bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of human mannan-binding lectin in Pichia pastoris (B171)
Authors: Kunkalla Kranthi , Rajagopalan Rema , Salvi Veena P. , Rawal Nenoo
Published: January 2023

Mannan-binding lectin (MBL), a pattern-recognition molecule binds to sugars present on the cell surface of microorganisms and activates the lectin pathway of complement. Three naturally occurring point mutations that occur in exon 1 of MBL 2 gene give rise to the structural variants of MBL and are correlated with impaired immune response. Wild type human MBL A and its variant, MBL B (Gly to Asp) were expressed in the yeast, Pichia pastoris. The yeast system has several advantages such as easy to manipulate, less expensive and generally gives high expression levels. Western blot analysis under reduced conditions of rMBL A and rMBL B expressed in the yeast culture medium exhibited a molecular size similar to that of native MBL (32 kDa) purified from human plasma (hMBL). Analysis under nonreduced conditions showed rMBL A and rMBL B to form higher order oligomers (monomers to hexamers) as observed for hMBL. Functional analysis of the rMBL molecules ability to bind sugars measured by ELISA, upon extensive dialysis of the culture medium, indicated that both rMBL A and rMBL B bind to mannan. Functional studies related to complement activation are currently being investigated. The estimated expression level of rMBL A and rMBL B (~0.3mg/L) indicates that the yeast system may be a better expression system of rMBL than the insect cell system (~0.2mg/L).

Research supported by NIH grant HL073804
Proteome-wide profiling of the CD4 and humoral immune responses to vaccinia in humans reveals immunodominant antigens and limited T-B cell correlation (B184)
Authors: Jing Lichen , Davies D Huw , Chong Tiana M , Chun Sookhee , Felgner Philip L , Koelle David M
Published: January 2023

The aims were to identify potential immunodominant CD4 T-cell antigens, estimate within-subject CD4 diversity, and investigate CD4 T-B cell concurrence. Each of 205 predicted vaccinia ORFs was transcribed and translated in vitro. Serial PBMC and sera were obtained from vaccinated subjects. Antibody binding assays used protein-spotted arrays. CD4 proliferation assays used bulk PBMC-derived responders and autologous APC. Overall, CD4 and antibody responses were above background for 145 (70%) and 62 (30%) ORFs, respectively. The mean number of ORFs recognized per specimen by CD4 cells was 55 (range, 20 to 83). The ORFs recognized most frequently by CD4 cells were L4R, D13L, A10L, I3L, H3L, WR149, WR148, and A3L, while for antibodies, the most frequently detected responses were to A10L, B2R, H3L, WR148, I1L, D13L, D8R, and H5R. The CD4 immunodominant ORFs were mostly virion structural proteins and had late expression kinetics during virus replication. Each ORF immunodominant for antibody responses also frequently elicited CD4 responses, while antibody responses were not detected to many other CD4 antigens. This is the first proteome-wide screen of CD4 responses to a complex pathogen using full-length protein antigens. Immunodominant CD4 antigens identified using this technology may be rational subunit vaccine components for microbial pathogens requiring T-cell help for protection. First two authors contributed equally to the work.
Primary B-lymphocytes Preferentially Expand Allogeneic FoxP3+ CD4 T Cells (B19)
Authors: Chen Xinjian , Guo Xiaohua , Jensen Peter E
Published: January 2023

Antigen presentation by B-lymphocytes can result in immunity as well as tolerance. The potential impact of B cell-mediated tolerance and underlying mechanisms are incompletely understood. Using primary B cells as APC and allogeneic CD4 T cells as responder cells in mixed leukocyte reactions (MLR), we find that B cells, though weaker allo-stimulators than dendritic cells, preferentially expand Foxp3+ CD4 T (nTr) cells in the absence of any exogenous cytokines, such as IL-2 or TGF-beta. Partial blockade of class II MHC-TCR interaction further enhances the expansion of nTr cells partially through conversion of Foxp3- cells to Foxp3+ cells, but expansion is reduced by increasing the ratio of B cells to responder T cells or by provision of exogenous IL-2 or anti-CD28. Gamma irradiation completely abrogates the ability of B cells to expand isolated nTr cells, but exogenous IL-2 or anti-CD28 can partially restore this function. The B cell-expanded nTr cells express high levels of Foxp3 and are highly inhibitory in an antigen-specific manner. Taken together, our results suggest that

primary B cells are optimally equipped to preferentially expand nTr cells,this property is related to the modest allo-stimulatory function of B cells, anda low level of T cell stimulation favors nTr cell generation.
Single Intranasal Immunization with Recombinant Adenovirus-Based Vaccine Induces Protective Immunity against Respiratory Syncytial Virus Infection (B192)
Authors: Yu Jaerang , Kim Sol , Chang Jun
Published: January 2023

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses. Here, a recombinant replication-deficient adenovirus-based vaccine, rAd/3×G, expressing the soluble core domain of G glycoprotein spanning amino acids 130 to 230, was engineered by codon optimization and tandem repetition for higher level expression, and evaluated for its potential to be used as an RSV vaccine in murine model. A single intranasal but not intramuscular immunization with rAd/3xG provided potent protection against RSV challenge at an improved level over inoculation of a recombinant vaccinia virus expressing whole G protein (vvG). Strong mucosal secretory IgA responses were induced by single intranasal immunization but not by intramuscular administration or immunization of vvG, while comparable serum IgG responses were observed in each group. Interestingly, rAd/3xG immunization did not induce detectable CD4 T-cell response whereas vvG priming strongly elicited CD4 T-cell response against I-Edrestricted G epitope (amino acids 183 to 195). RSV-induced weight loss was significantly lower in rAd/3xG-immunized group than vvG-immunized group. Thus, our data demonstrate that a single intranasal administration of rAd/3xG elicits beneficial protective immunity and represents a novel vaccine regimen capable of providing mucosal protection against RSV infection.
Mining for Treasures: Systematic Profiling of 8-, 9-, 10-, and 11-mer HLA-B*0702-restricted CD8+ T cell epitopes from the Influenza A/Puerto Rico/8/34 (H1N1) Virus Hemagglutinin with Potential Application in Vaccine Development (B193)
Authors: Buchli Rico , VanGundy Rodney S. , Plompen Sonja , Rennels Aaron D. , Ede Nicholas J. , Hildebrand William H.
Published: January 2023

