bluuebunny/crossref_metadata_2025 huggingface dataset:
Plant polyphenols induce differential regulation of CD11b expression and function on monocytes and γδ T cells (97.11)
Authors: Graff Jill C , Jutila Mark A
Published: January 2023

Leukocyte adhesion and migration is partially mediated by CD11b/CD18 (MAC-1, CR3). Earlier studies have demonstrated a role for green tea polyphenols in down-regulating CD11b on monocytes and CD8+ T cells. We have recently shown a stimulatory effect of unripe apple polyphenols (APP) on γδ T cells. Thus, we compared the effect of APP on γδ T cell and monocyte CD11b expression. Purified bovine monocytes and monocyte-depleted peripheral blood lymphocytes were cultured with APP for 24 hours. CD11b levels decreased on monocytes in response to APP. In contrast, a subset of γδ T cells responded to APP by up-regulating CD11b. Consistant with the changes in surface CD11b expression, APP treated γδ T cells showed increased adherence to plastic, whereas monocyte adhesion was reduced. The affects of APP on monocytes could be inhibited by anti-CD11b mAb, whereas the mAb had no effect on the APP response in γδ T cells, suggesting the two cell types respond to APP via different mechanisms. Polyphenols are typically thought of as anti-inflammatory agents, however, these data, as well as other ongoing studies, indicate they have a pro-inflammatory effect on γδ T cells. In vivo, plant polyphenols may enhance γδ T cell migration to sites of inflammation, where they can enact rapid immunoregulatory and innate-like responses.

Funded by: NIH –NIAID DAIT-BAA-03 -41
ICAM-1 expressed on neutrophils is critically involved in vaccine-induced protection in a murine model of acute pneumonia (97.3)
Authors: Priebe Gregory P , Walsh Rebecca L , Cederroth Terra A , Pier Gerald B
Published: January 2023

Murine neutrophils are not known to express ICAM-1. In studying the mechanism of protection against P. aeruginosa pneumonia following immunization with a live-attenuated vaccine in a model of acute pneumonia, we detected ICAM-1 expression on lung neutrophils after challenge. We hypothesized that neutrophil ICAM-1 elicited homotypic adhesion as well as enhanced bacterial killing. C3H/HeN mice immunized intranasally (IN) with an aroA deletion mutant of P. aeruginosa strain PAO1 (PAO1ΔaroA) or E. coli (as control) at weekly intervals with 108, 5 x 108, and 109 CFU were challenged IN 3 weeks later with a cytotoxic variant of PAO1. Lung leukocytes and BAL cells were analyzed by flow cytometry, and decreased migration due to homotypic adhesion was assessed using Transwells. We also tested the impact of ICAM-1 blockade on bacterial clearance by IN instillation of anti-ICAM-1 antibody prior to challenge of immunized mice. PAO1ΔaroA-immune mice had more ICAM-1+ neutrophils in BAL 18 h after challenge compared with E. coli-immune controls (mean 74% vs. 37%, p < 0.01). Non-immune mice had low levels of ICAM-1+ neutrophils. BAL neutrophils from PAO1ΔaroA-immune mice transmigrated significantly less well, and blockade of ICAM-1 prior to challenge increased bacterial CFU in PAO1ΔaroA-immune mice. These data suggest that vaccine-induced neutrophil ICAM-1 plays an important role in the adaptive immune response in the lung.
Initiation of Adaptive-Innate Immune Interaction Sequences in Contact Sensitivity Responses (98.5)
Authors: Kish Danielle D. , Fairchild Robert L.
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to cutaneous sensitization and subsequent challenge with hapten. Hapten-primed effector CD8 T cell recruitment into the challenged skin to mediate CHS requires prior CXCL1/KC and CXCL2/MIP-2 directed neutrophil infiltration into the challenge site. The mechanisms inducing neutrophil chemoattractant production and the dynamics of neutrophil-CD8 T cell infiltration into the challenge site during the elicitation of CHS to dinitrofluorobenzene (DNFB) were investigated. Following DNFB challenge, CXCL1/KC and CXCL2/MIP-2 were produced within 3–6 h at 10-fold higher levels in sensitized vs. non-sensitized mice. This early chemokine production was dependent on hapten-specific CD8 T cells. At 6 h post-challenge of sensitized but not naïve mice, vascular endothelial cells (EC) in the challenge site stained positively for CXCL1/KC protein as well as for hapten. Although DNFB-primed CD8 T cells induced early CXCL1/KC production, the effector T cells did not infiltrate the parenchymal tissue of the challenge site at 6 h post-challenge but did 12 h later. In contrast, neutrophils infiltrated the challenge site at 6 h post-challenge and this was dependent on hapten-primed CD8 T cell induction of CXCL1/KC production. In vitro, CD8 T cells also stimulated hapten-presenting EC to produce KC. These results indicate hapten challenge to elicit CHS results in EC presentation of hapten to primed CD8 T cells. The T cells stimulate EC production of neutrophil chemoattractants that direct the neutrophil recruitment required for the subsequent infiltration of the effector CD8 T cells to mediate the allergic response.
Migration and expansion of dendritic cells improves cross-presentation of tumor antigens in virotherapy (98.8)
Authors: Edukulla Ramakrishna , Woller Norman , Mundt Bettina , Kuehnel Florian , Kubicka Stefan
Published: January 2023

Triggering of dendritic cell (DC) migration into tumor tissue and subsequent expansion of DCs may be useful to improve cross-presentation of tumor antigens during virotherapy.

Appropriate syngenic mouse models were established for lung carcinoma cells CMT64 and KLN205.Crystal violet staining showed that CMT64 and KLN205 are permissive for replication of the oncolytic telomerase-dependent adenovirus hTert-Ad.

