Peripheral Immune Cell Profiles Research Articles

Disease-associated immune cell endotypes in anti-MDA5-positive dermatomyositis using unbiased hierarchical clustering.

Clinical and prognostic features of Anti-MDA5-Positive Dermatomyositis (MDA5+ DM) are diverse. This study aimed to examine the peripheral immune cell profiles of patients with MDA5+ DM, identify disease endotypes related to the heterogeneous manifestations and prognosis, and guide individualized therapy regimen. This inpatient cohort included 123 patients with MDA5+ DM. Unsupervised hierarchical clustering analysis was used to derive disease endotypes from the circulating immune cell profiles on admission. Clinical symptoms, laboratory test results, inpatient treatments, and disease outcomes were then analyzed among the identified endotypes. Three disease endotypes in MDA5+ DM were identified from peripheral immune cell profiles. Endotype1 had the highest percentages of CD4+ T cells and monocytes, and the lowest percentage of neutrophils; Endotype2 had the highest percentage of B cells; Endotype3 had the highest percentage of CD8+ T cells and NK cells. Clinical and prognostic heterogeneity of the endotypes were revealed. Endotype1 had the lowest 3-month mortality with the high incidence of periungual capillary changes. Endotype2 and Endotype3 had higher prevalence of rapidly progressive interstitial lung disease (RPILD) and mortality at 3 months than Endotype1. Meanwhile, Endotype3 had higher pneumocystis jiroveci and CMV viremia cases with significantly elevated of activated CD8+ T cells and multiple cytokines than Endotype1. Clustering analysis of peripheral immune cell profiles identified three different endotypes in MDA5+ dermatomyositis. Endotpye2 and 3 showed higher RPILD, 3-month mortality, pneumocystis jiroveci and CMV viremia.

Pituitary-Adrenal Axis and Peripheral Immune Cell Profile in Long COVID.

In Long COVID, dysfunction in the pituitary-adrenal axis and alterations in immune cells and inflammatory status are warned against. We performed a prospective study in a cohort of 42 patients who suffered COVID-19 at least 6 months before attending the Long COVID unit at Althaia Hospital. Based on Post-COVID Functional Status, 29 patients were diagnosed with Long COVID, while 13 were deemed as recovered. The hormones of the pituitary-adrenal axis, adrenocorticotropin stimulation test, and immune cell profiles and inflammatory markers were examined. Patients with Long COVID had significantly lower EuroQol and higher mMRC scores compared to the recovered individuals. Their symptoms included fatigue, myalgia, arthralgia, persistent coughing, a persistent sore throat, dyspnoea, a lack of concentration, and anxiety. We observed the physiological levels of cortisol and adrenocorticotropin in individuals with or without Long COVID. The results of the adrenocorticotropin stimulation test were similar between both groups. The absolute number of neutrophils was lower in the Long COVID patients compared to recovered individuals (p < 0.05). The total count of B lymphocytes remained consistent, but Long COVID patients had a higher percentage of mature B cells compared to recovered participants (p < 0.05) and exhibited a higher percentage of circulating resident memory CD8+ T cells (p < 0.05) and Treg-expressing exonucleases (p < 0.05). Our findings did not identify adrenal dysfunction related to Long COVID, nor an association between adrenal function and clinical symptoms. The data indicated a dysregulation in certain immune cells, pointing to immune activation. No overt hyperinflammation was observed in the Long COVID group.

Repeated iv anti-CD20 treatment in multiple sclerosis: Long-term effects on peripheral immune cell subsets.

Repeated intravenous administration of anti-CD20 depleting monoclonal antibodies 6 months apart is among the highly effective treatment options in multiple sclerosis (MS). Here, we aimed to investigate peripheral immune cell subset depletion kinetics following either rituximab (RTX) or ocrelizumab (OCR) infusions in people with MS (pwMS). We studied pwMS treated de-novo with either RTX (n = 7) or OCR (n = 8). The examinations were scheduled before the initiation of anti-CD20 therapy and every 12 weeks for up to 15 months. Immunophenotyping of immune cell subsets in peripheral blood was performed by multiparametric fluorescence cytometry. A significant, persistent decrease of CD19+ B cells was observed already with the first anti-CD20 infusion (p < 0.0001). A significant proportional reduction of memory B cells within the B-cell pool was achieved only after two treatment cycles (p = 0.005). We observed a proportional increase of immature (p = 0.04) and naive B cells (p = 0.004), again only after the second treatment cycle. As for the peripheral T-cell pool, we observed a continuous proportional increase of memory T helper (TH) cells/central memory TH cells (p = 0.02/p = 0.008), while the number of regulatory T cells (Treg) decreased (p = 0.007). The percentage of B-cell dependent TH17.1 central memory cells dropped after the second treatment cycle (p = 0.02). No significant differences in the depletion kinetics between RTX and OCR were found. Peripheral immune cell profiling revealed more differentiated insights into the prompt and delayed immunological effects of repeated intravenous anti-CD20 treatment. The observation of proportional changes of some pathogenetically relevant immune cell subsets only after two infusion cycles deserves further attention.

