bluuebunny/crossref_metadata_2025 huggingface dataset:
A model for mapping the fate of CD8+ T cells that have expressed granzyme B (GzmB)
Authors: Bannard Oliver Michael , Fearon Douglas
Published: June 2021

Commitment to effector differentiation of the CD8+ T cell may be associated with senescence. To define when this occurs, it is necessary to indelibly mark a cell during development and follow its fate without artificial interventions. We have generated a BAC transgenic mouse line that expresses a tamoxifen‐regulated Cre/ERT2 recombinase controlled by transcriptional elements of the effector gene, Gzmb. Crossing these mice with the R26R‐EYFP Cre reporter line permits conditional, permanent expression of EYFP in cells that had transcribed Gzmb. Stimulation in vitro under GzmB‐inducing conditions caused expression of EYFP in up to 50% of GzmB+ CD8+ T cells from these mice, only in GzmB+ cells, and only with tamoxifen. Influenza infection caused tamoxifen‐dependent EYFP expression by 21% and 31% of nucleoprotein (NP)‐specific CD8+ T cells in the spleen and lung, all of which were GzmB+. Six weeks post‐infection the splenic number decreased to 15%, all EYFP+ cells were now GzmB−, and were CD62L high or low. After secondary infection without tamoxifen, the number of EYFP+ CD8+ T cells in the lung increased ∼100‐fold, indicating a capacity for clonal expansion despite prior expression of GzmB. Thus, we now have a model with which we can map the fate of CD8+ T cells that have transcribed Gzmb at a specified time during repetitive or persistent viral infections, irrespective of continued expression of the gene. Wellcome Trust.
The role of GITR in the anti‐influenza CD8 memory T cell response
Authors: Snell Laura M , Lin Gloria HY , Riccardi Carlos , Pandolfi Pier P , Watts Tania H
Published: June 2021

Tumor necrosis receptor associated factor 1 (TRAF1) is an adaptor protein recruited to several costimulatory tumor necrosis factor receptor (TNFR) superfamily members, including 4‐1BB, CD30 and glucocorticoid‐induced TNF receptor family‐related gene (GITR). Work from our lab has shown that in the absence of TRAF1, a 7–8 fold defect in antigen‐independent memory CD8 T cell survival resulted 3 weeks following transfer into naïve mice. Under similar conditions, the lack of 4‐1BBL results in a 2–3 fold defect, suggesting that other than 4‐1BB at least 1 other TRAF1‐linked TNFR family member may contribute to CD8 T cell survival. Here we show that IL‐15, a cytokine involved in memory T cell survival, induces the co‐expression of GITR and 4‐1BB on CD8 memory T cells, suggesting a possible synergy between these receptors. In order to test the role of GITR on CD8 memory T cells in vivo we infected wildtype and GITR−/− mice intranasally with influenza A/X31. Whereas similar numbers of influenza specific CD8 T cells were observed in the lungs and draining lymph nodes of WT and GITR−/− mice at the peak of the primary response, reduced numbers were seen in the spleen of GITR−/− mice at this timepoint. When mice were challenged with influenza A/PR8 30 days after priming, GITR−/− mice showed decreased numbers of influenza specific CD8 T cells in the lung with a similar trend observed in draining LN. This defect is being investigated further using a transgenic model where GITR is absent only on CD8 T cells. This study was supported by a grant from the Canadian Institute of Health Research to T.H.W. L.M.S. is the recipient of a doctorate research award from the Fonds de la recherche en santé du Québec.
B cell selection is a function of the post‐natal thymus
Authors: Abuattieh Mouhammed Omar , Platt Jeffrey L , Cascalho Marilia
Published: June 2021

Children with severe heart disease early in life may undergo cardiac transplantation in infancy. Attendant to that procedure, the thymus is removed in whole or part and mature T cells are depleted. These manipulations should cause the T cell repertoire to be severely contracted. Surprisingly, however, those who undergo cardiac transplantation in infancy are not immunodeficient suggesting that the T cell compartment adapts in early life to absence of the thymus and depletion of T cells. Whether the “adapted” T cell compartment provides optimal help for antibody production and development and/or maintenance of B cell memory is not known. To answer that question we studied mice subjected to removal of the thymus and/or T cell depletion. The number and phenotype of T cells in adult thymectomized mice was comparable to controls. Thymectomy did not perturb the development of B cells or T‐independent type 2 antibody responses. In contrast, thymectomy did profoundly impair B cell memory as depicted by decreased production of long‐lived antibody secreting cells in the bone marrow, and decreased antibody affinity maturation. Antibodies produced by thymectomized mice were encoded by heavy chain genes with CDR3 regions longer by 2 or 3 amino‐acids than the sham operated controls. Our findings indicate an unprecedented role for the adult thymus in B cell memory and in the selection of the peripheral B cell repertoire.
Maintenance of Plasma Cell Pool is Independent of Memory B cells
Authors: Ahuja Anupama , Anderson Shannon M. , Khalil Ashraf , Shlomchik Mark
Published: June 2021

Humoral memory to an Ag is maintained for several decades in the form of memory B cells and serum Ab. In fact, plasma cells (PC) that secrete Ab are known to be long‐lived and could be solely responsible for maintaining the long‐lived Ab titers. Alternatively, continuous differentiation of memory cells into long‐lived PCs as a result of non‐specific stimulation could account for humoral memory. To test these possibilities, we used a novel mouse model system to deplete Ag‐specific memory B cells with an anti‐hCD20 mAb and determined the effect on the PC compartment over 16 weeks. Using a combination of surface markers, we demonstrated that memory B cells remained depleted over the course of the experiment. However, despite absence of memory cells for an extended duration, PC numbers in spleen and bone marrow did not decline. This indicates that the PC compartment does not require a significant contribution from memory B cells for its maintenance and instead that PCs are sufficiently long‐lived to maintain Ab titers over a long period without renewal. This settles an important controversy in B cell biology and has implications for the design of vaccines and for B cell depletion therapy in patients. Supported by NIH AI43603 and AR44077 to MJS; and Arthritis Foundation Postdoctoral Fellowship to AA.
Repertoire‐based selection during B cell development in polyclonal mice
Authors: Carey John , Moffatt‐Blue Chantelle , Watson Lisa , Gavin Amanda , Feeney Ann
Published: June 2021

