Gene gun-mediated IL-12 gene therapy induces antitumor effects in the absence of toxicity: a direct comparison with systemic IL-12 protein therapy.

Using three murine tumor models, we compared the antitumor efficacy and certain physiological effects of an in vivo interleukin-12 (IL-12) gene therapy protocol and a systemic IL-12 protein therapy protocol. An IL-12 cDNA gene construct was administered in situ into skin tissue via gene gun delivery, and recombinant IL-12 protein was administered subcutaneously at a dose of 1 microgram/mouse/treatment. Both treatment regimes induced a comparable level of regression of established intradermal MethA sarcomas. In B16 melanoma and P815 mastocytoma models, antitumor efficacy of IL-12 protein therapy appeared to be slightly higher than that of IL-12 gene therapy; however, the protein therapy protocol in this comparative study resulted in a high level of mortality of mice. It was also demonstrated that IL-12 gene therapy, in contrast to the IL-12 protein therapy, was not associated with weight loss, splenomegaly, increased Ly6 antigen expression in the spleen, or visible signs of toxicity, such as fur ruffling and lethargy. Moreover, serum levels of interferon-gamma (IFN-gamma) induced in response to IL-12 gene therapy were 300-1000 times lower than those induced by the systemic IL-12 protein administration. Together, these results suggest that gene gunmediated in vivo delivery of IL-12 cDNA may be considered as a safer alternative to IL-12 protein therapy for certain human cancers.

DNA cancer vaccines: a gene gun approach.

A wide variety of approaches, all using gene transfer, have been tested experimentally as alternative means to vaccinate against cancer, either prophylactically or therapeutically. These include both ex vivo and in vivo gene transfer to tumour and/or non-tumour cells, using both viral and non-viral vectors. The transferred DNA has varied widely as well, including genomic or cDNA encoding tumour-associated or oncofoetal antigens, cytokines, histocompatibility molecules, and costimulatory molecules. Several of these approaches have been applied in human clinical trials. This review summarizes those approaches, then compares and evaluates various methods using cytokine DNA in conjunction with autologous tumour cells, with particular emphasis on particle-mediated gene transfer via a gene gun. Finally, prospects and needs for further development are discussed.

Immune responses induced by intramuscular or gene gun injection of protective deoxyribonucleic acid vaccines that express the circumsporozoite protein from Plasmodium berghei malaria parasites.

The circumsporozoite protein (CSP) is a target for effector Ab and cell mediated immunity against malaria parasites; DNA vaccination can induce both types of effector response. The immunogenicity and efficacy of two DNA plasmids expressing different amounts of Plasmodium berghei CSP were evaluated by immunizing BALB/c mice i.m. or epidermally and by varying the number of immunizations (one to three doses) and the interval between immunizations. Expanding the interval gave the strongest effect, increasing efficacy and antibody boosting, and, in the case of epidermal vaccination, promoting a switch in CSP-specific IgG isotypes from IgG1 to a balance with IgG2a. The strongest humoral immune response and the greatest level of protection were induced by vaccinating epidermally with high expresser plasmid, using a gene gun to administer three doses at 6-wk intervals. For this group, the mean, repeat-specific, prechallenge antibody titer among mice not infected after challenge was significantly higher than that in infected mice, but the mean prechallenge titers for antibody reactive with whole sporozoites were not significantly different. The interval-dependent induction of IgG2a antibodies by epidermal vaccination contradicts the widely held belief that antibody responses induced by this method are restricted to those that are Th2 dependent.

Dual expression of human leukocyte antigen molecules and the B7-1 costimulatory molecule (CD80) on human melanoma cells after particle-mediated gene transfer.

