Comprehensive flow cytometric reference intervals of leukocyte subsets from six study centers across Europe.

A group of European FOCIS Centers of Excellence adapted panels of the Human Immunophenotyping Consortium (HIPC) for whole blood analysis. Using four core panels [T/regulatory T cell/B/natural killer (T/Treg /B/NK) and myeloid cells] the main leukocyte populations were analyzed in a clinical-diagnostic setting in a harmonized manner across different platforms. As a first step, the consortium presents here the absolute and relative frequencies of the leukocyte subpopulations in the peripheral blood of more than 300 healthy volunteers across six different European centers.

Blood immune cell biomarkers in lung cancer.

Characterization of host immune cell parameters prior to treatment is expected to identify biomarkers predictive of clinical outcome as well as to elucidate why some patients fail to respond to immunotherapy. We monitored blood immune cells from 58 patients with non-small- cell lung cancer (NSCLC) undergoing surgery of the primary tumor and from 50 age-matched healthy volunteers. Complete leukocyte blood count, the number of circulating dendritic cells (DC), HLA-DRlow monocytes and several lymphocytic subpopulations were determined by eight-color flow cytometry. Furthermore, the prognostic value of the immune cell parameters investigated was evaluated by patients' survival analysis. Compared to the control group, blood of NSCLC patients contained more neutrophils resulting in a higher neutrophil-to-lymphocyte ratio (NLR), but a lower number of blood DC, in particular of plasmacytoid DC (pDC), natural killer (NK) cells and naive CD4+ and CD8+ T cells. Furthermore, a higher frequency of CD4+ regulatory T cells (Treg) and HLA-DRlow monocytes was detected, and smoking had a significant impact on these values. HLA-DRlow monocytes were positively correlated to the number of neutrophils, monocytes and NLR, but negatively associated with the number of pDC and naive CD4+ T cells. The frequency of Treg, HLA-DRlow monocytes and naive CD4+ and CD8+ T cells as well as the ratios of CD4/HLA-DRlow monocytes and HLA-DRlow monocytes/pDC correlated with patient's overall survival. Next to Treg, HLA-DRlow monocytes and naive T cells represent prognostic markers for NSCLC patients and might be useful for monitoring of patients' responses to immunotherapies in future studies.

Molecular mechanisms of HLA class I-mediated immune evasion of human tumors and their role in resistance to immunotherapies.

Although the human immune system can recognize and eradicate tumor cells, tumors have also been shown to develop different strategies to escape immune surveillance, which has been described for the first time in different mouse models. The evasion of immune recognition was often associated with a poor prognosis and reduced survival of patients. During the last years the molecular mechanisms, which protect tumor cells from this immune attack, have been identified and appear to be more complex than initially expected. However, next to the composition of cellular, soluble and physical components of the tumor microenvironment, the tumor cells changes to limit immune responses. Of particular importance are classical and non-classical human leukocyte antigen (HLA) class I antigens, which often showed a deregulated expression in cancers of distinct origin. Furthermore, HLA class I abnormalities were linked to defects in the interferon signaling, which have both been shown to be essential for mounting immune responses and are involved in resistances to T cell-based immunotherapies. Therefore this review summarizes the expression, regulation, function and clinical relevance of HLA class I antigens in association with the interferon signal transduction pathway and its role in adaptive resistances to immunotherapies.

c-Myc and EBV-LMP1: two opposing regulators of the HLA class I antigen presentation machinery in epithelial cells.

BACKGROUND: Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) up-regulates the human leukocyte antigen (HLA) class I antigen presentation machinery (APM). This appears counterintuitive with immune evasion in EBV-associated tumours like nasopharyngeal carcinoma (NPC). METHODS: Latent membrane protein 1-transfected epithelial cell lines were used as a model system to study the impact of LMP1 and c-Myc on HLA class I components. The expression of components of the HLA class I APM, c-Myc and Ki-67 was analysed in LMP1+ and LMP1- NPC by immunohistochemistry. RESULTS: In epithelial cells, LMP1 up-regulated HLA class I APM. This effect could be counteracted by c-Myc, which itself was up-regulated by LMP1 apparently through IL6 induction and Jak3/STAT3 activation. Studies of NPC biopsies revealed down-regulation of HLA class I APM expression. No difference was observed between LMP1+ and LMP1- NPC. However, expression of Ki-67 and c-Myc were up-regulated in LMP1+ tumours. CONCLUSION: These findings raise the possibility that c-Myc activation in NPC might antagonise the effect of LMP1 on HLA class I expression thus contributing to immune escape of tumour cells.

