Lymphocyte Cytokine Production Research Articles

Effect of Collagen Peptides on Function of Mouse T Lymphocytes under Simulated Microgravity

To understand the effect of collagen peptides on the function of mouse lymphocytes under simulated microgravity. The splenocytes of mice were isolated, and the rotary cell culture system was used to simulate the microgravity. The T lymphocytes were stimulated with mitotic agents, concanavalin A (ConA), and the cells were treated with different concentrations of collagen peptides. The proliferation of lymphocytes and the levels of cytokines in the supernatant were detected. Simulated microgravity could inhibit the proliferation of spleen T lymphocytes and decrease the level of cytokines in the supernatant. Collagen peptides could promote the lymphocyte proliferation and cytokine production in cells cultured under simulated microgravity. Collagen peptides may attenuate the inhibitory effect of simulated microgravity on T lymphocytes by regulating the cell proliferation and the secretion of cytokines.

TRAF1 Deficiency in Macrophages Drives Exacerbated Joint Inflammation in Rheumatoid Arthritis.

The tumor necrosis factor receptor-associated factor 1 (TRAF1) plays a key role in promoting lymphocyte survival, proliferation, and cytokine production. Recent evidence showed that TRAF1 plays opposing roles in monocytes and macrophages where it controls NF-κB activation and limits pro-inflammatory cytokine production as well as inflammasome-dependent IL-1β secretion. Importantly, TRAF1 polymorphisms have been strongly linked to an increased risk of rheumatoid arthritis (RA). However, whether and how TRAF1 contributes to RA pathogenesis is not fully understood. Moreover, investigating the role of TRAF1 in driving RA pathogenesis is complicated by its multifaceted and opposing roles in various immune cells. In this study, we subjected wildtype (WT) mice to the collagen antibody-induced arthritis (CAIA) model of RA and injected them intra-articularly with WT- or TRAF1-deficient macrophages. We show that mice injected with TRAF1-deficient macrophages exhibited significantly exacerbated joint inflammation, immune cell infiltration, and tissue damage compared to mice injected with WT macrophages. This study may lay the groundwork for novel therapies for RA that target TRAF1 in macrophages.

HIV immunological non-responders are characterized by extensive immunosenescence and impaired lymphocyte cytokine production capacity.

Immunological non-responders (INR) are people living with HIV (PLHIV) who fail to fully restore CD4+ T-cell counts despite complete viral suppression with antiretroviral therapy (ART). INR are at higher risk for non-HIV related morbidity and mortality. Previous research suggest persistent qualitative defects. The 2000HIV study (clinical trials NTC03994835) enrolled 1895 PLHIV, divided in a discovery and validation cohort. PLHIV with CD4 T-cell count <350 cells/mm3 after ≥2 years of suppressive ART were defined as INR and were compared to immunological responders (IR) with CD4 T-cell count >500 cells/mm3. Logistic and rank based regression were used to analyze clinical data, extensive innate and adaptive immunophenotyping, and ex vivo monocyte and lymphocyte cytokine production after stimulation with various stimuli. The discovery cohort consisted of 62 INR and 1224 IR, the validation cohort of 26 INR and 243 IR. INR were older, had more advanced HIV disease before starting ART and had more frequently a history of non-AIDS related malignancy. INR had lower absolute CD4+ T-cell numbers in all subsets. Activated (HLA-DR+, CD38+) and exhausted (PD1+) subpopulations were proportionally increased in CD4 T-cells. Monocyte and granulocyte immunophenotypes were comparable. INR lymphocytes produced less IL-22, IFN-γ, IL-10 and IL-17 to stimuli. In contrast, monocyte cytokine production did not differ. The proportions of CD4+CD38+HLA-DR+ and CD4+PD1+ subpopulations showed an inversed correlation to lymphocyte cytokine production. INR compared to IR have hyperactivated and exhausted CD4+ T-cells in combination with lymphocyte functional impairment, while innate immune responses were comparable. Our data provide a rationale to consider the use of anti-PD1 therapy in INR.

