Matti Kaartinen

Flow cytometric analysis of apoptotic subpopulations with a combination of Annexin V‐FITC, propidium iodide, and SYTO 17

BACKGROUND We have previously characterized apoptotic cell death induced in a follicular lymphoma cell line, HF-1, after triggering via the B-cell receptor (BCR) or treatment with Ca(2+) Ionophore A23187. We analyzed the kinetics of apoptosis induced by these two treatments, as two alternative models of classical apoptosis, by flow cytometry using a novel combination of cytofluorometric stains. METHODS Cells were stained with a combination of Annexin V-FITC, propidium iodide (PI), and SYTO 17 and analyzed by a two-laser flow cytometry system using 488-nm argon and 633-nm HeNe air-cooled lasers. RESULTS In both apoptotic models, the first apoptotic cells were detected by SYTO 17 staining. The alteration in SYTO 17 staining intensity was followed by an increased uptake of PI. Finally, the apoptotic cells were labeled with Annexin V in BCR-induced apoptosis. On the contrary, on treatment with Ca(2+) Ionophore A23187, cells became positive for Annexin V earlier than for PI. CONCLUSIONS The novel cytofluorometric dye, SYTO 17, discriminates apoptotic alterations before Annexin V and PI. PI also discriminates apoptotic alterations before the loss of plasma membrane asymmetry by BCR but not by Ca(2+) Ionophore A23187-induced apoptosis. Finally, the combination of these three cytofluorometric dyes allows effective detection of apoptotic subpopulations and ordering of apoptotic events by flow cytometry.

Determination by ellipsometry of the affinity of monoclonal antibodies

The reaction between monoclonal antibodies and surface-immobilised hapten was studied by ellipsometry, a method allowing absolute measurement of the surface concentration of proteins. Monoclonal antibodies against 2-phenyloxazolone were used and their affinity for the antigen in solution was determined by calculations of the equilibrium constant from data obtained by measuring fluorescence quenching of the hapten due to antibody binding. The binding rate of antibody to surface-immobilised hapten and the dissociation rate of the complex were measured by ellipsometry. The equilibrium constant of the heterogeneous antigen-antibody reaction was determined by a Scatchard plot. The affinity of the antibodies for the antigen was found to be higher in the heterogeneous than in the homogeneous reaction by a factor which varied between different monoclonal antibodies.

Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain.

Fab fragments of IgG1 and IgG3 subclass antibodies which bind to 2-phenyloxazolone (Ox) were produced in Escherichia coli. The signal sequences of the Fd and L chains were correctly processed, the fragments were secreted into the periplasmic space and released into the culture medium upon prolonged cultivations. The yields of active Ox IgG1 and Ox IgG3 Fab fragments after one-step purification from the culture medium by affinity chromatography were 2 micrograms/ml and 0.5 micrograms/ml, respectively. The majority of the purified Ox IgG1 Fab was properly assembled, but in the case of Ox IgG3, the preparation was found to consist of a complete L chain and C-terminally degraded fragments of the Fd chain. A deletion up to the interchain disulfide bond in the first constant domain (CH1) of the Ox IgG3 Fd chain led to proper assembly of the truncated Fab fragment. The production level of the truncated fragment was comparable to that of the Ox IgG1 Fab and its hapten-binding activity similar to that of the idiotype monoclonal antibody. The temperature stability of the Ox IgG1 Fab was similar to that of the intact antibody. However, both of the Ox IgG3 Fab fragments showed reduced stability, suggesting that the CH1 domain contributes significantly to the thermal stability of the Fab fragment.

The Somatic Hypermutation Activity of a Follicular Lymphoma Links to Large Insertions and Deletions of Immunoglobulin Genes

A biopsy specimen from a patient with follicular lymphoma was divided into two fragments. DNA was extracted from one fragment and a 1.2 kb region of the functional heavy chain (IgH) gene was amplified, cloned and sequenced (eight clones). From the other fragment a cell line (HF‐1) was started. The IgH gene region was amplified from the cell line, and sequenced without cloning. The nine sequences obtained could be arranged into a genealogical tree where the individual sequences differed from the deduced ancestor by 16–29 single nucleotide changes, some also by an insertion and/or a deletion. It is apparent that the sequence alterations were caused by somatic mutations during the growth of the lymphoma.

Two novel human B-cell lymphoma lines of lymphatic follicle origin: Cytogenetic, molecular genetic and histopathological characterisation

Abstract:  Two B‐cell lines, designated as HF‐1 and HF‐4, were characterised. The cell lines have complicated karyotype abnormalities including a 14;18 translocation and an 8q24 breakpoint originating from t(2;8)(p11;q24) (HF‐1) or t(1;8)(p21:q24) (HF‐4). The lines have BCL2 rearrangement and they are positive for CD19, CD20, CD22, CD39. HF‐1 is also positive for IgG, and HF‐4 is positive for IgM and IgD. On Northern blot analyses, the 2.6‐kb and 4.2‐kb transcripts corresponding to the major transcripts of CMYC and BCL2, respectively, were seen. In Western blot as well as in FACS (fluorescence‐activated cell sorting) analysis the presence of BCL2 protein in the both HF‐1 and HF‐4 cells was demonstrated. The cell lines are expected to serve as an important tool in the study of the chromosomal mechanism activating cellular oncogenes, the somatic hypermutation mechanism of antigen‐activated B cells and the apoptosis of B cells.

