Immune Stimuli Research Articles

Effects of sex and pro-inflammatory cytokines on context discrimination memory

In learning and memory tasks, immune overactivation is associated with impaired performance, while normal immune activation is associated with optimal performance. In one specific domain of memory, context discrimination memory, peripheral immune stimulation has been shown to impair performance on the context-object discrimination memory task in male rats. In order to evaluate potential sex differences in this task, as well as potential mechanisms for the memory impairment, we evaluated the ability of peripheral immune stimulation to impair task performance in both males and females. Next, we examined whether treatment with interleukin-1 receptor antagonist (IL-1ra), a receptor antagonist for the pro-inflammatory cytokine interleukin (IL)−1β, was able to rescue the memory deficit. We examined microglial morphology in the hippocampus and cytokine mRNA and protein expression in the hippocampus and the periphery. Male rats displayed memory impairment in response to LPS, and this impairment was not rescued by IL-1ra. Female rats did not have significant memory impairments and IL-1ra administration improved memory following inflammation. A subset of cytokines and chemokines were increased only in LPS-treated males. Inflammation alone did not alter microglia morphology, but IL-1ra did in certain sub-regions of the hippocampus. Together, these results indicate that sex differences exist in the ability of a peripheral immune stimulus to influence context discrimination memory and specific cytokine signals may be altered in impaired males. This study highlights the importance of sex differences in response to inflammatory challenges, especially related to memory impairments in context discrimination memory.

A nanoadjuvant that dynamically coordinates innate immune stimuli activation enhances cancer immunotherapy and reduces immune cell exhaustion.

Although conventional innate immune stimuli contribute to immune activation, they induce exhausted immune cells, resulting in suboptimal cancer immunotherapy. Here we suggest a kinetically activating nanoadjuvant (K-nanoadjuvant) that can dynamically integrate two waves of innate immune stimuli, resulting in effective antitumour immunity without immune cell exhaustion. The combinatorial code of K-nanoadjuvant is optimized in terms of the order, duration and time window between spatiotemporally activating Toll-like receptor 7/8 agonist and other Toll-like receptor agonists. K-nanoadjuvant induces effector/non-exhausted dendritic cells that programme the magnitude and persistence of interleukin-12 secretion, generate effector/non-exhausted CD8+ T cells, and activate natural killer cells. Treatment with K-nanoadjuvant as a monotherapy or in combination therapy with anti-PD-L1 or liposomes (doxorubicin) results in strong antitumour immunity in murine models, with minimal systemic toxicity, providing a strategy for synchronous and dynamic tailoring of innate immunity for enhanced cancer immunotherapy.

Loss of the Immunomodulatory Transcription Factor BATF2 in Humans Is Associated with a Neurological Phenotype.

Epilepsy and mental retardation are known to be associated with pathogenic mutations in a broad range of genes that are expressed in the brain and have a role in neurodevelopment. Here, we report on a family with three affected individuals whose clinical symptoms closely resemble a neurodevelopmental disorder. Whole-exome sequencing identified a homozygous stop-gain mutation, p.Gln19*, in the BATF2 gene in the patients. The BATF2 transcription factor is predominantly expressed in macrophages and monocytes and has been reported to modulate AP-1 transcription factor-mediated pro-inflammatory responses. Transcriptome analysis showed altered base-level expression of interferon-stimulated genes in the patients' blood, typical for type I interferonopathies. Peripheral blood mononuclear cells from all three patients demonstrated elevated responses to innate immune stimuli, which could be reproduced in CRISPR-Cas9-generated BATF2-/- human monocytic cell lines. BATF2 is, therefore, a novel disease-associated gene candidate for severe epilepsy and mental retardation related to dysregulation of immune responses, which underscores the relevance of neuroinflammation for epilepsy.

Use of CRISPR/CAS9 Technologies to Study the Role of TLR in Dendritic Cell Subsets.

