²First Affiliated Hospital，Nanjing Medical University，Nanjing 210029)

摘 要 为探讨CD45蛋白酪氨酸磷酸酶在γδ T细胞发育过程中的作用，观察CD45基因敲除变种鼠体内树突状表皮T细胞（DETC）等γδ T细胞。以原位免疫标记法检查小鼠表皮内DETC的形状、数量及免疫表型；以RT-PCR测定表皮细胞中Vγ3TCR mRNA之表达水平；FCM检测DETC在表皮细胞中以及另一组γδ T细胞、Vγ2 T细胞在淋巴结细胞中所占比例。结果显示，CD45缺失鼠与野生型鼠相比，上述各项指标仍无明显差异。提示CD45分子虽在αβ T细胞发育过程中起重要作用，但对包括DETC及Vγ2 T细胞在内的γδ T细胞发育成熟可能并非必需。

中图号 R392.11

Abstract To investigate the role of CD45 protein tyrosine phosphatase in γδ T cell development，we exa-mined whether Vγ3 dendritic epidermal T cells （DETC），a subset of γδ T cells uniquely reside in the murine epidermis were altered in the CD45-gene -deficient mice．In situ immunolabelling on epidermal sheets demonstrsted that the CD45-deficient mice had a normal density and immunophenotype of Vγ3 DETC in comparison to the wild-type control mice．RT-PCR revealed that similar levels of Vγ3 TCR mRNA were present in the epidermis of both CD45-deficient mice and wild-type controls．FCM showed no significant difference in the proportion of Vγ3 T cells in the epidermal cells between the two genotypes．In addition， the frequency of Vγ2 T cells， another subset of γδT cells in lymph nodes was normal in CD45-deficient mice．These results indicate that althouh CD45 is crucial for the development of αβ Τ cells，it might be not necessary for the thymic maturation of γδ T cells including Vγ3 DETC and Vγ2 T cells．

Key words CD45 protein tyrosine phosphatase （CD45 PTPase）， Dendritic epidermal T cells （DETC）， γδ T cell， T cell generation

CD45 is a transmembrane protein tyrosine phosphatase （PTPase） expressed on all leukocytes，and plays a pivotal role in T cell development．T cells can subdivide into two distinct li-neages，αβ T cell and γδ T cell．In the shaping of the αβT cell repertoire，CD45 has been demonstra-ted to play an important role in its positive and negative selection processes， but the role of CD45 in the generation of γδ T cells remains unclear．The dendritic epidermal T cells （DETC） resident in the murine skin belong to the γδ T cell lineage and express TCR Vγ3，so they are also termed Vγ3 DETC^［1］．In this study， we examined Vγ3 DETC in CD45-deficient mice by in situ immunolabelling， FCM and RT-PCR to investigate the role of CD45 in the development of γδ T cells．

MATERIALS AND METHODS

Mice and antibodiesCD45-gene-targeted mutant mice（－／－mice）^［2］ were kindly supplied by Dr． TW Mak （University of Toronto Canada）．C57BL／6 mice were used as a wild-type control（＋／＋mice）．All mice were used at 8～12 weeks of age．Monoclonal antibodies（mAbs）：anti-Vγ3 TCR，Vγ2，TCR，γδ TCR，anti-Vγ3 TCR／FITC，anti-Thy-1．2／PE，anti-CD32／CD16（to block FcrR Ⅱ／Ⅲ），etc （Pharmingen， USA）．Anti-CD3，CD4，CD8，CD45， anti-Thy-1．2，anti-CD3／PE，Goat anti-rat IgG／biotin，Goat anti-hamster IgG／biotin，Goat anti-mouse IgM／biotin and streptavidin／FITC，Extravidin／TRITC（Serotec Canada and Sigma，USA）．

