Background
Several hydrolyzed cow's milk (CM) formulas are available for avoidance of allergic reactions in CM‐allergic children and for prevention of allergy development in high‐risk infants. Our ...aim was to compare CM formulas regarding the presence of immunoreactive CM components, IgE reactivity, allergenic activity, ability to induce T‐cell proliferation, and cytokine secretion.
Methods
A blinded analysis of eight CM formulas, one nonhydrolyzed, two partially hydrolyzed (PH), four extensively hydrolyzed (EH), and one amino acid formula, using biochemical techniques and specific antibody probes was conducted. IgE reactivity and allergenic activity of the formulas were tested with sera from CM‐allergic patients (n = 26) in RAST‐based assays and with rat basophils transfected with the human FcεRI, respectively. The induction of T‐cell proliferation and the secretion of cytokines in Peripheral blood mononuclear cell (PBMC) culture from CM allergic patients and nonallergic individuals were assessed.
Results
Immune‐reactive α‐lactalbumin and β‐lactoglobulin were found in the two PH formulas and casein components in one of the EH formulas. One PH formula and the EH formula containing casein components showed remaining IgE reactivity, whereas the other hydrolyzed formulas lacked IgE reactivity. Only two EH formulas and the amino acid formula did not induce T‐cell proliferation and proinflammatory cytokine release. The remaining formulas varied regarding the induction of Th2, Th1, and proinflammatory cytokines.
Conclusion
Our results show that certain CM formulas without allergenic and low proinflammatory properties can be identified and they may also explain different outcomes obtained in clinical studies using CM formulas.
Immune responses to citrullinated peptides have been described in autoimmune diseases like rheumatoid arthritis (RA) and multiple sclerosis (MS). We investigated the post-translational modification ...(PTM), arginine to citrulline, in brain tissue of MS patients and controls (C) by proteomics and subsequently the cellular immune response of cerebrospinal fluid (CSF)-infiltrating T cells to citrullinated and unmodified peptides of myelin basic protein (MBP). Using specifically adapted tissue extraction- and combined data interpretation protocols we could establish a map of citrullinated proteins by identifying more than 80 proteins with two or more citrullinated peptides in human brain tissue. We report many of them for the first time. For the already described citrullinated proteins MBP, GFAP, and vimentin, we could identify additional citrullinated sites. The number of modified proteins in MS white matter was higher than control tissue. Citrullinated peptides are considered neoepitopes that may trigger autoreactivity. We used newly identified epitopes and previously reported immunodominant myelin peptides in their citrullinated and non-citrullinated form to address the recognition of CSF-infiltrating CD4
T cells from 22 MS patients by measuring proliferation and IFN-γ secretion. We did not detect marked responses to citrullinated peptides, but slightly more strongly to the non-modified version. Based on these data, we conclude that citrullination does not appear to be an important activating factor of a T cell response, but could be the consequence of an immune- or inflammatory response. Our approach allowed us to perform a deep proteome analysis and opens new technical possibilities to analyze complex PTM patterns on minute quantities of rare tissue samples.
IgE sensitization to cockroach allergens is associated with development of allergic diseases, such as asthma. To understand the relevance of different cockroach allergens for diagnosis and ...immunotherapy, a comprehensive analysis of IgE antibody levels and T cell reactivity to an expanded set of cockroach allergens and their relationship to disease was performed in a cohort of USA cockroach sensitized patients. IgE antibody levels to recombinant chitinase and hemocyanin were measured for 23 subjects by custom-made ImmunoCAPs and compared with IgE levels to eight cockroach allergens we previously reported for the same cohort.
