Rauli Franssila

A Defect of Regulatory T Cells in Patients with Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a monogenic recessive disease characterized by autoimmunity against multiple tissues, offers a unique possibility to study the breakdown of self-tolerance in humans. It is caused by mutations in the autoimmune regulator gene (AIRE), which encodes a transcriptional regulator. Work using Aire−/− mice suggests that Aire induces ectopic expression of peripheral Ags and promotes their presentation in the thymus. We have explored reasons for the difference between the comparatively mild phenotype of Aire-deficient mice and human APECED patients. We provide evidence that, unlike in the Aire−/− mice, in the patients a key mediator of active tolerance, the CD4+CD25+ regulatory T (Treg) cell subset is impaired. This was shown by significantly decreased expression of FOXP3 mRNA and protein, decreased function, and alterations in TCR repertoire. Also, in the normal human thymus a concentric accumulation of AIRE+ cells was seen around thymic Hassall’s corpuscles, suggesting that in the patients these cells may be involved in the observed Treg cell failure. In Aire−/− mice the expression of FoxP3 was normal and even increased in target tissues in parallel with the lymphocyte infiltration process. Our results suggest that a Treg cell defect is involved in the pathogenesis of APECED and emphasize the importance of active tolerance mechanisms in preventing human autoimmunity.

Contrast sensitivity as a function of spatial frequency, viewing distance and eccentricity with and without spatial noise

Using computer graphics and a two-alternative forced-choice method we measured threshold contrast as a function of viewing distance, spatial frequency, and eccentricity for gratings with and without added, white two-dimensional spatial noise. Our experiments showed that in spatial noise contrast sensitivity was independent of viewing distance as long as contrast sensitivity was lower with noise than without. With increasing spatial frequency (f) the grating area (A) was reduced in order to keep the relative grating size (Af2) constant. At all spatial frequencies the test gratings thus had the same amount of detail and contour. Noise spectral density was reduced in direct proportion to grating area in order to keep the physical signal-to-noise ratio constant. An increase in spatial frequency was thus accompanied with reductions in grating area and noise spectral density similar to those produced by a corresponding increase in viewing distance. In agreement, contrast detection in spatial noise was found to be independent of spatial frequency as long as contrast sensitivity was lower with noise than without. The effect of increasing eccentricity on visual performance can be compensated for by reducing the viewing distance (M-scaling). Hence, without M-scaling the effect of increasing eccentricity is similar to that of increasing viewing distance. In agreement, we found that contrast sensitivity in spatial noise was independent of eccentricity as long as contrast sensitivity was lower with noise than without.

T Helper Cell–Mediated In Vitro Responses of Recently and Remotely Infected Subjects to a Candidate Recombinant Vaccine for Human Parvovirus B19

T cell proliferation to human parvovirus B19 antigen was measured in 6 patients with recent B19 infection (1 with pneumonia and pleuritis), 1 patient with symptoms persisting >180 days after onset, 18 nonsymptomatic subjects with remote B19 immunity, and 12 B19-seronegative control subjects. Recombinantly expressed virus-like particles (VP1/2 capsids), a candidate B19 vaccine, were used as antigen. Virus-specific T helper cell proliferation was detectable in all the recently infected patients and in most (17/18) of the remotely infected subjects but not in the seronegative control subjects. The B19-specific T cell responses, in general, were most vigorous among the recently infected patients. However, such strong B19-specific proliferation was not confined within the acute phase, as 28% (5/18) of the remotely infected healthy individuals had B19-specific reactivity persisting at acute-phase levels, apparently for years or decades. These data indicate that B cells recognizing the VP1/2 capsids receive class II-restricted help from CD4(+) T lymphocytes.

Erythroid Progenitor Cells Expanded from Peripheral Blood without Mobilization or Preselection: Molecular Characteristics and Functional Competence

Background Continued development of in-vitro procedures for expansion and differentiation of erythroid progenitor cells (EPC) is essential not only in hematology and stem cell research but also virology, in light of the strict erythrotropism of the clinically important human parvovirus B19. Methodology/Principal Findings We cultured EPC directly from ordinary blood samples, without ex vivo stem cell mobilization or CD34+ cell in vitro preselection. Profound increase in the absolute cell number and clustering activity were observed during culture. The cells obtained expressed the EPC marker combination CD36, CD71 and glycophorin, but none of the lymphocyte, monocyte or NK markers. The functionality of the generated EPC was examined by an in vitro infection assay with human parvovirus B19, tropic for BFU-E and CFU-E cells. Following infection (i) viral DNA replication and mRNA production were confirmed by quantitative PCR, and (ii) structural and nonstructural proteins were expressed in >50% of the cells. As the overall cell number increased 100–200 fold, and the proportion of competent EPC (CD34+ to CD36+) rose from <0.5% to >50%, the in vitro culture procedure generated the EPC at an efficiency of >10 000-fold. Comparative culturing of unselected PBMC and ex vivo-preselected CD34+ cells produced qualitatively and quantitatively similar yields of EPC. Conclusions/Significance This approach yielding EPC directly from unmanipulated peripheral blood is gratifyingly robust and will facilitate the study of myeloid infectious agents such as the B19 virus, as well as the examination of erythropoiesis and its cellular and molecular mechanisms.

