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Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: (A) CD28+ and CD28− RO+ CD8 T cells were isolated from peripheral blood mononuclear cells, labeled with CFSE, and aliquots cultured with a mixture of 3 different allogeneic B cell lines. After 96 hours, the cells were collected, washed and the dilution of CFSE by the CD8 T cells was analyzed in a histogram as an indication of proliferation. Representative results from a single experiment of 6 different experiments having similar results in each are shown. (B) Cumulative data of proliferating memory CD28+ vs. CD28− memory CD8 T cells from the 6 different experiments is shown with mean percent proliferation of each T cell population ± SD. *p < 0.01

Article Snippet: PBMC were isolated by Ficoll-Hypaque separation (IsoPrep, Robbins Scientific Corporation, Sunnyvale, CA) and processed to isolate the CD8 memory T cells (CD8 + CD45RO + CD45RA − CD56 − CD57 − ) by negative selection using the Human CD8 + Memory T Cell Isolation Kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Isolation, Labeling, Cell Culture

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: CD28+ and CD28− RO+ CD8 T cells were isolated from peripheral blood mononuclear cells, labeled with CFSE, and aliquots cultured with or without a mixture of 3 different allogeneic B cell lines in the absence or presence of the indicated amounts of recombinant IL-15. After 96 hours, the cells were collected, washed and the dilution of CFSE by the CD8 T cells was analyzed in a histogram as an indication of proliferation. Representative results from a single experiment of 4 different experiments having similar results in each are shown.

Article Snippet: PBMC were isolated by Ficoll-Hypaque separation (IsoPrep, Robbins Scientific Corporation, Sunnyvale, CA) and processed to isolate the CD8 memory T cells (CD8 + CD45RO + CD45RA − CD56 − CD57 − ) by negative selection using the Human CD8 + Memory T Cell Isolation Kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Isolation, Labeling, Cell Culture, Recombinant

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: CD28+ and CD28− RO+ CD8 T cells were isolated from peripheral blood mononuclear cells, labeled with CFSE, and aliquots cultured with a mixture of 3 different allogeneic B cell lines in the absence or presence of 10 ng/ml of recombinant IL-15. At the indicated time after culture initiation, the cells were collected, washed and the dilution of CFSE by the CD8 T cells was analyzed in a histogram as an indication of proliferation. Representative results from a single experiment of 4 different experiments having similar results in each are shown.

Article Snippet: PBMC were isolated by Ficoll-Hypaque separation (IsoPrep, Robbins Scientific Corporation, Sunnyvale, CA) and processed to isolate the CD8 memory T cells (CD8 + CD45RO + CD45RA − CD56 − CD57 − ) by negative selection using the Human CD8 + Memory T Cell Isolation Kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Isolation, Labeling, Cell Culture, Recombinant

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: (A) CD28+ and CD28− RO+ CD8 T cells were isolated from peripheral blood mononuclear cells, labeled with CFSE, and aliquots cultured with or without a mixture of 3 different allogeneic B cell lines in the absence or presence of 10 ng/ml of recombinant IL-15, IL-2 or IL-7. After 96 hours, the cells were collected, washed and the dilution of CFSE by the CD8 T cells was analyzed in a histogram as an indication of proliferation. Representative results from a single experiment of 4 different experiments having similar results in each are shown. (B) Cumulative data of proliferating memory CD28+ vs. CD28− memory CD8 T cells from the 4 different experiments is shown with mean percent proliferation of each T cell population ± SD. *p < 0.001 for CD28− cells with allogeneic B cells plus IL-15 vs. all other groups and **p < 0.01 for CD28+ cells with allogeneic B cells plus IL-15 vs. T cells cultured with B cells only. (C) CD28− RO+ CD8 T cells were isolated from peripheral blood mononuclear cells, labeled with CFSE, and aliquots cultured with or without a mixture of 3 different allogeneic B cell lines in the absence or presence of the indicated concentrations of recombinant IL-15, IL-2 or IL-7. After 96 hours, the cells were collected, washed and the dilution of CFSE by the CD8 T cells was analyzed and the proliferation observed reported as in Figures 2 and ​and3.3. Representative results from a single experiment of 3 different experiments having similar results in each are shown.

