New advances in CMV and immunosenescence.

Immunosenescence, defined as the age-associated dysregulation and dysfunction of the immune system, is characterized by impaired protective immunity and decreased efficacy of vaccines. An increasing number of immunological, clinical and epidemiological studies suggest that persistent Cytomegalovirus (CMV) infection is associated with accelerated aging of the immune system and with several age-related diseases. However, current evidence on whether and how human CMV (HCMV) infection is implicated in immunosenescence and in age-related diseases remains incomplete and many aspects of CMV involvement in immune aging remain controversial. The attendees of the 4th International Workshop on "CMV & Immunosenescence", held in Parma, Italy, 25-27th March, 2013, presented and discussed data related to these open questions, which are reported in this commentary.

Intense antiextracellular adaptive immune response to human cytomegalovirus in very old subjects with impaired health and cognitive and functional status.

Human aging is characterized by expanded and altered adaptive immune responses to human CMV (HCMV). It is unclear whether this expansion has its origins in age-related homeostatic disturbances or viral reactivation, whether anti-CMV immune surveillance may still be effective, and what are the consequences of this expanded immune response for health and longevity. We conducted an observational cross-sectional study in groups of HCMV-seropositive subjects aged >or=65 y of variable health status to compare the intensity of Ab responses against HCMV with those against EBV and with CD4(+) and CD8(+) T cell proinflammatory effector responses directed to HCMV-derived pp65 and immediate-early protein 1 synthetic peptides. Ab responses to HCMV, but not to EBV, and anti-HCMV CD4(+), but not CD8(+), T cell responses were more intense in elderly subjects aged >or=85 y in poor health and were inversely correlated with markers of functional activity and cognitive function. Therefore, humoral and CD4(+) T cell anti-HCMV responses were specifically intensified in advanced aging associated with comorbidity and cognitive and functional impairments. Such a distinctive pattern of adaptive immunity indicates that immune responses targeting the extracellular phase of HCMV are increased in these elderly subjects and could represent an indirect effect of localized and undetectable HCMV reactivation. This study demonstrates that the oldest subjects in poor health with physical and mental impairment express intense functional immune responses to extracellular HCMV and suggests that they may be at risk for direct pathogenic effects by HCMV reactivation as well as indirect pathogenic effects linked to proinflammatory anti-HCMV effector responses.

Prospective study of natural killer cell phenotype in recurrent hepatitis C virus infection following liver transplantation.

BACKGROUND/AIMS: Graft re-infection invariably occurs after liver transplantation (OLT) for chronic hepatitis C and disease progression is unpredictable. We prospectively examined peripheral blood mononuclear cells (PBMC) subsets and natural killer (NK) cell receptors (NKRs) in patients with recurrent hepatitis C post-OLT. METHODS: PBMC were obtained at baseline and at different time points after OLT. NKRs were identified using monoclonal antibodies by flow cytometry. RESULTS: The proportions of NK, natural T (NT), total and gammadelta T cells were significantly reduced (p<0.01) 7 days post-transplant, probably as a result of graft repopulation. NKG2D+ NK cells were significantly higher compared with healthy controls (p<0.01), declined post-OLT and subsequently returned to baseline values. This, together with a progressive increase in the proportion of CD94/NKG2C+ NK cells over time (p< or = 0.01), appeared to be related to hepatitis C recurrence. There was a statistically significant correlation between expression of the natural cytotoxicity receptors (NCRs) and ALT (p<0.05), supporting the hypothesis that NK cells participate in the necroinflammatory process. CONCLUSIONS: The data are compatible with homing of immune cells to the liver allograft after surgery, most of which return to pre-OLT levels. HCV recurrence may cause variations in selected NKRs expression akin to other viral infections.

Combination of radiofrequency ablation and immunotherapy.

The enhancement of immune response against tumor antigens has shown some efficacy when used as a single mode of systemic treatment in patients with late stage disease. Novel strategies of active immunotherapy could be more effective in patients with less advanced disease who receive standard therapies supporting concomitant stimulation of the immune system. Radio-Frequency Ablation (RFA) is a minimally invasive technique which is used as standard local therapy of primary and metastatic liver tumors. Tumor ablation by RFA induces effects important for boosting anti-tumor immune responses. Tumor cell necrosis generates a permanent immunogenic source of tumor antigens. These antigens can be uptaken, processed and presented by dendritic cells for effective immunization without requirement for ex vivo antigen loading. Further immune activation can be originated by RFA through induction of heat shock proteins on tumor cells, acute phase response which causes the release of pro-inflammatory cytokines, and mobilization of antigen presenting cells and effector lymphocytes. Thus, RFA can facilitate immune responses to tumor antigens driven by active immunotherapy. On the other hand, immunotherapy is expected to eradicate residual disease after RFA and prevent tumor recurrences. The combination of RFA and active immunotherapy may well have synergistic effects for cancer treatment.