The Immunomodulatory Effects of Herbal Supplements in Poultry

Purpose: The general purpose of the study was to explore the immunomodulatory effects of herbal supplements in poultry.&#x0D; Methodology: The study adopted a desktop research methodology. Desk research refers to secondary data or that which can be collected without fieldwork. Desk research is basically involved in collecting data from existing resources hence it is often considered a low cost technique as compared to field research, as the main cost is involved in executive’s time, telephone charges and directories. Thus, the study relied on already published studies, reports and statistics. This secondary data was easily accessed through the online journals and library.&#x0D; Findings: The findings reveal that there exists a contextual and methodological gap relating to immunomodulatory effects of herbal supplements in poultry. Preliminary empirical review revealed that herbal extracts enhanced immune parameters, such as lymphocyte proliferation and cytokine production, suggesting their potential to improve poultry health and disease resistance. The diverse mechanisms of action of phytochemicals underscored the complexity of herbal supplements' effects on poultry immunity, while considerations such as formulation and environmental factors influenced their efficacy. Overall, while further research is warranted to optimize formulations and deepen our understanding of mechanisms, herbal supplements show considerable promise as alternatives to conventional additives in poultry production, offering holistic and sustainable approaches to enhancing poultry health and welfare.&#x0D; Unique Contribution to Theory, Practice and Policy: The Systems Biology theory, Ecological Systems theory and Ethnopharmacology theory may be used to anchor future studies on immunomodulatory effects of herbal supplements in poultry. The study provided recommendations that contributed to theory, practice, and policy in poultry production and animal health. The findings advanced theoretical knowledge by elucidating the complex interactions between phytochemicals and the avian immune system. In practice, recommendations supported the incorporation of herbal supplements into poultry diets to promote immune health and disease resistance, benefiting poultry producers, veterinarians, and feed manufacturers. Moreover, policy recommendations advocated for regulatory frameworks supporting the use of herbal supplements and education initiatives aimed at enhancing stakeholder awareness and understanding. Collaboration between academia, industry, and government agencies was recommended to further research and innovation, while international cooperation facilitated knowledge exchange and harmonization of standards.&#x0D; Keywords: Immunomodulatory, Herbal Supplements, Poultry, Phytochemicals, Immune Function, Sustainable, Regulation, Collaboration, International Cooperation

Abstract 2505: Immune dynamics and response predictors to regorafenib, ipilimumab, and nivolumab (RIN) treatment in chemotherapy resistant microsatellite stable (MSS) metastatic colorectal cancer (mCRC)

Abstract Background: In our previous study, RIN therapy in MSS mCRC patients, excluding those with liver metastases, yielded a notable 36.4% objective response rate. RIN treatment was observed to remodel the local tumor microenvironment by enhancing immune infiltration, especially in responders. Systemic immunity, as assessed through peripheral blood analysis, showed that responders had lower baseline immune activity, which increased markedly post-treatment. Patients with liver metastases demonstrated a reduced CD4/CD8 T cell ratio, indicative of immunosenescence. Methods: Building on our prior research, we analyzed blood samples from 29 chemotherapy-resistant MSS mCRC patients undergoing RIN therapy, collected at baseline and after two consecutive treatment cycles. Two 28-marker spectral flow cytometry panels were used to assess immune senescence and cytokine production in lymphocytes. Additionally, T cell receptor repertoires and transcriptional profiles were analyzed using RNA sequencing from sorted non-naïve T cells. Results: The study unveiled distinct immunological profiles in MSS mCRC patient responders vs non-responders to RIN treatment. Responders demonstrated increased numbers of newly expanding CD8 T cell clones on-treatment, indicative of an active immune responses. In contrast, non-responders displayed evidence of increased DNA damage in circulating lymphocytes (T, B, and NK cells), as marked by elevated γ-H2AX levels. We also identified divergent cytokine production profiles patterns at baseline: T cells in non-responders demonstrated increased capacity for production of IL-2, IL-10, IL-4/-13, and IL-22, while production of IFN-γ in both T cells and NK cells correlated with improved outcomes. Notably, patients with liver metastases exhibited greater frequencies of senescent T cells, characterized by increased expression of senescence markers (SA-βGal, CD57, Granzyme B/K) and a reduction in CD27 and CD28 in CD4 T cells, along with an accumulation of terminally differentiated CD8 T cells both pre- and post-treatment. Additionally, RNA sequencing revealed an upregulation of FOXO1 related genes in non-naive T cells of responder patients, which is consistent with its important role for this pathway in attenuating senescence and maintenance of stem-like properties in T cells. Conclusions: The effectiveness of RIN therapy is associated with baseline functional potential of circulating T cells and an on-treatment expansion of T cell clonotypes. Pre-existing and persistent features of senescence and DNA damage in circulating lymphocytes may be a major barrier to RIN treatment efficacy in MSS colorectal cancer and should be validated further in future studies. Citation Format: Jian Ye, Chongkai Wang, Colt Egelston, Weihua Guo, Peter Lee, Marwan Fakih. Immune dynamics and response predictors to regorafenib, ipilimumab, and nivolumab (RIN) treatment in chemotherapy resistant microsatellite stable (MSS) metastatic colorectal cancer (mCRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2505.

