Reticulocytes in donor blood units enhance red blood cell alloimmunization.

Although red blood cell (RBC) transfusions save lives, some patients develop clinically-significant alloantibodies against donor blood group antigens, which then have adverse effects in multiple clinical settings. Few effective measures exist to prevent RBC alloimmunization and/or eliminate alloantibodies in sensitized patients. Donor-related factors may influence alloimmunization; thus, there is an unmet clinical need to identify which RBC units are immunogenic. Repeat volunteer blood donors and donors on iron supplements have elevated reticulocyte counts compared to healthy non-donors. Early reticulocytes retain mitochondria and other components, which may act as danger signals in immune responses. Herein, we tested whether reticulocytes in donor RBC units could enhance RBC alloimmunization. Using a murine model, we demonstrate that transfusing donor RBC units with increased reticulocyte frequencies dose-dependently increased RBC alloimmunization rates and alloantibody levels. Transfusing reticulocyte-rich RBC units was associated with increased RBC clearance from the circulation and a robust proinflammatory cytokine response. As compared to previously reported post-transfusion RBC consumption patterns, erythrophagocytosis from reticulocyte-rich units was increasingly performed by splenic B cells. These data suggest that reticulocytes in a donated RBC unit impact the quality of blood transfused, are targeted to a distinct compartment, and may be an underappreciated risk factor for RBC alloimmunization.

Retention of functional mitochondria in mature red blood cells from patients with sickle cell disease.

Sickle cell disease (SCD) is an inherited blood disorder characterized by sickled red blood cells (RBCs), which are more sensitive to haemolysis and can contribute to disease pathophysiology. Although treatment of SCD can include RBC transfusion, patients with SCD have high rates of alloimmunization. We hypothesized that RBCs from patients with SCD have functionally active mitochondria and can elicit a type 1 interferon response. We evaluated blood samples from more than 100 patients with SCD and found elevated frequencies of mitochondria in reticulocytes and mature RBCs, as compared to healthy blood donors. The presence of mitochondria in mature RBCs was confirmed by flow cytometry, electron microscopy, and proteomic analysis. The mitochondria in mature RBCs were metabolically competent, as determined by enzymatic activities and elevated levels of mitochondria-derived metabolites. Metabolically-active mitochondria in RBCs may increase oxidative stress, which could facilitate and/or exacerbate SCD complications. Coculture of mitochondria-positive RBCs with neutrophils induced production of type 1 interferons, which are known to increase RBC alloimmunization rates. These data demonstrate that mitochondria retained in mature RBCs are functional and can elicit immune responses, suggesting that inappropriate retention of mitochondria in RBCs may play an underappreciated role in SCD complications and be an RBC alloimmunization risk factor.

Past and Future of the Molecular Characterization of the T Cell Repertoire: Some Highlights of Eli Sercarz's Contributions.

The contribution of Eli E. Sercarz to immunology and immunopathology has been remarkable and achieved many milestones in the understanding of the processes of the mechanisms fine-tuning immune responses. A part of his work was dedicated to the study of the deep complexity of the lymphocyte T cell repertoire and its importance during the physiologic development and disease, such as clonal heterogeneity of T cell responses. Starting from these studies, under his mentoring, we had the opportunity to implement the spectratyping method and apply it to human and experimental autoimmune diseases, obtaining intriguing results. The open question of this brief review is the possible role of this fine and complex technique, the immunoscope analysis, in the era of the big data and omics.

S100B Protein as a Therapeutic Target in Multiple Sclerosis: The S100B Inhibitor Arundic Acid Protects from Chronic Experimental Autoimmune Encephalomyelitis.

