bluuebunny/crossref_metadata_2025 huggingface dataset:
Langerhans cells of human skin are the natural antigen‐presenting cells for CD1c antigen presentation
Authors: Sieling Peter Allan , Becker Todd , Ochoa Maria‐Teresa , Legaspi Annaliza , Rea Thomas , Modlin Robert
Published: June 2021

CD1 proteins are a family of MHC‐like polypeptides that are distinguished from MHC proteins through their presentation of lipids rather than peptides to T cells. Although the expression of CD1 proteins resembles that of MHC class II, i.e. mostly on professional antigen presenting cells, the natural antigen presenting cell population(s) for human CD1c‐restricted T cells have not been identified. CD1c‐restricted T cells derived from leprosy patients were potently activated by LCs derived from the epidermis of healthy donors and their in vitro‐derived counterparts, LC‐like dendritic cells (LC‐like DCs). In contrast, although DCs isolated from the dermis of healthy skin and monocyte‐derived DCs presented antigen to CD1c‐restricted T cells, their level of antigen presentation was greater than two‐fold less effective than LCs. Our data indicate that Langerhans cells from normal human epidermis are natural antigen presenting cells for CD1c‐restricted T cells and suggest that LCs are programmed for CD1c antigen presentation to T cells against cutaneous pathogens such as M. leprae.s
Staphylococcal enterotoxin B can activate NKT cells independent of Cd1d
Authors: Rieder Sadiye Amcaoglu , McKallip Robert , Nagarkatti Prakash , Nagarkatti Mitzi
Published: June 2021

Staphylococcal enterotoxin B (SEB) is a superantigen that can activate 5 to 30% of the T cell population and unlike conventional antigens, it directly binds to the MHC Class II molecules and activates T cells with variable TCR β chain such as Vβ8. Such activation triggers massive cytokine release that leads to rash, fever, and can often lead to multi‐organ failure, coma and death. The precise role of NKT cells in the cytokine production and toxicity induced by SEB is not clear. In this study, we investigated the role of NKT cells following SEB immunization. We found that SEB administration in vivo triggered Vascular Leak Syndrome (VLS) in the lungs of wild‐type (WT) mice while it was significantly decreased in Cd1d−/−mice lacking NKT cells. This was reversed when WT spleenocytes were injected into the Cd1d−/−mice. Cd1d−/−mice also exhibited decreased cytokine production in response to SEB, particularly IL‐4. When bone marrow derived dendritic cells (DCs) from WT and Cd1d−/−mice were tested for their ability to activate NKT cells, we noted that DCs from Cd1d−/−mice were able to activate NKT cells. Together, our studies demonstrate that NKT cells can be directly activated independent of CD1d. Thus, the plethora of cytokines produced following SEB can also be attributed to initial NKT cell activation (Supported in part by NIH grants: AI053703, ES09098, AI058300, DA016545, HL058641 and P01AT003961).
TLR ligands efficiently convert tolerogenic CD4‐8‐ DC into immunogenic ones stimulating long‐lasting antitumor immune response
Authors: Zhang Xueshu , Xiang Jim
Published: June 2021

Different dendritic cell (DC) subsets play distinct roles in immune responses. CD4−8−DC secreting transforming growth factor (TGF)‐β stimulate CD4+ regulatory T type 1 (Tr1) cell responses leading to antitumor immune tolerance. CD4−8−DC could be converted into inducing Th1 rather Tr1 by being cultured for another 16–24 hrs and pulsed with 1 mg/ml OVA protein. In this study we investigate whether TLR ligands, agonist anti‐CD40 and their combinations could more efficiently convert CD4−8−DC to induce Th1 response. We found that CpG, LPS and anti‐CD40 alone or CpG+anti‐CD40, but not LPS+anti‐CD40 stimulate those DCs to express similar higher level of CD40, CD54, CD80, CD86 and I‐Ab after 8hrs culture than newly isolated DCs; Among the above groups, CpG group showed the strongest capacity of stimulating the proliferation of OT1 CD8 and OTII CD4 T cells and induced 100% animal protection from tumor challenge with highest OVAI specific CTL ratio and in vivo cytotoxicity; LPS+anti‐CD40 group was the weakest in every above aspect and could not provide any animal protection. Taken together, our findings indicate that CpG 8 hrs treatment could convert tolerogenic DC into immunogenic ones provoking strong antitumor immunity; and TLR ligands plus anti‐CD40 combination should be carefully tested before using as adjuvant for DC maturation. Thus our results might have great impact on designing the future tumor vaccine.
Polycytokine responses by CD8+ T lymphocytes to KSHV/HHV8 lytic and latency proteins
Authors: Lepone Lauren , Knowlton Emilee , Rappocciolo Giovanna , Piazza Paolo , Jais Mariel , Rinaldo Charles
Published: June 2021

Objectives: Based on our previous findings that dendritic cells (DC) are required to reveal epitopes of human herpesvirus 8 (KSHV/HHV8) glycoprotein B (gB) (Blood 99:3360, 2002), we determined CD8+ T cell responses to multiple lytic and latency viral proteins.

Methods: T cells were stimulated with DC from HHV8 seropositive, HLA A*0201 donors that were loaded with overlapping peptides derived from gB and K8.1 lytic proteins, and K12 and LANA latency proteins, and screened for IFNγ production by ELISPOT. Positive peptides were then assessed for production of IFNγ, IL2, TNFα, MIP1β and CD107a by flow cytometry.

Results: Our DC‐T cell model revealed 5 gB, 4 K8.1, 2 K12 and 5 LANA HLA A*0201 epitopes. The HHV8 lytic and latency proteins induced predominately IFN‐γ, IL2 and CD107a.

