Frequency Of Immune Cells Research Articles

P0179 Spatial immune profiling of Crohn’s Disease fistula carcinomas - Defining a distinct cancer subtype

Abstract Background Fistula formation is a common and debilitating complication of Crohn’s disease (CD). Although rare, CD-associated fistula carcinomas present significant diagnostic and prognostic challenges. This study aims to identify disease-specific immune cell subsets in CD-associated fistula carcinomas. Methods The study analyzed tissue samples from 10 CD patients with fistula carcinomas, 7 patients with CD-associated fistulas, and 6 patients with sporadic colorectal cancer (CRC). In the tumor samples, the main tumor, infiltration front, and non-involved areas were included. Using a 36-marker panel, the immune landscape was characterized through imaging mass cytometry. The samples were processed, stained, and analyzed for immune cell compositions, cell-cell interactions, and spatial microenvironments. Results The immune infiltrate observed in fistula carcinomas displayed similarities to both CD fistulas and CRC. Fistula-carcinoma samples exhibited elevated levels of neutrophils, B cells, and CD163high macrophages. CRC main tumor samples exhibited a higher presence of intraepithelial CD8+ lymphocytes and CD163low macrophages. The highest levels of Cleaved Caspase-3 were observed in CRC main tumor samples, which exhibited a positive correlation with CD163low macrophages and cytotoxic T cells. Conversely, a negative correlation was observed between cleaved Caspase-3 and cytotoxic T cells in fistula-carcinoma main tumor samples. The analysis of cellular microenvironments and dimensionality reduction clustering based on immune cell frequencies revealed that fistula carcinomas exhibit a unique combination of immune cell characteristics derived from both CD fistulas and CRC. Conclusion The immune landscape of CD-associated fistula carcinomas exhibits characteristics of both CD fistulas and CRC, indicating a complex pathogenesis driven by chronic inflammation. These findings suggest that fistula carcinomas constitute a distinct cancer subtype, necessitating further research to develop targeted therapeutic strategies.

Protection Conferred by Gallid Alphaherpesvirus 2 Vaccines Against Immunosuppression Induced by Very Virulent Plus (vv+) Marek's Disease Virus Strains in Commercial Meat Type Chickens.

Very virulent plus Marek's disease virus (vv+MDV) induces severe immunosuppression in commercial chickens. In this study, we evaluated how three Gallid alphaherpesvirus 2 (GaHV-2) vaccines (CVI-988, rMd5-BAC∆Meq, and CVI-LTR) protected against two negative outcomes of vv+MDV infection: (1) reduced viability and frequency of immune cells in the spleen and (2) decreased efficacy of the CEO (chicken embryo origin) vaccine against infectious laryngotracheitis challenge. At 25 days post-infection with vv+MDV 686, all vaccines are protected against the reduced viability of splenocytes. However, there were differences in the frequency of splenic immunophenotypes among groups. Compared to the uninfected control, the frequency of B cells was reduced in the CVI-988/686 group but not in the rMd5-BAC∆Meq/686 and CVI-LTR/686 groups. T cell subset frequencies showed no difference between the negative controls and CVI-988/686; however, there was a reduction in activated CD4+ T cells in the rMd5-BAC∆Meq/686 group and in activated CD4+, activated CD8+, and γδ+ T cells in the CVI-LTR/686 group. We also demonstrated that the three vaccines protected against MDV-induced tumors, but only rMd5-BAC∆Meq and CVI-LTR protected against the negative impact of vv+MDV 648A strain on CEO vaccine efficacy. Our findings demonstrate important differences in the biology and/or mechanisms of protection of these vaccines.

Circulating immune biomarkers correlating with response in patients with metastatic renal cell carcinoma on immunotherapy.

As multiple front-line immune checkpoint inhibitor (ICI)-based combinations are approved for metastatic renal cell carcinoma, biomarkers predicting for ICI responses are needed past clinical prognostication scores and transcriptome gene expression profiling. Circulating markers represent opportunities to assess baseline and dynamic changes in immune cell frequency and cytokine levels while on treatment. We conducted an exploratory prospective correlative study of 33 patients with metastatic clear cell renal cell carcinoma undergoing treatment with ICIs and correlated changes in circulating immune cell subsets and cytokines with clinical responses to treatment. Cell frequencies and cytokine levels were compared between responders and non-responders using unpaired parametric t tests, using a pre-specified level of significance of p<0.05. Classical monocyte subsets (CD14+ CD16-), as well as seven cytokines (IL-12/23 p40, macrophage inflammatory protein-1a, macrophage inflammatory protein-1b, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, IL-8, and TNF-alpha) were higher at baseline for responding versus non-responding patients. Dynamic changes in thymus- and activation-regulation chemokine (TARC), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) also correlated with patients with ICI response. In summary, macrophage activating agents were observed to be important in ICI response and may highlight the importance of the innate immune response in ICI responses.