Immune profiling in patients with glioblastoma treated with VT1021 in a phase I/II expansion study.

2021 Background: The cyclic peptide VT1021 is a first-in-class therapeutic agent that has been shown to inhibit tumor growth via stimulation of thrombospondin-1 (TSP-1) and reprogramming the immune tumor microenvironment (TME) in preclinical models. Subsequently, VT1021 has been tested in a phase I/II clinical study in solid tumors (NCT03364400) and has advanced to a phase II/III clinical study in glioblastoma (NCT03970447). VT1021 has demonstrated promising single-agent clinical activity against recurrent glioblastoma (rGBM) in a phase I/II expansion study. Among 22 evaluable subjects with rGBM, 3 had complete response (CR), 1 had partial response (PR), and 6 had stable disease (SD) with an average study duration of over 120 days. One subject has been on VT1021 treatment for &gt; 900 days with no measurable lesion left. The overall disease control rate (DCR) was 45%. Here, we sought to examine the peripheral immune cell profile(s) in response to VT1021 and explore the association between these profiles and clinical responses in rGBM subjects. Methods: In the phase I/II expansion study, 22 evaluable rGBM subjects received VT1021 at a dose of 11.8 mg/kg (twice per week intravenously) until disease progression. Peripheral blood samples were collected from all evaluable subjects; immune cell profiles were analyzed by flow cytometry. Results: The immune profiles following VT1021 treatment on day 1 did not vary significantly among rGBM subjects, regardless of response. Intriguingly, after long-term VT1021 treatment (day 53), our analysis of the immune profiles revealed beneficial and sustained changes in three cytotoxic T Cell (CTL) parameters and PD-L1+ MDSCs that correlated with response. Specifically, CR/PR subjects showed a 20% increase in total CTLs (Pre 33.79% vs. 6hr 40.42%), a 66% increase in proliferating CTLs (Pre 1.37% vs. 6hr 2.28%) and a 63% increase in CTL/Treg ratio (Pre 7.85 vs. 6hr 12.81) on day 53 of VT1021 treatment. In contrast, SD and PD subjects exhibited no change or decrease in these three CTL parameters, respectively. We also observed that CR/PR subjects experienced a sustained decrease in PD-L1+ MDSCs after VT1021 treatment (day 1 Pre 95.96% vs. day 53 Pre 92.66% and day 53 6hr 88.06%). Conversely, the sustained decrease of PD-L1+ MDSCs after VT1021 treatment was not observed in SD and PD subjects. Conclusions: Here we report that the durable responses of peripheral immune cells to VT1021 may be associated with better clinical outcomes in rGBM subjects. These findings are consistent with the MOA of TSP-1 to reprogram the TME by stimulating immune and inflammatory cell functions. While the interpretation of our data is limited by the lack of a control arm and the small sample size, the results provide a signal and warrant continued analysis of these parameters in the ongoing phase II/III trial.

