bluuebunny/crossref_metadata_2025 huggingface dataset:
MicroRNAs mediate autoimmune disease pathology (137.3)
Authors: Smith Kristen M. , Liu Chang-gong , Taccioli Cristian , Williams Jessica , Kithcart Aaron , Mavrikis Gina , Shawler Todd , Whitacre Caroline C.
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE), studied as a model for multiple sclerosis (MS), is mediated by autoreactive CD4+ T cells. MicroRNAs (miRNAs) are a class of small, noncoding RNAs that regulate gene expression posttranscriptionally. Aberrant expression of miRNAs has been implicated in various disease processes. Recent evidence indicates that miRNAs are central to immune function and play a role in T cell responsiveness to antigen stimulation. We hypothesized that certain miRNAs would be differentially expressed in mice with EAE, and that these miRNAs represent critical mediators of autoimmune disease pathology. We utilized microarray technology, followed by PCR verification, to evaluate miRNA expression level at varying times during the EAE disease course. RNA was obtained from the lymph nodes and spleen during the acute and effector phases of EAE, and compared to animals receiving adjuvant alone. We found that several miRNAs are up- or downregulated during EAE and expression is temporally distinct. These data indicate that miRNAs represent a critical component of EAE pathology, and may be useful therapeutic targets or biomarkers in MS and other autoimmune diseases. Supported by NIH grants T32 GM068412, NS 48316 and AI 064320.
Quantitative and functional gender-biased differences in regulatory and CD8+ suppressor T cells in SLE patients (137.35)
Authors: Singh Ram P , Christina Charles , Grossman Jannifer , Taylor Mihaela , Mccurdy Deborah , Furst Daniel , Dinesh Ravi , Ranganath Veena , Louie James , Gupta Smita Rani , Grewal Iqbal , McEarchern Julie , La cava Antonio , Bevra Hahn
Published: January 2023

The goal of the present study was to study the quantity and function of regulatory and suppressor T cells, and the effect of sex hormones on T reg functions in healthy individuals and in SLE patients of both genders. Immunophenotyping of PBMC from SLE patients (n =27) indicated significantly reduced numbers of CD4+CD25hi Foxp3+ T cells (p<0.0002) and CD8+Foxp3+ suppressor T cells (p<0.005) as compared to healthy matched controls (n =15) and autoimmune disease controls RA patients (n=25). Moreover, both CD4+CD25hi and CD8+CD25hi subsets of males had 3-4-fold higher Foxp3 mRNA compared to females. The in vitro proliferative potential of CD4+T regulatory cells and CD8+T suppressor cells were defective in CFSE staining experiments in SLE patients as compared to the healthy controls (p<0.001). Foxp3 expression was higher in males than in female PBMC with and without stimulation of with recombinant TGFb (p<0.06). In regard to the role of sex hormones, stimulation of PBMCs with b estradiol (30pg/ml) decreases Foxp3 expression in healthy female but not in age matched healthy male. At higher doses (60, 150pg/ml) estrogen has little effect in both the sexes. Estrogen decreases Foxp3 mRNA expression in both female and male SLE patients (p<0.05). Estrogen decreases Foxp3 protein expression in CD4+CD25- T cells from SLE patients.

An Inhibitor of bestradiol (ERa) increased apoptosis in male SLE patients only. These data suggest that estrogen affect the T regulatory compartment in healthy females and and further decreases Foxp3 mRNA in SLE patients of both genders.
Immunophenotypic Characterization of Th17 Cells in Human Peripheral Blood (137.38)
Authors: Li Linda , Yang Xifeng , Chen Benjamin Pin-Wen , Struss Peter E. , Perna Avi , Wei Bing-Yuan , Jiang Nan , Kraft Brad , Lay Gene
Published: January 2023

IL-17-secreting T cells represent a distinct lineage of CD4+ T effector cells (Th17) that is essential in the pathogenesis of numerous inflammatory and autoimmune diseases. Although the functionality of Th17 cells has been extensively studied, the phenotypic characteristics are still not well understood. In this study, we investigated the surface marker expression patterns of the Th17 cells. Human peripheral blood mononuclear cells (from healthy donors) were stimulated with PMA/Ionomycin (in the presence of monensin). The cells were then harvested and multi-color immunofluorescent staining was performed with a combination of fluorochrome-conjugated monoclonal antibodies to CD4, IL-17, and a panel of cell surface markers and analyzed by flow cytometry. Our results show that about 0.2% to 0.6% of CD4+ cells in healthy individuals produce IL-17. The CD4+ IL-17+ cells express high levels of integrin αL, α4, α6, β1 and β2, but not αM, αX and α2. All of the Th17 cells have high expression of CD45RO, CD50, CD58, CD95, CD150, CD229 and negative expression of CD25 and CD45RA. In addition, the Th17 cells do not express CD38 and its counter receptor CD31. We then further analyzed chemokine receptors expression on Th17 cells, and found that the Th17 cells express CCR6 (which is the same as reported by others), CCR7 and CXCR4, but not CCR1, CCR4, CXCR3 and CXCR6. More interestingly, IL-17+ cells can be divided into two subpopulations by CD27 (CD27+ Th17 and CD27- Th17). The functional differences of these Th17 subsets remain to be elucidated.
Oxazolone-Induced Delayed Type Hypersensitivity (DTH) Reaction in Adult Yucatan pigs: Validation of the Model with Cyclosporine A (CsA) (137.39)
Authors: nuhaily Samer , Yei Soonpin , Lopez Carlos , Incardona Francesca , Esber Henry J , Damaj Bassam B
Published: January 2023

