An Unfinished Canvas: Teacher Preparation, Instructional Delivery, and Professional Development in the Arts

Based on surveys, interviews, and secondary data analyses, identifies deficiencies in teacher preparation, instruction, and development in the arts in California, and recommends minimum training requirements and support for professional development

Disc Large Homolog 1 Is Critical for Early T Cell Receptor Micro Cluster Formation and Activation in Human T Cells

T cell activation by antigen involves multiple sequential steps, including T cell receptor-microcluster TCR-(MC) formation, immunological synapse formation, and phosphorylation of mediators downstream of the TCR. The adaptor protein, Disc Large Homolog 1 (DLG1), is known to regulate proximal TCR signaling and, in turn, T cell activation, acting as a molecular chaperone that organizes specific kinases downstream of antigen recognition. In this study, we used knockdown and knockout technologies in human primary T cells and a human T cell line to demonstrate the role of DLG1 in proximal T cell signaling. High-end confocal microscopy was used for pictorial representation of T cell micro-clusters and colocalization studies. From all these studies, we could demonstrate that DLG1 functions even earlier than immunological synapse formation, to regulate T cell activation by promoting TCR-MC formation. Moreover, we found that DLG1 can act as a bridge between the TCR-ζ chain and ZAP70 while inhibiting binding of the phosphatase SHP1 to TCR-ζ. Together, these effects drive dysregulation of T cell activation in DLG1-deficient T cells. Overall, the activation and survival status of T cell is a critical determinant of effective vaccine response, and DLG1-mediated T cell signaling events can be a driving factor for improving vaccine-designing strategies

Disc Large Homolog 1 Is Critical for Early T Cell Receptor Micro Cluster Formation and Activation in Human T Cells

T cell activation by antigen involves multiple sequential steps, including T cell receptor-microcluster TCR-(MC) formation, immunological synapse formation, and phosphorylation of mediators downstream of the TCR. The adaptor protein, Disc Large Homolog 1 (DLG1), is known to regulate proximal TCR signaling and, in turn, T cell activation, acting as a molecular chaperone that organizes specific kinases downstream of antigen recognition. In this study, we used knockdown and knockout technologies in human primary T cells and a human T cell line to demonstrate the role of DLG1 in proximal T cell signaling. High-end confocal microscopy was used for pictorial representation of T cell micro-clusters and colocalization studies. From all these studies, we could demonstrate that DLG1 functions even earlier than immunological synapse formation, to regulate T cell activation by promoting TCR-MC formation. Moreover, we found that DLG1 can act as a bridge between the TCR-ζ chain and ZAP70 while inhibiting binding of the phosphatase SHP1 to TCR-ζ. Together, these effects drive dysregulation of T cell activation in DLG1-deficient T cells. Overall, the activation and survival status of T cell is a critical determinant of effective vaccine response, and DLG1-mediated T cell signaling events can be a driving factor for improving vaccine-designing strategies

NF kappa B regulator Bcl3 controls development and function of classical dendritic cells required for resistance to Toxoplasma gondii.

The atypical IκB family member Bcl3 associates with p50/NF-κB1 or p52/NF-κB2 homodimers in the nucleus, and positively or negatively modulates transcription in a context-dependent manner. In mice lacking Bcl3 globally or specifically in CD11c+ cells, we previously reported that Toxoplasma gondii infection is uniformly fatal and is associated with an impaired Th1 immune response. Since Bcl3 expression in dendritic cells (DC) is pivotal for antigen presentation and since classical DCs (cDC) are major antigen presenting cells, we investigated the role of Bcl3 specifically in cDCs in vivo by crossing Zbtb46 cre mice with Bcl3flx/flx mice. Bcl3flx/flx Zbtb46 cre mice were as susceptible to lethal T. gondii infection as total Bcl3-/- mice and generated poor Th1 immune responses. Consistent with this, compared to wildtype controls, splenic Xcr1+ Bcl3-deficient cDC1 cells were defective in presenting Ova antigen to OT-I cells both for Ova257-264 peptide and after infection with Ovalbumin-expressing T. gondii. Moreover, splenic CD4+ and CD8+ T cells from infected Bcl3flx/flx Zbtb46 cre mice exhibited decreased T. gondii-specific priming as revealed by both reduced cytokine production and reduced T. gondii-specific tetramer staining. In vitro differentiation of cDCs from bone marrow progenitors also revealed Bcl3-dependent cDC-specific antigen-presentation activity. Consistent with this, splenocyte single cell RNA seq (scRNAseq) in infected mice revealed Bcl3-dependent expression of genes involved in antigen processing in cDCs. We also identified by scRNAseq, a unique Bcl3-dependent hybrid subpopulation of Zbtb46+ DCs co-expressing the monocyte/macrophage transcription factor Lysozyme M. This subpopulation exhibited Bcl3-dependent expansion after infection. Likewise, by flow cytometry we identified two T. gondii-induced hybrid subpopulations of Bcl3-dependent cDC1 and cDC2 cells both expressing monocyte/macrophage markers, designated as icDC1 and icDC2. Together, our results indicate that Bcl3 in classical DCs is a major determinant of protective T cell responses and survival in T. gondii-infection