S100B is an astrocytic protein behaving at high concentration as a damage-associated molecular pattern molecule. A direct correlation between the increased amount of S100B and inflammatory processes has been demonstrated, and in particular, the inhibitor of S100B activity pentamidine has been shown to ameliorate clinical scores and neuropathologic-biomolecular parameters in the relapsing-remitting experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. This study investigates the effect of arundic acid (AA), a known inhibitor of astrocytic S100B synthesis, in the chronic experimental autoimmune encephalomyelitis, which is another mouse model of multiple sclerosis usually studied. By the daily evaluation of clinical scores and neuropathologic-molecular analysis performed in the spinal cord, we observed that the AA-treated group showed lower severity compared to the vehicle-treated mice, particularly in the early phase of disease onset. We also observed a significant reduction of astrocytosis, demyelination, immune infiltrates, proinflammatory cytokines expression and enzymatic oxidative reactivity in the AA-treated group. Overall, our results reinforce the involvement of S100B in the development of animal models of multiple sclerosis and propose AA targeting the S100B protein as a focused potential drug to be considered for multiple sclerosis treatment.

In Vitro and Ex Vivo Methodologies for T-Cell Trafficking Through Blood-Brain Barrier After TLR Activation.

This chapter describes ex vivo isolation of human T cells and of naïve splenocytes respectively collected from multiple sclerosis patients and healthy controls and experimental autoimmune encephalomyelitis-affected mice. After the magnetic sorting of naïve and activated T helper lymphocytes, we provide details about the cell cultures to measure the interaction with extracellular matrix proteins using standard cell invasion or hand-made in vitro assays, upon different stimuli, through Toll-like receptor(s) ligands, T-cell activators, and cell adhesion molecules modulators. Finally, we describe the methods to harvest and recover T cells to evaluate the properties associated with their trafficking ability.

Reticulocytes in donor RBC units enhance RBC alloimmunization.

Although red blood cell (RBC) transfusions save lives, some patients develop clinically-significant alloantibodies against donor blood group antigens, which then have adverse effects in multiple clinical settings. Few effective measures exist to prevent RBC alloimmunization and/or eliminate alloantibodies in sensitized patients. Donor-related factors may influence alloimmunization; thus, there is an unmet clinical need to identify which RBC units are immunogenic. Repeat volunteer blood donors and donors on iron supplements have elevated reticulocyte counts compared to healthy non-donors. Early reticulocytes retain mitochondria and other components, which may act as danger signals in immune responses. Herein, we tested whether reticulocytes in donor RBC units could enhance RBC alloimmunization. Using a murine model, we demonstrate that transfusing donor RBC units with increased reticulocyte frequencies dose-dependently increase RBC alloimmunization rates and alloantibody levels. Transfusing reticulocyte-rich RBC units was associated with increased RBC clearance from the circulation and a robust proinflammatory cytokine response. As compared to previously reported post-transfusion RBC consumption patterns, erythrophagocytosis from reticulocyte-rich units was increasingly performed by splenic B cells. These data suggest that reticulocytes in a donated RBC unit impact the quality of blood transfused, are targeted to a distinct compartment, and may be an underappreciated risk factor for RBC alloimmunization.

Liposome-based nanoparticles impact on regulatory and effector phenotypes of macrophages and T cells in multiple Sclerosis patients.

Current available treatments of Multiple Sclerosis (MS) reduce neuroinflammation acting on different targets on the immune system, but potentially lead to severe side effects and have a limited efficacy in slowing the progression of the disease. Here, we evaluated in vitro the immunomodulatory potential of a new class of nanoparticles - liposomes, constituted by a double-layer of phosphatidylserine (PSCho/PS), and double-faced, with an outer layer of phosphatidylserine and an inner layer of phosphatidic acid (PSCho/PA), either alone or in the presence of the myelin basic protein (MBP) peptide (residues 85-99) (PSCho/PS-MBP and PSCho/PA-MBP). Results showed that PSCho/PS are equally and efficiently internalized by pro- and anti-inflammatory macrophages (M1 and M2 respectively), while PSCho/PA were internalized better by M2 than M1. PSCho/PS liposomes were able to inhibit the secretion of innate pro-inflammatory cytokine IL-1ß. PSCho/PS liposomes expanded Tregs, reducing Th1 and Th17 cells, while PSCho/PA liposomes were unable to dampen pro-inflammatory T cells and to promote immune-regulatory phenotype (Treg). The ability of PSCho/PS liposomes to up-regulate Treg cells was more pronounced in MS patients with high basal expression of M2 markers. PSCho/PS liposomes were more effective in decreasing Th1 (but not Th17) cells in MS patients with a disease duration >3 months. On the other hand, down-modulation of Th17 cells was evident in MS patients with active, Gadolinium enhancing lesions at MRI and in MS patients with a high basal expression of M1-associated markers in the monocytes. The same findings were observed for the modulation of MBP-driven Th1/Th17/Treg responses. These observations suggest that early MS associate to a hard-wired pro-Th1 phenotype of M1 that is lost later during disease course. On the other hand, acute inflammatory events reflect a temporary decrease of M2 phenotype that however is amenable to restauration upon treatment with PSCho/PS liposomes. Thus, together these data indicate that monocytes/macrophages may play an important regulatory function during MS course and suggest a role for PSCho/PS and PSCho/PS-MBP as new therapeutic tools to dampen the pro-inflammatory immune responses and to promote its regulatory branch.

