anti-CMV Immune Response Research Articles

Antigen-specific γδ T cells contribute to cytomegalovirus control after stem cell transplantation.

γδ T cells support the immunological control of viral infections, in particular during cytomegalovirus (CMV) reactivation in immunocompromised patients after allogeneic hematopoietic stem cell transplantation. It is unclear how γδ T cells sense CMV-infection and whether this involves specific T cell receptor (TCR)-ligand interaction. Here we summarize recent findings that revealed an adaptive-like anti-CMV immune response of γδ T cells, characterized by acquisition of effector functions and long-lasting clonal expansion. We propose that rather CMV-induced self-antigen than viral antigens trigger γδ TCRs during CMV reactivation. Given that the TCRs of CMV-activated γδ T cells are often cross-reactive to tumor cells, these findings pinpoint γδ T cells and their γδ TCRs as attractive multipurpose tools for antiviral and antitumor therapy.

Intérêt de l'exploration de l'immunité cellulaire dans l'infection à cytomégalovirus par les tests IGRAs dans la prise en charge des patients transplantés d'organes solides

AbstractCytomegalovirus (CMV) infection represents the most frequent infectious complication in solid organ transplantation for which the risk is mainly stratified according to the anti-CMV serological status of the recipient (R) and the donor (D) before transplantation. There are two strategic options for antiviral treatment, universal prophylaxis or preemptive therapy. As T-cell response mediated by CD8 and CD4 lymphocytes is the major mechanism of the anti-CMV immune response, multiple IGRAs tests (IFN-γ Release Assays) have been developed: the Elisa test, QuantiFERON®-CMV and the ELIspot tests: T-Spot® CMV and T-Track® CMV These assays are based on the measurement of IFN-γ secretion by specific memory T cells after their stimulation by CMV peptides. This review presents the usefulness of IGRAs tests, in addition to the anti-CMV serological status data, for a better stratification of patients at risk before transplantation. It also discusses the benefit of monitoring of viral replication control by T effectors in order to optimize antiviral therapeutic management during post-transplant monitoring in addition to virological data.

Revisiting the Role of γδ T Cells in Anti-CMV Immune Response after Transplantation

Gamma delta (γδ) T cells form an unconventional subset of T lymphocytes that express a T cell receptor (TCR) consisting of γ and δ chains. Unlike conventional αβ T cells, γδ T cells share the immune signature of both the innate and the adaptive immunity. These features allow γδ T cells to act in front-line defense against infections and tumors, rendering them an attractive target for immunotherapy. The role of γδ T cells in the immune response to cytomegalovirus (CMV) has been the focus of intense research for several years, particularly in the context of transplantation, as CMV reactivation remains a major cause of transplant-related morbidity and mortality. Therefore, a better understanding of the mechanisms that underlie CMV immune responses could enable the design of novel γδ T cell-based therapeutic approaches. In this regard, the advent of next-generation sequencing (NGS) and single-cell TCR sequencing have allowed in-depth characterization of CMV-induced TCR repertoire changes. In this review, we try to shed light on recent findings addressing the adaptive role of γδ T cells in CMV immunosurveillance and revisit CMV-induced TCR reshaping in the era of NGS. Finally, we will demonstrate the favorable and unfavorable effects of CMV reactive γδ T cells post-transplantation.

Understanding human γδ T cell biology toward a better management of cytomegalovirus infection.

Cytomegalovirus (CMV) infection is responsible for significant morbidity and mortality in immunocompromised patients, namely solid organ and hematopoietic cell transplant recipients, and can induce congenital infection in neonates. There is currently an unmet need for new management and treatment strategies. Establishment of an anti-CMV immune response is critical in order to control CMV infection. The two main human T cells involved in HCMV-specific response are αβ and non-Vγ9Vδ2 T cells that belong to γδ T cell compartment. CMV-induced non-Vγ9Vδ2 T cells harbor a specific clonal expansion and a phenotypic signature, and display effector functions against CMV. So far, only two main molecular mechanisms underlying CMV sensing have been identified. Non-Vγ9Vδ2 T cells can be activated either by stress-induced surface expression of the γδT cell receptor (TCR) ligand annexin A2, or by a multimolecular stress signature composed of the γδTCR ligand endothelial protein C receptor and co-stimulatory signals such as the ICAM-1-LFA-1 axis. All this basic knowledge can be harnessed to improve the clinical management of CMV infection in at-risk patients. In particular, non-Vγ9Vδ2 T cell monitoring could help better stratify the risk of infection and move forward a personalized medicine. Moreover, recent advances in cell therapy protocols open the way for a non-Vγ9Vδ2 T cell therapy in immunocompromised patients.

