Changes in HCMV immune cell frequency and phenotype are associated with chronic lung allograft dysfunction.

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Persistent CD8 T Cell Marks Caused by the HCMV Infection in Seropositive Adults: Prevalence of HLA-E-Reactive CD8 T Cells.

This study investigated the frequency and peptide specificity of long-lasting HCMV-specific CD8 T cells in a cohort of 120 cytomegalovirus seropositive (HCMV+) healthy carriers with the aim of deciphering the relative contribution of unconventional HLA-E- versus conventional HLA-A2-specific CD8 T cells to long-term T cell memory expansion in HCMV immunity. The presence of HCMV-specific CD8 T cells was investigated by flow cytometry using five MHC/peptide tetramer complexes (HLA-A2/pp65, HLA-A2/IE1 and three different HLA-E/UL40). Here, we report that 50% of HCMV+ healthy individuals possess HCMV-specific CD8 T cells, representing ≥0.1% of total blood CD8 T cells years post-infection. Around a third (30.8%) of individuals possess HLA-A2-restricted (A2pp65 or A2IE1) and an equal proportion (27.5%) possess an HLA-E/UL40 CD8 T response. Concomitant HLA-E- and HLA-A2-reactive CD8 T cells were frequently found, and VMAPRTLIL peptide was the major target. The frequency of HLA-E/VMAPRTLIL among total blood CD8 T cells was significantly higher than the frequency of HLA-A2pp65 T cells (mean values: 5.9% versus 2.3%, p = 0.0354). HLA-EUL40 CD8 T cells display lower TCR avidity but similar levels of CD3 and CD8 coreceptors. In conclusion, HLA-E-restricted CD8 T cells against the VMAPRTLIL UL40 peptide constitute a predominant subset among long-lasting anti-HCMV CD8 T cells.

Do variations in the HLA-E ligand encoded by UL40 distinguish individuals susceptible to HCMV disease?

Human cytomegalovirus (HCMV) is carried lifelong by ∼80 % of adults worldwide, generating distinct disease syndromes in transplant recipients, people with HIV (PWH) and neonates. Amino acids 15-23 encoded by the HCMV gene UL40 match positions 3-11 of HLA-A and HLA-C, and constitute a "signal peptide" able to stabilise cell surface HLA-E as a restriction element and a ligand of NKG2A and NKG2C. We present next generation sequencing of UL40 amplified from 15 Australian renal transplant recipients (RTR), six healthy adults and four neonates, and 21 Indonesian PWH. We found no groupwise associations between the presence of multiple sequences and HCMV burden (highest in PWH) or HCMV-associated symptoms in neonates. Homology between UL40 and corresponding HLA-C and HLA-A peptides in 11 RTR revealed perfect matches with HLA-C in three individuals, all carrying HCMV encoding only VMAPRTLIL - a peptide previously associated with viremia. However indices of the burden of HCMV did not segregate in our cohort.

Intraocular human cytomegaloviruses of ocular diseases are distinct from those of viremia and are capable of escaping from innate and adaptive immunity by exploiting HLA-E-mediated peripheral and central tolerance.

Human cytomegalovirus (HCMV) infections develop into CMV diseases that result in various forms of manifestations in local organs. CMV-retinitis is a form of CMV disease that develops in immunocompromised hosts with CMV-viremia after viruses in the peripheral circulation have entered the eye. In the HCMV genome, extensive diversification of the UL40 gene has produced peptide sequences that modulate NK cell effector functions when loaded onto HLA-E and are subsequently recognized by the NKG2A and NKG2C receptors. Notably, some HCMV strains carry UL40 genes that encode peptide sequences identical to the signal peptide sequences of specific HLA-A and HLA-C allotypes, which enables these CMV strains to escape HLA-E-restricted CD8+T cell responses. Variations in UL40 sequences have been studied mainly in the peripheral blood of CMV-viremia cases. In this study, we sought to investigate how ocular CMV disease develops from CMV infections. CMV gene sequences were compared between the intraocular fluids and peripheral blood of 77 clinical cases. UL40 signal peptide sequences were more diverse, and multiple sequences were typically present in CMV-viremia blood compared to intraocular fluid. Significantly stronger NK cell suppression was induced by UL40-derived peptides from intraocular HCMV compared to those identified only in peripheral blood. HCMV present in intraocular fluids were limited to those carrying a UL40 peptide sequence corresponding to the leader peptide sequence of the host's HLA class I, while UL40-derived peptides from HCMV found only in the peripheral blood were disparate from any HLA class I allotype. Overall, our analyses of CMV-retinitis inferred that specific HCMV strains with UL40 signal sequences matching the host's HLA signal peptide sequences were those that crossed the blood-ocular barrier to enter the intraocular space. UL40 peptide repertoires were the same in the intraocular fluids of all ocular CMV diseases, regardless of host immune status, implying that virus type is likely to be a common determinant in ocular CMV disease development. We thus propose a mechanism for ocular CMV disease development, in which particular HCMV types in the blood exploit peripheral and central HLA-E-mediated tolerance mechanisms and, thus, escape the antivirus responses of both innate and adaptive immunity.

