bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
The transfer of B6 splenocytes into BDF1 (B6-&gt;F1) mice results in donor anti-host cytotoxic T cell elimination of host B cells whereas transferring DBA parental splenocytes (DBA-&gt;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-&gt;F1 mice develop mild lupus long term. The present study compared pfp-&gt;F1 vs gld-&gt;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-&gt;F1 mice exhibited significantly better elimination of B cells vs pfp-&gt;F1 mice however both groups were significantly impaired compared to B6-&gt;F1 mice. Both pfp-&gt;F1 and gld-&gt;F1 females were defective and did not differ significantly. Long term, female DBA-&gt;F1 mice exhibited severe renal disease compared to all other groups. Disease was mild and comparable in both male and female gld-&gt;F1 and pfp-&gt;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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.