The Immune Response Against Human Cytomegalovirus Links Cellular to Systemic Senescence.

Aging reflects long-term decline in physiological function and integrity. Changes arise at a variable pace governed by time-dependent and -independent mechanisms that are themselves complex, interdependent and variable. Molecular decay produces inferior cells that eventually dominate over healthy counterparts in tissues they comprise. In a form of biological entropy, progression from molecular through cellular to tissue level degeneration culminates in organ disease or dysfunction, affecting systemic health. To better understand time-independent contributors and their potential modulation, common biophysical bases for key molecular and cellular changes underlying age-related physiological deterioration must be delineated. This review addresses the potential contribution of cytomegalovirus (CMV)-driven T cell proliferation to cellular senescence and immunosenescence. We first describe molecular processes imposing cell cycle arrest, the foundation of cellular senescence, then focus on the unique distribution, phenotype and function of CMV-specific CD8+ T cells in the context of cellular senescence and “inflammaging”. Their features position CMV infection as a pathogenic accelerant of immune cell proliferation underlying immune senescence. In human immunodeficiency virus (HIV) infection, where increased inflammation and exaggerated anti-CMV immune responses accelerate immune senescence, CMV infection has emerged as a major factor in unhealthy aging. Thus, we speculate on mechanistic links between CMV-specific CD8+ T-cell expansion, immune senescence and prevalence of age-related disorders in HIV infection.

A role of impaired neonatal T cell activation upon late CMV detection

Objective: examine signs of impaired neonatal T cell activation upon late cytomegalovirus (CMV) DNA detection in the blood and urine.Materials and methods. 147 neonates with non-specific clinical symptoms were enrolled to the study. Peripheral blood lymphocyte subsets were immunophenotyped by using anti-human antibody panel to stain for surface markers CD3, CD4, CD8, CD20, CD3+CD28-, CD3+CD28+, CD3–CD28+, CD20+CD40+, CD28, CD40 (IMMUNOTECH, France) for further analysis on a laser flow cytometer Beckman Coulter Epics XL II. CMV infection in 123 neonates was verified by PCR, whereas it was negative in 24 cases. Further, 24 children aged 1.5–3 months negative for CMV DNA during the first month of life were later shown to contain it in the blood and urine samples as well as elevated anti-CMV IgG antibodies allowing to verify CMV infection. Results. It was found that upon late CMV DNA detection, amount of CD3+CD28+ T cells in peripheral blood was directly related to count of total CD3+ T cells regardless of CD28 expression. Moreover, the peak percentage of CD3+CD28+ (70–80%) T cells ranged within 85–90%. Whereas children upon early CMV detection the maximum percentage of CD3+CD28+ T cells was associated with lowered CD3+ T cell count (45–55%), it was also paralleled with high CD28 expression (higher than 3%). Importantly, T cells co-expressing CD28+ activation marker necessary for co-stimulation represent one of significant parameters used in diagnostics. Down-modulated CD28 expression abrogating costimulatory signaling and resulting in T cell anergy contributes to developing immunological failure in neonatal CMV infection. The data obtained confirmed importance of cell-cell immune and non-immune interactions for executing anti-CMV immune response within the first months of life. Finally, it is proposed to predict developing CMV infection depending on percentage of CD3+ and CD28+CD3+ T cell subsets in neonates manifested with non-specific clinical signs upon late CMV DNA detection.

Clinically Relevant Immune Responses against Cytomegalovirus: Implications for Precision Medicine.

Immune responses to human cytomegalovirus (CMV) can be used to assess immune fitness in an individual. Further to its clinical significance in posttransplantation settings, emerging clinical and translational studies provide examples of immune correlates of protection pertaining to anti-CMV immune responses in the context of cancer or infectious diseases, e.g., tuberculosis. In this viewpoint, we provide a brief overview about CMV-directed immune reactivity and immune fitness in a clinical context and incorporate some of our own findings obtained from peripheral blood or tumour-infiltrating lymphocytes (TIL) from patients with advanced cancer. Observations in patients with solid cancers whose lesions contain both CMV and tumour antigen-specific T-cell subsets are highlighted, due to a possible CMV-associated “bystander” effect in amplifying local inflammation and subsequent tumour rejection. The role of tumour-associated antibodies recognising diverse CMV-derived epitopes is also discussed in light of anti-cancer immune responses. We discuss here the use of anti-CMV immune responses as a theranostic tool—combining immunodiagnostics with a personalised therapeutic potential—to improve treatment outcomes in oncological indications.

