CD45 MOLECULE AND GENERATION OF EPIDERMAL γδ T CELLS FROM MIC
作者：Cai Qinchao (蔡钦朝)1， Wang Binghe(王秉鹤)2s:%t), 百拇医药
单位：s:%t), 百拇医药
关键词：词 CD45蛋白酪氨酸磷酸酶；树突状表皮T细胞；γδT细胞；T细胞发育s:%t), 百拇医药
免疫学杂志990202(1Department of Biochemistry，Anhui College of Traditional Chinese Medicine，Hefei 230038s:%t), 百拇医药
2First Affiliated Hospital，Nanjing Medical University，Nanjing 210029)s:%t), 百拇医药
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。s:%t), 百拇医药
中图号 R392.11s:%t), 百拇医药
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 generationn6#3j, 百拇医药
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.n6#3j, 百拇医药
MATERIALS AND METHODSn6#3j, 百拇医药
Mice and antibodies CD45-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 sheets Epidermal 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.im;#, 百拇医药
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.im;#, 百拇医药
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.im;#, 百拇医药
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.-c@5d7, 百拇医药
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.
RESULTS68kvp, 百拇医药
Morphology，density and phenotype of Vγ3 TCR cells are normal in the epidermis of CD45-deficient mice The 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).68kvp, 百拇医药
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.68kvp, 百拇医药
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.68kvp, 百拇医药
Expression of TCR Vγ3 mRAN is normal in CD45-deficient mice The 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， respectively. No significant difference in the level of TCR Vγ3 mRNA was detected between the genotypes.
Fig 1 RT-PCR analysis of TCR Vγ3 mRNA from spidermal cells1oi5&, 百拇医药
Lane 1：CD45 -/-mice1oi5&, 百拇医药
Lane 2：CD45 +/+mice1oi5&, 百拇医药
Lane 3，4：negative controls(liver and thymus)1oi5&, 百拇医药
248 bp：TCR Vγ31oi5&, 百拇医药
540 bp：β-action1oi5&, 百拇医药
Proportion of Vγ3 DETC in epidermal cells is normal in CD45-deficient mice We detected the Thy-1 and Vγ3 TCR double-positive cells by two-color FCM.As shown in Fig 2，epidermal cells suspensions from CD45-deficient mice contained normal proportions of Vγ3 TCR+/Thy-1+ double-positive cells (DETC) as compared with the wild-type mice.
1oi5&, 百拇医药
Fig 2 Two-color FCM anslysis of the epidermal cells1oi5&, 百拇医药
a，c：from CD45+/+mice b，d：from CD45-/-mice1oi5&, 百拇医药
To examine whether CD45 molecules affect other subsets of γδ T cells，we detected Vγ2 T cells in lymph nodes by two-color FCM.About 1.2%～2.3% of lymph node cells were Vγ2 TCR+/CD3+ double-positive cells in both CD45-/-mice and the +/+mice.No significant difference in the frequency of Vγ2 cells was detected between these genotypes.
DISCUSSIONS{$v%fu, 百拇医药
αβ T cells and γδ T cells arise from common progenitors and segregate before the onset of TCR gene rearrangements[6].The ontogenesis of αβ T cells is clear.They develop from double negative immature cells in the thymus， then progress to a double positive stage，finally undergo a selection process to become mature single positive T cells. However， the ontogenesis of γδ T cells remain unclear and may not involve the double positive stage.{$v%fu, 百拇医药
CD45 is one of the most abundant leukecyte cell surface glycoproteins.As a transmembrane PTPase，it plays important roles in lymphocyte activation and thymic maturation.Antigen receptor induced singnal transduction in both T cell and B cell lines is crucially dependent on the transmembrane PTPase.To better investigate the in vivo role of CD45 in the development of lymphocytes，Kishihara et al[2]generated CD45-deficient mice by gene disruption of CD45-exon 6.In these mutant mice，B lymphocytes develop normally but T lymphocyte development is impaired.Thymocyte maturation is blocked at the transitional stage from immature double positive to mature single positive cells，and thus the number of peripheral T cells dramatically decreased. These observations suggest that CD45 PTPase plays a crucial role in the development of αβ T cells. But the role that CD45 plays in development of γδ T cells remains unclear.