DTH reactions are an important arm of the immune system and their deregulation results in several auto-immune and inflammatory diseases. Although rodents are widely used for the validation of novel anti-inflammatory drugs, it represents a challenge since their immune system is much less diverse and complex then the human immune system. We describe here and validate the first DTH swine model induced by Oxazolone using CsA. The results show a dramatic decrease in the ear inflammation in CsA treated pigs. In particular, this drug reduced the redness, inhibited the accumulation of ear fluid and inflammatory cells, as well as the accumulation and/or the release of the inflammatory mediators, CRP, IL-2 and IL-8 in the inflamed ears. Additionally, dosing with CsA significantly inhibited the levels of CRP, IL-2 and IL-8 in the sera of sensitized pigs, and also inhibited the in vitro proliferation of T cells collected from their spleens or PBMCs, particularly when these cells were stimulated in vitro with PMA plus ionomycin. Furthermore, CsA also inhibited the in vitro IL-2 released from PBMCs or splenocytes with or without stimulation with PMA plus ionomycin. Although CsA tended to reduce the ear thickness developed in mice and rats, this reduction did not reach significance, suggesting that pigs represent a better and more suitable model for screening the anti-inflammatory activity of compounds.
Anti-CD3 antibody ameliorates experimental autoimmune uveitis (EAU) by increase in IL-10 and TGF-1-secreting regulatory T cells and down-regulation of both Th1 and Th17 T cells (137.41)
Authors: KE YAN , Jiang Guomin , Bian Yang , Wang Yali , Kaplan Henry J. , Shao Hui
Published: January 2023

One of the major goals for the immunotherapy of autoimmune diseases is the induction of regulatory T cells that mediate immunologic tolerance. In this study, we wanted to determine whether treatment of anti-CD3 mAb could suppress EAU by up-regulating regulatory T cells. EAU was induced in B10RIII by immunization with IRBP161-180 and anti-CD3 mAb was injected at various doses before immunization or at the disease onset. We have found that anti-CD3 Abs significantly suppressed the severity of the disease when administered both prior to disease induction and at disease onset. Ab treatment selectively suppressed the activation and expansion of pathogenic T cells, but enhanced division and functions of regulatory T cells in vivo, since the frequency of regulatory cells of both CD1d-restricted natural killer T (iNKT) cells and CD4+Foxp3+ T cells was increased in CD3mAb treated mice. The inhibition of pathogenic T effector cells might be mediated directly by anti-CD3 mAb and indirectly by regulatory T cells. By releasing TGF-¦ Â and IL-10, regulatory T cells inhibited differentiation of Th17 and Th1 cells by antagonizing ROR¦Ã T and T-bet function. Moreover, Disease suppression also occurred following adoptive transfer of T cells from anti-CD3-treated animals to naïve mice immunized with IRBP161-180. Our results demonstrate that anti-CD3 Ab is potent inhibitor for established autoimmune uveitis.
Autoantibody-mediated regulation on B cell responses by functional anti-CD22 autoantibodies in patients with systemic sclerosis (137.42)
Authors: Fujimoto Manabu , Odaka Maki , Hasegawa Minoru , Takehara Kazuhiko
Published: January 2023

Objectives. Studies have demonstrated that B cells play important roles in systemic sclerosis (SSc), especially through the gCD19/CD22 autoimmune loop h. In this study, we examined the presence and functional property of circulating autoantibodies reacting with CD22.

Methods. Serum samples from 10 tight skin (TSK/+) mice and 50 SSc patients were assessed for anti-CD22 autoantibodies by enzyme-linked immunosorbent assays using recombinant mouse or human CD22. The association between anti-CD22 antibodies and clinical features was also investigated in SSc patients. Furthermore, the influence of SSc serum including anti-CD22 autoantibodies for CD22 tyrosine phosphorylation was examined by western blotting using phospho-tyrosine specific antibodies reacting with four major tyrosine motifs of CD22 cytoplasmic domain.

Results. Anti-CD22 autoantibodies were positive in 80% of TSK/+ mice and in 22% of SSc patients. Patients positive for anti-CD22 antibodies showed significantly higher modified Rodnan skin thickness score compared with patients negative for anti-CD22 antibodies. Furthermore, anti-CD22 antibodies from patients' sera were capable of reducing phosphorylation of all four CD22 tyrosine motifs, while sera negative for anti-CD22 antibodies did not affect CD22 phosphorylation.

Conclusions. A subset of SSc patients possessed autoantibodies reacting with a major inhibitory B cell response regulator, CD22. Since these antibodies can interfere CD22-mediated suppression onto B cell activation in vitro, SSc B cells produce functional autoantibodies that can enhance their own activation. This unique regulation may contribute to the autoimmune aspect of SSc.
Frequency of acetylcholine receptor specific B cells correlates with experimental myasthenia gravis severity (137.5)
Authors: Allman Windy R. , Saini Shamsher S. , Christadoss Premkumar
Published: January 2023

Myasthenia gravis (MG) is a condition in which neuromuscular transmission is impaired by antibodies (Abs) specific for muscle acetylcholine receptor (AChR). Anti-AChR Abs can be detected in the serum of patients with MG, but serum anti-AChR Abs level do not correlate to disease severity. In this study, we developed a flow cytometric assay for the detection of peripheral blood AChR-specific B cells to characterize B cell phenotypes associated with experimental autoimmune myasthenia gravis (EAMG). Alexa-conjugated AChR was used as a probe for AChR-specific B cells. B cells were characterized as B220+MHCII+. Female mice had significantly more B cells than males. However, there were no differences in the frequencies of antigen-specific AChR+ B cells between male and female mice with EAMG. Mice with EAMG have significantly elevated frequencies of AChR+IgG2+ B cells but not AChR+IgM+ B cells compared to healthy controls. AChR+IgG2+ B cells have significant correlations to clinical grade of disease and loss of limb muscle strength. Now we can identify the frequency of AChR specific B cells in MG which could be used as a marker for disease severity. This research has been supported by AFM and MGFA.
Novel mouse NK progenitors in lymph node: developmental origin and functional contributions. (138.1)
Authors: Luther Claudia , Senger Laura , Takei Fumio
Published: January 2023

