An HLA-E-targeted TCR bispecific molecule redirects T cell immunity against Mycobacterium tuberculosis.

Peptides presented by HLA-E, a molecule with very limited polymorphism, represent attractive targets for T cell receptor (TCR)-based immunotherapies to circumvent the limitations imposed by the high polymorphism of classical HLA genes in the human population. Here, we describe a TCR-based bispecific molecule that potently and selectively binds HLA-E in complex with a peptide encoded by the inhA gene of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans. We reveal the biophysical and structural bases underpinning the potency and specificity of this molecule and demonstrate its ability to redirect polyclonal T cells to target HLA-E-expressing cells transduced with mycobacterial inhA as well as primary cells infected with virulent Mtb. Additionally, we demonstrate elimination of Mtb-infected cells and reduction of intracellular Mtb growth. Our study suggests an approach to enhance host T cell immunity against Mtb and provides proof of principle for an innovative TCR-based therapeutic strategy overcoming HLA polymorphism and therefore applicable to a broader patient population.

Platelets accumulate in lung lesions of tuberculosis patients and inhibit T-cell responses and Mycobacterium tuberculosis replication in macrophages.

Platelets regulate human inflammatory responses that lead to disease. However, the role of platelets in tuberculosis (TB) pathogenesis is still unclear. Here, we show that patients with active TB have a high number of platelets in peripheral blood and a low number of lymphocytes leading to a high platelets to lymphocytes ratio (PL ratio). Moreover, the serum concentration of different mediators promoting platelet differentiation or associated with platelet activation is increased in active TB. Immunohistochemistry analysis shows that platelets localise around the lung granuloma lesions in close contact with T lymphocytes and macrophages. Transcriptomic analysis of caseous tissue of human pulmonary TB granulomas, followed by Gene Ontology analysis, shows that 53 platelet activation-associated genes are highly expressed compared to the normal lung tissue. In vitro activated platelets (or their supernatants) inhibit BCG-induced T- lymphocyte proliferation and IFN-γ production. Likewise, platelets inhibit the growth of intracellular macrophages of Mycobacterium (M.) tuberculosis. Soluble factors released by activated platelets mediate both immunological and M. tuberculosis replication activities. Furthermore, proteomic and neutralisation studies (by mAbs) identify TGF-ß and PF4 as the factors responsible for inhibiting T-cell response and enhancing the mycobactericidal activity of macrophages, respectively. Altogether these results highlight the importance of platelets in TB pathogenesis.

B-Cell Receptor Signaling Is Thought to Be a Bridge between Primary Sjogren Syndrome and Diffuse Large B-Cell Lymphoma.

Primary Sjogren syndrome (pSS) is the second most common autoimmune disorder worldwide, which, in the worst scenario, progresses to Non-Hodgkin Lymphoma (NHL). Despite extensive studies, there is still a lack of knowledge about developing pSS for NHL. This study focused on cells' signaling in pSS progression to the NHL type of diffuse large B-cell lymphoma (DLBCL). Using bulk RNA and single cell analysis, we found five novel pathologic-independent clusters in DLBCL based on cells' signaling. B-cell receptor (BCR) signaling was identified as the only enriched signal in DLBCL and pSS peripheral naive B-cells or salivary gland-infiltrated cells. The evaluation of the genes in association with BCR has revealed that targeting CD79A, CD79B, and LAMTOR4 as the shared genes can provide novel biomarkers for pSS progression into lymphoma.

Emerging Roles of Cells and Molecules of Innate Immunity in Alzheimer's Disease.

The inflammatory response that marks Alzheimer's disease (neuroinflammation) is considered a double-edged sword. Microglia have been shown to play a protective role at the beginning of the disease. Still, persistent harmful stimuli further activate microglia, inducing an exacerbating inflammatory process which impairs ß-amyloid peptide clearance capability and leads to neurotoxicity and neurodegeneration. Moreover, microglia also appear to be closely involved in the spread of tau pathology. Soluble TREM2 also represents a crucial player in the neuroinflammatory processes. Elevated levels of TREM2 in cerebrospinal fluid have been associated with increased amyloid plaque burden, neurodegeneration, and cognitive decline in individuals with Alzheimer's disease. Understanding the intricate relationship between innate immunity and Alzheimer's disease will be a promising strategy for future advancements in diagnosis and new therapeutic interventions targeting innate immunity, by modulating its activity. Still, additional and more robust studies are needed to translate these findings into effective treatments. In this review, we focus on the role of cells (microglia, astrocytes, and oligodendrocytes) and molecules (TREM2, tau, and ß-amyloid) of the innate immune system in the pathogenesis of Alzheimer's disease and their possible exploitation as disease biomarkers and targets of therapeutical approaches.

