bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel nanosize protein-carrier for the development of adjuvant- free nasal vaccine (39.33)
Authors: Yuki Yoshikazu , Nochi Tomonori , Mejima Mio , Kataoka Nobuhiro , Takahashi Haruko , Sawada Shinichi , Kohda Tomoko , Kazaki Shunji , Akiyoshi Kazunari , Kiyono Hiroshi
Published: January 2023

Most of inactivated or subunit type vaccines are poor immunogens to both systemic and mucosal immune compartments when given mucosally without mucosal adjuvant. We developed an effective intracellular vaccine delivery system with self-assembled nanosize cationic hyrogels (nanogel), which composed of a polysaccharide with ethylendiamine group of cholesteryl group-bearing pullulan. When hold protein antigen in a segregated nanomatrix, the nanogel acts as artificial chaperones, which protect against the aggregation of denature proteins and assist in the refolding of protein after release. In this study, we demonstrated that nasally administered nanogel holding a subunit type of C. botulinum antigen (BoHc-nanogel) was effectively taken up by nasophalynx-associated lymphoid tissue and induced brisk levels of antigen-specific systemic and mucosal immune responses even in the absence of mucosal adjuvant. In contrast, nasal administration with commercialized cationic liposome (Pro-ject) holding the same amount of BoHc without adjuvant induces antigen-specific low systemic and no mucosal antibody immune responses. In addition, mice nasally immunized with BoHc-nanogel protected not only systemically challenged but also nasally challenged botulinum toxin. These data suggest that nanogel-based mucosal vaccination is a novel strategy for the induction of protective immunity without the use of mucosal adjuvant.
Alveolar macrophages control the quality of CD4+ T lymphocytes in the respiratory tract upon recombinant adenoviral mucosal vaccination (39.34)
Authors: Acosta-Ramirez Elizabeth , Xing Zhou , Schwendener Reto , Wang Jun
Published: January 2023

Infectious diseases are acquired through mucosa surfaces making mucosal vaccination important for their control. Previously, using a recombinant adenoviral-based (Ad) vaccine we have demonstrated that a single mucosal but not parenteral immunization confers protection against intracellular bacterial challenge in the lung. Here, we examined the mechanisms regulating induction of OVA-specific CD4 T-cell responses at mucosal and systemic compartments in Balb/C mice after intranasal (i.n.) or intramuscular (i.m.) vaccination with an Ad expressing OVA. We found that i.n. vaccination induced local OVA-specific T cell activation whereas i.m. vaccination induced both local and systemic T-cell activation despite of the dosage used for vaccination. Remarkably, i.n. immunization induced a characteristic proliferative kinetic different from the one induced by i.m. vaccination. Furthermore, i.n. vaccination induced a highly polarized Th1 response while i.m. immunization induced a mixed Th1/Th2 response. Of importance, in vivo depletion of alveolar macrophages led to similar T-cell proliferation kinetics and comparable cytokine production in mice receiving i.n. and i.m. vaccination. Our data suggests that alveolar macrophages provide a special microenvironment at the mucosal site, which control the quantity and quality of CD4 T memory cells and protective immunity against mucosal infectious challenges.
Effects of growth phase and substrate on immunomodulatory activity of lactobacilli (39.37)
Authors: Cooper William , Masotti Adriana , Wagar Lisa , Champagne Claude , Buckley Nicole , Forrester Sean , Green-Johnson Julia
Published: January 2023

Immunomodulatory effects of lactic acid bacteria vary with strain and may vary with growth phase and medium. The ability of different lactobacilli strains (Lactobacillus helveticus R0052, L. rhamnosus R0011, L. rhamnosus GG) at different growth phases to modulate macrophage and intestinal epithelial cell cytokine production following a proinflammatory challenge was examined. Soy and milk ferments were also prepared with strains shown to grow well in soy milk (Bifidobacteria longum R0175 and Lactobacillus helveticus R0052). Modulation of cytokine production by human macrophage cell lines (U937 and THP-1) and intestinal epithelial cells (HT-29) induced by Tumor Necrosis Factor αwas assayed by ELISA for interleukin-8 (IL-8) production. Strain-dependent differences were observed in the ability of spent MRS broths from log versus stationary growth phase, and of soy and milk ferments to down-regulate IL-8 production by challenged U937 cells. Overall, variation in immunomodulatory activity of these lactic acid bacteria reflects not only strain variation but potentially also differences in growth phase and substrate.

Funded by NSERC, Lallemand Inc. and the Canadian Space Agency.
Effects of dietary algal supplementation on bovine immunity (39.50)
Authors: Green-Johnson Julia M , Johnson Kathy , Masotti Adriana , Wagar Lisa , Fillmore Sherry , Glover Kathleen , MacLaren Leslie , Enright Cathy , Fredeen Alan
Published: January 2023

Marine algae are primary producers of long chain omega-3 fatty acids and can serve as novel dairy feed supplements with the added potential to influence bovine immunity. Our objective was to assess the immunomodulatory impact of dietary supplementation with different algal types (macroalgae and microalgae). Effects of dietary algal supplementation were examined using a crossover design and a 28 day feeding period with each algal type. Primary immunization with Keyhole Limpet Hemocyanin (KLH) was carried out 8 days into the trial, with secondary immunization 14 days later. KLH-specific serum IgG responses were significantly higher in cows receiving macroalgae-supplemented feed than in cows receiving microalgae-supplemented feed. In contrast, cows receiving microalgae-supplemented feed showed significantly higher levels of macrophage oxidative burst activity in response to PMA challenge in vitro. These results suggest that dietary supplementation with algal products has immunomodulatory potential and also indicates that the effects on cellular and humoral immunity are dependent on the type of algae used.

