bluuebunny/crossref_metadata_2025 huggingface dataset:
Dose-response immunomodulatory effects of glucocorticoid on gene expression of cytokine and stress hormone receptors in human PBMC (57.18)
Authors: Xiang Lianbin , Marshall, Jr. Gailen
Published: January 2023

Previous studies demonstrate Th1/Th2 cytokine balance is altered with stress hormone equivalent dexamethasone (Dex) levels in human PBMC cultures. The modulatory effects of Dex on gene expressions of FoxP3, IFN-γ receptors (IFN-γR), IL-4 receptors (IL-4R), glucocorticoid receptors (GR), and beta 2 adrenergic receptors (β2AR) were investigated. PBMC from 17 normal humans were cultured with 10-7, 10-8, or 10-9M Dex (mimics high, low, and non stressed states) for 24 hrs (short term) and 11 days (long term). Gene expression changes were assessed by real-time PCR. Results showed mRNA expressions of FoxP3, GR and IL-4R were decreased, whereas β2AR and IFN-γ R were upregulated with various concentrations of Dex (24 hrs). After 11 days, FoxP3 level remained down-regulation, whereas GR and β2AR levels returned to control levels in all cultures. In contrast, IL-4R expression increased (11 days) in all Dex concentrations. This resulted in decreased IFN-γR/IL-4R from 24 hrs (favoring Th1) to 11 days (favoring Th2) with Dex. This suggests that short term Dex exposure (acute stress) can initiate immunoregulatory reactions (i.e. Th1 based) that may be protective against many infectious challenges, possibly through a down-regulation of GR resulting in less suppressive effects of steroids and increased sensitivity to catecholamines. Longer term Dex exposure (chronic stress) may ultimately alter host response resulting in increased susceptibility to infections and/or hypersensitivity states.
Immune mechanisms associated with altered infant CD4+ T cell function (57.20)
Authors: dela Pena Myra Grace , Wicks Megan , Rao Kalyan , Abel Kristina
Published: January 2023

Infant, when compared to adult, CD4+ T cell responses are characterized by a T helper 2 bias and reduced IFN-γ production. We hypothesized that the (1) expression of genes important in TCR signaling is age-dependent and (2) transcription factors involved in cytokine signaling (e.g. signal transducer and activator of transcription (STATs), are differentially activated in infant and adult CD4+ T cells. Naïve and differentiated CD4+ T cells from 2 to 6 months old infant rhesus macaques were FACS-purified, stimulated with anti-CD3 and anti-CD28 antibodies, and analyzed by a Rhesus Th1/2/3 PCR array. The results showed an age-dependent increase in genes associated with Th1 development (e.g. T-bet). Next, human and/or rhesus cord and adult blood samples were stimulated with IL-2, IFN-γ or IL-4 and analyzed by Phosflow for the phosphorylation of STAT5, 1, or 6, respectively. While both infant and adult CD4+ T cells expressed a high percentage of phosphorylated STAT6 positive cells, adult CD4+ T cells had significantly higher frequencies of phosphorylated STAT5 and STAT1 positive CD4+ T cells. Thus, our data indicate that mechanisms of T cell activation differ between infant and adult CD4+ T cells. Defining these differences in distinct signaling pathways may provide novel and critical insights into pediatric HIV vaccine and therapeutics design.
Nogo B Receptor is expressed in a subset of T cells; is AmNogoB a cytokine? (57.21)
Authors: Valle-Rios Ricardo , Burkhardt Amanda , Hevezi Peter , Zlotnik Albert
Published: January 2023

The Reticulon/Nogo is a large family of proteins, which share the C-terminal region of approximately 200 aa, called the Reticulon homology domain (RHD). Many of these molecules have been identified in the Central Nervous System and that is where most studies have focused so far. The best characterized gene of the Reticulon family is RTN4. It is composed of three isoforms: RTN4/Nogo A, RTN4/Nogo B and RTN/Nogo C. Nogo B plays an essential role in vascular remodeling and appears to be important in angiogenesis and metabolic processes. Interestingly, the amino terminal region of Nogo B (amNogo B), can be a soluble peptide that promotes vascular adhesion and migration of endothelial cells by binding the recently reported Nogo B receptor (NgBR). Using a comprehensive database of gene expression in the human body, we made the unexpected observation that the cells exhibiting the strongest expression of NgBR include activated CD4+ and CD8+ T cells. We have confirmed that NgBR is expressed in a subpopulation of activated T cells expressing the activation marker CD69 in mouse, indicating the existence of a new subpopulation of activated T cells that are NgBR+CD69+. Since amNogoB is present in plasma, it should be able to bind this new subpopulation of activated T cells via NgBR and therefore it may play a role in modulating T cell activation. We are currently studying the functional consequences of the amNogoB:NgBR interaction in activated T cells.
MicroRNA-29 stabilizes interferon-gamma mRNA by antagonizing AU-rich element-mediated decay (57.3)
Authors: Savan Ram , Legiewicz Michal , McFarland Adelle , Schwerk Johannes , Bindewald Eckart , Orr Selinda , Ramakrishnan Karthika , Yalamanchili Rajesh , Kronfli Anthony , McVicar Daniel , Carrington Mary , Anderson Stephen , Shapiro Bruce , LeGrice Stuart , Young Howard
Published: January 2023

