BACKGROUND:Renal transplant recipients (RTRs) tend to mount weaker immune responses to vaccinations, including vaccines against the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).METHODS:Humoral immunity was assessed using anti-receptor binding domain (RBD) and neutralizing antibodies (NAb) serum levels measured by ELISA, and cellular immunity was assessed using T-, B-, NK, natural killer-like T (NKT)-cell subpopulations, and monocytes measured by flow cytometry, and also specific T-cell immunity, at predefined time points after BNT162b2 vaccination, in 57 adult RTRs.RESULTS:Administration of three booster doses was necessary to achieve anti-RBD and NAb protective levels in almost all patients (92.98%). Ab production, at several time points, was positively correlated with the corresponding renal function and inversely correlated with hemodialysis vintage (HDV) and treatment with mycophenolic acid (MPA). A gradual rise in several cell subpopulations, including total lymphocytes (p = 0.026), memory B cells (p = 0.028), activated CD4 (p = 0.005), and CD8 cells (p = 0.001), was observed even after the third vaccination dose, while a significant reduction in CD3+PD1+ (p = 0.002), NKT (p = 0.011), and activated NKT cells (p = 0.034) was noted during the same time interval. Moreover, SARS-CoV-2-specific T-cells were present in 41% of the patients who were unable to develop Nabs, and their positivity rates four months after the second dose were in inverse correlation with monocytes (p = 0.045) and NKT cells (p = 0.01).CONCLUSIONS:SARS-CoV-2-specific T-cell responses preceded the humoral ones, while two booster doses were needed for this group of immunocompromised patients to mount a protective immune response.

Introduction: Kidney transplant recipients show poor humoral immune response following SARS-CoV-2 vaccination. Knowledge of vaccine-induced cellular immunity and its association with antibody titers is limited. Method: SARS-CoV-2–specific neutralizing antibodies (Nab) were measured with chemiluminescence immunoassay in 53 naive kidney transplant recipients after the 2nd dose and before and after the 3rd dose of BNT162b2 mRNA vaccination. Nab were also measured in 23 Covid-19-recovered renal transplant patients before vaccination. Anti-HLA Abs were detected using Luminex to estimate any increase in HLA sensitization after vaccination. In 39 vaccinated patients with no Nab detected after the 2nd dose, SARS-CoV-2–specific T cells response was studied using interferon (IFN)-γ ELISpot analysis before and one month after the 3rd dose. Results: One month after the 2nd dose, only 15% of the naive patients demonstrated positive Nab and only 10% preserved them four months later. After the 3rd dose responders increased to 70%, and Nab was preserved in a repeat measurement four months later. Antibody titers after the 3rd dose, had a median value of 9.6 times above the cut-off value; however, they decreased to six times (p=0.003) four months later. Regarding anti-HLA Abs, neither MFI nor HLA specificities increased after vaccination, and none of the patients developed dnDSAs. Of the Covid-19-recovered transplant recipients, 74% demonstrated positive Nab with a median value three times above the cut-off value. In a repeat measurement before vaccination (median time 5 months later), 55% of recovered patients retained their Nab. SARS-CoV-2–specific T cells response was demonstrated in 41% of patients with nonresponse after the 2nd dose. The 3rd dose improved cellular immunity in these transplant recipients, as a specific T cell response was demonstrated in 64% of patients, with a significant increase (p=0.001) in specific T lymphocyte titers. Conclusion: Vaccination against SARS-CoV-2 appears to be safe regarding the development of anti-HLA Abs in kidney transplant recipients. The humoral immune response improved after the 3rd dose in naive patients, with Nab induced alike to Covid-19-recovered patients, although antibody titers declined four months later. Cellular immunity was present in many patients with primary humoral nonresponse before the 3rd dose and further improved after the 3rd dose, in parallel with Nab induction.

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.

