Giovanni Almanzar

Long-Term Cytomegalovirus Infection Leads to Significant Changes in the Composition of the CD8+ T-Cell Repertoire, Which May Be the Basis for an Imbalance in the Cytokine Production Profile in Elderly Persons

ABSTRACT In spite of the present belief that latent cytomegalovirus (CMV) infection drives CD8+ T-cell differentiation and induces premature immune senescence, no systematic studies have so far been performed to compare phenotypical and functional changes in the CD8+ T-cell repertoire in CMV-infected and noninfected persons of different age groups. In the present study, number, cytokine production, and growth potential of naïve (CD45RA+ CD28+), memory (CD45RA− CD28+), and effector (CD45RA+ CD28− or CD45RA− CD28−) CD8+ T cells were analyzed in young, middle-aged, and elderly clinically healthy persons with a positive or negative CMV antibody serology. Numbers and functional properties of CMVpp65495-503-specific CD8+ T cells were also studied. We demonstrate that aging as well as CMV infection lead to a decrease in the size of the naïve CD8+ T-cell pool but to an increase in the number of CD8+ effector T cells, which produce gamma interferon but lack substantial growth potential. The size of the CD8+ memory T-cell population, which grows well and produces interleukin-2 (IL-2) and IL-4, also increases with aging, but this increase is missing in CMV carriers. Life-long latent CMV infection seems thus to diminish the size of the naïve and the early memory T-cell pool and to drive a Th1 polarization within the immune system. This can lead to a reduced diversity of CD8 responses and to chronic inflammatory processes which may be the basis of severe health problems in elderly persons.

CD25-Expressing CD8+ T Cells Are Potent Memory Cells in Old Age

We have recently described an IL-2/IL-4-producing CD8+CD25+ nonregulatory memory T cell population that occurs in a subgroup of healthy elderly persons who characteristically still have a good humoral response after vaccination. The present study addresses this specific T cell subset and investigates its origin, clonal composition, Ag specificity, and replicative history. We demonstrate that CD8+CD25+ memory T cells frequently exhibit a CD4+CD8+ double-positive phenotype. The expression of the CD8 αβ molecule and the occurrence of signal-joint TCR rearrangement excision circles suggest a thymic origin of these cells. They also have longer telomeres than their CD8+CD25− memory counterparts, thus indicating a shorter replicative history. CD8+CD25+ memory T cells display a polyclonal TCR repertoire and respond to IL-2 as well as to a panel of different Ags, whereas the CD8+CD25− memory T cell population has a more restricted TCR diversity, responds to fewer Ags, and does not proliferate in response to stimulation with IL-2. Molecular tracking of specific clones with clonotypic primers reveals that the same clones occur in CD8+CD25+ and CD8+CD25− memory T cell populations, demonstrating a lineage relationship between CD25+ and CD25− memory CD8+ T cells. Our results suggest that CD25-expressing memory T cells represent an early stage in the differentiation of CD8+ cells. Accumulation of these cells in elderly persons appears to be a prerequisite of intact immune responsiveness in the absence of naive T cells in old age.

Tumor-Associated Embryonic Antigen-Expressing Vaccines that Target CCR6 Elicit Potent CD8+ T Cell-Mediated Protective and Therapeutic Antitumor Immunity

Despite its potency, the wider use of immunotherapy for B cell malignancies is hampered by the lack of well-defined tumor-specific Ags. In this study, we demonstrate that an evolutionarily conserved 37-kDa immature laminin receptor protein (OFA-iLRP), a nonimmunogenic embryonic Ag expressed by a variety of tumors, is rendered immunogenic if targeted to the APCs using the CCR6 ligands MIP3α/CCL20 and mDF2β. The CCR6 targeting facilitated efficient Ag cross-presentation and induction of tumor-neutralizing CTLs. Although the Ag targeting alone, without activation of dendritic cells (DCs), is proposed to induce tolerance, and MIP3α does not directly activate DCs, the MIP3α-based vaccine efficiently induced protective and therapeutic antitumor responses. The responses were as strong as those elicited by the OFA-iLRP fusions with moieties that activated DCs and Th1-type cytokine responses, mDF2β, or mycobacterial Hsp70 Ag. Although the same cDNA encodes the dimerized high-affinity mature 67-kDa mLRP that is expressed in normal tissues to stabilize the binding of laminin to cell surface integrins, the vaccines expressing OFA-iLRP elicited long-term protective CD8+ T cell-mediated memory responses against syngeneic B cell lymphoma, indicating the potential application of these simple vaccines as preventive and therapeutic formulations for human use.