Tight regulation of interferon-gamma (IFN-g) expression is critical for an optimal immune response. IFN-g mRNA levels are attenuated by AU-rich element (ARE)-mediated decay (AMD) via the tristetraprolin (TTP) complex. Here we show that miR-29 targets the 3'untranslated region of IFN-g mRNA and unlike the conventional inhibitory function of miRNAs, increases IFN-g protein expression by stabilizing the mRNA. Through analysis of RNA structure derived by chemoenzymatic probing, we demonstrate that the AMD function is blocked by a miR-29 induced RNA conformational switch that makes the TTP binding site inaccessible. This effectively results in competition between the miRNA-induced silencing complex and TTP for post-transcriptional control of IFN-g gene expression. Our proposed role of miR-29 in stabilizing IFN-g provides a novel molecular mechanism of miRNA regulation and presents new targets for the manipulation of the immune system in the treatment of human diseases.
Type I interferon and pro-inflammatory cytokine synthesis induced by Toll-like receptor signaling in dendritic cells is regulated by estrogen receptor alpha (57.7)
Authors: Kovats Susan , Bentley Caroline , Turner Sean , Roach Kimberly , Wilburn Stephanie , Khan Sohaib
Published: January 2023

Estrogen receptors (ER) regulate target gene expression by forming complexes with chromatin-modifying coregulators and transcription factors. Indeed, ER alter the activity of NF-κB and C/EBPβ, which are crucial regulators of cytokine gene expression. To test the hypothesis that ER alpha (ERα) signaling regulates pro-inflammatory cytokine and type I interferon production by CD11c+ dendritic cells (DC), we developed a novel line of mice, bearing CD11c-Cre and a conditional allele of ERα, in which ERα deficiency is restricted to CD11c+ cells. Isolated ERα+ or ERα-/- splenic DC were stimulated by Toll-like receptor (TLR) ligands in steroid-deficient medium with physiological levels (1 nM) of estradiol, followed by assessment of cytokine RNA and protein synthesis. ERα deficiency significantly reduced the synthesis of Ifna and Il6 RNA and protein by plasmacytoid DC triggered via TLR7 with influenza virus or via TLR9 with unmethylated CpG-rich DNA. In conventional DC stimulated via TLR9, ERα deficiency led to reduced levels of Il1a, Csf2, Ifnb, Nos2 and Ifng RNA and IL-1α and GM-CSF protein. Taken together, these data show that cell autonomous ERα signaling in DC promotes type I interferon and pro-inflammatory cytokine synthesis induced by pathogen-specific nucleic acids. Thus, estradiol-bound ERα may interact with transcription factors activated by TLR 7 and 9 signaling to increase transcription of cytokine genes, thereby regulating the ability of DC to promote pathogen immunity.
Listeria monocytogenes inhibits innate immunity by driving regulatory B10 cell expansion (58.10)
Authors: Weimer Eric , Horikawa Mayuka , Tedder Thomas
Published: January 2023

Regulatory B cells, including the CD1dhiCD5+ IL-10-competent regulatory B cell subset called B10 cells, significantly influence immune responses. In this study, B10 cell regulatory functions were examined during L. monocytogenes infection. Five days post-infection, B10 cells had expanded ≥2-fold in the spleen, lymph nodes, and blood of wild type mice. Remarkably, CD19-/- mice that lack B10 cells were more resistant to L. monocytogenes infection when compared with wild type mice. Furthermore, the adoptive transfer of wild type CD1dhiCD5+ B cells, but not CD1dloCD5- B cells, inhibited bacterial clearance in CD19-/- mice. To confirm the importance of endogenous B10 cell expansion in wild type mice, a monoclonal antibody that preferentially depletes B10 cells was given to mice before Listeria infection. B10 cell depletion lowered bacteria numbers in the spleen by two-logs when compared with control antibody-treated mice. Furthermore, monocytes from infected mice produced 2-3 fold more IFN-γ and TNF-α after B10 cell depletion. Taken together, these results argue that endogenous B10 cells negatively regulate monocyte activation and IFN-γ production, which are both critical for early Listeria clearance. As a consequence of lowered bacterial load, the proliferation of antigen-specific CD4+ T cells was reduced 5-fold in mice that were depleted of B10 cells. Thus, endogenous B10 cells negatively regulate innate immune responses during Listeria infection.
Immunization with heat-killed T. cruzi prior to T. cruzi infection causes increased cardiac inflammation and decreased parasitosis (58.11)
Authors: Bonney Kevin , Taylor Joann , Engman David
Published: January 2023

Chagas’ heart disease (CHD), caused by the protozoan parasite Trypanosoma cruzi, is a potentially fatal myocarditis often associated with cardiac-specific autoimmunity. Immunization with heat-killed T. cruzi (HKTC) induces polyantigenic autoimmunity similar to that observed during T. cruzi infection, as well as acute cardiac damage. However, HKTC-immunized mice do not develop myocarditis or the robust Th1/Th17-skewed immune response observed in T. cruzi-infected mice. We hypothesized that HKTC immunization inhibits inflammatory Th1 and Th17 responses and myocarditis by inducing a regulatory response (Treg). We infected mice that had been immunized with HKTC or PBS for 28 days with T. cruzi, and assessed the development of myocarditis and Th responses through 21 days post-infection. HKTC-immunized, infected mice developed more cardiac inflammation and lower cardiac parasitosis than controls, and a reduced Th1 response late in infection. This suggested that prior exposure to HKTC may actually suppress regulatory responses in infected mice. To assess the role of Tregs in these responses, we treated mice with neutralizing anti-CD25 antibody, and analyzed the development of myocarditis and Th responses following Treg depletion. Our findings suggest that autoimmunity may contribute to the pathogenesis of CHD, which has implications for the efficacy of antigen-based vaccines. We are continuing to investigate the mechanism(s) by which HKTC modulates the inflammatory response.
A possible TLR2-medited immunity to experimental cysticercosis caused by Taenia crassiceps (58.3)
Authors: Terrazas Luis I , Reyes Jose Luis , Gonzalez-Gonzalez Marisol I. , Satoskar Abhay R.
Published: January 2023

