bluuebunny/crossref_metadata_2025 huggingface dataset:
Memory cells derived from homeostatic proliferation defer to true memory during infection
Authors: Cheung Kitty , Goldrath Ananda
Published: June 2021

In the absence of antigen, CD8+ T cells derived from lymphopenia induced homeostatic proliferation (HP) acquire many phenotypic markers and functional characteristics typical of true memory CD8+ T cells. To understand better the basis of competition in the memory compartment, we explored the differences between these two populations. We performed a mixed adoptive transfer of HP memory‐like T cells and true memory CD8+ OT‐I T cells into the same recipient, followed by infection with L. monocytogenes expressing ovalbulmin. In the context of the mixed transfer, we observed differences that were not apparent in single transfers of each cell type alone. When in competition with one another, we found that the HP memory‐like T cells underwent reduced expansion and failed to form an equivalent amount of secondary memory compared to the true memory CD8+. The HP memory‐like T cells also preferentially localized to the lymph nodes, while the population of true memory T cells dominated in all the other tissues, including the spleen, lung, liver, and bone marrow. These results point to the existence of previously unknown differences between HP memory‐like and true memory CD8+ T cells arising in the context of competition. Future experiments will focus on elucidating the nature of the defective immune response by the HP memory‐like CD8+T cells.
B cells fail to mature or form LZ despite GC formation in CD19‐deficient mice
Authors: Myers Riley C , Carter Robert H
Published: June 2021

Germinal Centers (GC) are crucial to humoral immunity. T cells and follicular dendrtic cells (FDC) work in concert to promote antigen (Ag) specific B cell survival and maturation into antibody forming cells. CD19 is required for proper B cell activation and normal humoral responses. Therefore, we used CD19‐deficient mice to delineate the events leading to GC formation. Mice lacking CD19 do not form GC in the spleen or draining lymph nodes (DLN) in response to SRBC, and as a result, fail to activate FDC in order to receive survival signals. When wild‐type B cells were adoptively transferred into CD19‐deficient mice, GC formation and FDC activation were rescued. In contrast, GC form and FDC are activated in the mesenteric lymph nodes (MLN) of CD19‐deficient mice, presumably due to constant stimulation by Ag. Despite the presence of GC and activated FDC, GC B cells in the MLN of CD19‐deficient mice fail to downregulate IgD and CD38 compared to wild‐type GC B cells, indicating defects in maturation. Furthermore, the light zone (LZ) within these GC is almost nonexistent. Collectively, these results show that splenic and DLN FDC are inactive in CD19‐deficient mice after SBRC challenge, but despite B cell and FDC activation in the MLN of CD19‐deficient mice, CD19 is still required for GC B cells to fully mature and enter the LZ. Supported by NIH R01AI42265.
Defective influenza‐specific B cell responses in mice lacking expression of ST6Gal‐I
Authors: Onami Thandi M. , Zeng Junwei , Joo Hye‐Mee , Wrammert Jens P. , Ahmed Rafi , Sangster Mark Y.
Published: June 2021

Post‐translational modification of proteins, such as glycosylation can affect the localization of cell surface glycoproteins on the cell membrane, impacting cell signaling and function. ST6Gal‐I is a glycosyltransferase expressed by T and B cells that catalyzes the addition of alpha2,6 sialic acid to galactose, a modification typically found on N‐linked glycoproteins. In this study, we show that in contrast to activated T cells, ST6Gal‐I expression remains high on plasma blasts and germinal center B cells following viral infection. To determine the in vivo role of the loss of this enzyme during viral infection, we infected ST6Gal‐I null mice with influenza A/HK x31. We demonstrate that loss of ST6Gal‐I expression results in similar infectivity in the lung at day 3, but significantly reduced influenza‐specific IgM and IgG levels in the serum, as well as significantly reduced antibody‐forming cells (AFCs) in the MedLN and spleen in the acute phase of influenza infection. Following the acute phase of the response, we measured similar overall levels of influenza‐specific antibody in the serum and similar numbers of influenza specific memory B cells. These studies suggest that loss of ST6Gal‐I expression significantly impairs the acute viral specific B cell immune response and that changes in surface carbohydrate modification may affect B cell differentiation in vivo.
A Novel Model of IVIG Immunosuppression: Activation of Natural Regulatory T cells by IgG‐Derived Peptides
Authors: Rivera Daniel S , Martin William , Wambre Erik , Van Overvelt Laurence , Scott David W , Knopf Paul M , Moingeon Philippe , De Groot Anne S
Published: June 2021

Background. Intravenous IgG (IVIG) is widely used to suppress immune responses in autoimmune diseases, allergy and transplantation. However, the exact mechanism responsible for its immunosuppressive/tolerogenic activity remains unknown. We hypothesized that the immunosuppressive effect of IVIG may be due to the presence of T cell epitopes in IgG that are capable of activating natural regulatory T cells (Tregs), leading to the suppression of T effector immune responses.

Methods and Results. Using the EpiMatrix algorithm, we identified three highly promiscuous MHC Class II T‐cell epitopes in both the heavy and light chain constant domains of IgG. These epitopes were shown to bind multiple HLA Class II molecules with high affinity and specifically upregulated FoxP3 expression in CD4+CD25high T cells. Co‐administration with various antigens led to suppression of effector T cell responses an increase in IL‐10 and CTLA‐4 expression.

Discussion. We believe that these data point to a previously undescribed mechanism for the immunosuppressive activity of IVIG. In this model, regulatory T‐cell epitopes (Tregitopes) in IgG are co‐processed and co‐presented with immunogenic epitopes derived from an antigen. The IgG Tregitopes activate a subset of natural regulatory T cells that can, at low dose of antigen, tip the resulting immune response towards tolerance rather than immunogenicity.

