bluuebunny/crossref_metadata_2025 huggingface dataset:
Intrathymic Role for Leptin in the Prevention of Thymic Atrophy
Authors: Gruver Amanda Louise , Sempowski Gregory D
Published: June 2021

Thymic atrophy is highly inducible by stress. Prolonged thymus atrophy can contribute to T cell deficiency or prevent immune recovery after acute T cell depletion, to which no treatment is currently available. Leptin is an endogenous metabolic hormone used therapeutically for obesity. We have shown that leptin induces thymopoiesis during lipopolysaccharide (LPS) induced stress by increasing thymus weight and TCR gene rearrangement. We have also shown that leptin blunts corticosteroid and pro‐inflammatory cytokine responses to LPS. The role of leptin within the thymus however is currently unknown. We now report that the leptin receptor predominantly co‐localizes to thymic medulla, with long and short isoforms being expressed. We further demonstrated that leptin protects against LPS‐induced apoptosis of CD4+CD8+ DP thymocytes and increases proliferation in CD4−CD8− DN thymocytes. Alternatively, we evaluated the endotoxin response in the absence of leptin or its receptor. We found that LPS induced substantial thymic atrophy in leptin deficient ob/ob mice while only induced mild thymic atrophy in leptin receptor deficient db/db mice. Both ob/ob and db/db mice had severely reduced cytokine responses to LPS, suggesting duel anti‐ vs pro‐inflammatory roles of leptin. Taken together, these data strongly suggest multiple roles of leptin in modulating thymopoiesis and inflammation. Supported by AG025150.
Role of CD4 T cell help in the differentiation of memory CD8 T cells
Authors: Choo Daniel
Published: June 2021

The role of CD4 T cell help (TH) in the generation of memory CD8 T cells has been much debated over the past several decades. TH are thought to aid in the “programming” of CD8 T cells as memory cells generated in the absence of TH results in defective secondary expansion. Despite progress, it is not known when and how TH influences the generation of optimal memory cells. To address this, we controlled the duration of TH during various phases of the immune response (naïve to effector; effector to memory; throughout the response; or never). As shown by others, in the complete absence of TH, memory differentiation was impaired. Interestingly, TH during the initial programming (naïve to effector) alone was not sufficient as transferring effector CD8 T cells from wild‐type animals into infection matched CD4 −/− animals resulted in defective memory. However, when the same cells were transferred into naïve CD4 −/− animals, memory differentiation was normal suggesting that the environment in infected CD4 −/− animals may be impeding memory differentiation. In support, proliferation of CFSE labeled CD8 memory sensor cells into infected CD4 −/− animals following the effector phase suggested impaired Ag clearance. Our data shows that, contrary to current paradigm, TH are not involved in programming CD8 T cell differentiation, but rather aid in the conditioning of the environment for optimal memory formation.
ICOS costimulation regulates the development of effector memory CD4 T cells
Authors: Moore Tamson V. , Clay Bryan , Bandukwala Hozefa , Blaine Kelly , Shilling Rebecca , Sperling Anne I.
Published: June 2021

Inducible costimulator (ICOS) is a member of the CD28/B7 family of costimulatory molecules. ICOS costimulation has been shown to enhance T‐helper responses in vivo, particularly Th2 responses. ICOS is upregulated upon T cell activation and is highly expressed on memory phenotype CD4 T cells, but the role of ICOS costimulation on the development and survival of memory CD4 T cells is unclear. To address the influence of ICOS costimulation on the development of memory cells, we utilized an adoptive transfer model to track the development of memory cells from naïve ICOS−/− and wild‐type precursors. The ICOS−/− T cells had an initial defect in proliferation and differentiation. Interestingly, the initial contraction of the ICOS−/− population was slower than that of the wild‐type population, but was more prolonged so that fewer memory cells developed from ICOS−/− precursors. Thus, the ICOS−/− T cells produced significantly less memory CD4 T cells, specifically effector memory (EM) cells. In support of these findings, mice lacking ICOS costimulation accumulated polyclonal endogenous EM CD4 T cells at a significantly slower rate than wild‐type mice. These data suggest that ICOS costimulation selectively enhances the development of the EM cells in vivo, probably by enhancing proliferation and proliferation‐associated differentiation.
The synergistic effect of Hemokinin 1 in the activation of mature B lymphocytes
Authors: wang wei , Zhang Yu
Published: June 2021

Hemokinin 1 (HK‐1) is a substance P‐like peptide of the tachykinin family. Previous studies have demonstrated that, by promoting the proliferation and survival of lymphoid precursors, HK‐1 is involved in lymphopoiesis. The current study was undertaken to investigate the potential role of HK‐1 in the activation of mature B lymphocytes. HK‐1 alone had no significant effect on mature B cells. On the other hand, it was able to synergize with many of those classical B cell stimulators in their induction of B cell activation. When B cells were stimulated with suboptimal concentrations of LPS, anti‐IgM or CD40, addition of HK‐1 significantly increased cell proliferation, expression of CD80/86, and the frequency of Ig‐secreting cells as well as the absolute amount of secreted Ig. Intriguingly, even more potent synergistic effects were observed with memory B cells. Thus, we suggest that HK‐1 may be a co‐stimulatory factor in the activation of naïve B cells and in the maintenance of B cell memory.
Transcriptional regulation of natural IgM secretion by a novel B‐1 cell population in the bone marrow
Authors: CHOI YOUN SOO , BAUMGARTH NICOLE
Published: June 2021