Expression, regulation and function of the ISGylation system in prostate cancer.

The androgen receptor (AR) plays a crucial role in the modulation of prostate cell proliferation and is involved in the development and progression of prostate cancer (PCa). An understanding of the complex regulation of AR provides novel treatment options for PCa. Here, we show (i) that the ubiquitin-like modifier, interferon-stimulated gene 15 (ISG15), and most enzymes involved in ISG15 conjugation were upregulated in tumor samples versus in non-malignant tissues of PCa patients and (ii) that the expression of these components significantly differed between tumors in patients treated with and without androgen ablation. Using PCa cell lines as in vitro models, the specific androgen-mediated, AR-dependent regulation of the ISGylation components was confirmed. In addition, the ISGylation system controls AR mRNA and protein expressions, as overexpression of Ube1L as a limiting ISGylation factor in the AR(+) androgen-sensitive PCa cell line, LNCaP, results in significant AR upregulation, accompanied by an increased proliferation even under androgen deprivation. Accordingly, Ube1L knockdown decreased the AR expression. Thus, this study describes for the first time the modulation of AR expression by ISGylation components, which affects the proliferation of PCa cells, thereby providing evidence for a novel function of the ISGylation system in malignant transformation.

Overexpressed vs mutated Kras in murine fibroblasts: a molecular phenotyping study.

Ras acts in signalling pathways regulating the activity of multiple cellular functions including cell proliferation, differentiation, and apoptosis. Amino-acid exchanges at position 12, 13, or 61 of the Kras gene convert the proto-oncogene into an activated oncogene. Until now, a direct comparison of genome-wide expression profiling studies of Kras overexpression and different Kras mutant forms in a single assay system has not been carried out. In our study, we focused on the direct comparison of global gene expression effects caused by mutations in codon 12 or 13 of the Kras gene and Kras overexpression in murine fibroblasts. We determined Kras cellular mRNA, Ras protein and activated Ras protein levels. Further, we compared our data to the proteome analysis of the same transfected cell lines. Both overexpression and mutations of Kras lead to common altered gene expression patterns. Only two genes, Lox and Col1a1, were reversely regulated in the Kras transfectants. They may contribute to the higher aggressiveness of the Kras codon 12 mutation in tumour progression. The functional annotation of differentially expressed genes revealed a high frequency of proteins involved in tumour growth and angiogenesis. These data further support the important role of these genes in tumour-associated angiogenesis.

Expression and regulation of non-classical HLA-G in renal cell carcinoma.

Under physiological conditions, the non-classical major histocompatibility complex class Ib molecule human leukocyte antigen G (HLA-G) is selectively expressed in placental trophoblasts, thymus and cornea. In pathological situations, HLA-G expression was frequently found in tumour cells of distinct origin, thereby allowing these tumour cells to escape immune surveillance. Although HLA-G expression occurs at a relatively high frequency in renal cell carcinoma (RCC) of the clear cell subtype, the molecular mechanisms of its aberrant expression in RCC has not yet been determined. Therefore, the constitutive and cytokine-mediated HLA-G expression as well as its mode of regulation was investigated. In addition to HLA-G-specific mRNA expression, membrane-bound and soluble/shed HLA-G protein was determined. Eight of 14 RCC cell lines analysed (57%) exhibited HLA-G-specific transcripts, whereas only 6 of 14 RCC cell lines (43%) expressed HLA-G protein, suggesting a post-transcriptional control of HLA-G in some cases. Treatment of RCC cell lines with either interferon-gamma or interleukin-10, respectively, increased HLA-G-specific mRNA and protein in six of eight HLA-G(+) RCC lines (75%), but not in HLA-G(-) RCC cells. A 5'-aza-2-deoxycytidine (5-Aza-dC)-mediated demethylation of the HLA-G promoter DNA resulted in an enhanced HLA-G expression in four of six RCC cell lines, whereas a de novo induction of HLA-G was only observed in one HLA-G(-) RCC cell line on treatment with 5-Aza-dC. Thus, there exist multiple mechanisms controlling HLA-G expression in RCC, which might also have an impact on the development of RCC-specific immunotherapies.

Loss of interferon-gamma inducibility of the MHC class II antigen processing pathway in head and neck cancer: evidence for post-transcriptional as well as epigenetic regulation.