Natural killer (NK) cells are widely distributed in various tissues. Among them, lymph node (LN) NK cells have been shown to differ from spleen NK cells in their phenotype and function. About ~20% of LN NK cells have rearranged TCRγ genes, express CD127+ (IL-7Rα), and they are thought to derive from the thymus. However, LN NK cells from nude mice are normal in number and phenotype, suggesting that the majority of LN NK cells are thymus-independent. We have recently found a novel cell population in the LN of C57/BL6 mice that is lineage (LIN)-negative but expresses the pan NK cell marker CD49b (DX5). LIN-CD49b+ cells are also found in the bone marrow (BM), but they differ from the LN counterparts. BM LIN-CD49b+ cells are Sca-1locKithiCD127- and include common lymphoid progenitors and other immature hematopoietic progenitors whereas LN LIN-CD49b+ cells are Sca-1+cKit-CD127+. When cultured on OP9 stroma cells, LN LIN-CD49b+ cells acquire the NK cells markers NK1.1, CD122 and Ly49 as well as cytotoxic function and IFN-γ production. They also retain CD127 expression. The precursor frequency of NK progenitors among LIN- CD49b+ cells in LN and BM are 1/40 and 1/13, respectively. Thus, LN LIN- CD49b+ cells seem to represent a novel NK progenitor that migrates from the BM to the LN for final differentiation into unique subsets of mature NK cells.

This work is supported by a grant from the German Research Council (DFG) and the National Cancer Institute of Canada.
Identification of the branchpoint between B and T cell development through MiDReG (138.17)
Authors: Inlay Matthew A. , Bhattacharya Deepta , Sahoo Debashis , Seita Jun , Karsunky Holger , Serwold Tom , Plevritis Sylvia , Dill David , Weissman Irving L.
Published: January 2023

Early hematopoietic development proceeds in a stepwise manner through progenitor intermediates. One such intermediate, the common lymphoid progenitor (CLP), can give rise to all lymphoid but not myeloid lineages in vivo, but has been suggested to preferentially give rise to B cells. Thus it remains unclear whether CLPs represent a node where lymphoid fate decisions are made, an intermediate en route to B cell commitment, or a mixture of the two. To determine if the CLP population contains both B-committed and uncommitted lymphoid progenitors, we used a novel algorithm called MiDReG (Mining Developmentally Regulated Genes) on over 2000 publicly-available mouse expression arrays to predict surface proteins with differential expression during the transition from fully multipotent progenitors to committed B cells. One of these markers bisects CLPs into two distinct populations, which we call CLPa and CLPb. Upon adoptive transfer, CLPa have robust B, T, NK, and DC output, while CLPb produce almost exclusively B cells. Thus, this surface protein marks the earliest identified step at which B cell commitment is established in vivo, with CLPa residing at the branchpoint where the B cell lineage separates from the other lymphoid fates. These results highlight the utility of MiDReG to identify novel stages of hematopoietic development. M.I. is funded by a fellowship from CIRM.
Role of stem cell factor and bone marrow derived fibroblasts in airway remodeling. (140.1)
Authors: Dolgachev Vladislav , Ullenbruch Matthew R , Lukacs Nicholas W , Phan Sem H
Published: January 2023

Recent evidence suggests involvement of bone marrow derived fibroblasts in airway remodeling in asthma, but their role and the mechanism of recruitment remain unclear. Stem cell factor (SCF), a key factor in the propagation of hematopoietic stem cells, is important in airway remodeling. To test the hypothesis that SCF is involved in the recruitment and differentiation of these bone marrow derived progenitor cells, GFP bone marrow chimera mice were created. These mice were then sensitized and chronically challenged with cockroach antigen (CRA) to induce chronic airway disease. Fluorescence microscopy revealed the influx of significant numbers of GFP expressing fibroblasts in these airways, which was confirmed by flow cytometric analysis for cells co-expressing GFP and collagen I. These cells preferentially expressed c-kit, IL-31 receptor and telomerase reverse transcriptase when compared to lung-derived fibroblasts. Interestingly, SCF stimulated IL-31 receptor expression in bone marrow cells, while IL-31 strongly induced telomerase reverse transcriptase expression in fibroblasts. Treatment with neutralizing antibodies to SCF significantly reduced airway remodeling and suppressed recruitment of these bone marrow-derived cells to the lung. Thus SCF in conjunction with IL-31 may play a significant role in airway remodeling by promoting the recruitment of bone marrow-derived fibroblast precursors into the lung with the capacity to promote lung myofibroblast differentiation.
A new compound isolated from Vitex rotundifolia Linn fil., pyranopyran-1,8-dione, suppresses airway epithelial cell inflammatory responses in a murine model of asthma (140.14)
Authors: Lee Heekyung , Keum Dongjune , Kim Haejeong , Shin Min-Kyu , Hong Moo-Chang , Kim Yangseok , Bae Hyunsu
Published: January 2023