Ferritin Metabolism Reflects Multiple Myeloma Microenvironment and Predicts Patient Outcome.

Multiple myeloma (MM) is a hematologic malignancy with a multistep evolutionary pattern, in which the pro-inflammatory and immunosuppressive microenvironment and genomic instability drive tumor evolution. MM microenvironment is rich in iron, released by pro-inflammatory cells from ferritin macromolecules, which contributes to ROS production and cellular damage. In this study, we showed that ferritin increases from indolent to active gammopathies and that patients with low serum ferritin had longer first line PFS (42.6 vs. 20.7 months and, p = 0.047, respectively) and OS (NR vs. 75.1 months and p = 0.029, respectively). Moreover, ferritin levels correlated with systemic inflammation markers and with the presence of a specific bone marrow cell microenvironment (including increased MM cell infiltration). Finally, we verified by bioinformatic approaches in large transcriptomic and single cell datasets that a gene expression signature associated with ferritin biosynthesis correlated with worse outcome, MM cell proliferation, and specific immune cell profiles. Overall, we provide evidence of the role of ferritin as a predictive/prognostic factor in MM, setting the stage for future translational studies investigating ferritin and iron chelation as new targets for improving MM patient outcome.

Phosphatidylserine Liposomes Reduce Inflammatory Response, Mycobacterial Viability, and HIV Replication in Coinfected Human Macrophages.

Chronic immune activation is the key pathogenetic event of Mycobacterium tuberculosis-human immunodeficiency virus (HIV) coinfection. We assessed the therapeutic value of phosphatidylserine-liposome (PS-L) in an in vitro model of M. tuberculosis-HIV coinfection. PS-L reduced nuclear factor-κB activation and the downstream production of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 in bacille Calmette-Guérin-infected macrophages and of TNF-α and IL-1ß in M. tuberculosis-infected and M. tuberculosis-HIV-coinfected macrophages. Importantly, a significant reduction of intracellular M. tuberculosis viability and HIV replication were also observed. These results support the further exploitation of PS-L as host-directed therapy for M. tuberculosis-HIV coinfection.

Tumor-Derived Small Extracellular Vesicles Induce Pro-Inflammatory Cytokine Expression and PD-L1 Regulation in M0 Macrophages via IL-6/STAT3 and TLR4 Signaling Pathways.

Tumor-associated macrophages play a key role in promoting tumor progression by exerting an immunosuppressive phenotype associated with the expression of programmed cell death ligand 1 (PD-L1). It is well known that tumor-derived small extracellular vesicles (SEVs) affect the tumor microenvironment, influencing TAM behavior. The present study aimed to examine the effect of SEVs derived from colon cancer and multiple myeloma cells on macrophage functions. Non-polarized macrophages (M0) differentiated from THP-1 cells were co-cultured with SEVs derived from a colorectal cancer (CRC) cell line, SW480, and a multiple myeloma (MM) cell line, MM1.S. The expression of PD-L1, interleukin-6 (IL-6), and other inflammatory cytokines as well as of the underlying molecular mechanisms were evaluated. Our results indicate that SEVs can significantly upregulate the expressions of PD-L1 and IL-6 at both the mRNA and protein levels and can activate the STAT3 signaling pathway. Furthermore, we identified the TLR4/NF-kB pathway as a convergent mechanism for SEV-mediated PD-L1 expression. Overall, these preliminary data suggest that SEVs contribute to the formation of an immunosuppressive microenvironment.

HLA-E-restricted CD8+ T Lymphocytes Efficiently Control Mycobacterium tuberculosis and HIV-1 Coinfection.