All work was funded by EpiVax Inc.
CD4+FoxP3+ Tregs are activated by injury
Authors: Tajima Goro , MacConmara Malcolm P. , O’Leary Fionnuala , Delisle Adam J. , Mannick John A. , Kuchroo Vijay K. , Lederer James A.
Published: June 2021

Enhanced CD4+CD25+ regulatory T cell (Treg) potency occurs following injury suggesting that Tregs may play a natural role in controlling the host response to injury. This study addresses whether injury can activate Tregs. To accomplish this, we used a FoxP3‐GFP knock‐in mouse as a tool to accurately identify and measure phenotypic changes in FoxP3‐expressing Tregs in sham versus injured mice. FoxP3‐GFP mice were anesthetized and underwent sham or burn injury. At days 1, 3, and 7 after injury, mice were sacrificed and cells were prepared from the injury site, lymph nodes draining the injury site, and spleen. Cells were stained to detect CD4 T cells and counterstained to detect changes in CD25, CD69, CD62L, ICOS, PD‐1, and CTLA‐4 expression levels. We found that injury caused a significant increase in the percentage of FoxP3+ Tregs in the lymph nodes draining the injury site at all time points after injury. In contrast, significant increases in Tregs were not detected until 7 days after injury at the site of injury and in the spleen. The results of experiments testing for phenotypic changes in Tregs after injury showed that injury induced increased expression of CD25, CD62L‐low, ICOS, PD‐1, and CTLA‐4 on lymph node and spleen FoxP3+ Tregs as early as 1 day after injury. At 7 days after injury, FoxP3+ CD4 T cells showed further signs of activation and expressed significantly higher levels of ICOS and CTLA4. We confirmed that injury activates Tregs by performing an adoptive transfer of FoxP3‐GFP+ cells into CD4−/− mice and showing that Tregs from burn mice expressed an activated phenotype. Thus, Tregs are activated by injury and injury can increase the percentage of Tregs at the site of injury, the draining lymph nodes and spleen. Therefore, the enhanced Treg function that occurs after injury might be due to the injury‐induced activation of FoxP3+ Tregs.
The PTPN22 allelic variant associated with autoimmunity impairs B cell signaling
Authors: Arechiga Adrian F. , Buckner Jane H.
Published: June 2021

PTPN22 is a gene encoding the protein tyrosine phosphatase Lyp in humans. A missense mutation in the gene, changing residue 1858 from a cytosine to a thymidine (1858 C/T), is associated with the development of multiple autoimmune disorders. The mechanism by which the 1858T variant contributes to the development of autoimmunity remains elusive. Although studies have demonstrated that Lyp has an inhibitory effect on T cell receptor mediated signaling pathways its role in B cell signaling has not been examined. In this study, we examine the impact of the 1858T variant on B lymphocyte function. B lymphocytes from healthy subjects expressing the variant allele were compared to those of healthy subjects without the variant. When B cells were stimulated via the B‐cell receptor, we observed impaired BCR signal transduction among subjects possessing the variant allele. This defect in signaling is characterized by a deficit in proliferation, diminished calcium mobilization and decreased phosphorylation of key signaling proteins including Syk, which we further demonstrates interacts with Lyp. These findings suggest that the PTPN22 1858T variant alters BCR signaling and implicates B cells in the mechanism by which the PTPN22 1858T variant contributes to autoimmunity. This work was supported by funds from the Juvenile Diabetic Research Foundation (the Center for Translational Research at Benaroya Research Institute).
TCR Dβ sequence dictates the amino acid composition of CDRβ3 in mature T cells
Authors: Schelonka Robert Lewis , Kapoor Pratibha , Zhaung Yingxin , Sleckman Barry , Schroeder Harry W
Published: June 2021

The coding sequence of the TCR diversity gene segment is highly conserved. Human Dβ1 is base for base identical in sequence to that of trout Dβ1. We have previously shown that the sequence of immunoglobulin diversity sequences control the Ig CDR‐H3 amino acid repertoire and can influence B cell development, antibody production, and resistance to infection. To test the role of TCR Dβ sequence in regulating TCR CDRβ3 diversity, we targeted a 129 ES cell that had previously undergone Dβ2‐Jβ2 deletion and replaced the germline Dβ1 with a sequence encoding charged amino acids in all three reading frames. After obtaining germline transmission, we cloned and sequenced TCRβ transcripts from the blood of mice heterozygous for the TCR Dβ beta 1 mutation. Approximately one half of the transcripts had identifiable mutant TCR Dβ1 sequence. These sequences were enriched for charged amino acids and created a repertoire distinct and separate from the repertoire expressed by those transcripts encoded by wild‐type Dβ as well as those expressed by wild‐type littermate controls. These studies demonstrate that T cells use conserved diversity gene segment sequence to delimit the TCRβ repertoire.
The p85α regulatory subunit of phosphatidylinositol 3‐kinase critically modulates class switch DNA recombination
Authors: Xu Zhenming , Zhang Jinsong , Ho Yin‐Wei , Ho Yin‐Wei , Al‐Qahtani Ahmed , Pone Egest , Zan Hong , Casali Paolo
Published: June 2021

Somatic hypermutation (SHM) and class switch DNA recombination (CSR) are central to the maturation of the antibody response. The PI 3‐K signaling plays an important role in the B cell development, proliferation and survival. PI 3‐K was also suggested to negatively regulate CSR by inhibiting AID expression and/or promoting plasmacytoid differentiation. Here, we directly addressed the role of PI 3‐K in CSR and, thereby, the maturation of the T‐dependent antibody response, using mice that are deficient in p85α, the predominant Class I PI 3‐K regulatory subunit. We found that the p85α‐deficiency did not significantly alter proliferation of in vitro stimulated B cells, but significantly decreased CSR to IgG1 or other isotypes, mainly due to a decreased proportion of switched B cells that had undergone five or more divisions. While B cell receptor (BCR)‐crosslinking increased the overall degree of CSR inp85α−/− B cells, it did not rescue the defective CSR in p85α−/− B cells at high division numbers, suggesting a BCR signaling‐independent function of PI 3‐K in CSR. Accordingly, CSR in p85α−/− mice immunized with a T‐dependent antigen was impaired, leading to a three‐ to five‐fold decrease of antigen‐specific IgG1, as compared to their wildtype counterparts. Thus, p85α and, therefore, PI 3‐K signaling, plays an important role in CSR and an effective antibody response.

