Autologous human monocyte-derived dendritic cells genetically modified to express melanoma antigens elicit primary cytotoxic T cell responses in vitro: enhancement by cotransfection of genes encoding the Th1-biasing cytokines IL-12 and IFN-alpha.

DNA-based immunization strategies designed to elicit cellular antitumor immunity offer an attractive alternative to protein- or peptide-based approaches. In the present study we have evaluated the feasibility of DNA vaccination for the induction of CTL reactivity to five different melanoma Ags in vitro. Cultured, monocyte-derived dendritic cells (DC) were transiently transfected with plasmid DNA encoding human MART-1/Melan-A, pMel-17/gp100, tyrosinase, MAGE-1, or MAGE-3 by particle bombardment and used to stimulate autologous PBMC responder T cells. CTL reactivity to these previously identified melanoma Ags was reproducibly generated after two or three stimulations with genetically modified DC. Co-ordinate transfection of two melanoma Ag cDNAs into DC promoted CTL responders capable of recognizing epitopes from both gene products. Coinsertion of genes encoding the Th1-biasing cytokines IL-12 or IFN-alpha consistently enhanced the magnitude of the resulting Ag-specific CTL reactivity. Importantly, DC transfected with a single melanoma Ag cDNA were capable of stimulating Ag-specific CTL reactivity restricted by multiple host MHC alleles, some of which had not been previously identified. These results support the inherent strengths of gene-based vaccine approaches that do not require prior knowledge of responder MHC haplotypes or of relevant MHC-restricted peptide epitopes. Given previous observations of in situ tumor HLA allele-loss variants, DC gene vaccine strategies may elicit a greater diversity of host therapeutic immunity, thereby enhancing the clinical utility and success of such approaches.

I-region restriction of alloantigen recognition: alloantigen expressed on the surface of a hybridoma cell must be presented in the context of self class II major histocompatibility complex determinants for recognition by a dual-reactive T-cell clone.

A helper-T-lymphocyte clone, designated A10, proliferated in response to both hen egg ovalbumin (OVA) presented in the context of self I-Ak and to the alloantigen I-As. The alloantigen source could be provided by irradiated H-2s spleen cells and also by paraformaldehyde-fixed H-2s spleen cells. However, for fixed allogeneic spleen cells to stimulate proliferation of the cloned cells, it was necessary to add irradiated syngeneic I-Ak-bearing spleen cells, as fixed H-2s spleen cells added, by themselves, to A10 cells were nonstimulatory. We have extended these findings by generating a monoclonal hybridoma cell which expressed the I-As allodeterminant. Similar to our results with fixed allogeneic spleen cells, this source of alloantigen could stimulate A10 cells to proliferate only if irradiated syngeneic spleen cells were added to the cultures. These proliferative responses were effectively inhibited by anti-I-Ak monoclonal antibody (mAb) and by anti-I-As mAb. Furthermore, the response of A10 cells to the alloantigen-bearing hybridoma cells were also inhibited by the anti-L3T4 mAb GK1.5. Collectively, these data indicate that, in some situations, alloreactivity may be mediated by self class II major histocompatibility complex restriction of alloantigen-driven proliferation.

Cell surface structures involved in T cell activation.

We have discussed four specific models which provide different kinds of information about the requirements for T cell activation. The first utilized a CTL clone designated L3, which is reactive specifically with Ld alloantigen, to study the involvement of the associative recognition structure Lyt-2 in cytolysis. The apparent requirements for activation of this CTL clone differ depending on whether the target cells bear specific alloantigen or are hybridoma cells which express on their cell surface a clonotypic antibody which reacts specifically with the L3 T cell receptor for antigen. When the antigen receptor reacts with alloantigen on the allogeneic target cell, cytolysis is inhibited by anti-Lyt-2 antibody. However, when the clonotypic antibody of the target cell reacts with the antigen receptor of the T cell, cytolysis is much less inhibited by anti-Lyt-2 antibody. The antigen receptor seems to be responsible for the specificity of both these interactions but the avidity of the interaction between CTL and target cell seems to differ in the two situations. Evidence that participation of the L3T4 associative recognition structure on HTL is less important for cloned T cells which have higher affinity antigen receptors was provided by the second model system which used cloned HTL selected for optimal responses to different concentrations of nominal antigen. Proliferative responses of those clones which responded to lower antigen concentrations were less readily inhibited by anti-L3T4 mAb. Evidence provided by these two model systems is consistent with the concept that associative recognition structures are of lesser importance for T cell activation for those T cells which have higher affinity antigen receptors. In the third model system, we have identified several monoclonal antibodies which augment proliferative response of cloned T cells to sub-optimal amounts of IL-2, probably by reacting with the antigen receptor or with the associated Leu-4/T3 structure. The reactivity patterns of these antibodies indicate that several different epitopes are being recognized. Some appear to be clonotypic although they do not block functional activity of the clone with which they react. Others react with all T clones which we have tested. Several of these react with a cell surface antigen which is expressed at about the same level as the clonotypic structures: these antibodies may react with the murine equivalent of the human Leu-4/T3 molecular complex. One of the "pan-T cell" antibodies which augments IL-2-induced T cell proliferation appears to react with Thy-1; this antibody is similar to one described recently by Gunter et al. (1984).(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence implicating I region-restricted antigen presentation in alloantigen and nominal antigen recognition by a dual-reactive helper T lymphocyte clone.

