Matthias Ballmaier

A New Population of Myeloid-Derived Suppressor Cells in Hepatocellular Carcinoma Patients Induces CD4+CD25+Foxp3+ T Cells

BACKGROUND & AIMSnSeveral studies have shown that development of hepatocellular carcinoma (HCC) generates a number of immune suppressive mechanisms in these patients. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that have been shown to inhibit T-cell responses in tumor-bearing mice, but little is known about these cells in humans owing to a lack of specific markers. In this study, we have investigated the frequency and function of a new population of MDSC denoted here as CD14(+)HLA-DR(-/low) in HCC patients. We have also identified a novel, MDSC-mediated immune regulatory pathway in these patients.nnnMETHODSnWe have directly isolated and characterized MDSCs for phenotype and function from peripheral blood (n = 111) and tumor (n = 12) of patients with HCC.nnnRESULTSnThe frequency of CD14(+)HLA-DR(-/low) cells in peripheral blood mononuclear cells (PBMC) from HCC patients was significantly increased in comparison with healthy controls. CD14(+) HLA-DR(-/low) cells were unable to stimulate an allogeneic T-cell response, suppressed autologous T-cell proliferation, and had high arginase activity, a hallmark characteristic of MDSC. Most important, CD14(+)HLA-DR(-/low) cells from HCC patients induced a CD4(+)CD25(+)Foxp3(+) regulatory T-cell population when cocultured with autologous T cells.nnnCONCLUSIONnCD14(+)HLA-DR(-/low) cells are a new population of MDSC increased in blood and tumor of HCC patients. We propose a new mechanism by which MDSC exert their immunosuppressive function, through the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells in cocultured CD4(+) T cells. Understanding the mechanism of action of MDSC in HCC patients is important in the design of effective immunotherapeutic protocols.

Thrombopoietin Is Essential for the Maintenance of Normal Hematopoiesis in Humans

Abstract: Recently, we and others could define the molecular cause of the rare disease congenital amegakaryocytic thrombocytopenia (CAMT) as mutations in the c‐mpl gene (Blood 97: 139, 2001). We proposed that c‐mpl mutations are the cause not only for the hypomegakaryocytic thrombocytopenia, but also for the development of an aplastic anemia (AA) in patients with CAMT. The effects of thrombopoietin (TPO) on early multipotent hematopoietic progenitors were elucidated by a recent series of in vitro and in vivo studies. Like CAMT patients, mice lacking the TPO receptor c‐Mpl demonstrate a major reduction of early hematopoietic progenitor cells of all lineages. However, these mice achieve a normal marrow cellularity and composition, despite the lack of megakaryocytes. On the other hand, the incidence of development of aplastic anemia in CAMT is not clear owing to difficult and not consistent diagnosis of this disease.

Grafting of thrombopoietin‐mimetic peptides into cystine knot miniproteins yields high‐affinity thrombopoietin antagonists and agonists

Thrombopoietin is the primary regulator of platelet production. We exploited two naturally occurring miniproteins of the inhibitor cystine knot family as stable and rigid scaffolds for the incorporation of peptide sequences that have been shown to act as high‐affinity thrombopoietin antagonists. Several miniproteins that antagonistically block thrombopoietin‐mediated receptor activation were identified using a microscale reporter assay. Covalent miniprotein dimerization yielded potent bivalent c‐Mpl receptor agonists with EC50 values in the low nanomolar or picomolar range. One selected miniprotein‐derived thrombopoietin agonist was almost as active as natural thrombopoietin with regard to stimulation of megakaryocyte colony formation from human bone marrow mononuclear cells, and elicited doubling of platelet counts in mice. Our data suggest that dimeric cystine knot miniproteins have considerable potential for the future development of small and stable receptor agonists. This approach may provide a promising strategy for pharmaceutical interference with other receptors activated by ligand‐induced dimerization.

Mutations in the gene encoding neutrophil elastase (ELA2) are not sufficient to cause the phenotype of congenital neutropenia

Mutations in the ELA2 gene encoding human neutrophil elastase have been reported recently to be involved in the aetiology of both, cyclic (CyN) and congenital neutropenia (CN). We analysed the correlation between the occurrence of ELA2 mutations and the neutropenic phenotype in a family with two children affected with CN. The two children harboured the same heterozygous mutation in the ELA2 gene that was inherited from their unaffected father. We conclude that ELA2 mutations are not the single cause of CN although they might be a necessary prerequisite for the expression of the neutropenic phenotype in a subgroup of CN patients.