Supported by NIH grants AI 45011, AI 60573 and AR 40908.
Characterization of B16 mFL‐expanded DC populations in peripheral and GI lymphoid tissues
Authors: Papenfuss Tracey L. , Singh Sanjay , Whitacre Caroline C. , Boyaka Prosper N.
Published: June 2021

In vivo expansion of murine DCs can be accomplished by either human Flt3‐ligand (hFL) or murine FL (mFL). Recent work shows that mFL DCs more accurately model resting DCs. Furthermore, while mFL‐secreting B16 cells (B‐mFL) are used for in vivo expansion, it remains unclear whether tumor burden influences the DC generation. In this study, we evaluated the kinetics/phenotype of mFL DCs in vivo in peripheral and GI lymphoid tissues. Injection of 1x106 B‐mFL cells rapidly expanded DCs in both the peripheral lymph nodes (PLN) and spleen (SPL), which were markedly increased in size. MHC class II expression (PLN and SPL) and CD86 (PLN) decreased over time, as well as, the number of CD8a+ DCs within the SPL. We then refined the use of B‐mFL cells with mitomycine (Mit) treatment to prevent tumor growth. Mice injected with 2x106 B‐mFL cells (+) Mit showed increased weight of SPL (55–90%) and mesenteric LN (MLN) (20–30%). Cell yield was increased in the PLN and MLN (5–6x over controls) with a 1.5–2 fold increase in SPL and Peyer's patches (PP). mFL increased the yield of CD11c+ DCs most dramatically in the MLN (5–7x) and PLN (3–5x) and less so in the SPL (1.2–1.9 fold) with minimal effect on PP DCs. These studies demonstrate that B‐mFL cells (+)Mit is a viable means to expand DCs in peripheral and GI lymphoid tissues. Supported in part by NMSS FG1555‐A‐1 and NIH K01RR022198‐01A1.
Gene expression profiling identifies STAT3‐ as a novel pathway for immunomodulation by cholera toxin adjuvant
Authors: Lycke Nils Y
Published: June 2021

Cholera toxin (CT), an enterotoxin elaborated by Vibrio cholerae, is an efficient mucosal and systemic adjuvant. However, the pleiotropic effects associated with CT are still far from understood and both pro‐ and anti‐inflammatory activities have been reported. Here we have investigated global gene expression patterns following exposure to CT in murine B cells using microarrays to dissect possible immunomodulatory pathways that could explain its adjuvant function. Unlike many other adjuvants, we found that CT‐regulated genes were relatively few, but representing a wide range of functional groups. The transcriptome hosted, among others, up‐regulated genes encoding transcription factors, G‐proteins, cell cycle regulators and co‐stimulatory molecules. We focused on STAT3 and found that at 8h of exposure to CT, 7–8‐fold increased gene expression and protein production levels were recorded. At this time the B cells also showed increased sensitivity to IL‐10 and IL‐6 induced STAT3 phosphorylation. Moreover, after i.v injections with CT isolated splenic B cells exhibited increased STAT3 protein production and level of phosphorylation. The modulating effect of IL‐6 and IL‐10 on B cell differentiation and antibody production was significantly affected by prior CT exposure and directly related to intracellular STAT3 levels.
Progesterone polarization of dendritic cell function varies during the estrus cycle
Authors: Butts Cherie L , Belyavskaya Elena , Sternberg Esther M
Published: June 2021

Elevated concentrations of steroid hormones in females can shift an immune response from a TH1 to TH2 or tolerogenic outcome. Dendritic cells (DCs) drive a variety of immune responses and may be the primary targets of steroid hormone actions. We previously showed that progesterone inhibits TH1‐promoting DC function and that these effects are mediated through the progesterone receptor (PR). As PR is differentially expressed during the estrus cycle, we wanted to determine the effects of progesterone on splenic and uterine DCs at the specific stages and how this might affect responses to pathogens. LPS‐stimulated DCs from females in metaestrus and diestrus were more sensitive to progesterone compared to those at proestrus and estrus and exhibited less robust T cell proliferative capacity in vivo. This effect correlated with PR expression and was reversed by the PR antagonist RU486. Furthermore, fewer DCs from females in metaestrus and diestrus expressed CCR6 compared to those in proestrus and estrus, indicating a limited ability to migrate, which was consistent with a reduced number of uterine DC at these stages. These data show that progesterone profoundly affects DC function in female rodents, depending on the stage of the estrus cycle and PR status, and that differences in susceptibility to infection and disease in females could be associated with modulated DC function.
Critical Amino Acid Residues in the B subunit of Type IIb Enterotoxin (LT‐IIb‐B5) for TLR2 Binding and Immunostimulation
Authors: Liang Shuang , Hosur Kavita B. , Nawar Hesham F , Connell Terry D. , Hajishengallis George
Published: June 2021