B‐1 cells are an important source for much of the polyreactive natural antibodies (Ab) that are secreted in the absence of antigenic challenge to protect against pathogens. However, the molecular pathways underlying their differentiation to Ab‐secreting cells are incompletely understood. Contradictory data exist as to whether the transcriptional regulators that control differentiation of conventional B cells to Ab‐generating plasma cells (paired box protein 5a (PAX‐5a), B lymphocyte induced maturation protein 1 (BLIMP‐1), and X‐box binding protein 1 (XBP‐1)) also control B‐1 cell differentiation. Using Ig‐allotype chimeric mice, in which B‐1 cells and their Abs are distinguished from conventional B cells, we identified a novel IgM‐secreting B‐1 cell population in the bone marrow (BM). Together with splenic B‐1 cells, these cells are the major source of natural IgM. While B‐1 cells are abundant in the peritoneal cavity (PerC), there they secrete little IgM. Intracytoplasmic staining and qRT‐PCR analysis of FACS‐purified B‐1 cells from BM, spleen and PerC revealed a clear correlation between high levels of expression for BLIMP‐1, XBP‐1 and sXBP‐1 and the ability of B‐1 cells to secrete IgM. In contrast to conventional B cells, however, B‐1 cells retained high levels of PAX‐5a. The data point to novel regulatory pathways controlling differentiation of natural IgM‐secreting B‐1 cells.

This work was supported by NIH/NIAID AI051354.
Sphingosine 1‐phosphate and interleukin‐4 signaling requirements for peripheral conversion of naïve T cells to adaptive CD4+25+ regulatory T cells
Authors: Marquez Christopher Phillip , Huang Mei‐Chuan , Wang Wengang , Goetzl Edward J.
Published: June 2021

Sphingosine 1‐phosphate (S1P) mediates the development and activities of regulatory T cells (Treg) of several types through its G protein‐coupled receptors. In T cells, type 1 (S1P1) and type 4 (S1P4) G protein‐coupled receptors are quantitatively predominant. As the dominant functional receptor, S1P1 is responsible for the direct chemotactic response of CD4 T cells to S1P and inhibition of chemotactic responses to chemokines. S1P1 also mediates part of the inhibitory effect of S1P on T cell proliferation in S1P1–transgenic (TG) mice. S1P4 exerts immunosuppression by its effects on T cell proliferation and cytokine production. The local cytokine environment induces naïve CD4+ T helper cells (Th) to differentiate towards T helper 1 (Th1), Th2, Th17 and Treg phenotypes. This study explores the roles of physiological concentrations of S1P and interleukin‐4 (IL‐4) on naïve T cell differentiation in vitro into Treg and Th17 cells. We found that the absence of IL‐4 suppresses peripheral conversion of naïve splenic Th cells to Tregs and concurrently enhances their conversion to Th17 cells, which alters fundamental immune responses. Supported by Minority Access to Research Careers (MARC), Howard Hughes Medical Institute (HHMI), and RO‐1 grant HL31809 from the National Institutes of Health.
α2‐Macroglobulin (a2M) Mediates T Cell Suppression via a Peptide Sequence in its Cytokine‐Binding Site.
Authors: Koo Peter H. , Khol Matthew
Published: June 2021

a2M family proteins are innate immune/physiologic regulators. Monoamine‐reacted a2M (MA‐a2M) is much more inhibitory to T cells than native a2M (N‐a2M). These proteins form stable complexes with placental protein‐14 (PP14; a potent immunosuppressive cytokine) and TGF‐β, and enhance PP14's ability to inhibit T‐cell growth and IL2 production. To further study their role in T cell regulation, we employed a cloned T‐helper cell line (HT), which requires IL2 or IL4 for growth. In contrast to N‐a2M, MA‐a2M is consistently and potently inhibitory to IL4‐promoted HT growth. TGF‐β1 dose‐dependently inhibits IL4‐promoted but stimulates IL2‐promoted HT‐growth. But as a2M and TGF are combined, MA‐a2M dose‐dependently neutralizes whereas N‐a2M stimulates TGF's regulatory activities on HT cells. To delineate mechanism of a2M action, we tested two recombinant GST‐a2M‐peptide fusion proteins, N591‐N774 (FP3) and N775‐N1080 (FP4), which respectively encode the peptide sequences within and outside of a2M's cytokine binding site. FP3 itself potently and dose‐dependently inhibits IL4‐promoted HT cell growth, and adds to the immunosuppressive activity of TGF‐β1; but FP4 can not. Neither FP3 nor FP4 can block the immunosuppressive activity of MA‐a2M or TGF‐β1. We conclude that a peptide segment within the a2M's cytokine‐binding site may mediate T cell suppression, and it is not the site that neutralizes TGF‐β1 activity.
Inhibition of NFAT2 expression by FOXP3
Authors: Cron Randy Q. , Wells Andrew D. , Torgerson Troy R.
Published: June 2021

FOXP3 is critical for the development and suppressive function of CD4+CD25+ regulatory T cells (TREG). FOXP3 represses transcription of cytokine genes regulated by the Nuclear Factor of Activated T cells (NFAT). Various mechanisms have been proposed by which FOXP3 mediates these effects. We identified novel FOXP3 regulated genes by microarray analysis using inducible cell lines, and confirmed that WT FOXP3 inhibits NFAT2 expression and transcription of the inducible NFAT2 promoter in primary CD4 T cells by real‐time RT‐PCR and reporter gene analysis, respectively. Moreover, NFAT2 protein expression is markedly diminished in activated CD4+CD25+FOXP3+ TREG compared to CD4+CD25negFOXP3neg T cells. Chromatin immunoprecipitation shows that FOXP3 binds to the NFAT2 promoter and significantly decreases the inducible binding of NFAT1 to the NFAT2 promoter. Overexpression of NFAT2 by retroviral transduction restores expression of IL‐2 in FOXP3‐expressing T cells. These data suggest that FOXP3 functions not only to suppress the first wave of NFAT‐mediated transcriptional responses but may also affect sustained NFAT‐mediated responses and effector T cell development through suppression of inducible NFAT2 transcription. Thus, FOXP3 works to inhibit effector cell function via inhibition of NFAT2, a master regulator of cytokine function and effector cell lineage commitment.
Modulation of CD4+Foxp3+ regulatory T cell responses in the lung during an acute heterologous LCMV infection in influenza‐immune mice
Authors: Kraft Anke Renate Maria , Wlodarczyk Myriam , Selin Liisa K
Published: June 2021