Identification of T cell epitopes is of crucial importance for the development of cancer and viral T cell eliciting vaccines. Here, we define CD8+ T cell epitopes using a novel high-throughput system for the synthesis and screening of peptide libraries that include all dominant epitope lengths (8–11mers) for MHC class I molecules. These representative peptide libraries are tested for their ability to bind various MHC class I molecules in a competitive manner. This comprehensive screening strategy begins with the synthesis of an overlapping truncated peptide library containing mixes of four peptides with a common C terminus, but having a length of 8, 9, 10, or 11 residues. Following the initial screen, positive hits are resolved into individual epitope responses. In order to identify HLA-B*0702-restricted epitopes, a peptide library was prepared for the influenza A virus Hemagglutinin (HA) protein. Screening results revealed 63 high/medium affinity epitopes at various sizes. A number of these putative influenza epitopes coincide with directly discovered influenza epitopes as well as influenza epitopes reportedly recognized by CTL. In conclusion, this study demonstrates that the rapid and methodical screening of T cell immune epitope libraries can help in detecting and monitoring infections as well as enabling immune intervention strategies with respect to treatment or prevention of the disease.
Control of viremia and preservation of intestinal CD4+ T cells in SHIVsf162P3 infected macaques intravenously challenged with pathogenic SIVmac251 (B197)
Authors: Pahar Bapi , Piatak Michael , Wang Xiaolei , Lifson Jeffrey , Montefiori David C , Ling Binhua , Lackner Andrew , Veazey Ronald
Published: January 2023

Attenuated vaccines in primates have provided better protection than most approaches to date. We hypothesized that SHIVsf162P3 infection might “immunize” macaques against intravenous challenge with a highly pathogenic virus. Twelve rhesus macaques of Chinese (Ch) and 2 of Indian (In) origin were intravaginally infected with SHIVsf162P3 and followed until their level of virus had subsided to undetectable or very low levels in plasma. SHIV vaccinated macaques were challenged intravenously with either 100, 10TCID50 SIVmac251 or plasma with high viral loads. Four “naïve” Ch macaques were similarly challenged with SIVmac251 as controls. Remarkably, Ch macaques “immunized” with SHIVsf162P3 were able to control viral replication of pathogenic SIVmac compared to naive (non-immunized) Ch controls. Viral loads after challenge in immunized Ch macaques ranged from undetectable to 104 in plasma compared to105 to 107 in naïve Ch controls. Indian macaques had higher levels of virus than Ch-macaques. but had better levels of protection compared to naïve challenged macaques. Control (naïve) macaques had rapid depletion of intestinal CD4+ T cells but levels remained fairly stable in immunized macaques. Control of viral load in “immunized” macaques did not correlate with the presence of neutralizing antibody and/or cell-mediated immune responses. Combined, these data suggest that effective control of SIV replication through immunization may be possible, but correlates of resistance have yet to be determined.
UltraSensitive Human Cytokine 10-Plex for the Luminex xMAP™ Platform (B200)
Authors: Bhakta Sheetal , Liu Nina , Fu Yubin , Brodey Mary Michael , Wang Jimin
Published: January 2023

Profiling low concentrations of Th1/Th2 and proinflammatory cytokines can yield valuable information about immune system status in clinical and subclinical disease states. To facilitate cytokine profile analysis, Invitrogen has recently developed an UltraSensitive Cytokine 10-Plex Assay for the Luminex xMAP™ Platform. Analytes include human GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and TNF-α. This assay system is validated for high content, high sensitivity serum, plasma, and tissue culture medium applications. Through careful choice of antibody pairs and use of proprietary buffer formulations, we are able to achieve an improvement in sensitivity of approximately 10-fold compared to our conventional cytokine assays for the Luminex xMAP™ platform. Sensitivities for all 10 analytes are 1 picogram per milliliter or less. Other performance characteristics, including assay precision, recovery, linearity of dilution, and calibration to NIBSC reference preparations, are similar to our conventional assays.
Co-expression of apoptosis-related molecules on activated CD8+CD38+ T-cells is associated with HIV-1 disease progression (B213)
Authors: Ríos-Olivares Eddy , Rodríguez José W. , Rodríguez Luis A. , Nair Madhavan , Hunter Robert
Published: January 2023

HIV-1 disease progression has been associated with a remarkable increase of CD8+CD38+ T-cells. It has been documented that a significant distribution of HIV-specific CD8+ T-cells resides in the CD8+CD38+ T-cell sub-population. The failure of HIV-specific CD8+CD38+ T-cells to control HIV-1 infection has been attributed to several mechanisms including apoptosis. However, the relationship between the CD38 expression and molecular events involved in CD8+ T-cell apoptosis is not well understood. We evaluated the expression of two membrane-associated apoptosis-related molecules: CXCR4 and CD95, and two cytoplasm-associated apoptosis-related molecules: Bcl-2 and active caspase-3 in 41 HIV-1 positive patients and 15 HIV-1 negative individuals. HIV-1 infection alters the level of expression of CD38, CD95, CXCR4, Bcl-2, and active caspase-3. A positive correlation was established between CD95, CXCR4, and active caspase-3 expression with low CD4 count and high plasma viremia and CD38 expression. The majority of activated CD8+CD38+ T-cells were apoptotic since they expressed active caspase-3 and the rest of these cells were susceptible to become apoptotic since they co-expressed CD95 and CXCR4. We suggest that one of the most likely HIV-mediated apoptosis mechanisms is via CD95 and CXCR4 induction through the caspase cascade despite Bcl-2 expression. This provides another explanation of why HIV-1 infection is not fully contained by HIV-specific CD8+CD38+ T-cells.

