bluuebunny/crossref_metadata_2025 huggingface dataset:
An essential role of invariant natural killer T cells in the pathogenesis of eosinophilic esophagitis (59.1)
Authors: Mishra Anil , Rajavelu Priya , Rayapudi Madhavi , Zhu Xiang
Published: January 2023

Experimental eosinophilic esophagitis (EE) is a recently described chronic allergic inflammatory disorder whose basic pathogenesis is not well understood. Herein, we focus on the hypothesis that iNKT cell responses are essential for the initiation and progression of EE. We show that iNKT cells and their specific chemokine CXCL16 are induced in human and experimental EE. CXCL16 is primarily derived from esophageal epithelial cells and correlates with the levels of esophageal eosinophils. Notably, iNKT cell-deficient CD1d-null mice are protected from the induction of experimental EE; whereas, NK cell depleted mice exhibit full disease following allergen challenge. Interestingly, in vivo activation of human iNKT cells by the α-galactosylceramide analog PBS57 is sufficient to induce esophageal eosinophilia. Mechanistically, PBS57 exposed human iNKT cells produce IL-5 and IL-13 via STAT5 activation. Taken together, these findings provide evidence that iNKT cells have an essential pathogenic role in the initiation and progression of human and experimental EE, providing new opportunities for therapeutic strategies.
Specific comensal bacteria modulate epithelial glycosylaion (59.5)
Authors: Goto Yoshiyuki , Umesaki Yoshinori , Benno Yoshimi , Kiyono Hiroshi
Published: January 2023

Commensal bacteria constantly reside in our mucosal sites, especially in the intestine and create a mutually beneficial condition of symbiosis with the host. The physical and molecular crosstalk between host and gut microbiota is established at the surface of intestinal epithelial layer. It has been reported that specific epithelial carbohydrate chains, fucoses, are involved in the attachment of bacteria and viruses and ablation of these fucoses seems to be associated with the onset of Crohn’s disease. In spite of this important role, the mechanism of the induction and regulation of epithelial fucoses is not fully understood. Here, we show that specific commensal bacteria, segmented filamentous bacteria (SFB), physiologically induce fucoses on the ileal epithelial cells. To analyze the role of commensal bacteria in the induction of fucosylated epithelial cells (F-ECs), we firstly checked germfree mice. F-ECs were not found in germfree mice, while a large number of F-ECs were observed in the ileum of SPF mice. When we analyzed the ileal bacterial population to identify bacteria responsible for the induction of F-ECs, SFB were dominant in the ileum. Mono-colonization of SFB led to the development of F-ECs in germfree mice. Furthermore, F-ECs were abolished in SFB deficient mice bred in Jackson laboratory, indicating that SFB physiologically induce F-ECs. Our findings provide the basis of mutually beneficial system between the host and commensal bacteria.
Increased expression of HSP70, HSP90 and P53 in tissue of patients with periodontal and gingivitis deseases (59.6)
Authors: Sanchez-Rodriguez Sergio , López-Martínez Lucio , Hernandez-Rodriguez Alejandra , Ramirez-Santoyo Rosa , Barbosa-Cisneros Olga , Aguilera-Galaviz Luis , Lopez-Luna Maria
Published: January 2023

Periodontal disease (PD) is a degenerative and progressive inflammatory disease induced by supragingival plaque bacteria, which affects the tooth supporting tissue such as periodontal ligament, cementum and alveolar bone. The host response to infection by periodontal bacteria may be related to a process of acute humoral response or cellular response. The expression of heat shock proteins (Hsp) and P53 is important in apoptosis, cell cycle control in cellular homeostasis and stress are overexpressed. The baseline of this study was to determine the expression of Hsp70, Hsp90 and P53 in the tissue of patients with chronic periodontal disease and gingivitis. The oral tissues were obtained by surgery, and quantified the amount of protein and characterized by SDS-PAGE, Western blot. To detect the expression of Hsp70, Hsp90 and p53 were using monoclonal antibodies. Hsp 70 detecction by RT-PCR were performed using Super ScriptTM One-Step and densitometric analysis. Results: There was an increase of Hsp70 and 90, and a slight increase in p53 levels. By PCR Hsp70 expression was increased in initial and progresive periodontitis and in a low level in advanced.
In vitro and in vivo role of SIgA in the interaction of commensals bacteria with the intestinal epithelium (59.9)
Authors: Rol Nicolas , Mathias Amandine , Favre Laurent , Benyacoub Jalil , Corthesy Blaise
Published: January 2023

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal microorganisms in the gastrointestinal environment. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion, increased the level of nuclear translocation of NF-kappaB and production of epithelial polymeric Ig receptor as compared to bacteria alone, whereas transepithelial electrical resistance remained stable. Following administration in mouse ligated ileal loops containing a Peyer’s patch, it was found that SIgA bound to Lactobacillus or Bifidobacterium is involved in translocation to underlying dendritic cells residing in the subepithelial dome (SED) region. This phenomenon was mediated by passage across M cells, and required exogenously delivered or endogenous SIgA, as free bacteria was not observed in the SED region. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in its complex cross-talk with the intestinal epithelium.
Histamine and lipopolysaccharide induce endothelial cell bone morphogenetic protein 2 (60.1)
Authors: Walia Damandeep , Vimala Raveendran Vineesh , Zhou Jianping , Sharma Mukut , Stechschulte Daniel , Dileepan Kottarapat
Published: January 2023

