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. 2016 Nov 29;113(48):13827-13832.
doi: 10.1073/pnas.1609118113. Epub 2016 Nov 10.

Graft Versus Self (GvS) Against T-cell Autoantigens Is a Mechanism of Graft-Host Interaction

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Free PMC article

Graft Versus Self (GvS) Against T-cell Autoantigens Is a Mechanism of Graft-Host Interaction

Nora Mirza et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Graft-versus-host disease (GVHD) represents the major nonrelapse complication of allogeneic hematopoietic cell transplantation. Although rare, the CNS and the eye can be affected. In this study, manifestation in the retina as part of the CNS and T-cell epitopes recognized by the allogeneic T cells were evaluated. In 2 of 6 patients with posttransplantation retina diseases and 6 of 22 patients without ocular symptoms, antigen-specific T-cell responses against retina-specific epitopes were observed. No genetic differences between donor and recipient could be identified indicating T-cell activation against self-antigens (graft versus self). Transplantation of a preexisting immunity and cross-reactivity with ubiquitous epitopes was excluded in family donors and healthy individuals. In summary, an immunological reaction against retina cells represents a mechanism of graft-versus-host interaction following hematopoietic cell transplantation.

Keywords: T-cell epitope; allogeneic hematopoietic cell transplantation; autoimmunity; graft-versus-host disease; retina.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. S1.
Fig. S1.
Approach of the study. Blood samples of patients were obtained before start of conditioning regimen and after HCT during follow-up visits. PBMCs and DNA were isolated. High polymorphic regions of Retina protein genes were amplified by PCR and sequenced. Peptides according to known SNP and newly identified SNP were predicted with EpiToolKit and SYFPEITHI (MHC class I) or designed as 17 mers (MHC class II). Immunogenicity of MHC class I peptides was analyzed by in vitro priming of CD8+ T cells harvested from HD. Memory T-cell responses in PBMCs from patients and family donors were analyzed by IFN-γ ELISpot and ICS after peptide stimulation.
Fig. 1.
Fig. 1.
T-cell responses after HCT in patients with PS diseases. (A–D) Patient 1: fundus photography (A) showing optic nerve atrophy with very narrow vessels. (B) MRI scan (Left) of the brain revealed a periventricular lesion where a stereotactic biopsy was taken. Histologic workup (Right) including immunohistochemistry showed a CD8+ atypical vasculitis without any evidence of meningeal disease relapse. (C) T-cell response after stimulation of 250,000 prestimulated (12 d) PBMCs per well with retGC-derived peptides (1 µg/mL) detected by IFN-γ ELISpot and sequencing (D) of donor and recipient DNA (GUCY2D exon 2). (E–G) Patient 5: fundus photography (E) revealing a massive, centrally located CMV retinitis with detection of vitreous cells (cloudiness of the picture). T-cell responses were evaluated (F) after stimulation of 500,000 prestimulated (12 d) PBMCs per well with retGC-derived MHC class II peptides (2.5 µg/mL) detected by IFN-γ ELISpot and sequencing (G) of donor and recipient DNA (GUCY2D exon 2). CD, cluster of differentiation; OD, oculus dexter; OS, oculus sinister.
Fig. 2.
Fig. 2.
T-cell responses after HCT in patient 7. CD4+ T-cell responses after stimulation of 500,000 prestimulated (12 d) PBMCs per well with the GCAP-1 and the GCAP-2derived MHC class I predicted peptide detected by (A) IFN-γ ELISpot (peptides at 1 µg/mL) and (B) ICS (peptides at 10 µg/mL). (A) T cells were analyzed over a period of 17 mo (before HCT until 17 mo after HCT). IFN-γ ELISpot (Upper) revealed changing of the T-cell reactivity against one peptide but not against the corresponding variant peptide, which were not detectable before HCT. Before HCT, only very limited PBMC counts were available due to the myelodysplastic syndrome. The bar graph presents changes in the T-cell reactivity (absolute spot counts in ELISpot) normalized on the negative control (incubation with irrelevant peptide) over time. (B) ICS revealed T-cell activation indicated by TNF (far left and third panel from left) and IFN-γ (second panel from left and far right panel) production on incubation with the A variant peptides (panels first row), but not with the variant peptide with difference in one amino acid (panels second row). The response was mediated by CD4+ T cells. As controls, incubation with HIV-derived epitopes are presented in the lower two rows for CD4+ (third row) and CD8+ (fourth row) responses. (C) Sequencing of the GUCA1A (Upper) and GUCA1B (Lower) genes for donor (Left) and recipient (Right) showing the same genetic sequence and, hence, revealing no SNP.
