bluuebunny/crossref_metadata_2025 huggingface dataset:
CCR1 expression and signal transduction by murine BMMC results in secretion of TNF-α, TGFβ-1 and IL-6 (98.10)
Authors: Fifadara Nimita , Aye Cho Cho , Raghuwanshi Sandeep , Richardson Ricardo , Fukuda Ken , Ono Santa
Published: January 2023

Chemokine receptors are important co-stimulatory molecules found on many blood cells. Chemokine receptor 1 (CCR1) is one of the important chemokine receptor identified in different immune cells known for its function in various diseases. The expression and functions of chemokine receptors on mast cells has been quite controversial. In this study, we report for the first time that murine bone marrow-derived mast cells (BMMC) express mRNA and protein for CCR1. BMMC cultured in the presence of murine recombinant stem cell factor and murine interleukin-3 expressed CCR1 after 5-6 weeks. Co-stimulation of CCR1 by its ligand macrophage inflammatory protein 1α (MIP-1α) and the IgE receptor FceRI enhanced BMMC degranulation compared to FceRI stimulation alone (85% vs. 54%, p < 0.0001) and Ca2+ influx. We report for the first time that mBMMCCCR1+ cells endogenously express neurokinin receptor-1 and intercellular adhesion molecule-1. We also observed significant increases in mast cell secretion of key growth factors, cytokines and chemokine mediators upon CCR1- FceRI co-stimulation. These factors include transforming growth factor β-1, tumor necrosis factor-α, ciliary neurotrophic factor and the cytokine interleukin-6. Taken together, our data indicate that CCR1 plays a key role in BMMC function. These findings contribute to our understanding of mechanisms for immune cell trafficking during inflammation.
The effect of interleukin-32 gamma on the differentiation of osteoclasts from CD14+ monocytes (98.15)
Authors: Lee Chang-Keun , Kim Yong-Gil , Kim Soo-Hyun , Lee Sang-Heon , Yoo Bin
Published: January 2023

IL-32 induced various cytokines including TNF-a in monocytes and differentiation of monocytes into macrophage-like cells. However, the effect of IL-32 on differentiation of monocytes into osteoclasts is still unknown. Our study was designed to evaluate whether IL-32 affects on osteoclasts differentiation and how this cytokine modulates the receptor activator NF-κB ligand (RANKL) and osteoprotegerin (OPG) on fibroblast-like synoviocytes (FLS).

CD14+ monocytes were isolated from healthy volunteers and FLS were isolated from synovium of RA patients. To test of osteoclastogenic effect, IL-32γ (0 as control, 25, 50, 100-ng/ml) or IL-17 (20-ng/ml) were treated in the monocytes culture system and Osteologic disks® with M-CSF and sRANKL for 2-week. To evaluate RANKL and OPG changes in FLS by IL-32γ or IL-17 (0, 3, 5-day), the mRNA expression and protein level were evaluated using RT-PCR and ELISA.

After 2-week culture, the osteoclasts count and the resorbed area on Osteologic disks® were increased significantly in 50-ng/ml IL-32γ group more than those in IL-17 group (p < 0.01 and p < 0.05). Increased level of RANKL and decreased level of OPG were observed prominently in IL-32γ stimulated group compared with IL-17 stimulated group at 5-day.

Our results suggest IL-32γ is a potent osteoclastogenic cytokine and could make more favorable condition for osteoclastogenesis in RA synovium via RANKL and OPG modulation in FLS.
CXCL11 and CXCL12 mediate matrix metalloproteinase expression by breast cancer cells through CXCR7 (98.2)
Authors: Mosley Lasharon  D , Sharma Praveen K , Singh Rajesh , Singh Shailesh , Lillard, Jr. James W
Published: January 2023

Chemokines and their receptors have been implicated in organ-specific homing of metastatic breast cancer (BrCa) cells. Stromal cell-derived factor-1 (SDF-1; CXCL12), a homeostatic chemokine, has been suggested to promote BrCa progression. Interferon-inducible T cellα-chemoattractant (I-TAC; CXCL11), an inflammatory chemokine, is elevated following IL-1, TNF-α, IL-6, and/or IFN-γ stimuli, which are increased during breast tumor burden. In this study, we focused on CXCL12 and its receptors, CXCR4 and CXCR7, as well as CXCL11 and its receptors, CXCR3 and CXCR7. RT-PCR and ImageStream analysis demonstrated that MCF-7 and MDA-MB-231 cell lines differentially express CXCR3, CXCR4, and CXCR7. CXCL11 stimulation modulated matrix metalloproteinase (MMP) expression via CXCR7 >> CXCR3 interaction; similarly, CXCL12 increased MMP-1, MMP-13, MMP-2, and MMP-9 mRNAs and active protein expression in a largely CXCR7-dependent fashion. These CXCR7-required events were significantly altered by selective Gαi protein subunit and Gβ/γ protein subunit(s) inhibition. Taken together, these studies provide novel information regarding the cellular and molecular mechanisms involved in CXCR7-mediated BrCa progression. [This study was supported by funds from the James Brown Graham Cancer Center, Smith & Lucille Gibson Endowment. and National Institute of Health Grants CA092078, CA086359, and MD00525.]
Nanonized black soybean enhances immune response in senescence-accelerated mice (98.20)
Authors: Jeng Kee Ching G. , Chan Yin-Ching , Wu Chia-Chuan , Chan Kung-Chi , Lin Yo-Giao , Liao Jiunn-Wang
Published: January 2023