The aim of this study was to determine if human melanoma cells could be molecularly modified by particle-mediated gene transfer with a "gene gun", using genes for interferon-gamma (IFN-gamma), the B7-1 costimulatory molecule (CD80), and human leukocyte antigen (HLA)-A2, to augment expression of both HLA molecules and B7-1. Established and early passage melanoma cells transfected with human IFN-gamma complementary DNA (cDNA) produced IFN-gamma (50-5,000 pg/mL). The biological effect of this IFN-gamma transgene included an upregulation, or de novo appearance, of HLA expression. These melanoma cells had no detectable baseline surface expression of the B7-1 costimulatory molecule, but 8% to 31% of these cells became B7-1 positive with no selection procedure after gene transfer with human B7-1 cDNA. After combination gene transfer with cDNAs for both IFN-gamma and B7-1, 9% to 33% of these cells expressed both HLA-DR and B7-1. In combination gene transfer experiments with cDNAs for both HLA-A2 and B7-1, dual expression of HLA-A2 and B7-1 was achieved in 10% to 17% of the melanoma cells. Thus, the molecular modification of human melanoma cells to increase expression of both HLA and B7-1 can be achieved by particle-mediated gene delivery and presents a promising strategy to stimulate antimelanoma T-cell immunity. Key words: Melanoma; T cells; B7-1 costimulatory molecule (CD80); major histocompatibility complex.

Nonviral and viral delivery of a human immunodeficiency virus protective gene into primary human T cells.

Because AIDS has been refractory to traditional pharmacologic interventions, alternative approaches have been developed. Although the introduction of specific antiviral genes into T leukemia cells can provide relative resistance to human immunodeficiency virus (HIV) replication, the testing of such genes against primary viral isolates in human CD4+ lymphocytes has been limited, and safety questions remain regarding gene delivery into cells from HIV-infected patients. In this report, we evaluate the efficacy of a transdominant mutant protein, Rev M10, against cloned and primary HIV isolates in human peripheral blood lymphocytes and describe different methods of gene transfer into peripheral blood lymphocytes from HIV-infected individuals. We show that gold microparticles can mediate stable Rev M10 gene transfer into these cells. Introduction of Rev M10 by these techniques conferred resistance to HIV infection in vitro to cloned and clinical isolates. Nonviral delivery of HIV protective genes will facilitate the development of gene therapy for AIDS and the analysis of viral and cellular gene expression in human T lymphocytes.

Linkage analysis of maturity-onset diabetes of the young (MODY): genetic heterogeneity and nonpenetrance.

We have analyzed the inheritance of maturity-onset diabetes of the young (MODY) on chromosome 20 in a large multigeneration family, the R.-W. family, and in two other MODY families. Of the four branches of the R.-W. pedigree which have been studied, two have documented early onset of non-insulin-dependent diabetes mellitus (NIDDM), while there is no evidence of early onset in the other two branches. The early-onset branches have apparently inherited the same D20S16 allele from the affected parent, while another D20S16 allele was inherited in the two branches without evidence of early onset. A test for homogeneity, the M-test, using the results of two-point linkage analysis with D20S16 indicates heterogeneity between early- and late-onset branches of the R.-W. family (P less than or equal to .014). In addition, analysis strongly suggests that MODY as expressed in the EDI and WIS families is unlinked to loci on chromosome 20 (P less than or equal to .018-.004). Comparable results are seen when the data are analyzed by the HOMOG program. Three polymorphic loci-D20S16, D20S17, and ADA--show no recombination with the MODY locus when two-point linkage analysis is used in the early-onset branches of the family. The multipoint lod score in the early-onset branches of the R.-W. family is 10.16, with the most likely location being between D20S4 and D20S17. Multipoint linkage analysis using the CHROMPICS option of the program CRI-MAP has been used to follow inheritance of the MODY disease locus. This analysis has identified two cases of possible nonpenetrance in the early-onset branches of the family (odds of at least 156:1), as determined by the appearance of apparent isolated double crossovers at the MODY locus in these unaffected individuals.

HLA and insulin-dependent diabetes. A protective perspective.