Dendritic cells (DCs) have a significant role in coordinating both innate and adaptive immunity by serving as sentinels that detect invaders and initiate immune responses to eliminate them, as well as presenting antigens to activate adaptive immune responses that are specific to the antigen and the context in which it was detected. The regulation of DC functions is complex and involves intracellular drivers such as transcription factors and signaling pathways, as well as intercellular interactions with adhesion molecules, chemokines, and their receptors in the microenvironment. Toll-like receptors (TLRs) are crucial for DCs to detect pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways that lead to DC maturation and education in bridging with adaptive immunity, including the upregulation of MHC class II expression, induction of CD80, CD86, and CD40, and production of innate cytokines. Understanding the TLR pathways that DCs use to respond to innate immune stimuli and convert them into adaptive responses is important for new therapeutic targets identification.We present a novel platform that offers a fast and affordable CRISPR-Cas9 screening of genes that are involved in dendritic cells' TLR-dependent activation. Using CRISPR/Cas9 screening to target individual TLR genes in different dendritic cell subsets allows the identification of TLR-dependent pathways that regulate dendritic cell activation and cytokine production. This approach offers the efficient targeting of TLR driver genes to modulate the immune response and identify novel immune response regulators, establishing a causal link between these regulators and functional phenotypes based on genotypes.

Open multi-organ communication device for easy interrogation of tissue slices.

Here, we have developed an open multi-organ communication device that facilitates cellular and molecular communication between ex vivo organ slices. Measuring communication between organs is vital for understanding the mechanisms of health regulation yet remains difficult with current technology. Communication between organs along the gut-brain-immune axis is a key regulator of gut homeostasis. As a novel application of the device, we have used tissue slices from the Peyer's patch (PP) and mesenteric lymph node (MLN) due to their importance in gut immunity; however, any organ slices could be used here. The device was designed and fabricated using a combination of 3D printed molds for polydimethylsiloxane (PDMS) soft lithography, PDMS membranes, and track-etch porous membranes. To validate cellular and protein transfer between organs on-chip, we used fluorescence microscopy to quantitate movement of fluorescent proteins and cells from the PP to the MLN, replicating the initial response to immune stimuli in the gut. IFN-γ secretion during perfusion from a naïve vs. inflamed PP to a healthy MLN was quantitated to demonstrate soluble signaling molecules are moving on-chip. Finally, transient catecholamine release was measured during perfusion from PP to MLN using fast-scan cyclic voltammetry at carbon-fiber microelectrodes to demonstrate a novel application of the device for real-time sensing during communication. Overall, we show an open-well multi-organ device capable of facilitating transfer of soluble factors and cells with the added benefit of being available for external analysis techniques like electrochemical sensing which will advance abilities to probe communication in real-time across multiple organs ex vivo.

Itaconate promotes a wound resolving phenotype in pro-inflammatory macrophages

Pathological conditions associated with dysfunctional wound healing are characterized by impaired remodelling of extracellular matrix (ECM), increased macrophage infiltration, and chronic inflammation. Macrophages also play an important role in wound healing as they drive wound closure by secretion of molecules like transforming growth factor beta-1 (TGF-β). As the functions of macrophages are regulated by their metabolism, local administration of small molecules that alter this might be a novel approach for treatment of wound-healing disorders. Itaconate is a tricarboxylic acid (TCA) cycle-derived metabolite that has been associated with resolution of macrophage-mediated inflammation. However, its effects on macrophage wound healing functions are unknown. In this study, we investigated the effects of the membrane-permeable 4-octyl itaconate (4-OI) derivative on ECM scavenging by cultured human blood monocyte-derived macrophages (hMDM). We found that 4-OI reduced signalling of p38 mitogen-activated protein kinase (MAPK) induced by the canonical immune stimulus lipopolysaccharide (LPS). Likely as a consequence of this, the production of the inflammatory mediators like tumor necrosis factor (TNF)-α and cyclooxygenase (COX)-2 were also reduced. On the transcriptional level, 4-OI increased expression of the gene coding for TGF-β (TGFB1), whereas expression of the collagenase matrix metalloprotease-8 (MMP8) was reduced. Furthermore, surface levels of the anti-inflammatory marker CD36, but not CD206 and CD11c, were increased in these cells. To directly investigate the effect of 4-OI on scavenging of ECM by macrophages, we developed an assay to measure uptake of fibrous collagen. We observed that LPS promoted collagen uptake and that this was reversed by 4-OI-induced signaling of nuclear factor erythroid 2–related factor 2 (NRF2), a regulator of cellular resistance to oxidative stress and the reduced glycolytic capacity of the macrophage. These results indicate that 4-OI lowers macrophage inflammation, likely promoting a more wound-resolving phenotype.

