Désirée Kunkel

How αβ T cells deal with induced TCRα ablation

On deletion of the gene encoding the constant region of the T cell antigen receptor (TCR)α chain in mature T cells by induced Cre-mediated recombination, the cells lose most of their TCR from the cell surface within 7–10 days, but minute amounts of surface-bound TCRβ chains are retained for long periods of time. In a situation in which cellular influx from the thymus is blocked, TCR-deficient naïve T cells decay over time, the decay rates being faster for CD8+ cells (t1/2 ≈ 16 days) than for CD4+ cells (t1/2 ≈ 46 days). TCR+ naïve cells are either maintained (CD8+) or decay more slowly (CD4+; t1/2 ≈ 78 days.) Numbers of TCR-deficient memory T cells decline very slowly (CD8+ cells; t1/2 ≈ 52 days) or not at all (CD4+ cells), but at the population level, these cells fail to expand as their TCR+ counterparts do. Together with earlier data on T cell maintenance in environments lacking appropriate major histocompatibility complex antigens, these data argue against the possibility that spontaneous ligand-independent signaling by the αβTCR contributes significantly to T-cell homeostasis.

Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis

Glucocorticoids mediate their therapeutic actions mostly by genomic effects via cytosolic receptors, but some effects are too rapid to be mediated by changes at the genomic level. The detailed mechanisms of these nongenomic actions are still unclear. Membrane‐bound glucocorticoid receptors (mGCR) have been suggested to be involved, although their physiological existence in humans so far is hypothetical. For the first time we demonstrate the existence of mGCR on monocytes and B cells obtained from healthy blood donors using high‐sensitivity immunofluorescent staining. Immunostimulation with lipopolysaccharide increases the percentage of mGCR‐positive monocytes, which can be prevented by inhibiting the secretory pathway. Overexpression of the human glucocorticoid receptor α alone is not sufficient to enhance mGCR expression. These in vitro findings are consistent with our clinical observation that in patients with rheumatoid arthritis the frequency of mGCR positive monocytes is increased and positively correlated with disease activity. We conclude that mGCR are 1) indeed physiologically present in healthy blood donors, but remained unidentified by conventional techniques due to their small number per cell and 2) actively up‐regulated and transported through the cell after immunostimulation. These receptors may reflect a feedback mechanism of the organism upon immunostimulation and/or play a role in pathogenesis.—Bartholome, B., Spies, C. M., Gaber, T., Schuchmann, S., Berki, T., Kunkel, D., Bienert, M., Radbruch, A., Burmester, G.‐R., Lauster, R., Scheffold, A., Buttgereit, F. Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up‐regulated after in vitro stimulation and in patients with rheumatoid arthritis. FASEB J. 18, 70–80 (2004)

Mutations in PYCR1 cause cutis laxa with progeroid features.

Autosomal recessive cutis laxa (ARCL) describes a group of syndromal disorders that are often associated with a progeroid appearance, lax and wrinkled skin, osteopenia and mental retardation. Homozygosity mapping in several kindreds with ARCL identified a candidate region on chromosome 17q25. By high-throughput sequencing of the entire candidate region, we detected disease-causing mutations in the gene PYCR1. We found that the gene product, an enzyme involved in proline metabolism, localizes to mitochondria. Altered mitochondrial morphology, membrane potential and increased apoptosis rate upon oxidative stress were evident in fibroblasts from affected individuals. Knockdown of the orthologous genes in Xenopus and zebrafish led to epidermal hypoplasia and blistering that was accompanied by a massive increase of apoptosis. Our findings link mutations in PYCR1 to altered mitochondrial function and progeroid changes in connective tissues.

Impairment of the intestinal barrier is evident in untreated but absent in suppressively treated HIV-infected patients

Background and aims: Impairment of the gastrointestinal mucosal barrier contributes to progression of HIV infection. The purpose of this study was to investigate the effect of highly active antiretroviral therapy (HAART) on the HIV-induced intestinal barrier defect and to identify underlying mechanisms. Methods: Epithelial barrier function was characterised by impedance spectroscopy and [3H]mannitol fluxes in duodenal biopsies from 11 untreated and 8 suppressively treated HIV-infected patients, and 9 HIV-seronegative controls. The villus/crypt ratio was determined microscopically. Epithelial apoptoses were analysed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) and caspase-3 staining. Tight junction protein expression was quantified by densitometric analysis of immunoblots. Mucosal cytokine production was determined by cytometric bead array. Results: Only in untreated but not in treated HIV-infected patients, epithelial resistance was reduced (13 (1) vs 23 (2) Ω cm2, p<0.01) and mannitol permeability was increased compared with HIV-negative controls (19 (3) vs 9 (1) nm/s, p<0.05). As structural correlates, epithelial apoptoses and expression of the pore-forming claudin-2 were increased while expression of the sealing claudin-1 was reduced in untreated compared with treated patients and HIV-negative controls. Furthermore, villous atrophy was evident and mucosal production of interleukin 2 (IL2), IL4 and tumour necrosis factor α (TNFα) was increased in untreated but not in treated HIV-infected patients. Incubation with IL2, IL4, TNFα and IL13 reduced the transepithelial resistance of rat jejunal mucosa. Conclusions: Suppressive HAART abrogates HIV-induced intestinal barrier defect and villous atrophy. The HIV-induced barrier defect is due to altered tight junction protein composition and elevated epithelial apoptoses. Mucosal cytokines are mediators of the HIV-induced mucosal barrier defect and villous atrophy.