Conclusions: This is the first demonstration that HHV8 lytic and latency proteins induce polycytokine production by CD8+ T cells. These targeted regions of the virus could be critical in HHV8 immunopathogenesis and vaccine development. (U01‐AI35041, R01 CA82053)
Expression and characterization of loop‐ and helix‐forming epitopes of measles virus hemagglutinin in plants
Authors: Sulima Sergey , Musiychuk Konstantin , Bouche Fabienne B. , Muller Claude P. , Yusibov Vidadi , Mett Vadim
Published: June 2021

The number of cases of acute Measles Virus (MV) in the United States plummeted 98% since 1963, when a live attenuated vaccine has been introduced. However, such a vaccine requires constant refrigeration to preserve potency. More importantly, the presence of maternal antibodies interferes with effectiveness of the vaccine before ~9 months of age. These reasons present a major impediment for the vaccination of young children, especially in developing countries. Here, we report the transient expression of two MV hemagglutinin epitopes in Nicotiana benthamiana. These loop‐ (L) and helix‐ (H) forming epitopes are not recognized by maternal antibodies, have been shown to generate a protective immune response in mice, and could be used to develop a vaccine for infants before the age of 9 months. The epitopes were fused to the surface loop of the thermostable enzyme of Clostridium thermocellum, and produced in plants through a combination of viral and bacterial vectors. Purified antigens were characterized by SDS‐PAGE followed by immunoblotting. Immunological testing showed that the L‐ and H‐epitopes are recognized by L‐ and H‐specific antibodies, respectively, thus successfully mimicking natural antigenic properties.
Effects of interferon‐γ and Francisella tularensis elicited soluble factors on macrophage antigen processing
Authors: Katkere Bhuvana , Wilson Justin E , Drake James R
Published: June 2021

Francisella tularensis is an intracellular pathogen that survives and replicates within the cytoplasm of macrophages (MØ) upon escape from the phagosome. In interferon‐γ (IFN‐γ) activated MØ, F.tularensis is unable to escape the phagosome and is directed to the endosomal‐lysosomal degradation pathway. IFN‐γ also up‐regulates MHC class II expression, but it is as yet unclear how it influences MØ antigen processing. In a cell line where class II expression is under the control of an IFN‐γ non‐responsive promoter, it was observed that IFN‐γ activation results in changes in the antigen processing and presentation pathway, leading to altered the T‐cell activation and cytokine production. Furthermore, IFN‐γ suppresses the fluid‐phase antigen uptake, but fails to alter Fc‐receptor‐mediated antigen uptake. These results suggest that IFN‐γ differentially alters the antigen uptake, to target antigen for differential processing and presentation. Interestingly, supernatant from F. tularensis infected MØ contain one or more soluble factors, which suppresses fluid‐phase antigen uptake, perhaps to alter antigen processing and presentation to skew T‐cell responses.

PO1 # AI‐056320
Two different human Th17 cells defined by chemokine receptor expression
Authors: Sato Wakiro , Aranami Toshimasa , Yamamura Takashi
Published: June 2021

Objective: IL‐17‐producing CD4+ T cells (Th17 cells) have emerged as a T cell subset that may be pivotal in the pathogenesis of autoimmune diseases. We previously reported that human Th17 cells are distinct from Th1 cells in their chemokine receptor expression; Th17 cells as CCR2+CCR5−, whereas Th1 cells as CCR5+ (J. Immunol. 178:7525–7529, 2007). But other reports demonstrated that human Th17 cells are CCR4+CCR6+. We analyzed the relationship between the two populations.

Method: Healthy human PBMCs (n = 6) were stained with CCR2, CCR4, CCR5 and CCR6 as well as CD45RA and CD4 and analyzed by FACS. CCR2+CCR5− and CCR4+CCR6+ memory CD4+ T cell subpopulations were sorted by FACS. Their IL‐17‐producing capacity was measured by ELISA.

Result: The frequency of CCR2+CCR5− subpopulations within memory CD4+ T cells was 2.2+/−2.0%, whereas that of CCR4+CCT6+ was 16.8+/−6.9%. 24.7+/−8.8% of CCR2+CCR5− cells were CCR4+CCR6+, whereas only 3.0+/−2.5% of CCR4+CCR6+ cells were CCR2+CCR5−. The IL‐17 production level of both subpopulations was generally similar.

Conclusion: CCR2+CCR5− cells and CCR4+CCR6+ cells basically do not overlap, suggesting that each population may function at different inflammatory conditions.
Functional characterization of chicken proinflammatory cytokine IL‐17D
Authors: Lillehoj Hyun Soon , Hong Yeong Ho , Han Jae Young , Kim Jin Kyu
Published: June 2021

In order to characterize the proinflammatory cytokine IL‐17D, full‐length cDNA was cloned from a testis cDNA library and its biological function investigated. The full‐length chicken IL‐17D (chIL‐17D) cDNA consisted of a 348 nucleotide sequence encoding an open reading frame of 116 amino acids with a predicted molecular mass of 17.0 kDa. Comparison of the deduced amino acid sequence of chIL‐17D with homologous proteins from human, mouse and opossum revealed 64%, 53% and 76% identity respectively, including six conserved cysteine residues present in the mammalian polypeptides. The chIL‐17D gene transcript was expressed in a wide range of tissues, and highest levels were in pancreas, thymus and lung. Following Eimeria maxima infection, levels of the chIL‐17D mRNA were up‐regulated in the intestinal jejunum, bursa, lung, and spleen but decreased in the thymus. Infected chickens also expressed greater levels of chIL‐17D mRNA in CD4+, CD8+ and TCR1+ intestinal intraepithelial lymphocytes while decreased expression was seen in TCR2+ cells. Treatment of CHCC‐OU2 fibroblasts with chIL‐17D recombinant protein induced the expression of IL‐6 and IL‐8. Collectively, these results suggest that chL‐17D has structural and functional similarities to mammalian IL‐17Ds and that it plays an important role in local gut innate immune responses during experimental coccidiosis.
Human T cells that are able to produce IL‐17 express the chemokine receptor CCR6
Authors: Singh Satya , Zhang Hongwei , Foley John , Hedrick Michael , Farber Joshua
Published: June 2021

