A rapid HLA-DRB1*04 subtyping method using PCR and DNA heteroduplex generators.

We describe here a rapid polymerase chain reaction (PCR)-based method for the identification of HLA-DRB1*0401-*0412 alleles. The method is based on the generation of specific DNA heteroduplex patterns between PCR products derived from selective group-specific amplification of the various DRB1*04 alleles and PCR products derived from two synthetic DNA heteroduplex generator (DHG) molecules following non-denaturing polyacrylamide minigel electrophoresis. One DHG was designed to detect DRB1*0401, *0405, *0407, *0408, and *0409 alleles, whilst the other was designed to detect DRB1*0402, *0403, *0404, *0406, *0410, *0411, and *0412 alleles. Characteristic heteroduplex patterns were obtained for all DRB1*04 alleles tested both in homozygous and heterozygous situations. Both DHG and PCR-SSP (sequence-specific primer) typing were performed on 41 DRB1*04 positive DNAs from Singaporean Chinese blood donors and complete concordance in results was obtained. HLA-DRB1*0403, *0405, and *0406 were found to account for 95% of the DRB1*04 alleles in the population studied. The DHG technique described is technically simple and rapid since it essentially involves only two PCR amplifications per individual subtyping. The technique is particularly useful for resolving DRB1*04 combinations which are indistinguishable by PCR-SSO (sequence-specific oligonucleotide) or PCR-SSP subtyping.

Immunotherapy of human cancers.

Advances in molecular biology technologies have significantly facilitated the identification of functional genes which cause, promote or control a variety of human diseases. Through recombinant DNA and polymerase chain reaction technologies, individual genes responsible for specific diseases have been identified, and consequently, the prospect that these diseases might be "cured" through replacement of the defective genes by their normal counterparts become distinct possibilities. Therefore, the goal of gene therapy is to apply this technology to the treatment of human diseases. In addition to its logical role for the correction of inherited diseases caused by a missing or defective gene product, gene therapy also holds promise for treatment of acquired disorders such as human cancer through the introduction of genes whose products have been implicated in controlling the growth of cancer. In this report, we present our results on the introduction of allogeneic major histocompatibility complex genes into cancer cells as an approach to increase the host's immune response against cancer. Various gene delivery strategies have been optimized for the introduction of DNA into various human tumour cells and these data are presented.

Generation of allo-reactive cytotoxic T lymphocytes by particle bombardment-mediated gene transfer.

Mature T lymphocytes comprise functionally distinct subsets with discrete roles in the regulation of the immune response. The cellular basis of the anti-tumor effect is now understood to involve the activation and expansion of tumor-specific cytotoxic T lymphocytes (CTL). To immuno-potentiate the generation of CTL, we have employed the biolistic system for the genetic immunization of mice. Here, we report the efficient generation of anti-H-2Kb allo-reactive CTL by particle acceleration-mediated genetic immunization of mouse spleen cells with H-2Kb DNA. The insertion and expression of exogenous gene into host spleen cells following in situ genetic inoculation to effect the generation of a cellular immune response may permit novel alternative strategies for immunotherapy.

Acquisition of immunogenicity by AKR leukemic cells following DNA-mediated gene transfer is associated with the reduction of constitutive reactive superoxide radicals.