TLR2 is a key molecule required for innate immunity and is involved in the recognition of a wide range of pathogens from bacteria, fungi, parasites and viruses. Its role on immunity to cestode infections is poorly understood. Here the role of TLR2 was evaluated in a Taenia crassiceps infection model. Compared with wild-type mice, TLR2 knockout (KO) mice failed to produce significant levels of inflammatory cytokines in both sera and their spleens during the first two weeks of Taenia infection. TLR2 KO mice developed a Th2-dominant whereas wild-type mice developed a Th1-dominant immune response after Taenia infection. Associated with the insufficient production of early inflammatory cytokines and lack of Th1-dominant adaptive immunity, TLR2 KO mice developed significantly higher numbers of parasites whereas WT mice appeared to be resistant. TLR2 KO mice developed more amounts of alternatively activated macrophages. These results suggest that TLR2-dependent signaling pathway is not only required for inflammatory cytokine production in the early phase of host response to T. crassiceps infection but also that plays a critical role in the development of Th1-adaptive immunity, both of which may be essential for limiting infection and reducing pathology during experimental cysticercosis. Collectively, these studies indicate that TLR2 is involved in T. crassiceps recognition and subsequent innate immune activation.
Defective neutrophil function underlies the absence of T cell recruitment into challenge sites during elicitation of contact sensitivity in gld/perforin-/- mice (60.7)
Authors: Kish Danielle , Gorbachev Anton , Fairchild Robert
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell mediated response to hapten sensitization and challenge of the skin. Hapten-primed CD8 T cell infiltration into the skin challenge site is not observed until 12-16 h post-challenge and requires prior neutrophil infiltration and activation within the site. The neutrophil activities required to direct this T cell infiltration to mediate CHS responses are undefined. Although primed CD8 T cells do not express FasL or perforin, CHS responses to 2, 4-dinitrofluorobenzene (DNFB) are absent in gld/perforin-/- mice. ELISPOT analyses indicated equivalent priming of DNFB-specific CD8 cells in sensitized wild-type (WT) and gld/perforin-/- mice. Whereas DNFB-primed CD8 T cells from WT or gld/perforin-/-mice transferred CHS responses to naïve WT recipients, WT CD8 T cells did not transfer the response to gld/perforin-/- mice. Neutrophil infiltration into the challenge site of sensitized gld/perforin-/- and WT mice was equivalent at 6 h post-challenge but T cell infiltration was absent in the challenge site of gld/perforin-/- mice at 18 h. This T cell infiltration and the CHS response were restored by transferring WT neutrophils i.v. to sensitized gld/perforin-/- mice at challenge. Neutrophils from sensitized WT mice expressed high levels of FasL and perforin mRNA. These results indicate that neutrophil expression of FasL and perforin required to direct effector CD8 T cell recruitment into the skin challenge site to mediate CHS responses.
B cells have a dual effect on pulmonary fibrosis induced by bleomycin in a mouse model (61.11)
Authors: Hamaguchi Yasuhito , Tedder Thomas , Fujimoto Manabu
Published: January 2023

CD20 antibody immunotherapy effectively treats non-Hodgkin’s lymphoma and autoimmune diseases. However, the contributions of B cells to the pathophysiology of pulmonary fibrosis remains unknown. Therefore, the contributions of B cells to the initiation and progression of pulmonary fibrosis was quantified using the mouse model of bleomycin-induced fibrosis. B cell depletion before the induction of lung fibrosis substantially exacerbated disease symptoms. By contrast, B cell depletion during the inflammatory phase of pathology dramatically suppressed disease symptoms. Similarly, pulmonary fibrosis was also significantly worse in genetically B cell-deficient μMT mice when compared with wild type mice. The adoptive transfer of wild type B cells into μMT mice before disease initiation significantly reduced pulmonary pathology in μMT mice. Thus, B cells have important regulatory and pathogenic roles during the immunopathogenesis of lung fibrosis. These findings parallel published observations in the EAE model of multiple sclerosis and the NZBxW F1 mouse model of lupus, where a regulatory subpopulation of endogenous B cells (B10 cells) significantly reduces disease severity during early disease onset through the production of IL-10. Thereby, B cell depletion for the treatment of fibrosis may depend on the relative contributions and the timing of these opposing B cell activities during the course of disease.
Positive selection during affinity maturation relies on T cell derived BLyS (61.3)
Authors: Goenka Radhika , Mathews Andrew , O'Neill Patrick , Scholz Jean , Leonard Warren , Stohl William , Cancro Michael
Published: January 2023

Protective humoral immunity relies on high affinity B cell clones that are generated and selected during the germinal center (GC) reaction. To determine whether BLyS family member(s) play a role in this process, we assessed their distribution within the GC. We show that majority of the GC is devoid of BLyS despite the presence of ample BLyS in the adjacent follicular regions. The paucity of BLyS correlates with down-regulation of TACI receptor on GC B cells and likewise follicular B cells in TACI-deficient mice bind less BLyS. TACI down-regulation on follicular B cells is mediated by IL-21 in a surrogate thymus-dependent stimulation. Furthermore, some BLyS staining is detectable in the light zone associated by T Follicular Helper cells (TFH). The expression of BLyS by TFH is crucial for appropriate GC evolution, since efficient affinity maturation fails in mixed bone marrow chimeras where T cells are deficient in BLyS. We conclude that positive selection during affinity maturation relies on restricted sources of BLyS on GC TFH that provide survival signals to preserve high affinity clones.
Dendritic cell-B cell interactions in the marginal zone drive extrafollicular antibody responses. (61.4)
Authors: Chappell Craig , Draves Kevin , Giltiay Natalia , Clark Edward
Published: January 2023

