The Edward Jenner Institute for Vaccine Research, Compton, Berkshire RG20 7NN, UK
Correspondence to: E. Tchilian; E-mail: elma.tchilian{at}jenner.ac.uk
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Abstract |
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Keywords: CD45, CD45 isoforms, CD45 polymorphism, viral infection
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Introduction |
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In spite of extensive data linking expression of different CD45 isoforms to lymphocyte function and conservation of N-terminal alternative splicing in fish, birds and mammals, there is very little understanding of the role of CD45 isoforms. It is clear that a large extracellular domain is required for TcR signaling in transfected cell lines (12), but no specific ligand has been found for CD45, although various interactions with lectin like molecules have been reported (1316). The formation of homo- and heterodimers by CD45 isoforms has been proposed as another mechanism that may regulate CD45 phosphatase activity (17). Recently a series of transgenic mice expressing single low molecular weight CD45RB and CD45R0 isoforms under the control of the vav promoter has been constructed (18). These mice show that either isoform is effective in restoring T, but not B cell development and function.
We and others have taken a different approach to understanding the function of CD45 by seeking to identify polymorphisms of human CD45, define their phenotypic and functional characteristics and study disease associations. The most extensively studied polymorphism is the C77G transversion of exon 4 (19,20). This is a point mutation in a splice silencer region of the exon, and individuals with the 77G allele are unable to splice out the exon normally (21,22). Activated/memory lymphocytes in these individuals continue to express both CD45RA and CD45R0 isoforms in contrast to the normal pattern of low molecular weight CD45R0 expression. We have reported an excess of C77G individuals in a cohort of HIV patients (odds ratio 3.7) (23,24) and others have reported associations of C77G with autoimmune hepatitis (25) and systemic sclerosis (26). Multiple sclerosis has also been reported to be associated with C77G in Germany and Italy (27,28) although no association has been shown in Swedish and North American cohorts (29,30). These disease associations are relatively weak but the studies are difficult to perform because of the low frequency of the C77G allele (less than 2%) in England, Germany, Sweden, Italy and North America (24). Extensive functional studies have not been reported but these individuals do have an increased proportion of activated T cells (31), supporting the idea that they may have subtly altered immunological function. Homozygotes have not been described.
Several other rare polymorphisms have been reported (24,32,33) but the first common polymorphism to be described is A138G in exon 6, which is present at a high frequency in the Far East (34). Individuals homozygous for the 138G variant have more CD45R0 cells and these cells have a phenotype characteristic of more differentiated T cells, expressing less CD62L, CD27, CD28 and CCR7, more CD95 and high levels of CD11a. The immunomodulatory effects of the A138G polymorphism are reflected in disease associations, since cohorts of Japanese patients with HBV infection or autoimmune Graves' thyroiditis show reduced numbers of 138G individuals compared to controls (relative risks of 2.7 and 2.2 respectively).
All of these data indicate that a quite subtle alteration in alternative splicing of CD45 can result in readily measurable phenotypic and functional effects, as well as influencing infectious and autoimmune disease susceptibility. These data have prompted us to construct transgenic mouse models that can be used in the future to investigate the biochemical effects of abnormal CD45 isoform expression. Since the most common CD45 variants are characterized by either increased CD45RA (exon 4 C77G) or CD45R0 expression (exon 6 A138G) we constructed Tg mice expressing single murine CD45RABC or CD45R0 isoforms on an exon 9-targeted CD45/mouse background. Here we show that the level of single CD45 isoform expression is critical for development and function of T cells. We also show that whereas mice expressing different single isoforms at comparable levels function equally well, altered CD45 expression affects lymphocyte phenotype and turnover.
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Methods |
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Flow cytometric analysis
The following reagents and antibodies were used to stain cell suspensions: monoclonal antibodies against mouse pan-CD45CyChrome (30-F11), CD3PE (17A2), CD4FITC (GK1.5), CD8PerCP (53-6.7), CD69biotin (H1.2F3), CD44PE (IM7), Ly6Cbiotin (AL-21), CD11aPE (2D7), CD122PE (Tm-ß1), CD132PE (4G3), CD25PE (PC61) (all from BD Biosciences, Oxford, UK), CD62LPE (MEL-14, Caltag, Silverstone, UK). Isotype matched mAbs were used as controls. 10 000 or 50 000 events per sample were collected on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and analysed using WinMDI software.
