CD28null Cells Research Articles

#3555 LYMPHOCYTE SENESCENCE: A CROSS-SECTIONAL STUDY COMPARING SYSTEMIC LUPUS ERYTHEMATOSUS AND END STAGE RENAL DISEASE

Abstract Background and Aims Immunosenescence, as a consequence of ageing or chronic inflammation, encompasses a spectrum of lymphocyte characteristics, including the presence or absence of certain lymphocyte surface molecules. Aim of the present study was to evaluate the immunosenescent effect of two different forms of chronic inflammation, Systemic Lupus Erythematosus (SLE), a chronic autoimmune disease and end stage renal disease (ESRD), a chronic inflammatory disorder. Method Certain lymphocyte surface molecules, including CD31, CD45RA, CCR7, CD28, CD57, for T, and IgD, CD27 for B lymphocytes, were analyzed by flow cytometry, and distinct naïve, active and senescent lymphocyte subtypes were determined, in SLE and ESRD patients. Results were compared to healthy controls (HC) of similar age, gender and nationality. Results Lymphopenia was significant in both SLE and ESRD patients, compared to HC, affecting B cells 75.4(14.4–520.8), 97(32–341) and 214(84–576)cells/μL, respectively, P < 0.0001, and CD4+ cells 651.2(71.1–1478.2), 713(234–1509) and 986(344–1591)cells/μL, respectively, P < 0.0001. The allocation of B cell subpopulations was remarkably different between SLE and ESRD patients, with the SLE showing a clear shift to senescent (IgD-CD27-) 11.75(2.3–74.2)% vs. 8.1 (1.7–35), and ESRD patients, to naïve subpopulations, compared to HC 69.9(1.1–92)% vs. 62 (4.5–86.9)%, P = 0.019. Instead, senescent subtypes of CD4+ lymphocytes, were reduced in SLE, compared to ESRD and HC, 3.2 (0.1–42.2)%, 6 (0.4–56.4)% and 3.5 (0.3–19.5)% for CD4+CD28null cells and 1.2 (0.1–23.9)%, 2.5 (0–51.9)% and 1.9 (0–17.9)%, for CD4+CD28-CD57+ cells. CD8+ lymphocyte subtypes presented a complete redistribution, in favor of CD8+CD45RA-CCR7+ (central memory), 53(1.8–92.4)%, 52.2(0.1–91.5)% and 23.4(0.1–92)%, respectively, and lowering the terminally differentiated populations (CD45RA+CCR7-), 2.8 (0–58.1)%, 7.8 (0.2–54.4)% and 12.8(0–71.6), respectively. CD8+CD28null cells were significantly reduced in SLE, compared to ESRD and HC, 31.9(1.1–87.2)%, 44.4(14–89.7)% vs. 34.5(6.4–72.3)%, respectively, P = 0.04. Conclusion Senescent phenotype of B lymphocytes predominated in SLE patients, while ESRD patients showed a completely different profile, with increased senescence involving mainly CD8+ lymphocytes.

Senescent CD4+CD28null cells are increased in chronic hyperuricemia, show aberrant effector phenotypes, and are reversed after allopurinol therapy: a proof-of-concept pilot study.

To characterize CD4+CD28null cells in chronic hyperuricemia and investigate whether allopurinol could restore CD28 expression and the balance of T helper phenotypes. Asymptomatic individuals with chronic hyperuricemia and ultrasonographic findings evocative of urate deposition in the joints. Age- and gender-matched normouricemic individuals were also studied. Oral allopurinol at 150mg/day for 4weeks, followed by 300mg/day through week 12. Color-flow cytometry on peripheral blood mononuclear cells (PBMC) with antibodies against CD4, CD28, T-bet (Th1), GATA-3 (Th2), and RORγt (Th17). Six patients (five men, median age of 53years) and seven controls were studied. At baseline, hyperuricemic patients had more CD4+CD28null/CD4+ cells than normouricemic subjects (36.8% vs. 6.1%; p = 0.001), with a predominance of T-bet+ cells (98.5% vs. 6.6%; p = 0.001) and few RORγt+ cells (0.7% vs. 89.4%; p = 0.014). In hyperuricemic patients, the number of CD4+ cells/10,000 PBMC was similar before and after allopurinol (3378 vs. 3954; p = 0.843). Conversely, CD4+CD28null cells decreased from 36.8% (23.0-43.7) to 15.8% (4.7-28.1; p = 0.031). CD4+CD28nullT-bet+ cells decreased from 98.5% (95.0-99.4) to 88.3% (75.2-98.9; p = 0.062), CD4+CD28nullGATA-3+ cells increased from 0% (0-4.0) to 2.8% (0.1-15.6; p = 0.156), and CD4+CD28nullRORγt+ cells increased from 0.7% (0.4-7.0) to 4.5% (1.3-28.1; p = 0.031). The CD4+CD28null cell subset is abnormally expanded in chronic hyperuricemia, despite the absence of overt urate-related disease. Allopurinol may partially restore CD28 expression on CD4+ cells while enhancing the homeostatic balance of T helper phenotypes. ClinicalTrials.gov, number NCT04012294.