Oxidative stress can alter the antigenicity of immunodominant peptides

APCs operate frequently under oxidative stress induced by aging, tissue damage, pathogens, or inflammatory responses. Phagocytic cells produce peroxides and free‐radical species that facilitate pathogen clearance and can in the case of APCs, also lead to oxidative modifications of antigenic proteins and peptides. Little information is available presently about the consequences of such modifications on the immune response. To model oxidative modification of an immunodominant antigenic peptide, we oxidized the methionine residue of the human CMV pp65495–503 (NLVPMVATV) peptide. Such modifications of an antigenic peptide can affect MHC binding or TCR recognition. Using binding and dissociation assays, we demonstrate that oxidative modification of the CMVpp65495–503 peptide leads to a decreased binding of the pMHC complex to the TCR, whereas binding of the peptide to the MHC class I molecule is not impaired. Additionally, we show that CD8+ T cells have a decreased proliferation and IFN‐γ production when stimulated with oxidized CMVpp65495–503 peptide. Spectratyping the antigen‐binding site of the TCR of responding T cells demonstrates that the CMVpp65495–503 and the CMVoxpp65495–503 peptides preferentially stimulate BV8 T cells. Sequencing of this dominant BV family reveals a highly conserved CDR3 amino acid motif, independent of the mode of stimulation, demonstrating the recruitment of the same T cell clonotypes. Our results suggest that oxidative modification of antigenic peptides may affect T cell responses severely by binding T cell clones with different affinity. This may lead to an altered immune response against infectious agents as well as against tumor or autoantigens under oxidative stress conditions.

Immunodominant peptides from conserved influenza proteins--a tool for more efficient vaccination in the elderly?

ZusammenfassungInfluenza-spezifische zytotoxische CD8+ T-Zellen stellen einen wesentlichen Bestandteil bei der Überwindung einer Infektion dar, insbesondere in Hochrisikogruppen wie älteren Personen. Eine Aktivierung dieser Zellen durch eine Impfung wäre deshalb eine wichtige Voraussetzung um einen besseren Schutz für diese Altersgruppe zu erreichen. Deshalb haben wir die Häufigkeit, den Phänotyp und die Funktion von Influenza M158–66 Peptid-spezifischen CD8+ T-Zellen in drei verschiedenen Altersgruppen (27 ± 1 Jahre; 46 ± 3 Jahre und 72 ± 2 Jahre) ex vivo und nach in vitro Stimulation untersucht. Bei Personen ab einem Alter von 38 Jahren stellten wir eine geringere Anzahl von M158–66-spezifischen CD8+ T-Zellen fest. Diese Zellen exprimierten CD28, CD62L und waren entweder CD45RAlowCD45ROlow oder CD45RA-CD45RO+, produzierten aber kein Perforin. Kein Unterschied zeigte sich beim Phänotyp von Influenza-spezifischen CD8+ T-Zellen zwischen den drei Altersgruppen. Trotz der zu Beginn geringen Anzahl von M158–66-spezifischen CD8+ T-Zellen in älteren Personen, konnte diese Population nach in vitro Stimulation mit dem M158–66-Peptid signifikant vergrößert werden. Diese M158–66-spezifische CD8+ T-Zellen produzierten Perforin und hatten einen CD45RO+CD28+CD62L+/− Phänotyp. Unsere Ergebnisse zeigen, dass trotz einer geringen Anzahl M158–66-spezifischer CD8+ T-Zellen bei älteren Personen, diese Zellen nach in vitro Stimulation mit einem Peptid gut expandieren und in Perforin-produzierende Effektor-T-Zellen differenzierbar sind. M158–66-Peptid oder andere immundominante Peptide aus konservierten Influenzaproteinen könnten somit einen wichtigen Bestandteil zukünftiger Influenzaimpfstoffe darstellen um älteren Personen einen verbesserten Schutz vor Influenza zu bieten, insbesondere beim Auftreten einer Influenza Pandemie.SummaryInfluenza-specific CD8+ T cells are important for the clearance of infection especially in high risk groups such as elderly persons. Activation of these cells by immunization might therefore be a useful tool for a better protection of this specific age group. We therefore analyzed the frequency, phenotype and function of CD8+ T cells with specificity to the influenza M158–66 peptide in young, middle-aged and elderly persons ex vivo and after in vitro stimulation. Significantly lower numbers of M158–66-specific CD8+ T cells were detected in the middle-aged and elderly compared to young donors. M158–66-specific CD8+ T cells were either CD45RAlowCD45ROlow or CD45RA-CD45RO+, expressed CD28 and CD62L and did not produce perforin. There was no difference in the phenotype of influenza-specific CD8+ T cells between the three age groups. Despite the initially low numbers of M158–66-specific CD8+ T cells in the older age groups, these cells could be expanded in vitro following peptide stimulation. They also acquired a CD45RO+CD28+ CD62L+/− phenotype and produced perforin. Our results demonstrate that although initially low in number, M158–66-specific CD8+ T cells from elderly persons can be propagated and differentiated into perforin producing effector cells upon appropriate stimulation. M158–66 peptide or other immunodominant peptides derived from conserved influenza proteins could therefore be useful in future influenza vaccines in order to render elderly persons better protected against disease, in particular in the case of an influenza pandemia.

