bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the immunogenicity of an alphavirus replicon particle vaccine expressing the SARS-CoV spike (S) glycoprotein in non-human primates (B191)
Authors: Reap Elizabeth A , Watson Aubrey D , Norberg Pamela K , Cecil Chad , Dornsife Ronna E , Kamrud Kurt I , Smith Jonathan
Published: January 2023

We previously evaluated the immunogenicity and efficacy of a virus-like replicon particle (VRP) vaccine expressing the SARS CoV S glycoprotein in mice. SARS S VRP were highly immunogenic and conferred protection upon challenge. To extend that work we have evaluated the immune responses induced by SARS S VRP in macaques immunized at 0, 4 and 24 weeks. PBMC were assayed at two week intervals by ELISPOT and ICS. Post prime, both CD4 and CD8 T cells were found that secrete IFN- γ, IL-2 and TNF- α. The percentage of CD4 cells secreting IFN- γ, IL-2 and TNF- α was substantially more than the percentage of CD4 cells secreting IL-4 post prime indicating a Th1 bias. Post boost, the number of CD4 cells secreting all measured cytokines were equivalent indicating that the SARS S VRP induced a balanced Th1/Th2 response after 2 inoculations. By week 8 a measured decline in the percentage of cytokine-secreting CD8 cells was observed. There were no significant differences in the numbers of T cells secreting cytokines with 2 different VRP dosages given as measured by ICS, however a dose response was seen by ELISPOT. Neutralizing antibodies (NAb) to SARS CoV were measured in sera following post prime and boost inoculations. Three of 4 animals in the low dose group had measurable NAb post boost (GMT = 101) whereas all 4 animals in the high dose group had serum NAb after two doses (GMT = 190). These data provide support to further development of a SARS S VRP vaccine.

This study was supported by NIH grant #UC1-AI62582
Development and Qualification of a Human IFN-gamma ELISPOT Assay for Testing T Cell Responses to an HIV gag DNA Vaccine (B203)
Authors: Chen Hong , Cohen Michael , Rappaport Ruth , Wu Hong-Yin
Published: January 2023

Cell-mediated immunity is believed to be critical for the control and clearance of viral infections. However, frequencies of antigen-specific human T cells are rather low, quantitation of these cells requires assays that exhibit robust sensitivity, specificity, and reproducibility. In this study, a peptide-induced human IFN-gamma ELISPOT assay has been developed and qualified for testing T cell responses to an HIV gag DNA vaccine. We optimized the number of cells per well, the concentrations of HIV gag peptide pools used for stimulation, and the incubation time of cells with the stimulants. We found that overnight-cultured PBMCs, rather than immediately thawed PBMCs, yielded a greater number of spots. The assay is specific, as it detected IFN-gamma but not IL-2 or granzyme B secretion by stimulated PBMCs. The assay results were reproducible and robust across a wide range of cell concentrations. Coefficients of variation of 25% or less were observed for intra-assay replicates from samples containing more than 50 spot-forming cells. Furthermore, the use of cryopreserved PBMC samples allowed direct comparison within a single assay run of samples from multiple time points during the course of a vaccination regimen, minimizing potential run-to-run assay variability. These results indicate that the ELISPOT assay was reliable and suitable for direct ex vivo quantitation of antigen-specific T cells in HIV vaccine trials.
IDRs as Novel Immunomodulators (B208)
Authors: Guarna Marta , Yang Haiyan , Glavas Natalie , Deng Yanshen , Dullaghan Edie , Mookherjee Neeloffer , Bishop Jennifer , Waldbrook Matt , Donini Oreola , Scott Monisha , Gold Michael R. , Finlay Brett , Hancock Robert E. W. , North John R.
Published: January 2023

Inimex Innate Defence Regulators (IDRs) are novel, synthetic immunomodulatory peptides that protect against infections by selectively activating the innate immune system while regulating inflammation. The multi-faceted effects of IDRs are mediated primarily by monocytes and macrophages. These cells respond to IDRs by selectively increasing the expression of cell surface receptors, and cytokines and chemokines (MCP-1, MCP-3 and CCL-5) which trigger the recruitment and activation of immune cells to the site of the infection. In addition, IDRs control inflammation by enhancing the expression of the anti-inflammatory cytokine IL-10, and down-regulating the release of pro-inflammatory cytokines TNF-α and IL-6 in response to pathogen-associated stimuli. As a result, IDRs selectively activate the immune system without concomitant up-regulation of inflammatory responses. This combination of IDR effects is a distinctive quality of these agents since they are able to maintain a balance between immunostimulatory and inflammatory responses to an invading pathogen.

Haiyan Yang is the recipient of an NSERC Industrial R&D Fellowship.

Funded in part by a grant from the FNIH and the CIHR through the Grand Challenges in Global Health initiative
Atorvastatin treatment decreases brain Amyloid-β and serum TNF-α levels in Alzheimer’s disease transgenic mice (B214)
Authors: Kao Patricia F. , Gupta Eric K. , O’Barr Stephen A.
Published: January 2023

Alzheimer’s Disease (AD) is the most common form of dementia affecting over 14 million elderly worldwide. AD, which has no single cause, is characterized neuropathologically by deposits of Amyloid-β (Aβ) peptides, neurofibrillary tangles, reactive astrocytosis, activation of microglial cells, and deficits in the cholinergic system. Since inflammatory markers are increased markedly in AD brain, it is likely that they participate directly in the destruction of brain cells, and may predict the development of AD years before clinical symptoms appear. Non-steroidal anti-inflammatory drug (NSAID) therapies have shown clinical benefit in treating AD. Likewise, it has been hypothesized that the anti-inflammatory properties of statins may alter AD pathology, independent of their cholesterol lowering effect.

