bluuebunny/crossref_metadata_2025 huggingface dataset:
The function of HSP90 in MHC class II presentation (106.31)
Authors: Deffit Sarah , Blum Janice
Published: January 2023

A role for heat shock proteins (HSP) in promoting antigen presentation via the MHC class I pathway is well established and appears to play a key role in host recognition of tumors and transformed cells. Studies suggest that some HSP may promote the presentation of cytoplasmic antigens via the class I pathway, as well as selecting cytoplasmic antigens for presentation in the context of MHC class II molecules. A role for intracellular HSP90 as well as HSC70 in regulating the presentation of a cytoplasmic autoantigen, glutamic acid decarboxylase (GAD) has been demonstrated in B lymphoblasts. Modulating intracellular expression of HSP90 alpha in human B lymphoblasts altered class II presentation of GAD epitopes. Yet, diminished cellular HSP90 alpha expression did not alter the steady state levels of GAD antigen, or disrupt the trafficking of this native antigen from the cytoplasm to membraneous intracellular organelles. This may suggest HSP90 acts after antigen processing to promote epitope transit into endosomal/lysosomal compartments. Prolonged knockdown of HSP90 alpha in B cells, lead to an increase in the expression of HSP90 beta subunit. Studies are currently underway to examine whether the activation state of B cells influences cytoplasmic antigen presentation as well as the role of HSP90 co-factors in regulating this pathway for class II presentation.
Increased heterogeneity of MHC-II antigen presentation by B cells from older individuals (106.33)
Authors: Clark Heather , Banks Richard , Jones Leola , Burant Christopher , Canaday David
Published: January 2023

Why many current vaccines are less effective in older individuals is not fully understood. Immunosenescence of antigen presenting cells may contribute to this. We compared the function of peripheral blood B-cells from 10 young (age 22-45) and 11 old subjects (age 62-92) using a novel T-cell hybridoma system that specifically measures the B-cell receptor (BCR)-mediated MHC-II antigen presentation. We compared whole antigen (anti-BCR) that requires uptake and processing with peptide that can be directly presented on the cell surface. The mean magnitude of BCR-mediated antigen presentation was the same in older and younger subjects. Older subjects, however, showed increased heterogeneity of BCR-mediated antigen presentation. As a result, 5 of 11 geriatric subjects demonstrated inferior processing and presentation at all concentrations of anti-BCR antigen compared to the results from all younger subjects studied. The magnitude and variability of peptide that is presented but does not require processing was the same between groups. Surface BCR and HLA-DR expression showed no significant difference between groups. In contrast, both antigen processing and presentation function by blood monocytes had similar magnitude and variability between the older and younger subjects. These data suggest that poor B-cell antigen processing resulting in diminished presentation in some older individuals may contribute to poor vaccine responses.
Global evaluation of the role of ubiquitin in MHC Class I-restricted antigen processing. (106.35)
Authors: Goodenough Elliot , Eisenlohr Laurence
Published: January 2023

The multicatalytic proteasome plays an indispensable role in cellular proteolysis and in the production of most MHC class I-restricted antigenic peptides. Although ubiquitinylation was originally considered to be necessary for directing substrates to proteasomal destruction, growing evidence supports a role for ubiquitin-independent degradation as a substantial portion of intracellular proteolysis. To explore the importance of ubiquitin in MHC class I-restricted antigen processing, we have employed an inducible vaccinia virus system to overexpress wild-type and dominant-negative lysineless forms of ubiquitin (Ub) in mammalian cells. We have previously shown that abrogation of polyubiquitination in this manner inhibited epitope presentation from endoplasmic reticulum-targeted model antigens but had little impact on antigen presentation from cytosolic proteins. We are now seeking to elucidate the selective role of ubiquitin in MHC class I antigen processing by global evaluation of MHC class I-associated peptide display in the presence or absence of polyubiquitination, using mass spectrometric identification of peptides and comparative analysis of parent protein families.
Phenotypic and functional characterization of human blood BDCA3+ subsets (106.4)
Authors: Shih Tiffany , Dai Jihong , Fitzgerald-Bocarsly Patricia
Published: January 2023

There has been a recent focus on human blood DCs expressing BDCA3 (CD141). However, these cells are not fully characterized. Our study aimed to understand whether they constitute a homogeneous population or if they are related to two other DC subsets: plasmacytoid DCs (BDCA2+) and myeloid DCs (BDCA1+). We constructed multi-color panels for surface phenotyping of blood BDCA3+ DCs by flow cytometric analysis. We observed that lineage- PBMC heterogeneously distribute into BDCA3hi and BDCA3int subsets, with the latter subset expressing higher levels of CCR5 and CXCR4. Surface phenotyping of lineage- PBMC for the BDCA2 (found on pDC) and BDCA3 surface antigens yielded a subset of BDCA2+/BDCA3int cells, representing 15-30% of BDCA2+/lin- pDC. To assess functional significance of this population, we stimulated PBMC with TLR9 agonists CpGA or HSV-1 and stained for intracellular IFN-α and TNF-α in cells expressing BDCA2 and/or BDCA3. The BDCA2+/BDCA3int subpopulation produced IFN-α and TNF-α and upregulated the transcription factor IRF-7 more strongly than the BDCA2+/BDCA3- subpopulation, while the BDCA2-/BDCA3+ subpopulation constitutively expressed the lowest level of IRF-7 and barely produced IFN-α. However, similarly lower levels of TNF-α were found in the BDCA2-/BDCA3+ and BDCA2+/BDCA3- subpopulations when compared to the BDCA2+/BDCA3int subpopulation. These results suggest that the BDCA2+/BDCA3int subset has major functions associated with classical pDCs.
Describing the Peptide Binding Specificity of HLA-C (106.41)
Authors: Rasmussen Michael , Harndahl Mikkel , Nielsen Morten , Buus Søren , Stryhn Anette
Published: January 2023