Fig. S2.
Fig. S2.
T-cell responses after HCT in patients 9 and 11. (A–C) Patient 9: Strong CD4+ T-cell reactivity after stimulation of 500,000 presensitized (12 d) PBMCs per well with the GCAP-1derived peptides in (A) IFN-γ ELISpot (peptides at 1 µg/mL) and (B) ICS (peptides at 10 µg/mL). Antigen-specific T cells against the HLA A*0301-restricted variant peptide GUCA1A 47A (NLSPSASQY) could be detected 12 and 13 mo after HCT, which were not detectable before HCT. No reactivity could be detected against the alternate HLA A*0301-restricted peptide GUCA1A 47B (NLSLSASQY). The bar graph (A) presents changes in the T-cell reactivity (absolute spot counts in ELISpot) normalized on the negative control (incubation with irrelevant peptide) over time. (C) Sequencing of exon 3 of the GUCA1A gene for donor and recipient with detection of a sequence homology coding for the GUCA1A 47A variant peptide. (D–F) Patient 11: T-cell reactivity after stimulation of 500,000 presensitized (12 d) PBMCs per well with the GCAP-2derived peptides in (D) IFN-γ ELISpot (peptides at 1 µg/mL). Antigen-specific T cells against the pooled HLA A*0101restricted peptide pair GUCA1B 133A (QTEQGQLLT) and GUCA1B 133B (QTEQDQLLT) could be detected 7 mo after HCT which were not detectable before HCT. Due to limited availability of PBMCs, this reaction could not be further discriminated between the two peptides. The bar graph (E) presents changes in the T-cell reactivity (absolute spot counts in ELISpot) normalized on the negative control (incubation with irrelevant peptide) over time. (F) Sequencing of exon 3 of the GUCA1B gene for donor and recipient with detection of a sequence homology coding for the GUCA1A 133A (QTEQGQLLT) variant peptide.
Fig. S3.
Fig. S3.
T-cell responses after HCT in patient 12. T-cell reactivity after stimulation of 500,000 (donor: 250,000) presensitized (12 d) PBMCs per well with retGC-, GCAP-1, and GCAP-2derived peptides in (A) IFN-γ ELISpot (peptides at 1 µg/mL). Antigen-specific T cells against the HLA A*0201-restricted peptides GUCY2D 44A (LLQPPALSAV) and 44B (LLQPPALSSV), the HLA A*0201-restricted GUCA1A 152A (SLEEFIEGV) and 152B (SLEGFIEGV), the pooled HLA A*0201-restricted peptide pair GUCA1B 131A (ELQTEQGQLL) and 131B (ELQTEQDQLL), as well as the pooled HLA A*0201-restricted GUCA1B 152A (LVDENGDGQL) and 152B (LVDDNGDGQL) peptides could be detected 5 and 6 mo after HCT, which were not detectable before HCT in the patient and in the donor sample. The bar graph (B) presents changes in the T-cell reactivity (absolute spot counts in ELISpot) normalized on the negative control (incubation with irrelevant peptide) over time. (C) Changes in T-cell reactivity (absolute spot counts in ELISpot) normalized to the donor sample stimulated with the respective peptides. In case of measurement of single or fewer peptides, means were calculated of all included peptides (in peptide pools used in earlier samples) and normalized to the donor sample. (D) Sequencing of both donor (Left) and recipient (Right) DNA. (Top) Sequencing of exon 2 of the GUCY2D gene with detection of a heterozygous SNP coding for the GUCY2D 44A peptide (LLQPPALSAV) in the donor and a heterozygous gene sequence coding for the GUCY2D 44A (LLQPPALSAV) and 44B (LLQPPALSSV) peptides. (Second Panels from Top) Sequencing of exon 6 of the GUCA1A gene for donor and recipient with detection of a sequence homology coding for the GUCA1A 152A (SLEEFIEGV) variant peptide. (Second Panels from Bottom) Sequencing of exon 3 of the GUCA1B gene for donor and recipient with detection of a sequence homology coding for the GUCA1B 131A (ELQTEQGQLL) variant peptide. (Bottom) Sequencing of exons 3 and 4 of the GUCA1B gene for donor and recipient with detection of a sequence homology coding for the GUCA1B 152A (LVDENGDGQL) variant peptide.
Fig. S4.
Fig. S4.
