bluuebunny/crossref_metadata_2025 huggingface dataset:
The key to the blood brain door: Differential signaling of the adenosine receptor subtypes regulates lymphocyte entry into the central nervous system at the choroid plexus (95.12)
Authors: Mills Jeffrey H , Mueller Cynthia , Bynoe Margaret S
Published: January 2023

Lymphocyte entry into the central nervous system (CNS) is a highly regulated process which serves to promote tissue repair (when required) while protecting against potentially fatal inflammation. As the formation of extracellular adenosine (catalyzed from ATP by CD39 and CD73) during inflammation serves as a negative-feedback signal to prevent excessive cell damage, our results suggest that adenosine receptor (AR) signaling also regulates CNS lymphocyte infiltration. We have recently shown that CD73 and AR signaling are required for CNS lymphocyte entry during experimental autoimmune encephalomyelitis (EAE), the animal model of autoimmune inflammatory disease Multiple Sclerosis. We now show that CD39, CD73, and the A1- and A2A-AR subtypes are found clustered in the CNS only at the choroid plexus, a structure which regulates lymphocyte migration from the blood to the cerebrospinal fluid. Moreover, while A1- and A2A-AR activation have functionally opposing cellular effects (cAMP studies), mice given A2AAR antagonists are protected against EAE and CNS lymphocyte infiltration while A1AR-/- mice develop severe EAE. Additionally, AR signaling can induce lymphocyte transmigration across an in vitro choroid plexus transwell barrier while A2AAR activation promotes the upregulation of ICAM-1. Therefore, we conclude that AR signaling at the choroid plexus mediates CNS lymphocyte entry. AI 57854 and NS 063011
T cell migration to synovial joints is not necessary for the development of proteoglycan-induced arthritis (95.5)
Authors: Egelston Colt , Angyal Adrienn , Glant Tibor T , Mikecz Katalin
Published: January 2023

Rheumatoid arthritis (RA) is largely mediated by autoantibodies, the production of which is supported by autoreactive T cells. Previous reports in RA and its animal models also suggest that T cells present in the joints can locally propagate arthritis. To identify T cells migrating to the joints before and during arthritis development, we transferred fluorescence-labeled T cells and unlabeled antigen-presenting cells from BALB/c mice with proteoglycan-induced arthritis (PGIA) to syngeneic immunodeficient mice, and then monitored the recruitment of donor T cells in the ankle joints of the recipients using in vivo two-photon microscopy, immunohistochemistry, and flow cytometry. Despite the development of arthritis in the recipient mice, we found very few donor T cells in their joints after cell transfer. Furthermore, treatment of recipient mice with FTY720, an inhibitor of lymphocyte egress from lymphoid organs, which effectively depleted T cells in both peripheral blood and joints, failed to inhibit PGIA development. In contrast, arthritis did not occur in recipient mice after injection with donor cells depleted in T cells in vitro prior to transfer. Our results suggest that while lymphoid organ-homing T cells are required for the adoptive transfer of PGIA, the few T cells that migrate to the joints do not play a significant role in arthritis development.

Work supported by the NIH and the Grainger Foundation.
T cells regulate interstitial lung disease via expression of integrins (95.9)
Authors: Luzina Irina G. , Todd Nevins W. , Atamas Sergei P.
Published: January 2023

Interstitial lung disease, a combination of pulmonary inflammation and fibrosis, is deadly and occurs idiopathically or as a complication of autoimmune rheumatic diseases (scleroderma, rheumatoid arthritis, poly- and dermatomyositis, lupus). T cells often accumulate in the lungs of such patients. Previous reports suggested that αV-containing integrins may activate TGF-β and facilitate fibrosis. Whether pulmonary T cells utilize such integrins in interstitial lung disease is not clear. Expression of integrins αVβ3 (ITGAVB3) or αVβ5 (ITGAVB5) on pulmonary T cells was observed by double-stain immunofluorescence confocal microscopy in six out of six patients with scleroderma lung disease, as well as in the animal models of pulmonary CCL18 overexpression that manifests in selective T lymphocytic infiltration of the lungs and T cell-dependent pulmonary fibrosis. Flow cytometry of bronchoalveolar lavage T cells confirmed expression of integrins on more than 10% (up to 50%) of pulmonary T cells in 14 of 25 scleroderma patients and in all CCL18-overexpressing mice; Q-PCR experiments confirmed elevated integrin mRNAs in purified BAL T cells. There was no expression of ITGAVB3 or ITGAVB5 on T cells from the lungs of healthy volunteers or control mice. Injections of anti-ITGB3 blocking antibody or genetic deficiency of ITGB3 protected mice from CC18-induced inflammation and fibrosis. Jurkat cells that were forced to overexpress ITGAVB3 or ITGAVB5 induced Smad2 phosphorylation and nuclear translocation, and increases in collagen and α-smooth muscle actin expression in co-cultures with primary lung fibroblasts. Blocking anti-TGF-β antibodies attenuated such regulation in cell co-cultures. Thus, expression of integrins on pulmonary T cells contributes to interstitial lung disease by promoting T cell infiltration, TGF-β activation, and fibrosis.
Th1 fidelity vs. Th1/Th17 plasticity are dictated in vivo by pathogen lifestyle, exogenous cytokines and prior expression of IFN-gamma (96.1)
Authors: Curtis Meredith M , Way Sing Sing , Rowell Emily , Urdahl Kevin , Wilson Christopher B
Published: January 2023