Human leukocyte antigen (HLA) presents peptides to T-cells for immune scrutiny. Whereas HLA-A and -B have been described in great detail, HLA-C has received much less attention. Here, to increase the coverage of HLA-C and the accuracy of the corresponding tools, we have generated HLA-C molecules; peptide-binding assays, data and predictors; and tetramers; representing the most prevalent HLA-C molecules. We have combined positional scanning combinatorial peptide library (PSCPL) with a homogenous high-throughput dissociation assay and generated specificity matrices for 11 different HLA-C molecules. We find preference for hydrophobic residues at the peptide C-terminus for all HLA-C molecules. Most molecules were found to have an additional strong anchor at P2 or P3, with auxiliary anchor observed at P1, P2, P3, and P7. The binding affinity is measured for peptides fitting the specificity matrix for 5 HLA-C molecules and for all, but one, molecule we find a high frequency of binders, >70%, among these peptides. To extend the examined peptide space, we use bioinformatic prediction tools to search for additional binders. Finally, we update our prediction tool, NetMHCpan, with the HLA-C affinity data and show that the predictive performance for HLA-C molecules now is increased to a level comparable withthat of HLA-A and -B. These novel HLA-C molecules and predictors are successfully used to generate HLA-C tetramers and validate HLA-C-restricted T cell responses.
MHC Class II immunoproteomic analysis of Chlamydia trachomatis and C. muridarum infected murine dendritic cells reveals overlapping immunodominant epitopes (106.7)
Authors: Karunakaran Karuna , Yu Hong , Jiang Xiaozhou , Moon Kyung-Mee , Shen Caixia , Foster Leonard , Brunham Robert
Published: January 2023

Protective immunity to Chlamydia is T cell-mediated, requiring MHC class II presented antigens. Immunodominant T cell antigens are potential human Chlamydia vaccine candidates. An immunoproteomic approach was undertaken in which Chlamydia-derived peptides presented by MHC class II molecules on the surface of infected murine dendritic cells (DCs) were identified by tandem mass spectrometry. DCs from both Balb/c and C57BL/6 mice were infected with C. muridarum and DCs from C57BL/6 were infected with C. trachomatis and C. muridarum. DCs from Balb/c infected with C. muridarum yielded 14 MHC class II binding peptides from 11 different C. muridarum proteins. Only one of the 11 proteins was shared in common with 16 C. muridarum proteins that generated MHC class II binding peptides using DCs from C57BL/6 mice. However, C57BL/6 DCs infected with C. trachomatis serovar D yielded 60 MHC class II binding peptides derived from 27 proteins, 10 of which were shared between the C. muridarum and C. trachomatis proteome. Among the 10 overlapping proteins six were tested for protective immunity and all conferred protection in mice against C. muridarum genital tract infection using DDA/MPL adjuvant. Three of the protective proteins belonged to the polymorphic membrane family of proteins, which generated MHC binding epitopes at distinct sequences within the proteins. We conclude that T cell immunodominance is determined by both MHC allelic selection and inherent properties of pathogen proteins.
Autophagy in dendritic cells modulates CD4+ T cell response during RSV infection (106.9)
Authors: Reed Michelle , Morris Susan , Lukacs Nicholas
Published: January 2023

Severe respiratory syncytial virus (RSV) infections cause significant morbidity among susceptible individuals, especially infants. During viral infection, dendritic cells (DC) prime appropriate adaptive immune responses through antigen presentation, cytokine production and costimulation of T cells. Previous studies suggest that macroautophagy, an intracellular process delivering cytoplasmic constituents to endosomes for degradation, functions in intracellular pathogen surveillance and antigen delivery for presentation by DC. RSV infection of bone marrow-derived dendritic cells (BMDC) stimulates autophagosome formation, while siRNA knockdown of LC3 prior to RSV infection reduces innate cytokine production. Pulmonary DCs from RSV-infected LC3b-deficient mice show dysregulated maturation through altered MHC class II and costimulatory molecule expression. BMDC cultured from LC3b-deficient mice skew primary and secondary T cell responses toward Th17, while decreasing expression of IFNγ and Th2 cytokines. These results suggest that virally-induced autophagy in DC modulates the host response to RSV infection through altered activation of CD4+ T cells.
MAP kinase phosphatase 5 regulates innate immune response to influenza via type I interferon (108.14)
Authors: Zhang Yongliang , Teh Hong Ying , Flavell Richard , Dong Chen
Published: January 2023

MAP kinase phosphatases (MKP), also known as dual specificity phosphatases, are major negative regulators of MAP kinases and play important roles in immunity. Previously, we have shown that MAP kinase phosphatase 5 (MKP5), one member of MKP protein family, plays critical roles in both innate and adaptive immune responses. However, the role of this protein in anti-viral immune responses has not been well studied. To investigate the role of MKP5 in innate immune response against influenza, wild-type (WT) and MKP5 knockout (KO) mice were infected with A/Putero Rico/8/34 (PR8, H1N1) viruses. We found that viral titers in the lung of MKP5 KO mice are significantly lower on day 3 and day 5 post-infection than those in the lung of WT mice, which is associated with increased expression of IFN-beta in the lung and IFN-beta concentrations in the BAL from KO mice compared with those from WT mice. Overexpression of MKP5 in macrophages inhibits IFN-beta promoter activity as well as its expression. Macrophages from MKP5 KO mice produced significantly higher amount of IFN-alpha and IFN-beta in response to PR8 virus infection than cells from WT mice. The regulation of type I interferon by MKP5 was further investigated. Our study demonstrated that MKP5-regulated signaling plays an important role in regulating type I interferon and innate immune responses against virus infection.
CCR5-deficiency abrogates microglial activation and neuronal injury in HIV gp120-transgenic mice. (108.5)
Authors: Kaul Marcus , Maung Ricky , Sejbuk Natalia , Medders Kathryn , Desai Maya , Russo Rossella , Williams Roy
Published: January 2023