Allelic polymorphism of mouse Igh-J locus, which encodes immunoglobulin heavy chain joining (JH) segments

The mouse genome contains four functional JH genes, which encode immunoglobulin heavy chain joining segments. The JH gene cluster is located a few kilobases 5′ from the constant region genes (C genes) on chromosome 12. The polymerase chain reaction (PCR)-technique was used to amplify DNA stretches from mouse genome of approximately 1 340 nucleotides in length containing all four JH genes (Igh-J locus). PCR products were directly used as templates in Sangers dideoxy-sequencing, and sequences were determined. Twelve inbred mouse strains belonging to ten different Igh-C haplotypes were studied. The strains were: BALB/c, C58/J, RIII, DBA/2, CE, RF, CBA, NZB/J, AKR, C57BL/10, SJL, and A/J. Five allelic forms of the Igh-J locus were found among these strains. The A/J mouse has an allele (e) which differs from the BALB/c allele (a) by 15 nucleotides. C57BL and SJL have the allele (b) with eight differences from BALB/c. The CBA allele (j) has two differences, and the CE allele (f) has a single nucleotide difference compared with the BALB/c sequence. Based on the JH, variable (V) and constant (C) region sequences we conclude that independent reshuffling of VH,JH, and CH gene clusters occurred during the evolution of Mus musculus.

A cross-over chromosome combiningIg heavy chain genes of two mouse strains

Two congenic strains of mice were bred in Konstanz that bear theIg heavy chain allotype gene of the C57BL/6 (Ig-1b) in a BALB/c background genome. One line (CB-8K) underwent eight backcross generations to BALB/c before sister-brother mating was initiated. For the other line (CB-16KN) backcrossing was continued for eight further generations, then a homozygousIg-1b/Ig-1b strain was produced by sister-brother mating. Both lines were tested for four VH markers of the BALB/c and one of the C57BL parent. The CB-16KN strain expressed the C57BL marker VHNPb together with the C57BL allotype marker, and failed to express the three BALB/c markers, VHDEX, VHS117, VHphOx and VHNPa. It thus resembled the CB-20 strain.Strain CB-8K expressed the VHNPb marker of C57BL but also all the four BALB/c markers that were tested. The strain appeared more heterogeneous than the CB-16KN strain, and a subline was bred from two exceptional mice that did not express the VHNPb marker. This subline (CB-8KN) expressed the BALB/c marker VHNPa regularly, and was negative for the VHNPb marker. It thus resembles the BAB-14 line.The crossing over event thus must have happened in one of the two meioses, which led to the CB-8K line. As BAB-14 is derived in an analogous manner to a branch of the backcross of Potter and Lieberman, which ended up in CB-20, the unexpected finding is discussed that two independent crossing over events (in CB-8KN and BAB 14) within theIg heavy chain gene region have taken place at approximately the same stage of two breeding programs.

The alternative binding site for protein A in the Fab fragment of immunoglobulins

Twenty‐six new human or murine monoclonal immunoglobulins (IgM, IgA, murine IgGl or human IgG3) with a known V‐region sequence were tested for alternative (non‐Fc) binding to Staphylococcal protein A. Seven of them did not bind at all. Four immunoglobulins (all mouse IgGl) were bound but easily eluted (at pH 6). They were probably bound via the Fc part. All eleven were classified as negative for alternative binding. Fifteen immunoglobulins were found to bind more firmly; they came off the protein A column at pH 4–3 (alternative binders).

The same few V genes account for a majority of oxazolone antibodies in most mouse strains

The early primary anti-phenyloxazolone antibodies of 12 mouse strains were studied by determining proportions of two defined subsets id495 (the classical phOx idiotype) and id350. Id495-positive antibodies bear an H chain encoded by VHOx1 gene (family Q52) and an L chain usually coded for by VKOx1 but occasionally by other VK genes. Id350-positive antibodies are encoded by a VK gene VK45.1, and usually by a VH gene of the S107 family. All 12 strains (representing nine H-chain and four kappa-chain haplotypes) produced id350-positive anti-phOx antibodies. While id495 is the predominant major subset in the BALB/c response (originally studied), id350 seems to be the predominant subset of early anti-phOx antibodies in the mouse species. The combined proportion of the two subsets varied from ca. 50 to almost 100% of the total in all strains except C57BL.

Combinatorial association of V genes: One VH gene codes for three non-cross-reactive monoclonal antibodies each specific for a different antigen (phOxazolone, NP or GAT)

Two anti-phenyloxazolone (phOx3) and one anti-GAT MAbs from C57BL mice are shown to be coded by VH gene 186.2. This gene has been found earlier to code for several anti-NP (NNP) antibodies (Bothwell et al., 1981) and anti-GT antibodies (Rocca-Serra et al., 1983; Carmack and Pincus, 1986). The L chain partner of the VH 186.2 gene is different in anti-NP and anti-GAT antibodies (Bothwell et al., 1981; Rocca-Serra et al., 1983; Carmack and Pincus, 1986); in anti-phOx antibodies two new unrelated kappa chain V regions were found. Both of the new VK genes involved code frequently for anti-phOx antibodies in BALB/c mice but then with different VH genes. We tested five 186.2-coded antibodies for cross-reactions. Four antibodies were specific, one bound only to NNP, one only to phOx and two only to GT (GAT). The fifth antibody (anti-phOx) bound also to NNP, GAT and ABA-HOP though probably with a low affinity. This is the first demonstration that one V gene can code for three different antibody specificities. It emphasizes the role of the combinatorial element in antibody diversity.