TLS and immune cell profiling: immunomodulatory effects of immunochemotherapy on tumor microenvironment in resectable stage III NSCLC.

The use of programmed death-1 (PD-1) inhibitors in the neoadjuvant setting for patients with resectable stage III NSCLC has revolutionized this field in recent years. However, there is still 40%-60% of patients do not benefit from this approach. The complex interactions between immune cell subtypes and tertiary lymphoid structures (TLSs) within the tumor microenvironment (TME) may influence prognosis and the response to immunochemotherapy. This study aims to assess the relationship between immune cells subtypes and TLSs to better understand their impact on immunotherapy response. This study initially compared the tertiary lymphoid structures (TLSs) density among patients who underwent immunochemotherapy, chemotherapy and upfront surgery using 123 tumor samples from stage-matched patients. Multiplex immunohistochemistry (mIHC) was employed to analyze the spatial distribution of PD-L1+CD11c+ cells and PD1+CD8+ T cells within TLSs. Cytometry by time-of-flight (CyTOF) was used to assess immune cell dynamics in paired biopsy and resection specimens from six patients who underwent immunochemotherapy. Key immune cells were validated in newly collected samples using flow cytometry, mIHC, and in vitro CAR-T cells model. Patients who underwent neoadjuvant chemotherapy or immunochemotherapy exhibited increased TLSs compared to those who opted for upfront surgery. The TLS area-to-tumor area ratio distinguished pCR+MPR and NR patients in the immunochemotherapy group. Spatial analysis revealed variations in the distance between PD-L1+CD11c+ cells and PD1+CD8+ T cells within TLSs in the immunochemotherapy group. CyTOF analysis revealed an increase in the frequency of key immune cells (CCR7+CD127+CD69+CD4+ and CD38+CD8+ cells) following combined therapy. Treatment responders exhibited an increase in CCR7+CD4+ Tcells,whereas CD38+CD8+ T cells were associated with compromised treatment effectiveness. Immunochemotherapy and chemotherapy increase TLSs and granzyme B+ CD8+ T cells in tumors. The TLS area-to-tumor ratio distinguishes responders from non-responders, with PD-L1+ dendritic cells near CD8+PD-1+ T cells linked to efficacy, suggesting that PD-1 inhibitors disrupt harmful interactions. Post-immunochemotherapy, CD8+ T cells increase, but CD38+CD8+ T cells show reduced functionality. These findings highlight the complex immune dynamics and their implications for NSCLC treatment.

FC04 Dynamic KC-neutrophil-fibroblast communication network centred on IL-36/TNFSF15 responses characterizes inflammatory responses in generalized pustular psoriasis

Abstract Generalized pustular psoriasis (GPP) is a severe psoriatic subtype characterized by epidermal neutrophil infiltration, often occurring in acute, potentially life-threatening episodes. However, the characterization of the immune microenvironment in GPP lesions compared with plaque psoriasis remains largely unknown. Here, we used single-cell RNA profiling to interrogate the transcriptomes of ∼60 000 single cells from GPP lesional skin (n = 13) and healthy adult skin (n = 4), combined with spatial RNA sequencing. GPP was associated with major shifts in multiple cell populations, including, in particular, increased frequency of myeloid immune cells and supraspinous keratinocytes. We identified a unique myeloid-derived neutrophil subset characterized by the absence of CASP8. CASP8− neutrophils were accompanied by high expression of inflammatory pathway genes, including RIPK1, NF-KB1, IL1B, CXCL1, and CXCL8 in a stable disease state and were found at low levels in an inactive state in healthy skin. Spatial mapping of these genes illustrated the neutrophil transition from pre-inflammation to a pro-inflammation state through casting off CASP8. In silico and in vitro co-culture studies demonstrated that the communication network between IL36G+ spinous KCs and neutrophils is enlarged in the GPP lesions, with TNFSF15 (TL1A) released from neutrophils exaggerating the inflammatory crosstalk. While there was a slight increase in Th17 cells, there was greater prominence of CD8+ cytotoxic T cells highlighted by upregulated GZMB, CCL5, NKG7, and PRF1 expression, hinting at a cytotoxicity tendency rather than inflammation response in GPP lesions. Notably, a subset of S100A9+ fibroblasts in GPP was observed to contribute to the amplification of the GPP immune network through the transition to a pro-inflammatory state, connected by ligand–receptor interactions with other spatially proximate immune cells, including Th17 cells, CD8+ Tc cells, neutrophils, and IL1B+ macrophages. In sum, these data provide an in-depth view of immune cell participation and highlight the role of neutrophil–KC–fibroblast crosstalk in GPP pathogenesis.