Targeting Ag directly to dendritic cell (DC) subsets through the use of monoclonal antibodies (mAb) is increasingly being employed as a means to generate CD4 and CD8 T cell responses. However, the ability of individual DC subsets to raise primary Ab responses following Ag targeting is not well characterized. Using the hapten 4-hydroxy-3-nitrophenyl (NP) coupled to mAbs that recognize DC-specific DEC205 or DCIR2, we found that MZ-associated DCIR2+ DCs, but not DEC205+ DCs, were able to induce robust T cell- and CD40-dependent anti-NP Ab responses following injection of soluble Ag. Anti-NP Ab responses were restricted to the IgG1 sub-class but failed to either persist over time or undergo affinity maturation, despite the addition of TLR or RLR agonists. Using adoptive transfers with NP-specific B cells, we show that the unique ability of DCIR2+ DCs to induce Ab responses was associated with rapid, MHC class II-independent activation of Ag-specific B cells that occurred specifically upon Ag delivery to DCIR2. Importantly, we found that NP-specific B cells accumulated in MZ-associated bridging channels in close association with CD11c+ cells as early as 24hr following Ag delivery to DCIR2+ DCs. Together, these results reveal a previously unrecognized role for DC-B cell interactions in splenic bridging channels, and provide a novel mechanism for the initiation of T-dependent extrafollicular Ab responses.
TLR9 induces germinal center light zone expansion and augments B cell fate decisions (61.8)
Authors: Rookhuizen Derek , Sha William , DeFranco Anthony
Published: January 2023

Toll-like receptor 9 (TLR9) signaling regulates inflammatory processes in response to unmethylated CpG motifs that are enriched in viral, bacterial and protozoan DNA. While its roles in dendritic cell (DC) maturation, class switch recombination, and polyclonal B cell proliferation have been extensively studied, the importance of TLR9 in T-dependent germinal center (GC) B cell processes is unclear. To address this problem in vivo, we linked a biotinylated form of the model antigen NP-chicken gamma globulin (NP-CGG) with either biotin-CpG or biotin-nonCpG control (Ctrl) oligos using streptavidin and generated CpG- and Ctrl-conjugates, respectively. As early as 7dpi in the footpad of C57BL/6 mice, we observed TLR9-induced affinity maturation and a selective expansion of GC B cells in the light zone (LZ) where B cells receive survival signals from T follicular helper cells (Tfh) and selection for high affinity clones and distinct B cell fates occurs. We then looked for evidence of TLR9-enhanced selection and found increases in the GC to plasma cell transition as well as a robust increase in GC B cell CD38 expression that signifies memory B cell formation. We conclude that TLR9 selectively enhances light zone processes such as B cell fate decisions and enrichment for high affinity clones. The relationship between LZ expansion and fate decision is a future focus of this project.
Induction of antigen-specific B cell responses to pathogens — a balancing act! (61.9)
Authors: Baumgarth Nicole , Priest Stephen
Published: January 2023

Complex developmental and immune regulatory programs ensure that B cells respond to foreign but not to self-antigens. However, B cell responses can also be triggered non-specifically by mitogens, such as TLR-agonists, which seems counterintuitive and even dangerous. Furthermore, our studies showed a strong, albeit transient and highly specific increase of TLR3 and TLR7 in B cells that had accumulated in regional lymph nodes within 48h after infection with influenza virus, causing enhanced responsiveness of B cells to TLR7 agonists. This increase was induced by direct stimulation of B cells via the type I IFNR. While these observations provide a mechanism for the observed enhancing effects of type I IFN on antiviral B cell responses, they raise the question of how self-reactive responses are avoided, given that TLR7 overexpression has been directly linked to enhanced self-reactivity and autoimmune diseases. We hypothesize that signals must exist that counterbalance this increased sensitivity of B cells to TLR7 signals during influenza infection. Our data now suggest that type I IFN itself might provide such a counterbalance by suppressing B cell cell-cycle progression. Together our data indicate a dual role for type I IFN in differentially regulating specific and non-specific B cell responses after infection. They also raise questions about the role and function of the increased levels of type I IFN observed in patients with certain autoimmune diseases such as SLE.
Mitf family inhibition perturbs B cell homeostasis (62.14)
Authors: Lopez Ocasio Maria , Huan Chongmin , Roman Christopher
Published: January 2023

B cell activation is essential for recruitment of the appropriate antigen-specific B cells into an immune response and must be tightly controlled to avoid autoantibody production. The transcription factor Mitf, a member of the Mitf family of transcription factors that includes TFE3, TFEB and TFEC, is known to be important in restraining B cell activation by acting as a repressor of IRF4, itself needed for plasma cell development. However, the Mitf mutation caused osteopetrosis due to defective Mitf-dependent osteoclast development, and thus necessitated the evaluation of Mitf mutant B cells via adoptive transfer of Mitf-mutant stem cells into a normal host. In addition, the contribution of the other MiT family members that are also expressed in B cells was not evaluated. To address these issues, we have developed a single transgene model for B cell specific MiT family inactivation. The transgene encodes an artificial trans-dominant negative protein that forms inactive heterodimers with all four MiT family members. In preliminary data, we observed that mice that express this TDN protein in B cell lineage exhibit a number of B cell defects including splenomegaly, elevated numbers of spontaneous GCs and plasma cells, and perturbations to different B cell subsets. These data point to B cell subset specific dependency on the MiT family and possibly reveal the contribution of other MiT family members.
MicroRNA-mediated repression of XBP1: a novel mechanism for regulation of a UPR transcriptional activator (62.15)
Authors: Byrd Andrew , Brewer Joseph
Published: January 2023