Supported by NIH/RCMI Grant # G12-RR03035.
The role of MHC-II in the maintenance of memory B cell function (B22)
Authors: Shimoda Michiko , Koni Pandelakis Andreas
Published: January 2023

By using conditional, B cell-restricted MHC-II-deficient mice (IA-B mice), we previously showed that antigen-specific memory B cells could be established but they then lost MHC-II expression due to ongoing cd19Cre-mediated deletion of the conditional iab allele, without significant loss in memory cell number. In this study, we examined whether or not MHC-II deficient memory B cells are functionally competent to differentiate into plasma cells. IA-B mice carrying a 4-hydroxy-3-nitro-phenyl-acetyl (NP) hapten-specific VH transgene (QM) were immunized with NP-coupled chicken gamma globulin plus alum. QM/IA-B mice generated NP+IgG+ memory B cells (~0.1% of total spleen cells), which subsequently lost MHC-II during maintenance. When cultured in the presence of 3T3 feeder cells with cytokines and various stimuli, both MHC-II(+) and MHC-II(−) NP+ memory B cells purified from QM/IA-B mice produced anti-NP IgG antibody upon LPS stimulation. However, anti-NP IgG production of MHC-II(−) NP+ memory B cells upon polyvalent B cell receptor stimulation with NP−Ficoll was significantly reduced compared to that of MHC-II(+) NP+ memory B cells. These results suggest that expression of MHC-II on memory B cells is required for maintenance of their capacity to differentiate into plasma cells in response to antigen stimulation.

This work was supported by NIAMS AR052470 (M.S.) and NIAID AI47379 (P.A.K.).
Immunoglobulin mutation traits in humans with AID deficiency (B220)
Authors: Longo Nancy S. , Satorius Colleen , Durandy Anne , Lipsky Peter E.
Published: January 2023

Germinal center (GC) B cells express high levels of activation induced cytidine deaminase (AID) which targets WRC (W=A/T, R=A/G) motifs generating a uracil:guanidine mismatch. During somatic hypermutation (SHM) 76% of the mutations are attributed to AID and error-prone repair utilizing polymerase eta (POL η). To characterize the remainder background mutations, 754 nonproductive V(D)J rearrangements from three AID−/− patients were analyzed. The mutation frequency was 20-fold less than normals (0.09% vs 2%) but 5x10e4-fold more than non-B cells. Reduced RGY (6% vs 27%) and WRC (6% vs 15%) motif mutations and decreased replacements in complementarity determining regions were attributed to the lack of AID. Reduced WA motif mutations (12% vs 23%) suggested POL η activity was decreased. An increased percentage of C>T and G>A transitions suggested that uracil DNA-glycosylase (UNG) activity was reduced even though UNG is upregulated in normal GC B cells and large GCs occur in AID−/− patients. The mutation pattern suggests that AID-independent C substitution contributes a small proportion of SHM but lacks the targeting provided by AID. Finally, low level of SHM clearly can develop in the absence of AID, however this mechanism is inefficient at altering the binding capacity of Ig heavy and light chain genes.

IRP, NIAMS, NIH
Apoptosis of Gr-1+ granulocytes in the lung promotes hypersensitivity pneumonitis by inducing dendritic cell maturation (B24)
Authors: hwang sujin , Chung Doohyun
Published: January 2023

Hypersensitivity pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to inhaled particulate antigens. HP is characterized with lymphocytic alveolitis, granuloma formation and fibrosis, and categorized as a Th1 disease. However, it remains unclear whether apoptosis in the lung contributes to the development of HP.

To investigate the functional role of the Fas-mediated apoptosis in HP, Fas(lpr) or FasL(gld)-deficient and B6 mice were challenged with Saccharopolyspora rectivirgula (SR) intranasally. lpr and gld mice attenuated HP in terms of histological alteration, CD4/CD8 ratio in bronchoalveolar lavage fluids (BALF), and SR-specific immune responses compared with B6 mice. The blockade of apoptosis in B6 mice using caspase inhibitor attenuated HP. In tunnel assay, the apoptosis was not found in respiratory epithelial cells whereas the apoptosis of immune cells, especially Gr-1+ granulocytes in BALF were prominent. Moreover, the depletion of Gr-1+ granulocytes attenuated SR-induced HP in B6 mice. B6 mice produced high levels of IFN-g, TNF-a and TGF-b whereas lpr or gld mice showed high levels of IL-4 production in the lung compared with B6 mice. The apoptotsis of Gr-1+granulocytes promoted pulmonary dendritic cell (DC) maturation and the production of MIP-1a and MCP-1 by DC, which contributed to recruiting inflammatory cells in the lung during SR-induced HP.