Funded by the Atlantic Innovation Fund.
Stratification of antigen-presenting cells within the normal cornea (39.57)
Authors: Knickelbein Jared E. , Watkins Simon C. , Hendricks Robert L.
Published: January 2023

The composition and location of professional antigen presenting cells (APC) varies in different mucosal surfaces. The cornea, long considered an immune privileged tissue devoid of APCs is now known to host a network of bone marrow-derived cells. Here, we utilized transgenic mice that express EGFP from the CD11c promoter in conjunction with immunohistochemical staining to demonstrate an interesting stratification of APCs within non-inflamed murine corneas. CD11c+ DCs reside in the basal epithelium, many seemingly embedded in the basement membrane. Most DCs express MHC class II on at least some dendrites, which extended up to 50 µm in length and traversed up 20 µm tangentially nearly to the apical surface of the epithelium. The DC density diminished from peripheral to central cornea. Beneath the DCs and adjacent to the stromal side of the basement membrane reside CD11c-CD11b+ presumed macrophages that expressed low levels of MHC class II. Finally, MHC class II- CD11b+ cells filled the remainder of the stroma. This highly reproducible stratification of bone marrow-derived cells is suggestive of progression from APC to barrier function.
Expression of indoleamine 2,3-dioxygenase (IDO) in multiple myeloma promotes the generation of regulatory T cells: Investigations into the role of hepatocyte growth factor (40.1)
Authors: Rutella Sergio , Bonanno Giuseppina , Corallo Maria , Mariotti Andrea , Procoli Annabella , Majolino Ignazio , De Cristofaro Raimondo
Published: January 2023

Multiple myeloma (MM) is a plasma cell (PC) malignancy with a unique ability to evade immunosurveillance through the induction of APC dysfunction and the expansion of regulatory T cells (Treg). Hepatocyte growth factor (HGF) is elevated in MM and confers an unfavorable prognosis. We have previously shown that HGF induces the tryptophan (Trp)-degrading enzyme indoleamine 2,3-dioxygenase (IDO) in human monocytes.

In this study, IDO-1 was detected in OPM-2, MOLP-8 and LP-1 MM cell lines but not in HUNS-1 cells. Kynurenine (KYN) production and Trp depletion were evidenced in supernatants of IDO-1+ MM cells, suggesting that IDO-1 may be functional. When challenged with exogenous HGF, IDO- HUNS-1 cells up-regulated IDO-1 mRNA 6-fold on average. IDO-1+ MM cells promoted the conversion of allogeneic CD4+CD25- T cells into bona fide CD4+FoxP3+ Tregs.

We also detected IDO-1 expression in 6 out of 10 PC samples from patients with MM. Higher percentages of CD4+FoxP3+ Tregs were measured in IDO-1+ compared with IDO-1- MM. Intriguingly, serum KYN levels were higher in IDO-1+ compared with IDO-1- MM, suggesting that IDO-1 may mediate the in vivo expansion of Tregs through immunosuppressive KYN. Finally, serum HGF was over-represented in IDO-1+ compared with IDO-1- MM, providing indirect clues in favor of in vivo HGF effects on IDO-1 expression.

Collectively, these data point to HGF as a potential inducer of IDO-1 in MM. Whether this will impact on the ability of the immune system to control disease progression remains to be prospectively investigated in larger cohorts of patients.
Activation of anti-tumor NK cells by gamma delta T lymphocytes through 4-1BB engagement (40.26)
Authors: Chapoval Andrei , Maniar Amudhan , Zhang Xiaoyu , Gastman Brian R , Pauza C. David , Strome Scott E
Published: January 2023

Emerging evidence suggests that gamma delta (γd) T cells play a critical role in bridging innate and adaptive immune responses, yet the mechanisms responsible for these effects are uncertain. In this study we show that isopentenyl pyrophosphate (IPP) activated γd T lymphocytes induce cytolytic function of Ig stimulated NK cells. These "costimulated" NK cells up regulate the CD54 and CD69 markers of cellular activation and also display enhanced cytotoxicity against squamous cell carcinoma of the head and neck (SCCHN). Based on our findings that "co stimulation" of Ig primed NK cells by γδ T lymphocytes requires cell-to-cell contact, we performed a comprehensive expression analysis of TNFSF members expressed on the surface of IPP activated γδ T lymphocytes, and found that these cells express high levels of 4-1BB ligand. Stimulation of Ig primed NK cells with 4-1BBL but not mock transfected P815 cells, induces up regulation of CD54 and CD69 and blockade of 4-1BB/4-1BBL interaction by soluble 4-1BB fusion protein decreased activation of NK co-cultured with γδ T lymphocytes. In addition, we observed that NK cells cultured with Ig/γδ T lymphocytes up regulate NKG2D and anti-NKG2D mAb blockade partially abrogates the cytolytic effects of these "costimulated" NK cells against SCCHN. Taken in concert, our data indicate that γδ T lymphocytes induce activation of Ig primed NK cells through coordinated 4-1BB -4-1BBL interactions and up regulation of targeted cell death pathways.
Chronic alcohol consumption decreases NK cells in the peripheral lymph nodes and exacerbates B16BL6 melanoma metastasis into the draining lymph nodes (40.5)
Authors: Zhang Hui , Meadows Gary G.
Published: January 2023