The unfolded protein response (UPR), a multi-faceted signaling system emanating from the endoplasmic reticulum (ER) membrane, plays a critical role in the differentiation of B-lymphocytes into antibody-secreting plasma cells. Specifically, XBP1(S), a UPR transcriptional activator generated by IRE1-mediated splicing of Xbp1(u) mRNA, mediates expansion of the ER, up-regulates expression of many secretory pathway genes, and is required for normal antibody production. However, a full understanding of the molecular mechanisms regulating expression of Xbp1 is still lacking. Using bioinformatics analysis, we identified a microRNA (miR-30c-2*) predicted to target Xbp1 mRNA. Indeed, luciferase reporter assays demonstrated that miR-30c-2* targets a site in the 3’-untranslated region of Xbp1 mRNA in a sequence-specific manner. Over-expression of miR-30c-2* attenuated expression of both Xbp1 and XBP1(S) target genes, consistent with diminished expression of XBP1(S) protein. Furthermore, our studies revealed that expression of miR-30c-2* is up-regulated during UPR activation, concomitant with Xbp1, suggesting a feedback mechanism for XBP1(S) homeostasis. Finally, preliminary studies provided evidence linking expression of miR-30c-2* with specific UPR signaling events. These data provide the first link between microRNA and the UPR and, importantly, implicate miR-30c-2* in the regulation of Xbp1 and plasma cell differentiation.
Chromatin conformations and contacts that support Tcra/d locus rearrangement (62.2)
Authors: Shih Han-Yu , Olcott William , Krangel Michael
Published: January 2023

The Tcra/d locus rearranges in DN thymocytes to assemble Tcrd genes and in DP thymocytes to assemble Tcra genes. We used 3D-FISH to show that the 3’ portion of the locus is contracted in both DN and DP thymocytes, whereas the 5’ portion is contracted in DN but decontracts in DP thymocytes. We proposed that the fully contracted conformation in DN thymocytes allows dispersed Vαs to be used in a single round of Vδ-Dδ-Jδ rearrangement, whereas the 3’-contracted, 5’-decontracted conformation in DP thymocytes allows for multiple rounds of Vα-to-Jα rearrangement initiating with 3' Vαs. For high resolution analysis, we used the 3C method to detect molecular interactions between different sites in the locus. The Tcra enhancer (Eα) activates the T early α promoter (TEA) to target 5’Jαs for initial rearrangement. Eα also activates proximal Vαs over 500 kb. We detected molecular interactions between Eα and TEA, between Eα and proximal Vαs, and between different proximal Vαs, in DP but not DN thymocytes. All pairwise interactions depended on Eα. With TEA deleted, Eα also interacted with downstream Jαs. We propose that Eα nucleates a chromatin hub that supports transcription and ordered Vα-to-Jα recombination. However, deletion of Eα does not impact Tcra/d locus 3’end contraction as measured by 3D-FISH. Thus, overall locus conformation is independent of Eα, but this conformation may facilitate Eα interactions with distant sites.
Regulatory B10 cells activated in vivo transiently produce IL-10 before differentiating into antibody-secreting cells with a diverse BCR repertoire (62.4)
Authors: Bryant Jacquleyn , Candando Kathleen , Maseda Damian , Smith Susan , Weaver Casey , Dilillo David , Tedder Thomas
Published: January 2023

B cells with potent IL-10-dependent regulatory functions (B10 cells) expand in mice and humans during inflammation and autoimmunity. In this study, the fate of B10 cells following in vivo expansion was examined using two distinct il-10 reporter strains: Tiger mice that express cytoplasmic GFP and 10BiT mice that express cell surface Thy1.1. B10 cells from Tiger mice express cytoplasmic IL-10 and GFP with similar kinetics following stimulation in vitro and in vivo. By contrast, B10 cell Thy1.1 expression in 10BiT mice was delayed relative to IL-10 production and persisted even after B10 cells had lost the capacity to produce IL-10. Thy1.1 also served as a marker for B10 cells that expressed plasma cell-associated transcription factors and had the capacity to differentiate into CD138+B220LO antibody-secreting plasma cells in vivo and in vitro. Antibodies secreted by B10 cells included broadly reactive “natural antibodies” and autoantibodies, and were antigen-specific in immunized mice. At the molecular level, B10 cells predominantly expressed a germline-encoded IgM antibody repertoire with diverse VH and VL gene utilization. These results indicate that B10 cells can differentiate following IL-10 secretion to produce broadly-reactive germline-encoded antibodies that may function to rapidly clear antigens and further reduce inflammation and immunopathology.
Repertoire restriction of immunoglobulin DH gene segments impairs T cell regulation of a thymus-dependent immune response (62.7)
Authors: Lemke Hilmar , Trad Ahmad , Tanasa Radu , Zemlin Michael , Schroeder Harry
Published: January 2023

The significance of DH gene segments for the thymus-dependent (TD) immune response to the hapten 2-phenyloxazolone (phOx) was investigated in two mouse strains with a single DH gene segment that was derived from a modified DFL16.1 gene. In DD-DmuFS and in DD-iD mice, the central portion of the altered DH gene segment predominantly encodes for hydrophobic or charged amino acids, respectively. Compared with the response of BALB/c wild-type mice, the restriction of DH repertoire in both mutant strains caused drastic changes of the anti-phOx response. (i) The humoral anti-phOx antibody response was significantly reduced. (ii) Class switch recombination (CSR) was severely impaired. (iii) There was no restriction of VH/VL gene usage during CSR in the primary response and during further immune maturation. (iv) There was no selection of a dominant clonotype. (v) CSR did not select B cell clones with shorter and more uniform CDR-H3 lengths, as observed in BALB/c wild-type mice. These data confirm those obtained in wild-type mice and corroborate the view that CDR-H3-directed T cell action is important for clonal selection during CSR and immune maturation.
Upregulation of Bcl6 expression in Follicular Helper T cells (TFH) requires both dendritic and antigen-specific B cell help. (63.10)
Authors: Weinstein Jason , Poholek Amanda , Hernandez Sairy , Nowyhead Heba , Bertino Sarah , Craft Joseph
Published: January 2023