Immune cell subpopulations as risk factors for atrial fibrillation: The Cardiovascular Health Study and Multi-Ethnic Study of Atherosclerosis

Atrial fibrillation (AF) is the most common treatable cardiac arrhythmia. Risk factors for AF are difficult to modify, and interventions to prevent AF in clinical trials have failed to date. Inflammation is an important pathway for the development of AF, and recent studies suggest that activation of specific innate and adaptive immune responses, such as intermediate monocytes1 and highly cytotoxic CD4+CD28null cells,2,3 may play a role in AF pathogenesis. However, it remains unclear whether immune activation is a result or a cause of AF.

Circulating T Cells and Cardiovascular Risk in People With and Without HIV Infection

BackgroundLower CD4+ cell count in people with HIV infection (PWH) is associated with increased cardiovascular disease (CVD) risk. Whether subsets of CD4+ T helper cells are linked with CVD is unclear. ObjectivesThe aim of this study was to explore the association between peripherally circulating CD4+ T cell subsets and incident CVD. MethodsData from 1,860 participants (1,270 PWH) without prevalent CVD from the VACS (Veterans Aging Cohort Study), a prospective, observational cohort of veterans with and without HIV infection, were analyzed. T cell subsets were quantified in baseline samples using flow cytometry. Incident CVD events were identified using International Classification of Diseases-9th Revision and International Classification of Diseases-10th Revision diagnosis and procedure codes. Participants were followed from baseline date (2005-2006) to the first of CVD incidence, death, or September 30, 2016. Cox proportional hazards regression was used to model associations between these T cell subsets and the risk for incident CVD while adjusting for demographics and other CVD risk factors. ResultsThe median participant age at baseline was 51.6 years. Most were male (94%) and of Black race (69.1%). There were 344 incident CVD events (219 in PWH) during follow-up (median 9.8 years). In PWH, higher proportions (per SD increment) of T helper type 17 cells (adjusted HR: 1.19; 95% CI: 1.08-1.31), T effector memory cells re-expressing CD45RA (adjusted HR: 1.19; 95% CI: 1.07-1.34), and CD28null cells (adjusted HR: 1.18; 95% CI: 1.03-1.34) were significantly associated with an increased risk for incident CVD. Among those without HIV infection, no T cell subsets were significantly associated with CVD. ConclusionsAmong PWH, T helper type 17 cells, senescent cells, and CD4+ T effector memory cells re-expressing CD45RA were significantly associated with incident CVD that was not explained by CVD risk factors.

Methotrexate improves endothelial function in early rheumatoid arthritis patients after 3 months of treatment

BackgroundEndothelial dysfunction contributes to increased cardiovascular (CV) disease in rheumatoid arthritis (RA). Angiogenic T cells (Tang) are a key regulator of vascular function via their interaction with endothelial progenitor cells (EPCs). Methotrexate (MTX) has been associated to reduced CV disease risk, but its effects on endothelial homeostasis have been poorly explored. We investigated MTX effects on endothelial homeostasis in early, treatment-naïve RA patients.MethodsFifteen untreated, early RA patients and matched healthy controls (HC) were enrolled. RA patients with long-standing disease in remission or low disease activity treated with MTX for at least 6 months were selected as controls. Circulating CD28+ and CD28null Tang cell, endothelial microparticle (EMP), EPC and soluble vascular cell adhesion molecule (sVCAM)-1 levels were measured.ResultsTang percentage was higher in early RA than in HCs and significantly increased after 3-month MTX treatment. Tang cells in RA were characterized by higher percentage of CD28null and lower CD28-positive cells than HCs. MTX restored a Tang cell phenotype similar to HCs. Altered sVCAM-1, EMP and EPC were restored to levels similar to HCs after a 3-month MTX. Biomarker levels after 3 months of MTX were not different to those of patients with long-standing treatment.ConclusionsMTX has a positive effect on Tang, sVCAM-1, EPCs and EMPs in RA. Restoration of imbalance between CD28 + and CD28null Tang by MTX may be one of the mechanisms underlying its favourable effects on endothelial dysfunction. These effects seem to be long-lasting and independent from systemic inflammation reduction, suggesting a direct effect of MTX on the endothelium.

