bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of Aryl Hydrocarbon Receptor activation by TCDD on IL-12R signaling and generation of regulatory T cells (47.9)
Authors: Marshall Nikki B , Kerkvliet Nancy I
Published: January 2023

We have previously shown that allo-reactive donor CD4+ T cells are converted into CD25High Foxp3-negative regulatory T cells during an acute Graft-vs-Host Response (aGVHR) in mice treated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). Activation of the Aryl Hydrocarbon Receptor (AhR) in donor CD4+ T cells is required for full suppression of aGVHR and generation of regulatory T cells. CD4+ T cells exposed to TCDD during aGVHR exhibit a significant increase in IL-12Rb2 mRNA and protein as well as phosphorylated-STAT4 expression. We used IL-12Rb2-deficient (KO) donor T cells to determine whether increased IL-12R signaling was important for the generation of TCDD-induced regulatory T cells. CD25 protein expression was not changed on IL-12Rb2 KO cells compared to wild-type control (WT), however significantly fewer IL-12Rb2 KO T cells expressed phosphorylated-STAT4. A marked decrease in expression levels of phosphorylated-STAT1 and STAT5 was also observed in both WT and IL-12Rb2 KO donor T cells from TCDD-treated mice compared to vehicle control. Although IL-12Rb2 deficiency does not appear to affect the generation of donor-derived CD25High Foxp3-negative regulatory T cells during aGVHR in TCDD-treated mice, current studies focus on the effects of TCDD and altered IL-12R signaling on Foxp3-positive regulatory T cell generation.
Roles for Id3 in T Cell Development and Autoimmunity (48.17)
Authors: Mahlios Josh M , Zhuang Yuan
Published: January 2023

T cell development is a tightly regulated process that requires a number of known transcription factors, one of which is Inhibitor of Differentiation 3 (Id3). Here we find that Id3 plays an important role during thymopoiesis by preventing the development of an autoimmune condition similar to that of primary Sjogren's Syndrome. In the absence of a functional Id3 protein, thymocyte development is dysregulated and leads to the production of T cells capable of inducing symptoms associated with Sjogren's Syndrome. The T cell repertoire is altered in a manner in which large numbers of γδ T cells are produced, raising the question as to which subset(s) of T cells might be responsible for the induction of autoimmunity. Through the use of various genetic knockout mouse models, we have identified αβ rather than γδ T cells as an important population required for many of the phenotypic abnormalities associated with Sjogren's Syndrome.
IL-6 may interfere with the development of functional adaptive Tregs in NOD mice (48.19)
Authors: Azeem Nabeel , Triplett Ashley S , Parnell Sarah , Zhao Yuan , Alard Pascale , Kosiewicz Michele M
Published: January 2023

The cytokine milieu dictates whether TGF-β induces naïve CD4 cells to become adaptive regulatory cells or pro-inflammatory Th17 cells. We have found previously that while NOD CD4+CD25- cells convert into CD4+CD25+Foxp3+ cells in vivo, these cells are unable to regulate. The conversion defect in NOD mice appears to reside in the APC, and not the T cell, compartment, and transfer of B6.g7 or Idd3/5 DCs can reconstitute effective conversion in NOD mice. We report here preliminary results indicating that NOD DCs cultured with BDC2.5 CD4+CD25- cells, antigen and varying doses of TGF-β are less effective mediators of conversion into Foxp3+CD4+ cells than B6.g7 or Idd3/5 DCs. Furthermore, NOD DCs produce more IL-6 than either B6.g7 or Idd3/5 DCs both constitutively and after LPS stimulation in vitro. Interestingly, spleen and LNs (including pancreatic) from NOD mice contain higher (albeit small) numbers of IL-17+CD4+ cells than control mice. Taken together, these data could indicate that IL-6 produced by APC in NOD mice may interfere with the conversion of naïve CD4+ cells into functional adaptive regulatory T cells by driving these cells toward an Th17-like pathway. Funded by NIH R01 AI064421.
Cyclosporine A and CTLA-4Ig differentially inhibit the effector phase of the DTH response in two separate mouse strains (48.20)
Authors: MacLeod Heather , Goodwin Debbie , Damphousse Christy , Nickerson-Nutter Cheryl
Published: January 2023

Delayed type hypersensitivity (DTH) is classically defined as inflammation involving antigen presentation, cytokine production and T cell activation. Both the Balb/c strain and the autoimmune-prone B6D2F1 strain develop a DTH response. However, DTH in the Balb/c strain is inhibited by the immunosuppressive agent cyclosporine A (CsA), which blocks NFAT activity, whereas DTH in the B6D2F1 strain is not. Balb/c mice generate a traditional Th1 cytokine response that is inhibited by CsA, while B6D2F1 mice produce lower levels of Th1 cytokines and not all are reduced by CsA. Since T cell activation can also occur by costimulation through CD28, we hypothesized that the B6D2F1 DTH effector response may involve activation through CD28 that is not affected by CsA. To test this hypothesis, CTLA-4Ig, which blocks CD28, was administered with and without CsA at the effector phase. Our data indicate that to inhibit DTH in the B6D2F1 strain, both CD28 and NFAT activity must be simultaneously blocked, since neither CsA nor CTLA-4Ig alone were effective. In contrast, CsA is sufficient to block DTH in Balb/c mice indicating that costimulation through CD28 is not involved in the effector phase of the DTH response in this strain. Our results indicate that the pathology of the DTH response is different in Balb/c compared to B6D2F1 mice, implying that different etiologies are involved in the resultant inflammation.
Analysis of Treg defects in female (NZBxNZW)F1 mice with established disease (48.21)
Authors: Tucker Colleen F , Parnell Sarah A , Alard Pascale , Kosiewicz Michele M
Published: January 2023

