bluuebunny/crossref_metadata_2025 huggingface dataset:
Cμ→Cδ Class Switch Recombination and IgD Production Contribute to Mucosal Immunity
Authors: Chen Kang , He Bing , Santini Paul A , Chiu April E , Chadburn Amy , Xu Weifeng , Cerutti Andrea , Cattalini Marco , Plebani Alessandro
Published: June 2021

IgD has remained an enigmatic isotype since its discovery in 1965. Although assumed to have evolved recently, its discovery in amphibians and bony fishes suggests that IgD arose much earlier and has evolutionarily important functions. IgD is usually expressed by B cells through alternative splicing of an RNA containing the VDJ exon and the Cu and C6 genes. Yet, some B cells express exclusively IgD after class switch recombination (CSR) from Cμ to Cδ. We detected abundant IgD+IgM− B cells in human respiratory mucosa. Mucosal IgD+IgM− B cells expressed virtually exclusively λ light chain, exhibited a partial plasmablastic phenotype and secreted IgD across the epithelium. They also expressed AID and contained extrachromosomal Sμ‐σδ switch circles, two hallmarks of ongoing IgD CSR. In vitro, IgD CSR and secretion occurred after exposure of B cells to either T cell‐dependent (CD40L) or ‐independent (BAFF or APRIL) signals. In vivo, IgD CSR did not require functional follicles and T cell help via CD40L. Together with prior studies showing the polyreactivity of IgD+IgM− B cells, our data suggest that IgD CSR takes place to provide the respiratory mucosa with additional extrafollicular and follicular layers of immune protection. Finally, by showing elevated numbers of circulating IgD+IgM− plasmablasts in children with diseases associated with hyper IgD production, our findings functionally link IgD with inflammation.
West Nile Virus Infection: A Model for Human Epitope Immunodominance
Authors: McMurtrey Curtis , Schafer Freda , Bardet Wilfred , Buchli Rico , Eckard Annette , Piazza Paolo , Hansen Ted , Bramson Jonathan , Rinaldo Charles , Hildebrand William
Published: June 2021

Cytotoxic lymphocytes (CTL) have been shown to play an important role in the clearance of West Nile Virus (WNV), however no human CTL epitopes have been identified. Using a comparative mass spectrometric approach we identified six HLA‐A*0201 restricted viral‐derived peptides from five different viral proteins (Env, NS2b, NS3, NS4b, and NS5) that are presented during infection. All WNV peptide epitopes were of high affinity for HLA‐A*0201 and the epitope sequences were highly conserved among reported viral isolates. When these epitopes were tested with PBMC from WNV infected patients a hierarchy of reactive epitopes emerged. In the majority of the infected patients tested (65%), a peptide from the envelope glycoprotein of WNV was the immunodominant epitope. Furthermore 82.5% of the patients reacted to more than one epitope such that a clear hierarchy emerges among WNV CTL epitopes. This pattern is reiterated in animal models. In summary, we report the detection of endogenous WNV class I HLA peptide epitopes, we characterize these epitopes recognition by CTL, and we identify a pattern of immunodominance that is consistent among humans and animal models.
Compromised CD8 T cell recall responses in the aged mice
Authors: Decman Vilma , DiMenna Lauren J. , Ertl Hildegund C J , Wherry E. John
Published: June 2021

Loss of effective immune responses with age has a fundamental impact on health and survival. As a consequence, the elderly are more susceptible to infections. To investigate the regulation of anti‐viral T cell responses during aging, we compared the generation and maintenance of effector and memory CD8 T cells in young versus aged mice during LCMV or influenza‐virus infection. By examining both systemic and local immune responses, we demonstrated that aged mice were able to mount primary anti‐viral CD8 and CD4 T cell responses. Aged mice, however, had higher morbidity and mortality compared to young mice and the generation of the effector CD8 T cell responses was delayed. Interestingly, many of antigen‐specific memory CD8 T cells in the aged mice were poorly functional. Nearly half of these antigen‐specific cells did not produce IFNγ and most did not make IL2 upon antigen stimulation. Most dramatically, virus‐specific memory CD8 T cells from aged mice had major defects in proliferative capacity in vivo upon rechallenge infection.

Thus, many virus‐specific memory CD8 T cells in aged mice appear to have functional defects and poor proliferative potential of these memory CD8 T cells may underlie poor protective immunity in vivo.

These studies were funded by grants from AHA, NIH (HHSN266200500030C) and the Ellison Medical Foundation.
Aged Environment Impairs Specific CD8 T Cell Response to Viral Infection
Authors: Jiang Jiu , Bennett Andrew J. , Murasko Donna M.
Published: June 2021

It is well known that the immune response is diminished with aging. However, the mechanisms are still largely unclear. In this study, we have shown that the lymphoid microenvironment of the aged mouse inhibits Ag‐specific CD8 T cell response to virus infection. First, the specific CD8 T cell response to virus infection was delayed and decreased in the aged mice. Second, using adoptive transfer of virus specific transgenic CD8 T cells, we demonstrate that the aged environment inhibited the clonal proliferation and expansion of the specific CD8 T cells during virus infection. Third, a component of the microenvironment influencing CD8 T cells expansion appears to be IFN‐I. Not only is generation of functional IFN‐I in serum lower in aged than in young mice after virus infection, but antibody to IFN‐I inhibited specific CD8 T cell response to virus in young, but not aged, mice. Lastly, while the function of DCs of aged appears intact, there is a decreased number of DCs in aged mice resulting in poor priming of the Ag‐specific CD8 T cell response. Taken together, the results of this study suggest that elements of the aged environment, such as cytokines and APCs, play important roles in the alteration of specific CD8 T cell responses to virus infection. This work was supported by National Institute of Health Grant AG14913 to D.M.M.
Dendritic cells undergo maturation following respiratory vaccinia virus infection
Authors: Beauchamp Nicole Marie , Alexander‐Miller Martha
Published: June 2021

