Christiane Steeg - Academia.edu (original) (raw)

Papers by Christiane Steeg

Parasite Immunology, Jun 7, 2012

About 225 million malaria cases have been reported worldwide in 2009, and one-third of the world'... more About 225 million malaria cases have been reported worldwide in 2009, and one-third of the world's population is infected with parasitic helminths. As helminths and Plasmodium are co-endemic, concurrent infections frequently occur. Helminths have been shown to modulate the host's immune response; therefore, pre-existing helminth infections may interfere with the efficient immune response to Plasmodium. To study the interaction between helminths and Plasmodium, we established a murine model of co-infection using the gastrointestinal nematode Strongyloides ratti and Plasmodium yoelii. We show that a pre-existing Strongyloides infection slightly enhanced peak parasitemia and weight loss in P. yoelii-infected BALB ⁄ c mice, while disease progression was not altered in co-infected C57BL ⁄ 6 mice. The Plasmodiuminduced IFN-c production and final clearance of Plasmodium infection were not affected by S. ratti co-infection in both C57BL ⁄ 6 and BALB ⁄ c mice. Interestingly, the T helper cell (Th) 2 response induced by S. ratti was significantly suppressed upon P. yoelii co-infection. This suppressed Th2 response, however, was still sufficient to allow expulsion of S. ratti parasitic adults. Taken together, we provide evidence that simultaneous presence of helminth and protist parasites does not interfere with efficient host defence in our co-infection model although changes in Th responses were observed.

PLOS Pathogens, Nov 1, 2016

<p>To determine mechanisms involved in the suppression mediated by PD1<sup>+</sup&... more <p>To determine mechanisms involved in the suppression mediated by PD1⁺CTLA4⁺CD4⁺ T cells, we conducted suppression assays in transwell systems and in the presence of blocking antibodies for IL10R, TGFβ, CTLA4 and PDL1. The net (stimulated minus spontaneous) proliferation counts for CD4⁺ T cells (ratio 1:0), stimulated with anti-CD3/28, were set as 100%. Proliferation results for other cell culture conditions are expressed as percentage of net proliferation counts of CD4⁺ T cells (ratio 1:0). A) 5 x10⁴ CD4⁺ T cells were stimulated with anti-CD3/28 and cultured with/without an equal number of PD1⁺CTLA4⁺CD4⁺ T cells, separated by a 0.2μm transwell membrane. P = 0.75, using a Wilcoxon matched pairs test (n = 3). B) 2.5 x10⁴ CD4⁺ T cells were stimulated with anti-CD3/28 and cultured with/without an equal number of PD1⁺CTLA4⁺CD4⁺ T cells (abbreviated with PD1⁺) and with/without addition of 10μg/ml blocking antibodies for IL10R, TGFβ, CTLA4 and PDL1 (n = 4). *, overall P = 0.01. Statistical significance was determined using a Friedman test with Dunn´s multiple comparisons test.</p

PLOS Pathogens, Nov 1, 2016

<p>PBMC from patients with acute malaria were stimulated with iRBC and addition of Brefeldi... more <p>PBMC from patients with acute malaria were stimulated with iRBC and addition of Brefeldin A/Monensin for 18h. Stimulation without antigen (medium only) and with uninfected red blood cells (uRBC) served as negative controls. PHA was used as positive control. A) Dotplots show intracellular staining for IFNγ and IL10 in response to medium only (left), iRBC (middle) and PHA (right) after gating for CD4⁺ T cells. One representative donor with acute malaria out of 11 is shown. Gating strategies for cytokine production are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005909#ppat.1005909.s009&quot; target="_blank">S7 Fig</a>. B) IFNγ⁺ and IL10⁺ CD4⁺ T cells were further analyzed for expression of CTLA4 and PD1. One representative patient out of 11 is shown. C) PBMC were stimulated for 120hrs with iRBC and with/without addition of blocking antibodies against CTLA4 and PDL1. IL10 and IFNγ were measured in culture supernatants by ELISA. Fold-increase of cytokine production was calculated by dividing the net iRBC-specific cytokine production with blockade of CTLA4/PDL1 by net-cytokine production without antibody blockade. P = 0.004 (IFNγ) and P = 0.016 (IL10), using the Wilcoxon matched pairs test on net-cytokine concentration results.</p

PLOS Pathogens, Nov 1, 2016

<p>A) Whole blood samples of malaria patients were analyzed for <i>ex-vivo</i> ... more <p>A) Whole blood samples of malaria patients were analyzed for <i>ex-vivo</i> expression of intracellular Ki67 and Tbet on CD4⁺ and PD1⁺CTLA4⁺CD4⁺ T cells. Dotplots show one representative donor out of 5. Scatter plots show the frequency of Ki67 expression on the assessed CD4⁺ T cells (left) and PD1⁺CTLA4⁺CD4⁺ T cells (right) for all malaria patients included in the analysis (N = 12). *** P < 0.001. B) PBMC of malaria patients were labeled with CFSE and stimulated with/without iRBC. Cells were first gated for CD4⁺ T cells. Proliferating cells were further analyzed for expression of CTLA4 and PD1. The assay is shown here for one representative malaria patient out of three. The other two patients are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005909#ppat.1005909.s008&quot; target="_blank">S6 Fig</a>. C) PBMC were stimulated with iRBC with/without the addition of 10μg/ml anti-PDL1 and anti-CTLA4 and ³H uptakes measured after 120hrs of culture. Proliferation indices were calculated by dividing ³H counts of stimulated cells by ³H counts of unstimulated cells. A proliferation index > 2 was considered to be a positive response. (n = 11).</p