This article presents a model for the HLA effect in insulin-dependent diabetes mellitus (IDDM) that is almost the mirror image of a model suggested by Nepom. In the Nepom model, the products of certain HLA alleles are associated with IDDM because they bind and present a specific peptide or peptides so as to induce an immune response to pancreatic beta-cells; certain other alleles can protect against IDDM if they compete strongly for binding of the diabetogenic peptide. My model focuses instead on the failure of the immune system to maintain tolerance to pancreatic beta-cells. I suggest that the HLA alleles negatively associated with IDDM (e.g., DR2 and DQw1) produce products with high affinity for certain beta-cell peptide or peptides needed to establish and maintain tolerance to beta-cells, whereas the alleles that are common in IDDM (e.g., DR3, DR4, and DQw8) produce products that have low affinity for the tolerogenic peptide or peptides or that bind the peptide or peptides in the wrong orientation or configuration for establishing tolerance. I also discuss the multiplicity of HLA loci, alleles, and amino acids contributing to IDDM and the fact that the associations of specific loci, alleles, and even genotypes with IDDM depend not only on their intrinsic properties but also on various population parameters.

The cryptic HLA-DQA2 ("DX alpha") gene is expressed in human B cell lines.

Although there are two gene pairs in the HLA-DQ region, DQA1/B1 and DQA2/B2, the latter pair have never been found to be expressed. The DQA2 and DQB2 genes appear to be normal at the DNA sequence level, there being no indication that they should be pseudogenes, and normal splicing occurs when DQA2 is included in a retroviral vector. The putative amino acid sequences of DQA2 and DQB2 have been nearly invariant, raising the questions of how these genes have been maintained normal-appearing and nearly monomorphic if they are not expressed and, if they are expressed, in what cell types are they expressed. We present Northern blot, nuclease protection assay, and mRNA sequence data that demonstrate the existence of spliced DQA2 mRNA in human B lymphoblastoid cell lines. In contrast, we found no evidence of DQB2 mRNA in the same cell lines, nor of DQA2 mRNA in the monocyte line U937 or in activated T cells.

T cell defined HLA epitopes and T cell receptor polymorphism in insulin dependent diabetes mellitus.

T cell defined epitopes on class II HLA molecules (epitopes distinguishable by T cells but not by antibodies) seem to be important determinants of IDDM susceptibility/resistance. Although HLA-DR4 is associated with IDDM in many populations, DR4-positive HLA haplotypes vary greatly (relative risk from greater than 10 to less than 1). This variation seems to depend on both the DQ allele and T cell defined subtypes of the DR4 allele. These IDDM associated alleles at the two loci (DQB1 and DRB1) are not correlated with each other in the healthy population, so they clearly are independent risk factors. HLA-DR2 has universally been associated with lack of IDDM, and seems to be protective. However, not all DR2 haplotypes protect, and the protection or lack of protection correlates with T cell defined subtypes of DR2. In this case, however, the DR2 subtypes do correlate with DQ alleles, so it is unclear which locus (loci) is (are) actually affecting the disease process. It may be significant that, for both DR2 and DR4, only the more protective subtypes have arginine at amino acid position 71. Other portions of the DR beta chain are clearly important, however. Although TCR alpha and beta seemed to be promising candidates for additional IDDM susceptibility genes, in fact the various TCR alpha and beta haplotypes are equal, or nearly equal, with regard to IDDM susceptibility. The importance of HLA alleles in IDDM susceptibility, and the lack of importance of TCR alpha and beta alleles, may be due to the different means by which the HLA and TCR molecules achieve antigen binding diversity: HLA molecules by multiple loci and allelic diversity, and TCR molecules by the tremendous diversity that can be generated from a single TCR allele during T cell maturation.

Human genes for the immunoglobulin gamma-chain (Gm) allotypes and for the T cell receptor alpha-chain are distantly linked or unlinked.