Control of murine cytomegalovirus infection by γδ T cells.

Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of γδ T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of γδ T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 αβ-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1-/- mice lacking any T- and B-cells. γδ T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1-/- mice after adoptive transfer. γδ T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the γδ T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused Vγ1 and Vγ2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add γδ T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that γδ T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by γδ T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described.

Direct and Indirect Effects of Cytomegalovirus-Induced γδ T Cells after Kidney Transplantation.

Despite effective anti-viral therapies, cytomegalovirus (CMV) is still associated with direct (CMV disease) and indirect effects (rejection and poor graft survival) in kidney transplant recipients. Recently, an unconventional T cell population (collectively designated as Vδ2neg γδ T cells) has been characterized during the anti-CMV immune response in all solid-organ and bone-marrow transplant recipients, neonates, and healthy people. These CMV-induced Vδ2neg γδ T cells undergo a dramatic and stable expansion after CMV infection, in a conventional “adaptive” manner. Similarly, as CMV-specific CD8+ αβ T cells, they exhibit an effector/memory TEMRA phenotype and cytotoxic effector functions. Activation of Vδ2neg γδ T cells by CMV-infected cells involves the γδ T cell receptor (TCR) and still ill-defined co-stimulatory molecules such as LFA-1. A multiple of Vδ2neg γδ TCR ligands are apparently recognized on CMV-infected cells, the first one identified being the major histocompatibility complex-related molecule endothelial protein C receptor. A singularity of CMV-induced Vδ2neg γδ T cells is to acquire CD16 expression and to exert an antibody-dependent cell-mediated inhibition on CMV replication, which is controlled by a specific cytokine microenvironment. Beyond the well-demonstrated direct anti-CMV effect of Vδ2neg γδ T cells, unexpected indirect effects of these cells have been also observed in the context of kidney transplantation. CMV-induced Vδ2neg γδ T cells have been involved in surveillance of malignancy subsequent to long-term immunosuppression. Moreover, CMV-induced CD16+ γδ T cells are cell effectors of antibody-mediated rejection of kidney transplants, and represent a new physiopathological contribution to the well-known association between CMV infection and poor graft survival. All these basic and clinical studies paved the road to the development of a future γδ T cell-based immunotherapy. In the meantime, γδ T cell monitoring should prove a valuable immunological biomarker in the management of CMV infection.

Influence of Production Affecting Cytokine Genetic Variants on Allogeneic Hematopoietic Cell Transplantation Outcomes

Introduction:High resolution human leukocyte antigen (HLA) matching is regarded as a prerequisite for the clinical success of allogeneic haematopoietic cell transplantation (HCT). However, studies have reported immunogenetic factors other than HLA also plays an important role in preventing and controlling post HCT complications, in particular graft-versus-host disease (GVHD) and infections. Cytokines and cytokine receptors, which are the key regulators of the immune surveillance against infections and/or allo-immune responses, are important candidates. The genetic control of cytokine production is evidenced by polymorphisms in cytokine gene regulatory regions resulting in low, moderate, or high cytokine production. Here, we evaluated the prognostic significance of cytokine genetic variants known to impact cytokine production on different outcomes of allogeneic HCT.Materials and Methods:A total of 793 subjects were accrued, including 360 adult allo-HCT donors (discovery cohort; n=240, and validation cohort; n=120), 382 HCT allo- recipients and 50 healthy individuals. All subjects of the discovery cohort and healthy individuals were analyzed for 22 single nucleotide variants located in the regulatory and/or exonic regions of 13 cytokine or cytokine receptor gene using sequence-specific primer based assay. All subjects of the validation cohort were genotyped SNPs located in the IL10 and IL1R gene promoter region by direct sequencing. . CMV specific immune response was assessed by stimulating PBMNCs from healthy individuals with CMV lysate and CMV peptide-pp65 followed by enumeration of IFN-g producing and CD107a expressing (degranulating) MNCs, T cells and their subsets.Results:Cytokine gene variants of donors and not that of recipients appear to influence post HCT complications. Two of these variant system- SNPs leading to low production of IL10 and high production of IL1R showed strong correlation with GVHD and posttransplant CMV infections respectively.Allo-HCT recipients who have received graft from donors carrying low IL-10 producing gene variants (AA) at IL-10 -1082G/A and (ATA/ATA at IL-10 -1082G/A, -819C/T and -592C/A) have high incidence of significant GVHD (defined as grade II-IV acute GVHD and/or chronic GVHD requiring systemic immunotherapy) (P=0.008, HR= 2.8, Figure 1; and P=0.03, HR= 1.8 respectively) compared to recipients who have received graft from donors carrying high producer IL-10 gene variants.Allo-HCT recipients who have received graft from donors carrying low IL-1R producing genotype (-1970 CC) showed high incidence of CMV reactivation (p=0.01; HR=2.1, Figure 2) in comparison to the HCT recipients receiving grafts from donors carrying high IL1-R producing genotype (-1970 GG). Further, the in-vitro culture analysis in healthy individuals showed that IL1R+ cells have 3-5 fold stronger anti-CMV immune responses (IFNg+ and/or CD107a+ cells) in comparison to IL1R- cells.Conclusions:Genetic predisposition to low IL-10 production is a strong predictor of GVHD while that tohigh production of IL-1R confers strong protection against CMV reactivation after allogeneic HCT. Thefindings implicate the importance of prospective assessment of cytokine gene variants to improve allogeneic HCT donor selection. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

