ALBERT

Description: Recent findings are reviewed from observations in the field on the generation of the {delta}13C signal in shells of live (Rose Bengal stained) benthic foraminifera, and end up with implications for the interpretation of fossil signatures. The {delta}13C values of calcite tests of preferentially epifaunal foraminifera principally reflect the {delta}13C of dissolved inorganic carbon (DIC) of ambient seawater, whereas infaunal species record a porewater signal, both with an offset from equilibrium calcite. Species occupying the deepest average living depth in the sediment usually exhibit lowest {delta}13C test values, but {delta}13C values of conspecific specimens at a single site do not decrease with increasing subbottom depth and decreasing porewater {delta}13CDIC. Organic carbon fluxes to the sediment surface are generally reflected by infaunal species such that lowered {delta}13C values coincide with high fluxes, but even strictly epifaunal species may reflect seasonally pulsed phytodetritus supply by depleted test {delta}13C. In high-productivity environments, however, where dissolved oxygen and sedimentary carbonate contents are low, benthic foraminiferal tests show 13C enrichment probably due to carbonate-ion undersaturation. Ontogenetic increase in {delta}13C values of certain infaunal species suggests a slow-down of metabolic rates during test growth and decreasing fractionation with age. At sites of active methane discharge {delta}13C values of infaunal species reflect low pore water {delta}13CDIC values, documenting active methane release in the sediment, whereas lowered {delta}13C values of strictly epifaunal species are most probably the result of incorporation of 13C depleted methanotrophic biomass.

Description: Down-regulation of immune responses by regulatory T (Treg) cells is an important mechanism involved in the induction of tolerance to allo-antigens (Ags). Recently, a novel subset of Ag-specific T-cell receptor (TCR)αβ+ CD4-CD8- (double-negative [DN]) Treg cells has been found to be able to prevent the rejection of skin and heart allografts by specifically inhibiting the function of antigraft-specific CD8+ T cells. Here we demonstrate that peripheral DN Treg cells are present in humans, where they constitute about 1% of total CD3+ T cells, and consist of both naïve and Ag-experienced cells. Similar to murine DN Treg cells, human DN Treg cells are able to acquire peptide–HLA-A2 complexes from antigen-presenting cells by cell contact-dependent mechanisms. Furthermore, such acquired peptide-HLA complexes appear to be functionally active, in that CD8+ T cells specific for the HLA-A2–restricted self-peptide, Melan-A, became sensitive to apoptosis by neighboring DN T cells after acquisition of Melan-A–HLA-A2 complexes and revealed a reduced proliferative response. These results demonstrate for the first time that a sizable population of peripheral DN Treg cells, which are able to suppress Ag-specific T cells, exists in humans. DN Treg cells may serve to limit clonal expansion of allo-Ag–specific T cells after transplantation.

Description: In eukaryotic cells the phospholipid phosphatidylserine (PS) is restricted to the inner plasma membrane leaflet. This lipid asymmetry which is maintained by the concerted action of phospholipid transport proteins is mainly lost during apoptosis. Here, we demonstrate that primary human CD8+ cytotoxic T lymphocytes (CTL) expose PS upon T-cell receptor (TCR)-mediated antigen recognition: antigen-specific CTL, recognizing the HLA-A2 binding Melan-A peptide, demonstrated a marked exposure of PS as determined by annV-FITC staining, a highly specific PS-binding protein, after 4 h of stimulation with Melan-A-loaded antigen presenting cells (APC). PS exposure was Ag-specific (〉80% annV-positive T cells) but control peptide (gp100)-pulsed and unpulsed APC also induced a slight background PS exposure on the CTL. To follow more precisely the fate of annV-positive CTL after antigen-specific stimulation, we labeled Melan-A-specific CTL with the membrane dye PKH-26 and isolated all annV+ PKH-26+ CTL 4 h after stimulation with peptide-pulsed APC cells by cell sorting. Results demonstrate that the annV-positive T-cell population is heterogenous: while the annV-high T-cell population retained the annV-high phenotype and consisted of apoptotic T cells, the annV-intermediate(int) T-cell population revealed a constant decrease of annV binding and became annV-negative at 54 to 72 h after stimulation. Using three independent assays for apoptosis we found that annV-int T cells are propidium iodide negative, do not exhibit DNA strand brakes and contain no active caspase 3. In contrast, annV-int CTL revealed a strongly activated phenotype indicated by an upregulation of CD69 expression and downregulation of the TCRαβ chain expression. Fluorescence microscopic analysis demonstrated that PS is distributed inhomogenously over the plasma membrane and concentrated in membrane lipid raft domains at the immunological synapse. By studying the activity of PS transport proteins using a fluorescence-labeled PS analogue, we determined a constitutive outward transfer of PS molecules in Melan-A-specific CTL. The constitutive PS outward transport was not further accelerated after antigen re-stimulation. In sharp contrast, the inward transporting flippase was strongly inhibited in stimulated CTL resulting in an accumulation of PS molecules on the cell surface. Shielding of exposed PS by annexinV protein during antigen recognition diminished cytokine secretion, activation and cell-cell clustering of antigen-specific CTL. In summary, our data demonstrate for the first time that externalized PS on antigen-stimulated CTL is linked to T-cell activation and probably involved in cell-cell contact formation at the immunological synapse.