(NZBxNZW)F1 (BWF1) is a mouse model of SLE in which females develop nephritis by 36 wks. Sick female BWF1 mice have considerably more Foxp3+ Tregs than age-matched non-sick females and males, but these Tregs are unable to control disease. BrdU uptake by CD4+ (non-Treg) cells was increased in sick female mice, suggesting that CD4 activation/proliferation was not well-controlled in vivo in these mice. However, in in vitro analyses of cells from sick females, no defects in Treg function, responder cell sensitivity to suppression or APC activation of Tregs were detected. CD103+ Tregs, a memory/effector subset, were also increased in sick mice. Vβ TCR usage by CD103+ Tregs differed between sick and non-sick mice suggesting the Treg TCR repertoire, and possibly the antigen specificity of Tregs, could be altered in sick mice. Expression of molecules that affect lymphocyte trafficking was also analyzed. In sick mice, no increases in E-cadherin, the CD103 ligand expressed by activated B cells and epithelium, or other molecules associated with lymphocyte sequestration (e.g., CD62L) in lymphoid organs were detected. However, CXCR3, which is involved in trafficking to inflamed sites, was decreased in CD103+ Tregs from sick mice. These data suggest that features of Tregs other than their inherent regulatory function could be altered in sick mice; however, it is unclear whether this is a cause or an effect of inflammation. Funded by LRI.
EAE induced by a non-Th1/Th17 effector is GM-CSF dependent (48.6)
Authors: Kroenke Mark A , Segal Benjamin M
Published: January 2023

IL-12 and IL-23 modulated CD4+ T cells both have encephalitogenic properties and are capable of inducing experimental autoimmune encephalomyelitis (EAE). Although IL-12 polarized Th1 cells do not make IL-17, it is possible that IL-17 comes into play downstream and is essential for disease pathogenesis. In order to address this possibility, we transferred Th1 cells into IL-17 receptor deficient (IL-17RA -/-) mice. We show that the encephalitogenicity of Th1 cells is not compromised in the absence of IL-17A/F signaling. Furthermore, the adoptive transfer of IL-12 or IL-23 polarized IFNγ -/- CD4+ T cells into wild-type mice is known to induce atypical EAE. Not surprisingly, IL-23 modulated IFNγ -/- T cells lose their encephalitogenic properties upon transfer into IL-17RA -/- mice. In contrast, the transfer of IL-12 modulated IFNγ -/- CD4+ T cells into IL-17RA -/- mice resulted in conventional EAE, demonstrating an absolute requirement for IL-17A/F signaling in atypical EAE. In this model, conventional EAE was completely abrogated by administration of GM-CSF neutralizing antibodies, indicating that GM-CSF can mediate the effector phase of EAE in the absence of IFNγ and IL-17.
Rheumatoid factor autoimmunity in the NZM2410 model combines antigen-specific and polyclonal activation (49.16)
Authors: Niu Haitao , Zhou Zhenhai , Sobel Eric S , Shlomchik Mark J , Morel Laurence
Published: January 2023

The rheumatoid factor (RF) specific AM14 BCR tg model has shown that the activation of autoreactive B cell is antigen (Ag) specific and it bypasses the germinal center (GC) in the MRL/lpr mouse. To characterize the activation pattern of AM14 Id+ (Id+) RF B cells in the Fas sufficient NZM2410 model, we have bred the AM14 HC tg on NZM2410-derived B6.Sle1.Sle2.Sle3 (TC) and B6 mice in the presence or absence of the cognate Ag IgG2aa (TC.IgHa AM14, TC.IgHb AM14, B6.IgHa AM14, and B6.IgHb AM14). Id+ RF cells were relatively excluded from the marginal zone in TC.IgHa AM14 mice and preferentially differentiated into plasmablasts with secretion of Id+ RF in TC.IgHa AM14 as compared to B6.IgHa AM14 mice. Id+ RF secretion was however found in some TC.IgHb AM14 mice, indicating that polyclonal activation can activate autoreactive B cells in this model. BrdU labeling showed that the majority of the Id+ RF secreting cells are proliferating and immunofluorescence showed that the Id+ RF plasmablasts are extrafollicular and bypasses GC in TC.IgHa AM14 mice. In conclusion, Id+ RF B cells break tolerance due to a combination of Ag specific and polyclonal activation bypassing GC on the TC background.

This study is supported by NIH/NIAID R01-A058150
A Novel HLA Shared Epitope VHYLA in Rheumatoid Arthritis (49.5)
Authors: Freed Brian M , Schuyler Ron , Lambert Cherie , Aubrey Michael
Published: January 2023

Although rheumatoid arthritis has long been associated with HLA shared epitopes, a systematic search for epitopes has never been reported. We have developed a computer program that can analyze large HLA data sets and detect all shared epitopes comprising from one to five amino acids. We analyzed high resolution data from the International Histocompatibility Working Group, which included 639 subjects with rheumatoid arthritis and 429 controls, for all combinations of up to five amino acids among HLA-DRB1 positions 9-86. We compared this analysis with a traditional allele analysis and the prevalence of the QRAA epitope. Statistical significance was determined using a Chi-square analysis with a Bonferroni correction. The allele analysis identified three HLA alleles that were positively associated with RA, DRB1*0401 (p=2x10-9), *0404 (p=0.006) and *0405 (p=3x10-5) and five alleles that were resistant, DRB1*0701 (p=0.007), *0801 (p=0.037), *1101 (p=0.037), *1301 (p=0.04) and *1302 (p=0.004). DRB1*0101 (p=0.24) and *0102 (p=0.421) were not associated with RA in this data set. The single amino acid analysis identified V11 (p=10-16), H13 (p=10-16), Y37 (p=10-5), L67 (p=3x10-9), Q70 (p=0.0001) and A74 (p=0.0007) as RA-susceptibility markers. In addition, S11 (p=4x10-7), S13 (p=2x10-6), I67 (p=3x10-7) and D70 (p=5x10-9) were RA-resistance markers. The combination of these positions yielded a VHYLA11,13,37,67,74 epitope that was more strongly associated with RA (p=4x10-22) than QRAA70,71,73,74 (p=9x10-10) or QKAA70,71,73,74 (p=3x10-10). VHYLA is associated with DRB1*0401,*0404,*0405,*0408, *0409 and *0410, but not with DRB1*01 alleles, consistent with the allele analysis in this data set.
Macrophage migration inhibitory factor promotes resistance to glucocorticoids in experimental autoimmune encephalomyelitis (50.12)
Authors: Forsthuber Thomas G. , Salazar Elisabeth , Yang Yuye
Published: January 2023