Association between Immunosenescence, Mitochondrial Dysfunction and Frailty Syndrome in Older Adults

Aging is associated with changes in the immune system, increased inflammation and mitochondrial dysfunction. The relationship between these phenomena and the clinical phenotype of frailty is unclear. Here, we evaluated the immune phenotypes, T cell functions and mitochondrial functions of immune cells in frail and robust older subjects. We enrolled 20 frail subjects age- and gender-matched with 20 robust controls, and T cell phenotype, response to immune stimulation, cytokine production and immune cell mitochondrial function were assessed. Our results showed that numbers of CD4+ and CD8+ T cells were decreased in frail subjects, without impairment to their ratios. Memory and naïve T cells were not significantly affected by frailty, whereas the expression of CD28 but not that of ICOS was decreased in T cells from frail subjects. T cells from robust subjects produced more IL-17 after CD28 stimulation. Levels of serum cytokines were similar in frail subjects and controls. Mitochondrial bioenergetics and ATP levels were significantly lower in immune cells from frail subjects. In conclusion, we suggest that changes in T cell profiles are associated with aging rather than with frailty syndrome; however, changes in T cell response to immune stimuli and reduced mitochondrial activity in immune cells may be considered hallmarks of frailty.

Degradation of HDAC10 by autophagy promotes IRF3-mediated antiviral innate immune responses.

Histone deacetylases (HDACs) play important roles in immunity and inflammation. Through functional screening, we identified HDAC10 as an inhibitor of the type I interferon (IFN) response mediated by interferon regulatory factor 3 (IRF3). HDAC10 abundance was decreased in mouse macrophages in response to innate immune stimuli and was reduced in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) compared with that in PBMCs from healthy donors. Deficiency in HDAC10 in mouse embryonic fibroblasts and in mice promoted the expression of genes encoding type I IFNs and of IFN-stimulated genes (ISGs), leading to enhanced antiviral responses in vitro and in vivo. HDAC10 bound in a deacetylase-independent manner to IRF3 in uninfected cells to inhibit the phosphorylation of IRF3 at Ser396 by TANK-binding kinase 1 (TBK1). Upon viral infection, HDAC10 was targeted for autophagy-mediated degradation through its interaction with LC3-II. Consequently, IRF3 phosphorylation was increased, which resulted in enhanced type I IFN production and antiviral responses. Our findings identify a potential target for improving host defense responses against pathogen infection and for treating autoimmune disease.

Expression profile and molecular function of beclin-1 in Epinephelus akaara in response to immune stimuli and oxidative stress

Beclin-1, the mammalian ortholog of the yeast autophagy-related gene 6 (Atg 6), is a key regulator of autophagy. A variety of health and disease conditions in mammals are intricately related to the broad spectrum of beclin-1 functions. Nevertheless, few studies have investigated the role of beclin-1 in fish. In this study, we identified and cloned the beclin-1 cDNA (EaBECN-1) of Epinephelus akaara (red-spotted grouper) and carried out in silico analysis, tissue-specific expression analysis, immune challenge experiment, and in vitro analysis of its roles against viral infection and oxidative stress. The open reading frame was 1344 bp long and encoded 447 amino acids with a molecular weight of 51.2 kDa. Beclin-1 consisted of a conserved N-terminal BH3 and APG6 domains, and shared more than 88% identity with other vertebrates, according to a pairwise sequence alignment. EaBECN-1 expression profile analysis in E. akaara revealed that it is mostly expressed in the blood. Moreover, transcriptional modulation of EaBECN-1 was observed following stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly (I:C)), and nervous necrosis virus. During the viral hemorrhagic septicemia virus challenge, increased viral gene expression was observed at 12 h post-infection in FHM cells ectopically expressing EaBECN-1, and decreased thereafter at 24 h post-infection compared to control cells. However, increased antiviral gene expression at 12 and 24 h confirmed the antiviral function of EaBECN-1. Furthermore, EaBECN-1 overexpression protected the cells against H2O2-mediated apoptosis, as evidenced by the MTT assay, analysis of mRNA expression levels of apoptotic genes, and AO-EtBr staining. Overall, our study demonstrated the protective role of EaBECN-1 against viral pathogenesis and oxidative stress through autophagy, increasing our understanding of the role of beclin-1 in fish.

Mechanical high-intensity focused ultrasound creates unique tumor debris enhancing dendritic cell-induced T cell activation.