Peripheral immune cell profiling of double-hit lymphoma by mass cytometry

BackgroundDouble-hit or Triple-hit lymphoma (DHL/THL) is a subset of high-grade B cell lymphoma harboring rearrangements of MYC and BCL2 and/or BCL6, and usually associate with aggressive profile, while current therapies tend to provide poor clinical outcomes and eventually relapsed. Further explorations of DHL at cellular and molecular levels are in demand to offer guidance for clinical activity.MethodsWe collected the peripheral blood of DHL patients and diffused large B cell lymphoma (DLBCL) patients from single institute and converted them into PBMC samples. Mass cytometry was then performed to characterize these samples by 42 antibody markers with samples of healthy people as control. We divided the immune cell subtypes based on the expression profile of surface antigens, and the proportion of each cell subtype was also analyzed. By comparing the data of the DLBCL group and the healthy group, we figured out the distinguished immune cell subtypes of DHL patients according to their abundance and marker expression level. We further analyzed the heterogeneity of DHL samples by pairwise comparison based on clinical characteristics.ResultsWe found double-positive T cells (DPT) cells were in a significantly high percentage in DHL patients, whereas the ratio of double-negative T cells (DNT) was largely reduced in patients. Besides, CD38 was uniquely expressed at a high level on some naïve B cells of DHL patients, which could be a marker for the diagnosis of DHL (distinguishing from DLBCL), or even be a drug target for the treatment of DHL. In addition, we illustrated the heterogeneity of DHL patients in terms of immune cell landscape, and highlighted TP53 as a major factor that contributes to the heterogeneity of the T cells profile.ConclusionOur study demonstrated the distinct peripheral immune cell profile of DHL patients by contrast to DLBCL patients and healthy people, as well as the heterogeneity within the DHL group, which could provide valuable guidance for the diagnosis and treatment of DHL.

Probiotic supplementation has sex-dependent effects on immune responses in association with the gut microbiota in community-dwelling older adults: a randomized, double-blind, placebo-controlled, multicenter trial.

Probiotics have been suggested as potent modulators of age-related disorders in immunological functions, yet little is known about sex-dependent effects of probiotic supplements. Therefore, we aimed to investigate sex-dependent effects of probiotics on profiles of the gut microbiota and peripheral immune cells in healthy older adults. In a randomized, double-blind, placebo-controlled, multicenter trial, healthy elderly individuals ≥ 65 yrs old were administered probiotic capsules (or placebo) for 12 wk. Gut microbiota was analyzed using 16S rRNA gene sequencing and bioinformatic analyses. Peripheral immune cells were profiled using flow cytometry for lymphocytes (natural killer, B, CD4+ T, and CD8+ T cells), dendritic cells, monocytes, and their subpopulations. Compared with placebo, phylum Firmicutes was significantly reduced in the probiotic group in women, but not in men. At the genus level, sex-specific responses included reductions in the relative abundances of pro-inflammatory gut microbes, including Catabacter and unclassified_Coriobacteriales, and Burkholderia and unclassified Enterobacteriaceae, in men and women, respectively. Peripheral immune cell profiling analysis revealed that in men, probiotics significantly reduced the proportions of dendritic cells and CD14+ CD16- monocytes; however, these effects were not observed in women. In contrast, the proportion of total CD4+ T cells was significantly reduced in women in the probiotic group. Additionally, serum lipopolysaccharide-binding protein levels showed a decreasing tendency that were positively associated with changes in gut bacteria, including Catabacter (ρ = 0.678, P < 0.05) and Burkholderia (ρ = 0.673, P < 0.05) in men and women, respectively. These results suggest that probiotic supplementation may reduce the incidence of inflammation-related diseases by regulating the profiles of the gut microbiota and peripheral immune cells in healthy elders in a sex-specific manner.

Translational potential of JAX humanized APOE mice: Peripheral biomarkers of pathology

AbstractBackgroundAlzheimer’s Disease (AD) drives the global rising incidence of neurodegenerative dementia. Disease progression includes cerebral aggregation of amyloid‐beta plaques and immune activation. Many AD animal models carry mutations that cause an early onset of these phenotypes, but the mutations are rare in human populations; most cases are idiopathic and late‐onset AD (LOAD). Another approach utilizing a transgenic APOE animal model may better model clinical populations. APOE4 is the predominant genetic risk factor for LOAD and confers increasing risk for LOAD in allelic fashion, relative to normative APOE3. This study investigates the JAX humanized APOE knock‐in mouse model of LOAD’s translational potential for peripheral biomarkers of pathology in aged, male and female mice.MethodAged mice of both sexes (23‐25 months old; 39M/37F) with APOE3/3,APOE3/4, andAPOE4/4 genotypes underwent blood plasma amyloid‐beta (Aß) measurement. A subset of these animals (30M/28F) additionally received peripheral immunophenotyping of whole blood via flow cytometry. Two‐way ANOVA and post‐hoc t‐tests were conducted to determine data significance.ResultsFemale mice had lower Aß42 plasma levels as well as a decreased Aß42/40 ratio, suggesting higher levels of the aggregate‐prone Aß42 protein in the brain, but there were no observed APOE genotype effects. Blood plasma Aß40 levels did not differ by genotype or sex. Whole blood flow cytometry showed an elevation in CD8+ memory T cells in APOE4/4 mice relative to APOE3/3 andAPOE3/4 mice. Aged female mice had a peripheral immune cell profile consistent with a more reactive and dysfunctional immune system than genotype‐matched aged males: higher levels of CD11b+ B cells, CD4+ helper T cells, and CD8+ cytotoxic T cells along with a lower percentage of naïve CD4+ and CD8+ T cells.ConclusionIn mice aged to resemble a ∼70‐year‐old human population, these results suggest increased brain levels of the aggregate‐prone Aß42 isoform and a reactive peripheral immune system in female APOE mice. This sex difference is consistent with the increased prevalence of LOAD in women. However, the specificity of APOE4 genotype effects may define the translational potential of this humanized APOE mouse model of late‐onset Alzheimer’s Disease.Research supported by T32AG061897, R01AG057931, and RF1AG059093 to RDB.