We investigated the contribution of human leukocyte antigen A2 (HLA-A2) and HLA-E-restricted CD8+ T cells in patients with Mycobacterium tuberculosis and human immunodeficiency virus 1 (HIV-1) coinfection. HIV-1 downregulates HLA-A, -B, and -C molecules in infected cells, thus influencing recognition by HLA class I-restricted CD8+ T cells but not by HLA-E-restricted CD8+ T cells, owing to the inability of the virus to downmodulate their expression. Therefore, antigen-specific HLA-E-restricted CD8+ T cells could play a protective role in Mycobacterium tuberculosis and HIV-1 coinfection. HLA-E- and HLA-A2-restricted Mycobacterium tuberculosis-specific CD8+ T cells were tested in vitro for cytotoxic and microbicidal activities, and their frequencies and phenotypes were evaluated ex vivo in patients with active tuberculosis and concomitant HIV-1 infection. HIV-1 and Mycobacterium tuberculosis coinfection caused downmodulation of HLA-A2 expression in human monocyte-derived macrophages associated with resistance to lysis by HLA-A2-restricted CD8+ T cells and failure to restrict the growth of intracellular Mycobacterium tuberculosis. Conversely, HLA-E surface expression and HLA-E-restricted cytolytic and microbicidal CD8 responses were not affected. HLA-E-restricted and Mycobacterium tuberculosis-specific CD8+ T cells were expanded in the circulation of patients with Mycobacterium tuberculosis/HIV-1 coinfection, as measured by tetramer staining, but displayed a terminally differentiated and exhausted phenotype that was rescued in vitro by anti-PD-1 (programmed cell death protein 1) monoclonal antibody. Together, these results indicate that HLA-E-restricted and Mycobacterium tuberculosis-specific CD8+ T cells in patients with Mycobacterium tuberculosis/HIV-1 coinfection have an exhausted phenotype and fail to expand in vitro in response to antigen stimulation, which can be restored by blocking the PD-1 pathway using the specific monoclonal antibody nivolumab.

Detailed characterization of human Mycobacterium tuberculosis specific HLA-E restricted CD8+ T cells.

HLA-E presented antigens are interesting targets for vaccination given HLA-Es' essentially monomorphic nature. We have shown previously that Mycobacterium tuberculosis (Mtb) peptides are presented by HLA-E to CD8+ effector T cells, but the precise phenotype and functional capacity of these cells remains poorly characterized. We have developed and utilized in this study a new protocol combining HLA-E tetramer with intracellular staining for cytokines, transcription factors and cytotoxic molecules to characterize these cells in depth. We confirm in this study the significantly increased ex vivo frequency of Mtb-peptide/HLA-E-TM+ CD8+ T cells in the circulation of patients with active tuberculosis (TB). HLA-E restricted CD8+ T cells from TB patients produced more IL-13 than cells from controls or subjects with latent tuberculosis infection (LTBI). Compared to total CD8+ T cells, HLA-E restricted cells produced more IFNγ, IL-4, IL-10, and granulysin but less granzyme-A. Moreover, compared to "classical" Mtb specific HLA-A2 restricted CD8+ T cells, HLA-E restricted CD8+ T cells produced less TNFα and perforin, but more IL-4. In conclusion, HLA-E restricted- Mtb specific cells can produce Th2 cytokines directly.

Buffy coat-derived platelets cryopreserved using a new method: Results from a pivotal clinical trial on thrombocytopenic patients with acute leukaemia.

The administration of cryopreserved platelets (PLTs) may overcome the limits of platelet shortage and availability, especially during some seasons or in specific contexts like rural areas. After in vitro validation studies, ad hoc prepared buffy coat-derived pooled platelet concentrates (BC-PLTs), treated with dimethyl sulphoxide (DMSO) and cryopreserved (CRY BC-PLTs) at -80 °C with a modified Valeri method, were transfused in patients with severe thrombocytopenia secondary to chemotherapy for acute leukaemia (AL). Five inpatients were enrolled in the pivotal clinical trial NCT02032134: 4 males and 1 female with a mean age of 71 years (range: 65-80). Four patients were diagnosed with acute myeloid leukaemia and 1 had acute lymphoblastic leukaemia.Transfusion of one Unit of CRY BC-PLTs resulted effective in active bleeding control in two patients without any adverse reaction or concomitant antihaemorrhagic therapies. CRY BC-PLTs met the currently accepted criteria for cryopreserved PLTs, their transfusion in patients with AL was safe. (Clinical trial: NCT02032134).

