Deletion of the CD4 silencer element supports a stochastic mechanism of thymocyte lineage commitment.

The mechanism of T cell lineage commitment remains controversial; to examine it we deleted the CD4-silencer element in the germ line of a mouse using a combination of gene targeting and Cre/LoxP-mediated recombination. We found that these mice were unable to extinguish CD4 expression either in immature thymocytes or mature CD8+ cytotoxic T cells (CTLs), which resulted in the development of major histocompatibility complex class II-restricted double-positive CTLs in the periphery. This finding strongly supports a stochastic over an instructive mechanism of coreceptor down-regulation.

Characterization of genotype-specific epitopes of the HN protein of mumps virus.

The SBL-1 strain of mumps virus, grouping with genotype A on the basis of the small hydrophobic protein gene sequence, was grown in the presence of three different monoclonal antibodies. The monoclonal antibodies were directed against the haemagglutinin-neuraminidase (HN) protein and they inhibited haemagglutinating activity and infectivity of the virus. The HN genes of the nine neutralization-escape virus mutants were sequenced and the predicted amino acid sequences were compared with that of the parental virus. Amino acid substitutions were found at positions 269, 352 and 354, respectively, of the 582 amino acid long protein. The three monoclonal antibodies did not react with 35 virus strains isolated in Stockholm during the years 1970 to 1985. Thirteen and four of the strains were found to belong to the D and C genotypes, respectively. A type-specific neutralization antibody response was also found in sera of rabbits hyperimmunized with purified virions of genotype A and D. The genotype-specific difference in neutralizing activity in mice and rabbits was not corroborated by an overall difference in the amino acid sequence of the HN protein of the different genotypes. Further studies are needed to explore the efficacy of mumps virus vaccines for protection against homologous and heterologous genotypes of mumps virus.

Lysis of allogeneic and syngeneic target cells by primary cytotoxic T lymphocytes induced in vitro by Drosophila melanogaster cells.

Incubation of unprimed splenocytes directly with synthetic peptides induces CD8+ primary CTL in vitro. The ability of peptide-pretreated LPS blasts or H-2Db-expressing Drosophila melanogaster cells to induce peptide-reactive primary CTL was analysed using synthetic peptides corresponding to distinct and overlapping amino acids in SV40 T antigen Db-restricted T-cell epitopes I, II-III, and V. Peptide-pretreated, but not untreated, LPS blasts induced strong peptide-reactive CTL in splenocytes of naive C57 BL/6 mice. The reactivity of these CTL was indistinguishable from that of CTL induced by direct stimulation of spleen cells with peptides. In contrast, effector cells induced by untreated or peptide-pretreated drosophila cells expressed equivalent cytolysis both against untreated and peptide-pretreated syngeneic target cells. They also strongly lysed the allogeneic (H-2d) P815 target cells. The cytolytic activity was expressed by CD8+ T lymphocytes. However, the lack of specificity and restriction was probably due to stimulation of LAK cells as well. The CTL-inducing potential of the drosophila cells was stable at 46 degrees C. Peptide-activated splenocytes cultured for 1 month in medium supplemented with spent medium from drosophila growth culture were 75% B cells and 25% CD4+ T cells. Taken together, these results indicated that LPS blasts are efficient APC for induction of primary CTL and that drosophila cell-derived molecule(s) are capable of inducing broad-reactive effector cells and may also assume the function of leukocyte growth factor(s).

Interaction of in vitro- and in vivo-generated cytotoxic T cells with SV40 T antigen: analysis with synthetic peptides.

Virus-specific cytotoxic T cells recognize antigens in the form of peptides (8 or 9 amino acids long) bound to MHC class-I molecules. Exposure of unprimed murine splenocytes to synthetic peptides of viral antigens elicits primary CTL in vitro. The fine specificity of such CTL as well as the correlation between binding affinity of peptides to class-I molecules and CTL induction was analysed using synthetic peptides corresponding to overlapping and distinct amino-acid residues in SV40 T antigen (Tag) Db-restricted T-cell epitopes I, II-III, and V. The peptides induced cross-reactive CD8+ primary CTL in splenocytes of naive C57 BL/6 mice. This reactivity was seen regardless of the peptides allelic anchor motifs or their abilities to stabilize empty class-I molecules. However, none of the primary CTL and CTL lines lysed Tag-expressing cells. In contrast, CTL generated in vivo by immunizing mice with Tag-expressing cells recognized endogenously processed Tag as well as synthetic peptides. The peptides recognized by these CTL depended on the intracellular concentration of Tag antigen in the immunizing cells. The reactivity of these CTL was peptide specific as shown by a functional peptide competition assay. Moreover, three peptides bound to and were recognized in the context of both Kb and Db molecules. These results have revealed a flexible disposition of MHC class-I molecules with regard to peptide binding and also reflected lack of correlation between binding affinity to class-I molecules and the capacity of peptides to induce primary CTL or to serve as potential targets. The significance of these findings in relation to identifying major T-cell epitopes using allele specific peptide motif and in vitro maintained CTL clones is discussed.

