Efficient transduction of hemopoietic CD34+ progenitors of human origin using an original retroviral vector derived from Fr-MuLV-FB29: in vitro assessment.

A novel retroviral vector has been designed based on a Friend-murine leukemia virus (Fr-MuLV) FB29 strain. The latter has been selected according to characteristics of pathogenicity in mice where it induces a disease of the haemopoietic system affecting all lineages. Higher infectivity has also been demonstrated as compared to other strains. In accordance with these findings, the amphotropic producer clone used in this study carrying along the neomycine resistance gene (FOCH-Neo), harbors viral titers over 10(7) cfu/ml. To investigate the potential of genetically engineering hematopoietic precursors, CD34+ progenitors were selected from cord blood, bone marrow, and peripheral blood mobilized stem cells (patients + solid tumors) and transduced with FOCH-Neo. High transduction rates were achieved using virus supernatant and minimal doses of hematopoietic growth factors during pretransduction and transduction steps. A polymerase chain reaction (PCR) assay investigating the presence of both neomycin-encoding and viral vector sequences tested positive in 45-90% of granulocyte-macrophage colony-forming units (CFU-GM) generating cells (bone marrow and peripheral blood derived cells) following transduction. An average of 35% colonies showed resistance to G418. Such levels of transduction proved reproducible using only supernatants harboring over 10(7) cfu/ml. In those experiments where long-term in vitro cultures could be maintained over 5 weeks (all cord blood and 5 among 23 PBSC), efficient transduction of long-term culture initiating cell (LTC-IC) hematopoietic progenitors was demonstrated on the basis of both resistance to G418 and virus integration. In the latter case, the PCR assay tested positive in as much as 35-60% of late unselected CFU-colonies. This novel retroviral vector harbors interesting features toward genetic modification of hematopoietic progenitors.

A human minor histocompatibility antigen which appears to segregate with the major histocompatibility complex.

We obtained a cell line (So1) from a patient who rejected a T-depleted allogeneic BMT. Cytotoxic activity by cell-mediated lympholysis was found using So1 as effector and EBV-transformed donor B cells as targets, but no lysis of the patient's pretransplantation cells and of an unrelated HLA-nonidentical subject was observed, suggesting it was related to recognition of a minor transplantation antigen which could have contributed to rejection of the graft. To define the HLA-restricting element(s), cell-mediated lympholysis experiments were performed with several B cell lines as targets. So1 lysed only targets sharing an HLA-B44 antigen with the patient, thus demonstrating that the minor transplantation antigen recognized was restricted by HLA-B44. The absence of lysis against the patient's pretransplantation cells may be related to the absence of the minor antigen, suggesting that the patient's cytotoxic lymphocytes able to recognize a minor transplantation antigen on the donor cells contributed to the rejection of the HLA-identical graft. Mendelian segregation of this minor antigen was found in familial studies. Lysis was observed with cells from members of 2 families who had an association of HLA-B44 antigen in the haplotype and the minor antigen, whereas in 2 other HLA-B44-positive families, no lysis was found, probably because this minor antigen was absent. Furthermore, these family studies: (1) demonstrated that this minor antigen segregates with the MHC, suggesting its localization on chromosome 6; and (2) showed a close relationship between the minor antigen and HLA-B44, strongly suggesting a linkage disequilibrium between the minor antigen and its restriction antigen B44.

Linkage analysis in spinal muscular atrophy, by six closely flanking markers on chromosome 5.

The proximal spinal muscular atrophies (SMA) represent the second most common autosomal recessive disorder, after cystic fibrosis. The gene responsible for chronic SMA has recently been mapped to chromosome 5q by using genetic linkage studies. Among six markers mapping to this region, five were shown to be linked with the SMA locus in 39 chronic SMA families each containing at least two affected individuals. Multilocus analysis by the method of location score was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between loci D5S6 and D5S39. The genetic distances between these two markers are estimated to be 6.4 cM in males and 11.9 cM in females. Since meiosis were informative with D5S39 and D5S6 in 92% and 87% of SMA families, respectively, it is hoped that the present study will contribute to the calculation of genetic risk in SMA families.

Production of a monoclonal antibody (2B44) reactive on a shared epitope on dendritic reticulum cells, smooth muscle cells of vessels and Reed-Sternberg cells.

