Human basophils are a source of - and are differentially activated by - IL-31.

BACKGROUND: Basophils are important effector cells involved in the pathogenesis of inflammatory skin diseases including chronic urticaria which is associated by increased IL-31 serum levels. So far the effects of IL-31 on human basophils are unknown. OBJECTIVE: To analyse the functional role of IL-31 in basophil biology. METHODS: IL-31 expression was evaluated in skin samples derived from chronic spontaneous urticaria patients. Oncostatin M receptor (OSMR), IL-31 receptor A (RA) and IL-31 protein expressions were analysed on human basophils from healthy donors. Basophil responses to IL-31 were assessed for chemotaxis, externalization of CD63 and CD203c as well as the release of histamine, IL-4 and IL-13. RESULTS: IL-31RA and OSMR were expressed on human basophils. IL-31 was strongly expressed in the skin of patients with chronic spontaneous urticaria and was released from isolated basophils following either anti-IgE, IL-3 or fMLP stimulation. IL-31 induced chemotaxis and the release of IL-4 and IL-13 which was specifically inhibited by anti-IL-31RA and anti-OSMR. Conversely, IL-31 had no effect on CD63 and CD203c externalization or histamine release. CONCLUSIONS AND CLINICAL RELEVANCE: Human basophils are a source of -and are activated by - IL-31 with the release of pro-inflammatory cytokines and the induction of chemotaxis indicating an important novel function of IL-31 in basophil biology.

Monitoring of adenovirus (ADV)-specific T cells in a boy with ADV pneumonia and disseminated disease after lung transplantation.

Human adenovirus (ADV) infections are the cause of severe morbidity and mortality in transplant recipients. Cidofovir (CDV) is the current standard antiviral treatment. We report the case of a 3-year-old boy after lung transplantation with severe ADV sepsis, who was monitored for ADV-specific T cells during his disease and recovery. A strong increase in ADV-specific T cells was accompanied by resolution of ADV in blood and bronchoalveolar lavage fluid. Antiviral treatment with CDV was individually adapted according to anti-ADV immune responses, which provides a new method for tailoring antiviral treatment in lung transplant recipients.

Regulation of melanocortin 1 receptor in allergic rhinitis in vitro and in vivo.

BACKGROUND: α-melanocyte-stimulating hormone (α-MSH) was shown to inhibit allergic airway inflammation and exert suppressive effects on human basophils. OBJECTIVE: This study aims to extend our current knowledge on the melanocortin 1 receptor (MC1R) expression in nasal tissue of patients with allergic rhinitis (AR) and functional effects of α-MSH in human basophils especially from patients with allergic rhinitis. METHODS: MC1R expression before and after nasal allergen provocation was studied in nasal mucosal tissue of AR patients and in a mouse model of allergic airway inflammation using immunofluorescence. In vitro regulation of the MC1R and CD203c surface expression on whole-blood basophils of patients with AR and controls was assessed with flow cytometry. Functional effects of α-MSH on isolated basophils were analysed regarding apoptosis with flow cytometry and chemotaxis using a Boyden chamber assay. RESULTS: We detected an accumulation of MC1R-positive basophils in nasal mucosa tissue of patients with AR 24 h after nasal allergen provocation. Such accumulation was not present in mucosa sections from healthy controls. In mice with allergic airway inflammation, we found a clear accumulation of MC1R-positive basophils in the nasal tissue compared to control mice. MC1R expression was inducible in AR patients and controls by stimulation with anti-IgE. α-MSH inhibited anti-IgE and grass pollen induced upregulation of CD203c, but had no effect on chemotaxis or apoptosis of basophils in vitro. CONCLUSIONS AND CLINICAL RELEVANCE: MC1R-positive basophils accumulate in the nasal mucosa of patients with AR after nasal allergen provocation. Since α-MSH suppresses proinflammatory effector functions in human basophils via the MC1R, it constitutes an interesting novel target for modulating the allergic inflammatory response.

Human basophil chemotaxis and activation are regulated via the histamine H4 receptor.