Glucocorticoid (GC) treatment is the standard of care for acute attacks of Multiple sclerosis (MS). However, while initially highly efficacious in inducing remission of MS, GCs eventually loose efficacy and overall do not prevent progression of the disease. GCs have the unique property of inducing the release of Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine with broad effects on the immune response. Thus, we hypothesized that MIF could play an important role in promoting disease by its unique ability to counter-regulate the immunosuppressive effects of GCs. Testing this question, we found that treatment of MIF knockout mice (KO) with GCs was substantially more efficacious as compared with GC-treatment of Wt mice and resulted in a substantial delay in disease onset and severity of EAE. Histological and immunofluorescence studies showed that inflammatory infiltrates were dramatically reduced in MIF KO mice. Furthermore, thymus and spleen cells from MIF KO mice showed higher extend of apoptosis upon incubation with GCs as compared with Wt mice. The results suggest that the blockade of MIF in combination with GC administration could be a promising strategy for the treatment of MS. Supported by grants NS052177 (NIH) and RG3499 (NMSS).
Inhibition of T cell activation by the phytochemical piperine (50.35)
Authors: Doucette Carolyn D , Rodgers Gemma , Liwski Robert , Hoskin David W
Published: January 2023

Piperine, a major alkaloid of the fruits of black and long pepper plants, exhibits anti-inflammatory activity both in vitro and in vivo. Although the effect of piperine on T cell function is not yet known, we hypothesized that piperine might inhibit T cell processes involved in inflammation. We therefore examined the effect of piperine on T cell proliferation, activation marker expression, and cytokine production. Cellular proliferation was determined by tritiated-thymidine incorporation and Oregon Green® 488 staining while activation marker expression was assessed by flow cytometry. Cytokine production and intracellular signalling pathways were examined by ELISA and Western blot analysis, respectively. Piperine inhibited T cell proliferation in a dose-dependent manner without affecting T cell viability. Piperine also inhibited expression of the T cell activation markers CD25, CD69, and CTLA-4, as well as production of the cytokines IFN-γ, IL-2, IL-4, and IL-17. Phosphorylation of extracellular signal-regulated kinase (ERK) and inhibitor of κBα (IκBα) was diminished in T cells stimulated in the presence of piperine, indicating that piperine affects signalling pathways involved in T cell activation. Collectively, these data suggest that piperine warrants further investigation as a possible immunosuppressive agent for the treatment of T cell-mediated inflammation. Supported by NSERC and CIHR.
T cell receptor-mediated protein geranylgeranylation controls transforming growth factor-beta-induced Foxp3 expression by regulating the induction of Smad6/7 (50.40)
Authors: Kim Yong Chan , Kim Kee Kwang , Shevach Ethan M
Published: January 2023

TGF- β plays a critical role as a differentiation factor for the generation of Foxp3+ induced T regulatory cells (iTregs) from naïve T cells. To evaluate the potential role of the mevalonate pathway in the control of iTreg development, we have used the mevalonate pathway inhibitor, Simvastatin. When CD4+Foxp3- T cells were activated with anti-CD3/CD28, the addition of Simvastatin increased the induction of Foxp3+ T cells. A marked synergistic induction of Foxp3 expression was observed when Simvastatin was added to the induction cultures with a suboptimal concentration of TGF-β. The action of Simvastatin was not mediated by enhancement of Smad3 phosphorylation, but was associated with marked inhibition of induction of the Smad 6/7 proteins. The synergistic effect was mediated at the level of mRNA transcription and was associated with increased DNA demethylation of the Foxp3 promoter. The effects of Simvastatin and TGF-βon the induction of Foxp3 expression were reproduced by the addition of an inhibitor of protein geranylgeranylation, but not an inhibitor of protein farnesylation. These studies demonstrate that protein geranylgeranylation represents of one of the key regulatory steps in the generation of iTregs by TGF-β. Pharmacologic manipulation of the mevalonate pathway may represents one mechanism for the induction of iTregs in vivo.
Blockade of the CD30/CD30L pathway inhibits renal disease in young, SLE-prone NZB/W F1 mice (50.41)
Authors: Rottman James  B , Hu Yi-Ling , Willis Cynthia
Published: January 2023