Recently， Kawai et al[7]reported that the development of Vγ3 DETC was impaired in CD45 PTPase-deficient mice as well as in p561ck protein tyrosine kinase (PTKase)-deficient mice. Vγ3 DETC are a subset of γδT cells uniquely residing in the murine epidermis.In 1983，Tschachler et al[8]demonstrated that in addition to Langerhans cells murine epidermis harbours a population of Thy-1+，MHC class Ⅱ+ dendritic cells. With the demonstration that they express TCR composed of Vγ3，Jγ1， CR1/Vδ1，Dδ2，Jδ2，Cδ gene segment，these cells were named dendritic epidermal T cells (DETC) or Vγ3 DETC..)ewn4(, 百拇医药
In contrast to the finding of Kawai et al，out study showed no significant difference in the density，morphology and phenotype of Vγ3 DETC between CD45-deficient mice and wild-type mice.The discrepancy between the two groups may be related to the experimental protocol.In our study，for direct immunolabelling we incubated the epidermal sheets with FITC-conjugated anti-Vγ3 TCR mAb overnight at room temperature，while Kawai et al incubated the sheets for only 1h at 37°C.Secondarily，the staining of Vγ3 DETC was weaker and the dendritic processes were less ob-vious in the direct immunolabelling technique than in indirect immunolabelling.
It is believed that both PTKase and PTPase play important roles in the signal transduction during thymocyte development，but they act at different time points.Unlike the block at the transitional stage from double positive cells to single positive cells in CD45 PTPase-deficient mice，the block in p561ckPTKase-deficient mice occurs at an early stage，before positive and negative selection processes commence[9].Therefor the maturation of both αβ T cell and γδ T cells are impaired after p561ck gene disruption.However， CD45 only affect the development of αβ T cells.|4h%{^, 百拇医药
作者简介 第一作者：男，56岁，本科，副教授|4h%{^, 百拇医药
REFERENCE|4h%{^, 百拇医药
1 Asarnow DM，Thomas G，LeFrancois L，et al.Distinct antigen receptor repertoires of two classes of murine epithelium-associated T cells.Nature，1989，341：60|4h%{^, 百拇医药
2 Kishihara K，Penninger J，Wallace VA，et al.Normal B lymphocyte development but impaired T cell maturation in CD45- exon 6 protein tyrosine phosphatase-deficient mice.Cell，1993，74：143|4h%{^, 百拇医药
3 Erb KJ，Hanke T，Schimpl A，et al.Impaired survival of T cell receptor Vγ3 cells in interleukin-4 transgenic mice.Eru J Immunol，1995，25：1442
4 Chomczynski P，Sacchi N.Single step method of RNA isolation by acid guanidium thiocyanate- phenol-chloroform extraction.Anal Biochem，1987，162：156m?w, 百拇医药
5 Kondo S，Kono T，Sauder DN，et al.IL-8 gene expression and production in human keratinocytes and their modulation by UVB.J Invest Dermatol，1993，101：690m?w, 百拇医药
6 Petrie HT，Scollay R，Shortman K.Commitment to the T cell receptor-αβ or-γδ lineages can occur just prior to the onset of CD4 and CD8 expression among immature thymocytes.Eur J Immunol，1992，22：2185m?w, 百拇医药
7 Kawai K，Kishihara K，Molina TJ，et al.Impaired development of Vγ3 dendritic epidermal T cells in p561ck protein tyrosine kinase-deficient and CD45 protein tyrosine phosphatase-deficient mice.J Exp Med， 1995，181：345m?w, 百拇医药
8 Tschachler E，Schuler G，Hutterer J，et al.Expression of Thy-1 antigen by murine epidermal cells.J Invest Dermatol，1983，81：282m?w, 百拇医药
9 Molina TJ，Kishihara K，Siderovski DP，et al.Profound block in thymocyte development in mice lacking p561ck.Nature，1992，357：161m?w, 百拇医药
(1997-10-10收稿；1998-06-09修回)(Cai Qinchao (蔡钦朝)1， Wang Binghe(王秉鹤)2)
单位：s:%t), 百拇医药
关键词：词 CD45蛋白酪氨酸磷酸酶；树突状表皮T细胞；γδT细胞；T细胞发育s:%t), 百拇医药
免疫学杂志990202(1Department of Biochemistry，Anhui College of Traditional Chinese Medicine，Hefei 230038s:%t), 百拇医药
2First Affiliated Hospital，Nanjing Medical University，Nanjing 210029)s:%t), 百拇医药
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。s:%t), 百拇医药
中图号 R392.11s:%t), 百拇医药
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 generationn6#3j, 百拇医药
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.n6#3j, 百拇医药
MATERIALS AND METHODSn6#3j, 百拇医药
Mice and antibodies CD45-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 sheets Epidermal 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.im;#, 百拇医药
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.im;#, 百拇医药
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.im;#, 百拇医药
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.-c@5d7, 百拇医药
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.
