Distinctive phenotype for HLA-E- versus HLA-A2-restricted memory CD8 αβT cells in the course of HCMV infection discloses features shared with NKG2C+CD57+NK and δ2-γδT cell subsets

International audienceThe human cytomegalovirus (HCMV) triggers both innate and adaptive immune responses, including protective CD8+ αβT cells (CD8T) that contributes to the control of the infection. In addition to CD8T restricted by classical HLA class Ia molecules, HCMV also triggers CD8T recognizing peptides from the HCMV UL40 leader peptide and restricted by HLA-E molecules (HLA-EUL40 CD8T). This study investigated the frequency, phenotype and functions of HLA-EUL40 CD8T in comparison to the immunodominant HLA-A2pp65 CD8T upon acute (primary or secondary infection) or chronic infection in kidney transplant recipients (KTR) and in seropositive (HCMV+) healthy volunteer (HV) hosts. The frequency of hosts with detected HLA-EUL40 CD8T was similar after a primary infection (24%) and during viral latency in HCMV+ HV (26%) and equal to the frequency of HLA-A2pp65 CD8T cells in both conditions (29%). Both CD8T subsets vary from 0.1% to >30% of total circulating CD8T according to the host. Both HLA-EUL40 and HLA-A2pp65 CD8T display a phenotype specific of CD8+ TEMRA (CD45RA+/CCR7-) but HLA-EUL40 CD8T express distinctive level for CD3, CD8 and CD45RA. Tim3, Lag-3, 4-1BB, and to a lesser extend 2B4 are hallmarks for T cell priming post-primary infection while KLRG1 and Tigit are markers for restimulated and long lived HCMV-specific CD8T responses. These cell markers are equally expressed on HLA-EUL40 and HLA-A2pp65 CD8T. In contrast, CD56 and PD-1 are cell markers discriminating memory HLA-E- from HLA-A2-restricted CD8T. Long lived HLA-EUL40 display higher proliferation rate compared to HLA-A2pp65 CD8T consistent with elevated CD57 expression. Finally, a comparative immunoprofiling indicated that HLA-EUL40 CD8T, divergent from HLA-A2pp65 CD8T, share the expression of CD56, CD57, NKG2C, CD158 and the lack of PD-1 with NKG2C+CD57+ NK and δ2-γδT cells induced in response to HCMV and thus defines a common immunopattern for these subsets

EARLY EXPANSION OF TEMRA CD8 WITH INNATE-LIKE FUNCTION IDENTIFIES KIDNEY TRANSPLANT RECIPIENTS AT HIGH-RISK OF GRAFT FAILURE

Lola Jacquemont and Gaëlle Tilly are co-authorsInternational audienceAs CD8 TEMRA cells are associated with higher risk of long-term graft dysfunction, in this study, we evaluate if the monitoring of CD8-related biomarkers could improve the prognostic capacities of a clinical-based scoring system (Kidney Transplant Failure Score; KTFS). We also characterize the functionality of TEMRA and especially their reactivity upon donor-specific stimulation. 286 kidney-transplant recipients prospectively enrolled were followed for more than 8-years. 51 return in dialysis. We demonstrate that the frequency of early memory CD8 cells (EM) and TEMRA measured at 1-year post-transplantation is correlated with the risk to return in dialysis during time. For patients at high-risk of long-term graft dysfunction (according to KTFS), the use of one-year TEMRA frequency allows the discrimination of patients that will lose their graft from those that will not. Donor-specific reactivities from TEMRA and EM were similar with an early expression of CD25+CD69+CD107a+ and the high secretion of pro-inflammatory and cytotoxic molecules. Importantly, we identify an innate-like signature of TEMRA, with more than 5-fold higher expression of FCGR3A (CD16) by TEMRA as compared to NAIVE and EM. Cross-linking of CD16 triggers the secretion of TNFa and IFNg by TEMRA and their cytotoxic function and was further enhanced by the provision of IL-15. Finally, we demonstrate TEMRA and not EM display in vitro Antibody Dependent Cell Cytotoxicity conferring to TEMRA features of both adaptive and innate-like immunity and showing that anti-HLA antibodies, a major risk factor for long-term allograft outcome, could activate TEMRA in a TCR-independent manner leading to the inflammatory response

