CD4+ T cells from IRF-1-deficient mice exhibit altered patterns of cytokine expression and cell subset homeostasis.

Interferon regulatory factor-1 (IRF-1) is a member of a family of transcription factors that regulate an array of genes involved in cell growth, differentiation, and death. Analysis of cytokine expression by stimulated CD4+ cells from IRF-1(-/-) and IRF-1(+/+) mice revealed that IRF-1 deficiency resulted in an elevated production of Th2-related cytokines and a compensatory decrease in the expression of naive cell- and Th1-related cytokines. The altered cytokine profiles of IRF-1(-/-) cells could be explained, in part, by a shift in the representation of subsets of CD4+ cells; IRF-1(-/-) mice exhibited a decreased percentage of naive cells (a major source of IL-2) but increased numbers of memory or effector cells (the source of Th2-related cytokines). We analyzed purified, phenotypically matched memory/effector cells from IRF-1(-/-) and IRF-1(+/+) mice and found that the increased Th2:Th1 cytokine ratio was still evident in the IRF-1(-/-) group, thus suggesting that IRF-1 is involved in the polarization of the cytokine repertoire in CD4+ cells. Our data indicate that IRF-1 plays an important role in the maintenance of CD4+ cell subset homeostasis and in the expression of cytokines by naive and memory/effector cells.

Apoptosis induced by HIV infection in H9 T cells is blocked by ICE-family protease inhibition but not by a Fas(CD95) antagonist.

Infection of human CD4-positive T lymphocytes by human immunodeficiency virus type 1 (HIV-1) is thought to lead to death of infected cells by apoptosis, although one recent report questions this conclusion. Here we demonstrate that HIV-1-induced apoptosis of the H9 human T cell line is blocked by peptide inhibitors of IL-1 beta converting enzyme (ICE)-family proteases, but not by the antagonistic M3 anti-Fas Ab. Apoptosis occurred in all phases of the cell cycle, not selectively in G2 as a consequence of vpr-mediated cell cycle arrest. We conclude that apoptosis accounts for all cell death related to HIV-1 infection of the human CD4-positive cell line H9, requires an ICE-like protease but is not Fas mediated, and occurs in all phases of the cell cycle.

A radiation hybrid map of 15 loci on the distal long arm of chromosome 4, the region containing the gene responsible for facioscapulohumeral muscular dystrophy (FSHD).

A physical map of 4q35 was constructed through radiation hybrid analysis of 134 clones generated from the cell line HHW416, a chromosome 4-only human-hamster somatic cell hybrid. This subtelomeric region contains the as-yet-unidentified gene responsible for facioscapulohumeral muscular dystrophy. The most likely order of 15 loci within 4q35 was determined. The loci ordered on this radiation hybrid map include both genes and polymorphic loci, as well as monomorphic loci which cannot be placed on a genetic linkage map. The physical distance spanning these loci was estimated to be approximately 4.5 Mb, by using a kilobase/centiray conversion factor derived from 4p16.3 marker analysis through the same set of radiation hybrids. The comparison of this physical map to establish genetic maps suggests that this region is smaller than initially estimated and that recombination rates are increased near the telomere.

Tandem linkage of genes coding for leukemia inhibitory factor (LIF) and oncostatin M (OSM) on human chromosome 22.

Leukemia-inhibitory factor (LIF) and oncostatin M (OSM) are members of a family of structurally similar growth factors presenting overlapping and specific functions. Although the genes coding for IL-6, CSF3 and CNTF are scattered in the human and mouse genome, human LIF and OSM genes have conserved synteny in the course of evolution. Through isolation of a YAC and a cosmid clone containing both LIF and OSM we demonstrate that the two genes are linked in tandem on human chromosome 22q12, separated by 16 kilobases of intervening genomic DNA and transcribed in the same head-to-tail orientation. The close physical linkage between LIF and OSM genes brings new evidence of their evolutionary relationship.

Structure and linkage of the D2 dopamine receptor and neural cell adhesion molecule genes on human chromosome 11q23.

The gene encoding the D2 dopamine receptor (DRD2) is located on human chromosome 11q23 and has been circumstantially associated with a number of human disorders including Parkinson's disease, schizophrenia, and susceptibility to alcoholism. To determine the physical structure of the DRD2 gene, we utilized cosmid cloning, isolation of yeast artificial chromosomes (YACs), and pulsed-field gel electrophoresis to construct a long-range physical map of human chromosome 11q23 linking the genes for the DRD2 and neural cell adhesion molecule (NCAM). The D2 dopamine receptor gene extends over 270 kb and includes an intron of approximately 250 kb separating the putative first exon from the exons encoding the receptor protein. The resulting physical map spans more than 1.5 mb of chromosome band 11q23 and links the DRD2 gene with the gene encoding the NCAM located 150 kb 3' of the DRD2 gene and transcribed from the same DNA strand. We additionally located the sites of at least four hypomethylated HTF islands within the physical map, which potentially indicate the sites of additional genes. High-resolution fluorescent in situ suppression hybridization using cosmid and YAC clones localized this gene cluster between the ApoAI and STMY loci at the interface of bands 11q22.3 and 11q23.1.