AIDS patients and mice expressing the envelope glycoprotein gp120 of human immunodeficiency virus-1 (HIV-1) as transgene in the brain (gp120tg) share key features of viral neuropathology, such as a decrease of neurites and synapses, pronounced astrocytosis, and activated microglia and macrophages. Virus-associated or soluble HIV-1 gp120 interacts with CD4 and co-receptors CCR5 and CXCR4 leading to infection and/or cellular signaling. In the present study we crossed HIVgp120tg with CCR5KO mice. Microarray analysis of brain tissue from 6 month-old mice showed that HIV-1 gp120 affected the expression of about 800 genes in the presence of CCR5 but of only 51 genes in the absence of the co-receptor. Differentially expressed genes in CCR5 wild type (wt)/gp120tg mice compared to non-transgenic controls primarily belong in the nervous and immune systems. CCR5KO/gp120tg mice and non-transgenic CCR5KO controls differed mostly in expression of an astrocytic protein and factors of the innate immune system. Using quantitative fluorescence and deconvolution microscopy, we assessed neuronal injury and glial cell activation in brain sections. CCR5wt/gp120tg mice displayed a significant reduction in neuronal dendrites and pre-synaptic terminals besides microglial activation (increased Iba1 immuno-reactivity) in comparison to all of the three other genotypes. Thus, in our model CCR5 is required for HIV-1 gp120 to activate microglia and to cause damage to neuronal processes and synapses.
Aging-associated alterations in IL-7 receptor signaling and inflammation promote declining B-lymphopoiesis and increased leukemogenesis (109.9)
Authors: Henry Curtis , Marusyk Andriy , Zaberezhnyy Vadym , Casas Matias , Adane Biniam , Merz Andrea , Serkova Natalie , DeGregori James
Published: January 2023

The accumulation of oncogenic mutations over time is thought drive increased cancer incidence with age. Alternatively, we hypothesize that aging reduces cellular fitness in stem and progenitor pools, leading to increased selection for oncogenic mutations and increased cancers. We have explored how aging affects the fitness of B-progenitor cells and leukemogenesis. The fitness of B-progenitors declines with age, as defined by competitive transplantation assays. Analyses of aged B-progenitors revealed increased expression of inflammatory genes, and decreased expression of genes involved in B-lineage determination, B-cell signaling, nucleotide synthesis, and other metabolic pathways. These changes correlated with decreased interleukin-7 receptor (IL-7R) mediated signaling, reduced production of ATP and other nucleotides, and perturbations in the TCA cycle in aged B-progenitors. Decreasing IL-7R signaling and inducing inflammation in young mice impaired B-lymphopoiesis, accompanied by aging-like gene expression profiles. Importantly, neutralization of IL-7 significantly increased selection for the Bcr-Abl oncogene in B-progenitor pools, coinciding with restored signaling. Similarly, Bcr-Abl is adaptive within B-progenitor pools from aged but not young mice. These studies reveal roles for IL-7R signaling and inflammation in declining B-lymphopoiesis in old age, and demonstrate how the selection for Bcr-Abl in aged lymphopoietic backgrounds can drive leukemogenesis.
MiR-155 modulates effector and memory CD8 T cell differentiation in response to murine γ herpes virus. (110.13)
Authors: Tsai Ching-Yi , Zhang Weijun , Allie S. , Usherwood Edward
Published: January 2023

MicroRNAs (miRNAs) have emerged as a major regulatory network with profound effects on many physiological processes. They are particularly important for determining cell fate decisions. Several processes mediate CD8 T cell differentiation. While many studies have revealed the importance of transcriptional and epigenetic regulation, it is likely that miRNAs also contribute to this programming. miRNAs are critical in determining B and CD4 T cell lineage, yet little is known about their effect on CD8 T cell differentiation. Studies with in vitro skewed central memory CD8 T cells show a down regulation of miR-155 relative to skewed effector memory cells. Using murine gammaherpesvirus 68 as a model, we examined the influence of miR-155 on CD8 differentiation in vivo. Our results showed lowered CD8 responses in the absence of miR-155 accompanied by a phenotypic skewing towards a memory precursor phenotype at the acute phase, which resulted in more central memory cells. The resulting memory cells in the absence of miR-155 were as functionally competent as their wild type counterparts, as revealed by their ability to recall to the same antigen. Our data indicate the importance of mIR-155 in effector differentiation, which may be a critical piece of the puzzle in CD8 differentiation giving useful information for vaccine research.
Itk and antigen specific CD8+ T cell memory differentiation (110.8)
Authors: Huang Fei , Huang Weishan , Bai Yuting , Chew Tina , Briggs Jessica , August Avery
Published: January 2023