Evaluation of an improved DNA probe for diagnosis of pertussis.

A Bordetella pertussis specific subclone, pRZ61, of a Bordetella genus-specific clone, pB23, was evaluated on nasopharyngeal aspirates of 179 patients with suspected pertussis. Hybridization was performed directly after spotting or after 1-3 days of preculture of the nylon membranes on solid culture medium. A direct comparison of the two probes was obtained by reprobing with the subclone the same membranes that had been hybridized with the parent probe. pRZ61 detected 50% of the serologically defined cases of pertussis, that is, had the same sensitivity as standard culture. Specificity as compared with serology was close to 100%. The increasing sensitivity and the corresponding decreasing specificity after preculture noted for pB23 was not seen with the subclone. The study showed that the improved probe represents a rapid diagnostic method in pertussis.

Identification of a DNA fragment in the genome of Bordetella pertussis carrying repeated DNA sequences also present in other Bordetella species.

A DNA fragment (3.8 kbp) which hybridized to repeated sequences in the genome of Bordetella species has been cloned from Bordetella pertussis chromosomal DNA. Eleven subclones were constructed from this fragment. They exhibited distinct inter-species hybridization patterns in genomic blots of each of the Bordetella used in the study. One subclone revealed intraspecies variability among B. pertussis strains and another did not hybridize to B. parapertussis. The 3.8 kbp DNA fragment possesses a middle sequence surrounded by repeated sequences organized in an opposite symmetrical orientation. The external inverted repeats of it hybridized to a 680 bp DNA sequence which is located about 800 bp upstream of the pertussis toxin operon. The novel structural organization of the 3.8 kbp fragment suggests the possibility that this DNA segment is an IS-like element which may have an important function in the expression of virulence determinants in Bordetella bacteria.

In vitro and in vivo stimulation of murine lymphocytes by human respiratory syncytial virus strains.

Respiratory syncytial virus (RSV) strains of subtype A (A2, WV9894, and WV12138) and of subtype B (WV1293, WV4843, and WV6873) are mitogenic in vitro for unprimed BALB/c spleen cells. The virus also triggered splenocytes in vitro to secrete immunoglobulins. Plaque-purified and UV-irradiated materials of both RSV subtypes produced comparable levels of DNA synthesis. Infectious materials of both subtypes also induced pronounced responses. Lymphocyte activation with UV-inactivated RSV strain A2 was dose-dependent and maximal responses occurred after 4-5 days of incubation. The virus preparations were mitogenic for spleen cells depleted of T lymphocytes by treatment with anti-Thy 1.2 and complement and for lymphocytes of congenitally athymic mice (nu-nu). They were also mitogenic for highly purified T lymphocytes separated by panning of spleen cells on anti-mouse Ig-coated Petri dishes, suggesting that both B and T lymphocytes respond to the mitogenic activity of RSV. Moreover, mice infected intranasally with RSV strain A2 generated local as well as peripheral cellular and humoral responses.

The role of serologically defined epitopes on mumps virus HN-glycoprotein in the induction of virus-dependent cell-mediated cytotoxicity. Analysis with monoclonal antibodies.