Regionally restricted modulation of Sam68 expression and Arhgef9 alternative splicing in the hippocampus of a murine model of multiple sclerosis.

Multiple sclerosis (MS) and its preclinical models are characterized by marked changes in neuroplasticity, including excitatory/inhibitory imbalance and synaptic dysfunction that are believed to underlie the progressive cognitive impairment (CI), which represents a significant clinical hallmark of the disease. In this study, we investigated several parameters of neuroplasticity in the hippocampus of the experimental autoimmune encephalomyelitis (EAE) SJL/J mouse model, characterized by rostral inflammatory and demyelinating lesions similar to Relapsing-Remitting MS. By combining morphological and molecular analyses, we found that the hippocampus undergoes extensive inflammation in EAE-mice, more pronounced in the CA3 and dentate gyrus (DG) subfields than in the CA1, associated with changes in GABAergic circuitry, as indicated by the increased expression of the interneuron marker Parvalbumin selectively in CA3. By laser-microdissection, we investigated the impact of EAE on the alternative splicing of Arhgef9, a gene encoding a post-synaptic protein playing an essential role in GABAergic synapses and whose mutations have been related to CI and epilepsy. Our results indicate that EAE induces a specific increase in inclusion of the alternative exon 11a only in the CA3 and DG subfields, in line with the higher local levels of inflammation. Consistently, we found a region-specific downregulation of Sam68, a splicing-factor that represses this splicing event. Collectively, our findings confirm a regionalized distribution of inflammation in the hippocampus of EAE-mice. Moreover, since neuronal circuit rearrangement and dynamic remodeling of structural components of the synapse are key processes that contribute to neuroplasticity, our study suggests potential new molecular players involved in EAE-induced hippocampal dysfunction.

Immunopathology of SARS-CoV-2 Infection: A Focus on T Regulatory and B Cell Responses in Children Compared with Adults.

While the clinical impact of COVID-19 on adults has been massive, the majority of children develop pauci-symptomatic or even asymptomatic infection and only a minority of the latter develop a fatal outcome. The reasons of such differences are not yet established. We examined cytokines in sera and Th and B cell subpopulations in peripheral blood mononuclear cells (PBMC) from 40 children (<18 years old), evaluating the impact of COVID-19 infection during the pandemic's first waves. We correlated our results with clinical symptoms and compared them to samples obtained from 16 infected adults and 7 healthy controls. While IL6 levels were lower in SARS-CoV-2+ children as compared to adult patients, the expression of other pro-inflammatory cytokines such as IFNγ and TNFα directly correlated with early age infection and symptoms. Th and B cell subsets were modified during pediatric infection differently with respect to adult patients and controls and within the pediatric group based on age. Low levels of IgD− CD27+ memory B cells correlated with absent/mild symptoms. On the contrary, high levels of FoxP3+/CD25high T-Regs associated with a moderate−severe clinical course in the childhood. These T and B cells subsets did not associate with severity in infected adults, with children showing a predominant expansion of immature B lymphocytes and natural regulatory T cells. This study shows differences in immunopathology of SARS-CoV-2 infection in children compared with adults. Moreover, these data could provide information that can drive vaccination endpoints for children.