Accumulation of CD28null Senescent T-Cells Is Associated with Poorer Outcomes in COVID19 Patients.

Coronavirus disease 2019 (COVID-19), a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes infectious disease, and manifests in a wide range of symptoms from asymptomatic to severe illness and even death. Severity of infection is related to many risk factors, including aging and an array of underlying conditions, such as diabetes, hypertension, chronic obstructive pulmonary disease (COPD), and cancer. It remains poorly understood how these conditions influence the severity of COVID-19. Expansion of the CD28null senescent T-cell populations, a common phenomenon in aging and several chronic inflammatory conditions, is associated with higher morbidity and mortality rates in COVID-19. Here, we summarize the potential mechanisms whereby CD28null cells drive adverse outcomes in disease and predispose patients to devastating COVID-19, and discuss possible treatments for individuals with high counts of CD28null senescent T-cells.

MO016CD28 AS A MARKER OF T CELL IMMUNOSENESCENCE IN PATIENTS ON HEMODIALYSIS

Abstract Background and Aims Reduced expression of CD28 molecule on CD4+ and CD8+ cells is a potential marker of senescence and has also been described in end stage renal disease (ESRD). Aim of the present study was to evaluate changes of CD28 expression on CD4+ and CD8+ cells in ESRD patients, and to assess possible effects of ageing and HD. Method Flow cytometric analysis was performed in 96 patients [M/F 54/42 Age 56.6(18-81)yrs] on HD to evaluate the expression of CD28 antigen on CD4+ and CD8+ cells. Proportions and absolute numbers of CD4+CD28null and CD8+CD28null cells were estimated, and results were correlated to patients age and time on HD. Patients with malignant or systemic disease were excluded, as those with inadequate HD (KT/V<1,2), or recent (<3months) infection. Results were compared to those of 27 healthy controls, similar age, sex, and race. Results HD patients had significantly reduced lymphocyte count (p<0.001), percentage and total count of CD3+ cells (p=0.04, p<0.0001, respectively) and CD4+ cells (p=0.005, p<0.001, respectively). Percentage of CD4+CD28null and CD8+CD28null cells were both increased in patients compared to controls (p=0.009, p=0.01, respectively). However, although changes in CD8CD28null cells were evident early, even in <2yrs, similar changes in CD4CD28null cells, appeared only after ≥2yrs on HD, (p=0.004). Age of the patients had significant correlation with CD8+CD28null cells (r=0.4, p=0.001). In the older groups (>40yrs) patients had significantly increased CD3+CD28null, CD4+CD28null and CD8+CD28null cells, compared to older controls (p=0.002, p=0.003, p=0.01, respectively). Conclusion CD28 expression is significantly reduced on CD4+ and CD8+ cells in HD patients. CD8+CD28null expression is significantly affected early, both by age and HD, while CD28 expression on CD4+ seems to be more stable, as CD4+CD28null cells increase in older patients, after long time on HD.

MO161DIABETIC NEPHROPATHY AS AN ADDITIONAL FACTOR CAUSES DISTURBANCES IN T CELL IMMUNITY IN CHRONIC KIDNEY DISEASE PATIENTS

Abstract Background and Aims Chronic kidney disease (CKD) and Diabetes Mellitus (DM), are chronic inflammatory conditions, and may both affect T-cell immunity. However, our knowledge about alterations of specific T cell subpopulations, as well as the further modifications occurring after dialysis initiation, is limited. The present study aimed to investigate the changes of T lymphocyte subtypes in CKD patients due to Diabetic Nephropathy (DN-CKD) and compare to nonDN-CKD patients. Furthermore, the effect of dialysis, was also estimated in both groups. Method In 30 DN-CKD, 30 nonDN-CKD patients and 25 healthy individuals, cytometric analysis of T cell subpopulations was performed, including CD4+, CD8+, CD4+CD28null, CD8+CD28null, Natural Killer cells and Tregs. Measurements were performed at the day started (T0) on either hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD) and repeated six months later (T6). Results At time T0, CD4+, NK cells and Tregs were significantly reduced (p=0.001, p=0.01, p<0.0001, respectively), while CD28null cells were increased (p=0.005) in CKD patients compared to controls. CD4CD28null and CD8CD28null (%) cells were increased in DN-CKD compared to nonDN-CKD (p=0.03, p=0.02, respectively). At time T6, a significant increase in total CD28null cells were noticed in DN patients (p=0.05) but not in nonDN patients. More interestingly, DN-CKD patients who started on HD showed a significant increase in CD4CD28null cells [from 30(14-100) to 52.7(15-203), p=0.02], CD8CD28null cells [from 130(47-400) to 212(25-1192), p=0.02] and NK cells [from 137(56-275) to 266(103-456), p=0.01], while those DN-CKD started on CAPD showed a significant reduction in the percentage of CD4CD28null cells [from 9.8(2.9-19.8) to 6.9(1.3-+18), p=0.05]. Conclusion Patients with CKD due to DN are more likely to have impaired T cell immunity, mainly regarding CD28null cells, which further deteriorates after initiation of HD. Instead, CAPD method seems to ameliorate those changes.