Previous in vitro studies have shown that dendritic cells (DC) fail to undergo maturation following vaccinia virus (VV) infection. In spite of this finding, in vivo studies suggest a role for direct presentation of viral antigen in the generation of a virus‐specific CD8+ T cell response. These seemingly contradictory findings raise the question of how DC maturation is regulated in vivo. Using a model of respiratory VV infection, we find that infected DC are capable of up‐regulating expression of both CD80 and CD86. Thus in contrast to what has been suggested by in vitro studies, in vivo, VV infected, mature DC are candidates for T cell activation. Infected DC exhibiting a mature phenotype are present in both CD8+ and CD8− DC populations, suggesting both subsets can contribute to T cell activation. In addition, a subpopulation of non‐infected DC, both CD8+ and CD8−, are triggered to undergo maturation. Certainly the CD8+ DC are potential contributors to activation via cross‐presentation of antigen. Results from initial studies of naïve T cell activation by non‐infected CD8+ DC support this interpretation. These data provide the first analysis of VV infection of defined DC subsets in vivo following respiratory infection and the subsequent effect on their maturation and activating potential, as well as novel insights into our understanding of the generation of the adaptive immune response.
EBV‐encoded LMP1 indirectly activates the Jak/STAT pathway through production of IFN‐γ
Authors: Vaysberg Maria , Lambert Stacie L , Krams Sheri M , Martinez Olivia M
Published: June 2021

Epstein Barr virus (EBV) is associated with multiple human malignancies including post‐transplant lymphoproliferative disease (PTLD) B cell lymphomas. LMP1 is an EBV‐encoded protein expressed in PTLD tumors that can drive tumor growth through constitutive activation of NFκB, Erk, p38, Akt and JNK. We have shown that EBV+ B cell lines from PTLD patients express LMP1 and have a constitutively active Jak/STAT pathway. Dysregulated Jak/STAT activation has been observed in several B cell malignancies. Here we determine the role of the viral protein LMP1 in STAT activation. NGFR‐LMP1 chimeric proteins were expressed in EBV‐Burkitt's lymphoma lines to permit inducible LMP1 signaling. LMP1 induced STAT/DNA complexes but only after 24hr of LMP1 activation suggesting that STAT activation is indirect. STAT1 was the predominant STAT activated by LMP1. Inhibition of cytokine secretion prior to LMP1 activation abolished STAT binding indicating a secreted protein is responsible for STAT activation. IFNs are known inducers of the STAT pathway and pan cytokine luminex assay revealed that LMP1 can induce IFNγ but not IFNα. Moreover, IFNγ receptor blockade prior to LMP1 activation dramatically decreased STAT1 activation. Together, these data demonstrate that LMP1 does not activate the Jak/STAT pathway through direct interaction with JAK but rather through an indirect pathway involving IFNγ induction.
Characterization of the cytoplasmic domain of CD28 in T cell activation and the regulation of HIV transcription.
Authors: Henderson Andrew J , Yang Polung , August Avery , Natarajan Malini
Published: June 2021

T cell activation requires signaling from the T cell receptor and the costimulatory receptor CD28. Protein tyrosine phosphorylation plays a crucial role in CD28 signaling. In order to understand the molecular basis for CD28 signaling and its ability to mediate HIV transcription, we have used a chimeric receptor approach. The cytoplasmic tail of CD28, with its four tyrosines (Y) at positions 173, 188, 191 and 200, is fused to the extracellular receptor of CD8, generating a CD8/28 chimeric receptor. We show that the tyrosines in the cytoplasmic tail of CD28 have different functions. Y173 inhibits T cell activation and HIV transcription, whereas, in the absence of the inhibitory signal Y188, Y191 and Y200 have increase HIV transcription. Signals recruited by Y191 and Y200 have redundant functions, are required to overcome the Y173 inhibition and are sufficient for activating HIV transcription, NF‐κB and Vav phosphorylation. Y188 activates Vav but does not induce HIV transcription. How these positive and negative signals are coordinated to influence HIV transcription will be discussed.
Zinc deficiency suppresses alveolar macrophage function in in vivo models of both chronic alcohol ingestion and HIV‐1 transgenic expression
Authors: Joshi Pratibha C , Guidot David M
Published: June 2021

Alcohol abuse and HIV‐1 infection independently affect many individuals and render them susceptible to severe pulmonary infections. Alcoholics and HIV‐1 infected individuals are known to be deficient in immunostimulatory micronutrient zinc. Although zinc deficiency is known to have adverse effects on the ciliated epithelium within the upper airway, there is very little known about zinc homeostasis within the alveolar space. In this study, we separately examined alveolar zinc levels and the effect of inadequate zinc microenvironment on alveolar macrophage immune function in chronic alcohol‐fed and hemizygous NL4‐3Δgag/pol transgenic rats. Alveolar epithelial lining fluid from each rat model was significantly zinc deficient. Alveolar macrophages from these rats had significantly fewer granulocyte‐macrophage colony stimulating factor (GM‐CSF) receptors, and significantly reduced capacity to phagocytose bacteria. Zinc supplementation increased pulmonary zinc levels, GM‐CSF receptor expression, and significantly improved phagocytic function of alveolar macrophages in both the models. These data suggest an important role for pulmonary zinc in these two clinical settings that are associated with pneumonia.
HIV‐1‐Infected Peripheral Blood Mononuclear Cells (PBMC) Promote Neutrophil Survival and Induce GM‐CSF‐Dependent Expression of HLA‐DR and IFN‐γ‐Dependent Expression of CD64
Authors: Fu Jun , Riggs Emily , Thomas Larry
Published: June 2021

