEMA7A KO 4T1 mammary tumor bearing mice showed decreased tumor growth and metastasis compared to WT. Collectively, we show a role for not only a tumor-derived, but also stromal-derived SEMA7A in hypoxia-mediated mammary tumor progression.
Host-protective anti-tumor immune responses induce immunoediting of indolent but not quiescent dormant tumor cells
Authors: Manjili Masoud H , Payne Kyle K , Keim Rebecca , Graham Laura , Idowu Michael , Wan Wen , Toor Amir , Wang Xiang-Yang Shawn , Bear Harry D
Published: January 2023

Two major barriers to cancer immunotherapy include tumor-induced immune suppression mediated by myeloid-derived suppressor cells (MDSCs), and poor immunogenicity of the tumor expressing self-antigens. To overcome these barriers, we used adoptive immunotherapy (AIT) by means of tumor-sensitized T cells and CD25+ NKT cells combined with Decitabine (Dec). Dec enhanced immunogenicity of the tumor, and the presence of CD25+ NKT cells rendered T cells resistant to remaining MDSCs. This combinatorial therapy significantly prolonged survival of animals bearing metastatic tumor cells. However, AIT eventually induced tumor immunoediting and subsequent tumor escape and progression. We hypothesized that conventional cancer therapeutics result in residual dormant tumor cells with a heterogeneous sensitivity to immunoediting; we therefore sought to identify the characteristics of dormant tumor cells that are resistant to immunoediting. We used Adriamycin (ADR) chemotherapy or radiation therapy (RT) which simultaneously induces tumor cell death and tumor dormancy. Resultant dormant cells became refractory to additional doses of ADR or RT, but they remained sensitive to tumor-reactive immune cells. Importantly, we discovered that dormant tumor cells contained indolent cells that expressed low levels of Ki67 (Ki67+/low) and quiescent cells that were Ki67 negative (Ki67−). Whereas the former were prone to tumor immunoediting and escape, the latter did not demonstrate immunoediting. Our results suggest that administration of immunotherapy during quiescent tumor dormancy could overcome tumor relapse and prevent disease progression to advanced stage cancer.
‘PD-1 related effector functions rely on the presence/absence of CD28 co-stimulatory molecule in Ag-specific CD8+ T cell clones isolated from melanoma patients’
Authors: Palermo Belinda , Franzese Ornella , Di Donna Cosmo , Panetta Mariangela , Gualtieri Novella , Soriani Antonella , Foddai Maria Laura , Santoni Angela , Nisticò Paola
Published: January 2023

The relationship between PD-1 expression and T-cell effector functions is still unclear, although crucial in the new era of immunotherapy targeting the PD-1/PDL-1 pathway. We have recently reported that Melan-A-specific CD8+ T-cells isolated from melanoma patients treated with combined chemo-immunotherapy (dacarbazine before peptide Melan-A/gp100-vaccination), show an enlarged T-cell repertoire, CD28− PD-1+AKT+ phenotype with high anti-tumor polyfunctionality sustained by ICOS (Franzese et al, OncoImmunology, in press). Herein, we have analyzed the gp100-specific CD8+ T-cell clones isolated from the same patients. Cells have been characterized for gp-100 specificity, and TCR beta-chain sequencing analysis evidenced that they possess an oligoclonal repertoire. The relationship among inhibitory receptors (PD-1, TIM-3 and LAG-3), co-stimulatory molecules (CD27 and CD28) and effector functions of T cells has been studied. Taking advantage from a battery of Melan-A- and gp100-specific T-cell clones, here we show that gp100-CD8+ T cells with a CD28+ PD-1+AKT+ phenotype are unable to kill gp100-expressing melanoma cells, differently from data obtained with Melan-A-specific clones showing a CD28− PD-1+ AKT+ phenotype, and able to lyse tumor cells with high efficiency. Studies are in progress to identify the biochemical pathways involved in the control of functionality of PD-1-expressing CD8+ T cells.
To clone tumor specific T cell receptor in cancer patients after immunotherapy with patient-specific antigens
Authors: Han Yanyan , Long Jiangting , Zhou Xiangjun
Published: January 2023

Background

Adoptive T cell transfer was recently often used to treat cancer patients who have failed in standard therapies. An immunotherapy named MASCT (Multiple Antigen Stimulating Cellular Therapy) was applied to cancer patients, which is composed of patient-specific antigen pulsed dendritic cells (DCs) and autologous tumor specific T lymphocytes.

Methods

Patients were treated by the autologous T cells stimulated with mature DCs pulsed with multiple tumor antigens. The tumor antigen specific immune responses were examined during the treatment, and the antigens were selected accordingly. The tumor specific T cell receptor (TCR) would be further cloned from these patients who have demonstrated both immunological and clinical responses.