Is pre-transplant sensitization against angiotensin II type 1 receptor still a risk factor of graft and patient outcome in kidney transplantation in the anti-HLA Luminex era? A retrospective study

International audienceWe aimed to assess the correlation of anti-angiotensin II type 1 receptor antibodies (anti-AT1R-Abs) before transplantation on a multicentric cohort of kidney transplant recipients (2008-2012), under tacrolimus and mycophenolate mofetil (MMF), screened by Luminex technology for anti-HLA immunization. Anti-AT1R antibody levels were measured by ELISA in pretransplantation sera of 940 kidney recipients from three French centers of the DIVAT cohort. Multivariable Cox models estimated the association between pretransplant anti-angiotensin II type 1 receptor antibodies and time to acute rejection episodes (ARE) or time to graft failure. Within our cohort, 387 patients (41.2%) had pretransplant AT1R-Abs higher than 10 U/ml and only 8% (72/970) greater than 17 U/ml. The cumulative probability of clinically relevant (cr)-ARE was 22.5% at 1 year post-trans-plantation [95% CI (19.9-25.4%)]. The cumulative probability of graft failure and patient death were 10.6% [95% CI (8.4-13.3%)] and 5.7% [95% CI (4.0-8.1%)] at 3 years post-transplantation, respectively. Multivariate Cox models indicated that pretransplant anti-AT1R antibody levels higher than 10 U/ml were not significantly independently associated with higher risks of acute rejection episodes [HR = 1.04, 95% CI (0.80-1.35)] nor with risk of graft failure [HR = 0.86, 95% CI (0.56-1.33)]. Our study did not confirm an association between pretransplant anti-AT1R antibody levels and kidney transplant outcomes

EARLY EXPANSION OF TEMRA CD8 WITH INNATE-LIKE FUNCTION IDENTIFIES KIDNEY TRANSPLANT RECIPIENTS AT HIGH-RISK OF GRAFT FAILURE

Lola Jacquemont and Gaëlle Tilly are co-authorsInternational audienceAs CD8 TEMRA cells are associated with higher risk of long-term graft dysfunction, in this study, we evaluate if the monitoring of CD8-related biomarkers could improve the prognostic capacities of a clinical-based scoring system (Kidney Transplant Failure Score; KTFS). We also characterize the functionality of TEMRA and especially their reactivity upon donor-specific stimulation. 286 kidney-transplant recipients prospectively enrolled were followed for more than 8-years. 51 return in dialysis. We demonstrate that the frequency of early memory CD8 cells (EM) and TEMRA measured at 1-year post-transplantation is correlated with the risk to return in dialysis during time. For patients at high-risk of long-term graft dysfunction (according to KTFS), the use of one-year TEMRA frequency allows the discrimination of patients that will lose their graft from those that will not. Donor-specific reactivities from TEMRA and EM were similar with an early expression of CD25+CD69+CD107a+ and the high secretion of pro-inflammatory and cytotoxic molecules. Importantly, we identify an innate-like signature of TEMRA, with more than 5-fold higher expression of FCGR3A (CD16) by TEMRA as compared to NAIVE and EM. Cross-linking of CD16 triggers the secretion of TNFa and IFNg by TEMRA and their cytotoxic function and was further enhanced by the provision of IL-15. Finally, we demonstrate TEMRA and not EM display in vitro Antibody Dependent Cell Cytotoxicity conferring to TEMRA features of both adaptive and innate-like immunity and showing that anti-HLA antibodies, a major risk factor for long-term allograft outcome, could activate TEMRA in a TCR-independent manner leading to the inflammatory response

HCMV triggers frequent and persistent UL40- specific unconventional HLA-E-restricted CD8 T-cell responses with potential autologous and allogeneic peptide recognition