We used adenoviral expression of Macrophage Inflammatory Protein 1α (MIP 1α) to recruit DCs into hTert-Ad infected tumors and Ad-Flt3L to expand DCs within the tumor nodules during virotherapy.

Compared with virotherapy alone or virotherapy with expression of a single CC chemokine, expression of both MIP1α and Flt3L during virotherapy significantly increases infiltration of DCs and T cells, as shown by histochemistry. Interferon-γ, Pentamer staining and CTL assay showed that the combination of cytokines improves cross-presentation of tumor antigens and induces an antitumoral immune response during virotherapy. Combination of DC vaccination and viroimmunotherapy showed nearly complete regression of the subcutaneous tumors

In conclusion mobilizing of DCs to virally infected tumors during virotherapy improves cross-presentation of tumor antigens and induces antitumoral immune response which facilitates an effective viroimmunotherapy of established tumors.
Accumulation of antigen-specific B and T cells in ectopic lymphoid tissue after immunization (99.11)
Authors: Weinstein Jason Scott , Nacionales Dina C , Lee Pui Y , Kelly-Scumpia Kindra M , Sobel Eric , Satoh Minoru , Reeves Westley H
Published: January 2023

Objective: Ectopic lymphoid tissue is associated with autoantibody production. Although reminiscent of authentic secondary lymphoid tissue, it is unclear if it can serve as a site of cognate T-B cell interactions leading to autoimmunity. This question was examined in mice with ectopic lymphoid tissue induced by tetramethylpecadentane (TMPD).

Methods: B6 and BALB/c mice were treated i.p. with TMPD followed 3 mo later by s.c. immunization with NP-KLH or NP-OVA. NP specific B cells were identified by analysis of VH gene usage, flow cytometry, and ELISPOT. OVA transgenic T cells from DO.11 mice were transferred i.v. into BALB/c mice 3 d before immunizing with NP-OVA. OVA-specific T cells were identified by flow cytometry and proliferating B and T cells by BrDU staining.

Results: 12 d after immunizing with NP-KLH, 90% of H chain genes from ectopic lymphoid tissue of B6 mice bore V186.2 or analogous NP-specific VH sequences. B cells stained by NP-PE were identified in ectopic lymphoid tissue by flow cytometry and secretion of anti-NP antibodies shown by ELISPOT. The ectopic lymphoid tissue and draining lymph nodes also had numerous OVA-specific T cells 7 d after immunization with NP-OVA and there was a progressive increase of BrDU+ T and B cells.

Conclusion: The data suggest that following immunization, antigen-specific B and T cells undergo a local germinal center-like reaction within ectopic lymphoid tissue.
Using the chicken feather as a window into cell-mediated tissue responses (99.12)
Authors: Erf Gisela F. , Lockhart Becky , Bowen Olivia T. , Bateman Kristin , Finley ReGina C.
Published: January 2023

Cell-mediated immune (CMI) responses are typically tested by injecting the skin and monitoring the skin reaction. In chickens, various integumental tissues are suitable to measure the CMI reaction, including the wattle, wing web, toe web and foot pad. Using the Smyth line chicken vitiligo model to study the spontaneous autoimmune destruction of pigment cells in feathers, we have found that one growing feather-tip (3 mm diameter by 8 mm length) yields generous amounts of material for down-stream gene-expression-, histological-, and cell population-analyses. Moreover, growing feathers are easily accessible for microinjection, removal of a feather is minimally invasive, and feathers are very suitable for repeated sampling during an immune response. To investigate the potential of the feather as a window into tissue immune responses, time-course studies were conducted comparing leukocyte infiltration in wattles, wing webs and growing feathers in response to injection of vehicle, PHA or LPS, and during the recall response to Mycobacterium butyricum. Based on histology and immunohistochemical staining, leukocyte infiltration profiles over a 72 hour period were similar in all three tissues tested. Hence, the feather provides excellent opportunity to monitor and assess CMI responses and immunomodulation strategies in situ and via down-stream analyses.

Support: Arkansas Experiment Station Animal Health Grant
Abdominal lymphatic pump treatment increases leukocyte count and flux in thoracic duct lymph (99.13)
Authors: Hodge Lisa , King Hollis H , Williams Arthur , Reder Stephanie , Belavadi Tejaswi , Simecka Jerry , Stoll Scott , Downey H. Fred
Published: January 2023

Studies suggest that lymphatic pump techniques (LPT) enhance immunity and resistance to infection, but direct evidence of this has not been documented. In this study, the immediate effects of LPT on thoracic duct lymph flow and leukocyte output was measured. Lymph flow was measured and lymph was collected at baseline and during LPT. The baseline leukocyte count was 4.8 ± 1.7 x 106 cells/ml, and LPT increased leukocytes to 11.8 ± 3.6 x 106cells/ml (P<0.05). While macrophages, neutrophils, total lymphocytes, T cells and B cell numbers were similarly increased during LPT, their relative percentage in lymph was unaltered by LPT; however, IgA antibody forming B cells increased from 5.8% at baseline to 17% during LPT. Furthermore, LPT enhanced lymph flow 4-fold. Leukocyte flux was computed from the product of lymph flow and cell count. LPT enhanced leukocyte flux from 8.2± 4.1 x 106total cells per min to 60 ± 25 x 106cells per min. In summary, LPT significantly increased both thoracic duct lymph flow and leukocyte count, so lymphatic leukocyte flux was markedly enhanced. In addition, our data suggests that LPT can act preferentially on mucosal tissues, revealing a source of these mobilized leukocytes. Enhanced mobilization and lymphatic transport of immune cells during abdominal compression is likely an important mechanism responsible for the increased immune responses of patients treated with LPT.
Developmental and antigen-driven switches in the trafficking receptors of FoxP3+ regulatory T cells (99.2)
Authors: Kim Chang H , Lee Jeeho , Kang Seung G
Published: January 2023