Results

A human papilloma virus (HPV) positive cervical cancer patient with bone metastasis on the right sacroiliac joint have repeatedly received treatment of MASCT. Specific responses against tumor antigens were detected by IFNγ ELISPOT assay, such as telomerase, CEA, and HPV18/58. Based on patient’s specific immune response, we adjusted her antigen peptide pool by saving the antigens, which had induced specific responses and removed the peptides that did not. The adjusted antigen pool clearly further boosted the specific responses. Moreover, this patient was evaluated as partial remission after MASCT treatment with patient-specific antigens. Therefore, we are now trying to clone the CEA and telomerase specific TCRs from this patient.

Conclusion

Our study provides a promising method to clone tumor specific TCRs from cancer patients, who have shown enhanced immunological responses as well as clinical benefits after immunotherapy with patient-specific antigens.
Development of LAMP-based vaccine for cashew allergy immunotherapy
Authors: Shen Wei , Marketon Anthony , Su Yan , Macauley Juliete , Heiland Teri
Published: January 2023

Tree nut allergy is the second most common triggers of anaphylaxis behind peanut in children and adults. Proactive therapies to decrease reaction severity are currently not available. Here, we utilized Lysosomal-associated membrane protein-1 (LAMP-1) as a targeting signal to make novel DNA vaccines against three major cashew allergens (Ana O1, Ana O2, Ana O3). Briefly, the chimeric constructs encoding the DNA sequences of Ana O1 or Ana O2 or Ana O3 or multivalent plasmid fused to LAMP-1 were synthesized and administrated to BALB/c mice through a needle-free delivery method (Biojector). By ELISA analysis, it showed that LAMP-Ana O 1/Ana O 2/Ana O 3 immunized mice produced high titers of IgG2a and moderate titers of IgG1 antibodies against each antigen. In contrast, IgE response was reduced in the vaccine immunized mice. Moreover, induction of IFN-γ and reduction of allergen-stimulated Th2 cytokine IL-4 were observed in vitro in splenocyte cells of LAMP-Ana O1/Ana O2/Ana O 3 treated mice after antigen stimulation, suggesting a predominant Th1 type immune response was elicited via DNA immunization. To further explore the therapeutic effect of this vaccine, we pre-sensitized the C3H/HeJ mice by oral gavage of protein of cashew extracts, and then immunized the mice with either single LAMP-Ana O plasmids or multivalent plasmid. LAMP-Ana O 1/Ana O 2/Ana O 3 treated mice exhibited strong IgG2a response and decreased IgE level, while control LAMP-vector treated mice demonstrated low IgG2a titer and higher IgE level accompanied by elevation of plasma histamine and IL-4 producing basophils in the blood. These results indicate that immunotherapy with LAMP-Ana O1/Ana O 2/Ana O 3 is effective, and may be a potential approach for treating cashew allergy.
Global dysregulation of gene expression in macrophages from different chicken haplotypes correlates with disease resistance
Authors: Drechsler Yvonne , Irizarry Kris , Griggs Lisa , Kopulos Renee , Keeler Calvin , Boettger Cynthia , Carr Thomas , Clark Jory , Bryden Randall , Downs Eileen , Collisson Ellen
Published: January 2023

Disease resistance and susceptibility has been associated with the major histocompatibility complex (MHC) in several species. This study aimed to better characterize the molecular basis for dramatically different nitric oxide production and associated immune function between the B2 and the B19 haplotype chicken macrophages in response to IFNγ stimulation, previously shown by our laboratory. A next generation RNA sequencing approach was employed to analyze gene expression from purified macrophages from each haplotype group (B2 vs. B19) across 9 time points spanning seven days. Ultimately, our data shows global dysregulation of gene expression in B19 haplotypes correlating with increased disease susceptibility of this haplotype. Our results demonstrate that transcription factors (TF) exhibit divergent expression between B2 and B19 haplotype cells both prior to and after stimulation. Our results also reveal that miRNAs exhibit divergent expression between B2 and B19 haplotype cells both, prior to, and after stimulation with IFNγ. In contrast to TFs, miRNAs can modulate gene product levels post-transcriptionally. We also identify ubiquitin-related enzymes and factors that exhibit divergent expression between B2 and B19 haplotype cells. Some of our results indicate that snoRNAs exhibit divergent expression between B2 and B19 haplotype cells, which is noteworthy because snoRNAs can modulate genes epigenetically. Taken together our results suggest that the global dysregulation of gene expression observed in B19 haplotypes can at least in part be attributed to differential epigenetic regulation.