A new lineage of effector/memory CD4+ T cells has been identified whose signature products are IL‐17 cytokines and whose differentiation requires the nuclear receptor, RORγt. These Th17 cells are critical effectors in mouse models of autoimmune disease. We have analyzed the association between chemokine receptor expression and IL‐17 production for human T cells. Activating cord blood (naïve) CD4+ T cells under conditions driving Th17 differentiation led to preferential induction of CCR6, CCR9 and CXCR6. Despite these data, we found no strong correlation between the production of IL‐17 and expression of CCR9 or CXCR6. By contrast, virtually all IL‐17‐producing CD4+ T cells, either made in our in vitro or found in peripheral blood, expressed CCR6. Compared with CD4+CD45RO+CCR6− cells, CD4+CD45RO+CCR6+ cells contained at least 100‐fold more IL‐17A mRNA and secreted 100‐fold more IL‐17 protein. The CCR6+ cells showed a similar enrichment in mRNA for RORγt. CCR6 was likewise expressed on all IL‐17‐producing CD8+ PBL. CCR6 has been associated with the trafficking of T, B, and dendritic cells to epithelial sites, but has not been linked to a specific T cell phenotype. Our data reveal a fundamental feature of IL‐17‐producing human T cells and a novel role for CCR6, suggesting both new directions for investigating IL‐17‐related immune responses and possible targets for preventing inflammatory injury. Research support: NIAID, NIH
Determination of the functional biology of IL‐25
Authors: Saenz Steven A. , Zaph Colby , Cua Daniel J. , Kastelein Robert A. , Artis David
Published: June 2021

Interleukin (IL)‐25 (IL‐17E) is a recently described member of the IL‐17 cytokine family. Recent studies have demonstrated key functions of IL‐25 in TH2 cytokine‐mediated host protective immunity and in the inhibition of IL‐17‐mediated inflammation. Despite a better understanding of its biological relevance, little is known about the cellular sources and targets of IL‐25 or the factors that regulate its expression. Using mice that express β‐galactosidase from the IL‐25 locus (IL25+/lacZ mice), we identified that gut‐resident CD4+ and CD8+ T cells are the major producers of IL‐25 in vivo. To address what cytokines might drive the differentiation of IL‐25‐producing T cells, in vitro cultures of cells from IL25+/lacZ mice were polyclonally stimulated in the presence of a variety of cytokines. Increased IL‐25 expression was observed in the presence of IL‐4, IL‐6, and TNFα. A better understanding of the regulatory pathways controlling the expression of IL‐25 may provide the rationale for targeted therapeutics for multiple inflammatory diseases.
LFA‐1 engagement‐induced T cell cytokine mRNA stabilization involves a Rac, p38 MAP kinase, MK2 cascade
Authors: Rao Gautham K. , Ramgolam Vinod S. , DeGregorio Scott D. , Chi Hongbo , Flavell Richard A. , Gaestel Matthias , Collinge Mark , Bender Jeffrey R.
Published: June 2021

We have demonstrated that engagement of the β2 integrin, lymphocyte function‐associated antigen‐1 (LFA‐1), results in stabilization of Th1 mRNA transcripts containing 3′‐untranslated region AU‐rich elements (AREs), including those encoding IFN‐γ and TNF‐α, requiring an induced nuclear‐to‐cytosolic translocation of the ARE‐binding protein HuR. We now show that LFA‐1 engagement by its ligand, ICAM‐1, results in PI‐3 kinase (PI3K)‐dependent activation of the Rho family GTPases Rac‐1 and ‐2 in HSB‐2 T cells. Pretreatment with the PI3K inhibitor LY294002, or Rac‐1/2 knockdown, resulted in attenuation of LFA‐1‐induced TNF‐α mRNA stabilization. LFA‐1‐stimulated HuR translocation and IFN‐γ mRNA stabilization was absent in T cells isolated from the Rac target MKK3 gene‐deleted mice. Moreover, LFA‐1‐stimulated activation of p38 and its target, MK2, are required for induced TNF‐α mRNA stabilization, which was abrogated by the p38 inhibitor SB203580, and absent in T cells obtained from MK2−/− mice. These results demonstrate that a sequentially activated PI3K, Rac, MKK3, p38, MK2 cascade drives T cell LFA‐1‐induced Th1 cytokine mRNA stabilization. (Supported by NIH grant RO1 HL043331 and the Raymond and Beverly Sackler Foundation)
Novel NF‐κB‐mediated regulation of IFN‐α production by plasmacytoid dendritic cells upon stimulation of CpG‐A and CpG‐B
Authors: Cui Chongwei , Chung Eugene , George Thaddeus C , Singh Sukhwinder , Fitzgerald‐Bocarsly Patricia
Published: June 2021

Two distinct types of CpG oligodeoxynucleotides have been identified that differ in their capacity to stimulate plasmacytoid dendritic cells (PDC): CpG‐A, but not CpG‐B, induces high amounts of IFN‐α, whereas CpG‐B induces PDC maturation. Here, we demonstrate that upon the inhibition of NF‐κB activation by SN50 or NBD, CpG‐B induced high amounts of IFN‐α, while CpG‐A‐induced IFN‐α was decreased. In addition, CpG‐B‐induced maturation was inhibited by NF‐κB inhibition. Using the new ImageStream imaging flow cytometry technology, we found that CpG‐B has a stronger ability to induce NF‐κB translocation in PDC than CpG‐A. Although both CpG‐A and CpG‐B‐induced NF‐κB translocation were inhibited by SN50, the inhibitory effect on CpG‐B‐induced translocation was more pronounced than with CpG‐A. Given this novel role for NF‐κB signaling in IFN‐α production, we tested whether there is a correlation between NF‐κB and IFN‐regulatory factor 7 (IRF‐7), the master regulator of IFN‐α production, activation. In contrast to the inability of CpG‐B to induce IRF‐7 translocation, we found that CpG‐A‐induced IRF‐7 translocation occurred at 5–6 hrs after stimulation. However, with the inhibition of NF‐κB translocation, CpG‐B induced high levels of IRF‐7 translocation after 1hr. These results suggest that NF‐κB plays an inverse role in the regulation of IFN‐α production by PDC upon stimulation with CpG‐A or CpG‐B.

