Mutation in the class II trans-activator leading to a mild immunodeficiency.

The expression of MHC class II molecules is essential for all Ag-dependent immune functions and is regulated at the transcriptional level. Four trans-acting proteins control the coordinate expression of MHC class II molecules: class II trans-activator (CIITA), regulatory factor binding to the X box (RFX)-associated protein; RFX protein containing ankyrin repeats, and RFX5. In humans, defects in these genes result in MHC class II expression deficiency and cause combined immunodeficiency. Most patients with this deficiency suffer from severe recurrent infections that frequently lead to death during early childhood. We investigated three sisters, now ages 21, 22, and 24 years, in whom MHC-II deficiency was detected. Even though the eldest sibling was asymptomatic and the other two had only mild immunodeficiency, none of the three class II isotypes was expressed on T cell blasts, fibroblasts, EBV B cell lines, or epidermal dendritic cells. Residual HLA-II expression was detected in fresh PBMC. Somatic complementation identified the disease as CIITA deficiency. A homozygous T1524C (L469P) substitution was found in the coding region of the CIITA cDNA and was shown to be responsible for the defect in MHC-II expression. This missense mutation prevents the normal functioning of MHC-II but does not lead to the nuclear exclusion of the L469P CIITA. Transfection experiments demonstrated that the CIITA L469P mutant had residual MHC class II trans activation activity, which might explain the unusual clinical course of the patients studied. This study shows that an attenuated clinical phenotype or an asymptomatic clinical course can be observed in patients despite a profound defect in the expression of MHC class II genes. The frequency of the inherited MHC class II deficiency might thus be underestimated.

Founder effect for a 26-bp deletion in the RFXANK gene in North African major histocompatibility complex class II-deficient patients belonging to complementation group B.

Expression of major histocompatibility complex (MHC) class II genes is controlled at the transcriptional level by at least four trans-acting genes, CIITA, RFXANK, RFX5, and RFXAP. Defects in these regulatory genes result in the absence of MHC class II molecule expression and, thereby, cause a combined immunodeficiency. MHC class II deficiency is inherited as an autosomal recessive trait. Since the first description of the disease, about 70 patients from 50 families have been reported. Forty-three of these families have been classified into four complementation groups: A, B, C, and D. In the largest group, B, the majority of patients are of North African origin. In two of these patients, the same mutation in the RFXANK gene (752delG-25) was identified. We performed a mutation analysis in 20 additional patients belonging to complementation group B and detected the 752delG-25 mutation in 17. All of these patients are of North African origin. A founder effect for this mutation was documented, since all tested patients, except one, display a common haplotype spanning the RFXANK locus.

In a novel form of IFN-gamma receptor 1 deficiency, cell surface receptors fail to bind IFN-gamma.

Complete IFN-gamma receptor ligand-binding chain (IFNgammaR1) deficiency is a life-threatening autosomal recessive immune disorder. Affected children invariably die of mycobacterial infection, unless bone marrow transplantation is undertaken. Pathogenic IFNGR1 mutations identified to date include nonsense and splice mutations and frameshift deletions and insertions. All result in a premature stop codon upstream from the segment encoding the transmembrane domain, precluding cell surface expression of the receptors. We report herein two sporadic and two familial cases of a novel form of complete IFNgammaR1 deficiency in which normal numbers of receptors are detected at the cell surface. Two in-frame deletions and two missense IFNGR1 mutations were identified in the segment encoding the extracellular ligand-binding domain of the receptor. Eight independent IFNgammaR1-specific mAb's, including seven blocking antibodies, gave recognition patterns that differed between patients, suggesting that different epitopes were altered by the mutations. No specific binding of (125)I-IFN-gamma to cells was observed in any patient, however, and the cells failed to respond to IFN-gamma. The mutations therefore cause complete IFNgammaR1 deficiency by disrupting the IFN-gamma-binding site without affecting surface expression. The detection of surface IFNgammaR1 molecules by specific antibodies, including blocking antibodies, does not exclude a diagnosis of complete IFNgammaR1 deficiency.

Partial interferon-gamma receptor signaling chain deficiency in a patient with bacille Calmette-Guérin and Mycobacterium abscessus infection.

Complete deficiency of either of the two human interferon (IFN)-gamma receptor components, the ligand-binding IFN-gammaR1 chain and the signaling IFN-gammaR2 chain, is invariably associated with early-onset infection caused by bacille Calmette-Guérin vaccines and/or environmental nontuberculous mycobacteria, poor granuloma formation, and a fatal outcome in childhood. Partial IFN-gammaR1 deficiency is associated with a milder histopathologic and clinical phenotype. Cells from a 20-year-old healthy person with a history of curable infections due to bacille Calmette-Guérin and Mycobacterium abscessus and mature granulomas in childhood were investigated. There was a homozygous nucleotide substitution in IFNGR2, causing an amino acid substitution in the extracellular region of the encoded receptor. Cell surface IFN-gammaR2 were detected by flow cytometry. Cellular responses to IFN-gamma were impaired but not abolished. Transfection with the wild-type IFNGR2 gene restored full responsiveness to IFN-gamma. This is the first demonstration of partial IFN-gammaR2 deficiency in humans.

