Questionnaire
PWWG Conclusion II:
RT-PCR Procedure
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Nomenclature
Materials available
for Workshop
Analysis
RT-PCR primers
Antibodies
Pr. Edgardo D. Carosella, Chair, Paris - France
Dan Geraghty, Co-Chair, Seattle - USA
Coordinators: A. Arnaiz-Villena, R. Fauchet, G. B. Ferrara, M. Le Discorde, Ph. Moreau, P. Paul, N. Rouas-Freiss, A. Ishitani
This component will address specific questions that are fundamental to the understanding of the biology of the HLA-E, -F, -G antigens. Below we have outlined four major goals. Interested participants are requested to contact the person coordinating each of the specific aims presented below.
Specific Aim 1) HLA-E, -F and -G Polymorphism. Previous work has left our knowledge of this subject with conflicting results concerning the polymorphism of HLA-E and G and only a small effort has focused on HLA-F. We propose to define a target population of appropriate size and diversity according to input from interested parties and consultations with experts from the anthropology section. Suitable primers have been identified and DNA from these samples will be isolated and distributed to interested labs for direct PCR sequence analysis. Participation in this aim will therefore necessarily be restricted to labs with access to ABI (or equivalent) equipment. Once obtained, chromatograms will be assembled from participating sites and analyzed. In this way we will have 1) obtained definitive, direct, raw data relating to HLA-E, F, G polymorphism that can be directly examined for data quality and 2) sampled a sufficiently diverse set of individuals to achieve high confidence that nearly all allelic variants will have been identified.
Coordinator: A. Arnaiz-Villena
Specific Aim 2) HLA-G and -E antibodies and transfectants. We proposed to assemble all available antibodies specific for HLA-G and HLA-E (see Table 1) as well as cell lines and transfectant cells (see Table 2) to decide upon, from a consensus of four interested laboratories, 2 to 3 well-defined protocols for histochemical staining and cytofluorometry (see pre-workshop group conclusions I). These validated antibodies are used to study HLA-G and HLA-E expression in normal and pathological cell lines and tissues (see Specific Aim 4). Laboratory studies began in February 2000, following distribution of antibodies for Aim 4.
Coordinator: N. Rouas-Freiss
Specific Aim 3) Validation of RT-PCR tools and protocols for analysis of HLA-G, E and F transcription : alternative splicing and semi-quantitative analysis (see pre-workshop group conclusions II). Specific primers and probes were validated on cells used for antibodies validation.
Coordinators: Ph. Moreau, and M. Le Discorde
Specific Aim 4) HLA-G expression in normal and pathological cell lines and tissues. This specific aim is to establish the expression pattern of HLA-G in placental and tumor tissues and cell lines. The antibodies and protocols validated as mentioned above, are distributed to interested laboratories and raw data will be assembled for analysis and scoring by a subset of the participants (to be defined). This study is intended to help clarify the extent of HLA-G expression both within and outside placental and tumor tissues and cell lines. Laboratory studies began in March 2000. Coordinators: R. Fauchet, G. B. Ferrara, and P. Paul
For a better understanding between scientific groups we would like to define NOMENCLATURE of HLA-G MOLECULES. The nomenclature of the HLA-G proteins accepted by the International Histocompatibility Working Group is:
- a. HLA-G membrane-bound proteins : HLA-G1 to-G4.
b. Soluble proteins : HLA-G5 to -G7.
c. Other soluble forms coming from shedding or dead cells which correspond to HLA-G1, -G2, -G3 or -G4 are named with the same nomenclature that membrane form following by a " s " : HLA-G1s, -G2s, -G3s or -G4s.
This nomenclature must be respected in all abstracts and presentations in the next International Workshop Meeting.
PRE-WORKSHOP GROUP CONCLUSIONS
All tools and protocols are available in Human Immunology 61:1177-1195, 2000.