BACKGROUND: Abnormalities of the major histocompatibility complex (MHC) antigens by tumour cells impair the cellular immune response and promote tumour evasion from immune surveillance. So far, studies analysing the MHC class II expression levels in head and neck cancer have been limited. OBJECTIVES: Therefore, we investigated the constitutive and interferon (IFN)-gamma-regulated expression profiles of MHC class II antigen processing machinery (APM) in various head and neck cancer cell lines and also analysed the MHC class II expression in head and neck cancer lesions. METHODS: Using immunohistochemistry, flow cytometry, and reverse transcriptase-polymerase chain reaction analyses we investigated the expression pattern of various components of the MHC class II APM in biopsies and cell lines from head and neck cancers. Furthermore, we analysed the class II transactivator (CIITA) and HLA-DR promoter activity in head and neck cancer cells by transient transfection of specific luciferase promoter constructs and finally studied the methylation pattern of the CIITA promoter using methylation sensitive restriction enzymes. RESULTS: Head and neck cancer cell lines analysed in vitro lacked constitutive MHC class II surface expression. Despite the IFN-gamma-mediated induction at the mRNA level, six out of 10 cell lines did not show any relevant MHC class II surface expression. This phenomenon might be attributed to a post-transcriptional dysregulation of specific MHC class II APM components. One cell line displayed a loss of IFN-gamma-induced CIITA-expression that corresponded to impaired MHC class II surface expression, which could be linked to hypermethylation of the IFN-gamma-responsive CIITA-promoter IV. In vivo, immunohistochemistry analyses of 35 patients revealed that about 86% of head and neck cancer tissues exhibit a negative or only marginally positive staining, whereas 14% displayed a heterogeneous or highly positive MHC class II surface expression. There was no statistical correlation between tumour differentiation and the MHC class II expression in head and neck cancer lesions. CONCLUSIONS: Taken together these results suggest a high frequency of MHC class II abnormalities in head and neck cancer in vitro and in vivo, which could occur at different steps of the antigen processing pathway. This information may have a significant impact on practical and clinical aspects of tumour immunotherapeutic strategies.

A novel HLA-B*57 (B*5714) allele in a healthy male Caucasian individual.

A novel human leucocyte antigen (HLA)-B57 (HLA-B*5714) allele has been identified in a male Caucasian individual from Middle Europe using single allele-specific sequencing strategy. This allele is identical to the HLA-B*570101 allele except for two point mutations in exon 3 at codon 138 (ACG-->ACC) with no amino acid change [persisting threonine (T)] and at codon 171 (TAC-->CAC), resulting in an amino acid change from tyrosine (Y) to histidine (H).

Soluble CD30 serum level--an adequate marker for allograft rejection of solid organs?

The CD30 molecule, a 120 kDa cell surface glycoprotein, is a member of the tumor necrosis factor receptor (TNF-R) superfamily and was originally identified on the surface of Reed-Sternberg cells and anaplastic large cell lymphomas in Hodgkin's disease patients. In addition to lymphoproliferative disorders the expression of CD30 was found in both activated CD8+ and CD4+ Th2 cells which lead to the activation of B-cells and consequently to the inhibition of the Th1-type cellular immunity. The membrane-bound CD30 molecule can be proteolytically cleaved, thereby generating a soluble form (sCD30) of about 85 kDa. Low serum levels of soluble CD30 were found in healthy humans, whereas increased sCD30 serum concentrations were detected under pathophysiological situations such as systemic lupus erythematosus, rheumatoid arthritis, certain viral infections and adult T cell leukaemia/lymphoma. In addition, it has recently been suggested that pre- or post-transplant levels of sCD30 represent a biomarker for graft rejection associated with an impaired outcome for transplanted patients. We here review (i) the current knowledge of the clinical significance of sCD30 serum levels for solid organ transplantations and (ii) our own novel data regarding inter- and intra-individual variations as well as time-dependent alterations of sCD30 levels in patients. (iii) Based on this information the implementation of sCD30 as predictive pre-transplant or post-transplant parameter for solid organ transplantation is critically discussed.

Analysis of HLA class I-II haplotype frequency and segregation in a cohort of patients with advanced stage ovarian cancer.