In a mouse model of respiratory viral infection it has been previously shown that influenza (flu)‐immune compare to naïve mice infected with LCMV have enhanced viral load, severe lung pathology and an altered cytokine profile. We noted that more CD4+Foxp3+ regulatory T (Treg) cells were present in lungs of these flu‐immune mice compared to naïve or LCMV‐immune mice. Therefore, we questioned, whether a modulation in the normal balance of Treg and effector T cell responses might be contributing to these altered responses in flu‐immune mice infected with LCMV. We examined immune responses both at the site of the T cell activation (mLN) and the peripheral site of virus entry (lung). Flu‐immune mice had enhanced pro‐inflammatory cytokine and chemokine levels in the lungs and a higher frequency of Treg cells through out LCMV infection. In the mLN the Treg cell population in flu‐immune mice had altered kinetics compared to naïve mice infected with LCMV. Whereas Treg cells followed the same kinetics as CD4+ and CD8+ T cells in the mLN of acute LCMV‐infected mice (peak at day 3, decline by day 7), in flu‐immune mice the Treg cells persisted at higher levels, until day 9 after LCMV infection. The presence of increased Treg cells in flu‐immune lungs and the skewed Treg cell kinetics in subsequent heterologous LCMV respiratory infections may be contributing to increasing virus load and the enhanced lung pathology.
CD127 is a robust surface marker for the enumeration of natural Treg cells
Authors: Vidal Jason G. , Boyce Christopher S. , Hingorani Ravi , Li Li
Published: June 2021

Natural T regulatory cells (nTregs) may play an important role in down regulating acute GVHD and in regulating severe autoimmune diseases. Expression of CD127 and CD25 on CD4 nTregs correlates with expression of FoxP3 in the same cells. Advantages of using CD127 (surface) over FoxP3 (intracellular) to identify nTregs include that nTregs can be more efficiently sorted (compared to sorting CD25++) for in vitro assays. Also, CD 127 allows for direct immune monitoring ex vivo in peripheral blood mononuclear cells (PBMC) or Whole Blood (WB). We describe feasibility and assay performance of an immune monitoring method using CD127 for detecting nTregs and are currently evaluating a direct absolute count assay in WB. We demonstrate the intra‐assay precision and feasibility of a CD4/CD25/CD127 (hIL7R‐M21.3) cocktail and/or FoxP3 for nTreg enumeration. PBMC were evaluated by flow cytometry in normal donors. We identified nTreg populations via FoxP3 staining or by the phenotype CD4+CD25+/++CD127dim for comparison. For CD4+CD25+/++CD127dim cells the % positive mean = 6.8, with range of 5.5–8.9 and SD=1.3. CD4+CD25+/++CD127dim nTregs showed intra‐assay precision of a mean %CV = 6.14, median %CV= 4.3, range 0.7–10.2 (N>8). Finally CD4+CD25+/++CD127dim results correlated to FoxP3+ (N>4). CD127 is a robust surface marker for the evaluation of nTregs. CD127 is for research use only and is not intended for diagnostic purposes.
Multiparameter flow cytometric analysis of phosphorylated cell signaling proteins expressed by regulatory T cells (Tregs)
Authors: Sisouvanthong Chad , Ernst David , Li Li , Wang Xiao , Chen Jie , Shih Jing Ping , Huang Joseph
Published: June 2021

Multicolor immunofluorescent staining and flow cytometry are invaluable tools for analyzing the correlated expression of multiple functional molecules expressed by individual cells. These tools are important for determining the nature of rare cell types within heterogeneous primary cell samples, e.g., peripheral blood mononuclear cells. We applied these tools to study regulatory T cells (Tregs) because of their important roles in immunity. We developed new methods and antibodies to characterize signaling molecule profiles involved in human and mouse Treg development and function. Different methods for primary cell preparation were compared since no universal buffer system existed that allowed for the direct staining of CD4, CD25, CD127, FoxP3 and various phosphorylated cell signaling proteins that are coexpressed by Tregs. By using appropriate buffers, fluorescent antibody clones for staining CD4, CD25, and FoxP3, and flow cytometric gating strategies, we analyzed the patterns of phosphorylated STAT molecules expressed by Tregs. Interestingly, we found that phosphorylated STAT5 was upregulated in Tregs (induced by IL‐2). This is in accord with its role as an essential transcription factor that binds to the FoxP3 gene promoter region and initiates transcription. In contrast, phosphorylated STAT1 (IFNα) and STAT3 (IL‐6) levels were down regulated in agreement with other labs.
The parasite Nippostrongylus brasiliensis induces multiple regulatory pathways that control increases of IL‐17 expression and associated pathology in the lung
Authors: Liu Zhugong , Lin Hongxia , Liu Qian , Mousavi Gity , Gause William C.
Published: June 2021

Infection with the Nippostrongylus brasiliensis (Nb) induces a highly polarized Th2 response typically associated with elevations in IL‐4 and IL‐13. In these studies we examined the regulation of associated IL‐17 expression. Migrating Nb larvae in the lung induce considerable pathology, which is rapidly resolved. Infection induced elevated Th2 cytokine expression in the lung, and local IL‐17 gene expression and protein production were also increased. CD4+ T cells and γδT cells were identified as two major cellular sources of IL‐17. Nb infected IL‐4 knockout (KO) mice expressed significantly higher IL‐17 compared to wide type (WT) controls. Nb infection increased CD25+FoxP3+ Treg cells in the lung, which expressed elevated IL‐10 compared to Treg cells purifed from naïve mice. After anti‐CD25 depletion, Nb infection further increased local IL‐17 expression in IL‐4KO mice, suggesting that in addition to IL‐4, Treg cells modulate IL‐17 expression during parasite infection. Alternatively activated macrophages isolated Nb infected mice express dramatically increased IL‐10. In vivo IL10 blockade further increased lung IL‐17 expression. The elevated IL‐17 expression was associated with increased inflammatory cell infiltration in the lung. These data suggest that parasite infection elicits multiple independent mechanisms that together regulate local IL‐17 levels and control the severity of inflammation.
The downstream enhancer, Ed, positively regulates Ig kappa gene expression and somatic hypermutation
Authors: Xiang Yougui , Garrard William T.
Published: June 2021