This study investigated the effect of soybean supplementation on the immune response in the senescence-accelerated-prone mice (SAMP8) and -resistant mice (SAMPR1, control). Male SAMP8 mice (6 mo old) were supplemented with or without nanonized (Nano-soy) or microparticled (Micro-soy) black soybeans (n = 8/group), respectively for 12 wk. Human PBMC and murine splenocytes were stimulated with mitogens and cytokines were determined by RT-PCR and/or ELISA. The results showed that food intake was similar among all groups. Isoflavone (daidzin and genistin) intake was higher in the Nano-soy group than the Micro-soy group. The lymphoproliferation and production of IL-2 and IFN-γ in the Nano-soy group had a significantly higher (P < 0.05) than those in the control and Micro-soy groups. The Nano-soy group mice reached these cytokine levels similar to SAMR1 mice. This result was consistent with the in vitro data that daidzein (a metabolite of daidzin), at a concentration of 10 μM, increased IL-2, IL-4, and IFN-γ production from PHA-stimulated PBMC (P < 0.05). However at higher dose (> 50 μM), daidzein only reduced IL-10 and IFN-γ levels, whereas genistein reduced levels of the IL-2, IL-4, IL-10, IFN-γ mRNA and protein. This suggests that the Nano-soy supplementation improved immune response in SAMP8 mice which may be attributable to higher daidzin content in the diet.
Activation of NK-cell cytokine production via anti-CD48 (98.29)
Authors: Sinha Suwan Kumar , Gao Ning , Yuan Dorothy
Published: January 2023

In addition to the killing of transformed and virally infected cells NK cells also modulate immune responses via secreted cytokines or interaction with other lymphocytes. We have previously reported that B cells can induce IL-2 propagated NK cells to produce IL-13 in culture. The NK cell activation is dependent on B cells surface CD48 and requires CD244 (2B4) expression on NK cells, suggesting that CD48 on B cells can provide a positive signal for activation of NK cells via 2B4. Interestingly, we now show that a similar activation of NK cells can be achieved by interaction with immobilized anti-CD48 in the absence of B cells. CD48 is a glycoprotein found on most lymphocytes, including B and NK cells, but it has no intracellular region to transduce signals on its own. NK cells from 2B4-CD2 double knockout mice do not respond to immobilized anti-CD48 whereas absence of only CD2 does not affect IL-13 production. These results suggest that modulation of CD48 on NK cells can alter the nature of 2B4 signaling in NK cells. To gain some insight into the mechanism, we performed immuno-staining which revealed that immobilized anti-CD48 leads to clustering of CD48 on the surface of NK cells and 2B4 co-localizes with the former. Furthermore, the clustering by anti-CD48 was impaired in NK cells from 2B4-CD2 KO mice. These findings suggest a new relationship between CD48 and 2B4 in NK cells that may impact positive vs. negative signaling via 2B4.
CXCL13 blockade abrogates bone metastasis by impairing beta-3 integrin activation and matrix metalloproteinase expression by prostate cancer cells (98.4)
Authors: El-Haibi Christelle P , Singh Rajesh , Sharma Praveen K , Singh Shailesh , Lillard, Jr. James W
Published: January 2023

Prostate cancer (PCa) is the second leading cause of cancer morbidity and mortality among men in the United States. Mortality is not due to primary tumor growth but rather from complications caused by metastasis to vital organs. It is well known that PCa cells preferentially metastasize to bone; however, the molecular, cellular, and anatomic bases of this spread remain obscure. Chemokines and their receptors are thought to play key roles in this multifactorial process. We have recently shown that CXCR5 expression positively correlates with prostate tumor staging, and that PCa cell lines (LNCaP, C4-2B, and PC3) differentially express CXCR5 unlike their normal counterparts (PrEC and RWPE-1). By virtue of its presence in the bone microenvironment during disease and/or tumor burden, we hypothesized that CXCL13 (the ligand for CXCR5) is critical for bone metastasis and prostate tumor progression. We demonstrate CXCL13 is present in bone marrow of xenograft tumor-bearing mice following intracardiac injection of PC3 cells. Luciferase-expressing PC3 cells established skeletal metastases, but CXCL13 blockade significantly delayed prostate tumor formation and spread to bones, than compared to mice treated with control antibody. Immunohistochemical staining of tumors and viscera excised from experimental versus control groups revealed differential matrix metalloproteinase and integrin expression as well as tumor cell phenotype, which were presumably modulated by CXCL13-CXCR5 interactions. In conclusion, the CXCL13-CXCR5 axis partially supports PCa bone metastasis.
Brain-derived neurotrophic factor BDNF is involved in MP4-induced experimental autoimmune encephalomyelitis (99.11)
Authors: Kuerten Stefanie , Javeri Sita , Rodi Michael , Addicks Klaus , Tary-Lehmann Magdalena , Lehmann Paul V
Published: January 2023

In multiple sclerosis and its animal model EAE, the role of BDNF is still not understood. Results have been ambiguous with some studies pointing to a neuroprotective role while others implied neurodestructive components. The use of gene-modified mice could largely help shed more light on the mechanisms BDNF plays in the disease. Thus far, however, such studies have not been done. Our results show significantly attenuated disease in terms of onset and severity in BDNF-deficient B6.129S4-BdnftmJae/J compared to wild-type mice. Strikingly, while wild-type mice displayed a classical TH1 cytokine profile in response to MP4, levels of antigen-specific T cells secreting IFN-γ and IL-2 were undetectable in the BDNF-deficient mice. In addition, lesion frequencies in the CNS were lower in these mice. Demyelination was also less severe. Analysis of the cellular composition of infiltrates revealed significantly higher numbers of B cells and CD8+ T cells in the wild-type mice. Our results suggest an important role for BDNF in the pathogenesis of MP4-induced EAE, which points to a more disease sustaining than neuroprotective function. Our initial immunological and histological studies suggest the involvement of this factor not only in mediating peripheral T cell responses, but also CNS pathology itself. Further studies are needed to dissect the mechanisms underlying our preliminary data.
Role of Cdk4 in immunological tolerance and in pancreatic beta cell mass homeostasis in T1D (99.14)
Authors: mora conchi , Garcia Ainhoa , Marzo Nuria , Altirriba Jordi , Gomis Ramon , Martín Javier , Ortega Sagrario
Published: January 2023

