Stereoselective syntheses of (E)-α,β-didehydroamino acid and peptide containing its residue utilizing oxazolidinone derivative

金沢大学理工学域Reaction of methyl N-Boc-N-phenoxycarbonylglycinate with various aldehydes afforded the corresponding cis-4,5-oxazolidinone derivatives, which were effectively converted to (E)-α,β-didehydroamino acids by means of a base. Furthermore, N-deprotection of the oxazolidinone derivatives and subsequent coupling reaction with Boc-amino acid furnished the corresponding dipeptides, which were transformed to dipeptide containing α,β-didehydroamino acid with high E selectivity

CIN85 drives B cell responses by linking BCR signals to the canonical NF-κB pathway

CIN85 transduces B cell receptor signals to IKK-β, and its expression in B cells is essential for T cell–independent type II antibody responses in mice

The Role of BACH2 in T Cells in Experimental Malaria Caused by Plasmodium chabaudi chabaudi AS

BTB and CNC Homology 1, Basic Leucine Zipper Transcription Factor 2 (BACH2) is a transcription factor best known for its role in B cell development. More recently, it has been associated with T cell functions in inflammatory diseases, and has been proposed as a master transcriptional regulator within the T cell compartment. In this study, we employed T cell-specific Bach2-deficient (B6.Bach2ΔT) mice to examine the role of this transcription factor in CD4+ T cell functions in vitro and in mice infected with Plasmodium chabaudi AS. We found that under CD4+ T cell polarizing conditions in vitro, Th2, and Th17 helper cell subsets were more active in the absence of Bach2 expression. In mice infected with P. chabaudi AS, although the absence of Bach2 expression by T cells had no effect on blood parasitemia or disease pathology, we found reduced expansion of CD4+ T cells in B6.Bach2ΔT mice, compared with littermate controls. Despite this reduction, we observed increased frequencies of Tbet+ IFNγ+ CD4+ (Th1) cells and IL-10-producing Th1 (Tr1) cells in mice lacking Bach2 expression by T cells. Studies in mixed bone marrow chimeric mice revealed T cell intrinsic effects of BACH2 on hematopoietic cell development, and in particular, the generation of CD4+ and CD8+ T cell subsets. Furthermore, T cell intrinsic BACH2 was needed for efficient expansion of CD4+ T cells during experimental malaria in this immunological setting. We also examined the response of B6.Bach2ΔT mice to a second protozoan parasitic challenge with Leishmania donovani and found similar effects on disease outcome and T cell responses. Together, our findings provide new insights into the role of BACH2 in CD4+ T cell activation during experimental malaria, and highlight an important role for this transcription factor in the development and expansion of T cells under homeostatic conditions, as well as establishing the composition of the effector CD4+ T cell compartment during infection

The adaptor molecule CD2AP in CD4 T cells modulates differentiation of follicular helper T cells during chronic LCMV infection

<div><p>CD4 T cell-mediated help to CD8 T cells and B cells is a critical arm of the adaptive immune system required for control of pathogen infection. CD4 T cells express cytokines and co-stimulatory molecules that support a sustained CD8 T cell response and also enhance generation of protective antibody by germinal center B cells. However, the molecular components that modulate CD4 T cell functions in response to viral infection or vaccine are incompletely understood. Here we demonstrate that inactivation of the signaling adaptor CD2-associated protein (CD2AP) promotes CD4 T cell differentiation towards the follicular helper lineage, leading to enhanced control of viral infection by augmented germinal center response in chronic lymphocytic choriomeningitis virus (LCMV) infection. The enhanced follicular helper differentiation is associated with extended duration of TCR signaling and enhanced cytokine production of CD2AP-deficient CD4 T cells specifically under T_H1 conditions, while neither prolonged TCR signaling nor enhanced follicular helper differentiation was observed under conditions that induce other helper effector subsets. Despite the structural similarity between CD2AP and the closely related adaptor protein CIN85, we observed defective antibody-mediated control of chronic LCMV infection in mice lacking CIN85 in T cells, suggesting non-overlapping and potentially antagonistic roles for CD2AP and CIN85. These results suggest that tuning of TCR signaling by targeting CD2AP improves protective antibody responses in viral infection.</p></div

<i>Cd2ap</i>-deficiency enhances T_FH differentiation in a cell-intrinsic manner.