We examined the ability of HIV‐1 infected PBMC to influence neutrophil function. Incubating neutrophils with supernatant of PHA‐activated PBMC that had been cultured with HIV‐1 for six days (HIV‐1+ culture supernatant) significantly increased neutrophil survival at 48 hr. Depletion of the HIV‐1 virions by centrifugation did not alter the pro‐survival activity of the HIV‐1+ culture supernatant. The HIV‐1+ culture supernatant also induced expression of HLA‐DR and CD64 by neutrophils. The induction of HLA‐DR paralleled induction of HLA‐DR expression by GM‐CSF. The HIV‐1+ culture supernatant, however, did not induce neutrophil expression of CD80, CD86, or CD40. The HIV‐1+ culture supernatant stimulated concentration‐dependent increases in neutrophil HLA‐DR and CD64 at supernatant concentrations containing 5 to 50 pg GM‐CSF/ml and 3 to 30 pg IFN‐γ/ml, respectively. Neutralizing anti‐GM‐CSF antibody inhibited HLA‐DR expression and neutralizing anti‐IFN‐γ antibody inhibited CD64 expression induced by the HIV‐1+ culture supernatant. These results indicate that HIV‐1‐infected PBMC can prolong neutrophil survival and potentially influence neutrophil immune activity through the production of GM‐CSF and IFN‐γ. Supported by NIH Grant R21 DE070170
Different effects of HBV and its viral proteins on TRAIL‐induced apoptosis and their distinct mechanisms
Authors: Du Juan , Liang Xiaohong , Chen Youhai , Liu Yugang , Cui Min , Han Lihui , Liu Suxia , Qu Zhonghua , Zhang Zhiyong , Sun Wensheng
Published: June 2021

TNF‐related apoptosis‐inducing ligand (TRAIL) has recently been implicated in the death of hepatocytes during HBV infection, but the mechanisms are not well understood. To determine the role of TRAIL in HBV infection, we examined the effects of HBV genome and its viral proteins on TRAIL‐induced apoptosis. TUNEL assay showed HBV genome or HBx protein significantly increased TRAIL‐induced apoptosis through upregulating Bax expression. Similarly, C‐terminally truncated middle hepatitis B surface protein (MHBs(t)) sensitized hepatocytes to TRAIL‐induced apoptosis through involving ERK2. By contrast, other HBV proteins played an opposite role in TRAIL‐induced apoptosis although the mechanisms remain to be elucidated. These results establish different HBV proteins have different effects on TRAIL‐induced hepatocyte apoptosis. If the pro‐apoptotic proteins, such as HBx, are dominant, HBV infected hepatocytes may die as a consequence and fulminant hepatitis may develop. While, if the anti‐apoptotic viral proteins prevail, the infected hepatocytes may not undergo apoptosis and chronic HBV infection may ensue. These findings clarify the roles of imbalanced apoptosis in the pathogenesis of hepatitis B infection. This work is supported by the Natural Science Fund of China (No.30772031, No.30700973).
CpG‐containing immunostimulatory sequences (ISS) exhibit potent inhibition of HCV replication in HCV replicon line
Authors: Heeke Darren Scott , Martins Eduardo , Livingston Brian , Van Nest Gary , Marshall Jason
Published: June 2021

Only 50% of HCV‐infected patients respond to current treatment and many subjects experience adverse effects stemming from systemic IFN‐a administration, underlining the need for safer and more efficacious therapies. Exposure of Huh‐7 replicon cells, an in vitro HCV replication system, to supernatants from human PDCs stimulated with IFN‐a‐inducing CpG‐C ISS also displayed a potent ability to inhibit HCV replication that correlated with the quantity of IFN‐a present in the supernatants. To more closely mimic the in vivo situation, co‐cultures of replicon cells and human PDCs were established. In this case, HCV replication was also inhibited by direct ISS treatment, and optimal inhibition was achieved using lower doses of ISS than were necessary via PDC‐supernatant transfer. Type I IFN blockade during the co‐culture was insufficient to completely inhibit HCV replication, suggesting that other ISS‐induced factors contribute to controlling HCV replication. Furthermore, the dose of efficacious ISS could be further reduced when administered together with the antiviral drug ribavirin, suggesting that effective combination therapy might be achieved with low doses of ISS. These results suggest that a novel combination therapy of ISS and ribavirin could exhibit potent HCV suppressive activity while avoiding adverse events associated with high levels of systemic IFN‐a.
Modulation of lung environment and new T cell responses by flu‐primed CD4 memory T cells
Authors: Jelley‐Gibbs Dawn M , McKinstry K. Kai , Strutt Tara , Dibble John P , Swain Susan L
Published: June 2021

Infection of mice with influenza (flu) viruses generates CD4 memory T cells that reduce the accumulation of newly primed CD4 and CD8 T cells in the lungs during subsequent infections, while allowing normal T cell priming in lymphoid organs. Since the modulation of new T cell responses by CD4 memory T cells is not due to competition for antigen, we assayed for classical regulatory T cell populations within the CD4 memory T cell pool. While there was an increase in classical Treg and Tr1 phenotype T cells after flu infection, the CD4 memory T cells did not need to express CD25, GITR, or IL‐10 to carry out their modulatory functions. Gene expression in the lungs of flu infected mice adoptively transferred with CD4 memory T cells, versus naïve CD4 T cell transfer, revealed a reduced expression of genes associated with type‐1 inflammation and increased expression of genes associated with type‐2 inflammation. Thus, we are exploring whether CD4 memory T cells prevent accumulation of newly primed T cells in the lungs due to a reduced expression of type‐1 chemoattractants or active suppression of newly primed T cell survival in a type‐2 biased lung environment. However, we have not ruled out the possibility that the modulatory CD4 memory T cells may be recruiting classical regulatory T cells to the lungs during flu infection. Determining the mechanism by which CD4 memory T cells modulate newly primed T cell responses at sites of infection, and the impact this has on the generation of subsequent memory T cell responses, will yield important insights into methods for inducing protective T cell memory to a broad range of pathogens. Funding provided by NBC‐AI057158‐Lipkin.
Influenza‐specific CD4 effectors utilize perforin for cytolytic function and protection in vivo
Authors: Brown Deborah M , Kamperschroer Cris , Roberts Deborah , Swain Susan L
Published: June 2021