Elements related to heterogeneity of antibody-dependent cell cytotoxicity in patients under trastuzumab therapy for primary operable breast cancer overexpressing Her2.

Preliminary results from a pilot trial on trastuzumab's mechanism of action against operable breast tumors overexpressing Her2 suggested a role for antibody-dependent cell cytotoxicity (ADCC). To examine factors affecting ADCC intensity and variability, we extended this study to the phenotypic and functional analysis of circulating mononuclear cells in 18 patients. ADCC was induced by trastuzumab therapy in 15 of 18 patients (83%). Inability to develop ADCC in three patients did not depend on inadequate levels of trastuzumab because further increase in its concentration in vitro was ineffective. Rather, susceptibility to develop ADCC was fairly predicted by test with trastuzumab before therapy and was correlated to the number of lymphocytes coexpressing CD16 and CD56. Phenotypic analysis at the end of ADCC evaluating down-regulation of CD16, and up-regulation of CD69 and CD107a, confirmed that natural killer (NK) cells and CD56(+) T cells were involved in productive engagement of trastuzumab. Also, the killing efficiency of CD16(+) lymphocytes was influenced by 158 V/F polymorphism of Fc gamma RIII (CD16), whereas variations of CD247 on NK cells were consistent with trends between ADCC before and after therapy. Complete pathologic response was observed in one patient showing ADCC of outstanding intensity, whereas four cases of partial response showed intermediate ADCC; none of the three patients unable to mount ADCC had significant tumor regression. These data indicate that quantity and lytic efficiency of CD16(+) lymphocytes are major factors for ADCC induction by trastuzumab, and confirm that breast cancer responses to short-term trastuzumab monotherapy may depend on involvement of the ADCC mechanism.

Massive load of functional effector CD4+ and CD8+ T cells against cytomegalovirus in very old subjects.

A progressive, systemic, and low-grade proinflammatory status is one of the major characteristics of immunosenescence. Emerging data suggest a possible contribution of CMV, known to chronically infect a large proportion of humans, lifelong from newborns to centenarians. To test this hypothesis, we evaluated functional T cell responses to two CMV immunogenic proteins, pp65 and IE-1, in 65 chronically infected subjects aged 25-100 years. PBMC were stimulated with mixtures of peptides spanning the entire sequence of both proteins, and Ag specificity and magnitude of intracellular IFN-gamma- and TNF-alpha-positive cells were then analyzed within both CD4+ and CD8+ T cells. Results indicate that pp65 and, to a lesser extent, IE-1 constitute major Ags against which aged people target functionally efficient T cell effector responses with massive production of Th1 cytokines and exhibition of CD107a degranulation marker. As a result, the production of IFN-gamma induced in T cells by both Ags was seven to eight times greater in very old than in young subjects. The comparative analysis of pp65-specific responses in these very long-term carriers revealed a reciprocal relationship between CD4+ and CD8+ producing IFN-gamma in the same individuals. These results indicate that CMV represents an important pathogen responsible for a strong immune activation in human aging. Such a remarkable burden of effector CD4+ and CD8+ T cells may be necessary to protect the elderly from CMV endogenous reactivation, but can turn detrimental by giving a substantial contribution to the proinflammatory status that accompanies the main age-related diseases.

Radiofrequency thermal ablation of hepatocellular carcinoma liver nodules can activate and enhance tumor-specific T-cell responses.

Radiofrequency thermal ablation (RFA) destroys tumoral tissue generating a local necrosis followed by marked inflammatory response with a dense T-cell infiltrate. In this study, we tested whether hepatocellular carcinoma thermal ablation can induce or enhance T-cell responses specific for hepatocellular carcinoma-associated antigens. Peripheral blood mononuclear cells derived from 20 patients with hepatocellular carcinoma were stimulated before and a month after RFA treatment with autologous hepatocellular carcinoma-derived protein lysates obtained before and immediately after RFA treatment. The effect of thermal ablation on memory T-cell responses to recall antigens [tetanus toxoid, protein purified derivative (PPD), Escherichia coli] was also assessed. T-cell reactivity was analyzed in an IFN-gamma enzyme-linked immunospot assay and by intracellular IFN-gamma staining. Treatment was followed by a significant increase of patients responsive either to tumor antigens derived from both the untreated hepatocellular carcinoma tissue (P < 0.05) and the necrotic tumor (P < 0.01) and by a higher frequency of circulating tumor-specific T cells. T-cell responses to recall antigens were also significantly augmented. Phenotypic analysis of circulating T and natural killer cells showed an increased expression of activation and cytotoxic surface markers. However, tumor-specific T-cell responses were not associated with protection from hepatocellular carcinoma relapse. Evidence of tumor immune escape was provided in one patient by the evidence that a new nodule of hepatocellular carcinoma recurrence was not recognized by T cells obtained at the time of RFA. In conclusion, RFA treatment generates the local conditions for activating the tumor-specific T-cell response. Although this effect is not sufficient for controlling hepatocellular carcinoma, it may represent the basis for the development of an adjuvant immunotherapy in patients undergoing RFA for primary and secondary liver tumors.