It is unknown whether faithful and heritable commitment of CD4 T cells to an effector lineage occurs in vivo or whether plasticity of T cells to adopt different effector functions in vivo is the norm. To address this question, we examined commitment of naïve antigen-specific CD4 T cells generated during intracellular bacterial (Listeria monocytogenes, Lm) or viral (LCMV) infections. The robust TH1-polarizing environment induced by these infections yielded memory CD4 T cells stably committed to the TH1 lineage. The intracellular lifestyle of the microbe and the resultant cytokine milieu induced in response to this lifestyle were key determinants of TH1 fidelity. Infection of wild-type mice with mutant Lm that could not escape the phagocytic vacuole and replicate in the cytosol induced CD4 T cells with a mixed TH1/ TH17 response and decreased effector lineage fidelity, as did infection with wild-type Lm of mice deficient for both IL-12 and type I IFN signaling. Thus, memory CD4 T cells generated in strong TH1-polarizing environments in vivo are strongly committed to this effector lineage. Commitment is highly dependent on innate immune cytokines induced in response to entry into and replication of Lm within the cytosol, and, as we also show, on prior expression of Ifng by these memory T cells. Supported by grants from the NIH and March of Dimes.
Differential modulation of regulatory T cell function by pro-inflammatory cytokines (96.13)
Authors: Raimondi Giorgio , Thomson Angus W
Published: January 2023

Little is known regarding the modulation of regulatory T cell (Treg) activity. Maturing dendritic cells (DC) secrete a cocktail of cytokines (R-cocktail) that liberates T cells from Treg suppression. IL-6 is one component, but others remain unknown. We performed microarray analysis of maturing DC and identified IL-12p40, TNFα, and Ebi3 as candidates. We developed a DC-free in-vitro suppression assay to test their function. As expected, addition of supernatant from maturing DC allowed T cells to resist suppression. Use of IL-6 increased T cell resistance significantly. Its combination with IL-12 or TNFα increased T cell resistance further. IL-12 and TNFα were interchangeable, suggesting redundant activity. The R-cocktail exerted its effect on T cells since a short pre-exposure before co-culture released them from suppression. On the other hand, Treg pre-exposure increased their suppressive capacity, indicating that the effect exerted on T cells was dominant. Unexpectedly, when we tested Ebi3, in the form of IL-27, we observed sustained Treg suppressive activity, an effect that was exerted specifically on Treg, as indicated by the pre-exposure experiment. This observation helps clarify the reported anti-inflammatory capacity of IL-27. Thus, pro-inflammatory cytokines exert distinct effects on T cells and Treg, promoting counter-balancing activities used by the immune system to finely tune its reactivity.
Tec family non-receptor tyrosine kinase ITK is important for the development of skin-specific intraepithelial T cells through regulation of their skin-homing property (96.17)
Authors: Xia Mingcan , Qi Qian , August Avery , Xiong Na
Published: January 2023

Proper localization of immune cells during the development is critical for their optimal functions. Murine skin-specific intraepithelial T cells (sIELs) are a unique tissue-specific innate T cell population that function in the first line of defense to maintain the local tissue integrity. The sIELs originate from their fetal thymic progenitors that specifically express canonical Vγ3+ TCR. Although it has been shown that the expression of skin-homing molecules, such as CCR10, is associated with the TCR-mediated cellular selection of the progenitors for their development into sIELs, molecules that mediate the selection-associated homing molecule expression is not fully understood. Here we report that ITK-null mice have a defective sIEL development due to impaired expression of the homing molecules in the fetal thymic sIEL progenitors. In the absence of ITK, the fetal thymic Vγ3+ cells failed to upregulate the expression of the chemokine receptors important for their thymus-exiting and skin-homing, including S1P1 and CCR10, which was associated with a gradient accumulation of the Vγ3 cells in the fetal thymus and a severe defect in their seeding of the skin. On the other hand, the ITK deficiency did not affect the TCR- or IL-15 receptor-mediated proliferation of the progenitor cells. These results indicate that ITK-mediated signaling is critical for the acquirement of proper homing property by the sIEL progenitors for their peripheral localization.
CCR6 regulates the migration and effector function of Th17 cells in the gut (96.2)
Authors: Wang Chuanwu , Kang Seung G , Lee Jee H , Kim Chang H
Published: January 2023

T helper cells that produce IL-17 (Th17 cells) play important roles in regulation of immunity to bacteria and fungi and can cause organ-specific inflammation. The intestine is a tissue where Th17 cells are highly enriched, but migration and trafficking receptors of Th17 cells to the intestine have been unclear. It has been established that CCR6 is the signature chemokine receptor for Th17 cells but the role of this trafficking receptor in Th17 cell trafficking to the intestine has not been determined. We report here that Th17 cells preferentially migrate to the intestine and associated-lymphoid tissues, and CCR6 is the homing receptor important for Th17 cell migration to certain tissue microenvironments of the intestine where its ligand CCL20 is highly expressed. We determined the function of CCR6 of Th17 cells by comparing the migration and effector function of Th17 cells derived from wild type and CCR6-deficient mice. CCR6-deficient Th17 cells were defective in migration to Peyer's patches and small intestine but their migration to the large intestine was normal. Surprisingly, administration of CCR6-deficient Th17 cells into severe combined immunodeficiency (SCID) mice led to excessive intestinal inflammation. Following the administration of Th17 cells, we found that Th17 cells fail to propagate in the gut in the absence of CCR6 and transdifferentiate into inflammatory Th1 cells. In addition, CCR6 deficiency led to aberrantly wide-spread effector T cells in the inflamed intestine of the SCID mice. Finally, we found that the expression of CCR6 by Th17 cells is induced by TGF-β1 but suppressed by IL-2 in vitro and in vivo. Taken together, we conclude that CCR6 is a key trafficking receptor that regulates the migration and effector function of Th17 cells in the intestine.
CC chemokine receptor 4 is required for optimal Th1 memory but not IL-17+ gamma/delta T cell effector responses during Mycobacterium bovis infection (96.5)
Authors: Chensue Stephen W. , Stolberg Valerie R. , Chiu Bo-Chin , Schmidt Brian M. , Kunkel Steven L. , Sandor Matyas
Published: January 2023

