A study of immune tolerance control in physiological models and in immune-mediated diseases

Immune response is based on a complex molecular and cellular network able to ensure protection against pathogens and simultaneously maintain tissue homeostasis. Multiple immunoregulatory processes are physiologically involved in preventing potentially deleterious immune reactions against self tissues. The key role of regulatory immune cell populations, as represented by CD4+CD25+Foxp3+ Treg cells, in induction and maintenance of immunological tolerance has been largely demonstrated. Aim of this study is to investigate on cell-dependent control of immune response in physiological conditions as well as in the context of immune-mediated diseases, also addressing the possibility to modulate deranged immune effectors. With this purpose we focused: i. a human model of autoimmunity, as represented by a subgroup of patients affected by Myelodysplastic Syndrome (MDS), a hematological disorder characterized by immune-mediated selection and expansion of pathological stem precursors; ii. human and animal models of pharmacological and metabolic immune-modulation; iii. the functional analysis of a T cell population, characterized by the co-expression of CD3 and CD56 molecules, whose quantitative defect has been observed in autoimmune diabetes. Immune-response has been largely recognised as a finely tuned micro-site process. Thus, the possibility to focus cell-mediated immune tolerance control in Bone Marrow (BM), the microenvironment in which immune-mediated selection of pathological stem precursor takes place, represent a powerful analysis tool to investigate on MDS pathogenesis. Our study of BM T cell repertoire revealed an inverse correlation between BM Treg levels, activation status and BM clonal expansion of CD8+ T lymphocytes in MDS patients. Thus, BM Treg were proposed to represent a key element for the control of the deranged immune effectors in an inflammatory microenvironment. Cross talk between immune response and metabolism is still largely undefined. Particularly, Treg availability has been observed in vitro to specifically depend on the oscillatory activity of the mammalian Target Of Rapamycin (mTOR), a Serine/Threonine kinase playing a key role in regulating cell growth and metabolism in response to nutritional cues. The employment of mTOR pharmacological inhibition for the control of tumour cell growth has been largely described. We found that dosage and administration schedule of the mTOR inhibitor Everolimus, able to ensure mTOR oscillatory activity, is relevant to induce immune-tolerance rather than inhibition of cell growth in a model of tolerance induction, as represented by allogeneic kidney transplant. Our study also addressed the possibility to use nutraceutical compounds, by us selected for their immune-modulating effects in a veterinary model of chronic infection, to control immune effector activity in vitro. Our data are conceivable with the possibility to employ these substances as pharmacological co-adjuvants to modulate pro-inflammatory activity in contexts of altered immune homeostasis. Co-expression of CD3 and CD56 molecules identifies a lymphocyte population whose functional activity is largely undefined. A severe reduction of this cells has been associated with the extent of β-cell loss in patients affected by type 1 diabetes. We found that CD3+CD56+ lymphocytes, by us named TR3-56, represent a distinct subgroup of T lymphocytes, able to preferentially modulate effector function of cytotoxic T cells. Indeed, the co-culture of TR3-56 with CD8+ effector cells mediates significant inhibition of their cytotoxic activity and IFN- γ production. No effects were observed when cytotoxic T cells were cultured with NK, CD4 or CD8 T lymphocytes. A contact-dependent mechanism has been observed to underlie immune-modulating activity of TR3-56 cells. A better knowledge of cell mediated processes involved in immune-tolerance control is expected to significantly improve the availability of innovative immune-modulating strategies, thus ameliorating clinical management of immune-mediated disorders

Bone marrow CD3+ CD56+ regulatory T lymphocytes (TR3 -56 cells) are inversely associated with activation and expansion of bone marrow cytotoxic T cells in IPSS-R very-low/low risk MDS patients

Background Emergence of dysplastic haematopoietic precursor/s, cytopenia and variable leukaemia risk characterise myelodysplastic syndromes (MDS). Impaired immune-regulation, preferentially affecting cytotoxic T cells (CTL), has been largely observed in MDS. Recently, we described the TR3-56 T cell subset, characterised by the co-expression of CD3 and CD56, as a novel immune-regulatory population, able to modulate cytotoxic functions. Here, we address the involvement of TR3-56 cells in MDS pathogenesis/progression. Objectives To analyse the relationship between TR3-56 and CTL activation/expansion in bone marrow (BM) of very-low/low-risk MDS subjects. Methods Peripheral blood and BM specimens, obtained at disease onset in a cohort of 58 subjects, were analysed by immune-fluorescence and flow cytometry, to preserve the complexity of the biological sample. Results We observed that a trend-increase of BM TR3-56 in high/very-high MDS stage, as compared with very-low/low group, associates with a decreased activation of BM resident CTL; significant correlation of TR3-56 with BM blasts has been also revealed. In addition, in very-low/low-risk subjects the TR3-56 amount in BM inversely correlates with the presence of activated BM CTL showing a skewed V beta T-cell repertoire. Conclusions These data add TR3-56 to the immune-regulatory network involved in MDS pathogenesis/progression. Better knowledge of the immune-mediated processes associated with the disease might improve MDS clinical management

An Open Question in the COVID-19 Pandemic: Can Humans Transmit the Disease to Pets and Vice Versa?

