Journal: The Journal of Biological Chemistry

Article Title: Magnesium transporter 1 (MAGT1) deficiency causes selective defects in N- linked glycosylation and expression of immune-response genes

doi: 10.1074/jbc.RA119.008903

Figure Lengend Snippet: Mg2+ regulates glycosylation and plays a unique role in T-cell–mediated immunity, especially in immune protection against EBV. A, flow cytometry histograms of surface expression of NKG2D, CD70, HLA-DR, and CD5 in cycling T cells from HCs cultured either in cRPMI for 5 days, dRPMI for 5 days, or cells that were cultured in dRPMI for 3 days followed by the addition of Mg2+ (0.5 mm) back into the dRPMI for 2 days. B, quantification of the MFI in A. Error bars represent the standard error of the mean of eight independent experiments, and p values were calculated with a paired t test. C, representative Western blot analysis of NKG2D, CD70, TCR-β, and β-actin in cycling T cells from a HC as described in A. Numbers at left indicate kDa standards. Glycosylation patterns are shown at right: fully-glycosylated (2); partially-glycosylated (1); and unglycosylated (0). Data are mean of eight (A and B) or are representative of three (C) independent replicates. D, schematic diagram for killing pathway with NK cells (top) and EBV-721.221(target; bottom) cells. E, flow cytometry analysis of surface expression of NKG2D in EBV-specific NK cells from a HC with cRPMI and dRPMI. F, quantification of the MFI in F. G, percent lysis of autologous EBV-LCLs by EBV-specific NK cells from HC with a different dose of the Mg2+ with significance determined by one-way ANOVA. n indicates the number of independent samples. H, quantification from G. Data are representative of three independent biological replicates. NS, nonsignificant.

Article Snippet: The following antibodies were used: MAGT1 (Mer catalog no. 17-1, 1:50 dilution, produced by Merck); TUSC3 (Mer 85 catalog no. 38-1, affinity-purified, produced by Merck) ( Fig. S6 ) 8 ; β-actin (ab20272, Abcam); NKG2D (D-20, sc-9621, Santa Cruz Biotechnology); tubulin (AA2, 05-661, Merck Millipore); TCR-β (H-197, sc-9101, Santa Cruz Biotechnology); CERS2 (A303-193A, Bethyl Laboratories, Inc.); ribophorin I (C-15, sc-12164, Santa Cruz Biotechnology); COP1 (PA1-061, Thermo Fisher Scientific); EEA1 (C45B10, 3288 Cell Signaling); STT3B (HPA036646, Sigma); OST 48 (Santa Cruz Biotechnology); Na,K-ATPase α1 (D4Y7E, 23565, Cell Signaling).

Techniques: Glycoproteomics, Flow Cytometry, Expressing, Cell Culture, Western Blot, Lysis

Journal:

Article Title: Control of Cytomegalovirus in Bone Marrow Transplantation Chimeras Lacking the Prevailing Antigen-Presenting Molecule in Recipient Tissues Rests Primarily on Recipient-Derived CD8 T Cells

doi:

Figure Lengend Snippet: Establishment of chimerism in an HvG setting of MHC class I-mismatched BMT after incomplete lymphohematoablative conditioning. BALB/c-H-2dm2 recipients (uninfected [BMT] and infected with murine CMV on the day of BMT [BMT + CMV]) were γ irradiated with a sublethal dose of 6 Gy and then reconstituted with the indicated graded numbers of BMC derived from BALB/c donors. (A) Kaplan-Meier survival plots for groups of 20 mice. The asterisk marks the conditions of BMT under which all subsequent experiments were performed. (B) Origin of BMC in the repopulated recipient BM measured for 6-week survivors. Femoral BMC of three mice were pooled for analysis. Positive expression of Ld identifies donor (BALB/c)-derived BMC. The histograms are based on the analysis of 10,000 BMC. RCN, relative cell number. Expression of Ld (abscissa) is given in log FITC fluorescence (FL-1) intensity. (C) Analysis of chimerism among T cells and T-cell subsets derived from the spleens of 6-week survivors. Lymphocytes derived from three spleens were pooled for analysis. Individual testing of aliquots taken before forming the pools gave essentially the same results (not depicted). Three-color fluorescence (FL) analyses were performed for the marker combinations FITC (FL-1)-Ld plus PE (FL-2)-TCR α/β plus RED613 (FL-3)-CD4 or RED613 (FL-3)-CD8. A first gate was set on lymphocytes; a second gate was set on α/β T cells positive in FL-2 (top), on CD4 T cells simultaneously positive in FL-2 and FL-3 (center), and on CD8 T cells simultaneously positive in FL-2 and FL-3 (bottom). FL-1 histograms are shown for 10,000 gated cells. For chimeric cell populations, the percentage of donor-derived, Ld-positive cells is indicated. Controls of Dd expression in the chimeras and of Ld expression in BALB/c-BALB/c isochimeras gave single peaks with positive FL-1 (not shown); these controls were made to make sure that we were not misled by MHC class I-negative cells and by Ld-low/high-expressing cells, respectively.