Marginal zone (MZ) B cells resemble fetally‐derived B1 B cells in their innate‐like rapid responses to blood‐borne pathogens but the basis for this is unknown. H‐CDR3 sequence analysis reveals that 20% of MZ B cells in C57BL/6 mice possess fetally‐derived and fetal‐type BCR which lack N regions (N−). Using bone marrow chimeras, with adult TdT+/+ and TdT−/− donor cells, we demonstrate preferential repertoire‐based selection of N− fetal‐type B cells into the MZ, indicating a functional importance for these specificities. Furthermore, selection events in bone marrow and splenic transitional compartments show differential selection for these N+ and N− H‐CDR3 repertoires, with the N− repertoire being more enriched in Igλ expression. Entry into the splenic T1 compartment favors the N− repertoire. In contrast, the T3 population, which is enriched in anergic and autoreactive B cells, preferentially selects the adult‐type, N‐region containing, and Igκ repertoires. Our data suggest repertoire‐based bifurcations early and late in B cell differentiation and suggest novel differentiation pathways for MZ B cell development, based on H‐CDR3 and light chain differences.
Extracellular adenosine production is essential for Treg‐mediated Th cell suppression
Authors: Kurtz Courtney C. , Alam Mohammad S. , Ernst Peter B.
Published: June 2021

Background: Adenosine is an important inflammatory mediator that suppresses proliferation and cytokine production in helper T cells (Th). Two enzymes, CD39 and CD73, expressed on the surface of several cell types including Treg, synthesize adenosine. Expression of these enzymes on Tregs is suggested to play a role in their suppressive ability. Methods: In order to determine whether CD73 activity is involved in Treg‐mediated suppression, we assessed the ability of Treg from CD73−/− mice to suppress proliferation of wildtype and CD73−/− effector T cells (Teff) in vitro. Cytokine expression was examined in Th cells from wildtype and CD73−/− mice stimulated in vitro. To test the function of CD73 on Treg in vivo, Treg from CD73−/− mice were adoptively transferred with wildtype Teff into RAG1−/− recipients. Results: CD73−/− Treg were unable to suppress proliferation of either wildtype or CD73−/− effector T cells in vitro. In addition, production of inflammatory cytokines by CD73−/− Th cells was significantly greater than wildtype. Adoptive transfer of Treg from CD73−/− mice attenuated colitis to a lesser degree than wildtype Treg. Conclusions: These data indicate that extracellular adenosine production by CD73 plays an important role in suppressing Th cell proliferation and inflammatory cytokine production in vitro and inflammation in vivo. Supported by NIH R21 AI069880 and T32 AI007496‐12.
Human FOXP3+ T regulatory cells suppress mouse T cell activation by targeting mouse dendritic cells via a human LFA‐1/mouse ICAM‐1 mediated interaction
Authors: Tran Dat , Glass Deborah , Shevach Ethan
Published: June 2021

We have developed a novel in vitro T suppressor cell assay in which the responders are mouse CD4+CD25‐ T cells (mCD4+) stimulated by mouse T‐depleted splenocytes and soluble anti‐mouse CD3, and the T suppressors are human CD4+FOXP3+CD25hi cells (nTreg) activated with plate‐bound anti‐human CD3/CD28. Human nTreg were almost as efficient as mouse nTreg in suppressing the responses of mCD4+ cells. Suppression was not abrogated by neutralizing human IL‐10 or TGFbeta, and was not observed when responders and suppressors were separated in a transwell. Cell contact between the human nTreg and the mouse cells was required as blocking human CD11a or CD18 completely abrogated suppression. Mouse mCD4+ cells deficient in ICAM‐1 were suppressible, suggesting that the nTreg targeted the mouse dendritic cells through the binding of human LFA‐1 with mouse ICAM‐1. Mouse nTreg could not suppress human CD4+ T cells most likely due to the fact that mouse LFA‐1 could not bind to human ICAM. Under suboptimal stimulatory conditions, hIL‐2 was required for activation of nTreg suppressor function. Since the human IL‐2R does not bind mouse IL‐2, IL‐2 consumption is ruled out as a mechanism of suppression. Our results indicate that one of the mechanisms of nTreg‐mediated suppression functions across species and that this cross‐species assay should prove to be a valuable tool for studying the mechanisms of action of human nTreg.
Dynamics of Tolerance induction by B‐cell delivered Gene Therapy
Authors: Matheu Melanie , Su Yan , Parker Ian , Cahalan Michael , Scott David W.
Published: June 2021

B cells, functioning as APC's, can provide signals to T cells either activating immunity or inducing a specific hyporesponsive state (tolerance). Our laboratory has shown that B cells, when retrovirally transduced with peptide‐IgG, are highly tolerogenic for epitopes fused with the IgG. In this system, a DNA sequence encoding a full‐length protein or an immunodominant epitope is inserted into the N‐terminus of the murine IgG1 heavy chain. After injection into immunocompetent mice, peptide‐IgG transduced B cells are able to induce specific tolerance to the IgG associated antigen. This gene therapy approach has been applied to several animal models of autoimmune diseases, including EAU, EAE, and diabetes, as well as in hemophilia. In these disease models, specific tolerance was observed in both Th1 and Th2 responses. Moreover, our data indicate that the IgG heavy chain is critical for efficient tolerance induction and trafficking to endosomal processing pathways. We recently reported that activation with LPS, anti‐IgM or CD40L led to efficient tolerogenic presentation, but CpG‐activated B cells were not tolerogenic. While IL‐10 production by LPS‐activated B cells may play a role, analysis by two‐photon microscopy suggests that the LPS‐stimulated B cells interact with specific T cells longer, thus facilitating tolerogenic signals. We also propose that IgG promotes tolerance by co‐presentation of regulatory epitopes. (Supported by NIH grants AI035622 and GM41514)