In situ tumor ablation releases a unique repertoire of antigens from a heterogeneous population of tumor cells. High-intensity focused ultrasound (HIFU) is a completely noninvasive ablation therapy that can be used to ablate tumors either by heating (thermal (T)-HIFU) or by mechanical disruption (mechanical (M)-HIFU). How different HIFU ablation techniques compare with respect to their antigen release profile, their activation of responder T cells, and their ability to synergize with immune stimuli remains to be elucidated. Here, we compare the immunomodulatory effects of T-HIFU and M-HIFU ablation with or without the TLR9 agonist CpG in the ovalbumin-expressing lymphoma model EG7. M-HIFU ablation alone, but much less so T-HIFU, significantly increased dendritic cell (DC) activation in draining lymph nodes (LNs). Administration of CpG following T- or M-HIFU ablation increased DC activation in draining LNs to a similar extend. Interestingly, ex vivo co-cultures of draining LN suspensions from HIFU plus CpG treated mice with CD8+ OT-I T cells demonstrate that LN cells from M-HIFU treated mice most potently induced OT-I proliferation. To delineate the mechanism for the enhanced anti-tumor immune response induced by M-HIFU, we characterized the RNA, DNA and protein content of tumor debris generated by both HIFU methods. M-HIFU induced a uniquely altered RNA, DNA and protein profile, all showing clear signs of fragmentation, whereas T-HIFU did not. Moreover, western blot analysis showed decreased levels of the immunosuppressive cytokines IL-10 and TGF-β in M-HIFU generated tumor debris compared to untreated tumor tissue or T-HIFU. Collectively, these results imply that M-HIFU induces a unique context of the ablated tumor material, enhancing DC-mediated T cell responses when combined with CpG.

Transcriptomic profiling implicates PAF1 in both active and repressive immune regulatory networks

BackgroundSitting at the interface of gene expression and host-pathogen interaction, polymerase associated factor 1 complex (PAF1C) is a rising player in the innate immune response. The complex localizes to the nucleus and associates with chromatin to modulate RNA polymerase II (RNAPII) elongation of gene transcripts. Performing this function at both proximal and distal regulatory elements, PAF1C interacts with many host factors across such sites, along with several microbial proteins during infection. Therefore, translating the ubiquity of PAF1C into specific impacts on immune gene expression remains especially relevant.ResultsAdvancing past work, we treat PAF1 knockout cells with a slate of immune stimuli to identify key trends in PAF1-dependent gene expression with broad analytical depth. From our transcriptomic data, we confirm PAF1 is an activator of traditional immune response pathways as well as other cellular pathways correlated with pathogen defense. With this model, we employ computational approaches to refine how PAF1 may contribute to both gene activation and suppression. Specifically focusing on transcriptional motifs and regulons, we predict gene regulatory elements strongly associated with PAF1, including those implicated in an immune response. Overall, our results suggest PAF1 is involved in innate immunity at several distinct axes of regulation.ConclusionsBy identifying PAF1-dependent gene expression across several pathogenic contexts, we confirm PAF1C to be a key mediator of innate immunity. Combining these transcriptomic profiles with potential regulatory networks corroborates the previously identified functions of PAF1C. With this, we foster new avenues for its study as a regulator of innate immunity, and our results will serve as a basis for targeted study of PAF1C in future validation studies.

Dynamics and immunomodulation of cognitive deficits and behavioral changes in non-severe experimental malaria.

Data recently reported by our group indicate that stimulation with a pool of immunogens capable of eliciting type 2 immune responses can restore the cognitive and behavioral dysfunctions recorded after a single episode of non-severe rodent malaria caused by Plasmodium berghei ANKA. Here we explored the hypothesis that isolated immunization with one of the type 2 immune response-inducing immunogens, the human diphtheria-tetanus (dT) vaccine, may revert damages associated with malaria. To investigate this possibility, we studied the dynamics of cognitive deficits and anxiety-like phenotype following non-severe experimental malaria and evaluated the effects of immunization with both dT and of a pool of type 2 immune stimuli in reversing these impairments. Locomotor activity and long-term memory deficits were assessed through the open field test (OFT) and novel object recognition task (NORT), while the anxiety-like phenotype was assessed by OFT and light/dark task (LDT). Our results indicate that poor performance in cognitive-behavioral tests can be detected as early as the 12th day after the end of antimalarial treatment with chloroquine and may persist for up to 155 days post infection. The single immunization strategy with the human dT vaccine showed promise in reversal of long-term memory deficits in NORT, and anxiety-like behavior in OFT and LDT.