BACKGROUND: IgE-mediated cross-linking of FcεRI results in the release of mediators stored in basophil granules, such as histamine and proteases, and in the de novo synthesis of sulfidoleukotrienes. OBJECTIVE: In this study, we investigated the role of the histamine receptors, in particular that of the histamine H4 receptor (H4R), in modulating human basophil function. METHODS: The mRNA expression of the histamine receptors was measured by real-time PCR. Migration of basophils was assessed using the modified Boyden chamber technique. The expression levels of CD63 and CD203c on the cell surface and the sulfidoleukotriene release were determined by flow cytometry and ELISA, respectively. RESULTS: We could show that highly purified basophils express the H1R, H2R, and H4R but not the H3R mRNA. Human basophils expressed higher H4R mRNA levels as compared to the expression levels of the H1R (P < 0.01). Histamine and the H4R agonist ST-1006 initiated active migration of basophils (P < 0.001). A significant reduction in FcεRI cross-linking-mediated surface expression of CD63 and CD203c was observed on basophils after pre-incubation with histamine or the specific H4R agonist ST-1006 (P < 0.01). The synthesis and release of sulfidoleukotrienes from basophils after activation with different stimuli, by FcεRI cross-linking or by stimulation with hymenoptera venom allergens, were significantly reduced by histamine or the H4R agonist ST-1006 (P < 0.05-0.001). CONCLUSION: These data imply that the H4R regulates IgE-dependent processes in human basophils and provides a novel function of the H4R preventing an overwhelming immune reaction by engagement of a negative feedback loop.

Granulocyte colony-stimulating factor impairs CD8(+) T cell functionality by interfering with central activation elements.

Besides mobilizing stem cells into the periphery, granulocyte colony-stimulating factor (G-CSF) has been shown to influence various types of innate and adaptive immune cells. For example, it impairs the effector function of cytotoxic T lymphocytes (CTLs). It is assumed that this effect is mediated indirectly by monocytes, regulatory T cells and immunomodulatory cytokines influenced by G-CSF. In this study, isolated G-CSF-treated CD8(+) T cells were stimulated antigen-dependently with peptide-major histocompatibility complex (pMHC)-coupled artificial antigen-presenting cells (aAPCs) or stimulated antigen-independently with anti-CD3/CD28 stimulator beads. By measuring the changes in interferon (IFN)-γ and granzyme B expression at the mRNA and protein level, we showed for the first time that G-CSF has a direct effect on CD8(+) CTLs, which was confirmed based on the reduced production of IFN-γ and granzyme B by the cytotoxic T cell line TALL-104 after G-CSF treatment. By investigating further elements affected by G-CSF in CTLs from stem cell donors and untreated controls, we found a decreased phosphorylation of extracellular-regulated kinase (ERK)1/2, lymphocyte-specific protein tyrosine kinase (Lck) and CD3ζ after G-CSF treatment. Additionally, miRNA-155 and activation marker expression levels were reduced. In summary, our results show that G-CSF directly influences the effector function of cytotoxic CD8(+) T cells and affects various elements of T cell activation.

Modulation of heme oxygenase-1 by metalloporphyrins increases anti-viral T cell responses.

Heme oxygenase (HO)-1, the inducible isoform of HO, has immunomodulatory functions and is considered a target for therapeutic interventions. In the present study, we investigated whether modulation of HO-1 might have regulatory effects on in-vitro T cell activation. The study examined whether: (i) HO-1 induction by cobalt-protoporphyrin (CoPP) or inhibition by tin-mesoporphyrin (SnMP) can affect expansion and function of virus-specific T cells, (ii) HO-1 modulation might have a functional effect on other cell populations mediating effects on proliferating T cells [e.g. dendritic cells (DCs), regulatory T cells (T(regs)) and natural killer cells] and (iii) HO-1-modulated anti-viral T cells might be suitable for adoptive immunotherapy. Inhibition of HO-1 via SnMP in cytomegalovirus (CMV)pp65-peptide-pulsed peripheral blood mononuclear cells (PBMCs) led to increased anti-viral T cell activation and the generation of a higher proportion of effector memory T cells (CD45RA(-) CD62L(-)) with increased capability to secrete interferon (IFN)-γ and granzyme B. T(reg) depletion and SnMP exposure increased the number of anti-viral T cells 15-fold. To test the possibility that HO-1 modulation might be clinically applicable in conformity with good manufacturing practice (GMP), SnMP was tested in isolated anti-viral T cells using the cytokine secretion assay. Compared to control, SnMP treatment resulted in higher cell counts and purity without negative impact on quality and effector function [CD107a, IFN-γ and tumour necrosis factor (TNF)-α levels were stable]. These results suggest an important role of HO-1 in the modulation of adaptive immune responses. HO-1 inhibition resulted in markedly more effective generation of functionally active T cells suitable for adoptive T cell therapy.

