bluuebunny/crossref_metadata_2025 huggingface dataset:
Prolyl isomerase Pin1 controls TGF-β-Smad signaling and pulmonary fibrosis (142.12)
Authors: Shen Zhong-Jian , Braun Reudi , Xie Qifa , Chu Haiyan , Love Robert , Stodola Levi , Malter James
Published: January 2023

Normal pulmonary function requires precise control over the content and composition of the extracellular matrix (ECM). During the pathogenesis of lung fibrosis, a net increase in ECM content displaces epithelial cells and causes severe reductions of pulmonary function. This process is often associated with elevated levels of TGF-β1 which enhances ECM production by fibroblasts. However, the signaling mechanisms that coordinate ECM production remain unclear. Here, we show that Pin1, a peptidyl-prolyl isomerase which isomerizes pSer/pThr-Pro peptide bonds, reciprocally controls the production of pro- and anti-fibrogenic ECM. Pin1 knockout mice treated with bleomycin, which induces pulmonary fibrosis by elevating TGF-β1, showed significantly attenuated expression of collagens (I, III and V), TIMPs (TIMP1 and 4) and fibrogenic cytokines (TGF-β1, IL-1β and CTGF) but increased MMPs (MMP2 and 3). In primary lung fibroblasts, Pin1 was required for TGF-β1-induced phosphorylation and subsequent nuclear transactivation of Smad3. These functions were mediated by TGF-β1 dependent interactions of Pin1 with inhibitory Smad6. In Pin1 knockout cells, Smad6 remained predominantly cytoplasmic, likely attenuating Smad3 phosphorylation and transcriptional activity. These data support and extend our previous observations that Pin1 blockade in vivo significantly reduced total collagen deposition in the lungs of animal models of asthma and lung transplantation.
β-tubulin-induced autoimmune hearing loss exacerbates in IL-10-deficient mice and restores by IL-10 gene transfer (143.10)
Authors: Zhou Bin , Zhou Yixuan , Habiby Kermany Mohammad , Cai Qing , Yoo Tai
Published: January 2023

IL-10 is a potent immunoregulatory cytokine in homeostatic regulation of autoreactive T cell repertoire, experimental autoimmune hearing loss (EAHL) is characterized by Th1 cell-mediated disorder plus down-regulation of IL-10. We hypothesized that endogenous levels of IL-10 function to regulate the severity of EAHL, and exogenous of IL-10 would abrogate EAHL. BALB/c (WT) and homozygous IL-10-deficient mice were underwent β-tubulin immunization to develop EAHL; some β-tubulin-immunized IL-10-deficient mice were intramuscular injected IL-10 DNA. EAHL developed progressively in both WT and IL-10-deficient mice immunized with β-tubulin, and severity of hearing loss in IL-10-/- mice was significantly greater than that in WT animals. However, the hearing function was significantly improved in IL-10 DNA administration group than that in the control group, ABR thresholds are equivalent to naive controls, and histological analysis of cochlear cross section was similar to those of naïve controls. IL-10 DNA administration suppressed autoreactive T cells and reduced expression of IFN-γ, the proportion of regulatory T cells (Treg) of IL-10-treated mice was significantly increased in comparison with control mice. Therefore lack of IL-10 exacerbates hearing loss, exogenous administration of IL-10 improved hearing function. Thus, IL-10 is capable of controlling autoimmune reaction severity by suppressing Th1-type inflammatory production and enhancing the percentage of Treg cells.
The Coagulation Factors, Thrombin and Activated Protein C, can influence the Pathogenesis of Experimental Autoimmune Encephalomyelitis (EAE) (143.26)
Authors: Alabanza Leah , Bynoe Margaret
Published: January 2023

There is accumulating evidence linking coagulation factors with immune-mediated inflammation. Thrombin, the central enzyme in the coagulation cascade, and the anticoagulant, activated protein C (APC), have been implicated in influencing both systemic and central nervous system (CNS) inflammation. Thrombin is known to mediate pro-inflammatory effects while APC can confer anti-inflammatory properties. We set out to investigate the roles of both thrombin and APC in the pathogenesis of Experimental Autoimmune Encephalomyelitis (EAE), the animal model for multiple sclerosis. We examined the mRNA expression profiles of several coagulation factors as EAE progressed. We observed that mRNA expression of coagulation factors that activate thrombin are increased in the brain during acute EAE. Conversely, the mRNA expression of protein C is suppressed as EAE develops. Given that the major pathological component of EAE is the breakdown of the blood brain barrier, we utilized an in vitro system to directly assess thrombin’s effects on the blood brain barrier. We observed that treatment with thrombin upregulated the mRNA expression of adhesion molecules and chemokines in brain endothelial cells. In addition, thrombin increased the migration of cells across the in vitro human endothelial barrier. These data suggest that coagulation factors are potential contributors to the pathogenesis of EAE and multiple sclerosis.
Enhanced regulatory T cell suppressive function within Drak2-deficient mice aids in the resistance to autoimmunity(143.28)
Authors: Hernandez Jeniffer , Walsh Craig
Published: January 2023