EP16.02-017 Predictive Value of Peripheral Blood Immune Cell Profiling in EGFR-T790M Mutant Lung Adenocarcinoma

Peripheral immune cell profiles have been shown to correlate with clinical outcomes in cancer patients. Exploring the peripheral blood immune cell profile of lung adenocarcinoma (LUAD) patients will likely be a biomarker for predicting the efficacy of osimertinib.

The immune cell profile of the developing rat brain

Little is known about the peripheral immune cell (PIC) profile of the developing brain despite growing appreciation for these cells in the mature nervous system. To address this gap, the PIC profile, defined as which cells are present, where they are located, and for how long, was examined in the developing rat using spectral flow cytometry. Select regions of the rat brain (cerebellum, hippocampus, and hypothalamus) were examined at embryonic day 20, and postnatal days 0, 7 and 16. At their peak (E20), PICs were most abundant in the cerebellum, then the hippocampus and hypothalamus. Within the PIC pool, monocytes were most prevalent in all regions and time points, and shifted from being majority classical at E20 to non-classical by PN7. T cells increased over time, and shifted from majority cytotoxic to T-helper cells by PN7. This suggests the PIC profile transitions from reactive to adaptive and surveilling in the second postnatal week. NK cells and mast cells increased temporarily, and mast cells were restricted to the hippocampus and hypothalamus, suggesting they may play a specific role in the development of those regions. Mimicking a viral infection by administration of Poly I:C increased the influx of PICs into the neonatal brain, particularly of NK cells and in the case of males only, non-classical monocytes. This work provides a map for researchers as they study immune cell contributions to healthy and pathological brain development.

Integrated multi-omic characterization of congenital heart disease.

The heart, the first organ to develop in the embryo, undergoes complex morphogenesis that when defective results in congenital heart disease (CHD). With current therapies, more than 90% of patients with CHD survive into adulthood, but many suffer premature death from heart failure and non-cardiac causes1. Here, to gain insight into this disease progression, we performed single-nucleus RNA sequencing on 157,273 nuclei from control hearts and hearts from patients with CHD, including those with hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot, two common forms of cyanotic CHD lesions, as well as dilated and hypertrophic cardiomyopathies. We observed CHD-specific cell states in cardiomyocytes, which showed evidence of insulin resistance and increased expression of genes associated with FOXO signalling and CRIM1. Cardiac fibroblasts in HLHS were enriched in a low-Hippo and high-YAP cell state characteristic of activated cardiac fibroblasts. Imaging mass cytometry uncovered a spatially resolved perivascular microenvironment consistent with an immunodeficient state in CHD. Peripheral immune cell profiling suggested deficient monocytic immunity in CHD, in agreement with the predilection in CHD to infection and cancer2. Our comprehensive phenotyping of CHD provides a roadmap towards future personalized treatments for CHD.

Immune response to SARS-CoV-2 vaccination in relation to peripheral immune cell profiles among patients with multiple sclerosis receiving ocrelizumab

BackgroundVaccination has proven to be effective in preventing SARS-CoV-2 transmission and severe disease courses. However, immunocompromised patients have not been included in clinical trials and real-world clinical data point to...