A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection.

The immunogenetic basis of severe infections caused by bacille Calmette-Guérin vaccine and environmental mycobacteria in humans remains largely unknown. We describe 18 patients from several generations of 12 unrelated families who were heterozygous for 1 to 5 overlapping IFNGR1 frameshift small deletions and a wild-type IFNGR1 allele. There were 12 independent mutation events at a single mutation site, defining a small deletion hotspot. Neighbouring sequence analysis favours a small deletion model of slipped mispairing events during replication. The mutant alleles encode cell-surface IFNgamma receptors that lack the intra-cytoplasmic domain, which, through a combination of impaired recycling, abrogated signalling and normal binding to IFNgamma exert a dominant-negative effect. We thus report a hotspot for human IFNGR1 small deletions that confer dominant susceptibility to infections caused by poorly virulent mycobacteria.

Genetic and molecular definition of complementation group D in MHC class II deficiency.

Four complementation groups, A, B, C and D, have been described among cell lines defective in the coordinate expression of MHC class II genes. These include cell lines established from patients affected with MHC class II deficiency and experimentally generated mutant cell lines. Group D, in contrast to the other groups, was for a long time represented only by the 6.1.6 mutant cell line. The gene responsible for the defect in this group, RFXAP , recently was cloned and found to be mutated in the 6.1.6 cell line and in three patients. Here we report fusion experiments in several new HLA class II-deficient patients, completing the classification of the majority of known patients into the four complementation groups. Patients from five unrelated families were classified in complementation group D, while nine others fall into complementation groups A and B. None of the patients defined a new complementation group. Full correction of MHC class II expression was obtained in cells from patients belonging to group D by transfection with the RFXAP cDNA. The RFXAP coding region was found to be mutated in all patients. Mutations were found to be recurrent since only three different mutations have been found in the eight unrelated families reported to date.

Partial interferon-gamma receptor 1 deficiency in a child with tuberculoid bacillus Calmette-Guérin infection and a sibling with clinical tuberculosis.

Complete interferon-gamma receptor 1 (IFNgammaR1) deficiency has been identified previously as a cause of fatal bacillus Calmette-Guérin (BCG) infection with lepromatoid granulomas, and of disseminated nontuberculous mycobacterial (NTM) infection in children who had not been inoculated with BCG. We report here a kindred with partial IFNgammaR1 deficiency: one child afflicted by disseminated BCG infection with tuberculoid granulomas, and a sibling, who had not been inoculated previously with BCG, with clinical tuberculosis. Both responded to antimicrobials and are currently well without prophylactic therapy. Impaired response to IFN-gamma was documented in B cells by signal transducer and activator of transcription 1 nuclear translocation, in fibroblasts by cell surface HLA class II induction, and in monocytes by cell surface CD64 induction and TNF-alpha secretion. Whereas cells from healthy children responded to even low IFN-gamma concentrations (10 IU/ml), and cells from a child with complete IFNgammaR1 deficiency did not respond to even high IFN-gamma concentrations (10,000 IU/ml), cells from the two siblings did not respond to low or intermediate concentrations, yet responded to high IFN-gamma concentrations. A homozygous missense IFNgR1 mutation was identified, and its pathogenic role was ascertained by molecular complementation. Thus, whereas complete IFNgammaR1 deficiency in previously identified kindreds caused fatal lepromatoid BCG infection and disseminated NTM infection, partial IFNgammaR1 deficiency in this kindred caused curable tuberculoid BCG infection and clinical tuberculosis.

Two complementation groups account for most cases of inherited MHC class II deficiency.

MHC class II immuno-deficiency is a rare autosomal recessive disease due to a defect in transacting genes, which control the expression of the entire family of MHC alpha and beta class II genes. Previous analyses classified cells from eight MHC class II-deficient patients and four experimental mutant cell lines into four complementation groups, pointing to the existence of a large number of regulatory genes. We conducted fusion experiments with cell lines from two-thirds of all known patients and found that two complementation groups accounted for 20 of the 22 cases studied. These two complementation groups correspond closely to two ethnic groups: most patients of north African origin were classified into one group, while all patients originating from Spain were classified into a second main group. This suggests the existence of restricted number of ancestor mutations leading to this disease.