Increased proportions of functionally impaired regulatory T cell subsets in systemic sclerosis

Treg abnormalities have been implicated in the pathogenesis of systemic sclerosis (SSc). Treg subpopulations and their cytokines, IL-10 and TGF-β in the peripheral blood of early stage SSc patients were investigated. We hypothesized that epigenetically regulated methylation of the FOXP3 promoter and enhancer regions are altered in Tregs of SSc patients, which might be involved in the T cell imbalance. CD4+CD25+Foxp3+CD127- Treg cells were significantly elevated in patients with diffuse cutaneous SSc and in patients with anti-Scl-70/RNA-Pol-III autoantibody positivity and with lung fibrosis. Increased CD62L+ Treg cells were present in all SSc subgroups. The production of immunosuppressive cytokines by both CD127- and CD62L+ Tregs was diminished. We observed reduced methylation of Treg specific FOXP3 enhancer regions, and elevated FOXP3 gene expression in active SSc cases with negative correlation in the frequency of CD62L+IL-10+ Tregs. Our data indicate an inappropriate distribution and cytokine production of Treg cells in early form SSc.

Hypermethylation of FOXP3 Promoter and Premature Aging of the Immune System in Female Patients with Panic Disorder

Immunological abnormalities associated with pathological conditions, such as higher infection rates, inflammatory diseases, cancer or cardiovascular events are common in patients with panic disorder. In the present study, T cell receptor excision circles (TRECs), Forkhead-Box-Protein P3 gene (FOXP3) methylation of regulatory T cells (Tregs) and relative telomere lengths (RTLs) were investigated in a total and subsamples of 131 patients with panic disorder as compared to 131 age- and sex-matched healthy controls in order to test for a potential dysfunction and premature aging of the immune system in anxiety disorders. Significantly lower TRECs (p = 0.004) as well as significant hypermethylation of the FOXP3 promoter region (p = 0.005) were observed in female (but not in male) patients with panic disorder as compared to healthy controls. No difference in relative telomere length was discerned between patients and controls, but significantly shorter telomeres in females, smokers and older persons within the patient group. The presently observed reduced TRECs in panic disorder patients and FOXP3 hypermethylation in female patients with panic disorder potentially reflect impaired thymus and immunosuppressive Treg function, which might partly account for the known increased morbidity and mortality of anxiety disorders conferred by e.g. cancer and cardiovascular disorders.