Changes in lymphocytes, neutrophils and immunoglobulins in year-1 cladribine treatment in multiple sclerosis

Cladribine is a synthetic purine nucleoside analogue, approved for the treatment of active multiple sclerosis (MS). Cladribine mechanism-of-action encompasses selective cytotoxicity on circulating autoreactive B and T lymphocytes, which are thought to drive inflammatory demyelination and neuro-axonal loss in MS ( Compston and Coles, 2008 Compston A. Coles A. Multiple sclerosis. Lancet. 2008; 372: 1502-1517https://doi.org/10.1016/S0140-6736(08)61620-7 Abstract Full Text Full Text PDF PubMed Scopus (3270) Google Scholar ). Looking at clinical trial results, during cladribine treatment, there is a marked and long-lasting CD19 B-cell depletion and a rather modest T-cell depletion ( Baker et al., 2017 Baker D. Herrod S.S. Alvarez-Gonzalez C. et al. Both cladribine and alemtuzumab may effect MS via B-cell depletion. Neurol. Neuroimmunol. NeuroInflamm. 2017; 4: e360 Crossref PubMed Scopus (71) Google Scholar ; Stuve et al., 2019 Stuve O. Soerensen P. Leist T. et al. Effects of cladribine tablets on lymphocyte subsets in patients with multiple sclerosis: an extended analysis of surface markers. Ther. Adv. Neurol. Disord. 2019; 12: 1-6 Crossref Scopus (40) Google Scholar ; Comi et al., 2019 Comi G. Cook S. Giovannoni G. et al. Effect of cladribine tablets on lymphocyte reduction and repopulation dynamics in patients with relapsing multiple sclerosis. Mult. Scler. Relat. Disord. 2019; 29: 168-174 Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar ). Ideally, cladribine only targets memory B cells ( Moser et al., 2020 Moser T. Schwenker K. Seiberl M. et al. Long-term peripheral immune cell profiling reveals further targets of oral cladribine in MS. Ann. Clin. Transl. Neurol. 2020; 7: 2199-2212 Crossref PubMed Scopus (14) Google Scholar ), while plasma cells should remain unaffected ( Jacobs et al., 2018 Jacobs B.M. Ammoscato F. Giovannoni G. et al. Cladribine: mechanisms and mysteries in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry. 2018; 89: 1266-1271 Crossref PubMed Scopus (25) Google Scholar ), with normal immunoglobulin (Ig) levels, though never explored.

Multimarker scores of Th1 and Th2 immune cellular profiles in peripheral blood predict response and immune related toxicity with CTLA4 blockade and IFNα in melanoma

Neoadjuvant therapy with ipilimumab in combination with high dose IFNα was evaluated in patients with locally/regionally advanced melanoma in a previously reported clinical trial [NCT01608594]. In this study, peripheral immune cell profiling was performed in order to investigate the underlying mechanisms of tumor immune susceptibility and resistance. Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 28) were collected at baseline and then at 6-weeks, 3-months and 12-months. High complexity (14-color) flow cytometry, designed to detect key immunological biomarkers was used to evaluate the frequencies of immune cell subsets. Statistical significance was determined using R-package employing Kruskal's test. We found that higher levels of Th1 cells at baseline (defined as CD45RA- CCR6- CXCR3+ CCR4-) correlated with the preoperative radiological response (p = 0.007) while higher Th2 cells (defined as CD45RA- CCR6- CXCR3- CCR4+) were associated with progressive disease (p = 0.009). A multimarker score consisting of higher levels of Th1 cells and CD8+ central memory T-cells was associated with pathologic complete response (pCR) (p = 0.041) at surgical resection. On the other hand, high TIM3 expression on T-cells correlated with gross viable tumor (p = 0.047). With regard to immune related toxicity, higher levels of phenotypically naive (defined as CCR7+CD45RA+) and effector memory (defined as CCR7-CD45RO+) CD8+ T-cells (p = 0.014) or lower levels of Th2 cells were associated with lower toxicity (p = 0.024). Furthermore, a multimarker score consisting of higher CD19+ and CD8+ cells was associated with lower toxicity (p = 0.0014). In conclusion, our study yielded mechanistic insights related to the immune impact of CTLA4 blockade and IFNα and potential biomarkers of immune response and toxicity.

Long-term peripheral immune cell profiling reveals further targets of oral cladribine in MS.