T cell activation (Ox40/PDL-1 expression) of PBMCs stimulated with peptide pools derived from 11 German cockroach proteins, including nine official cockroach allergens, plus chitinase and vitellogenin, was determined by flow cytometry. IgE prevalences to chitinase (17%) and hemocyanin (44%) were comparable to values for the other eight allergens that we previously reported (21-57%). Hemocyanin (Bla g 3), was a major allergen (one to which more than 50% of patients with an allergy to its source react) for a sub-group of 15 highly cockroach-sensitized subjects (IgE > 3.5 kU
/L: 53%). Chitinase was officially named as new allergen Bla g 12. Cockroach-specific IgE levels in plasma showed excellent correlation with the sum of 10 allergen-specific IgE (r = 0.94, p < 0.001). T cell reactivity to 11 proteins was highly variable among subjects, the highest being for vitellogenin, followed by Bla g 3. The main finding was that cockroach allergen-specific IgE and T cell reactivity patterns were unique per subject, and lacked immunodominant allergens and correlation with clinical phenotype/disease severity in the studied cohort. Knowing the subject-specific B/T cell reactivity profiles to a comprehensive panel of cockroach allergens will contribute to diagnosis of cockroach allergy and will be important for planning and assessing allergen immunotherapy outcomes, according to the allergen content in therapeutic cockroach extracts.
Cockroach allergy is associated with the development of asthma. The identification of cockroach allergens, which began in the 1990 s, is an ongoing process that has led to the current listing of 20 ...official allergen groups in the WHO/IUIS Allergen Nomenclature database. The function and structure of some of these allergens has been determined and define their natural delivery into the environment and their allergenicity. Analysis of antigenic determinants by X-ray crystallography and rational design of site-directed mutagenesis led to the identification of IgE binding sites for the design of molecules with reduced IgE reactivity and T cell modulatory capacity. New developments in recent years include component analyses of B and T cell reactivity and a recent cockroach immunotherapy trial, CRITICAL, that will contribute to understand the immune response to cockroach and to define future directions for cockroach allergy diagnosis and immunotherapy.
•Cockroach (CR) allergy is associated with the development of asthma.•CR extracts for diagnosis and immunotherapy (IT) show high molecular heterogeneity.•Subjects have unique B and T cell reactivity profiles (no immunodominant allergens).•Crystallographic analyses of CR allergens contribute to design molecules for IT.•CR IT trial CRITICAL and new experimental approaches will inform future therapies.
•Comparative evaluation of antibody screening tests after SARS-CoV-2 infection or vaccination.•Excellent test performance in very young and very old subjects (6 to 99 years).•Parallel occurrence of ...N-specific antibodies with nABs in recovered subjects.•Positive Viramed-Test results correspond with T-cell reactivity.•Viramed-Test is suitable for differential diagnosis of SARS-CoV-2 infection and/or vaccination.
Detection of seroconversion after SARS-CoV-2-infection or vaccination is relevant to discover subclinical cases and recognize patients with a possible immunity.
Test performance, effects of age, time-point of seroconversion and immune status regarding neutralizing antibodies (NAbs) and T-cell-reactivity were investigated.
Two antibody assays (Viramed-Test for S/N-specific IgG, Roche-Test for N-specific IgA, -M, -G) were evaluated with classified samples. In total, 381 subjects aged 6-99 years, who had either recovered from the disease or had been vaccinated, were screened for SARS-CoV-2-specific antibodies. This screening was part of an open observational study with working adults. Additionally, children and adults were analyzed in a longitudinal COVID-19 study in schools. For immunity evaluation, virus neutralization tests and ELISpot tests were performed in a subgroup of subjects.
Viramed revealed a slightly lower test performance than Roche, but test quality was equally well in samples from very young or very old donors. The time-point of seroconversion after the respective immunization detected by the two tests was not significantly different. N-specific antibodies, detected with Roche, highly correlated with NAbs in recovered subjects, whereas a positive Viramed-Test result was paralleled by a positive ELISpot result.
Viramed-Test was not as sensitive as Roche-Test, but highly specific and beneficial to distinguish between recovered and vaccinated status. For both tests correlations with humoral and cellular immunity were found. Of note, the expected early detection of IgA and IgM by the Roche-Test did not prove to be an advantage over IgG testing by Viramed.