In this current study, we treat huAPP R1.40 YAC transgenic mice, a model of Alzheimer’s disease, with therapeutic doses of atorvastatin. Mice either receive a control dose of vehicle, a low dose of atorvastatin or a high dose of atorvastatin via oral gavage. After 12 weeks, animals are sacrificed and levels of Aβ and inflammatory markers, such as IL-1β, IL-6 and TNF-α, were measured. We find that Aβ levels in membrane fractions of the brain are reduced while cytosol fractions show no change. In serum, TNF-α levels decrease with atorvastatin treatment.
EVIDENCE FOR THE EXISTENCE OF AN ALTERNATIVE B CELL DEVELOPMENT PATHWAY (B3)
Authors: Kumar Ritu , Fossati Valentina , Deriva Alessandra , Martensson Lill , Snoeck Hans Willem
Published: January 2023

During B-cell development rearrangement of immunoglobulin heavy and light chain genes occurs sequentially, separated by a pre-BCR checkpoint where the quality of the heavy chains is tested by pairing with a surrogate light chain (SLC). Here we demonstrate the existence of a second B-cell development pathway, that is characterized by a high expression of Sca1 and LFA1, is less dependent on SLC expression and does not have a late pre-B stage. This pathway becomes predominant with age and in conditions of lymphopenia, and is biased towards marginal zone B cells. However, cell fate switches occur between marginal zone and follicular B cells, likely to maintain marginal zone homeostasis. FO B cells derived directly from MZ B cells are distinguished by a higher expression of Sca1. Thus, an alternative, and perhaps ancestral, B-cell development pathway exists that is biased toward to the generation of MZ B cells.
Induction of Chemokines through LTB4 Receptors, BLT1 and BLT2, in Primary Human Monocytes (B47)
Authors: Sun Binggang , Huang Jing-Feng , Dai Heng , Lars Karlsson , Fourie Anne
Published: January 2023

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation. The biological functions of LTB4 are mediated through two homologous receptors with high and low affinities, namely, BLT1 and BLT2. Using human primary monocytes, we are trying to understand the functions of LTB4 in atherosclerosis from a transcriptional activation perspective. We have also examined the contributions from each receptor. Primary human monocytes were treated with LTB4 and total RNA prepared and analyzed by microarray. Of particular interest, among genes upregulated were 4 chemokines including CCL2/MCP-1, CCL7/MCP-3, CXCL2/Groβ and CXCL3/Groγ. Follow up analysis with real-time PCR and ELISA showed time- and dose-dependent induction of these chemokines. The induction of MCP-1 is Gi-coupled since PTX effectively blocked the induction. BLT2 antagonist LY255283 dose-dependently inhibited the induction of MCP-1 gene expression, while BLT1 agonist, U75302 did the opposite. Selective inhibitors of Ca2+ signaling, PI3K, tyrosine kinase and ERK1/2, but not JNK, effectively blocked the LTB4-induced MCP-1 production. To understand the relative contributions of each receptor, we used receptor specific agonist U75302 for BLT1 and 12(S)-HETE for BLT2. We showed that both agonists induced the expression of all four genes, suggesting overlapping roles of the two receptors in induction of gene expression of these chemokines. More interestingly, 12(S)-HETE greatly increased the induction of MCP-1, MCP-3 and CXCL2 by U75302, suggesting synergistic effect between the two receptors.
The Regulation of CCL19 Gene Expression in Human Antigen Presenting Cells (B49)
Authors: Pietila Taija E , Veckman Ville , Lehtonen Anne , Osterlund Pamela , Julkunen Ilkka
Published: January 2023

CCL19 chemokine plays an important role in regulating DC traffic and recruitment of naïve T cells to the vicinity of activated DCs and macrophages. We have analyzed the regulation of CCL19 gene expression in human monocyte-derived DCs/macrophages infected with Salmonella enterica or Sendai virus. Computer analysis of the CCL19 promoter identified two putative NF-κB binding sites and one interferon stimulated response element (ISRE). Transcription factor binding experiments demonstrated that Salmonella or Sendai virus infection increased the binding of NF-κB proteins (p50, p52, p65, and RelB) to both CCL19 promoter NF-κB elements. Salmonella or Sendai virus infection also increased the binding of multiple IRFs, as well as STAT1 and STAT2, to the ISRE site.

The role of NF-κB and various IRF family members in transcriptional control of CCL19 gene was further studied in transfection experiments. The expression of NF-κB dimers readily activated CCL19 promoter. The expression of IRF1, IRF3, or IRF7 proteins was also able to activate the promoter in the presence of Sendai virus infection. CCL19 promoter constructs containing mutated NF-κB and/or ISRE sites were weakly activated. Our data also suggests that c-Jun and c-Fos proteins binding to target AP-1 elements in CCL19 promoter plays a role in transcriptional control of CCL19. Further work focuses on the regulation of CCL19 in human macrophages and myeloid DCs.
A Role for IL-27/IL-27R Signaling in Prevention of Acute Intestinal Inflammation (B53)
Authors: Troy Amy E. , Saris Christiaan J M , Hunter Christopher A , Artis David
Published: January 2023