Alteration in gut microbiota is associated with immune imbalance in Graves' disease.

Graves' disease (GD), characterized by immune aberration, is associated with gut dysbiosis. Despite the growing interest, substantial evidence detailing the precise impact of gut microbiota on GD's autoimmune processes remains exceedingly rare. This study was designed to investigate the influence of gut microbiota on immune dysregulation in GD. It encompassed 52 GD patients and 45 healthy controls (HCs), employing flow cytometry and enzyme-linked immunosorbent assay to examine lymphocyte and cytokine profiles, alongside lipopolysaccharide (LPS) levels. Gut microbiota profiles and metabolic features were assessed using 16S rRNA gene sequencing and targeted metabolomics. Our observations revealed a disturbed B-cell distribution and elevated LPS and pro-inflammatory cytokines in GD patients compared to HCs. Significant differences in gut microbiota composition and a marked deficit in short-chain fatty acid (SCFA)-producing bacteria, including ASV263(Bacteroides), ASV1451(Dialister), and ASV503(Coprococcus), were observed in GD patients. These specific bacteria and SCFAs showed correlations with thyroid autoantibodies, B-cell subsets, and cytokine levels. In vitro studies further showed that LPS notably caused B-cell subsets imbalance, reducing conventional memory B cells while increasing naïve B cells. Additionally, acetate combined with propionate and butyrate showcased immunoregulatory functions, diminishing cytokine production in LPS-stimulated cells. Overall, our results highlight the role of gut dysbiosis in contributing to immune dysregulation in GD by affecting lymphocyte status and cytokine production.

Screening of Ca2+ Influx in Lymphocytes.

The Ca2+ ion is an important second messenger in lymphocytes, similarly to its function in other mammalian cells. The generation of long-lasting intracellular Ca2+ elevations is essential for Ca2+-dependent gene transcription, proliferation, differentiation, and cytokine production in lymphocytes. Since store-operated Ca2+ entry (SOCE) is considered the predominant mode of Ca2+ influx in lymphocytes, the activation and function of lymphocytes can be generally predicted by monitoring SOCE. A method suitable for dynamic monitoring of Ca2+ influx using fura-2 labeling in lymphocytes is introduced in this chapter. Using this technique, large-scale screening of the activation status of primary or cultured lymphocytes can be realized.

Genetic profile of adaptive immune traits and relationships with parasite resistance and productivity in Scottish Blackface sheep

Gastrointestinal (GI) parasites cause significant production losses in grazing ruminants which can be mitigated by breeding animals resistant to disease. Lymphocyte cytokine production and parasite-specific Immunoglobulin A (IgA) are adaptive immune traits associated with immunity to GI parasites. To explore the utility of these traits for selective breeding purposes, this study estimated the genetic parameters of the immune traits in sheep and assessed their relationship with disease and productivity traits. Whole blood stimulation assays were performed on 1 040 Scottish Blackface lambs at two months of age in 2016–2017. Blood was stimulated with either pokeweed mitogen (PWM), a non-specific activator of lymphocytes, and Teladorsagia circumcincta (T-ci) larval antigen to activate parasite-specific T lymphocytes. The type of adaptive immune response was determined by quantifying production of cytokines interferon-gamma (IFN-γ), interleukin (IL)-4, and IL-10, which relate to T-helper type (Th) 1, Th2 and regulatory T cell responses, respectively. Serum T-ci specific IgA was also quantified. Heritabilities were estimated for each immune trait by univariate analyses. Genetic and phenotypic correlations were estimated between different immune traits, and between immune traits vs. disease and productivity traits that were recorded at three months of age. Disease phenotypes were expressed as faecal egg counts (FEC) of nematode parasites (Strongyles and Nematodirus), faecal oocyst counts (FOC) of coccidian parasites, and faecal soiling score; production was measured as lamb live weight. Significant genetic variation was observed in all immune response traits. Heritabilities of cytokine production varied from low (0.14 ± 0.06) to very high (0.77 ± 0.09) and were always significantly greater than zero (P < 0.05). IgA heritability was found to be moderate (0.41 ± 0.09). Negative associations previously identified between IFN-γ production and FOC, and IL-4 production and strongyle FEC, were not evident in this study, potentially due to the time-lag between immune and parasitology measures. Instead, a positive genetic correlation was found between FOC and PWM-induced IFN-γ production, while a negative genetic correlation was found between FOC and T-ci induced IL-10. Live weight was negatively genetically correlated with IFN-γ responses. Overall, IFN-γ and IL-4 responses were positively correlated, providing little evidence of cross-regulation of Th1 and Th2 immunity within individual sheep. Furthermore, T-ci specific IgA was highly positively correlated with PWM-induced IL-10, indicating a possible role for this cytokine in IgA production. Our results suggest that while genetic selection for adaptive immune response traits is possible and may be beneficial for parasite control, selection of high IFN-γ responsiveness may negatively affect productivity.