T cells lacking the serine/threonine kinase Drak2 have a diminished activation threshold, suggesting that Drak2 serves as a quiescence factor to maintain tolerance to weak self-antigens. Drak2-/- mice are resistant to experimental autoimmune encephalomyelitis (EAE). Given the ability of regulatory T cells (Treg) to prevent autoimmunity, we have investigated the potential that Drak2 may contribute to the generation or function of Treg. We have observed a significant increase in the ratio of Treg relative to conventional T cells (Tconv) in Drak2-/- mice. The increased proportion of Treg observed may be a consequence of enhanced differentiation of Drak2-/- Tconv toward induced Treg. Indeed, culture of naïve Drak2-/- Tconv with anti-CD3 alone resulted in the induction of a significant population of Treg. In vitro suppression assays demonstrate Drak2-/- Treg to have a greater suppressive function on a per cell basis. To determine if Drak2-/- Treg have a greater suppressive function than wildtype Treg in vivo, wildtype and Drak2-/- MOG-reactive T cells with or without Treg were adoptively transferred into Rag2-/-xγc-/- mice. In accordance with our in vitro results, Rag2-/-xγc-/- mice that received Drak2-/- T cells with Treg were protected from EAE whereas mice receiving Drak2-/- T cells depleted of Treg succumb to disease. These findings demonstrate that Drak2 modulates regulatory T cell function and highlight the potential that Drak2 inhibition may alleviate autoimmune diseases.
Nuclear expression of CXCR5 is asscociated with expansion of spleen follicular T helper cells in autoimmune BXD2 mice (143.30)
Authors: Ding Yanna , Wang John , Xie Shutao , Hsu Hui-Chen , Mountz John
Published: January 2023

CXCR5+ follicular T helper (Tfh) cells primarily help B cells differentiate into antibody producing plasma cells and memory B cells. The objective of this study is to determine how CXCR5+ Tfh cells are involved in the pathogenic germinal center (GC) response in autoimmune BXD2 mice. FACS analysis showed that there was 2.5 and 1.5 fold more ICOS+CXCR5+ and IL21+CD4+ T cells, respectively, in the spleens of BXD2 mice compared to B6 mice. Confocal microscopy examination revealed that ICOS+CXCR5+CD4+ T cells were mainly clustered in GCs and located adjacent to CD21/CD35+ follicular dendritic cells in spleens of BXD2 mice. Surprisingly, CXCR5 was mainly detected inside the nucleus but not on cell membrane of CD4 T cells in GC light zone. Western blot analysis confirmed that there was higher expression of CXCR5 in the nucleus compared to the cytoplasm of CD4 T cells from BXD2 mice. Stimulation of purified CD4 T cells from BXD2 mice with CXCL13, the ligand of CXCR5, caused a time-dependent increased expression of nuclear CXCR5 and increased cell proliferation. CXCR5+CD4+T cells from BXD2 mice also expressed higher levels of BrdU and lower levels of Fas. Nuclear CXCR5 expression co-localized with Ki67+ cells in vivo. Our study indicates that there was expansion of Tfh cells in BXD2 mice.A unique feature of these Tfh cells was the increased intra-nuclear expression of CXCR5 which may increase proliferation or inhibit apoptosis of Tfh to facilitate the autoreactive GC responses.
CD8+ T suppressor cells protect from Lupus-like autoimmunity (143.31)
Authors: McPHee Caroline , Bubier Jason , Sproule Thomas , Wettstein Peter , Morse Herbert , Roopenian Derry
Published: January 2023

Systemic lupus erythematosus (SLE) inflicts damage via autoantibody complexes that result in multiorgan damage and renal failure. The BXSB.Yaa mouse is a well-established model of SLE. We show that genetic disruption of multiple components of the MHC I axis greatly accelerates autoimmune disease in this model. Mice lacking both CD8 and IL15 succumb to SLE-like disease earliest (mean age 14 wks), implying a protective role for CD8 T cells and potentially natural killer cells dependent on IL15. BXSB.Yaa mice develop an expanded population of CD8 T cells with increased expression of both CD122 and IL15Rα, which we propose is due to increased expression of IL15R on these cells. Neither Foxp3 nor CD25 are elevated, and IL10 deficient BXSB.Yaa mice fail to show accelerated disease. However, lytic function appears significant as BXSB.Yaa mice deficient in perforin show decreased survival. TCR analysis failed to suggest a biased CD8 T cell repertoire, suggesting that the cellular targets of suppression are not antigenically discrete. Overall, the results suggest the existence of potent MHC I-dependent suppressor cell population(s) that normally protect BXSB.Yaa mice from an even more aggressive form of SLE-like disease. Moreover, the results support a model in which suppression is unlikely to be mediated by cytokine regulation but instead by lytic mechanisms. Harnessing this inherent regulatory cell population could provide a novel strategy to treat SLE and related syndromes.
Peripheral deficiency of Foxp3+ Regulatory T cells in SOCS-1 knockout mice associated with peri-lethal death. (143.33)
Authors: Collins Erin , Jager Lindsey , Benitez Patrick , Lau Kenneth , Holdstein Kaitlin , Johnson Howard , Larkin III Joseph
Published: January 2023

Suppressor of cytokine signaling-1 (SOCS-1) knockout (KO) mice succumb to a T cell dependent autoimmune disease, with a 100% mortality rate, within 21 days after birth. We have observed that the onset of autoimmune disease coincided with a peripheral deficiency of Foxp3+ regulatory T cells (Tregs), despite enhanced thymic development of Tregs. Strikingly, adoptive transfer of SOCS-1 sufficient CD4+ cells into SOCS-1 KO mice, combined with administration of a peptide previously shown to partially restore SOCS-1 function (SOCS-1 KIR) significantly delayed peri-lethal morbidity. Moreover, examination of SOCS-1 KO mice receiving adoptive transfers revealed enhanced Treg peripheral populations compared to wild-type. In addition, a distinct reduction in activated T lymphocytes was observed in mice receiving treatment. Significantly, these results establish a correlation between Foxp3+ Tregs and SOCS-1 in immune system regulation and prevention of autoimmune disease.
CD4+CD25+ T regulatory cells in SJL mice regulate development of Th1 and Th2 immunity (143.38)
Authors: Hussain Shabbir , Kirwin Stefanie , Stohlman Stephen
Published: January 2023

