Background. Frequencies of alloreactive T cells determined by limiting dilution assays (LDA) may not adequately reflect the donor-reactive immune status in transplant recipients. To reevaluate LDA frequencies, we developed a flow cytometry test for direct determination of alloreactive T-cell frequencies and compared these frequencies with classical LDA estimates of frequencies. Methods. For determination of frequencies by flow cytometry, peripheral blood lymphocytes (or lymphocytes taken from primary mixed lymphocyte culture) were stimulated with either Epstein-Barr virus–transformed lymphoblastoid cell lines or T cell–depleted spleen cells and stained for intracellular interferon (IFN)-γ production and CD69. In lung transplant recipients, frequencies of IFN+ alloreactive T cells were compared with LDA frequencies, that is, cytotoxic T lymphocyte precursors and helper T lymphocyte precursors. Results. With flow cytometry, alloreactive T cells were detected after overnight allostimulation as IFN-γ+ CD69bright cells (range, 0.1–0.58% and 0.1–0.66% of total CD4 and CD8 cells, respectively). Frequencies increased 25-fold or more when lymphocytes were prestimulated in primary mixed lymphocyte culture before testing. After lung transplantation, mean donor-specific IFN+ CD8 T-cell frequencies did not decrease as mean donor-specific LDA cytotoxic T lymphocyte precursor frequencies, whereas no difference was seen in pretransplantation samples or third-party–specific frequencies at both time points. Mean frequencies of IFN+ CD4 did not differ from helper T lymphocyte precursors at both time points, but frequencies did not correlate. Conclusions. The flow cytometry test allows a direct measurement of alloreactive T-cell frequencies and demonstrates a discrepancy between donor-specific IFN+ CD8 T-cell frequencies and LDA CLTp after transplantation. This may be a result of the existence of “functional diverse” alloreactive T cells or of activation-induced cell death of donor-reactive T cells during long (LDA) culturing, which is avoided in the flow cytometry test.
Background The inability of seasonal influenza vaccines to effectively protect against infection with antigenically drifted viruses or newly emerging pandemic viruses underlines the need for development of cross-reactive influenza vaccines that induce immunity against a variety of virus subtypes. Therefore, potential cross-protective vaccines, e.g., whole inactivated virus (WIV) vaccine, that can target conserved internal antigens such as the nucleoprotein (NP) and/or matrix protein (M1) need to be explored. Methodology/Principal Findings In the current study we show that a WIV vaccine, through induction of cross-protective cytotoxic T lymphocytes (CTLs), protects mice from heterosubtypic infection. This protection was abrogated after depletion of CD8+ cells in vaccinated mice, indicating that CTLs were the primary mediators of protection. Previously, we have shown that different procedures used for virus inactivation influence optimal activation of CTLs by WIV, most likely by affecting the membrane fusion properties of the virus. Specifically, inactivation with formalin (FA) severely compromises fusion activity of the virus, while inactivation with β-propiolactone (BPL) preserves fusion activity. Here, we demonstrate that vaccination of mice with BPL-inactivated H5N1 WIV vaccine induces solid protection from lethal heterosubtypic H1N1 challenge. By contrast, vaccination with FA-inactivated WIV, while preventing death after lethal challenge, failed to protect against development of disease and severe body weight loss. Vaccination with BPL-inactivated WIV, compared to FA-inactivated WIV, induced higher levels of specific CD8+ T cells in blood, spleen and lungs, and a higher production of granzyme B in the lungs upon H1N1 virus challenge. Conclusion/Significance The results underline the potential use of WIV as a cross-protective influenza vaccine candidate. However, careful choice of the virus inactivation procedure is important to retain membrane fusion activity and full immunogenicity of the vaccine.
Objective Patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and granulomatosis with polyangiitis (Wegener's) (GPA) have a 3–20‐fold increased risk of herpes zoster compared to the general population. The aim of this study was to evaluate if susceptibility is due to decreased levels of cellular and/or humoral immunity to the varicella‐zoster virus (VZV). Methods A cross‐sectional study of VZV‐specific immunity was performed in 38 SLE patients, 33 GPA patients, and 51 healthy controls. Levels of IgG and IgM antibodies to VZV were measured using an in‐house glycoprotein enzyme‐linked immunosorbent assay (ELISA). Cellular responses to VZV were determined by interferon‐γ (IFNγ) enzyme‐linked immunospot (ELISpot) assay and carboxyfluorescein succinimidyl ester (CFSE) dye dilution proliferation assay. Results Levels of IgG antibodies to VZV were increased in SLE patients as compared to healthy controls, but levels of IgM antibodies to VZV were not. Antibody levels in GPA patients did not differ significantly from levels in healthy controls. In response to stimulation with VZV, decreased numbers of IFNγ spot‐forming cells were found among SLE patients (although not GPA patients) as compared to healthy controls. Proliferation of CD4+ T cells in response to stimulation with VZV was decreased in SLE patients but not GPA patients. Conclusion SLE patients have increased levels of IgG antibodies against VZV, while cellular immunity is decreased. In GPA patients, antibody levels as well as cellular responses to VZV were comparable to those in healthy controls. These data suggest that increased prevalence of herpes zoster in SLE patients is due to a poor cellular response. Vaccination strategies should aim to boost cellular immunity against VZV.