Mycobacterial heat shock protein 65 (mbHSP65)-induced atherosclerosis: Preventive oral tolerization and definition of atheroprotective and atherogenic mbHSP65 peptides

Objective: The aim of this study was to identify atherogenic and atheroprotective peptides of bacterial HSP60 [taking mycobacterial HSP65 (mbHSP65) as a potent paradigmatic representative] that could be used as candidates for an orally applied tolerizing vaccine against atherosclerosis. Methods: ApoE � /� mice were immunized with mbHSP65 protein or peptides, given mbHSP65 orally and then kept either on chow or high cholesterol diet. Atherosclerosis was assessed by en face and immunohistological analysis. Anti-HSP autoantibodies were detected by ELISA. The number and in vitro suppressive function of splenic and lymph node regulatory T cells (Tregs) were analyzed by flow cytometry. Specific T cell reactivity against mbHSP65 protein or peptides was assessed by proliferation assay.

Contribution of CD8+ T cells to inflammatory cytokine production in systemic sclerosis (SSc)

Abstract Only limited attention has been paid to the role of CD8 + T cells in the etiopathogenesis and progression of systemic sclerosis (SSc). CD8 + T cells may have autoantigen-specific and pro-inflammatory but also immunomodulatory properties. To investigate the differentiation of CD8 + T cells, staining of cell surface factors and of chemokine receptors were performed. In addition, the cytokine-producing ability of circulating CD8 + T cells and their sensitivity to suppression by regulatory T cells (Tregs) were compared between patients with diffuse (dcSSc) or limited cutaneous SSc (lcSSc) and healthy individuals. We identified CD8 + T cells as producers of pro-inflammatory type-2 cytokines with a significant contribution of memory CD8 + T cells. Memory CD8 + T cells of SSc patients stayed unaltered after suppression with autologous Tregs. Expression of chemokine receptors was significantly correlated with intracellular cytokine production in CD8 + T cells with a clear dichotomy of type 1 and type 2 cytokines. High levels of intracellular cytokines, such as interleukin-(IL)-4, IL-13 and tumor-necrosis-factor-alpha (TNFalpha) were positively associated with the presence of Scl-70 or anti-centromere antibodies and negatively with the administration of glucocorticoids. Administration of glucocorticoids was positively associated with higher IFNgamma production. Lack of anti-centromere antibodies and therapy with methotrexate were positively associated with higher intracellular IL-10 production. CD8 + T cells may significantly contribute to inflammation in SSc. Our findings suggest to not only focus on T helper cells in the development of therapeutic strategies but also to consider the role of CD8 + T cells in the etiopathogenesis and perpetuation of SSc.

Expression of 11beta-hydroxysteroid-dehydrogenase type 2 in human thymus.

11beta-hydroxysteroid-dehydrogenase type 2 (11β-HSD2) is a high affinity dehydrogenase which rapidly inactivates physiologically-active glucocorticoids to protect key tissues. 11β-HSD2 expression has been described in peripheral cells of the innate and the adaptive immune system as well as in murine thymus. In absence of knowledge of 11β-HSD2 expression in human thymus, the study aimed to localize 11β-HSD2 in human thymic tissue. Thymic tissue was taken of six healthy, non-immunologically impaired male infants below 12months of age with congenital heart defects who had to undergo correction surgery. 11β-HSD2 protein expression was analyzed by immunohistochemistry and Western blot. Kidney tissue, peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVEC) were taken as positive controls. Significant expression of 11β-HSD2 protein was found at single cell level in thymus parenchyma, at perivascular sites of capillaries and small vessels penetrating the thymus lobuli and within Hassalls bodies. The present study demonstrates that 11β-HSD2 is expressed in human thymus with predominant perivascular expression and also within Hassalls bodies. To our knowledge, this is the first report confirming 11β-HSD2 expression at the protein level in human thymic tissue underlining a potential role of this enzyme in regulating glucocorticoid function at the thymic level.