Physical inactivity exacerbates pathologic inflammatory signalling at the single cell level in patients with systemic lupus

Physical activity is an adjunctive therapy that improves symptoms in people living with systemic lupus erythematosus (SLE), yet the mechanisms underlying this benefit remain unclear. We carried out a cohort study of 123 patients with SLE enrolled in the California Lupus Epidemiology Study (CLUES). The primary predictor variable was self-reported physical activity, which was measured using a previously validated instrument. We analyzed peripheral blood mononuclear cell (PBMC) single-cell RNA sequencing (scRNA-seq) data available from the cohort. From the scRNA-seq data, we compared immune cell frequencies, cell-specific gene expression, biological signalling pathways, and upstream cytokine activation states between physically active and inactive patients, adjusting for age, sex and race. We found that physical activity influenced immune cell frequencies, with sedentary patients most notably demonstrating greater CD4+ T cell lymphopenia (Padj=0.028). Differential gene expression analysis identified a transcriptional signature of physical inactivity across five cell types. In CD4+ and CD8+ T cells, this signature was characterized by 686 and 445 differentially expressed genes (Padj<0.1). Gene set enrichment analysis demonstrated enrichment of proinflammatory genes in the TNF-α signalling through NF-kB, interferon-γ (IFN-γ), IL2/STAT5, and IL6/JAK/STAT3 signalling pathways. Computational prediction of upstream cytokine activation states suggested CD4+ T cells from physically inactive patients exhibited increased activation of TNF-α, IFN-γ, IL1Β, and other proinflammatory cytokines. Network analysis demonstrated interconnectivity of genes driving the proinflammatory state of sedentary patients. Findings were consistent in sensitivity analyses adjusting for corticosteroid treatment and physical function. Taken together, our findings suggest a mechanistic explanation for the observed benefits of physical activity in patients with SLE. Specifically, we find that physical inactivity is associated with altered frequencies and transcriptional profiles of immune cell populations and may exacerbate pathologic inflammatory signalling via CD4+ and CD8+ T cells. This work was supported by the US National Institutes of Health (NIH) (R01 AR069616, K23HL138461-01A1, K23AT011768) the US CDC (U01DP0670), and the CZ Biohub.

Neuropathological and cerebrospinal fluid correlates of choroid plexus inflammation in progressive multiple sclerosis

AbstractAmong the intrathecal inflammatory niches where compartmentalized inflammation persists and plays a pivotal role in progressive multiple sclerosis (MS), choroid plexus (CP) has recently received renewed attention. To better characterize the neuropathological/molecular correlates of CP in progressive MS and its potential link with other brain inflammatory compartments, such as perivascular spaces and leptomeninges, the levels, composition and phenotype of CP immune infiltration in lateral ventricles of the hippocampus were examined in 40 post‐mortem pathologically confirmed MS and 10 healthy donors, using immunochemistry/immunofluorescence and in‐situ sequencing. Significant inflammation was detected in the CP of 21 out of the 40 MS cases (52%). The degree of CP inflammation was found correlated with: number of CP macrophages (R: 0.878, p = 1.012 x 10‐13) and high frequency of innate immune cells expressing the markers MHC‐class II, CD163, CD209, CD11c, TREM2 and TSPO; perivascular inflammation (R: 0.509, p = 7.921 x 10‐4), and less with meningeal inflammation (R: 0.365, p = 0.021); number of active lesions (R: 0.51, p: 3.524 x 10‐5). However, it did not significantly correlate with any clinical/demographic characteristics of the examined population. In‐situ sequencing analysis of gene expression in the CP of 3 representative MS cases and 3 controls revealed regulation of inflammatory pathways mainly related to ‘type 2 immune response’, ‘defense to infections’, ‘antigen processing/presentation’. Analysis of 78 inflammatory molecules in paired post‐mortem CSF, the levels of fibrinogen (R: 0.640, p = 8.752 x 10‐6), PDGF‐bb (R: 0.470, p = 0.002), CXCL13 (R: 0.428, p = 0.006) and IL15 (R: 0.327, p = 0.040) were correlated with extent of CP inflammation. Elevated fibrinogen and complement deposition were found in CP and in underlying subependymal periventricular areas, according to “surface‐in” gradient associated with concomitant prominent microglia activation. CP inflammation, predominantly characterized by innate immunity, represents another key determinant of intrathecal, compartmentalised inflammation persisting in progressive MS, which may be possibly activated by fibrinogen and influence periventricular pathology, even without substantial association with clinical features.

Impact of breastfeeding and formula feeding on immune cell populations and blood cell parameters: an observational study.