Supported by AI26806.
TNF‐α synthesis is differentially regulated by NFATc1 and NFATc2 in T cells
Authors: Kaminuma Osamu , Kitamura Fujiko , Kitamura Noriko , Miyatake Shoichiro , Hiroi Takachika
Published: June 2021

The NFAT family transcription factors play important roles in T cell cytokine synthesis; however, the functional differences among NFAT members have not been fully elucidated. We investigated the relative contribution of NFATc1 and NFATc2 to transactivation of cytokine genes in T cells. Enforced expression of NFATc2 but not NFATc1, especially its short isoform, augmented TNF‐α synthesis in human T cells at the gene transcription level, whereas both NFATs enhanced IL‐2 expression. Moreover, a reduction of the shortest NFATc1 isoform using RNA interference technology failed to diminish TNF‐α expression. The promoter activity of the NFATbinding site in the TNF‐α gene was upregulated by NFATc2 but not by NFATc1, whereas both NFATs equivalently associated with this region. Messenger RNA expression study employing NFATc1/NFATc2 chimeric molecules revealed that the enhancing activity of NFAT on the TNF‐α gene was lost by truncation of its Cterminal transactivation domain. Furthermore, this domain derived from NFATc2 behaved as a dominant negative against the NFAT site in TNF‐α promoter‐dependent transcriptional activity. In conclusion, the C‐terminal transactivation domain in NFAT is crucial for TNF‐α gene expression in human T cells.
A cytokine known as MIF or GIF undergoes posttranslational modification and suppresses CD4 cell‐dependent immune responses
Authors: Kim Mijwa , Janssen Edith , Black Roy A , Sugie Katsuji
Published: June 2021

MIF is believed to be a key proinflammatory cytokine that activates the innate immunity. CD74/CD44, CXCR2 and CXCR4 have been reported to be MIF receptors. However, recombinant MIF demonstrates no cytokine activity when contaminating LPS is rigorously removed. The susceptibility of MIF−/− mice to endotoxin shock varies among reports. Here we show that this cytokine, posttranslationally modified and secreted from CD4 cells, suppresses Th effector generation. The MIF protein was also designated GIF. GIF/MIF−/− mice showed augmented T‐dependent antibody formation, especially of IgE isotype, and germinal center reactions although T‐independent antibody formation was normal. GIF−/− mice demonstrated enhanced allergic airway inflammation. More IL‐4‐ and IFN‐γ‐secreting CD4 cells were detected in GIF−/− than +/+ mice after Ova immunization. GIF−/− dendritic cells and macrophages exhibited normal antigen‐presenting capacity and responsiveness to TLR ligands. However, GIF−/− CD4 cells produced enhanced amounts of IL‐4 in vitro. GIF undergoes cysteinylation at C60 in the course of secretion. CD25‐CD4+ cells were the major source of cysteinylated GIF. rGIF modified at C60 inhibited IL‐4 production in vitro, whereas unmodified GIF had no effect. Thus non‐Treg CD4 cells posttranslationally modify and secrete this cytokine as a negative feedback of Th differentiation. Supported by NIH RO1 AI56211.
STAP‐2 regulates integrin‐mediated T‐cell adhesion through protein degradation of FAK
Authors: Matsuda Tadashi , Ikeda Osamu , Sekine Yuichi
Published: June 2021

Signal transducing adaptor protein‐2 (STAP‐2) is a recently identified adaptor protein that contains Pleckstrin and Src homology 2‐like domains as well as a YXXQ motif in its C‐terminal region. Our previous studies demonstrated that STAP‐2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, we find that STAP‐2‐deficient splenocytes or T‐cells exhibit enhanced cell adhesion to fibronectin (FN) after phorbol myristate acetate‐treatment, and that STAP‐2‐deficient splenocytes contain the increased protein contents of focal adhesion kinase (FAK). Furthermore, overexpression of STAP‐2 induces a dramatic decrease in the protein contents of FAK and integrin‐mediated T‐cell adherence to FN in Jurkat T‐cells via the degradation of FAK. Regarding the mechanism for this effect, we find that STAP‐2 associated with FAK and enhanced its degradation, proteasome inhibitors blocked FAK degradation, and STAP‐2 recruits an endogenous E3‐ubiquitin ligase, Cbl, to FAK. These results reveal a novel regulation mechanism for integrin‐mediated signaling in T‐cells via STAP‐2, which directly interacts with and degrades FAK.
Signaling through the PSGL‐1 cytoplasmic domain to activate β2‐integrin‐mediated slow rolling of neutrophils
Authors: Miner Jonathan J , Xia Lijun , Yago Tadayuki , Shao Bojing , Klopocki Arkadiusz G , Setiadi Hendra , McEver Rodger P
Published: June 2021

Interactions of selectins with the leukocyte mucin P‐selectin glycoprotein ligand‐1 (PSGL‐1) mediate rolling on vascular surfaces. Engagement of PSGL‐1 during neutrophil rolling on P‐ or E‐selectin activates integrin LFA‐1 to slow rolling on ICAM‐1 by a pathway that requires the tyrosine kinase Syk. Based primarily on studies of cell lines, the cytoplasmic domain has been suggested to localize PSGL‐1 in uropods of polarized cells, to be required for rolling, and to mediate signaling through Syk or the lipid kinase PI3Kδ. To study the functions of the cytoplasmic domain in primary leukocytes, we generated mice expressing PSGL‐1 without the cytoplasmic domain. Surprisingly, leukocytes from these mice localized PSGL‐1 normally to microvilli, uropods, and lipid rafts, and they rolled on P‐selectin under flow. However, neutrophils expressing PSGL‐1 without the cytoplasmic domain did not exhibit LFA‐1‐mediated slow rolling on P‐selectin and ICAM‐1. PSGL‐1 signaling to slow rolling of wild‐type neutrophils required lipid rafts, Src family kinases, Syk, and the Tec kinase Btk, but not PI3K. These data demonstrate that the cytoplasmic domain of PSGL‐1 is dispensable for membrane localization and for rolling on P‐selectin, but is required to initiate a Syk‐dependent, PI3K‐independent pathway to activate LFA‐1 to slow neutrophil rolling on ICAM‐1.
THE ROLE OF GRANULOCYTE‐MACROPHAGE COLONY STIMULATING FACTOR (GM‐CSF) IN TNBS‐COLITIS MODEL
Authors: Khajah Maitham Abbas , McCafferty Donna‐Marie
Published: June 2021