Allogeneic and xenogeneic anti-tumor effect of callithrix jacchus natural killer cells is dependent on NKp30 and B7-H6 interaction.

Natural Killer (NK) cells mount a fast and efficient immune response against tumor cells and are currently a major focus in the development of anti-cancer cell-based therapies. Due to major differences between the murine and human NK cell receptor system, a non-human primate model would be helpful to evaluate the efficiency of NK-cell based therapies prior to clinical applications. In humans, B7-H6 has been shown to facilitate the elimination of lymphoma cells through the interaction with its receptor NKp30. The common marmoset (Callithrix jacchus) is a new world monkey readily used in biomedical research due to its easy management and proximity to humans. In this study, we demonstrated the expression of B7-H6 antigen in marmoset B-lymphoblastoid cell lines. In addition, a method was established to isolate B- or NK-cells from peripheral blood of marmosets with purities of up to 97%We detected the expression of B7-H6 in lymphoma cells and for the first time in leukemic blasts of human acute myeloid leukemia (AML). Marmoset NK cells were shown to lyse marmoset B lymphoblastoid cell line (B-LCL) cells by up to 28.4% and human B-LCL cells by up to 20%. This effect was abrogated when the NK cells were pre-treated with an anti-NKp30 specific antibody. Also, marmoset NK cells were able to lyse primary leukemic AML cells and lymphoma cells by up to 8.3 and 20.3%respectively. Stimulation of marmoset NK cells with recombinant B7-H6 induced phosphorylation of ERK1/2 and proliferation rates. Furthermore, the secretion of IL-1ß, IL-8, IFN-γ and TNF-α was significantly increased upon B7-H6 stimulation. In conclusion, we demonstrated that non-human primate NK cells have similar mechanisms for the lysis of tumor cells as human NK cells. Thus, this animal model constitutes a very promising tool for the development and evaluation of novel NK-cell based therapies.

The new HLA-C variant HLA-C*05:26 is likely to be structurally identical to the C*05:01P alleles.

The novel allele HLA-C*05:26 differs from HLA-C*05:01 by the non-synonymous amino acid exchange Gly16Ser.

The new HLA-B*58:21 allele is predicted to be functionally similar to the B*58:01P group of alleles.

The new human leukocyte antigen (HLA)-B*58:21 allele differs from B*58:01:01 by an amino acid exchange at codon 90.

Closing the gap: discrimination of the expression profile of HLA questionable alleles by a cytokine-induced secretion approach using HLA-A*32:11Q.

Matching of human leukocyte antigen (HLA) alleles between donors and recipients plays a major role in hematopoietic stem cell transplantation (HSCT). Null or questionably expressed HLA allelic variants are a major issue in HLA matching, because the aberrant expression of such alleles can have a major impact on the outcome of HSCT and/or its complications such as graft-versus-host disease. The goal of this study was to investigate the potential of a recently developed cytokine-induced secretion assay to differentiate the expression levels of HLA-A*32:11Q (questionable) into a null (N) or low (L) expression variant. An amino acid mutation at position 164 of HLA-A*32:11Q disrupts the disulfide bridge in the α2 domain. HLA-A*32:11Q is not detectable by standard microlymphocytotoxicity assay. To this end, we cloned soluble HLA-A*32:11Q and a reference allele (HLA-A*32:01) into expression vectors and transfected/transduced HEK293 and K562 cells. Allele-expressing K562 cells were simultaneously transfected/transduced with a ß2-microglobulin (B2M)-encoding vector to ensure the intact HLA structure with B2M. After treatment with proinflammatory cytokines, secreted soluble HLA molecules were determined by enzyme-linked immunosorbent assay in the supernatant and intracellular accumulation of the recombinant proteins by flow cytometry. HLA-A*32:11Q was nearly undetectable in untreated transfectants. Cytokine treatment increased the secretion of HLA-A*32:11Q to detectable levels and resulted in intracellular accumulation of the allele. There was no difference in mRNA transcription between the A*32 alleles. On the basis of these results, we recommend reclassification of HLA-A*32:11Q as a low expression (L) variant.