Taken together, these results suggest that the apoptosis of Gr-1+ granulocytes promotes SR-induced HP by inducing DC maturation and chemokine production. The blockade of apoptosis in the lung may be a useful therapeutic modality for HP.
Adjuvant Regulates Antigen-Specific Helper T Cell Evolution (B27)
Authors: Malherbe Laurent , McHeyzer-Williams Louise J , Panus Joanne F , McHeyzer-Williams Michael G
Published: January 2023

Antigen-specific helper T cells are the cognate regulators of multiple developmental checkpoints in adaptive immunity. Importantly, TCR-pMHCII interactions also control the evolution of antigen-specific Th cells in ways that remain poorly understood. Here, we demonstrate that typical depot producing adjuvants drive clonal dominance through the action of their adjuvant vehicle. In contrast, non-depot adjuvants re-set the selection threshold to propagate clones expressing high affinity TCR. Furthermore, oil in water formulations that readily disperse antigen-adjuvant aggregates in vivo also impact the magnitude of clonal expansion and substantially exaggerate the penetrance of high affinity clones. Finally, reducing antigen dose does not impact the affinity threshold for clonal selection arguing against a mechanism driven by inter-clonal competition in vivo. These findings reveal vaccine adjuvants as critical regulators of a TCR affinity-based clonal selection mechanism that underpins the evolution of adaptive immunity.
Signaling from the B cell antigen receptor (BCR) induces VH replacement in human immature B cells (B33)
Authors: Liu Jing , Fan Run , Yu Zhihong , Raikwar Himanshu P , Su Kaihong , Zhang Zhixin
Published: January 2023

VH replacement occurs through RAG-mediated secondary IgH recombination between a cryptic RSS (cRSS) from a previously rearranged VH gene and a 23-bp RSS from an upstream VH gene. Our previous studies have shown that VH replacement occurs in human bone marrow immature B cells and contributes to about 5% of the primary B cell repertoire. However, how VH replacement is regulated is not clear. The human EU12 cells, which undergo spontaneous VH replacement, are sensitive to BCR stimulation. BCR crosslinking strongly induces double stranded DNA breaks at the VH3 cRSS border in the μHC+ EU12 cells as measured by semi quantitative ligation-mediated PCR (LM-PCR) and real time LM-PCR, suggesting the induction of VH replacement. The VH3 to VH1 VH replacement excision circles are also induced upon BCR crosslinking. In addition, ongoing VH replacement was found in the newly emigrated immature B cells from peripheral blood of three healthy donors, which can be further induced by BCR crosslinking. These results provide the first evidence that VH replacement in human immature B cells can be regulated by BCR mediated signaling.
Transcriptional repression of the MHC-class I-related Fc receptor for IgG by interferon-gamma through activation of signal transducer and activator of transcription (Stat)-1 (B34)
Authors: Liu Xindong , Ye Lilin , Zhu Xiaoping
Published: January 2023

Expression of the MHC genes has been previously shown to be enhanced at the transcriptional or posttranscriptional level following exposure to cytokine IFN-gamma. However, we here reported that IFN-gamma down regulated the constitutive expression of the FcRn that functions to transport IgG across polarized epithelial cells and protect IgG from degradation. Intestinal epithelial cell, endothelial cell, and macrophage-like THP-1 or fresh isolated human monocytes exposure to IFN-gamma resulted in significantly-decreased human FcRn expression, as assessed by real-time PCR and western blot. Chromatin immunoprecipitation and gel mobility shift assays identified that Stat-1 bound to an IFN-g activation site (GAS) in FcRn promoter region. Functional analysis of luciferase reporter gene under the control of FcRn promoter showed that IFN-gamma had no effect on luciferase expression in JAK- and Stat-deficient cell lines. The unresponsive to IFN-gamma repression was rescued by over-expression of JAK or Stat1, revealing that Stat1-Jak signaling pathway was necessary and sufficient to mediate the repression of IFN-gamma. Furthermore, the inhibitory effects of IFN-gamma responsiveness were selectively reversed or blocked by site-directed mutagenesis of a cis-acting GAS element, and by over-expression of the Stat1 inhibitor, PIAS1, or of the dominant –negative Stat-1 Y701F containing a mutation at Tyr-701. IFN-gamma stimulation dampened down bidirectional transport of IgG across polarized Calu-3 lung epithelial monolayer. Taken together, our results indicate that down-regulation of FcRn gene expression by IFN-gamma may involve direct interaction of activated Stat-1 with the general transcriptional machinery that drives constitutive FcRn expression.
Defective development of follicular, marginal zone and B1 B cells in interferon regulatory factor 4 deficient mice (B4)
Authors: Pathak Simanta , Ma Shibin , Trinh Long , lu runqing
Published: January 2023

Interferon regulatory factor 4 (IRF4) is an immune system specific transcription factor. Previous studies have demonstrated that IRF4 and its closest relative IRF8 control light chain locus activation and are critical in the pre-B cell development. In this report, the role of IRF4 in the development of follicular, marginal and B1 B cells was further investigated. Our results show that the number of follicular B cells is significantly reduced while the marginal zone and B1 B cell population are dramatically increased in the IRF4 deficient mice. Transplantation study supports a B cell intrinsic role of IRF4 in regulating FO and MZ B cell development. Kinetic study reveals that the distorted FO and MZ B cell development in the absence of IRF4 is a result of accelerated MZ B cell production and reduced FO B cell generation. Molecular analysis further reveals that transitional B cells deficient in IRF4 express lower level of Bcl2. Ectopic expression of a Bcl2 transgene partially restore the normal development of follicular and marginal zone B cells in the IRF4 deficient mice. Thus, IRF4 maybe a novel transcriptional regulator of follicular, marginal zone and B1 B cell development and homeostasis.
Auranofin, immunosuppressive drug, inhibits the MHC class I and MHC class II pathway of antigen presentation in dendritic cells (B41)
Authors: Kim Kyungjae , Han Shinha , Kim Hyunyul , Kim Kwanghee , Kwon Jeunghak , Lee Chong-Kil
Published: January 2023