The pentameric B subunit of Type IIb heat‐labile enterotoxin of E. coli (LT‐IIb‐B5) can elicit TLR2‐dependent immunostimulatory activity and is a potential mucosal adjuvant. The upper region of the B pentamer contains a large hydrophobic surface. We identified 4 hydrophobic residues in this region that are involved in its interaction with TLR2. Specifically, point mutations rendering these residues hydrophilic all resulted in molecules that failed to bind TLR2. However, these mutants kept the ability to bind to ganglioside GD1a, indicating that the structure of the mutants was preserved. The mutants lost immunostimulatory function since wild‐type LT‐IIb‐B5, but not the mutants, induced TLR2‐mediated NF‐κB activation and cytokine production. Wild‐type LT‐IIb‐B5 moreover displayed the capability to induce up‐regulation of Class II MHC and co‐stimulatory molecule expression on dendritic cells, whereas the mutants lacked these capabilities. Furthermore, maturation of dendritic cells by LT‐IIb‐B5 could boost helper T cell proliferation and IL‐2 production, which may be one of the mechanisms through which LT‐IIb‐B5 fulfills its potential adjuvant function.
Effector CD4+ T lymphocytes resolve acute herpes simplex virus (HSV‐1) infection at both genital and neuronal sites
Authors: Johnson Alison J. , Chu Chin‐Fun , Milligan Gregg N.
Published: June 2021

In primary infection, CD8+ T cells are important for clearance of infectious HSV from sensory ganglia. We present evidence for CD4+ T cell‐mediated clearance of infectious HSV‐1 from genital tract and neuronal tissues. HSV‐specific CD4+ T cells were present in genital and neuronal tissues coincident with HSV‐1 clearance. Neuronal CD4+ T cells secreted IFN‐γ, TNF‐α, IL‐2 or IL‐4 after stimulation with HSV antigen. By adoptively transferring CD4+ T cells to Rag1−/ − mice, we showed that CD4+ T cells were sufficient for clearance of HSV‐1 from the genital tract. Compared to controls that did not receive T cells, CD4‐recipient mice had dramatically lower viral titers in the spinal cord and sensory ganglia, suggesting CD4+ T cells were active in clearing infectious HSV‐1 from neuronal tissue. To examine possible mechanisms by which CD4+ T cells resolve the neuronal HSV‐1 infection, CD4+ T cells from either perforin‐ or FasL‐deficient mice were adoptively transferred to Rag1−/ − mice. Clearance of infectious virus from genital and neuronal tissues was not significantly different in mice receiving these deficient T cells, compared to mice receiving wild‐type cells. These results suggest CD4+ T cells are important for resolution of primary HSV‐1 infection at both genital and neuronal sites, possibly via a non‐lytic mechanism. Supported by NIH grants AI42815, AI054444. Vale‐Asche Predoctoral Fellowship to AJJ.
Kinetic Analysis of a Complete Poxvirus Transcriptome Reveals a Novel Class of Genes
Authors: Assarsson Erika , Greenbaum Jason A. , Sundstrom Magnus , Schaffer Lana , Pasquetto Valerie , Oseroff Carla , Tscharke David , Sidney John , Grey Howard , Head Steven , Peters Bjoern , Sette Alessandro
Published: June 2021

Vaccinia virus is the prototypic orthopoxvirus and was the vaccine used to eradicate smallpox, yet the expression profiles of many of its genes remain unknown. Using a genome tiling array approach, we simultaneously measured the expression levels of all 223 annotated vaccinia virus genes during infection and determined their kinetics. Almost all of the genes were transcribed, and for 62 of these, this is the first empirical evidence of expression. Most remarkably, classification of the genes by their expression profiles revealed 35 genes exhibiting immediate‐early expression. Although a similar kinetic class has been described for other virus families, this is the first demonstration of its existence in orthopoxviruses. Despite expression levels higher than for genes in the other 3 kinetic classes, the functions of more than half of these remain unknown. Additionally, genes within each kinetic class were spatially grouped together in the genome. This genome‐wide picture of transcription alters our understanding of how orthopoxviruses regulate gene expression.

This work was supported by National Institutes of Health grants ROI‐AI‐56268 and HHSN266200400124C.
The missing link in the transfer of antigen between the dendritic cells after HSV infection.
Authors: KUMARAGURU UDAY , Woolard Stacie N , Nandakumar Subhadra
Published: June 2021

NK cells can be recruited to inflamed tissues and meet with the immature DC within a short period of time after HSV infection. Evidence from a recent study suggests that antigen bearing DC (CD205+CD11c+CD11b‐CD8a‐) that migrate to the lymphoid organs do not prime the T cells. They transfer the antigen to lymphoid resident DCs (CD205+CD11c+CD11b‐CD8a+) which prime a potent T cell response. The mechanism of transfer is still not clear. We believe the missing link in enabling this transfer is the NK cell. We propose that the NK cell uses the TNF receptor apoptosis inducing ligand (TRAIL) to deliver the death signal to the antigen bearing migratory iDC.

We used ex vivo and in vivo assays to assess the role of NK‐TRAIL. DC and NK isolated from mice infected on FITC painted skin were used. They were cocultured in the presence of anti‐TRAIL antibody or control IgG. The skin DCs were analyzed for apoptosis and uptake by LN‐ DC by gating on FITC positive cells. NK cells isolated from TRAIL knock out bone marrows were assessed for their ability to lyse HSV infected migratory DCs. The LN‐DCs were then assessed for their ability to stimulate T cells in vitro and in vivo. Results suggest a series of events, which include DC‐induced NK‐cell proliferation, NK‐mediated killing of antigen bearing DCs, NK‐dependent DC maturation and cross‐presentation.

