Silvia Della Bella

Immunosenescence and vaccine failure in the elderly

An age-related decline in immune responses in the elderly results in greater susceptibility to infection and reduced responses to vaccination. This decline in immune function affects both innate and adaptive immune systems. A meeting of experts in immunology and gerontology in Paris, France, in April 2008, considered current understanding of immunosenescence and its clinical consequences. Essential features of immunosenescence include: reduced natural killer cell cytotoxicity on a per cell basis; reduced number and function of dendritic cells in blood; decreased pools of naive T and B cells; and increases in the number of memory and effector T and B cells. In particular, an accumulation of late differentiated effector T cells, commonly associated with cytomegalovirus infection, contributes to a decline in the capacity of the adaptive immune system to respond to novel antigens. Consequently, vaccine responsiveness is compromised in the elderly, especially frail patients. Strategies to address the effects of immunosenescence include ensuring that seroprotective antibody levels against preventable infectious diseases are maintained throughout adulthood, and improving diet and exercise to address the effects of frailty. New vaccines are being developed, such as intradermal and high-dose vaccines for influenza, to improve the efficacy of immunization in the elderly. In the future, the development and use of markers of immunosenescence to identify patients who may have impaired responses to vaccination, as well as the use of end-points other than antibody titers to assess vaccine efficacy, may help to reduce morbidity and mortality due to infections in the elderly.

Human defensins activate monocyte-derived dendritic cells, promote the production of proinflammatory cytokines, and up-regulate the surface expression of CD91

Defensins are endogenous defense peptides with well‐defined antimicrobial activity against a broad spectrum of pathogens including bacteria, fungi, viruses, and parasites. Several lines of evidence suggest that defensins might also contribute to the regulation of host innate and adaptive immunity, but their immunomodulatory functions are still poorly understood. Herein, we studied the impact of human defensins on multiple functions of DCs, which are a central player in all immune responses, bridging innate and adaptive immunity. We challenged DCs differentiated in vitro from human moDCs with HNP‐1 α‐defensin or HBD‐1. HNP‐1 and HBD‐1 were chemotactic for moDCs. Both defensins promoted the activation and maturation of moDCs, as assessed by up‐regulation of surface expression of the costimulatory molecules CD80, CD86, and CD40, the maturation marker CD83, and HLA‐DR. HNP‐1 and HBD‐1 also enhanced the production of the proinflammatory cytokines TNF‐α, IL‐6, and IL‐12p70 but did not affect the production of the regulatory cytokine IL‐10. According to these stimulatory effects, HNP‐1 and HBD‐1 increased the allostimulatory activity of moDCs significantly. Finally, HNP‐1 and HBD‐1 promoted the up‐regulation of CD91 on the DC surface. CD91 is a scavenger receptor involved in the recognition of multiple ligands including defensins, thus suggesting that defensins may amplify their own effects through the activation of an autocrine loop. Taken together, our observations may provide new insight into the immunomodulatory properties of human defensins and may aid the exploration of new therapeutic strategies to potentiate antimicrobial and antitumor immunity.

Decrease and dysfunction of dendritic cells correlate with impaired hepatitis C virus-specific CD4+ T-cell proliferation in patients with hepatitis C virus infection

Through the production of stimulatory and suppressive cytokines, dendritic cells (DCs) regulate virus‐specific immune responses that are crucial to virus eradication. To explore a possible role of DCs in the persistence of hepatitis C virus (HCV) infection, in this study we analysed peripheral blood DCs (PBDCs) in patients with chronic hepatitis C (CHC) compared with those in both healthy seronegative (HSN) controls and a group of subjects who had spontaneously resolved infection, defined as healthy HCV‐seropositive (HSP), and we evaluated the relationships between PBDCs and HCV‐specific CD4+ T‐cell reactivity. The number of PBDCs, their immunophenotype and expression of regulatory cytokines were evaluated by flow cytometry on whole‐blood samples. HCV‐specific CD4+ T‐cell activation, proliferation and cytokine production were evaluated in cultures of peripheral blood mononuclear cells (PBMCs) stimulated in vitro with HCV peptides. We found that PBDCs from CHC subjects were numerically reduced and showed lower interleukin‐12 (IL‐12) and higher IL‐10 expression than those from HSN controls. PBDCs from HSP subjects were similar to those from HSN controls. HCV‐specific CD4+ T‐cell proliferation was less frequent and vigorous in CHC than in HSP patients and was directly related to the number of PBDCs and their IL‐12 production but inversely related to their IL‐10 production. Taken together, these results seem to suggest that cytokines of DC origin contribute to the regulation of HCV‐specific immunity in CHC patients and indicate that PBDCs may represent a novel non‐invasive tool for immune monitoring of these patients.