FoxP3+ regulatory T cells play important roles in immune regulation and tolerance. There is an increasing body of evidence that the migration ability of FoxP3+ T cells is important for their regulatory functions at effector tissue sites. We investigated the two different trafficking receptor switches of FoxP3+ T cells occurring in the thymus and secondary lymphoid tissues. The first trafficking receptor switch in the thymus is developmentally programmed: Precursors of FoxP3+ cells undergo the first trafficking receptor switch from CCR8/CCR9 to CXCR4 and then finally to CCR7, generating mostly homogeneous CD62L+CCR7+ FoxP3+ T cells. The recent thymic emigrant CD62L+CCR7+ FoxP3+ T cells are programmed to migrate to secondary lymphoid tissues. The CD62L+CCR7+ FoxP3+ T cells undergo the second switch in trafficking receptors in an antigen-dependent manner. This second switch involves down-regulation of CCR7 and CXCR4 but up-regulation of a number of memory/effector type homing receptors, resulting in generation of heterogeneous FoxP3+ T cell subsets expressing various combinations of trafficking receptors including CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 and CXCR5. FoxP3+ cells undergo the second switch to selected non-lymphoid tissue homing receptors at highly accelerated rates. This results in generation of FoxP3+ T cells with unconventionally efficient migratory capacity to major non-lymphoid tissues such as intestinal lamina propria and bone marrow. Importantly, this accelerated switch of FoxP3+ T cells is conserved in both men and mice. The two switches in homing receptors are though to be important for effective distribution and differentiation-dependent effector functions of FoxP3+ regulatory T cells.
Where do blood-borne cells enter the brain? (99.9)
Authors: Lampson Lois A. , Tripp Cara A.
Published: January 2023

The blood-brain barrier stops passive entry of proteins, but not living cells. Both tumor cells and leukocytes can enter the brain, and it is attractive to exploit blood-borne leukocytes to attack blood-borne metastases or pathogens. However, more needs to be known about where different cell types enter, lodge, and grow.

METHODS. In 6u sections of rat brain, inert beads, blood-borne metastases of marked tumor, and T cells were stained histochemically or by common antibodies.

RESULTS.

Tumor, T cells, and inert beads can all enter vessels of the brain proper, choroid plexus (within the ventricles), and meninges. However, the relative importance varies with the cell type.Metastases in the brain proper were common for one tumor line, and rare for the other. Both lines metastasized to the choroid plexus.In contrast, the choroid plexus was not, in general, a favored site of T cell entry. T cells were more likely to enter at cerebral vessels.When T cells did accumulate in the ventricles, they were most concentrated in the narrow foramen of Monro.

DISCUSSION. The traffic patterns of other populations (T cell subsets, B cells, monocytes) must now be compared. In seeking to exploit blood-borne leukocytes against blood-borne tumor or pathogens, the optimal responding cell type may vary with the target site.
Enumeration of double stranded DNA breaks in developing thymocytes in young and aged mice (B1)
Authors: Hesse Jill , Faulkner Matthew F , Durdik Jeannine M
Published: January 2023

Aging at the cellular and molecular level has been shown to involve a decline in the immune system. In spite of this decline, the aged thymus is still functional in both thymocyte development and output of mature naïve T cells producing a small but significant population. During V(D)J recombination double stranded breaks (DSB) are created in the DNA of developing thymocytes. Complete and accurate resolution of the DSB is critical to the proper function and survival of cells. In our studies we utilized gamma phosphorylated-H2A.X(γH2A.X) to estimate the amount of DSB that are created and/or persist in thymocytes. H2A.X phosphorylation is the earliest ATM-dependent response to DSB, and it is speculated that the γH2A.X may help keep broken DNA ends together. For quantitation it is import to note that many H2AX molecules are phosphorylated in response to one DSB. The amplification of the signal allows us to visualize foci. We find cells from aged thymus have more DSB than cells from the young thymus. This increase in breaks may be the result of more breaks being created or breaks persisting due to slow or lack of DSB repair. By utilizing fluorescent staining and confocal microscopy we were able to further investigate the number of cells with DSBs and compare different thymocyte subpopulations. Here we show the enumeration of γH2A.X foci in thymocyte subpopulations in both young and aged mice.
IRF4 Expression Defines Th2 Populations Secreting High Levels of IL-10 (B102)
Authors: Ahyi Ayele-Nati Nathalie , Chang Hua-Chen , Kaplan Mark H
Published: January 2023

We have recently identified the ETS family transcription factor PU.1 as regulating heterogeneity in Th2 populations. To define additional factors that might contribute to Th2 heterogeneity, we examined the PU.1 interacting protein IFN-regulatory factor (IRF)-4, a transcription factor expressed in lymphocytes and macrophages. When Th2 cells are separated based on levels of IL-10 secretion, IRF4 expression segregates into the subset of Th2 cells expressing high levels of IL-10. To investigate the role of IRF4 in cytokine heterogeneity, Th2 cells were infected with retrovirus expressing IRF4. The cells overexpressing IRF4 secreted significantly higher levels of IL-10 compared to cells infected with a control vector while other Th2 cytokines were not affected. To understand the mechanism by which IRF4 regulates IL-10 expression in various Th2 cell subpopulations we used DNA pull down assays as well as co-immunoprecipitation assays to determine transcription factors that interact with IRF4. Our data shows that PU.1, IRF4 and NFATc2 form a complex in Th2 nuclear extract. The role of these complexes and their contribution towards Th2 heterogeineity will be further defined.
Toll-like receptor 4 signaling in lung dendritic cells is required for the induction of T helper cell type 1 responses but dispensable for type 2 (B105)
Authors: Tan Anna Marie , Bottomly Kim
Published: January 2023

