Effects of rapid antigen degradation and VEE glycoprotein specificity on immune responses induced by a VEE replicon vaccine.

Genetic vaccines are engineered to produce immunogens de novo in the cells of the host for stimulation of a protective immune response. In some of these systems, antigens engineered for rapid degradation have produced an enhanced cellular immune response by more efficient entry into pathways for processing and presentation of MHC class I peptides. VEE replicon particles (VRP), single cycle vaccine vectors derived from Venezuelan equine encephalitis virus (VEE), are examined here for the effect of an increased rate of immunogen degradation on VRP vaccine efficacy. VRP expressing the matrix capsid (MA/CA) portion of SIV Gag were altered to promote rapid degradation of MA/CA by various linkages to co-translated ubiquitin or by destabilizing mutations and were used to immunize BALB/c mice for quantitation of anti-MA/CA cellular and humoral immune responses. Rapid degradation by the N-end rule correlated with a dampened immune response relative to unmodified MA/CA when the VRP carried a glycoprotein spike from an attenuated strain of VEE. In contrast, statistically equivalent numbers of IFNgamma(+)T-cells resulted when VRP expressing unstable MA/CA were packaged with the wild-type VEE glycoproteins. These results suggest that the cell types targeted in vivo by VRP carrying mutant or wild type glycoprotein spikes are functionally different, and are consistent with previous findings suggesting that wild-type VEE glycoproteins preferentially target professional antigen presenting cells that use peptides generated from the degraded antigen for direct presentation on MHC.

Phenotype of B cells responding to the thymus-independent type-2 antigen polyvinyl pyrrolidinone.

We have determined the origin and cell surface phenotype of B cells producing antibody in response to immunization with the non-self TI-2 antigen polyvinyl pyrrolidinone (PVP). We report that the responding cells are derived from precursors in adult bone marrow and display the phenotype characteristic of B-1 cells. By use of allotype marked chimeric mice, constructed by reconstituting lethally irradiated recipients with adult bone marrow and peritoneal B-1 lymphocytes of recognizably different Ig allotypes, immunized with 1 microgram PVP, we found that although a substantial part of the total IgM produced in these chimeras bore the allotype of the transferred peritoneal B-1 cells, essentially all of the anti-PVP IgM expressed the allotype of the adult bone marrow. Fifteen of 16 hybridomas derived from a normal PVP-immune adult mouse bore N nucleotides at the V-D and D-J junctions of their heavy chain CDR3 regions, indicating their origin from precursors in the adult bone marrow. By use of ELISA spot analysis, we found the cells responding to PVP to be localized in the spleens of normal immunized mice. We then used multiparameter flow cytometric sorting to determine the cell surface phenotype of these cells. We found that the cells producing anti-PVP were greatly enriched in a small subpopulation with the phenotypic characteristics of B-1 cells; they were B220intermediate, CD5low, IgMhigh, IgDlow, CD43+ and CD23-. This subpopulation was also enriched for all cells producing IgM, regardless of specificity (the so-called 'spontaneous' antibody). We conclude that the B-1 phenotype is more likely a marker for a state of differentiation than for a discrete lineage of B cells.

Rous sarcoma virus-induced tumours in mice. II. Contribution of H-2 and non-H-2 alloantigen barriers to tumour immunogenicity in vivo.

The features of the immune recognition of a murine fibrosarcoma induced by Rous sarcoma virus were tested in histocompatible and histoincompatible mice. No evidence of a genetic regulation of spontaneous reactivity to tumour-associated antigens was found in various histocompatible F1 hybrids. Incompatibility in multiple minor histocompatibility antigens triggers a host reaction incapable of causing tumour rejection in some cases. The growth rate of incipient tumours is unaffected, whereas that of already visible tumour masses is significantly delayed. Admixture to the challenge of inactivated leukocytes bearing the same minor histocompatibility antigens as the tumour triggers a significantly stronger reaction. The reaction of hosts incompatible in the H-2K or H-2DL regions is quite efficient. However, the intensity of the immune reaction of H-2DLs antigens displayed by tumour cells is markedly dependent on the alleles of genes located in the central regions of the H-2 complex.

Rous sarcoma virus-induced tumors in mice--I. Macrophage-mediated natural cytotoxicity.

Lymphoid cells from normal untreated A.SW mice showed a cell-mediated cytotoxicity (CMC) towards Rous sarcoma virus-induced tumor lines in a 48-hr microcytotoxicity assay in vitro, using 3[H]-thymidine prelabeled tumor cells. The tissue distribution of this natural CMC shows high levels of activity in the peritoneal cavity, intermediate levels in bone marrow and spleen, and low levels in lymph node, peripheral blood and thymus. Natural CMC of unstimulated resident peritoneal cells against tumor cells was also found in several strains of mice. This was not dependent on the donor's age, since similar levels appeared in mice aged 1-40 weeks. Passage through nylon wool columns or adherence to the plastic surface of resident peritoneal cells removed cytotoxic activity. By contrast, adherent cell displayed significant CMC levels. Treatment with carbonyl iron powder and magnet decreased peritoneal cell cytotoxicity when performed once and abolished it when repeated. All these results suggest that in mice, natural cytotoxicity against Rous sarcoma virus-induced tumors is primarily mediated by macrophages or macrophage-like cells.