PLOS Pathogens, Nov 1, 2016

<p>Cell cultures were set up by stimulating CD4⁺ T cells with anti-C... more <p>Cell cultures were set up by stimulating CD4⁺ T cells with anti-CD3/28 (A and B), iRBCs (C) or tetanus toxoid (D) with/without addition of FACS sorted PD1⁺CTLA4⁺CD4⁺ T cells (ratio 1:0, 1:1, 1:0.5). ³H-Thymidine uptake was measured after 120 hrs of culture to assess cell proliferation. Cultures were performed in triplicates unless there were insufficient cells, and values represent mean + SE. A) shows the absolute proliferation counts per minute for 2.5 x 10⁴ anti-CD3/28 stimulated CD4⁺ T cells (ratio 1:0), 5x 10⁴ CD4⁺ T cells (ratio 2:0), 2.5 x 10⁴ PD1⁺CTLA4⁺CD4⁺ T cells (0:1) and 2.5 x 10⁴ CD4⁺ T cells with equal (ratio 1:1) or half the numbers (ratio 1:0.5) of PD1⁺CTLA4⁺CD4⁺ T cells for one representative malaria patient out of 7. Adding twice the number of CD4⁺ T cells, 5 x 10⁴ cells, (2:0), was included as a control condition to exclude higher cell numbers as a cause for suppressed cell proliferation. B) summarizes the results for all malaria patients included in this experiment (n = 7 for condition 1:0 and 1:1). The net (stimulated minus spontaneous) proliferation counts for 2.5 x 10⁴ CD4⁺ T cells (1:0), stimulated with anti-CD3/28, were set as 100%. Proliferation results for other cell culture conditions are expressed as percentage of net proliferation counts of CD4⁺ T cells (ratio 1:0). Statistical analysis was conducted using the Wilcoxon matched pairs test on net counts per minutes (*, P = 0.016). C and D) For antigen-specific stimulation, 10⁵ CD4⁺ T cells were stimulated with equal numbers of irradiated feeder cells and with/without equal numbers of PD1⁺CTLA4⁺CD4⁺ T cells. The diagrams show one representative suppression assay of 2, using iRBC (C) or tetanus toxoid as specific antigen (D).</p

PLOS Pathogens, Nov 1, 2016

<p>A) Blood samples from acute malaria patients and healthy controls were analyzed <i&gt... more <p>A) Blood samples from acute malaria patients and healthy controls were analyzed <i>ex-vivo</i> for the expression of PD1 and intracellular CTLA4 on CD4⁺ T cells by flow cytometry. Dotplots show one representative donor out of 31 (malaria patients) or 19 (healthy controls). B) Scatter plots show the frequency of CTLA4⁺ (left), PD1⁺ (middle) and PD1⁺CTLA4⁺ cells (right) as percentage of CD4⁺ T cells for all analyzed donors. Horizontal bars represent means. ***, P < 0,0001 (t-test with Holm-Sidak correction for multiple comparisons). Gating strategies for CD4⁺ T cells and CTLA4⁺ and PD1⁺CD4⁺ T cells are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005909#ppat.1005909.s005&quot; target="_blank">S3 Fig</a>. C) Correlation analysis between the expression levels of PD1 and intracellular CTLA4 on CD4⁺ T cells for all malaria patients analyzed (N = 31). The linear regression line is shown. R = 0.83, P < 0,001 (Pearson correlation). D) Kinetics of CTLA4 (left) and PD1 (right) expression in CD4⁺ T cells at the time of diagnosis (day 1) and over the time-course of the malaria treatment and follow-up. Three malaria patients are shown. E) Relationship between <i>P</i>. <i>falciparum</i> parasitemia at time of diagnosis and expression of CTLA4 (left) and PD1 (right) on CD4⁺ T cells at the same time point. The linear regression lines are shown. R = 0.16 and R = 0.17 (Pearson correlation). F) The bar graphs shows the frequency of CTLA4⁺ and PD1⁺ cells as percentage of CD4⁺ T cells for donors with known uncomplicated malaria (n = 13) and severe, cerebral malaria (n = 3). *, P = 0.024 and NS, P = 0.073 (unpaired t-test). G) CD4⁺ T cells and PD1⁺CTLA4⁺CD4⁺ T cells in malaria patients were analyzed <i>ex vivo</i> for the expression of Foxp3 and CD25. Dotplots are shown for one representative patient. The scatter plot shows the frequency of Foxp3⁺ and of Foxp3⁺CD25⁺ cells as percentage of the assessed CD4⁺ and PD1⁺CTLA4⁺ CD4⁺ T cells of all patients included in this analysis (n = 12). H) The mean fluorescence intensity of CTLA4 expression was compared between Foxp3⁺ and Foxp3^- CTLA4⁺ CD4⁺ T cells of malaria patients. The dotplot shows the expression of CTLA4 and Foxp3 on CD4⁺ T cells of one representative malaria patient. The scatter plot shows the geometric mean fluorescence intensity of CTLA4 of Foxp3⁺ and of Foxp3^-CTLA4⁺ CD4⁺ T cells of all patients included in the analysis (n = 12). Horizontal bars represent means. **, P = 0.002 (paired t-test).</p