The transcription factor B cell CLL/lymphoma 6 (Bcl6) was recently found to be both necessary and sufficient for TFH cell development. However, the factor(s) that promotes the upregulation of Bcl6 expression in TFH cells remains incompletely known. Here we explored the roles of B and dendritic cells (DC) in the upregulation of Bcl6 in TFH cells. Antigen-specific, Thy1-mismatched, TCR transgenic CD4 T cells were co-transferred with Ag-specific or polyclonal B cells into CD19-/- or wild type hosts. Upon immunization of CD19-/- mice with NP-OVA, Bcl6 expression increased in donor T cells compared to unimmunized controls, despite the absence of functional B cells. Although Bcl6 expression was not equivalent to T cells transferred to CD19-intact mice post immunization, upon transfer into CD19-/- animals expression of T cell surface markers was consistent with emigration towards the T-B border, albeit without expression of TFH cell markers and follicular entry. The transfer of Ag-specific B cells, but not their polyclonal counterparts, to CD19-/- hosts restored Bcl6 expression to levels approximating that observed from immunized wild type controls, and restored the TFH cell phenotype with follicular entry. Bcl6 was not upregulated in T cells transferred to mice depleted of DCs, followed by immunization. These data demonstrate that CD4 T cells need signals from both DCs and from Ag-specific B cells to fully upregulate Bcl6 and drive TFH cell development and function.
Priming with very low affinity peptide ligands gives rise to CD8+ T cell effectors with enhanced function but with greater susceptibility to TGFβ-mediated suppression (63.15)
Authors: Guevara-Patino Jose-Alejandro , O’Sullivan Jeremy , Zloza Andrew , Kohlhapp Frederick , Moore Tamson , Lacek Andrew , Dulin Nickolai
Published: January 2023

While the effects of TCR affinity and TGFβ on CD8+ T cell function have been studied individually, the manner in which TCR affinity dictates susceptibility to TGFβ-mediated suppression remains unknown. To address this issue we utilized OVA altered peptide ligands (APLs) of different affinities in the OT-I model. We demonstrate that, while decreased TCR ligand affinity initially results in weakened responses, such interactions prime the resultant effector cells to respond more strongly to cognate antigen upon secondary exposure. Despite this, responses by CD8+ T cells primed with lower affinity TCR ligands are more effectively regulated by TGFβ. Susceptibility to TGFβ-mediated suppression is associated with downregulation of RGS3, a noncanonical negative regulator of TGFβ signaling, but not expression of TGFβ receptors I/II. These results suggest a novel tolerance mechanism whereby CD8+ T cells are discriminately regulated by TGFβ according to the affinity of the ligand on which they were initially primed.
Induction of autoimmune pathology linked to asymmetric T cell division (63.21)
Authors: King Carolyn , Koehli Sabrina , Hausmann Barbara , Schmaler Mathias , Palmer Ed
Published: January 2023

Immunological tolerance, defined as the ability to distinguish between self and nonself, begins in the thymus where T cells undergo development. The affinity between T cell receptor (TCR) and pMHC has long been established as a major determinant guiding thymocyte selection and recent data revealed a universal threshold affinity used by all thymocytes with class I MHC restricted TCRs to initiate self tolerance. Nevertheless, the prevalence of autoimmune disease indicates that self reactive T cells can be activated in the periphery. Whether the affinity threshold established during thymic selection plays a role in the maintenance of peripheral self tolerance has not been established. Here we show that only high affinity ligands (above the threshold for negative selection) are able to induce disease in a mouse model of autoimmune diabetes. Immunization with high affinity peptides results in more efficient T cell-APC conjugate formation, T cell proliferation, VLA-4 upregulation and ultimately pancreatic infiltration. Importantly, higher affinity peptides are more efficient at inducing asymmetric cell division resulting in a greater number of fully differentiated effector T cells. In contrast, stimulation with low affinity peptides results in fewer asymmetric divisions and fewer effector T cells capable of promoting disease. These findings underscore the critical importance of TCR in directing T cell fate and maintaining peripheral T cell tolerance.
Inter-follicular germinal center B cell and T follicular helper cell development precedes follicular Tfh maintenance (63.23)
Authors: Kerfoot Steven , Yaari Gur , Patel Jaymin , Johnson Kody , Gonzalez David , Kleinstein Steven , Haberman Ann
Published: January 2023

We identify the interfollicular (IF) zone of the lymph node as the site where germinal center B cell and T follicular helper (Tfh) cell differentiation initiates. For the first two days post-immunization, antigen-specific T and B cells remained confined within the IF zone, where they participated in long-lived interactions and upregulated Bcl-6, which controls the differentiation of both cell types. During this time, T cells also acquired the Tfh markers CXCR5, PD-1 and GL7. While T cell immigration into the follicle interior occurred three days post immunization, responding B cells remained largely restricted to the IF zone and the area adjacent to the sub capsular sinus at this timepoint. B cell entry into the follicle interior, and formation of nascent germinal centers, did not occur until a day later. Notably, in the absence of cognate B cells, PD-1hi Tfh cells still formed and migrated to the follicle. However, without such B cells, PD-1, ICOS and GL7 expression was not maintained on T cells that nevertheless persisted within the follicle. Thus, cognate B cells are not required for Tfh cell differentiation in the IF zone, but are instead required for the long-term maintenance of the activated Tfh phenotype.
Type I interferon promotes PD-1 transcription and limits the duration of T cell-mediated immunity (63.24)
Authors: Chikuma Shunsuke , Terawaki Seigo , Shibayama Shiro , Hayashi Tamon , Yoshida Takao , Okazaki Taku , Honjo Tasuku
Published: January 2023