Background: GM‐CSF is a hematopoietic cytokine that promotes neutrophil proliferation and maturation, promotes survival, and primes them for enhanced functions. It is produced by a variety of cell types including endothelial cellsand macrophages. Aim: in this study, we examined the role of GM‐CSF in a chemically‐induced model of colitis; 2,4,6‐trinitrobenzene sulphonic acid (TNBS). Methods: colitis was induced by a single intrarectal injection of TNBS(4mg, 20% ethanol) at day 1. Animals were treated with a single i.p injection of neutralizing anti‐GM‐CSF antibody (100μg/mouse) on day 1, or multiple injections on day 1,2,3, and 4, and killed on day 5 post‐induction of colitis. Control mice were injected with i.p NaCl. The role of GM‐CSF in neutrophil chemotaxis in vitro was also examined. Results: single injection of GM‐CSF antibody did not change the macroscopic score of inflammation and MPO activity between antibody treated and control mice. In contrast, by using multiple injections of GM‐CSF antibody, TNBS injection induced a significant increase in the macroscopic score of inflammation and colonic MPO activity in control mice, but mice treated with anti‐GM‐CSF antibody had a significantly reduced score. Real time‐PCR showed significantly enhanced mRNA expression of GM‐CSF in the colon at day 3 and 7 post‐induction of colitis compared to saline treated mice. A significant increase in WT neutrophil chemotaxis toward GM‐CSF was seen in the under‐agarose and the Boyden chamber assay. This neutrophil chemotactic response was PI3K dependent. Conclusion: 1‐GM‐CSF induces neutrophil chemotaxis in vitro through a PI3K‐dependent pathway. 2‐TNBS induces acute inflammation in mice in vivo, and GM‐CSF neutralization ameliorates colitis severity.

3‐GM‐CSF may play a role in neutrophil recruitment to the gut.
Molecular effects to human regulatory T cells by treatment with T cell activation inhibitor and TNF blockade
Authors: Suzuki Katsuya , Setoyama Yumiko , Suzuki Miyuki , Keiko Yoshimoto , Kameda Hideto , Tsuzaka Kensei , Amano Koichi , Takeuchi Tsutomu
Published: June 2021

Objective: Regulatory T cell (Treg) has a critical role for human autoimmunity. However, it has not been known how clinically effective therapeutic agents for autoimmune diseases, such as T cell activation inhibitor and TNF blockade affect to Treg in molecular level.

Methods: Human CD4+T cells were purified and then co‐cultured with these agents (tacrolimus or infliximab) under anti‐CD3/CD28 antibody stimulation. The cells were harvested and FOXP3+Tregs and its related molecules were analyzed by flow cytometry. Next, DNA microarray analysis of CD4+CD25+CD127‐Treg by in vitro treatment was performed.

Results: Tacrolimus significantly reduced CD62L low expressed inducible Tregs in dose dependent manner. In DNA microarray analysis, we have identified significantly up and down regulated 1,021 (tacrolimus) and 621 (infliximab) genes. Gene ontology analysis has also revealed significantly affected 15 gene groups, such as multicellular organismal process in both treatments.

Conclusions: Immunological therapeutic intervention obviously affects to human peripheral Tregs in vitro. Its clinical significance should be required further studies in vivo.
Longitudinal study of B cell depletion and recovery in mice after fucose‐free anti‐CD19 antibody treatment
Authors: Wang Yue , Gallagher Sandra , Groves C , Sims GP , Kuta E , Rowe D , Ward E , Mittereder N , Carlesso G , Cheng L , Cook K , Tedder TF , Damschroeder M , DallAcqua W , Kiener P , Coyle AJ , Herbst R
Published: June 2021

Depletion of B cells by anti‐CD20 has generated clinical benefits in autoimmune diseases, however, the depletion is sometimes incomplete or transient. Compared to CD20, CD19 is expressed by a broader range of B cells. Therefore, an anti‐CD19 antibody has been developed as an alternative B cell depleting agent. This antibody, anti‐CD19‐aFuc, is engineered with BioWa's Potelligent® Technology to be free of fucose at the Fc portion, which enhances the binding affinity to Fc receptors and exhibits increased ADCC activity. In vivo, the anti‐CD19‐aFuc antibody effectively depleted the majority of B cells in human CD19 transgenic mice. The depletion of circulating B cells was sustained for >10 weeks after receiving 250ug anti‐CD19‐aFuc antibody. An additional 4~6 weeks were required for the recovered B cells to reach similar quantity and maturity as in IgG control‐treated animals. Serum BAFF levels increased after depletion of B cells and returned to base line when B cells were fully recovered. Whereas serum immunoglobulins steadily increased in the control mice, the increases were blocked by the treatment with anti‐CD19‐aFuc. In summary, treatment of mice with anti‐CD19‐aFuc resulted in profound and sustained depletion of B cells and potently inhibited antibody responses. Targeting B cells with anti‐CD19 may be a more effective strategy for the treatment of a number of B cell malignancies and certain autoimmune disorders.
Role of IL‐21R in the pathogenesis of murine models of SLE
Authors: Andreyeva Tatyana A , Guay Heath , Damphousse Christie , Bloom Laird , Olland Stephane , Brown Tom , Nickerson‐Nutter Cheryl , Collins Mary , Young Debbie
Published: June 2021

IL‐21 is a pleiotropic type I cytokine that is secreted by activated CD4 T cells. IL21 has been found to induce apoptosis in resting B cells but promotes class switch recombination and plasma cell differentiation of activated B cells. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a break down in both T and B cell tolerance, and the development of anti‐nuclear antibodies. Generation of high titers of IgG antibodies reactive with dsDNA coincides with disease progression. Thus, factors that promote B cell activation and antibody production, such as IL‐21, may be important for the development of SLE. We examined the role of IL‐21R in the cGVHD model of lupus‐like disease using IL‐21R deficient mice. Cells from bm12 mice were transferred into C57Bl/6 mice or into IL21R KO mice on the C57Bl/6 background to determine whether the lack of IL‐21R in the host would affect antibody production, immune complex deposition, and kidney pathology. All of these parameters were decreased in the IL21R KO recipient mice. We also show that treatment of MRL‐Faslpr mice with an antagonistic anti‐IL‐21R antibody significantly reduced anti‐dsDNA IgG serum antibody levels and prevented IC deposition and kidney pathology in these mice. Therefore, blockade of the IL‐21 pathway is a potential therapeutic target for the treatment of human lupus.
IL‐4 and IL‐10 Synergize to Suppress Mast Cell Development and IgE Receptor Expression
Authors: Kolawole Motunrayo , Speiran Kelly , Bailey Daniel , Ryan John
Published: June 2021