IL-27 is a member of the IL-12 cytokine family initially implicated in promoting Th1 cell differentiation. IL-27 is elevated in the intestines of inflammatory bowel disease patients and in a murine model of this disease, although the influence of IL-27 on intestinal inflammation remains unclear. To address this, we employed IL-27R−/− mice and the dextran sodium sulfate (DSS) model of acute intestinal inflammation. Following exposure to 5% DSS, IL-27R−/− mice exhibited early and severe disease, as defined by increased weight loss, increased severity of edema and inflammatory lesions in the colon, and increased diarrhea and intestinal bleeding. This pathology lead to high mortality in IL-27R−/− mice during the first five days of DSS treatment. Cytokines IL-6 and IL-17 were elevated in IL-27R−/− mice, while other pro-inflammatory cytokines such as IFNγ remained unchanged. IL-17 was produced both by CD4+ T cells and by non-T cells. Finally, the lamina propria of DSS-treated IL-27R−/− mice contained increased frequencies of innate immune cells following treatment. These results indicate that endogenous IL-27-IL-27R signaling can regulate both innate and adaptive immune cells and contribute to prevention of severe intestinal inflammation.
Assessing function of skin homing T cell selectin-ligands via selective knockdown with siRNA (B62)
Authors: Thieu Khanh P , King Sandy , Fuhlbrigge Robert C
Published: January 2023

The recruitment of T cells to skin is a key feature of inflammatory diseases such as psoriasis and eczema. Skin homing T cells express cutaneous lymphocyte-associated antigen (CLA), a carbohydrate epitope that binds to E-selectin on postcapillary venules and mediates lymphocyte tethering and rolling in shear flow. E-selectin ligand (ESL) activity and CLA epitope expression have been described on P-selectin glycoprotein ligand-1 (PSGL-1) and leukosialin (CD43). CLA+ PSGL-1 serves as both a P-selectin ligand (PSL) and an ESL, whereas CLA+ CD43 is only an ESL. Although their ESL functions appear redundant, the true relative contribution of PSGL-1 and CD43 to ESL activity in human CLA+ T cells has not yet been evaluated.

Using siRNA, we inhibited expression of PSGL-1, CD43, or both on CLA+ T cells by 70–80%, as measured by flow cytometry. PSGL-1 knockdown resulted in a 70% reduction of PSL but relatively unchanged ESL activity, as assessed by shear flow binding assays. CD43 knockdown did not signficantly impact either PSL or ESL activity. Surprisingly, knockdown of both PSGL-1 and CD43 did not noticeably reduce ESL activity nor CLA expression.

As predicted from animal models, PSGL-1 is the major PSL on human CLA+ T cells but serves a redundant role as an ESL. The high residual amounts of CLA expression and ESL activity remaining after knockdown of both PSGL-1 and CD43 suggest that an uncharacterized ESL distinct from PSGL-1 and CD43 exists and may represent a signficant pool of ESL function on skin homing T cells.
Regulation of Toll Like Receptor-3 mediated inflammatory responses by glucocorticoid receptors: Role of MAP Kinases (B66)
Authors: BHATTACHARYYA SANDIP , MUGLIA LOUIS J.
Published: January 2023

Exaggerated activation of toll like receptors (TLRs) often causes chronic inflammatory disorders. Glucocorticoids are a mainstay of therapy for the treatment of an array of inflammatory diseases. Although macrophages are the integral part of TLR-mediated immune response, the crucial mediators present in macrophages to regulate the complex proinflammatory intracellular signaling are yet to be fully delineated. To explore the role of glucocorticoids in this context, we generated mice (MGRKO) with conditional deletion of glucocorticoid receptor (GR) in macrophages. We show that in vivo activation of TLR-3 by Poly (I:C) caused elevated serum TNF-α (2.5 fold, p<0.01), IL-6 (3.5 fold, p<0.01), IL-12 (3 fold, p<0.01) in MGRKO mice as compared to control mice. In vitro studies with cultured macrophages showed that pretreatment with dexamethasone (Dex) significantly inhibited TLR-3 induced expression of TNF-α (3.4 fold), IL-6 (3.5 fold), and IL-12 (4.3 fold) in the control but no effect in GR-deficient macrophages. We observed that among the major MAP kinases JNK was the crucial target for GR after TLR-3 activation. This hypothesis was validated by the use of specific inhibitors to MAP Kinase pathways. In contrast, after TLR-4 activation Dex selectively suppressed p38 MAPK with no effect on JNK. Collectively, we identify JNK as an integral part of GR-driven regulation of TLR-3 mediated inflammatory responses.

This work was supported by grants from NIH/NIAD to Louis J Muglia.
Characterization of a novel, chronic inflammatory response following spinal cord injury with flow cytometry (B67)
Authors: Beck Kevin Douglas , Nguyen Hal X , Galvan Manuel , Salazar Desiree L , Anderson Aileen J
Published: January 2023

Traumatic spinal cord injury results in a breakdown of the blood brain barrier allowing inflammatory cells to enter the cord, potentially mediating both damage and repair. Several studies have described the acute cellular inflammatory response, however our study quantifies inflammatory cells up to 90 days post injury, revealing a previously uncharacterized chronic inflammatory phase. We developed a rapid, debris-minimizing flow cytometry method, allowing for accurate quantification of cellular inflammation in a defined area of the injured spinal cord. We validated the technique by confirming its ability to distinguish differences in cellular inflammation between mild, moderate, and severe injuries. Using this method, we quantified inflammatory cells in segments T8-T10 following a 200 kd contusion injury to T9 in female Sprague-Dawley rats. We confirm and expand upon the published timecourse for both neutrophil and macrophage/microglial infiltration of the injured spinal cord by assaying daily for the first 10 days post injury and over wider intervals up to 90 days post injury. Interestingly, we were able to detect novel chronic inflammation months after injury, with the number of macrophages at 60 days exceeding that at 7 days, the previously published peak of activity. Further experimentation is needed to test whether the chronic inflammatory response aids remyelination or regeneration of damaged axons.
Naïve T cells activated in the liver by hepatocytes express a phenotype consistent with death by neglect (B69)
Authors: Bertolino Patrick Jerome , Holz Lauren , O’Reilly Lorraine , Benseler Volker , McCaughan Geoffrey
Published: January 2023

