Classical and non-classical HLA molecules and p16(INK4a) expression in precursors lesions and invasive cervical cancer.

OBJECTIVES: Viruses and tumour cells may regulate the expression of HLA molecules on the cell surface to escape immune system surveillance. Absence of classical HLA class I molecules may impair the action of specific cytotoxic cells, whereas non-classical HLA class I molecules may regulate innate and adaptive immune cells. We assess here the possible associations between classical/non-classical class I HLA and p16(INK4a) molecule expression in cervical biopsies of women infected with HPV, stratified according to grade of the lesion and HPV type. STUDY DESIGN: Cervical biopsies (N=74) presenting cervical intraepithelial neoplasia grade 1 (CIN1) (n=31), CIN2-3 (n=19), and invasive cancer (n=14) were evaluated alongside 10 normal cervical specimens. RESULTS: HLA-A/B/C/G staining was observed in the early stages of HPV infection. A significant association was detected between HLA-A/B/C staining and HPV16/18 infection (OR=0.12, 95%CI: 0.0163-0.7899; p=0.04). HLA-E expression increased with the progression of the lesion (chi(2)-test for trend=4.01; p=0.05), and a significant association was found between HLA-E staining and HPV16/18 infection (OR=11.25, 95%CI: 2.324-54.465; p=0.003). Irrespective of the grade of the lesion, HLA-A/B/C staining and p16(INK4a) presented a good concordance (Kappa: 0.67). CONCLUSIONS: HLA-E overexpression seemed to be associated with invasive cancer and HPV16/18 infection.

Cellular co-localization of intron-4 containing mRNA and HLA-G soluble protein in melanoma analyzed by fluorescence in situ hybridization.

HLA-G5, -G6, and -G7 soluble isoforms of the immunosuppressive HLA-G molecule are produced from the splice variants of the primary HLA-G mRNA transcript containing intron-4 that encodes a specific 21 amino acids tail. In particular, HLA-G5 interacts with the inhibitory ILT2/4 and KIR2DL4 receptors that are expressed on immune cells. Acquisition of soluble HLA-G in the microenvironment may turn a HLA-G non-expressing cell into a HLA-G-bearing one. To address the question of how to distinguish cells that express soluble HLA-G generated by alternative splicing from those that have acquired HLA-G, we have developed a method capable of detecting intron-4 containing mRNA and protein in situ simultaneously. M8 melanoma cell line either transfected or not with HLA-G5 cDNA was analyzed by indirect immunofluorescence confocal microscopy using double staining with a HLA-G intron-4 digoxygenin labeled probe along with a monoclonal antibody directed against the 21 amino acid tail. The combined fluorescence in situ hybridization was also used on the HLA-G-positive choricarcinoma cell line JEG-3. This method would be helpful to follow-up bona fide HLA-G expression in a heterogeneous cell population and to elucidate the mechanisms underlying soluble HLA-G mediated immune modulation in physiological conditions such as pregnancy and pathophysiological situations such as cancer.

HLA-G*0105N null allele encodes functional HLA-G isoforms.

Expression of the nonclassical HLA class I antigen, HLA-G, is associated with immune tolerance in view of its role in maintaining the fetus in utero, allowing tumor escape, and favoring graft acceptance. Expressed on invasive trophoblast cells, HLA-G molecules bind inhibitory receptors on maternal T lymphocytes and NK cells, thereby blocking their cytolytic activities and protecting the fetus from maternal immune system attack. The HLA-G gene consists of 15 alleles, including a null allele, HLA-G*0105N. HLA-G*0105N presents a single base deletion, preventing translation of both membrane-bound (HLA-G1) and full-length soluble isoforms (HLA-G5) as well as of the spliced HLA-G4 isoform. The identification of healthy subjects homozygous for this HLA-G null allele suggests that the HLA-G*0105N allele may generate other HLA-G isoforms, such as membrane-bound HLA-G2 and -G3 and the soluble HLA-G6 and -G7 proteins, which may substitute for HLA-G1 and -G5, thus assuming the immune tolerogeneic function of HLA-G. To investigate this point, we cloned genomic HLA-G*0105N DNA and transfected it into an HLA-class I-positive human cell line. The results obtained indicated that HLA-G proteins were indeed present in HLA-G*0105N-transfected cells and were able to protect against NK cell lysis. These findings emphasize the role of the other HLA-G isoforms as immune tolerogeneic molecules that may also contribute to maternal tolerance of the semiallogenic fetus as well as tumor escape and other types of allogeneic tissue acceptance.