HLA-E restricted cytomegalovirus UL40 peptide polymorphism may represent a risk factor following congenital infection.

BACKGROUND: Congenital cytomegalovirus immunopathogenesis is largely unknown and multifactorial due to the complex interactions between viral, maternal, placental, and child factors. Polymorphisms in the HLA-E binding UL4015-23 peptide mimics HLA-E complexed peptides from certain HLA-A, -B, -C and -G alleles, which regulate the cellular immune response driven by natural killer-cells (NK) and CD8 + T cells. The aim of this study was to compare UL4015-23 peptides distribution in congenital CMV and the counterpart HLA Class I peptides in a healthy cohort to investigate risk factors and markers for cCMV disease. In this 10-year retrospective study, the UL40 gene was directly sequenced from 242 clinical samples from 199 cases of congenital CMV (166 children and 33 pregnant or breast feeding women). Distribution of HLA-E binding UL4015-23 peptides was analyzed and compared to those of HLA Class I observed in a cohort of 444 healthy individuals. RESULTS: Nineteen different HLA-E binding UL4015-23 peptides were found. Three of them (VMAPRTLIL, VMAPRTLLL, VMAPRTLVL) were found in 88.3% of UL40 and 100% of HLA Class I of healthy individuals. In contrast, 15 of them (10.7%) were not found in HLA Class I. The VMAPRTLFL peptide was found in 1% of UL40 and all HLA-G alleles. Significant differences in peptide (VMAPRTLIL, VMAPRTLLL, VMAPRTLVL, other UL4015-23 peptides, other HLA Class I peptides) distribution between UL4015-23 from congenital CMV and HLA-A, -B, -C and -G from healthy individuals were found. CONCLUSIONS: Our findings suggest that a mismatch between UL4015-23 peptides and HLA Class I peptides between children and mothers might play a role in congenital CMV disease, and it may account for differences in outcome, morbidity and sequelae.

Corrigendum: Intraocular human cytomegaloviruses of ocular diseases are distinct from those of viremia and are capable of escaping from innate and adaptive immunity by exploiting HLA-E-mediated peripheral and central tolerance.

[This corrects the article DOI: 10.3389/fimmu.2022.1008220.].

Association between chronic lung allograft dysfunction and human Cytomegalovirus UL40 peptide variants in lung-transplant recipients.

Natural-Killer cells play an important role in the pathogenesis of chronic lung allograft dysfunction (CLAD) in lung-transplant recipients. Activating NKG2C+ and inhibitory NKG2A+ NK cells proliferate in response to human Cytomegalovirus (HCMV) infection via the presentation of virally encoded UL40 peptides on HLA-E molecules. We aimed to clarify whether infection with HCMV strains carrying different UL40 peptide variants is associated with the development of CLAD. We included 82 lung-transplant recipients, 18 patients developing CLAD and 64 matched control patients without CLAD. In all patients 1 episode of high-level HCMV-replication occurred. HCMV UL40 variants and Natural-Killer-cell proliferation with distinct UL40 peptides were assessed. The VMTPRTLIL variant was significantly overrepresented in patients developing CLAD (p < 0.0001) and lead to a significantly lower proliferation of inhibitory NKG2A+ cells, compared to the VMAPRTLIL, VMAPRTLVL and VMAPRTLLL variants (p < 0.0001). Thus, HCMV UL40 variants may contribute to development of CLAD over the NK cell response.

Extent of Cytomegalovirus Replication in the Human Host Depends on Variations of the HLA-E/UL40 Axis.