The CC chemokine receptor 4 (CCR4) is a widely expressed receptor that in regard to T cell biology was purported to be a Th2 restricted receptor, however recent evidence suggests that this receptor is expressed by memory/effector T cells of different subsets. In an ongoing investigation, we tested the participation of CCR4 in the response to mycobacterial pulmonary infection using C57BL/6 CCR4 knockout (CCR4-/-) mice and monitoring effector cell populations by flow cytometric detection. Initial studies indicated CCR4 agonist expression occurred in lungs following I.T. infection with 105 M. bovis BCG. Compared to CCR4+/+ controls, CCR4-/- mice displayed delayed bacterial clearance and impaired accumulation of interferon-γ (IFNγ) producing CD4+ T (Th1) cells in lungs. In contrast, IL-17+ cells which were largely γ/δ T cells were unaffected in CCR4-/- mice. The CCR4-/- appeared to generate Th1 cells normally in draining lymphoid tissue but these did not accumulate optimally in lungs. Moreover, CCR4-/- also displayed abrogated granuloma recall responses when lungs were challenged with mycobacterial Ag coated beads. These findings further support the notion that CCR4 is important for optimal Th memory responses, including Th1-mediated responses. Supported by NIH-NIAID grant AI43460.
Positive cooperativity between BCR and IL4 receptor signaling pathways. (97.13)
Authors: Aflakiyan Nooshin , Rao Kanury Venkata Subba
Published: January 2023

The BCR-triggered responses of mature and transitional immature B cells differ at both the biochemical and functional level. It was previously shown that BCR cross-linking in vitro results in death of early transitional (T1) immature B cells whereas mature B cells survive and start proliferation. In this study, we show that in Ch1 cell line which are a model system for immature mouse B cells, Surface IgM (sIgM) cross-linking leads to cell cycle arrest. we found that this immature B cells do enter, but fail to completely progress through G1 phase of the cell cycle following BCR engagement. Interestingly, IL-4, which by itself does not stimulate proliferation of immature B cells, could rescue these cells from G0 /G1 arrest. Consistent with this idea, IL-4 can reverse effect of anti-Ig Abs to promote cell cycle progression of immature B cells. To investigate the different signaling pathways involved in these processes, CH1 cells were stimulated with anti-IgM alone, with IL4 alone, or with a combination of both IL4 and anti-IgM. Following this western blot strategy, protein/DNA transcription factor arrays and microarray data were employed to monitor the evolution of the signaling cascades. we have analysed the role of the MAP kinase families (MAPK) cascades, which are required for growth-factor-stimulated replication and for differentiation of B cells. All the intermediate signal transducers and the distal transcription modulators were studied to understand how intracellular signals from these two distinct receptors integrate to make the conesquence cell-fate decision.
NOD1 Engagement Leads to Increased IL12/IL23 p40 and decreased IL-10 Expressions in Sarcoidosis as Compared to Healthy Subjects (98.17)
Authors: Samavati Lobelia , Du Wenjin , Rastogi Ruchi , Nuñez Gabriel
Published: January 2023

Sarcoidosis is a systemic inflammatory disorder of unknown etiology affecting barrier organs like lung and skin. The presence of granuloma and its associated inflammation may lead to organ dysfunction.

Two NLR family members, NOD1 and NOD2, trigger immune defenses in response to bacterial peptidoglycans (PGNs). Interaction between these sensors and microbial moieties leads to up-regulation of cytokines involved in innate and adaptive immunity. We hypothesize that granuloma formation in sarcoidosis is due to an abnormal sensing of specific microbial constituents through NOD1, leading to an aberrant induction of pro-inflammatory and/or a decreased anti-inflammatory cytokines.

Methods: We assessed the functional responses of whole blood or isolated PBMC of patients with established diagnosis of sarcoidosis in response to NOD1 and TLR agonists and compared to those of healthy subjects. Pro- and anti-inflammatory cytokines were measured at the gene and protein levels.

Our results demonstrate that subjects with sarcoidosis expressed several cytokines including IL-1β, IL-8 and IL-6 significantly higher at baseline as compared to healthy controls. Secondly we show for the first time that patients with sarcoidosis respond to NOD1 stimulation and to co-stimulation of NOD1 and TLR4 receptors with a significant increase of IL12/IL23 p40 and decrease of IL-10 expressions at both the gene and protein levels.
Expression and function of bovine CCL28 (98.28)
Authors: Wilson Eric , Distelhorst Kathryn , Hughes Mellissa , Griffith Shannon , Nygaard Tyler , Voyich Jovanka
Published: January 2023

Bovine mastitis represents the most costly disease of the dairy industry. In an effort to better understand natural host defense mechanisms against mastitis causing pathogens we have begun to characterize the bovine chemokine CCL28 (bCCL28). CCL28 is a dual function chemokine which has been shown to mediate migration of antibody secreting cells to mucosal tissues; this chemokine has also been shown to function as an antimicrobial protein. We have found that expression of bCCL28 mRNA in bovine mucosal tissues is highly variable with highest expression in exocrine glands, including mammary gland and milk. Additionally, we demonstrate that recombinant bCCL28 has antimicrobial activity against mastitis causing microorganisms, including Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae. These results demonstrate that bCCL28 is expressed in mucosal tissues and exhibits potent antimicrobial activity in vitro, suggesting that this dual function chemokine may serve an important role in bovine immunity.
OPN expression by epithelial tubular cells regulates NK cell-mediated kidney ischemia reperfusion injury (98.30)
Authors: Zhang Zhu-Xu , Shek Kelvin , Wang Shuang , Huang Xuyan , Yin Ziqin , Sun Hongtao , Liu Weihua , Garcia Bertha , Rittling Susan , Jevnikar Anthony
Published: January 2023

Renal ischemia reperfusion injury (IRI) is a major cause of acute injury in both native and transplanted kidneys. This type of kidney injury is considered an antigen-independent inflammatory condition that involves multiple factors leading to tubular and endothelial dysfunction. We have recently found that NK cells induce apoptosis in tubular epithelial cells (TEC) and contribute to renal IRI. Kidney can express osteopontin (OPN) during injury. Therefore we examined the role OPN in NK cell function and kidney IRI.