In solid tumors, human leucocyte antigen (HLA)-A2 has been suggested to be a risk factor and a negative prognostic factor. The HLA-A2 allele in Scandinavia has a high prevalence; it decreases with latitude and also with ovarian cancer mortality in Europe. Furthermore, an association of the HLA-A2 allele with severe prognosis in serous adenocarcinoma of the ovary in stages III-IV was found. Thirty-two unrelated Swedish women with relapsing or progressive ovarian cancer were analysed for the genotypes at the HLA-A, HLA-B, HLA-Cw, and HLA-DRB1 loci by the polymerase chain reaction/sequence-specific primer method. The frequencies of HLA alleles of healthy Swedish bone marrow donors provided by the coordinating centre of the Bone Marrow Donors Worldwide Registries, Leiden, the Netherlands were used as controls. When this cohort of epithelial ovarian cancer patients was compared with healthy Swedish donors, the frequency of HLA-A1 and HLA-A2 gene/phenotype appears, although not statistically significant, to be increased in patients with ovarian carcinoma, while HLA-A3 was decreased. HLA-A2 homozygotes were twofold higher in patients. The A2-B8 haplotype was significantly increased (corrected P value). A2-B5, A2-B15, A2-DRB1*03, A2-DRB1*04, A2-B15-Cw3, and A2-B8-DRB1*03 had odds ratio as well as the level of the lower confidence interval above 1 and significant P value only when considered as single, non-corrected analysis. HLA-B15 and HLA-Cw3 were only present in HLA-A2-positive patients showing that the HLA-A2-HLA-Cw3 and HLA-B15 haplotypes were segregated. In this selected cohort with advanced disease, there are indications of an unusual overrepresentation of HLA class I and II genes/haplotypes as well as segregation for the HLA-A2-HLA-Cw3 and HLA-B15 haplotypes. These findings are presented as a descriptive analysis and need further investigations on a larger series of ovarian cancer patients to establish prognostic associations.

A novel HLA-A*33 (A*3309) allele in a Caucasian family that differs at protein position 66 from HLA-A*3308.

A novel human leucocyte antigen (HLA)-A33 (HLA-A*3309) allele has been identified in a Caucasian family from Middle Europe using single allele-specific sequencing strategy. This allele is identical to the HLA-B*3308 allele except for one point mutation in exon 2 at codon 66 (AAA-->AAT), resulting in an amino acid change from lysine (K) to asparagine (N).

A novel HLA-B*35 (B*3570) allele of a Caucasian family differing with one amino acid mismatch from HLA-B*3503 allele in exon 4.

A novel human leucocyte antigen (HLA)-B35 (HLA-B*3570) allele has been identified in a Caucasian family from Middle Europe using single allele-specific sequencing strategy. This allele is identical to the HLA-B*3503 allele except for one point mutation in exon 4 at codon 188 (CAC-->CGC), resulting in an amino acid change from histidine to arginine.

A novel HLA-A*0119 allele of a Caucasian family containing a mutation in a highly conserved region.

A novel human leucocyte antigen (HLA)-A (HLA-A*0119) allele has been identified in two individuals of a Caucasian family from Middle Europe using a single-allele-specific sequencing strategy. This allele is identical to the HLA-A*0101 allele except for one point mutation in the highly conserved codon 92 (TCT --> GCT) resulting in an amino acid change from serine to alanine.

Comparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA-based HLA crossmatch procedure.

The detection of donor-specific anti-HLA antibodies by standard procedures such as complement-dependent cytotoxicity assay (CDC) or flow cytometric (FACS) analysis is limited by its low sensitivity and the quality of the donor cells. Therefore, an ELISA-based technique was employed using solid phase-immobilized monoclonal antibodies to capture HLA class I or class II molecules of the donor, respectively. In this HLA class I and class II antibody monitoring system (AMS) the donor-specific anti-HLA antibodies from the sera of recipients bind to the HLA molecules of the donor which have been immobilized by monoclonal antibodies (mAb) recognizing non-polymorphic epitopes. Upon binding of donor-specific anti-HLA antibodies they are recognized by secondary enzyme-conjugated anti-human immunoglobulin (Ig) antibodies. A newly established modification of the standard protocol allows the differentiation between bound antibodies of the IgG and IgM isotype. Furthermore, this assay was adapted for investigating small amounts of solid tissue of donors from whom no other cells (e.g. from blood) were available. We here provide an overview of the classical crossmatch methods with their advantages and limits. In addition, the design of the novel AMS-ELISA is described in terms of quality and sensitivity of the approach using exemplary cases of different application. The selected cases show that the AMS-ELISA represents a valuable tool for the post-transplantation monitoring of donor-specific anti-HLA antibodies during reaction crisis, after transfusion reactions and in particular cases of tissue transplantations lacking single cells.