Correlation of T cell response and bacterial clearance in human volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination with Ty21a-based Salmonella vaccines

Background: Helicobacter pylori remains a global health hazard, and vaccination would be ideal for its control. Natural infection appears not to induce protective immunity. Thus, the feasibility of a vaccine for humans is doubtful. Methods: In two prospective, randomised, double-blind, controlled studies (Paul Ehrlich Institute application nos 0802/02 and 1097/01), live vaccines against H pylori were tested in human volunteers seronegative for, and without evidence of, active H pylori infection. Volunteers (n = 58) were immunised orally with Salmonella enterica serovar Typhi Ty21a expressing H pylori urease or HP0231, or solely with Ty21a, and then challenged with 2×105 cagPAI− H pylori. Adverse events, infection, humoral, cellular and mucosal immune response were monitored. Gastric biopsies were taken before and after vaccination, and postchallenge. Infection was terminated with antibiotics. Results: Vaccines were well tolerated. Challenge infection induced transient, mild to moderate dyspeptic symptoms, and histological and transcriptional changes in the mucosa known from chronic infection. Vaccines did not show satisfactory protection. However, 13 of 58 volunteers, 8 vaccinees and 5 controls, became breath test negative and either cleared H pylori (5/13) completely or reduced the H pylori burden (8/13). H pylori-specific T helper cells were detected in 9 of these 13 (69%), but only in 6 of 45 (13%) breath test-positive volunteers (p = 0.0002; Fisher exact test). T cells were either vaccine induced or pre-existing, depending on the volunteer. Conclusion: Challenge infection offers a controlled model for vaccine testing. Importantly, it revealed evidence for T cell-mediated immunity against H pylori infection in humans.

Reduced Peripheral and Mucosal Tropheryma whipplei-Specific Th1 Response in Patients with Whipple’s Disease

Whipple’s disease is a rare infectious disorder caused by Tropheryma whipplei. Major symptoms are arthropathy, weight loss, and diarrhea, but the CNS and other organs may be affected, too. The incidence of Whipple’s disease is very low despite the ubiquitous presence of T. whipplei in the environment. Therefore, it has been suggested that host factors indicated by immune deficiencies are responsible for the development of Whipple’s disease. However, T. whipplei-specific T cell responses could not be studied until now, because cultivation of the bacteria was established only recently. Thus, the availability of T. whipplei Twist-MarseilleT has enabled the first analysis of T. whipplei-specific reactivity of CD4+ T cells. A robust T. whipplei-specific CD4+ Th1 reactivity and activation (expression of CD154) was detected in peripheral and duodenal lymphocytes of all healthy (16 young, 27 age-matched, 11 triathletes) and disease controls (17 patients with tuberculosis) tested. However, 32 Whipple’s disease patients showed reduced or absent T. whipplei-specific Th1 responses, whereas their capacity to react to other common Ags like tetanus toxoid, tuberculin, actinomycetes, Giardia lamblia, or CMV was not reduced compared with controls. Hence, we conclude that an insufficient T. whipplei-specific Th1 response may be responsible for an impaired immunological clearance of T. whipplei in Whipple’s disease patients and may contribute to the fatal natural course of the disease.

Impaired Immune Functions of Monocytes and Macrophages in Whipple's Disease

BACKGROUND & AIMS Whipples disease is a chronic multisystemic infection caused by Tropheryma whipplei. Host factors likely predispose for the establishment of an infection, and macrophages seem to be involved in the pathogenesis of Whipples disease. However, macrophage activation in Whipples disease has not been studied systematically so far. METHODS Samples from 145 Whipples disease patients and 166 control subjects were investigated. We characterized duodenal macrophages and lymphocytes immunohistochemically and peripheral monocytes by flow cytometry and quantified mucosal and systemic cytokines and chemokines indicative for macrophage activation. In addition, we determined duodenal nitrite production and oxidative burst induced by T whipplei and by other bacteria. RESULTS Reduced numbers of duodenal lymphocytes, increased numbers of CD163(+) and stabilin-1(+), reduced numbers of inducible nitric synthase+ duodenal macrophages, and increased percentages of CD163(+) peripheral monocytes indicated a lack of inflammation and a M2/alternatively activated macrophage phenotype in Whipples disease. Incubation with T whipplei in vitro enhanced the expression of CD163 on monocytes from Whipples disease patients but not from control subjects. Chemokines and cytokines associated with M2/alternative macrophage activation were elevated in the duodenum and the peripheral blood from Whipples disease patients. Functionally, Whipples disease patients showed a reduced duodenal nitrite production and reduced oxidative burst upon incubation with T whipplei compared with healthy subjects. CONCLUSIONS The lack of excessive local inflammation and alternative activation of macrophages, triggered in part by the agent T whipplei itself, may explain the hallmark of Whipples disease: invasion of the intestinal mucosa with macrophages incompetent to degrade T whipplei.