CD30 and CD30L are interacting members of the TNFR and TNF family, respectively. CD30 is expressed by subsets of activated T and B cells, whereas CD30L is expressed by subsets of activated T cells, macrophages and dendritic cells. The CD30 / CD30L interaction modulates T cell activation, germinal center formation and antibody isotype class switching. Given the importance of this pathway to B cell function, we tested the hypothesis that blockade of the CD30 / CD30L pathway would decrease or abrogate autoantibody formation and renal disease in the NZB/W F1 murine model of systemic lupus erythematosis (SLE). Young (5 mo) NZB/W F1 mice were treated with 300 μg CD30L blocking (M15-N297A) or blocking / depleting (M15-IgG2a) antibody weekly for 3 months during the onset of autoimmunity. Both treatments a) reduced the onset and severity of proteinuria, b) significantly decreased the severity of renal histologic scores, c) decreased glomerular immune complex and complement deposition and d) these effects were associated with reduced production of anti-DNA antibodies of the IgG, but not IgM isotype. CD30 / CD30L blockade may be a viable approach for the treatment of SLE.
Treatment with Cl-amidine, a peptidyl arginine deiminase (PAD) inhibitor, significantly reduces collagen-induced arthritis (CIA) (50.6)
Authors: Willis Van C , Gizinski A , Banda N K , Knuckley B , Cordova K , Luo Y , Causey C P , Levitt B , Glogowska M , Benbarak M , Chandra P , Kulik L , Arend W P , Robinson W H , Thompson P R , Holers V M
Published: January 2023

Antibodies to citrullinated protein antigens (ACPA) are specific for rheumatoid arthritis (RA). Post-translational citrullination of epitopes to which ACPA are directed is catalyzed by PADs. We have previously shown that ACPA develop in CIA and contribute to joint damage. In this study we used Cl-amidine, a small molecule that specifically inhibits all PAD enzymes, to determine whether limiting PAD activity can modify CIA. CIA and collagen antibody-induced arthritis (CAIA, a model of the antibody-mediated effector pathway of CIA) were induced in DBA/1j mice using standard methods. Mice received either no treatment, vehicle alone (PBS), or 1, 10 or 50 mg/kg/day Cl-amidine by daily IP injection. Cl-amidine reduced CIA disease activity scores by 42%, 53% and 55% in the respective treatment groups (1, 10, 50 mg/kg/day) as well as C3 deposition, IgG1 and IgG2a anti-mouse CII Abs and epitope spreading to citrullinated epitopes. Conversely, Cl-amidine treatment had no effect on the development of CAIA. These results suggest that PADs are essential components in the development of inflammatory arthritis. This work was funded by an ACR REF WOR Grant.
Analysis of the performance of peptide predicting methods using MHC class I peptide sequencing in the C57BL/6 mouse (78.11)
Authors: Delgado Julio , Escobar Hernando , Crockett David , Reyes-Vargas Eduardo , Jensen Peter
Published: January 2023

MHC class I peptide-binding motifs can defined by sequencing of peptides naturally bound to MHC class I molecules. More recently MHC class I-peptide binding motifs have be defined on the basis of quantitative MHC-peptide binding assays in vitro using peptide libraries. The information on peptide binding motifs obtained using binding assays have been useful to develop numerous bioinformatics-based tools to predict the binding of peptides to MHC class I molecules. To date few studies have analyzed the performance of these bioinformatics tools to predict peptides determined by sequencing of peptides eluted directly from MHC class I molecules. In this study we performed sequencing of endogenous peptides eluted from H2Kb and Db molecules expressed in the C57BL/6 mouse. Using data from 280 H2-class I-bound peptides we identified novel preferred anchor residues located in H2Kb and H2Db-associated peptides. When comparing the predicting affinity performance of 3 bioinformatics tools we found that between 60 to 95% of peptides eluted from H2Kb and H2Db molecules were correctly classified as binders by these 3 predicting algorithms, suggesting that these bioinformatics tools are reliable and efficient for prediction of naturally processed MHC class I ligands. These results expand our knowledge about H2 class I binding specificities and provide the identity of positions that can be used for better characterization of H2 class I peptide-binding motifs. Similar studies using sensitive methods for sequencing large numbers of peptides from other MHC molecules are needed to establish more precise motif information that can be used for the training of bioinformatics tool employed in the prediction of CD8 T-cell epitopes
A molecular understanding of how the immunomodulatory E3-19K protein from adenovirus interferes with the class I antigen presentation pathway (78.13)
Authors: Bouvier Marlene , Fu Jie
Published: January 2023

Adenoviruses (Ads) cause persistent infections. The Ad E3-19K protein binds and retains class I MHC molecules in the endoplasmic reticulum (ER), suppressing the cell-surface presentation of viral peptides. The molecular mechanism of E3-19K-mediated class I retention is mostly uncharacterized. This knowledge is important to better understand Ad pathogenicity, and to gain insights into the susceptibility of the class I antigen presentation pathway to viral interferences.

Using biophysical and cell-based approaches, we showed for the first time that E3-19K associates with immature (nascent) and mature (peptide-filled) class I molecules. We also showed that E3-19K does not compete with the class I assembly proteins for binding onto class I molecules. Importantly, immature class I sequestered by E3-19K can still bind peptides. Together, these results suggest that Ads have evolved to interfere with the early and late stages of the class I antigen presentation pathway. We also showed that E3-19K associates with HLA-A and -B molecules, but not HLA-C molecules. This locus specificity may provide a functional advantage to Ads by inactivating T-cell receptors, while avoiding activation of NK receptors. Finally, we showed that residue 56 in MHC and residue 93 in E3-19K are highly critical for class I association with E3-19K. Overall, our results provide a molecular understanding of Ad pathogenicity. Funded by NIH AI65943.
Adapter-mediated substrate selection by a viral immune evasion molecule (78.15)
Authors: Corcoran Kathleen M , Wang Xiaoli , Lybarger Lonnie
Published: January 2023