We have previously reported that in a mouse model of allergic sensitization, low level inhaled LPS (LPSlow) signaling through TLR4 was necessary to induce Th2 responses while high level LPS (LPShigh) induced Th1 responses to inhaled antigen. The mechanism by which LPS signaling resulted in Th2 sensitization involved the activation of antigen-containing dendritic cells (DC). It remains unclear whether induction of these responses is dependent on TLR signaling in DCs, other lung cells, or in both. Here we report that antigen/LPShigh sensitized chimeric mice specifically lacking TLR4 signaling in the hematopoietic compartment (HPC) but sufficient for TLR4 in the stromal compartment (SC+HPC-) do not mount Th1 responses following antigen challenge while SC-HPC+ mice do. Strikingly, however, SC+HPC- mice mount exaggerated Th2 responses. Stromal TLR4 signaling in SC+HPC- mice leads to upregulation of the Th2-inducing Notch ligand Jagged-1 in DCs but not the Th1-inducing ligand Delta-4. In coculture, DCs activated by the stromal response to LPS polarize naïve antigen specific CD4 T cells to produce Th2 cytokines. These studies demonstrate that the TLR4-mediated response of stromal cells, through conditioning of DCs, is sufficient to induce robust allergic inflammatory responses to inhaled antigen containing LPS and define a novel role for lung stromal cells in the TLR4-dependent induction of allergic Th2 responses.
Priming with Inhaled Antigen: the Role of CCR2 (B106)
Authors: Wilkinson Kathryn Nancy , Bottomly Kim
Published: January 2023

We have developed a murine asthma model in which inhalation of OVA and low levels of LPS results in a Th2 response via a TLR4 dependent mechanism. Unlike asthma models that initiate a response via an intra-peritoneal injection of OVA and alum, our model uses a natural route of exposure and therefore provides insight into how pulmonary Th2 responses are initiated. Asthmatic responses that involve priming via an intra-peritoneal injection of OVA and alum are reported to be CCR2 independent. In contrast, in our model CCR2−/− mice show reduced airway inflammation and antigen specific antibody production. It is known that CCR2−/− mice have decreased recruitment of monocytes to sites of inflammation. In addition, it is known that under inflammatory circumstances CCR2+ monocytes can mature into tissue dendritic cells. In our asthma model, we find that inhalation of OVA and LPS induces recruitment of dendritic cells into the lung. We are currently determining if these recruited dendritic cells contribute to initiation of an asthmatic response and if their absence is responsible for the decreased responses observed in CCR2 knockout mice.
Cathepsin S in a Murine Model of Allergic Asthma (B108)
Authors: Weicker Sean , Cromlish Wanda , Lamontagne Sonia , Gauthier Jacques-Yves , Desmarais Sylvie , Oballa Renata , Ouellet Marc , Percival M. David , Black Cameron , Guimond Alain , Frossard Nelly , O’Neil Gary , Slipetz Deborah , Tan Christopher
Published: January 2023

Cathepsin S (Cat S), expressed predominately on antigen presenting cells, has been proposed as a therapeutic target for asthma. We used genetic and pharmacological tools to investigate the role of Cat S in murine models of allergic asthma. Mice null for Cat S were protected from OVA-induced pulmonary inflammation, but exhibited no protection from OVA-induced airway hyper-reactivity. To determine the role of Cat S during the challenge phase, we identified a potent and selective Cat S inhibitor, Compound A (Cpd A, IC50 mCat S = 0.6 nM, ≥470 fold selective vs mCat B, K, L), which inhibited antigen presentation in a mouse cell-based assay (IC50 = 44 nM). The prodrug of Cpd A, Cpd B, gave excellent plasma levels of Cpd A when dosed in mice by gavage, or in food. In vivo competition in mice with an irreversible pan-selective cysteine cathepsin probe showed that Cpd B gave selective inhibition of lung and spleen Cat S at 1 mpk, but lost selectivity at 50 mpk. Cpd B was dosed in mice (10, 100 mpk in food) over 4 days of the challenge period in the murine ovalbumin model. Both doses had no effect on bronchoalveolar lavage infiltrating cells, despite showing high levels of Cat S inhibition. Thus, Cat S inhibition in a therapeutic mode does not attenuate airway inflammation in antigen sensitised mice suggesting that anti-Cat S therapy would not be an effective asthma treatment.
Role of CCR2+ Monocyte-derived Cells in LPS-induced Acute Lung Injury (B113)
Authors: Chen Yu-Hsun , Lin Kaifeng Lisa , Gunn Michael Dee
Published: January 2023

Leukocytes are believed to cause the lung damage associated with acute lung injury (ALI) but the specific cell types involved have not been clearly demonstrated. Using flow cytometry, we examined the inflammatory cell types that arise in lung after intranasal administration of LPS. We find that LPS results in the accumulation of neutrophils, inflammatory monocytes, exudative macrophages (exMACs), monocyte-derived DC (moDC), and lymphocytes during the acute phase (days 2–3) of ALI. In comparison to WT mice, CCR2-deficient mice given LPS display significantly reduced numbers of lung monocytes, exMACs, and moDC. CCR2-deficient mice also display reduced accumulation of neutrophils and lymphocytes, suggesting that inflammatory monocyte-derived cells promote the accumulation of other cell types. Relative to WT mice, CCR2-deficient mice display decreased weight loss and recover more quickly after LPS challenge. These findings suggest that CCR2+ monocyte-derived inflammatory DC and macrophages play a central role in mediating the lung injury and morbidity associated with LPS-induced acute lung injury.
Intestinal double positive CD4+CD8+ T cells of neonates are highly activated memory cells with higher proliferative capacity compared to single positive CD4+/CD8+ T cells (B115)
Authors: Pahar Bapi , Wang Xiaolei , Lackner Andrew A , Veazey Ronald S
Published: January 2023

