Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function
- Michigan Medicine, University of Michigan, United States
- Emory University School of Medicine, United States
- Emory University, United States
- Sirona Genomics, Immucor, Inc., United States
Figures
Figure 1
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 …
Figure 2
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)…
Figure 3
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 …
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) …
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 …
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 …
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 …
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 …
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 …
Figure 7
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 …
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- https://doi.org/10.7554/eLife.36341.013