MM and YS contributed equally to this work
Identification of a New Gene in the Mouse MHC Class II Gene Cluster
Authors: Chen Lili , David Chella , Jensen Peter , He Xiao
Published: June 2021

MHC Class II molecules are essential in antigen presentation and selection of the CD4+ T cell repertoire. They are mainly expressed on professional Antigen Presenting Cells (APC), i.e. B lymphocytes, dendritic cells and macrophages. Activated human T lymphocytes also express MHC Class II but MHC Class II expression is not detected on activated mouse T cells. HLA‐DR3 can be expressed as a transgene on CD4+ T cells in complete MHC Class II knock‐out (AEo) mice, but not in I‐Ab knock‐out (Abo) mice, suggesting that a gene, which could modify the expression of HLA‐DR3, might exist between I‐A and I‐E. Expression sequence tag (EST) databases were searched using the flank regions between I‐A and I‐E and a new potential gene was identified. No similar sequence was found in the human genome database. Expression in multiple mouse tissues, but not in human cell lines, was verified by RT‐PCR. However, this gene was found to be expressed in B6 mouse B cells and two mouse B cell lines, but not in T cells, indicating that it might not be related to the expression of HLA‐DR3. Transduction of this gene into the LG2 cell line had no effect on MHC II expression. In summary, we report the discovery of a new gene between the I‐Aa and I‐Eb1 region in C57BL/6 mice and its function remains unknown. (Supported by NIH‐AI30554)
Understanding the spread of mutations during somatic hypermutation
Authors: Unniraman Shyam , Schatz David G.
Published: June 2021

Somatic hypermutation of antibody genes is initiated by the deamination of cytosines to uracils in DNA by activation induced deaminase (AID). However, the process that leads from the initiating deamination event at cytosines to mutations at other residues remains poorly understood. Using a novel transgenic mouse model system, here, we demonstrate that a single cytosine on the top (non‐template) strand is sufficient to recruit AID and lead to mutations of both upstream and downstream A/T residues up to 29 bp away. In contrast, although cytosines on the bottom strand are targeted by AID, this does not lead to substantial mutation of neighboring residues. This strand asymmetry is eliminated in mice that are deficient in mismatch repair, indicating that error‐prone mismatch repair preferentially targets top strand uracils during somatic hypermutation. We are currently testing the influence of other repair factors on the orientation and extent of the spread. Research funded by Howard Hughes Medical Institute.
Maternal asthma causes gene expression and epigenetic changes in neonatal dendritic cells
Authors: Nguyen Phuong‐son , Fedulov Alexey , Kobzik Lester
Published: June 2021

In a mouse model of maternal transmission of asthma risk (J. Immunol 2003, 170:1683), baby mice of asthmatic (As), but not normal (Nrm), mothers show increased susceptibility to allergy. Adoptive transfer to normal baby mice of dendritic cells (DCs) harvested from asthma‐susceptible but allergen‐naïve neonates reproduces the increased susceptibility to asthma. To identify molecular mechanisms, we performed genomic and epigenomic profiling of neonatal DCs. BALB/c mice were sensitized by 2 i.p. injections of ovalbumin (OVA) in alum and repeatedly challenged with OVA aerosols (As) prior to mating with Nrm males. Splenic CD11c+ DCs of 14‐day old allergen‐naïve offspring from these As and Nrm mothers were isolated using magnetic beads. Gene expression profiles of total cell RNA were obtained using Illumina gene chip microarrays. Genomic DNA methylation wa profiled using Switchgear epigenomic chip arrays. We identified 30 genes with significantly different RNA expression, (0.5 – 2 fold). Analysis of epigenomic data identified up to 300 chromosomal regions with significantly different methylation status, (2 – 10 fold). Clustering methods and pathway analysis identified several interrelated gene groups that merit further study. Maternal asthma causes multiple significant gene expression and epigenetic changes in neonatal dendritic cells. ES 00002
Catecholamine effects on the expression of microRNAs in BALB/c mouse thymocytes and S49 cells
Authors: Koduvayur Sujatha P. , Cohen Rhonna L. , Chambers Donald A.
Published: June 2021

MicroRNAs (miRNA) have been associated with cellular differentiation and neoplastic progression as well as viral and bacterial activation of the immune system. This laboratory aims to understand how stress signals affect immune function. Here we investigate the effect of the stress hormone norepinehprine (NE) on the expression of miRNAs in BALB/c mouse thymocytes and S49 thymic lymphoma cells derived from BALB/c mice. Cells were treated with NE for 30′ and 2 hrs and miRNA expression was detected by reverse transcription followed by qPCR. Results indicate constitutive expression of 5 selected miRNAs in the presence or absence of NE in S49 cells and thymocytes. The expression levels of miR16 and miR181a are expressed 10 fold more in thymocytes, in contrast miR128b is expressed 10 fold less. MiR92 is expressed 2 fold less in S49 cells and miR142‐5p levels are unchanged in both cell types. That the expression of these miRNAs is unaffected by NE, argues that the levels of miRNAs may be conserved independent of this stress signal. Supported by NIH.
A functional polymorphism in ITPK(C) and its impact in T cell‐mediated pathogenesis of Kawasaki disease
Authors: Franco Alessandra , Shimizu Chisato , Watson Virginia , Tremoulet Adriana , Onouchi Yoshihiro , Burns Jane
Published: June 2021

Kawasaki disease (KD) is a self‐limited, acute vasculitis that is the most common cause of acquired heart disease in children. Death by coronary artery aneurysms occurs in up to 25% of untreated children.