The mouse Ig kappa locus has three known enhancers: the MAR/intronic enhancer (MEi), the 3′enhancer (E3′) and the further downstream enhancer (Ed). Previous studies have shown that both MEi and E3′ enhancers are required for maximal gene rearrangement of the locus, and that E3′ is also required for maximal expression and somatic hypermutation (SHM). To functionally elucidate Ed in vivo, we generated knockout mice with a targeted germ line deletion of Ed. Ed deleted homozygous mice (Ed−/−) have moderately reduced numbers of kappa expressing B cells and correspondingly increased numbers of lambda expressing B cells in spleen. Ed−/− mice also have decreased kappa mRNA expression in resting and T‐cell dependent activated splenic B cells and reduced kappa chains in sera. However, our analysis indicates that kappa gene rearrangement is normal in Ed−/− mice. In addition, our results show that Ed−/− mice exhibit reduced SHM in the kappa gene J‐C intronic region in Peyer's patch germinal center B cells. We conclude that Ed positively regulates Ig kappa gene expression and SHM, but not gene rearrangement.

Research supported by grants GM29935 and AI067906 from NIH and I‐0823 from the Robert A. Welch Foundation.
Understanding the role of neural crest cells in congenital birth defects such as Treacher Collins Syndrome
Authors: Trainor Paul A
Published: June 2021

Neural crest cells are a stem and progenitor cell population that gives rise to the majority of the bone, cartilage, connective and peripheral nerve tissues in the head and face. Not surprisingly, craniofacial anomalies which account for approximately one third of all congenital birth defects, arise largely through defects in neural crest cell patterning. Treacher Collins Syndrome is a rare human congenital disorder of craniofacial development which is characterised by numerous developmental anomalies including hypoplasia of the facial bones, middle and external ear defects and cleft palate. We have generated a mouse model of Treacher Collins Syndrome via null mutation of Tcof1 and through a combination of whole embryo culture together with cell lineage tracing and transplantations demonstrate that Tcof1 acts cell autonomously and that haploinsufficincy of Tcof1 induces massive neuroepithelial apoptosis. This in turn severely depletes the neural crest progenitor cell population, leading to insufficient numbers of migrating neural crest cells, which fail to proliferate appropriately and consequently results in hypoplasia of the cranioskeletal elements characteristic of Treacher Collins Syndrome. Tcof1 encodes a nucleolar protein known as treacle which is essential for normal ribosome biogenesis.
A genome wide association study of systemic lupus erythematosus (SLE) by SLEGEN, the International SLE Genetics Consortium.
Authors: Harley John B. , Alarcon‐Riquelme Marta E , Criswell Lindsey A , Jacob Chaim O. , Kimberly Robert P. , Moser Kathy L. , Tsao Betty P. , Vyse Timothy J. , Langefeld Carl D.
Published: June 2021

Systemic Lupus Erythematosus (SLE) is a pleomorphic, systemic autoimmune disorder with a strong genetic component (λS~30). The first 720 cases and 2337 controls, all female and self‐reported as European‐derived were divided into two subsets and genotyped at 317,501 markers. The more consistently significant markers were genotyped in third and fourth cohorts of 1739 and 1932 additional European‐derived female subjects for a total of 6728 participants. The most powerful association spans 7.05 mb across the HLA region, where 93 SNPs have p<10‐6 with the minimum p<10–54 at 31.82 mb in the MSH5 gene. Four additional associations are identified in ITGAM, KIAA1542, PXK, an anonymous marker at 1q25, and the known IRF5, all at odds ratios >1.2 and p<10‐7 in the aggregate collection. The results also support STAT4, FCGR2A, and PTPN22 as additional genetic risk factors.
PKA dependent regulation of Foxo1 and downstream targets in murine S49 thymic lymphoma cells
Authors: Davis Daniel Hal , Cohen Rhonna , Chambers Donald A.
Published: June 2021

Previous investigations revealed the presence of a norepinephrine (NE)/beta‐2‐adrenergic receptor/cAMP/PKA‐dependent mRNA decay system in T lymphocytes. The Fox family of transcription factors is involved in regulating lymphocyte physiology. Foxo1 is associated with the suppression of T cell proliferation and apoptosis. We explored whether treatment with PKA‐selective cAMP analogues (8Br‐cAMP or 8CPT‐cAMP), Forskolin, and NE could regulate BALB/c murine thymocyte cell lines, S49wt and S49kin‐ (PKA deficient), by affecting the genetic expression of Foxo1 and downstream targets Bim, Cyclin G2, and Fasl. Real‐time PCR studies revealed a 2–3 fold upregulation of Foxo1 mRNA in S49wt, but not kin‐cells with NE, 8Br‐cAMP or 8CPT‐cAMP 2 hrs after treatment. Foxo1 activation related to increase Bim, Cyclin G2, and Fasl mRNA expression in S49wt cells. Treatment with an Epac‐selective cAMP analogue had no effect. These results suggest that cAMP regulation of Foxo1 and downstream targets is acting via PKA, as opposed to the alternative Epac GEF pathway. These data imply PKA modulates increased Foxo1 mRNA expression and Fox gene‐mediated regulation of lymphocyte physiology.ted regulation of lymphocyte physiology. Supported by the NIH.
HuR protein and Thy‐1 mRNA decay in mouse S49 lymphosarcoma cells
Authors: LaJevic Melissa Dawn , Davis Daniel Hal , Cohen Rhonna L. , Chambers Donald A.
Published: June 2021

