Changes in CD4+ cells’ miRNA expression following exposure to HIV-1

Background: MiRNAs inhibit HIV-1 expression by either modulating host innate immunity or by directly interfering with viral mRNAs. Here, we investigated the miRNA profile that discriminates different classes of HIV-1 infected patients from multiple exposed uninfected individuals. Methods: The expression levels of 377 miRNAs were selectively analyzed in CD4+ cells isolated from whole blood of HIV-1 \ue9lite LTNP (\ue9LTNP), naive, and multiply exposed uninfected individuals (MEU). MiRNA extraction was performed by the mirVana miRNA Isolation Kit (Ambion) and their expression was subsequently examined by real-time PCR-based arrays. The expression of miRNAs was also determined in primary culture of CD4+T cells and monocyte-macrophages infected in vitro by R5 strains. Expression of Dicer and Drosha was evaluated by real-time PCR. Results: We only considered miRNAs that were expressed in the 70% of patients of at least one class and varied by at least 1 log10 from healthy controls. Out of 377 miRNAs, 26 were up-regulated, while 88 were down-regulated. Statistical analysis showed that 21 miRNAs significantly differentiated \ue9LTNP from MEU and 23 miRNAs distinguished naive from MEU, while only 1 (miR-155) discriminated \ue9LTNP from naive. By hierarchical clustering of the miRNAs according to patient class, \ue9LTNP clustered with naive whereas all MEU subjects grouped together. The Dicer and Drosha expression in the patient classes correlated with miRNA profile changes. Among miRNAs differentially expressed in patient classes, 32 were detected in in vitro infection model: the most of the up-regulated miRNAs were expressed in monocyte-macrophages, whereas the most of the down-regulated miRNAs were expressed in T lymphocytes. Conclusions: These findings support that miRNA profile could be the result not only of a productive infection, but also of the exposure to HIV products that leave a signature in immune cells. These data provide some intriguing issues relative to the development of HIV vaccines targeting viral proteins

The Conjugation of Antibodies for the Simultaneous Detection of Surface Proteins and Transcriptome Analysis at a Single-Cell Level

Peer reviewe

Redistribution of CD8+ T cell subsets in metastatic renal cell carcinoma patients treated with anti-PD-1 therapy

Renal-cell carcinoma (RCC) is responsible for the majority of tumors arising from the kidney parenchyma. Although a progressive improvement in median overall survival has been observed after the introduction of anti-PD-1 therapy, many patients do not benefit from this treatment. Therefore, we have investigated T cell dynamics to find immune modification induced by anti-PD-1 therapy. Here, we show that, after therapy, RCC patients (5 responders and 14 nonresponders) are characterized by a redistribution of different subsets across the memory T cell compartment

Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

Background: Ewing's sarcoma (ES) is an aggressive cancer affecting children and young adults. We pre-clinically demonstrated that mesenchymal stromal/stem cells (MSCs) can deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) against primary ES after local injection. However, ES is often metastatic calling for approaches able to support MSC targeting to the ES multiple remote sites. Considering that the disialoganglioside GD2 is expressed by ES and to optimise MSC tumour affinity, bi-functional (BF) MSCs expressing both TRAIL and a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR) were generated and challenged against ES. Methods: The anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were tested in several in vitro ES models. Tumour targeting and killing by BF MSCs was further investigated by a novel immunodeficient ES metastatic model characterized by different metastatic sites, including lungs, liver and bone, mimicking the deadly clinical scenario. Findings: In vitro data revealed both tumour affinity and killing of BF MSCs. In vivo, GD2 tCAR molecule ameliorated the tumour targeting and persistence of BF MSCs counteracting ES in lungs but not in liver. Interpretation: We here generated data on the potential effects of BF MSCs within a complex ES metastatic in vivo model, exploring also the biodistribution of MSCs. Our BF MSC-based strategy promises to pave the way for potential improvements in the therapeutic delivery of TRAIL for the treatment of metastatic ES and other deadly GD2-positive malignancies

Monocytic population in chronic lymphocytic leukemia shows altered composition and deregulation of genes involved in phagocytosis and inflammation