B lymphocyte stimulation
B cells were stimulated by placing 4 x 105 splenic cells in a round bottom 96-well plate in IMDM (10% FCS, 105 M ß-mercaptoethanol, Life Technologies) in the presence of the following mitogens: F(ab')2 fragment of goat anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA) at 10 µg/ml, anti-CD40 (clone 1C10, R&D Systems, Abingdon, UK) at 5 µg/ml and Escherichia coli lipopolysaccharide (LPS, Sigma, Poole, UK) at 5 µg/ml. Cells were harvested at 72 h after a 12 h pulse with 1 µCi of [3H]thymidine per well.
Influenza virus infection
The HKx31 (H3N2) strain of influenza virus was grown in the allantoic cavity of 10-day-old embryonated hen eggs and stored at 70°C until use. Mice were anaesthetized by intra-peritoneal injection of 2,2,2-tribromoethanol (Avertin) and infected intranasally with 30 µl of phosphate-buffered saline containing 5000 EID50 (50% egg infectious dose) of influenza virus. On days 9 and 30 post-infection, mice were sacrificed and mediastinal lymph nodes, spleens and serum were collected. Cytotoxic T cells (CTL) were generated in bulk cultures as previously described (36). The presence of influenza virus was assessed by injecting 0.1 ml volumes of homogenized lung tissue from individual mice (and 10-fold dilutions) into the allantoic cavities of 10-day-old embryonated hen eggs (obtained from the Poultry Production Unit, Institute for Animal Health, UK). Allantoic fluids were tested 4872 h later for haemagglutinating activity using chicken red blood cells. Influenza virus-specific serum antibody titres were determined as described previously (37). Purified influenza virus for coating ELISA plates was obtained from SPAFAS Laboratories (Preston, CT) and goat anti-mouse isotype-specific reagents conjugated to alkaline phosphatase were from Southern Biotechnology Associates Inc. (Birmingham, AL).
LCMV infection
Stocks of lymphocytic choriomeningitis virus (LCMV) Armstrong 53b, a clone triple plaque purified from ARM CA 1371 (38) were prepared by growth on baby hamster kidney cells, and titers were determined by plaque assay on Vero cells as previously described (38). Mice were infected with LCMV by intraperitoneal (i.p.) inoculation of 2 x 105 plaque forming units (p.f.u.) of virus in a 200 µl volume. Viral titers in the spleen and liver of LCMV-infected animals were determined by plaque assay on Vero cells. The CD8+ T cell response induced following LCMV infection was assessed by antibody and tetramer staining using PE-labeled tetrameric complexes of H2-Db bound to the LCMV GP-13341 peptide (ProImmune, Oxford, UK), and CD8Cy5 antibody.
Turnover of T and B cells
The turnover of T and B cell subsets was analysed as previously described (39). Mice underwent thymectomies at 56 weeks of age. After surgery, the mice were left for 1 month and then given sterile drinking water containing 0.8 mg/ml bromodeoxyuridine (BrDU, Sigma) for 7 days. Cell suspensions from lymph nodes or spleen were surface stained using for T cells CD4PE, CD8Cy5, CD44biotin or CD122biotin and for B cells IgMPE and IgDbiotin. Biotinylated antibodies were followed by streptavidinCyChrome (BD Biosciences). After surface staining the cells were washed, resuspended in cold 0.15 M NaCl and fixed by dropwise addition of cold 95% ethanol. The cells were incubated for 30 min on ice, washed with PBS, then incubated with PBS containing 1% paraformaldehyde and 0.01% Tween 20 for 1 h. Cells were pelleted, then incubated with 50 Kunitz units DNase I (Sigma) in 0.15 M NaCl, 4.2 mM MgCl2, for 10 min. After washing, the cells were incubated with FITC-conjugated anti-BrDU mAb (Beckton Dickinson) and analysed on a FACSCalibur flow cytometer.
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Results |
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Altered phenotype of mature peripheral T lymphocytes in CD45 Tg mice
The percentages and absolute number of mature CD4 and CD8 T cells were restored to normal in mesenteric, mediastinal and peripheral lymph nodes of both CD45RABChi and CD45R0hi mice (Table 1). However, numbers of T cells detected in the spleens of either CD45RABChi or CD45R0hi mice were reduced to 50% of that of wild type for each subset. In the CD45R0lo mice, 3-fold fewer CD4 and CD8 cells were detected in lymph nodes and spleens compared to the CD45R0hi mice (Table 1).