Chronic alcohol consumption decreases NK cells in the spleen (SP) and compromises their release from the bone marrow (BM). NK cells in the lymph nodes (LN) are important to T cell priming and polarization. Little is known as to how alcohol consumption affects NK cells in the LN. To address this issue, female C57BL/6 mice were given 20% (w/v) alcohol in drinking water for 3-6 months. The percentage, number, phenotype and functions of NK cells in the peripheral LN were studied. Alcohol consumption decreased the percentage and numbers of NK cells in the LN without altering the degree of NK cell apoptosis. Ly49H+, Ly49D+ and Ly49G2+ NK cells also decreased in the LN. The decrease in these cell populations was more pronounced in the SP, and there were no changes in the BM. The results from normal SP cell transfer experiments indicated a time-dependent decrease in accumulation of NK cells into the LN of alcohol consuming as compared to water drinking mice. Alcohol consumption increased the percentage of CXCR3+CD3- and decreased CD62L+CD3-NK cells in the SP and BM. The percentage of CXCR3+CD3+ T cells and CD62L+CD3+ T cells did not change. Consistent with the decrease of NK cells in the LN, alcohol consumption enhanced melanoma metastasis in the draining LN. The results indicate that decreased NK cells in the LN are associated with decreased CD62L+ NK cells in the SP and BM, which impairs NK cell migration to the LN.

Supported by R01 AA07293.
Peritoneal B cells responding to CD40 ligation resist macrophage-mediated suppression (40.8)
Authors: Swider Adam , Sacavage Steve , Deeney Brian , Kozlowski Megan , Riggs James E.
Published: January 2023

Although cells essential for anti-tumor immunity exist within tumors they often fail to prevent cancer progression. The immunosuppressive tumor microenvironment, notable for a high macrophage to T cell ratio (Mö:T), inhibits T cell activation primarily by Arg and Trp catabolism. We have assessed the impact of high M ö ratios on B cell (Mö:B) activation in a model culture system. LPS and F(ab')2 anti-IgM failed to activate B cells at high Mö:B ratios unless cyclooxygenase was inhibited by indomethacin. In contrast, B cells activated by CD40 ligation were resistant to Mö-mediated suppression. Compared with spleen (SP) B cells, peritoneal cavity (PerC) B cells responding to CD40 ligation were particularly resistant to suppression. PerC B cells from B-1 B cell subset-deficient BALB.xid mice were also resistant to Mö-mediated suppression. The CD40 response, however, was suppressed in cultures where both B (áCD40) and T (áCD3) cells were activated illustrating that suppression by amino acid catabolism overrides the CD40 response of B cells in high Mö:B environments. These results are relevant to the development of strategies designed to reverse the immune suppression characteristic of many tumors. Supported by NIH AREA R15-AI060356-01.
Identification of molecular checkpoints in patients with prostate cancer immunized with PSA146-154 peptide vaccine (41.24)
Authors: Perambakam Supriya , Xie Hui , Edassery Seby , Peace David J
Published: January 2023

The objective of the current study is to delineate genes that may be differentially expressed in responding versus non responding patients in the context of a completed PSA146-154 peptide vaccine protocol. 28 HLA-A2+ prostate cancer patients with high risk, local disease or metastatic, hormone sensitive disease in remission were vaccinated on weeks 1, 4, and 10 with peptide plus GM-CSF or peptide loaded autologous DC. Before and after PBMC were assessed for specific tetramer response and gene expression profiles per Affymetrix whole human genome arrays. Clinical status was evaluated per serial serum PSA. 13 of 27 patients achieved stable serum PSA (1 not evaluable), of whom 9 patients developed specific tetramer responses at six months post first vaccination. Thus the detectable immunity was associated with lower risk of PSA progression (p=0.02). Comparison of array data between responders and non responders indicated that 114 of 54,675 genes were differentially expressed (p<0.001). Notably VEGF-A, LIF, TNFRSF21 and IFNK, pertaining to the cytokine-cytokine receptor interaction pathway were predicted to be impacted (Impact factor=5.19). Taqman gene assays indicated that the expression of VEGF-A relative to GAPDH was greater in non responding compared to responding patients. Hence, VEGF-A is one of the likely candidates to impede vaccine efficacy, possibly by mediating its effects on antigen presenting cells.
Acetal-modified Dextran: An Acid-sensitive, Biodegradable Material for Effective Microparticulate Antigen Delivery (41.53)
Authors: Cohen Joel A , Broaders Kyle E , Beaudette Tristan T , Bachelder Eric M , Fréchet Jean MJ
Published: January 2023

Polymer-based microparticle systems capable of delivering proteins to antigen-presenting cells (APCs) of the immune system have been investigated for the enhancement of vaccines and immunotherapy. The size of the particles in these systems can be controlled to passively target APCs , without being taken up by other cells in the body. We have developed a new concept in acid-sensitive microparticles based on the use of a water-soluble polysaccharide, dextran, that can be rendered insoluble in aqueous solutions by protecting the hydroxyl groups with acid-sensitive acetals. The resulting polymer is soluble in organic solvents used to generate protein loaded particles by a variety of methods in which the modified polymer is condensed around the encapsulated antigen. Once administered, the acetals rapidly hydrolyze under the acidic conditions present in the phagosomes of APCs to provide a solubility switch that enables particle degradation and release of encapsulated antigen in a controlled manner. We have recently demonstrated the ability to control the rate of particle degradation over a range of minutes to several hours at pH 5, with interesting results observed in how degradation rate affects the presentation of model antigens by APCs to T cells in vitro, as well as how degradation rate may affect the processing mechanism of the released antigen within APCs.
Alphavirus replicon particles expressing melanocyte differentiation antigens are effective vaccines for melanoma (41.55)
Authors: Avogadri Francesca , Merghoub Taha , Hirschhorn Daniel , Maughan Maureen , Morris John , Houghton Alan N
Published: January 2023

Melanocyte differentiation antigens are attractive candidates for melanoma immunotherapy, but because they are expressed by malignant cells as well as their normal counterpart eliciting effective immune responses is challenging.