The MHC class I antigen presentation pathway is a frequent target of virus-encoded immune evasion molecules. Proteins of the viral K3 family are E3 ubiquitin ligases that downregulate immune modulatory molecules. Further, it has been established that the K3 family has cellular homologs (Membrane-Associated RING-CH proteins), some of which act to regulate immune molecules via ubiquitination. The mK3 molecule of γ-herpesvirus 68 has been shown to utilize ER-associated degradation pathways to degrade MHC class I, thereby inhibiting antigen presentation. Here, we have explored the basis for specific targeting of class I for ubiquitination by mK3. We provide direct evidence that adapter proteins (TAP/tapasin, in this case) in the ER membrane position the RING-CH domain of mK3 to permit ubiquitination of the cytosolic tail of the class I heavy-chain. This was accomplished by generating a novel substrate for mK3 which could be ubiquitinated only when placed within the proper orientation within the TAP complex. These findings demonstrate an essential role of adapter proteins in ubiquitin ligase substrate selection, with implications for substrate selection by other K3-related E3 ligases within the immune system.
Post-transcriptional regulation of MARCH1 expression (78.16)
Authors: Jabbour Maurice , Fares Hanna , Lybarger Lonnie
Published: January 2023

In antigen presenting cells (APC) ubiquitination regulates the trafficking of immune modulators (e.g. MHC II). MARCH1 (membrane-associated RING-CH), a newly identified ubiquitin E3 ligase expressed in APC, ubiquitinates MHC II and prevents efficient surface expression. Following LPS-induced maturation of dendritic cells (DC), MARCH1 mRNA is downregulated, permitting MHC II redistribution to the cell surface. Here, we show that MARCH1 expression is also regulated at the post-transcriptional level. In primary DC and APC cell lines, MARCH1 is degraded with a half-life of <30 min. MARCH1 degradation appears to occur mostly, if not exclusively, in lysosomes, since inhibiting lysosomal activity stabilized MARCH1 protein levels. Similar stabilization was observed when MARCH1-expressing cells were treated with cysteine protease inhibitors. Mutational analyses of MARCH1 defined discrete domains required for destabilization, proper localization, and functional interaction with substrates. Together, these data suggest that MARCH1 expression is regulated at a post-transcriptional level by trafficking to endocytic compartments where MARCH1 is proteolysed. The short half-life of MARCH1 permits very rapid changes in the levels of the protein in response to changes in the mRNA, resulting in efficient induction of MHC class II and CD86 once APC receive maturational signals. Supported by Arizona Biomedical Research Commission
Paradoxical influence of the quantity of antigen expressed by the antigen donor cell on the efficiency of cross priming (78.30)
Authors: Kelly III John B , Srivastava Pramod K
Published: January 2023

Cross-priming is a critical component of T cell responses to cancers and viruses, and involves transfer of antigen from antigen donor cells to the antigen presenting cells. In spite of the centrality of antigen in this process, the influence of the quantity of antigen expressed by the antigen donor cell on the efficiency of cross-priming remains un-examined. Here, we have created a novel system where the model antigen ovalbumin is expressed in P815 (d haplotype) cells under the control of an inducible promoter. However, even in un-induced condition, a very low level of ovalbumin can be detected using highly sensitive methods. We have used titrated quantities of un-induced and induced cells, expressing vastly different quantities of ovalbumin, and have monitored cross-priming of the endogenous anti-SIINFEKL T cell response quantitatively, using in vivo cytolytic T lymphocytes assays. The experiments show, paradoxically, that minute quantities of antigen expressed by a large number of cells are far more effective at cross-priming than exponentially larger quantities of antigen expressed by fewer cells. The results point to the existence of cell-associated factors that enhance the availability of antigen in the antigen donor cells to the antigen presenting cells by orders of magnitude.
Intravenous immunoglobulin (IVIG) inhibit B cell-mediate antigen presentation (78.35)
Authors: Paquin Proulx Dominic , Aubin Éric , Lemieux Réal , Bazin Renée
Published: January 2023

IVIg are therapeutic preparations of IgG prepared from the plasma of thousands of donors and are currently used to treat more than one hundred autoimmune or inflammatory diseases, though their mechanisms of action are not precisely known in any of these diseases. Previous work from our laboratory done on human B cells revealed that IVIg interact with MHC class I and II molecules. These interactions led us to hypothesize that IVIg might interfere with B cell-mediated antigen presentation. In the present work, we used an in vitro antigen presentation system with an OVA-specific T cell line (DO11.10) and showed that IVIg inhibited OVA presentation by B cells. IVIg did not decrease OVA internalization or MHC II expression on B cells, suggesting that IVIg inhibited antigen presentation by interfering with either antigen processing or loading of peptides on MHC II molecules. We further showed, using the OVA peptide 323-339 specific for the TCR expressed by DO11.10 T cells which can be loaded directly on MHC II molecules, that IVIg significantly reduced OVA 323-339 presentation, suggesting that the mechanism by which IVIg inhibit OVA presentation by B cells is by competing for peptide occupancy of MHC II molecules. This study extends our previous observations on the spontaneous internalization of IVIg inside living cells by showing a measurable effect of internalized IVIg on the normal cellular process of antigen presentation.
Possible role of class I myosins in molecule segregation at B cell microvilli structures (78.37)
Authors: Maravillas-Montero José  Luis , Gillespie Peter G. , Patino-Lopez Genaro , Shaw Stephen , Santos-Argumedo Leopoldo
Published: January 2023

Cell cytoskeleton plasticity is now recognized as an important element of cell to cell and cell to antigen interactions. It has been shown that T cells and dendritic cells use shape changes in order to establish effective contacts between them. B lymphocytes also show cytoskeleton rearrangements during antigen recognition. The B cell surface change upon activation with LPS or anti-CD40 plus IL-4; these stimuli induce the generation of long thin microvilli, longer than those observed in resting conditions. These structures are formed by a central actin filaments bundle with bridging associated proteins that links them to plasma membrane. One of these proteins are class I myosins, which are molecular motors that interacts both with actin fibers and plasma membrane phosphoinositides; Myo1c and Myo1g are importantly expressed in B cells and appears to increase upon activation. By using live-cell microscopy we determined that they are present across microvilli structures and seems to be involved in their formation. Finally, our data suggest a role for microvilli by segregating molecules such as MHC-II, LFA-1, CD40, etc. possibly mediated by class I myosins.