In order to assess the activation state of lymphocytes in CD45Tg mice, the surface expression of a panel of lymphocyte antigens was analysed in CD4 and CD8 cells of lymph node (not shown) and spleen (Fig. 1B). CD3 expression was reduced on both CD4 and CD8 cells in CD45RABChi and CD45R0hi Tg mice. Cell surface markers of memory CD8 T cellsCD44, CD11a, Ly6C and CD122were upregulated on CD8 cells in both Tg mice in lymph nodes and spleen, while CD4 Tg cells showed increased expression of CD44 and CD11a. The proportion of cells expressing the lymph node homing adhesion molecule CD62L was decreased in CD45RABChi and CD45R0hi Tg mice compared to CD45+/+ controls. Neither the CD4 or CD8 subset showed evidence of recent TCR activation, both expressing low amounts of CD25 and CD69 (data not shown), indicating that these cells have been previously activated and now have a memory/effector phenotype. CD45R0lo mouse T cells showed a similar activated phenotype to T cells of CD45R0hi and CD45RABChi mice (data not shown). Taken together these results show that the levels of CD45 Tg expression are important for restoration of cell number and that the cells in the periphery in all Tg lines are memory/effector phenotype.
Bone marrow development and peripheral B cell maturation in CD45 Tg mice
The total number of nucleated bone marrow cells was comparable in all of the CD45 mice. In the CD45R0hi and CD45R0lo lines the transgene was expressed on a proportion of the CD43+/low progenitor cells and on all cells expressing IgM and IgD. Interestingly in CD45RABChi mice a proportion of IgM and IgD cells did not express the transgene although all of the T cells were transgene positive. Similar heterocellular patterns of CD45 Tg expression have been reported previously with Tg mice expressing a single murine CD45R0 isoform (18). Both murine CD45 transgenes were also expressed on a proportion of the bone marrow myeloid Gr-1+ cells and were absent on erythrocytes as determined by staining with TER119 (data not shown).
All CD45 Tg mice had higher numbers of B cells in LN and spleen, although the increase was not statistically significant for lymph nodes of CD45RABChi or CD45R0hi mice (Table 2). IgM+ B cells in lymph nodes and spleen expressed the CD45R0 transgene; however in CD45RABChi mice, CD45 was not detected on a high proportion of IgM+ cells (70%) (Fig. 2A). We examined the expression of IgM and IgD in lymph nodes and spleen in CD45+/+, CD45/, CD45RABChi and CD45R0hi mice (Fig. 2B). In CD45/ and CD45Tg mice the transition from immature intermediate (population II: IgMhi IgDhi) to mature (population I: IgMloIgDhi) B cells was impaired. There is a large increase in the number of immature population II cells, while the mature population I is decreased in all CD45 Tg and CD45/ mice. A similar distribution and phenotype was observed in CD45R0lo (data not shown). In agreement with previous observations neither the CD45RABC or CD45R0 transgenes restored signaling following anti-IgM antibody crosslinking in vitro, while all of the CD45 Tg B cells proliferated as well as wild type to CD40 and LPS activation (Fig. 2C).
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Normal anti-influenza T and B cell responses in CD45 Tg mice
To examine the immune responses of CD45RABChi and CD45R0hi mice in vivo, we infected these mice with x31 influenza virus. Acute phase influenza virus specific immune responses were analysed at day 9 and memory responses at day 36. The ability to generate CTL responses after infection with influenza virus was tested in mediastinal lymph nodes and spleen by in vitro restimulation assays. CD45/ mice did not mount CTL responses, while both CD45RABChi and CD45R0hi mice showed normal CTL responses against influenza virus in spleen and mediastinal lymph nodes, 9 and 36 days after the initial infection (Fig. 3A). These results were further supported by data indicating complete clearance of virus from the lungs (a CD8 T cell dependent process) by day 9 in both CD45RABChi and CD45R0hi Tg and wild-type mice, while in CD45/ mice virus titres of 7 x 104 to 1 x 105 EID50 were detected. We investigated antibody responses to influenza virus in CD45Tg mice at day 9 as an indicator of a primary response. All the mice were able to mount IgM (data not shown) and IgG responses (Fig. 3B) and there was no consistent difference between CD45+/+ or CD45 transgenic mice, showing that all of the mice were capable of isotype switching. These results indicated that the CD4 cells in CD45 Tg mice are able to provide effective help. At day 36 both CD45Tg mouse lines also maintained good IgG responses (Fig. 3B).
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LCMV infection
Since the most profound defect in both CD45 Tg lines was the partial block in development of the CD8 lineage and reduced number of CD8 cells in the spleen, we next examined immune responses after acute infection with lymphocytic choriomeningitis virus (LCMV), a well-characterized model for studying systemic CD8 T cell immunity. Infection of CD45+/+ mice with LCMV Armstrong results in expansion of a strong virus-specific CD8 T cell response, peaking at day 8 post-infection in the spleen.