Vascular calcification is one of the complications of chronic inflammatory diseases. It is a complex and multifaceted disease and is recognized as a pathobiological process sharing many features of bone remodeling. BMP2 is a powerful osteogenic morphogen which belongs to TGFβ superfamily and plays a central role in bone remodeling and calcification of atherosclerotic plaques. We have previously shown that histamine (HIS) and LPS independently and synergistically induce endothelial cell inflammatory responses and contribute to vascular inflammation. Our hypothesis is that HIS and LPS stimulate BMP2 expression in endothelial cells. Human vascular endothelial cells were incubated with HIS (10µM), LPS (100 ng/ml) or HIS+LPS for different time periods and the mRNA and protein expressions were determined by qRT-PCR and Western Blot analyses. HIS and LPS induced BMP2 mRNA expression by 4-fold and 3-fold respectively. HIS-induced BMP2 expression was completely abrogated by cetirizine, a histamine receptor 1 (H1R) antagonist, indicating that histamine effect is mediated through H1R. Western blot analysis showed upregulation of BMP2 by HIS, LPS or HIS+LPS as early as 2 hrs post treatment which sustained upto 24 h. These results suggest that HIS and LPS play a role in the regulation BMP2 expression in chronic inflammation and infection.
Sphingosylphosphorylcholine stimulates CCL2 production and ICAM expression from human umbilical vein endothelial cells (60.2)
Authors: Bae Yoe-Sik , Lee Ha Young , Lee Sun Young , Kim Sang Doo , Kim Hak Jung , Jung Young Su , Baek Suk-Hwan
Published: January 2023

We investigated the functional role of sphingosylphosphorylcholine (SPC), a component of high density lipoprotein (HDL) particles, in human umbilical vein endothelial cells (HUVECs). SPC stimulation induced production of the CCL2 chemokine in a PTX-sensitive G-protein-dependent manner. SPC treatment caused the activation of NF-κB and AP-1, which are essential for SPC-induced CCL2 production, and also induced the activation of three MAPKs, ERK, p38 MAPK, and JNK. p38 MAPK or JNK by specific inhibitors caused a dramatic decrease in SPC-induced CCL2 production. The Jak/STAT3 pathway was also activated upon SPC stimulation of HUVECs. Pretreatment with a Jak inhibitor blocked not only SPC-induced p38 MAPK and JNK activation but also NF-κB and AP-1 activation. Our results suggest that SPC stimulates HUVECs, resulting in Jak/STAT3-, NF-κB-, and AP-1-mediated CCL2 production. We also observed that SPC stimulated expression of the adhesion molecule ICAM-1 in HUVECs. Taken together, we suggest that SPC may contribute to atherosclerosis; thus SPC and its as yet unidentified target receptor offer a starting point for the development of a treatment for atherosclerosis.
Pigment epithelium-derived factor as an inflammatory factor — a new role for an old protein (60.4)
Authors: Chavan Sangeeta , Hudson LaQueta , Li Jian Hua , Ochani Mahendar , Katz David , Tracey Kevin
Published: January 2023

Obesity is a major risk factor for insulin resistance, type 2 diabetes mellitus, and atherosclerosis. A chronic inflammatory state plays a pathogenic role in the development and progression of obesity-related disorders. Adipose tissue is infiltrated with macrophages; however the factors mediating the inflammatory response are not yet clear. Here we have identified and characterized a mediator of inflammation that is secreted by adipocytes. Pigment epithelium-derived factor (PEDF), a non-inhibitory serine protease inhibitor, is expressed by differentiated adipocytes. PEDF induces dose dependent increase in expression of proinflammatory cytokines (TNF, IL1 and IL6) in macrophages. The PEDF receptor, adipose triglyceride lipase (ATGL), is expressed in macrophages, and inhibitors of ATGL attenuated PEDF mediated responses indicating importance of PEDF-ATGL interaction in PEDF mediated inflammatory responses. Analysis of downstream effects demonstrated that p38 and ERK1/2 kinases are phosphorylated following PEDF treatment and consistently, inhibitors of p38 and ERK1/2 kinases attenuate PEDF induced cytokine production. PEDF administration induced increased serum levels of TNF and IL6 in mice, while neutralizing PEDF attenuated inflammatory responses. These studies suggest that PEDF secreted by adipocytes contributes to onset and maintenance of chronic inflammation observed in obesity and may serve as a therapeutic target for treatment of obesity related disorders.
Extracellular adenosine as a master regulator of immune cell migration into the central nervous system via induction of CX3CL1 (60.9)
Authors: Mills Jeffrey , Alabanza Leah , Mahamed Deeqa , Bynoe Margaret
Published: January 2023

Having the ability to modulate lymphocyte entry into the central nervous system (CNS) would benefit patients with neuroinflammatory diseases. We have previously shown that extracellular adenosine regulates CNS entry of lymphocytes during experimental autoimmune encephalomyelitis (EAE), the animal model for the CNS inflammatory disease multiple sclerosis. For instance, while extracellular adenosine levels are vastly increased following inflammatory cellular damage (from the hydrolysis of released cytoplasimic ATP by CD39 and CD73), mice lacking CD73 or given adenosine receptor (AR) antagonists have significantly reduced CNS lymphocyte entry during EAE. We now show through detailed genetic studies that AR signaling regulates lymphocyte migration into the CNS though induction of CX3CL1, a specialized chemokine that acts as both an adhesion molecule and chemoattractant for lymphocytes, monocytes, and NK cells. We show that AR signaling is necessary and sufficient to induce CNS expression of CX3CL1 (as compared to over 40 other chemokines and adhesion molecules). AR regulation of CX3CL1 is critical for EAE progression, as daily anti-CX3CL1 treatments prevent CNS lymphocyte infiltration during EAE. Importantly, AR signaling induces CX3CL1 expression on the choroid plexus, a known CNS entry point for lymphocytes. As AR signaling can promote lymphocyte transmigration across a choroid plexus transwell barrier, we conclude that adenosine is a master regulator of CNS lymphocyte entry.
Blimp1 and two naturally present dinucleotide repeats regulate the expression of mouse CS1 gene. (62.13)
Authors: Dongre Prachi , Mathew Porunelloor
Published: January 2023