Auranofin (AF) is an orally administered, gold-based, anti-arthritic agent that has been used as a drug for the treatment of rheumatoid arthritis since the late 1970s. The precise mechanism of its anti-arthritic action is unclear. Therefore, this study examined the ability of AF to immunomodulate the antigen presenting cells (APCs), dendritic cells (DCs). The DCs were cultured in the presence of AF and examined for their ability to present exogenous antigens in association with the major histocompatibility complexes (MHC). This study examined whether or not AF (5 ~ 40 μM/ml) can inhibit the cross-presentation of DCs. DC2.4 cells (H-2Kb) or bone marrow-derived DCs (BM-DCs) generated from the BM cells of C57BL/6 mouse (H-2Kb) were cultured in the presence of AF with OVA-microspheres, and the amount of OVA peptide-class I MHC complexes was measured by T cell hybridoma, B3Z, that recognizes the OVA (257–264 : SIINFEKL)-(H-2Kb) complex and expresses β-galactosidase. The inhibitory activity of AF appeared to be due not only to its ability to inhibit the phagocytic activity of DCs but also to suppress the expression of MHC molecules on DCs. AF decreased the level of IL-2 production when the effects of AF on CD4+ T cells were examined. The secretion of TNF-α, IL-1β and IL-6 was measured in cell supernatants using ELISA when RAW264.7 was exposed to AF for 24 hrs. AF decreased production and pro-inflammatory cytokine, TNF-α, IL-1β, IL-6 and nitric oxide (NO) in macrophages. These results provide an understanding of the mechanism of immunosuppressive activity and anti-inflammatory effects of AF in relation to their actions on APCs.
Expression and regulation of IL-22 in the IL-17-producing CD4+ T lymphocytes (B46)
Authors: Chung Yeonseok , Yang Xuexian , Chang Seon Hee , Dong Chen
Published: January 2023

IL-22 is a novel cytokine in the IL-10 family that functions to promote innate immunity of tissues against infection. Although CD4+ helper T lymphocytes (TH) were found as a source of IL-22, the regulation of this cytokine has been poorly understood. Here, we show that IL-22 is expressed at both mRNA and protein levels by a novel subset of TH cells that also makes IL-17. IL-22 and IL-17 were found to be coordinately regulated by TGFbeta and IL-6 during TH differentiation by real-time PCR as well as ELISA analysis. However, IL-22 does not regulate TH differentiation; exogenous IL-22 or an IL-22 antagonist had no effect on TH differentiation. These data demonstrate a novel cytokine expressed by IL-17-producing T cells, and suggest interaction and synergy of IL-22 and IL-17 signaling pathways in tissue inflammation and autoimmune diseases.

This work was in part supported by National Institutes of Health (to C.D.). S.H.C receives a post-doctoral fellowship from the Arthritis foundation and C. D. is a Cancer Research Institute Investigator and a MD Anderson Cancer Center Trust Fellow.
IL-10 and natural regulatory T cells: two independent anti inflammatory mechanisms in HSV induced ocular immonopathology (B63)
Authors: Sarangi Pranita Pragnyadipta , Suvas Susmit , Sehrawat Sharvan , Kim Bumseok , Rouse Barry T
Published: January 2023

Our past studies have shown a protective role for both CD4+CD25+Foxp3+ natural regulatory T cells (nTregs) as well as IL-10 in Herpes Simplex Virus (HSV) induced Stromal Keratitis (SK). Among several other mechanisms, IL-10 has been shown to be involved in the nTreg mediated suppression. We have shown in vitro that blocking of IL-10 could partially block the Treg mediated suppression of activated effectors isolated from SK lesions. But it is not known whether in the in vivo conditions, IL-10 and nTreg influence the SK pathology independently or in concert. Interestingly, IL-10−/− animals depleted of nTregs prior to ocular HSV infection had more severe SK lesion as compared to undepleted IL-10−/− animals. These results show the presence of two independent anti inflammatory mechanisms in SK. Along this line, by using mice expressing GFP-Foxp3 we could not show CD4+GFP-Foxp3+IL-10+ cells in the DLNs and spleens of infected wild-type (WT) mice although animals depleted of nTregs had higher levels of IL-10 in the DLNs post infection. Furthermore, by using WT, IL-10−/− and nTreg depleted WT and IL-10−/− animals we have compared the relative importance of IL-10 and nTregs in reducing SK pathology. Although disease severity was significantly higher in IL-10−/− and both the nTreg depleted groups as compared to WT, highest severity was found in the IL-10−/− mice depleted of nTregs. Intracellular cytokine staining with UV-HSV and polyclonal stimulation of the cells isolated from infected corneas, DLN and spleens revealed that IL-10 has more profound anti inflammatory effects in SK as compared to nTregs and that the later could act in an IL-10 independent manner.
TGF-β Induces Microvascular Endothelial Cell Migration and PP-2A Inactivation (B65)
Authors: Walsh Jarrett Elbert , Young M. Rita I.
Published: January 2023

An increasing number of anti-tumor therapies have focused on angiogenesis pathways. Targeting various aspects of angiogenesis has been shown to disrupt tumor growth and reduce tumor progression and recurrence. Our studies show that treatment of microvascular endothelial cells with TGF-β leads to the reduction in activity of the protein phosphatase PP-2A, which results in increased motility. The mechanism by which TGF-β signaling results in PP-2A inactivation remains unknown. Our studies will show that neither change in mRNA nor protein levels of PP-2A subunits is associated with this decreased activity. However, TGF-β induced changes in ceramide levels may play a role in reduced activity. Furthermore, the role of TGF-β signaling on the protein inhibitors of PP-2A, I1PP2A and I2PP2A, will be demonstrated. The goal of these studies is to identify mechanisms by which TGF-β signaling induces PP-2A inactivation and, in turn, increased cellular motility in the tumor microenvironment. Elucidation of these pathways will provide novel cellular targets to interfere with tumor angiogenesis and metastasis.
TGF-β-induced TCR-Tg/NOD regulatory T cells suppress both autoimmune diabetes transfer and islet graft destruction in an antigen dependent manner (B75)
Authors: Tonkin Daniel , Bradley Brenda , Barbour Gene , Haskins Kathryn
Published: January 2023

