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Knowledge on immunogenicity of the bivalent Omicron BA.4/5 vaccine in dialysis patients and the effect of a previous infection is limited. Therefore, vaccine-induced humoral and cellular immunity was analyzed in dialysis patients and immunocompetent controls with and without prior infection. In an observational study, 33 dialysis patients and 58 controls matched for age, sex and prior infection status were recruited. Specific IgG, neutralizing antibody activity and cellular immunity towards the spike-antigen from parental SARS-CoV-2 and Omicron-subvariants BA.1, BA.2 and BA.4/5 were analyzed before and 13-18 days after vaccination. The bivalent vaccine led to a significant induction of IgG, neutralizing titers, and specific CD4+ and CD8+ T-cell levels. Neutralizing activity towards the parental strain was higher than towards the Omicron-subvariants, whereas specific T-cell levels towards parental spike and Omicron-subvariants did not differ indicating substantial cross-reactivity. Dialysis patients with prior infection had significantly higher spike-specific CD4+ T-cell levels with lower CTLA-4 expression compared to infection-naive patients. When compared to controls, no differences were observed between infection-naive individuals. Among convalescent individuals, CD4+ T-cell levels were higher in patients and neutralizing antibodies were higher in controls. Vaccination was overall well tolerated in both dialysis patients and controls with significantly less adverse events among patients. In conclusion, our study did not provide any evidence for impaired immunogenicity of the bivalent Omicron BA.4/5 vaccine in dialysis patients. Unlike in controls, previous infection of patients was even associated with higher levels of spike-specific CD4+ T cells, which may reflect prolonged encounter with antigen during infection. [ABSTRACT FROM AUTHOR]

For vulvar cancers, radiotherapy is targeting cancer cells, but also affects the host immune system. As this may affect treatment outcome, in this prospective study, we characterized the individual T cell immune milieu induced by surgery and adjuvant radio +/− chemotherapy (aRT) systemically in the blood of vulvar cancer patients and found increased frequencies of Interleukin (IL)‐17‐producing CD4+ and CD8+ T cells after aRT while frequencies of Th1 and perforin‐producing CD8+ killer cells were strongly diminished. Phenotypic characterization revealed enhanced expression of the ectonucleotidase CD39 on Th17 and Tc17 cells as well as CD8+ perforin+ cells after aRT. Furthermore, the aRT cohort exhibited increased proportions of Programmed Cell Death Protein (PD‐1) expressing cells among Th1 and CD8+ perforin+ cells, but not among Th17 and Tc17 cells. High post‐therapeutic levels of Th17 and Tc17 cells and low proportions of Th1 and CD8+ perforin+ cells expressing PD‐1 was associated with reduced recurrence free survival on follow‐up. In conclusion, our study defines individual therapy‐induced changes in the cellular immune milieu of patients and their association with cancer relapse. Our results may help to explain differences in the individual courses of disease of vulvar cancer patients and suggest PD‐1 and IL‐17 as targets for immunotherapy in vulvar cancer. [ABSTRACT FROM AUTHOR]

Post‐transplant cytomegalovirus (CMV) infections and increased viral replication are associated with CMV‐specific T‐cell anergy. In the ATHENA‐study, de‐novo everolimus (EVR) with reduced‐exposure tacrolimus (TAC) or cyclosporine (CyA) showed significant benefit in preventing CMV infections in renal transplant recipients as compared to standard TAC + mycophenolic acid (MPA). However, immunomodulatory mechanisms for this effect remain largely unknown. Ninety patients from the ATHENA‐study completing the 12‐month visit on‐treatment (EVR + TAC n = 28; EVR + CyA n = 19; MPA + TAC n = 43) were included in a posthoc analysis. Total lymphocyte subpopulations were quantified. CMV‐specific CD4 T cells were determined after stimulation with CMV‐antigen, and cytokine‐profiles and various T‐cell anergy markers were analyzed using flow cytometry. While 25.6% of MPA + TAC‐treated patients had CMV‐infections, no such events were reported in EVR‐treated patients. Absolute numbers of lymphocyte subpopulations were comparable between arms, whereas the percentage of regulatory T cells was significantly higher with EVR + CyA versus MPA + TAC (p = 0.019). Despite similar percentages of CMV‐specific T cells, their median expression of CTLA‐4 and PD‐1 was lower with EVR + TAC (p < 0.05 for both) or EVR + CyA (p = 0.045 for CTLA‐4) compared with MPA + TAC. Moreover, mean percentages of multifunctional CMV‐specific T cells were higher with EVR + TAC (27.2%) and EVR + CyA (29.4%) than with MPA + TAC (19.0%). In conclusion, EVR‐treated patients retained CMV‐specific T‐cell functionality, which may contribute to enhanced protection against CMV infections. [ABSTRACT FROM AUTHOR]

