A mosaic de novo duplication of 17q21-25 is associated with GH insensitivity, disturbed in vitro CD28-mediated signaling, and decreased STAT5B, PI3K, and NF-κB activation.

OBJECTIVE: The established causes of GH insensitivity include defects of the GH receptor and STAT5B. The latter condition is also characterized by severe immunodeficiency. A recent case with short stature, GH resistance, and immunodeficiency due to an IκB mutation suggests that the NF-κB pathway may interact with STAT5B signaling. DESIGN: Here, we present a case of a short child with several congenital anomalies as well as GH insensitivity and mild immunodeficiency associated with a mosaic de novo duplication of chromosome 17q21-25, suggesting that overexpression of one of the duplicated genes may be implicated in GH resistance. METHODS AND RESULTS: In vitro studies on blood lymphocytes showed disturbed signaling of the CD28 pathway, involving NF-κB and related proteins. Functional studies on cultured skin fibroblasts revealed that NF-κB activation, PI3K activity, and STAT5 phosphorylation in response to GH were suppressed, while the sensitivity to GH in terms of MAPK phosphorylation was increased. An in silico analysis of the duplicated genes showed that MAP3K3 and PRKCA are associated with the NF-κB pathway. Baseline MAP3K3 expression in T-cell blasts (TCBs) was normal, but PRKCA expression in TCBs and fibroblasts was significantly higher than that in control cells. CONCLUSIONS: We conclude that the 17q21-25 duplication is associated with GH insensitivity and disturbed STAT5B, PI3K, and NF-κB signaling, possibly due to PRKCA mRNA overexpression.

Effect of amino acid substitutions in the human IFN-γR2 on IFN-γ responsiveness.

Patients with interferon-γ receptor (IFN-γR) null mutations have severe infections with poorly pathogenic Mycobacteria. The IFN-γR complex involves two IFN-γR1 and two IFN-γR2 chains, in which several amino acid substitutions, some linked to disease and some apparently naturally occurring, have been described. We developed a model system to study functional effects of genetic variations in IFN-γR2. We retrovirally transduced wild-type IFN-γR2 and IFN-γR2 carrying presently known amino acid substitutions in various human cell lines, and next determined the IFN-γR2 expression pattern as well as IFN-γ responsiveness. We determined that the T58R, Q64R, E147K and K182E variants of IFN-γR2 are fully functional, although the Q64R variant may be expressed higher on the cell membrane. The R114C, T168N and G227R variants were identified in patients that had disseminated infections with non-tuberculous Mycobacteria. Of these genetic variants, T168N was confirmed to be completely non-functional, whereas the novel variant G227R, and the previously reported R114C, were partial functional. The impaired IFN-γ responsiveness of R114C and G227R is mainly due to reduced receptor function, although expression on the cell membrane is reduced as well. We conclude that the T58R, Q64R, E147K and K182E variants are polymorphisms, whereas the R114C, T168N and G227R constitute mutations associated with disease.

Mycobacterium bovis BCG-itis and cervical lymphadenitis due to Salmonella enteritidis in a patient with complete interleukin-12/-23 receptor beta1 deficiency.

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare disorder with predisposition to severe, sometimes lethal, disease caused by otherwise poorly virulent, non-tuberculous environmental mycobacteria and poorly virulent salmonellae. In patients with MSMD, mutations have been identified in five genes that encode for the proteins IL-12/IL-23p40, IL-12/ IL-23Rbeta1, IFN-R1, IFN-gammaR2 and STAT1. These proteins play important roles in the type-1 cytokine pathway, which is crucial for human host defence against intracellular pathogens such as mycobacteria and salmonellae. We report a girl with mild Mycobacterium bovis Bacille Calmette-Guérin (BCG) disease and Salmonella enteritidis cervical lymphadenitis. Despite treatment, she has remained a fecal carrier of S. enteritidis for the past 14 years. She was found to have complete IL-12/IL-23Rbeta1 deficiency. A homozygous r.518G>C IL12RB1 mutation was identified, leading to a non-functional R173P substitution in the IL-12/IL-23Rbeta1 protein. This mutation abrogated IL-12/IL-23Rbeta1 cell-surface expression and resulted in complete lack of T cell responsiveness to both IL-12 and IL-23.