Article Snippet: PBMC were isolated by Ficoll-Hypaque separation (IsoPrep, Robbins Scientific Corporation, Sunnyvale, CA) and processed to isolate the CD8 memory T cells (CD8 + CD45RO + CD45RA − CD56 − CD57 − ) by negative selection using the Human CD8 + Memory T Cell Isolation Kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Isolation, Labeling, Cell Culture, Recombinant

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: (A) CD28+ and CD28− RO+ CD8 T cells were labeled with CFSE and aliquots cultured with allogeneic B cells plus 10 ng/ml of recombinant IL-15. After 96 hours, the cultured cells were stained with fluorochrome labeled antibodies to detect expression of CD25, CD127, CD215 and ICOS on gated T cells that were (blue line) or were not (red line) proliferating as assessed by CFSE dilution. Representative results from a single experiment of 5 different experiments having similar results in each are shown. (B) CD28− memory CD8 T cells were isolated, labeled with CFSE, and aliquots cultured with pooled allogeneic B cells in culture media with 10 ng/ml IL-15. After 24 hours, the IL-15 supernatant was removed and replaced with media alone or media with 10 ng/ml IL-2. IL-2 was also added without removal of IL-15. All cells were analyzed for proliferation at 96 hours after culture initiation. Representative results from a single experiment of 4 different experiments having similar results in each are shown. (C) CD28+ and CD28− RO+ CD8 T cells were isolated, labeled with CFSE, and aliquots cultured with or without allogeneic B cell lines in the presence of 10 ng/ml IL-15. After 48 or 72 hours, the cells were collected and whole cell RNA isolated for PCR analysis of IL-15 receptor α chain. The results indicate the mean relative quantitation (RQ) of IL-15Rα in the memory CD8 T cells cultured alone or with allogeneic B cells ± SD for 4 individual samples. *p < 0.01 for expression of CD28− vs. CD28+ memory T cells.

Article Snippet: PBMC were isolated by Ficoll-Hypaque separation (IsoPrep, Robbins Scientific Corporation, Sunnyvale, CA) and processed to isolate the CD8 memory T cells (CD8 + CD45RO + CD45RA − CD56 − CD57 − ) by negative selection using the Human CD8 + Memory T Cell Isolation Kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Labeling, Cell Culture, Recombinant, Staining, Expressing, Isolation, Quantitation Assay

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: (A) CD28+ and CD28− RO+ CD8 T cells were labeled with CFSE and aliquots cultured with allogeneic B cell lines with 10 ng/ml of recombinant IL-15. After 96 hours, the cultured cells were stained with fluorochrome labeled antibodies to detect expression of CD107a expression. The CFSE dilution patterns were used to gate the T cells into non-proliferating (gate A), early proliferating (gate B) and late proliferating (gate C) cells and the expression levels of CD107a on T cells in each gate were determined. Representative results from a single experiment of 4 different experiments having similar results in each are shown. (B) CD107a expression was compared by overlaying histograms of late proliferating CD28− (red) and CD28+ (green) memory CD8 T cells following 96 hours of culture with allogeneic B cells plus IL-15. (C) After 96 hours, supernatants were removed from cultures of CD28+ and CD28− RO+ CD8 T cells with allogeneic B cells plus 10 ng/ml IL-15. Production of IFN-γ and TNF-α was tested by ELISA and expressed as mean concentration ± SD for 4 samples per group. *p < 0.01 for cultures with IL-15 vs. those without IL-15.