Cdk4 plays a central role in perinatal pancreatic β cell replication and is also highly expressed in healthy, adult human pancreatic beta cells. We have previously reported that, when the hyperactive mutant form of Cdk4, Cdk4R24C replaces the wild type gene, it causes β cell hyperplasia without promoting hypoglycemia in a non-autoimmune-prone mouse strain. We found out that when the mutant Cdk4R24C knockin model is backcrossed onto the NOD genetic background, diabetes is exacerbated due to the expression of the mutated Cdk4R24C form in the NOD immune system too, as in the absence of NODCdk4R24C lymphocytes (NOD/SCID model), βcells become resistant to adoptively transferred autoimmune attack.

Since the knockin model does not allow discriminating between the role of Cdk4 in either β cells or in the immune system, we have designed a model where overexpression of Cdk4 is restricted specifically to NOD β cells. We have obtained transgenic lines in the NOD genetic background in which the Rat Insulin Promoter (RIP) drives the expression of either Wild Type (WT) Cdk4 (Cdk4wt) or its mutant, hyperactive form, (Cdk4R24C), leaving the lymphocytic compartment presumably intact.

This experimental approach will allow us to determine whether β cell hyperproliferation induced by Cdk4 overexpression specifically on pancreatic β cells, balances the autoimmune attack against β cells inherent to the NOD mice, and leads to an steady-state of long-lasting imunological tolerance.
Enhanced activation of Notch signaling in peripheral blood T helper cells of patients with rheumatoid arthritis (99.21)
Authors: jiao Zhijun , Wang Wenhong
Published: January 2023

Expression of Notch homologues in local inflammation issue of rheumatoid arthritis (RA) and cultured synoviocytes has been reported, but the expression profile of Notch-related molecules in peripheral lymphocyte in RA remains unclear. Here, the expression of Notch receptors in peripheral lymphocytes of RA patients was assessed by both flow cytometry and real-time polymerase chain reaction (PCR). The expression of Notch target gene HES-1, regulatory gene NUMB and Notch-ICD was determined by PCR and immunoblot. There was an increased expression of Notch 2, Notch 3 and Notch 4 in T helper cells from active RA patients among which increased Notch 3 was mainly expressed by activated T cells. Notably, Notch 3-expressing T cells decreased in inactive RA patients and the level was similar to that of healthy controls (HC). T helper cells from RA patients exhibited increased expression of target gene HES-1 but decreased expression of negative modulation gene NUMB of Notch signaling. There was also an increased nuclear translocation of Notch-ICD in active RA disease. The present study demonstrate that T helper cells from RA patients display significantly altered expression profile of Notch receptors and enhanced activation of Notch signaling compared with those from HC.

This study was supported by grants from the Natural Science Foundation of China (30872335) and Society Development Foundation of Zhenjiang (SH2008035).
Absence of sphingosine kinase 1 alters progression of TNF-alpha induced arthritis (99.25)
Authors: Baker DeAnna , Obeid Lina , Gilkeson Gary
Published: January 2023

Changes in sphingolipid levels can alter cellular functions. Sphingosine 1 phosphate (S1P) and hTNF stimulates COX-2 and PGE2 in fibroblast-like synoviocytes (FLS). In vitro, hTNF and S1P both produce more COX-2 and PGE2 than either separately. Blocking sphingosine kinase 1(SphK1) decreases COX-2 and PGE2 after hTNF stimulation. Our hypothesis is mice overexpressing hTNF without functional SphK1 (hTNF/SphK1-/-) will have less synovial inflammation than mice that overexpress hTNF with functional SphK1 (hTNF/SphK1+/+). Transgenic hTNF mice were crossed with SphK1-/- mice, genotyped and observed, while histological sections were collected to evaluate disease activity. FLS were isolated from the knee joints of WT and SphK1-/- mice, cultured, and stimulated with hTNF. hTNF/SphK1-/- mice had significantly lower arthritis scores, less inflammatory infiltrates and preserved joint spaces compared to hTNF/SphK1+/+ mice. hTNF-stimulated FLS from SphK1-/- mice produced less PGE2 than WT mice. Deletion of SphK1 significantly delays progression and severity of hTNF induced arthritis, synovial proliferation and inflammatory infiltrates. SphK1-/- FLS produced less PGE2 in response to hTNF. Therefore, inhibiting SphK1 is a potential novel therapeutic agent. The project described was supported by Grant Number R01GM062887 from the National Institute of General Medical Sciences and a grant from the ACR REF.
Anti-Inflammatory Properties of IL-35 Promote Induction of Regulatory CD4+T Cells To Suppress Collagen II-Induced Arthritis (CIA) (99.27)
Authors: Kochetkova Irina , Golden Sarah , Callis Gayle , Pascual David W.
Published: January 2023