OR45: INTERLEUKIN-1 RECEPTOR (IL1-R): ROLE IN ANTI-CYTOMEGALOVIRUS (CMV) IMMUNE RESPONSE AND PROTECTION AGAINST CMV REACTIVATION AFTER ALLOGENEIC HEMATOPOETIC CELL TRANSPLANTATION

Aim An immunocompromised state after hematopoietic cell transplantation (HCT) leads to a deficient anti-cytomegalovirus (CMV) immune response. This often culminates into episodes of posttransplant CMV reactivation and complications. Cytokines and their receptors act as chief mediators of anti-viral immune response. Genetic control of cytokine production is evidenced by polymorphisms in cytokine gene regulatory regions resulting in low, moderate, or high cytokine production profiles. In the present investigation, we assessed whether gene variants of cytokine or their receptors influences post HCT CMV complications and anti-CMV immune response. Methods A total of 240 allogeneic HCT donors and 50 healthy individuals were genotyped for 22 single nucleotide variants located in the regulatory and/or exonic regions of 13 cytokine or cytokine receptor genes. CMV specific immune response was assessed by stimulating PBMNCs from healthy individuals with CMV lysate and CMV peptide-pp65 followed by enumeration of IFN-γ producing and CD107a expressing (degranulating) MNCs, T cells and their subsets. Results Allogeneic HCT recipients receiving grafts from donors carrying low IL-1R producing genotype (-1970 CC) had high incidence of CMV reactivation (p = 0.01; Hazard ratio = 2.1, Fig. 1 ) in comparison to the HCT recipients receiving grafts from donors carrying high IL1-R producing genotype (-1970 TT). Further, the analysis in healthy individuals showed that IL1R+ cells have 3-5-fold stronger anti-CMV immune response (IFNγ+ and/or CD107a+ cells) in comparison to IL1R- cells. Conclusions High IL-1R producing gene variants confer strong protection against CMV reactivation after allogeneic HCT. The conclusions are further supported by the finding that anti-CMV immune response is dominated by IL1R+ MNCs.

Induction of innate immune signatures following polyepitope protein-glycoprotein B-TLR4&9 agonist immunization generates multifunctional CMV-specific cellular and humoral immunity

Recent studies have suggested that a successful subunit human cytomegalovirus (CMV) vaccine requires improved formulation to generate broad-based anti-viral immunity following immunization. Here we report the development of a non-live protein-based vaccine strategy for CMV based on a polyepitope protein and CMV glycoprotein B (gB) adjuvanted with TLR4 and/or TLR9 agonists. The polyepitope protein includes contiguous multiple MHC class I-restricted epitopes with an aim to induce CD8+ T cell immunity, while gB is an important target for CD4+ T cell immunity and neutralizing antibodies. Optimal immunogenicity of this bivalent non-live protein vaccine formulation was dependent upon the co-administration of both the TLR4 and TLR9 agonist, which was associated with the activation of innate immune signatures and the influx of different DC subsets including plasmacytoid DCs and migratory CD8-DEC205+CD103-CD326- langerin-negative dermal DCs into the draining lymph nodes. Furthermore these professional antigen presenting cells also expressed IL-6, IL-12p70, TNFα, and IFNα which play a crucial role in the activation of adaptive immunity. In summary, this study provides a novel platform technology in which broad-based anti-CMV immune responses upon vaccination can be maximized by co-delivery of viral antigens and TLR4 and 9 agonists which induce activation of innate immune signatures and promote potent antigen acquisition and cross-presentation by multiple DC subsets.

