1. Immunology and Inflammation
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Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function

  1. Jie Geng
  2. John D Altman
  3. Sujatha Krishnakumar
  4. Malini Raghavan  Is a corresponding author
  1. Michigan Medicine, University of Michigan, United States
  2. Emory University School of Medicine, United States
  3. Emory University, United States
  4. Sirona Genomics, Immucor, Inc., United States
Research Article
Cite this article as: eLife 2018;7:e36341 doi: 10.7554/eLife.36341
7 figures, 1 data set and 1 additional file

Figures

Peptide-deficient conformers of HLA-B molecules have different thermostabilities.

(A) Peptide-deficient HLA-B can be prepared by cleavage of engineered HLA-B (LZ-ELBM) with a specific enzyme and peptide-loaded versions by subsequent incubation with specific peptide as described in the scheme. (B) Representative SDS (left panels) and native (right panels)-PAGE gels showed cleavage and loading of B*18:01, B*35:01, B*44:02, and B*51:01 molecules with peptides DEVASTHDW (DW9), HPVGEADYFEY (HY11), EEIPDFAFY (EY9) and VPYEPPEV (VV8), respectively. (C) Averaged (n ≥ 3 replicates) normalized thermal shift assays were performed with peptide-deficient conformers of B*18:01, B*35:01, B*44:02 and B*51:01 molecules. B*18:01 and B*35:01 are more stable than B*44:02 and B*51:01.

https://doi.org/10.7554/eLife.36341.003
CD8-dependent binding of peptide-deficient HLA-B*35:01 tetramers to CD8+ T cells.

Uncleaved B*35:01 tetramer poorly stained CD8+ T cells (A and F), whereas peptide-deficient B*35:01 tetramers stained most (over 70%) CD8+ T cells from B*35:01-positive donors (such as Donor 111) (B) or B*35:01-negative donors (such as Donor 133) (G) in a manner sensitive to blockage by anti-CD8 (Clone SK1) (C and H). CD4+ T cells were, in comparison, poorly stained by peptide-deficient B*35:01 tetramers and the staining cannot be blocked by anti-CD8 (D, E, I and J). Peptide-deficient B*35:01 tetramer staining data from 10 tested donors (mean ± SEM of one to two assays) are shown in (K and L).

https://doi.org/10.7554/eLife.36341.004
Preferential binding of peptide-deficient conformers of HLA-B*35:01 to CD8.

(A) Primary NK cells (CD3-CD56+) from Donor 115 were stained with peptide-deficient B*35:01 tetramers, demonstrating specific binding to the CD8+ NK cell fraction (left panel). NK cell staining by peptide-deficient B*35:01 tetramers was blocked by anti-CD8 (right panel). Representative data are shown based on two experiments each with four donors. (B) Primary NK cells from different donors have different CD8+ fractions and CD8-dependent binding of peptide-deficient B*35:01 tetramer to NK cells is proportional to the CD8+ fraction of NK cells among tested donors. The mean ± SEM of two experiments for each donor are shown. (C) Binding of SA-bead immobilized peptide-deficient or peptide-filled B*35:01 to the indicated concentrations of CD8-FITC. Proteins pulled-down were analyzed by SDS-PAGE gel and fluorimaging. (D) Quantified binding signals are plotted following background subtraction. Data are representative of four experiments.

https://doi.org/10.7554/eLife.36341.005
Figure 4 with 1 supplement
Binding of peptide-deficient conformers of HLA-B*35:01 to CD8 enhances cell adhesion.

HLA-B*35:01 and HLA-B*35:01-CD8 null were expressed by retroviral infection in the TAP1-deficient cell line, SK19. Similar levels of HLA-I in either peptide-deficient (A) or peptide-filled (B) versions were detected on the cell surface by flow cytometry. The peptide-deficient conformers can partly be blocked by the HLA-B*35:01-specific peptide (HPV) but not control peptide (HGV), which are indicated by reduced HC10 staining (C) and enhanced W6/32 staining (D). The mean ± SEM of two experiments are shown. Confocal microscopy (E–H) was used to test cell adhesion between SK19 cells expressing HLA-B*35:01 or HLA-B*35:01-CD8 null and a CTL line A2-AL9. A2-AL9 was incubated with preattached and CFSE-labeled SK19 cells (green) infected with retroviruses lacking HLA-B (E), or encoding HLA-B*35:01 (F and G) or HLA-B*35:01-CD8 null (H). For G, SK19-HLA-B*35:01 cells were preloaded with peptide HPV (100 μM). Cells were washed, fixed and stained with anti-CD8 (red) before analysis. Representative data are shown. Flow cytometry was used as a more quantitative assessment to test cell adhesion between SK19 cells expressing HLA-B*35:01 or HLA-B*35:01-CD8 null and CTL lines, A2-AL9 or B8-RL8 (I). CFSE and CD8 double positive cells were quantified as percentages of total SK19 cells. The condition with SK19 cells lacking HLA-B was subtracted as background. The mean ± SEM of three experiments are shown. Statistical analyses were undertaken using one-way ANOVA analysis with Fisher’s LSD test. *p<0.05, **p<0.01.

https://doi.org/10.7554/eLife.36341.006
Figure 4—figure supplement 1
No direct activation of peptide-deficient HLA-B*35:01 tetramers on CTL activation.

Peptide-deficient HLA-B*35:01 tetramers (40 μg/ml) were incubated with primary CD8+ T cells (A–C), CTL line A2-AL9 (D–F) or CTL line B8-RL8 (G–I). Intracellular IFN-γ was tested with flow cytometry as CD8+ T cell activation marker. Untreated cells (A, D and G) or cells treated with PMA + ionomycin (C), cognate HLA-A*02:01-AL9 tetramer (F) or HLA-B*08:01-RL8 tetramer (I) were used as negative and positive controls, respectively. Representative data from n ≥ 3 independent experiments are shown.

https://doi.org/10.7554/eLife.36341.007
Figure 5 with 1 supplement
Clustering of peptide-deficient HLA-I in cognate peptide-induced immunological synapses.