The liver has unique immunological properties. We have proposed that these properties are associated with the unique ability of the hepatic tissue to support primary activation of CD8+ T cells independently of lymphoid tissues. The fate of T cells activated in the liver however remains a matter of debate. Our results indicate that liver-activated cells die prematurely due to lower expression of cytokines and cell survival genes. In contrast, recent studies in a mouse liver transplantation model indicate that liver-activated T cells mature into full effector cells able to recirculate into lymph nodes (LN) and become tolerised at a latter stage through other mechanisms. In this latter model, it is not clear whether full effector T cells originate from recirculating T cells activated in LN or whether intrahepatic inflammation changes the phenotype of T cells activated locally. To address specifically the recirculation patterns, phenotype and fate of naïve CD8+ lymphocytes activated in the liver and LN in the absence of injury, we used transgenic mouse models in which the antigen is expressed at only one of these sites. Liver-activated cells down-regulated CD62L and thus, were unable to recirculate to LN during the first 3 days. Importantly, these cells were found to express lower levels of CD25 and CD54 than LN-activated cells. Simultaneously anti-apoptotic proteins were upregulated in liver-activated cells whereas this did not occur in LN-activated cells. Thus, liver-activated CD8+ T cells do not recirculate to the LN at early time points and are rather programmed to undergo premature death in vivo thus contributing to the liver tolerance effect.
Blockage of of Gi linked G-protein coupled receptor (GPCR) signaling reduces local and inhibits systemic tissue injury in the mesenteric IR model. (B74)
Authors: Moratz Chantal M , Egan Ryan , Zacharia Athina , Tsokos George
Published: January 2023

Ischemia-reperfusion (IR) is a frequent clinical entity encountered in diverse fields such as vascular surgery, trauma, myocardial infarction, hemorrhage, stroke, and multi-organ failure. The acute inflammatory response following an ischemic insult instigates pathology in local and remote organs leading to significant morbidity and mortality. IR induces injury in a biphasic pattern. The acute phase is characterized by oxidative tissue injury resulting in activation of intracellular damage pathway and antibody-mediated complement activation. The sub acute phase is characterized by leukocyte infiltration. It has been shown previously in several models of IR that complement inhibitors reduce tissue injury. In this study the role of G-protein-coupled receptor signaling was examined. Studies in the mesenteric IR model a global Gi inhibitor, pertussis toxin, attenuated local intestinal tissue injury and production of LTB4 and PGE2. In these animals the systemic inflammatory response was also inhibited, assessed by decrease lung tissue injury and blocked cellular infiltrate. Next specific receptor inhibitors such as blocking antibodies to examine the effectiveness of inhibiting chemokine receptors such as CXCR2 or blocking adhesion molecules such as ICAM-1 or P-selectin were utilized. The data indicate specific chemokine receptors are more efficient in inhibiting tissue damage. Comparison of these inhibitors will be useful in developing therapeutics which can control deleterious effects of systemic inflammation after an initial trauma.

USAMRAA W81xWH0610485
Fatal autoimmune disease in scurfy mice is initiated and sustained by conventional effector CD4+ T cells and not by cells that express mutant Foxp3 gene and follow the developmental program of regulatory CD4+ T cells. (B81)
Authors: Kraj Piotr Jan , Kuczma Michal , Mazurek Maciej , Ignatowicz Leszek
Published: January 2023

To determine features of natural regulatory T cells (Treg) controlled by Foxp3 we investigated a subset of CD4+ T cells identified in scurfy mice expressing transcript of mutant, non-functional allele of the Foxp3 gene (Foxp3scurfy+ cells). Foxp3scurfy+ cells poorly responded to TCR stimulation in vitro, produced low level of cytokines including Il-2 and remained dependent on exogenous Il-2 for proliferation. In vivo Foxp3scurfy+ cells did not expand relative to other CD4+ T cells and expressed lower levels of activation markers than conventional CD4+ T cells. Thus, Foxp3scurfy+ cells retained the properties of Treg cells despite losing suppressor function suggesting that Foxp3 is not necessary for the development of Treg cells and that the developmental program followed by these cells is not superseded by inflammatory environment. In contrast, conventional CD4+ T cells expressed highly activated phenotype and produced very high level of Il-2 and inflammatory cytokines. In conclusion, the loss of Foxp3 by Treg cells does not convert them into aggressive, self-reactive T cells. Our results identify conventional CD4+ T cells as the population that initiates and sustains autoimmune disease.
Therapeutic effect of Jagged1 DNA vaccine on collagen-induced arthritis (B86)
Authors: Kijima Mika , Yasutomo Koji
Published: January 2023

Notch signaling plays an important role in functional differentiation of mature T cells. Those findings have allowed us to examine whether modification of Notch signaling has therapeutic effects on immune-associated diseases. We here examined whether DNA vaccine of Jagged1, one of the Notch ligands, is able to prevent collagen-induced arthritis (CIA) in mice. The CIA was induced in male DBA/1J mice by immunizing bovine collagen type II. Those mice were vaccinated with extracellular region of Jagged1 (soluble form: sJagged1) gene three times every one week starting at 2 days before initial immunization. The mice vaccinated with sJagged1 showed reduced clinical score and disease incidence compared with control mice. Histological analysis also showed reduced infiltration of inflammatory cells in the joints by sJagged1-treated mice. The vaccination with sJagged1 did not affect the numbers of regulatory T cell and production of IL-10 from T cells. The coimmunization of OVA and full length Jagged1 reduced the number of OVA specific and granzyme B producing CD8+ T cells. These results indicate that Jagged1 suppresses antigen-specific CD8+ T cell functions resulted in reduced CIA score, suggesting that Jagged1 has the therapeutics effects on autoimmune diseases by suppressing T cell functions.
Anti-desmoglein autoantibody repertoire in pemphigus (B89)
Authors: Qian Ye , Clarke Stephen H , Diaz Luis A
Published: January 2023

Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are autoimmune skin blistering diseases characterized by pathogenic autoantibodies against desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3), respectively. Fogo selvagem (FS) is the endemic form of PF. To study the etiology of PV and FS, hybridomas were generated from the B cells of 5 PV patients and 7 FS patients. The V genes of both H and L chain of the Dsg-specific autoantibodies from these hybridomas were sequenced and analyzed for the H and L chain pairing of the autoantibodies, the diversity of H and L chain V gene usage, and the extent of somatic mutation. The number of somatic mutations in the expressed genes ranged from zero to 42, and the replacement to silent mutation (R/S) ratio was much higher in CDRs than in FWRs of both IgM and IgG hybridomas from FS patients. The R/S ratio was also much higher in CDRs than in FWRs of IgG hybridomas from PV patients. In addition, clonally related sets of anti-Dsg1 or anti-Dsg3 hybridomas were identified from individual FS or PV patients based on VH-D-JH junctional sequence identity that reveals ongoing somatic mutation during clonal expansion. Thus, our evidence suggests that the anti-Dsg autoimmune response in both PV and FS is antigen selected.
The regulatory role of naturally occurring CTLA-4 splice variants:the suppressive function of short splice variant 1/4 WT CTLA-4 (B91)
Authors: Zhang Zheng , Zhu Chen , Xiao Sheng , Chung Denise , Wang Yue , Rainbow Dan , Wicker Linda S , Sharpe Arlene H. , Kuchroo Vijay K.
Published: January 2023

Cytotoxic T lymphocyte Antigen 4 (CTLA-4) is an important cell surface molecule that inhibits T cell activation. Four splice variants of CTLA-4 have been identified, indicating the regulatory complexity of CTLA-4. In this study, we characterized the biology of the shortest splice variant 1/4WT CTLA-4 and its functional correlation to full length (FL CTLA-4). 1/4WT CTLA-4 mRNA was found in quiescent CD4+ T cells and upregulated after immunization with MOG35-55 peptide. The kinetic expression of 1/4WT was opposite to FL CTLA-4. To test functions of 1/4WT CTLA-4, we transduced CD4 T cells with a retroviral vector coexpressing 1/4WT CTLA-4 and the green fluorescent protein (GFP). FL CTLA-4 was downregulated on the cell surface by 1/4WT overexpression, which consequently enhanced antigen specific T cell responses by enhancing T cell proliferation and cytokine production in vitro. This functional correlation between 1/4WT and FL CTLA-4 had profound influence on effector CD4 T cell function. Mice adoptively transferred with MOG35-55 specific CD4 T cells containing ectopically expressed 1/4WT CTLA-4 showed dramatically exacerbated EAE disease, increased CD4 T cell proliferation, and increased IFN gamma and IL-17 production. These data suggest that 1/4WT CTLA-4 plays an important role in regulating T cell activation by affecting expression and/or function of FL CTLA-4.
15-deoxy-12,14-prostaglandin J2 and curcumin regulate the expression of toll-like receptors in autoimmune T lymphocytes (B97)
Authors: Chearwae Wanida , Mo Caiqing , Bright John J
Published: January 2023

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease model for multiple sclerosis (MS). Toll-like receptors (TLR), expressed primarily on innate immune cells, play critical roles in the pathogenesis of EAE. We have shown recently that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and curcumin ameliorate EAE through modulating inflammatory signaling pathways in T lymphocytes. In this study we have examined the effects of 15d-PGJ2 and curcumin on the expression of TLRs in T cells in EAE. We induced EAE in SJL/J and C57BL/6 mice by immunization with PLPp139-151 and MOGp35-55 antigen respectively. The mice were treated with DMSO, 15dPGJ2 or curcumin on every other day and the spleen cells isolated on day 14 were used fresh or after culture with antigen for 3 days. The cells were stained with TLR4, TLR9, CD4 or CD8 antibodies and analyzed by flow cytometry. Our results show that naïve CD4+ and CD8+ T cells express detectable levels of TLR4 and TLR9 and that increase significantly following induction of EAE. Interestingly, in vivo or in vitro treatment with 15d-PGJ2 or curcumin differentially regulate the frequency and intensity of TLR4 and TLR9 expression in CD4+ and CD8+ T cells in EAE. Although the exact mechanisms are not known, the regulation of TLR expression in T lymphocytes by 15d-PGJ2 and curcumin suggests new therapeutic targets in the treatment of T cell-mediated autoimmune diseases.
The Rate of Absorption of Ragweed Pollen Material from the Nose
Authors: Cohen Milton B. , Ecker E. E. , Breitbart J. R. , Rudolph J. A.
Published: January 2023

A Prausnitz-Küstner reaction was performed with the serum of a patient highly sensitive to ragweed on 50 non-atopic subjects and 12 patients with asthma, hay fever, or vasomotor rhinitis, nine of whom gave skin reactions by scratch tests. The patients were not naturally sensitive to ragweed. Twenty-four hours later the pollen was blown into both nostrils of the non-atopic and atopic subjects and the sites of injections observed for the appearance of redness and wheal formation. The absorption time of the non-atopic subjects varied from fourteen minutes to thirty-one minutes, while that of the 9 sensitive subjects giving a skin reaction showed a marked delay, ranging from forty-seven minutes to no reaction after two hours. The other three with negative skin tests reacted like the non-atopics.
Parenteral Denaturization of Foreign Proteins
Authors: Boone T. H. , Manwaring W. H.
Published: January 2023