IL-35, a novel heterodimeric cytokine (IL-12p35 subunit plus Epstein-Barr virus-induced gene 3 [EBI3]), had been reported to expand regulatory CD4+CD25+ T cells and ameliorate CIA in mice. IL-35 was expressed as a single chain polypeptide, and the recombinant cytokine was detected by Western blot using anti-EBI3 or anti-IL-12 mAbs. To assess its activity, BALB/c mice were adoptively transferred with DO11.10 CD4+ T cells, challenged s.c. with OVA in IFA, and treated every day for 5 days with IL-35, resulting in diminished IgG2a and IgG2b anti-OVA Abs, as well as suppressed DTH responses. To exploit IL-35's therapeutic potential, B6 mice were induced with CIA, and upon the onset of disease (day 21), mice received 2.0 μg/mouse every 2 days until day 39. All mice in the control group developed arthritis by day 28 post-induction, whereas none of IL-35-treated mice had any clinical symptoms. Restimulated sorted CD4+ T cells from LNs of protected mice showed decreased IFN-γ and IL-17 production. FACS analysis revealed enhancement of regulatory CD4+ CD25+ T cells, of which, half co-expressed CD39, an ectonucleoside triphosphate diphosphohydrolase. Cell-sorted CD39+ CD4+ T cells from IL-35-treated mice could suppress CII-specific proliferation by CD39- CD4+ T cells via enhanced IL-10 production. These findings suggest the stimulation by IL-35 for the induction of regulatory CD4+ T cells. Supported by NIH R01 AI78938.
Oral administration of the tolerogenic peptide pCons induces CD4+CD25+Foxp3+ regulatory T cells in mesenteric lymph nodes (99.28)
Authors: Skaggs Brian J , Connolly Jay , LaCava Antonio , Hahn Bevra H
Published: January 2023

Purpose: We wanted to examine whether pCons, a 15 amino acid peptide based on the VH regions of murine anti-DNA antibodies that delays nephritis and anti-DNA antibodies in NZB/NZW F1 (BWF1) mice when administered i.v. or subcutaneously, is also efficacious when given orally.

Methods: Ten-week old lupus-prone BWF1 mice were fed different concentrations of pCons (25μg/100μg/250μg) by gavage for 5 days every four weeks. Control animals were fed saline. Both the L- and D- amino acid forms of pCons were given. After twelve weeks, four mice in each group were killed to examine the regulatory T cell populations in the spleen and mesenteric lymph nodes (MLN) by flow cytometry. Progression of lupus is monitored by proteinuria and the presence of anti-DNA antibodies in peripheral blood.

Results: MLN Treg were increased 2-fold in the 250μg D-pCons group. No increase in CD4+ Treg and CD8+ Ts was observed in splenocytes, and no increase in Treg or Ts populations were observed in the lower doses of each peptide. Interestingly, anti-DNA antibody titers and proteinuria were lower in L-pConsed mice vs. D-pCons and saline fed mice.

Conclusions: Oral administration of D-pCons increases MLN Treg populations in preclinical BWF1 mice. However, oral L-pCons appears to delay SLE disease onset better than D-pCons. These studies could shed light on oral tolerance in SLE, as well as the development of a potential orally-based lupus therapy.
Migration of marginal zone precursor B cells into the T-cell zone promoted by interferon-alpha induces a T-dependent germinal center response (99.33)
Authors: Hsu Hui-Chen , Wang John , Wu Qi , Li Jun , Yang PingAr , Li Hao , Mountz John D
Published: January 2023

The immunopathogenic mechanisms by which IFNα triggers and promotes autoimmune B-cell responses have not been well characterized. In the spleens of autoimmune BXD2 mice, we found very high numbers of plasmacytoid dendritic cells (pDCs) in the marginal zone (MZ) that produce high levels IFNα. There is a larger population of MZ precursor (MZP) B cells, defined as being IgMhighCD21high and CD23high in BXD2 mice as compared to normal B6 mice. MZP B cells express high levels of ICOS ligand, CD80 and CD86, but low levels of ICOS. MZP B cells are extremely efficient antigen presenting cells (APCs) to induce Ag specific CD4 T cell activation more effectively compared to follicular (FO) and conventional CD23low MZ B cells in the spleens of BXD2 mice. IFNα induced higher levels of CD69 expression by MZP B cells and lead to greater suppression of the S1P induced chemotactic response by MZP B cells as compared to conventional MZ and FO B cells. Knockout of IFNαR in BXD2 mice abrogated the development of the spontaneous autoreactive GCs, and this is associated with significant reduction of IFNα-induced CD69 upregulation in MZP B cells and dramatic suppression of MZP migration into the T-cell zone following toll-like receptor 9 stimulation in vivo. Our results indicate that IFNα− producing pDCs that are localized primarily in the MZ of the spleen can promote a T dependent humoral response through their regulation of the migration of high APC function MZP B cells in the spleens of autoimmune BXD2 mice.
Impact of clinical and demographic features in influenza vaccination responses in human systemic lupus erythematosus (99.38)
Authors: Crowe Sherry R , Air Gillian M , Thompson Linda F , James Judith A
Published: January 2023

The goal of this study is to understand intrinsic and extrinsic defects in influenza vaccination responses in systemic lupus erythematosus (SLE) patients. SLE is a prototypic autoimmune disease characterized by high titers of autoantibodies. We enrolled 72 patients to evaluate anti-influenza antibody responses, autoantibody titers and disease activity after vaccination. In addition, detailed clinical and demographic information was collected. Individuals received the current influenza vaccine and sera were collected at baseline, 2, 6, and 12 weeks post vaccination. Based upon post-vaccination antibody responses, relative antibody affinities and hemaglutination inhibition levels, patients were grouped into "high" and "low" responders. Surprisingly, the high responder group was enriched in older subjects and African Americans. Patients with high anti-influenza antibodies had an increased frequency of discoid rash and anti-nRNP autoantibodies, while patients with low antibody responses had an increase in hematological criteria and anti-aPL autoantibodies. Interestingly, several patients had increased autoantibody titers following vaccination, but there were no significant associated increases in disease flare. In summary, specific demographic, clinical, and autoantibody features were associated with poor antibody responses to influenza vaccination in SLE patients. NIH: NO1-AI-50026
Enhanced selection of high affinity DNA-reactive B cells following cyclophosphamide treatment (99.40)
Authors: Jeganathan Venkatesh , Kawabata Daisuke , Ramanujam Meera , Davidson Anne , Grimaldi Christine M , Diamond Betty
Published: January 2023

A major goal for the treatment of SLE patients with cytotoxic therapies is the induction of long term remission. There is, however, a paucity of information concerning the effects of these therapies on the reconstituting B cell repertoire. Since there is recent evidence suggesting that B cell lymphopenia might attenuate negative selection of autoreactive B cells, we elected to investigate the effects cyclophosphamide (CY) on the selection of the re-emerging B cell repertoire in transgenic mice that express the H chain of an anti-DNA antibody (R4A-Tg).