Since the genes for two of the T cell receptor (TCR) chains (alpha and delta) and for the immunoglobulin (Ig) heavy chains (alpha, gamma, delta, epsilon and mu) are all on the long arm of human chromosome 14, it is of interest to determine if they are genetically linked and, if so, how strongly. We tested genetic polymorphisms of the TCR alpha-locus and Ig gamma-locus in 19 families comprising 107 members, and asssessed the frequency of recombination between the two loci. The results rule out strong linkage: all recombination fractions below 0.26 gave lod scores less than -2 (odds less than 1:100). Combining the data on maternal and paternal meioses, the most likely recombination fraction was 0.5 (nonlinkage).

HLA-DQA2 (DX alpha) polymorphism and insulin dependent diabetes.

A certain HLA-DQA2 locus TaqI fragment, DX alpha"U", has been reported to be associated with insulin-dependent diabetes mellitus (IDDM). Reports of various studies in this vein have ranged from stating that the association of DQA2"U" with IDDM exists even among subjects positive for HLA-DR3 and -DR4 to stating that the association of DQA2"U" with diabetes can be attributed to linkage disequilibrium between the DQA2"U" and some component(s) on the affected haplotypes. Using a synthetic 97-base probe corresponding to a portion of an intron of DQA2, in a Southern blot analysis of IDDM and control subjects from Wisconsin, we were able to confirm the association of DQA2"U" with diabetes. However, among DR3 subjects there was no significant association between DQA2"U" and diabetes (p = 0.26). Although there was a (nonsignificant) association of IDDM with DQA2"U" among DR4-positive subjects (p = 0.14), this can be completely attributed to linkage disequilibrium between DQA2"U" and DQw8. We also sequenced most of the second exon (corresponding to the alpha 1 domain of the DQA2 glycoprotein) from five individuals that were homozygous for either DQA2"U" or DQA2"L." The only polymorphisms observed were a "silent" mutation at position 36 and one example of a difference that would result in a change of amino acid at position 41.

Allelic T-cell receptor alpha complexes have little or no influence on susceptibility to type 1 diabetes.

We performed a multiple-affected-sib study to determine if T-cell receptor alpha-chain alleles affect susceptibility to insulin-dependent diabetes mellitus. Restriction fragment length polymorphisms were used to follow the segregation of allelic T-cell receptor alpha complexes within the families. The segregation of T-cell receptor alpha alleles in 29 multiplex families revealed no significant tendency for affected sibs to share T-cell receptor alpha-chain alleles more often than would be expected by chance alone (p greater than 0.2). In contrast, the same type of analysis for HLA alleles easily detected the well-known linkage of insulin-dependent diabetes mellitus susceptibility to the HLA complex (p = 0.003). We suggest that the importance of HLA alleles in insulin-dependent diabetes mellitus susceptibility and the lack of importance of T-cell receptor alpha alleles result from the different strategies by which HLA and T-cell receptor molecules achieve antigen-binding diversity: multiple loci and allelic diversity in the case of HLA; combinatorial, junctional, and N-region diversity in the case of the T-cell receptor. In this paper we also describe three new restriction fragment length polymorphisms of the T-cell receptor alpha complex and a new method for testing the significance of linkage in multiple-affected-sib studies.

Correlation of structure with T cell responses of the three members of the HLA-DRw52 allelic series.

A third allele at the DRB3 locus, DRw52c, represents an intermediate sequence between DRw52a and DRw52b and may have arisen by a gene conversion-like event. The recognition of cells bearing these molecules by a number of alloreactive and antigen-specific DR-restricted T cell clones was analyzed. On the basis of a theoretical model of HLA class II structure, distinct amino acid clusters have been identified as motifs controlling TCR recognition. These are located both in the cleft and in the alpha-helical edge of the MHC class II recognition platform. Motifs shared between two alleles may restrict public T cell clones.

A diabetes-susceptible HLA haplotype is best defined by a combination of HLA-DR and -DQ alleles.