Lymph node cells from a patient with Hodgkin's disease (HD) were cultured without Epstein-Barr virus (EBV) or leukine adjuvant. A cell line (719-AB) emerged from the culture after four weeks. The cell line express CD20 (79%), CD 21 (30%), CD30 (63%), CD 35 (61%) antigens and weakly CD25 (19%). using Southern Blot technique, the existence of specific EBV DNA and polyclonal immunoglobulin genes rearrangement were observed in the cell line. In order to obtain a monoclonal antibodies (MoAb), mice Balb/C were immunized with this cell line. The splenic cells suspension of immunized animals were fused with the mouse myeloma NS1. Antibody IgM kappa from secreting clones 2B44 was studied using both indirect immunofluorescence with labeled anti-mouse immunoglobulin and immunohistochemistry based on alkaline phosphatase/antiphosphatase complex (APAAP) and ModAMeX technique on a panel of normal or pathological cells. Normal peripheral lymphocytes, monocytes, polymorphonuclear cells, and erythrocytes, did not react. The MoAb 2B44 recognized the dendritic reticulum cells and the smooth muscle cells of vessels on frozen section and paraffin section from HD or reactive lymph nodes. On specially processed paraffin sections (ModAMeX) Reed-Sternberg cells (RSC) were reactive with 2B44 MoAb (in 2 cases out of 5 tested). The molecular weight of the antigen recognized by 2B44 MoAb is of 37 kd. The description of a new epitope shared by different histological components might be of interest for defining a new cluster and better understanding the nature of RSC.

Nucleotide sequence of the HLA-A26 class I gene: identification of specific residues and molecular mapping of public HLA class I epitopes.

A cosmid clone bearing an HLA class I gene has been isolated from a human genomic library by hybridization to a class I-specific probe. This clone encodes the HLA-A26 molecule characterized by immunologic reagents on murine transfected L cells. Nucleotide sequencing of the A26 allele has been performed, and the deduced amino acid sequence was compared with previously published HLA class I sequences. Amino acid sequence homologies between HLA-A26 molecules and members of the HLA-AW19 cross-reactive group were observed and allowed us to demonstrate that residue Q144 is the only critical residue involved in the binding of the 4E monoclonal antibody defining an epitope common to all HLA-B, -C, and -Aw19 alleles. This study also permitted designation of a V residue at position 189 in the third domain as possibly involved in the binding of the B1-23-2 monoclonal antibody. Furthermore, we located clusters of variability in reference to the three-dimensional structure of the HLA-A molecules, i.e., the ninth residue of the first beta-strand domain, the upper surface of the first helical region, and both beta and alpha structures of the alpha 2 domain.

Phenotype and function of T lymphocytes infiltrating the skin during graft-versus-host disease following allogeneic bone marrow transplantation.

Seven lymphocyte populations were expanded from skin samples of patients with acute or chronic GVHD following allogeneic genotypically identical BMT. After amplification without in vitro antigenic stimulation or addition of mitogens, 5 of the 7 cell lines showed a large majority of mature CD4+ T cells (in contrast to published immunopathological data). One cell line showed an equal number of CD4+ and CD8+ cells, and another a predominance of CD4+ cells along with a large number of cells with a phenotype suggestive of non-MHC-restricted CTLs. After in vitro antigenic stimulation, various cytotoxicity patterns were seen: specific antihost cytotoxicity was seen in half the cell lines, NK activity was seen in 5 of the 7 lines, and a strong LAK activity was seen in 1 of the 7 cell lines. These results point to a diversity of cytotoxic effectors involved locally in GVHD and emphasize the need for further study of these local events. The cell lines established now constitute basic functional material for the in vitro study of cellular and humoral interactions at the site of GVHD lesions.

Chromosomal assignment of two myosin alkali light-chain genes encoding the ventricular/slow skeletal muscle isoform and the atrial/fetal muscle isoform (MYL3, MYL4).