International audienceImmune response against human cytomegalovirus (HCMV) includes a set of persistent cytotoxic NK and CD8 T cells devoted to eliminate infected cells and to prevent reactivation. CD8 T cells against HCMV antigens (pp65, IE1) presented by HLA class-I molecules are well characterized and they associate with efficient virus control. HLA-E-restricted CD8 T cells targeting HCMV UL40 signal peptides (HLA-E[UL40]) have recently emerged as a non-conventional T-cell response also observed in some hosts. The occurrence, specificity and features of HLA-E[UL40] CD8 T-cell responses remain mostly unknown. Here, we detected and quantified these responses in blood samples from healthy blood donors (n = 25) and kidney transplant recipients (n = 121) and we investigated the biological determinants involved in their occurrence. Longitudinal and phenotype ex vivo analyses were performed in comparison to HLA-A*02/pp65-specific CD8 T cells. Using a set of 11 HLA-E/UL40 peptide tetramers we demonstrated the presence of HLA-E[UL40] CD8 αβT cells in up to 32% of seropositive HCMV+ hosts that may represent up to 38% of total circulating CD8 T-cells at a time point suggesting a strong expansion post-infection. Host's HLA-A*02 allele, HLA-E *01:01/*01:03 genotype and sequence of the UL40 peptide from the infecting strain are major factors affecting the incidence of HLA-E[UL40] CD8 T cells. These cells are effector memory CD8 (CD45RA[high]RO[low], CCR7-, CD27-, CD28-) characterized by a low level of PD-1 expression. HLA-E[UL40] responses appear early post-infection and display a broad, unbiased, Vβ repertoire. Although induced in HCMV strain-dependent, UL40[15-23]-specific manner, HLA-E[UL40] CD8 T cells are reactive toward a broader set of nonapeptides varying in 1-3 residues including most HLA-I signal peptides. Thus, HCMV induces strong and lifelong lasting HLA-E[UL40] CD8 T cells with potential allogeneic or/and autologous reactivity that take place selectively in at least a third of infections according to virus strain and host HLA concordance

A composite score associated with spontaneous operational tolerance in kidney transplant recipients

International audienceNew challenges in renal transplantation include using biological information to devise a useful clinical test for discerning high- and low-risk patients for individual therapy and ascertaining the best combination and appropriate dosages of drugs. Based on a 20-gene signature from a microarray meta-analysis performed on 46 operationally tolerant patients and 266 renal transplant recipients with stable function, we applied the sparse Bolasso methodology to identify a minimal and robust combination of six genes and two demographic parameters associated with operational tolerance. This composite score of operational tolerance discriminated operationally tolerant patients with an area under the curve of 0.97 (95% confidence interval 0.94-1.00). The score was not influenced by immunosuppressive treatment, center of origin, donor type, or post-transplant lymphoproliferative disorder history of the patients. This composite score of operational tolerance was significantly associated with both de novo anti-HLA antibodies and tolerance loss. It was validated by quantitative polymerase chain reaction using independent samples and demonstrated specificity toward a model of tolerance induction. Thus, our score would allow clinicians to improve follow-up of patients, paving the way for individual therapy

KiT-GENIE, the French genetic biobank of kidney transplantation

International audienceKiT-GENIE is a monocentric DNA biobank set up to consolidate the very rich and homogeneous DIVAT French cohort of kidney donors and recipients (D/R) in order to explore the molecular factors involved in kidney transplantation outcomes. We collected DNA samples for kidney transplantations performed in Nantes, and we leveraged GWAS genotyping data for securing high-quality genetic data with deep SNP and HLA annotations through imputations and for inferring D/R genetic ancestry. Overall, the biobank included 4217 individuals (n = 1945 D + 2,272 R, including 1969 D/R pairs), 7.4 M SNPs and over 200 clinical variables. KiT-GENIE represents an accurate snapshot of kidney transplantation clinical practice in Nantes between 2002 and 2018, with an enrichment in living kidney donors (17%) and recipients with focal segmental glomerulosclerosis (4%). Recipients were predominantly male (63%), of European ancestry (93%), with a mean age of 51yo and 86% experienced their first graft over the study period. D/R pairs were 93% from European ancestry, and 95% pairs exhibited at least one HLA allelic mismatch. The mean follow-up time was 6.7 years with a hindsight up to 25 years. Recipients experienced biopsy-proven rejection and graft loss for 16.6% and 21.3%, respectively. KiT-GENIE constitutes one of the largest kidney transplantation genetic cohorts worldwide to date. It includes homogeneous high-quality clinical and genetic data for donors and recipients, hence offering a unique opportunity to investigate immunogenetic and genetic factors, as well as donor-recipient interactions and mismatches involved in rejection, graft survival, primary disease recurrence and other comorbidities

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

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