Cytomegalovirus (CMV) represents a major cause of infectious complications after transplantation. Recently, chronic infections with lymphocyte choriomeningitis virus (LCMV), HIV or HCV were shown to ...be associated with functionally exhausted T cells characterized by high expression of the programmed death (PD)‐1 molecule and altered cytokine expression patterns. We therefore hypothesized that functional exhaustion of CMV‐specific CD4 T cells may determine impaired CMV control in patients after renal transplantation. In viremic transplant recipients, a significantly higher proportion of CMV‐specific CD4 T cells was PD‐1 positive (median 40.9%, 17.0–88.7%) as compared to nonviremic transplant patients (8.8%, 0.8–80.5%), dialysis patients (8.8%, 0–36.7%) or controls (3.2%, 0.3–15.4%, p < 0.0001). In line with functional impairment, PD‐1‐positive T cells produced significantly less IFNγ as compared to PD‐1‐negative T cells (p < 0.0001). Moreover, unlike controls or nonviremic patients, CMV‐specific T cells from viremic patients showed a significant loss of IL‐2 production (p < 0.0001). Interestingly, functional anergy of CMV‐specific CD4 T cells was reversible in that antibody‐mediated blockade of PD‐1 signaling with its ligands PD‐L1/‐L2 led to an up to 10‐fold increase in CMV‐specific proliferation. In conclusion, expression of PD‐1 defines a reversible defect of CMV‐specific CD4 T cells that is associated with viremia, and blocking PD‐1 signaling may provide a potential target for enhancing the function of exhausted T cells in chronic CMV infection.
This prospective study found that PD‐1 and loss of IL‐2 expression defines a reversible defect of CMV‐specific CD4 T‐cells that is associated with viremia after transplantation, and blocking PD‐1 signaling may provide a potential target for enhancing the function of exhausted T‐cells in chronic CMV‐infection.
Antigen complexity represents a major challenge for scoring CD4+ T cell immunogenicity, a key hallmark of immunity and with great potential to improve vaccine development. In this chapter, we provide ...a comprehensive picture of a pipeline that can be applied to virtually any complex antigen to overcome different limitations. Antigens are characterized by Mass Spectrometry to determine the available protein sources and their abundances. A reconstituted in vitro antigen processing system is applied along with bioinformatics tools to prioritize the list of candidates. Finally, the immunogenicity of candidate peptides is validated ex vivo using PBMCs from HLA-typed individuals. This protocol compiles the essential information for executing the whole pipeline while focusing on the candidate epitope prioritizing scheme.
Background
Solid organ transplant recipients are at high risk to develop (complicated) herpes zoster (HZ). Booster vaccination could prevent HZ. However, end-stage renal disease (ESRD) patients show ...poor immunological responses to vaccinations. We studied the effect of a live attenuated VZV booster vaccine on VZV-specific B and T cell memory responses in ESRD patients and healthy controls. NL28557.000.09,
www.toetsingonline.nl
Methods
VZV-seropositive patients, aged ≥50 years, awaiting kidney transplantation, were vaccinated with Zostavax
®
. Gender and age-matched VZV-seropositive potential living kidney donors were included as controls. VZV-specific IgG titers were measured before, at 1, 3 and 12 months post-vaccination. VZV-specific B and T cell responses before, at 3 months and 1 year after vaccination were analysed by flow-cytometry and Elispot, respectively. Occurrence of HZ was assessed at 5 years post-vaccination.