Article Snippet: PBMC were isolated by Ficoll-Hypaque separation (IsoPrep, Robbins Scientific Corporation, Sunnyvale, CA) and processed to isolate the CD8 memory T cells (CD8 + CD45RO + CD45RA − CD56 − CD57 − ) by negative selection using the Human CD8 + Memory T Cell Isolation Kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Labeling, Cell Culture, Recombinant, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: (A) Peripheral blood mononuclear cells (PBMC) were labeled with CFSE cultured with irradiated pooled allogeneic B cells +/− 10 ng/ml IL-15 in the presence of absence of 100 ug/ml CTLA-4Ig. Proliferation of the (CD3+) T cells was analyzed by flow cytometry 96 after initiation of the cultures. (B) Proliferation of CD28+ versus CD28− memory CD8 T cells alone or in response to allogeneic B cells plus 10 ng/ml IL-15 in the presence or absence of 100 ug/ml CTLA-4Ig. (C) Cumulative data from 4 separate experiments indicating the ability of IL-15 to confer resistance of alloantigen-reactive memory CD8 T cell or peripheral blood T cell proliferation to CTLA-4Ig. *p < 0.01 compared to all cultures with CTLA-4Ig but without IL-15. (D) Isolated CD28+ memory CD8 T cells were labeled with CFSE and cultured with pooled allogeneic B cells in the presence or absence of 10 ng/ml IL-15. After 96 hours, the cultured cells were washed and stained with anti-CD28 mAb and the expression of CD28 on non-proliferating and proliferating CD8 T cells was determined by flow cytometry. In the third (far right)panel, the expression of CD28 was compared on proliferating CD28+ memory CD8 T cells following 96 hours of cultures with the allogeneic B cells +/− IL-15 by overlaying the histograms from each of the other two panels.

Article Snippet: PBMC were isolated by Ficoll-Hypaque separation (IsoPrep, Robbins Scientific Corporation, Sunnyvale, CA) and processed to isolate the CD8 memory T cells (CD8 + CD45RO + CD45RA − CD56 − CD57 − ) by negative selection using the Human CD8 + Memory T Cell Isolation Kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Labeling, Cell Culture, Irradiation, Flow Cytometry, Isolation, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Allorecognition of HLA-C Mismatches by CD8 + T Cells in Hematopoietic Stem Cell Transplantation Is a Complex Interplay between Mismatched Peptide-Binding Region Residues, HLA-C Expression, and HLA-DPB1 Disparities

doi: 10.3389/fimmu.2016.00584

Figure Lengend Snippet: Heterogenous alloresponse tested by in vitro mixed lymphocyte reaction (MLR)/flow cytometry assays in 63 responder/stimulator cell combinations that were characterized by a single HLA-C mismatch . The mismatched HLA-C allele of the stimulator cells and the %ΔCD137 + CD8 + cells induced after restimulation at day 14 are indicated (see ). The cutoff of 2% ΔCD137 + CD8 + cells is indicated by the dashed line. Activation of CD8 + NK cells (ranging between 0.2 and 1.2% CD8 + CD56 + CD137 + cells, results not shown) was not taken into consideration. Overall, 24 different shared HLA-A~B~DRB1~DQB1 haplotypes were tested (data not shown). Twelve MLRs were repeated but dotted as single mean %ΔCD137 + CD8 + values. Black dots represent MLRs with HLA-C MMs located in α1/α2 domains and HLA-DPB1 MMs. Blue triangles represent MLRs with HLA-C MMs located in α1/α2 domains and matched HLA-DPB1. Open circles represent MLRs with C*03:03/03:04 MMs and C*03:04/03:03 MMs, all HLA-DPB1 MMs. Open triangles represent MLRs with HLA-C MMs located outside α1/α2 domains (C*02:29/02:02, C*02:02/02:29) and matched HLA-DPB1.

Article Snippet: The percentage of CD137 + PKH-2 − CD8 + CD56 − viable T cells was quantified by flow cytometry using APC-labeled anti-human CD8a, PerCP/Cy5.5 anti-human CD56 (BioLegend, Fell, Germany), and FITC-labeled anti-human CD137 (Milteny Biotec, Bergisch-Gladbach, Germany) antibodies, as well as APC- and FITC-labeled murine IgG1 isotype controls (BD Bioscience, Allschwil, Switzerland).

Techniques: In Vitro, Flow Cytometry, Activation Assay

Journal: Frontiers in Immunology

Article Title: Allorecognition of HLA-C Mismatches by CD8 + T Cells in Hematopoietic Stem Cell Transplantation Is a Complex Interplay between Mismatched Peptide-Binding Region Residues, HLA-C Expression, and HLA-DPB1 Disparities

doi: 10.3389/fimmu.2016.00584

Figure Lengend Snippet: Box and whisker plots of HLA-C mRNA expression quantified by RT-PCR in stimulator cells at day 0 inducing a negative versus a positive (cutoff = 2% ΔCD137 + CD8 + cells) alloresponse .