Stress is associated with immune dysfunction. Our laboratory has shown that the neurotransmitter norepinephrine (NE) increases the rate of Thy‐1 mRNA decay in S49 T lymphoma cells through a classical β2AR/AC/cAMP/PKA pathway, identified an ARE (a sequence associated with message regulation) in the 3’UTR of Thy‐1 mRNA and at least 10 ARE binding proteins for the Thy‐1 mRNA ARE including AUF1, HuR, TIAR, KSRP, and Hsc70. This study investigates the relationship between HuR expression and cAMP mediated Thy‐1 decay by knocking down HuR with short hairpin RNA (shRNA). S49 cells were stably transfected with a vector containing either a nonspecific scrambled shRNA (scr) or an shRNA specific for HuR. Whole cellular HuR protein levels detected by immunoblotting and mRNA levels detected by qPCR showed a 50% decrease in the expression of HuR in the knockdown cells compared to the scr cells. This decrease of HuR expression in the HuR shRNA cells did not alter the basal levels of Thy‐1 mRNA, but did correlate with an increase in Thy‐1 message decay after 4 hrs of treatment with a cAMP analogue. These results suggest a stabilizing role for HuR in cAMP mediated Thy‐1 mRNA decay. Supported by NIH.
Are the KIR genes actively silenced prior to their tissue‐specific activation? A KIR intronic promoter produces spliced antisense transcripts in human ES cells
Authors: Anderson Stephen Kent , Li Hongchuan , Sharma Neeraj , Wright Paul W , Woll Petter , Kaufman Dan
Published: June 2021

The KIR genes are in a silent methylated state in NK progenitors prior to their selective activation by developing NK cells. We have recently identified an additional antisense transcript originating in the second KIR intron. The KIR2DL1 antisense transcript is composed of three exons that contain: (1) a segment from intron 2; (2) exon 2 plus flanking intron sequences; (3) exon 1 plus the bidirectional promoter region. The composition of the spliced antisense KIR transcript (KIRaS) suggests that it functions to silence KIR gene expression. KIRaS was detected in human embryonic stem (ES) cells, an embryonal carcinoma (EC) cell line (Ntera2), and the HEK293 cell line, but not NK, T, or B cell lines. In vitro promoter analysis revealed that KIRaS promoter activity is only detected in the cell lines where transcript is present. Promoter activity is controlled by the MZF‐1 transcription factor that is expressed in totipotent hematopoieticcells and myeloid progenitors. KIR gene activation appears to be under the control of four distinct promoter units: distal forward; proximal forward or reverse; distal antisense. The potential interplay of the multiple KIR promoters and the timing of their expression during development will be discussed.

Funded by NCI Contract N01‐CO‐12400
Enhanced RNAi‐1 (Eri‐1) regulates miRNA homeostasis, rRNA processing, and lymphocyte effector functions
Authors: Ansel Karl Mark , Pastor William A , Rath Nicola , Lapan Ariya Dararutana , Thai To‐Ha , Rao Anjana , Heissmeyer Vigo H
Published: June 2021

Helper T cell cytokine production and effector cell differentiation are influenced by endogenous RNA interference (RNAi). Northern blot analysis revealed that many miRNAs are rapidly downregulated following antigen stimulation of B and T cells. The murine orthologue of C. elegans Eri‐1 (also known as Thex1), an exonuclease that degrades short‐interfering RNA (siRNA), is upregulated in activated T cells, and cells lacking Eri‐1 exhibited increased miRNA expression and siRNA function. However, activation‐induced miRNA downregulation still occurs in the absence of Eri‐1. Eri‐1‐deficient helper T cells are biased to differentiate into Th2 effectors, and Eri‐1 deficiency in B cells leads to reduced germinal center responses. In addition to regulating RNAi, Eri‐1 catalyzes 5.8S ribosomal RNA 3′ end processing. In its absence, ribosomes are assembled with 5.8S rRNA extended by 1–2 nucleotides, and Eri‐1 can process these aberrant ribosomes to the wildtype form in vitro and in cells. Surprisingly, Eri‐1 remains associated with ribosomal subunits after 5.8S processing is complete. Further investigation is needed to clarify the relationship between the molecular functions of Eri‐1 in ribosome biogenesis and miRNA regulation, and their contribution to the defective functions of Eri‐1‐deficient lymphocytes.
Phylogenetic and Functional Analyses Identify Ets‐1 as an Important Regulator of the Th2 Cytokine Gene Locus
Authors: Strempel Jannine M , Grenningloh Roland , Ho I‐Cheng , Vercelli Donata
Published: June 2021

The Th2 cytokine gene locus has emerged as a remarkable example of coordinated gene expression, the regulation of which appears to be rooted in an extensive array of cis‐regulatory regions. Using a computational approach that integrated multi‐species (n=11) sequence comparisons with algorithm‐based transcription factor (TF) binding site predictions, we sought to identify evolutionarily conserved non‐coding regions (ECRs), and motifs shared among them, which may underlie coregulation. Twenty‐two TF families were predicted to have binding sites in at least two Th2 ECRs. The ranking of these shared binding motifs according to their distribution and relative frequency pointed to a regulatory hierarchy among the TF families. Indeed, GATA sites were the most prevalent and widely distributed, consistent with the known role of GATA3 as a Th2 master switch. Sites for ETS‐domain proteins were also predicted within several Th2 ECRs, and the majority of these sites supported Ets‐1 binding in vitro. Analysis of Ets‐1 binding in vivo is ongoing. Interestingly, the expression of IL5, IL13 and IL4, but not IL10, was significantly decreased in Th2 cells generated from Ets‐1‐deficient mice. Collectively, our data suggest that Ets‐1 binding to motifs throughout the Th2 locus may contribute to the regulation of Th2 cytokine genes.
Blimp‐1 directly represses il2 and the gene encoding il2 transcriptional activator Fos
Authors: Martins Gislaine A. , Cimmino Luisa , Calame Kathryn
Published: June 2021