Breastfeeding is associated with improved health outcomes in infancy and throughout adulthood as breast milk encompasses diverse immune-active factors that affect the ontogeny of the immune system in breastfed (BF) infants. Nevertheless, the impact of infant feeding on the immune system is poorly understood, and a comprehensive understanding of immune system development in human infants is lacking. In this observational study, we addressed the effects of different infant feeding approaches on cell populations and parameters in the peripheral blood of infants to gain insight into the innate and adaptive arms of the immune system. Using flowcytometric analysis, we performed complete blood counts and immunoprofiling of peripheral blood collected from BF and formula-fed (FF) infants at different ages. Our results showed that the blood of BF infants had a higher frequency of leukocytes and erythrocytes in early infancy. The hemoglobin concentration was enhanced in BF infants. However, the platelet count was comparable regardless of feeding regimen. We observed immunophenotypic differences between the two populations of infants, mirrored by improved frequency of innate and adaptive immune cells in BF infants.

Mass cytometry reveals cellular correlates of immune response heterogeneity to SARS-CoV-2 vaccination in the elderly

Heterogeneity in vaccine response, particularly in vulnerable populations like the elderly, represents a significant public health challenge. We conducted an in-depth examination of immune cell profiles before and after SARS-CoV-2 vaccination utilizing mass cytometry in a cohort of healthy Norwegian seniors (65–80 years). We have demonstrated that higher pre-vaccination frequencies of CD27+IgD- class-switched memory B cells and subsets of CD27-CD24+CD38+ transitional B cells were associated with a robust vaccine response. Post-vaccination, high responders exhibited increased frequencies of IFN-γ+CD4+ T cells with antigen recall and a concurrent decrease in CCR6(+) TH cell subset frequencies compared to low responders. The presence of a γδ T cell subset displaying polyfunctional cytokine responses was also associated with better vaccine response in the elderly. This in-depth profiling sheds light on inherent differences in immune cell frequencies and functions that may offer insights for targeted vaccination strategies in older populations.

Brain temperature, brain metabolites, and immune system phenotypes in temporal lobe epilepsy.

Epileptogenesis is linked to neuroinflammation. We hypothesized that local heat production caused by neuroinflammation can be visualized non-invasively invivo via brain magnetic resonance spectroscopic imaging (MRSI) and MRSI-thermometry (MRSI-t) and that there is a relationship in patients with temporal lobe epilepsy (TLE) between MRSI-t and brain metabolites choline and myo-inositol and between neuroimaging and cellular and serum biomarkers of inflammation. Thirty-six (36) participants, 18 with temporal lobe epilepsy (13 females) and 18 age-matched healthy controls (nine females), were enrolled prospectively and underwent MRSI/MRSI-t; TLE participants also provided blood samples. Temperature was measured using creatine as a reference metabolite. Analysis of Functional NeuroImages 3dttest++ tool was used to analyze voxel-level group differences in temperature, choline, and myo-inositol. Associations with immune cell subsets, cytokines, and chemokines related to inflammation were quantified using correlation coefficients with significant relationships as noted. Patients with TLE showed elevated temperature, choline, and myo-inositol in the temporal lobes. Higher brain temperature was associated with higher levels of cytokines and chemokines, including GM-CSF, TNF, IL-1β, and IL - 12p70, and lower frequency of immune cells including CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and classical monocytes. Higher choline was associated with higher levels of the cytokines including LT-α, IL-13, and IL-4, and higher myo-inositol was associated with a higher frequency of CD4+ T-cell and CD19+ B-cell subsets and higher levels of cytokines and chemokines including LT-α, IL-13, and CCL3. This study, for the first time, showed that in temporal lobes of patients with TLE temperature and metabolite changes correlate with cellular and serum biomarkers of inflammation. Our results provide support for further development of MRSI-t as a measure of neuroinflammation in epilepsy and potentially other neurological disorders and as an investigative and clinical tool. Neuroinflammation is associated with excessive heat production which can be visualized with magnetic resonance spectroscopic imaging and thermometry (MRSI-t). We prospectively investigated the relationship between MRSI-t and cellular and serum measures of peripheral inflammation in patients with temporal lobe epilepsy (TLE); we compared the results of MRSI-t in patients with TLE to healthy controls. We showed a relationship between the temperature elevations in TLE and elevations of various measures of peripheral inflammation. Our results support further development of MRSI-t as a measure of neuroinflammation in epilepsy and potentially other neurological disorders and as an investigative and clinical tool.

An immune signature of postoperative cognitive decline: a prospective cohort study.