ObjectivesTo expand the knowledge about the immunological consequences of cladribine (CLAD), a pulsed immune reconstitution therapy approved for active multiple sclerosis (MS), beyond the known short‐term effects on peripheral immune cell subsets.MethodsIn this study, we characterized depletion and restitution kinetics as well as cytokine profiles of peripheral immune cell subsets in 18 patients with MS following treatment with oral CLAD. The methods involved blood collection prior to CLAD and every three months over a period of 24 months, and extensive characterization of various immune cells subsets by multiparametric flow cytometry.ResultsWe found a selectivity of CLAD towards central memory T cells and memory B cells and detected a hyper‐repopulation of maturing B cells. Counts of classical (−65%) and various nonclassical TH17 cells (−84% to −87%) were markedly reduced 24 months after treatment start, and were comparable with depletion rates of class‐switched memory B‐cell phenotypes (−87% to −95%). The nadir of TH cells was more pronounced in the second treatment year. We observed a proportional surge of CD20 T‐cell subsets and an expansion of regulatory T, B and NK cells. Natural killer T cells (NKT) were only depleted in year two and did not recover.InterpretationPeripheral immune cell profiling revealed more differentiated insights into the immunological effects of CLAD. While some immune cell subsets expanded, we also observed additive depleting effects after the second treatment course. Further studies are required to elucidate whether these changes are paramount for the consistent and prolonged disease‐modifying effect of CLAD.

Intrastriatal injection of preformed alpha-synuclein fibrils alters central and peripheral immune cell profiles in non-transgenic mice

Parkinson’s disease (PD) is characterized by the accumulation of alpha-synuclein (α-syn) inclusions, the major component of Lewy bodies. Extracellular α-syn aggregates act as a damage-associated molecular pattern (DAMP) and the presence of autoantibodies against α-syn species in the cerebrospinal fluid and the serum of PD patients implicate the involvement of innate and adaptive immune responses. In non-transgenic (Tg) mice, intrastriatal injection of preformed fibril (PFF) α-syn results in widespread pathologic α-syn inclusions in the CNS. While the PFF model has been broadly utilized to study the mechanistic relationship between α-syn transmission and other neuropathological phenotypes, the immune phenotypes in this model are not clearly demonstrated. This study aimed to characterize the immune phenotypes during pathologic α-syn propagation by utilizing PFF α-syn–injected non-tg mice. Here, we showed that pathologic α-syn inclusions are prevalent in various brain regions and the gut at 5 months post injection (p.i.), preceding the degeneration of dopaminergic neurons in substantia nigra (SN). We discovered a distinct inflammatory response involving both activation of microglia and astrocytes and infiltration of B, CD4+ T, CD8+ T, and natural killer cells in the brain at 5 months p.i. Moreover, PFF α-syn–injected mice display significant alterations in the frequency and number of leukocyte subsets in the spleen and lymph nodes with minimum alterations in the blood. Our data provide primary evidence that intracerebral-initiated synucleinopathies in non-tg mice alter immune cell profiles both in the CNS and peripheral lymphoid organs. Furthermore, our data provides support for utilizing this mouse model to assess the mechanistic connection between immune responses and synuclein pathology.

Peripheral immune cell profile and activation status in chronic HCV mono- and HCV/HIV co-infected patients