Description: It is well established, that the curative potential of allogeneic peripheral blood stem cell transplantation (allo PBSCT) is due to immunocompetent donor T cells inducing potent anti-neoplastic effects against host tumor cells. This reaction, which is termed graft-versus-leukemia (GVL) effect, is clinically effective against a number of different hematologic malignancies such as myeloid and lymphoid leukemias. Despite great efforts of allo PBSCT in treatment of CML, the 5-year survival rate of AML patients after allo PBSCT is only about 30% due to relapsing disease. The recurrent disease is inefficiently controlled by the immune system, due most likely to the various immune escape mechanisms described for AML blasts including upregulation of anti-apoptotic molecules. Since cytotoxic T lymphocytes (CTL) and natural killer cells are the cells responsible for eliminating leukemic blasts, the most important effector molecule is Granzyme B (GrB). Misdirected GrB is quenched by its specific physiological inhibitor Protease Inhibitor-9 (PI-9) leading to inactivation of GrB. PI-9 expression by tumour cells can be used to escape immune surveillance and its presence has been shown for different tumors e.g. melanoma, colon carcinoma and lymphoma. Despite other regulators, interferon-γ (IFN-γ) has been shown to upregulate PI-9 expression in hepatocytes. Here, we wanted to investigate the expression of PI-9 in primary AML blasts and its regulation by IFN-γ. Using CD34+ positive magnetic selection, we isolated primary blasts with a purity of 〉90% from 20 AML patients with different FAB subtypes. For detection of PI-9 expression by Western Blotting, whole cell lysates were made from freshly purified blasts and after 24 h +/− 200 IU/ml IFN-γ. In some patients, PI-9 expression was confirmed by FACS analysis with an anti- PI-9 specific monoclonal antibody. Here we describe for the first time, that PI-9 is constitutively expressed in 16/20 (80%) of AML blasts. Treatment of AML blasts with IFN-γ could upregulate PI-9 expression in a dose-dependent manner (2–2,000 IU/ml) and strong expression of PI-9 was detectable in 6/18 patients within 4–5 h after IFN-γ exposure. Of note, a mild upregulation of PI-9 upon 24 h incubation w/o IFN-γ could be detected in 4/18 (22%) patients. We conclude, that cytokines such as IFN-γ which are secreted during the cytokine storm of acute graft-versus-host disease can contribute to the development of immune escape mechanisms in AML blasts.

Description: Abstract 4490 Background Chronic graft-versus host disease (cGvHD) can result in a scleroderma-like disease with dermal, pulmonary and gastrointestinal fibrosis. The histopathological hallmark of sclerodermatous chronic GvHD is accumulation of extracellular matrix proteins by pathologically activated fibroblasts, caused at least in part by the profibrotic cytokines TGF-β and PDGF. It was shown recently that the family of abl kinases plays an important role in the activation of matrix synthesis. Here we investigated whether imatinib and nilotinib, both targeting specifically TGF-β and PDGF signaling pathways by inhibiting the tyrosine kinase activity of c-abl and PDGF receptors, could reduce cGvHD in a murine model. Material and Methods Recipient mice (BALB/c, H2d) received total body irradiation (2×6 Gy), followed by 1×10∧6 bone marrow cells and 2×10∧6 splenocytes from B10.D2 donors (H2d, multiple minor mismatches). Recipients receiving syngeneic bone marrow and spleen cells served as control. The treatment of transplanted mice started 12 to 14 days post transplantation. Imatinib (150 mg/kg) was given per os once daily and nilotinib (37.5 mg/kg) was administrated per os twice daily until 6 weeks after bone marrow transplantation. Observed skin changes were documented using a clinical score. After 6 weeks dermal thickness was determined by staining with hematoxylin and eosin and activated fibroblasts by using immunohistochemistry for alpha smooth muscle actin. Collagen concentration in afflicted skin was analyzed with the hydroxyproline assay. Results Untreated mice receiving an allogeneic transplant, in contrast to syngeneic controls, showed clinical features of cutaneous cGvHD, including skin lesions with alopecia and a lower mobility, in addition to diarrhea and weight loss until 6 weeks after alloBMT. Treatment of recipients with imatinib and nilotinib resulted in serum concentrations comparable to those achieved in humans by standard dosing. Application of imatinib or nilotinib completely prevented the development of cutaneous cGvHD including alopecia and skin ulceration, and was effective in preventing weight loss. Compared to untreated recipients dermal thickness was decreased by 100 ± 8 % (p = 0.001) with imatinib and by 54 ± 14 % (p = 0.016) with nilotinib. The numbers of myofibroblasts were reduced by 84 ± 16 % (p = 0.001) with imatinib and by 87 ± 18 % (p = 0.002) with nilotinib. Furthermore, the collagen content in afflicted skin was reduced by 83 ± 15 % with imatinib and by 94 ± 14 % with nilotinib. Conclusions The combined inhibition of c-abl and PDGFR by imatinib or nilotinib was effective for the prevention of sclerodermatous chronic GvHD. The data support recent clinical observations of the potential effect of imatinib in fibrotic chronic graft-versus-host disease. Disclosures: Spriewald: Novartis: Research Funding. Off Label Use: Imatinib and Nilotinib as anti-fibrotic drug in the prevention and treatment of chronic graft-versus-host disease.. Distler:Novartis: Research Funding.