Background T-cell immunity is affected in end stage renal disease (ESRD). However, whether this happens at pre- or post-dialysis stage and what is the impact of different renal replacement methods, remains unclear. We investigated the alterations of T-cell subtypes in patients at pre-dialysis ESRD and their further changes during dialysis. Methods CD4+, CD8+, CD4 + CD28null and CD8 + CD28null T-cells were analysed in 40 ESRD patients at two different time points, (a) the day started on dialysis (ESRD-T0) and (b) 6 months later (ESRD-T6), while being on haemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). Twenty-five age matched healthy volunteers served as controls. Results CD4+ and CD8+ T-cells were significantly reduced in ESRD-T0 patients compared to controls, 604 (105-3551) vs 943 (584-1867)mu/L,P= .001, and 352 (103-1561) vs 422.4 (263-1453)mu/L,P= .05, respectively. However, proportions of CD4 + CD28null and CD8 + CD28null cells were significantly increased, 6.4 (0.3-30)% vs 2.7 (0.1-7.8)%,P= .04 and 58.2 (12.8-85.4)% vs 39 (7.8-57.1)%,P= .01, respectively. Proportion of CD4 + CD28null cells showed significant correlation with serum CRP (r= .4,P= .04) and albumin levels (r= -.5,P= .007) in ERSD patients. ESRD-T0 patients with cardiovascular disease (CVD) had increased CD4 + CD28null and CD8 + CD28null proportions, 8.6 (1-30)% vs 2.1 (0.1-19.8)%,P= .04 and 62.5 (12.8-85.4)% vs 45.5 (5.7-73.7)%,P= .02, respectively, compared to those without. Six months later, both CD4 + CD28null and CD8 + CD28null T-cells were increased in HD compared to CAPD patients, by +110.11 (-27.1 to 311.4)% vs -28.1 (-100 to 30)%,P= .003 and +55.23 (-29.06 to 197.93)% vs -8.34 (-54.99 to 66.72)%,P= .05, respectively. Conclusions CD4 + CD28null and CD8 + CD28null T-cells are increased at pre-dialysis ESRD, and correlate with chronic inflammatory markers and the presence of CVD. Dialysis methods seem to have different impact on these subpopulations.

Lymphocyte apoptosis, as a programmed mechanism of lymphocyte death, is essential in maintaining homeostasis and balance between inflammatory and immune reactions. Disturbances in the apoptotic progress, leading to fragmented lymphocytes, “late apoptotic” cells, may result in immunodeficiency, oncogenesis, atheromatosis, etc. Aim of the present study was to investigate the lymphocyte apoptotic progress in End Stage Renal Disease (ESRD) and the effect of dialysis. The study included patients on ESRD; measurements were performed at the first day of dialysis (T0) and repeated 6 months later (T6), while being on dialysis. Total lymphocytes and B lymphocytes (CD19+) were gated and stained with Annexin V to detect apoptotic cells; early and late apoptotic cells were quantified. The results were compared to age-matched healthy control group. ESRD patients had reduced lymphocyte and B cell count, 1550±592μ/L vs. 2692±690μ/L, p<0.001 and 120.4±80μ/L vs. 321.7±184.7μ/L, p=0.002, respectively, compared to controls. There was an increase in total lymphocytes and B cells, being on later apoptotic stages (LAS) in ESRD-T0 compared to controls, 0.3±0.8% vs. 0.06±0.1%, and 0.04±0.08% vs. 0.01±0.03%, respectively, although differences did not reach statistical significance. After 6 months on dialysis, a reduction was noticed in the population of lymphocytes on LAS, 0.18±0.2% from 0.34±0.8%, while there was an increase of B cells on LAS, 0.1±0.2% from 0.02±0.07, with subsequent alterations in total numbers of apoptotic cells were also evident Late apoptotic changes affecting total and particularly B lymphocytes happen in ESRD, and initiation of dialysis seem to cause further alterations, which may be implicated in the increased morbidity and mortality of disease

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.

Abstract Background and Aims Tregs regulate immune responses and maintain self-tolerance, while Natural Killer cells (NK) are cytotoxic lymphocytes critical for both adaptive and innate immunity. The presence of chronic kidney disease (CKD) is often characterized by disturbances in immune responses, including dysregulation between tolerance and cytotoxicity. Aim of the present study was to evaluate the influence of CKD on the Tregs (CD4+CD25+FoxP3+) and NK cells, as well as the effect of hemodialysis (HD) and peritoneal dialysis (PD) on these subtypes. Method A total of 40 CKD Stage V patients and 15 healthy volunteer subjects were recruited into our study. Peripheral Tregs and NK cells were isolated from whole blood samples using flow cytometry, and the results were compared to those of age and gender matched controls. The immunologic profile of CKD patients was repeated 6 months after HD initiation (n=20) and PD (n=20) in order to investigate the effect of different dialysis methods. Results Patients with CKD had significantly reduced populations of CD4+CD25+ FoxP3+ Tregs (47.5± 28.8 vs 71.5±24.7) p=0.01, while there was no difference in NK cells between patients and controls (16.4±8.1% vs. 13.4±9.1%), p=NS, respectively. There were no differences between patients started on HD or PD in terms of clinical and laboratory parameters. Frequencies and total numbers of Tregs were significantly reduced after 6 months of HD, from 7.6±2.1% to 5.2±2.3%, p=0.009, and from 53.6±26K/μL to 36.9±19K/μL, p=0.02, respectively, while showed no differences on PD patients, from 5.3±3.3% to 5.6±2%, p=NS and 33.2±27K/μL to 45.6±28K/μL, p=NS, respectively. NK cells were increased in HD patients, not reaching statistical significance (245±156K/μL to 261±128K/μL). Frequencies of NK cells were positively correlated with CRP (r=0.56, p=0.009) and Intimal media thickness (IMT) (r=0.48, p=0.03) Conclusion The results of our study show that initiation of hemodialysis (HD) seems to reduce the expression of CD4+CD25+FoxP3+ T cells while peritoneal dialysis (PD) doesn’t seem affect this cell population. The immune alterations observed in hemodialysis patients may have a clinical impact on chronic inflammation and atheromatosis.