We have found that TEC expressed high levels of OPN in vitro and in vivo after injury. Kidneys in OPN-/- mice had less severity of IRI compared to wild-type mice (P<0.05). Interestingly, recombinant OPN could activate NK cells that express high levels of perforin and granzyme and could mediate TEC apoptotic death. Importantly, we have found that NK cell migrate towards OPN protein as well as OPN-producing TEC in the transwell assay. Whereas, less NK cell migration was seen towards OPN-/- TEC (P<0.01). Further more, Kidneys in OPN-/- mice had less NK cell infiltration after IRI compared to WT mice (P<0.02). Taken together, our study results support a previously unrecognized role for TEC expression of OPN in NK cell-mediated kidney injury. TEC expression of OPN promotes early kidney inflammatory and IRI, and limiting OPN expression may improve kidney function and graft survival.
CXCL13-CXCR5 interactions support prostate cancer cell migration and invasion in a PI3K p110-, SRC- and FAK-dependent fashion (98.5)
Authors: El-Haibi Christelle P , Johnson Paul R , Sharma Praveen K , Suttles Jill , Singh Shailesh , Lillard, Jr. James W
Published: January 2023

We have previously shown that CXCR5 is significantly expressed by the prostate cancer (PCa) tissue than compared to high grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. We have also demonstrated PCa cell lines significantly express functional CXCR5, which promote cell invasion, migration, and active matrix metalloproteinase expression. In the present study, we elucidate the molecular mechanisms of PCa cell migration and invasion mediated by CXCL13-CXCR5. Using phospho-specific antibody microarray and Western blot analyses, we have delineated the pathways involved in CXCL13-CXCR5 signaling by PCa cell lines. For the first time, we show Gα, Gβ, and Gγ protein isoforms are differentially expressed by normal prostatic epithelial cell lines (PrEC and RWPE-1) versus PCa cell lines (PC3, DU145, LNCaP, and C4-2b). We also illustrate LNCaP cells express PI3K-p110α, -p110β, and -p110δ, while PC3 cells express PI3K-p110α, -p110β, -p110γ, and DOCK2. Migration and invasion assays revealed that both PC3 and LNCaP cell lines invade and migrate toward CXCL13 in a PI3K-dependent fashion. Src-FAK were required for this CXCL13-mediated migration and invasion. PC3 cells were capable of exploiting both PI3K-p110α/β /γ- and PI3K-p110γ-integrin β3-mediated events for migration and invasion, while LNCaP cells were only able to use PI3K-p110α/β /δ-dependent mechanisms for progression. In conclusion, the diversity of G protein and PI3K p110 isoforms along with Src- and FAK-signaling dictate the potential of PCa cell lines to migrate and invade in response to CXCL13.
Adipocyte culture medium increases metastasis of MDA-MB-231 cells through production of CCL20 (98.6)
Authors: Yang Young , Park Jeong-Su
Published: January 2023

We tried to identify chemokine that involved in communication between adipose tissues and breast cancers. In order to examine whether adipocyte culture medium (adipocyte-CM) affects the secreted chemokine profile of tumor cells, because adipocytes, as stromal cells, stimulate progression or metastasis of breast cancer cells, and chemokines secreted from tumor cells are involved in these processes, MDA-MB-231 cells were treated with adipocyte-CM and preadipocytes-CM. CCL20 expression was dramatically increased, and an NF-κB blocker completely inhibited adipocyte culture medium-induced CCL20 expression in MDA-MB-231 cells. We showed that adipocyte culture medium increased the production of TNF-α in MDA-MB-231 cells, which stimulated CCL20 expression in an autocrine fashion. Our data also showed that CCL20 increased the migration and invasiveness of MDA-MB-231 cells, but did not affect the proliferation of these cells. Adipocyte-CM stimulates migration and metastasis of breast cancer cells through the increase of CCL20 production, indicating that adipose tissue positively regulates microenvironment of breast tumours.

This work was supported by the Biogreen 21 project (20070301034031) of Rural Development Administration.
Dual role of CD8+ T cells in PLPp91-110 induced experimental autoimmune encephalomyelitis in HLA-DR3.Abo transgenic mice (99.1)
Authors: Mangalam Ashutosh K , Luckey David , Smart Michelle , Pease Larry , Rodriguez Moses , David Chella
Published: January 2023

MS is an inflammatory demyelinating disease of CNS with a presumed autoimmune origin. Prevalent data from animal model of disease, experimental autoimmune encephalomyelitis (EAE) suggests that myelin-specific CD4 T-cells play a major role in disease. Role of CD8 T-cells in MS/EAE has been enigmatic as it has been assigned either regulatory or pathogenic role in disease. Previously we have shown that PLPp91-110 peptide can induce inflammatory neurological disease in HLA-DR3 transgenic (Tg) mice. In order to understand the role of CD8 T cells in this humanized model, we generated HLA-DR3 Tg mice lacking CD8 T cells (DR3.CD8-/-). These mice showed earlier onset and more severe disease when compared to CD8 sufficient HLA-DR3 Tg mice indicating that CD8 T-cells plays a regulatory role. However, pathological analysis of DR3.CD8-/- mice with EAE showed less inflammation and demyelination in CNS compared to DR3.CD8 sufficient mice suggesting a second pathogenic role of CD8 T cells. Thus, our data suggests both a regulatory as well as a pathogenic role of CD8 T cells in EAE and possibly in MS. We have characterized both subsets in detail and show that CD8+CD122- T cells are regulatory subset of CD8 T cells and suppress CD4 T cells by inhibiting antigen presentation pathway, while CD8+CD122- T cells are pathogenic subset. Thus ours is the first study to show a dual role of CD8 T cells in the onset, incidence, progression, severity and pathogenesis of EAE/MS.
Characterizing the pathophysiology of Systemic Lupus Erythematosis disease progression in the BXSB Yaa CD8-/-, IL15 -/- mouse model (99.16)
Authors: McPhee Caroline , Bubier Jason , Morse Herbert , Foreman Oded , Roopenian Derry
Published: January 2023