Buffy coat-derived platelets cryopreserved using a new method: Results from in vitro studies.

Cryopreservation for the long-term storage of platelets (PLTs) is a useful method to overcome the limits of platelet shortage. This is an in vitro prospective study to evaluate the count, viability, and function of buffy coat-derived pooled platelet concentrates (BC-PLTs), treated with dimethyl sulphoxide (DMSO) and cryopreserved (CRY BC-PLTs) at -80 °C with a modified Valeri method. PLTs were stored in 6% DMSO with a patented kit. Overall, 49 BC-PLTs from 245 healthy volunteer donors were prepared, cryopreserved, and analysed before and after 3, 6, and 9 months of storage. In flow cytometry, a statistically significant reduction in CD 42b (92.7 ± 4.29% at T0 vs. 23.6 ± 27.5% at T3, 16.38 ± 12.54% at T6, and 17.3 ± 9.6% at T9) and PAC-1 (1.9 ± 1.34% at T0 vs. 0.62 ± 0.4% at T3, 0.63 ± 0.83% at T6, and 0.49 ± 0.48% at T9) was observed after storage. CRY BC-PLTs showed a good and stable endogenous thrombin generation potential (nM min): 529.25 ± 98.64 at T0 vs. 533.04 ± 103.15 at T9 months. CRY BC-PLTs showed a good viability in vitro, according to currently accepted criteria for cryopreserved PLTs.

Inflammation and the coagulation system in tuberculosis: Tissue Factor leads the dance.

Mycobacterium tuberculosis, the causative agent of tuberculosis, drives the formation of granulomas, structures in which both immune cells and the bacterial pathogen cohabit. The most abundant cells in granulomas are macrophages, which contribute as both cells with bactericidal activity and as targets for M. tuberculosis infection and proliferation during the entire course of infection. The mechanisms and factors involved in the regulation and control of macrophage microenvironment-specific polarization and plasticity are not well understood, as some granulomas are able to control bacteria growth and others fail to do so, permitting bacterial spread. In this issue of the European Journal of Immunology, Venkatasubramanian et al. [Eur. J. Immunol. 2016. 46: 464-479] show that mice lacking the tissue factor gene in myeloid cells have augmented M. tuberculosis growth and increased inflammation in the lungs. This suggests that tissue factor, an initiator of coagulation, is important for the generation of fibrin, which supports granuloma formation. This article demonstrates for the first time the involvement of tissue factor in inducing effective immunity against M. tuberculosis, and sheds new lights on the complex interplay between host inflammatory response, the coagulation system, and the control of M. tuberculosis infection.

Human CD8+ T-cells recognizing peptides from Mycobacterium tuberculosis (Mtb) presented by HLA-E have an unorthodox Th2-like, multifunctional, Mtb inhibitory phenotype and represent a novel human T-cell subset.

Mycobacterial antigens are not exclusively presented to T-cells by classical HLA-class Ia and HLA-class II molecules, but also through alternative antigen presentation molecules such as CD1a/b/c, MR1 and HLA-E. We recently described mycobacterial peptides that are presented in HLA-E and recognized by CD8+ T-cells. Using T-cell cloning, phenotyping, microbiological, functional and RNA-expression analyses, we report here that these T-cells can exert cytolytic or suppressive functions, inhibit mycobacterial growth, yet express GATA3, produce Th2 cytokines (IL-4,-5,-10,-13) and activate B-cells via IL-4. In TB patients, Mtb specific cells were detectable by peptide-HLA-E tetramers, and IL-4 and IL-13 were produced following peptide stimulation. These results identify a novel human T-cell subset with an unorthodox, multifunctional Th2 like phenotype and cytolytic or regulatory capacities, which is involved in the human immune response to mycobacteria and demonstrable in active TB patients' blood. The results challenge the current dogma that only Th1 cells are able to inhibit Mtb growth and clearly show that Th2 like cells can strongly inhibit outgrowth of Mtb from human macrophages. These insights significantly expand our understanding of the immune response in infectious disease.