T cells primed in female SJL mice secrete cytokines associated with a Th1 phenotype. By contrast, T cells primed in age-matched males secrete cytokines associated with a Th2 phenotype. Gender-dependent differential activation of Th2 versus Th1 T cells correlates with increased CD4+CD25+ T regulatory cells (Treg) in males compared to females. Treg from males and females exhibit equivalent in vitro T cell suppression. However, T cells primed in males depleted of CD4+CD25+ T cells preferentially secrete IFN-g and decreased IL-4 and IL-10 compared to CD4+CD25+ T cells sufficient males suggesting that Treg influence subsequent antigen specific cytokine secretion. Treg from males exhibited increased CTLA-4 and CD62L expression and preferentially secrete IL-10. These data suggest that the increased frequency of IL-10 secreting Treg in male SJL may contribute to resistance to autoimmune disease by favoring development of Th2 immune response.
Both perforin (pfp) and Fas ligand (FasL) contribute to control of autoreactive B cells and retard lupus like disease in parent-into-F1 mice. (143.45)
Authors: Puliaiev Maksym , Puliaev Roman , Puliaeva Irina , Soloviova Kateryna , Via Charles
Published: January 2023

The transfer of B6 splenocytes into BDF1 (B6->F1) mice results in donor anti-host cytotoxic T cell elimination of host B cells whereas transferring DBA parental splenocytes (DBA->F1) results in a lupus like disease. Using perforin (pfp) or FasL (gld) defective donor T cells we have shown that both pathways are critical for host B cell elimination at two weeks and pfp->F1 mice develop mild lupus long term. The present study compared pfp->F1 vs gld->F1 mice at both two weeks and 10-12 weeks for both males and females to determine if FasL or pfp was more important in controlling B cell hyperactivity and preventing lupus like disease. At two weeks, male gld->F1 mice exhibited significantly better elimination of B cells vs pfp->F1 mice however both groups were significantly impaired compared to B6->F1 mice. Both pfp->F1 and gld->F1 females were defective and did not differ significantly. Long term, female DBA->F1 mice exhibited severe renal disease compared to all other groups. Disease was mild and comparable in both male and female gld->F1 and pfp->F1 mice as measured by proteinuria (1+ or 2+) and autoantibodies. These results demonstrate that both pfp and FasL play important and relatively equivalent roles in controlling autoreactive B cell expansion and progression to lupus-like disease.
Sex-related differences in regulatory T cell function are estrogen and B7-H1 dependent (143.53)
Authors: Lin Pei-Yi , Sun Lishi , Wierman Margaret , Tekmal Rajeshwar , Pierce Anson , Daniel Benjamin , Curiel Tyler
Published: January 2023

Sex-based immune differences generally include a greater autoimmune disease risk in females. Regulatory T cells (Tregs) control peripheral tolerance and defend against autoimmunity. B7-H1 is a co-signaling molecule with an incompletely understood role in peripheral tolerance. Tregs in female B7-H1-/- (KO) mice are poorly functional in vitro compared to male KO or wild type (WT). Transfer of KO male Tregs protected Rag1-/- mice from colitis in vivo but KO female Tregs did not. Differences did not depend on known B7-H1 ligands. CD4+CD25- T cells from KO females converted into phenotypically and functionally normal Tregs with IL-2+TGF-β in vitro, suggesting no significant cell-autonomous defects in Treg differentiation or function. Treg function in sexually immature KO females was equivalent to adult WT females, but Treg function in KO females declined by 8 weeks, just after estrus onset. Oöpherectomized mice receiving estrogen maintained defective Treg function, whereas mice receiving placebo had functional Tregs. B7-H1-mediated Treg functional defects significantly reduced female protection from anti-CTLA4 induced autoimmunity and resulted from altered B7-H1-dependent mTOR and PTEN signals. Dendritic cell B7-H1 corrected KO female Treg defects in vivo. These data demonstrate a previously unknown sex-dependent relationship between estrogen, B7-H1, mTOR signaling, and Treg function that corrected predict important sex-dependent differences to immune insults and immunotherapies.
Targeted mutagenesis induced by AID (144.10)
Authors: Li Jia
Published: January 2023

Activation-induced cytidine deaminase (AID) is an essential enzyme for immunoglobulin somatic hypermutation, gene conversion, and class switch recombination in immune cells. Somatic hypermutation is induced by AID mediated cytidine deamination around the V regions of immunoglobulin genes in germinal center B cells. It has been shown that AID also can induce somatic mutations outside Ig loci and in non-lymphoid cells. Our present study attempts to use AID as a target specific mutagen. We propose to confine the mutagenesis function of AID in certain DNA regions by fusing AID with a specific DNA binding protein. We choose the Cre recombinase as the DNA binding protein in the test system. It is hypothesized that Cre-AID fusion protein should promote AID mediated mutagenesis around the loxp site, the binding site of Cre. A reporter system was built by placing a single loxp site next to a GFP expression cassette, which contains stop condon in the middle of GFP coding sequence. If the stop codon is reversely mutated into non-stop codon by AID, GFP signal can be detected. This reporter system would allow us to evaluate whether Cre mediated DNA binding could enhance AID mediated mutagenesis around the loxp site. This experimental strategy has the potential to be used in the directed mutagenesis when Cre is further modified to inactivate the recombinase activity but retain loxp DNA binding activity.
Production and Characterization of High Affinity Rabbit Monoclonal Antibodies to Amyloid β 40 (Aβ40) and Amyloid β 42 (Aβ42) Peptides (144.2)
Authors: Mehta Pankaj , Barshatzky Marc , Patrick Bruce , Mehta Sangita , Potempska Anna , Miller David
Published: January 2023