Article Snippet: L d was labeled as described above; labeling of D d was performed directly with FITC-conjugated mouse MAb anti-mouse H-2D d MAb (IgG2a; clone 34-5-85, catalog no. 9009F; Cedarlane). (ii) T-cell receptor (TCR) α/β was labeled with phycoerythrin (PE)-conjugated hamster anti-mouse TCR α/β MAb (clone H57-597, catalog no. 01305A; Pharmingen). (iii) Labeling of surface CD8 and CD4 was performed with duochrome (RED613)-conjugated rat anti-mouse CD8a MAb (IgG2a; clone 53-6.7, catalog no. 19870-021; Gibco BRL) and CD4 (IgG2a; clone H129.19, catalog no. 19862-028; Gibco BRL), respectively.

Techniques: Infection, Irradiation, Derivative Assay, Expressing, Fluorescence, Marker

Journal:

Article Title: Control of Cytomegalovirus in Bone Marrow Transplantation Chimeras Lacking the Prevailing Antigen-Presenting Molecule in Recipient Tissues Rests Primarily on Recipient-Derived CD8 T Cells

doi:

Figure Lengend Snippet: Preferential recruitment of CD8 T cells to the infected lungs and control of pulmonary infection. (A) Three-color cytofluorometric analysis of lung infiltrate lymphocytes in the combination FITC (FL-1)-CD8 plus PE (FL-2)-TCR α/β plus RED613 (FL-3)-CD4. A lymphocyte gate was set in the FSC-versus-SSC plot, representing cell size and cell granularity, respectively (upper left); a second gate was set on positive FL-2 to select cells expressing TCR α/β (lower left). (Right) Expression of CD4 and CD8 among the FL-2-positive α/β T cells, shown in a two-dimensional dot plot of FL-3 and FL-1 fluorescence, respectively. The percentages of CD4 and CD8 T cells as well as the CD8/CD4 ratio are indicated. The analysis of the subpopulations was based on 20,000 α/β T cells. (B) Kinetics of lung infiltration. For independent but analogous transplantations, CD8/CD4 ratios within pulmonary α/β T cells were determined in weekly intervals. Each closed circle represents the analysis at the indicated time point of a particular transplantation. Data refer to a pool of pulmonary infiltrate cells derived from five recipients. The median values for the independent transplantations are indicated by dashes; the shaded area indicates the range of CD8/CD4 ratios observed in uninfected recipients under otherwise corresponding conditions. n.t., not tested. (C) Kinetics of murine CMV replication in the lungs. Virus titers in the lungs of infected recipients were determined for the transplantations shown in panel B. Closed circles represent the median values of the virus titers of three recipients per transplantation and time point. The median values for the independent transplantations are marked by dashes. The dotted line indicates the detection limit (DL) of the plaque assay. n.t., not tested.

Article Snippet: L d was labeled as described above; labeling of D d was performed directly with FITC-conjugated mouse MAb anti-mouse H-2D d MAb (IgG2a; clone 34-5-85, catalog no. 9009F; Cedarlane). (ii) T-cell receptor (TCR) α/β was labeled with phycoerythrin (PE)-conjugated hamster anti-mouse TCR α/β MAb (clone H57-597, catalog no. 01305A; Pharmingen). (iii) Labeling of surface CD8 and CD4 was performed with duochrome (RED613)-conjugated rat anti-mouse CD8a MAb (IgG2a; clone 53-6.7, catalog no. 19870-021; Gibco BRL) and CD4 (IgG2a; clone H129.19, catalog no. 19862-028; Gibco BRL), respectively.

Techniques: Infection, Expressing, Fluorescence, Transplantation Assay, Derivative Assay, Plaque Assay

Journal:

Article Title: Control of Cytomegalovirus in Bone Marrow Transplantation Chimeras Lacking the Prevailing Antigen-Presenting Molecule in Recipient Tissues Rests Primarily on Recipient-Derived CD8 T Cells

doi:

Figure Lengend Snippet: Chimerism among CD8 T cells in pulmonary infiltrates. Pulmonary lymphocytes were isolated at the peak of infiltration after BMT (6 Gy, 107 BMC) and infection. Three-color cytofluorometric analysis was performed for the combination FITC (FL-1)-Dd or FITC (FL-1)-Ld plus PE (FL-2)-TCR α/β plus RED613 (FL-3)-CD8. A gate was set on lymphocytes, and the analysis was restricted to α/β T cells by a second gate set on positive FL-2. (A and C) Two-dimensional dot plots of FL-3 (CD8) versus FL-1 (Dd [A] and Ld [C]) for 20,000 α/β T cells. CD8-positive cells are marked by a frame. (B and D) Histograms of FL-1 for the framed FL-2- and FL-3-positive CD8 T cells. The percentages of Ld-negative, recipient-derived and Ld-positive, donor-derived cells are indicated.