Visualization of peptide presentation following oral application of antigen in normal and Peyer's patches‐deficient mice

Orally applied antigens generate systemic unresponsiveness by induction of anergy and deletion of specific T cells at high antigen doses, and induction of regulatory T cells at low doses of antigen. These different immune reactions have been attributed to different types of antigen‐presenting cells (APC) and/or different secondary lymphoid organs participating in the induction of the immune response. We used high‐sensitivity immunofluorescence to directly identify for the first time the cells presenting orally applied antigen in vivo. At low peptide doses (<1 mg) peptide presentation was exclusively detected on dendritic cells (DC) of the Peyers patches (PP) and mesenteric lymph nodes (mLN). At high doses (>1 mg) peptides were presented systemically and by all types of APC but presentation was still maximal on DC of the PP (up to 65%). Nevertheless, at limiting antigen doses T cell activation in the gut‐associated lymphoid tissue occurs preferentially in the mLN but not in PP. PP‐deficient mice have the same frequencies of peptide‐presenting cells in mLN, peripheral lymph nodes and spleen and activation of naive T cells in vivo is not affected. Therefore, PP are not critical for antigen presentation as well as for T cell activation in response to orally applied soluble antigens.

Gut Mucosal FOXP3+ Regulatory CD4+ T Cells and Nonregulatory CD4+ T Cells Are Differentially Affected by Simian Immunodeficiency Virus Infection in Rhesus Macaques

ABSTRACT The gastrointestinal tract represents a major site for human and simian immunodeficiency virus (HIV and SIV) replication and CD4+ T-cell depletion. Despite severe depletion of mucosal CD4+ T cells, FOXP3+ regulatory CD4+ T cells (Treg) are highly increased in the gut mucosa of chronically HIV-infected individuals and may contribute to HIV pathogenesis, either by their immunosuppressive function or as a significant target cell population for virus production. Little is known about the susceptibility of mucosal Treg to viral infection and the longitudinal effect of HIV/SIV infection on Treg dynamics. In this study, we determined the level of SIV infection in Treg and nonregulatory CD4+ T cells (non-Treg) isolated from the colon of SIV-infected rhesus macaques. The dynamics of mucosal Treg and alterations in the mucosal CD4+ T-cell pool were examined longitudinally. Our findings indicate that mucosal Treg were less susceptible to productive SIV infection than non-Treg and thus were selectively spared from SIV-mediated cell death. In addition to improved survival, local expansion of Treg by SIV-induced proliferation of the mucosal CD4+ T-cell pool facilitated the accumulation of mucosal Treg during the course of infection. High frequency of mucosal Treg in chronic SIV infection was strongly related to a reduction of perforin-expressing cells. In conclusion, this study suggests that mucosal Treg are less affected by productive SIV infection than non-Treg and therefore spared from depletion. Although SIV production is limited in mucosal Treg, Treg accumulation may indirectly contribute to viral persistence by suppressing antiviral immune responses.

Diversity of Clonal T Cell Proliferation Is Mediated by Differential Expression of CD152 (CTLA-4) on the Cell Surface of Activated Individual T Lymphocytes

Inhibitory effects of CD152 (CTLA-4) engagement during T cell activation have been described. To date, such effects could only be correlated to CD152 expression at the population level because expression of CD152 on the cell surface is too low to be assessed by conventional immunofluorescence on the single cell level. In this study, we use magnetofluorescent liposomes for the immunofluorescent detection of surface CD152-expressing CD4+ T cells and show that, despite the fact that nearly all cells express intracellular CD152, only a fraction of 12% of activated T cells expresses surface CD152 at any given time point. Surface CD152+ T cells appear with similar kinetics after primary or secondary activation in vitro. However, the frequency of surface CD152+ T cells 48 h postactivation is 2-fold higher during secondary activation. Surface expression of CD152 is independent of the proliferative history of an activated T cell. Instruction of T cells for surface expression of CD152 rather depends on the time elapsed since the onset of activation, with a maximum at 48 h, and requires less than 12 h of Ag exposure. CD152− T cells, when isolated by cell sorting and restimulated, continue to proliferate. CD152 blockade has no effect on their proliferation. Isolated surface CD152+ T cells do not proliferate upon restimulation unless CD152 is blocked. CD152 thus acts directly and autonomously on individual activated and proliferating T lymphocytes. Due to its heterogeneous expression on the cell surface of activated Th cells, CD152 might diversify the T cell response.