Postoperative cognitive decline (POCD) is the predominant complication affecting patients over 60 years old following major surgery, yet its prediction and prevention remain challenging. Understanding the biological processes underlying the pathogenesis of POCD is essential for identifying mechanistic biomarkers to advance diagnostics and therapeutics. This study aimed to provide a comprehensive analysis of immune cell trajectories differentiating patients with and without POCD and to derive a predictive score enabling the identification of high-risk patients during the preoperative period. Twenty-six patients aged 60 years old and older undergoing elective major orthopedic surgery were enrolled in a prospective longitudinal study, and the occurrence of POCD was assessed 7 days after surgery. Serial samples collected before surgery, and 1, 7, and 90 days after surgery were analyzed using a combined single-cell mass cytometry and plasma proteomic approach. Unsupervised clustering of the high-dimensional mass cytometry data was employed to characterize time-dependent trajectories of all major innate and adaptive immune cell frequencies and signaling responses. Sparse machine learning coupled with data-driven feature selection was applied to the presurgery immunological dataset to classify patients at risk for POCD. The analysis identified cell-type and signaling-specific immune trajectories differentiating patients with and without POCD. The most prominent trajectory features revealed early exacerbation of JAK/STAT and dampening of inhibitory κB and nuclear factor-κB immune signaling responses in patients with POCD. Further analyses integrating immunological and clinical data collected before surgery identified a preoperative predictive model comprising one plasma protein and 10 immune cell features that classified patients at risk for POCD with excellent accuracy (AUC=0.80, P =2.21e-02 U -test). Immune system-wide monitoring of patients over 60 years old undergoing surgery unveiled a peripheral immune signature of POCD. A predictive model built on immunological data collected before surgery demonstrated greater accuracy in predicting POCD compared to known clinical preoperative risk factors, offering a concise list of biomarker candidates to personalize perioperative management.

IL15/IL15Rα complex induces an anti-tumor immune response following radiation therapy only in the absence of Tregs and fails to induce expansion of progenitor TCF1+ CD8 T cells.

This work seeks to understand whether IL15-incorporating treatments improve response to radiotherapy and uncover mechanistic rationale for overcoming resistance to IL15 agonism using novel therapeutic combinations. Orthotopic tumor models of PDAC were used to determine response to treatment. IL15-/- and Rag1-/- mouse models were employed to determine dependence on IL15 and CTLs, respectively. Flow cytometry was used to assess immune cell frequency and activation state. Phospho-proteomic analyses were used to characterize intracellular signaling pathways. We show that the combination of radiation therapy (RT) and an IL15/IL15Ra fusion complex (denoted IL15c) fails to confer anti-tumor efficacy; however, a CD8-driven anti-tumor immune response is elicited with the concurrent administration of an aCD25 Treg-depleting antibody. Using IL15-/- and Rag1-/- mice, we demonstrate that response to RT + IL15c + aCD25 is dependent on both IL15 and CTLs. Furthermore, despite an equivalent survival benefit following treatment with RT + IL15c + aCD25 and combination RT + PD1-IL2v, a novel immunocytokine with PD-1 and IL2Rβγ binding domains, CTL immunophenotyping and phospho-proteomic analysis of intracellular metabolites showed significant upregulation of activation and functionality in CD8 T cells treated with RT + PD1-IL2v. Finally, we show the immunostimulatory response to RT + PD1-IL2v is significantly diminished with a concurrent lack of TCF+ CD8 T cell generation in the absence of functional IL15 signaling. Our results are illustrative of a mechanism wherein unimpeded effector T cell activation through IL2Rβ signaling and Treg inhibition are necessary in mediating an anti-tumor immune response.

Characterising how a single bout of exercise in people with myeloma affects clonal plasma cell and immune effector cell frequency in blood, and daratumumab efficacy in vitro

Multiple myeloma is a haematological cancer characterised by the accumulation of clonal plasma cells in the bone marrow and is commonly treated with daratumumab, an anti-CD38 monoclonal antibody immunotherapy. Daratumumab often fails to induce stringent complete responses, due in part to resistance to antibody-dependent cellular cytotoxicity (ADCC) exerted by natural killer (NK)-cells and monocytes. Exercise bouts undertaken by healthy people induce lymphocytosis in blood, including to NK-cells and B-cells, but the effects of exercise are unknown in myeloma patients. In addition, whether exercise mobilises plasma cells has not been adequately investigated, and as such the potential impact of exercise on daratumumab treatment is unclear. In this exploratory pilot study, n = 16 smouldering multiple myeloma participants enrolled and n = 9 completed the study which comprised a bout of cycling 15% above anaerobic threshold for ∼30-min, with blood samples collected pre-, immediately post-, and 30-min post-exercise. Peripheral blood mononuclear cells were isolated from blood samples and incubated with the RPMI-8226 plasmacytoma cell line, with or without the presence of daratumumab to determine specific lysis using a calcein-release assay. Daratumumab-mediated cell lysis increased from 18.8% to 23.2% pre- to post-exercise, respectively (p < 0.001), owing to an increased frequency of CD3−CD56+CD16+ NK-cells (+348%), HLA-DR+CD14dimCD16+ monocytes (+125%), and HLA-DR+CD14+CD32+ monocytes (+41%) in blood (p < 0.01). However, overall, total plasma cells (CD38+CD138+) nor clonal plasma cells (CD38brightCD138+CD45−/dimCD19− with light-chain restriction) increased in blood (p > 0.05). Notably, we observed a 305% increase in NK-cells expressing CD38, the daratumumab target antigen, which might render NK-cells more susceptible to daratumumab-mediated fratricide – whereby NK-cells initiate ADCC against daratumumab-bound NK-cells. In conclusion, exercise modestly improved the efficacy of daratumumab-mediated ADCC in vitro. However, plasma cells were largely unchanged, and NK-cells expressing CD38 – the daratumumab target antigen – increased in blood. Future research should consider the optimal timings of exercise during daratumumab treatment in myeloma to avert exacerbation of daratumumab-mediated NK-cell lysis.