Reduced IL-7 Responsiveness Defined by Signal Transducer and Activator of Transcription 5 Phosphorylation in T Cells May Be a Marker for Increased Risk of Developing Cytomegalovirus Disease in Patients after Hematopoietic Stem Cell Transplantation

Cytomegalovirus (CMV) reactivation may lead to CMV disease associated with high morbidity and mortality in patients after hematopoietic stem cell transplantation (HSCT); the identification of clinically relevant markers may aid in the identification of patients at increased risk for developing CMV-associated complications. We evaluated the phosphorylation of signal transducer and activator of transcription 5 (STAT5) in CD4+ T cells, CD8+ T cells, and TCRγδ T cells in response to stimulation with IL-7 or IL-2 after HSCT by analyzing blood samples taken monthly 1 to 6 months after HSCT. Patients were monitored weekly with a quantitative PCR from the time of engraftment for CMV viral load in whole blood until at least day 100 after HSCT. We identified a correlation between clinical outcome regarding CMV replication and the ability to respond to IL-7 and IL-2 defined by STAT5 phosphorylation (pSTAT5). Patients with recurrent or prolonged CMV replications had significantly lower pSTAT5 upon stimulation of T cells with either IL-7 or IL-2 at time points 1 through 3 than those without CMV replication (P < .05). This was also found after stimulation of CD8+ T cells at time point 2 (P < .05). We conclude that reduced responses to IL-7, reflected by pSTAT5, may represent a clinically relevant functional biomarker for individuals at increased risk for CMV reactivation; our data may also aid in designing better strategies to improve anti-CMV immune responses without increasing the risk of developing graft-versus-host disease.

Distinct IFN-Gamma Dependent Effect of the TLR5 Agonist, Flagellin On NK and CD4+ T Cell Immunity Against CMV

AbstractAbstract 255 Background:Opportunistic CMV infection remains a major clinical complication in allogeneic BMT (allo-BMT) recipients. We previously published that peri-transplant treatment of highly purified flagellin, a TLR5 agonist extracted from S. typhimurium reduced GvHD in lethally irradiated allo-BMT recipients by reducing early activation and proliferation of donor T cells. Flagellin-treated recipients had enhanced lymphoid immune reconstitution and were protected from lethal MCMV infection. CMV initiates innate immunity through the activation of Toll-like receptors (TLR) 9, 3 and 2 but direct enhancement of anti-CMV immunity through TLR5 has not been studied. Methods:To further explore the effects of a TLR5 agonist on anti-CMV immunity, we infected flagellin-treated C57BL/6 (B6), TLR5KO and Myd88KO mice (B6 background) with sub lethal dose (1×105pfu/mouse i.p) of MCMV. Anti-CMV immunity of NK and T-cells were determined by measuring surface activation markers on the NK and T-cells. Flagellin-treated and MCMV infected IL-12KO, IFN-γRKO and IDO KO mice were used to study the role of IL-12, IFN-γ and IDO on anti-CMV immunity. The relationship between the MCMV lethality and flagellin signaling in hematopoietic or epithelial tissues expressing TLR5 was further investigated by generating radiation chimeras, using WT mice engrafted with TLR5 KO bone marrow (BM) and TLR5 KO mice engrafted with WT B6 BM. Chimeric recipients with WT or TLR5KO hematopoietic cells (or the reverse) were then treated with flagellin or PBS and infected with a sub lethal dose of MCMV infection. Results:Without MCMV infection, flagellin treatment significantly increased numbers of NK cells (p= 0.001) and KLRG1+ NK cells (p=0.009) in WT B6 mice compared with the PBS-treated control mice. Following MCMV infection (day 3), there were significantly increased numbers of splenic NK cells (p= 0.01) and KLRG1+ NK cells (p=0.0008) in flagellin-treated mice compared with the control mice. Flagellin-treatment also enhanced viral clearance, tending to decrease liver viral loads in WT B6 mice. In contrast, MCMV-infected and flagellin-treated TLR5 KO or Myd88KO mice had significantly increased liver viral loads (p=00001) compared with the flagellin- and PBS-treated B6 mice on day 3 post MCMV infection. While flagellin-treatment increased the numbers and activation status of NK cells, it decreased the numbers of activated (CD69+, ICOS+ and KLRG1+) CD4+ T cells on day 3 post MCMV infection. On day 10 post MCMV infection, enhanced numbers of splenic CD8+ T cells and CMV-specific tetramer+ CD8+ T cells (p<0.05) were detected in flagellin-treated mice compared with controls. The numbers of splenic CD4+ T cells increased >2 fold (p= 0.008) associated with significantly increased numbers of splenic CD4+CD25+ Foxp3+ regulatory T cells (p<0.05) on day 10 post MCMV infection in flagellin-treated mice compared with the control mice. Flagellin-induced anti-CMV immunity required IFN-γ signaling as flagellin-treated IFN-γRKO mice had markedly increased susceptibility to MCMV infection whereas flagellin-treated IL-12 KO and IDO KO mice had significantly reduced liver viral loads compared to the PBS-treated IL-12 KO and IDO KO mice on day 10 post MCMV infection. To explore interaction of flagellin with TLR5+ host hematopoietic cells and TLR5+host gut epithelium on CMV immunity, we next infected radiation chimeras expressing either TLR5 on hematopoietic cells or on gut epithelium with sub lethal i.p dose of MCMV. Flagellin-treated TLR5 KO BM → B6 radiation chimeras (TLR5+ host epithelium) had increased mortality compared with other groups following MCMV infection indicating that TLR5+ hematopoietic cells are required for protective immunity against CMV infection.In summary, flagellin caused selective early increased activation of NK cells and reduced activation of CD4+ T cells in presence of MCMV infection. The protective effect of flagellin on anti-CMV immune responses are IFN-γ dependent but independent of IL-12 and IDO. The TLR5+ hematopoietic cells are required for the protective immunity against CMV infection. These data suggest the clinical importance of flagellin in modulating both innate and adaptive immunity against CMV infection and also as an alternative GvHD prophylaxis in allo-BMT recipients. Disclosures:No relevant conflicts of interest to declare.