IgG subclass response to human parvovirus B19 infection.

BACKGROUND IgG antibodies are essential to immunity against human parvovirus B19 and can neutralize infection both in bone marrow cell cultures infected in vitro and in chronically infected immunosuppressed individuals. OBJECTIVES To assess the levels and response kinetics of IgG subclasses towards individual structural proteins of human parvovirus B19. STUDY DESIGN Subclasses of IgG for capsid proteins VP1 or VP2 were quantified by EIA using monoclonal antibodies in 30 acutely infected and 30 convalescent patients, as well as in 32 remotely infected and 20 non-infected controls. RESULTS In all groups of seropositive individuals the predominant subclass for either structural protein was IgG1. Subclass IgG3 was associated with acute infection. By contrast, IgG4 appeared months after infection, and occurred specifically towards VP1. The ratio of VP1-specific subclasses IgG3 and IgG4 provided a diagnostic test for recent infection with a specificity of 98% and a sensitivity of 97%. CONCLUSIONS Comparative measurement of VP1-specific IgG3 and IgG4 is useful in diagnosis. The IgG4 results point to long-term expression of immunologically active VP1 and to T-cell help of T(h)2 type for B-cells recognizing VP1.

CD4+ T Helper Cell Responses against Human Bocavirus Viral Protein 2 Viruslike Particles in Healthy Adults

Abstract Background. Human bocavirus (HBoV) was recently described as a new member of the Parvoviridae family, and its possible association with respiratory illness in infants has been discussed. To date, HBoV genomes have been detected worldwide in respiratory tract samples obtained from children with pulmonary diseases, whereas only limited data on virus-specific immunity are available, mainly because of the lack of recombinant viral antigens. Methods. HBoV viruslike particles (VLPs) were produced in insect cells and characterized by electron microscopy and cesium chloride gradient centrifugation. HBoV viral protein 2 (VP2)-specific antibodies and CD4+ T helper cell responses were analyzed by enzyme-linked immunsorbent assay and enzyme-linked immunospot assay. Results. VP2 capsid proteins of HBoV were produced in insect cells infected with a recombinant baculovirus, and the formation of icosahedral VLPs (diameter, 21–25 nm; sedimentation density, 1.33 g/cm3) was demonstrated. A significant increase in secretion of VP2-specific interferon-γ was detected in cultures of peripheral blood mononuclear cells obtained from 69 healthy adults found to be positive for HBoV-specific immunoglobulin G antibodies, compared with control stimulations. In parallel, T cell responses against identically expressed parvovirus B19 VP2 VLPs were frequently observed in the individuals studied, without there being obvious cross-reactions between HBoV and parvovirus B19. Conclusions. Data suggest the presence of HBoV-specific immune responses in adults and strongly support a high prevalence of HBoV among humans.

Critical flicker frequency to red targets as a function of luminance and flux across the human visual field

When the number of cells (cones at eccentricities 0-10 deg and ganglion cells above 10 deg) stimulated at various retinal locations was kept constant by enlarging the stimulus area with increasing eccentricity in the temporal visual field (M-scaling), CFF to red stimuli with dark surround increased as a single function of photopic luminous flux, collected by ganglion-cell receptive-field centres and calculated by multiplying Riccos area with retinal illuminance at each eccentricity studied. The increase of CFF with the logarithm of photopic flux could be best explained by the Collins logarithmic law, the Kelly square-root law was almost equally good and the Ferry-Porter law was poorest. Adopting the general formulation of Corwin and Dunlap (Vision Research, 27, 2119-2123, 1987) the exponent of CFF is 0, 0.5, and 1 for the Collins, Kelly and Ferry-Porter laws, respectively. The exponent that best explained our results was found to be 0-0.3.