Low level IGF-1 and common variable immune deficiency: an unusual combination.

A relation between growth hormone (GH) deficiency and immunoglobulin deficiency has been suggested previously in a few cases. We describe a patient with an insulin-like growth factor 1 (IGF-1) deficiency and common variable immune deficiency and briefly review earlier publications on the possible interaction between IGF-1 and the immune system. IGF-1 is the downstream mediator of GH. In this patient, GH and IGF-1 levels were both low. The GH response to a GH-releasing hormone test was normal whereas no subsequent IGF-1 response was seen. In our cohort of 14 patients with hypogammaglobulinaemia, two turned out to have slightly decreased IGF-1 serum levels and one patient with a thymoma had an increased IGF-1 level. Even though IGF-1 may be connected to B lymphocyte differentiation, in this patient we hypothesise there is a common impairment in the IGF-1 and IgG pathways.

HLA class II restricted T-cell receptor gene transfer generates CD4+ T cells with helper activity as well as cytotoxic capacity.

Both cytotoxic T cells and helper T cells are important in immune responses against pathogens and malignant cells. In hematological malignancies which express HLA class II molecules, immunotherapy may be directed to HLA class II restricted antigens. We investigated whether it is possible to engineer HLA class II restricted T cells with both antigen-specific cytolytic activity and the capacity to produce high amounts of cytokines. CD4+ and CD8+ peripheral-blood-derived T cells were retrovirally transduced with the HLA class II restricted minor histocompatibility antigen dead box RNA helicase Y (DBY)-specific TCR. The TCR-transduced CD4+ T cells exerted DBY-specific cytolytic activity, produced Th0, Th1, or Th2 cytokines, and proliferated upon DBY-specific stimulation. TCR-transduced CD8+ T cells exerted cytolytic activity which equaled the level of cytolytic activity of the TCR-transferred CD4+ T cells. Cotransfer of CD4 enhanced the cytolytic activity of the TCR-transduced CD8+ T cells, but introduction of CD4 was not sufficient to generate DBY-specific CD8+ T cells with the capacity to produce high amounts of cytokines. In this study, we demonstrated the feasibility to engineer T cells with antigen-specific cytolytic activity, as well as the ability to produce significant amounts of cytokines, by TCR transfer to CD4+ T cells.

Dual HLA class I and class II restricted recognition of alloreactive T lymphocytes mediated by a single T cell receptor complex.

The alloreactive human T cell clone MBM15 was found to exhibit dual specificity recognizing both an antigen in the context of the HLA class I A2 molecule and an antigen in the context of the HLA class II DR1. We demonstrated that the dual reactivity that was mediated via a single clonal T cell population depended on specific peptide binding. For complete recognition of the HLA-A2-restricted specificity the interaction of CD8 with HLA class I is essential. Interestingly, interaction of the CD8 molecule with HLA class I contributed to the HLA-DR1-restricted specificity. T cell clone MBM15 expressed two in-frame T cell receptor (TCR) Valpha transcripts (Valpha1 and Valpha2) and one TCR Vbeta transcript (Vbeta13). To elucidate whether two TCR complexes were responsible for the dual recognition or one complex, cytotoxic T cells were transduced with retroviral vectors encoding the different TCR chains. Only T cells transduced with the TCR Valpha1Vbeta13 combination specifically recognized both the HLA-A2(+) and HLA-DR1(+) target cells, whereas the Valpha2Vbeta13 combination did not result in a TCR on the cell surface. Thus a single TCRalphabeta complex can have dual specificity, recognizing both a peptide in the context of HLA class I as well as a peptide in the context of HLA class II. Transactivation of T cells by an unrelated antigen in the context of HLA class II may evoke an HLA class I-specific T cell response. We propose that this finding may have major implications for immunotherapeutic interventions and insight into the development of autoimmune diseases.

UTY gene codes for an HLA-B60-restricted human male-specific minor histocompatibility antigen involved in stem cell graft rejection: characterization of the critical polymorphic amino acid residues for T-cell recognition.