Sialic acid–modified der p 2 allergen exerts immunomodulatory effects on human PBMCs

BackgroundHouse dust mite (HDM) extract based allergen immunotherapy (AIT) to treat HDM allergy is substantially effective but still presents some safety and efficacy concerns that warrant improvement. Several major allergen-based approaches to increase safety and efficacy of AIT have been proposed. One of them is the use of the group 2 allergen, Der p 2. ObjectiveIn this study, we investigated the immunomodulatory effects of sialic acid-modified major allergen recombinant Der p 2 (sia-rDer p 2) on PBMCs from healthy volunteers. MethodsWe activated PBMCs with anti-CD3/CD28 antibodies and incubated them at 37°C for 6 days in the presence or absence of either native rDer p 2 or α2-3 sialic acid-modified rDer p 2 (sia-rDer p 2). We assessed the changes in CD4+T cell activation and proliferation by flow cytometry and changes in T lymphocyte cytokine production in cell culture supernatant by ELISA. ResultsWe observed that, PBMCs treated with sia-rDer p 2 presented with a markedly decreased expression of CD69 and an increased abundance of LAG-3+ lymphocytes compared to cells treated with rDer p 2. Moreover, PBMCs treated with sia-rDer p 2 showed a reduced production of IL-4, IL-13 and IL-5 and displayed a higher IL-10/IL-5 ratio compared to rDer p 2-treated PBMCs. ConclusionWe demonstrate that α2-3 sialic acids-modified rDer p 2 might be a safer option than native rDerp2 for Der p 2-specific AIT. This is most relevant in the early phase of AIT that is often characterized by heightened Th2 responses, as sia-rDer p 2 does not enhance the production of Th2 cytokines.

Reduced regulatory effects of bone marrow-derived mesenchymal stem cells on activated T lymphocytes and Th1/Th2 cytokine secretion in children with aplastic anemia.

Acquired aplastic anemia (AA) is a recognized immune-mediated disorder and abnormally activated T lymphocyte-mediated bone marrow destruction is considered to be its main pathogenesis. Whether abnormal activation of T lymphocytes would also damage bone marrow-derived MSCs remains to be further studied. The aim of this study was to analyze the extent of T lymphocyte activation and the levels of Th1/Th2 cytokines of AA patients, and to explore the immunomodulatory effects of BM-MSCs on IL-2-stimulated T lymphocyte activation and cytokine production in vitro by means of transwell co-culture assay and flow cytometry measurement. The intermediate (CD25+) activated T cells were dominant in peripheral blood, while the early (CD69+) and late (HLA-DR+) activated T cells were predominant in bone marrow. Severe AA patients have an obviously higher proportion of CD3+CD8+CD69+ T cells than NSAA cases. The levels of IL-2 and IL-6 in AA patients were slightly elevated and INF-γ was mildly decreased in comparison with normal individuals. BM-MSCs derived from AA could not effectively inhibit the IL-2-induced activation of T cells with higher proportions of CD25+CD3+CD4+, CD69+CD3+CD4+ and CD25+CD3+CD8+ T cells after co-culture, and they showed a decreased ability to balance the Th1/Th2 cytokine production. Moreover, they had less robust osteogenic differentiation and more prone to adipogenic differentiation. We concluded that abnormally excessive T cell activation accompanied by abnormal cytokine secretion may impair the function of BM-MSCs in children with aplastic anemia.

Structural characterization and immunomodulatory activity of a novel polysaccharide from Panax notoginseng.