At this time-point we observed a marked increase in both the total number (not shown since half of each spleen was used for virus titration) and the percentage (Fig. 4A) of CD8+ T cells in the spleen of CD45+/+ mice. There was also a dramatic expansion of CD8+ T cells in the spleen of CD45 transgenic mice and we observed some (although a much smaller) increase in the percentage of CD8+ T cells in the spleen of CD45/ mice (Fig. 4A). Tetramer staining (data not shown) revealed that 15.9% (mean of three mice) of splenic CD8+ T cells were specific for the Db-restricted LCMV GP3341 epitope in CD45+/+ mice at day 8 post-infection, the transgenic lines varying from 6 to 27%. The percentage of tetramer positive CD8 cells in uninfected control mice varied from 2% in CD45+/+ to 20% in CD45/ mice, reflecting the difficulty of tetramer staining the very low percentage of splenic T cells in these mice and inevitable high background. However, the total number of GP3341-specific CD8 T cells in the spleen of CD45/ and CD45 Tg mice was lower than in CD45+/+ mice because of their reduced overall numbers of splenic CD8+ T cells. The total number of LCMV-specific CD8 T cells in the spleen of CD45+/+ mice on day 8 post-infection was estimated to be 45 times the number in CD45RABChi and CD45R0hi mice, and 1020 times the number in CD45ROlo and CD45/ mice.
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Turnover of T and B cells
We analysed the turnover of T and B cell subsets by administering the DNA precursor BrDU in the drinking water and then examining the surface markers on BrDU labeled cells. Our results show a trend toward increased turnover of T cells in CD45RABChi and CD45R0hi mice (Table 3). This is apparent in both spleen and lymph nodes, and the difference between the CD45 Tg and CD45+/+ mice is more pronounced in CD8 than in CD4 T cells. Most of the proliferating cells in the CD45Tg mice were CD44hi or CD122hi (data not shown). The increased turnover in CD45 Tg mice was due to increased proportion of CD44hi or CD122hi cells in these mice. Although the turnover rate within CD44hi or CD122hi populations is similar in CD45+/+, CD45RABChi and CD45R0hi mice.
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Discussion |
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Recently, polymorphisms have been described in exon 4 (C77G, C59A) and exon 6 (A138G) of human CD45 that show significant disease associations as well as phenotypic and functional effects (24,27,32,34). These polymorphisms affect alternative splicing of the molecule, suggesting that subtle abnormalities and disturbance of the balance of different isoforms may be important for normal CD45 function. In order to explore this we generated transgenic mice expressing single murine CD45RABC or CD45R0 isoforms under the control of the CD2 promoter, giving good levels of expression in T cells.
Our results show that in mice with high level Tg expression, either CD45RABC or CD45R0 isoforms are capable of restoring thymic differentiation, peripheral T cell function and immune responses to influenza virus and LCMV. In contrast, CD45R0lo Tg mice with much lower transgene expression show a severe block in thymic development, have very few peripheral T cells and reduced response to LCMV. In sum, high and low molecular weight CD45 isoforms restore equally well the development, activation and distribution of lymphocyte but the level of expression of a single isoform is critical for these processes. Whether more discriminating assays of T cell function, for example responsiveness to autoantigens, would reveal differences between mice with equivalent surface expression of different isoforms, remains to be determined.
Two other goups have constructed CD45 single isoform transgenic mice. In the first set, the CD45RABC and CD45R0 transgenes were under the control of the lck promoter confering high expression in the thymus and restoring thymic differentiation. However, the expression of the transgenes was at a low level in the periphery (40) and differences between mice with different isoforms, particularly the poorer T cell function in mice with expression of CD45RABC isoform, are hard to interpret because the transgenes were expressed at different levels and the CD45 knockout mice used (exon 6 deleted) have residual low level CD45 expression.
In a set of more recently described single isoform mice, high level expression of low molecular weight CD45R0 and CD45RB transgenes was obtained using a vav promoter, on a truly CD45 null background (18). In these experiments, thymic development and peripheral T cell numbers are largely restored except in lines with low transgene expression. Interestingly even in mice with high level CD45 transgene expression, equivalent to the total amount of CD45 expressed in wild-type mice, B cell development is still abnormal, with persistence of increased numbers of immature cells.