Rejection of a graft after human leukocyte antigen (HLA)-identical stem cell transplantation (SCT) can be caused by recipient's immunocompetent T lymphocytes recognizing minor histocompatibility antigens on donor stem cells. During rejection of a male stem cell graft by a female recipient, 2 male (H-Y)-specific cytotoxic T lymphocyte (CTL) clones were isolated from peripheral blood. One CTL clone recognized an HLA-A2-restricted H-Y antigen, encoded by the SMCY gene. Another CTL clone recognized an HLA-B60-restricted H-Y antigen. In this study UTY was identified as the gene coding for the HLA-B60-restricted H-Y antigen. The UTY-derived H-Y antigen was characterized as a 10-amino acid residue peptide, RESEEESVSL. Although the epitope differed by 3 amino acids from its X-homologue, UTX, only 2 polymorphisms were essential for recognition by the CTL clone HLA-B60 HY. These results illustrate that CTLs against several H-Y antigens derived from different proteins can contribute simultaneously to graft rejection after HLA-identical, sex-mismatched SCT. Moreover, RESEEESVSL-specific T cells could be isolated from a female HLA-B60+ patient with myelodysplastic syndrome who has been treated with multiple blood transfusions, but not from control healthy HLA-B60+ female donors. This may indicate that RESEEESVSL-reactive T cells are more common in sensitized patients.

DFFRY codes for a new human male-specific minor transplantation antigen involved in bone marrow graft rejection.

Graft rejection after histocompatibility locus antigen (HLA)-identical stem cell transplantation results from the recognition of minor histocompatibility antigens on donor stem cells by immunocompetent T lymphocytes of recipient origin. T-lymphocyte clones that specifically recognize H-Y epitopes on male target cells have been generated during graft rejection after sex-mismatched transplantation. Previously, 2 human H-Y epitopes derived from the same SMCY gene have been identified that were involved in bone marrow graft rejection. We report the identification of a new male-specific transplantation antigen encoded by the Y-chromosome-specific gene DFFRY. The DFFRY-derived peptide was recognized by an HLA-A1 restricted CTL clone, generated during graft rejection from a female patient with acute myeloid leukemia who rejected HLA-phenotypically identical bone marrow from her father. The identification of this gene demonstrates that at least 2 genes present on the human Y-chromosome code for male-specific transplantation antigens.

Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification.

With the ongoing progress in human genome projects, many genes are discovered whose function and/or expression pattern are not known. Most of these genes are expressed in relatively low abundance compared to housekeeping genes such as elongation factor-1alpha and beta-actin. Gene expression is studied by Northern blot assays or by semiquantitative PCR methods. Another method is the visualization of transcripts in tissue or cell cultures by fluorescence in situ hybridization (FISH). However, for low-abundance RNA detection, this method is hampered by its limited detection sensitivity and by the interference of background signals with specific hybridization signals. Background signals are introduced by nonspecific hybridization of probe sequences or nonspecific binding of antibodies used for visualization. To eliminate background signals derived from both sources and to benefit from the peroxidase-driven tyramide signal amplification (TSA), we directly conjugated horseradish peroxidase (HRP) to oligodeoxynucleotides (ODNs) and used these probes to study in the bladder cancer cell line 5637 the expression of various cytokine genes which, according to Northern hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) assays, are expressed at levels up to 10,000-fold less than abundantly expressed housekeeping genes. The results show that reduction of probe complexity and the limited use of immunocytochemical detection layers strongly reduces noise signals derived from nonspecific binding of nucleic acid probe and antibodies. The use of the HRP-ODNs in combination with TSA allowed detection of low-abundance cytokine mRNAs by FISH.

Generation of dendritic cells expressing bcr-abl from CD34-positive chronic myeloid leukemia precursor cells.