The importance of virus structural proteins for the induction of virus-dependent cellular cytotoxicity (VDCC) was studied by means of monoclonal antibodies raised in mice against mumps virions. Antibodies against the viral glycoprotein bearing the haemagglutination and neuraminidase activities (HN) inhibited VDCC but not the natural cytotoxicity (NK) displayed by the lymphocytes in the absence of virus. Antibodies to the fusion factor, the membrane protein or the nucleoprotein were inactive. These results confirmed our previous conclusion, that the only viral component required for VDCC induction is the HN protein. To clarify the role of this protein in VDCC further, the inhibitory activity of 13 HN-specific monoclonals, all of IgG isotype and directed against 9 distinct determinants, was studied in detail. Seven antibodies reacting with 3 different determinants of the peptide moiety of the HN protein were strongly inhibitory. The remaining antibodies, specific for 5 additional peptide epitopes, had intermediate or weak inhibitory effects. One carbohydrate specific anti-HN antibody was inactive although its antigen-binding capacity was of the same magnitude as that of a good inhibitory antibody. The anti-HN antibodies inhibited VDCC regardless of their IgG subclass. Moreover, VDCC inhibition was not correlated with the capacity of the antibodies to inhibit haemagglutination, haemolysis, neuraminidase activity, or the infectivity of the virus. These results suggest that full expression of VDCC requires the interaction of more than one of the serologically defined structures of the HN polypeptide with virus receptors on the lymphocytes and probably also on the target cells. These structures may be different at least in part from those involved in other known biological activities of the virus. Treatment of lymphocytes with virus increases both the number of target-binding cells (TBC) and the number of cytotoxic effector cells. However, when treated under conditions which gave optimal VDCC inhibition, none of the inhibitory antibodies reduced the virus-mediated increase in TBC. This indicates either that the anti-HN antibodies decreased the efficiency of effector-target cell interaction necessary for VDCC induction, or that they blocked a post-binding step required for triggering of cytotoxicity.

Virus-dependent cellular cytotoxicity in vitro. Mechanisms of induction and effector cell characterization.

When human peripheral blood lymphocytes were incubated with 51Cr-labelled tissue culture cells (T24 bladder carcinoma cells or Chang liver cells), their natural cytotoxicity (NK) usually stopped after 8 h of incubation. The 51Cr release induced by lymphocytes treated with small amounts of live or ultraviolet-inactivated mumps virus was strongly enhanced and lasted longer. When the lymphocytes were fractionated by Percoll gradient centrifugation, the highest NK activity was found in the low-density fraction enriched in large granular lymphocytes, whereas that of the T-cell-enriched high-density fractions was low. In contrast, the virus-dependent cellular cytotoxic (VDCC) activity was more evenly distributed between these fractions. However, there was a difference between the target cells in that the T24 cells were more susceptible to the cytotoxicity of lymphocytes in the high-density fractions than the Chang cells. Studies of Percoll fractions in the single-cell agarose assay showed that virus treatment increased the proportion of both target binding cells and killer cells in all fractions. Moreover, in the high-density fractions the increase in the number of killer cells was greater than that in binding cells, suggesting that the enhanced target cell killing induced by the virions reflected both increased binding and effector cell activation. Surface marker analysis of unfractionated lymphocytes indicated that the number of T3+ effector cells was greater than that of the HNK-1+ effector cells, regardless of whether the lymphocytes were treated with virus or not. However, for both NK and VDCC, the T3 to HNK-1 distribution ratio on the effector cells was 5-8:1 for T24 and 2:1 for Chang. Taken together, the results indicate that both NK and VDCC effector cells are phenotypically heterogeneous and that the target cells may play an active role in the recruitment of those effector cells that are most efficient in that system. The enhancement of lymphocyte cytotoxicity primarily reflects effector cell recruitment.

Mumps virus-induced enhancement of the in vitro cytotoxicity of cord blood lymphocytes.

Purified lymphocytes from the umbilical cord of healthy donors (CBL) displayed lower natural cytotoxicity (NK) and antibody-dependent cellular cytotoxicity (ADCC) than peripheral blood (PBL) from adult donors. In contrast, CBL treated with small amounts of UV-inactivated or live mumps virions expressed the same level of enhanced cytotoxicity (virus-dependent cytotoxicity (VDCC)) against non-infected target cells as PBL. For individual CBL donors there was no correlation between the level of NK and VDCC, indicating involvement of partly distinct effector cell populations. The heterogeneity of the effector cells active in VDCC was confirmed by cell fractionation experiments. The major CBL effector cells in NK and ADCC were found in 'non-T' lymphocyte fractions and/or in fractions containing cells with high-avidity receptors for IgG. In contrast, CBL fractions consisting of about 100% lymphocytes bearing T-cell markers and depleted of Fc gamma R+ cells were strongly cytotoxic in VDCC when T24 cells (human bladder carcinoma) were the targets. With two other target cell types of similar susceptibility to VDCC, the cytotoxic activity of T-cell-containing fractions was less pronounced, indicating that the target cells play an active role in effector cell selection. The surface marker profiles of the VDCC effector cells were the same for CBL and adult PBL. Incubation of CBL with UV-inactivated virions usually gave no significant stimulation of DNA synthesis above that seen in virus-free controls. Taken together, our results suggest that neither specific recognition of viral antigen by T cells nor mitogenic effects of viral material are involved in VDCC generation.

The role of viral glycoproteins in mumps virus-mediated antibody-dependent cellular cytotoxity in vitro.