Erythroblasts secrete the nonclassical HLA-G molecule from primitive to definitive hematopoiesis.

The initial steps of primitive hematopoiesis and endothelial vascular formation in the human embryo remain to be defined. Here, we report the identification of a novel marker, namely the nonclassical HLA-G class I molecule, which targets both primitive erythroid cells of the yolk sac and endothelial cells from developing vessels. Moreover, HLA-G was present in its soluble form in the erythropoietic lineage in all organs sustaining primitive to definitive erythropoiesis (ie, aorta-gonad-mesonephros, liver, spleen, and bone marrow). The alternatively spliced transcript coding the soluble HLA-G5 molecule was detected in erythroid cells. The corresponding intron 4-retaining 37-kDa HLA-G5 isoform was secreted from the erythroid progenitor stage to the reticulocyte but was lost in mature erythrocytes and in endothelial cells from differentiated vessels. This study constitutes the first description of an HLA class I antigen expression on the primitive erythroid lineage and provides a way of seeking both primitive and definitive erythropoiesis using HLA-G5. This new marker, previously known by its immunotolerogeneic properties, may be involved in erythroid differentiation, angiogenesis, or both.

The 14 bp deletion-insertion polymorphism in the 3' UT region of the HLA-G gene influences HLA-G mRNA stability.

Human leukocyte antigen (HLA)-G molecules are generated by an alternative splicing of the primary transcript of the gene and display specialized function in regulating the immune response. Although HLA-G gene polymorphism is low, it may influence levels of protein expression and, in some cases, has been associated with pregnancy diseases. The HLA-G gene exhibits 14 alleles generating six proteins with minor variations and a null allele. HLA-G allelic variants may be also characterized by a 14 bp deletion-insertion polymorphism located in the 3' UT region of the HLA-G gene. The presence of the 14 bp insertion is known to generate an additional splice whereby 92 bases are removed from the start of exon 8. To analyze the effect of this deletion on the stability of HLA-G mRNAs, we performed actinomycin D treatments on both JEG-3 choriocarcinoma cell line and M8 melanoma cell line transfected with HLA-G*010102 allele. We observed that HLA-G mRNAs having the 92-base deletion are more stable than the complete mRNA forms, suggesting that this region may be involved in the mechanisms controlling post-transcriptional regulation of HLA-G molecule associated with allelic variants.

Expression of HLA-G in human cornea, an immune-privileged tissue.

Human leukocyte antigen (HLA)-G retains the capacity to modulate immune responses, favoring the establishment of tolerance in solid-tissue allotransplants. To better understand the mechanisms that promote corneal allograft survival, we investigated whether HLA-G was an immunoregulatory factor involved in corneal immunology. We therefore sought HLA-G expression in corneal tissues. Corneal transplantation consists in replacing the center of a diseased cornea with normal corneal tissue. Two corneal parts are not used in such surgery: diseased central corneal tissue and peripheral normal cornea. For this study, we used healthy corneas obtained from deceased donors and diseased corneas obtained from patients with pseudophakic bullous keratopathy or keratoconus who had undergone corneal transplantation. Immunohistochemical analysis carried out on the cryopreserved corneas showed a positive immunohistochemical staining with anti-HLA-G, anti-HLA-A, -B, and -C, and anti-HLA class I monoclonal antibodies. Staining was obtained for keratocytes, epithelial cells, and endothelial cells from both healthy and pathologic human corneas, revealing the presence of HLA class I proteins, including HLA-G. HLA-G transcripts were detected in normal cornea by reverse transcriptase-polymerase chain reaction with a classical pattern of alternative splicing. The detection of HLA-G protein in adult corneas leads to the conclusion that this protein may contribute to the maintenance of the privileged immune status of cornea.

HLA-G molecules: from maternal-fetal tolerance to tissue acceptance.

Over the past few years, HLA-G, the non-classical HLA class I molecule, has been the center of investigations that have led to the description of its specific structural and functional properties. Although located in the HLA class I region of chromosome six, the HLA-G gene may be distinguished from other HLA class I genes by its low polymorphism and alternative splicing that generates seven HLA-G proteins, whose tissue-distribution is restricted to normal fetal and adult tissues that display a tolerogeneic function toward both innate and acquired immune cells. We review these points, with special emphasis on the role of HLA-G in human pathologies, such as cancer, viral infection, and inflammatory diseases, as well as in organ transplantation.