The transcriptional repressor B‐lymphocyte‐induced maturation protein 1 (Blimp‐1) was initially studied for its crucial role in plasmacytic differentiation. More recent studies, however, have demonstrated that Blimp‐1 is expressed in T lymphocytes and plays important roles for the function of these cells. T‐cell specific deletion of Blimp‐1 (CKO mice) disrupts the homeostasis of CD4+ and CD8+ T cells, resulting in accumulation of antigen‐experienced cells in the periphery and culminating in the spontaneous development of colitis. We have previously reported that lack of Blimp‐1 in T cells results in increased production of IL‐2. We now present evidence that Blimp‐1 represses transcription of the il2 gene in CD4+ T cells by acting in at least two different ways: Blimp‐1 directly represses il2 and the gene encoding il2‐activator Fos, as demonstrated by chromatin immunoprecipitation (ChIP) assays in primary CD4+ T cells. Accordingly, IL‐2 and Fos steady‐state mRNA levels are elevated in Blimp‐1 deficient CD4+ T cells compared to Blimp‐1 sufficient cells. The implications of this regulation of IL‐2 by Blimp‐1 in vivo are currently been investigated and preliminary evidence suggests that Blimp‐1 is a critical component of a recently reported IL‐2 auto‐regulatory circuit that controls the development of effector responses.
Association Between BclI SNP of the Glucocorticoid Receptor (GCR) And Immune Response to Stress
Authors: Xiang Lianbin , Olivier Jacob , Ben Kevin Del , Ferguson Lee M , Marshall Gailen D
Published: June 2021

Stress influences immunity through various mechanisms including immunoregulatory elements. Previous studies show associations between high stress levels and altered immune balance such as in TH1/TH2 (IFNγ, IL‐4, IL‐10) cytokine production. Such immunoregulatory imbalances are associated with altered serum cortisol levels and can be reproduced with glucocorticoids (GC) in vitro. GC act through the GCR for which several SNPs have been described. The GCR SNPs BclI and N363S are associated with hyperreactivity to GC. We examined the associations between GCR SNPs and cytokine responses. 86 normal volunteers were stratified into four groups by k‐means clustering using Perceived Stress Score (PSS) and immunologic variables. We examined numbers of CD4+CD25+FoxP3+ (Treg) and CD4+ cells producing TH1/TH2 cytokines by intracellular flow cytometry. DNA was extracted from peripheral blood and SNP determined by PCR and RFLP analysis. Clusters were categorized by PSS into high stress (HS‐19.2±3.5, n=15), low stress (LS‐8.3±4.7, n=7) and two groups of typical stress (TS‐12.6±5.0, n=37 and 12.7±6.3, n=27). The HS cluster had decreased Treg (2.0±0.6%) and TH1 (22.1±4.4%) compared to the LS cluster with higher Treg (3.5±1.1%) and TH1 (33.4±6.7). There were no cluster differences in IL‐4 and IL‐10. BclI (but not N363S) SNP showed a trend in C/C homozygosity in 66.7% (10/15) of the HS compared to 28.6% (2/7) of the the LS cluster (χ2(1)=2.79, p=0.095). These results suggest that genetic variation, such as GCR BclI SNP, may participate in regulation of immune response to stress.
Characterization of invasion pattern of Shigella flexneri in the small intestine of infant mice
Authors: Shim Doo‐Hee , Kweon Mi‐Na
Published: June 2021

Shigella spp. invades the large intestinal epithelium and promotes a strong inflammatory response in humans. A recent study revealed that 4‐day‐old mice were infected with invasive Shigella strains but not 5‐day‐old mice. We therefore attempted to clarify the underlying mechanism that induces inflammation in the small intestine of infant mice but not of adult mice. Balb/c mice of different ages were orally administered 5 × 109 CFU of Shigella flexneri 5a (M90T) strain and analyzed for colonization and physiopathological features. Unexpectedly, we found that 4‐, 7‐, and 14‐day‐old mice, but not 21‐day‐old mice, showed tissue destruction and PMN infiltration in the small intestine at 6 h following Shigella infection, mimicking human shigellosis. Interestingly, Shigella mainly invaded epithelial cells and crypt region but not the M cells in the small intestine. Further, high numbers of Shigella were mainly colonized in the lamina propria but not in Peyer's patches in the small intestine, and the Shigella colonization gradually decreased with ageing. We are now attempting to determine the crucial signaling mechanism of Shigella infection in the epithelium and crypt region of the small intestine of infant mice.
In situ imaging studies reveal different responses by naïve and memory CD8 T cells to late antigen presentation by lymph node DC after influenza infection
Authors: Cauley Linda S. , Khanna Kamal M. , Lefrancois Leo
Published: June 2021

The lung draining lymph nodes (DLN) remain reactive for several weeks after pulmonary influenza virus infection while processed viral antigens continue to be presented to virus‐specific CD8 T cells. Scanning confocal microscopy has been used to analyze the response of naïve and memory influenza‐specific CD8 T cells to early and late antigen presentation. Naïve CD8 T cells responded to antigen that was in the DLN until at least 60 days after infection, but lost the ability to become fully differentiated effector cells as the time after infection increased. Many of the responding naïve T cells formed large stable clusters of dividing cells in the paracortex of the DLN where they remained in close association with DCs for up to a week after transfer. Central/memory cells also reached the DLN after the day 30 transfers, but showed no sign of a response to late antigen presentation in vivo or in vitro. In contrast endogenous NP‐specific CD8 T cells in the DLN exhibited signs of a response to late antigen presentation, but were phenotypically consistent with effector/memory cells that had been recruited to the DLN earlier in the response. These data show that naïve and memory CD8 T cells use distinct mechanisms to detect antigen‐bearing APC in some situations and that viral antigen continues to play an instructive in T cell migration long after infectious virus has been eliminated. The local changes in T cell migration have important implications for protective immunity since some acutely activated T cells can be retained near the site of infection, while other virus‐specific central/memory CD8 T cells are protected from exhaustion during late antigen‐presentation.