Patients with pre-existing comorbidities and immunosuppression are at greater risk for SARS-CoV-2 infection and severe manifestations of COVID-19. This also includes cancer patients, who are shown to have a poor prognosis after infection. Here, we describe the case of a 72-year old male patient with B-cell depletion after maintenance treatment with rituximab for non-Hodgkin-lymphoma who had a prolonged COVID-19 course and initial false negative test results. Our case highlights the diagnostic pitfalls in diagnosing COVID-19 in B-cell depleted patients and discuss the role of B-cell depletion in the course and treatment of COVID-19. Furthermore, we investigated peripheral blood monocytes and SARS-CoV-2 specific T cells in our patient. In conclusion, our case report can help physicians to avoid diagnostic pitfalls for COVID-19 in hemato-oncological patients under chemoimmunotherapy and tries to explain the role of B-cell depletion and SARS-CoV-2 specific T cells in this context. [ABSTRACT FROM AUTHOR]

Background: Micro(mi)RNAs are increasingly recognized as central regulators of immune cell function. While it has been predicted that miRNAs have multiple targets, the majority of these predictions still await experimental confirmation. Here, miR-34a, a well-known tumor suppressor, is analyzed for targeting genes involved in immune system processes of leucocytes. Methods: Using an in-silico approach, we combined miRNA target prediction with GeneTrail2, a web tool for Multi-omics enrichment analysis, to identify miR-34a target genes, which are involved in the immune system process subcategory of Gene Ontology. Results: Out of the 193 predicted target genes in this subcategory we experimentally tested 22 target genes and confirmed binding of miR-34a to 14 target genes including VAMP2, IKBKE, MYH9, MARCH8, KLRK1, CD11A, TRAFD1, CCR1, PYDC1, PRF1, PIK3R2, PIK3CD, AP1B1, and ADAM10 by dual luciferase assays. By transfecting Jurkat, primary CD4+ and CD8+ T cells with miR-34a, we demonstrated that ectopic expression of miR-34a leads to reduced levels of endogenous VAMP2 and CD11A, which are central to the analyzed subcategories. Functional downstream analysis of miR-34a over-expression in activated CD8+ T cells exhibits a distinct decrease of PRF1 secretion. Conclusions: By simultaneous targeting of 14 mRNAs miR-34a acts as major hub of T cell regulatory networks suggesting to utilize miR-34a as target of intervention towards a modulation of the immune responsiveness of T-cells in a broad tumor context. [ABSTRACT FROM AUTHOR]

• Cell-mediated immunity assays help resolve CMV status when passive antibodies exist. • Flow cytometry detected CMV-specific CD4+Tcells useful to assign CMV status pre-SOT. • CD27-CD28-CD4 + T cell analysis lacks specificity in assignment of CMV status pre-SOT. • CMV DNA is rarely detected in blood/urine of CMV-seropositive adults awaiting SOT. Background/objectives: Determination of Cytomegalovirus (CMV) status in solid organ transplant (SOT) candidates is essential to stratify risk of post-transplant CMV disease. Passive transfusion-acquired antibodies can make serologic determination of CMV status unreliable. We evaluated 3 assays, not affected by passive antibodies (PA), in assignment of CMV status: quantification of CMV-specific CD4 + T-cells (CMV-TC) and exhausted CD27-CD28- CD4 + T-cells, and detection of CMV DNA with Nucleic Acid Amplification Testing (NAAT). Study design: We enrolled 50 adults awaiting SOT and 50 immunocompetent age-matched controls, and collected a throat swab, urine, saliva and blood sample on each. Using flow cytometry CD4 + T-cells were phenotypically analyzed for expression of CD27 and CD28 and CMV-specific CD4 + T-cells were identified by CD69 expression and intracellular IFN-γ quantification after stimulation with CMV-antigen lysate. CMV NAAT was performed on all specimens using real-time PCR. CMV serology (CMV IgG) was determined by enzyme immunoassay. Subjects were considered to have potential PA if they received blood products within 2 months of collection. Results: The CMV-TC assay discriminated between CMV-seropositive and seronegative SOT candidates without PA well (sensitivity 79%, specificity 93%) while the CD27-CD28-CD4 + T-cell assay had good sensitivity (86%) but specificity of 74%. Detection of CMV DNA was uncommon in CMV-seropositive SOT candidates (2/21). Conclusions: Given its high specificity, the CMV-TC assay is valuable in confirming true-positive CMV status in seropositive SOT candidates with PA, while use of CD27-CD28-CD4 + T-cell analysis is limited by moderate specificity. Detection of CMV DNA is of limited value in assignment of CMV status in adults. [ABSTRACT FROM AUTHOR]