We suggest the following sequence of events to take place after HSV infection. HSV prevents maturation of DCs, but upon arrival at the lymphoid organ stimulate the NK cells, they reciprocate by killing them through TRAIL‐induced apoptosis. The resulting inflammatory milieu than aids the lymphoid resident DC to take up the antigen bearing dead DC, processes and cross‐presents the viral epitope resulting in optimized immune activation.
CD28/B7 CO‐STIMULATION REGULATES MEMORY CD4 T CELL RESPONSES TO INFLUENZA VIRUS INFECTION
Authors: Teijaro John Ross , Ndejembi Modesta , Chandran Smita , Farber Donna
Published: June 2021

The host immune response to many viral infections is often accompanied by immunopathology. We have established that memory CD4 T cells specific for influenza virus hemagglutinin (HA) facilitates viral clearance in the context of increased weight loss and lung mononuclear cell infiltration. Work in the lab demonstrated that the CD28/B7 co‐stimulatory pathway alters cytokine production, expansion and subset distribution of antigen‐specific memory CD4 T cells upon recall. We hypothesized that modulation of the memory CD4 T cell response by blocking CD28/B7 might optimize protection to influenza virus challenge. To test this hypothesis we adoptively transferred HA ‐specific memory CD4 T cells into naive BALB/c mice, which were treated with CTLA4Ig or IgG2a control and infected with influenza virus PR8 (A/PR8/34). Interestingly, CTLA4Ig treatment modestly inhibited CD4 memory‐mediated viral clearance; however, CTLA4‐Ig treated hosts exhibited significant reduction in weight loss. Treatment with CTLA4Ig also resulted in a shift of the memory CD4 T cells to a predominantly CD62Lhi central memory phenotype accompanied by inhibition of recruitment of memory and endogenous CD4 T cells to infected lungs. These results suggest that blocking the CD28/B7 co‐stimulatory pathway controls memory‐mediated responses to influenza by limiting pathology while maintaining protection.
Comparative analysis of genes induced by respiratory syncytial virus and dsRNA in primary human bronchial epithelial cells
Authors: Limmon Gino V , McCann Kelly L , Ducharme Danica M , Imani Farhad
Published: June 2021

The primary target of respiratory viruses such as respiratory syncytial virus (RSV) is the respiratory epithelium. Accordingly epithelial cells may play a pivotal role in virus‐induced lung inflammation and in the initiation of innate and subsequently adaptive immune responses. Presence of double‐stranded RNA (dsRNA) during viral infections is a potent inducer of inflammatory cytokines and antiviral responses and dsRNA has been extensively used as a viral mimetic. In this study, we compared the gene expression profiles of RSV‐infected or dsRNA treated human primary bronchial epithelial cells (PHBE). Our transcriptomic analysis showed that RSV induced up‐regulation of 561 genes in PHBE cells while treatment with dsRNA upregulated 77 genes. Our comparison showed that 44 genes were commonly upregulated by dsRNA treatment and RSV infection. The mRNA and protein expression of select genes were confirmed with real‐time RT‐PCR and bioplex assay. Further analysis of these common upregulated genes revealed that they could be functionally grouped in multiple response pathways such as inflammation, apoptosis, transcription regulation, cell signaling, and cell differentiation. This high‐throughput gene expression study offers a comprehensive view of transcriptional changes induced by dsRNA and RSV in human epithelial cell.
Cross‐presentation of Influenza virus vaccine antigens to CD8+ T cells in humanized mice
Authors: Yu Chun I , Gallego Mike , Marches Florentina , Zurawski Sandra , Ramilo Octavio , Zurawski Gerard , Garcia‐Sastre Adolfo , Banchereau Jacques , Palucka A. Karolina
Published: June 2021

Critical to the development of novel vaccines is the availability of in vivo models of the human immune system that permit testing of vaccine efficacy. Here, we used NOD/SCID beta2m −/− immunodeficient mice which, when engrafted with human CD34+ hematopoietic progenitors, develop all subset of human dendritic cells (DCs) and B cells. T cells and their subsets can be reconstituted by adoptive transfer. We show that these mice can generate recall CD8+ T cell responses upon exposure to seasonal influenza virus vaccines i) live attenuated trivalent vaccine, i.e. FluMist; or ii) killed trivalent vaccine, i.e., Fluzone. CD8+ T cells specific to at least two influenza antigens FluM1 and NS1 can be detected in the blood of mice vaccinated with FluMist but not in mice vaccinated with Fluzone. Specific T cells are also present in the spleen and peripheral tissues (lung) demonstrating that human T cells can utilize murine signals for trafficking and extravasation. Upon short‐term ex vivo antigen exposure CD8+ T cells produce IFN‐g and express surface CD107 consistent with their acquisition of effector function. Response to vaccination is dependent upon reconstitution of the human hematopoietic system. Thus, vaccine antigens can be cross‐presented by endogenous human antigen presenting cells in humanized mice. Therefore, this model might be useful for testing vaccines oriented towards generation of cellular immunity.
CTA1‐3M2e‐DD; a universal influenza vaccine
Authors: Lycke Nils Y , Eliasson1 Dubravka Grdic , Saelens Xavier
Published: June 2021

At present no vaccine against potentially pandemic influenza A virus infections exists. In the present study we provide compelling evidence that a targeted fusion protein based on the CTA1‐DD adjuvant and containing tandem repeats of the conserved M2e ectodomain epitope, CTA1‐3M2e‐DD, confers strong protective immunity against a lethal challenge infection with influenza A virus in mice. Intranasally immunized mice survived the challenge and showed a dramatic reduction in lung viral load and less morbidity, as assessed by loss of body weight and temperature decrease. The adjuvant component was highly effective for mucosal immunizations and readily promoted specific serum IgG1 and IgG2a as well as mucosal IgA antibody levels. Importantly, we found a hitherto unknown MHC class II restricted T cell epitope in the M2e peptide, resulting in strong anti‐M2e IFNg T cell responses, which may have significant impact on the efficacy of this universal anti‐influenza A virus vaccine. The CTA1‐3M2e‐DD stimulated stronger responses than the single CTA1‐M2e‐DD construct and comparable responses to a preparation of M2e‐virus like particles based on Hepatitis B virus core subunits. Taken together, CTA1‐3M2e‐DD combines a molecular adjuvant, a universal influenza A vaccine antigen and a targeting domain into a single recombinant protein.
Persistence of Infection Contributes to Heterologous Protection against Ehrlichiosis
Authors: Thirumalapura Nagaraja , Crossley Emily , Walker David H , Ismail Nahed
Published: June 2021