In dogs, India ink blockade reduces the rate of parenteral alien protein denaturization fully 80 per cent. The capillary endothelium is, therefore, presumably the dominant factor in such denaturization.
Signaling by the serine/threonine kinase Drak2 in T cells (35.23)
Authors: Newton Ryan H. , Walsh Craig
Published: January 2023

A role for Drak2 in negative regulation of T cell activation has been well established, yet the manner in which Drak2 mediates downstream events to limit the spurious activation of T cells remains to be elucidated. While T cells from mice deficient in Drak2 exhibit a decreased activation threshold and hyper-respond to sub-optimal stimuli, the proliferative capacity of these cells is diminished as result of a deficiency in survival. A resistance to the induction of EAE in Drak2-deficient mice underscores a dependence on negative regulatory molecules to enforce the requirement of co-stimulation to engage an effective immune response. Biochemical data tracking Drak2 activation has revealed that protein kinase D (PKD) is required for Drak2 signaling. PKD has been shown to respond to the release of calcium from intracellular stores as well as to oxidative stress, two events coupled to T cell activation through the processes of IP3R-mediated ER-calcium depletion and increased mitochondrial respiration. The signaling requirements of Drak2 similarly include both store-operated calcium entry and the production of reactive oxygen species. The data presented indicate that Drak2 and PKD constitute a signaling axis that defines a novel role for PKD in T cell activation and further sheds light on how Drak2 orchestrates decreased responsiveness as a negative regulatory molecule.
The SNARE protein syntaxin 7 is required for immunological synapse formation in cytotoxic T lymphocytes (35.24)
Authors: Pattu Varsha , Qu Bin , Becherer Ute , Matti Ulf , Schwarz Eva , Marshall Misty , Krause Elmar , Hoth Markus , Rettig Jens
Published: January 2023

Cytotoxic T lymphocytes (CTLs) kill target cells by secretion of cytotoxic components such as perforin and granzymes which are contained in lytic granules. Fusion of lytic granules occurs at the contact zone between the target cell and the CTL, the immunological synapse (IS). T cell receptor (TCR) enrichment at the IS is one of the key early events of IS formation. Soluble NSF attachment receptor (SNARE) proteins are required for all fusion events in cells, but the individual SNARE proteins that are important for CTL function are not yet known. We identified syntaxin 7 in CTLs by reverse transcriptase PCR and immunocytochemistry. We found that syntaxin 7 is localised to the IS with a similar distribution to the lytic granule marker - perforin. We then blocked the function of syntaxin 7 by deleting its transmembrane domain and observed a complete loss of TCR accumulation at the IS, indicating that no IS is formed. These results imply that syntaxin 7 is required for IS formation in CTLs. Further studies using specific endosome markers will help us determine the mechanism of syntaxin 7 function in CTLs.

Source of research support - GRK1326
Characterization of SNARE proteins involved in granule exocytosis of cytotoxic T lymphocytes (35.25)
Authors: Marshall Misty R , Pattu Varsha , Qu Bin , Hoth Marcus , Rettig Jens
Published: January 2023

Cytotoxic T lymphocytes (CTLs) exert their cytotoxic activity through the polarized secretion of cytotoxic granules at the immunological synapse (IS). Because soluble NSF attachment receptor (SNARE) proteins mediate numerous intra- and intercellular fusion events and are essential effectors of exocytosis of synaptic granules in neurons, we examined the contribution of specific SNARE proteins to the formation of the IS. To determine the functional significance of specific SNAREs, our lab has examined the expression profile of more than 40 SNARE proteins by RT-PCR and quantitated various protein levels of these SNAREs in resting and activated CTLs. We have found that the expression profile of some SNARE proteins is up or down-regulated upon CTL maturation. In conjunction with these studies, we have also analyzed the sub-cellular localization and mobilization following IS formation of all SNAREs that are expressed in CD8+ T-cells for which specific antibodies are available. Some but not all SNARE proteins are translocated towards the immunological synapse and some co-localized with cytotoxic lytic granules at the immunological synapse. We found that syntaxin 4, syntaxin 6, VAMP 3, and VAMP 8, among other proteins, seem to polarize and colocalize with cytotoxic granule markers at the immune synapse suggesting they play important roles in cytotoxic granule secretion.

Grant/ other support: DFG RE 1092/6-1
HS1 and Itk cooperate to regulate actin remodeling and Ca2+ signaling in T cells (35.28)
Authors: Carrizosa Esteban , Gomez Timothy S. , Labno Christine M. , Chang Briana L. , Klos Dehring Deborah A. , Zhu Peimin , Freedman Bruce D. , Billadeau Daniel D. , Burkhardt Janis K.
Published: January 2023

Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We have shown that the actin regulatory protein HS1 is required for the stabilization of F-actin and Vav1 at the immunological synapse (IS) and for efficient Ca2+ responses. The Tec family kinase Itk also regulates actin responses, in part by facilitating the localization of Vav1 at the IS, suggesting that these proteins act in the same pathway. Using video microscopy, we found that T cells lacking Itk or HS1 behaved similarly, extending unstable actin-rich protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk's SH2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, HS1 failed to localize to the IS. Since Itk is required for PLCγ1 phosphorylation and Ca2+ store release, we examined the Ca2+ signaling pathway in HS1 knockout T cells in greater detail. T cells lacking HS1 exhibited abnormal Ca2+ store release. These cells showed normal phosphorylation but altered localization of PLCγ1. We conclude that Itk-dependent recruitment of HS1 is required for actin stabilization at the IS, and directs the localization of PLCγ1.