Here we evaluated an alphavirus replicon vector system as a potential melanoma immunotherapy. Use of propagation-defective virus-like replicon particles (VRP) based on an attenuated strain of Venezuelan equine encephalitis (VEE) virus is especially attractive because VRP express heterologous proteins to high levels and target expression to dendritic cells. VRP vaccines have been shown to elicit both humoral and cellular immune responses to the vectored gene products in many animal disease models and in phase I clinical trials.

The efficacy of VRP expressing the melanocyte differentiation antigens tyrosinase, gp100 or TRP-2/DCT was analyzed against challenge with the poorly immunogenic and highly aggressive B16 murine melanoma. TRP-2/DCT-expressing VRP were the most effective in delaying tumor occurrence when compared to the other antigens tested. Although both TRP2/DCT-specific CD8+ T cells and IgG are induced, the data suggest that B-cells may play a more important role in the effector phase of tumor protection.

These studies demonstrate that VRP vaccines present a promising approach for melanoma immunotherapy.
Immunological evaluation of patients with osteosarcoma received adjuvant treatment with specific Transfer Factor. (41.63)
Authors: Rodriguez-Flores Azucena , Castillo-Juarez Paola , Vazquez Dotor , Gonzalez-Guzman R , Rico-Martinez G. , Estrada-Parra Sergio
Published: January 2023

Osteosarcoma is a malignant bone tumor with a tendency to metastasis. Based on the immune response and the degree of progress in primary injury Levin developed a classification for osteosarcoma ranging from Phase I to Phase IV associated with the loss of the immune response of cytotoxic cell type and primarily to the development of metastases. Transfer Factor is a polypeptide with a molecular weight of about 6 kilodalton present in dialyzable leukocyte extract the human capacity for immunoregulation.

The present study is conducting a pilot clinical trial evaluating the different populations and lymphocyte subpopulations in peripheral blood at baseline and post-surgical type, neoadjuvant chemotherapy and specific transfer factor.

So far 16 patients have been assessed, of which it was found that 62.5 percent have decreased the number of lymphocyte cells, T lymphocytes, 44 percent and 68.8 percent total cytotoxic and helper response at baseline compared to healthy individuals. After treatment we observed that 63 percent of patients increased the number of T lymphocytes, 75 percent increased their cytotoxic T lymphocytes and 50 per cent of these cells cooperating. The 87.5% of patients come back to phase I of Levins classification
A novel human-mouse chimeric death receptor (DR5) molecule mediates TRA-8 induced apoptosis in mouse cell lines (41.9)
Authors: Li Jun , Yang Ping-Ar , Hsu Hui-Chen , Mountz John D.
Published: January 2023

DR5 is a pro-apoptotic protein considered as a target for cancer and autoimmune therapy. An anti-human DR5 antibody, TRA-8, has been shown to induce apoptosis in various cells. The purpose of this study is to generate a humanized mouse DR5 molecule that can be used to study the DR5 mediated apoptosis and immune responses using TRA-8 in a mouse.

A chimeric DR5 consisting of a human extracellular domain, a mouse transmembrane and intracellular domain was subcloned downstream of a 1kb, 3kb putative promoters, and a 3 kb first intron of mouse DR5. Transfection was performed by using Lipofectamine and nucleofection. Chimeric DR5 expression was determined by FACS and Western blotting. Cell viability was estimated by the ATPLite.

Maximal cell surface expression was seen with the 1 kb promoter in NIH3T3 and 4T1 cells. Longer promoter with first intron conferred authentic expression. TRA-8 enhanced apoptosis with efficacy varying from 15% (shorter promoter, high DR5 expression, high cell death with/without TRA-8) to 60% (longer promoter plus the first intron, authentic DR5 expression, low cell death without TRA-8, high cell death with TRA-8).

This novel DR5 molecule with regulatory elements confers its authentic expression and enables TRA-8 induced apoptosis in mouse cells. It will lead to the generation of a transgenic mouse for analysis of the TRA-8 apoptotic mechanism, therapeutic response in both autoimmune diseases and cancer.
Multicolor flow cytometry using only Qdot® conjugated primary antibodies (42.12)
Authors: Jaron Shulamit , Godfrey William L.
Published: January 2023

Qdot® nanocrystals allow researchers to add more parameters to their flow cytometry experiments by filling in the spectral landscape around common organic fluorochrome conjugates. The availability of Qdot® nanocrystals in multiple emission wavelengths and ever more Qdot® conjugates, allows the creation of a multicolor panel consisting solely of Qdot® conjugates. We show the effectiveness of using only Qdot®-conjugated antibodies in a typical 8-color phenotyping panel. Experiments were performed using standard staining protocols with human peripheral blood leucocytes. Analysis was performed on an LSR II flow cytometer using 405 nm and 488 nm lasers equipped with octagon optical pathways. Attention is paid to experimental set-up including the selection of appropriate filters to minimize within-laser and cross-laser compensation. The fluorescence from Qdot® 525, 605, 655, 705, and 800 nanocrystal conjugates were easily isolated from adjacent channels due to the narrow emission profiles of the nanocrystals. More care was needed in the filter selection and compensation between 565 and 585, 625 and 605, and 625 and 655 nanocrystal conjugates. By selecting the right combination of laser/detector and filters, compensation can be minimized. These results demonstrate the ease of performing a multicolor experiment with Qdot® nanocrystal conjugates using no more than two lasers.