Regulatory T cells (Tregs) are understood to fall into two categories: natural Tregs and induced Tregs. Induced Tregs can be produced by activating CD4 T cells in the presence of TGF-β. Such induced Tregs are of great promise for immunotherapy, but it has been unclear if they require stimulation by antigen in order to suppress in vivo. We have investigated the limits of induced Tregs in the NOD mouse model of type I diabetes. We produced TGF-β-induced Tregs from the 6.9 TCR-Tg/NOD mouse that are specific for islet autoantigen. In some experiments we used a congenic mouse, the NOD.C6, that specifically lacks the antigen for the 6.9 TCR-Tg T cells but not for other islet-specific T cells. These Tregs express Foxp3, and produce much less IFN-γ than non-Tregs. We have shown that induced Tregs can prevent transfer of diabetes by effector T cells in NOD mice, but fail to protect in NOD.C6 mice that lack the Treg antigen. These Tregs decrease inflammatory cytokine production and macrophage infiltration in the pancreas. In human patients with diabetes, induced Tregs might be used to protect islet grafts from immune destruction, and we have demonstrated that our induced Tregs can prevent islet graft destruction in NOD mice. In addition, we have grafted islets from the NOD.C6 mouse. By using these antigen-free islets, we have conclusively demonstrated that induced Tregs require activation by their antigen in the islet graft in order to protect.
TCRbeta formation: Vbeta accessibility is not the limiting or regulated step (B8)
Authors: Carpenter Andrea C , Bassing Craig H.
Published: January 2023

The T cell receptor β (TCRβ) locus contains three gene segment families, variable (V), diversity (D) and joining (J), each of which contributes one segment to form a TCRβ exon. In DN2 stage thymocytes, a Dβ segment joins a Jβ segment on both alleles, while in the DN3 stage Vβ segments join with previously formed DJβ complexes. Formation of a productive (protein encoding) VDJβ complex on one allele is theorized to prevent Vβ segments from joining with DJβ complexes on the other allele, leading to TCRβ allelic exclusion. To elucidate mechanisms that regulate Vβ joining and TCRβ allelic exclusion, we generated and analyzed mice with a recombination reporter cassette consisting of the Dβ1 and Jβ1.1 gene segments inserted into the endogenous Vβ14 segment. We found Vβ14DJrecombination initiated concurrent with Vβ14 rearrangement, suggesting local chromatin environment determines the developmental timing of Vβ14 rearrangement. Our analysis of Vβ14DJ/WT and Vβ14DJ/DJ αβ T cell hybridomas revealed that Vβ14DJrecombination occurs at a substantially higher frequency than Vβ14 joining. Moreover, our initial findings indicate that Vβ14DJrecombination is not inhibited by expression of a productive VDJβ complex. Collectively, these data suggest that modulation of Vβ14 recombinational accessibility is neither the rate-limiting or regulated step in joining of Vβ14 segments with DJβ complexes.

Training Grant: 5-T32-Al055428
The type II anti-diabetic activity of an Aloe formulation in DIO mice (B80)
Authors: Kim Kyungjae , Kim Kwanghee , Kim Hyunyul , Kwon Jeunghak , Han Shinha , Lee Chong-Kil
Published: January 2023

Aloe is known to have various medical effects. These include healing damaged skin, as well as anti-diabetic, anti-cancer, and anti-arthritis activity. This study evaluated the anti-hyperglycemic effect of an Aloe formulation in DIO (Diet induced obesity) mice. The aloe formulation (AF) reduced body weight gain, fasting blood glucose, plasma triglyceride, the size of fat cells, and the amount of neutral fat deposits in the liver and improved insulin action. The AF treated group showed a reduction in the size of epididymal fat cells compared with the untreated group, and a lower amount of neutral fat deposits in the liver. AF exerted beneficial effects on lipogenesis related genes, such as SREBP1a, FAS, GPAT, and SCD-1 expressions in liver. These results suggest that AF is useful as an anti-diabetic treatment for Type II diabetes patients.
Spontaneous pulmonary hypertension in mice associated with OX40 ligand overexpression (B82)
Authors: Rabieyousefi Moloud , Zhang Ming-Cai , Soroosh Pejman , Ishii Naoto , Sugamura Kazuo , Ono Masao
Published: January 2023

[Background] Ligation of OX40 receptor on T cells provides potent co-stimulatory signals associated with memory T cell generation, Ag-specific T cell migration, and enhanced inflammatory responses. OX40L is expressed on antigen presenting cells and vascular endothelial cells. Previous studies have shown that OX40L transgenic mice (OX40L-Tg) spontaneously developed ulcerative colitis-like disease and undetermined lung disease, which were evident in C57BL/6 (B6) but not BALB/c (BALB) genetic background.

[Objective] The aim of this study is histopathological and genetic characterizations of the lung disease in OX40L-Tg.

[Methods] We generated 351 OX40L-Tg from (BALB x B6) x B6 OX40L-Tg backcrosses. Severity of pulmonary arterial lesion was evaluated with microscopic examinations (0-1-2 grades). Genome-wide scan and other genetic analyses were carried out based on microsatellite polymorphisms.