Abstract Background and Aims End-stage renal disease (ESRD) is linked to immunodeficiency, which makes a significant contribution to morbidity and mortality. Disturbances in innate and adaptive immunity have been described in patients on dialysis, although their association with the therapy itself is yet to be defined. The present study aimed to assess the impact of dialysis on B cell subpopulations Method B cells (CD19+) and their subsets B1a (CD19+CD5+), naive (CD19+CD27−), memory (CD19+CD27+), (CD19+BAFFR+) and (CD19+IgM+), were quantified using flow-cytometry of in the peripheral blood of ESRD patients, the first day on dialysis (T0), and repeated 6 months later (T6). The results were compared to age-matched healthy control group. Exclusion criteria were age <18 or>75 years, active autoimmune or chronic inflammatory disease, medical history of malignancy, corticosteroids or immunosuppresive treatment for the last 12 months Results Pre dialysis ESRD patients had reduced lymphocyte count (1527±646μ/L vs. 2459±520μ/L, p<0.001) and B cell (CD19+) count (82.7±59.5μ/L vs. 177.6±73.8μ/L, p<0.001) compared to controls, whereas the percentages of B cell subsets were not particularly affected, except for B1a subset which presented a significant increase (4.1±3.6% vs. 0.7±0.7% p<0.001). In 17 patients who had a follow-up sample 6 months later, the percentage of most subsets was reduced (CD19+CD5+: 1.02±0.8% from 3.6±4.6%, p=0.015, Naive: 40±22.3% from 61±17.4%, p=0.001, CD19+BAFF+:75.8±12.6% from 82.1±9.1%, p=0.04,), apart from memory B cells percentage, which was increased (49.4±52.1% from 32,9±35,5%, p=0.01) and CD19+ IgM+ percentage, which was unaffected . Conclusion A significant reduction of almost all subsets of B cells was noticed in patients with ESRD on pre-dialysis stage. Furthermore, the initiation of renal replacement therapy may be linked to further alterations in B cells subpopulations, especially at their early stages.

INTRODUCTION AND AIMS:Aliskiren as an add-on therapy to losartan further reduced proteinuria among patients with diabetic nephropathy, yet the ALTITUDE study designed to investigate the potential renal protection benefits using this combination was prematurely terminated due to increased adverse events in the aliskiren arm.The potential long-term efficacy of aliskiren in non-diabetic chronic kidney disease (CKD) is unknown.METHODS: This open-label, prospective, randomized, controlled trial investigated the renoprotective potential and safety of aliskiren added to an angiotensin II receptor blocker in non-diabetic CKD stages 3-4 patients in a University teaching hospital in Hong Kong.Eligible patients receiving an ARB at the maximal dose were randomly assigned aliskiren or conventional treatment to achieve BP under 130/80 mmHg.The coprimary outcomes were doubling of baseline serum creatinine (sCr) or a 40% reduction in estimated glomerular filtration rate (eGFR); and incident end-stage renal disease (ESRD).Secondary endpoints included cardiovascular events, hyperkalemia and death.RESULTS: 76 patients were randomized: 37 to aliskiren (27 male, mean age 55.0611.1 y), and 39 to control (27 male, mean age 55.069.4 y).There was no difference in baseline demographics, sCr (194661 vs. 215665 lmol/l, P¼0.15), eGFR (31.969.0 vs. 27.769.0ml/min/1.73m 2 , P¼0.05), and urine protein-to-creatinine ratio for treatment vs. control subjects.After a follow-up of 144 weeks, four patients in intervention group and seven patients in control group reached the composite endpoint of doubling of sCr or ESRD (10.8% vs. 18.0%,P¼0.377).The number of cardiovascular events was 4 (10.8%) vs. 1 (2.6 %), P¼0.147.Hyperkalemia was encountered in 7 (18.9%) vs. 2 (5.1%) patients (P¼0.063).There was no difference in the rate of eGFR change or proteinuria between the 2 groups.CONCLUSIONS: Aliskiren conferred no additional renoprotective benefit nor increased adverse events except for more hyperkalemia in non-diabetic CKD patients.