T cells are critically involved in the pathogenesis of KD, as suggested by the infiltration of CD8+ cytotoxic T‐cells (CTL) in the coronary arterial walls of autopsy samples.

Has been recently discovered a novel functional single nucleotide polymorphism (SNP) (itpkc_3) located in intron 1 in the inositol 1,4,5‐triphosphate kinase (ITPK)C gene on Chromosome 19 responsible for the reduction of the expression of the ITPKC kinase that catalyzes the phosphorylation of Ins(1,4,5)P3 to Ins(1,3,4,5)P4.

We are currently studying the impact of the ITPKC locus in influencing the generation of pro‐inflammatory vs. regulatory T cells (Tregs). If we can understand the biologic effect of this SNP and its influence on disease susceptibility and outcome, this will be the first breakthrough in correlating a genotype with a phenotype in KD. Understanding this connection will have very important therapeutic implications since Cyclosporin A (CsA) specifically inhibits T cell activation affecting IL2 signaling through binding to calcineurin, which is an important downstream substrate of IP3 in the TCR‐NFAT signaling pathway.
Toll‐like receptor 2 and 4 sensing in Helicobacter felis infections: implications for an alternative sensing mechanism
Authors: Durham Carolyn G. , Lorenz Robin G.
Published: June 2021

Helicobacter felis is a Gram‐negative bacterium, implying the innate immune system sensing is via Toll‐like receptor (TLR) 4. Yet, previous data shows TLR4 is not directly responsible for H. felis recognition. Instead, in vitro studies show TLR2 is critical in recognizing Helicobacter LPS. The project's hypothesis is if TLR2 is critical to recognition of Helicobacter, then mice engineered to be deficient in this molecule should have an altered disease process. C57BL/6 mice deficient in TLR2, TLR4, or both (TLR2/4 double knockout) were infected with H. felis and evaluated four and twelve weeks after infection. Using wildtype C57BL/6 as controls, the knockouts and controls were compared to mock infected animals of the same genotype. Surprisingly, histological inflammation in the TLR2, 4 and 2/4 knockout mice appeared similar to the infected C57BL/6 controls, indicating these recognition molecules were not critical in inflammation induction. However, IFNγ, IL‐17, and TNFα mRNA levels elevated in the C57BL/6 mice post‐infection were not elevated in the TLR2/4‐deficient stomachs, implying in vivo recognition of Helicobacter is independent of TLR2 and 4 and other cytokines mediate this inflammation.
IFN‐γ induces IL‐23 producing DC‐like macrophage via suppression of endogenous IL‐10 signaling
Authors: Kamada Nobuhiko , Hisamatsu Tadakazu , Okamoto Susumu , Kobayashi Taku , Hibi Toshifumi
Published: June 2021

Recently, it has become evident that intestinal macrophages in normal intestine show anti‐inflammatory phenotypes, and play a central role in regulation of immune responses against commensal bacteria. On the other hand, we have reported that intestinal macrophages in patients with Crohn's disease (CD) revealed IL‐23‐producing pro‐inflammatory phenotypes. In this study, we have demonstrated that IFN‐γ, which contributes to the pathogenesis of CD, affected the macrophage differentiation. IFN‐γ conditioned macrophages produced larger IL‐23, and co‐expressed some DC markers on their cell surface. As a mechanism of such abnormal macrophage differentiation, we demonstrated that IFN‐γ suppressed the IL‐10 production associated with M‐CSF stimulation on macrophages. Interestingly, lamina propria conditioned media from patients with CD, which contained large amount of IFN‐γ, also suppressed the M‐CSF‐induced IL‐10 signaling, and blocking of IFN‐γ from conditioned media could rescue this suppression. Collectively, up‐regulated mucosal IFN‐γ in patients with CD induces abnormal differentiation of intestinal macrophage with IL‐23 producing DC‐like phenotypes via suppression of endogenous IL‐10 signaling. (This work was supported in part by grants‐in‐aid from the Japanese Ministry of Education, Culture, and Science and Keio Unversity Medical Fund)
Protective Anti‐Lipopolysaccharide Monoclonal IgA Antibodies Interferes with Salmonella Motility and Invasion of Epithelial cells, Independent of Bacterial Agglutination
Authors: Mantis Nicholas John , Forbes Stephen , Easchmann Marissa , Bumpus Tia
Published: June 2021

Mucosal immunity to enteric bacterial pathogens, including Salmonella enterica serovar Typhimurium (S.Typhimurium) and Shigella flexneri is mediated by secretory IgA (SIgA) antibodies directed against bacterial lipopolysaccharide (LPS). It has long been assumed that these antibodies protect intestinal epithelial cells from microbial colonization and invasion by simply promoting bacterial agglutination and entrapment in mucus. In this study, we challenge this paradigm by demonstrating that protective, serotype‐specific, anti‐LPS monoclonal IgA antibodies (IgA mAbs) at sub‐agglutinating concentrations interfere with bacterial motility and type three‐mediated secretion of proteins necessary for invasion of epithelial cells. These effects occurred within minutes of antibody exposure and at IgA concentrations sufficient to coat less than 5% of the bacterial LPS. By scanning electron microscopy we observed anti‐LPS IgA mAbs induced dramatic morphological changes in the surface topology of the bacterial outer membranes, suggesting motility arrest and inhibition of type‐three secretion may be the result of physical changes in the LPS layer. These data reveal a novel and previously unrecognized capacity of SIgA to effectively “disarm” microbial pathogens on mucosal surfaces and render them unable to colonize or invade host intestinal epithelial cells. This work was supported by New Investigator funds from the Wadsworth Center, New York State Department of Health.
Inflammation Is Attenuated In Germ‐free C57BL/6 Mice Infected with Helicobacter felis
Authors: Schmitz Julia M , Schoeb Trenton R , McCracken Vance J , Lorenz Robin G
Published: June 2021