This work was supported by the Damon Runyon Foundation (KMK) and National Institutes of Health grants AI065895 and AI071213. (LSC), DK45260 and AI41576 (LL).
Nasal Cholera Toxin Up‐regulates Mucosal DCs And B1 B Cells Interactions For The Induction Of Innate IgA CSR.
Authors: KATAOKA KOSUKE , Fujihashi Keiko , Sekine Shinichi , Kobayashi Ryoki , Sims Rebekah , Ito Hiroo , McGhee Jerry R , Shizukuishi Satoshi , Fujihashi Kohtaro
Published: June 2021

Our previous study showed that nasal immunization with TNP‐LPS plus cholera toxin (CT) as mucosal adjuvant elicited increased levels of mucosal TNP‐LPS‐specific secretory (S)‐IgA Ab responses through an IL‐5‐IL‐5 receptor interplay between CD4+ T cells and B1a B cells in murine submandibular glands (SMGs) and nasal passages (NPs). Thus, RT‐PCR assay revealed that NALT, NPs and SMGs of mice given nasal TNP‐LPS plus CT contained AID, αCT and Iμ‐Cα transcripts. In this study, we tested our hypothesis that DCs play a central role in the induction of IgA class switching recombination (CSR) by mucosal B1 B cells for the enhancement of T cell‐independent S‐IgA Ab responses. When cells from NPs and SMGs were subjected to flow cytometric analysis, DCs of mice given nasal TNP‐LPS plus CT expressed higher levels of co‐stimulatory molecules than those of mice given TNP‐LPS alone, even though the numbers of DCs in NPs and SMGs were unchanged. Interestingly, higher levels of APRIL‐specific mRNA was expressed by DCs in NPs and SMGs, while B cells in SMGs and NPs showed significant levels of TACI‐ and BCMA‐specific mRNA. These findings suggest that DCs activated by nasal CT induce B cell IgA CSR through TACI and APRIL/BMCA interactions. This work is supported by NIH grants DE 12242, AG 025873, AI 18958 and AI 043197 as well as by Grants‐in‐Aid (C‐17592179, C‐19592403) from the Ministry of Education, Culture, Sports and Science of Japan.
CCL5 modulates pneumococcal surface protein A (PspA) peptide‐specific T helper cell responses
Authors: SINGH RAJESH , SINGH SHAILESH , SINGH UDAI P , BRILES DAVID E , LILLARD JAMES W
Published: June 2021

Pneumococcal surface protein A (PspA) is a cell surface protein present on Streptococcus pneumonia. We have previously shown that CCL5 blockade alters PspA‐specific humoral and cellular pneumococcal immunity, during S.pneumoniae strain EF3030‐induced carriage. Here we show that CCL5 blockade decreases the number of IFNγ+ and increases the number of IL10+CD4+ T cells in the spleen and nasal tract (NT) during the recognition phase of the pneumococcal adaptive immune response. Residues 287 to 378 of PspA caused the greatest cytokine secretion and proliferation recall responses by splenic and NT CD4+T cells isolated from F1 (B6 × BALB/c) mice. CCL5 inhibition significant reduction in the capacity for IFNγ, but not TNFα or IL4, secretion by mucosal and systemic PspA peptide specific CD4+T cells. CCL5 blockade also resulted in a significant increase in PspA peptide specific IL 10 producing CD4+ T cells compared to control Ab treated mice. In support CCL5 deficiency resulted in a higher number of CCL5+ CD11b+ CD11c+and IL‐10+CD11b+cells, but lower number of CCR5+ CD11b+ CD11c−cells in the spleen compared to similarly challenged mice that received control Ab. Together, these changes corresponded with the transition from pneumococcal carriage to lethal pneumonia. These data suggest that CCL5 is an essential factor for the induction and maintenance of protective pneumococcal immunity.
Pregnancy Modulates Adoptively Transferred Experimental Autoimmune Encephalomyelitis (EAE)
Authors: Song Fei , Gienapp Ingrid , Shawler Todd , Williams Jessica , Smith Kristen , Kithcart Aaron , Whitacre Caroline C
Published: June 2021

Pregnancy affects the clinical course of autoimmune disease such as multiple sclerosis (MS), Rheumatoid arthritis and lupus. In this study, we determined the immunoregulatory role of pregnancy on the effector phase of murine experimental autoimmune encephalomyelitis (EAE), a model for studying MS. We used an adoptive transfer system with myelin basic protein (MBP) peptide NAc1‐11 activated Th1 or Th17 MBP T cell receptor (TCR) transgenic (Tg) donor cells. Transfer of activated Th1 cells to recipients during mid pregnancy resulted in EAE with a delayed onset and reduced clinical severity. No significant differences in EAE were observed when Tg cells transferred into late pregnant or post partum recipients compared with virgin controls. Mice during mid pregnancy produced less Th1 cytokines (IFN‐gamma and IL‐2) but increased IL‐10. In contrast, mice during post partum produced more Th1 and Th17 cytokines. No differences were noted in Foxp3 regulatory cells or Th2 cytokine production. These results suggest that mid pregnancy provides an immunoregulatory environment that suppresses Th1‐induced EAE (Supported by NIH grant AI 43376).
The role of PD‐1 during maternal T cell responses to fetal antigen during pregnancy
Authors: Taglauer Elizabeth Susan , Yankee Thomas M , Petroff Margaret G
Published: June 2021

