Frequency of unconventional HLA-E_UL40 CD8 T-cell responses compared to conventional HLA-A*02_pp65 CD8 T-cell responses in HCMV⁺ kidney transplant recipients and healthy volunteers.

<p>PBMCs were isolated from freshly or prospectively harvested at M12 post-transplantation blood samples issued from healthy donors (HV) or from kidney transplant recipients (KTR), respectively. <i>Ex vivo</i> detection of HLA-E_UL40 CD8 T and HLA-A*02_pp65 CD8 T cells was performed using flow cytometry by selecting CD3⁺ CD8α⁺ TCRγδ^- tetramer⁺ cells on PBMCs. Detection threshold was 0.1% of total CD8 αβT cells and kidney transplant recipients and healthy volunteers bearing ≥0.1% of HLA-E_UL40 CD8 T cells (in blue) or ≥0.1% HLA-A*02_pp65 CD8 T cells (in red) were considered as positive. Detection of both types of CD8 T-cell responses are indicated in violet. Absence of detection is shown in light grey in HCMV^- recipients and dark grey for HCMV⁺ hosts. Data shown are the number of individuals that display anti-HCMV CD8 T-cell responses. Frequencies of the CD8 T-cell subsets were calculated among subgroups for all (total), non HLA-A*02 and HLA-A*02 individuals and expressed as percentages (%).</p

Sequences of UL40_15-23 peptide in the infecting HCMV strains.

<p>Sequences of UL40_15-23 peptide in the infecting HCMV strains.</p

Patient’s characteristics.

<p>Patient’s characteristics.</p

HLA-A*02 allele and HLA-E genotype influence the development of HLA-E_UL40-specific CD8 T cells in HCMV⁺ individuals.

<p>(A) HLA-A*02 allele frequency and (B) genotype distribution were investigated in HCMV^- (n = 39) <i>versus</i> HCMV⁺ (n = 105) individuals including a total of 144 healthy volunteers and kidney transplant recipients (left panels) and in HCMV⁺ host with (+, n = 31) or without (-, n = 74) HLA-E_UL40 CD8 T-cell response (right panels). (C) HLA-E*01:01 and HLA-E*01:03 allele frequency and (D) HLA-E genotype distribution were investigated in HCMV^- (n = 35) <i>versus</i> HCMV⁺ (n = 96) individuals of the cohort (left panels) and in HCMV⁺ host with (+, n = 30) or without (-, n = 66) HLA-E_UL40 CD8 T-cell response (right panels). <i>P</i> values were calculated using appropriate statistical tests: Fisher’s exact test for allele frequencies and Chi-square test for genotype distribution analysis.</p

Potential cross-recognition of autologous and allogeneic HLA-I signal peptides by HLA-E_UL40 CD8 T cells.

<p>PBMCs were isolated from freshly or prospectively collected blood samples at M12 post-transplantation issued from healthy donors (HV, n = 25) or from kidney transplant recipients (KTR, n = 119), respectively. <i>Ex vivo</i> detection of HLA-E_UL40 CD8 T and HLA-A*02_pp65-specific CD8 T cells was performed using flow cytometry by selecting CD3⁺ CD8α⁺ TCRγδ^- tetramer⁺ cells on PBMCs. Eight different HLA-E_UL40 tetramers were used independently. (A) Percentage of circulating anti-HCMV CD8 T cells in blood detected using the various HLA-E_UL40 (blue) and HLA-A*02_pp65 (in red) tetramers in HV and KTR. For each tetramer/peptide, the number of individuals with a given CD8 T-cell response is indicated. (B) Diversity and magnitude of the HLA-E_UL40 CD8 T-cell responses in KTR and HV. HLA-E_UL40 CD8 T-cell responses appear in blue and colour intensity is proportional to the percentage of HLA-E_UL40 CD8 T cells. (C) Classification of the HLA-E_UL40 CD8 T-cell responses in HCMV⁺ hosts according to possible recognition of self (orange), donor-specific allogeneic (green) or both (violet) (n = 31, 23 KTR and 8 HV). Grey boxes show HLA-I signal peptides which are not derived from the recipient, nor from the donor. Asterisks indicate peptides with underestimated information due to a lack of HLA-C genotyping.</p

HCMV strain-dependent variability of UL40_15-23 sequences and HCMV strain-specific HLA-E_UL40 T-cell response in hosts.