It has been shown that Itk, a Tec kinase family member, is important for T cell development and differentiation. CD4+ and CD8+ T cells in Itk-/- mice display innate memory phenotype. Since Itk-/- CD8+ T cells preferentially develop into innate memory T cells in thymus, we aimed to determine whether naïve Itk-/- CD8+ T cells also preferentially differentiate into memory T cells after primary response to foreign antigens. To study the intrinsic and extrinsic effects of Itk in naïve CD8+ T cell differentiation into memory T cells, we used an OVA-Listeria monocytogenes infection model in which naïve OTI Rag1-/- T cells or OTI Itk-/- Rag1-/- T cells were adoptively transferred into Itk-/- or WT recipient mice. We found that naïve OTI ITK-/- Rag1-/- T cells have comparable ability to WT OTI Rag-/- T cells in memory T cell differentiation after a 5-week infection in WT hosts. In contrast, WT OTI Rag1-/- T cells are deficient in differentiating into memory T cells when placed in Itk-/- hosts. Although Itk affects the CD8+ T cell development in the thymus, its absence does not affect antigen specific CD8+ memory T cell differentiation. However, Itk-/- mice have an impaired environment for naïve CD8+ T cells differentiation into memory T cells during L. monocytogenes infection. These results suggest that Itk expression in cells other than CD8+ T cells is critical for supporting the antigen specific CD8+ T cell memory differentiation.
Immune ontogeny of CD4 T cells (111.28)
Authors: dela Pena Myra Grace , Rao Kalyan , Abel Kristina
Published: January 2023

The knowledge of immune ontogeny is limited. Our goal is to define molecular mechanisms that underlie the T helper 2-biased response of infant CD4 T cells. Age-dependent differences in cytokine signaling were determined by (i) measuring the cytokine profile of CD4 T cells after PMA/ionomycin stimulation, (ii) quantitation of cytokine receptors on CD4 T cells, and (iii) Phosflow analysis of cytokine-specific transcription factors using human cord (n=5) and adult (n=5) blood samples and longitudinal samples of infant rhesus macaques from birth to 1 year of age (n=20). Antigen-experienced T cells increased throughout the first year. Despite apparent phenotypic maturation, infant compared to adult CD4 T cells continued to show reduced cytokine responsiveness. In particular, IL-2 and TNF-α producing CD4 T cells were detectable as early as 1 week, but IFN-γ producing CD4 T cells remained rare. Lower IFN-γ production was associated with fewer IFN-γ receptors on infant compared to adult CD4 T cells. Furthermore, only 1.00%±1.66% of infant, but 15.00%±10.09% of adult CD4 T cells responded with STAT1 phosphorylation to IFN-γ stimulation (P<0.003). Similar results were obtained for STAT5 after IL-2 stimulation (P< 0.0001). In contrast, there was no difference in the activation of STAT6 in response to IL-4, consistent with similar levels of IL-4Rα on infant and adult CD4 T cells. Thus, distinct molecular mechanisms in cytokine signaling contribute to the Th2-biased response in infants.
Notch ligands control the human αβ/γδ lineage choice through receptor-specific induction of differential Notch signal strength. (111.29)
Authors: Taghon Tom , Van de Walle Inge , Waegemans Els , De Medts Jelle , De Smet Greet , De Smedt Magda , Vandekerckhove Bart , Kerre Tessa , Leclercq Georges , Wang Tao , Plum Jean
Published: January 2023

Differential Notch signal strength influences the αβ versus γδ T-cell lineage decision in both mouse and human. Here, we show that the Notch ligands Delta-like-4, Jagged1 and Jagged2, differentially impact this lineage decision in human. Whereas Delta-like-4 supports both αβ and γδ T-cell development, Jagged1 induces αβ-lineage differentiation and Jagged2 primarily γδ T-cell development. Jagged2 induces the strongest Notch signal in human thymocytes as a result of interactions with both Notch1 and Notch3, whereas Delta-like-4 only binds Notch1. In agreement, Notch3 is a stronger activator of Notch target genes and only supports γδ-lineage development, whereas Notch1 is a weaker activator supporting both αβ and γδ T cell development. Blockage of Notch3 activation in Jagged2 cocultures rescues αβ-lineage differentiation, showing the importance of this interaction for driving γδ-lineage differentiation. Further analysis revealed that Jagged2-mediated Notch3 activation blocks αβ-lineage differentiation as a result of inhibition of TCR-β chain expression. Consistently, TCR-αβ T cell development is restored in Jagged2 cocultures by providing immature human T cell progenitors with a TCR-β chain. Our results show that Notch ligands control the human αβ/γδ lineage choice through receptor-specific induction of differential Notch signal strength and that Jagged2-mediated Notch3 activation drives human γδ-lineage differentiation as a result of inhibition of αβ-lineage differentiation.
Homing of hematopoietic progenitors to the thymus is profoundly reduced after irradiation (111.32)
Authors: Zhang Shirley , Manna Sugata , Zlotoff Daniel , Bhandoola Avinash
Published: January 2023

After bone marrow transplantation (BMT), T cells are among the last of the hematopoietic lineages to recover, but the reasons for this delay are poorly understood. Previous work on T lineage reconstitution after BMT assumed progenitor supply to the thymus was not limiting; therefore research focused on intrathymic defects as potentially underlying delayed T cell recovery. We have recently discovered that delivery of progenitors to the thymus is limiting for T cell reconstitution after BMT. We have developed an assay that directly measures settling of intravenously injected progenitors within the thymus. Using this short-term settling assay, we have found that irradiated mice exhibit dramatically reduced thymic settling by intravenously injected progenitors. Directed irradiation of the thymus alone also showed reduced thymic settling. We suggest that one defect in T cell reconstitution after BMT is due to defective progenitor homing to the thymus, as survival of progenitors in the post-irradiation environment appears intact.
Methyl-CpG binding by the Mi-2/NuRD complex is required for transcriptional repression in B cells. (111.33)
Authors: Dege Carissa , Ramirez Julita , Hagman James
Published: January 2023