Article Snippet: The percentage of CD137 + PKH-2 − CD8 + CD56 − viable T cells was quantified by flow cytometry using APC-labeled anti-human CD8a, PerCP/Cy5.5 anti-human CD56 (BioLegend, Fell, Germany), and FITC-labeled anti-human CD137 (Milteny Biotec, Bergisch-Gladbach, Germany) antibodies, as well as APC- and FITC-labeled murine IgG1 isotype controls (BD Bioscience, Allschwil, Switzerland).

Techniques: Whisker Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Allorecognition of HLA-C Mismatches by CD8 + T Cells in Hematopoietic Stem Cell Transplantation Is a Complex Interplay between Mismatched Peptide-Binding Region Residues, HLA-C Expression, and HLA-DPB1 Disparities

doi: 10.3389/fimmu.2016.00584

Figure Lengend Snippet: Scatter plot of HLA-C expression [mean fluorescence intensity (MFI) values according to Ref. ( , )] and alloreactivity (%ΔCD137 + CD8 + cells) . A label is plotted for each stimulator allele at the top of the graph, indicating its corresponding MFI value. The linear regression through the data points is shown in red, and the confidence interval is in yellow (coefficient = 0.004, p = 0.637).

Article Snippet: The percentage of CD137 + PKH-2 − CD8 + CD56 − viable T cells was quantified by flow cytometry using APC-labeled anti-human CD8a, PerCP/Cy5.5 anti-human CD56 (BioLegend, Fell, Germany), and FITC-labeled anti-human CD137 (Milteny Biotec, Bergisch-Gladbach, Germany) antibodies, as well as APC- and FITC-labeled murine IgG1 isotype controls (BD Bioscience, Allschwil, Switzerland).

Techniques: Expressing, Fluorescence

Journal: Frontiers in Immunology

Article Title: Allorecognition of HLA-C Mismatches by CD8 + T Cells in Hematopoietic Stem Cell Transplantation Is a Complex Interplay between Mismatched Peptide-Binding Region Residues, HLA-C Expression, and HLA-DPB1 Disparities

doi: 10.3389/fimmu.2016.00584

Figure Lengend Snippet: Box and whisker plots of the number of mismatched residues at HLA-C between the stimulator and the responder alleles and alloreactivity defined as negative or positive (cutoff = 2% ΔCD137 + CD8 + cells) . The boxes correspond to the interquartile range, the median is the thick line inside the box, and whiskers extend up to observations that are outside the box for less than 1.5 times the interquartile range. No outliers to these limits were observed. The observations are also plotted individually with information on two predictor variables: matching at HLA-DPB1 is indicated by two different colors (blue for matched and red for mismatched HLA-DPB1), while variation at position 116 is indicated by the shape of the dots (a reversed triangle for matched and a circle for mismatched).

Article Snippet: The percentage of CD137 + PKH-2 − CD8 + CD56 − viable T cells was quantified by flow cytometry using APC-labeled anti-human CD8a, PerCP/Cy5.5 anti-human CD56 (BioLegend, Fell, Germany), and FITC-labeled anti-human CD137 (Milteny Biotec, Bergisch-Gladbach, Germany) antibodies, as well as APC- and FITC-labeled murine IgG1 isotype controls (BD Bioscience, Allschwil, Switzerland).

Techniques: Whisker Assay

Journal: Frontiers in Immunology

Article Title: Allorecognition of HLA-C Mismatches by CD8 + T Cells in Hematopoietic Stem Cell Transplantation Is a Complex Interplay between Mismatched Peptide-Binding Region Residues, HLA-C Expression, and HLA-DPB1 Disparities

doi: 10.3389/fimmu.2016.00584

Figure Lengend Snippet: Scatter plot of the number of mismatched residues at HLA-C between the stimulator and the responder allele and alloreactivity (%ΔCD137 + CD8 + cells) . Information on several predictor variables is plotted: matching at HLA-DPB1 is indicated by two different colors (blue for matched and orange for mismatched HLA-DPB1), mRNA expression of the stimulator HLA-C allele is indicated by the size of the dots, while variation at position 116 is indicated by the shape of the dots (a reversed triangle for matched and a circle for mismatched). The cutoff value considered for positive/negative alloreactivity is shown by the red dotted line.