The new natural compound, pyranopyran-1,8-dione (PPD), was isolated from Vitex rotundifolia L. To elucidate the mechanism by which the anti-asthmatic responses of PPD occurred in vitro, A549 cells were stimulated with TNF-¥á, IL-4 and IL-1¥â to induce the expression of chemokines and adhesion molecules involved in eosinophil chemotaxis. PPD treatments reduced the expression of eotaxin, IL-8, IL-16 and VCAM-1 mRNA significantly. Additionally, PPD reduced eotaxin secretion and significantly inhibited eosinophil migration toward A549 medium. In addition, PPD treatment suppressed the phosphorylation of p65 and ERK1/2, suggesting that it can inhibit the MAPK/NF-¥ê b pathway. To clarify the anti-inflammatory and antiasthmatic effects of PPD in vivo, mice were sensitized and challenged with OVA and then examined for the following typical asthmatic reactions: an increase in the number of eosinophils in BALF; the presence of Th2 cytokines such as IL-4 and IL-5 in the BALF; the presence of allergen-specific IgE in the serum; and a marked influx of inflammatory cells into the lung. Taken together, our results revealed that PPD exerts profound inhibitory effects on the accumulation of eosinophils into the airways while reducing the levels of IL-4, IL-5, and IL-13 in the BALF.
Anti-Malarial Drug Artesunate Attenuates Allergic Airway Inflammation in A Murine Asthma Model (140.18)
Authors: Cheng Chang , Ho Eugene WX , Chu Kai Ling , Wong WS Fred
Published: January 2023

Artesunate is a derivative of artemisinin isolated from a Chinese herb Artemisia annua L. It is used clinically for the treatment of malaria. There are recent evidences supporting an immune-regulatory role by artesunate. We investigated potential anti-inflammatory actions of artesunate in a mouse asthma model. BALB/c mice sensitized and challenged with OVA developed airway eosinophilia and mucus hypersecretion, and elevations in serum IgE level and in bronchoalveolar lavage (BAL) fluid cytokine level. Artesunate significantly inhibited OVA-induced increases in BAL fluid eosinophil count, and IL-5 and IL-13 levels. It also reduced the serum level of OVA-specific IgE. Histologic analysis reveals that artesunate suppressed OVA-induced inflammatory cell infiltration and airway mucus production. Besides, artesunate markedly attenuated OVA-induced mRNA expression of ICAM-1, VCAM-1, Muc5ac and chitinases. Furthermore, artesunate inhibited TNF-α-induced phosphorylation of phosphoinositide 3-kinase (PI3K) and its downstream Akt and p70 S6 kinase, as well as nuclear factor (NF)-κ B DNA binding activity in normal human bronchial epithelial cells. Taken together, our results clearly demonstrate anti-inflammatory effects of artesunate in a mouse asthma model, and it may act by inhibiting PI3K/Akt signaling pathway. (This work was supported by a BioMedical Research Council grant BMRC06/1/21/19/443)
Reduction of serum IgE by depletion of the IgE B cell lineage - a novel approach for the treatment of allergic diseases (140.7)
Authors: Wu Lawren C , Brightbill Hans D , Jeet Surinder , Lin Zhonghua , Yan Donghong , Zhou Meijuan , Tan Martha , Nguyen Allen , Yeh Sherry , Delarosa Donnie , Leong Steven R , Wong Terence , Chen Yvonne , Jackman Janet , Dennis Mark S , Chuntharapai Anan , DeForge Laura , Meng Y Gloria , Xu Min , Lee Wyne P , Refino Canio J , Balazs Mercedesz
Published: January 2023

Anti-IgE antibody targeting of serum IgE is an efficacious treatment for asthma and other allergic diseases. Currently, therapeutic anti-IgE antibodies neutralize free serum IgE, but do not affect IgE production. We have developed monoclonal antibodies that disrupt IgE production by binding to the M1' domain of human membrane IgE, a 52 amino acid extracellular segment on human IgE-switched B cells and plasmablasts that is not present in serum IgE. Using genetically modified mice that contain the human M1' domain knocked into the mouse IgE locus, we demonstrate that our anti-M1' antibodies reduce serum IgE and IgE plasma cells, but not other immunoglobulin isotypes, in vivo. Anti-M1' antibodies are effective when delivered prophylactically and therapeutically in mouse immunization, asthma, and Nippostrongylus infection models. Moreover, the anti-M1' antibodies are active on primary human cells in vivo, reducing serum IgE and IgE plasma cells in a human PBMC-SCID mouse model. In vitro and in vivo studies indicate that anti-M1' antibodies trigger apoptosis of IgE B cells and may therefore abolish IgE memory. Anti-M1' targeting of IgE B cells is a promising new therapy for the treatment of asthma and allergy.
Fullerene C70 derivatives inhibit eosinophilia associated with chronic asthma (140.8)
Authors: Norton Sarah K , Conrad Daniel H , Kepley Christopher L
Published: January 2023

Fullerenes are nanospheres of carbon that are extremely stable and highly reactive with oxygen free radicals. While native fullerenes are insoluble in water, the carbon cage can be derivatized by the addition of polar chemical groups, thus generating water soluble molecules. Water soluble fullerene derivatives have been shown to inhibit mast cell degranulation and cytokine release (J. Immunol. 179: 665-672), demonstrating a potential role in controlling mast cell diseases such as allergic asthma. In order to observe the effects of derivatized fullerenes on chronic features of asthma in vivo, we used a murine model of asthma pathogenesis. Mice were sensitized to ovalbumin by intraperitoneal injection and then challenged by directly introducing the protein into the lungs. Fullerene treated mice did not exhibit any acute toxicity, and when compared to PBS or vehicle treated controls, showed a significant reduction in airway eosinophilia. The data also suggest a reduction of IL-4, IL-5, and KC in the bronchoalveolar lavage fluid of fullerene treated mice, but further investigation is necessary. Finally, we are investigating the effect of fullerenes on B cell IgE production in vitro as a possible mechanism of inhibition. Together, these results suggest that fullerenes can control chronic inflammation characteristic of asthma pathogenesis, indicating the potential for their use in the treatment of chronic asthma.
The T Cell Cytolytic Molecules FasL and TRAIL are Required for Thymic GVHD (141.14)
Authors: Lu Sydney X , Na Il-Kang , Goldberg Gabrielle L , King Christopher G , Smith Marsinay , Suh David , Rao Uttam , Yim Nury , Holland Amanda M , Penack Olaf , Jenq Robert R , Ghosh Arnab , Houghton Alan , van den Brink Marcel RM
Published: January 2023

Thymic graft-versus-host-disease (tGVHD) is an important contributor to impaired T cell reconstitution in patients with GVHD. We analyzed molecules relevant for T cell trafficking and cytolytic function in tGVHD.