Functional repertoire of dendritic cells generated in granulocyte macrophage‐colony stimulating factor and interferon‐α

Monocyte‐derived dendritic cells (DCs) generated in granulocyte macrophage‐colony stimulating factor GM‐CSF) and interleukin‐4 (IL‐4–DCs) are used to enhance antitumor immunity in cancer patients, although recent evidence suggests that their functional repertoire may be incomplete; in particular, IL‐4–DCs appear unable to induce type 2 cytokine‐producing T helper (Th) cells. To assess whether type 1 interferon (IFN) could replace IL‐4 and generate DCs with a more complete repertoire, we characterized in detail DCs generated from human monocytes cultured with GM‐CSF and IFN‐α (IFN–DCs). We found that IFN‐α induces DC differentiation more efficiently than IL‐4, yielding similar numbers of DCs in a shorter time and that this differentiation persists upon removal of cytokines. Although IFN–DCs had a more mature immunophenotype than IL‐4–DCs, showing higher expression of CD80, CD86, and CD83, they still preserved comparable endocytic and phagocytic capacities and responsiveness to maturation stimuli. IFN–DCs had strong antigen‐presenting capacity, inducing intense proliferation of T cells to alloantigens or influenza virus. Moreover, IFN–DCs produced lower levels of IL‐12p70 and higher levels of IFN‐α, IL‐4, and IL‐10 than IL‐4–DCs. As a consequence of this different pattern of cytokine secretion, IFN–DCs induced T cells to produce type 1 (IFN‐γ) and type 2 (IL‐4 and IL‐10) cytokines, and as expected, IL‐4–DCs induced only Th1 differentiation. As immune responses with extreme Th1 bias are considered inadequate for the induction of optimal, systemic antitumor immunity, the ability of IFN–DCs to promote more balanced cytokine responses may suggest the advisability to consider these cells in the development of future, DC‐based immunotherapy trials.

Peripheral Blood Endothelial Progenitors as Potential Reservoirs of Kaposi's Sarcoma-Associated Herpesvirus

Background The cellular reservoirs of Kaposis sarcoma-associated herpesvirus (KSHV) and the exact nature of the putative KSHV-infected circulating precursor of spindle cells of Kaposis sarcoma (KS) still remain poorly defined. Because KS spindle cells are thought to be of endothelial origin, and because mature endothelial cells do not sustain persistent KSHV-infection, our attention was focalized on circulating hematopoietic precursors able to differentiate into endothelial lineage. Methods and Findings Late endothelial progenitor cells (late-EPCs) were cultured from the peripheral blood mononuclear cells of 16 patients with classic KS. The presence and load of KSHV genomes were analyzed by real-time polymerase chain reaction in DNA extracted from cells and supernatants of late-EPC cultures obtained from 7 patients. Endothelial colonies cultured from the peripheral blood of KS patients were found to satisfy all requisites to be defined late-EPCs: they appeared from the CD14-negative fraction of adherent cells after 11–26 days of culture, could be serially expanded in vitro, expressed high levels of endothelial antigens but lacked leukocyte markers. Late-EPC cultures were found to harbor KSHV-DNA at variable levels and to retain the virus after multiple passages in cells as well as in supernatants, suggesting that a quote of KSHV lytic infection may spontaneously occur. Lytic phase induction or hypoxia could amplify virus release in supernatants. Conclusion Our results suggest that circulating endothelial progenitors from KS patients are KSHV-infected and support viral productive replication and may therefore represent potential virus reservoirs and putative precursors of KS spindle cells.

Application of six-color flow cytometry for the assessment of dendritic cell responses in whole blood assays

Analysis of peripheral blood dendritic cells (PBDCs) is increasingly reaching clinical relevance in a wide range of pathologies, in which investigating the capacity of DC subsets to respond adequately to specific stimuli may aid the comprehension of underlying immunopathologic mechanisms. The evaluation of PBDC responses directly challenged in whole blood (WB) samples offers many advantages over other methods that require DC isolation and culture, but it is limited in multiparametric analysis, currently based on 3- or 4-color assays. Therefore, in this study we developed a 6-color assay dedicated to the analysis of PBDC responses upon WB stimulation. We incubated WB samples with ligands to toll-like receptors (TLRs) with a clear-cut distribution on myeloid DCs (mDCs) or plasmacytoid (pDCs) and analyzed DC responses in terms of upregulation of activation/maturation markers, as well as production of a wide range of regulatory cytokines. Four colors were used to gate on mDCs and pDCs that were identified as lineage-/HLA-DR+/CD11c+ and lineage-/HLA-DR+/CD123+, respectively, and two further colors were used to analyze either the surface expression of CD80, CD86, CD40 or CD83, or the intracellular accumulation of IL-12, tumor-necrosis factor (TNF)-alpha, interferon (IFN)-alpha, IL-6, IL-10 or IL-4. With this method, we could directly compare in the same flow cytometric tube the responses of mDCs and pDCs to TLR stimulation, and investigate the reciprocal coexpression of distinct activation markers or regulatory cytokines. We suggest that the 6-color WB assay presented here may represent a novel tool for investigating the complex biology of DCs.