We have employed the DNA-mediated gene transfer method to introduce the allogenic major-histocompatibility-complex(MHC)-class-I gene H-2Kb into the K36.16 tumor cells, H-2k, in order to generate tumor-specific immunity. The acquisition of immunogenicity by the H-2Kb-transformed clones following gene transfer is associated with the reduction of constitutive reactive superoxide radicals. When the levels of cellular superoxide for the H-2Kb-positive immunogenic clones were determined, they were significantly lower (30 to 60%) than that of the parental K36.16 tumor cells. This reduction of superoxide in the H-2Kb-transformed cells was associated with a significant increase in the level of Cu-Zn superoxide dismutase (SOD) and GPX I, together with a reduction in the DNA-binding form of the NF-kappa B transcription factor. The K36.16 parental tumor cells were also found to be relatively more resistant to the cytotoxic effects of hydrogen peroxide in vitro. To further support the role of superoxide anion radicals in tumorigenesis, in vivo depletion of glutathione promoted the tumorigenicity of the H-2Kb-transformed clones in (AKR/J x C57BL/6/J) F1 mice, whereas SOD was able to reduce their tumorigenicity. In addition, the presence of R-sulfoxine (BSO) in spleen-cell cultures in vitro abolished the ability of the immune lymphocytes to develop into tumor-specific cytotoxic T lymphocytes (CTL). These observations support the concept that oxidative processes in tumor cells may have a strong influence on the host response against tumors.

Promotion of tumor growth by transfecting antisense DNA to suppress endogenous H-2Kk MHC gene expression in AKR mouse thymoma.

Many AKR spontaneous thymomas are reported to express different amounts of the major histocompatibility complex class I H-2Kk molecules. Moreover, H-2Kk-deficient AKR tumor cells are found to be more malignant when compared to tumor cells that express abundant levels of the H-2Kk molecules. To corroborate further the role of H-2Kk in tumorigenesis of AKR leukemia, we have, in this study, expressed antisense H-2Kk RNA in a high-H-2Kk-expressing and poorly tumorigenic AKR thymoma cell line 369. The down-regulation of H-2Kk molecules in the transfected 369 clones rendered them more tumorigenic in syngeneic AKR/J mice. The increase in oncogenicity correlates well with a concomitant reduction in their susceptibility to tumor-specific cytotoxic T lymphocytes in vitro. These results suggest the relevance of H-2Kk molecules in the immune surveillance of AKR tumors.

Regulation of HLA class I gene expression in human colorectal carcinoma.

The under-representation of HLA antigens in human tumors is usually associated with a poor prognosis. In this report, the expression of HLA class I genes in human colorectal carcinomas was studied using HLA-A and HLA-B locus-specific probes. Over 50% of the colorectal carcinomas studied showed a reduction in the amount of steady state HLA class I mRNA. For some carcinomas, non-coordinated regulation of the HLA-A and HLA-B genes was observed. The mechanism of HLA suppression was investigated and is most likely due to the presence of transcriptional regulatory elements.

Tumor rejection mediated by transfection with allogeneic class I histocompatibility gene.

Non-self class I histocompatibility Ag can act as strong alloantigens and be recognized as distinct targets by CTL. To study the possibility of using allograft rejection to generate tumor-specific immunity, we have introduced an allogeneic class I histocompatibility gene, the H-2Kb gene, into a k haplotype tumor, K36.16, by DNA-mediated gene transfer. The K36.16 tumor grows readily and does not confer protective immunity in AKR mice. A total of 37 H-2Kb-transfected K36.16 clones (Kb/K36.16) was isolated and studied individually. The Kb/K36.16 clones were found to differ significantly in the amount of the exogenous H-2Kb antigens expressed on their cell surface. Moreover, as a result of the transfection, the level of expression of the endogenous H-2Dk Ag was also altered when compared to that of the parental K36.16 tumor cells. All the Kb/K36.16 clones that were positive for the H-2Kb Ag were rejected by the semisyngeneic AKR mice. Moreover, some of these Kb/K36.16 clones were also rejected by syngeneic (AKR x C57BL/10)F1 mice. In consequence of immunization with the Kb/K36.16 clones, the AKR and F1 mice were able to survive a subsequent challenge of the wild-type, unmodified, parental K36.16 tumor cells. More importantly, some of these Kb/K36.16 clones demonstrated an active and specific immunotherapeutic effect, and they were able to eradicate the growth of the parental K36.16 tumor cells in AKR mice. This observation therefore reinforces the feasibility of using DNA-mediated gene transfer as a molecular approach to abrogate tumor growth.