Our laboratory previously demonstrated that CD4 effectors generated in vitro could promote survival against a highly pathogenic influenza virus via an antibody independent mechanism that involved class II restricted, perforin‐mediated cytotoxicity. The present study set out to determine whether influenza specific CD4 cells could acquire cytolytic activity in vivo and the role of perforin in CD4 T cell responses to influenza. Surprisingly, influenza PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung, but not the draining LN. These cells expressed granzyme B (GrB), were CD62L low and required perforin to lyse peptide pulsed targets. CD4 cells isolated from the lungs of infected mice were able to confer protection to a lethal dose of influenza PR8; however, cells isolated from the DLN were more effective at promoting survival. This was attributed to the ability of DLN CD4 cells to further differentiate to cytolytic GrB+ cells upon re‐infection. Lethally infected mice given flu specific CD4 cells deficient in perforin showed greater weight loss and slower time to recovery compared to mice given wildtype flu specific CD4 cells, suggesting that CD4 mediated cytolytic activity may play a role in protection to lethal infection. Studies are underway to determine whether perforin is required for B cell independent protection mediated by CD4 effectors in B cell deficient mice. Supported by PHS grants AI‐0672, AG‐21600 and the Trudeau Institute.
Separation of pulmonary and systemic aspects of respiratory syncytial virus vaccine‐enhanced disease
Authors: Castilow Elaine , Varga Steven M.
Published: June 2021

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. Prior vaccination with a formalin‐inactivated (FI) RSV vaccine resulted in enhanced morbidity and mortality upon natural RSV infection that was characterized by an extensive pulmonary mononuclear cell infiltrate and eosinophilia. Vaccination of BALB/c mice with a recombinant vaccinia virus expressing the attachment protein of RSV (vvG) results in pulmonary eosinophilia mimicking the response of the FI‐RSV vaccine recipients. The G protein elicits a CD4 T cell response that is comprised of a mixture of Th1 and Th2 cells which predominately express the VÎ214 T cell receptor. We have utilized STAT6‐ and STAT4‐deficient mice to examine the role of IL‐4/IL‐13 and IL‐12 signaling in mediating individual aspects of RSV vaccine enhanced‐disease. We demonstrate that STAT6 is necessary for the development of pulmonary eosinophilia, but that systemic disease is unaltered in the absence of STAT6. In contrast, we find that STAT4 is necessary for systemic disease but not for pulmonary eosinophilia. In both STAT6‐ and STAT4‐deficient mice, the magnitude of inflammation remained unaltered. Our results suggest that RSV vaccine‐enhanced pulmonary injury and systemic disease are mediated by distinct pathways. This work was supported by the NIH (to SMV) and the American Heart Association (to EMC).
Critical requirement for 4‐1BBL for protection from severe but not mild respiratory Influenza A infection in mice
Authors: Lin Gloria Hoi Ying , Sedgmen Bradley J. , Snell Laura M. , Watts Tania H.
Published: June 2021

In view of intensifying concerns about pandemic Influenza virus, there is considerable interest in the mechanisms that control respiratory Influenza virus infection. Here we sought to determine the importance of 4‐1BBL‐mediated costimulation during mild versus severe respiratory Influenza virus infection in mice. During infection with the mildly virulent Influenza A/HKx31 virus, regardless of route of infection or epitope studied, 4‐1BBL is completely dispensable for the primary CD8 T cell response. However, 4‐1BBL is required for maximal recall responses, previously attributed to a requirement for 4‐1BBL in the maintenance of T cell memory. In contrast, during primary intranasal infection with Influenza A/PR8, which leads to severe respiratory disease in mice, the absence of 4‐1BBL results in decreased CD8 T cell responses in the lung, increased viral load and increased mortality. Thus 4‐1BBL plays a key role in protective immunity in the respiratory tract during severe Influenza disease. These results show that costimulation requirements are greater during severe as compared to mild respiratory Influenza virus infection.
EFFECTS OF COSTIMULATION ON INFLUENZA SPECIFIC CD8+ T CELLS DURING LATE ANTIGEN PRESENTATION
Authors: Suarez Jenny Elizabeth , Mohni Kareem N , Redman Jason M , Vella Anthony , Mittler Robert S , Cauley Linda S
Published: June 2021

CD8T cells are important for controlling influenza infection. We have shown that processed viral antigens remain in the lung draining lymph nodes (DLN) more than a month after pulmonary infection. Transfer experiments show that naïve nucleoprotein (NP)‐specific CD8+T cells respond to this late antigen presentation but do not become fully differentiated “effector” T cells as shown by a limited ability to make IFN‐γ. Based on this phenotype we hypothesize that immature DC contribute to late antigen presentation, but do not provide optimal costimulation. To test this naïve NP‐specific cells were transferred one month after infection and 24h later the recipients were treated with either: (1) LPS to promote DC maturation or (2) antibodies to B7H1 to block PD‐1 interactions in vitro. Our results show that the numbers of endogenous NP‐specific CD8+T cells in the lungs and spleens increased ∼4 fold after LPS treatment. A larger percentage of the transferred NP‐specific CD8+T cells also became fully differentiated “effector” cells that reached other tissues including the lungs after LPS treatment, however there was no change when antibodies to B7H1 were used. When partially‐activated NP‐specific CD8T cells from the DLN were transferred to naïve mice they responded to reinfection by becoming “effector” T cells and long‐lived memory T cells. The long term goal of these studies is to augment protection by enhancing CD8T cell activity during late antigen presentation.
The Cytokine and Chemokine Response to Influenza Virus Infection in Neonatal Mice
Authors: Lines Janet Louise , Hollifield Melissa , Cauley Linda , Garvy Beth A.
Published: June 2021