Detection and characterization of "chimeric" yeast artificial chromosome clones by fluorescent in situ suppression hybridization.

"Chimeric" yeast artificial chromosomes (YACs) are clones containing two or more noncontiguous segments of DNA and represent the most common artifact found in total genomic YAC libraries currently used for large-scale genome mapping. These YACs create spurious mapping information that complicates the construction of YAC contigs and leads to erroneous maps during chromosome walks. The presence of these artifactual clones necessitates laborious and time-consuming characterization of each isolated YAC clone, either by comparison of the physical map of the YAC with the corresponding source genomic DNA, or by demonstrating discrepant chromosomal origins for the two ends of the YAC by hybridization or polymerase chain reaction (PCR). Here, we describe a rapid and sensitive method for the assessment of YAC colinearity by fluorescence in situ suppression hybridization (FISSH) by utilizing fluorescein-12-dUTP for labeling YAC clones. We have analyzed 51 YACs and found that 43% (22 out of 51) are chimeric and significantly larger (302 kb) than colinear ones (228 kb). One of the 51 YAC clones (2%) examined contains portions of three chromosomes and 2 (4%) seem to map to a chromosome different than that of the identifying STS. FISSH analysis offers a straightforward visualization of the entire YAC insert on the chromosomes and can be used to examine many YACs simultaneously in few days.

Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast.

Yeast artificial chromosomes (YACs) provide a powerful tool for the isolation and mapping of large regions of mammalian chromosomes. We developed a rapid and efficient method for the isolation of DNA fragments representing the extreme ends of YAC clones by the insertion of a rescue plasmid into the YAC vector by homologous recombination. Two rescue vectors were constructed containing a yeast LYS2 selectable gene, a bacterial origin of replication, an antibiotic resistance gene, a polylinker containing multiple restriction sites, and a fragment homologous to one arm of the pYAC4 vector. The 'end-cloning' procedure involves transformation of the rescue vector into yeast cells carrying a YAC clone, followed by preparation of yeast DNA and transformation into bacterial cells. The resulting plasmids carry end-specific DNA fragments up to 20 kb in length, which are suitable for use as hybridization probes, as templates for direct DNA sequencing, and as probes for mapping by fluorescence in situ hybridization. These vectors are suitable for the rescue of end-clones from any YAC constructed using a pYAC-derived vector. We demonstrate the utility of these plasmids by rescuing YAC-end fragments from a human YAC library.

Two distinct mechanisms account for the immune response (Ir) gene control of the T cell response to pigeon cytochrome c.

Previous experiments have demonstrated that the immune response of MHC congenic mice to pigeon cytochrome c is under Ir gene control. Expression of I-E-encoded gene products influences both the magnitude and fine specificity of the Th cell response to pigeon cytochrome c and phylogenetic derivatives. Results of those experiments implicate both determinant selection and repertoire selection as mechanisms of Ir gene control in this system. In this report we have compared the TCR expressed in pigeon cytochrome c-reactive Th cells from B10.A(I-Ek), B10.A(5R) (I-Eb), and B10.S(9R) (I-Es) mice. The B10.A(5R) strain is a low responder to pigeon cytochrome c, but in response to moth cytochrome c this strain produces T cells which respond to pigeon or moth cytochrome c on B10.A APC. These cells are phenotypically identical to the predominant clonal phenotype seen in the B10.A response to pigeon cytochrome c. In this report, we show that the B10.A and B10.A(5R) pigeon cytochrome c-reactive T cells express essentially identical T cell receptors. These results, coupled with recent studies reporting a relatively low affinity for I-Eb molecules by pigeon cytochrome c peptides compared with moth cytochrome c peptides, strongly argue that the immune response defect in the B10.A(5R) strain is due to a defect in Ag presentation (determinant selection). In contrast, B10.A and B10.S(9R) strains are high responders to pigeon cytochrome c. Both strains produce T cell clones which are capable of responding to cytochrome c presented by either B10.A or B10.S(9R) APC in vitro. We show that, even in T cells with this MHC restriction degeneracy, the TCR expressed in the two strains are different. Because the APC of both strains can clearly present the cytochrome c Ag, we conclude that the differential expression of the TCR in the responses is due to a T cell repertoire selection difference in the two strains. Thus, for the response to one Ag in three MHC congenic strains, there exists evidence that both determinant selection and repertoire selection can be mechanisms of Ir gene control of an immune response.