We demonstrated previously that the mb-1 (Igα; Cd79a) promoter is demethylated progressively during its activation in early B cell development. Demethylation of promoter CpGs is initiated by binding of the transcription factor, Early B Cell Factor 1 (EBF1). EBF1 activates the accessibility and demethylation of mb-1 promoter DNA, which allows for binding by Pax5 prior to transcription. This process is inhibited by Mi-2/NuRD chromatin remodeling complexes. This was evidenced by knockdown of the ATPase subunit of Mi-2/NuRD, Mi-2β (CHD4), which potentiated effects of EBF1 and Pax5. Recent work in the laboratory has focused on other components of the Mi-2/NuRD complex to assess its association with methylated DNA. The Mi-2/NuRD complex contains the Methyl Binding Domain proteins 2 or 3 (MBD2 or MBD3). MBD2 has the ability to bind methylated cytosines, while MBD3 binds hydroxymethylated cytosines. Similar to depletion of Mi-2β, knockdown of MBD2 using shRNA potentiated mb-1 transcriptional activation in the presence of EBF1 and Pax5. This increased activation was similar to that obtained using 5-azacytidine, which reduces cytosine methylation by inhibiting DNA methyltransferases. In contrast, depletion of MBD3 had no effect on EBF1/Pax5-dependent promoter transcription. Our data suggest that methyl-cytosine binding by Mi-2/NuRD complexes containing MBD2 is important for transcriptional attenuation of the mb-1 gene in B cells.
Defective Flt3L driven DC development in the absence of Ikaros (111.35)
Authors: Brugmann William , Winandy Susan
Published: January 2023

Ikaros null (Ik-) mice have impaired dendritic cell (DC) development. This defect is recapitulated when Ik- bone marrow (BM) is cultured with Flt3L, a DC growth factor. Ik- BM cultures generate significantly fewer DCs than their wild type (WT) counterparts. This phenotype is specific to Flt3L-driven DC developments as Ik- GM-CSF cultures generate normal numbers of DCs. Time course gene expression analyses of Flt3L-cultures show that genes important for DC development such as Irf4, Irf8 and Spib are variably under-expressed in both Ik- progenitors and mature DCs, with Spib being the most affected. These data demonstrate that low expression of DC genes is not solely due to decreased DC differentiation and suggest that Ikaros may regulate their expression, which we are now exploring. The DCs that develop in Ik- Flt3L cultures are also qualitatively different from their WT counterparts. They have an activated phenotype, implicating Ikaros in regulation of DC function as well. Enforced expression of full-length Ikaros (Ik1) in Ik- BM cannot restore Flt3L-driven DC development, suggesting a different Ikaros isoform may be needed for DC rescue. Studies now underway will define Ikaros isoform expression during DC development and determine if enforced expression of discrete Ikaros isoforms or the Flt3 receptor itself can restore Flt3L-driven DC development in Ik- BM. These studies will place Ikaros relative to Flt3 signals during DC development and define its molecular role.
FAK regulates progenitor B cell growth and retention in their bone marrow microenvironments (111.39)
Authors: Park Shin-Young , Wolfram Peter , Manis John , Beggs Hilary , Nombela-Arrieta Cesar , Pivarnik Gregory , Silberstein Leslie
Published: January 2023

Progenitor B cells reside in complex bone marrow microenvironments where they receive signals for growth and maturation. Here we have conditionally targeted in B cells Focal Adhesion Kinase (FAK), an integrator of cell extrinsic signals including CXCL12 and VCAM-1. The numbers of progenitor (pro-B and pre-B) and immature B cells are reduced by 30-40% in CD19-Cre Fakfl/fl mice. When cultured in methylcellulose with IL-7 ± CXCL12, Fak deleted pro-B cells undergo increased cell division and cell cycle progression, which correlates with down-regulation of four cell cycle regulatory genes, i.e. Cdk5rap1, Cdkn2a, E2f2, and Sfn. However, the increased cell division is accompanied by increased apoptosis, ultimately yielding fewer B cells and colonies. Utilizing quantitative laser scanning cytometry of longitudinal sections of femoral bone marrow, we show that Fak deletion disrupts the non-random gradient distribution of pro-B cells, which are preferentially localized in endosteal regions of the metaphyses and diaphysis. Increased numbers of pro-B cells are detected in the periphery of Fak deleted mice, and mobilization of pro-B cells induced by the inflammatory antigen NP-CGG-alum is increased 3-fold in Fak-deleted versus wild type mice. Collectively, these studies illustrate the importance of FAK in regulating progenitor B cell homeostasis and maintenance of their spatial distribution in bone marrow niches.
The AP-1 transcription factor Fra-1 is a negative regulator of B2B cell proliferation and of immunoglobulin class switch (111.49)
Authors: Groetsch Bettina , Lang Christiane , Neubert Kirsten , Brachs Sebastian , Zech Christine , Derer Anja , Luther Julia , Voehringer David , Wagner Erwin , Schett Georg , Mielenz Dirk , David Jean-Pierre
Published: January 2023