FcγRIV is required for IgG2c mediated enhancement of RBC alloimmunization.

Passive immunization with anti-D can prevent maternal alloimmunization to RhD thereby preventing hemolytic disease of the fetus and newborn. Unexpectedly, anti-D fails in some cases and some monoclonal anti-D preparations paradoxically enhances alloimmunization. The underlying mechanisms modulating humoral alloimmunization by anti-D are unknown. We previously reported that IgG antibody subclasses differentially regulate alloimmunity in response to red blood cell (RBC) transfusions in a mouse model; in particular, IgG2c significantly enhanced RBC alloantibody responses. Initial mechanistic studies revealed that IgG2c:RBC immune complexes were preferentially consumed by the splenic dendritic cell (DC) subsets that play a role in RBC alloimmunization. The deletion of activating Fc-gamma receptors (FcγRs) (i.e., FcγRI, FcγRIII, and FcγRIV) on DCs abrogated IgG2c-mediated enhanced alloimmunization. Because DCs express high levels of FcγRIV, which has high affinity for the IgG2c subclass, we hypothesized that FcγRIV was required for enhanced alloimmunization. To test this hypothesis, knockout mice and blocking antibodies were used to manipulate FcγR expression. The data presented herein demonstrate that FcγRIV, but not FcγRI or FcγRIII, is required for IgG2c-mediated enhancement of RBC alloantibody production. Additionally, FcγRI is alone sufficient for IgG2c-mediated RBC clearance but not for increased alloimmunization, demonstrating that RBC clearance can occur without inducing alloimmunization. Together, these data, combined with prior observations, support the hypothesis that passive immunization with an RBC-specific IgG2c antibody increases RBC alloantibody production through FcγRIV ligation on splenic conventional DCs (cDCs). This raises the question of whether standardizing antibody subclasses in immunoprophylaxis preparations is desirable and suggests which subclasses may be optimal for generating monoclonal anti-D therapeutics.

A TLR/CD44 axis regulates T cell trafficking in experimental and human multiple sclerosis.

In the pathogenesis of autoimmune disorders, the modulation of leukocytes' trafficking plays a central role, still poorly understood. Here, we focused on the effect of TLR2 ligands in trafficking of T helper cells through reshuffling of CD44 isoforms repertoire. Concurrently, strain background and TLR2 haplotype affected Wnt/ß-catenin signaling pathway and expression of splicing factors. During EAE, mCD44 v9- v 10 was specifically enriched in the forebrain and showed an increased ability to bind stably to osteopontin. Similarly, we observed that hCD44 v7 was highly enriched in cells of cerebrospinal fluid from MS patients with active lesions. Moreover, TLRs engagement modulated the composition of CD44 variants also in human T helper cells, supporting the hypothesis that pathogens or commensals, through TLRs, in turn modulate the repertoire of CD44 isoforms, thereby controlling the distribution of lesions in the CNS. The interference with this mechanism(s) represents a potential tool for prevention and treatment of autoimmune relapses and exacerbations.

Recovering or Persisting: The Immunopathological Features of SARS-CoV-2 Infection in Children.