Nasally delivered chitosan-coated poly(lactide-co-glycolide) encapsulating honeybee venom enhances T helper 1-related immunity against Salmonella Typhimurium infection in pigs.

Salmonella Typhimurium is a significant zoonotic concern for human food poisoning and a substantial economic burden in the swine industry. We previously reported that nasally delivered chitosan-coated poly(lactide-co-glycolide) (PLGA) encapsulating honeybee venom (CP-HBV) could enhance CD4+ T helper 1 (Th1)-related immune responses in healthy pigs. Building upon these findings, the current study aimed to investigate the protective immune enhancement by nasally delivered CP-HBV in pigs challenged with S Typhimurium. 36 healthy, 4-week-old, female, Landrace X Yorkshire X Duroc pigs. 36 pigs were allocated into 3 groups: CP-HBV (n = 16), control (n = 16), and healthy baseline control (n = 4). CP-HBV and control groups were challenged with S Typhimurium 7 days post-treatment. Pigs from the healthy control group were sacrificed at 0 days postinfection (DPI), and 4 pigs from each of the control and CP-HBV groups were sacrificed at 1, 2, 4, and 7 DPI. Salmonella shedding, immune cell frequencies, cytokines, and transcriptional factor expression levels were measured. The CP-HBV group exhibited lower bacterial shedding and an enhanced Th1-related immune response characterized by an upregulation of CD4+ T cells and CD4+ Interferon-γ+ T cells, accompanied by increased expression of Th1-related cytokines and reduced expression of regulatory T cells and immunosuppressive cytokines compared to the control group. CP-HBV is a promising strategy for controlling Salmonella infections in pigs and improving public health.

Unsupervised clustering of brain tumor leukocytes to reveal atypical lymphoid cell subsets.

25 Background: The brain tumor immune microenvironment (TiME) is characterised by suppression of tumor-infiltrating leukocytes. The lymphocyte niche activity is particularly downregulated within this TiME milieu. Methods: Here, using data-driven approaches in flow cytometry and RNA sequencing analysis, we aimed at dissecting leukocyte cell types infiltrating human brain tumor TiME. Results: Unsupervised clustering provided T lymphocyte subsets including double-negative (CD3+CD4- CD8-) T cells (DNT). Our DNT phenotype was validated with an independent dataset, suggesting DNT to be compatible with γδ T cell phenotype. In our cohort, myeloid immune cell frequencies were associated with malignancy type as previously reported, while a gliosarcoma notably presented unexpectedly high lymphocyte frequencies. Consistently, double-positive (CD4+ CD8+) T cells (DPT) had higher frequency in the gliosarcoma than other tumors. The flow cytometry results were supported by transcriptome patterns and the activity of immune-related genes in these tumors. Conclusions: Our findings feature the benefit of data-driven approaches for brain TiME lymphocyte analysis, and show that primary brain tumors can entail atypical lymphocyte subsets like DNTs and DPT cells. Unbiased lymphocyte characterisation can potentially outline novel targets for immune checkpoint blockade therapy.

Frequency of CD4+CD25 + (High) Foxp3+ Regulatory T cells (Tregs) in Peripheral Blood of Pediatric Patients with Autism