Description: Transfer of peripheral T cells into lymphopenic hosts results in a burst-like proliferation, called endogenous proliferation (EP) and an antigen-independent proliferation, called homeostatic proliferation (HP). Formerly both were named lymphopenic or homeostatic proliferation. T cells that undergo either endogenous or homeostatic proliferation acquire an activated phenotype, develop the ability to produce Interferon-gamma (IFN-g) and to lyse target cells specifically, but only homeostatic proliferating T cells acquire a CD44bright CD62Lbright phenotype. We developed a murine model to distinguish effects of both types of lymphopenia-associated proliferation. In this model we were able to induce polyclonal populations of syngenic CD4+ or CD8+ peripheral T cells (derived from C57BL/6 mice) to proliferate homeostatically (after transfer into lymphopenic RAG−/− mice) whereas only EP occurred when transferring into mice that inherit only an additional single CD8+ T cell clone (transfer into the non-lymphopenic P14/RAG−/− mice). CFSE dilution was observed from CD4+ or CD8+ T cells transferred into RAG−/− mice, whereas HP was inhibited when transferring the same cells into P14/RAG−/− mice, whereas EP occurred in both recipients. Independent of CFSE dilution T cells developed CD44bright phenotype after transfer into RAG−/− as well as in P14/RAG−/− mice as early as day 5, whereas the host P14tg CD8+ clone kept its naïve phenotype. Applying our model on the CD4+ T-cell-induced colitis model, we found HP to play a pivotal role in induction of colitis. Despite the notion of EP being triggered by antigens from the bowel, colitis was only induced when HP occurred, independent of the occurrence of EP. In detail CD4+ T cells induced colitis 10 weeks after transfer into RAG−/− mice (EP and HP). However neither RAG−/− mice transplanted with CD8+ T cells nor P14/RAG−/− mice transplanted with CD4+ T cells (only EP) developed any signs of colitis. In addition the treatment with or without antibiotics during these 10 weeks did not influence on the induction of colitis. We therefore conclude that the lymphopenia-mediated colitis after transfer of polyclonal CD4+ T cells correlates only with the occurrence of homeostatic proliferation. Furthermore the absence of regulatory CD4+CD25+ T cells within the suppressing P14 tg CD8+ clone indicates HP-induced colitis to be mediated by competition for cytokines or self-MHC stimulus. These findings might be of important relevance for research concerning chronic inflammatory bowel disease and donor lymphocyte induced colitis.

Description: Regulatory T lymphocytes play an important role in the maintenance of immune tolerance to self antigens and are involved in downregulating immune responses in autoimmunity, transplant rejection and tumor immunity. Numerous studies have demonstrated the existence of distinct T cell subsets with immunoregulatory properties. Recently, a novel subset of TCRαβ+ CD4− CD8− (double-negative, DN) T cells has been characterized to specifically suppress immune responses in both mice and humans. Here we demonstrate for the first time that human DN T cells are highly potent suppressor cells of allogeneic CD4+ or CD8+ T cell responses after priming with allogeneic antigen presenting cells (APC). A prerequisite for the immunosuppressive activity is the repetitive priming with allogeneic dendritic cells whereas stimulation with artificial APCs has no effect. Using a transwell system we could show that the suppressive activity against allogeneic immune responses, mediated by DN T cells, requires cell contact. In contrast to murine DN T cells, which eliminate effector T cells via a fas/fasL or perforin/granzyme pathway, human DN T cells suppress the proliferation of alloreactive T cells in an active manner. Taken together, our data indicate that human DN T cells possess strong immunosuppressive effects on alloreactive CD4+ or CD8+ T cells. DN T cells may serve to limit clonal expansion of alloantigen-specific T cells after allogeneic peripheral stem cell transplantation.