Antimony Resistant <i>Leishmania donovani</i> but Not Sensitive Ones Drives Greater Frequency of Potent T-Regulatory Cells upon Interaction with Human PBMCs: Role of IL-10 and TGF-β in Early Immune Response

<div><p>In India the sand fly, <i>Phlebotomus argentipes</i>, transmitted parasitic disease termed kala-azar is caused by <i>Leishmania donovani</i> (LD) in humans. These immune-evading parasites have increasingly developed resistance to the drug sodium antimony gluconate in endemic regions.</p><p>Lack of early diagnosis methods for the disease limits the information available regarding the early interactions of this parasite with either human tissues or cell lineages. We reasoned that peripheral blood mononuclear cells (PBMCs) from healthy human beings could help compare some of their immune signatures once they were exposed for up to 8 days, to either pentavalent antimony sensitive (Sb^S-LD) or resistant (Sb^R-LD) <i>Leishmania donovani</i> isolates.</p><p>At day 2, PBMC cultures exposed to Sb^S-LD and Sb^R-LD stationary phase promastigotes had four and seven fold higher frequency of IL-10 secreting monocyte-macrophage respectively, compared to cultures unexposed to parasites. Contrasting with the CD4⁺CD25⁻CD127⁻ type-1 T-regulatory (Tr1) cell population that displayed similar features whatever the culture conditions, there was a pronounced increase in the IL-10 producing CD4⁺CD25⁺CD127^low/− inducible T-regulatory cells (iTregs) in the PBMC cultures sampled at day 8 post addition of Sb^R-LD.</p><p>Sorted iTregs from different cultures on day 8 were added to anti-CD3/CD28 induced naïve PBMCs to assess their suppressive ability. We observed that iTregs from Sb^R-LD exposed PBMCs had more pronounced suppressive ability compared to Sb^S-LD counterpart on a per cell basis and is dependent on both IL-10 and TGF-β, whereas IL-10 being the major factor contributing to the suppressive ability of iTregs sorted from PBMC cultures exposed to Sb^S–LD. Of note, iTreg population frequency value remained at the basal level after addition of genetically modified Sb^R-LD lacking unique terminal sugar in surface glycan.</p><p>Even with limitations of this artificial <i>in vitro</i> model of <i>L. donovani</i>-human PBMC interactions, the present findings suggest that Sb^R-LD have higher immunomodulatory capacity which may favour aggressive pathology.</p></div

GalT Knock down parasites (KDSb^R-LD) behaves like Sb^S-LD isolates.

<p><b>A</b>. Production of IL-10, <b>B</b>. TGF-β from KDSb^R, Sb^S and Sb^R-Sup. For TGF-β measurement the culture supernatants were acidified followed by neutralization, the level of TGF-β was measured by ELISA. <b>C</b>. Suppression assay performed in presence of neutralizing IL-10 antibody and/or rLAP using iTreg from KDSb^R-LD-PBMC at day eight. Data were analysed by Mann-Whitney test and two-tailed paired Student t test, and levels of significance are indicated by P values.</p

Isolated iTregs are active and shows pronounced suppressive ability.

<p><b>A</b>. Percentage suppression analysed after co-culturing isolated iTreg cells (described earlier) at day eight with autologous freshly isolated PBMC at different Treg: responder ratio. Expression of GARP/LRRC32 in iTreg cells. <b>B</b>. mRNA expression level of LRRC32 gene in sorted iTreg cells with median values indicated and <b>C</b>. iMFI values was calculated by multiplying the frequency of GARP protein expressing iTregs and their mean fluorescence intensity. Data were analyzed by the Mann-Whitney test, and levels of significance are indicated by P values. Groups Sb^S-iTreg and Sb^R-iTreg were compared to control iTregs. “*” indicates 0.0001</p

IL-10 producing Tr1 and iTReg cells are induced in response to Sb^S and Sb^R-LD.

<p>Percentages of IL-10 producing Tr1 and iTreg cells from Sb^S-LD-PBMC and Sb^R-LD-PBMC at early and late time point are represented. <b>A, B</b>. Percentage of IL10⁺Tr1 cells on day two, <b>C</b>. Real-Time RTPCR of IL-10 mRNA level in sorted population of Tr1 cells on day two. <b>D, E</b>. Percentage of IL10⁺Tr1 cells on day eight, <b>F</b>. Real-Time RTPCR of IL-10 mRNA level in sorted population of Tr1 cells on day eight. <b>G, H</b>. Percentage of IL10⁺iTreg cells on day two, <b>I</b>. Real-Time RTPCR of IL-10 mRNA level in sorted population of iTeg cells on day two. <b>J, K</b>. Percentage of IL10⁺iTreg cells on day eight, <b>L</b>. Real-Time RTPCR of IL-10 mRNA level in sorted population of iTreg cells on day eight. Dot plots indicate one representative data set. Data were analyzed by the Mann-Whitney test, and levels of significance are indicated by P values.</p

Median values of IL-10 production (pg/ml) at different time points.

<p>The median values of IL-10 production at different time points are tabulated. Freshly isolated PBMCs were incubated with different Sb^S and Sb^R-LD isolates and culture supernatants (Sb^S and Sb^R-Sup) were isolated on day- one, two, three and eight, and IL-10 level (pg/ml) was measured by ELISA/CBA.</p