Ig isotype switching in B lymphocytes. Isolation and characterization of clonal variants of the murine Ly-1+ B cell lymphoma, CH12, expressing isotypes other than IgM.

We have selected and cloned variant cells from the murine B cell lymphoma, CH12, that produce a variety of other Ig isotypes in addition to or in place of the original IgM and IgD. Variants were selected by flow cytometry and automated cloning and isotype production was analyzed by membrane immunofluorescence and ELISA of culture fluids. Variants have been isolated that produce the single isotypes IgA, IgG2b, and IgG3, as well as variants that produce more than one isotype simultaneously, i.e., IgM, IgD, and IgA; IgG2b and IgA; IgG3 and IgA. All isotypes have been seen as cell surface proteins and all except IgD have been found in culture supernatants. All isotypes display the same idiotype and Ag-binding specificity for phosphatidyl choline as the original IgM and all are translated from the same VDJH and VJ kappa gene assemblies. Production of more than one isotype by a variant clone is due to simultaneous production of all the isotypes by each cell within the clone. The finding that the variants producing more than one isotype are all tetraploid suggests the interesting possibility that each isotype is derived from an independently switching chromosome. All isotype variants can be stimulated by LPS to secrete the appropriate Ig isotype at an increased rate similar to the IgM expressing parent. The variants differ in stability; some have remained stable for more than 9 months in culture, whereas other have undergone further isotype switching. The facts that some isotypes have not been seen, that multistep switching has occurred, and that many variants produce IgA in addition to another isotype are discussed in relation to current notions of isotype switching mechanisms.

Ig isotype switching in B lymphocytes. A method for estimating isotype switch frequency in cloned B cell lymphomas.

We have developed a method for enumerating the frequency of Ig isotype switching in clones of B cells. The method adapts Poisson statistics to analyze the distribution of amounts of switched isotype produced by multiple subclones of cells and thus enables one to estimate the probability that a single cell will switch isotype in one cell generation. We have applied this method to determine the spontaneous switch frequency of two Ly-1+ B cell lymphomas of B10-H-2aH-4bp/Wts mice. Both CH12.LX and CH27.LX switch from IgM to IgA at very high frequencies (1 - 5 x 10(-3) switch events per cell division) and from IgM to IgG at low but detectable frequencies (10(-4) - 10(-5) switch events per cell division). Cloned IgG variants of CH12.LX switch to IgA at the same frequency as the IgM-producing cells. Bacterial lipopolysaccharide has a strong inhibitory effect on isotype switching by CH12.LX. Possible explanations for the observed preference for switching to IgA are discussed.

Ig isotype switching in B lymphocytes. The effect of T cell-derived interleukins, cytokines, cholera toxin, and antigen on isotype switch frequency of a cloned B cell lymphoma.

The murine B cell lymphoma CH12.LX, which bears cell surface IgM specific for the phosphatidyl choline epitope of sheep red blood cells, is capable of spontaneous isotype switching in vitro. Switching to IgG3, IgG1, IgG2b, and IgA has been observed and variants expressing those isotypes have been isolated and cloned. We have developed a procedure for precise numerical evaluation of the frequency of switching to the several isotypes to which CH12.LX can switch. We have used a modified Poisson method which can distinguish between treatments which change isotype switch frequency and those which affect, in an isotype-specific fashion, growth or secretion rates of cells which have already switched. In this report we examine the effect of several cytokines, cholera toxin, hydroxyurea, and antigen on the isotype switch frequency of CH12.LX. The strongest effect observed was that of transforming growth factor-beta, which increases switch frequency 40-fold to an absolute switch frequency of 0.04 switch events (from IgM to IgA expression) per cell division. Interleukin-4 (IL-4) and cholera toxin also increase the switch frequency of CH12.LX while IL-5, IL-6 (with or without antigen), antigen (SRBC) alone, interferon-gamma, or hydroxyurea have no effect. We have shown that none of the cytokines studied change the relative frequency of switching to the available isotypes, only the absolute frequency of switching. We infer from this that the factors tested do not 'instruct' CH12.LX to switch to a particular isotype, but rather they deliver a 'go' signal to cells committed to switching to IgA at high frequency, rarely to IgG3, IgG1, or IgG2b, and never to IgG2a or IgE.

Immune recognition of tumor cells in vivo. I. Role of H-2 gene products in T lymphocyte activation against minor histocompatibility antigens displayed by adenocarcinoma cells.

Challenges from ADK-1t adenocarcinoma cells of BALB/c (H-2d, Mlsb) mouse origin are rejected by DBA/2 (H-2d, Mlsa) mice on the basis of differences in a limited number of minor histocompatibility antigens. This T lymphocyte-dependent reaction is highly specific, and efficiently triggered only by Ia+ leukocytes infiltrating the tumor mass. ADK-1t challenges depleted of Ia+ infiltrating BALB/c leukocytes grow and kill DBA/2 mice, whereas the simultaneous injection of Ia+-inactivated BALB/c leukocytes induces tumor rejection. The expression of Mlsb-incompatible determinants on the Ia+ BALB/c leukocyte membrane is irrelevant in the induction of this efficient T lymphocyte reaction against BALB/c minor histocompatibility antigens. By contrast, a critical requirement is H-2 matching between the Ia+ leukocytes and the recipient mice at the inductive phase of the reaction.