Objective: To generate high affinity rabbit monoclonal antibodies (RmAbs) to human Aβ40 and Aβ42, and develop an ELISA to measure low concentrations of free plasma Aβ. Background: Quantitation of Aβ40 and Aβ42 in CSF or plasma has been used to aid the daignosis and monitor the progression of probable Alzheimer disease (AD). Design/Methods: Rabbits were immunized with C-terminal part of Aβ40 (nh2-IGLMVGGVV-oh) and Aβ42 (nh2-GLMVGGVVIA-oh), respectively (Miller et al., 2007, Amyloid). Spleens were sent to Epitomics Inc., which generated hybridomas. The clones were examined for specificity, sensitivity and affinity using ELISA and immunoblotting. Results: Rmab 5-139 specific for Aβ40 and 1-11-3 for Aβ42 were chracterized. Inhibition ELISA showed that 1 nM Aβ40 inhibited 50% of binding between 5-139 and Aβ40, whereas, 300 nM Aβ42 showed no inhibition. Similiarly, 0.5 nM Aβ42 inhibited binding of Aβ42 to 1-11-3; whereas 1500 nM Aβ40 showed no inhibition. The Kd of 5-139 was 4 nM and 1-11-3 was 0.7 nM. On immunoblots 5-139 detected 1 fmole of Aβ40 but 200 fmole of Aβ42 showed no reactivity. 1-11-3 detected 1 fmole of Aβ42; whereas, 200 fmole of Aβ40 showed no reactivity. In sandwich ELISA 1-11-3 detected Aβ42 at 4fmole/ml; whereas 2.4 pmole /ml of Aβ40 showed no reactivity. Conclusions/Revelance: The RmAbs possessed high affinity, sensitivity and specificity. Low levels of plasma Aβ42 were measurable without interference by high levels of Aβ40.
Selective activation of antigen-specific T cells by nano-particles (144.22)
Authors: Lo Ying-Chun , Edidin Michael , Powell Jonathan
Published: January 2023

We hypothesized that the spatial differences of surface TCR between naïve and previously activated cells might be exploited to enable anti-CD3 coated quantum dot (Invitrogen, Qdots) to selectively activate antigen specific cells. CD4+ Rag -/- TCR transgenic T cells specific for Pigeon Cytochrome c were stimulated +/- peptide with or without anti-CD3 Qdots and evaluated for proliferation. Anti-CD3 Qdots markedly enhanced proliferation of T cells previously incubated with low dose peptide, but did not drive proliferation of T cells that were incubated in medium alone, though these cultured T cells responded to soluble anti-CD3. We repeated this experiment using CD4+ 6.5 TCR transgenic T cells, specific for hemagglutinin (HA). These mice are Rag+/+ allowing us to simultaneously evaluate the ability of anti-CD3 Qdots to activate both the 6.5+ and 6.5- T cells. Soluble anti-CD3 resulted in proliferation even in the absence of peptide. When cells were first incubated with low dose HA peptide as expected only the 6.5+ T cells proliferated. Addition of anti-CD3 Qdots to such cultures led to a marked enhancement of proliferation of the 6.5+ T cells but not of the 6.5- T cells in a single culture. In similar experiments using CD8+ TCR transgenic T cells specific for HA, once again, only the HA specific CD8+ T cells responded to the anti-CD3 Qdots. Experiments to exploit these findings in vivo in models of infection and tumor immunity are underway.
Extracting cell-type-specific gene expression differences from complex tissues (144.30)
Authors: Shen-Orr Shai , Tibshirani Robert , Khatri Purvesh , Gaidarski Alexander , Bodian Dale , Staedtler Frank , Perry Nicholas , Hastie Trevor , Sarwal Minnie , Davis Mark , Butte Atul
Published: January 2023

Blood is a complex tissue made up of many cell-types, each with its own functional attributes and molecular signature. Yet, the proportions of any given cell-type in the blood can vary markedly, even between normal individuals. This results in a significant loss of sensitivity in gene expression studies of blood cells and great difficulty in identifying the cellular source of any perturbations. Ideally, one would like to perform differential expression analysis between patient groups for each of the cell-types within a tissue but this is impractical and prohibitively expensive. Here we present a statistical methodology which, given microarray data from two groups of biological samples and the relative cell-type frequencies of each sample, estimates in a virtual manner the gene expression data for each cell-type at a group level, and uses these to identify differentially expressed genes at a cell-type specific level between groups. To validate the utility of csSAM, we apply our method to whole blood gene expression measurements of stable and acute rejection renal transplant patients. csSAM was able to identify hundreds of genes that were upregulated in monocytes of the acute rejection patients, as well as in other cell-types, that were undetectable at the whole blood level. Our methodology is widely applicable and enables the analysis of gene expression in complex tissues at a much higher resolution than previously possible.
Large Scale T cell receptor repertoire sequencing of murine CD8+ T cells. (144.5)
Authors: Buntzman Adam , Vincent Benjamin , Steele Shaun , Walsh Jesse , Kepler Thomas , Frelinger Jeffrey
Published: January 2023

Theoretically, mice can produce 1015 possible αβ T cell receptors (TCRs), with over 106 TCR clonotypes present at a given time. Data driven estimates based on a rigorous analysis of this massive complex repertoire have not been done due to the cost of obtaining a large set of TCR sequences. The advent of next generation DNA sequencing platforms (Illumina, SOLiD, 454) has brought TCR sequence analysis into the realm of possibility. Matching the platform with the TCR template’s unique characteristics is essential. Each T cell rearranges its genome to generate a unique TCR (VDJ recombination), with nongermline bases enzymatically produced at the junctions. Attempts to “assemble” TCR sequences from short reads (35bp, Illumina) that do not span the recombination junctions have introduced an assembly conundrum. However, the 454 pyrosequencing platform’s 250bp sequence reads generate full TCR clonotypes with no need for overlap assembly. Herein, we provide the first large scale C57Bl/6 mouse TCR β repertoire on a long read platform (Roche 454). To illustrate our methods strength, we have generated hundreds of thousands of TCRs from multiple B6 mice. To estimate the procedures error rates we have sequenced TCRs from a recombinase deficient TCR transgenic mouse (P14). This technique enables organism level analysis of T cell lineage specification, selection, and regulation during development and in disease states (infectious, autoimmune, graft and vaccine responses).
A means to study details of complex antigen mixture neutralizations when limited to antibodies unresponsive to Fc region dependent technologies for immunoprecipitation. (144.6)
Authors: Price Joseph , Sanny Charles
Published: January 2023