Recombinant generation of two fragments of the rat complement inhibitory factor H [FH(SCR1-7) and FH(SCR1-4)] and their structural and functional characterization in comparison to FH isolated from rat serum.

Factor H (FH) is the predominant soluble inhibitor of the complement system. With a concentration of 200-800 microg/ml in human and rat plasma it acts as a cofactor for the soluble factor I (FI)-mediated cleavage of the component C3b to iC3b. Furthermore it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of approximately 60 amino acid (aa) residues. Two functional fragments of FH comprising the SCR1-4 or SCR1-7 were generated using either the Baculovirus system or stably transfected human embryonal kidney cells, respectively. These fragments, as well as FH purified from rat serum, were first analyzed for their relative molecular weights (Mr) using non-reducing or reducing SDS-PAGE. The Mr of the FH variants differed by about 20% depending on the experimental conditions employed. Only the Mr of proteins separated under reducing conditions were in accordance with the MW calculated from the aa sequence. Analyses of the glycosylation patterns using PAS-staining showed a lack of staining of the recombinant variants (SCR1-4 and SCR1-7) in contrast to FH(SCR1-20) from serum. Using a complement hemolysis assay (CH50-assay) all three variants exhibited a molar complement inhibitory activity of FH(1-20)/FH(1-7)/FH(1-4) of about 3/1/1. These data support the postulated model of FH bearing three binding sites for its ligand C3b, from which one is located in the SCR1-4, whereas the other two are located in the SCR8-20.

Cellular immunity to the Her-2/neu protooncogene.

Her-2/neu (HER-2) is a 185-kDa receptor-like glycoprotein that is overexpressed by a variety of tumors such as breast, ovarian, gastric, and colorectal carcinomas. Overexpression of this oncogene is directly associated with malignant transformation of epithelial cells. The frequency of HER-2 overexpression varies among the different types of cancers, but universally represents a marker of poor prognosis. The critical role of HER-2 in epithelial oncogenesis as well as its selective overexpression on malignant tissues makes it an ideal target for immunotherapy. Antibodies and T cells reactive to HER-2 are known to naturally occur in patients with HER-2 positive tumors, confirming the immunogenicity of the molecule. Both antibodies as well as T cells reactive to HER-2 have been utilized for immunotherapy of HER-2 positive tumors. The "humanized" monoclonal antibody Herceptin has been tested in several clinical trials and found to be an effective adjuvant therapy for HER-2 positive breast and ovarian cancer patients. However, the frequency of patients responding to Herceptin is limited and a majority of patients initially responding to Herceptin develop resistance within a year of treatment. The use of vaccination strategies that generate T cell responses with or without accompanying antibody responses may serve to mitigate the problem. Various strategies for generating T cell-mediated responses against HER-2 are currently being examined in animal models or in clinical trials. The potential advantages of the various approaches to immunotherapy, their pitfalls, and the mechanisms by which HER-2 positive tumors can evade immune responses are discussed in this review.

Longitudinal analysis of the T-cell receptor (TCR)-VA and -VB repertoire in CD8+ T cells from individuals immunized with recombinant hepatitis B surface antigen.

Recent studies have suggested that vaccination induces alterations in the T cell receptor (TCR) repertoire. We investigate the diversity of the TCR repertoire after immunization with a recombinant hepatitis B surface vaccine in seven healthy subjects in CD8+ T cells in peripheral blood lymphocytes. Cellular immune responses were monitored over time by sorting CD8 T cells followed by TCR-VA and -VB complementarity determining region 3 (CDR3) analysis. Frequency of individual VB families was determined by flow cytometry. TCR-VA/VB repertoires obtained from CD8+ T cells drawn after vaccination were compared to the TCR repertoire determined prior to vaccination. Monoclonal TCR transcripts could be detected exclusively in CD8+, but not in CD4+ T cells. Such monoclonal TCR transcripts were either stable in some individuals, or could only be detected at certain time points after vaccination. Sorting of monoclonal TCR-VB3+ T cells, which constituted up to 5% of the CD8+ T cell population from one individual, revealed that this T cell clone recognizes an epitope provided by the recombinant hepatitis B vaccine presented by MHC-class I on autologous antigen-presenting cells. Examination of the structural anatomy, defined by the TCR, and the frequency of T cells responding to the immunizing antigen may be helpful to provide surrogate markers to monitor cellular immune responses induced by protein antigens utilized for vaccination.