In all eukaryotes, myosin plays a major role in the maintenance of cell shape and in cellular movement; in association with actin and other contractile proteins it is also a major structural component of the muscle sarcomere. Several isoforms of myosin alkali light chain have been identified, associated with different muscle types. We have recently localized the gene encoding the fast skeletal muscle alkali light-chain isoforms MLC1F and MLC3F (HGM symbol, MYL1) to human chromosome 2q32.1-qter (Cohen-Haguenauer 1988). We present here the chromosomal assignment of two loci encoding the ventricular muscle isoform MLC1V (equivalent to the slow skeletal muscle isoform MLC1Sb) and the atrial muscle isoform MLC1A (equivalent to the fetal isoform MLC1emb) using a panel of 25 independent man-rodent somatic cell hybrids. The MLC1V gene (HGM symbol, MYL3) was mapped to human chromosome 3 using a human full-length cDNA probe that hybridizes to a single major human TaqI2.8-kb fragment. The MLC1A probe (HGM symbol, MYL4) was a 360-bp mouse cDNA fragment that gave a distinct signal with human DNA using low stringency conditions of hybridization and washings and after presaturation of the Southern blots with rodent DNA. A single PstI 7.8-kb fragment gives an intense signal, and its presence correlates with the presence of chromosome 17 among the hybrids. These data are in keeping with the localizations of the MLC1V gene to mouse chromosome 9, and of the MLC1A gene to mouse chromosome 11, which share some markers in common with human chromosomes 3 and 17 respectively.

Localization of the acetylcholine receptor gamma subunit gene to human chromosome 2q32----qter.

The nicotinic acetylcholine receptor of skeletal muscle (CHRN in man, Acr in mouse) is a transmembrane protein composed of four different subunits (alpha, beta, gamma, and delta) assembled into the pentamer alpha 2 beta gamma delta. These subunits are encoded by separate genes which derive from a common ancestral gene by duplication. We have used a murine full-length 1,900-bp-long cDNA encoding the gamma subunit subcloned into M 13 (clone gamma 18) to prepare single-stranded probes for hybridization to EcoRI-digested DNA from a panel of human x rodent somatic cell hybrids. Using conditions of low stringency to favor cross-species hybridization, and prehybridization with rodent DNA to prevent rodent background, we detected a single major human band of 30-40 kb. The pattern of segregation of this 30-40 kb band correlated with the segregation of human chromosome 2 within the panel and the presence of a chromosomal translocation in the distal part of the long arm of this t(X;2)(p22;q32.1) chromosome allowing the localization of the gamma subunit gene (CHRNG) to 2q32----qter. The human genes encoding the gamma and delta subunits have been shown to be contained in an EcoRI restriction fragment of approximately 20 kb (Shibahara et al., 1985). Consequently, this study also maps the delta subunit gene (CHRND) to human chromosome 2q32.1----qter. In the mouse, the Acrd and Acrg genes have been shown to be linked to Idh-1, Mylf (IDH1 and MYL1 in humans, respectively) and to the gene encoding villin on chromosome 1. Interestingly, we have recently localized the human MYL1 gene to the same chromosomal fragment of human chromosome 2. These results clearly demonstrate a region of chromosomal homoeology between mouse chromosome 1 and human chromosome 2.

MHC related genetic susceptibility to Hodgkin's disease.

The present report describes 4 Caucasoid families, HLA genotyped, with at least 2 affected siblings suffering from Hodgkin's disease. The affected sibling pairs were identical (2 shared haplotypes) in 2 families and haploidentical (1 shared haplotype) in the 2 others. These results together with the data already published provide evidence for a distortion of the segregation of HLA haplotypes: from a total of 43 pairs of siblings reported, the observed repartition is 22, 15, 6 (2, 1, 0 shared haplotypes respectively) instead of 10.75, 21.5, 10.75 (mendelian repartition). The excess of identical siblings pairs (51% instead of 25%) (p less than 10(-5) confirms the existence of a genetic linkage between the chromosomal HLA region and the susceptibility to the disease.

Erythema multiforme is associated to HLA-Aw33 and DRw53.

Erythema multiforme is an acute eruption of the skin and mucous membranes of various aetiologies. Forty-one unrelated patients were HLA typed for 53 specificities of the HLA-A, B, C, DR and DQ series. Frequencies of Aw33 and DRw53 were significantly increased: Aw33, 17.0% in patients vs 2.8% in controls (corrected p = 0.01, relative risk = 7.2); DRw53, 70.7% in patients vs 30.5% in controls (corrected p = 0.0005, relative risk = 5.5).

Study of cis and trans interactions between extended HLA-haplotypes in insulin-dependent diabetes.