The Crofab™ antivenin, an ovine Fab IgG fragment product, thus lacking an Fc region for typical immunochemical physical separations, is the sole antivenin used in American for treatment of the thousands of life threatening Crotalid envenomations each year, but has unexplained clinical failures. We postulated that by removing the immune complexes and unreacted antibody from neutralization mixtures, thus leaving only the unreacted venom components, that high resolution chromatographic analysis of in vitro binding studies could be developed. So we evaluated biotinylation of the ovine Fab antivenin, although successful biotin conjugation of Crofab™ or any ovine Fab fragments has not been reported, and reduced activity of biotinylated ovine Fab2 had been reported. Our results show with varying coupling reaction mass ratios tested, up to a 75% coupling yield and a ratio of 2-3 biotin per Fab. By SE-HPLC analysis, the biotinylated antivenin did not lose binding activity. RP-HPLC showed differential neutralization of various venom components. This innovative method provides a means to conduct a much more detailed analysis of any ovine Fab antivenin neutralization or similar antibody-complex antigen mixtures, and suggests that Fab forms of other species of antivenin, or other species’ antibodies which cannot be immunoprecipitated with Fc region dependent technologies might be responsive to this approach, leading to detailed analyses of neutralizations.
Prophylactic use of flagellin to reduce Graft-vs-Host Disease in allogeneic HSCT recipients: a novel therapeutic approach (145.21)
Authors: Hossain Mohammad , Gewirtz Andrew , Roback John , Waller Edmund
Published: January 2023

To control Graft-vs-Host Disease (GvHD) in allogeneic hematopoietic stem cell transplant (HSCT) recipients, we tested whether flagellin, a bacterial protein that agonistically binds with TLR5 can modulate allo-reactivity of donor T-cells in murine HSCT. Administration of flagellin (50μg/mouse i.p.) 3 hrs before 11Gy irradiation and 24 hrs after allogeneic HSCT {5 million splenocytes and 5 million T cell depleted bone marrow (BM) cells} in H-2K → H-2b model significantly reduced GvHD and had 33% death by 132 days post transplant versus severe acute GvHD and 100% early mortality among control HSCT recipients that received PBS. Flagellin-treated recipients had 100% donor chimerism with both naïve and memory T cells in their blood and BM and the cell numbers were similar to a normal B6 mice. The numbers of B, NK and stem cells per BM were also found similar with that of normal B6 mouse. Flagellin-treated CB6F1 recipients in allogeneic parent to F1 model also showed similar immune reconstitution without GvHD within 70 days post transplant and had higher levels CMV specific CD8+ T cells in spleen on day 10 after i.p. MCMV infection compared to control recipients and survived. Generation of new donor BM- and spleen-derived T cells and regulatory T cells were maintained through functional thymus. Thus, flagellin protected allogeneic HSCT recipients from lethal GvHD, established central and peripheral immune reconstitution, and protected recipients from opportunistic CMV infection.
Ex vivo expansion of antigen specific Human Regulatory T-cells for the prevention or treatment of Graft-vs.-Host Diseases (145.26)
Authors: Veerapatharan Anandharaman , Pidala Joseph , Beato Francisca , Anasetti Claudio
Published: January 2023

Evidence from animal studies has demonstrated that allogeneic regulatory T (Treg) cells expanded ex vivo can be used as effective therapeutic tools in the treatment of allograft rejection and graft-vs.-host disease. However, translating Treg-based therapies from animal models of autoimmunity to human clinical trials requires methods for the isolation and expansion of cells with regulatory function. Currently, no effective approach has been established for selective expansion of human allospecific Treg ex vivo that are anticipated to be more potent and exert more selective immunoregulation than polyclonal Treg. In this study, we show the selective expansion of alloantigen specific human CD4+CD25+CD127- Tregs. CD4+CD25+CD127- Treg were freshly isolated, labeled with CFSE and stimulated by MHC (HLA)-mismatched APC’s, in the presence of IL-2 and IL-15 to amplify the proliferative responses, and sirolimus to suppress other T cells. The expanded Tregs with low CFSE content were sorted by flow cytometry and exhibited suppressive function against CD4 T cell responses. Experiments are underway to selectively expand the antigen specific Tregs with indirect allospecificity. Our data suggest that the antigen specific Tregs can be expanded for the development of Treg cell-based immunotherapy in humans and provides a platform for the selective expansion of Tregs against minor histocompatability antigens to prevent graft-vs.-host disease while sparing graft-vs. -leukemia effects.
Repression of NFAT1 protein expression by MicroRNA-184 in umbilical cord blood (UCB) CD4+ T-cells as a potential mechanism underlying lower incidence of acute graft-vs-host disease (aGVHD) (145.36)
Authors: Weitzel R. , Lesniewski Mathew , Laughlin Mary
Published: January 2023