Description: Cancer-induced tolerance involves multiple immunosuppressive pathways, which subvert adaptive immune responses against tumor cells. Extensive phenotypic characterization of NK cells from tumor-bearing mice led us to the observation of a consistent expansion of c-kit-expressing NK cells (Kit+NK cells) compromising the NK cell arm of tumor immunosurveillance. In naïve animals, Kit+NK cells (CD3−NK1.1+CD117+) represent about 3.3±0.7% of blood, 4.8±1.4% of spleen and 13.2±2.4% of lymph node NK cells. In tumor-bearing animals, percentages and absolute numbers of Kit+NK cells increased to levels that mediate inhibitory effects on mature NK cells. Purified Kit+NK cells failed to produce IFNγ or GM-CSF in response to IL-2 and could not promote DC maturation in contrast to conventional Kit−NK cells. Moreover, adoptive transfer of Kit+ (but not Kit−) NK cells into mice injected with CT26 colon carcinomas or B16F10 melanomas promoted the establishment of lung metastases. Micro array comparison of CD3−NK1.1+Kit+ and CD3−NK1.1+Kit− cells revealed profound differences in their transcriptomes. Two major sets of genes involved in tolerance (B7-H1, CTLA4, Lag3, Hmox1) or tissue repair and bone marrow homeostasis (Cxcl2, CD81, CD63, Csf2) were markedly up-regulated in Kit+NK cells. Among these, B7-H1 appeared particularly intriguing as we found that Kit+NK cells killed Kit−NK cells in a B7-H1/PD-1-dependent manner. Moreover, IL-18 produced by CT26 or B16F10 tumors converts Kit− into Kit+NK cells endowed with immunoablative functions in lymph nodes. Upon tumor inoculation, Kit+ NK cells, which upregulate B7-H1, accumulate in lymphoid organs in an IL-18R/MyD88-dependent manner and directly kill conventional NK cells, thereby promoting the progression of NK-controlled cancers. Importantly, IL-18R−/− mice lost the development of Kit+NK cells during tumor progression. Neutralization of IL-18 by RNA interference in tumors or systemically by IL-18-binding protein (IL-18BP) reduced the expansion of Kit+NK cells, stimulated the NK cell-dependent immunosurveillance and significantly reduced tumor growth. The inflammatory cytokine IL-18 is known to accumulate in cancer-bearing patients but its pathophysiological role still remains unclear. Here, we show that IL-18 is a major tumor-derived immunosuppressant affecting the innate arm of tumor immunosurveillance. In summary, these data suggest that IL-18 promotes the expansion of Kit+NK cells endowed with immunosuppressive functions that could act as negative regulator of general NK responses.

Description: Several cell-based immunotherapy strategies have been developed to specifically modulate T cell–mediated immune responses. These methods frequently rely on the utilization of tolerogenic cell–based antigen-presenting cells (APCs). However, APCs are highly sensitive to cytotoxic T-cell responses, thus limiting their therapeutic capacity. Here, we describe a novel bead-based approach to modulate T-cell responses in an antigen-specific fashion. We have generated killer artificial APCs (κaAPCs) by coupling an apoptosis-inducing α-Fas (CD95) IgM mAb together with HLA-A2 Ig molecules onto beads. These κaAPCs deplete targeted antigen-specific T cells in a Fas/Fas ligand (FasL)–dependent fashion. T-cell depletion in cocultures is rapidly initiated (30 minutes), dependent on the amount of κaAPCs and independent of activation-induced cell death (AICD). κaAPCs represent a novel technology that can control T cell–mediated immune responses, and therefore has potential for use in treatment of autoimmune diseases and allograft rejection.

Description: In eukaryotic cells the phospholipid phosphatidylserine (PS) is restricted to the inner plasma-membrane leaflet. This lipid asymmetry, which is maintained by the concerted action of phospholipid transport proteins, is mainly lost during apoptosis. Here, we demonstrate that primary human CD8+ cytotoxic T lymphocytes (CTLs) expose PS on T-cell receptor (TCR)–mediated antigen (Ag) recognition. In contrast to PS externalization on apoptotic cells, activation-induced PS exposure is less pronounced and reversible. Fluorescence microscopic analysis revealed that PS is distributed nonhomogenously over the plasma membrane and concentrated in membrane lipid raft domains at the immunologic synapse. By studying the activity of PS transport proteins using a fluorescence-labeled PS analogue, we found that activation of CTLs inhibited the flippase-mediated inward-directed PS transport without affecting the outward transport. Shielding of exposed PS by annexin V protein during Ag recognition diminished cytokine secretion, activation, and cell-to-cell clustering of Ag-specific CTLs. In summary, our data demonstrate for the first time that externalized PS on Ag-stimulated CTLs is linked to T-cell activation and probably involved in cell-to-cell contact formation at the immunologic synapse.