From the study of HLA, A, B, C, DR, Bf and C4A, C4B alleles in 287 insulin-dependent diabetes mellitus patients and 108 controls, comparisons were made between 424 diabetic and 216 normal extended haplotypes. In the "cis" situation (haplotype), the highest relative risks (RR) for IDDM were borne by multiloci allelic associations, mainly DR/complement alleles, rather than by DR3 or DR4 considered alone. Susceptibility was strongly associated with two extended haplotypes (Aw30, Cw5, B18, C4BQ0, C4A3, BfF1, DR3 and A2, Cw3, B15, C4Bx, C4A3, BfS, DR4) or their smaller segments. Two haplotypes, S31 associated with DR2 or DR5 and F31 associated with DRw6 or DR7 had a protective effect. In the "trans" situation (opposite haplotype) the large excess of DR3/DR4 heterozygotes was not the only distortion observed. An excess of DR1 (57%) and of C4BQ0 (40%) was noted among non DR3, non DR4 haplotypes in diabetics compared to normal individuals (26% and 23%, respectively, P less than 0.01, 0.05). Homozygotes for DR3 or DR4 were not increased, and other homozygotes were decreased compared to controls. The protective antigens HLA DR2, DR5 and DR7 seemed not to be distributed randomly: their putative protective effect was not observed in the case of combination with DR1 or a B18, DR3 haplotype. DR2 was never found homozygous or combined with DR5. These results suggest that susceptibility to IDDM is generated by both cis and trans interactions between genes or gene products of the HLA region.

Isolation of a human major histocompatibility complex class I gene encoding a nonubiquitous molecule expressed on activated lymphocytes.

The human major histocompatibility complex is a multigene family containing at least 20 class I genes. Included within this family are the loci encoding the highly polymorphic HLA-A, -B, and -C antigens present at the surface of most nucleated cells. The large number of genes detected with class I probes by Southern blot analysis and the existence of serological reagents defining nonubiquitous, non-HLA-A,B,C class I antigens suggest that products other than HLA-A,B,C antigens are encoded within the class I gene family. These products might be the human counterparts of the murine Qa and TL antigens. In order to identify non-HLA-A,B,C genes, we have developed a probe, JF11, located in noncoding regions flanking the HLA-A locus. This probe detects only a limited number of class I genes and does not detect HLA-A,B,C-associated restriction fragments on Southern blots. This probe was used to screen a human cosmid library. Some of the cosmids isolated with this probe were then transferred into mouse fibroblasts expressing human beta 2-microglobulin. One of the transfectants specifically reacts with one alloantiserum (HA2) that detects HLA class I molecules specific to HLA-A2-positive, phytohemagglutinin-activated T cells and not found on resting T or B cells. Data presented in this paper provide evidence for the isolation and expression of a class I gene encoding a nonubiquitous class I antigen that could be a human analogue of the murine Qa antigens.

An influenza A virus-specific and HLA-DRw8-restricted T cell clone cross-reacting with a transcomplementation product of the HLA-DR2 and DR4 haplotypes.

The clone TA10 is a T3+ T4+ T8- proliferative and cytolytic human T cell clone. This clone has been shown to be specific for the hemagglutinin of influenza A Texas virus and restricted by an HLA class II molecule associated with the DRw8-Dw8.1 phenotype. Here we show that TA10 and all of its subclones can also react with eight HLA-DRw8 negative, Epstein-Barr virus (EBV)-transformed cell lines or phytohemagglutinin blasts in the absence of influenza antigens. All of these cell lines are HLA-DR2/DR4 with a classic DR2 long haplotype. The only nonreactive HLA-DR2/DR4 cell line observed bears a DR2 short haplotype. Only heterozygous HLA-DR2/DR4 but not parental DR2 or DR4 EBV-transformed cell lines can be recognized by TA10, indicating that the cross-reacting determinant is a transcomplementation product between HLA-DR2 and HLA-DR4 haplotypes. DR-specific, but not DQ- or DP-specific monoclonal antibodies, inhibit in the proliferation assay and in the chromium release test both the DRw8-Dw8.1-restricted and the anti-DR2/DR4 reactions. These results show that HLA-DR-restricted, anti-viral human T cell clone can evidence cross-reactivity for allospecific class II molecules of the major histocompatibility complex, and human CTL can recognize transcomplementation products of class II HLA genes. In addition, the results suggest that a beta-chain coded for by an HLA-DR gene and associated with an alpha-chain coded for by a still unidentified but possibly HLA-DQ gene constitute this functional transcomplementation product.