The reconstituting B cell repertoire contained an increased frequency of mature, high affinity DNA-reactive B cells as determined by single cell PCR analysis, and the mice expressed increased levels of serum anti-DNA antibodies. This coincided with a significant increase in levels of BAFF, a B cell survival factor. Treatment with BAFF-R-Ig, a BAFF blocking agent or with DNase, to reduce exposure to autoantigen, limited the expansion of high affinity DNA-reactive B cells during B cell reconstitution. These studies suggest that during B cell reconstitution, negative selection of high affinity DNA-reactive B cells is impaired by increased BAFF. B cells escaping negative selection are positively selected by autoantigen. BAFF blockade following treatment with B cell depleting agents may be useful for the successful long-term remission of SLE.
Increased Expression of BCMA on Systemic Lupus Erythematosus B Cells is Associated with Enhanced Response to CpG (99.43)
Authors: Kim Jinoh , Gross Jane A , Dillon Stacey R , Elkon Keith B
Published: January 2023

BLyS and APRIL are members of the TNF superfamily that regulate B cell survival and autoreactivity, and can exist as homotrimers or BLyS/APRIL heterotrimers (HT). To further understand BLyS and APRIL function in SLE, we examined the expression profiles of their receptors (BAFF-R, TACI and BCMA) and their functional effects on B cell subsets. Whereas BAFF-R expression on SLE B cells was significantly lower compared to its levels on healthy control B cells (MFI of control vs SLE, naïve=1387 vs 929, p=0.002; memory=1290 vs 840, p=0.003), BCMA expression was substantially higher on SLE B cells (MFI of control vs SLE: 18 vs 28, p=0.038). This was most pronounced in the memory B cell subset (MFI of control vs SLE=19 vs 29, p=0.008) but was also evident on certain subsets of CD27- B cells. BCMA+ cells were mostly IgD+ and tended to be larger than BCMA-negative cells, and had higher CD19 and CD86 expression, indicating a greater degree of activation. FACS sorted BCMA+ B cells proliferated and differentiated much more potently than BCMA- B cells following CpG stimulation. CpG mainly induced the proliferation of BCMA+ B cells and increased BCMA expression on total B cells. These findings may help to explain the observed expansion of DNA-reactive SLE B cells in the presence of BLyS, APRIL or HT.
An inducible antigen system to investigate CD8 T cell tolerance (99.46)
Authors: Jellison Evan R , Turner Michael J , Lefrançois Leo
Published: January 2023

The induction of peripheral tolerance is critical for deterrence of autoimmunity. Most self-reactive T cells are deleted during development in the thymus. However, this deletion process is not absolute and self-reactive T cells can escape to the periphery. These self-reactive cells must either be deleted or regulated in the periphery to prevent autoimmunity. To study these processes, we generated mice in which antigen expression (the ovalbumin-derived peptide SIINFEKL) can be tightly controlled in small intestinal epithelial cells or dendritic cells by administration of doxycycline. Our system provides the unique ability to examine endogenous T cell responses to tissue-specific and developmentally regulated self antigen. Importantly, the transgenic mice are ignorant of antigen prior to induction. This result indicated that AIRE-mediated tolerance was not operating in our system. Antigen induction in either the small intestine or dendritic cells caused tissue-specific activation of transferred TCR transgenic OT-I T cells. Antigen induction also resulted in activation and expansion of endogenous SIINFEKL-specific CD8 T cells. Following antigen induction, an impairment of the SIINFEKL-specific response to infection with vesicular stomatitis virus expressing ovalbumin was observed, suggesting an antigen-specific regulation of the immune response. Characterization of immune responses in these antigen-inducible mice will provide key insights into mechanisms controlling tolerance versus autoimmunity in the intestinal mucosa. Supported by NIH grant DK57932
Kainate receptor activation may serve as a novel mechanism of B cell activation in autoimmunity (99.47)
Authors: Sturgill Jamie Lynn , Conrad Daniel H
Published: January 2023

Glutamate, the major excitatory neurotransmitter in the CNS, has recently been implicated in a variety of diseases. For example, it has been shown that patients with HIV, certain cancers, epilepsy, and autoimmune illnesses such as SLE all have elevated levels of glutamate in the periphery. While glutamate receptor signaling has been examined in T cells, there are currently no published observations on the effects of glutaminergic stimuli on B cells. Therefore, we performed a novel study to examine the effects of glutamate on B cells and thus humoral immunity. Both human B cell lines as well as primary B cells were first examined for the presence of glutamate receptors via RT-PCR and Western Blot. We identified the presence of the kainate receptor (KAR), a specific subtype of ionotropic glutamate receptor thought to be exclusively resident in the CNS. We found that KAR activation increased ADAM10 mRNA which is in agreement with receptor activity in the CNS. We also observed a significant corresponding increase in CD23 released into the supernatant as ADAM10 has been identified as the protease responsible for CD23's ectodomain shedding. Remarkably, we also detect a significant increase in cellular proliferation and Ig synthesis. Thus, we report for the first time the unique presence of a neurotransmitter receptor and that activation of this receptor could serve as a novel mechanism for B cell activation in autoimmunity.
Galectin-3 deficiency reduces the severity of experimental autoimmune encephalomyelitis (99.5)
Authors: Lukic Miodrag L , Jiang Hui-Rong , Al Rasebi Zakeya , Mensah-Brown Eric , Shahin Allen , Xu Damo , Fukada Sandra , Liu Fu-Tong , Liew Foo
Published: January 2023