Passive antibodies, maternal or transfusion-acquired, make serologic determination of pretransplant cytomegalovirus (CMV) status unreliable. We evaluated 3 assays unaffected by passive antibodies, in assignment of CMV infection status in children awaiting solid organ transplant and in controls: (1) CMV nucleic acid amplification testing (NAAT), (2) quantification of CMV-specific CD4+ T cells, and (3) quantification of CD27-CD28-CD4+ T cells. Our results highlight that CMV NAAT, from urine and oropharynx, is useful in confirming positive CMV status. Detection of CMV-specific CD4+ T cells was sensitive and specific in children >18 months but was less sensitive in children <12 months. CD27-CD28-CD4+ T cells are not likely useful in CMV risk stratification in children. [ABSTRACT FROM AUTHOR]

Background: Research with primary human white blood cell (WBC) subpopulations requires high quantity, quality, and functionality of peripheral blood mononuclear cells (PBMCs) as a source to further characterize cellular subpopulations such as T and B lymphocytes, monocytes, or natural killer cells. Apart from buffy coats derived from whole blood, residual blood from preparative hemapheresis kits are used as a source for PBMCs, but knowledge on the yield and functionality of cells from different devices is limited.Study Design and Methods: We evaluated quantity and quality of PBMCs isolated from apheresis kits of two apheresis devices (AMICUS, Fenwal; and Trima Accel, Terumo BCT), the latter being our standard source for many years. PBMCs derived from Trima or AMICUS were tested for yield and subtype composition by flow cytometry. Functionality was assessed by cytokine induction of CD4+ and CD8+ T cells and by degranulation. Moreover, cytotoxic activity of natural killer cells was quantified by a real-time killing assay.Results: Mean numbers of isolated cells were 5.5 ± 2.4 × 108 for AMICUS, and 10.3 ± 6.4 × 108 for Trima Accel, respectively. The proportion of WBC subtypes corresponded to well-known numbers from whole blood, with minor differences between the two apheresis systems. Likewise, minor differences in cytokine induction were found in stimulated CD4+ or CD8+ T cells. Finally, PBMCs derived from the two systems showed comparable cytotoxic activity.Conclusion: PBMC derived from residual blood of the AMICUS and Trima Accel apheresis devices serve as an economic and easily accessible source for functional PBMCs with comparable quantity and quality to PBMCs derived from whole blood. [ABSTRACT FROM AUTHOR]

VZV‐reactivation may lead to symptomatic central nervous system (CNS) diseases, but identification of VZV as causative pathogen of CNS‐diseases is challenging. This study was performed to characterize VZV‐specific T cells from cerebrospinal fluid (CSF) and blood of patients with active CNS‐disease and to determine whether this may improve differential diagnosis. 27 patients with pleocytosis in the CSF were recruited and classified into three groups (10 VZV‐related, 10 non‐VZV‐related, 7 unclear). VZV‐specific CD4+ T cells were quantified in CSF and blood after simultaneous stimulation with a VZV‐antigen lysate and detection of cytokines (IFN‐γ, IL‐2, TNF‐α) and CTLA‐4. Polyclonal stimulation served as positive control. VZV‐specific CD4+ T‐cell frequencies were highest in both CSF (p = 0.0001) and blood (p = 0.011) of patients with VZV‐infection, and were enriched at the site of infection (p = 0.002). While cytokine‐expression profiles only showed minor differences between the groups, CTLA‐4‐expression levels on VZV‐specific T cells from CSF and blood were significantly increased in VZV‐related CNS‐infections (p = 0.0002 and p<0.0001) and clearly identified VZV‐related CNS‐diseases (100% sensitivity and 100% specificity). Polyclonally stimulated T cells did not show any quantitative and phenotypical differences between the groups. Increased frequency and CTLA‐4‐expression of VZV‐specific T cells from CSF or blood are specifically found in patients with VZV‐related CNS‐infection. [ABSTRACT FROM AUTHOR]