Human cytomegalovirus (HCMV) may cause severe infections in lung transplant recipients (LTRs). In response to HCMV infections, a subset of NKG2C+ NK cells expands, which limits HCMV replication and is characterized by high expression of the activating NKG2C/CD94 and absence of the inhibitory NKG2A/CD94 receptor. Both receptors bind to HLA-E, which is stabilized by HCMV-encoded UL40 peptides. HLA-E and UL40 occur as different genetic variants. In this study, we investigated the interplay between the human NK cell response and the infecting HCMV-UL40 strain, and we assessed the impact of HCMV-UL40 and of donor- and recipient-encoded HLA-E*0101/0103 variants on HCMV replication after lung transplantation. We included 137 LTRs displaying either no or low- or high-level (>1,000 copies/ml plasma) viremia. HCMV-UL40 and HLA-E*0101/0103 variants were determined. UL40 diversity was investigated by next-generation sequencing. UL40 peptide-dependent NK cell cytotoxicity was assessed by flow cytometry. Donor-encoded HLA-E*0101/0103 was significantly associated with development of high-level viremia after transplantation (P = 0.007). The HCMV-UL40 variant VMAPRTLIL occurred significantly more frequently in highly viremic LTRs, and the variant VMTPRTLIL occurred significantly more frequently in low-viremic LTRs (P = 0.004). This difference was associated with a better inhibition of NKG2A+ NKG2C- NK cells by VMAPRTLIL (P < 0.001). In LTRs with repeated high-level viremic episodes, HCMV strains with UL40 variants displaying low affinity to the patients' HLA-E variant emerged over time. The HLA-E-UL40 axis has a substantial impact on the level of HCMV replication in LTRs. The interplay between UL40 peptide variants, the recipient HLA-E status, and the activation of inhibitory NKG2A+ NKG2C- cells is of major importance for development of high-level viremia after lung transplantation.IMPORTANCE Infection with human cytomegalovirus (HCMV) is associated with substantial morbidity in immunosuppressed patients and after congenital infections. Therefore, development of a vaccine against HCMV is a main public health priority. Revealing the complex interaction between HCMV and host responses, is of utmost importance for understanding viral pathogenesis and for vaccine design. The present data contribute to the understanding of HCMV-specific host immune responses and reveal specifically the interaction between HLA-E and the virus-encoded UL40 peptide, which further leads to a potent NK cell response. We demonstrate that this interaction is a key factor for reduction of virus replication in immunosuppressed patients. We further show that distinct naturally occurring HCMV-UL40 variants reduce the activation of a specific subpopulation of host NK cells and thereby are associated with high-level viremia in the patients. These findings will allow the characterization of patients at risk for severe HCMV infection and contribute to strategies for HCMV vaccine development.

Elusive Role of the CD94/NKG2C NK Cell Receptor in the Response to Cytomegalovirus: Novel Experimental Observations in a Reporter Cell System.

Human cytomegalovirus (HCMV) infection promotes the differentiation and persistent expansion of a mature NK cell subset, which displays high surface levels of the activating CD94/NKG2C NK cell receptor, together with additional distinctive phenotypic and functional features. The mechanisms underlying the development of adaptive NK cells remain uncertain but some observations support the involvement of a cognate interaction of CD94/NKG2C with ligand(s) displayed by HCMV-infected cells. To approach this issue, the heterodimer and its adaptor (DAP12) were expressed in the human Jurkat leukemia T cell line; signaling was detected by transfection of a reporter plasmid encoding for Luciferase (Luc) under NFAT/AP1-dependent control. Engagement of the receptor by solid-phase bound CD94- or NKG2C-specific monoclonal antibodies (mAbs) triggered Luc expression. Moreover, reporter activation was detectable upon interaction with HLA-E+ 721.221 (.221-AEH) cells, as well as with 721.221 cells incubated with synthetic peptides, which stabilized surface expression of endogenous HLA-E; the response was specifically antagonized by soluble NKG2C- and HLA-E-specific mAbs. By contrast, activation of Jurkat-NKG2C+ was undetectable upon interaction with Human Fetal Foreskin Fibroblasts (HFFF) infected with HCMV laboratory strains (i.e., AD169, Towne), regardless of their differential ability to preserve surface HLA-E expression. On the other hand, infection with two clinical isolates or with the endotheliotropic TB40/E strain triggered Jurkat-NKG2C+ activation; yet, this response was not inhibited by blocking mAbs and was independent of CD94/NKG2C expression. The results are discussed in the framework of previous observations supporting the hypothetical existence of specific ligand(s) for CD94/NKG2C in HCMV-infected cells.