CS1 (CD319) is a member of the SLAM family of receptors, expressed on NK cells, T cells, B cells, DCs, etc. It induces NK cell cytotoxicity and is up regulated on B cell activation. CS1 is aberrantly up regulated on B cells in multiple myeloma and a therapeutic humanized anti-CS1 mAb HuLuc63 (Elotuzumab) is in Phase III of clinical trials. Recently, a unique population of CS1high B cells, implicated as major producers of pathogenic auto antibodies was detected in high SLE disease activity index (SLEDAI) patients. Regression analysis indicated direct correlation between the percentage of CS1high B cells and SLEDAI. With the available information on the pathogenic outcome of CS1 up-regulation on B cells, it is vital to study the molecular basis of CS1 expression. Here, we discuss the transcriptional regulation of mouse CS1 gene. According to our data, mouse CS1 promoter binds to Blimp1, mainly a trans-repressor and the master regulator of B cell maturation. Interestingly, mCS1 promoter also bears two unique dinucleotide repeat sequences, (TC) n=48 and (AG) n=42, sensitive to S1 nuclease, indicating presence of two DNA triplexes. Reporter assays indicate a dual regulatory role of Blimp1, as activatory at -1241bp and inhibitory at -704bp on mCS1 promoter and a trans-activating effect of the dinucleotide repeats. This is the first study to demonstrate dual regulation by Blimp1 and the role of unique dinucleotide repeats in transcriptional regulation of a SLAM family member.
Shortening of complementarity determining region 3 of the T cell receptor α and β chains during thymocyte development (62.3)
Authors: Matsutani Takaji , Ogata Masaki , Suzuki Ryuji , Itoh Tsunetoshi , Nishimoto Norihiro
Published: January 2023

T cell receptor (TCR) diversity depends mainly on the hypervariable complementarity determining region 3 (CDR3) α and β loops generated by non-germline-encoded nucleotide insertions and/or deletions. To reveal alteration of CDR3 length during thymocyte development, we analyzed CDR3α and β length distributions in thymic CD4+CD8+ (DP), CD4+CD8- (CD4SP) and CD4-CD8+ (CD8SP) T cells from C57BL/6 (H-2b), C.B10 (BALB/c congenic, H-2b) and BALB/c (H-2d) mice. The CDR3α and β lengths shortened in both CD4SP and CD8SP cells of all strains tested and the extent of shortening varied considerably depending on V segment use. The CDR3α length shortened to the same extent between CD4SP and CD8SP cells whereas the shortening of CDR3β was more considerable in CD4SP than in CD8SP for C57BL/6 and C.B10, but the shortening was only slight for Balb/c. There was no correlation in the extent of CDR3 shortening between CD4SP and CD8SP cells. Interestingly, there was a good correlation in the extent of CDR3β shortening but not of CDR3α in CD8SP among different strains of mice, independent of MHC haplotype. These results suggest that the extent of CDR3 shortening is primarily determined by germline-encoded V segments and partially affected by allelic variation of MHC class I but not of MHC class II. This shows that T cells with shorter CDR3α and β lengths are preferably selected for the functional TCR repertoire, providing an intriguing insight into the interactions of the TCR and p-MHC ligand.
Profiling the repertoire of TCRB usage in induced and natural Treg cells (62.5)
Authors: Carlson Christopher , Livingston Robert , Sherwood Anna , Desmarais Cindy , Warren Edus , Robins Harlan
Published: January 2023

In our recent research, we have found that a subset of all possible T cell receptor beta (TCRB) rearrangements is produced more frequently than expected in unrelated individuals. This subset appears to be defined by rearrangements that are near to germline, with relatively few non-templated nucleotides inserted at the V-D and D-J junctions within complementarity determining region 3. The degree of overlap between TCRB repertoire between individuals does not appear to be dramatically HLA context-dependent. Other groups have described several subsets of regulatory CD4 T cells (Treg), including both thymus-derived natural Tregs (nTregs) and peripherally induced Tregs (iTregs). While both subtypes express the FOXP3 transcription factor that defines them as regulatory T cells, it has been asserted that the expression of a second transcription factor (Helios) can differentiate nTregs (Helios+) from induced Tregs (Helios-). We hypothesize that the nTreg population is likely to be enriched for near-germline TCRB rearrangments, as recurrently produced TCRB that predispose to the nTreg fate are likely to be shared between individuals. We will compare and contrast the TCRB repertoire in three cellular populations within several individuals: CD4+ FoxP3- Helios-, CD4+ FoxP3+ Helios-, and CD4+ FoxP3+ Helios+.
Kinetics of c-Fos induction depends upon strength of TCR stimulation. (63.13)
Authors: Padhan Kartika , Rajat Varma
Published: January 2023