<p>(A) Schematic of experimental strategy for the generation of mixed BM chimeras and subsequent analysis of anti-LCMV responses. (B) Contribution of CD45.2 <i>Cd2ap</i>^F/F or <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F cells to B cells, activated CD4 T cells and T_FH cells in the mixed BM chimeras 22 days after LCMV-c13 infection. Representative Data from 2 independent experiments (n = 4 of each genotype) are shown with means and standard deviations. Statistical difference was tested by Student’s <i>t</i>-test. (C) A volcano plot showing differentially expressed genes between Cd2ap-deficient (KO, n = 2) and -sufficient (WT, n = 2) T_FH cells harvested from mixed BM chimeras 22 days after infection. Genes that were differentially expressed by >2-fold are shown.</p

<i>Cd2ap</i> deficiency has a minimal impact on T_FH differentiation and GC B cell responses following immunization with SRBCs.

<p>(A, B) Flow cytometric analysis of expression of PD-1 and CXCR5 on pre-gated CD4⁺ B220⁻ T cells (A) and GL7 and Fas expression on CD19⁺ B220⁺ B Cells (B) 12 days following SRBC immunization. (C-E) Numbers and frequencies of total CD4 T cells and CXCR5⁺ PD-1⁺ T_FH in the spleen of <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F and control <i>Cd2ap</i>^F/F mice 12 days after immunization with SRBCs. (F-H) Numbers and frequencies of total B cells and Fas⁺ GL7⁺ GC B cells in the spleen of <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F and control <i>Cd2ap</i>^F/F mice 12 days after immunization with SRBCs. Data are pooled from 2 independent experiments shown as means and standard deviation.</p

<i>Cin85</i>-Deficiency results in defective clearance of LCMV-c13 and is correlated with decreased neutralizing antibody titer.

<p>(A, B) Analysis of LCMV abundance in plasma in <i>Cin85</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cin85</i>^F/F mice as determined by LCMV <i>gp</i> transcript levels (A) or focus forming assay (B) at day 80. Horizontal lines indicate median. The limit of detection is shown by dashed lines. Statistical significance was tested by Mann Whitney U-test. (C) Frequencies of Fas⁺ GL7⁺ B220⁺ GC B cells at day 35 after LCMV-c13 infection. (D) anti-LCMV IgG antibody titers of plasma from <i>Cin85</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cin85</i>^F/F mice 30 days after LCMV-c13 infection. (E) Neutralizing activity of plasma from <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F mice 80 days after LCMV-c13 infection. Reduction in focus forming units (FFU) in the presence of 1:5 dilution of plasma compared to untreated control (% Neutralization) is shown with mean and standard deviation. Student <i>t</i>-test. Data combined from 3 independent experiments are shown with n = 3–4 mice per genotype.</p

<i>Cd2ap</i>-deficiency causes sustained TCR signaling specifically in T_H1 cells <i>in vitro</i>.

<p>(A) Schematic of experimental strategy for culture of CD4 T cells. (B) Concentration of IFN-γ or IL-4 in supernatant measured by ELISA 24 hours after re-stimulation of polarized <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F T_H1 cells and T_H2 cells with plate-bound anti-CD3/anti-CD28 antibodies. (C) Immunoblotting showing phosphorylation of MEK1/2 in polarized <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F T_H1 or T_H2 cells following re-stimulation with plate-bound anti-CD3/anti-CD28 for the indicated times at 37°C. Anti-CD2AP, -CIN85 and ERK were used as control. Data are representative of three experiments. (D) Downregulation of surface TCR of <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F T cells that were polarized under indicated conditions. Cells were stimulated with plate-bound anti-CD3 and surface TCR levels were quantitated at the indicated time points. Data are representative of three experiments. (E, F) Intracellular staining for IFN-γ and TNF-α of polarized <i>Cd2ap</i>^F/F and <i>Cd4</i>-cre⁺ <i>Cd2ap</i>^F/F T_H1 cells after re-stimulation with PMA and Ionomycin or plate-bound anti-CD3 and anti-CD28 for 4 or 24 hours in the presence of Brefeldin A for the last 2 hours before harvest. Representative plots (E) and statistical analyses with means and standard deviations (F) from three experiments are shown.</p