Treatment of human peripheral blood lymphocytes with live or UV-inactivated mumps virions enhances antibody-mediated cellular cytotoxicity (ADCC), reflected by increased target cell lysis in a 51Cr-release assay or an increased number of plaque-forming cells on monolayers of bovine erythrocytes (Eb) in the presence of anti-Eb antibodies. Virus treatment of the Eb targets causes a similar enhancement. The role of viral glycoproteins in ADCC enhancement was investigated by using a panel of monoclonal antibodies raised in mice against mumps virions. Most of the lymphocytes bound mumps virions, as ascertained by indirect immunofluorescence. A high proportion of virus-treated lymphocytes also formed rosettes with Eb. Anti-HN antibodies inhibited rosetting to various degrees. Although antibodies with high haemagglutination inhibition titres were most efficient inhibitors, antibodies without this serological activity were also inhibitory. Anti-F antibodies were only weakly inhibitory, and anti-NP antibodies had no effect. Anti-HN antibodies also abrogated target cell lysis in the 51Cr-release assay and effector cell recruitment in the ADCC plaque assay by inhibiting virus-mediated Eb-lymphocyte interactions both at the target cell and at the effector cell level. Anti-F or anti-NP antibodies were only weakly or not at all inhibitory. The results suggest that virus-mediated enhancement of ADCC is caused by the HN glycoprotein, primarily (although perhaps not exclusively) by its improvement of the effector cell-target cell contacts necessary for the efficient execution of target cell lysis.

Virus-mediated induction in human lymphocytes of antibody-independent cytotoxicity (VDCC) and enhancement of antibody-dependent cytotoxicity (ADCC) against natural killer-resistant tumor target cells.

The effect of Parotis virus on the in vitro cytotoxicity of human lymphocytes against NK-resistant mouse mastocytoma cells was studied. In the 51Cr-release assay, treatment of lymphocytes with virus induced a rapid cytotoxicity in the absence of anti-P815 antibody (virus-dependent cellular cytotoxicity, VDCC) and strongly enhanced antibody-dependent cytotoxicity (ADCC). At the effector cell level, virus treatment was found to increase the frequency of target-binding cells (TBC) as well as the proportion thereof mediating VDCC and/or ADCC, indicating recruitment of active effector cells. The recruited cells were heterogeneous but contained a major fraction bearing the T-cell-associated antigen T3. Virus was found to decrease rather than to increase the recycling capacity of the cytotoxic lymphocytes, suggesting that VDCC induction and ADCC enhancement were due to a virus-mediated improvement of effector cell-target cell interactions. VDCC and ADCC enhancement may be of protective importance in early phases of virus infection as well as for the production of nonspecific tissue injuries associated with viral disease.

Virus-induced enhancement of lymphocyte-mediated antibody-dependent cytotoxicity (ADCC) in vitro.

The effect of Parotis virus on antibody-dependent cellular cytotoxicity in vitro (ADCC) of human lymphocytes was investigated in a 51Cr-release assay and, at the effector cell level, in an ADCC plaque assay. Target cells were bovine or chicken erythrocytes, which are not susceptible to natural cytotoxicity (NK) of human lymphocytes. They were not killed when incubated with virus-treated lymphocytes in the absence of antibodies. Treatment of the lymphocytes or the target cells with small amounts of virus, however, resulted in a very significant enhancement of ADCC. The same results were obtained with live or UV-inactivated virus, suggesting that enhancement was a passive phenomenon not requiring infection. Enhancement was already significant after 3 hr of incubation, indicating that it was independent of endogenously released interferon. Enhancement of ADCC by virus was due to effector cell recruitment rather than due to the increase of the cytotoxic potential of the individual K cell. The highest frequency of effector cells was present in Percoll fractions enriched in large granular lymphocytes (LGL). Virus treatment resulted in recruitment of effector cells carrying T cell markers such as the T3 antigen (OKT3+), receptors for sheep erythrocytes, or Fc receptors for IgM. In contrast, the absolute number of K cells carrying the HNK-1 marker (Leu-7) or receptors for C3 fragments was not changed by the virus. It is concluded that Parotis virus enhances ADCC by improving effector cell-target cell contacts, resulting in recruitment of effector cells with T cell characteristics. Recruitment is accompanied by a significant reduction of the antibody concentration needed for ADCC induction. This virus-mediated enhancement of ADCC may be of importance for protection of the host in the early phases of a virus infection in which the amounts of anti-viral IgG antibodies capable of inducing cellular cytotoxicity may yet be very small.