Engagement of NKp30 on Vδ1 T-cells induces the production of CCL3, CCL4 and CCL5 and suppresses HIV-1 replication

Natural cytotoxicity receptors (NCRs) were originally identified as specific natural killer cell activating receptors that, on binding to their endogenous ligands, trigger the killing of tumor cell targets. We recently described the differentiation of a novel subset of NCR(+) Vδ1 T cells characterized by a remarkably high cytolytic potential against cancer cells. Here we demonstrate that the engagement of NKp30, one of the NCRs expressed de novo on Vδ1 T cells after stimulation, triggers the production of high levels of CCL3/MIP-1α, CCL4/ MIP-1β, and CCL5/RANTES but not of CXCL12/SDF-1. In turn, this NKp30-induced secretion of cc-chemokines is able to significantly suppress the replication of a CCR5 tropic strain of HIV-1 in CD4(+)/CCR5(+) infected PM1 cell lines. This experimental evidence disclosing an unanticipated antiviral function of NCR(+) Vδ1 T cells opens new avenues for understanding the pathogenic role and for manipulating the function of γδ T cells in HIV-1 infection.

Inflammation and preterm birth.

Preterm birth is the leading cause of neonatal morbidity and mortality. Although the underlying causes of pregnancy‐associated complication are numerous, it is well established that infection and inflammation represent a highly significant risk factor in preterm birth. However, despite the clinical and public health significance, infectious agents, molecular trigger(s), and immune pathways underlying the pathogenesis of preterm birth remain underdefined and represent a major gap in knowledge. Here, we provide an overview of recent clinical and animal model data focused on the interplay between infection‐driven inflammation and induction of preterm birth. Furthermore, here, we highlight the critical gaps in knowledge that warrant future investigations into the interplay between immune responses and induction of preterm birth.

Speeding of pulmonary VO2 on-kinetics by light-to-moderate-intensity aerobic exercise training in chronic heart failure: Clinical and pathophysiological correlates

BACKGROUND Pulmonary VO2 on-kinetics during light-to-moderate-intensity constant-work-rate exercise, an experimental model mirroring energetic transitions during daily activities, has been shown to speed up with aerobic exercise training (AET) in normal subjects, but scant data are available in chronic heart failure (CHF). METHODS AND RESULTS Thirty CHF patients were randomized to 3 months of light-to-moderate-intensity AET (CHF-AET) or control (CHF-C). Baseline and end-protocol evaluations included i) one incremental cardiopulmonary exercise test with near infrared spectroscopy analysis of peak deoxygenated hemoglobin+myoglobin concentration changes (Δ[deoxy(Hb+Mb)]) in vastus lateralis muscle, ii) 8 light-to-moderate-intensity constant-work-rate exercise tests for VO2 on-kinetics phase I duration, phase II τ, and mean response time (MRT) assessment, and iii) circulating endothelial progenitor cell (EPC) measurement. Reference values were obtained in 7 age-matched normals (N). At end-protocol, phase I duration, phase II τ, and MRT were significantly reduced (-12%, -22%, and -19%, respectively) and peak VO2, peak Δ[deoxy(Hb+Mb)], and EPCs increased (9%, 20%, and 98%, respectively) in CHF-AET, but not in CHF-C. Peak Δ[deoxy(Hb+Mb)] and EPCs relative increase correlated significantly to that of peak VO2 (r=0.61 and 0.64, respectively, p<0.05). CONCLUSIONS Light-to-moderate-intensity AET determined a near-normalization of pulmonary VO2 on-kinetics in CHF patients. Such a marked plasticity has important implications for AET intensity prescription, especially in patients more functionally limited and with high exercise-related risk. The AET-induced simultaneous improvement of phase I and phase II, associated with an increase of peak peripheral oxygen extraction and EPCs, supports microcirculatory O2 delivery impairment as a key factor determining exercise intolerance in CHF.

Treatment with belimumab restores B cell subsets and their expression of B cell activating factor receptor in patients with primary Sjogren’s syndrome

OBJECTIVE The aim of this study was to investigate the biological effects of belimumab on B cells in the first phase II open-label trial with belimumab in patients with primary SS (pSS) (BELISS). METHODS Peripheral blood B cell subsets and their B cell activating factor-receptor (BAFF-R) expression were analysed by multicolour flow cytometry in 10 pSS patients either before or after 24 and 52 weeks of therapy with belimumab. Serum BAFF levels were analysed by ELISA. RESULTS At baseline, pSS patients showed a significant increase in circulating B cells compared with healthy donors matched for age and sex, with a predominant expansion of transitional and naive B cell subsets. pSS patients also showed higher serum BAFF levels and lower B cell BAFF-R expression. Therapy with belimumab in pSS patients induced a significant reduction in transitional and naive B cell subsets to levels similar to those observed in healthy donors. Furthermore, belimumab normalized BAFF-R expression in all B subsets comprised within the memory compartment. The restoration of B cell frequency and subset composition in response to belimumab was also associated with a decrease in serum levels of Ig, RF, ANAs, and with an increase in the C4 complement fraction. All of these belimumab-mediated effects were observed after 24 weeks of therapy and maintained until the end of the therapeutic protocol. CONCLUSION Taken together, our findings show that targeting BAFF with belimumab is successful in normalizing B cell frequency, phenotype and functions in pSS. TRIAL REGISTRATION clinicaltrials.gov; https://clinicaltrials.gov/; NCT01008982.