[Results] Pulmonary arterial lesion characterized by intimal and medial thickening that was observed in B6 but not BALB OX40L-Tg. Pulmonary hypertension (PH) in OX40L-Tg was shown by the elevation of right ventricular systolic pressure. We identified a novel susceptibility locus to the PH with suggestive linkage (P=0.0012, LOD=1.8) on chromosome 5.

[Conclusion] Augmented OX40-OX40L interaction predisposes mice to spontaneous onset of PH under the influence of particular genetic background.
The regulation of parathyroid hormone expression in thymus by Gcm2-dependent and Gcm2-independent pathways (B83)
Authors: Liu Zhijie , Chen Lizhen , Manley Nancy
Published: January 2023

The promiscuous gene expression of tissue-restricted self-antigens (TRA) in medullary thymic epithelial cells (mTEC) is required for the negative selection to establish the central tolerance. The mechanism of the promiscuous gene expression is still not clear. Two models have been proposed: Mosaic model proposed that many different tissue cells within the medulla express corresponding TRAs following the rules of tissue-specific regulation. Permissive model proposed that some mTECs can express TRAs differently compared to the tissue-specific regulation rules.

Parathyroid hormone (Pth) is one of TRAs expressed in thymus. We explored the mechanism of the regulation of thymic Pth expression in mouse. We found the major thymic Pth expression was from some clusters of misplaced parathyroid cells. In Gcm2 null mutants, thymic Pth expression was greatly reduced due to the ablation of these misplaced parathyroid cells, suggesting a Gcm2-dependent pathway functioning in these cells. Using genetic marker technique, we sorted Foxn1-expressing TECs, and demonstrated that TECs were another source of thymic Pth expression. Thymic Pth expression level was similar in the purified TECs from Gcm2−/− and wildtype, suggesting this expression was regulated by a Gcm2-independent pathway. Our results support the permissive model. It will be interesting to know if this mechanism is also used by other TRAs.
Type I interferon regulation of Th cell responses in SLE (B84)
Authors: Mangini Alyson J. , Meyers John A. , Nagai Taro , vanSeventer Gijs , vanSeventer Jean Maguire
Published: January 2023

Type I interferons (IFNs) can have contrasting effects on adaptive Th1 responses. IFN-α/β are well recognized anti-viral agents that promote anti-viral Th1 immune responses. In contrast, IFN-β therapy provides a beneficial effect in multiple sclerosis patients, in part, by suppressing Th1 type autoimmune responses. In systemic lupus erythematosus (SLE), autoimmune complex-induced, plasmacytoid dendritic cell (pDC)-derived IFN-α is thought to be a major disease effector. To investigate how type I IFNs regulate Th cell autoimmune inflammatory responses in SLE, we established a model of human peripheral blood mononuclear cell (PBMC) stimulation. In this model, the effects of continuous type I IFN exposure are mimicked by pretreating PBMC with either recombinant type I IFN or the pDC-TLR9 agonist, CpG-A, and stimulation of autoreactive Th cells is mimicked by stimulation with the superantigen, SEA. Type I IFN neutralization with B18R, a vaccinia virus-derived soluble type I IFN receptor, is used to specifically identify effects of CpG-A-induced type I IFN. Our results indicate that pretreatment with rIFN-α, rIFN-β, or CpG-A-induced type I IFN inhibits SEA-stimulated IFN-γ secretion by healthy donor PBMC. Preliminary experiments demonstrate that, when compared to serum from control individuals, some SLE patient sera similarly inhibit IFN-γ secretion by normal PBMC. In addition, co-pretreatment of PBMC with B18R and serum from several SLE patients reveals a type I IFN-mediated inhibitory effect on IFN-γ secretion. Our findings suggest that in SLE circulating type I IFNs may suppress Th cell-mediated tissue injury by inhibiting inflammatory cytokine-secreting autoreactive Th cells
Genetically controlled preferential loss of myelin-associated glycoprotein (MAG) coincides with severity and relapsing course of experimental autoimmune encephalomyelitis in H-2b/s mice (B85)
Authors: Skndric Dusanka , Zhou Weili , Dai Rujuan
Published: January 2023

Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) are demyelinating diseases of central nervous system (CNS). Four types of demyelinating lesions and associated distinct immunopathogenetic mechanisms reflect heterogeneity of clinical MS. It is unknown if demyelinating patterns in EAE recapitulate those found in MS. We investigated patterns of demyelination in genetically related H-2band H-2b/s mice following immunization with H-2b restricted immunodominant epitope of myelin oligodendrocyte protein (MOG35–55). We measured loss of myelin proteins specifically, MOG, myelin associated glycoprotein (MAG), myelin basic protein (MBP), and levels of neurofilament (NF160), and DNA-binding apoptosis regulatory protein (PARPp80) in demyelinating lesions of EAE mice. While loss of MOG was apparent in both strains during acute disease, it was significantly higher in H-2b/s compared to H-2b mice. Conversely, profound depletion of MAG, accompanied with decreased NF160, and elevated PARPp80 levels were exclusively found in severely relapsing H-2b/s mice. Levels of MBP remained relatively unchanged in both strains. Our data show that preferential loss of MAG, which is suggestive of distal oligodendrogliopathy, associates with reduction of axonal neurofilament, signs of apoptosis, and severe relapsing disease in H-2b/s mice. We propose that pattern of demyelination in H-2b/s mice resembles the type III MS lesion.
Human cardiac myosin (HCM) activates Toll-like receptors 2, 7 and 8 (B87)
Authors: Zhang Ping , Alvarez Kathy M. , Heuser Janet S. , Cunningham Madeleine W.
Published: January 2023