Macrophages reside in tissues infiltrated by chronic lymphocytic leukemia B-cells and the extent of infiltration is associated with adverse prognostic factors. Blood monocyte population was studied by flow cytometry and whole-genome microarrays. A mixed lymphocyte reaction was performed to evaluate T cell proliferation in contact with monocytes from patients and normal donors. Migration and gene modulation in normal monocytes treated with leukemia were also evaluated. Chronic lymphocytic leukemia patients showed an increase in the absolute number of monocytes compared to normal controls (792+/-86 cells/mL vs. 485+/-46 cells/mL, p=0.003). Higher number of nonclassical CD14+CD16++ and Tie-2 expressing monocytes (TEMs) was also detected in patients. Furthermore, we performed a gene expression analysis of monocytes in chronic lymphocytic leukemia patients, showing up-regulation of RAP1GAP and down-regulation of tubulins and CDC42EP3, which would be expected to result in impairment in phagocytosis. We also detected gene alterations such as the down-regulation of PTGR2, a reductase able to inactivate the prostaglandin E2, indicating an immunosuppressive activity. Accordingly, T cell proliferation was inhibited in contact with monocytes from patients compared to normal controls. Finally, normal monocytes in vitro increased migration and up-regulated CD16, RAP1GAP, IL-10, IL-8, MMP9 and down-regulated PTGR2 in response to leukemic cells or conditioned media. In conclusion, altered composition and deregulation of genes involved in phagocytosis and inflammation were found in blood monocytes obtained from chronic lymphocytic leukemia patients, suggesting that leukemia-mediated 'education' of immune elements may also include the establishment of a skewed phenotype in monocyte/macrophage population

Novel genetic association of TNF-α-238 and PDCD1-7209 polymorphisms with long-term non-progressive HIV-1 infection.

About 2-5% of HIV-1-infected subjects, defined as long-term non-progressors (LTNPs), remain immunologically stable for a long time without treatment. The factors governing this condition are known only in part, and include genetic factors. Thus, we studied 20 polymorphisms of 15 genes encoding proinflammatory and immunoregulatory cytokines, chemokines and their receptors, genes involved in apoptosis, and the gene HCP5. METHODS: We analyzed 47 Caucasian LTNPs infected for >9 years, compared with 131 HIV-1-infected Caucasian patients defined as 'usual progressors'. The genotypes were determined by methods based upon PCR, and the statistical analysis was performed by univariate logistic regression. RESULTS: The well-known CCR5Δ32 del32 allele, the cell death-related TNF-α-238 A and PDCD1-7209 T alleles, and HCP5 rs2395029 G, a non-coding protein associated with the HLA-B*5701, were found positively associated with the LTNP condition. No association was observed for other single nucleotide polymorphisms (SDF-1-801, IL-10-592, MCP-1-2518, CX3CR1 V249I, CCR2V64I, RANTES-403, IL-2-330, IL-1β-511, IL-4-590, FASL IVS3nt-169, FAS-670, FAS-1377, FASL IVS2nt-124, PDCD1-7146, MMP-7-181, and MMP7-153). CONCLUSIONS: The novel genetic associations between allelic variants of genes TNF-α-238 and PDCD1-7209 with the LTNP condition underline the importance of host genetic factors in the progression of HIV-1 infection and in immunological preservation

Circulating mucosal-associated invariant T cells identify patients responding to anti-PD-1 therapy

Immune checkpoint inhibitors are used for treating patients with metastatic melanoma. Since the response to treatment is variable, biomarkers are urgently needed to identify patients who may benefit from such therapy. Here, we combine single-cell RNA-sequencing and multiparameter flow cytometry to assess changes in circulating CD8+ T cells in 28 patients with metastatic melanoma starting anti-PD-1 therapy, followed for 6 months: 17 responded to therapy, whilst 11 did not. Proportions of activated and proliferating CD8+ T cells and of mucosal-associated invariant T (MAIT) cells are significantly higher in responders, prior to and throughout therapy duration. MAIT cells from responders express higher level of CXCR4 and produce more granzyme B. In silico analysis support MAIT presence in the tumor microenvironment. Finally, patients with >1.7% of MAIT among peripheral CD8+ population show a better response to treatment. Our results thus suggest that MAIT cells may be considered a biomarker for patients responding to anti-PD-1 therapy

Altered pathways of keratinization, extracellular matrix generation, angiogenesis, and stromal stem cells proliferation in patients with systemic sclerosis