Novel Immune Cell Subsets Exhibit Different Associations With Vascular Outcomes in Chronic Kidney Disease Patients-Identifying Potential Biomarkers.

Background and Aims: Alterations in novel immune cell subsets, such as angiogenic T cells (Tang), senescent T cells (CD4+CD28null), and monocyte subsets are associated with impaired vascular homeostasis in several inflammatory conditions. However, mediators underlying vascular deterioration in chronic kidney disease (CKD) are poorly characterized. This study assessed their role in the vascular deterioration of CKD using a broad spectrum of surrogate markers ranging from altered functionality to overt calcification.Methods: Tang (CD3+CD31+CXCR4+), CD4+CD28null cells, and monocytes [CD14/CD16 subsets and angiotensin-converting enzyme (ACE) expression] were measured in peripheral blood by flow cytometry in 33 CKD stage 5 patients undergoing peritoneal dialysis (CKD5-PD) and 15 healthy controls (HCs). Analyses were replicated in a hemodialysis cohort. Vascular surrogate markers (including adventitial vasa vasorum, pulse wave velocity, intima-media thickness, and vascular calcification) were assessed by appropriate imaging methods.Results: In CKD5-PD, decreased Tang levels (p < 0.001) were unrelated to clinical features or traditional cardiovascular (CV) risk factors but correlated negatively with troponin T levels (r = −0.550, p = 0.003). Instead, CD4+CD28null frequency was increased (p < 0.001), especially in those with vascular calcifications. Quantitative and qualitative differences were also observed within the monocyte pool, a shift toward CD16+ subsets and ACE expression being found in CKD. Equivalent results were observed in the replication cohort. Each subset associated distinctly with adverse vascular outcomes in univariate and multivariate analyses: while Tang depletion was linked to poor vascular function and subclinical atherosclerosis, increases in CD4+CD28null were associated with overt vascular thickening and calcification. Monocytes were not independently associated with vascular outcomes in CKD patients.Conclusions: Novel T cell and monocyte subsets are altered in CKD. Altered T-cell subpopulations, but not monocytes, exhibited distinct associations with different vascular outcomes in CKD. Tang are emerging biomarkers of subclinical vascular deterioration in CKD.

CD28null and Regulatory T Cells Are Substantially Disrupted in Patients with End-Stage Renal Disease Due to Diabetes Mellitus.

Background: End-stage renal disease (ESRD) is associated with alterations in T-cell immunity, including increased CD28null and reduced regulatory T cells (Tregs). However, whether immune disturbances are due to ESRD or primary disease is not yet clear. As diabetes mellitus is the leading cause of ESRD, we evaluated its impact on the immune profile of ESRD patients. Methods: CD28null, Tregs, and natural killer cells were initially analyzed by flow cytometry in 30 predialysis ESRD patients due to diabetes (DM), 30 non-DM (NDM), and 25 healthy controls. Measurements were repeated after 6 months on hemodialysis (HD) or peritoneal dialysis (CAPD). Results: The percentage of CD4 + CD28null cells, CD8 + CD28null cells, and Tregs showed significant differences in DM, NDM, and controls; mean rank 33.71 vs. 25.68 vs. 18.88, p = 0.006, 37.79 vs. 28.82 vs. 17.08, p = 0.008, and 20.79 vs. 26.12 vs. 41.33, p = 0.001, respectively. DM vs. NDM had increased CD4 + CD28null and CD8 + CD28null cells, 11.5% (1.5%–24%) vs. 4.1% (0–42.3%), p = 0.02 and 61.3% (24%–76%) vs. 43% (5.7%–85%), p = 0.04, respectively. After 6 months on HD but not CAPD, DM showed a significant further increase in CD4 + CD28null cells, from 30 (14–100) to 52.7 (15–203), p = 0.02; and CD8 + CD28null cells, from 137 (56–275) to 266 (103–456), p = 0.01. Conclusions: Diabetes mellitus affects T-cell subtypes even at predialysis stage, though changes become more prominent after commencement on HD.