Supported by CONACyT, project 56836
CLEC4F, A Kupffer Cells Specific Marker, Is Critical for Presentation of Alfa-Galactoceromide to NKT Cells (78.38)
Authors: Hsieh Shie-Liang Edmond , Yang Chi-Ya
Published: January 2023

CLEC4F, a member of C-type lectins, was firstly purified from rat liver extract with high binding affinity to fucose, galactose and N-acetylgalactosamine, and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we generated mAbs to detect the expression of murine CLEC4F (mCLEC4F) in vivo, and investigated its function using mCLEC4F-deficient mice. The anti-mCLEC4F mAbs bind to 293T cells transfected with mCLEC4F cDNA as determined by flow cytometry, and the binding specificity of anti-mCLEC4F mAbs is further confirmed in mCLEC4F-deficient mice. Histochemical staining showed that mCLEC4F is only expressed on F4/80+ cells localized in liver, and is undetectable in bone marrow, spleen, lymph nodes, or other tissues in adult mice. However, mCLEC4F is detected in the liver of embryonic day 11.5 (E11.5), which is 1.5 day earlier than the formation of liver (E10) and is 3.5 day earlier than the formation of bone marrow (E15-16). Moreover, recombinant mCLEC4F.Fc binds to alpha-galactoceramide in a Ca++-dependent manner, and both galactose and ceramide can partially inhibit CLEC4F.Fc binding to alpha-galactoceramide. Interestingly, mCLEC4F-deficient (mCEC4F k/o) mice produced far less cytokines than wild type littermates after intravenous injection ofalpha-galactoceramide. This suggests that mCLEC4F is not only a specific marker for Kupffer cells, but is also critical for the presentation of glycolipid antigen to NKT cells.
Eraap knockdown in CT26 alters antigenic response to the tumour invivo (78.39)
Authors: Reeves Emma , Bailey Ian , Elliott Tim , James Edd
Published: January 2023

Regulatory T cells (Tregs) play a pivotal role in the suppression of tumour antigen responses. It is well established that balb/c mice depleted of Tregs, using the anti-CD25 antibody pc61, are capable of rejecting the colon carcinoma line CT26. Mice challenged with CT26 in these circumstances have also been shown to reject a further challenge with CT26 or a second tumour line. The antigens responsible for this rejection are only partially characterised, with MHC class 1 antigens known to be restricted to all three H2-D molecules. Our laboratory has identified a Dd restricted antigen, GSW11, which is largely responsible for the cross protective response.

Investigation into the processing of GSW11 revealed a sensitivity to over-processing by Eraap. In an attempt to increase the immunogenicity we generated Eraap knockdown CT26 using a plasmid knockdown system. These CT26 derivatives with reduced Eraap mRNA and protein levels were found to stimulate a peptide specific hybridoma more than wild type CT26. In addition, balb/c mice challenged with Eraap knockdown CT26 gained an 80% protection verses 0% mice injected with wild type CT26. Further experimentation is being undertaken to elucidate the exact mechanisms behind this protection.
The threshold for negative selection and regulatory T-cell development is defined by peptide-MHC density (78.41)
Authors: Atibalentja Danielle F , Unanue Emil R
Published: January 2023

High avidity TCR/pMHC binding results in the deletion of thymocytes by negative selection. However, for natural regulatory T-cell (TR) precursors, high avidity TCR/pMHC interaction leads to differentiation and escape from negative selection. The process of diverting thymocytes into alternative pathways of development is incompletely understood. In this study, we consider the role that pMHC density may have in this process. Using two TCR transgenic mice against differentially presented Hen egg white lysozyme (HEL) epitopes, we evaluated negative selection and Foxp3+CD4SP T cell development in response to graded amounts of intravenously injected HEL. In both mice, thymocyte sensitivity to negative selection and TR induction depended on antigen dose and reflected the level of presentation of their cognate epitopes. The absolute increase in TR occurred at doses of HEL lower than the dose required for complete negative selection of conventional CD4SP. TR induction was inefficient in the absence of TCRα gene rearrangement and was not a result of TR subset expansion. Our data shows that pMHC density provides an important signal in the routing of TR precursors away from negative selection towards development as regulatory T-cells.
Characterization and Functional Analysis of Human IL15-induced Dendritic Cells (78.43)
Authors: Okada Starlyn L , Han Shuhong , Chang Lung-Ji
Published: January 2023

Dendritic cells (DCs) are vital to innate and adaptive immune responses. Human monocyte-derived DCs are commonly generated in vitro via IL4 and GM-CSF (IL4 DCs). IL15 has also been shown to induce DC differentiation (IL15 DCs). Mature IL15 DCs are reported to express Langerin cell surface markers and induce a strong CD8 T cell response; however, functional differences between these two DC subsets have yet to be fully characterized. In this study, we report significant variations between the two DC subsets in morphology as well as expression of surface and secreted molecules involved in antigen presentation, co-stimulation and innate immune functions. Despite higher expression of HLA class I, HLA-DR, CD1a, DC-SIGN, CD80, CD86 and CD83 by mature IL4 DCs, IL15 DCs induced higher NK cell proliferation and stronger antigen-specific primary and memory immune responses as determined by IFN-g secretion and CD107a degranulation of autologous lymphocytes. IL15 DCs also secreted very high levels of cytokines and chemokines such as IL6, IL8, IL15, IFN-g, TNFa, CCL1 and MCP-1. Intracellular flow cytometry analysis showed that T and NK cells stimulated with IL15 DCs exhibited increased amounts of activated STAT5 and nuclear NFkB molecules. Our results indicate that IL15 DCs may play a pivotal role in the rapid development of both innate and cognate immune responses. Supported by Vectorite Biomedica Inc. and Yongling Foundation.
B Lymphocyte-Induced Maturation Protein-1/Positive Regulatory Domain I-Binding Factor 1 regulates two amino peptidases associated with antigen processing (78.9)
Authors: Piskurich Janet F , Mayorov Vladimir I
Published: January 2023