Background: After kidney transplantation, immunosuppressive therapy causes impaired cellular immune defense leading to an increased risk of viral complications. Trough level monitoring of immunosuppressants is insufficient to estimate the individual intensity of immunosuppression. We have already shown that virus-specific T cells (Tvis) correlate with control of virus replication as well as with the intensity of immunosuppression. The multicentre IVIST01-trial should prove that additional steering of immunosuppressive and antiviral therapy by Tvis levels leads to better graft function by avoidance of over-immunosuppression (for example, viral infections) and drug toxicity (for example, nephrotoxicity). Methods/design: The IVIST-trial starts 4 weeks after transplantation. Sixty-four pediatric kidney recipients are randomized either to a non-intervention group that is only treated conservatively or to an intervention group with additional monitoring by Tvis. The randomization is stratified by centre and cytomegalovirus (CMV) prophylaxis. In both groups the immunosuppressive medication (cyclosporine A and everolimus) is adopted in the same target range of trough levels. In the non-intervention group the immunosuppressive therapy (cyclosporine A and everolimus) is only steered by classical trough level monitoring and the antiviral therapy of a CMV infection is performed according to a standard protocol. In contrast, in the intervention group the dose of immunosuppressants is individually adopted according to Tvis levels as a direct measure of the intensity of immunosuppression in addition to classical trough level monitoring. In case of CMV infection or reactivation the antiviral management is based on the individual CMV-specific immune defense assessed by the CMV-Tvis level. Primary endpoint of the study is the glomerular filtration rate 2 years after transplantation,secondary endpoints are the number and severity of viral infections and the incidence of side effects of immunosuppressive and antiviral drugs. Discussion: This IVIST01-trial will answer the question whether the new concept of steering immunosuppressive and antiviral therapy by Tvis levels leads to better future graft function. In terms of an effect-related drug monitoring, the study design aims to realize a personalization of immunosuppressive and antiviral management after transplantation. Based on the IVIST01-trial, immunomonitoring by Tvis might be incorporated into routine care after kidney transplantation. [ABSTRACT FROM AUTHOR]

The evaluation of delayed-type hypersensitivity immune responses by the tuberculin skin test and, more recently, by ex vivo IFN-γ release assays (IGRAs) form the basis for the risk assessment for the development of tuberculosis (TB) in close contacts of infective index patients and in immunocompromised hosts. In contrast, immunodiagnosis has little value for the diagnosis of active TB so far. A report in this issue of the European Journal of Immunology [Eur J Immunol 2012. 42: 2844-2850] provides new insight into the phenotypic characteristics and selective recruitment of Ag-specific T cells to the site of the infection, as analyzed by flow cytometry, which may provide new opportunities for the immune-based diagnosis of TB in the future. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV) infection is the most frequent viral complication in patients after allogeneic stem cell transplantation. As CMV replication is tightly controlled by the cellular arm of specific immunity, the kinetics of CMVspecific T cells in association with individual reactivation episodes were prospectively analyzed in 40 allogeneic transplant recipients in a routine clinical setting and evaluated as determinant of impaired CMV control. Antigen-specific CD4 and CD8 T cells were quantified directly from whole blood using intracellular cytokine staining after specific stimulation and MHC class I multimers, respectively. Highly dynamic intraindividual changes of CMV-specific CD4 T cells were observed in patients experiencing CMV viremia. Episodes of CMV reactivation were associated with a drop of CMV-specific CD4 T cells that reincreased after viral clearance (p,0.0001). Furthermore, levels of CMV-specific CD4 T cells at the onset of viremia inversely correlated with peak viral load thereafter (p = 0.02). In contrast, CMV-peptide specific CD8 T cells did not show any association with viremia (p = 0.82). Interestingly, therapeutic dosages of cyclosporine A and corticosteroids led to a dosedependent reduction of CMV-specific T-cell functions, indicating a causal link between intensified immunosuppressive treatment and CMV reactivation. In conclusion, levels of CMV-specific CD4 T cells inversely correlate with reactivation episodes and may represent a valuable measure to individually guide antiviral therapy after stem cell transplantation. [ABSTRACT FROM AUTHOR]

Background: Cytomegalovirus (CMV) seronegative recipients (R-) of kidney transplants (KT) from seropositive donors (D+) are at higher risk for CMV replication and ganciclovir(GCV)-resistance than CMV R(+). We hypothesized that low CMV-specific T-cell responses are associated with increased risk of CMV replication in R(+)-patients with D(+) or D(-) donors. Methods: We prospectively evaluated 73 consecutive KT-patients [48 R(+), 25 D(+)R(-)] undergoing routine testing for CMV replication as part of a preemptive strategy. We compared CMV-specific interferon-γ (IFN-γ) responses of CD4+CD3+ lymphocytes in peripheral blood mononuclear cells (PBMC) using three different antigen preparation (CMV-lysate, pp72- and pp65-overlapping peptide pools) using intracellular cytokine staining and flow cytometry. Results: Median CD4+ and CD8+T-cell responses to CMV-lysate, pp72- and pp65-overlapping peptide pools were lower in D(+)R(-) than in R(+)patients or in non-immunosuppressed donors. Comparing subpopulations we found that CMV-lysate favored CD4+- over CD8+-responses, whereas the reverse was observed for pp72, while pp65-CD4+- and -CD8+-responses were similar. Concurrent CMV replication in R(+)-patients was associated with significantly lower T-cell responses (pp65 median CD4+ 0.00% vs. 0.03%, p = 0.001; CD8+ 0.01% vs. 0.03%; p = 0.033). Receiver operated curve analysis associated CMV-pp65 CD4+ responses of > 0.03% in R(+)-patients with absence of concurrent (p = 0.003) and future CMV replication in the following 8 weeks (p = 0.036). GCV-resistant CMV replication occurred in 3 R(+)-patients (6.3%) with pp65- CD4+ frequencies < 0.03% (p = 0.041). Conclusion: The data suggest that pp65-specific CD4+ T-cells might be useful to identify R(+)-patients at increased risk of CMV replication. Provided further corroborating evidence, CMV-pp65 CD4+ responses above 0.03% in PBMCs of KT patients under stable immunosuppression are associated with lower risk of concurrent and future CMV replication during the following 8 weeks. [ABSTRACT FROM AUTHOR]