Peripheral blood and thymic double positive CD4+CD8+ (DP) T cells from neonates have been described in humans and a number of animals, but the function and immunophenotypic characteristics of other tissue-derived DP T cells are not clearly understood. Here we demonstrate the phenotypic characteristics of DP cells in 6 different tissues including thymus from normal neonatal rhesus macaques (Macaca mulatta) between 0–21 days of age. In general, DP T cells of neonates have higher percentages of memory markers (CD28+CD95+, CD45RA−) compared to single positive (SP) CD4+ and CD8+ T cells. In addition, intestinal DP T cells increase as the animals’ age and levels of CD62L decrease with age suggesting that DP cells proliferate and are activated with maturity and/or antigen exposure. Consistent with this, intestinal DP T cells in neonates express higher levels of activation markers and CCR5 expression. Furthermore, DP T cells have higher rates of proliferation compared to SP CD4+ and SP CD8+ T cells as determined by BrdU labelling techniques. Finally, DP T cells produce higher levels of cytokine production in response to mitogen stimulation compared to SP CD4+ or SP CD8+ T cells. Collectively, these findings demonstrate that intestinal DP T cells of neonates are proliferating, effector cells and are likely involved in regulating immune as compared to the immature DP T cells in the thymus. As in adults, these intestinal DP T cells may be important target cells for early HIV infection in neonates due to their activation, high expression of CCR5, and memory phenotype.
CD137 crosslinking mediates effector T cell death and immune suppression during LCMV infection (B126)
Authors: Zhang Benyue , Mittler Robert S.
Published: January 2023

CD137 (4-1BB) is a member of TNFR superfamily that acts as a costimulatory receptor and plays a role in inducing T cell proliferation, effector function and apoptosis. CD137 is expressed on activated T cells and several other cell lineages e.g. dendritic cells, granulocytes, NK cells and macrophages. Agonist α-CD137 mAbs enhance T cell proliferation, eradicate tumors, suppress humoral immunity and reverse autoimmune disease. Here, we show that α-CD137 can suppress both CD4 and CD8 T cells in an LCMV Armstrong infection model. Suppression only occurs when α-CD137 is injected within 48 hrs of infection. Anti-CD137 injected, LCMV Armstrong infected mice developed elevated levels of IL-10 and prolonged TNF-α production, resulting in decreased virus-specific CD8 T cells and chronic infection like the LCMV Clone 13. By day 8 P.I., both CD4 and CD8 T cells in the treated mice underwent extensive cell death and this correlated with increased levels of TNF-α-dependent Fas and FasL expression on T cells. Anti-CD137-mediated suppression of anti-vial immunity was blocked by injection of a neutralizing anti-TNF-α into IL-10 deficient mice. T cells in LCMV infected, α-CD137 treated Fas KO mice did not undergo apoptosis and CD8 T cell responses were normal, suggesting that suppression is enforced through TNF-Fas-dependent apoptosis. The cell target of α-CD137 responsible for initiating suppression is still under investigation. Our data suggest CD137 signaling at the early stage of virus infection can negatively regulate host anti-viral immunity by inducing T cell deletion through apoptosis.
Respiratory syncytial virus blocks MHC Class-II surface translocation in human myeloid dendritic cell (B127)
Authors: Xu Chuang , Gill Michelle Ann , Bajwa Gagan , Xue Yaming , Pallazo Adam , Banchereau Jacques , Connolly John E.
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants and young children. Antigen presentation by dendritic cells (DCs) is critical for the establishment of antiviral immunity and DCs are frequent target of viral immune evasion. Here we show that primary blood mDCs exposed in vitro to RSV failed to upregulate MHC Class-II. Similar results were observed in mDCs isolated from the nasal mucosa of RSV infected infants. MHC Class-II levels remained low despite the apparent upregulation of other markers of DC maturation such as MHC Class-I, CD83, CD86 and CD40. This inhibition could not be explained by the reduced bio-synthesis of MHC Class-II as class II transactivator (CIITA) level remained unchanged and the total cellular HLA-DR was comparable to flu exposed mDC. Furthermore, SDS stability assay showed similar level of MHC Class-II peptide complex between the two viral treatments, indicating RSV exposure did not block Class-II loading. Consistent with the reduced surface expression, confocal microscopy demonstrated a selective blockade of MHC Class-II surface translocation by RSV. These results demonstrate that RSV blocks DC antigen presentation by inhibiting Class-II peptide complex transport to the surface and suggest a mechanism of viral immune evasion.

