Immune 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-EUL40) have recently emerged as a non-conventional T-cell response also observed in some hosts. The occurrence, specificity and features of HLA-EUL40 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-EUL40 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-EUL40 CD8 T cells. These cells are effector memory CD8 (CD45RAhighROlow, CCR7-, CD27-, CD28-) characterized by a low level of PD-1 expression. HLA-EUL40 responses appear early post-infection and display a broad, unbiased, Vβ repertoire. Although induced in HCMV strain-dependent, UL4015-23-specific manner, HLA-EUL40 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 life-long lasting HLA-EUL40 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.
Although anti-HLA antibodies (Abs) cause most antibody-mediated rejections of renal allografts, non-anti-HLA Abs have also been postulated to contribute. A better understanding of such Abs in ...rejection is needed.
We conducted a nationwide study to identify kidney transplant recipients without anti-HLA donor-specific Abs who experienced acute graft dysfunction within 3 months after transplantation and showed evidence of microvascular injury, called acute microvascular rejection (AMVR). We developed a crossmatch assay to assess serum reactivity to human microvascular endothelial cells, and used a combination of transcriptomic and proteomic approaches to identify non-HLA Abs.
We identified a highly selected cohort of 38 patients with early acute AMVR. Biopsy specimens revealed intense microvascular inflammation and the presence of vasculitis (in 60.5%), interstitial hemorrhages (31.6%), or thrombotic microangiopathy (15.8%). Serum samples collected at the time of transplant showed that previously proposed anti-endothelial cell Abs-angiotensin type 1 receptor (AT1R), endothelin-1 type A and natural polyreactive Abs-did not increase significantly among patients with AMVR compared with a control group of stable kidney transplant recipients. However, 26% of the tested AMVR samples were positive for AT1R Abs when a threshold of 10 IU/ml was used. The crossmatch assay identified a common IgG response that was specifically directed against constitutively expressed antigens of microvascular glomerular cells in patients with AMVR. Transcriptomic and proteomic analyses identified new targets of non-HLA Abs, with little redundancy among individuals.
Our findings indicate that preformed IgG Abs targeting non-HLA antigens expressed on glomerular endothelial cells are associated with early AMVR, and that
cell-based assays are needed to improve risk assessments before transplant.
Alloreactive T cells play a key role in mediating graft-vs-host disease and allograft rejection, and recent data suggest that most T cell alloreactivity resides within the CD4 T cell subset. ...Particularly, T cell responses to herpesvirus can shape the alloreactive repertoire and influence transplantation outcomes. In this study, we describe six distinct EBV-specific CD4(+) T cell clones that cross-reacted with EBV-transformed lymphoblastoid cell lines (LCLs), dendritic cells, and endothelial cells expressing MHC class II alleles commonly found in the population. Allorecognition showed exquisite MHC specificity. These CD4(+) T cell clones efficiently killed dendritic cells or LCLs expressing the cross-reactive allogeneic MHC class II molecules, whereas they did not kill autologous LCLs. Endothelial cells expressing the proper allogeneic MHC molecules were poorly killed, but they induced high-level TNF-alpha production by the EBV-specific CD4(+) T cell clones. As already proposed, the strong alloreactivity toward LCLs suggest that these cells could be used for selective depletion of alloreactive T cells.
The endothelial cell (EC) protein C receptor (EPCR) negatively regulates coagulation and inflammation. Factors and mechanisms regulating the expression of cell-bound EPCR and the release of soluble ...(s) EPCR are still unclear.
We investigated the reciprocal regulation of membrane-bound and sEPCR upon inflammation using primary cultures of vascular EC. The impact of 2 parameters, gender and EPCR gene A3 haplotype, on sEPCR plasma basal level and endothelial expression was examined by Elisa and flow cytometry.
Exposure of EC to tumor necrosis factor α causes a rapid downregulation of membrane-associated EPCR expression without affecting markedly the spontaneous release of sEPCR by EC. In a cohort of 100 healthy donors, we show that males express significantly higher basal sEPCR in plasma than females (194 ± 12 vs. 145 ± 9 ng/ml, respectively, p<0.01). Both gender and EPCR A3 haplotype affect sEPCR plasma levels but have no apparent effect on EPCR expression by EC. No quantitative correlation between cellular expression and circulating blood sEPCR was observed, suggesting that endothelial expression may not reflect the plasma level.
Male gender is another parameter with A3 haplotype associated with elevated sEPCR levels in blood, and both parameters may contribute to selective regulatory mechanisms of EPCR release upon inflammation.
After kidney transplantation, donor-specific antibodies against human leukocyte antigen donor-specific antibodies (HLA-DSAs) drive antibody-mediated rejection (ABMR) and are associated with poor ...transplant outcomes. However, ABMR histology (ABMRh) is increasingly reported in kidney transplant recipients (KTRs) without HLA-DSAs, highlighting the emerging role of non-HLA antibodies (Abs).