Influenza virus is an important cause of mortality and morbidity in children. However, little data exists on the neonatal immune response to influenza virus. We studied intranasal influenza virus infection in adult and 2 day‐old mice, and found that the LD10 for pups is 10 fold lower than for adults. We also observed an interstitial distribution of T cells in the lungs of influenza virus infected pups. By 14 days post infection, the numbers of CD4 and CD8 T cells in the lung interstitia were comparable between pups and adults. However, in pups, T cells were absent from the airways. We therefore determined the mRNA levels of chemokines in lungs from infected pups and adults. Interestingly, in pups, CCL5 (RANTES) expression was elevated, while CXCL9 (MIG) expression was reduced by over 10 fold compared to adult mice. We previously found that TGFβ is constitutively expressed in neonatal lungs, so we examined TGFβ in the context of influenza virus infection. Total TGFβ levels dropped after infection in adults, but remained elevated in pup lungs throughout infection. This corresponded to reduced IFNγ. Consistent with this cytokine profile, a lower level of pup T cells expressed markers of activation. Together, these data suggest that the immune response to influenza infection in neonates is hindered by inappropriate expression of cytokines and chemokines.

Supported by an AHA predoctoral fellowship.
Persistent MCMV infection drives continuous production of short‐lived effector CD8 T cells
Authors: Snyder Christopher M. , Cho Kathy , Morrison Elizabeth , Hill Ann B.
Published: June 2021

Murine cytomegalovirus is a beta herpesvirus that establishes chronic infection in mice driving a huge CD8+ T cell response that is maintained for the life of the host with little or no evidence of T cell exhaustion. The MCMV‐specific CD8 T cell population can be divided into 2 categories based on antigen specificity: “inflating” and “non‐inflating”. We wished to understand how these two populations were driven and addressed this with a series of adoptive transfer experiments. Non‐inflating T cells crash after acute infection and persist by slow division during chronic infection with a central memory‐like phenotype, behaving as if the virus were cleared. In contrast, inflators resemble effector memory T cells and increase dramatically in number shortly after acute infection. Our data suggest that inflation results from a combination of cell division and recruitment of naive T cells. At late times however, the rate of inflation slows and the large effector memory population is maintained. Strikingly, most inflated cells at these time points no longer divide and have a short life‐span (t1/2 ~45–60 days) that is independent of viral antigen, similar to what has been described as short‐lived effectors. Our data suggest that the overall population is maintained by recruitment of naïve cells that join this short‐lived pool. Yet there is real memory within this population since new infection drives extensive, rapid division.
Development of a Multiplexed HPV L1 Virus‐Like Particle Type‐Specific Immunoglobulin Subclass and Isotyping Assay
Authors: Opalka David William , Matys Katie Marie , Green Tina , Antonello Joseph , Radley David , Sikkema Daniel , Barr Eliav , Esser Mark Thomas
Published: June 2021

GARDASIL™ has been proven in clinical trials as a highly effective vaccine for preventing HPV 6‐, 11‐, 16‐, or 18‐related pre‐cancerous lesions and genital warts. In this study, we compared the HPV‐type specific subclass and isotype humoral immune responses following vaccination to those induced by HPV infection. Sera collected from vaccinees at month 7 and month 60 post‐vaccination, naturally infected and negative subjects were tested to determine serum levels of HPV VLP‐specific IgG, IgG1, IgG3, IgG4 and IgA using a Luminex based, direct binding, subclass and isotyping assay method. The same serum samples were also evaluated for their levels of antibodies to type‐specific, neutralizing epitopes on the VLPs using a competitive immunoassay designed to measure type‐specific, neutralizing antibodies. Vaccination with GARDASIL™ induced high serum levels of VLP‐specific IgG, IgG1, IgG3, IgG4 and IgA antibodies, and such antibodies to VLP 6, 11, 16 and 18 were also detected at Mo 60 in a high proportion of subjects. These levels were comparable to or greater than antibody levels associated with subjects naturally infected with HPV. Importantly, serum anti‐L1, HPV IgA levels induced by vaccination may provide evidence of mucosal immunity that could protect the anogenital tract from HPV infection.
Anti‐viral Effects of Phosphatidylethanolamine‐targeting agents
Authors: Soares M. Melina , Mims Susan , Barbero Gustavo , Li Shuzhen , Thorpe Philip E
Published: June 2021

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are segregated to the inner leaflet of the plasma membrane of resting mammalian cells. We have previously shown that PS is exposed on the outer surface of virus‐infected cells and on enveloped viruses that bud out of these virus‐infected cells. The internal positioning of PS is maintained by aminophospholipid translocase, suggesting that this transporter is inhibited during viral infection. Because aminophospholipid translocase tranports both PS and PE from the outer to the inner leaflets of the plasma membrane, we hypothesized that PE would also be exposed on the outer surface of virus‐infected cells and on enveloped viruses. If so, the external PE could serve as a target for anti‐viral therapy. To target PE, we modified the 19‐amino‐acid PE‐binding tetracyclic peptide duramycin by conjugating it to biotin (DLB). The modification reduces the hemolytic activity and toxicity of the peptide but retains its PE binding abilities. DLB neutralizes multiple viruses in vitro. DLB also shows therapeutic efficacy in a lethal murine model for human cytomegalovirus. Our study demonstrates the promise of using PE as a target for broad‐spectrum anti‐viral drugs.
Yersinia pestis elicits growth‐condition dependent patterns of dendritic cell IL12p40 production and CCL19‐dependent chemotaxis
Authors: Robinson Richard T. , Khader Shabaana A. , Smiley Stephen T. , Cooper Andrea M.
Published: June 2021