Thymic output and cellularity were inversely proportional to numbers of mature donor T cells in the allograft, suggesting that tGVHD severity was inversely associated with thymic function. Upon adoptive transfer of CFSE-labeled donor T cells, we noted that thymus-infiltrating alloreactive donor T cells were largely fast-proliferating and highly activated. These T cells infiltrated the thymus within one week after allogeneic bone marrow transplantation.

We analyzed T cells in tGVHD with mice deficient for certain molecules, and found that the trafficking molecules CCR9, β7 integrin, and PSGL-1 were all partially required for tGVHD, while L-selectin and αE integrin subunit may be dispensable. Similarly, we examined the role of T cell cytolytic pathways for tGVHD, and found that FasL and tumor necrosis factor associated apoptosis inducing ligand (TRAIL) were required for tGVHD, but that perforin and TNF were dispensable.

As host thymic stroma may be an important target for donor alloreactive T cells, we assessed the expression of the death receptors Fas and DR5 in thymic stroma from normal and irradiated mice, and found that endothelium and fibroblasts, as well as epithelial cells upregulated the expression of both Fas and DR5 after irradiation. FLIP, which is a negative regulator of Fas ligand and TRAIL mediated apoptosis, was also downregulated after irradiation.

Our study suggests that TRAIL, which has previously been shown to be important in graft-versus-tumor activity, may also be important for mediating GVHD.
Simultaneous bone marrow and tissue transplantation in rats treated with a nonmyeloablative conditioning regimen promote tolerance to composite tissue allografts (141.18)
Authors: Ramsey Deborah M. , Bozulic Larry D. , Wu Shengli , Adamson Larry A. , Rahhal Dina N. , Swearingen Bruce J. , Xu Hong , Ildstad Suzanne T.
Published: January 2023

Approaches to safely induce graft acceptance through bone marrow chimerism and limited immunosuppression are currently being pursued. Here we evaluated the effect of nonmyeloablative conditioning on the establishment of tolerance and mixed chimerism in recipients of bone marrow transplantation (BMT) and reconstructive tissue allotransplantation (RTA). Twenty Wistar-Firth rats were treated with a short course of immunosuppressive therapy (anti-ab-TCR mAb, tacrolimus, and anti-lymphocyte serum). One day prior to BMT, rats received total body irradiation followed by transplantation of heterotopic osteomyocutaneous flaps taken from hind limbs of ACI rats. Following BMT, rats were monitored for rejection and mixed chimerism (>2% donor cells). Fifteen rats rejected their RTA, and five rats accepted the graft. Mixed chimerism was detected in peripheral blood at one month after RTA, but chimerism was lost in all transplant recipients by four months. After 1.5 years post-RTA, the number of donor cells in spleen, mesenteric lymph node, recipient bone marrow and thymus was <2%. To determine the tolerance mechanism, ten rats were given donor skin grafts at one year post-RTA and examined for anti-donor antibodies by flow cross-match assay. Acceptors of RTA were tolerant of donor skin and no anti-donor antibodies were detectable, which points to a deletional mechanism. These studies suggest that nonmyeloablative conditioning can establish long-term tolerance and indefinite allograft survival in rats. (Research supported by a grant from the US Department of Defense).
MHC class I restriction in DiGeorge anomaly patients after allogeneic thymus transplantation (141.22)
Authors: Li Bin , Li Jie , Devlin Blythe H , Markert Mary Louise
Published: January 2023

Purpose: The aim of this study is to determine whether antigen specific T cells are restricted to host or donor MHC after human allogeneic thymus transplantation for complete DiGeorge anomaly. Methods: Tetramers composed of HLA-A*0201, B*0801, or B*0702 with an Epstein Barr virus (EBV) peptide or HLA-B*0702 with a respiratory syncytial virus (RSV) peptide were used in flow cytometry to detect CD8 T cells restricted to these specificities. After thymus transplantation, DiGeorge anomaly subjects were screened and identified for infection by EBV and RSV. Healthy adults serologically positive for EBV or RSV were identified. EBV and RSV specific CD8 T cells were detected by flow cytometry. Results: We found binding of recipient CD8 T cells to tetramers in 4 of 4 subjects with host HLA:EBV peptide and in 1 subject with host HLA:RSV peptide after thymus transplantation. Donor-restriction was not seen in 5 of 5 subjects receiving thymus unmatched for the tested specificity. This implies that host cortical epithelium is present in the donor thymus to effect host selection, that host selection can occur on cell types other than cortical epithelium (such as on thymocytes), or that host selection can result from initial restriction on totally disparate MHC molecules on donor cortical epithelium. We are assessing host HLA expression in thymic epithelial cell cultures derived from allograft biopsies. The presence of host thymic epithelium could explain the HLA restriction to host seen in the tetramer studies.