Introduction: Polysaccharides are important components of Panax notoginseng that contribute to its immunomodulatory ability. This study aimed to isolate polysaccharides from notoginseng and investigate the structural feature and potential immunomodulatory activity. Methods: The polysaccharide was isolated from notoginseng by anion exchange and gel permeation chromatography. Its preliminary structure was characterized by Fourier transform infrared (FT-IR) spectroscopy, gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. The immunoregulatory function was further investigated in cyclophosphamide induced immunosuppressive mice, murine splenocytes and macrophages. Results: A novel homogeneous polysaccharide (PNPB1) was isolated from notoginseng with the molecular weight of 9.3 × 105 Da. Monosaccharide composition analysis indicated that PNPB1 consisted of Glc (88.2%), Gal (9.0%), Ara (2.4%) and trace GlcA, with the major backbone of (1→4)-linked α-Glcp, (1→6)-linked β-Glcp, and (1, 4→6)-linked β-Glcp. The polysaccharide was found to significantly enhance murine body weight, improve their thymus and spleen indices and increase the white blood cells (WBC). PNPB1 significantly enhanced splenic lymphocyte proliferation, NO and cytokine (TNF-α, IL-2, IL-10 and IFN-γ) production, as well as the phagocytosis and TLR2 expression of peritoneal macrophages, indicating potent immunoenhancement effect. Discussion: These findings provide a theoretical basis for elucidating the structure and immune activity of notoginseng polysaccharides.

Titanium Dioxide Nanoparticles Modulate Systemic Immune Response and Increase Levels of Reduced Glutathione in Mice after Seven-Week Inhalation

Titanium dioxide nanoparticles (TiO2 NPs) are used in a wide range of applications. Although inhalation of NPs is one of the most important toxicologically relevant routes, experimental studies on potential harmful effects of TiO2 NPs using a whole-body inhalation chamber model are rare. In this study, the profile of lymphocyte markers, functional immunoassays, and antioxidant defense markers were analyzed to evaluate the potential adverse effects of seven-week inhalation exposure to two different concentrations of TiO2 NPs (0.00167 and 0.1308 mg TiO2/m3) in mice. A dose-dependent effect of TiO2 NPs on innate immunity was evident in the form of stimulated phagocytic activity of monocytes in low-dose mice and suppressed secretory function of monocytes (IL-18) in high-dose animals. The effect of TiO2 NPs on adaptive immunity, manifested in the spleen by a decrease in the percentage of T-cells, a reduction in T-helper cells, and a dose-dependent decrease in lymphocyte cytokine production, may indicate immunosuppression in exposed mice. The dose-dependent increase in GSH concentration and GSH/GSSG ratio in whole blood demonstrated stimulated antioxidant defense against oxidative stress induced by TiO2 NP exposure.

Delayed Microbial Maturation Durably Exacerbates Th17-driven Asthma in Mice.

Microbial maturation disrupted by early-life dysbiosis has been linked with increased asthma risk and severity; however, the immunological mechanisms underpinning this connection are poorly understood. We sought to understand how delaying microbial maturation drives worsened asthma outcomes later in life and its long-term durability. Drinking water was supplemented with antibiotics on Postnatal Days 10-20. To assess the immediate and long-term effects of delaying microbial maturation on experimental asthma, we initiated house dust mite exposure when bacterial diversity was either at a minimum or had recovered. Airway hyperresponsiveness, histology, pulmonary leukocyte recruitment, flow cytometric analysis of cytokine-producing lymphocytes, and assessment of serum IgG1 (Immunoglobulin G1) and IgE (Immunoglobulin E) concentrations were performed. RT-PCR was used to measure IL-13 (Interleukin 13)-induced gene expression in sequentially sorted mesenchymal, epithelial, endothelial, and leukocyte cell populations from the lung. Delayed microbial maturation increased allergen-driven airway hyperresponsiveness and Th17 frequency compared with allergen-exposed control mice, even when allergen exposure began after bacterial diversity recovered. Blockade of IL-17A (Interleukin 17A) reversed the airway hyperresponsiveness phenotype. In addition, allergen exposure in animals that experienced delayed microbial maturation showed signs of synergistic signaling between IL-13 and IL-17A in the pulmonary mesenchymal compartment. Delaying microbial maturation in neonates promotes the development of more severe asthma by increasing Th17 frequency, even if allergen exposure is initiated weeks after microbial diversity is normalized. In addition, IL-17A-aggravated asthma is associated with increased expression of IL-13-induced genes in mesenchymal, but not epithelial cells.