Patients with a relapse of chronic myeloid leukemia (CML) after allogeneic bone marrow transplantation can be successfully treated with blood mononuclear cells from the original bone marrow donor. However, the antileukemic effect of this treatment is often accompanied by graft-versus-host disease (GVHD). Treatment with cytotoxic T-lymphocyte (CTL) lines or clones that are specifically generated against leukemic antigen-presenting cells from the patient, may separate antileukemic effects from GVHD. In this report we demonstrate that after culturing CD34-positive cells purified from bone marrow of patients with chronic phase CML in medium containing human serum, GM-CSF, TNF alpha, and IL-4 up to 28% of the cultured cells were dendritic cells, characterized by morphology, phenotypic analysis, and their efficient capacity to stimulate allogeneic T lymphocytes. The expression of HLA and costimulatory molecules and the stimulatory capacity of the dendritic cell-enriched cell suspensions were optimal between days 7 and 10 after onset of the cultures. Fluorescence in situ hybridization revealed that all cultured dendritic cells contained the CML specific t(9;22) translocation. PCR analysis showed expression of the translocation specific bcr-abl mRNA. These leukemic dendritic cells may enhance the induction and proliferation of CTL lines and clones with more specificity for the leukemic cells.

Human cytotoxic CD8+ T-lymphocyte clones engraft in severe combined immunodeficient (SCID) mice but show diminished function.

Mice with severe combined immunodeficiency (SCID) provide an in vivo model for studying interactions between human tumor cells and effector cells of the immune system. We studied the behavior of human alloreactive cytotoxic T lymphocytes (CTLs) in SCID mice, including the migration pattern of CD8+ or CD4+ CTL clones to various murine tissues, their engraftment in the absence or presence of recombinant human interleukin 2 (rhIL-2) compared with the engraftment of lymphokine activated killer (LAK) cells, and the in vitro as well as the in vivo function of the engrafted CTL clones. The polymerase chain technique using T-cell-receptor-gamma specific primers revealed the presence of human CD8+ CTL clones in the blood, lungs, and liver of mice receiving rhIL-2 for 14 days, for at least 18 days after intravenous inoculation. Human T cells were transiently detected in the bone marrow, lymph nodes, thymus, and spleen. Although the three CD8+ CTL clones and two CTL lines tested required rhIL-2 for their engraftment, the four LAK cell populations also engrafted in the absence of rhIL-2. In contrast to CD8+ T cells, only low frequencies (<1%) of CD4+ cells could be detected in the blood of the SCID mouse. Engrafted human T cells recovered from the murine blood showed absent or diminished cytotoxicity in vitro against human target cells in five or six experiments, respectively. In addition, when the antitumor activity of engrafted CD8+ clones was investigated in vivo using a xenotransplantation model of human B-cell lymphoma in SCID mice, no significant prolongation of the mean survival time of six treated animals was observed compared with animals treated with rhIL-2 alone. Our results illustrate that although in vitro primed and cultured human CD8+ T cells engraft in SCID mice, their in vitro and in vivo function is impaired.

Anti-CD45 and anti-CD52 (Campath) monoclonal antibodies effectively eliminate systematically disseminated human non-Hodgkin's lymphoma B cells in Scid mice.

Severe combined immunodeficient (Scid) mice inoculated with the human (t(14;18)-positive B cell lines DoHH2 and BEVA develop lethal systemically disseminated lymphoma (de Kroon et al., Leukemia 8:1385, and Blood 80 [suppl 1]:436). These models were used to study the therapeutic effect of rat-anti-human CD52 (Campath-1G) or CD45 monoclonal antibodies (mAbs) on systemically disseminated tumor cells and on tumor cells present in solid tumor masses. Both mAbs were effective in inhibiting growth of systemically disseminated malignant cells. When treatment with anti-CD52 or anti-CD45 mAbs at a dose of 30 micrograms/mouse/d for 4 days was started 24 hours after intravenous inoculation of human DoHH2 or BEVA cells, a 3-log kill of tumor cells was observed as measured by prolonged survival. After treatment, surviving animals injected with high numbers of BEVA cells showed tumor masses in liver, kidney, and mesenteric lymph nodes. In contrast to nontreated animals, however, only low numbers of malignant cells were found in peripheral blood, and bone marrow was free of tumor cells. Similarly, after mAb treatment of mice inoculated subcutaneously (sc) with DoHH2 cells, no tumor cells could be found in the bone marrow, and few DoHH2 cells could be detected in the peripheral blood, spleen, liver, kidney, or lung. In contrast, tumor cells present in subcutaneous tumors and axillary lymph nodes were relatively unaffected by mAb therapy. The presence of rat immunoglobulin (Ig) could be demonstrated on surviving tumor cells. The presence of murine macrophages in areas in these tumors that were depleted of DoHH2 cells suggested that the mAb-mediated antitumor effect observed in the Scid mouse model is mediated by cellular mechanisms. Apparently these mechanisms were not sufficient to eliminate the fast-growing tumor cells present in the protected sites. Our results indicate that treatment with anti-CD52 or anti-CD45 mAbs potentially may be useful as adjuvant immunotherapy for systemically disseminated B cell lymphoma.