AP-1 transcription factors are immediate early genes that sense changes in the cellular environment to control differentiation, proliferation and apoptosis. Their functions in B cells are poorly described and were only suggested by the pro-apoptotic role of JunB. Fra-1(FosL1), an interaction partner of JunB, has been firstly described in activated B cells. Here, we confirm that Fra-1 becomes up-regulated in activated murine primary B cells that prompted us to analyze B cell development and humoral immune responses following Fra-1 gain and loss of function in vivo. Mice over-expressing Fra-1 that displayed a reduced B2 B cell compartment failed to mount any IgG response and did not form germinal centers. Bone marrow transfer in mice lacking B cells demonstrated that these phenotypes were B cell autonomous. B cell-specific Fra-1 deficient mice developed enhanced secondary responses, further reinforcing a cell autonomous key role for Fra-1 in B cell function. In vitro experiments showed that sorted fra-1tg B2 cells that express all relevant genes required for plasma cell differentiation and class switching were unable to class-switch, do not proliferate and die after activation. Thus, we identified Fra-1 as negative regulator of IgG production and class switch that functions independently of chromatin remodeling and class switch gene expression. Our work therefore suggests that Fra-1 acts as a negative regulator of B2 B cell proliferation, which is required for Ig class switch.
All-trans retinoic acid-induced antimicrobial activity against M. tuberculosis is dependent on NPC2-mediated regulation of cellular cholesterol (112.1)
Authors: Liu Philip , Kim Elliot , Wheelwright Matthew , Krutzik Stephan
Published: January 2023

One key function of the innate immune response is the rapid detection and elimination of microbial invaders, which can be influenced by micronutrients. A role for vitamin A in the host defense against Mycobacterium tuberculosis has been suggested through epidemiological and in vitro studies; however, the antimicrobial mechanism remained unclear. Here, we demonstrate that treatment of M. tuberculosis-infected human monocytes with either all-trans retinoic acid (ATRA) or 1,25-dihydroxyvitamin D, the biologically active forms of vitamin A and vitamin D respectively, resulted in antimicrobial activity against the pathogen. Comparison of the ATRA and 1,25D3 elaborated gene expression profiles revealed that ATRA distinctly triggered a lipid metabolism and efflux pathway in human monocytes. Accordingly, stimulation with ATRA but not 1,25D3 led to decreased cellular cholesterol content and altered intracellular lipid distribution, which were both dependent upon induction of NPC2. Knockdown of NPC2 expression resulted in the loss of ATRA- but not 1,25D3-induced antimicrobial activity against M. tuberculosis. These results demonstrate that ATRA-triggered host defense is mediated through NPC2-dependent regulation of cellular cholesterol content.
Lipocalin 2 mediated hypoferremia protects against endotoxin- induced sepsis in mice (112.11)
Authors: Vijay-Kumar Matam , Aitken Jesse , Srinivasan Gayathri , Sashidharamurthy Rangaiah , Jones Dean , Gewirtz Andrew
Published: January 2023

Sepsis is characterized by hypoferremia, a primitive protective mechanism to limit iron-induced oxidative stress. Lipocalin-2 (Lcn2) which is highly upregulated during inflammation and has been shown to transport iron under physiologic conditions. Accordingly, we hypothesized that such upregulation of Lcn2 serves to protect against sepsis associated oxidative stress. Lcn2 deficient mice (Lcn2KO) and their WT littermates were given E. coli LPS (20mg/kg BW) i.p. and monitored for mortality. Serum TNFα, IL-18, LDH, aminotransferases, iron, glutathione and cysteine were analyzed. Splenocyte apoptosis was assayed by TUNEL, caspase 3 cleavage and flow cytometry. In WT mice, LPS-induced systemic Lcn2 peaked at 24h and returned to basal levels by 48h. All Lcn2KO died by day 8 while only 20% mortality was observed in WT mice. In addition, Lcn2KO exhibited substantial elevation of every parameter tested including both pro- and anti-inflammatory cytokines. Further,Lcn2KO splenocytes exhibited extensive apoptosis as measured by caspase 3 cleavage, TUNEL staining and flow cytometry. More importantly, when compared to WT mice, Lcn2KO mice failed to exhibit LPS-induced hypoferremia at early time points and exhibited severe hepatic oxidative stress. Our data demonstrate that Lcn2 facilitates LPS-induced hypoferremia and serves as a host protective factor by reducing oxidative stress. Given Lcn2 multifunctionality, it may be developed as a potential therapeutic agent to treat sepsis.
Epithelial SPLUNC1 mediated antimicrobial activity against Klebsiella pneumoniae (112.14)
Authors: Di Y.Peter , Birru Rahel
Published: January 2023

Short PLUNC protein 1 (SPLUNC1) is an epithelial antimicrobial protein that is highly expressed in the nasal, pharyngeal, and lung epithelia. We have recently shown that in a transgenic mouse model (Scgb1a1-hSPLUNC1) over-expressing human SPLUNC1 is critical in pulmonary innate immune response against P. aeruginosa. The objective of this study was to identify differentially expressed transcripts using microarray analysis to understand the genetic basis of how SPLUNC1 regulates innate immune response in host against Klebsiella pneumoniae, an insidious organism that causes invasive respiratory infections. Scgb1a1-hSPLUNC1 transgenic (TG) mice were challenged with K. pneumoniae and displayed significant resistance to bacterial infection than wild-type (WT) mice. Categorization of the differentially expressed genes showed that those preferentially involved in host immune responses were activated extensively and TG mice exhibited more robust response at early time points after infection than WT mice. Ingenuity pathway analysis results suggested several key inflammatory regulatory genes are regulated by the over-expression of hSPLUNC1. TG mice displayed enhanced bacterial clearance that was accompanied by a decrease in neutrophil infiltration and cytokine levels that resulted in enhanced mouse survival against K. pneumoniae-induced respiratory infection. Our data indicate that SPLUNC1 is a crucial component of mucosal immune defense against pulmonary infection with K. pneumoniae.
Pathogen array reveals innate immune defense against molecular mimicry (112.16)
Authors: Stowell Sean , Arthur Connie , McBride Ryan , Smith David , Paulson James , Cummings Richard
Published: January 2023