IL‐4 and IL‐10 are co‐expressed by Th2 cells and have the potential to regulate the mast cell allergic response. One means by which mast cell homeostasis can be regulated is at the level of progenitor development. We have previously found that IL‐4 or IL‐10 can induce apoptosis in developing mouse mast cells. In the current study we demonstrate that these cytokines have synergistic activity, potently killing developing mast cells. Both cytokines also reduced IgE receptor expression on developing mast cells, and suppressed IgE‐mediated cytokine production or histamine release. The related cytokines IL‐13 and IL‐22 had similar activities, though generally less potent than IL‐4 or IL‐10. Lastly, we found that endogenous IL‐4 and IL‐10 may regulate mast cell development, since bone marrow from either IL‐4‐deficient or IL‐10‐deficient mice yielded mast cells with higher IgE receptor expression levels than their wild type counterparts. Our data demonstrate that IL‐4 and IL‐10 control mast cell homeostasis in part by regulating progenitor survival and IgE receptor expression. Supported by NIH grant 1R01 AI59638.
Evaluation of proapototic transgenes to use in combination with Flt3L in an immune‐stimulatory gene therapy approach for Glioblastoma multiforme (GBM)
Authors: Candolfi Marianela , King Gwendalyn D , Muhammad AKM G , Puntel Mariana , Kroeger Kurt M , Liu Chunyan , Ahlsadeh Gabrielle E , Lerner Jonathan , Lee Sharon , Foulad David , Sato Katsuaki , Lowenstein Pedro R , Castro Maria G
Published: June 2021

We showed that Flt3L recruits and activates APCs within GBM. We tested adenoviral vectors expressing pro‐apoptotic transgenes [HSV1‐thymidine kinase (Ad‐TK), TNF‐á (Ad‐TNF‐á), FasL (Ad‐FasL) or TRAIL (Ad‐TRAIL)], that would release tumor antigens to be up taken by APCs recruited by Ad‐Flt3L for GBM immunotherapy. Ad‐TK, Ad‐FasL and Ad‐TRAIL exerted GBM cell death in vitro, but only Ad‐TK and, at a lesser extent Ad‐FasL, improved survival of GBM‐bearing rats. Also, Ad‐TRAIL and Ad‐FasL were very neurotoxic, while Ad‐TK did not induce any side effects. Thus, we treated large intracranial GBM in rats with Ad‐TK+Ad‐Flt3L, which significantly improved survival and induced antitumor immunological memory. Ad‐TK+Ad‐Flt3L induced intratumoral recruitment of APCs with features of immature pDCs (CD3−, CD4+, CD45R+ cells that were CD86− and MHCII−). In summary, TK is a very safe and efficient pro‐apoptotic strategy to induce GBM regression when combined with Ad‐Flt3L. Our results warrant further development of this approach for human GBM Phase I trials.

Support: NIH/NINDS R01 NS44556.01, R21‐NSO54143.01; UO1 NS052465.01; RO3 TW006273‐01 to M.G.C.; RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309‐01, and R21 NS047298‐01 to P.R.L; F32 NS058156.01 to M.C.
Cancer vaccine enhanced non‐tumor‐reactive CD8+ T cells exhibit a distinct molecular program associated with ‘division arrest anergy’
Authors: Beyer Marc , Mallmann Michael R. , Karbach Julia , Zander Thomas , Eggle Daniela , Classen Sabine , Debey‐Pascher Svenja , Jäger Elke , Schultze Joachim L.
Published: June 2021

Vaccination of cancer patients against tumor antigens can induce peptide‐specific but non‐tumor‐reactive (NTR) CD8+ T cells. We used NY‐ESO‐1 peptide‐specific CD8+ T cell clones to assess the molecular programs associated with lack of tumor‐reactivity. TCR sequences revealed that NTR and tumor‐reactive (TR) T cells are derived from distinct progenitors. NTR T cells are characterized by low avidity TCRs and lack of NY‐ESO‐1 tetramer binding. Most striking, NTR T cells are characterized by a molecular program associated with division arrest anergy with elevated expression of the inhibitory molecule p27kip1, high expression of CD25, CD7, and CD69 and modulated expression of coreceptors. NTR T cells also exhibit elevated expression of inhibitory molecules and reduced levels of transcription factors responsible for T cell activation. In vivo monitoring of the NTR CD8+ T cell clone using a TCR‐specific PCR revealed high prevalence of NTR T cell clones with even increased levels during vaccination.

In vivo expansion of NTR T cells that can only be quantified by combining classical T cell cloning approaches with quantitative PCR adds another level of complexity to tumor host interaction. Certainly, these novel findings need to be considered when developing future adoptive T cell transfer approaches and cancer vaccines.