Lower expression of the transcription factor NFAT1 in UCB CD4+ T-cells compared to adult blood (AB) results in reduced inflammatory cytokine production after stimulation, in part underlying its advantages in unrelated transplant. Fractionation and RT-PCR reveal that NFAT1 is translationally repressed in UCB during early stimulation, which may be due to microRNA. microRNA queries reveal that miR-184 is strongly predicted to bind to the NFATc2 3’ untranslated region (UTR). In UCB, insertion of the miR-184 binding site into the luciferase 3’ UTR reduced expression by 62% and the full 3’ UTR by 40%. Both these effects were reversible by cotransfection with excess of blocking antisense to miR-184. The UTR did not impact luciferase expression in AB CD4+ T-cells, but both luciferase expression and NFAT1 protein were reduced in AB by a miR-184 mimic in separate analyses, also reversible by cotransfection of antisense. This antisense increased NFAT1 protein expression in UCB CD4+ T-cells as measured by Western Blot without reducing NFATc2 mRNA. qRT-PCR confirmed that miR-184 is expressed 58.4-times greater in UCB than in AB CD4+ T-cells, possibly due to methyl-CpG DNA-binding proteins. We also demonstrated that the effects of miR-184 on NFAT1 are sufficient to alter transcription of the NFAT-dependent cytokine IL-2. This work may hold implications for understanding GVHD, neonatal lymphocyte activation, and suggest miR-184 as a useful biomarker or target of ex vivo graft analysis.
Cyclin-dependent kinase 2 (CDK2) opposes T cell anergy and promotes cardiac allograft rejection (145.41)
Authors: Chunder Neelanjana , Wang Liqing , Hancock Wayne , Wells Andrew
Published: January 2023

T cell differentiation and anergy are linked to the cell cycle, but the molecular basis of this relationship is not understood. Cyclin-dependent kinases (CDK) represent a major class of cell cycle proteins that could potentially regulate T cell function. Using a combination of dominant-negative, shRNA knock-down, and genetic deletion approaches, we show that the TCR/CD28 complex is specifically coupled to the il2 gene through CDK2. Additionally, a chemical genetic approach using CD4+ T cells expressing an analog-sensitive mutant of CDK2 resulted in dose-dependent inhibition of IL-2 by a specific analog inhibitor. Importantly, transient inhibition of CDK2 activity resulted in a stable state of anergy. T cells lacking CDK2 exhibited defective in vivo alloimmune responses, and mice deficient for CDK2 failed to reject cardiac allografts under conditions of limiting CD28 costimulation. Conversely, deletion of the CDK inhibitory protein p27kip1 resulted in resistance to T cell anergy and failed induction of transplantation tolerance in response to costimulatory blockade. These results suggest that CDK2 is a component of a direct signaling pathway from TCR/CD28 to the il2 gene that is required for anergy avoidance, and implicate CDK2 as a potential target for immunosuppressive therapies that may promote immunological tolerance.
Alloreactive CD8 T cells require cell-intrinsic PD-1 and upregulate Nr4a and Egr family members during deletional tolerance in vivo (145.43)
Authors: Lucas Carrie , Sykes Megan
Published: January 2023

Objective: To characterize deletional tolerance of peripheral alloreactive CD8 T cells induced by anti-CD40L and allogeneic BMT. Methods: B6 mice received 3Gy TBI on Day -1 and 2mg anti-CD40L ip prior to B10.A BMT on Day 0. For CD8 transfer, KbDb KO mice were given 6-9x10^6 purified, recipient-type CD8 cells on Day 0. For deletion and microarray studies, synchimeras containing 10-20% 2C TCR Tg (donor-reactive) cells among CD8 T cells were used. Results: Using adoptive transfer, we determined that the inhibitory PD-1 receptor is required on CD8 T cells for tolerance (6/7 vs. 0/8 chimeras with WT vs. KO CD8s). 2C cells in animals treated with blocking anti-PD-1 initially disappeared at a similar rate to those in tolerized mice. On Day 6 the frequency of apoptotic 2C cells in tolerant controls was equivalent to that in anti-PD-1-treated mice (p = 0.2) and greater than that in mice with no mAb (p = 0.03). Following complete rejection of donor BM, 2C cells in anti-PD-1-treated mice expanded (p = 0.004 vs. tolerant at Day 42). Microarray analysis on sorted 2C+ CD8 T cells isolated on Day 6 post-BMT revealed significantly higher transcript levels for the apoptosis-associated Nr4a family members Nur77 (7.3 fold, p = 0.01), Nurr1 (23 fold, p = 0.01), and Nor1 (9 fold, p = 0.007) and the tolerance-associated Egr family members Egr-1 (4 fold, p = 0.01), Egr-2 (15 fold, p = 0.001), and Egr-3 (8 fold, p = 0.04) in tolerant compared to rejecting mice.
Determining the extent to which clinically effective treatment, ustekinumab or etanercept, reverses the molecular disease profile of psoriatic skin: comparisons of lesional, non-lesional and normal skin(34.5)
Authors: Brodmerkel Carrie , Li Katherine , Baribaud Frédéric , Suarez-Farinas Mayte , Krueger James
Published: January 2023

ACCEPT, a randomized, active-controlled study, compared the efficacy of etanercept and ustekinumab in 903 moderate-severe plaque PsO pts through wk12. Skin bx were performed in a subset of pts at baseline(BL), wk1 & wk12. Microarray analyses(Affymetrix U133+2 array) comparing nonlesional skin(NL,n=85) to lesional skin(n=85) at BL showed several thousand probe sets differentially expressed(>2fold change FDR,p<0.05) in lesional skin. 25 healthy skin bx were analyzed. Comparison of NL to healthy skin showed a series of lesional genes dysregulated in NL(DEFB4,S100A7A,CCL18,SERPINB3). Analyses to understand the impact of p40 cytokine(IL12/23)or TNFα blockade on resident and inflammatory cells and on expression of gene circuits that may drive chronic immune activation and inflammation in skin were done. Analyses to understand residual molecular profile or “molecular scar” following 12wk of tx were completed. Pts responding to each agent(≥PASI75,ETA n=21,UST n=19)had signif changes in ∼4000 transcripts compared to untreated lesions, indicating signif resolution of pathological gene circuits back to NL levels. However, genes such as DEFB4, S100A7, CCL18 & SERPINB3 though reduced to levels similar to NL were not reduced to that of healthy skin by either tx unlike ADAM10 & HSD3B1. Elucidation of molecular pathways which remain dysregulated following effective tx may provide insight into pathological mechanisms that remain active despite appearance of clinical and histologic resolution.
Th17 cells contribute to the exacerbated autoimmune arthritis in CCR2-deficient mice (34.6)
Authors: Liu Peng , Tarrant Teresa , Rampersad Rishi , Vallanat Christopher , Quintero-Matthews Tatiana , Patel Dhavalkumar , Fong Alan
Published: January 2023