Objective: Systemic sclerosis is characterized by endothelial dysfunction, autoimmunity abnormalities, and fibrosis of the skin and internal organs. The pathogenetic mechanisms underlying systemic sclerosis vasculopathy are still not clarified. A complex cellular and extracellular network of interactions has been studied, but it is currently unclear what drives the activation of fibroblasts/myofibroblasts and the extracellular matrix deposition. Methods: Using RNA sequencing, the aim of the work was to identify potential functional pathways implied in systemic sclerosis pathogenesis and markers of endothelial dysfunction and fibrosis in systemic sclerosis patients. RNA-sequencing analysis was performed on RNA obtained from biopsies from three systemic sclerosis patients and three healthy controls enrolled in our University Hospital. RNA was used to generate sequencing libraries that were sequenced according to proper transcriptomic analyses. Subsequently, we performed gene set enrichment analysis of differentially expressed genes on the entire list of genes that compose the RNA-sequencing expression matrix. Results: Gene set enrichment analysis revealed that healthy controls were characterized by gene signatures related to stromal stem cells proliferation, cytokine–cytokine receptor interaction, macrophage-enriched metabolic network, whereas systemic sclerosis tissues were enriched in signatures associated with keratinization, cornification, retinoblastoma 1 and tumor suppressor 53 signaling. Conclusion: According to our data, RNA-sequencing and pathway analysis revealed that systemic sclerosis subjects display a discrete pattern of gene expression associated with keratinization, extracellular matrix generation, and negative regulation of angiogenesis and stromal stem cells proliferation. Further analysis on larger numbers of patients is needed; however, our findings provide an interesting framework for the development of biomarkers useful to explore potential future therapeutic approaches

Successful immunosenescence and the remodelling of immune responses with ageing.

In recent decades, major theoretical and technological advances have been achieved in the field of immunology. These have allowed the scientific community to analyse the immune system in a much more sophisticated manner than was possible even 20 years ago. Moreover, great theoretical changes have also occurred in gerontology - in particular, the hypothesis has been put forward that ageing and diseases are two different phenomena, and that successful ageing, i.e. ageing in good psychophysical conditions, is really possible for most humans and animals. Immunosenescence was then carefully investigated, either in selected healthy people of advanced age or in the oldest old people, such as healthy centenarians. The main results showed that most immune parameters are indeed well preserved even at this far advanced age. This paper deals with some of the most important theoretical problems of immunosenescence. An immunological tenet was that the most important phenomenon of immunosenescence is the involution of the thymus. In most textbooks and papers it is taken for granted that the thymus starts its involution immediately after puberty. When people aged 60-65 were considered old, it was not difficult to think that they could live for the rest of their life with a fully involuted thymus. The findings on centenarians challenge this tenet, as they have only a small reduction of T lymphocytes, and a relatively normal number of virgin and memory T cells, together with a functional T cell repertoire. Other observations reported here on centenarians, concerning the activity of B lymphocytes and the cytokine network, as well as those on the well-preserved innate immunity and the cells' capability of undergoing proliferation after appropriate stimuli, suggest that complex immune changes occur with age, but also indicate that we have to modify our attitude, to grasp the new scenario which is emerging. Immunosenescence can no longer be considered as a unidirectional deterioration, and this complex phenomenon is much better described by terms such as 'remodelling', 'reshaping' or 'retuning'

Expansion of plasmablasts and loss of memory B cells in peripheral blood from COVID-19 patients with pneumonia

Studies on the interactions between SARS-CoV-2 and humoral immunity are fundamental to elaborate effective therapies including vaccines. We used polychromatic flow cytometry, coupled with unsupervised data analysis and principal component analysis (PCA), to interrogate B cells in untreated patients with COVID-19 pneumonia. COVID-19 patients displayed normal plasma levels of the main immunoglobulin classes, of antibodies against common antigens or against antigens present in common vaccines. However, we found a decreased number of total and na\uefve B cells, along with decreased percentages and numbers of memory switched and unswitched B cells. On the contrary, IgM+ and IgM 12 plasmablasts were significantly increased. In vitro cell activation revealed that B lymphocytes showed a normal proliferation index and number of dividing cells per cycle. PCA indicated that B-cell number, naive and memory B cells but not plasmablasts clustered with patients who were discharged, while plasma IgM level, C-reactive protein, D-dimer, and SOFA score with those who died. In patients with pneumonia, the derangement of the B-cell compartment could be one of the causes of the immunological failure to control SARS-Cov2, have a relevant influence on several pathways, organs and systems, and must be considered to develop vaccine strategies