Background: Due to similar routes of viral transmission, chronic HCV and HIV co-infection is frequent. Since it has been reported that both viruses perturbs immune homeostasis, our aim was to evaluate the peripheral immune cell moiety in HCV mono- and HCV/HIV co-infected patients to determine if co-infection alters HCV immune profile. Methods & Materials: Liver biopsies (47 = HCV treatment naïve, 13 = HCV/HIV only HAART) and concomitant blood samples were analysed. In peripheral blood: 1) frequency of total T cells(CD3 + ), T helper(Th, CD3 + CD4 + ), cytotoxic T lymphocytes(CTL, CD3 + CD8 + ) and Natural Killer cells(NK, CD3-CD56 + CD16 + ); 2) CTL and Th differentiation status [naïve (CD27 + CD45RA + ), central-memory (CD27 + CD45RA-), effector-memory(CD27-CD45RA-), effector(CD27-CD45RA + ) and activated (DR + )]; 3) frequencies of Th17(CD4 + IL17A + ), Th1(CD4 + IFNg + ) and Treg(CD4 + CD25hiFoxp3 + ) and 4) NK and CTL degranulation activity (CD107a) were evaluated by FACS. Non-infected donors (n = 33) were included. Inflammatory activity and fibrosis were assessed in liver biopsies using the modified Knodell scoring system and METAVIR. Results: While HCV patients did not display differences in lymphocyte frequencies compared to donors, co-infected patients showed the lowest Th(donor vs HCV/HIV p < 0.001, HCV vs HCV/HIV p < 0.001), the highest CTLs(donor vs HCV/HIV p < 0.001, HCV vs HCV/HIV p < 0.001) and the lowest NK(donor vs HCV/HIV p < 0.01, HCV vs HCV/HIV p < 0.05) frequencies. Absolute numbers confirmed these results. Moreover, only Th17 absolute numbers were diminished in co-infection (donor vs HCV/HIV p < 0.05). Regarding T cell differentiation status in HCV patients compared to donors: Th cells showed low naïve (p < 0.01) but high effector memory (p < 0.05) and activated cells (p < 0.01) frequencies; CTL showed low naïve (p < 0.01) but high activated cell (p < 0.01) frequencies. Although, HCV/HIV displayed a similar profile to HCV, Th and CTL activation was higher in HCV/HIV than HCV (p < 0.001). Degranulation activity of CTL and NK cells showed no differences between groups. Concerning to liver damage, any of the evaluated immune cell parameters displayed association with fibrosis or hepatitis severity. Conclusion: Although in chronic HCV infection the lymphocyte frequency was not modified, an altered T cell differentiation profile was evident. The co-infected condition distorted lymphocyte frequencies and enhanced T cell activation. This deteriorated immune profile may condition T cell response against antigens, including vaccines.

ELEVATED CENTRAL AND PERIPHERAL INFLAMMATORY PROFILES IN A POPULATION AT RISK FOR ALZHEIMER’S DISEASE

Mid-life obesity and hypertension, two interconnected healthcare epidemics, increase the risk of cognitive decline and age-related neurodegeneration. These vascular risk factors are most common in women and African Americans, who are also at higher risk for Alzheimer's disease (AD). Elevated inflammatory signaling cascades are a primary characteristic of obesity and hypertension. Peripheral inflammation has been linked to cognitive decline and neurodegeneration. However, the mechanistic links remain unknown. Here we tested the hypothesis that obesity and hypertension are mechanistically linked to AD through a multistage process that involves dysregulation of the renin angiotensin system (RAS), systemic inflammation and a heightened peripheral immune response, followed by increased immune cell trafficking into the brain thereby leading to chronic inflammation. Cognitively normal, middle aged participants with a parental history of Alzheimer's disease were enrolled. Blood and CSF were centrifuged for isolation of immune cells for immunophenotyping by multicolor flow cytometry; supernatant biofluids were collected for measurement of inflammatory factors by multiplexed immunoassays. Deep immunophenotyping analysis of CSF revealed that individuals with high blood pressure displayed altered brain immune cell profiles, including elevated central memory T cells, compared to those with normal blood pressure. This same T cell population was altered in obese (BMI >30) compared to non-obese individuals (BMI 18.5–23.9). Altered immune cell profiles were also detected in participants who suffered from disturbed sleep, a common complaint of those who suffer from Alzheimer's disease. Profiling immune cell populations in the CSF is a novel approach in the neurodegeneration field that may reveal opportunities for therapeutic intervention with targeted immunomodulatory therapies. AD risk factors associated with hypertension correlate with altered central and peripheral immune cell profiles in cognitively normal middle-aged participants. Our results demonstrate that immune cell dysfunction is detectable in cognitively intact hypertensive individuals at risk for AD and suggest that RAS-targeted interventions may afford therapeutic benefit to delay onset of AD.

Immune modulation associated with vascular endothelial growth factor (VEGF) blockade in patients with glioblastoma.