Human monocytotropic ehrlichiosis (HME) is an emerging life‐threatening tick‐transmitted disease caused by the obligately intracellular bacterium, Ehrlichia chaffeensis. HME is modeled in mice using Ehrlichia muris, which causes persistent infection, and Ixodes ovatus Ehrlichia (IOE), which is either acutely lethal or cleared depending on the dose and route. Primary sublethal IOE infection does not protect mice against secondary IOE challenge, but persistent E. muris infection confers heterologous immunity against IOE. The objective of this study is to determine the role of bacterial persistence in the maintenance of protective immunity against IOE. E. muris‐infected mice were treated with doxycycline or left untreated and then challenged with an ordinarily lethal dose of IOE. The cytokine production profiles of splenic memory T cell subsets were assessed by flow cytometry. Doxycycline treatment affected neither the numbers of antigen‐specific, IFN‐γ‐producing CD4+ and CD8+ T cells nor the bacterial burden following IOE challenge. However, spleens from treated mice had fewer numbers of effector memory (CD44highCD62Llow) CD8+ T cells and produced less TGF‐β1. Histology and TUNEL assay revealed greater numbers of inflammatory cells and more extensive apoptosis in the liver of treated mice following IOE challenge. These results suggest that the persistence of E. muris infection contributes to protective heterologous immunity, possibly by inducing the accumulation of regulatory cells at the sites of infection.
Substance P and CGRP are involved in the formation and maintenance of microabscesses in chronic infection with Nocardia brasiliensis
Authors: SOTO JUAN MANUEL SOLIS , Serrano J A , Ramirez J C Segoviano , Kouri J B , Carmona M C Salinas
Published: June 2021

Actinomycetoma is caused by several pathogens; the main causal agent in Mexico is Nocardia brasiliensis. It is characterized by an infiltration of several inflammatory cell populations and the formation of microabscesses. To explore pathogenetic aspects of the immune response we injected N. brasiliensis in footpads of BALB/c mice. Seven, fifteen and thirty days postinfection immunocytochemical and ultrastructural studies were performed to compare presence and distribution of substance P and calcitonin gene‐related peptide (CGRP) during the formation of microabscesses. Analysis of serial skin sections showed colocalization of these immunomodulators in some macrophages (CD14 immunoreactive) at the periphery of the microabscesses. Substance P was also present in a few macrophages of the dermis, outside the microabscesses. Ultrastructural studies showed a grain of bacteria in the center of the microabscess. Surrounding the bacteria a layer of polymorphonuclear cells were present. Around the polymorphonuclear cell layer several lymphocytes (CD3) and macrophages were producing the cytokines IFN‐gamma, IL‐1beta, IL‐10 and IL‐4.

The distribution of substance P and CGRP suggests that they act as immunomodulators of the formation and maintenance of the microabscesses during chronic infection with N. brasiliensis.

This work was supported by grants from PROMEP 103.5/05/2485 and PAICYT SA1420‐06 (UANL).
Comparative studies on the immunogenicity of theronts, tomonts and trophonts of Cryptocaryon irritans in grouper
Authors: Li Anxing , Bai Jianshan , Xie Mingquan , Zhu Xingquan
Published: June 2021

Cryptocaryon irritans is a ciliated obligate parasite of marine fish, causing white spot disease in tropical and subtropical regions. Recent studies have shown that fish can mount immune response against C. irritans and acquire protection. The present study compared the protective immunity in grouper (Epinephelus coioides) against C. irritans induced by theronts, trophonts and tomonts via intraperitoneal (i.p.) injection, and tentatively identified an immobilization antigen. Fish immunized with theronts produced higher levels of serum antibody against C. irritans than fish immunized with trophonts or tomonts. Significantly higher level of immune protection was achieved in fish immunized with theronts, but not in fish immunized with tomonts or trophonts. Western blot analysis using polyclonal immobilization antibodies of rabbit immunized with theront revealed an immunoreactive band of the protective (immobilization) antigens of C. irritans, the size of which is approximately 34 kDa. The sera of mice immunized with the protein could immobilize theronts of C. irritans. These results demonstrated that the theront stage of C. irritans elicited stronger protective immunity than the trophont and tomont stages in grouper. The tentative identification of protective antigen provides the solid foundation for the development of a defined vaccine against C. irritans.
CD16 is detrimental in sepsis by inhibiting E. coli phagocytosis and enhancing inflammation through a negative FcRγ signaling
Authors: Silva Fabiano Pinheiro da , Aloulou Meryem , Skurnik David , Benhamou Marc , Chiamolera Murilo , Velasco Irineu , Andremont Antoine , Verbeek Sjef , Launay Pierre , Monteiro Renato C
Published: June 2021