W e designed a non-HLA Ab detection immunoassay (NHADIA) using HLA class I and II-deficient glomerular endothelial cells (CiGEnC
HLA) that had been previously generated through CRISPR/Cas9-induced
and
gene disruption. Flow cytometry assessed the reactivity to non-HLA antigens of pretransplantation serum samples from 389 consecutive KTRs. The intensity of the signal observed with the NHADIA was associated with post-transplant graft histology assessed in 951 adequate biopsy specimens.
W e sequentially applied CRISPR/Cas9 to delete the
and
genes to obtain a CiGEnC
HLA clone. CiGEnC
HLA cells remained indistinguishable from the parental cell line, CiGEnC, in terms of morphology and phenotype. Previous transplantation was the main determinant of the pretransplantation NHADIA result (
<0.001). Stratification of 3-month allograft biopsy specimens (
=298) according to pretransplantation NHADIA tertiles demonstrated that higher levels of non-HLA Abs positively correlated with increased glomerulitis (
=0.002), microvascular inflammation (
=0.003), and ABMRh (
=0.03). A pretransplantation NHADIA threshold of 1.87 strongly discriminated the KTRs with the highest risk of ABMRh (
=0.005, log-rank test). A multivariate Cox model confirmed that NHADIA status and HLA-DSAs were independent, yet synergistic, predictors of ABMRh.
The NHADIA identifies non-HLA Abs and strongly predicts graft endothelial injury independent of HLA-DSAs.
Human cytomegalovirus (HCMV) infection is common and often severe in lung transplant recipients (LTRs), and it is a risk factor associated with chronic lung allograft dysfunction (CLAD). The complex ...interplay between HCMV and allograft rejection is still unclear. Currently, no treatment is available to reverse CLAD after diagnosis, and the identification of reliable biomarkers that can predict the early development of CLAD is needed. This study investigated the HCMV immunity in LTRs who will develop CLAD.
This study quantified and phenotyped conventional (HLA-A2pp65) and HLA-E-restricted (HLA-EUL40) anti-HCMV CD8
T (CD8 T) cell responses induced by infection in LTRs developing CLAD or maintaining a stable allograft. The homeostasis of immune subsets (B, CD4T, CD8 T, NK, and γδT cells) post-primary infection associated with CLAD was also investigated.
At M18 post-transplantation, HLA-EUL40 CD8 T responses were less frequently found in HCMV
LTRs (21.7%) developing CLAD (CLAD) than in LTRs (55%) keeping a functional graft (STABLE). In contrast, HLA-A2pp65 CD8 T was equally detected in 45% of STABLE and 47.8% of CLAD LTRs. The frequency of HLA-EUL40 and HLA-A2pp65 CD8 T among blood CD8 T cells shows lower median values in CLAD LTRs. Immunophenotype reveals an altered expression profile for HLA-EUL40 CD8 T in CLAD patients with a decreased expression for CD56 and the acquisition of PD-1. In STABLE LTRs, HCMV primary infection causes a decrease in B cells and inflation of CD8 T, CD57
/NKG2C
NK, and δ2
γδT cells. In CLAD LTRs, the regulation of B, total CD8 T, and δ2
γδT cells is maintained, but total NK, CD57
/NKG2C
NK, and δ2
γδT subsets are markedly reduced, while CD57 is overexpressed across T lymphocytes.
CLAD is associated with significant changes in anti-HCMV immune cell responses. Our findings propose that the presence of dysfunctional HCMV-specific HLA-E-restricted CD8 T cells together with post-infection changes in the immune cell distribution affecting NK and γδT cells defines an early immune signature for CLAD in HCMV
LTRs. Such a signature may be of interest for the monitoring of LTRs and may allow an early stratification of LTRs at risk of CLAD.
Tumor-derived soluble factors, including soluble HLA molecules, can contribute to cancer immune escape and therefore impact on clinical course of malignant diseases. We previously reported that ...melanoma cells produce, in vitro, soluble forms of the non-classical MHC class I molecule HLA-E (sHLA-E). In order to investigate sHLA-E production by various tumors and to address its potential value as a tumor-associated marker, we developed a specific ELISA for the quantification of sHLA-E in biological fluids.
We developed a sHLA-E specific and sensitive ELISA and we showed that serum sHLA-E levels were significantly elevated (P<0.01) in melanoma patients (n = 127), compared with healthy donors (n = 94). sHLA-E was also detected in the culture supernatants of a wide variety of tumor cell lines (n = 98) including melanomas, kidney, colorectal and breast cancers. Cytokines regulation of sHLA-E production by tumor cells was also carried out. IFN-γ, IFN-α and TNF-α were found to upregulate sHLA-E production by tumor cells.