Human CD8 T lymphocytes recognize Mycobacterium tuberculosis antigens presented by HLA-E during active tuberculosis and express type 2 cytokines.

CD8 T cells contribute to protective immunity against Mycobacterium tuberculosis. In humans, M. tuberculosis reactive CD8 T cells typically recognize peptides associated to classical MHC class Ia molecules, but little information is available on CD8 T cells recognizing M. tuberculosis Ags presented by nonclassical MHC class Ib molecules. We show here that CD8 T cells from tuberculosis (TB) patients recognize HLA-E-binding M. tuberculosis peptides in a CD3/TCR αß mediated and CD8-dependent manner, and represent an additional type of effector cells playing a role in immune response to M. tuberculosis during active infection. HLA-E-restricted recognition of M. tuberculosis peptides is detectable by a significant enhanced ex vivo frequency of tetramer-specific circulating CD8 T cells during active TB. These CD8 T cells produce type 2 cytokines upon antigenic in vitro stimulation, help B cells for Ab production, and mediate limited TRAIL-dependent cytolytic and microbicidal activity toward M. tuberculosis infected target cells. Our results, together with the finding that HLA-E/M. tuberculosis peptide specific CD8 T cells are detected in TB patients with or without HIV coinfection, suggest that this is a new human T-cell population that participates in immune response in TB.

Janus-faced liposomes enhance antimicrobial innate immune response in Mycobacterium tuberculosis infection.

We have generated unique asymmetric liposomes with phosphatidylserine (PS) distributed at the outer membrane surface to resemble apoptotic bodies and phosphatidic acid (PA) at the inner layer as a strategy to enhance innate antimycobacterial activity in phagocytes while limiting the inflammatory response. Results show that these apoptotic body-like liposomes carrying PA (ABL/PA) (i) are more efficiently internalized by human macrophages than by nonprofessional phagocytes, (ii) induce cytosolic Ca(2+) influx, (iii) promote Ca(2+)-dependent maturation of phagolysosomes containing Mycobacterium tuberculosis (MTB), (iv) induce Ca(2+)-dependent reactive oxygen species (ROS) production, (v) inhibit intracellular mycobacterial growth in differentiated THP-1 cells as well as in type-1 and -2 human macrophages, and (vi) down-regulate tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-1ß, IL-18, and IL-23 and up-regulate transforming growth factor (TGF)-ß without altering IL-10, IL-27, and IL-6 mRNA expression. Also, ABL/PA promoted intracellular killing of M. tuberculosis in bronchoalveolar lavage cells from patients with active pulmonary tuberculosis. Furthermore, the treatment of MTB-infected mice with ABL/PA, in combination or not with isoniazid (INH), dramatically reduced lung and, to a lesser extent, liver and spleen mycobacterial loads, with a concomitant 10-fold reduction of serum TNF-α, IL-1ß, and IFN-γ compared with that in untreated mice. Altogether, these results suggest that apoptotic body-like liposomes may be used as a Janus-faced immunotherapeutic platform to deliver polar secondary lipid messengers, such as PA, into phagocytes to improve and recover phagolysosome biogenesis and pathogen killing while limiting the inflammatory response.

Integrated Analysis of Single-Cell and Bulk RNA Sequencing Data Reveals Memory-like NK Cell Subset Associated with Mycobacterium tuberculosis Latency.

Natural killer (NK) cells are innate-like lymphocytes that belong to the family of type-1 innate lymphoid cells and rapidly respond to virus-infected and tumor cells. In this study, we have combined scRNA-seq data and bulk RNA-seq data to define the phenotypic and molecular characteristics of peripheral blood NK cells. While the role of NK cells in immune surveillance against virus infections and tumors has been well established, their contribution to protective responses to other intracellular microorganisms, such as Mycobacterium tuberculosis (Mtb), is still poorly understood. In this study, we have combined scRNA-seq data and bulk RNA-seq data to illuminate the molecular characteristics of circulating NK cells in patients with active tuberculosis (TB) disease and subjects with latent Mtb infection (LTBI) and compared these characteristics with those of healthy donors (HDs) and patients with non-TB other pulmonary infectious diseases (ODs). We show here that the NK cell cluster was significantly increased in LTBI subjects, as compared to patients with active TB or other non-TB pulmonary diseases and HD, and this was mostly attributable to the expansion of an NK cell population expressing KLRC2, CD52, CCL5 and HLA-DRB1, which most likely corresponds to memory-like NK2.1 cells. These data were validated by flow cytometry analysis in a small cohort of samples, showing that LTBI subjects have a significant expansion of NK cells characterized by the prevalence of memory-like CD52+ NKG2C+ NK cells. Altogether, our results provide some new information on the role of NK cells in protective immune responses to Mtb.