Relationship Between T-Cell Exosomes and Cellular Subsets in SLE According to Type I IFN-Signaling.

Objective: To quantify the levels of circulating exosomes derived from T-cells and monocytes and their possible associations with leukocyte subpopulations and cytokine milieu in Systemic Lupus Erythematosus (SLE).Methods: Total circulating exosomes (CD9+-Ex) and those derived from T-cells (CD3+-Ex) and monocytes (CD14+-Ex) were quantified by flow cytometry in 82 SLE patients and 32 controls. Leukocyte subsets and serum cytokines were analyzed by flow cytometry or by immunoassays. IFN-score was evaluated by real time RT-PCR in whole blood samples from a subgroup of 73 patients and 24 controls.Results: Activation markers (IFNR1 and BLyS) on monocytes, neutrophils and B-cells correlated inversely with circulating exosomes (CD9+-Ex, CD3+-Ex, and CD14+-Ex) in controls but directly with CD3+-Ex in patients (all p < 0.05). Although CD9+-Ex were increased in SLE, no differences were found in CD3+-Ex, supporting that exosome content accounts for this opposite role. Interestingly, CD4+CD28null cells correlated with CD3+-Ex in patients and controls, and displayed similar associations with leukocyte subsets in both groups. Additionally, CD3+-Ex correlated in patients with the expression of CD25 in CD4+CD28null cells. Furthermore, the activated status of this senescent subset was related to IFNα serum levels in controls and to IFN-score in SLE patients. Finally, patients presenting high IFN-score, in addition to elevated CD25+CD28null cells associated with the activation of myeloid cells, displayed higher levels of inflammatory cytokines and chemokines.Conclusion: Our results support a relationship between T-cell exosomes and cellular subsets in SLE according to type I IFN-signaling, which could amplify chronic immune activation and excessive cytokine/chemokine response.

Increase in inflammatory T cell subsets in atrial fibrillation: the missing link underlying inflammation in AF

Abstract Background The precise role of inflammation in the development and perpetuation of atrial fibrillation (AF) is yet to be fully uncovered. T lymphocytes have pivotal roles in orchestrating inflammation. Specialised subsets of lymphocytes either promote or prevent inflammation. We are investigating a unique subset of lymphocytes, the CD4+CD28null T cells that expand in patients with chronic inflammation. These cells secrete high levels of pro-inflammatory cytokines and have cytolytic function. CD4+CD28null T cells are normally maintained under control by regulatory T cells (Treg), a specialised subset of T lymphocytes with suppressive function that maintain immune homeostasis and prevent pathogenic immune responses. The role of CD4+CD28null and Treg cells has not been investigated in AF. Purpose We hypothesised that in AF the balance between pro-inflammatory and regulatory T lymphocytes is skewed in favour of inflammatory T cells, which may sustain inflammation in AF. Methods Circulating CD4+CD28null T lymphocytes and Tregs were quantified by flow cytometry in paroxysmal and persistent AF patients and healthy controls (n=30). Inflammatory cytokines were quantified in serum and the function of T lymphocyte subsets was investigated using ex vivo functional assays. Results CD4+CD28null T lymphocytes were significantly increased in the circulation of AF patients compared to controls. Of note, a higher proportion of patients with persistent AF showed an increase in inflammatory CD4+CD28null T lymphocytes compared to patients with paroxysmal AF. A marked reduction in Treg cells was present in AF patients compared to controls. Functional assays showed that IL-7 and IL-15 cytokines were responsible for CD4+CD28null T lymphocyte expansion in AF patients. Conclusions We show that patients with AF have marked changes in T lymphocytes subsets: pro-inflammatory CD4+CD28null T cells increase significantly, whilst anti-inflammatory Tregs are significantly reduced. We show for the first time that the cytokines IL-7 and IL-15 are the main drivers of CD4+CD28null T cell expansion in AF patients. These novel findings may reveal novel therapeutic strategies (e.g. cytokine blockade) to re-establish the balance between pro- and anti-inflammatory mechanisms at work in AF to improve patient outcomes. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation

The IMMENSE Study: The Interplay Between iMMune and ENdothelial Cells in Mediating Cardiovascular Risk in Systemic Lupus Erythematosus.