Yersinia pestis is the causative agent of bubonic plague. At the temperature of Y. pestis natural flea vector (26°C), Y. pestis expresses a profile of genes and lipopolysaccharide variants that are distinct from those expressed in a mammalian host (37°C). As dendritic cells (DCs) are targeted by Y. pestis, we examined whether DC production of IL‐12p40 ‐ a critical component of both cellular and humoral immunity ‐ was differentially stimulated by Y. pestis grown at 26°C versus Y. pestis grown at 37°C (hereafter referred to as Y.pestis‐26° and Y. pestis‐37°, respectively). Using IL12p40‐reporter bone‐marrow derived DCs (BMDCs) we demonstrate that Y.pestis‐26° is capable of eliciting a >25‐fold increase in IL12p40 production above unstimulated controls in a dose‐dependent manner. This IL12p40 response, however, was much attenuated among BMDCs stimulated with Y. pestis‐37°. As IL12p40‐homodimer contributes to DC chemotaxis, we also examined the chemotactic ability of Y.pestis‐26° versus Y. pestis‐37° stimulated BMDCs. Stimulation of BMDCs with Y.pestis‐26° significantly increased their chemotaxis towards CCL19 in a dose‐dependent manner, whereas the CCL19‐dependent chemotaxis of Y. pestis‐37° stimulated BMDCs was only minimally increased. Thus, Y.pestis‐26° and Y. pestis‐37° differentially stimulate BMDCs, with the Y. pestis‐37° being associated with diminished IL12p40‐production and chemotaxis.
SOCS3 and IL‐10 anti‐inflammatory activity in Lyme disease
Authors: Dennis Vida , Kaushal Deepak , Dixit Saurabh , Mehra Smriti , Singh Shree , Philipp Mario
Published: June 2021

We have shown that mouse J774 macrophages incubated with IL‐10 and Borrelia burgdorferi spirochetes augmented their SOCS3 expression levels. This correlated with the IL‐10 inhibition of expression of several pro‐inflammatory cytokines. We hypothesized that SOCS3 induced by B. burgdorferi and IL‐10 is functionally important in the control of inflammation in Lyme disease, with SOCS3 acting as a mediator of the IL‐10 anti‐inflammatory activity in macrophages. To begin to address this hypothesis, we silenced the socs3 gene in J774 macrophages with SOCS3 small interfering RNA to determine the effect of silencing on the IL‐10 inhibition of pro‐inflammatory mediators induced by B. burgdorferi. Transfected cells were co‐stimulated with IL‐10 and B. burgdorferi; RNA samples were collected and subjected to genome‐wide transcriptomics using mouse oligonucleotide microarrays. These studies revealed that the socs3 gene is up‐regulated (~40‐fold) in cells stimulated with IL‐10 and B. burgdorferi. In the presence of SOCS3, IL‐10 down‐modulated several pro‐inflammatory mediators in B. burgdorferi‐stimulated cells. However, silencing of the socs3 gene in cells resulted in a marked reversal of the down‐modulatory effect exerted by IL‐10 on many of these inflammatory mediators including cytokines, chemokines, IFN‐inducible, apoptotic and TNF ligand/receptor genes. This study demonstrates that SOCS3 in part mediates the IL‐10 anti‐inflammatory effect in macrophages stimulated with B. burgdorferi. This work was supported by National Institutes of Health grants R21AI073356 and RR00164 and the Centers for Disease Control and Prevention grant U01‐CI000153.
Intracellular Release of Granzyme B Drives a Rapid Listeriolysin O‐induced T Cell Apoptosis
Authors: Carrero Javier A , Vivanco Hector , Unanue Emil R
Published: June 2021

The Listeria monocytogenes protein listeriolysin‐O (LLO) is a pore‐forming protein essential for bacterial virulence. LLO's major role is to allow L. monocytogenes entry into the cytosol. Recent work has shown that it also induces apoptosis of activated lymphocytes. We studied the mechanism of induction of apoptosis by LLO at the cellular level. CD4+ T cells were generated from either normal mice or mice granzyme deficient mice and treated with LLO. LLO induced apoptosis was dependent on the expression of granzyme B. In the absence of granzyme apoptosis was dramatically reduced in magnitude. Neutralization of intracellular acidification using chloroquine inhibited the rapid apoptotic death. Mice deficient in granzyme were more resistant to infection and displayed reduced apoptosis in the splenic white pulp. Thus, LLO's pore‐forming activity in acidic organelles releases proteins, such as granzymes, into the cytosol, to initiate an apoptogenic cascade that causes death from within and this process modulates the in vivo infection.
The nature of the association between polysaccharide and protein antigens in an intact bacterium versus a related conjugate vaccine critically determines the ability to generate memory for the polysaccharide‐specific IgG response
Authors: Colino Jesus , Chattopadhyay Gouri , Sen Goutam , Chen Quanyi , Lees Andrew , Canaday David H. , Rubtsov Anatoly , Torres Raul , Snapper Clifford M.
Published: June 2021