Induction of immediate early genes (like Fos, Jun) in response to T cell receptor stimulation with different MHC-peptide (MHC-p) dose and potency is not well understood. Our aim is to study the kinetics of c-Fos induction in T cells in response to antigen dose and potency. Our model system is AND TCR transgenic mice, whose T cells are specific for moth Cytochrome-C (MCC) peptide. We study the impact of MHC-p potency using altered peptide ligands (APLs). To study c-Fos induction we crossed AND mice to c-Fos-EGFP transgenic mice that express a fusion protein of c-Fos and EGFP driven by the Fos promoter. We observed that upon stimulation of TCR, expression of c-Fos starts after ~30 min, peaks at 3hrs and plateaus for another 2hrs. Strong agonists induce c-Fos even without co-stimulatory molecules where as weak agonists fail to do so. Co-stimulation enhances the percentage of cells responding as well as the level of c-Fos expression. Highering or lowering the peptide dose reduces the expression level of c-Fos. Inhibiton of Calcineurin has no effect on c-Fos expression. Experiment with MAPK inhibitors indicates that all the three MAP Kinases- ERK, p38 and JNK regulate c-Fos induction. ERK primarily regulate c-fos expression in the case of strong agonists (MCC and L98A) where as JNK primarily regulates c-fos expression in case of weak agonists (K99A). We are looking at other regulators of c-Fos induction in response to APLs.
Suppression of Cytokine Signaling 4 (SOCS4) is a novel immunomodulatory molecule in T cells (63.14)
Authors: Luckey Megan , Hong Changwan , Feigenbaum Lionel , Park Hyun
Published: January 2023

Preventing excessive cytokine signaling is critical for a functional immune system. Molecules of the Suppressor Of Cytokine Signaling (SOCS) family play a crucial role in regulating cytokine response, and here we show that SOCS4 is a novel SOCS family member that regulates immune function in T cells. We found that SOCS4 mRNA is highly expressed in the thymus, and specifically in pre-selection CD4, CD8 double positive (DP) thymocytes. TCR signaling significantly downreglated both SOCS4 mRNA and protein expression in DP thymocytes, which also correlated with lower SOCS4 expression in mature thymocytes and in peripheral T cells. Furthermore, T lineage specific overexpression of SOCS4 resulted in impaired T cell development in the thymus, which was presumably induced by a developmental block at the DN stage. Interestingly, SOCS4 overexpression did not result in inhibition of proximal cytokine receptor signaling such as STAT5 or STAT3 phosphorylation as would be expected from other conventional suppressors of cytokine signaling such as SOCS1 or SOCS3. These data suggest that SOCS4 might utilize another mechanism to modulate T cell reactivity. To asses this question, we have generated SOCS4 transgenic mice expressing a transgenic TCR. Preliminary results show further impairment in T cell development in these mice which detailed mechanisms are currently under investigation. Collectively, this study uncovers SOCS4 as a new immunomodulatory molecule in T cells.
The role of deltex1 in regulating T cell tolerance (63.20)
Authors: Hsiao Huey-Wen , Lai Ming-Zong
Published: January 2023

Deltex1 (DTX1) is an E3 ubiquitin ligase and is suggested to be a mediator of Notch signaling. Our previous study found that DTX1 supressses T cell activation and promotes MEKK1 degradation. In this study, we found that T cell activation was partially suppressed by DTX1 with either inactive mutation of RING finger domain or deletion of WWE domain. In addition, we identified another suppressive mechanism of DTX1 by upregulation of Gadd45β, an inhibitor of JNK activation. DTX1 may promote the expression of Gadd45β in the absence of RF domain or WWE domain. Therefore, DTX1 inhibit T cell activation by either E3-dependent or E3-independent mechanisms. To further investigate the physiological function of DTX1 in lymphocyte development and activation, Dtx1-deficient mice were generated. T cell development in thymus and spleen was normal. B cell development in bone marrow was not affected but margine zone B cell population in spleen was reduced in Dtx1-deficient mice. T cell proliferation, cytokine production and MAPK activation were enhanced in Dtx1-deficient T cells. In addition, enhanced autoantibody production, elevated immune complex deposition in kidney and increased leukocyte infiltration in lung and liver were observed in aged Dtx1-deficient mice. These results suggest that DTX1 plays a role in T cell tolerance induction.
CTLA4Ig abrogates allergic airway inflammation through a novel B7-mediated IFNγ/STAT1/NO signaling pathway (63.22)
Authors: Deppong Christine , Bricker Traci , Boomer Jonathan , Green Jonathan
Published: January 2023

Optimal T cell activation requires both TCR signaling and costimulation through CD28. In vivo use of the drug CTLA4Ig (Abatacept) interrupts B7:CD28 binding and has been an effective therapeutic strategy for chronic inflammatory disease. The presumed mechanism of action of CTLA4Ig is that it prevents the initiation of CD28 signaling. However, previous studies in our laboratory have demonstrated that in a murine model of allergic airway inflammation, CTLA4Ig can function by a novel CD28-independent, NOS2-dependent mechanism. In this study we further dissected this novel mechanism of CTLA4Ig by looking at both in vivo and in vitro models. We now demonstrate through the use of bone marrow chimeras and knockout mice that this in vivo mechanism requires IFNγ and STAT-1 mediated signaling. Furthermore, we used an in vitro coculture system of splenocytes and bone marrow derived macrophages to establish that CTLA4Ig binding to B7 on splenocytes induces IFNγ secretion. This cytokine stimulates the bone marrow derived macrophages to activate STAT-1 dependent NO production, which inhibits T cell proliferation. This data establishes a novel mechanism for immunosuppression by CTLA4Ig. In contrast to accepted dogma, CTLA4Ig inhibits ongoing inflammation not solely by preventing CD28-mediated costimulation, but by a novel mechanism whereby B7 ligation leads to the induction of an IFNγ/STAT1/NO mediated signaling pathway and suppression of T lymphocytes.
Multiple receptor-ligand interactions direct activation of epidermal γδ T cells in response to keratinocyte damage (63.7)
Authors: Witherden Deborah , Garijo Olivia , Rieder Stephanie , Komori H. , Havran Wendy
Published: January 2023