Semaphorin 7A inhibits platelet production from CD34+ progenitor cells.

BACKGROUND: The multifunctional protein semaphorin 7A (Sema7A) may have regulatory effects on blood cell differentiation via its receptors ß1-integrin and plexin C1. As thrombocytopenia can be treated with transfusion of ex vivo CD34(+) cell-derived megakaryocytes, we investigated the effect of Sema7A on differentiation of CD34(+) progenitor cells into megakaryocytes and platelets. METHODS: Megakaryocytes and platelets were differentiated with a specific cytokine cocktail (CC) from CD34(+) progenitor cells in the presence or absence of Sema7A. Expression of cell markers CD41, CD42a and CD61 or detection of the activation of the signal mediator focal adhesion kinase (FAK) was performed by flow cytometry, cytokine secretion by Luminex technology, and megakaryocyte cell density and morphology by microscopic studies. Sema7A levels in vivo were assessed by real-time PCR and ELISA in hematological patients undergoing chemotherapy. RESULTS: CD34(+) progenitor cells expressed the receptors for Sema7A. Expression of CD41, CD42a and CD61 was markedly reduced in the presence of Sema7A, after CC-dependent platelet production from CD34(+) progenitor cells. As revealed by microscopic analysis, megakaryocyte cell density was significantly lower in the presence of Sema7A as compared with controls. Blocking of CD29 abrogated the Sema7A-mediated inhibition. Sema7A activated FAK in CD34(+) progenitor cells and significantly increased secretion of the proinflammatory cytokines IL-6, IL-8 and GM-CSF. Finally, Sema7A levels were up-regulated in 50% of patients after chemotherapy. CONCLUSIONS: Sema7A markedly reduces the production rates of megakaryocytes and platelets from CD34(+) progenitor cells. Hence, up-regulation of Sema7A may be a major risk factor for a reduced platelet repopulation after hematopoietic stem cell transplantation.

The new HLA allele, HLA-A*03:57, differs from HLA-A*03:01 by two amino acids at positions 76 and 77 in the α2 domain affecting the pocket F of the peptide-binding groove.

HLA-A*03:57 has two amino acid exchanges in exon 2 at adjacent positions of the peptide-binding groove.

HLA-B*08:01:08- joining the fold of silent alpha-1 proline mutations in HLA-B.

The sequence of HLA-B*08:01:08 differs from other HLA-B*08:01 alleles by at least two synonymous nucleotide exchanges.

The novel allele HLA-B*35:167 differs from HLA-B*35:03:01 by the amino acid exchange Val152Glu.

HLA-B*35:167 allele differs from HLA-B*35:03:01 and HLA-B*35:70 by an amino acid exchange at position 152.

The composition of the F pocket in HLA-A*74 generates C-terminal promiscuity among its bound peptides.

In this study we sequenced the bound peptides from three alleles belonging to the HLA-A*74 group (HLA-A*74:04, A*74:06 and A*74:07) that are distinguished by four polymorphic residues within the peptide-binding region. Our data illustrates that A*74:04 exhibits preference for L, M or I at P2 and L, S or P at PΩ, while for A*74:07 the P2 anchor prefers L, P or I and the PΩ anchor S, P, L. In contrast A*74:06 features a P2 anchor motif of S or L, while a PΩ anchor could not be defined; however, a preference for polar residues S, T, Q or the charged residue R at the PΩ position could be detected.

Modulatory role of calreticulin as chaperokine for dendritic cell-based immunotherapy.