Assessment of antigen-specific T-cell responses has been greatly facilitated by development of ELISPOT and intracellular cytokine flow cytometry (CFC) assays. The use of autologous antigen presenting cells transfected with in vitro transcribed RNA as stimulators allows in principle quantification of antigen-specific T-cells independent of the knowledge of the epitopes. We describe here a cytokine secretion assay that enables simultaneous assessment of both antigen-specific CD4+ as well as CD8+ T-cells directly from clinical samples without the need for generation of dendritic cells. To this aim, bulk PBMCs were electroporated with RNA encoding the antigen fused to trafficking signal sequences derived from a MHC class I molecule and used as stimulators. With human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) as antigen we show that for measuring ex vivo T-cell responses in ELISPOT and CFC such stimulators are superior or at least equivalent to a pool of overlapping peptides representing the entire pp65 sequence as well as to untagged pp65 encoding RNA. This approach avoids the time consuming generation of dendritic cells as immune stimulators and, in particular when used in the context of the CFC, is robust, broadly applicable and fast. [ABSTRACT FROM AUTHOR]

Abstract: Several prevalent and life-threatening agents enter the organism via the mucosa. In this case, a mucosal cellular immune response is essential for protection and is therefore considered the main objective of vaccination. The frequency of antigen-specific CD4+ and CD8+ T cells can be determined directly in human whole blood by a combination of surface marker and intracellular cytokine staining. Immune cells primed in the mucosal compartment also migrate through the blood and can be identified by expression of the gut-specific homing receptor α4β7. Simultaneously, these lymphocytes can be functionally characterized regarding their differentiation status by analysis of CD45RO and CD27 expression and effector functions by measuring intracellular perforin or granzyme B content. Thus, the technique described in the paper is a powerful tool for clinical monitoring of the total cellular immune response to complex antigens during infection or vaccination. [Copyright &y& Elsevier]

Tuberculin skin testing underestimates a high prevalence of latent tuberculosis infection in hemodialysis patients. Background. Identification of latent Mycobacterium tuberculosis infection in hemodialysis patients is hampered by reduced sensitivity of the established tuberculin skin test. We investigated whether in vitro quantitation of purified protein derivative (PPD)–specific T cells using a rapid 6-hour assay may represent an alternative approach for detecting latent infection. Methods. One hundred and twenty-seven hemodialysis patients and 218 control patients (blood donors, health care workers, and control patients) were analyzed. Specific T cells toward PPD and early secretory antigenic target-6 (ESAT-6), a protein expressed in Mycobacterium tuberculosis but absent from M. bovis bacillus Calmette-Guerin (BCG) vaccine strains, were flow cytometrically quantified from whole blood, and results were compared with skin testing. Results. Compared to blood donors, a high proportion of both health care workers (48.6%) and hemodialysis patients (53.5%) had PPD-specific Th1-type CD4 T-cell reactivity with similar median frequencies of PPD-specific T cells (0.17%; 0.06–3.75% vs. 0.26%; 0.06–4.12%, respectively). In contrast, skin test reactivity was significantly reduced in hemodialysis patients. Whereas 85.7% of control patients with PPD reactivity in vitro were skin test–positive, the respective percentage among hemodialysis patients was 51.4% ( P= 0.007). Among individuals with PPD reactivity in vitro, ∼50% had T cells specific for ESAT-6. Conclusion. Unlike the skin test, measurement of PPD reactivity by in vitro quantitation of PPD-specific T cells was unaffected by uremia-associated immunosuppression. This whole-blood assay may thus be a valuable alternative to skin testing, and detection of ESAT-6–specific T cells could moreover allow distinction of latent M. tuberculosis infection from BCG-induced reactivity to PPD. The assay is well suited for clinical use and may facilitate targeting of preventative therapy in high-risk individuals. [ABSTRACT FROM AUTHOR]

Analyzes the adenovirus-specific T cell responses from healthy individual and long-term renal transplant recipients. Use of flow-cystometric, standard proliferation and enzyme-linked immunosorbent assays; Domination of CD4 T cells in the adenovirus-specific immunity; Decrease in the frequency of adenovirus-specific T cells.