T-cell responses after HCT in patient 20. T-cell reactivity after stimulation of 250,000 (before HCT: 125,000 for peptides, PHA: 62,500) presensitized (12 d) PBMCs per well with the retGC derived peptides in (A) IFN-γ ELISpot (MHC class I peptides at 1 µg/mL, MHC class II peptides at 2.5 µg/mL). Antigen-specific T cells could be detected 5 mo after HCT against the pooled GUCY2D peptides including the MHC class II peptides GUCY2D 52A (LLQPPALSAVFTVGVLG) and 52B (LLQPPALSSVFTVGVLG), as well as the MHC class II peptides GUCY2D 782A (DQAPVECILLMKQCWAE) and 782B (DQAPVECIHLMKQCWAE). These cells were not detectable before HCT and not anymore at 14 mo after HCT. Due to limited availability of PBMCs, this reaction could not be further discriminated between the four peptides. The bar graph (B) presents changes in the T-cell reactivity (absolute spot counts in ELISpot) normalized on the negative control (incubation with irrelevant peptide) over time. (C) (Upper) Sequencing of exon 2 of the GUCY2D gene for donor and recipient with detection of a heterozygous sequence homology coding for the GUCY2D 52A (LLQPPALSAVFTVGVLG) and 52B peptides (LLQPPALSSVFTVGVLG). (Lower) Sequencing of exon 12 of the GUCY2D gene for donor and recipient with detection of a sequence homology coding for the GUCY2D 782A (DQAPVECILLMKQCWAE) variant peptide.
Fig. S5.
Fig. S5.
T-cell responses after HCT in patient 23. (A) CD4+ T-cell responses after stimulation of 500,000 prestimulated (12 d) PBMCs per well with retGC-derived MHC class II peptides detected by IFN-γ ELISpot (2.5 µg/mL) (A) and ICS (10 µg/mL) (B). As a control presensitized (12 d) donor-derived PBMCs (500,000 PBMCs per well) were stimulated with the respective peptides (2.5 µg/mL) in IFN-γ ELISpot (A). Antigen-specific T cells against the MHC class II peptides GUCY2D 52A (LLQPPALSAVFTVGVLG) and 52B (LLQPPALSSVFTVGVLG) could be detected between 4 and 10 mo after HCT, which were not detectable before HCT in the patient and in the donor sample. T-cell reactivity was not detectable anymore at 12 mo after HCT. The bar graph (C) presents changes in the T-cell reactivity (absolute spot counts in ELISpot) normalized on the negative control (incubation with irrelevant peptide) over time. (D) Changes in T-cell reactivity (absolute spot counts in ELISpot) normalized to the donor sample stimulated with the respective peptides. (E) Sequencing of the GUCY2D exon 2 in donor, the recipient, and a reference DNA without a SNP revealed a sequence homology coding for the GUCY2D 52B (LLQPPALSSVFTVGVLG) variant peptide between donor and recipient. For comparison, the reference sequence coding for the GUCY2D 52A peptide (LLQPPALSAVFTVGVLG) is provided.
Fig. 3.
Fig. 3.
In vitro priming of peptide-specific CD8+ T cells. DCs based in vitro priming experiments were performed for all MHC class I peptides and measured in ICS. Two exemplified peptide pairs are shown. (A) GUCA1A 144: Weak T-cell reactivity could be induced against the B-variant (DVNGDGEFSL) but not against the A-variant peptide (DVNGDGELSL) as indicated by TNF and IFN-γ production. (B) GUCY2D 18: Very strong T-cell reactivity could be induced against both peptide variants as indicated by TNF and IFN-γ production detected by flow cytometry. (C) Summary of identification of retina-specific T-cell epitopes: antigen-specific T cells could be detected in patients only (n = 2), patients and in vitro priming (n = 6), and in vitro priming only (n = 30). Seventeen peptides could not be confirmed as epitopes. (D) Classification of the intensity of CD8 T-cell responses into very weak >0.05–0,1%, weak >0.1–0.5%, intermediate >0.5–1%, strong >1–3%, and very strong T-cell responses >3% (percentage is referred to all CD4-negative gated cells).
Fig. S6.
Fig. S6.
Summary of the mechanisms of grafthost interaction. Graft-versus-host interaction was previously believed to be mediated by direct recognition of differences in HLA types and the respective expressed self-peptides (Left) in case of HLA-mismatched HCT or by recognition of sequence variant MHC-restricted peptides based on genetic SNP between donor and recipient serving as miHAG (Center). The data reported in this article indicate a mechanism of graft–host interaction. Herein, recipient cells expression self-sequence based peptides (Right) can be recognized by allogeneic T cells (GvS).

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