SARS-CoV-2 infection apparently emerged in China in December 2019, causing the disease known as COVID-19,which can cause severe damage to vital organs (Ackermann et al, 2020). Spillover of SARS-CoV- 2 from bats to humans has been hypothesized (Ackermann et al, 2020). The virus spike protein is the main determinant of viral tropism because it is responsible for binding to the angiotensin converting enzyme 2 (ACE2) and subsequent entry of SARSCoV- 2 to host cells in humans and several animal species (Sun et al, 2020). Therefore, it is reasonable to hypothesize that the spike proteineACE2 receptor complex may represent evolutionary exploitation to overcome speciesbarriers to infection, thushighlighting the zoonotic origin and transmission of the virus

Superoxide Dismutase-1 intracellular content in T lymphocytes associates with increased Regulatory T Cell level in Multiple Sclerosis subjects undergoing immune-modulating treatment.

Reactive oxygen species (ROS) participate in the T-cell activation processes. ROS-dependent regulatory networks are usually mediated by peroxides, which are more stable and able to freely migrate inside cells. Superoxide dismutase (SOD)-1 represents the major physiological intracellular source of peroxides. We found that antigen-dependent activation represents a triggering element for SOD-1 production and secretion by human T lymphocytes. A deranged T-cell proinflammatory response characterizes the pathogenesis of multiple sclerosis (MS). We previously observed a decreased SOD-1 intracellular content in leukocytes of MS individuals at diagnosis, with increasing amounts of such enzyme after interferon (IFN)-b 1b treatment. Here, we analyzed in depth SOD-1 intracellular content in T cells in a cohort of MS individuals undergoing immune-modulating treatment. Higher amounts of the enzyme were associated with increased availability of regulatory T cells (Treg) preferentially expressing Foxp3-exon 2 (Foxp3-E2), as described for effective Treg. In vitro administration of recombinant human SOD-1 to activated T cells, significantly increased their IL-17 production, while SOD-1 molecules lacking dismutase activity were unable to interfere with cytokine production by activated T cells in vitro. Furthermore, hydrogen peroxide addition was observed to mimic, in vitro, the SOD-1 effect on IL-17 production. These data add SOD-1 to the molecules involved in the molecular pathways contributing to re-shaping the T-cell cytokine profile and Treg differentiation

Adaptive and Innate Cytotoxic Effectors in Chronic Lymphocytic Leukaemia (CLL) Subjects with Stable Disease

Chronic lymphocytic leukaemia (CLL) is characterised by the expansion of a neoplastic mature B cell clone. CLL clinical outcome is very heterogeneous, with some subjects never requiring therapy and some showing an aggressive disease. Genetic and epigenetic alterations and pro-inflammatory microenvironment influence CLL progression and prognosis. The involvement of immune-mediated mechanisms in CLL control needs to be investigated. We analyse the activation profile of innate and adaptive cytotoxic immune effectors in a cohort of 26 CLL patients with stable disease, as key elements for immune-mediated control of cancer progression. We observed an increase in CD54 expression and interferon (IFN)-γ production by cytotoxic T cells (CTL). CTL ability to recognise tumour-targets depends on human leukocyte antigens (HLA)-class I expression. We observed a decreased expression of HLA-A and HLA-BC on B cells of CLL subjects, associated with a significant reduction in intracellular calnexin that is relevant for HLA surface expression. Natural killer (NK) cells and CTL from CLL subjects show an increased expression of the activating receptor KIR2DS2 and a reduction of 3DL1 and NKG2A inhibiting molecules. Therefore, an activation profile characterises CTL and NK cells of CLL subjects with stable disease. This profile is conceivable with the functional involvement of cytotoxic effectors in CLL control

T cell activation induces CuZn Superoxide Dismutase (SOD)-1) intracellular re-localization, production and secretion

Reactive Oxygen Species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H2O2), more stable and able to freely diffuse through cell membranes. CuZn Superoxide dismutase (SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H2O2 and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H2O2 generation are involved in T Cell Receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the “non T” cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors

An Open Question: Is It Rational to Inhibit the mTor-Dependent Pathway as COVID-19 Therapy?