Specific T cell immunity in patients with active tuberculosis is associated with a decrease in multifunctionality. However, it is unknown whether cytokine profiles differ in patients with primary infection and those with prior contact. We therefore used intravesical immunotherapy with attenuated live Bacille Calmette–Guérin (BCG) in patients with urothelial carcinoma as a model to characterise the induction of systemic immunity towards purified protein derivate (PPD) and to study whether cytokine profiles differ depending on pre-existing immunity. Eighteen patients with non-muscle invasive bladder cancer were recruited during the BCG-induction course. Fifty-four healthy individuals served as controls. Interferon (IFN)-γ and interleukin (IL)-2 producing PPD-specific CD4 T cells were analysed longitudinally before each instillation using a rapid flow-cytometric whole blood immunoassay. Baseline levels of IFN-γ producing PPD-specific T cells were comparable to controls. T cells showed a 5-fold increase to 0.23% by week 2/3, and further increased 8-fold by week 4/5 (to 0.42%, p=0.0007). Systemic immunity was induced in all patients, although the increase was less pronounced in patients with pre-existing immunity. As in active TB, cytokine profiling during therapy revealed a lower percentage of multifunctional IFN-γ/IL-2 double-positive T cells compared to controls (60.2% vs. 71.9%, p=0.0003). Of note, when comparing patients with and without pre-existing immunity, cytokine profiles in patients with primary immunity were shifted towards IL-2 single producing T cells (p=0.02), whereas those in patients with pre-existing immunity were shifted towards IFN-γ single-positivity (p=0.01). In conclusion, systemic T cell responses were induced after BCG-therapy, and their kinetics and cytokine profile depended on pre-existing immunity. Decreased functionality is a typical feature of specific immunity in both patients with active tuberculosis and BCG-therapy. Among patients with active infection, a shift towards IL-2 or IFN-γ single-positive cells may allow distinction between patients with primary infection and cases with boosted immunity after prior contact, respectively. [ABSTRACT FROM AUTHOR]

Objectives: Influenza vaccination was used to assess whether induction of immunity or side effects are influenced by the timing of the last training session before vaccination. Methods: Forty-five healthy athletes (36 male, 23 ± 8 yr, ≥5 training sessions per week, predominantly national competition level) were vaccinated with the tetravalent influenza vaccine; blood samples were collected immediately before and 1, 2, and 26 wk after vaccination. Athletes were randomly assigned to vaccination within 2 h after the last training session versus after 24–26 h. Influenza-specific T cells were quantified after stimulation with the vaccine based on intracellular cytokine staining. Antibodies (IgA, IgG, IgM) were quantified by enzyme-linked immunosorbent assay and neutralization assay. Participants documented resulting side effects and training restrictions using a standardized diary. Results: Both groups showed an increase in influenza-reactive CD4 T-cell levels, which peaked 1 wk after vaccination (fold changes to baseline; median (interquartile range), 3.7 (3.0–5.4; P < 0.001) in the 2-h group; 4.6 (2.8–7.4; P < 0.001) in the 26-h group) with no difference between groups (P = 0.52). Influenza-specific antibodies showed a significant increase after vaccination in both groups (at least 1.4-fold, each P < 0.001, no group differences; P = 0.24–0.97 for different antibody types). Only antibodies toward the Brisbane strain showed a trend toward significant differences in neutralization titers between groups (4-fold (2–17.8) in the 2-h group, 16-fold (4–32.9) in the 26-h group; P = 0.06), whereas other specificities did not differ (P = 0.16–0.72). No intergroup differences were found for side effects; no athlete reported a loss of training time due to the vaccination or its side effects. Conclusion: Infection prophylaxis in elite athletes by influenza vaccination seems to be effective and safe. Timing of vaccination after prior training does not seem to require specific constraints. [ABSTRACT FROM AUTHOR]