The microbiome regulates diverse biological processes, including general metabolism and immunity, within a given individual. However, the examination of immunological factors responsible for regulating this superorganism remains difficult. In order to overcome these current limitations, we developed a microbial glycan microarray from a diverse range of microbial species. Serological comparison between humans and mice demonstrated striking differences in reactivity toward the antigenic determinants of different species, strongly suggesting unique exposure and/or distinct immunological responses to microbial flora between organisms. More importantly, innate immune lectins, galectin-4 and galectin-8, displayed exquisite specificity for bacteria displaying self-like antigens, while failing to bind related strains with non-self-like antigens. Galectin binding to these bacteria caused death accompanied by alterations in membrane integrity, whereas binding to mammalian cells expressing similar structures had no effect on cell viability. These results demonstrate that microbial glycan microarrays provide a new platform to explore the distinct contributions of different immunological factors to a wide variety of organisms and strongly suggest that innate immune lectins exist that provide unique protection against microbial molecular mimicry.
Role of microRNAs in immune response to infection (112.18)
Authors: Mehta Manali , Liu Philip , Teles Rosane , Modlin Robert
Published: January 2023

Regulation of microRNAs has been shown to be critical in immune cell development as well as the progression of certain cancers. However, their role in immune response to microbial infection has remained elusive. We chose to use Mycobacterium leprae, the causative agent of leprosy, as a model to determine the potential roles of microRNA in pathogenesis. The immune response to M. leprae has been well-characterized as presenting as a spectrum, ranging from a more contained Th1 response in tuberculoid patients to a more disseminated Th2 response in lepromatous patients, providing a good system for examining the correlation of microRNAs and type of immune response. We performed mRNA and microRNA microarray analysis on 6 T-lep and 6 L-lep lesions to determine whether microRNA disregulation and subsequent downregulation of key immune genes contributes to the outcome and morbidity of infection. We discovered 7 microRNAs in T-lep and 28 microRNAs in L-lep lesions and cross-referenced them in MiRNA and Genes Integrated Analysis (MAGIA) web tool with mRNA gene transcripts that were significantly downregulated in the same lesions. Preliminary analysis of Th1 and Th2 gene targeting shows microRNAs more highly expressed in L-lep lesions may preferentially target proinflammatory genes, which could explain the bias in immune response to infection.
Small-molecule BET inhibitor JQ1 subdues inflammation in vitro and protects mice from LPS-induced shock (112.19)
Authors: Belkina Anna , Denis Gerald
Published: January 2023

New treatment strategies are greatly needed for both acute and chronic inflammatory diseases. A novel triazolediazepine-fused ring compound JQ1 was recently reported to disrupt interactions between acetylated chromatin and dual bromodomains of BET proteins Brd4 and Brd2. Brd2 knockdown mice demonstrate reduced inflammatory responses, and various Brd2 shRNA-expressing cells cannot fully respond to inflammatory stimuli. Our HYPOTHESIS was that JQ1 disrupts BRD interactions to ablate inflammatory responses in a similar manner. RESULTS. We treated RAW264.7 cells and BMDMs with JQ1, stimulated them with LPS, and assayed mRNA of cytokine genes and cytokine levels in culture medium (TNFa, IL-6, IL-10, MCP-1). We stimulated mouse B cells with E. coli LPS and Pam3CSK4 to see the effect of JQ1 on TLR-mediated response. Finally, we subjected C57BL/6 mice to a lethal dose of LPS and treated them with 50 mg/kg of JQ1. We recorded survival and measured cytokine response in the serum 20,40,60,90,120, and 240 min after LPS injection. We found that JQ1 treatment dramatically reduces inflammatory responses in vitro and in vivo, downregulating expression and production of key NFkB-dependent cytokines. In B cells, JQ1 reduced proliferative effects of TLR2 and TLR4 stimulation. 90% of JQ1-treated mice survived endotoxic shock while 0% survived in the non-treated group. JQ1 ablated serum inflammatory cytokines within 1 hr. Thus JQ1 is a potent, novel, epigenetically-acting anti-inflammatory.
SLAM is a microbial sensor which regulates bacterial phagosome functions in macrophages by recruiting a Vps34/beclin 1/UVRAG complex (112.8)
Authors: Ma Chunyan , Wang Ninghai , Detre Cynthia , Wang Guoxing , O’Keeffe Michael , Terhorst Cox
Published: January 2023

Phagocytosis is a pivotal process by which macrophages eliminate microorganisms upon recognition by pathogen sensors. Surprisingly, the self-ligand cell surface receptor Slamf1 functions not only as a co-stimulatory molecule but also as a microbial sensor controlling killing of Gram-negative bacteria by macrophages. Slamf1 regulates activity of the Nox2 enzyme and phagolysosomal maturation upon entering the phagosome, by recruiting the enzyme Vps34, which produces PI3P. Here we report that in mouse macrophages, Slamf1 interacted with Vps34, Beclin 1 and UVRAG in E. coli-containing phagosomes. However this interaction was also found without induction of phagocytosis in macrophages, transfectant HEK293 cells and human chronic lymphocytic leukemia cells. Removal of the second ITAM motif in the cytoplasmic tail of Slamf1 seemed to enhance the interaction of Slamf1 with Beclin 1, while Slamf1 tailless totally abolished the interaction. Both the BD and CCD domains of Beclin 1 were required for efficient binding to Slamf1. Slamf1 did not interact with Atg14L or Rubicon, which also complex with Vps34 and Beclin 1 in autophagy. We conclude that Slamf1 recruits a subset of Vps34 associated proteins, which is involved in membrane fusions.
Naturally processed HLA class I-restricted epitopes inform targets of protective CD8+ T cell-mediated immunity (113.11)
Authors: Gilchuk Pavlo , Spencer Charles , Conant Stephanie , Niu Xinnan , Erickson John , McAfee Kristi , Oseroff Carla , Hadrup Sine , Bennink Jack , Hildebrand William , Edwards Kathryn , Crowe, Jr James , Williams John , Buus Søren , Sette Alessandro , Schumacher Ton , Link Andrew , Joyce Sebastian
Published: January 2023