A diverse repertoire of specifically-trimmed peptides is presented by the Major Histocompatibility Complex Class I (MHCI) pathway of antigen presentation for immune surveillance by CD8+ cytotoxic T cells. These peptides are generated in the cytosol, then trimmed by amino peptidases to meet the binding requirements of MHCI. Two endoplasmic reticulum amino peptidases, ERAP1 and ERAP2, perform this function in human cells. Positive Regulatory Domain I-Binding Factor 1 (PRDI-BF1) called B Lymphocyte Induced Maturation Protein-1 (BLIMP-1) in mice drives the differentiation of B lymphocytes into plasma cells. We demonstrated that PRDI-BF1/BLIMP-1 regulates the MHCII pathway of antigen presentation by repressing MHC class II transactivator (CIITA) transcription. We now show that PRDI-BF1/BLIMP-1 plays a key role in modulating the MHCI pathway. Functional promoter analyses and protein/DNA binding studies demonstrate that both full-length PRDI-BF1/BLIMP-1 and a truncated form of this transcription factor called PRDI-BF1β, expressed in myeloma cells and associated with resistance to chemotherapy in diffuse large B-cell and T-cell lymphoma, mediate repression of ERAP1 and ERAP2. Repression occurs through a site nearly identical in sequence to a site for repression of CIITA. IFN-γ or ectopic expression of IRF-1 up regulates ERAP expression even in PRDI-BF1/BLIMP-1-expressing cells. The goal of this work is to uncover pathways useful in therapeutic approaches to improve the ability of the immune system to recognize and destroy PRDI-BF1-expressing tumors.

Supported by grants from the National Cancer Institute (RO1CA102203) and MEDCEN Community Health Foundation of Central Georgia.
Immune response and clinical reaction to food proteins with high vs. low/non allergenic potential in an adjuvant-free mouse model (79.17)
Authors: Gangur Venu , Parvataneni Sitaram , Gonipeta Babu , Tempelman Rob
Published: January 2023

Predicting allergenic potential of novel proteins present in foods such as genetically modified foods is a major challenge facing regulatory agencies and biotech industries. Here we tested the hypothesis that the adjuvant-free mouse model that we have previously reported (Birmingham et al Int Arch Allergy Immunol 2007;144(3):203-10) will be useful to distinguish human food proteins with high vs. low/non allergenic potential. Three readouts were used for testing food allergenicity: 1) systemic specific IgE antibody response to transdermal food protein exposure; 2) clinical symptoms of systemic anaphylaxis to oral food protein challenge; and 3) drop in rectal temperature (hypothermia) to oral food protein challenge. The following food proteins were tested for allergenicity: food proteins with high allergenic potential (cashew nut, milk), food proteins with low/non allergenic potential (kidney bean, Pinto bean); and a food protein with unknown history of allergenicity (Amaranth seed protein). We found that whereas, both cashew nut and milk tested positive for all three readouts of allergenicity, Pinto bean and kidney bean although induced specific IgE antibodies, did not cause systemic anaphylaxis and hypothermia. Interestingly, Amaranth seed protein induced not only a robust IgE response but also elicited clinical symptoms of systemic anaphylaxis as well as hypothermia. These data demonstrate that: 1) further testing with additional food proteins is necessary to evaluate the predictive utility of this mouse model for allergenicity testing; and 2) further studies are needed to evaluate the relationship between circulating food specific IgE antibodies and clinical sensitization for oral food induced systemic anaphylaxis. Funding: US EPA STAR Grant#R833133
SCF and IL-25 induced Th2 associated pathophysiology in chronic cockroach antigen induced asthma (79.19)
Authors: Petersen Bryan , Dolgachev Vladislav , Lukacs Nicholas W
Published: January 2023

Asthma is a chronic inflammatory airway disease with a nationwide prevalence of ~6%. It is associated with goblet cell metaplasia and airway hyperreactivity (AHR). Models of allergen-induced asthma exhibit increased levels of stem cell factor (SCF) and the accumulation of myeloid derived SCF receptor (c-kit)+ cells in the airway. Anti-SCF antibody treatments into the airway attenuate Th2 cytokine responses and reduce IL-25 responsiveness in CRA challenged mice in a T cell independent manner. Additional data have identified that an IL-25R+ myeloid cell accumulates in the lung during chronic allergen responses and contributes to Th2 cytokine production. When W/Wv mice were sensitized and challenged with allergen, AHR and mucous hypersecretion responses were significantly attenuated along with overall inflammatory responses. A significant reduction in IL-25R+ myeloid cells was a prominent feature that correlated with reduced Th2 cytokine responses. Subsequent experiments will further define the responses using adoptive transfer of wildtype myeloid progenitor cells to define whether the IL-25R+ myeloid cell population is reconstituted along with Th2 cytokine responses and pathophysiology.