Results
26 patients and 27 donors were included. Median VZV-specific IgG titers were significantly higher at all time-points post-vaccination in patients (mo 1: 3104 IU/ml 1967-3825, p<0.0001; mo 3: 2659 1615-3156, p=0.0002; mo 12: 1988 1104-2989, p=0.01 vs. pre: 1397 613-2248) and in donors (mo 1: 2981 2126-3827, p<0.0001; mo 3: 2442 2014-3311, p<0.0001; mo 12: 1788 1368-2460, p=0.0005 vs. pre: 1034 901-1744. The patients’ IgG titers were comparable to the donors’ at all time-points. The ratio VZV-specific B cells of total IgG producing memory B cells had increased 3 months post-vaccination in patients (0.85 0.65-1.34 vs. pre: 0.56 0.35-0.81, p=0.003) and donors (0.85 0.63-1.06 vs. pre: 0.53 0.36-0.79, p<0.0001) and remained stable thereafter in donors. One year post-vaccination, the percentage of CD4+ central memory cells had increased in both patients (0.29 0.08-0.38 vs. 0.12 0.05-0.29, p=0.005) and donors (0.12 0.03-0.37 vs. 0.09 0.01-0.20, p=0.002) and CD4+ effector memory cells had increased in donors (0.07 0.02-0.14 vs. 0.04 0.01-0.12, p=0.007). Only 1 patient experienced HZ, which was non-complicated.
Conclusion
VZV booster vaccination increases VZV-specific IgG titers and percentage VZV-specific memory T-cells for at least 1 year both in ESRD patients and healthy controls. VZV-specific memory B cells significantly increased in patients up to 3 months after vaccination. Prophylactic VZV booster vaccination prior to transplantation could reduce HZ incidence and severity after transplantation.
SUMMARY
Previous studies have established that inactivated mycobacteria are potent and selective activators of Vγ9+/Vδ2+ human γ/δ T cells. Here we have analysed the proliferative response of human ...γ/δ T cells to five serologically distinct groups of streptococci. While heat‐inactivated streptococci of all five serogroups tested (A, B. C, D and F) induced a strong proliferative response in peripheral blood mononuclear cells (PBMC), only groups A, B and C elicited a selective activation of Vγ9+γ/δ T cells in 10 (serogroup B) or 11 (serogroups A and C) of 11 tested healthy individuals. In striking contrast, groups D and F streptococci failed to activate γ/δ T cells in nine of 11 donors and induced only a weak γ/δ T cell response in two additional individuals. Depletion of Vγ9+ T cells before culture completely eliminated all γ/δ T cell responses to streptococci. These data indicate that groups A, B and C (but not D or F) streptococci can be included in the growing list of selective ligands for Vγ9+/Vδ2+ human γ/δ T cells.
T follicular helper (TFH) cells are a heterogeneous subset of immunocompetent T helper (TH) cells capable of augmenting B cell responses in lymphoid tissues. In transplantation, exposure to ...allogeneic tissue activates TFH cells increasing the risk of the emergence of de novo donor-specific HLA-antibodies (dnDSA). These can cause antibody-mediated rejection (AMR) and allograft loss. Follicular regulatory T (TFR) cells counteract TFH cell activity. Here, we investigated the implications of TFH and TFR cells on dnDSA formation after renal transplantation (RTX). Considering TFH cells to be CXCR5+ and IL-21+, we found by flow cytometry that patients with dnDSA produced IL-21 more abundantly compared to healthy volunteers. In in vitro alloreactivity assays, patients with dnDSA featured an enhanced alloreactive TH cell pool in response to donor-specific HLA antigens. Besides, longitudinal investigations suggested enhanced alloreactivity shortly after transplantation increasing the risk of dnDSA development. Taken together, in spite of continuous immunosuppression we report a strong IL-21 response in TFH cells and an expanded reservoir of donor-specific memory TH cells in patients with dnDSA. This warrants further investigations if aberrant TFH cell activation may precede the formation of dnDSA promoting AMR.
•CNI are in principle potent suppressors of IL-21 producing TFH cells•TFH cells show unaltered frequencies after RTX in patients with DSA•TFH cell cytokine profile is not different comparing patients with DSA versus without DSA•Patients with dnDSA featured an enhanced alloreactive Th cell pool in response to donor-specific HLA antigens
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