Article Snippet: L d was labeled as described above; labeling of D d was performed directly with FITC-conjugated mouse MAb anti-mouse H-2D d MAb (IgG2a; clone 34-5-85, catalog no. 9009F; Cedarlane). (ii) T-cell receptor (TCR) α/β was labeled with phycoerythrin (PE)-conjugated hamster anti-mouse TCR α/β MAb (clone H57-597, catalog no. 01305A; Pharmingen). (iii) Labeling of surface CD8 and CD4 was performed with duochrome (RED613)-conjugated rat anti-mouse CD8a MAb (IgG2a; clone 53-6.7, catalog no. 19870-021; Gibco BRL) and CD4 (IgG2a; clone H129.19, catalog no. 19862-028; Gibco BRL), respectively.

Techniques: Isolation, Infection, Derivative Assay

Journal:

Article Title: Control of Cytomegalovirus in Bone Marrow Transplantation Chimeras Lacking the Prevailing Antigen-Presenting Molecule in Recipient Tissues Rests Primarily on Recipient-Derived CD8 T Cells

doi:

Figure Lengend Snippet: Absence of MHC class I Ld-specific, HvG-directed cytolytic activity in the lungs of mixed chimeras. Pulmonary infiltrate lymphocytes isolated at the peak of lung infiltration were tested for ex vivo cytolytic activity at the indicated effector-to-target (E/T) cell ratios on target cells expressing Ld (A; parental P815 mastocytoma), Ld plus B7-1 (B; transfectant P815-B7), and Ld plus Fas (C; transfectant P815-Fas). Insets show the cytofluorometric analyses verifying the expression of B7-1 and Fas by the respective transfectants, as well as their absence on parental P815. FL, log FITC fluorescence intensity. The total CTL activity contained in the tested lymphocyte population was determined by the TCR redirected lysis assay using a MAb directed against TCR α/β bound to Fc receptors that are expressed constitutively by P815 (D). All target cells expressed surface Ld as detected by cytofluorometry, and all were lysed by a CTL line specific for Ld (not shown).

Article Snippet: L d was labeled as described above; labeling of D d was performed directly with FITC-conjugated mouse MAb anti-mouse H-2D d MAb (IgG2a; clone 34-5-85, catalog no. 9009F; Cedarlane). (ii) T-cell receptor (TCR) α/β was labeled with phycoerythrin (PE)-conjugated hamster anti-mouse TCR α/β MAb (clone H57-597, catalog no. 01305A; Pharmingen). (iii) Labeling of surface CD8 and CD4 was performed with duochrome (RED613)-conjugated rat anti-mouse CD8a MAb (IgG2a; clone 53-6.7, catalog no. 19870-021; Gibco BRL) and CD4 (IgG2a; clone H129.19, catalog no. 19862-028; Gibco BRL), respectively.

Techniques: Activity Assay, Isolation, Ex Vivo, Expressing, Transfection, Fluorescence, Lysis

Journal:

Article Title: Control of Cytomegalovirus in Bone Marrow Transplantation Chimeras Lacking the Prevailing Antigen-Presenting Molecule in Recipient Tissues Rests Primarily on Recipient-Derived CD8 T Cells

doi:

Figure Lengend Snippet: Cytolytic activity of sorted recipient-derived and donor-derived CD8 T cells. (A) Pulmonary lymphocytes were isolated at the peak of infiltration, and CD8-positive cells were enriched by immunomagnetic selection. (Top) The efficacy of the positive selection was monitored for an aliquot of the cells by a two-color cytofluorometric analysis of CD8 (PE, FL-2) versus Ld (FITC, FL-1) expression performed for 20,000 cells with no preselection of gates. (Bottom) The majority of the CD8 T cells were labeled for Ld only and were then sorted into Ld-negative and Ld-positive sets. The efficacy of the sorting was monitored for 2,000 cells of each set by two-color cytofluorometric analysis of TCR α/β (PE, FL-2) and Ld (FITC, FL-1) expression performed with no preselection of gates. (B) The cytolytic activity of pulmonary CD8 T cells was determined by TCR α/β redirected lysis at the indicated effector-to-target (E/T) cell ratios. (Left) Cytolytic activity of the chimeric CD8 T-cell population after immunomagnetic purification but before the cytofluorometric sorting; (right) cytolytic activity of the sorted cells. For comparison, the presort activity is included for the part of the titration marked by dotted lines.

Article Snippet: L d was labeled as described above; labeling of D d was performed directly with FITC-conjugated mouse MAb anti-mouse H-2D d MAb (IgG2a; clone 34-5-85, catalog no. 9009F; Cedarlane). (ii) T-cell receptor (TCR) α/β was labeled with phycoerythrin (PE)-conjugated hamster anti-mouse TCR α/β MAb (clone H57-597, catalog no. 01305A; Pharmingen). (iii) Labeling of surface CD8 and CD4 was performed with duochrome (RED613)-conjugated rat anti-mouse CD8a MAb (IgG2a; clone 53-6.7, catalog no. 19870-021; Gibco BRL) and CD4 (IgG2a; clone H129.19, catalog no. 19862-028; Gibco BRL), respectively.

Techniques: Activity Assay, Derivative Assay, Isolation, Selection, Expressing, Labeling, Lysis, Purification, Titration