The Journal of Immunology

The reactivity of Lyt-2+ or L3T4+ T cells stimulated with either mutant class I or class II MHC a... more The reactivity of Lyt-2+ or L3T4+ T cells stimulated with either mutant class I or class II MHC alloantigens was studied. Whereas stimulation with class I MHC antigens induced only Lyt-2+ T cells to proliferate and to secrete IL 2, stimulation with class II MHC alloantigens induced L3T4+ but not Lyt-2+ T cells. When the frequencies of precursors of IL 2-secreting T lymphocytes (IL 2TL-p) were determined by limiting dilution analyses, class I MHC-reactive Lyt-2+ T cells displayed frequencies (f = 1/200) as high in magnitude as those within class II MHC-reactive L3T4+ (f = 1/100). Clonally developing IL 2TL of either T cell subset were antigen-specific, as shown in split-culture experiments. Whereas L3T4+ helper TL could be induced to specific IL 2 secretion over a long time period (days 3 to 9), Lyt-2+ TL showed a marked time optimal on day 4; thereafter, the number of TL colonies inducible to secrete IL 2 decreased steadily. IL 2 production and IL 2TL-p frequencies of unseparated T ...

European Journal of Immunology, 1995

The protease dipeptidylpeptidase IV (CD26) provides an alternative activation pathway for T lymph... more The protease dipeptidylpeptidase IV (CD26) provides an alternative activation pathway for T lymphocytes and is involved in several aspects of T cell function. Activation via CD26 requires the expression of the T cell receptor (TcR)/CD3 complex. Here we have investigated the role of the TcR ζ chain for T cell activation via CD26. T cell hybridomas expressing TcR with various deletions in the CD3 ζ chain were transfected with a CD26 cDNA and the response of the transfected cells to anti‐CD26 monoclonal antibodies was tested. Our data show that the ζ chain is essential and that at least one YXXL motif in the cytoplasmic tail of the ζ chain is required for CD26‐mediated signaling. Other TcR components do not replace the ζ chain.

Immunobiology, 1987

We have determined the frequencies and specificities of MHC-reactive and MHC-restricted cytotoxic... more We have determined the frequencies and specificities of MHC-reactive and MHC-restricted cytotoxic T lymphocyte precursors (CTL-p) in mitogen (ConA)-activated splenocytes of normal unprimed mice. The limiting dilution (LD) system supported the growth of one out of three Lyt2+ T cell blasts. The generated CTL-populations lysed blast cell targets specifically as determined by split well analyses. MHC-gene product expression was necessary for lysis to occur, since MHC-negative F9 teratocarcinoma cells were not lysed. The frequency determinations and split well analyses revealed: 1) equally high numbers (approximately 1/100) of CTL-p that generated specific allo-MHC or self-MHC reactive CTL populations, 2) high frequencies of CTL-p which recognized hapten (TNP) or minor H (MH)-antigens in the context of self MHC or allo-MHC determinants. The results are discussed with respect to antigen, restriction and receptor specificities of mitogen-activated unprimed T cell blasts.

Clinical and Experimental Immunology, 2006

Summary The protozoan parasite Trypanosoma cruzi circulates in the blood as trypomastigotes and i... more Summary The protozoan parasite Trypanosoma cruzi circulates in the blood as trypomastigotes and invades a variety of cells to multiply intracellularly as amastigotes. The acute phase triggers an immune response that restricts the proliferation of the parasite. However, parasites are able to persist in different tissues causing the pathology of Chagas’ disease. Natural killer (NK) cells play an important role in innate resistance to a variety of pathogens. In the present study we demonstrate that NK cells trigger trypanocidal mechanisms in infected L929 cells that are critically dependent on inducible nitric oxide (NO) synthase (iNOS) induction which is, to a major degree, triggered by interferon (IFN)-γ provided by NK cells. This work provides a more detailed analysis of how NK cells as a part of the innate immune system participate in the control of parasites that reside intracellularly in fibroblast-like L929 cells.

Malaria, caused by infection with Plasmodium falciparum (Pf), can progress to severe disease with... more Malaria, caused by infection with Plasmodium falciparum (Pf), can progress to severe disease with high lethality. Observations from studies in malaria-endemic areas and in murine malaria models indicate that a strong pro-inflammatory T cell response contributes to severe malaria. An optimal regulation[for full text, please go to the a.m. URL]

Cellular Immunology, 1995

Regulatory T cells (Treg) have been shown to restrict vaccine-induced T cell responses in differe... more Regulatory T cells (Treg) have been shown to restrict vaccine-induced T cell responses in different experimental models. In these studies CD4+CD25+ Treg were depleted using monoclonal antibodies against CD25, which might also interfere with CD25 on non-regulatory T cell populations and would have no effect on Foxp3+CD252 Treg. To obtain more insights in the specific function of Treg during vaccination we used mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT) receptor-eGFP fusion protein under the control of the foxp3 gene locus (depletion of regulatory T cell mice; DEREG). As an experimental vaccine-carrier recombinant Bordetella adenylate cyclase toxoid fused with a MHC-class I-restricted epitope of the circumsporozoite protein (ACT-CSP) of Plasmodium berghei (Pb) was used. ACT-CSP was shown by us previously to introduce the CD8+ epitope of Pb-CSP into the MHC class I presentation pathway of professional antigen-presenting cells (APC)...