089 3D Computational Modelling of Immune Interaction and Epidermal Homeostasis in Psoriasis

Psoriasis is a chronic inflammatory skin disease characterised by immune cytokine stimulated epidermal hyperproliferation. Previous modelling studies of psoriatic skin have been limited to 2D models and lacked cell-cell interactions. We developed the first 3D agent-based model of epidermal cell dynamics to gain insights into complex spatio-temporal changes, in particular how immune cytokine stimuli induces hyperproliferation altering epidermal cell division and structure in psoriasis. The agent-based model was implemented in state-of-art LAMMPS open-source, massively parallel simulator.

Functional immunophenotyping of children with critical status asthmaticus identifies differential gene expression responses in neutrophils exposed to a poly(I:C) stimulus

The host immune response to a viral immune stimulus has not been examined in children during a life-threatening asthma attack. We determined whether we could identify clusters of children with critical asthma by functional immunophenotyping using an intracellular viral analog stimulus. We performed a single-center, prospective, observational cohort study of 43 children ages 6–17 years admitted to a pediatric intensive care unit for an asthma attack between July 2019 to February 2021. Neutrophils were isolated from children, stimulated overnight with LyoVec poly(I:C), and mRNA was analyzed using a targeted Nanostring immunology array. Network analysis of the differentially expressed transcripts for the paired LyoVec poly(I:C) samples was performed. We identified two clusters by functional immunophenotyping that differed by the Asthma Control Test score. Cluster 1 (n = 23) had a higher proportion of children with uncontrolled asthma in the four weeks prior to PICU admission compared with cluster 2 (n = 20). Pathways up-regulated in cluster 1 versus cluster 2 included chemokine receptor/chemokines, interleukin-10 (IL-10), IL-4, and IL-13 signaling. Larger validation studies and clinical phenotyping of children with critical asthma are needed to determine the predictive utility of these clusters in a larger clinical setting.

Hemophagocytic Lymphohistiocytosis.

Hemophagocytic Lymphohistiocytosis.

Lipopolysaccharide distinctively alters human microglia transcriptomes to resemble microglia from Alzheimer's disease mouse models.

ABSTRACTAlzheimer's disease (AD) is the most common form of dementia, and risk-influencing genetics implicates microglia and neuroimmunity in the pathogenesis of AD. Induced pluripotent stem cell (iPSC)-derived microglia (iPSC-microglia) are increasingly used as a model of AD, but the relevance of historical immune stimuli to model AD is unclear. We performed a detailed cross-comparison over time on the effects of combinatory stimulation of iPSC-microglia, and in particular their relevance to AD. We used single-cell RNA sequencing to measure the transcriptional response of iPSC-microglia after 24 h and 48 h of stimulation with prostaglandin E2 (PGE2) or lipopolysaccharide (LPS)+interferon gamma (IFN-γ), either alone or in combination with ATPγS. We observed a shared core transcriptional response of iPSC-microglia to ATPγS and to LPS+IFN-γ, suggestive of a convergent mechanism of action. Across all conditions, we observed a significant overlap, although directional inconsistency to genes that change their expression levels in human microglia from AD patients. Using a data-led approach, we identify a common axis of transcriptomic change across AD genetic mouse models of microglia and show that only LPS provokes a transcriptional response along this axis in mouse microglia and LPS+IFN-γ in human iPSC-microglia. This article has an associated First Person interview with the first author of the paper.

Individualised computational modelling of immune mediated disease onset, flare and clearance in psoriasis.

Despite increased understanding about psoriasis pathophysiology, currently there is a lack of predictive computational models. We developed a personalisable ordinary differential equations model of human epidermis and psoriasis that incorporates immune cells and cytokine stimuli to regulate the transition between two stable steady states of clinically healthy (non-lesional) and disease (lesional psoriasis, plaque) skin. In line with experimental data, an immune stimulus initiated transition from healthy skin to psoriasis and apoptosis of immune and epidermal cells induced by UVB phototherapy returned the epidermis back to the healthy state. Notably, our model was able to distinguish disease flares. The flexibility of our model permitted the development of a patient-specific “UVB sensitivity” parameter that reflected subject-specific sensitivity to apoptosis and enabled simulation of individual patients’ clinical response trajectory. In a prospective clinical study of 94 patients, serial individual UVB doses and clinical response (Psoriasis Area Severity Index) values collected over the first three weeks of UVB therapy informed estimation of the “UVB sensitivity” parameter and the prediction of individual patient outcome at the end of phototherapy. An important advance of our model is its potential for direct clinical application through early assessment of response to UVB therapy, and for individualised optimisation of phototherapy regimes to improve clinical outcome. Additionally by incorporating the complex interaction of immune cells and epidermal keratinocytes, our model provides a basis to study and predict outcomes to biologic therapies in psoriasis.

