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Volume 45, Issue 2, Pages (August 2016)

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1 Volume 45, Issue 2, Pages 280-291 (August 2016)
Cell-Extrinsic MHC Class I Molecule Engagement Augments Human NK Cell Education Programmed by Cell-Intrinsic MHC Class I  Jeanette E. Boudreau, Xiao-Rong Liu, Zeguo Zhao, Aaron Zhang, Leonard D. Shultz, Dale L. Greiner, Bo Dupont, Katharine C. Hsu  Immunity  Volume 45, Issue 2, Pages (August 2016) DOI: /j.immuni Copyright © 2016 Elsevier Inc. Terms and Conditions

2 Immunity 2016 45, 280-291DOI: (10.1016/j.immuni.2016.07.005)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3 Figure 1 Development and Characterization of an HLA Class I-Expressing Humanized Mouse Model (A) HLA-Bw4 expression on murine splenocytes. Dashed gray histogram represents B27 Tg+ mice, and the solid black histogram represents Tg− mice. (B) Engraftment of human cells from CD34+ stem cells in B27 Tg+ (squares) or Tg− mice (circles). Data are representative of 3 donors in a total of 22 mice. (C) Accumulation of NK cells developing in B27 Tg+ (squares) and Tg− (circles). Data are representative of 3 donors in a total of 22 mice, from which serial blood samples were obtained (∗p < 0.05). (D) Representative staining of splenic NK cells for conserved activating receptors CD16, NKG2D, and NKp46 on NK cells developing in humanized mice and NK cells from healthy human donors. Mouse samples are derived 8 weeks after CD34+ stem cell transplantation to a Tg− mouse. (E) Representative staining for KIR molecules on splenic NK cells developing in humanized mice and comparison to NK cells from healthy human donors. Mouse samples are derived 7.5 weeks after CD34+ stem cell transplantation to a Tg− mouse. (F–M) Comparison of receptor expression among NK cells raised in B27 Tg+ and Tg− animals, 8 weeks after CD34+ transplant. (F) CD16+(%); (G) NKp46+(%); (H) NKG2D+(%); (I) NKG2A+(%); (J) KIR2DL1+(%); (K) KIR2DL2+ or KIR2DL3+(%); (L) KIR3DL1+(%); (M) CD57+(%). Bars compare means ± SEM. ∗p < 0.05; ∗∗p < Data are representative of three independent experiments each with a minimum of two CD34+ donors and a minimum of two Tg− and two B27 Tg+ mice per donor. Bars represent means ± SEM. Please see also Figure S1. Immunity  , DOI: ( /j.immuni ) Copyright © 2016 Elsevier Inc. Terms and Conditions

4 Figure 2 Human NK Cells Derived in Humanized Mice Are Functionally Competent and Educated by HLA Class I Molecules Available from Hematopoietic and Stromal Sources (A) Representative flow cytometry plots demonstrating reactivity of KIR3DL1+ and KIR3DL1− NK cells derived in humanized mice 8 weeks after transplant of CD34+ human stem cells to a Tg− mouse. For comparison, NK cells from the peripheral blood of an HLA-Bw4+ donor are shown. (B) Proportion of the total NK cell population degranulating in response to challenge with K562 cells. (C) Response of KIR3DL1+ NK cells to K562 target cell stimulation. Lines join NK cells derived from the same CD34+ stem cell donor. (D) Degranulation of KIR3DL1+ (KIR2DL−) and KIR3DL1− NK cells derived from HLA-Bw4+ or HLA-Bw4− donors, in B27 Tg+ or Tg− littermates. (E) Degranulation of KIR2DL2+ or KIR2DL3+ NK cells derived from HLA-C1+ or -C1− donors. Bars represent means ± SEM. ∗p < 0.05; ∗∗p < 0.01. Bars represent means ± SEM. Please see also Figure S2. Immunity  , DOI: ( /j.immuni ) Copyright © 2016 Elsevier Inc. Terms and Conditions