Systemic Lupus Erythematosis (SLE) is a common autoimmune disease affecting mainly women and people of non Caucasian descent. This disease varies in severity and presentation but death due to renal failure is the most common outcome. The BXSB Yaa mouse has been a widely accepted model for the study of SLE. In an attempt to further illuminate the pathogenesis of this disease, CD8 and interleukin 15 were genetically removed from this model. The resulting congenic mouse develops an SLE like disease at an accelerated rate, succumbing to disease in half the time of the BXSB Yaa (3 months versus 6 months of age). Immunoglobulin levels are high at an early age in these mice. A loss of architecture and overwhelming plasmablast cell populations are seen histologically in the spleen and lymph nodes. These plasmablast cells were also found in the gastric mucosa, lung parenchyma, and renal tissues. Through immunoflorescence (IFA) and flow cytometry of splenic tissue, a number of these cells appear to be B220 negative and CD138 positive. There also appears to be an association with CD5, CD49b, and CD43. IFA has also revealed a loss of normal orientation between B220 labeled and CD4 labeled cells in the spleen and lymph nodes of these mice as well as many CD138 positive cells throughout these tissues. Further characterization of these cells is being pursued as well as age related studies in these mice to further define the pathogenesis of SLE.
Persistence of FOXP3 expression is impaired in RR-MS Tregs (99.3)
Authors: Schneider Anya , Long Sarah Alice , Kita Mariko , Buckner Jane H
Published: January 2023

Impaired Treg function may contribute to the development of Multiple Sclerosis (MS). FOXP3 is required for Treg function and its expression is maintained by the common γ-chain cytokines through phosphorylation of STAT5 (pSTAT5). We examined the pSTAT5 levels of CD4+CD25+Treg from relapsing remitting MS and control subjects in response to low-dose IL-2. We found that, despite equivalent levels of CD25, pSTAT5 was lower in RR-MS subjects (p= 0.0013), indicating a mechanism by which FOXP3 expression and Treg function may be lost in MS. To further examine the expression and persistence of FOXP3 in these subjects we generated adaptive Tregs and followed FOXP3 expression in these cells over time. We found that the initial induction of FOXP3 was similar to that of controls, but by day 10 of culture FOXP3 expression in CD4+CD25+ T cells was significantly lower (p<0.0001). FOXP3 expression could not be rescued by IL-2, IL-7 or IL-2/IL-7 in combination. In addition, cell surface expression of CD122 and CD132 is significantly decreased on RR-MS CD4+ CD25+ T cells. Taken together we conclude that the impaired persistence of FOXP3 expression in aTreg may impact the number and function of Tregs in individuals with RR-MS. This loss of FOXP3 may be due to decreased pSTAT5 in response to IL-2 and IL-7 which may be due to decreased levels of the IL-2R and IL-7R complexes. This work was supported by grants from the JDRF33-2008-398 and DFG108312-1.
Full Activation of Autoreactive B Cells Requires Helper Signals from Plasmacytoid Dendritic Cells (99.32)
Authors: ding chuanlin , Yan Jun
Published: January 2023

Systemic lupus erythematosus (SLE) is characterized by dysregulated autoreactive B cell activation. Previous studies demonstrate that plasmacytoid dendritic cells (pDCs) play a pivotal role in the pathogenesis of SLE. This study was undertaken to investigate the mechanisms of interactions between pDCs and autoreactive B cells. After co-culture of anti-small nuclear ribonucleoprotein particle (snRNP) B cells with activated pDCs via toll-like receptor (TLR)-7 or -9 agonist, we found that pDCs could significantly enhance autoreactive B cell proliferation and autoantibody production. In addition, autoreactive B cell activation via BCR engagement was also augmented by activated pDCs. This in vitro enhancement effect was further demonstrated by an in vivo B cell adoptive transfer experiment, demonstrating that autoreactive B cell proliferation was significantly decreased in MyD-88-deficient mice compared to that in wildtype mice. Neutralization of IFN-α and IL-6 could abrogate partially enhanced B cell activation via pDCs. Transwell studies demonstrated that pDCs could provide activation signal to B cells in a cell-to-cell contact manner. The involvement of the ICAM-LFA-1 pathway, but not the CD40L-CD40 pathway, was discovered to mainly contribute to this effect. These data suggest that pDCs regulate autoreactive B cell activation by soluble factors as well as cell-to-cell direct interactions. (This work was supported by an Arthritis Foundation Postdoctoral Fellowship)
Effects of expression Fli1 transcription factor on lupus disease development in NZM2410 mice (99.35)
Authors: Zhang Xian , Mathenia Jeremy , Reyes-Cortes Emmanuel , Molano Ivan , Gilkeson Gary
Published: January 2023

The Fli1 gene is a member of the ets family of transcription factors that is expressed in hematopoietic cells. Two-fold overexpression of Fli1 protein in transgenic mice results in the development of a lupus-like disease. Expression of Fli1 in systemic lupus erythematosus (SLE) patients and animal models of lupus is higher compared to normal controls. Here we examined the effects of reduced Fli1 expression on disease development in NZM2410 mice, an animal model of SLE.

