Possible association between HLA antigens and the response to interferon in Japanese patients with chronic hepatitis C

Correlation between the major histocompatibility complex class I antigens (HLA-A, -B and -C) and the elimination from serum of hepatitis C virus in patients with chronic hepatitis C has not been understood. We analyzed HLA phenotypes and their relationship to the efficacy of interferon treatment. Of the 172 patients who were treated with 9 million units of interferon-α2a three times a week for 6 months, 54 patients were responders and 118 patients were non-responders. No significant difference was observed between the 172 patients and 199 healthy subjects with regard to the frequencies of HLA-A, -B and -C antigen phenotypes. However, HLA-B55, B62, CW3 and CW4 frequencies were significantly higher in responders than in non-responders to the interferon treatment. CW4 was found to link with B62, but other phenotypes were independent each other. Patients with HLA B55, B62 and CW3 had a significantly lower viral load, and showed a better response to interferon. These results suggest that HLA system does not have an influence on the evolution towards chronicity of the disease due to hepatitis C virus, but HLA B55, B62 or CW4, and CW3 may be a virus quantity-regulating factors which then affect to response to the interferon treatment, indicating that these HLA antigens in conjunction with a viral peptide is a key target antigen for cytotoxic T lymphocytes in patients with chronic hepatitis C.     

Analysis of human class II antigens by cloned cytolytic T cell reagents: a study using HLA loss mutant lymphoblastoid cell lines and monoclonal antibodies detecting the HLA-DP product(s)

We have utilized cloned T cell reagents and ionizing radiation-induced mutants of an HLA heterozygous lymphoblastoid cell line (LCL) to investigate the determinants detected by the cell-mediated lympholysis (CML) assay. Cells of an LCL clone, 721.501, an HLA haplotype loss mutant expressing the HLA-A2-Cw1-Bw51-DR1-Dw1-DQw1-DPw2-GLO haplotype were used as sensitizing cells for responder cells in vitro. "Cloned" reagents were generated by single-cell deposition of cells of a bulk reagent primed against 721.501 cells. Those clones were screened for cytolytic activity against HLA loss mutant targets (derived from LCL 721) of four different categories; HLA-A2 loss only, A2-Cw1-Bw51 loss, A2-Cw1-Bw51-DR1-DQw1 loss, and the entire HLA haplotype loss. Of 196 clones tested, 36 were cytolytic, including three anti-A2, five anti-Bw51/Cw1, 12 anti-DR1/DQw1, 13 anti-DP region associated with DPw2, and three of undetermined specificity, based on cytolytic patterns against the HLA loss mutant targets. Of 25 anti-HLA class II lytic clones, 23 (92%) fitted the characteristics of helper cell-independent cytolytic T cells (HITc), whereas only two of eight (25%) anti-class I clones were HITc. The 13 anti-DP region clones were divided into three subgroups defined by blocking by anti-FA and not Tü39 monoclonal antibodies (MoAb), by Tü39 and not anti-FA, and by both MoAbs.     

HLA class II restriction repertoire of antigen-specific T cells. I. The main restriction determinants for antigen presentation are associated with HLA-D/DR and not with DP and DQ

In order to study the HLA class II restriction repertoire in antigen presentation to T cells, T lymphoblasts (T-LB) of ten different HLA class II donors were generated by a simple and rapid technique; peripheral blood lymphocytes (PBL) were restimulated in vitro with purified protein derivative (PPD) or tetanus toxoid (TET), and then propagated in interleukin-2 containing conditioned medium (IL2-CM). These T-LB appeared to be antigen specific and devoid of alloreactivity. Antigen was presented to these T-LB by allogeneic irradiated PBL as antigen-presenting cells (APC) in 179 combinations. T-LB proliferative responses were restricted mainly by determinants associated with HLA-DR and not with -DP or -DQ; in 102 fully DR mismatched T-LB/APC combinations matching for DP or DQ determinants had no significant influence on T-LB responses. For PPD, preferential DR1 restriction was observed, and the results suggest a preferential DRw11 vs. DRw12 restriction for TET. Moreover, DRw13 may be associated with low anti-PPD T-LB responsiveness.     

Monoclonal antibody (B27M2) subdividing HLA-B27

Because HLA-B27 shows strong association with ankylosing spondylitis, it was of interest to produce murine monoclonal antibodies to study this antigen in detail. The first such anti-B27 antibody, anti-B27M1, reacted with 100% of B27 + cells and cross-reacted with homozygous B7 cells. In the present report a second monoclonal antibody, anti-B27M2, is described which subdivides HLA-B27 into two variants. Among healthy B27⁺ individuals, 87% were B27M1⁺ and B27M2⁺, and 13% were B27M1⁺ but B27M2⁺ by standard lymphocytotoxicity assays. The specificity of B27M2 antibody for the HLA-B27 molecule was confirmed with blocking studies using F(ab′)₂ fragments of HLA alloantibodies. Both the B27M2⁺ and B27M2 variants of HLA-B27 bred true in family studies. Unlike B27M1, B27M2 antibody did not react with B7 but did react with the rare Bw47 allele.For antibody binding studies Epstein-Barr virus-transformed B lymphoblastoid cell lines were derived from normal donors KCA (by lymphocytotoxicity shown to be B27⁺, B27M2⁺. VC (B27⁺, B27M1⁺, B27M2^?), and WH (B27^?, B27M1^?, B27M2^?). Each cell line bound equivalent amounts of W6/32 (monoclonal anti-HLA-ABC): KCA and VC bound similar amounts of anti-B27M1, but only KCA bound substantial anti-B27M2 antibody. These data are consistent with a model in which all B27 antigens possess a B27M1 epitope: whereas most, but not all, possess an additional and distinct epitope, B27M2. Although the relation of these genetic variants to disease susceptibility remains to be determined, the availability of epitope-specific monoclonal antibodies should help to refine our understanding of the structure and function of HLA molecules.     

