Association of -318 C/T and +49 A/G cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms with a clinical subset of Italian patients with systemic sclerosis.

Systemic sclerosis (SSc) is a complex and heterogeneous autoimmune disorder with a multi-factorial pathogenesis. Like other autoimmune disorders, the possible role of specific cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms in predisposing to SSc has been hypothesized, but it remains controversial. CTLA-4 promoter (-318C/T) and exon 1 (+49 A/G) polymorphisms have been analysed in 43 Italian females with SSc and in 93 unrelated matched healthy controls by a newly designed tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method. No significant association has been found with either polymorphisms.Nevertheless, SSc patients without concomitant Hashimoto's thyroiditis (HT) were carrying both the -318T allele (P = 0.031) and the +49 G allele (P = 0.076) more frequently than SSc patients with HT [defined by positivity for anti-thyroperoxidase (TPO) and anti-thyroglobulin (TGA) autoantibodies] than controls. Haplotype analysis confirms this association (P = 0.028), and suggests the predominant role of the -318T, whereas that of the +49 G, if any, seems weak. Thus, in Italian SSc patients the CTLA-4 -318C/T promoter polymorphism appears to be associated with the susceptibility to develop SSc without thyroid involvement. Larger studies are needed to confirm these findings and to clarify whether the -318C/T polymorphism is the functional responsible or whether it reflects the presence of another linked genetic element in the same chromosomal region.

Expression of CTLA-4 in nonhuman primate lymphocytes and its use as a potential target for specific immunotoxin-mediated apoptosis: results of in vitro studies.

T-cell-mediated immunoregulation is one of the main mechanisms implicated in induction and maintenance of transplantation tolerance. In this regard, deletion or modulation of xeno/alloantigen-specific T cells, as well as blocking of their interactions with other cell populations, are currently being pursued for tolerance induction in humans as well as nonhuman primates. In order to investigate whether cytotoxic T-lymphocyte antigen-4 (CTLA-4) may represent a suitable target for a T cell depletion approach in nonhuman primate models, we analysed CTLA-4 expression in peripheral blood mononuclear cells (PBMCs) from nonhuman primates and the potential role of two anti-CTLA-4 saporin-conjugated immunotoxins. The analysis was performed in PBMCs from 8 cynomolgus monkeys from Philippines and from Mauritius both at protein level by flow cytometry and at transcriptional level by RT-PCR. In addition, the apoptotic role of the immunotoxins was investigated. The results showed that CTLA-4 was expressed at variable levels depending on the origin of the cynomolgus monkeys and the resting or activated cell condition. CTLA-4 was not expressed on resting Mauritius PBMCs and showed a lower up-regulation upon PMA/PHA activation compared to the Philippines PBMCs that expressed CTLA-4 also before activation. Two CTLA-4 RNA transcripts (672 and 550 bp) were detected with levels variations after cell stimulation. Two anti-CTLA-4 immunotoxins induced in vitro apoptosis of activated PBMCs from both sources of cynomolgus monkeys. This is the first report that documents CTLA-4 expression both at protein and transcriptional level by nonhuman primate PBMCs and provides novel perspectives of xeno/allograft rejection immunotherapy based on CTLA-4 targeting.

Deletion analysis of chromosome 13q14.3 and characterisation of an alternative splice form of LEU1 in B cell chronic lymphocytic leukemia.

Heterozygous and homozygous deletions of chromosome 13q14.3 are found in 50% of patients with B cell CLL, suggesting the presence of one or more tumour suppressor genes within the deleted region. To identify candidate genes from the region, we constructed a map of 13q14.3 using a combination of genomic and cDNA library screening. The incidence of deletions in CLL patients was 51.5% encompassing a 265 kb region of minimal deletion (RMD) telomeric to markers D13S319. Two CpG islands were identified within the RMD, the telomeric of which is fully methylated whilst the more centromeric is unmethylated. A novel transcript was identified within the RMD that represents an alternative splice version of Leu1. The nine exons of this transcript span a genomic of 436 kb with exon 1 of Leu1 being the common first exon. The remaining exons were shown to be more frequently deleted than Leu1 itself. All splice forms of this transcript were detectable by RT-PCR but Leu1 detected the most abundant message on Northern blotting. Sequence analysis failed to reveal inactivating mutations in patients with heterozygous deletion of 13q14.3, although a polymorphic T to A variant was identified within exon 1 of Leu1 in leukemic and normal controls. As no mutations have been detected for Leu1 or any other transcript so far described, we cannot exclude the existence of control elements within the RMD that may regulate expression of genes lying in this region.

Heterogeneity of VH-JH gene rearrangement patterns: an insight into the biology of B cell precursor ALL.

Oligoclonal B cell proliferation, as defined by the presence of more than one leukemic clone, has been detected in approximately 20% to 30% of patients with acute lymphoblastic leukemia (ALL) using PCR or Southern blotting. An accurate assessment of these populations is required to avoid false negative measurements of minimal residual disease (MRD) in follow-up bone marrow (BM) samples of ALL patients. In this study, we analysed 29 ALL patients with two or more immunoglobulin heavy (IGH) chain gene rearrangements in the presentation samples using IGH fingerprinting PCR and sequence analysis. Thirty-nine (51%) of 76 sequences (from 15 patients), shared no VNDNJ homology (ie different CDR3 regions). In the remaining 14 patients, at least two related VH sequences were identified in each patient (identical DNJ sequences). Numerical abnormalities of chromosome 14 was detected in 10 patients. Eight patients were analysed at presentation and relapse. In four of them, expansion of a minor presentation-clone was detected at relapse while the major presentation clone disappeared, confirming 'subclonal evolution'. Finally, in our cohort of patients, the presence of related or unrelated IGH clones did not influence overall survival.