Supported by Life Technologies.
Numbers matter: quantitative and dynamic analysis of the formation of a mature immunological synapse using imaging flow cytometry (42.15)
Authors: Ahmed Fariyal , Friend Sherree , George Thaddeus C , Barteneva Natasha , Lieberman Judy
Published: January 2023

Activation of T lymphocytes by antigen-presenting cells (APC) results in the formation of an immunological synapse. Following intercellular contact with the target cell, key signaling and adhesion molecules polarize within minutes to hours to the T cell-APC interface. We use multispectral imaging flow cytometry, a new technology which combines flow cytometry with imaging, to visualize and quantify the recruitment of signaling molecules CD3ε and Lck during the evolution of a mature immune synapse. We use two independent analysis methods to show that the dynamics of Lck and CD3ε recruitment are comparable; however, CD3ε exhibits longer residence times (> 8 min) following Ca++ triggered activation in the synapses of thousands of T cell-macrophage conjugates.
Engineering XID(Btk) site specific Homing Endonucleases for gene repair in hematopoietic stem cells (42.19)
Authors: Wang Yupeng , Jarjour Jordan , Havranek Jim , Baker David , Scharenberg Andrew M. , Rawlings David J.
Published: January 2023

X-linked agammaglobulinemia (XLA), a human immunodeficiency caused by mutations in Btk, represents an ideal candidate target for HSC gene therapy. However, current gene replacement methods carry risks of insertional mutagenesis as well as gene silencing. In contrast, gene repair induced by site specific DNA double strand breaks (DSB) offers a novel means to treat genetic disease with reduced risk and the potential to utilize endogenous gene expression control elements. LAGLIDADG Homing Endonucleases (LHEs) have been shown to efficiently induce site specific DSB and markerless modification of genes without toxicity. Due to the limited number of LHEs currently available, engineering site specific LHEs represents a critical step for enabling technology for DSB-induced gene modification.

In the current study we have begun to generate a site specific variant of the LHE I-AniI, targeted to recognize a unique mutation in the mouse BTK gene in the XID model of human XLA. Our approaches combine: protein computational design with random mutation and selection using yeast library surface display; or alternatively, iterative somatic hyper-mutation and selection within DT40 B cell library targeted to the light chain locus. Using these combinatorial methods we have generated a panel of I-AniI variants with novel DNA binding and cleavage specificity towards the XID site. Preliminary characterization of a subset of these I-AniI variants will be presented. Ultimately, new I-Anil variants and donor DNA repair template will be delivered into HSC derived from XID/Tec-/- mice; and evaluated for capacity to re-engraft and reconstitute B lineage development and function in vivo.
A proinflammatory program of perinatal plasmacytoid dendritic cells activate natural killer (NK) cells to damage the bile duct epithelium in experimental biliary atresia (43.2)
Authors: Saxena Vijay , Shivakumar Pranavkumar , Meithke Alex G , Sabla Gregg E , Chougnet Claire , Bezerra Jorge A
Published: January 2023

Biliary atresia results from an NK cell-mediated inflammatory injury of the bile duct epithelium with onset in the early postnatal period. Based on key immunoregulatory roles of dendritic cells (DC), we hypothesized that early neonatal DC activation triggers an effector immune response that results in biliary atresia. We tested this hypothesis in a mouse model of rotavirus-induced biliary atresia. Following rotavirus challenge on day 1 of life, viral immunogenic protein NSP3 was detected in hepatic PDCA-1+ plasmacytoid DC (pDC) but not CD11c+ conventional DC (cDC) within 3 days of infection. Hepatic pDC increased 3 fold, while non-pDC (CD11b+CD11c+ cells) decreased 2.5 fold. Rotavirus-naïve perinatal hepatic pDC and cDC spontaneously expressed B7.1/2 (2-3 fold higher than adults), with a much higher increased expression in B7.1/2, IFNγ, and IL-12p40 by pDC after rotavirus challenge. IL-15 was spontaneously expressed by naïve perinatal pDC and increased dramatically after rotavirus infection, but in vitro activation of NK cells required co-culture with both pDC and cDC. Most notably, ablation of pDC decreased the liver population of NK cells and prevented the mucosa injury and obstruction of neonatal bile ducts, leading to improved symptoms and long-term survival. Thus early activation of pDC due upon viral exposure regulates neonatal duct injury and obstruction in biliary atresia.
Adoptive transfer of CD4 T cells into T cell deficient Mycobacterium avium infected mice results in a fatal Th1 disease that mimics human Immune Reconstitution Inflammatory Syndrome (IRIS) (43.6)
Authors: Barber Daniel L. , Mayer Katrin D. , Antonelli Lis R. , Sher Alan
Published: January 2023

IRIS is the paradoxical worsening of pathology in HIV patients after initiation of anti-retroviral therapy (ART). Patients with severe CD4 T cell loss and concurrent mycobacteria infection display a high risk of developing IRIS after ART. To study the mechanism(s) underlying the pathogenesis of IRIS, we have developed a mouse model that focuses on a fundamental feature of the clinical scenario: CD4 T cell reconstitution in the context of concurrent mycobacteria infection. Both WT and TCRα-/- mice survive for many months after Mycobacterium avium infection. However, when TCRα-/- mice are infected with M. avium and then inoculated with CD4 T cells from naïve mice several weeks later, they develop a rapid wasting disease and succumb as early as day 12 post-transfer. IFNγ-/- CD4 T cells induce much less disease than WT CD4 T cells, and TNFα neutralization delays its onset. TCR Tg CD4 T cells specific for irrelevant Ag's cause no disease indicating that this is an Ag driven T cell response. Interestingly, both self-Ag and M. avium specific CD4 T cells induce rapid mortality after transfer into TCRα-/- M. avium infected mice. This suggests that (1) the combination of lymphopenia and concurrent infection triggers the reconstituting CD4 T cells to mount dysregulated Ag-driven Th1 responses that lead to fatal wasting and (2) IRIS-inducing CD4 T cells can be specific for either self or mycobacterial antigens.