This study was supported by NIH grant R01 AI 47040 and grant FD-R-002602 from the FDA Office of Orphan Products Development.
Interruption in Dendritic Cells Paracrine Delivery of IL-2 to Treg Cells Impairs Their Function. (141.30)
Authors: Nasr Mohamed E , Kulhankova Katarina , Rouse Todd , Field Elizabeth H
Published: January 2023

Regulatory T cell plays an important role in controlling immunity to self and foreign antigens. CD4+CD25+ require T cell receptor and IL-2 receptor activation for in vivo / in vitro expansion and function, but the source of IL-2 remains unknown. In identifying the important paracrine source of IL-2 for Treg cells, we concentrated on the dendritic cell as the paracrine source, because in vitro imaging studies demonstrated fine localization of CD25 at the membrane interface between RD6 and dendritic cells. We used ELISPOT assay to detect IL-2 production by the freshly isolated splenic and bone marrow dendritic cells Dendritic cells secrete a small fraction of IL-2 early in culture, CpG-B enhances the IL-2 production of DCs. Coculture with regulatory cells reduces the level of detectable IL-2.We examined the direct effect of dendritic cell IL-2 on Treg function, CD4+CD25+ cells suppressed CD4+CD25- effectors cells when stimulated with WT dendritic cells, but not when stimulated with IL-2KO DCs. Anti-CD25 abrogates the function of both Treg and RD6 co-cultured with WT DCs, suggesting that CD25 is required for delivery of IL-2 into the Tregs for processing and the addition of high doses of exogenous IL-2 can restore the inhibitory function of Treg and RD6 cells co-cultured with IL-2 -/- dendritic cells. These data suggest that DC IL-2 is essential for Treg function.
Contact-dependent suppression pathways in non-human primate Tregs expanded ex vivo in the presence of Rapamycin (141.36)
Authors: Johnson Jennifer Lynn , Hennessy Emily A , Pahuja Anil , Graham Melanie , Blazar Bruce , Hering Bernhard , Bansal-Pakala Pratima
Published: January 2023

Regulatory T cell therapy offers a safe and effective alternative to achieve long-term graft survival by establishing a state of operational tolerance in the transplant recipient. Prior to use in human clinical trials, Treg therapy must be tested in non-human primate (NHP) pre-clinical models. To assess tolerogenic potential of Treg therapy, we used CD3/CD28 costimulation and frequent, low doses of IL-2 to expand natural CD4+CD25hi Tregs purified from NHP peripheral blood, with and without rapamycin. We achieved 500-800 fold expansion in 21 days and maintained high levels of CD25 and Foxp3.The ex vivo expanded Tregs were capable of inhibiting CD4 proliferation to anti-CD3 and allostimulation by 90%. Preliminary analyses of potential regulatory mechanisms employed by ex vivo expanded Tregs show high expression of CTLA4 and Granzyme B but little or no expression of TGF-b and IL-10 suggesting that these Tregs may function primarily through contact-dependent pathway. To our knowledge this is the first report of potential suppressive pathways used by ex vivo expanded NHP Tregs. Further investigations into suppressive molecules used by freshly isolated Tregs and whether ex vivo expansion selectively induced CTLA4 and Granzyme B are underway. These data establish the feasibility of using ex vivo expanded Tregs as part of a tolerogenic protocol in NHP model of islet transplantation.
Induction of Regulatory T-cells by Donor-Specific Blood Transfusion in the Absence or Presence of Liver Allograft Transplantation (141.43)
Authors: Grisham Matthew B. , Abe Yuta , Ostanin Dmitry V. , Zibari Gazi
Published: January 2023

Donor-specific blood transfusion (DST) has been shown to be effective at inducing tolerance and long-term survival in animals transplanted with MHC-mismatched tissue. The objective of this study was to ascertain how DST alone or in combination with liver allograft transplantation (LTx) affects the generation and function of regulatory T-cells in vitro and in vivo. We found that DST alone generates CD4+ T-cells that when transferred into naïve recipients suppress liver allograft rejection despite expressing little or no Foxp3 at the time of adoptive transfer. In addition, we present the novel finding that DST plus LTx (but not DST alone) increases the formation of Foxp3-expressing regulatory T-cells (Tregs) that suppress alloantigen-induced activation in vitro as well as induce long term tolerance to liver allografts when adoptively transferred into naïve recipients prior to LTx. Finally we demonstrate that virtually all Foxp3-expressing Tregs reside within the CD4+CD45RC- population whereas these Tregs are equally distributed between the CD4+CD25+ and CD4+CD25- populations. Taken together, our data suggest that DST administration, in the absence of allograft transplantation, induces the formation of CD4+ T-cells that are not Tregs themselves but develop into fully functional Foxp3+ Tregs or help to induce the formation of Foxp3+ Tregs following allograft transplantation.
Species Variation in the Mechanisms of Mesenchymal Stem Cell-Mediated Immunosuppression (141.45)
Authors: Ren Guangwen , Zhang Liying , Su Juanjuan , Zhao Xin , Ling Weifang , Roberts Arthur I , Shi Yufang
Published: January 2023

Bone marrow derived mesenchymal stem cells (MSCs) hold great promise for treating immune disorders, although the immunoregulatory mechanisms remain largely controversial. Here we show that the mechanisms of MSC-mediated immunosuppression vary in different species. Under the same culture conditions, immunosuppression by MSCs derived from human or monkey was mediated by indoleamine 2, 3 dioxygenase (IDO), while immunosuppression by mouse MSCs depended on nitric oxide (NO). When the expression levels of inducible nitric oxide synthase (iNOS) and IDO were examined in mouse and human MSCs stimulated with their respective inflammatory cytokines, we found that human MSCs expressed high levels of IDO, but very low levels of iNOS, while mouse MSCs expressed high levels of iNOS and very little IDO. Except the executor molecules, moreover, the human and mouse MSCs shared some similarities. Like in mouse MSCs, immunosuppression by human MSCs was not intrinsic, but inducible by inflammatory cytokines. Additionally, IDO-mediated immunosuppression by human MSCs is also chemokine-dependent. These findings help to explain the apparently conflicting reports in the literatures and provide critical information to allow better application of human MSCs in treating immune disorders.
Modulation of extracellular redox remodeling by regulatory T cells (33.11)
Authors: Yan Zhonghua , Garg Sanjay , Kipnis Jonathan , Banerjee Ruma
Published: January 2023