Th17 cells have been implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis (RA). Chemokine receptor CCR2 mediates leukocyte trafficking to sites of inflammation and is considered to be an important proinflammatory factor in RA. However, as an unexpected paradox, mice lacking CCR2 (CCR2-/-) develop an accelerated and enhanced arthritis in the collagen induced arthritis model (CIA). To investigate the underlying mechanism(s), we examined the role of Th17 cells in CCR2-/- mice with CIA. We found that the number of Th17 cells was increased significantly in draining lymph nodes of CCR2-/- CIA mice (845±144) compared to WT controls (289±136, p=0.017). In contrast, Th1 cells were not significantly different between groups (485±171 vs. 516±86 for WT, p=0.88). Consistently, CCR2-/- CIA mice had elevated levels of sera IL-17 (84.4±15.5 pg/ml) and IL-17 mRNA in arthritic joints (3.43±0.61 fold) compared to WT mice (45±9.7 pg/ml in the serum and 0.21±0.05 fold in the joint, p=0.04 and 0.009 respectively). Furthermore, Th17-favored cytokines IL-6 and IL-1β were up-regulated in the serum of CCR2-/- animals (IL-6: 366.1±54.5 pg/ml vs. 58.7±13.3 pg/ml for WT, p=0.0004; IL-1β: 189.6±21.9 pg/ml vs. 106.4±21.3 pg/ml for WT, p=0.01). These data suggest that Th17 cells contribute to the pathogenesis of the aggravated phenotype of autoimmune arthritis in CCR2-/- mice. Mice deficient in CCR2 may serve as a model for evaluations of anti-Th17 therapies against RA.
Regulating human Th17 cells via differential expression of IL-1 receptor (34.7)
Authors: Lee Won-Woo , Kang Seong Wook , Choi Jihoon , Lee Seung-Hyun , Shah Kamini , Eynon Elizabeth , Flavell Richard , Kang Insoo
Published: January 2023

In humans, IL-1β is essential for developing Th17 cells. However, little is known about the relationship of IL-1 receptor (IL-1R) expression and Th17 cell differentiation. We report the presence of two distinct CD4+ T cell populations with and without expression of IL-1RI that correlates with the capacity to produce IL-17 in human naive and memory CD4+ T cells. A combination of T cell receptor (TCR) triggering and IL-1β augmented IL-17 production from IL-1RI+ memory CD4+ T cells. IL-1RI+ memory CD4+ T cells in peripheral blood had increased gene expression of IL17, RORC and IRF4 even before TCR triggering, suggesting that the programmed effect of IL-1β in these cells. Of interest, IL-1β also promoted IL-17 production from TCR-stimulated IL-1RI- memory CD4+ T cells, which was likely secondary to TCR-mediated up-regulation of IL-1RI. Adding IL-1β to TCR triggering resulted in production of IL-17 from IL-1RI+ but not from IL-1RI- naive CD4+ T cells. While CD4+ T cells from umbilical cord blood did not express IL-1RI, the cytokines IL-7, IL-15 and TGF-β up-regulated IL-1RI expression on naive CD4+ T cells, suggesting that IL-1RI+ naive CD4+ T cells develop in periphery. Furthermore, IL-17 production from the cytokine-treated IL-1R1- naive CD4+ T cells was induced by IL-1β and this induction was blocked by IL-1R antagonist. These results indicate that human Th17 cell differentiation is regulated via differential expression of IL-1RI which is controlled by IL-7 and IL-15.
IFNβ: an anti-inflammatory cytokine which inhibits dendritic cell migration and proinflammatory cytokine production (35.1)
Authors: Yen Jui-Hung , Kong Weimin , Ganea Doina
Published: January 2023

IFNβ is an approved therapeutic option for the treatment of autoimmune disease, MS/EAE. However, the molecular mechanisms underlying the effects of IFNβ in MS/EAE are not fully understood. In EAE, migrating CD4+ T cells and dendritic cells (DC) are essential for development of clinical symptoms. Expressions of CCR7 and MMP-9 are required for DC migration from the inflammatory site to draining lymph nodes for antigen presentation and activation of naïve T cells and to the CNS during chronic neuroinflammation. Our results show IFNβ inhibits CCR7 expression, MMP-9 production, and migration of DCs matured with different stimuli, including LPS, Poly I:C, TNFα+IFNα, or TNFα+IL-1β+IL-6 in the presence of PGE2. STAT-1 activation is required for the IFNβ-mediated inhibition of CCR7 and MMP-9 expression, and subsequent DC migration. In addition, IFNβ inhibits the production of the proinflammatory cytokines IL-12 and IL-23, whereas promoting the expression of the antinflammatory cytokine IL-10. In vivo administration of IFNβ results in doneregulation of CCR7 and MMP-9 expression, while upregulation of IL-10 production in splenic CD11c+ DC. There is reduced in vivo migration of exogenous DC treated with IFNβ, and of endogenous DC exposed to FITC in mice inoculated with IFNβ. In conclusion, our results provide novel molecular mechanisms for the beneficial effects of IFNβ in MS/EAE through the inhibition of mature DC migration and proinflammtory cytokine production.
Targeting the function of extracellular cyclophilins as a novel approach for the treatment of Rheumatoid Arthritis (RA). (35.13)
Authors: Jurjus Rosalyn , Gloor Kayleen , Herzog Dallen , Berman Dana , Constant Stephanie
Published: January 2023