Work supported by NIAID Intramural Research Program.
Local intrahepatic antigen presentation induces dysfunctional antiviral CD8+ T cell responses (44.19)
Authors: Dolina Joseph S , Lukens John R , Kim Taeg S , Hahn Young S
Published: January 2023

Immune responses to pathogens in the liver are uniquely characterized by a tolerogenic feature as a result of constant exposure to bacterial constituents and food-derived antigens. Little is known about how the immune dysregulation in the liver contributes to persistent viral infection. Specifically, it is important to understand how the site of antigen delivery impacts the quality of CD8+ T cells either trafficked to or generated within the liver. In this study, we show that subcutaneous (SC) adenovirus infection results in the trafficking of CD8+ T cells to the liver with effective antiviral function. In contrast, when antigen is delivered directly to the liver during intravenous (IV) adenovirus infection, intrahepatic CD8+ T cell responses are defective in their ability to produce inflammatory cytokines and lyse target cells. Further, these intrahepatic CD8+ T cells express elevated levels of PD-1, downregulate TCR, and exhibit increased apoptosis. IV adenovirus infection distinctively results in local intrahepatic antigen presentation responsible for limiting the CD8+ T cell immune response. These findings not only have applications for expanding the field of knowledge on chronic liver infections, but they also demonstrate that the delivery route of vaccine can orchestrate the quality of immune mediators present in the liver. NIH Grants DK063222 and Training Fellowship T32 AI007496 supported this work.
An activating receptor's critical role in supporting NK cell proliferation for survival during viral infections (44.2)
Authors: Lee Seung-Hwan , Kim Kwang-Sin , Fodil-Cornu Nassima , Vidal Silvia M , Biron Christine A
Published: January 2023

Activating receptors, including the Ly49H molecule recognizing MCMV, can stimulate NK cell cytotoxicity and proliferation. Ly49H induction of NK cell cytotoxicity contributes to defense against MCMV, but its in vivo importance in stimulating NK cell expansion remains unclear. To define Ly49H's role in supporting cell proliferation during infection, experiments were carried out evaluating NK cell responses in Ly49h-/-, perforin 1 (Prf1)-/- and wild type (wt) B6 mice. NK cell numbers were similar in uninfected mice, and either deficiency alone resulted in high MCMV burdens. In the absence of Ly49h, however, NK cell numbers declined, with remaining populations having an immature phenotype. In the absence of Prf1, NK cells expanded and had higher rates of proliferation, eccentric phenotypes, and Ly49H expression on nearly all cells. Expansion was abolished, with negative consequences for survival, in mice deficient for both Ly49H and Prf1 (Ly49h-/-Prf1-/-), and NK cell depletions in Prf1-/- mice decreased survival during infection. The results prove that Ly49H can drive dramatic NK cell expansion during challenges with MCMV. Moreover, by dissecting functions for proliferation from cytotoxicity, they demonstrate a previously unappreciated critical role for activating receptors in keeping NK cells present during viral infection. Supported by NIH CA41268 and the Canadian Institutes of Health Research MOP-7781.
Poor anti-viral immunity in aged mice is associated with defects in CD8 T cell recall responses (44.5)
Authors: Decman Vilma , DiMenna Lauren J. , Laidlaw Brian J. , Ertl Hildegund C.J. , Wherry E.  John
Published: January 2023

Aging leads to an increased susceptibility to infectious diseases. To investigate the age-related changes in T cell memory we compared anti-viral CD8 T cell immunity in young versus aged mice following systemic (lymphocytic choriomeningitis virus; LCMV) or local (influenza virus) viral infections. The number of memory CD8 T cells formed was similar or only modestly different between aged and young animals, though in some cases changes in immunodominance and cytokine production were apparent. Both influenza virus-specific and LCMV-specific memory CD8 T cells from aged mice, however, had major substantially reduced proliferative capacity upon secondary infection. As a result, aged mice displayed increased morbidity and mortality upon viral rechallenge. The studies described herein demonstrate that the defects intrinsic to aged CD8 T cells are at least partially responsible for diminished immunity of aged mice. Thus, the strategies that accelerate the induction of T cell responses or that overcome proliferative deficiencies may prove beneficial.

These studies were funded by grants from AHA, NIH (HHSN266200500030C) and the Ellison Medical Foundation.
NK cell Activation during Acute Hepatitis C Virus Infection (45.18)
Authors: Pelletier Sandy , Bédard Nathalie , Drouin Christian , Khakoo Salim , Bruneau Julie , Shoukry Naglaa H.
Published: January 2023

Genetic studies have implicated the inhibitory natural killer (NK) cell receptor KIR2DL3 and its human leukocyte antigen C group 1 (HLA-C1) ligand in resolution of hepatitis C virus (HCV) infection but a functional correlation was not established. To examine the role of NK cells during acute HCV, we have analyzed longitudinally the phenotype and function of NK cells from a cohort of patients following HCV exposure. Three groups with different infectious outcome were identified: spontaneous resolvers (SR), chronic evolution (C) and exposed un-infected (EU) (n=10 in each group). We have observed increased NK cell cytotoxicity (%CD107a+) in all groups suggesting NK cell activation following exposure but no correlation was established with infectious outcome. Nevertheless, we observed decreased expression of KIR2DL1, associated with highest NK cell inhibition, in SR. Furthermore, the highest IFN-γ production was observed in KIR2DL3+ NK cells, the least inhibited population, in all groups. Our results suggest that NK cell direct cytotoxicity might not be implicated in HCV clearance but that less NK cell inhibition is more frequent in SR patients. These results confirm the important role of host genetic background in NK cell function and provides the first demonstration of such a functional correlation in an in vivo infection setting.