Supported by NIH F31-AI071385 to EC and NIH R01-A1065644 to JKB
Inhibition of T cell function in vitro and in vivo using small molecule antagonists of PIM kinases (35.33)
Authors: Jackson Leila J , Wright Dale , Gross Stefan , Robinson John , Marmsater Fredrik P , Allen Shelley , Munson Mark , Carter Laura L
Published: January 2023

The PIM family consists of three highly conserved serine/threonine kinases whose activity is regulated at the level of expression. PIM kinase expression has been shown to be stimulated by a variety of cytokines, hormones, and mitogens and it functions in the regulation of cellular proliferation, differentiation, apoptosis and tumorigenesis. The role of PIM-induced oncogenic transformation has been extensively studied in hematopoietically derived tumors; however the function of PIM in normal immune cells has yet to be fully defined. We sought to elucidate the role of PIM in activation of primary murine T cells by utilizing a highly selective, small molecule PIM inhibitor. We found that inhibition of PIM activity resulted in decreased anti-CD3 and antigen-induced CD4+ T cell proliferation. These effects were observed under Th0, Th1, Th2 and Th17 polarizing conditions, and were not due to increased cell death, but appear to be mediated by a G0/G1 cell cycle arrest. Inhibition of PIM during polarization prevented the production of Th1, Th2, and Th17 effector cytokines suggesting that PIM expression is required for naïve T cell differentiation. Consistent with the in vitro data, mice treated with the PIM inhibitor prior to immunization had a significant reduction in ex vivo recall responses. These results support a key role for PIM in the activation and differentiation of murine CD4+ T cells.
Biochemical evidence for bivalent T cell antigen receptor expression in primary T cells (35.42)
Authors: Schrum Adam G. , Turka Laurence A. , Palmer Ed
Published: January 2023

The αβ T cell antigen receptor (TCR)/CD3 complex is currently viewed as a monovalent receptor with the subunit composition αβγδε2ζ2. This model has emerged from among at least five different potential stoichiometries that were proposed in previous years. Recently, the case for the accepted stoichiometry has been supported by experiments involving co-immunoprecipitation (IP) followed by Western blot, and by blue native (BN)-PAGE. We have revisited the issue of TCR/CD3 stoichiometry by examining immunoprecipitated complexes by flow cytometry (IP-FCM), a technique which allows a quantitative estimate of the subunit constituents in native multi-protein complexes. Together with size-exclusion chromatography, the data supports the conventional stoichiometry, but provides evidence that the copy number may be two, making the TCR/CD3 a precisely bivalent complex, [αβγδε2ζ2] x 2. The expression of a bivalent TCR/CD3 complex has implications regarding possible mechanisms by which the interaction with antigen may trigger signaling.

Research support: Adam G. Schrum, start-up funds from the Mayo Foundation; Laurence A. Turka, grants AI41521 and AI 43620 from NIH; Ed Palmer, grants from the Swiss National Science Foundation.
A Monoclonal Antibody that inhibits IgE binding to the High affinity IgE receptor recognizes a glycolipid (36.11)
Authors: Guiraldelli Michel F , Berenstein Elsa H , Siraganian Reuben P
Published: January 2023

The mAb BD6 is a monoclonal IgM that binds to the surface of RBL-2H3 cells and inhibits the binding of IgE to FcεRI, without reacting with this receptor. By cloning, mAb BD6 binding was resulted by the expression of the alpha1,3-galactosyltransferase gene. In binding assays on RBL-2H3 and alpha1,3-galactosyltransferase transfected PEAK cells there was partial competition between mAb BD6 and IB4, a galactose binding lectin. Both galactose and melibiose decreased the binding of mAb BD6 in a dose dependent manner on RBL-2H3 cells and abolished its binding on alpha1,3-galactosyltransferase transfected PEAK cells. MAb BD6 recognized a low molecular weight band by immunoblotting suggesting that it may be binding a lipid. This was confirmed by blotting lipids from RBL-2H3 cells separated by thin layer chromatography. By sucrose gradient analysis mAb BD6 bound to the lipid rafts fractions. RBL-2H3 variants were isolated completely deficient in mAb BD6 binding; these cells were also deficient in the ganglioside GD1b and had low expression of ganglioside GM1. However, these cells deficient in mAb BD6 binding and GD1b ganglioside still had normal FceRI induced degranulation. These results demonstrate that mAb BD6 inhibits IgE binding and reacts with a glycolipid present in lipid rafts; furthermore the ganglioside GD1b is not essential for degranulation.

Supported by the Intramural Research Program, NIDCR, NIH.
Novel dendritic cell (DC)-stimulating antibody, B7-DCXAb, effectively downregulates allergen-specific IgE, IgG1, Th2 cytokines, and allergic inflammation (36.13)
Authors: Pease Larry R , Checkel James L , Iijima Koji , Radhakrishnan Suresh , Van Keulen Virginia P , McClimon Brad J , Kobayashi Takao , Plager Douglas A , Kita Hirohito
Published: January 2023