Specific immunoglobulin G4 correlates with Th2 cytokine reduction in patients with allergic asthma treated by Dermatophagoides pteronyssinus subcutaneous immunotherapy

BackgroundThe modulations of lymphocyte subsets and cytokine production due to subcutaneous allergen immunotherapy (SCIT) are not fully clarified. ObjectiveWe investigated the changes in T-lymphocyte subsets and serum Dermatophagoides pteronyssinus-specific immunoglobulin G4 (Der-p sIgG4), as well as cytokine production during Der-p SCIT, in patients with allergic asthma. MethodsThis study involved 20 patients with allergic asthma who were receiving 156-week Der-p SCIT and 20 patients without SCIT (non-SCIT). We measured symptom and medication scores (SMS), serum Der-p sIgG4 levels, CD4+CD25+Foxp3+ T regulatory (Treg), CD4+IL-4−IFN-γ+ T-helper (Th) 1, and CD4+IL-4+IFN-γ− Th2 lymphocyte percentages in peripheral blood mononuclear cells (PBMCs) with/without Der-p extract stimulation at weeks 0, 4, 12, 16, 52, 104, and 156. Cytokine release inhibition assays were performed by incubation with serum from SCIT and non-SCIT patients, Der-p allergen, and PBMCs. Levels of interleukin (IL)-4, IL-5, IL-10, IL-13, IL-17, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β1 were evaluated in supernatant. ResultsWe found that SCIT patients had significantly lower SMS after week 52. Der-p sIgG4 levels in SCIT patients significantly increased at week 16 compared with non-SCIT subjects. CD4+IL-4+IFN-γ− Th2% in SCIT patients showed a significant decrease from weeks 104–156 compared with week 0, while no change was observed in CD4+CD25+Foxp3+ Treg and CD4+IL-4−IFN-γ+ Th1 percentages. IL-5, IL-13, IL-4, IL-17, and TNF-α levels in supernatant of PBMCs cultured with serum of SCIT patients after 16 weeks showed significant lower levels compared with non-SCIT patients, and showed significant reverse associations with Der-p sIgG4 levels. ConclusionSCIT induced Dep-p sIgG4 may be involved in downregulating Th2 cytokine production in Der-p allergic asthma patients.

Quantifying the Detrimental Effects of Multiple Freeze/Thaw Cycles on Primary Human Lymphocyte Survival and Function

The use of cryopreserved peripheral blood mononuclear cells is common in biological research. It is widely accepted that primary cells are rendered unusable by several freezing cycles, although this practice might be very helpful when the biological material is valuable and its re-collection is impractical. To determine the extent to which primary cells undergoing repeated freezing cycles are comparable to one another and to fresh samples, we evaluated overall lymphocyte viability, their proliferation and cytokine production capabilities, as well as the levels of 27 cell subtypes in ten human peripheral blood mononuclear cells frozen for five years and repeatedly thawed. As expected, we observed a progressive increase in cell death percentages on three rounds of thawing, but the frequency of the main lymphocyte subsets was stable across the three thawings. Nevertheless, we observed a significant reduction of B cell frequency in frozen samples compared to fresh ones. On repeated thawings and subsequent conventional stimulation, lymphocyte proliferation significantly decreased, and IL-10, IL-6, GM-CSF, IFN-gamma, and IL-8 showed a trend to lower values.

2072. Of Mice and Men: HIV-induced CD38 expression on CD8+ T lymphocytes contributes to mitochondrial dysfunction and chronic inflammation despite antiretroviral therapy