Article Snippet: The percentage of CD137 + PKH-2 − CD8 + CD56 − viable T cells was quantified by flow cytometry using APC-labeled anti-human CD8a, PerCP/Cy5.5 anti-human CD56 (BioLegend, Fell, Germany), and FITC-labeled anti-human CD137 (Milteny Biotec, Bergisch-Gladbach, Germany) antibodies, as well as APC- and FITC-labeled murine IgG1 isotype controls (BD Bioscience, Allschwil, Switzerland).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Allorecognition of HLA-C Mismatches by CD8 + T Cells in Hematopoietic Stem Cell Transplantation Is a Complex Interplay between Mismatched Peptide-Binding Region Residues, HLA-C Expression, and HLA-DPB1 Disparities

doi: 10.3389/fimmu.2016.00584

Figure Lengend Snippet: Mixed lymphocyte reactions (MLRs) between different responders and the same HLA-C MM stimulator . Each panel represents MLRs between cells of three (A) or two (B–D) responders and one stimulator. MLRs of each panel were done in parallel (i.e., at the same time) for each of the four experiments (A–D) and represent four different HLA-C MMs: the mismatched HLA-C alleles of the responder and the stimulator are indicated below each panel. Alloresponses are given as %ΔCD137 + CD8 + cells (2% cutoff indicated by the dashed line). All pairs tested in (A–C) were HLA-DPB1 mismatched. In experiment (D) , the two pairs (one positive, one negative) were HLA-DPB1 matched.

Article Snippet: The percentage of CD137 + PKH-2 − CD8 + CD56 − viable T cells was quantified by flow cytometry using APC-labeled anti-human CD8a, PerCP/Cy5.5 anti-human CD56 (BioLegend, Fell, Germany), and FITC-labeled anti-human CD137 (Milteny Biotec, Bergisch-Gladbach, Germany) antibodies, as well as APC- and FITC-labeled murine IgG1 isotype controls (BD Bioscience, Allschwil, Switzerland).

Techniques:

Journal: Frontiers in Immunology

Article Title: Allorecognition of HLA-C Mismatches by CD8 + T Cells in Hematopoietic Stem Cell Transplantation Is a Complex Interplay between Mismatched Peptide-Binding Region Residues, HLA-C Expression, and HLA-DPB1 Disparities

doi: 10.3389/fimmu.2016.00584

Figure Lengend Snippet: Mixed lymphocyte reactions (MLRs) between different HLA-C MM stimulators and the same responder . Each panel represents MLRs between responder cells isolated from the same individual and HLA-C MM stimulator cells from two to three different individuals. MLRs of each panel were performed in parallel (i.e., at the same time) for each experiment and represent four different HLA-C MM: the mismatched HLA-C alleles of the responder and the stimulator are indicated below each panel. Alloresponses are given as %ΔCD137 + CD8 + cells (2% cutoff indicated by the dashed line). All pairs were DPB1 incompatible except two pairs [dashed bars in (A,C) ]. (E) shows the correlation between HLA-C mRNA expression of the stimulator cells and the induced alloresponse (%ΔCD137 + CD8 + cells): r = 0.42, p = 0.056. Triangles correspond to HLA-DPB1-matched pairs. All other MLRs were HLA-DPB1 mismatched. Colors in (E) correspond to those used in (A–D) .

Article Snippet: The percentage of CD137 + PKH-2 − CD8 + CD56 − viable T cells was quantified by flow cytometry using APC-labeled anti-human CD8a, PerCP/Cy5.5 anti-human CD56 (BioLegend, Fell, Germany), and FITC-labeled anti-human CD137 (Milteny Biotec, Bergisch-Gladbach, Germany) antibodies, as well as APC- and FITC-labeled murine IgG1 isotype controls (BD Bioscience, Allschwil, Switzerland).

Techniques: Isolation, Expressing