Rational design of CD8+ T cell (TCD8)-based vaccines requires knowledge of the immunogenic and protective epitopes presented during infection, information which is currently lacking. Using the clinically successful smallpox vaccine as a model, ~200 novel naturally processed vaccinia viral peptides presented by HLA-A*0201 and -B*0702 molecules were identified and characterized. Humans showed a variegated response to these determinants, reminiscent of the hierarchic response seen in HLA class I transgenic mice. Importantly, multiple TCD8 epitopes were commonly recognized by humans and mice and identified potential targets for protective TCD8-mediated immunity. After acute infection, both dominant and subdominant TCD8 specificities exhibited all of the immunologic features necessary for protection. However, early and efficient presentation of immune determinants during infection ensured protective responses, regardless of dominance, such that subunit vaccination targeting subdominant or recessive TCD8 specificities conferred protection against lethal poxvirus challenge. Hence, an in-depth knowledge of naturally processed T cell epitopes coupled with the identification of TCD8-based targets that protect from lethal poxviral infection are essential for rational vaccine design.
CpG-E1E2 glycosylation-mutants of Hepatitis C Virus enhance specific cellular immune response and neutralizing antibodes (113.14)
Authors: Zhang Xiao-Lian , Ren Yushan , Lin Miao , Min Yuanqin
Published: January 2023

Hepatitis C virus (HCV) infects 170 million people world wide, and about 80% of the infected individuals develop chronic hepatitis with a risk of progression to cirrhosis and hepatocellular carcinoma. In contrast to hepatitis A and B virus, there is no vaccine for HCV. Thus, the development of an idea vaccine against hepatitis C remains a high prority goal. N-linked glycosylation of viral proteins have been implicated in immunicity. HCV neucleocapsids consist of two highly glycosylated envelop protein E1 and E2, in which the E1 contains 5 N-linked glycans and the E2 has 11 N-glycosylation sites. These oligose chains are essential for correct folding, assemble, and antigenic variation of virus and important for inducing immune response. In this study, the effects of the N-linked glycosylation of HCV E1-E2 fusion protein, a naturally poor immunogen, on the induction of specific immune response were examined. Different E1E2 glycosylation mutants were constructed into pVAX -1 and were used as DNA vaccine. We found that some fusion E1E2 mutants and CpG-E1E2 mutants could enhance much higher specific cellular and humoral reesponses than the wild type of E1E2 in the immunized BALB/c mice. Specific mutants were determined and could be as a HCV candidcate vaccine for its higher induction of specific cellular and neutralizing antibody immune responses.
Different memory T cell phenotypes are elicited by Ad5 and rare adenoviruses (113.2)
Authors: Penaloza-MacMaster Pablo , Borducci Erica , McNally Anna , Simmons Nate , Iampietro Justin , Provine Nick , Teigler Jeff , Tan Wendy , Ahmed Rafi , Barouch Dan
Published: January 2023

We have compared T cell memory phenotypes and immune protection elicited by common adenoviruses serotype 5 (Ad5) versus rare adenoviruses serotypes (Ad26, Ad35, and Ad48). For these comparative studies, we immunized mice with non-replicating adenovirus vectors expressing the lymphocytic choriomeningitis virus (LCMV) GP, and challenged with chronic LCMV Cl-13. Our comparative data show that Ad5-GP generates an increased magnitude of LCMV GP-specific CD8 T cell responses (compared to that generated by rare adenoviruses). However, GP-specific CD8 T cells elicited by Ad5-GP immunization express inhibitory PD-1, and produce reduced amounts of cytokines, suggesting qualitative defects in memory CD8 T cells. This unexpected expression of PD-1 may also reflect antigen-persistence of Ad5. Moreover, Ad5-GP vaccinated mice also showed increased numbers of T regulatory cells after an anamnestic challenge with LCMV Cl-13. These data suggest that alternative serotype Ad vectors may offer substantial benefits over Ad5 as vaccine vectors in eliciting optimal T cell memory to chronic viruses such as HIV, HBV or HCV.
Multi-epitope display on virus-like particles (113.26)
Authors: Tyler Mitchell , Tumban Ebenezer , Chackerian Bryce
Published: January 2023

Virus-like particles (VLPs) are a flexible vaccine platform for the highly immunogenic display of peptides derived from pathogens or other vaccine targets. When designing vaccines for pathogens, it is often necessary to provoke the broadest possible immune response in order provide protection against diverse strains or serotypes. We have been interested in developing VLPs that display multiple antigenic targets. Using a recombinant PP7 bacteriophage VLP platform, we genetically inserted peptides derived from the minor capsid protein (L2) of different human papillomavirus (HPV) types and hypothesized that antibodies produced in response to vaccination will be more broadly protective than that of VLPs displaying one target only. Peptides derived from the L2 protein of multiple HPV types were displayed on the surface of recombinant PP7 bacteriophage VLPs either alone or with that of another type. Mice were immunized and the reactivity of produced antibodies was measured by through endpoint ELISA against peptides derived from an array of different HPV types as well as an in vivo heterologous HPV pseudovirus (PsV) challenge. We found that certain combinations of targets produced an antibody response with higher reactivity for heterologous HPV types than VLPs with one target alone and protected from in vivo PsV infection. Thus, displaying multiple targets on VLPs may be useful in developing broadly reactive vaccines.