γδ T cells represent a major T cell population in epithelial tissues where they function in maintenance of the epithelium and provide a crucial first line defence against epithelial insults. Despite their importance, little is known about the molecular mechanisms involved in their activation and function. γδ T cells resident in the epidermis are a prototypic epithelial γδ T cell population and are an essential component of the wound healing process in the skin. We have exploited this population to identify molecules involved in the complex cell-cell interactions between epithelial resident γδ T cells and the neighboring cells they support. In the skin, keratinocyte damage is a primary trigger of γδ T cell activation, however the molecular interactions regulating this activation are unknown. Here we confirm a vital role for TCR-ligand interactions in the initiation of the γδ T cell response to keratinocyte damage. In addition, we identify two receptor-ligand pairs crucial for an effective γδ T cell response both in vitro and in vivo. Interactions between JAML and CAR provide costimulatory signals for γδ T cell activation and signals through CD100 and plexin B2 are responsible for γδ T cell morphology changes in response to wounding. Disruption of these interactions delays the γδ T cell-mediated wound healing response. Our results provide insight into the molecular mechanisms of γδ T cell activation and describe two novel receptor-ligand pairs vital to this process.
How we achieve tolerance for our mothers: evidence for a layered immune system in humans (64.1)
Authors: Mold Jeffrey , Venkatasubrahmanyam Shivkumar , Burt Trevor , Michaelsson Jakob , Weinberg Kenneth , Nixon Douglas , Stoddart Cheryl , McCune Joseph
Published: January 2023

The developing fetal and neonatal immune system is known to be exceedingly prone to tolerance induction following exposure to foreign antigens. In mice this has been partially attributed to the absence of a functional adaptive immune system prior to birth. By contrast, the adaptive immune system is highly developed in human beings as early as the second trimester of fetal gestation. Nonetheless, evidence for intrauterine tolerance to non-inherited maternal alloantigens has been observed in clinical studies suggesting that the human fetal immune system is also prone to tolerance induction. In these studies we provide the first evidence for a mechanism underlying the development of immunological tolerance in human beings involving the generation of fetal CD4+CD25+ regulatory T cells (TReg) in the fetal peripheral lymphoid tissues. Additionally we provide evidence that the tolerogenic properties of the fetal adaptive immune system are in part attributed to the presence of a unique population of fetal T cells distinct from those found after birth. Our findings suggest that fetal hematopoietic stem cells (HSC) give rise to phenotypically and functionally distinct T cells than those derived from adult HSC. These findings have significant implications for many different clinical settings including intrauterine infections, transplantation, and autoimmunity.
The cKrox transgene affects the development and function of multiple T cell types. (64.10)
Authors: Twu Yuh-Ching , Tong Jade , Teh Hung-Sia
Published: January 2023

The zinc finger transcription factor cKrox is crucial for CD4 T cell development. Mice lacking a functional cKrox gene lack CD4 T cells. Furthermore, in cKrox-deficient mice, developing T cells expressing a MHC class II-restricted TCR are redirected into the CD8 T cell lineage. We investigated the effect of the cKrox transgene on the development and function of two other types of T cells, namely self-specific CD8 T cells and CD4 T regulatory cells. In TCR transgenic mice that express a male-specific TCR (H-Y TCR), the only CD8 T cells that developed in male TCR transgenic mice are self-specific CD8 T cells. These H-Y TCR+ self-specific CD8 T cells are characterized by high expression of CD44, CD122, Ly6C, 1B11, NK cell markers and proliferative response in response to IL-2 or IL-15. We found that the cKrox transgene converted these self-specific CD8 T cells into CD4 T cells. Furthermore, the converted CD4 T cells lost the characteristics of these self-specific CD8 T cells described above. Notably, the converted self-specific CD8 T cells express genes that are characteristic of CD4 T cells (GATA-3 and IL-2). The numbers of CD4 T regulatory cells were significantly higher in cKrox transgenic mice and these cells expressed higher suppressor activity compared to CD4 T regulatory cells from control mice. These studies indicate that the cKrox transgene affects the development and function of multiple T cell types. This work is supported by the Canadian Cancer Society.
The public T cell repertoire is continuously generated over the lifespan of the individual. (64.11)
Authors: Kondo Kayo , Maverakis Emanual
Published: January 2023

It has been demonstrated that syngeneic mice can respond to antigenic challenge using the same “public” T cell repertoire. This is at odds with the apparent low probability of the same TCR being generated in multiple individuals. To investigate this, two public T cell responses were studied. One response was generated from a germline VDJ recombination lacking N additions and was specific to a foreign antigen, hen egg white lysozyme p106-116, in the context of I-Ed. The second was an autoreactive public response specific to the N-terminus of myelin basic protein, MBP:Ac1-9, presented in the context of I-Au. These two model systems yielded the same results. Both young and very old animals responded to antigenic challenge using the same public T cell response. To determine if this public response could be generated over time, naïve mice underwent CD4+ T cell depletion with antibody and then were allowed to reconstitute their CD4+ T cells. Following antigen priming these mice responded using the characteristic public T cell repertoire. In another experiment, naïve animals underwent total immune ablation via lethal irradiation followed by immune reconstitution via syngeneic bone marrow transplantation. Again, identical public T cell expansions were noted following antigen challenge. Together, these results demonstrate that naive mice, regardless of age, respond to antigen using similar T cells and that these can be continuously regenerated.
Regulation of gene expression during thymocyte differentiation by Protein Phosphatase 1 (64.16)
Authors: Dovat Sinisa , Li Zhanjun , Payne Kimberly , Song Chunhua
Published: January 2023