The presence of plasmin receptors on three mammary carcinoma MCF-7 sublines.

We studied plasmin receptors on 3 MCF-7 sublines: MCF7, MCF7R which was derived by transfection with v-Ha-ras oncogene, and MCF7MF which has been studied for the secretion of procathepsin D in the presence of estrogen. All 3 sublines bound plasmin (Pli) with a much higher affinity than plasminogen (Pg). The number of binding sites was increased about 4-fold by weak proteolytic pretreatment of tumor cells. Transfection by v-Ha-ras oncogene did not apparently change the affinity of Pli binding sites (KD 27-26 nM) and increased their number very slightly (3,800 against 3,200 fmoles/mg protein). However, this number was 5 times higher for MCF7MF (15,000 fmoles/mg protein) and the affinity was 4 times lower (106 nM). MCF7 and MCF7R sublines have previously been reported to be non-invasive in the in vitro invasion assay system (embryo chick heart muscle) but tumorigenic and metastatic in nude mice. In contrast, the MCF7MF subline has been shown to be invasive in both in vivo and in vitro invasion systems. Thus, the number of Pli binding sites at the MCF7 tumor-cell surface may be associated directly or indirectly with tumor-cell invasiveness.

Solubilization of the plasmin receptor from human carcinoma cells.

We could solubilize the plasmin receptor from the human colonic tumor cell line SW1116, using various detergents. Among these, Triton X100 and Sodium dodecyl sulfate were the most efficient. The solubilized receptor retained its plasmin binding activity, as judged by dot blot tests. After electrophoresis of the solubilisate in SDS polyacrylamide gels and transfer to nitrocellulose, specific plasmin fixation was still observed on autoradiograms as a sharp band in the 50K area. This band was not altered by treatment of the solubilisate with beta-mercaptoethanol.

Limited proteolysis of tumor cells increases their plasmin-binding ability.

Mild proteolytic treatment of SW1116 tumor cells with trypsin or plasmin increases their plasmin-binding ability considerably by increasing the number of binding sites without altering their affinity. This mechanism may be operative for increasing the concentration of active plasmin at the surface of tumor cells. C-terminal lysine residues are involved in plasmin binding to cells, since treatment of cells with carboxypeptidase B decreases this binding by 50%.

Immunohistochemical and biochemical study of a cathepsin B-like proteinase in human colonic cancers.

An immunohistological study was carried out on 51 human colorectal adenocarcinomas and eight samples of histologically normal colonic mucosa removed far from tumors, using anti-rabbit cathepsin B and anti-human cathepsin B immunoglobulins. Positive reactions were obtained on tumor cells and macrophage-like cells. However, as these immunoglobulins could not discriminate between cathepsin B and cathepsin B-like proteinases, and as they cross-reacted with cathepsins H and L, a partial characterization of the proteinase activities was performed in order to identify the type of enzyme present in the positive cells. The levels of cathepsins H and L were very low in extracts of colorectal tumors and normal colonic mucosa. A peculiar cathepsin B-like proteinase activity with pH optimum at 6.8 was found in tumor extracts together with the lysosomal cathepsin B, whereas normal colonic mucosa showed only cathepsin B activity (pH optimum, 6.0). These results indicate that lysosomal cathepsin B is responsible for staining of macrophage-like cells found in the lamina propria of colonic mucosa and in the peritumoral stroma. Immunohistochemical staining of colonic tumor cells observed in 29/51 cases seems on the other hand to be primarily due to a cathepsin B-like proteinase. Three colonic tumor cell lines, Colo-205, HT-29, and SW-1116, were also studied using the same methods. These cells produced a latent cathepsin B-like proteinase which, after activation, was similar to that found in tumor extracts. This latent proteinase was detected mainly in the culture media. The cultured colonic tumor cells, after staining by anti-cathepsin B antibodies, showed strongly positive granules. In conclusion, this work demonstrates that malignant colonic cells are the source of a cathepsin B-like proteinase, with optimal activity near neutrality. Its secretion into the extracellular space indicates furthermore, that it may be an important component of the "proteinase cascade" associated with tumor invasion and metastasis.

Receptor for plasmin on human carcinoma cells.

We have shown that cells from human tumor cell line SW 1116 have receptors for plasmin and plasminogen. These receptors are the same for the proenzyme and the enzyme, but they have a much higher affinity for plasmin (Kd = 6 X 10(-8) M) than for plasminogen (Kd = 5 X 10(-6) M). Plasminogen binding was strongly increased by preincubation of the tumor cells with urokinase and was inhibited by anti-urokinase serum. Because free plasmin is rapidly neutralized in vivo, it is likely that, under physiological conditions, plasminogen is bound by tumor cells and partially transformed into plasmin by urokinase already present at the surface of these cells. Bound plasmin retains its enzymatic activity, which demonstrates that its binding does not involve the enzyme's active site.