Galectin-3 (Gal-3) is a member of β-galactoside-binding lectin family and plays an important role in inflammatory. However, the precise role of Gal-3 in autoimmune diseases remains obscure. We have investigated the functional role of Gal-3 in experimental autoimmune encephalomyelititis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. Gal-3 deficient (Gal-3-/-) mice developed significantly milder EAE and markedly reduced leukocyte infiltration in the CNS compared with similarly treated wild-type (WT) mice. Gal-3-/- mice also contained fewer monocytes and macrophages but more apoptotic cells in the CNS than did WT mice. Following Ag stimulation in vitro, lymph node cells from the immunized Gal-3-/- mice produced less IL-17 and IFN-γ than did those of the WT mice. In contrast, Gal-3-/- mice produced more serum IL-10, IL-5 and IL-13 and contained higher frequency of Foxp3+ regulatory T cells in the CNS than did the WT mice. Furthermore, bone marrow-derived dendritic cells from Gal-3-/- mice produced more IL-10 in response to LPS or bacterial lipoprotein than did WT marrow-derived dendritic cells. Moreover, Gal-3-/- dendritic cells induced AG-specific T cells to produce more IL-10, IL-5 and IL-12, but less IL-17 than did WT dendritic cells. Taken together, our data demonstrate that Gal-3 plays an important disease-exacerbating role in EAE through its multifunctions roles in preventing cell apoptosis and increasing IL-17 and IFN-γ synthesis, but decreasing IL-10 production.
Kinetic differences in the activation of CD4+ T cells during the initiation of tolerance and autoimmunity (99.50)
Authors: Katzman Shoshana , Villarino Alejandro , O'Gorman William , Nolan Gary , Abbas Abul
Published: January 2023

The initial encounter of a T cell with its antigen-presenting cell (APC) defines a major regulation point, relaying information to the responding T cell. We have assessed the interaction of T cells with APCs under conditions of tolerance and activation, by evaluating the response of auto-reactive CD4+ T cells upon recognition of self-antigen. In this model DO11.10 CD4+ T cells specific for OVA peptide (323-339) are transferred into transgenic mice, which express OVA as a systemically secreted soluble protein (sOVA-Tg). Previous studies in the lab have shown that in a "full" sOVA host, the auto-reactive T cells become tolerant, while in an "empty" or lymphopenic sOVA Rag-/- host, T cells respond with uncontrolled activation and host mice develop autoimmunity. Using 2-photon microscopy we have evaluated the velocity and movement during the initial encounter of T cells with APCs under conitions of tolerance or activation. Our data suggests that under both conditions the antigen specific cells initially stop on APCs, this interaction is retained in sOVARag-/- (autoimmune) mice but lost in sOVA (tolerant) hosts. In addition, the initial activation through the T cell receptor, as determined by pS6 staining, is comparable during early activation. This signal is retained in sOVARag-/- hosts but in contrast rapidly declines in sOVA hosts. These data show that the loss of early activation may define the tolerant phenotype. Understanding the interactions of self-reactive T cells with APCs presenting self-antigen is key to understanding the induction of tolerance vs. immunity and how this balance is maintained is integral for the design of therapies in the future. Research Support-NIH (F32)
Skin Tumor Responsiveness to IL-2 Treatment Correlates to CD8 Treg Expansion in an Immunocompetent Mouse Model. (100.15)
Authors: Foureau David , McKillop Iain , Jones Chase , Amin Asim , White Richard , Salo Jonathan
Published: January 2023

CD8 Tregs play an important role in graft tolerance and autoimmune disease status. Interleukin-2 (IL-2) induces Foxp3 expression by activated human CD8 T cells in vitro, and expands circulating CD8 Foxp3+ T cells in melanoma patients. IL-2 is FDA approved for the treatment of advanced melanoma, yet positive outcomes are limited. The aim of this study was to determine the phenotype and distribution of CD8 Foxp3 expressing T cells in skin tumor-bearing mice receiving IL-2. In normal or skin tumor-bearing mice, Foxp3 expressing CD8 T cells were a rare but naturally occurring cell subset. Primarily located in skin-draining lymph nodes CD8 Foxp3+ T cell expressed both activated CTL (CD28+, CD44+) and Treg (CTLA4+, PD1lo, NKG2Alo) markers. Following high dose IL-2 treatment, a dramatic increase in CD8 Treg prevalence was observed in a subcutaneous-tumor model that is non-responsive to IL-2 treatment (B16-F10 melanoma cell line). Furthermore, distribution analysis in this (B16-F10) model demonstrated increased CD8 Treg expression in the circulation and tumor-draining lymph nodes. Conversely, in the IL-2 responsive MCA-205 fibrosarcoma-bearing mouse model no significant changes in CD8 Treg were measured, either in the bloodstream or within the tumor-draining lymph nodes. In conclusion, this study characterized CD8 Foxp3+ T cells (CD8 Treg) as a new immunosuppressive cell subset, the expansion of which was associated with skin tumor that failed to respond to IL-2 treatment.
Mechanical disruption of the primary tumor using magnetic beads inhibits metastasis of breast cancer. (100.24)
Authors: Bouchlaka Myriam , Wilkins Danice , Fuchs Alan , Evrensel Cahit , Murphy William
Published: January 2023