PD-1 is a co-receptor for T lymphocytes that provides feedback inhibition of T-cell activation. Although PD-1’s expression on T cells is known to be activation dependent, the factors that determine the timing, intensity, and duration of PD-1 expression in immune reactions are not fully understood. To address this question, we analyzed a conserved 5’-flanking region of the PD-1 gene, and identified a putative interferon-stimulation response element (ISRE), which was responsible for PD-1 transcription in the 2B4.11 T-cell line. Consistent with this finding, activation by interferon (IFN) alpha enhanced both the induction and maintenance of PD-1 expression on TCR-engaged primary mouse T cells through an association of interferon-responsive factor-9 (IRF9) to the ISRE. Indeed, PD-1 expression on antigen-specific CD8+ T cells was augmented by IFN alpha in vivo. We also demonstrated that IFN alpha administration in combination with PD-1 blockade in tumor-bearing mice effectively augmented the anti-tumor immunity. Thus we propose that strong innate inflammatory responses promote primary T-cell activation and their differentiation into effector cells, but also cause an attenuated T-cell response in sustained immune reactions, at least partially through type I interferon-mediated PD-1 transcription.
T follicular helper cells require cognate B cell interactions for acquisition of full effector function (63.4)
Authors: Barnett Lisa , Goenka Radhika , Silver Jonathan , O'Neill Patrick , Hunter Christopher , Cancro Michael , Laufer Terri
Published: January 2023

Follicular helper T cells (TFH) are a unique subset of CD4+ helper T cells that are critical effectors of thymus-dependent humoral immune responses, yet much about their development in vivo remains unclear and controversial. The requirements for different antigen presenting cell subsets in TFH formation are not yet fully understood. By transiently ablating or restricting MHCII antigen presentation to CD11chi dendritic cells (DCs), we show that cognate, MHCII dependent interactions with DCs are both necessary and sufficient for the formation of CXCR5+, ICOS+ TFH cells that express the key transcription factor Bcl6, which governs their differentiation. However, TFH cells that do not undergo TCR-MHCII-dependent interactions with B cells lack a small subset of GL-7+ PD1hi cells found in the germinal center (GC) and GCs fail to form. These TFH exhibit a significant reduction in their ability to produce IL-21 when compared to TFH cells generated in the presence of cognate B cell interactions. Consistent with these data, in vitro priming of naïve T cells by B cells, but not DCs, induces optimal IL-21 production, which, in turn, induces a GC B cell transcriptional profile. Thus, this study supports a multi-step model for priming effector TFH in which DCs activate CD4+ T cell towards a poised intermediate which can then interact with responding B cells to sustain the GC status of both cell types.
Modulation of mucosal effector and regulatory T cell responses by Helicobacter pylori (63.6)
Authors: Carbo-Barrios Adria , Climent Montse , Muite Kevin , Viladomiu Monica , Pedragosa Mireia , da Fonseca Isabel , Humedas Merce , Lu Pinyi , Horne William , Hontecillas Raquel , Bassaganya-Riera Josep
Published: January 2023

Helicobacter pylori is a gram-negative, microaerophilic bacterium that colonizes the gastric mucosa and a leading cause of gastritis, peptic ulcer and gastric cancer. While dysregulated immune responses contribute to H. pylori-mediated pathogenesis, its immunoregulatory mechanisms are incompletely understood. To characterize the mechanisms of immune modulation by H. pylori we have performed dose-titration and time-course studies in mice infected with H. pylori strain 26695. Our dose-titration results indicate an increase in the percentages of INFγ+Tbet+ Th1 cells, lymphocytic infiltration into the gastric lamina propria, and disease activity with increasing concentrations of the H. pylori infectious dose (i.e., 106, 108 and 1010 cfu/ml). The profiling of T cell responses in the gastric lymph nodes (GLN) over time illustrates an initial increase in CD4+INFγ+Tbet+ (Th1 cells) on day 7 post-infection followed by a predominant peak of CD4+IL10+ T cells and smaller but significant peak of CD4+IL17+ T cells on day 14 post-infection. In conclusion, gastric H. pylori infection in mice induced mucosal effector Th1 and Th17 responses that were paralleled by strong regulatory responses, thereby lending support to importance of characterizing effector and regulatory networks to better comprehend microbial pathogenesis at the site of infection.
Foxg1 is necessary for thymic epithelial cell differentiation. (64.13)
Authors: Condie Brian , Wei Qiaozhi
Published: January 2023

Thymic epithelial cells (TECs) are an essential component of the thymic microenvironment and are necessary for the production of normal T cells. Cortical (cTEC) and medullary TEC (mTEC) subtypes have different functions and distinct gene expression profiles. However, the transcription regulatory network controlling the specification and differentiation of TECs is poorly understood. We have initiated a screen for additional transcription factors that are likely to be required for TEC development. This screen identified Foxg1 as a candidate regulator of thymic epithelial cell differentiation. We have found that Foxg1 is expressed in TECs before the onset of TEC differentiation and continues to be expressed in them throughout fetal development. Analysis of Foxg1 mutant mouse embryos revealed that TEC differentiation initiates normally but then becomes abnormal at E13.5-E14.5. Foxn1, which is required for the initiation of TEC differentiation, is expressed throughout fetal development in Foxg1 mutant TECs suggesting that Foxg1 activity is not necessary for Foxn1 expression. Overall, our results suggest that Foxg1 may regulate TEC differentiation independently of or in collaboration with Foxn1.
A genetically selective inhibitor reveals ZAP-70 kinase-dependent checkpoints in T cell development (64.15)
Authors: Au-Yeung Byron , Cheng Debra , Zhang Chao , Shokat Kevan , Weiss Arthur
Published: January 2023