The goal of our research is to define signal transduction pathways that regulate thymocyte differentiation. We have previously reported that Protein Phosphatase 1 (PP1) directly dephosphorylates and regulates the activity of the Ikaros protein - a master regulator of lymphocyte differentiation (Popescu et al. J Biol Chem 2009 284:13869). We extended our studies to determine whether PP1-mediated dephoshorylation of Ikaros controls Ikaros’ function in regulating the expression of the terminal deoxynucleotidyl transferase (TdT) gene during thymocyte differentiation. An Ikaros mutant that is unable to interact with PP1 (IK 465/7A), and that is subsequently hyperphosphorylated, was compared to the wild type Ikaros for DNA-binding affinity toward the TdT promoter and for ability to repress transcription of the TdT gene using a luciferase reporter assay. Chromatin immunoprecipitation assays showed that the loss of PP1-mediated dephosphorylation of Ikaros leads to decreased Ikaros DNA-binding affinity for the D’ regulatory element of the TdT promoter in vivo. The IK 465/7A mutant also failed to repress transcription of TdT in the luciferase reporter assay, suggesting that Ikaros’ interaction with PP1 and subsequent dephosphorylation is essential for its function as repressor of TdT transcription. Results suggest that the PP1 signal transduction pathway regulates thymocyte differentiation by controlling the function of Ikaros and transcriptional repression of the TdT gene.
Visualizing acute negative selection in the thymus (64.3)
Authors: Dzhagalov Ivan , Melichar Heather , Robey Ellen
Published: January 2023

Negative selection in the thymus ensures that only the functional and not the useless or harmful thymocytes will be exported into the circulation. The behavior of cells undergoing negative selection has been visualized only recently. However, the steady-state model used did not allow the precise reconstruction of the timeline of the events. To circumvent this drawback, we developed a model for visualizing synchronous negative selection following peptide injection, while avoiding the induction of non-specific cell death due to peripheral T cell activation. We observed that transgenic thymocytes slowed down significantly after the injection of their cognate peptide. This occurred very early, possibly within minutes of the injection. In addition, we established the time-course of cell activation and apoptosis induction in the system. Our data suggest that while stopping of the thymocytes is an event immediately downstream of the T cell receptor signaling, committing to death takes much longer time and most likely requires integration of multiple stimuli.
Identifying molecules that “fine-tune” T cell antigen receptor signaling during thymocyte selection. (64.5)
Authors: Pinkhasov Julia , Lee Jan , El-Khoury Dalal , Lee LiQi , Hwang SuJin , Lesourne Renaud , Zhang Yongqing , Weng Nan-Ping , Love Paul
Published: January 2023

The prevailing view of thymocyte selection is that the fate of developing cells is dictated exclusively by the affinity/avidity of the expressed TCR for self-MHC/self-ligand. We hypothesize that there is significant plasticity in the signaling processes that control T cell development and that the ability to ‘fine-tune’ the TCR signaling response, through the regulated expression of molecules that positively or negatively impact the TCR signaling response operates to maximize the TCR repertoire. One example of a ‘fine-tuning’ molecule that was previously characterized is the cell surface protein CD5. We found that CD5 is an inhibitory tuning receptor, and its surface expression is directly and proportionately regulated by the affinity of the TCR for selecting ligand. In order to identify additional candidates for ‘TCR-tuning’, we employed a TCR transgenic experimental system where TCR expression was fixed on all developing thymocytes and where the affinity of the positively selecting TCR/self-MHC/self-ligand interaction could be altered by changing the MHC haplotype. Thymocytes that successfully underwent selection under conditions of relatively low or relatively high avidity interactions were analyzed by microarray. These experiments identified CD5 as well as several additional potential tuning molecules not previously thought to play a role in ‘TCR-tuning’. Our results provide important new insights into the genetic and molecular processes that control thymocyte selection.
Diethylstilbestrol and the methoxychlor metabolite, HPTE, alter viability and differentiation of embryonic C57BL/6 thymocytes (64.9)
Authors: Mourad Faraj , Escalante Priscilla , Sortillon Don , Leung Liu Lucie , Pierce Katy , Broussard Christine
Published: January 2023

Endocrine disrupting chemicals (EDCs) with estrogenic activity are ubiquitous in the environment. Studies of select EDCs have suggested that human exposure alters the immune system leading to increases in infection and autoimmune disease frequency. How these alterations occur is unclear. Exogenous and pregnancy-related increases in estrogen have been shown to induce atrophy and alterations in thymocyte development, suggesting a potential mechanism for EDC alteration of the immune system. Diethylstilbestrol (DES), a synthetic estrogen once prescribed to pregnant women, and HPTE, the metabolite of the once widely applied pesticide Methoxychlor (a replacement for DDT) are EDCs of interest. Using an in vitro assay, our research probed whether HPTE and DES alter thymocyte development in embryonic C57Bl/6 mice and at what concentrations. We also examined the mechanism by which DES and HPTE affect thymocyte differentiation. Our preliminary data show a dose-dependent decrease in thymocyte viability and alterations in thymocyte population distribution. Over the range of doses tested, increasing DES and HPTE concentration led to significant decreases in CD4+ intermediate and DP T-cells. Furthermore, we designed a time course experiment to probe the cell death mechanism induced by DES and HPTE using Annexin V and PI staining. Our work suggests that one mechanism of action of DES and HPTE on embryonic thymocytes is induction of cell death through apoptosis.
Quantifying immunoreactivity of a dual-labeled imaging agent by near-infrared flow cytometry (65.17)
Authors: Hall Mary , Aldrich Melissa , Azhdarinia Ali , Robinson Holly , Haviland David , Sevick-Muraca Eva
Published: January 2023