Activated human hepatic stellate cells induce myeloid derived suppressor cells from peripheral blood monocytes in a CD44-dependent fashion

BACKGROUND & AIMSnMyeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells associated with the suppression of immunity. However, little is known about how or where MDSCs are induced and from which cells they originate. The liver is known for its immune regulatory functions. Here, we investigated the capacity of human hepatic stellate cells (HSCs) to transform peripheral blood monocytes into MDSCs.nnnMETHODSnWe cultured freshly isolated human monocytes from healthy donors on primary human HSCs or an HSC cell-line and characterized the phenotype and function of resulting CD14(+)HLA-DR(-/low) monocytes by flow cytometry, quantitative PCR, and functional assays. We analyzed the molecular mechanisms underlying the induction and function of the CD14(+)HLA-DR(-/low) cells by using blocking antibodies or knock-down technology.nnnRESULTSnMature peripheral blood monocytes co-cultured with HSCs downregulated HLA-DR and developed a phenotypic and functional profile similar to MDSCs. Only activated but not freshly isolated HSCs were capable of inducing CD14(+)HLA-DR(-/low) cells. Such CD14(+)HLA-DR(-/low) monocyte-derived MDSCs suppressed T-cell proliferation in an arginase-1 dependent fashion. HSC-induced development of CD14(+)HLA-DR(-/low) monocyte-derived MDSCs was not mediated by soluble factors, but required physical interaction and was abrogated by blocking CD44.nnnCONCLUSIONSnOur study shows that activated human HSCs convert mature peripheral blood monocytes into MDSCs. As HSCs are activated during chronic inflammation, the subsequent local induction of MDSCs may prevent ensuing excessive liver injury. HSC-induced MDSCs functionally and phenotypically resemble those isolated from liver cancer patients. Thus, our data suggest that local generation of MDSCs by liver-resident HSCs may contribute to immune suppression during inflammation and cancer in the liver.

HIV Infection Is Associated with a Preferential Decline in Less-Differentiated CD56dim CD16+ NK Cells

ABSTRACT HIV-1 infection is characterized by loss of CD56dim CD16+ NK cells and increased terminal differentiation on various lymphocyte subsets. We identified a decrease of CD57− and CD57dim cells but not of CD57bright cells on CD56dim CD16+ NK cells in chronic HIV infection. Increasing CD57 expression was strongly associated with increasing frequencies of killer immunoglobulin-like receptors (KIRs) and granzyme B-expressing cells but decreasing percentages of cells expressing CD27+, HLA-DR+, Ki-67+, and CD107a. Our data indicate that HIV leads to a decline of less-differentiated cells and suggest that CD57 is a useful marker for terminal differentiation on NK cells.

Implications of mutations in hematopoietic growth factor receptor genes in congenital cytopenias.

Abstract: Mutations in the genes of hematopoietic growth factor receptors as a cause of congenital cytopenia, such as congenital amegakaryocytic thrombocytopenia (CAMT) or severe congenital neutropenia (CN), are discussed. There are striking differences in the relevance of receptor mutations in these diseases. CAMT is a rare disease characterized by severe hypomegakaryocytic thrombocytopenia during the first years of life that develops into pancytopenia in later childhood. In patients with CAMT, we found inherited mutations in c‐mpl, the gene coding for the thrombopoietin receptor, in 8 out of 8 cases. The type of mutation seems to correlate with the clinical course seen in the patients. Functional studies demonstrated defective thrombopoietin (TPO) reactivity in hematopoietic progenitor cells and platelets in CAMT patients. CN is a group of hematopoietic disorders characterized by profound, absolute neutropenia due to a maturation arrest of myeloid progenitor cells. About 10% of all patients develop secondary MDS/leukemia. The malignant progression is associated with acquired nonsense mutations within the G‐CSF receptor gene that lead to the truncation of the carboxy‐terminal cytoplasmic domain of the receptor protein involved in maturation of myeloid progenitor cells. This seems to be one important step in leukemogenesis in CN patients. CAMT is caused by inherited mutations in c‐mpl, the gene for the thrombopoietin receptor, which lead to reduced or absent reactivity to TPO. In contrast, mutations in the G‐CSF receptor in CN are acquired and are most probably connected with progression of the neutropenia into MDS/leukemia as a result of a loss of differentiation signaling.