The tyrosine kinase ZAP-70 is critical for the activation of signal pathways downstream of the TCR. We previously generated a transgenic mouse expressing a ZAP-70 mutant (ZAP-70AS) that is selectively inhibited by a PP1 analog, called 3-methyl benzyl PP1 (3-MB-PP1). This system enables the rapid and specific inhibition of ZAP-70 catalytic activity. We used a combination of ZAP-70 inhibitor treatment with fetal thymic organ culture (FTOC) to determine when ZAP-70 kinase activity is required for proper T cell development. Our results show that treatment of ZAP-70AS FTOC with 3-MB-PP1 impairs development of the CD4 CD8 double-negative 4 (DN4) subset, indicating that ZAP-70 has an important role in pre-TCR signaling and TCR β-chain selection. This block is not complete, allowing generation of double-positive (DP) thymocytes, possibly due to expression of the related kinase Syk. However, ZAP-70 kinase activity is fully required for positive selection, as characterized by DP cell upregulation of surface levels of TCR, CD5 and CD69. Here we show that ZAP-70 kinase activity has both partial and full requisite roles in T cell development.
β-catenin acts upstream of RORγt in double positive thymocyte survival regulation (64.17)
Authors: Wang Ruiqing , Xie Huimin , Sun Zuoming
Published: January 2023

CD4+CD8+ double positive (DP) thymocytes are subject to positive and negative selection, and thus the lifespan of DP cells determines T cell repertoire. Previously, we have identified RORγt as a critical player in the regulation of DP thymocyte survival. However, the molecular mechanisms responsible for RORγt-mediated survival remain unknown. Here we demonstrated that β-catenin/TCF acts upstream of RORγt to enhance DP thymocyte survival via up-regulation of anti-apoptotic factor Bcl-xL. Activation of TCF-1 pathway by transgenic expression of a constitutively active β-catenin leads to increase of RORγt level and enhanced DP thymocyte survival via upregulation of Bcl-xL, whereas DP thymocytes from Tcf-1-/- mice undergo accelerated apoptosis and have significantly reduced RORγt and Bcl-xL. More importantly, forced expression of RORγt in Tcf-1-/- thymocytes leads to rescue of massive apoptosis and restoration of Bcl-xL level. We further demonstrated that β-catenin/TCF-1 activates RORγt promoter. Our data thus suggest that β-catenin/TCF-1 acts upstream of RORγt in the regulation of Bcl-xL expression and DP thymocyte survival.
FLVCR, a heme export protein, is required for T cell development beyond the CD4+CD8+ double-positive stage (64.18)
Authors: Philip Mary , Zaballa Alexandra , Phelps Susan , Abkowitz Janis
Published: January 2023

Heme is essential for every mammalian cell; however, free heme can induce free radical formation and cellular damage and cells must carefully regulate heme levels. The feline leukemia virus subgroup C receptor (FLVCR) exports heme from cells. Deletion of Flvcr caused progressive anemia in neonatal and adult mice (Science 319:825-8, 2008). We developed a transplant model in which developing lymphocytes lacked FLVCR while erythroid cells expressed FLVCR, preventing anemia, and found that CD4 and CD8 peripheral T cells were severely decreased while B cell numbers were only mildly affected. We analyzed thymic development and found that although there were similar numbers of CD4-CD8- double-negative thymocytes (DN) and CD4+CD8+ double-positive thymocytes (DP), there was a dramatic decrease in the number of CD4+ and CD8+ single positive thymocytes (SP) in FLVCR KO-transplanted mice. Because sensitive quantification of intracellular free heme is challenging, we measured the relative expression of a heme-inducible gene, heme oxygenase-1 (hmox-1) and found higher hmox-1 expression in FLVCR KO thymocytes compared to controls, suggesting that Flvcr deletion leads to higher intracellular free heme in developing T cells. While heme is required for erythroid function, there is no known specific role for heme in T cell development. Studies are underway to determine whether direct heme toxicity arrests T cell development, or whether excess heme alters T cell developmental gene expression.
IL-18 and IL-12 promote development of early thymic progenitors and can substitute for IL-7 in short-term OP9-DL4 co-cultures (64.7)
Authors: Tan Chibing , Marino Julie , Taylor Ashlee , Wren Jonathan , Teague T. Kent , Van De Wiele C. Justin
Published: January 2023

Determining the most effective parameters for in vitro propagation of T cell precursors is critical for realizing the promise of many envisioned T cell-based immunotherapies. To this end, the consequences of culturing ETP and DN2 thymocytes with IL-18 and IL-1β in the context of the OP9-DL4 co-culture model were evaluated after inference of a hypothesized role for these molecules in thymopoiesis based on results obtained via a novel microarray/literature mining algorithm. Sort-purified mouse ETPs and DN2 thymocytes were cultured on OP9-DL4 stromal layers ±IL-18, IL-1β, IL-12 and/or IL-7 for one week. IL-18 and IL-12 alone promoted proliferation and differentiation of ETPs to the DN3a stage and these cytokines could compensate for absence of IL-7. When IL-18 was used in conjunction with IL-7, synergistic proliferation was observed. IL-1β proved to be deleterious to development and was not overcome by inclusion of IL-7. To evaluate the developmental potential of the derived pre-T cells, culture-generated DN3a-like cells were sort-purified then cultured for an additional 8 days. In these assays, the pre-T cells demonstrated differentiation to the DP stage. Overall, these results indicate that IL-18 and IL-12 can positively impact T cell development and should prove useful in strategies to augment in vitro T cell development in the OP9-DL4 system.