Research support: The Dana Foundation, CIHR, FRSQ, NCRTP-HepC
Mucosal Epithelial Cells Provide the Critical Link for Dendritic Cell-mediated Amplification of HIV (45.19)
Authors: Fontenot Danielle R , He Hong H , Hanabuchi Shino , Nehete Pramod N , Ma Zhong-min , Courtney Amy N , Miller Christopher J , Sun Shao-Cong , Liu Yong-Jun , Sastry K. Jagannadha
Published: January 2023

An estimated 30-40% of all new HIV-1 infections occur in women through the vaginal mucosa even though it carries a lower transmission probability per exposure rate (1/200 - 1/2,000) than both rectal and mother to child transmission (1/10 - 1/300). Paradoxically, the mucosal epithelial cells express low to negligible levels of the HIV receptors and the mucosal tissue microenvironment is laden with numerous antiviral factors. We obtained in vitro and in vivo evidence of HIV manipulating the mucosal microenvironment to sustain and increase infection by inducing genital mucosal epithelial cells to produce thymic stromal lymphopoietin (TSLP), a cytokine known to activate DC to induce robust CD4+ T cell homeostatic expansion. We provide molecular evidence for the involvement of NFκB signaling pathway in HIV-induced TSLP expression. In rhesus macaques we observed dramatic increases in TSLP expression concurrent with increases in DC and CD4+ T cell numbers, and viral replication in the vaginal tissues within the first two weeks after vaginal SIV exposure. Our studies demonstrate that HIV-mediated TSLP production by mucosal epithelial cells is a critical trigger for DC-mediated amplification of HIV-infection in activated CD4+ T cells. The cross talk between mucosal epithelial cells and DC, mediated by HIV induced TSLP, is a novel mechanism important for AIDS pathology that should be explored for therapeutic potential.
Rat coronavirus infection of alveolar type I cells causes neutrophil recruitment followed by resolution of infection (45.28)
Authors: Miura Tanya A. , Funk Joel , Holmes Kathryn V. , Mason Robert J.
Published: January 2023

Viral infections in the lung are an important cause of morbidity and mortality in humans worldwide. In order to design strategies for vaccination and therapy, we need to understand the molecular mechanisms that initiate and regulate effective antiviral immune responses in the lung. This requires animal models in which in vitro mechanistic studies can be correlated with infections in vivo. We study rat coronavirus (RCoV) infection in rats and cultures of primary differentiated rat alveolar epithelial cells as a model for the early events in respiratory coronavirus pathogenesis. Intratracheal inoculation of adult rats with RCoV resulted in infection of the lung, primarily in alveolar type I cells. Infection caused transient weight loss and focal lesions and recruitment of neutrophils in the lung, followed by clearance of virus and resolution of inflammation. Thus, adult rats mounted an effective innate immune response to RCoV infection in the lung. In agreement, in primary cultures of differentiated rat alvoelar type I cells, RCoV replicated and induced expression of neutrophil chemotactic CXC chemokines. This in vitro model will elucidate the molecular interactions between RCoV and alveolar epithelial cells that initiate and regulate the innate antiviral immune response in the lung.

This work was supported by NIH grants AI-059576 and HL-029891, and NCRR/NIH grant P20 RR015587.
Epitope-Specific Regulatory T cells Differentially Modulate the CD8 T Cell Response to Respiratory Syncytial Virus in Mice (45.29)
Authors: LIU JIE , RUCKWARDT TRACY , CHEN MAN , JOHNSON TERESA , GRAHAM BARNEY
Published: January 2023

RSV infection is the leading cause of lower respiratory disease in infants and young children, accounting for >120,000 hospitalizations each year in the US alone. Although CD8 T cell effector function is key to virus clearance, CD4 T cells coordinate the immune response and can lead to immunopathology. Therefore, they play a central role in the immunity and immunopathogenesis of RSV infection.

To facilitate in depth evaluation of RSV-induced CD4 T cell responses we identified two new I-Ab- restricted dominant CD4 T cell epitopes, M213-223 and M227-37, in RSV M and M2 that also contain well characterized CD8 T cell epitopes. CD4 T cells from lung and spleen of RSV-infected CB6F1 mice produce different patterns of IL-2, IFN-γ and other cytokines after peptide stimulation. Novel tetrameric peptide-I-Ab complexes identified 0.5% and 1.6% RSV M- and M2-specific CD4 T cells in lungs of the infected mice. Peptide-stimulated CD4 T cells produced different patterns of regulation on CD8 T cell responses to RSV M and M2 epitopes in vitro. The regulatory function of the CD4 T cell correlated with the level of FoxP3 expression in epitope-specific CD4 T cells Our study demonstrates that the biophysical properties of the epitope may dictate the functional properties of the CD4 T cell response. Understanding how epitope structure influences T cell function will provide new insight into viral pathogenesis and may improve vaccine design.
Liver cCD8a+DC from Mice Immunized with Gamma-irradiated Plasmodium berghei Sporozoites Mediate the Induction of Intrahepatic Effector CD8+ T cells against Liver-Stage Malaria (45.30)
Authors: Krzych Urszula , Jobe Ousman , Schwenk Robert
Published: January 2023