Heat shock proteins (HSPs) play a regulatory role for maturation of antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages. Whereas HSP70 has been shown to enhance the maturation of human DCs via a nuclear factor kappa-B (NF-κB)-dependent pathway, the regulatory role of calreticulin (CRT), which is a HSP with similar functions to HSP70, is not well studied. To investigate the role of CRT as adjuvant in cell activation and co-stimulatory responses we determined the effects of CRT on human APC maturation in comparison to that of HSP70. To facilitate eukaryotic endotoxin-free CRT protein expression, three different methods were compared. We demonstrate that CRT induces the maturation of human DCs and increases the production of proinflammatory cytokines via the NF-κB pathway. CRT-mediated maturation was qualitatively similar to that induced by HSP70. Interestingly, priming of monocytes with HSPs showed an even more prominent effect on maturation than exposure of immature DCs to these compounds. A higher expression of CD86, CD83 and CCR7 on mature DCs were found in response to CRT. Our data provide novel insights into the role of extracellular HSPs as chaperokines in the processes of APC generation and may thus be useful to improve adoptive immunotherapy.

Regulation of HLA class II expression prevents allogeneic T-cell responses.

Human leukocyte antigen (HLA) class II molecules are polymorphic heterodimers that present peptides to CD4+ T-cells. The HLA-DM molecule contributes to assemble HLA class II-peptide complexes. We investigated the effect of silencing either HLA-DR or HLA-DM expression in the allogeneic T-cell responses. The delivery of HLA-DR- or HLA-DM-specific short hairpin RNAs (shRNAs) in a monocytic cell line caused a decrease by up to 85% and 75% at the respective mRNA level. Allogeneic T-cells stimulated with HLA-DM-silenced monocytes decreased to 30% granzyme B mRNA and interferon gamma (IFN-γ) production in comparison with T-cells stimulated with monocytes expressing a non-specific shRNA. By contrast, HLA-DR-silenced monocytes did not induce proliferation, up-regulation of granzyme B mRNA levels or high IFN-γ secretion by allogeneic T-cells vs HLA-DR expressing cells. Direct targeting of HLA-DR expression prevented more efficiently an allogeneic T-cell response in comparison with the knockdown of the expression of HLA-DM molecules. Silencing the expression of HLA-DR molecules might contribute to the development of new allogeneic cell-based therapeutic approaches.

The amino acid variation of HLA-A*02:182 in a highly conserved region is predicted to be functionally similar to HLA-A*02:01:01:01 allele.

We here describe the identification of the novel human leukocyte antigen allele HLA-A*02:182 which has been detected in a potential bone marrow donor. The new allele differs from the sequence of HLA-A*02:01:01:01 only by a non-synonymous nucleotide exchange of Guanin (G) → Cytosin (C) at position 199 in exon 3 replacing amino acid (AA) Arginine (Arg, R) by Threonine (Thr, T) in codon 157. Since the HLA-A*02:01:01:01 allele differs from A*02:182 only at AA position 157, it is assumed that the protein structures of these alleles are highly similar. A mismatch between HLA-A*02:01:01:01 and HLA-A*02:182 is predicted to have a very low allogeneic potential in hematopoietic stem cell transplantation.

Polymorphism between HLA-A*0301 and A*0302 located outside the pocket F alters the PΩ peptide motif.

The human leukocyte antigen (HLA)-A*03 group has more than 90 known members and is one of the largest families of HLA class I alleles, with the most common variant being HLA-A*0301. In this study, we determined the peptide-binding motif of the highly frequent Sudanese allele A*0302 and compared it with the previously published peptide-binding motif of A*0301. The two alleles differ only at two distinct residues Glu152Val and Leu156Gln, which are predicted to be part of specificity pockets D, C and E and thus in contact with the peptide. Soluble recombinant A*0302 was expressed, affinity purified and the bound peptides were then eluted and analysed by mass spectrometry. The peptide-binding motif of A*0302 differs significantly from the previously published HLA-A*0301 and the Glu152Val/Leu156Gln mismatches appear to have a significant impact on the peptide-binding features of A*0302 and A*0301.