Successful anti-tumor effects are weakened by removal of the tumor antigen pool (i.e. surgery) or use of cytoreductive and immunosuppressive therapies (i.e. chemotherapy and radiation). We hypothesize that mechanical disruption of the tumor in situ will augment immune responses due to antigen release. Iron particles were injected as magneto-rheological fluid (MRF) into an orthotopic primary breast cancer and followed by application of a magnetic field to exert force and shear stress on the tumor. This will result in disruption of the tumor’s architecture and induction of classical “danger signals”: necrotic death, release of tumor antigen with activation of a localized immune response. To promote systemic immune responses we combined MRF treatments with immunotherapy (IT) at suboptimal dosages. Tumors treated with daily application of magnetic field showed increased necrotic cell death, recruitment of activated DCs (i.e. MHC II+, CD83+) to the draining LNs with production of proinflammatory cytokines/chemokines. We then combined MRF treatment with IT to determine if systemic anti-tumor effects could be achieved. Not only have we observed suppression of tumor growth on the contralateral side treated with MRF and IT but also we saw absence of metastasis to the bone marrow of animals treated with either MRF and IT or MRF and magnetic field alone. These data suggest that MRF and magnetic field application to the primary tumor synergize with immunotherapy in disseminated cancer.
Identification of an inhibitory ganglioside responsible for immune suppressive effects associated with ovarian cancer (100.27)
Authors: Oelke Mathias , Webb Tonya J. , Giuntoli, II Robert L. , Lopez Pablo H.H. , Li Xiangming , Schnaar Ronald , Tsuji Moriya , Schneck Jonathan
Published: January 2023

Tumors often use mechanisms to prevent or suppress immune recognition. One such mechanism is the shedding of immune-suppressive glycolipids into the local microenvironment. Previously we reported that pretreatment of CD1d expressing stimulator cells with ascites fluid from ovarian cancer patients inhibited the activation of NKT cells, one of the earliest lines of defense in an anti-cancer immune response. Here we identify the inhibitory factor responsible, ganglioside GD3, which we isolated from the polar lipid fraction of ovarian cancer-associated ascites. GD3 inhibited CD1d-mediated NKT cell activation directly, without antigen processing. Purified GD3 binds with high affinity to CD1d, even when compared to the well-established experimental CD1d activator, α-galactosylceramide. Mechanistically we show that factors in ascites fluid as well as purified GD3 bind directly to the CD1 antigenic binding site, and this presumably displaces endogenous NKT cell ligands. These data indicate that shedding of GD3 by ovarian cancer tumors is a mechanism of immune evasion that inhibits the activation of an anti-tumor NKT cell response.
Inflammation-Induced Myeloid-Derived Suppressor Cells have enhanced resistance to apoptosis. (100.31)
Authors: Chornoguz Olesya , Grmail Lydia , Sinha Pratima , Artemenko Konstantin , Zubarev Roman , Ostrand-Rosenberg Suzanne
Published: January 2023

The purpose of this study is to deduce the influence of inflammation on apoptosis of Myeloid-Derived Suppressor cells. Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression. MDSC induced in heightened inflammatory environments with high levels of IL-1β and/or IL-6 accumulate more rapidly, are more potent suppressors of T cell activation, produce more IL-10, and have a greater ability to down-regulate macrophage production of IL-12, as compared to MDSC that develop in environments with lower levels of these cytokines. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory vs. less inflammatory settings. Proteomic pathway analysis identified Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by down-regulating Fas-mediated MDSC apoptosis. Using cytochrome C and activated caspase 3 and caspase 8 as indicators of apoptosis, confocal microscopy and flow cytometry demonstrated that inflammation-induced MDSC have enhanced resistance to Fas-mediated apoptosis. These results suggest that an inflammatory environment protects MDSC against extrinsic-induced apoptosis resulting in MDSC with a longer in vivo half-life and may explain why MDSC accumulate more rapidly and to higher levels in inflammatory settings.
Coordination of hepatic and myeloid signal transducer and activator of transcription 3 in governing liver carcinogenesis and regeneration (100.36)
Authors: Wang Hua , Park Ogyi , Lafdil Fouad , Yin Shi , Horiguchi Norio , Gao Bin
Published: January 2023

Activation of signal transducer and activator of transcription 3 (STAT3) occurs virtually in most liver diseases, but how STAT3 links liver regeneration and cancer remains elusive. Using cell-specific STAT3 deletions mouse strains, we have investigated the interplay of myeloid and hepatic STAT3 in controlling liver regeneration induced by partial hepatectomy (PHx) and hepatocarcinogenesis induced by diethylinitrosamine (DEN). We show that in DEN model hepatic STAT3 plays an important role in mediating a cytoprotective action and enhancing tumor growth, while myeloid STAT3 also significantly affects tumor growth via its anti-inflammatory effect. Furthermore, STAT3 is activated in myeloid cells after PHx and its conditional deletion leads to enhanced innate inflammatory response and liver regeneration. Conditional deletion of STAT3 in both hepatocytes and myeloid cells results in strong elevated inflammatory response, activation of STAT1, apoptosis of hepatocytes and a dramatic reduction in survival after PHx, whereas additional global deletion of STAT1 protects against these effects. These data show that the interplay of myeloid and hepatic STAT3 signaling is essential to prevent acute liver failure during liver regeneration through tempering a strong innate inflammatory response mediated by STAT1 signaling. Secondly, orchestral crosstalk between hepatic and myeloid STAT3 enables survival of mutagenized cells and subsequent tumor promotion in DEN-induced hepatocarcinogenesis.
Role of human lymphocyte activation gene 3 in tumor infiltrating lymphocytes (101.12)
Authors: Bruno Tullia , Noonan Kimberly , Grosso Joseph , Durham Nicholas , Goldberg Monica , Getnet Derese , Yen Hung Rong , Pyle Kristin , Borrello Ivan , Pardoll Drew , Drake Charles
Published: January 2023