We investigated whether human IgM antibody, B7-DCXAb, which binds to B7-DC and enhances DCs' antigen uptake and retention, migration, and cytokine production, improves the efficacy of allergen immunotherapy. Mice were sensitized by intraperitoneal injection of OVA or house dust mite (HDM) antigens plus alum and then treated by intravenous B7-DCXAb injection intranasal or sublingual administration of antigen. OVA-sensitized mice developed anti-OVA IgE and IgG1, Th2-type splenocyte responses to OVA; marked airway eosinophilic inflammation was observed after intranasal OVA challenge. When these mice were treated by intravenous B7-DCXAb plus intranasal OVA, anti-OVA IgE and IgG1 decreased by 75~95% within 1 week; this decrease persisted for >8 weeks. Anti-OVA IgG2a decreased transiently. Splenocyte production of IL-4, IL-5, and IL-13 in response to OVA was abolished; no increases in IFN-g or IL-17 were noted. In OVA-challenged mice, airway eosinophilia and lung levels of IL-4, IL-5 and IL-13 were reduced by >90%. Immunotherapeutic effects were also observed with intravenous B7-DCXAb plus sublingual OVA, but not with intravenous B7-DCXAb alone. Furthermore, HDM-sensitized mice were treated successfully by intravenous B7-DCXAb plus intranasal HDM. B7-DCXAb injection plus mucosal administration of antigen effectively treats allergic responses and may provide novel strategy for allergen immunotherapy.
From horses to high school - bringing Pigeon Fever studies into the classroom (37.3)
Authors: Haus Mary , Pollock Roberta
Published: January 2023

Pigeon Fever is an equine disease caused by Corynebacterium psuedotuberculosis. It manifests itself in three forms; external abscesses, internal abscesses, and ulcerative lymphangitis. The hypothesis is that the different manifestations of the disease are due to differences in TH1 versus TH2 responses in infected horses. ELISA tests were used to determine the antibody response to phospholipase D (PLD), the antigen produced by the bacteria. Current diagnostic tools state that horses with internal abscesses have high titers to PLD and horses with external abscesses have low titers. Our results show that while most horses with internal abscesses show high antibody responses to PLD, many horses with external abscesses and some uninfected horses also show high titers to PLD. Another possible tool for diagnosing and studying this disease is by typing monoclonal antibodies to determine the class and subclasses of anti-PLD antibodies being produced to test whether TH1 or TH2 induced antibodies predominate. These monoclonal antibodies have been made by R. Pollock and her colleagues and some have been typed; additional typing of these antibodies will be done by an Advanced Placement Biology class. In doing so, students will not only be able to learn about the immune response in the context of this disease, but actually contribute to an ongoing "real" research project.
Case Study That Focuses On Viral Replication That Enables Students To Understand How Easily Infection Is Transmitted (37.5)
Authors: Jarigese Kevin
Published: January 2023

Case Study That Focuses On Viral Replication That Enables Students To Understand How Easily Infection Is TransmittedKevin J. Jarigese, MST, Science Teacher, West Mesa High School, 6701 Fortuna Road NW, Albuquerque, NM 87121.

This case study is an educational unit developed for beginning biology students. In this unit, students will review an actual case study of a woman who works in a nursing home that is exposed to influenza virus. Through student group evaluations of behavioral issues, evaluation of the effects of vaccines on the epidemiology, and how infectious diseases may spread, students will develop a diagnosis and evaluate courses of action to be taken by patients and health care professionals. A focus on the issues of scientific literacy and the blending of concepts, history, and philosophy will be worked into the case study to help students evaluate and understand the scientific issues of our time.
Interactive computer simulations for teaching tumor immunology (37.8)
Authors: Dokken Nels , Habig Jeff , Otter Tim , Pennell Christopher A
Published: January 2023

We have created computer models to teach principles of cell biology, experimental design and data analysis associated with tumor immunology. The series of models presented here introduce two key concepts in tumor immunology. The first is that cancer arises from the accumulation of mutations, which results in uncontrolled proliferation and spread. The second concept is immune surveillance, which posits that in healthy individuals tumors are recognized and eliminated by the immune system before progressing to clinical disease. We illustrate both concepts by modeling interactions between tumor cells and immune effector cells. Students can study the effects of mutations on tumor cell survival and proliferation, and understand the mechanisms of immune evasion by monitoring gene expression and intracellular resources, and by analyzing metabolic regulatory networks that control emergent cell behaviors for each cell type or lineage. Because of their versatility, ease of use, Internet availability, and compatibility with readily available computing equipment, these models are well suited for a discovery-based learning approach as well as other teaching methods in secondary and college environments. More information about using simulations in education is available at http://www.cdres.com/education.
A predominant role for p75TNFR in TNF-induced CCL1 production (38.12)
Authors: Thomas Heather J , Lipsky Brian , Mozaffarian Afsi , Born Teresa
Published: January 2023

The proinflammatory activities of TNF are primarily mediated through p55TNFR, whereas little is known about the specific functions of p75TNFR. A preponderance of the literature, as well as our expression testing, suggests T cell populations uniquely tend to express more p75TNFR than p55TNFR protein, and the role of p75TNFR in murine thyomcyte development is well established. For these reasons, we studied the murine cytotoxic T cell line CT6 in search of novel consequences of p75TNFR engagement. These cells express high levels of p75TNFR and little p55TNFR. TLDA analysis showed upregulation of the chemokine CCL1/TCA-3 in response to TNF stimulation. ELISA experiments confirmed a striking TNF-induced secretion of CCL1 in these cells. CCL1 induction could be induced by agonistic p75TNFR-specific antibodies, and the TNF-induced response could be inhibited by p75TNFR blocking antibodies. Consistent with data in CT6 cells, CCL1 could be detected in the serum of animals, and serum levels increased 2-3 fold four hours following injection with TNF. This induction was maintained in p55TNFR knockout animals. Our data suggests that induction of the chemokine CCL1/TCA-3 in response to TNF stimulation is driven primarily by p75TNFR signaling. A better understanding of p75TNFR-dependent CCL1 induction will likely provide more insight into the protective or pathological role of p75TNFR in immune disease.