5 Figure 3 Donor and Recipient HLA Class I Molecules Both Contribute to NK Cell Education (A–C) NK cells derived in patients 200–365 days after HLA mismatched HCT were challenged using K562 cells, and degranulation was quantified. NK cells exclusively expressing (A) KIR3DL1, (B) KIR2DL1, (C) KIR2DL2, or KIR2DL3 were compared. The presence or absence of (A) the KIR3DL1 ligand, HLA-Bw4, (B) the KIR2DL1 ligand, HLA-C2, or (C) the KIR2DL2 and KIR2DL3 ligand, HLA-C1, in the engrafting donor unit and recipient are indicated. Bars indicate mean ± SEM for a minimum of four donor-recipient pairs each. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < Data are compiled from three independent trials, and each bar represents a minimum of four donor-patient pairs. Immunity  , DOI: ( /j.immuni ) Copyright © 2016 Elsevier Inc. Terms and Conditions

6 Figure 4 Adoptively Transferred NK Cells Maintain Reactive Potential in Tg− Mice and Gain Function in B27 Tg+ Mice (A) Identification of NK cells in the peripheral blood, primary and secondary lymphoid tissues 2 weeks after adoptive transfer of mature NK cells. (B) Representative proliferation of NK cells from the same donor in Tg− or B27 Tg+ mice, segregated by tissue of harvest. Black histograms represent cells recovered from mice and gray histograms represent unproliferated NK cells pre-transfer. (C) Degranulation response of KIR3DL1+ NK cells in response to K562 stimulation before adoptive transfer (input) or following transfer to B27 Tg+ or Tg− animals. Bars represent means ± SEM and are compared using repeated-measures ANOVA comparing the average reactivity of NK cells derived from the same donor. ∗p < 0.05. (D) Fold-change in responsiveness of cells from HLA-Bw4+ or HLA-Bw4− donors after transfer to B27 Tg+ mice. A fold-change of one indicates that NK cell responsiveness was equivalent before and after adoptive transfer. Means are compared using Student’s t test; ∗∗∗∗p < (E) Relative change in responsiveness comparing NK cells from the same donor in B27 Tg+ and Tg− mice. Bars represent the mean fold increase ± SEM and are compared using repeated-measures ANOVA. ∗∗p < Data are representative of three independent trials, each with two to three human donors and a minimum of two Tg− and two B27 Tg+ mice per donor. Bars represent means ± SEM. Please see also Figure S3. Immunity  , DOI: ( /j.immuni ) Copyright © 2016 Elsevier Inc. Terms and Conditions

7 Figure 5 All trans Sources of Cognate HLA-Bw4 Enhance KIR3DL1+ NK Cell Function (A) Schematic representation of mouse-to-mouse bone marrow chimeras and subsequent adoptive transfer of mature NK cells. Total bone marrow collected from B27 Tg+ or Tg− mice was used to reconstitute B27 Tg+ or Tg− mice after lethal irradiation. After complete reconstitution of the mouse hematopoietic compartment, NK cells isolated from healthy human donors were adoptively transferred to quartets of mice that had each permutation of B27 Tg+ or Tg− bone marrow and stromal cells. (B) Data represent the reactivity of KIR3DL1+ NK cells before and 2 weeks after transfer into the indicated reconstituted bone marrow chimeric mice. R and D represent recipient stroma and donor bone marrow HLA-Bw4 status, respectively; + and − indicate B27 Tg+ and Tg−, respectively. Matched symbols represent the average reactivity of NK cells from the same human donors adoptively transferred to bone marrow chimeric mice. Black filled symbols represent HLA-Bw4− human donors; open symbols indicate HLA-Bw4+ donors. NK cells from each of three representative donors were transferred to a minimum of two mice for each donor x recipient combination. ∗p < 0.05 by paired samples t test, comparing NK cells derived from the same human donor. (C) Fold change in KIR3DL1+ NK cell reactivity compared with reactivity before transfer where y = 1 indicates the same responsiveness before and after transfer to mouse recipients. All recipient and donor combinations encoding HLA-B∗27:05 in at least one compartment enhanced NK cell reactivity above baseline ∗p < Data are representative of three human donors, with each donor used to implant at least one of each of the four chimeric configurations. Bars represent means ± SEM. Immunity  , DOI: ( /j.immuni ) Copyright © 2016 Elsevier Inc. Terms and Conditions