This work was supported by NIH grant HL59178
Microarray analysis of the gene expression profile of HMC-1 mast cells following Schizonepeta tenuifolia treatment (80.14)
Authors: Sohn Sung-Hwa , Ko Eunjung , Kim Yong , Lee Ki-Heung , Kim Yangseok , Shin Minkyu , Hong Moochang , Bae Hyunsu
Published: January 2023

Objective: This study was conducted to evaluate the anti-inflammation mechanism of Schizonepeta tenuifolia (ST) extract and ST purified chemicals on PMA plus A23187-induced stimulation of HMC-1 human mast cells. Methods: In the present study, the anti-inflammatory effects of ST, Rosmarinic acid (RA), Pulegone (PG), and 2¥á,3¥á,24-thrihydrooxylen-12en-28oic acid (TH) on stimulated HMC-1 cells were then evaluated by analyzing IL-6, IL-8, and TNF-¥á secretion. In addition, the effects of ST, RA, PG, and TH on gene expression profiles in stimulated HMC-1 cells were evaluated by microarray and real time RT-PCR (RTRP). Results: ST, RA, PG, and TH treated HMC-1 cells were significantly suppressed pro-infalmmatory cytokines in dose dependent manner. In addition, the results of the microarray and RTRP revealed that ST regulates several pathways, including cytokine-cytokine receptor interaction (CCRI), MAPK, and the Toll-like receptor (TLR) signaling pathways. Conclusions: ST may have potential efficacy for the treatment of inflammation disease through anti-inflammation activity that occurs via inhibition of the CCRI, MAPK, and TLR signaling pathways.
Activation of Bovine NK cells through Toll-like receptors (81.1)
Authors: Mayer Sandra V , Golde William , Estes Don Mark
Published: January 2023

Toll like receptors (TLRs) play a critical role in the immediate detection and control of infection in mammals and are very important for initiating a series of signaling events which result in proinflammatory gene expression. Many of these receptors are type I transmembrane proteins that belong to the interleukin-1 receptor family. There are 13 TLRs already identified in mammals, 10 in humans, 10 in bovine and 13 in mice. The TLRs main functions include secretion of type 1 interferon, proinflamatory cytokines, chemokines and expression of co-stimulatory molecules. The aim of this study was to verify intracellular perforin and gamma interferon production, granzyme, perforin transcription and extra cellular gamma interferon secretion in bovine NK cells treated with TLRs ligands such as TLR2, TLR3, TLR4, TLR5 and TLR9. The cells were sorted by magnetic cell sorting with the NKp46 antibody and 24 hours later they were stimulated for 72 hours with the TLR ligands mentioned above. Our preliminary results demonstrated that flagellin (6/6) and PPD (5/6) are the most inducers of intracellular interferon gamma production followed by LPS (4/6) and CpG (4/6). When we analyzed the intracellular perforin production we verified an increase in the cells treated with CpG (4/6) or LPS (3/6). These results suggest that bovine NK cells might be activated by these TLRs ligants, otherwise further studies are needed to elucidate this hypothesis.

Keywords: Toll like receptors, bovine, NK cells.
Role of CD16 and 2B4 in activation of cytolytic and cytokine release by bovine NK cells (81.2)
Authors: Mayer Sandra V , Golde William , Scherer Charles FC , Endsley Mark , Estes Don Mark
Published: January 2023

Natural Killer cells (NK) are important in both antiviral, bacterial and tumor defense and constitute a major effector population of the innate immune defense. Activation of NK cells by specific cell surface expressed proteins in infected or tumor cells can induce the production of IFNg and cytotoxicity through release of cytotoxic granules such as perforin and granulysin. Bovine NK cells were recently phenotypically characterized but selective activation via cell surface receptors is not fully understood. Treatments using either inhibitory or stimulatory receptors that activate NKs can be used as a target for vaccine formulation against important bovine and human pathogens. In the present study we used lymph node-derived bovine NK cells that were activated with different conditions including activation via CD244 AND CD16 resulting in increasing IFNg and Perforin. Our studies demonstrate that specific in vitro activation conditions of purified bovine NK cells results in a rapid response via secretion of IFNg and cytotoxic granules. Insight into selective NK functional activities can be used in the development of better vaccines against rapidly replicating pathogens such as foot and mouth disease virus.

keywords: NK cells, Bovine, CD16 Receptor, CD244 Receptor
Changes in levels of Sema4A mRNA in developing thymocyte subsets in C57 mice (81.5)
Authors: Chatterjea Devavani , Linder Grace , Matthes David
Published: January 2023

Since their discovery in 1993, the semaphorin family has expanded to include over twenty different types of the proteins with known guidance and developmental roles ranging from heart morphogenesis and vertebrate growth to immuno-regulation. These functions of semaphorins as adaptors of peripheral immune responses in vertebrates have led us to ask whether these molecules may be important for developing T cells in the mouse thymus. We previously showed by RT-PCR that Sema4A mRNA is expressed in the thymus of 4 wk-old C57BL/6 mice. Our immuno-histochemistry studies showed that Sema4A protein is expressed in the thymus of these mice and appears localized to the medullary region. Our current findings show that by quantitative real time PCR analysis, CD4-CD8- (DN) cells as well as CD4+ and CD8+ (SP ) cells express Sema4A mRNA at 2-2.5 fold higher levels compared to CD4+CD8+ (DP) cells in the thymus of C57BL/6 mice. Taken together, our results show that levels of Sema4A mRNA fluctuate during the staged development of thymocytes. In particular, the expression pattern on DN and SP but not DP cells suggests that Sema4A may be used as an adaptor molecule in the expansion of DP cells during positive selection and naïve SP cells once again express Sema4A mRNA prior to their exit from the thymus upon which this molecule serves its known accessory role in antigen-mediated peripheral activation and maturation.