Research support: Sofja Kovalevskaja Award (Alexander von Humboldt‐Foundation)
Nicotine impairs cancer vaccines
Authors: Nouri‐Shirazi Mahyar , Guinet Elisabeth , Zeng Menghua
Published: June 2021

Smokers constitute a majority of patients with cancer and hence, are frequently subjected to vaccination. Vaccines, designed to enhance host immunity against infectious agents and neoplastic cells, depend on functional dendritic cells (DCs), the body's key immune cells that are pivotal in the initiation immune responses. Our recent studies indicate that the presence of nicotine in the microenvironment severely affects the properties of DCs and impairs their ability to induce the development of effective immune responses. These observations raised some doubts on the ability of cancer vaccines to generate sufficient immune responses in smokers in order to mediate tumor regression or prevent tumor occurrence. Thus, in this study, we investigated whether the adverse effects of nicotine on DC system decrease the effectiveness of cancer vaccines in tumor‐bearing mice exposed to nicotine. We found that the vaccine effectively controls the tumor growth in control mice. However, the same vaccine provides little tumor regression in nicotine‐exposed mice as evidenced by a larger tumor mass. We also found that compared to control mice, lymphoid cells isolated from nicotine‐exposed mice produce up to 50% less IFN‐γ after re‐exposure to tumor antigen and exhibit markedly reduced CTL killing activity against inoculated tumor cells. Collectively, our data suggests that vaccines provide only partial protection in nicotine‐exposed individuals. Philip Morris USA Inc., and Philip Morris International.
All in one: VLP‐MUC1 vaccine for prevention and treatment of epithelial tumors
Authors: Pejawar‐Gaddy Sharmila , Viscidi Raphael , Bossis Ioannis , Finn Olivera J.
Published: June 2021

A promising target for immunotherapy, both for prophylactic and therapeutic vaccines, is the tumor antigen MUC1. Normally, the MUC1 glycoprotein is expressed at low levels on epithelial cells. However, MUC1 is hypoglycosylated and overexpressed on most adenocarcinomas of breast, pancreas, colon, ovary and lung. The goal of this project is to test a safe, practical and potentially very effective MUC1 vaccine using bovine papillomavirus like particles (BPV VLP) as a MUC1 delivery vehicle. BPV contains 2 capsid proteins, L1 and L2. L1 protein is made up of 12 β strands, 6 loops and 5 α helices and can self‐assemble into VLP. To enable conjugation of MUC1 and VLP, substitutions were made in the loop regions of the L1 molecule. Successful VLP formation following loop substitutions was achievable only in the HI loop where eight amino acids were replaced with negatively charged glutamic acid residues followed by a cysteine. Further, the MUC1 peptide was preceded by seven positively charged arginine and one cysteine residue. These substitutions allow the formation of a polyionic and a covalent bond that strengthens the conjugation between VLP and MUC1. Our preliminary results show that VLP‐MUC1 can cause the maturation of DC and that these DC can process and present the MUC1 peptide to a MUC1 specific T cell hybridoma. The potential of this vaccine to elicit an immune response, as well as tumor rejection is currently being evaluated.
Role of CD30/CD30L Mediated Signaling in the Cross‐talk of Natural Killer Cells and Dendritic Cells
Authors: Simhadri Vijaya Lakshmi , Hansen Hinrich P , Simhadri Venkateswara Rao , Reiners Katrin , Engert Andreas , Von Strandmann Elke Pogge
Published: June 2021

Natural killer (NK) cells are involved in innate immunity and also contribute to adaptive immunity, mediated through the cross‐talk with immature dendritic cells (iDCs). NK cells and iDCs play a major role at the site of inflammation. The activation of NK cells and maturation of iDCs is partially characterized on the molecular level, depends on the release of proinflammatory cytokines and receptor‐ligand interactions.

Activated NK cells express CD30, and its ligand CD30L (CD153) is expressed on iDCs. We describe that engagement of CD30L contributes to the activation and maturation of dendritic cells.

Stimulation of monocyte derived iDCs with immobilized CD30‐Fc results in the release of pro‐inflammatory cytokines TNFalpha, IL‐6 and IL‐8, and can be blocked by CD30 antibodies. Engagement of CD30L on iDCs leads to Reactive Oxygen Species (ROS) signaling; and the release of TNFalpha is blocked by MAP kinase inhibitors. Intriguingly, the interaction induced the expression of CD83, a specific marker for mature dendritic cells. We postulate that CD30‐CD30L interaction contributes to DC‐maturation, based on the induction of cytokine release.

The better understanding of the molecules involved in the NK‐DC cross‐talk will provide a basis for the development of immunotherapeutics modulating the activity of NK cells and DCs.

Research was supported by Deutsche Forschungsgemeinschaft (DFG) (STR 530/5‐1).
Particulate Yeast‐derived Beta‐Glucan Stimulates Anti‐tumor T Cell Responses via Modulating Tumor Microenvironment toward Th1 Response
Authors: Yan Jun , Liu Jingjing , Li Bing , Hansen Richard
Published: June 2021

β‐Glucans are glucose polymers produced by a variety of plants and microorganisms. Previous studies demonstrated that soluble yeast β‐glucans could bind to the lectin site of complement receptor 3 (CR3) and prime leukocyte CR3 to elicit cytotoxicity of iC3b‐opsonized tumors. In this study, we investigated the mechanism of action for particulate yeast β‐glucan in tumor immunotherapy. Both in vitro and in vivo studies showed that particulate β‐glucan, but not soluble β‐glucan, stimulated DCs for up‐regulation of surface accessory molecules and production of cytokines such as IL‐12 and TNF‐α. Orally administered particulate β‐glucan treatment was shown to induce enhanced antigen‐specific CD4 and CD8 T cell responses. Oral particulate β‐glucan treatment also activated tumor‐infiltrating DCs and modulated cytokine profile within the tumor milieu, predominately upregulation of IL‐12 and IFN‐γ secretion. Tumor‐bearing mice treated with particulate β‐glucan alone had a significant reduced tumor burden with respect to mice treated with saline. The enhanced anti‐tumor responses correlated with increased IFN‐γ‐producing T cells and augmented CD8 T cell‐mediated cytotoxic activity. Taken together, we demonstrate that particulate β‐glucan has its unique function in eliciting potent anti‐tumor adaptive immune responses via regulating the tumor microenvironment. (This work was supported by NIH/NCI RO1 CA86412)
Integrin alpha V polymorphisms and haplotypes were associated with susceptibility to chronic hepatitis B and hepatocellular carcinama in Korean population
Authors: Kwack KyuBum , Yi Cheong‐Ho , Kim Min‐Ho , Cheong Jae Youn , Cho Sung Won , Kang Jason Jongho , Yang Song‐Ju , Lee Seung Ku
Published: June 2021