A helper T lymphocyte clone, designated A10, was generated from spleen cells of a B10.A mouse and demonstrated reactivity to both the nominal protein antigen hen egg ovalbumin (OVA) presented by I-Ak-bearing antigen-presenting cells (APC) and irradiated I-As-bearing spleen cells in the absence of OVA. A stimulatory signal was delivered to the cloned cells by syngeneic spleen cells that were exposed to OVA and then fixed with paraformaldehyde. However, paraformaldehyde-fixed allogeneic spleen cells that bear the I-As determinant recognized by a monoclonal antibody (mAb) were unable, by themselves, to stimulate the A10 cells. The inability of fixed allogeneic spleen cells to stimulate was rectified by the addition of irradiated I-Ak-positive spleen cells, suggesting that at least in this situation, alloantigen must be presented to the alloreactive A10 cells. Further evidence supporting this proposal for class II-restricted presentation of alloantigen included the observations that 1) irradiated I-Ak-negative spleen cells were unable to present the fixed H-2s spleen cells, 2) the anti-I-As mAb blocked the stimulatory signals delivered by irradiated H-2s spleen cells and by fixed H-2s spleen cells plus irradiated syngeneic spleen cells, 3) some anti-I-Ak mAb preparations were able to inhibit stimulation by OVA and by fixed H-2s spleen cells plus irradiated syngeneic spleen cells, and 4) an anti-L3T4a mAb effectively blocked all three stimulatory signals. These data suggested that alloreactivity can be mediated by an antigen-presentation process similar to that proposed for nominal peptide antigen presentation, and that alloantigen in the form of paraformaldehyde-fixed allogeneic spleen cells is recognized in the context of self-determinants before a stimulatory signal can be delivered.

Assessment of the tracheal ring bioassay for detection of the cystic fibrosis serum factor.

We have examined the effects of serum from cystic fibrosis patients, healthy human volunteers and from guinea pigs on ciliary activity of guinea pig tracheal ring explants after 48 hours in culture. Sera from 9 out of 10 cystic fibrosis patients produced ciliostasis. This is a significantly greater percentage than the 7 out of 21 serum samples from healthy volunteers that produced ciliostosis. Ninety-two percent of the explants in guinea pig serum had unaltered ciliary activity, illustrating the importance of intrinsic control in the bioassay design. These results suggest that the guinea pig tracheal ring bioassay may be of value as a means of identifying the presence of the ciliotoxic factor in cystic fibrosis serum for research use but is a poor discriminator for diagnostic purposes. Modification such as rinsing the tracheal mucosa with sterile medium and a new chamber for the microscopic observation of the tissue have simplified the assay.

Impaired proliferative response to B-lymphocyte activators in common variable immunodeficiency.

The cell-mediated immune responses of 39 patients with common variable immunodeficiency (CVI) were studied in vitro, using Staphylococcus aureus and Escherichia coli prepared as whole cells and Candida albicans extract. These microbial activators wee found to require intact B-lymphocyte function for normal proliferative response. The patient group was observed to have significantly depressed lymphocyte responses compared wit those of controls studied in parallel (P less than 0.01). Negative lymphocyte response to one activator and strongly positive response to another were found in individual patients. Examination of patients' lymphocyte response to S. aureus and E. coli in association with serum IgG levels demonstrated that a rough correlation could be drawn, showing that patients with serum IgG less than 125 mg/dl had markedly lower (P less than 0.01) lymphocyte responses than those with serum IgG greater than 300 mg/dl. No similar correlation with phytohaemagglutinin activation was observed. Since depressed lymphocyte responses did not correlate with reduced B-cell number in these patients, intrinsic B-lymphocyte deficiency was indicated. These preparations of microbial activators are potentially useful tools in exploring lymphocyte subpopulation functions in primary immunodeficiency diseases.