Expression levels of HLA-I on the surface of activated CD4+ T cells isolated from PBMCs of three healthy donors were assessed by flow cytometry after staining with HC10 (A), confirming that activated lymphocytes express peptide-deficient conformers of HLA-I molecules. The mean ± SEM of two replicates for each donor are shown. CTL line B8-RL8 was incubated with activated CD4+ T cells from Donor 25 (carrying B*08:01 and B*35:01) loaded with peptide RL8 (B, C, D and E) or not (F and G). Cells were fixed and stained with anti-CD8 and W6/32 or HC10 before analysis by confocal microscopy. Peptides were used at a concentration of 100 μM. Anti-CD8 staining (B, D and F) were shown in red and W6/32 (C) or HC10 (E and G) in green. Arrowheads indicate peptide-deficient conformers of HLA-I clustering at the interface between CD4+ T cells and CTL line. The intensity of HLA-I staining of the CD4+ T cells at the interface was compared with the membrane at a noncontact area and plotted as the fold increase above background (H). The results with a total of 20–30 conjugates (mean ± SEM) per condition are shown. CD8 clustering was derived from 30 conjugates under conditions shown in D. Statistical analyses were undertaken using a one-way ANOVA analysis with Fisher’s LSD test. ***, p<0.001; ****, p<0.0001.

https://doi.org/10.7554/eLife.36341.008
Figure 5—figure supplement 1
Clustering of peptide-deficient HLA-I in cognate peptide-induced immunological synapses.

CTL line B8-RL8 was incubated with activated PBMCs from Donor 25 (carrying B*08:01 and B*35:01) loaded with peptide RL8 (A–D) or not (E and F). Cells were fixed and stained with anti-CD8 and W6/32 or HC10 before analysis by confocal microscopy. Peptides were used at a concentration of 100 μM. Anti-CD8 staining (A, C and E) is shown in red and W6/32 (B) or HC10 (D and F) in green. Arrowheads indicate peptide-deficient conformers of HLA-I clustering at the interface between PBMCs and CTL line. The intensity of HLA-I staining of the PBMCs at the interface was compared with the membrane at a noncontact area and plotted as the fold increase above background (G). The results with a total of 39 conjugates (for B and D) or 10 conjugates (for F) per condition are shown. CD8 clustering was derived from 39 conjugates as condition shown in C. The mean ± SEM was shown. Statistical analyses were undertaken using one-way ANOVA analysis with Fisher’s LSD test. ****, p<0.0001.

https://doi.org/10.7554/eLife.36341.009
Figure 6 with 1 supplement
HLA-B*35:01 peptide-deficient conformers enhance cognate peptide-induced lysis of target cells by CD8+T cell activation.

CTL line A2-AL9 specifically killed activated primary CD4+ T cells (expressing HLA-A*02:01 and HLA-B*35:01 and used as antigen presenting cells) loaded with cognate peptide AL9 but not non-cognate peptide SL9 (A). Peptides that binds specifically to HLA-B*35:01 (YPL, HPN, HPV and FPT) could reduce AL9-induced cell lysis. Representative data with CD4+ T cells from Donor 24 are shown in Panel A and statistical analyses from Donors 24 and 168 in Panels B and C. Cell lysis assays were also performed with another CTL line B8-RL8 and activated primary CD4+ T cells (carrying HLA-B*08:01 and B*35:01) from Donors 25 and 132 loaded with cognate peptide RL8 were used as target cells (D and E). (B–E) The mean ± SEM of three independent assays are shown. Statistical analyses were undertaken using one-way ANOVA analysis with Dunnett test. *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001. (F–I) Blocking of HLA-B*35:01 peptide-deficient conformers on the surface of CD4+ T cells by B*35:01-specific peptides (YPL, HPN, HPV and FPT), but not nonspecific control peptide (TW10 or HGV) was confirmed by flow cytometry after staining cells with HC10. The mean ± SEM of two to three independent experiments are shown.

https://doi.org/10.7554/eLife.36341.010
Figure 6—figure supplement 1
Cell lysis by CTL line A2-AL9 of activated primary CD4+ T cells expressing HLA-A*02:01.

Lysis was induced by pulsing cells with the cognate peptide AL9 but not the non-cognate peptide SL9. Cell lysis assay was performed at different effector: target cell ratios 1:1, 5:1 and 20:1, as described in the Materials and methods section. Representative data from n = 3 independent experiments are shown.

https://doi.org/10.7554/eLife.36341.011
Peptide-deficient conformers of MHC-I molecules enhance CTL activation.

CTL activation is generally induced by the recognition of a specific peptide-MHC-I complex on the surface of antigen presenting cells (APC) by a T cell receptor (TCR) and co-receptor CD8. In this study, we show that peptide-deficient conformers of MHC-I molecules have increased binding to CD8 compared with peptide-filled conformers and therefore enhance the cell-cell contact between APC and CTL. Peptide-deficient conformers of MHC-I molecules are enriched in the immunological synapse and augment CTL activation.

https://doi.org/10.7554/eLife.36341.012

Data availability

The data that support the findings of this study are openly available in Dryad at https://doi.org/10.5061/dryad.543pp71.

The following data sets were generated
  1. 1
    Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function
    1. Geng J
    2. Altman JD
    3. Krishnakumar S
    4. Raghavan M
    (2018)
    Available at Dryad Digital Repository under a CC0 Public Domain Dedication.

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