This work is supported by grants from Baylor Health Care Systems Foundation, the Children’s Medical Center of Dallas Foundation to MG, and the National Institutes of Health (K08 AI059379-02 to MG, U19 AIO57234 to JB).
Exaggerated sickness behavior and brain proinflammatory cytokine expression in aged mice in response to intracerebroventricular lipopolysaccharide (B13)
Authors: Henry Christopher J. , Huang Yan , Dantzer Robert , Johnson Rodney W. , Godbout Jonathan P.
Published: January 2023

Age-associated changes in glial reactivity may predispose individuals to exacerbated neuroinflammatory cytokine responses that are permissive to cognitive and behavioral complications. The purpose of this study was to determine if aging is associated with an exaggerated sickness response to central innate immune activation. Our results show that intracerebroventricular (i.c.v) administration of lipopolysaccharide (LPS) caused a heightened proinflammatory cytokine response (IL-1β, IL-6, and TNFα) in the cerebellum 2 h post i.c.v. injection in aged mice compared to adults. This amplified inflammatory profile was consistent with a brain region-dependent increase in reactive glial markers (MHC class II, TLR2 and TLR4). Moreover, LPS caused a prolonged sickness behavior response in aged mice that was paralleled by a protracted expression of brain cytokines in the cerebellum and hippocampus. Finally, central LPS injection caused amplified and prolonged IL-6 levels in the periphery of aged mice. Collectively, these data establish that activation of the central innate immune system leads to exacerbated neuroinflammation and prolonged sickness behavior in aged as compared to adult mice.
Hyper-induction of cytokines by avian influenza A (H5N1): The role of signaling pathways and Toll-like receptors (B131)
Authors: Hui Kenrie Pui Yan , Cheung CY , Ng HY , Wu HS , Peiris JSM
Published: January 2023

Avian influenza virus A/HK/483/97 (H5N1) is associated with unusually severe human disease. H5N1 viruses hyper-induce pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-α] and interferon beta [IFN-β]) and macrophage-tropic chemokines from primary human macrophages in vitro and this is paralleled by elevated levels of these cytokines in patients sera. It is therefore important to understand the mechanisms underlying this H5N1 induced differential hyper-induction of cytokines. Unlike H1N1, H5N1 virus strongly activated interferon regulatory factor 3 (IRF3). siRNA mediated gene silencing of IRF3 led to a dramatic reduction of the gene expression of IFN-β in H5N1-infected cells but only a partial decrease in TNF-α gene expression. Thus the differential activation of IRF3 may explain the hyper-induction of IFN-β, but does not fully explain the hyper-induction of TNF-α. Similarly, gene silencing of toll-like receptor 3 (TLR3) did not completely abrogate the TNF-α response. The significance of these findings in the pathogenesis of human H5N1 disease will be discussed.
A novel gammaherpesvirus secreted protein induces Vβ4+ CD8+ T cell lymphocytosis during latency (B132)
Authors: Evans Andrew George , Moser Janice M. , Krug Laurie T. , Speck Samuel H.
Published: January 2023

Despite the important contribution of T cells to the control of lytic viral infection, relatively little is known about their role during latent herpesvirus infection. Murine gammaherpesvirus 68 (γHV68) induces a CD8+ T cell response that persists throughout latency, is characterized by cells bearing a Vβ4+ T cell receptor, and is independent of MHC Class Ia and β2-microglobin expression in vivo. It has been proposed that an unknown viral superantigen or T cell stimulatory ligand could be responsible for their activation. We have demonstrated that the novel γHV68 gene, M1, is required for stimulation of Vβ4+ CD8+ T cells and their interferon-γ (IFN-γ) production. Examination of M1-deficient viral mutants has revealed a role for these T cells in the IFN-γ-dependent control of viral reactivation from latency in C57Bl/6 mice and the development of CD8+ T cell-mediated fibrosis during persistent γHV68 infection of IFN-γ receptor-deficient (IFN-γR−/−) mice. Analysis of recombinant M1 expression reveals that the mature protein is fully secreted, and is capable of stimulating IFN-γ production from Vβ4+ CD8+ T cells ex vivo. Studies are underway to explore the mechanism, be it either non-classical (Class Ib) MHC:peptide presentation or superantigen-like CD8+ T cell activation, by which M1 is capable of eliciting such an enigmatic T cell response.

This work was supported by NIH grants R01 CA95318, CA52004, CA58524, and AI58057.
Expression of Cytokine Genes and Signal Transduction during Orientia tsutsugamushi Infection (B134)
Authors: Yun Ji-Hyun , Goo Jeong-Eun , Koh Young-Sang
Published: January 2023

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus. TNF-α and IFN-β mRNAs were induced after infection with O. tsutsugamushi. Western blot analysis shows that O. tsutsugamushi-stimulation leads to phosphorylation of ERK1/2, JNK1/2, and p38 MAPKs. Selective inhibitors of ERK1/2, JNK1/2, p38 MAPKs could all completely prevent TNF-α secretion. Host TNF-α production via p38 & JNK pathways by this bacterium was found to be regulated by post-transcriptional mechanism. In contrast, ERK pathway mainly control the transcription step of TNF-α gene regulation. Orientia inactivation by heat did not abolish induction of TNF-α production. However, heat inactivation of bacterium or treatment of host cell with cytochalasin D led to a complete loss of IFN-β induction, suggesting requirement of internalization and escape into cytoplasm by bacteria for host IFN-β response. Selective inhibitors of ERK1/2, p38 MAPKs, or NF-κB could all completely prevent IFN-β induction. In conclusion, our data suggest that distinct mechanism of pathogen detection is involved in activation of TNF-α and IFN-β genes in response to Orientia tsutsugamushi infection.

This study was supported by Brain Korea 21 (BK21) program in 2007.
Age-related Differences in Pulmonary Cytokine Response to Respiratory Syncytial Virus Infection: Modulation by Anti-inflammatory and Antiviral Treatment (B135)
Authors: Boukhvalova Marina S. , Yim Kevin C. , Kuhn Katie H. , Hemming John P. , Prince Gregory A. , Porter David D. , Blanco Jorge C.G.
Published: January 2023

BACKGROUND: Respiratory syncytial virus (RSV) is the major cause of severe lower respiratory tract infection in infants and young children. Recently RSV has also been recognized as a serious health risk in the elderly, but the pathogenesis of RSV infection in the elderly remains unknown.