<p>(A, B) Genomic DNAs isolated from HCMV positive blood samples in HCMV⁺ transplant recipients (n = 25) were sequenced for the identification of UL40 protein (amino acids 1–221) provided by the circulating HCMV strains. (A) Amino acid variability, expressed as a number of amino acid variants, within the HLA-E-binding peptide (UL40_15-23, shown in red) among the sequence for HCMV UL40 signal peptide (UL40_1-37, shown in grey). A total of 32 UL40 sequences from 25 hosts were analysed. UL40 protein sequence from the Merlin HCMV clinical strain was used as reference. Positions 1 to 9 of residues in the HLA-E-binding peptide (UL40_15-23) are indicated. (B) Sequence LOGO of the UL40_15-23 HLA-E-binding peptide from 25 transplanted hosts. The height of the letter is proportional to the frequency of each amino acid in a given position (P1 to P9). Major anchor residues for binding in the HLA-E peptide groove are indicated in blue. Red letters highlight the important variability observed in position 8 of the HLA-E-binding peptide. Grey boxes correspond to a constitutive deletion of the corresponding amino acid in the UL40 sequence from the infecting viral strain. (C) Representative dot plot analyses showing the detection of strain-specific anti-UL40 HLA-E-restricted CD8 T-cell responses in 4 KTRs (KTR#026, #105, #108 and #109). Frequencies (%) of the HLA-E_UL40-specific T cells among total circulating αβ CD8 T cells are indicated.</p

Time course analysis of the HLA-E_UL40 and HLA-A*02_pp65 CD8 T-cell anti-HCMV responses upon infection and patterns of activation markers.

<p>(A) Time course analysis of the HLA-E_UL40 and HLA-A*02_pp65 CD8 T-cell responses according to the HCMV viremia. PBMCs prospectively collected from M0 and M13 (#109) post-transplantation were retrospectively processed for the concomitant detection and quantification of anti-HCMV HLA-E_UL40 and HLA-A*02_pp65 CD8 T-cell responses upon infection. Three representative patterns of anti-HCMV CD8 T cell responses in 3 KTR (KTR#107, #108 and #109) are represented. (B) Analysis of T-cell activation. Expression of CD69 (left panel) and PD-1 (right panel) analysed on blood samples from KTR#107, #108 and #109. Facs histogram overlays represent the % of expression for the activation markers CD69 and PD-1 among CD3⁺ CD8α⁺ TCRγδ^- tetramers⁺ cells, for HLA-E_UL40 (in blue) and HLA-A*02_pp65 (in red) anti-HCMV CD8 T-cell responses at M6 post-transplantation. (C) Comparative analysis of CD69 (left panel) and PD-1 (right panel) expression on HLA-E_UL40 (n = 4 hosts) and HLA-A*02_pp65 (n = 8 hosts) CD8 T cells investigated at M6 post-transplantation. <i>P</i> values were calculated using a Mann Whitney test.</p

Diversity of HLA-E_UL40 CD8 TCR Vβ repertoire and peptide recognition.

<p>HLA-E_UL40 CD8 T cells were sorted from PBMCs for 5 KTR (#104, #105, #107, #108 and #109) and amplified <i>in vitro</i>. (A) Diversity of the HLA-E_UL40-specific TCR Vβ repertoire. Percentages indicate the ratio of individual Vβ chains by the total repertoire for each patient. (B) Specificity of peptide recognition toward UL40 and HLA-I peptides. HLA-E_UL40-specific CD8 T cells were stimulated for 5h with one of the eleven HLA-E_UL40 tetramers and the percentage of TNF-producing cells among total CD8α cells was determined. Red arrows indicate the HCMV UL40 peptide provided by the infecting strain. Recognition of HLA-Ia signal peptides derived from autologous (orange bars), transplant-specific allogeneic (green bars) or both (purple bars) is shown. Grey bars indicate recognition of peptides that do not match with UL40 from the infecting strain or with HLA-Ia signal peptides not expressed by donors or hosts. Asterisks indicate samples with missing data for HLA-C genotype.</p

Demographic and baseline characteristics of primary and second transplants performed in the DIVAT network between January 1996 and November 2010.

<p>BMI, body mass index; PRA, panel reactive antibody; HLA, human leukocyte antigen; CMV, cytomegalovirus; EBV, Epstein-Barr virus.</p>*<p>p<0.05.</p

Multivariate Cox model analysis of acute rejection episode (ARE)-free time risk factors (N = 3103).

<p>CI, confidence interval; BMI, body mass index; PRA, panel reactive antibody; HLA, human leukocyte antigen.</p