Patients with systemic lupus erythematosus (SLE) have a significant increase in cardiovascular (CV) risk although they display a preserved number of circulating angiogenic CD3+CD31+CXCR4+ T cells (Tang), a subpopulation of T cells which promotes repair of damaged endothelium. This happens due to the concomitant expansion of a Tang subset with immunosenescent features, such as the loss of CD28. Therefore, the aim of this study was to elucidate the interplay between Tang subpopulations and endothelial cells in a group of young SLE patients without previous cardiovascular events. Twenty SLE female patients and 10 healthy controls (HCs) were recruited. Flow cytometric analysis of endothelial progenitor cells (EPCs) and Tang subsets were performed and serum levels of interleukin (IL)-6, -8, matrix metalloproteinase (MMP)-9 and interferon (IFN)-γ were measured. Human umbilical vein endothelial cells (HUVECs) proliferation and pro-inflammatory phenotype in response to subjects’ serum stimulation were also evaluated. Results showed that the percentage of Tang and EPC subsets was reduced in SLE patients compared with HCs, with a marked increase of senescent CD28null cells among Tang subset. SLE disease activity index-2000 (SLEDAI-2K) was inversed related to Tang cells percentage. Furthermore, IL-8 serum levels were directly correlated with the percentage of Tang and inversely related to the CD28null Tang subsets. We indirectly evaluated the role of the Tang subset on the endothelium upon stimulation with serum from subjects with a low percentage of Tang CD3+ cells in HUVECs. HUVECs displayed pro-inflammatory phenotype with up-regulation of mRNA for IL-6, intercellular adhesion molecule (ICAM)-1 and endothelial leukocyte adhesion molecule (ELAM)-1. Cell proliferation rate was directly related to IL-8 serum levels and EPC percentage. In highly selected young SLE patients without previous CV events, we found that the deterioration of Tang compartment is an early event in disease course, preceding the development of an overt cardiovascular disease and potentially mediated by SLE-specific mechanisms. The overcome of the CD28null subset exerts detrimental role over the Tang phenotype, where Tang could exert an anti-inflammatory effect on endothelial cells and might orchestrate via IL-8 the function of EPCs, ultimately modulating endothelial proliferation rate.

First report of expansion of CD4+/CD28 null T-helper lymphocytes in adult patients with idiopathic autoimmune hemolytic anemia

CD28 null T helper (Th) cells are rare in healthy individuals, but they are increased in various inflammatory and immune-mediated diseases. In this study, we determined the size of the CD4+/CD28 null T lymphocyte compartment in the peripheral blood of 40 autoimmune hemolytic anemia (AIHA) patients (idiopathic and secondary) and 20 healthy control subjects, using tri-color flow cytometry. The frequency and absolute count of CD4+/CD28 null T helper (Th) cells was significantly higher in idiopathic AIHA patients, compared to healthy controls (p = 0.001 and 0.001, respectively) and to patients with secondary AIHA (p = 0.04 and 0.01, respectively). The percentage of CD4+/CD28 null Th cells was also negatively correlated to the hemoglobin (Hb) level (p = 0.03). These findings demonstrate, for the first time, the expansion of this phenotypically-defined population of T lymphocytes in patients with idiopathic AIHA and indicate that it likely plays an etiological role in the development of this disease. However, establishing the use of this marker for diagnosis or monitoring treatment of such patients needs further studies.

CD8+CD28null T Lymphocytes are Associated with the Development of Atrial Fibrillation after Elective Cardiac Surgery.

Postoperative atrial fibrillation (POAF) is assumed as a complex and multifactorial interaction of different pathogenic factors. Data suggests an inflammatory process as the main trigger of this specific type of atrial fibrillation. CD8+ T lymphocytes that lack the surface protein CD28 were found to be crucially involved in chronic inflammatory processes within the cardiovascular system. Of utmost interest, these so-called CD8+CD28null T cells are known to present with autoaggressive behavior and deleterious cytotoxic effects on human tissue. A total of 129 patients undergoing elective cardiac valve and/or coronary artery bypass graft surgery were enrolled. Fluorescence-activated cell sorting was performed to investigate lymphocyte subsets. Patients were stratified in two subgroups according to patients developing POAF (n = 60) and individuals free of POAF (n = 69). Comparing patients developing POAF to individuals free of POAF, the fraction of CD8+ lymphocytes was significantly higher in individuals developing POAF (30.5% [POAF] vs. 25.7% [no-POAF]; p = 0.021). Interestingly, also the fraction of CD8+CD28null T lymphocytes was significantly higher in the POAF subgroup (66.7% [POAF] vs. 61.6% [non-POAF]; p = 0.043). Multivariate logistic regression proved that the fraction of CD8+CD28null cells is a strong and independent prognosticator for the development of POAF with an adjusted odds ratio per 1 standard deviation of 3.21 (95% confidence interval 1.01-10.18; p = 0.048). We found that cytotoxic CD8+CD28null T lymphocytes proved to be a strong and independent predictor for the development of POAF after elective cardiac surgery. Our results potentially indicate an autoimmune impact of this preexisting, highly cytotoxic T cell subset in the pathogenesis of POAF.