Phenotypically and functionally distinct subsets contribute to the expansion of CD56-/CD16+ natural killer cells in HIV infection.

Objective:Chronic HIV infection has been associated with activation and increased turnover of natural killer (NK) cells as well as with disturbed homeostasis of the NK cell compartment, including loss of CD56+ NK cells and accumulation of dysfunctional CD56−/CD16+ NK cells. We performed a comprehensive phenotypical and functional characterization of this population. Design:A cross-sectional study was performed to analyze CD56−/CD16+ NK cells from 34 untreated HIV-infected and 15 seronegative individuals. Methods:NK cells were analyzed by flow cytometry. Degranulation was assessed by measuring their expression of CD107a after stimulation with K562 cells, interleukin-12 and interleukin-15. Results:CD56−/CD16+ NK cells are heterogeneous and composed of two populations, namely CD122−/CCR7+ cells and CD122+/CCR7− cells. We show that expanded CD122+ but not CCR7+ cells in HIV-seropositive individuals are characterized by expression of senescence marker CD57 similarly to CD56dim/CD16+ NK cells along with expression of KIRs, CD8, perforin and granzyme B. Despite expression of perforin and granzyme B, CD57 expressing cells exhibited less numbers of degranulating cells as measured by CD107a, indicating their functional impairment. However, there was no correlation between expansion of total CD56−/CD16+ NK cells or the distinct subpopulations and viral load or CD4 cell count. Conclusion:These data indicate that expansion of CD56−/CD16+ cells in HIV infection is driven by a distinct subset within this population with high expression of terminal differentiation marker with a phenotype resembling CD56dim/CD16+ NK cells.

The Wilms’ tumor suppressor Wt1 activates transcription of the erythropoietin receptor in hematopoietic progenitor cells

The Wilms tumor protein Wt1 is required for embryonic development and has been implicated in hematologic disorders. Since Wt1 deficiency may compromise the proliferation and differentiation of erythroid progenitor cells, we analyzed the possible role of the transcriptionally active Wt1 isoform, Wt1(—KTS), in regulating the expression of the erythropoietin receptor (EpoR). Wt1 and EpoR were coexpressed in CD117+ hematopoietic progenitor cells and in several hematopoietic cell lines. CD117+ cells of Wtl‐deficient murine embryos (Wt1−/−) exhibited a significantly lower proliferation response to recombinant erythropoietin than CD117+ cells of heterozygous (Wt1+/−) and wild‐type littermates (Wt1+/−). EpoR expression was significantly diminished in hematopoietic progenitors (CD117+) that lacked Wt1, and the erythroid colony‐forming capacity was reduced by more than 50% in fetal liver cells of Wt1‐deficient embryonic mice. Wt1(—KTS) significantly increased endogenous EpoR transcripts in transfected cells. The proximal EpoR promoter of human and mouse was stimulated more than 10‐fold by Wt1(—KTS) in transiently cotransfeced K562 erythroleukemia cells. A responsible cis‐element, which is highly conserved in the EpoR promoter of human and mouse, was identified by mutation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. In conclusion, activation of the EpoR gene by Wt1 may represent an important mechanism in normal hematopoiesis.—Kirschner, K. M., Hagen, P., Hussels, C. S., Ballmaier, M., Scholz, H., Dame, C. The Wilms tumor suppressor Wt1 activates transcription of the erythropoietin receptor in hematopoietic progenitor cells. FASEB J. 22, 2690–2701 (2008)

Exogenous HIV-1 Vpr disrupts IFN-α response by plasmacytoid dendritic cells (pDCs) and subsequent pDC/NK interplay

HIV Vpr is known for its immunomodulatory capacities including its impairment of NK cell functions. However, the role of pDCs in this context remains elusive. We show that synthetic Vpr substantially inhibits type I IFN production by pDCs without inducing apoptosis in pDCs. Furthermore, we found that exogenous Vpr compromises subsequent pDC/NK interplay as shown by diminished IFN-gamma production by NK cells. Thus, Vpr-mediated dysregulation of IFN-alpha and IFN-gamma production affects key components of the innate immune response supporting an essential role of Vpr in HIV pathogenesis.