HLA-DR4 is associated with insulin-dependent diabetes mellitus (IDDM) in many populations. Many recent studies suggest that the DR4 effect is really due to DQ3.2, an allele of the nearby DQB1 locus. We used T cell clones, MAb, and allele-specific oligonucleotides to test IDDM and control subjects for DR4 subtypes (Dw4, Dw10, Dw13, and Dw14) and for DR4-associated DQB1 alleles (DQ3.1 and DQ3.2). We find that (a) IDDM is approximately equally associated with alleles of the DRB1 locus (Dw4 and Dw10, combined relative risk, RR = 6.4) and the DQB1 locus (DQ3.2, RR = 5.9); and (b) there is significant interaction, in a statistical sense, between these DR and DQ alleles in IDDM. The only IDDM-associated DR4 haplotypes were those carrying the IDDM-associated alleles at both loci (RR = 12.1); haplotypes with Dw4 or 10 but not DQ3.2, or vice versa, had a RR less than 1. Alternative explanations include: (a) that susceptibility requires specific allelic products of both DR and DQ loci; (b) that the combination of certain DR and DQ alleles marks haplotypes with the true susceptibility allele at a third locus; or (c) that Dw4 and 10 mark haplotypes with an allele at another locus that interacts with DQ3.2. As discussed, this third locus is unlikely to be DQA1 (DQ alpha). The data thus are not easily reconciled with an exclusive effect of HLA-DQ. This information increases our ability to predict IDDM by genetic typing: in the population studied, heterozygotes DR3/[DQ3.2, Dw4] or DR3/[DQ3.2, Dw10] had a relative risk of 38.0 and an absolute risk of 1 in 15.

Lack of interaction of HLA and IgG heavy chain allotypes in insulin-dependent diabetes mellitus.

Because of conflicting previous reports showing the presence or absence of Gm-HLA interaction in insulin-dependent diabetes mellitus (IDDM), we report results for a group of Wisconsin families having 2 or more siblings with IDDM. Although this study is very similar to one by Field et al., who found HLA-Gm interaction in IDDM, we find no evidence for such an interactive effect p = 0.33). We discuss published data on HLA-Gm interaction in IDDM, and conclude that overall there is little reason to postulate such an interaction.

T-cell-defined DR4 subtypes as markers for type 1 diabetes.

In most populations studied, HLA-DR4, a DRB1 (formerly DR beta I) allele, is increased in frequency among patients with insulin-dependent diabetes mellitus (IDDM). Using T-cells, one can distinguish five subtypes of DR4 (Dw4, Dw10, Dw13, Dw14, and Dw15). Two of these (Dw4 and Dw10) are IDDM-associated in the populations studied here. Therefore, Dw4 and Dw10 could be causative or merely markers for a linked diabetes allele. If they are causative, one might expect them to share some unique structural element or at least to associate consistently with IDDM in different populations. Published sequence data show no structural element unique to Dw4 and Dw10; moreover, the associations of these DR4-Dw subtypes with diabetes vary considerably in different populations. Thus the DRB1 locus probably cannot account for the DR4 association in IDDM. The strong linkage disequilibrium between IDDM and Dw4 and Dw10 suggests that the diabetes susceptibility locus should be in the vicinity of the DR region or the DQ region of the HLA complex. Alternative hypotheses are discussed, relating DR- and DQ-region alleles to IDDM. We further postulate that the evolutionary event that produced the Dw10 allele occurred on a Dw4 haplotype that happened to carry a diabetes susceptibility allele at another locus.

Functional polymorphism of the HLA-DR beta III chain.