The capsular polysaccharide type 14 (PPS14) covalently linked to pneumococcal surface protein A (PspA) [conjugate] induces a memory IgG anti‐PPS14 response, in contrast to intact Streptococcus pneumoniae type 14 (Pn14). Nevertheless, the IgG anti‐PPS14 response to both conjugate and Pn14 are CD4+ T cell dependent. Pn14, in contrast to conjugate, fails to induce substantial B or T cell memory for the IgG anti‐PPS14 response, although both induce similar levels of PspA‐specific memory. Using lsc−/− mice we demonstrate that the IgG anti‐PPS14 response to Pn14 is dependent on marginal zone B (MZB) cells whereas the same response to conjugate utilizes follicular B (FB) cells. Nevertheless, conjugate linked to the surface of a non‐encapsulated Pn, depleted of PspA, can induce IgG anti‐PPS14 memory, but is now dependent on MZB. These data suggest that (i) the particulate nature of the immunogen is not the critical determinant for PPS14‐specific memory generation, and (ii) IgG PPS14‐specific memory can be generated in MZB, as well as FB cells. These data suggest instead, that the critical factor in the failure of Pn14 to generate PPS14‐specific memory may be the non‐covalent association of the bacterial proteins with PPS‐14, and subsequent failure to generate sufficient CD4+ T cell help for PPS14‐specific B cells.
T cell independent B cell response to an RNA‐binding viral protein (Vaccinia E3)
Authors: Moyron‐Quiroz Juan Enrique , Crotty Shane
Published: June 2021

How the innate immune system impact and shape the adaptive immune system is not totally understood; the adjuvant properties of the molecules dedicated to the recognition of Pathogen‐Associated Molecular Patterns (PAMPs) is currently subject to intense research. Using mice deficient in MHC II, we found that all but one of the IgG antibody specificities induced after VACV infection are dependent on CD4 T cell help, the only exception is E3, a double stranded RNA (dsRNA) binding protein to which antibodies of the IgG3 isotype can be detected as early as day 5 post‐infection. The anti‐E3 IgG response is not dependent of MyD88, the adaptor protein involved in the signaling of all know Toll‐like receptors (TLR) but TLR3. However, TRIF deficient mice have a severely diminished antibody response against E3; TRIF is the adaptor for TLR3. TLR3 is a member of the TLR family of PAMPs receptors and recognizes dsRNA inducing the production of type I interferons. As both TLR3 and E3 bind dsRNA, we hypothesize that E3‐dsRNA complex is bridging the signals of the BCR with the signals of TLR3, resulting in a T cell independent IgG response.
Identification of a B cell epitope of enterohemorrhagic Escherichia coli O157:H7 EspA
Authors: Mao Xu‐Hu , Yu Shu , Gu Jiang , Wang Qingxu , Zou Quanï¼□ming
Published: June 2021

Backgrounds

Enterohaemorrhagic E. coli (EHEC) O157:H7 is a primary food‐borne bacterial pathogen capable of posing serious challenge to the public health worldwide. EspA (E. coli secreted protein A) plays an important role in the attaching/effacing lesion. Determination of EspA epitopes will be facilitate to understanding of the multipler pathogenicity as well as the developing of vaccine anainst O157:H7.

Methods

Anti‐EspA mAbs were produced by hybridoma technique. Epitope mapping was carried out by truncated EspA protein immuno‐blotting with mAb, and overlapping peptides were synthesized to identify the antigenicity and immuogenicity by competitive inhibition ELISA, dot‐blotting and mice immune. Immune‐fluorescence assay was performed to review the ability of antibody's blocking the actin polymerization and pedestal formation.

Results

Three mAbs against EspA, named 1H10,2A3 and 3E5 respectively, were obtained. The epitope recognized by 1H10 was located in 100–120aa, but 2A3 and 3E5 were detected to recognize conformational epitopes possibly. The conjugate of epitope‐BSA could produce high titer antibodies immunized with mice, the polyclonal sera aginst E100–120 and 1H10 could inhibitate the host cell actin rearrangement dramaticly.

Conclusions

A novel B cell epitope in EspA, E100–120 was identified by the 1H10, and the epitopes recognized by 2A3 and 3E5 were possible conformationally.
Characterization of the Antibody Response to Two Distinct Neutralizing Epitopes on Human Papilloma virus Type 6 Virus‐Like Particle
Authors: Matys Katie , Opalka David , Brown Martha , Smith Judith , Brownlow Michelle , Green Tina , Sikkema Daniel , Bryan Janine , Esser Mark Thomas , Bryna Janine
Published: June 2021

Human papillomaviruses (HPV) infect epithelial cells, including the epithelium of mucous membranes. GARDASIL™ was approved for the prevention of genital warts, vaginal, vulvar and cervical cancer caused by HPV types 6, 11, 16 and 18. A competitive Luminex immunoassay (cLIA) has been developed to measure serum levels of virus‐like particle (VLP) type‐specific antibodies. The cLIA uses neutralizing, type‐specific, R‐phycoerythrin‐labeled monoclonal antibodies to monitor the antibody response to type‐specific, neutralizing epitopes on the VLPs. Antibody responses to two distinct neutralizing epitopes on VLP‐6 were characterized using monoclonal antibodies H6.M48 and H6.1C2. Both the H6.M48 and H6.1C2 mAbs bind to HPV‐6 VLPs. Competition assays showed that the H6.M48 and H6.1C2 bind to type‐specific, overlapping, neutralizing epitopes. Avidity analysis shows that the H6.1C2 binds to the HPV 6 VLP with greater avidity than the H6.M48 mAb. Sera from individuals with confirmed HPV‐6 infection, a history of genital warts or sera from females who had been vaccinated with GARDASIL were tested in competition experiments with both of the mAbs. Results show that the H6.1C2 mAb binds to a more immunodominant epitope than the H6.M48 mAb as recognized by serum antibodies from both vaccinees and naturally infected individuals as indicated by a high prevalence in such antibodies in sera collected.
Chimeric Solid Tissue Grafts Diminish Allospecific Immunity
Authors: Saban Daniel Raphael , Chauhan Sunil K , Dana Reza
Published: June 2021

Purpose: All layers of a solid tissue graft do not contribute equally to rejection. In the cornea, corneal graft epithelium immunizes the host since it bears the crux of the antigenicity, while corneal graft endothelium is the critical target in rejection. We therefore hypothesized that chimeric corneal grafts, consisting of allodisparate epithelium (EPI) versus stroma/endothelium (S/E), can diminish allospecific immunity to graft S/E, and whether this can promote allograft survival.