T cell activation and proliferation requires a reducing microenvironment that is provided by dendritic cells (DC). Naturally occurring CD4+CD25+ regulatory T cells (Treg) can suppress autoimmunity by inhibiting CD4+CD25- effector T cells (Teff). The mechanism is not fully understood. In this study, we have tested the hypothesis that inhibition by Treg cells of DC-induced extracellular redox remodeling is a component of the Treg immunosuppressive mechanism. We demonstrate that the increase in extracellular Cys concentration upon co-culture of DCs with Teff cells results from the system xc--dependent import of cystine, its conversion to glutathione, which is subsequently exported and degraded by γ-glutamyltranspeptidase and a dipeptidase, to furnish Cys. Suppression of DC-dependent Teff cell proliferation by Treg cells is correlated with a significant diminution in extracellular Cys concentration and is abrogated by addition of exogenous Cys. The decrease in the extracellular redox potential during T cell activation is associated with an increase in the levels of exofacial surface thiols in both DCs and T cells, which is diminished in the presence of Treg cells. This study elucidates the pathway of thiol release by DCs in response to T cell stimulation, demonstrates that Treg cells interfere with the process of extracellular redox remodeling and identifies a novel mechanism for immunomodulation.
Characterization of isoquinolinium salt derivatives as potent inhibitors of transcription factor NFAT (33.20)
Authors: Park Jungchan , Kim Jung-Ho , Lee Mina
Published: January 2023

Transcription factor NFAT plays critical roles in gene expression during the immune response. NFAT activation is regulated by calcineurin (CaN), a target of the immunosuppressive drugs such as cyclosporin A (CsA) and FK-506. CsA blocks phosphatase activity of CaN, which results in inactivation of NFAT. In search for novel immunosuppressive drug candidates, we have screened an organic chemical library and identified several isoquinolinium salt derivatives as novel inhibitors of NFAT. These compounds inhibited proliferation of peripheral blood mononuclear cell (PBMC) and T-lymphocytes by a mixed-lymphocyte reaction. Their inhibitory effect was specific to transcription factor NFAT, but in contrast with CsA, they neither affect the phosphatase activity of CaN nor nuclear localization of NFAT upon ionomycin stimulation. The DNA binding assay reveals that these compounds strongly attenuated DNA binding activity of NFAT. Taken together, these results indicate that these novel compounds inhibit NFAT activity without any significant change in CaN activity, which is a different mechanism from that of CaA, and suggest as potential candidates for immunosuppression
Effect of vitamin C on the activation of T cell in Gulo(-/-) mice (33.23)
Authors: Maeng Hyung Gun , Kang Jae Seung , Kim Hye Min , Lee Na Eun , Kong Joo Myoung , Bae Se Yeon , Kim Hang Rae , Hwang Young Il , Lee Wang Jae
Published: January 2023

It is known that vitamin C has anti-viral and anti-tumor activity via the activation of immune cells including T and NK cells. Even though the mechanisms of vitamin C on the activation of NK cells were extensively studied, but the effect of vitamin C on the activation of T cells is poorly understood, especially in vivo system. Therefore, we compared the surface marker and cytokine profiles of T cells from Gulo (-/-) mice, which cannot synthesize endogenous vitamin C, upon vitamin C depletion. First, we examined the phenotype and cytokine secretion of naïve T cells from wild type C57BL/6 mice, and vitamin C-depleted and supplemented Gulo(-/-) mice. Interestingly, the number of regulatory T cells were increased in vitamin C depleted Gulo(-/-) mice. Next, we investigated the changes on activated T cells in Gulo(-/-) mice upon vitamin C depletion. As a result, the expression of activation markers, CD25 and CD69, were relatively low, but there was no change on the level of IFN-gamma production. Taken together, when there was insufficient amount of vitamin C, the immune system might be suppressed by the increase of regulatory T cell population. In addition, T cells are not effectively activated when antigens were administered in vivo. Therefore, our results suggest that vitamin C maintains cell-mediated immunity via the suppression of the regulatory T cell development and plays a pivotal role during the process of T cells activation.
Loss of CD28 expression in human T cells: possible causes and downstream effects (33.24)
Authors: Parish Stanley T , Wu Jennifer , Sekhon Rekha , Effros Rita B
Published: January 2023

High proportions of CD8+CD28- T cells are present during HIV infection and aging. These cells, which have features of replicative senescence, are associated with decreased vaccine responses, early mortality, and progression to AIDS. To identify the underlying mechanisms for CD28 downregulation, we first analyzed caspase-3, which inhibits the CD28 promoter. Both CD28- T cells tested immediately ex vivo and cells that reached replicative senescence in vitro have significantly greater caspase-3 levels than their CD28+ counterparts. This increase is reversed in cultures in which CD28 expression is maintained by inhibiting TNFα. We next addressed the possible role of adenosine, which is released from metabolically active cells, and which, when added to long-term cultures, downregulates CD28. We showed that adenosine deaminase (ADA), which converts adenosine to inosine, is more highly expressed on CD28+ vs. CD28- T cells. These data suggest multiple regulatory pathways for CD28 gene suppression. Irrespective of the precise mechanism, once cells lose CD28 expression, they show decreased transcription of numerous glycolysis pathway enzymes, consistent observations on ex vivo T cells from older persons. Our data, which help elucidate the complexity of CD28 downregulation and its downstream consequences, may lead to therapeutic strategies to prevent accumulation of senescent T cells in vivo. (NIH AG023720).
Heat shock factor 1 is activated in lymphoid cells as a consequence of antigen recognition and is critical for cell cycle progression during an immune response (33.25)
Authors: Gandhapudi Siva K , Murapa Patience , Sarge Kevin D , Woodward Jerold G
Published: January 2023