Integrins and ECM proteins are highly expressed in chronic liver disease including chronic hepatitis (CH), liver cirrhosis, and hepatocellular carcinoma (HCC). Integrins are cell surface receptors for the extracellular matrix (ECM) proteins, which has been identified as regulators of cancer progression and potent candidate for a cancer treatment. In this study, we investigated whether single nucleotide polymorphisms (SNPs) of integrin alpha V (ITGAV) are associated with CH and HCC in Korean population. Thirteen SNPs of ITGAV were genotyped using Illumina Golden‐gate genotyping assays by allele specific extension. We found that ITGAV intron SNP, 3′ untranslated region SNP, and three haplotypes were significantly associated with the susceptibility of CH or HCC in genetic models (P=0.017~0.048). Whereas, the ht 1 was significantly associated with insusceptibility in CH (P=0.015) but was not HCC. Our results suggest that ITGAV SNPs and haplotype might be associated with the susceptibility of CH and HCC.
Mediating factors in the relationship between ethnicity and infant feeding intentions
Authors: Nommsen‐Rivers Laurie Ann , Chantry Caroline J. , Dewey Kathryn G.
Published: June 2021

Our objective was to determine if modifiable factors mediate the often‐reported relationship between ethnicity and breastfeeding (BF) practices. We interviewed 485 nulliparae at 34–40 wk gestation regarding: exposure to BF by others (BFE): never, a few times, often; comfort with idea of BF (BFC) and idea of formula‐feeding (FFC): very uncomfortable=1, to very comfortable=4; and BF self‐efficacy (BFSE). The Infant Feeding Intentions (IFI) Scale measured strength of intentions to exclusively BF. Subjects were 42% white, 23% Hispanic, 16% black, 13% Asian and 6% mixed. In a cumulative odds logit model with education level (ED), the adjusted odds (AOR [95% CI]) of being in a higher IFI category were 0.55 [0.32–0.93] for black vs. white women (not significant for other ethnicities). Black women had higher levels of FFC (AOR [CI] 1.94 [1.13–3.31]), but similar levels of BFC (1.33 [0.74–2.38]). Overall, increased BFE was predictive of higher BFC and BFSE, and lower FFC (p<.0001 for all). FFC, BFC, BFSE and ED explained 42% of variation in IFI (p<.0001), with FFC the strongest predictor: AOR of higher IFI level tripled with each successive decrease in FFC level (p<.001). Ethnicity was not significant when added to this model. Thus, modifiable factors mediate the relationship between ethnicity and IFI. Campaigns stressing risks of FF may be more effective than those stressing BF benefits for increasing BF rates.
Rice consumption is associated with a greater nutrient density, lower blood pressure, decreased body weight, smaller waist circumference and a reduced risk for metabolic syndrome in adults
Authors: Fulgoni Victor L , Fulgoni Sally A , Upton Julie L , Moon Maggie
Published: June 2021

The association between rice consumption, dietary intake and physiological parameters was examined in adults using data from the National Health and Nutrition Examination Survey (1999–2004). Rice consumers (n=1,840) were defined as individuals who reported consuming at least 14 g of white rice, brown rice, or rice flour during a 24‐hour dietary recall. When compared to non‐consumers, adult (19–50 years) rice consumers had significantly (p<0.001) lower total fat (82 vs 90 g), saturated fat (26 vs 30 g), and added sugar intake (23 vs 27 tsp). Subjects consuming rice had a greater intake of over 12 essential vitamins and minerals, including iron (18 vs 16 mg), total folate (692 vs 541 DFE), vitamin B6 (2.1 vs 1.9 mg), niacin (27 vs 24 mg), and greater intakes of total grains (8 vs 7 servings), total fruits (1.1 vs 0.9 servings) and legumes (0.21 vs 0.10 servings) relative to non‐consumers. Rice consumers had significantly (p<0.01) lower body weights (79 vs 81 kg), smaller waist circumferences (92.6 vs 94.4 cm), and lower systolic (115 vs 117 mm Hg) and diastolic (72 vs 73 mm Hg) blood pressure in comparison to non‐consumers. In addition, rice users had a 21% reduced risk of metabolic syndrome (p=0.029). Therefore, consumption of rice in adults was associated with better nutrient intakes and increased intake of several important food groups. In addition, rice consumption was associated with lower systolic and diastolic blood pressure, decreased body weights, and smaller waist circumferences, all of which are important factors in reducing the risk of metabolic syndrome. Supported by the USA Rice Federation.
Omega‐3 fatty acid food intakes of urban Midwestern Latino women
Authors: Lora Karina Regina , Lewis Nancy M
Published: June 2021

The purpose of this study is to document on omega‐3 fatty acids (ALA, EPA, DPA, DHA) food intakes of urban Midwestern Latino women as part of the validation of a culturally appropriate food frequency questionnaire to measure omega‐3 fatty acid food intakes in this population. Fifty apparently healthy Latino women, aged 20 to 50 y, who were not pregnant or lactating at the time of the study, born in a Latin American country and living in an urban Midwestern city served as participants. The women were interviewed to complete a sociodemographic and a food frequency questionnaire (FFQ) containing 194 items. The FFQ developed from one‐on‐one interviews included 14 prepared ethnic dishes. Fifty two percent of the women were in the 20 to 30 y range, 58% were born in Mexico, 42% had high school education or less, 88% reported Spanish to be the language spoken at home, and 52% had lived in the U.S. from 0 to 5 years. Daily ALA, EPA, DPA, DHA and total omega‐3 intake (mean ± SD) in grams of the participants were 1.87 ± 0.94, 0.05 ± 0.04, 0.02 ± 0.02, 0.10 ± 0.08, and 2.04 ± 1.02 respectively. Principal omega‐3 food sources were beef, tacos dorados, vegetable oil, walnuts, canola oil, mayonnaise and salmon. Daily ALA and total omega‐3 intake were negatively correlated with age, daily EPA intake was positively correlated with household income, and daily DPA intake was positively correlated with language spoken at home (P<0.05). Daily DPA and DHA intake were positively correlated with household income (P<0.01). These Latino women had higher mean intake than the Adequate Intake for ALA for women >19 y. Household income, an indicator of socioeconomic status, was related to intake of EPA, DPA and DHA. Supported by Tobacco Funds.