Specific pathogen free (SPF) C57BL/6 mice infected with Helicobacter felis develop gastric cancer after 12 – 15 months. Unpublished studies using denaturing gradient gel electrophoresis (DGGE) indicated the presence of additional bacterial types in H. felis infected mice relative to mock mice. We hypothesize that changes that occur in the stomach during progression to gastric cancer, including increased pH and changes in the mucous layer, allow other bacteria to colonize the stomach and perhaps contribute to cancer progression. To test whether such bacteria could affect the progression to cancer, germ free (GF) B6 and SPF B6 mice were mock infected or infected with H. felis for sixteen weeks. Stomachs were analyzed by RT‐PCR and ELISAs for cytokine expression, DGGE for analysis of bacterial populations, and immunohistochemistry for histological analysis. A trend toward decreased inflammation (mean score of 6.3 on a scale of 0–9) and epithelial tissue destruction was observed in infected GF.B6 mice compared to SPF B6 mice (mean score of 9). To further evaluate the additive effect of other bacteria on the progression to gastric cancer, SPF and GF B6 mice will be infected for a year. The function of the additional bacteria in the SPF B6 mice remains to be elucidated but could be evaluated by adding specific bacteria identified by DGGE analysis. [NIH T32 AI07041, R01 DK59911 and ACS]
A subunit type of botulinum mucosal vaccine effectively induces protective immunity in non‐human primates
Authors: Nochi Tomonori , Yuki Yoshikazu , Mejima Mio , Ono Fumiko , Katakai Yuko , Shibata Hiroaki , Kohda Tomoko , Kozaki Shunji , Terao Keiji , Kiyono Hiroshi
Published: June 2021

Mucosally infected Clostridium Botulinum produces deadly poisonous exotoxin in the aero‐digestive tract and causes botulism. In spite of its high pathogenicity, effective vaccine against C. Botulinum has not been developed. We here prepared subunit antigen, C‐terminal half of heavy chain of botulinum neurotoxin (Hc), by E. coli expression system as a botulinum mucosal vaccine. Nasally immunized Hc induced high level of botulinum neurotoxin‐specific immune responses in both systemic and mucosal compartments of non‐human primates even in the absence of any mucosal adjuvant. Furthermore, immunized non‐human primates survived without any clinical sign after challenging with 25,000 LD50 dose of botulinum neurotoxin, while non‐immunized primates caused severe botulism within 3 hours. These results clearly show that mucosal vaccination with Hc is a novel strategy for the induction of protective immunity against botulism, which is naturally occurred or stemmed from acts of bioterrorism.
The PD‐1 and IL‐10 pathways play a down‐modulatory role in LP‐BM5 retrovirus‐induced murine immunodeficiency syndrome
Authors: Green Kathy Ann , Okazaki Taku , Honjo Tasuku , Cook W. James , Green William R.
Published: June 2021

Pathology due to the immune system's response to viral infections represents a delicate balance between inhibition of viral pathogenesis and regulation of protective immunity. In C57BL/6 mice, the retroviral isolate LP‐BM5 induces splenomegaly, hypergammaglobulinemia, B‐ and T‐cell immunodeficiency, and increased susceptibility to pathogens and terminal B‐cell lymphomas. We have found that B6.PD‐1 (Programmed death‐1) and B6.IL‐10 knockout mice (k.o.) are more susceptible to LP‐BM5‐induced disease than wild type (w.t.) B6 mice. LP‐BM5 infected PD‐1 k.o.'s and IL‐10k.o.'s, as compared to infected w.t. mice, developed increased: splenomegaly, hypergammaglobulinemia, and immunodeficiency; susceptibility to lower doses of LP‐BM5; and exaggerated disease early post infection. There was no significant difference in retroviral loads for infected PD‐1 k.o., IL‐10 k.o., and wt. mice. By reconstitution experiments, PD‐1+ B cells appear crucial in limiting LP‐BM5 induced disease in w.t. mice. Also, infected w.t. mice have increased CD11b+IL‐10+ spleen cells while infected PD‐1 k.o.'s, though showing an even greater expansion of CD11b+ cells, did not show an equivalent increase in IL‐10+ cells. We conclude that PD‐1/PD‐L interactions and IL‐10 are important in moderating the effects of LP‐BM5 induced disease in w.t. B6. Supported by NIH grant CA50157.
The relationship study between the changes of the frangible lymphocyte and the degree of destroyed liver function in fulminant viral hepatitis
Authors: Wang Haibin
Published: June 2021