Alterations in expression and synthesis of glycolipid antigens in human colonic tumor cell lines.

Incubation of human intestinal SW1116 tumor cells in serum-free medium containing butyric acid reduced their capacity to synthesize gastrointestinal carcinoma-associated (GICA) glycolipid antigen 4- to 8-fold as determined by a radioimmunobinding assay using anti-GICA monoclonal antibody:high-performance thin-layer chromatography; autoradiography; and densitometry. The structure of GICA has been described as a sialylated Lea glycolipid (J. L. Magnani, B. Nilsson, M. Brockhaus, D. Zopf, Z. Steplewski, A. Koprowski, and V. Ginsburg. J. Biol. Chem., 257: 14365-14369, 1982). Tritiated fucose incorporation into GICA was reduced per cell (7-fold), per mg protein (5-fold), and per mg lipid (4-fold). A purified organically soluble glycolipid fraction from control SW1116 cells contained more Lewis antigen than did butyrate-treated cells as determined by the high-performance thin-layer chromatography radioimmunobinding assay. Incorporation of radioactivity from [3H]fucose and guanosine diphosphate [14C]fucose into Lewis antigens in butyrate-treated cells was 2- to 3-fold lower than in control cells. HT29 cells carry the blood type of the original donor, Blood Group A. Isotope incorporation into A glycolipid antigen was reduced 2- to 8-fold upon exposure to butyrate. Commensurate with these results was a dramatic reduction in cell population-doubling rate. We propose that synthesis of these fucolipid antigens is associated more with dividing, undifferentiated tumor cell populations. The diminution in antigen levels may derive from diminished cells' capacity for fucosylation in the presence of butyrate.

Purificaton and characterization of a new antigen from human polymorphonuclears.

A new antigen was isolated from human tissue extracts. By immunofluorescence it was found only in granulocytic cells both in the cytoplasm and on the surface of these cells. It is an alpha-globulin having a molecular weight of approximately 47,000. It is unglycosylated and does not bind to various lectins. Due to its electrophoretic mobility and its cellular localization, it was named alpha-G (for granulocytes) globulin.

Proteases in human tumors.

We carried on an investigation of proteases associated to human tumors by immunohistological techniques. Most of our work dealt with the plasmin system (plasminogen, its two activators and the two plasmin inhibitors, a 2 antiplasmin and a 2 macroglobulin). We found an antigen reacting with anti plasminogen serum in all the 30 colorectal adenocarcinomas we studied by immunofluorescence. This antigen was mainly plasminogen, as we could not detect active plasmin by a histochemical technique on sections of the same tumors. However it is likely that plasminogen present at the surface of tumor cells and on the contour of tumor foci, mainly in invasive areas, can be converted into plasmin, which in turn plays an important role in the degradation of basement membrane antigens. This makes easier tumor invasiveness and release of isolated tumor cells, ready for metastasizing. Plasmin action is likely of short duration and within short range, as this enzyme is rapidly neutralized by its inhibitors, both present at high concentration in the peritumoral stroma. Cathepsin B was characterized by immunoperoxidase method in many colorectal tumors. However our results are preliminary, so we cannot draw any conclusion on the role of this enzyme in tumor invasiveness.

A study of gastrointestinal cancer-associated antigen (GICA) in human fetal organs.

The gastrointestinal cancer-associated antigen (GICA) characterized by 1116 NS 19-9 monoclonal antibody was studied in human fetal organs by immunohistology and immunofixation on nitrocellulose sheets. It was found in all the gastrointestinal tracts of fetuses and newborns. Other fetal organs, except biliary and pancreatic ducts, were negative. Immunohistological data and enzymatic studies led us to conclude that GICA is predominantly a mucin-type glycoprotein in fetal organs.

Lymphoid cells in lymph nodes and peripheral blood of patients with squamous cell carcinoma of the head and neck.

Fifty-five patients with squamous cell carcinoma of the head and neck were evaluated immunologically by measuring the level of T cells (E-RFC) and high affinity subset T cells (E-29) in the peripheral blood and peritumorous lymph nodes. A significant decrease (p less than 0.05) in mean percentage of E-29 was observed in cancer patient peripheral blood. In peritumorous lymph nodes, there was no difference in terms of total T cells or of high affinity subset T cells, as compared to non-malignant lymph nodes, or between tumor-free and metastatic lymph nodes. Macrophage content was much higher in metastatic than in tumor-free lymph nodes (p less than 0.05) and these macrophages frequently appeared to be more active when tested in phagocytosis of sheep red blood cells sensitized with IgG or IgM + C.