Among the various molecules that regulate T cell function, lymphocyte activation gene 3 (LAG- 3) has garnered significant interest. LAG-3 is expressed on activated T cells, B cells, NK cells, tumor infiltrating lymphocytes (TILs), and plasmacytoid dendritic cells. We previously showed that LAG-3 was relatively over-expressed on HA-specific transgenic T cells rendered anergic in vivo by encounter with cognate self antigen. In this system, regulatory activity could be functionally blocked with a LAG-3 specific monoclonal antibody (Huang et al). Observations in our lab using LAG-3 knockout mice demonstrate that CD8 T cells undergo enhanced homeostatic proliferation in vivo if LAG-3 is absent. Currently, we are conducting studies to understand the role of LAG-3 in human cancer. Via microarray, we compared CD4+25+GITR+ (Treg) T cells from the prostate to CD4+25-45RA+ (naïve) T cells from the peripheral blood. We found LAG-3 and CTLA-4 to be relatively upregulated in prostate infiltrating T regulatory cells. Also, at the expression level, we observed variable levels of LAG-3 expression on patient CD8 tumor infiltrating T lymphocytes (TILs). At a functional level, we have shown that a human anti-human LAG-3 antibody enhances T cell proliferation and cytokine function in a mixed lymphocyte reaction. Further studies that are currently underway suggest that LAG-3 could be a promising candidate for enhancing anti-tumor immunotherapy in a clinical setting.
Beta7 integrin deficiency suppress cutaneous tumor formation (101.15)
Authors: Kai Hiromichi , Kadono Takafumi , Ohmatsu Hanako , Tomita Manabu , Asano Yoshihide , Sugaya Makoto , Sato Shinichi
Published: January 2023

Beta7 integrin, a cell adhesion molecule, associates either with the alpha4 or with the alphaE integrin subunit. Alpha4beta7 integrin is expressed on most leucocytes and is essential for their migration to gut-associated lymphoid tissues by interacting with its primary ligand, MAdCAM-1. Although involvement of beta7 integrin in intestinal immunity is well-established, little has been known about the engagements of beta7 integrin in cutaneous immune responses, especially cutaneous anti-tumor immunity. In this study, we injected B16 murine melanoma cells into the skin superficial to the ear cartilage of beta7 integrin-deficient mice. The tumor formation in the ears from beta7 integrin-deficient mice were reduced compared to that from C57BL/6 mice. Local cytokine mRNA expression levels were measured by real-time PCR in the ear tumors, and IFN-gamma mRNA levels tended to increase in beta7 integrin-deficient mice compared with wild-type mice. To evaluate leukocyte subsets in the tumors, immunohistochemical analysis of the ear tumor was performed. Numbers of infiltrating CD4+Tcells per unit area were decreased in beta7 integrin-deficient mice compared with wild-type mice. Overall, these results indicate an important role of beta7 integrin in tumor immunity.
Comparative screening of human antibody libraries derived from autoimmune vs. cancer patients for affinity reagents directed against tumor-associated antigens (101.20)
Authors: Zhao Aizhi , Cheng Yi , Coukos George , Siegel Don , Scholler Nathalie
Published: January 2023

In our previous study we showed that scFv against tumor vasculature marker could be isolated from a yeast-display scFv library derived from an autoimmune patient (TTP). However, scFv library derived from cancer patients may yield more diverse and higher affinity reagents against tumor-associated markers. To address this question, we constructed a yeast display scFv library from PBMC and ascites B cells of 5 ovarian cancer (OC) patients. To clone the antibody repertoires into the yeast display vector, we designed a new set of primers capable of amplifying all antibody sub-families and we optimized a three-fragment gap repair method using VH, VL and linearized vector. While scFv assemblage by overlap PCR was not performing well, the recombination of VH, VL and linearized vector in yeast yielded 1x108 transformant per electroporation. Sequencing of 50 clones demonstrated that all of them contained different full-length scFv inserts. We are currently screening the OC scFv library with human TEM1 protein, to compare the frequency and affinity of the scFv against tumor markers when retrieved from OC scFv library vs. TTP scFv library. In conclusion, our findings so far indicate that three-fragment gap repair is a valid and cost-effective strategy to construct large yeast-display libraries. We anticipate that the comparative screening of the TTP and OC libraries using tumor markers may give indications about the repertoire and anti-tumoral specificity of cancer patient antibodies.
In vivo impact of proteasome- and TAP-dependent MHC class II processing. (130.12)
Authors: Robinson Tara , Eisenlohr Laurence
Published: January 2023

In order to be recognized by CD4+ T cells, viral protein antigens must be degraded intracellularly and resultant peptide fragments loaded onto MHC class II molecules. Classically, class II antigen processing is thought to occur via endosomal proteases and loaded onto MHC molecules in a late endosomal compartment with the assistance of the chaperone molecule H2-M. Previously, our lab has demonstrated that the H2-IEd-restricted epitope NA79-93 of the influenza A/PR/8/34 neuraminidase protein must be degraded by the proteasome and requires the presence of TAP- both molecules previously associated exclusively with the MHC class I pathway. Furthermore, this pathway is largely inaccessible when the source of viral antigen is an inactivated, non-replicating virus. A substantial fraction of the CD4 T cell response is directed towards epitopes processed via this endogenous pathway. In the current study we extend this work to examine how endogenously processed epitopes 1) affect T cell priming, and 2) contribute to providing protection from viral challenge. Differences in CD4 T cell function and the restricted repertoire elicited in the absence of endogenously processed class II epitopes contribute to differences noted after adoptive transfer and challenge with influenza. These results may provide important insights into rational vaccine design.