32 cases confirmed with fulminant hepatitis and 66 cases with chronic hepatitis have been studied. 100 μl lymphocytes (1×106 /ml) and 100 μl tumor cells(1×105 /ml) mixed with 100 μl self‐serum at 37° C for 5 min to 30 min. The lymphocyte attached with tumor cell is undestroyed anything can be regarded as negative frangible lymphocyte. Less than 30 percent of lymphocyte attached with tumor cells is destroyed will be conformed as low‐grade frangible lymphocyte, between 30 percent to 60 percent regard as mid‐grade, and more than 60 percent as severe‐grade mode. The frangible lymphocyte is detected in fulminant hepatitis and other groups like that in SARS in 2003 years. 3 person's lymphocytes show severe‐grade frangible and 9 people display mid‐grade in 46 adult individuals. Contrarily There is high of 46.9 percent of cases with fulminant hepatitis show severe‐grade frangible lymphocytes, only 4 cases is negative and 75 percent is recovered in one months. The proportion of frangible lymphocytes in fulminant hepatitis is highest, and significantly higher than that in chronic hepatitis. The phenomenon of frangible lymphocytes may be an important characteristic of an innate immunology reaction of the body.
Differential role of lung dendritic cells and macrophages against influenza virus infection
Authors: Song Joo‐Hye , Seo Sang‐Uk , Kweon Mi‐Na
Published: June 2021

Influenza virus is a major cause of epidemic acute respiratory tract infections that have a high rate of mortality. Antigen‐presenting cells (APC), including dendritic cells (DC) or macrophages, may play a central role in eliciting innate and adaptive immune responses to influenza virus infection in the lung. We attempted to clarify the role of lung APC for protection of influenza virus infection. Balb/c and C57BL/6 mice were infected intranasally with a virulent A/PR8 influenza virus (105 PFU) and lung APC were analyzed 48 hr later. Interestingly, CD11c+B220+mPDCA+Gr‐1+ cells (pDC), CD11c+B220−CD11b+Gr‐1− (cDC), and CD11c−F4/80−Mac‐3+ cells (Macrophage) mainly infiltrated into the lung 48 hr following influenza virus infection. In contrast, no significant changes were detected in numbers of CD11c+F4/80+Mac‐3+ cells (Alveolar Macrophages). Among the lung APC, cDC represents primary cells to stimulate T cell proliferation by influenza hemagglutinin. In addition, pDC generate IFN‐γ producing T helper cells while AM and M∅ generate Th17 cells. Our studies demonstrate that each APC has a differential role in CD4 T cell differentiation during influenza virus infection.
Altered Immune Response in 2B4 Knock‐Out Mice Persistently Infected with LCMV
Authors: Waggoner Stephen N. , Mathew Porunelloor , Kumar Vinay , Welsh Raymond M.
Published: June 2021

The signaling lymphocyte activation molecule (SLAM) family receptor, 2B4, has been implicated in regulation of lymphocyte activation in vitro and in vivo. We and others have recently observed that virus‐specific CD8 T cells display sustained, high‐level expression of 2B4 during persistent, but not acute, LCMV infection in mice. Therefore, we hypothesized that 2B4 may play a role in exhaustion of virus‐specific CD8 T cells during chronic virus infection. In contrast to the lymphopenia and T cell clonal exhaustion observed during persistent LCMV infection of wild type mice, persistently infected 2B4‐deficient mice displayed altered liver pathology and marked splenomegaly characterized by increased numbers of all lymphocytes, including CD8 T cells. There were also enhanced numbers of splenic CD8 T cells with a naïve phenotype (CD62L high, CD44 low) and a concomitant increase in the frequency of double‐positive (CD4,CD8) cells within the thymus. Lysis of LCMV peptide‐labeled CTL targets in vivo was markedly reduced in the absence of 2B4, and viral loads were elevated in the spleen and liver. We propose that the absence of 2B4‐induced regulatory signals results in reduced activation of LCMV‐specific CD8 T cells, which poorly control virus replication and resist depletion during viral infection.

NIH T32 AI07349‐18
A novel therapeutic HBV vaccine induces potent surface‐ and core‐specific immunogenicity in mice rhesus macaques
Authors: Gesner Marianne L , Heeke Darren S , Martins Eduardo , Livingston Brian S , Van Nest Gary , Marshall Jason D
Published: June 2021

HBV infects approximately 350 million people worldwide, and carriers are at increased risk for cirrhosis and hepatocellular carcinoma. Current HBV therapies do not eradicate HBV and have limited long‐term efficacy. We have developed a novel immunotherapeutic vaccine for treatment of chronic HBV infection. The vaccine, composed of HBsAg (HBV surface antigen), HBcAg (HBV core antigen) and a proprietary adjuvant, was designed to induce virus‐specific cellular immune responses such as are typically correlated with HBV clearance. Mice immunized with the vaccine exhibited robust cellular and humoral immune responses to both HBcAg and HBsAg. Cell‐mediated responses, as determined by ELISPOT and intracellular cytokine staining (ICS), demonstrated that CD8 T cell responses were dominated by HBcAg‐specific immunity, while the majority of the CD4 response was HBsAg‐directed. The vaccine also elicited strong anti‐HBsAg and anti‐HBcAg immunoglobulin responses in both IgG1 and IgG2a isotypes. In rhesus macaques, 4 monthly immunizations induced high frequencies of antigen‐specific IFN‐g/IL‐2‐secreting T cells in response to HBsAg and HBcAg overlapping peptide pools as measured by ELISPOT and ICS. Vaccinated rhesus also exhibited >105 mIU/mL anti‐HBsAg Ig, after only two immunizations, and substantial levels of anti‐HBcAg Ig as well. These data suggest this dual‐antigen immunotherapeutic vaccine may be valuable in the treatment of chronic HBV infection.
A striking difference in cell‐mediated immune response to an inactivated vaccine against West Nile virus between yearling and adult horses
Authors: Zhang Yuwen W , Houseright Ruth A , Davis Elizabeth G
Published: June 2021