• Regular training in elite athletes does not adversely affect vaccination responses. • Influenza-specific T-cell responses are more pronounced in elite athletes. • Influenza-specific neutralizing antibodies are more pronounced in elite athletes. • Local side-effects after vaccination were mild and did not differ in both groups. Compliance of elite athletes with vaccination recommendations is low mainly based on concerns about side-effects and perceived poor vaccine efficacy due to continued physical training. We therefore employed seasonal influenza vaccination to investigate the effect of regular physical training on vaccine-induced cellular and humoral immunity in elite athletes and controls. Lymphocyte subpopulations and vaccine-specific T-cells were quantified and functionally characterized from 45 athletes and 25 controls before, and 1, 2 and 26 weeks after vaccination. Moreover, influenza-specific antibodies and their neutralizing function were quantified. Both groups showed a significant increase in vaccine-reactive CD4 T-cell levels which peaked one week after vaccination (p < 0.0001). The increase was significantly more pronounced in athletes (4.1-fold) compared to controls (2.3-fold; p = 0.0007). The cytokine profile changed from multifunctional T-cells co-producing IFNγ, IL-2 and TNFα to cells with restricted cytokine expression. This change in functionality was associated with a significant increase in CTLA-4 expression (p < 0.0001), which again was more pronounced in athletes. Likewise, the increase in neutralizing antibodies was stronger in athletes (p = 0.004 for H1N1; p = 0.032 for H3N2). In conclusion, both groups mounted a strong vaccine-specific cellular and humoral immunity after standard vaccination. The more pronounced increase in specific T-cells and neutralizing antibodies indicates that high frequency and intensity of training enhance vaccine-responses in elite athletes. [ABSTRACT FROM AUTHOR]

A lung and kidney transplant recipient underwent cytomegalovirus (CMV) primary infection with a UL97 mutation. Combined monitoring of viral load and CMV-specific CD4 T-cells allowed reduction of treatment duration with foscarnet, and illustrates how knowledge on the individual immunocompetence towards CMV may be used to individualize duration of antiviral treatment. [ABSTRACT FROM AUTHOR]

Highlights: [•] Vaccine responses are hampered in patients with end-stage renal failure. [•] Patients and controls show induction of specific T-cells after 1ry vaccination. [•] Induction of specific T-cells in controls is less pronounced after 2ry vaccination. [•] Patients show impaired maintenance of memory precursor cells over time. [•] Re-vaccination kinetics in patients resemble those of vaccination-naïve controls. [ABSTRACT FROM AUTHOR]

AbstractGenetically modified lymphoblastoid cell lines (LCL) have been shown to be an attractive alternative source of antigen-presenting cells for cancer vaccination in vitro. We tested their application in patients with pancreatic cancer in a phase I clinical trial. As a model tumor antigen, we selected the point-mutated (codon 12) Ki-Rasp21 oncogene (muRas) frequently (?85%) present in pancreatic adenocarcinoma. Autologous LCLs were established in vitroby spontaneous outgrowth from peripheral blood lymphocytes of seven pancreatic carcinoma patients and were genetically modified with an episomal Epstein-Barr virus (EBV)–based expression vector to express muRas (muRas-LCL). Weekly vaccinations with subcutaneous injection of 5×106muRas-LCL were done. In six of seven patients, therapeutic vaccination elicited a T-cell response with an increase in the frequency of muRas-specific precursor cytotoxic T lymphocytes in the peripheral blood and positive delayed-type hypersensitivity reactions at the injection site. Besides local reactions and flu-like symptoms, there were no signs of toxicity and no acute EBV infection, onset of EBV-associated lymphoma, or other severe complications. A clinical response (stable disease) was observed for a short time period (2–4 months) in four of seven patients (57%), mostly in earlier tumor stages. Our results indicate that LCL presenting genetically modified antigen represent a valuable and easily available tool for in vivoautologous tumor vaccination. LCL can be transfected with any known tumor antigen and therefore should be further clinically investigated. [ABSTRACT FROM AUTHOR]

Abstract: Background: Serological analysis of the infection status with the human cytomegalovirus (CMV) may be inaccurate after transfusion of blood products due to the variable content of CMV-specific antibodies. Objectives: In this situation, analysis of cellular immunity may represent a more accurate parameter to assign the individual CMV-infection status. This hypothesis was assessed in a sequence of clinically defined events where a CMV-seronegative patient received human immunoglobulins before AB0 incompatible transplantation of a graft from his CMV-seropositive mother and developed CMV-primary infection thereafter. Study design: Humoral immunity was analyzed using ELISA, and CMV-specific CD4 T-cells were flow-cytometrically quantified using intracellular cytokine staining after a 6h-stimulation with a CMV-antigen lysate. Results: Prior to transplantation, both CMV-specific antibody-titers and T-cell frequencies were below detection limit. After plasma infusion, the patient was temporarily seropositive but remained T-cell negative indicating passive immunity. CMV-specific T-cells became stably detectable after graft-related primary infection, thereby confirming a truly positive infection status. Conclusion: This case provides an instructive proof of principle to show that CMV-specific CD4 T-cells may serve as an accurate marker to define the true CMV-infection status in situations where serological testing is limited by the presence of passively administered antibodies. [Copyright &y& Elsevier]