Differential transcriptional and posttranscriptional regulation of gene expression of the colony-stimulating factors by interleukin-1 and fetal bovine serum in murine fibroblasts.

Colony-stimulating factors (CSF) are important factors in the proliferation and differentiation of hematopoietic progenitor cells (HPC), and in the survival and activation of mature blood cells. Interleukin-1 (IL-1) combined with fetal bovine serum (FBS) strongly induces the expression of macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), and granulocyte-macrophage-CSF (GM-CSF) in fibroblasts. Here, we report on the regulation of CSF gene expression in murine fibroblasts following IL-1 and FBS stimulation. We demonstrate that 10T1/2 murine fibroblasts induced by FBS or IL-1 accumulate M-CSF messenger RNA (mRNA). G-CSF mRNA expression was induced by IL-1, and not by FBS. For GM-CSF expression, induction with both FBS and IL-1 was required. Blocking studies with actinomycin-D showed that active transcription is essential for accumulation of all three CSF mRNAs. After blocking protein synthesis with cycloheximide, IL-1- or FBS-induced M-CSF expression and IL-1 plus FBS-induced GM-CSF expression still occurred and was increased. IL-1-induced G-CSF expression was completely prevented in these cells by pretreatment with cycloheximide, illustrating that, for this effect, intermediate protein synthesis was required. The half-lives of M-CSF transcripts were not substantially altered by addition of IL-1, FBS, or FBS plus IL-1. Using nuclear run-on assays, we demonstrated that the transcription rate of M-CSF was increased up to 20-fold by the addition of FBS, IL-1, or FBS plus IL-1. After blocking protein synthesis with cycloheximide, IL-1-or FBS-induced increase in M-CSF transcription rate was also observed. GM-CSF transcription increased up to fourfold after induction with FBS or IL-1. G-CSF transcription rate was not altered by FBS or IL-1. Our results indicate that M-CSF expression induced by FBS or IL-1 in these fibroblasts is primarily regulated at the transcriptional level. GM-CSF expression appears to be regulated both transcriptionally and posttranscriptionally, and G-CSF expression is regulated mainly at the posttranscriptional level.

Effects of FSH and IGF-I on immature rat Sertoli cells: inhibin alpha- and beta-subunit mRNA levels and inhibin secretion.

Effects of follicle-stimulating hormone (FSH) and insulin-like growth factor-I (IGF-I) on inhibin production by cultured Sertoli cells from 21- to 23-day-old rats were studied. The expression of inhibin alpha- and beta-subunit mRNAs, and inhibin immunoreactivity and in vitro bioactivity were estimated. Using a cDNA probe corresponding to the alpha-subunit of bovine inhibin, specific hybridization with a 1.5-1.7 kilobase (kb)mRNA species was observed. Addition of FSH to the cultured Sertoli cells for 24 h markedly increased the level of this mRNA in a dose-dependent way. IGF-I had no effect on the intensity of the hybridization. Using a cDNA probe corresponding to the beta B-subunit of human inhibin, 3.5 and 4.2 kb mRNA species were detected. FSH and IGF-I had no effect on the hybridization signal. No hybridization was observed with a cDNA probe corresponding to the beta A bovine inhibin subunit. Inhibin activity was detected in cells and medium by immunoassay, and in the medium by in vitro bioassay. FSH stimulated both immunoreactivity and in vitro bioactivity, whereas IGF-I had no effect at all. The present effect of FSH on inhibin alpha-subunit mRNA expression in cultured Sertoli cells indicates that regulation of inhibin production by FSH includes an effect at the transcriptional level. However, this does not exclude additional translational and posttranslational effects.