RESULTS68kvp, 百拇医药
Morphology，density and phenotype of Vγ3 TCR cells are normal in the epidermis of CD45-deficient mice The 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).68kvp, 百拇医药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.68kvp, 百拇医药
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.68kvp, 百拇医药
Expression of TCR Vγ3 mRAN is normal in CD45-deficient mice The 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， respectively. No significant difference in the level of TCR Vγ3 mRNA was detected between the genotypes.Fig 1 RT-PCR analysis of TCR Vγ3 mRNA from spidermal cells1oi5&, 百拇医药
Lane 1：CD45 -/-mice1oi5&, 百拇医药
Lane 2：CD45 +/+mice1oi5&, 百拇医药
Lane 3，4：negative controls(liver and thymus)1oi5&, 百拇医药
248 bp：TCR Vγ31oi5&, 百拇医药
540 bp：β-action1oi5&, 百拇医药
Proportion of Vγ3 DETC in epidermal cells is normal in CD45-deficient mice We detected the Thy-1 and Vγ3 TCR double-positive cells by two-color FCM.As shown in Fig 2，epidermal cells suspensions from CD45-deficient mice contained normal proportions of Vγ3 TCR+/Thy-1+ double-positive cells (DETC) as compared with the wild-type mice.
1oi5&, 百拇医药Fig 2 Two-color FCM anslysis of the epidermal cells1oi5&, 百拇医药
a，c：from CD45+/+mice b，d：from CD45-/-mice1oi5&, 百拇医药
To examine whether CD45 molecules affect other subsets of γδ T cells，we detected Vγ2 T cells in lymph nodes by two-color FCM.About 1.2%～2.3% of lymph node cells were Vγ2 TCR+/CD3+ double-positive cells in both CD45-/-mice and the +/+mice.No significant difference in the frequency of Vγ2 cells was detected between these genotypes.
DISCUSSIONS{$v%fu, 百拇医药
αβ T cells and γδ T cells arise from common progenitors and segregate before the onset of TCR gene rearrangements[6].The ontogenesis of αβ T cells is clear.They develop from double negative immature cells in the thymus， then progress to a double positive stage，finally undergo a selection process to become mature single positive T cells. However， the ontogenesis of γδ T cells remain unclear and may not involve the double positive stage.{$v%fu, 百拇医药
CD45 is one of the most abundant leukecyte cell surface glycoproteins.As a transmembrane PTPase，it plays important roles in lymphocyte activation and thymic maturation.Antigen receptor induced singnal transduction in both T cell and B cell lines is crucially dependent on the transmembrane PTPase.To better investigate the in vivo role of CD45 in the development of lymphocytes，Kishihara et al[2]generated CD45-deficient mice by gene disruption of CD45-exon 6.In these mutant mice，B lymphocytes develop normally but T lymphocyte development is impaired.Thymocyte maturation is blocked at the transitional stage from immature double positive to mature single positive cells，and thus the number of peripheral T cells dramatically decreased. These observations suggest that CD45 PTPase plays a crucial role in the development of αβ T cells. But the role that CD45 plays in development of γδ T cells remains unclear.
Recently， Kawai et al[7]reported that the development of Vγ3 DETC was impaired in CD45 PTPase-deficient mice as well as in p561ck protein tyrosine kinase (PTKase)-deficient mice. Vγ3 DETC are a subset of γδT cells uniquely residing in the murine epidermis.In 1983，Tschachler et al[8]demonstrated that in addition to Langerhans cells murine epidermis harbours a population of Thy-1+，MHC class Ⅱ+ dendritic cells. With the demonstration that they express TCR composed of Vγ3，Jγ1， CR1/Vδ1，Dδ2，Jδ2，Cδ gene segment，these cells were named dendritic epidermal T cells (DETC) or Vγ3 DETC..)ewn4(, 百拇医药
In contrast to the finding of Kawai et al，out study showed no significant difference in the density，morphology and phenotype of Vγ3 DETC between CD45-deficient mice and wild-type mice.The discrepancy between the two groups may be related to the experimental protocol.In our study，for direct immunolabelling we incubated the epidermal sheets with FITC-conjugated anti-Vγ3 TCR mAb overnight at room temperature，while Kawai et al incubated the sheets for only 1h at 37°C.Secondarily，the staining of Vγ3 DETC was weaker and the dendritic processes were less ob-vious in the direct immunolabelling technique than in indirect immunolabelling.
It is believed that both PTKase and PTPase play important roles in the signal transduction during thymocyte development，but they act at different time points.Unlike the block at the transitional stage from double positive cells to single positive cells in CD45 PTPase-deficient mice，the block in p561ckPTKase-deficient mice occurs at an early stage，before positive and negative selection processes commence[9].Therefor the maturation of both αβ T cell and γδ T cells are impaired after p561ck gene disruption.However， CD45 only affect the development of αβ T cells.|4h%{^, 百拇医药
作者简介 第一作者：男，56岁，本科，副教授|4h%{^, 百拇医药
REFERENCE|4h%{^, 百拇医药
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(1997-10-10收稿；1998-06-09修回)(Cai Qinchao (蔡钦朝)1， Wang Binghe(王秉鹤)2)