Variation between inbred mice of susceptibility to experimental Trypanosoma cruzi infection has f... more Variation between inbred mice of susceptibility to experimental Trypanosoma cruzi infection has frequently been described, but the immunogenetic background is poorly understood. The outcross of the susceptible parental mouse strains C57BL/6 (B6) and DBA/2 (D2), B6D2F1 (F1) mice, is highly resistant to this parasite. In the present study we show by quantitative PCR that the increase of tissue parasitism during the early phase of infection is comparable up to day 11 between susceptible B6 and resistant F1 mice. A reduction of splenic parasite burdens occurs thereafter in both strains but is comparatively retarded in susceptible mice. Splenic microarchitecture is progressively disrupted with loss of follicles and B lymphocytes in B6 mice, but not in F1 mice. By genotyping of additional backcross offspring we corroborate our earlier findings that susceptibility maps to three loci on Chromosomes 5, 13 and 17. Analysis of gene expression of spleen cells from infected B6 and F1 mice with m...

Frontiers in Immunology

In Plasmodium falciparum malaria, CD8 + T cells play a double-edged role. Liver-stage specific CD... more In Plasmodium falciparum malaria, CD8 + T cells play a double-edged role. Liver-stage specific CD8 + T cells can confer protection, as has been shown in several vaccine studies. Blood-stage specific CD8 + T cells, on the other hand, contribute to the development of cerebral malaria in murine models of malaria. The role of CD8 + T cells in humans during the blood-stage of P. falciparum remains unclear. As part of a cross-sectional malaria study in Ghana, granzyme B levels and CD8 + T cells phenotypes were compared in the peripheral blood of children with complicated malaria, uncomplicated malaria, afebrile but asymptomatically infected children and non-infected children. Granzyme B levels in the plasma were significantly higher in children with febrile malaria than in afebrile children. CD8 + T cells were the main T cell subset expressing granzyme B. The proportion of granzyme B + CD8 + T cells was significantly higher in children with complicated malaria than in uncomplicated malaria, whereas the activation marker CD38 on CD8 + T cells showed similar expression levels. This suggests a pathogenic role of cytotoxic CD8 + T cells in the development of malaria complications in humans.

Scientific Reports

The Acknowledgements section in this Article is incomplete. "We thank Eric Fomevor, Isaac Yaw Asa... more The Acknowledgements section in this Article is incomplete. "We thank Eric Fomevor, Isaac Yaw Asamoah and Evans Gawu for their technical and logistical help during the study. We especially thank the children and their parents at St Michael's Hospital and Jachie Primary School for participating in the study. " should read: "We thank Eric Fomevor, Isaac Yaw Asamoah and Evans Gawu for their technical and logistical help during the study. We especially thank the children and their parents at St Michael's Hospital and Jachie Primary School for participating in the study. The work was funded by grants of the Collaborative Research Center

Scientific reports, Jan 9, 2018

A common helper-like innate lymphoid precursor (CHILP) restricted to the innate lymphoid cells (I... more A common helper-like innate lymphoid precursor (CHILP) restricted to the innate lymphoid cells (ILC) lineage has been recently characterized. While specific requirements of transcription factors for CHILPs development has been partially described, their ability to sense cytokines and react to peripheral inflammation remains unaddressed. Here, we found that systemic increase in Flt3L levels correlated with the expansion of Lineage (Lin)α4β7 precursors in the adult murine bone marrow. Expanded Linα4β7 precursors were bona fide CHILPs as seen by their ability to differentiate into all helper ILCs subsets but cNK in vivo. Interestingly, Flt3L-expanded CHILPs transferred into lymphopenic mice preferentially reconstituted the small intestine. While we did not observe changes in serum Flt3L during DSS-induced colitis in mice or plasma from inflammatory bowel disease (IBD) patients, elevated Flt3L levels were detected in acute malaria patients. Interestingly, while CHILP numbers were stable...

Scientific reports, Jan 19, 2018

The immune response of malaria patients is a main factor influencing the clinical severity of mal... more The immune response of malaria patients is a main factor influencing the clinical severity of malaria. A tight regulation of the CD4 T cell response or the induction of tolerance have been proposed to contribute to protection from severe or clinical disease. We therefore compared the CD4 T cell phenotypes of Ghanaian children with complicated malaria, uncomplicated malaria, asymptomatic Plasmodium falciparum (Pf) infection or no infection. Using flow cytometric analysis and automated multivariate clustering, we characterized the expression of the co-inhibitory molecules CTLA-4, PD-1, Tim-3, and LAG-3 and other molecules implicated in regulatory function on CD4 T cells. Children with complicated malaria had higher frequencies of CTLA-4 or PD-1 CD4 T cells than children with uncomplicated malaria. Conversely, children with uncomplicated malaria showed a higher proportion of CD4 T cells expressing CD39 and Granzyme B, compared to children with complicated malaria. In contrast, asymptom...

PLoS pathogens, 2016

In acute Plasmodium falciparum (P. falciparum) malaria, the pro- and anti-inflammatory immune pat... more In acute Plasmodium falciparum (P. falciparum) malaria, the pro- and anti-inflammatory immune pathways must be delicately balanced so that the parasitemia is controlled without inducing immunopathology. An important mechanism to fine-tune T cell responses in the periphery is the induction of coinhibitory receptors such as CTLA4 and PD1. However, their role in acute infections such as P. falciparum malaria remains poorly understood. To test whether coinhibitory receptors modulate CD4+ T cell functions in malaria, blood samples were obtained from patients with acute P. falciparum malaria treated in Germany. Flow cytometric analysis showed a more frequent expression of CTLA4 and PD1 on CD4+ T cells of malaria patients than of healthy control subjects. In vitro stimulation with P. falciparum-infected red blood cells revealed a distinct population of PD1+CTLA4+CD4+ T cells that simultaneously produced IFNγ and IL10. This antigen-specific cytokine production was enhanced by blocking PD1/P...