We generated Fli1 knockout heterozygous mice (Fli1+/-; Fli-1 homozygous knockout is embryonic lethal). Compared to wild-type (Fli1+/+) mice, we found that expression of Fli1 protein in Fli1+/- mice was reduced by half. Autoantibodies, Urinary albumin excretion and serum immunoglobulin concentrations were measured by ELISA.

Fli1+/- NZM2410 mice survived significantly longer than Fli1+/+ NZM2410 mice. At the age of 52 weeks, 81% Fli1+/- NZM 2410 mice survived, whereas only 35% of Fli1+/+ mice survived. There were significant low urinary albumin excretions in Fli1+/- NZM2410 mice compared to Fli1+/+ NZM2410 mice at the age of 34 weeks. Compared with Fli1+/+ NZM2410 mice, Fli1+/- NZM2410 mice have significantly decreased levels of IgG anti-dsDNA and glomerular antigen (GA) antibodies at the age of 34 weeks.

Our results suggest that the expression of Fli1 plays an important role in disease development in NZM2410 mice.
Src-like adaptor protein (SLAP) deficiency leads to decreased autoantibody production (99.37)
Authors: Peterson Lisa K , Shaw Laura A , Dragone Leonard L
Published: January 2023

Background: Intrinsic defects in lymphocyte signaling have been implicated in the pathogenesis of autoimmunity. SLAP has been shown to influence BCR levels and signaling in developing B cells. We hypothesize that SLAP deficiency will enhance the negative selection of autoreactive B cells leading to decreased autoantibody production.

Methods: To test this hypothesis we immunized SLAP-deficient and wild-type BALB/c mice with a peptide mimitope for dsDNA that leads to the development of dsDNA antibodies and a systemic lupus erythematosus-like autoimmune disease. In addition, we used a second model where we generated SLAP-deficient mice expressing an autoreactive BCR heavy chain 3H9H/E56R (56R) that binds dsDNA to determine how SLAP deficiency impacts BCR repertoire selection and autoantibody production.

Results: SLAP-deficiency led to decreased serum levels of dsDNA antibodies in both experimental systems with a 6-fold reduction in the mimitope treated mice and a 3-fold reduction in dsDNA antibodies in the SLAP-deficient 56R mice. Flow cytometric analysis of bone marrow B cell subsets in SLAP-deficient 56R mice revealed a 27% reduction in fraction D, 37% in E and a 2-fold reduction in F, with a 19% increase in lambda usage compared to controls.

Conclusions: These data support the hypothesis that SLAP deficiency enhances negative selection of autoreactive B cells leading to decreased autoantibody production.
IL-17 activates the classical NF-κB signaling pathway to upregulate chemokine signal inhibitor molecules RGS16 and RGS13 in autoimmune B cells of BXD2 mice (99.49)
Authors: Xie Shutao , Hsu Hui-Chen , Wang John , Wu Qi , Li Jun , Li Hao , Mountz John D.
Published: January 2023

We have previously reported that IL-17 upregulated the expression of rgs13 and rgs16 to attest the migration response of B cell to CXCL12. This specific effect of IL-17 on B cells is associated with the development of spontaneous germinal center in the spleens of autoimmune BXD2 mice. This study is to determine if NF-κB signaling is involved in IL-17 mediated upregulation of RGS proteins in B cells. Western blotting and confocal imaging analyses both showed that IL-17 stimulation of B cells from BXD2 mice resulted in rapid (5 min) activation of the classical NF-κB pathway which can be characterized by phosphorylation of p65 (P-p65) and translocation of P-p65 to the nuclei. Significantly higher level of activated P-p65 was found in BXD2 B cells than in B6 and BXD2-Il17r-/- B cells (>4.0-fold, p<0.01). Expression of the endogenous p50 subunit of NF-κB in BXD2 B cells was also higher than in B6 B cells (5.0-fold, p<0.01) but was surprisingly lower than in BXD2-Il17r-/- B cells (2.5-fold, p<0.01). The activated NF-κB pathway by IL-17 in B cells correlated with the upregulation of RGS16 and RGS13 in both protein and mRNA levels. Inhibition of P-p65 significantly down-regulated the expression of rgs genes (6.0-fold), and released the B cell migration arrest induced by IL-17 stimulation. These data suggest that IL-17 directly upregulates the expression of RGS through activating the classical NF-κB pathway in autoreactive BXD2 B cells.
Taenia crassiceps infection and its antigens abrogate Experimental Autoimmune Encephalomyelitis, a role for alternatively activated macrophages (AAMφ) (99.51)
Authors: Reyes Jose L , Espinoza Arlett F , Terrazas Luis I
Published: January 2023

Helminth infections induce strong immunoregulation and may modulate subsequent pathogenic challenges. Taenia crassiceps causes chronic infections, induces Th2-biased responses and recruits AAMΦ with suppressive ability. To test the influence of this cestode on autoimmunity, we infected C57BL/6 mice with 40 T. crassiceps metacestodes, 8 weeks later Experimental Autoimmune Encephalomyelitis (EAE) was induced with MOG35-55 on infected and uninfected mice. It was found that T. crassiceps-infected mice delayed the onset of EAE; only 50% of them displayed symptoms and those were significantly less severe than uninfected mice. They also had decreased MOG-specific spleen cell proliferation, low IL-17 production and low leukocyte infiltration in the spinal cord. Levels of TNFα in sera and transcripts for TNFα and iNOS in CNS were lower in previously infected mice. AAMΦ markers Ym1, Relm-α and Arg1 were over-expressed in CNS of EAE T. crassiceps-bearing mice, whereas T-bet was expressed in EAE mice. Injection of 50μg of soluble T. crassiceps Ag at the onset of EAE abrogated the symptoms. Infection and Ag induced AAMΦ expressing PDL1 & PDL2 which suppressed anti-MOG specific cell proliferation; such effects were inhibited in mice receiving chlodronate-loaded liposomes. Thus, T. crassiceps' immune-regulation decreases EAE severity by dampening T cell proliferation and migration to CNS with a key role for AAMΦ.
Tim-1 expressed on dendritic cells enhances effector T cell responses and inhibits development and function of regulatory T cells (99.9)
Authors: Xiao Sheng , Kuchroo Vijay K
Published: January 2023