METHODS: Dynamics of pulmonary cytokine response (including IFN-gamma, IL-4, IL-10, IL-6, MCP-1, and GRO mRNA) during acute RSV infection were investigated in young (<2 months old) and aged (>9 months old) cotton rats S.hispidus. Therapeutic treatments that diminish viral replication (antiviral antibody) and pulmonary pathology (anti-inflammatory corticosteroid) in RSV-infected animals were used to evaluate contribution of virus replication and inflammation to the development of RSV disease with respect to age.

RESULTS: The time of the peak expression of majority of cytokines was shifted with respect to age. Antiviral and anti-inflammatory treatments had similar effect on cytokine expression in aged and young animals. GRO mRNA transcripts were more abundant in the lungs of aged animals.

CONCLUSIONS: The present work reports the age-related delay in the pulmonary cytokine response to RSV, imbalance in chemokine production with respect to age and underscores different components of RSV pathogenesis with respect to their molecular signature.
In situ location of effector and memory CD8 T cells (B137)
Authors: Dauner Joseph G. , Williams Ifor R. , Jacob Joshy
Published: January 2023

CD8 T cells are critical for the clearance of intracellular pathogens. Following viral infection, naïve CD8 T cells differentiate into effector cells that target and eliminate infected cells. Upon viral clearance, a majority of effector cells die, while a small fraction survives to establish a memory population. Upon future exposures to the same pathogen, these memory T cells are able to rapidly respond and efficiently eliminate the infection. Although much is known about the activation and differentiation of CD8 T cells, the precise in situ location of effector and memory CD8 T cells is not well characterized. Here we present an in situ analysis of the localization of effector and memory CD8 T cells during the murine immune response to acute infection with LCMV. We utilized a transgenic mouse model system in which effector and memory CD8 T cells are irreversibly tagged with yellow fluorescent protein (YFP). We tracked YFP-positive effector and memory CD8 T cells in relation to LCMV-infected cells. Following LCMV infection, activated CD8 T cells were first observed within the T cell zones. As the immune response progressed, effector CD8 T cells migrated to the red pulp regions. Upon viral clearance, memory CD8 T cells localized to the T cells zones. Interestingly, CD44hi memory-phenotype CD8 T cells in naïve mice exhibited identical in situ localization as the memory CD8 T cells observed following LCMV infection.
Direct inhibition of CD4+ T cells by Mycobacterium tuberculosis (B139)
Authors: Mahon Robert Norman , Rojas Roxana E. , Drage Michael G. , Fulton Scott A. , Harding Clifford V. , Boom W. Henry
Published: January 2023

For MTB to survive in the face of robust innate and adaptive immune responses multiple immune evasion mechanisms are required. These mechanisms and the MTB molecules responsible for them are beginning to be elucidated. Apart from interfering with the innate and adaptive immune functions of macrophages, recent studies suggest that MTB also can directly modulate CD4+ T cell function. The current study was undertaken to identify MTB molecules and mechanisms for direct inhibition of CD4+ T cell activation. Murine CD4+ T cells were purified from spleens by a combination of negative selection with mAb-coated beads and flow sorting. Purified T cells were activated for 24–48 hrs. with plate-bound anti-CD3 and soluble anti-CD28 mAbs with whole bacilli or subcellular fractions of MTB. Multiple measures of activation were used including IL-2 production by ELISA, proliferation by thymidine incorporation, and cell surface receptor expression by flow cytometry. MTB bacilli decreased T cell activation by all three measures. Fractionation identified a lipophilic fraction of the MTB cell wall as a major inhibitor of anti-CD3/CD28 mediated activation. T cell metabolic activity was unchanged in the presence of the lipophilic fraction. Activation of CD4+ T cells by PMA/ionomyocin was not affected by MTB. Thus lipophilic molecules in the MTB cell wall directly inhibit CD3/CD28 mediated CD4+ T cell activation by interfering with proximal T cell signaling events.
The Role of CD8+ T cells in the Development of Oral Tolerance (B14)
Authors: Arnaboldi Paul M. , Roth-Walter Franziska , Mayer Lloyd
Published: January 2023

The role of CD4+ vs. CD8+ T cells in the generation of low dose oral tolerance is still controversial. To address the roles of these cells in the setting of an intact immune system C57BL/6 mice were fed PBS, OVA, or molar equivalents of MHC Class I (SIINFEKL) or MHC Class II (OVA(323–339)) peptides by gastric lavage followed by immunization with OVA in both hind-leg footpads. Mice were sacrificed and cells from the spleen and draining lymph nodes were restimulated in-vitro with OVA. Mice fed OVA or SIINFEKL before OVA immunization displayed significantly reduced cell proliferation and cytokine (IFN-γ and IL-2) production compared to mice fed PBS. In contrast, feeding OVA(323–339) prior to OVA immunization resulted in elevated levels of IFN-γ and IL-2 upon in-vitro restimulation. However, tolerance was achieved if mice were both fed and immunized with OVA(323–339). The induction of cytokine mRNA (IFN-γ, TGFβ and IL-10) in jejunum, ileum, Peyer’s patches and spleen was then assessed 6h after feeding OVA or peptides. IFN-γ and IL-10 were induced in the jejunum and ileum but not PPs with either OVA or peptides but these cytokines were only seen in the spleen after OVA or OVA(323–339) feeding. These results suggest that the stimulation of CD8+ T cells in the intestine following antigen feeding may be a key step in the induction of low dose oral tolerance.
Analysis of dendritic cell maturation following respiratory infection with vaccinia virus (B140)
Authors: Beauchamp Nicole Marie , Alexander-Miller Martha , Alexander-Miller Martha
Published: January 2023