Abstract Background Antiretroviral treatment (ART) has increased the life expectancy of people with HIV (PWH), however, comorbidities are higher in PWH than HIV-negative individuals. This is partly due to accelerated cellular aging, a consequence of CD38 expression, mitochondrial dysfunction, and inflammation. However, the role of CD38-expressing T cells on accelerated cellular aging in PWH is not completely understood. Methods The proportion of cytotoxic CD38+ CD8+ T lymphocytes from peripheral blood mononuclear cells (PBMCs) of PWH and HIV negative-individuals was measured. PBMCs from PWH on ART were cultured with or without gag-specific peptide stimulation and cytokine production was detected by flow cytometry. Mitochondrial function from T cells of PWH on ART and healthy donors was compared. Last, we generated a humanized (NSG) mouse model and infected mice with HIV (Bal strain) to determine the proportion of CD38+ T cells and cytokine production. Results Mean CD38 expression on CD8+ T cells was significantly different between PWH on ART and HIV-negative individuals (Figure 1). In PWH, gag-specific peptide stimulation of CD8+ T cells significantly increased CD38 expression (Figure 2A) and augmented secretion of IFNγ+ and TNFα+ (Figure 2B). There was a higher expression of CD38 among CD8+ T cells that produced IFNγ and TNFα (Figures 2C). T cells in PWH have altered mitochondrial function (Figures 2D-2F). Finally, HIV-infected NSG mice had T cell changes consistent with HIV infection (Figure 3A), a higher proportion of CD38+ T cells with an activated phenotype (Figures 3B and 3C), and significantly higher levels of inflammatory cytokines (Figure 3D). Figure 1.CD38 expression in PWH and HIV-negative individuals.Mean %CD38+CD8+ T cells in people with HIV (PWH) on and off antiretroviral therapy (ART) and HIV-negative individuals. Statistical significance was tested by Kruskal-Wallis test with post hoc analysis (Dunn’s Multiple comparison test). ****, P value&amp;lt; 0.0001.Figure 2.Cytokine production in response to gag stimulation from CD38+CD8+ T cells and mitochondrial function in T cells of PWH. Figure 2. (A) Frequency of CD38+ cells in gag-peptide stimulated CD8+ T cells and unstimulated cells. (B) Frequency of IFNγ and TNFα producing CD8+ T cells in gag-stimulated and unstimulated cells. Proportion of CD38+ cells among (C) IFNγ-/IFNγ+ and TNFα-/TNFα+ gag-peptide stimulated CD8+ T cells. Statistical significance was tested by paired t test. (D) Mitochondrial mass was determined by measuring the mean fluorescence intensity (MFI) of mitochondria-specific dye mitotracker green (MTG) among CD8+ T lymphocytes. (D) Frequency of mitochondrial superoxide positive lymphocytes and level of mitochondrial superoxide (MFI) were determined by staining the CD8+ T lymphocytes with mitosox red dye. (F) Mitochondrial membrane depolarization was determined by the red to green ratio of MFIs of mitochondrial membrane potential-dependent dye JC-1 among the CD8+ T lymphocytes. Statistical significance was tested by Mann-Whitney test. *, P value&amp;lt; 0.05; **, P value&amp;lt; 0.01; ***, P value&amp;lt; 0.001; ns – not significant. PWH=People with HIV. HD= Healthy Donors. Figure 3.Lymphocyte changes, CD38 expression, and cytokine production in HIV-infected NSG mice.(A) Frequency of CD4+ and CD8+ T lymphocytes and comparison of ratio of the frequencies of CD4+ to CD8+ T cells between control and HIV infected humanized mice. Statistical significance determined by Mann-Whitney test. (B) Frequency of CD38+ and CD38+HLADR CD4+ (A) and CD8+ (B) T cells in HIV- infected humanized mice compared to control. Statistical significance determined by Mann-Whitney test. (D)Cytokine measurements of HIV-infected humanized mice and controls. Statistical significance was tested by independent t test. *, P value&amp;lt; 0.05; **, P value&amp;lt; 0.01; ns – not significant. HLADR= Human Leukocyte Antigen – DR isotype, IFN=Interferon, IP=Inducible Protein, MCP=Monocyte Chemoattractant Protein, TNF=Tumor Necrosis Factor, IL=Interleukin. Conclusion ART decreases CD38 expression on CD8+ T cells, but not to the levels of HIV-negative individuals. In vitro, T cells of PWH have altered mitochondrial function, and HIV-specific stimulation augments CD38 expression on CD8+ T cells and contributes to a proinflammatory response. These findings translate to a humanized mouse model, where HIV infection upregulates CD38 expression and cytokine production. Together, these data suggest that CD38 is a potential therapeutic target for mitigating the mitochondrial dysfunction and chronic inflammation that drive cellular aging and comorbidities in PWH. Disclosures Poonam Mathur, DO, MPH, American Academy of HIV Medicine: Honoraria|Med Learning Group: Speaker/presenter|NACCME: Advisor/Consultant|NKGP Pharma: Advisor/Consultant Shyam Kottilil, MD, PhD, Arbutus Pharmaceuticals: Grant/Research Support|Gilead: Grant/Research Support|Merck: Grant/Research Support|Regeneron Pharmaceuticals: Advisor/Consultant|Silverback Therapeutics: Advisor/Consultant|The Liver Company: Advisor/Consultant|Yufan Biotechnologies: Advisor/Consultant.

Metabolomic profile in patients with primary warm autoimmune haemolytic anaemia.