Tim-1 has been reported to be expressed on CD4+ T cells upon activation and to regulate Th2 responses. Here we report that Tim-1 is constitutively expressed on dendritic cells (DCs), the key instigators of adaptive immune responses. Tim-1 engagement in DCs induces a signaling cascade with NF-kB acivation, and promotes their maturation, resulting in upregulation of costimulatory molecules and production of proinflammatory cytokines. Such DCs enhance effector T cell (especially Th17) responses but inhibit de novo generation of Foxp3+ regulatory T cells (Treg). In vivo when given at the induction of immune response, agonistic anti-Tim-1 antibody enhances immunogenic function of DCs, decreases suppressive function of Foxp3+ Treg, and substantially increases proinflammatory Th17 responses as well as Th1 responses. The anti-Tim-1 treatment induces more severe experimental autoimmune encephalomyelitis (EAE) in susceptible mice, and also breaks tolerance and induces disease in the EAE-resistant strain of mice. These findings indicate that Tim-1 has an important role in the innate immune system in that Tim-1 signaling in DCs decreases immune regulation by Treg and promotes effector T cell responses.
Erythropoietin increases macrophage-mediated suppression in a model of the tumor microenvironment (100.1)
Authors: Riggs James , Orlowski Michelle
Published: January 2023

Erythropoietin (EPO), a glycoprotein that increases RBC production, is used to reverse chemotherapy-induced anemia in cancer patients. Unfortunately some patients have experienced accelerated tumor growth and decreased survival rates. We have been modeling the in vivo tumor microenvironment by culturing murine peritoneal cavity (PerC) cells in vitro. Many tumors have significant leucocyte composition including increased representation of myeloid suppressor cells. When PerC T cells are activated in a macrophage-rich environment such as this their proliferation is suppressed. Interferon-gamma (IFN-g)-triggered inducible nitric oxide synthase (iNOS) expression by macrophages drives T cell suppression via arginine catabolism. Addition of 1-methyl arginine (1-MA), a competitive inhibitor of iNOS, recovers the T cell response. We have studied EPO’s impact upon T cell activation in the PerC macrophage model. Exogenous EPO increased T cell suppression by C57BL/6J (wildtype) macrophages and this effect could be blocked by the addition of 1-MA. EPO’s enhanced suppressive effect was not evident with macrophages from iNOS or IFN-g receptor knockout mice. Studies of BALB/c PerC T cells, which were less susceptible to iNOS-mediated suppression, revealed that EPO had no effect upon T cell activation. These results suggest that exogenous EPO can increase T cell suppression and does so by acting through an IFN-g/iNOS-triggered myeloid cell pathway. Supported by NIH AREA R15-CA136901-01.
Lymphotoxin induces tumor evasion through TGF-beta signaling (100.10)
Authors: Park Chang-Eon , Kang Hyung-Sik
Published: January 2023

While major histocompatibility class I (MHC class I) expression was inhibited in mice administrated with pIRES-LTab plasmid, it was elevated in the spleen and liver of lymphotoxin(LT)a-/- and LTbR-/- mice. Consistent with this, the expression of genes that upregulated MHC class I was downregulated in mice administrated with pIRES-LTab plasmid, but it was upregulated in the spleen and liver of LTa-/- and LTbR-/- mice. In addition, gene expression of Fas and FasL was also regulated by LT in vitro and in vivo. Whereas gene expression of TGF-b receptor was upregulated in the large intestine of LTa-/- and LTbR-/- mice, its expression was increased in mice immunized with pIRES-LTab plasmid as a DNA adjuvant. Administration of pIRES-LTab plasmid prolonged the survival rate of mice challenged with human papillomavirus (HPV)-transformed mouse TC-1 tumor cells, and delayed the growth rate of the TC-1 cells. Gene expression of smad3 and smad4 was increased in colon and small intestine of LTa-/- and LTbR-/- mice. These results collectively suggested that MHC class I expression was regulated by LT-induced upregulation of TGF-b expression, and subsequently tumor evasion might be induced by suppression of MHC class I.
Epidermal expression of oncogenic ras subverts cytotoxic inflammation by skewing infiltrating myeloid cells towards an immunosuppressive phenotype. (100.16)
Authors: Gunderson Andrew , Mohammed Javed , Glick Adam
Published: January 2023

Cancer development is frequently accompanied by inflammation and immune activation. The nature of this inflammatory response, however, is non-uniform and can consist of cells with opposing functions. To characterize the innate immune response to neoplastic epithelial tissue, we used an epidermally-restricted transgenic mouse model of oncogenic H-Ras expression in which epidermal hyperplasia and premalignant progression induces aberrant myelopoesis and cutaneous hyperinflammation associated with the formation of myeloid resident epithelial microabscesses. Antibody mediated depletion of neutrophils concurrent with transgene activation ablated neutrophil infiltration and microabscesses resulting in increased epidermal thickening relative to isotype controls. Neutrophils isolated from mice expressing ras for 1 week killed keratinocytes in vitro, suggesting that the in vivo myeloid response to oncogenic ras expression is initially a cytotoxic one. Simultaneously, there is cutaneous upregulation of genes implicated in the activation, function and recruitment of myeloid derived suppressor cells (MDSC) such as GMCSF, GCSF, MMP9, VEGF, S100A8/9, CCL2, and IL1β. This coincides with upregulation of IL4Rα on myelocytes, increased percentage of Tregs, and decreased effector T cell responses from 1 to 2 weeks post H-Ras expression. These data suggest that the acute cytotoxic neutrophil response is replaced by a suppressor myeloid phenotype that could enhance neoplastic progression.
Anaplastic plasmacytomas associated with NFATc2 deficiency in aged B6 mice (100.4)
Authors: Modiano Jaime , Highfill Sarah , Matise Ilse , Morse Herbert , Jubala Cristan , Coffey David , O'Sullivan Gerard , Bellgrau Donald
Published: January 2023