P0297CHANGES OF CD4CD28NULL AND CD8CD28NULL T CELLS IN CHRONIC KIDNEY DISEASE AND THE IMPACT OF DIFFERENT DIALYSIS MODALITIES

Abstract Background and Aims Chronic Kidney Disease (CKD) affects both the innate and adaptive immunity, although clinical consequences, and the effect of different renal replacement methods on those alterations remain unclear. The purpose of this prospective observational study was to investigate the alterations of T cell immunity in CKD patients, as well as the effect of different dialysis methods on T lymphocyte subtypes. Method T cell subpopulations namely CD3+CD4+, CD3+CD8+, CD4+CD28- and CD8CD28- cells, were isolated from whole blood samples using flow cytometry in 40 CKD patients at predialysis state (ESRD-T0). The immunological profile was repeated six months later after initiation of renal replacement therapy (hemodialysis (HD) or peritoneal dialysis (CAPD)). Fifteen age and gender matched healthy individuals served as controls. Results Both CD4+ and CD8+ T cells were significantly reduced in ERSD-T0 patients compared to controls, 604(105-3551) vs. 943(584-1867) μ/L, p=0.001, and 352(103-1561) vs. 422.4(263-1453) μ/L, p=0.05. The percentage of both CD4+CD28null and CD8+CD28null cells, 6.4(0.3-30) % vs. 2.7(0.1-7.8) %, p=0.04 and 58.2(12.8-85.4) % vs. 39(7.8-57.1) %, p=0.01 was increased in ERSD-T0 patients comparing to controls. Furthermore the percentage of CD4+CD28null cells correlated with CRP (r=0.4, p=0.04) and serum albumin levels (r=-0.5, p=0.007), while, significant differences were noticed between patients with and without cardiovascular disease, regarding both, CD4+CD28null and CD8+CD28null cells, 8.6(1-30) % vs. 2.1(0.1-19.8) %, p=0.04 and 62.5(12.8-85.4) vs. 45.5(5.7-73.7), p=0.02, respectively. Changes in the population of CD4+CD28null after 6 months on dialysis showed significant differences between HD and CAPD methods, 110.11(-27.1 to 311.4)% vs. -28.1(-100 to 30)%, respectively, p=0.003, as were in CD8+CD28null cells, 55.23(-29.06 to 197.93)% vs. -8.34(-54.99 to 66.72)%, respectively, p=0.05. Conclusion CKD seem to affect specific T cell subtypes, at a pre-dialysis stage, and levels correlate with chronic inflammatory markers and the presence of CVD. These disturbances are further enhanced in HD, while they are alleviated in CAPD.

CD4+CD28null T cells are expanded in moderately active systemic lupus erythematosus and secrete pro-inflammatory interferon gamma, depending on the Disease Activity Index.

Pathogenic CD4+CD28null cells are characterized by inflammatory cytokine synthesis and tropism to the inflamed tissues. Recent studies showed the involvement of CD28null T cells in a severe clinical outcome of lupus. However, their role in moderately active disease is still unresolved. We examined the levels of circulating CD4+CD28null cells and CD8+CD28null suppressor T cells. We also compared the CD4+CD28null and CD4+CD28+ T-cell functional properties, including the expression of interferon gamma (IFN-γ) and Ki67 among systemic lupus erythematosus (SLE) patients (n = 20) and healthy controls (n = 20). All the patients were under immunosuppressive treatment and exhibited moderate SLE activity (median SLE Disease Activity Index (SLEDAI) = 6). In patients, we found elevated CD4+CD28null and unchanged levels of suppressor CD8+CD28null T cells. There was no difference between patients and controls in IFN-γ and Ki67-expressing CD4+, CD4+CD28+, and CD4+CD28null T cells, except for higher IFN-γ levels in CD4+CD28+ T cells in SLE. In each studied group, we observed a higher preponderance of IFN-γ- and Ki67-expressing cells among CD4+CD28null T cells and lower levels of IFN-γ in CD4+CD28null T cells compared to the CD28+ subset. Similarly, Ki67 intensity was decreased in healthy CD4+CD28null cells, whereas in patients, comparably high expression was observed in both subsets. IFN-γ intensity in CD4+CD28null T cells correlated with SLEDAI. SLE with a moderately active clinical course is characterized by peripheral blood expansion of CD4+CD28null T cells and a normal abundance of suppressor CD8+CD28null T cells. The demonstration that these pathogenic CD4+ T cells, despite the lack of CD28, maintain the ability to produce pro-inflammatory IFN-γ positively correlated with disease activity as well as relatively high proliferative capacity may suggest their potentially predictive role in SLE flares.