The role of PAF in immunopathology: From immediate hypersensitivity reactions to fungal defense.

Platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) was discovered when the mechanisms involved in the deposition of immune complex in tissues were being scrutinized in the experimental model of rabbit serum sickness. The initial adscription of PAF to IgE-dependent anaphylaxis was soon extended after disclosing its release from phagocytes stimulated by calcium mobilizing agents, formylated peptides, and phagocytosable particles. This explains why ongoing research in the field turned to the analysis of immune cell types and stimuli involved in PAF production with the purpose of establishing its role in pathology. This was spurred by the identification of the chemical structure of PAF and the enzymic mechanisms involved in its biosynthesis and degradation, which showed commonalities with those involved in eicosanoid production and the Lands' cycle of phospholipid fatty acid remodeling. The reassignment of PAF function in immunopathology is explained by the finding that the most robust mechanisms leading to PAF production are associated with opsonic and non-opsonic phagocytosis, depending on the cell type. While polymorphonuclear leukocytes exhibit opsonic phagocytosis, monocyte-derived dendritic cells show a marked preference for non-opsonic phagocytosis associated with C-type lectin receptors. This is particularly relevant to the defense against fungal invasion and explains why PAF exerts an autocrine feed-forwarding mechanism required for the selective expression of some cytokines.

SARS-CoV-2 pneumonia and bacterial pneumonia patients differ in a second hit immune response model

Secondary infections have been shown to complicate the clinical course and worsen the outcome of critically ill patients. Severe Coronavirus Disease 2019 (COVID-19) may be accompanied by a pronounced cytokine release, and immune competence of these patients towards most pathogenic antigens remains uncompromised early in the disease. Patients with bacterial sepsis also exhibit excessive cytokine release with systemic hyper-inflammation, however, typically followed by an anti-inflammatory phase, causing immune paralysis. In a second hit immune response model, leukocyte activation capacity of severely ill patients with pneumonia caused by SARS-CoV-2 or by bacteria were compared upon ICU admission and at days 4 and 7 of the ICU stay. Blood cell count and release of the pro-inflammatory cytokines IL-2, IFNγ and TNF were assessed after whole-blood incubation with the potent immune stimulus pokeweed mitogen (PWM). For comparison, patients with bacterial sepsis not originating from pneumonia, and healthy volunteers were included. Lymphopenia and granulocytosis were less pronounced in COVID-19 patients compared to bacterial sepsis patients. After PWM stimulation, COVID-19 patients showed a reduced release of IFNγ, while IL-2 levels were found similar and TNF levels were increased compared to healthy controls. Interestingly, concentrations of all three cytokines were significantly higher in samples from COVID-19 patients compared to samples from patients with bacterial infection. This fundamental difference in immune competence during a second hit between COVID-19 and sepsis patients may have implications for the selection of immune suppressive or enhancing therapies in personalized medicine.

UPLC-HDMSE to discover serum biomarkers in adults with type 1 diabetes

Type 1 diabetes (T1D) is a complex disease with metabolic and functional changes that can alter an individual's proteome. An LC-MS/MS analytical method, in an HDMSE system, was used to identify differentially expressed proteins in the high abundance protein-depleted serum of T1D patients and healthy controls. Samples were processed in Progenesis QI for Proteomics software. A functional enrichment of the proteins was performed with Gene Ontology and ToppGene, and the interactions were visualized by STRING 11.5. As a result, 139 proteins were identified, 14 of which were downregulated in the serum of patients with T1D compared to controls. Most of the differentially expressed proteins were shown to be involved with the immune system, inflammation, and growth hormone stimulus response, and were associated with the progression of T1D. Differential protein expression data showed for the first-time changes in CPN2 expression levels in the serum of patients with T1D. Our findings indicate that these proteins are targets of interest for future investigations and for validation of protein biomarkers in T1D.