Several clusters of class II HLA genes contribute to variation in human antigen-presenting capacity. In the HLA-DR cluster, most of the variation is due to the highly polymorphic DR beta I gene. Recent work by others has shown some nucleotide and implied amino acid sequence variation in DR beta III chains, but this variation is not known to be functionally significant. We show here that two proliferating human T-cell clones define three allelic variants of DR beta III (assignment to DR beta III based on blocking of proliferation by selected monoclonal antibodies). Thus, the DR beta III locus encodes at least three alleles that are distinguishable by human T cells and most probably contribute to the human antigen-presenting repertoire. The three DR beta III alleles subdivide the "supertypic" HLA antigen DRw52 into subtypes provisionally called DRw52.1-52.3. The DR3 haplotypes studied to date have been either DRw52.1 or 52.2; DR5 haplotypes have all (23 of 23) been 52.2; DRw6 haplotypes have included all three DRw52 subtypes, nearly half being 52.3. Our data, combined with other published data, imply that DRw8 must either have a fourth DRw52 subtype or be DR beta III null.

Differential expression of T cell differentiation antigens and major histocompatibility antigens on activated T cells during the cell cycle.

In this report we have analyzed cell cycle-related fluctuations of both quantity and density of the T cell differentiation antigens, CD3 (T3), CD4 (T4) and CD8 (T8), as well as the major histocompatibility complex (MHC) antigens on the cell surface of activated T cells. Phytohemagglutinin-activated T cells cultured for 3 days with or without conditioned medium or for 10 days with conditioned medium and mixed lymphocyte culture-derived T cell clones were used for the analysis. Correlated measurements of the surface antigen quantity (immunofluorescence), DNA content (dye Hoechst 33342), and cell size (light scatter), not influenced by synchrony induction methods and cell fixation, were performed by dual-beam flow cytometry. Our results demonstrate that the T cell differentiation antigens, CD3, CD4 and CD8, and class I MHC antigens are increased in density in the G1 phase for all activated T cells tested. In contrast, class II MHC antigens are increased in density in the G2 phase of activated T cells maintained with conditioned medium. Since it is known that the T cell differentiation antigens and class I MHC antigens on activated T cells are necessary for proliferation of T cells, our study suggests that this effect is more significant in the G1 phase. The cell cycle changes in expression of class I and class II MHC antigens, but not of the T cell differentiation antigens, appear to be mediated by soluble factors, probably including interferon-gamma, which could produce a differential increase of class I and class II MHC antigens on G2 phase cells.

A minor subset of HLA-DR3 haplotypes is preferentially increased in type 1 (insulin-dependent) diabetes.

We have been using human T-lymphocyte clones specifically sensitized to detect leucocyte antigens of Type 1 (insulin-dependent) diabetic patients in the hope of detecting novel HLA antigens associated with Type 1 diabetes. We previously described two such clones which define a new class II HLA antigen, Boston-1 (BO1). BO1 is found mainly on cells of persons with particular HLA-DR antigens and, of potential significance for diabetes, BO1 identifies a distinctive subset of DR3 haplotypes. We report here that BO1+ DR3 haplotypes are overrepresented in Type 1 diabetes. That is, significantly more of the DR3-positive subjects are BO1-positive in the patient group (31%) than in the control group (8%), suggesting that a diabetes-susceptibility gene may be more common on the BO1+ than on the BO1- DR3 haplotypes. Alternative interpretations are also discussed.

A particular subset of HLA-DR4 accounts for all or most of the DR4 association in type I diabetes.

Two human T-lymphocyte clones, derived from a mixed leukocyte culture (MLC) with stimulating cells from a type I diabetic patient, define a subset of HLA-DR4, tentatively called "DR4S." In testing of 69 random type I diabetic subjects and 69 random controls, 79% (37/47) of DR4-positive patients, but only 44% (8/18) of DR4-positive controls, had DR4S (P less than 0.01). The relative risk of type I diabetes for DR4S+ individuals was 8.8, while that for DR4+ DR4S- individuals was only 1.0. Thus, in the population tested, DR4S accounts for all or most of the increased frequency of HLA-DR4 in type I diabetes.