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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 γκT cells) postprimary 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 κ2-γdT cells. In CLAD LTRs, the regulation of B, total CD8 T, and d2+gdT cells is maintained, but total NK, CD57+/NKG2C+ NK, and d2-gdT 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 HCMVspecific HLA-E-restricted CD8 T cells together with post-infection changes in the immune cell distribution affecting NK and gdT 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 CMVviremia 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 HLAA and HLA-C allotypes, which enables these CMV strains to escape HLA-Erestricted 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. [ABSTRACT FROM AUTHOR]

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 CMVviremia 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 HLAA and HLA-C allotypes, which enables these CMV strains to escape HLA-Erestricted 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) is an important pathogen that infects the majority of the population worldwide, yet, currently, there is no licensed vaccine. Despite HCMV encoding at least seven Natural Killer (NK) cell evasion genes, NK cells remain critical for the control of infection in vivo. Classically Antibody-Dependent Cellular Cytotoxicity (ADCC) is mediated by CD16, which is found on the surface of the NK cell in a complex with FcεRI-γ chains and/or CD3ζ chains. Ninety percent of NK cells express the Fc receptor CD16; thus, they have the potential to initiate ADCC. HCMV has a profound effect on the NK cell repertoire, such that up to 10-fold expansions of NKG2C+ cells can be seen in HCMV seropositive individuals. These NKG2C+ cells are reported to be FcεRI-γ deficient and possess variable levels of CD16+, yet have striking ADCC functions. A subset of HCMV cell surface proteins will induce robust antibody responses that could render cells susceptible to ADCC. We will consider how the strong anti-HCMV function of NKG2C+ FcεRI-γ-deficient NK cells could potentially be harnessed in the clinic to treat patients suffering from HCMV disease and in the development of an efficacious HCMV vaccine.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Society for Histocompatibility and Immunogenetics. All rights reserved.)

Human cytomegalovirus (HCMV) is an important pathogen that infects the majority of the population worldwide, yet, currently, there is no licensed vaccine. Despite HCMV encoding at least seven Natural Killer (NK) cell evasion genes, NK cells remain critical for the control of infection in vivo. Classically Antibody-Dependent Cellular Cytotoxicity (ADCC) is mediated by CD16, which is found on the surface of the NK cell in a complex with FceRI-? chains and/or CD3? chains. Ninety percent of NK cells express the Fc receptor CD16; thus, they have the potential to initiate ADCC. HCMV has a profound effect on the NK cell repertoire, such that up to 10-fold expansions of NKG2C+cells can be seen in HCMV seropositive individuals. These NKG2C+cells are reported to be FceRI-? deficient and possess variable levels of CD16+, yet have striking ADCC functions. A subset of HCMV cell surface proteins will induce robust antibody responses that could render cells susceptible to ADCC. We will consider how the strong anti-HCMV function of NKG2C+ FceRI-γ-deficient NK cells could potentially be harnessed in the clinic to treat patients suffering from HCMV disease and in the development of an efficacious HCMV vaccine. [ABSTRACT FROM AUTHOR]

Natural killer (NK)-cytotoxic T lymphocytes (CTL) are a subset of CD8+ cytolytic T lymphocytes that express human leukocyte antigen (HLA) class I–specific inhibitory receptors. They are detectable as monoclonal expansions in the blood of cytomegalovirus (CMV)-seropositive individuals displaying particular HLA-Cw allotypes. Similar to NK cells, they are capable of killing various allogeneic tumor cell lines, a function referred to as “NK-like activity.” The mechanism underlying this unusual functional property has recently been clarified. Via their T-cell receptor, NK-CTL recognize the nonclassical HLA class I molecule HLA-E, which is characterized by a limited polymorphism and by the ability to bind peptides derived from the leader sequence of various HLA class I alleles as well as from few viral proteins. The analysis of the T-cell receptor avidity revealed that NK-CTL recognize with high avidity a CMV UL40-derived peptide. The HLA-E–restricted recognition of CMV by NK-CTL may represent an important immunologic strategy in defenses against this virus. Indeed, unlike conventional CTL, NK-CTL mediated lysis is apparently not affected by the downregulation of major histocompatibility complex class I that occurs during CMV infection. Because the CMV UL40-derived peptide is identical to the one generated from the leader sequence of various HLA-Cw alleles, NK-CTL are also able to display an “HLA-E–dependent alloreactivity” against allogeneic target cells expressing appropriate HLA-Cw alleles. This broad ability to recognize and kill allogeneic cells may pose serious problems in transplantation. [Copyright &y& Elsevier]