Role of IL-15 in the Stimulation of Human CD8+ CMV-Reactive Human T Cells in a Hu-PBL Mouse Model Preconditioned with Self-Differentiated Human Dendritic Cells Programmed with Lentiviral Vectors

Abstract 2966IL-15 is known to be a key factor in the homeostatic stimulation of T cells and maintenance of central memory lymphocytes involved in long-lasting immune responses. In addition, IL-15 may enhance NK cell function to protect against infection and relapse after allo-HCT. Thus, IL-15 ectopic overexpression provided by dendritic cell (DC) immunotherapy after bone marrow transplantation or donor lymphocyte infusion may direct prompt and durable immune responses against leukemia relapse or viral reactivation.Here, we show that overnight transduction of monocytes with tricistronic lentiviral vectors (LV) co-expressing GM-CSF/IL-4/IL-15 induced their differentiation into highly viable DC that expressed the major DC markers CD209 (75%), CD86 (95%), HLA-DR (90%) for up to 21 days in culture. These so-called SmartDC/IL-15 maintained stable secretion of several inflammatory and Th1 cytokines (TNF α, MCP-1, IL-1 β, IL-8). A single in vitro priming of T cells obtained from CMV seropositive donors with autologous SmartDC/IL-15 loaded with CMV-pp65 peptides led to expansion of higher frequencies pp65 tetramer-positive CTLs as compared with SmartDC that were not engineered to express IL-15 (26.5% vs. 16.5% respectively). Importantly, IL-15 expression in Smart-DCs led to the enrichment of T cells with effector memory (80% vs. 65%) and central memory (8% vs. 6%) as compared with SmartDC controls. SmartDCs/IL-15 presented high viability in vivo (>6 weeks) in immune deficient NOD.Rag1—/—.IL2r γ—/— (NRG) mice. We further evaluated the effects of SmartDCs/IL-15 on lymphatic and anti-CMV immune reconstitution in a humanized peripheral blood lymphocyte (Hu-PBL) mouse model. NRG mice preconditioned with unloaded or pp65-loaded SmartDCs/IL-15 were infused 7 days later with autologous luciferase-labeled T cells for optical imaging analysis. At day 21 after fLUC-T cells infusion, mice injected with pp65-loaded SmartDCs/IL-15 showed higher bioluminescence signal from expanded T cells in spleens (3-fold) and injection site (2-fold) as compared with control SmartDCs. Human T cells recovered from spleens of SmartDC-IL15 showed enhanced frequencies of effector memory (58% vs. 30%) and central memory (5.6% vs. 4%) phenotype as compared with SmartDC controls.Remarkably, bioluminescent T cells co-localized with the site of SmartDC/IL-15 injection in a pp65-dependent manner and enhanced the bio-distribution of human T cells towards other rudimentary lymphatic structures found in NRG mice. Anti-pp65 reactivity in vivo was confirmed by flow cytometry analyses by tetramer staining in peripheral blood. Thus, SmartDCs/IL-15 induced accelerated anti-CMV immune responses and triggered lymphatic regeneration and repopulation in vivo, which may be explored clinically to enhance homeostatic and antigen-dependent immune reconstitution after transplantation. Disclosures:No relevant conflicts of interest to declare.