West Nile virus (WNV) infection may result in meningitis and/or encephalitis in humans and horses, especially aged and immunosuppressed individuals. Vaccine recommendations for horses involve an initial series followed by boosters 1–2 times annually, depending on the product used. Neonatal animals and humans are defective in generating effective T helper (Th) type 1 immunity against intracellular infections and are susceptible to atopic diseases. Since foals have been found to be interferon gamma (IFN‐γ)‐deficient we surmised that young horses may have different immune responses to WNV vaccination compared to adults. We investigated cell‐mediated immune responses to an inactivated WNV vaccine in yearling and adult horses by examining the ex vivo production of IFN‐γ and interleukin (IL)‐4 by T lymphocyte subsets. In yearling horses, cellular immunity is characterized by both Th1 and Th2 immune responses with a Th1‐bias. In adult horses, cell‐mediated immunity is represented by a pronounced Th1 response. Such a striking difference in the cellular immune response to WNV vaccination between yearling and adult horses may result from defective IL‐12 production by professional antigen‐presenting cells since defective IL‐12 production is common in neonatal mice and yearling children.
Antiviral effect of Alloferon on Kaposi's sarcoma‐associated herpesvirus
Authors: Lee Na Eun , Kang Jae Seung , Kim Jee Eun , Jung Da Jung , Lee Seung Koo , Bae Seyeon , Kong Joo Myoung , Lee Seona , Shin Dong Hoon , Hwang Young‐il , Lee Wang Jae
Published: June 2021

Kaposi's sarcoma‐associated herpesvirus (KSHV) is a human gammaherpesvirus that is associated with the development of Kaposi's sarcoma and primary effusion lymphoma. KSHV establishes two alternate genetic life cycle programs upon infection of its host cells, which maintain a latent infection in infected cells but undergo lytic reactivation in response to various stimuli. In this study, we used BCBL‐1, persistent KSHV‐infected B lymphoma cell line, where the lytic and latent cycle of KSHV was regulated by TPA treatment. Alloferon is an immunomodulating peptide originating from insects which has been successfully developed in clinical trials as antiviral drug in Russia and showed remarkable effect on prevention of recurrent HSV infection. However, its specific mechanism remains to be clarified. We investigated the effect of alloferon on the regulation of lytic reactivation in viral infected cells. We found that alloferon effectively suppressed the down‐regulation of cell proliferation induced by TPA treatment. It suggests that the lytic reactivation of KSHV within BCBL‐1 can be regulated by alloferon. We next investigated the specific intracellular target of alloferon. Alloferon regulated the expression level of RTA as lytic switch protein, and that of ORF45 and vIRF2, which inhibit the production of type I IFNs. Therefore, we suggests that alloferon may be an effective antiviral agent on KSHV infection.
Identification of cellular composition and architectural organization of tuberculous granulomas in cynomolgus macaques
Authors: Mattila Joshua T.
Published: June 2021

Infection by Mycobacterium tuberculosis (Mtb), the agent of human tuberculosis (TB), induces formation of leukocyte‐rich granulomas that prevent bacterial dissemination. The cellular composition and organization of granulomas may have important implications on individual's ability to contain Mtb, yet these factors have not been exhaustively examined. To better understand granuloma architecture, we used laser‐scanning confocal microscopy to visualize granulomas from Mtb‐infected cynomolgus macaques, a primate model of human TB. Granulomas were classified into two broad categories: solid cellular granulomas entirely composed of cells and caseous granulomas with centers composed of acellular necrotic material. Granuloma morphology was organized into three regions: a periphery of normal tissue abutting the granuloma, a ring‐like transition zone between the periphery and center, and a central region of caseum or cells depending on the granuloma type. Cell types within each region were consistent between examined granulomas, with lymphocytes macrophages predominating. Cells including B cells and NK cells were also observed. Numerous cells, including CD3+ lymphocytes, were observed to express TNF‐□ and IFN‐□ in the transition zone. The granuloma periphery included IL‐10 expressing CD3+ lymphocytes and CD68+ macrophages. These results suggest a variety of cell‐types are present in predictable places within granulomas and region‐specific cytokine expression may enhance anti‐Mtb responses in the granuloma and downregulate immune responses outside the granuloma.
Pseudomonas aeruginosa toxin reduces MHC class I antigen presentation
Authors: Bomberger Jennifer Melinda , Bangia Naveen , O'Toole George , Green William , Stanton Bruce
Published: June 2021

Pseudomonas aeruginosa is an opportunistic pathogen critical to the pathogenesis of nosocomial infections, chronic obstructive pulmonary disease (COPD), ventilator‐associated pneumonia (VAP) and Cystic Fibrosis (CF). Cif, a bacterial toxin secreted by laboratory and clinical isolates of P. aeruginosa, inhibits the intracellular trafficking of a variety of ABC transporters. Cif accesses epithelial cells via the lipid raft machinery and traffics retrograde to the ER. In the current study, we tested the hypothesis that Cif regulates the transporter associated with antigen processing (TAP), thereby regulating antigen presentation by MHC class I. Cif reduced TAP1 abundance and MHC class I plasma membrane expression in a time‐ and concentration‐dependent manner in polarized human airway epithelial cells, concurrent with an increase in Cif‐mediated ubiquitination of these proteins. Proteosome inhibitors prevented Cif‐induced degradation of TAP1 and MHC class I molecules. Furthermore, Cif decreased MHC class I presentation of peptide in a time‐dependent manner in a T‐cell tumor line. These observations predict that Cif, by promoting the degradation of TAP1 and reducing the presentation of peptide‐loaded MHC class I to the plasma membrane, will reduce the ability of cytotoxic T lymphocytes to eliminate viral/bacterial infections, severely compromising the immune defenses of the lung in patients with CF, COPD and VAP.
Anti RSV immunoglobulin isotype selection requires dsRNA‐activated protein kinase (PKR)
Authors: Minor Radiah Ann Corn , Croom Brittany , Imani Farhad
Published: June 2021