Poxvirus infections have the potential to induce significant pathology. Clearance of these infections requires the generation of a potent adaptive immune response. A key contributor to this process is the dendritic cell (DC). Using a recombinant vaccinia virus (VV) expressing enhanced green fluorescent protein (eGFP), infected DCs were tracked following respiratory infection. CD8+ and CD8− subsets of DCs harvested from the mediastinal lymph node (MLN) and lung were analyzed for the expression of eGFP and the maturation markers CD80 and CD86. EGFP+ cells were found within both subsets. Although the absolute number of eGFP+ CD8+ DCs was lower, on average, than the eGFP+ CD8− DCs, they were similarly infected on a percentage basis. EGFP+ cells appeared to undergo maturation, showing increased expression of CD80 and CD86. These results suggest that following infection of the respiratory tract, VV has the ability to provide a maturation signal to infected DC. As a result these cells may contribute directly to the elicitation of a T cell response.

This work was supported by NIH grant P01 AI60642.
Lewis Lung Carcinoma-derived CCL22 recruitment of T regulatory cells (B148)
Authors: Mailloux Adam William , Lathers Deanne M R , Young M Rita I
Published: January 2023

Two aspects of cancer progression that contribute to malignancy are the ability of the tumor to metastasize to remote locations in the body, and the ability to evade effective host anti-tumor immune responses. It has been shown that T regulatory cells (Treg) can inhibit immune effector cell function and aid in anti-tumor immune evasion. Several types of tumors have elevated Treg numbers, and it is believed that tumors may actively recruit these cell types. Treg migration is induced by several chemokines and their receptors such as CCL22 and CCR4. Here, we use flow cytometry to analyze CCR4 expression on T cell populations and show that Tregs express more CCR4 than any other T-cell type. We also used ELISA to compare CCL22 secretion levels in normal and Lewis Lung Carcinoma (LLC) bearing lung tissue. While high basal levels of CCL22 are secreted from normal lung tissue, even higher levels are secreted by LLC bearing lung tissue. In addition, we used media conditioned on these tissues in transwell migration assays to measure preferential Treg chemoattraction. Normal lung tissue induced high levels of selective Treg chemoattraction, while LLC bearing lung selectively attracted even greater numbers of Tregs. These data suggest that normal lung tissue may be able to recruit elevated levels of immune inhibitory Tregs through the secretion of CCL22 prior to metastasis, and that once LLC metastasis occurs, both CCL22 and Treg levels increase even further.
Long-term inhibition of tumor growth by T cells targeting cancer stroma alone: persistent elimination of tumor-promoting myeloid suppressor cells (B149)
Authors: zhang bin , zhang Yi , Schietinger Andrea , Bowerman Natalie , Fu Yang-Xin , Kranz David , Rowley Donald , Schreiber Hans
Published: January 2023

The growth of solid tumors depends on tumor stroma. Targeting tumor stromal cells might provide an effective strategy to inhibit tumor growth. A single adoptive transfer of CD8+ T cells which only recognize tumor-Ag-loaded stromal cells, but not the cancer cells because of MHC restriction, caused a long-term equilibrium between tumor growth and elimination. The T cells persistently destroyed at least two subpopulations of myeloid-derived suppressor cells (MDSCs) in the tumor stroma: CD11b+ F4/80+ and CD11b+ Gr1+ cells. Using high affinity TCR tetramers, we found that both subpopulations captured tumor Ag from surrounding cancer cells. Epitopes on the captured Ag make these cells targets for Ag-specific T cells. These two subpopulations suppressed T cell proliferation and promoted tumorogenesis. By adding Gr1+ immature myeloid cells that expressed irrelevant MHC-I molecules, T cell-mediated inhibition of tumor growth was abrogated. Therefore, recognition and elimination of these MDSC by T cells may significantly contribute to effective control of tumor growth. These findings indicate targeting MDSCs by T cells may complement conventional cancer treatments.
Small molecular MHC-loading enhancers (MLE) amplify immune responses by targeting pocket 1 of class II MHC proteins (B150)
Authors: Gupta Shashank , Hoepner Sabine , Dickhaut Katharina , Rupp Bernd , Kühne Ronald , Freund Christian , Jung Günther , Falk Kirsten , Roetzschke Olaf
Published: January 2023

We have shown that certain organic compounds can amplify immune responses by catalyzing the peptide-loading of human class II MHC molecules. Notably, the effect was evident only for an allelic subset of HLA-DR molecules expressing glycine at the dimorphic position b86. The residue forms the floor of the conserved pocket P1, which is located in the peptide binding site of MHC molecule. The transient occupation of this pocket by adamantane derivatives stabilizes the peptide-receptive conformation, permitting rapid antigen loading. On ‘adamantyl-susceptible’ MHC molecules these structural transitions are detectable by conformation-specific antibodies. As ‘MHC-loading enhancer’ (MLE) these compounds may be useful molecular tools to amplify the immune response. In experimental tumour models MLE compounds have already been successfully used as ‘adjuvant’ to enhance the CD4+ T cell response against several tumour antigens. The observation, however, that the ligand repertoire can be affected through polymorphic sites may also imply that environmental factors could modulate allergic or autoimmune reactions by this mechanism. Preliminary in vitro data with the celiac disease associated HLA-DQ2 and in vivo data obtained in the MS animal model EAE suggest that MLE compounds could act as risk factors acting in a strictly allele-selective manner.