The role of Vδ2-negative γδ T cells during cytomegalovirus reactivation in recipients of allogeneic stem cell transplantation

AbstractReactivation of cytomegalovirus (CMV) remains a serious complication after allogeneic stem cell transplantation, but the role of γδ T cells is undefined. We have studied the immune reconstitution of Vδ2negative (Vδ2neg) γδ T cells, including Vδ1 and Vδ3 subsets and Vδ2positive (Vδ2pos) γδ T cells in 40 patients during the first 24 months after stem cell transplantation. Significant long-term expansions of Vδ2neg but not Vδ2pos γδ T cells were observed during CMV reactivation early after transplantation, suggesting direct involvement of γδ T cells in anti-CMV immune responses. Similarly, significantly higher numbers of Vδ2neg γδ T cells were detected in CMV-seropositive healthy persons compared with seronegative donors; the absolute numbers of Vδ2pos cells were not significantly different. The expansion of Vδ2neg γδ T cells appeared to be CMV-related because it was absent in CMV-negative/Epstein-Barr virus-positive patients. T-cell receptor-δ chain determining region 3 spectratyping of Vδ2neg γδ T cells in healthy subjects and patients showed restricted clonality. Polyclonal Vδ2neg cell lines generated from CMV-seropositive healthy donors and from a recipient of a graft from a CMV-positive donor lysed CMV-infected targets in all cases. Our study shows new evidence for role of γδ T cells in the immune response to CMV reactivation in transplantation recipients.

Variable viral clearance despite adequate ganciclovir plasma levels during valganciclovir treatment for cytomegalovirus disease in D+/R- transplant recipients

BackgroundValganciclovir, the oral prodrug of ganciclovir, has been demonstrated equivalent to iv ganciclovir for CMV disease treatment in solid organ transplant recipients. Variability in ganciclovir exposure achieved with valganciclovir could be implicated as a contributing factor for explaining variations in the therapeutic response. This prospective observational study aimed to correlate clinical and cytomegalovirus (CMV) viral load response (DNAemia) with ganciclovir plasma concentrations in patients treated with valganciclovir for CMV infection/disease.MethodsSeven CMV D+/R- transplant recipients (4 kidney, 2 liver and 1 heart) were treated with valganciclovir (initial dose was 900-1800 mg/day for 3-6.5 weeks, followed by 450-900 mg/day for 2-9 weeks). DNAemia was monitored by real time quantitative PCR and ganciclovir plasma concentration was measured at trough (Ctrough) and 3 h after drug administration (C3h) by HPLC.ResultsFour patients presented with CMV syndrome, two had CMV tissue-invasive disease after prophylaxis discontinuation, and one liver recipient was treated pre-emptively for asymptomatic rising CMV viral load 5 weeks post-transplantation in the absence of prophylaxis. CMV DNAemia decreased during the first week of treatment in all recipients except in one patient (median decrease: -1.2 log copies/mL, range: -1.8 to 0) despite satisfactory ganciclovir exposure (AUC0-12 = 48 mg·h/L, range for the 7 patients: 40-118 mg·h/L). Viral clearance was obtained in five patients after a median of time of 34 days (range: 28-82 days). Two patients had recurrent CMV disease despite adequate ganciclovir exposure (65 mg·h/L, range: 44-118 mg·h/L).ConclusionsValganciclovir treatment for CMV infection/disease in D+/R- transplant recipients can thus result in variable viral clearance despite adequate ganciclovir plasma concentrations, probably correlating inversely with anti-CMV immune responses after primary infection.

Anti CMV Immunotherapy with Donor Antigen Specific Lymphocytes Selected with GMP HLA Class 1 Tetramer Reagents.