Toll-like receptor (TLR) activation has been implicated as a link between innate immunity and adaptive immunity in autoimmune diseases including myocarditis. Human cardiac myosin (HCM) has long been known as an autoantigen in myocarditis. The role of HCM on innate immunity has not been studied. To test the hypothesis that HCM activates TLRs, HEK 293 cells transfected with human (h) TLR2, 3, 4, 5, 7, 8, and 9 were treated with HCM or a cryptic HCM peptide S2-16 for 72h. IL-8 levels in supernatants were measured by ELISA. HCM or S2-16 treatment resulted in 2–4 times more IL-8 from hTLR2, 7 and 8 transfected cell lines compared with media. HEK control cells treated with HCM or S2-16 showed same level of IL-8 as media. In addition, skeletal muscle myosin treated hTLR transfected cell lines showed no difference in IL-8 levels from media. To confirm this observation, human monocytes were isolated and treated with HCM and S2-16. Supernatant was harvested 24 h later and IL-8 was measured by ELISA. Both HCM and S2-16 treatment resulted in a dose-dependent increase of IL-8. Similar increase in IL-8 in human monocytic THP-1 cell line was also observed. The finding that HCM activates innate immunity through TLR 2, 7, and 8 is novel, and links innate and adaptive immunity in autoimmune myocarditis. Exposure of HCM during viral infection or after apoptosis or necrosis in the heart may lead to chronic stimulation of TLRs and autoimmune heart disease.
A role for mast cells in EAE: W/Wv, Wsh, and CD34−/− mice are fully susceptible to disease induction using standard protocols (B92)
Authors: Bennett Jami L , Blanchet Marie Renee , Kubes Paul , McNagny Kelly
Published: January 2023

EAE is a murine model of multiple sclerosis involving destruction of myelin by the immune system. Mast cells, known for a role in asthma and allergy, are reportedly required for EAE induction in mice. Our lab has reported that mature mast cells express the surface marker CD34. To further understand the role of mast cells in EAE, we utilized CD34−/− mice which have functional mast cells in the tissues, but are defective in their mobilization. We asked whether mast cells seeded in development were sufficient for EAE induction. Previous studies of mast cells in EAE used W/Wv mice, where reconstitution with WT-mast cells restored susceptibility to EAE. It was possible that CD34−/− mice were protected from EAE due to the decreased peripheral mobility of mast cells, or, seeding of CNS- or vascular-localized mast cells during development could be sufficient for their function. Here we compare clinical disease in WT, CD34−/−, W/Wv, and Wsh mice. We find CD34−/− mice are susceptible to induction of EAE, consistent with suggestions that mast cells act remotely in this model. Surprisingly, however, we also find both W/Wv and Wsh mice susceptible to disease in our hands. This result was confirmed by other laboratories. Interestingly, analysis of infiltrating leukocytes in the CD34−/− CNS shows increased mast cells vs. WT. We are now investigating whether inflammatory factors have bypassed the requirement for cKit on mast cells in EAE.
‘Type B’ T cells escape T cell tolerance in HLA-DRB5 transgenic mice (B93)
Authors: Forsthuber Thomas G. , Murphey Cathi , Kawamura Kazuyuki
Published: January 2023

Multiple sclerosis (MS) is associated with the MHC class II genes HLA-DR2 and HLA-DRB5. The role of these MHC II molecules in the pathogenesis of MS has remained unresolved, but it is conceivable that these molecules fail to induce T cell tolerance. We have investigated T cell tolerance to MBP in HLA-DR2, -DRB5, and –DR4 transgenic mice. Our results showed profound T cell tolerance to MBP in all three tg mouse lines. However, ‘type B’ T cells recognizing MBP85-99 in an altered conformation escaped tolerance in HLA-DRB5 tg mice. These T cells responded only to the MBP85-99 peptide, but not to the native MBP protein. Importantly, these T cells induced severe EAE upon adoptive transfer into naïve recipients. Thus, the data suggest that ‘type B’ T cells could escape T cell tolerance in MS patients with the HLA-DR2 haplotype and contribute to the disease.
Expression of Leukotrienes is Increased in Human MS and in a Mouse Adoptive Transfer Model of MS (B98)
Authors: Mendoza Lisa M. , King Beverly , Rosser Mary , Almond Cosima , Hidalgo ToniRose , Schaefer Caralee , Mahmoudi Mithra , Harvey Susan , Kirkland Thomas , Guilford William , Vogel David , Lassmann Hans , Reder Anthony , Parkinson John , Halks-Miller Meridith
Published: January 2023

Leukotrienes are well established as mediators of inflammation, however little is known regarding their role in the pathogenesis of multiple sclerosis (MS). In humans, elevated LTA4-h levels were reported in acute inflammatory lesions of a single primary progressive (PP) patient, while increased mRNA and protein levels of 5-lipoxygenase were seen in both PP-MS and relapsing remitting (RR) patients. LTB4 levels were higher in the cerebrospinal fluid of patients with active MS compared to controls, while levels of cysteinyl leukotrienes LTC4, LTD4, and LTE4 were not different. Beyond these preliminary disease association data, little is known about role of leukotrienes in MS. BLT1, the high affinity receptor for LTB4, has recently been reported to be expressed on both human and mouse antigen-experienced T lymphocytes. We show here that encephalitogenic T cells specific for a myelin antigen express functional BLT1. When transferred into naïve recipients these T cells are sufficient to induce disease. In addition, peripheral blood T cells from RR-MS patients express altered levels of the BLT1 receptor as compared to normal healthy controls. In the CNS, we show that demyelinating plaques in human MS brain tissues have up-regulated levels of LTA4-h and BLT1 by immunohistochemistry suggesting involvement of this pathway in the pathogenesis of MS and highlighting potential new targets for therapeutic intervention.