Pathogen‐induced immune dysregulation is largely responsible for the morbidity and mortality associated with sepsis. Sepsis also involves inefficient bacterial clearance by phagocytic cells. Here we show that FcRγ, the ITAM‐bearing signal transduction subunit of the Fc receptor family, plays a deleterious role in sepsis. FcRγ−/− mice show increased survival during peritonitis, due to markedly increased E. coli phagocytosis and killing and to lower production of the pro‐inflammatory cytokine TNFα. The FcRγ‐associated receptor that inhibits E. coli phagocytosis is FcγRIII (CD16), and its absence protects mice from sepsis. CD16 binds E. coli, and this interaction induces FcRγ phosphorylation, SHP‐1 recruitment and PI3‐K dephosphorylation. Decreased PI3‐K activity inhibits E. coli phagocytosis and increases TNFα production through TLR4. We identified the phagocytic receptor negatively regulated by FcRγ on macrophages as the class A scavenger receptor MARCO. E. coli‐CD16 interaction induces the recruitment of SHP‐1 to MARCO, thereby inhibiting E. coli phagocytosis. Thus, by binding CD16, E. coli triggers an inhibitory FcRγ pathway that both impairs MARCO‐mediated bacterial clearance and activates TNFα secretion. This mechanism could be the target of new therapeutic approaches in sepsis. Granted by Inserm, ANR‐Mime and FAPESP.
Uropathogenic E. coli subverts innate immune function of bladder epithelial cells
Authors: Trama Jason Peter , Hilbert David , Pascal Kristen , Libby Erika , Mordechai Eli , Adelson Martin
Published: June 2021

Urinary tract infections (UTI) are a serious cause of morbidity in the United States. Although uropathogenic E. coli (UPEC) accounts for 80–90% of UTI, UPEC colonization of the bladder is poorly understood. We hypothesize that evasion of the immune response plays a role in UPEC pathogenesis. Co‐culture of T24 bladder epithelial cells with non‐pathogenic E. coli (NPEC) resulted in induction of IL6 secretion, whereas UPEC co‐culture did not. A mixed UPEC/NPEC culture reduced IL6 secretion by T24 cells at least two‐fold compared to NPEC alone, suggesting that the inhibitory effect is dominant. To test this possibility, we exposed T24 cells to a mixture of UPEC and Toll‐like receptor (TLR) agonists. We found that UPEC reduced IL6 secretion in response to FSL‐1, Poly(I:C), and lipopolysaccharide (LPS). One possible mechanism of suppression is that UPEC LPS antagonizes TLR4 signaling. However, we found that LPS purified from both NPEC and UPEC induced secretion of IL6 to similar levels, and expression of a dominant negative TLR4 allele had no effect on inhibition. UPEC actively suppresses innate immune function in bladder epithelial cells, preventing secretion of cytokines in response to the bacteria themselves or to other TLR stimuli. This suppression is at least partially mediated by an LPS‐ and TLR4‐independent pathway. Subversion of the innate immune response likely plays an important role in UPEC pathogenesis.
Bacterial pore‐forming toxin effects on myeloid DC are mediated through purinergic receptors
Authors: Chu Jessica , Thomas Louis Michael , Watkins Simon C. , Salter Russell D.
Published: June 2021

Cholesterol‐dependent cytolysins (CDC), a class of gram‐positive bacterial exotoxins, bind cholesterol in cell membranes and oligomerize to form pores. While a small numbers of pores can be repaired by cells, the formation of many pores leads to cell death. Differences in susceptibility to CDC have been observed for various immune cells, suggesting potential differences downstream of CDC‐induced pore formation. In this study, we have addressed the mechanism by which exposure to CDC induces pores in myeloid lineage cells relative to their toxic effects. Using a cell line derived from fetal mouse skin with myeloid dendritic cell properties, we generated a toxin‐resistant cell line by selection with increasing amounts of CDC family member tetanolysin O. Using this variant cell line combined with pharmacologic inhibitor studies in primary myeloid cells, we show a process in which CDC‐induced pores allow rapid release of intracellular adenosine triphosphate (ATP) into the surrounding environment followed by activation of the cell surface ATP receptor P2X7, and subsequent pore formation and IL‐1beta release. Our study demonstrates that bacterial toxins can trigger an endogenous host cell inflammatory response that might both protect the host and induce tissue damage when excessive. Supported by R01 AI57168 and 5T32CA082084‐09 grants.
Selective CD4 T cell help for B cell and antibody responses to vaccinia
Authors: Crotty Shane , Moutaftsi Magdalini , McCausland Megan , Quiroz Juan Moyron , Garboczi David , Felgner Philip , Sette Alessandro
Published: June 2021

Selective CD4 T cell help for B cell and antibody responses to vaccinia

It remains unclear what parameters determine which pathogen proteins are targeted by the host antibody response. Peptide vaccination of mice with individual MHC II restricted vaccinia virus epitopes revealed that after vaccinia (smallpox vaccine) infection CD4 T cell help to B cells was surprisingly non‐transferable to B cells specific for different vaccinia virus proteins, including other virion surface or core proteins. This restriction had a direct impact on neutralizing antibody titers. Furthermore, large scale analysis of the antibody and CD4 T cell response specificities recognized during a vaccinia virus infection revealed that many of the CD4 T cell responses (identified in an unbiased screen) targeted viral virion proteins that were also targets of antibody responses, consistent with a deterministic linkage between the specificities (P < 0.0009). We then tested the deterministic linkage model by using this knowledge to efficiently predict new vaccinia virus MHC II epitopes (830% increase in identification efficacy). In contrast to the standard intermolcular help model, these data indicate that individual proteins are the primary unit of immunological recognition for a large virus, and therefore MHC II restriction at the protein level is a key selective event for the antiviral antibody response, which is likely of great relevance for vaccine development to large pathogens.
Diversity of neutralizing antibody responses in human smallpox vaccinees
Authors: RAFII‐EL‐IDRISSI MOHAMMED BENHNIA , McCausland M , Su Hua‐Poo , Singh K , Hoffmann J , DAVIES D. HUW , FELGNER P , HEAD S , SETTE A , GARBOCZI D , Crotty S
Published: June 2021