Effect of statins on atherosclerosis in chronic kidney disease patients guided by CD4+CD28nullT cells over duration of six months

Abstract Objectives Chronic kidney disease and atherosclerosis are considered to be inflammatory process in which T cells and cytokines participate. This study determines the effect of statin therapy as an anti-inflammatory agent on the level of CD4+CD28null T lymphocyte population, and subsequently on atherosclerosis in patients with chronic renal disease. Methods We recruited 90 chronic kidney disease patients. The patients were divided into three groups according to carotid intimal medial thickness (CIMT) as an indicator of atherosclerosis. Two groups (group A in whom CIMT above 0.95 mm and B in whom CIMT below 0.95 mm) were given statin (atorvastatin 20mg) while the third group (group C in whom CIMT below 0.95 mm) continue only on the conservative treatment for CKD patients. CD4+CD28null T cells was measured in the three groups at the beginning of the study and after 6 months of statin therapy. Results CD4+CD28null T cells was decreased in statin groups (group A and B) when compared to no-statin group (group C) at the end of the study. Multivariable regression analysis for the effect of statin therapy showed that statin can independently increase the percentage of decrease both CD4+CD28null cells at the end of our study (p-value &amp;lt;0.0001). Conclusion Our study demonstrates that statins reduce CD4+CD28null T cells in CKD patients especially with atherosclerosis suggesting that statins may help in altering the inflammatory process that lead to atherosclerosis.

Low-density granulocytes and monocytes as biomarkers of cardiovascular risk in systemic lupus erythematosus.

The aim was to evaluate the most relevant cell populations involved in vascular homeostasis as potential biomarkers of SLE-related cardiovascular disease (CVD). Low-density granulocytes (LDGs), monocyte subsets, endothelial progenitor cells, angiogenic T(Tang) cells, CD4+CD28null and Th1/Th17 lymphocytes and serum cytokine levels were quantified in 109 SLE patients and 33 controls in relationship to the presence of subclinical carotid atheromatosis or cardiovascular disease. A second cohort including 31 recent-onset SLE patients was also included. Raised monocyte and LDG counts, particularly those LDGs negative for CD16/CD14 expression (nLDGs), in addition to the ratios of monocytes and nLDGs to high-density lipoprotein-cholesterol (HDLc) molecules (MHR and nLHR, respectively), were present in SLE patients with traditional risk factors or subclinical atheromatosis but not in those who were CV-free, thus revealing their value in the identification of patients at risk of CVD, even at the onset of disease. Accordingly, nLDGs were correlated positively with carotid intima-media thickness (cIMT) and with inflammatory markers (CRP and IL-6). A bias towards more differentiated monocyte subsets, related to increased IFN-α and IL-17 serum levels, was also observed in patients. Intermediate monocytes were especially expanded, but independently of their involvement in CVD. Finally, CD4+CD28null, Th17 and Th1 lymphocytes were increased, with CD4+CD28null and Th17 cells being associated with cIMT, whereas endothelial progenitor and Tang cell levels were reduced in all SLE patients. The present study highlights the potential use of MHR and nLHR as valuable biomarkers of CVD risk in SLE patients, even at diagnosis. The increased amounts of nLDGs, monocytes, Th17 and senescent-CD28null subsets, coupled with reduced pro-angiogenic endothelial progenitor cells and Tang cells, could underlie the development of atheromatosis in SLE.

Peripheral Blood B and T Cell Profiles in Children with Active Juvenile Idiopathic Arthritis.

Juvenile idiopathic arthritis (JIA) is one of the most common autoimmune diseases in children. Our study aimed to evaluate the peripheral blood B and T lymphocyte subpopulations in children with JIA. This case-control study included 20 children with JIA as well as 20 healthy children with matching age and sex as a control group. All patients included in the study were in activity as determined by visual analog scale. In addition to complete clinical evaluation, basic investigations, peripheral blood B and T lymphocyte subpopulations were done to all participants by flow cytometry. JIA patients displayed a significant decrease in IgM memory B lymphocytes, switched memory B lymphocytes, and total memory B lymphocytes when compared to the healthy controls. The percentages of naïve B lymphocytes were significantly increased in JIA patients than in controls. Total T lymphocytes, CD8+CD28null cells, and CD4+CD28null cells were significantly increased in JIA patients as compared to controls. In conclusion; JIA patients have an alteration in both B and T lymphocytes with the predisposition of memory cells which may have a role in sustaining the JIA disease activity.