Expansion of human lymphocyte populations expressing specific immune reactivities. III. Specific colonies,either cytotoxic or proliferative,obtained from a population of responder cells primed in vitro. Preliminary immunogenetic analysis

Human alloreactive cell lines were maintained in culture over prolonged period of some using conditioned medium. Primed lymphocyte typing reactivity was observed in for only 1 mo, but these T cell lines have remained for more than 7 mo cytotoxic.Using as growth promoter an irradiated autologous feeder consisting of irradiated blood lymphocytes and the lectin leucoagglutinin, we have derived by limiting in vitro primed allogeneic combinations, primary colonies for primary immune reactivities either cytotoxic (the rarest observed) or PLT the colonies). Furthermore, each monofunctional primary colony when tested for PLT reactivity on a panel of unrelated PBL, always showed a restricted specificity when the original primed population. The PLT reactivity of each of the primary lasting in contrast to their growth potential. The CML reactivity of the primary the T cell lines, was long lasting as was their growth potential.     

Lack of correlation between serum cholesterol levels, lymphocyte plasma membrane fluidity and mitogen responsiveness in young and aged chickens

Chickens were studied in an attempt to demonstrate correlations between serum lipid levels and peripheral blood lymphocyte (PBL) plasma membrane fluidity and mitogen responsiveness: (a) in the laying hen; (b) during aging; and (c) following dietary manipulation of serum cholesterol of young and aged chickens. The membrane fluidity of PBL from laying hens was significantly greater than that of immature birds. However, no direct correlation was found between serum lipid levels, nor the serum free cholesterol/phospholipid (FC/Pl) mole composition and PBL membrane fluidity in any of the age-groups tested. Likewise, no correlation was found either between serum FC/Pl mole ratio or membrane fluidity and mitogen responsiveness of PBL from birds up to 5 years of age nor was there any evidence for a decline in mitogen responsiveness up to this age. Supplementation of diets with 1% cholesterol induced hypercholesterolemia, mainly in the very low density lipoprotein (VLDL) fraction, but membrane fluidity and mitogen responsiveness remained unaffected.     

A simple micromethod for MLC and CML with low numbers of murine lymph node and peripheral blood lymphocytes

A series of 6 hybrid clones are described which produced antibodies directed to murine IgM. These were derived by immunizing rats with W279 B-lymphoma cells and then fusing the immune spleen cells to enzyme-deficient murine plasmacytoma cell lines. Immunoenzymatic techniques were used to select clones producing desirable antibodies to cell surface or soluble antigens. Antibodies from 3 of the clones (293.14, 414.18 and 307.5) preferentially bound to idiotypic determinants of W279 immunoglobulin whereas the remaining 3 clones made antibodies which bound to various myeloma and normal cell surface IgM molecules. One IgM-specific clone (287.2) produced antibody of relatively low avidity. The two other clones (81.20 and 331.12) which secreted anti-mu antibodies appeared to be directed to determinants which differed in glutaraldehyde lability and expression in F(ab')2 preparations.     

Lung cancer neovascularisation: Cellular and molecular interaction between endothelial and lung cancer cells

Background

Novel vascular-independent conduits have been observed in some cancers. These have been variously described as vasculogenic mimicry, mosaic vessel formation, vascular co-option and intratumour embryonic-like vasculogenesis. Despite lung cancer being the most common cancer worldwide, there is little information on its neovascularisation or the pathways involved.

Methods

An in vitro model involving co-cultures of microvascular lung endothelial cells and squamous or adenocarcinoma lung cancer cells was developed to assess their angiogenic interaction. Cells were incubated and examined by phase contrast microscopy and by immunocytochemistry in both mono- and co-cultures. Cultured cells and lung cancer tissue sections were assessed for new tumour vessel formation, expression of the endothelial marker CD31 and morphology.

Results

Lung tumour cells and endothelial cells interacted morphologically via pseudopodia and used alternative pathways to generate new vessels. Co-culturing microvascular endothelial and squamous carcinoma cells led to endothelial cells surrounding tumour cells and the tumour cells being incorporated into vessel walls. Co-culturing endothelial and adenocarcinoma cells resulted in cellular contact and the formation of tumour cell bridges around clusters of endothelial cells. These adencocarcinoma cells became strongly positive for CD31. Tumour tissue section studies supported the in vitro findings.

Conclusion

Lung carcinoma cells when co-cultured with lung endothelial cells modify their cellular and molecular features that encourage alternative means of providing blood supply. The mechanisms underpinning these non-angiogenic processes need to be further investigated and should be considered when anti-tumour therapeutic interventions are being considered.