DataSheet_1_Human gut microbiota-reactive DP8α regulatory T cells, signature and related emerging functions.pdf

In mice, microbiota-induced Tregs both maintain intestinal homeostasis and provide resistance to immuno-pathologies in the adult. Identifying their human functional counterpart therefore represents an important goal. We discovered, in the human colonic lamina propria and blood, a FoxP3-negative IL-10-secreting Treg subset, which co-expresses CD4 and CD8α (hence named DP8α) and displays a TCR-reactivity against Faecalibacterium prausnitzii, indicating a role for this symbiotic bacterium in their induction. Moreover, supporting their role in intestinal homeostasis, we previously reported both their drastic decrease in IBD patients and their protective role in vivo against intestinal inflammation, in mice. Here, we aimed at identifying the genomic, phenotypic and functional signatures of these microbiota-induced Tregs, towards delineating their physiological role(s) and clinical potential. Human F. prausnitzii-reactive DP8α Treg clones were derived from both the colonic lamina propria and blood. RNA-sequencing, flow cytometry and functional assays were performed to characterize their response upon activation and compare them to donor- and tissue-matched FoxP3+ Treg clones. DP8α Tregs exhibited a unique mixed Tr1-like/cytotoxic CD4+ T cell-profile and shared the RORγt and MAF master genes with mouse gut microbiota-induced FoxP3+ Tregs. We revealed their potent cytotoxic, chemotactic and IgA-promoting abilities, which were confirmed using in vitro assays. Therefore, besides their induction by a Clostridium bacterium, DP8α Tregs also partake master genes with mouse microbiota-induced Tregs. The present identification of their complete signature and novel functional properties, should be key in delineating the in vivo roles and therapeutic applications of these unique human microbiota-induced Tregs through their study in pathological contexts, particularly in inflammatory bowel diseases.</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

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

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

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

Patient’s characteristics.

<p>Patient’s characteristics.</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

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