Differentiation, phenotype, and function of interleukin-17-producing human Vγ9Vδ2 T cells.

In healthy adults, the major peripheral blood γδ T-cell subset expresses the Vγ9Vδ2 TCR and displays pleiotropic features. Here we report that coculture of naive Vγ9Vδ2 T cells with phosphoantigens and a cocktail of cytokines (IL-1-ß, TGF-ß, IL-6, and IL-23), leads to selective expression of the transcription factor RORγt and polarization toward IL-17 production. IL-17(+) Vγ9Vδ2 T cells express the chemokine receptor CCR6 and produce IL-17 but neither IL-22 nor IFN-γ; they have a predominant terminally differentiated (CD27(-)CD45RA(+)) phenotype and express granzyme B, TRAIL, FasL, and CD161. On antigen activation, IL-17(+) Vγ9Vδ2 T cells rapidly induce CXCL8-mediated migration and phagocytosis of neutrophils and IL-17-dependent production of ß-defensin by epithelial cells, indicating that they may be involved in host immune responses against infectious microorganisms. Accordingly, an increased percentage of IL-17(+) Vγ9Vδ2 lymphocytes is detected in the peripheral blood and at the site of disease in children with bacterial meningitis, and this pattern was reversed after successful antibacterial therapy. Most notably, the phenotype of IL-17(+) Vγ9Vδ2 T cells in children with meningitis matches that of in vitro differentiated IL-17(+) Vγ9Vδ2 T cells. Our findings delineate a previously unknown subset of human IL-17(+) Vγ9Vδ2 T lymphocytes implicated in the pathophysiology of inflammatory responses during bacterial infections.

Genome-based in silico identification of new Mycobacterium tuberculosis antigens activating polyfunctional CD8+ T cells in human tuberculosis.

Although CD8(+) T cells help control Mycobacterium tuberculosis infection, their M. tuberculosis Ag repertoire, in vivo frequency, and functionality in human tuberculosis (TB) remains largely undefined. We have performed genome-based bioinformatics searches to identify new M. tuberculosis epitopes presented by major HLA class I supertypes A2, A3, and B7 (covering 80% of the human population). A total of 432 M. tuberculosis peptides predicted to bind to HLA-A*0201, HLA-A*0301, and HLA-B*0702 (representing the above supertypes) were synthesized and HLA-binding affinities determined. Peptide-specific CD8(+) T cell proliferation assays (CFSE dilution) in 41 M. tuberculosis-responsive donors identified 70 new M. tuberculosis epitopes. Using HLA/peptide tetramers for the 18 most prominently recognized HLA-A*0201-binding M. tuberculosis peptides, recognition by cured TB patients' CD8(+) T cells was validated for all 18 epitopes. Intracellular cytokine staining for IFN-γ, IL-2, and TNF-α revealed mono-, dual-, as well as triple-positive CD8(+) T cells, indicating these M. tuberculosis peptide-specific CD8(+) T cells were (poly)functional. Moreover, these T cells were primed during natural infection, because they were absent from M. tuberculosis-noninfected individuals. Control CMV peptide/HLA-A*0201 tetramers stained CD8(+) T cells in M. tuberculosis-infected and noninfected individuals equally, whereas Ebola peptide/HLA-A*0201 tetramers were negative. In conclusion, the M. tuberculosis-epitope/Ag repertoire for human CD8(+) T cells is much broader than hitherto suspected, and the newly identified M. tuberculosis Ags are recognized by (poly)functional CD8(+) T cells during control of infection. These results impact on TB-vaccine design and biomarker identification.