Parasite Immunology, Jun 7, 2012

About 225 million malaria cases have been reported worldwide in 2009, and one-third of the world'... more About 225 million malaria cases have been reported worldwide in 2009, and one-third of the world's population is infected with parasitic helminths. As helminths and Plasmodium are co-endemic, concurrent infections frequently occur. Helminths have been shown to modulate the host's immune response; therefore, pre-existing helminth infections may interfere with the efficient immune response to Plasmodium. To study the interaction between helminths and Plasmodium, we established a murine model of co-infection using the gastrointestinal nematode Strongyloides ratti and Plasmodium yoelii. We show that a pre-existing Strongyloides infection slightly enhanced peak parasitemia and weight loss in P. yoelii-infected BALB ⁄ c mice, while disease progression was not altered in co-infected C57BL ⁄ 6 mice. The Plasmodiuminduced IFN-c production and final clearance of Plasmodium infection were not affected by S. ratti co-infection in both C57BL ⁄ 6 and BALB ⁄ c mice. Interestingly, the T helper cell (Th) 2 response induced by S. ratti was significantly suppressed upon P. yoelii co-infection. This suppressed Th2 response, however, was still sufficient to allow expulsion of S. ratti parasitic adults. Taken together, we provide evidence that simultaneous presence of helminth and protist parasites does not interfere with efficient host defence in our co-infection model although changes in Th responses were observed.

PLOS Pathogens, Nov 1, 2016

<p>To determine mechanisms involved in the suppression mediated by PD1<sup>+</sup&... more <p>To determine mechanisms involved in the suppression mediated by PD1⁺CTLA4⁺CD4⁺ T cells, we conducted suppression assays in transwell systems and in the presence of blocking antibodies for IL10R, TGFβ, CTLA4 and PDL1. The net (stimulated minus spontaneous) proliferation counts for CD4⁺ T cells (ratio 1:0), stimulated with anti-CD3/28, were set as 100%. Proliferation results for other cell culture conditions are expressed as percentage of net proliferation counts of CD4⁺ T cells (ratio 1:0). A) 5 x10⁴ CD4⁺ T cells were stimulated with anti-CD3/28 and cultured with/without an equal number of PD1⁺CTLA4⁺CD4⁺ T cells, separated by a 0.2μm transwell membrane. P = 0.75, using a Wilcoxon matched pairs test (n = 3). B) 2.5 x10⁴ CD4⁺ T cells were stimulated with anti-CD3/28 and cultured with/without an equal number of PD1⁺CTLA4⁺CD4⁺ T cells (abbreviated with PD1⁺) and with/without addition of 10μg/ml blocking antibodies for IL10R, TGFβ, CTLA4 and PDL1 (n = 4). *, overall P = 0.01. Statistical significance was determined using a Friedman test with Dunn´s multiple comparisons test.</p

PLOS Pathogens, Nov 1, 2016

<p>PBMC from patients with acute malaria were stimulated with iRBC and addition of Brefeldi... more <p>PBMC from patients with acute malaria were stimulated with iRBC and addition of Brefeldin A/Monensin for 18h. Stimulation without antigen (medium only) and with uninfected red blood cells (uRBC) served as negative controls. PHA was used as positive control. A) Dotplots show intracellular staining for IFNγ and IL10 in response to medium only (left), iRBC (middle) and PHA (right) after gating for CD4⁺ T cells. One representative donor with acute malaria out of 11 is shown. Gating strategies for cytokine production are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005909#ppat.1005909.s009&quot; target="_blank">S7 Fig</a>. B) IFNγ⁺ and IL10⁺ CD4⁺ T cells were further analyzed for expression of CTLA4 and PD1. One representative patient out of 11 is shown. C) PBMC were stimulated for 120hrs with iRBC and with/without addition of blocking antibodies against CTLA4 and PDL1. IL10 and IFNγ were measured in culture supernatants by ELISA. Fold-increase of cytokine production was calculated by dividing the net iRBC-specific cytokine production with blockade of CTLA4/PDL1 by net-cytokine production without antibody blockade. P = 0.004 (IFNγ) and P = 0.016 (IL10), using the Wilcoxon matched pairs test on net-cytokine concentration results.</p

PLOS Pathogens, Nov 1, 2016

<p>A) Whole blood samples of malaria patients were analyzed for <i>ex-vivo</i> ... more <p>A) Whole blood samples of malaria patients were analyzed for <i>ex-vivo</i> expression of intracellular Ki67 and Tbet on CD4⁺ and PD1⁺CTLA4⁺CD4⁺ T cells. Dotplots show one representative donor out of 5. Scatter plots show the frequency of Ki67 expression on the assessed CD4⁺ T cells (left) and PD1⁺CTLA4⁺CD4⁺ T cells (right) for all malaria patients included in the analysis (N = 12). *** P < 0.001. B) PBMC of malaria patients were labeled with CFSE and stimulated with/without iRBC. Cells were first gated for CD4⁺ T cells. Proliferating cells were further analyzed for expression of CTLA4 and PD1. The assay is shown here for one representative malaria patient out of three. The other two patients are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005909#ppat.1005909.s008&quot; target="_blank">S6 Fig</a>. C) PBMC were stimulated with iRBC with/without the addition of 10μg/ml anti-PDL1 and anti-CTLA4 and ³H uptakes measured after 120hrs of culture. Proliferation indices were calculated by dividing ³H counts of stimulated cells by ³H counts of unstimulated cells. A proliferation index > 2 was considered to be a positive response. (n = 11).</p