The smallpox vaccine (vaccinia virus, VACV) is the prototypic vaccine, yet the key antibody targets in humans remain unclear. We set out to identify a stereotypic, dominant, mature virion (MV) neutralizing antibody target in humans; but we have instead found that diversity is a defining characteristic of the human antibody response to VACV. We show H3 is the most immunodominant VACV neutralizing antibody target in humans, as determined by correlation analysis of MV neutralizing antibody titers to IgG of individual viral protein specificities. However, using multiple experimental approaches it was determined that while human anti‐H3 IgG is sufficient for neutralization of vaccinia, anti‐H3 antibodies are not required in vaccinated humans (or mice) for neutralization of VACV. We also detected positive correlations between D8, A27, D13, A14, and L1 IgG and virus neutralization activity in vaccinees. Importantly, no single one of these responses was consistently observed in all individuals. For example, L1, which is an immunodominant target in mice, is infrequently observed in immunized humans, but some individuals do have strong anti‐L1 IgG responses, and those antibodies possess virus neutralization activity. Our results suggest that VACV succeeds in driving strong neutralizing antibody responses to multiple viral proteins in vaccinees. We propose this is a fundamental attribute of the smallpox vaccine.
Comparisons of the regulatory function of ex vivo expanded polyclonal or antigen‐specific non‐human primate T regulatory cells
Authors: Johnson Jennifer Lynn , Pahuja Anil , Graham Melanie , Hering Bernhard , Blazar Bruce , Murtaugh Michael , Bansal‐Pakala Pratima
Published: June 2021

T regulatory (Treg) cells play a critical role in controlling autoimmunity, suppressing allo‐ and xenogenic immune responses and preventing transplant rejection. To assess therapeutic potential of ex vivo expanded Tregs in islet transplant, we established Treg expansion in non‐human primates. Work in mice shows that Tregs expanded against a specific antigen better suppress response to that antigen. In this study, we isolated and expanded polyclonal and antigen‐specific Tregs from NHP and evaluated their regulatory capabilities.

Tregs were isolated from rhesus macaque PBMCs and expanded with irradiated pig splenocytes or anti‐CD3/CD28 coated microbeads and IL‐2. FoxP3 expression at day 21 was 98% for both groups. Polyclonal stimulated Tregs (PST) underwent more robust expansion, compared to antigen specific Tregs (AST). AST more readily suppressed T effectors stimulated with specific xeno‐antigen when compared to PSTs.

Our data suggest that polyclonal and antigen specific expanded NHP Tregs maintain their regulatory activity. The success of these ex vivo experiments suggest that Tregs can be used as part of immunomodulatory protocol paving the way to their use in clinical transplantation.
NFAT1 Regulatory Mechanisms in UCB‐Derived CD4+ T‐cells: Implications for Graft vs. Host Disease (GVHD)
Authors: Weitzel Richard Patrick , Lesniewski Mathew , Laughlin Mary J
Published: June 2021

The transcription factor nuclear factor of activated T‐cells 1 (NFAT1) is a key regulator of both active immune responses and T‐cell anergy. We have previously demonstrated lower expression of NFAT1 protein but not its mRNA transcript in umbilical cord blood (UCB) CD4+ cells following primary stimulation when compared to cells obtained from adult blood (AB). We hypothesize a critical role for NFAT1 and NFAT1‐dependent factors in the increased proliferation, decreased cytokine production seen in UCB, and lower incidence of GVHD following UCB transplantation. We have demonstrated via RNAi‐mediated knockdown and RT‐PCR analysis of AB‐derived primary T‐cells that expression of a Th1‐like cytokine profile is dependent on expression of NFAT1, and that knockdown of NFAT1 results in a transcriptional profile similar to that seen in UCB T‐cells with respect to key cytokines such as IFN‐γ, and TNF‐α and signal transducers such as CTLA4 following stimulation. Time point analyses of NFAT1 transcript and protein levels under normal, knockdown, and induced expression conditions suggest a crucial regulatory role for post‐transcriptional events which may uniquely occur in UCB or AB‐derived T‐cells in vivo. Overall, our data indicates that regulation of NFAT1 and its subsequent downstream effects may constitute a key difference between T‐cells of adult and neonatal origin.
Gene expression of mesenchymal stem cell (MSC) involved in the regeneration of injured liver tissue
Authors: Cho Kyung‐Ah , Ju Sun‐Young , Ryu Kyung‐Ha , Woo So‐Youn
Published: June 2021

Mesenchymal stem cells (MSCs) are potential cell source for the development of therapeutic product. Recent studies reported that MSCs transplantation to the damaged organ including heart, liver and kidney result in restore of damaged tissue. Because it is not clearly revealed the mechanism involved in the regeneration process, we focused the therapeutic potential of MSCs in injured liver, and investigated the gene expression profiles of MSCs in presence of injured liver cell and normal liver cell using a microarray containing 40,000 genes. For inducing liver cell regeneration, we co‐cultured MSCs with CCl4 induced injured liver cell and normal liver cell, respectively. The cells were obtained from C57BL6 female mice. After 48 hrs of coculturing, MSCs were collected and RNA was extracted for microarray analysis. The data were normalized with 8 categories such as signal transduction, cell cycle, response to stress and immune response. Expressed genes were various under each conditions. For example, in presence of injured liver cells, MSCs highly expressed several genes involved in angiogenesis including fibroblast growth factor 1, prostaglandin‐endoperoxide synthase 2 and Tenascin C. Genes involved in cellular physiolosical process such as TYSP‐like 3, sentrin specific peptidase 6, activin A receptor type IC, intestinal cell kinase and pantothenate kinase also highly expressed. Our research may provide information on the differential molecular mechanisms regulating the MSC properties in regeneration of injured liver tissue.