Autoimmune haemolytic anaemia (AIHA) is a rare clinical condition with immunoglobulin fixation on the surface of erythrocytes, with or without complement activation. The pathophysiology of AIHA is complex and multifactorial, presenting functional abnormalities of T and B lymphocytes that generate an imbalance between lymphocyte activation, immunotolerance and cytokine production that culminates in autoimmune haemolysis. In AIHA, further laboratory data are needed to predict relapse and refractoriness of therapy, and thus, prevent adverse side-effects and treatment-induced toxicity. The metabolomic profile of AIHA has not yet been described. Our group developed a cross-sectional study with follow-up to assess the metabolomic profile in these patients, as well as to compare the metabolites found depending on the activity and intensity of haemolysis. We analysed the plasma of 26 patients with primary warm AIHA compared to 150 healthy individuals by mass spectrometry. Of the 95 metabolites found in the patients with AIHA, four acylcarnitines, two phosphatidylcholines (PC), asymmetric dimethylarginine (ADMA) and three sphingomyelins were significantly increased. There was an increase in PC, spermine and spermidine in the AIHA group with haemolytic activity. The PC ae 34:3/PC ae 40:2 ratio, seen only in the 12-month relapse group, was a predictor of relapse with 81% specificity and 100% sensitivity. Increased sphingomyelin, ADMA, PC and polyamines in patients with warm AIHA can interfere in autoantigen and autoimmune recognition mechanisms in a number of ways (deficient action of regulatory T lymphocytes on erythrocyte recognition as self, negative regulation of macrophage nuclear factor kappa beta activity, perpetuation of effector T lymphocyte and antibody production against erythrocyte antigens). The presence of PC ae 34:3/PC ae 40:2 ratio as a relapse predictor can help in identifying cases that require more frequent follow-up or early second-line therapies.

IRF2 is required for development and functional maturation of human NK cells.

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells.

Abstract 15471: Neonatal Mesenchymal Stem Cells Rejuvenate Cardiac Myocardium by Regulating Macrophage Polarization Revealed by Single Cell Sequencing After Ischemia-Reperfusion Injury in Rats

Introduction: Stem cells are an emerging therapy for cardiovascular disease. The immune system is modulated by stem cell administration, therefore we sought to characterize the immune environment following stem cell administration. Hypothesis: Neonatal cardiac mesenchymal stem cells (nMSC) improved heart function through an acute sterile immune response characterized by the temporal and regional of macrophages. Methods: nMSC were isolated from healthy neonatal myocardium. Brown Norway rats underwent ischemia-reperfusion injury (IRI) by temporary ligation of left anterior descending artery for 45 minutes. Following closure of the chest cavity, neonatal mesenchymal stem cells (nMSCs), dead nMSCs (dnMSCs), or placebo was administered by intra-coronary injection. At day five heart was collected for single cell suspensions. CD45 positive selection was performed and fed into the Chromium Single Cell Gene Expression workflow (Pleasanton, CA). Single cell sequencing analysis was completed using the Seurat package on RStudio v1.3.1093 (Boston, MA). Echocardiography was completed on post-operative days 7 and 28 for rats not sacrificed for single cell sequencing. Results: Our results demonstrate that nMSC treatment after IRI injury can significantly recover left ventricular ejection fraction. Single-cell sequencing analysis of CD45+ cells identified multiple unique CD68+ clusters. Notably, macrophage cluster 2 primarily consisted of cells from the nMSC treated group, whereas the macrophage cluster 1 was primarily cells from the dnMSC and placebo groups. Gene ontology analysis of macrophage cluster 2 demonstrated that this cluster primarily consisted of macrophages strongly associated with various aspects of the activated immune response, including lymphocyte activation and cytokine production. Conversely, macrophage cluster 1 gene ontology analysis revealed a weaker immune response signature. Conclusions: nMSCs are a promising therapeutic candidate for the treatment of myocardial infarction. The mechanism of this therapy is likely related to macrophage activation that further enhances the immune response in the impacted and at-risk myocardium following IRI injury.

Tick feeding modulates the human skin immune landscape to facilitate tick-borne pathogen transmission.

During cutaneous tick attachment, the feeding cavity becomes a site of transmission for tick salivary compounds and tick-borne pathogens. However, the immunological consequences of tick feeding for human skin remain unclear. Here, we assessed human skin and blood samples upon tick bite and developed a human skin explant model mimicking Ixodes ricinus bites and tick-borne pathogen infection. Following tick attachment, we observed rapidly occurring patterns of immunomodulation, including increases in neutrophils and cutaneous B and T cells. T cells upregulated tissue residency markers, while lymphocytic cytokine production was impaired. In early stages of Borrelia burgdorferi model infections, we detected strain-specific immune responses and close spatial relationships between macrophages and spirochetes. Preincubation of spirochetes with tick salivary gland extracts hampered accumulation of immune cells and increased spirochete loads. Collectively, we showed that tick feeding exerts profound changes on the skin immune network that interfere with the primary response against tick-borne pathogens.