T-helper Cell-Mediated Proliferation and Cytokine Responses against Recombinant Merkel Cell Polyomavirus-Like Particles

The newly discovered Merkel Cell Polyomavirus (MCPyV) resides in approximately 80% of Merkel cell carcinomas (MCC). Causal role of MCPyV for this rare and aggressive skin cancer is suggested by monoclonal integration and truncation of large T (LT) viral antigen in MCC cells. The mutated MCPyV has recently been found in highly purified leukemic cells from patients with chronic lymphocytic leukemia (CLL), suggesting a pathogenic role also in CLL. About 50–80% of adults display MCPyV-specific antibodies. The humoral immunity does not protect against the development of MCC, as neutralizing MCPyV antibodies occur in higher levels among MCC patients than healthy controls. Impaired T-cell immunity has been linked with aggressive MCC behavior. Therefore, cellular immunity appears to be important in MCPyV infection surveillance. In order to elucidate the role of MCPyV-specific Th-cell immunity, peripheral blood mononuclear cells (PBMC) of healthy adults were stimulated with MCPyV VP1 virus-like particles (VLPs), using human bocavirus (HBoV) VLPs and Candida albicans antigen as positive controls. Proliferation, IFN-γ, IL-13 and IL-10 responses were examined in 15 MCPyV-seropositive and 15 seronegative volunteers. With the MCPyV antigen, significantly stronger Th-cell responses were found in MCPyV-seropositive than MCPyV-seronegative subjects, whereas with the control antigens, the responses were statistically similar. The most readily detectable cytokine was IFN-γ. The MCPyV antigen tended to induce stronger IFN-γ responses than HBoV VLP antigen. Taken together, MCPyV-specific Th-cells elicit vigorous IFN-γ responses. IFN-γ being a cytokine with major antiviral and tumor suppressing functions, Th-cells are suggested to be important mediators of MCPyV-specific immune surveillance.

Trichodysplasia spinulosa-Associated Polyomavirus (TSV) and Merkel Cell Polyomavirus: Correlation between Humoral and Cellular Immunity Stronger with TSV

Merkel Cell Polyomavirus (MCV) is a common infectious agent likely to be involved in the pathogenesis of most Merkel cell carcinomas (MCC). Trichodysplasia spinulosa-associated polyomavirus (TSV), which exhibit high seroprevalence in general population, has been detected in trichodysplasia spinulosa (TS) skin lesions suggesting an etiological role for this disease. Previous studies have shown strong MCV-specific T-cell responses, while no data exist on T-cell immunity against TSV. In order to characterize Th-cell immunity against TSV, and to allow comparisons with the MCV-specific Th-cell immunity, we studied TSV-specific proliferation, IFN-γ, IL-10 and IL-13, and MCV-specific IFN-γ and IL-10 responses in 51 healthy volunteers, and in one MCC patient. Recombinant TSV and MCV VP1 virus-like particles (VLPs) were used as antigens. A significant correlation was found between virus-specific Th-cell and antibody responses with TSV; with MCV it proved weaker. Despite significant homology in amino acid sequences, Th-cell crossreactivity was not evident between these viruses. Some subjects seronegative to both TSV and MCV exhibited Th-cell responses to both viruses. The agent initially priming these Th-cells remains an enigma. As CD8+ cells specific to MCV T-Ag oncoprotein clearly provide an important defense against established MCC, the MCV VP1-specific Th-cells may, by suppressing MCV replication with antiviral cytokines such as IFN-γ, significantly contribute to preventing the full process of oncogenesis.

Granzyme B mediated function of Parvovirus B19-specific CD4(+) T cells.

A novel conception of CD4+ T cells with cytolytic potential (CD4+ CTL) is emerging. These cells appear to have a part in controlling malignancies and chronic infections. Human parvovirus B19 can cause a persistent infection, yet no data exist on the presence of B19‐specific CD4+ CTLs. Such cells could have a role in the pathogenesis of some autoimmune disorders reported to be associated with B19. We explored the cytolytic potential of human parvovirus B19‐specific T cells by stimulating peripheral blood mononuclear cell (PBMC) with recombinant B19‐VP2 virus‐like particles. The cytolytic potential was determined by enzyme immunoassay‐based quantitation of granzyme B (GrB) and perforin from the tissue culture supernatants, by intracellular cytokine staining (ICS) and by detecting direct cytotoxicity. GrB and perforin responses with the B19 antigen were readily detectable in B19‐seropositive individuals. T‐cell depletion, HLA blocking and ICS experiments showed GrB and perforin to be secreted by CD4+ T cells. CD4+ T cells with strong GrB responses were found to exhibit direct cytotoxicity. As anticipated, ICS of B19‐specific CD4+ T cells showed expected co‐expression of GrB, perforin and interferon gamma (IFN‐γ). Unexpectedly, also a strong co‐expression of GrB and interleukin 17 (IL‐17) was detected. These cells expressed natural killer (NK) cell surface marker CD56, together with the CD4 surface marker. To our knowledge, this is the first report on virus‐specific CD4+ CTLs co‐expressing CD56 antigen. Our results suggest a role for CD4+ CTL in B19 immunity. Such cells could function within both immune regulation and triggering of autoimmune phenomena such as systemic lupus erythematosus (SLE) or rheumatoid arthritis.