The failure to reject the semiallogenic fetus suggests that maternal T lymphocytes are regulated by potent mechanisms in pregnancy. PD‐1, a CD28 family receptor, is expressed on activated T cells and may be involved in establishing peripheral tolerance. Here we tested the role of PD‐1 in maternal tolerance to the fetus. No significant differences in fetal viability were observed between syngenic (n=8) and allogenic pregnancies (n=10) in PD‐1−/− mothers. We further characterized the function of PD‐1 on maternal T cells using adoptive transfer of naïve OT‐I cells into female mice carrying ovalbumin (ova)‐expressing or wild type (WT) fetuses. CFSE‐labeled spleen cells from OT‐I mice were transferred at mid‐gestation into females bred to ova‐transgenic or WT males. Three days post‐transfer, cells from uterus‐draining lymph nodes were stained for OT‐I markers and PD‐1 and analyzed by flow cytometry. Transferred OT‐I cells proliferated and upregulated PD‐1 in females carrying ova‐expressing fetuses. We next transferred either OT‐I/PD‐1+/− or OT‐I/PD‐1−/− cells, and in the absence of PD‐1 there was a significant increase in the percentage of OT‐I cells detected in lymph nodes (p = 0.003, n = 3). These results suggest that PD‐1 aids in controlling maternal T cell responses to fetal antigen, and that pregnancy involves mechanisms in addition to PD‐1 to maintain maternal tolerance to the semiallogenic fetus. Support: NIH HD045611.
An edema toxin‐based sublingual vaccine promotes immunity against both anthrax toxin and yersinia pestis antigens
Authors: Boyaka Prosper N , Duverger Alexandra , Tang Wei‐Jen , Leppla Stephen H
Published: June 2021

Our separate studies have shown that nasal co‐administration of anthrax edema toxin (EdTx) derivative [i.e., protective antigen (PA) plus edema factor (EF) mutant N447S], enhances mucosal IgA and systemic immunity to both PA and co‐administered unrelated protein antigen. However, while both native and mutant derivatives of EdTx as transcutaneous adjuvants promoted IgG (serum and saliva) responses systemic to either PA or co‐administered antigen they failed to induce IgA responses. This study examined whether sublingual application of EdTx could induce mucosal IgA Ab responses against both anthrax PA and Yersinia pestis F1‐V antigens. Mice immunized by sublingual application of EdTx plus recombinant F1‐V developed significant levels of antigen (i.e., F1‐V and PA)‐specific IgA Abs in the serum, saliva, and vaginal secretion. The adjuvant activity of EdTx given by the sublingual route induced IgG responses characterized by IgG1>IgG2b>>IgG2a, IgG3. Interestingly, sublingual immunization with F1‐V and cholera toxin as adjuvant promoted IgG responses with the characteristics of a mixed Th1/Th2 responses (i.e., IgG1>IgG2a, IgG2b>IgG23. Our results indicates that EdTx derivatives represent a new class of adjuvant that could be employed for the development of multivalent transcutaneous vaccines (Supported in part by NIH Grant: AI 43197).
CXCL10 blockade protects mice from cyclophosphamide‐induced cystitis
Authors: Sakthivel Senthilkumar K , Singh Udai P , Singh Shailesh , Taub Dennis D , Lillard James W
Published: June 2021

New intervention and prevention strategies are greatly needed for cystitis, which is a chronic, relapsing, and severely debilitating disease of the urinary bladder. Our study shows that the serum levels of monokine‐induced by interferon‐γ (IFN‐γ) (MIG/CXCL9), IFN‐γ‐inducible protein‐10 (IP‐10/CXCL10), and IFN‐γ‐inducible T cell α chemoattractant (I‐TAC/CXCL11) are elevated in patients with cystitis. This clinical feature closely correlates with cyclophosphamide (CP)‐induced murine cystitis, in which the serum levels of these CXCR3 ligands and local T helper type 1 (Th1) cytokines were also increased. We demonstrate that CXCR3 as well as CXCL9 and CXCL11 mRNA levels were up regulated at sites of urinary bladder inflammation, while CXCR3 and CXCL9 transcripts were elevated in the iliac lymph nodes following CP treatment. The number of CD4+ T cells, mast cells, neutrophils, natural killer (NK) cells, and NKT cells were increased at systemic and mucosal sites, after CP treatment in mice. Most importantly, we show that CXCL10 blockade impeded the development of cystitis in CP‐treated mice, decreased the local production of CXCR3 ligands as well as Th1 cytokines and lowered their corresponding serum levels. Urinary bladder CD4+ T cell, mast cell, neutrophil, and NKT cell infiltrates were also reduced following anti‐CXCL10 antibody (Ab) treatment of mice with cystitis. The present study provides new and important information regarding the cellular and molecular mechanisms of cystitis.
Trans‐presentation of IL‐15 by intestinal epithelial cells drives development of CD8 alpha alpha T cells
Authors: Ma Lisa J. , Schluns Kimberly S.
Published: June 2021

IL‐15, which is trans‐presented by IL‐15Rα+ cells to neighboring IL‐2Rβ/γC+ cells, is crucial for the development of intestinal intraepithelial lymphocytes (IEL). Parenchymal cells have a major role in the trans‐presentation of IL‐15 to IELs; however, the specific identity of this cell type is unknown. Since trans‐presentation requires cell‐cell contact, we hypothesize that intestinal epithelial cells (IEC) are the parenchymal cell trans‐presenting IL‐15 to IELs. To determine whether IECs trans‐present IL‐15 to developing IELs, we developed a mouse model with IL‐15Rα expressed exclusively by the IECs. This model was generated by driving IL‐15Rα under the Villin promoter and crossing to the IL‐15Rα−/− background. Analysis of these transgenic mice detected high levels of IL‐15Rα by the IECs. Among the IELs, Thy1‐ CD8αα TCRαβ and TCRγδ subsets were restored to normal or higher levels as compared to control and IL‐15Rα−/− mice. The increases in cell number did not appear to be the result of cell expansion as no differences in proliferation rate was detected among transgenic, control, or IL‐15Rα−/− mice. Collectively, this study demonstrates that IL‐15Rα expression by IECs alone is completely sufficient to direct all the IL‐15‐mediated events driving the development of CD8αα IELs. This research is supported by NIH grant AI070910 and the MDACC Trust Fellowship.