Elevated levels of extracellular cyclophilins A and B are present in the synovial fluid of RA patients and have been reported to correlate with disease severity. Based on the findings that cyclophilins have potent chemotactic properties for various subsets of pro-inflammatory leukocytes, we propose that extracellular sources of cyclophilins may contribute to the development of RA pathology by promoting leukocyte recruitment into synovial spaces and tissues. Using the collagen-induced arthritis (CIA) mouse model of RA, we tested the impact of treating CIA mice with non immunosuppressive analogs of cyclosporine A (CsA) to inhibit the function of cyclophilins. Our findings demonstrate that treatment with non-immunosuppressive CsA leads to a significant reduction in CIA clinical disease severity, with corresponding reductions in histopathology and levels of pro-inflammatory cytokines and enzymes within the joints. Inhibiting the function of cyclophilin A might provide a novel approach for reducing RA-mediated joint inflammation.
Didox (Dihydroxybenzohydroxamic acid) ameliorates macrophage AhR activation, CYP1A1/AhR Induction, and oxidative stress profiles induced by PCB-126 (35.16)
Authors: Rice Charles , Gallichio Vincent , Matsebatlela Thabe
Published: January 2023

Exposure to AhR-activating xenobiotics is associated with oxidative stress and inflammation. For example, PCB-126 is a potent and well-studied AhR-ligand and inducer of CYP1A1/2 and Phase II metabolic enzymes. Intracellular ROI increases in cells within a few hrs of exposure, long before mRNA transcription and protein induction. Thereafter, levels of ROI continually rise for several days due to a high affinity of PCB-126 for the AhR and recalcitrant metabolism. After a 24 hr exposure of RAW264.7 murine macrophages to 1 µM PCB-126 we show (using qRT PCR) that expression levels of GSH-peroxidase and SOD genes are suppressed, while expression levels of of NADPH-oxidase genes are elevated. As expected, CYP1A1 gene/protein expression is markedly induced and associated with elevated superoxide anion and H2O2 production. In search of strong, but non-toxic antioxidants that may ameliorate AhR-associated ROI and inflammation, we found that Didox (3,4-Dihydroxybenzohydroxamic acid) is very potent. Several GSH-peroxidase genes and GSH reductase, peroxiredoxins, thioredoxin reductase, all three types of SODs, and catalase are elevated by Didox. Furthermore, Didox reverses oxidative stress gene profiles initiated by PCB-126. Of particular note, Didox mimics LPS in that exposure suppresses the expression of both CYP1A1 and AhR genes. We postulate that Didox may have significant potential in ameliorating the toxicity of several classes of AhR-activating immunotoxic compounds.
Blimp1 is not required for all Ig secretion and J-chain expression in nurse shark plasma cells (36.11)
Authors: Doremus Caitlin , Dooley Helen , Ohta Yuko , Flajnik Martin
Published: January 2023

The J-Chain is involved in multimerization of IgM. The oldest animals with Ig isotypes, the cartilaginous fish, express IgM in two forms: a pentameric (19S) form, attached to J-chain, and a monomeric (7S) form. By in situ hybridization adult nurse sharks have splenic IgM-producing plasma cells that are either J-chain positive, and presumed to secrete pentameric IgM, or are J-chain negative 7S secretors. Neonatal nurse sharks produce pentameric IgM, and all plasma cells express J-chain. In addition, some neonatal shark plasma cells, found in discrete splenic locations, express a germline joined isotype, IgM1gj. We hypothesize that during B cell development either 19S producers “switch” to become 7S producers by shutting down J-chain expression, or the two populations develop from separate lineages, with the 7S, J-chain-negative lineage arising later in development than the 19S and IgM1gj lineages. Identification of factors controlling J-chain expression, in shark B cells is necessary to clarify which B cell development pathway is correct. In mammals, Blimp-1 is required for development and maintenance of J-chain expression and immunoglobulin secretion in plasma cells. However, in situ hybridization data show that Blimp-1 is not expressed in neonatal IgM secreting cells, or in half of adult shark secretory cells, presumably the 19S producers. Our data imply that J-chain expression and antibody secretion functions of some shark plasma cells are not controlled by BLIMP1.
Interleukin-2 Controls flt3L-dependent Development and Phenotype of Conventional and Plasmacytoid Dendritic Cells (36.19)
Authors: Lau-Kilby Annie , Kretz Cosima , O'Shea John , Trinchieri Giorgio , Tarbell Kristin
Published: January 2023

DC development requires the ligand for FMS-like tyrosine kinase 3 receptor (flt3L), but little is known regarding other cytokines that may control this process. In our study, we have addressed the effect of IL-2 on DC development. We have shown that the addition of IL-2 to flt3L-stimulated bone marrow (BM) cultures can block the development of both pDCs (CD11c+SiglecH+) and cDCs (CD11c+CD11b+), while expanding the populations of NK/NKT/T cells (CD3+/-NK1.l+/-). By sorting for DC precursors that are Lineage- flt3+ CD11c-, we confirm that IL-2 blocks DC development by acting directly on DC precursors, and not indirectly via its effect on NK/NKT/T cells. The inhibition of DC development by IL-2 is dependent on both the IL-2R alpha and beta chains, and STAT5. IL-2 treatment of DC precursors leads to a decrease in the expression of various transcription factors crucial for DC development. In addition, our ongoing studies indicate that the presence of IL-2 during flt3L-dependent DC development also alters the phenotype of DCs. Together our data has indicated that IL-2 inhibits the development of DCs, and suggests IL-2 can also alter DC phenotype. This has important implications in the therapeutic use of IL-2, as well as our current understanding on factors that can lead to the loss of immune tolerance in various autoimmune diseases.