Vascular endothelial growth factor (VEGF), in addition to being pro-angiogenic, is an immunomodulatory cytokine systemically and in the tumor microenvironment. We previously reported the immunomodulatory effects of radiation and temozolomide (TMZ) in newly diagnosed glioblastoma. This study aimed to assess changes in peripheral blood mononuclear cell (PBMC) populations, plasma cytokines, and growth factor concentrations following treatment with radiation, TMZ, and bevacizumab(BEV). Eleven patients with newly diagnosed glioblastoma were treated with radiation,TMZ, and BEV, following surgery. We measured immune-related PBMC subsets using multi-parameter flow cytometry and plasma cytokine and growth factor concentrations using electrochemiluminescence-based multiplex analysis at baseline and after 6weeks of treatment. The absolute number of peripheral blood regulatory T cells (Tregs) decreased significantly following treatment. The lower number of peripheral Tregs was associated with a CD4+ lymphopenia, and thus, the ratio of Tregs to PBMCs was unchanged. The addition of bevacizumab to standard radiation and temozolomide led to the decrease in the number of circulating Tregs when compared with our prior study. There was a significant decrease in CD8+ cytotoxic and CD4+ recent thymic emigrant T cells, but no change in the number of myeloid-derived suppressor cells. Significant increases in plasma VEGF and placental growth factor (PlGF) concentrations were observed. Treatment with radiation, TMZ, and BEV decreased the number but not the proportion of peripheral Tregs and increased the concentration of circulating VEGF. This shift in the peripheral immune cell profile may modulate the tumor environment and have implications for combining immunotherapy with anti-angiogenic therapy.

Abstract A46: Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 and B16 mouse melanoma

Abstract Background: The underlying cause for the failure of immune checkpoint blockade is the overwhelming, persistent and multifocal immune suppression in the tumor microenvironment. This is due to the absence of pre-existing antitumor Teff because of the action of important upstream immune checkpoints that recruit tumor infiltrating myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and M2 macrophages, which also produce potent immunosuppressive cytokines (e.g., TGF-beta and IL-10). Phosphatidylserine (PS) is an upstream immune checkpoint that drives global immunosuppression. Previous work has shown that PS targeting agents can override PS-driven immunosuppression and re-program the tumor microenvironment from immunosuppressive to immunosupportive, break tumor immune tolerance, and elicit potent de novo antitumor T-cell immunity. Methods: In the present study, the antitumor effect of the combination of a PS-targeting antibody with antibodies that inhibit the immune checkpoints programmed death receptor-1 (PD-1) or cytotoxic T-lymphocyte associated antigen (CTLA-4) in the K1735 mouse melanoma model was examined. Tumor-bearing mice were treated with each antibody alone or the combination at 5 to 10 mg/kg, twice a week. Tumor growth, survival, as well as intra-tumoral and peripheral immune cell profiles after treatment were assessed by FACS and IHC. Results: Here we demonstrate that combination of PS-targeting antibody (mch1N11) with anti-PD-1 or anti-CTLA-4 antibodies significantly improved therapeutic efficacy in mice bearing K1735 melanoma compared to single agent treatment. Similar results were seen in the B16 mouse melanoma model. Importantly, we found that the level of MDSCs in the spleen was significantly reduced by combination treatment, 9.9% in the combination treated group, as compared with 18.27% and 14.69% in anti-PD-1 (p&amp;lt;0.0005) or control C44 (p&amp;lt;0.05) treated group, respectively. In addition, combination therapy had the highest ratio of tumor-infiltrating immune effector to suppressor cells and the most IL-2 and IFNγ-secreting T cells in the spleen in response to non-specific stimulation. The percentage of IFNγ-secreting CD8+ T cells in the combination group was 5.85%, as compared with 4.1% and 4.0% in anti-PD-1 or control C44 group, respectively (p&amp;lt;0.05). The percentage of IL-2-secreting CD8+ T cells in the combination group was 2.4%, as compared with 1.29% and 1.17% in anti-PD-1 or control C44 group, respectively (p&amp;lt;0.05). The percentage of IL-2-secreting CD4+ T cells in the combination group was 6.5%, as compared with 2.9% and 3.2% in anti-PD-1 or control C44 group, respectively (p&amp;lt;0.05). Finally, combination of PS-targeting with downstream immune checkpoint blockade did not induce any observable toxicity following multiple treatment doses. Conclusions: Our findings indicate that the combination of PS-blockade with blockade of downstream immune checkpoints (e.g., PD-1 and CTLA-4) represents a promising strategy to enhance the efficacy of cancer immunotherapy. Citation Format: Bruce Freimark, Jian Gong, Dan Ye, Van Nguyen, Shen Yin, Rich Archer, Chris Hughes, Jeff Hutchins, Jeff Hutchins, Alan Schroit, Rolf Brekken, Xianming Huang. Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 and B16 mouse melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A46.

Peripheral immune cell profiles in fingolimod treated multiple sclerosis patients following short term drug interruption (P1.155)

Peripheral immune cell profiles in fingolimod treated multiple sclerosis patients following short term drug interruption (P1.155)