Our transgenic mice were constructed using yet another promoter, that of human CD2. These mice show expression intermediate between the two other sets of transgenics discussed above but nevertheless the data presented in this paper add several novel observations to those reported previously. First we show that the highest molecular weight CD45RABC isoform is able to restore both thymic differentiation and peripheral T cell function equally as well as low molecular weight CD45R0. Furthermore, restoration of T cell function is demonstrable in vivo in demanding anti-viral response requiring both generation of CTL and their entry into the lung and spleen. Second we measured the turnover of T and B cells and show increased proliferation of CD8 and a block in B cell development in CD45 Tg lines, although no differences in the turnover between the CD45RABChi and CD45R0hi Tg mice were detected. We also show that even in mice with an intermediate to high level of CD45, entry to or survival of T cells in the spleen may be compromised [as reported previously (40)], suggesting that either even higher level expression of a single isoform or expression of a combination of isoforms is required.
In contrast, following influenza virus or LCMV infection, virus specific T cells are readily detected in the spleen. Entry and/or survival of these activated cells is presumably less CD45 dependent and migration of activated CD8 Tg cells into the lung (after influenza virus) or liver (after LCMV) were unaffected in both CD45 Tg mice (data not shown) further suggesting that in vivo activated CD45Tg CD8 cells can migrate into the spleen and non-lymphoid tissues.
All of our Tg mice exhibited a block in peripheral B cell maturation. Even transgenic mice expressing high/intermediate levels of CD45RABC, the dominant isoform of normal B cells, fail to generate normal numbers of mature B cells. Although a caveat to this is that the CD45RABC mice express the transgene on only a proportion of B cells at all stages of differentiation. A similar lack of mature B cells has been reported in transgenic mice expressing CD45R0 or RB isoforms at levels approximating to the total amount of CD45 of all isoforms expressed on wild-type cells (18). Interestingly, in spite of the block in maturation of peripheral B cells both CD45RABChi and CD45R0hi mice developed an IgG response to influenza virus and LCMV (data not shown). This is in accord with data showing that despite signaling abnormalities, CD45/ mice have normal immunoglobulin levels and B cell responses to T cell-dependent stimuli in vivo if CD45+/+ helper T cells are provided (18).
Our mice also show another abnormality not previously described in CD45 transgenics, that the peripheral T cells have an activated phenotype with higher CD44 and lower CD62L expression. At first we considered that this was mostly likely because the mice had lower peripheral T cell numbers because of difficulties in thymic differentiation and the activated phenotype was a consequence of homeostatic mechanisms regulating cell numbers. This notion was supported by the increased turnover of the very few T cells in CD45KO mice (data not shown). However, although lower peripheral T cell numbers may partially account for the altered proliferation in CD45RABChi and CD45R0hi mice, this does not appear to be the whole answer. First, even single positive CD4 and CD8 thymocytes show the activated phenotype and in the periphery, the CD45RABChi and CD45R0hi mice, which have similar peripheral T cell numbers to wild-type mice have increased proportions of cells with an activated phenotype. In B cells, although there are normal or increased numbers of peripheral B cells and overall B cell proliferation is not altered in CD45/ or Tg mice, the phenotype of the proliferating B cells in the CD45/ or Tg mice is more immature than in wild-type mice. We therefore suggest that normal differentiation and homeostasis of T and B cells requires not only a threshold level of total CD45 but also expression of correct combinations of isoforms.
This suggestion is supported by human data. Individuals with either the C77G or A138G polymorphisms have increased numbers of T cells with a memory/activated phenotype (31,34). This is particularly striking in the case of C77G, because in these individuals the defect in splicing prevents generation of memory/effector cells expressing only low molecular weight isoforms (CD45R0 and CD45RB). It is also notable that the increased proportion of memory/activated cells is more apparent among CD8 than CD4 cells in both humans with the variant alleles and mice with altered expression of CD45 isoforms.
At present, the mechanisms underlying the altered phenotype and function of lymphocytes of humans with variant CD45 alleles or mice with altered expression of CD45 isoforms at the cell surface are not known. However, homo-dimerization of CD45 molecules or cis or trans interactions with other cell surface molecules or soluble ligands, have been suggested to be possible mechanisms for regulating the phosphatase activity (12,17) and an altered balance of different isoforms at the cell surface could well affect these interactions. Whatever the mechanisms, mice with normal levels of surface expression of CD45, but an altered balance of different isoforms, will provide models to investigate both the biochemical events leading to an activated phenotype of lymphocytes and the immunological mechanisms underlying the disease associations of CD45 variants in humans.
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Acknowledgements |
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Notes |
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Transmitting editor: H. R. MacDonald
Received 28 April 2004, accepted 28 June 2004.
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References |
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