PLOS Pathogens, Nov 1, 2016

<p>Cell cultures were set up by stimulating CD4⁺ T cells with anti-C... more <p>Cell cultures were set up by stimulating CD4⁺ T cells with anti-CD3/28 (A and B), iRBCs (C) or tetanus toxoid (D) with/without addition of FACS sorted PD1⁺CTLA4⁺CD4⁺ T cells (ratio 1:0, 1:1, 1:0.5). ³H-Thymidine uptake was measured after 120 hrs of culture to assess cell proliferation. Cultures were performed in triplicates unless there were insufficient cells, and values represent mean + SE. A) shows the absolute proliferation counts per minute for 2.5 x 10⁴ anti-CD3/28 stimulated CD4⁺ T cells (ratio 1:0), 5x 10⁴ CD4⁺ T cells (ratio 2:0), 2.5 x 10⁴ PD1⁺CTLA4⁺CD4⁺ T cells (0:1) and 2.5 x 10⁴ CD4⁺ T cells with equal (ratio 1:1) or half the numbers (ratio 1:0.5) of PD1⁺CTLA4⁺CD4⁺ T cells for one representative malaria patient out of 7. Adding twice the number of CD4⁺ T cells, 5 x 10⁴ cells, (2:0), was included as a control condition to exclude higher cell numbers as a cause for suppressed cell proliferation. B) summarizes the results for all malaria patients included in this experiment (n = 7 for condition 1:0 and 1:1). The net (stimulated minus spontaneous) proliferation counts for 2.5 x 10⁴ CD4⁺ T cells (1:0), stimulated with anti-CD3/28, were set as 100%. Proliferation results for other cell culture conditions are expressed as percentage of net proliferation counts of CD4⁺ T cells (ratio 1:0). Statistical analysis was conducted using the Wilcoxon matched pairs test on net counts per minutes (*, P = 0.016). C and D) For antigen-specific stimulation, 10⁵ CD4⁺ T cells were stimulated with equal numbers of irradiated feeder cells and with/without equal numbers of PD1⁺CTLA4⁺CD4⁺ T cells. The diagrams show one representative suppression assay of 2, using iRBC (C) or tetanus toxoid as specific antigen (D).</p

PLOS Pathogens, Nov 1, 2016

<p>A) Blood samples from acute malaria patients and healthy controls were analyzed <i&gt... more <p>A) Blood samples from acute malaria patients and healthy controls were analyzed <i>ex-vivo</i> for the expression of PD1 and intracellular CTLA4 on CD4⁺ T cells by flow cytometry. Dotplots show one representative donor out of 31 (malaria patients) or 19 (healthy controls). B) Scatter plots show the frequency of CTLA4⁺ (left), PD1⁺ (middle) and PD1⁺CTLA4⁺ cells (right) as percentage of CD4⁺ T cells for all analyzed donors. Horizontal bars represent means. ***, P < 0,0001 (t-test with Holm-Sidak correction for multiple comparisons). Gating strategies for CD4⁺ T cells and CTLA4⁺ and PD1⁺CD4⁺ T cells are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005909#ppat.1005909.s005&quot; target="_blank">S3 Fig</a>. C) Correlation analysis between the expression levels of PD1 and intracellular CTLA4 on CD4⁺ T cells for all malaria patients analyzed (N = 31). The linear regression line is shown. R = 0.83, P < 0,001 (Pearson correlation). D) Kinetics of CTLA4 (left) and PD1 (right) expression in CD4⁺ T cells at the time of diagnosis (day 1) and over the time-course of the malaria treatment and follow-up. Three malaria patients are shown. E) Relationship between <i>P</i>. <i>falciparum</i> parasitemia at time of diagnosis and expression of CTLA4 (left) and PD1 (right) on CD4⁺ T cells at the same time point. The linear regression lines are shown. R = 0.16 and R = 0.17 (Pearson correlation). F) The bar graphs shows the frequency of CTLA4⁺ and PD1⁺ cells as percentage of CD4⁺ T cells for donors with known uncomplicated malaria (n = 13) and severe, cerebral malaria (n = 3). *, P = 0.024 and NS, P = 0.073 (unpaired t-test). G) CD4⁺ T cells and PD1⁺CTLA4⁺CD4⁺ T cells in malaria patients were analyzed <i>ex vivo</i> for the expression of Foxp3 and CD25. Dotplots are shown for one representative patient. The scatter plot shows the frequency of Foxp3⁺ and of Foxp3⁺CD25⁺ cells as percentage of the assessed CD4⁺ and PD1⁺CTLA4⁺ CD4⁺ T cells of all patients included in this analysis (n = 12). H) The mean fluorescence intensity of CTLA4 expression was compared between Foxp3⁺ and Foxp3^- CTLA4⁺ CD4⁺ T cells of malaria patients. The dotplot shows the expression of CTLA4 and Foxp3 on CD4⁺ T cells of one representative malaria patient. The scatter plot shows the geometric mean fluorescence intensity of CTLA4 of Foxp3⁺ and of Foxp3^-CTLA4⁺ CD4⁺ T cells of all patients included in the analysis (n = 12). Horizontal bars represent means. **, P = 0.002 (paired t-test).</p