8 Figure 6 cis Interactions between KIR and Cognate HLA Class I Molecules Maintain NK Cell Education (A) Representative decrease in HLA class I molecule expression 48 hr after shRNA-mediated silencing of HLA class I mRNA expression. (B) Responsiveness of NK cells exhibiting a single KIR type to stimulation with K562 cells following mock infection or shRNA-mediated HLA class I silencing. Bars compare means ± SEM. ∗∗∗∗p < (C) CD107a expression in response to stimulation with HLA class I− K562 cells by NK cells in a representative donor exhibiting HLA-C1 and HLA-Bw4, but not HLA-C2, with and without silencing of β2m in triplicate samples. NK cells predicted to be educated based on donor HLA class I molecules are shown in bold and underlined. (D) Percent loss of reactivity among educated or non-educated NK cells after HLA class I silencing. Educated and uneducated cells are compared. Bars represent means ± SEM and are compared by Student’s t test. ∗∗∗∗p < ; ∗p < Data in (C) represent one donor with four replicates of silencing in KIR+ NK cells. For (B) and (D), data are collected from 34 independent samples in five independent trials. Bars represent means ± SEM. Immunity  , DOI: ( /j.immuni ) Copyright © 2016 Elsevier Inc. Terms and Conditions

9 Figure 7 HLA Class I Molecules Acquired from Neighboring Cells Augment NK Cell Reactivity Human HLA-Bw4− NK cells labeled with cell trace violet (CTV) were transferred to B27 Tg+ or Tg− mice for 2 days. (A) Representative flow cytometry plot depicting murine CD45 expression among CTV+ human NK cells. (B) Overlaid histograms represent murine CD45+ staining on CTV− (murine) cells (gray shaded histograms) and on CTV+ NK cells before (dotted black histograms) or after (solid black histograms) 2 days in vivo. (C) HLA-Bw4 staining on HLA-Bw4− NK cells before transfer and after transfer to Tg− or B27 Tg+ mice. HLA-Bw4 staining of NK cells from a healthy human donor exhibiting HLA-B∗27:05 is shown for comparison. (D) The phenotype of CD56+CTV+ NK cells after exposure to Tg− or B27 Tg+ mice is shown in representative flow cytometry plots demonstrating uptake of CD45 and HLA-Bw4 by transferred NK cells. Populations are gated on CTV+ human NK cells. (E) CD107a response to K562 target cells by KIR3DL1+ NK cells from HLA-Bw4− donors 2 days following transfer to B27 Tg+ or Tg− mice, segregated based on acquisition of HLA-Bw4. ∗∗p < Bar graphs represent two independent HLA-Bw4− donors and a total of 34 mice. (F) Staining for HLA-Bw4 on lymphocytes collected from human patients 1 year after HCT (shaded gray histograms). HLA-Bw4-positive (dashed histograms) and HLA-Bw4− (solid black histograms) donors are shown for comparison. Labels indicate the donor → recipient HLA-Bw4 status. (A–E) are derived from two experiments, each with two independent human NK cell donors and transplants into pairs of Tg− or B27 Tg+ mice. Samples shown in (F) are representative of four independent donors. Bars represent means ± SEM. Immunity  , DOI: ( /j.immuni ) Copyright © 2016 Elsevier Inc. Terms and Conditions


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