In view of the broad tumor tissue release of HLA-E and its up-regulation by inflammatory cytokines, sHLA-E should be studied for its involvement in immune responses against tumors. Interestingly, our results demonstrated a positive association between the presence of serum sHLA-E and melanoma. Therefore, the determination of sHLA-E levels, using ELISA approach, may be investigated as a clinical marker in cancer patients.
There is an unmet need for robust and clinically validated biomarkers of kidney allograft rejection. Here we present the KTD-Innov study (ClinicalTrials.gov, NCT03582436), an unselected ...deeply phenotyped cohort of kidney transplant recipients with a holistic approach to validate the clinical utility of precision diagnostic biomarkers. In 2018–2019, we prospectively enrolled consecutive adult patients who received a kidney allograft at seven French centers and followed them for a year. We performed multimodal phenotyping at follow-up visits, by collecting clinical, biological, immunological, and histological parameters, and analyzing a panel of 147 blood, urinary and kidney tissue biomarkers. The primary outcome was allograft rejection, assessed at each visit according to the international Banff 2019 classification. We evaluated the representativeness of participants by comparing them with patients from French, European, and American transplant programs transplanted during the same period. A total of 733 kidney transplant recipients (64.1% male and 35.9% female) were included during the study. The median follow-up after transplantation was 12.3 months (interquartile range, 11.9–13.1 months). The cumulative incidence of rejection was 9.7% at one year post-transplant. We developed a distributed and secured data repository in compliance with the general data protection regulation. We established a multimodal biomarker biobank of 16,736 samples, including 9331 blood, 4425 urinary and 2980 kidney tissue samples, managed and secured in a collaborative network involving 7 clinical centers, 4 analytical platforms and 2 industrial partners. Patients' characteristics, immune profiles and treatments closely resembled those of 41,238 French, European and American kidney transplant recipients. The KTD-Innov study is a unique holistic and multidimensional biomarker validation cohort of kidney transplant recipients representative of the real-world transplant population. Future findings from this cohort are likely to be robust and generalizable.
Rabbit-generated antithymocyte globulins (ATGs), which target human T cells, are widely used as immunosuppressive agents during treatment of kidney allograft recipients. However, ATGs can induce ...immune complex diseases, including serum sickness disease (SSD). Rabbit and human IgGs have various antigenic differences, including expression of the sialic acid Neu5Gc and α-1-3-Gal (Gal), which are not synthesized by human beings. Moreover, anti-Neu5Gc antibodies have been shown to preexist and be elicited by immunization in human subjects. This study aimed to assess the effect of SSD on long-term kidney allograft outcome and to compare the immunization status of grafted patients presenting with SSD following ATG induction treatment.
We analyzed data from a cohort of 889 first kidney graft recipients with ATG induction (86 with SSD SSD(+) and 803 without SSD SSD(-)) from the Données Informatisées et Validées en Transplantation data bank. Two subgroups of SSD(+) and SSD(-) patients that had received ATG induction treatment were then assessed for total anti-ATG, anti-Neu5Gc, and anti-Gal antibodies using ELISA assays on sera before and after transplantation.
SSD was significantly associated with long-term graft loss (>10 years, P = 0.02). Moreover, SSD(+) patients exhibited significantly elevated titers of anti-ATG (P = 0.043) and anti-Neu5Gc (P = 0.007) IgGs in late post-graft samples compared with SSD(-) recipients.
In conclusion, our data indicate that SSD is a major contributing factor of late graft loss following ATG induction and that anti-Neu5Gc antibodies increase over time in SSD(+) patients.
This study was funded by Société d'Accélération du Transfert de Technologies Ouest Valorisation, the European FP7 "Translink" research program, the French National Agency of Research, Labex Transplantex, the Natural Science and Engineering Research Council of Canada, and the Canadian Foundation for Innovation.
The 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-E
CD8T). This study investigated the frequency, phenotype and functions of HLA-E
CD8T in comparison to the immunodominant HLA-A2
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-E
CD8T was similar after a primary infection (24%) and during viral latency in HCMV+ HV (26%) and equal to the frequency of HLA-A2
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-E
and HLA-A2
CD8T display a phenotype specific of CD8
TEMRA (CD45RA
/CCR7
) but HLA-E
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-E
and HLA-A2
CD8T. In contrast, CD56 and PD-1 are cell markers discriminating memory HLA-E- from HLA-A2-restricted CD8T. Long lived HLA-E
display higher proliferation rate compared to HLA-A2
CD8T consistent with elevated CD57 expression. Finally, a comparative immunoprofiling indicated that HLA-E
CD8T, divergent from HLA-A2
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.
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