Different contribution of EBV and CMV infections in very long-term carriers to age-related alterations of CD8+ T cells.

Aging is accompanied by a complex dynamics of CD8+ T cell subsets whose origin is unclear. To evaluate the impact of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) chronic infections on CD8+ T cells in far advanced age, we studied CD8+ T cells frequencies and phenotype in nonagenarians and centenarians by HLA-A*0201- and HLA-B*0702-tetramers incorporating epitopes specific of both viruses along with viral replication. The results demonstrate that EBV and CMV infections induce quantitatively and qualitatively different CD8+ T-cell responses in advanced aging. The frequency and absolute number of CD8+ T cells specific for one lytic and two latent EBV-epitopes, were relatively low and mostly included within CD8+ CD28+ cells. By contrast, CMV infection was characterized by highly variable numbers of CD8+ T cells specific for two differently restricted CMV-epitopes that, in some subjects, were strikingly expanded. Moreover, the great majority of anti-CMV CD8+ T cells did not bear CD28 antigen. Notwithstanding the expansion of CMV-specific CD8+ lymphocytes, CMV-DNA detection in blood samples was invariably negative. Altogether, we suggest that CMV, but not EBV, can sustain chronic activation of the HLA-class I restricted effector arm in elderly that might have detrimental effects on age-associated diseases.

Reconstitution dynamics of plasmacytoid and myeloid dendritic cell precursors after allogeneic myeloablative hematopoietic stem cell transplantation.

Dendritic cells (DCs) are fundamental for immunity. We investigated reconstitution of plasmacytoid DC (PDC) and myeloid DC (My-DC) precursors in the first 2 months after allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Circulating DCs were monitored from the earliest phase of hematopoietic reconstitution in 43 children given standard therapy to prevent graft-versus-host disease (GVHD) and either treated or untreated with granulocyte colony-stimulating factor (G-CSF) after HSCT. In patients without GVHD, both My-DCs and PDCs reached consistently high absolute values during the initial phase. Time of engraftment did not differ between My-DCs and PDCs, regardless of administration of G-CSF. Treatment with G-CSF (1) accelerated early recovery of My-DC absolute numbers; (2) was associated with lower numbers of both My-DCs and PDCs in the later phase; and (3) significantly reduced the proportion of interleukin-12 (IL-12)-secreting cells. In some patients who developed acute GVHD, we found high numbers of circulating DC precursors during the early phase of this complication. However, treatment with steroids invariably induced rapid decrease of PDCs. Altogether, these data provide an evaluation of DC release after Allo-HSCT, indicate that postgrafting administration of G-CSF impairs the appearance of IL-12-producing DCs, and suggest that DC homeostasis may be disrupted at onset of GVHD.

T-cell dynamics after high-dose chemotherapy in adults: elucidation of the elusive CD8+ subset reveals multiple homeostatic T-cell compartments with distinct implications for immune competence.

Recovery of total T cell numbers after in vivo T-cell depletion in humans is accompanied by complex perturbation within the CD8+ subset. We aimed to elucidate the reconstitution of CD8+ T cells by separate analysis of putative naïve CD95- CD28+, memory CD95+ CD28+ and CD28- T cell compartments after acute maximal depletion by high-dose chemotherapy (HD-ChT) in women with high-risk breast cancer. We found that recovery of putative naïve CD8+ CD95- CD28+ and CD4+ CD95- CD28+ T cells, was compatible with a thymus-dependent regenerative pathway since their recovery was slow and time-dependent, their values were tightly related to each other, and their reconstitution patterns were inversely related to age. By analysing non-naïve T cells, a striking diversion between putative memory T cells and CD28- T cells was found. These latter increased early well beyond normal values, thus playing a pivotal role in total T-cell homeostasis, and contributed to reduce the CD4 : CD8 ratio. In contrast, putative memory T cells returned to values not significantly different from those seen in patients at diagnosis, indicating that this compartment may recover after HD-ChT. At 3-5 years after treatment, naïve T cells persisted at low levels, with expansion of CD28- T cells, suggesting that such alterations may extend further. These findings indicate that CD28- T cells were responsible for 'blind' T-cell homeostasis, but support the notion that memory and naïve T cells are regulated separately. Given their distinct dynamics, quantitative evaluation of T-cell pools in patients undergoing chemotherapy should take into account separate analysis of naïve, memory and CD28- T cells.