Criteria for agent batch release used in clinical diagnostics include validation of biological activity. The Lindmo assay (LA) has been used conventionally to quantify immunoreactivity of radiolabeled antibodies (Ab), however, this method does not provide consistently accurate results. We developed and validated a near-infrared (NIR) flow cytometry (FC) assay for quantifying immunoreactivity of a dual-labeled Ab for use in human and small animal nuclear and NIR fluorescence imaging. Ab specific for epithelial cell adhesion molecule (EpCAM) was conjugated to DOTA-NHS-ester (DOTA), labeled with IRDye 800CW, and further labeled with 64Copper (Cu) or nonradioactive Cu prior to reacting with human prostate cancer (PC3) cells for testing by LA or FC, respectively. Immunoreactivity was 76% and 73%, respectively, by LA and FC. Ab without Cu yielded 79% immunoreactivity by FC. No significant differences were found among these levels of activity (P > 0.05), indicating that NIR FC is an equivalent method for measuring immunoreactivity and that Cu-labeling does not significantly affect the Ab’s ability to bind to its target. Immunoreactivity was significantly reduced when increasing dye-to-protein (D/P) ratios were tested by NIR FC. A similar trend was observed in the LA. Nonspecific, control Ab did not bind PC3 cells. In summary, NIR FC is a validated method with specificity and sensitivity equivalent to the LA for quantifying immunoreactivity of NIR-labeled agents.
Development of an high efficiency peripheral B lymphocyte immortalization tool. (65.18)
Authors: Razanajaona-Doll Diane , Zhu Ying , Clear Olivier , Laurant Stéphanie , Pin Jean-Jacques
Published: January 2023

The low proportion of peripheral B cells (5-10% of PBMC) and their poor capacity to enter cell cycle limited the study of the overall B repertoire. A prerequisite is an in vitro amplification and immortalization of B cells. To date, B cell immortalization was mainly relying on the CD40 system and/or EBV transformation; however, these methods targeted only 1% and 1/106 cells of the starting B cells, respectively. Dendritics company has both optimized previous methods and developed new tools aiming at increasing immortalization efficiency: the irradiated fibroblasts of the CD40 system were replaced by inert reagents, the EBV transformation capacity was significantly increased, a novel monoclonal antibody inducing B cell proliferation was generated, a specific B cell growth booster reagent was developed and a 2 stimulation/transformation combined culture steps, starting with PBMC, was set up. With the Dendritics process, up to 20% of the starting B cell population was transformed within less than 2 months. As proofs of concept, cultures were prolonged, allowing the isolation of very rare specific antibody secreting cells such as anti-HPA5b or anti-Chikungunya virus B cells. Genomic, transcriptomic and proteomic studies of the overall B cell repertoire will be facilitated by this innovative tool. This new Dendritics process was packaged as a transferable, ready to use, and easy to handle product labeled Human Blood B Booster kit which marketing is currently under study.
In situ peptide-MHC-II tetramer staining of antigen-specific CD4+ T cells in tissues (65.37)
Authors: Dileepan Thamotharampillai , Kim Hyeon , Moon James , Linehan Jonathan , Jenkins Marc , Cleary Patrick , Skinner Pamela
Published: January 2023

The invention of peptide-MHC-tetramer (pMHC) technology to label antigen-specific T cells has lead to a greatly enhanced understanding of T lymphocyte biology. Although in situ staining of antigen specific CD8+ T cells with pMHCI tetramers has been well characterized and permits visualization and characterization of antigen-specific CD8+ T cells relative to other cell types in stained undisrupted tissue sections, similar staining of antigen specific CD4+ T cells in situ with pMHCII tetramers has been elusive. Thus far, detection of antigen specific CD4+ T cells with pMHCII tetramers has been limited to cells isolated from blood or disrupted tissues that are analyzed by flow cytometry. In this report, we describe our development of an in situ pMHCII tetramer staining method to directly visualize antigen-specific CD4+ T cells in tissues. We used an infectious model system, where Group A streptococcus (GAS) expressing a surrogate peptide (2W) fused to the M1 protein was used to inoculate C57BL/6 mice. NALT from these mice were used in optimizing the in situ staining of 2W:I-Ab specific CD4+ T cells. Parallel flow cytometric analysis was performed to validate the findings of in situ staining. The results showed pMHCII tetramer stained cells in GAS-2W but not GAS infected mice. Thus, we have developed a pMHCII in situ staining method, and this novel methodology can be used to study the localization, abundance and phenotype of antigen specific CD4+ T cells in tissues.
A novel Quantum Dot/peptide-MHC probe to study recognition of self by T cells (65.38)
Authors: Anikeeva Nadia , Gakamsky Dimitry , Sykulev Yuri
Published: January 2023