Immunization with radiation (γ)-attenuated Plasmodia sporozoites (γ-spz) confers sterile and long-lasting immunity against malaria liver-stage infection. In the P. berghei γ-spz model, protection is linked to liver CD8+ T cells that express an effector/memory (TEM) phenotype, (CD44hiCD45RBloCD62Llo ), and produce IFN-γ. However, neither the antigen presenting cells (APC) that activate these CD8+ TEM cells nor the site of their induction have been fully investigated. Because conventional (c)CD8α+ DC (a subset of CD11c+ DC) are considered the major inducers of CD8+ T cells, in this study we focused primarily on cCD8α+ DC from livers of mice immunized with Pb γ-spz and asked whether the cCD8α+ DC might be involved in the activation of CD8+ TEM cells. We demonstrate that multiple exposures of mice to Pb γ-spz lead to a progressive and nearly concurrent accumulation in the liver but not the spleen of both the CD11c+NK1.1- DC and CD8+ TEM cells. Upon adoptive transfer, liver CD11c+NK1.1- DC from Pb γ-spz-immunized mice induced protective immunity against sporozoite challenge. Moreover, in an in vitro system, liver cCD8α+ DC induced naïve CD8+ T cells to express the CD8+ TEM phenotype and to secrete IFN-γ. The in vitro induction of functional CD8+ TEM cells by cCD8α+ DC was inhibited by anti-MHC class I and anti-IL-12 mAbs. These data suggest that liver cCD8α+ DC present liver-stage antigens to activate CD8+ TEM cells, the pre-eminent effectors against pre-erythrocytic malaria. These results provide important implications towards a design of anti-malaria vaccines. Supported by a grant from NIH and USAMRMC.
RICK/RIP2 Promotes Inflammation and Lethality after Gram-Negative Bacterial Infection in Mice Stimulated with Lipopolysaccharide (45.4)
Authors: Park Jong-Hwan , Kim Yun-Gi , Nunez Gabriel
Published: January 2023

RICK, a serine-threonine kinase, functions downstream of the pattern-recognition receptors Nod1 and Nod2 to mediate NF-kB and MAPK activation in response to specific microbial stimuli. However, the function of RICK in the recognition and host defense of Gram-negative bacteria remains poorly understood. We report here that infection of wild-type and RICK-deficient macrophages with Pseudomonas aeruginosa and Escherichia coli, elicited comparable activation of NF-kB and MAPKs as well as secretion of pro-inflammatory cytokines. However, production of IL-6 and IL-1b induced by these Gram-negative bacteria was impaired in RICK-deficient macrophages when the cells were previously stimulated with LPS or E. coli. The diminished pro-inflammatory response of RICK-deficient macrophages to bacteria was associated with reduced activation of NF-kB and MAPKs. Importantly, mutant mice deficient in RICK were less susceptible than wild-type mice to P. aeruginosa infection when the animals were previously stimulated with LPS. The reduced lethality of RICK-deficient mice infected with P. aeruginosa was independent of pathogen clearance, but was associated with diminished production of pro-inflammatory molecules in vivo. These results demonstrate that RICK contributes to the induction of pro-inflammatory responses and susceptibility to Gram-negative bacteria after exposure to LPS, a condition that is associated with reduced TLR signaling.
Poly-gamma-D-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule of Bacillus anthracis in guinea pigs and rabbits (45.8)
Authors: Rhie Gi-eun , Lee Deog-Yong , Ha Hyun-Joon , Park Jungchan , Chun Jeong-Hoon , Oh Hee-Bok , Yoo Cheon-Kwon
Published: January 2023

Anthrax is a lethal infectious disease caused by spore-forming Bacillus anthracis. The two major virulence factors of B. anthracis are exotoxin and the poly-gamma-D-glutamic acid (PGA) capsule. The three components of exotoxin, protective antigen (PA), lethal factor and edema factor act in binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of anthrax vaccine. In this study, we report that PA-PGA conjugates induce expression of anti-PA, anti-PGA and toxin neutralizing antibodies in guinea pigs and completely protect guinea pigs against a 50xLD50 challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA 15mer offers passive protection to guinea pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role.

This work was supported by Korea National Institute of Health Grants (H. H and G. R.).
Differential requirements of MHC and of DCs for endogenous proliferation of different T cell subsets in vivo (46.16)
Authors: Min Booki , Do Jeong-su
Published: January 2023

T cells transferred into severe lymphopenic hosts undergo rapid proliferative responses that are distinct from conventional homeostatic proliferation, known as 'endogenous proliferation'. Unlike homeostatic proliferation, cytokines such as IL-7 is dispensable, yet TCR interaction with peptide:MHC complexes is believed to be essential for this process to occur. However, cell types inducing the proliferation have not been defined. In this study, we provide evidence that CD11c+ conventional DCs play pivotal roles in inducing endogenous proliferation of both naive and memory phenotype CD4 T cells via TCR-MHC II interaction. T cells transferred into MHC II-/- TCRβ-/- or CD11c+ DC-deleted TCRβ-/- recipients failed to undergo endogenous proliferation. By contrast, CD8 T cell endogenous proliferation was independent of MHC I or CD11c+ DCs. Interestingly, depleting both B cells and DCs was able to abrogate the proliferation of naive CD8 T cells in TCRβ-/- environments. However, memory phenotype CD8 T cell proliferation was not abrogated by the absence of both B cells and DCs. Taken together, these results strongly suggest that depending on the T cell lineages as well as the differentiation status different mechanisms operate during endogenous proliferation, revealing unexpected in vivo complexity of T cell proliferation under lymphopenic conditions.