Immunotoxins containing recombinant anti-CTLA-4 single-chain fragment variable antibodies and saporin: in vitro results and in vivo effects in an acute rejection model.

Immunotoxins containing recombinant human-derived single-chain fragment variable (scFv) reagents (83 and 40) against CTLA-4 (CD152) linked to saporin, a ribosome-inactivating protein, were prepared and tested on CD3/CD28-activated T lymphocytes, MLRs, CTLA-4-positive cell lines, and hemopoietic precursors. Immunotoxins induced apoptosis in activated T lymphocytes and were able to specifically inhibit MLR between T lymphocytes and dendritic cells. The 83-saporin immunotoxin also inhibited the T cell activation in an MLR between T lymphocytes and an EBV-positive lymphoblastoid B cell line. Toxicity tests on hemopoietic precursors showed little or no effects in inhibiting colonies' growth. As the 83 scFv Ab was reactive also with activated mouse T lymphocytes, 83-saporin was tested in a model of tumor rejection consisting of C57BL/6 mice bearing a murine H.end endothelioma cell line, derived from DBA/2 mice. The lymphoid infiltration due to the presence of the tumor was reduced to a high extent, demonstrating that the immunotoxin was actually available and active in vivo. Thus, taking the results altogether, this study might represent a new breakthrough for immunotherapy, showing the possibility of targeting CTLA-4 to kill activated T cells, using conjugates containing scFv Abs and type 1 ribosome-inactivating protein.

Detection of minimal residual disease in B-lymphoproliferative disorders: a three step SSCP-PCR method.

The most recent therapeutic approaches can improve the outcome of B-cell neoplasia. By PCR analysis we amplify tumor specific DNA sequences of clonal IgH rearrangement from a limited number of malignant cells against a background of normal B cells. Recently described PCR based techniques for tracking minimal residual disease (MRD) in B lymphoproliferative disorders have given promising but discordant results, with significant variations in the sensitivity and specificity of the procedures. We have developed a three step single strand conformational polymorphism polymerase chain reaction (SSCP-PCR) strategy which is able to detect clonal malignant cells in B lymphoproliferative disorders at a frequency as low as 1 in 10(6) cells. Since this method is simple, rapid, reliable and as specific as ASO-PCR, it could be especially useful in monitoring patients affected by B lymphoproliferative disorders in complete haematological and immunophenotypic remission.

Investigation of HLA class I downregulation in breast cancer by RT-PCR.

Downregulation of HLA class I antigen expression has been reported in a significant proportion of primary breast carcinomas suggesting an escape mechanism from CTL mediated lysis leading to tumor dissemination and metastasis. We have previously reported the biochemical and immunohistochemical analysis of HLA total class I (W6/32 mAb), alpha-chain (Q1/28,TP25.99 mAbs) and beta(2)-microglobulin (Namb-1 mAb) subunits expression in 25 primary breast carcinomas. This study at protein level resulted in the observation of three different HLA class I expression patterns by both techniques: high, low, and absent downregulation patterns. To better characterize the HLA class I antigens downregulation we extended such analysis also at RNA level by RT-PCR using HLA-A, HLA-B, HLA-C, and beta(2)-microglobulin specific primers either in breast cancer or normal tissues derived from the same patient. None (100%) of the alpha-chain genes analyzed in patient tumor tissues showed significant reduction of expression. In 10 patients out of 25 (40%) the beta(2)-microglobulin gene showed complete loss of expression compared with the corresponding normal tissue counterpart, which showed a constitutive expression, whereas in 2 patients (12.5%) its expression was comparable with the normal counterpart. Sequence analysis at genomic level revealed no defects affecting beta(2)-microglobulin gene in those patients showing lack of expression. Also TAP1 and TAP2 genes expression were investigated in order to confirm or exclude involvement of the MHC class I molecules assembling machinery. The RT-PCR approach mainly confirmed our beta(2)-microglobulin biochemical analysis indicating that in breast cancer specimens it is possible to address the HLA class I gene downregulation as a phenomenon occurring at post-transcriptional level mainly affecting the beta(2)-microglobulin gene expression.

Melanomas and melanoma cell lines do not express HLA-G, and the expression cannot be induced by gammaIFN treatment.

HLA-G is an effective ligand of natural killer (NK) inhibitory receptors, HLA-G transcripts have been detected in several human tumors, and cytokines like gamma interferon (IFN) enable HLA-G molecules to be expressed. These findings are particularly upsetting in case of melanomas: IFN treatment is frequently included in melanoma therapeutic protocols, and downregulation of classical class I molecules occurs in nearly half of these tumors. Therefore, a melanoma cell downregulating classical class I and de novo expressing HLA-G, either constitutively or upon IFN treatment, is probably a stealthy target for the immune system, having inhibited both the cytotoxic T lymphocyte (CTL) and the NK activity. To elucidate this point we have investigated the expression of HLA-G molecules in 45 melanoma cell lines before and after gammaIFN treatment. Analysis was performed by immunofluorescence and flow cytometry, using the anti-HLA-G MoAbs 87G and G233, by Western blot, using the anti-HLA-G MEM/G1 MoAb and PAG1 antiserum, and by RT-PCR analysis. In addition, 8 melanoma tissues from patients free from therapy and 6 nevi were studied by immunohistochemistry using the 87G MoAb. No evidence was gathered of HLA-G expression, neither constitutive nor, in cell lines, after gammaIFN treatment. We therefore conclude that HLA-G expression is an uncommon event in melanomas, and that a therapy including IFNs cannot harm the patient by inducing the de novo expression of HLA-G molecules at least in its G1 isoform.