Abstract Background Immune system dysfunctions have been observed in autistic spectrum of disorders (ASD). Autoimmunity may have a role in ASD. In addition, skewed cytokine responses, differences in total numbers and frequencies of immune cells and their subsets, neuroinflammation, both adaptive and innate immune dysfunction, altered levels of immunoglobulins and the presence of autoantibodies in some autistic children were reported. Objective To measure the frequency of CD4+CD25 + (High) Foxp3+ Tregs in peripheral blood of children with autism in relation to the disease severity. Patients and Methods This cross-sectional study involved 30 ASD patients were enrolled from the Psychiatry and Development clinic, Children’s Hospital, Ain Shams University, after excluding children with concomitant allergic diseases or infections. Another 30 healthy children were included as a control group. Severity of ASD and associated convulsions or impaired intelligence were demonstrated. The frequency of CD4+CD25 + (High) Foxp3+ Tregs was assessed using flow cytometry. Data collected were coded, tabulated and analyzed using Microsoft Excel software Statistical analysis (SPSS) (version 21, Chicago, IL, USA). Results Thirty patients with primary ASD were recruited, including 24 males (80%) and 6 females (20%) with mean age of 6.62 ± 2.11 years. Mild illness was diagnosed in 20 patients (66.7%) and severe illness was defined in 10 patients (33.3%)%). CD4+CD25 + (High) Foxp3+ Tregs were significantly lower among autistic children compared to healthy control (mean percentage in patients 15.41 ± 32.1%, mean percentage in control 49.2 ± 42.6%, P-value &amp;lt;0.001). The markers were not correlated with disease severity, epilepsy status or IQ level (P = 0.716, 0.729, 0.472, respectively). However,CD4+CD25 + (High) Foxp3+ Tregs were significantly lower among male patients compared to females(Median in males 1.25 (0-96), Median in females 2.25 (1.5-84.2). CD4+CD25 + (High) lymphocytes less than 3.15 cells were found to be 80% sensitive and 73.5% specific for ASD probability (AUC was 0.816). Whereas percentage of FOXP3 less than 2.55% showed 73.3% and 76.7% sensitivity and specificity, respectively (AUC was 0.753). Conclusion CD4+CD25 + (High) FOXP3, the master promotor of Tregs, was detected in lower levels in ASD Egyptian children. A level of expression lower than 2.5% is both sensitive and specific for ASD diagnosis. We recommend considering CD4+CD25 + (High) FOXP3 as a future therapeutic target in children with ASD to achieve early, efficient management.

A unified metric of human immune health.

Immunological health has been challenging to characterize but could be defined as the absence of immune pathology. While shared features of some immune diseases and the concept of immunologic resilience based on age-independent adaptation to antigenic stimulation have been developed, general metrics of immune health and its utility for assessing clinically healthy individuals remain ill defined. Here we integrated transcriptomics, serum protein, peripheral immune cell frequency and clinical data from 228 patients with 22 monogenic conditions impacting key immunological pathways together with 42 age- and sex-matched healthy controls. Despite the high penetrance of monogenic lesions, differences between individuals in diverse immune parameters tended to dominate over those attributable to disease conditions or medication use. Unsupervised or supervised machine learning independently identified a score that distinguished healthy participants from patients with monogenic diseases, thus suggesting a quantitative immune health metric (IHM). In ten independent datasets, the IHM discriminated healthy from polygenic autoimmune and inflammatory disease states, marked aging in clinically healthy individuals, tracked disease activities and treatment responses in both immunological and nonimmunological diseases, and predicted age-dependent antibody responses to immunizations with different vaccines. This discriminatory power goes beyond that of the classical inflammatory biomarkers C-reactive protein and interleukin-6. Thus, deviations from health in diverse conditions, including aging, have shared systemic immune consequences, and we provide a web platform for calculating the IHM for other datasets, which could empower precision medicine.

Interactions between immune cell types facilitate the evolution of immune traits.

An essential prerequisite for evolution by natural selection is variation among individuals in traits that affect fitness1. The ability of a system to produce selectable variation, known as evolvability2, thus markedly affects the rate of evolution. Although the immune system is among the fastest-evolving components in mammals3, the sources of variation in immune traits remain largely unknown4,5. Here we show that an important determinant of the immune system's evolvability is its organization into interacting modules represented by different immune cell types. By profiling immune cell variation in bone marrow of 54 genetically diverse mouse strains from the Collaborative Cross6, we found that variation in immune cell frequencies is polygenic and that many associated genes are involved in homeostatic balance through cell-intrinsic functions of proliferation, migration and cell death. However, we also found genes associated with the frequency of a particular cell type that are expressed in a different cell type, exerting their effect in what we term cyto-trans. The vertebrate evolutionary record shows that genes associated in cyto-trans have faced weaker negative selection, thus increasing the robustness and hence evolvability2,7,8 of the immune system. This phenomenon is similarly observable in human blood. Our findings suggest that interactions between different components of the immune system provide a phenotypic space in which mutations can produce variation with little detriment, underscoring the role of modularity in the evolution of complex systems9.

Flt3 agonist enhances immunogenicity of arenavirus vector-based simian immunodeficiency virus vaccine in macaques.

Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens are capable of inducing efficacious humoral and cellular immune responses in nonhuman primates. Several studies have evaluated the use of immune modulators to further enhance vaccine-induced T-cell responses. The hematopoietic growth factor Flt3L drives the expansion of various bone marrow progenitor populations, and administration of Flt3L was shown to promote expansion of dendritic cell populations in spleen and blood, which are targets of arenaviral vectors. Therefore, we evaluated the potential of Flt3 signaling to enhance the immunogenicity of arenaviral vaccines encoding SIV immunogens (SIVSME543 Gag, Env, and Pol) in rhesus macaques, with a rhesus-specific engineered Flt3L-Fc fusion protein. In healthy animals, administration of Flt3L-Fc led to a 10- to 100-fold increase in type 1 dendritic cells 7 days after dosing, with no antidrug antibody (ADA) generation after repeated dosing. We observed that administration of Flt3L-Fc fusion protein 7 days before arenaviral vaccine increased the frequency and activation of innate immune cells and enhanced T-cell activation with no treatment-related adverse events. Flt3L-Fc administration induced early innate immune activation, leading to a significant enhancement in magnitude, breadth, and polyfunctionality of vaccine-induced T-cell responses. The Flt3L-Fc enhancement in vaccine immunogenicity was comparable to a combination with αCTLA-4 and supports the use of safe and effective variants of Flt3L to augment therapeutic vaccine-induced T-cell responses.IMPORTANCEInduction of a robust human immunodeficiency virus (HIV)-specific CD4+ and CD8+ T-cell response through therapeutic vaccination is considered essential for HIV cure. Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens have demonstrated strong immunogenicity and efficacy in nonhuman primates. Here, we demonstrate that the immunogenicity of arenaviral vectors encoding SIV immunogens can be enhanced by administration of Flt3L-Fc fusion protein 7 days before vaccination. Flt3L-Fc-mediated increase in dendritic cells led to robust improvements in vaccine-induced T- and B-cell responses compared with vaccine alone, and Flt3L-Fc dosing was not associated with any treatment-related adverse events. Importantly, immune modulation by either Flt3L-Fc or αCTLA-4 led to comparable enhancement in vaccine response. These results indicate that the addition of Flt3L-Fc fusion protein before vaccine administration can significantly enhance vaccine immunogenicity. Thus, safe and effective Flt3L variants could be utilized as part of a combination therapy for HIV cure.

Elucidating the immune active state of HR+HER2- MammaPrint High 2 early breast cancer.

506 Background: The 70-gene signature, MammaPrint (MP), classifies patients (pts) with early-stage breast cancer (EBC) as having an UltraLow, Low, High 1 (H1), or High 2 (H2) risk of distant recurrence. I-SPY 2 showed that pts with MP H2, HR+HER2- tumors have significantly higher response rates to neoadjuvant chemotherapy + immunotherapy relative to pts with MP H1 tumors. Expression-based immune deconvolution provides more detailed information about immune cell function beyond conventional methods enumerating tumor infiltrating lymphocytes. To elucidate the underlying biology that mediates immune therapy response, we performed in silico analysis of full transcriptome data to characterize immune cell frequencies and antigen presentation in HR+HER2-, MP High Risk EBC from pts enrolled in FLEX. Methods: The prospective, observational FLEX Study (NCT03053193) includes stage I-III pts with EBC who received MP testing and consented to full transcriptome and clinical data collection. Women with HR+HER2-, and MP High Risk invasive ductal EBC were included (n = 2916). Tumors were stratified into MP H1 or MP H2. The R-package Limma was used to preprocess gene expression data. The gene signature-based method xCell was used to determine immune cell abundances for each group based on enrichment score. Expression of genes involved in “antigen processing and presentation KEGG pathway”, and immune regulation, were evaluated between groups. Differences in immune cell frequency and gene expression were determined by a t-test with an adjusted p-value &lt; 0.05. Results: MP classified 79% (n=2292) of HR+HER2- EBC as H1 and 21% (n=624) as H2. Pts with H2 EBC were more likely to be ≤ 50 years (32%) compared to pts with H1 tumors (23%). Although low ER staining (1-10%) tumors were more frequent in H2 vs H1 (15% vs 1%), a majority of H2 tumors (75%) had ER staining &gt; 10%. Nearly 30% of H2 tumors were Grade 1 or 2, whereas 22% of H1 tumors were Grade 3. H2 tumors had significantly higher frequency of antigen presenting cells (APCs) including activated dendritic cells and macrophages, CD4+ memory T cells, CD8+ T cells, memory B cells and plasma cells relative to H1 tumors. The genes expressing PD-1 and PDL-1 and genes involved in antigen processing, including B2Mand TAP1/2, and presentation, i.e., major histocompatibility (MHC) class I ( HLA-A, -B, -F) and class II molecules ( HLA-DM, -DQ), were significantly upregulated in H2 vs H1. Conclusions: H2 tumors exhibited a heighted immune active state compared to H1 tumors among HR+HER2- EBC pts. The presence of APCs and increased antigen presentation, which are critical in eliciting T- and B-cell activation may explain the improved response rates to immunotherapy observed in H2 tumors. Selecting pts based on grade or ER% alone may exclude pts likely to benefit from immune therapy. These data support ongoing trials evaluating the response and benefit of adding immunotherapy to standard of care treatment regimens in MP H2 tumors. Clinical trial information: NCT03053193 .