Respiratory syncytial virus (RSV) is the most common cause of severe respiratory tract infections in infants and young children world wide. A great deal of research has been focused on understanding the immune response to RSV in hopes of developing a safe and effective vaccine. Previous experiments by our lab showed that the double‐stranded RNA‐activated protein kinase (PKR) was required for RSV and dsRNA induction of inflammation. In the present study, we have focused on the role of PKR in adaptive immunity. Wild‐type and PKR‐deficient mice were infected with RSV (2 × 107 pfu/mouse) and after 1, 7 and 14 days, the levels of serum immunoglobulins were determined. Our data showed that over time, the total levels of IgG2A and IgG1 within the serum of WT but not PKR‐deleted mice increased. Furthermore, western blot analysis on extracts from RSV infected cells, showed that serum from infected WT mice contained significantly greater levels of RSV reactive immunoglobulins than serum from PKR‐deleted mice. The IgG2A isotype significantly contributed to the increase in reactivity. Further investigation of the defect in IgG2A expression in PKR‐deleted mice revealed that the deficit was not due to differences in the levels of IFN‐γ but involved defects in the level of T‐bet in stimulated B lymphocytes. Since T‐bet is a transcription factor for IgG2A class switching, our data suggest that an optimal RSV‐specific IgG2A response requires PKR through a mechanism that involves T‐bet.
The role of anti‐dengue antibodies in the pathogenesis of Dengue disease
Authors: Lei Huan‐Yao
Published: June 2021

Dengue disease includes from mild dengue fever to severe dengue hemorrhagic fever (DHF), which is an important health problem in tropical or sub‐tropical areas. The immunopathogenesis of DHF is initiated by aberrant immune activation and autoantibody production caused by virus infection. There is a molecular mimicry between dengue antigens and self‐proteins. The anti‐dengue antibody cross‐react with platelet and endothelial cell, and would trigger the subsequent dysfunction of endothelial cells and hemorrhage during the acute infection. The antibody‐dependent enhancement theory plays a central role in the dengue disease. The enhancing antibody is dependent on the types of the target cells and the specificity of the enhancing antibody. The enhancing antibody can be either anti‐prM or anti‐E antibody. For anti‐E Ab‐mediated enhancement on monocytic cells, it can be concentration‐dependent: enhancing at sub‐neutralization level or enhance regardless of concentration by Fc and FcR interaction. For anti‐prM Ab‐mediated enhancement, it enhanced the dengue virion binding on both FcR or non‐FcR bearing cells with dual specificity. The anti‐dengue antibodies seem to be either enhancing antibody or the pathogenic antibody, and play a major role in the DHF pathogenesis.
Apoptotic Cells Induce Immunosuppression through Dendritic Cells: Critical Roles of Interferon‐gamma and Nitric Oxide
Authors: Ren Guangwen , Zhao Xin , Das Gorbardhan , Zhang Liying , Shi Yufang
Published: June 2021

Apoptotic cells induce immunosuppression through unknown mechanisms. To identify the underlying molecular mediators, we examined how apoptotic cells induce immunoregulation by dendritic cells (DC). We found that administration of DC exposed to apoptotic cells (DCap) strongly inhibited expansion of lymphocytes in draining lymph nodes (dLNs) in vivo and subsequent antigen‐specific activation of dLN lymphocytes ex vivo. Unexpectedly, DCap supported T cell activation to a similar extent as normal DC in vitro, leading to proliferation and IL‐2 production, except DCap did not support T cell production of IFNgamma. Surprisingly, when DCap were co‐cultured with normal DC, they completely lost their ability to support T cell activation, an effect reversed by anti‐IFNgamma or inhibitors of inducible nitric oxide synthase (iNOS). As expected, exposure to apoptotic cells rendered DCap capable of producing much more nitric oxide (NO) in response to exogenous IFNgamma than normal DC. Furthermore, DCap from iNOS−/− or IFNgammaR1−/− mice were not inhibitory. Therefore, the IFNgamma‐induced production of NO by apoptotic cell‐sensitized DC plays a key role in apoptotic cell‐mediated immunosuppression.
Atopy and Th2‐biased immune responses exacerbate rather than promote corneal allograft survival
Authors: Niederkorn Jerry Young , Chen Peter W , Beauregard Clay , Mellon Jessamee , Stevens christina , Mayhew Elizabeth
Published: June 2021

It is believed that corneal allograft rejection is Th1‐mediated and that directing alloimmune responses toward a Th2 pathway will down‐regulate Th1 responses and promote allograft acceptance. We tested the effect of two Th2‐mediated diseases ‐ allergic conjunctivitis and airway hyperreativity (AHR) ‐ on corneal allograft survival. Allergic conjunctivitis and AHR were induced with short ragweed pollen (SRW) or ovalbumin (OVA). C57BL/6 corneal allografts were transplanted to BALB/c mice with ongoing allergic conjunctivitis or AHR. Mice with conjunctivitis induced with SRW or OVA had exacerbated allograft rejection compared to controls (100% vs 50% rejection; median survival time [MST] = 17 vs. 54 days and 95% vs. 50% rejection; MST = 18 vs. 26 days). SRW‐induced AHR or OVA‐induced AHR exacerbated corneal allograft rejection compared to vehicle controls (94% vs 50% rejection; MST = 21 vs 50 days; and 90% vs. 50% rejection; MST = 20 vs. 56 days). In other experiments, SRW challenge was suspended and mice were challenged 30 days later. Exacerbation of corneal allograft rejection was transient, as there was no difference between SRW‐treated mice and untreated controls (50% rejection; MST >50 days). Thus, atopic diseases are important risk factors in corneal allograft rejection. Tilting the alloimmune response toward a Th2 pathway not only fails to promote corneal allograft survival, but exacerbates immune rejection. Supported by NIH grants EY007641 and EY016664 and Research to Prevent Blindness.