In December 2019, a novel coronavirus infection appeared in China (Wuhan City and Hubei Province), causing the first cases of abnormal severe pneumonia. Since then, the SARS-Cov2 infection has become pandemic and the correlated coronavirus disease (COVID-19) has been showing a plethora of pathophysiological manifestations that do not exclusively reduce COVID-19 to the occurrence of severe acute respiratory distress. Although the immunological responses against SARS-Cov2 remain poorly defined it is of note that the critical phase of COVID-19 currently appears, at least in some critical pathophysiological aspects, as a sort of autoimmune disease or as immune response hypersensitivity. Consequently, many authors have proposed various therapeutic approaches based on the modulation/inhibition of abnormal immune response in COVID-19. Recently, the effects of Tocilizumab administration has seemed to indicate that inhibition of the Interleukin (IL)-6 receptor (IL-6R) may result in the recovery of critical COVID-19 patients in the advanced post-alveolitic phase, when extensive pulmonary fibrosis is accompanied by a diffuse interstitial inflammation apparently sustained by a described exacerbated cytokine storm. Such evidence highlights the critical relevance of controlling the IL-6/IL-6R pro-inflammatory pathway in the pathophysiology of COVID-19 in order to mitigate the adverse immune response that is a determinant of the most serious and undesirable phase of SARS-Cov2 infection.In particular, hyper-reactivity was described as a major feature of the critical phase of COVID-19, broadly due to the hyperacute inflammatory context that leads to pulmonary interstitial disease and severe acute respiratory distress

HLA-E and HLA class I molecules on bone marrow and peripheral blood polymorphonuclear cells of myelodysplatic patients.

Relevance of immune-dysregulation for emergence, dominance and progression of dysplastic clones in myelodysplastic syndromes (MDS) was suggested, but valuable or predictive criteria on this involvement are lacking. We previously reported that reduced T-regulatory cells (Treg) and high CD54 expression on T cell identify a sub-group of patients in whom an immune-pathogenesis might be inferred. Here, we suggest the occurrence of immune-selection of dysplastic clones in a subgroup of MDS patients, with reduced HLA-I and HLA-E on PMN, and propose that an altered immune profile might represent a valuable criterion to classify Low/Int-1 patients on the basis of immune-pathogenesis of MDS

Circulating regulatory T cells (Treg), leptin and induction of proinflammatory activity in obese Labrador Retriever dogs.

Over-nutrition and obesity have been associated with impaired immunity and low-grade inflammation in humans and mouse models. In this context, a causal role for unbalanced T regulatory cell (Treg)-dependent mechanisms has been largely suggested. Obesity is the most common nutritional disorder in dogs. However, it is not defined whether canine obesity may influence circulating Treg as well as if their number variation might be associated with the occurrence of systemic inflammation. The present study investigated the immune profile of healthy adult obese dogs belonging to the Labrador Retriever breed, in comparison with the normal weight counterpart. Indeed, obesity has been described as particularly evident in this dogs. With this purpose, 26 healthy dogs were enrolled and divided into two groups based on body condition score (BCS): controls (CTR: BCS 4–5) and obeses (OB: BCS ≥ 7). Our data indicate that adult obese Labrador Retrievers are characterised by the inverse correlation between leptin serum concentration and circulating Treg (CD4+CD25highFoxp3+) levels. In addition, an increased number of cytotoxic T cell effectors (CD3+CD8+) and a higher IFN-γ production by cytotoxic T lymphocytes were observed in OB group. These results may provide new insights into the immunological dysregulation frequently associated to obesity in humans and still undefined in dog

Reduced regulatory T cells (Treg) in bone marrow preferentially associate with the expansion of cytotoxic T lymphocytes in low risk MDS patients.

The myelodysplastic syndromes (MDS) include clonal bone marrow (BM) disorders characterised by the emergence/dominance of dysplastic progenitors in the context of ineffective haematopoiesis, peripheral cytopenias and increased risk of acute myeloid leukaemia (AML). The link between immune dysregulation and MDS has been suggested . Autoimmune attack to normal precursors as well as the activity of bystander T cells, recruited during an immune‐response against dysplastic antigens, were hypothesised as relevant for the selection of dysplastic clones that are able to escape immune‐mediated damage. The involvement of Natural Killer cells was also described