Selection of amino acid sequences in the beta chain of the T cell antigen receptor.

The induction of an immune response in mammals is initiated by specifically reactive T lymphocytes. The specificity of the reaction is mediated by a complex receptor, part of which is highly variable in sequence and analogous to immunoglobulin heavy- and light-chain variable domains. The functional specificity of the T cell antigen receptor is, however, markedly different from immunoglobulins in that it mediates cell-cell interactions via the simultaneous recognition of foreign antigens and major histocompatibility complex-encoded molecules expressed on the surface of various lymphoid and nonlymphoid cells. The relation between the structure of the receptor and its functional specificity was investigated by analyzing the primary sequences of the receptors expressed by a series of T lymphocyte clones specific for a model antigen, pigeon cytochrome c. Within this set of T lymphocyte clones there was a striking selection for amino acid sequences in the receptor beta-chain in the region analogous to the third complementarity-determining region of immunoglobulins. Thus, despite the functional differences between T cell antigen receptors and immunoglobulin molecules, analogous regions appear to be important in determining ligand specificity.

The influence of MHC gene products on the generation of an antigen-specific T-cell repertoire.

We have previously described the B10.A pigeon cytochrome c-specific response in terms of clonal phenotypes and T-cell receptor (TcR) gene usage. All B10.A T-cell clones studied respond to antigen in association with syngeneic B10.A APCs and cross-react to antigen in association with one or two allogeneic variants of the I-E-encoded MHC molecules. In congenic strains of mice expressing these allogeneic MHC alleles [B10.A(5R) and B10.S(9R)], pigeon cytochrome c-specific T cells exhibit very similar MHC cross-reactivities. Our goal was to determine whether the same MHC cross-reactive T-cell clones were expressed in each appropriate strain, or whether each T-cell repertoire was unique. The results indicate that identical V alpha-J alpha and V beta-J beta combinations were expressed by the major pigeon cytochrome c-specific response phenotype in B10.A and B10.A(5R) mice. Previous functional data supports this overlap in expressed T-cell clones. B10.A and B10.S(9R) mice exhibit similar response phenotypes to pigeon cytochrome c but express distinctly different TcR genes. The results of these studies support the existence of at least two different mechanisms in determining MHC-linked immune response polymorphisms.

The molecular basis of alloreactivity in antigen-specific, major histocompatibility complex-restricted T cell clones.

We have studied the relationship between major histocompatibility complex (MHC)-restricted antigen recognition and alloreactivity by examining T cell receptor (TCR) alpha and beta gene expression in cytochrome c-specific, Ek alpha:Ek beta (Ek)-restricted helper T cell clones derived from B10.A mice. The clones could be segregated on the basis of four distinct alloreactivity patterns. Clones cross-reactive for three different allogeneic la molecules (As alpha:As beta [As], Ab alpha:Ab beta [Ab], Ek alpha: Eb beta [Eb]) expressed the same V alpha and V beta gene segments, generating the distinct alloreactive specificities via unique V alpha-J alpha and V beta-D beta-J beta joining events. Ek alpha:Es beta (Es)-alloreactive B10.A clones expressed the same V alpha, J alpha, and V beta segments as an Es-restricted, Ek-alloreactive, cytochrome c-specific, H-2-congenic B10.S(9R) clone. This homology between TCRs mediating allorecognition of la molecules and recognition of the same la molecules as restriction elements associated with nominal antigen suggests that MHC-restricted recognition and allorecognition represent differences in the affinity of the TCR-MHC molecule interaction.

Correlations between T-cell specificity and the structure of the antigen receptor.

The derived amino-acid sequences of the heterodimeric antigen receptors expressed by a series of murine T-cell clones are presented. A comparison of the receptor sequences indicates that several mechanisms for generating receptor diversity can influence T-cell specificity, including junctional diversity, combinatorial joining, and combinatorial chain associations.

Immunologic aspects of the prostate.

Our experiments involving the use of the Dunning R3327 adenocarcinoma as an animal model of prostatic cancer as well as clinical studies on the immunocompetence of prostatic cancer patients are described. Utilizing the Dunning tumor, we have demonstrated that this transplantable adenocarcinoma of the rat prostate was similar to human prostatic cancer with respect to its macroscopic and microscopic appearances, growth rate, growth differential in male and female recipients, and some of its metastatic potential. Cryosurgery was capable of destroying the primary tumor as it can in man. Both antibody and cellular immune responses could be produced against antigens associated with the tumor cells. Tumor-bearing rats treated by cryosurgery in combination with BCG were capable of producing an antitumor immunity that protected them from rechallenge. Clinical studies of prostatic cancer patients showed a diminished in vitro immunity, but the responses of the cancer patients were not significantly different from those of patients with benign prostatic disease.