Background. The profile of cellular immunological responses of children across the spectrum of COVID-19, ranging from acute SARS-CoV-2 infection to full recovery or Long COVID, has not yet been fully investigated. Methods. We examined and compared cytokines in sera and cell subsets in peripheral blood mononuclear cells (B and regulatory T lymphocytes) collected from four distinct groups of children, distributed as follows: younger than 18 years of age with either acute SARS-CoV-2 infection (n = 49); fully recovered from COVID-19 (n = 32); with persistent symptoms (Long COVID, n = 51); and healthy controls (n = 9). Results. In the later stages after SARS-CoV-2 infection, the cohorts of children, both with recovered and persistent symptoms, showed skewed T and B subsets, with remarkable differences when compared with children at the onset of the infection and with controls. The frequencies of IgD+CD27− naïve B cells, IgD+IgM+ and CD27−IgM+CD38dim B cells were higher in children with recent infection than in those with an older history of disease (p < 0.0001 for all); similarly, the total and natural Tregs compartments were more represented in children at onset when compared with Long COVID (p < 0.0001 and p = 0.0005, respectively). Despite the heterogeneity, partially due to age, sex and infection incidence, the susceptibility of certain children to develop persistent symptoms after infection appeared to be associated with the imbalance of the adaptive immune response. Following up and comparing recovered versus Long COVID patients, we analyzed the role of circulating naïve and switched B and regulatory T lymphocytes in counteracting the evolution of the symptomatology emerged, finding an interesting correlation between the amount and ability to reconstitute the natural Tregs component with the persistence of symptoms (linear regression, p = 0.0026). Conclusions. In this study, we suggest that children affected by Long COVID may have a compromised ability to switch from the innate to the adaptive immune response, as supported by our data showing a contraction of naïve and switched B cell compartment and an unstable balance of regulatory T lymphocytes occurring in these children. However, further prospective immunological studies are needed to better clarify which factors (epigenetic, diet, environment, etc.) are involved in the impairment of the immunological mechanisms in the Long COVID patients.

Translational Research in the Era of Precision Medicine: Where We Are and Where We Will Go.

The advent of Precision Medicine has globally revolutionized the approach of translational research suggesting a patient-centric vision with therapeutic choices driven by the identification of specific predictive biomarkers of response to avoid ineffective therapies and reduce adverse effects. The spread of "multi-omics" analysis and the use of sensors, together with the ability to acquire clinical, behavioral, and environmental information on a large scale, will allow the digitization of the state of health or disease of each person, and the creation of a global health management system capable of generating real-time knowledge and new opportunities for prevention and therapy in the individual person (high-definition medicine). Real world data-based translational applications represent a promising alternative to the traditional evidence-based medicine (EBM) approaches that are based on the use of randomized clinical trials to test the selected hypothesis. Multi-modality data integration is necessary for example in precision oncology where an Avatar interface allows several simulations in order to define the best therapeutic scheme for each cancer patient.

Haemophilus parasuis (Glaesserella parasuis) as a Potential Driver of Molecular Mimicry and Inflammation in Rheumatoid Arthritis.

Background: Haemophilus parasuis (Hps; now Glaesserella parasuis) is an infectious agent that causes severe arthritis in swines and shares sequence similarity with residues 261-273 of collagen type 2 (Coll261-273), a possible autoantigen in rheumatoid arthritis (RA). Objectives/methods: We tested the presence of Hps sequencing 16S ribosomal RNA in crevicular fluid, synovial fluids, and tissues in patients with arthritis (RA and other peripheral arthritides) and in healthy controls. Moreover, we examined the cross-recognition of Hps by Coll261-273-specific T cells in HLA-DRB1*04pos RA patients, by T-cell receptor (TCR) beta chain spectratyping and T-cell phenotyping. Results: Hps DNA was present in 57.4% of the tooth crevicular fluids of RA patients and in 31.6% of controls. Anti-Hps IgM and IgG titers were detectable and correlated with disease duration and the age of the patients. Peripheral blood mononuclear cells (PBMCs) were stimulated with Hps virulence-associated trimeric autotransporter peptide (VtaA10755-766), homologous to human Coll261-273 or co-cultured with live Hps. In both conditions, the expanded TCR repertoire overlapped with Coll261-273 and led to the production of IL-17. Discussion: We show that the DNA of an infectious agent (Hps), not previously described as pathogen in humans, is present in most patients with RA and that an Hps peptide is able to activate T cells specific for Coll261-273, likely inducing or maintaining a molecular mimicry mechanism. Conclusion: The cross-reactivity between VtaA10755-766 of a non-human infectious agent and human Coll261-273 suggests an involvement in the pathogenesis of RA. This mechanism appears emphasized in predisposed individuals, such as patients with shared epitope.