Heat shock factor 1 (HSF1) is the dominant transcription factor controlling the cellular response to a variety of cellular stress signals. We now demonstrate that HSF1 becomes activated to the DNA binding form and trans-activates a large number of genes in lymphoid cells strictly as a consequence of receptor activation in the apparent absence of stress. Furthermore, this activation stabilizes cell cycle progression, becoming increasingly critical in the face of stress. EMSA assays revealed the DNA binding form of HSF1 in activated lymphocytes, but not in naïve cells at 37oC. Microarray analysis comparing WT and KO gene expression in T cells activated at 37oC revealed a diverse set of 307 genes significantly regulated by HSF1 in non-stressed T cells. HSF1-/- T or B cells proliferated normally under benign conditions of anti-CD3 or LPS stimulation in vitro. However, proliferation was severely compromised under conditions mimicking a robust immune response (SEB induced T cell activation) or stress (39oC). Western blotting revealed that the block in proliferation was associated with an inability to up-regulate cyclin A resulting in a block in cell cycle progression. Finally, hematopoetic stem cell proliferation from HSF1-/- mice, but not WT mice was also attenuated at elevated temperatures, indicating that HSF1 is a major regulator of hematopoetic cell cycle progression under stress.
T cells can be activated by physical forces acting on T cell receptor complex (33.4)
Authors: LI YA-CHEN , Chen Bing-Mae , Wu Pei-Chun , Roffler Steve R.
Published: January 2023

T cell receptor (TCR) are recognized by major histocompatibility complex (MHC) which binds antigenic peptides to activate T cell for prevention of infections and cancers. The T cell activation cascades that result in T cell proliferation, differentiation and cytokine secretions are well studied in the past years. However, the mechanism of T cell receptor triggering is still unclear so far. In this study, we expressed single- chain antibodies (scFv) or single- chain MHC (scMHC) molecules on artificial antigen presenting cell (APCs) and micromanipulating single T cells that bind to the engineered APCs. We found the mechanical force can initiate T cell activation via TCR signaling. In addition, increasing the dimensions of intercellular adhesion molecules-1 (ICAM-1) on the APCs can promote T cell activation by elongating TCR ligands. Our data indicated that ICAM-1 and leukocyte function-associated antigen-1 (LFA-1) interaction may play an important role to regulate the force in TCR triggering.
Monte Carlo model elucidates mechanisms of B cell affinity discrimination and its modulation by B cell immune synapse formation (34.1)
Authors: Raychaudhuri Subhadip , Tsourkas Philippos
Published: January 2023

B cells are known to use their signaling mechanism to recognize antigens over a wide range of affinities (10 6 -

10 10 M -1) and generate a graded response that depends on the affinity of antigen binding, a phenomenon known as affinity discrimination. However, the molecular mechanism behind such affinity discrimination is not clear. We have developed a computational model of B cell signaling that reveals that a minimum threshold time of BCR-antigen binding is needed for such affinity discrimination to occur. For a given antigenic affinity, our numerical experiments give rise to stochastically varying levels of activated signaling molecules, such as phosphorylated BCR ITAMs and Syk molecules, as is the case in single cell experiments. Recent experiments have revealed that B cell signaling is coupled with the immunological synapse pattern - an ordered protein segregation structure consisting of BCR/Antigen and LFA-1/ICAM-1 molecules that forms at the cell-cell contact area. We used our Monte Carlo computer model to show that synapse formation modulates the affinity discrimination in B cells.
PGE2 Upregulates Activation-Induced Cytosine Deaminase (AID) in Human B Lymphoblasts (34.12)
Authors: Mongini Patricia K. A. , Chiorazzi Nicholas , Haque Shabirul , Trott Joshua
Published: January 2023

AID is expressed in mature B cells during activation and in some non-lymphoid tissues during inflammation and malignancy. Elevated COX-2 & PGE2 characterize all the above and may be involved. We here confirm that PGE2 enhances AID in activated human B2 cells (J Immunol. 178:35.4, 2007 abstract) and explore the mechanism. AID was assessed, by immunoblotting and two-color flow cytometry of CFSE-labeled cells, following activation by anti-IgM:anti-CD21:dex + IL-4 + BAFF w/wo PGE2 or agonists/antagonists of PGE2 receptors (EP1, EP2, EP3, and EP4). PGE2 added at physiological doses augmented AID. EP2 was involved on the basis of (a) AID-augmenting effects of an EP2-specific agonist and (b) concordant upregulation of AID and EP2 on replicating blasts. Furthermore, PGE2-mediated elevation of AID was thwarted by inhibitors of PKA, downstream of EP2. To examine whether autocrine PGE2 synthesis contributes to AID, replicating cells were assessed for mPGE2-1 - an enzyme that converts COX-2-generated PGH2 to PGE2. Elevated mPGE2-1 in progeny and COX-2 inhibitor-reduced AID both suggest that autocrine PGE2 promotes AID. Additionally, functional AID (on the basis of IgG+ progeny) was elevated and reduced by exogenous PGE2 and COX-2 inhibitors, respectively. Thus PGE2, elaborated by classic inflammatory cells or generated during activation, augments AID expression/function in EP2-expressing human B lymphoblasts. NIH R01 AI52189; M01 General Clinical Research Center grant RR018535; and Karches Foundation