Abstract 4492▪Patients undergoing allogeneic haematopoietic stem cell transplants frequently develop infections that are responsible for a high rate of morbidity and mortality during first 6 months post transplantation. The occurrence of CMV infection might reach 70% to 80% of transplanted patients; this rate is increased in HSC transplantations conditioned by in vivo and/or in vitro immunosuppressive treatment. Prophylactic treatment of CMV infection by ganciclovir or foscarnet, though proven to be efficient, faces complications such as myelotoxicity, nephrotoxicity and/or the development of virus resistance. The control of CMV reactivation is achieved in healthy individuals by CD4 +and CD8 +T lymphocytes specifically directed against CMV. After allograft, it has been demonstrated that restoration of anti-CMV immune responses is efficient but all the more delayed when conditioning is heavier. Furthermore, it has been demonstrated that the transfer of donor T lymphocytes specifically directed against CMV was able to prevent and even more to control viral reactivation. Several strategies have been described to generate anti-CMV CD8+ cytotoxic T lymphocytes (CTL) in vitro, by using as antigen-presenting cells (APCs) CMV-infected fibroblasts (Walter, NEJM 1995, 333: 1038), or EBV-transformed B cells retrovirally transduced to express CMV pp65 protein (Sun, Blood 1999, 94: 3242). However, the length of process and the presence of potentially dangerous viral particles in these trials limit their clinical utilisation. Recent progresses in technology resulting from the HLA tetramer technique have enabled to develop direct and fast enrichments of anti-CMV CTL sufficient to inject one or several doses of these lymphocytes (Cobbod abstract 155, ASH 2004).In order to reach the regulatory requirements for ancillary products to be used in an anti CMV immunotherapy protocol, a tetramer selection system and its associated production process compatible with cGMP has been developed. The developed manufacturing process (Protein Expert) is based on: - the E. coli production of the different components, HLA201 heavy chain and β2 microglobulin as well as streptavidin, the chemical synthesis of the pp65 peptide; - the production and biotinylation of the monomers-the production of the tetramers using recombinant streptavidin covalently linked to magnetic beads (Miltenyi Biotec ®) compatible with clinical applications.Seven immunomagnetical selections on MS column of HLA A2-pp65 positive tetramer cells from healthy blood donors were performed with these GMP reagents. Respective means of CD8 tetramer +cells in starting and selected fractions were 2.5 %(+/-3.1) and 99 %(+/-0.6).ExptStartSelectedCD8 tetramer+ cells (%)CD8 tetramer+ cells (10e5))CD3 + cells (%)CD8 tetramer+ cells (%)CD8 tetramer+ cells (10e5)CD8 tetramer+ cells yield (%))10,541,3189991,7311728,457,46999912,216230,320,5793990,6410540,391,3791991,6811253,358,599899,55,846764,472,798982,48670,140,27999,80,29116Mean2,53,292,499,03,5109,3SD3,13,47,10,64,229,6Starting and selected cells were restimulated with CMV or irrelevant peptide and evaluated for interferon gamma secretion (respectively 5.4%+/5.2 and 71.8%+/-14.7) and CD107a expression (6.1%+/-2.9 and 56.2%+/-32.7).The GMP reagents used for selection demonstrated a very good efficacy for enrichment of HLA A2-pp65 positive tetramer CD8 cells with high purity and yield. Selected cells were shown to be antigen specific and cytotoxic. This procedure scaled up on Clinimacs device could provide in a very short time, sufficient CD8 tetramer positive cells for very selective adoptive immunotherapy after stem cells transplant. Disclosures:No relevant conflicts of interest to declare.

Long-term expansion of effector/memory Vδ2− γδ T cells is a specific blood signature of CMV infection

AbstractThe ability of human γδ T cells to develop immunologic memory is still a matter of debate. We previously demonstrated the involvement of Vδ2− γδ T lymphocytes in the response of immunosuppressed organ recipients to cytomegalovirus (CMV). Here, we demonstrate their ability to mount an adaptive immune response to CMV in immunocompetent subjects. Vδ2− γδ T-cell peripheral blood numbers, repertoire restriction, and cytotoxicity against CMV-infected fibroblasts were markedly increased in CMV-seropositive, compared with CMV-seronegative, healthy persons. Whereas Vδ2− γδ T cells were found as naive cells in CMV− patients, they virtually all exhibited the cytotoxic effector/memory phenotype in CMV+ patients, which is also observed in transplanted patients challenged with CMV. This long-term complete remodeling of the Vδ2− γδ T-cell population by CMV predicts their ability to exhibit an adaptive anti-CMV immune response. Consistent with this, we observed that the secondary response to CMV was associated with a faster γδ T-cell expansion and a better resolution of infection than the primary response. In conclusion, the increased level of effector-memory Vδ2− γδ T cells in the peripheral blood is a specific signature of an adaptive immune response to CMV infection of both immunocompetent and immunosuppressed patients.

348: Chronic GvHD decreases anti-CMV immune responses in allogeneic BMT: Defect in T-cell homing

Backgound: The major complications of allogeneic bone marrow transplantation are relapse, acute and chronic Graft-vs-Host Disease (aGvHD; cGvHD) and opportunistic infections, especially cytomegalovirus (CMV). In this study, we investigated the effect of cGvHD on susceptibility to murine CMV infection following allogeneic BMT, focusing on the contribution of cGvHD to functional immunodeficiency.