We have assembled soluble pMHC proteins on fluorescent nanoparticles, Quantum Dots (QD), to probe membrane associated events and downstream signaling followed by TCR engagement on live T cells. A unique architecture of QD/pMHC probe let us to demonstrate CD8-dependent strong binding of non-cognate QD/pMHC that did not induce any detectable Ca2+ flux in T cells unless at least one pMHC complex per dot was cognate [1]. In contrast, cognate and non-cognate pMHC assembled on Streptavidin scaffold into the tetramer did not show detectable binding to CTL and Ca2+ mobilization. Consistent with these data, we have found very different distribution of the separating distances between pMHC ligands in the two different oligomers as established by analysis of Foster Resonance Energy Transfer (FRET) between MHC bound peptides labeled with either donor or acceptor fluorophores. We have also shown that close proximity between pMHC on QD but not the valency of the probe endows QD/pMHC with unique properties. We propose to utilize this novel tool to study responses of autoimmune T cells recognizing self pMHC as well as the nature of tonic signal required for maintenance of quiescent T cells. [1]. Anikeeva et al. (2006) PNAS, 103:16846
Profiling antigen-specific T cell phenotypes with heavy metal labeled peptide-Major Histocompatibility Complex tetramers and single cell mass-spectroscopy (CyTOF) (65.40)
Authors: Newell Evan , Sigal Natalia , Bendall Sean , Nolan Garry , Davis Mark
Published: January 2023

The direct detection of antigen-specific T cells using fluorescently tagged pMHC-tetramers is widely used in basic and clinical immunology, allowing the unperturbed assessment of T cell phenotypes by concurrent staining with surface and/or intracellular markers. However, the number of T cell specificities and phenotypic markers that can be assessed in a single sample is limited by fluorescence spectral overlap. We recently devised a way to label pMHC tetramers with heavy metals that allows for detection using single-cell mass spectroscopy (Cytometry via Time of Flight - CyTOF). The ability to use 30+ different isotopic labels in this system greatly extends the number of T cell specificities, phenotypic markers and intracellular cytokines that can be simultaneously evaluated. Using this approach, we find that CD8+ T cells from human blood display a much broader diversity of phenotypes and cytokine production capabilities than previously appreciated. Although the phenotypes for different pathogen specific T cells are diverse, cells specific for a given antigen display a distinct subset of phenotypes, which differs depending on the antigen specificity and the nature of the antigen. High-content single-cell phenotypic profiling of antigen-specific T cells should allow for more accurate predictions of T cell response outcomes. Application of CyTOF-based combinatorial tetramer staining, as we previously described for fluorescent labels, will extend the utility of this approach.
Performance of Luminex and MSD high-sensitivity multiplex cytokine assays across multiple lots and laboratories (65.6)
Authors: Breen Elizabeth , Reynolds Sandra , Cox Christopher , Jacobson Lisa , Magpantay Larry , Mulder Candice , Dibben Oliver , Margolick Joseph , Bream Jay , Sambrano Elise , Martínez-Maza Otoniel , Sinclair Elizabeth , Borrow Persephone , Landay Alan , Rinaldo Charles , Norris Phillip
Published: January 2023

Circulating cytokine levels can provide valuable information about immune status, but often require high-sensitivity assays. Multiplex assays hold great promise, especially when limited sample volumes are available. Four high-sensitivity cytokine multiplex assays (BioRad, BioSource/Invitrogen, Linco/Millipore, Meso Scale Discovery [MSD]) were evaluated for ability to detect circulating levels of cytokines, and for lot and laboratory variability. Assays were performed in six different laboratories utilizing archived serum from HIV-uninfected and -infected subjects from the Multicenter AIDS Cohort Study (MACS) and the Women’s Interagency HIV Study (WIHS), and commercial plasma samples spanning initial HIV viremia. In a majority of serum samples IL6, IL8, IL10, and TNFα were detectable with at least three kits, while IL1β was clearly detected with only one kit. No single multiplex panel detected all cytokines, and there were highly significant differences (p<0.001) between laboratories and/or lots with all kits. The kits generally detected similar cytokine patterns over the onset of HIV viremia. This multi-site comparison suggests that current multiplex assays vary in their ability to measure serum and/or plasma levels of cytokines, and may not be sufficiently reproducible for repeated determinations over a long-term study or in multiple laboratories, but may be useful for longitudinal studies in which relative, rather than absolute, changes in cytokines are important.
CD28: a novel therapeutic target for the treatment of multiple myeloma (66.10)
Authors: Murray Megan , Nair Jayakumar , Lee Kelvin
Published: January 2023

Multiple myeloma (MM) is a plasma cell neoplasm which resides in the bone marrow (BM) and is critically dependent upon the BM microenvironment (BMM) for its survival. It has been observed that CD28 is expressed on MM cells and that expression levels correlate with worsening progression and worse prognosis. We have also observed that ligation of CD28 protects MM from chemotherapy. Taken together, these data imply that CD28 is a critical pro-survival molecule for the MM. The ligands for CD28, CD80 and CD86, are expressed on antigen-presenting cells, particularly dendritic cells (DC). We have observed that DCs infiltrate myelomatous portions of patient BM biopsies. We therefore hypothesize that it is the DC in the BMM which is providing the pro-survival signal to the MM via a CD28:CD80/86 interaction. Co-culture of murine or human DC with MM increased MM survival in melphalan. We then treated MM/DC co-cultures with CTLA4-Ig (which blocks CD80/86) and cultured them with or without melphalan. In the presence of melphalan, DC alone increased the survival of MM cells from 30% to 75%; however, addition of CTLA4-Ig completely abrogated this protection. We observed similar results using a CD28 blocking antibody rather than CTLA4-Ig. Mechanistically, such pro-survival signals often involve regulation of apoptosis. We have preliminary data which suggest that CD28 ligation decreases the levels of the pro-apoptotic molecule Bim, suggesting a mechanism for CD28-mediated survival.