NFATc2 is an intrinsic negative regulator of lymphocyte proliferation that promotes T cell anergy, cell cycle arrest, and possibly differentiation to the Fox-P3+ T regulatory phenotype. NFATc2 knockout (KO) mice show systemic peripheral expansion of lymphocytes with reduced thresholds for T cell activation. Unlike NFATc2 KO in mixed (H2b X H2bc) or H2d backgrounds, homozygous NFATc2 B6 KO mice did not develop splenomegaly, but instead developed systemic lymphadenopathy that was most pronounced in mesenteric and submandibular lymph nodes. The mice retained a reduced threshold of activation for both B- and T-cells. When compared to wild type and heterozygous littermates, mice had greater frequency of mixed lymphocytic infiltrates in kidney, liver, and salivary glands. Six of 11 NFATc2 KO mice that were allowed to reach 16 to 20 months of age developed anaplastic plasmacytomas in the mesenteric lymph nodes, a phenotype that was not seen in 17 age matched WT or heterozygous KO littermates. This suggests NFATc2 deficiency facilitates development of plasma cell tumors in the H2b background. We propose this may be driven by chronic antigenic stimulation and is due to poorly restrained intrinsic negative regulation and not due to extrinsic immunologic defects. These mice represent a valuable resource to explore mechanisms that account for progression from inflammation to malignancy. SLH and JFM contributed equally to this work.
Murine Hepatocellular Carcinoma Stem Cells Express Pluripotency-associated Transcription Factors and are Sensitive to Immune Mediated Apoptosis. (101.16)
Authors: de Kluyver Rachel , Stauffer Jim , Brooks Alan , Shanker Anil , Sayers Tom
Published: January 2023

Current evidence supports the view that cancer stem cells (CSCs) constitute the primary pathogenic component of both metastases and minimal residual disease and that elimination of CSCs is required for cure. Cell surface markers have been used to identify CSCs but fail to identify CSC in all tumors from the same tissue or in all patients. Therefore there is a need to define and test more reliable markers of CSCs. Oct-4, Sox-2 and Nanog transcription factors are key regulatory factors in maintenance of pluripotency and have been demonstrated to be robust intracellular biomarkers for tissue and cancer stem cells. A GFP-based reporter driven by a promoter responsive to Nanog or Oct-4 was used to mark pluripotent tumor cells from in vitro cultured, cancer cell lines (breast cancer- 4T1.2), and from autochthonous hepatocellular carcinoma. The molecular profile of primary Akt/β-Cat-induced murine HCC recapitulates that observed in poor prognosis Ep-CAM+/AFP+ human HCC. Ep-CAM and CD133 and Nanog and Oct-4 are independent CSC markers. Directly ex vivo, murine HCC stem cells express abundant CD1d and MHC class II in addition to TNF-R2, lymphotoxin-β receptor (LTβR) and DR5. The functional significance of CD1d, MHC class II, TNF-R2, LTβR and DR5 expression will be assessed in future studies. This study will provide valuable proof-of-principle data to the merits of CSCs as targets for curative anti-cancer therapies.
In vivo analysis of cellular sentinels for extracellular Heat Shock Protein-peptide complexes. (101.2)
Authors: Messmer Michelle , Binder Robert
Published: January 2023

Heat shock proteins are expressed in all cells and chaperone protein and peptide cargoes within cells. Based on their abundance and intracellular localization, HSPs in the extracellular environment have been described as indicators of cellular/membrane damage. Thus, the immune system is designed to detect HSPs in the extracellular environment (either released from dying cells or by immunization) by cell surface receptors such as CD91. Our goal is to identify the subset(s) of antigen presenting cells that detect extracellular HSPs and initiate the immune response. By introducing fluorescent labeled HSPs into mice, cells in the draining lymph node that engage extracellular HSP can be tracked. Our data demonstrates that the CD4+CD11c+ dendritic cells are the predominant antigen presenting cell that internalizes the HSP. The ability of these cells to cross-present HSP-chaperoned peptides and provide co-stimulation is under investigation. These studies will be important for understanding how endogenous HSPs act to alert the immune system of cellular damage and for optimizing immunization regimens with HSP-based vaccines.
CD5 knock out mice display a strong antitumor immune response associated with tumor infiltration by hyperactivated tumor-reactive CD8+ T lymphocytes (101.21)
Authors: Mami-Chouaib Fathia , Franciszkiewicz Katarzyna , Raman Chander , Tabbekh Mouna
Published: January 2023

We have previously reported the TCR inhibitory molecule CD5 impairs reactivity of tumor-specific PBL-derived T-cell clones against the cognate target and controls their susceptibility to activation-induced cell death (AICD) triggered by tumor cells. In this report, we compared the antitumor T-cell response developed against the B16F10 melanoma engrafted in CD5-deficient and wild-type (wt) C57BL/6 mice. Our results indicate that CD5 knock out mice elicit a delayed tumor growth as compared to wt mice, which is associated with tumor infiltration by more activated tumor-reactive T lymphocytes. Our data also indicate that tumor suppression in CD5-deficient mice is transient and that tumor flare up correlates with increase in AICD of tumor-infiltrating CD8+ T cells. Our data suggest that tumor T lymphocyte infiltration occurs at early stages of cancer development and that tumor-mediated AICD is most likely involved in the induction of T-cell tolerance to malignant cells.