In situ Immunolabelling on epidermal sheetsEpidermal sheets obtained from the ears of mice were indirectly immunolabelled with anti-Vγ3 TCR（incubated with anti- Vγ3 TCR mAbs，secondary antibody／biotin，then streptavidin／FITC）．Alternatively，epidermal sheets were directly labelled with anti-Vγ3 TCR／FITC．Positive labelled dendritic epidermal cells were counted in 10 random fields with the aid of a micrometer grid．

For double immunolabelling， epidermal sheets were incubated with mAbs（anti-CD3，CD4，CD8，Thy-1， γδ TCR）， secondary antibody／biotin， Extravidin／TRITC as above， and finally double labelled with anti-Vγ3 TCR／FITC．

Flow cytometry （FCM）Epidermal cell suspensions were prepared and subjected to a 15min density gradient centrifugation．Cell（10⁶） were incubated for 5min on ice with anti-CD32／CD16（to block FcrR Ⅱ／Ⅲ），then reacted with anti-Vγ3 TCR／FITC for 45min on ice．After washing，the cells were reacted with anti-Thy1．2／PE for ano-ther 45min on ice for two color immunofluorescence．

Lymph node cell suspensions were prepared from the inguinal and axillary lymph nodes．Cells were double labelled as the epidermal cells mentioned above，but with anti-Vγ2 TCR／FITC and anti-CD3／PE instead of anti Vγ3 TCR／FITC and anti-Thy-1．2-PE to examine Vγ2 T cells， another subset of γδ T cells located mainly in the secondary lymphoid organs．

Two-color immunofluorescence was analyzed on a coulter EPICS75L flow cytometer for the epidermal cells and lymph node cells．A minimum of 20 000 cells were analyzed for each sample．

Reverse transcriptese-polymerase chain reaction （RT-PCR）The primers^［3］for TCR Vγ3：5’-TGTGCACGTGTACCAACTGA-3’sense），5’-CAGAGGGAATTACTATGAGC-3’antisense） and the primers for β-actin：5’-GTGGGCCGCTCTAGGCACCAA-3’sense），5’-CTCTTTGATGTCACGCACGATTTC-3’antisense） were synthesized by Dolton Chemical Laboratories Inc（Canada）． Total RNA were extracted from epidermal cells suspensions， and from liver and thymus homogenized in liquid nitrogen（as controls ）by single-step method of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction as described^［4］．cDNA synthesis and amplification were performed essentially as previously des-cribed^［5］．PCR product was electrophoresed on 1．6％ agalose gel containing EtBr and was photographed under UV light．For relative semi-quantification， photo-negatives were used for densitometric scanning． The densitometry values of Vγ3 TCR was normalized to that of the house-keeping gene，β-action．

RESULTS

Morphology，density and phenotype of Vγ3 TCR cells are normal in the epidermis of CD45-deficient miceThe indirect immunolabelling of epidermal sheets demonstrated that there was no significant difference in morphology and density of Vγ3 TCR^＋ cells between CD45—／—mice and the ＋／＋mice （417±52 cells／mm² vs 423±41 cells／mm²，±s，P＞0．05）．

The epidermal sheets stained with direct immunolabelling technique showed that the labelling of Vγ3 TCR^＋ cells was weaker and the dendritic processes were less obvious than those with indirect immunolabelling．

Phenotypic analysis of Vγ3 TCR^＋ cells with double immunolabelling demonstrated that these cells were CD3^＋，CD4^－，CD8^－，Thy-1^＋ and γδ TCR^＋ in both genotype mice，confirming that they were DETC．

Expression of TCR Vγ3 mRAN is normal in CD45-deficient miceThe results of RT-PCR showed that abundant TCR Vγ3 transcripts were present in the epidermis from both CD45—／—mice and the ＋／＋mice， but not in the liver or thymus（Fig 1）．The relative amount of PCR products （TCR Vγ3／β-actin） determined by scanning of negative films using a laser densitometer were 0．62±0．08 and 0．68±0．07（±s， P＞0．05） in the epidermis of CD45－／－mice and the ＋／＋ mice，<