The Journal of Immunology

The reactivity of Lyt-2+ or L3T4+ T cells stimulated with either mutant class I or class II MHC a... more The reactivity of Lyt-2+ or L3T4+ T cells stimulated with either mutant class I or class II MHC alloantigens was studied. Whereas stimulation with class I MHC antigens induced only Lyt-2+ T cells to proliferate and to secrete IL 2, stimulation with class II MHC alloantigens induced L3T4+ but not Lyt-2+ T cells. When the frequencies of precursors of IL 2-secreting T lymphocytes (IL 2TL-p) were determined by limiting dilution analyses, class I MHC-reactive Lyt-2+ T cells displayed frequencies (f = 1/200) as high in magnitude as those within class II MHC-reactive L3T4+ (f = 1/100). Clonally developing IL 2TL of either T cell subset were antigen-specific, as shown in split-culture experiments. Whereas L3T4+ helper TL could be induced to specific IL 2 secretion over a long time period (days 3 to 9), Lyt-2+ TL showed a marked time optimal on day 4; thereafter, the number of TL colonies inducible to secrete IL 2 decreased steadily. IL 2 production and IL 2TL-p frequencies of unseparated T ...

European Journal of Immunology, 1995

The protease dipeptidylpeptidase IV (CD26) provides an alternative activation pathway for T lymph... more The protease dipeptidylpeptidase IV (CD26) provides an alternative activation pathway for T lymphocytes and is involved in several aspects of T cell function. Activation via CD26 requires the expression of the T cell receptor (TcR)/CD3 complex. Here we have investigated the role of the TcR ζ chain for T cell activation via CD26. T cell hybridomas expressing TcR with various deletions in the CD3 ζ chain were transfected with a CD26 cDNA and the response of the transfected cells to anti‐CD26 monoclonal antibodies was tested. Our data show that the ζ chain is essential and that at least one YXXL motif in the cytoplasmic tail of the ζ chain is required for CD26‐mediated signaling. Other TcR components do not replace the ζ chain.

Immunobiology, 1987

We have determined the frequencies and specificities of MHC-reactive and MHC-restricted cytotoxic... more We have determined the frequencies and specificities of MHC-reactive and MHC-restricted cytotoxic T lymphocyte precursors (CTL-p) in mitogen (ConA)-activated splenocytes of normal unprimed mice. The limiting dilution (LD) system supported the growth of one out of three Lyt2+ T cell blasts. The generated CTL-populations lysed blast cell targets specifically as determined by split well analyses. MHC-gene product expression was necessary for lysis to occur, since MHC-negative F9 teratocarcinoma cells were not lysed. The frequency determinations and split well analyses revealed: 1) equally high numbers (approximately 1/100) of CTL-p that generated specific allo-MHC or self-MHC reactive CTL populations, 2) high frequencies of CTL-p which recognized hapten (TNP) or minor H (MH)-antigens in the context of self MHC or allo-MHC determinants. The results are discussed with respect to antigen, restriction and receptor specificities of mitogen-activated unprimed T cell blasts.

Clinical and Experimental Immunology, 2006

Summary The protozoan parasite Trypanosoma cruzi circulates in the blood as trypomastigotes and i... more Summary The protozoan parasite Trypanosoma cruzi circulates in the blood as trypomastigotes and invades a variety of cells to multiply intracellularly as amastigotes. The acute phase triggers an immune response that restricts the proliferation of the parasite. However, parasites are able to persist in different tissues causing the pathology of Chagas’ disease. Natural killer (NK) cells play an important role in innate resistance to a variety of pathogens. In the present study we demonstrate that NK cells trigger trypanocidal mechanisms in infected L929 cells that are critically dependent on inducible nitric oxide (NO) synthase (iNOS) induction which is, to a major degree, triggered by interferon (IFN)-γ provided by NK cells. This work provides a more detailed analysis of how NK cells as a part of the innate immune system participate in the control of parasites that reside intracellularly in fibroblast-like L929 cells.

Malaria, caused by infection with Plasmodium falciparum (Pf), can progress to severe disease with... more Malaria, caused by infection with Plasmodium falciparum (Pf), can progress to severe disease with high lethality. Observations from studies in malaria-endemic areas and in murine malaria models indicate that a strong pro-inflammatory T cell response contributes to severe malaria. An optimal regulation[for full text, please go to the a.m. URL]

Cellular Immunology, 1995

Regulatory T cells (Treg) have been shown to restrict vaccine-induced T cell responses in differe... more Regulatory T cells (Treg) have been shown to restrict vaccine-induced T cell responses in different experimental models. In these studies CD4+CD25+ Treg were depleted using monoclonal antibodies against CD25, which might also interfere with CD25 on non-regulatory T cell populations and would have no effect on Foxp3+CD252 Treg. To obtain more insights in the specific function of Treg during vaccination we used mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT) receptor-eGFP fusion protein under the control of the foxp3 gene locus (depletion of regulatory T cell mice; DEREG). As an experimental vaccine-carrier recombinant Bordetella adenylate cyclase toxoid fused with a MHC-class I-restricted epitope of the circumsporozoite protein (ACT-CSP) of Plasmodium berghei (Pb) was used. ACT-CSP was shown by us previously to introduce the CD8+ epitope of Pb-CSP into the MHC class I presentation pathway of professional antigen-presenting cells (APC)...