Methods: Donor corneal layers were separated for reassembly into chimeric allografts (C3H/He EPI + C57BL/6 S/E, or v.v.), and orthotopically placed onto BALB/c hosts. In vitro recall stimulation of host T cells with APC allomatched to graft S/E was used to measure allospecific immunity. Graft survival was also evaluated, via slit‐lamp biomicroscopy.

Results: Relative to conventionally allografted hosts, T cells from chimeric engrafted hosts showed reduced levels of proliferation, IFN‐γ secretion, and CD4+ IFN‐γ+ frequencies to graft S/E. Also, an increase in graft survival of chimeric allografts (80%) over conventional allografts (30%) was observed.

Conclusions: Chimeric allografts diminish allospecific immunity to graft S/E. This is important because graft endothelium is the critical target in corneal graft rejection, and thus diminishing alloimmunity to this tissue can apparently enhance survival substantially.
Stimulation of antigen specific CD4+ T‐cells by B‐cells loaded with mycobacterial antigens.
Authors: Ramos Bernardo Villarreal , Mead Elinor , Siddiqui Nazneen , McAualay Martin , Coffey Tracey , Monaghan Paul , Hawes Pippa , Capinos Charles Scherer , Endsley Janice , Estes Don Mark
Published: June 2021

Protection against tuberculosis (TB) is thought to be mediated by Th1‐type responses. Development of antibody responses during TB is associated with disease progression. Development of antibody responses requires the interaction between B‐and T‐cells. To date, the role of B‐cells in the immune response to mycobacteria is not clear. Understanding the role of B‐cells in the immune response to mycobacteria and in particular, their possible role in the development of pathogenic responses, may help develop vaccines that avoid the induction of pathogenic responses and better diagnostics. In this work, we have used the bovine model to study the ability of B‐cells to stimulate mycobacteria‐specific T‐cell responses. We will present evidence that B‐cells are capable of presenting mycobacteria antigens to antigen‐specific CD4+ T‐cells and that the nature of the T‐cell response stimulated by B‐cells differs markedly from T‐cell responses stimulated by macrophages. The implications of the stimulation of CD4+ T‐cell responses by B‐cells, compared to T‐cell responses induced by macrophages will be discussed.
CD4+ CD25+ Foxp3+ Porcine Natural Regulatory T Cells Induced by Helminth Infection Display a Functionally Suppressive Immunomodulatory Phenotype
Authors: Reece Joshua J , Kohn Kristen , Ets Hillevi , Urban Joseph F , Dawson Harry D
Published: June 2021

CD4+/CD25+/Foxp3+ natural regulatory T cells (nTregs) limit immune responses to helminth infection and reduce inflammatory responses in rodent models. The phenotype of human nTregs is fairly well defined; but their role in immunity is largely unknown. We hypothesized that the phenotype of pig nTregs would be closer to human nTregs as compared to rodents due to greater overall immunological similarity. nTregs were characterized in pigs infected with Ascaris suum. We observed that the basal level of circulating nTregs in PBMCs closely resembled that of humans with approximately 2% of CD4+ T cells expressing CD25 (IL‐2Ra) and Foxp3. Like human nTregs, Con A‐stimulation of PBMCs resulted in higher FoxP3 mRNA and protein expression. Ascaris infection induced a consistently higher amount of PBMC CD4+/CD25+/FOXP3+ cells at 7, 14 and 21 days after infection as larvae migrated through the liver, lungs, and small intestine. Surface expression of CD25 was significantly higher in nTregs from Ascaris‐infected pigs. Infection with Ascaris also enhanced the transcription of CTLA4, PD1, and ICOS. Significant Ascaris‐induced enhancement of Con A‐stimulated PBMC mRNA expression of Foxp3, IL‐10 and TGF‐beta was also observed. These data provide the first characterization of nTregs in the pig during helminth infection and support a role for activated Tregs during Ascaris infection of its natural host. Funded by ARS/USDA.
A Study on Relations between Biomarkers and Risk of Diabetes
Authors: Liu Shumei
Published: June 2021

To identify diabetes‐related biomarkers, we recruited 1074 participants with multistage, randomized cluster sampling scheme based on economic levels. Interviewer‐administered questionnaires were employed for collecting data on health, physical activity and dietary intakes. Blood samples were tested in participants available. The overall prevalence of diagnosed diabetes, based on fasting glucose and 2‐h post glucose load (OGTT), was 2.8%, including 2 % for newly diagnosed diabetes. As risk factors, obesity (esp. central adiposity), levels of hemoglobin and serum ferritin, and dyslipidemia affected diabetes at different levels when those factors were treated as monofactorial variables. Higher levels of hemoglobin, serum ferritin, total cholesterol, triglyceride and low density lipoprotein cholesterol, and lower level of high density lipoprotein cholesterol increased risk of prediabetes/diabetes. Persons with normal hemoglobin had higher risk to suffer from diabetes than those with anemia and furthermore the pattern scores of “Diabetes risk dietary pattern” were closely related to levels of hemoglobin. Although higher level of serum ferritin increased risk of diabetes, this effect appeared independent of the dietary pattern identified. In addition, the study showed that dyslipidemia was an important risk factor of prediabetes and diabetes, and the pattern deteriorated dyslipidemia.