Abstract: Background: Donor-derived transmission of hepatitis B virus (HBV) may cause serious complications after transplantation. To date, transplantation from HBV-infected donors to HBV-infected recipients seems feasible, although this is recommended with prophylaxis with specific drugs and antibodies only, whereas pre-emptive strategies are rarely used. Objectives: Here, we assessed the success of transplantation of kidneys from a chronically HBV-infected deceased donor (HBs-antigen positive, anti-HBc positive, HBV-DNA positive) to two recipients with cleared HBV-infection (HBs-antigen negative, anti-HBc positive, anti-HBs >100IU/l) where risk-assessment was performed using a pre-emptive approach in the absence of prophylaxis. Study design: Pre-emptive monitoring included assessment for evidence of infection by analysis of liver enzymes, viral load, and humoral and cellular immunity against HBV and CMV. Results: In line with undetectable HBV-load, HBc-specific T-cell frequencies remained stable (mean 0.46±0.10% and 0.06±0.03%), whereas CMV-specific T-cell frequencies in one patient showed dynamic changes that coincided with CMV-viremia. Likewise, HBV-specific antibody titres were stable. Liver enzymes demonstrated absence of liver-cell injury and renal function was good (creatinine 1.8 and 0.8mg/dl at last follow-up after 39 and 38 months, respectively). Conclusions: When combined with careful HBV-monitoring, kidneys from HBV-infected donors may be transplanted into HBV-immune recipients without the need for specific prophylaxis. [ABSTRACT FROM AUTHOR]

Besides iatrogenic immunosuppression, endogenous suppression by regulatory T cells (Tregs) may also mediate inhibition of effector T cells after transplantation. Here we determined the effect of common immunosuppressive drug regimens on both Treg and effector T cells. Tregs and cytomegalovirus (CMV)-specific T cells were quantified in 88 renal transplant recipients, 58 hemodialysis patients, and 22 controls. T cell dynamics were longitudinally assessed within 20 weeks after transplantation. The number of Tregs was quantified by measurement of CD25 and/or FOXP3-positive cells and by functional assays. CMV-specific T cells were quantified by stimulation-induced intracellular cytokine analysis. Treg frequencies in transplant recipients were significantly lower compared to those in hemodialysis patients and controls. These lower Treg levels were associated with a less pronounced suppression of effector function. Treg levels decreased within the first weeks after transplantation and remained low in the long term. In contrast, although decreased at early post-transplant, long-term levels of CMV-specific T cells normalized to levels found in hemodialysis patients and controls. These studies suggest that there is an initial decrease of Tregs and effector T cells as a consequence of a direct inhibitory effect of immunosuppressive drugs. In the long term, persistently low Treg levels may favor normalization of effector T cells to ensure sufficient pathogen control.Kidney International (2009) 76, 557–566; doi:10.1038/ki.2009.198; published online 3 June 2009 [ABSTRACT FROM AUTHOR]

Background: Tumor-infiltrating lymphocytes (TIL) play an important role in the pathogenesis of renal cell carcinoma. Characterization of TIL requires efficient isolation procedures, especially in early stage disease when the tumor is of small in size. Conventional methods for isolating TIL are based on enzymatic tissue digestion, most frequently with collagenase. Collagenase isolation is limited by poor cell recovery, altered expression of cell-surface molecules, and impaired TIL-functionality. To overcome these limitations, we developed and optimized conditions for a robust collagenase-free mechanical procedure for improved isolation of TIL from renal cell carcinoma samples.Methods: TIL from tumor samples and T cells from peripheral blood were collected from 12 patients undergoing partial or radical nephrectomy. Samples were subjected to an enzymatic reference protocol and to a newly established mechanical isolation protocol. After viability staining, TIL-subpopulations were quantified and phenotyped by immunohistochemistry and flow-cytometric analysis, and were compared to characteristics of peripheral blood T cells. As a marker for TIL-functionality, T-cell cytokine induction was quantified after polyclonal stimulation.Results: We show that this new technique is rapid and allows identification of CD4 and CD8 T-cell subpopulations including CD4, CD8, and regulatory T cells expressing anergy markers such as programmed death-1 (PD-1) or B- and T-lymphocyte attenuator. When compared to the reference protocol involving collagenase digestion, the yield of TIL after mechanical isolation was higher and the expression of cell-surface markers was better preserved. Moreover, although antitumor activity was not assessed, mechanically isolated TIL are at least equally functional as T cells from peripheral blood, as polyclonal stimulation induced cytokines such as interferon-γ and tumor necrosis factor-α in both TIL and T cells from peripheral blood.Conclusion: The mechanical procedure may be applied as a robust and rapid alternative to collagenase digestion for isolation of high amounts of phenotypically and functionally intact TIL from fresh tumor samples. [ABSTRACT FROM AUTHOR]