Variation between inbred mice of susceptibility to experimental Trypanosoma cruzi infection has f... more Variation between inbred mice of susceptibility to experimental Trypanosoma cruzi infection has frequently been described, but the immunogenetic background is poorly understood. The outcross of the susceptible parental mouse strains C57BL/6 (B6) and DBA/2 (D2), B6D2F1 (F1) mice, is highly resistant to this parasite. In the present study we show by quantitative PCR that the increase of tissue parasitism during the early phase of infection is comparable up to day 11 between susceptible B6 and resistant F1 mice. A reduction of splenic parasite burdens occurs thereafter in both strains but is comparatively retarded in susceptible mice. Splenic microarchitecture is progressively disrupted with loss of follicles and B lymphocytes in B6 mice, but not in F1 mice. By genotyping of additional backcross offspring we corroborate our earlier findings that susceptibility maps to three loci on Chromosomes 5, 13 and 17. Analysis of gene expression of spleen cells from infected B6 and F1 mice with m...

Frontiers in Immunology

In Plasmodium falciparum malaria, CD8 + T cells play a double-edged role. Liver-stage specific CD... more In Plasmodium falciparum malaria, CD8 + T cells play a double-edged role. Liver-stage specific CD8 + T cells can confer protection, as has been shown in several vaccine studies. Blood-stage specific CD8 + T cells, on the other hand, contribute to the development of cerebral malaria in murine models of malaria. The role of CD8 + T cells in humans during the blood-stage of P. falciparum remains unclear. As part of a cross-sectional malaria study in Ghana, granzyme B levels and CD8 + T cells phenotypes were compared in the peripheral blood of children with complicated malaria, uncomplicated malaria, afebrile but asymptomatically infected children and non-infected children. Granzyme B levels in the plasma were significantly higher in children with febrile malaria than in afebrile children. CD8 + T cells were the main T cell subset expressing granzyme B. The proportion of granzyme B + CD8 + T cells was significantly higher in children with complicated malaria than in uncomplicated malaria, whereas the activation marker CD38 on CD8 + T cells showed similar expression levels. This suggests a pathogenic role of cytotoxic CD8 + T cells in the development of malaria complications in humans.

Scientific Reports

The Acknowledgements section in this Article is incomplete. "We thank Eric Fomevor, Isaac Yaw Asa... more The Acknowledgements section in this Article is incomplete. "We thank Eric Fomevor, Isaac Yaw Asamoah and Evans Gawu for their technical and logistical help during the study. We especially thank the children and their parents at St Michael's Hospital and Jachie Primary School for participating in the study. " should read: "We thank Eric Fomevor, Isaac Yaw Asamoah and Evans Gawu for their technical and logistical help during the study. We especially thank the children and their parents at St Michael's Hospital and Jachie Primary School for participating in the study. The work was funded by grants of the Collaborative Research Center

Scientific reports, Jan 9, 2018

A common helper-like innate lymphoid precursor (CHILP) restricted to the innate lymphoid cells (I... more A common helper-like innate lymphoid precursor (CHILP) restricted to the innate lymphoid cells (ILC) lineage has been recently characterized. While specific requirements of transcription factors for CHILPs development has been partially described, their ability to sense cytokines and react to peripheral inflammation remains unaddressed. Here, we found that systemic increase in Flt3L levels correlated with the expansion of Lineage (Lin)α4β7 precursors in the adult murine bone marrow. Expanded Linα4β7 precursors were bona fide CHILPs as seen by their ability to differentiate into all helper ILCs subsets but cNK in vivo. Interestingly, Flt3L-expanded CHILPs transferred into lymphopenic mice preferentially reconstituted the small intestine. While we did not observe changes in serum Flt3L during DSS-induced colitis in mice or plasma from inflammatory bowel disease (IBD) patients, elevated Flt3L levels were detected in acute malaria patients. Interestingly, while CHILP numbers were stable...

Scientific reports, Jan 19, 2018

The immune response of malaria patients is a main factor influencing the clinical severity of mal... more The immune response of malaria patients is a main factor influencing the clinical severity of malaria. A tight regulation of the CD4 T cell response or the induction of tolerance have been proposed to contribute to protection from severe or clinical disease. We therefore compared the CD4 T cell phenotypes of Ghanaian children with complicated malaria, uncomplicated malaria, asymptomatic Plasmodium falciparum (Pf) infection or no infection. Using flow cytometric analysis and automated multivariate clustering, we characterized the expression of the co-inhibitory molecules CTLA-4, PD-1, Tim-3, and LAG-3 and other molecules implicated in regulatory function on CD4 T cells. Children with complicated malaria had higher frequencies of CTLA-4 or PD-1 CD4 T cells than children with uncomplicated malaria. Conversely, children with uncomplicated malaria showed a higher proportion of CD4 T cells expressing CD39 and Granzyme B, compared to children with complicated malaria. In contrast, asymptom...

PLoS pathogens, 2016

In acute Plasmodium falciparum (P. falciparum) malaria, the pro- and anti-inflammatory immune pat... more In acute Plasmodium falciparum (P. falciparum) malaria, the pro- and anti-inflammatory immune pathways must be delicately balanced so that the parasitemia is controlled without inducing immunopathology. An important mechanism to fine-tune T cell responses in the periphery is the induction of coinhibitory receptors such as CTLA4 and PD1. However, their role in acute infections such as P. falciparum malaria remains poorly understood. To test whether coinhibitory receptors modulate CD4+ T cell functions in malaria, blood samples were obtained from patients with acute P. falciparum malaria treated in Germany. Flow cytometric analysis showed a more frequent expression of CTLA4 and PD1 on CD4+ T cells of malaria patients than of healthy control subjects. In vitro stimulation with P. falciparum-infected red blood cells revealed a distinct population of PD1+CTLA4+CD4+ T cells that simultaneously produced IFNγ and IL10. This antigen-specific cytokine production was enhanced by blocking PD1/P...